Selective Sample Handling and Detection in High-Performance Liquid Chromatography, Part B (Journal of Chromatography Library)

JOURNAL OF CHROMATOGRAPHY LIBRARY-

volume 396

selective sample handling and detection in high-performance liquid chromatography part B

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JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 3 9 B

selective sample handling and detection in high-performance liquid chromatography partB

edited by K. Zech Byk Gulden Pharmaceuticals, Byk Guldenstrasse2, P. 0. Box 6500,

7750 Konstanz, F.R. G. and

R. W. Frei

'

Department of Analytical Chemistry, Free University, De Boelelaan 1083, 108 1 HV Amsterdam, The Netherlands

ELSEVIER Amsterdam - Oxford - N e w York - Tokyo 1989

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 2 1 1, 1000 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.

ISBN 0-444-88327-4 (Vol. 398) ISBN 0-444-41 6 16- 1 (Series)

0 Elsevier Science Publishers B.V., 1989 All rights reserved. No part of this publication may 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 permission of the publisher, Elsevier Science Publishers B.V./ Physical Sciences & Engineering Division, P.O. Box 330, 1000 AH Amsterdam, The Netherlands. Special regulations for 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 may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred t o the wblisher. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. 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

CONTENTS

L i s t o f Contributors

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

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

Preface

XI

1

PRECONCENTRATION AND CHROMATOGRAPHY ON CHEMICALLY MODIFIED

CHAPTER I

SILICAS WITH COMPLEXATION PROPERTIES

.

1

Introduction 1.1

1.2 2

.

1.3

4

.

.

2.2

5

.......

s u p p o r t ... .............. ................

8

Preconcentration using l i q u i d - s o l i d e x t r a c t i o n i q u i d - s o l i d ex r a c t i o n

.........

S t a t i o n a r y phases f o r Choice o f a

5 6

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

Chemically m o d i f i e d s i l i c a s 2.1

3

Bagnoud and W . H a e r d i ) ................. ...............................................

(J.L. Veuthey. M.A.

S u r f a c e m o d i f i c a t i o n .. .............. ................ C r o s s - l i n k e d f u n c t i o n a groups t o s i l cas ............

6

8 9 10

P r e c o n c e n t r a t i o n o f i n o r g a n i c compounds u s i n g c h e l a t i n g s i 1i c a s

11

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

12

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

3.1

Metal preconcentration

3.2

Advantages and disadvantages o f c h e l a t i n g s i l i c a s i n

3.3

............................... p r e c o n c e n t r a t e d m e t a l s .............. .....

preconcentrating metals

16

Analysis o f

16

The p r e c o n c e n t r a t i o n o f o r g a n i c compounds u s i n g c h e l a ing

..... ............................................. 5 . C h e l a t i n g s i l i c a s i n chromatography .................. ..... 5.1 M e t a l s e p a r a t i o n chromatography ................. ..... 5.2 Ligand exchange chromatography ........................ 5.3 Enantiomer s e p a r a t i o n ................................ 6 . Conclusion ................................................ References ..................................................... s i 1i c a s

18 21 21 23 27 29 29

CHAPTER I 1 SAMPLE HANDLING I N I O N CHROMATOGRAPHY (P.R. Haddad)

. 2. 1

Introduction

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

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

.......................... 2.1 Sample c o l l e c t i o n ..................................... 2.2 E x t r a c t i o n methods .................................... 2.3 Sample d i g e s t i o n ...................................... 2.4 Combustion methods .................................... 3 . Sample cleanup methods ..................................... Sample c o l l e c t i o n and d i s s o l u t i o n

33 33

37 37 38 39 40 42

VI

3.1 3.2 4

.

Introduction Filtration

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

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

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

3.3 Chemical m o d i f i c a t i o n o f t h e sample Contamination e f f e c t s 4.1 I n t r o d u c t i o n 4.2 Contamination from p h y s i c a l h a n d l i n g o f t h e sample ... 4.3 Contamination from f i l t r a t i o n devices and c a r t r i d g e columns 4.4 Contamination from chromatographic hardware components 4.5 Contamination o f t h e column

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

. .

53 54

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

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

79 79

Introduction

........................... a n a l y s i s ...................

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

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

6

44 53

55 56 59 61 61 62 62 74 76 76 76 77

5 . Sample handling f o r u l t r a - t r a c e 5.1

42 44

5.2 Use o f l a r g e i n j e c t i o n volumes 5.3 Use o f p r e c o n c e n t r a t i o n columns 5.4 Use of d i a l y t i c p r e c o n c e n t r a t i o n methods M a t r i x e l i m i n a t i o n methods

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

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

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

6.1

Introduction

6.2 6.3

On-column m a t r i x e l i m i n a t i o n

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

Post-column m a t r i x e l i m i n a t i o n

7 Conclusion References

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

CHAPTER 111 WHOLE BLOOD SAMPLE CLEAN-UP FOR CHROMATOGRAPHIC ANALYSIS

.

1

2

.

.

(U C h r i s t i a n s and K.-Fr. Introduction

.

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

E x t r a c t i o n procedures f o r blood samples

2.1

3

Sewing)

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

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

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

85 85

2.2

Solid-liquid

93

2.3

Column-switching

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

96

Blood-sample p r e p a r a t i o n and HPLC a n a l y s i s o f SandimmunR

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

3.2 3.3

96 96

Blood sample p r e p a r a t i o n f o r SandimmunR ( c y c l o s p o r i n e ) measurement

.

82

Liquid-liquid extraction

(cyclosporine) 3.1 I n t r o d u c t i o n

4

82

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

106

Chromatographic a n a l y s i s o f SandimmunR ( c y c l o s p o r i n e ) and i t s m e t a b o l i t e s

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

115

Trouble shooting i n development o f blood-sample clean-up

................................................. References ..................................................... procedures

126 127

VII CHAPTER I V

RADIO-COLUMN LIQUID CHROMATOGRAPHY (A.C. Veltkamp)

............................................ 1. I n t r o d u c t i o n ............................................... 1.1 The use o f r a d i o i s o t o p e s i n chemical a n a l y s i s ......... 2

.

1.2

C h a r a c t e r i s t i c s o f radio-column l i q u i d chromatography

134 136

1.3

O b j e c t i v e s and o u t l i n e

141

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

P r i n c i p l e s o f s c i n t i 1 l a t i o n count ing chromatography

2.1

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

n column l i q u i d

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

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

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

P r i n c i p l e s o f flow-through y.counting

2.3

P r i n c i p l e s o f R-counting i n column l i q u i d

2.4

chromatography Data a n a l y s i s

2.5

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

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

............................................. 3 . O p t i m i z a t i o n parameters f o r flow-through &counting ........

.

Introduction

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

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

3.2

The c o u n t i n g e f f i c i e n c y E; general aspects

3.3

The background count r a t e CPM(b); general aspects

3.4

.....

...................................... ....................................... ............................................... Preparation. p u r i f i c a t i o n . i d e n t i f i c a t i o n ............. D i s t r i b u t i o n and metabol sm of exogenic compounds .....

Other parameters

3.5 Special methods Applications 4.1 4.2

141 145 150 157

Commercially a v a i l a b l e flow-through r a d i o a c t i v i t y

detectors

3.1

141

I n t r o d u c t o r y comments on n u c l e a r r a d i a t i o n and detection

2.2

4

133 134

4.3

D i s t r i b u t i o n and metabol sm o f endogenic compounds

4.4

Radioassays

............. ............................. 4.5 Miscellaneous ........... ............................. 5 . Concluding remarks ........... ............................. References .....................................................

....

161 162 162 162 165 168 170 177 177 186 188 189 197 200 202

MODERN POST-COLUMN REACTION DETECTION I N HIGH-PERFORMANCE

CHAPTER V

LIQUID CHROMATOGRAPHY

. .

(H

. Jansen

and R.W.

Frei)

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

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

1

General i n t r o d u c t i o n

2

Types o f post-column r e a c t o r s

208 208 211

2.2

.............................. Open t u b u l a r r e a c t o r s ................................. Packed bed r e a c t o r s ...................................

212

2.3

Segmented stream t u b u l a r r e a c t o r s

212

2.1

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

211

VIII 3 5

5.1

................................ ............. ........... I n t r o d u c t i o n ..........................................

5.2

The use o f i m m o b i l i z e d enzymes i n post-column

.

Choice o f r e a c t i o n d e t e c t o r

213

.

A p p l i c a t i o n s o f post-column r e a c t i o n d e t e c t i o n New approaches t o post-column r e a c t i o n d e t e c t i o n

214 216 216

.

4

.............................................. O t h e r s o l i d - p h a s e c h e m i s t r i e s .........................

216

The use o f e l e c t r o c h e m i c a l reagent p r o d u c t i o n

241

reactors

5.3 5.4

5.5

The use o f photochemical and thermo i n i t i a t e d reactions

5.6

Miniaturization

.........

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

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

5.7 H o l l o w f i b e r s as post-column r e a c t o r s 6 Conclusions References

.

CHAPTER V I

.

1

.

2

.

242 245 252 254 255

NEW LUMINESCENCE DETECTION TECHNIQUES

.

V e l t h o r s t and R.W. F r e i ) ................. ...............................................

(C G o o i j e r . N.H.

260

Introduction

260

...... .......................... i n CL ............................

Chemiluminescence d e t e c t i o n w i t h s o l i d s t a t e r e a c t o r s

264

2.1

D e t e c t i o n based on CL and BL

264

2.2 2.3 2.4

Solid state reactors H 0 d e t e c t i o n by p e r o x y o x a l a t e CL 2 2 Use o f t h e s o l i d TCPO r e a c t o r f o r d e t e c t i o n of

2.5

Quenched peroxyoxal a t e chemi luminescence d e t e c t i o n

2.6

Concluding remarks

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

f 1uorophores

3

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

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

232

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

267

....

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

L i q u i d phase phosphorescence d e t e c t i o n 3.1 Fundamental aspects o f phosphorescence 3.2 3.3

New developments i n phosphorimetry Experimental a s p e c t s

3.4

I n d i r e c t phophorescence d e t e c t i o n

266

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

.................................. ..................... 3.5 A l t e r n a t i v e phosphorophores/luminophores .............. 3.6 Concluding remarks .................................... References .....................................................

283 285 295 296 296 300 304 308 324 328 329

CHAPTER V I I CONTINUOUS SEPARATION TECHNIQUES I N FLOW-INJECTION ANALYSIS

.

1 2

.

(M

. V a l c a r c e l and M . D . Luque de Cast ro) ................... ...............................................

Introduction

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

335 335

Gas-liquid interfaces

338

2.1

338

Gas-diffusion

IX

2.2 Distillation .......................................... 2.3 Hydride generation 3 Gas-solid interfaces 4 . Liquid-liquid interfaces ................................... 4.1 Extraction ............................................ 4.2 Dialysis .............................................. 5 . Solid-1 iquid interfaces .................................... 5.1 I o n exchange 5.2 Adsorptive preconcentration 5.3 Precipitation and dissolution 6 HPLC-FIA association ....................................... 6.1 Pre-column assemblies 6.2 Post-column assemblies ................................ 7 Final remarks .............................................. References .....................................................

343 343 345 347 347 355 359 359 361 367 369 371 373 376 378

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

383

.

.

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

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

.

S u b j e c t Index

Journal o f Chromatography Library (other volumes in the series) ........

391

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XI

LIST OF CONTRIBUTORS

P rof. Dr. W. H aerd i, D r . J.L. Veuthey, D r . M.A. Bagnoud Department o f I n o r g a n i c , A n a l y t i c a l and A p p l i e d Chemistry U n i v e r s i t y o f Geneva 30 9.E. Ansermet 1211 Geneva 4 SWITZERLAND

P r o f . D r . R.W. F r e i D r . H. Jansen P h i l i p s L i g h t i n g b.v. P.O.Box 8 OD 20 5600 JM Eindhoven THE NETHERLANDS

A ssociat e P r o f . P.R. Haddad Department o f A n a l y t i c a l Chemistry U n i v e r s i t y o f New South Wales P.O. Box 1 Kensington N.S.W. 2033 AUSTRAL I A

P r o f . D r . N.H. V e l t h o r s t , D r . C. G o o i j e r , P r o f . Dr. R.W. F r e i Department o f General and A n a l y t i c a l Chemistry Free U n i v e r s i t y a t Amsterdam De B o e l elaan 1083 1081 HV Amsterdam THE NETHERLANDS

Prof. D r . K.-Fr. Sewing and D r . U. C h r i s t i a n s Medi z i n i s c h e Hochschul e Hannover Zentrum Pharmakologie und T o x i k o l o g i e A b t e i l u n g A1 lgemeine Pharmakologie P.O. Box 61 01 80 3000 Hannover F.R.G.

D r . M. V a l c h r c e l , M.D. Luque de C a s t r o Departamento de Q uimica A n a l i t i c a U n i v e r s i d a d de Cordoba F a c u l t a d de Ciencias Dr. M. V a l c a r c e l 14004 Cordoba SPAIN

D r . A.C. Veltkamp E C N S t i c h t ing Energieonderzoek Centrum Nederland Researc hcent rum P.O. Box 1 1755 ZG P e t t e n THE NETHERLANDS

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PREFACE On 29 January 1989,

d u r i n g t h e c o m p l e t i o n o f t h i s second volume,

P r o f e s s o r Roland F r e i d i e d a f t e r an i l l n e s s o f s e v e r a l months. Through h i s death I have l o s t a dear f r i e d ,

t o whom I am g r a t e f u l f o r many

s c i e n t i f i c s t i m u l i . Roland F r e i ' s c o n t r i b u t i o n s t o t h e f i e l d o f s e l e c t i v e sample h a n d l i n g and d e t e c t i o n i n HPLC w i l l be missed n o t o n l y by m y s e l f b u t a l s o by many c o l l e a g u e s w i t h s i m i l a r i n t e r e s t s . I t was f o r t u n a t e t h a t we shared an i n t e r e s t i n sample p r e p a r a t i o n , w i t h , emphasis

on

environmental

samples

and,

i n Amsterdam,

i n Constance,

on

the

biological

samples a r i s i n g from pharmaceutical r e s e a r c h . T h i s book i s t h e second and p r o v i s i o n a l l y l a s t p a r t o f a two-volume project.

It follows

t h e p r e v i o u s l y expressed view t h a t t h e h a n d l i n g ,

s e p a r a t i o n and d e t e c t i o n o f complex samples should b e c o n s i d e r e d as an i n t e g r a t e d , i n t e r c o n n e c t e d process. On t h e b a s i s o f t h i s p h i l o s o p h y we choose t h e c o n t r i b u t i o n s ,

which we hope w i l l

perhaps even more so t h a n i n P a r t A leads

to

a simplification

of

-

convince t h e reader

-

t h a t o p t i m a l sample p r e p a r a t i o n

detection o r

reduced

demands

on

the

s e p a r a t i o n process. The r e v e r s e o f t h i s i s a l s o shown i n d e t a i l . I n accordance w i t h t h e aims o f t h i s book, we have t r i e d once a g a i n t o p u t t h e emphasis on chemical p r i n c i p l e s and have, t h e r e f o r e , suppressed, as f a r as p o s s i b l e , a d i s c u s s i o n o f t h e equipment r e q u i r e d . T h i s r e f l e c t s o u r o p i n i o n t h a t t h e l i m i t i n g f a c t o r i n t h e a n a l y s i s o f complex samples i s incomplete knowledge o f t h e u n d e r l y i n g c h e m i s t r y r a t h e r t h a n

the

a v a i l a b l e hardware. T h i s l a c k o f knowledge i s becoming more e v i d e n t as t h e demands f o r l o w e r d e t e c t i o n l i m i t s grow, as r e s o l v i n g complex m a t r i x problems r e q u i r e s even more u n d e r s t a n d i n g o f t h e chemical

interaction

between t h e substance t o be analysed and t h e s t a t i o n a r y phase. Thus, a p a r t f r o m one c h a p t e r d e a l i n g w i t h c h e m i c a l l y m o d i f i e d s i l i c a s , t h e main theme o f t h i s book i s developed i n t h r e e c h a p t e r s on sample p r e p a r a t i o n and t h r e e on d e t e c t i o n . The

first

chapter

outlines

concentration

and

chromatography

c h e m i c a l l y m o d i f i e d s i l i c a s w i t h complexing p r o p e r t i e s .

on

Examples o f t h e

use o f t h e s e phases w i t h o r g a n i c and i n o r g a n i c compounds a r e g i v e n . Chapter I 1

is

the

first

o f t h r e e c o n t r i b u t i o n s d e a l i n g w i t h sample

2

preparation. following

I n particular,

critical

ion

questions

chromatography

regarding

sample

clearly

exposes

preparation.

Is

the the

prepared sample r e p r e s e n t a t i v e o f t h e m a t e r i a l t o be analysed? How can contamination be avoided, which r e s u l t s from t h e u b i q u i t o u s n a t u r e of t h e compound t o be analysed? What i s t h e b e s t s e p a r a t i o n procedure t o use i n order t o avoid t e d i o u s sample p r e p a r a t i o n ? I n Chapter I 1 1 t h e processing o f whole blood ( r a t h e r than plasma o r serum) f o r drug a n a l y s i s i s described.

The a n a l y s i s o f c y c l o s p o r i n e and

i t s m e t a b o l i t e s , an e s p e c i a l l y d i f f i c u l t case, demonstrates how comprehensive t h e o p t i m i s a t i o n o f sample p r e p a r a t i o n must be i n o r d e r t o successfully described.

perform t h e a n a l y s i s .

Several

other

examples

are

also

Chapter I V deals w i t h radio-column l i q u i d chromatography and i n troduces t h e o t h e r theme o f t h i s book: s e l e c t i v e d e t e c t i o n methods. The widespread use o f r a d i o i s o t o p e s r e q u i r e s a h i g h degree o f p u r i f i c a t i o n d u r i n g t h e manufacture o f t h e compounds, as w e l l as h i g h l y accurate d e t e c t i o n methods i n b i o l o g i c a l and biochemical s t u d i e s . Chapter V continues t h e theme o f s e l e c t i v e d e t e c t i o n w i t h an overview o f post-column r e a c t i o n d e t e c t i o n ,

The use of

immobilised enzymes i n

post-column r e a c t o r s o r so c a l l e d 'pumpless' r e a c t o r systems f o r o n - l i n e reagent generation a f t e r t h e chromatographic s e p a r a t i o n s t e p i s discussed i n detail.

Various examples of t h e s e p a r a t i o n o f b i o l o g i c a l compounds

show how t h e p r o d u c t i o n o f electrochemical

reagents and photochemical

r e a c t i o n d e t e c t i o n have increased t h e s e l e c t i v i t y o f t h e d e t e c t i o n .

This

has l e d t o more economical a n a l y t i c a l systems.

1 uminescence d e t e c t i o n techniques i s The use o f immobilised fluorophores o r t h e

S e l e c t i v e d e t e c t i o n employing o u t l i n e d i n Chapter V I .

coup1 i n g t o photochemical r e a c t i o n s leads t o h i g h l y s e l e c t i v e d e t e c t i o n systems, which again g r e a t l y s i m p l i f y t h e sample handling. The volume concludes w i t h a r e v i e w of t h e use o f continuous s e p a r a t i o n techniques

in

flow

injection

analysis.

It

demands

that

a

strong

i n t e r d i s c i p l i n a r y dependence between sample h a n d l i n g and separation i n t h i s area i s e s s e n t i a l . The completion o f t h i s second volume marks t h e achievement o f t h e aims f i r s t s e t o u t by Professor F r e i i n t h e p r e f a c e t o P a r t A. It i s thus a f i t t i n g conclusion t o t h i s preamble t o quote these words: "By t h e n a t u r e o f i t s c o n t e n t , and w r i t t e n as i t i s by experienced p r a c t i t i o n e r s , t h e book should be useful t o i n v e s t i g a t o r s i n many areas

of

application.

Each chapter

includes

sufficient

references

t o the

l i t e r a t u r e t o serve as a v a l u a b l e s t a r t i n g p o i n t f o r more d e t a i l e d i n v e s t i g a t i o n . The s t r o n g emphasis on sample h a n d l i n g makes t h e book unique i n many ways and i t should prove u s e f u l t o t h e environmental s c i e n t i s t as w e l l as t o i n v e s t i g a t o r s from t h e c l i n i c a l , pharmaceutical and b i o a n a l y t i c a l f i e l d s

.

"

F i n a l l y , I would l i k e t o thank t h e authors f o r t h e i r c o n t r i b u t i o n s , many f r i e n d s f o r s t i m u l a t i n g d i s c u s s i o n s , D r . M. Galvan f o r l i n g u i s t i c assistance and Mrs. G. Bader and C. Jantke f o r t h e p r e p a r a t i o n o f t h e camera ready manuscript.

In p a r t i c u l a r , I am extremely g r a t e f u l t o my

w i f e , who took over most o f t h e e d i t o r i a l work f o l l o w i n g Roland's death.

September 1989

K. Zech

(Constance, F. R .G. )

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5 CHAPTER I

PRECONCENTRATION AND CHROMATOGRAPHY ON CHEMICALLY MODIFIED SILICAS W I T H COMPLEXATION PROPERTIES J.L. VEUTHEY, M.A.

BAGNOUD and W. HAERDI

1. 1.1 1.2 1.2.1 1.2.2 1.3 2. 2.1 2.2 3. 3.1 3.2

Introduction Preconcentration using l i q u i d - s o l i d e x t r a c t i o n S t a t i o n a r y phases f o r l i q u i d - s o l i d e x t r a c t i o n P u r i t y o f preconcentration supports Other f a c t o r s i n f l u e n c i n g t h e c h o i c e o f s u p p o r t s Choice o f a s u p p o r t Chemically modified s i l i c a s Surface modification C r o s s - l i n k e d f u n c t i o n a l groups t o s i l i c a s P r e c o n c e n t r a t i o n o f i n o r g a n i c compounds u s i n g c h e l a t i n g s i l i c a s Metal p r e c o n c e n t r a t i o n Advantages and disadvantages of c h e l a t i n g s i l i c a s i n preconcentrating metals 3.3 Analysis o f preconcentrated metals 4. The p r e c o n c e n t r a t i o n of o r g a n i c compounds u s i n g c h e l a t i n g s i 1icas 5. C h e l a t i n g s i l i c a s i n chromatography Metal s e p a r a t i o n chromatography 5.1 5.2 L i gand exchange chromatography L i gand exchange p r i n c i p l e s 5.2.1 5.2.2 Types o f s t a t i o n a r y phases 5.2.2.1 Metal s i l i c a s 5.2.2.2 Complexing metal s i l i c a s 5.3 Enantiomer s e p a r a t i o n 6. Conclusion References

1. INTRODUCTION

In t h e p a s t 20 y e a r s numerous s t u d i e s have been made t o develop s o l i d s u r f a c e s c o n t a i n i n g complexing s i t e s f o r v a r i o u s a p p l i c a t i o n s . The f i r s t complexing metals

surfaces

from

found

a p p l i c a t i o n s i n preconcentrating

n a t u r a l media

(ref.

1).

Through

improved

transition

cross-linking

t e c h n i q u e s , complexing groups w i t h f a s t e r exchange k i n e t i c s were bound t o t h e support.

I t t h e n became p o s s i b l e t o use t h e s e s u p p o r t s i n chroma-

tography ( r e f . 2 ) . More r e c e n t l y , o t h e r a p p l i c a t i o n s i n c l u d e :

-

t h e use o f t h e s e supports i n heterogeneous c a t a l y s i s a t t h e complexing m e t a l s i t e s ( r e f . 3)

-

r e t a i n i n g o r g a n i c s f r o m a i r o r w a t e r ( r e f . 4) peptide synthesis ( r e f . 5 ) i m m o b i l i z i n g enzymes ( r e f . 6 ) .

6

These a p p l i c a t i o n s have o n l y been made p o s s i b l e a f t e r having t e s t e d a number o f d i f f e r e n t types of supports. F o r example, t h e f i r s t supports employed f o r metal r e t e n t i o n were d i v i n y l benzene p o l y s t y r e n e copolymer r e s i n s . The development o f o t h e r supports such as s i l i c a t e s and cellulose,

have r e c e n t l y replaced these r e s i n s .

Cross-1 inked s i l i c a t e

supports have been h i g h l y developed and have found a wide range of a p p l i c a t i o n s , e s p e c i a l l y i n chromatography, due t o t h e i r low c o s t and t h e f a c i l i t y o f b i n d i n g a wide range o f f u n c t i o n a l groups. This chapter w i l l discuss t h e use o f these supports i n l i q u i d - s o l i d e x t r a c t i o n s , t h e types o f supports a v a i l a b l e and a c e r t a i n number of considerations which should be made concerning t h e i r a p p l i c a t i o n . PRECONCENTRATION USING LIQUID-SOLID EXTRACTION

1.1

The

analysis

of

inorganic,

organic,

or

organometallic

species

necessitates, f o r t h e most p a r t , a p u r i f i c a t i o n s t e p t o e l i m i n a t e a l a r g e p r o p o r t i o n o f unwanted products.

E x t r a c t i o n techniques seem t o be t h e

most a p p r o p r i a t e i n f u l f i l l i n g t h i s need. These techniques, i n a d d i t i o n t o clean-ups, o f f e r t h e advantage o f reducing t h e i n i t i a l sample volume, thus concentrating i t . S e l e c t i v e l y e x t r a c t i n g a compound o r c l a s s o f compounds from a complex m i x t u r e w h i l e reducing t h e f i n a l sample volume b e f o r e a n a l y s i s i s c a l l e d "preconcentration" o r "enrichment". This technique i s very u s e f u l when compounds a r e found a t t r a c e l e v e l s . H i g h l y measured p r e c a u t i o n s must be taken by t h e a n a l y s t d u r i n g t r a c e analysis. A s l i g h t contamination o f t h e e x t r a c t i n g s o l v e n t , f o r example, may c o n t r i b u t e t o t h e e r r o r s contaminations

may

revealed

be preconcentrated

i n the f i n a l as

much

as

analysis the

since

compound

of

i n t e r e s t . Consequently, i t i s necessary t o f i n d techniques which reduce contamination r i s k s keeping t h e number o f o p e r a t i o n s t o a s t r i c t minimum. L i q u i d - s o l i d e x t r a c t i o n , which c o n s i s t s o f r e t a i n i n g s o l u t e s i n a l i q u i d phase on a s o l i d support ( f i l t e r , column, suspension) f u l f i l l these prer e q u i s i t e s b e t t e r than any o t h e r technique. These e x t r a c t i o n p r i n c i p l e s are n o t e x c l u s i v e t o l i q u i d phases alone b u t a r e a l s o a p p l i c a b l e t o gas phases as we1 1.

1.2

STATIONARY PHASES FOR LIQUID-SOLID EXTRACTION

Four d i f f e r e n t types of supports e x i s t f o r l i q u i d - s o l i d e x t r a c t i o n , namely ( r e f . 7 ) : 1.) 2.)

s y n t h e t i c and foamed p l a s t i c r e s i n s i l i c a and alumina

7

cellulose a c t i v a t e d carbon

3.) 4.)

A 1 1 o f these supports have advantages and disadvantages,

hence t h e

choice o f t h e support depends on t h e t y p e o f a n a l y s i s needed.

Several

c r i t e r i o n a r e necessary f o r t h i s choice. For example, t h e p u r i t y of t h e support i s a very important f a c t o r . Consideration should a l s o be g i v e n t o c e r t a i n p r o p e r t i e s such as t h e r i g i d i t y o f t h e support o r t h e ease of a n a l y s i s o r even t h e c o s t o f t h e support, a l l o f which p l a y an i m p o r t a n t r o l e i n t h i s choice. 1.2.1

PURITY OF PRECONCENTRATION SUPPORTS

Trace a n a l y s i s n e c e s s i t a t e s t h e use of h i g h p u r i t y p r e c o n c e n t r a t i o n supports. Although s i l i c a t e s have been o b t a i n e d w i t h a h i g h degree of p u r i t y , s p e c i a l a t t e n t i o n should be g i v e n t o those which have been chemically m o d i f i e d as i m p u r i t i e s may have been i n t r o d u c e d from reagents used d u r i n g t h e chemical m o d i f i c a t i o n ( f o r example, a l k o x y s i l a n e treatment o f s i l i c a t e g e l s ) .

i m p u r i t i e s due t o

Consequently they r e q u i r e a

pretreatment s t e p t o e l i m i n a t e these t r a c e compounds. S t a t i o n a r y phases may be p r e t r e a t e d u s i n g e i t h e r a Soxhlet e x t r a c t i o n o r simply prewashed w i t h an o r g a n i c s o l v e n t o r an a p p r o p r i a t e m i n e r a l acid. 1.2.2

OTHER FACTORS INFLUENCING THE CHOICE OF SUPPORTS

One important p h y s i c a l p r o p e r t y o f these supports i s t h e i r r i g i d i t y . E l u t i o n i s u s u a l l y performed under a p p l i e d pressures deforming t h e support thus

changing

t h e i r physicochemical

properties.

supports having h i g h e r r i g i d i t y a l l o w h i g h e r e l u t i o n f l o w

I n general, r a t e s t o be

obtained thus making a n a l y s i s t i m e s h o r t e r . For instance, divinylbenzene p o l y s t y r e n e r e s i n s (Chelex 100) t e n d t o s w e l l , r e t r a c t o r become deformed upon c o n t a c t of t h e m o b i l e phase. These problems may be minimized when they

a r e mixed w i t h

s i l i c a gel

of

approximately t h e same p a r t i c l e s i z e . Foamed p l a s t i c s , on t h e o t h e r hand, a r e r i g i d and remain so even a t h i g h e l u t i o n f l o w r a t e s . A t lower e l u t i o n f l o w r a t e s , s i l i c a t e s , c o n t r o l l e d pore glasses (CPG) and c r o s s - l i n k e d c e l l u l o s e s c o n t a i n i n g c h e l a t i n g s u b s t i t u t e n t s are n o t deformed e i t h e r and may be u t i l i z e d f o r p r e c o n c e n t r a t i o n as w e l l as f o r h i g h performance chromatography. Another f a c t o r a f f e c t i n g

t h e choice of

a n a l y s i s which can be performed.

After

a support

i s t h e t y p e of

preconcentration,

a n a l y s i s of

r e t a i n e d products u s i n g e i t h e r neutron a c t i v a t i o n o r X-ray fluorescence may be done d i r e c t l y on s i l i c a s ,

resins o r cellulose.

However,

direct

8

a n a l y s i s o f CPG o r foamed p l a s t i c s i s n o t recommended and e l u t i o n i s necessary. Ligand exchange k i n e t i c s a l s o p l a y s a very i m p o r t a n t r o l e i n t h e choice o f a support. Resins, f o r example, have very slow exchange k i n e t i c s as opposed t o those o f m o d i f i e d s i l i c a t e s . This d i f f e r e n c e may be a t t r i b u t e d t o t h e s t r u c t u r e i t s e l f o f t h e supports, s i n c e i n f a c t , t h e cross-bonded f u n c t i o n a l groups o f t h e r e s i n a r e p r a c t i c a l l y i n a c c e s s i b l e whereas those o f t h e m o d i f i e d s i l i c a t e s l i e a t t h e surface.

CHOICE OF A SUPPORT

1.3

Among t h e f o u r d i f f e r e n t types o f s o l i d supports employed f o r t h e extraction o r preconcentration o f t r a c e organics o r inorganics, s i l i c a s o f f e r t h e most a t t r a c t i v e p r o p e r t i e s and consequently a r e r e c e i v i n g i n creasing i n t e r e s t f o r t h e f o l l o w i n g reasons: 1.) t h e i r low c o s t

2.)

they may be used d i r e c t l y o r m o d i f i e d through t h e p h y s i c a l o r

3.)

they a r e a p p l i c a b l e t o t h e e x t r a c t i o n o f o r g a n i c as w e l l as

4.)

they a r e used n o t o n l y f o r p r e c o n c e n t r a t i o n purposes b u t f o r

chemical adsorption o f c h e l a t i n g agents i n o r g a n i c c o n s t i t u e n t s from l i q u i d o r gas phase systems chromatographic

separation

o f organic o r mineral

components

as

we1 1

5.)

they have f a s t l i g a n d exchange k i n e t i c s . The f o l l o w i n g discussion w i l l focus on complexing s i l i c a s and w i l l

cover a number o f t h e i r a p p l i c a t i o n s . order,

With r e s p e c t t o c h r o n o l o g i c d l

i t w i l l be necessary t o f i r s t mention t h e synthesis

complexing

silicas,

subsequently

their

use

for

o f these

preconcentration

a p p l i c a t i o n s , and f i n a l l y , t h e i r use as chromatography supports. C e r t a i n s i l i c a s , such as t h e c r o s s - l i n k e d a l k y l - c h a i n s i l i c a s , which a r e r e a d i l y employed i n l i q u i d chromatography, w i l l n o t be discussed here s i n c e they have been thoroughly described i n numerous a r t i c l e s ( r e f s . 8-10). 2.

CHEMICALLY M O D I F I E D SILICAS S i l i c a s can e a s i l y be modified t o o b t a i n a wide range o f supports o f

differing

properties.

Numerous

factors

may

influence

the

chemical

m o d i f i c a t i o n o f s i 1 i c a t e s making t h e i r q u a n t i t a t i v e s y n t h e s i s d i f f i c u l t An e n t i r e monography

(ref.

11) t r e a t s t h e s u b j e c t o f s i l i c a s ,

.

their

physico-chemical p r o p e r t i e s and t h e i r r e a c t i o n mechanisms. Chemical m o d i f i c a t i o n o f s i l i c a s i s p r i n c i p a l l y accomplished by one of

9

two methods: a.)

a r e a c t i o n c a l l e d " s u r f a c e m o d i f i c a t i o n " between an organosilane and t h e s i 1 i c a t e

b.)

o r by h y d r o l y t i c polycondensation o f organosilanes. Surface m o d i f i c a t i o n i s by f a r t h e most o f t e n employed as i t i s a much

s i m p l e r procedure. difficult,

I n contrast,

the

second method

is

found

u n c o n t r o l l a b l e , and g i v e s r i s e t o undefined supports.

to

be

Conse-

quently, t h i s d i s c u s s i o n w i l l focus on s u r f a c e m o d i f i c a t i o n . 2.1

SURFACE MODIFICATION Several types o f r e a c t i o n s between v a r i o u s o r g a n i c groups -R

and

s i l i c a s are employed t o modify these supports, namely: 1.)

-R may be d i r e c t l y bonded t o t h e s i l i c i u m atom, S i - R

2.)

-R may be bonded t o t h e s i l i c a t e v i a a heteroatom

a. Si-0-R b. S i - N H - R c . Si-0-Si-R Among t h e f o u r d i f f e r e n t

procedures o f c r o s s - l i n k i n g organics,

-R,

onto s i l i c a s , Si-0-Si-R i s t h e most o f t e n u t i l i z e d . The s i l o x a n e bond i s , in e f f e c t , t h e most s t a b l e one and consequently i s t h e most s u i t a b l e f o r a p p l i c a t i o n s i n complex systems such as n a t u r a l a q u a t i c systems. Problems such as h y d r o l y s i s and s y n t h e s i s a r e encountered w i t h t h e t h r e e o t h e r types o f bonds. C r o s s - l i n k i n g o f t h e o r g a n i c -R group through a s i l o x a n e bond may be accomplished u s i n g organosilanes

such as RnSiX4-n

(l 5.5. I n blood one f r a c t i o n o f drugs i s bound t o plasma p r o t e i n s and t h e o t h e r blood components and t h e o t h e r f r a c t i o n i s free. By d e p r o t e i n a t i o n and e x t r a c t i o n t h e p r o t e i n bonds must be broken o r t h e recovery may be decreased. A decrease i n recovery can a l s o occur if t h e compounds o f i n t e r e s t a r e c o - p r e c i p i t a t e d o r p h y s i c a l l y entrapped i n the protein precipitate.

Basic drugs can be e x t r a c t e d from b l o o d

w i t h o u t p r i o r procedures by t h e use of a p p r o p r i a t e b u f f e r s o l u t i o n s w i t h a pH ranging from 6 t o 14. U s u a l l y t h e pH t o be chosen i s 3 u n i t s above t h e pKa because then more than 99% of t h e b a s i c drug i s i n i t s u n i o n i z e d form and can be e x t r a c t e d i n t o an o r g a n i c solvent.

Due t o t h e i r i o n i c

s t r e n g t h s these b u f f e r s o l u t i o n s cause p r o t e i n d e n a t u r a t i o n w i t h minimal

loss o f t h e drug (refs. a d j u s t i n g t h e pH < 5.5.

2,6,7). A c i d i c drugs can be e x t r a c t e d a f t e r The low pH causes p r o t e i n p r e c i p i t a t i o n w i t h t h e

r i s k o f c o - p r e c i p i t a t i n g t h e compounds of i n t e r e s t . For l i q u i d - l i q u i d p u r i f i c a t i o n o f blood samiles f o u r s t r a t e g i e s a r e used: 1. The drug i s converted i n t o i t s i o n i z e d form by changing t h e pH and can be e x t r a c t e d i n t o an aqueous phase. The o r g a n i c l a y e r i s removed and discarded. I n a second s t e p t h e l i p o p h i l i c , u n i o n i z e d form o f t h e drug i s r e - c o n s t i t u t e d by changing t h e pH i n t o t h e o p p o s i t e d i r e c t i o n and t h e drug can be back-extracted i n t o an organic s o l v e n t ( F i g . 2, s i d e columns)

.

86

2. The drug is dissolved in an aqueous/organic solvent, e.g. water/ acetonitrile, and the interfering compounds are removed by washing the sample with a lipophilic solvent, that is not miscible with the aqueous layer e.g. hexane. Compounds of interest dissolved in a lipophilic solvent can also be purified by washing with an aqueous solution (Fig. 1, middle column).

Liquid-liquid extraction can be combined with solid-liquid purification steps: 1. The extracted sample is spread on a TLC plate. After development the circle of silica adsorbing the compounds of interest is scraped off the plate and the silica gel is extracted (ref. 8). 2. Interfering materials are removed by adsorption on a solid phase or after liquid-liquid extraction the compounds of interest are adsorbed on a solid phase and thus separated from interfering materials.

Fig. 1 i s a schematic flow-diagram of steps used for liquid-liquid sample clean-up of basic and acidic drugs. The diagram demonstrates the way of blood sample preparation for GC and HPLC analysis. For reversed phase chromatography the aqueous back-extracts can be directly injected into the HPLC system The side columns of the diagram show purification steps by basic or acidic back-extraction, the middle column the removal of interfering materials with an organic solvent, inmiscible with the aqueous layer containing the compounds of interest, in which these materials are insoluble. Table 1 shows an overview of blood sample extraction strategies with subsequent chromatographic analysis. The extraction procedures 1 isted are used for sample preparation prior to chromatographic analysis (HPLC, GC, TLC). Usually the authors describe the extraction from several different kinds of biological matrices but only procedures and values for blood sample preparation and analysis are listed here. The procedures are divided into the four main steps as discussed above. In addition de Silva (refs. 9 and 10) describes schematically the extraction of various 1,4-benzodiazepines and Foerster et al. (ref. 11) the extraction of multiple acidic and neutral drugs from blood. Basic back-extraction describes the following procedure: the sample is acidified and the drugs are extracted into an aqueous phase. Then the pH is raised and the drugs are re-extracted into an organic solvent. If the solvents used for extraction and back-extraction are identical the solvent used for back-extraction is not mentioned.

a7

blood sample c o n t a i n i n g a c i d i c druas

blood sample c o n t a i n i n g

rn

I

protei n precipitation (pH < 5.5)

protein denaturation

I

extraction into’ organic solvent

e x t r a c t i o n i n t o organic s o l v e n t shake,

I centrifuge

I

shake, c e n t r i f u g e

I I

I

d i s c a r d aqueous 1ayer

d i s c a r d a ueous l a y e r

pH r a i s e d > 7 + organic s o l v e n t

+ orga’nic s o l v e n t

decrease pH < 5.5 + organic s o l v e n t

shake,

shake, c e n t r i f u g e

shake, c e t r i f u g e

I centrifuge I

9

I I

I I

d i s c a r d organic l a y e r

discard organic layer

discard organic layer

decrease pH < 5.5 + organic s o l v e n t

HPLC,‘ GLC

increase PH > 7 + organic s o l v e n t

I

I

I I

I

shake, c e r h r i f u g e

shake, 2ent r i f uge

I

I I

d i s c a r d a ueous l a y e r

d i s c a r d aqueos l a y e r evaporate o r g a n i c l a y e r -evaporate reconstitution

9

organic layer

I

HPLC. GLC Fig. 1

L i q u i d - l i q u i d clean-up procedures f o r blood sample a n a l y s i s .

rABLE I

m m

Liquid-liquid extraction of blood samples

Substance ( s ) Acebutolol

l-a acetyl-methadol Acetazol amide

Ant i pyrine Atenolol

pKa Ref. Deproteinization/ pH adjustment

Extraction

Purification/ Derivatization

%

rec. Analysis/ Detection

det. .g/l 10

distilled water, 2 N NaOH

ethylacetate

acidic backextraction

n.r. HPLC / UV (240 nm)

pH 9.2

n-butylchloride

basic back-extraction with CHCl,

>90 GC / MS

5

7.2 14 9.0

acetate buffer pH 5

CH C1 /diethylether methylation with /2zprGpanol (6/4/2) trimethylphenylammonium hydroxide

80- Gd3NiECD 90

25

15

filtration, ethanol

9095 HPLC / UV 9.9 (254nm)

6

9.4 12

13

9.6 16

0.1

N ammoniumhydroxide

CH2C12/n-pentane (50/50) n-butanol/cyclohexane (70/30)

basicbackextraction

55

GC/63NiECD

Bacmecillinam

6.8 17

CH3C1 /hexane (1/9)

Barbiturates

4.0 18

CH2C1

acidic back-extrac- 83- GC/FID tion, methylation 113

Barbiturates

4.0 19

acetone/ether (50/50)

derivatization with methyl iodide and KJO,

Benzodi azepine derivative

20

phosphate buffer PH 9

diethyl ether/ CH3C1 (70/30)

96- HPLC/UV 104 (230 nm)

70

GC/FID

925 HPLC/UV 5.4 (230 nm)

10 0.6 100 16 ng/l

50

TABLE I

(continued) 21

Benzodiazepine derivatives Butaperazine

1

Carbamazepine

22

Carprofen

23

Chloroprocaine

8.7 24

distilled water, ammonium hydroxide

diethyl ether

70- HPLC / UV 100 (240 nm)

n.r.

distilled water, sodium carbonate

n-pentane/ isopropanol (97/3)

82- HPLC/UV 95

11)

hexane/ 1-pentanol (90/10)

basic back-extraction 85in CHC13 105

GC/NSD

10

nMol /1

Chl oroquine

Chloroquine

8.4 27 10.8

8.4 28 10.8

Cimetidine

6.8 29

distilled water, dipotassium hydrogen phosphate pH 9.5

CH2C12

acidic extraction into the aqueous phase

7397

HPLC/UV (254 nm)

3-4

deionized water, 0.001 N HC1

hexane

basic backextraction

95103

GC/NSD

5-15

freeze and thaw, 1 N NaOH pH 9.0

1-octanol

basic back-extraction 98in ethanol 106

HPLC/UV (228 nm)

50

cn

W

TABLE

I

(continued)

L o

0

Clonazepam

1.5 10.5

30

b o r a t e b u f f e r pH 10

isoamyl a l c o h o l / hexane (10/90)

hydrolysis

3550

GC/ECD

5

Debri soqu ine

11.9

31

d i s t i l l e d water

diethyl ether

b a s i c back-extraction n.r. i n cyclohexane

GC/FID o r NSD

3

Diazepam

3.3

32

1 M phosphate b u f f e r pH 7.0

diethyl ether

b a s i c back-extraction 91116

HPLC/UV (240 nm)

20 30

Diazepam

3.3

33

phosphate b u f f e r pH 7.0

n-heptane

b a s i c back-extraction 9095

GC/63NiECD

sodium hydroxide

diethyl ether

93.9 HPLC/FD(285/ 1 430 nm)

n-heptane

98- GC/TCD 100

D i py r idamol e

6.4

Enf 1urane

34 35

50

4.1 pMo 1

/1 Flestolol

4.0 36

acetonitrile

acetoni t r i 1e/ CH2C12 ( U 5 )

acidic extraction i n t o aqueous phase

38

HPLC/UV (229 nm)

10

Tetrahydrocannabinol

10.6

37

2 N HC1 (pH 4)

hexane/ iso-amyl a1coho1 (98/2)

a c i d i c back-extract i o n i n t o hexane

n.r.

TLC/FD

0.4

116-9-tetrahydrocannabinol

10.6 10.4

38

CHC13

f i l t r a t i o n , TLC

98- GC/MS 100

diethyl ether

e x t r a c t i o n o f the organic l a y e r i n t o a c e t o n i t r i l e and phosphoric a c i d

85

Hydroxychloroquine

39

d i s t i l l e d water, ammonia (pH 13)

0.5

HPLC/FD 1 (337/370 nm)

TABLE I

(continued)

Imipramine

9.5 40

ammonium hydroxide

butanol/hexane (20/80)

92.5 HPLC/FD 25 (240/370nm)

Ketamine

7.5 41

ammonium hydroxide pH 10.1

CHC13: isopropanol (75:25) isopropyl acetate

n.r. GC/FI

100

86% GC/MS 9

3

Mef 1 oquine

42

freeze and thaw

Mef 1 oquine

43

phosphate buffer pH 7.4

ethyl acetate

Mefloquine

44

0.2 N H2S04

diethyl ether

1005 HPLC/UV 9.9

wash acidified sample with ether, derivati zation

935 GC/63Ni ECD 9.7 and FID

50

1

Methadone

8.3

45

4 M Na2C03

1-chlorobutane

basic back-extraction 935 GC/FID into CHC13 2

5

Morphine

8.0

46

phosphate buffer pH 8.7 - 9.0

ethyl acetate

aluminium oxide, deri vatization

1

47

acetate buffer pH 5 , B-glucuronidase

ethylacetatelisopropanol (90/10)

basic back-extraction 81

7.9 48

1 M carbonate buffer pH 10

diethyl ether

distilled water, 5M HC1 glacial acetic acid

9.9

Morphine-3glucuronide Naloxone Pentacaine Phenobarbital , Phenytoin, Primidone

49 7.4 50 8.3

83

GUb5Ni ECO HPLC/ECD

0.5

acidic extraction in aqueous phase

78+ HPLC/UV 3.2 (214 nm)

1

1,2 dichloroethane

heptane, Nap C03 fi 1 trati on

75- G U M S 92

5

CHC13

basic back-

90- HPLC/UV 110 (254 nm)

extraction

100 200

ID

300

c

TABLE I

W

(continued)

N

Phentolamine

7.7

51

1 M amnonium hydroxide

diethyl ether

acidic extraction in aqueous phase

83

Promethazine

9.1

52

borax buffer pH 10

n-heptane/isopentanol (99/1)

basic backextraction

97- GC/NSD 99

5

Propranolol

9.5

53

5 N NaOH

isoamyl-alcohol: nheptane (1.5: 98.5)

n.r. HPLC/FD

5

54

4.8 M KC1 pH 6.1

benzene

98 5 GC/ECD 8.9

2

glacial acetic acid

CHC13

90- GC/FID 110

1000

phosphate buffer pH 5.5

CH2C1

n.r. HPLC/UV (290 nm)

200

67- HPLC/UV 90 (225 nm)

0.03 ppm

Pyramidobenzazepine Theophylline Thiopental

c1 55 8.1

56

Tocainide

7.8 57

1 N NaOH, destilled water

CH3Cl

Warfarin

5.0 58

5 N HC1

CHCl

deri varization

fi 1 tration , wash with aqueous sodium pyrophosphate

HPLC/UV (280 nm)

HPLC/UV (270 nm)

ECD: electron capture detector, FD: fluorescence detector, FID: flame ionization detector, GC: gas chromatography HPLC: high performance 1 iquid chromatography, MS: mass spectrometry, NSD: nitrogen selective detector, TCD: thermal conductivity detector, TLC: thin layer chromatography, UV: ultraviolet absorbance detector, n. r. : not reported.

15

Another f o rm o f l i q u i d - l i q u i d e x t r a c t i o n i s t h e use o f s i l i c a m a t e r i a l l i k e E x t r e l u t R ( r e f s . 59-62). Though t h e e x t r a c t i o n columns c o n t a i n s o l i d phase m a t e r i a l , t h e b a s i c p r i n c i p l e i s a l i q u i d - l i q u i d e x t r a c t i o n . E x t r a c t i o n w i t h diatomaceous e a r t h obeys t h e same b a s i c mechanism ( r e f s . 63-65).

Silica

gels

are

porous

carrier

materials.

Water

molecules

d i s t r i b u t e on t h e s u r f a c e of t h e s i l i c a g e l and become t h e s t a t i o n a r y phase. Compounds a r e d i s s o l v e d i n t h e w a t e r phase and a r e e l u t e d f rom t h e columns by o r g a n i c s o l v e n t s , u n m i s c i b l e w i t h wat er. Such columns can be used a t a pH range f r o m 1-13. A f t e r p r o t e i n p r e c i p i t a t i o n by a c i d o r b u f f e r t h e aqueous b l o o d sample i s p u l l e d by vacuum t h r o u g h t h e column ( r e f s . 61,62). S i l i c a g e l can a l s o be used f o r sample p u r i f i c a t i o n ( r e f s . 46,59) by a bs or b i n g i n t e r f e r i n g m a t e r i a l s from b l o o d w i t h o u t absorbing t h e components t o be e l u t e d . 2.2

SOL ID-L I Q U ID EXTRACTION Several methods f o r t h e e x t r a c t i o n o f compounds f rom b l o o d have been

r e p o r t e d u s i n g s o l i d s o r b e n t s as an a l t e r n a t i v e t o l i q u i d - l i q u i d extraction.

F or t h e e x t r a c t i o n of b l o o d samples t h e use o f s o l i d phase

m a t e r i a l has t h e f o l l o w i n g advantages ( r e f s . 66,67): 1. The f o r m a t i o n o f emulsions d i s t u r b i n g e x t r a c t i o n i s avoided. 2. L i t t l e volume o f s o l v e n t s a r e necessary. 3. A c i d i c drugs can be e x t r a c t e d w i t h h i g h r ecovery. 4. F a t t y a c i d s , t h e i r e s t e r s and c h o l e s t e r o l a r e n o t c o - e x t r a c t e d .

The s o l i d - l i q u i d e x t r a c t i o n procedures o f b l o o d samples can be d i v i d e d i n t o 4 main s t e p s : 1. hemolysis, d e p r o t e i n a t i o n and pH-adjustment,

2. a d s o r p t i o n o f t h e compounds of i n t e r e s t on t h e s o l i d phase m a t e r i a l , 3. p u r i f i c a t i o n b y washing t h e adsorbent w i t h l i p o p h i l i c o r h y d r o p h i l i c solvents, 4. e l u t i o n o f t h e drugs f r o m t h e adsorbent, 5. volume r e d u c t i o n and i f necessary d e r i v a t i z a t i o n . L i q u i d - l i q u i d e x t r a c t i o n of a c i d i c drugs i s sometimes complicat ed by the co-extraction

o f 1 i p i d s and 1 i p o p r o t e i n s .

Co-extraction

of

these

compounds i s l e s s i n s o l i d - l i q u i d e x t r a c t i o n and t h u s f u r t h e r clean-up st e ps 1 ike b a c k - e x t r a c t i o n s

o f t e n r e d u c i n g recovery a r e u s u a l l y n o t

r e q u i r e d . For s o l i d - l i q u i d e x t r a c t i o n t h e f o l l o w i n g s o l i d sorbent s a r e used :

94 1. Bonded phase s i l i c a gels, a l s o a v a i l a b l e as pre-packed disposable columns ( r e f s . 68-70), 2. anion and c a t i o n exchange r e s i n s ( r e f . 71), 3. n o n - i o n i c r e s i n s l i k e a c t i v a t e d charcoal ( r e f . 72) and A m b e r l i t e XAD-2 ( r e f s . 73-75)

.

