Preparative Liquid Chromatography

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JOURNAL OF CHROMATOGRAPHYLIBRARY - volume 38

preparative liquid chromatography

This Page Intentionally Left Blank

JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 38

preparative liquid chromatography edited by Brian A. Bidlingmeyer Waters Chromatography Division of Millipore, 34 Maple Street, Milford, MA 01 757, U.S.A.

ELSEVlER Amsterdam - Oxford - New York - Tokyo

1987

ELSEVIER SCIENCE PUBLISHERSB.V. Sara Burgerhartstraat 25 P.O. Box 21 1, lo00 AE Amsterdam, The Netherlands Distributors for the United States and Canada: ELSEVIER SCIENCE PUBLISHING COMPANY INC. 655,Avenue of the Americas New York, NY 10010, U S A .

First edition 1987 Second impression 1989 Third impression 1991

ISBN 0-444-42832-1 0 Elsevier Science PublishersB.V., 1987 All rights reserved. No part of this publication rnay be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written per-mission of the publisher, Elsevier Science Publishers E.V./ Academic PublishingDivision, P.O. Box 330, loo0 AH Amsterdam, The Netherlands. Special regulationsfor readers in the USA -This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication rnay be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the publisher. No responsibility is assumed by the Publisherfor any injury and/or damage to persons or property as a matter of products liability, negligenceor otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer. This book is printed on acid-free paper. Printed in The Netherlands

V CW-EHTS Journal o f Chromatography L I b r a r y (other volum6s I n t h e s e r l e s )

XI

Contr lb u t o r s

xi11

Preface

XIV

Adcnwle d p e n t s

1.

S t r a t e g l e s for Successful P r e p a r a t l v e L l q u i d Chromatography (P.D. McDonald and B.A. B I d l I n p y e r ) Introduction Planning t h e separation Achieving a separation Column l o a d a b t l i t y Scallng up a separation P r e p a r a t l v e LC packlngs and eluents separation system 1.7 P r e p a r a t i v e LC system c o n f i g u r a t i o n s 1.8 Conclusion 1.9 Acknowledgments 1.10 References

1.1 1.2 1.3 1.4 1.5 1.6

2.

2.9 3.

a

Introduct ion Layers f o r PTLC Sample appl i c a t i o n Development D e t e c t l o n o f zones Removal o f substances from t h e l a y e r A p p l i c a t l o n s o f PTLC Transfer o f r e s u l t s from TLC t o p r e p a r a t i v e l i q u i d chromatography References

I n c r e a s l n g the E f f l c l e n c y o f Very Large Scale Packed Bed Chroaatographlc Separatlons (P.C. Wankat)

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.

- Choosing

Introduction I n e f f i c i e n c i e s i n p r e p a r a t i v e e l u t i o n Chromatography Counter-current systems Simulated moving bed systems Hybrid methods: combine SMB and e l u t i o n chromatography Other methods f o r large-scale chromatography Acknowledgment Nomenclature References

P r e p a r a t l v e L l q u l d Chromatography I n the P h a m c e u t l c a l I n d u s t r y (A. Wehrl 1)

4.1 4.2 4.3 4.4 4.5 4.6

Introduction Major areas of p r e p a r a t i v e LC a p p l i c a t i o n i n pharmaceutical i n d u s t r y Chromatographic systems used Apparatus and examples Summary References

1 3 4 11 19 44

P r e p a r a t l v e T h l n Layer Chromatography (J. S h e m and B. F r l e d )

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

V II

55 81 94 94 95 105 105 106 109 111 113 115 117 122 125 129 129 131 134 138 144 147 148 149 149 153 154 154 157 159 179 180

VI 5.

6.

P r e p a r a t i v e Liquid Chromatography f o r t h e Synthetic Chemist (J.K. W h l t e s e l l )

183

5.1 5.2 5.3 5.4 5.5

183 187 198 200 20 1

Biochemical Applications of P r e p a r a t i v e L i q u l d Chromatography (W.S. Hancock and R.L. Prestldge) 6.1 6.2

6.3 6.4 6.5 6.6 6.7

7.

Introduction General requirements f o r amino a c i d and p o l y p e p t i d e separations P r e p a r a t i v e s e p a r a t i o n s o f amino a c i d s and p e p t i d e s Use o f reversed phase t h i n l a y e r chromatography f o r t h e monitoring o f p r e p a r a t i v e separations Preparative separations o f p r o t e i n s Other c l a s s e s o f b i o c h e m i c a l s References

The D i r e c t Preparative Resolutlan o f E n a n t i m e r s by L i q u i d Chromatography on C h l r a l S t a t l o n a r y Phases (W.H. P i r k l e and B.C. Hamper) 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8

a.

Introduction Computer s i m u l a t i o n s The r e a l w o r l d Practical matters References

Introduction Chromatographic methods f o r t h e i s o l a t i o n o f enantiomers Chromatographic p r o p e r t i e s o f c h i r a l s t a t i o n a r y phases (CSPS ) CSPs from n a t u r a l l y o c c u r r i n g m a t e r i a l s S y n t h e t i c CSPs Conclusions Addendum References

Preparative S i z e Excluslon Chromatography (G.L. 8.1 8.2 8.3 8.4 8.5 8.6

Index

Hagnauer)

Introduction I n s t r u m e n t a t i o n , columns and column packing m a t e r i a l s General requirements and s p e c i a l t e c h n i q u e s Optimization o f t h e preparative separation Applications References

203 203 204 211 222 223 23 0 23 1

235 23 5 23 7 24 0 242 248 281 28 1 283 289 289 293 298 312 3 24 33 1 335

VII

JOURNAL OF CHROMATOGRAPHY LIBRARY A Series of Books Devoted to Chromatographic and Electrophoretic Techniques and their Applications Although complementary to the Journal of Chromatography, each volume in the Library Series is an important and independent contribution in the field of chromatography and electrophoresis. The Library contains no material reprinted from the journal itself.

Other volumes in this series Volume 1

Chromatography of Antibiotics (see also Volume 26) by G.H. Wagman and M.J. Weinstein

Volume 2

Extraction Chromatography edited by T . Braun and G. Ghersini

Volume 3

Liquid Column Chromatography.A Survey of Modern Techniques and Applications edited by 2. Deyl, K. Macek and J. Janik

Volume 4

Detectors in Gas Chromatography by J . SevEik

Volume 5

Instrumental Liquid Chromatography.A Practical Manual on High-Performance Liquid Chromatographic Methods (see also Volume 27) by N.A. Parris

Volume 6

Isotachophoresis.Theory, Instrumentation and Applications by F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen

Volume 7

Chemical Derivatization in Liquid Chromatography by J.F. Lawrence and R.W. Frei

Volume 8

Chromatography of Steroids by E. Heftmann

Volume 9

HPTLC - High Performance Thin-Layer Chromatography edited by A. Zlatkis and R.E. Kaiser

Volume 10

Gas Chromatography of Polymers by V.G. Berezkin, V.R. Alishoyev and I.B. Nemirovskaya

Volume 11

Liquid Chromatography Detectors (see also Volume 33) by R.P.W. Scott

Volume 12

Affinity Chromatography by J. Turkovh

Volume 13

Instrumentation for High-Performance Liquid Chromatography edited by J.F.K. Huber

Volume 14

Radiochromatography.The Chromatography and Electrophoresis of Radiolabelled Compounds by T.R. Roberts

Volume 15

Antibiotics. Isolation, Separation and Purification edited by M.J. Weinstein and G.H. Wagman

VIII

Volume 16

Porous Silica. Its Properties and Use as Support in Column Liquid Chromatography by K.K. Unger

Volume 17

76 Years of Chromatography - A Historical Dialogue edited by L.S. Ettre and A. Zlatkis

Volume 18A

Electrophoresis. A Survey of Techniques and Applications. Part A: Techniques edited by Z. Deyl

Volume 18B

Electrophoresis. A Survey of Techniques and Applications. Part B: Applications edited by Z. Deyl

Volume 19

Chemical Derivatization in Gas Chromatography by J. Drozd

Volume 20

Electron Capture. Theory and Practice in Chromatography edited by A. Zlatkis and C.F. Poole

Volume 21

Environmental Problem Solving using Gas and Liquid Chromatography by R.L. Grob and M.A. Kaiser

Volume 22A

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part A: Fundamentals edited by E. Heftmann

Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. Part B: Applications edited by E. Heftmann

Volume 23A

Chromatography of Alkaloids. Part A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte

Volume 23B

Chromatography of Alkaloids. Part B: Gas-Liquid Chromatography and High-Performance Liquid Chromatography by R. Verpoorte and A. Baerheim Svendsen

Volume 24

Chemical Methods in Gas Chromatography by V.G. Berezkin

Volume 25

Modern Liquid Chromatography of Macromolecules by B.G. Belenkii and L.Z. Vilenchik

Volume 26

Chromatography of Antibiotics Second,Completely Revised Edition by G.H. Wagman and M.J. Weinstein

Volume 27

Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods Second, Completely Revised Edition by N.A. Parris

Volume 28

Microcolumn High-Performance Liquid Chromatography by P. Kucera

Volume 29

Quantitative Column Liquid Chromatography. A Survey of Chemometric Methods by S.T.Balke

IX

Volume 30

Microcolumn Separations. Columns, Instrumentation and Ancillary Techniques edited by M.V. Novotny and D. Ishii

Volume 31

Gradient Elution in Column Liquid Chromatography.Theory and Practice by P. Jandera and J. ChurLEek

Volume 32

The Science of Chromatography.Lectures Presented at the A.J.P. Martin Honorary Symposium, Urbino, May 27-31,1985 edited by F.Bruner

Volume 33

Liquid Chromatography Detectors. Second, Completely Revised Editon by R.P.W. Scott

Volume 34

Polymer Characterization by Liquid Chromatography by G. Glockner

Volume 35

Optimization of Chromatographic Selectivity. A Guide to Method Development by P.J. Schoenmakers

Volume 36

Selective GM Chromatographic Detectors by M. Dressler

Volume 37

Chromatography of Lipids in Biomedical Research and Clinical Diagnosis edited by A. Kuksis

Volume 38

Preparative Liquid Chromatography edited by B.A. Bidlingmeyer


XI

CONTR I BUTORS D r . Brian A. Bidlingmeyer, Waters Chromatography D i v i s i o n o f M i l l i p o r e , 34 Maple Street, M i l f o r d , MA 01757, USA D r . Bernard Fried, Department o f Biology, Lafayette College, Easton, PA, 18042, USA

D r . Gary L. Hagnauer, Polymer Research Division, U.S. Army Materials Technology Laboratory, Watertown, MA 02172, USA

D r . Bruce C . Hamper, The Roger Adams Laboratory, School o f Chemical Sciences, University of I l l i n o i s , Urbana, IL, 61801, USA D r . William S. Hancock, Department o f Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, New Zealand D r . P a t r i c k D. McDonald, Waters Chromatography D i v i s i o n of M i l l i p o r e , 34 Maple Street, M i l f o r d , MA 01757, USA D r . William H. P i r k l e , The Roger Adams Laboratory, School of Chemical Sciences, University of I l l i n o i s , Urbana, IL, 61801, USA D r . Ross L. Prestidge, Department o f Pathology, Auckland University School o f Medicine, Auckland, New Zealand

D r . Joseph Sherma, Department o f Chemistry, Lafayette College, Easton, PA, 18042, USA D r . P h i l l i p C . Wankat, School o f Engineering, Purdue University, West Lafayette, I N 47907, USA

D r . A. Wehrli, Sandoz Ltd., Pharmaceutical D i v i s i o n , Pre-Clinical Research, 4002 Basle, Switzerland

D r . James K . Whitesell, Department of Chemistry, The University o f Texas a t Austin, Austin, Texas 78712 USA


XI11

The c m e r c i a l b i r t h o f modern l i q u i d chromatography (LC) occurred a t the 1969 P i t t s b u r g h Conference when the f i r s t " L i q u i d Chromatograph" was introduced.

With

r o o t s reaching back t o amino a c i d analyzers and gel permeat i o n chromatographs o f the l a t e 1950's and e a r l y 1960's, the new LC i n s t r u m e n t a t i o n o f f e r e d new working techniques t o s c i e n t i s t s needing separations p r i o r to f i n a l analyses.

The

phenomenal growth o f LC as a l a b o r a t o r y took i n the years f o l l o w i n g t h a t f i r s t product i n t r o d u c t i o n i s w e l l known. Progress i n chemistry has always r e q u i r e d b o t h a n a l y s i s and i s o l a t i o n o f i n d i v i d u a l compounds i n m i x t u r e s .

I t i s , t h e r e f o r e , no s u r p r i s e t h a t t h e e a r l y

successes i n u s i n g LC for a n a l y s i s went hand-in-hand w i t h the development o f p r e p a r a t i v e LC

for i s o l a t i o n and p u r i f i c a t i o n .

I n the e a r l y 1970's, one o f the

key proponents o f Prep LC was Nobel Laureate D r . R.B. Woodward o f Harvard, who wrote i n an a r t i c l e d e s c r i b i n g the synthesis o f v i t a m i n 812, " t h e power o f these h i g h pressure l i q u i d chromatographic methods h a r d l y can be imagined by the chemis who has not had experience w i t h them . . . " (see Chapter 1 . 1 ) . D r . Woodward's comnents a r e reminiscent o f an e a r l i e r o b s e r v a t i o n o f the man who many b e l i e v e i s the e a r l i e s t chromatographer. M . S . Tswett.

Around the t u r n o

the century Tswett wrote "An e s s e n t i a l c o n d i t i o n for a l l f r u i t f u l research i s to have a t o n e ' s disposal s a t i s f a c t o r y technique."

Today, Prep LC has become a

" s a t i s f a c t o r y technique" i n the l a b o r a t o r y ; and as we increase our a c t i v i t i e s i n the management o f chemical composition, LC w i l l increase i t s r o l e i n advancing sc i ence.

I t i s the e d i t o r ' s b e l i e f t h a t separation development i s based m a i n l y upon experience and proceeds most e f f e c t i v e l y when i t i s c a r r i e d out w i t h focus on a p r a c t i c a l appl i c a t i o n .

Appl i c a t ion focus, coupled w i t h an understanding o f

s t r a t e g y and the i n t e r r e l a t i o n s h i p between the input and output o f the instrumentation, enables a s t r a i g h t f o r w a r d approach t o i s o l a t i o n and p u r i f i c a t i o n problems.

I t i s to t h i s p h i l o s o p h i c a l ideal t h a t t h i s book i s t a r g e t e d .

Chapters

1 and 2 a r e mainly discussions o f s t r a t e g y u s i n g a p p l i c a t i o n s o n l y t o i l l u s t r a t e points.

Chapters 3 t o 8 are a p p l i c a t i o n focused i n t h e i r e n t i r e t y .

I t i s hoped

that the researcher w i l l f i n d t h i s book b e n e f i c i a l i n a c h i e v i n g a speedy s o l u t i o n

to the most c h a l l e n g i n g p u r i f i c a t i o n requirements. BRIAN A . BIDLINGMEYER Waters Chromatography D i v i s i o n Mil

I i p o r e Corporation

XIV

ACKNOWLEDCMENTS S i n c e r e thanks i s extended t o t h e i n d i v i d u a l a u t h o r s f o r t h e i r c o o p e r a t i o n and c o n t r i b u t l o n s t o t h i s work.

I would a l s o l i k e t o acknowledge

w i t h g r a t i t u d e t h e many c o l l e a g u e s who o f f e r e d h e l p f u l comments and t e c h n i c a l d i s c u s s i o n s d u r i n g t h e p r e p a r a t i o n o f t h i s book.

O f p a r t i c u l a r assistance i n

p r o o f r e a d i n g t h e c h a p t e r s were C r a i g Dorschel, Jim O b e r h o l t z e r , Chuck Phoebe, Carmen Santasania, Michael Swartz, Tom T a r v i n , Mike Tomany and Vince Warren. A s p e c i a l thank-you i s extended t o Janet Newman whose s k i l l and p a t i e n c e i n

t h e p r e p a r a t i o n o f t h e f i n a l m a n u s c r i p t s made i t a l l p o s s i b l e .

1

CHAPTER 1

STRATEGIES FOR SUCCESSFUL PREPARATIVE LIQUID CHROMATOGRAPHY

Patrick D. McDonald and Brian A. Bidlingmeyer Waters Chromatography Division, Millipore Corporation 34 Maple Street, Milford, MA 01757, U.S.A.

CONTENTS 1.1

INTRODUCTION

1.2

PLANNING THE SEPARATION 1.2.1 Defining the Problem

1.2.1.1 Preparative or Analyiical? 1.2.1.2 Nature of the Sample? 1.2.1.3 Practical Considerations? 1.2.2 Senlng Goals 1.2.3 A Preparative LC Separation Scheme 1.3 ACHIEVING A SEPARATION 1.3.1 1.3.2 1.3.3 1.3.4 1.4

Origin of the Separation Factor Origin of the Plate Number Relationship of Q and N to Resolution How Much Resolution Is Necessary in Preparative LC?

COLUMN LOADABILITY 1.4.1 Load vs. Overload 1.4.2 Column Load - a Qualitative Model 1.4.3 Relationship Between Separation Parameters and Load 1.4.3.1 Load VS. Q

1.4.3.2 1.4.3.3 1.4.3.4 1.4.4 Effect of 1.4.4.1 1.4.4.2

Load vs. Efficiency Load vs. Retention Time Loadin Recycle Mode Load on Peak Shape Peak Shape Changes with Increased Sample Load Additional Causes of Peak Shape Distortion

2 1.5

SCALING UP A SEPARATION 1.5.1 Scaleup from Analytical LC

1.5.1.1 General Considerations 1.5.1.1.1 Packing chemistry 1.5.1.1.2 Packing history 1.5.1.1.3 Columngeometry 1.5.1.1.4 Sample concentration 1.5.1.2 A Step-byGtep Example 1.5.2 Scaleup from TLC 1.5.3 Scaleup of Gradient Elution Separations 1.6

PREPARATIVE LC PACKINGS AND ELUENTS- CHOOSING A SEPARATION SYSTEM 1.6.1 Stationary Phase ConsMerations

1.6.1.1 Chemical Properties 1.6.7.1.1 Separation mechanisms 1.6.1.1.2 Adsorption vs. partition 1.6.1.1.3 Chemical stabilliy and same contaminatbn 1.6.1.1.4 Stationary phase contamination 1.6.1.2 Physical Properties 1.6.1.2.1 Particle size, shape anddensity 1.6.1.2.2 Pore size, pore volume, and surfacearea 1.6.1.2.3 Choice of phases based on pore sizehlume analysis 1.6.2 Mobile Phase Considerations

1.6.2.1 Chemical and Physical Propetties 1.6.2.1. 1 General ansiderations 1.6.2.1.2 Eluent strength 1.6.2.1.3 Selectivity differences among equieluotropic mobile phases 1.6.2.2 Practical Guidelines 1.6.2.2.1 Solvent purity 1.6.2.2.2 Mobile phase preparation 1.6.2.2.3 Column equilibratbn 1.6.2.2.4 Detector compatibil/iy 1.6.2.2.5 Step gradients 1.6.2.2.6 Sanple ~ l u b i l i r y 1.6.2.3 Safety 1.7

PREPARATIVE LC SYSTEM CONFIGURATIONS 1.7.1 Preparative LC Columns

1.7.1.1 Column Design 1.7.1.2 Packed B8dStNCt~re 1.7.1.2.1 Larger diameter colrmns 1.7.1.2.2 Packing methods 1.7.2 Eluent and Sample Delivery Systems 1.7.2.1 Tuftwleni vs. Laminar Fbw

1.7.2.2 R e c ~ l esystem des@n 1.7.3 Detection 1.7.4 Sample Collection and Recovery 1.7.5 Automation 1.8

CONCLUSION

1.9 ACKNOWLEDGMENTS 1.10 REFERENCES

3

1.1 INTRODUCTION Taking Things Apart & Putting Things Together was the title of both the American Chemical Society's Centennial Exhibit (1 876-1976) and centennial commemorative volume [l]. Certainly it is appropriate to define chemistry in this way, since separation and synthesis form the foundation for progress in our understanding of the chemical world within and around us. Each time a new separations technology emerges, it soon is applied to the solution of significant problems, thereby advancing the frontiers of chemical science. Such a situation occurred in the early 1970's when Professor Robert B. Woodward at Harvard University pioneered in the use of the then infant technique of modern liquid chromatography (LC) in the total synthesis of Vitamin 812 [ref.2]. At times, even the most creative of synthetic chemists encounters a key separation problem. Professor Woodward described his predicament as follows: "...we were now faced with the problem that we could lose the stereochemical integrity of our substances at the three centers mentioned. And that createdproblem of separation. ... If we look at the heptamethylbisnorcobyrinates,leaving stereochemistry undefined at the three centers which I have discussed, we then clearly faced problems of stereochemistry, and of course, associated problems of the separation of very closely related molewles." [3]

The resolution of these separation problems were made possible using LC. Again, in Professor Woodwards own words: "Here I shouM say that of absohrtely crucial importance to all of our further wo& has been our taking up the use of high pressure liquid or liquid-liquid chromatography to effect the very difficult separations with which we were faced from this point onwards. The power Of these hlgh pf8SSUre llquld chromatographlc methods hardly can be lmaglned by the chemlst who has not had exporlence wlth them; they represent relatively simple instrumentation and I am certain that they will be indispensablein the laboratory of every organic chemist in the very near future.'[rl]

Professor Woodward came rapidily to rely upon LC. The degree to which he depended upon it as a standard tool is indicated in statements such as: "The cobyric acid was crystalline, and It was ldentlcal In all respects, mOSt partlcularly In llquld chromatographlc behavlor, wlth cobyrlc acld derlved from natural sources." [S,emphasis op. cit. ]

Professor Woodwards colleague and collaborator in these pursuits, Professor Albert Eschenmoser of the EidgenBssische Technische Hochschule (ETH) in Zurich, Switzerland, also praised the preparative capability of LC in his own account of the 812 synthesis: "...this presented difficulties not of principle, but rather of an experimental nature. That these difficulties were successfully overcome with the aid of high-pressure liquid chromatography,which appeared at precisely the r@htmoment, proved to be one of the first illustrations of the efficacy of this new chromatographic separation procedure in organic synthesis." 161

4

Indeed, following Woodwards and Eschenmoser's first reports of success with the modern techniques of preparative LC, many workers have continued to use this powerful methodology for rapidly isolating quantities of purified materials for a variety of end uses. Several reviews have appeared in the recent literature on theory, materials, and methods [7-381.In this chapter, we will discuss efficient ways to use the power and versatility of preparative LC to isolate, enrich, and purify components in samples of interest.

1.2 PLANNING THE SEPARATION 1.2.1 Defining the Problem 1.2.1.1 Preparative or Analytical?

Before any separation is attempted, careful planning must be done to insure the best possible outcome. A separation is usually either analytical or preparative in purpose. Analytical and preparative separations differ significantly in goals, even when they are performed on the same scale. Not only the choice of methodology and apparatus but also the attitude of the chromatographer must be carefully matched to the task at hand. There are many commonly held misconceptions about preparative LC [Fig. 1.11. These notions should be dispelled by the discussion that follows in this chapter.

from its original matrix

Fig. 1.1. Common Misconceptions about Preparative LC.

5

In general, analytical LC is used to learn something qualitative or quantitative about a given sample: e.g., a characteristic elution profile or "fingerprint"; how many components are present; the presence or absence of a particular component; how much of a component is present; the relative concentrations of components of interest [see Table 1.I]. When coupled with the variety of powerful detection and data handling devices available today, analytical LC also can be used to establish or confirm the identity of certain components of interest. And now that sophisticated, selective detection schemes, based on unique systems and sensors, chemical modification of sample components, andlor deconvolution techniques, are available, information can be obtained from a sample without the need for complete resolution of its important components. Some forms of detection may be destructive to the sample. Usually, the sample components are discarded in an appropriate manner after the separation has been performed. In contrast, preparative LC is used to isolate, enrich, or purify one or more components of a given sample which are collected for some subsequent application: e.g., further synthesis; use as a reference standard; assay or testing by physical, chemical or biological procedures; sale as a commercial product [see Table 1.11. Detectors are used to monitor the progress of the separation, make decisions on how to fractionate the effluent stream, or to locate components of interest in specific fractions that have been collected. Often, in large scale separations, detection is done on a small portion of the sample which is split from the main stream [on-line] or removed from various fractions [off-line]. Obviously, resolution must be adequate to permit isolation of materials at the desired degree of purity. Throughout this chapter, the term "preparative LC" will be used in the general sense. Most often, as indicated in Table 1.1, preparative LC separations done in a laboratory encompass sample sizes in the range of 0.1 to 100 grams. When appropriate, preparative LC separations done at a scale smaller or larger than this will be denoted by the respective terms "micropreparative LC" and "macropreparative LC" [see Table 1.11. 1.2.1.2 Nature of the sample?

Once the need for a preparative separation has been identified, the characteristics of the sample must be reviewed in order to set appropriate goals and plan a successful separation scheme. 0 Composition - Is the sample composition known or unknown? 0 Matrix -What is the chemical/physical nature of the matrix? 0 Complexity- Is the sample a relatively simple mixture of a few components (e.g.,a synthetic product) or a complex mixture of multiple components (e.g., a natural product)? 0 Properties - Are the components in the sample similar or dissimilar in chemical

6

I I

TABLE 1.1 A Comparlson of Analytical and Preparative LC. ANALYTICAL LC

To obtain Qualitativeor Quantitative Informatbn about Sample.

Maximum Peak Capacity (#of components separatedor measuredlunit time)

Basls of Comparlson

PURPOSE

PRACTICAL OBJECTIVE

TY PlCAL SAMPLE SIZE

As light as possible; typical range

To Isolate, Enrich, or Purify Sample Components.

Maximum Throughput (Amount of material purified/ unit time)

Micropreparative: s 100 mg Preparative: 0.1 to 100 g Macropreparative: 20.1 kg

COLUMN LOAD

As heavy as possible; typical range 0.001 to 0.1 gram of sample/gram of packing

DEGREE OF SEPARATION

Matched to requirements for level of purity & recovery. Often, moderate resolution is sufficient.

io-lo

to10-3gramofsanple/ gram of packing

Matchedto sophisticatbn of detectbn technique. Baseline separation is typical.

PREPARATIVE LC

Small volume (< 5-15 ml) Small particle (3-2@), expensive packing Excess separatbn power relative to need.

TYPICAL COLUMN CHARACTERISTICS

Volume scaled to sample size (milliliterto multiliier) Large particle (20-10Q.~), economical packing Separation power matched to requirements.

Required, often with high sensitivily and wide linear dynamic range.

DETECTOR

Desirable,to assay fractions onor off-line; extra range at bw sensitivity.

DISPOSITION OF SAMPLE

Sample fractions are collected; mobile phase may be recycled.

usually sample is discarded along with mobile phase.

7

composition, solubility, and other properties? 0 Phase - Is the sample a liquid, a solid, or a mixture of soluble and insoluble materials? 0 Concentration -'What is the concentration of the desired compound(s) in the sample matrix (e.g., trace quantity, single predominant component, one of several major components)? 1.2.1.3

Practical Considerations?

0 Quantify - How much sample must be separated? 0 System Capacity - How does the sample quantity compare with the capacity of the separation tools available to do the job? 0 Value - How precious is the sample? 0 Cost Benefit - What value is added to the important components, once enriched or purified, relative to the economics of performing the separation? 0 Frequency - How often must the separation be repeated in the future? P Safety - What are the hazards in dealing with the sample and the separation methods? 1.2.2 SETTING GOALS Once the nature and scope of the problem have been defined, realistic goals for the separation must be set. It is easy to say that each and every component must be isolated in pure state, with no loss of material, in an hour's time at a cost of one dollar per gram of sample. But such a set of goals may be impossible to achieve. Furthermore, it may not really be necessary to meet each goal as stated. If some level of impurity and loss of sample can be tolerated, the separation might be achieved in minimal time at reasonable cost, and the products may still meet the needs for which they were isolated. Preparative LC separations have at least five interdependent elements: purity, quanfity, time, difficulty, and cost [Fig. 1.21. To achieve an optimum result, inevitably, one or more of these variables must be compromised to a certain degree. Each element will be considered briefly now. Later in this chapter, their interrelationships will be examined in more depth.

Purity is an absolute. A substance is either pure or impure. There is no middle ground. Chemists and biochemists, however, use the term "purity" and related phrases (e.g., "degree of purity", "x percent pure", "degree of activity") in a relative, not an absolute, sense. "Purity" is generally determined on the basis of the ability to detect impurity or lowered activity in a sample using available analytical techniques. The higher the purity level that is required, the more the other preparative LC elements must be compromised, especially if the separation is a difficult one.

8

DIFFICULTY

Fig. 1.2. Some Dimensions 01 Preparative LC

When setting goals for purity in preparative LC,the end use of the purified material must be considered. Synthetic intermediates may be useful at 85-95% purity (tools such as routine NMR or IR spectra may only detect impurities above the 5% level). Trace level natural products might be identified when enriched to 50-75% concentration. Analytical reference standards or drugs for animal testing may need to be 99+% pure. Some bioactive compounds may not need to be enriched further but rather have a trace amount of potent toxin removed. The purity of most bioactive molecules is measured in units of activity, not mass. Activity level is usually compared to that of the active principle in its original source. In this case, "purity" is represented by a maximum and constant level of activity. Quantity requirements have several implications. If the concentration of the component of interest is low within its matrix, then large amounts of sample must be processed to yield the desired quantity of enriched compound. The quantity of sample that must be processed determines the separation technique and scale of apparatus to be used. For valuable components, recovered yields should be high relative to the total amount processed. If only small scale equipment is available and a large scale separation need be done only once, then the sample may be divided into manageable portions and chromatographed in multiple runs. If preparative or macropreparative LC separations must be done more frequently, especially on a commercial production basis, then an investment in large scale hardware may be justified. Micropreparative LC may be done with existing analytical LC instrumentation.

9

Time is a key factor in maximizing throughput, a principal objective of preparative LC (see Table 1. I ) . Modern technology has dramatically reduced the time necessary to carry out a large scale chromatographic separation from days or hours to minutes. In setting a time goal, the chromatographer must not only plan for optimization of the separation conditions, but also for sample preparation, system set-up and equilibration, and recovery of eluate components at each stage of the separation scheme. Difficulty can be heightened by the complexity of the sample, the number of components that must be isolated, disparate concentrations of important compounds, and, especially, similarities between compounds that provide little basis for separation. Difficult separations cost more money, take more time, and may place limits on the quantity that can be processed and the degree of purity of the isolated products. The best way to deal with a complex separation is to break it down into several simpler stages and spend time optimizing conditions for the most difficult separation steps. Liquid chromatography should be viewed as only one of many separation tools at the chemist's disposal to deal with difficult samples. Cost becomes a significant concern as the scale of the separation increases, especially when the difficulty factors or requirements for purity, recovery, or minimum process time escalate. Long-lived columns and mobile phase recovery/recycling ,must be considered when packing and solvent costs increase. Obviously, labor intensive steps have a significant budget impact. But total separation time is a significant factor today in the productivity of academic research programs as well as commercial operations. Competitive advantage in both private and public arenas has great value, though it may be hard to quantify. A PREPARATIVE LC SEPARATION SCHEME Defining the problem and setting goals are the first steps in planning a separation scheme to obtain the desired result. As outlined in Fig. 1.3, a potentially formidable task can be broken down into a logical sequence of very manageable stages. There are some key points to keep in mind when deciding how to go about achieving any preparative LC separation. 1.2.3

Point 1: Chromatography is a relatively expensive separation tool and should be used only when necessary. If, for example, a solid or liquid phase extraction or crystallization or combination thereof will achieve the desired separation, then a chromatographic step may be obviated. Point 2: A good strategy seeks to fractionate large samples into classes or groups

of components relatively quickly and economically using crude separations. Then, individual components are isolated by more refined methods. Point 3: Any sample to be chromatographed needs to be in a solution of appropriate concentration and solvent composition.

10

Analytlcal System

& Modify Mobile Phase

Preparatlve System

Collect Fractlons

Analytlcal System

IC..lrLI. Flnal Workup and lsolatlon I

Fig. 1.3. A separation scheme for preparative LC.

Crystallization

- Solvent Removal ~

I

I

11

Point 4: Techniques used to put the sample into solution and remove insoluble matter may concurrently eliminate large, extraneous portions of the matrix, thereby reducing both the complexity and scale of the ultimate separation problem. Point 5: Every stage, properly planned, often adds an order of magnitude more separation power and speed to a multi-dimensional separation scheme. For example, a preparative LC step gradient can rapidly apportion a large, heterogeneous sample into 6-8 fractions spanning a wide polarity range. Each fraction can then be further refined by an additional LC separation under conditions tailored to the isolation of individual compounds (see Section 1.6.2.2.5 and Table 1.a). Point 6: The time it takes to optimize selectivity and retention is time well spent if the preparative LC separation is difficult, large-scale, or frequently run. (see Section 1.3) Point 7: Analytical LC is the ideal tool for system optimization: thin layer chromatography (TLC) can also be used (see Section 1.5). Once developed, the analytical system also serves as a means to determine scale-up parameters as well as to analyze preparative LC fractions for purity after the large scale separation has been completed. Point 8: While preparative LC solves one separation problem, it also creates another. Indeed, sample components may be isolated in individual fractions, but they are still in solution at a concentration significantly lower than that when they were injected. The dilution factor is dependent upon the column volume and the degree to which the component is retained. Usually, the solvent must be removed to recover the compound of interest in the dry state. Further work-up ( e . ~ .recrystallization) , may be desirable to remove contaminants in the compound of interest arising from the large volume of mobile and/or stationary phases used, an often overlooked but inevitable outcome of certain preparative LC separations (see Section 1.6).

1.3 ACHIEVING A SEPARATION In many practical ways, preparative LC is simpler than analytical LC. Those less gifted in manual dexterity may find larger sample vessels and equipment easier to work with than microsyringes and miniature plumbing. However, preparative LC is often done under conditions with separation efficiency lower than that typical of analytical LC. Thus, many chromatographers may find it less mentally demanding to crack a desirable analytical separation with the sledgehammer of sheer column horsepower. To optimize preparative LC, one often compensates for limited column efficiency with a greater understanding of how to achieve a good chromatographic separation by physicochemical means. Herein lies one of the premier questions in the realm of chromatographic science: Which is more important to a successful separation: separation factor or plate number?

12

The usual reply, of course, is tempered by attitude and experience. The real answer is that both parameters are critical to success with the relative importance of each being dependent upon the specific circumstances involved. 1.3.1 Origin of the Separatlon Factor Chromatography is a dynamic equilibrium process. At the molecular level, it involves the distribution of solute molecules between a mobile phase and a stationary phase. A tiny difference in the relative affinities of similar, though distinct, solutes for the two phases, when multiplied by the multitude of phase transfers that occur during the passage of molecules through a chromatographic column, accounts for the measurable difference in retention between sample components. Selecting an optimum combination of mobile and stationary phases to maximize the relative differences between their affinities for sample components will have the most significant effect on the success of any preparative LC separation. A detailed description of the thermodynamic and kinetic treatments of the chromatographic process can be found in several reference works and need not be repeated here [39-471. Two points, though, must be emphasized: First, the separation factor, a,is derived from equilibrium theory and represents the ratio of two mass distribution ratios, Dm2/Dm, (see Fig. 1.4), measured under identical conditions, for a given pair of compounds. Second, it is quite easy to determine a either from static equilibrium measurements or chromatographic experiments. The mass distribution ratio, Dm, in turn, is:

Dm =

amount of compound in stationary phase

=k'

amount of compound in mobile phase

While the IUPAC Commission on Analytical Nomenclature has recommended using Dm [48], most chromatographers continue to represent the ratio in eqn. (1.1) by the term capacity factor, k', a throwback to the development of the theory of gas chromatography. While it is habitually used, the term capacity factor can be misleading in preparative LC since k' is inversely proportional to sample capacity (see Section 1.4.2). Therefore, in the present chapter, to help attune our understanding of preparative LC, we will use both terms as a reminder that k' actually describes the distribution of a sample component between the two phases of a chromatographic system.

13

........................................................................................ ............................................................................v2

.........

(1111.

-Retention

+' Q

Volumes

V1 ........................................................................................................

i111~ (1111

I

1

..

Peak 2

v)

c -. 0

n v)

0

K

At

Peak 1

..

.. ............................

Number of Hold-up Volumes

b Elution Volume

Mass Distribution Ratio

a,,,=

D,, = -2-

(1.3)

Fig. 1.4. Calculations Based on an Analytical Elution Curve

Both k' (Dm) and a are calculated from measurements made on a chromatogram as illustrated in Fig. 1.4. The unretained peak represents a marker solute that passes through the chromatographic system without interacting in any significant .way with the stationary phase. The volume of mobile phase required to elute this compound from the column, VM, or hold-up volume, is equal to the volume of mobile phase contained by the chromatographic system from point of injection to point of detection [injector, column packing (between and within particles), column end fittings, connection tubing, and detector cell]. Retention volumes V1 and V2, the respective volumes of mobile phase required to elute compounds 1 and 2 from the column, are measured from point of injection to the respective peak maxima. Each peak in the chromatogram represents directly or indirectly the concentration of a solute in the mobile phase at the detection point, a short but sometimes significant distance downstream from the outlet of the chromatographic bed (see Section 1.7). If the flow of mobile phase (volume per unit time) through the system is constant, then the volume axis in a chromatogram can also be represented as a time axis. If the chart drive is also constant, then either volume or time corresponds to the measured distance along the x-axis. In practice, constancy of volume flow rate and chart speed as well as proportionality to concentration and linearity of detector response must be verified, not assumed.

14

In the model chromatogram computer-drawn in Fig. 1.4, each peak has the shape of a Gaussian distribution curve, an ideal, well-defined mathematical description of the probability that a molecule will elute at a certain volume in an LC system. While the peak maximum, used to calculate VM or Vn, represents the elution point of highest probability, note that molecules elute well before and after this point. Even when the detector signal at a given sensitivity setting has returned to baseline (zero response), the tail of the Gaussian curve continues invisibly, never actually reaching zero concentration. This latter point, often forgotten by chromatographers, must not be overlooked in preparative LC where peak tailing is usually more pronounced and non-Gaussian in shape (see Section 1.4.4). The magnitude of a peak’s tail, while not necessarily apparent in a chromatogram, must be considered when planning sample fraction collection and post-LC run workup to reach desired purity levels. It should be re-emphasized that since a is a ratio (by convention, greater than 1) of two specified mass distribution ratios, it relates directly to the chromatographic process whereby molecules of sample compounds are dynamically partitioned between mobile and stationary phases. This distribution process ultimately determines the respective location of each solute (peak maximum) in the elution profile. Peak width is not taken into account by the separation factor a. 1.3.2 Origin of the Plate Number Peak resolution, R,, is a mathematical function of both relative peak location and average peak width for a given pair of eluting solutes. In units of volume,

RS’

v2 - v1 0.5 (W1 + W2)

where W1 and We are the respective widths, 0.5(W1 +We) is the average width, and (V2 - V1) is the distance between the centers of peaks 1 and 2. In analytical LC, it has become convenient to assume that peaks have Gaussian shapes. Whether or not this assumption accurately represents nature, it simplifies the mathematics of LC theory greatly. A Gaussian distribution is symmetrical about a mean value, and its width can be measured readily in units of 0 , the standard deviation from the mean. Referring to Fig. 1.5, the width of a Gaussian curve between its inflection points, located at 60.7% of the peak height, is 20. If tangents, drawn to either side of the curve at these inflection points, are extended to the baseline, they intersect a baseline distance of 40, equivalent to the width of the curve at 13.4% of peak height above baseline. Similarly other points exist which represent widths of 30, 50, etc. Early in the development of chromatographic theory, a performance indicator was sought by which various LC systems could be compared. Ideally, this indicator had to be dimensionless, independent of scale, and easily measured. The term theoretical

15

plate number, N, was borrowed from distillation theory and defined, not as a physical column feature as in distillation, but in terms of a statistical function, the population variance (02)~

which relates the narrowness of a peak (6) to its elution volume (eqn. 1.5). It is possible to use statistical moments to calculate N, independent of peak shape, but unless the peak response can be converted to a digital signal and processed by a computer, most chromatographers prefer the convenience of manual measurement methods as shown in Fig. 1.5. With these latter methods, the theoretical plate number is tied to a Gaussian peak shape model [49].

FQ. 1.5. Methods of Calculating Theoretical Plate Number N from Gaussian Peak Measurements.

When a peak no longer can be approximated by a Gaussian distribution, then a theoretical plate number cannot be calculated using eqn. 1.6. Since the latter situation is commonplace in preparative LC (see Section 1.4.4), plate counts calculated by any of the methods shown in Fig. 1.5 are virtually meaningless when comparing preparative LC system performance under heavily loaded conditions. Furthermore, performance under light (analytical) load conditions, due to system design, may not give a true indication of performance under preparative LC sample loads (see Section 1.7.1).

16

Thus, while the separation factor, a,is derived from equilibrium thermodynamics, the theoretical plate number, N, arises from a statistical treatment which takes both peak location and peak width into account. Whereas a is primarily affected by the physicochemical characteristics of the mobile and stationary phases and solutes, many factors can significantly alter N, as shown in Fig 1.6. Further discussion of these factors can be found in many fine texts on LC [39-47, 50-591. 1.3.3 Relationship of a and N to Resolution If, in a chromatogram such as shown in Fig. 1.4, it is assumed that peaks 1 and 2 are Gaussian and have approximately equal values of N, then by rearranging and combining eqns. 1.2, 1.3, and 1.4 and substituting the result into eqn. 1.6, the effective theoretical plate number, Ne, can be expressed in terms of resolution, capacity factor, k'l (mass distribution ratio, D,.,,l), and the separation factor, a:

2K1 ( a - 1)

I2

This formula can be further simplified by assuming that the widths of each peak are the same, an assumption only valid for closely spaced peaks representing similar elution volumes, detector responses and quantities of solutes. When rearranged, this produces the well-known resolution equation, eqn. 1.8.

As can be seen from eqn. 1.8, by far the most significant factor in good resolution is a. A change in a from 1.1 to only 1.2 doubles R,; going from 2 to 4 triples resolution. Larger values of a provide increased resolution which, in turn, permits heavier sample loads (see Section 1.4.1). Increasing the capacity factor (k') from near 0 to 2 also has a dramatic effect on R,, but at k' values much beyond 5, the effect diminishes greatly while solvent consumption increases significantly. Since Rs depends upon the square root of column efficiency, doubling the effective plate number only increases the resolution by a factor of 1.4; a ten-fold increase in N yields only a 3.2-fold rise in R., It is also important to realize that increasing N significantly usually corresponds to higher back pressure, higher cost, and lower capacity, all undesirable in preparative LC. Thus, maximizing the separation factor between components of interest is the single most important srep that can be taken to optimize a preparative LC separation.

17

Fig. 1.6. Some of the Factors Which Affect the Theoretical Plate Number, N.

1.3.4 How Much Resolution Is Necessary in Preparative LC? As mentioned in Table 1.1, the typical goal in analytical LC is baseline resolution between peaks of interest. This corresponds to Rs 2 1.5. In practice, if R, 2 1.O, then adequate peak integration and quantitation can usually be achieved. But if Rs = 0.6, a separation is barely apparent, as shown in Fig. 1.7. Yet, it is still possible to isolate pure

compounds in this situation.

18

Cut Point A

B

C

4

100% c

o

*

m

z

?OOYo Empound 2

*

88% Compound 1 i 88% Compound 2 12% Compound 2 i 12% Compound 1 Fg. 1.7. How to Obtain Pure Compounds When Apparent Resolution is Incomplete.

If the goal was simply to enrich the concentration of compounds 1 and 2 in respective fractions, then collecting the portion of the eluting material prior to cut point B (refer to Fig. 1.7) and subsequent to cut point B in separate containers yields compounds at purity levels of 88% (for Gaussian peaks). To obtain each compound in high purity, cut points A and C are used. The fraction of mixed sample between these two cut points can be collected and reprocessed, or recycled (see Section 1.4.3.4). As Rs increases, the purity level at cut point B rises dramatically (see Table 1.2); e.g., 95% purity can be obtained at R, = 0.8, still well below the resolution typically sought in analytical LC. The example shown in Fig. 1.7 is for a lightly loaded column and a moderately difficult separation. For an easier separation (larger a)with a sample load increased to the point where Rs has dropped to 0.6, the underlying peak shapes will deviate significantly from Gaussian distributions with steeper fronts and more pronounced tails (see Section 1.4.4). This will permit even more of pure compound 1to be collected, but will reduce the amount of pure compound 2 that can be obtained beyond the point where the tail of the actual peak 1 has dropped to baseline. Later in this chapter it will be shown how to take such a separation and obtain complete recovery of both compounds in high purity using the technique of recycle (Section 1.4.3.4). Sample

19

Table 1.2 Purity Obtained at Various Values of R

(refer to Fig. 1.7)

Resolution, [RJ

Purity at Cuf Point B’

0.5

84.0% 88.0% 92.0% 95.0% 98.0% 99.4%

0.6

0.7 0.8 1 .o 1.25

‘Equal amounts of each compound; Gaussian peak shape. Illustrationscan be found in ref. 60 and in Chmter 5.

recovery also depends upon elution order and relative concentrations of each component. This is well illustrated in Chapter 5 in this book and in ref.60.

1.4

COLUMN LOADABILITY

1.4.1 Load vs. Overload The foremost question in the mind of a prospective preparative LC practitioner is: “How much sample can I load into the system and still be successful in achieving the desired separation?” A system is often dismissed as “overloaded” when load levels exceed the region wherein performance ceases to be mathematically predictable, isotherms become non-linear, and equations fail to describe adequately non-Gaussian peak shapes. Yet, in spite of these obstacles, chemists can still be successful in separating components of their samples by following certain practical guidelines. In some discussions of chromatography, overload has been defined as the point at which sample size causes a component’s capacity factor, k’, (mass distribution ratio, Dm) to decrease by 10% (as measured by earlier elution of the peak apex). As shall be shown here, successful preparative LC can be done at load levels far exceeding this point. A much more practical definition of overload is: that loading which no longer permits the isolation of product at the desired purity or recovery levels. With few exceptions, before the advent of modern, high performance liquid chromatography, column LC was a preparative technique. Chemists such as Kuhn, Lederer, and Winterstein who revived Tswett’s dormant method in the early 1930’sand Reichstein and coworkers who standardized elution LC methodologies, particularly as applied to steroid separations, had to develop guidelines for sample loading via practical experimentation with large numbers of samples over a period of more than a decade [61]. Martin and Synge pioneered the chromatographic concept of a theoretical

20

plate with their development of liquid-liquid partition chromatography [62]. Adsorption

LC was put on much firmer theoretical ground in the decade following Stahl's standardization of the technique of thin layer chromatography (TLC) [39,501. Well before any separation factor had ever been proposed or calculated, chromatographers discovered by trial and error that, for a fixed sample size, more difficult separations require a significantly higher weight ratio of packing material to sample than do easy separations. As shown in Table 1.3, the empirical guidelines developed in the 1940's agree quite well with the loadings proposed in the 1960's based on a knowledge of linear adsorption isotherms and standardized adsorbents.

Table 1.3 A Comparison of a with Preparative LC Sample Load Guidelines Developed in the 1940's and 1960's.

a

Dlfflculty of Separatlon

22.0

easy

21.5

moderate

21.3

difficult

60A.

It must be emphasized that sample load is dependent upon most of the same parameters listed in Fig. 1.6 which influence N. Thus, any guidelines which are published for the convenience of the chromatographer are just that - guidelines. Experimentation is still the best way to determine what load is practical for a given situation. The structure and properties of a molecule, the manner and orientation of its interaction with the mobile and stationary phases in the presence of other sample components, and the geometry and properties of the active phases (especially bonding or attractive interactions with solvent molecules and the activity, surface area, pore dimensions, etc., of an adsorbent), among other considerations, are all critical to determining an optimal sample size which maximizes throughput in preparative LC.

21

1.4.2 Column Load - a Qualitatlve Model Chromatography, by definition, is a dynamic process, since one of the two partitioning phases is in motion. The concept of dynamic equilibrium is hard to grasp mathematically, as well as mentally, so it is often easier to approximate it by taking "snapshots" at various stages and then integrating them into a model representative of the whole image. Let's consider now a snapshot view of sample molecules entering a chromatographic bed. Hopefully, from this simple picture will emerge a better understanding of some of the many complicating variables which govern the chromatographic process. Our model is illustrated for the case of a relatively small organic compound undergoing adsorption LC on a silica gel column. It is generally believed that, in chromatography on silica gel, the "active sites" at which adsorption occurs are the silanol (-Si-O-H) groups on the silica surface. These sites are the termini of the silicon-oxygen polymeric structure, -(Si02)n- , which is built of tetrahedral subunits. Some silanols are close enough to each other to attract a hydrogen atom from a neighboring silanol group (hydrogen-bonded silanols); other silanols stand alone (free, unbound silanols). The addition of small amounts of water to silica produces a more homogeneous surface by forming hydrogen bonds with free silanols and partially hydrogen-bonded silanols, creating, in effect, a stationary interfacial layer of water molecules one or more molecules thick. This is the usual condition when chromatography is done in a silica bed, since silica adsorbs moisture from the air as well as from incompletely dried solvent mixtures. Small amounts of water, once adsorbed, are very difficult to remove chromatographically. And, in fact, it is usually desirable to have a small amount of water present (3-5% by weight) to simplify column equilibration, aid reproducibility, reduce irreversible adsorption, and increase sample capacity and recovery [64] (see Section 1.6.1.1.4). Many attempts have been made to measure quantitatively the number of silanol groups on the surfaces of various types of silica. For chromatographic quality silica, the number of silanol groups per 100 square angstroms of surface is thought to be between 2 and 6. The value differs, depending upon many experimental variables and the technique used for the measurement [65]. Let's assume that the injected sample molecule has a compact size and shape (MW 250) with one significantly interactive functional group such that potentially 3 molecules can be adsorbed onto every 100 square angstroms of silica surface. Since 1 meter = 10'0 A, then (3 molecules +. 100 A2) x (1010A/m)2 = 3 x 10'8 molecules can be adsorbed on every square meter of silica surface. With a column that contains 325 grams of silica with a surface area of 300 m2/gram, then the bed might adsorb as many as (3 x 10'8 molecules/m2) x (300 m2/g) x (325 g) = 3.12 x molecules of the compound. Dividing by Avogadro's Number, this potential adsorption capacity is 0.52 moles or 730 grams of the compound at its assumed molecular weight of 250.

22

Further suppose that the capacity factor, k', (mass distribution ratio, )D , of this compound is 2; i.e., twice as many molecules are adsorbed on the stationary phase as reside in the mobile phase at any given instant under equilibrium conditions. This means that the column could potentially contain 195 grams of compound (130 grams adsorbed and 65 grams at equilibrium in the mobile phase) at one time. If k' were increased to 10, then the capacity would drop to (130 g adsorbed + 13 g in mobile phase) = 143 grams, while if k' were 0.1, then presumably (130 + 1300) = 1430 grams of compound could be contained at full capacity. In view of this potentially enormous capacity for sample, the model must be developed a bit further to explain the fact that typical sample loads for separations done on a column of this sort are only 1-10 grams. Each molecule of a compound with k' greater than zero spends a portion of its residence time in the chromatographic bed adsorbed onto the stationary phase. Another type of molecule will spend either more or less time on the stationary phase, depending upon the relative magnitude of the attractive forces of the stationary and mobile phases for that particular molecule. This attraction may be chemical, mechanical, or electrical in nature, or some combination of these forces. The difference between the attractive forces of the silica for two different types of molecules is extremely small, yet large relative to the absolute magnitude of the forces involved. So, for one molecule to be selectively retarded and thus separated from another in its passage through the silica bed, it must move back and forth between the silica surface and the mobile phase many, many times such that the tiny differential between mass transfer times for each type of molecule, thus multiplied, may result in a significant difference in measurable retention time. If it were only necessary to separate two molecules, one from another, then the problem would be simple. But a single gram of sample might contain lo2' molecules (within an order of magnitude or so, depending upon molecular weight), and this requires the model to incorporate statistical factors (kinetics) and bulk property effects (thermodynamics) to explain the complex interactions and competition between molecules and the mechanism of the chromatographic process. Details are best described elsewhere [e.g., 39,401. Injecting a batch of molecules into a silica bed creates an initial chaos that takes time and space to sort out. Both sample and eluent molecules compete to occupy available active surface sites; displace one another; take the paths of least resistance (though still tortuous) into, around, and possibly through porous silica particles; bypass some adsorption sites in favor of others; and, in general, do not take full advantage of the entire silica surface available to them. It takes a certain volume of silica bed for a given quantity of sample, injected in a particular concentration, to reach equilibrium distribution between stationary and mobile phases. This volume of bed, is, in fact, the original definition of a chromatographic "plate" [62]. If this bed segment were the entire silica bed, then the column would be said to contain

23

one plate. If this segment represented only 1/300th or 1/1000th of the silica bed, then the column would contain 100 or 1000 "plates", respectively. At a point where the sample size is very small (analytical load levels), the innate efficiency of the column (number of "plates" or plate dimension) is determined, and limited, by factors other than sample size, principally mechanical parameters such as particle size, method of packing, and column design (see Section 1.4.3.2). But, in preparative LC, plate dimension (length or height, which for a given diameter of cylinder specifies volume) is principally determined by the quantity and concentration of sample injected into the bed. The smaller the differences between the each phase's attractive forces for two kinds of molecules, the more difficult the separation, and the more times adsorption-desorption must occur for a separation to be achieved. To provide for sufficient interaction of a hard-to-separate sample with the silica surface, the sample size should be decreased appropriately. If, even by reducing the effective plate volume in this manner, the desired separation cannot be achieved, then the plate number (innate column efficiency) must be increased to allow sufficient residence time for the requisite number of molecular transfers to occur between mobile and stationary phases. This can usually be done in one of the following ways: (a) decreasing particle size in a column of the same dimensions (which severely limits sample size); or (b) increasing effective column length (which permits larger samples to be loaded but increases separation time and solvent consumption) by: 1. packing a longer column 2. coupling columns together in series 3. using recycle. If, in advance, the number of plates required for a given separation were known, then the sample size that could be separated in a column bed of given dimensions could be estimated. Unfortunately, it is very difficult to arrive at an exact numerical value of plate dimension upon injection in preparative LC. And this dimension only increases -never decreases- as the sample molecules pass through the column bed, due to various bandspreading mechanisms. And as the sample components separate into individual, often overlapping, ever broadening bands, the initial plate volume becomes nearly impossible to measure. Thus, to bring our model to the point of estimating column load, some further assumptions must be made. Instead of measuring the plate volume upon injection in the preparative LC mode, the volume of each eluting peak can be measured along with its retention volume from an analytical chromatogram (light load conditions). An effective theoretical plate number for each component can be calculated with eqn. 1.7. Such plate numbers, Ne, for various separation factors and resolution values are tabulated in Table 1.4. Returning to our example above, by dividing the total potential adsorption capacity for the 325-gram silica column by the number of plates required for a compound, the

24

Table 1.4 Number of Effective Theoretical Plates Required for Each Component at Various Separation Factors.

Other conditions: ki = 2 0 ; a = 16 (tangent method, see Fig. 1.5)

Table 1.5 Sample Load (gramslcomponent) for Different Column Diameters (d ) at Various Separation Factors.

-

Load Der comDonent in grams 2.5 5.7 8 0.8 1 .o

1 .o 0.6 0.3 0.1 0.07 0.04 0.016 0.008 0.002 0.0005

2 1 0.46 0.18 0.10 0.06 0.02 0.01 0.003 0.001

10

52 103 30 6 60 15 29 2.9 11 6 1 .I 7 3 0.64 4 0.36 2 1.5 0.8 0.15 0.7 0.07 0.4 0.2 0.02 0.1 0.05 0.005 0.025 10 I 63 I 325 I 640 6.4 Weight of Packing in Each Column

lOcm

161 93 46 18 10

6 2.4 1.1 0.3 0.08 7000 g

Other conditions: k i = 2.0 : a = 16 (tangent method, see Fig. 1 S ) :all mlumns are 30 cm in length, packed with silica gel of 300 sq.mlg surface area; = 0.7; see text for compound characteristics.

25

sample load which can be accomodated is estimated. Thus, for a mixture of two components, a*,,= 1.3, each of MW 250 as above, and k', = 2, to be separated at a desired resolution of 0.7, (195 grams/237 plates) = 0.8 grams of the first component and (1 80/237) = 0.8 grams of the second component can be injected for a total sample load of 1.6 grams. The innate column efficiency required would be at least 2 x 237 = 474 plates, a value easily achievable at high flow rates with a large particle packing at reasonable cost and relatively low back pressure. Sample load per component for other separation factors and sizes of columns is tabulated in Table 1.5. Keep in mind that, as the type of compound or adsorbent changes, these numbers will vary. But experiments over the last decade have proven these guidelines to be a good starting point for estimating sample load for preparative adsorption LC separations [66].More often than not, these estimates will be safely on the low side, so that in order to truly maximize throughput, further experiments beyond these initial conditions must be performed to establish actual loading limits in a specific situation.

1.4.3 Relationship Between Separation Parameters a n d Load 1.4.3.1 Load vs. a

In Section 1.3.3, the importance of the separation factor, a,in obtaining the highest possible resolution in LC separations was emphasized. By examining the data in Tables 1.4 and 1.5. the dramatic effect on innate column efficiency requirements and sample load becomes clear. A graphical representation of the relation between a and the difficulty of a separation, directly proportional to load and/or column efficiency requirements, is shown in Fig. 1.8. By comparing the three representations of chromatograms in Fig. 1.8 with a values of 2.0, 1.3 and 1. I , respectively, the effect of a on throughput is dramatically illustrated. Additional comparisons can be made using the opposing scales shown at the top of Fig. 1.8. If, for example, by choosing an alternate stationary and mobile phase combination, a can be increased from 1.05 to only 1.2, then the load can be increased nearly 15-fold. Or, the load might be kept at the same level, but the column length reduced instead in the same proportion, thereby saving expense in packing materials, solvent consumption, and time needed to complete the separation. In similar fashion, increasing a from 1.2 to 2.0 affords another 15-fold increase in load, throughput, or corresponding savings. Higher values of a mean that larger particle packing materials can be used. These generally are much less expensive, operate at reduced back pressures (AP, which is inversely proportional to the square of particle diameter), and put less severe demands upon equipment design (see Section 1.7). Thus, time spent in maximizing the separation factor, a,can provide many important benefits in preparative LC.

26

. ..:.: : I : .

i:

%

%

a = 1.1

k; = 2

L

Fig. 1.8. Relationship Between Separation Factor [a] and Difficulty of Separation. In the three artificial representations of chromatograms,the solid-lined triangles indicate peaks at lighter loads while the dotted lines delineate the apparent peak separations under similar conditions but with a ten-fold increase in sample load. The approximate resolution values given correspond to the latter more heavily loaded situations. Note: In the illustrations of LC separations, for simplicity, no allowance has been made for skewed peak shapes or shitts in peak maxima with increased load.

1.4.3.2 Load vs. Efficiency From the discussions in Sections 1.3.2 and 1.4.2, it should be apparent that there are two types of efficiency that concern a preparative chromatographer:

21

lnnate column efficiency which is determined. by the mechanics and fluid dynamics of the packed bed structure, hardware design, packing material properties, etc., and; Separation efficiency which is affected significantly by the nature and quantity of the sample and the physicochemical characteristics of the separation system. Plate number, N, is used as a measure of both types of efficiency, but the former is usually determined under ideal, the latter under actual, circumstances. Innate column efficiency, as mentioned above, is measured under lightly loaded conditions where the adsorption or partition isotherm is linear (see Section 1.4.4). Any column used in preparative LC should have an innate efficiency, as measured in an analytical mode (light sample load), as high as possible for its particular combination of geometry and packing characteristics. A rule of thumb is that the length or height of a plate, h, in an efficient column is approximately twice the diameter (dp) of the average particle with which the column is packed. Thus, a 30 cm long column filled with lop packing should contain about 15,000 plates under ideal conditions [h = 2dp = 2 x lop = 20p or 0.002 cm; 30 cm + h = 15,000]; loop particles in the same column should yield 1500 plates [30 cm -L (2 x 0.01 cm) = 15001. Ways in which this ideal number is reduced by the multitude of variables shown in Fig. 1.6 are discussed in many reference texts and will not be elaborated here [39-47, 50-591. Typical preparative LC columns often have plate heights in the range of 2dp to 1Odp. The key point however is this: a column should be chosen, designed, and packed to perform the requisite separation in the most efficient and economical manner possible, and, whatever particle size or column dimension is used, this column should be as inherently efficient as possible. Separation efficiency, in contrast to innate efficiency, is measured under actual preparative LC conditions. For large values of a,this effective plate number will be much smaller under heavy loads than the innate plate number for the column being used. As a approaches unity, however, the load must be decreased to achieve separation and the two types of efficiency will near each other in value. The two types of efficiency are illustrated by the data plotted in Fig. 1.9 [ref.66]. In these experiments, increasing weights of a test compound, p-dimethoxybenzene [k' = 2.0 in chloroform], were injected into a silica gel column packed with l o p particles. The innate efficiency, denoted by the point at which line A intersects the vertical axis, was in excess of 6500 plates, measured at a flow rate (2.0 ml/min) far from the optimal value, though typical of what might be used in practice. As the injected quantity was increased, the effective plate count eventually began to decrease, slowly at first, and then at a linear rate denoted by line C (slope = 1 decadeldecade on a log-log basis). According to traditional guidelines, this column would be deemed overloaded at about 0.02 mg. However, it can be seen that the rate of loss of efficiency is still less than the rate of load increase up to 0.2 mg, 10 times the traditional overload point. And, if the separation factor is high enough, of course, still larger quantities can be injected.

28

Additional data plotted in Fig. 1.9 contrasts the effect of volume overload with mass overload (see Sections 1.5.1.1.4 and 1.6.2.1.2). Note that when the sample is injected in 0.1% concentration, the measured efficiency beyond 0.01 mg (10 pL volume) begins to take on lower values than when the corresponding mass is injected in higher concentration. The effect of sample volume begins to take hold at higher loads for 1% and 10% solutions and may explain the plateau region (line B) which provides an inflection point between the two parallel linear regions (lines C and D).

p-Dimethoxybenzene MW 138 OMe Concentration in Chloroform: 0 loo/a[~lume] 0 1% 0.1%

I

I 10

!

0.001

[Each point represents average of 2-4 measurements]

1 2olumn: pPORASILm, 4.2 mm ID x 30 cm -low Rate: 2.0 mVmin .inear Velocity: 0.26 cwsec (measured with hexane marker) letection Method: Refractive Index I

I

0.01

0.1

I

1 .o

\

9

0

I

10

Sample Load [mg] Fig. 1.9. Decline of Plate Count with Increasing Sample Load at Varying Sample Concentrations [log-log plot].

As mentioned in the development of the qualitative load model in Section 1.4.2, a change in the type of molecule will greatly influence the sample load and separation efficiency. This is illustrated by the data in Fig. 1.10 [ref.66]. The lines A, B, C, D and points representing pdimethoxybenzene are identical to those in Fig. 1.9. Notice that the much larger tristearin molecule, with its three polar ester groups which can interact with the silica surface separately or in combination, causes the same column, measured at light loads, to have a innate efficiency about 8-fold lower than the corresponding value of N for the smaller aromatic molecule. Tristearin, in this solvent, also has a much larger k' (Dm) value at light loads; this value drops off dramatically as the load increases. Above 10 mg, the measured efficiency becomes very erratic, due to the very low values and greatly distorted peak shapes, but seems to be entering a plateau

29

region similar to the one for the smaller molecule delineated by line B. C

D

\

0

A Concentration: 10% Wvolurne in Other conditions as in Fig. 1.9

0

2 v)

Q)

c (P

1 02 ,

E

1

p-Dirnethoxybenzene MW138 0 Tristearin MW 841 0

0.4

!

I

I

I

0.01

0.1

1 .o

10

10

I

100

Sample Load [mg] Fig. 1.10. Decline of Plate Count with Increasing Sample Load at Varying Sample Molecular Weights [log-log plot].

The general shape of the curves (excluding the plateaus) in Figs. 1.9 and 1.I0was predicted by James L. Waters in the early 1970's tref.671, before this data was obtained experimentally. Waters also predicted that, if a larger particle packing were used, the column would have a correspondingly lower value ofinnate efficiency at light loads, but efficiency would remain constant over a wider range of load, eventually dropping off and joining the corresponding data from the smaller particle column on the same linear line, all other variables being equal. The experiment which corroborated Waters' hypothesis is shown in Fig. 1.1 1. The data for the 1O-micron pPorasiP column and the lines A,B,C, and D are identical to those in the two previous figures. Notice that for an approximately ten-fold increase in particle size (to 125 p), the loading increases more than 100-fold. This means that for difficult separations, the best strategy to accomodate sizable samples of simple mixtures of lower MW compounds is to use larger particle packings configured in longer columns (or multiple columns and/or recycle), rather than shorter, small particle columns with similar overall innate efficiency (though more innate efficiency per unit length). In 1974, this finding ran contrary to then conventional practice. But more recent experiments by other groups now reinforce this conclusion [68-701.

30

0 pPORASILW, 1 Op,4.2 mm ID x 30 cm

C

1 04-

(Surface Area: 325 sq. rrrlgram) 2 rnVmin (linear velocity = 0.26 crrrlsec) PORASIL@A, 125p,7.9 mm ID x 61 cm (Surface Area: 350 sq.Wgram) 6 mVmin (linear vebcii = 0.26crrrlsec)

0

p-Dimethoxybenzene, 10% in Chloroform Other conditions as in Fig. 1.9.

1 03,

E 0

0

c? v) a

c 0

2 z 10,

-

10 0.01

I

0.1

I

1 .o

I

10

1 100

Sample Load [mg, normalized for column volume] Fig. 1 .I 1. Decline of Plate Count with Increasing Sample Load with Different Particle Size Packings [log-log plot].

1.4.3.3 Load

vs. Retention Time

Throughput, or the amount of material purified per unit time, is the practical objective of preparative LC [see Table 1.11. Load and time are the two principal parameters which determine throughput for a given separation. And, like so many other variables that have been discussed up to this point, they are interdependent in ways which make compromise necessary when seeking to optimize a separation system (Fig. 1.2). As defined in Section 1.3.1 and illustrated in Fig. 1.4, volume can be expressed directly as retention time if the flow rate of the mobile phase (volume per unit time) and the system volume remain constant throughout the separation. This qualification is important since, for example, pump output may falter or the chromatographic bed may shrink or swell dynamically (e.g., like some ion exchange packings in a salt gradient). In any case, in larger scale preparative LC, the time it takes to perform a separation, fully eluting all the components of interest and preparing the column for reuse (by washing, reequilibrating, etc.), contributes to at least two significant economic factors: costs for labor and solvent consumption. Separation time, in turn, is determined by a cascade of variables beginning with the

31

thermodynamic properties of the LC system. The distribution ratio of solute between stationary and mobile phases (k') determines the volume/time required to elute that solute from the chromatographic bed (see Section 1.3.1). Though smaller k' values permit increased load in adsorption LC (Section 1.4.2), increasing k' up to about 5 may provide increased resolution (Section 1.3.3). When optimizing the separation factor, a, mobile and stationary phase combinations are chosen first to maximize the ratio of k' values and secondarily adjusted to seek the smallest practical values of k' which permit good loadability at acceptable resolution while minimizing solvent consumption and overall separation time. Unfortunately, in many cases where separations are difficult (a c 1.3), increased time and solvent consumption are usually the price one must pay to achieve the desired results. Given a quantity of sample that must be separated, either the separation must be repeated several times with a small load on a smaller volume column (higher efficiency per unit length) or run once at full load' on a larger volume column (same overall efficiency but larger capacity, see Section 1.4.3.2). . Even in the latter case which is usually optimal, more time may be required to process the necessary amount of sample. Flow rate is the operational variable which most influences time. For easy separations (a2 2) a high flow rate is chosen to make the separation as fast as possible while working within the constraints of acceptable system back pressure (AP) and practical solvent handling capabilities. As a becomes smaller, flow rate may have to be decreased to maintain a good compromise betwaen column dimensions, load, and separation efficiency requisite for the desired degree of resolution and solute recovery. Thus, indirectly, the choice of a specific flow rate is linked to the thermodynamics of a separation ( a and retention time). Other flow rate considerations which affect both time and scale-up of preparative LC separations will be discussed in Section 1.5.1. One key point must be emphasized: with the capabilities of modern preparative LC systems, it is not only desirable but entirely practicable to perform preparative LC separations on a time scale which is identical to that for corresponding analytical LC runs. It is this speed, coupled with heavy loading capacity and separation power (selectivity and efficiency), that makes modern preparative LC so impressive (see Section 1.1). The challenge to the chromatographer is not simply to do more work in less time, but to find ways to accomplish tasks that could never have been done before: to isolate previously unknown, labile compounds; to streamline or simplify synthetic sequences; to improve product performance; to open up new areas of investigation, etc.

Load in Recycle Mode Recycle is a technique used in preparative LC to increase the effective length of a chromatographic bed while avoiding the expense of purchasing and operating longer columns or additional column sections. In practice, it is accomplished by adding a valve to the fluid system to permit the eluent, or some designated portion thereof, to be directed from the column outlet back into the column inlet (see Section 1.7.2.2). 1.4.3.4

32

Complex samples, especially those with strongly retained components that may require step or continuous gradients for elution, are not usually amenable to the recycle technique. These types of samples are best fractionated in the early stages of a separation scheme by class-specific techniques, as mentioned in Section 1.2.3. Then, in the later stages of an isolation protocol, a compound-specific method such as recycle LC can be a very powerful tool to handle low a separations (11.3) with many fewer compounds present in the sample. A dramatic illustration of this separation power emerged from the total synthesis of Vitamin 812 by Prof. Woodward's team [71]. At one stage, it became necessary to isolate the intermediate cobyrinic acid ester, shown in Fig. 1.12, from its neo analog, differing from the normal ester only in configuration at position 13. Recycle LC on silica separated the two epimers to baseline in seven passes through the 10' long column bank, equivalent to a seventy-foot column.

A OD uv 254 nm

A

0

.

.

,

-

.

.

.

.

.

.

.

.

-

20

4a

60

84

loo

120

14a

160

180

m

220

240

260

.

-

280

300

TIME. MINUTES

Fig. 1.12. One of the first dramatic examples of recycle in modern LC: recycle separation of normal- and neo-dicyanoheptamethylcobyrinates. Conditions: column: five - 0.125" I.D. x 24" sections in series, total length = 1 0 , CorasilQ II silica, 37-75p pellicular packing; mobile phase: hexanehnethyl acetatel isopropanoVmethanol-HCN (220:60:10:10);flow rate: 1 mUmin (reproduced with permission from ref.71).

A second example of the capability of recycle to separate closely related compounds with an a value nearing 1.0 is shown in Fig. 1.13. Following acetylation of 26-hydroxycholesteroI (2 mg) isolated from human aortal tissue, the 25s-epimer (10% of mixture) of 26-hydroxycholesteroI-3,26-diacetatewas separated from the 25R-epimer ~ columns [72]. Isolation of the in seven passes through a pair of 30 cm long, 1 0 silica

25S-epimer, the minor component in the mixture, was favored by its lower polarity,

33

causing it to elute earlier. If the elution order had been reversed, then the 25Sepimer would have required a few more passes to fully separate from the non-Gaussian tail of the predominant 25R-epi mer peak.

0

1

2

3

4

5

6

7

Time (hrs.) Fig. 1.13. Recycle of an extremely low a mixture of epimers. Conditions: column: pPorasil, two - 0.39 cm I.D. x 30 cm in series, 60 cm total length; sample: 26-hydroxycholesteroI-3,26-diacetate from human aorta, 25.5-epimer is less polar and elutes ahead of more polar 25R-epimer; detector: refractive index (reproducedwith permission from ref.72).

In the preceeding two examples, the difficulty of the separations precluded heavy sample loads, and the isolation time was measured in hours. Apparently, the researchers were, in each case, content with the initial success in separating a pair of compounds so similar in structure. Further attempts to optimize the selectivity, retention time, or throughput were not needed to achieve their experimental goals. Beyond the advantages of recycle in dealing with very difficult separations, it has the potential, especially when a 2 1.3, to permit great increases in sample load on the same column with good resolution and recovery while delivering considerable savings in solvent cost and separation time. This is illustrated by the model experiments summarized in Fig. 1.14 [ref.73]. A moderate load (100 mg each) of two test components (a= 1.3) on a large particle column gave a resolution of 0.8 (Fig. 1.14a). Doubling the column length to 60 cm by adding another column unit in series yielded nearly baseline separation (Rs = 1.3) at the same load (Fig. 1.14b). A ten-fold increase in load (1 ghetained component) with an additional 60 cm of column (total length = 120 cm) also resulted in baseline resolution on a single pass (Fig. 1 . 1 4 ~ ) .However, 3 passes through a single 30 cm column produced nearly the same separation in less time with nearly a three-fold savings in solvent consumption (Fig. 1.14d). Notice also that, with the high volume flow rates used in the larger diameter column, the time scale is equivalent to that of an analytical LC separation.

34

a

TIME lmml

I Other Conditions: 'column: one or more PrepPAKTM500 Silica Cartridges (5.7 cm ID x 30 cm, hold-up volume 500 mL, 75p mean particle diameter) in series; "sample: standard mixture of hexane (1% w/v), butyl acetate, and ethyl acetate (each 10% w/v, a = 1.3) in mobile phase (methylene chloride); detector: refractive index; '*'resolution: estimated on third pass through column by comparison with standard curves [60](chromatograms reproduced with permission from ref.73).

-

Fig. 1.I4 a,b,c,d. Demonstration of increased throughput and decreased solvent consumption possible with recycle in preparative LC also shows effect of column length and sample load on resolution.

35

A technique for making recycle LC an even more effective tool is peak shaving. In this method, a valve is used during each pass: (a) to direct unwanted components to waste; (b) to collect portions of pure sample components of interest from peak fronts and tails; and (c) to recycle the remainder of incompletely separated sample mixtures back through the column. The concept is demonstrated in Fig. 1.15 (refer also back to Section 1.3.4 and Fig. 1.7). A larger quantity of Compound 1 can be collected in high purity on the first pass, while the quantity of purified Compound 2 collected on the first cycle will be reduced from the ideal if Compound 1 is present in higher concentration and/or tails significantly (deviates from the ideal Gaussian shape). The amount of peak tailing will be a function of sample load and the LC system being used (see Section 1.4.4).

RECYCLE

A

COLLECT

(heavier load, with allowance for Deak tailing)

Fig. 1.15. Using the peak shaving-recycle technique

The benefits of peak shaving-recycle are shown in Fig. 1.16 [ref.74]. As the heavy sample load is reduced on each successive pass by shaving, the column's effective separation efficiency increases, thereby reducing the number of passes necessary to separate and recover the total sample. Alternatively , running the lightly loaded system six times could also purify all of the 3 gram sample but at only one third the throughput with nearly four times the solvent consumption. Peak shaving-recycle can also be used to advantage in very difficult separations. The separation shown in Fig. 1. I 7 was performed on a 3 7 - 7 5 ~reversed phase packing in a three section column (9' total length). This follows the strategy outlined in Section 1.4.3.2; a longer column packed with larger particles was used to provide both increased capacity and separation efficiency. In turn, both capacity and efficiency were

36

I-

16 MINUTES

+

0.5 g Sample

Fig. 1.16. Throughput advantage of peak shaving-recycle technique. Conditions: column: 5.7 cm ID x 30 cm; flow rate: 500 mumin; Heavily Loaded Run: sample size: 3.0 g; separation time: 16 minutes; solvent Consumption: 6.3 L; throughput: 0.19 g/min; Lightly Loaded Run: sample size: 0.5 g (6 injections for 3.0 g total sample); separation time: 48 minutes (for 3.0 9); solvent consumption: 24 L (for 3.0 g); throughput: 0.06 amin (for 3.0 9). (Chromatograms reproduced with permission from ref.74.)

further augmented by peak shaving-recycle. Three hundred milligrams of three positional isomers (1, 2, 3),products of a Bamford-Stevens elimination on dehydroisoandrosterone-17-tosylhydrazone, were thus isolated and identified by NMR and mass spectrometry [75]. Then 100 mg of the first isomer, suspected on the basis of tlc and analytical LC separations to be the side chain cleavage product from an in vifro adrenal mitochondria1 incubation of a 20-aryl analog of (20S)-20-hydroxycholesterol, was admixed with the unknown radiolabeled incubation product. This mixture was chromatographed in a selective liquid-liquid partition system and then recycled in the same system shown in Fig. 1.1 7. Isolation of Compound 1 and further crystallization to constant specific activity strongly suggested the identity of the unknown as 17-methyl-18-norandrosta-5,13(17)dien-3P-01 (Compound 1). This was ultimately proven by mass spectrometry on the product (1 pg) of a larger scale incubation [75]. Thus, the preparative LC peak shavingrecycle technique was used here in two ways: first, to purify synthetic standards for structure elucidation; and second, to confirm, in sequence with other chromatographic separations and analytical techniques (reverse isotope dilution analysis, mass spectrometry), the identity of a biological transformation product isolated at the femtomole level.

37

3

w (I

0

,

~ ~ . - r r ~ + c ~ ~ t ~ ~ c + r i c t n - t c + w ~ c t ~ ~ c t m - c ~ c w - - c c + w -

Clearly, the five examples just illustrated show the practical benefits of recycle in preparative LC systems where it can be used to advantage: higher throughput, lower separation time, lower solvent consumption, minimum column investment. Recycle works best for mixtures of only a few closely related compounds [76-851. Strongly retained components should be removed first by means of other crude separation methods (see Section 1.2.3 and Fig. 1.3). Some additional practical considerations of recycle will be reviewed in Section 1.7.2.2. Theoretical treatments which substantiate the benefits of recycle have been published elsewhere [86,87].

Effect of Load on Peak Shape A picture is worth a thousand words. To a chromatographer, a graphical representation of detector response or effluent concentration - a chromatogram - can deliver immediate visual insight into system status and the nature of the separation process. As aforementioned, preparative LC should be done under heavily loaded conditions, which then result in chromatograms displaying peaks whose shape is far from the Gaussian ideal shown in Fig. 1.5. Often, peak skewing can be attributed to large sample size. But, there are other deleterious reasons for distorted peak shapes, and these can often be deduced from a combination of tangible (chromatogram) and intangible (knowledge of sample and LC system properties) evidence. Before reviewing several other possible causes of peak distortion in Section 1.4.4.2, first consider the effect of large sample size on an adsorption process. 1.4.4

38

1.4.4.1

Peak Shape Changes with Increased Sample Load

Referring to eqn. 1.I in Section 1.3.1, the amount of sample in the stationary or mobile phase can be expressed as the product of sample concentration (Cs or C,, resp.) and phase volume (V, or V,, resp.):

The mass distribution ratio D (), or k' is related to the ratio of volumes of stationary and mobile phases by the distribution constant, KD (also called the partition coefficient). A plot of Cs versus C, for a given compound and adsorption system under equilibrium conditions at constant temperature and pressure should give a straight line with a slope equal to KD (Fig. 1.1 8). The curve shown in Fig. 1.I 8, representing a Langmuir isotherm [88,89], deviates from linearity, however, at a point when either the sample adsorption

A Concave

==-'-

. Linear (Gaussian peak shape)

CS

Fig. 1.18. Plot of Langmuir Isotherms (Description of corresponding chromatographic peak shape given in parentheses).

capacity of the stationary phase or the sample solubility in the mobile phase becomes limited. In the former situation, the isotherm becomes convex; in the latter case, the isotherm assumes a concave shape. Convex isotherms are the most common in adsorption LC and result in peak tailing at higher sample concentrations. Conversely, concave isotherms cause fronting of chromatographic peaks. Peak tailing due to a convex isotherm takes the form of peak asymmetry beginning at the apex and continuing to the baseline. As shown schematically in Fig. 1.19, using a triangle defined by the baseline and tangents to each side, the peak shape at low solute concentration is like an isosceles triangle. As the concentration increases into the non-linear region of the isotherm, the peak shape gradually assumes the form of a right triangle. In this situation, since K, is decreasing, the molecules in the band center tend

39

to remain longer in the mobile phase and migrate faster than those at lower concentration in the band extremities, thereby steepening the peak front and broadening the tail. The vertical leg of the nearly right triangle elutes first at an apparent k' much earlier in time than the actual k' calculated using the position of the peak apex under lightly loaded conditions. For a concave isotherm, the inverse situation occurs, with a broad peak front culminating in a steep tail (the right triangle

W In C

0

a In

d

Fig. 1.19. Triangular representationof band shape as sample size is increased.

shape is reversed). It is important to note that peak area, not peak height, is proportional to concentration in the non-linear region, so long as the detector response remains linear. However, if quantitation must be done based on detector output, the response linearity must be checked in the appropriate range. Looking at broadly tailing peaks and imagining how they overlap might leave a chromatographer, used to analytical LC, wondering how pure sample components might ever be isolated under non-linear isotherm conditions. Examples of success are found in Figs. 1.14d, 1.16, and 1.17 where the peak shaving-recycle technique was used to advantage. But until one gains experience with such separations, a tenuous faith in being able to achieve the desired results might lead to overly conservative column loading and solvent flow rates, thereby reducing throughput significantly. The experiment shown in Fig. 1.20 [ref.90] demonstrates what actually happens during a heavily loaded preparative LC run. Understanding this situation should allay

40

any uneasy feelings about the outcome of a similar separation and instill confidence in one's ability to do successful preparative LC with large sample loads. A 14.4 gram mixture of propylparaben (A) and butylparaben (B) was separated in less than 1 1 minutes, but with little apparent resolution indicated by a slight shoulder on the rear side of a broadly tailed peak (not shown in Fig. 1.20). The total column effluent was collected in 10 mL fractions. Aliquots of each fraction were analyzed for the concentration of each paraben using reversed phase analytical LC with UV detection.

0.15 r

O.O5

t I

I'

O1

2

Fig. 1.20. Actual concentrationprofiles underlying a heavily loaded preparative separation of parabens with minimal apparent resolution. Conditions: sample: A = Propylparaben, B = Butylparaben; sample load: 7.2 g each of A 8 B dissolved in 180 mL mobile phase; column: 5.7 cm I.D. x 30 cm, PrepPAKdOO C18 Cartridge; mobile phase: methanollwater (70:30vk); flow rate: 200 mUmin (one hold-up volume/2.5 min); temperature: ambient; fraction size: 10 mL.

Fig. 1.20 is a plot of UV absorbance for each component in each fraction, shown as individual, overlapping concentration profiles, versus elution volume. Dotted curves show the cumulative percent of each component as it eluted from the column. Examination of the data in Fig. 1.20 shows that as much as 40% of propylparaben (Compound A, 2.8 grams) could have been collected in pure form (>99%) by shaving the front of the peak up to approximately 2.3 hold-up volumes, and 20% of the amount of butylparaben injected (Compound B, 1.4 grams) likewise could have been recovered in >99% purity from the tail of the peak at an elution point greater than 3.5 hold-up volumes. If the overlapping portion between 2.3 and 3.5 hold-up volumes had been recycled, additional pure material could have been collected in similar fashion. A decision on where to collect fractions would have been much easier on the second pass since the 30% reduction in sample load plus increased effective column length would have increased resolution considerably (from less than 0.6 to nearly 0.8, with a

41

pronounced valley between peak centers). Suppose 95% purity were sufficient; then 55% of A and 65% of B could have been collected in a single pass at that purity level. These quantities may have proven sufficient for subsequent use, in which case the overlapped region could then have been discarded in an appropriate manner. Or, alternatively, recycle of the overlapped portion with peak shaving would enable complete recovery of the injected sample components at the desired purity, even if not all of the separated portions of A and B were removed on the first or second pass. Fig. 1.20 also illustrates an important phenomenon that aids the success of heavily loaded preparative LC separations - frontal displacement. If a sample component as it passes through a chromatographic bed is followed closely by a high concentration of a more strongly retained compound, molecules of the latter will compete with and displace the first component from adsorption sites. If the concentration is high enough, the displacement by the second component may become even more significant than the desorption caused by mobile phase molecules. In either case, the effect is to sharpen or narrow the band of the earlier eluting compound and increase the amount of that compound that can be collected in pure form from an incompletely resolved mixture. Thus, it is best to choose a separation system in which the desired component elutes first, especially if it is present in a small amount relative to the potential displacing compound. A discussion of the use of frontal displacement as a technique to separate materials, particularly on a macropreparative scale is beyond the scope of this chapter [91,92]. It is worth repeating some key points of strategy which have emerged from our discussion of the peak shape for heavily loaded preparative LC systems: 0 Take advantage of the higher concentration of solute present in the steep front of a broadly tailed peak by shaving and collecting as much pure compound as possible from this region. 0 Try to choose a combination of stationary and mobile phases which will cause the desired component in a mixed pair to elute first, especially if it is present in much lower concentration. Q When possible, use the peak shaving-recycle technique to advantage for full mass recovery of sample components at desired purity levels. 0 All of the above works best when a is maximized for greatest throughput.

Additional Causes of Peak Shape Distortion As mentioned at the outset of Section 1.4.4, situations other than heavy sample loading can lead to peak shape distortion. Some of these are illustrated in Fig. 1.21 by representations of peak shapes. Peak 1 is a heavily loaded component of the type discussed at length above with a convex, non-linear adsorption isotherm. A similar shape in mirror image would be seen for a concave isotherm. Such a shape is to be expected if maximum throughput is to be obtained in preparative LC. Peak 2 is a more or less symmetrical peak for a lightly loaded component under 1.4.4.2

42

linear isotherm conditions as normally encountered in analytical LC. If such a peak appears in a preparative LC separation at a detector sensitivity setting adjusted for heavily loaded components, it may indicate the presence of a structurally unrelated component in low concentration but with an exceptionally strong detector response uncharacteristic of the other kinds of compounds present in a mixture. Peak 3 exhibits a nearly normal shape except for pronounced tailing which doesn't begin until the rear, later-eluting side nears the baseline. This is characteristic of compounds which have a strong or multi-mode interaction with the stationary phase, e.g., basic compounds on silica gel in normal phase solvent systems. It may be that the compound can exist in two or more forms, depending upon pH or solvent composition or temperature, with one form predominant, e.g., anionic and neutral forms of an organic acid: anomers of a monosaccharide, etc.. Or, the separation mechanism may be complex with competing modes, e.g., liquid-liquid partition and adsorption. Or the separation may be complicated by a prior, undetected change in a portion of the stationary phase which creates heterogeneous modes of solute interaction, e.g., partial hydrolysis of a bonded silica phase, exposing hydrophilic sites on an otherwise hydrophobic surface.

Non-linear

Normal Peak

Faulty injection Two solutes

Mixed mode

Insolubility in mobile phase

Incomplete equilibrium

Fig. 1.21. Guide to peak shape as a visual indicator of the nature of the separation process and equipment status.

Peak 4 looks somewhat like a mirror image of Peak 3. A shape like that of Peak 4 usually indicates that some or all of the solute mixture has limited solubility in the mobile phase. Prolonged fronting on such a peak may indicate that the sample has precipitated at the inlet of the chromatographic bed and is slowly being dissolved. Separation in such cases is usually very poor. It may be necessary to dissolve such a sample in another solvent which has better ability to keep the sample in solution but can be injected in reasonable quantity into the separation system without interfering with the

43

resolution of the peaks (see Section 1.6.2.2.6). Peak 5 is typical of a mechanical problem. The apparent incomplete resolution of two components may in fact be due to a faulty injection, injector system, or column inlet distributor which introduced a sample into the chromatographic bed in two somewhat separated portions. Or, another possibility is the presence of a defect (a void or channel) in the column which may cause a separation of the sample into two or more portions. In this case, the column should be tested for bed integrity under lightly loaded conditions, and, if a defect is found, the column should be repacked or replaced. Peak 6 shows a simple change in slope at a point on its tail (or front). This is usually the telltale sign that a second, incompletely resolved component is present within this peak envelope. In such cases, peak shaving-recycle or the use of a longer or more efficient column or a reduction in sample load or, best of all, the further optimization of the separation system to maximize a will enhance the capability to isolate both components of this mixture. Peak 7 is most often encountered with the use of refractive index detectors and liquid-liquid partition separation mechanisms. In such a case, the actual compound has eluted in a relatively narrow peak with a front side as indicated. But the actual tail is hidden by the prolonged drift down to baseline since the sample component has disrupted the equilibrium between stationary and mobile phases that existed prior to its elution from the column. A differential refractive index detector is very sensitive to minute changes in solvent composition and thus tracks the return of the separation system to an equilibrium baseline condition. In such a case, fractions should be collected across the peak and monitored for the presence of sample. When no sample is found in later fractions, even though the detector response has not yet reached baseline, then the separation is finished and the run can be terminated to conserve solvent and time. In summary, broad tailing of chromatographic peaks in preparative adsorption LC can usually be traced to sample loads in the non-linear region of the adsorption isotherm. But there are several other causes of deviation from symmetrical peak shape which may indicate that there is a problem in the chromatographic system for which corrective steps should be taken. Developing the ability to recognize these situations will not only increase the chances for successful preparative separations but also enhance one's ability to optimize conditions and equipment rapidly for maximum throughput and benefit. If ever in doubt about the interpretation of a peak shape, it is wise to have collected fractions throughout the separation. Then each fraction can be examined, often by analytical LC using the same mobile/stationary phase system, to determine what has happened. Appropriate fractions can then be combined or discarded as necessary. In preparative LC, it is far better to have the option to combine smaller eluate fractions as appropriate than to have to reprocess larger fractions due to the inadvertent remixing of separated components during sample collection.

44

1.5 SCALING UP A SEPARATION The stages of a separation scheme are outlined in Fig. 1.3 and discussed in Section 1.2.3. Before doing a large-scale preparative LC run with its attendant materials expense and labor cost, the separation first should be optimized and tested on a small scale, if at all possible. The best methodology for this purpose is analytical LC. Thin layer chromatography (TLC) can also be used successfully in some situations. If scaling up is to be straightforward, then the small scale system should have as many parameters as possible in common with the large scale system that will be used. This concordance of parameters is especially important in partition and gradient separations; it is somewhat less so when simple adsorption separations are to be done. 1.5.1 Scaleup from Analytical LC Analytical LC is the best scaleup tool because it affords the best opportunity to mimic the larger scale separation conditions in a controlled manner. But it is not always perfect in this regard. Some of the potential concerns and points to consider are

outlined below. 7.5.7.7 General Considerations 7.5.7.7.7 Packing chemistry ideally, there should be no difference between the analytical LC and preparative LC packings except, perhaps, for particle size. Any chemical or physical modifications to the surface such as bonding, heat treatment, washing, etc., should be performed identically on both analytical and preparative packings and verified by analytical measurements as well as chromatographic performance tests. Because these treatments are often done on different scales, identical performance under all conditions for surface-modified materials is always an ideal, but not always a reality. Even simply reducing the particle size of otherwise unmodified inorganic substrates such as silica and alumina or organic polymer matrices such as polystyrene or polyacrylate may not guarantee uniformity of chromatographic selectivity between the two sizes of the "same" material. For example, grinding up larger particles to make smaller ones potentially may expose new surfaces which differ in physicochemical character from that of the surface originally accessible in the larger particles. Most often, any observed differences between analytical and preparative LC runs which can be attributed to packing chemistry do not impair the ability to achieve the desired result. Should significantly different results be expected because of the choice of stationary phases for the small and large scale runs, suitable experiments done on small columns filled with each phase may be performed to determine what system adjustments might be necessary for scaleup.

45

1.5.1.1.2 Packing hlstory Proper phase equilibration and treatment of both analytical and preparative packings is very important for successful scaleup as well as reproducible results. This is especially true, and potentially time-consuming, in liquid-liquid partition separations on surface active substrates and in liquid-solid adsorption systems using multi-component mobile phases which contain one or more minor constituents (lo% w/v) is greatest when scaling up to very large

46

preparative LC columns. This, however, can also lead to a larger plate volume, excessive band broadening, and reduced resolution, all symptoms of mass overload (see Section 1.4.2). At normal sample concentrations (99.9%. (Reproduced wkh permission from ref.73.)

50

Step 6. Collect fractions. Five fractions were collected during each preparative LC run as indicated in Fig. 1.24. Step 7. Check purity on analytical system. As shown in Fig. 1.25, the analytical LC system developed in Step 3 above was also used to monitor the purity of the collected fractions from the preparative LC runs. Steps 8-10. Combine appropriate fractions; Final workup and isolation; Maximum yield of pure materials. A total of 12.6 grams of compound A (pooled fractions #4 and 5) and 3.3 grams of B (fraction #2) was recovered from the two preparative LC runs, following solvent evaporation at a purity level greater than 99%. Since the sample was not costly and the quantities of pure components isolated were sufficient for subsequent synthetic steps, fractions #1, 3, and 5 were discarded. Nor were these fractions investigated for potential trace components, since none were apparent during the analytical LC runs and since this possibility was not germane to the research problem being investigated.

1.5.2 Scaleup from TLC There are significant differences between TLC and LC that may make scaleup from TLC to preparative LC less than straightforward. First, common TLC plates use silica packings which are higher in surface area (500-700 m2/g) and smaller in average pore diameter (60 A) than typical LC packings (300 rn2/g, 100 A, resp.). Thus, samples are generally more strongly retained on silica TLC plates than on analytical silica LC columns. Second, a TLC separation is actually a gradient separation. As mobile phase crawls up a silica TLC plate by capillary action, the more polar constituent in the solvent (even a few ppm of dissolved water) is adsorbed by the dry plate and moves more

slowly than the less polar component. Depending upon the degree of retention of the sample components, they will be more or less affected by this gradient; the resultant selectivity may be different on TLC from that on an LC column with the same mobile phase. Third, a TLC plate usually has less separation horsepower than a typical LC column. Fortunately, this usually works to the chromatographer's advantage during scale-up. But, in going directly from TLC to large preparative columns, it may be disconcerting to see sample components that were not known to exist before doing LC. So, be prepared for the unexpected if analytical LC cannot be done beforehand. Fourth, solvent consumption in TLC is small since sample components are allowed to migrate only a short distance along the chromatographic bed, rather than being fully eluted from the plate. Furthermore, separation is aided, though potentially elution is limited, by the decrease in the velocity of the mobile phase as it moves up the plate against the force of gravity.

51

Fifth, unless precautions are taken, TLC may subject samples to potential photochemical, oxidative, or chemical degradation, due to the manner in which components are spread on a thin, potentially catalytic layer of adsorbent and the time of exposure if development times are long due to the nature of the solvent(s) and characteristics of the layer used (thickness, particle size, etc.). For a review of TLC principles and techniques, see refs. 93-94 and references cited therein. Preparative TLC is also discussed in Chapter 2 in this book. In TLC, the relative degree of migration of sample components is indicated by the Rf value: R

Distance traveled by center of sample zone

=

(1.12)

Distance traveled simultaneously by mobile phase Using the origin (the point at which the sample is spotted on a plate) and positions of the sample spots and solvent front at the time sample migration is terminated, the Rf value is conveniently calculated from simple distance measurements in TLC. Rf can be correlated to a k'.value in elution LC (see Fig. 1.4) using the following relation: Rf = l/(K

+ 1)

or

k' = (1 - Rf)/ Rf

(1.13)

Due to the differences between TLC and elution LC previously described, eqn. 1.13 cannot always be used to predict k' values accurately for an analytical LC separation from a corresponding TLC run using the same mobile phase. Hara [95] found for a series of relatively non-polar to moderately polar steroids and solvents ranging in polarity from pentane to ethyl acetate that a better estimate of k' values for LC separations from TLC runs is obtained by eqn. 1.14 where b = 1.5: be Rf = l/(K + 1) or k'= [ l - (be Rf)]/(b* Rf)

(1.14)

Using eqn. 1.14 with b = 1.5 seems adequate to predict k' values for a wide variety of simple organic compounds on silica columns under liquid-solid adsorption LC conditions. For instance, using the data cited earlier in Section 1.5.1.2, Step 3, for the hydroxytetrahydrophenanthrenes, allowing for some difference between 0.75% and 1YO ethanol in chloroform, eqn. 1.14 predicts reasonably well from the TLC data the k' values for A and B in the second LC trial separation. Another example in shown in Fig. 1.26. A mixture of cholesterol esters was well [ref.96]. As indicated by the actual separated on silica TLC in benzene-hexane (50:50) R f and k' values in Fig. 1.26, eqn. 1.14 (with b = 1.5) closely predicts the corresponding k' data obtained from analytical LC on silica, run using the same mobile phase. Scaleup of the TLC separation to a 10 gram preparative LC run was readily accomplished. Fraction purity was also established by TLC; analytical LC separations are shown for comparison.

52

b

a

W

1 1 1 1 1 4 8

0

TIME (min)

C Fration A

I ,'us1

TIME (minl

TIME Irninl

Fig. 1.26. A comparison of TLC and analytical LC scale up to a preparative LC run for the separation of cholesterol esters. Fig. 1.26a: Analytical separation of mixture by TLC 8 LC; Fig. 1.26b: Prep LC; Fig. 1.26~:TLC 8 LC of prep LC Fraction A; and Fig. 1.26d: TLC 8 LC of prep LC Fraction C. General conditions: sample: equal mixture by weight of cholesterol benzoate (1) and cholesterol phenylacetate (2), 10% wlv in mobile phase; mobile phase for all analytical LC, TLC, and prep LC: benzene/hexane (50:50 v/v). Analytical TLC Conditions: plate: 5 x 20 x 0.025 cm precoated Silica Gel 60; development time: 40 min; distance from origin to solvent front: 16.3 cm; visualization: phosphomolybdic acid (10% in ethanol); Rf values 8 separation factor: (1) 5 = 0.36, k'calculated from eqn. 1.14 = 0.85; (2) Rf = 0.21, k' calculated from eqn. 1.14 = 2.17; ARf = 0.15, a = 2.6. Analytical LC Conditions: column: pPorasil lop silica, 4.2 mm ID x 30 cm; flow rate: 2.0 mUmin; detector: refractive index, 8x; analysis time: 3.5 min; capacity 8 separation factors: (1) k' = 0.82; (2) k = 2.06; cx = 2.5. Prep LC Conditions: column: one PrepPAK-500 silica cartridge, 5.7 cm ID x 30 cm; flow rate: 350 mumin: detector: refractive index; sample size: 10 grams in 100 mL mobile phase; time analysis: column preparation: 1.5 min; column wetting 8 equilibration: 5.0 min; sample preparation 8 loading: 2.0 min; separation 8 fraction collection: 8.0 min; Total elapsed time: 16.5 min. Analysis of Purity: Fraction A: cholesterol benzoate, 1, ~99.9%by analytical LC; Fraction B: cholesterol by analytical LC. (Reproduced with permission from ref.96.) phenylacetate, 2, ~ 9 9 . 4 %

53

TLC can also be used to predict the use of stepwise elution conditions for preparative LC separations as shown in Fig. 1.27 [ref.97]. Three TLC systems using mobile phases of progressively increasing polarity were compared for the separation of a pair of glycol monoesters. Based on this information, the large separation factors, and the relationship between c1 and load in Table 1.5, nearly 100 grams of the mixture was separated in only 8 minutes in a three-step gradient. After analysis by TLC and combination and workup of fractions as shown, 14.8 grams of A and 54 grams of B were obtained in high purity.

111

Analvtlcal TLC Mobile phase* RfA RfB

I

MP 1 MP2

MP3

I

Preparatlve LC

1a

0.49

0.08

11

0.65

0.30

4.3

0.90

0.64

5.1

MP1= CH2CI2 MP 2 = HexanelEtOAc (7030 v&] MP 3 = HexanelEtOAc (50:50 v&)

Sample Sample Ratio Sample Size Flow Rate

Pair of Glycol Monoesters N0 30170 9Egin1000mLofMP1 500 mUmin (0.5 holckrp volumelmin) Column 5.7 cm I.D. x 60 cm, two PrepPAK500 Silica Cartridges in series Gradient steps 1 hold-up volume (1000 mL) MP 1 1 hold-up volume (1000 mL) MP 2 2 hold-up volumes (2000 mL) MP 2 Fraction Size 200 mL Solvent Consumption 5 liters 6.9 Umin (amt. Wrifiedkepn. time) Throughput

-

Monltorlng Sample Recovery by Anelytlcel TLC [Conditions: 20 x 20 cm Silica Plate; Solvent: MP 2; Fraction Numbers are shown below origin spof for each aliquot; Recovered sample amounts from cornbingd fractions as indicated.]

B

. . . . . . . . . . . . Yr. 1

2

3

4

+--

5678910111216

14.8g 29.1 g

Fig. 1.27. A large monoesters (971.

r)

20

54 g

a step gradient separation scaled up from TLC data; separation of a pair of glycol

54

More information and examples on scaling up TLC separations to preparative LC systems are presented in ref.98. The successful results illustrated above are for separations based largely on a liquid-solid adsorption LC mechanism. Separations by liquid-liquid partition and other mechanisms are not so easily scaled up from TLC to preparative LC in the same manner. In these cases, judicious use of analytical LC as a scaling tool is preferred. 1.5.3 Scaleup of Gradient Elution Separatlons In the world of small molecule separations by analytical LC, gradient elution has become a valuable tool for examining complex mixtures or groups of compounds with disparate chromatographic polarities. Advances in instrumentation, particularly solvent delivery and mixing systems, detector design, and microprocessor-based controllers, have made possible a higher level of reproducibility in continuous gradient separation schemes. Micropreparative LC separations performed using analytical LC equipment can take advantage of these sophisticated gradient capabilities. However, once the scale of separation moves into the preparative and macropreparative LC range, the availability, sophistication, and ability of gradient forming and controlling devices in handling large volumes of mobile phase at higher flow rates while duplicating small scale gradient conditions diminishes greatly. Therefore, it is usually recommended that an appropriate sequence of isocratic and/or stepwise gradient elution conditions be used in such situations, if at all possible (see Section 1.6.2.2.5). The resulting benefits may include operational simplicity, reduced capital investment, tighter process control, and, often, lower processing costs. Remember also that the goals of analytical and preparative LC differ (see Table 1.1). It may be that a gradient separation can be used to advantage to shorten analysis times or provide for the quantitation of two or more compounds of widely differing polarity from a common sample matrix in a single chromatographic run. But to use similar conditions for the isolation of one or more of these compounds on a large scale might not be optimal. Review carefully the goals of the preparative separation scheme. Consider the points made in Section 1.2. The capability to process quickly heavier sample loads in a stepwise gradient coupled with the higher resolution possible for pairs of components, thus isolated, under subsequent isocratic conditions may provide a superior alternative to a continuous gradient purification scheme. With the growing importance of micropreparative LC as a purification tool for macromolecules of biological importance, however, it has become necessary to provide means for scaleup of continuous gradient separations. This is true especially for newer separations relying on hydrophobic or mixed mode interactions with high performance stationary phases. Those classical ion exchange, gel permeation, and affinity methods traditionally performed in larger diameter tow pressure or gravity-driven column systems may well be supplanted by these *modern methodologies, as appropriate materials and equipment become available. Very recent product introductions by LC equipment

55

manufacturers augur well for future large scale gradient capability, but experience to date with such systems is very limited. Successful scaleup of gradient systems is simply a matter of common sense combined with the same guidelines proposed in this chapter for isocratic and stepwise gradient preparative LC separations. Care should be taken to reproduce the gradient slope or curve shape, taking into account any differences in column geometry (length as well as volume), packing chemistry (Section 1.5.1), mode of gradient generation, pre-column mixing characteristics, and gradient delay volume. In all cases, a separation relies on the distribution ratio of a compound between stationary and mobile phases (k’ or DM). A continuous gradient changes the k’ value in a predictable manner approximated by an integrated series of small isocratic steps. At the same time, increasing solvent strength during elution compresses a sample band. This results in narrowed peaks and decreased peak tailing, even under heavily loaded conditions, though a corresponding increase in resolution does not necessarily follow. A full discussion of gradient elution and scaleup thereof is beyond the scope of this chapter. The reader is referred to several reviews and recent references for more information [41-43, 46-47, 57-58, 99-1051.

1.6 PREPARATIVE LC PACKINGS AND ELUENTS CHOOSING A SEPARATION SYSTEM

-

As stated in Section 1.3.3,the most important step in developing a preparative LC separation is maximizing the selectivity or separation factor, a. This is done by choosing carefully a combination of stationary and mobile phases with chemical and physical properties appropriate for the sample component(s) which must be purified. Much has been written on this subject [e.g., 40-45, 47, 50-59, 99, 106 and references therein] and will not be repeated here. Instead, some ideas, insights, and experiences will be shared to guide the reader in selecting the best route to maximum a.

Statlonary Phase Considerations Achieving the desired separation is first and foremost; optimizing the separation factor is second; minimizing costs and operational difficulties ranks a close third in the hierarchy of goals which govern the choice of a preparative LC stationary phase. Many mixtures can be separated successfully by more than one LC system; given such options, handling and economic considerations may uttimately determine which system should be used. On the other hand, some isolation procedures may never prove optimal, since sample properties may necessitate critical compromises between various system parameters. In these cases, make every attempt to choose a stationary phase based primarily on its ability to contribute to an optimal separation. If there is no prior experience to serve as a guide, then use a review of available packings such as ref.7 1.6.1

56

and refer to manufacturers' literature to locate materials which meet the separation scheme requirements. Apart from the initial purchase price, don't overlook the labor and materials cost to pack and equilibrate the column, the stability of the packing, and the frequency and cost of regeneration or replacement of spent packing. Any investment or expense should be justified based on the value of the product(s), once it has been isolated at the desired purity level and in the appropriate quantity. 1.6.1.1 ChemlC8l Propertles 1.6.1.1.1 Separatlon mechanisms The two principal categories of chromatographic separation mechanisms are attractive lnteraction with and non-atfractlve permeatlon of the stationary phase. Attractive interaction mechanisms include: general or nonspecific adsorption onto solid surfaces; partition into a liquid layer phase; electronic attraction to a polar, ionic, or z-electron rich surface functionality or phase component; and structure-specific affinity for target functionalities on a surface. Non-attractive permeation mechanisms include separation by: size andlor shape in solution; and/or ionic state. Common terminology in nearly every LC treatise groups these various mechanisms under the general headings: liquid-solid adsorption; liquid-liquid partition; ion exchange or charge transfer; affinity; gel permeation (including subsets: size exclusion and gel filtration); and ion exclusion chromatography, respectively. It is now widely recognized, however, that classification of a particular packing under one of these general separation modes may belie the fact that more than one of the mechanisms just listed may be operating competitively or synergistically in any given separation system. When planning a particular separation, try first to predict which separation mechanism(s) might be used to exploit the differences in molecular structure, size, shape, or charge of the compounds to be isolated and then choose a stationary phase accordingly. For example, silica, with the geometry and polarity of its surface, is a good choice for compounds which differ only in the position of a fairly polar functional group (such as the hydroxytetrahydrophenanthrenes in Fig. 1.22) or in the structure near the polar end of a fairly non-polar molecule (such as the cholesterol esters in Fig. 1.26). Alumina has a unique selectivity for aromatic hydrocarbons [50]. Large biomolecules such as proteins with similar primary structures may have different effective sizes in solution or differences in net charge which suggest the use of a nonadsorptive gel permeation (gel filtration) packing or a weak ion exchange packing, respectively. An extensive review of available preparative LC packings and their properties has recently been published [7], and many of the LC texts previously cited contain detailed discussions on each type of separation mechanism [e.g.. ref.421. Over the course of chromatographic history nearly every conceivable material has been used at one time or another as a LC stationary phase, including charcoal, granulated sugar, powdered bone, tea leaves, and laundry detergent [57l. However, the general utility of LC became widespread only after the technique had been

51

standardized (see Section I .4.1). Part of this process included restricting stationary phases to those few suitable and universally available materials which could be manufactured economically with reproducible properties. For such packings, chromatographers turned to allied technologies normally used for the production of dessicants, catalyst supports, fillers, and other surface-active, mechanically stable powders. From this search first came alumina, and finally silica emerged as the preeminent LC stationary phase [50, 511. To this day, silica, in all its modern refinement, stands as probably the best choice for a preparative LC stationary phase when separating small organic molecules (MW ~ 2 0 0 0 ) . Its particle shape, surface properties, and pore structure can be tailored to a variety of needs. It has a high surface activity which can be modified easily by coating with water or other agents or by covalent bonding (see Sections 1.6.1.1.2 and 1.6.1.1.3). It is wetted by nearly every potential mobile phase and is inert to most compounds. It has good capacity for adsorption as well as absorption. It offers high sample capacity with high purity and excellent mechanical strength at reasonably inexpensive cost. Most important, silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior, based upon extensive history, is generally predictable and reproducible. When contemplating larger scale preparative LC, if possible, one should first explore the possibility of using a solid phase adsorption mode on underivatized silica gel with all the attendant benefits of low cost, versatility, mechanical strength, good LC separation properties, and ease of scaleup and use. 1.6.1.1.2 Adsorption vs. partition Among the various separation modes in LC, the one with probably the greatest possible number of phase combinations, and therefore, the greatest potential for selectivity, namely, liquid-liquid partition LC, is also the mode least practiced in LC laboratories. Traditional liquid-liquid partition involves passing a less polar mobile phase through a bed packed with a high volume, low surface area, inert support (such as diatomaceous earth) on which has been coated a polar stationary liquid phase [107, 1081. Reversing the polarity of the phases, with the less polar phase coated on the support and the more polar liquid as the mobile phase, is an aiternate variation of the technique. These two situations have been termed normal phase and reversed phase chromatography, respectively. A simple three*omponent, two-phase system of isooctane-methanol-water, in various proportions, is a highly selective separation tool for a large variety of compounds [e.g., see ref. 75 and citations therein, also ref. 1091. In such a system, methanol acts to increase the concentrations of isooctane in the aqueous phase and water in the organic phase, respectively. When passing the mobile phase through the supported stationary phase under gravity or low pressure, a portion of the stationary phase liquid is normally swept out of the column bed in the first hold-up volume with small amounts eluting thereafter. Using higher pressure to decrease separation time

58

also magnifies the rate of stationary phase loss. One approach to overcoming the problem of stationary phase bleed, which makes the liquid-liquid system incompatible with common detection techniques in analytical LC, has been to bond covalently the stationary phase onto the surface of the solid support. The refinement of such bonded phases, especially octadecylsilyloxy-silica ( C I ~ ) ,has been one of the most significant factors in the tremendous growth of analytical LC applications in the last 15 years. It has been estimated that between 60-90% of all analytical LC separations are done on a "reversed phase" bonded silica packing [54, 110-11I]. Today the term reversed phase usually designates a separation done using a silica packing whose surface has been made hydrophobic via chemical modification. Unfortunately, just as the terminology has narrowed in meaning, so, too, a bonded phase limits the stationary phase options available to a chromatographer for designing a separation system. Furthermore, not all analytical LC bonded phases have suitable preparative LC counterparts, identical in selectivity, though larger in particle size; this makes scaleup less than straightfotward (Section 1.5.1 .I). Fortuitously, though the number of stationary phase choices is limited, many analytical LC separations can be accomplished on a bonded silica column packing. Such success has made C18 overwhelmingly the first choice when developing a new analytical LC method. However, this has lead many chromatographers to overlook other stationary phase options which may be better suited for larger scale preparative LC applications. For example, unmodified silica gel is much less expensive than bonded silica packings and can do many separations as well as, if not better than, bonded silicas using eluents typical of either normal or reversed phase systems [112-1141. The latter systems clearly operate by a combination of adsorption and partition mechanisms, though the details are not well understood. Even bonded phases, such as C18,will adsorb some amount of the organic solvent from an aqueous-organic mobile phase mixture, creating a liquid stationary phase in situ [40, 54, 110-1111. The nature of this adsorbed layer may change with the concentration of organic solvent in the mobile phase. Thus, it has been observed that a mixture of steroidal compounds, previously separated by traditional liquid-liquid partition chromatography, when injected into a c18 column, elutes in "normal" phase order with a 60:40 methanoVwater eluent but in "reversed-phase" order when the methanol/water proportion is changed to 40:60 [ref.l15]. Such a reversal in elution order would be unlikely if solid phase adsorption (hydrophobic interaction) were the only mechanism operative in this chromatographic system. Thus, the following points merit emphasis: c3 Having done an analytical separation on a reversed phase bonded silica does not always provide the optimal basis for scaleup to a preparative LC system, pending an analysis of mobile and stationary phase considerations (selectivity, cost, lifetime,

59

stability, etc.; see also Sections 1.6.1.2 and 1.6.2 below). CICareful equilibration of any system is especially important if a partition mechanism is implicated. Slight shifts in solvent composition of the mobile phase, due to inadequate mixing, temperature changes, imbibition in situ by the solid phase, etc., can cause significant differences in the eluant composition and selectivity (see Section 1.6.2.2.3). 1.6.1.1.3 Chemical stability and sample contamination As previously pointed out in Section 1.2.3 (Point 8), solving one separation problem often creates another. Preparative LC brings the sample into contact with a large amount of stationary phase, thereby creating the potential for sample contamination. Contaminants from stationary phase sources usually differ considerably in chemical composition from sample components. In some situations, such impurities can be tolerated. Other times, their removal is mandatory. Fortunately, the differences between sample and impurity may simplify any further processing step(s), permitting the use of crude techniques such as crystallization, selective precipitation, or solid-phase adsorption. Sample contaminants arising from stationary phases are likely t o result from chemical instability or degradation of the packing or from co-elution of impurities residing in the packing matrix. The former situation is likely to occur with any bonded silica packing or ion exchange packing (either polymer resin- or silica-based). For example, nearly all silica-based bonded phases available today are made with a siloxane bond, -3-0-Si-, as the link between the silica matrix and the surface functionality. While this bond is thermally stable (permitting use of certain bonded phases in gas chromatography (GC) applications), the reactions used to form it are reversible [116,117]. This often overlooked characteristic imparts a measure of hydrolytic instability which becomes significant under acidic or basic conditions. It often happens that conditions that favor hydrolysis of the bonded phase (e.g., peptide purification on C18 with an aqueous mobile phase containing trifluoroacetic acid at pH 2-3) also favor retention of the hydrolysis product on the packing ( e . g . , octadecyldimethylsiianol is retained on C18 in aqueous solution), thereby forming in situ a coated phase with separation properties similar to those of the original bonded phase. Thus, the first time, separation occurs as predicted, but when the packing is subsequently washed with organic solvent to "clean" the column, the adsorbed phase is removed, leaving behind a changed surface with a different selectivity ( e . g . , less hydrophobic/more hydrophilic due to a higher percentage of accessible unbonded silanol groups). Strong anion exchange packings also exhibit some degree of chemical instability. Opening a container of such a packing usually releases a strong fishy odor due to the free tertiary amine released during hydrolytic cleavage of the quaternary amino functionality. Though anion-exchangers are usually stored in a more stable salt form,

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e.g., chloride or acetate, the presence of moisture initiates conversion to the less stable hydroxide form of the exchanger in situ, which creates the potential for more rapid degradation of the packing, irreversible loss of ion exchange capacity, and sample contamination. One way to avoid the problems associated with instability or degradation of surface-bonded chemical functionalities is to use nonfunctionalized inorganic or organic materials such bare silica or crosslinked polystyrene. But, even these packings can contribute to sample contamination. Polymer matrices, even those which are highly crosslinked, may contain small amounts of low molecular weight oligomers and other compounds used or formed during synthesis of the polymer. These components may be trapped within the polymer matrix and take considerable time to diffuse to the surface where they can be swept away by mobile phase. Silica in the dry state is a very active adsorbent; when improperly stored, it might not only pick up moisture but also various organic materials from the environment (oils, smoke constituents, chemical vapors, etc.) which may elute under the conditions of a preparative LC separation. Some workers attempt to clean "dirty" silica by heating it in air to several hundred degrees Centigrade, hoping to "burn off" organic contamination. In the process, they often cause deposits of carbon to form on the surface, thereby permanently modifying the packing's selectivity and hydrophilic nature. Silica also dissolves, albeit very slowly, in aqueous media. A simple way to remove most of the dissolved or suspended fine particle silica from a sample is to evaporate the contaminated fraction to dryness in a glass vessel. Then, redissolve the sample in a suitable solvent; the silica adheres to the glass wall of the vessel and remains behind [118]. Thus, no matter whether the stationary phase to be used is unmodified or surface-functionalized, the following is recommended: 0 Don't assume that the name of a functional group describes the true surface chemistry of a modified LC packing; e.g., analytical and preparative "c18" packings may differ dramatically in selectivity, depending upon the methods of substrate pre-treatment, synthesis, post-bonding treatment, column equilibration, and upon column history . 0 Use a packing that is uniform in quality from lot to lot and free of surface contamination. 0 If cleaning is necessary, it should be done in a non-destructive, cost-effective manner just prior to use. 0 Beware of changes in packing selectivity with washing and use; make appropriate adjustments in separation conditions for subsequent runs and/or change to a fresh column.

1.6.1.1.4 Stationary phase contamination Just as the sample may be contaminated by the stationary phase, so too, the

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stationary phase may be spoiled by the sample and/or the mobile phase. Stationary phase fouling becomes more difficult to deal with in larger columns. If the column packing cannot be "cleaned" effectively and restored to its original state, either in sifuor after treatment following removal from the column, then some or all of the packing may have to be discarded and replaced. This adds to processing costs and system downtime. The cost of cleaning or reactivating a column may often be higher than the cost of packing replacement if large amounts of expensive solvent must be used to remove strongly bound contaminants which affect selectivity andlor capacity (see Section 1.6.2.2.3). Common causes and cures of stationary phase contamination are: D Adsorption of water from wet mobile phase or sample injection solvents onto dry, active surfaces such as those of silica or alumina. This is one of the most common forms of stationary phase contamination and may prolong the time for column equilibration even the first time a fresh column is used. If possible, it is best to use a stationary phase which already contains a small amount of water, e.g., 3-5% by weight for silica [64]. Then, for the preparation of mobile phases and sample solutions, use organic solvents which contain small amounts of water; the quantities of water present in typical LC grade solvents are usually sufficient [66]. This facilitates column equilibration and separation reproducibility. If a super-active stationary phase is needed for the separation of very non-polar compounds, then extremely dry materials must be used with handling means appropriate for moisture-sensitive substances. Both stationary and mobile phases may have to be discarded after use if dryness cannot be maintained or if the means to reactivate the separation system is costly or time-consuming. Finding an alternate mode of separation which obviates the need for highly active stationary or mobile phases may be desirable in a few situations. 0 Irreversible binding of soluble, more strongly retained, sample components to the packing under the conditions of elution. These substances may or may not be washed off the packing by a stronger mobile phase (see Section 1.6.2.1.2 for a discussion of eluent strength). They are best removed in a preliminary solid-phase adsorption step prior to the preparative LC run. D Clogging of particle pores and interstitial space between particles and within porous bed retainers (frits, screens, etc.) by insoluble particulate or colloidal materials in the sample matrix. These should be removed prior to separation by a suitably effective filtration step. 0 Precipitation of one or more sample components within the bed. This may be due to injection of the sample in a solvent other than the mobile phase (see Section 1.6.2.2.6) or to the separation of certain sample components, one of which had helped to solubilize another, into different zones within the bed; this occasionally happens in natural product isolations [119]. The former case can be avoided by testing the result of mixing a bit of sample injection solution with the mobile phase before running a preparative LC separation. The latter situation may be harder to predict;

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experience with similar samples is the best guide. 0 Microbial growth induced by the nature of the sample and/or phase system. This should be avoidable by any one of several means: minimizing the time of the separation; adding suitable inhibitors to the column during use and/or storage; maintaining sterile conditions during the separation; or, discarding the column packing after use rather than storing and reusing it. D Reaction of one or more sample components with the packing (e.g., Schiff base formation) or catalytic degradation of sample components induced by the packing (e.g., condensation reactions of aldehydes or ketones initiated by dry, active alumina) followed by irreversible adsorption of the higher molecular weight degradation products. These situations should be avoided by predicting in advance the potential chemical complications; then, either keep flow rates high and column residence times short, add reaction inhibitors to the mobile phase, or else, if possible, use stationary phases which cannot induce or promote such reactions. Physical Properties Ideally, the random walk taken by a solute molecule through a chromatographic bed should be equal in length and rate (and therefore, time) to that stroll taken by any other identical molecule through the same bed [39]. Of course, this ideal is not achieved in practice. Given the current state of the art in making particles and packing them into beds, it is probable that the ideal random walk will never be achieved in a chromatographic bed fabricated from particles. However, until an alternative becomes practical, using particulate beds remains a convenient way to practice chromatography. Key physical considerations in designing a preparative LC bed include particle size, shape, porosity (pore size, shape, and distribution), accessible surface area, mechanical stability, cost, and availability. 1.6.1.2

1.6.1.2.1 Particle size, shape, and density The importance of particle diameter (dp) to innate column efficiency has been described in Section 1.4.3.2. Keep in mind that while it is convenient to have a single numerical value for dp to use in various equations, in reality, dp is represented by a paflick? size distribution or range of diameters. It has been found by experience of many workers that the optimum compromise between column efficiency and packing cost is obtained from packings where the dp range from low to high varies by no more than a factor of 1.5 (for dp 5 15p) to 2 (for dp 2 -2Op); e.g., 8-12 p, 10-15p, 20-40p, 50-100p,etc. [ref.120 and citations therein]. One must be cautious in interpreting results in the published literature, however, since findings depend a great deal on experimental conditions, packing technique, and column hardware design, important variables which are not always controlled in an appropriate manner (see Section 1.7.1). Particle shape, particle density, and mechanical strength play key roles in bed permeability (resistance to flow, back pressure) as well as bed stability and efficiency.

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Though generally grouped into two classifications, irregular and regular (e.g., spherical), there are almost as many particle shapes as there are types of particles. For example, irregular silica particles resemble shards of broken glass; rounded silica can be produced by smoothing the rough edges of irregular particles; spherical or spheroidal (ovate) silicas are usually made by direct synthesis. Hydroxylapatite usually takes the form of flat plates though some newer modifications have cubic or spheroidal shapes. Cellulose may be in the shape of fibers, microcrystalline rods, or spheres. Porous polymer particles may resemble popcorn balls, or irregular fragments thereof after grinding. Clearly, a bed densely packed with flat plates of minimum porosity will have very little interstitial volume through which mobile phase can flow; A P will be much higher in such a column and preparative LC throughput will be severely limited by the necessary reduction in flow rate. Similar limitations result from packing columns with friable, fragile, or compressible materials. In such cases, as flow is increased, A P rises exponentially (normally, A P increases directly in proportion to flow rate), due to loss of interstitial space caused by compression of particles or shifting the location of fines or particle fragments, usually generated during column packing or periods of bed operation at high flow rates. For these reasons, packings with good mechanical strength and more rounded (as opposed to flat or fibrous) shape are preferred for preparative and macropreparative LC applications. Available packings which meet the requirements of strength, shape, and reproducible selectivity are usually made from inorganic substrates such as silica and alumina or highly crosslinked organic polymers such as poly(styrene4ivinylbenzene) [7]. When dp is less than about 10-15p,spherical particle shape is believed to have the advantages of being easier to pack into an efficient bed (though the higher particle density often typical of spherical materials may also contribute to this advantage) and of producing beds with lower resistance to flow. As dp increases, ease of packing depends more on particle density and less on particle shape, and pressure drop (AP) becomes a somewhat less critical variable. For optimum bed permeability, of course, fines should be removed from preparative LC packings before use. Monodisperse packings with dp variations of less than 10-20% within a particular range, though perhaps more costly to manufacture, can minimize flow resistance in beds where AP is critical. Also, they may enhance success in packing particles with dp slop. But, with good packing technique and larger size particles for preparative separations, a broader distribution of dp may give equivalent, if not higher, innate efficiency, probably because the amount of space and channel width between larger particles is reduced by the intermingling of smaller particles, thereby making the interstitial fluid velocity more uniform throughout the bed [120,121].Some controlled experiments with newly available packings and equipment are needed to verify this hypothesis; unfortunately, various experimental results reported in the literature are. for one reason or another, inconclusive.

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In large beds formed from particles with dp 2 20p, irregular particles generally have equivalent chromatographic performance to, but a significant cost advantage over, spherical particles. However, depending upon the nature of the material used, spherical shape may impart slightly more mechanical stability to particles, for better resistance to added weight from above when bed heights are increased or for reduced particle attrition when flow rates are accelerated. But these properties should be verified experimentally before investing in a more expensive packing material. Pore size, pore volume, and surface area There is still some mystery and mythology surrounding the role of pores within stationary phase particles in the chromatographic process. The majority of a packing's surface area and pore volume usually results from the smallest pores within the matrix (c40A in diameter). Unfortunately, these are the pores that are least useful for chromatographic separations 1501. They are also the pores that are generally inaccessible to reagents used for surface modification, thereby adding to the heterogeneity of a functionalized packing surface. Pore shapes have been described variously as "ink bottles", "bubbles", "channels", etc. Mathematical description of anything other than a circular cross section becomes more difficult and typically requires simplifying assumptions. Pores may be openly interconnected within a matrix or isolated as separate cells. No matter what a pore's shape and network status is, it will never be penetrated by sample molecules in a mobile phase unless it opens onto and forms part of the accessible surface of a particle. Thus, only a small percentage of pores actually participate directly in the separation process [122,123]. This realization led to the development for analytical LC of pellicular packings and small dp, totally porous packings wherein the diffusion path length of sample molecules into or out of stationary phase pores is minimized [42 and references cited therein]. For preparative LC applications where higher capacity is important, larger dp, higher surface area, totally porous packings are typically used. With this type of material, however, it is more probable that a few molecules might diffuse deeply enough into the particle matrix to get trapped and not find their way back outside in time to travel with the remainder of the sample zone. At best, this causes excessive band broadening; at worst, this leads to irreversible adsorption and stationary phase contamination. For silica-based, preparative LC packings, the best compromise between pore dimensions, accessible surface area, and sample capacity for small organic molecules seems to be: pore range: 4 0 % below 50 A, 250% above 80-90 A (see below); surface area: 200-350 m2/gram (B.E.T. N2 adsorption) [124]. This experience runs counter to the traditional inexpensive, larger particle, irregular silicas used for years in open column and low pressure preparative LC. These were derived from dessicant grades of silica produced cheaply on large commercial scale and optimized for water adsorption with a high population of small pores (cc50 A) and correspondingly high 7.6.7.2.2

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surface areas (400-700 m2/gram and beyond) [50]. Separation of large molecules such as polypeptides and proteins by hydrophobic interaction on silica-based packings requires a different set of properties. Three approaches seem to work for analytical separations: (a) pore range: 2 50% above 150-200 A; surface area: 20-100 m2/gram [ref.l25]; (b) pore range: 2 50% below 50 A; surface area: >400 m2/gram [ref.l25]; or (c) pore range: almost totally non-porous, pore volume < 0.05 mugram; surface area: 25-30p; slurry packing is preferred for particles below 20-25p in diameter. Lower pressure slurry packing may also be required for larger diameter stationary phase materials if they must be pre-swollen by wetting before packing into column beds, e.g., polymeric ion exchangers. Earlier methods of packing preparative LC columns often combined the process of filling a tube with the application of force by various means to cause the bed to settle into a densely packed configuration. Probably the best of the dry packing methods devised is the rotate-tap-pour (RTP) procedure [186,187].While slowly rotating the column and continuously pouring dry packing material into the center of the tube, the side of the tube is tapped at the upper level of the packing only. At the surface, the packing moves as a fluid, while the particles underneath settle into a closely packed configuration, in much the same way as sand becomes packed under the influence of waves passing over a beach surface. If the pouring is not continuous, visible discontinuities appear in the bed which affect efficiency. Also, boucing or vibrating the column (as is commonly done with GC columns) disrupts the packing structure of the bed, rather than settling it, thereby reducing homogeneity and efficiency [185].Addition of baffles to larger diameter columns has been advocated to aid in radial remixing of sample zones as a means to increase efficiency, but, since these devices also served to make the bed more difficult to pack, any potential efficiency gain was usually neutralized by packing inhomogeneity [188]. Though dry packing methods are cheaper, more convenient, and less hazardous for larger columns, dry packing normally does not create beds quite as dense and efficient as slurry packing processes, all other things being equal [186].Slurry packing methods usually combine the settling of particles with the application of pressure in an axial direction paraallel to the direction of flow through the bed. This force may be generated by external fluid pumps [42 and references therein] or by a piston integral to the column structure [189,190]. A more recent technique combines an effective dry filling process with the application of pressure on a flexible column wall in a direction perpendicular to the axis of flow to create a very uniform bed cross-section, eliminating both wall channeling, bridging, and internal voids within the packed bed structure [73,121,191,192].Termed radial compression, this process is claimed by the manufacturer to have the advantages of

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convenience and reproducibility in forming homogeneous packed beds for preparative LC as well as of being able to remove voids formed in situ, thereby prolonging the life of the column. Furthermore, the use of cartridges permits rapid "repacking" of columns or switching to columns with different selectivity for subsequent steps in a LC separation scheme. A comparison of columns packed by radial and axial compression methods under controlled conditions has demonstrated the effectiveness of the former technique in producing higher column efficiencies and preparative LC throughput with less time and effort expended [193]. Eluent and Sample Delivery Systems Some desirable features for the means of introducing mobile phase and sample solutions into the chromatographic bed include the following: Q An inert pump capable of delivering solvents at suitable pressures and reasonably constant flow rates in the range up to about one hold-up volume per minute, thereby permitting compatibilty with samples, solvents, columns, and detectors as well as achieving separations on a time scale comparable to corresponding analytical LC separations. A pump should have low internal volume for recycle capability (see Section 1.7.2.3) and easy maintenance and servicing characteristics. It should be capable of drawing solvent from a reservoir of any volume. CI An inert sample injection system capable of withstanding reasonable system pressure, delivering quantitative amounts of sample, and handling volumes up to about 20% of the column hold-up volume. For large scale preparative LC systems, a combination of suitable valves, pump, and sample reservoir is usually constructed; automtatic control of injection, collection, and solvent delivery systems may be desirable for repetitive injections in a batch processing mode. 0 An inert mobile phase reservoir capable of continuous mixing with a capacity of about 20-40 times the column hold-up volume to permit a sufficient mobile phase supply for column equilibration, separation, and collection of the last component of interest (k' 5-10). In the above descriptions, the word inert means not affected by the samples or solvents used in any way that would lead to component wear, weakening, corrosion, or failure or to contamination of the sample or fluid stream. Clearly, only those materials of construction which come into contact with the sample/fluid path need be inert. And it may not be possible or desirable to build a fluid handling system of r,,aterials which are inert to all possible samples or mobile phases while maintaining other necessary characteristics such as an adequate pressure rating, ease of machining or fabrication, mechanical strength, or reasonable cost. For example, a plastic pump may work well with biological samples and aqueous buffers, but bind due to swelling or other dimensional or chemical changes caused by organic solvents. When the operational back pressure (AP) of a preparative LC system increases by an order of magnitude, e.g., from 100 psig to 1000 psig, then the difficulty of meeting 1.7.2

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design requirements for seals, fittings, wear tolerances, and mechanical strength of system components may escalate by a similar factor. Stationary phase particle size, bed density, column dimensions, mobile phase viscosity, and volumetric flow rate are some of the key parameters which determine AP. Once these are matched to the separation goals (Section 1.2.2), then the design requirements for the LC system may be set and appropriate hardware components may be chosen and assembled. In constructing higher capacity preparative LC systems, fluid delivery components are usually chosen first on the basis of the materials of construction, AP capability, and flow rate range. Often uniformity of flow delivery and flow pulsation become secondary considerations which are compromised in order to meet the primary requirements just mentioned. Reasonably constant flow rate (+ -5% or better) is important if the separation needs to be repeated predictably and/or automated; it is somewhat less critical if many fractions are to be collected and analyzed off-line. Pump pulsation, as in analytical LC systems, should be minimized if the on-line detector used, with or without a stream splitter, has a characteristic sensitivity to flow pulsations which would interfere with analysis of the effluent stream. With some industrial pumps converted for preparative LC use, pulsation can be extreme, with intermittant pressure surges which can lead potentially to premature degradation of column and system performance. In such cases, some pulse dampening device appropriate for the pump flow rate and AP must be used. Unfortunately, the band spreading characteristics of most pulse-dampeners preclude the use of a recycle mode as shown in Fig. 1.29 (see Section 1.7.2.2). It is best to consult the manufacturers of fluid handling equipment for preparative LC applications for their recommendations (see Table 1.9).

Turbulent vs. Laminar Flow Within channels through packed beds of both analytical and preparative LC columns, solvent flow is laminar, with pressure drop directly proportional to flow rate. In a laminar flow regime, viscous forces are strong relative to inertial forces moving the liquid through a channel causing a characteristic parabolic flow profile with fluid velocity being slower near the walls and faster in the center of the channel. When the channel diameter and fluid velocity increase, a transition to turbulent flow is possible with inertial forces being predominant. The resultant flow profile is much flatter with better radial mixing; however, the pressure drop increases dramatically, being exponentially proportional to flow rate in turbulent flow [41]. Whether flow is turbulent or laminar can be predicted by the Reynolds number (Re), a dimensionless parameter which equals the product of channel diameter, fluid linear velocity, and ratio of density to viscosity: 1.7.2.1

Re = [p (g/cm3)/q (poise)] x [linear velocity (cm/sec)] x [tube I.D. (cm)]

(1.16)

When Re is below 2000, laminar flow predominates. The transition to turbulent flow occurs at Re values between 2000 and 5000.

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The issue of turbulent flow becomes significant in preparative LC systems wherein the I.D. of transport tubing is larger than that’of analytical LC systems. For example, with dichloromethane (p/q = 300),flow is turbulent at 100-500mumin in tubing of 0.093 I.D. (Re = 13,560at 500 mumin; 271 2 at 100 mumin). Water (p/q = 100)does not enter turbulent flow in this dimension of tubing until nearer the 500 mumin flow rate (Re = 4490 at 500 mumin; 900 at 100 mUmin). The advantage of turbulent flow in transport tubing is reduced band spreading; the disadvantage is higher system operating pressure. Therefore, when designing a preparative LC system, it may be desirable to use minimum lengths of transport tubing designed for turbulent flow with a column (always under laminar flow) and pump capable of handling the additional back pressure.

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1.7.2.2 Recycle System Design To realize the advantages of peak shaving and recycle for preparative LC separations illustrated in Section 1.4.3.4, a system such as that shown in Fig. 1.29can be used. The key feature is the selection valve which permits alternate redirection of eluate flow back through the pump and column for recycle and collection of fractions. In designing such a system, a means must be provided to flush out the various pieces of transport tubing when not being used, perhaps with an air pump, so that when the next fraction is directed back into that section of tubing, cross contamination with a previously collected fraction does not occur. An alternative system design for doing recycle is shown in Fig. 1.31. This system was originally used for gel permeation chromatography [194-I971. It is less convenient for larger scale preparative LC systems since at least two columns are required. These columns must be matched in efficiency and performance in order to benefit from the use of recycle; this may be more difficult in very large scale columns, especially after they

have been reused several times. Another potential disadvantage of this system is that solvent is being consumed continuously, while in the system shown in Fig. 1.29,during periods of recycle, solvent is being conserved. It is possible to estimate the number of cycles possible with recycle in a particular LC system by using a simple rule of thumb. Assuming that the maximum peak spreading that can occur before the tail will catch the front of a band is equal to a ratio of elution volume to peak width at baseline of 3:1,then, using eqn. 1.6 in Fip. 1.5,the minimum number of plates, N, necessary for one cycle = 16(3/1)2= 144 plats. Divide the total column plate count (see Section 1.4.2,esp. Table 1.4)by 144 to estimate the number of cycles possible without shaving or collection. For example, if a column has an innate efficiency of 500 plates, then about 3 cycles are possible. By selective shaving of mass during subsequent passes, the peak width is thereby reduced, and the number of cycles possible can be extended further.

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Six-Port Valve

+

Fig. 1.31. A Schematic Representation of a Preparative LC System Designed for the Technique of Alternate Column Recycle. After the portion of eluate to be recycled enters Column #2. then the valve position is switched to the setting indicated by the shaded arrows. Then, if further recycle is desired, the band is allowed to re-enter Column #1, and the valve is switched back to the initial position. In either position, the portion of eluate not being recycled can be diverted to a fraction collector or to waste by another valve connected as shown to Port #4.

1.7.3 Detection If the ideal detector for preparative LC were to exist, it would have some of the following features: 0 The ability (a) to detect universally all compounds with equal response to mass or molar quantity present or (b) to detect specifically only those compounds of interest in series with another detector which has characteristic (a). 0 Have a wide dynamic response range, with the high sensitivity required for trace components and small quantities present after several recycles with peak shaving, as well as the reduced sensitivity to accomodate high concentrations injected into heavily loaded columns. 0 Be insensitive to flow noise, pressure changes, temperature changes, and solvent composition changes (gradients) at any flow rate. 0 Have long term baseline stability for continuous operation. 0 Have no restrictions on solventhobile phase/sample compatibility. 0 Be insensitive to bubbles, small particles, etc. 0 Be easily serviceable, reliable, and require few adjustments, if any.

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D Have a cell design capable of use on-line at high flow rates and typical system operating pressures. Now that the litany of features of the ideal detector has been recited, it must be realized that no detection system available today can meet all these needs. Traditionally, the refractive index detector, because of its nearly universal response to most classes of compounds and its wide dynamic range which favors lower sensitivity, has been the preparative LC detector of choice. A UVNisible detector can be used if it has multi-wavelength adjustment capability so that it can be offset from the wavelength of maximum absorbance for the compound(s) of interest. Also, a shorter path length cell is used typically to deal with high concentrations of sample components with high extinction coefficients. Keep in mind that any detector can become overloaded by large sample concentrations, producing non-linear response and skewed peaks (see Fig. 1.23,for example). If the same separation is scaled up directly to a preparative LC column with sufficient innate efficiency, though lower than that of the smaller column, the sample concentration at any corresponding point in the eluting peak will be diluted due to increased band width, and detector response may remain linear throughout the chromatogram. An example of this phenomenon is seen in Fig. 1.24(compare with Fig.

1.23). UV detectors can serve to monitor gradient runs, but they also limit the composition of mobile phases to reasonably UV-transparent components. A combination of detectors such as UV and RI in series can occasionally deliver more information about sample composition than can a single detector. For example, strongly UV-absorbing trace impurities might be monitored while the major compounds of interest are detected at low sensitivity by RI changes. Both UV and RI detector cells are limited in the flow rate and pressure drop that can be tolerated. For this reason, in larger scale preparative LC systems which employ high flow rates, a stream splitter, as depicted in Fig. 1.29,is often used. When adjusted properly, a splitter diverts a small portion (s-l%) of the total eluent flow, within the range typical of analytical LC, to the detector cell in real time so that decisions on when to collect fractions, etc., can be made while the separation is being monitored. Also, a splitter permits the use of a normal analytical cell, minimizes back pressure on the cell walls and fittings, and does not change the detector response to sample concentration in the eluate. If total sample recovery is required, then the effluent from the detector can be collected and/or recombined with the majority of the column effluent stream before it arrives at the primary fraction collection device. Sometimes, neither an in-line detector nor a detectorkplitter combination is used at all. In such cases, fractions are collected and analyzed off-line by a suitable method, e.g., bioassay or liquid scintillation or reaction with a suitable visualization reagent (often done after spotting aliquots on a TLC plate). Of course, if the assay method is destructive to the sample, then only a portion of each fraction is sacrificed while the

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remainder is processed in an appropriate manner for sample recovery. Sample Collection and Recovery Preparative LC done on analytical LC systems can often take advantage of semi-automatic fraction collection systems which hold test tubes or vials. Larger scale systems require the use of collection devices which can divert flow into large containers by means of an automatic flow divider/funnel type mechanism and lengths of tubing. This type of collector has more parts to keep clean but can be set up to accommodate any suitably sized container. Remember, when in doubt, it is much better, to collect many small fractions during the entire course of a run and recombine them later as appropriate after analysis. Otherwise, the preparative LC run may have to be repeated. Rotary evaporation and flash distillation devices are most commonly used to recover purified sample components and mobile phases. Remember to take suitable precautions to minimize loss of labile or volatile compounds. Batch or column solid phase extraction can also be used for sample recovery. This works well with aqueous eluate mixtures from which removal of large quantities of water by evaporation or freeze drying is slow or which contain additional salts or buffer components which must be separated from the sample. To begin a typical solid phase isolation protocol, first reduce the eluent strength of the combined eluate fractions (e.g., a solution of a hydrophobic compound in an aqueous-rganic, buffered mobile phase) by diluting them with more water, being careful not to precipitate the sample of interest. Then pass the mixture through a bed of a hydrophobic packing such as C18-bonded silica (which has been prewet with organic solvent, followed by washing with clean water). The compound is adsorbed while the buffered mobile phase passes through the bed. Wash out any remaining polar compounds with additional water or dilute water-organic solvent mixture equal in composition to the loading solvent but minus the buffer components. Then use a water-miscible organic solvent such as methanol to wash the desired component off the bed in a small volume (1 -2 hold-up volumes). This solution can then be easily filtered, evaporated, and/or recrystallized to recover the sample. This process for sample recovery is analogous to a technique called trace enrichment which is often used to concentrate organics from dilute aqueous sample matrices onto the head of a column containing a hydrophobic or reversed phase LC packing [42, 1441. 1.7.4

1.7.5 Automation A full discussion of the automation of preparative LC systems is beyond the scope of this chapter. Commercial systems are available which can automate solvent delivery, gradient generation, sample introduction, separation monitoring, and fraction collection (check with companies listed in Table 1.9, among others). Much work has been done with production scale LC systems, especially in the areas of continuous chromatographic processes, overlapping injections, and other alternate system

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designs. For more details, see references 31, 188, 198-212, Chapter 3 in this book, and citations therein. The next generation of powerful personal computers and computer work stations is certain to have profound impact on the ways in which preparative LC systems, including sample and solvent recovery, are controlled and operated, even from locations remote from hazardous laboratory or plant environments, just as centralized, total management of analytical laboratory instrument systems is now rapidly becoming co mmonplace.

1.8 CONCLUSION It is hoped that the foregoing discussion of strategies, guidelines, and hints for achieving successful preparative LC separations will lay the foundation for reading the following chapters in this volume and for enabling the reader to harness the power of large scale LC to accomplish specific separations of interest and importance.

1.9

ACKNOWLEDGMENTS

Sincere thanks are expressed to: Carla J. Clayton for her extensive, expert bibliographic searches and reference retrieval using technology for on-line access to computerized databases; Dr. Charles Phoebe for a critical reading of the manuscript; our colleagues, many of whom are referenced herein, for allowing us to relate some unpublished, hitherto proprietary research results; and especially, James Waters and Burleigh Hutchins for their inspiring support of ours and other pioneering efforts to create a new philosophy as well as a new technology for doing large laboratory scale preparative LC.

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1.10 REFERENCES The published references cited in this chapter and additional references which should place the history and development of preparative LC in perspective as well as provide models for future applications are categorized in Table 1.I 0. Table 1.10 Some references on Preparative LC Topic

General LC references Preparative LC reviews Preparative LC theory Column design & preparation Stationary phases Mobile phases & solvents Liquid-solid adsorption LC Reversed-phase LC Liquid-liquid partition LC Gel permeation LC Ion exchange LC Affinity LC Gradient elution LC Trace enrichment Micropreparative LC Macropreparative LC Thin-layer chromatography Instrument design Sample modification Recycle in preparative LC 4pplications of preparative LC in: Life science

Reference Numbers'

[39-49, 52-53,55-59,106,173,213-21 51 [9-13, 16,20-25,28,30,32-36,2841 [8,18-19,26-27,29,37-38,50,60,68,69-70, 86-87,89,91-92,95,141-143,177,210-211, 268-279,2861 [61,73,120-121,182,184,186-187,189-193, 212,279-289,2961 [7,50-51,112-114,1 1 7,123,125-127,135, 155,184,245,259,287-288,290-296,3031 [137-139,147-155,157,160,163,2971 [50-51, 54,61,95,109,285,3061 [54,101,110-111,154,2291 [62,107-109,2501 [78,130-134,180-181, 194,196,251-256,294, 3061 [IOI,228,257-259, 294,3051 [I 01, 204,206,260-2641 [99-105, 229,2941 [I 44,2301 [299-3011 [31,180-181,188,198-205,207-210,2121 [93-95, 98,266-267,3041 [169-172, 174-176,178-179,194-197,2891 12651 [72-87,194-1971 [58,75,83,100-101,105,121,125,175,18 201,206.21 1, 216-230,244,256,259,294, 231,232,284,3061

NOTE: References are listed in numerical order at the end of Chapter 1.

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99 120 C. Dewaele and M. Verzele, J. Chromatogr., 260 (1983) 13-21. 121 H.C. Jordi. U.D. Neue, H.M. Quinn, and C.W. Rausch, in G.L. Hawk, P.B. Champlin, R.F. Hutton, and C. Mol (Eds.), Biol./Biomed. Appl. Liq. Chromatogr., 111,3rd Intl. Symp., Boston. MA, Oct. 11-12, 1979, Marcel Dekker. New York. 1981, w.327-346. 122 Ref. 51, chap. 2. 123 L.C. Hansen and T.W. Gilbert, J. Chromatogr. Sci., 12 (1974) 464-472 124 P.D. McDonald and R.L. Vivilecchia, unpublished work, 1974. 125 H. Engelhardt and H. Mirller, Chrornatographia, 19 (1985) 77-84. 126 K.K. Unger, G. Jilge, R. Janzen, H. Giesche, and J.N. Kinkel, Chromatographia, 22 (1986) 379-380. 127 K.K. Unger, G. Jilge, J.N. Kinkel, and M.T.W. Hearn, J. Chromatogr., 359 (1986) 61-72. 128 H.S.Schultz and M. Murphy, private communication, 1984. 129 P.D. McDonald, in Waters Sourcebook for Chromatography Columns and Supplies, Waters chromatography Division, Millipore Corporation, Milford, MA, 1985, p. 6. 130 W.W. Yau. J.J. Kirkland, and D.D. Bly, Modem Size-Exclusion Lquid Chromatography, Practice of Gel Permeation and Gel Filtration Chromatography, Wileylnterscience, New York,1979. 131 B.G. Belenkii and L.Z. Vilenchik, Modern Liquid Chromatography of Macromolecules, Elsevier, Amsterdam, 1983. 132 P.L. Dubin (Ed.), Aqueous Size-Exclusion Chromatography, Elsevier, Amsterdam, 1987. 133 T. Kremmer and L. Boross, Gel Chromatography, Theory, Methodology, Applications, Wiley-Interscience, New York, 1979. 134 J. Janca (Ed.), Steric Exclusion Liquid Chromatography of Polymers, Marcel Dekker, New York, 1984. 135 F.V. Warren, Jr., and B.A. Bidlingmeyer, Anal. Chem., 56 (1984) 950-957. 136 Ref. 40, pp. 240-241. 137 J.T. Przybytek (Ed.), High Purity Solvent Guide, 2nd edn., Burdick & Jackson Laboratories, Inc., Muskegon, MI, 1982. 138 L.R. Snyder, J.L. Glajch, and J.J. Kirkland, J. Chromatogr., 218 (1981) 299-326. 139 J.L. Glajch, J.J. Kirkland, and L.R. Snyder, J. Chromatogr., 238 (1982) 269-280. 140 Ref. 109, p. 249. 141 R.A. Barford. R. McGraw, and H.L. Rothbart, J. Chromatogr., 166 (1978) 365-372. 142 B. Coq, G. Cretier. and J. Rocca. Anal. Chem., 54 (1982) 2271-2277. 143 H. Poppe and J.C. Kraak, J. Chromatogr., 255 (1983) 395-414. 144 B.A. Bidlingmeyer, LC, 2 (1984) 578, 580. 145 P.D. McDonald, unpublished experiment, 1980. 146 P.D. McDonald, unpublished experiment, 1973. 147 J.A. Riddick, W.B. Bunger, and T.K. Sakano, Organic Solvents: Physical properties and methods of purification, 4th edn., Wiley-Interscience, New York, 1986. 148 N.E. Spingarn, C.T. Garvie-Gould, and L.L. Vuolo, Anal. Chem., 53 (1981) 565-566. 149 L. Carlsen, H. Egsgaard, and J.R. Andersen, Anal. Chem., 51 (1979) 1593-1595. 150 H. Kiesele, Anal. Chem., 52 (1980) 2230-2232. 151 Reagent Chemicals, 7th edn., American Chemical Society. Washington, D.C., 1986. 152 J.L. Glajch. J.J. Kirkland, and W.G. Schindel, Anal. Chem., 54 (1982) 1276-1279. 153 P.L. Bartlett, Chemtech, 10 (1980) 354. 154 H. Engelhardt and G. Ahr, Chromatographia, 14 (1981) 227-233. 155 R.A. Bredeweg. L.D. Rothman, and C.D. Pfeiffer, Anal. Chem., 51 (1979) 2061-2063. 156 P.D. McDonald, unpublished experimental details, 1973. 157 M. Karstadt, Harvard Med. School Health Letter, 11(August,1986) 5-7. 158 ACS Committee on Chemical Safety, Safety in Academic Chemistry Laboratories, 3rd edn., American Chemical Society, Washington, D.C., 1979. 159 L. Bretherick (Ed.), Hazards in the Chemical Laboratoty, 3rd edn., The Royal Society of Chemistry, London, 1981.

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220 221 222 223 W.E. Cohn, in E. Heftmann (Ed.), Chromatography, 3rd edn.. Van Nostrand-Reinhold, New York, 1975, pp. 714-743. 224 J. Rivier, R. McClintock, R. Galyean, and H. Anderson, J. Chromatogr., 288 (1984) 303-328. 225 C.A. Bishop, D.R.K. Harding. L.J. Meyer, W.S. Hancock, andM.T.W. Hearn, J. Chromatogr., 192 (1980) 222-227. 226 K.M. Patel, J.D. Morrisett, and J.T. Sparrow, J. Lipid Res., 20 (1979) 674-677. 227 R . Shaw and W.H. Elliott, J. Chromatogr.. 177 (1979) 289-295. 228 M.N. Schmuck, K.M. Gooding, and D.L. Gooding, J. Lq. Chromatogr.. 7 (1984) 2863-2874. 229 M.T.W. Hearn, J. Liq. Chromatogr. 3 (1980) 1255-1276. 230 R. Dixon, R. Evans, and T. Crews, J. Lq. Chromatogr., 7 (1984) 177-190. 231 C. Oliemqn, D. Voskamp, and M. Verhage. in G.L. Hawk (Ed.), 4th Symp. BiologicaVBiomedical Appl. Liq. Chromatogr., Amsterdam, April 28-29, 1981, Marcel Dekker, New York, 1982, pp. 1-12. 232 V.R. Meyer, J. Chromatogr., 316 (1984) 113-124. 233 N.G. Iconornu, Pharm. Delt., Epistem. Ekdosis, 9 (1983) 31-42. 234 I.W. Wainer (Ed.), Liquid Chromatography in Pharmaceutical Development: An Introduction, Aster Pub. Corp., New York, 1985. 235 M.E. Wall, M.C. Wani, and H. Taylor, Cancer Treat. Rep., 60 (1976) 1011-1030.

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236 K.H. Kubeczka, in A. Baerheim-Svendsenand J.J.C. Scheffer (Eds.), Proc. 15th Int. Symp. Essent. Oils Aromat. Plants, 1984, NijhoffNunk, Dordrecht, Netherlands, 1985, pp. 107-126. 237 G. Tiiel and H. Wagner, in Stud. Org. Chem. (Amsterdam), 11 (1982) 299-310. 238 M. Dong, I. Schmeltz, and D. Hoffmann, J. Chromatogr., 150 (1978) 269-272. 239 P. Shenbagamurlhi, F. Naider, J.M. Becker, and AS. Steinfeld, J. Chromatogr., 256 (1983) 117-125. 240 P. Shenbagarmrthi, H.A. Smith, J.M. Becker, and F. Naider, J. Chromatogr.. 245 (1982) 133-137. 241 R.K. Tiebach and M. Schramm, Chromatographia, 13 (1980) 403-407. 242 K.P. Hupe, H. Lauer, and K. Zech, Chromatographia, 13 (1980) 413-420. 243 J.D. Warlhen, Jr., J. Liq. Chromatogr., 3 (1980) 279-286. 244 K.M. Patel and J.T. Sparrow, J. Chromatogr., 150 (1978) 542-547. 245 L.K.T. Lam, C. Yee, A. Chung, and L.W. Wattenberg, J. Chromatogr., 328 (1985) 422-424. 246 V.A. Davankov, A.A. Kurganov, and A1.S. Bochkov, Adv. Chromatogr. (N.Y.), 22 (1983) 71-1 16. 247 W. Giger, M. Ahel. and C. Schaffner, Comm. Eur. Communities, [Rep.] EUR, EUR 8518, Anal. Org. Micropollut. Water, (1984) 91-109. 248 H. Schindlbauer and M. Zander, Erdoel Kohle, Erdgas, Petrochem., 37 (1984) 206-21 1. 249 P. Pei, J. Britton, Jr., and S. Hsu, J. Liq. Chromatogr., 6 (1983) 627-645. 250 K. Hostettman, M. Hostettmann-Kaldas,and K. Nakanishi, J. Chromatogr., 170 (1979) 355-362. 251 K.A. Boni, in R.R. Myers and J.S. Long (Eds.), Treatise Coat., Vol. 2(2). Marcel Dekker, New York, 1976, pp. 67-121. 252 A.R. Cooper, A.J. Hughes, and J.F. Johnson, J. Appl. Polymer Sci., 19 (1975) 435-450. 253 J-Y. Chuang and J.F. Johnson, Separation Sci., 10 (1975) 161-165. 254 Y. Kato, T. Kametani, K. Furukawa, and T. Hashimto, J. Polymer Sci., Polymer Phys. Edn., 13 (1975) 1695-1703. 255 A.R. Cooper. A.J. Hughes, and J.F. Johnson, Chromatographia, 8 (1975) 136-142. 256 0. Mikes, High Performance Liquid Chromatography of Biopolymers and Biooligomers, Parts A & B, Elsevier, Amsterdam, 1987. 257 D. Naden and M. Streat (Eds.), Ion Exch. Technol., Ellis Hotwood, Chichester, UK, 1984. 258 H.F. Walton (Ed.), Ion-ExchangeChromatography, Halsted Press, New Yo&, 1976. 259 H.A. Chase, in D. Naden and M. Streat (Eds.), Ion Exch. Technol., Howood, Chichester, UK, 1984, pp. 400-406. 260 P.D.C. Dean and J. N.B. Walker, Bochem. Soc. Trans., 13 (1985) 1055-1058. 261 H.A. Chase, J. Chromatogr., 297 (1984) 179-202. 262 J. TulkovA, Affinity Chromatography, Elsevier, Amsterdam, 1978. 263 I.M. Chaiken, M. Wilchek, and I. Parikh (Eds.), Affinity Chromatography and Biological Recognition, Academic Press, Orlando, 1983. 264 H. Schott, Affinity Chromatography, Template Chromatography of Nucleic Acids and Proteins, Marcel Dekker, New Yolk. 1984. 265 J.F. Lawrence and R.W. Frel, Chemical Dedvatizatbnin Liquid Chromatography, Elsevier, Amsterdam, 1976. 266 M. Canale, S. Bistarini, and M. Merler, Arch. Toxicol.. 37 (1977) 143-147. 267 W. Jost, H.E. Hauck, and F. ElsenbelR, Fresenius' 2. Anal. Chem., 318 (1984) 300-301. 268 G. Cretier and J.L. Rocca, Chromatographia, 20 (1985) 461-465. 269 K.P. Hupe and H. Lauer, J. Chromatogr., 203 (1981) 41-52 270 V.R. Meyer, poster paper, 10th Int. Symp. Column Liq. Chromatogr., San Francisco, May, 1986. 271 A.W.J. DeJong, J.C. Smh, H. Poppe, and J.C. Kraak, Anal. P a . (London), 17 (1980) 508-512. 272 G. Cretier and J.L. Rocca, Chromatographia, 18 (1984) 623-627. 273 E. Geeraert and M. Verzele, Chromatographia, 12 (1979) 50-51. 274 J.P. Larmann, R.C. Williams, and D.R. Baker, Chromatographia, 8 (1975) 92-96. 275 A. Wehrli, U. Hermann, and J.F.K. Huber, J; Chromatogr., 125 (1976)'59-70. 276 K.J. Bombaugh and P.W. Almquist, Chromatographia, 8 (1975) 109-118.

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105

PREPARATIVE TH IN LAYER CHROMATOGRAPHY Joseph Sherma Department o f Chemistry and Bernard F r i e d Department o f B i o l o g y L a f a y e t t e Col lege, Easton, PA

2.1

18042

INTRODUCTION

2.2

LAYERS FOR PTLC

2.3

SAMPLE APPLICATION

2.4

DEVELOPMENT

2.5

DETECTION OF ZONES

2.6

REMOVAL OF SUBSTANCES FROM THE LAYER

2.7

APPLICATIONS OF PTLC 2.7.1 2.7.2 2.7.3 2.7.4

Fats and L i p i d s Drugs and Pharmaceuticals Dyes and Pigments Miscellaneous Compounds

2.8

TRANSFER OF RESULTS FROM TLC TO PREPARATIVE LIQUID CHROMATOGRAPHY

2.9

REFERENCES

2 . 1 INTRODUCTION

P r e p a r a t i v e t h i n l a y e r chromatography (PTLC) i s used t o s e p a r a t e and i s o l a t e amounts o f m a t e r i a l l a r g e r than a r e normal for a n a l y t i c a l TLC. q u a n t i t i e s processed range from 10 mg t o g r e a t e r t h a n 1 gram.

The

I n preparative

TLC, m a t e r i a l s t o be separated a r e o f t e n a p p l i e d as l o n g s t r e a k s , r a t h e r t h a n spots, i n t h e sample a p p l i c a t i o n zone.

A f t e r development, s p e c i f i c components

may be recovered by s c r a p i n g t h e s o r b e n t l a y e r f r o m t h e p l a t e i n t h e r e g i o n of I n t e r e s t and e l u t i n g t h e separated m a t e r i a l from t h e s o r b e n t u s i n g a s t r o n g

106

The m a t e r i a l t h a t i s r e c o v e r e d from t h e l a y e r may r e q u i r e f u r t h e r

solvent.

p u r i f i c a t i o n b y TLC or o t h e r c h r o m a t o g r a p h i c m e t h o d s , or t h e p u r i t y may be adequate for i d e n t i f i c a t i o n and s t r u c t u r e d e t e r m i n a t i o n by elemental a n a l y s i s

or s p e c t r o m e t r y , fo r u s e i n b i o l o g i c a l a c t i v i t y or c h e m i c a l s y n t h e s i s s t u d i e s , or fo r use as s t a n d a r d r e f e r e n c e m a t e r i a l f o r c o m p a r i s o n w i t h unknown s a m p l e s . The p r o c e d u r e s f o r PTLC a r e g e n e r a l l y s i m i l a r t o a n a l y t i c a l TLC, t h e m a j o r d i f f e r e n c e b e i n g t h e use o f t h i c k e r l a y e r s .

PTLC i s f a s t e r a n d more

c o n v e n i e n t t h a n c l a s s i c a l c o l u m n c h r o m a t o g r a p h y , and i t i s much l e s s e x p e n s i v e and l e s s c o m p l i c a t e d t h a n h i g h p e r f o r m a n c e column l i q u i d c h r o m a t o g r a p h y .

The

e q u i p m e n t and o p e r a t i o n a l s k i l l s a r e s i m p l e t o m a s t e r a n d e a s y t o a p p l y .

The

use o f s m a l l amounts o f s o l v e n t ; ease o f e s t i m a t i n g p a r a m e t e r s b y a n a l y t i c a l TLC; p o s s i b i l i t y o f m u l t i p l e d e v e l o p m e n t t o o b t a i n s h a r p e r s e p a r a t i o n s ; d i r e c t d e t e c t i o n on t h e l a y e r ; e a s y r e m o v a l o f s e p a r a t e d zones from t h e p l a t e ; a n d t h e a b i l i t y t o r u n r e f e r e n c e compounds s i m u l t a n e o u s l y u n d e r i d e n t i c a l c o n d i t i o n s to a s s i s t i n l o c a t i n g the d e s i r e d m a t e r i a l a r e d i s t i n c t advantages f o r PTLC.

R e s o l u t i o n o n l a y e r s and i n columns s h o u l d be c o m p a r a b l e i f

e q u i v a l e n t l o a d i n g (sample t o sorbent r a t i o ) i s used.

Possible disadvantages

of PTLC a r e t h e d e c o m p o s i t i o n o f l a b i l e compounds when i n c o n t a c t w i t h a i r a n d l i g h t on t h e exposed l a y e r s u r f a c e a n d t h e p r e s e n c e of e x t r a c t e d l a y e r i m p u r i t i e s or f i n e s o r b e n t p a r t i c l e s w i t h t h e r e c o v e r e d compound. PTLC o n l a y e r s 1 mm t h i c k was r e p o r t e d b y R i t t e r a n d Meyer i n 1962 E l l . E a r l i e r p r e p a r a t i v e work [ e . g . ,

21, a l t h o u g h t e r m e d TLC, was a c t u a l l y

p e r f o r m e d o n a d s o r b e n t b a r s u s e d as columns or o n a n a l y t i c a l l a y e r s a f t e r column c h r o m a t o g r a p h y [ 3 , 4 1 .

PTLC has a l s o been c a r r i e d o u t on c o n i c a l [ 5 1

and c y l i n d r i c a l [ 6 1 l a y e r s , on l a y e r - c o a t e d b e l t s [71, i n l a y e r s s u p p o r t e d o n a s t a i n l e s s s t e e l f r a m e w o r k [ 8 1 , and w i t h o t h e r s p e c i a l a p p a r a t u s e s and l a y e r

forms.

However, p r e p a r a t i v e a p p l i c a t i o n s of TLC a r e most o f t e n p e r f o r m e d on

r e g u l a r , f l a t t h i n l a y e r s with increased thickness, and i t i s t h i s v a r i a t i o n

o f TLC t h a t w i l l be d i s c u s s e d m a i n l y i n t h i s c h a p t e r .

The f o l l o w i n g

s u b s e c t i o n s w i 11 f o c u s o n t h e e x p e r i m e n t a l t e c h n i q u e s u s e d t o accommodate l a r g e r samples i n TLC and w i l l a l s o i n c l u d e t h e p r i n c i p l e s i n v o l v e d i n t h e s c a l e - u p o f a n a l y t i c a l TLC, where a p p r o p r i a t e .

2 . 2 LAYERS FOR PTLC P r e p a r a t i v e l a y e r s a r e made i n t h e l a b o r a t o r y or a r e p u r c h a s e d commercially precoated.