Blood samples must be prepared f o r e x t r a c t i o n on bonded phase s i l i c a gel columns by hemolysis and p r o t e i n p r e c i p i t a t i o n

b e f o r e sucked by

vacuum through t h e e x t r a c t i o n columns. The columns a r e p r e v i o u s l y primed w i t h t h e same s o l v e n t , as used f o r e l u t i o n o f t h e drugs from t h e columns, i n o r d e r t o remove i n t e r f e r i n g substances. With a p o l a r s o l v e n t ( u s u a l l y water) t h e c o n d i t i o n s used f o r e x t r a c t i o n a r e e s t a b l i s h e d by l o a d i n g t h e columns with p o l a r groups. The drugs a r e r e t a i n e d by t h e column and can f u r t h e r be p u r i f i e d by washing t h e adsorbed m a t e r i a l s w i t h l i p o p h i l i c o r h y d r o p h i l i c s o l v e n t s i n which t h e y have a small p a r t i t i o n c o e f f i c i e n t . The columns are a l s o s u i t a b l e f o r an e x t r a c t i o n by i o n - p a i r chromatography ( r e f . 68) and can be cleaned and reused. The l i f e span of e x t r a c t i o n columns used f o r e x t r a c t s from blood i s s h o r t e r than f o r those from serum o r plasma,

s i n c e l a r g e amounts o f blood components

like

l i p o i d s and l i p o p r o t e i n s a r e c o - e x t r a c t e d and can p l u g t h e columns. The columns should n o t be c o n f r o n t e d w i t h s o l v e n t s w i t h a pH > 9. The ext r a c t i o n procedure w i t h disposable s o l i d phase e x t r a c t i o n columns can be automated u s i n g e x t r a c t i o n systems such as an advanced automated sample processing u n i t (AASP,

Varian, Walnut Creek, CA, USA). The p r e - e x t r a c t e d

blood samples a r e a u t o m a t i c a l l y loaded on t h e e x t r a c t i o n columns, p u r i f i e d and i n j e c t e d i n t o t h e HPLC-system ( r e f s . 76,77). I o n i c and non-ionic r e s i n s can be added t o t h e b l o o d sample i n ext r a c t i o n columns ( r e f s . 78-80), bags ( r e f .

i n capsules ( r e f . 69),

78) o r as r e s i n s l u r r y ( r e f s . 74-75).

i n nylon f a b r i c

Anion exchange r e s i n s

are s u i t a b l e f o r t h e e x t r a c t i o n of a c i d i c drugs such as b a r b i t u r a t e s , sal i c y l a t e s and phenylbutazone, c a t i o n exchange r e s i n s f o r t h e e x t r a c t i o n of basic drugs such as q u i n i d i n e , chlorpromazine, s t r y c h n i n e , and morphine. Charcoal i s i n e f f e c t i v e i n b i n d i n g most b a s i c drugs except s t r y c h n i n e and proved v a l u a b l e i n b i n d i n g n o n - i o n i c o r g a n i c compounds l i k e gluthetimide,

meprobamate and carbromal

(ref.

71).

I o n exchange

r e s i n s are a l s o used t o remove i o n i c i m p u r i t i e s from b l o o d samples ( r e f . 81)

.

The a n i o n i c r e s i n Amber1 i t e XAD-2, i n t r o d u c e d i n t o pharmacological and t o x i c o l o g i c a l a n a l y s i s by F u j i m o t o e t a l . ( r e f . 82) i s a nonpolar styrene-divinylbenzene copolymer w i t h a p a r t i c l e s i z e o f 50-100 p ( r e f .

66).

It a l l o w s t h e e x t r a c t i o n of

deproteination

( r e f . 80).

The

drugs from b l o o d w i t h o u t preceeding

resin

s l u r r y i s prepared by washing t h e

95 r e s i n subsequently w i t h water, methanol and acetone. The r e s i n i s s t o r e d i n water o r a b u f f e r s o l u t i o n ( r e f s . 66,73,75).

A f t e r adsorption o f the

compounds o f i n t e r e s t t h e XAD-2 p a r t i c l e s a r e f i l t e r e d and e x t r a c t e d w i t h an organic s o l v e n t . S c h l i c h t e t a l . ( r e f . 66) and I b r a h i m e t a l . ( r e f . 79) described t h e e x t r a c t i o n o f several drugs from b l o o d samples u s i n g XAD-2 r e s i n s , Ford e t a l . t h e e x t r a c t i o n o f a c i d i c drugs from blood u s i n g CI8 bonded s i l i c a columns ( r e f . 67) and Missen e t a l . ( r e f . 75) compared t h e e x t r a c t i o n of benzodiazepines w i t h various r e s i n s . The e x t r a c t i o n o f drugs from b l o o d using s o l i d phase m a t e r i a l s i s acquainted w i t h some disadvantages t h a t must be taken i n t o account. 1. The e x t r a c t i o n may g i v e v a r i a b l e r e c o v e r i e s

o f the e l u t i n g solvent

and

due t o t h e pH and n a t u r e

t h e sorbent.

2 . The r e s i n s and column m a t e r i a l s loose t h e i r a d s o r p t i o n e f f i c i e n c y t h e

more o f t e n t h e y are reused. 3. The f r i t s and t h e column m a t e r i a l can b e plugged by n o t s u f f i c i e n t l y

d e p r o t e i n i z e d samples o r i f t h e columns a r e reused t o o o f t e n . Since i t i s o f t e n n o t p o s s i b l e t o perform ' d i g i t a l chromatography' on t h e e x t r a c t i o n columns, an i n t e r n a l standard may h e l p t o c o r r e c t recovery o f a compound and t o make e x t r a c t i o n more r e l i a b l e and r e p r o d u c i b l e . A good i n t e r n a l standard should 1. s t r u c t u r a l l y be as s i m i l a r t o t h e compound o f i n t e r e s t as p o s s i b l e , 2. have t h e same d i s t r i b u t i o n c o e f f i c i e n t s i n organic s o l v e n t s ,

3. have t h e same b i n d i n g c h a r a c t e r i s t i c s t o t h e blood compounds as t h e compound o f i n t e r e s t , 4. have a r e t e n t i o n time i n chromatographic a n a l y s i s c l o s e t o t h e compound o f i n t e r e s t , 5. be c l e a r l y separated from t h e compound o f i n t e r e s t d u r i n g a n a l y s i s , 6. have t h e same p r o p e r t i e s concerning t h e d e t e c t i o n system used. The i n t e r n a l standard i s added i n a known amount t o t h e sample p r i o r t o sample p r e p a r a t i o n and a n a l y s i s . A good i n t e r n a l standard i s a b l e t o e l i m i n a t e t h e b i a s caused by losses and compensates random e r r o r s d u r i n g e x t r a c t i o n o r a n a l y s i s ( r e f . 83). F i g . 2 shows a f l o w - c h a r t of s o l i d - l i q u i d e x t r a c t i o n procedures.

If

XAD-2 m a t e r i a l f o r t h e e x t r a c t i o n o f n e u t r a l and b a s i c drugs i s used i t can be renounced a t t h e d e p r o t e i n a t i o n s t e p ( r e f . 80). The p u r i f i c a t i o n s t e p can a l s o be performed a f t e r e l u t i o n o f t h e drugs from t h e s o l i d sorbent u s i n g l i q u i d - l i q u i d e x t r a c t i o n .

2-3

COLUMN-SWITCHING On-line sample preparation using column-switching has been applied to plasma, serum and urine samples and is discussed in detail i n Volume I. Blood sample analysis requires a preceeding purification step and is basically equal to analysis in plasma, serum or urine. Column-switching techniques for cyclosporine blood samples are described in part 3 . 2 . 3 of this chapter.

3. 3.1

BlOOD SAMPLE PREPARATION AND HPLC ANALYSIS OF SandimmunR (CYLOSPORINE) INTRODUCTION SandimmunR (Cyclosporine A, cyclosporine, Sandoz OL 27-400 N) is an immunosuppressive agent and i t s application after organ transplantation has proved to be of great value (refs. 84-85). Due to its narrow therapeutic range and its pharmacokinetic properties, blood level monitoring is mandatory. (ref. 86). Simultaneous measurement of the parent compound and the cyclosporine metabolites in blood by HPLC is of great clinical relevance, since the

97

commercially available and commonly used monoclonal radioimmuno assay (RIA) kits (Sandoz) (ref. 86) measure the parent compound or all metabolites to an mostly unknown extent. With HPLC it is possible to determine the metabolites and to quantify each of the metabolites separately. This will be of special value if one or more of the metabolites prove to be responsible for the cyclosporine adverse effects especially nephrotoxicity. Cyclosporine is a neutral, lipophilic and cyclic undecapeptide with a molecular weight of 1202.6. All its amino acids are S-configurated except D-alanine in position 8 (Fig. 1). Amino acids in positions 1, 3, 4, 6, 9, 10, and 11 are N-methylated. The amino acid in position 1 is a O-hydroxilated, N-methylated and unsaturated C9-amino acid. The tertiary structure of cyclosporine is an antiparallel R-pleated sheet conformation. Its partition coefficient octanol/water is 120/1. The cyclosporine molecule lacks of chromophoric substituents, making UV-detection more unspecific and demanding more extensive extraction procedures. The molar absorption coefficient at the wave-length maximum (195 nm) is 66 000 l/mol x cm. It shows good solubility in alcohols, ether, acetone and chlorinated hydrocarbons and poor solubi1 i ty in water and saturated hydrocarbons (refs. 88-91). Cyclosporine is metabolized by microsomal cytochrome P450 (ref. 92) in the liver to more than 30 metabolites (ref. 93). The structures of the metabolites 1, 8, 9, 10, 13, 16, 17, 18, 21, 25, 26 (refs. 94 and 95), 203-218 (ref. 96) and two aldehyde metabolites (ref. 97) have been elucidated. All metabolites retain the cyclic undecapeptide structure and prove to be more hydrophilic than the parent compound. The reactions involved in cyclosporine degradation are demethylation, hydroxilation, oxidation and cyclization (Table 11). Choice of the bioloaical matrix (ref. 98) For routine drug monitoring cyclosporine is usually measured in blood. However, the question of the biological matrix is still under discussion. 58% of cyclosporine are bound to the erythrocytes in blood, 10 to 20% to the lymphocytes. In plasma cyclosporine is bound to lipoproteins, preferentially those of high and low density (refs. 99-103). The free fraction is 1-1.5% at 37OC (ref. 104). The distribution between blood and plasma is temperature dependent and is lowered from 37OC to room temperature (refs. 99, 105-108). Binding of cyclosporine to the lipoproteins i s also temperature dependent being highest at body temperature and decreasing linearly with lower temperature. The cyclosporine metabolites 1 and 17 are associated with the erythrocytes

98 (>go%) ( r e f s . 109-111). The r e l a t i v e d i s t r i b u t i o n i n b l o o d i s constant u n t i l c y c l o s p o r i n e c o n c e n t r a t i o n s > 1000 ~ / 1 . Furthermore t h e r e l a t i o n between c o n c e n t r a t i o n i n b l o o d and plasma v a r i e s s i g n i f i c a n t l y w i t h t h e hematocrit ( r e f s . 112 and 113). The reasons f o r choosing blood as t h e b i o l o g i c a l m a t r i x are: 1. There a r e no t e c h n i c a l problems because o f t h e temperature dependent

d i s t r i b u t i o n between e r y t h r o c y t e s and plasma. 2. Measurement i s independent o f t h e hematocrit.

3. Plasma l e v e l s a r e considerably increased i n hemolysed b l o o d samples. The choice o f t h e a n t i c o a g u l a n t used f o r c y c l o s p o r i n e blood samples proved t o be o f importance. I n r o u t i n e h e p a r i n i z e d specimens s t o r e d > 1 days c o n t a i n small blood c l o t s .

Since c y c l o s p o r i n e i s bound t o a g r e a t

percentage t o t h e corpuscular blood components c l o t t i n g causes a decrease i n t h e c o n c e n t r a t i o n measured and t h e c l o t s p l u g t h e e x t r a c t i o n columns i n solid-phase e x t r a c t i o n procedures ( r e f s . 4, 98, 114-116). The

methods

developed

for

the

quantitative

determination

of

cyclosporine and i t s m e t a b o l i t e s i n blood cover almost t h e whole spectrum o f blood sample p r e p a r a t i o n s t r a t e g i e s .

99

I

CH2

I

AA8 Fig. 3

AA2

AAll AA1 -

AAlO

CH3

CH3

I

I

AA 7

CH-OH

I

AA6 -

I CH2

CH3

I

I

AA5

Structural formula of cyclosporine.

AA4

100

TABLE I 1

Structures o f the cyclosporine (Cs) metabolites, hitherto characterized (refs. 94-96) with their molecular weights.

I R

R1

R2

H

CH3

1 8

OH

CH3

CH3 CH3

OH

CH20H

CH3

9 10 13

OH

CH3

H

H

OH

OH

CH3

CH3

OH

H

16 17

OH

CH3

CH3

H

H H

CH20H CH20H

CH3

H

H

1234.64 1218.64

CH3

H

H AA1:cyclization

1218.64

H H

CH3 CH20H

H

H

H

1188.62

25

H

H

H

1204.64

26

OH

CH20H

CH3

H

H AA1:cyclization

1234.62

203-218

H

COOH

CH3

H

H

1232.62

Metabolite cs

18 21

modifications weight

R3

R4

H H

H

1202.64

H

1218.64 1234.64

hydroxyl ated and N-demethyl ated derivative of cs OH

1220.62 1234.64 1204.62

TABLE 111 Characteristics of various HPLC procedures for quantitative determination of cyclosporine

Extraction Ref. matrix preparation

extraction

117 plasma + water urine

diethyl ether

118 plasma

cv

HPLC clean-up recovery column elution det.-limit (pg/1 1

-

76+5% 10455%

RP8

gradient

extraction identical with ref. 117

n.r.

RP8

isocratic

5

119 blood + distilled plasma water

diethyl ether

hexane

74% 49%

RP18

isocratic

25

9.2

120 serum

C18 cartridge (Sep-Pak, Waters)

water/ 90+10% methanol

RP18

isocratic

n.r.

n.r.

step gradient

25

3

RP18

gradient

50

77.356%

TMS

isocratic

100

n.r.

83-99%

RP8 isocratic ultrasphere

31

3.6-

methanol

121 plasma heat (55OC), blood freeze + thaw

column-switching

122 serum

CN cartridge (Baker)

123 plasma

Clin Elut

91.9+0.9% RP8, RP18

water/ 50-70% methanol

20

comments

%

n.r.

4.4 derivatization o f cyclosporine with 2naphthyleneselenylchloride

9.314.1

cartridge (Fisher) 124 plasma acidification diethyl ether blood (HC1)

NaOH

6.0

TABLE I I I 125

blood serum

Tris-buffer pH 9.8

126/ plasma 127 blood

81 blood

c

(conti nued)

0 N

diethyl ether/ aceton- 34.7% C N cartridge nitrile/ (Baker) water, hexane

acetonitri le/water, column-switching freeze + thaw acetonitrile acetonitrile

128

blood freeze+thaw diethyl ether plasma buffer pH 10

129

serum

phosphoric colum-switching acid in acetonitri le

130

blood

freeze+thraw charcoal slurry acetonitrile ethyl acetate

131 blood,

plasma 132

blood

-

diethyl ether

10% isoC18 cartridge propanol in (Baker Bond) acetonitri le

RP8

isocratic

25

21

RP8, RP18

columnswitching

5 15

0.511.1

hexane, 90%+5% Dowex ion exchange resin

RP18

isocratic

25

acidifi- 74% cation, 85% hexane

RP18

isocratic

25

n.r.

RP8, RP18

columnswitching

n.r.

RP18

isocratic

50

CN

isocratic 100

6.04

isocratic

n.r.

hexane

100%

80%

alkalized 96+6% acidi fi ed met h ano 1 70%

methanol

automated sample preparation

0.38.0

86+108% C N

50

7.0

2.512.5

8.6

modification of refs. 126, 127

TABLE 111

(continued)

110 blood acetonitrile/ CN cartridie serum, water (Bond Elut ) plasma (30/70,v/v)

acetonitrile/ acetic acid

133 blood

acetonitrile/ C18cartridfle di methy 1 (Bond Elut ) sulfoxide

acetoninitrile water, hexane

134 blood

--

acidification, hexane

n.r.

135 blood

acetonitrile columnswitching

acetonitrile/ water

75~3% Ultrfl- columnpore switching RPSC (Altex) RP18, 3 vm

136 blood

HC1

NAOH

diethyl ether

diethyl ether

90% 98%

CN

isocratic

15

75-

CN

i socrati c 96%

3 ccm

3.8- extraction with ad12.5 vanced automatic sample unit AASP (Varian), normal phase chromatography 5.6

determination of metabolite 1, 17, 18, 21

TABLE

II I

(conti nued)

I.46 blood,

diethyl ether

silica 45~2% cartridge (Sep Pak, Waters), acetylacetate/ hexane

RP18

isocratic

1020

6

diethyl ether

heptane, 70% NaOH , hexane

RP8, 3 Pm

isocratic

25

5.311.5

acetoni- 47trile/ 95% water

CN isocratic 15a1 ternat i vely 25 RP 8, silica gel semi-preparative isolation of metabolites

acetoni- 73trile/ 85% water, hexane

RP8

plasma

147 blood

148 blood

HC1

acetonitrile/ CN cartridRe water (30/70) (Bond Elut )

149 blood, acetonitrile/ C8 extraction 150 bile, water(30/70) columns 151 urine

n.r.: not reported, RP: reversed phase.

gradient

25

7.1 determination of 9.6 metabolite 1 , 8, 13, 17, 18, 21, 25, 26, 203-218 and 1 yet unidentified metabolite

5.6 12.6

determination o f metabolite 1, 8, 9, 10, 13, 16, 17, 18, 21, 25, 26, 203-218 and 7 yet unidentified metabolites

106

BLOOD SAMPLE PREPARATION FOR SANDIMMUN~ (CYCLOSPORINE) MEASUREMENT 3.2 3.2.1 LIQUID-LIQUID EXTRACTION PROCEDURES A l l methods published u n t i l now use

c y c l o s p o r i n e C o r 0 as i n t e r n a l

standard. The e x t r a c t i o n procedures c o n s i s t o f f o u r steps:

1. hemolysis and d e p r o t e i n a t i o n , 2. e x t r a c t i o n o f cyclosporine, 3. sample p u r i f i c a t i o n , 4. volume r e d u c t i o n and t r a n s f e r i n t o t h e m o b i l e phase Hemolysis was achieved by r a p i d thawing and f r e e z i n g ( r e f . adding d i s t i l l e d water ( r e f . c h l o r i c a c i d (137).

119), a c e t o n i t r i l e ( r e f .

129) o r by

81) and hydro-

I n r o u t i n e a n a l y s i s c y c l o s p o r i n e was e x t r a c t e d from b l o o d by d i e t h y l methyl-t-butyl ether (ref. e t h e r ( r e f s . 117,119,124,127,128,136-138),

142) and a c e t o n i t r i l e ( r e f . 81). The advantage o f m e t h y l - t - b u t y l e t h e r over d i e t h y l e t h e r a r e i t s r e s i s t a n c e t o peroxide f o r m a t i o n and c l e a n e r e x t r a c t s than obtained by d i e t h y l e t h e r e x t r a c t i o n ( r e f . 142). The use of

acetonitrile

combines

s o l v e n t and i t s p r o t e i n

i t s p r o p e r t i e s as

an e f f e c t i v e e x t r a c t i o n

p r e c e p i t a t i n g potency

(ref.

81). Since t h e

e x t r a c t s c o n t a i n i n t e r f e r i n g l i p o p h i l i c m a t e r i a l and a c i d i c , b a s i c and i o n i c contamination ( r e f .

81) which may cause damage t o t h e column,

p u r i f i c a t i o n steps are required. P u r i f i c a t i o n was achieved by washing t h e sample w i t h hexane o r heptane ( r e f s . 119,128,134,137,139,142)w i t h a c i d i c and basic s o l u t i o n s ( r e f s . 124,128,131,134,142) o r by adding i o n exchange r e s i n s ( r e f . 81). A f t e r e v a p o r a t i o n o f t h e p u r i f i e d l a y e r and resuspension i n t h e mobile phase, some methods use a second p u r i f i c a t i o n s t e p by e x t r a c t i n g i n t e r f e r i n g substances w i t h a f i n a l wash ( r e f s .

hexane o r heptane

137, 142). Back e x t r a c t i o n o f c y c l o s p o r i n e from an aqueous

phase by changing pH i s n o t p o s s i b l e because o f i t s chemical p r o p e r t i e s . Thus t h e organic l a y e r c o n t a i n i n g c y c l o s p o r i n e i s washed by b a s i c and a c i d i c s o l u t i o n s and t h e aqueous l a y e r has t o be discarded i n e i t h e r case. G f e l l e r e t . a l . ( r e f . 118) used a d e r i v a t i z a t i o n o f c y c l o s p o r i n e w i t h 2-naphthylselenylchloride t o improve t h e d e t e c t i o n l i m i t . The method o f Sawchuck and C a r t i e r ( r e f .

119) i n t r o d u c e d a hexane

washing s t e p i n t o c y c l o s p o r i n e a n a l y s i s and many l a t e r p u b l i s h e d l i q u i d l i q u i d e x t r a c t i o n methods used a m o d i f i c a t i o n o f t h i s e x t r a c t i o n p r o cedure ( r e f s . 128,137,139,142). Blood, d i s t i l l e d water and t h e i n t e r n a l standard Cyclosporine D were g i v e n i n t o a c e n t r i f u g e tube. D i e t h y l e t h e r was added and t h e sample shaken and c e n t r i f u g e d . The aqueous phase was discarded and t h e organic l a y e r was evaporated. The sample was taken up

107 i n methanol and was washed w i t h hexane t w i c e . The hexane l a y e r s were removed, t h e aqueous l a y e r was b a s i f i e d w i t h NaOH and c y c l o s p o r i n e was e x t r a c t e d by d i e t h y l e t h e r . The d i e t h y l e t h e r phase was evaporated and t h e re ma ining m a t e r i a l s were r e c o n s t i t u t e d w i t h t h e m o b i l e phase. Most

of

these

extraction

Cy c los porin e D o r C.

procedures

use

an

internal

st andard:

Cyclosporine D i s cyclosporine w i t h v a l i n

and

c y c l o s p o r i n e C w i t h t h r e o n i n e as amino a c i d 2 (F ig. 3 ) . These c y c l o s p o r i n e d e r i v a t i v e s r e p r e s e n t o n l y a s m a l l m o d i f i c a t i o n o f t h e whole molecule. They

have

distribution

coefficients

in

organic

solvents

equal

to

c y c l o s p o r i n e and almost t h e same UV-absorbing p r o p e r t i e s . The use of t h e s e i n t e r n a l s t a ndar d s f o r t h e q u a n t i f i c a t i o n o f c y c l o s p o r i n e m e t a b o l i t e s i s critical

(ref.

152).

The b e h a v i o r d u r i n g e x t r a c t i o n

i s considerably

d i f f e r e n t f rom t h e m e t a b o l i t e s as shwon i n b i l e i n r e f . 151 and Table V . 3.2.2 SOLID-LIQUID EXTRACTION PROCEDURES U n t i l now a l l column e x t r a c t i o n

methods described f o r c y c l o s p o r i n e i n -

cl uded 5 s t e ps : 1. Hemolysis o f t h e c o r p u s c u l a r b l o o d i n g r e d i e n t s and d e p r o t e i n a t i o n , 2. sample l o a d i n g on t h e e x t r a c t i o n column, 3. sample p u r i f i c a t i o n , 4. e l u t i o n o f c y c l o s p o r i n e and i t s m e t a b o l i t e s f rom t h e e x t r a c t i o n column,

5 . volume r e d u c t i o n f o r HPLC a n a l y s i s . Yee e t a l . ( r e f . 122) used a p r o t e i n p r e c i p i t a t i o n s t e p w i t h a c e t o n i t r i l e c o n t a i n i n g t h e i n t e r n a l s t a n d a r d C y c l o s p o r i n e D. The sample was t h e n p u l l e d by vacuum through a prepacked d i s p o s a b l e cyanopropyl column, b e i n g washed w i t h a c e t o n i t r i l e and w a t e r . The column was washed w i t h methanol/ wat e r 40/60 ( v / v ) and c y c l o s p o r i n e was e l u t e d u s i n g methanol. Kates e t a l . ( r e f . 125) combined a d i e t h y l e t h e r e x t r a c t i o n w i t h p u r i f i c a t i o n on

prepacked d i s p o s a b l e cyano columns.

Blood samples were

a d j u s t e d a t pH 9.8 and e x t r a c t e d w i t h d i e t h y l e t h e r . D i e t h y l e t h e r was evaporated, t h e sample d i s s o l v e d i n methanol/water was d i l u t e d w i t h w a t e r and drawn t hro ug h t h e column w i t h water. The sample was subsequent ly cleaned by a c e t o n i t r i l e / w a t e r 25/75 ( v / v ) and hexane was t hen e l u t e d f r o m t h e column w i t h methanol. The method developed i n o u r l a b o r a t o r y

(refs.

149,150,151,

Fig.

4)

uses 3 m l g l a s e x t r a c t i o n columns f i l l e d w i t h 25-40 p RP 8 m a t e r i a l R (L iC hro pre p , Merck, Darmstadt, FRG). The i n t e r n a l st andard C y c l o s p o r i n e D was d i s s o l v e d

in

a c e t o n i t r i l e / w a t e r (pH 3.0) 50/50 ( v / v ) a t a concen-

108

tration of 10 @/ml. 25 pl of the internal standard solution were pipetted into a 10 ml centrifuge tube. Subsequently 1 ml blood and 2.1 ml acetonitrile/water (pH 3.0) (30/70 v/v) were added. Each sample was vortexed for 20 s and centrifuged for 5 min at 2 500 rpm. The supernatant was pulled by vacuum through the extraction columns. The extraction columns were previously primed with 3 ml acetonitrile and 3 ml water. The samples were washed with 3.2 ml acetonitrile /water (pH 3.0) (20/80 V/V) and with 0.5 ml hexane. The column was dried by sucking air through it for 1 min. To elute cyclosporine and its metabolites the extraction column was set into a diethyl ether cleaned 10 ml centrifuge tube and 2 ml dichloromethane was centrifuged through the extraction columns (700 rpm, 5 min). Dichloromethane was evaporated and the remaining materials were taken up in 300 pl acetonitrile/ water (pH 3.0) (50/50 v/v). 500 d hexane were added and the sample was vortexed for 10 s. Phases were separated and 100 ml of the aqueous phase were injected into the HPLC system. This extraction procedure is a modification of the method pub1 ished by Lensmeyer and Fields (ref. 110). The first step of the extraction procedure consists of adding a mixture of acidified water (pH 3.0) /acetonitrile (30/70 v/v) resulting in a final acetonitrile concentration of 20% in the sample. According to ref. 110 gross protein precipitation occurs at a final acetonitrile concentration of more than 21%. At the acetonitrile concentrations reached in the sample blood cells are hemolysed and some protein blood components precipitate. The recovery is considerably lower at a higher pH of the dilution mixture. The recovery drops to about 20% when gross protein precipitation occurs due to plugged extraction columns. Critical conditions for gross protein precipitation are high temperatures over 25OC as reached when centrifuging the sample in a warm centrifuge. Another reason for a decreased recovery is the extraction of deep frozen or samples stored at +4OC for more than 1 week. The first step also adjusts the sample to the conditions required for column extraction. After centrifugation the supernatant which has a clear red color is given onto the extraction columns. We chose no commercially available disposable prepacked columns but refillable glas columns with removable teflon frits for the following reasons: 1. To reduce costs of external column extraction procedures the ex-

traction columns are reused. The more often they are reused the more reproducibility and recovery decrease and the chance of loosing a

109 sample because o f a plugged column increases. A f t e r a n a l y s i s t h e s o l i d phase o f t h e g l a s s columns i s removed and t h e f r i t s reusable f o r a t l e a s t t h r e e times a r e cleaned by u l t r a - s o u n d i n a c e t o n i t r i l e . 2. One o f t h e main problems o f c y c l o s p o r i n e a n a l y s i s a r e i n t e r f e r i n g m a t e r i a l s l i k e p l a s t i c s o f t e n e r s which have s i m i l a r chromatographic and s p e c t r a l p r o p e r t i e s l i k e c y c l o s p o r i n e i t s e l f . I t has been r e p o r t e d t h a t i n t e r f e r i n g m a t e r i a l can be leached from t h e e x t r a c t i o n columns. Glass i s an i n e r t m a t e r i a l . A ' d i g i t a l chromatography' o f c y c l o s p o r i n e on t h e e x t r a c t i o n columns i s

n o t p o s s i b l e r e s u l t i n g i n v a r i a b l e r e c o v e r i e s o f 7 0 4 5 % i n o u r system. I n our method t h i s v a r i a b i l i t y can be compensated by u s i n g an i n t e r n a l standard (Table I V ) . The columns a r e f i l l e d w i t h 100 mg RP 8 s o l i d phase m a t e r i a l . V a r i a t i o n o f t h e packing volume up t o 50% does n o t i n f l u e n c e recovery. pH adjustment o f t h e sample t o an a c i d pH increases r e t e n t i o n on t h e columns e s p e c i a l l y o f t h e c a r b o x y l a t e d m e t a b o l i t e 203-218.

After

l o a d i n g c y c l o s p o r i n e and i t s m e t a b o l i t e s o n t o t h e e x t r a c t i o n columns, t h e

(80/20 v / v ) m i x t u r e , decreasing t h e amount o f p o t e n t i a l l y i n t e r f e r i n g m a t e r i a l . During t h i s s t e p t h e recovery i s n o t reduced when t h e water i s a c i d i f i e d . The n e x t sample i s washed w i t h an a c e t o n i t r i l e / w a t e r (pH 3.0)

step, washing t h e column w i t h hexane i s t o remove l i p o h i l i c i m p u r i t i e s . c y c l o s p o r i n e and i t s m e t a b o l i t e s a r e almost i n s o l u b l e i n hexane. Up t o t h i s s t e p t h e s o l v e n t s are sucked through t h e column by vacuum. To e l u t e t h e compounds o f i n t e r e s t from t h e e x t r a c t i o n column t h e columns a r e s e t i n c e n t r i f u g e tubes.

The e l u e n t dichloromethane i s c e n t r i f u g e d t h r o u g h

t h e e x t r a c t i o n columns and t h e e l u a t e c o n t a i n i n g c y c l o s p o r i n e and i t s metabolites

i s collected a t

the

bottom o f t h e c e n t r i f u g e

tube.

In

c o n t r a s t t o o t h e r e l u e n t s l i k e methanol o r a c e t o n i t r i l e dichloromethane can f a s t e r be evaporated, The amount o f c o e l u t e d i n t e r f e r i n g m a t e r i a l i s equal t o an e l u t i o n by o t h e r s o l v e n t s . For evaporation o f dichloromethane an apparatus equipped w i t h g l a s s tubes f o r n i t r o g e n i n s u f f l a t i o n should be used, s i n c e p l a s t i c tubes a r e a p o t e n t i a l source o f p o l l u t i o n of t h e sample w i t h p l a s t i z i s e r s (Fig.

10). The f i n a l hexane wash used i n o u r

method removed i n t e r f e r i n g m a t e r i a l , stemming from t h e l a b o r a t o r y equipment. This step was n o t e s s e n t i a l b u t i t made e x t r a c t i o n more re1 i a b l e . With s l i g h t m o d i f i c a t i o n s t h e method c o u l d be adapted t o analyse u r i n e and b i l e samples. 1 m l u r i n e was p i p e t t e d i n t o a 10 m l c e n t r i f u g e t u b e and 300 pl a c e t o n i t r i l e were added. A f t e r c e n t r i f u g a t i o n t h e supernatant

110 was passed through t h e e x t r a c t i o n columns. 1 m l o f a b i l e sample and 2 m l a c e t o n i t r i l e / w a t e r (pH 3.0) acetonitrile/water

(pH 3.0)

discarded.

loading

After

(30/70 v/v) were washed w i t h 3 m l hexane. The phase was separated and t h e hexane l a y e r cyclosporine

and

its

metabolites

on

the

e x t r a c t i o n columns the e x t r a c t i o n procedure was continued as described f o r blood samples.

1 m l blood + 25 pl i n t e r n a l standard (containing 250 ng cyclosporine D) + 2.1 m l a c e t o n i t r i l e / w a t e r (pH 3.0) 30/70 (v/v)

I I e x t r a c t i o n column

+ 3.2 m l a c e t o n i t r i l e + 3.2 m l water (pH 3.0)

shake 20 s (vortex-mixer) c e n t r i f u g e 5 min, 2500 rpm

I I

I draw supernatant through t h e e x t r a c t i o n column

I

-

+ 3.2 m l a c e t o n i t r i l e / w a t e r (pH 3.0) (20/80 v/v) + 0.5 m l hexane d r y column by a i r stream

I

c e n t r i f u g e 2.0 m l dichloromethane through t h e column

I

remove column m a t e r i a l teflon f r i t s

I clean

I evaporate e l u a t e a t 50°C under a stream o f n i t r o g e n

I + 0.3 m l a c e t o n i t r i l e / w a t e r (pH 3.0)

(50/50 v/v)

+ 0.5 m l hexane

I shake 20 s (vortex-mixer) c e n t r i f u g e 2 min, 2500 rpm

I i n j e c t 75 pl i n t o HPLC-system Fig. 4

Extraction o f cyclosporine and i t s metabolites from blood by s o l i d l i q u i d e x t r a c t i o n ( r e f s . 149, 150, 151)

111

Modifications for the extraction of urine samples 1 ml urine + 25 pl internal standard

+ 300

pl

acetonitrile

I vortex mix (40s) centrifuge 2 min 2500 x g

I (extraction continued like blood) Modifications for the extraction o f bile samples 1 ml bile + 25 pl internal standard + 2.1 ml acetonitrile/water (pH 3.0) + 2 ml hexane

(30/70 v/v)

I vortex mix (1 min) centrifuge 2 min 2500 x g

I discard hexane layer

suck acetonitrile/water phase through extraction columns

I (extraction continued like blood) Reproducibility, linearity and detection limit of the method used in our laboratory are listed in Table I V . TABLE I V

Calibration curve, detection limit and CV of the method described above Calibration curve range checked

blood bile urine

0-3

mg/l 0-6 mg/l 0-30 mg/l

r 1.o

0.989 0.996

detection limit 25 c9/1 50 @/1 50 d l

cv 6.3% 7.2% 12.3%

112

The CV includes the variation of the cyclosporine metabolites. The recovery in blood ranged from 72-85% with an average of 79.2%. The recoveries of the metabolites 8 , 26, 17 and the internal standard Cyclosporine D are shown i n table V. The cyclosporine metabolites, the parent compound and the internal standard differ i n their lipophilic properties. Thus, the possibility must be taken into account that the recoveries of these compounds are not identical during the extraction procedure. In the table it is shown that the recovery of the internal standard i s signi fi cantly (p 0 ) , t h e y a r e n o r m a l l y r e f e r r e d t o as e i t h e r y-or & d e t e c t o r s , m a i n l y because o f l a r g e d i f f e r e n c e s i n E f o r t h e two t y p e s o f r a d i a t i o n . The c hoic e o f d e t e c t o r f u r t h e r depends on t h e s t a t e o f t h e sample (gaseous, l i q u i d o r s o l i d ) t o b e counted, t h e n u c l e a r e n e r g i e s o f t h e r a d i o i s o t o p e s i n v o l v e d and, w i t h m i x t u r e s o f y - e m i t t i n g i s o t o p e s , a b i l i t y t o d i s c r i m i n a t e between d i f f e r e n t y-energies.

the

For t h e l a t t e r ,

Ge (L i) spectroscopy i s n o r m a l l y p r e f e r r e d above NaI ( T l ) because o f i t s s u p e r i o r e n e r g y - r e s o l v i n g power ( F i g . 1). De t e rminat io n o f & r a d i o a c t i v i t y

i n l i q u i d s i s r e l a t i v e l y inconvenient

because o f ( i ) t h e small range o f B p a r t i c l e s (see T able 111) which f o r e f f i c i e n t d e t e c t i o n n e c e s s i t a t e s c l o s e c o n t a c t between sample and d e t e c t o r medium, and (ii)t h e c o n t i n u e o u s energy d i s t r i b u t i o n (F ig. 4A, B,

C).

The I3 s p e c t r a a r e s p e c i f i e d by t h e maximum end p o i n t energy

E (max) , and r a d i o i s o t o p e s a r e c o r r e s p o n d i n g l y c l a s s i f i e d as ' l o w ' ( 3 H I 6'Ni),

'medium' (14C,

35S) o r ' h i g h ' (32P, 3 6 C l ) energy e m i t t e r s . As a

consequence, i n d i v i d u a l q u a n t i f i c a t i o n i n m i x t u r e s o f p u r e & e m i t t e r s

is

possible only f o r isotopes w i t h l a r g e differences i n E as i s t h e case 3 32 3 14' i n t h e d u a l - i s o t o p e d e t e r m i n a t i o n o f , f o r example, H/ C o r H/ P. 8 - s c i n t i 11a t i o n c o u n t i n g Low t o medium energy

a r e n o r m a l l y determined by l i q u i d o r heterogeneous ( s o l i d ) s c i n t i l l a t i o n c o u n t i n g (LSC and HSC, r e s p e c t i v e l y ) . I n LSC ( r e f s . scintillator

0's

18, 19), t h e sample i s t h o r o u g h l y mixed w i t h a l i q u i d c o c k t a i l . The c o c k t a i l i s u s u a l l y based on t o l u e n e ,

pseudocumene o r dioxane t o which f l u o r o f o r s such as 1. 4-diphenyloxazole (PPO)

and/or

1.4-bis-2(5-phenyloxazolyl)benzene(POPOP)

and s o l u b i 1 i z e r s

have been added, t h e i r n a t u r e depending on t h e sample t y p e t o be i n c o r p o ra t e d: a l c o h o l s o r n o n - i o n i c d e t e r g e n t s such as T r i t o n X-100 can be used

144 as solubilizers for aqueous samples. The sovent molecules absorb the incident nuclear energy to form an electronically excited state. This energy is transferred to the fluorofor (the scintillator) which returns to its electronic ground-state by emission of photons (scheme 1). Within several nanoseconds, 20-30 (depending on the incident C-energy) photons are produced per desintegration which are isotropical ly emitted. This enables coincidence counting techniques to be used (Fig. 10): The sample is placed between two opposite PM tubes, the signals of which are fed to an electronic circuit and checked for coincidence within a pre-set coincidence time. Coincident pulses are counted only. To some extent it is thus possible to distinguish between scintillation pulses and one-photon events, such as chemi luminescence. The overaleffect of the coincidence circuit i s to lower the background count rate, CPM(b). The coincidence time may be chosen from several tens to hundreds of ns, depending on the type of scintillator and sample. Too short times, however, may adversely affect the counting efficiencies, and a compromise between optimum E and CPM(b) must be found experientally. Energy pulse height analysis is another means to improve the E/CPM(b) ratio, by setting lower and upper energy discriminators of the window. The lower discriminator is used for reducing low-energy back-ground pulses. Pulse height analysis is also used in dual-isotope detection with two-energy windows chosen, in which counts are collected (Fig. 4A; see also refs. 20, 127). The activity from both isotopes is determined after correction for counts, collected from the high-energy isotope in the low-energy window (cross-over correction). The same detection device can be used for HSC. In HSC, the sample is in contact with solid scintillator, for example granular anthracene, inorganic glasses such as yttrium silicate or europium-doped calcium fluoride or plastic scintillator, doped with organic fluorofors. The C-spectra from the glasses (see Fig. 4C) are distinctly different form spectra recorded in LSC, with the maxima shifted to higher energy. This facilitates discriminating radioactivity signals and low-energy pulses arising from PM darkcurrent or chemi luminescence. Cerenkov counting High energy 0's with Eb(max) of over about 0.3 MeV can be detected by scintillation and also at satisfactory efficiencies (0.2 < E < 0.4) via 24 Cerenkov radiation, produced on the interaction of these R ' s ( Na, 32P, 36Cl, 40K) with solvents of high dielectric constant such as, e.g., water

(refs.

18, 19). The wavelength range o f t h i s t y p e o f r a d i a t i o n i s from i n t o the v i s i b l e , but with largest i n t e n s i t y i n the

the u l t r a v i o l e t

short-wavelength

region.

Therefore,

t h e PM tubes should p r e f e r a b l y be

equipped w i t h UV-transparent q u a r t z windows. can be added t o t h e sample,

Alternatively,

fluorofors

t h e f u n c t i o n o f which i s t o accept t h e

Cerenkov r a d i a t i o n and s h i f t t h e emission wavelength t o h i g h e r values i n o r d e r t o match t h e maximal s e n s i t i v i t y o f t h e PM tubes. Cerenkov c o u n t i n g has t h e obvious advantage t h a t no s c i n t i l l a t o r c o c k t a i l i s needed and t h e sample can be recovered unmodified. 40-

,..- 30-

'E

m

-0 ;

20-

$

1

s

3 10

-

, OLS,

Scheme 1

2.2

-

Coupled energy t r a n s f e r and energy l e v e l diagram f o r t h e components i n a t e r n a r y l i q u i d s c i n t i l l a t i o n system (Reproduced w i t h permission from r e f . 19, Amersham, England, 1977).

PRINCIPLES OF FLOW-THROUGH y-COUNTING As w i l l be i l l u s t r a t e d below i n s e c t i o n 4.1 i t f o l l o w s t h a t y -column

l i q u i d chromatography i s used p r i m a r i l y f o r 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 purposes i n t h e s y n t h e s i s o f r a d i o t r a c e r s . a c t i v i t i e s loaded on t h e column,

Due t o t h e h i g h

flow-through y - d e t e r m i n a t i o n

becomes

s t r a i g h t f o r w a r d , a l l o w i n g t h e use o f f l o w c e l l volumes i n t h e o r d e r of 10

4 . Thereby, t h e chromatographic i n t e g r i t y i s preserved w h i l e m a i n t a i n i n g s u f f i c i e n t p r e c i s i o n i n c o u n t i n g r e s u l t s . As o n l y one r a d i o i s o t o p e has t o be detected, energy r e s o l u t i o n i s o f l e s s concern. I n these s i t u a t i o n s , s c i n t i l l a t i o n c o u n t i n g (SC) i s adequate.

146

I

i

n

I

I

I

I

I

I

I I

I

I I

I

Fig. 4A, B Liquid scintillation spectra o f 3 H (A) and 14C ( B ) , recorded for unquenched and quenched samples on a Philips PW 4700 scintillation counter.

147

C ? $

1

7-

0

----I

IW

50

260 ----

150

250

Pulsa Heiphl (Channel)

F ig. 4C

CaF (Eu) s c i n t i l l a t i o n spectrum f o r 14C, recorded w i t h o u t COi n c f d e n c e ( A ) , t h r o u g h c o i n c i d e n c e (0) and a n t i - c o i n c i d e n c e (X) ( R e p r i n t e d from t h e J . Chromatogr., E l s e v i e r , f rom r e f . 21).

.

T h a l l i u m - a c t i v a t e d sodium i o d i d e (NaI(T1)) i s by f a r t h e most p o p u l a r s c i n t i l l a t o r ( r e f . 22).

I t i s r e l a t i v e l y i nexpensive, r e a d i l y a v a i l a b l e

i n d i f f e r e n t geometries and i s a p p l i c a b l e t o a broad range o f y-energies a t s a t i s f a c t o r y c o u n t i n g e f f i c i e n c i e s . The p e n e t r a t i o n power o f t h e y-rays i n t h e c r y s t a l i n c r e a s e s w i t h i n c r e a s i n g energy, and high-energy 7's may escape f r o m t h e m a t e r i a l w i t h o u t g e n e r a t i o n o f s c i n t i l l a t i n g p u l ses. Large c r y s t a l s i z e s , however, a r e i n c o n v e n i e n t because o f t h e ext ens i v e s h i e l d i n g necessary t o reduce t h e background count r a t e , CPM(b). A c y l i n d r i c a l 7.6 values

x 7.6 cm NaI(T1) c r y s t a l can y i e l d e f f i c i e n c i e s w i t h

0.10-0.30

of

for

energies

of

90-600

keV,

with

an

optimum

performance a t 90-160 keV. Flow c e l l s can b e c o n s t r u c t e d f r o m PTFE w i t h low y-energies (up t o 100 keV) o r s t a i n l e s s - s t e e l c a p i l l a r i e s w i t h h i g h e r y - e n e r g i e s . They s h o u l d be f i x e d p r e f e r a b l y i n t h e c e n t r a l d e p l e t i o n ( w e l l ) o f t h e c r y s t a l f o r h i g h and r e p r o d u c i b l e c o u n t i n g geometry ('471 I c o u n t i n g ) . Because o f t h e h i g h p e n e t r a t i o n power o f y - r a d i a t i o n , t h e c r y s t a l should be l e a d shielded

from

artificial capi 11a r i es

band

.

the

environment.

broadening

Improper

originating

shielding

f rom

activity

may

result

in

in

connecting

An i l l u s t r a t i v e b l o c k diagram o f t h e apparatus i s g i v e n i n F i g . 5 . A f t e r i n j e c t i o n , t h e i n j e c t e d p l u g i s f i r s t transported through a

148 stainless-steel flow cell which measures the total radioactivity of the injected sample. Subsequently, separation takes place and the column eluate is led through a second flow cell to monitor the radiogram. By comparison of the signals from the first and second flow cell, the recovery of activity from the column can be established. The principle can also be used to indicate detector overload resulting from deadtime losses in the radioactivity detector. It is therefore recommended that the count rate in the flow cell should not exceed 10,000 CPS.

Fig. 5

Apparatus for flow-through NaI (T1)-scintillation counting with pre- and post-column y-counting. 1 = injector, optional: 2 = fraction collector, 3 = strip-chart recorder, 4 = ratemeter, 5 = multi-or single-channel analyzer, 6 = frequency/voltage converter. (Pinciple adapted from ref. 22).

In Fig. 6, an HPLC chromatogram of rare-earth radionuclides i s given with flow-through NaI(T1) counting. The system was used in the investigation o f the short-lived fission products of 252Cf. Some alternatives for flow-through NaI (Tl) detection have been published. Simonnet et al. (ref. 24) adapted a commercially available flow-through radioactivity detector for the determination of lZ5I-labeled proteins. It was made of a 0.76 mm i.d. polyethylene capillary,

149 p o s i t i o n e d i n a 20 m l sample v i a l f i l l e d w i t h l i q u i d s c i n t i l l a t o r

in

f r o n t o f two c o i n c i d e n t PM t u b e s ( e x t e r n a l SC). A c o u n t i n g e f f i c i e n c y of 0.42 was found a t CPM(b) = 465. It i s a n t i c i p a t e d t h a t t h i s p r i n c i p l e can be a p p l i e d f o r d e t e c t i o n o f o t h e r low-energy

- o r X-ray

emitters.

I PI

I 0

I

I

I

I

I

I

I

1

2

3

4

5

6

7

8

T ~ m e(nun)

F ig. 6

Flow-through+i$I(Tl) radiogram o f s h o r t - l i v e d pro duc t s o f C f . Column, Aminex A-9 (150 x pH- and c o n c e n t r a t io n - g r a d i e n t e l u t i o n w i t h a c i d f ro m 0.65 M (pH 3.6) t o 0.95 M (pH (Re prin t e d w i t h p e r m i s s i o n f r o m t h e J . E l s e v i e r , r e f . 23).

rare earth fisaion 3.2 mm, a t 95 C); w h y d r o x y i s o b u t y r ic 4.8), 1.0 ml/min. Radioanal. Chem.,

Nowadays most commercial f l o w - t h r o u g h d e t e c t o r s have p r o v i s i o n f o r e x t e r n a l SC, which a r e n o r m a l l y s p e c i f i e d f o r 1251 and 9gmTc o n l y . The f l o w c e l l s a r e made o f PTFE c a p i l l a r y , encapsuled i n l i q u i d , p l a s t i c o r NaI(T1) s c i n t i l l a t o r .

Few papers on t h e i r use have been published.

A

c o u n t i n g e f f i c i e n c y o f 0.05 o n l y has been c a l c u l a t e d f o r t h e 511 keV a n n i h i l a t i o n r a d i a t i o n of 13N w i t h a p l a s t i c s c i n t i l l a t o r f l o w c e l l ( r e f . 25) a t CPM(b) = 60. Th i s p r i n c i p l e was a l s o used i n t h e d e t e r m i n a t i o n o f homogeneous [ m o n ~ - ~ ~ ~ I - T y rand l ~ ] - [ m ~ n o - ~ ~ - ~ I - T y r ~ ~ - ] - g l u c a( ag fot ne r HPLC s e p a r a t i o n ) ( F i g . 25). Langstrom and

Lundqvist

stainless steel f o i l

(ref.

(thickness,

27)

0.07

constructed

a flow

cell

from

mm) surrounded by 1.5 mm t h i c k

p l a s t i c s c i n t i l l a t o r f o r t h e d e t e r m i n a t i o n o f high-energy p o s i t r o n e m i t t e r s , which f o r "C

r e s u l t e d i n E = 0.40 a t CPM(b) < 180.

150 Flow-through y -determination based on semiconductor detectors is rather rare (refs. 28, 29). Needham and Delaney (ref. 28) utilize a small-volume CdTe semiconductor (2.6 x 2.6 x 2.0 mm housed in a 5 mm i.d. x 12 mm long aluminium cylinder). The compactness of this detector device even allowed positioning against the heat exchanger coil of an RI detector. Unfortunately, the counting efficiencies are rather low and decrease rapidly with increasing energy; E values of 0.10, 0.02 and 0.013 are found for the 93, 185 and 300 keV photopeaks of 67Ga, respectively, and of 0.24 for the 140 keV photopeak of 99mTc. For this isotope, the relative quantitative sensitivity RSQ (as defined in ref. 30) was 30-fold worse as compared to that in NaI(T1) detection. This was attributed to the better counting geometry of the flow cell in the NaI(T1)-well. 2.3 PRINCIPLES OF 8-COUNTING IN COLUMN LIQUID CHROMATOGRAPHY 2.3.1 INTRODUCTION Except for the most important radioisotopes for biomedical and pharmaceutical research, notably 3H, 14C, 35S and 32P, are pure (100%) 8-emitters. Their relatively long half-lifes facilitate the radiosynthesis of a wide variety o f compounds with no time restrain on subsequent experiments, which makes them excellent radiotracers in recovery and metabolic profiling studies of organic compounds. Developments in 8-column liquid chromatography up to 1976 have been reviewed by Roberts (ref. 12). Until then, most workers used off-line LSC. Flow-through detectors, when available, were assembled largely from readily available electronic components, such as high-voltage supply, PM tubes, amplifiers, pulse height channel analyzers and coincidence circuits. The remaining equipment (flow cells, light-tight housings) were home-made. In the following sections, basic principles and apparatus for 8determination in column liquid chromatography are given. A more detailed treatment of performances and alternative methods is given in section 3. 2.3.2 OFF-LINE LIQUID SCINTILLATION COUNTING (LSC) In off-line LSC, successive eluate fractions are collected in sample vials on a regular time basis. After thorough mixing of the fractions or of aliquots of the fractions with LC, the vials are placed on a tray and counted separately. Plotting of the counts/fraction or the count rate/fraction versus the fraction number gives the radioactivity distribution (radiogram) in the column eluate.

151

Despite its relatively simple operation and good counting performance, the total procedure of collection, counting and data analysis is rather laborious and difficult to automate. Moreover, the preservation of the chromatographic resolution strongly depends on the fraction volume chosen, as is illustrated in Fig. 7 and refs. 25, 32, 33, 34. Small fraction columes are beneficial in this respect, but increased counting times/fraction are required to maintain precision in the counting results, and the total analysis time may become immoderate. For these reasons, it may be concluded that, in general, off-line LSC is not applicable to high-efficiency HPLC, especially when high sample throughputs are required. It has further been shown in reversed-phase HPLC, that collection of small (< 0.2 ml) aqueous fractions becomes more capricious if increasing percentages of water are present in the eluent (ref. 35). This has been explained by fluctuations in surface tension of the droplets, which led to significant differences in the collected volume/fraction. Some of these difficulties can at least partially be avoided by collecting total peak volumes. For this, the output of any type of flow-through detector may be triggered to recognize the beginning and end of an eluting peak, which controls an automatic fraction collector. However, the number and positions of peaks normally identified by liquid chromatography detectors are by no means representative for the actual radioactivity distribution in the chromatogram, as can be seen from Figs. 2, 9 and 11. Of course, no problems in selectivity exist when using flow-through radioactivity counting for this purpose, and most commercial &detectors have a provision for controlling the fractionation of column eluates. 2 . 3 . 3 FLOW-THROUGH LSC

The apparatus needed for flow-through LSC is shown in Fig. 8. A reference detector is used for the determination of mass (if specific activities have to be determined) and facilitates the development of separation conditions. A splitter is included if part of the eluate must be recovered unmodified. In most applications, reversed-phase HPLC is used (see section 4). Efficient mixing of the aqueous eluate with the organic scintillator cocktail then is of crucial importance for reproducible and efficient counting. Pulse-free pumping of the cocktail to the eluate is accomplished by using a packed 'dummy' column or pressure resistor in the scintillator solvent line. In some examples, mixing is accomplished in a low-volume mixing chamber with magnetic stirrer (refs. 36-38), but it has

152 now been accepted that small-bore (0.25 mn i.d.) T-pieces work equally well, giving efficient mixing with virtually no extra column peak broadening under normal conditions. Under more demanding circumstances, such as 3H counting in eluents o f high ionic strength or with high water contents, a specially designed high-efficiency mixing unit may be helpful (ref. 39). Depending on the type of eluent, the mixing ratio scintillator /eluate and the mixing efficiency, typical E values are 0.20-0.35 for 3H and 0.50-0.90 for 14C. Typical background count rates vary from 10 to 60 CPM.

I"

121 10

la1

8 6

4 2

_____

1

2

3

- Time lrninl

Fig 7a-c

-

4

5

(a) Flow-through LSC of 14C-labeled amino acids. Column, Brownlee RP-18 Spheri-5 (100 x 4.6 mm); eluent, methanol/ water (5/95, v/v) pH 4.2 with 0.02 M acetate buffer containing 0.03 M sodium hexylsulphonate, 1.0 ml/min; liquid scintillator, 1.0 mjjmin; flow cell volume, 63 4; sample (b, c) represent corresponding frequency, 0.6 s simulations for off-lipe detection with sample frequencies of 3.0 (b) and 6.0 (c) s- , respectively; at eluent flow rate of 1.0 ml/min, this corresponds to fraction volumes of 0.05 and 0.1 ml, respectively. (Reproduced by permission from Eur. Chromatogr. News, John Wiley & Sons, ref. 31).

.

153

SCINTILLATOR PUMP

REFERENCE DETECTOR

1

-

ELUENT

B-DETECTOR

;

MIXING COIL

I

Fig. 8

WASTE

Apparatus for flow-through liquid scintillation counting. 1 = injector, 2 = fraction collector. For detailed diagram of 8detector, see Fig. 10

The flow cell in the 8-detector consists of a spiral made out of wound 1 ight-transparent material (normally PTFE), which is closely held between two PM tubes for coincidence counting. Dilution of eluate with scintillator allows cell columes Vd to be somewhat larger as compared to cell columes commonly used in, e.g., UV or RI detectors, while maintaining most of the chromatographic integrity. This is illustrated in Fig. 9 , in which vd = 1.0 ml at Fe = 1.0 ml/min and Fs = 4.0 ml/min. 2 . 3 . 4 FLOW-THROUGH HETEROGENEOUS SCINTILLATION COUNTING (HSC)

Compared to LSC, the set-up for flow-through HSC is considerably simpler because no LS-pump or eluate splitting is needed (Fig. 10). Three approaches may be distinguished: (1) the flow cell is constructed of UVtransparant glass or PTFE capillary, packed with finely divided, solid scintillator; (2) it is made of plastic scintillator capillary; or (3) it is constructed from a PTFE capillary encapsuled in a liquid or plastic scintillator for external SC. Little information on 8 counting with the latter two approaches is available. However, due to the small range of 8 particles in liquids as compared to the dimensions of these cells, counting efficiencies are relatively low. For instance, with a 0.7 mm i.d. NE102A plastic scintillator cell, an efficiency of 0.057 has been claimed for 14Clabeled solvents (ref. 41). The corresponding efficiency for 14C-labeled gaseous carbon dioxide was 0.58. Plastic scintillators are not inert to oxidising acids and most organic solvents, such as acetonitrile and tetrahydrofuran. Their use is therefore restricted to qualitative SC with

154 aqueous eluates. The performance for higher energy 0 ’ s from 32P is significantly better; values of over 0.70 may then be obtained (ref. 41).

A

Fig. 9

Representative UV (A) and radiogram (6) o f tyrosine containing metabolites of enkephalin. (B) was obtained after incubation o f an hyogenate of rat-astrocytes with added carboxypeptidase A and [ H-Tyrlenkephalin. Column, Spherisorb ODs I1 (60 x 4.1 mm); gradient elution with mobile phases consisting o f methanol and citric acid/phosphate buffer to which 0.3 mM sodium n-octanesulphonate was added; detection, UV (254 nm) and flow-through LSC, respectively; flow cell volume of the radioactivity detector was 1 ml; peaks: 1 = Tyr, 2 = Tyr-Gly-Gly, 3 = Tyr-Gly, 4 = Tyr-Gly-Gly-Phe, 5 = Met-enkephalin and 6 = Leu-enkephalin. (Reprinted with permission from the J. Chromatogr., Elsevier, ref. 40).

Of the heterogeneous modes, the packed cell (1) guarantees the most intense contact between eluate and scintil lator and, thus, optimum geometric counting efficiencies. This approach was first used in amino acid analyzers, with anthracene- (ref. 42) or POPOP-packed cells (ref. 43). The main disadvantages o f these types o f scintillators are their solu-

155

bility in organic solvents and the relatively low (< 0.02) counting efficiencies for 3H. These materials have now been replaced by inorganic scintillator glasses, such as cerium-activated lithium silicate or yttrium silicate, or salts such as europum-doped calcium fluoride. The new types of scintillators are virtually inert to most solvents used in column liquid chromatography. It is, however, recommended to avoid pH values of less than 2 or greater than 8 . Furthermore, the calcium fluoride material is normally not compatible with solvents containing ammonia. Depending on the particle size and the packing density of the cell, counting efficiencies are around 0.05 ( 3 H), 0.70 ( 14C) and 0.90 ( 32PI. Fig. 11 shows that a relatively large cell volume can be used without deterioration of the chromatographic resolution in the radiogram as obtained from the reference detector. In this case, the HSC cell has a volume of 0.6 ml, and is packed with yttrium silicate particles.

SAMPLE

Fig. 10 Schematic diagram for flow-through heterogeneous scintillation counting. AMP = Amplifier, SCA = Single-Channel Analyser, MCA = Multi-Channel Analyser. (Reprinted from the J. Chromatogr., Elsevier, ref. 21).

156 2.3.5 FLOW-THROUGH CERENKOV COUNTING To the author's knowledge, o n l y one paper dealing w i t h flow-through Cerenkov counting has been published ( r e f . 45). This i s r a t h e r s u r p r i s i n g i n view o f some d i s t i n c t advantages o f t h i s technique i n t h e determination o f high-energy 8's (see s e c t i o n 2.1).

In t h e paper, inorganic

pyrophosphate (PPi) produced i n a c e l l c u l t u r e medium was determined by incubation w i t h r a d i o a c t i v e orthophosphate ( 32 Pi). I n t r a - and e x t r a c e l l u l a r 32PPi

was measured using a weak anion exchange HPLC separation of

from Pi and other phosphor-containing compounds, w i t h flow-through Cerenkov counting a t E > 0.99 (Fig. 12). PPi

ATP

1

'(002)

2

Fig. 11 HPLC-UV and HPLC-radioactivity p r o f i l e s o f a p e r c h l o r i c a c i d extracf40f human lymphoblastic c e l l l i n e MOLT-3 a f t e r incubation with C-uridine f o r 2 h. The extracted nucleotides were separated on P a r t i s i l - 1 0 SAX r a d i a l l y compressed modules (100 x 8 mm) a t 2.0 ml/min; d e t e c t i o n was by UV (254 nm) and flowthrough HSC, respectively. For t h e l a t t e r , the f l o w c e l l was packed w i t h modified y t t r i u m s i l i c a t e granules, r e s u l t i n g i n an empty volume o f 0.6 m l . Under these conditions, a d e t e c t i o n l i m i t o f 5 Bq was found ( a t E = 0.43); peaks: 1 = UDP-Nacetylglucosamine and/or UDP-N-acetylgalactosamine, 2 = UDPglucose and/or UDP-galactose. (Reprinted w i t h permission from t h e J . Chromatogr., E l s e v i e r , r e f . 44).

157

a

n

a

0.5 01

03 0 N ID

02

W

01

lOOr

250

125

0

0

1

2

3

1

time

5

6

7

8

9

10

[rninl

F i g . 12 Chromatographic s e p a r a t i o n o f phosphate-containing compounds. Column, radial-pack Bondapak-NH (100 x 8 mm); m o b i l e phase, 0.1 M c i t r i c a c i d ( b u f f e r A) and M c i t r i c a c i d c o n t a i n i n g 0.1 M potassium sulphate p l u s 0.02 M magnesium s u l p h a t e (6) ( g r a d i e n t e l u t i o n ) ; d e t e c t i o n bY2UV (260 nm; up e r t r a c e ) and flow-through Cerenkov counting o f P (lower t r a c e r . (Re r i n t e d by permission from Anal. Biochem., Academic Press r e f . 4 5 r

03

2.4.

DATA ANALYSIS

2.4.1

DEFINITIONS OF DETECTION LIMITS

Relatively

few

papers

have

been

concerned

with

definitions

of

d e t e c t i o n l i m i t s i n flow-through c o u n t i n g i n terms o f chromatographic parameters and t h e p r o p e r t i e s o f t h e d e t e c t o r device, such as t h e c o u n t i n g

30, 32, 46-49). Algorithms d e r i v e d by Sieswerda e t a l . ( r e f . 46) and K l e i n and Hunt ( r e f . 47) a r e b r i e f l y summarized i n t h i s section. F o r convenience, symbols and a b b r e v i a t i o n s as used by these authors have been changed t o

efficiency,

c o u n t i n g t i m e and background count

rate

(refs.

conformity. A more fundamental treatment on d e t e c t i o n 1 i m i t s i n r a d i o a c t i v i t y d e t e r m i n a t i o n can be found i n r e f s . 50 and 51. The n e t peak area Cs ( i n counts) i s given by:

i n which cs+b represents t h e t o t a l counts c o l l e c t e d i n t h e peak and cb t h e t o t a l background counts c o l l e c t e d i n t i m e Tb ( i n min). Tw i s t h e t o -

158 t a l peak counting t i m e ( i n min), which i n flow-through c o u n t i n g equals t h e base peak w i d t h ( i n min). SD ( C s ) , i s I f i t i s assumed t h a t t h e standard d e v i a t i o n i n C, determined by t h e s t a t i s t i c s of t h e r a d i o a c t i v e decay alone, SD(Cs) can be c a l c u l a t e d from Poisson s t a t i s t i c s , and f o l l o w s from:

This assumption i s v a l i d f o r low l e v e l s o f a c t i v i t y only. A t h i g h a c t i v i t y l e v e l s , c o n t r i b u t i o n s from o t h e r sources, such as v a r i a t i o n s i n i n j e c t i o n volume, c o u n t i n g considered as w e l l .

efficiency

or

counting

time

should

be

Sieswerda ( r e f . 46) then defines t h e p r e c i s i o n as t h e s i g n a l - t o - n o i s e r a t i o i n C,, i n C,,

which i s t h e r e c i p r o c a l o f t h e r e l a t i v e standard d e v i a t i o n Combining eq. (3) and (4) y i e l d s :

RSD(Cs).

SD(Cs)

RSD(Cs) =

--

1 [-x

‘bxTw (1 + -x ‘sxTb

CS

Since Cs = becomes :

TW

(1

E x DPM(s) x Td and Cb

1

=

+

0.5

+)I

Tb

CPM(b) x Tb, from eq.

CPM(b)xTw

ExTdxDPM( s)

( 5 ) RSD(Cs)

TW

x (1 +

RSD(Cs) = [

(5)

(I++)IO*~ E x Td x DPM(s)

(6)

Tb

Assuming Tb >> Tw, eq. (6) s i m p l i f i e s t o :

1 RSD(Cs) = [

CPM(b) x Tw x (1

E x Td x DPM(s)

+ I)-

Oe5

(7)

E x Td x DPM(s)

From eq (7) d e t e c t i o n l i m i t s have been c a l c u l a t e d as f u n c t i o n of Td and RSD(Cs), they have been p l o t t e d i n F i g . values f o r Tw and CPM(b).

E x

1 3 , assuming t y p i c a l

159

s -

0001

001

01

10

EiTd

F i g . 13 D e t e c t i o n l i m i t s ( i n DPM) vs. ExTd, c a l c u l a t e d f rom eq. (7) a t RSD(C,) = 0.33, 0.10 and 0.05, w i t h CPM(b) = 30 CPM and Tw = 0.5 min.

K l e i n and Hunt ( r e f . 47) i n t e r p r e t t h e d e t e c t i o n l i m i t as t h e a c t i v i t y DPM ( m i n ) t h a t must b e p r e s e n t i n t h e e l u t i n g peak i n o r d e r t o o b t a i n a peak h e i g h t c oun t r a t e o f t w i c e t h e background count r a t e . Assuming a t r i a n g u l a r peak shape, an e x p r e s s i o n f o r DPM (min) i s d e r i v e d : CPM(b) x [(Fex S + Fs)] x [Tw + vd/ (Fex

s

+ F,)]

DPM(min) =

(8) SXEXVd

i n which S i s t h e f r a c t i o n of t h e e l u a t e t r a n s p o r t e d t hrough t h e r a d i o a c t i v i t y d e t e c t o r . Td t h u s f o l l o w s f r o m Td = Vd/[Fe x S + Fs].

160

At this point it should be noted that in practice, Td is the Only parameter which the analytical chemist can vary at will to improve precision in the measurement. Methods other than off-line counting that are intended to improve precision via Td have been compiled in sections 3.4 and 3.5. 2.4.2 OATA ACQUISITON,PROCESSING AND PRESENTATION In order to adequately represent the shape of an eluting peak it is normally advised to collect 20-30 data points (samples) in the peak. For peak widths of about 0.5 min, this requires sample frequencies of 1 s to be used. However, the total number of counts accumulated per sample decreases with decreasing sample frequency, which results in noisy signals.

I

CAR.

[PAR.

20

I

-TIME

-

Fig. 14 Radiograms of the 14C-labeled pesticides carbaryl (1.0 Bq) and parathion (0.8 Bq). Chromatographic conditions, see Fig. 3; raw (A) and filtered (by Fast Fourier Transform; B) data. (Reproduced from ref. 7; see also refs. 89-93). Therefore, the use of data filtering techniques is of prime importance in low-level flow-through radioactivity counting in order to be able to recognize small peaks recorded at short sampling frequency. An electronic filter for radiochromatography has been described (ref. 52). With the introduction of data collection, storage and hand1 ing by personal computers, the use of filtering algorithms has eliminated much of the need for electronic filtering. Filtering can thus be performed in real time during

the collection of data samples or, alternatively, after storage of the raw data. The latter is preferred, mainly because it allows more sophisticated filtering programmes such as Savinsky-Golay moving average (ref. 53) or Fast Fourier Transform (FFT) (ref. 54) to be used. Fig. 14 illustrates the effectiveness o f FFT on a detection limit determination of 14C-labeled pesticides with flow-through LSC. No peak broadening occurs after filtering, allowing a more simple peak detection algorithm to be used.

2.5. COMMERCIALLY AVAILABLE FLOW-THROUGH RADIOACTIVITY DETECTORS At present, at least six detectors for flow-through &counting are commercially available (Table IV). Most of these systems can be purchased with an (optimal) LS-pump, eluent-splitter system, built-in micro processor, software packages for control ling data collection and data handling of digital and analogue signals, flow cells with volumes ranging from 0.05 to about 1.5 ml and different types of solid scintillators. TABLE IV Commercially available flow-through radioactivity detectors TYPe ( s )

Manufacturer

MODEL 171/170

Beckman Instruments, Inc., Fullerton, CAI USA

LB 506 (A, 6, D)

Berthold Lab., Wildbad, FRG

FLO-ONE (IC, CR, CT, BD)

Radiomatic Instruments and Chemical Co., Inc. Tampa, F1, USA

ISOFLO

Nuclear Enterprizes Limited, Edinburgh, GB

BETACORD 1208

LKB WALLAC, Turku, Finland

RAMONA radioactivity detectors Isomess Isotopenmessgerate, Straubenhardt, FRG IN/US, Fairfield, NJ, USA

B-MAT

Depending on the configuration, the costs of these instruments vary between Hfl. 20,000-25,000* for basic set-ups (without LS-pump), splitter and computer system, and one analogue output from a ratemeter provided) to about Hfl. 40,000-60,000for more complex systems. These prices are, of course, subject to variation and give rough indications only. *(Hfl

. 1,000 = about $500)

162

3.

3.1

OPTIMIZATION PARAMETERS FOR FLOW-THROUGH 8-COUNTING INTRODUCTION

I n t h i s section, t h e o p t i m i z a t i o n of E, CPM(b) and Td i s considered i n more d e t a i l . I t should be stressed t h a t most o f t h e v a r i a b l e s t h a t can be used t o improve t h e counting r e s u l t s are t o some extent i n t e r r e l a t e d ; t h i s requires c a r e f u l s e l e c t i o n o f t h e operating conditions. I n section 3.5, some a l t e r n a t i v e methods f o r monitoring o f &labeled compounds i n column l i q u i d chromatography are discussed, most of which deal w i t h t h e increase i n s e n s i t i v i t y v i a Td by decoupling separation from counting 3.2

.

THE COUNTING EFFICIENCY E; GENERAL ASPECTS I n flow-through SC, E i s determined by the product o f t h r e e indepen-

dent e f f i c i e n c i e s : t h e e f f i c i e n c y w i t h which t h e s c i n t i l l a t o r / e l u a t e mixture converts t h e 8-energy i n t o photons ( t h e i n t r i n s i c e f f i c i e n c y ) , the e f f i c i e n c y w i t h which these photons reach t h e photocathode of the PM tubes (the geometric e f f i c i e n c y )

,

and t h e e f f i c i e n c y o f t h e e l e c t r o n i c

c i r c u i t ( i n c l u d i n g the PM tubes) t o convert the photons i n t o e l e c t r i c a l pulses t h a t pass through t h e coincidence c i r c u i t . The coincidence time and t h e s e t t i n g s o f t h e lower and upper pulse height discriminators may be chosen t o improve E. However, increasing t h e coincidence time o r decreasing t h e lower pulse height d i s c r i m i n a t o r may adversely a f f e c t t h e background count r a t e . set-up,

To characterize a given

i t therefore i s n o t s u f f i c i e n t t o quote E but r a t h e r t h e Figure

of M e r i t , FM, defined as: FM = E2/CPM(b)

(9)

E can be s p e c i f i e d under dynamic f l o w conditions o r a t zero flow. Differences i n dynamic and s t a t i c E values have been reported ( r e f s . 35, 55-57). Dynamic e f f i c i e n c i e s appear t o be somewhat smaller. One explanat i o n f o r t h i s phenomenon i s t h e decrease i n e f f e c t i v e f l o w c e l l volume w i t h increasing f l o w r a t e , as observed by Van Nieuwkerk e t a l . ( r e f . 58); i n other words, t h e actual dynamic and s t a t i c e f f i c i e n c i e s then are about equal. For the determination o f dynamic e f f i c i e n c i e s ,

i t i s more convenient

t o perform plug i n j e c t i o n s (without column i n s t a l l e d ) f o r greater choice i n r a d i o a c t i v e standard,

Possible radiochemical i m p u r i t i e s then do n o t

i n t e r f e r e w i t h the measurement.

This a l s o f a c i l i t a t e s t h e recording o f

gradient curves y i e l d i n g E as a f u n c t i o n o f the gradient parameters which

163 are normally needed when combining gradient elution with flow-through LSC . It turns out that the strongest influence the user can have on E is via the intrinsic efficiency, and experimental data from flow-through LSC and HSC on this subject are given in the next two sections. 3.2.1 E in LSC Scintillator cocktails, especially formulated for dynamic flow counting have become available only recently. Their main properties are fast sample incorporation and high sample hold capacity. With these scintillators, LS/eluate rations of about 3-5 will normally give satisfactory performance. Nitrogen purging of the scinti 1 lator may reduce oxygen quenching by about 50% (ref. 59). Some precautions with respect to the eluent composition should be taken. Halogenated solvents such as chloroform and dichloromethane act as strong quenchers of the LS fluorescence and must be avoided. The applicability of types of solvents to LSC may be judged from table V, which gives the relative quenching properties of some aliphatic substituents. TABLE V Relative quenching properties in LSC of aliphatic groups1 Diluter

Mild quencher

Strong quencher

-H

-CH=CHR

-F

-c1

-0CO.COR - I > Br

-NH2 -OH

-COOH

-NHR -NO2 -CHO -SH=SR -C12 -C13

'Reproduced by permission of Friedr. Vieweg & Sohn, ref. 59. E can further be affected by gradient elution. Roberts and Fields

(ref. 35) determined dynamic counting efficiencies for 3H with acetonitrile-water gradients at an LS/eluent ratio of 3 . Values of 0.21, 0.13 and 0.22 were found for 0.60 and 100% (v/v) acetonitrile, respectively. Webster and Whaun (ref. 57) examined the effect of methanol

164 or salt gradients on static efficiencies for 14C in RP-HPLC. NO significant influence on E was found for the methanol-water gradient (0-35% methanol in 0.01 M phosphate buffer, pH 5.6, which is in accordance with the observation made by Causey et al. (ref. 60). Salt gradient elution resulted i n a slight decrease in E from 91 to 77%, with a 0.01-0.7 M phosphate buffer at pH values of 3.4 and 4.3,respectively. Finally E can be influenced by the injected sample. This was illustrated in ref. 61, where preconcentration of a 900 ml pool of hamster urine on a conical pre-column resulted in heavy quenching by endogeneous material in parts of the radiogram. These extreme conditions, though, are not likely to be met in routine work. 3.2.2 E I N HSC The influence of scintillator particle size on E has been studied for various materials (refs. 21, 62-64). Some results are given in Table VI. Mutual comparison of the scintillators is hampered because o f differences in mesh sizes (or mesh-size distributions) and packing densities of the cells. TABLE V I Counting efficiencies o f solid scintillators for 3 H and 14C Particle size Im Mesh

Type

3H1

14C1

Ref.

0.44 0.57 0.62 0.16(D) 0.40(D) 0.38(S) 0.62 0.71 0.20(S) 0.57(S)

62 62 62 67 68 64 64 64 21 21 63 63

~~

Ce(Li)

EuCaF2

'0

=

90-125 63-90 38-63 250-350 250-350

60-80 140-160 160-180 150 150-250 45-100 100-150

dynamic, S

=

static

0.018-0.014 0.041-0.044 0.069-0.062

O.O35(S) 0.086 ( S )

165 Nevertheless,

i t can be concluded t h a t E increases w i t h decreasing

mesh sizes. A t t h e same time, however, t h e pressure build-up, d P , across the c e l l increases. A P can be approximated from t h e well-known equation: A P =

[ rjx LL]/[Ko x Td x dpZ] (bar)

I n which L i s t h e c e l l length ( i n m),

q t h e eluent v i s c o s i t y ( i n Kg/(m x

s ) ) , d the s c i n t i l l a t o r p a r t i c l e s i z e ( i n m) and KO t h e p e r m e a b i l i t y P constant ( w i t h values o f 0.001-0.002). S u b s t i t u t i n g t y p i c a l values o f L = 0.04 m, TI= 0.001 kg/(m x s ) , KO = 0.0015, Td = 10 s and d = 2 5 ~ 1 0 - ~ m P 3P i s 1.7 bar. The

lower

limit

of

d i s f u r t h e r determined by t h e adsorption P behaviour o f t h e s c i n t i l l a t o r m a t e r i a l , which becomes e s p e c i a l l y evident a t increasing s p e c i f i c surface areas, A t present, counting e f f i c i e n c i e s of over 0.80 and 0.11 have been claimed by some manufacturers f o r 14C and 3H, respectively. For 3H, apart from few exceptions ( r e f s . 39, 55, 651, such impressive f i g u r e s have n o t been confirmed i n t h e l i t e r a t u r e as y e t . Although w i t h HSC i t i s normally assumed t h a t E i s n o t a f f e c t e d by t h e eluent composition, some evidence o f t h e opposite i s a v a i l a b l e . Giersch ( r e f . 66) determined t h e counting e f f i c i e n c i e s o f 14C-labeled sugars and corresponding esters w i t h a l i t h i u m glass s c i n t i l l a t o r . With an aqueous s a l t gradient o f 5-400 mM phosphate, E was 0.22,

which increased t o 0.39

f o r a c e t o n i t r i l e / w a t e r (80/20, v/v, pH = 3.0). The d i f f e r e n c e was a t t r i b u t e d t o a l t e r a t i o n s i n t h e p a r t i t i o n c o e f f i c i e n t s o f t h e analytes between mobile

phase and s c i n t i l l a t o r

surface,

and r e l a t i v a t e s

HSC

counting e f f i c i e n c i e s c a l c u l a t e d from equation (1). F i n a l l y , compounds i n t h e eluent may absorb photons emitted from t h e s c i n t i l l a t o r . Colour quenching by p-nitrophenol has been observed by Mackey e t a l .

( r e f . 62).

Mori reported c o l o u r quenching from reagents

used i n the post-column n i n h y d r i n d e r i v a t i z a t i o n o f amino acids (ref. 67) and the bromocresolpurple d e r i v a t i z a t i o n o f c a r b o x y l i c acids (ref. 68).

3.3

THE BACKGROUND COUNT RATE CPM(b); GENERAL ASPECTS I d e a l l y , a s t a b l e background countrate should be observed,

which

o r i g i n a t e s from the thermally generated PM dark c u r r e n t , cosmic rays and 40 r a d i o a c t i v i t y present i n t h e glass envelope o f t h e PM tubes ( K, 232Th and 238U) ( r e f . 69). I n p r a c t i s e , a number o f user-determined sources c o n t r i b u t e t o CPM(b) as w e l l . These include t h e l a b o r a t o r y environment and i n t e r f e r i n g processes i n the f l o w c e l l of t h e r a d i o a c t i v i t y monitor. Examples from the f i r s t category

are

external

r a d i o a c t i v e sources,

166 stray light, spikes from electric apparatus and temperature fluctuations. Proper lead shielding can give a 3-fold reduction in CPM(b) (ref. 70). Connections to and from the detector should be light-tight. For this the use of stainless-steel or blackened PTFE capillary is recommended. Dark counts from the PM tubes can be reduced substantially by cooling, but only one example of this effect is found in the literature on radiocolumn liquid chromatography (ref. 63). Contributions to CPM(b) arising from cross-talk of two opposite PM tubes can be diminished by using paper masks (ref. 62). The second category is perhaps of more importance because it refers more to the daily routine. Radioactive contamination in the flow cell is the most abundant example in this class, but chemiluminescence and phosphorescence have also been shown to add to CPM(b). Although onephoton events, these processes may pass the coincidence circuit if the pulse rate exceeds the coincidence resolving time and thus be counted (ref. 59). Differences in the pulse-height distribution of luminescence and scintillator fuorescence enable corrections to be made for pulses not originating from scintillator fluorescence (ref. 71) and some radioactivity monitors have provisions to perform such correction. From the above reflections it can be argumented that CPM(b) should be specified under realistic conditions, i .e. between successive measurements with the flow cell filled with the scintillator-eluate mixture. It is good practice to run 'blank' chromatograms, prefereably at short sample frequency to observe spikes and thus be able to identify possible background sources. The following sections summarize some observations described in the literature on background contributions arising in the flow cell. 3.3.1 CPM(b) in flow-through LSC Chemiluminescence may occur after mixing alkaline solvents or ethers with liquid scintillators (refs. 19, 30, 36, 55, 59). The effect is especially pronounced for dioxane-based cocktails. Peroxides in ether or dioxane are believed to play a major role. It can be suppressed by cooling of the LS/eluate mixture to 10 OC before entering the flow cell (refs. 30, 36) or electronically corrected for after pulse height analysis. Because of the relatively short lifetimes of most chemiluminescence processes, increasing the time lapse between mixing and counting may also be used. Radioactive contamination of the flow cell is not expected to be a major problem, yet adsorption of radiolabeled peptides (ref. 351, pesticides

167 (ref. 72) and anions (refs. 73, 74) on PTFE-capillary flow cells has been observed. 3.3.2. CPM(b) I N FLOW-THROUGH HSC Contamination of packed cells has been observed for a large variety of solutes, some of which are collected in Table VII. These observations are not always consistent. For instance, Schutte (ref. 77) explained the observed adsorption of 14C-labeled nucleotides on calcium fluoride by the low solubility of calcium-salts of nucleotides, whereas Nakamura and Koizumi (ref. 21) found no adsorption for the same types of materials. TABLE VII

Observations o f adsorption on solid scintillator particles

Sc i nti1 1 ator

Samp 1 e

Yt2Si05 CeLi

phospholipids sugars

CeLi CeLi EuCaF2 EuCaF2 EuCaF2 EuCaF2

Remedy

HN03 flush (20%) hot detergent and 0.1 M HC1 flush carboxylic acids repacking after 50 inj. phosphates, acetates repacking polymers -glucuronides methanol, water or detergent flush ri bonucleotides si 1 anizat ion nucleotides --

Ref.

75 66 68 62 63 76

44 77

In general, HSC i s not compatible with solutes having molecular weights larger than about 600, such as proteins and polymers. The increase in CPM(b) observed with these solutes is explained both by filtering processes and the adsorption of radioactivity to the scintillator due to the relatively large number of reactive sites in these molecules (ref. 78). As a general rule one should avoid injecting radioactive samples of unknown identity or samples with radiolabeled solutes with large differences in polarity. Before starting flow experiments, the adsorption behavior of the solutes should be established using test-tubes filled with the scintillator powder (ref. 39). These test tubes also facilitate developing washing procedures for the packed cell. Other precautions that can be taken are presaturation of the scintillator with non-labeled analogs or silanization of the scintillator surface (ref. 44).

168 Some o f t h e disadvantages i n HSC may be circumvented by t h e p r i n c i p l e introduced by Rucker e t a l . (ref. 78), who employed f l o w c e l l s f i l l e d w i t h a x i a l l y aligned 0.1 mm i.d.

Ce(Li) glass f i b e r s . Computer models

were developed f o r t h e p r e d i c t i o n of t h e geometric counting e f f i c i e n c i e s o f R-emitters.

Except f o r 3H, t h e p r e d i c t e d values agreed w e l l w i t h t h e

experimental counting e f f i c i e n c i e s . For 14C and 32P, E values o f 0.55 and 0.93 were found, respectively. For 3 H, E was o n l y 0.001 (predicted 0.10). The system was compared w i t h a HSC c e l l packed w i t h Yt2Si05 powder ( w i t h

< 25 m). As compared t o t h e packed c e l l , s u b s t a n t i a l reductions i n P pressure build-up and contamination were observed using t h e glass f i b e r c e l l (Table V I I I ) . No radiograms demonstrating t h e f l o w c h a r a c t e r i s t i c s of t h e g l a s s - f i b e r c e l l were given as y e t . d

3.4

OTHER PARAMETERS

3.4.1 SELECTING THE FLOW CELL VOLUME vd I N LSC Some authors simply r e l a t e t h e maximum permitted Vd t o the minimum time A W , observed between two neighbouring peaks o f i n t e r e s t i n t h e reference detector (refs. 47, 55). Vd(max) i s then c a l c u l a t e d according to:

Obviously, only those peaks are detected which r e f e r t o radiolabeled products. For instance, i n metabolism studies o n l y the parent compound and i t s metabolites are labeled and t h e r e f o r e detected i n t h e radiogram. I t may then be worthwhile t o optimize r e s o l u t i o n o f r a d i o a c t i v e peaks,

which allows l a r g e r c e l l volumes t o be used. A l t e r n a t i v e l y , Vd(max) may be r e l a t e d t o the e f f e c t i v e base peak w i d t h Tw ( r e f . 30). For most p r a c t i c a l purposes, a Tw/Vd r a t i o o f about 10 i s acceptable w i t h respect t o e x t r a column peak broadening ( r e f . 79). Vd(max) then becomes: Vd(max) = [0.4 x Vo x (1 x k')]/[X

x NOs5] ( m l )

(12)

in which X i s t h e eluate f r a c t i o n i n t h e e l u a t e / s c i n t i l l a t o r mixture. S u b s t i t u t i n g t y p i c a l values o f N = 3000, Vo = 0.5 m l and X = 0.25,

maxi-

mum permitted f l o w c e l l volumes o f 0.030 and 0.073 m l are calculated f o r

k ' = 1.0 and 4.0, respectively. The c r i t e r i a given above do n o t i n c l u d e losses i n r e s o l u t i o n caused by

poor flow dynamics in the mixing-T, connecting capillaries or flow cell. To minimize the extra-column peak broadening, eluate segmentation techniques can be used. These are treated in section 3.5. 3.4.2 SELECTING THE FLOW CELL VOLUME Vd IN HSC The criteria for Vd(max) given in section 3.4 may be adapted to flowthrough HSC if additional band broadening from the packed cell can be neglected. To some extent the packed cells can be treated as packed-bed reactors. For analytes that do not adsorb to the scintillator particles (no retention), the added variance in peak volume uW arising from axial molecular diffusion and convective mixing in a cylindrical packed cell can in that case be approximated by: uw =

[(h x d x Td2)/L]Oa5 (ml)

P

in which h is the reduced plate height (2 < h < 6 ) . From this equation it follows that it is advantageous to use long, small-bore capillaries rather than short, large-bore ones in constructing the cell. Substituting m and L = 0.04 m, the typical values of Td = 1.0 s , h = 4, d = 25 x P added variance is 0.05 s. A 20-fold decrease i n Fe(Td = 20 s) gives uw = 1.0 s, which still seems quite acceptable for most applications. Large differences between experimental and theoretical values for uw may be explained by adsorption of the analyte on the scintillator material, resulting in asymmetric tailing peaks (ref. 80). 3.4.3 REPRODUCIBILITY AND LINEARITY Limited data are available on reproducibilities and linear dynamic ranges in flow-through R-counting. For high levels of activity, statistical and background fluctuations can be neglected (refs. 46, 60, 81) and reproducibility is expected to be determined primarily by E. This has not been thoroughly evaluated yet. Both for E and Td, pulse-free pumping of solvents and (with LSC) efficient mixing of eluent and scintillator are probably the most important requirements. RSD values, as determined from repetitive injections of high 14C-levels, can be as low as 1-2% for the LSC mode (refs. 57, 60) and 2.2% for the HSC mode (ref. 82). The effect of replacing packed cells on reproducibility must also be considered; repacking a single HSC cell resulted in static 14C E of 0.716

170 2 0.003 (n = 3), while packing of five HSC cells of about the same geometric dimensions resulted in static 14C E values of 0.688 f. 0.024 (ref. 62). The linear dynamic response is determined primarily by the dead time of the electronic circuit and the maximum number o f counts per sample frequency which the computer can accumulate (ref. 83). A linearity of up to about 4x103 Bq/peak can easily be obtained (ref. 44).

TABLE V I I I Adsorption (%) on granular Yt2Si05 and fiber scinti 1 1 ators 1

Compound

Eluting solvent

Glycine

0.5 M acetate 50% ethanol benzene 50% ethanol 0.01 M phosphate 0.01 M phospahte 50% ethanol 0.01 M phosphate

61 ucose

Cholesterol UDP

ATP Inul in Insulin Cytochrome C

Retained % Yt2S i O5 Fiber 0.5 1.6 2.1 382 5EI2 1.4 832 86'

0.7 0.2 1.4 1 .o 9.9 0.1 24 84

Reproduced by permission of Friedr. Vieweg & Sohn, ref. 78 Irreversible sorption 3.5

SPECIAL METHODS Bakay (refs. 74, 84-86) suppressed peak broadening in flow-through LSC of 14C-labeled amino acids by using post-column gel segmentation. For this, a polyacrylamide gel is pumped by motor-driven high-pressure syringes to the scintil lator/eluate mixture. The benefits of segmentation on the clearance of the flow cell is clearly demonstrated in Fig. 15. Schutte (ref. 77) used air segmentation, but data of its effect on suppresion of peak broadening were not given. Snyder (ref. 87) suggested the usage of post-column segmentation and buffer storage systems for decoupl ing separation and counting steps. In addition, in comparing post-column reactors (open tubular, liquid- or gassegmented flow and packed bed), Scholten et al. (ref. 88) showed that when dealing with capillaries with internal diameters o f over 0.3 mm and residence time of over about 20 s , segmentation techniques should be

171 applied in order to prevent peak broadening. Van Nieuwkerk (ref. 89) adapted the principle of solvent segmentation and extraction of aqueous column eluates for subsequent storage of the segmented stream in a capillary storage loop (Fig. 1 6 ) . During the separation, the analytes are extracted from the aqueous eluate into water-immiscible LS plugs. The LS plugs act both as detection medium and segmentator. The segmented flow is transported through a flow-through 8-detector for recording the direct radiogram and subsequently stored in a capillary storage loop. After storage of the complete chromatogram the contents of the loop are re-introduced into the detector at low flow rates by turning the switching valve. These flow rates can be selected according to the counting time Td and, therefore, the sensitivity needed, independently from the separation (reverse radiogram).

A

WITHOUT SPACER USING Iml FLOW CELL

0 w i i n SPACER USING I mi FLOW CELL

XI# 40”l 4ooo

3000 c

2 a c

2000

a U’

1000

L

0

~~

0

1

2

3 YIfIUTES

4

5

0

I

2

3

4

MINUTES

Fig. 15 Tracings of radioactivity of samples containing 14C-labeled amino acids. Samples were injected at 0.5 min intervals, without (A) and with (B) gel segmentation. Total flow rate of column eluate and liquid scintillator is 2.5 ml/min to which the gel spacer is added at 0.044 ml/min ( B ) . (Reprinted with permission from Anal. Biochem., Academic Press. ref. 84) The principle was applied in the determination of 14C-pesticides (refs. 7, 90) and 14C-amino acids (ref. 91). For extractable analytes, a 0.75 mm i.d. stainless-steel capillary was used as storage loop, and it was shown that segmentation effectively suppressed peak broadening in the

172 c a p i l l a r i e s , p e r m i t t i n g Td values o f over 5 min t o be used (see a l s o Figs. 3 and 14).

I I

injector

NdUn AnJvticel 662

C 0

I

U

m n

interface

~si-n/z computer

dual disk driua

Fig. 16 Schematic diagram o f HPLC equipment w i t h flow-through B-detector and e x t r a c t ion/segmentat ion/storage system. (Reproduced w i t h permission from Chromatographia, F r i e d r . Vieweg and Sons, ref.

90.) For non-extractable analytes,

the stainless-steel

capillary led t o

s i g n i f i c a n t peak broadening, which was explained by aqueous w e t t i n g on the inner w a l l o f t h e s t a i n l e s s - s t e e l c a p i l l a r y and t r a n s p o r t o f t h e

non-

extracted analytes through t h e aqueous f i l m . This e f f e c t was suppressed by using a 0.80

mm i.d.

PTFE loop instead.

For t h e

non-extracted

analytes, as compared t o corresponding d i r e c t measurements, a s i g n i f i c a n t decrease in E was observed i n t h e reverse measurements. This could be explained by the increase i n the volumes o f t h e aqueous and s c i n t i l l a t o r segments during t h e t r a n s p o r t and storage i n t h e c a p i l l a r i e s . consequence, reverse mode.

less & p a r t i c l e s In this

As a

reach t h e s c i n t i l l a t o r segments i n t h e

case, reverse

counting

efficiencies

could be

173 improved by homogenizing the contents of the storage loop just before reintroduction into the 8-detector while still maintaining the peak broadening suppression effect. A second scintillator pump was installed for this purpose for the addition of water-miscible LS to the contents of the storage loop (Fig. 17). For some selected amino acids the performance of the system for non-extractable analytes is illustrated in Fig. 18. Alternatively, the principle can be adapted for non-extractable analytes by using post-column ion-suppression or ion-pair extraction techniques as shown by Veltkamp et al. (refs. 92, 93), thereby avoiding the need for a second scintillator pump. This was demonstrated in the determination of the 14C-labeled, amine-containing pharmaceuticals remoxipride (ref. 92) and urapidil (ref. 93). Urapidil and its main metabolites (Fig. 19) were separated on a cyano-bonded phase, using an aqueous eluent consisting of acetonitrile/water (12/88, v/v, pH = 2.2). Under these conditions, the analytes are non-extractable because of protonation of the amine substituents, which resulted in counting efficiencies for 14C of less than 0.05 for the direct radiogram. Addition of the ion-pair reagent sodium dodecylbenzenzesulphonate to the water-immiscible LS increased the counting efficiency to over 0.80 (Figs. 20, 21). The principle was applied in the determination of urapidil and its main metabolites in rat plasma. An additional advantage of using water-immiscible LS for flow-through LSC in reversed-phase systems is that water is excluded from the scintillator segments, and that the LS/eluate ratio may be chosen such as to optimize the sensitivity E x Td. Extraction into the scintillator plugs, and thus E, improves at high ratios. At the same time, however, the total flow rate Ft increases, and Td decreases proportionally (and vica versa). This is shown in Fig. 21 for remoxipride. Repetitive injections of the analyte were made at Fe = 1.0 ml/min while varying the scintillator flow rate F,. For extractable compounds, the sensitivity was normally at its maximum at Fs/Fe = 0.2 (ref. 31). This compares favourably to water-miscible scintillators for which ratios of over 3 are recommended in order to obtain a homogeneous phase (refs. 30, 59). Baba et al. (refs. 48, 94, 95) developed a flow-through synchronized accumulating radioisotope detector for GC and HPLC. It consists of five flow cells connected in series. The signals from each cell are synchronized and accumulated. As a result, a five-fold increase in sensitivity is obtained. Additional peak broadening in the fifth cell was negligible as compared to the first cell with Vd = 1.1 ml each, Fe = 1.5 ml/min and Fs = 8.3 ml/min (Fig. 22). In the HSC mode considerable peak

174

broadening took place with yttrium silicate packed cells with volumes of 0.39 ml each (ref. 48), which hampered synchronization of the signals. The high cost of the counting equipment required for data collection of five different cells will probably prevent other workers to adapt this principle.

SOLVENT FILTER

STwlAOE CACIILARV lPTFEl

HOMOOLNlZlNO CAPILLARY

lREVE118€0

FLOW1

Fig. 17 Schematic diagram of HPLC equipment with flow-through 8-detector and segmentation/storage system, adapted for the determination o f radiolabeled, non-extractable analytes. (Reproduced from J. Chromatogr., Elsevier, ref. 91).

175

R-direct FFT-fllter

I

L 200

100

L

n V

I

I

I Rieverse

200-

FFT-1 ilter

150100-

50-

0

-. 20

40

60

TIME (MINI

Fig. 18 Radiograms of 14C-labeled amino acids. Chromatographic conditions, see Fig. 7; sample, alanine (65 Bq), valine (77 Bq), isoleucine (82 Bq) and leucine (41 Bq); upper trace, radiogram recorded during separation at T = 0.03 min; lower trace, radiogram recorded after storage o f the complete chromatogram by reintroduction of the contents of the storage loop into the 8-detector at lower flow rate (Td = 0.15 min); both traces filtered by FFT. (Reproduced from J . Chromatogr., Elsevier, ref. 91).

Karmen et al. (ref. 96) adapted an automatic micro-fraction collector for fractionation of the column eluate on filter paper and subsequent autoradiography. Fractions with volumes of up to 0.30 ml are collected into wells, formed in non-wetting fluorocarbon film. After evaporation to near-dryness, the remaining spots are transferred to a filter paper placed over the wells by using a vacuum technique. Depending on the volatility of the fractions, the time needed to evaporate and quantitatively transfer the spots to the paper is about 5-10 min. The volume of the remaining spots was controlled by the addition of 0.0005% glycerol to the eluent. The performance of the system was tested i n the determination o f selected 14C-labeled amino acids, with 0.125 ml fractions and at Fe = 1.0 ml/min (Fig. 23). The sensitivity and the linear dynamic range was strongly determined by the exposure time of

176 the filter paper to the X-ray photographic film. The widest linear range (250-5000 DPM/spot) was obtained for a 6 h exposure time. Exposure for 7 2 h permitted the determination o f 5-80 DPM/spot. With this method, a high sample throughput may be obtained even for extended exposure times at moderate costs. The reproducibility was satisfactory. Since autoradiography is normally used for qualitative purposes only (ref. 97), care should be taken when quantitatively interpreting autoradiographs with large differences in acti vi ty/spot

.

ir

Fig. 19 Structural formulae of 14C-labeled pesticides (parathion and carbaryl) and pharmaceuticals (remoxipride and urapidil and main metabolites). The positions of the labels are indicated by the dsteri sk.

177

I

1

.6

Ibl

1°1

103,

Fig. 20

t

cpm

Re pre s e n t a t i v e UV chromatogram (269 p$) o f u r a p i d i l and i t s main C - u r a p i d i l (730 Bq) under m e t a b o l i t e s (A) and radiograms o f n o n - e x t r a c t i v e (B) and e x t r a c t i v e (C) c o n d i t i o n s . Column, P i e r c e Cyano S p h e r i - 5 (100 x 4.6 mm); e l u e n t , a c e t o n i t r i l e / w a t e r (15/85, v / v ) , pH = 2.2 w i t h p h o s p h o r i c a c i d , 1.0 ml/min; w a t e r - i m m i s c i b l e l i q u i d s c i n t i l l a t o r , Ready-Solv NA, 0.2 ml/min. F o r F ig. ZOC, 1.0 mM sodium dodecylbenzenesulphonate was added t o t h e s c i n t i 11a t o r .

4.

APPLICATIONS

4.1

PREPARATION, PURIFICATION, IDENTIFICATION

I n most experiments i n v o l v i n g t h e use o f r a d i o i s o t o p e s , t h e v a l i d i t y o f t h e r e s u l t s i s s t r o n g l y dependent on t h e radiochemical p u r i t y o f t h e compound used, which i s d e f i n e d as t h e t o t a l r a d i o a c t i v i t y p r e s e n t as t h e n u c l i d e o f i n t e r e s t i n a s p e c i f i c chemical form. Furthermore, knowledge

o f t h e s p e c i f i c a c t i v i t y i s normally also required. Repeated a n a l y s i s j u s t p r i o r t o t h e use o f t h e s t o r e d m a t e r i a l i s recommended because, a p a r t f r o m i t s expected chemical and m i c r o b i o l o g i c a l decomposition, i t s r a d i o c h e m i c a l p u r i t y decreases upon s t o r a g e due t o r a diolysis. I n

particular

R-labeled

compounds

are

prone

t o radiolysis

178 because the 0 ' s emitted form high density spurs of reactive radicals in the surrounding matrix. A detailed discussion on radiolysis can be found in ref. 98.

"1

7'

k

so04

LintH.to!-

-

Fig. 21 14C-peak area (in counts) as function o f the scintillator flqy Crate. Chromatographic conditions, see Fig. 20; sample, remoxipride (600 Bq); liquid scintillator, water-immiscible (+), water-immiscible with 1.0 mM sodium dodecylbenzenesulphonate (0) and water miscible ( Y ) . (Reproduced by permission from Eur. Chromatogr. News, John Wiley & Sons, Ref. 31.) Of the various methods for purification, isolation and identification, HPLC is the most convenient because of its superior selectivity, easy operation (which is important from the standpoint of safety in handling high levels of radioactivity), and speed of analysis which even allows the purification of compounds labeled with shortlived isotopes. Examples have been collected in Table IX, some of which are described below in more detail, with emphasis on the benefits o f using HPLC in the procedures. Chasko and Thayer (ref. 99) considerably simplified isolation of cyclotron-produced 13N-labeled nitrite or nitrate from water targets by using a reductor, a concentrator and two analytical columns in series, packed with copperized cadmium and Partisi 1-10 SAX anion exchanger, respectively (Fig. 24A, B ) . As compared to the alternative method of rotary evaporation, an increase in concentration of the radiolabeled material of at least 10-fold is obtained, with the additional advantages o f

179

less sample handling and higher radiochemical purities of the material. Boothe et al. (ref. 100) used RP-HPLC with mobile phases containing the ion-pairing reagent n-octylamine for separation of anions labeled with "C, 13N or 18F. A significant influence o f carrier addition on the elution patterns was observed. For example, NCA 18F- only eluted form the column after the addition of carrier F-. These observations are consistent with results by other workers (refs. 8-10).

5

4

6

7

8

7

h a l f w i d t h (m) f l r l t Cel I

flfth

d

16 rnl"

cell

A

19.1+0.8

22.4+0.4

B

25.9tl.0

40.7+3.4

Fig. 22 Peak broadening using a synchronized accumulating radioisotope detector in liquid (upper trace) and heterogeneous (lower trace) scintillation counting. For explanation, see text. (Reprinted from J . Chromatogr., Elsevier, ref. 48).

In the determination of specific activities of radiohalogenides, Kloster and Laufer (ref. 101) used pre-column derivatization of the halides with 2-naphthol in the presence o f the oxidizing agent chloramine-T and subsequent HPLC. The detection limit o f the halonaphthol reaction products was 0.5 pmol (at 220 nm), which allowed the determination of specific activities of NCA radioiodine or radiobromine using only 5% of the total radioactivity.

A

0 a2

TABLE IX

Preparation, purification and identification in radio-CLC

Sample(s)

Column and mobile phase

13N-anions ( 13NO3-, 13N0 -, 13NH4+) radioanions (llC, 13N, "F, 82Br, 1311, 99m~c) radio-halogenides (38Cl, 8oBr, 82Br, 1281, 13h) lZ5r, lZ5~-proteins radioanions (13N, 77Br, lZ81O3-, mixture) 1251-monoiodoglucagon lZ5I/ 14C-pept i des 99mTc-diphosphonates 14C-S-adenosylmethionine 14C-ch 1orofo m , 14C-di bromoethane 14C-proteinhydrosylate 14C-butoprozi ne

f.t.

=

flow-through; 0.1.

=

off-line

Detection'

Ref.

Partisil-10 SAX, 30 mM phosphate buffer (pH = 3.0) f.t. C18, 0.01 M octylamine (aqueous; 4.5 < pH < 6.5) f.t.NaI(T1)

100

TSK LS-222, acetone/sodiumnitrate (1.5 N) (l/l,v/v) f.t.GM

108

Sephadex 6-25 YEW AX-1, 4 mM Na2C03/NaHC03 58' n-propanol/phosphate buffer (lOmM, pH

external LSC f.t .Ge( i) = 2.5) externa plastic C18, acetonitrile/trifluoroacetic acid (0.05%), 0.1. Aminex A28, 0.7 M sodium acetate f.t.NaI T1) C18, methanol/phosphate buffer (45/55, v/v) f. t .HSC C18, mathanol/water (75/25, v/v) f.t LSC SCX (preperative), lithium/sodium citrate, gradient f.t.HSC C8, methanol/dichloromethane/water (100/35/15, v/v/v) + 0.5% diethylamine (v/v) 0.1.

.

99

24 29 26 103 106 109 110 111

112

1 W

5 .. U

W u)

Fig. 23

W

E-I

0

U

CL

W

z

J




could

be

[3 H-14 Clglucuronide-metabolite

HPLC on a p o l a r amino-cyano bonded phase column was used i n s e p a r a t i n g the radiolabeled

glucuronide

conjugates

from UDP[14C]GA,

with

flow-

through LSC. An example i s g i v e n i n F i g . 31, where 3 H-androsterone was used as substrate.

For most o f t h e s u b s t r a t e s t e s t e d , t h e corresponding

glucuronide conjugates e l u t e d between 8-10 min a f t e r i n j e c t i o n , and c o u l d be detected a t t h e 100-200 pmol l e v e l . Flow-through r a d i o a c t i v i t y det e r m i n a t i o n considerably s i m p l i f i e d t h e procedure, and t h e authors t h e r e f o r e suggest t h a t t h e assay can be extended i n t h e i n v e s t i g a t i o n of many o t h e r compounds. R e l a t i v e l y few papers a r e concerned w i t h t h e use o f r a d i o i s o t o p e s i n p r e - o r post-column d e r i v a t i z a t i o n r e a c t i o n s f o r HPLC, probably because o f t h e hazards i n v o l v e d i n h a n d l i n g h i g h l e v e l s o f r a d i o a c t i v i t y and t h e small masses o f radiochemicals a v a i l a b l e , which make i t d i f f i c u l t t o obtain satisfactory reaction y i e l d s a t a t high specific a c t i v i t i e s . Dietrich

et

al.

(ref.

131)

adapted

t h e method

of

c h a r a c t e r i z a t i o n o f DNA adducts by 32P-postlabeling based on t h e r e a c t i o n o f DNA w i t h t h e chemical ( i . e .

Randerath

for

(Scheme 2).

the

It i s

c a r c i n o g e n i c agent)

under study f o l l o w e d by enzymatic h y d r o l y s i s o f t h e r e s u l t i n g m o d i f i e d DNA t o form deoxyribonucleoside-3'-phosphates, which a r e subsequently

194 radiolabeled by r e a c t i o n w i t h 32P-ATP i n t h e presence o f T4 polynucleotide kinase. The r e s u l t i n g [3' ,5'-32P]biphosphates are charact e r i z e d by HPLC e i t h e r d i r e c t l y o r a f t e r enzymatic h y d r o l y s i s w i t h Nuclease 1 t o [5'-32P]monophosphates. Figs. 32A-C g i v e chromatograms of products obtained a f t e r r e a c t i o n o f 3'-dGMP w i t h t h e e t h y l a t i n g agent d i e t h y l s u l f a t e (DES) followed by 32P-postlabel i n g w i t h UV and radioa c t i v i t y detection by flow-through LSC.