F o r homemade l a y e r s , s p e c i a l s o r b e n t s t h a t a r e

s u p p l i e d b y some f i r m s f o r PTLC a r e u s u a l l y u s e d .

The m o s t p o p u l a r l a y e r

t h i c k n e s s f o r PTLC i s 0 . 5 - 2 . 0 mm (500-2000 pm), and t h e s i z e o f t h e p l a t e s i s

107

usually 5 x 20, 10 x 20, 20 x 20. 2 0 x 40, or even 20 x 100 cm. The conditions for successful separations on preparative plates include a homogeneous layer, regularly applied sample, and a well-saturated developing chamber. The latter is important because a substance moves in the layer with changing velocity according to the rate of evaporation of the solvent. It moves faster on the surface of the layer and more slowly on the portion of the layer close to the support plate. This effect is minimized in a completely saturated chamber. Preparative plates are made with spreading devices or applicators, preferably with adjustable slit-width. The amount of the sorbent applied changes from case to case. For silica gel, it takes 20-25 g per plate of 20 x 2 0 cm dimensions for a layer 1 mm thick. There is danger that thick layers may crack during drying. Therefore, the amount of water in the sorbent suspension or paste should be decreased, the amount of binder (gypsum) increased, drying time increased, and drying assisted by infrared radiation from above C91. To avoid cracking of thick layers, other workers have allowed plates to stand overnight before oven drying at 8OoC C101. The optimum layer thickness for PTLC has been a point of debate by different workers. In general, increased thickness leads to greater sample capacity but reduced resolution of zones. Stahl and co-workers 1 1 1 1 predicted non-uniform development speeds related to layer depth and poorer separations on layers of greater thickness than 0.5-1.0 mm, while Honegger C91 suggested optimal layers in the 1-3 mm range. Halpaap C121 has found that 2 mm layers are suitable, but above this thickness separations are degraded. The critical point is that resolution, sample complexity, sample load, and layer thickness are interrelated and the load and thickness should be chosen to yield at least a 3 mm clean distance on either side of the zone of interest. Thicker layers will take up larger amounts of mobile phase and will require longer drying periods between multiple developments. Layers of 5-10 mm thickness, prepared from special silica gel with increased binder, have been found suitable only for crude separations [121. Loose layers o f binder-free silica gel and alumina have been employed in mm thickness on horizontal or slightly inclined plates C13.141. Because experimental problems are encountered with these loose layers during sample application and zone detection, bound layers are preferred by most workers. 1-3

108

Silica gel and alumina powders containing finely ground particles to improve adhesion, but no foreign binder, are commercially available for making preparative layers, as are sorbents containing calcium sulfate binder (P series). Sorbents may contain 254 nm o r 254 and 366 nm phosphors to aid zone detection by fluorescence quenching. The manufacturer's instructions for slurrying the powders and drying and activating the layers must be carefully followed to avoid pitting, cracking, and flaking of the layers. Only silica gel or alumina layers have had significant use for preparative TLC to date. Detailed directions and precautions for coating of preparative layers of different thickness are available in the chapter by Halpaap C121. Precoated, commercial, silica gel plates with 500, 1000, 1500, and 2000pm layer thickness are available with either proprietary organic compounds ("hard plates") or gypsum as the binder, with or without flourescent indicators. Because of their convenience, precoated plates are most widely used today for PTLC. Preparative plates are available in various sizes, 10 x 20, 2 0 x 20, and 2 0 x 40 cm being most popular. Wider plates are advantageous because of higher loading capacity, longer plates because of increased development distance. However, because of slow development times and the resultant zone diffusion with increased plate length, multiple developments on a 20 cm plate are usually preferred. Alumina, microcrystalline cellulose, fibrous cellulose, and C18 reversed phase precoated preparative plates are also available, but silica gel has been most widely used, by far. Two unique plates have been designed to improve the results obtained in PTLC. The Whatman Linear preparative plate contains an inert preadsorbent strip along the lower edge of a 1000 pm silica gel or chemically bonded C 1 8 reversed phase layer to simplify sample application. The Analtech Taper Plate contains a wedge-shaped silica gel layer ranging in thickness from 300 pm at the bottom to 1700 pm at the top, with an adjacent 700 pm preadsorbent layer for sample application. Improved resolution of zones compared to conventional preparative TLC plates is claimed for the Taper Plate because o f solvent and zone migration effects during development that are similar to those realized in radial (circular) TLC. The cross-sectional area traversed by the solvent front increases during development on the tapered layer, so the crosssectional flow per unit area is highest at the bottom of the layer and decreases toward the solvent front. As a result, the back o f a zone moves faster than the zone front, causing a focusing or concentration effect during development and reducing band broadening.

109 2.3

SAMPLE APPLICATION

Up t o 5 mg o f sample has been a p p l i e d t o a 2 0 x 2 0 cm a n a l y t i c a l l a y e r

so t h a t i f t h i s q u a n t i t y i s s u f f i c i e n t for t h e p u r p o s e a t hand, t h i c k e r Overloading of the a n a l y t i c a l p l a t e w i l

p r e p a r a t i v e l a y e r s a r e n o t needed.

be e v i d e n t from t h e appearance o f t a i l e d or o t h e r w i s e d i s t o r t e d z o n e s . A n a l y t i c a l p l a t e s w i t h an i n e r t p r e a d s o r b e n t or d i s p e n s i n g a r e a (Whatman L i n e a r - K or A n a l t e c h ) can be l o a d e d w i t h more sample t h a n s t a n d a r d a d s o r b e n t plates without loss i n resolution. As a r u l e o f thumb, t h e c a p a c i t y o f a PTLC p l a t e i n c r e a s e s a s t h e s q u a r e

root of t h i c k n e s s w i t h l i t t l e or n o d e g r a d a t i o n o f s e p a r a t i o n ; a 1000 pm l a y e r w i l l , t h e r e f o r e , h a v e t w i c e t h e l o a d i n g c a p a c i t y as a 2 5 0 prn l a y e r .

The

l o a d i n g can be i n c r e a s e d beyond t h i s amount i f d e t e r i o r a t i o n o f t h e s e p a r a t i o n The a c t u a l w e i g h t t h a t can be s p o t t e d w i l l depend u p o n t h e

can be t o l e r a t e d .

s i z e o f t h e p l a t e , t h e l a y e r t h i c k n e s s , t h e s o r b e n t used, and t h e s p e c i f i c sample t o be s e p a r a t e d .

As an example, Honegger [91 a p p l i e d 5-25 mg of s a m p l e

per m i l l i m e t e r of l a y e r thickness for a

20 x 20 cm p l a t e o f s i l i c a g e l G.

B e f o r e a p r e p a r a t i v e p r o c e d u r e , t h e o p t i m u m c a p a c i t y o f t h e l a y e r c a n be empirically tested:

a s o l u t i o n o f m i x t u r e t o be s e p a r a t e d i s a p p l i e d o n t o t h e

p l a t e i n s e v e r a l c o n c e n t r a t i o n s , and d e t e c t i o n i s c a r r i e d out a f t e r d e v e l o p m e n t i n o r d e r t o see w h i c h i s t h e l a r g e s t c o n c e n t r a t i o n a t w h i c h t h e required separation w i 1 1 take place. The sample s h o u l d be d i s s o l v e d i n a v o l a t i l e s o l v e n t t h a t i s a s n o n p o l a r as p o s s i b l e , a t a c o n c e n t r a t i o n t h a t a l l o w s t h e sample components t o be a d s o r b e d o n t h e c o a t i n g and n o t d e p o s i t e d as c r y s t a l s .

Crystal overloading

can g i v e b a d l y d i s t o r t e d bands o r s p o t s s i n c e t h e c o m p o n e n t s ' r a t e s of d i s s o l v i n g i n t h e m o v i n g c a r r i e r s o l v e n t become a l i m i t i n g f a c t o r [ 1 5 1 .

A

c o n c e n t r a t i o n o f 2-10% i s t y p i c a l . I t i s recommended t h a t p l a t e s be prewashed b y d e v e l o p m e n t w i t h a s o l v e n t

p r i o r t o s p o t t i n g t o m i n i m i z e i m p u r i t i e s t h a t may be r e c o v e r e d a l o n g w i t h t h e compound o f i n t e r e s t d u r i n g t h e e l u t i o n s t e p . prewashed w i t h c h l o r o f o r m - m e t h a n o l

S i l i c a gel plates are usually

( 1 : l v / v ) or e t h y l e t h e r c o n t a i n i n g 1%

ammonia o r a c e t i c a c i d , d e p e n d i n g upon w h e t h e r t h e s u b s e q u e n t m o b i l e p h a s e i s a c i d i c or b a s i c . predevelopment.

P l a t e s can be l e f t i n t h e t a n k o v e r n i g h t f o r t h i s The p l a t e s a r e t h e n d r i e d i n a n o v e n or vacuum d e s i c c a t o r .

Samples a r e u s u a l l y a p p l i e d as a n a r r o w s t r e a k ( 3 - 5 mm h i g h ) a c r o s s t h e p l a t e , 2 . 5 cm from t h e l o w e r e d g e .

Streak a p p l i c a t i o n i s n o t o n l y convenient

110

f o r l o a d i n g l a r g e r amount o f sample onto t h e l a y e r , b u t r e s o l u t i o n o f s t r e a k s i s u s u a l l y s u p e r i o r to t h a t o b t a i n e d from i n i t i a l s p o t s .

Manual a p p l i c a t i o n

o f up t o 1 0 m l o f s o l u t i o n can be a c h i e v e d using a s y r i n g e or o r d i n a r y p i p e t and a s t r a i g h t edge as a g u i d e .

The s t r e a k s h o u l d be a s s t r a i g h t and n a r r o w

as p o s s i b l e a n d s h o u l d t e r m i n a t e a t l e a s t 1-3 cm i n from e a c h s i d e o f t h e p l a t e t o a v o i d "edge e f f e c t s " t h a t o f t e n make t h e s o l v e n t move f a s t e r or s l o w e r ( u s u a l l y t h e f o r m e r ) a t t h e edges t h a n i n t h e c e n t e r 14-18 cm o f 2 0 x 20 cm p l a t e .

U n l e s s t h e flow and t h e r a t e o f movement of t h e p i p e t a r e

c a r e f u l l y c o n t r o l l e d , t h e sample a p p l i c a t i o n w i l l be i r r e g u l a r . Repeated sample a p p l i c a t i o n s a r e u s u a l l y made t o d e p o s i t t h e d e s i r e d amount o f s o l u t i o n . streaks.

I t i s i m p o r t a n t t o c o m p l e t e l y remove t h e s o l v e n t b e t w e e n

I f t h e sample s o l v e n t i s n o t removed b e t w e e n s t r e a k s , i t w i l l " w i c k "

i n t o t h e p l a t e , r e s u l t i n g i n a w i d e sample s t r e a k t h a t i s n o t u n i f o r m i n concentration across i t s v e r t i c a l dimension [151.

For a s i m i l a r r e a s o n , t h e

s t r e a k i n g m e t h o d u s e d m u s t n o t gouge or damage t h e a d s o r b e n t c o a t i n g .

Gouging

w i l l i n t e r f e r e w i t h t h e r e g u l a r s t r a i g h t l i n e movement o f c a r r i e r , m a k i n g l a t e r component r e c o v e r y d i f f i c u l t [15,161.

I f t h e s t r e a k becomes e x c e s s i v e l y

b r o a d ( h i g h ) d u r i n g sample a p p l i c a t i o n , i t c a n be s h a r p e n e d by a p r e d e v e l o p m e n t for a d i s t a n c e o f 1-2 cm w i t h a v e r y p o l a r s o l v e n t , a f t e r w h i c h t h e s o l v e n t i s t h o r o u g h l y e v a p o r a t e d and d e v e l o p m e n t i s c a r r i e d o u t w i t h an a p p r o p r i a t e c h r o m a t o g r a p h i c m o b i l e phase [ 1 7 1 .

Honegger [ 9 1 a p p l i e d samples

t o a V-shaped g r o o v e , 1-2 mm w i d e and h a l f t h e d e p t h o f t h e l a y e r .

In this

t e c h n i q u e , c a r e must b e t a k e n n o t t o remove t h e s o r b e n t a l l t h e way t o t h e g l a s s p l a t e , w h i c h c o u l d i n t e r f e r e w i t h m o b i l e phase movement.

Small, round

s p o t s have a l s o been a p p l i e d s i d e b y s i d e a c r o s s t h e o r i g i n t o m i m i c a s t r e a k , b u t t h i s o p e r a t i o n i s t e d i o u s a n d t i m e consuming a n d may l e a d t o n o n u n i f o r m i t y t h a t w i l l harm r e s o l u t i o n between components w i t h s i m i l a r l y RF v a l u e s . Less c a r e i n s t r e a k i n g can be t o l e r a t e d i f a p p l i c a t i o n i s made t o a PTLC l a y e r w i t h an i n e r t p r e a d s o r b e n t a r e a (Whatman L i n e a r - K or A n a l t e c h ) .

The

p r e a d s o r b e n t l a y e r i s made o f an i n e r t n o n a d s o r b i n g m a t e r i a l w h i c h s e r v e s a s a " h o l d i n g " zone f o r sample components u n t i l d e v e l o p m e n t b e g i n s .

Soluble

compounds m i g r a t e w i t h t h e s o l v e n t f r o n t t h r o u g h t h e p r e a d s o r b e n t l a y e r , t h u s improving r e s o l u t i o n .

Although less-careful

a p p l i c a t i o n i s t o l e r a t e d on

p r e a d s o r b e n t p l a t e s , t h e p o s s i b i l i t y o f h i g h l o c a l c o n c e n t r a t i o n s and o v e r l o a d i n g e f f e c t s s h o u l d n o t be d i s c o u n t e d , e s p e c i a l l y f o r c r u d e s a m p l e s . Optimum r e s u l t s can b e e x p e c t e d if c a r e f u l , e v e n a p p l i c a t i o n o f samples i s made t o p r e a d s o r b e n t p l a t e s .

An a d d i t i o n a l a d v a n t a g e o f t h e s e p l a t e s i s t h a t

some c l e a n u p may be a c h i e v e d due t o p r e c i p i t a t i o n o f i n s o l u b l e s u b s t a n c e s from t h e sample o n t h e p r e a d s o r b e n t .

111

The design and construction of a number of PTLC applicators have been described [18-201 and commercial streaking pipets and more or less automatic streaking devices are available from several manufacturers.

Fig. 2 . 1 .

Camag Linomat 111 automatic sample applicator couite;y of Camdg Scientific I n c . ) .

(Photograph

Fig. 2.1 shows the Camag Linomat 111, which can apply by spraying up to 495 p1 sample from a preloaded 500 p1 syringe in a band 1-3 mm wide and up to 199 mm long. Movement of the syringe plunger and the plate as well as the nitrogen gas flow are all automatically controlled to apply a preselected sample volume and band length. For analytical TLC, 2-99 p1 can be applied from a 100 p1 syringe in the form of shorter bands. of

Samples are applied to loose layers as drops placed near to each other, or the sample is sprayed through a suitable mask. Difficulty in applying samples without disturbing the layer is a major reason loose layers have not been widely used. 2 . 4 DEVELOPMENT

Development is usually carried out in the ascending mode as in analytical TLC using a solvent system (mobile phase) previously established as optimum for a given separation. It is possible to find potentially suitable solvent -

112

F o r an

systems i n t h e TLC l i t e r a t u r e f o r a w i d e v a r i e t y o f compounds.

e x t e n s i v e l i s t i n g o f m o b i l e phases c o u p l e d w i t h s e l e c t e d s o r b e n t s f o r Prior to

p a r t i c u l a r compounds see Zweig and Sherma [ 2 1 1 and K i r c h n e r [ 2 2 1 .

PTLC, s o l v e n t s s h o u l d b e t e s t e d w i t h a n a l y t i c a l p l a t e s o r m i c r o s c o p e s l i d e s M o d i f i c a t i o n b y t r i a l and e r r o r p r o c e d u r e s may be needed f o r systems t h a t i n i t i a l l y were o p t i m a l f o r a n a l y t i c a l TLC s e p a r a t i o n s .

This i s e s p e c i a l l y

t r u e where m u l t i p l e d e v e l o p m e n t i s t o be u s e d . Because o f i t s t h i c k e r l a y e r , a PTLC p l a t e w i l l more q u i c k l y d e p l e t e s o l v e n t i n a chamber t h a n w i l l an a n a l y t i c a l p l a t e .

An a d d i t i o n a l amount o f

t h e s o l v e n t may be t o be added t o t h e chamber d u r i n g d e v e l o p m e n t . t h e c o v e r o f some t a n k s ( e . g . ,

Kontes) i s p r o v i d e d for t h i s .

A hole i n

C a r e s h o u l d be

t a k e n when a d d i n g s o l v e n t t o a v o i d d i s t u r b i n g t h e l i q u i d t h a t i s a l r e a d y p r e s e n t i n the tank [151.

As i n a n a l y t i c a l TLC, t h e m o b i l e p h a s e s h o u l d be

p r e p a r e d from t h e p u r e s t g r a d e o f s o l v e n t s a v a i l a b l e . A c c o r d i n g t o K i r c h n e r [ 2 2 1 , S t a h l [ 2 3 1 , a n d H a l p a a p [12.171,

the

r e s o l u t i o n o f complex m i x t u r e s c a n be i m p r o v e d b y m u l t i p l e d e v e l o p m e n t w i t h l e s s p o l a r s o l v e n t s t h a t i n i t i a l l y g i v e low RF v a l u e s . be e v a p o r a t e d a f t e r e a c h r u n .

The s o l v e n t s h o u l d

Subsequent d e v e l o p m e n t s a r e done i n t h e same

d i r e c t i o n e i t h e r w i t h t h e same or a d i f f e r e n t m o b i l e phase.

F o r some

s e p a r a t i o n s , marked i m p r o v e m e n t may o c c u r i f t h e s o l v e n t m i g r a t e s up t o 1 0 times through the l a y e r .

From a p r a c t i c a l s t a n d p o i n t , 2 t o 3 s e p a r a t e

ascending developments a r e u s u a l l y performed.

A f t e r d e t e c t i o n and e l u t i o n

from t h e p r e p a r a t i v e p l a t e , f r a c t i o n s n o t c o m p l e t e l y r e s o l v e d d u r i n g m u l t i p l e

d e v e l o p m e n t c a n be r e c h r o m a t o g r a p h e d o n t h i n n e r a n a l y t i c a l l a y e r s .

Halpaap

[ 1 2 1 has p r o v i d e d e x t e n s i v e t a b u l a r and g r a p h i c d a t a s h o w i n g t h e a d v a n t a g e s o f m u l t i p l e a s c e n d i n g d e v e l o p m e n t i n PTLC. I n PTLC, 20 x 20 cm p l a t e s a r e d e v e l o p e d s i n g l y i n t h e u s u a l s a n d w i c h or r e c t a n g u l a r chambers.

PTLC chambers a r e a v a i l a b l e t o accommodate l a r g e r 2 0 x

40 cm p l a t e s , and m u l t i p l e p l a t e s can be d e v e l o p e d i n a s p e c i a l c h r o m a t o t a n k

or m u l t i - s a n d w i c h a s s e m b l y .

F i g . 2 . 2 shows a Desaga p r e p a r a t i v e s e p a r a t i n g

chamber e q u i p p e d w i t h t h e Desaga m u l t i p l e r a c k . accommodate up t o t e n p l a t e s o f 20 x 40 cm

T h i s a p p a r a t u s can

or t w e n t y 2 0 x 2 0 cm p l a t e s .

The

chamber i s c l o s e d b y a g l a s s l i d w i t h a f i l l i n g u n i t for t h e s o l v e n t . C o n t r a r y t o t h e a d v i c e o f H a l p a a p E l 2 1 on m u l t i p l e a s c e n d i n g d e v e l o p m e n t , a r e c e n t t e c h n i c a l b u l l e t i n o n PTLC s u g g e s t s t h e use o f o n l y one d e v e l o p m e n t 1241.

H a l p a a p ' s a d v i c e was based o n u s i n g homemade p l a t e s , whereas t h e

t e c h n i c a l b u l l e t i n r e f e r r e d o n l y t o commercial p l a t e s .

However, r e g a r d l e s s of

t h e t y p e o f p l a t e u s e d , . t h e d e v e l o p m e n t p r o c e d u r e i s most l i k e l y t o be d i c t a t e d b y ' t h e c o m p l e x i t y a n d q u a n t i t y o f t h e sample c h r o m a t o g r a p h e d .

113

fig. 2.2.

Desaga preparative c,epdrating chambel- and w l t i p l e rack (Photograph courtejy of Whatman, a distributor of Desaga pr-oducts).

Although the prime mode of development in PTLC i s ascending, Horobin C251 has used continuous descending elution in a tank with an upper solvent reservoir. 2.5

DETECTION OF ZONES

If the compounds o f interest are naturally colored, they can be located simply by eye in daylight. Likewise, fluorescent compounds can be detected by viewing under UV light. A PTLC plate with florescent material (usually marked " F " or "UV" on commercial plates) will show the separated substances as dark zones on a bright background when examined under 2 5 4 or 366 nm UV light if the compounds absorb at or near these wavelengths (fluorescence quenching). Commercial fluorescent impregnating agents such as zinc silicate are generally insoluble in the solvents used to elute compounds and should not add significant contaminants.

Perhaps the best chemical detection procedure for PTLC is the use o f iodine vapor in a closed chamber. This procedure will visualize compounds o f numerous chemical classes as light or dark brown zones on a light tan background. Usually the iodine vapor can be evaporated without any change in the compounds o f interest. Zones should be marked by outlining them with a pencil, needle, or scalpel, before the iodine evaporates leaving the substance

114

of i n t e r e s t i n v i s i b l e .

P r e c a u t i o n s h o u l d be t a k e n w i t h t h e i o d i n e p r o c e d u r e ,

s i n c e a l t e r a t i o n o f some compounds may o c c u r .

F o r i n s t a n c e , Nichaman e t a l .

[ 2 6 1 showed t h a t t h e r e i s a d e c r e a s e i n u n s a t u r a t e d l i p i d s when i o d i n e i s u s e d as t h e d e t e c t i n g r e a g e n t , p r e s u m a b l y due t o t h e i o d i n a t i o n o f d o u b l e b o n d s . B a r r e t t [ 2 7 1 has p r o v i d e d an e x c e l l e n t r e v i e w o n t h e use o f t h e i o d i n e p r o c e d u r e a l o n g w i t h o t h e r r e v e r s i b l e or n o n d e s t r u c t i v e r e a g e n t s u s e d f o r detection. The f l u o r e s c e i n - b r o m i n e t e s t w i l l d e t e c t u n s a t u r a t e d a n d o t h e r compounds t h a t r e a c t r e a d i l y w i t h bromine [281.

I n t h i s procedure, the solvent-free

PTLC p l a t e i s s p r a y e d w i t h a s o l u t i o n o f 0.05% f l u o r e s c e i n i n w a t e r .

The

p l a t e i s t h e n exposed t o b r o m i n e v a p o r ; t h e f l u o r e s c e i n i s c o n v e r t e d t o e o s i n , e x c e p t where compounds a r e l o c a t e d t h a t t a k e up t h e b r o m i n e , t h u s l e a v i n g t h e fluorescein with i t s original yellow color. A n o t h e r u s e f u l t e c h n i q u e uses w a t e r a s a s p r a y r e a g e n t E29.301.

The

s i l i c a gel i s sprayed w i t h water u n t i l the l a y e r i s t r a n s l u c e n t ; w a t e r - i n s o l u b l e compounds show up as w h i t e opaque s p o t s a g a i n s t a d a r k background.

S h a r p e r zones c a n be h a d b y l e t t i n g t h e s a t u r a t e d p l a t e d r y u n t i l

t h e zones become c l e a r .

Compounds t h a t a r e r a d i o a c t i v e can be l o c a t e d o n a

p r e p a r a t i v e l a y e r b y means o f a u t o r a d i o g r a p h y or a G e i g e r c o u n t e r [ 3 1 1 . F o r l o o s e l a y e r s , a n a r r o w s t r i p of m o i s t e n e d p a p e r may be a p p l i e d against the l a y e r [321.

The s t r i p i s removed, and a d h e r i n g p a r t i c l e s of

sorbent are sprayed w i t h a s u i t a b l e reagent.

A l t e r n a t i v e l y , a narrow s t r i p of

p l a t e coated w i t h s i l i c a i s pressed a g a i n s t the s i d e of the p r e p a r a t i v e l a y e r

t o o b t a i n a " p r i n t " , w h i c h i s t h e n d e t e c t e d i n t h e u s u a l way [ 3 3 1 . Numerous s p e c i f i c d e t e c t i o n r e a g e n t s h a v e been s u g g e s t e d t o i d e n t i f y compounds s e p a r a t e d b y p a p e r c h r o m a t o g r a p h y and TLC [ 2 1 - 2 3 1 . r e a g e n t s can b e u s e d i n PTLC as f o l l o w s :

Most o f t h e s e

I f d e s t r u c t i v e or n o n r e v e r s i b l e

r e a g e n t s a r e r e q u i r e d f o r d e t e c t i o n , s m a l l s p o t s o f sample s h o u l d b e a p p l i e d

to t h e o u t e r s i d e margins o f t h e p l a t e .

The m a r g i n s a r e t h e n s p r a y e d w i t h t h e

r e q u i s i t e r e a g e n t , a f t e r c o v e r i n g or m a s k i n g t h e m a j o r s t r e a k e d p o r t i o n o f t h e p l a t e w i t h g l a s s , c a r d b o a r d , or f o i l , and s c r i b i n g v e r t i c a l c h a n n e l s b e t w e e n t h e s t r e a k s and s p o t s .

The d e s i r e d s u b s t a n c e s a r e t h e n v i s i b l e o n each s i d e

o f t h e p l a t e a n d s e r v e as a g u i d e f o r s c r i b i n g h o r i z o n t a l l i n e s a c r o s s t h e c e n t e r o f t h e p l a t e t o o u t l i n e t h e a r e a s t h a t c o n t a i n t h e compounds of interest.

An a l t e r n a t i v e p r o c e d u r e i s t o s t r e a k t h e sample a c r o s s most o f t h e

l a y e r and t o use t h e o u t e r edges o f t h e s t r e a k e d sample, o u t s i d e of t h e mask, as a g u i d e a r e a t o be s p r a y e d . A c o m m e r c i a l d e a l e r ( A n a l t e c h ) s e l l s

115

p r e p a r a t i v e p l a t e s t h a t a r e p r e s c o r e d one i n c h i n from e a c h s i d e

The s a m p l e

s t r e a k i s e x t e n d e d o n t o t h e s c o r e d a r e a s , and f o l l o w i n g d e v e l o p m e n t t h e s i d e p o r t i o n s a r e snapped f r e e .

A f t e r d e t e c t i o n i s made, t h e edge s t r i p s a r e

p l a c e d n e x t t o t h e c e n t e r s e c t i o n s t o mark t h e a r e a s f o r c o l l e c t i o n .

These

p l a t e s e l i m i n a t e t h e chance o f s p r a y e d r e a g e n t w i c k i n g i n t o t h e c e n t e r o f t h e p l a t e and a l s o a n y u n d e s i r a b l e e f f e c t s t h a t h e a t i n g m i g h t c a u s e , i f h e a t i s r e q u i r e d d u r i n g t h e v i s u a l i z a t i o n p r o c e s s [151.

The a f o r e m e n t i o n e d p r o c e d u r e s

can be u s e d w i t h a n y p l a t e s , i f a g l a s s c u t t e r i s a v a i l a b l e . Heating of p r e p a r a t i v e p l a t e s d u r i n g the chromatographic process should be a v o i d e d so t h a t s u b s t a n c e s a r e r e c o v e r e d i n an u n a l t e r e d s t a t e . 2.6

REMOVAL OF SUBSTANCES FROM THE LAYER

The m a t e r i a l l o c a t e d i n t h e s c r i b e d a r e a o f t h e p l a t e i s r e c o v e r e d by r e m o v i n g t h e a d s o r b e n t zone, e l u t i n g t h e s u b s t a n c e from t h e a d s o r b e n t w i t h a s u i t a b l e s o l v e n t , and s e p a r a t i n g t h e r e s i d u a l adsorbent.

The f i n a l s t e p

involves concentrating the solution, usually b y evaporation. The o u t l i n e d a r e a s o f t h e l a y e r c o n t a i n i n g t h e s u b s t a n c e s o f i n t e r e s t a r e s c r a p e d o f f down t h e b a c k i n g o f t h e p l a t e w i t h a s p a t u l a , s c a l p e l , r a z o r b l a d e , or c o m m e r c i a l s c r a p e r .

The l o o s e n e d a d s o r b e n t i s t r a n s f e r r e d to a

s h e e t o f w e i g h i n g p a p e r or f i l t e r p a p e r and p l a c e d i n a g l a s s v i a l or c e n t r i f u g e tube.

A f e w d r o p s o f w a t e r can be added t o t h e p l a t e t o h e l p

d i s p l a c e components from t h e a d s o r b e n t .

To r e d u c e t h e l o s s o f f i n e s i l i c a

p a r t i c l e s b y f l a k i n g d u r i n g t h e s c r a p i n g and t r a n s f e r s t e p s , t h e p l a t e c a n be wetted w i t h a f i n e spray of ethanol.

The s o l v e n t used t o e l u t e t h e compound

s h o u l d be a d e q u a t e l y p o l a r and m i s c i b l e w i t h t h e component.

M e t h a n o l i s not

recommended, s i n c e s i l i c a g e l and some o f i t s i m p u r i t i e s a r e s o l u b l e i n t h i s solvent.

A c e t o n e , e t h a n o l , c h l o r o f o r m , or t h e s o l v e n t u s e d f o r t h e TLC

d e v e l o p m e n t a r e good c h o i c e s for s o l u t e r e c o v e r y .

The u s e of w a t e r s h o u l d be

avoided since i t i s d i f f i c u l t to evaporate d u r i n g t h e c o n c e n t r a t i o n step.

The

f o l l o w i n g f o r m u l a h a s been u s e d t o d e t e r m i n e t h e r e q u i r e d volume of s o l v e n t when t h e TLC m o b i l e phase i s s e l e c t e d f o r e l u t i o n : s o l v e n t volume = (1.0 - R F ) ( 1 0 ) ( v o l u m e o f t h e s c r a p i n g s ) . The s o l v e n t o f c h o i c e i s added t o t h e t u b e w i t h t h e s c r a p i n g s , a n d t h e t u b e i s a g i t a t e d on a V o r t e x m i x e r or shaken b y hand f o r u p t o 5 m i n u t e s .

The

c o n t e n t s o f t h e t u b e a r e a l l o w e d t o s e t t l e b y g r a v i t y or t h e t u b e i s c e n t r i f u g e d , and t h e s u p e r n a t a n t s o l u t i o n i s removed b y d e c a n t a t i o n , f i l t r a t i o n , or w i t h t h e a i d o f a p i p e t .

To a s s u r e t h a t a l l s i l i c a g e l

p a r t i c l e s a r e e x c l u d e d from t h e s o l u t i o n , t h e f i l t e r i n g medium s h o u l d be a b l e

116

t o r e t a i n 2 pm p a r t i c l e s (most TLC s i l i c a g e l p a r t i c l e s a r e i n t h e 5-20 pm range.).

A d d i t i o n a l s o l v e n t can be added t o t h e s c r a p i n g s and t h e e x t r a c t i o n

repeated; t h e s o l u t i o n s a r e t h e n combined. An a l t e r n a t i v e method used t o remove scraped adsorbent zones t a k e s advantage o f t h e p r i n c i p l e o f vacuum s u c t i o n .

Mottier and P o t t e r a t C341

a p p l i e d vacuum s u c t i o n t o remove spots from loose l a y e r p l a t e s by a p p l y i n g a vacuum t o one end of a 6-8 mm g l a s s t u b e t h a t was c o n s t r i c t e d t o h o l d a p l u g o f cotton.

The adsorbent was sucked i n t o t h e tube and h e l d by t h e c o t t o n

p l u g . Glass wool can be used i n p l a c e of t h e c o t t o n p l u g [351 or an asbestos mat supported by a f i n e mesh s t a i n l e s s s t e e l screen can be employed C361. Other workers have used g l a s s d i s c s t o r e t a i n t h e adsorbent C37,381.

More

r e c e n t l y , Dekker [391 has d e s c r i b e d an apparatus for t h e i s o l a t i o n o f compounds f r o m l a y e r s by e l u t i o n and d i r e c t M i l l i p o r e f i l t r a t i o n , and P l a t t [401 designed a zone c o l l e c t o r t h a t used vacuum t o t r a n s f e r separated zones

from t h i n layers d i r e c t l y to l i q u i d s c i n t i l l a t i o n v i a l s . A homemade c o m b i n a t i o n s c r a p e r - c o l l e c t o r can be made from a Pasteur p i p e t

(225 mm x 0.7 mm o . d . 1 .

The p i p e t i s c u t w i t h a f i l e 60 mm f r o m t h e t o p and

65 mm f r o m t h e t i p , t o achieve a 100 mm l o n g p i p e t t h a t i s plugged w i t h g l a s s wool.

The p i p e t t i p i s a t t a c h e d t o a vacuum pump o r an a s p i r a t o r , and t h e t o p

i s used t o scrape t h e zone on t h e l a y e r c o n t a i n i n g t h e compound o f i n t e r e s t . Scraping and vacuum c o l l e c t i o n i s t h u s accomplished s i m u l t a n e o u s l y . i s removed from t h e vacuum and used as a column. small v i a l ( i . e . ,

The p i p e t

The column i s p l a c e d i n a

60 mm x 10 mm i . d . , 8 m l c a p a c i t y ) .

The v i a l s h o u l d be

covered w i t h aluminum f o i l and t h e column pushed t h r o u g h t h e f o i l .

The

e l u t i o n s o l v e n t i s a l l o w e d t o p e r c o l a t e t h r o u g h t h e column.

I f the glass w o o l p l u g i n t h e column i s s u f f i c i e n t l y t i g h t , most o f t h e s i l i c a g e l w i l l be

r e t a i n e d i n t h e column. Various commercial d e v i c e s a r e a v a i l a b l e f o r t h e removal and r e c o v e r y o f adsorbent from t h e l a y e r . R e p r e s e n t a t i v e sample r e c o v e r y tubes from Whatman a r e shown i n F i g . 2.3.

These d e v i c e s come i n d i f f e r e n t s i z e s and can

accommodate d i s c s o f medium o r coarse p o r o s i t y . Once a s o l u t i o n o f t h e m a t e r i a l i s a v a i l a b l e , f r e e of t h e adsorbent, t h e s o l v e n t i s evaporated t o c o n c e n t r a t e t h e sample.

T h i s s h o u l d be done a t low

temperature and p r e f e r a b l y under an i n e r t stream o f gas such as n i t r o g e n so t h a t t h e sample i s n o t decomposed or o t h e r w i s e a l t e r e d .

117

Fig. 2 . 3

2.7

Sample r e c o v e r y t u b e s

( P h o t o g r a p h c o u r t e s y o f Whatman)

APPLICATIONS OF PTLC

PTLC i s u s e d w i d e l y i n t h e p h a r m a c e u t i c a l i n d u s t r y .

M a n u f a c t u r e r s o f TLC

p l a t e s have i n f o r m e d us t h a t a s i g n i f i c a n t p o r t i o n o f t h e i r s a l e s o f p r e p a r a t i v e p l a t e s i s t o pharmaceutical companies.

The use o f PTLC i n

i n d u s t r y i s o f t e n p r o p r i e t a r y , and c u r r e n t l i t e r a t u r e i n PTLC i s n o t i n d i c a t i v e o f the widespread a p p l i c a t i o n of t h i s procedure. Most usage o f PTLC i n t h e l a t e 1 9 7 0 ' s and i n t h e 1 9 8 0 ' s i s b a s e d o n t h e a v a i l a b i l i t y o f c o m m e r c i a l PTLC p l a t e s .

Several manufacturers have r e p o r t e d

t h a t PTLC p l a t e s a l e s c o m p r i s e u p t o 15% o f a l l o f t h e i r TLC p l a t e s a l e s . We have a l s o been i n f o r m e d t h a t l e s s t h a n 5% ( p r o b a b l y 1-2%) o f a l l c u r r e n t u s e r s make t h e i r own l a y e r s .

Those who p r e p a r e t h e i r own p l a t e s o f t e n

use s o r b e n t m i x t u r e s t h a t a r e n o t r e a d i l y a v a i : a b l e

from c o m m e r c i a l s u p p l i e r s .

I n r e v i e w i n g t h e l i t e r a t u r e on PTLC we h a v e been s e l e c t i v e r a t h e r t h a n exhaustive.

Most a p p l i c a t i o n s i n v o l v e s e p a r a t i o n s of l i p o p h i l i c compounds o n

. s i l i c a gel layers.

I n f o r m a t i o n o n PTLC o f h y d r o p h i l i c compounds i s l e s s

a b u n d a n t , and s i g n i f i c a n t a p p l i c a t i o n s i n t h i s f i e l d have been g i v e n where applicable.

S i n c e some m a n u f a c t u r e r s s u p p l y p r e c o a t e d p r e p a r a t i v e p l a t e s w i t h

sorbentr o t h e r than s i l i c a g e l , i n c l u d i n g v a r i o u s types of c e l l u l o s e , f u t u r e a p p l i c a t i o n s w i l l p r o b a b l y be e x t e n d e d t o i n c l u d e more h y d r o p h i l i c compounds.

118 2.7.1

Fats and Lipids Up t o 20 mg o f l i p i d s can be a p p l i e d a s a band on 20 x 20 cm s i l i c a g e l G When l i p i d s c o n s i s t m a i n l y o f t r i g l y c e r i d e s , up t o

p l a t e s , 0.5 mm t h i c k [ 4 1 1 .

5 0 mg can be a p p l i e d t o a 0.5 mm t h i c k s i l i c a g e l p l a t e 1411.

S o l v e n t systems

f o r t h e s e p a r a t i o n o f m o s t commonly o c c u r r i n g n e u t r a l ( s i m p l e ) l i p i d s f o r PTLC a r e h e x a n e - d i e t h y l e t h e r - f o r m i c a c i d (80:20:2 v / v ) or b e n z e n e - d i e t h y l e t h e r - e t h y l a c e t a t e - a c e t i c a c i d (80:10:10:0.2

v / v ) [411.

A f r e q u e n t l y used

m o b i l e phase, p e t r o l e u m e t h e r - d i e t h y l e t h e r - a c e t i c a c i d ( 8 0 : 2 0 : 1

or 7 0 : 3 0 : 1

v / v ) , i s e f f e c t i v e for t h e s e p a r a t i o n o f commonly o c c u r r i n g n e u t r a l l i p i d s i n animal p l a n t t i s s u e s .

For a general d i s c u s s i o n of n e u t r a l l i p i d separations,

see C h a p t e r 1 4 i n t h e b o o k b y F r i e d a n d Sherma [ 4 2 1 . S i n c e t h e above s o l v e n t s y s t e m s do n o t p r o v i d e u n e q u i v o c a l s e p a r a t i o n o f d i g l y c e r i d e s from f r e e s t e r o l s , t h e f o l l o w i n g p r o c e d u r e i s i m p o r t a n t i n PTLC [431.

Up t o 20 mg o f n e u t r a l l i p i d s c a n be s e p a r a t e d o n 0 . 5 mm t h i c k 20 x 20

cm s i l i c a g e l G p l a t e s u s i n g t h e d u a l s o l v e n t s y s t e m of S k i p s k i e t a l . [ 4 4 1 . I n t h i s p r o c e d u r e a s c e n d i n g d e v e l o p m e n t i s f i r s t done w i t h t h e m o b i l e p h a s e i s o p r o p y l e t h e r - a c e t i c a c i d ( 9 6 : 4 v / v ) f o l l o w e d b y a second a s c e n d i n g development ( a f t e r t h e p l a t e i s d r i e d ) i n p e t r o l e u m e t h e r - d i e t h y l ether-acetic a c i d (9O:lO:l

v/v).

Neutral l i p i d s are visualized w i t h iodine.

The o r d e r o f m i g r a t i o n (low t o h i g h RF) i s d i g l y c e r i d e s , f r e e s t e r o l s , a n d tr i g l y c e r i d e s

.

The t r i g l y c e r i d e f r a c t i o n o f a n i m a l t i s s u e s ( p a r t i c u l a r l y i n v e r t e b r a t e s ) may c o n t a i n g l y c e r y l e t h e r s .

I s o l a t i o n of t h e t r i g l y c e r i d e f r a c t i o n by

p r e p a r a t i v e TLC and r e c h r o m a t o g r a p h y i n h e x a n e - d i e t h y l e t h e r ( 9 5 : s v / v ) may i n d i c a t e t h e presence of g l y c e r y l e t h e r s 1451.

A r e c e n t t e c h n i q u e f o r t h e PTLC o f t r i g l y c e r i d e s o f o i l s and f a t s from c o m m e r c i a l samples i n v o l v e d s e p a r a t i o n o f t h e l i p o p h i l i c f r a c t i o n o n s i l i c a g e l G u s i n g t h e m o b i l e phase p e t r o l e u m e t h e r - a c e t o n e ( 1 0 0 : 8 v l v ) [ 4 6 1 . V a r i o u s t e c h n i q u e s have been r e p o r t e d f o r t h e PTLC o f s t e r o l s from o t h e r n o n s a p o n i f i a b l e l i p i d s [301. chloride-acetone ( 9 : l v/v). t h i s system.

S i l i c a g e l H p l a t e s a r e developed w i t h methylene The m o b i l i t y o f a l l f r e e s t e r o l s i n s i m i l a r i n

V i s u a l i z a t i o n i s a c h i e v e d u n d e r UV l i g h t a f t e r s p r a y i n g w i t h

0.005% aqueous Rhodamine

B. F o r t h e s e p a r a t i o n o f u n s a t u r a t e d s t e r o l s

( s t a n o l s ) . PTLC on s i l i c a g e l H p l a t e s i m p r e g n a t e d w i t h 25% s i l v e r n i t r a t e has been used [ 4 7 1 .

F o l l o w i n g two a s c e n d i n g d e v e l o p m e n t s w i t h

chloroform-benzene ( 1 : l v / v ) ,

two s e p a r a t e bands s h o u l d be o b t a i n e d .

PTLC

separation of i n t e s t i n a l ( f e c a l ) s t e r o l s i n t o t h r e e classes o n F l o r i s i l p l a t e s

119

d e v e l o p e d w i t h d i e t h y l e t h e r - h e p t a n e ( 5 5 : 4 5 v / v ) has been r e p o r t e d 147, 481. The s l o w e s t m o v i n g band c o n t a i n e d c h o l e s t e r o l , p h y t o s t e r o l s , and r i n g saturated 5-a-sterols;

t h e m i d d l e band c o n t a i n e d c o p r o s t a n o l a n d r i n g

s a t u r a t e d 5-O-phytosterol

d e r i v a t i v e s ; and t h e f a s t e s t m o v i n g band c o n t a i n e d

c o p r o s t a n o n e and 3 - k e t o d e r i v a t i v e s o f t h e p h y t o s t e r o l s . Up t o 20 mg o f f a t t y a c i d m e t h y l e s t e r s h a v e been s e p a r a t e d o n 0.5 mm s i l i c a g e l p l a t e s w i t h a m o b i l e phase o f d i e t h y l e t h e r - h e x a n e ( 4 : l v / v ) [ 4 1 1 . Bands were d e t e c t e d u s i n g a 2 , 7 - d i c h l o r o f l u o r e s c e i n s p r a y a n d r e c o v e r e d by e l u t i n g t h e s c r a p e d a d s o r b e n t i n a column w i t h d i e t h y l e t h e r or A l t e r n a t i v e l y , u p t o 10 mg of f a t t y a c i d

chloroform-methanol ( 9 : l v l v ) .

e s t e r s were s e p a r a t e d o n a 20 x 20 cm p l a t e c o a t e d w i t h a 0.5 mm 1 a y e r . o f s i l i c a g e l c o n t a i n i n g 10% ( w / w ) [411. di-, e i the

s i l v e r n i t r a t e ( a r g e n t a t i o n chromatography)

U s i n g t h i s p r o c e d u r e , e s t e r s c a n be s e p a r a t e d i n t o s a t u r a t e d mono-, tri-, tetra-,

p e n t a - , and h e x a n o i c f r a c t i o n s w i t h a m o b i l e p h a s e o f

h e x a n e - d i e t h y l e t h e r ( 9 O : l O v l v ) or h e x a n e - d i e t h y l e t h e r ( 4 0 : 6 0 v l v ) .

V i sua i z a t i o n and e l u t i o n t e c h n i q u e s a r e as d e s c r i b e d i n t h e p r e v i o u s

d i scu s i o n o f f a t t y a c i d methyl e s t e r s .

Preparative separations of f r e e f a t t y

a c i d s and t h e i r m e t h y l e s t e r s can a l s o be a c c o m p l i s h e d u s i n g r e v e r s e d p h a s e PTLC.

These s e p a r a t i o n s a r e o f t e n c a r r i e d o u t o n s i l i c o n i z e d s i l i c a g e l w i t h

a c e t o i t r i l e - m e t h a n o l - w a t e r as t h e m o b i l e p h a s e [ 4 1 1 . F a t t y a c i d s and n o n a p o n i f i a b l e l i p i d s were e x t r a c t e d from tobacco b y hexane, and t r e a t m e n t of t h e e x t r a c t w i t h d i a z o m e t h a n e y i e l d e d f a t t y a c i d methyl e s t e r s .

The m e t h y l e s t e r s were s e p a r a t e d from h y d r o c a r b o n s and s t e r o l s

b y PTLC 1491. G a n g l i o s i d e s , w h i c h a r e o f i n t e r e s t s i n c e t h e y p l a y a r o l e i n human l i p i d o s e s , were s e p a r a t e d p r e p a r a t i v e l y i n t o mono-,

di-,

and

t r i s i a l o g a n g l i o s i d e s o n l a y e r s o f s i l i c a g e l w i t h t h e m o b i l e phase chloroform-methanol-2.5

N ammonia ( 6 0 : 4 0 : 9 v / v )

[501.

Considerable i n f o r m a t i o n i s a v a i l a b l e on t h e preparative separation of p h o s p h o l i p i d s (complex l i p i d s ) .

A c c o r d i n g t o C h r i s t i e C411. up t o 1 0 mg o f

p h o s p h o l i p i d s c a n be s e p a r a t e d o n 0 . 5 mm l a y e r s (20 x 20 cm) o f s i l i c a g e l u s i n g v a r i o u s m o b i l e phases c o n t a i n i n g c h l o r o f o r m - m e t h a n o l - w a t e r .

This

p r o c e d u r e i s u s u a l l y s a t i s f a c t o r y f o r t h e more common p h o s p h o l i p i d s of a n i m a l tissues.

H a l a a p [ 1 2 1 u s e d v a r i o u s m o b i l e phases c o n t a i n i n g e i t h e r

chloroform-methanol-acetic

a c i d or c h l o r o f o r m - m e t h a n o l - a m m o n i a

to separate

p r e p a r a t i v e l y t h e more common p h o s p h o l i p i d s from a n i m a l t i s s u e on homemade 2 mm l a y e r s o f s i l i c a g e l PF254

+

366.

120

P r e p a r a t i v e techniques u s e f u l for the separation of lysophosphatidyl chol ine, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine. and v a r i o u s c e r e b r o s i d e s have been a c c o m p l i s h e d b y F r i e d and S h a p i r o [ 5 1 1 . The use o f p r e c o a t e d 2 0 x 20 cm s i l i c a g e l 60 p l a t e s i n a t a n k s a t u r a t e d w i t h chloroform-methanol-water

(65:25:4 v / v ) proved very successful i n these

separations.

2.7.2 Drugs and Pharmaceuticals Up t o 2 g o f a s t e r o i d m i x t u r e d i s s o l v e d i n c h l o r o f o r m - m e t h a n o l ( 9 8 : 2 v / v ) was s e p a r a t e d o n 1 . 5 mm l a y e r s o f s i l i c a g e l HF254 u s i n g t h e m o b i l e phase c h l o r o f o r m - m e t h a n o l ( 9 O : l O v l v ) . nm UV l i g h t .

The components were v i s u a l i z e d i n 2 5 4

A f t e r f i v e separate ascending developments i n t h i s m o b i l e phase,

t h e h y d r o c o r t i s o n e f r a c t i o n ( u p p e r b a n d ) was c l e a r l y s e p a r a t e d from p r e d n i s l o n e ( l o w e r band) [121. A m i x t u r e of n i t r o g e n - c o n t a i n i n g drugs c o n s i s t i n g of p-aminobenzoic a c i d ,

p h e n a c e t i n , and n i c o t i n a m i d e d i s s o l v e d i n c h l o r o f o r m - m e t h a n o l ( 1 : l

v / v ) was

s e p a r a t e d o n a n a l u m i n a PF254 l a y e r b y two s e p a r a t e d e v e l o p m e n t s i n chloroform-methanol (95:5 v / v ) .

A d i s t i n c t separation of t h e t h r e e f r a c t i o n s

was o b s e r v e d b y v i s u a l i z a t i o n u n d e r s h o r t w a v e UV l i g h t [ 1 2 1 . To p r e p a r a t i v e l y s e p a r a t e c a f f e i n e from a c o f f e e e x t r a c t d i s s o l v e d i n chloroform-methanol ( 1 : l was u s e d .

v / v ) , t h e m o b i l e phase h e p t a n e - c h l o r o f o r m ( 3 0 : 7 0 v / v )

The a d s o r b e n t was s i l i c a g e l PF254, and c a f f e i n e was v i s u a l i z e d

u n d e r s h o r t w a v e UV l i g h t [ 1 2 1 . Up t o 5 g o f o p i u m a l k a l o i d s e x t r a c t e d i n c h l o r o f o r m - m e t h a n o l ( 1 : l v / v ) was s e p a r a t e d o n s i l i c a g e l

PF254 u s i n g t h r e e m o b i l e p h a s e s :

h e p t a n e - c h l o r o f o r m ( 3 0 : 7 0 v / v ) , p u r e c h l o r o f o r m , and c h l o r o f o r m - m e t h a n o l (9O:lO v l v ) .

S e p a r a t i o n o f p a p a v e r i n e , n o s c a p i n e , t h e b a i n e , c o d e i n e , and

m o r p h i n e was o b s e r v e d u n d e r UV l i g h t [ 1 2 1 . 5 5 ~ - S a t u r a t e dand A - u n s a t u r a t e d s t e r o i d a l k a l o i d s and s a p o g e n i n s were s e p a r a t e d o n s i l i c a g e l G or a l u m i n a G l a y e r s i m p r e g n a t e d w i t h AgN03 b y d e v e l o p m e n t w i t h c h l o r o f o r m - m e t h a n o l ( 9 O : l O or 9 5 : 5 v / v ) or w i t h chloroform-ethyl ether-acetic acid (97:2.5:0.5 v / v ) .

The s t e r o i d s were

d e t e c t e d b y s p r a y i n g w i t h 10% aqueous K B r , d r y i n g , a n d t h e n t r e a t i n g w i t h a s o l u t i o n of 0 . 2 g o f i o d i n e and 0 . 4 g o f K I i n 100 m l o f w a t e r [ 5 2 1 . PTLC was used t o s e p a r a t e v a r i o u s n a r c o t i c s and b a r b i t u r a t e s from human tissues f o l l o w i n g autopsy.

S i l i c a g e l p l a t e s c a p a b l e o f h a n d l i n g 5 0 t o 100 mg

121

of samples were used. Mobile phases consisted of various combinations o f chloroform and acetone C531. De Zeeuw [ 5 4 1 has described the use of TLC with vapor-programmed development for the preparative isolation of sulfonamides. 2.7.3

Dyes and Pigments

Kirchner [ 8 1 used PTLC to separate a mixture of six dyes o n thick homemade layers of silica gel with 20% gypsum added as the binder. Multiple ascending development was carried out with various combinations o f benzene-chloroform and benzene to separate Yellow 06, Sudan I , Sudan 111, Sudan 11, Methyl Red, and Crystal Violet (high to low RF). Sudan I1 dye was also purified on silica gel with a 20% gypsum binder by two ascending developments with benzene-chloroform (2O:l v/v). A PTLC technique using ascending continuous flow development in a sandwich type chamber was used to separate a mixture of the dyes butter yellow, Sudan Red, and indophenol C551. Chlorophyll fractions from plant leaves were extracted with chloroform, and extracts were placed as bands on 2 mm layers of silica gel PF254 C121. Zones were separated using multiple development in the ascending direction with the mobile phase petroleum ether-chloroform (1:l v/v). After several developments, separated chlorophyll and non-chlorophyll bands were visible in daylight and under UV light. Zones were recovered by typical elution procedures. Doss [ 5 6 1 has reported the PTLC of porphyrin methyl esters. Separations of the dicarboxylic porphyrin esters from proto-, deutero-, and hematoporphyrin IX were obtained o n silica gel or alumina plates with benzene-ethyl acetate-methanol solvent systems. M i sce I I aneous Compounds PTLC of purines was carried out on ready-made cellulose plates C571. Golankiewicz and Antkowiak [ 5 8 1 used PTLC to separate synthetic oligonucleotides on DEAE-cellulose. This procedure proved more effective than column chromatography in regard to better separation of the desired products and elimination of side products in the separation o f di- and trinucleoside phosphates. PTLC was done on reversed phase silanized silica gel plates t o purify deoxyr i boo1 igonucleot ides C591. 2.7.4

122

PTLC was used t o separate f l a v o n o i d s i n green pea l e a v e s .

S i l i a gel G

p l a t e s impregnated w i t h a c e t a t e , b o r a t e , molybdate, or, p r e f e r a b l y , t u n g s t a t e i o n s f a c i l i t a t e d these s e p a r a t i o n s C601.

Xanthones. t r i t e r p e n e s , and saponins

were i s o l a t e d by p r e p a r a t i v e c e n t r i f u g a l , r a d i a l TLC u s i n g t h e Chromatotron Xanthone aglycones were separated i n a p p r o x i m a t e l y 0.5 h r from

Model 7924.

crude Gentiana e x t r a c t s by c e n t r i f u g a l TLC on s i l i c a g e l GFZs4 and e l u t i o n w i t h c h l o r o f o r m o r chloroform-methanol f o l l o w e d by d e t e c t i o n i n UV l i g h t [611. PTLC has been used t o s e p a r a t e t h e v a r i o u s forms o f v i t a m i n B6 i n c h i c k embryos C621.

A r e v i e w o f t h e use o f PTLC w i t h carbohydrates has been

presented by Wing and B e M i l l e r C631.

The use o f polyamide l a y e r s i n PTLC has

been reviewed by Wang and Hang C641.

2.8 TRANSFER OF RESULTS FROM TLC TO PREPARATIVE LlOUlD CHROMATOGRAPHY

A s t r a t e g y f o r t r a n s f e r i n g TLC t o p r e p a r a t i v e LC was b r i e f l y d i s c u s s e d i n Chapter 1.5.2.

Another r e p o r t 1651 d i s c u s s e s t h e t r a n s f e r of r e s u l t s f r o m

a n a l y t i c a l s i l i c a g e l TLC t o p r e p a r a t i v e s i l i c a g e l LC for t h e purpose o f s e l e c t i n g an a p p r o p r i a t e m o b i l e phase and l o a d i n g f o r t h e column s e p a r a t i o n o f interest.

T h i s s e c t i o n o u t l i n e s t h e elements o f t h e t r a n s f e r s t r a t e g y C651.

I . RF values d e s i g n a t e m i g r a t i o n i n TLC: d i s t a n c e o f spot movement

RF =

d i s t a n c e o f s o l v e n t f r o n t movement

RF values a r e i n c r e a s e d by r a i s i n g t h e p o l a r i t y o f t h e m o b i l e phase and decreased by l o w e r i n g i t s p o l a r i t y .

2. The optimum r e t e n t i o n range for a p r e p a r a t i v e column s e p a r a t i o n i s 3-8 c o l umn volumes .

3 . The r e l a t i o n s h i p between RF and column volume i s : C.V. =

1 RF

T h e r e f o r e , t h e column volume range o f 3-8 corresponds t o an RF range o f a p p r o x i m a t e l y 0.30-0.15,

which i s optimum f o r small sample l o a d s ( F i g . 2 . 4 ) .

123

1

I

1

1

0

1

2

3

I

1

4 Cob""

5

I

1

s

r

L

a

1

,

9

0

"0l"ll.l

R e l a t i o n s h i p between RF values i n TLC and e l u t i o n i n

Fig. 2.4.

p r e p a r a t i v e L C f o r a m o b i l e phase w i t h i n ( A ) and o u t s i d e of ( 6 ) t h e optimum p r e p a r a t i v e rang?

(Courtesy of Waters

Chromatoylaphy D i v i s i o n o f Mi11iDoi.e).

T h i s r e l a t i o n s h i p i s n o t e x a c t because o f d i f f e r e n c e s i n t h e technique's o f column LC and TLC and v a r i a t i o n s i n t h e p r o p e r t i e s o f TLC and LC s i l i c a g e l s .

G e n e r a l l y , LC column volumes w i l l be equal t o

or l e s s t h a n t h e v a l u e c a l c u l a t e d from t h e above e q u a t i o n . 4. The e l u t i o n volume i s a l s o dependent upon t h e sample l o a d and s o l v e n t

used t o s o l u b i 1 i z e t h e sample.

5. The q u a n t i t y o f sample t h a t can be l o a d e d o n t o a p r e p a r a t i v e column i s a f u n c t i o n o f t h e s e p a r a t i o n a c h i e v e d by TLC.

A m o b i l e phase

p r o v i d i n g a more complete s e p a r a t i o n a l l o w s a l a r g e r amount o f sample t o be loaded o n t o a column t h a t w i l l be developed w i t h t h a t s o l v e n t . For example, f o r ARF = 0.15, a 20 g sample can be loaded, w h i l e f o r

RF

= 0.10, o n l y 5-10 g can be separated.

6. RF v a l u e s should be a d j u s t e d toward 0.10-0.15

fo r i n c r e a s e d sample

loads and more d i f f i c u l t s e p a r a t i o n s (ARF < 0.07).

7 . E t h y l a c e t a t e i s a f a v o r a b l e p r e p a r a t i v e LC s o l v e n t because i t p r o v i d e s good s o l u b i l l t y f o r many samples, m a i n t a i n s column l i f e ,

124

e q u i l i b r a t e s q u i c k l y , and i s e a s i l y d i s t i l l e d from c o l l e c t e d The amount o f e t h y l a c e t a t e i n t h e TLC m o b i l e phase i s

fractions.

a d j u s t e d t o o b t a i n a p o l a r i t y t h a t moves t h e s o l u t e s w i t h i n t h e 0.15-0.35

RF r a n g e .

C o r r e l a t i o n f r o m TLC t o p r e p a r a t i v e LC i s good

when t h e m o b i l e phase c o n t a i n s s o l v e n t s e q u a l t o , o r l e s s p o l a r t h a n , ethyl acetate. 8 . W i t h more p o l a r s o l v e n t s , t h e a f f i n i t y b e t w e e n s i l i c a g e l and t h e s o l v e n t becomes g r e a t e r and c o r r e l a t i o n from TLC t o p r e p a r a t i v e LC i s less reliable.

The e q u i l i b r a t e d p r e p a r a t i v e column s o l v e n t m i x t u r e

s h o u l d c o n t a i n o n l y 10% t o 20% as much o f t h e p o l a r component i n i t i a l l y u s e d i n t h e TLC s e p a r a t i o n f o r c o r r e l a t i o n t o o c c u r . R e p r o d u c i b l e c o r r e l a t i o n o f a n a l c o h o l i c TLC s o l v e n t s y s t e m r e q u i r e s c a r e f u l p r e c o n d i t i o n i n g o f t h e p r e p a r a t i v e c o l u m n and s o l v e n t . 9. I f s p o t s t a i l d u r i n g TLC, t h e cause may be o v e r l o a d i n g , or t h e s o l u t e s may b e e x t r e m e l y p o l a r , a c i d i c , or b a s i c . b a s i c s o l u t e s i s a f f e c t e d b y pH.

The s e p a r a t i o n o f a c i d or

This e f f e c t i s tested by p l a c i n g a

s m a l l b e a k e r o f ammonium h y d r o x i d e i n s i d e t h e chamber d u r i n g development.

I f t h e RF v a l u e s change s i g n i f i c a n t l y or t a i l i n g i s

e l i m i n a t e d due t o t h e p r e s e n c e o f NH3 v a p o r , t h e s o l u t e s a r e b a s i c and a base s h o u l d be i n c o r p o r a t e d i n t h e p r e p a r a t i v e s o l v e n t m i x t u r e

t o p r e v e n t a d s o r p t i o n o f t h e compound i n t o t h e s i l i c a g e l .

If there

i s n o s i g n i f i c a n t change i n R F , t h e compound i s n o t b a s i c , and a n e u t r a l or a c i d i c

p r e p a r a t i v e LC s o l v e n t w i l l be needed.

10. The u s e o f a s t e p g r a d i e n t t o s e p a r a t e complex m i x t u r e s o n a

p r e p a r a t i v e LC c o l u m n may be r e q u i r e d .

T h i s w i l l be i n d i c a t e d i f t h e

t h i n l a y e r c h r o m a t o g r a m c o n t a i n s some compounds o f i n t e r e s t t h a t c a n n o t be m a i n t a i n e d i n t h e o p t i m u m 0 . 1 5 - 0 . 3 5 compounds a r e i n t h i s p r e p a r a t i v e r a n g e .

RF r a n g e w h i l e o t h e r

I n t h i s case, t h e mobile

phase t h a t moves t h e l e a s t p o l a r compounds betwee'n RF 0 . 1 5 a n d 0 . 3 5 i s t h e i n i t i a l column s o l v e n t .

The more p o l a r components w i l l be

c l o s e t o t h e o r i g i n on t h e t h i n l a y e r chromatogram w i t h t h i s m o b i l e phase.

The second c o l u m n s o l v e n t i s a t h i n l a y e r m o b i l e phase t h a t

moves t h e p o l a r compounds i n t o t h e 0 . 1 5 - 0 . 3 5

RF r a n g e .

I f the

sample c o m p l e x i t y r e q u i r e s , a d d i t i o n a l s o l v e n t s t e p s may be d e t e r m i n e d i n a s i m i l a r manner. The d e s i g n o f TLC m o b i l e phases h a v i n g s p e c i f i c e l u t i o n s t r e n g t h a n d r e s o l v i n g power was d i s c u s s e d b y F r i e d and Sherma [661.

The a p p r o a c h i n v o l v e s

125

b l e n d i n g s o l v e n t s t o a c h i e v e t h e r e q u i r e d RF v a l u e s f o r t h e compounds o f i n t e r e s t a n d t h e n s u b s t i t u t i n g o t h e r s o l v e n t s i n a p p r o p r i a t e amounts t o m a i n t a i n t h e s o l v e n t s t r e n g t h b u t change t h e i n t e r a c t i o n s o f t h e c h r o m a t o g r a p h i c s y s t e m and i n c r e a s e r e s o l u t i o n .

Reference [651 contains

d e t a i l e d i n f o r m a t i o n and i l l u s t r a t i v e examples t h a t , when c o m b i n e d w i t h t h e procedures i n r e f e r e n c e [661, w i l l a l l o w a s y s t e m a t i c approach t o p r e p a r a t i v e LC s o l v e n t d e s i g n b y TLC .

S i m i l a r c o n s i d e r a t i o n s should be a p p l i c a b l e t o

p r e p a r a t i v e LC on c h e m i c a l l y bonded r e v e r s e d p h a s e columns, i f t h e o p p o s i t e r e l a t i o n s h i p s among p o l a r i t y , s o l v e n t s t r e n g t h , and m i g r a t i o n o r d e r a r e k e p t i n mind. O t h e r s t u d i e s o f T L C - p r e p a r a t i v e column LC r e l a t i o n s h i p s h a v e been made, b u t none a r e r e p o r t e d i n t h e same d e t a i l as t h e one d e s c r i b e d a b o v e .

For

e x a m p l e , S o c z e w i n s k i and Wawrzynowicz [ 6 7 1 i n t r o d u c e d l a r g e volumes o f s a m p l e s o l u t i o n from t h e edge o f t h i n l a y e r and t h e n e l u t e d c o n t i n u o u s l y u s i n g a h o r i z o n t a l s a n d w i c h chamber w i t h a g l a s s d i s t r i b u t o r .

These c o n d i t i o n s w e r e

s i m i l a r t o column c h r o m a t o g r a p h y w i t h t h e same s o r b e n t - e l u e n t s y s t e m . authors a l s o described a modified

These

TLC t e c h n i q u e f o r two d i m e n s i o n a l

preparative separations.

2.9 REFERENCES

1. 2. 3. 4. 5. 6 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

F . J . R i t t e r a n d G . M. M e y e r , N a t u r e , 193 ( 1 9 6 2 ) 941. J . M. M i l l e r and J . G. K i r c h n e r , A n a l . Chem., 23 ( 1 9 5 1 ) 4 2 8 .

J. M. M I I l e r and J . G. K i r c h n e r , A n a l . Chem., 24 ( 1 9 5 2 ) 1480. D. Hahn and H . Fuchs, H e l v . Chim. A c t a , 4 5 ( 1 9 6 2 ) 261. K . B r e n d e l , R . S . S t e e l e a n d E. A . D a v i d s o n , J . C h r o m a t o g r . , 3 0 ( 1 9 6 7 ) 232. D. M. J o r d a n , J . C h r o m a t o g r . , 57 ( 1 9 7 1 ) 427 and 6 3 ( 1 9 7 1 ) 4 4 2 . R . V i s s e r . A n a l . Chim. A c t a , 38 ( 1 9 6 7 ) 1 5 7 . J . G. K i r c h n e r , J . C h r o m a t o g r . , 6 3 ( 1 9 7 1 ) 45. C. G. Honegger, H e l v . Chim. A c t a , 45 ( 1 9 6 2 ) 1409. 8 . P: K o r z u n , L . Dorfman a n d S. M. B r o d y , A n a l . Chem., 35 ( 1 9 6 3 ) 9 5 0 . H . K. Mangold, H. H . 0. S c h m i d t and E . S t a h l , T h i n L a y e r C h r o m a t o g r a p h y , i n D. G l i c k ( E d . ) , Methods o f B i o c h e m i c a l A n a l y s i s , Vol. 12, I n t e r s c i e n c e , 1964, p . 393. H . Halpaap, P r e p a r a t i v e T h i n Layer Chromatography, i n I . Smith (Ed.), C h r o m a t o g r a p h i c a n d E l e c t r o p h o r e t i c T e c h n i q u e s , Vol. 1, 3 r d e d i t i o n , C h a p t e r 34, I n t e r s c i e n c e , 1969, p . 834. E . A . M i s t r y u k o v . J . C h r o m a t o g r . . 9 ( 1 9 6 2 ) 311. J . D a v i d e k and J . B l a t t n a , J . C h r o m a t o g r . , 7 ( 1 9 6 2 ) 2 0 4 . H . R. F e l t o n , P r e p a r a t i v e T h i n Layer Chromatography, A n a l t e c h Technical R e p o r t No. 7903. P. 0. Box 7558, Newark, DE 19711, 1978. H . R . F e l t o n , P r e p a r a t i v e TLC, i n J . C. T o u c h s t o n e ( E d . ) , Advances i n T h i n L a y e r C h r o m a t o g r a p h y - C l i n i c a l and E n v i r o n m e n t a l A p p l i c a t i o n s , C h a p t e r 2, W i l e y - I n t e r s c i e n c e , 1982. H . H a l p a a p . Chem.-1ng.-Tech., 35 (1963) 488. E . S. A l h a d e f f . J . Chem. Educ., 46 ( 1 9 6 9 ) 249. J. M. Walsh, J. Chem. Educ., 48 ( 1 9 7 1 ) 4 1 5 .

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40 41. 42, 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

60. 61. 62.

H. C. P r i c e , J . Chem. Educ., 51 (1974) 267. G . Zweig and J . Sherma, Handbook of Chromatography, Vol. 1 , CRC Press, Boca Raton, FL, 1972, p . 784. J . G. K i r c h n e r , T h i n Layer Chromatography, 2nd e d i t i o n , W i l e y - I n t e r s c i e n c e , New York. p. 1137. E . S t a h l , T h i n Layer Chromatography, 2nd e d i t i o n , S p r i n g e r - V e r l a g , B e r l i n , p. 1041. Quantum I n d u s t r i e s T e c h n i c a l B u l l e t i n No. 625, P r e p a r a t i v e P l a t e Recovery Techniques, 1977, 3 pp. (Quantum i s now p a r t o f Whatman. I n c . ) . R. W. Horobin, J . Chrornatogr., 38 (1968) 508. 2. Nichaman, C. C . Sweeley, N. M. Oldham and R . E . Olson, J . L i p i d Res.. 4 (1963) 484. G. C. B a r r e t t , Adv. Chromatogr.. 11 (1974) 146. J . G. K i r c h n e r , J . M . M i l l e r and G. J . K e l l e r , A n a l , Chem.. 23 (1951) 420. R . J . G r i t t e r and R. J . A l b e r s , J . Chromatogr.. 9 (1962) 392. M. E . Tate and C . T . Bishop, Can. J . Chem., 40 (1962) 1043. P . E . Schulze and M. Wenzel, Angew. Chem. I n t . Ed. Engl.. 1 (1962) 580. E . A . M i s t r y u k o v , J . Chromatogr., 9 (1962) 311. M. Dobiasova J . L i p i d R e s . , 4 (1963) 481. M. M o t t i e r and M. P o t t e r a t . A n a l . Chim:Acta, 13 (1955) 46. M. 8eroza and T. P. McGovern, Chemist-Analyst, 52 (1963) 82. M. A. M i l l e t t , W . E. Moore and J . F . Saemen. A n a l . Chem.. 36 (1964) 491. 8. G o l d r i c k and J . H i r s c h , J . L i p i d Res.. 4 (1963) 482. J . S. Matthews. A . L. Pereda-V and A . A g u i l e r a - P . , J . Chromatogr.. 9 (1962) 331. D . Dekker, J . Chromatogr.. 168 (1979) 508. S. C. P l a t t . A n a l . Biochem.. 91 (1978) 357. W . W. C h r i s t i e , L i p i d A n a l y s i s , I s o l a t i o n S e p a r a t i o n , I d e n t i f i c a t i o n and S t r u c t u r a l A n a l y s i s o f L i p i d s . Pergamon Press, O x f o r d , 1973, p. 338. El. F r i e d and J . Sherma, Thin l a y e r Chromatography - Techniques and A p p l i c a t i o n s . Marcel Dekker, I n c . , New York, 1982. p . 308. 6. F r i e d and J . B o d d o r f , J . P a r a s i t o l . , 64 (1978) 174. V . P. S k i p s k i , A . F . Smolowe, R. C. S u l l i v a n and M. B a r c l a y , Biochim. Biophys. A c t a , 106 (1965) 386. F . Snyder, I n A . N i e d e r w i e s e r and G . P a t a k i (Eds.), Progress i n T h i n Laver Chromatoqraphv - . - and R e l a t e d Methods, Science Pub., I n c . , Ann Arbor, 1971, p . 105. D. Chobanov, R. Taradziska and R. Chobanova, J . Am. O i l Chem. SOC., 53 (1976) 48. J . B a r r e t t , G. P. C a i n and D. F a i r b a i r n , J . P a r a s i t o l . , 56 (1970) 1004. T . A. M i e t t i n e n , E . H. Ahrens and 5. M. Grundy, J . L i p i d Res., 6 (1965) 411. J . J . E l l i n g t o n . P . F. Schlotzhauer and A . I.Schepartz, J . Am. O i l Chem. Soc.. 55 (1978) 572. R . Ledeen, J . A m e r . O i l Chem. SOC., 43 (1966) 57. 6. F r i e d and I.L . S h a p i r o , J . P a r a s i t o l . , 65 (1979) 243. H . Roensch and K . S c h r e i b e r , J . Chromatogr., 30 (1967) 149. S. Goenechea. F r e s e n i u s ' Z . Anal. Chem., 225 (1966) 30. R . A . De Zeeuw, Sep. P u r i f . Methods, 6 (1977) 129. S. Hara, S . Yamazaki and H . I c h i k a w a , Chem. I n d . (London), 46 (1969) 1657. M. Doss, Z . K l i n . Chem. K l i n , Biochem., 9 (1970) 197. K. Doerner and H . Manzke, Z . K l i n , Chem. K l i n , Biochem., 9 (1971) 57. K. Golankiewicz and J . Antkowiak, A c t a Biochem. Pol., 21 (1974) 17. H. M. Hsiung, R. Brousseae, J . M i c h n i e w i c z and 5 . A . Narang, N u c l e i c Acids Res., 6 (1979) 1371. D . 6. Harper and H . Smith, J . Chromatogr.. 41 (1969) 138. K . Hostettmann. t4. Hostettmann-Kaldas and 0. S t i c h e r . J . Chromatogr., 202 (1980) 154. M . A. Smith and L. 5 . D i e t r i c h , Biochem. Biophys. A c t a . 230 (1971) 262.

127 63. 64. 65. 66. 67.

R. Wing and J . N . B e M i l l e r . Methods C a r b o h y d . Chem., 6 ( 1 9 7 2 ) 60. K . - T . Hang a n d I . 5 . Y . Wang, J . C h r o m a t o g r . , 2 4 ( 1 9 6 6 ) 4 5 8 . P . Rahn and M. Woodman, Amer. L a b . , 1 3 ( 2 ) (1981) 9 6 . B . F r i e d and J . Sherma, T h i n L a y e r C h r o m a t o g r a p h y - T e c h n i q u e s and A D D l i c a t i o n s . M a r c e l D e k k e r . I n c . . New York. Second E d i t i o n , 1986, C h a p t e r 6. E . S o c c z e w i n s k i and T . Wawrzynowicz, J . C h r o m a t o g r . , 2 1 8 ( 1 9 8 1 ) 7 2 9 .


129

CHAPTER 3 INCREASING THE EFFICIENCY OF VERY LARGE SCALE PACKED BED CHROMATOGRAPHIC SEPARAT IONS P h i l l i p C. Wankat School of Chemical E n g i n e e r i n g Purdue Un i v e r s i t y West L a f a y e t t e , I N 47907

INTRODUCTION

3.1 3.2

INEFFICIENCIES I N PREPARATIVE ELUTION CHROMATOGRAPHY

3.3

COUNTER-CURRENT SYSTEMS

3.4

SIMULATED MOVING BED (SMB) SYSTEMS

3.5

HYBRID METHODS: COMBINE SMB AND ELUTION CHROMATOGRAPHY

3.6

OTHER METHODS FOR LARGE-SCALE CHROMATOGRAPHY

3.7

ACKNOWLEDGEMENT

3.8

NOMENCLATURE

3.9

REFERENCES

3 . 1 INTROOUCTION

L a r g e s c a l e l i q u i d c h r o m a t o g r a p h y i s n o t a new method.

For e x a m p l e ,

S p e d d i n g e t a l . [ I 1 r e p o r t e d o n u s e o f p r e p a r a t i v e i o n exchange c h r o m a t o g r a p h y i n t h e M a n h a t t a n p r o j e c t , S h e a r o n and Gee [ 2 1 c o m m e r c i a l l y s e p a r a t e d c a r 0 e n e , x a n t h o p h y l l and c h l o r o p h y 1 o n b e d s o f a c t i v a t e d c a r b o n , a n d Ek [31 d e s c t b e d commercial g e l permeation chromatography equipment. applications large scale s e p a r a t i o n method.

Despite these e a r l y

i q u i d c h r o m a t o g r a p h y has n o t become a s t a n d a r d

I n f a c t , i t i s s t i l l news when a l a r g e s c a l e u n i t i s

constructed. R e c e n t l y t h e r e h a v e been s e v e r a l r e p o r t s o f l a r g e s c a l e l i q u i d chromatographs.

For e x a m p l e , Seko e t a l . [ 4 1 r e p o r t e d on p i l o t p l a n t

e x p e r i m e n t s and t h e d e s i g n of a l a r g e s c a l e s y s t e m t o s e p a r a t e p a r a , m e t a a n d o r t h o x y l e n e s and e t h y l b e n z e n e u s i n g an u n s p e c i f i e d z e o l i t e a d s o r b e n t a n d a n unspecified desorbent.

One of t h e p r o p o s e d l a r g e s c a l e s y s t e m s w o u l d p r o d u c e

130

70,000 t o n l y e a r o f p-xylene and would have an adsorbent charge o f 270 t o n s . E l u t i o n chromatography was used w i t h r e c y c l e of a p-xylene-ethylbenzene A d i s t i l l a t i o n column was used t o r e c o v e r p-xylene f r o m t h e

mixture. desorbent.

H e i k k i l a C51 r e p o r t e d on s e v e r a l o p e r a t i n g systems u s i n g i o n

e x c l u s i o n chromatography t o s e p a r a t e sugars from molasses.

Commercial columns

range from 2.0 t o 12.0 meters h i g h and from 0.5 t o 4 . 0 meters i n d i a m e t e r .

A

t y p i c a l u n i t ( f o r Amino GmbH) processes 60,000 m e t r i c t o n s l y e a r o f molasses i n seven columns each 3 . 6 m i n d i a m e t e r and 12 m h i g h . e l u a n t and r e c y c l e i s employed.

Water i s used as t h e

Many o t h e r l a r g e - s c a l e a p p l i c a t i o n s a r e i n

use b u t have n o t been r e p o r t e d i n t h e l i t e r a t u r e .

Many s m a l l e r s c a l e

a p p l i c a t i o n s a r e r e p o r t e d i n t h e o t h e r c h a p t e r s o f t h i s book. For these l a r g e s c a l e systems i t i s g e n e r a l l y agreed t h a t t h e f o l l o w i n g design c r i t e r i a a r e i m p o r t a n t : 1.

H i g h l y s e l e c t i v e packing, a > 1.5 and p r e f e r a b l y w i t h a > 2 ( p r o b a b l y a s p e c i a l l y formulated packing).

2.

High c a p a c i t y p a c k i n g .

3.

Very t i g h t l y s i e v e d p a c k i n g , p r e f e r a b l y r i g i d s p h e r i c a l p a r t i c l e s .

In

broad d i s t r i b u t i o n packings t h e e f f i c i e n c y (HETP) i s determined by t h e l a r g e r p a r t i c l e s and t h e p r e s s u r e d r o p by t h e small p a r t i c l e s .

Since

APlL i s i n v e r s e l y p r o p o r t i o n a l t o d2 and t h e mass t r a n s f e r P 2 l i m i t i n g p o r t i o n o f HETP i s p r o p o r t i o n a l t o ,,d, i t i s important t o have narrow p a r t i c l e d i s t r i b u t i o n s . 4.

A p a c k i n g method which produces a u n i f o r m p a c k i n g .

5.

Good l i q u i d d i s t r i b u t i o n across t h e column.

6.

L i m i t e d m i x i n g and zone broadening o u t s i d e t h e column.

7.

C o n t r o l o f p r o d u c t w i t h d r a w a l s based e i t h e r on t i m e or p r e f e r a b l y by c o n c e n t r a t i o n measurements.

E n g i n e e r i n g has r e c e n t l y progressed where one can, w i t h c a r e and p r o p r i e t a r y knowledge. meet these s t r i n g e n t r e q u i r e m e n t s .

Some u s e f u l h i n t s on how t o do

t h i s a r e g i v e n by Seko e t a l . [ 4 1 , and H e i k k i l a [ 5 1 .

131 D e s p i t e these advances, l a r g e s c a l e chromatography i s n o t e f f i c i e n t .

In

t h i s c h a p t e r we w i l l l o o k a t ways t o improve t h e e f f i c i e n c y by changing t h e o p e r a t i n g scheme.

These new methods assume t h a t we can s a t i s f y t h e

r e q u i r e m e n t s l i s t e d above.

3 . 2 INEFFICIENCIES I N PREPARATIVE ELUTION CHROMATOGRAPHY

By i t s v e r y n a t u r e e l u t i o n chromatography does n o t e f f i c i e n t l y u t i l i z e e i t h e r the sorbent material o r the solvent. I n a n a l y t i c a l a p p l i c a t i o n s these I n l a r g e - s c a l e a p p l i c a t i o n s where t o n s o f

i n e f f i c i e n c i e s are unimportant.

m a t e r i a l a r e b e i n g handled these i n e f f i c i e n c i e s become c r i t i c a l and make e l u t i o n chromatography a r e l a t i v e l y expensive s e p a r a t i o n scheme. The i n e f f i c i e n c i e s i n an e l u t i o n chromatograph can be e x p l a i n e d by c o n s i d e r i n g t h e c o n c e n t r a t i o n p r o f i l e s i n t h e column.

These p r o f i l e s a r e

shown s c h e m a t i c a l l y i n F i g . 3 . 1 f o r t h r e e d i f f e r e n t t i m e s .

A feed w i t h t h r e e

s o l u t e s which we d e s i r e t o r e c o v e r s e p a r a t e l y i s i l l u s t r a t e d , b u t t h e i d e a s can be extended t o o t h e r s i t u a t i o n s .

Some o f t h e r e g i o n s i l l u s t r a t e d may n o t

occur i n a p a r t i c u l a r example. I n t h e r e g i o n s marked 1 , no s o l u t e i s p r e s e n t . n o t d o i n g any u s e f u l s e p a r a t i o n .

O b v i o u s l y these r e g i o n s a r e

The adsorbent and s o l v e n t r e q u i r e d t o f i l l

these r e g i o n s i s b e i n g " s t o r e d " f o r use a t a l a t e r t i m e .

A chromatographic

column i s an expensive s t o r a g e v e s s e l .

I n t h e r e g i o n s marked 2 a l l t h e

solutes are a t the feed concentration.

Again no u s e f u l s e p a r a t i o n i s

occurring.

I n r e g i o n 4 o n l y two s o l u t e s a r e p r e s e n t b u t t h e y a r e a t t h e i r

feed c o n c e n t r a t i o n .

T h i s r e g i o n i s a l s o n o t d o i n g any u s e f u l s e p a r a t i v e work.

I n t h e r e g i o n s marked 3, 3 ' and 3" o n l y one s o l u t e p l u s s o r b e n t and s o l v e n t are present.

Since t h e s o l u t e i s a t t h e feed c o n c e n t r a t i o n i n r e g i o n s 3 n o

separation i s occurring. the sorbent. wave.

I n r e g i o n 3 ' t h e p u r e s o l u t e i s b e i n g removed from.

This i s u s e f u l .

Regions 3" a r e t h e l e a d i n g edge o f a s o l u t e

I n these r e g i o n s s o l v e n t or desorbent i s b e i n g removed from pure

solute.

This i s a l s o u s e f u l .

The s e p a r a t i o n o f s o l u t e s from each o t h e r i s o c c u r r i n g i n t h e r e g i o n s marked 5. T h i s i s t h e f r a c t i o n of t h e column which i s d o i n g t h e d e s i r e d s e p a r a t i v e work. remixing.

I n t h e r e g i o n marked M separated s o l u t e s A and C a r e

This i s Obviously undesirable.

T h i s r e g i o n can be e l i m i n a t e d by

feeding pulses f u r t h e r apart, b u t t h i s w i l l increase the s i z e of regions 1.

132 A

2 s 3" I II

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CONC.

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AXIAL DISTANCE Fig. 3.1

Schematic of concentration p r o f i l e s i n s i d e a p r e p a r a t i v e e l u t i o n chromatography column.

Key

A

133

The s i z e o f r e g i o n 1 i s a l s o i n c r e a s e d when e x t r e m e p r o d u c t p u r i t y i s required.

T h i s p r o b l e m i s u s u a l l y managed b y a l l o w i n g t h e s e p a r a t i o n z o n e s

b e t w e e n A and B a n d between B a n d C t o m i g r a t e t o t h e c o l u m n o u t l e t . P a r t i a l l y separated m a t e r i a l i s then recycled.

The A-B r e c y c l e s h o u l d b e

i n p u t b e f o r e t h e f e e d p u l s e and t h e B-C r e c y c l e a f t e r t h e f e e d p u l s e .

Further

s u b d i v i s i o n s of t h e r e c y c l e ( i n s t e a d of c o m p l e t e l y m i x i n g i t ) can a l s o b e helpful.

However, r e c y c l e r e d u c e s o n l y r e g i o n 1 and does n o t e f f e c t t h e o t h e r

i n e f f i c i e n t r e g i o n s 2 , 3 and 4 . What can be done t o make t h e c o l u m n more e f f i c i e n t ?

Any p r o c e d u r e w h i c h

d e c r e a s e s d i s p e r s i o n and mass t r a n s f e r r a t e l i m i t a t i o n s ( i n c r e a s e N) w i l l h e l p some.

When N i n c r e a s e s t h e s i z e s o f t h e s e p a r a t i o n zone 5, d e s o r p t i o n z o n e s

3 ' , and a d s o r p t i o n zones 3" a l l d e c r e a s e ( t h e r e i s l e s s z o n e s p r e a d i n g ) .

Then

t h e n e x t f e e d p u l s e c a n be i n p u t s o o n e r and r e g i o n 1 i s d e c r e a s e d i n s i z e . T h i s approach i s i n h e r e n t i n o u r l i s t o f d e s i g n c r i t e r i a for l a r g e s c a l e chromatography.

T h e r e a r e p r a c t i c a l l i m i t s t o how much p a c k i n g d i a m e t e r c a n

be d e c r e a s e d i n a l a r g e s c a l e s y s t e m .

P r e s s u r e d r o p s and p a c k i n g c o s t s w i l l

i n c r e a s e as p a r t i c l e d i a m e t e r d e c r e a s e s . A v e r y l a r g e number o f s t a g e s ( p l a t e s ) i s a b s o l u t e l y n e c e s s a r y when t h e

s e l e c t i v i t y i s c l o s e t o one.

When s e l e c t i v i t y i s much h i g h e r (2.0 or a b o v e ) ,

t h e number o f s t a g e s r e q u i r e d i s much l e s s .

Then c h e a p e r , l a r g e r d i a m e t e r

p a c k i n g s w i t h l o w e r p r e s s u r e d r o p s can be u s e d .

With h i g h s e l e c t i v i t i e s t h e

i n h e r e n t c h a r a c t e r i s t i c s o f p r e p a r a t i v e e l u t i o n d e v e l o p m e n t become much m o r e i m p o r t a n t t h a n zone s p r e a d i n g . To e x p l o r e t h i s l e t ' s l o o k a t a n i d e a l c o l u m n w i t h a n i n f i n i t e number o f s t a g e s and h e n c e n o zone s p r e a d i n g .

The c o n c e n t r a t i o n s f o r a n i d e a l s y s t e m

when t h e A-B s e p a r a t i o n i s most d i f f i c u l t a r e shown i n F i g . 3 . 2 .

Since t h e r e

i s no zone s p r e a d i n g , e a c h component r e m a i n s a t i t s f e e d c o n c e n t r a t i o n . t h a t r e g i o n s 5 , 3 ' a n d 3" a l l become v e r t i c a l l i n e s . r e g i o n s w i t h o u t u s e f u l s e p a r a t i o n d o not d i s a p p e a r .

Note

Unfortunately, the R e g i o n 2 i s unchanged.

A b o u t o n e h a l f o f e a c h r e g i o n 3 ' a n d 3" i s c o n v e r t e d t o r e g i o n 3 ( p u r e s o l u t e a t the feed concentration).

The s e p a r a t i o n r e g i o n s , S , a r e r e d u c e d t o a

v e r t i c a l l i n e a n d t h e r e m a i n d e r become r e g i o n s 3 and 4.

Region 1 can be

r e d u c e d s i g n i f i c a n t l y , b u t s i n c e r e g i o n s 3 and 4 grow t h e n e t r e s u l t i s a modest improvement i n f e e d c a p a c i t y . The e x a c t amount o f t i m e we c a n f e e d i s e a s i l y c a l c u l a t e d fo r t h i s i d e a l chromatograph.

With n o zone b r o a d e n i n g and e x a c t l y p e r f e c t s e p a r a t i o n , e a c h

s o l u t e w i l l t a k e a t i m e equal to t h e f e e d p u l s e t o e x i t t h e column.

Hith

134

CONC.

#I

I

I

I

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ji IFB-, I

I

I II

I 1 1

I

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AXIAL DISTANCE F i g . 3.2.

Schematic o f c o n c e n t r a t i o n p r o f i l e s i n s i d e an i d e a l p r e p a r a t i v e e l u t i o n chromatography column. spreading).

I n f i n i t e number o f stages ( n o zone

Same k e y as F i g . 3 . 1 .

t h r e e components t h e b e s t we can d o i s t o i n p u t f e e d 113 o f t h e t i m e .

This

a l s o r e q u i r e s t h a t t h e A-6 and 6 - C r e s o l u t i o n s be e x a c t l y t h e same which w i l l occur i f K6 = 112 ( K A be l e s s than 113.

t

KC).

I n F i g . 3.2 t h e maximum f e e d p e r i o d w i l l

I n g e n e r a l , w i t h n-components

the highest f r a c t i o n of time

we can i n p u t f e e d i n t o t h e column and achieve p e r f e c t s e p a r a t i o n i s l / n . and w i l l u s u a l l y be l e s s .

Zone b r o a d e n i n g w i l l i n c r e a s e b o t h t h e adsorbent and

the solvent requirements. The c o n c l u s i o n i s t h e r e a r e l i m i t a t i o n s i n t h e e l u t i o n method o f o p e r a t i o n r e g a r d l e s s o f t h e number o f stages i n t h e column.

A d d i t i o n a l improvements

r e q u i r e s w i t c h i n g to an e n t i r e l y d i f f e r e n t o p e r a t i n g method.

We w i l l no

l o n g e r have c l a s s i c a l e l u t i o n chromatography. 3.3 COUNTER-CURRENT SYSTEMS

When a b i n a r y s e p a r a t i o n i s d e s i r e d , a c o u n t e r - c u r r e n t system can be used

t o e l i m i n a t e t h e r e g i o n s which a r e d o i n g no s e p a r a t i o n . i n F i g . 3.3.

This i s i l l u s t r a t e d

Now t h e s o l i d s and l i q u i d move c o u n t e r - c u r r e n t l y

I n zones I and I 1 we w i s h t o s e p a r a t e s o l u t e s A and 6 .

t o each other..

I n zone 111 s o l u t e 6

i s desorbed from t h e s o r b e n t so t h a t t h e s o r b e n t can be r e c y c l e d . Zone I V i s o p t i o n a l . Zone I V removes s o l v e n t from s o l u t e A so t h a t t h e A p r o d u c t i s more concentrated.

T h i s reduces t h e l o a d on t h e downstream s e p a r a t o r s which

r e c o v e r s o l u t e from s o l v e n t .

For t h e moment we w i l l assume we can meet o u r

l i s t of seven e n g i n e e r i n g c o n d i t i o n s and t h a t we can u n i f o r m l y move t h e s o l i d s i n p l u g flow.

136

Solids

1 -

1 t IV

-A+S

I

-A

4- B

II -B+S

1"IT 1

Recycle

Solvent or Desorbent F i g . 3.3.

C o u n t e r - c u r r e n t system.

A schematic o f t h e expected s e p a r a t i o n i s shown i n F i g . 3.4.

Since t h e c o u n t e r - c u r r e n t process o p e r a t e s a t steady s t a t e , these r e s u l t s a r e c o n s t a n t f o r a l l times a f t e r t h e i n i t i a l s t a r t u p .

One way t o l o o k a t t h i s i s t h a t we

have t a k e n t h e d e s i r e d s e p a r a t i o n i n an e l u t i o n chromatograph ( F i g . 3.1) and " f r o z e n " i t i n t i m e and space by moving t h e p a c k i n g i n a downward p l u g flow. I f we d o t h i s p r o p e r l y none of t h e "dead" r e g i o n s ( 1 , 2 , 3 or 4 ) o c c u r and t h e r e m i x i n g r e g i o n , M, w i l l n o t o c c u r .

The n e t r e s u l t w i l l be a s h o r t e r column

u s i n g l e s s adsorbent, and l e s s s o l v e n t .

Thus t h e produc s w i l l be l e s s

d i l u t e d and downstream s e p a r a t o r s w i l l be s m a l l e r .

This saves b o t h c a D i t a l

and o p e r a t i n g expenses. The c o u n t e r - c u r r e n t process can be analyzed by a s i m p l e r e t e n t i o n argument

C61.

R e f e r r i n g t o F i g s . 3 . 3 and 3 . 4 . i t i s c l e a r t h a t we d e s i r e s o l u t e A t o

move up i n zone I and I 1 w h i l e s o l u t e B moves down i n these zones. separate t h e two s o l u t e s .

This w i l l

To r e c y c l e s o l i d s we need t o have s o l u t e B desorbed

i n zone 111 and move up i n t h i s zone.

To r e c y c l e s o l v e n t , s o l u t e A must be

136

t

CONC.

Solven

Schematic o f c o n c e n t r a t i o n p r o f i l e s i n s i d e a c o u n t e r - c u r r e n t

F i g . 3.4.

separator.

s o r b e d i n zone I and move down i n t h i s zone.

These c o n d i t i o n s c a n b e w r i t t e n

as.

> O "LA

(3.1)

cc4

and

> o

> 0 > P B PBcc3

>

ccl

%

(3.2)

i s t h e v e l o c i t y o f s o l u t e A i n zone I o f t h e c o u n t e r c u r r e n t

where

s y s t e m and so f o r t h .

For l i n e a r isotherms these v e l o c i t i e s a r e e a s i l y

d e t e r m i n e d from t h e r e t e n t i o n t i m e s o b t a i n e d i n e l u t i o n c h r o m a t o g r a p h y .

I n an

e l u t i o n chromatograph t h e s o l u t e v e l o c i t i e s a r e ,

(3.3) L

where

t Ri i s t h e r e t e n t i o n t i m e and

L i s t h e column l e n g t h .

This solute

v e l o c i t y m u s t be d e t e r m i n e d a t t h e same r e l a t i v e i n t e r s t i t i a l f l u i d v e l o c i t y

with respect to the s o l i d .

The a p p r o p r i a t e f l u i d v e l o c i t y c a n be d e t e r m i n e d

as,

V

=

Vsuper E

+ Vsolid

(3.4)

137

where v is the interstitial fluid velocity with respect to the solid, E is the porosi tY, Vsuper is the superficial fluid velocity, and Vsolid is the solids velocity. If K i values are known, we can also calculate the solute velocities in elution chromatography from,

(3.5) where Ki is the distribution constant, Vs is the volume of stationary phase and Vm is the volume of mobile phase. An observer will see a solute velocity 'cci

=

i'

-

'solid

(3.6)

in the counter-current system. Equations (3.1) and (3.2) can be satisfied by varying fluid velocities in the different zones, changing the K i values with changing desorbent concentrations or both. Since the feed is input between zones I and 11, we must have v I > vII. If the selectivity is close to 1.0 it is difficult t o simultaneously have 'Acc2 > 0 and 'Bccl c 0. Thus relatively high selectivities are necessary for the counter-current system if we desire a significant feed rate. For a given feed rate appropriate solvent and product rates can be calculated so that equations (3.1) and ( 3 . 2 ) are satisfied. Note that Fig. 3.4 has no "dead" regions. If we could make an ideal counter-current system with no zone spreading, regions 3'. 3" and S would all become vertical lines. In this ideal case each packed region could be very thin without affecting the feed rate. The theoretical maximum ratio for feedlsolvent is 1.0 for linear systems when desorbent does not change K values. Somewhat less than this is required to satisfy equation (3.1) and (3.2). Zone broadening increases the amount of adsorbent needed but will have little effect on the solvent requirements. The advantages o f the counter-current process are: 1 ) It is a steady state operation, not batch. Thus switching o f product streams is not required. 2 ) It is more efficient and thus uses less adsorbent and less solvent. 3 ) The downstream separators will be smaller and process less material since products are not as dilute. There are three major disadvantages. First, the counter-current process is a steady-state binary separator. If we desire to separately recover three

138

components two columns w i l l be r e q u i r e d .

We c o u l d t a k e A as one p r o d u c t and a

m i x t u r e of B and C as t h e o t h e r p r o d u c t i n t h e f i r s t column. m i x t u r e would be separated i n t h e second column.

Then t h e B-C

Second, t h e d e v i c e i s q u i t e

complex. F i n a l l y , i n a p r a c t i c a l sense u n t i l r e c e n t l y we d i d not know how t o move s o l i d s i n a u n i f o r m p l u g flow w i t h o u t m i x i n g . Thus developing a device w i t h reasonable HETP v a l u e s i s d i f f i c u l t .

Research on d e v e l o p i n g t r u l y

coun t e r-c urrent chromatography has been r e v i e w e d by Barker [ 7 1 , Rendell [81. Sussman and Rathore 191. Sussman 1103 and Barker e t a l . [11,121.

One

prom is ing method i s t o use t h e m a g n e t i c a l l y s t a b i l i z e d f l u i d i z e d bed system [131 developed by Exxon.

Moving bed systems have been e x t e n s i v e l y employed

f o r i o n exchange [14,151,

b u t many fewer stages a r e r e q u i r e d .

The problem o f moving t h e s o l i d has been overcome by u s i n g packed beds and s i m u l a t i n g c o u n t e r - c u r r e n t movement.

S i m u l a t e d moving bed (SMB) systems a r e

i n i n d u s t r i a l use and w i l l be d i s c u s s e d n e x t . 3 . 4 SIMULATED MOVING BED SYSTEMS

The idea of s i m u l a t i n g c o u n t e r - c u r r e n t m o t i o n by changing t h e l o c a t i o n o f feed and pro duc t streams i s an o l d i d e a t h a t can be t r a c e d a t l e a s t as f a r back as t he Shanks system f o r l e a c h i n g soda ash i n England d u r i n g t h e 1840's [ r e f . 161.

T his s i m u l a t e d c o u n t e r - c u r r e n t system i s s t i l l used f o r l e a c h i n g

[161, f o r a d s o r p t i o n and i o n exchange systems t o remove one or more s o l u t e s 117,181. and for chromatographic s e p a r a t i o n s .

uct

t , to t z F i g . 3.5.

t 2

to t a

t 3 to

t4

Method of s i m u l a t i n g c o u n t e r - c u r r e n t m o t i o n

139 The method f o r s i m u l a t i n g a moving bed (SMB) i s

l l u s t r a t e d i n f i g . 3.5.

A

s e r i e s o f packed beds a r e arranged w i t h plumbing i n between s e c t i o n s t o a l l o w a d d i t i o n or w i t h d r a w a l o f m a t e r i a l between each sec i o n . i n s i d e a s i n g l e column o r w i t h a s e r i e s o f columns.

T h i s can be done

Every few minutes t h e

l o c a t i o n o f a l l p r o d u c t s and feeds a r e moved u p one s e c t i o n ( i n t h e d i r e c t i o n o f f l u i d flow).

I n F i g . 3.5 t h i s o c c u r s a t t i m e s tl,t2.t3.t4 e t c .

An

o b s e r v e r a t t h e p r o d u c t p o r t would see s o l i d s move downwards a t these t i m e s w h i l e f l u i d c o n t i n u o u s l y goes upward.

Thus t h i s o b s e r v e r sees an i n t e r m i t t e n t

c o u n t e r - c u r r e n t flow o f s o l i d s and f l u i d .

Since the s o l i d s r e a l l y are

s t a t l o n a r y , t h i s i s a s i m u l a t e d c o u n t e r - c u r r e n t or s i m u l a t e d moving bed (SMB) process.

To s i m u l a t e t h e system shown i n F i g . 3.3 a r e c y c l e l i n e connects t h e

t o p and b o t t o m of t h e column.

When a g i v e n p o r t l o c a t i o n reaches t h e t o p of

t h e column, i t switches t o t h e bottom.

The apparatus i s t r e a t e d l i k e a l o o p

or donut.

How c l o s e i s t h i s s i m u l a t i o n t o a t r u l y c o u n t e r - c u r r e n t system?

Liapis

and R i p p i n [181 s t u d i e d t h e q u e s t i o n t h e o r e t i c a l l y and f o u n d t h a t w i t h f o u r packed s e c t i o n s i n a s i n g l e zone t h e SMB i s a b o u t 98% as e f f i c i e n t as a t r u l y c o u n t e r - c u r r e n t process f o r t h e chemical system t h e y s t u d i e d . The modern a p p l i c a t i o n o f s i m u l a t e d counter c u r r e n t t e c h n i q u e s t o l i q u i d chromatography u s i n g t h e systems shown i n F i g . 3.3 and 3.5 was f i r s t developed by Broughton and h i s coworkers a t U n i v e r s a l O i

Products 119-311.

This

s i m u l a t e d c o u n t e r - c u r r e n t process (SORBEX) was f i r s t c o m m e r c i a l i z e d f o r s e p a r a t i o n o f 1 i n e a r p a r a f f i n s f r o m branched c h a i n and c y c l i c hydrocarbons (Molex) u s i n g 5A m o l e c u l a r s i e v e s C19-211.

O t h e r commercial a p p l i c a t i o n s

i n c l u d e p u r i f i c a t i o n o f p-xylenes (Parex) 1231, o l e f i n s e p a r a t i o n (Olex) 1221, and s e p a r a t i o n o f f r u c t o s e f r o m g l u c o s e (Sarex) [26-281.

Pilot plant

s e p a r a t i o n s have been demonstrated f o r : p a r a and meta c r e s o l f r o m an isomer m i x , p-cymene

or m-cymene from an isomer m i x t u r e , ethylbenzene from

C8-aromatics,

b u t e n e - I from C4 o l e f i n - p a r a f f i n m i x t u r e ;

p - d i i s o p r o p y l b e n z e n e f r o m an isomer m i x t u r e , O-pinene from a p i n e n e m i x t u r e and p-diethylbenzene from an isomer m i x t u r e [25,29,301.

A l l of these studies

a r e reviewed by de Rosset e t a l . 1311. The U n i v e r s a l 011 p r o d u c t s systems use a s p e c i a l l y designed r o t a r y v a l v e t o c o n t r o l l i q u i d f l o w s to and from t h e column.

Other m a n i f o l d systems can be used.

The types o f s e p a r a t i o n which a r e achieved a r e i l l u s t r a t e d i n F i g s . 3 . 6 and 3.7.

F i g . 3.6 shows p i l o t - p l a n t s c a l e r e s u l t s f o r t h e commercial Sarex system

140

[281. On a d r y s o l i d s b a s i s , t h e f e e d was 41.3 w t . % f r u c t o s e , t h e f r u c t o s e p r o d u c t i s 91 w t . % f r u c t o s e and 91.5% of t h e f r u c t o s e was r e c o v e r e d (remember o p e r a t i o n i s a t steady s t a t e ) .

I n F i g . 3.6 t h e more s t r o n g l y

adsorbed f r u c t o s e moves down t h e column i n zones I and I 1 and up t h e column i n zone 111.

D e i o n i z e d water was t h e s o l v e n t and a m o l e c u l a r s i e v e z e o l i t e was

t h e adsorbent. F i g . 3.7 shows p i l o t p l a n t s c a l e r e s u l t s f o r p u r i f i c a t i o n o f para-xylene f r o m a mixed C8-aromatic feed.

[251.

The p-xylene was most s t r o n g l y adsorbed

The l i m i t i n g s e l e c t i v i t y i n F i g . 3.7 was 1 . 6 between p-xylene and e t h y l

benzene.

Commercial systems a r e more s e l e c t i v e .

A system w i t h 24 small

chromatographic columns was used t o s i m u l a t e c o u n t e r - c u r r e n t m o t i o n .

de

Rosset e t a l . C251, used e l u t i o n chromatography r e s u l t s t o p r e d i c t o p e r a t i n g c o n d i t i o n s f o r t h e SMB system. used as desorbents.

Commercially t o l u e n e or p - d i e t h y l b e n z e n e a r e

A t y p i c a l SMB system f o r p-xylene p u r i f i c a t i o n would

produce 100,000 m e t r i c t o n s l y e a r o f p-xylene p r o d u c t of g r e a t e r t h a n 99.3% p u r i t y and a r e c o v e r y f r o m 96.7 t o 99.7% (depending on t h e desorbent u s e d ) . These a r e l a r g e chromatographic systems!

-

t

0 FRUCTOSE A DEXTROSE

2 0

E

P 5

8n

33

I

ZONE IV

F i g . 3.6.

Compositions i n column from SareX p i l o t p l a n t ( F i g . 8 from N e u z i l and Jensen. r e f . 2 8 ) .

141

OJ -

Q

f

0

v,

+ 0

c

C

W 0 L

a"

0

I

R

Fig. 3 . 7 .

2

4

6

0

F Sample Serial

E

D

Purification of p-xylene from mixed C aromatic feed in Parex a pilot plant (Fig. 6 from de Rosset et al., ref. 25).

Broughton et al. [231, used a staged model to analyze the Parex system. They predicted that the SMB needs only 1125 of the adsorbent inventory o f an elution chromatographic system, and 112 the desorbent is required. This latter requirement is important since it means that downstream distillation columns will be significantly smaller. Exact details of the elution chromatography system they were comparing the SMB to were not given. It apparently did not utilize recycle. An optimized elution chromatograph will be much closer to the SMB system. This is not surprising since the SMB can be considered as a complicated application of column switching and recycle techniques. Unfortunately, the SMB process and the elution chromatography system o f Seko et al. [41, cannot be directly compared since different adsorbents were used.

One group, de Rosset et al. [25, 29, 311, reported on the purification o f ethylbenzene from mixed xylenes in a pilot plant system. This study is of interest since it is the first published study where the least adsorbed material is the main product. Compared to elution chromatography the authors expected to need 114 as much adsorbent and 1/2 as much desorbent. A simple retention argument [61 can be used to analyze the SMB process shown in Figs. 3.3 and 3.5. Now the solids d o not really move. Instead the

142 average v e l o c i t y o f p o r t movement i s t h e i m p o r t a n t v a r i a b l e .

This i s ,

pport = ’ p o r t / tp o r t

(3.7)

where 1

p o r t i s t h e p a c k i n g h e i g h t between p o r t s and tporti s t h e t i m e between switches of p o r t s . To have s o l u t e A c o n c e n t r a t e between zone I and I V ( s e e F i g . 3.3) we want:

(3.8) where t h e s o l u t e v e l o c i t i e s can be determined from r e t e n t i o n times d e t e r m i n e d by e l u t i o n chromatography as i n eqns. ( 3 . 3 ) or ( 3 . 5 ) . f o r t h e slower moving s o l u t e s we want t o have t h e s o l u t e e f f e c t i v e l y move down i n zone I and 11 and up i n 111.

pB3

These c o n d i t i o n s a r e ,

’ p p o r t ’ pB1 ’ pB2

(3.9)

The h i g h e r t h e s e l e c t i v i t y t h e e a s i e r i t w i l l be t o s i m u l t a n e o u s l y s a t i s f y e q u a t i o n s ( 3 . 8 ) and ( 3 . 9 ) .

Equations ( 3 . 8 ) and ( 3 . 9 ) a r e e s s e n t i a l l y t h e same

c o n d i t i o n s as equations (3.1) and ( 3 . 2 ) .

A more d e t a i l e d t h e o r y i n c l u d i n g

mass t r a n s f e r e f f e c t s i s p r e s e n t e d by Ruthven [321. Many o t h e r a p p l i c a t i o n s o f SMB systems have been s t u d i e d .

Ishikawa e t a l .

[331 and H i r o d a and Shioda 1341 developed SMB systems f o r s e p a r a t i o n s o f f r u c t o s e and glucose.

These systems use o n l y one column per each zone t o

approximate t h e system shown i n f i g . 3 . 3 .

S i n c e each zone i s n o t a c l o s e

a p p r o x i m a t i o n t o c o u n t e r - c u r r e n t m o t i o n , these systems a r e designed f o r o n l y p a r t i a l s e p a r a t i o n o f the sugars. commercially.

Techniques s i m i l a r t o these a r e used

In a s e r i e s o f papers Barker and h i s coworkers s t u d i e d

a p p l i c a t i o n s of SMB methods t o g a s - l i q u i d chromatography [35-361. The SMB process has a l s o been extended t o s i z e e x c l u s i o n chromatography b y Barker e t a l . [11,121.

They used t e n 5.1 cm I.D. by 70 cm l o n g g l a s s columns

packed w i t h 200-400 mm S p h e r o s i l XOB075 t o o b t a i n narrow m o l e c u l a r w e i g h t f r a c t i o n s of D e x t r a n 40.

The d e x t r a n C o n c e n t r a t i o n p r o f i l e a l o n g t h e e n t i r e

l e n g t h o f t h e apparatus i s shown i n f i g . 3.8 [ r e f . 121. concentration e x l s t s i n the center sections. glcm3 and t h e f e e d flow r a t e was 7.9 cm 3 /min. times t h e f e e d f l o w r a t e . and 27000 f o r p r o d u c t 2.

Note t h a t a v e r y h i g h

The f e e d c o n c e n t r a t i o n was 0 . 2 1 E l u e n t f l o w r a t e was 5.66

The mean m o l e c u l a r w e i g h t was 95000 for p r o d u c t 1 . S e p a r a t i o n was n o t complete and t h e r e was

c o n s i d e r a b l e o v e r l a p i n t h e m o l e c u l a r weight d i s t r i b u t i o n s of t h e p r o d u c t s .

143

I Feed

t-

-

t

0.5 0.6

F7

E

-:: 0 2 0 0 C

f

s

016-

.-

c C

5

; 0.12 -

v)

W

0

u

5

c X

0.08

P

-

a

(I04 010

v

I

1

I

70

140

210

Product 1

F i g . 3.8.

I

I

1

I

280

350

I

420

I

490

Distance from Product 1 Outlet (cm)

630 &

560

700

Prodict 2

D e x t r a n c o n c e n t r a t i o n a n d mean Kd v a l u e s f o r s i z e e x c l u s i o n SMB (Fig. 7 f r o m Barker e t a l . , r e f . 1 2 ) .

B e f o r e l o o k i n g a t o t h e r a l t e r n a t i v e s , i t w i l l be h e l p f u l t o summarize a d v a n t a g e s and d i s a d v a n t a g e s o f SMB systems compared t o e l u t i o n chromatography.

I d e a l l y , t h e SMB s y s t e m does not have r e g i o n s where n o

separation i s occurring.

Thus t h e SMB uses l e s s a d s o r b e n t , l e s s d e s o r b e n t a n d

operates a t higher solute concentrations.

The p r o c e s s g i v e s a s t e a d y s t a t e

product.

The SMB p r o c e s s has been u s e d c o m m e r c i a l l y i n v e r y l a r g e s c a l e

systems.

D i s a d v a n t a g e s a r e t h a t t h e SMB i s c o n s i d e r a b l y more complex t h a n

e l u t i o n chromatography.

S i n c e we s t i l l must meet o u r l i s t o f s e v e n d e s i g n

c r i t e r i a , considerable care i s r e q u i r e d i n d e s i g n i n g the system to p r e v e n t e x c e s s i v e m i x i n g between segments o f columns or between c o l u m n s . l a r g e s e l e c t i v i t y i s r e q u i r e d for easy o p e r a t i o n . binary separator. mixed C

8

A fairly

The m e t h o d i s e s s e n t i a l l y a

I f we d e s i r e d p u r e p - x y l e n e a n d p u r e e t h y l b e n z e n e from a

f e e d ( s e e F i g . 3 . 7 ) two c o m p l e t e SMB s y s t e m s w o u l d b e r e q u i r e d .

When s h o u l d one p l a n t o use e l u t i o n c h r o m a t o g r a p h y and when s h o u l d o n e u s e an SMB?

N a t u r a l l y , t h e answer depends o n e c o n o m i c s , b u t some g u i d e l i n e s c a n

be p r o p o s e d .

I n r e l a t i v e l y s m a l l s c a l e systems t h e e l u t i o n c h r o m a t o g r a p h ' s

s i m p l i c i t y seems t o make i t t h e p r o c e s s o f c h o i c e .

I n l a r g e s c a l e systems

where o n l y one p u r e component i s d e s i r e d and i t i s e i t h e r m o s t s t r o n g l y or l e s s s t r o n g l y s o r b e d t h e SMB w i l l p r o b a b l y be c h e a p e r .

Currently, the choice

f o r i n t e r m e d i a t e s c a l e systems and for c e n t e r c u t s i n i n t e r m e d i a t e a n d l a r g e

s c a l e systems i s n o t c l e a r .

144 3.5

HYBRID METHODS:

COMBINE SMB AND ELUTION CHROMATOGRAPHY

The l i s t o f a d v a n t a g e s and d i s a d v a n t a g e s show t h a t t o a l a r g e e x t e n t e l u t i o n c h r o m a t o g r a p h y and SMB c h r o m a t o g r a p h y a r e c o m p l e m e n t a r y . c a n ' t do the o t h e r can.

What o n e

Thus i t i s r e a s o n a b l e t o e x p l o r e t h e p o s s i b i l i t y o f

c o m b i n i n g t h e s e two methods.

Wankat and h i s c o w o r k e r s [ 3 7 - 4 2 1 have e x p l o r e d

some o f t h e s e h y b r i d p o s s i b i l i t i e s . M o v i n g f e e d p o i n t c h r o m a t o g r a p h y [37-40.421 u s e s t h e a p p a r a t u s shown i n F i g . 3.9.

S o l v e n t i s c o n t i n u o u s l y f e d t o t h e b o t t o m of t h e s y s t e m .

The

system o p e r a t e s as a n e l u t i o n c h r o m a t o g r a p h e x c e p t t h e l o c a t i o n t o i n p u t t h e

t

Products

Feed

Solvent F i g . 3.9.

Apparatus for moving feed p o i n t chromatography

145

f e e d i s moved up t h e column w h i l e t h e f e e d i s added t o t h e system. f e e d p u l s e , e l u t i o n development i s used. p o r t movement i s g i v e n by eqn ( 3 . 7 ) .

A f t e r the

The average v e l o c i t y of t h e f e e d

For a b i n a r y system t h i s average f e e d

p o r t v e l o c i t y shou1.d l i e between t h e two s o l u t e v e l o c i t i e s , (3.10) w i t h more t h a n two s o l u t e s e q u a t i o n (3.10) can be s a t i s f i e d f o r t h e most d i f f i c u l t separations.

S a t i s f y i n g eqn (3.10) w i l l m i n i m i z e u n p r o d u c t i v e r e g i o n

2 and reduce r e g i o n 1 i n F i g . 3.1.

The s o l u t e bands w i l l be more c o n c e n t r a t e d

and t h e p r o d u c t w i l l e x i t sooner s i n c e t h e l a s t p a r t o f t h e f e e d i s i n p u t h i g h e r i n t h e column.

The i n i t i a l t h e o r e t i c a l p r e d i c t i o n s were done by Wankat

[371 and was extended t o two-dimensional

systems [381.

McGary and Wankat C401 s t u d i e d t h e s e p a r a t i o n o f naphthalene, anthracene and pyrene w i t h a 80-100 mesh p o l y v i n y l p y r o l l i d o n e r e s i n u s i n g 2-propanol a s t h e solvent.

The system used f o u r columns 5 cm l o n g f o l l o w e d by a 30.4 cm column.

R e s u l t s f o r normal e l u t i o n chromatography and f o r t h e moving f e e d s y s t e m a r e shown i n F i g . 3.10.

Note t h a t t h e moving f e e d p o i n t system has n a r r o w e r , more Also,

c o n c e n t r a t e d peaks w i t h b e t t e r r e s o l u t i o n between d i f f e r e n t components.

t h e pyrene e x i t s t h e column e a r l i e r which would a l l o w t h e n e x t peak t o be i n p u t sooner.

I n F i g . 3.10 t h e d o t t e d l i n e i s t h e t r a c e o f t h e UV r e c o r d e r .

reduced c o n c e n t r a t i o n i s C/C,

where C,

The

i s the concentration o f solute

a c t u a l l y e n t e r i n g t h e column ( a f t e r m i x i n g w i t h s o l v e n t ) .

Since solute f e d a t

d i f f e r e n t times can o v e r l a p , t h e reduced c o n c e n t r a t i o n can be g r e a t e r t h a n 1 . 0 i n F i g . 3.10b.

For i d e n t i c a l r e s o l u t i o n s an i n c r e a s e i n t h r o u g h p u t f r o m 90 t o

300% was observed f o r themoving f e e d p o i n t system compared to e l u t i o n chromatography. systems.

Recycle was n o t used, b u t c e r t a i n l y c o u l d be used w i t h b o t h

A l i n e a r d i s p e r s i o n model was used t o model t h e systems.

With one

a d j u s t a b l e parameter ( P e c l e t number, HETP or number o f s t a g e s ) agreement between t h e o r y and experiment was good.

The chromatogram shown i n F i g . 3.10b

i s t h e sum o f t h e Gaussian peaks f o r each f e e d l o c a t i o n i n F i g . 3 . 9 . Very r e c e n t l i n e a r and n o n l i n e a r e q u i l i b r i u m model s i m u l a t i o n s [ 4 2 1 showed t h a t e s s e n t i a l l y t h e same improvement can be o b t a i n e d by v e r y r a p i d l y a d d i n g a l l f e e d t o t h e b o t t o m o f t h e column.

For example, for t h e system shown i n

F i g . 3.9 a l l of t h e f e e d would be i n t r o d u c e d i n t o t h e bottom o f t h e column i n t h e same t i m e p e r i o d as one of t h e 5 f e e d p u l s e s . t h a t i n F i g . 3.9 b u t f o r 115 t h e t i m e .

The feed r a t e i s 5 t i m e s

I g n o r i n g mass t r a n s f e r and d i s p e r s i o n ,

t h i s simple " s h o r t p u l s e " method g i v e s e s s e n t i a l l y t h e same r e s u l t s as an

A

B

1.0

F i g . 3.10. Chromatograms f o r s e p a r a t i o n o f n a p h t h a l e n e , anthracene and pyrene

for 2 5 m i n u t e f e e d p u l s e s .

A . E l u t i o n chromatography (Same as F i g .

6 from McGary and Wankat, r e f . 4 0 ) .

8 . Moving f e e d p o i n t

chromatography (Same as F i g . 7 from McGary and Wankat, r e f . 4 0 ) Dotted l i n e i s t r a c e o f U . V . recorder.

147 o p t i m i z e d moving f e e d . due t o r e d u c e d d i l u t i o n .

Thus t h e improvement seen i n m o v i n g f e e d a p p e a r s t o be A l t h o u g h t h e r e may b e d i f f i c u l t i e s w i t h p r e s s u r e

d r o p , t h e " s h o r t p u l s e " method i s s i m p l e r and i s recommended. The m o v i n g f e e d p o i n t s y s t e m r e d u c e s u n p r o d u c t i v e r e g i o n s 1 a n d 2 i n F i g . 3 . 1 . The SMB i s a b l e t o d o t h i s p l u s remove u n p r o d u c t i v e r e g i o n s 3 and 4 a n d f u r t h e r reduce r e g i o n 1 .

The SMB does t h i s b y m o v i n g b o t h f e e d a n d p r o d u c t l i n e s .

It

makes sense t o t r y m o v i n g t h e p r o d u c t w i t h d r a w a l l i n e s i n a n e l u t i o n chromatography system. s w i t c h i n g methods.

T h i s can be c o n s i d e r e d a s a n a d a p t a t i o n o f c o l u m n

T h e o r e t i c a l c a l c u l a t i o n s for a s i n g l e case for the

a n t h r a c e n e , n a p h t h a l e n e , p y r e n e s y s t e m s p r e d i c t e d a 69.5% i n c r e a s e i n t h r o u g h p u t w i t h t h e same r e s o l u t i o n f o r a m o v i n g p r o d u c t w i t h d r a w a l c h r o m a t o g r a p h compared t o e l u t i o n c h r o m a t o g r a p h y [ 4 1 1 .

Nonlinear simulations

[ 4 2 1 showed s i m i l a r or g r e a t e r i n c r e a s e s i n t h r o u g h p u t p l u s l e s s d i l u t i o n o f s l o w m o v i n g components.

M o v i n g w i t h d r a w a l i s m o s t u s e f u l for f e e d s w i t h a v e r y

b r o a d r a n g e o f a f f i n i t i e s , and i s one way o f s o l v i n g t h e " g e n e r a l e l u t i o n problem".

The methods have n o t been t r i e d e x p e r i m e n t a l l y .

The h y b r i d p r o c e s s e s a r e n o t n e a r l y as d e v e l o p e d as e l u t i o n c h r o m a t o g r a p h y and SMB s y s t e m s .

However, s i n c e t h e h y b r i d t e c h n i q u e s a r e not l i m i t e d t o

b i n a r y systems and a r e more e f f i c i e n t t h a n e l u t i o n c h r o m a t o g r a p h y , one or more o f t h e h y b r i d methods may become u s e f u l f o r l a r g e - s c a l e c h r o m a t o g r a p h y .

The

l i k e l y s i t u a t i o n s where t h e h y b r i d s y s t e m s w i l l be u s e d a r e f o r i n t e r m e d i a t e s i z e s and for c e n t e r c u t s i n i n t e r m e d i a t e and l a r g e s c a l e s e p a r a t i o n s . 3.6

OTHER METHODS FOR LARGE-SCALE CHROMATOGRAPHY

A v a r i e t y o f o t h e r o p e r a t i n g methods have b e e n s t u d i e d for u s e i n

large-scale chromatography. these approaches.

I n t h i s s e c t i o n we w i l l b r i e f l y r e v i e w some o f

More c o m p r e h e n s i v e r e v i e w s o n t h e s e methods h a v e been

p u b l i s h e d [7-11,43,441. C e n t r i f u g e s have been u s e d f o r medium s c a l e c h r o m a t o g r a p h i c s y s t e m s [8,45-481.

A l a y e r o f p a c k i n g i s l a i d down i n a b a s k e t t y p e c e n t r i f u g e .

E l u t i o n development i n t h e r a d i a l d i r e c t i o n i s used.

The a d v a n t a g e o f t h e s e

s y s t e m s i s t h a t a r e l a t i v e l y l a r g e t h r o u g h p u t c a n be o b t a i n e d i n a s m a l l s y s t e m . Continuous, steady, s t a t e two-&imensional a n n u l u s was f i r s t s u g g e s t e d b y M a r t i n [ 4 9 1 .

chromatography u s i n g a r o t a t i n g S i n c e t h e n , many p e o p l e h a v e

e x p l o r e d t h i s s y s t e m w i t h e i t h e r a r o t a t i n g a n n u l u s or a s e r i e s of c o l u m n s i n a

148 E a r l y e x p e r i m e n t a l equipment i s d i s c u s s e d by Svensson

G a t l i n g gun arrangement. e t a l . 1501.

A p p l i c a t i o n s f o r s i z e e x c l u s i o n chromatography a r e r e v i e w e d by

N i c h o l a s and Fox 1511.

A modern h i g h p r e s s u r e c o n t i n u o u s chromatograph was

developed by a group a t Oak Ridge [52,531. o f one-dimensional and two-dimensional [541.

The r e l a t i o n s h i p between a v a r i e t y

s e p a r a t i o n s was e x p l o r e d by Wankat

The r e v i e w s by Jamrack [431, B a r k e r [71, Rendell

[lo],

Sussman and

Rathore 191, and Sussman [ l o 1 d i s c u s s t h i s and o t h e r two-dimensional Various moving bed systems have been e x p l o r e d .

geometries.

Movement o f s o l i d s i s common

i n i o n exchange [14,151 and has been t r i e d f o r l i q u i d chromatography [7-101. G e n e r a l l y these methods have n o t been v e r y successful for l i q u i d chromatography because o f e x c e s s i v e m i x i n g and s o l i d s a t t r i t i o n . s o l i d i n a tube which r o t a t e s [7-121.

An a l t e r n a t e i s t o pack t h e

T h i s approach i s m e c h a n i c a l l y q u i t e

complex and a p p a r e n t l y has been made o b s o l e t e by t h e SMB.

Jamrack [431

discusses use of endless b e l t s which t r a v e l from one t a n k t o t h e o t h e r t o g i v e counter-current f l o w . A v e r y d i f f e r e n t approach t o l a r g e - s c a l e chromatography i s t o use changes i n

thermodynamic v a r i a b l e s such as temperature t o f o r c e t h e s e p a r a t i o n .

Rotating

h e a t e r s have been used i n gas chromatography i n t h e chromatothermagraphy process [55,561.

A v a r i e t y o f c y c l i c processes u s i n g changes i n a

thermodynamic v a r i a b l e such as p a r a m e t r i c pumping and c y c l i n g zone a d s o r p t i o n have been developed [431.

Most o f t h e processes r e m v e a l l s o l u t e s t o g e t h e r

a l t h o u g h multicomponent s e p a r a t o r s have been d e v i s e d 157,581.

A l l of these a l t e r n a t e processes can be made t o work.

However, t h e y have

been employed f o r o n l y s p e c i a l i z e d a p p l i c a t i o n s and have n o t become s t a n d a r d s e p a r a t i o n methods.

I n my o p i n i o n e l u t i o n p r e p a r a t i v e chromatography,

s i m u l a t e d c o u n t e r - c u r r e n t systems and h y b r i d s o f t h e s e two methods a r e much more l i k e l y t o be e x t e n s i v e l y used i n l a r g e - s c a l e a p p l i c a t i o n s . 3.7

ACKNOWLEDGMENT

T h i s work was p a r t i a l l y supported b y NSF g r a n t s CPE-8006903 and CPE-8211835.

T h i s a s s i s t a n c e o f Norman L1 and H a r r y P e t h e r t o n i n o b t a i n i n g

papers and p a t e n t s i s g r a t e f u l l y acknowledged.

149

3 . 8 NOMENCLATURE

solutes

A,B,C

d i s t r i b u t i o n c o e f f i c i e n t f o r s i z e e x c l u s i o n chromatography

Kd

s o l u t e conc. i n s t a t i o n a r y p h a s e l s o l u t e conc. i n m o b i l e phase

Ki

por t

p a c k i n g h e i g h t between p o r t s

L

column l e n g t h

N

number o f stages

t

time t i m e between s w i t c h e s o f p o r t s

:port Ri PA'"B'Vc'Pi Pcc

Ppor t

r et e n t i o n time s o l u t e v e l o c i t i e s , eqn ( 3 . 3 and 3 . 5 ) s o l u t e v e l o c i t i e s i n c o u n t e r - c u r r e n t systems, eqn ( 3 . 6 ) average v e l o c i t y o f p o r t s w i t c h i n g , eqn ( 3 . 7 ) i n t e r s t i t i a l f l u i d v e l o c i t y w i t h respect to s o l i d

V

"super "sol i d s

superficial f l u i d velocity superficial solids velocity volume m o b i l e phase

"H

volume s t a t i o n a r y phase

"S

a x i a l d i s t a n c e i n column

2 aBA

s e l e c t i v i t y = K /K B A

E

porosity

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2. 3. 4. 5. 6. 7.

Spedding, F . H . , E . I.Fulmer, T . A . B u t l e r , E . M . Gladrow, M . Gobush, P. E . P o r t e r , J . E . Powell and J . M . W r i g h t , The S e p a r a t i o n o f Rare E a r t h s by I o n Exchange. 111. P i l o t P l a n t Scale S e p a r a t i o n s , J . Am. Chem. SOC., 69, 2812 (1947). Shearon, W . H . and 0. F . Gee, Carotene and C h l o r o p h y l l - Commercial Chromatographic P r o d u c t i o n , I n d . Eng. Chem., 42, 218 (1949). Ek. L . , Process B i o c h e m i s t r y , 3 ( 9 ) . 25 ( 1 9 6 8 ) . Seko, M . , H . Takeuchi and T.Inada, Scale-up f o r Chromatographic S e p a r a t i o n o f p-xylene and ethylbenzene. I n d . Eng. Chem. Prod. Res. Develop., 21, 656 ( 1 9 8 2 ) . H e i k k i l a , H . , S e p a r a t i n g Sugars and Amino Acids With Chromatography , Chemical E n g i n e e r i n g , 50 (Jan. 24, 1983). Wankat, P. C . , O p e r a t i o n a l Techniques f o r A d s o r p t i o n and I o n Exchange , Proceedings Corn R e f i n e r s A s s o c i a t i o n 1982 S c i e n t i f i c Conference, L i n c o l n s h i r e , I L , June 16-18, 1982, p. 119-167. B a r k e r , P.E., Continuous Chromatographic R e f i n i n g i n E . 5. P e r r y and C . J . Van O s s ( e d s . ) , Progress i n S e p a r a t i o n and P u r i f i c a t i o n , v o l . 4 , Wiley, NY 1971, p, 325.

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R e n d e l l , M . , The Real F u t u r e f o r Large-Scale Chromatography , Process E n g i n e e r i n g , ( A p r i l 1975)., p. 66. Sussman, M. V. and R . N . S . Rathore, Continuous Modes o f Chromatography , 9. Chromatographia, 8 , 55 ( 1 9 7 5 ) . 10. Sussman, M . V . , Continuous Chromatography , Chemtech, 6. 260 ( 1 9 7 6 ) . 1 1 . B a r k e r , P. E . . F. J . E l l i s o n and B. W . H a t t , Continuous Chromatography o f Macromolecular S o l u t e s , i n R . Epton ( e d . ) , Chromatography o f S y n t h e t i c and B i o l o g i c a l Polymers, v o l . 1 , E l l i s Horwood, C h i c h e s t e r Eng., 1978, p . 2 18-239. 12 B a r k e r , P. E . , F . J . E l l i s o n and B. W. H a t t , A New Process f o r t h e Continuous F r a c t i o n a t i o n of D e x t r a n , I n d . Eng. Chem. Process Des. Develop., 1 7 , 302 (1978). 13. Rosensweig, R . E., Magnetic S t a b i l i z a t i o n o f t h e S t a t e o f U n i f o r m F l u i d i z a t i o n , I n d . Eng. Chem. Fundam., 18, 260 (1979). 14. S l a t e r , M. J . , Recent I n d u s t r i a l - S c a l e A p p l i c a t i o n s o f Continuous Resin I o n Exchange Systems , J . Separ. P r o c . T e c h n o l . , 2, ( 3 ) 2 (1981). 15. S t r e a t , M . Recent Developments i n Continuous I o n Exchange , J . Separ. Proc. T e c h n o l . , 1, ( 3 ) 10 (1980). 16. T r e y b a l , R. E . , Mass T r a n s f e r O p e r a t i o n s , 3 r d ed., McGraw-Hill, NY, 1980, c h a p t . 13. 17. K i n g , C. J . , S e p a r a t i o n Processes, 2nd ed., McGraw-Hill. NY, 1980, p . 172-175. 18. L i a p i s , A. I. and D.W.T. R i p p i n , The S i m u l a t i o n o f B i n a r y A d s o r p t i o n i n Continuous C o u n t e r - c u r r e n t O p e r a t i o n and a Comparison W i t h O t h e r O p e r a t i n g Modes , AIChE J o u r n a l , 25, 455 ( 1 9 7 9 ) . 19. Broughton, D . B., and D. B . Carson, The Molex Process , P e t r o l e u m R e f i n e r , 38, ( 4 ) 130 (1959). 20. Broughton. D. B. and C. G . Gerhold, Continuous S o r p t i o n Process Employing F i x e d Beds o f Sorbent and Moving I n l e t s and O u t l e t s , U.S. P a t e n t No. 2,985,589, May 23, 1961. 21. Broughton, D . B . , Molex: Case H i s t o r y o f a Process , Chem. Eng. P r o g . , 64, ( 8 ) 60 (1968). 22. Broughton. D. B. and R. C . Berg, O l e f i n s by D e h y d r o g e n a t i o n - E x t r a c t i o n , Hydrocarbon Processing, 48, ( 6 ) 115 (1969). 23. Broughton. D. B., R. W . N u e z i l , J . M. P h a r i s and C . S . B r e a s l e y , The Parex Process f o r Recovering Paraxylene , Chem. Eng. Prog. 6 6 ( 9 ) , 70 (1970). 24. F i c k e l , R. G . , Continuous A d s o r p t i o n - A Chemical E n g i n e e r i n g Tool i n G . H . Cummings ( e d . ) , AIChE Symposium S e r . , 69(135), 65 (1973). 25. de Rosset. A . J . , R . W.Neuzi1 and D. J . Korous, L i q u i d Column Chromatography as a P r e d i c t i v e Tool f o r Continuous C o u n t e r - c u r r e n t A d s o r p t i v e S e p a r a t i o n s , I n d . Eng. Chem. P r o c . Des. Develop., 15, 261 (1976). 26. B i e s e r , H . J . and A . J . de Rosset, Continuous C o u n t e r - c u r r e n t S e p a r a t i o n o f Saccharides w i t h I n o r g a n i c Adsorbents , D i e S t a r k e , 29, ( 1 1 ) 392 ( J a h r g 1977) ( I n E n g l i s h ) . 27 N e u z i l , R . W . and J . W . P r i e g n i t z . Process for Separ'ating a Ketose f r o m a Aldose by S e l e c t i v e A d s o r p t i o n , U . S . P a t e n t NO. 4,024,331. (May 1 7 , 1977). 28. N e u z i l , R . W . , and R . H . Jensen. Development o f t h e Sarex P r o c e s s f o r t h e S e p a r a t i o n o f Saccharides , paper 22d, AIChE m e e t i n g , P h i l a d e l p h i a , PA, (June 6, 1 9 7 8 ) . 29. de Rosset, A. J . , R. W. N e u z i l , D. G. T a j b l and J . M. Braband, S e p a r a t i o n o f Ethylbenzene from Mixed Xylenes by Continuous A d s o r p t i v e P r o c e s s i n g , Separat. S c i . Technology, 15, 637 (1980). 30. N e u z i l , R . W . , D. H. Rosback, R. H. Jensen, J . R. Teague and A . J . de Rosset, An Energy-saving S e p a r a t i o n Scheme , Chemtech, 10, 498 (Aug. 1980). 8

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31. de Rosset. A. J., R. W. Neuzil and D. B. Broughton, Industrial Applications of Preparative Chromatography , in A. E. Rodrigues and D. Tondeur (eds.), Percolation Processes, Theory and Applications, Sijthoff and Noordhoff, Alphen aan den Rijn, Netherlands 1981, p. 249-281. 32. Ruthven, D . M . , Principles of Adsorption and Adsorption Processes, Wiley-Interscience, N Y , 1984, Chapt. 12. 33. Ishikawa, H.. H. Tanabe and K. Usui, Process of the Operation of a Simulated Moving Bed , U.S. Patent No. 4,182,633 (Jan. 8, 1980). 34. Hiroda, T. and K. Shroda. A Method for the Elimination of Oligosaccharides , Japanese Patent Office Patent Journal, Kokai Patent No. SHO 55 El9801 - 48400, (April 7, 1980). 35. Barker, P. E. and R. E. Deeble, Production Scale Organic Mixture Separation Using a New Sequential Chromatographic Machine , Anal. Chem., 45, 1121 (1973). 36. Barker, P. E., M. I . Howari and G. A. Irlam, The Separation of Fatty Acid Esters by Continuous Gas-Liquid Chromatographic Refining , J. Separ. Proc. Technol., 2 (2), 33 (1981). 37. Wankat, P. C., Improved Efficiency in Preparative Chromatographic Columns Using a Moving Feed , Ind. Eng. Chem. Fundam., 16, 468 (1977). 38. Wankat. P. C., Increasing Feed Throughput in Preparative Two-Dimensional Separations , Separation Sci., 12, 553 (1977). 39. Wankat, P. C. and P. M. Ortiz, Moving Feed Point Gel Permeation Chromatography: An Improved Preparative Technique , Ind. Eng. Chem. Process Des. Develop., 21, 416 (1982). 40. McGary, R. H. and P. C. Wankat, Improved Preparative Liquid Chromatography: The Moving Feed Point Method , Ind. Eng. Chem. Fundamentals, 22, 10 (1983). 41. Wankat, P. C., Improved Preparative Chromatography: Moving Port Chromatography , Ind. Eng. Chem. fundamentals, 23, 256 (1984). 42. Geldart, R.W., N.H.L. Hang and P.C. Wankat, Non-Linear Analysis of Moving Withdrawal and Moving Port Chromatography , American Chemical Society Meeting, New York City, April 16. 1986. 43. Jamrack, W. D . , Rare Metal Extraction by Chemical Engineering Methods, Pe.rgarnon Press, NY. 1963, chapt. 3. 44. Wankat, P. C., Cyclic Separation Techniques , in A. E. Rodrigues and D . Tondeur (eds.), Percolation Processes, Theory and Applications, Sijthoff and Noordhoff, Alphen aan den Rijn, Netherlands, (19811, p. 443-516. 45. Hopf, P. P., Radial Chromatography In Industry , Ind. Eng. Chem.. 39, 938 ( 1947). 46. Mitchell, H. L . , W . G. Shrenk and R. E . Silker, Preparation o f Carotene Concentrates from Dehydrated Alfalfa Meal , Ind. Eng. Chem., 4 5 , 415 (1953). 47. Heftmann, E . , J.M. Krochta, D . F . Farkas and S. Schwimmer, The Chromatofuge, An Apparatus for Preparative Rapid Radial Column Chromatography , J. Chromatog., 66, 365 (1972). 48. Delaney, R.A.M., J . K. Donnelly and R. D. Kearney, Industrial Applications of Gel Filtration. 1 . Whey, Process Biochemistry. 8. (3) 13 (March 1973). 49. Martin, A.J.P., Summarizing Paper , Disc. faraday SOC.. 7, 332 (1949). 50. Svensson, H . , C.-E Agrell, S.-0. Dehlen and L. Hegdahl. An Apparatus for Continuous Chromatographic Separation , Science Tools, The LKB Instrument Journal, 2 , (2) 17 (1955). 51. Nicholas, R . S. and J . 6 . Fox, Continuous Chromatography Apparatus, 1 1 1 , Application , J . Chromatog.. 4 3 , 61 (1969). 52. Canon, R. M., J . M. Begovich and W. G. Sisson, Pressurized Continuous Chromatography , Separat. Sci, Technol., 15. 655 (1980). 53. Begovich, J . M., C. H . Byers and W. G. Sisson, A High Capacity Pressurized Continuous Chromatograph , Separ. Sci. Technol., 18, 1167 (1983).

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Wankat, P. C . , The R e l a t i o n s h i p Between One-Dimensional and Two-Dimensional S e p a r a t i o n Processes , AIChE J o u r n a l , 23, 859 (1977). Z h u k h o v i t s k i i , A . A . , Some Developments i n Gas Chromatography i n t h e Butterworth, USSR, i n Gas Chromatography 1960. R.P.W. S c o t t ( e d . ) . London, England 1960, p. 293. Tudge, A . P . , S t u d i e s i n Chromatographic T r a n s p o r t . 111 Chromathermography , Canadian J . P h y s i c s , 40, 557 ( 1 9 6 2 ) . F o o , S. C., K . H . Bergstrom and P. C. Wankat, Multicomponent F r a c t i o n a t i o n by D i r e c t , Thermal Mode C y c l i n g Zone A d s o r p t i o n , I n d . Eng Chem. Fundamental, 19, 86 (1980). Nu, Xiang-Zhi and P. C . Wankat, Continuous Multicomponent P a r a m e t r i c Pumping , I n d . Eng. Chem. Fundam., 22, 172 (1983).

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CHAPTER 4 PREPAflAT IVE t IQU ID CHROMATOGRAPHY IN THE PHAWCJlCEUTICAL INDUSTRY A. WehrIi Sandor Ltd, Pharmaceutical Division, P r e c l i n i c a l Research 4002 Basle (Switzerland)

4.1

INTRODUCTION

4.2

MAJOR AREAS OF PREPARATIVE LC APPLICATION I N PHARMACEUTICAL INDUSTRY 4.2.1 4.2.2 4.2.3 4.24

4.3

LC LC LC LC

In Searching New Pharmaca In Biological Testing In Testing S t a b i l i t i e s o f Drugs and Product ion

CHROMATOGRAPHIC SYSTEMS USED 4.3.1 4.3.2 4.3.3 4.3.4

4.4

Preparative Preparative Preparative Preparat ive

Adsorption Chromatography Bonded Phase Chromatography 4 . 3 . 2 . 1 Reversed Phase Chromatography 4 . 3 . 2 . 2 Normal Phase Chromatography Ion Exchange Chromatography Gel Permeation Chromatography and Other Techniques

APPARATUS AND EXAMPLES 4.4.1 4.4.2

4.4.3

Analytical Scale Preparative LC (mg-scale) 4 . 4 . 1 . 1 Apparatus 4 . 4 . 1 . 2 Example Laboratory Scale Preparative LC (9-scale) 4 . 4 . 2 . 1 Apparatus Columns and Equipment Constructed f o r 4.4.2.1.1 Separations up t o 50 Bars Columns and Equipment Constructed f o r 4.4.2.1.2 Separations between 50 and 100 Bars 4 . 4 . 2 . 2 Examples 4 . 4 . 2 . 3 Some Trends I n Laboratory Scale LC Repet i t i ve Cyc Ie Operat ion 4.4.2.3.1 4.4.2.3.2 Gradient Elution and Recycling Production Scale LC 4 . 4 . 3 . 1 Apparatus 4 . 4 . 3 . 2 Examples

4.5

SUMMARY

4.6

REFERENCES

154

4.1

INTRODUCTION The r e v i v a l o f l i q u i d chromatography b e g i n n i n g i n 1970, l e a d i n g t o HPLC.

induced the s t u d y o f p r e p a r a t i v e s e p a r a t i o n s and advanced w i t h i n r e c e n t y e a r s i n t o one o f t h e techniques most w i d e l y used i n t h e p h a r m a c e u t i c a l i n d u s t r y [ 1 , 2 1

I t accompanies a d r u g t h r o u g h i t s course o f m a n u f a c t u r e .

T h i s course i s a l o n g

one and i n c l u d e s t h e f o l l o w i n g s t e p s : o Search f o r and p r e p a r a t i o n of new compounds, accompanied by p u r i f i c a t i o n and measurement o f p h y s i c a l and chemical p r o p e r t i e s

o pharmacological t e s t s ( a c t i v i t y and t o x i c i t y ) o small s c a l e p r o d u c t i o n o measurement o f b i o c h e m i c a l parameters ( a d s o r p t i o n , d i s t r i b u t i o n , metabolism, e x c r e t i o n ) o volunteer t e s t s o f o r m u l a t i o n s t u d i e s and s t a b i l i t y t e s t s of t h e substance

o l i m i t e d c l i n i c a l t r i a l f o r comparison w i t h t h e b e s t e x i s t i n g remedies o p i l o t p l a n t and l a r g e s c a l e p r o d u c t i o n o finishing On t h i s r o u t e a n a l y t i c a l and p r e p a r a t i v e chromatography has r e c e n t l y been a p p l i e d t o the study o f numerous problems a s s o c i a t e d w i t h these s t e p s . 4 . 2 MAJOR AREAS OF PREPARATIVE LC APPLICATION IN PHARMACEUTICAL INDUSTRY 4 . 2 . 1 P r e p a r a t i v e LC I n Searching New Pharmaca New pha-maca a r e f o u n d by chemical d e r i v a t i z a t i o n o f known p h a r m a c e u t i c a l s

or by i s o l a t i o n o f n a t u r a l p r o d u c t s . chromatography.

Both t e c h n i q u e s need p r e p a r a t i v e l i q u i d

For pharmaceutical compounds, found by s y n t h e s i s o f chemical

d e r i v a t i v e s , p u r i t y i s advantageous.

I t i s a l s o governed by n a t i o n a l laws.

Therefore, s y n t h e s i s of pure p r o d u c t s p l a y a fundamental r o l e i n p h a r m a c e u t i c a l industry.

To a t t a i n t h i s g o a l , s i d e r e a c t i o n s and t h e s t r u c t u r e s o f

s i d e - p r o d u c t s should be known i n o r d e r t o o p t i m i z e t h e s y n t h e s i s .

For

s t r u c t u r a l i d e n t i f i c a t i o n t h e i s o l a t i o n o f t h e unknown b y - p r o d t r t s i s necessary.

T h i s i s p o s s i b l e by expanding a n a l y t i c a l LC t o t h e p r e p a r a t i v e

mg-scale 131.

When t h i s i s done, t h e i s o l a t i o n o f by-products and t h e i r

subsequent i d e n t i f i c a t i o n by q u a l i t a t i v e methods a r e v e r y e f f e c t i v e [ 4 1 .

155

The same p r e p a r a t i v e LC a p p r o a c h t h e c o l l e c t i o n o f m a t e r i a l ( m g - s c a l e ) f o r s t r u c t u r e i d e n t i f i c a t i o n or s t r u c t u r e e l u c i d a t i o n i s a p p l i e d i n r e s e a r c h l a b o r a t o r i e s when s e a r c h i n g f o r and i s o l a t i n g new s u b s t a n c e s from p l a n t s [ 5 1 , from f e r m e n t a t i o n b r o t h s [ 6 1 or from a n i m a l s C71.

Once t h e e n d - p r o d u c t has been i s o l a t e d or s y n t h e s i z e d , p h y s i c a l and c h e m i c a l p r o p e r t i e s must be a c c u r a t e l y measured and a n a l y t i c a l s t a n d a r d s e s t a b l i s h e d , so t h a t any s u b s e q u e n t b a t c h e s t h a t have t o be made, can be shown a s t o be o f comparable q u a l i t y .

T h e r e f o r e , q u a n t i t i e s i n t h e gram r a n g e a r e a l s o s e p a r a t e d

and p u r i f i e d b y p r e p a r a t i v e LC i n p h a r m a c e u t i c a l l a b o r a t o r i e s .

To a t t a i n s u c h

amounts, a p p a r a t u s , columns a n d s t r a t e g y h a v e t o be a d j u s t e d a c c o r d i n g t o t h e preparative requirements.

4 . 2 . 2 Preparative LC I n Biological Testing

A f t e r t h i s f i r s t s t e p o f r e s e a r c h , t h e compound i s p a s s e d o v e r t o t h e p h a r m a c o l o g i s t f o r a n i m a l t e s t i n g i n o r d e r t o a s s e s s t h e u t i l i t y o f new d r u g s , e s p e c i a l l y i n r e l a t i o n to e x i s t i n g comparable medicines.

Besides these

p h a r m a c o l o g i c a l t e s t s i t w i l l be n e c e s s a r y t o l o o k i n t o t h e b i o c h e m i s t r y a n d t o x i c o l o g y o f t h e compound.

T h i s means t h a t t h a t t h e a b s o r p t i o n , t h e

d i s t r i b u t i o n , t h e e x c r e t i o n and t h e m e t a b o l i c changes i n v i v o a r e o f f u n d a m e n t a l interest.

T h e r e f o r e , two m a i n q u e s t i o n s m u s t be answered:

1 ) how i s t h e

compound a l t e r e d b y t h e v a r i o u s enzyme s y s t e m s i n t h e b o d y a n d 2 ) a r e a n y o f t h e s e m e t a b o l i t e s t o x i c , i n a c t i v e or more a c t i v e t h a n t h e o r i g i n a l compound? These q u e s t i o n s c a l l for n g - i s o l a t i o n a n d f o r i d e n t i f i c a t i o n o f compound structure.

The c l a s s i c way o f d o i n g t h i s has u s u a l l y i n v o l v e d p r e p a r a t i v e

chromatography [El. 4 . 2 . 3 Preparative LC I n Testing S t a b i l i t i e s of Drugs

When t h e m e t a b o l i s m and t h e o t h e r a n i m a l e x p e r i m e n t s h a v e been s u c c e s s i v e l y c o m p l e t e d , t h e n e x t s t e p i s t h e b i g one o f t r a n s f e r r i n g t h e compound t o human pharmacology.

The e x p e r i m e n t s a r e u s u a l l y c a r r i e d out o n v o l u n t e e r s , r e c r u i t e d

from t h e s t a f f o f t h e Company or e l s e w h e r e .

A t t h i s stage, p r e l i m i n a r y

f o r m u l a t i o n s t u d i e s must be c a r r i e d o u t t o f i n d t h e m o s t s u i t a b l e form i n w h i c h t h e doses c a n be g i v e n t o t h e p a t i e n t .

T h i s work i n v o l v e s a t f i r s t s t u d i e s o f

s u b s t a n c e s t a b i l i t y i n t h e form chosen for a d m i n i s t r a t i o n d u r i n g t h e c l i n i c a l trial.

156

Such long-term studies of t h e stability o f t h e d r u g s and of t h e kinetic studies o n the decomposition process a r e important as is t h e separation and subsequent identification of decomposition products. For these purposes preparative liquid chromatography in t h e mg-range o f f e r s a rapid method for collecting fractions which contain unknown decomposition products. T h e s e fractions are used f o r further identification a s described in t h e previous section o n metabolites [91. 4 . 2 . 4 Preparative Liquid Chromatography and Production

When all these preclinical hurdles have been o v e r c o m e , t h e last phase of t h e work involving clinical trial, application in patients, and s t u d i e s of large scale production c a n be started. In this study the purification s t e p plays a central role and is a major segment of t h e chemical engineering field. T h e classical techniques such as distillation, extraction, crystallization, etc., are well established in the pharmaceutical industry. In addition t o these techniques. however, there a r e many o t h e r s . O n e o f t h e newer techniques, not so familiar in chemical engineering, is preparative liquid chromatography. It is especially used in large scale production o f drugs manufactured by f e r m e n t a t i o n processes [lo]. Reviewing this course of manufacturing a pharmaceutical product w e c a n conclude that preparative liquid chromatography f i n d s wide-spread application in the separation of substances in t h e pharmaceutical industry. It is mainly used in searching f o r n e w compounds, biological testing, testing stabilities of d r u g s and in production. T h e quantities t o be isolated r a n g e as shown f r o m p g . u p t o kilograms. Depending o n t h e amount of material needed in t h e pharmaceutical industry preparative liquid chromatography can b e classified into: 1 ) analytical preparative scale 1 iquid chromatography, 2 ) laboratory scale 1 iquid chromatography and 3 ) production s c a l e 1 iquid chromatography. In research and development laboratories, LC is applied t o t h e c o l l e c t i o n of material for structure identification o r structure e l u c i d a t i o n (mg amounts) f o r such uses as 1 ) searching f o r new effective substances in plants o r animals, 2 ) isolating and separating reaction o r degradation products a n d 3 ) investing metabolism. Quantities in the gram range a r e separated and purified by laboratory scale preparative LC. Fractions a r e collected f o r chemical, phys cal and biological tests. Quantities in t h e multigram/kilogram r a n g e u s e produc ion scale preparative LC in the production stage a s a purification method.

157 4.3

CHROMATOGRAPHIC SYSTEMS USED P r e p a r a t i v e l i q u i d chromatography has found wide a p p l i c a t i o n

pharmaceutical i n d u s t r y .

n the

The reason i s t h a t today t h e s e p a r a t i o n e f f i c i e n c y o f Examples o f t h e

a wide range o f drugs i s well-known and e s t a b l i s h e d .

chromatographic s e p a r a t i o n o f p u r e compounds i n c l u d e a n a l g e s i c s , a n a e s t h e t i c s , h y p n o t i c s and s e d a t i v e s , p s y c h o s t i m u l a n t s , a n t i convul s a n t s , a n a l e p t i cs, parasympatholyt ic s , a n t i h i stamines, a n t i t u s s i ves , a n t i c o a g u l a n t s , c a r d i o v a s c u l a r drugs, d i u r e t i c s , hormones, v i t a m i n s , a n t i b i o t i c s , sulfonamides, a n t i n e o p l a s t i c s and o t h e r s .

Such substances can be separated whether t h e y o c c u r i n t a b l e t s ,

cough m i x t u r e s , creams o r o i n t m e n t s , o i l based p r e p a r a t i o n s , body f l u i d s , f e r m e n t a t i o n b r o t h , or s y n t h e t i c m i x t u r e s [11.12,1,3,141. I n a c c o m p l i s h i n g t h e s e o a r a t i o n / p u r i f i c a t i o n o f t h i s wide range of compounds,

v a r i o u s modes o f LC must be used.

T h i s means t h a t a d s o r p t i o n

chromatography 1151, bonded phase chromatography [161, i o n exchange chromatography [171 and s i z e e x c l u s i o n chromatography C181 a r e w e l l e s t a b l i s h e d i n p h a r m a c e u t i c a l l a b o r a t o r i e s , as a r e t h e t e c h n i q u e s o f l i q u i d - l i q u i d chromatography E l 9 1 and a f f i n i t y chromatography 1201. 4 . 3 . 1 Adsorption Chromatography

A d s o r p t i o n chromatography has been used q u i t e e x t e n s i v e l y i n t h e pharmaceutical i n d u s t r y .

The m a t e r i a l s were a l u m i n a and s i l i c a .

m a t e r i a l s h y d r o x y l groups on t h e s u r f a c e cause t h e a d s o r p t i o n .

On b o t h Therefore, the

water c o n t e n t or t h e c o n t e n t o f p o l a r substances i n t h e s t a t i o n a r y phase i s an i m p o r t a n t f a c t o r i n a d s o r p t i o n chromatography.

For t h i s r e a s o n i t i s necessary

t o assay t h i s c o n t e n t c a r e f u l l y i n o r d e r t o o b t a i n r e p r o d u c i b l e s e p a r a t i o n s . This can be d i f f i c u l t i n p r e p a r a t i v e l i q u i d chromatography, because a change o f t h e m o b i l e phase or i t s water c o n t e n t r e q u i r e s some t i m e t o e q u i l i b r a t e a chromatographic system.

T h i s i s f o r example t h e reason why t h e g r a d i e n t e l u t i o n

technique i s n o t recommended for p r e p a r a t i v e a d s o r p t i o n chromatography.

Despite

a l l t h i s , a d s o r p t i o n m a t e r i a l s a r e s t i l l , because o f t h e i r l o w c o s t s , v e r y o f t e n used i n p r e p a r a t i v e l i q u i d chromatography. 4 . 3 . 2 Bonded Phase Chromatography

The p r i n c i p a l disadvantage o f a d s o r p t i o n chromatography - l o n g e q u i l i b r a t i o n time - i s e l i m i n a t e d i n bonded phase chromatography.

Such column p a c k i n g s have

good chemical i n e r t n e s s . e x c e l l e n t s t a b i l i t y and s h o r t r e c o n d i t i o n i n g t i m e s i n g r a d i e n t e l u t i o n or column c l e a n i n g procedures [211.

They a l l o w a g r e a t freedom

158 o f c h o i c e w i t h r e s p e c t t o m o b i l e phases and t h e i r c o m p o s i t i o n . found i n r e v e r s e d phase and normal phase p a c k i n g s .

They can be

Such phases a r e e x p e n s i v e

and, t h e r e f o r e , a r e used i n p r e p a r a t i v e l i q u i d chromatography m o s t l y on t h e small s c a l e . 4 . 3 . 2 . 1 Reversed Phase Chromatography I n r e c e n t y e a r s , t h e m a j o r i t y o f t h e p u b l i s h e d work r e l a t i n g t o a p p l i c a t i o n s i n t h e pharmaceutical i n d u s t r y has r e f e r r e d to r e v e r s e d phase chromatography because such columns have n o t o n l y t h e advantages mentioned above, b u t a l s o p r o v i d e a wide range o f e l u t i o n s t r e n g t h i n s i m p l e b i n a r y m i x t u r e s such as methanol-water o r a c e t o n i t r i l e - w a t e r .

Therefore, a l o t o f pharmaceutical

p r o d u c t s can be chromatographed w i t h success on columns f i l l e d w i t h r e v e r s e d phase m a t e r i a l s . 4 . 3 . 2 . 2 Normal Phase Chromatography D i f f e r e n t p o l a r f u n c t i o n a l groups can a l s o be used f o r p r e p a r a t ve l i q u i d chromatography today.

These groups a r e permanently bonded o n t o t h e s u r f a c e of

porous s i l i c a , as a r e t h e r e v e r s e d phase groups. amino-, d i o l - , cyano- and n i t r o - g r o u p s .

The most f r e q u e n t y f o u n d a r e

These phases, m o s t l y used

mode, a r e s i m i l a r t o a d s o r p t i o n chromatography.

n t h e norma

They a r e s t a b l e t o h y d r o l y s i s

and have - compared w i t h a d s o r p t i o n chromatography - a s h o r t e q u i l i b r a t i o n time.

Consequently such phases can be r a p i d l y c l e a n e d and e q u i l i b r a t e d , l i k e

t h e above mentioned r e v e r s e d phase packings, and a r e t h e r e f o r e used i n p r e p a r a t i v e l i q u i d chromatography i f t h e y o f f e r equal o r b e t t e r performance compared t o a d s o r p t i o n chromatography. 4 . 3 . 3 Ion Exchange Chromatography I n t h e p a s t , i o n exchange chromatography had been w i d e l y used f o r s e p a r a t i o n s i n pharmaceutical l a b o r a t o r i e s . importance i s decreasing.

Except i n p e p t i d e i s o l a t i o n ,

its

The reason f o r t h i s may be t h e t i m e consuming

procedure for methods development and t h e s m a l l range o f components which a r e p r e d e s t i n e d for good i o n exchange s e p a r a t i o n s i n complex p h a r m a c e u t i c a l m i x t u r e s 4 . 3 . 4 Gel Permeation Chromatography and Other Techniques With a few e x c e p t i o n s g e l permeation chromatography as w e l l as o t h e r chromatographic techniques, such as l i q u i d - l i q u i d chromatography or a f f i n i t y chromatography, have found o n l y l i t t l e a t t e n t i o n i n p h a r m a c e u t i c a l p r e p a r a t i v e applications.

I f used, these methods a r e a p p l i e d to t h e i s o l a t i o n o f n a t u r a l

p r o d u c t s , as for example p e p t i d e s and p r o t e i n s .

159 4.4 APPARATUS AND EXAMPLES

A s a l r e a d y mentioned, p r e p a r a t i v e LC i s used i n t h e p h a r m a c e u t i c a l i n d u s t r y

for s e v e r a l purposes.

We have d i s t i n g u i s h e d between a n a l y t i c a l - ,

and p r o d u c t i o n - s c a l e chromatography.

laboratory-

T h i s c l a s s i f i c a t i o n can be m a i n t a i n e d when

speaking about apparatus and examples. 4 . 4 . 1 Analytical Scale Preparative LC (mg-scale) 4 . 4 . 1 . 1 Apparatus

P r e p a r a t i v e a n a l y t i c a l LC I s t h e area i n which - i f necessary - h i g h q u a l i t y s e p a r a t i o n s a r e s c a l e d up i n such a way t h a t a q u a n t i t y o f substance i s o b t a i n e d s u f f i c i e n t l y pure t o c a r r y o u t compound i d e n t i f i c a t i o n or s t r u c t u r e elucidation. required.

For such a p p l i c a t i o n s t h e b e s t p o s s i b l e s e p a r a t i o n power i s

I n t h e l a s t decade t h i s was achieved on columns, chosen w i t h n a r r o w

bores (3-24 mm i . d . ) and small p a r t i c l e s i z e s (3-10 pm)[221.

However,

experiments have shown t h a t a n a l y t i c a l columns a r e l i m i t e d t o t h e s e p a r a t i o n o f m i l l i g r a m q u a n t i t i e s o f samples because o f t h e r e l a t i v e l y small c a p a c i t y o f System c a p a c i t y f o r most p r a c t i c a l purposes can be a p p r o x i m a t e l y

these systems.

f r o m 0.1 up t o 1 mg o f s o l u t e p e r gram o f t h e t o t a l porous p a c k i n g , as shown i n F i g . 4.1.

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R e l a t i o n between r e l a t i v e r e s o l u t i o n and l o a d a b i l i t y l g p a c k i n g .

160 Nowadays, s e p a r a t i o n s such as t h a t shown i n F i g . 4.1 a r e performed w i t h a n a l y t i c a l i n s t r u m e n t s , p e r m i t t i n g flow r a t e s up t o and i n c l u d i n g 30 m l l m i n a t 300 b a r .

These a n l y t i c a l i n s t r u m e n t s a l l o w t h e o p e r a t i o n o f a n a l y t i c a l columns

w i t h 4 mm I . D . as w e l l as columns with 2 5 mm i . D . , up t o a column l e n g t h of 25-50 cm.

f i l l e d w i t h small p a r t i c l e s

C o n s i d e r a t i o n o f t h e volume g a i n e d w i t h such

s c a l e d up columns and t h e system c a p a c i t y o f s o l u t e p e r gram o f t h e p a c k i n g shows t h a t i t i s p o s s i b l e t o separate 100 mg of a main s o l u t e i n a n o t - t o o complex m i x t u r e or 1 mg o f a 1 p e r c e n t by-product i n a s i n g l e r u n on such a chromatographic system. 4 . 4 . 1 . 2 Example

S e p a r a t i o n s on a n a l y t i c a l columns w i t h subsequent p u r i f i c a t i o n and i d e n t i f i c a t i o n a r e a m a t t e r o f r o u t i n e i n many l a b o r a t o r i e s of t h e pharmaceutical i n d u s t r y .

The f r a c t i o n s c o n t a i n i n g t h e separated components a r e

e x t r a c t e d o r re-chromatographed i n t o c h l o r o f o r m o r i n t o a n o t h e r s o l v e n t , which w i l l then be evaporated under n i t r o g e n , and t h e pg t o mg amounts o f c o l l e c t e d

mass a r e used f o r s t r u c t u r e e l u c i d a t i o n w i t h NMR, I R and mass s p e c t r o m e t r y .

The

f i n a l s t e p i n t h e i d e n t i f i c a t i o n i s a comparison o f t h e s p e c t r a l and r e t e n t i o n d a t a w i t h r e f e r e n c e d a t a fo r t h e presumed compounds [231. Most i d e n t i f i c a t i o n problems i n t h e p h a r m a c e u t i c a l i n d u s t r y do n o t need t h e above mentioned t o t a l s t r u c t u r e e l u c i d a t i o n equipment.

Prior knowledge o f t h e

n a t u r e o f ' t h e sample a l l o w s a t e n t a t i v e i d e n t i f i c a t i o n a n d / o r a s t r u c t u r e confirmation.

Examples of t h i s use a r e i n s y s t e m a t i c i n v e s t i g a t i o n s o f p l a n t ,

animal o r f e r m e n t a t i o n e x t r a c t s , i n t h e c o n t r o l o f s y n t h e t i c p r o d u c t s and edducts i n m e t a b o l i c s t u d i e s , e t c .

When t h i s can or must be done, i t may

s u f f i c e t o c o l l e c t enough m a t e r i a l f o r mass s p e c t r a l or NMR c o n f i r m a t i o n o f t h e s t r u c t u r e expected.

Since mass s p e c t r o m e t r y needs about 1000 t i m e s l e s s

substance f o r a spectrum .than o t h e r s p e c t r o s c o p i c methods, o f f - l i n e LC-MS, u s i n g small b o r e packed a n a l y t i c a l columns, i s a p p l i e d i n some cases as an a n a l y t i c a l p i l o t i d e n t i f i c a t i o n t o o l C241. F i g . 4.2 shows t h e chromatogram o f a c l a v i c e p s purpurea f e r m e n t a t i o n e x t r a c t u s i n g an a n a l y t i c a l column ( 4 mm i . 0 . ) packed w i t h a bonded phase L i C h r o s o r b RP-8 column and developed w i t h g r a d i e n t e l u t i o n .