6

25 20 T

0

r X

E n u

15

-

10

I

m -

2 5 a

1-0

I

5

10

15

I

I

20

25

30

35

40

I

45

min

Fig. 31 HPLC assay of UDP-glucuronosyltransferase a c t i v i t towards androsterone, using UDP-glucuronic a c i d (UDP-GAY as COsubstrate. Column, P a r t i s i l - 5 PAC (Polar Amino-Cyano) (260 X 4.5 mm); gradient e l u t i o n from 100% a c e t o n i t r i l e t o 100% 0.01 M TBAHS; detection by flow-through l i q u i d s c i n t i l l a t i o n counting: incubations, performed s e p a r a t e l y j contained e i t h e r unlabeled androsterone and UDP-14C-GA o r H-androsterone and unlabeled UDP-GA (---); peak i d e n t i f i c a t i o n , (A) androsterone; 1 = androsterone glucuronide; 2 = glucuronic acid: 3 and 4, u n i d e n t i f i e d ; 5 = UDP-GA (UDP = u r i d i n e diphospho). Reprinted by permission from Anal. Biochem., Academic Press, r e f . 130). Hakam e t a l . ( r e f . 132) used 3H-labeling i n t h e simultaneous determin a t i o n o f mono- and poly-ADP-ribose. The c i s - d i o l s o f these molecules a r e w p r t i v.e l.,v n ~." i A.--" i 7 e d" hv,n n d a t e treatment hv,rerliiction "-l .---. r -e,r i,.,"""" ". .."-....,..- fnllnwerl .-. .-..-- . - - - - - .-. . w .. .i t-.h. 3 sodium- H-borohydride. The t r i t i a t e d products were separated by RP-HPLC

--

w i t h o f f - l i n e LSC (Fig. 33), and could be detected a t t h e subpicomol l e v e l . I n p r i n c i p l e , t h i s r e s u l t can be improved by using borohydride w i t h higher s p e c i f i c a c t i v i t y .

195 A G C T B

Nd-44

Scheme 2

1

DNA Modifying Agent

1

1 ) Micrococcal Nuclease 2) Spleen Phosphodiesterase

1

Y"P ATP T4 Polynucleotide Kinase

1

Nuclease P1

Outline of the DNA postlabeling method. (Reprinted with permission from Chromatographia, Friedr. Vieweg and Sons, ref. 131).

(A)

I

~

10

20

TIME (Min.)

Fig. 32.

30

10

20

TIME (Min.)

10 20 TIME (Min.)

HPLC characterization of products QPtained from the reaction of dGMP with diethyl sulphate and P postlabeling (Scheme 2) as deoxyribonucleoside-5'-phosphates ( A ) , as deoxyribonucleoside-3,5' -phosphates (B) and as deoxyri bonucleoside-5' phosphates (C). Column, Spherisorb ODs-2 (250 x 4.6 m); gradient elution from 0-4096 ( v / v ) methanol in 0.075 M potassium phosphate (pH 3.0); detection by UV (at 260 nm, A) and flow-through liquid scintillation counting (B, C); peaks: a = 3'-dGMP, a' = 3',5'-dGbisP, a" 5'-dGMP; b = N-7 ethyl-3'-dGMP, b'= N-7 ethyl-3',5'-dGbisPI b" = N-7 ethyl-5'-dGMP; c = ethyl ester of 3'-dGMP, c' = ethyl ester of 3 ' .5'-dbisP. (Reprinted by permission from Chromatographia, Friedr. Vieweg & Sohn, ref. 131).

-

196

35

Fig. 33 RP-HPLC analysis of products obtained after 3 H-borohydride reduction of poly(ADP-ribose) (B). Before reduction, the cisdiols in the ribose are sel tively oxidised by using eriodate. In (6) , in vitro-generated ‘$-labeled poly(ADP-ribose! was used to determine whether all riboje compounds are tritiated. In (A), the products obtained after H-borohydride reduction of AMP is given, which is used as primary external standard. Column, Ultrasphere ODS (250 x 4.6 mm); gradient elution from 100% 0.10 M potassium phosphate (pH 4.25) to 100 % methanol/O.lO M potassium phosphate (20/80, v/v) to 100% acetonitrile/l.O M urea (50/50, v/v); dual isotope detection by off-line liquid scintillation counting of 0.75 ml fractions. (Reprinted from J. Chromatogr., Elsevier, ref. 132).

Banerjee and Steimers (ref. 133) developed a method for indirect detection of anions in ion chromatography, based on the use o f 35S-sulfate in the eluent. When an anion elutes, a simultaneous decrease in sulfate concentration occurs; hence, the decrease in radioactivity s i gnal in collected fractions can be used to quantitate eluting anions. Chloride was used as test analyte with plug injections. With a mobile phase containing 3.4 nM sulfate (13.4GBq/mmol), the detection limit o f

197

a.

C 1 - was about 0.1

I n p r i n c i p l e , t h e d e t e c t i o n l i m i t s can be brought

down t o the low nanogram region when using higher s p e c i f i c a c t i v i t i e s . For c a t i o n analysis, t h e use o f 45Ca i s suggested. I t i s very u n l i k e l y , however, t h a t t h e p r i n c i p l e w i l l be adapted by other workers because a l t e r n a t i v e s f o r i o n determination which a r e n o t based on r a d i o a c t i v i t y detection are r e a d i l y available. I t i s worth mentioning here t h a t t h e s e l e c t i v i t y o f many radioassay

techniques

can

be

significantly

improved

by

using

column

liquid

chromatography i n sample clean up p r i o r t o t h e actual assay o f t h e pooled f r a c t i o n s , f o r example i n a c t i v a t i o n analysis and HPLC-RIA ( r e f s . 159-163). 4.5

MISCELLANEOUS I n t h i s section a p p l i c a t i o n s on radio-column l i q u i d chromatography

have been c o l l e c t e d i n which radiolabeled compounds are used as t r a c e r s f o r which no change i n the chemical expected (Table

from o f the compound i s t o be

XIII).

Radiotracers are excellent i n t e r n a l standards t h a t a r e i d e n t i c a l t o the compound t o be determined except f o r t h e r a d i o l a b e l . They are most frequently applied i n optimizing sample preparation steps, based on one radiolabeled t r a c e r only. The advantages are best exploited, however, by using mixtures o f radiolabeled i n t e r n a l standards f o r radio-CLC. Depending on t h e s p e c i f i c a c t i v i t i e s ,

t h e mass o f t h e standard can

normally be neglected as compared t o t h e mass t o be determined. The added signal i n t h e reference detector may then be neglected. The amounts of a c t i v i t y may be chosen as h i g h as t o a l l o w s a t i s f a c t o r y q u a n t i t a t i o n of the

radioactivity

by

flow-through

radioactivity

counting.

This

was

i l l u s t r a t e d by Frey and Frey (ref. 143) i n t h e simultaneous determination o f prednisone, prednisolone and 6-hydroxyprednisolone i n u r i n e samples using RP-HPLC, w i t h 3H-labeled prednisolone and 6-hydroxycortisol as i n t e r n a l standards. The r a d i o a c t i v i t y i n t h e HPLC e f f l u e n t was monitored by flow-through HSC (Fig. 34).

Benedict ( r e f . 144) developed a clean-up

procedure f o r t h e determination of catecholamines i n u r i n e and plasma samples.

14C-labeled catecholamines were used t o c o r r e c t f o r

losses

during the sample treatment step and t o i d e n t i f y e l u t i n g peaks a f t e r HPLC.

198

0.

MINUTES 8 16

1 ' 1 '

0

8

24

32

'

16 24 MNUTES

32

Fig. 34 HPLC chromatogram of prednisone ( P ) , prednisolone (Po), 68tjydroxycortjsol (68-HC) and 68-hydroxyprednisolone (68-Po) , with H-Po and H-6B-HC) as internal standards. Column LiChrosorb SI-60 (250 x 3.2 mn); mobile phase, hexane/diethyl ether/tetrahydrofuranlethanollgl aci a1 acid (55.9/31/6.5/2.3/0.3) ; detection by UV (254 nm; lower trace) and flow trough heterogeneous scintillation counting (upper trace). (Reprinted from J. Chromatogr., Elsevier, ref. 143). A second type o f radiotracer work for CLC is the study on the chromatographic behaviour of analytes, i .e. the study of retention characteristics of proteins (refs. 145-147) and Gd-DTPA (ref. 148) on reversed-phase packings, phospholipids on normal-phase packings (ref. 331, progesteronesuccinyltyrosine methyl esters on Sephadex LH-20 (refs. 149, 150) or alkali and transition metal ions on a silica-supported zirconium phosphate ion exchanger (ref. 151). In all examples, radionuclides were used to facilitate detection. 140La was used by Cassidy et al. (ref. 152) to identify the sources o f memory effects in the HPLC of lanthanides on dynamically generated ion exchangers. Apart from the normal peak broadening, major sources for tailing found in eluting peaks were sorption on metal surfaces and retention within sample loops (Fig. 35).

199 By using PTFE loops and tubing, most of the tailing could be eliminated, which resulted in cross-contamination of collected fractions of less than 0.006%. Finally, examples of radio-column liquid chromatography can be found in biochemical transport studies. Josic et al. (ref. 153) determined the kinetics of intracellular uptake of iron by ferritin, for which 59Fe was used as tracer. In this case, ferritin-bound and free radioactivity were separated by ion-exchange HPLC (Fig. 36). Boulieu et al. (ref. 154) studied the transport of the purine base hypoxanthine in human erythrocytes using 14C-hypoxanthine. This enabled the determination of the analyte at physiological concentrations in erythrocytes and incubation medium. Radio-HPLC with flow-through LSC confirmed that no metabolism of the analyte took place during the experiments.

RETENTION VOLUME

Fig. 35 Removal rate of 140La from sample loops:(A) metal loop and 0.01N HNO eluent, (6) metal loop and 0.01 N ehydroxyisobutyric acid at a H 4.6, (C) teflon loop and 0.01 N HNO eluent; flow rate, 2 ml/min. Curves (B) and (C) are shifted qrom origin for visibility (Reprinted by permission from Anal. Chem., American Chemical Society, ref. 152).

200

1

PP

Fig. 36 ,&n-exchange HPLC of K562 cell lysate after incubation with Fe-transferrin. Column, Mono Q (50 x 5 mm); salt gradient elution from 5 mM Hepes (pH 6.0) (buffer A) with 1 M NaCl (buffer B); after the salt gradient, 0.5 ml of 60% acetic acid was injected (arrow HAc); Ft = ferritin-bound radioactivity; detection by UV (280 nm) and off-line NaI(T1) scintillation counting of 0.5 ml fractions. (Reprinted by permission from Anal. Biochem., Academic Press, ref. 153) 5.

CONCLUDING REMARKS Compared to the flow-through mode, off-line counting in radio-column liquid chromatography is inherently more sensitive, with better control over the precision in counting results and the background count rate. The sensitivity of the off-line procedure can be obtained by using different methods which are also based on the decoupling of the separation and counting step, for example by the deposition of the column eluate on fluorocarbon films followed by autoradiography (ref. 961, or by using flow-programming as in refs. 89-93. The principle of the latter is based on solvent segmentation of the column eluate and temporary storage of the segmented stream in a capillary storage loop. After reintroduction of the contents of the loop and flow-programming, counting times of over 5 min can be used in selected regions of the radiogram, which is comparable with counting times comnonly used in off-line determinations. The chromatographic integrity, however, is somewhat better preserved i n f low-through counting.

20 1 When comparing flow-through LSC and HSC, i n t h e authors'view, t h e liq u i d mode i s t h e more v e r s a t i l e because o f lower r i s k s o f ( r a d i o a c t i v e ) contamination o f t h e f l o w c e l l and t h e absence o f r e s t r i c t i o n s on t h e t y pe of samples. I n addtion, f o r 3 H t h e c o u n t i n g e f f i c i e n c y n o r m a l l y i s b e t t e r by a f a c t o r o f 3-5 f o r t h e l i q u i d mode. The r a d i o l a b e l e d s o l u t e s can sometimes be recovered from t h e l i q u i d s c i n t i l l a t o r . For example, Devine ( r e f .

158)

used Sep-pak

androstenedione and c a r o t i n e :

cartridges

f o r t h e recovery o f

14C-

Q u a n t i t a t i v e recovery c o u l d be o b t a i n e d

provided t h e p o l a r i t y o f t h e s c i n t i l l a t o r compounds and t h e s o l u t e s o f i n t e r e s t were d i f f e r e n t and no detergents had been used i n t h e s c i n t i 11a t o r . The main drawback

of

flow-through

scintillator/eluate ratios of

at

LSC o r i g i n a t e s

from t h e

high

least three normally required w i t h

corresponding h i g h f l o w r a t e s ( s h o r t c o u n t i n g time) i n t h e r a d i o a c t i v i t y detector; t h i s r e s u l t s i n h i g h volumes o f r a d i o a c t i v e waste. For e x t r a c t a b l e analytes, however, i t has been shown t h a t r a t i o s o f about 0.2 can be a p p l i e d when u s i n g post-column

extraction

techniques

with

a

water-immiscible l i q u i d s c i n t i l l a t o r ( r e f . 31). The a p p l i c a t i o n s presented i n s e c t i o n 4 show t h a t

column

liquid

chromatography i s o f g r e a t value i n t h e p r e p a r a t i o n and c h a r a c t e r i z a t i o n of

r a d i o l a b e l e d solutes.

I n addition,

t h i s chromatographic technique

becomes i n c r e a s i n g l y important as sample pretreatment s t e p i n o r d e r t o f u r t h e r improve s e l e c t i v i t y o f w e l l - e s t a b l i s h e d r a d i o - a n a l y t i c a l methods ( r e f s . 159-163).

Examples a r e found i n a c t i v a t i o n a n a l y s i s (Fig. 1) and

HPLC-RIA. Continuous a t t e n t i o n i s however, a l s o g i v e n t o t h e development

o f non-radio HPLC procedures as an a l t e r n a t i v e t o radioanalyses ( r e f s . 166-171). For example, Stevens e t a l . ( r e f . 170) s t u d i e d t h e s o r p t i o n and d e s o r p t i o n o f t h e p r e s e r v a t i v e c h l o r h e x i d i n e on s o f t c o n t a c t lenses by radioassay o r , a l t e r n a t i v e l y ,

by HPLC-UV. The former procedure i n v o l v e s

t h e c a t a l y t i c o x i d a t i o n o f t h e l e n s m a t r i x which c o n t a i n s 14C-chlorh e x i d i n e t o 14C-carbon d i o x i d e and water which a r e trapped i n a c o c k t a i l and analyzed by l i q u i d s c i n t i l l a t i o n counting. Although t h e s e n s i t i v i t y , accuracy and p r e c i s i o n of

b o t h methods were s a t i s f a c t o r y ,

t h e HPLC

procedure has t h e advantages o f being more s e l e c t i v e , e a s i l y automatable and a l l o w i n g m o n i t o r i n g o f a n a l y t e degradation. The combination of

column l i q u i d chromatography and r a d i o a c t i v i t y

d e t e c t i o n has proven t o be an indispensable t o o l

i n many areas,

in

p a r t i c u l a r i n t h e e l u c i d a t i o n o f complex biomedical problems, such as t h e d i s t r i b u t i o n and metabolism o f endogenous compounds i n b i o l o g i c a l samples ( r e f . 173). F u t u r e work should be focussed on t h e combination of o n - l i n e preconcentration o f r a d i o l a b e l e d s o l u t e s f o r radio-column l i q u i d chroma-

202 tography, thereby rendering flow-through q u a n t i t a t i o n f e a s i b l e . There can be no doubt t h a t t h e use of flow-through r a d i o a c t i v i t y detectors which are now commercially a v a i l a b l e from several manufacturers - w i l l stimulate the u t i l i z a t i o n of radioisotopes i n t h e various areas of research mentioned above.

-

6. ACKNOWLEDGEMENTS

The author would l i k e t o thank Prof. O r . U.A.Th. Brinkman, Prof. D r . 1r.H.A. Das and Prof. Or. R.W. F r e i f o r s t i m u l a t i n g discussions and t h e i r many h e l p f u l suggestions i n t h e preparation o f t h i s chapter. REFERENCES 1 H.J.M. Bowen, Chemical Applications o f Radioisotopes, Muthuen i3 CO. Ltd, London (1969). 2 W. Geary, Radiochemical Methods, published on b e h a l f o f ACOL, London, by John Wiley & Sons, Chichester (1986). 3 D.D. Breimer, Rational Selection o f Methods f o r Therapeutic Drug Monitoring. I n : Therapeutic Relevance o f Drug Analysis, F.A. DeWolff, H. M a t t i e and 0.0. Breimer (Eds.), Martinus Nijhoff, The Hague (1979) pp. 9-21. 4 H.C. Dorn, Anal. Chem., 56 (1984) 747A-758A. 5 H.F. Haas and V. Krivan, Fresenius Z. Anal. Chem., 324 (1986) 13-18. 6 C.L. Holder, H.C. Thompson, Jr, A.B. Gosnell, P.H. Siitonen, W.A. Korfmacher, C.E. C e r n i g l i a , D.W. M i l l e r , D.A. Casciano and W. S l i k k e r , Jr., J. Anal. Toxicol., 11 (1987) 113-121. 7 A.C. Veltkamp, H.A. Das, U.A. Th. Brinkman and R.W. F r e i , p o s t e r presented a t t h e 9 t h I n t e r n a t i o n a l Symposium on Column L i q u i d Chromatography, Edinburgh (1985). 8 G. K l o s t e r and P. Laufer, I n t . J. Appl. Radiat. I s o t . , 35 (1984) 545. 9 D.S. Wilbur, Development o f No-Carrier-Added Radiopharmaceuticals w i t h the Aid o f Radio-HPLC. In: A n a l y t i c a l and Chromatographic Techniques i n Radi opharmaceutical Chemistry , D.M. W i e l and, M.C. Tobes and T.J. Mangner (Eds.), Springer-Verlag, New York, 1986, Ch. 11. 10 T.J. Manger, P o t e n t i a l A r t i f a c t s i n t h e Chromatography of Radiopharmaceutic a l s. I n : A n a l y t i c a l and Chromatographic Techniques i n Radiopharmaceutical Chemistry, D.M. Wieland, M.C. Tobes and T.J. Manger (Eds.), Springer-Verlag, New-York, (1986), Ch. 13. 11 T.J. Beugelsdijk and D.W. Knobeloch, J. Liq. Chromatogr., 9 (1986) 3093-3131. 12 T R Roberts , Radioc hromat ograp hy , t h e C hromat ograp hy and Electrophoresis o f Radiolabel l e d Compounds, Elsevier, Amsterdam, (1978), Ch. 6. 13 P.C. White, Analyst, 109 (1984) 976-979. 14 R.P.W. Scott, L i q u i d Chromatography Detectors, Elsevier, Amsterdam (1986) 15 H.G. Barth, W.E. Barber, C.H. Lochmuller, R.E. Majors and F.E. Regnier, Anal. Chem., 58 (1986) 211R-250R. 16 H.G. Barth, W.E. Barber, C.H. Lochmuller, R.E. Majors and F.E. Regnier, Anal. Chem. , 60 (1988) 387R-435R. 17 D.M. Wieland, M.C. Tobes and T.J. Mangner (Eds.), A n a l y t i c a l and Chromatographic Techniques i n Radiopharmaceut ic a l Chemistry , Springer-Verlag, New York, 1986.

..

.

203 18

G. Simonnet and M. Oia, Les Mesures de R a d i o a c t i v i t e a 1'Aide des Compteurs a S c i n t i l l a t e u r Liquide, E d i t i o n s Eyrolles, Paris (1977)

p . 19.

19

C.T. Peng, Sample Preparation i n L i q u i d S c i n t i l l a t i o n Counting, Review 17, The Radiochemical Centre, Amersham, Bucks., England

20 21 22

M.J. Kessler, J. Liq. Chrornatogr., 5 (1982) 313-325. Y. Nakamura and Y. Koizumi, J. Chromatogr., 333 (1985) 83-92. C.A. Mathis, R.M. Jones and J.H. Chasko, Overall Radio-HPLC Design. In: A n a l y t i c a l and Chromatographic Techniques i n Radiopharmaceutical Chemistry, D.M. Wieland, M.C. Tobes and T.J. Mangner (Eds.), Springer-Verlag, New-York (1986) Ch. 6. J.D. Baker, R.J. Gehrke, R.C. Greenwood and D.M. Meikrantz, J. Radioanal. Chem., 74 (1982) 117-124. F. Simonnet, J. Combe and G. Simonnet, I n t . J. Appl. Radiat. ISOtOp., 38 (1987) 571-572. E. Nieves, K.C. Rosenspire, S. Filc-DeRicco and A.S. Gelbard, J Chromatogr., 383 (1986) 325-337. V. Pingoud, J. Chromatogr., 331 (1985) 125-132. 6. Langstrom and H. Lundqvist, Radiochem. Radioanal. L e t t e r s , 41

23 24 25 26 27

(1977).

.

(1979) 375-381.

28 29 30 31

33

R.E. Needham and M.F. Delaney, Anal. Chem., 55 (1983) 148-150. K. Susuki, I n t . J . Appl. Radiat. Isot., 35 (1984) 801-804. D.R. Reeve and A. Crozier, J. Chromatogr., 137 (1977) 271-282. A.C. Veltkamp, H.A. Das, R.W. F r e i and U.A.Th. Brinkman, Eur. J. Chromatogr. News, l ( 2 ) (1987) 16-21. B.F.H. Drenth, T. Jagersma, F. Overzet, R.T. Ghijsen and R.A. de Zeeuw, Meth. Surv. Biochem. Anal., 12 (1983) 75-80. H.Alam, J.B. Smith, M. J. S i l v e r and D. Ahern, J. Chromatogr., 285

34 35

M.J. Kessler, J. Chromatogr. Sci., 20 (1982) 523-527. R.F. Roberts and M.J. F i e l d s , J. Chromatogr., Biomed. Appl.,

32

(1982) 218-222. 342

(1985) 25-33.

40 41

J.A. Hunt, Anal. Biochem., 23 (1968) 289-300. D. Westerlund, J . C a r l q v i s t , M. Ovesson and B. G. Pring, Acta Pharm. Suec., 21 (1984) 93-102. H.A. Dugger and B.A. Orwig, Drug. Metab. Rev., lO(2) (1979) 247-269. S.W. Wunderley, poster presented a t t h e 11th I n t e r n a t i o n a l Symposium on Column L i q u i d Chromatography, Amsterdam (1987). H. Lentzen and R. Simon, J. Chromatogr., 389 (1987) 444-449. Brochure 126L, Nuclear Enterprises, Edinburgh, Great B r i t a i n ,

42

S.J. Potashner, N. Lake and J.D.

36 37 38 39

(1980). Knowles, Anal. Biochem., 112 (1981)

82-89. 43 44 45 46

G.B. Sieswerda and H.L. Polak, J. Radioanal. Chem., 11 (1972) 49-58. 0. de Korte, Y.M.T. Marijnen, W.A. Haverkort, A.H. van Gennip and D. Roos, J. Chromatogr., 415 (1987) 383-387. A.P.A. Prins, E. K i l j a n , R.J. van de Stadt and J.K. van de Korst, Anal. Biochem., 152 (1986) 370-375. G.B. Sieswerda. H. PoDoe and J.F.K. Huber, Anal. Chim. Acta, 78

..

(1975) 343-3581 47

F.K.

Hunt, I n t . J. Appl. Radiat. I s o t . , 32 (1981)

K l e i n and C.A.

669-671. 48

S. Baba. Y. Suzuki. Y. Sasaki and M. Horie, J . Chromatogr.,

(1987) i57-164. 49

50 51

392

'

H.J. van Nieuwkerk, Thesis: On-line Radiometry i n High-Performance L i q u i d Chromatography using a Storage Loop, Petten, The Netherlands, 1987, pp. 68-71. L.A. Currie, Anal. Chem., 40 (1968) 586-593. P.R. Bevington, Data Reduction and E r r o r Analysis f o r t h e Physical Sciences, McGraw-Hill Book Co., New York, (1969).

204 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

71 72 73

74 75 76

77 78 79 80 81 82 83

84 85 86 a7

D.J. Malcolme-lawes, S. Massey and P. Warwick, J. Radioanal. Chem., 57 (1980) 335-361. A. Savitsky and M.J.E. Golay, Anal. Chem., 36 (1964) 1627-1639. G.J. de Groot, Trends i n Anal. Chem., 4 (1985) 134-137. B.M. Frey and F.J. Frey, Clin. Chem., 28 (1982) 689-692. M.J. Kessler, J. Chromatogr., 255 (1983) 209-217. H.K. Webster and J.M. Whaum, J. Chromatogr., 209 (1981) 283-292. H.J. van Nieuwkerk, A.C. Veltkamp, H.A. Das, U.A.Th. Brinkman and R.W. F r e i , J. Radioanal. Nucl. Chem., 100 (1986) 165-176. N.G.L. Harding, Y. Farid, M.J. Stewart, J. Shepherd and D. N i c o l l , Chromatographia 15 (1982) 468-474. A.G. Causey, B. Middleton and K. B a r t l e t t , Biochem. J., 235 (1986) 343-350. H.M. Ruijten, P.H. van Amsterdam and H. de Bree, J. Chromatogr., 314 (1984) 183-191. L.N. Mackey, P.A. Rodriguez and F.B. Schroeder, J. Chromatogr., 208 (1981) 1-8. K. Shirahashi, 6. Izawa, Y. Murano, Y. Muramastu and K. Yoshihara, J. Radioanal. Nucl. Chem., Letters, 86 (1984) 1-10. L.J. Everett, Chromatographia, 15 (1982) 445-448. M.H. Simonian and M.W. Capp, Chromatogram 8 (1987) 5-6 (HPLC newsletter published by Beckman Inc. , F u l l e r t o n , CA, USA). C. Giersch, J . Chromatogr., 172 (1979) 153-161. S. Mori, Plant & C e l l Physiol., 23 (1982), 703-708. S. Mori, Agric. B i o l . Chem., 45 (1981) 1881-1884. B.H. Candy, Rev. Sci. Instrum., 56 (1985) 183-193. K. Strandgarden and P.O. Gunnarson, poster presented a t t h e 11th I n t e r n a t i o n a l Symposium on Column L i q u i d Chromatography, Amsterdam (1987). G. Dietzel, G I T Fachz. Lab., 28 (1984) 909-913. B. Detjens, K. Figge and H. Martinen, G I T Fachz. Lab., 22 (1978) 578-585. A.D. Nunn and A.R. Fritzberg, in: A n a l y t i c a l and Chromatographic Techniques i n Radi opharmaceut i c a l Chemistry D.M. W i e l and, M.C. Tobes and T.J. Mangner (Eds.), Springer-Verlag, New York, 1986, p. 116. B. Bakay, E.Nissinen and L. Sweetman, Anal. Biochem., 86 (1978) (65-77). M.A. Clark, T.M. Conway and S.T. Crooke, J. Liq. Chromatogr., 10 (1987) 2707-2719. P.W. Albro, J. Lorenzo and J. Schroeder, LC. Liq. Chromatogr. HPLC Mag., 2 (1984) 310-312. L. Schutte, J. Chromatogr., 72 (1972) 303-309. T.L. Rucker, H.H. Ross and G.K. Schweitzer, Chromatographia, 25 (1988) 31-36. J.C. Sternberg, i n J.C. Giddings and R.A. K e l l e r (Eds.), Advances i n Chromatography, Vol. 2, Dekker, New York, USA, 1966, p. 205. L. Nondek, U.A.Th. Brinkman and R.W. F r e i , Anal. Chem., 54 (1983) 1466-70. P.R. Bewington, Data Reduction and E r r o r Analysis f o r the Physical Sciences, McGraw-Hill Book Co., New York, 1969, p. 78. D.W. Roberts, Meth. Surv. Biochem. Anal., 12 (1983) 81-88. C.A. Mathis, R.M. Jones and J.H. Chasko, Overall Radio HPLC Design. In: A n a l y t i c a l and Chromatographic Techniques i n Radiopharmaceutical Chemistry, D.M. Wieland, M.C. Tobes and T.J. Manger (Eds.), Springer-Verlag, New York, (1986) p. 133-137. B. Bakay, Anal. Biochem., 63 (1975p 87-98 B. Bakay, Clin. Chem., 21 (1975) 1212-1216. B. Bakay, in: Liquid S c i n t i l l a t i o n Counting. H.A. Crook and P. Johnson, Vol 4, p. 133, Heyden Press, London, 1977. L.R. Snyder, J. Chromatogr., 149 (1978) 653-668.

.

.

88

89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117

118 119 120 121

A.H.M.T. S c h o l t e n , U.A. Th. Brinkman and R.W. F r e i , Anal. Chem., 54 (1982) 1932-38. H.J. van Nieuwkerk, Thesis: O n - l i n e Radiometry i n High-Performance L i q u i d Chromatography u s i n g a Storage Loop, Pet t en, The Net herlands (1987). H.J. van Nieuwkerk, H.A. Das, U.A. Th. Brinkman and R.W. F r e i , Chromatographia, 19 (1984) 137-144. H.J. van Nieuwkerk, H.A. Das, U.A.Th., Brinkman and R.W. F r e i , J. Chromatogr., 360 (1986) 105-117. A.C. Veltkamp, H.A. Das, R.W. F r e i and U.A.Th. Brinkman, J. Chromatogr., 384 (1987) 357-369. A.C. Veltkamp, H.A. Das, R.W. F r e i and U.A.Th. Brinkman, J. Pharm. Biomed. Anal. 6 (1988) 609-622. S. Baba, Y. Shinohara, H. Sano, T. Inoue, S. Masuda and M. Kurono, J. Chromatogr., 305 (1984) 119-126. S. Baba, M. H o r i e and K. Watanabe, J. Chromatogr., 244 (1982) 57-64. A . Karmen, G. M a l i k i n , L. F r e u n d l i c h and S. Lam, J. Chromatogr., 349 (1985) 267-274. H.C. T r e u t l e r and K. Fr e y e r , Z f I - M i t t . , 103 (1985) 18-32. E.A. Evans, S e l f - d e c o m p o s i t i o n o f Radiochemicals: P r i n c i p l e s , C o n t r o l , Observations and E f f e c t s , Review 16, Amersham I n t e r n a t i o n a l Ltd., Amersham, Bucks., England, 1976. J.H. Chasko and J.R. Thayer, I n t . J. Appl. Radiat . I s o t . , 32 (1981) 645-649. T.E. Boothe, A.M. Emran, R.D. Fi n n , P.J. K o t h a r i and M.M. Vora, J. Chromatogr., 333 (1985) 269-275. G. K l o s t e r and P. L a u f e r , J. Lab. Comp. Radioph., 20 (1983) 1305-1315. A.E. B o l t o n , R a d i o i o d i n a t i o n Techniques, Review 18, Amersham. Bucks., England (1985). R.C. Judd and H.D. C a l d w e l l , J. L i q . Chromatogr., 8 (1985) 1109-1120. S.J. Wagner and M.J. Welch, J. Nucl. Med., 20 (1979) 428. D.J. Hnatowich, R.L. C h i l d s , D. L a n t e i g n e and A. N a j a f i J. Immun. Meth., 65 (1983) 147-157. M.V. M i k e ls o n s and T.C. P i n k e r t o n , Anal. Chem. , 58, (1986) 1007-1013. J.A.G.M. van den Brand, H.A. Das, 6. G. Dekker and C.L. de L i g n y , I n t . J . Appl. R a d i a t . I s o t . , 33 (1982) 917. D. I s h i i , A. H i r o s e and I. H o r i u c h i , J. Radioanal. Chem., 45 (1978) 7-14. J. Vockova and V. Svoboda, J. Chromatogr., 410 (1987) 500-503. D.A. W e lls , G.A. Garbolas and G.A. D i q e n i s , J. Chromatogr., 356 (1986) 367-371. I.Kleinmann, V. Svoboda and J. Vockova, J. Chrornatogr., 411 (1987) 335-344. B.F.H. D re n t h and R.A. de Zeeuw, I n t . J. Appl. Radiat . I s o t . , 33 (1982) 681-683. S.K. Mauldi n , F.A. Richard, M.Plescia, S.D. Wyrick, A. Sancar and S.G. Chaney, Anal. Biochem., 157 (1986) 129-143. K.J. Hoffman, Drug Metab. Disp., 14 (1985) 341-348. L. W eidolf , J . Chromatogr., 343 (1985) 85-97. K.O. Vollmer, W. Klemisch and A. von Hodenberg, Z. N a t u r f o r s c h . , C: B i o s c i . , 41 (1986) 115-125. J.E. Coutant, R.J. Barbuch, D.K. S a t o n i n and R.J. Cregge, Biomed. Environ. Mass Spectrom. , 14 (1987) 325-330. C. Meinard, P. Bruneau and M. Roche, J. Chromatogr., 349 (1985) 105- 108. G.L. Lamoureux and D.G. Rusness, J. Agr. Food. Chem., 35 (1987) 1-7. M.J. Arnaud, Drug Metab. Oisp., 13 (1985) 471-478. G.A. Kyerematen, L.H. T a y l o r , J.O. d e B e t hizy and E.S. V e s e l l , J. Chromatogr., 419 (1987) 191-203.

206 122

126 127

I . Plakunov, T.A. Smolarek, D.L. Fischer, J.C. Wiley J r . and W.M. Bai r d , Carci nogenesi s , 8 (1987) 59-66. T.J. Monks, S.S. Lau, R.J. Highet and J.R. G i l l e t t e , Drug. Metab. Disp., 13 (1985) 553-559. N.J. Parks, K.A. Krohn, C.A. Mathis, J.H. Chasko, K.R. Geiger, M.E. Gregor and N.F. Peek, Science, 212 (1981) 58-60. R.S. Chapkin, V.A. Ziboh, C.L. Marcel0 and J.J. Voorhees, J. L i p i d Res., 27 (1986) 945-954. P.I. Lundmo and A. Sunde, J. Chromatogr., 308 (1984) 289-294. D.L. T r i b b l e , M.R. Glover and J.D. Lambeth, J. Chromatogr., 414

128 129

G.Bonn, J. Chromatogr., 387 (1987) 393-398. C. Abeijon, J.M. Capasso and C.B. Hirschberg, J. Chromatogr., 360

130

M.W.H;

123 124 125

(1987) 411-416. (1986) 293-297. Coughtrie, B. Burchell and J.R.

Bend, Anal. Biochem.,

159

(1986) 198-205. 131 132 133 134 135

M.W. D i e t r i c h , W.E. Hopkins, K.J. Asbury and W.P. Ridley, Chromatographia, 24 (1987) 545-551. A. Hakam, J. McLick and E. Kun, J. Chromatogr., 359 (1986) 275-284. S. Banerjee and J.R. Steimers, Anal. Chem., 57 (1985) 1476-1477. V.L. Wilson, R.A. Smith, H. Autrup, H. Krokan, D.E. Musci, Ngoc-Nga-Thi Le, J. Longeria, D. Ziska and C.C. H a r r i s , Anal. Biochem., 152 (1982) 275-284. L. Janocko and R.B. Hochberg, J. S t e r o i d Biochem., 24 (1986)

1049-1052. 136

138 139 140 141 142 143 144 145 146

E.W. Bergink, P.S.L. Janssen, E.W. T u r p i j n and J. van der Vies, J. Steroid Biochem., 22 (1985) 831-836. L.D. Fairbanks, A. Goday, G.S. M o r r i s , M.F.J. Brolsma, H.A. Simmonds and T. Gibson, J. Chromatogr., 276 (1983) 427-432. R . T . M a r s i l i and H. Ostapenko, J. Agr. Food Chem., 35 (1987) 304-308. E. Nissinen, J. Chromatogr., 342 (1985) 175-178. K.L.L. Fong and B.Y.H. Hwang, Biochem. Pharmac., 32 (1983) 2781-2786. S.R. Childers, Neurochem. Res., 11 (1986) 161-171. F.A. Hommes and L. Moss, Anal. Biochem., 154 (1986) 100-103. B.M. Frey and F.J. Frey, J. Chromatogr., 229 (1982) 283-292. C.R. Benedict, J. Chromatogr. , 385 (1987) 369-375. G. Raspi, A. LO Moro and M. S p i n e t t i , Ann. Chim., 77 (1987) 525-532. F.L. de Vos, D.M. Robertson and M.T.W. Hearn, J. Chromatogr., 392

147

P.C.

137

(1987) 17-32. Sadek, P.W. Carr.,

L.D.

Bowers and L.C. Haddad, Anal. Biochem.,

153 (1986) 359-371. 148 149 150 151 152 153

M.M. Vora, S . Wukovnig, R.D. Finn, A.M. Emran, T.E. Boothe and P.J. Kothari , J. Chromatogr. , 369 (1986) 187-192. G. Toth and J. Zsadanyi, J. Chromatogr., 329 (1985) 264. G. Toth, J. Chromatogr., 404 (1987) 258-260. L. van So and L. S z i r t e s , J. Radioanal. Nucl. Chem., 99 (1986) 55-60. R.M. Cassidy, F.C. M i l l e r , C.H. Knight, J.C. Roddick and R.W. S u l l i v a n , Anal. Chem., 58 (1986) 1389-1394. D. Josic, E. Mattia, G. Ashwell and J. van Renswoude, Anal. Biochem.,

152 (1986) 42-47. 154

158 159

R. Boulieu, C. Bory, P. Baltassat and C. Gonnet, J. L i q . Chromatogr., 7 (1984) 1013-1021. H. van den Berg, P.G. Boshuis and W.H.P. Schreurs, J. Agr. Food Chem., 34 (1986) 264-268. B.A. Tomkins and W.H. G r i e s t , J. Chromatogr., 386 (1987) 103-110. S.T. I n g a l l s , P.E. Minkler, C.L. Hoppel and J.E. Nordlander, J. Chromatogr., 299 (1984) 365-376. P.C. Devine and B.V. Milborrow, J. Chromatogr., 325 (1985) 323-326. J.J. Fardv, G. D. McOrist and T.M. Florence, Anal. Chim. Acta, 159

160

G.V.

155 156 157

(1984) 1991209. Iyengar, J. Radioanal. Nucl. Chem.,

110 (1987) 503-517.

20 7 161 162 163 164 165 166 167 168 169 170

171 172 173

E. G e l p i , Trends i n Anal. Chem., 4 (1985) XIII-XIV. O.B. Holland, M. R i s k , H. Brown, K. Komes, P. Dube and C. Swann, J. Chromatogr., 385 (1987) 393-396. G.H. F r i d l a n d and D.M. D e s i d e r i o , L i f e S c i . , 41 (1987) 809-812. J.J. P r a t t , Ann. C l i n . Biochem., 23 (1986) 251-276. P.K.F. Yeung, J.W. Hubbard, B.W. Baker, M.R. Looker and K.K. Midha, J. Chromatogr., 303 (1984) 412-416. J. Krska, G.M. Addison and S.D. Soni, Ann. C l i n . Biochem., 23 (1986) 340-345. 0. L a n t t o , Clin. Chem., 28 (1982) 1129-1132. G.J. Peters, E. Laurensse, A. Leyva and H.M. Pinedo, Anal. Biochem., 161 (1987) 32-38. P. Hjemdahl, M. Daleskog and T. Kahan, L i f e Sci., 25 (1979) 131-138. L.E. Stevens, J.R. Durrwachter and 0.0. H e l t o n , J. Pharm. Sci., 75 (1986) 83-86. F. Overzet and R.A. de Zeeuw, J. Pharma. Biomed. Anal., 2 (1984) 3-17. J.A. van d e r K r o g t , C.F.M. van Valkenburg and R.D.M. B e l f r o i d , J. Chromatogr. Biomed. Appl., 427 (1988) 9-17. E. Buncel and J.R. Jones (Eds.), I s o t o p e s i n t h e P h y s i c a l and Biomedical Sciences, Volume 1 L a b e l l e d Compounds ( P a r t A), E l s e v i e r , Amsterdam, 1987.

208 CHAPTER V

MODERN POST-COLUMN RE CT ON DETECTION LIQUID CHROMATOGRAPHY

HIGH-PERFORMANCE

H. JANSEN and R. W. FREI

1. 2. 2.1 2.2 2.3 3. 4. 5. 5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.3.1 5.2.3.2 5.2.3.3 5.2.3.4 5.2.3.5 5.2.3.6 5.2.3.7 5.2.3.8 5.2.3.9 5.2.4 5.3 5.3.1 5.3.2 5.3.2.1 5.3.2.2 5.3.2.3 5.4 5.5 5.6 5.7 6.

General i n t r o d u c t i o n Types o f post-column r e a c t o r s Open t u b u l a r r e a c t o r s Packed bed r e a c t o r s Segmented stream t u b u l a r r e a c t o r s Choice o f r e a c t i o n d e t e c t o r A p p l i c a t i o n s o f post-column r e a c t i o n d e t e c t i o n New approaches t o post-column r e a c t i o n d e t e c t i o n I n t r o d u c t ion The use o f immobilized enzymes i n post-column r e a c t o r s Introduction Enzyme i m n o b i l i z a t i o n A p p l i c a t i o n s o f immobilized enzymes E a r l y work Urease Hydroxysteroid dehydrogenases A c e t y l c h o l i n e esterase and c h o l i n e oxidase Glucuronidase and glycosidase Xanthine oxidase L-Amino a c i d oxidase Cholesterol o x i dase A1 k a l i n e phosphatase Concluding remarks Other solid-phase c h e m i s t r i e s Introduction A p p l i c a t i o n s o f s o l id-phase c h e m i s t r i e s E a r l y work C a t a l y t i c s o l id-phase c h e m i s t r i e s N o n - c a t a l y t i c s o l id-phase c h e m i s t r i e s The use o f electrochemical reagent p r o d u c t i o n The use o f photochemical and thermo i n i t i a t e d r e a c t i o n s Miniaturization Hollow f i b e r s as post-column r e a c t o r s Conc 1us ions References

1.

GENERAL INTRODUCTION I n many a n a l y t i c a l l a b o r a t o r i e s a l l over t h e world, h i g h performance

l i q u i d chromatography (HPLC) i s used f o r t h e a n a l y s i s o f a wide v a r i e t y

o f samples. A p p l i c a t i o n s i n c l u d e , e.g.,

environmental a n a l y s i s , c l i n i c a l

a n a l y s i s , food a n a l y s i s , pharmaceutical a n a l y s i s and q u a l i t y c o n t r o l . I n many cases, t r a c e l e v e l c o n c e n t r a t i o n s o f t h e a n a l y t e s o f i n t e r e s t a r e

of t h e o f t e n inadequate s e n s i t i v i t y and/or s e l e c t i v i t y i n t h e d e t e c t i o n process i n HPLC needed f o r t r a c e a n a l y s i s i n

encountered.

Because

209 comp 1ex m a t r i c e s (b io l o g i c a l f 1u ids , heav i1y contaminated env ironmenta 1 samples) methods have been developed t o overcome t h i s problem. Two powerf u l approaches can be d i s t i n g u i s h e d : The use o f small precolumns f o r s e l e c t i v e o n - l i n e sample handling.

-

With t h e proper s e l e c t i o n o f sorbents, clean-up and t r a c e enrichment can be achieved. An e x c e l l e n t review showing t h e p o s s i b i l i t i e s o f t h i s

-

method was published r e c e n t l y ( r e f . 1). The use o f chemical d e r i v a t i z a t i o n techniques w i t h t h e goal o f improving t h e d e t e c t a b i l i t y o f t h e compounds o f i n t e r e s t .

C u r r e n t l y , t h e b e s t and most r e l i a b l e d e t e c t o r s f o r HPLC t h a t a r e w i d e l y used a r e UV-VIS, fluorescence and e l e c t r o c h e m i c a l d e t e c t o r s . I t i s d e s i r a b l e t o use these d e t e c t o r s f o r t r a c e a n a l y s i s which i s p o s s i b l e when s u i t a b l e chemical d e r i v a t i z a t i o n techniques a r e i n c o r p o r a t e d t o convert t h e a n a l y t e s o f i n t e r e s t w i t h t h e i r o r i g i n a l l y poor d e t e c t i o n p r o p e r t i e s i n t o compounds t h a t can be detected w i t h h i g h s e n s i t i v i t y w i t h these detectors. Apart from an increase i n d e t e c t a b i l i t y , t h e d e r i v a t i z a t i o n s t e p can a l s o improve t h e s e l e c t i v i t y o f t h e t o t a l a n a l y t i c a l method. i.e.,

The d e r i v a t i z a t i o n can be c a r r i e d o u t i n t h e precolumn mode,

b e f o r e t h e separation takes p l a c e and u s u a l l y o f f - l i n e ,

recent advances revival

of

i n automation o f pre-column

pre-chromatographic

on-1 i n e d e r i v a t i z a t i o n

techniques.

although

l a b e l l i n g has caused a

However,

t h e m a j o r i t y of

procedures a r e c a r r i e d o u t i n t h e post-column

mode. Both approaches have t h e i r i n h e r e n t advantages and disadvantages which have been discussed p r e v i o u s l y ( r e f . 2) and t h e c h o i c e f o r one of t h e two modes w i l l g e n e r a l l y depend a l o t on t h e a n a l y t i c a l problem t h a t i s t o be solved. This paper w i l l deal w i t h post-column d e r i v a t i z a t i o n . HPLC-system w i t h an o n - l i n e r e a c t i o n 1. Pump 1 i s used f o r t h e m o b i l e phase supply. A f t e r separation o f t h e compounds i n t h e sample on t h e a n a l y t i c a l column, t h e reagent i s added w i t h pump 2 v i a a s u i t a b l e m i x i n g device. The combined streams a r e passed through t h e post-column r e a c t o r , p r o v i d i n g t h e d e s i r e d c.q. r e q u i r e d holdup t i m e f o r t h e r e a c t i o n t o occur, and f i n a l l y through t h e d e t e c t o r . Several advantages o f post-column r e a c t i o n can be given: - As opposed t o d e r i v a t i z a t i o n p r i o r t o t h e s e p a r a t i o n , t h e r e a c t i o n does n o t have t o y i e l d a s i n g l e d e t e c t a b l e species, s i n c e t h e r e a c t i o n products a r e n o t separated anymore. A

schematic

diagram o f

an

d e t e c t o r can be found i n Fig.

-

The r e a c t i o n does n o t have t o go t o completion and t h e r e a c t i o n products do n o t have t o be s t a b l e .

As l o n g as t h e r e a c t i o n i s r e -

producible, i t can, i n p r i n c i p l e , be used.

2 10

-

The technique can be used with different detectors in series, i.e., an UV-detector directly at the column outlet followed by a fluorescence detector after a post-column reactor. Because the compounds are separated in their original form it is possible to use separation procedures from the literature.

sampling valve analytical column

D--i

T -piece post -column reactor detector

Fig. 1

General schematic of an HPLC system with reaction detector.

There are also distinct disadvantages of the technique: - Additional pumps are needed for reagent supply. A stable, nonpulsating flow is required. - Mixing problems can be encountered, e.g., when the mobile phase and the reagent have very different viscosity. - The addition of reagent gives a dilution o f the mobile phase. - Frequently, the mobile phase needed for the separation is not the best medium for the derivatization reaction. Therefore, compromises must be made for those cases. - The post-column reactor gives a contribution to total band broadening with a resultant loss in chromatographic resolution. - In some cases, the excess of reagent interferes in the detection process. When using post-column reaction detection, it i s necessary to have a reactor suitable i n terms of chemical resistance, holdup time, band broadening etc. for the reaction involved. In principle, three basically

different reactor types can be distinguished, viz. straight, coiled or knitted open tubular reactors (OTRs), packed bed reactors (PBRs) and segmented stream tubular reactors (SSRs). Band broadening can, as far as OTRs and PBRs are concerned, be predicted by the use of simple equations as will be outlined below. Huber et al. have published a good comparison between OTRs and PBRs (ref. 3). Special attention was given to the loss of resolution caused by the reactor and to the mixing process. 2.

TYPES OF POST-COLUMN REACTORS

2.1

OPEN TUBULAR REACTORS

The simplest post-column reactor is a piece of stainless steel or PTFE tubing. Coiling or knitting is attractive since it effects an increase in radial mass transfer resulting in a decrease in band broadening (refs. 4, 5). A good discussion of different types of these deformed OTRs can be found in ref. 6. Band broadening in time units in open tubes is described by tr t

0

:d

'

0.5

1

= (x

96

'

D,

In this equation, x (0 < x < 1) accounts for the reduced band broadening for coiled or knitted as compared to straight ( x = 1) tubes, tr ist the mean residence time in the reactor, dt is the tube inner diameter and D, is the molecular diffusion coefficient in the carrier (mobile phase + reagent) stream. A thorough investigation o f the influence of coiling has been given (refs. 5, 6). T o demonstrate the influence of coiling of open tubular reactors, x is calculated to be 0.14 by using the equations given (ref. 5) for the following set of parameters, which are typical for a post-column reactor coupled to a 4.6 mm I.D. chromatographic column: p (solvent density) = 103 kg/m3 , 7 (viscosity) = kg/m.s, h (coil to tube diameter ratio) = 0.01, 0, = lo-' m2/s, F (total volumetric flow m. For a 30 s residence time, rate) = 30 . 10- 9 m3/ s , and dt = 500 . ut is calculated to be 3 . 3 s which is lower by 62 % when compared with the value for a straight tube. It is possible to get less band broadening by using smaller inner diameter tubing but a larger pressure drop is the price that must be paid. Pressure drop (dp) in the tubular reactor is described by the poiseuille equation

212

For the set o f parameters given above, p is calculated to be 0.9 bar, m which is fully acceptable but a reduction in tube diameter to 200 . would lead to a pressure drop of over 200 bar for the same residence time. PACKED BED REACTORS Packed bed reactors are glass or stainless-steel columns that are packed with small, inert nonporous glass beads. They can be considered as chromatographic columns operated under non-retention conditions. Therefore, band broadening in these reactors essentially follows the theory for HPLC columns, which yields:

2.2

h stands for the reduced plate height with values

o f between 2 and 6 roughly dependent on the packing quality, d is the particle diameter and P L is the reactor length. From Eq. 3 it can be concluded that reducing the particle size and increasing the reactor length while keeping the residence time unchanged will result in less band broadening. It is important not to have excessive pressure drop over the reactor. The pressure drop is given by . L

Ap

2 (4)

=

. 2 '

tr

dp

where KO is the permeability constant which has a value o f 0.001 to 0.002. SEGMENTED STREAM TUBULAR REACTORS Using this type of post-column reactor, the mixed mobile phase + reagent stream is segmented either with gas (air) bubbles or with

2.3

213 non-miscible s o l v e n t plugs. I f t h e r e i s no c a r r y - o v e r from one segment of another, which can be achieved by t h e proper c h o i c e o f t h e r e a c t o r m a t e r i a l ( r e f . 7 ) , t h e a x i a l d i s p e r s i o n i s determined by t h e s i z e o f t h e segment. With s u f f i c i e n t l y small segments, band broadening can be k e p t very low. Band broadening u s i n g t h i s t y p e o f r e a c t i o n d e t e c t i o n system i s u s u a l l y determined mainly by t h e phase separator o r debubbler, needed t o c r e a t e a homogeneous f l o w through t h e d e t e c t o r , r a t h e r than t h e r e a c t o r i t s e l f . The t h e o r e t i c a l treatment of band broadening i n segmented stream r e a c t o r s i s r a t h e r complex. Pressure drop over a gas segmented stream r e a c t o r should be low due t o c o m p r e s s i b i l i t y o f t h e gas bubble hence, r e l a t i v e l y l a r g e i n n e r diameter t u b i n g (0.8 t o 1 mm I.D.)

i s commonly

used i n combination w i t h 4.6

mm I.D. a n a l y t i c a l columns. For s o l v e n t segmented systems h i g h e r back pressures can be t o l e r a t e d hence, a h i g h e r

f l e x i b i l i t y e x i s t s f o r t h e c h o i c e o f c o i l geometries and some degree of miniaturization i s possible ( r e f . 9). 3.

CHOICE OF REACTION DETECTOR For t h e e f f e c t i v e use o f post-column r e a c t i o n d e t e c t i o n systems, i t i s

o f outmost

importance t o have a r e a c t o r attuned t o t h e p a r t i c u l a r r e a c t i o n c o n d i t i o n s . The choice o f one o f t h e r e a c t o r types g i v e n above

i s mainly i n f l u e n c e d by t h e r e a c t i o n t i m e needed. One can s t a r t c a l c u l a t i n g band broadening and pressure drop f o r open t u b u l a r and packed bed r e a c t o r s u s i n g Equations 1 through 4. For open t u b u l a r r e a c t o r s , band broadening w i l l be r a t h e r h i g h f o r l o n g e r r e a c t i o n times (above ca.

1

min.) when pressure drop should n o t be t o o high. Therefore, t h i s r e a c t o r t y p e i s commonly recommended f o r s h o r t e r r e a c t i o n times.

i.e.,

below

about 30 s . With a k n i t t e d r e a c t o r , somewhat l o n g e r r e a c t i o n times, up t o several minutes according t o Engelhardt ( r e f s . 4, 6 ) , a r e f e a s i b l e . reactions w i t h intermediate kinetics,

For

packed bed r e a c t o r s a r e t o be

prefered. They a r e mainly used f o r r e a c t i o n times between about 0.5 and 5 minutes. I n t h i s regions, they o f f e r an a t t r a c t i v e compromise between band broadening and pressure drop.