Four major components o f t h e

sample were separated i n t h i s mode. I n o r d e r t o enhance t h e c o n c e n t r a t i o n o f t h e components, 2 p1 of t h e f r a c t i o n c u t a t t h e peak maximum i s p l a c e d on a whisker w i t h t h e a i d of a s y r i n g e , as shown i n F i g . 4.3.

T h i s d r o p l e t i s then evaporated i n a warm To g e t an in-beam

n i t r o g e n stream t o p r e v e n t o x i d a t i o n p r i o r t o a n a l y s i s .

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P l a c i n g a d r o p l e t of e l u e n t on a w h i s k e r .

8-spectrum l e s s than a p p r o x i m a t e l y 10 ng s o l u t e i s necessary.

T h i s means c a .

1-10 p1 o f t h e c e n t r a l f r a c t i o n c u t condensed on t h e w h i s k e r i s i n some c a s e s

s u f f i c i e n t for such a mass-spectrum.

A t f i r s t , t h e E l e c t r o n beam ( E B ) - s p e c t r a

o f each m a i n peak was r e c o r d e d i n t h e f e r m e n t a t i o n e x t r a c t e x p e r i m e n t .

The

s p e c t r a o f peaks A . B , D and E were e a s y t o i n t e r p r e t and i d e n t i f i c a t i o n was p o s s i b l e by comparing t h e s p e c t r a w i t h a r e f e r e n c e l i b r a r y . The f o u r t h s p e c t r u m , t h e o n e o f peak C. seemed t o be an E B - m i x t u r e s p e c t r u m , t h e r e f o r e we made a f i e l d d e s o r p t i o n (FD) a n a l y s i s w i t h an a d d i t i o n a l 5 p1 o f t h i s peak f r a c t i o n .

As demonstrated i n F i g . 4.4a. f i e l d d e s o r p t i o n g i v e s such

h i g h molecular i o n i n t e n s i t i e s t h a t m i x t u r e a n a l y s i s i s p o s s i b l e based o n FD-spectra.

I n o u r e x p e r i m e n t s i t i s shown t h a t peak C i s a m i x t u r e o f two

compounds. The s t r u c t u r e e l u c i d a t i o n based on such FD s p e c t r a a l o n e a n d o n E B - s p e c t r a

o f a m i x t u r e i s u n a c h i e v a b l e or r i s k y .

B u t t h e s e a p p a r e n t d i s a d v a n t a g e s c a n be

m a s t e r e d b y CAMS ( C o l l i s i o n a l A c t i v a t i o n MS-MS)[251,

where e a c h i o n c a n be

e f f e c t i v e l y s e p a r a t e d b y means of a t r i p l e s t a g e or a l i n k e d scan c o n v e n t i o n a l mass s p e c t o m e t e r and a n a l y s e d a f t e r i m p a c t s w i t h n e u t r a l t a r g e t gas i n d u c i n g fragmentations of the separated i o n . necessary for i d e n t i f i c a t i o n .

This fragmentation gives the information

Such CAMS-spectra o f t h e FDM' peak 1 and p e a k 2

a r e shown i n s p e c t r a 2 and 3 ( F i g . 4 . 4 b ) .

A s t h e s p e c t r a d e m o n s t r a t e , more

f r a g m e n t s and t h e r e f o r e more i n f o r m a t i o n i s o b t a i n e d .

T h i s i n f o r m a t i o n was

163

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LC-PEAK C FO-SPECTRUM

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100

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400

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FD-MS spectrum ( a ) and FD-CA-MS s p e c t r a o f LC-peak C ( b ) .

s u f f i c i e n t for a t e n t a t i v e i d e n t i f i c a t i o n o f t h e two compounds as C, = e r g o k r y p t i n and Cg = e r g o s i n i n , two well-known e r g o t a l k a l o i d s .

The r e s u l t s

were v e r i f i e d by comparison w i t h r e f e r e n c e s p e c t r a . T h i s experiment demonstrates t h a t such an MS-MS tool can be of c o n s i d e r a b l e importance for crude e x t r a c t a n a l y s i s and t h a t such a m i x t u r e a n a l y s i s combined w i t h LC w i l l f i n d q u i t e a number o f a p p l i c a t i o n s i n t h e f u t u r e .

164 4.4.2

Laboratory Scale Preparative LC (9-scale)

4.4.2.1 Apparatus

Another a r e a o f p r e p a r a t i v e column l i q u i d chromatography i s on t h e l a b o r a t o r y - s c a l e , where l o t s t o be separated l i e between some mg and a few grams.

I n a n a l y t i c a l p r e p a r a t i v e a p p l i c a t i o n s t h e chromatographs d e l i v e r flow

v e l o c i t i e s up t o 5 cm.sec-’

and have t o work up t o 300 b a r s .

p r e s s u r e used i n a n a l y t i c a l work may be 100 b a r s .

The mean

I n l a b o r a t o r y - s c a l e as w e l l

as i n p r o d u c t i o n s c a l e one i s chromatographing i n a t r a n s i t i o n r e g i o n between 1 and 100 bars.

The equipment i n use can be d i v i d e d i n t o two groups: 1 ) d e v i c e s

up t o 50 bars and 2) devices between 50 and 100 b a r s . 4 . 4 . 2 . 1 . 1 Columns and Equipment Constructed for Separations up to 50 Bars. Today

i n the pharmaceutical area many p r e p a r a t i v e LC a p p l i c a t i o n s on t h e l a b o r a t o r y s c a l e up t o 50 bars a r e performed i n g l a s s columns.

These columns w i t h s t a n d l i m i t e d

pressure and a whole a r r a y o f a u x i l i a r y hardware i s a v a i l a b l e ( i n j e c t i o n p o r t s , j a c k e t s , columns, column c o n n e c t i o n s , e t c . ) . These columns a r e b e s t s u i t e d for f i l l i n g w i t h o u t t h e suspension t e c h n i q u e and a r e t h e r e f o r e m o s t l y a p p l i e d w i t h s t a t i o n a r y phases which do n o t g i v e h i g h back pressure.

To r u n such columns low p r e s s u r e equipment i s r e q u i r e d .

Home-made

systems composed o f a pump, a l o o p i n j e c t o r and a d e t e c t o r can be found i n many l a b o r a t o r i e s .

Some a r e combined w i t h a low p r e s s u r e g r a d i e n t d e v i c e ,

others w i t h a f r a c t i o n c o l l e c t o r . A g l a s s column as used i n o u r l a b o r a t o r i e s i s d e s c r i b e d i n F i g . 4.5 [ r e f . 261

The column i t s e l f i s c o n s t r u c t e d o f g l a s s t u b i n g o f 2 . 5 , 5 or 7 . 5 cm I . D . and 60 cm i n length.

The maximum f i l l i n g o f t h i s column i s a p p r o x i m a t e l y 1 kg, which

corresponds t o a packed bed h e i g h t o f 50 cm. O t h e r l a b o r a t o r i e s work i n t h i s p r e s s u r e range w i t h commercial equipment. I n 1975, f o r example, Waters A s s o c i a t e s i n t r o d u c e d t h e PrepLClSystem 500 p r e p a r a t i v e l i q u i d chromdtograph 1271.’ The i m p o r t a n t advantage i n t h e d e s i g n o f t h i s i n s t r u m e n t i s t h e use o f a d i s p o s a b l e , pre-packed column w i t h a p l a s t i c wall.

T h i s c a r t r i d g e i s f i l l e d i n a c y l i n d r i c a l compression chamber and w i l l be

r a p i d l y compressed p r o r to sample i n j e c t i o n .

T h i s procedure compresses t h e

column packing i n t o a h i g h l y e f f i c i e n t chromatographic bed.

(See Chapter 1 for

a more thorough d i s c u s i o n ) .

The Prep-PAK 500 s i l i c a c a r t r i d g e s a r e 5 . 7 cm i n

diameter and 30 cm i n l e n g t h .

The e f f e c t i v e column l e n g t h can be i n c r e a s e d by

c o n n e c t i n g such c a r t r dges i n s e r i e s .

The flow r a t e i s a d j u s t a b l e between 0.05

and 0.5 l l m i n t o meet t h e s e p a r a t i o n c o n d i t i o n s s e l e c t e d .

The maximum o p e r a t i n g

165

F i g . 4.5.

P r e p a r a t i v e LC-column, g l a s s c o n s t r u c t i o n .

p r e s s u r e i s a d j u s t a b l e to 30 b a r . w i t h a syringe.

The sample i s loaded d i r e c t l y o n t o t h e column

The sample i n t r o d u c t i o n system p e r m i t s t h e l o a d i n g o f v i r t u a l l y

any s i z e o f sample.

The d e t e c t o r used i s a d i f f e r e n t i a l r e f r a c t o m e t e r o p t i m i z e d

for p r e p a r a t i v e o p e r a t i o n . 4 . 4 . 2 . 1 . 2 Columns and Equipment C o n s t r u c t e d f o r S e p a r a t i o n s Between 50 and 100

Bars.

When working as above mentioned i n t h e r e g i o n up to 50 b a r s , medium

r e s o l u t i o n i s r e q u i r e d I n most s e p a r a t i o n s . supports > 30 pm i n diameter a r e used.

I n such cases columns f i l l e d w i t h

Such columns can be homogeneously

dry-packed down t o t h e mentioned p a r t i c l e - s i z e .

When w o r k i n g w i t h p r e s s u r e s

166

h i g h e r than 50 b a r s , medium t o h i g h r e s o l u t i o n i s a must and i s o f t e n r e q u i r e d i n pharmaceutical i n d u s t r y .

T h i s r e s o l u t i o n can be reached by e l o n g a t i o n o f t h e

-

column l e n g t h or by u s i n g f i n e r p a r t i c l e s than 30 pm. Under t h e c o n d i t i o n s mentioned two problems have t o be s o l v e d - f i r s t , t h e c o n s t r u c t i o n o f t h e whole equipment has t o r e s i s t t h i s p r e s s u r e and, second, t h e column has t o be f i l ed by a i d of t h e s l u r r y t e c h n i q u e . These two problems have been addressed by J o b i n Yvon i n t h e Prepmatic L q u i d Chromatograph, which enables columns to be packed and samples t o be chromatographed (see Chapter 1.7).

The key t o e f f e c t i v e performance i n d e a l i n g

Flow of excess d w n t

I

Piston on down-stroke onsmon

Piston In up-stroke powtion

a

b

C

d

F i g . 4.6.

Four steps t o i l l u s t r a t e t h e method o f p a c k i n g t h e s t a t i o n a r y phase

i n t h e Chromatospac-Prep 100.

167 w i t h these two t a s k s l i e s i n t h e d e s i g n o f a column ( 1 m x 8 cm), f i t t e d w i t h a piston.

Upward movement o f t h e p i s t o n compacts t h e column bed t h o r o u g h l y and

makes p a c k i n g a good column a r e l a t i v e l y s i m p l e o p e r a t i o n , as i l l u s t r a t e d i n Fig. 4.6.

A s l u r r y o f t h e p a c k i n g m a t e r i a l i n t h e m o b i l e phase i n a s o l v e n t i s

poured i n t o t h e t o p o f t h e open column ( a ) .

The sample i n l e t head i s t h e n

r e p l a c e d on t h e t o p o f t h e column ( b ) and t h e p i s t o n i s moved upwards t o compress t h e bed w h i l e d r i v i n g excess o f s o l v e n t o u t t h r o u g h t h e end o f t h e column.

The f i n a l pressure on t h e p i s t o n i s m a i n t a i n e d t h r o u g h o u t t h e

chromatographic process ( c ) .

T h i s system i s c l a i m e d t o have t h e advantage o f

p r o v i d i n g homogeneous and r e p r o d u c i b l e p a c k i n g t o o p e r a t e w i t h e l u t i o n and column e x t r u s i o n [281. 4 . 4 . 2 . 2 Examples

T y p i c a l examples f o r t h e l a b o r a t o r y s c a l e s e p a r a t i o n s o f pharmaca a r e t h e p r o d u c t i o n o f h i g h l y p u r i f i e d substances f o r c h e m i c a l , p h y s i c a l o r b i o l o g i c a l t e s t s i n t h e mg t o t h e g range, and t h e enrichment or p u r i f i c a t i o n o f s y n t h e s i z e d p r o d u c t s , o f p l a n t s and o f f e r m e n t a t i o n e x t r a c t s . A f i r s t example [291 i l l u s t r a t i n g such a s e p a r a t i o n w i l l be shown i n F i g . 4.7.

The example demonstrates an a n a l y t i c a l s e p a r a t i o n and a l a r g e d i a m e t e r

column s e p a r a t i o n .

Chromatogram ( a ) i s t h e a n a l y t i c a l s e p a r a t i o n i n a

b

10 Fig. 4 . 7 .

20

30

40 [min]

10

20

30

40

[min]

P u r i f i c a t i o n o f pyranosides (glucomanno- and g a l a c t o p y r a n o s i d e ) . a)

A n a l y t i c a l separation, recalculated.

b)

Preparative separation w i t h overload.

Column, 40 x 7 cm, s i l i c a g e l ; m o b i l e phase, e t h y l a c e t a t e l n - h e p t a n e ; sample w e i g h t , 5 g . R e p r i n t e d f r o m [261 w i t h p e r m i s s i o n .

168

10.2 E

Q

C

I

1

0.01 E

0.1 E

L J J L

I

A I

1

i

i

Fig. 4.8.

A

i?ti

C

0

io

i4

is

iz tCR

1

i

h

iz

rb

io

h

is

* iz t[min]

Enrichment of 3-nitrophenacetin reaction products with the aid of column-strategy. a) Analytical and preparative chromatogram of nitrophenacetin mixture. Column, 250 x 4.6 mm and 250 x 16 mm i . D . ; stationary phase, LiChrosorb SI 100, d = 10 p;mobile phase, P n-hexane-dichloromethane-acetonitrile-water (195:780:24:0,1); flow rate, 2,s and 30 ml/min. b) and c) Column, 250 x 4.6 mm and 250 x 16 mm 1 . D . ; stationary phase, LiChrosorb RP-8, d = 10pm; mobile P phase, w a t e r - a c e t o n i t r i l e - t r i e t h y l a m i n e (870:124:6); flow rate, 2.5 and 30 ml h i n .

169 non-overloaded c o n d i t i o n

Chromatogram ( b ) shows t h e same system i n a

p r e p a r a t i v e mode u s i n g a

arge-diameter,

low r e s o l u t i o n glass-column o p e r a t e d i n

an o v e r l o a d e d c o n d i t i o n .

These examples demonstrate t h a t a l o t of a p p l i c a t i o n s

o f preparative l a b scale

i q u i d chromatography a l l o w o v e r l o a d i n g i n o r d e r t o

reach h i g h e r c a p a c i t y and g r e a t e r t h r o u g h p u t a t t h e expense o f r e s o l u t i o n . T h e r e f o r e , low p r e s s u r e systems w i t h a s m a l l e r e f f i c i e n c y a r e s t i l l i n demand and, as mentioned, i n use i n a number o f l a b o r a t o r i e s . I t i s the separation of f o u r

Another example [301 i s shown i n F i g . 4 . 8 . peaks.

The chromatography was c a r r i e d o u t i n t w o s t e p s .

A t f i r s t t h e sample

was d i v i d e d i n t o two groups o f compounds a s shown i n chromatogram ( a ) i n t h e figure.

T h i s chromatogram a l s o demonstrates t h e s u p e r p o s i t i o n o f two n e a r l y Fractions

r e c t a n g u l a r peaks i n each group on t h e LiChrosorb-60 system chosen.

o f t h e column e f f l u e n t corresponding t o t h e two groups o f compounds were c o l l e c t e d manually and t r a n s f e r r e d as samples t o a more e f f i c i e n t L i C h r o s o r b RP-8 system as shown i n chromatogram ( b ) and ( c ) ,

so t h a t a complete s e p a r a t i o n

o f a l l f o u r compounds was achieved i n t h i s manner.

The t o t a l enrichment and

s e p a r a t i o n t i m e f o r a l l t h r e e chromatographic s e p a r a t i o n s was two h o u r s . Concerning such chromatographic experiments i t can be s t a t e d :

In a

chromatographic s e p a r a t i o n o f a m i x t u r e i t i s t h e aim t o f i n d a column f o r which t h e s m a l l e s t s e l e c t i v i t y c o e f f i c i e n t t h a t o c c u r s i s as l a r g e as p o s s i b l e . Furthermore, a good s t r a t e g y has t o be chosen.

I f t h i s i s done, as l a r g e a

sample as p o s s i b l e has t o be p l a c e d on t h e column so as t o i n c r e a s e t h e t h r o u g h p u t per chromatogram. The t h i r d example, F i g . 4 . 9 , w i l l demonstrate t h e s t r a t e g y o f m a x i m i z i n g t h r o u g h p u t u s i n g s e v e r a l steps i n s e l e c t i v i t y .

I n t h i s example i t was

advantageous t o use f o r c e d p r e s s u r e p r e p a r a t i v e s c a l e LC r a t h e r t h a n c o n v e n t i o n a l g r a v i t y f l o w for t h e s e p a r a t i o n o f p u r e c o n s t i t u e n t s from a c r u d e p l a n t e x t r a c t c o n t a i n i n g t e r p e n o i d s and a l k a l o i d s . T h i s example was p u b l i s h e d by Hostettmann [311 and shows t h e s e p a r a t i o n o f t h e crude hexane e x t r a c t o f f a g a r a chalybea ( r u t a c e a e ) on a Waters PrepLClSystem 500 u s i n g s i l i c a g e l column c a r t r i d g e s ( 3 0 cm x 5 . 7 c m ) . The f i r s t s t e p was t o remove the small amount o f p o l a r m a t e r i a l from t h e b u l k o f the e x t r a c t by f i l t r a t i o n through s i l i c a g e l w i t h ether:hexane 1 : l . T h i s a l s o avoided column c o n t a m i n a t i o n .

A s shown i n t h e scheme, t h e f i l t r a t e

was submitted t o p r e p a r a t i v e LC upon which t h e l e a s t p o l a r f r a c t i o n s 1 ( a b o u t 1 g ) and 2 (850 g) were o b t a i n e d i n s i x m i n u t e s .

A f t e r peak 2, t h e s o l v e n t system

was switched t o t h e more p o l a r e t h y l acetate/hexane 1:4 m i x t u r e , and t h e

170

Crud. heron. oxtract Fagoro chalyboo

a.50 filtration

(510,) Pro -LC o t b r 1 hPuono

30

I.9

EIOAc/horona

I mixlure

2 germacrone

( 1 g)

(U50 rng)

3 dihydrochclcrylhrina (120 mo)

1 I4

6 N-mclhylllindenim (123 mr)

I mixlure I IN0 mg) 5 pure compound

(40mg)

F i g . 4.9.

S e p a r a t i o n o f a crude hexane e x t r a c t o f f a g a r a chalybea on a Waters PrepLC System/500 u s i n g s i l i c a g e l c a r t r i d g e s and f o r c e d p r e s s u r e . R e p r i n t e d from C311 w i t h p e r m i s s i o n .

remainder was c o l l e c t e d i n one f r a c t i o n .

T h i s f r a c t i o n was c o n c e n t r a t e d , and a

second stage o f p r e p a r a t i v e LC w i t h t h e above s o l v e n t system was c a r r i e d o u t . This r e s u l t e d i n t h e s e p a r a t i o n o f f o u r f r a c t i o n s : (40 mg), and 6 (125 mg) i n 25 m i n u t e s .

3 (120 mg). 4 (180 mg), 5

C o n s i d e r i n g t h a t t h e whole i s o l a t i o n

process r e q u i r e d 2-3 hours, whereas c o n v e n t i o n a l t e c h n i q u e needs about two weeks, we can s t a t e t h a t such chromatography can be a g r e a t s i m p l i f i c a t i o n o f t h e i s o l a t i o n technique . These a r e t h r e e examples i l l u s t r a t i n g l a b s c a l e LC i n p h a r m a c e u t i c a l industry.

I n t h e l i t e r a t u r e t h e r e a r e a l o t of o t h e r a p p l i c a t i o n s t o be f o u n d .

Most o f them can be c l a s s e d w i t h t h e examples shown i n F i g . 4.10.

They a r e : 1 )

s e p a r a t i o n o f c l o s e l y e l u t i n g compounds, 2 ) p u r i f i c a t i o n o f r e l a t i v e l y

171

A BY-PRODUCT

---- - -

- --

SEVERAL COPPOUNDS I N A SIF’PLE MIXTURE

,c

A TRACE COMPOUND _--

L . _ _ _ _ ----

F i g . 4.10.

Chromatograms i l l u s t r a t i n g

---

I N A COWLEX MIXTURE

s o l a t i o n problems i n p r e p a r a t i v e LC.

w e l l - s e p a r a t e d main compounds o r enrichment o f by-products and 3 ) i s o l a t i o n o f compounds i n a complex m a t r i x . I n t h e t h i r d case, t h e minor compound must f i r s t be e n r i c h e d by one or more steps.

This enrichment i s accomplished by o v e r l o a d i n g t h e column and c o l l e c t i n g

f r a c t i o n s i n t h e ’ a r e a o f expected r e t e n t i o n o f t h e d e s l r e d compound. These c o l l e c t e d f r a c t i o n s can then be pooled, c o n c e n t r a t e d and r e i n j e c t e d on d i f f e r e n t systems u n t i l i s o l a t i o n i s reached.

172 A much s i m p l e r case i s t h e p u r i f i c a t i o n o f a r e l a t i v e l y w e l l separated main compound or t h e enrichment o f by-products.

The b e s t w i l l be first t o o p t i m i s e

Then t h e sample l o a d has t o be

the r e t e n t i o n i n t h e k ' range up t o 1 0 .

increased up t o the p o i n t where peaks a r e o v e r l a p p i n g and t h r e a t e n t o d i s t u r b . A t t h i s p o i n t t h e m a t e r i a l w i l l be c o l l e c t e d .

I n t h e l a s t case two or more c l o s e l y e l u t i n g compounds have t o be i s o l a t e d . I n t h i s s i t u a t i o n i t i s u s e f u l t o i n c r e a s e t h e r e s o l u t i o n ( b y s e l e c t i v i t y or efficiency)

or e l s e a r e c y c l i n g technique can be employed.

4 . 4 . 2 . 3 Some Trends i n L a b o r a t o r y S c a l e LC [32] 4.4.2.3.1 R e p e t i t i v e Cycle Operation.

I n p r e p a r a i v e chromatography t h e

f a i t o r s o f sample s i z e and r e t e n t i o n a r e combined i n t h e s o - c a l l e d p r o d u c t i o n r a t e , which i s t h e amount o f separated compounds number o f t h e o r e t i c a l p l a t e s .

e r u n i t t i m e for a g i v e n

The r e l a t i o n s h i p between t h i s p r o d u c t i o n r a t e

(xB) and band broadenina ( R / R m a x )

i s known and i n d i c a t e d i n F i g . 4 . 1 1 .

The graph shown i s w e l l s u i t e d t o i l l u s t r a t e t h e s i t u a t i o n . t h e r e i s a f l o a t i n g border between a r e a A and C.

Naturally,

But each a r e a can be extended

a) by diameter i n c r e a s e s , and b ) by r e p e a t i n g t h e chromatograms (or s e p a r a t i o n cycles).

Both can n a t u r a l l y be combined t o g e t h e r and a l s o p r o v i d e d w i t h an

a u t o m a t i c c o n t r o l system.

t

0

t

I

0

I

F i g . 4.11.

These c o n t r o l systems for p e r f o r m i n g programmed

I

I

10

100

S c a l i n g up o f LC.

I

1 '000

b

I

10'000

xB

173

----------PUMP

DAMPING DEVICE

PRESSURE MONITORING AND SAFETY DEVICE

rj-t-, I

I I

I I I I

I

INJECTION DEVICE

I I I I 1 I I

COLUMN

I

I F i g . 4.12.

8

FRACTION COLLECTION

I

Scheme o f an automated b a t c h system.

s e p a r a t i o n s can be d i v i d e d i n t o t h r e e groups: 1 ) t i m e c o n t r o l o n l y (system w i t h o u t feedback o f t h e a c t u a l s e p a r a t i n g sequence), 2 ) c o n c e n t r a t i o n dependent c o n t r o l (system i n which t h e program i s a f f e c t e d by t h e s e p a r a t i o n sequence), and 3 ) time and c o n c e n t r a t i o n dependent c o n t r o l .

The most r e l i a b l e system i s

p r o b a b l y t h a t w i t h t i m e and c o n c e n t r a t i o n dependent c o n t r o l .

F i g . 4.12 shows a b l o c k diagram f o r such a t y p e of a u t o m a t i c b a t c h chromatograph f i t t e d w i t h a c o n t r o l system, which i s used i n our l a b o r a t o r i e s .

174 4 . 4 . 2 . 3 . 2 Gradient Elution and Recycling.

Techniques such as g r a d i e n t e l u t i o n

and column s w i t c h i n g may be i n t e r e s t i n g supplements i n p r e p a r a t i v e instrumentation.

I n p a r t i c u l a r , a g r a d i e n t e l u t i o n apparatus can s o l v e t h e

general e l u t i o n problem and i s t h e r e f o r e v e r y h e l p f u l i n many s e p a r a t i o n s o f drugs. R e c y c l i n g may a l s o h e l p and o f t e n overcomes d i f i c u l t i e s i n r e s o l u t o n . The s i m p l e s t concept o f such a r e c y c l i n g i n s t r u m e n t p aces t h e pump, t h e sampling system, t h e column and t h e d e t e c t o r i n a c l o s e d l o o p ( F i g . 4.13)

RESERVOIR

I

COLLECTOR

I

PUMP

SAMPLE INTRODUCTION DEVICE

F i g . 4.13.

R e c y c l i n g arrangement.

The equipment f o r r e c y c l i n g o p e r a t i o n d i f f e r s from c o n v e n t i o n a l l i q u i d chromatographic equipment i n t h a t i t imposes a double c a r r i e r s o l v e n t i n l e t t o t h e pump (one from t h e c a r r i e r r e s e r v o i r and t h e o t h e r from t h e r e c y c l e ) and a s p l i t t i n g v a l v e a t t h e d e t e c t o r e x i t f o r r e c y c l i n g or c o l l e c t i n g p a r t i c u l a r f r a c t i o n s of a chromatogram.

T h i s a d d i t i o n a l v a l v e i s necessary because a

r e c y c l i n g system i s a c l o s e d system w i t h a f i n i t e volume.

Hence, t h e

fast-moving m a t e r i a l w i 1 1 e v e n t u a l l y o v e r t a k e t h e slower moving m a t e r i a l and re-mix.

T h i s v a l v e must be p r o v i d e d so t h a t t h e o p e r a t o r can remove a p o r t i o n

of p o s s i b l y m i xed components b e f o r e peak o v e r l a p p i n g can o c c u r . The s e p a r a t i o n of m e t a b o l i t e s o f a u r i n a r y e x t r a c t made by c o n n e c t i n g t h e o u t l e t o f an UV d e t e c t o r t o a Waters S o l v e n t D e l t v e r y System w i t h s m a l j - d i a m e t e r

175

tubing is shown in Fig. 4.14. Fig. 4.14a demonstrates the starting chromatogram and the separation drawn in Fig. 4.14b was obtained by using eight cycles at 9 ml/min and a 25 cm x 8 mm i.D. column. The silica used was Merckosorb Si 100, 5 pm in diameter. Because o f the good separation, it was possible t o collect the two compounds without any loss of material.

E

0

I

c

t

CYCLE

2

4

6

8

Fig. 4.14. Separation by recycling: a) Original chromatogram. The sample in question is an extract o f metabolites in plasma. b) Separation by recycling o f substances 2 0 and 21.

4.4.3

Production Scale LC

Apparatus In order to increase the capacity o f such preparative laboratory work, cy;le operation combined with progressive column diameter [331 (as shown in Fig. 4.11) or continuous chromatography 1341 permits the scaling up to production scale 4.4.3.1

176 throughputs.

I t i s q u i t e c l e a r t h a t such a u t o m a t i c d e v i c e s , which a r e i n use i n

development l a b o r a t o r i e s today, w i l l be a p p l i e d and expanded t o t h e pharmaceutical p r o d u c t i o n s c a l e i f n e c e s s i t y and economy a r e proved. But today i n most f a c t o r i e s , compounds or compound groups which have t o be e n r i c h e d o r cleaned a r e separated i n one r u n . have diameters between 2 . 5 cm and about 1 m.

The columns a r e made of g l a s s and They a r e f i l l e d w i t h s i l i c a g e l ,

A l 2 O 3 or c e l l u l o s e (or i t s d e r i v a t i v e s ) t o a h e i g h t o f 50 t o 250 cm,

p a r t i c l e s of a p p r o x i m a t e l y 100 pm d i a m e t e r .

I n most cases t h e p a r t i c l e s i z e i s

based on p r a c t i c a l c o n s i d e r a t i o n s , such as p e r m e a b i l i t y or f a c i l i t a t i o n and s e c u r i t y (hazard of i n h a l a t i o n ) o f t h e p a c k i n g method. Columns w i t h t h i s low s e p a r a t i o n e f f i c i e n c y , compared w i t h t h o s e o f a n a l y t i c a l chromatography, a r e s t i l l i n use f o r s i m p l e s e p a r a t i o n s as e . g . group s e l e c t i v e separation, size separation, e t c . t h i s purpose i s shown i n F i g . 4.15.

P r o d u c t i o n s c a l e equipment used f o r

I n t h i s b l o c k d e s i g n t h e r e a r e two p a r t s

ELUENT

lIF====Tl

SOLVENT

I

RESERVOIRS

REVERSING

REGENERATOR

VALVE

CONCENTRATING PLANT

r

mmm CONCENTRATED FRACTIONS

F i g . 4.15.

FRACTION DISTRIBUTOR

FRACTION COLLECTING TANKS

B l o c k diagram o f a p r o d u c t i o n s c a l e LC-plant used f o r group s p e c i f i c separations.

177

The two

shown w h i c h were n o t s p e c i a l l y m e n t i o n e d i n t h e p r e v i o u s s e c t i o n s . items a r e

r e s p e c t i v e l y : 1 ) t h e s o l v e n t column r e g e n e r a t i o n a n d 2 ) t h e s a m p l e

d i s t r i b u t i o n system.

B o t h p l a y a n i m p o r t a n t r o l e i n p r e p a r a t i v e LC.

Whether a s o l v e n t s h o u l d be p u r i f i e d has to be d e c i d e d b y t h e u s e r . G e n e r a l l y , s o l v e n t r e g e n e r a t i o n or p u r i f i c a t i o n i s a c h i e v e d i n p r e p a r a t i v e LC i n three steps: 1.

by freeze d r y i n g

2.

b y r e d i s t i11a t i o n

3.

b y p a s s i n g t h r o u g h a s e l e c t i v e column ( p o s s i b l y a p r e - c o l u m n ) .

I n p r e p a r a t i v e LC a f t e r f r e e z e d r y i n g t h e r e c o v e r e d s o l v e n t s a r e i n m o s t c a s e s p u r e enough f o r p r e p a r a t i v e s e p a r a t i o n s i n l a b o r a t o r y - or p r o d u c t i o n s c a l e . ( I n a n a l y t i c a l p r e p a r a t i v e LC v e r y o f t e n f u r t h e r p u r i f i c a t i o n i s n e c e s s a r y . ) The n e c e s s i t y f or c l e a n i n g and r e c o n d i t i o n i n g t h e column p a c k i n g depends o n t h e sample i n t r o d u c e d . Samples t h a t have n o t been p r e f r a c t i o n e d o f t e n have too l a r g e a n e l u t i o n volume and r e p e t i t i v e c y c l i c s e p a r a t i o n o f compounds i s n o t p o s s i b l e , e x c e p t ' i f t h e c o l u m n i s p u r i f i e d and r e c o n d i t i o n e d .

The e c o n o m i c f e a s i b i l i t y o f p u r i f y i n g

and r e c o n d i t i o n i n g i s t h e r e f o r e d e p e n d e n t upon t h e c o s t s o f t h e s o l v e n t a n d s t a t i o n a r y phase. O f t e n i t i s p r e f e r a b l e t o r e p l a c e t h e c o l u m n p a c k i n g i n s t e a d

of reconditioning. I f one f e e d s a column w i t h a d i a m e t e r l a r g e r t h a n 20 mm, a good d i s t r i b u t i o n o f t h e sample o v e r t h e c r o s s - s e c t i o n o f t h e c o l u m n i s e s s e n t i a l . point-injection,

Using

t h e column w i l l be c e n t r a l l y o v e r l o a d e d a n d o f t e n n o t i c e a b l e

t a i l i n g or f r o n t i n g i s o b s e r v e d .

I f one w i s h e s t o o b t a i n a h i g h l o a d i n g , o n e

has t o s p r e a d t h e sample homogeneously o v e r t h e whole c r o s s - s e c t i o n ,

as

d e m o n s t r a t e d i n F i g . 4 . 1 6 , where t h e d i s t r i b u t i o n b a f f l e s ( f i v e m e t a l t u b e s ) , t h e g l a s s s p h e r e s and t h e s i e v e p l a t e s a l l o w a homogeneous d i s t r i b u t i o n o f t h e sample ( s e e C h a p t e r 1.7 for a d i s c u s s i o n o f t h e g e n e r a l a p p r o a c h ) . 4 . 4 . 3 . 2 Examples

F i g . 4 . 1 7 i l l u s t r a t e s t h a t e v e n l a r g e r q u a n t i t i e s o f d r u g s c a n be i s o l a t e d w i t h columns o f l a r g e r i n t e r n a l d i a m e t e r s (20 cm).

Here a m i x t u r e o f

p r o s t a g l a n d i n s was s e p a r a t e d i n t o p u r i f i e d components o n a l o n g i t u d i n a l l y compressed 100 x 20 c m - i . D .

column o f s i l i c a g e l .

Although, t h i s i s n o t a " v e r y

p r e t t y " chromatogram. v e r y l a r g e amounts o f h i g h l y p u r i f i e d ( e . g . p r o s t a g l a n d i n e E) were i s o l a t e d i n a s i n g l e r u n [ 3 5 1 .

150 g o f

Columns o f t h i s i n t e r n a l

d i a m e t e r can be used t o p r e p a r e k i l o g r a m q u a n i t i t i e s o f p u r i f i e d m a t e r i a l s w i t h i n a reasonable t i m e .

178 MOBILE PHASE

-

SIEVE PLATE

DISTRIBUTION BAFFLES

GLASS SPHERES

COLUMN

Fig. 4.16.

Dosing distributor.

PG E

1 Fig. 4.17.

2

3

4 h

Large-scale separation o f prostaglandins. Column, 100 x 20 cm, silicagel (Woelm); mobile phase, ethylacetatelhexane. Reprinted with permission f r o m [351.

179

4 ,t t

t

F i g . 4.18.

t

t

T

! t

Automatic r e p e t i t i v e p r e p a r a t i v e s e p a r a t i o n of d i a s t e r e o m e r i c carbamates.

Column, 122 x 6 . 4 cm. a c i d i c a l u m i n a ; m o b i l e p h a s e , R e p r i n t e d from [ 3 6 1 w i t h p e r m i s s i o n

benzene; sample w e i g h t , 1 g.

Such i s o l a t i o n s , of c o u r s e , can be a s s i s t e d b y a u t o m a t i c p r e p a r a t i v e LC. As t h e l a s t example shows, such r e p e t i t i v e LC c a n be u s e d t o i s o l a t e v e r y l a r g e amounts o f h i g h l y p u r i f i e d components.

F i g . 4 . 1 8 shows t h e r e p e t i t i v e

s e p a r a t i o n o f d i a s t e r e o m e r i c c a r b a m a t e s on a c o l u m n o f a c i d i c a l u m i n a ( a b o u t o n e hour r e q u i r e d per i n d i v i d u a l r u n ) .

This [361 system separates c l e a r l y t h e

diastereoisomeres (a = 1.37) [ r e f . 361.

T h i s i s a l s o an e x a m p l e i l l u s t r a t i n g

t h a t w i t h r e p e t i t i v e s e p a r a t i o n gram t o k i l o g r a m q u a n t i t i e s o f m i x t u r e s w i t h s m a l l s e l e c t i v i t i e s c a n a l s o be p r o c e s s e d . 4 . 5 SUMMARY

The r e v i v a

o f l i q u i d c h r o m a t o g r a p h y ( 1 9 7 0 ) , l e a d i n g t o HPLC, i n d u c e d t h e s t u d y

o f p r e p a r a t i v e s e p a r a t i o n s and b r o u g h t a b o u t a number o f r u l e s a n d a p p l i c a t i o n s i n

p h a r m a c e u t i c a l i n d u s t r y , w h i c h can be c l a s s i f i e d a s : - analytical preparative - l a b o r a t o r y s c a l e and

-

production scale preparative.

180

The amounts required are nanograms to kilograms. In the research and development laboratories LC is applied t o the collection o f material for structure identification o r structure elucidation (requirement o f substance [mgl as e.g. when searching for new effective substances in plants o r animals. For these applications analytical instrumentation is used. Quantities in the gram range are also separated and purified by preparative column liquid chromatography. They are collected for chemical, physical o r biological tests. To attain such amounts, apparatus, columns and strategy have t o be adjusted according t o preparative requirements. Preparative column liquid chromatography is presently seldom used in the production stage, except as a cleaning method o r sometimes as a means for the separation o f main components on large diameter columns. Considering the stormy development of liquid chromatographic instrumentation, phases and methods, o n e is inclined t o predict an increase in the importance o f preparative liquid chromatography in the pharmaceutical industry. 4.6 REFERENCES

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20.

E. Geevaert, M. Verzele, Chromatographia, 1 1 (1978) 6 4 0 . L . S . Ettre, Chromatographia, 12 (1979) 302. A. Wehrli, 2 . Anal. Chem., 277 (1975) 289. K.P. Hupe, H . H . Lauer. Chromatographia, 13 (1980) 413. D.G.I. Kingston, J. Nat. Prod., 4 2 (1979) 237. N . Neuss, et. al., Helv., 63 (1980) 1119. C.J.O.R. Morris, P. Morris, Separation Methods in Biochemistry, Pitman Publishing, London, 1976. F. Overzet, HRC and CC, 5 (1982) 604. G.K. Poochikian. J.A. Kelly, J. Pharm. Sci., 7 0 (1981) 182. G. Schmid, C . Goelker, in P. Prave, et. al. (Ed.), Handbuch der Biotechnologie, Akad. Verlagsges., Wiesbaden, 1982. pp. 235-242. E. Heftmann, Chromatography Part A and 6, Elsevier Scientific Publishing, Amsterdam, 1983. L . Fishbein, in E. Heftmann (Ed.). Chromatography Elsevier Scientific Publishing, Amsterdam, 1983, pp 8-287-8-320. K. Wessely, K. Zech. HPLC in Pharmaceutical Analyses, hp, 1979. J.E. Fairbrother and others, Quarterly Reviews, in Pharmaceutical Journals. K.K. Unger, Porous Silica, Elsevier Scientific Publishing, Amsterdam, 1979. E. Gruschka, Bonded Stationary Phases in Chromatography, Ann Arbor Science, Ann Arbor, Michigan, 1974. J.S. Fritz, D.T. Gjerde, G. Pohlandt, Ion Chromatography, Huthig-Verlag, Heidelberg, 1982. W . W . Yau, J.J. Kirkland and D.D. Bly. Modern Size-Exclusion Chromatography, Wi ley-Interscience, New York, 1979. L . R . Snyder and J.J. Kirkland, Introduction t o Modern LC, John Wiley, New York, 1979. J. Turkova, Affinity Chromatography, Elsevier Scientific Publishing, Amsterdam, 1978.

181

21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

A. Wehrli, J.C. Hildenbrand, H.P. Keller, R. Stampfli, R.W. Frei. J . Chromatogr., 149 (1978) 199. J.A. Biesenberger, M. Tan, I. Dudevani, T. Maurer, Polymer Sci., B 9 (1971) 353. D.R. Barker, R.A. Henry, R.C. Williams, D.R. Hudson, J . Chromatogr.,83 (1973) 233. A. Wehrli, Th. Dublin, Ch. Quiquerez, Chromatographia. 7 (1982) 414. R.G. Cooks, (Ed.) Collision Spectroscopy, Plenum Press, New York, 1978. H. Loibner, G. Seidel, Chromatographia, 12 (1979) 169. B.A. Bidlingmeyer, J. Meili, Chemie-Technik, 8 (1979) 169. R. Rosset, Analusis, 5 (1977) 253. J.N. Little, R . L . Cotter, J.A. Prendergast, P.D. McDonald, J . Chromatogr., 126 (1976) 439. A. Wehrli., U. Hermann, J . F . K . Huber, J. Chromatogr., 125 (1976) 59. K . Hostettmann, M.J. Pettel, I. Kubo, K . Nakanishi, Helv., 60 (1977) 670. A. Wehrli, in Instrumentation for HPLC, J.F.K. Huber (Ed.) Elsevier Scientific Publishing, Amsterdam, 1978, pp. 93-111. J. Porath, Biotechol. Bioing. Symp.. 3 (1972) 145. P.E. Barker. C.M. Chuah, Chem. Ing. Tech., 53 (1981) 987. Jobin Yvon, Division d'Instruments S.A. W.H. Pirkle, R.W. Anderson, J. Org. Chem., 39 (1974) 3901.


183

PREPARATIVE 1IQU ID CHROMTOCRAPHY FOR THE SYNTHET IC CHEMIST James K . Whitesell Department of Chemistry The University of Texas a t Austin Austin, Texas 78712

5.1

INTRODUCTION

5.2

COMPUTER SIMULATIONS

5.3

THE REAL WORLD

5.4

PRACTICAL HATTERS

5.5

REFERENCES

5.1

I NTRODUCTI ON

The monumental advances t h a t have t a k e n p l a c e d u r i n g t h e l a s t two decades i n t h e r e a l m o f s y n t h e t i c c h e m i s t r y and e s p e c i a l l y i n t h e t o t a l s y n t h e s i s o f complex n a t u r a l p r o d u c t s have been g r e a t l y f a c i l i t a t e d by t h e i n t r o d u c t i o n o f modern s e p a r a t i o n methods.

I t i s thus n o t s u r p r i s i n g t h a t i t i s t h e s y n t h e t i c

chemist who i s b o t h t h e f i r s t t o e x p l o i t and t h e f i r s t t o e x t o l t h e v i r t u e s o f new advances i n s e p a r a t i o n s c i e n c e .

I t i s the I n t e n t of t h i s chapter to

d e t a i l p r a c t i c a l methods f o r t h e a p p l i c a t i o n of s t a t e - o f - t h e - a r t

technology i n

1 i q u i d chromatography f o r t h e t y p e s of s e p a r a t i o n s t y p i c a l l y encountered b y

the synthetic chemist. The most f r e q u e n t l y used s e p a r a t i o n t e c h n i q u e i s l i q u i d / s o l i d p a r t i t i o n chromatography w i t h s i l i c a g e l as t h e s o l i d s u p p o r t , a system t h a t can be used w i t h a wide range o f compounds and one t h a t i s r e l a t i v e l y i n e x p e n s i v e and simple t o o p e r a t e .

L i q u i d chromatography has e v o l v e d o v e r t h e y e a r s from t h e

e a r l y use o f open columns f i l l e d w i t h s t a t i o n a r y phase, t o l a y e r s spread o n g l a s s p l a t e s , t o c l o s e d and p r e s s u r i z e d columns of s t a i n l e s s steel..

Early a t t e m p t s t o m a i n t a i n reasonable flow r a t e s w i t h f i n e r p a r t i c u l a t e s i l i c a - g e l i n v o l v e d t h e use o f open, g l a s s columns w i t h gas p r e s s u r e p r o v i d e d a t t h e t o p

184 o f a n a t t a c h e d s o l v e n t r e s e r v o i r , a t e c h n i q u e a p p a r e n t l y d i s c o v e r e d by many b u t f o r m a l i z e d by S t i l l [ 1 1 and r e f e r r e d t o as " f l a s h chromatography".

Such a

technique i s cl'early l i m i t e d by t h e n a t u r e o f t h e column m a t e r i a l to, a t most, a few atmospheres o f p r e s s u r i z a t i o n .

The use o f v e r y l a r g e p l a t e s (20 x 80

cm) and t h i c k l a y e r s o f s i l i c a g e l ( 0 . 2 i n . ) p r o v i d e d r e a s o n a b l e r e s o l u t i o n when a s u i t a b l e means of n o n - d e s t r u c t i v e v i s u a l i z a t i o n was a v a i l a b l e , b u t was e x t r e m e l y t i m e consuming and t e d i o u s f o r m u l t i g r a m samples.

Dry packing of

s i l i c a gel intended for t h i c k l a y e r chromatography i n t o a n y l o n tube i m i t a t e d t h e r e s o l u t i o n a v a i l a b l e from l a y e r s [ 2 , 3 1 ,

b u t w i t h a considerable increase

i n t h e q u a n t i t i e s t h a t c o u l d b e p r o c e s s e d a t o n e t i m e , and a d e c r e a s e i n effort.

F i n a l l y , t h e use of metal columns and a l a r g e v a r i e t y of mechanical

s o l v e n t p r e s s u r i z a t i o n systems b r o u g h t t h e a r t t o i t s p r e s e n t s t a t e . The needs o f t h e s y n t h e t i c c h e m i s t for s e p a r a t i o n s c i e n c e a r e q u i t e demanding and i n c l u d e r e q u i r e m e n t s f o r b o t h a n a l y t i c a l and p r e p a r a t i v e t e c h n i q u e s w i t h l i m i t a t i o n s imposed b y s o l v e n t c o s t a n d t o x i c i t y t h a t must be considered for m u l t i g r a m s e p a r a t i o n s .

Whereas an o p t i m a l , a n a l y t i c a l

s e p a r a t i o n f o r a p a r t i c u l a r m i x t u r e m i g h t be a c h i e v e d w i t h a r e v e r s e d phase, C18 column w i t h w a t e r - a c e t o n i t r i l e as m o b i l e p h a s e , t h e c o s t o f t h e C18 m a t e r i a l and t h e t o x i c i t y o f a c e t c n i t r i l e w o u l d p r o h i b i t use o f t h e s y s t e m f o r large scale separations.

These r a t h e r s t r i c t l i m i t a t i o n s h a v e l e d many

s y n t h e t i c g r o u p s t o t h e same, " u n i v e r s a l " s e p a r a t i o n system:

s i l i c a g e l as

s t a t i o n a r y phase w i t h an a p p r o p r i a t e m i x t u r e of e t h y l a c e t a t e and hexane ( S k e l l y - 6 ) as t h e e l u e n t .

A s w i l l be d e v e l o p e d , t h e l i m i t a t i o n s i n r e s o l u t i o n

t h a t may be imposed b y a s t r i c t a d h e r e n c e t o any g i v e n s e p a r a t i o n s y s t e m c a n be c i r c u m v e n t e d i n m o s t c a s e s .

I t s h o u l d be c l e a r t h a t t h e r o u t i n e

a p p l i c a t i o n of a system i n v o l v i n g r e l a t i v e l y i n e x p e n s i v e m a t e r i a l s and non-toxic,

e a s i l y p u r i f i e d , a n d s t a b l e s o l v e n t s t h a t can be r e c o v e r e d and

r e u s e d has d e f i n i t e a d v a n t a g e s w h i c h become a l l t h e more d e s i r a b l e when p r e p a r a t i v e needs a r e c o u p l e d w i t h t h e r e q u i r e m e n t t h a t t h e r e be s i m u l t a n e o u s l y a r a p i d , s i m p l e , and i n e x p e n s i v e means f o r q u a l i t a t i v e a n a l y s i s t h a t p a r a l l e l s as c l o s e l y as p o s s i b l e t h e p r e p a r a t i v e s e p a r a t i n g s y s t e m . O v e r t h e y e a r s t h e r e has been a c o n t i n u a l demand f o r e v e r i n c r e a s i n g r e s o l v i n g a b i l i t y t o k e e p pace w i t h t h e enhanced c o m p l e x i t y o f and r e s u l t i n g s u b t l e t i e s o f d i f f e r e n c e s between m o l e c u l e s u n d e r i n v e s t i g a t i o n .

The two m o s t

i m p o r t a n t f a c t o r s i n f l u e n c i n g t h e q u a l i t y o f s e p a r a t i o n a r e t h e s i z e and u n i f o r m i t y of s i z e o f t h e s o l i d phase p a r t i c l e s , b u t the method of f o r m a t i o n and hence t h e n a t u r e o f t h e s o l i d p h a s e , s p h e r i c a l v e r s u s i r r e g u l a r , a l s o p l a y s a r o l e C41.

U n f o r t u n a t e l y , t h e use o f f i n e r p a r t i c l e s o f u n i f o r m s i z e

185

accompanied by an increase in the head pressure required to maintain a given rate of flow of the mobile phase. The same disadvantage is associated with the use of longer columns to increase separation: each additional column length doubles the pressure and all systems, from flash chromatography t o modern pumping systems are limited in this regard by the technology used to transport the solvent and the nature o f the column material. Modern liquid chromatography pumping systems o f the reciprocating piston type offer a relatively simple solution to this dilemma: multiple passes o f the sample through the same column provide the enhanced resolution of many columns but the back pressure o f only one. Such recycling combined with peak shaving can ultimately provide practical, preparative scale separations even in cases where resolution by classical techniques.such as thick layer chromatography would be impractical. Recycling, and especially the accompanying peak shaving, require that there be available some form of real time method of analysis of the flowing stream. Unfortunately, many synthetic chemists fail to realize the advantages inherent in the use of a real time detector and rely instead on analysis o f a a large number o f fractions collected either by hand or with an automated fraction collector. When thin layer chromatographic analysis is used to provide a qualitative picture o f fraction purity this procedure becomes extremely tedious and, as well, very inaccurate. This is especially true for very poorly resolved mixtures where indeed no apparent resolution might be seen by routine thin layer analysis. What is more, there is a great tendency to err on the side of excessive purity since decisions are made o n a fraction by fraction basis as opposed to the sum collected for each of the components. While a fraction close to the center might contain 20% o f a minor component this is not necessarily an indication that it should not be included in the combined fraction for that material since it is the overall purity level of the combined material which is important. The most frequently heard argument in favor of analysis of individual fractions obtained with a fraction collector is the lack o f a truly universal detection system. The only analysis method currently available which can be consistently relied upon to detect a wide range of compounds is the refractive index detector. Unfortunately such systems inherently rely upon the measurement of a difference in refractive index between the solvent alone and the solvent containing the sample. When the refractive index o f the solvent system is quite close to the components being analyzed this method can be

186

q u i t e i n s e n s i t i v e and i s q u i t e l i m i t e d i n many cases f o r a c c u r a t e a n a l y s i s on small q u a n t i t i e s .

On t h e o t h e r hand, t h e r e l a t i v e l y h i g h c o n c e n t r a t i o n s used

i n p r e p a r a t i v e s e p a r a t i o n methods almost always r e s u l t s i n s u f f i c i e n t d e t e c t o r response f o r q u a l i t a t i v e a n a l y s i s o f t h e degree o f s e p a r a t i o n .

Indeed, we

have never been m i s l e d i n o u r 8 years o f e x p e r i e n c e u s i n g o n l y r e f r a c t i v e index d e t e c t o r s for p r e p a r a t i v e s e p a r a t i o n s on s c a l e s from 500mg t o 509. The use o f any modern s e p a r a t i o n tool t h a t i n v o l v e s an o n g o i n g a n a l y s i s of t h e e f f l u e n t r e q u i r e s a f i r m u n d e r s t a n d i n g o f t h e f a c t o r s t h a t c o n t r i b u t e to t h e e f f e c t i v e r e s o l u t i o n o f a m i x t u r e and a c l e a r p i c t u r e o f t h e degree of s e p a r a t i o n t h a t i s t o be expected from a g i v e n chromatographic t r a c e . U n f o r t u n a t e l y , t h e s y n t h e t i c chemist i s u s u a l l y p o o r l y t r a i n e d i n t h i s regard.

The degree o f r e s o l u t i o n o f two (or more) components o f a m i x t u r e i s

a combination o f t h e a b s o l u t e s e p a r a t i o n o f t h e peak c e n t e r s and t h e degree of spreading i n h e r e n t i n each ( b o t h measured on a common s c a l e , eg. t i m e , e l u e n t volume).

C l e a r l y , i f e i t h e r f a c t o r were optimum then t h e o t h e r c o u l d be

ignored.

I f t h e peaks were i n f i n i t e l y narrow, then any r e s o l u t i o n o f t h e

peaks would l e a d t o q u a n t i t a t i v e s e p a r a t i o n , or i f t h e peaks were i n f i n i t e l y separated (one component m o b i l e , t h e o t h e r n o t ) t h e n t h e degree o f s p r e a d i n g would be u n i m p o r t a n t .

R e a l i s t i c a l l y , both f a c t o r s p l a y a r o l e .

peak spreading i s due m a i n l y to two f a c t o r s :

The e x t e n t of

t h e use o f a porous s t a t i o n a r y

phase i n which t h e e n t i r e p a r t i c l e i s a v a i l a b l e as w e l l as t h e o c c u r r e n c e o f v o i d s between p a r t i c l e s .

Various molecules o f a p a r t i c u l a r component e i t h e r

r a c e ahead t h r o u g h t h e v o i d s w i t h no i n t e r a c t i o n w i t h s t a t i o n a r y phase, o r t r a i l behind as t h e y d i f f u s e t o t h e i n t e r i o r and then back o u t o f t h e particles.

The d i s t r i b u t i o n o v e r t i m e i n t h e f l o w i n g steam t h a t r e s u l t s from

t h i s b e h a v i o r i s r o u g h l y Gaussian.

The use o f u n i f o r m l y s i z e d s t a t i o n a r y

phase m a t e r i a l g r e a t l y reduces t h e v o i d volume w h i l e v e r y s m a l l p a r t i c l e s minimizes t h e spreading t h a t o c c u r s because some molecules t o u c h o n l y on t h e surface while others simultaneously are d i f f u s i n g through the i n t e r i o r of t h e particles.

O b v i o u s l y , and u n f o r t u n a t e l y , t h e s m a l l e r t h e p a r t i c l e and t h e

more u n i f o r m l y i t i s packed t h e g r e a t e r i s t h e p r e s s u r e r e q u i r e d t o m a i n t a i n a

given solvent f l o w r a t e .

Thus, even i f i t were p r a c t i c a l t o manufacture

e c o n o m i c a l l y e x t r e m e l y f i n e , u n i f o r m p a c k i n g , p r a c t i c a l pumping t e c h n o l o g y i s not available.

S i l i c a g e l w i t h an average s i z e o f 5p o v e r a v e r y narrow range

i s a v a i l a b l e for a n a l y t i c a l use, w h i l e m a t e r i a l i n t h e range o f 37-55p i s t h e f i n e s t m a t e r i a l t h a t can be p r a c t i c a l l y used f o r p r e p a r a t i v e s e p a r a t i o n s .

187 The degree of a b s o l u t e s e p a r a t i o n o f t h e peak c e n t e r s i s c o n t r o l l e d b y a l a r g e number o f parameters, t h e most i m p o r t a n t o f which a r e t h e n a t u r e o f t h e components themselves ( s e l e c t l v i t y ) , t h e s t a t i o n a r y and m o b i l e phases ( c a p a c i t y f a c t o r ) , and t h e l e n g t h o f t h e column ( e f f i c i e n c y ) .

This l a s t

f a c t o r i s t h e most e a s i l y c o n t r o l l e d and has a d i r e c t l y p r o p o r t i o n a l e f f e c t : d o u b l i n g t h e column l e n g t h leads t o t w i c e t h e d i s t a n c e between t h e peaks centers.

On t h e o t h e r hand, a d o u b l i n g o f t h e column l e n g t h l e a d s o n l y t o

s p r e a d i n g of t h e peaks by a f a c t o r o f 1.414 (square root o f 2 ) , a d i r e c t r e s u l t o f t h e Gaussian d i s t r i b u t i o n o f t h e peak.

The r e a d e r i s r e f e r r e d t o

Chapter 1.3 f o r a more thorough d i s c u s s i o n o f r e s o l u t i o n and c o n t r i b u t i o n s t o t h e s e p a r a t i o n o f peaks. 5.2

COMPUTER SIMULATIONS

The c o m b i n a t i o n o f these two e f f e c t s i s g r a p h i c a l l y i l l u s t r a t e d i n F i g . 5.1, a computer s i m u l a t i o n of t h e r e s o l u t i o n t o be expected by u s i n g one, two, t h r e e and f o u r i d e n t i c a l columns.

The d o t t e d l i n e s r e p r e s e n t t h e i n d i v i d u a l

components w h i l e t h e s o l i d l i n e r e p r e s e n t s t h e sum o f b o t h .

Of course, i t i s

o n l y t h i s l a t t e r , c o m b i n a t i o n t r a c e t h a t i s produced by t h e d e t e c t o r .

The

f i g u r e i n c l u d e s s i m u l a t i o n s for m i x t u r e s r a n g i n g i n r a t i o from 1 : l t o 1 O : l . w i t h t h e h i g h e r r a t i o s b e i n g more r e p r e s e n t a t i v e o f t y p i c a l t h o s e produced by the synthetic chemist.

U n f o r t u n a t e l y , t h e enhanced r e s o l u t i o n achieved i s t o

some e x t e n t masked by t h e peak spreading t h a t g i v e s t h e f a l s e i m p r e s s i o n t h a t r e s o l u t i o n i s decreasing.

However, t h e i m p o r t a n t parameter i s n e i t h e r t h e

peak w i d t h nor t h e s e p a r a t i o n between peaks b u t r a t h e r t h e i r r a t i o .

Provided

underneath each computed t r a c e i s t h e p u r i t y o f each component, or "peak",

if

two f r a c t i o n s were t a k e n about t h e p o i n t where t h e i n d i v i d u a l component t r a c e s c r o s s , i n d i c a t e d by t h e t i c k mark.

Those chemists w i t h o u t e x t e n s i v e

chromatographic t r a i n i n g almost always e s t i m a t e a l e v e l o f p u r i t y lower t h a n t h a t shown.

For example, w h i l e t h e f i r s t t r a c e for each m i x t u r e shows n o

outward i n d i c a t i o n o f s e p a r a t i o n , a simple m i d p o i n t d i v i s i o n would l e a d t o two f r a c t i o n s each o f 82% p u r i t y f r o m t h e o r i g i n a l 1 : l m i x t u r e .

The use o f two

columns i n s e r i e s p r o v i d e s a p u r i t y l e v e l o f 91%, and t h r e e wou d r e s u l t i n 94% p u r i t y , a c c e p t a b l e f o r many purposes i n t o t a l s y n t h e s i s .

A

would be

a n t i c i p a t e d , t h e p u r i t y l e v e l i n c r e a s e s f o r t h e major component as t h e o r i g i n a l m i x t u r e i s made unequal, w h i l e t h e p u r i t y o f t h e minor component p a r a l l e l s the decline i n i t s o r i g i n a l concentration.

w

n

I\

I/

Pu

M I N v42

n

PEM

M I W 942 94

PEM

M I T V

Tex 98

F i g . 5.2.

741 74

411 b1

RcoIlERl 472 47

E f f e c t o f s h a v i n g o v e r l a p p e d peaks upon t h e p u r i t y o f t h e c o l l e c t e d fractions.

Peak C o n c e n t r a t i o n r a t i o 1 : l .

94

89

190

There a r e many f a c t o r s which combine t o l i m i t t h e enhancement o f r e s o l u t i o n t h a t can be o b t a i n e d s i m p l y by t h e use o f a d d i t i o n a l columns.

I n a d d i t i o n to

t h e obvious c o s t o f t h e columns and p a c k i n g , t h e s o l v e n t r e q u i r e m e n t i n c r e a s e s and t h e c o n c e n t r a t i o n o f samples decreases, c o m p l i c a t i n g i s o l a t i o n o f p u r i f i e d materials.

C r i t i c a l l y , t h e p r e s s u r e r e q u i r e d t o m a i n t a i n a g i v e n flow r a t e

increases p r o p o r t i o n a l l y w i t h each a d d i t i o n a l l e n g t h o f column, u n t i l e i t h e r the l i m i t o f t h e column m a t e r i a l or t h e p r e s s u r i z a t i o n system i s exceeded. The s o l u t i o n t y p i c a l l y taken i s t o employ as l o n g a column (or c o m b i n a t i o n o f columns) as p o s s i b l e and t o s e p a r a t e t h e m i x t u r e i n t o t h r e e f r a c t i o n s , w i t h t h e m a t e r i a l from the m i d d l e b e i n g e i t h e r d i s c a r d e d or r e t a i n e d f o r f u r t h e r chromatographic p u r i f i c a t i o n .

The e f f e c t o f " s h a v i n g " h i g h l y pure f r a c t i o n s

i n t h i s f a s h i o n f r o m t h e head and t a i l i s i l l u s t r a t e d i n F i g . 5 . 2 . The t o p t h r e e t r a c e s i l l u s t r a t e t h e r e s u l t o f s e p a r a t i o n i n t o o n l y two f r a c t i o n s , as i n F i g . 5 . 1 , w h i l e i n t h e b o t t o m t h r e e , p o i n t s f o r s h a v i n g were s e l e c t e d so as t o o b t a i n 94, 96, and 98% p u r i t i e s o f t h e a p p r o p r i a t e component i n t h e f i r s t and t h i r d f r a c t i o n s .

The r e c o v e r i e s o f t h e two

components i n these o u t e r f r a c t i o n s i s a l s o p r o v i d e d .

Again, most chemists

would g r e a t l y underestimate e i t h e r t h e l e v e l o f p u r i t y o r t h e degree o f r e c o v e r y t o be expected under these c i r c u m s t a n c e s .

For m o s t s y n t h e t i c

purposes 96% p u r i t y i s s a t i s f a c t o r y and indeed, m o s t a n a l y t i c a l t o o l s used i n a r o u t i n e f a s h i o n by t h e s y n t h e t i c chemist (eg. ' H and 13C NMR) would n o t d e t e c t 4% c o n t a m i n a t i o n s i n c e t h e i m p u r i t i e s a r e g e n e r a l l y q u i t e s i m i l a r i n s t r u c t u r e . Note t h a t i n t h e l e a s t w e l l r e s o l v e d case, o b t a i n i n g t h e h i g h e s t l e v e l o f p u r i t y (98%) r e s u l t s i n an u n a c c e p t a b l e 22% r e c o v e r y , w h i l e i n t h e b e s t S e p a r a t i o n i l l u s t r a t e d , 81% of each component can be recovered wtth t h i s p u r i t y . The s i m u l a t i o n s i n F i g . 5 . 2 a r e f o r an equal m i x t u r e o f two, c l o s e l y r e l a t e d substances, a r e l a t i v e l y r a r e (as w e l l as embarrassing) s i t u a t i o n f o r t h e s y n t h e t i c chemist who expends c o n s i d e r a b l e e f f o r t i n t h e d e s i g n o f schemes t o c o n t r o l t h e course o f r e a c t i o n s t o f a v o r t h e d e s i r e d outcome. cases a r e i l l u s t r a t e d i n F i g s . 5.3, 5 . 4 and 5.5

More t y p i c a l

where t h e two components a r e

i n 2:1, 4:l and 1 O : l r a t i o s . The f o r e g o i n g d i s c u s s i o n a p p l i e s t o any s e p a r a t i o n t e c h n i q u e t h a t p r o v i d e s a r e a l t i m e a n a l y s i s o f t h e f l o w i n g stream as i t emerges from t h e column, and t h e s y n t h e t i c chemist who wishes t o t a k e f u l l advantage o f t h e c a p a b i l i t i e s of modern f a c i l i t i e s for chromatographic s e p a r a t i o n would be w e l l a d v i s e d t o s t u d y c a r e f u l l y t h e r e s o l u t i o n o b t a i n e d under t h e v a r i o u s s i m u l a t i o n I n t h e absence o f a c l e a r p e r s p e c t i v e of t h e

conditions i n Figs. 5 . 2 - 5 . 5 .

n

n

PEW

PEW 1 2

RRITY 961 96

m 22

REcDllERy

-1 61

w PEW 2

f l g . 5.3.

RRITY 941 94

PVIIW 9.z

m

RcDIlERI 461

u

Effect o f shaving overlapped peaks upon the purity o f the collected fractions. Peak concentration ratio 2.1.

1 2

2

941

961

94

n6

961 96

941 81

I(0 N

2

Fig. 5.4.

. ... 94

47

Effect of shaving overlapped peaks upon the purity o f the collected fractions. Peak concentration ratio 4.1.

2

2

A ,,

pEIy(

1 2

RRITY 96X

REtDIlERv 9n

2

F i g . 5.5

E f f e c t o f s h a v i n g o v e r l a p p e d peaks upon t h e p u r i t y o f t h e c o l l e c t e d fractions.

Peak c o n c e n t r a t i o n r a t i o 1O:l.

194

d e g r e e of p u r i f i c a t i o n t h a t c a n be o b t a i n e d w i t h a n y g i v e n o v e r l a p of components, t h e u s u a l r e s p o n s e i s to e r r on t h e c o n s e r v a t i v e s i d e l e a d i n g t o sample p u r i t i e s f a r i n e x c e s s of t h a t r e q u i r e d and c o r r e s p o n d i n g l y p o o r recoveries.

While l a r g e m i d d l e f r a c t i o n s c o n t a i n i n g s u b s t a n t i a l q u a n t i t i e s of

b o t h components can be r e p r o c e s s e d , c o n s i d e r a b e a d d i t i o n a l e f f o r t i s r e q u i r e d t o d o so i n m o s t c i r c u m s t a n c e s .

Many e a r l y , commerci a1 1 i q u l d c h r o m a t o g r a p h

p r o v i d e d p r e s s u r e from a

t r a p p e d volume of s o l v e n t w i t h e i t h e r e x t e r n a l gas p r e s s u r e ( n i t r o g e n ) or a mechanism s i m i l a r t o a l a r g e s y r i n g e p r o v i d i n g t h e p u s h .

M o s t modern

c h r o m a t o g r a p h i e s use r e c i p r o c a t i o n p i s t o n pumps t h a t d r a w from a s o l v e n t r e s e r v o i r a t atmospheric pressure.

Such i n s t r u m e n t a t i o n p r o v i d e s a u n i q u e

o p p o r t u n i t y for p r e p a r a t i v e s e p a r a t i o n s s i n c e i t i s q u i t e f e a s i b l e t o r e c y c l e t h e e f f l u e n t from t h e c o l u m n v i a t h e pump for a second p a s s t h r o u g h t h e c o l u m n

[51.

The p r o c e s s can t h e n be r e p e a t e d , w i t h e a c h s u b s e q u e n t pass r e s u l t i n g i n

a n i n c r e a s e o f t h e s e p a r a t i o n b e t w e e n peaks r e l a t i v e t o t h e w i d t h s .

The

e f f e c t i s p r e c i s e l y t h e same a s t h a t o b t a i n e d b y u s i n g a d d i t i o n a l c o l u m n s , e q u a l to t h e number o f r e c y c l e s , e x c e p t t h a t n e i t h e r t h e c o s t o f t h e columns and p a c k i n g nor t h e b a c k p r e s s u r e i s i n c r e a s e d t h r o u g h r e c y c l i n g .

I t should

be n o t e d t h a t enhancement i n r e s o l u t i o n o c c u r s t h r o u g h r e c y c l i n g o n l y i f t h e m i x i n g volume p r e s e n t i n t h e pump h e a d s , i n j e c t o r , a n d a s s o c i a t e d p l u m b i n g i s r e l a t i v e l y s m a l l compared w i t h t h e volume o f s o l v e n t i n w h i c h t h e p e a k i s eluting.

The p e a k s p r e a d i n g t h a t o c c u r s from t h i s dead volume can o f t e n be a

l i m i t i n g f a c t o r i n t h e u s e o f r e c y c l i n g w i t h s m a l l b o r e , a n a l y t i c a l columns b u t i s u n i m p o r t a n t i n m u l t i g r a m , l a r g e volume s y s t e m s .

An expanded d i s c u s s i o n

of r e c y c l e occurs i n Chapter 1.7. M u l t i p l e r e c y c l i n g i s u l t i m a t e l y l i m i t e d b y t h e f a c t t h a t peak w i d t h s a r e i n c r e a s i n g w i t h e a c h s u b s e q u e n t p a s s t h r o u g h t h e c o l u m n u n t i l t h e l e a d i n g edge o v e r t a k e s t h e t r a i l i n g edge, h o p e l e s s l y m i x i n g t h e f a s t e r a n d s l o w e r m o v i n g components.

T h i s s i t u a t i o n i s o f t e n a r r i v e d a t a f t e r only o n e r e c y l e w i t h

l a r g e sample s i z e s t h a t o v e r l o a d t h e c a p a c i t y o f t h e column. solution exists.

A simple

B o t h t h e l e a d i n g a n d t r a i l i n g edges a r e removed, or s h a v e d ,

o n each p a s s a n d o n l y t h e p o o r l y r e s o l v e d , m i d d l e f r a c t i o n c o n t a i n i n g b o t i components i s r e c y c l e d t h r o u g h t h e c o l u m n .

Even w i t h p o o r l y r e s o l v e d

components, s h a v i n g c a n r e s u l t i n f r a c t i o n s r e p r e s e n t i n g h i g h p u r i t y m a t e r i a l s as i l l u s t r a t e d i n F i g . 5.5.

Such a p r o c e s s o f s h a v i n g two o u t s i d e f r a c t i o n s

o f h i g h l y p u r e m a t e r i a l and d i r e c t l y r e c y c l i n g t h e p o o r l y r e s o l v e d m i d d l e f r a c t i o n t h r o u g h t h e column c a n be u s e d r e p e a t e d l y u n t i l t h e q u a n t i t y r e m a i n i n g i n t h e m i d d l e f r a c t i o n becomes u n i m p o r t a n t or t h e p a t i e n c e o f t h e o p e r a t o r i s expended.

195

Fig. 5.6.

Computer s i m u l a t i o n o f r e s o l u t i o n f o r f o u r p a s s e s t h r o u g h a column.

Peak c o n c e n t r a t i o n r a t i o 4 : l .

F i g . 5.6 i l l u s t r a t e s t h i s concept for four passes o f a 4 . 1 m i x t u r e t h r o u g h a column where t h e d e g r e e of r e s o l u t i o n i s t h e same a s t h e worst c a s e s i n F i g s 5.5.

The r e c o v e r y l e v e l s i n d i c a t e d a r e f o r e a c h p a s s o n l y , w h i l e t h e t o t a l

recovery of p u r i f i e d m a t e r i a l i s i n d i c a t e d below i n Table 5.1. TABLE 5 . 1

T o t a l Recovery 2

3

4

component 1

52

80

92

95

component 2

38

77

86

90

a f t e r pass

1

-

196 A f t e r f o u r passes, a t o t a l o f 95% o f t h e major component c o u l d be o b t a i n e d w i t h a 98% p u r i t y .

The shaving p o i n t s f o r t h e minor component f r a c t i o n s

were s e l e c t e d so as t o p r e v e n t any p o s s i b l e o v e r l a p o f t h e t r a i l i n g edge w i t h t h e more mobile m a t e r i a l , r a t h e r t h a n t o maximize e i t h e r t h e p u r i t y or t h e recovery o f t h i s material.

Nonetheless, t h e combined f o u r f r a c t i o n s o f t h e

minor component would have a p u r i t y o f 86% and r e p r e s e n t a 90% r e c o v e r y of this material.

I t should be c l e a r t h a t t h e g r e a t e r t h e amount o f m a t e r i a l

t h a t i s removed i n each shaving o p e r a t i o n , t h e b e t t e r .

Again, i t i s

recommended t h a t t h e shaving p o i n t s i l l u s t r a t e d i n F i g s . 5.2-5.5 be s t u d i e d carefully. On f i r s t i n s p e c t i o n i t m i g h t be f e l t t h a t t h e same o v e r a l l e f f e c t c o u l d be o b t a i n e d by t h e more c l a s s i c a l approach o f t a k i n g a m i d d l e f a c t i o n which i s then c o n c e n t r a t e d and s e p a r a t e l y rechromatographed.

However

such an

approach would n o t make use o f t h e moderate s e p a r a t i o n o f t h e components t h a t

i s apparent i n t h e m i d d l e f r a c t i o n .

T h i s can be e a s i l y seen w i t h t h e more

d i f f i c u l t s e p a r a t i o n i l l u s t r a t e d i n F i g . 5.7, where t h e i n i t i a l m i x t u r e c o n t a i n s equal amounts o f b o t h components.

The t o t a l r e c o v e r i e s a f t e r each

pass a r e compiled i n Table 5.2, f r o m which i t can be seen t h a t 92% o f each component can be o b t a i n e d i n 98% p u r i t y a f t e r f o u r passes. TABLE 5.2

T o t a l Recovery

1

2

3

4

component 1

22

63

83

92

component 2

22

63

83

92

a f t e r pass

N o t i c e t h a t t h e l e v e l o f r e c o v e r y a t 98% p u r i t y for each r e c y c l e ( s e e F i g .

5.7) jumped from 22% f o r t h e f i r s t pass t o 53, '55, and 59% for t h e second, t h i r d and f o u r t h passes, d r a m a t i c a l l y i l l u s t r a t i n g t h e enhanced r e s o l u t i o n t h a t r e s u l t s a f t e r t h e m i d d l e f r a c t i o n has passed t h r o u g h t h e column t w i c e . Were t h e m i d d l e f r a c t i o n n o t d i r e c t l y r e c y c l e d b u t i s o l a t e d by c o n c e n t r a t i o n and s e p a r a t e l y rechromatographed, each r u n would appear as t h e f i r s t t r a c e w i t h a r e c o v e r y o f 22% o f each component p r e s e n t .

Eleven s e p a r a t e

chromatographic r u n s would be r e q u i r e d t o a c h i e v e t h e same r e c o v e r y of 92% w i t h a p u r i t y l e v e l o f 98% as o b t a i n e d from t h e t h r e e r e c y c l e s ( f o u r passes) i l l u s t r a t e d i n F i g . 5.7. C l e a r l y , a compound would have t o be e x c e p t i o n a l l y v a l u a b l e t o w a r r a n t eleven runs t o achieve reasonable p u r i t y and r e c o v e r y l e v e l s and i n most cases t h e s e p a r a t i o n would be d e c l a r e d too d i f f i c u l t and n o t even a t t e m p t e d .

But

197

PEAK

PURITY 90%

I

22

M I T V

PEAK

RECOVERY

mz m

1

2

PEAK I 2

M I T V

PEM

PURITY

13% 53

RECDVERV

mz m

I

S?%

S?

RECOVERY

ssx

982 90

2

Fig. 5.7.

RECOVERY 22%

m

2

ss

Computer s i m u l a t i o n o f r e s o l u t i o n by c o l l e c t i n g f r a c t i o n s and rechromatographing t h e o v e r l a p p e d f r a c t i o n .

the savings i n h e r e n t i n t h e use o f r e c y c l i n g combined w i t h s h a v i n g go beyond. the o b v i o u s .

O f prime importance i s t h e f a c t t h a t s o l v e n t i s consumed o n l y

when m a t e r i a l i s a c t u a l l y b e i n g c o l l e c t e d : stream i s reused.

d u r i n g recycle, the e f f l u e n t

Because s a t i s f a c t o r y s e p a r a t i o n can u l t i m a t e l y

be achieved even w i t h v e r y poor s e p a r a t i o n s by m u l t i p l e r e c y c l i n g , a h i g h e r l e v e l o f o v e r l o a d i n g can be t o l e r a t e d , r e s u l t i n g i n savings o f b o t h column m a t e r i a l and s o l v e n t . I t i s i m p o r t a n t t o keep i n mind t h a t t h e f u n c t i o n of s h a v i n g i s not t o c o l l e c t m a t e r i a l b u t r a t h e r t o p r e v e n t t h e l e a d i n g edge o f t h e chromatogram

from o v e r t a k i n g t h e t r a i l i n g m a t e r i a l d u r i n g t h e s p r e a d i n g t h a t o c c u r s w i t h recycling.

I t i s t h e m u l t i p l e r e c y c l i n g t h a t p r o v i d e s t h e enhanced

198 r e s o l u t i o n , making p r a c t i c a l l a r g e scale separations of p o o r l y r e s o l v e d materials.

The more p o o r l y t h e m a t e r i a l s a r e r e s o l v e d , t h e l e s s t h e t o t a l

m a t e r i a l s w i l l s p r e a d w i t h e a c h p a s s a n d t h e g r e a t e r t h e number o f r e c y c l e s t h a t can be a c h i e v e d b e f o r e s h a v i n g i s r e q u i r e d .

5 . 3 THE REAL WORLD

The f o r e g o i n g d i s c u s s i o n s were b a s e d o n t h e c o m p u t e r s i m u l a t e d d i s p l a y s i n Figs. 5.1-5.7.

T h e r e a r e many f a c t o r s w h i c h d e g r a d e p e r f o r m a n c e i n a c t u a l

chromatographic s i t u a t i o n s b u t t h e most important a r e those which impart asymmetry t o t h e peak s p r e a d i n g .

E s p e c i a l l y i n t h e c a s e where t h e d e s i r e d

component i s t h e s l o w e r moving, non-Gaussian d i s t r i b u t i o n s w i l l l e a d t o a g r e a t e r d e g r e e o f c o n t a m i n a t i o n and h e n c e l o w e r r e c o v e r i e s p e r c y c l e for a given desired p u r i t y l e v e l .

Nonetheless, the importance of t h e concept of

r e c y c l i n g combined with s h a v i n g s t i l l h o l d s . F i g s . 5.8 and 5.9 a r e a c t u a l c h r o m a t o g r a p h i c t r a c e s of s e p a r a t i o n s e f f e c t e d u s i n g t h e Waters Pr.epLC/System 500 p r e p a r a t i v e l i q u i d c h r o m a t o g r a p h .

It is

i m p o r t a n t to n o t e t h a t the c r i t i c a l f a c t o r here i s n o t t h e t e c h n o l o g y used b u t r a t h e r t h e i m p l e m e n t a t i o n o f t h e c o n c e p t of r e c y c l i n g combined w i t h p e a k shaving.

We have s u c c e s s f u l l y u s e d t h e same c o n c e p t for s m a l l e r s c a l e

Recycle

IWaste KI Collect

1

IEI

2

"

433 1

Fig., 5.8.

P r e p a r a t i v e s e p a r a t i o n o f two e p l m e r i c k e t o n e s .

o f a 1 : l m i x t u r e were i n j e c t e d .

T h i r t y - f i v e grams

199 s e p a r a t i o n s u s i n g 318 i n . by 2 f t . columms w i t h a Water's Model 6000A s o l v e n t d e l i v e r y system a t 9 . 9 m l l m i n .

f i g . 5 . 8 shows t h e s e p a r a t i o n o f a p p r o x i m a t e l y

359 of a 1 : l m i x t u r e of t h e two e p i m e r i c ketones f o r t h e s y n t h e s i s o f i r i d o i d terpenes.

1 and 2. t h a t

we have employed

The degree of s e p a r a t i o n on s i l i c a g e l

w i t h an a p p r o p r i a t e m i x t u r e of e t h y l a c e t a t e l h e x a n e i s such t h a t t h e components cannot be f u l l y r e s o l v e d on a 3 i n . t h i n l a y e r p l a t e and appear as an h o u r g l a s s . The e l u e n t was c o l l e c t e d through t h e s o l v e n t f r o n t u n t i l t h e samples began t o emerge ( d u r i n g pass 1 ) and t h e n t h e t o t a l sample was r e c y c l e d .

Some

o v e r l a p o f t h e t a i l i n g and l e a d i n g edges can be seen between pass 1 and 2 as t h e o r i g i n a l b a s e l i n e was n o t r e - e s t a b l i s h e d , and a s m a l l , mixed f r a c t i o n was taken a t t h i s p o i n t .

The more m o b i l e , exo k e t o n e was t h e n c o l l e c t e d up t o t h e

top o f t h e f i r s t peak, t h e middle m a t e r i a l r e c y c l e d , and t h e endo k e t o n e c o l l e c t e d f r o m t h e t o p o f t h e second peak.

Again, a mixed f r a c t i o n was t a k e n

between pass 2 and 3, and t h e process r e p e a t e d two more t i m e s .

The sharp

d i s l o c a t i o n s i n t h e t r a c e i n t h e m i d d l e o f pass 3 and between 3 and 4 a r e due t o i n c r e a s e s i n d e t e c t o r s e n s i t f v i t y a t these p o i n t s .

The f i n a l m i d d l e

f r a c t i o n i n pass 4 was n o t r e c y c l e d b u t c o l l e c t e d and combined w i t h t h e o t h e r mixed f r a c t i o n s .

Note t h a t t h e s e n s i t i v i t y d u r i n g pass 4 i s f i v e t i m e s

g r e a t e r than d u r i n g t h e f i r s t pass, and thus r e l a t i v e l y l i t t l e m a t e r i a l i s i n v o l v e d i n t h i s l a s t , mixed f r a c t i o n .

The combined f r a c t i o n s o f endo and exo

ketone amounted t o 1 3 . 8 and 15.39. r e s p e c t i v e l y . r e p r e s e n t i n g a t o t a l r e c o v e r y i n these f r a c t i o n s o f 83%.