For r e a c t i o n times l o n g e r than 5

minutes a l s o as an a l t e r n a t i v e t o packed bed r e a c t o r s segmented stream r e a c t o r s are used. Reaction times up t o 20 minutes have been r e p o r t e d . This r e a c t i o n t i m e can be considered as an upper p r a c t i c a l l i m i t

in

post-column r e a c t i o n d e t e c t o r systems. Apart from band broadening and pressure drop c o n s i d e r a t i o n s , o t h e r aspects might have t h e i r i n f l u e n c e on t h e r e a c t o r choice. For i n s t a n c e , an open t u b u l a r r e a c t o r can be p r e f e r e d f o r i t s simple c o n s t r u c t i o n ,

f o r t h e f a c t t h a t c l o g g i n g problems a r e

almost never observed o r because an a l k a l i n e r e a c t i o n medium would cause

214

the glass particles in a packed bed reactor to dissolve. On the other hand, segmented stream reactors must be used when reactions on liquidliquid interfaces are used for the derivatization. As an example, ion-pair formation can be carried out. With this technique an ion-pair is extracted to one phase while the excess of reagent, i.e., the counter ion, remains in the other phase. This post-column ion-pairing is part of the so-called post-column extraction detection techniques. Another example of post-column extraction is extracting the analyte to the segmenting solvent thereby separating it from interferring compounds. After phase separation, the segmentation solvent phase is guided through the detector. Post-column extraction and ion-pairing techniques are in their appearance closely related to post-column reaction techniques and have been reviewed in the recent past (refs. 8, 9). Several papers dealing with the optimal choice of reaction detectors and giving a more detailed theoretical treatment of the various reactor types have appeared during the last years (e.9. refs. 3, 5, 6, 8, 10) and are suggested for additional reading. 4.

APPLICATIONS OF POST-COLUMN REACTION DETECTION In Table I , a list is given of some of the possibilities of postcolumn reaction detection. The table is intended to give an idea of the broad applicability in various analytical fields and problem areas rather than to give a complete review of all the applications published during the past few years. Examples of the use of the different reactor types discussed above and coupled to different detection modes are included in the table. Though not always the best choice in terms of band broadening, it appears from the literature that the open tubular reactor is most frequently used for post-column derivatization. This is most probably a result of the simplicity o f construction and of the reliability during operation.

215

TABLE I

Some a p p l i c a t i o n s o f post-column r e a c t i o n d e t e c t o r s i n HPLC

Compounds A f 1atoxins

Aldehydes and ketones Amino a c i d s Ant hraqui nones

Reagent (s)

I2 semicarbazide hydrochloride o-phthal a1 dehyde

Catechol amines

sodium d i t h i o n i t e / sodi um hydroxide borate b u f f e r (PH 10) NaOH, o-phthal aldehyde Ethylenediamine

Ch 1oroan i1ines

R F1uram

Phenols

Hydroperoxides

p o t a s s i um f e r r i c y a n i de 4 -am in oan t ipy r ine acetyl acetonel ammon ia NaOH, phenanthrenequi none NaI

&Lac t am a n t i b i o t ics

Barbiturates Carbamates

Formaldehyde

Reactor

Detection*

Reference

OTR

F

11

PBR

E

12

OTR PBR OTR

F F VIS

13 5 14

no r e a c t o r UV

15

OTR

F

16

SSR

F

17

OTR, PBR F and SSR no r e a c t o r V I S

18 19

20

knitted OTR OTR

VIS and F F

21

PBR

uv

22

o-phthalaldehyde

OTR

F

23

Organosul phur compounds

Pd( I I ) - c a l c e i n

SSR

F

24

Penici 11 i n s

sodium hydroxide/ mercury ( I I) c h l o r i d e / e t h y l ened i ami n e t e t r a a c e t i c acid

OTR

uv

25

OTR

E

26

OTR OTR OTR OTR

E F UV and V I S F

27 28 29 30

SSR

F

31

Guani d i nes

Pros tag1 andi ns

Br2

Reducing carbohydrates

2-cyanoacetamide

Thiamine and thiamine phosphate e s t e r s

various reagents p o t a s s i um f e r r i c y a n i de/ sodium hydroxide

Vitamin K,

sodium borohydride

J

*

F: Fluorescense E: Electrochemical UV: U l t r a v i o l e t absorption

VIS: V i s i b l e l i g h t absorption

216 5. 5.1

NEW APPROACHES TO POST-COLUMN REACTION DETECTION

INTRODUCTION Post-column

reaction detection

in

its

straight

forward

form

as

discussed above has developed i n t o a powerful method o f d e t e c t i o n f o r various analytes and i s used i n many a n a l y t i c a l l a b o r a t o r i e s . Since t h e a d d i t i o n a l hardware needed i s q u i t e simple, problems i n operating an HPLC/reaction detection system are seldom encountered. However, t h e r e are s i t u a t i o n s i n which conventional r e a c t i o n d e t e c t i o n i s n o t successful o r where other approaches y i e l d b e t t e r r e s u l t s , lower operation Cost, simpler

systems

or

other

advantages.

These

facts

have

stimulated

researchers t o develop new techniques i n post-column r e a c t i o n d e t e c t i o n f o r l i q u i d chromatography.

There are a number o f d i s t i n c t trends

in

research being done a t present times. These trends, demonstrated w i t h several applications, w i l l be discussed below and include a) t h e use of immobilized enzymes i n post-column reactors;

b) t h e use o f solid-phase

chemistries (other than reactions on immobilized enzymes), i.e., c a t a l y s i s and the use of s o l i d supported reagents; c) t h e use o f e l e c t r o chemical reagent production; d) t h e use o f photochemical and thermo i n i t i a t e d reactions;

e) m i n i a t u r i z a t i o n ,

as a l o g i c a l

necessity when a

microbore column i s p r e f e r r e d o r when expensive and/or h i g h l y t o x c chemicals and solvents are used, and f) t h e use o f hollow f i b e r s as pos

-

column reactors.

5.2 THE USE OF IMMOBILIZED ENZYMES I N POST-COLUMN REACTORS 5.2.1 INTRODUCTION Enzymes are a t t r a c t i v e for use i n r e a c t i o n d e t e c t i o n systems because o f t h e i r inherent s e l e c t i v i t y . When the product formed i n the enzymatic reaction i s w e l l detectable, an i n t e r e s t i n g h i g h l y s p e c i f i c a n a l y t i c a l method can be obtained, Much research i s being done on the use of i m mobilized

enzymes

since,

as

cited

below,

there

are

some

distinct

advantages as compared t o enzymes i n s o l u t i o n : the e l i m i n a t i o n o f enzyme s o l u t i o n pumps and mixing u n i t s and, hence,

-

-

-

the reduction o f cost and the absence o f mixing and d i l u t i o n problems the p o s s i b i l i t y o f working w i t h enzymes t h a t would otherwise i n t e r f e r e i n the detection process immobilized enzymes can be re-used. immobilization o f t e n improves the stabi 1it y o f t h e enzymes.

storage

p r o p e r t i e s and the pH

There are many methods f o r immobilizing enzymes on a wide v a r i e t y o f supports ( r e f s . 32-34). Supports t h a t are widely used include agarose,

217

cellulose, polyacrylamide, nylon, glass, silica and ion exchangers. Binding can take place either by physical adsorption or by covalent attachment. Physical adsorption has the disadvantage that loss of enzyme due to desorption can take place. For this reason, covalent attachment is generally preferred.The binding capacity of a support is an important parameter to consider. High surface area is advantageous. On the other hand, this should not be the result of very small pore diameters. Relatively large pore diameters (> ca. 40 nm) are needed for enzymes to enter into the pores. ENZYME IMMOBILIZATION Two types of enzyme reactors can be found in the literature, viz. a wall-coated open tubular reactor and a reactor packed with enzyme immobilized on beads. A major disadvantage of the first reactor type is its unfavourable ratio of active surface to mobile phase volume, leading to low activity per unit volume. For this reason, the latter reactor type i s primarily used in combination with HPLC. The first type is sometimes used in flow injection analysis. Good mechanically stable packed-bed reactors can be obtained when the enzyme is bound on glass, silica or alumina particles. Other materials are quite soft and can easily be deformed in flow systems, leading to high pressure drop and excessive peak broadening due to voids. Small particles should be used if possible for reasons of band broadening as discussed before. Besides the band broadening that occurs in any packed bed reactor, the reaction itself on the enzymatic layer may give rise to band broadening. This phenomenon has been discussed in the literature (refs. 35-37). No additional band broadening due to the reaction is observed either when reactant and product move at the same velocity through the reactor or when the reaction occurs instantaneously. When using enzymes in a post-column reactor, it is necessary to be careful with the mobile phase composition. Since enzymes are usually active in only a relatively small pH range and usually can not withstand high organic modifier concentration, development of a separation with a mobile phase directly compatible with the enzyme reactor is mandatory. If this is not possible, an adaptation of the mobile phase by adding a make-up flow, e.g., a buffer, prior to entering the reactor is possible. However, this has the disadvantage that an additional pump is needed and that dilution occurs. Therefore, direct coupling is prefered if possible.

5.2.2

2 18 5.2.3 5.2.3.1

APPLICATIONS OF IMMOBILIZED ENZYMES EARLY WORK

Preliminary work i n t h e use o f immobilized enzymes i n post-column reaction d e t e c t i o n systems was done by Schlabach and Regnier ( r e f . 38). A column w i t h glucose-6-phosphate dehydrogenase immobilized on glass beads was used t o react glucose-6-phosphate w i t h the formation o f NADH which i s detected f l u o r i m e t r i c a l l y . 5.2.3.2

UREASE

A schematic diagram of

an HPLC-system equipped w i t h an enzymatic

reactor i s given i n Fig. 2. The system i s used f o r t h e determination of urea and ammonia, which i s based on i o n - p a i r HPLC w i t h o n - l i n e postcolumn d e r i v a t i z a t i o n

on immobilized urease ( r e f . 39). In t h e urease

solid-phase reactor (SPR), urea i s q u a n t i t a t i v e l y converted i n t o ammonia, which reacts w i t h o-phthalaldehyde

and i s detected by

fluorescence

monitoring. The method i s very s p e c i f i c due t o t h e inherent s e l e c t i v i t y

o f the enzyme combined w i t h t h e s e l e c t i v i t y o f t h e OPA reaction. This i s shown i n Table I 1 which gives t h e response f o r compounds r e l a t e d t o urea t h a t are present i n t h e samples from an urea p l a n t f o r which t h e method was developed. Later, t h e same method was adapted t o t h e determinati

n of

urea and ammonia i n u r i n e and i n serum Fig. 3. An o f f - l i n e sample pretreatment procedure w i t h an anion exchanger was needed t o e l i m i n a t e n t e r f e r i n g amino acids ( r e f . 40).

TABLE I 1 Response o f by-products r e l a t i v e t o response o f urea (Reprinted from r e f . 39) Compound

S t r u c t u r a l formula

Urea

H2N

-$

R e l a t i v e response

100

-NH2

0 B iuret

H2N

- f - 0 -F - NH2 0

Guani d i ne

H2N

H

0.29

O

- F - NH2

0

NH Cyanamide

H2N

- CNI

1.5

Dicyandiamide

H2N

- F - t/ - C=N

0.64

NH

H

jn Pump

219

pulse"damper

guard column

Q

l

pressure trans ducer Sampling

valve

R P 18 anal column

urease SPR

wlse damper OPA reactor OPA reagent

fluorimeter

'.a 'I

waste

Fi g . 2

integrator

0

recorder

Schematic diagram o f an HPLC/urease reactor/OPA r e a c t o r / f lu ore s c e n c e d e t e c t o r system. R e p r i n t e d f rom r e f . 39.

220

urine 1000 x diluted without sample pretreatment

urine 1000x diluted with sample oretreatment

without sample pretreatment

with sample pretreatment

UREA

AMMON I A

AMMONIA

x '112

(1

r r L 4-

JL

X1'P

4

8

i

L-

(a)

0

xl

r

I

I

0

-

X l

r-

4

,

,

,

0 0 time (min )

(b)

4

(C )

I

,

8

I

,

0

,

4

,

,

8

(d)

Fig. 3

(a) Chromatogram o f a 1000-fold d i l u t e d u r i n e sample w i t h o u t p r e treatment procedure. Column, 5 - p n Spherisorb ODs-2; e l u e n t , 0.05 M potassium phosphate b u f f e r (pH 6.9) w i t h 0.005 M s o d i m o c t y l s u l phonate. Urease-SPR, s t a i n l e s s - s t e e l column packed w i t h immobilized urease. Fluorescence d e t e c t i o n . Note: X 1/12 means t h a t t h e actual peak h e i g h t i s t w e l v e times t h e peak h e i g h t as i n d i c a t e d i n t h e f i g u r e . (b) As F i g . 3a but with a urine sample cleaned by pretreatment procedure. (c) As F i g . 3a, b u t w i t h o u t urease-SPR. (d) As F i g . 3b, b u t w i t h o u t urease-SPR. Reprinted from r e f . 40.

5.2.3.3

HYOROXYSTEROID DEHYDROGENASES

Several papers discuss t h e use o f 3 w h y d r o x y s t e r o i d dehydrogenase i n a post-column r e a c t o r f o r t h e d e t e r m i n a t i o n o f b i l e acids. A f t e r t h e e a r l y work by Okuyama e t a1 ( r e f . 41) methods f o r t h e r o u t i n e d e t e r m i n a t i o n o f b i l e acids i n serum were developed.

B i l e a c i d s r e a c t w i t h NAD i n t h e

enzyme column w i t h t h e f o r m a t i o n o f NADH; NADH i s r e a c t e d w i t h phenazine methosulphate s o l u t i o n which i s followed by electrochemical d e t e c t i o n a t + 0.1 V ( r e f . 42). D i r e c t o x i d a t i o n o f NADH was n o t successful due t o t h e

h i g h e r p o t e n t i a l needed (+ 0.33 V ) which c o n s i d e r a b l y decreased t h e

221 s e l e c t i v i t y o f t h e method. Another approach i s t h e f l u o r e s c e n c e d e t e c t i o n o f NADH ( r e f . 43). The a u t h o r s succeeded i n t h e use o f t h e same a l k a l i n e pH (9.7) f o r b o t h s e p a r a t i o n and enzyme r e a c t i o n . Besides t h e e l u e n t pump t h e system needs a second pump f o r NAD a d d i t i o n , b u t compared t o e l e c t r o chemical

detection

cellulose

(ref.

beads were

42)

chosen

one as

pump

less

support

is

required.

because o f

Spherical

their

chemical

s t a b i l i t y a t a l k a l i n e pH. L a t e r , t h e system was equipped w i t h an o n - l i n e sample p r e t r e a t m e n t system t o e l i m i n a t e t e d i o u s manual p r e c e s s i n g s t e p s and make aut o ma t i c c o n t r o l 1 p o s s i b l e ( r e f . 44). Now, NAD was added t o t h e m o b i l e phase so t h a t a s i n g l e pump system was obt ained. Hayashi e t a l . ( r e f . 45) succeeded i n t h e s e p a r a t i o n o f t h e b i l e a c i d s i n r a t b i l e . Rats possess s ev era l p e c u l i a r b i l e a c i d s which c o m p l i c a t e t h e s e p a r a t i o n . A C8 column was used f o r s e p a r a t i o n and a second pump was used f o r a d d i t i o n o f NAD and f o r pH a d a p t a t i o n a f t e r t h e s e p a r a t i o n . A chromatogram o f a r a t b i l e e x t r a c t i s g i v e n i n F i g . 4. Another h y d r o x y s t e r o i d dehydrogenase was used i n a s i m i l a r a n a l y t i c a l system ( r e f . 46). A column packed w i t h 3 8 , 1 78-h y dro x y s t e ro i d dehydrogenase immobi 1 i z e d on g l a s s beads was used t o react

J

5 -3 8-h y dr o x y s t e r o i d s u l p h a t e s w i t h NAD w i t h t h e f o r m a t i o n o f NADH

which was d e t e c t e d by f l u o r e s c e n c e m o n i t o r i n g . The method was s u c c e s s f u l f o r serum a n a l y s i s and good c o r r e l a t i o n between t h e HPLC method and r a d i o immuno assay was found. 5.2.3.4

ACETYLCHOLINE ESTERASE AND CHOLINE O X I D A S E

A method f o r d e t e r m i n a t i o n of c h o l i n e and a c e t y l c h o l i n e i n neuronal

t i s s u e was i n t r o d u c e d by Damsma e t a l . choline

are

ad a pt a t io n,

separated

by

means

of

(ref.

47).

cation

C h o l i n e and a c e t y l -

exchange

HPLC.

W it hout

t h e m o b i l e phase e n t e r s t h e r e a c t o r packed w i t h a c e t y l -

c h o l i n e s t e r a s e and c h o l i n e o x i d a s e co-immobil i z e d on sepharose beads. The latter

enzyme

releases

hydrogen

peroxide

f rom c h o l i n e ,

the

former

co nv ert s a c e t y l c h o l i n e t o c h o l i n e . The hydrogen p e r o x i d e formed i s de t e c t e d e l e c t r o c h e m i c a l l y . A v e r y s i m i l a r method was d e s c r i b e d by Asano e t al.

( r e f . 4 8 ) , t h e d i f f e r e n c e b e i n g t h e use o f g l a s s beads as t h e

su pport f o r i m m o b i l i z i n g t h e enzyme.

Instead o f d e t e c t i n g t h e evolved

hydrogen p e r o x i d e e l e c t r o c h e m i c a l l y , t h e r e i s a l s o t h e p o s s i b i l i t y t o use pe rox y ox alat e chemiluminescence ( r e f . 49). Three pumps were needed t o p r o v i d e f o r t h e o p t i m a l media f o r s e p a r a t i o n , enzymatic r e a c t i o n and chemiluminescence d e t e c t i o n . Only s t a n d a r d m i x t u r e s were analyzed. L a t e r , a more e l e g a n t system w i t h chemiluminescence d e t e c t i o n was p u b l i s h e d ( r e f . 50).

The system has an enzyme r e a c t o r coupled d i r e c t l y t o t h e

analytical

column

and

uses

solid

phase c h e m i s t r i e s

in

the

chemi-

222

luminescence detection step. Determination of chol ine and acetylcholine in urine and in serum (Fig. 5) was reported. Due to the selectivity of the chemiluminescence detection system, the sample pretreatment was simplified. An alternative immobilization method is the use of adsorption on an anion exchange cartridge (refs. 51, 52). This is a very simple way of preparing a reactor; just by injection of an enzyme solution on the cartridge at the proper pH. If low ionic strength mobile phases are used the reactor can be used for ca. 3 weeks before it needs to be reloaded. Problems arise when the reaction product that is to be detected is strongly retained on the ion exchanger. Strong retention o f hydrogen peroxide formed in a reactor containing glucose oxidase immobilized on an ion exchanger was reported by Van Zoonen et al. (ref. 5 3 ) .

Fig. 4

Typical chromatogram of bile acids extracted from normal rat bile. Peaks: 1 = a -muricholic acid; 2 = 0-muricholic acid; 3 = cholic acid; 4 = ursodeoxycholic acid; 5 = hyodeoxycholic acid; 6 = chenodeoxycholic acid; 7 = deoxycholic acid; 8 = 50-androstane-3% 1 1 4 170-trio1 (internal standard); = unknown. Fluorescence detection. Reprinted from ref. 45.

223

@

@

Ch Ch

In

0

I

X

e V

~

0

4

8

t(min)-

Fig. 5

(a) Chromatogram o f a d e p r o t e i n a t e d pooled serum sample, (b) serum sample spiked w i t h 200 pmole o f Choline and A c e t y l c h o l i n e . Chemiluminescence d e t e c t i o n . R e p r i n t e d w i t h permission from r e f . 50.

5.2.3.5

GLUCURONIDASE AND GLYCOSIDASE

End products

from metabolic

pathways

i n plants

and

animals

are

d e t e c t a b l e by means o f immobilized enzyme post-column r e a c t i o n d e t e c t i o n systems. R-glucuronidase from bovine l i v e r was immobilized on agarose and on g l a s s

beads

glycosides w i t h

and was

used

electrochemical

for

post-column

detection o f

cleavage o f the phenolic

phenoiic compounds

formed ( r e f . 54). A f t e r enzymatic cleavage, lower d e t e c t i o n p o t e n t i a l s can be used. Though t h e system can work w i t h a s i n g l e pump, t h e s e n s i t i v i t y can be improved by changing t h e pH p r i o r t o t h e enzymatic

reaction.

Almost t h e same system was used f o r t h e d e t e r m i n a t i o n o f g l u c u r o n i d e conjugates f o r fenoldopam, an a n t i h y p e r t e n s i v e agent i n human plasma and u r i n e ( r e f . 55). The diastereomeric fenoldopam glucuronides a r e cleaved by t h e enzyme and t h e r e s u l t a n t catechol moiety ca be d e t e c t e d e l e c t r o chemically. Chromatograms obtained w i t h t h e system a r e g i v e n i n F i g . 6.

224

The reactor containing the enzyme immobilized on glass beads is coupled directly between the analytical column and the detector. Dalgaard and Brimer (refs. 56,57) developed a method of analysis for xyanogenic glycosides. Glycosidase from Helix pomatia immobilized on glass beads was used in the post-column reactor. After the enzymatic reaction, base i s added and the evolved cyanide is detected amperometrically at a silver electrode. The reactions involved are Enzymatic reaction: R1-

9

9

Glycosidase C-- CN + H 2 0 LR1-- C-CN I b l y OH

+ GlyOH

C1 eavage by base :

9

Rl-C--CN AH

+ OH- > -

R I R ~ C O+ CN- + H 2 0

Anodic reaction : Ag(CN)Z + e-

Ag + 2CN-

A Fig. 6

Chromatograms of plasma extracts from a human subject before (A) and 0.5 h after (B) oral administration of fenoldopam. The concentrations of 1(R)- and 1(S)-fenoldopam-7-0-R-glucuronide are 156 and 160 ng/nl, resprectively. Electrochemical detectiori. Reprinted from ref. 55.

Introduction of a split between the analytical column and the reactor was used to create a longer residence time in the reactor, useful for slower reacting substrates. The method was used for the determination of

225 cyanogenic glycosides i n human u r i n e , serum and i n crude p l a n t t i s s u e e x t r a c t s (see Fig. 7 ) .

C IS

A

I

I

s

1

L

L

J

0 3 6 9 12 15 182; 2Lmin

~

I

I

I

L

'

J

C 3 6 9 12 15 l @ 2 1 ~ 1 1 n

Fig. 7

Chromatograms o f cyanogenic g l y c o s i d e s i n p l a n t e x t r a c t s a f t e r s u i t a b l e d i l u t i o n . (A) S . n i g r a L ( d i l u t i o n f a c t o r 50); (B) P. Lauracerasus L.; (C) T. baccata L. ( d i l u t i o n f a c t o r 50). Amygdalin added as i n t e r n a l standard ( i s ) . Electrochemical d e t e c t i o n . Reprinted from r e f . 57.

5.2.3.6

XANTHINE OXIDASE

The simultaneous d e t e r m i n a t i o n o f hypoxanthine

(Hyp)

and Xanthine

(Xan) i n b i o l o g i c a l f l u i d s i s important f o r v a r i o u s pharmacological and p h y s i o l o g i c a l reasons. An HPLC method w i t h o n - l i n e post-column enzymatic d e r i v a t i z a t i o n was developed ( r e f s . 58, 59). Hyp and Xan were o x i d i z e d by t h e enzyme: x a n t h i ne Xan + H202

Hyp + H20 t O2 oxidase x a n t h i ne Xan t H20 t

O2

u r i c a c i d + H202 oxidase

226 The resulting uric acid was detected by UV-absorption at 290 nm. However this detection mode did not offer the selectivity and sensitivity needed for serum analysis. An alternative was found by using fluorescence monitoring of the product formed in the reaction between H202 and p-hydroxyphenylacetic acid on immobilized peroxidase (ref. 60). A reactor with immobilized catalase was used in the reagent stream prior to the mixing in order to remove H202 and thereby reduce background fluorescence. A chromatogram obtained with this method applied to urine analysis is given in Fig. 8 and demonstrates the high selectivity and sensitivity of this approach. Later, the system was modified to be able to also determine inosine (ref. 61). Four enzyme reactors and three pumps were needed. A schematic diagram of the experimental set-up is given in Fig. 9. In the first reactor inosine is converted into Hyp by immobilized purine nucleoside phosphorylase. A1 1 four enzymes were immobi 1 ized on glass beads. Although the system is claimed to work reliably, the detection approach looks quite complicated for routine analysis. a h

u 0

Fig. 8

10

20

30

mi*

L

I

I

0

10

20

I

I

30

Chromatograms of a urine extract from a normal subject obtained by the present method (see text) ( A ) , when the immobilized xanthine oxidase reactor i s removed from the system ( B ) , and with UV absorbance at 254 nm when all reactors are removed (C). Reprinted from ref. 60.

*in

227

Fig. 9

Flow diagram o f an HPLC system f o r Hyp, Xan and i n o s i n e . (1) Eluent pump, (2) i n j e c t o r , (3) guard column, (4) a n a l y t i c a l column, ( 5 ) immobilized p u r i n e nucleoside phosphorylase r e a c t o r , (6) immobilized xanthine oxidase r e a c t o r , (7) immobilized p e r oxidase r e a c t o r , (8) fluorescence d e t e c t o r , (9) pump f o r pH adaptation, (10) reagent pump, (11) immobilized c a t a l a s e r e a c t o r , (12) i n t e g r a t o r . Reprinted w i t h permission from r e f . 61.

5.2.3.7

L-AMINO A C I D OXIDASE

Another

system

i n which

H202

i s detected v i a

homovanillic

acid

r e a c t i o n on coimmobil i z e d peroxidase was i n t r o d u c e d f o r t h e s e l e c t i v e detection o f

L-amino

acids

(ref.

62).

The immobilized L-amino

acid

oxidase catalyses t h e s t e r e o - s e l e c t i ve deami n a t i o n o f L-amino acids: L-amino a c i d +

O2

+ H20

9

2-keto a c i d + NH3 + H202

0-amino acids do n o t r e a c t a n d ' w i l l n o t be detected.

F l a v i n e adenine

d i n u c l e o t i d e was added i n t h e i m m o b i l i z a t i o n process i n o r d e r t o o b t a i n a more s t a b l e and h i g h l y a c t i v e r e a c t o r . The use of s t e r e o - s e l e c t i v e enzymes i n t h e d e t e c t i o n process offers attractive alternative t o chiral

separations.

Unfortunately,

an

in this

system t h e f a c t t h a t t h e m o b i l e phase should be compatible w i t h t h e enzyme r e a c t o r r e s u l t e d i n i n s u f f i c i e n t r e s o l u t i o n o f some o f t h e amino acids. An a l t e r n a t i v e i n t h e d e t e c t i o n o f amino acids a f t e r t h e enzymatic reaction i s t o monitor the conductivity o f the reactor e f f l u e n t ( r e f . 63). The System works w i t h a s i n g l e pump b u t , compared w i t h fluorescence detection ( r e f . 62), l i m i t s o f detection are i n f e r i o r .

228 5.2.3.8. CHOLESTEROL OXIDASE Cholesterol oxidase immobilized on g l a s s beads was used i n t h e detection

of

cholesterol

and some a u t o - o x i d a t i o n

products

(ref.

64).

Cholesterol has o n l y moderate UV a b s o r p t i o n a t a s h o r t wavelength whereas t h e o x i d a t i o n product formed i n t h e enzymatic r e a c t i o n can be d e t e c t e d w i t h h i g h a b s o r p t i v i t y a t 241 nm. A h i g h flow o f b u f f e r was added t o t h e column e f f l u e n t t o reduce t h e ethanol

content p r i o r t o e n t e r i n g t h e

enzyme r e a c t o r . The optimum d i l u t i o n r a t i o was found by b a l a n c i n g t h e loss o f s e n s i t i v i t y due t o t h e d i l u t i o n versus t h e decreased conversion e f f i c i e n c y w i t h increased ethanol c o n t e n t i n t h e r e a c t o r . I n o r d e r t o reduce t h e flow r a t e through t h e r e a c t o r f o r l o n g e r r e a c t i o n time, p a r t o f t h e f l o w was s p l i t o f f .

UV-monitoring a t 241 nm w i t h t h e r e a c t o r was

found t o y i e l d ca. 4 times h i g h e r s e n s i t i v i t y than a t 211 nm w i t h o u t reactor. 5.2.3.9

ALKALINE PHOSPHATASE

Inorganic phosphate r e s u l t i n g from i n o s i t o l b i s - and t r i p h o s p h a t e s and o t h e r organic phosphates i n a r e a c t o r packed w i t h immobilized a l k a l i n e phosphatase was

detected

by

reacting with

a molybdate s o l u t i o n

2s

described by Meek and N i c o l e t t i ( r e f . 65). The enzyme was immobilized by simple adsorption on a hydrophobic support.

No mobile phase a d a p t a t i o n

between t h e anion exchange a n a l y t i c a l column and t h e enzyme r e a c t o r was needed. The method was demonstrated w i t h t e s t m i x t u r e s b u t t h e authors claim s u f f i c i e n t s e n s i t i v i t y f o r t i s s u e analysis. 5.2.4

CONCLUDING REMARKS

Up t o now,

immobilized enzymes i n post-column

r e a c t o r s a r e almost

uniquely used f o r t h e d e r i v a t i z a t i o n o f m o s t l y p o l a r a n a l y t e s t h a t can be separated on t h e a n a l y t i c a l column w i t h a h i g h l y aqueous mobile phase. I n t h i s way, t h e enzymatic r e a c t i o n can t a k e p l a c e under c o n d i t i o n s t h a t a r e s i m i l a r t o those i n nature. Although immobilized enzymes a r e r e p o r t e d t o be more s t a b l e than enzymes i n s o l u t i o n they can u s u a l l y n o t w i t h s t a n d h i g h m o d i f i e r concentration. On t h e o t h e r hand, t h e r e a r e examples of a c t i v e enzymes i n s o l u t i o n s w i t h a r e l a t i v e l y h i g h c o n t e n t o f o r g a n i c modifier. b i l e acids

I n t h e system described by Okuyama f o r t h e d e t e r m i n a t i o n o f (ref.

41)

a 22% a c e t o n i t r i l e s o l u t i o n f l o w s through t h e

r e a c t o r and a 80% a c e t o n i t r i l e s o l u t i o n was used i n a f l o w i n j e c t i o n a n a l y s i s system w i t h immobilized glucose oxidase ( r e f . 53). Ethanol a t h i g h c o n c e n t r a t i o n (17.3%) was pumped through t h e r e a c t o r w i t h i m m o b i l i z e d c h o l e s t e r o l oxidase ( r e f . 64). However, most a n a l y t i c a l systems

229 w i t h an immobilized enzyme r e a c t o r c o n t a i n o n l y a few p e r cent o f o r g a n i c m o d i f i e r and p r e f e r a b l y a l c o h o l s r a t h e r than a c e t o n i t r i l e s i n c e o t h e r w i s e t h e enzymes l o s e a c t i v i t y i n e i t h e r a r e v e r s i b l e o r i n an i r r e v e r s i b l e The i n f l u e n c e o f v a r i o u s o r g a n i c s o l v e n t s i n d i f f e r e n t concect r a t i o n s on r e a c t i o n k i n e t i c s was i n v e s t i g a t e d by Bowers and Johnson f o r

way.

66). When p-nitrophenylglucuronide i s used as t h e s u b s t r a t e , i n c r e a s i n g t h e methanol content r e s u l t s f i r s t in an increase i n a c t i v i t y up t o ca. 10% methanol f o l l o w e d by a steep decrease whereas w i t h e s t r i o l - 3 - g l u c u r o n i d e as t h e s u b s t r a t e t h e r e i s o n l y a decrease i n r e a c t i o n r a t e upon i n c r e a s i n g t h e methanol content. The r e a c t i o n r a t e on t h e enzyme was a l s o i n v e s t i g a t e d w i t h e t h a n o l , a c e t o n i t r i l e and e t h y l e n e g l y c o l as t h e o r g a n i c c o s o l v e n t (see Fig. 10). The above r e s u l t s reveal t h a t an enzyme can r e a c t i n d i f f e r e n t ways when i t i s brought i n c o n t a c t w i t h non-natural compounds. Also t h e s u b s t r a t e t o be converted i s an important parameter. Other enzymes b e i n g i n v e s t i g a t e d i n 2 s i m i l a r way can show very d i f f e r e n t behaviour. T h i s makes i t imp o s s i b l e t o p r e d i c t whether a c e r t a i n m o b i l e phase needed f o r 2 p a r t i c u l a r separation w i l l be compatible w i t h t h e immobilized enzyme r e a c t i o n . It i s very questionable whether such a p r e d i c t i o n w i l l be p o s s i b l e i n t h e future. For t h e time being, i n each p a r t i c u l a r a n a l y t i c a l system i n v o l v i n g an immobilized enzyme some t r i a l and e r r o r work w i l l be needed. However, t h e compiled a p p l i c a t i o n s above (see Table 111) c l e a r l y r e v e a l t h a t t h e r e are d i s t i n c t p o s s i b i l i t i e s f o r s u c c e s s f u l l y c o u p l i n g HPLC w i t h immobilized enzyme r e a c t o r s and i t i s l i k e l y t h a t more a p p l i c a t i o n s w i l l be published i n t h e coming years. immobilized 8-glucuronidase

(ref.

230

TABLE 111

The use of immobilized enzymes in post-column reactors

Anal yte( s)

Glucose-bphosphate

Enzyds)

Idil i z e d on

Product detected

Detection*

Ref.

gucosed-phosphate

glass

WDH

F

30

dehydrogenase urea

urease

silica

NH

after 3: d e n vat izat ion with OPA reagent

F

39, 40

B i l e acids

3 a-hydroxysteroid dehydrogenase

glass

WDH

F

41

81 l e

3 a-hydroxystemid dehydrogenase

glass

NADH, a f t e r

E

42

B i l e acids

3 a-hydroxysteroid dehydrogenase

eel l u lose

derivatization with phenazine nrthosulphate NADH

F

43, 44

B i l e acids

3 a-hydroxysteroid dehydrogenase

glass

NADH

F

45

-3C-hydroxysteroid sulphates

30,1713-hydroxysteroid dehydrogmase

g Lass

NADH

F

46

Choline and acetylcholine

acetylchol inesterase and choline oxidase

sepharose

H 0

E

47

Choline and acetylcholine

acetylcholinesterase and choline oxidase

glass

no

E

48

Choline and acetylcholine

acetylcholinesterase and choline oxidase

silica

H O

CL

49

Choline and acetylcholine

acetylcholincsterase and choline oxidase

sepharose

CL

50

Choline and acetylcholine

choline oxidase and cholinesterase

H 0

E

51

Acetylcholine

choline oxidase and cholinesterase

i o n exchange

H 0

E

52

0-glucuronidase

agarose, glass phenolic

E

54

E

55

d

acids

5

Phenolic glycosides

ion exchange resin

resin

2 2

2 2

2 2

“O2

2 2

2 2

conrxwnds Glucuronide con] ugates of fenoldupam

R-glucuronidase

glass

fenoldcpm

23 1

TABLE I 1 1

(Continued)

Analytds)

Enzyme($)

Inrnobilized on

Product detected

Detection*

Ref.

Cyanqeni c glycosides

glycosidase

glass, s i l i c a

cyanide, f o r d a f t e r basic cleavage of product from enzyme reaction

E

56, 57

Hypoxanthi ne and xanthine

xanthine oxidase

g Lass

u r i c acid

uv

58, 59

Hypoxanth i ne and xanthine

xanthine oxidase

glass

“24. reaction .after with

F

60

F

61

F

62

p-hydroxypheny 1acetic acid on imnwbi l i z e d w r o x i dase

22: :At tit

Hypoxanthine, xanthine and inos ine

xanthine oxidase and purine nucleoside phosphorylase

glass

L-amino acids

L-amino acid oxidase

glass

L-amino acids

L-amino acid

glass

change i n the i o n i c strenght created by the react im

C

63

Cholesterol and autooxidation products

cholesterol oxidase

glass

oxidized

uv

64

:nositol bis- and triphosphates and other organic phosphates

alkaline phosphatase

UV

65

XF:

E: CL: UV: C:

i h p-hydroxypheny 1acetic acid on imnwbi l i z e d peroxidase H20z, .after reaction with honovanillic acid on coimmobi lized peroxidase

cholesterol

Fluorescence Electrochemical Chemi luminescence U l t r a v i o l e t absorption Conductivity

pheroxyacetyl- Inorganic phoscellulose

phate a f t e r reaction with a mlybdate solution

232

% Acetonitrile

% Ethanol

% Ethylene Glycol

Fig. 10 Normalized reaction rate as a function of vol. percent organic cosolvent. In the methanol panel, curves A and B represent the behaviour observed for immobilized and soluble enzyme, respectively, with p-nitrophenyl glucuronide substrate, while curve C represents the reaction of estriol-3-glucuronide with immobilized enzyme. The acetonitrile and ethanol panels are the results of p-nitrophenyl glucuronide hydrolysis by immobilized 8-glucuronidase. The ethylene glycol panel illustrates the difference observed between the immobilized (A) and soluble (B) enzyme with p-nitrophenyl glucuronide substrate. In all cases the buffer was 0.1 mol/l phosphate, pH. 6.7. Reprinted from ref. 66. 5.3 OTHER SOLID-PHASE CHEMISTRIES 5.3.1 INTRODUCTION In common reaction detection, reagent solution is added to the column effluent by means of a pump as outlined before. An alternative is to introduce the chemicals needed for the derivatization reaction in a heterogeneous way. The reactor, usually a short column packed with active material, can act as a catalyst or provide reagents for the reaction. In the latter case the reactants can come directly in contact with the solid

reagent o r reagent i s d i s s o l v e d g r a d u a l l y b e f o r e t h e r e a c t i o n takes place. The r e a c t o r has t o be recharged p e r i o d i c a l l y . The former approach i s i n t e r e s t i n g s i n c e t h e r e a c t o r keeps i t s a c t i v i t y w i t h o u t t h e need f o r reloading,

provided no c a t a l y s t p o i s o n i n g takes place. The use of

im-

m o b i l i z e d enzyme i n a post-column r e a c t o r as o u t l i n e d i n t h e p r e v i o u s s e c t i o n i s an example o f t h i s approach. T h i s s e c t i o n w i l l discuss t h e o t h e r uses

of

solid-phase

i n the f i e l d o f

chemistries

post-column

reaction detection. 5.3.2

APPLICATIONS OF SOLID-PHASE CHEMISTRIES

5.3.2.1

EARLY WORK

The f i r s t a p p l i c a t i o n s of solid-phase chemistry i n a post-column r e a c t o r were published by Studebaker e t a l . ( r e f s . 67, 68). The method i s designed f o r t h e d e t e c t i o n o f t h i o l s , d i s u l f i d e s and p r o t e o l y t i c enzymes. I n each case, t h e compound of i n t e r e s t releases a d e t e c t a b l e species from t h e packing m a t e r i a l i n a column downstream from t h e a n a l y t i c a l column. The

d e t e c t i o n system

for

disulfides

is

outlined

in

Fig.

11.

The

d i s u l f i d e s r e l e a s e t h i o l s i n t h e upper r e a c t o r and t h e t h i o l s r e l e a s e t h e d e t e c t a b l e species which i s i n i t i a l l y bound t o t h e polymer i n t h e l o w e r r e a c t o r . L a t e r , t h e system was m o d i f i e d by M i l l o t e t a l . ( r e f . 63), t h e main d i f f e r e n c e being t h e use o f s i l i c a i n s t e a d o f a polymer as t h e support i n o r d e r t o minimize band broadening.

5.3.2.2

CATALYTIC SOLID-PHASE CHEMISTRIES

The concept o f s o l id-phase c a t a l y s i s i n post-column d e r i v a t i z a t i o n was f i r s t introduced f o r the determination

o f non-reducing

carbohydrates

( r e f s . 29, 79, 71). Several s t r o n g l y a c i d i c c a t i o n exchangers were used as t h e c a t a l y s t f o r conversion o f t h e non-reducing carbohydrates

into

reducing

Best

carbohydrates which

a r e d e t e c t a b l e by

r e s u l t s were obtained u s i n g 4% c r o s s - l i n k e d r e a c t o r (6 cm i n length)

several

polystyrene

was operated a t 85

OC

means. resins.

t o ascertain

The 10G%

conversion. The separations were c a r r i e d o u t a t a sulphonic a c i d t y p e 2+ (Ca ) c a t i o n exchanger w i t h water as t h e mobile phase. A thorough i n v e s t i g a t i o n o f band broadening i n t h i s r e a c t o r t y p e was p u b l i s h e d by Nondek e t a l .

( r e f . 35). Apart from t h e band broadening t h a t occurs i n

any packed bed, a d d i t i o n a l band broadening i s observed when r e a c t a n t and product have d i f f e r e n t r e t e n t i o n i n t h e r e a c t o r column. A mathematical model was proposed t h e v a l i d i t y o f which was t e s t e d w i t h t h e decocp o s i t i o n o f diacetone alcohol on alumina and t h e c a t a l y t i c h y d r o l y s i s of

1-naphthyl-N-methylcarbamate/Carbaryl) on a s t r o n g l y b a s i c anion ex-

234

changer. Later, the reactivity of other N-methylcarbamates on the ion exchange resin and the applicability t o residue analysis were investigated (refs. 36, 72). Upon decomposition of the N-methylcarbamates, methylamine is splitt off which, after labelling with o-phthalaldehyde (OPA), i s detected by fluorescence monitoring. The reactor is operated at high temperature (100 OC or slightly above) in order t o keep reacticjn band broadening low and to obtain high conversion. The method was applied to the analysis of river water samples (ref. 36) and, after having been combined with on-1ine preconcentration and clean-up, to the analysis of heavily polluted water samples with detection limits well below 1 ppb (ref. 72). FROM ANALYTICPL COLUMN

RSSR'

j

FLOW

R'SSD

OPTICAL DETECTOR

Fig. 11 Diagram of the solid-phase apparatus for detection of disulfides. RSSR' represents a disulfide in the eluate. P-S is a polymer with a bound thiol, P-SSD is a polymer with a bound detectable species. Reprinted from ref. 68.

235

5 3 - 2 a3 NON-CATALYTIC SOLID-PHASE CHEMISTRIES The use o f n o n - c a t a l y t i c solid-phase c h e m i s t r i e s has r e s u l t e d i n d i f f e r e n t approaches i n t h e f i e l d o f post-column r e a c t i o n d e t e c t i o n . The e a r l i e s t p u b l i c a t i o n d e a l t w i t h t h e use o f a column packed w i t h z i n c powder ( r e f . 73). By pumping an a c i d i c m o b i l e phase through t h i s column, hydrogen i n s t a t u nascendi i s produced t h a t s p l i t s o f f i o d i n e from i o d i n a t e d thyronines. The i o d i d e i o n was detected by means o f a c a t a l y t i c p r i n c i p l e based on t h e i o d i d e - c a t a l y z e d r e a c t i o n o f chloramine-T and N , N'-tetramethyldiaminodiphenylmethane i n an a i r segmented t u b u l a r r e a c t o r . Chromatograms o f plasma e x t r a c t s a r e g i v e n i n Fig. 12. The z i n c column should be repacked d a i l y . Another i n t e r e s t i n g use o f a r e a c t o r packed w i t h z i n c was i n t r o d u c e d by Sigwardson and B i r k s ( r e f . 74). N i t r o p o l y aromatic hydrocarbons (nitro-PAHs) were on-1 i n e reduced t o t h e c o r r e s ponding amino-PAHs t h a t c o u l d be detected w i t h h i g h s e n s i t i v i t y u s i n g peroxyoxalate chemi luminescence d e t e c t i o n . The r e a c t o r can be placed e i t h e r b e f o r e o r a f t e r t h e a n a l y t i c a l column so t h a t t h e a n a l y t e s e l u t e e i t h e r as t h e amino-PAHs o r as t h e nitro-PAHs. useful f o r i d e n t i f i c a t i o n . carbon b l a c k e x t r a c t s .

This was found t o be

The method was a p p l i e d t o t h e a n a l y s i s of

3

-+---+ 0 5 ~

rnin

4

t

+

0

5

10

mm

F i g . 12 Chromatographic d e t e r m i n a t i o n o f i o d i n a t e d t h y r o n i n e s w i t h c a t a l y t i c d e t e c t i o n . Column, C-18; mobile phase, methanol-water (67:33) p l u s 0.05% o f methanesulphonic acid; f l o w - r a t e , 0.5 m l / min; d e t e c t i o n wavelength, 600 nm. (A) Determination o f t o t a l T ( t h y r o x i n e ) . I n j e c t i o n volume, 50 m l o f e t h a n o l i c serum efttract. 1, Free h a l i d e ions; 2, 10 ng o f T ( t r i - i o d o t h y r o n i n e , i n t e r n a l standard); 3,8 ng o f T (6) S t e r e a - s p e c i f i c d e t e r m i n a t i o n L-T i n serum a f t e r d e h v a t i z a t i o n . I n j e c t i o n volume, 30 m l . 1, Fr%e h a l i d e ions; 2, 10 ng o f T ( i n t e r n a l standard); 3 and 4, n o t i d e n t i f i e d ; 5, L-Leu-L-T co?respondin t o 7 ng of L-T ) ; 6, L-Leu-D-T4 (corresponding 7 ng o f D - T j . R e p r i n t e d f r o 4 r e f . 73.

.

td

236 Krull et al. (refs. 75-78) described various reactors containing sol id-supported reagents for on-1 ine reduction and oxidation. Most of the applications, e.g., the on-line reduction o f aldehydes to the corresponding alcohols using solid supported borohydride (ref. 75) and the oxidation of primary and secondary alcohols, aldehydes and ketones using an anion exchanger in the permanganate form (ref. 78), deal with precolumn derivatization using difference chromatography to study the extent of reaction. This i s mainly done for identification purpose since no significant changes in detector response are reported which limits, though possible and described (e.9. for the post-column reduction of a variety of aldehydes, ketones and acid chlorides on solid-supported borohydride in normal-phase HPLC) (ref. 76), the applicability in post-column derivatization. All reactors described by Krull et al. can be used for hundreds o f analyses before they loose activity. Solid-phase reactors containing either lead dioxide or manganese dioxide precipitated on silica were compared for use in the pcst-column oxidation of catecholamines to the respective adrenochromes (ref. 79). This was followed by homogeneous reduction to the fluorescent trihydroxyindoles. The two reactors yield similar results in all respects and allow for a simpler, more economic and more reliable post-column reaction system as compared to an all homogeneous approach. The number of analyses possible before depletion of the reagent occurs is more than 300 for both reactors. The same lead dioxide reactor was used to oxidize lower oxidation state chromium ions to chromate which is detected by complexation with 1,5-diphenylcarbazide (ref. 80). As applications, wastewaters and samples from a steel company were analyzed. A solid-phase reactor used in a sulphate selective post-column derivatization system was developed for the analysis of wastewaters of the potato starch industry (ref. 81). The solid-phase reactor is packed with a mixture of silica and barium chloranilate. The solubility of barium sulphate is less than that of barium chloranilate. The sulphate eluting from the ion exchange analytical column will precipitate as barium sulphate and an equivalent amount of the highly coloured acid chloranilate ion is released:

-:OS

+

BaC6C1204 + Ht

+ ByS04 J+ HC6C1204-

Irth et al. (ref. 82) developed a post-column solid-phase derivatization for the selective detection of thiram and disulfiram. These

23 7 t h i u r a m d i s u l f i d e s undergo c o m p l e x a t i o n i n a v e r y s h o r t ( 4 mm i n l e n g t h ) c a r t r i d g e - t y p e r e a c t o r packed w i t h f i n e l y d i v i d e d m e t a l l i c copper t o form a c oloure d copper complex w i t h an a b s o r p t i o n maximum a t 435 nm. The

post-column

complexation

enhances

the

selectivity

of

the

a n a l y t i c a l method w i t h almost t h e same s e n s i t i v i t y as UV d e t e c t i o n a t 254 nm. The post-column r e a c t o r was found t o cause o n l y l i t t l e a d d i t i o n a l band broadening s i n c e no r e t e n t i o n t a k e s p l a c e and t h e r e a c t o r i s s h o r t . The method was demonstrated w i t h t h e d e t e r m i n a t i o n o f t h i r a m i n surf ace wat e r and w i t h t h e d e t e r m i n a t i o n o f d i s u l f i r a m i n u r i n e ( F i g . s h o r t precolumn was used f o r o n - l i n e t r a c e enrichment.

13).

A

When nanogram

amounts o f a n a l y t e a r e i n j e c t e d i n t h e a n a l y t i c a l system,

t h e copper

r e a c t o r can be used f o r more t h a n 200 analyses. Another use o f s o l i d - s u p p o r t e d r e a g e n t was i n t r o d u c e d by Jansen e t a l . ( r e f . 83). The a n a l y t i c a l system c o n t a i n s an anion exchange column i n t h e hydroxy form i n s e r t e d para1 l e l t o t h e i n j e c t o r and a n a l y t i c a l column ( F i g . 14). One p a r t o f t h e a c e t a t e - c o n t a i n i n g m o b i l e phase f l o w s t h r o u g h t h e i n j e c t i o n v a l v e and a n a l y t i c a l column t o achieve t h e s e p a r a t i o n , t h e o t h e r p a r t f l o w s through t h e anion-exchange column where t h e a c e t a t e i o n causes t h e r e l e a s e o f i o n exchanger-bound hydroxide i o n s . F i n a l l y , t h e a l k a l i n e stream from t h e anion-exchange column i s recombined w i t h t h e a n a l y t i c a l column e f f l u e n t . The r e s u l t a n t a l k a l i n e d e t e c t i o n medium i s fav oura ble f o r t h e UV d e t e c t i o n o f b a r b i t u r a t e s a t 254 nm. O nly one pump i s needed f o r t h e s e p a r a t i o n and t h e post-column pH m o d i f i c a t i o n .

A

low-

c o s t l a r g e p a r t i c l e i o n exchanger was used s i n c e t h e anion exchange column does n o t c o n t r i b u t e t o band broadening. Both t h e d e t e r m i n a t i o n c f b a r b i t u r a t e s i n u r i n e and i n plasma were shown. The anion-exchange c o l m n should be regenerated a f t e r a p p r o x i m a t e l y 17 hours use. B a s i c a l l y t h e same a n a l y t i c a l system was adapted f o r a f l a t o x i n d e t e r m i n a t i o n ( r e f . 8 4 ) . Now, t h e p a r a l l e l column was packed w i t h s o l i d i o d i n e and a k n i t t e d open t u b u l a r r e a c t o r was mounted between t h e m i x i n g T -piece and t h e flu o re s c enc e d e t e c t o r . m o b i l e phase;

Iodine i s only p a r t l y soluble

i n t h e aqueous

t h e r e f o r e , t h e p a r a l l e l column can be used i n t h e ana-

l y t i c a l system f o r t h e d e l i v e r y o f a s a t u r a t e d i o d i n e s o l u t i o n o v e r l o n g p e r i o d s of t i m e b e f o r e r e f i l l i n g i s necessary. For optimum response,

a

s p l i t t i n g r a t i o of ca. 30 t o 1 was used. I o d i n e attachment t o t h e double bond of a f l a t o x i n B1 and G I makes them about as f l u o r e s c e n t as a f l a t o x i n B2 and G2. As i n t h e system w i t h t h e p a r a l l e l i o n exchanger, o n l y one

h i g h - q u a l i t y pump i s needed making l o w - c o s t post-column d e r i v a t i z a t i o n p o s s i b l e w i t h o u t t h e need f o r a d a i l y p r e p a r a t i o n o f t h e i o d i n e s o l u t i o n as was necessary b e f o r e ( r e f . 11). The method was s u c c e s s f u l l y a p p i i e d t o

238

the analysis of peanut butter extracts as shown in Fig. 15. The parallel column approach was also found to be applicable in chemiluminescence detection (ref. 85). Bis-2,4,6-trichlorophenyloxalate (TCPO) is added from a solid reagent bed and the fluorophore is immobilized on glass beads packed in a flow cell. Hydrogen peroxide generated photochemically by quinone analytes is measured.

6

4

2

Time (min)

0

t-

Fig. 13 Determination of disulfiram in urine. HPLC conditions: analytical column packed with 5 m Hypersil ODs; pre-columc packed with 5-m LiChrosorb RP-18; eluent, acetonitrile-aqueous acetate buffer (10 mM, pH 5.0) (65:35); detection wavelength, 435 nm (0.02 a.u.f.s.). Pre-concentration of 1.0 ml of urine spiked with 87 ppb of disulfiram (sample stabilized with 10 mM EDTA-citrate). Reprinted from ref 82.

239

flow rcstr

anion exch. COI

splittingT

mixing T

uv flow rcstr

in) valve

anal

COI

Fi g. 14 Experimental s e t - u p o f a p a r a l l e l column d e r i v a t i z a t i o n system. R e p r i n t e d from r e f . 83.

F ig. 15

Chromatogram o f an e x t r a c t o f eanut b u t t e r s p i k e d (8 p p b ) , AfG (8ppb), A f B ( 4 ppb! and AfG 9 4 ppb);

AfBl

with

---_--- Chromatogkam o f an u n s t i k e d peanut b u t t & e x t r a c t . C ondit io n s : a n a l y t i c a l column packed w i t h 5-p LiChrosorb RP-18, p a r a l l e l column packed w i t h s o l i d i o d i n e , k n i t t e d open t u b u l a r reactor, fluorescence detection. Reprinted w i t h permission from r e f . 84.