A n a l y s i s o f these f r a c t i o n s by a n a l y t i c a l LC

showed t h a t each was i s o m e r i c a l l y pure t o a t l e a s t t h e l e v e l o f 9 9 : l . The s e p a r a t i o n i l l u s t r a t e d above demonstrates n o t o n l y t h e e f f e c t i v e n e s s o f r e c y c l i n g b u t a l s o t h e r e s u l t o f o v e r c a u t i o u s shaving.

The o v e r l a p o f t h e

t a i l i n g edge o f pass one and t h e l e a d i n g edge o f pass two was t h e d i r e c t r e s u l t o f f a i l u r e t o shave m a t e r i a l f r o m t h e f r o n t o f t h e peak d u r i n g pass one, and had t h e t a i l i n g edge been shaved d u r i n g t h e f i r s t pass, t h e n t h e o v e r l a p between passes 2 and 3 c o u l d have been avoided.

While t h e f i r s t pass

e x h i b i t s no outward s i g n s o f s e p a r a t i o n , some m a t e r i a l o f h i g h p u r i t y c o u l d have been removed a t t h a t t i m e .

Nonetheless, a v e r y r e s p e c t a b l e r e c o v e r y o f

m a t e r i a l o f v e r y h i g h p u r i t y was o b t a i n e d i n a t o t a l chromatographic tir

25 m i n u t e s . employed.

of

A l t e r n a t i v e l y , a l e s s p o l a r s o l v e n t system c o u l d have been The e f f e c t o f decreasing s o l v e n t p o l a r i t y i n a system such as e t h y l

acetate-hexane i s e s s e n t i a l l y t h e same as t h a t observed ( F i g . 5.1) by us n i l a d d i t i o n a l column l e n g t h : i f t h e peak i s r e t a i n e d t w i c e as l o n g t h e h a l f - h e i g h t w i d t h w i l l be i n c r e a s e d by 2 .

200

3

Fig. 5.9. Preparative separation of isomeric aldehydes. injected.

S e v e n grams w e r e

A n even m o r e difficult separation is illustrated in Fig. 5.9 f o r t h e regioisomeric aldehydes 2 and 4. The difference between these isomers is structurally quite subtle a n d , as a result, they a r e o n l y poorly resolved e v e n using analytical LC and n o resolution can be seen by T L C analysis. The sample, approximately 7 9 , was slightly enriched ( - 2 : l ) in t h e desired isomer 4. Since o n l y the trailing component was desired, shaving w a s restricted t o this component. After six passes, a total of 2 . 7 9 of 4 had been collected, t h e remainder saved f o r further separation. T h e purity of t h i s material w a s excellent (>98%) as can be seen f r o m the analytical trace.

5 . 4 PRACTICAL MATTERS

Large scale separations require large quantities o f solvent, regardless o f the methods and technology employed. Where t h e individual solvent components are stable in storage, it is quite practical t o recover and r e u s e t h e solvent mixtures. Both t h e undeslred fractions and t h e distillate f r o m concentration of desired fractions can be combined and distilled through a s h o r t , packed column. Analysis of the mixture by g a s chromatography provides m o r e than

201

sufficient accuracy by simply comparing peak height ratios with a calibration curve made from analysis of standard mixtures. If all recovered solvent, regardless of ratio, is combined before distillation, then the distillates will tend to a median solvent ratio that will rarely be close to that desired. Large quantities of either the more o r less polar solvent will thus be required to adjust the ratio to that needed. The result is that the quantity o f recovered solvent grows faster than that which is lost in handling. A better system involves segregation o f the solvent to be recovered into two or, better, three categories such as less than 5.1 between 5:l and 1O:l. and greater than 1O:l. Individual batches o f recovered solvent are then often found to be sufficiently close to the desired ratio that adjustment is not required. Large scale chromatography a1 so involves dedication of correspondingly large quantities of silica gel. Economical operation again requires reuse of the stationary phase. This presents no problems as long as all materials placed on the column are removed after each separation. This necessitates that samples are prepurified to remove very polar impurities that would otherwise collect at the beginning of the column, quickly raising the back pressure to unusable levels. This can be easily accomplished by a simple "filtration" of the sample through a short glass column of silica gel using the same solvent system that will be used to effect the more careful chromatography to follow. The silica gel used in this initial, crude separation need not be of high quality but this stage should not be hurried by, for instance, forcing the solvent through the column. Each separation should be followed by flushing the column with at least two column volumes of the more polar solvent which can be distilled and reused for the same purpose. This column cleanup will reduce the possibility of cross contamination from one sample to the next.

5.5

REFERENCES

W.C. Still, M. Kahn and A . Mitra, J . Org. Chem., 43 (1978)2923 6. Loev and K.M. Snader, Chem. Ind. (London) 1965, 15. 6 . Loev and M.M. Goodman, Chem. Ind. (London) 1967. 2026. L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography, John Wiley and Sons, Second Edition, New York, 1979, p. 178. 5. K. Conroe, Chromatographia, 8 (1975)119.

1. 2. 3. 4.


203

B IOCHEMICAL APPL ICAT IONS OF PREPARAT IVE 1IQUID CHROMATOGRAPHY William S. Hancock Department o f Chemistry, Biochemistry and Biophysics, Massey University, Palmerston North, New Zealand. and Ross L . Prestidge Department o f Pathology, Auckland University School o f Medicine, Auckland, New Zealand.

CONTENTS 6.1

INTRODUCTION

6.2

GENERAL REOUIREMENTS FOR AMINO ACID AND POLYPEPTIDE SEPARATIONS 6.2.1 6.2.2 6.2.3 6.2.4

Nature of the Stationary Phase Nature of the Mobile Phase Nature o f the Organic Modifier Some Comments on What Constitutes a Preparative Separation

6.3

PREPARATIVE SEPARATIONS OF AMINO ACIDS AND PEPT IDES

6.4

USE OF REVERSED PHASE THIN LAYER CHROMATOGRAPHY FOR THE MONITORING OF PREPARATIVE SEPARATIONS

6.5

PREPARATIVE SEPARATIONS

6.5.1 6.5.2

OF PROTEINS

Reversed Phase Separations Other Separation Modes

6.6

OTHER CLASSES OF BIOCHEMICALS

6.7

REFERENCES

6.1

INTRODUCTION

It is o f t e n necessary t o prepare large a m o u n t s o f a single biochemical c o m p o n e n t , purified t o homogeneity, f o r further biological o r chemical studies. In many cases, liquid chromatography (LC) is becoming the preparative technique

204

o f choice.

For example, i n t h e sequence d e t e r m i n a t i o n o f a p r o t e i n , LC has made

A r e c e n t r e v i e w by Bhown and B e n n e t t

an impact i n a l l aspects o f t h e procedure.

[ 1 1 d e s c r i b e d t h e use o f g e l f i l t r a t i o n chromatography f o r t h e i s o l a t i o n o f a pure p r o t e i n sample and for t h e i s o l a t i o n o f l a r g e p r o t e i n fragments a f t e r p a r t i a l cleavage o f t h e p o l y p e p t i d e c h a i n .

Reversed phase s e p a r a t i o n s have

proven t o be p a r t i c u l a r l y s u i t a b l e f o r t h e i s o l a t i o n o f m i l l i g r a m amounts o f pure p e p t i d e s f r o m t h e complex m i x t u r e s t h a t a r e formed by d i g e s t i o n o f t h e p r o t e i n w i t h proteases such as t r y p s i n and c h y m o t r y p s i n .

Reversed phase LC i s

a l s o found t o be a v e r y s u i t a b l e t e c h n i q u e f o r t h e p r e p a r a t i o n o f p r o t e i n s such as c e l l s u r f a c e a n t i g e n s and hormones and o t h e r s o l u b l e p r o t e i n s which a r e o n l y s y n t h e s i z e d i n v e r y small amounts i n b i o l o g i c a l systems.

C e r t a i n assay methods,

such as radioimmunoassays may n o t be t o t a l l y s p e c i f i c f o r a p a r t i c u l a r p e p t i d e o r p r o t e i n and thus these r e s u l t s a r e o f t e n c o n f i r m e d by p r e p a r a t i v e LC f o l l o w e d by a s p e c i f i c chromatographic t e c h n i q u e C21.

The use o f p r e p a r a t ve LC i n

b i o c h e m i s t r y i s a l r e a d y s i g n i f i c a n t , and i s r a p i d l y i n c r e a s i n g . T h i s r e v i e w w i l l , t h e r e f o r e , c o n c e n t r a t e on t h e p r e p a r a t i v e s e p a r a t i o n s o f amino a c i d , p e p t i d e and p r o t e i n samples.

The complex i o n i c s t r u c u r e s o f t h e s e

samples demonstrate w e l l t h e p o t e n t i a l p i t f a l l s i n t h e a p p l i c a t i o n o f c l a s s i c a l systems t o t h e s e p a r a t i o n o f b i o c h e m i c a l s .

I n i t i a l r e s u l t s i n the preparative

s e p a r a t i o n of o t h e r b i o c h e m i c a l s , such as l i p i d s , c a r b o h y d r a t e s and n u c l e i c a c i d s suggest t h a t many o f t h e techniques used f o r t h e s e p a r a t i o n o f p o l y p e p t i d e s w i l l indeed be o f general a p p l i c a t i o n .

The f i n a l s e c t i o n i n t h i s

r e v i e w w i l l c o n c e n t r a t e on p r e p a r a t i v e s e p a r a t i o n s o f these o t h e r c l a s s e s o f compounds. 6.2

GENERAL REQUIREMENTS FOR AMINO ACID AND POLYPEPTIDE SEPARATIONS Many of t h e d i f f i c u l t i e s encountered i n e a r l y a t t e m p t s a t t h e s e p a r a t i o n

o f p o l y p e p t i d e s by LC arose f r o m t h e d i f f e r e n c e s i n t h e chromatographic p r o p e r t i e s o f these complex samples when compared t o t h e t r a d i t i o n a l s e p a r a t i o n s o b t a i n e d w i t h low m o l e c u l a r w e i g h t samples.

I n f a c t the separation o f

p o l y p e p t i d e samples r e q u i r e s c a r e f u l a t t e n t i o n t o t h e n a t u r e o f t h e s t a t i o n a r y and m o b i l e phase, as w e l l as a r e q u i r e m e n t for s u i t a b l e i n s t r u m e n t a t i o n . 6 . 2 . 1 N a t u r e of t h e S t a t i o n a r y Phase

The n a t u r e o f t h e r e v e r s e d phase column i s o f t e n an i m p o r t a n t f a c t o r i n the s e p a r a t i o n o f an amino a c i d or p o l y p e p t i d e sample.

There a r e a number o f

205

p r o p e r t i e s o f t h e r e v e r s e d phase column t h a t may have a m a j o r e f f e c t on t h e sample r e s o l u t i o n w h i c h i s o b t a i n e d and, m o s t i m p o r t a n t l y i n p r e p a r a t i v e separations, on t h e recovery o f m a t e r i a l a f t e r separation.

For example

p a r a m e t e r s such as p o r e s i z e , n a t u r e o f t h e r e v e r s e d phase ( c h e m i c a l f u n c t i o n a l i t y as w e l l as t h e n a t u r e o f b o n d i n g t o t h e s i l i c a p a r t i c l e ) , l o a d i n g 2 o f phase ( p m o l e s p e r m ) , s u r f a c e a r e a o f t h e p a c k i n g m a t e r i a l a n d p a r t i c l e s i z e have been f o u n d t o be s i g n i f i c a n t [ 3 1 .

Probably t h e most i m p o r t a n t

determinant i n polypeptide separations i s t h e n a t u r e of t h e s i l i c a m a t e r i a l used i n m a n u f a c t u r e o f t h e r e v e r s e d phase.

I n a r e c e n t r e v i e w on p r o t e i n s e p a r a t i o n s

[ 4 1 i t was n o t e d t h a t , f o r example, b o t h R e s o l v e and Vydac s i l i c a s p r o v i d e d a n e x c e l l e n t base m a t e r i a l f o r t h e p r e p a r a t i o n o f C,8 r e v e r s e d p h a s e c o l u m n s f o r t h e s e p a r a t i o n o f p e p t i d e s and p r o t e i n s .

The d i f f e r e n c e s b e t w e e n t h e s e s i l i c a s

and o t h e r s may r e l a t e i n p a r t t o s m a l l v a r i a t i o n i n p h y s i c a l s t r u c t u r e or i n t h e l e v e l s o f t r a c e c o n t a m i n a n t s such as m e t a l i o n s .

A t present o u r knowledge of

the surface p r o p e r t i e s of s i l i c a preparations which g i v e a s u p e r i o r reversed phase p a c k i n g i s v e r y l i m i t e d and t h e c h o i c e o f a c o l u m n f o r a g i v e n s e p a r a t i o n i s almost completely e m p i r i c a l . M i c r o p a r t i c u l a t e s i l i c a presents a heterogeneous s u r f a c e w i t h s i l a n o l g r o u p s o f v a r y i n g a c c e s s i b i l i t y and a c i d i t y , w i t h a pKa r a n g e o f 5-6 [ r e f . 51.

The p r e s e n c e o f i n t r a m o l e c u l a r h y d r o g e n b o n d i n g i s p r o b a b l y t h e c a u s e o f

t h i s v a r i a t i o n o f pKa v a l u e s .

I n a d s o r p t i o n c h r o m a t o g r a p h y t h e more r e a c t i v e

s i l a n o l groups are d e a c t i v a t e d by treatment of the s i l i c a w i t h p o l a r s o l v e n t s such as w a t e r and m e t h a n o l [ 6 1 .

We have f o u n d t h a t i t i s j u s t a s i m p o r t a n t i n

p r e p a r a t i v e as i n a n a l y t i c a l s e p a r a t i o n s t o s e l e c t a p a c k i n g m a t e r i a l w h i c h a l l o w s h i g h l y s e l e c t i v e s e p a r a t i o n s as w e l l as h i g h ' s a m p l e l o a d i n g s .

It i s

probable t h a t e f f e c t i v e s i l a n o l group i n t e r a c t i o n s p l a y a v i t a l r o l e i n a l l o w i n g a g i v e n r e v e r s e d phase p a c k i n g t o e x h i b i t t h e s e d e s i r a b l e p r o p e r t i e s .

For

example, i n t h e s e p a r a t i o n o f h i g h l y complex p e p t i d e maps i t was f o u n d t h a t a s u p p o r t w i t h b o t h r e v e r s e d phase and p o l a r i n t e r a c t i o n s ( i o n i c a n d h y d r o g e n b o n d i n g i n t e r a c t i o n s ) a l l o w e d maximum r e s o l u t i o n o f t h e m i x t u r e 171. T h i s s y s t e m was shown t o e x h i b i t a mixed-mode mechanism, a n d i n g e n e r a l a mixed-mode i n t e r a c t i o n between t h e sample a n d t h e s t a t i o n a r y phase a l l o w e d t h e m o s t e f f e c t i v e s e p a r a t i o n o f p o l y p e p t i d e samples.

6.2.2 Nature of the Mobile Phase The a d d i t on o f s a l t s t o t h e m o b i l e p h a s e i s a l s o an i m p o r t a n t p r o c e d u r e

fo r m i n i m i z i n g

n t e r a c t i o n s between t h e s i l a n o l g r o u p s o f t h e c o l u m n a n d i o n i c

groups present

n the solute.

The s a l t can e i t h e r a c t a s a g e n e r a l e l e c t r o l y t e

206

t h e r e b y suppressing i o n i c i n t e r a c t i o n s , or i n c e r t a i n cases a more s p e c i f i c i n t e r a c t i o n may occur 181.

R e c e n t l y amine phosphate b u f f e r s have become p o p u l a r

i n t h e LC o f amino a c i d s , p e p t i d e s and p r o t e i n s [ 9 1 .

P a r t of t h e success of

these b u f f e r s can be a t t r i b u t e d t o t h e d e a c t i v a t i o n o f s i l a n o l groups by i o n i c and/or hydrogen bonding i n t e r a c t i o n s w i t h amines added t o t h e m o b i l e phase. Although a mobile phase o f 0.1% H3P04 was adequate f o r the s u c c e s s f u l chromatography o f t h e p e p t i d e Gly-Gly-T r , o t h e r p e p t i d e s such as Gly-Leu-Tyr r e q u i r e d a mobile phase which c o n t a i n e d an amine phosphate such as triethylammonium phosphate ( T E A P ) .

Fur hermore, t h e presence o f a r g i n i n e i n a

p e p t i d e p r e s e n t s an a d d i t i o n a l s t r o n g l y b a s i c s i t e due t o t h e g u a n i d i n o s i d e chain.

I t i s not s u r p r i s i n g , therefore

t h a t t h e p e p t i d e s Met-Arg-Phe,

and

A

i

0.0:

I

2

4

6

RETENTION TIME (min) F i g . 6.1.

The chromatography o f t h e p e p t i d e s Leu-Trp-Met-Arg ( A and 6) and Met-Arg-Phe (C and D ) on a Radial-Pak Resolve C18 column. The column had been s u b j e c t e d t o t h e methanol pre-wash. Each a n a l y s i s was c a r r i e d o u t on 50ug o f t h e m o b i l e phase. The O.D. s e n s i t i v i t y was i n c r e a s e d t w o - f o l d I n 6 and D t o a l l o w f o r t h e broader peak shape. The f l o w - r a t e was Lml/min. I n A and C t h e m o b i l e phase was 1% TEAP-isopropanol (80:20) w h i l e 6 and D used a m o b i l e phase o f 1% T E A P - a c e t o n i t r i l e (80:20). R e p r i n t e d w i t h p e r m i s s i o n o f authors, r e f . 7.

207

Leu-Trp-Met-Arg e x h i b i t poor peak shapes on a r e v e r s e d phase column even i n t h e presence o f 1% TEAP i n t h e m o b i l e phase ( s e e F i g . 6.1 El and D ) . replacement o f a c e t o n i t r i l e w i t h

The

sopropanol as t h e o r g a n i c m o d i f i e r , however,

d r a m a t i c a l l y improves t h e e l u t i o n p r o f i l e f o r these b a s i c p e p t i d e s (see F i g . 6 . 1 A and C).

Methanol does n o t have t h i s e f f e c t when i t i s used i n s t e a d o f A p o s s i b l e e x p l a n a t i o n i s t h a t t h e i s o p r o p a n o l , which has a

acetonitrile.

s i g n i f i c a n t non-polar r e g i o n , i s p a r t i c u l a r l y s u i t a b l e f o r p e n e t r a t i n g t h e r e v e r s e d phase s u p p o r t and f u r t h e r d e a c t i v a t i n g t h e s i l a n o l groups b y hydrogen bonding [ 3 1 .

L e w i s and De Wald [ l o 1 have found t h a t n-propanol

i s a l s o an

e x c e l l e n t solvent f o r polypeptide separations. The r e s u l t s shown i n F i g . 6 . 1 suggest t h a t a m o b i l e phase which c o n t a i n s a r e l a t i v e l y h i g h c o n c e n t r a t i o n of TEAP and i s o p r o p a n o l w i l l be p a r t i c u l a r l y s u i t a b l e f o r t h e chromatography o f b a s i c hydrophobic samples.

This conclusion

was f u r t h e r strengthened by t h e f o l l o w i n g example o f a d i f f i c u l t p r o t e i n separation.

A p o l i p o p r o t e i n s , prepared by removal o f t h e l i p i d components of

l i p o p r o t e i n s , are extremely s e n s i t i v e to adsorption t o glass surfaces 1111.

In

an a t t e m p t t o e s t a b l i s h an assay f o r t h e C - a p o l i p o p r o t e i n s ( c o n s t i t u t e n t s of Very Low D e n s i t y L i p o p r o t e i n s ) t h e i s o c r a t i c s e p a r a t i o n o f a m i x t u r e o f apolipoproteins, C-I,

( s e e Fig. 6.2).

C-11, and C-111, was examined on a pBondapak C18 column

I n t h i s s e p a r a t i o n a normal phase, r a t h e r t h a n a r e v e r s e d phase,

OD 210 n m

0

2

1

1

L

6

RETENTION TIME ( m h ) Fig. 6.2.

The e l u t i o n p r o f i l e o b t a i n e d for t h e i s o c r a t i c e l u t i o n o f t h e a p o l i p o p r o t e i n s C - I , C-I1 and C-111, on a pBondapak C18 column w i t h a m o b i l e phase which c o n s i s t e d of 0.1% p h o s p h o r i c a c i d , pH Z . O / a c e t o n i t r i l e (40:60) and a flow r a t e o f l . S m l / m i n . R e p r i n t e d w i t h permission o f authors, r e f . 11.

208

p a r t i t i o n mechanism was s u g g e s t e d b y t h e o b s e r v a t i o n s t h a t a d e c r e a s e i n a c e t o n i t r i l e c o n c e n t r a t i o n i n t h e m o b i l e phase c a u s e d a d e c r e a s e i n r e t e n t i o n t i m e , w h i c h i s t h e o p p o s i t e t o what i s o b s e r v e d f o r r e v e r s e d phase s e p a r a t i o n s . The e l u t i o n o r d e r o f apoC-1111 and apoC-I i s a l s o i n t h e o r d e r e x p e c t e d f o r t h e i n t e r a c t i o n of b a s i c residues of t h e p r o t e i n s w i t h the a c i d i c s i l a n o l groups o f t h e column L111.

r e c o v e r i e s (20-50%).

N o t s u r p r i s i n g l y t h i s s e p a r a t i o n was f o u n d t o g i v e p o o r I f t h e m o b i l e phase was m o d i f i e d from 0 . 1 % H3P04

t o 1% TEAP and i s o p r o p a n o l s u b s t i t u t e d f o r a c e t o n i t r i l e , t h e n t h e s e p a r a t i o n o f t h e C - a p o l i p o p r o t e i n s shown i n F i g . 6 . 3 was o b t a i n e d .

I n t h i s separation the

o r d e r o f e l u t i o n t h a t w o u l d b e e x p e c t e d for a r e v e r s e d phase r a t h e r t h a n a normal phase s e p a r a t i o n , as w e l l as e x c e l l e n t r e c o v e r i e s ( o v e r 80% f o r e a c h component), were o b s e r v e d .

A l s o a h i g h i o n i c s t r e n g t h was r e q u i r e d , as a

d e c r e a s e from 1% t o 0.1% i n t h e c o n c e n t r a t i o n o f TEAP c a u s e d a t w o - f o l d r e d u c t i o n i n r e c o v e r i e s o f t h e a p o p r o t e i n s , as w e l l as r e s u l t i n g i n poor peak shapes w i t h e x c e s s i v e t a i l i n g [ 1 1 1 .

These r e s u l t s emphasize t h e o c c u r r e n c e o f a

mixed-mode mechanism i n t h e s e p a r a t i o n o f p o l y p e p t i d e sample i n r e v e r s e d p h a s e LC.

A s w i l l be d e s c r i b e d l a t e r i n t h i s r e v i e w , t h e s e r e s u l t s h a v e a l l o w e d t h e

d e v e l o p m e n t o f a p r e p a r a t i v e s e p a r a t i o n o f m u l t i g r a m amounts o f t h e s e p r o t e i n s .

RETENTION TIME (minl

F i g . 6.3.

The e l u t i o n p r o f i l e o b t a i n e d from a c r u d e m i x t u r e o f C - a p o l i p o p r o t e i n s (1OOpg) o n a pBondapak p h e n y l c o l u m n w i t h a m o b i l e phase o f 1% t r i e t h y l a m m o n i u m p h o s p h a t e a n d a flow r a t e o f 1 . 5 m l / m i n . The g r a d i e n t c o n s i s t e d o f a 1 0 - m i n c o n c d v e g r a d i e n t of 0-37% a c e t o n i t r i l e (number 2 on t h e W a t e r s 660 S o l v e n t Programmer) and 20-min convex g r a d i e n t of 37-42% a c e t o n i t r i l e (number 8). R e p r i n t e d w i t h a u t h o r s p e r m i s s i o n , r e f 1 1 .

209

nowever d e s i r a b l e the p r o p e r t i e s o f amine phosphates may be i n the h i g h e f f i c i e n c y separation o f polypeptide samples, the use o f n o n v o l a t i l e mobile phases w i l l be l i m i t e d i n p r e p a r a t i v e separations as the samples must be f u r t h e r processed w i t h a d e s a l t i n g procedure a f t e r the p r e p a r a t i v e separation.

I n fact,

we have found t h a t many polypeptide samples have a considerable a f f i n i t y for these amine phosphates and t h a t complete removal o f the i o n i c m o d i f i e r may be extremely d i f f i c u l t .

The p r e p a r a t i v e separation o f these samples has become a

p r a c t i c a l method since the i n t r o d u c t i o n o f v o l a t i l e mobile phases ( s e e Table 6.1).

However, i t must be remembered t h a t mobile phase a d d i t i v e s other than

amine phosphates may not be as e f f e c t i v e as the amine phosphates i n reducing silanol-solute interactions.

Table 6 . 1 gives a general summary o f the

e f f e c t i v e n e s s o f d i f f e r e n t m o b i l e phases, although some v a r i a t i o n w i l l occur f o r solutes w i t h d i f f e r e n t s u s c e p t i b i l i t i e s t o the e f f e c t s o f s i l a n o l groups.

Table 6.1

I o n i c M o d i f i e r s Comnonly Used i n V o l a t i l e Mobile Phases for P r e p a r a t i v e HPLC o f P r o t e i n s Amnonium bicarbonate (0.02 t o

O.lM, pH 8.0)

T r i f luoroacet i c Acid (0.1%)

Amnonium Acid (0.1%) Amnonium Acetate (0.01 t o 0.05h4, pH 4.0) Triethylamnonium formate ( 0 . 0 4 , pH 3. 15) P y r i d i n e formate/acetate ( 0 . 5 to 2M, pH 3.5 t o 5.0)

6.2.3 Nature of the Organic Modifier Table 6 . 2 l i s t s the organic m o d i f i e r s that a r e comnonly used i n the separation of amino a c i d and polypeptide samples.

For many a n a l y t i c a l

separations, the chromatographic p r o p e r t i e s and UV transparency o f a c e t o n i t r i l e make i t the solvent of choice.

For p r e p a r a t i v e separations, however, methanol

i s o f t e n p r e f e r r e d , as i t i s both less t o x i c and less expensive than a c e t o n i t r i l e , and i t gives separations almost as good, p a r t i c u l a r l y for amino acids and p e p t i d e s .

210 Table 6 . 2 Organic M o d i f i e r s Used i n the Separation o f Amino Acid and Po Iypep t i de Samp I esa

Methanol (acetone, a c e t i c a c i d )

E thano I (acetone, acetaldehyde, ethy I acetate. benzene) lsopropanol , propanol (acetone, propanol)

D i oxane and t e t rahydro f ur an (perox ides) A c e t o n i t r i l e (aromatics)

aComnonly observed i m p u r i t i e s shown i n parentheses

For p r o t e i n samples, the requirement t h a t the solvent be v o l a t i l e i s l e s s important, because many p r o t e i n s are n o t s t a b l e t o solvent evaporation techniques.

Consequently i t i s p o s s i b l e t o use a group o f solvents which g i v e

very good p r o t e i n separations b u t are l e s s v o l a t i l e , i n c l u d i n g propanol, isopropanol, ethylene g l y c o l and 2-methoxyethanol.

These solvents can be

removed by l y o p h i l i z a t i o n , d i a l y s i s or h o l l o w - f i b r e f i l t r a t i o n , but they a r e n o t p a r t i c u l a r l y toxic, and may n o t need t o be removed f o r many a p p l i c a t i o n s .

6.2.4 !%me Comments on What Constitutes a Preparative Separation The d i s t i n c t i o n between a n a l y t i c a l and p r e p a r a t i v e separations i s i n f a c t a conceptual one.

I f the i n t e n t i o n i s t o o b t a i n a p u r i f i e d sample f o r f u r t h e r

study, the technique used may be said t o be p r e p a r a t i v e , whether the sample mass

i s measured.in kilograms or micrograms.

With some p r o t e i n hormones such as

i n t e r f e r o n , the separation o f a f e w m i l l i g r a m s o f p u r i f i e d p r o t e i n would supply s u f f i c i e n t m a t e r i a l f o r many c l i n i c a l experiments, and so could be s a i d t o be preparative i n t h i s sense.

The problems o f column capacity, solvent removal and

the evaluation o f product p u r i t y are common t o p r e p a r a t i v e chromatography a t a l l scales . With separations o f samples o f greater than a gram or so, however, a new class o f problems a r i s e s .

Because the chromatographic equipment becomes

l i m i t i n g , i t becomes i n c r e a s i n g l y important t o o p t i m i z e the separation i n terms

o f sample loading, r e s o l u t i o n of the sample peaks, and the u t i l i z a t i o n of solvent and t i m e .

A t t h i s scale, the resemblance between p r e p a r a t i v e

chromatography and the techniques of chemical engineering becomes obvious. I n F i g . 6 . 4 some commonly used column s i z e s are shown, together w i t h suggested loadings f o r polypeptide samples.

I t can be seen t h a t t h i s range o f

211

Flexibly walled polythene columns

Stainless steel columns

preparative

repaat~w semi-

cohn

sizehml

57

XI

Fig. 6.4.

6.3

amlytical

08. 0 5 " 10

10

10

5.1[

prepsdtive

25.

21x

305

25

09% 0 7 x 30-50 50

andytical

OL. 10-30

30-100 30-100 x)-100 10-100 3-x)

A d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e column s i z e s t h t a r e commonly used i n a n a l y t i c a l and p r e p a r a t i v e s e p a r a t i o n s o f a m i n o a c i d s , p e p t i d e s a n d p r o t e i n s . The p o l y e t h y l e n e columns a r e f l e x i b l e and a l l o w r a d i a l compression o f t h e column which i s a u s e f u l t e c h n i q u e f o r t h e s e p a r a t i o n o f t h e s e s o l u t e s ( s e e r e f . 7 for f u r t h e r d e t a i l s ) . The l o a d i n g l i m i t s a r e c o n s e r v a t i v e v a l u e s w h i c h may w e l l be e x c e e d e d i n f a v o r a b l e s e p a r a t i o n s .

PREPARATIVE SEPARATIONS OF AMINO ACIDS AND PEPTIDES

The use o f HPLC i n t h e p r e p a r a t i v e s e p a r a t i o n o f a m i n o a c i d s has b e e n v e r y limited.

G r a n t and C l i f f e [ 1 2 1 r e p o r t e d a s e p a r a t i o n o f s t a t i n e , an u n u s u a l

amino a c i d f o u n d i n p e p s t a t i n , from a l a n i n e and v a l i n e , u s i n g a 0.1% H3P04/MeOH s y s t e m a n d a r e v e r s e d phase p r e p a r a t i v e c o l u m n . A r e s o l u t i o n of racemic [I-"CI

v a l i n e u s i n g a c h i r a l m o b i l e p h a s e was

r e c e n t l y r e p o r t e d b y Washburn e t a l . [ 1 3 1 .

The m o b i l e phase c o n t a i n e d

L - p r o l i n e , c u p r i c a c e t a t e , and s o d i u m a c e t a t e , a n d g a v e a l m o s t b a s e l i n e

212 r e s o l u t i o n between D- and L - v a l i n e on a r e v e r s e d phase column. 4mg o f L - [ l - " C I

A l t h o u g h only

v a l i n e was p u r i f i e d i n a s i n g l e r u n , t h e v e r y h i g h s p e c i f i c

a c t i v i t y o f t h i s m a t e r i a l ( 2 C i l m m o l ) means t h a t s e v e r a l c l i n i c a l doses can be prepared i n t h i s way i n o n l y 50 m i n u t e s .

T h i s e l e g a n t t e c h n i q u e , f i r s t used by

G i l - a v e t a l . C141, should have many f u t u r e a p p l i c a t i o n s i n p e p t i d e s y n t h e t i c chemistry. The malondialdehyde adducts o f s e v e r a l amino a c i d s were s e p a r a t e d on an A m b e r l i t e XAD-4 column by P i e t r z y k and S t o d o l s [151.

Several hundred m i l l i g r a m s

of p u r i f i e d compounds c o u l d be r e c o v e r e d i n good y i e l d .

F i g . 6.5.

P r e p a r a t i v e s e p a r a t i o n o f crude l e u c i n e - e n k e p h a l i n amide 0-benzyl e t h e r ( V ) and d e b e n z y l a t e d p r o d u c t (IV) on a Phenyl P o r a s i l B column (0.7 x 60cm) w i t h 30% a c e t o n i t r i l e - w a t e r - 0 . 1 % phosphot.ic a c i d as t h e m o b i l e phase. The l o a d i n g was 4 mg and t h e f l o w r a t e was Zml/min. R e p r i n t e d w i t h p e r m i s s i o n o f a u t h o r s , r e f 2 3 .

A p o p u l a r a p p l i c a t i o n of p r e p a r a t i v e , r e v e r s e d phase LC i s i n t h e

s e p a r a t i o n o f s y n t h e t i c p e p t i d e s prepared by t h e s o l i d phase method [161.

Fig.

6 . 5 shows t h e p r e p a r a t i v e s e p a r a t i o n of t h e s y n t h e t i c p e p t i d e , l e u c i n e e n k e p h a l i n amide on a phenyl P o r a s i l 6 column.

Peak I V corresponded t o t h e

d e s i r e d p e p t i d e w h i l e peak V c o n t a i n e d a b y - p r o d u c t formed i n t h e c a t a l y t i c h y d r o g e n a t i o n o f t h e c o r r e s p o n d i n g 0-benzyl p r o t e c t e d p e p t i d e .

The p u r i f i e d

p e p t i d e was shown t o be homogeneous b y amino a c i d a n a l y s i s and a n a l y t i c a l LC.

213 The f i g u r e shows t h e a d v a n t a g e of r e v e r s e d p h a s e LC as a p u r i f i c a t i o n m e t h o d f o r s y n t h e t i c p e p t i d e s , as t h e s e p a r a t i o n was r a p i d and gave good r e s o l u t i o n . F i g . 6 . 6 shows t h e s e p a r a t i o n of a s y n t h e t i c p e n t a d e c a p e p t i d e o n a C 1 8 column w i t h a m o b i l e phase w h i c h c o n t a i n s t r i e t h y l a m m o n i u m p h o s p h a t e [ 7 1 .

This

example shows t h a t u s e f u l amounts o f p e p t i d e c a n be s e p a r a t e d o n an a n a l y t i c a l column and i n f a c t u p t o 50mg o f c r u d e s y n t h e t i c p e p t i d e s h a v e been s e p a r a t e d o n t h i s column.

A m a j o r d i s a d v a n t a g e o f t h e m o b i l e phase u s e d i s t h a t

triethylammonium phosphate i s n o n v o l a t i l e , which n e c e s s i t a t e s a s e p a r a t e d e s a l t i n g s t e p t o o b t a i n t h e p e p t i d e i n a s a l t - f r e e form.

S p a r r o w has p u r i f i e d

c r u d e s y n t h e t i c p e p t i d e s (5 t o 209 l o a d i n g s ) on t h e p r e p a r a t i v e C18 c a r t r i d g e s

E C

tn

N

N

4 0

2

L

6

8

10

12

14

16

18

20

Time ( mid F i g . 6.6.

The p u r i f i c a t i o n o f a s y n t h e t i c a n a l o g o f t h e 1-15 segment o f human a p o l i p o p r o t e i n C-I, i n w h i c h Phe-14 i s r e p l a c e d w i t h p - i o d o p h e n y l a l a n i n e i n t h e c h e m i c a l s y n t h e s i s . The m o b i l e p h a s e was p u r i f i e d 1% TEAP w i t h a l i n e a r g r a d i e n t o f a c e t o n i t r i l e ( s e e t h e dashed l i n e s ) . The f l o w - r a t e was l . S m l / m i n . The l o a d i n g s i n A t o C were 0.5,2 a n d 8 mg o f c r u d e p e p t i d e d i s s o l v e d i n 1% TEAP, 3M g u a n i d i n e h y d r o c h l o r i d e a t a c o n c e n t r a t i o n o f l O m g l m l . Peak 3 c o r r e s p o n d e d t o t h e d e s i r e d p e p t i d e , a n d t h i s f r a c t i o n from e a c h r u n was p o o l e d . The a n a l y s i s o f a n a l i q u o t o f t h i s pool i s shown i n D. . R e p r i n t e d w i t h p e r m i s s i o n o f a u t h o r s , r e f . 7 .

214

t h a t a r e used i n the Waters PrepLClSystem 500, w i t h a m o b i l e phase o f 1% triethylammonium

phosphate, pH 3 . 0 and g r a d i e n t s o f i s o p r o p a n o l [ 1 7 1 .

The

p u r i f i e d p e p t i d e was c o l l e c t e d , t h e pH o f t h e s o l u t i o n was a d j u s t e d t o 8 . 0 w i t h ammonium hydroxide, t h e n t h e i s o p r o p a n o l was p a r t i a l l y removed under vacuum and t h e aqueous b u f f e r l y o p h i l i s e d .

I n t h i s s t u d y up t o p e n t a p e p t i d e s were p u r i f i e d

w i t h a y i e l d o f g r e a t e r than 90%. P r o t e c t e d s y n t h e t i c p e p t i d e s can be p u r i f i e d e i t h e r by r e v e r s e d phase chromatography

[ l a ] , or

by chromatography on pre-packed s i l i c a columns [19,201.

F i g . 6 . 7 shows t h e p u r i f i c a t i o n o f a s y n t h e t i c p e n t a p e p t i d e A c - S e r - T h r - I l e - G l u (OBz1-pNO2)-Arg(NO2)OH.

I n t h i s case, where t h e p e p t i d e b e i n g p u r i f i e d has

no f r e e amino groups, i t i s n o t necessary t o add an i o n i c m o d i f i e r t o suppress i n t e r a c t i o n s between t h e s u r f a c e o f t h e p a c k i n g and t h e p e p t i d e .

Instead acetic

a c i d was used t o suppress t h e i o n i z a t i o n o f t h e t e r m i n a l c a r b o x y l group, making t h e p e p t i d e more nonpolar t o i n c r e a s e r e t e n t i o n .

F i g . 6.7A shows t h a t i n i t i a l

a t t e m p t s a t t h e p u r i f i c a t i o n b y chromatography on Sephadex LH-20 gave a s e p a r a t i o n t h a t was u n s a t i s f a c t o r y and slow.

The material. f r o m t h e column was

pooled (cross-hatched area i n F i g 6.7A). and a sample was a n a l y z e d by r e v e r s e d phase LC ( F i g . 6 . 7 6 ) .

I n t h i s case t h e s e p a r a t i o n was r a p i d , b e i n g complete i n

l e s s t h a n 7 minutes, w i t h good r e s o l u t i o n .

The b u l k o f t h e r e s i d u e from t h e

chromatogram i n F i g . 6.7A was then chromatographed p r e p a r a t i v e l y , t h e p u r i f i e d m a t e r i a l (cross-hatched area F i g . 6.7C) was c o l l e c t e d and t h e n s u b j e c t e d t o an a n a l y t i c a l separation. l a r g e peak ( F i g . 6 . 7 D ) .

The p u r i t y o f t h e sample i s e v i d e n t f r o m t h e s i n g l e The r e s u l t s show t h a t t h e r e i s no s i g n i f i c a n t loss o f

r e s o l u t i o n w i t h t h e use of t h e p r e p a r a t i v e system, an o b s e r v a t i o n c o n f i r m e d by subsequent a n a l y t i c a l d a t a .

This separation

i l l u s t r a t e s t h e advantages o f p r e p a r a t i v e LC ( e x c e l l e n t r e s o l u t i o n i n l e s s t h a n 30 min) over c l a s s i c a l chromatographic methods ( i n a d e q u a t e r e s o l u t i o n d e s p i t e a 10 hour s e p a r a t i o n ) .

I n a s i m i l a r manner Beyerman e t a l . [211 have p u r i f i e d

crude p r o t e c t e d s e c r e t i o n 30.839) on two c a r t r i d g e s of PrepPak C,8

( 5 . 2 x 50

cm) w i t h a m o b i l e phase c o n s i s t i n g o f methanol: dimethylformamide: w a t e r : a c e t i c a c i d , 48:38:13:1. G a b r i e l e t a l . C19,201 have develaped an i n e x p e n s i v e system t h a t u t 1 i z e s low pressures (50 t o 150 p s i ) and s i l i c a g e l 60 prepacked i n heavy g l a $ s w a l l columns.

The columns had 200 t o 400 t h e o r e t i c a l p l a t e s .

A typical purification

i n v o l v e d the chromatography o f Z-Phe-Phe-Trp-Lys(Boc)-Thr(tBu)-Ser(tBu)-Cys (tBu)-OtBu(474mg) with c h l o r o f o r m : methano1:acetic a c i d (8O:lO:Z) as t h e m o b i l e phase.

A v a r i e t y o f p e p t i d e s up t o t e t r a d e c a p e p t i d e s have been chromatographed

215

B 0.8 06 0.4

0.2 n

2

3

4

5

7

6

8

Time (hours)

'75

15

22.5

Time (minutes)

"

0

2

4

6

8

Time (minutes)

F i g . 6.7.

E l u t i o n p r o f i l e s obtained during the p u r i f i c a t i o n of a p e n t a p e p t i d e . Ac-Ser-Thr-Ile-Clu-(OBzl-p-NO~)-Arg-(NO~~OH. ( A ) p r o f i l e o b t a i n e d w i t h t h e Sephadex LH-20 system and a m o b i l e phase c o n s i s t i n g o f 1 p e r c e n t methanol i n e t h y l a c e t a t e , a g l a s s column (31cm b y 25cm) a t a flow r a t e o f 60ml/hour w i t h a h y d r o s t a t i c head o f l m :10ml o f t h e sample was loaded and a U v i c o r d u l t r a v i o l e t d e t e c t o r a t 280nm was used. ( 6 and D ) P r o f i l e s o b t a i n e d from a uBondapak-Fatty a c i d a n a l y s i s column (30cm l o n g and 4mm i n i n s i d e d i a m e t e r ) , w i t h a m o b i l e phase c o n s i s t i n g o f 50 p e r c e n t methanol and 50 p e r c e n t water, w i t h 1 p e r c e n t a c e t i c a c i d . T h i s column had a p l a t e number o f 2100. (C) P r o f i l e o b t a i n e d f o r t h e p e n t a p e p t i d e chromatographed on t h e p r e p a r a t i v e column. The p r e p a r a t i v e HPLC system c a n s i s t e d o f two columns (60cm l o n g and 7mm i n i n s i d e d i a m e t e r ) connected i n s e r i e s . The columns were packed w i t h s i l a n i z e d Bondapak Phenyl P o r a s i l B (37 t o 50um; Waters) by t h e t a p - f i l l method ( 1 ) . The m o b i l e phase c o n s i s t e d o f 40 p e r c e n t methanol and 60 p e r c e n t water, w i t h 1 p e r c e n t a c e t i c a c i d . Samples (2ml) made up i n t h e m o b i l e phase were loaded; t h e flow r a t e was 5ml/min, and t h e r e q u i r e d p r e s s u r e was 13.7 atm. The two columns had a p l a t e number o f 520, which, a l t h o u g h s i g n i f i c a n t l y lower t h a n t h e p l a t e number for t h e a n a l y t i c a l column, made no s i g n i f i c a n t d i f f e r e n c e to the separation obtained. .Reprinted w i t h permission o f authors, r e f . 18.

216

w i t h t h i s system.

L a r g e r p e p t i d e s may have l i m i t e d s o l u b i l i t y i n t h i

mob l e

phase, a l t h o u g h l e s s . s o l u b l e compounds may be l o a d e d i n s o l v e n t s s u c h as d i m e t h y l f o r m a m i d e or g l a c i a l a c e t i c a c i d w i t h o u t loss o f c o l u m n e f f i c e n c y . Kullman developed a s i m i l a r system for t h e p u r i f i c a t i o n of p r o t e c t e d intermediates i n the enzymatic synthesis of leucine-enkaphalin, w i t h a m o b i l e phase t h a t c o n s i s t e d o f m i x t u r e s o f d i c h l o r o m e t h a n e , e t h a n o l and a c e t i c a c i d r221. I n i n i t i a l s t u d i e s aimed a t e x t e n d i n g t h e e f f e c t o f p h o s p h o r i c a c i d s o l u t i o n s on t h e e l u t i o n o f p e p t i d e s from r e v e r s e d phase c o l u m n s , t h e u s e o f a c e t i c a c i d a n d t r i f l u o r o a c e t i c a c i d a s m o b i l e phase a d d i t i v e s was e x a m i n e d .

It

was o b s e r v e d t h a t a c e t i c a c i d gave b r o a d p e a k s w i t h s i g n i f i c a n t l y g r e a t e r r e t e n t i o n t i m e s [ 2 3 1 . a n d was g e n e r a l l y not s u i t a b l e f o r t h e a n a l y s i s o f peptides.

T r i f l u o r o a c e t i c a c i d , however, g a v e e x c e l l e n t r e s u l t s t h a t were

comparable t o those o b t a i n e d w i t h phosphoric a c i d s o l u t i o n s .

The m a j o r

d i s a d v a n t a g e s of t h i s m o b i l e phase when compared t o p h o s p h o r i c a c i d was t h e g r e a t e r a c i d i t y o f t h e f l u o r i n a t e d a c i d which r e s u l t e d i n decreased column l i f e t i m e and a somewhat g r e a t e r a b s o r b a n c e a t 210 nm.

A major advantage.

however, was t h e e x c e l l e n t v o l a t i l i t y o f t r i f l u o r o a c e t i c a c i d s o l u t i o n s w h i c h conveniently allowed preparative separations. Another factor t h a t f a c i l i t a t e s p r e p a r a t i v e separations i s t h e a v a i l a b i l i t y of commercial p r e p a r a t i v e l i q u i d chromatographs which a l l o w t h e p r o p o r t i o n a t e s c a l e - u p o f an a n a l y t i c a l s e p a r a t i o n .

An e x a m p l e o f t h i s s c a l e up

i s t h e p r e p a r a t i o n s e p a r a t i o n o f a 59 sample of t e t r a p e p t i d e L e u - ( G l y ) 3 o n a 5.7 cm x 30 cm PrepPak C18 c a r t r i d g e (75um) on a W a t e r s P r e p L C l S y s t e m 500 i n s t r u m e n t [241.

The m o b i l e phase c o n t a i n e d 0.05% t r i f l u o r o a c e t i c a c i d

d i s s o l v e d i n w a t e r : m e t h a n o l ( 9 5 : 5 ) a n d a flow r a t e o f 100 m l / m i n was u s e d ( b a c k p r e s s u r e 100 p s i ) .

F i g . 6 . 8 a shows t h e a n a l y t i c a l p r o f i l e o f t h e c r u d e p r o d u c t

from a s o l u t i o n s y n t h e s i s of t h e t e t r a p e p t i d e and F i g . 6 . 8 b shows t h e p r o f i l e o b t a i n e d f o r an a u t h e n t i c sample. F i g . 6 . 9 shows t h e e l u t i o n p r o f i l e o b t a i n e d for a 59 sample o n t h e preparative instrument.

F i g . 6.10 g i v e s t h e e l u t i o n p r o f i l e for t h e

corresponding a n a l y t i c a l separations of f r a c t i o n s from the p r e p a r a t i v e run.

The

r e s u l t s shown i n F i g . 6 . 1 0 i n d c a t e t h a t t h e b a c k end o f t h e m a i n peak e l u t i n g a t approximately 8 min. i n the p r e p a r a t i v e r u n contained p u r e m a t e r i a l .

In

a d d i t i o n f u r t h e r p u r e m a t e r i a l was o b t a i n e d from f r a c t i o n 8 w h i c h was c o l l e c t e d from t h e back e n d o f t h e m a t e r a l r e c y c l e d f r o m t h e f r o n t e n d of t h e m a i n p e a k . Subsequent e x p e r i m e n t s showed a p p r o x i m a t e l y 95% [ r e f . 241.

h a t t h e r e c o v e r y o f p u r l f i e d m a t e r i a l was

217

2

1

6

8

6

0

Time I minl

0

4

2

Time ( m i n i

Fig. 6.8.

The e l u t i o n p r o f i l e o f t h e c r u d e s y n t h e t i c L - L e u ( G l y ) 3 ( a ) a n d the commercially o b t a i n e d standard L-Leu(Gly)3 ( b ) . C h r o m a t o g r a p h i c c o n d i t i o n s : Column, pBondapak c 1 8 ; m o b i l e p h a s e , w a t e r - 0 . 0 5 t TFA, pH 2 . 3 ; f l o w - r a t e , 1 . 5 m l l m i n . R e p r i n t e d w i t h permission of authors, r e f . 24.

A l a t e r p u b l i c a t i o n [251 d e s c r i b e d t h e p u r i f i c a t i o n o f t h e f o l l o w i n g s y n t h e t i c

p e p t i d e s i n up t o 59 l o a d i n g s p e r p r e p a r a t i v e r u n : G l y - G l y - O E t , G l y - G l y - G l u . Gly-Gly-Lys,

Pyr-His-Gly,

Met-enkephalin.

(Pro)3 and t h e p e n t a p e p t i d e s Leu- and

However, p o l a r p e p t i d e s a r e o f t e n n o t s u f f i c i e n t l y r e t a i n e d f o r

e f f i c i e n t s e p a r a t i o n s o n n o n - p o l a r columns when u s e d w i t h t r i f l u o r o a c e t i c a c i d , even i n t h e absence o f o r g a n i c m o d i f i e r i n t h e m o b i l e phase.

In the

a n a l y t i c a l s e p a r a t i o n o f a m i n o a c i d s and p o l y p e p t i d e s t h e u s e o f h y d r o p h o b i c i o n - p a i r i n g r e a g e n t s has become p o p u l a r L26.271.

A n i o n i c r e a g e n t s such as

a l k y l s u l p h o n a t e s , when added t o t h e m o b i l e p h a s e , can i n c r e a s e t h e o b s e r v e d r e t e n t i o n t i m e for a p o l y p e p t i d e sample.

A l k y l s u l p h o n a t e s a r e n o t s u i t a b l e for

p r e p a r a t i v e s e p a r a t i o n s , however, and t h u s we C281 and o t h e r s [ 2 7 1 i n t r o d u c e d a s e r i e s of p e r f l u o r o a l k a n o i c a c i d s .

Nhen t h e s e r e a g e n t s a r e added t o t h e m o b i l e

phase t h e r e t e n t i o n of t h e p o l y p e p t i d e was o b s e r v e d t o be d i r e c t l y r e l a t e d t o t h e c h a i n l e n g t h of t h e p e r f l u o r o a l k a n o i c a c i d C281. have been shown t o g i v e e x c e l l e n t peak shapes C281.

I n a d d i t i o n these reagents

218

-

0.8

0.7

-

0.6 -

0.5 -

E

0.4 -

0

m

N

0.3d 0

0.2 -

0.1

-

0

2

4

6

8

10

12

14

16

18

20

22

24

Time ( m i n l

Fig. 6.9.

The e l u t i o n p r o f i l e of t h e p r e p a r a t i v e p u r i f i c a t i o n o f l g c r u d e L-Leu(Gly)j. Chromatographic c o n d i t i o n s : column, Prep PAK-500/C18 c a r t r i d g e ; m o b i l e p h a s e , w a t e r - m e t h a n o l - T F A ( 9 5 : 5 : 0 : 0 . 0 5 ) , pH 2 . 3 ; f l o w - r a t e 1 0 0 m l / m i n . R e p r i n t e d w i t h p e r m i s s i o n of authors, r e f . 2 4 .

The u s e f u l n e s s of a m o b i l e phase c o n t a i n i n g 5mM of a p e r f l u o r o a l k a n o i c a c i d was d e m o n s t r a t e d b y t h e s u c c e s s f u l p u r i f i c a t i o n o f t h e p e p t i d e P y r - H i s - G l y (see F i g . 6.11).

F i g . 6 . 1 1 6 shows t h e s e p a r a t i o n a c h i e v e d when t h e s y n t h e t i c

p r o d u c t was c h r o m a t o g r a p h e d w i t h a m o b i l e phase w h i c h c o n t a i n e d 0.05% perfluoroacetic acid.

I n t h i s e l u t i o n p r o f i l e t h e r e i s a poor s e p a r a t i o n

between t h e t r i p e p t i d e and e a r l y e l u t i n g p o l a r i m p u r i t i e s .

Part A

shows t h e

same s e p a r a t i o n e x c e p t t h a t p e r f l u o r o b u t y r i c a c i d i s u s e d as t h e i o n - p a i r i n g reagent.

I n t h i s case P y r - H i s - G l y

late eluting impurities.

i s w e l l s e p a r a t e d f r o m e a r l y and

T h i s r e s u l t can be a t t r i b u t e d t o t h e i n c r e a s e d

r e t e n t i o n o f t h e s o l u t e o n t h e C 1 8 column when c o m p l e x e d w i t h t h e more l i p o p h i l i c ion-pairing reagent.

The p u r i f i e d m a t e r i a l t h a t was i s o l a t e d f r o m

219

002

-

0.01

L

5

6

8

9

007006

-

005-

E

= 2

003-

0

0.02

O

0.01 t

OOL-

0

0 . n

004

-

l

ihf

i

s

-

7

I

-

I

003-

.

002001%

I

L

Fig. 6.10. The analytical HPLC profiles of the collected fractions (1-9) from Chromatographic the preparative separation of crude L-Leu(Gly)j. conditions as in Fig. 6.8. Reprinted with permission o f authors, ref. 2 4 .

the center of the major peak as shown in Fig. 6.11A was subsequently shown to be pure by an analytical separation (Fig. 6.11C). In addition, the ammonium salt of the acid could be removed from the purified peptide by extraction with ether, a solvent in which most peptides are insoluble. Ammonium bicarbonate has been a popular solvent for the separation o f peptides by conventional chromatographic techniques due to its excellent volatility and the high solubility of many peptides in the buffer. The high apparent pH o f this mobile phase (7.7 to 8) precludes its use with siliconaceous supports packed in inflexible columns, due to the generation of column voids caused by dissolution of the silica. The radial compression that is used with the flexible-walled columns fitted in the Radial Compression Module from Waters Chromatography Division of Millipore circumvents this problem as any voids that may be generated are removed during column compression [ 7 1 . Provided the columr.

220

(P 9

1.0

B

0

OLkL=OaSOL 0

816243240

0 2 4

Time (min)

F i g . 6.11.

P r e p a r a t i v e s e p a r a t i o n o f Pyr-His-Gly. A l g sample o f t h e crude p r o d u c t was d i s s o l v e d i n t h e m o b i l e phase and i n j e c t e d onto t h e C18-column. A f l o w - r a t e o f 100ml/min was used f o r t h e p r e p a r a t i v e s e p a r a t i o n shown i n p a r t s A and B. I n p a r t B t h e m o b i l e phase was 0.05% p e r f l u o r o a c e t i c a c i d , p a r t s A and C p e r f l u o r o b u t y r i c a c i d ( 5 m M ) . The a n a l y t i c a l s e p a r a t i o n shown i n p a r t C was c a r r i e d out on a pBondapak c18 column, w i t h 5mM p e r f l u o r o b u t y r i c a c i d as t h e m o b i l e phase. The m a t e r i a l s t h a t were pooled and i s o l a t e d i n t h e p r e p a r a t i v e r u n s were shown by t h e s o l i d b a r . R e p r i n t e d w i t h p e r m i s s i o n o f a u t h o r s , r e f . 28.

i s washed w i t h water and then i s o p r o p a n o l each e v e n i n g , we have f o u n d t h a t an extended l i f e t i m e o f a t l e a s t 6 months C18 o r CN columns.

can be achieved with Radial-Pak Resolve

A s a f u r t h e r p r e c a u t i o n a guard column f i l l e d w i t h P o r a s i l B

C18 p a c k i n g m a t e r i a l i s used.

The guard column does' n o t degrade t h e

s e p a r a t i o n , b u t does a l l o w f o r a s i g n i f i c a n t i n c r e a s e i n column l i f e t i m e due b o t h t o removal o f contaminants f r o m t h e sample and m o b i l e phase and t o d i s s o l u t i o n of s i l l c a i n t h e guard column ( t h u s p a r t i a l l y p r e s a t u r a t i n g t h e m o b i l e phase w i t h s i l i c a ) . F i g . 6.12 shows t h e p u r i f i c a t i o n of 350 pg of t h e s y n t h e t i c p e p t i d e Leu-G1 y-Ser-Phe-Leu-Lys-Ser-Trp-Leu-Ser-A1

a-Leu-G1 y-G1 n-A1 a-Leu-Lys-A1 a [ 303.

221

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60 F i g . 6.12.

30

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b

The p u r i f i c a t i o n o f 350ug o f t h e p e p t i d e Leu-Gly-Ser-Phe-Leu-Lyswas accomplished u s i n g a Radial-Pak CN column w i t h a l i n e a r g r a d i e n t from 0 . 1 M NH4HCO t o i PrOH:CH3CN: 0.1 M NH4HC03 ( 3 : 3 : 4 ) a t a flow r a t e o? 1.Om;lmin. The sample was loaded i n t h r e e i n j e c t i o n s from a s o l u t i o n o f 5ml o f 6 M u r e a . P a r t A shows t h e e l u t i o n p r o f i l e f o r t h e crude p e p t i d e m i x t u r e . The area d e f i n e d by t h e s o l i d b a r was p o o l e d ( 2 m l ) , d i l u t e d t o 6ml w i t h 0 . 1 M NH4HC03, and rechromatographed u s i n g t h e same c o n d i t i o n as i n P a r t A . R e p r i n t e d w i t h p e r m i s s i o n o f a u t h o r s , r e f . 3b.

Set-Tri-Leu-Ser-Ala-Leu-Gly-Gln-Ala-Leu-Lys-Ala

P r i o r t o t h e LC s e p a r a t i o n , t h i s p e p t i d e had been p a r t i a l l y p u r i f i e d by g e l f i l t r a t i o n and i o n exchange chromatography.

The sample was l o a d e d i n 5 m l of

6 M u r e a as t h r e e a p p r o x i m a t e l y equal volumes t h r o u g h t h e sample i n j e c t o r (U6K, Waters).

The l a r g e peak was c o l l e c t e d (see b a r i n F i g . 6 . 1 2 ) and rechromato-

graphed as shown i n F i g . 6.128.

B e f o r e rechromatography t h e t r a p p e d peak ( 2 m l )

was d i l u t e d t o 6 m l w i t h 0.1 M NH4HC03 and l o a d e d as t h r e e a p p r o x i m a t e l y equal volumes u s i n g i d e n t i c a l c o n d i t i o n s as t h o s e i n F i g . 12A.

The p u r i f i e d

p e p t i d e s were c h a r a c t e r i z e d by amino a c i d a n a l y s i s - a t y p i c a l r e s u l t was Ser

3.0 ( 3 ) , G l u 3.1 ( 3 ) , A l a 2.9 ( 3 ) , Leu 5.0 (5). Phe 1.0 ( 1 ) . Lys 2.0 ( 2 ) and T r p 1.0 ( 1 ) .

The r e c o v e r y o f t h e p u r i f i e d p e p t i d e , as measured by amino a c i d

a n a l y s i s , was 88%.

222 6 . 4 USE OF REVERSED PHASE THIN LAYER CHROMATOGRAPHY FOR THE MONITORING OF PREPARATIVE SEPARATIONS

W i t h t h e a d v e n t of p r e p a r a t i v e r e v e r s e d p h a s e s e p a r a t i o n s , i t was n e c e s s a r y t o have a p r o c e d u r e t o a s s a y t h e p u r i t y o f t h e s e p a r a t e d f r a c t i o n s . S i n c e a n a d d i t i o n a l l i q u i d c h r o m a t o g r a p h sometimes i s n o t a v a i l a b l e t o m o n i t o r t h e s e p a r a t i o n , we have d e v e l o p e d a s i m p l e TLC m e t h o d [ 2 9 1 .

F i g . 6 . 1 3 shows use

o f t h e r e v e r s e d phase TLC s y s t e m t o follow t h e s e m i - p r e p a r a t i v e p u r i f i c a t i o n o f a s y n t h e t i c o c t a d e c a p e p t i d e . Leu-Gly-Ser-Phe-Leu-Lys-Ser-Trp(CHO)-Leu-Ser-Ala-

Leu-Gly-Gln-Ala-Leu-Lys-Ala.

The TLC r e s u l t s p r o v i d e d a r a p i d c h e c k o f t h e HPLC

s e p a r a t i o n and a l s o v e r i f i e d t h a t t h e o p t i c a l d e n s i t y peaks a c t u a l l y c o n s i s t e d

of peptide m a t e r i a l ( n i n h y d r i n r e a c t i v e ) .

T h i s p e p t i d e c o n t a i n e d a l a r g e number

of h y d r o p h o b i c r e s i d u e s ; t h e r e f o r e , i t was s t r o n g l y r e t a i n e d o n a r e v e r s e d p h a s e

E C

Roton tion timo (hours1 F i g . 6.13.

The s e m i - p r e p a r a t i v e p u r i f i c a t i o n o f a s y n t h e t i c o c t a d e c a p e p t i d e . Leu-G1 y-Ser-Phe-Leu-Lys-Ser-Trp(CH0)-Leu-Ser-A1 a-Leu-G1 y-G1 n-A1 a-LeuLys-A1 a . S o l v e n t A c o n s i s t e d o f aqueous t r i e t h y l a m m o n i um p h o s p h a t e ( 1 . 5 mM, pH 3.2) a n d s o l v e n t B o f p r o p a n - 2 - 0 1 - a c e t o n i t r i l e - a q u e o u s triethylammonium phosphate ( 7 . 5 mM), (40:40:20, v / v / v ) . The f l o w - r a t e was l . O m l / m i n a n d t h e g r a d i e n t ( A t o B ) shown b y t h e d o t t e d l i n e was u s e d . A 5mg sample o f t h e p e p t i d e d i s s o l v e d i n b u f f e r A (0.21111) was i n j e c t e d . The i n s e t shows t h e c o r r e s p o n d i n g r e v e r s e d p h a s e TLC s e p a r a t i o n of t h e f r a c t i o n s . R e p r i n t e d w i t h permission of authors, r e f . 29.

223 TLC p l a t e .

However, s a t i s f a c t o r y r e s u l t s were o b t a i n e d when t e t r a h y d r o f u r a n was

added t o t h e m o b i l e phase, and t h e n a r r o w s p o t s shown i n F i g . 6 . 1 3 were obtained.

D e s p i t e d i f f e r e n c e s i n t h e m o b i l e phases, t h e r e i s a c l e a r

c o r r e l a t i o n between t h e LC and TLC s y s t e m s ; for example, t h e e a r l y e l u t i n g m a t e r i a l i n p o o l A o f LC f r a c t i o n s gave t h e h i g h e s t R F i n t h e r e v e r s e d p h a s e TLC s y s t e m .

Thus a r e v e r s e d phase TLC s y s t e m based o n Whatman KC18F p l a t e s

and a m o b i l e phase w h i c h c o n t a i n e d 3% sodium c h l o r i d e and 0.2% s o d i u m d o d e c y l s u l f a t e c a n be u s e d f o r t h e m o n i t o r i n g o f f r a c t i o n s from a n LC s e p a r a t i o n .

6.5

PREPARATIVE SEPARATIONS OF PROTEINS Reversed Phase S e p a r a t i o n s

6.5.1

W h i l e t h e p r e p a r a t i v e s e p a r a t i o n of a m i n o a c i d s and s m a l l p e p t i d e s b y r e v e r s e d phase LC i s becoming an a c c e p t e d p r o c e d u r e , t h e s e p a r a t i o n o f p r o t e i n s by t h i s technique s t i l l r e q u i r e s c a r e f u l e v a l u a t i o n .

The b i o l o g i c a l a c t i v i t i e s

o f many p r o t e i n s a r e s e n s i t i v e t o d e n a t u r a t i o n b y e x t r e m e s i n pH, b y c o n t a c t

w i t h o r g a n i c s o l v e n t s or h i g h s a l t c o n c e n t r a t i o n s , b y a d s o r p t i o n onto g l a s s or h y d r o p h o b i c m o i e t i e s , or a t an a i r - w a t e r i n t e r f a c e [ 3 1 1 .

Therefore a j u d i c i o u s

c h o i c e o f r e v e r s e d phase columns w i t h a s u i t a b l e c h o i c e o f o r g a n i c m o d i f i e r (e.9.

i s o p r o p a n o l ) and o p t i m a l i o n i c m o d i f i e r c o n c e n t r a t i o n s ( e . g . 0.1%

t r i f l u o r o a c e t i c a c i d ) has a l l o w e d t h e s e p a r a t i o n o f p r o t e i n s w i t h r e t e n t i o n o f biological activity. A s was m e n t i o n e d f o r t h e s e c t i o n o n p e p t i d e s e p a r a t i o n s , t h e a d d i t i o n of an i o n i c m o d i f i e r i s o f t e n e s s e n t i a l f o r t h e s e p a r a t i o n o f p r o t e i n samples by r e v e r s e d phase LC.

The a d d i t i o n o f i o n i c m o d i f i e r s t o t h e m o b i l e p h a s e c a n a l s o

g i v e u s e f u l d i f f e r e n c e s i n t h e r e s o l u t i o n o f complex p r o t e i n m i x t u r e s .

The

i o n i c m o d i f i e r s m o s t w i d e l y u s e d i n c l u d e a c i d s such a s t r i f l u o r o a c e t i c 1 3 2 , 3 3 1 p h o s p h o r i c [ 91 a n d . h y d r o c h l o r i c L34.361; a n d t h e s a l t s o f o r g a n i c b a s e s , s u c h as t r i e t h y l a m i n e p h o s p h a t e [ 7 1 and p y r i d i n e f o r m a t e [ 3 7 , 3 8 1 .

I n general, the

e f f e c t o f an i o n i c m o d i f i e r i s m a g n i f i e d f o r a p r o t e i n r e l a t i v e t o a p e p t i d e sample.

T h i s m a g n i f i c a t i o n can be a t t r i b u t e d t o t h e p r e s e n c e o f a much l a r g e r

number o f i o n i z a b l e g r o u p s i n t h e p r o t e i n s a m p l e .

Therefore, t h e optimal choice

o f an i o n i c m o d i f i e r can be more c r u c i a l i n a p r o t e i n s e p a r a t i o n . Apolipoprotein A-I,

w h i c h has a m o l e c u l a r w e i g h t of: 28,000,

c a n be r e a d i l y

e l u t e d from a C 1 8 c o l u m n (VBondapak) w i t h a m o b i l e phase t h a t c o n t a i n s 1% t r i e t h y l a m m o n i u m p h o s p h a t e and a g r a d i e n t of 0 t o 40% i s o p r o p a n o l .

The s u b s t i t u t i o n of t r i e t h y l a m m o n i u m p h o s p h a t e w i t h 5mM b u t a n e s u l p h o n a t e r e s u l t s i n

224

Elution volume (I1 F i g . 6.14.

The p r e p a r a t i v e s e p a r a t i o n o f a p o l i p o p r o t e i n A-I on a PrepPak c 1 8 column. The p a r t i a l l y p u r i f i e d sample o f a p o l i p o p r o t e i n A-I ( 0 . 1 9 ) was d i s s o l v e d i n 20ml o f 3M guanidine-HC1, 1% t r i e t h y l a m m o n i u m phosphate, pH 3.2 and loaded on t o t h e column. The s e p a r a t i o n was achieved w i t h a g r a d i e n t , as shown by t h e d o t t e d l i n e , from 1 % triethylammonium phosphate, pH 3 . 2 t o 80% i s o p r o p a n o l and 20% o f the phosphate b u f f e r .

t h e p r o t e i n sample b e i n g r e t a i n e d i n d e f i n i t e l y on t h e phenyl column 1391. Also use o f t h e v o l a t i l e i o n - p a i r i n g r e a g e n t , t r i f l u o r o a c e t i c a c i d d i d n o t a1 low e l u t i o n o f t h e p r o t e i n sample.

The a n a l y t i c a l s e p a r a t i o n c o u l d be d i r e c t l y

scaled up t o t h e p r e p a r a t i v e p u r i f i c a t i o n shown i n F i g . 6.14 where a O.lmg sample of p a r t i a l l y p u r i f i e d a p o l i p o p r o t e i n A-I was chromatographed on a reversert phase p r e p a r a t i v e column (PrepPak C18).

Loading up t o 5mg o f t h i s

crude p r o t e i n m i x t u r e c o u l d be separated w i t h o u t a s e r i o u s loss o f s e p a r a t i o n efficiency. F i g u r e 6.15 shows t h e s e m i - p r e p a r a t i v e s e p a r a t i o n o f z i n c - f r e e i n s u l i n o n an a n a l y t i c a l C 1 8 column (pBondapak) [401.

T h i s r e s u l t has a l l o w e d t h e

development o f a p r e p a r a t i v e s e p a r a t i o n o f 59 i n s u l i n samples on a commercial i n s t r u m e n t . . Since t h e Waters PrepLC/System 500 has p r o v i s i o n for a second column i t can be seen t h a t i t i s p o s s i b l e t o r a p i d l y s e p a r a t e m u l t i g r a m amounts of p r o t e i n by p r e p a r a t i v e LC.

Sometimes r e c o v e r y i s i n f l u e n c e d by t r e a t m e n t o f t h e sample a f t e r separation.

T r y p s i n (lmg) has been p u r i f i e d on an a n a l y t i c a l column (pBondapak

C18) w i t h a m o b i l e phase t h a t c o n s i s t e d o f 0.1% t r i f l u o r o a c e t i c a c i d w i t h a g r a d i e n t of 0 t o 45% a c e t o n i t r i l e [411.

For c o n s i s t e n t r e c o v e r y o f t h e

225

L

1

-

1

x:

%i

E C

0

sd 0 -

-

4 0

2

4

0

2

4

6

Time ( minutes) Fig. 6.15:

The semi-preparative separation of zinc-free insulin (2mg) o n a pBondapak c18 column. A mobile phase of 40% acetonitrile: 1% triethylammonium phosphate, pH 4 . 5 was used a t a f l o w rate of 1 ml/min. This Figure was taken from ref. 40 with authors permission.

enzymatic activity, it was necessary t o promptly isolate the purified enzyme under extremely mild conditions. The acetonitrile was removed by evaporation under nitrogen at room temperature and then the aqueous trifluoroacetic acid was removed immediately by lyophilisatlon. A 57% recovery of protein was

226

achieved, w i t h t h e p u r i f i e d m a t e r i a l e x h i b i t i n g a h i g h e r s p e c i f i c a c t i v i t y t h a n t h e o r i g i n a l sample (151.5 vs 135.8 u n i t s h g r e s p e c t i v e l y ) . A v e r y s i m i l a r s e p a r a t i o n was r e p o r t e d by T i t a n 1 e t a l . 1421.

By a d d i n g 2mM CaC12 t o t h e

mobile phase, t h e s t a b i l i t y o f t r y p s i n and c h y m o t r y p s i n t o t r i f l u o r o a c e t i c a c i d was enhanced, and q u a n t i t a t i v e r e c o v e r i e s o f t h e a c t i v i t i e s o f b o t h enzymes were o b t a ined. I n a s i m i l a r manner L i and Chung [431 have p u r i f i e d a 700ug e x t r a c t o f human growth hormone on a C18 column ( A l l t e c h ) w i t h a m o b i l e phase o f 43% 2-propanol and w a t e r c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c a c i d .

A y i e l d o f 140ug

o f h i g h l y p u r i f i e d growth hormone was o b t a i n e d from t h i s p u r i f i c a t i o n .

Svoboda

and van Wyk [ 4 4 1 p u r i f i e d 50 t o 751119 o f an e x t r a c t o f somatomedin C on a 7.8mm x 30cm column (pBondapak C18) w i t h a l i n e a r g r a d i e n t from 20% t o 60% a c e t o n i t r i l e i n aqueous 0.1% t r i f l u o r o a c e t i c a c i d . Recent s t u d i e s by Lewis [71 and o t h e r s [38,451 have shown t h a t t h e p o r e s i z e of t h e s i l i c a used i n t h e manufacture o f t h e r e v e r s e d phase p a c k i n g m a t e r i a l may be c r u c i a l f o r t h e s e p a r a t i o n o f h i g h m o l e c u l a r w e i g h t p r o t e i n s .

A

r e c e n t comparison o f LiChrosorb RP-18 (100 A p o r e s ) w i t h Aquapore RP-300 ( 3 0 0 A pores) showed t h a t t h e l a t t e r column was n o t a b l y s u p e r i o r f o r t h e chromatography o f p r o t e i n s with a molecular w e i g h t exceeding 15,000 D , e.g. t h e s e p a r a t i o n of [14 C I - m e t h y l a t e d bovine p a n c r e a t i c t r y p s i n i n h i b i t o r , myoglobin and ovalbumin 1453.

R i v i e r C461 has r e c e n t l y r e p o r t e d t h a t t h e p r e p a r a t i v e p u r i f i c a t i o n o f a

s y n t h e t i c p r e p a r a t i o n o f c o r t i c o t r o p h i n r e l e a s t n g f a c t o r c o u l d be achieved w i t h a much g r e a t e r e f f i c i e n c y on Vydac C18 (300 A p o r e s ) t h a n on pBondapak C18 (125 A p o r e s ) .

U n f o r t u n a t e l y i n b o t h o f these two s t u d i e s t h e s i l i c a s were

d i f f e r e n t and t h e bonded phases were d i f f e r e n t so t h e r e can be no c o n c l u s i o n t o the e f f e c t o f pore size.

N e v e r t h e l e s s , t h e r e seems t o be a t r e n d toward w i d e r

pore s i l i c a s for p r o t e i n s e p a r a t i o n s ; and, a l t h o u g h some m a n u f a c t u r e r s have now produced r e v e r s e d phase columns prepared f r o m wide p o r e s i l i c a , t h e r e has been slower progress i n t h e a v a i l a b i l i t y o f p r e p a r a t i v e columns packed from these materials. I n a d d i t i o n , t h e t y p e o f s i l i c a used i n t h e p r e p a r a t i o n i s j u s t as i m p o r t a n t as t h e pore s i z e ; i n f a c t i t may be of c r u c i a l importance [471. r h e r e f o r e i t i s d i f f i c u l t t o p r e c i s e l y assess t h e e f f e c t o f p o r e s i z e i n a s t u d y such as t h a t o f N i l s o n e t a l . [451 compared.

where two d i f f e r e n t types o f s i l i c a s were

We d e s c r i b e d t h e use o f t h e Radial-Pak C18 column f o r t h e

s e m i - p r e p a r a t i v e s e p a r a t i o n (up t o 20mg) o f complex p e p t i d e and p r o t e i n mixtures.

F i g . 6.16 shows t h e s u c c e s s f u l chromatography of a m i x t u r e o f

C - a p o l i p o p r o t e i n s f r o m human v e r y low d e n s i t y l i p o p r o t e i n s (VLDL) u s i n g two

227

RLTENTKU T Y E

Fig. 6.16.

IKXRSI

RCTLNTKU TlYL IYIN I

The elution profiles o f a mixture o f C-apolipoproteins obtained from VLDL when chromatographed on a Radial-Pak Resolve c18 column and with different acetonitrile gradients (see dashed lines). An O.lmg sample of the protein mixture was dissolved in O.lml o f 1% TEAP, 6M guanidine hydrochloride and then chromatographed on the c18 column with an initial mobile phase of 1% TEAP, pH 3.2. Reproduced with permission o f authors, see ref. 7 .

different gradient programs. In this separation the Radial-Pak C18 (90 A pores) column clearly separated apolipoprotein C-I from C-I11 and C-11. 1.2 With the same chromatographic conditions, a pBondapak C18 (125 A pores) column gave a broad peak with no separation of the protein mixture. Here again is a separation based on two different silicas as well as two different bonding approaches. It is clear that more work needs to be done to define the specific nature o f protein interactions with reversed phase packings. Nevertheless, many excellent reversed phase separations are being done both on the analytical scale and on gram scale for proteins and polypeptides using a wide variety o f packings. For reversed phase separations of proteins, many are better carried out with shallow gradients rather than with an isocratic (i.e. fixed) level of solvent. The gradual increase in organic solvent concentration would serve to continually displace the protein molecules from adsorption sites before irreversible multi-point binding occurs. The separation o f apolipoproteins A-I shown in Fig. 6 . 1 4 used a gradient of isopropanol to elute the protein sample from the preparative column. Experiments directed at establishing an isocratic separation of this protein showed that the protein was either eluted in the void volume or bound irreversibly despite the use o f mobile phases that differed by 1% increments of isopropanol.

228

Reversed phase chromatography has proven to be s p e c t a c u l a r l y s u c c e s s f u l

for t h e p r e p a r a t i v e s e p a r a t i o n o f lymphokines 1481 such as t h e i n t e r l e u k i n s I L - 1 , I L - 2 and I L - 3 1491 and c o l o n y - s t i m u l a t i n g . f a c t o r s (CSF) [501.

These

hormones a r e produced by c e l l s o f t h e immune system, and mediate t h e c o n t r o l o f e v e r y aspect o f i m u n e f u n c t i o n . ( t y p i c a l l y 10-l'

-

They a r e a c t i v e a t v e r y low c o n c e n t r a t i o n s

10-12M) and a r e d i f f i c u l t t o p u r i f y because o f t h e i r

tendency t o adsorb t o g l a s s and p l a s t i c s u r f a c e s .

However, r e v e r s e d phase LC

u s i n g t h e T F A - a c e t o n i t r i l e system has been f o u n d t o g i v e e x c e l l e n t s e p a r a t i o n s o f I L - 2 , I L - 3 and granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r (GM-CSF) which can be separated w i t h b a s e l i n e r e s o l u t i o n and q u a n t i t a t i v e y i e l d .

A

t y p i c a l s e p a r a t i o n o f I L - 2 and I L - 3 from t h e m u r i n e T-lymphoma c e l l l i n e LBRM-33 C511 i s shown i n F i g . 6.17.

An i n t e r e s t i n g o b s e r v a t i o n i s t h a t t h e s e

i n t e r l e u k i n s a r e v e r y s t a b l e t o these e l u t i o n c o n d i t i o n s o f l o w a p p a r e n t pH and h i g h c o n c e n t r a t i o n s o f o r g a n i c s o l v e n t , and can be s t o r e d f o r s e v e r a l weeks i n t h e e l u t i o n b u f f e r a t 4OC w i t h o u t l o s s o f a c t i v i t y .

0.3 60 50 0.2

-I

7 1 2 460

40

IP n

2 0.1

0 0 '

/

00°

,

5

10

15

20

25

30

35

Fraction Number

F i g . 6.17.

P r e p a r a t i v e p u r i f i c a t i o n o f a m i x t u r e o f lymphokines o b t a i n e d from l e c t i n - s t i m u l a t e d c e l l s o f a murine T c e l l lymphoma c e l l l i n e , LBRM-33 c l o n e 5A4. The column used was a Waters Semi-prep C18 column. (30 x 0.78cm) w i t h a m o b i l e phase o f 0.1% t r i f l u o r o a c e t i c a c i d and a g r a d i e n t o f 0-70% a c e t o n i t r i l e ( c o n t a i n i n g 0.1% t r i f l u o r o a c e t i c a c i d ) o v e r 70 m i n u t e s . The flow r a t e was 2ml/min and 2ml f r a c t i o n s were c o l l e c t e d . I L - 3 a c t i v i t y was assayed by t h e p r o l i f e r a t i o n o f an IL-3-dependent c e l l l i n e (FDCP-2) where one u n i t of a c t i v i t y i s d e f i n e d as i n d u c i n g t h e uptake o f a half-maximal amount o f t r i t i a t e d t h y m i d i n e i n a 200 p1 c u l t u r e volume. I L - 2 a c t i v i t y was assayed by t h e p r o l i f e r a t i o n o f an IL-2-dependent c y t o t o x i c c e l l l i n e as measured by t h e cpm o f t r i t i a t e d thymidine incorporated.

229

6.5.2

Other S e p a r a t i o n Modes

Normal phase HPLC was suggested f o r p r o t e i n samples by Rubenstein C521 b u t does n o t appear t o have been w i d e l y used s i n c e t h a t t i m e .

The h i g h .

c o n c e n t r a t i o n s o f o r g a n i c s o l v e n t s r e q u i r e d i n t h e m o b i l e phase a r e p r o b a b l y n o t c o m p a t i b l e w i t h many p r o t e i n s . G e l f i l t r a t i o n chromatography i s i n c r e a s i n g i n p o p u l a r i t y due t o t h e

i n t r o d u c t i o n o f h i g h performance columns C53-561.

These columns can be used i n

n e u t r a l pH e l u e n t s , and t h e a d d i t i o n o f reasonable s a l t c o n c e n t r a t i o n s i s s u f f i c i e n t t o e l i m i n a t e i n t e r f e r e n c e from i o n exchange e f f e c t s .

However, i n

o r d e r t o a b o l i s h t h e hydrophobic p r o p e r t i e s o f t h e m a t r i x , i t may be n e c e s s a r y t o add a m o d i f i e r to t h e m o b i l e phase, such as 1% deoxycholate [531, 0.3% SDS [571, 5-10% propanol C541, 20% e t h a n o l [561, or 0.1% p o l y e t h y l e n e

g l y c o l [551.

W i t h i n these c o n s t r a i n t s , a r a p i d m o l e c u l a r weight-based

s e p a r a t i o n o f most p r o t e i n m i x t u r e s can be o b t a i n e d w i t h these columns.

The

major disadvantage o f h i g h performance g e l fi 1 t r a t i o n chromatography a t p r e s e n t i s t h e low r a t i o o f pore volume t o t o t a l column volume [581 o f t h e c u r r e n t

generation o f support materials.