240 Two systems w i t h TCPO a d d i t i o n from a s o l i d reagent bed, a dual pump design (Fig. 16a) and a p a r a l l e l column design ( F i g . 16b), r e s p e c t i v e l y , were compared and advantages and disadvantages were discussed. The dual pump has t h e advantage o f more f l e x i b l e f l o w r a t e r e g u l a t i o n b u t t h e s p l i t - f l o w has t h e advantage o f s i m p l i c i t y and economics. Much research i n post-column r e a c t i o n d e t e c t i o n has as goal t o render t h e equipment s i m p l e r and l e s s c o s t l y .

The development o f immobilized

enzyme r e a c t o r s and r e a c t o r s based on solid-phase c h e m i s t r i e s as o u t l i n e d i n t h e previous and i n t h i s s e c t i o n a r e c l e a r examples o f t h i s t r e n d since a l l

these systems c o n t a i n

a t l e a s t one pump l e s s than t h e i r

c l a s s i c a l homogeneous r e a c t i o n analogues o r p e r m i t r e a c t i o n types which a r e impossible by conventional means.

F u r t h e r approaches f o r pumpless

r e a c t i o n u n i t s are discussed i n t h e f o l l o w i n g s e c t i o n s .

b

F i g . 16 Schematic diagram o f t h e dual-pump system (a) and t h e s p l i t - f l o w system (b) f o r TCPO a d d i t i o n . Reprinted w i t h permission from r e f . 85.

241 5.4

THE USE OF ELECTROCHEMICAL REAGENT PRODUCTION

E lec t roc hemica l techniques can be used f o r on-1 i n e p r o d u c t i o n o f t h e reagent. One example i s t h e use of copper e l e c t r o d e s o r o t h e r s u i t a b l e metal e l e c t r o d e s a t which metal i o n s a r e generated. Complexing a n a l y t e s (amino a c i d s , d i c a r b o x y l i c a c i d s ) can t h e n be d e t e c t e d by amperometric techniques ( r e f s . 86, 8 7 ) . Another example o f o n - l i n e e l e c t r o c h e m i c a l reagent p r o d u c t i o n i s t h e use o f a m i c r o c o u l o m e t r i c p r o d u c t i o n c e l l downstream from t h e a n a l y t i c a l column i n which t h e p r o d u c t i o n o f bromine o r i o d i n e f ro m K B r o r K I d i s s o l v e d i n t h e m o b i l e phase i s e f f e c t e d . T h i s technique, i n t r o d u c e d by K i n g and K i s s i n g e r ( r e f . 88), i n v o l v e s r e a c t i o n of

bromine o r i o d i n e w i t h s u i t a b l e groups o f compounds such as un-

saturated

organics,

ganosulphur

phenolics,

methoxysubstituted

compounds and t h e decrease

det e c t e d a mp ero m e t r i c a l l y ( r e f s .

89,

90).

in

aromat ics

and

reagent c o n c e n t r a t i o n

A schematic

oris

diagram o f t h e

post-column system i s shown i n F i g . 17 and an a p p l i c a t i o n t o t h e d e t e r m i n a t i o n o f a m p i c i l l i n i n plasma i s g i v e n i n F ig. 18. The bromine o r i o d i n e produced i n t h e m i c r o c o u l o m e t r i c c e l l c o u l d a l s o be used f o r t h e d i r e c t oxidation o f the analyte t o y i e l d a fluorescent derivative. This p r i n c i p l e was demonstrated w i t h t h e d e t e r m i n a t i o n o f t h i o r i d a z i n e

in

plasma a f t e r o x i d a t i o n w i t h bromine ( r e f . 91) and w i t h t h e d e t e r m i n a t i o n o f a f l a t o x i n s i n c a t t l e f e e d f o l l o w i n g i o d i n e a d d i t i o n ( r e f . 9 2 ) . A more

d e t a i l e d d i s c u s s i o n on e l e c t r o c h e m i c a l r e a c t i o n t echniques f o r d e t e c t i o n i n HPLC can be found i n r e f . 93.

d--J; column

I

L _ - _ - _ - _ - - - - - ----J I production1 reaction detector cell COll waste

;

F i g . 17 Scheme o f t h e o n - l i n e r e a g e n t p r o d u c t i o n system: (A) a n a l y t e , (6) reagent (bromine), (R) p r e c u r s o r o f t h e reagent (bromide). R e p r i n t e d from r e f . 89.

242

\

u \ 0

F

. 18

5

2

10

L[min]

0

5 -+

10

15

t[min]

Chromatograms obtained with deproteinized plasma; (a) blank plasma; (b) plasma spiked with ampicillin (8 N/ml). Column: LiChrosorb RP-18; reagent production system as in Fig. 17. Reprinted with permission from ref. 90.

The extension of this principle to other reagents and particularly reactions involving noxious or unstable reagents offers interesting possibilities for the future.

5.5

THE USE OF PHOTOCHEMICAL AND THERMO INITIATED REACTIONS Instead of reagents, it is also possible to "pump" photons into a detection system and to explore the resulting reactions for detection purpose. Generally, some piece of UV-transparent tubing, usually teflon, is coiled or knitted (ref. 94) around a lamp. Also in this approach, a chemical modification of the analytes is achieved without having to use an additional reagent pump. Examples of photochemical derivatization have been discussed elsewhere (refs. 8, 95). In general, photochemical derivatization is not easy to control and the reaction products are not fully known. However, if the irradiation results in an increase in sensitivity and/or selectivity, this is not a drawback for use in a post-column system. In the more recent literature examples of better controlled photochemical derivatizations can be found. One way of improving the situation is to add small amounts of reagent to the mobile

243

phase to be able to conduct the reaction in the desired direction. An example is the photochemical reduction of Vitamin K1 (ref. 96). To ensure the conversion into the desired hydroquinone, a small amount of ascorbic acid is added t o the mobile phase and oxygen is carefully removed. The use of sensitizers is another means to expand the usefulness of photochemical reactions. A quinone can be used as sensitizer for the photochemical detection of reducing species (sugars, alcohols, aldehydes, etc.) (ref. 97). The resulting hydroquinone is detected fluorimetrically. Verbeke and Vanhee (ref. 98) used photochemical derivatization for the selective determination of diethylstilbestrol (DES). The photochemical reaction was followed by on-line oxidation to highly fluorescent products. The experimental set-up used is shown in Fig. 19. The metncd was successfully applied to the selective quantitation of DES residues a t the 1 ppb level in extracts of urine and animal tissues (Fig. 20).

lrom HPLC

A

~

”,

--C

-r ’

d

e

__

I

PHOTO REACTOR

I

r\

methanol-water

n-heplane phosphate buffer bisulphite HCI NaOH

:1

-

HEAT BATH 170C) I

COOL

FLUORIMETER

BATH

(15

I

CI k

I

rn

Fig. 19 Post-column reactor arrangement for the detection of DES: (1) methanol-water (65:35) (0.70 ml/min); (2) n-heptane (0.4 ml/min): (3) phosphate buffer, 0.02 M (0.1 ml/min); (4) hydrogen sulphite solution, 0.05 M (0.1 ml/min); ( 5 ) 3.7 HC1 (0.2 ml/min); (6) 4.8 M NaOH (0.2 ml/min: (7) organic solvent from phase separator (0.9 ml/min). Reprinted from ref. 98.

244

OES

I L

I

D

10

m

30

-

I 40

TWEIMIN'

Fig. 20 (a) Chromatogram of a DES-negative urine extract. (b) Chromatogram of an urine extract containing 1 ppb of DES. For details see Fig. 19. Reprinted from ref. 98.

An interesting extension of the use of photochemical techniques in HPLC was developed by De Ruiter et al. (ref. 99). Dansylated chlorophenols were, after separation on a RP-column, irradiated to knock off the dansyl group. This resulted in enhanced fluorescence since now, the fluorescence is not reduced anymore by inductive and intramolecular heavy atom effects originating from the chlorophenols. After sol id-phas? extraction and precolumn dansylation, chlorophenols can be determined at the ca. 100 ppt level in river water samples. Post-column thermolysis followed by photolysis and fluorescence detection was introduced for the trace-level determination o f ciprofloxacin and its metabolites in urine, serum/plasma, bile, faeces and tissue (ref. 100). Both the thermolysis step and the photolysis wer? optimized yielding fluorescence gain factors up t o 130 as compared t o

245 direct

fluorescence

detection,

i.e.,

without

the

thermolysis

and

p h o t o l y s i s step. Reaction times were very s h o r t , 2 s f o r t h e r m o l y s i s and 0.6 s f o r p h o t o l y s i s . Using t h i s d e t e c t i o n mode, o n l y minimal sample p r e p a r a t i o n , e x t r a c t i o n and/or d i l u t i o n , i s r e q u i r e d . Here, i t i s i n t e r e s t i n g t o mention t h a t another example o f t h e use of a temperature jump a f t e r t h e s e p a r a t i o n was r e p o r t e d by LePage and Rocha ( r e f . 101). They used t h e n i n h y d r i n r e a c t i o n f o r amino a c i d s and o t h e r amines which i s k i n e t i c a l l y slow a t ambient temperature. The reagent was added t o t h e mobile phase w i t h no r e a c t i o n t a k i n g p l a c e d u r i n g t h e separation. By a p p l y i n g a temperature jump a f t e r t h e column r e a c t i o n w i l l proceed a t a reasonable r a t e w i t h o u t n e c e s s i t a t i n g post-column a d d i t i o n o f reagents.

5.6

MINIATURIZATION

I n t h e p a s t decade, an i n c r e a s i n g i n t e r e s t has developed i n t h e use of narrow-bore separation columns (< 2 mm I.D.)

i n HPLC as evidenced by

several review p u b l i c a t i o n s and books i n t h i s f i e l d ( r e f s . 102

-

lG7).

These columns r e q u i r e a much lower v o l u m e t r i c f l o w r a t e than c o n v e n t i o n z l HPLC columns and savings i n s o l v e n t s , reagents and packing m a t e r i a l a r e obvious. I n o r d e r t o keep t h e chromatographic i n t e g r i t y o f t h e system, t h e sample volume must a l s o be reduced i n comparison t o conventional s c a l e HPLC, which can be an advantage when t h e sample volume i s l i m i t e d . Also t h e volumes o f t h e peaks e l u t i n g from a narrow-bore column a r e s m a l l e r than i n t h e case o f conventional s c a l e HPLC. T h i s r e s u l t s i n t h e necessity

t o use low volume

detectors.

When a post-column

reaction

d e t e c t i o n system i s used i n c o n j u c t i o n w i t h narrow-bore HPLC, a c a r e f u l design i s needed i n o r d e r t o a v o i d excessive band broadening and l o s s i n r e s o l u t i o n . Special a t t e n t i o n should be g i v e n t o t h e m i x i n g u n i t s used f o r t h e reagent a d d i t i o n and t o connections. These d i f f i c u l t i e s r e s u l t e d in

only

a

few

applications

of

post-column

reaction

detection

in

m i n i a t u r i z e d HPLC described i n t h e l i t e r a t u r e up t o now. H i r o s e e t a l , ( r e f , 108) were t h e f i r s t t o d e s c r i b e a t u b u l a r post-column r e a c t o r coupled t o a 0.5 mm I.D. a n a l y t i c a l column f o r r h e determination o f r a r e e a r t h metals by a c o l o u r r e a c t i o n w i t h x y l e n o i orange. Today t h i s post-column system can be considered as an o b s o l e t e device s i n c e a several-meter-long

tubular reactor o f r e l a t i v e l y large i n n e r diameter and a wide-bore m i x i n g u n i t were used. U n f o r t u n a t e l y , no

data on band broadening and d e t e c t i o n l i m i t s were given. Kucera and Umagat ( r e f . 109) described t u b u l a r post-column r e a c t o r s

246 f o r the

f a s t d e r i v a t i z a t i o n of

p r i m a r y amines w i t h o-phthalaldehyde

(OPA). A 30 cm x 1 mm I.D. a n a l y t i c a l column was used. The i n f l u e n c e s of t h e m i x i n g u n i t design and t h e pumps on m i x i n g n o i s e were c a r e f u l l y examined. The c o n t r i b u t i o n t o t o t a l band broadening o r g i n a t i n g from t h e t u b u l a r r e a c t o r was i n v e s t i g a t e d f o r d i f f e r e n t r e a c t o r l e n g t h s and i n n e r diameters. The authors' recommendation was t o zigzag t h e r e a c t o r t u b i n g i n o r d e r t o enhance r a d i a l m i x i n g due t o t h e secondary f l o w phenolnenon r e s u l t i n g i n lower band broadening.

K n i t t i n g t h e r e a c t o r as proposed by

Engelhardt and Neue ( r e f . 4) w i l l a l s o be very u s e f u l f o r t h i s purpose. The a n a l y s i s o f a m i x t u r e of primary amino a c i d s and a m i x t u r e of homologous n-alkylamines were presented as a p p l i c a t i o n s . Apffel e t a l . (ref.

110) used t h e same OPA r e a c t i o n i n t h e e v a l u a t i o n

of t u b u l a r and packed bed r e a c t o r s f o r narrow-bore HPLC from b o t h a t h e o r e t i c a l and an experimental p o i n t o f view. I n t h e model system, catecholamines were separated on a 1 mm I.D. a n a l y t i c a l column f o l l o w e d by OPA d e r i v a t i z a t i o n and fluorescence d e t e c t i o n . I n comparison w i t h t h e measurement o f t h e n a t u r a l fluorescence, t h e d e t e c t i o n l i m i t s were lowered by more than an o r d e r o f magnitude when u s i n g t h e post-column d e r i v a t i z a t i o n . The band broadening caused by t h e post-column system was acceptable b u t c o u l d have been

improved w i t h a b e t t e r m i x i n g u n i t .

Because o f t h e r e l a t i v e l y h i g h band spreading i n t h e m i x i n g u n i t , a Valco T-piece, no d i f f e r e n c e i n performance between a system w i t h a packed bed r e a c t o r ( p a r t i c l e s i z e 5 pn) and one w i t h a 100 pn I.D. t u b u l a r r e a c t o r could be found. Takeuchi e t a1 ( r e f . 111) described a solid-phase r e a c t o r c o n t a i n i n g 3mhydroxysteroid

dehydrogenase

immobilized

on

glass

beads

d e t e c t i o n o f b i l e acids a f t e r s e p a r a t i o n on a 0.26 mm I.D. column.

for

the

analytical

The 3mhydroxy group i n each b i l e a c i d was o x i d i z e d i n t h e

enzymatic r e a c t i o n , w h i l e NAD was reduced t o NADH which was subjected t o fluorescence monitoring.

Two experimental set-ups were compared ( f i g .

21). A d d i t i o n o f NAD t o t h e m o b i l e phase p r i o r t o t h e column avoided t h e need o f post-column baseline s t a b i l i t y .

reagent a d d i t i o n

and t h i s was found t o

improve

A chromatogram obtained w i t h t h e l a t t e r system i s

shown i n F i g . 22. Band broadening i n t h e immobilized enzyme r e a c t o r was relatively

large

because

sufficiently

small

glass

beads

were

not

a v a i l a b l e . The same equipment was used i n combination w i t h an o f f - l i n e t r a c e enrichment procedure f o r t h e d e t e r m i n a t i o n o f b i l e a c i d s i n s e w n ( r e f . 112).

247

rl 7

5

6

F i g . 21 Schematic diagrams o f t h e systems (see t e x t ) : (A) post-column m i x i n g system; (B) pre-mixing system. 1 = Pump (Micro Feeder); 2 = g r a d i e n t equipment; 3 = m i c r o v a l v e i n j e c t o r ; 4 = guard column; 5 = separation column; 6 = T-piece; 7 = immobilized enzyme column; 8 = spectrophotofluorimeter. R e p r i n t e d from r e f . 111. A d d i t i o n a l examples o f t h e use o f a solid-phase r e a c t o r i n post-column systems f o r narrow-bore HPLC can be found i n r e f . described

for

the determination o f

113. A system was pesticides v i a

N-methylcarbamate

separation on a 180 mm x 1.0 mm I . D . reversed-phase column, h y d r o l y s i s on an anion exchange r e s i n i n a solid-phase r e a c t o r k e p t a t 90 OC, and subsequent d e r i v a t i z a t i o n of t h e l i b e r a t e d methylamine w i t h I P A i n a v e r y s h o r t open t u b u l a r r e a c t o r ( F i g . 23). S i m i l a r equipment has been used f o r t h e r a p i d d e t e r m i n a t i o n o f urea and ammonia v i a t h e i r s e p a r a t i o n on a 40

mm x 1.0 mm I . D .

column, h y d r o l y s i s o f t h e urea on immobilized urease,

and f o l l o w e d by d e r i v a t i z a t i o n w i t h OPA i n a c o i l e d open t u b u l a r r e a c t o r . In this

system,

the analytical

column and t h e r e a c t o r were coupled

w i t h o u t connecting c a p i l l a r y ( F i g . 24). T h i s e l i m i n a t i o n o f a p o s s i b l e source o f band broadening

i s recommended i f column and r e a c t o r

are

operated a t t h e same temperature. Band broadening i n t h e m i x i n g u n i t c o u l d be kept s u f f i c i e n t y low by a s p e c i a l design (Fig. 25). This m i x i n g u n i t was constructed from two blocks; t h e upper one (B) contained t h e entrance and e x i t c a p i l l a r i e s (E)

t h a t ended i n a small groove

(D)

machined i n a PTFE p l a t e (C), supported by t h e lower p a r t (A). The same m i x i n g u n i t was used i n a m i n i a t u r i z e d v e r s i o n o f t h e s p l i t - f l o w

248 a n a l y t i c a l system f o r b a r b i t u r a t e s ( r e f . 83). A c r i t i c a l comparison between t h e conventional s c a l e (3 mm I.D. column) and t h e narrow bore (1 mm 1.13. column) system i s made i n r e f . 83. I t was found t h a t t h e concen-

t r a t i o n s e n s i t i v i t y i n narrow-bore system was lower than i n t h e conv e n t i o n a l s c a l e system. Although t h e peak broadening i n volume u n i t s i s l e s s i n t h e narrow-bore system, r e s u l t i n g i n more concentrated peaks i n comparison t o peaks e l u t i n g from t h e conventional s c a l e column i f t h e same mass i s i n j e c t e d on t h e column, t h i s does n o t y i e l d more d e t e c t o r s i g n a l s i n c e t h e peak i s now d e t e c t e d i n a c e l l w i t h a lower o p t i c a l p a t h length. Therefore, equal masses p u t on t h e two column types w i l l y i e l d roughly equal d e t e c t o r s i g n a l s . Since t h e mass a p p l i e d t o t h e narrow-bore column i s i n j e c t e d i n a low volume; t h e c o n c e n t r a t i o n s e n s i t i v i t y i s l e s s . A/B

100/0

5(3/50

0/100

I

0

1

2

T I 11. E l h \

Fig. 22

Separation o f b i l e a c i d s (each around 5 ng) by t h e p r e m i x i n g system Column; B i l e p a k , 250 x 0.26 mm 1.0. M o b i l e phase: g r a d i e n t p r o f i l e as i n d i c a t e d o f a c e t o n i t r i l e - 3 0 mM (A) o r 10 mM (B) potassium dihydrogen orthophosphate (pH 7.8) 10 mM potassium dihydrogen orthophosphate (pH 7.0) c o n t a i n i n g 6 mM NAD, 0.05% 2-mercaptoethanol and 1 mM EDTA, i n t h e p r o p o r t i o n s (A) 18:52:30 and (6) 35:35:30, each c o n t a i n i n g 0.1% ammonium carbonate. Samples: 1 = ursodeoxycholic a c i d , 2 = c h o l i c a c i d , 3 = g l y c o u r sodeoxycholic a c i d , 4 = g l y c o c h o l i c acid, 5 = tauroursodeoxycholic a c i d , 6 = t a u r o c h o l i c acid, 7 = chenodeoxycholic a c i d , 8 = deoxycholic a c i d , 9 = glycochenodeoxycholic a c i d , 10 = glycodeoxycholic a c i d , 11 = taurochenodeoxycholic a c i d , 12 = taurodeoxycholic a c i d , 13 = l i t h o c h o l i c a c i d , 14 = g l y c o l i t h o c h o l i c acid, 15 = t a u r o l i t h o c h o l i c a c i d . Wavelength o f detect i o n : e x c i t a t i o n 365 nm, emission 470 nm. Reprinted from r e f . 111.

-

249

1

2

aJ

m c 0 Cl m

f

L

0

3

4 4

aJ '13

L I

I

0

I

2 4

I

6

I

I

1

1

8101214

time(min1

F i g . 23

Chromatogram o f N-methylcarbamate p e s t i c i d e s . 1 = Methomyl, 2 = a l d i c a r b , 3 = propoxur, ca. 25 ng each; 180 mm x 1.0 mm I . D . column packed w i t h 5 Spherisorb ODs-2; 40 mm x 1.0 mn I.D. solid-phase r e a c t o r packed w i t h a s t r o n g anion exchange r e s i n ; 170 mm x 0.12 mm I . D . s t r a i g h t open-tubular r e a c t o r f o r OPAr e a c t i o n . Flow r a t e o f e l u e n t : 35.7 pl/min; f l o w r a t e o f OPA-reagent: 7.5 pl/min; fluorescence d e t e c t i o n . Reprinted w i t h permission from r e f . 113. /'I16

II

valco,connector\

I

I

flow direction-

F i g . 24

Construction o f t h e combination o f s h o r t a n a l y t i c a l column and urease-SPR (both 40 mm x 1.0 mm I.D., 1/16" O.D.) Reprinted w i t h permission from r e f . 113.

250

D

Fig. 25 Construction of the mixing unit. For explanation, see ex .. Reprinted with permission from ref. 113. miniaturized HPLC system with a tubular reactor was used with chemiluminescence detection for dansylated amino acids (ref. 114). The analytes are separated on a 250 mm x 1.0 mm I.D. column. The reagent, 1 mM bis(2,4,6-trichlorophenyl) oxalate in ethyl acetate-0.1 M H202 in acetone (1:3 v/v), was added at a relatively high flow-rate, thereby also working as a make-up flow. For this reason a cyclon-type mixing unit fGr conventional scale HPLC was used after a minor modification; a 0.1 mm I.D. stainless steel tube was inserted in the eluent inlet ((Fig. 26). The authors claim detection limits of 0.2 fmol. A chromatogram of four dansylated amino acids is given in Fig. 27. In a more recent publication (ref. 115), the authors replaced the 1 mm 1.0. column by a 2.1 mm I.D. column. This was done because two pump gradient elution on the 1 mm I.D. column was not successful due to pump irregularities, resulting in increased baseline noise. Van Vliet et al. (ref. 116) investigated the possibility of coupling a tubular post-column reactor to a 25 cm I.D. open tubular separation column. Using these extremely small bore columns, extra column volume is only tolerable on the nanoliter scale. Two A

251 s p e c i a l l y designed m i x i n g u n i t s ( F i g . 28) were proposed and e v a l u a t e d . 25 fl

I.D.

f u s e d s i l i c a t u b i n g was

used as

the reactor.

To make t h e

d e t e c t i o n i n a v e r y low volume, l a s e r - i n d u c e d f l u o r e s c e n c e was used. Even a t t h i s e x t r e m e l y low volume s c a l e , band b r o a d e n i n g i n t h e m i x i n g u n i t and r e a c t o r c o u l d be k e p t a t a l e v e l c o m p a t i b l e w i t h t h e column used. The O P A - d e r i v a t i z a t i o n o f a l a n i n e was g i v e n as an example.

T

F i g . 26

C y c l o n - t y p e m i x i n g u n i t f o r m i n i a t u r i z e d HPLC. R e p r i n t e d from r e f . 114.

W

In

Z

B

Ln

W

a: a: W

n

a:

(r

F i g . 27

1

1

1

I

0 15 30 RETENTION TIME (MIN

Chromatogram o f Dns-amino a c i d s , o b t a i n e d w i t h t h e m i c r o b o r e HPLC-chemi luminescence d e t e c t i o n system. The amount o f each amino a c i d i n j e c t e d was 3 fmol. E l u e n t f l o w - r a t e 0.03 m l / m i n . Reagent f l o w - r a t e 0.6 ml/min. R e p r i n t e d f r o m r e f . 114.

252

Fig. 28 Design of the mixing devices. 1 = Open-tubular column; 2 = reaction capillary; 3 = reagent delivery capillary; (A) based on a Valco 1/16-in zero dead volume connector, 4 = connector body, 5 = stainless-steel liner, 6 = PTFE ferrules, arrows indicate reagent flow; (B) based on Supelco butt-connector, 4 = vespel ferrule, 5 = connector body. Reprinted from ref. 116.

HOLLOW FIBERS AS POST-COLUMN REACTORS Hollow fiber membranes have been discussed for use as suppressors in i o n chromatography with conductometric detection and are commercialized by the Dionex Company (refs. 117-121). They can also be used as reactor and pumpless reagent addition device in post-column systems with fluorescence detection (ref. 122). The reactor was made of several parallel 325 lm I.D. sulphonated polyethylene hollow fibers, 8 inch in length each. This reactor was suspended in a container of the appropriate reagent. Permeation through the membrane should be small to avoid loss of analyte. The reagent concentration in the eluent can be made sufficiently

5.7

253 s t r o n g by u s i n g h i g h l y concentrated reagent s o l u t i o n i n t h e c o n t a i n e r . A t t e n t i o n should be given t o t h e choice o f s o l v e n t f o r t h e reagent as t h i s may have a b e n e f i c i a l o r a d e t r i m e n t a l i n f l u e n c e on t h e reagent f l u x through t h e membrane. Several model systems were discussed: t h e enhanced UV d e t e c t i o n o f n i t r o p h e n o l s and t h e enhanced fluorescence d e t e c t i o n o f phenols upon pH-adaptation, t h e fluorescence d e t e c t i o n o f amines a f t e r

d e r i v a t i z a t i o n w i t h fluorescamine and t h e n i n h y d r i n c o l o u r f o r m a t i o n w i t h amino acids.

A

6

- -

0

10

Time (rnin)

i i g . 29

20

0

10

20

Time (min)

Chromatograms o f c o n t r o l s a l i v a ( A ) and c o n t r o l s a l i v a s p i k e d w i t h amobarbital (B). 1 = h e x o b a r b i t a l (0.5 I g / m l , i n t e r n a l standard); 2 = amobarbital (0.5 a / m l ) . I n j e c t i o n volume: 50 m l . UV-detection a t 240 nm, 0.008 AUFS. Reprinted from r e f . 124.

Haginaka e t a l . ( r e f .

123) were t h e f i r s t t o d e s c r i b e t h e use o f a

h o l l o w f i b e r post-column r e a c t o r f o r t h e a n a l y s i s o f r e a l samples.

The

determination o f 8-lactamase i n h i b i t o r s ( c l a v u l a n i c a c i d and sulbactam) i n serum and u r i n e based on h o l l o w f i b e r pH adaptation and UV d e t e c t i o n

254 a t 270

-

280 nm was discussed. A 1.2 m l o n g h o l l o w f i b e r (0.3 mm

I.D.)

suspended i n a 1.0 M sodium hydroxide s o l u t i o n was used. The response of the

analytes

concentration.

was optimized

by changing

reactor

l e n g t h and

reagent

E s s e n t i a l l y t h e same a n a l y t i c a l system was used by these

authors f o r t h e d e t e r m i n a t i o n o f some s e l e c t e d b a r b i t u r a t e s whereby t h e determination o f amobarbital i n s a l i v a was g i v e n as t h e a p p l i c a t i o n ( r e f .

124). A 0.05 M ammonium hydroxide s o l u t i o n was taken as t h e reagent and t h e h o l l o w f i b e r r e a c t o r was o n l y 15 cm i n l e n g t h . T y p i c a l chromatograms obtained u s i n g t h i s method a r e g i v e n i n F i g . 29. Since t h e use o f h o l l o w f i b e r r e a c t o r s seems t o g i v e a wide range of p o s s i b i l i t i e s f o r simple reagent i n t r o d u c t i o n ,

i t i s l i k e l y t h a t more

a p p l i c a t i o n s w i l l be published i n t h e nearby f u t u r e . 6. CONCLUSIONS

HPLC i s a technique t h a t i s w i d e l y used i n many a n a l y t i c a l l a b o r a t o r i e s a l l over t h e world. When t h e HPLC s e p a r a t i o n i s combined with

on-line

post-column

reaction

detection

s e l e c t i v e a n a l y t i c a l systems can be obtained,

highly

sensitive

and

as r e f l e c t e d by a s t i l l

growing number o f p u b l i c a t i o n s d e a l i n g w i t h i n t e r e s t i n g a p p l i c a t i o n s . The success o f post-column r e a c t i o n d e t e c t o r s depends p r i m a r i l y on t h e proper choice o f t h e r e a c t o r i t s e l f , and where a p p l i c a b l e , on t h e m i x i n g u n i t used f o r reagent a d d i t i o n . If normal p r e c a u t i o n s a r e taken, t h e i n s e r t i o n o f a r e a c t i o n d e t e c t o r i n t o a HPLC system does n o t cause a s e r i o u s l o s s i n chromatographic r e s o l u t i o n ( l e s s than t w o - f o l d increase i n peak w i d t h i n most cases). Often, t h e l o s s i n r e s o l u t i o n w i l l be compensated by an increase i n s e l e c t i v i t y , hence one should n o t o n l y concentrate on keeping band broadening as low as p o s s i b l e s i n c e f r e q u e n t l y chemical s e l e c t i v i t y can be s u b s t i t u t e d

for

chromatographic

selectivity.

Sensitivity

ar;d

s e l e c t i v i t y are o f t e n improved t o such an e x t e n t t h a t sample pretreatment can be considerably s i m p l i f i e d and automated o r even omitted. Reaction d e t e c t i o n as such i s a l r e a d y a r e l a t i v e l y o l d technique t h a t

now, due t o i t s mature s t a t u s , i s used f o r many a n a l y t i c a l problems. Much i n t e r e s t e x i s t s f o r t h e newer developments i n t h e f i e l d o f r e a c t i o n d e t e c t i o n as discussed i n t h e present paper.

The use o f

immobilized

enzymes and, t o be more general , solid-phase c h e m i s t r i e s has r e s u l t e d i n simpler and more economical a n a l y t i c a l systems and o f t e n gave t h e p o s s i b i l i t y t o circumvent problems on c o m p a t i b i l i t y o f t h e reagent w i t h t h e d e t e c t i o n mode chosen, With e l e c t r o c h e m i c a l reagent p r o d u c t i o n and photochemical

r e a c t i o n d e t e c t i o n p o s t - c o l umn c h e m i s t r i e s

are possible

w i t h o u t t h e need f o r an a d d i t i o n a l reagent pump which, again, t h e economics o f t h e system.

improves

255 M i n i a t u r i z a t i o n o f r e a c t i o n d e t e c t o r has made them compatible f o r use w i t h narrow-bore (1 mm I . D . o r lower) a n a l y t i c a l columns. The major advantage o f m i n i a t u r i z e d r e a c t o r s being low reagent consumption hence p e r m i t t i n g t o work w i t h expensive m a t e r i a l s , reagents i s a l s o b e t t e r c o n t r o l l a b l e .

T o x i c i t y o f samples and

For such systems, t h e development

o f solid-phase r e a c t o r columns and o t h e r pumpless r e a c t i o n u n i t s seems promising because no m i x i n g o f column e f f l u e n t and reagent stream i s needed. Although p o s s i b l e , such m i x i n g i s somewhat d i f f i c u l t t o c o n t r o l i n m i n i a t u r i z e d HPLC. A very g e n t l e approach towards t h e m i x i n g problem i s t h e use o f h o l l o w f i b e r r e a c t o r s as i n t r o d u c e d o n l y a few years ago. With t h e ever i n c r e a s i n g developments i n membrane technologies,

rapid

development i n t h i s f i e l d seems t o be very l i k e l y . I n conclusion, i t can be s t a t e d t h a t post-column r e a c t i o n d e t e c t i o n has now been developed t o such a degree t h a t i t can b e used f o r many d i f f e r e n t types o f analyses, b o t h i n research and r o u t i n e l a b o r a t o r i e s . On t h e o t h e r hand t h e r e a r e s t i l l many d i f f e r e n t aspects f o r researchers t o work on t h e coming decade which w i l l no doubt r e s u l t i n even more i n t e r e s t i n g appl ic a t ions.

REFERENCES 1 M.W.F. Nielen, R.W. F r e i and U.A. Th. Brinkman, S e l e c t i v e Sample Handling and Detection ( E d i t o r s : R.W. F r e i and K. Zech), Vol. 1, E l sev ie r , Ams terdam, 1988. 2 R. W. F r e i , J. Chromatogr., 165, 75 (1979). 3 J.F.K. Huber, K.M. Jonker and H. Poppe, Anal. Chem. 52, 2 (1980). 4 H. Engelhardt and U.D. Neue, Chromatographia, 15 403 (1982). 5 R.S. Deelder, A.T.J.M. K u i j p e r s and J.H.M van den Berg, J. Chromatogr., 255, 545 (1983). 6 B. L i l l i g and H. Engelhardt, Reaction D e t e c t i o n i n L i q u i d Chroma tography, I.S. K r u l l ed., Marcel Dekker, New York, 1987, Ch. 1. 7 L. Nord and B. Karlberg, Anal. Chim. Acta, 164 233 (1984). 8 R.W. F r e i , Chemical D e r i v a t i z a t i o n i n A n a l y t i c a l Chemistry, Vol. 1 Chromatogra h y ' , R.W. F r e i and J.F. Lawrence eds., Plenum Press, New York, (1981!, Ch. 4 9 J.F. Lawrence, U.A. Th. Brinkman and R. W. F r e i , Reaction D e t e c t i o n i n L i q u i d Chromatography, I.S. K r u l l ed., Marcel Dekker, New York, (1987), Ch. 6. 10 J.H.M. van den Berg, R.S. Deelder and H.G.M. Egberink, Anal. Chim. Acta, 114, 91 (1980). 11 L.G.M.Th. T u i n s t r a and W. Haasnoot, J. Chromatogr., 282, 457 (1983). 12 C.J. L i t t l e , J.A. Whatley and A.D. Dale, J. Chromatogr., 171, 63 (1979). 13 P. Bohlen and R. Schroeder, Anal. Biochem., 126, 144 (1982). 14 N. Kiba, M. Takamatsu and M. Furusawa, J. Chromatogr., 328, 309 (1985) 15 C.R. C l a r k and J. Chan, Anal. Chem., 50, 635 (1978). 16 H.A. Moye, S.J. Scherer and P.A. S t . John, Anal. L e t t . , 10 1049 (1977) 17 G. Schwedt, Chromatographia, 10, 92 (1977).

. .

256 18 19 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

A.H.M.T. Scholten, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr., 218, 3 (1981). F.P. B i g l e y and R.L. Grob, J. Chromatogr., 350, 407 (1985). H. Engelhardt and R. K l i n k n e r , Chromatographia, 20, 559 (1985). M.D. Baker, H.Y. Mohammed and H. Veening, Anal. Chem., 53 1658 (1981). R.S. Deelder, M.G.F. K r o l l and J.H.M. van den Berg, J. Chromatogr., 125, 307 (1976). M.E. Rogers, M.W. Adlard, G. Saunders and G. H o l t , J. Chromatogr., 257, 91 (1983). C.E. Werkhoven-Goewie, W.M.A. Niessen, U.A.Th. Brinkman and R.W. F r e i , 3. Chromatogr., 203, 165 (1981). J.Haginaka and J. Wakai, Anal. Chem., 57, 1568 (1985). W.P. King and P.T. K i s s i n g e r , C l i n . Chem., 26 1484 (1980). S. Honda, T. Konishi and S. Suzuki, J. Chromatogr., 299 245 (1984). J.H.M. van den Berg, H.W.M. Horsels and R.S. Deelder, J. L i q . Chromatogr., 7 , 2351 (1984). P. Vratny, U.A.Th. Brinkman and R.W. F r e i , Anal. Chem., 57, 224 (1985) M. Kimura and Y. Itokawa, J. Chromatogr., 332, 181 (1985). A.J. Speek, J. S c h r i j v e r and W.H.P. Schreurs, J. Chromatogr., 301, 441 (1984) L.D. Bowers and W.D. B o s t i c k , Chemical D e r i v a t i z a t i o n i n A n a l y t i c a l Chemistry, Vol. 11, Separations and Continuous Flow Techniques, R.W. F r e i and J.F. Lawrence eds., Plenum, New York, 1982, Ch. 3. P.W. Carr and L.D. Bowers, Immobilized Enzymes i n A n a l y t i c a l and C l i n i c a l Chemistry, Wiley, New York (1980), Ch. 4 G.G. G u i l b a u l t , A n a l y t i c a l Uses o f Immobilized Enzymes, Marcel Dekker, New York (1984). L. Nondek, U.A.Th. Brinkman and R.W. F r e i , Anal. Chem., 54, 1466 (1983). L.Nondek, R.W. F r e i and U.A.Th. Brinkman, J. Chromatogr., 282 (1983) L.Nondek, Anal. Chem., 56 1192 (1984). T.D. Schlabach and F.E. Regnier, J. Chromatogr., 158, 349 (1978). H. Jansen, R.W. F r e i , U.A.Th. Brinkman, R.S Deelder and R.P.J. Snel 1 i n g s , J . Chromatogr., 325, 255 (1985). H. Jansen, E.G. van d e r Velde, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr. , 378, 215 (1986). S. Okuyama, N. Kokubun, S. Higashidate, 0. Uemura and Y. H i r a t a , Chem. L e t t . , 1443 (1979). S. Kamada, M. Maeda, A. Tsumji, Y. Umezawa and T. Kurahashi, J . Chromatogr., 239, 773 (1982). S. Hasegawa, R. Uenoyama, F. Takeda, J. Chuma and S. Baba, J. Chromatogr., 278, 25 (1983). S. Hasegawa, R. Uenoyama, F. Takeda, J. Chuma, K. Suzuki, F. Kamiyama, K. Yamazaki and S. Baba, J. L i q . Chromatogr., 7, 2267 (1984) M. Hayashi, Y. Imai, Y. Minami, S. Kawata, Y. Matsuzawa and S. Tarui, J. Chromatogr., 338, 195 (1985). M . 4 . Wu, K. Takagi, S. Okuyama, M. Ohsawa, T. Masahashi, 0. N a r i t a and Y. Tomeda, J . Chromatogr., 377, 121 (1986). G. Damsa, B.H.C. Westerink and A.S. Horn, J. Neurochem., 45, 1649 (1985). M. Asano, T. Miyanchi, T. Kato, K. F u j i m o r i and K. Yamamoto, J. L i q . Chromatogr., 9 , 199 (1986). K. Honda, K. Miyaguchi, H. Nishino, H. Tanaka, T. Yao and K. Imai, Anal. Biochem., 153, 50 (1986). P. van Zoonen, C. G o o i j e r , N.H. V e l t h o r s t , R.W. F r e i , J.H. Wolf, J. G e r r i t s and F. Flentge, J. Pharm. Biomed. Anal., 5, 485 (1987).

.

.

.

257 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

C. Eva, M. H a d j u c o n s t a n t i o u , N.H. N e f f and J.L. Meek, Anal. Biochem., 143, 320 (1984). J.L. Meek and C. Eva, J . Chromatogr., 317, 343 (1984). P. van Zoonen, I de Herder, C. G o o j j e r , N.H. V e l t h o r s t , R.W. F r e i , E. Kuntzberg and G. G u b i t z , Anal. L e t t . , 19, 1949 (1986). L. Dalgaard, L. Nordholm and L. B r i m e r , J. Chromatogr., 265, 183 (1983). V.K. Boppana, K.-L.L. Fong, J.A. Ziemniak and R.K. Lynn, J. Chromatogr., 353, 231 (1986). L. Dalgaard and L. Brimer, J. Chromatogr., 303, 67 (1984). L. B rimer and L. Dalgaard, J. Chromatogr., 303, 77 (1984). R. Tawa, M. K i t o and S. H i r o s e , Chem. L e t t . , 745 (1981). M. K i t o , R. Tawa, S. Takeshima and S. H i r o s e , J. Chromatogr., 231, 183 (1982). M. K i t o , R. Tawa, 5. Takeshima and S. H i r o s e , J. Chromatogr., 278, 35 (1983). M. K i t o , R. Tawa, S. Takeshima and S. H i r o s e , Anal. L e t t . , 18, 323 (1985). N. Kiba and M. Kaneko, J . Chromatogr., 303, 396 (1984). D.W, T a y l o r and T.A. Nieman, J. Chromatogr., 368, 95 (1986). L. Ogren, I . C s i k y , L. R i s i n g e r , L.G. N i l s s o n and G. Johansson, Anal. Chim. Acta, 117, 71 (1980). J.L. Meek and F. N i c o l e t t i , J. Chromatogr., 351, 303 (1986). L.D. Bowers and P.R. Johnson, Biochim. Biophys. Acta, 661, 100 (1981). J.F. Studebaker, S.A. Slocum and E.L. Lewis, Anal. Chem., 50 1500 (1978). J.F. Studebaker, J. Chromatogr., 185, 497 (1979). M.-C. M i l l o t , 6. S e b i l l e and J.-P. Mahieu, J. Chromatogr., 354, 155 (1986) P. V ra t n y , J . Ouhrabkova and J. Capikova, J. Chromatogr., 191, 313 (1980). P. V ra t n y , R.W. F r e i , U.A.Th. Brinkman and M.W.F. N i e l e n , J . Chromatogr., 295, 355 (1984). K.-S. Low, U.A. Th. Brinkman and R.W. F r e i , Anal. L e t t . , 17, 915 (1984). E.P. Lankmayr, 6. M a i c h i n , G. Knapp and F. Nachtmann, J. Chromatogr., 224,239 (1981). K.W. Sigvardson and J.W. B i r k s , J. Chromatogr., 316, 507 (1984). I.S. K r u l l , K.-H. X i e , S. Colgan, U. Neue, T. I zod, R. King and B. D idlingme y e r , J. L i q . Chromatogr., 6, 605 (1983). I.S. K r u l l , S. Colgan, K.-H. X i e , U. Neue, R. King and 6. B idlingme y e r , J. L i q . Chromatogr., 6, 1015 (1983). K.-H. X ie , S. Colgan and I . X . K r u l l , J . L i q . Chromatogr., 6, 125 (1983). K.-H. X ie, C.T. Santasania, I.S. K r u l l , U. Neue, B. Bidlingmeyer and A. Newhart, L. L i q . Chromatogr., 6, 2109 (1983). J. R ut e r, U.P. Kurz and 6. N e i d h a r t , J. L i q . Chromatogr., 8, 2475 (1985). J. R ut e r, U.P. F i s l a g e and 6. N e i d h a r t , Chromatographia, 19, 62 11984). ,--K. B r u n t , Anal. Chem., 57, 1338 (1985). H. I r t h , G.J. de Jong, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr., 370, 439 (1986). H. Jansen, C.J.M Vermunt, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr., 366, 135 (1986). H. Jansen, R. Jansen, U.A.Th. Brinkman and R.W. F r e i , Chromatographia, i n p r e s s .

.

I

81 82 83 84

258 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119

J.R. Poulsen, J.W. B i r k s , P. van Zoonen, C. G o o i j e r , N.H. V e l t h o r s t and R.W. F r e i , Chromatographia, 21, 587 (1986). W.Th. Kok, U.A. Th. Brinkman and R.W. F r e i , J. Chromatogr., 256, 17 (1983) W.Th. Kok, G. Groenendijk, U.A. Th. Brinkman and R.W. F r e i , J. Chromatogr., 315, 271 (1984). W.P. King and P.T. K i s s i n g e r , C l i n . Chem., 26, 1484 (1980). W.Th. Kok, U.A. Th. Brinkman and R.W. F r e i , Anal. Chim. Acta, 162, 19 (1984). W.Th. Kok, J.J. Halvax, W.H. Voogt, U.A. Th. Brinkman and R.W. F r e i , Anal. Chem., 57, 2580 (1985). W.Th. Kok, W.H. Voogt, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr. , 354, 249 (1986). W.Th. Kok, Th.C.H. van Neer, W.A. Traag and L.G.M.Th. T u i n s t r a , J. Chromatogr., 367, 231 (1986). W.Th. Kok, R.W. F r e i and U.A. Th.Brinkman, S e l e c t i v e Sample Handling and Detection ( E d i t o r s : R. W. F r e i and K. Zech), Vol. 1, E l s e v i e r , Amsterdam (1988). J.R. Poulsen, J.W. B i r k s , G. Gubitz, P. van Zonnen, C. G o o i j e r , N.H. V e l t h o r s t and R.W. F r e i , J. Chromatogr., 360, 371 (1986). I.S. K r u l l and W.R. LaCourse i n Reaction D e t e c t i o n , L i q u i d Chromatography, I.S. K r u l l ed., Marcel Dekker, New York, 1987, Ch. 7. M.F. Lefevere, R.W. F r e i , A.H.M.T. Scholten and U.A.Th. Brinkman, Chromatographia, 15, 459 (1982). M.S. Gandelman, J.W. B i r k s , U.A. Th. Brinkman and R.W. F r e i , J. Chromatogr., 282, 193 (1983). R. Verbeke and P. Vanhee, J. Chromatogr., 265, 239 (1983). C. de R u i t e r , J.F. Bohle, G.J. de Jong, U.A.Th. Brinkman and R.W. F r e i , Anal. Chem., i n press. H. S c h o l l , K. Schmidt and B. Weber, J. Chromatogr., 416, 321 (1987). J.N. LePage and E.M. Rocha, Anal. Chem., 55, 1360 (1983). R.P.W. S c o t t , J. Chromatogr., Sci., 18, 49 (1980). M. Novotny, Anal. Chem., 53, 1294A (1981). F.J. Yang, HRC&CC, 6, 348 (1983). P. Kucera, ed., Microcolumn High-Performance L i q u i d Chromatography, E l sevi e r S c i e n t if ic Pub1 is h i ng Company , Amsterdam, 1984. R.P.W. Scott, ed. , Small Bore L i q u i d Chromatography Columns: T h e i r P r o p e r t i e s and Uses, John Wiley & Sons, New York, 1984. H. Jansen, U.A. Th. Brinkman and R.W. F r e i , J. Chromatogr. Sci., 23 279 (1985). A. Hirose, Y. Iwasaki, I.Iwata, K. Ueda and D . I s h i i , HRC&CC, 4, 530 (1981) P. Kucera and H. Umagat, J. Chromatogr., 255, 563 (1983). J.A. Apffel, U.A. Th. Brinkman and R.W. F r e i , Chromatographia, 17, 125 (1983). 1. Takeuchi, S. S a i t o , D. I s h i i , J. Chromatogr., 258, 125 (1983). T. Takeuchi and 0. I s h i i , HRC&CC, 6, 571 (1983). H. Jansen, U.A.Th. Brinkman, R.W. F r e i , Chromatographia, 20, 453 (1985) K. Miyaguchi, K. Honda and K. Imai, J. Chromatogr., 316, 501 (1984). K. Miyaguchi, K. Honda, 1. Toyo'oka and K. I m a i , J. Chromatogr., 352, 255 (1986). H.P.M. van V l i e t , G.J.M. Bruin, J.C. Kraak and H. Poppe, 3 . Chromatogr. , 363, 187 (1986). T.S. Stevens, J.C. Davis and H. Small, Anal. Chem., 53, 1488 (1981). T.S. Stevens, G.L. Jewett, R.A. Bredeweg, Anal. Chem., 54, 1206 (1982) J. R i v i e l l o and C.A. Pohl, Dionex Technical Note 1983 and 1984, P i t t s b u r g h Conference.

.

.

.

.

259 120 121 122 123 124

R.M. Cassidy and B.D. Karcher, Reaction D e t e c t i o n i n L i q u i d Chromatography, I . $ . K r u l l ed., Marcel Dekker, New York, 1987, Ch. 3. D . T . Gjerde and J.S. F r i t z , I o n Chromatography, 2nd E d i t i o n , D r . A l f r e d H i i t h i g Verlag, Heidelberg, 1987 J.C. Davis, D.P. Peterson, Anal. Chem., 57, 771 (1985). J.Haginaka, J. Wakai and H. Yasuda, Anal. Chem., 59, 324 (1987). J. Haginaka and J. Wakai, J. Chromatogr., 390, 421 (1987)

260 CHAPTER V I

NEW LUMINESCENCE DETECTION TECHNIQUES

C. GOOIJER, N.H.

VELTHORST and R.W.

FREI

1. I n t roduct ion Chemiluminescence d e t e c t i o n w i t h s o l i d s t a t e reactors 2. Detection based on CL and BL 2.1 S o l i d s t a t e reactors i n CL 2.2 2.3 H O2 detection by peroxyoxalate CL 2.3.1 T i e TCPO reactor 2.3.2 Immobilized fluorophore 2.3.3 Coup1 ing w i t h photochemical reactors 2.3.4 Coupling w i t h IMERs 2.4 Use o f the s o l i d TCPO r e a c t o r f o r d e t e c t i o n o f fluorophores 2.5 Quenched peroxyoxalate chemi luminescence d e t e c t i o n On the mechanism o f peroxyoxalate CL 2.5.1 2.5.2 A p p l i c a b i l i t y o f quenched CL d e t e c t i o n The quenching a c t i o n 2.5.3 2.6 Concluding remarks L i q u i d phase phosphorescence d e t e c t i o n 3. Fundamental aspects o f phosphorescence 3.1 3.2 New developments i n phosphorimetry 3.2.1 Sol id-surface RTP 3.2.2 M i c e l l e - s t a b i l i z e d RTP RTP i n normal f l u i d s 3.2.3 3.2.4 Why RTP? Experimental aspects 3.3 Removal o f oxygen 3.3.1 Instrumentation 3.3.2 3.4 I n d i r e c t phosphorescence d e t e c t i o n 3.4.1 Sensitized ohosohorescence ' 3.4.1.1 I nt roduct ioh 3.4.1.2 Theoretical aspects 3.4.1.3 Appl i c a t i o n s 2.4.2 Quenched phosphorescence 3.4.2.1 I n t r o d u c t i o n 3.4.2.2 Theoretical aspects 3.4.2.3 Appl ic a t ions 3.5 A l t e r n a t i v e phosphorophores/luminophores 3.5.1 Immobi 1 ized phosphorophores 3.5.2 Rare earth metal ions as luminophores 3.6 Concluding remarks References 1.

INTRODUCTION

The use o f

luminescence d e t e c t i o n techniques

increasingly popular over

t h e years.

i n HPLC has become

Among these methods,

t h e most

frequently applied method i s fluorescence d e t e c t i o n due t o i t s s e n s i t i v i t y (refs.

1-3).

Furthermore i t can be n i c e l y adapted t o t h e demands of

miniaturized HPLC e s p e c i a l l y w i t h t h e help o f lasers as e x c i t a t i o n sources ( l a s e r induced fluorescence, LIF) Unfortunately, t h e number o f compounds

.

261 e x h i b i t i n g i n t e n s e n a t i v e fluorescence i s l i m i t e d . That i s t h e reason why chemical r e a c t i o n s p l a y an important r o l e i n HPLC fluorescence d e t e c t i o n : non- o r weakly f l u o r e s c e n t compounds a r e converted i n a p r e o r post-column mode i n t o h i g h l y f l u o r e s c e n t products. by chemical d e r i v a t i z a t i o n , ion-pair photochemical r e a c t o r ( r e f . 4 ) .

formation

or

T h i s can be done the

use

of

a

A w e l l known example o f chemical d e r i v a t i z a t i o n i s t h e r e a c t i o n o f Ophthalaldehyde (OPA) w i t h primary amino f u n c t i o n s i n a l k a l i n e media i n t h e presence o f t h e s t r o n g reducing agent 2-mercaptoethanol.

T h i s r e a c t i o n has

been a p p l i e d t o t h e d e t e c t i o n o f amino a c i d s and primary amines i n b i o logical fluids (refs.

5-8).

During t h e p a s t decade a l a r g e number of

reagents has been introduced f o r t h e r e a c t i o n d e t e c t i o n o f a wide v a r i e t y of compounds. Books and reviews on t h i s s u b j e c t a r e numerous ( r e f s . 9-17). With t h e i o n - p a i r formation method a n o n - f l u o r e s c i n g a n a l y t e forms an i o n - p a i r with a highly fluorescent

counter-ion.

Subsequently,

the apolar

i o n - p a i r i s e x t r a c t e d from t h e aqueous mobile phase t o an a p o l a r phase v i a s o l v e n t segmentation. The method can be used f o r b a s i c and a c i d i c a n a l y t e s under c o n t r o l l e d pH c o n d i t i o n s . ammonium type

drugs

(refs.

Several t e r t i a r y

18-20),

detergents

amine and quaternary (ref.

21)

and

alkyl-

sulphates and sulphonates ( r e f . 22) have been determined. With a photochemical r e a c t o r compounds a r e i r r a d i a t e d post-column

i n a quartz

or

t e f l o n c o i l by UV l i g h t and converted t o f l u o r e s c e n t r e a c t i o n products ( r e f s . 23, 24). Most a p p l i c a t i o n s have been i n t h e pharmaceutical area. I n p r i n c i p l e f l u o r e s c e n t a n a l y t e s can a l s o be d e t e c t e d v i a chemiluminescence (CL) o r bioluminescence (BL), provided t h a t t h e r e a r e chemical r e a c t i o n s which e f f i c i e n t l y

b r i n g them i n t h e e l e c t r o n i c a l l y

e x c i t e d s t a t e . A l o t o f research has been done i n t h i s f i e l d because i n chemiluminescence t h e l i g h t source can be e l i m i n a t e d from t h e d e t e c t i o n system ( r e f s . 3 , 25, 26). I n p r i n c i p l e , t h i s would be v e r y n i c e s i n c e t h e u l t i m a t e d e t e c t a b i l i t y i n fluorescence d e t e c t i o n i s l i m i t e d by background l i g h t and i t s n o i s e coming from t h e e x c i t a t i o n source ( r e f . 27). F o r a number o f compounds impressive r e s u l t s have been r e p o r t e d . However, i t i s obvious t h a t n o t i n a l l cases t h e CL d e t e c t i o n l i m i t s a r e s i g n i f i c a n t l y more favourable than those obtained v i a fluorescence d e t e c t i o n .

I n CL

d e t e c t i o n g e n e r a l l y background due t o c o m p e t i t i v e r e a c t i o n s o r i m p u r i t i e s f o r instance i n t h e e l u e n t determine t h e d e t e c t i o n l i m i t s t h a t can be reached ( r e f . 28). I n t h e present paper t h e main a t t e n t i o n i s t o t h e d e t e c t i o n o f nonf l u o r e s c e n t compounds v i a luminescence techniques o t h e r than fluorescence, i .e.,

1). Regarding chemiluminescence d e t e c t i o n i n l i q u i d chromatography, e s p e c i a l l y t h e s o - c a l l e d phosphorescence and chemiluminescence (Fig.

262

peroxyoxalate r e a c t i o n has g o t a l o t o f a t t e n t i o n i n t h e l i t e r a t u r e : s i n c e t h e f i r s t paper o f Kobayashi and Imai i n 1980 ( r e f . 29) more than 100 papers appeared on t h i s subject. A major c o n s t r a i n t i n a p p l y i n g CL i n r o u t i n e a n a l y s i s i s t h e r e l a t i v e complexity o f t h e equipment d e s p i t e of t h e f a c t t h a t t h e d e t e c t o r i t s e l f i s q u i t e simple. I n most cases more than two reagents have t o be added t o t h e a n a l y t e b e f o r e CL can be observed, so t h a t problems due t o m i x i n g and t o l i m i t e d s o l u b i l i t y and s t a b i l i t y of reagents may a r i s e . I n t h i s chapter t h e s o l i d - s t a t e r e a c t o r approach t o avoid these drawbacks i s discussed.

Use w i l l be made o f s o l i d o x a l a t e

( r e f s . 30, 31), immobilized f l u o r o p h o r e ( r e f . 32) and immobilized enzymes ( r e f . 33, 3 4 ) . I l l u s t r a t i v e f o r t h e eventual a p p l i c a b i l i t y o f t h e method i s t h e d e t e c t i o n o f c h o l i n e and a c e t y l c h o l i n e i n complex b i o l o g i c a l matrices (as u n d i l u t e d u r i n e and t h e d e p r o t e i n a t e d serum) w i t h o u t sample pretreatment ( r e f . 34).

- FLU

-___ -X-X-

Fig. 1

CL PHOS

S i m p l i f i e d diagram showing t h e Fluorescence, Chemiluminescence and Phosphorescence t r a n s i t i o n s . S and S1 are t h e lowest e l e c t r o n i c s i n g l e t s t a t e s and T1 'is t h e lowest e l e c t r o n i c t r i p l e t s t a t e o f t h e analyte.

Unl ike f 1 uorescence and chemi 1umi nescence,

phosphorescence i n t h e

l i q u i d s t a t e i s a r a r e phenomenon. A t a f i r s t s i g h t , t h i s f a c t severely l i m i t s t h e p o t e n t i a l a p p l i c a b i l i t y o f phosphorescence d e t e c t i o n i n HPLC. Nevertheless,

interesting

approaches ( r e f .

progress

has

been

advanced

following

two

35). The f i r s t makes use o f organized media, such as

m i c e l l a r s o l u t i o n s t o extend t h e number o f compounds t h a t do emit phosphorescence.

Such media

are

interesting

since

they

become

increasingly

important i n chemical separation problems. The second approach which w i l l be discussed e x t e n s i v e l y i n t h i s chapter i s focussed on phosphorescence o f

263

normal

l i q u i d s which can be u t i l i z e d as an i n d i r e c t d e t e c t i o n method

(refs.

2,36,37).

(refs.

38-41) and t h e quenched mode ( r e f s . 42-50).

Two modes can be d i s t i n g u i s h e d ,

i.e.,

the sensitized

I n most a p p l i c a t i o n s

developed thus f a r , t h e phosphorophore b i a c e t y l (2,3-butanedione)

i s added

t o t h e e l u e n t which needs t o be deoxygenated. Compounds a b l e t o quench t h e b i a c e t y l phosphorescence cause a decrease o f t h e monitored s i g n a l .

The

amount o f quenching i s dependent on t h e a n a l y t e , which i m p l i e s t h a t t h e method has an i n h e r e n t s e l e c t i v i t y i n i o n chromatography, s i n c e t h e UV absorption quenching

properties o f process.

t h e a n a l y t e s do n o t p l a y any

Furthermore,

unlike

indirect

UV

role i n the

absorption-

and

fluorescence d e t e c t i o n i n i o n chromatography, quenched phosphorescence i s n o t based on t h e displacement o f e l u e n t i o n s by a n a l y t e ions.

Instead i t

i s a dynamic process: t h e e f f i c i e n c y o f t h e phosphorescence i s reduced by t h e analyte. This d i f f e r e n c e has i n t e r e s t i n g consequences. The s e n s i t i z e d phosphorescence technique can be used f o r a n a l y t e s t h a t do absorb UV r a d i a t i o n b u t do n o t f l u o r e s c e because t h e y decay t o t h e first

excited t r i p l e t state.

I n presence o f b i a c e t y l

triplet-triplet

energy t r a n f e r from t h e a n a l y t e t o b i a c e t y l takes p l a c e a f t e r which t h e phophorescence o f t h e l a t t e r i s monitored. The s e n s i t i z e d phosphorescence method should be considered complementary t o UV and fluorescence detection. I n t h i s chapter t h e a p p l i c a b i l i t y o f b o t h s e n s i t i z e d and quenched phosphorescence w i l l be shown,

i n t h e o r g a n i c as w e l l as t h e i n o r g a n i c

f i e l d . An i n t e r e s t i n g aspect i s t h e r e l a t i v e ease compared t o fluorescence t o u t i l i z e time r e s o l u t i o n t o improve t h e r a t i o o f t h e phosphopescence s i g n a l and t h e background r a d i a t i o n . This i s n o t t o o complicated s i n c e t h e l i f t i m e s o f t h e phosphorescence s i g n a l i n t h e systems under c o n s i d e r a t i o n are i n t h e 0.1-1 msec range. furthermore a l t e r n a t i v e phosphorophores t o overcome some o f t h e disadvantages o f b i a c e t y l w i l l be t r e a t e d . The main disadvantages are t h a t b i a c e t y l i s p a r t o f t h e e l u e n t and, more i m p o r t a n t , t h a t t h e e l u e n t should be deoxygenated. I n one a l t e r n a t i v e method use i s made o f an immobilized phosphorophore packed i n t h e d e t e c t o r c e l l ( r e f . 49). I n another approach, r a r e e a r t h i o n s as Eu3+ and Tb3+ a r e u t i l i z e d as luminophores

(ref.

50).

They

emit

long-living

s o l u t i o n s w i t h o u t t h e need o f deoxygenation.

luminescence i n l i q u i d

Although i n a s t r i c t sense

luminescence o f r a r e e a r t h s i s no phosphorescence, i t i s a p p r o p r i a t e t o discuss

this

simi 1a r i t i e s .

technique

here

because

there

are

various

experimental

264 2. 2.1

CHEMILUMINESCENCE DETECTION WITH SOLID STATE REACTORS DETECTION BASED ON CL AND BL Various chemiluminescence (CL) and bioluminescence (BL) r e a c t i o n s have

been and a r e a p p l i e d i n a n a l y t i c a l chemistry ( r e f . 25).

O f course t h e y

commonly r e q u i r e more o r l e s s defined r e a c t i o n c o n d i t i o n s which g e n e r a l l y a r e n o t e q u i v a l e n t w i t h t h e o p t i m a l chromatographic circumstances i n LC. Hence f o r t h e development o f CL (and BL) as d e t e c t i o n technique i n HPLC, compatability i s a crucial point. W i t h i n t h i s c o n t e x t t h r e e CL r e a c t i o n s a r e t h e most important: (iso)luminol reaction;

reaction,

these w i l l

the

lucigenine reaction

the

and t h e peroxyoxalate

be discussed more e x t e n s i v e l y .

I n addition,

in-

c i d e n t a l l y use has been made o f t h e f i r e f l y BL r e a c t i o n , i . e . , f o r t h e isoenzymes o f c r e a t i n e kinase ( r e f . 51) and o f t h e l u c i f e r a s e b a c t e r i a l BL r e a c t i o n , i.e., f o r b i l e a c i d s producing NADH from NAD, which can be measured v i a b a c t e r i a l CL ( r e f . 2 5 ) . Furthermore, an areosol spray d e t e c t o r f o r LC based on ozon and s i n g l e t oxygen induced CL has been reported ( r e f s . 52, 53), a c e l l f o r e l e c t r o g e n e r a t e d CL ( r e f . 54) and a thermal energy analyzer CL d e t e c t o r ( r e f s . 5 5 , 56). To discuss t h e p o t e n t i a l o f t h e ( i s o ) l u m i n o l , t h e l u c i g e n i n e and t h e

peroxyoxalate r e a c t i o n f o r d e t e c t i o n i n LC i t should be r e a l i z e d t h a t t h e f i r s t two r e a c t i o n types d i f f e r e s s e n t i a l l y from t h e t h i r d one. Whereas f o r luminol and l u c i g e n i n e an e n e r g y - r i c h i n t e r m e d i a t e i s formed i n t h e e a r l y stage o f t h e r e a c t i o n which i t s e l f emits l i g h t , f o r peroxyoxalate t h e formation o f such an i n t e r m e d i a t e i s f o l l o w e d by energy t r a n s f e r t o a substance t h a t e v e n t u a l l y emits l i g h t . T h i s i m p l i e s t h a t t h e peroxyoxalate i n p r i n c i p l e can be invoked t o d e t e c t compounds w i t h n a t i v e fluorescence o r c a r r y i n g f l u o r e s c e n t l a b e l s . D e t e c t i o n o f f l u o r e s c e n t compounds v i a t h e ( i s o ) luminol o r t h e l u c i g e n i n e r e a c t i o n i s n o t a p p r o p r i a t e . The ( i s o ) l u m i n o l r e a c t i o n i s s c h e m a t i c a l l y d e p i c t e d as f o l l o w s :

0 + light (425 nm )

265 The r e a c t i o n r e q u i r e s s t r o n g a l k a l i n e c o n d i t i o n s ; t h e ( i s o ) l u m i n o l r e a c t s w i t h an o x i d a n t w h i l e metal i o n s c a t a l y z e t h e r e a c t i o n . T h i s i m p l i e s t h a t i n p r i n c i p l e i t can be used t o d e t e c t o x i d a n t s ( p o s s i b l y generated i n a post-column r e a c t i o n d e t e c t o r ) , metal i o n s (and metal c h e l a t i n g agents) and substances d e r i v a t i z e d w i t h ( i s o ) l u m i n o l . That c a t a l y z i n g metal i o n s (and i n d i r e c t l y a n a l y t e s t h a t reduce t h e f r e e metal i o n c o n c e n t r a t i o n ) can be detected i s r e a d i l y c l a r i f i e d . I n chemiluminescence t h e number o f e m i t t e d photons p e r second i s measured which i s p r o p o r t i o n a l t o t h e number of h i g h l y e n e r g e t i c indermediate compounds formed p r o second,

i n other

words t o t h e r e a c t i o n r a t e . The luminol CL r e a c t i o n has been f r e q u e n t l y a p p l i e d i n a n a l y t i c a l chemistry e s p e c i a l l y i n f l o w i n j e c t i o n a n a l y s i s , e.g.

f o r various c a t a l y z i n g metal c a t i o n s ( r e f s . 57-60), amino a c i d s and

proteins

acting

as

metal

chelating

agents

thus

suppressing

the

CL

i n t e n s i t y ( r e f s . 61-63) and hydrogenperoxide produced by enzymes ( r e f s . For HPLC d e t e c t i o n o n l y few a p p l i c a t i o n s o f l u m i n o l CL have been

64-69).

published. Metal i o n s have been determined a f t e r s e p a r a t i o n by i o n exchange ( r e f . 70); here c o m p a t a b i l i t y i s a s e r i o u s problem s i n c e metals are u s u a l l y separated under a c i d i c c o n d i t i o n s whereas t h e luminol CL r e q u i r e s h i g h pH values. The CL d e t e c t o r has been combined w i t h a photochemical r e a c t o r inducing photooxygenation o f a1 i p h a t i c a l c o h o l s , aldehydes, e t h e r s and saccharides under r e l e a s i n g o f H202 ( r e f . 71). Finally,

the

alkyl

substituted

i s o l u m i n o l , has been successfully and c a r o b o x y l i c a c i d s ( r e f . 72).

isoluminol,

N-(4-aminobutyl)-N-ethyl-

used f o r precolumn l a b e l i n g o f amines

The l u c i g e n i n e CL r e a c t i o n a l s o r e q u i r e s s t r o n g b a s i c s o l u t i o n s . Lucigenine r e a c t s both w i t h o x i d a n t s l i k e sodium p e r i o d a t e o r hydrogen peroxide as w i t h reductants

l i k e ascorbic

a c i d and glucose.

I n both

s i t u a t i o n s t h e e m i t t i n g species i s N-methylacridone:

Hence i t i s p o s s i b l e t o u t i l i z e l u c i g e n i n e CL t o measure b o t h o x i d a n t s and reductants.

For d e t e c t i o n i n HPLC i t has been used f o r a s c o r b i c and

dehydroascorbic a c i d , cortisol

f o r glucose,

( r e f s . 73-75).

creatinine,

heparin and s t e r o i d s as

Furthermore, carboxyl i c acids have been measured

266

indirectly after conversion to the p-nitrophenacyl esters (ref. 29). Obviously the peroxyoxalate C L reaction is applied by far the most extensively for detection in HPLC. Traditionally the reaction is presented in the following way, although results of various groups unambiguously show that this presentation cannot account for all experimental data (refs. 76-79):

[ :]

+

~

0

0

fluoNphnrr

-

fluoNphore' + 2C0,

I

Ar represents a substituted benzene nucleus: the most frequently used oxalate is TCPO, bis(2,4,6-trichlorophenyl) oxalate. Thusfar the peroxyoxalate C L reaction has been applied in HPLC for fluorescent or fluorescence-labeled compounds, for hydrogenperoxide (other oxidants are not appropriate) and for C L quenching analytes. The C L intensities appear to vary strongly with the fluorophore applied in the reaction so that not in all cases C L detection is more favourable than fluorescence detection. Furthermore detectability is generally limited by background luminescence, i .e., chemiluminescence due to impurities or competitive reactions observed when no fluorophore is added to the eluent. Interesting results have for instance been reported for polycyclic aromatic hydrocarbons (refs. 80-82) but also for dansyl-labeled compounds as amino acids, amines, catecholamines and steroids (refs. 28, 29, 83-88). Furthermore, likewise dansylation, other labeling reactions originally developed for fluorescence detection have been examined as the reactions with OPA, orthophthalaldehyde, and NBD, 7-nitrobenzo-2-oxa-l,3-diazole (refs. 86, 88). An approach specifically directed on C L is the use of amino-substituted aromatics which have extremely high CL efficiencies for labeling carboxylic acids (ref. 89), aldehydes and ketones (ref. 90). 2.2

S O L I D STATE REACTORS I N CL Solid state reactors have been introduced in C L detection for various

267 reasons. I n t h e f i r s t p l a c e t h e y can be used t o e x t e n t t h e a p p l i c a b i l i t y o f t h e CL r e a c t i o n under c o n s i d e r a t i o n .

An example i s t h e Z n - r e d u c t o r

t r a n s f e r r i n g nitro-PAHs t o amino-PAHs; whereas t h e f o r m e r have v e r y l o w CL e f f i c i e n c i e s i n t h e p e r o x y o x a l a t e r e a c t i o n , t h e l a t t e r can be d e t e c t e d e x t r e m e l y s e l e c t i v e and s e n s i t i v e

i n complex samples

(ref.

81).

Also

enzymes can be i m m o b i l i z e d f o r t h i s purpose, as f o r i n s t a n c e i m m o b i l i z e d enzyme r e a c t o r s (IMERS) p r o d u c i n g H202 t h a t i n t u r n can be measured v i a CL ( r e f s . 91, 9 2 ) . Thus v a r i o u s

i m p o r t a n t s u b s t r a t e s can i n p r i n c i p l e b e

analyzed; s u c c e s s f u l r e p o r t s have been p u b l i s h e d f o r g l u c o s e ( r e f s .

33,

65) and c h o l i n e l a c e t y l c h o l i n e ( r e f s . 34, 93). A l t e r n a t i v e l y , IMERS can b e u t i l i z e d t o enhance t h e CL i n t e n s i t y v i a t h e r e a c t i o n r a t e . As an example we mention i m m o b i l i z e d oxidase c a t a l y z i n g t h e l u m i n o l r e a c t i o n ( r e f . 9 4 ) . Obviously, a v e r y f a v o u r a b l e p r o p e r t y o f b o t h r e a c t o r t y p e s i s t h a t t h e y i n p r i n c i p l e can be used d u r i n g l o n g t i m e p e r i o d s . Nevertheless as a t h i r d p o s s i b i l i t y of a p p l y i n g s o l i d - s t a t e r e a c t o r s a l s o i m m o b i l i z e d r e a g e n t s t h a t a r e consumed have been used, f o r i n s t a n c e immobilized l u m i n o l ( r e f . 9 5 ) . Because o f t h e l a r g e s u r f a c e area a v a i l a b l e on s m a l l s o l i d p a r t i c l e s t h i s o f f e r s a way t o employ t h e s e r e a g e n t s a t much h i g h e r e f f e c t i v e c o n c e n t r a t i o n s t h a n a l l o w e d by s o l u b i l i t i e s . I n o u r l a b o r a t o r y we have u t i l i z e d a packed b e d - t y p e r e a c t o r f o r TCPO i n t h e p e r o x y o x a l a t e CL r e a c t i o n t o s i m p l i f y t h e e x p e r i m e n t a l

set-up

and t o

reduce i n s t a b i l i t y problems ( r e f . 26). T h i s compound b e i n g p o o r l y s o l u b l e i n t h e s o l v e n t s u s u a l l y a p p l i e d f o r p e r o x y o x a l a t e CL, i s s l o w l y d i s s o l v e d i n t h e c a r r i e r stream. I n some cases i m m o b i l i z e d CL r e a g e n t s a r e used t o q u a n t i t a t e species t h a t q u a n t i t a t i v e l y l i b e r a t e s t h e CL r e a g e n t from i t s s u p p o r t ( r e f s . 96, 97). As an example a d e t e c t i o n system f o r t h i o l s has been developed: a t h i o l - m o d i f i e d l u m i n o l i s bound on p o l y s a c c h a r i d e b u t r e 1eased a f t e r t h io l -d is u l f i d e i n t e r c h a n g e . F i n a l l y , i t i s emphasized t h a t i n t h e p e r o x y o x a l a t e CL t h e l u m i n e s c i n g compound can be s u c c e s s f u l l y bound on c o n t r o l l e d p o r e g l a s s o r s i l i c a . It i s n o t consumed d u r i n g t h e r e a c t i o n . I n o u r l a b o r a t o r y we have i m m o b i l i z e d

3-aminofluoranthene,

one

of

the

most

efficient

p e r o x y o x a l a t e system known a t t h e moment ( r e f . important

CL 32).

reagents

in

the

Immobilization i s

since

t h i s compound has c a r c i n o g e n i c p r o p e r t i e s . Another f a v o u r a b l e p o i n t i s t h a t t h e CL r e a c t i o n can be l o c a l i z e d i n t h e d e t e c t o r c e l l which i s packed w i t h i m m o b i l i z e d f l u o r o p h o r e . 2.3

H202 DETECTION BY PEROXYOXALATE CL I t i s w o r t h w h i l e t o improve H202 d e t e c t i o n methods i n FIA and t o

develop d e t e c t i o n methods i n HPLC n o t o n l y because H202 i s an i m p o r t a n t analyte

itself,

but

also

because

a c o u p l i n g can be made w i t h

photo-

chemical ( r e f s . 98, 99) and enzymatic r e a c t i o n s ( r e f s . 34, 92, 93). Development o f such coup1 i n g t e c h n i q u e s i s n o t o n l y i m p o r t a n t concerning s e n s i t i v i t y ; i n some cases t h e s e l e c t i v i t y parameter i s even more im port ant . F or example, i t i s t h e s e l e c t i v i t y o f t h e IMER CL combinat ion t h a t a l l o w s t h e q u a n t i t a t i o n o f c h o l i n e and a c e t y l c h o l i n e i n ext remely complex m i x t u r e s ( r e f s . 3 4 ) . Furthermore, because o f t h i s s e l e c t i v i t y t h e a d d i t i o n a l band broadening caused by t h e r e a c t o r s i s l e s s i m p o r t a n t t h a n usual i n HPLC d e t e c t i o n .

-

TCPO

HP2

@-

PMT

~_____ P

7

TCPO

FLU

L___--.J +

Fig. 2

2.3.1

"202

PMT

D e t e c t o r c o n f i g u r a t i o n s i n v e s t i g a t e d : (A) convent ional system; (B) system w i t h s o l i d - s t a t e TCPO r e a c t o r ; (C) system w i t h s epar a t e TCPO and i m n o b i l i z e d f l u o r o p h o r e r e a c t o r s ; (D) mixed r e a c t o r system (FLU = f l u o r o p h o r e , P = pump and PMT = phot om u l t i p l i e r tube). THE TCPO REACTOR ( r e f s . 30,31)

The s i g n i f i c a n t r e d u c t i o n i n c o m p l e x i t y o f H202 d e t e c t i o n which can be achieved by a p p l y i n g

solid-state

reactors

is

readily

conceived

from

Fig. 2: m a n i f o l d A i s t h e c o n v e n t i o n a l one; i n B a (hand packed) s o l i d - s t a t e TCPO r e a c t o r i s used, i n C and D b o t h a s o l i d - s t a t e TCPO r e a c t o r and a r e a c t o r w i t h 3 - a m i n o f l u o r a n thene immobilized on c o n t r o l l e d pore g las s (CPG) a r e a p p l i e d . I n B and C t h e TCPO r e a c t o r i s t h e same. I t i s a precolumn o f t h e t y p e designed i n ou r l a b by Goewie e t a1 ( r e f . c a r t r i d g e o f 4.6 mm I . D .

100) u s i n g a t e f l o n c o a t i n g

and 22 mm l e n g t h . Various experiment al parameters

f o r i t s use i n F I A have been e v a l u a t e d on t h e b a s i s o f c o n f i g u r a t i o n B, u t i l i z i n g peryl e n e as f l u o r o p h o r e Parameters

as reagent

purity

and aqueous a c e t o n i t r i l e as e l u e n t .

and a c e t o n i t r i l e

t o wat er

ratio

p l a y an

269 imp ort ant r o l e . To achieve a background s i g n a l as low and as s t a b l e as p o s s i b l e , s p e c i a l a t t e n t i o n has t o be p a i d t o reagent p u r i t y and t o t h e a c e t o n i t r i l e t o w a t e r r a t i o . Optimal r e s u l t s were o b t a i n e d by p a s s i n g a c e t o n i t r i l e ov er an alumina column p r i o r t o use and f u r t h e r m o r e b y a d j u s t i n g t h e c om p o s i t i o n o f H202 i n j e c t i o n p l u g t o t h e composit ion o f t h e c a r r i e r stream as much as p o s s i b l e . D e t e c t i o n l i m i t s o f 6 x lo-' M 3 0 2 (0.2 @/1) were achieved combined w i t h a l i n e a r dynamic range up t o 10 and a good r e p r o d u c i b i l i t y (R.S.D.

2.8% a t 2 x

M

M f o r 10 i n j e c t i o n s ) .

Large i n j e c t i o n volumes were r e q u i r e d u s i n g t h e r a t h e r b i g TCPO r e a c t o r . I n a c e t o n i t r i l e / w a t e r 80:20 t h i s b i g r e a c t o r c o u l d be operat ed a t l e a s t 8 hours.

Unfortunately,

l a r g e sample i n j e c t i o n volumes a r e r e q u i r e d which

i n d i c a t e s t h a t t h e r e a c t o r has a l a r g e dead volume so t h a t i t cannot be used i n s e r i e s w i t h t h e a n a l y t i c a l column i n an HPLC system. To reach t h e maximum CL s i g n a l i n FIA an i n j e c t i o n volume o f a t l e a s t 650 was necessary. O b v ious ly f o r combining t h e H202 d e t e c t i o n system w i t h HPLC a s m a l l e r TCPO r e a c t o r i s r e q u i r e d t o c i r c u m v e n t d e s a st rous band broadening.

t h i s p o i n t o f view c o n f i g u r a t i o n

From

D i s more a p p r o p r i a t e t h a n c o n f i g u r a t i o n

C. Here, a dual l a y e r d e t e c t o r c e l l i s a p p l i e d o f t y p i c a l l y 3 mm i n t e r n a l

diameter and 27 mm l e n g t h ; about 1/3 o f t h e c e l l i s f i l l e d w i t h TCPO. Since TCPO i s consumed d u r i n g t h e r e a c t i o n i t i s a p p r o p r i a t e t o p l a c e a f r i t between t h e TCPO and t h e luminophore l a y e r .

A l s o an i n l e t f r i t i s

necessary t o spread t h e f l o w e v e n l y o v e r t h e whole r e a c t o r . The two l a y e r c e l l can be used d u r i n g ca. 3 h c o n t i n u o u s o p e r a t i o n a t f l o w r a t e s o f about 1 m l m i n - l ( a c e t o n i t r i l e / w a t e r 80:20)

w i t h o u t excessive a d d i t i o n a l

band broadening caused by v o i d s i n t h e TCPO l a y e r . Repacking w i t h TCPO i s extreme ly simple.

I n F I A experiments a p p l y i n g m a n i f o l d D,

maximum CL

s i g n a l s were reached a t i n j e c t i o n volumes n o t l a r g e r t han 100 t o 150 pl. Presumably t h i s i s n o t o n l y due t o t h e s m a l l s i z e o f t h e TCPO r e a c t o r , b u t a l s o t o t h e l o c a l i z a t i o n o f t h e CL r e a c t i o n ( t h e l i g h t emission ) i n t h e d e t e c t o r c e l l . The l a t t e r i m p l i e s t h a t no band-broadening

i s caused by

d e t e c t i o n o f CL o c c u r r i n g i n t h e i n l e t and o u t l e t c a p i l l a r i e s o f t h e f l o w cell. Another i m p o r t a n t d i f f e r e n c e between t h e e xperiment al c o n f i g u r a t i o n s C and D i s t h e i r dependence on f l o w r a t e ( f o r 80% a c e t o n i t r i l e as a model eluent).

I n C t h e r e i s a s i g n i f i c a n t dependence, whereas i n D i t i s

n e g l i g i b l e . Most p r o b a b l y t h i s i s due t o t h e t i m e l a g i n C between t h e r e a c t i o n o f hydrogen p e r o x i d e w i t h TCPO i n t h e f i r s t r e a c t o r and t h e a c t u a l e x c i t a t i o n s t e p i n t h e second r e a c t o r . During t h i s t i m e i n t e r v a l some o f t h e i n t e r m e d i a t e formed i n t h e f i r s t r e a c t o r decomposes, so t h a t

270 t h e amount o f i n t e r m e d i a t e t h a t reaches t h e second r e a c t o r depends on t h e flow rate. To t e s t t h e s o l v e n t c o m p a t a b i l i t y o f t h e TCPO r e a c t o r , i n a d d i t i o n t o a c e t o n i t r i l e , t e t r a h y d r o f u r a n (THF) , acetone and methanol were s t u d i e d a1 1 i n mixtures o f 20% aqueous T r i s b u f f e r and 80% o r g a n i c s o l v e n t . THF i s unsuitable; i t g i v e s a very h i g h CL background, presumably because of peroxide formation.

Acetone can be a p p l i e d w i t h o u t any problem;

alike

a c e t o n i t r i l e t h e CL s i g n a l i s n o t i n f l u e n c e d by t h e f l o w r a t e . T h i s i s n o t t r u e f o r methanol; i n 80% aqueous methanol (even i n m a n i f o l d D) a s t r o n g flow-rate

dependence i s observed t h a t v a r i e s from one TCPO batch t o

another. I t has been shown t h a t t h i s dependence can be e l i m i n a t e d by addition o f 2,4,6-trichlorophenol (TCP) which i s a p r e c u r s o r i n t h e synthesis of TCPO. It has been suggested t h a t i n methanol TCPO undergoes s i d e r e a c t i o n s i n c o m p e t i t i o n w i t h t h e TCPO/H202 r e a c t i o n ; i f a r e p r o d u c i b l e TCP c o n c e n t r a t i o n i s maintained,

the side

reactions are

c o n t r o l l e d and t h e f l o w dependence i s suppressed. Concerning t h e optimal pH, f o r a c e t o n i t r i l e and acetone t h e r e i s no s i g n i f i c a n t e f f e c t on t h e s i g n a l between pH 7.5 and 10; i n methanol ( a p a r t from t h e r o l e o f TCP) t h e f l o w r a t e dependence a t pH

7.5 i s s i g n i f i c a n t l y lower than a t pH 9.5.

The f l o w independence of c o n f i g u r a t i o n D ( a p a r t from methanol) and t h e low dead volume o f t h e t w o - l a y e r c e l l i m p l i e s t h a t i t can be a p p r o p r i a t e l y a p p l i e d i n HPLC. On t h e o t h e r hand i t has been s u c c e s s f u l l y a p p l i e d f o r H202 q u a n t i t a t i o n

i n r a i n and c l o u d water samples by FIA; manual i n j e c t i o n s can be r e a d i l y performed because o f i t s low back pressure. F o r

t h i s type o f samples f i e l d m o n i t o r i n g i s e s s e n t i a l because very d i l u t e aqueous peroxide samples a r e unstable. H202, which i s b e l i e v e d t o be one o f t h e key intermediates i n t h e atmospheric decomposition c y c l e o f t h e sulphur oxides causing a c i d r a i n ,

i s present i n r a i n water i n concen-

M) i n p o l l u t e d areas t o

t r a t i o n s v a r y i n g from about 1 p~ 1-1 (3 x more than about 1 p~ m l - I

(3 x

M)

i n r e l a t i v e l y c l e a n areas.

The

present q u a n t i t a t i o n method based on t h e TCPO-CL system i n Fig. 2D g i v e s a d e t e c t i o n l i m i t o f 1.5 x 10-8M and a l i n e a r i t y over 6 o r d e r s o f magnitude; t h e R.S.D. i s 3% ( a t 17 jg 1- 1) and by manual i n j e c t i o n 40 samples can b e analyzed p e r hour. TO sum up t h e r e s u l t s f o r t h e TCPO r e a c t o r described i n t h i s s e c t i o n , i t w i l l be obvious t h a t t h e two l a y e r c e l l can be combined e a s i l y w i t h

HPLC. U n f o r t u n a t e l y , i t can o n l y be used d u r i n g a l i m i t e d t i m e p e r i o d , t y p i c a l l y 3 hours, b u t repacking i s q u i t e easy. It i s noted, however, t h a t a l s o t h e b i g g e r r e a c t o r (as used i n B and C) can be f r u i t f u l l y a p p l i e d i n HPLC i f i t i s placed i n a separate f l o w

line,

not i n l i n e with the

a n a l y t i c a l column. I n a set-up o f t h i s type, f o r m a t i o n o f voids i n t h e

TCPO r e a c t o r i s n o t so c r i t i c a l .

F r e q u e n t l y t h e s o l i d f i n e l y ground TCPO

i s mixed w i t h g l a s s beads o f 40-80 p d i a m e t e r s t o reduce backpressure. 2.3.2

IMMOBILIZED FLUOROPHORE ( r e f . 32) It i s obvious f r o m t h e s i m p l i f i e d r e a c t i o n scheme f o r p e r o x y o x a l a t e CL

(see s e c t i o n 2.1)

that

the

fluorophore

r e a c t i o n . From t h i s p o i n t o f view,

is

n o t consumed

during

the

immobilization i s q u i t e appropriate.

With res pec t t o t h e c h o i c e o f t h e f l u o r o p h o r e , i t i s well-known from CL measurements on f l u i d s s o l u t i o n s t h a t 3-aminofluoranthene (3-AF) i s one o f t h e most e f f i c i e n t f l u o r o p h o r i c compounds ( r e f . 81).

Immobilization of

t h i s compound i s n o t o n l y u s e f u l i n o r d e r t o e x t e n t t h e freedom of s o l v e n t choic e ( t h e s o l u b i l i t y o f t h e f l u o r o p h o r e i s no l o n g e r a l i m i t i n g f a c t o r ) , b u t e s p e c i a l l y because o f i t s t o x i c p r o p e r t i e s . Various f a c t o r s determine t h e CL s i g n a l t o n o i s e r a t i o t h a t can be reached i n a per o x y o x a l a t e system u t i l i z i n g immobilized 3-AF. F i r s t o f a l l , a f t e r coupling t o t h e s o l i d support t h e e l e c t r o n i c s t r u c t u r e o f t h e f l u o r o p h o r e should n o t have changed b a s i c a l l y . Only i f t h i s c o n d i t i o n i s f u l f i l l e d a CL e f f i c i e n c y approaching t h a t o f l i q u i d s t a t e 3-AF w i l l be a t t a i n a b l e . Secondly, t h e c o u p l i n g t o t h e s u p port must be r e a l i z e d by a spacer,

t h us c r e a t i n g a p s e u d o - l i q u i d s o l u t i o n f o r 3-AF.

Thirdly,

the

s o l i d s upport i t s e l f should be a p p l i c a b l e under HPLC c o n d i t i o n s w i t h o u t any problems. C e l l u l o s e f o r i n s t a n c e i s n o t s u i t a b l e , because i t s w e l l s i n aqueous a c e t o n i t r i l e and methanol t h u s c a u s i n g back pressure problems i n a HPLC set-up.

F i n a l l y , on t h e one hand a h i g h s u r f a c e coverage should be

r e a l i z e d t o enhance t h e CL s i g n a l , w h i l e on t h e o t h e r hand t h e transparancy o f t h e system f o r t h e outcoming luminescence l i g h t should n o t be reduced. A s uc c es s f u l

glass

i m m o b i l i z a t i o n procedure f o r 3-AF on c o n t r o l l e d p o r e

(CPG) and s i l i c a g e l

s c h e m a t i c a l l y g i v e n i n Fi g .

developed by G u b i t z e t a l . 3.

I n the f i r s t

(ref.

32)

is

s t e p a r e a c t i o n w i t h 3-

glycidoxypropyltrimethoxysilane i n t o l u e n e

i s performed producing a support c a r r y i n g l o n g c h a i n s e n d i n g w i t h an e poxide group. I n t h e second

s t e p t h e epoxide group r e a c t s w i t h t h e amino group o f 3-AF. Although f o r s i l i c a h i g h e r s u r f a c e coverages have been found, t h e h i g h e s t CL s i g n a l s have been obt a ined f o r CPG. Using CPG g l a s s beads o f 200-400 mesh, good s t a b i l i t y ov er l o n g p e r i o d s was o b t a i n e d . Poulson e t a l . have compared t h e CL e f f i c i e n c i e s o f immobilized 3-AF (on g l a s s beads) and l i q u i d s t a t e rubrene Sigvardson

et

al.

(ref.

81)

on

liquid

( r e f . 98). state

From r e s u l t s o f

efficiencies

of

flu oro phore s one would expect a s i g n i f i c a n t h i g h e r e f f i c i e n c y f o r 3-AF.

both

272 However,

for

immobilized 3-AF

the

difference

i s only

20

per

cent,

i n d i c a t i n g t h a t i m m o b i l i z a t i o n a f f e c t s t h e r o l e o f 3-AF i n peroxyoxalate CL

i n a n e g a t i v e way.

Nevertheless,

despite o f

this

reduction the

e f f i c i e n c y o f immobilized 3-AF i s very favourable i n comparison t o o t h e r f 1uorophores. 0

/ \

I

Si

~

OH + (CH,O), - Si - (CH,), - 0 - CH, - CH - CHI

I 0

I

I

I

I

/ \

S I - 0 - Si - (CH,), - 0- CH, - CH - CH2

-Si

Fig. 3 2.3.3

I

I

I I

- 0 - Si - (CH,), - 0 - CH,

-

RNH,

- CH - CH, - NHR

I

OH

I m m o b i l i z a t i o n o f 3-aminofluoranthene (R-NH ) on s i l i c a g e l and g l a s s beads, d e r i v a t i z e d w i t h 3-glycidoxyprgpyl t r i m e t h o x y s i lane. COUPLING WITH PHOTOCHEMICAL REACTORS ( r e f s . 98, 99)

Poulson e t a l . have coupled t h e peroxyoxalate CL system w i t h a p o s t column photochemical r e a c t o r producing hydrogenperoxide f o r d e t e c t i o n i n HPLC. I t i s emphasized t h a t t h e photochemical r e a c t o r r e q u i r e s methanol, a q u i t e i n t e r e s t i n g p o i n t i n view o f t h e l i t t l e s t a b i l i t y o f TCPO i n t h i s s o l v e n t (see s e c t i o n 2.3.1).

The a n a l y t e s were quinones, commonly used i n

t h e wood p u l p i n d u s t r y . The r e a c t i o n i s i n i t i a t e d by a p r o t o n t r a n s f e r from methanol t o t h e e x c i t e d quinone. r e a c t i o n pathways can be followed

Then, (as

i n presence o f oxygen v a r i o u s i s visualized

i n Fig.

4)

all

e v e n t u a l l y producing H202, w h i l e quinone i s n o t consumed. I n t h a t sense the reaction i s a photocatalytic

one;

i n practice

up t o

100 H202,

molecules are produced f o r any a n a l y t e molecule. Regarding s e l e c t i v i t y i t i s noted t h a t o n l y a l i m i t e d c l a s s o f compounds undergoes t h e above t y p e o f photooxygenation. has an i n h e r e n t detection.

Thus photochemical

selectivity,

a clear

r e a c t i o n d e t e c t i o n f o r quinones advantage over conventional

I n a f i r s t study t h e t w o - l a y e r TCPO r e a c t o r (see s e c t i o n 2.3.1)

UV

was

simply placed i n t h e main stream o f t h e a n a l y t i c a l system. As a photochemical r e a c t o r PTFE ( p o l y t e t r a f l u o r e t h y l e n e " t e f l o n " ) t u b i n g crocketed i n t o c y l i n d e r s t h a t f i t over a f l u o r e s c e n t

poster ("black")

lamp was

applied; t u b i n g o f d i f f e r e n t l e n g t h s p r o v i d e s d i f f e r e n t residence times f o r t h e analytes.

273

Q'

(TI)+ CHlCHzOH

I

Fig . 4

P o s s i b l e photochemical r e a c t i o n pathways f o r quinones (Q) i n t h e presence o f a l c o h o l s and oxygen l e a d i n g t o H202 ( r e f s . 98, 99).

To dis c us s t h e performance o f t h i s d e t e c t i o n system, one must c o n s i d e r

t h a t t h e peaks a r e a1 ready broadened by t h e post-column photochemical r e a c t o r so t h a t a d d i t i o n o f t h e CL d e t e c t o r c e l l causes r e l a t i v e l y l i t t l e e x t r a r e d u c t i o n i n chromatographic r e s o l u t i o n . Furthermore, i t should be remembered t h a t f o r r e a l samples some l o s s o f s e p a r a t i o n e f f i c i e n c y due t o band broadening caused by t h e d e t e c t o r i s l e s s i m p o r t a n t i f t h e d e t e c t i o n system i s more s e l e c t i v e ; i f t h e d e t e c t o r shows o n l y a response f o r a few compounds, s e p a r a t i o n problems can more e a s i l y be solved. O f course t h e band broadening caused by t h e photochemical res idenc e t ime.

Poulson e t a l .

r e a c t o r depends on

a p p l i e d a f l o w r a t e o f 0.76

the

m l min-'

methanol (95%); t h e n f o r a r e a c t o r o f 9.8 m l o n g t h e r e s i d e n c e t i m e i s 69 s and a band broadening o f 600 p12 was observed, w h i l e f o r a 29 m l o o p 2 r e a c t o r w i t h a re s i d e n c e t i m e o f 187 s t h e broadening was 1400 PI Under t hes e c o n d i t i o n s t h e c o n t r i b u t i o n o f t h e TCP0/3-AF d e t e c t o r c e l l

.

(see F i g . 2D) t o t h e band broadening i s 4000 4'. Though such a c o n t r i b u t i o n i s f a i r l y h i g h , good chromatograms c o u l d b e obt ained. I n f a c t , Poulson e t a l . o n l y l o s t a f a c t o r o f 5 i n s e n s i t i v i t y compared t o t h e c omp lic at e d t h r e e pump l i q u i d - p h a s e system (see F i g . 2 A ) . Unfortunately,

i n t h e set-up

under c o n s i d e r a t i o n , a f t e r about 2.5

hours o f use t h e broadening caused by t h e d u a l - l a y e r d e t e c t o r c e l l becomes more s erio us .

S i n c e t h e photochemical r e a c t o r r e q u i r e s h i g h methanol

c o n c e n t r a t i o n s , a f t e r t h a t t i m e v o i d f o r m a t i on i n t h e TCPO l a y e r occurs and t h e dead volume and t h u s t h e peak v a r i a n c e increases s i g n i f i c a n t l y w i t h use. Longer l i f e t i m e s a r e observed a t lower methanol c o n t e n t s , b u t t h i s i s as s oc iat e d w i t h a s t r o n g r e d u c t i o n o f t h e CL s i g n a l .

274 To avoid these band broadening problems, i n a subsequent study t h e TCPO r e a c t o r was placed o u t s i d e t h e p a t h o f t h e a n a l y t i c a l column e f f l u e n t

(see Fig. 5 ) . Obviously i n t h i s set-up t h e constant c o n t r i b u t i o n of t h e immobilized f l u o r o p h o r e c e l l t o t h e peak variance remains: i n a 3 cm x 1.5 mm

I.D.

cell

packed w i t h 40-80

p

CPG g l a s s

beads

(derivatized

or

u n d e r i v a t i z e d ) t h e peak variance i s 1200 p12. Besides i t i s noted t h a t t h e c e l l would be reduced s t r o n g l y if

variance caused by t h e fluorophore smaller g l a s s beads were applied.

Since t h e a n a l y t e does n o t pass through t h e TCPO reagent a d d i t i o n bed, t h e l i f e t i m e o f t h e bed can be extended by i n c r e a s i n g i t s c a p a c i t y w i t h o u t causing an increase i n peak variance. I n p r a c t i c e a bed o f 4.6 mm x 4 cm was used, packed each day w i t h a m i x t u r e o f f i n e l y ground TCPO and 40-80 f l glass beads, 70:30 by weight. Depending on t h e d e s i r e d l i f e t i m e , t h e TCPO charge v a r i e d from 50-250 mg; t h e remaining space a t t h e i n l e t s i d e

of t h e r e a c t o r was simply f i l l e d w i t h g l a s s beads. The g l a s s beads improve

t h e flow c h a r a c t e r i s t i c s ,

reduce back pressure,

and s i p l i f y packing by

decreasing t h e s t a t i c e l e c t r i c a l charge on t h e s o l i d TCPO p a r t i c l e s . Two c o n f i g u r a t i o n s were developed t o i n v e s t i g a t e t h e p o s s i b i l i t i e s of TCPO a d d i t i o n from o f f - p a t h s o l i d reagent beds. I n t h e f i r s t , a dual-pump design (Fig. 5a), choice o f t h e s o l v e n t d e l i v e r e d t o t h e reagent bed does n o t a f f e c t t h e HPLC separation. With t h i s system t h e s o l v e n t dependences o f t h e photochemical r e a c t i o n and chromatography a r e i s o l a t e d from t h e chemi luminescent response, addition conditions.

f a c i 1 it a t i n g t h e o p t i m i z a t i o n o f t h e reagent

I n t h e second, TCPO i s s o l u b i l i z e d from t h e reagent

bed w i t h o u t any a d d i t i o n a l reagent pump. To accomplish t h i s , t h e f l o w from t h e HPLC mobile phase pump i s s p l i t i n t o a reagent a d d i t i o n -

and a

chromatographic stream (Fig. 5b). Flow s p l i t t i n g reduces equipment demands a t t h e expense o f f l e x i b i l i t y i n choice o f reagent a d d i t i o n s o l v e n t . The f l o w r a t e through t h e reagent bed i s

an important

parameter i n t h e

o p t i m i z a t i o n o f these systems. Concentration o f TCPO i n t h e d e t e c t o r f l o w c e l l i s determined by i t s s o l u b i l i t y i n t h e reagent bed s o l v e n t ,

the

r e l a t i v e f l o w r a t e s o f t h e two s o l v e n t streams, and t h e r a t e o f reagent decomposition i n t h e s o l v e n t . Residence t i m e o f t h e reagent and t h e a n a l y t e i n t h e d e t e c t o r c e l l decreases as t h e t o t a l f l o w r a t e increases. Peroxyoxalate chemiluminescence i s l o n g l i v e d r e l a t i v e t o t h e residence time i n t h e HPLC d e t e c t o r c e l l so t h a t t h e e f f i c i e n c y o f l i g h t c o l l e c t i o n w i l l be reduced by h i g h t o t a l f l o w r a t e s .

275

In Table I some d e t e c t i o n l i m i t s o b t a i n e d w i t h t h e dual pump system a r e compared w i t h t h o s e o b t a i n e d w i t h l i q u i d - p h a s e

TCPO a d d i t i o n ,

combined

w i t h t h e i m m o b i l i z e d 3-AF d e t e c t o r c e l l o r w i t h l i q u i d - p h a s e r u b r e n e as f l u o r o p h o r e . I t i s obvious t h a t t h e r e s u l t s o b t a i n e d w i t h s o l i d - s t a t e TCPO a d d i t i o n combined w i t h i m m o b i l i z e d AF compare f a v o u r a b l y w i t h t h e o t h e r data.

TO WASTE

1

DUAL -PUYP SYSTEY PRESSURE YETER INJECTOR

I y HPLC PUYP

h

CONFLUENCE TEE,

- p f Clg COLUWY

1

n

FUSE DAYPEYER

~~

PHOTOCHEY ICAL REACTOR

BE0

?\TCPO

H16H PRESURESYRINCE PUYP

YOBILE PHASE

TO YASTE

t INJECTOR

-

-

CONfLUEYCE TEE \ RECORDER

A

Y I

PHOTOCHEYICAL REACTOR

\-

TCPOBED

i

M C i PRESSURE COLUYNS

YOBILE PHASE

Fig. 5

Schematic diagram o f t h e dual-pump system (a) and t h e s p l i t - f l o w system (b) ( r e f . 99). F u r t h e r d e t a i l s see t e x t .

276

TABLE I

Dual-pump system d e t e c t i o n l i m i t s (S/N = 3) i n picomoles on column. Solvent dependence and comparison t o l i q u i d phase reagent a d d i t i o n ( r e f s . 98, 99). S o l i d s t a t e TCPO a d d i t i o n w i t h immobilized f l u o r o p h o r e ~~

Compound phase

-

~

Reagent a d d i t i o n s o l v e n t

100% CH30H 80% CH30H

L i q u i d phase a d d i t i o n o f TCPO Immobilized

Liquid

fluorophore (3-AF)

fluorophore (rubrene)

100% CH3CN

2-methyl 1,4-naphthoquinone ( v i t a m i n K-3)

0.29

1.8

0.31

2.0

0.84

9,lO-anthraquinone

0.29

1.6

0.30

1.8

0.68

2-t-butylanthraqui none

0.24

1.3

0.24

1.5

0.53

The HPLC mobile phase i s 95% methanol a t a f l o w r a t e o f 0.76 ml/min i n a l l cases. I n t h e dual-pump system, t h e TCPO bed f l o w r a t e i s 0.42 ml/min. For l i q u i d phase a d d i t i o n o f TCPO i t i s d e l i v e r e d i n acetone a t a concent r a t i o n o f 0.92 g/1 w i t h a f l o w r a t e o f 0.28 ml/min. Rubrene, 45 mg/l i s t h e l i q u i d phase f l u o r o p h o r e and i s d e l i v e r e d i n 9 9 : l acetone: T R I S b u f f e r (pH = 8.0) a t 0.15 ml/min. The f i n a l b u f f e r c o n c e n t r a t i o n i s 0.5 mM.

6 some chromatograms a r e shown. The d e t e c t i o n l i m i t s a t t a i n a b l e w i t h t h e s p l i t - f l o w system a r e o n l y l i t t l e h i g h e r than f o r t h e dual-pump system. O f course t h e percentage o f water i n t h e e l u e n t i n t h e s p l i t - f l o w system p l a y s an important r o l e : on t h e one hand i t i n f l u e n c e s t h e r e t e n t i o n times, on t h e o t h e r hand t h e peak h e i g h t s i n c e t h e TCPO r e a c t i o n g i v e s lower CL s i g n a l s a t h i g h e r water contents. We conclude t h a t a d d i t i o n o f TCPO from s o l i d - s t a t e reagent beds has c l e a r advantages over l i q u i d - p h a s e d e l i v e r y . I t widens t h e range of r e a c t i o n s a p p l i c a b l e t o HPLC d e t e c t i o n by r e l a x i n g t h e s t a b i l i t y requirements o f t h e reagent i n t h e d e l i v e r y s o l v e n t . Even i n t h e case of p a r t i a l decomposition as i n methanol, t h e decreasing response over t i m e associated w i t h reagent breakdown d u r i n g l i q u i d - p h a s e a d d i t i o n i s n o t observed. Since t h e time spent i n t h e l i q u i d phase remains constant, t h e In

Fig.

l e v e l s o f reagent and reagent breakdown products i n t r o d u c e d t o t h e c e l l

also remain constant over t h e l i f e o f t h e reagent bed. Thus any reagent of l i m i t e d s o l u b i l i t y which e x h i b i t s a reasonable degree o f s t a b i l i t y i n s o l u t i o n i s amenable t o t h i s method o f a d d i t i o n .

277

I

10

e

")"r"

4 MINUTES

2

C

Fig. 6

Liquid chromatograms of quinones detected via the post-column photochemical reactor/peroxyoxalate CL detection system (ref. 99). The four peaks belong to menadione (5.9 pmol), anthraquinone (4.5 pmol), 2-methylanthraquinone (3.6 pmol) and 2-t-butyl anthraquinone (4.7 pmol). For chromatogram "C" the amounts injected are 2.5 times higher. a) Dual-pump system; 94% CH OH HPLC flow of 0.72 ml/min. TCPO is added with CH OH + T R I S h f f e r 99:l at 0.42 ml/min. b) Split-flow syztem; 94% CH OH HPLC flow of 0.73 ml/min and the reagent bed flow is 0.32 r?il/min. c) Split-flow system; 80% CH OH HPLC flow of 0.73 ml/min and the reagent bed flow is 0.32 r?il/min.

278 2.3.4

COUPLING WITH IMERs The a p p l i c a b i l i t y o f immobilized enzymes i n chemical a n a l y s i s has been

discussed thoroughly by L.D.Bowers

(ref,

91). The most obvious advantage

o f i m m o b i l i z i n g these b i o c a t a l y s t s i s t h a t they can be r e a d i l y separated from t h e r e a c t i o n m i x t u r e and t h u s reused. Other important aspects a r e t h a t , as a r e s u l t of i m m o b i l i z a t i o n , t h e enzymes may be more s t a b l e than t h e r e s o l u b l e analogues and a p p l i c a b l e i n s o l v e n t s c o n t a i n i n g o r g a n i c

O f course t h e l a t t e r p o i n t i s o f p a r t i c u l a r i n t e r e s t if immobilized enzymes are a p p l i e d as post-column r e a c t o r s (IMERs) i n HPLC experiments. Besides i t i s noted t h a t i n a f l o w system t h e apparent enzymic a c t i v i t y i s n o t o n l y dependent on t h e c a t a l y t i c r a t e of t h e enzyme b u t a l s o on nonenzymic f a c t o r s as mass t r a n s p o r t o f t h e s u b s t r a t e . Most o f t h e a p p l i c a t i o n s o f IMERs i n HPLC presented so f a r have d e a l t w i t h p o l a r n a t u r a l products which a r e e l u t e d from t h e HPLC system w i t h a low content o f organic s o l v e n t i n t h e mobile phase ( r e f . 92). As an example we r e f e r t o t h e q u a n t i t a t i o n o f urea and ammonia i n samples from an urea p l a n t and i n waste water samples ( r e f . 101). Immobilized urease degrades post-column u r e a i n t o arbon d i o x i d e and ammonia. The l a t t e r product subsequently reacted w i t h o-phthalaldehyde t o form a compound t h a t can be very w e l l q u a n t i t a t e d by f uorescence d e t e c t i o n :

modifier.