T h i s o f t e n r e q u i r e s t h e use o f two C53.551 or

more C54,551 columns i n tandem, w i t h a s s o c i a t e d h i g h c o s t s ; however, i t i s encouraging t h a t even such a d i f f i c u l t p r o t e i n as l y m p h o t o x i n C551 can be usefully purified. A t e c h n i q u e which i s o n l y i n t h e e a r l y stages o f e v a l u a t i o n i s h i g h

performance a f f i n i t y chromatography [59-661.

I n t h i s t h r o m a t o g r a p h i c mode, a

l i g a n d i s a t t a c h e d t o t h e column, and t h e sample separates i n terms of i t s b i o l o g i c a l a f f i n i t y f o r the immobilized ligand.

The r e s u l t s which have been

o b t a i n e d a r e p r o m i s i n g , and as t h e o p t i m i z a t i o n of s u p p o r t m a t e r i a l s C651 and l i g a n d c o u p l i n g methods 1661 proceeds, t h e method may be expected t o f i n d i n c r e a s i n g use i n t h e f u t u r e . Another most e x c i t i n g development i s t h e a p p l i c a t i o n of ion-exchange columns and t h e chromatofocusing techniques 1673.

A v a r i e t y o f p r o t e i n s were

separated by t h i s method u s i n g commercial chromatofocusing b u f f e r s .

The y i e l d s

o b t a i n e d a t a s e m i - p r e p a r a t i v e ( m i l l i g r a m ) s c a l e were e s s e n t i a l l y q u a n t i t a t i v e . and because o f t h e f o c u s i n g e f f e c t i n h e r e n t i n t h i s technique, t h e r e s o l u t i o n o f v a r i o u s p r o t e i n components was h i g h e r t h a n t h a t o f any o t h e r t e c h n i q u e , i n c l u d i n g r e v e r s e d phase LC.

230

6.6

OTHER CLASSES OF BIOCHEMICALS

The s u c c e s s f u l a p p l i c a t i o n o f LC t o p e p t i d e and p r o t e i n s e p a r a t i o n s does n o t appear t o be due t o any f a v o r a b l e p r o p e r t i e s o f these m a t e r i a l s .

On t h e

c o n t r a r y , o t h e r c l a s s e s o f compounds, and e s p e c i a l l y l i p i d s and n u c l e i c a c i d s , appear t o behave i n a much more p r e d i c t a b l e and t r a c t a b l e manner w i t h commonly used LC c o n d i t i o n s t h a n do p o l y p e p t i d e s .

Consequently t h e widespread use o f LC

i n the preparative separation of o t h e r classes of biochemicals merely awaits the investment o f t h e same amount of r e s e a r c h e f f o r t as has been i n v e s t e d i n t h e development o f p o l y p e p t i d e s e p a r a t i o n s . LC s e p a r a t i o n s o f n u c l e i c a c i d s have been e x t e n s i v e l y developed by Brown and o t h e r s 168.691 b u t p r e p a r a t i v e s e p a r a t i o n s a r e j u s t b e g i n n i n g t o appear i n t h e l i t e r a t u r e [69-721 w i t h t h e r e c e n t surge o f i n t e r e s t i n s y n t h e t i c oligonucleotides f o r genetic engineering.

I o n exchange, g e l f i l t r a t i o n and

r e v e r s e d phase s e p a r a t i o n s o f o l i g o n u c l e o t i d e s a r e now w i d e l y used as a f i n a l p u r i f i c a t i o n s t e p f o r s y n t h e t i c o l i g o n u c l e o t i d e p r i m e r s and probes.

Volatile

b u f f e r s such as triethylammonium a c e t a t e appear t o be v e r y s u i t a b l e f o r b o t h s i z e s e p a r a t i o n and r e v e r s e d phase modes [71 ,721. S y n t h e t i c l i p i d s [73,741, p h o s p h o l i p i d s [751 and s t e r o i d s [ 7 6 1 form a c l a s s o f compounds whose p r o p e r t i e s suggest t h a t p r e p a r a t i v e LC c o u l d be much more w l d e l y used than a t p r e s e n t .

The low p o l a r i t y o f m o s t l i p i d s enables t h e

use o f a d s o r p t i o n chromatography on b a r e s i l i c a columns 173,75,761 w i t h o r g a n i c solvent eluent mixtures.

For example Pate1 and Sparrow 1771 r e p o r t e d t h e

s e p a r a t i o n o f 59 o f crude egg y o l k p h o s p h a t i d y l c h o l i n e on s i l i c a g e l w i t h chloroform-methanol-water (60:30:4)

w i t h a r e c o v e r y o f 95% o f t h e s e p a r a t e d

For more p o l a r compounds such as l e u k o t r i e n e s 1741 and s t e r y l a c e t a t e s 1761, r e v e r s e d phase chromatography i s a1 so p o s s i b l e . The a v a i l a b i 1 i t y fractions.

o f s e v e r a l chromatographic modes for l i p i d s e p a r a t i o n s and t h e c l o s e a n a l o g i e s w i t h p r e v i o u s l y used chromatographic techniques suggest t h a t most p r e p a r a t i v e l l p l d s e p a r a t i o n s w i l l n o t be j u d g e d s u f f i c i e n t l y novel t o be p u b l i s h e d i n t h e i r own . r i g h t .

Consequently t h e chromatographic l i t e r a t u r e i s n o t expected t o

r e f l e c t t h e usage of LC i n l i p i d b i o c h e m l s t r y . A few p r e p a r a t i v e r e v e r s e d phase s e p a r a t ons o f c a r b o h y d r a t e s have been

r e p o r t e d [78-803 i n c l u d i n g one o f amino sugars 1801.

Both o c t a d e c y l s i l i c a

178,791 and amino [801 columns w i t h a c e t o n i t r i e-water m i x t u r e s have been used

231

I n g e n e r a l , b i o c h e m i c a l compounds o t h e r t h a n p o l y p e p t i d e s g i v e good r e s o l u t i o n and h i g h y i e l d s w i t h t h e v a r i o u s LC methods which have been investigated.

The use o f pH c o n t r o l or t h e a d d i t i o n o f i o n i c m o d i f i e r s i s n o t

generally required.

The p o t e n t i a l a p p l i c a t i o n o f p r e p a r a t i v e LC t o b i o c h e m i c a l

compounds i s such t h a t a b r i e f r e v i e w o f t h e e n t i r e f i e l d w i l l s h o r t l y be imposs ib l e .

6.7 1.

2. 3. 4.

5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19.

20. 21. 22.. 23. 24.

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235

CHAPTER 7 THE D IRECT PREPARAT IVE RESOLUTION OF ENANTIOMERS BY LIQUID CHROMATOGRAPHY ON CHIRAL STATIONARY PHASES William H. P i r k l e and Bruce C. Hamper The Roger Adam Laboratory

School of Chemical Sciences University of I l l i n o i s Urbana, I I I inois

CONTENTS 7.1

INTRODUCTION

7.2

CHROMATOGRAPHIC METHODS FOR THE ISOLATION OF ENANTIOMERS

7.3

CHROMATOGRAPHIC PROPERT I ES OF CHI RAL STAT IONARY PHASE (CSP)

7.4

CSPs FROM NATURALLY OCCURRING MATERIALS 7.4.1 7.4.2

7.5

Microcrystalline Cellulose Triacetate Potato Starch

SYNTHETIC CSPs 7.5.1 7.5.2 7.5.3 7.5.4 7.5.5 7.5.6 7.5.7 7.5.8 7.5.9 7.5.10

Development of Rationally Devised CSPs Chiral Crown Ethers Carbinol CSP Amino Acid DNBs Chiral Vinyl Polymers Polyamide CSPs Cyclodextrin Polymers TAPA CSPs Ligand Exchange Chromatography (LEC) Miscel laneous Liquid Chromatographic Methods

7.6

CONCLUSIONS

7.7

ADDENDUM

7.8

REFERENCES

7.1

I NTRODUCT I ON

Separation qf enantiomers poses one o f the more d i f f i c u l t p u r i f i c a t i o n problems encountered by chemists owing t o the inherent s i m i l a r i t y o f the components being separated [ l ] .

With the advent o f modern I i q u i d

chromatographic techniques [ 2 ] , researchers have a v a i l a b l e a powerful t o o l for separating even very s i m i l a r compounds.

W i t h i n the past few years, t h i s

236

methodology has been s u c c e s s f u l l y a p p l i e d t o t h e problem o f enantiomer separation.

A number of q u i t e i m p r e s s i v e enantiomer s e p a r a t i o n s have been

r e p o r t e d , many such a r e d e s c r i b e d i n r e v i e w b y L i n d n e r C31, Blaschke [41, Davankov [51, Audebert 161, and K r u l l [71.

I t has l o n g been a p p r e c i a t e d t h a t chromatography o f a racemate on a c h i r a l adsorbant m i g h t , i n p r i n c i p l e , r e s u l t i n t h e s e p a r a t i o n o f enantiomers C8,91. E a r l y a t t e m p t s t o e f f e c t such chromatographic r e s o l u t i o n s were u s u a l l y of m a r g i n a l success owing to b o t h t h e c h o i c e of c h i r a l adsorbant and to l i m i t a t i o n s i n chromatographic t e c h n o l o g y .

Only r e c e n t l y has t h e l i t e r a t u r e begun t o

suggest t h e p o s s i b i l i t y t h a t such d i r e c t r e s o l u t i o n s may become f a c i l e , u s e f u l and g e n e r a l .

Successful r e s o l u t i o n s cannot be a t t r i b u t e d s o l e l y t o improvements

i n chromatographic s k i l l o r hardware; g r e a t advances a r e underway i n t h e d e s i g n o f e f f e c t i v e c h i r a l s t a t i o n a r y phases ( C S P s ) .

There i s a growing awareness t h a t

one can r a t i o n a l l y d e s i g n CSPs f o r p a r t i c u l a r r e s o l u t i o n s and t h a t these CSPs can be bonded t o supports h a v i n g mechanical s t a b i l i t y and p a r t i c l e s i z e s compatible w i t h t h e h i g h o p e r a t i n g p r e s s u r e s o f modern L C . .

One can a n t i c i p a t e

t h a t f u t u r e workers w i l l have a v a i l a b l e a c o l l e c t i o n o f c h i r a l columns which w i l l make p o s s i b l e t h e d i r e c t r e s o l u t i o n o f a t r u l y l a r g e a r r a y o f compounds. Apart f r o m t h e obvious a n a l y t i c a l i m p l i c a t i o n s , such columns w i l l o f f e r a r e a d y source o f c h i r a l m a t e r i a l s o f known e n a n t i o m e r i c p u r i t y .

Such methodology i s

c e r t a i n t o be adopted r a p i d l y and w i l ! be used i n a v a r i e t y o f d i s c i p l i n e s . T h i s r e v i e w w i l l c o n s i d e r r e c e n t developments i n t h e l i q u i d chromatographic s e p a r a t i o n o f enantiomers on a p r e p a r a t i v e s c a l e .

For o u r purposes,

" p r e p a r a t i v e " s e p a r a t i o n s w i l l be those i n v o l v i n g

he c o l l e c t i o n o f t h e r e s o l v e d

m a t e r i a l s f o r subsequent u t i l i z a t i o n , whether t h s

solation affords

s u b - m i l l i g r a m or m u l t i - g r a m q u a n t i t i e s o f m a t e r i a l

.

Moreover, emphasis w i l l be

placed on methods which a l l o w t h e i s o l a t i o n o f e n a n t i o m e r i c a l l y p u r e , r a t h e r t h a t s l i g h t l y enriched m a t e r i a l s .

This w i l l l a r g e l y e l i m i n a t e discussion o f

many o f t h e h i s t o r i c a l l y s i g n i f i c a n t , b u t g e n e r a l l y i n e f f i c i e n t r e s o l u t i o n s covered i n o l d e r r e v i e w s 110-131.

* I n a sense, however, i t i s a r t i f i c i a l t o d i s t i n g u i s h between a n a l y t i c a l and p r e p a r a t i v e r e s o l u t i o n s , s i n c e t h e a n a l y t i c a l r u n s w i l l almost always be used i n t h e process o f o p t i m i z i n g c o n d i t i o n s f o r p r e p a r a t i v e r u n s .

237 7.2

CHROMATOGRAPHIC METHODS FOR THE ISOLATION OF ENANTIOMERS

C h r o m a t o g r a p h i c methods for o b t a i n i n g e n a n t i o m e r i c a l l y p u r e m a t e r i a l s f r o m e n a n t i o m e r i c a l l y i m p u r e m i x t u r e s may be c l a s s i f i e d as e i t h e r i n d i r e c t or D i r e c t s e p a r a t i o n e n t a i l s t h e use o f e i t h e r a c h i r a l s t a t i o n a r y p h a s e

direct.

(CSP) or a c h i r a l m o b i l e phase, w h i l e t h e i n d i r e c t m e t h o d r e q u i r e d a c h i r a l

d e r i v a t i z i n g agent t o convert t h e mixture of enantiomers i n t o a m i x t u r e of diastereomers.

D i a s t e r e o m e r s c a n d i f f e r i n many o f t h e i r p r o p e r t i e s i n c l u d i n g A l t h o u g h t h i s r e v i e w w i l l n o t c o v e r i n d i r e c t methods

chromatographic behavior.

[ 1 1 , 1 4 1 , we w i l l c o n t r a s t t h a t a p p r o a c h t o t h e d i r e c t method. I n t e r a c t i o n of enantiomers w i t h a c h i r a l s t a t i o n a r y phase g i v e s r i s e t o t r a n s i e n t d i a s t e r e o m e r i c a d s o r b a t e s w h i c h must d i f f e r i n t h e i r s t a b i i t y enantiomers a r e to separate.

f the

I n t h e i n d i r e c t method, t h e d i a s t e r e o m r i c

compounds may d i f f e r i n s t a b i l i t y , b u t such d i f f e r e n c e i s n o t e s s e n t i a l t o t h e chromatographic separation.

Enantiomers a l s o a f f o r d t r a n s i e n t d i a s t e r e o m e r i c

s o l v a t e s w i t h c h i r a l m o b i l e phases.

Here t h e s i t u a t i o n i s m o r e c o m p l e x , b u t

s t a b i l i t y d i f f e r e n c e s a r e u s u a l l y deemed i m p o r t a n t t o a n y r e s o l u t i o n n o t e d . I n p r i n c i p l e , d i r e c t methods a r e t o be p r e f e r r e d o v e r i n d i r e c t m e t h o d s , s i n c e d e r i v a t i z a t i o n and d e - d e r i v a t i z a t i o n may be b y p a s s e d .

This i s e s p e c i a l l y

advantageous f o r compounds h a v i n g low b a r r i e r s t o r a c e m i z a t i o n a n d w h i c h m i g h t be r a c e m i z e d d u r i n g c h e m i c a l m a n i p u l a t i o n .

Moreover, d i r e c t methods a r e

absolute i n t h a t t h e enantiomeric p u r i t y of t h e chromatographically separated bands i s n o t t i e d t o t h e e n a n t i o m e r i c p u r i t y o f a c h i r a l d e r i v a t i z i n g a g e n t . F o r example, t h e e n a n t i o m e r i c p u r i t y o f t h e a n t i p o d e s o b t a i n e d from a d i r e c t r e s o l u t i o n depends only o n t h e e x t e n t t o w h i c h t h e two b a n d s a r e s e p a r a t e d a n d i s not d i r e c t l y i n f l u e n c e d b y t h e e n a n t i o m e r i c c o m p o s i t i o n o f t h e s t a t i o n a r y phase.

I f t h e bands a r e c o m p l e t e l y s e p a r a t e d , t h e two s t e r e o i s o m e r s w i l l be

enantiomerically pure.

On t h e o t h e r hand, i f t h e c h i r a l d e r i v a t i z i n g a g e n t

employed for an i n d i r e c t r e s o l u t i o n i s o f l e s s t h a n t o t a l e n a n t i o m e r i c p u r i t y , t h e s e p a r a t e d d i a s t e r e o m e r s w i l l be o f t h e same d e g r e e o f e n a n t i o m e r i c impurity.

D e s p i t e t h e s e d i s a d v a n t a g e s , t h e i n d i r e c t method i s q u i t e u s e f u l a n d

i s f r e q u e n t l y employed.

C h i r a l d e r i v a t i z i n g agents which a f f o r d

c h r o m a t o g ' a p h i c a l l y s e p a r a b l e d i a s t e r e o m e r s h a v e been r e c e n t l y r e v i e w e d [ I 4 1 a n d research i n t h i s f i e l d i s ongoing.

238

Resolution of racemates o n chiral columns is an almost ideal w a y to o b t d i n modest quantities of each enantiomel In an enantiomerically pure state. The Scale o n which these resolutions ran b e conducted depends upon t h e size a n d efficiency o f the column employed and the degree o f "chiral recognition" a f f o r d e d by the CSP. Chiral recogiit.ion can o c c u r o n l y if tnere is a m i n i m u m of three simultaneous, stei eochemically dependent interaction> between the stationary phase and at least o n e of t h e solute enantiorners [151. The neceisarv interactions can be any o f the common intermoleculal interactions normally encountered, such as hydrogen bonding, electrostatic interactions. dipole-dipole alignments, n-n complexation, steric repulsions, and hydrophi Iic 01 hydrophobic intei.actions.

CSP

n

Solute Molecule

CSP

Solute Molecule

A'

D IIIIIIC

C 111111 M -)(

u fig. 7.1. T w o possible diasteromeric adsorbates each consisting of a chiral stationary phase (CSP) and o n e o f the enantiomeric s o l u t e molecules M and M*

A generalized CSP having three potential sites of interaction, A . 8 . and C is shown in Fig. 7.1. The molecular skeleton serves t o hold the thlre interaction sites in a distinct geometric array and protects the baLk f a c e of the ABC triangle from approach by solute. The solute molecule, M , coiltainc three complimentary sites A ' , B ' , and C ' , which interact with A , 6 , and C respectively during t h e fournation o f a diastereorneric adsorbate as shown in the figure o n the left. Its enantiomer, M I , can interact with t h e CSP such that only t w o of t h e potential interactions can o c c u r at a n y o n e instant. If a1 I three interactions a r e attractive in nature, enantiomer- M will form the m o r e

239 s t a b l e d i a s t e r e o m e r i c a d s o r b a t e a n d t h e r e f o r e be p r e f e r e n t i a l l y r e t a i n e d upon the CSP.

I f o n e of t h e i n t e r a c t i o n s i s r e p u l s i v e , such as t h e i n t e r a c t i o n

between A and A ' , M* w i l l be more s t r o n g l y r e t a i n e d .

D e t a i l e d knowledge o f t h e

n a t u r e o f t h e d i a s t e r e o m e r i c a d s o r b a t e s f o r m e d n o t only a l l o w s o n e t o d e t e r m i n e w h i c h s o l u t e s can b e r e s o l v e d o n a g i v e n CSP b u t a l s o a l l o w s c o r r e l a t i o n o f e l u t i o n o r d e r and s t e r e o c h e m i s t r y . I n d i s c u s s i n g c h i r a l r e c o g n i t i o n , i t i s u s e f u l t o c o n s i d e r whether t h i s p r o c e s s o c c u r s a t a s i n g l e c h i r a l s i t e ( i n d e p e n d e n t b e h a v i o r ) . or r e q u i r e s t h a t m u l t i p l e c h i r a l s i t e s be o r g a n i z e d i n some way so as t o a c t i n c o n c e r t (cooperative behavior).

Independent processes can o c c u r i n s o l u t i o n ,

c o o p e r a t i v e p r o c e s s e s c a n n o t ( u n l e s s t h e e n t i r e a s s e m b l y o f c h i r a l s i t e s c a n be organized i n solution).

Although the precepts of the three-point r u l e a r e s t i l l

v a l i d d u r i n g c o o p e r a t i v e b e h a v i o r , i t i s n o t a l w a y s a p p a r e n t a s t o what interactions are involved.

N e e d l e s s t o say, more t h a n t h r e e i n t e r a c t i o n s c a n be

involved. A number o f r e s e a r c h e r s h a v e employed c h i r a l m o b i l e p h a s e a d d i t i v e s ( C M P A s )

f o r t h e r e s o l u t i o n o f e n a n t i o m e r s on a c h i r a l c o l u m n s .

I n t h e l i g a n d exchange

mode, t h i s a p p r o a c h has p r o v e n t o be q u i t e e f f e c t i v e o n a n a n a l y t i c a l s c a l e f o r The CMPA m e t h o d i s a t t r a c t i v e

a m i n o a c i d s and o t h e r b i d e n t a t e l i g a n d s [16-191.

i n t h a t i s e n a b l e s t h e u s e r t o choose among many t y p e s o f r e a d i l y a v a i l a b l e columns.

From t h e p r e p a r a t i v e s t a n d p o i n t , t h e CMPA method h a s s e v e r a l i n t r i n s i c

disadvantages.

F i r s t , b u l k q u a n t i t i e s o f t h e CMPA w o u l d be r e q u i r e d , t h e amount

u s e d e q u a l l i n g or e x c e e d i n g t h e amount o f r a c e m a t e t o be r e s o l v e d .

Ultimately,

t h e c o l l e c t e d e n a n t i o m e r s m u s t be s e p a r a t e d from t h e CMPA [201. A s Davankov a n d Kurganov 1 2 1 1 have a p t l y p o i n t e d out, i t can b e d i f f i c u l t

t o a s c e r t a i n t h e mechanisms r e s p o n s i b l e for s o l u t e r e t e n t i o n i n t h e CMPA method.

I f we i m a g i n e a p a i r o f e n a n t i o m e r s , A a n d A* i n a s o l u t i o n c o n t a i n i n g

a CMPA, two e q u i l i b r i a can be w r i t t e n such t h a t A and A* e a c h form a d i f f e r e n t d i a s t e r e o m e r i c complex w i t h t h e CMPA and a n y o f t h e f i v e s p e c i e s c a n be retained.

H e r e , r e s o l u t i o n can b e a t t r i b u t e d t o a A

+

CMPA-EA - CMPAI 7

A*

+

CMPA

[A*-

CMPAI

240

combination o f ; a ) d i f f e r e n t i a l a d s o r b t i o n o f t h e d i a s t e r e o m e r i c complexes

[A-CMPAI and [ A * - C M P A I ,

analogous t o t h e s e p a r a t i o n o f d i a s t e r e o m e r i c

d e r i v a t i v e s , b ) s t r o n g a d s o r b t i o n o f t h e enantiomers A and A * w i t h t h e CMPA s e l e c t i v e l y " l i f t i n g " one enantiomer i n t o t h e m o b i l e phase t h r o u g h p r e f e r e n t i a l s o l v a t i o n and c ) t h e s t a t i o n a r y phase, by s t r o n g l y a d s o r b i n g t h e CMPA, a c t s as a c h i r a l s t a t i o n a r y phase and s e l e c t i v e l y r e t a i n s one enantiomer.

I n case b . t h e

p r e f e r e n t i a l l y s o l v a t e d enantiomer e l u t e s f i r s t ; i n case c i t e l u t e s l a s t . Hence, c o r r e l a t i o n o f e l u t i o n o r d e r s and a b s o l u t e c o n f i g u r a t i o n s may be complex owing t o u n c e r t a i n t y as t o which mechanism i s dominant.

I n some cases t h e CMPA

may be r e t a i n e d by t h e s t a t i o n a r y phase even a f t e r a d d i t i o n o f t h e CMPA has ceased.

Such " c o a t i n g " w i t h a CMPA a l l o w s one t o t r a n s f o r m an o r d i n a r y c h i r a l

column i n t o a de f a c t o CSP. 7.3

CHROMATOGRAPHIC PROPERTIES OF CHIRAL STATIONARY PHASES (CSPS)

While t h i s r e v i e w w i l l f o c u s on p r e p a r a t i v e s e p a r a t i o n s o f enantiomers, i t w i l l be u s e f u l t o d i s c u s s a n a l y t i c a l s e p a r a t i o n s as p r e l u d e s t o p r e p a r a t i v e

runs.

The r e c e n t development of CSPs e f f e c t i v e f o r p r e p a r a t i v e work can be

a t t r i b u t e d l a r g e l y t o r e s e a r c h done a t t h e a n a l y t i c a l l e v e l .

For example, t h e

s e p a r a b i l i t y o f enantiomers on a g i v e n CSP can be e v a l u a t e d q u i c k l y u s i n g a small q u a n t i t y o f racemate.

We1 1 separated enantiomers a r e p o t e n t i a l c a n d i d a t e s

f o r i n c o r p o r a t i o n i n t o " r e c i p r o c a l " CSPs which should r e s o l v e compounds o f t h e type used i n t h e i n i t i a l CSP ( v i d e i n f r a ) .

Any CSP which performs w e l l a t t h e

a n a l y t i c a l l e v e l can be used i n p r e p a r a t i v e columns.

Since t h e p r o p e r t i e s

d e s i r e d i n a p r e p a r a t i v e c h i r a l column a r e n o t u n l i k e those r e q u i r e d f o r any p r e p a r a t i v e s e p a r a t i o n , any t r e a t m e n t o f p r e p a r a t i v e l i q u i d chromatographic technique i s r e l e v a n t t o the s e p a r a t i o n o f e n a n t i o m e r s .

Here, we s h a l l t r e a t

s e v e r a l chromatographic parameters as t h e y s p e c i f i c a l l y ' a p p l y t o CSPs. The CSP must e x h i b i t a s u i t a b l e s e p a r a t i o n f a c t o r , a , f o r t h e two enantiomers, where a i s t h e r a t i o o f t h e c a p a c i t y f a c t o r o f t h e more h i g h l y r e t a i n e d enantiomer, k ' * . over t h a t o f t h e l e s s r e t a i n e d enantiomer, k I l . The a c t u a l a v a l u e r e q u i r e d for t h e r e s o l u t i o n o f a p a r t i c u l a r amount of sample w i l l depend on b o t h t h e s i z e and e f f i c i e n c y o f t h e column.

Typically,

p r e p a r a t i v e s e p a r a t i o n s i n v o l v e i n t e n t i o n a l sample o v e r l o a d of t h e column so as t o o b t a i n the g r e a t e s t amount o f r e s o l v e d m a t e r i a l i n t h e s h o r t e s t amount of

time.

Under these c o n d i t i o n s , b o t h t h e e f f i c i e n c y o f t h e column and the a v a l u e

can be d r a s t i c a l l y reduced compared t o t h e " s m a l l sample" s i t u a t i o n . Consequently, samples e x h i b i t i n g l a r g e a values a r e t h e e a s i e s t t o r e s o l v e i n

241

t h e " o v e r l o a d " c o n d i t i o n and a l l o w t h e g r e a t e s t o u t p u t o f m a t e r i a l . Samples e x h i b i t i n g small a v a l u e s may have t o be r e s o l v e d u s i n g "nonoverload" c o n d i t i o n s and perhaps r e c y c l e t e c h n i q u e s .

I n terms o f p r e p a r a t i v e s e p a r a t i o n o f

enantiomers. major advances w i l l come i n t h e form o f more e f f e c t i v e CSPs. The e f f i c i e n c y o f a g i v e n chromatographic system, measured i n t h e o r e t i c a l p l a t e s , i s h e a v i l y i n f l u e n c e d by hardware c o n s i d e r a t i o n s , b y t h e p a r t i c l e s i z e and s u r f a c e area o f t h e a d s o r b a n t , and by m o b i l e phase flow r a t e .

However,

k i n e t i c p r o p e r t i e s o f t h e s t a t i o n a r y phase a l s o c o n t r i b u t e t o t h e o v e r a l l e f f i c i e n c y o f t h e chromatographic process C221.

Ift h e a d s o r p t i o n

process i s slow, t h e chromatographic band w i l l t a i l .

a r e p a r t i c u l a r l y e v i d e n t when s o l u t e s a r e s t r o n g l y adsorbed. i m p l i c a t i o n s f o r t h e design o f CSPs.

-

desorption

Mass t r a n s f e r d i f f i c u l t i e s T h i s has

To i n c r e a s e t h e magnitude o f a, one must

i n c r e a s e t h e d i f f e r e n c e i n t h e s t a b i l i t i e s of t h e d i a s t e r e o m e r i c a d s o r b a t e s . T h i s w i l l be most e a s i l y accomplished when a d s o r p t i o n e n e r g i e s a r e l a r g e .

Thus,

d e s i g n c o n s i d e r a t i o n s which i n c r e a s e a may a d v e r s e l y a f f e c t band shape. E l e v a t e d temperatures w i l l improve band shapes b u t can be expected t o d i m i n i s h a values.

A t room temperature t h e flow r a t e fo r optimum e f f i c i e n c y may be l e s s

than t h e p r e p a r a t i v e chromatographer w i l l wish t o use.

The use of

m i c r o p r o c e s s o r c o n t r o l l e d p r e p a r a t i v e systems w i l l make r e p e t i t i v e , small s c a l e , " e f f i c i e n t " s e p a r a t i o n s f e a s i b l e s i n c e these can be conducted o v e r n i g h t w i t h o u t t h e a t t e n t i o n o f an o p e r a t o r . The i d e a l CSP w i l l e x h i b i t a h i g h sample l o a d i n g c a p a c i t y p e r gram o f s t a t i o n a r y phase w i t h o u t unreasonable decay o f e i t h e r t h e a v a l u e or chromatographic e f f i c i e n c y .

A chromatographic parameter which embodies b o t h o f

these f a c t o r s i s r e s o l u t i o n R S .

For t h e c o l l e c t i o n of two components, we a r e

most i n t e r e s t e d i n t h e r e l a t i o n s h i p between sample s i z e and r e s o l u t i o n , R S , d e f i n e d as t h e r a t i o o f t h e d i f f e r e n c e i n r e t e n t i o n times o f t h e two peaks t o t h e average base peak w i d t h .

( R e f e r t o Chapter 1.3 f o r more on r e s o l u t i o n . )

R S v a l u e s a l l o w f o r t h e comparison of r e s o l u t i o n e f f i c i e n c i e s and e s t i m a t i o n s

of the p u r i t i e s o f c o l l e c t e d m a t e r i a l s .

W i t h i n t h e l i n e a r c a p a c i t y range,

sample s i z e w i l l n o t degrade t h e r e t e n t i o n o r r e s o l u t i o n o f t h e components. Beyond t h i s range, an i n c r e a s e i n sample s i z e w i l l l e s s e n t h e r e s o l u t i o n R,, and t h e e f f i c i e n c y o f t h e column [ 2 3 1 .

For p r e p a r a t i v e work, i t i s u s e f u l t o

f i n d t h e l a r g e s t amount o f sample which s t i l l a l l o w s an R s v a l u e o f one or greater.

An R, v a l u e o f u n i t y i n d i c a t e s a 2% o v e r l a p o f t h e areas o f two

Gaussian peaks [21. greater resolution.

L a r g e r R,

v a l u e s i n d i c a t e l e s s o v e r l a p and c o n s e q u e n t l y

242

The capacity of a particular CSP for the resolution of racemic mixtures can be determined o n an analytical scale and used to approximate the conditions appropriate for larger preparative columns. A medium pressure chromatography system capable of resolving multigram quantities o f material [241 will typically employ larger particle size adsorbants (40-60pm) and exhibit lower backpressures (50-200 psi) than will its analytical counterpart. The larger irregular adsorbant particles'often have greater surface area than do the spherical particles being increasingly used for analytical columns. Hence, the preparative stationary phases will often exhibit larger loading capacities per gram than their analytical counterparts [251. The intermediate semi-preparative (10 mm i.d.) columns are typically just large analytical columns, being packed with small particle adsorbants and being used in analytical HPLC systems. Due to their size, they are limited to resolving mg quantities of racemates per pass Subsequent discussion will attempt to give insight into the relative merits of various CSPs as high performance adsorbants, based on their sample capacities, separation factors and efficiencies for stipulated racemates. Efficiency is especially important since a CSP must not only show a difference in affinity for the enantiomers, but it should exhibit good chromatographic properties as well. All too often authors fail to provide complete data concerning CSP column performance. Even so, direct comparisons are still somctimes Dossible. 7.4

CSPs FROM NATURALLY OCCURRING MATERIALS

The first CSPs were readily available chiral solids such as sugars, starch, cellulose and wool [10-131. While these materials are inexpensive and allow construction of large columns, they have, for the most part, been found to perform poorly as CSPs. Two polysaccarides, triacetylcellulose and potato starch are exceptions to this generalization. ne Cellulose T r i a c e t a t e Microcrystal 1 ine triacetylcellulose has been extensively used to resolve an evergiowing number of racemic mixtures. Hesse and Hagel have shown that the mode o f preparation i crucial to the effectiveness o f this CSP [26a,bl. Each D-glucose unit must be peracetylated, the greatest selectivity being obtained by "in situ" acetylation. Recrystallization must be avoided for it destroys the resolving ability of the CSP, presumably by altering the secondary structure of the natural polymer. Using their cellulose-derived CSP, Hesse and Hagel performed the first quantitative resolution o f Tr'dgers base, 1 (Table 7.1). 7.4.1

Microcrystal I

243

The Preparative Chromatographic Resolution of Chiral Compounds Containing Two Aryl Substituents On Microcrystalline Cellulose Triacetate.

TABLE 7.1

Sample mass

Racema te

Column dimensions L X i.d.(cm)

a

Rs

ref.

100 mg 50 mg

16 X 2 30 X 2.5

2.37 1.9

>1.0 3.9

26a 302

250 mg

40 X 3.1

2.63

>1.0

26b

1 mg

2 5 X 0.8

-

>l.O

29a

150 mg

60 X 2.5

-

1.0

29b

50 mg

30 X 2.5

2.1

3.8

30

50mg

30 X 2.5

1.5

1.6

30

50mg

30 X 2.5

1.2

1.3

30

50mg

30 X 2.5

3.7

6.0

30

50mg

30 X 2 . 5

1.2

1.4

30

0

R cFi3

0

6

7

R1

C02CH3

R2

H

C02CH-j

C02CH3

50mg

30 X 2.5

1.4

1.4

30

Br

Br

50mg

30 X 2.5

1.4

1.9

30

244

9

10

N

rv

M i c r o c r y s t a l l i n e t r i a c e t y l c e l l u l o s e o f 58pm p a r t i c l e s i z e i s s u i t a b l e as a chromatographic support and can withstand moderate pressures ( 2 - 1 1 b a r ) w i t h o u t bed collapse o r d i s r u p t i o n o f the polysaccharide s t r u c t u r e .

Ethanol i s m o s t

commonly employed as the mobile phase, although pentane, e t h e r and benzene have a l s o been used C27,281.

Lindner and Mannschreck C29al have prepared an

a n a l y t i c a l LC column c o n t a i n i n g small p a r t i c l e (5-10pm) m i c r o c r y s t a l l i n e c e l l u l o s e t r i a c e t a t e which can withstand pressures a s high as 195 b a r .

This

column has been used f o r the complete r e s o l u t i o n o f lmg q u a n t i t i e s o f

trans-1.2-diphenylcyclopropane,

3,

i n a s i n g l e pass (Table 7 . 1 ) .

Schlogl [301 has q u a n t i t a t i v e l y resolved compounds

4, 5 , 6 ,

and

2, each

of

which c o n t a i n two aromatic subst i t u e n t s , on 30 x 2.5 cm i . d . columns o f m i c r o c r y s t a l I i n e c e l l u l o s e t r i a c e t a t e (Table 7.1). Pentatetraene, diazi r idine,

9,

and benz0[2,2]metacyclophane,

lo, have

8.

a l s o been resolved, and

although the chromatographic parameters were n o t reported, the r e s o l u t i o n s were described as " n e a r l y basel ine" [27,31,32]. Compounds lacking a r y l subst i t u e n t s , such as the b a r b i t u a t e d e r i v a t i v e s ,

*-e,

have a l s o been resolved on

t r i a c e t y l c e l l u l o s e (Table 7.2). The amounts o f b a r b i t u a t e s r e s o l v a b l e i n a s i n g l e pass were between 160 and 300mg of racemate on a column (85 x 2.5 cm i . d . ) c o n t a i n i n g 205gm o f adsorbant ( F i g . 7.2) 1331. T r i a c e t y l c e l l u l o s e has been s u c c e s s f u l l y employed t o r e s o l v e compounds whose c h i r a l i t y i s l a b i l e since i t s t e m s from hindered r o t a t i o n about a

245

TABLE 7.2

Preparative Resolution o f Barbiturates on Microcrystalline Cellulose Triacetate. a

R1

Rac ema t e

a

H

/

v \CH3 0

a

Rs

wt (mgs) sample

C2H5

2.3

2.3

301

R2

C6H5

b

I-cyclohexenyl

CH3

1.7

1.5

160.6

c

I-cyclohexenyl

C2H5

2.0

1.7

205

d

cyc lohexy 1

C" 3

1.9

1.5

200

e

cyclohexyl

C2H5

2.4

1.7

200.2

1 la-e

a Column:

85 X 2.5 cm, Solvent: 95% ethanol; 2.5 bars pressure

-

-

13

12

14

N

15

Y

16

N

carbon-carbon or carbon-nitrogen single bond. Enamide 2 [ r e f . 341, hexadiene 13 [ref. 351, polarized alkene 14 [ref. 361. and N,N,N'.N'-tetramethyldithioozamide 15 [ref. 371, were partially resolved on triacetylcellulose.

246

1000

Fig. 7.2.

Resolution o f Methylphenobarbital.

ref. 33, p. 1001.

2500

2000

1500

Reprinted w i t h permission from

The racemate ( l l a ) was resolved chromatographically u s i n g

m i c r o c r y s t a l l i n e c e l l u l o s e t r i a c e t a t e as adsorbent (205mgs adsorbent, 85 X 2.5

cm. chromatography tube, 301mgs racemate using 95% ethanol as e l u e n t under a The a c t i v e acids obtained had a m.p. o f 22 0.645, ethanol) (125 mg) and 26.8O (C.

pressure o f 2 . 5 b a r s ) . -26.6O

(C,

IOOOC and a

0.670, ethanol) ( 1 2 7 mg).

Recycle techniques or shaving o f the chromatographic peaks would seem t o be required i f e n a n t i o m e r i c a l l y pure m a t e r i a l i s t o be obtained.

Although the

enantiomers were not completely separated i n the preceding instances, the method c l e a r l y provides an extremely m i l d method for the r e s o l u t i o n o f t h e r m a l l y l a b i l e compounds.

Coupled w i t h f r a c t i o n a l c r y s t a l l i z a t i o n , even p a r t i a l r e s o l u t i o n on

a c h i r a l column makes i t p o s s i b l e h i g h enantiomeric p u r i t y .

to o b t a i n some rather unusual compounds

For example, t e t r a o r g a n o t i n

3,has

enant iomer i c a l l y pure by t h i s combinat ion of techniques [381.

in

been obtained Recently, Schogl

has p a r t i a l l y resolved some racemic 1 , 1 , 2 , 2 - t e t r a k i s a r y l e t h a n e s having C2 symmetry [391. M i c r o c r y s t a l l i n e t r i a c e t y l c e l l u l o s e i s an example o f a useful " n a t u r a l " CSP having physical p r o p e r t i e s s u i t a b l e f o r chromatographic use avd showing reasonable s e l e c t i v i t y for c e r t a i n c h i r a l molecules.

U n f o r t u n a t e l y , the

247

mechanisms responsible for chiral recognition o n this CSP are not yet well understood. Hesse and Hagel C26a,bl have provided the most complete study of the relationship between the structure of the cellulose derivatives and the resultant chromatographic properties. Dissolution or purification of the microcrystalline CSP greatly reduces its ability to resolve enantiomers. This suggests the CSP exhibits "cooperative" rather than "independent" behavior (sec. 7.2) in the chiral recognition process, since recrystallization effects the secondary structure o f the natural polymer but not the individual glucose units. Although compounds analogous to those shown may prove to be resolvable on triacetylcellulose, no rules relating structure to resolvability have yet evolved for this CSP. It seems that solutes which can be resolved on this CSP are sought more or less empirically and that a certain degree o f luck is involved when success is attained. Potato Starch Potato starch has been used f o r the resolution of polar biaryl compounds such as 6,6'-dinitrodiphenic acid, g ,and the trimer and tetramers of orcinol, 18 and 19, respectively [40a,bl. I n the case o f trimer la, there i s a d.1-form and a meso form, the assignments being determined by the resolution of the d,l-form [40al. All three of the possible diastereomeric tetramers, 19, are resolvable on the CSP. Aqueous solvents are commonly employed as the mobile phase; the choice of buffer, pH and buffer concentration being crucial to the resolution process. Baseline resolutions have been obtained for as much as 500 mgs of one of the diastereomeric tetramers of orcinol, 19, o n a column (136 x 7.4.2

17

Y

HO

18

cv

248

5 . 2 cm i . d . )

c o n t a i n i n g p o t a t o s t a r c h us n g a s o d i u m - c i t r a t e b u f f e r a s t h e

m o b i l e phase.

P r e p a r a t i v e i s o l a t i o n mee s w i t h d i f f i c u l t i e s

however, owing t o

t h e t i m e n e c e s s a r y f o r t h e r e s o l u t i o n ( 4 5 d a y s ) , t h e low c o umn c a p a c i t y a n d t h e r e c o v e r y of t h e r e s o l v e d m a t e r i a l from l a r g e amounts o f aqueous b u f f e r .

I t i s p o s s i b l e t h a t an i m p r o v e d CSP c o u l d be d e r i v e d from p o t a t o s t a r c h b y simple chemical m o d i f i c a t i o n of t h e h y d r o x y l groups of t h e p o l y s a c c a r i d e . U n f o r t u n a t e l y , n o r e p o r t s h a v e emerged c o m p a r i n g t h e c h r o m a t o g r a p h i c p r o p e r t i e s of v a r i o u s l y m o d i f i e d p o t a t o s t a r c h e s . 7.5

SYNTHETIC CSPS

Development of Rationally Devised CSPs

7.5.1

S y n t h e t i c CSPs a r e s o - c a l l e d b e c a u s e , u n l i k e t h e CSPs d i s c u s s e d e a r l i e r ( s e c . 7 . 4 ) , t h e y do n o t u t i l i z e a n a t u r a l l y o c c u r r i n g m a t e r i a l f o r b o t h t h e c h r o m a t o g r a p h i c s u p p o r t and t h e s o u r c e o f t h e c h i r a l e n v i r o n m e n t .

Synthetic

CSPs can c o n s i s t o f c h i r a l m o l e c u l e s bonded t o i n e r t s u p p o r t s ( e . g . s i l i c a , p o l y m e r s ) , p o l y m e r i c m a t e r i a l s d e r i v e d from c h i r a l monomeric u n i t s or p o l y m e r i c m a t e r i a l s w h i c h a r e c h i r a l due t o t h e mode o f f o r m a t i o n ( i . e . t h e p r e s e n c e o f c h i r a l c a t a l y s t (or t e m p l a t e ) d u r i n g p o l y m e r i z a t i o n .

They may u t i l i z e a wide

v a r i e t y o f s u p p o r t m a t e r i a l s a n d p r o v i d e o p p o r t u n i t y for i n c o r p o r a t i o n o f a n a l m o s t i n f i n i t e number o f d i f f e r e n t c h i r a l m o l e c u l e s i n t o CSPs.

OH

Fig. 7.3.

+ H3N-C =N I

As i n s i g h t

H

P o s t u l a t e d t h r e e - p o i n t a t t r a c t i o n between a r g i n i n e a n d DOPA g i v e s

r i s e t o t h e p r e f e r e n t i a l r e t e n t i o n o f d-DOPA o n a c o l u m n c o m p r i s e d of 1-argfnine

R e p r i n t e d w i t h p e r m i s s i o n f r o m r e f . 41.

249

develops into the mechanisms of chiral recognition, specifically designed chiral molecules bonded to "state of the art" chromatographic supports will make possible the direct resolution of tens of thousands of racemates in a predictable fashion. Baczuk, et al. C411 prepared the first rationally designed CSPs intended specifically for the resolution of d,l-DOPA (Fig. 7 . 3 ) . These CSPs were comprised of 1-arginine bound to Sephadex, cellulose, or chloromethylated polystyrene. Sephadex proved best, affording a separation factor, a, of 1.60 and a resolution value, RS, of 0.9 for 20pg of racemic DOPA on a microbore column (300 x 2 . 8 mm i.d.). By fifty repetitive injections, enough resolved material was obtained for polarimetric measurements, verifying that complete resolution had occurred. The amino acid, I-arginine, was chosen for the CSP based on an understanding of the three-point rule (sec. 7 . 2 , Fig. 7.1). Space-filling models suggest that the d-isomer of DOPA. is better able to undergo three electrostatic interactions with 1-arginine, thereby result ng in its Dreferential retention on the CSP. 7.5.2

Chiral Crown Ethers The chromatographic resolution o f ammonium salts of amines, amino acids and amino esters has been accomplished on CSPs containing chiral crown ethers [ 4 2 , 4 3 1 . The chiral crown ethers, or host molecules, consist of a cyclic polyether incorporating two 1,l'-dinaphthyl units of knowr? configuration. These compounds were chosen on the basis of previous studies that demonstrated diastereoselective formation of host-guest complexes with racemic ammonium salts in two-phase extraction experiments 1441. Two CSPs were prepared employing similar chiral hosts, one attached to a silica support, 20, the other bonded to a chloromethyl polystyrene support, 21. Whereas 20 affords baseline resolutions of the methyl esters o f the ammonium salts of only bulky amino acids such as tyrosi ne, tryptophane, p-hydroxyphenylglyci ne and phenylalani ne, CSP 2 effectively resolves the perchlorate salts of many a-amino acids, either as the free acids or the methyl esters. In general, larger a values and better peak shapes were observpd for the polymer bound CSP.

A strong dependence was observed between separation factor, a, and the amount of sample introducted onto a column containing CSP 21 [ref. 4 3 1 . For the perchlorate salt of phenylglycine, a maximum a value was obtained for 1O.lmg o f material o n a column containing 9.5g of the CSP (Table 7 . 3 ) . For smaller and larger samples, the a value decreased. Intuitively, one might expect a values to decrease with greater sample loadings due t o column overload; however, surprisingly there is a decrease with small sample loadlngs. To gain insight into this phenomenon, one must realize that the

a,

250

y 3

OCH3

21

c1

retention of a n y component o n a chromatography column is t h e weighted time-average summation of all o p e r a t i v e retention processes. When t h e t w o solutes are enantiomers. many of t h e retention-solvation processes will be identical f o r each. O n l y a fraction of t h e overall retention processes actually differentiate between t h e enantiomers. In t h e case o f C S P s 2 and 21, host-guest complexation g i v e s r i s e t o t h e difference in retention of t h e enantiomers. A s t h e sample s i z e introduced i n t o t h e column is increased, t h e contribution of host-guest complexation t o t h e total retention of t h e enantiomers c a n decrease due t o saturation of all available host s i t e s . Consequently, t h e a value is "attenuated" by a greater contribution f r o m retention mechanisms which d o not afford chiral recognition. T h e decrease in a values at small s a m p l e mass must be due to a n entirely different phenomenon. It is reasonable t o a s s u m e that not all the host sites exhibit the same degree of chiral recognition. I f t h e m o s t retentive sites exhibit a lower degree of chiral recognition t h a n that do less retentive sites, "small" samples can exhibit reduced U-values as a greater portion of t h e retention occurs a t the less efficient sites. Preparative resolutions of t w o previously configurationally unknown a m i n o acid derivatives, 22 and 2J, w e r e performed on a c o l u m n (600 x 7.5 m m i.d.)

251

TABLE 7 . 3

R e s o l u t i o n of t h e P e r c h l o r a t e S a l t s of Amino A c i d s a n d Amino A c i d E s t e r s o n CSP ,?.la.

cioa

+H~HCHR~CO~R

wt, entry

22

R

CH3

23 24

R1

:ti3

mg

a

RS

%CH3CNICHCl

p-c h 1o r o p h e n y 1

9.5

8.5

2.2

10

p-carbomethoxyphenyl

9.5

12.6

2.3

10

phenyl

9.5

18.5

4.5

10

CH3

25

ti

phenyl

10.1

24.3

0.74

10

26

ti

methyl

1.6

1.5

0.21

4

-

a Column c o n s i s t e d o f 9.59 o f CSP 21 i n s t a i n l e s s s t e e l t u b e 600 X 7.5 mm i . d .

c o n t a i n i n g CSP

21

(Table 7.3).

A l t h o u g h 9.5mg samples a f f o r d b a s e l i n e

r e s o l u t i o n , l a r g e r samples ( 5 7 a n d 75mg) a f f o r d o v e r l a p p i n g b o n d s .

I n the

l a r g e r s c a l e r u n s , t h r e e f r a c t i o n s were c o l l e c t e d c o n t a i n i n g t h e l e a s t r e t a i n e d e n a n t i o m e r , a m i x t u r e , and t h e most r e t a i n e d e n a n t i o m e r . f r a c t i o n s from a number o f r u n s were combined a n d r e i n j e c t e d .

The m i d d l e

I n t h i s manner,

t h e p u r e e n a n t i o m e r s were o b t a i n e d f o r c o m p l e t e s p e c t r a l a n a l y s i s .

From t h e

c i r c u l a r d i c h r o i s r n s p e c t r a a n d t h e c h r o m a t o g r a p h i c o r d e r o f e l u t i o n of t h e enantiomers of

22

and

23,

t h e a b s o l u t e c o n f i g u r a t i o n s were a s s i g n e d .

In a

s i m i l a r f a s h i o n two r u n s o f 25mg e a c h o f t h e m e t h y l e s t e r o f p h e n y l g l y c i n e .

24, op).

a f f o r d e d 20mg o f t h e D i s o m e r (96.9% o p ) a n d 18mg o f t h e 1 - i s o m e r ( 9 9 % The p e r c h l o r a t e s a l t o f a l a n i n e ,

2 . was

tediously resolved by

c o l l e c t i n g f r a c t i o n s from f i v e r u n s o f 5mg e a c h and r e i n j e c t i n g t h e c o l l e c t e d f r a c t i o n s u n t i l 6 . l m g o f t h e 1 - e n a n t i o m e r ( 8 9 . 8 % o p ) and 5.0mg of t h e d - e n a n t i o m e r (95:2% o p ) were o b t a i n e d .

252

A l t h o u g h CSPs & aIn d

21

r e q u i r e a m o d e r a t e l y l e n g t h y s y n t h e t i c sequence t o

p r e p a r e , t h e y a r e s t a b l e and may be u s e d o v e r a l o n g p e r i o d o f t i m e .

The

l i f e - e x p e c t a n c y o f t h e s e CSPs a n d t h e s e p a r a t i o n f a c t o r s o b t a i n e d from p a r t i c u l a r , w a r r a n t t h e i n i t i a l t r o u b l e and expense o f p r e p a r i n g them.

21

in

The

CSP does, h o w e v e r , e x h i b i t low e f f i c i e n c y i n t e r m s of i t s c h r o m a t o g r a p h i c

behavior.

C h r o m a t o g r a p h i c bands a r e b r o a d a n d c o n s e q u e n t l y , e v e n w i t h l a r g e a

v a l u e s and low flow r a t e s , i t i s d i f f i c u l t t o o b t a i n c o m p l e t e s e p a r a t i o n s f o r T h i s may be due t o t h e ' k i n e t i c a s p e c t s of t h e h o s t - g u e s t

preparative runs.

r e l a t i o n s h i p ( i . e . slow adsorption

-

desorption).

C o n s e q u e n t l y , low flow r a t e

and r e l a t i v e l y s m a l l samples m u s t be u s e d t o o b t a i n maximum e f f i c i e n c y from t h e s e CSPs. 7.5.3

Carbinol CSP

The f i r s t o f t h e CSPs d e v e l o p e d by P i r k l e a n d c o - w o r k e r s was b a s e d on a n

p r i o r i c h i r a l r e c o g n i t i o n model t h a t a n t i c i p a t e d b o t h t h e s e p a r a t i o n and e l u t i o n orders of a s e r i e s of s u l f o x i d e enantiomers. f l u o r o a l c o h o l - d e r i v e d CSP,

27,

This

w i l l r e s o l v e s u l f o x i d e s , l a c t o n e s and

d e r i v a t i v e s o f a m i n e s , amino a c i d s , a l c o h o l s , h y d r o x y a c i d s a n d m e r c a p t a n s which c o n t a i n n - a c i d i c f u n c t i o n a l i t y [451. 2,4-dinitrophenyl

dodecylsulfoxide,

mm i . d . ) c o n t a i n i n g CSP

3

3 , has

P r e p a r a t i v e l y . 5mgs of been r e s o l v e d o n a c o l u m n ( 2 5 0 x 1 0

bonded t o 10 pm i r r e g u l a r s i l i c a p a r t i c l e s u s i n g

20% i s o p r o p y l a l c o h o l i n hexane as t h e m o b i l e p h a s e .

(a=1.26) was o b t a i n e d .

Baseline r e s o l u t i o n

Presumably, o t h e r racemates h a v i n g comparable a v a l u e s

c o u l d be r e s o l v e d o n a s i m i l a r (or l a r g e r ) s c a l e s i n c e t h i s was n o t a c o l u m n overload s i t u a t i o n .

Fz=:iOR

H

27

ry

28 h)

253 7 . 5 . 4 Amino Acids DNBs The f l u o r o a l c o h o l d e r i v e d CSP a l l o w e d t h e f a c i l e a s s e s s m e n t of o t h e r c h i r a l m o l e c u l e s a s p o t e n t i a l CSPs.

Chiral recognition i s reciprocal i n that a

s t a t i o n a r y phase a p p r o p r i a t e l y p r e p a r e d from a s i n g l e e n a n t i o m e r of a s o l u t e r e s o l v a b l e o n CSP

27

should separate t h e enantiomers of f l u o r o a l c o h o l analogs

LO.

I n t h i s manner, t h e 3 , s - d i n i t r o b e n z o y l of a c i d s were deemed s u i t a b l e as CSP p r e c u r s o r s .

d e r i v a t i v e s (DNB) o f a m i n o CSP

3 , prepared

from

3,5-dinitrobenzoylphenylglycine i o n i c a l l y bound t o y - a m i n o p r o p y l - s i l a n i z e d s i l i c a 1461, has p r o v e n h i g h l y s u c c e s s f u l and i s now c o m m e r c i a l l y a v a i l a b l e

[471. T h i s s t a t i o n a r y phase w i l l r e s o l v e t h e e n a n t i o m e r s o f many f u n c t i o n a l l y d i f f e r e n t c l a s s e s o f s o l u t e s [48,491. I t s ease i n p r e p a r a t i o n from r e a d i l y a v a i l a b l e c h i r a l m a t e r i a l s makes i t a n a t t r a c t i v e c a n d i d a t e f o r use i n l a r g e - s c g l e p r e p a r a t i v e systems.

29

rv

A 2 i n . x 30 i n . c o l u m n c o n t a i n i n g c h i r a l a d s o r b a n t

3

bonded t o 40pm

i r r e g u l a r s i l i c a a f f o r d e d r e s o l u t i o n s of m u l t i g r a m samples o f s t r u c t u r a l l y d i v e r s e c h i r a l compounds [501, r e p r e s e n t a t i v e e x a m p l e s o f w h i c h a r e shown i n T a b l e 7.4. T y p i c a l l y , one or more grams of r a c e m a t e i,s r e s o l v e d p e r p a s s . u t i l i z i n g a m o b i l e phase o f such p o l a r i t y t h a t t h e t o t a l t i m e r e q u i r e d than s i x hours.

s less

P r e p a r a t i v e r e s o l u t i o n o f o n e g r a m o f r a c e m a t e i s r o u t ne i n

cases where t h e a v a l u e i s a t l e a s t 1 . 3 , g r e a t e r a v a l u e s a l l o w i n g t h e

For example, a n e a r l y base1 ne r e sol u t o n was o b s e r v e d f o r 3 . 0 9 o f amide 33a ( F i g . 7.4). One r o u t i n e l y r e s o l u t i o n of g r e a t e r q u a n t i t i e s per pass.

o b t a i n s b o t h enantiomers i n a t l e a s t % e n a n t i o m e r i c excess ( e e ) .

Due t o

t h e t a i i n g of t h e c h r o m a t o g r a p h i c p e a k s , t h e f i r s t e l u t e d b a n d i s u s u a l l y o f g r e a t e r e n a n t i o m e r i c e x c e s s ( e . g . 99%) t h a n t h e second ( c a . 95% ee i s t y p i c a l , t h e a c t u a l v a l u e s b e i n g d e p e n d e n t o n t h e c u t p o i n t b e t w e e n t h e two peaks.

Seven grams o f amide &3J

(a=2.23)was r e s o l v e d i n a s i n g l e p a s s .

By

c o l l e c t i n g t h r e e f r a c t i o n s , t h e i n n e r or m i d d l e o n e c o n s i s t i n g of l e s s t h a n 5% o f t h e t o t a l m a t e r i a l , b o t h e n a n t i o m e r s were o b t a i n e d from t h e o u t e r f r a c t i o n s i n 99t% ee ( F i g . 7.4). S i g n i f i c a n t e n a n t i o m e r i c e n r i c h m e n t c a n b e

254

TABLE 7.4

P r e p a r a t i v e Multigram Resolutions o f Racemates on CSP 2ga.

X isopropyl alcohol i n entry

compound

R

w t , gms

a

hexane

H

1 .o

1.46

2

CH 3

1 .o

1.47

5

2.0

1.47

5

1 .o

1.86

5

1.2

1.56

10

1.1

1.68

10

- i sopropyl

3.0

2.17

-cyclohexyl

7.0

2.23

34

5.0

1.55

2

35

4.0

2.00

2

30a 30b 30c

CH 3 R

31 W

O

W

3

H H

32b

7 NF((CH2)8cH=CH2

33b

a The column (2 i n . x 30 i n . ) employed contained CSP 29 (40 pm p a r t i c l e s ) Flow r a t e was absorbance.

- 55mL/min.

D e t e c t i o n was accomplished by o b s e r v i n g UV

To o b t a i n two f r a c t i o n s o f h i g h p u r i t y (see t e x t ) a small

amount o f m a t e r i a l r e p r e s e n t i n g l e s s than 5% o f t h e t o t a l was c o l l e c t e d between the two chromatographic peaks and discarded.

255

obtained for even larger samples o f well-resolved compounds or for moderate quantities of materials exhibiting small a values. In the event of incomplete separation, material of high enantiomeric purity is obtainable by either employing recycling techniques or by simply collecting more fractions. By automation o f the chromatographic system, runs can be performed without operator intervention and the resolved materials can be automatically combined and concentrated. For example, 149 of racemic alcohol 30a was resolved in a total of 16 hours in four repetitive runs; fraction collection and sample introduction occurring according t o a preset program.

1

0

I

I

2 Time (hrs.) 1

I

3

1 1

I

I

I

I

I

I

I

0

1

2

3

4

5

6

7

Time (hrs.)

Fig. 7.4. The multigram preparative resolution of chiral amides 33a and 33b on a column ( 2 in. x 3 0 in.) containing CSP 29 (40 pm irregular particles; flow rate, 60ml/min; detection, UV-280nm). a) Resolution of 39 o f 33a (mobile phase, 2% isopropyl alcohol in hexane). b) Resolution o f 7g of 33b (mobile phase, 1% isopropyl alcohol in hexane).

In order t o perform chromatographic separations of multigram samples, a large scale chromatographic system i s required. It i s expedient to use a medium pressure system such as that employed for the preceding resolutions

256 The pump u s e d i n t h e a u t o m a t e d s y s t e m i s c a p a b l e o f flow r a t e s e x c e e d i n g 200 m l l m i n a t p r e s s u r e s up t o 200 p s i .

F o r t h e a f o r e m e n t i o n e d 2 i n x 30 i n

p r e p a r a t i v e c h i r a l columns, flow r a t e s o f c a . 6 0 m l l m i n were u t i l i z e d .

This

system, w h i c h has t h e a b i l i t y t o i n j e c t t h e s a m p l e , m o n i t o r t h e e f f l u e n t , d e t e c t and c o l l e c t c h r o m a t o g r a p h i c bands and d i s t i l l and r e u s e e l u t i n g s o l v e n t , has been p r e v i o u s l y d e s c r i b e d [ 2 4 1 .

F o r many a p p l i c a t i o n s , o n l y

m i l l i g r a m q u a n t i t i e s of r e s o l v e d m a t e r i a l may be r e q u i r e d .

I n these cases, a

s e m i - p r e p a r a t i v e column e m p l o y i n g s m a l l p a r t i c l e a d s o r b a n t s and w i d e l y a v a i l a b l e LC e q u i p m e n t i s more s u i t a b l e .

These o v e r s i z e d a n a l y t i c a l columns

t y p i c a l l y show much g r e a t e r c h r o m a t o g r a p h i c e f f i c i e n c i e s t h a n d o medium p r e s s u r e p r e p a r a t i v e columns a n d e n a b l e one t o s e p a r a t e m i l l i g r a m q u a n t i t i e s

of compounds e x h i b i t i n g a v a l u e s as s m a l l as 1 . 1 0 .

A g a i n , compounds

e x h i b i t i n g s m a l l e r a v a l u e s may be s e p a r a t e d b y r e c y c l i n g t e c h n i q u e s . A 10 mm X 250 mm s e m i - p r e p a r a t i v e

c o l u m n c o n t a i n i n g CSP

2

was f i r s t u s e d

t o o b t a i n e n a n t i o m e r i c a l l y e n r i c h e d sample o f c y c l i c s u l f o x i d e s for NMR and c i r c u l a r d i c h r o i s m s t u d i e s [511.

The s u p p o r t m a t e r i a l i n i t i a l l y employed was

i r r e g u l a r s i l i c a h a v i n g an a v e r a g e p a r t i c l e s i z e o f 10pm.

Consequently, t h i s

column i s n o t a s e f f i c i e n t as s u b s e q u e n t c o m m e r c i a l v e r s i o n s u t i l i z i n g 5pm s p h e r i c a l s i l i c a as s u p p o r t .

However, for t h e i n i t i a l a p p l i c a t i o n (NMR

d e t e r m i n a t i o n o f ' a b s o l u t e c o n f i g u r a t i o n s ) i t was d e s i r a b l e t o have e n r i c h e d samples i n w h i c h b o t h e n a n t i o m e r s a r e p r e s ' e n t i n c a . 2 : l r a t i o .

Since a t o t a l

r e s o l u t i o n was n o t d e s i r e d , s u i t a b l e e n r i c h e d samples o f s u l f o x i d e s

36

37

and

were o b t a i n e d b y i n t r o d u c i n g lOOmgs of t h e s u l f o x i d e i n t o t h e c o l u m n and c o l l e c t i n g two a p p r o x i m a t e l y e q u a l f r a c t i o n s .

Even w i t h t h i s c o m b i n a t i o n o f a

l a r g e sample s i z e , modest s e p a r a t i o n f a c t o r ( a c a . 1 . 2 5 ) and m o d e r a t e l y low e f f i c i e n c y c o l u m n ( v i d e s u p r a ) . samples of a l m o s t 50% ee were o b t a i n e d .

For

CD s p e c t r a , 5mgs o f e a c h r a c e m a t e were c o m p l e t e l y r e s o l v e d i n a s i n g l e p a s s . Sulfoxide j &

was r e s o l v e d on t h i s column b y i n t r o d u c i n g 7.51119 o f r a c e m a t e a n d

c o l l e c t i n g two f r a c t i o n s , t h e f i r s t c o n t a i n i n g 3.51119 o f t h e l e a s t r e t a i n e d e n a n t i o m e r ( 9 3 . 7 % ee) and t h e second c o n t a i n i n g 4.0mg o f t h e more r e t a i n e d enantiomer (72.2% ee). Q u a n t i t i e s between one and f i v e m i l l i g r a m s o f s u l f o x i d e s

38a-e

have a l s o

been r e s o l v e d for c h i r o p t i c p u r p o s e s u s i n g a n a n a l y t i c a l c o l u m n ( 2 5 0 x 4 . 6 mm i . d . 1 c o n t a i n i n g CSP sulfoxides

38a

and

2

a t t a c h e d t o s p h e r i c a l Spjn s i l i c a p a r t i c l e s C521.

m-g, b a s e l i n e

For

s e p a r a t i o n s o f t h e e n a n t i o m e r s were

o b t a i n e d u s i n g s u b - m i l l i g r a m samples o f r a c e m a t e ( T a b l e 7 . 5 ) .

As a r e s u l t ,

n e a r l y c o m p l e t e s e p a r a t i o n s were o b t a i n e d for o n e t o f i v e mg samples b y t h e j u d i c i o u s c h o i c e of t h e 2 - p r o p a n o l c o n t e n t o f t h e mobi.le p h a s e f o r a l l b u t

m, f o r

w h i c h o n l y b r o a d e n i n g o f t h e c h r o m a t o g r a p h i c peak was o b s e r v e d a f t e r

257 TABLE 7.5

n

38a-e

Resolution o f 2-Phenyl-2-alkyl-1, 2ga.

3-dithiolane-1-oxides o n CSP

entry

R

a

a

H

1.05

b

-CH3

NSb

C

-CH2CH3

1.06

d

-CH(CH3)2

1.26

e

cyc lohexyl

1.36

a The column ( 2 5 x 0.46 cm i.d.) contained CSP 2 9 o n 5 u m spherlcal particles (mobile phase, 5% isopropyl alcohol in hexane; f l o w r a t e , Z m l / m i n detector, UV-254nm). b N S indicates n o separation, however, peak broadening was observed.

n s-0

s+

n

o n e pass. By recycling compound $3J through the column f o u r times and t h e n collecting t w o f r a c t i o n s , enriched samples o f both enantiomers (25% ee) were obtained. Recently Weems and Yang [531 have employed an analytical column of t h i s type t o resolve unspecified quantities of the tetrahydrodiols 3. 40, a n d fi (a values o f 1.16, 1.18 and 1.17 respectively) for circular dichroism studies. These investigations clearly demonstrate that chiral analytical LC columns can efficiently resolve enough material f o r chiroptic evaluation.

258

TABLE 7.6

Comparison of the'Chromatographic Resolution o f 2,2,2-trif 1 uoro- 1 -(9-an thryl )ethanol , 30a, on an Anal yt ica 1 Column' Containing Chiral Stationary Phase 42 to the Injected Sample M a s s .

~~

Samples Mass

(mgs) d

~

Flow Rate b

(ml /mi n)

K'l

K'2

a

RS

nF

:n

0.001

1

8.18

11.76

1.44

4.26

3180

401 6

0.30

1

7.63

10.93

1.43

2.30

1058

1035

2.00

2

6.40

9.22

1.44

1.41

471

356

4.00

2

6.10

8.79

1.44

1.14

353

235

2

5.58

7.14

1.28

0.50

246

83

16.0

aThe analytical column was a Regis Pirkle Covalent Phenylglycine, 4.6mm i.d. X 250mm. Analysis was monitored by UV detection at 254, 290 or 364nm. bThe mobile consisted of 2% isopropyl alcohol in hexane. CSymbols n l and n2 refer to the determination of the number of theoretical plates e x h i b i t e d by the column based on t h e chromatographic peaks of the first and second eluted enantiomers respectively. Injection volumns were all less t h a n 20p1 with the exception of the last entry w h i c h was 7 5 ~ 1 .

39

5.

4Q

OH

%

259 The r e s o l u t i o n o f 2,2,2-trifluoro-1-(9-anthryl)ethanol,

=a,

a widely

u s e f u l c h i r a l s o l v a t i n g a g e n t f o r NMR s t u d i e s 1541, o n a n a l y t i c a l columns c o n t a i n i n g 3,5-dinitrobenzoylphenylglycine c o v a l e n t l y bonded t o aminopropylsilanized s i l i c a ,

2,has

been i n v e s t i g a t e d i n d e t a i l [551.'

The

a n a l y t i c a l d a t a was u s e d t o c l o s e l y d e t e r m i n e t h e o p t i m u m c o n d i t i o n s f o r t h e r e s o l u t i o n of lOOmgs o f @ on a n a n a l o g o u s s e m i - p r e p a r a t i v e c o l u m n .

The d a t a

i n T a b l e 7.6 were o b t a i n e d w i t h a c o m m e r c i a l l y a v a i l a b l e ( 2 5 0 x 4.6 mm i . d . ) a n a l y t i c a l column [ 4 7 1 .

The s e p a r a t i o n f a c t o r , a , i s not g r e a t l y i n f l u e n c e d

b y changes i n t h e sample mass o v e r g r e a t e r t h a n f i v e o r d e r s o f m a g n i t u d e .

The

d e c r e a s e i n a from 1 . 4 4 ( l p g s a m p l e ) t o 1 . 2 8 (16mg sample) i s due t o e x t r e m e mass o v e r l o a d a s i n d i c a t e d b y t h e s e v e r e d r o p i n p l a t e c o u n t , n .

Resolution,

R S , a n d t h e c a p a c i t y f a c t o r , k ' , a r e a f f e c t e d b y sample mass a n d b o t h

p a r a m e t e r s d e c r e a s e m a r k e d l y w i t h g r e a t e r amounts o f i n j e c t e d sample.

For

p r e p a r a t i v e work, t h e r e s o l u t i o n f a c t o r , R S , i s t h e most i m p o r t a n t s i n g l e i n d i c a t o r of the effectiveness of the resolution.

To r e s o l v e n o n - m i n u t e

q u a n t i t i e s o f r a c e m a t e , i t i s b e s t t o o p e r a t e t h e c o l u m n u n d e r mass o v e r l o a d c o n d i t i o n s , o p t i m i z i n g sample s i z e t o a c h i e v e a g i v e n R,.

For l a r g e

samples, t h e r e l a t i o n s h i p between t h e l o g o f t h e sample mass and t h e resolution factor

RS. was e m p i r i c a l l y f o u n d t o be n e a r l y l i n e a r .

By

e x t r a p o l a t i o n , i t was f o u n d t h a t a n R S v a l u e o f u n i t y s h o u l d be o b t a i n e d w i t h a 5.41119 sample o f r a c e m l c c a r b i n o l

30a

o n t h e a n a l y t i c a l column.

If the

peaks a r e G a u s s i a n a n d t h e two f r a c t i o n s a r e c o l l e c t e d a t t h e e q u a l p u r i t y c u t - p o i n t , b o t h e n a n t i o m e r s o f t h e c a r b i n o l s h o u l d be o b t a i n e d w i t h a p u r i t y o f 98% [ 2 , 5 6 1 .

T h i s i s an o v e r s i m p l i f i c a t i o n s i n c e f o r p r e p a r a t i v e work o n e

w i l l use sample o v e r l o a d c o n d i t i o n s and t h e r e w i l l i n e v i t a b l y be peak tailing.

Nonetheless, t h i s a p p r o x i m a t i o n serves as a reasonable s t a r t i n g

p o i n t for d e t e r m i n i n g t h e o p t i m a l c o n d i t i o n s for t h e p r e p a r a t i v e r u n . The d a t a from t h e a n a l y t i c a l c o l u m n was u s e d t o e s t i m a t e a p p r o p r i a t e c o n d i t i o n s f o r a l a r g e r d i a m e t e r s e m i - p r e p a r a t i v e c o l u m n ( 1 0 mm x 250 mm i . d . ) c o n t a i n i n g 4 . 7 t i m e s as much o f t h e CSP.

A l l of t h e c o n d i t i o n s for the

a n a l y t i c a l i n v e s t i g a t i o n s were d u p l i c a t e d ( t h e flow r a t e was i n c r e a s e d t o o b t a i n a c o m p a r a b l e l i n e a r flow v e l o c i t y ) and t h e sample s i z e was i n c r e a s e d b y a f a c t o r of 4.7.

I t was e x p e c t e d t h a t a 25mg sample s h o u l d g i v e r i s e t o a

R S v a l u e o f one.

As shown i n T a b l e 7 . 7 , t h e p r e p a r a t i v e r e s o l u t i o n p r o v e d

t o be s l i g h t l y more e f f i c i e n t , R S p r o v e d t o be 1.10.

P o r t i o n s o f t h e two

c o l l e c t e d f r a c t i o n s were a n a l y z e d on t h e a n a l y t i c a l c o l u m n a n d e a c h f o u n d t o be 98% e e .

A s i m i l a r r e s o l u t i o n o f 92mgs o f m a t e r i a l a f f o r d e d p o o r e r

o v e r a l l r e s o l u t i o n , a l t h o u g h t h e f i r s t e n a n t i o m e r was s t i l l o b t a i n e d i n 97% ee.

The g r e a t e s t t h r o u g h p u t o f m a t e r i a l was a c h i e v e d b y u s i n g a sample l a r g e

260

TABLE 7 . 7

P r e p a r a t i v e R e s o l u t i o n o f a C h i r a l A l c o h o l , 30a, upon a Semi - P r e p a r a t ive C o l umn. a

Xeec Injected Sample Mass (mg)

a

RS

F i r s t Eluted

Second E l u t e d

Enantiomer

E n a n t iome r

25

1.43

1-10

98

98

25b

1.31

0.76

>99.7

74

92

1.44

0.61

97

83

a A R e g i s P i r k l e C o v a l e n t P h e n y l g l y c i n e Column (1Omm i. d . X 250mm) was employed u s i n g 2% i s o p r o p y l a l c o h o l i n hexane a s t h e e l u e n t .

The flow r a t e

was 5 m l / m i n a n d d e t e c t i o n was a c c o m p l i s h e d b y UV a b s o r b a n c e a t 280nm.

bThe

sample i n j e c t i o n volume was I O m l , compared t o 4 0 0 ~ 1for a l l o t h e r r u n s . C

E n a n t i o m e r i c e x c e s s was d e t e r m i n e d c h r o m a t o g r a p h i c a l l y o n a n a n a l y t i c a l

column .

enough t o a f f o r d an R S o f c a . 0 . 6 , ' c o l l e c t i n g t h e f i r s t band and r e c y c l i n g t h e second e n a n t i o m e r t o a l s o o b t a i n i t i n h i g h e n a n t i o m e r i c p u r i t y .

For

compounds e x h i b i t i n g l a r g e r a v a l u e s , one can e x p e c t t o r e s o l v e e v e n more m a t e r i a l per pass.

The l i m i t a t i o n m o s t f r e q u e n t l y e n c o u n t e r e d may w e l l p r o v e

t o be l i m i t e d s o l u b i l i t y o f t h e r a c e m a t e i n t h e d e s i r e d volume o f i n j e c t i o n sol vent. For p r e p a r a t i v e w o r k , i t i s n o t a b s o l u t e l y n e c e s s a r y t o a p p l y samples i n small i n j e c t i o n values.

F o r l a r g e a v a l u e s , a volume o v e r l o a d may even be

advantageous 1571. The r e s o l u t i o n o f c h i r a l a l c o h o l 30a has been examined i n volume o v e r l o a d c o n d i t i o n s , a 25mg sample b e i n g i n t r o d u c e d i n l O m l o f s o l v e n t compared t o t h e 400pL i n j e c t i o n volume f o r t h e r u n d i s c u s s e d above. the a value and

Although

RS s u f f e r from t h e l a r g e r i n j e c t i o n volume, b y j u d i c i o u s

c h o i c e o f t h e c u t - p o i n t between t h e two f r a c t i o n s , t h e f i r s t e n a n t i o m e r was o b t a i n e d i n g r e a t e r t h a n 99% ee ( T a b l e 7 . 7 ) .

From t h e R S v a l u e o b t a i n e d

and t h e a p p e a r a n c e o f t h e chromatogram, i t seems e v i d e n t t h a t t h e two f r a c t i o n s w o u l d have been l e s s t h a n 98% ee h a d t h e e q u a l p u r i t y c u t - p o i n t been chosen.

261

The p r e p a r a t i v e r e s o l u t i o n o f c a r b i n o l column.

30a i s

n o t an i s o l a t e d case f o r t h i s

I n f a c t , e v e r y racemate so f a r i n v e s t i g a t e d which e x h i b i t s a

reasonable a v a l u e f o r a n a l y t i c a l s i z e samples can be p r e p a r a t i v e l y r e s o l v e d . The most i m p o r t a n t f e a t u r e s which i n f l u e n c e r e s o l v a b i l i t y ( i . e . R S ) w i l l be t h e a v a l u e , amount o f sample t o be r e s o l v e d and t h e s i z e o f t h e column. bonded t o 5pm s p h e r i c a l s i l i c a , CSP

42

When

c o n s i s t e n t l y e x h i b i t s a values which

a r e independent o f t h e sample s i z e up t o as much as 5mgs o f s o l u t e p e r gram o f adsorbent.

T h e r e f o r e , t h e amount o f m a t e r i a l r e s o l v a b l e on a p a r t i c u l a r

p r e p a r a t i v e column can be c l o s e l y e s t i m a t e d from t h e b e h a v i o r o f t h a t compound a t the a n a l y t i c a l l e v e l .

S t a t i o n a r y phase

2 also

exhibits favorable k i n e t

p r o p e r t i e s a1 l o w i n g t h e chromatographer t o use r e a s o n a b l y f a s t flow r a t e s . For t h e s e m i - p r e p a r a t i v e column d e s c r i b e d above, flow r a t e s between 5 and 8 ml/min have been employed and t h e m o b i l e phase p o l a r i t y a d j u s t e d t o a f f o r d complete p r e p a r a t i v e r u n s i n about one h o u r . T h i s same s e m i - p r e p a r a t i v e column has been used t o r e s o l v e s u l f o x i d e phthalide

43,

diazepinone

44

and h y d a n t o i n

45

( T a b l e 7.8).

37,

Interestingly,

w h i l e t h e a values observed f o r a l l b u t t h e s u l f o x i d e a r e h i g h e r t h a n f o r carbinol

E , no

more t h a n 25mgs of each c o u l d be a p p l i e d i f b a s e l i n e

r e s o l u t i o n was t o be o b t a i n e d .

I n o t h e r words, t h e r a t e a t which band shape

d e t e r i o r a t e s as sample s i z e i s i n c r e a s e d seems t o p a r a l l e l somewhat t h e magnitude o f a. A s a r e s u l t , t h e amount o f m a t e r i a l one can r e s o l v e f o r compounds e x h i b i t i n g h i g h e r a v a l u e s may be l e s s t h a n t h a t expected from t h e magnitude o f a o b t a i n e d from an a n a l y t i c a l r u n . diazepine

9 was

N e v e r t h e l e s s , 50mgs of

e f f e c t i v e l y r e s o l v e d by c o l l e c t i n g t h r e e f r a c t i o n s , t h e

m i d d l e f r a c t i o n c o n s i s t i n g o f l e s s t h a n 5% o f t h e t o t a l amount o f m a t e r i a l . The p u r i t i e s o f t h e f i r s t and l a s t f r a c t i o n s were e q u i v a l e n t , w i t h i n e x p e r i m e n t a l e r r o r , t o t h e p u r i t i e s o f t h e two f r a c t i o n s c o l l e c t e d from t h e I n terms o f o v e r a l l t h r o u g h p u t , i t i s advantageous t o

25mg r e s o l u t i o n .

i n t r o d u c e l a r g e samples and c o l l e c t a m i d d l e f r a c t i o n .

For v a l u a b l e

racemates, t h e m i d d l e f r a c t i o n may be rechromatographed. Sulfoxide

2

which e x h i b i t s a s e p a r a t i o n f a c t o r o f 1.21 on an a n a l y t i c a l

column c o n t a i n i n g column.

42,

has been r e s o l v e d on t h e analogous s e m i - p r e p a r a t i v e

T h i s r e s o l u t i o n demonstrates t h e advantages o f c o u p l i n g two r u n s t o

o b t a i n t h e maximum amount of m a t e r i a l i n t h e l e a s t amount o f t i m e ; a t e c h n i q u e p a r t i c u l a r l y u s e f u l f o r compounds e x h i b i t i n g small a v a l u e s .

I n order to

o b t a i n b o t h enantiomers i n g r e a t e r than 96% ee i n a s i n g l e r u n , o n l y 4mgs can be i n t r o d u c e d a t one t i m e .

However, 16mgs was p a r t i a l l y r e s o l v e d

(RS=0.48) y i e l d i n g t h e f i r s t enantiomer i n >99% ee and t h e second

262 TABLE 7 . 8

P r e p a r a t i v e R e s o l u t i o n o n a S e m i - p r e p a r a t i v e Column C o n t a i n i n g CSP 42

wt entry

compound

37

.

% ee H i g h Rf

% isopropyl Low Rf

a l c o h o l i n hexane

mgs.

a

16

1.19

0.48

>99

50.3

10

4

1.28

1.17

>99

96.3

10

%loa

1.18

0.76

>99

96.0

10

RS

0

CI

@

99.4

82.3

7.5

25

1.96

0.98

97

89

2

50

1.94

0.65

>9gb

8gb

2

7.5

a The Low Rf e n r i c h e d m a t e r i a l from t h e 16 mg r u n was rechromatographed t o o b t a i n t h e two enantiomers i n g r e a t e r t h a n 96% ee.

A s m a l l p o r t i o n o f t h e , v a l l e y r e g i o n between t h e

two chromatographic peaks r e p r e s e n t i n g t 5 X of t h e t o t a l mass was o m i t t e d to a f f o r d m r e highly enantiomerically enriched material.

42

hl

263 p a r t i a l l y e n r i c h e d of 50.3% e e .

By rechromatographing t h e p a r t i a l l y e n r i c h e d enantiomer, two new f r a c t i o n s w e r e o b t a i n e d ; t h e f i r s t enantiomer

(>99% e e ) and t h e more r e t a i n e d enantiomer (96% e e ) . Thus i n two coupled runs, 16mgs of m a t e r i a l was r e s o l v e d which would o t h e r w i s e have r e q u i r e d 4 s i n g l e r u n s of 4mg each t o o b t a i n t h e enantiomers i n t h e same degree o f p u r i t y .

I 0

I

I

I

I

I

I

I

1

10

20

30

40

50

60

70

80

Time (min 1 Fig. 7.5.

The p r e p a r a t i v e r e s o l u t i o n o f 95mgs o f 47 on a column 9250 x 10 mm

i . d . ) c o n t a i n i n g I - l e u c i n e d e r i v e d CSP 46 ( p a r t i c l e s i z e approx. 10pm); flow r a t e , 5ml/min; m o b i l e phase, 5% i s o p r o p y l a l c o h o l i n hexane; p r e s s u t e 800 psi).

A s i m i l a r s e m i - p r e p a r a t i v e column c o n t a i n i n g L - l e u c i n e - d e r i v e d

CSP

3

has

been prepared and f o u n d , as a r u l e , t o p r o v i d e l a r g e r s e p a r a t i o n f a c t o r s f o r d i a z e p i n o n e enantiomers. be r e s o l v e d on

46

diazepinones 44,

t h a n on

47,

and

Consequently, l a r g e r amounts o f t h e s e racemates can

42. For example, one hundred m i l l i g r a m samples o f 48 have each been r e s o l v e d on t h e l e u c i n e - d e r i v e d

s e m i - p r e p a r a t i v e column i n a t o t a l t i m e of one hour ( T a b l e 7 . 9 ) .

The b a s e l i n e

r e s o l u t i o n of

47,

be r e s o l v e d .

By comparing t h e r e s u l t s o b t a i n e d fo r t h e r e s o l u t i o n of

diazepinone

9, in

shown i n F i g . 7.5, suggests t h a t even l a r g e r samples c o u l d Tables 7.8 and 7.9. i t i s e v i d e n t t h a t t h r o u g h p u t p e r u n i t

t i m e i s i n f l u e n c e d h e a v i l y by t h e CSP d e s i g n .

F u r t h e r improvements i n CSP

d e s i g n c o u p l e d w t t h U t l l f Z a t i O n of improved s u p p o r t s can be expected to l e a d

to t h e development of q u i t e e f f e c t i v e c h i r a l s e m i - p r e p a r a t i v e columns.

264

No2

46 ry

TABLE 7 . 9

P r e p a r a t i v e R e s o l u t i o n o f Benziodiazepinones on a S e m i - p r e p a r a t i v e Column C o n t a i n i n g CSP 46a.

X ee entry

w t , mgs.

R

44

-CH(CH3)2

47

-CH20

48

-CH2CH2SCH3

a

RS

High Rf

Low Rf

100

3.10

0.56

>9gb

98. qb

95

3.14

0.93

>99

98.6

104

2.61

0.68

99.3

97.2

a The column (250 x 10 mm i . d . ) c o n t a i n e d CSP 46 on 10 pm i r r e g u l a r s i l i c a p a r t i c l e s ( f l o w r a t e , 5 mL/min; d e t e c t i o n UV 340 nm; m o b i l e phase, 5% isopropyl a1 coho1 i n hexane)

a l c o h o l i n hexane.

.

Mobi 1 e phase cons i s t e d o f 7.5% is o p r o p y l

A p o r t i o n o f t h e v a l l e y r e g i o n between t h e two peaks

( ~ 5 %o f t o t a l mass) was o m i t t e d t o a c h i e v e h i g h e r e n a n t i o m e r i c p u r i t i e s .

The CSPs

3, 42, and 46, a l l

prepared from t h e DNB d e r i v a t i v e s o f amino

a c i d s , can r e s o l v e l i t e r a l l y thousands o f racemic compounds [46-53,58-603;

the

scope and g e n e r a l i t y a1 l o w i n g t h e p r e p a r a t i v e r e s o l u t i o n o f many compounds which have n o t y e t been.resolved and i n v e s t i g a t e d . A number o f r e l a t e d amino

265

a c i d d e r i v e d CSPs which have n o t , t o o u r knowledge, been used for p r e p a r a t i v e All

r e s o l u t i o n deserve m e n t i o n owing t o t h e i r p o t e n t i a l f o r such r e s o l u t i o n s . o f these CSPs a r e p r e p a r e d from y-aminopropylsilanized-silica; a s u p p o r t m a t e r i a l which, a l t h o u g h p r o b a b l y n o t o p t i m a l , does a t l e a s t a l l o w t h e

c o n v e n i e n t s y n t h e s i s o f e f f i c i e n t columns e x h i b i t i n g a r e l a t i v e l y l a r g e number o f theoretical plates. The f i r s t CSP o f t h i s t y p e cons i s t e d o f N-3,5-di n i t r o p h e n y l -L-a1 a n i ne c o v a l e n t l y bound t o y - a m i n o p r o p y l s i l a n i z e d s i l i c a v i a an amide l i n k a g e .

This

CSP was employed f o r t h e r e s o l u t i o n o f h e p t a h e l i c e n e (a=1.06) and

l-aza[6lhelicene

(a=1.06) [ r e f . 611.

Even a t t h e a n a l y t i c a l l e v e l , t h e

r e s o l u t i o n f a c t o r , R S , was lower t h a n 0 . 6 f o r b o t h compounds.

N-Acyl

d e r i v a t i v e s o f amino a c i d s have found w i d e r use and g r e a t e r u t i l i t y i n t h e p r e p a r a t i o n o f CSPs. o f L-valine,

By comparing t h e r e s o l v i n g a b i l i t y o f N-acyl d e r i v a t i v e s

Hara e t . a l . found N-formyl-L-valylaminopropylsilanized-silica to

be b e s t f o r t h e r e s o l u t i o n o f d e r i v a t i z e d amino a c i d s [621.

Separation

f a c t o r s o f 1.2-1.38 were o b t a i n e d f o r the t - b u t y l e s t e r s f o r N - a c e t y l amino acids.

R e c e n t l y Akanya e t . a l . have compared CSPs d e r i v e d from v a r i o u s

N-formyl amino a c i d s ( l e u c i n e , i s o l e u c i n e , p h e n y l a l a n i n e , p r o l i n e , and v a l i n e ) and found t h e i s o l e u c i n e and v a l i n e d e r i v e d CSPs a f f o r d s e p a r a t i o n f a c t o r s as l a r g e as 1.6 f o r racemic N-acetyl l e u c i n e t - b u t y l e s t e r C631.

7.5.5 Chiral Vinyl Polymers Okamoto and co-workers have prepared a v i n y l polymer from t r i p h e n y l m e t h y l methacrylate,

9,h a v i n g

a h e l i c a l polymer c h a i n [641.

An i s o t a c t i c polymer

o f h i g h o p t i c a l r o t a t i o n i s o b t a i n e d by p o l y m e r i z a t i o n w i t h c h i r a l a n i o n i c c a t a l y s t s such as l i t h i u m (R)-N-(1-phenylethylanilide,)

(-)-spartein-butyllithium

complex.

or

The c h i r a l polymer o b t a i n e d i s u s e f u l f o r

t h e p r e p a r a t i o n o f CSPs which r e s o l v e c h i r a l a r o m a t i c hydrocarbons and compounds possessing a r o m a t i c groups a d j a c e n t t o c h i r a l c e n t e r s .

I t seems

l i k e l y t h a t t h e c o o p e r a t i v e b e h a v i o r o f t h e h e l i c a l a r r a y o f t r i p h e n y l m e hY1 groups g i v e s r i s e t o t h e c h i r a l r e c o g n i t i o n o f s u i t a b l e s u b s t r a t e s , poss b l Y by an i n t e r c a l a t i o n p r o c e s s . R e s o l u t i o n o f a racemic m i x t u r e o f hexahel cene on t h e o p t i c a l l y a c t i v e polymers o f

(+)

r o t a t i o n r e s u l t s i n r e t e n t i o n of the

enantiomer, which has t h e same h e l i c i t y as t h e s t a t i o n a r y phase. I n g e n e r a l , compounds which have r i g h t - h a n d e d or P h e l i c i t y w i t h r e s p e c t t o t h e i r

(+)

aroma t ic groups a r e p r e f e r e n t i a l l y r e t a i n e d on t h e CSP o f P h e l i c i t y [ 6 4 b l . The f r s t CSP prepared f r o m (+)polytriphenylrnethylmethacrylate.

9 , used

t h e f i n e y ground (20-44 pm p a r t i c l e s i z e ) i n s o l u b l e polymer as t h e s u p p o r t m a t e r i a l and a f f o r d e d b e s t r e s u l t s when methanol r a t h e r t h a n hexane, was used

266

as a m o b i l e phase.

F o r example, T r g g e r ' s base g i v e s an a v a l u e o f 1.37

(hexane) compared t o 1.77 ( m e t h a n o l ) .

An improved CSP can be prepared by

c o a t i n g d i phenylmethyl s i 1 a n i zed-s i1 ica w i t h lower m o l e c u l a r w e i g h t , s o l u b l e polymer

46.

A l t h o u g h a few cases were observed i n which t h e former CSP

a f f o r d s b e t t e r s e p a r a t i o n s , for t h e most p a r t , columns c o n t a i n i n g t h e s i l i c a based CSP e x h i b i t h i g h e r a v a l u e s , lower r e t e n t i o n t i m e s , and a g r e a t e r number of t h e o r e t i c a l p l a t e s 1 6 4 d l .

Comparison o f a few r e p r e s e n t a t i v e cases a r e

presented i n Table 7.10.

TABLE 7.10

R e s o l u t i o n o f Racemates on an A n a l y t i c a l Column C o n t a i n i n g CSP 49.

a v a l u e on column:

entry

compound

f ine 1 y ground

coated

po 1 yme ra

s i l i c a gel

b i4-naphthol

2.13

2.37

3

trans-stilbene oxide

2.17

5.21

1

Troger s base

1.77

--

50

hexahel icene

>13

> 50

b

a Column c o n s i s t e d o f i n s o l u b l e polymer 49 ground and s i z e d t o s m a l l p a r t i c l e s (20-44 urn) i n a column (250 x 4.6 mm i . d . ) ; m o b i l e phase, methanol; flow r a t e , 0.72 m l l m i n . Column c o n s i s t e d of s o l u b l e polymer 49 coated o n t o bonded phase s i l i c a i n a column (250 x 4 . 6 mm i . d . ) ;

C e r t a i n l y , CSP

9

methanol; flow r a t e , 0.5 mL/min.

o f f e r s t h e p o t e n t i a l for p r e p a r a t i v e r e s o l u t i o n of

s i g n i f i c a n t amounts of c e r t a i n racemates.

However, t o o u r knowledge, o n l y two

r e p o r t s o f p r e p a r a t i v e r e s o l u t i o n s on t h e CSP have yet appeared 165,663. p o l y n u c l e a r a r o m a t i c hydrocarbon, 1,4-dimethylhexahelicene,

50,

The

has been

r e s o l v e d by Nakazaki, e t . a1 u s i n g t h e f i n e l y ground polymer packed i n a g l a s s tube (50 mm x 12 mm i . d . )

[65al.

Hexane was employed as t h e m o b i l e phase t o

p a r t i a l l y r e s o l v e 400mgs o f racemate per r u n .

By combining t h e f i r s t e l u t i n g

267

f r a c t i o n s f r o m numerous r u n s ( a t o t a l o f 6.49 o f m a t e r i a l was e v e n t u a l l y i n t r o d u c e d i n t o t h e column)., 2.39 o f t h e (-)enantiornet- was o b t a i n e d i n a b o u t These workers have r e c e n t l y o b t a i n e d b e t t e r r e s u l t s for t h e

12% 0 . p .

r e s o l u t i o n o f h e l i c a l crown e t h e r s u s i n g t h e improved s i l i c a based CSP w i t h methanol as an e l u e n t [ 6 5 b l . The novel enantiomers o f bevel-gear compounds microporous s i l i c a g e l coated with CSP

9 [ref.

Ca-d

661.

have been r e s o l v e d on The a n a l y t i c a l

chromatograms shown i n F i g . 7 . 6 demonstrate almost complete s e p a r a t i o n o f and

a.P r e p a r a t i v e s e p a r a t i o n o f 0.5

51a

mgs o f s u b s t r a t e on a 250 x 7.2 mm

i . d . column (methanol f l o w r a t e = 3ml/min) p r o v i d e d t h e l e a s t r e t a i n e d enantiomers i n h i g h o p t i c a l p u r i t y . r e p e a t e d chromatography.

Compounds

The r e t a i n e d enantiomers were p u r i f i e d by and

u, however,

were d i f f i c u l t t o

o b t a i n i n e n a n t i o m e r i c a l l y e n r i c h e d f o r m even a f t e r r e p e a t e d chromatography owing t o small s e p a r a t i o n f a c t o r s .

Presumably, p r e p a r a t i v e amounts o f

compounds which e x h i b i t h i g h e r a v a l u e s ( e . g . h e l i c e n e s ) c o u l d be e a s i l y r e s o l v e d on t h i s CSP.

C"3

I

49 N

50 N

51 a-d N N N

268

Compound

I1

\.'

CH 2

c1

1-1

CH 2

H

c1

KO.

Compound No.

-43 -

., 0 0

U

V

C1

H CI.

H

x. 3

24

Elution volume (ml)

F i g . 7.6.

The r e s o l u t i o n o f a n a l y t i c a l samples o f d l i s o m e r s of 5 1 a - d .

Compounds 1-4 c o r r e s p o n d t o 51a, b , c a n d d r e s p e c t i v e l y .

Reprinted with

p e r m i s s i o n from r e f . 66.

Polymers c o n t a i n i n g c h i r a l c a v i t i e s w i t h f u n c t i o n a l groups arranged s t e r e o s p e c i f i c a l l y c a n be f o r m e d b y p o l y m e r i z a t i o n i n t h e p r e s e n c e o f a c h i r a l cavity.

U s i n g s u g a r d e r i v a t i v e s a s t e m p l a t e s , Wulff and c o - w o r k e r s p r e p a r e d a

c o p o l y m e r i c CSP u s i n g a c h i r a l 4 - v i n y l b o r o n i c e s t e r as o n e o f t h e monomer u n i t s and s u b s e q u e n t l y removed t h e c h i r a l t e m p l a t e b y h y d r o l y s i s t o a f f o r d c h i r a l c a v i t i e s c o n t a i n i n g b o r o n i c a c i d f u n c t i o n a l i t i e s 1671.

These p o l y m e r i c

CSPs c a n r e s o l v e t h e t e m p l a t e , 4 - n i t r o p h e n y l m a n n o p y r a n o s i d e , w i t h a maximum separation f a c t o r of 2 . 2 7 .

However, t h e low c a p a c i t y and e f f i c i e n c y of t h e

CSP l i m i t s i t s use f o r p r e p a r a t i v e w o r k .

Even w i t h t h e a n a l y t i c a l s e p a r a t i o n

n o t e d above, b a s e l i n e r e s o l u t i o n was n o t a c h i e v e d o w i n g t o t h e b r o a d n e s s of t h e peaks and t o a r a t h e r low t h e o r e t i c a l p l a t e c o u n t ( i . e . l e s s t h a n 100 theoretical plates/m). 7.5.6

P o l y a m i d e CSPs

P o l y m e t h y l a c r y l a m i d e and p o l y a c r y l a m i d e CSPs, p r e p a r e d from c h i a1 amines and a m i n o a c i d e t h e r s have b e e n st.own t o r e s o l v e c e r t a i n r a c e m i c p h a r m a c e u t i c a l s , a l l o f w h i c h c o n t a i n an amide or i m i d e f u n c t i o n a l g r o u p [ 4 1 . Racemates r e s o l v a b l e on t h e s e CSPs were s o u g h t i n a somewhat e m p i r c a l manner,

269

e a c h r a c e m a t e b e i n g t r i e d o n t h e v a r i o u s l y s u b s t i t u t e d p o l y a m i d e CSPs.

No

p a r t i c u l a r p o l y a m i d e CSP i s s u p e r i o r i n a l l c a s e s and, as B l a s c h k e s t a t e s , " I n e x p e r i m e n t s w i t h new r a c e m a t e s i t i s i m p o s s i b l e t o p r e d i c t on w h i c h a d s o r b a n t To f u r t h e r c o m p l i c a t e m a t t e r s , t h e mode of

t h e y w i l l be s o l v e d b e s t '"41.

p r e p a r a t i o n o f t h e p o l y m e r i c CSPs I s i n t e g r a l l y r e l a t e d t o t h e i r e f f e c t i v e n e s s i n terms o f c h i r a l r e c o g n i t i o n .

O p t i m i z a t i o n has been a c h i e v e d by t h e

s y s t e m a t i c v a r i a t i o n o f t h e c o n d i t i o n s f o r p o l y m e r i z a t i o n , t h e r e s o l v i n g power of the r e s u l t a n t polymer then b e i n g t e s t e d .

F o r p r e p a r a t i v e r e s o l u t i o n s , two CSPs,

2

and

53,

have been employed

s u c c e s s f u l l y for t h e b a s e l i n e r e s o l u t i o n o f racemates [33,68-701.

The

c h r o m a t o g r a p h i c r e s o l u t i o n o f 302mg o f N-benzoyl t y r o s i n e e t h y l e s t e r o n a column ( 6 5 0 x 25 mm i . d . ) c o n t a i n i n g 659 o f CSP h a v i n g an a v e r a g e o p t i c a l p u r i t y o f 81%.

o f 500mg o f t h a l i d o m i d e ,

2 . was

2

r e s u l t e d i n two f r a c t i o n s

The c o m p l e t e b a s e l i n e s e p a r a t i o n

p o s s i b l e o n a s i m i l a r s i z e column owing t o

t h e h i g h s e p a r a t i o n f a c t o r ( a = 2 . 0 ) o b s e r v e d f o r t h i s r a c e m a t e C691. P o l y a c r y l a m i d e 5 3 , p r e p a r e d from e t h y l p h e n y l a l a n a t e , has been u s e d f o r t h e r e s o l u t i o n of c h l o r t h a l i d o n e ,

55,

and oxazepam,

( 7 6 0 x 32 mm i . d . ) c o n t a i n i n g 2509 o f

3 , one

56

[ r e f . 701.

U s i n g a column

c a n r e s o l v e 530mg o f

chlorthalidone.

The p s y c h o p h a r m a c e u t i c a l oxazepam was a l s o r e s o l v e d o n t h e

e

I

C

H

C

H

3

# CH2- CH -C02E t I \c+

"0N

53 N

0

0 54 N

55 N

0

270

@---/

H

CI 56

H 57 N

N

C02CH3

same CSP. Because of t h e lower a value (1.2) noted, o n l y 46mg of this racemate could be resolved in a single pass. Nonetheless, this resolution superbly demonstrates t h e utility of t h e technique, f o r o x a z e p a m r a c e m i z e s so readily as t o almost preclude nonchromatographic methods (Fig. 7.7) [ref. 331.

' 1 1

I

I

2000

I

I

I

1

I

2500

I

I

I

I

I

30 00

Fig. 7.7. The chromatographic resolution of 45.6mgs of O x a z e p a m 56 o n 2 5 0 gms of Polyamide 53. Mobile phase consisted o f Toluene/Dioxane 1 : l . Reprinted with permission from ref. 33, p. 1005.

7.5.7 Cyclodextrin Polymers O-Cyclodextrins, cyclic oligosaccarides consisting of seven a-l,4-linked d-glucose units, f o r m s inclusion complexes with aromatic compounds in a q u e o u s

271

media. As 0-cyclodextrin is chiral, the inclusion of enantiomeric guests forms two diastereomeric complexes. The different physical properties of the diastereomers have been used to obtain enantiomerically enriched samples of the guest by fractional crystallization techniques [711. Water insoluble 8-cyclodextrin polymers can be used as CSPs for the resolution o f racemates. A column (820 x lOmm i.d.) containing large particle (100-400~) 8-cyclodextrin polymer was employed for the partial resolution of 100 mgs o f methyl mandelate [721. The low efficiency of the stationary phase gave rise to a poor overall separation although an a value of 1.09 was obtained. Recently, Zsadon, et. al., have resolved 8mgs of vincadifformine, 57, by using three O-cyclodextrin polymer columns (750 x 16 mm i.d. each) in tandem [731. Using citrate buffer (pH=4, flow rate 50ml/hr) a total of 2 2 hours was required to complete the separation.

7.5.8 TAPA CSPs

Optically active TAPA, 3,C2-(2,4,5,7-tetranitro-9-fluorenylideneaminooxy)propionic acid1 has been used for the preparation of CSPs which resolve chiral aromatic hydrocarbons C74-771. A silica o r alumina column treated in situ with TAPA affords a coated CSP which resolved hexahelicene ( a of 1 . 1 1 and 1.20 for silica and alumina support, respectively) and its derivatives [74,751. Using a coated alumina column, Wynberg et. al.. obtained baseline resolution of inherently chiral olefin 3 by elution with benzene:hexane (95:5). isolating sufficient material to determine the specific rotation of this previously unknown compound [761.

58 N

59 N

60 N

Gil-Av and coworkers have compared the performance of derivatives of TAPA and of coated or covalently bonded (by means of an amide bond to aminopropyl silanized-silica) CSPs [741. Highest efficiencies and best separations were

212

o b t a i n e d f o r t h e c o a t e d CSPs o f TAPA i t s e l f

14,000 t h e o r e t i c a l p l a t e s p e r

m e t e r b e i n g measured f o r n a p t h a l e n e ( m o b i l e phase h e x a n e , u = 0 . 2 5 cin/sec) on A l t h o u g h n o p r e p a r a i v e work has been r e p o r t e d u s i n g

a 200 x 2 . 3 mm c o l u m n .

t h i s column, good a n a l y t i c s e p a r a t i o n s have been o b t a i n e d . c a r c i n o g e n i c d e r i v a t i v e s o f b e n z o [ a l p y r e n e s . , s u c h as d i o l

M u t a g e n i c and

59,

have been

a n a l y t i c a l l y r e s o l v e d (a = 1 . 3 1 ) o n s i l i c a g e l c o v a l e n t l y l i n k e d t o

1771.

(R)-(-)-TAPA 7.5.9

,

Ligand Exchange C h r o m a t o g r a p h y (LEC)

L i g a n d exchange c h r o m a t o g r a p h y ( L E C ) has been e x t e n s i v e l y used t o a n a l y t i c a l l y s e p a r a t e a m i n o a c i d s and t h e i r d e r i v a t i v e s [ 5 1 .

These s t u d i e s

have l e d t o t h e p r e p a r a t i v e s e p a r a t i o n o f a m i n o a c i d s u s i n g c o n d i t i o n s f o u n d s u i t a b l e for a n a l y t i c a l runs.

E s s e n t i a l l y t h e r e a r e two modes o f LEC, t h e

f i r s t i n v o l v i n g t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f m e t a l c o o r d i n a t i o n complexes o n an a c h i r a l s t a t i o n a r y phase and t h e second c o n s i s t i n g o f a s t a t i o n a r y c h i r a l l i g a n d (CSP) f o r t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f m o b i l e l i g a n d s .

The

f i r s t mode r e q u i r e s a c h i r a l m o b i l e phase a d d i t i v e a n d has n o t been employed

for p r e p a r a t i v e work.

Hence, i t w i l l n o t be d i s c u s s e d ( v i d e s u p r a ) .

Numerous

c h i r a l l i g a n d s have been e l a b o r a t e d i n t o CSPs s u i t a b l e for LEC u s i n g v a r i o u s s u p p o r t m a t e r i a l s and t h e i r p o t e n t i a l a p p l i c a t i o n f o r p r e p a r a t i v e s e p a r a t i o n s e x p l o i t e d as d i s c u s s e d b e l o w . The d i r e c t s e p a r a t i o n o f e n a n t i o m e r s b y LEC depends o n t h e f o r m a t i o n o f d i a s t e r e o m e r i c c o o r d i n a t i o n complexes c o n s i s t i n g o f a f i x e d or s t a t i o n a r y l i g a n d , a m e t a l ion a n d a c h i r a l m o b i l e l i g a n d ( i . e . ,

t h e s u b s t r a t e t o be

resolved).

E n a n t i o s e l e c t i v i t y stems from t h e f o r m a t i o n o f m i x e d - l i g a n d

complexes.

These complexes m u s t d i f f e r s i g n i f i c a n t l y i n e n e r g y f o r

d i s c r i m i n a t i o n t o o c c u r and m u s t , m o r e o v e r , be k i n e t i c a l l y l a b i l e so as t o a f f o r d good c h r o m a t o g r a p h i c b e h a v i o r .

Consequently, t h e c h o i c e of t h e f i x e d

l i g a n d , m e t a l i o n , m o b i l e phase ( b u f f e r c o n c . , pH, m e t a l i o n c o n c . , e t c . ) a n d s u p p o r t m a t e r i a l a l l p l a y an i n t e g r a l p a r t i n t h e e f f i c i e n c y o f t h e chromatographic process.

The i m p o r t a n c e o f t h e s e p a r a m e t e r s on t h e s e p a r a t i o n

o f e n a n t i o m e r s b y LEC has r e c e n t l y been a d d r e s s e d i n a r e v i e w a r t i c l e b y Davankov [ 5 1 .

273

No.

-08{

Fig. 7 . 8 . Chromatography of 0.59 of dl-proline determined by measuring the optical rotation of eluent fractions. Reprinted with permission from ref. 78a

Three different support materials have been employed for analytical work and include polymeric supports such as polyacrylamides, polyacrylates, polyacrylesters and polystyrenes, silica bonded phases .and liphophilic stationary phases, prepared by coating an aliphatic bonded phase with a liphophilic chiral material. A s yet, preparative separations have been carried out almost exclusively on polymeric supports. However, the silica based materials offer many inherent advantages in terms of overall chromatographic efficiency and speed. The first report of a successful resolution by LEC utilized L-Proline attached t o chloromethylated polystyrene 61 [ref. 781. The prepared resin was saturated with Cut2 ions, packed into a column (475 x 9 mm i.d.). and employed for the baseline resolution of 500mg of dl-proline. The 1-isomer was eluted with water and the retained isomer eluted by switching t o 1N NH40H mobile phase ( F i g . 7 . 8 ) . Later, Jozefonvicz, et. al., reported the resolution of 160mg of dl-Proline using a similar CSP and column size [791. The time required for these resolutions was exceedingly long (typically 24-48 hours) owing to slow ligand exchange and the type of support material employed (large particle polymeric beads). A large one liter column containing 3009 of CSP 61 (I-hydroxyproline bonded to chloromethylated polystyrene) resolves 209 of dl-Proline o f 69 or

214

d l - t h r e o n i n e i n a s i n g l e pass 1801.

S m a l l e r s i z e d v e r s i o n s o f t h i s CSP have

been employed f o r t h e simultaneous r a d i o c h e m i c a l and e n a n t i o m e r i c r e s o l u t i o n

o f r a d i o c h e m i c a l l y l a b e l l e d amino a c i d s [ E l l .

I d e a l l y , t h e methods of

p u r i f i c a t i o n and r e s o l u t i o n o f l a b e l l e d compounds s h o u l d be app i c a b l e t o small amounts o f m a t e r i a l , be q u a n t i t a t i v e and r e q u i r e l i t t l e t me. Preparations i n v o l v i n g products of h i g h l y a c t i v e radiochemicals present f u r t h e r l i m i t a t i o n s i n terms of p o s s i b l e c o n t a m i n a t i o n and i n a c i v a t i o n o f t h e r e s o l v i n g agent by t h e e m i t t e d r a d i a t i o n .

T r i t i a t e d v a l i n e has been r e s o l ved

i n l e s s than two hours by maximizing t h e e f f i c i e n c y and m i n i m i z ng t h e t i m e required f o r a single run.

Since o n l y 0.1 t o 1.Omg q u a n t i t i e s were d e s i r e d a t

one t i m e , t h e e f f i c i e n c y o f t h e chromatographic process c o u l d be maximized a t t h e expense o f t h e amount o f m a t e r i a l r e s o l v e d .

D i r e c t chromatography

t h e r e f o r e o f f e r s an i d e a l method f o r t h e r e s o l u t i o n and p u r i f i c a t i o n o f r a d i o l a b e l l e d compounds. I n r e c e n t y e a r s many CSPs d e r i v e d f r o m p r o l i n e and i t s analogues have been prepared u s i n g s i l i c a s u p p o r t s .

The d i r e c t comparison o f these phases i s

c o m p l i c a t e d by t h e v a r i e t y o f c o n d i t i o n s employed.

The q u a l i t y o f t h e

s e p a r a t i o n s observed i s dependent on t h e c o m p o s i t i o n o f t h e m o b i l e phase, t h e mode o f p r e p a r a t i o n o f t h e CSP, t h e flow r a t e s employed, e t c .

For these

reasons, seemingly s i m i l a r s t a t i o n a r y phases prepared by two d i f f e r e n t workers may g i v e v a s t l y d i f f e r e n t r e s u l t s .

Use o f " s t a t e - o f - t h e - a r t "

support

m a t e r i a l s does seem t o r e s u l t i n h i g h e r chromatographic e f f i c i e n c i e s and s h o r t e r r u n t i m e s f o r t h e complete s e p a r a t i o n o f racemates.

Fortunately,

s y s t e m a t i c i n v e s t i g a t i o n s a r e b e i n g c a r r i e d o u t and t h e p o t e n t i a l f o r p r e p a r a t i v e s e p a r a t i o n s has been suggested.

G u b i t z and coworkers have developed a CSP c o n s i s t i n g of I-amino a c i d s bonded v i a 3-glycidoxypropyltrimethoxysilane t o a s i l i c a s u p p o r t [ 8 2 1 . CSPs

63-65

The

have been used t o r e s o l v e most t h e common amino a c i d s , t h e

1 - p i p e c o l i c a c i d CSP, 65. a f f o r d i n g the b e s t o v e r a l l r e s u l t s . Up t o 3600 t h e o r e t i c a l p l a t e s per meter have been observed f o r complexed I i g a n d s on a 25 cm column.

The f a c t o r s r e s p o n s i b l e f o r t h e success o f these phases a r e , f i r s t ,

275 t h e c a r b o x y l g r o u p o f t h e bonded a m i n o a c i d i s f r e e f o r complex f o r m a t i o n w i t h t h e m e t a l i o n and, second, t h e h y d r o x y l g r o u p i n t h e s i d e c h a i n , f o r m e d b y t h e opening of the epoxide. g i v e s r i s e to g r e a t e r c h i r a l r e c o g n i t i o n .

Although

t h e r o l e o f t h e h y d r o x y l g r o u p i s n o t c o m p l e t e l y u n d e r s t o o d , s i m i l a r phases t h a t l a c k t h i s h y d r o x y l a f f o r d l i t t l e or n o s e p a r a t i o n o f e n a n t i o m e r s .

The

c a p a c i t y o f t h e s e CSPs for p r e p a r a t i v e w o r k has been d e m o n s t r a t e d ; u p t o 1 . 5 mgs o f a m i n o a c i d h a s been s e p a r a t e d o n an a n a l y t i c a l c o l u m n (250 x 4.6 mm i . d . ) w i t h o u t s i g n i f i c a n t d e g r a d a t i o n o f k ’ va1ue.s or r e s o l u t i o n .

66 N

67 N

68 N 1 - P r o l i n e has a l s o been l i n k e d t o a m i n o p r o p y l - s i l a n i z e d s i l i c a , CSP an amide l i n k a g e [ 8 3 , 8 4 1 .

66.

via

A l t h o u g h t h i s s t a t i o n a r y p h a s e a f f o r d s low

s e p a r a t i o n f a c t o r s for t r y p t o p h a n , p h e n y l a l a n i n e and t y r o s i n e [ 8 3 1 , L i n d e r h a s f o u n d t h i s p h a s e t o be e f f e c t i v e for t h e r e s o l u t i o n o f d a n s y l d e r i v a t i v e s o f amino a c i d s .

S e p a r a t i o n f a c t o r s as l a r g e as 3.16 were o b t a i n e d f o r

d e r i v a t i z e d a s p a r t i c a c i d [841.

W i t h t h e e x c e p t i o n o f p r o l i n e , t h e common

amino a c i d s can be r e s o l v e d w i t h t h i s s y s t e m . p r e p a r e d p r o l i n e a n d v a l i n e based CSPs,

67

s e p a r a t i o n of d a n s y l d e r i v a t i z e d a m i n o a c i d s . and 1 . 8 were o b t a i n e d [851.

E n g e l h a r d t , e t . a l . , has

and @ r e s p e c t i v e l y , f o r t h e S e p a r a t i o n f a c t o r s b e t w e e n 1.0

R e c e n t l y , a h i s t i d i n e - d e r i v e d CSP bonded b y a n

amide l i n k a g e , has been shown t o r e s o l v e f r e e a m i n o a c i d s ( a o f 1 - 1 . 9 ) [ r e f . 861.

276 Two g r o u p s h a v e i n d e p e n d e n t l y p r e p a r e d CSP as t h e amine t o y - h a l o p r o p y l

69,

i n w h i c h 1 - p r o l i n e i s bonded

s i l a n i z e d s i l i c a L87.881.

Although the reports

have a d d r e s s e d t h e m e c h a n i s t i c a s p e c t s o f t h e CSPs, t h e s e p a r a t i o n f a c t o r s o b t a i n e d ( a b e t w e e n 1 and 4 ) s u g g e s t t h e p o s s i b i l i t y of p r e p a r a t i v e appl i c a t i o n .

69 N

70 N

C o a t e d CSPs o f f e r many a d v a n t a g e s f o r t h e p r e p a r a t i o n o f c h i r a l c o l u m n s . These i n c l u d e f a c i l e p r e p a r a t i o n from c o m m e r c i a l l y a v a i l a b l e c o l u m n s , t h e p o s s i b l e r e g e n e r a t i o n o f t h e c o l u m n , a n d , i f d e s i r e d , t h e r e t r i e v a l of t h e resolving agent.

By p r e p a r i n g l i p h o p h i l i c c h i r a l compounds, c o a t e d CSPs can

be p r e p a r e d f r o m common r e v e r s e phase columns c o n t a i n i n g a l i p h a t i c bonded phases.

Davankov has r e s o l v e d u n m o d i f i e d a m i n o a c i d s o n a s y s t e m composed o f

r e v e r s e phase p a c k i n g s c o a t e d w i t h N-alkyl-1-hydroxyproline

Lc,

[ r e f . 891.

These s y s t e m s h a v e a f f o r d e d t h e l a r g e s t s e p a r a t i o n f a c t o r s o b t a i n e d t h u s f a r

f o r LEC o f f r e e a m i n o a c i d s .

U s i n g r e v e r s e phase c o n d i t i o n s , t h e c o a t e d

columns d o n o t b l e e d a p p r e c i a b l y or a l t e r t h e i r s e l e c t i v i t y f o r a m i n o a c i d enantiomers.

Large separation f a c t o r s (up to 16.4 for d l - p r o l i n e ) ,

high

e f f i c i e n c i e s (3000 t h e o r e t i c a l p l a t e s f o r a 10 cm c o l u m n ) and low t o t a l t i m e r e q u i r e d f o r a n a l y t i c a l s e p a r a t i o n s (35 m i n ) s u g g e s t t h a t p r e p a r a t i v e a p p l i c a t i o n s o f t h e s e phases a r e p r a c t i c a l .

7.5.10 Miscellaneous Liquid Chromatographic Methods A f f i n i t y c h r o m a t o g r a p h y has not been employed f o r p r e p a r a t i v e s e p a r a t i o n of e n a n t i o m e r s . h o w e v e r , t h i s may be due t o t h e f a c t t h a t i t s a p p l i c a t i o n f o r t h e s e p a r a t i o n of e n a n t i o m e r s e v e n a t t h e a n a l y t i c a l l e v e l has been l a r g e l y overlooked.

I n 1973, S t e w a r d a n d D o h e r t y d e m o n s t r a t e d t h e s e p a r a t i o n o f t h e

a n t i p o d e s of d l - t r y p t o p h a n o n b o v i n e serum a l b u m i n (BSA) a t t a c h e d t o a g a r o s e

[901.

R e c e n t l y A l l e n m a r k and c o - w o r k e r s have e x t e n d e d t h e a p p l i c a t i o n o f t h i s

CSP t o i n c l u d e d e r i v a t i v e s o f t r y p t o p h a n e and c h i r a l s u l f u r compounds [911. The a l b u m i n - a g a r o s e s t a t i o n a r y phase e x h i b i t s low c a p a c i t i e s f o r t h e r a c e m a t e s and t h e o b s e r v e d s e p a r a t i o n f a c t o r s f o r d . 1 - 5 - h y d r o x y t r y t o p h a n

decrease

n o t i c a b l y w i t h i n c r e a s i n g sample s i z e , even i n w h a t i s c o n s i d e r e d t h e a n a l y t i c a l range.

R e c e n t l y an i m p r o v e d s t a t i o n a r y p h a s e i n w h i c h BSA i s

217

attached directly to silica (10pm) has been employed for the resolution of N-aroylamino acids (a's up to 3.6) and pharmacologically active sulfoxides [ 9 2 1 . This commercially available CSP may also provide higher mass capacities Numerous attempts have been made to obtain enantiomer resolutions on chiral ion exchange resins [131. The first o f these to result in a baseline resolution used a d-tartrate-form anion exchange resin for the resolution of threo-l-(p-nitrophenyl)-2-amino-l.3-propanediol. Ethyl acetate, as the mobile phase, afforded complete baseline separation of an analytical sample, larger sample sizes not being addressed [931. Yamagishi has found that liquid chromatography columns of clay-metal chelate adducts can partially resolve 2, 3-dihydro-2-methyl-5,6-diphenylpyrazine 71 [ref. 941. Ama7ingly enough, using the same chiral metal chelate adduct to coat the stationary phase, the clay support provided greater separation than more usual cation exchange resins (e.g.. Dowex or Sephadex). A s noted earlier (in sec. 7.5.4). the reciprocal nature of 1 : l chiral recognition processes can be used to develop new and novel stationary phases. In principle, if a CSP prepared from a single enantiomer of compound A can separate solute enantiomers B and B', a column constructed from a single enantiomer of B will separate the enantiomers of A and A*. In each case, the relative stability o f two diastereomeric complexes, A:B and A : B * , gives rise to the resolution. In practice, however, the two experiments are not exactly alike since chromatographically we are dealing with diastereomeric adsorbates rather than free complexes. The relative position of the support to the complex can alter the energetic difference and possibly even the sense of the difference between the two adsorbates [ 9 5 1 . However, the energetic differences between the complexes can be used as a first order approximation of the difference in stability between two analogous adsorbates.

71 N

By assessing the chromatographic separability of various racemates on a CSP, potential chiral molecules can be chosen as precursors to new second

generation CSPs.

This reiterative process may be carried o n to develop

278

TABLE 7.11

Comparative Liquid Chromatographic Separation of Racemates o n First and Third Generation Chiral Stationary Phases.a

separation f a c t o r , entry

R

X

78

'gHSCH2

79

'sH!i

80 81

a

CSP 2 7

C S P 72

C S P 73

CSP 74

C02CH3

1.10

2.04b

1.42'

4.73b

CH3

l.lgb

1.18

1.70'

1.50b

'gH5

C(CH3)2H

1 .3SC

1.72

1.53'

3.36b

C6H5CH2

CONHn-C4H9

1.52d

1.46b

1.98'

2.32b

aThe mobile phase consisted of 2-propanol :hexanes o f t h e following composition; b = 1 . 4 , c = 1.9, d = 1:19.

successful third and e v e n f o u r t h generation stationary phases. For example, beginning with fluoroalcohol-derived C S P 27, t h e separation f a c t o r s of derivatives of a m i n o a c i d s w e r e compared and suitable second generation C S P s 29, 9 and 46 w e r e prepared. Columns containing these C S P s w e r e found t o resolve not o n l y chiral alcohols, but a host of functionally different chiral molecules. Within each functional group, t h e racemate structure can b e optimized in a rational manner t o afford large separation f a c t o r s and a s i n g l e enantiomer o f an optimized compound elaborated t o afford a third generation CSP. Three such third g e n e r a t i o n CSPs, 72, 3 and 74, have been prepared and afford significantly larger separation factors for certain s o l u t e racemates t h a n the "first generation" C S P 11 (Table 7.11) [ref. 96-981. In f a c t , the third generation columns exhibit.selectivities f o r s o l u t e classes unique t o

279

72

73

N

N

X ( CH2)10-( CH2lI04 CH2ll0-

R CH(CH3I2 CH3 -CH3

-

-

74

75 76

R' CH3 -CH3 -H

-

-H each column.

Whereas CSP

e s t e r s and amides such as

2 i s superior 2 and E, and

for DNB d e r i v a t i v e s o f amino a c i d CSP

3

i s p a r t i c u l a r l y well suited

for the r e s o l u t i o n o f arnine d e r i v a t i v e s such as 3 . The d a t a presented here i s o n l y a r e p r e s e n t a t i v e sample o f the separation a b i l i t i e s of the t h i r d generation CSPs and already hundreds o f amines, amino a c i d s and amino a l c o h o l d e r i v a t i v e s have been resolved.

The improvement i n degree o f c h i r a l

r e c o g n i t i o n over e a r l i e r s t a t i o n a r y phases suggest that they w i l l be s u i t a b l e

for p r e p a r a t i v e c h i r a l columns; the o n l y l i m i t a t i o n being the a v a i l a b i l i t y of chiral precursors.

R e c e n t l y , b o t h P i r k l e and coworkers and 01, e t . a l . , have i n d e p e n d e n t l y developed CSPs

76

r e s p e c t i v e l y , prepared from t h e r e a d i l y a v a i l a b l e

and

c h i r a l 1-(a-naphthyl)

e t h y l amine C98.991.

These phases d i f f e r o n l y i n t h e

mode o f attachment t o s i l i c a and, s i m i l a r i n b e h a v i o r t o CSP d e r i v a t i v e s o f a c i d s , amines, amino a c i d s and amino a l c o h o l s . r u l e , CSP

2 affords

whereas CSPs

74, 75

of amino a c i d s . e s t e r on CSP

2

74,

resolve

As a general

h i g h e r s e p a r a t i o n f a c t o r s for DNB d e r i v a t i v e s o f amines and

3

a r e p r e f e r r e d f o r t h e r e s o l u t i o n of DNB d e r i v a t i v e s

The r e s o l u t i o n o f N-3,5-dinitrobenzoylphenylalanine methyl (Fig.7.9)

i s i n d i c a t i v e o f t h e h i g h e f f i c i e n c i e s b o t h i n terms

280

I

0

10

20

30

40

50

60

Time(min.1 Fig. 7.9.

The r e s o l u t i o n o f N-3,5-dinitrobenzoylphenylalanine m e t h v l e s t e r or

CSP 7 4 ( c o l u m n , 250 x 10 mm i . d . ; p a r t i c l e s i z e , 5pm; flow r a t p , 7rnl/mirm,

m o b i l e phase, 20% 2 - p r o p a n o l i n h e x a n e s ; d e t e c t i o n , UV 254nm)

of c h i r a l r e c o g n i t i o n ( a o f 4 . 7 3 ) a n d t h e o r e t i c a l p l a t e c o u n t ( 1 0 , 8 8 0 p l a t e s p e r meter) o b t a i n e d w i t h these CSPs.

The a v a i l a b i l i t y o f t h e c h i r a l

p r e c u r s o r s s h o u l d l e a d t o t h e c o n s t r u c t i o n o f a new g e n e r a t i o n o f p r e p a r a t i v e c h i r a l columns.

281

7.6

CONCLUS IONS

Successful systems have been developed for the direct preparative resolution of enantiomers by liquid chromatography. As one system will not be able t o solve all resolution problems, there is an on-going effort to develop new CSPs for the resolution of a wide array of solute classes and to further the understanding of chiral recognition processes. In this manner, it will be possible to choose the appropriate CSP for a particular resolution based o n an understanding of the underlying principles of chiral recognition. No doubt, CSPs will become commonplace in laboratories interested in evaluation and resolution of chiral compounds. There is a growing need for the facile separation of many biologically active chiral compounds for evaluation. Often, modest quantities of resolved material are sufficient for test purposes. Direct chromatographic resolution seems ideally suited to such needs. The resolution of intermediates in a synthetic route can also lead to the preparation of more complex chiral products. These recent publications clearly demonstrate the growth in interest in the direct chromatographic resolution of optical isomers both in chromatographic circles and in the scientific community at large.

7.7

ADDENDUM

After the preparation and review of this chapter, numerous new reports appeared which are relevant to this rapidly developing field. The CSP prepared from (+)-polytriphenylmethyl methacrylate (see sec. 7 . 5 . 5 ) now commercially available [I001 and has been employed by Tajiri, et al. El011 for the resolution of 2,2'-dimethyl-l.l'-biazulene and by Nakazaki, et al. El021 for the resolution of crown ethers incorporating helicene in the ring system. In both cases, circular dichroism spectra were obtained and the absolute configurations o f the resolved enantiomers thus assigned. In addition, p e r c h l o r o t r i p h e n y l a m i n e , the first example of an optically active Ar3Z type compound in which 2 is not a chiral center, has been baseline resolved on this CSP [1031. Previous attempts t o resolve this racemate on triacetylcellulose had led to enriched fractions o f not more than 1% ee. However, up to 6 mg may be resolved in one pass on an analytical column (30 X 2 . 2 cm i.d.) containing the aformentioned chiral vinyl polymer with only partial overlap of the enantiomers observed (a = 2 . 9 ) . is

282

Zsadon and coworkers El041 have made impressive improvements in the preparative application of their industrially prepared polymeric bead-supported B-cyclodextrin CSP (see sec. 7.5.7). By initial optimization of the analytical conditions of resolution of vincadifformine, 57. they have found suitable conditions for the resolution of 500mg of this racemate on a preparative size column (90 x 5 cm). A structural analog, (2)-quebrachamine, has also been resolved; the smaller separation factor allowing but 200mg of racemate to be chromatographed in a single pass. Recent 1 y , Mannschreck and co-worker s [ 1051 have reported the characterization o f microcrystalline triacetylcellulose and also determined the dependence of plate height, HEPT, on linear flow velocity, porosity, particle size and capacity factor of the solute. While such data has immediate ramifications for the analytical separation of enantiomers, it is also relevant to future preparative separations on similar high efficiency LC columns. The resolution of N-benzoylaminoacid esters, a-substituted phenylacetamides, N-substituted acetamides, benzoin derivatives and various drugs of pharmacetuical interest have been compared on triacetylcellulose and chiral polyamide CSPs C1061. Although no general trend was noted, nearly baseline resolutions were obtained for a few of these compounds, Seven helical phenanthrenes have been either partially or completely resolved on triacetylcellulose and the enriched materials used to determine enantiomerization barriers of these thermally labile compounds C1071. Partial resolution of a chiral azo dyestuff derived from mandelic acid was achieved on potato starch and also o n powdered silk, while n o noticeable separation was obtained on wool or cellulose C1081. Isaksson and coworkers [lo91 have reported a most notable separation of a racemic spiroacetal, the main pheromone component o f Adrena bees, on tracetylcellulose. Utilizing a column containing 1809 of CSP, they have resolved 140mg of trans, trans-2,8-dimethyl-l,7-dioxaspiro-C5,51-undecane in a single run, obtaining both enantiomers in greater than 98% ee. Chiral spiroacetals commonly occur as insect pheromone components, having been identified from bark beetles and wasps as well as bees. As such, this CSP offers promise for resolution of other spiroacetals of biological interest. The number of reports of new chiral stationary phases for liquid chromatography is truly phenomenal. For instance during the six month period of May through October 1985, n o less than twenty new phases were reported.

283 While these papers d e s c r i b e d o n l y t h e a n a l y t i c a l aspects o f these phases, t h e l a t t e r showed promise f o r p r e p a r a t i v e r e s o l u t i o n s as w e l l .

Hermansson has

developed a c h i r a l phase c o n s i s t i n g o f an i m m o b i l i z e d plasma p r o t e i n which i s u s e f u l f o r the r e s o l u t i o n o f some racemic drugs o r d r u g m e t a b o l i t e s [ l I O l . Human serum albumin has a l s o been i m m o b i l i z e d for e l a b o r a t i o n i n t o a CSP and has been used t o r e s o l v e oxepam e s t e r s w i t h s e p a r a t i o n f a c t o r s as l a r g e as 11.8 f o r oxepam hemisuccinate e s t e r [ 1 1 1 1 . Amino a c i d s and amino a c i d d e r i v a t i v e s have always been t h e f a v o r i t e s u b j e c t s o f most chromatographic r e s o l u t i o n s t u d i e s .

Consequently, t h e

m a j o r i t y o f new phases a r e developed t o r e s o l v e these compounds.

For example,

N-acyl amino a c i d e s t e r s a n d l o r amides have been r e s o l v e d by a d i a m i d e phase prepared f r o m 1 - v a l i n e [1121, a diamide phase prepared f r o m an amino a c i d and chrysanthemic a c i d , and a urea-amide CSP prepared f r o m 1 - v a l i n e [1131. r a t i o n a l approach t o CSP design has l e d P i r k l e and co-workers r e f i n e d phase based on t h e amide o f a 1 - ( 1 - a r y 1 ) a l k y l a m i n e

A

[I141 t o a

which n o t o n l y

a f f o r d s large separation f a c t o r s , but a l s o provides the l a r g e s t r e s o l u t i o n f a c t o r s ( R s ) noted t o d a t e f o r t h e enantiomers o f amino a c i d d e r i v a t i v e s . t h i s a r i s e s from a combination o f h i g h e n a n t i o s e l e c t i v i t y and chromatogi.aphic e f f i c i e n c y f o r t h i s CSP.

S i g n i f i c a n t improvements have a l s o been made on CSPs

f o r l i g a n d exchange chromatography ( s e e s e c . 7 . 5 . 9 ) o f u n d e r i v a t i z e d amino a c i d s which a l l o w b o t h improved e n a n t i o s e l e c t i v i t y and e f f i c i e n c y [115,1161. 7.8

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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100.


289

CHAPTER 8 PREPARATIVE S IZE DCLUSION CHROMATOGRAPHY Gary L. Hagnauer Polymer Research D i v i s i o n U.S. Army Materials Technology Laboratory Watertown, Massachusetts 02172-0001

8.1

INTRODUCTION

8.2

INSTRUMENTATION, COLUMNS AND COLUMN PACKING MATERIALS

8.3

GENERAL REQUIREMENTS AND SPECIAL TECHNIQUES 8.3.1 8.3.2 8.3.3 8.3.4 8.3.5 8.3.6 8.3.7 8.3.8 8.3.9 8.3.10

Mobile Phase - Eluent Sample Preparation Solvent Reservoir Solvent Delivery System Injector Columns and Column Packing Materials Monitor Fraction Collection and Recovery Refractionation and Recycling Temperature Control

8.4

OPTIMIZATION OF THE PREPARATIVE SEPARATION

8.5

APPLICATIONS

8.6

REFERENCES

8.1

INTRODUCTION

Size e x c l u s i o n chromatography

(SEC)

i s a I i q u i d chromatographic

technique which separates s o l u t e molecules according t o t h e i r s i z e i n solution.

It

IS

w i d e l y used as an a n a l y t i c a l procedure to i n v e s t i g a t e the

behavior o f small molecules i n s o l u t i o n , analyze chemical compositions, and determine the molecular weight

(MW) averages and molecular weight

d i s t r i b u t i o n s (MWD) of polymers.

The column i s packed w i t h porous p a r t i c l e s

and separation takes p l a c e as a r e s u l t o f the d i f f e r e n t i a l s o l u t e d i s r i b u t i o n o u t s i d e and w i t h i n the pores of the column packing m a t e r i a l .

Since d f f u s i o n

o f s o l u t e i n t o the pores i s u s u a l l y much f a s t e r than the l i n e a r veloc t y o f

the m o b i l e phase, d i f f u s i o n e q u i l i b r i u m o f the s o l u t e between the mob l e phase

290

and t h e p o r e s of t h e s t a t i o n a r y phase ( p a c k i n g ) may b e assumed.

Hence i f n o

a d s o r p t i o n or o t h e r i n t e r a c t i o n s w i t h t h e s t a t i o n a r y phase t a k e p l a c e , s e p a r a t i o n i s achieved by a p h y s i c a l s o r t i n g p r o c e s s such t h a t the r e t e n t i o n t i m e o r e l u t i o n volume ( V e ) o f a s o l u t e molecule depends upon t h e p o r e volume t o which i t has access.

For s o l u t e molecules h a v i n g d i f f e r e n t average

dimensions o r molar volumes, t h e l a r g e r molecule w i l l be excluded t o a g r e a t e r e x t e n t than t h e small one f r o m t h e p o r e s i n t h e column p a c k i n g and t h e r e f o r e w i l l e l u t e f i r s t and have a lower V e .

Consequently t h e e l u t i o n sequence o f

a s e r i e s o f sol.ute molecules i s p r e d i c t a b l e and, u n l i k e o t h e r t y p e s of l i q u i d chromatography (LC). t h e r e e x i s t b o t h an e l u t i o n volume minimum or v o i d volume (V

0

)

c o r r e s p o n d i n g t o t h e volume o f t h e m o b i l e phase i n t h e i n t e r s t i c e s

between t h e SEC column packing p a r t i c l e s and an e l u t i o n volume maximum (V,) which i s the sum o f Vo and t h e t o t a l l i q u i d volume c o n t a i n e d w i t h i n t h e pores o f the SEC p a c k i n g .

For polymers and c e r t a i n low MW homologs, i t has

been shown b o t h t h e o r e t i c a l l y and e x p e r i m e n t a l l y t h a t t h e e l u t i o n volume between V and Vm i s r e l a t e d l i n e a r l y t o t h e l o g a r i t h m o f t h e molar volume 0 Experimental d e s i g n o r MW o f the s o l u t e components, i . e . , log(MW) = f ( V e ) . and e s t a b l i s h i n g o p e r a t i n g c o n d i t i o n s t o o p t i m i z e s e p a r a t i o n s , i s g e n e t - a l l y much l e s s o f a problem w i t h SEC than w i t h o t h e r types o f LC. L i q u i d s i z e e x c l u s i o n , s t e r i c e x c l u s i o n , and g e l permeation chromatography a r e synonymous terms f o r SEC and a r e f r e q u e n t l y encountered i n l i t e r a t u r e due t o c o n v e n t i o n o r p e r s o n a l p r e f e r e n c e .

Another term, g e l

f i l t r a t i o n chromatography, f r e q u e n t l y i s a p p l i e d t o SEC s e p a r a t i o n s of b i o l o g i c a l molecules i n aqueous media u s i n g columns packed w i t h s o f t o r semi-rigid gel p a r t i c l e s . P r e p a r a t i v e SEC i s any a p p l i c a t i o n o f SEC which s p e c i f i c a l l y i n v o l v e s t h e chromatographic i s o l a t i o n o f one or more components ( o r f r a c t i o n s ) f r o m a m i x t u r e f o r purposes o f i d e n t i f i c a t i o n , p u r i f i c a t i o n , o r use i n o t h e r t y p e s of studies.

P r e p a r a t i v e SEC techniques may be d i v i d e d i n t o f o u r c a t e g o r i e s

determined by t h e s c a l e o f the s e p a r a t i o n o r f r a c t i o n s i z e -

(i)

Preparative-Analytical:

Standard a n a l y t i c a l i n s t r u m e n t a t i o n

and a n a l y t i c a l SEC columns ( 1 0 t o 50 cm l e n g t h and 0.5 t o 0.8 cm i n t e r n a l d i a m e t e r , I D ) a r e employed t o o p t i m i z e resolution.

S i n g l e or m u l t i p l e i n j e c t i o n s w i t h sample l o a d s

o f 5 t o 100 mg and i n j e c t i o n volumes o f 0.1 to 2 mL a r e g e n e r a l l y made t o a c q u i r e f r a c t i o n s o f s u f f i c i e n t s i z e ( 1 0 pg t o 10 mg) f o r s p e c t r o s c o p i c or o t h e r t y p e s o f chromatographic analyses.

F r a c t i o n c o l l e c t i o n i s u s u a l l y done m a n u a l l y .

291

(ii)

Semi-preparative: An analytical system as described above (i) is automated for multiple sample injection and fraction collection and/or operated at a higher mobile phase flow rate using an SEC column with a larger diameter (0.8 to 2 cm ID) than those ordinarily used for analytical separations. Larger injection volumes (0.1 to 10 mL) and sample loads (10 mg to 0.5 g) are used with larger diameter columns to obtain fractions weighing 10 mg to 0.2 g. Semi-preparative fractionations are often required to more fully identify the structures of isolated components, to purify major components for use as calibration standards and to clean-up samples in preparation for other types of analyses.

(iii)

Standard-Preparative: Large columns (length > 50 cm and 2 to 6 cm ID) with relatively low cost packing material (30 to 60 pm particle size) and special instrumentation to handle large amounts of mobile phase, inject large volumes (10 to 100mL), operate at high flow rates (10 to 140 mL/min), monitor solute concentrations and collect fractions are required. Single o r multiple injections with sample loadings ranging from 0.5 to 20 grams and fraction volumes of 10 to 200 mL (0.2 t o 2 g) are typical. Standard-preparative scale fractionations are run to clean-up or purify material, to obtain polymer fractions having different average M W ' s and narrow MWD's for structure-property studies and use as MW standards, and to isolate reaction products or intermediates for further study. Since standard-preparative scale operations are labor intensive and require large quantities of mobile phase, cost and safety factors deserve special attention.

(iv)

Large-Scale Preparative: In addition to special equipment described in ( i i i ) , automated operation with multiple sample injection, fraction collection, and solvent recovery capabilities are recommended. Cost, reliability, health and safety become of paramount. importance as the size of the operation increases. Large-scale separations usually involve the isolation of fractions in the range of 5 to several

292

h u n d r e d grams and a p p l i c a t i o n s i n c l u d e p o l y m e r f r a c t i o n a t i o n t o o b t a i n n a r r o w MWD samples f o r p r o p e r t y and p r o c e s s i n g s t u d i e s , p r o d u c i n g s t a n d a r d s , i s o l a t i o n of i n t e r m e d i a t e r e a c t i o n p r o d u c t s i n o r g a n i c s y n t h e s i s , a n d p r o d u c t i o n r u n s t o c l e a n - u p or p u r i f y l a r g e amounts o f a m a t e r i a l f o r r e s e a r c h or commerce. Column s i z e i s t h e p r i m a r y f a c t o r a f f e c t i n g t h e s c a l e o f p r e p a r a t i v e separations.

A s t h e i n t e r n a l d i a m e t e r o f a c o l u m n i s i n c r e a s e d , t h e sample

s i z e , i n j e c t i o n volume, and s o l v e n t flow r a t e may be i n c r e a s e d .

For

p r e p a r a t i v e separations i t i s u s u a l l y most d e s i r a b l e t o o p e r a t e t h e system a t t h e h i g h e s t sample l o a d i n g and flow r a t e p o s s i b l e w i t h o u t undue l o s s o f separation e f f i c i e n c y .

I n d e e d f o r w e l l - p a c k e d columns o f i d e n t i c a l l e n g t h ,

s e p a r a t i o n e f f i c i e n c y a s d e t e r m i n e d b y p l a t e c o u n t measurements o f t e n i m p r o v e s w i t h i n c r e a s i n g c r o s s - s e c t i o n a l a r e a , a s l o n g as a r e a s o n a b l e flow r a t e i s m a i n t a n e d and t h e columns a r e not o v e r l o a d e d w i t h s a m p l e .

I n general, both

samp 1 e s i z e and flow r a t e may b e i n c r e a s e d i n p r o p o r t i o n t o a c o l u m n ' s c r o s s - e c t i o n a l a r e a , r e g a r d l e s s o f t h e t y p e or s i z e o f t h e p a c k i n g p a r t i c es.

Because o f t h e h i g h c o s t of s m a l l d i a m e t e r

(
2 0 pm), rigid packing materials. Special procedures are usually necessary for packing semi-rigid and soft gel particles (see ref. 2, chapter 6.3). The same types o f packing materials used for analytical SEC may also be applied to preparative SEC. However, since speed and resolution are not as critical and because of cost, larger diameter particles (37 - 7 5 pm) are more commonly used for preparative SEC. The packings are available over a broad range of porosities from a number of manufacturers (Table 8 . 2 ) . (Table 8 . 2 should not be considered a complete listing as manufacturers and products are continually increasing.) The separation range and limitations of the packing

304

Table 8.2 Preparative S E C Packing Materials Suppl i er

Type Cross-linked, styrenedivinylbenzene copolymer gel (37 - 7 5 pm) Styragel

Waters (USA)

Porous silica beads Porasi 1

Waters (USA)

Spherosi 1

Gel Type SI

V i t-X

Corning Glass

Chrompack (Holland) Rhone-Poulenc (France) RSL (Belgium) S u p e l c o (USA) E. Merck (Germany, GFR)

Perkin-Elmer (USA)

Comments U s e o n l y with selected o r g a n i c solvents; not t o be used with acetone, alcohols o r aqueous solvents o r at pressures exceeding 600 psi

A v a i l a b l e a s untreated o r chemically deactivated, spherical particles ( 3 7 - 75 pm). Untreated spherical particles ( < 40 pm).

Untreated, irregularshaped particles.

Chemically deactivated, irregular-shaped particles (36 - 44 pm). Electro-Nucleonics (USA) Untreated o r glycerylmodified irregularshaped particles (37 14 vm).

305

TABLE 8.2 (Continued)

Type Polyacrylamide gel (acrylamide cross-linked with N,N'-methylene-bisacrylamide). Bio-Gel P2

Polysaccharide beads (Dextrans cross-linked with epichlorohydrin Sephadex G-25

Sephadex LH-20

Sulfonated, cross-linked styrene-divinylbenzene copolymer gel. Hydrogel

Suppl i er

Bio-Rad Labs (USA)

Comments

~

Neutral, hydrophilic gel particles (37 - 75 pm) for use with aqueous solvents; not recommended for use at pressures exceeding 100-1 50ps i

Relatively soft, gel particles (10 - 30 pin) for use in aqueous Pharmacia Fine Chemicals solvents, not recommended (USA) for use at pressures exceeding 150-200 p s i . A hydroxyl-propylated derivative o f Sephadex G-25 for use with pol-lr organic solvents; semirigid gel particles (25 100 pin), not recommended for use at pressures exceeding 150-200 psi.

Waters (USA)

Gel particles ( < 37 pm) for use with aqueous solvent pH 7 .

306 TABLE 8 . 2 ( C o n t i n u e d )

S u p p l ie r

Type

Comments

V in y 1a c e t a t e c o p o l yme r s

or EM g e l

.

~

Relatively soft, gel

E. Merck (Germany, FRG)

OR-PVA p a r t i c l e s (30 -

63 pm) f o r use w i t h a c e t o n e , a1 c o h o 1 s and other organic solvents; n o t recommended for u s e a t pressures exceeding 100-150 p s i .

m a t e r i a l s s h o u l d be c a r e f u l l y c o n s i d e r e d when e s t a b l i s h i n g o p e r a t i n g parameters for p r e p a r a t i v e separations.

To r e d u c e c o s t s and b r o a d e n t h e r a n g e

for s i z e s e p a r a t i o n , a s i n g l e p r e p a r a t i v e c o l u m n may be p r e p a r e d w i t h t h e packing m a t e r i a l arranged i n l a y e r s of d i f f e r e n t p o r o s i t i e s .

8.3.7

Monitor

The m o n i t o r i s a d e t e c t o r - r e c o r d e r s y s t e m f o r a n a l y z i n g t h e e l u a t e .

The

d e t e c t o r may be c o n n e c t e d e i t h e r i n s e r i e s or p a r a l l e l w i t h t h e c o l u m n o u t l e t and t h e r e c o r d e r c o n t i n u o u s l y d i s p l a y s d e t e c t o r r e s p o n s e v e r s u s e l u t i o n t i m e

or v o l u m e .

The same t y p e s o f d e t e c t o r s and s t r i p - t y p e r e c o r d e r s u s e d f o r

a n a l y t i c a l LC may a l s o be a p p l i e d t o m o n i t o r t h e e l u a t e i n p r e p a r a t i v e SEC. However, such d e t e c t o r s u s u a l l y r e q u i r e m o d i f i c a t i o n and t h e h i g h s e n s i t i v i t y n e c e s s a r y f o r a n a l y t i c a l work i s n e i t h e r r e q u i r e d n o r a l w a y s d e s i r a b l e f o r p r e p a r a t i v e SEC because o f t h e h i g h - v o l u m e flow r a t e s and h i g h sample concentrations. The p u r p o s e o f a m o n i t o r i s t o i n d i c a t e t h e r e s o l u t i o n and r e l a t i v e c o n c e n t r a t i o n s o f f r a c t i o n a t e d sample components and t o a s s i s t a n o p e r a t o r i n d e c i d i n g when or how f r e q u e n t l y t o c o l l e c t f r a c t i o n s .

I n p r a c t i c e , the

m o n i t o r a l e r t s an o p e r a t o r t o a l m o s t any p r o b l e m s o r m a l f u n c t i o n s t h a t d e v e l o p i n t h e p r e p a r a t i v e system by i n d i c a t i n g s h i f t s i n r e t e n t i o n times ( e l u t i o n v o l u m e s ) or o t h e r changes i n t h e chromatogram.

307 The d i f f e r e n t i a l r e f r a c t i v e i n d e x ( R I ) d e t e c t o r has t h e w i d e s t a p p l i c a b i l i t y a n d i s an i d e a l m o n i t o r for SEC where t h e c o m p o s i t i o n of t h e m o b i l e phase i s h e l d c o n s t a n t .

D e t e c t i n g components h a v i n g s m a l l r e f r a c t i v e

i n d e x i n c r e m e n t s ( w i t h R I v a l u e s n e a r t h a t o f t h e m o b i l e phase s o l v e n t ) or i n

low c o n c e n t r a t i o n s a r e t h e m a i n l i m i t a t i o n s o f R I d e t e c t o r s .

Small

f l u c t u a t i o n s i n t h e composition o f m o b i l e phases c o n t a i n i n g s o l v e n t m i x t u r e s and changes i n p r e s s u r e a n d t e m p e r a t u r e may a l s o cause d i f f i c u l t i e s i n R I detection. UV d e t e c t o r s w i t h s p e c i a l s h o r t p a t h l e n g t h ( 1 mm) c e l l s f o r p r e p a r a t i v e LC W a v e l e n g t h ( X ) s e l e c t a b l e or s c a n n i n g U V - v i s i b l e

are commercially available.

d e t e c t o r s a r e p a r t i c u l a r l y u s e f u l for m o n i t o r i n g s p e c i f i c t y p e s o f compounds w h i c h have a b s o r p t i o n maxima i n d i f f e r e n t s p e c t r a l r e g i o n s ( e . g . , compounds a t 'k > 300 nm).

colored

I n d e e d such d e t e c t o r s may be a d j u s t e d t o m o n i t o r a t

a w a v e l e n g t h d i f f e r e n t from t h a t o f t h e s o l u t e a b s o r p t i o n maxima t o d e c r e a s e s e n s i t i v i t y and t h e r e f o r e r e d u c e t h e l i k e l i h o o d o f o v e r l o a d i n g t h e d e t e c t o r ' s output signal. D e t e c t o r s s h o u l d be c o n s t r u c t e d of l a r g e b o r e (0.75 mm or l a r g e r I D ) t u b i n g

t o r e d u c e b a c k p r e s s u r e s g e n e r a t e d b y h i g h - v o l u m e f low r a t e s .

Although such

m o d i f i e d R I and UV d e t e c t o r s may be o b t a i n e d c o m m e r c i a l l y , t h e r e i s a n e e d t o i m p r o v e d e t e c t o r c e l l d e s i g n t o accommodate h i g h flow r a t e w i t h o u t c r e a t i n g large pressure d i f f e r e n t i a l s .

An a l t e r n a t e a p p r o a c h i s t o u s e a s t r e a m

s p l i t t e r between t h e column e x i t and t h e f r a c t i o n c o l l e c t o r t o d i r e c t a s m a l l f r a c t i o n o f t h e e l u a t e t o an a n a l y t i c a l d e t e c t o r .

I f such an a p p r o a c h i s

used, p r e c a u t i o n s a r e n e c e s s a r y t o p r o t e c t t h e c e l l from h i g h p r e s s u r e d i f f e r e n t i a l s and t o be c e r t a i n t h a t t h e r e l a t i v e flow r a t e or f r a c t i o n o f eluate being delivered to the detector i s constant.

Also t h e s y s t e m s h o u l d be

designed to ensure t h a t t h e composition of the e l u a t e e n t e r i n g the d e t e c t o r i s i d e n t i c a l t o t h a t b e i n g d e l i v e r e d t o t h e syphon or f r a c t i o n c o l l e c t o r .

8 . 3 . 8 Fraction Collection and Recovery Manual f r a c t i o n c o l l e c t i o n i s o f t e n s u f f i c i e n t f o r s i n g l e r u n f r a c t i o n a t i o n s and f o r i s o l a t i n g one or o n l y a f e w components.

However f o r

r e p e t i t i v e r u n s and when many f r a c t i o n s need t o be i s o l a t e d , f r a c t i o n c o l l e c t i o n s h o u l d be a u t o m a t e d .

S i n c e s m a l l s h i f t s i n flow r a t e h a v e a

s i g n i f i c a n t e f f e c t o n t h e r e t e n t i o n t i m e s o f SEC s e p a r a t e d components, t h e u s e

308 o f a syphon or some other volume-measuring device is highly recommended for

multiple run fractionations. Syphons frequently are employed with reel-type fraction collectors in custom-designed systems. The volume of the fraction depends upon the capacity of the syphon. The eluate feeds into the syphon which when filled empties into a container o n the fraction collector. Emptying o f the syphon then triggers a delayed-response signal for the reel to rotate and move another container into position to collect the next fraction. The Ana-Prep instrument used a photocell detector to signal the collection o f a pre-determined fraction volume from a holding chamber and a 40-port valve to distribute successive fractions to specified containers. The volume of the fraction to be collected depends on the type o r purpose of the fractionation and upon the characteristics of the column(s). In general, the maximum volume of eluate collected per run is equal approximately to the total liquid volume contained within the pores of the SEC packing; i.e., Vm-Vo. This volume also represents the maximum total dilution of the injected sample. For sample clean-up operations to remove either high or low MW contaminants, it may be desirable to collect a single, large fraction o f the sample eluting slightly after Vo or just before Vm. On the other hand, small fraction volumes may be required to isolate minor sample components in high purity. Regardless, it is advisable t o make a trial run and examine the fractions by analytical SEC before beginning a multi-run fractionation. Depending on the nature o f the solvent and solute, a number o f techniques may be applied for recovering solute fractions. For volatile solvents, solvent evaporation is the simplest and most direct method. Care should be taken not to overheat or cause fractions to become contaminated (e.g., with condensed water) during recovery. Large volumes of solvent can be efficiently and safely removed with a vacuum rotary evaporator. Freeze drying is an excellent technique for removing certain solvents (e.g., water, benzene, trichlorobenzene and dioxane) and converting solute fractions into a less dense form for easier handling. Fractions containing solvent additives (e.9.. antioxidant in THF) may need to be washed, re-dissolved and precipitated several times before they are pure. Also, solute fractions which are difficult to precipitate or tend to complex with the mobile phase solvent can be isolated by solvent-solvent extraction using separatory funnels. Solutes which crystallize can often be recovered after partial removal of the solvent by adding a nonsolvent or by lowering the temperature.

309 8 . 3 . 9 Refractionation and Recycling S o l u t e f r a c t i o n s o b t a i n e d b y p r e p a r a t i v e SEC may be r e f r a c t i o n a t e d t o improve t h e i r p u r i t y .

Oftentimes b e t t e r r e s u l t s are achieved by f i r s t

s e p a r a t i n g a sample i n t o r e l a t i v e l y l a r g e volume f r a c t i o n s a n d r e f r a c t i o n a t i n g each f r a c t i o n t h a n b y r u n n i n g a s i n g l e p r e p a r a t i v e s e p a r a t i o n a n d c o l l e c t i n g s m a l l volume f r a c t i o n s .

T h i s a p p r o a c h has been s u c c e s s f u l l y u s e d t o o b t a i n

n a r r o w MWD p o l y m e r f r a c t i o n s E8.91.

B r o a d MWD p o l y m e r s can b e s e p a r a t e d i n t o

h i g h , medium a n d low MW f r a c t i o n s w i t h n a r r o w e r MWD's t h a n t h e o r i g i n a l sample.

Upon r e f r a c t i o n a t i o n . d i f f e r e n t c o l u m n ( s ) and o p e r a t i n g p a r a m e t e r s

may t h e n be s e l e c t e d , i f n e c e s s a r y , t o o p t i m i z e t h e r e s o l u t i o n o f e a c h fraction. R e s o l u t i o n i n p r e p a r a t i v e SEC may a l s o be i m p r o v e d b y r e c y c l i n g t h e e l u a t e as i t l e a v e s t h e c o l u m n ( s ) t o t h e pump and p u m p i n g i t b a c k t h r o u g h t h e column(s).

T h i s p r o c e s s i n c r e a s e s t h e e f f e c t i v e column l e n g t h and, i n

p r i n c i p l e , may be r e p e a t e d u n t i l t h e sample b e i n g s e p a r a t e d o c c u p i e s t h e t o t a l R e c y c l i n g r e d u c e s t h e c o l u m n l e n g t h (amount o f p a c k i n g

volume o f t h e c o l u m n .

m a t e r i a l ) or number o f columns and t h e r e f o r e t h e c o s t r e q u i r e d t o a c h i e v e a p a r t icu a r s e p a r a t i o n . s o l v e n t i s needed.

Also, t h e r e i s n o r e - h a n d l i n g o f t h e s o l u t e and l e s s

By d e c r e a s i n g t h e column l e n g t h , t h e c o l u m n b a c k p r e s s u r e

i s lowe ed w h i c h s h o u l d enhance c o l u m n l o n g e v i t y and e n a b l e t h e u s e o f h i g h e r

flow r a es t o f u r t h e r r e d u c e o p e r a t i n g expenses and s e p a r a t i o n t i m e s . Band b r o a d e n i n g due t o i n s t r u m e n t a l f a c t o r s l i m i t s t h e number o f t i m e s a sample may be r e c y c l e d .

A l s o , a sample may have such a b r o a d d i s t r i b u t i o n

t h a t t h e e n t i r e p o r e volume i s o c c u p i e d d u r i n g t h e i n i t i a l c y c l e a n d l i t t l e improvement i n r e s o l u t i o n i s r e a l i z e d b y r e c y c l i n g .

I n s t r u m e n t band

b r o a d e n i n g i s a t t r i b u t e d t o d i f f u s i o n a n d a r i s e s from a v a r i e t y o f e f f e c t s . I n r e c y c l i n g , dead volume i n t h e pump and c o n n e c t i n g t u b i n g a n d f i t t i n g s a r e major sources of broadening.

C h r o m a t o g r a p h i c r e s o l u t i o n i s a l s o l o s t due t o

p u l s a t i o n s and m i x i n g as t h e e l u a t e p a s s e s t h r o u g h t h e pump.

Indeed the

d e s i g n o f t h e pump i s p e r h a p s t h e m o s t c r i t i c a l f a c t o r t o be c o n s i d e r e d i n a recycling operation.

As m e n t i o n e d i n s e c t i o n 8.3.4, p i s t o n - r e c i p r o c a t i n g

pumps a r e recommended.

The d u a l - h e a d r e c i p r o c a t i n g pump may e l i m i n a t e t h e

need f o r a p u l s e damper between t h e pump and c o l u m n and t h e r e f o r e f u r t h e r r e d u c e t h e dead v o l u m e .

However, i t i s i m p e r a t i v e t h a t t h e d i s p l a c e m e n t

volumes o f t h e p i s t o n s and check v a l v e s be k e p t a t a minimum a n d m i x i n g

310 chambers be e l i m i n a t e d i n t h e c o n s t r u c t i o n o f t h e pump.

Also, the pistons

s h o u l d be " t u n e d " t o d e l i v e r e q u a l v o l u m e s , a n d l e n g t h s o f t u b n g c o n n e c t i n g each p i s t o n chamber t o t h e i n l e t and o u t l e t o f t h e pump s h o u l d be i d e n t i c a l t o reduce mixing e f f e c t s .

I f a l o o p i n j e c t o r i s used, t h e i n j e c t v a l v e must b e

r e t u r n e d t o t h e l o a d p o s i t i o n a f t e r a sample i s i n j e c t e d a n d t h e l e n g t h o f bypass t u b i n g m u s t be as s h o r t as p o s s i b l e t o p r e v e n t band b r o a d e n i n g . R e c y c l i n g samples w i t h b r o a d d i s t r i b u t i o n s may be f a c i l i t a t e d b y s e l e c t i v e l y r e m o v i n g p o r t i o n s o f t h e e l u a t e a n d a d d i n g make u p s o l v e n t t o t h e system.

T h i s can be a c c o m p l i s h e d b y a s p e c i a l s w i t c h i n g v a l v e .

I n t h e case

o f p o l y m e r s , t a i l i n g h i g h MW a n d low MW f r a c t i o n s n e a r Vo a n d Vm, r e s p e c t i v e l y , may be removed i n t h i s way t o p e r m i t f u r t h e r r e c y c l i n g and b e t t e r r e s o l u t i o n o f i n t e r m e d i a t e MW f r a c t i o n s . Since r e c y c l i n g d i l u t e s s o l u t e f r a c t i o n concentrations, t h e s i z e of t h e f r a c t i o n volume grows as t h e number o f c y c l e s i s i n c r e a s e d . t i m e and e q u i p m e n t may be needed t o r e c o v e r t h e f r a c t i o n s .

Hence, a d d i t i o n a l Also, f r a c t i o n

c o l l e c t i o n becomes more c o m p l i c a t e d , e s p e c i a l l y when f r a c t i o n s m u s t be s e l e c t i v e l y removed a t c e r t a i n s t a g e s d u r i n g t h e r e c y c l i n g p r o c e s s . A u t o m a t i o n i s more d i f f i c u l t w i t h a d d i t i o n a l s w i t c h i n g v a l v e s t o c o n t r o l ; a n d when m u l t i p l e i n j e c t i o n s a r e made, r e p r o d u c i b l e e l u t i o n volumes ( t i m e s ) for s o l u t e f r a c t i o n s are an absolute necessity. The b e n e f i t s and p r o b l e m s i n t r o d u c e d must be c a r e f u l l y w e i g h e d when considering recycling. standard-preparative

I n h i s t h e s i s , Wang C211 d e s c r i b e s a

s c a l e r e c y c l e SEC a p p a r a t u s f o r p o l y m e r f r a c t i o n a t i o n .

He c o n c l u d e s t h a t t h e method o f f e r s a d v a n t a g e s i n o b t a i n i n g r e l a t i v e l y narrow MWD f r a c t i o n s b u t t h a t t h e e x p e r i m e n t a l d i f f i c u l t i e s a r e s i g n i f i c a n t .

He

s t a t e s t h a t i t s h o u l d be p o s s i b l e t o d e s i g n and c o n s t r u c t a n a u t o m a t e d a p p a r a t u s t h a t overcomes many o f t h e d i f f i c u l t i e s , b u t has r e s e r v a t i o n s t h a t t h e s m a l l amount o f a g i v e n MWD p o l y m e r f r a c t i o n o b t a i n e d b y r e c y c l i n g i s w o r t h t h e c o s t a n d e f f o r t compared t o u s i n g a s c a l e - u p ( l a r g e r c o l u m n ) p r e p a r a t i v e SEC method t o o b t a i n t h e same f r a c t i o n . On t h e o t h e r hand, Lesec and Q u i v o r o n [ 1 9 1 a r e h i g h l y s u c c e s s f u l i n a p p l y i n g r e c y c l i n g f o r t h e p r e p a r a t i v e SEC o f low MW compounds a n d c i t e low s o l v e n t c o n s u m p t i o n a n d . l o n g column l i f e as added a d v a n t a g e s . t h e i r a p p a r a t u s i s shown i s F i g . 8 . 2 . .

A diagram of

The s o l v e n t de1iver.y s y s t e m m e n t i o n e d

311

i n s e c t i o n 8 . 3 . 4 c o n s i s t s o f a low dead volume, d i a p h r a g m pump w i t h t h r e e heads s e t 120° o u t o f phase.

The sample i n j e c t o r i s p o s i t i o n e d i n p a r a l l e l t o

t h e s o l v e n t r e s e r v o i r between t h e pump and a s i x p o r t v a l v e f o r r e c y c l i n g .

In

the r e c y c l e p o s i t i o n , the r e c y c l e v a l v e channels the e l u a t e through the i n j e c t o r i n t o t h e pump; and i n t h e c o l l e c t p o s i t i o n t h e e l u a t e i s d i r e c t e d t o a fraction collector. 8 . 3 . 1 0 T e m p e r a t u r e Control

The i n j e c t o r , c o l u m n ( s ) and o t h e r p r e p a r a t i v e SEC components d i r e c t l y a f f e c t i n g sample s e p a r a t i o n s h o u l d be m a i n t a i n e d a t a c o n s t a n t t e m p e r a t u r e . The o p e r a t i n g t e m p e r a t u r e depends m a i n l y upon t h e s o l u b i l i t y c h a r a c t e r i s t i c s , v i s c o s i t y and v o l a t i l i t y o f t h e m o b i l e phase.

A h i g h t e m p e r a t u r e may be

r e q u i r e d t o r e n d e r samples s o l u b l e or t o r e d u c e t h e v i s c o s i t y o f t h e m o b i l e phase.

A l t e r n a t i v e l y , SEC components such as t h e pump, m o n i t o r a n d f r a c t i o n

c o l l e c t o r may n o t be d e s i g n e d f o r e l e v a t e d t e m p e r a t u r e

(>

6OoC) o p e r a t i o n .

Also, v o l a t i l i t y a n d s a f e t y c o n s i d e r a t i o n s may l i m i t t h e u s e o f c e r t a i n s o l v e n t s t o moderate temperatures

(

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