(

a -

NI l,),CO + I1,O

urew

co + 2N€13

CHO

NH, +

fluorescentconlpourtd

CHO

Coupling of post-column IMERs w i t h CL d e t e c t i o n i m p l i e s an a d d i t i o n a l c o m p a t i b i l i t y problem, s i n c e t h e o p t i m a l c o n d i t i o n s o f t h e CL r e a c t i o n do n o t match w i t h those o f t h e enzymatic r e a c t i o n .

For example, t h e l u m i n o l

r e a c t i o n r e q u i r e s s t r o n g l y a l k a l i n e c o n d i t i o n s (pH about 12), w h i l e f o r peroxyoxalate CL i n h i g h l y aqueous media t h e CL e f f i c i e n c y i s extremely low so t h a t h i g h organic m o d i f i e r c o n c e n t r a t i o n s a r e needed (e.g.,

80%

a c e t o n i t r i l e ) . Coupling of t h e l u m i n o l r e a c t i o n t o enzymatic r e a c t i o n s has been reported ( r e f s . 65,

102). S c o t t e t a l . have shown t h e p o t e n t i a l of t h e peroxyoxalate CL r e a c t i o n i n combination w i t h immobilized u r i c a s e f o r t h e determination o f u r i c a c i d ( r e f . 103).

279 One o f t h e u l t i m a t e goals o f t h e combination o f IMERs and HPLC i s t h e

I f the e l u a t e from t h e a n a l y t i c a l column f l o w s through t h e IMER, t h e enzyme causes a r e a c t i o n s e l e c t i v e f o r t h e s u b s t r a t e molecules l e a d i n g t o t h e formation o f products which can be detected by s u i t a b l e methods. It i s emphasized t h a t , d e s p i t e o f t h e s e l e c t i v i t y o f t h e IMER i n complex application o f

(natural)

group-specific

samples

and/or

as u r i n e o r

serum,

stereoselective

t h e eventual

enzymes.

detection o f

the

products formed i n t h e IMER may b e hindered by i n t e r f e r e n c e s . That i s t h e reason why combination w i t h t h e h i g h l y s e l e c t i v e CL d e t e c t i o n techniques i s interesting. I n o r d e r t o i n v e s t i g a t e t h e c o m p a t i b i l i t y o f immobilized oxidases w i t h the s o l i d - s t a t e peroxyoxalate CL d e t e c t i o n system, Van Zoonen e t a l . have t e s t e d ( t h e low c o s t enzyme) glucose oxidase as a model system ( r e f . 33). Two i m m o b i l i z a t i o n procedures f o r glucose oxidase were examined, i.e., i m m o b i l i z a t i o n on an ion-exchanger simply by e l e c t r o s t a t i c i n t e r a c t i o n according t o Meek e t a l .

(ref.

104)

and i m m o b i l i z a t i o n

v i a chemical

bonding on g l a s s beads f o l l o w i n g t h e g l u t a r a l d e h y d e method according t o Weetall ( r e f . 105). I n t h e l a t t e r method t h e g l a s s m a t r i x , a f t e r a c t i v a t i o n , i s coated w i t h an amino f u n c t i o n a l group and subsequently t h e f o l l o w i n g steps a r e c a r r i e d o u t :

The ion-exchanger support appeared t o be u n s u i t a b l e f o r i m m o b i l i z a t i o n

of

oxidases:

it

strongly

retains

the

formed

hydrogenperoxide.

Such

problems a r e n o t encountered f o r t h e IMER based on g l a s s beads. Two

FIA experimental set-ups were compared. I n t h e former a s i n g l e

f l o w l i n e was a p p l i e d and t h e f l o w composition was s i m p l y optimized f o r t h e CL r e a c t i o n , i.e., 80% aqueous a c e t o n i t r i l e c o n t a i n i n g a small amount of T r i s b u f f e r . Even f o r such a h i g h m o d i f i e r c o n c e n t r a t i o n i n an IMER o f 6 cm length and 3.0 mm

15% was

achieved.

I.D., a t pH

Rather

= 8.0 a glucose conversion as h i g h as s u r p r i s i n g l y t h e conversions a r e almost

independent o f f l o w r a t e i n t h e range between 0.3 and 1.5 m l m i n - l .

The

l i m i t o f d e t e c t i o n f o r glucose was 8 x 10-8M.

To avoid t h e entrance o f h i g h 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 s i n t h e IMER,

280 i n the second set-up a c e t o n i t r i l e was added according t o t h e make-up flow p r i n c i p l e . The aqueous f l o w containing T r i s b u f f e r passes trough t h e IMER a t a r a t e o f 0.3 m l min-' and combines w i t h an a c e t o n i t r i l e f l o w of 1.0 m l min-' before entering t h e s o l i d - s t a t e CL r e a c t o r c e l l . Under these conditions t h e maximum conversion o f about 50% was found (only R-D glucose i s converted) and w i t h a smaller IMER (length 0.4 cm) f o r glucose a LOD of 5 x M was achieved and l i n e a r range up t o M. These encouraging solid-state r e s u l t s i n d i c a t e the f e a s i b i l i t y o f t h e (oxidase) IMER peroxyoxal a t e CL combination f o r HPLC.

-

Honda e t a l . have applied t h e combination o f IMERs and l i q u i d s t a t e peroxyoxalate CL i n HPLC f o r tylcholine

the simultaneous

(ACh) and c h o l i n e (Ch)

(ref.

determination

o f ace-

A mixed bed r e a c t o r of

89).

immobilized acetylcholine esterase and cholineoxidase was applied enabling the f o l l o w i n g r e a c t i o n pathway f o r acetylcholine:

0

(CfI,),NCH2CH,OCCH, + HzO

acetylcholine

II

0

esterase

0 (CH,)3NCH,CHZOH

+ CH3COOtl

choline

The optimum pH f o r these enzymes i s from 8.1 t o 8.5 so t h a t t h e r e i s no pH problem f o r the a p p l i c a t i o n o f TCPO CL. Nevertheless, t h e HPLC set-up requires t h r e e pumps as shown i n F i g . 7. The separation o f c h o l i n e and acetylcholine i s based on paired-ion chromatography: t h e column i s an RP-18 column and t h e eluent (10 mM p h t h a l i c acid, 1.2 mM t r i e t h y l a m i n e and

76 mM sodiumoctanesulfonate pH adjusted t o 5.0 w i t h KOH) has a pH t o o low t o be applicable t o the IMER. Hence, a f t e r the separation column a flow of T r i s b u f f e r (pH = 8.5) was provided; n i t r a t e was used instead o f c h l o r i d e as a counterion because c h l o r i d e i s known t o quench t h e CL r e a c t i o n (see section 2.5). The t h i r d pump d e l i v e r s TCPO and t h e fluorophore perylene i n a mixture o f ethylacetate and acetone. Flow r a t e s were chosen so t h a t i n the CL reaction medium t h e f l o w has t h e composition ethy1acetate:acetone: buffer:eluate i n 15:45:4:8.

Due t o t h e low s o l u b i l i t y o f TCPO i n t h i s

medium i t s concentration i n the second a d d i t i o n l i n e could not be higher than 1.2 mM i n order t o prevent p r e c i p i t a t i o n e f f e c t s . Good r e s u l t s were obtained f o r standard solutions: d e t e c t i o n l i m i t s f o r Ch and ACh of about

1 pmol w i t h l i n e a r ranges from 10 pmol t o 10 nmol.

eluent

t--1

4

injector

P

TCPO

28 1

column

+ perylene

7

CL monitcr

Fig. 7

Schematical r e p r e s e n t a t i o n o f t h e e x periment al s e t - u p f o r t h e simultaneous d e t e r m i n a t i o n o f a c e t y l c h o l i n e and c h o l i n e v i a IMER-peroxyoxalate CL d e t e c t i o n , a c c o r d i n g t o Honda e t a l . ( r e f . 89). F u r t h e r d e t a i l s , see t e x t .

I t i s i n t e r e s t i n g t o compare t h i s approach, based on l i q u i d - s t a t e p e r -

oxy ox alat e CL, w i t h t h e s o l i d - s t a t e p r i n c i p l e a p p l i e d t o t h e same problem, i.e .

t h e simultaneous d e t e r m i n a t i o n o f ACh and Ch ( r e f . 34). A d e t a i l e d

b l o c k diagram o f t h e e x p e r i m e n t a l set-up i s present ed i n F ig. 8. Another s e p a r a t i o n p r i n c i p l e has been a p p l i e d , ( d e r i v e d f rom Damsma e t a l .

(ref.

106) based on a (home-packed) cation-exchange column f o r t h e s e p a r a t i o n o f ACh and Ch: t h e m o b i l e phase i s aqueous 0.05 M potassium phosphate (pH = 7.4) c o n t a i n i n g tetramethylamnoniumnitrate (again n i t r a t e i n s t e a d o f c h l o r i d e t o p r e v e n t CL quenching).

A precolumn was p l a c e d b e f o r e t h e

i n j e c t o r as a guard column and p u l s e dampener. The IMER (ACh e s t e r a s e and Ch oxidase c o v a l e n t l y bonded t o sepharose,

dimension 75 x 2.1 mm) was

d i r e c t l y coupled t o t h e a n a l y t i c a l column by means o f a v a l c o union. An a c e t o n i t r i l e make-up f l o w was a p p l i e d c o n t a i n i n g 18-crown-6, a crown e t h e r that

efficiently

conditions

forms

complexes

with

potassium

even 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

ions.

Under

these

o f pot assium phosphate

b u f f e r s can be mixed w i t h a c e t o n i t r i l e w i t h o u t p r e c i p i t a t i o n problems. Furthermore t h e a c e t o n i t r i l e f l o w c o n t a i n s t r i e t h y l a m i n e (TEA), causing a 10-f o ld improvement o f S/N r a t i o . Optimal f l o w r a t e s were 0.5 m l min-' f o r t h e chromatographic and 1.5 m l m i n - l f o r t h e make-up f l o w . E s s e n t i a l i s t h e use o f an e f f i c i e n t v o r t e x m i x e r because m i x i n g - n o i s e i s t h e main f a c t o r d e t e r m i n i n g t h e d e t e c t i o n l i m i t s t h a t can be reached. F or t h e CL d e t e c t i o n t h e t w o - l a y e r bed r e a c t o r o f s o l i d TCPO and 3-AF i m m o b i l i z e d on g l a s s beads was used.

282

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

apeour

QumQ

,.I,d\,>,,.,W

m i o n eichmgu

h"llrn.TM

Fig. 8

1W.R

Block diagram of the experimental set-up for the simultaneous determination of acetylcholine and choline via IMER-perox oxalate CL detection, according to Van Zoonen et al. (ref. 347. Further details see text.

Chromatograms of untreated urine samples spiked with ACh and Ch are presented in Fig. 9; similar pictures on deproteinated serum samples are shown in Fig. 10. The detection limits are comparable with those reported by Honda et al. (ref. 89). However, the solid state set-up is easier to handle since only one post-column pump is utilized. The TCPO layer has a lifetime of about 4 hours, but repacking is quite easy and can be performed with a microspatula. The IMER can be applied for several hundred samples: if it is used continuously for two weeks, the sensitivity is decreased by about 50%.

8

Fig. 9

(a) Chromatogram of an undiluted' urine sample, (b) chromatogram of a urine sample spiked with 20 pmol of Ch and ACh detected with the set-up depicted in Fig. 8 (ref. 34). Further details, see text.

Summarizing this section, it is concluded that the combination of IMERs and solid state peroxyoxalate reactors have potential for analysis

283 of complex samples e s p e c i a l l y because o f t h e h i g h s e l e c t i v i t y i n h e r e n t t o the

combination

of

rather

specific

enzymatic

and

chemiluminescence

r e a c t i o n s . Developments along these l i n e s have t o be a n t i c i p a t e d .

Fig. 10

2.4

(a) Chromatogram o f a d e p r o t e i n a t e d pooled serum sample, (b) serum sample spiked w i t h 200 pmol o f Ch and ACh detected w i t h t h e set-up depicted i n F i g . 8 ( r e f . 34). F u r t h e r d e t a i l s , see text. USE OF THE SOLID TCPO REACTOR FOR DETECTION OF FLUOROPHORES ( r e f .

107) Although

in

this

chapter

we

are

primarily

concerned

with

non-fluorescent compounds, i t i s a p p r o p r i a t e t o p o i n t o u t t h a t t h e s o l i d s t a t e TCPO r e a c t o r has been invoked s u c c e s s f u l l y f o r t h e d e t e c t i o n of f l u o r o p h o r i c compounds.

Compared t o t h e conventional

peroxyoxalate

CL

d e t e c t i o n system, a s i g n i f i c a n t s i m p l i f i c a t i o n has been reached w i t h o u t a s u b s t a n t i a l loss

i n sensitivity.

reduced experimental complexity, least

partially

circumvents

As noted above,

i n addition t o the

reagent a d d i t i o n from a s o l i d bed a t the

chemical

decomposition

problems

encountered i n l i q u i d - p h a s e a d d i t i o n o f o x a l a t e s . I n t h i s way a l a r g e r range o f s o l v e n t s can be u t i l i z e d , because TCPO i s d i s s o l v e d very s h o r t l y before i t i s a c t u a l l y used i n t h e CL r e a c t i o n . Two experimental

set-ups

have been examined w i t h t h e s o l i d TCPO

reagent bed s i t u a t e d p a r a l l e l t o t h e a n a l y t i c a l column i n o r d e r t o reduce band broadening (see F i g . 11). The s p l i t - f l o w system, u t i l i z i n g o n l y t h e mobile phase pump, can be employed i f t h e chromatographic s e p a r a t i o n can be achieved under c o n d i t i o n s matching those o f t h e CL r e a c t i o n ( i n t h e model system presented more than 80% a c e t o n i t r i l e ) . A h i g h l y s o p h i s t i c a t e d system t o r e g u l a t e t h e s p l i t r a t i o was n o t necessary; r e t e n t i o n times were r e p r o d u c i b l e w i t h i n 2% (over 14 chromatograms). The two pump system c o u l d be used very w e l l f o r mobile phase compositions w i t h a t l e a s t 50%

284 acetonitrile.

In

more aqueous media p r e c i p i t a t i o n o f TCPO i n t h e mixing

tee-piece was encountered. The CL signal i s l i n e a r l y dependent on t h e H202 concentration i n t h e eluent. However, if the concentrations become t o o high a l s o background i s increased. For p r a c t i c a l reasons such as corrosion o f pumps and s t a i n l e s s s t e e l p a r t s o f t h e system, concentrations higher than 10-1 M H202 were n o t u t i l i z e d . Under these conditions t h e performance o f the a n a l y t i c a l Column (Spherisorb ODs-5 i n the present example) remained constant f o r a t l e a s t a month. The TCPO reactor ( i n t h e present example a 6 cm, 3 mm I.D.

stainless

s t e e l column w i t h a mixture o f s o l i d f i n e l y ground TCPO and 40-80

rm

glass

beads) could be employed a t l e a s t 8 hours without any d r i f t s ; each day TCPO was d i r e c t l y added t o the reactor. A f t e r about 2 weeks t h e r e a c t o r should be repacked completely since a f t e r i m p u r i t i e s can accumulate in the reactor.

Fig. 11

a prolonged time of

use

Schematical representation of t h e two-pump system I and t h e single-pump system I1 f o r t h e d e t e c t i o n o f fluorescers based on peroxyoxalate chemiluminescence. As r e s t r i c t o r a 25 cm Spherisorb ODs-5 column was applied. The luminescence was measured w i t h a Kratos FS 970 fluorescence d e t e c t o r ( r e f . 107).

TABLE I 1

Comparison o f d e t e c t i o n l i m i t s (pg) obtained by Sigvardson e t a1 ( r e f . 80) and van Zoonen e t a1 ( r e f . 107).

.

.

Sigvardson e t a l . Ana 1y t e

SIN

=

2 ~

p e r y l ene 3-ami nofluoranthene 9,lO-dephenylanthracene anthracene tetracene benz (a) pyrene II2-benzanthracene

Van Zooner e t a l . 2 pump system SIN = 3

0.77 0.30*

s p l i t system SIN = 3

~

1.6 0.6 30 150 860 80 53

20 130 735 45 20.5

2.0 0.7 35 200 980 120

65

*Calculated from t h e t e x t I n Fig. 12a a chromatogram obtained by t h e s p l i t - f l o w method i s shown. I n Table I 1 d e t e c t i o n l i m i t s o f t h e both s o l i d TCPO a d d i t i o n systems (see i i g . 11) w i t h those o f t h e conventional CL system determined by Sigvardson e t al.

( r e f . 80) a r e compared.

I t i s obvious t h a t t h e s i m p l i f i e d system

provides no s u b s t a n t i a l l o s s o f s e n s i t i v i t y .

O f course t h e v a r i a t i o n i n

d e t e c t i o n l i m i t s f o r t h e fluorophores under i n v e s t i g a t i o n r e f l e c t s t h e i r CL e f f i c i e n c y i n t h e peroxyoxalate r e a c t i o n , which i s n o t o n l y determined by t h e energy o f t h e lowest e x c i t e d e l e c t r o n i c s t a t e b u t a l s o by t h e o x i d a t i o n p o t e n t i a l . As such, f o r i m m o b i l i z a t i o n on g l a s s

3-aminofluoranthene, t h e compound a p p l i e d beads (see s e c t i o n 2.3.2) i s t h e most

e f f i c i e n t CL f 1uorophore. QUENCHED PEROXYOXALATE CHEMILUMINESCENCE DETECTION

2.5

Various compounds, a l s o i f present a t low c o n c e n t r a t i o n s , a r e a b l e t o quench peroxyoxalate chemiluminescence ( r e f s . important

to

acquire

some

knowledge

about

108-110). the

Obviously i t i s

background

of

this

phenomenon i n o r d e r t o be aware of p o s s i b l e and p o t e n t i a l i n t e r f e r e n c e s when peroxyoxalate CL i s a p p l i e d f o r d e t e c t i o n purposes i n HPLC and F I A .

On

the

other

hand

it

is

interesting

to

examine

the

potential

of

peroxyoxalate CL f o r d e t e c t i o n o f these quenching compounds, e s p e c i a l l y if o t h e r e x i s t i n g d e t e c t i o n techniques have some disadvantages. I n t h i s s e c t i o n f i r s t some p o i n t s r e g a r d i n g new i n s i g h t s i n t o t h e mechanism o f t h e peroxyoxalate CL r e a c t i o n a r e discussed. Such a d i s c u s s i o n i s needed s i n c e i n t h e mechanistic s t u d i e s r e p o r t e d thus f a r no a t t e n t i o n has been p a i d t o quenching phenomena a t very low c o n c e n t r a t i o n l e v e l s . Subsequently, t h e a p p l i c a b i l i t y of Quenched CL d e t e c t i o n i s considered and f i n a l l y an i n t e r p r e t a t i o n o f t h e quenching process i s presented.

286

1

I

0

.

4

.

.

8

.

.

1

.

2

.

.

%

t(min) +

Fig. 12

2.5.1

Chromatogram obtained by t h e s p l i t - f l o w method f o r t h e determination o f fluorophores v i a s o l i d - s t a t e TCPO a d d i t i o n (ref. 107). Mobile phase compo$tion: 90% a c e t o n i t r i l e , 10% aqueous T r i s (5 mM, pH = 8.0), 10 M H202. I n j e c t i o n volume: 20 4. 1. 25 pg 3-aminofluoranthene 2. 2 ng anthracene 3. 300 pg 1,2-benzanthracene 4. 30 pg perylene 5. 149 pg benz(a)pyrene 6. 350 pg 9,lO-diphenylanthracene. ON THE MECHANISM OF PEROXYOXALATE CL

By now i t i s unambiguous t h a t t h e r e a c t i o n scheme o f t h e peroxyoxalate CL reaction presented i n section 2.1 does not account f o r a l l experimental data a v a i l a b l e i n the l i t e r a t u r e .

There are two obvious reasons why a

d i r e c t energy t r a n s f e r between t h e 1,2-dioxetanedione and the fluorophore i s extremely u n l i k e l y .

F i r s t of

all,

t h e i n t e n s i t y o f CL i s s t r o n g l y

dependent upon t h e e l e c t r o n e g a t i v i t y o f the a r y l group o f t h e o x a l a t e esters which excludes a common intermediate. Secondly, i t i s n o t o n l y t h e energy o f the lowest excited e l e c t r o n i c s t a t e o f the fluorophore t h a t determines the CL i n t e n s i t y ; a l s o i t s i o n i z a t i o n p o t e n t i a l plays a r o l e . Catherall, Palmer and Cundall have pub1 ished a d e t a i l e d k i n e t i c study using b i s (pentachloropheny1)oxalate

(PCPO)

as

oxal ate,

9,lO-diphenyl-

anthracene (DPA) as fluorophore and sodiumsalicylate as base ( r e f . 76). They observed t h a t the decay of CL i s independent o f t h e DPA concentration while the quantum y i e l d increases l i n e a r l y w i t h (DPA) reaches a maximum.

The rate-determining

step

and e v e n t u a l l y

i s probably a r e a c t i o n

between PCPO and t h e hydrogenperoxide anion OOH-(which explains t h e r o l e

of base catalysts) and does not involve the fluorophore. The lifetime of the intermediate formed in this step was found to be about 5 x which is an indication for an unstable compound and is not consistent with attempts to identify a relative stable dioxetane. The same authors compared the efficiencies of a number of fluorophores. They were able t o show the existence of a relationship between the normalized CL quantum yields (pL/Fwhere pLand + are the chemiluminescence and fluorescence quantum yields, respectively) and the oxidation potentials of the fluorophores pointing to an electrontransfer mechanism. Formally, the mechanism according to Catherall et al. can be represented as follows: PCPO+OOH-

+

X

PCPO+OOH-

-+

non CL products

x

+

non CL decay

X+F

+

XF

XF

+

F* + products

XF

+

F +products

+ +

F +hvF

F* F*

F +heat

(la)

(4b)

where (OOH-) is proportional to the initial concentrations of both H202 and PCPO and X is a reactive intermediate (ref. 76). Conventional kinetic treatment, assuming that the lifetimes o f X, XF and F* are short, gives for the intensity of chemiluminescence at any time, t, defined as

I = d-( h ~=) k4a [F*] t dt

the following expression

if oxalate i s in excess. [PCPO], is the oxalate ester concentration which is effectively constant with time and [H202], is the concentration of hydrogenperoxide at any time t. The @ s are the efficiencies of the reaction steps (4a), (3a) and (2b), respectively; @4a of course being equivalent to the fluorescence quantum yield F. The time dependence of

288 the hydrogenperoxide concentration can be w r i t t e n as

The number o f quanta emitted (QE) i s given by

=I m

QE

(9)

Itdt

0

so t h a t QE i s equal t o

wherein

la i s the e f f i c i e n c y o f

Thus, i n excess o f oxalate,

r e a c t i o n step ( l a ) .

TL i s

equal t o the product o f f o u r eff i c i e n c i e s (i.e., those o f steps ( l a ) , (Zb), (3a) and 4a)) and t h e i n i t i a l i.e., H202 concentration. The r o l e o f F i s r e a d i l y v i s u a l i s e d v i a

which approximates (kZb/kza) [F]

under conditions where kza >>4k2b

[Fl.

This i s generally met f o r fluorophore concentrations below 10- M since 3 1 k2b/k2a z 5 x 10 M- ; as noted before kZa-' i s about 5 x lom7 s ( r e f .

76). Therefore, a t low fluorophore concentrations QE ( t h e CL s i g n a l ) i s proportional t o [F]. Furthermore Eq. ( r e f . 10) shows the l i n e a r dependence These two r e l a t i o n s h i p s underline t h e between QE and [H2O2Io. a p p l i c a b i l i t y o f t h e peroxyoxalate CL r e a c t i o n f o r fluorophore and H2OZ detection purposes. As the key intermediate X I C a t h e r a l l e t a l . assume 3-pentachlorophenoxy-3-hydroxy-l ,2-di oxetanone, denoted as

0-0

R -0-C-C

I I

OH

I

R =

No

CI

CI

289

which r e a c t s w i t h F t o a r a d i c a l i o n p a i r

R-0-C-C

.

.

OH

t h a t subsequently undergoes t h e f o l l o w i n g sequence

More r e c e n t l y A l v a r e z e t a l . ( r e f . 77) p u b l i s h e d a d e t a i l e d k i n e t i c study on t h e same r e a c t i o n u t i l i z i n g TCPO as t h e o x a l a t e , t r i e t h y l a m i n e as t h e base, DPA as t h e f l u o r o p h o r e i n e t h y l a c e t a t e as t h e s o l v e n t .

Their

r e s u l t s s t r o n g l y suggest t h a t i n s t e a d o f a s i n g l e i n t e r m e d i a t e a t l e a s t two i n t e r m e d i a t e compounds XI

and X2 p r o d u c i n g t h e same s i n g l e t e x c i t e d

s t a t e o f DPA, p l a y a r o l e , a c o n c l u s i o n based on a t wo-pulse i n t e n s i t y / time p r o f i l e occuring a t lower concentrations o f triethylamine. A p o s s i b l e s t r u c t u r e f o r X2 i s t h e i n t e r m e d i a t e X of C a t h e r a l l e t a l . g i v e n above. A p o s s i b l e s t u r c t u r e f o r XI i s

HO

-0 -C

-C -0 -R

; :

which i n C a t h e r a l l ' s work i s a p r e c u r s o r f o r X . Thus an i m p o r t a n t consequence o f A l v a r e z ' s t u d y i s t h e e x i s t e n c e o f an a d d i t i o n a l pathway f o r t h e f lu o r o p h o r e . Finally,

i t s h o u l d be r e a l i z e d t h a t

i n HPLC and FIA

reaction frequently

s o l v e n t s p a r t i a l l y composed o f w a t e r a r e a p p l i e d w h i l e f u r t h e r m o r e e l u e n t s as methanol a r e v e r y i m p o r t a n t . I n t h e s e s o l v e n t composit ions a d d i t i o n a l

290 reaction pathways such as a direct reaction of the oxalate with H20 or methanol may complicate the establishment of a reaction mechanism. Thus, for practical reasons it i s relevant to evaluate aryl oxalates in terms of maximum chemiluminescence intensity (maximum of It), decay rate, SOlubility in different solvents, stability in presence of hydrogenperoxide and pH working range as has been done by Honda, Miyaguchi and Imai (ref. 111). They have shown that among others bis(2-nitrophenyl) oxalate, 2-NPO, has favourable properties: it is six times more soluble in acetonitrile than TCPO, has a reasonable stability in presence of H202 and is optimally applicable in the pH range 4-6. At this point we emphasize that it is not only the solubility of the oxalate that hampers the success of the peroxyoxalate CL detection in aqueous solvents. Apparently, the CL efficiency is very low in water, which is readily conceived in view of the mechanism discussed above: an immediate dissociation of the radical ion pair as probably occurs in water obviously prevents the formation of F*. APPLICABILITY OF QUENCHED CL DETECTION (refs. 109, 110) Since electron transfer from F to X is initiating the formation of F*, it is appropriate to examine whether other easily oxidizable (but nonfluorescent) compounds are able to consume X thus reducing the overall CL quantum yield. Van Zoonen et al. have shown that various compounds are able to induce quenching of peroxyoxalate CL, even if present at low concentrations (refs. 109, 110). Some examples are presented in Table 1 1 1 . They do obey a Stern-Volmer type relationship, i .e. 2.5.2

I / r = l + k [Q O Q

Q

wherein k is a constant o f quenching (in M- 1) , [Q] is the quencher concenQ tration (in M) and I /I is the ratio of the CL signals in absence and O Q presence of Q , respectively. Obviously k determines the sensitivity of Q Quenched CL detection (QCL) for a particular (quenching) analyte, so that the method has an inherent selectivity. On the other hand it should be realized that QCL is based on a decrease of luminescence. Hence the noise of the luminescence signal in absence of quencher should be reduced as much as possible to achieve a favourable signal to noise ratio for QCL. This can be reached most easily under experimental conditions where I, is high.

291 TABLE 111

D e t e c t i o n l i m i t s (S/N = 3) f o r quenched CL u s i n g 2-NPO as o x a l a t e , d e r i v e d f r o m chromatograms

Anal y t e

1.o.d.

(ng)

~

bromide

1.5

iodide

0.3

sulphit e

1.1

nitrite

0.3

p-isopropylaniline

1.4

N ,N-dimet hy 1an i1ine

0.6

N -e t h y l - m - t o l u i d i n e

1.o 8.0 1.o

N, N -d ipro p y l a n i 1i n e

thiourea N-a1 l y l t h i o u r e a

1.6

e t h y ny l t h i o u r e a

2.0

met h imazol e

0.4

The i n f l u e n c e o f t h e H202 c o n c e n t r a t i o n and t h e n a t u r e and concent r a t i o n o f f l u o r o p h o r e on k have been examined. [H202] does n o t i n f l u e n c e Q k f o r c o n c e n t r a t i o n s between lo-* and M. T his i m p l i e s t h a t i n hydro-

Q

genperoxide d e t e r m i n a t i o n s s t r a i g h t c a l i b r a t i o n 1 i n e s w i 11 be observed i n t h e presence o f quenchers. O f c o u r s e t h e a s s o c i a t e d slopes w i l l depend on t h e c o n c e n t r a t i o n s o f quenchers,

so t h a t st andard a d d i t i o n procedures

should be a p p l i e d t o c i r c u m v e n t s y s t e m a t i c e r r o r s . F or QCL, t h e a p p l i e d H202 c o n c e n t r a t i o n s h o u l d be as h i g h as p o s s i b l e i n o r d e r t o reach a h i g h

I,;

i n p r a c t i c e concentrations from In fluid

to

M are appropriate.

samples b o t h c o n c e n t r a t i o n and n a t u r e o f

oxidation potential) hardly influence k

Q’

F (i. e. ,

its

As an example t h e r e s u l t s f o r

v a r i a b l e p e r y l e n e c o n c e n t r a t i o n s a r e g i v e n i n Table I V .

I n view o f t h e

achiev able s i g n a l t o n o i s e r a t i o t h i s means t h a t a f l u o r o p h o r e w i t h a h i g h CL e f f i c i e n c y i s a p p r o p r i a t e f o r QCL, so t h a t (immobilized) 3 - a m i n o f l u o r anthene s hould be a good c h o i c e . U n f o r t u n a t e l y ,

t h e quenching c o n s t a n t

appears t o be a f f e c t e d by t h e i m m o b i l i z a t i o n procedure o f 3 - a m i n o f l u o r ant hene. The r e p r o d u c i b i l i t y between d i f f e r e n t batches f o r t h e s y n t h e s i s o f imm o biliz ed f l u o r o p h o r e w i t h r e s p e c t t o t h e quenching c o n s t a n t s i s r a t h e r poor. As a genera l t r e n d i t m i g h t be concluded t h a t s i l i a n i z a t i o n i n d r y t o l u e n e (see s e c t i o n 2.3.2)

g i v e s t h e most s u i t a b l e p r o d u c t f o r QCL, s i n c e

i t y i e l d s t h e h i g h e s t quenching c o n s t a n t s a t i n t e r m e d i a t e CL i n t e n s i t y .

292 Nevertheless, the poor reproducibility is not a very serious problem, since a single batch of immobilized 3-AF can be used over long periods of time. TABLE IV

Effect of the concentration of the fluorophore (perylene) on the quenching constant of peroxyoxalate CL (applying soljg state TCPO addition) measured with methimazole (5.2 x 10 M)

Concentration

1, in relative units

k

4 4 x 10-6

40500 10000 1200

44 48 42

4

Q in 103M-l

For the development of QCL detection both solid TCPO (in a dual-cell configuration or in a separate make-up flow line) and liquid-state 2-NPO were applied. The latter can be mixed with H202 in acetonitrile without decomposition problems. It was found that for 2-NPO the QCL peak heights (and thus the kQ-values) were about 10 times higher than for TCPO.

aqueous butter

"PI

c

--,

1

LEI3

Detector containing immobilized fluorophore

pump 2-NPO and hydrogen peroxide

Fig. 13

Experimental set-up for HPLC with Quenched Chemi luminescence detection (ref. 110).

This explains the experimental set-up for QCL detection, see Fig. 13. For the analytes under study aqueous mobile phases were applied (for instance the chromatograms of iodide, bromide, sulphite and nitrite were obtained with the mobile phase aqueous ammoniumbenzoate, 10 mM, pH 5 and PRPX-100 column) as the mobile phase at a flow rate of 1 ml min-' and a reagent flow of 0.05 M H202 and 8mM 2-NPO in acetonitrile at a rate of 1.2 ml min-l. Thus,CL detection method is fully compatible with aqueous separation systems as for instance commonly used in ion chromatography. As

293 such,

the

limits

of

detection

presented

in

Table

111

are

quite

i n t e r e s t i n g , i n d i c a t i n g t h e p o t e n t i a l o f QCL d e t e c t i o n . I t s s e l e c t i v i t y i s s a t i s f a c t o r y as can be seen f r o m F i g . 14 where a chromatogram o f s p i k e d u r i n e i s shown; no p r e t r e a t m e n t o r d i l u t i o n o f t h e samples was necessary t o d e t e c t methimazole and N - a l l y 1 t h i o r u r e a i n t h i s m a t r i x .

time (min)

6 4

1412108

F ig. 14

2.5.3

2

0

Chromatogram o f a u r i n e sample s p i k e d w i t h 100 ng of Na l l y l t h i o u r e a (peak 1) and 25 ng methimazole (peak 2 ) w i t h Quenched CL d e t e c t i o n ; i n j e c t i o n volume 20 pl; chromatographic c o n d i t i o n s : RP-18 column; m o b i l e phase-fqueous ammoniumbenzoate (10 mM, pH 5) a t f l o w - r a t e 0.8 m l min ; reage-qt f l o w , 0.05 M ( r e f . 110). H202 and 8 mM 2NP0 i n a c e t o n i t r i l e a t 1.2 m l min THE QUENCHING ACTION ( r e f . 112)

The r e s u l t s d e s c r i b e d i n s e c t i o n 2.5.2

o b t a i n e d d u r i n g development of

t h e QCL d e t e c t i o n method have t o be d i s c u s s e d w i t h i n t h e framework of t h e pero x y ox alat e mechanism o u t l i n e d i n s e c t i o n 2.5.1. l o w i n g p o i n t s have t o be considered:

Summarizing, t h e f o l -

(1) r e a d i l y o x i d i z a b l e compounds a r e e f f i c i e n t quenchers; (2) t h e quenching c o n s t a n t k depends on t h e c h a r a c t e r o f t h e a r y l g r o u p i n Q t h e o x a l a t e ( f o r 2-NPO k i s about 10 t i m e s h i g h e r t h a n f o r TCPO);

(3) t h e quenching c o n s t a n t k

Q Q

i s independent o f t h e n a t u r e and t h e

concentration o f the fluorophore i n t h e l i q u i d state; (4) f o r immo biliz e d f l u o r o p h o r e , t h e i m m o b i l i z a t i o n procedure a f f e c t s kQ; (5) t h e quenching c o n s t a n t k i s independent of t h e c o n c e n t r a t i o n of H202.

Q

294

Point (5) rules out that the action of Q is connected with the reaction between oxalate and H202 (or OOH-), i.e., step (1) in the reaction scheme. At a first sight, the combination of points (1) and ( 3 ) is rather puzzling; as F and Q have similar properties concerning electrontransfer, why is the quenching effect of Q not attributable to a competition between F and Q? Fortunately, this paradox can be readily solved on the basis of Eq. (11). In presence of Q, the magnitude o f 9 b wi 1 1 be reduced to

where k2c is the rate constant of the reaction

X +Q

3

X-'Q+' 3 non CL decay

However, since kpb and kZc will have a comparable magnitude, also under these conditions kZa is commonly by far the largest term in the denumerator of Eq. (13) and thus effectively qb, = q,,. Summarizing the previous paragraph in terms of Eq. ( l o ) , @la and $b are not affected by [Q]. The same holds for @4a, the quantum yield of fluorescence; this is evident since fluorescence is not quenched under the experimental conditions under consideration. Thus, in terms of the Catherall reaction scheme only one possible explanation remains: Q is i .e., the fraction of XF that particularly influencing efficiency 3a, leads to F*.This is not unreasonable in view of Catherall mechanism; before F* is produced, X-*' undergoes rearrangement while F" is not changing. Collision with an electron donating compound would immediately destroy F" which explains that readily oxidizable compounds are good quenchers, point (1). Furthermore the lifetime of the radical ion pair determines the efficiency of the collisional quenching; this is in-line with point (2) since the nature o f X determines the lifetime of XF. Finally, it will be obvious that this interpretation can also account for the fact that the fluorophore immobilization procedure influences k 9: quenching requires a coll ision between Q and the fluorophore radical cation in the ion pair. In other words, k will be influenced by the Q ability of Q to approach immobilized 3-AF, which of course depends on the detailed structure of the surface layer on the solid substrate.

295

CONCLUDING REMARKS The recent results obtained for peroxyoxalate chemiluminescence as a chromatographic detection method for non-fluorescent analytes indicate a significant progress. The main line is the selective and sensitive detection of hydrogenperoxide produced in a post-column photochemical or immobi 1 ized enzyme reactor. The peroxyoxal ate CL reaction does not only provide favourable detection limits for H202 compared to other CL reactions, but even more important, also a high degree of selectivity. Only a minor amount of compounds do affect the CL efficiency o f the reaction and furthermore, if quenchers are present, the signal remains linearly related to the hydrogenperoxide concentrations. Also post-column systems as IMERS and photochemical reactors producing H202 generally have a good selectivity. This explains why the combination of such reactors and peroxyoxalate CL detection allows the quantitation of analytes in very complex matrices without elaborate sample pretreatment as has been shown for choline and acetyl choline in urine and serum. Thus it is expected that further interesting applications will be realized for instance by applying group-specific enzymes. Of course an important aspect of the applicability o f the described system is the reduction in the complexity of the experimental set-up that has been realized following the solid-state approach. On the one hand this is achieved by the development of the immobilized fluorophore reactor. Since the fluorophore is not consumed during the reaction one of the most efficient fluorophores, i .e., 3-aminofluoranthene, can be used despite o f its toxic properties. Furthermore, the emission of luminescence is localized in the detector cell. On the other hand the application of a solid state oxalate (TCPO) reactor simplifies the system. A disadvantage of such a reactor is its limited lifetime since the oxalate is consumed. Nevertheless, the system is easy to handle and in practice it can be used over 8 hours without any reduction of chromatographic integrity provided that it i s positioned not in line with the analytical column but in a flow addition line. An important positive aspect of solid state reactors is that instability of reagents is of minor importance. This is illustrated by the fact that in methanol/water eluents good results have been obtained. Of course for any particular application the compatability of the CL reaction conditions and the chromatographic separation conditions including the conditions required by the post-column reactor had to be considered. As such it i s interesting that the examples presented indicate 2.6

296 t h a t aqueous eluents do not exclude t h e a p p l i c a b i l i t y o f peroxyoxalate CL. I n f a c t the most i n t e r e s t i n g a p p l i c a t i o n s reported thus f a r are those r e q u i r i n g aqueous mobile phases.

3. 3.1

LIQUID PHASE PHOSPHORESCENCE DETECTION FUNDAMENTAL ASPECTS OF PHOSPHORESCENCE

Phosphorescence was i d e n t i f i e d i n 1944 by Lewis and Kasha as t h e emission o f r a d i a t i o n from t h e lowest t r i p l e t s t a t e o f a molecule t o t h e s i n g l e t ground s t a t e (ref.

113). Fig.

15 shows a s i m p l i f i e d Jablonski

energy diagram o f an analyte molecule (denoted as An).

s2

S1

SO

Fig. 15

Jablonski energy diagram

( n r = non r a d i a t i v e )

-

> -

---- *

absorption ( I )

f 1uorescence ( f ) i n t e r n a l conversion ( i c )

M”G- v i b r a t i o n a l re1a x a t i on ( v r ) HHI-Ht)

w

intersystem crossing ( i s c ) phosphorescence (p)

I r r a d i a t i o n w i t h l i g h t o f a s u i t a b l e wavelength t r a n s f e r s An from i t s e l e c t r o n i c ground s t a t e So t o an e x c i t e d e l e c t r o n i c s t a t e

Sn (Eq. 141,

w h i l e the s i n g l e t spin s t a t e i s conserved, Then a n o n - r a d i a t i v e decay process takes place, eventually ending i n the lowest v i b r a t i o n a l l e v e l of the f i r s t e x c i t e d s i n g l e t s t a t e S1(Eq 15). The e f f i c i e n c y o f t h i s decay process,

comprising

internal

conversion

of

energy

and

vibrational

r e l a x a t i o n v i a c o l l i s i o n s w i t h t h e solvent molecules surrounding An,

is

generally hundred per cent. T r a n s i t i o n from the S1 s t a t e back t o the So s t a t e can occur e i t h e r r a d i a t i o n l e s s v i a i n t e r n a l conversion and subsequent v i b r a t i o n a l r e l a x a t i o n (Eq. 16) o r by emission o f r a d i a t i o n

297

(Eq. 17). Furthermore, bimolecular quenching via reactions with a quencher q is a possible decay pathway (Eq 18). And finally, intersystem crossing from the S1 state to the lowest excited triplet state T1 is another nonradiative decay of the S1 state (Eq. 19). Once the analyte has reached the T1 state transition to the So state can take place via three competitive pathways, i.e., radiationless (Eq. ZO), by emission of radiation (Eq. Zl), called phosphorescence and by a bimolecular quenching reaction with a quenching compound Q (Eq. 22); the capital Q is utilized to emphasize that compounds quenching efficiently a T1 state of the analyte do not necessarily quench the analyte in its S1 state and vice versa.

3

An(S0)

The rate constants knf(S1 + So) and kisc(S1 + TI) usually range from 105-107 and 106-109 s-', respectively (ref. 114). The rate constant of the fluorescence process kf is in the order of 107-109 s (ref. 114). According to the rules of quantum mechanics the phosphorescence process is strictly forbidden as two electronic states of different spin multiplicities are involved. Nevertheless this process can be observed for certain molecules, as a result of spin-orbit coupling. When this mechanism occurs a triplet state is not pure but has some singlet character and a singlet state has some triplet character. The result is a triplet-singlet transition probability unequal zero, which means that the rate constant of the

298

phosphorescence process k is 10-l-1O2 s-'. The efficiencies of fluorP escence (q), intersystem crossing ( qsc) and phosphorescence ( 8 ) can be P readily expressed in terms o f the rate constants of the reactions Eqs. (16) to (22). The fluorescence efficiency is given by

0 =

kf = Qf kisc+kf+knf+xkq[q1 q

It i s equal to the fluorescence quantum yield q,since it gives the probability that An after absorption of radiation emits fluorescence. On the contrary, the quantum yield of phosphorescence is the product of the efficiency of intersystem crossing (the probability for An to reach the T1-state) and the efficiency of phosphorescence, i .e.,

t,

wherein k. 8.

=

lSc

+z

1sc

kisc + kf + knf

kq [ql

9

and

k

It i s appropriate t o invoke lifetimes in Eqs. (23) and (24). The lifetime of fluorescence Tf is equal to the lifetime of An(S), so that

299 This l i f e t i m e should be d i s t i n g u i s h e d from t h e r a d i a t i v e l i f e t i m e o f An(SI)

which i s d e f i n e d as kf-';

o n l y decay p a t h f o r An(S1),

they a r e o n l y equal i f r a d i a t i o n i s t h e

af i s hundred p e r cent.

i n o t h e r words i f

S i m i l a r l y t h e phosphorescence e f f i c i e n c y can be w r i t t e n as

eP = 5 i n which

z

P

t h e l i f e t i m e o f phosphorescence, i s equal t o t h e l i f e t i m e o f P' An(T1). Analogously w i t h t h e fluorescence l i f e t i m e , T < k where k P P P i s t h e r a d i a t i v e l i f e t i m e o f phosphorescence. Generally, b i m o l e c u l a r quenching o f An(S1) i s n e g l i g i b l e , i m p l y i n g t h a t molecular fluorescence i n f l u i d s o l u t i o n s i s a q u i t e common phenomenon. This i s due t o t h e h i g h values o f t h e i n t r a m o l e c u l a r decay r a t e constants so t h a t u s u a l l y

-'

T

-'

despite o f the f a c t that k

can be as h i g h as t h e d i f f u s i o n a l - c o n t r o l l e d q constant. I f both %kq[q] and knf p l a y a m i n o r r o l e f o r t h e decay o f An [S1] combination o f Eqs. (23) and ( 2 5 ) r e v e a l s t h a t

e.1SC = I - @ f This c l e a r l y shows phosphorescence.

the

For phosphorescent

complementary

character

of

fluorescence

eisc must be r e l a t i v e l y high.

compounds

and This

e f f i c i e n c y depends on t h e amount o f s p i n - o r b i t c o u p l i n g which increases w i t h decreasing d i f f e r e n c e between t h e energies o f t h e T1 and So s t a t e s . Moreover i t can be enhanced by t h e i n t r o d u c t i o n o f heavy atoms i n t o t h e phosphorescent

compound

itself,

but

also

i n t o the

s o l v e n t molecules

( i n t e r n a l , r e s p e c t i v e l y , e x t e r n a l heavy atom e f f e c t ) . Besides i t i s noted t h a t no e f f e c t i s encountered i f i n absence o f heavy atoms approximates hundred per cent. I n c o n t r a s t w i t h fluorescence, solutions requires special prerequisite

that

molecular

eisc a l r e a d y

phosphorescence i n f l u i d

experimental circumstances.

Only under t h e

t h e c o m p e t i t i v e i n t r a - and i n t e r m o l e c u l a r d e a c t i v a t i o n

processes o f An(T1) are diminished as much as p o s s i b l e t h e r e i s a substan-

t i a l probability for a radiative transition.

C o n f i n i n g our a t t e n t i o n t o

b i m o l e c u l a r d e a c t i v a t i o n i t i s r e a d i l y seen t h a t commonly

10 -1 -1 a and 0 i s n e g l i g i b l e . I f f o r example k = 10 s-l and kQ = 10 M s P quencher c o n c e n t r a t i o n as low as !l a l r e a d y f u l f i l s Eq. (31). AS a r e s u l t phosphorescence i n homogeneous s o l u t i o n s w i l l be q u i t e e x c e p t i o n a l . Only f o r compounds w i t h a h i g h k value t h e r a d i a t i v e t r a n s i t i o n may be P a b l e t o compete s u c c e s s f u l l y w i t h b i m o l e c u l a r quenching p r o v i d e d t h a t t h e s o l v e n t i s thoroughly p u r i f i e d and deoxygenated. A second requirement i s t h a t t h e i n t r a m o l e c u l a r r a d i a t i o n l e s s decay process does n o t dominate. This e x p l a i n s why f r o z e n glassy samples have been a p p l i e d e x t e n s i v e l y i n phosphorimetry. The r a t e constant k

:

e s p e c i a l l y f o r compounds w i t h f l e nP x i b l e s t r u c t u r e s , can be much h i g h e r i n a f l u i d s o l u t i o n than i n a frozen and k nP P l e a d i n g t o a r e d u c t i o n o f T w h i l e t h e n e t e f f e c t on 0 i s u n p r e d i c t a b l e , P' P see Eq. (26).

solution.

3.2

The i n c o r p o r a t i o n o f heavy

both k

atoms enhances

NEW DEVELOPMENTS I N PHOSPHORIMETRY I n t h e l a s t decade i n t e r e s t i n g new developments i n phosphorimetry have

been r e a l i z e d , d i r e c t e d on t h e p o s s i b i l i t y t o circumvent t h e need of freezing

samples.

solution-sensitized escence.

3.2.1

They and

include

sol i d - s u r f a c e - ,

solution-quenched

mice1 l e - s t a b i 1ized-,

room temperature

phosphor-

SOLID-SURFACE RTP

The observation o f room temperature phosphorescence (RTP) from o r g a n i c molecules adsorbed on a s o l i d m a t r i x has been done several times i n t h e

115-117). The a n a l y t i c a l p o t e n t i a l o f t h i s technique was shown by Schulman and W a l l i n g i n 1982 ( r e f s . 118-119). They s t u d i e d a

sixties (refs.

number o f organic compounds adsorbed on s i l i c a , alumina and f i l t e r paper. Important

research

in this

field

i s carried out

i n the

groups

of

Winefordner and Hurtubise. A considerable number o f s o l i d s u b s t r a t e s has been used t o induce phosphorescence from adsorbed compounds;

t h e most promising seems t o be

120, 121). Beside several q u a l i t i e s of f i l t e r paper 119, 122-133), a l s o s i l i c a g e l ( r e f s . 134, 135), alumina ( r e f . 136), sodium a c e t a t e (refs. 137-141), p o l y a c r y l i c a c i d sodium c h l o r i d e

f i l t e r paper ( r e f s . (refs.

-

30 1 m i x t u r e s ( r e f s . 142-144) and c e l l u l o s e ( r e f . 145) have been t r i e d . RTP has a l s o been observed from compounds adsorbed on streched polymer f i l m s ( r e f . 146).All substrates g i v e r i s e t o a broad band background emission (400-600 nm) which o f t e n i n t e r f e r e s w i t h q u a n t i t a t i v e and q u a l i t a t i v e measurements ( r e f s . 147-148). The i n f l u e n c e s of phosphorophorelsubstrate combination, sample p r e p a r a t i o n ,

amount o f m o i s t u r e and oxygen present

(refs.

149,

150), optimum pH value and presence o f heavy atoms ( r e f s . 151, 155) on RTP have been s t u d i e d e x t e n s i v e l y . Furtheron t h e n a t u r e o f t h e phosphorophores u b s t r a t e i n t e r a c t i o n has been i n v e s t i g a t e d ( r e f s . 156-160). RTP spectra a r e very s i m i l a r i n shape t o LTP (low temperature phosphorescence) s p e c t r a although i n t e n s i t i e s and l i f e t i m e s can be s i g n i f i c a n t l y a f f e c t e d by t h e a c t u a l experimental c o n d i t i o n s .

RTP emission has been

observed from i o n i c organic compounds ( r e f s . 119, 134, 137-140, 1571, nonp o l a r p o l y n u c l e a r aromatic hydrocarbons (PAH's) ( r e f s . 121, 130, 132, 135,

153, 156, 159, 160) and compounds o f pharmaceutical ( r e f s . 127, 133, 153, 161, 162) and b i o l o g i c a l ( r e f s . 120, 128, 154, 161) i n t e r e s t . The r o t a t i n g h o l l o w drum developed by M i l l e r ( r e f s . 163) and t h e r o t a t i n g - m i r r o r phosphorescence as developed by Vo-Dinh e t a l . ( r e f . 164) made i t p o s s i b l e t o scan t h i n l a y e r chromatograms f o r phosphorescent compounds. L l o y d ( r e f . 165) described a f l o w

cell

packed w i t h

paper-derived l i n t t o d e t e c t tographic separation.

a m i x t u r e o f crushed q u a r t z

a n a l y t e s w i t h RTP a f t e r

and

l i q u i d chroma-

Although RTP has a poorer s e n s i t i v i t y than LTP t h e a n a l y t i c a l p r o cedure i s very simple. A d d i t i o n a l l y , a chromatographic s e p a r a t i o n can be performed on t h e s o l i d s u b s t r a t e b e f o r e t h e a n a l y s i s . A considerable g a i n i n s e n s i t i v i t y i n phosphorimetry was obtained by

t h e i n t r o d u c t i o n o f t h e pulsed source-time r e s o l v e d d e t e c t i o n technique ( r e f . 166). With t h i s technique i t i s p o s s i b l e t o analyze m i x t u r e s of phosphorphores. A f t e r a s h o r t e x c i t a t i o n source f l a s h , t h e phosphorescence emission i s measured, a f t e r a c e r t a i n delay t i m e td, d u r i n g a g a t i n g t i m e t

9'

By

this

approach,

the

phosphorescence s i g n a l

can be

temporally

d i s c r i m i n a t e d from r a p i d l y decaying species (e.g., f l u o r e s c e n t i m p u r i t i e s ) and source l i g h t s c a t t e r . Because of t h e i r e x c e l l e n t temporal c h a r a c t e r i s t i c s t h e use o f pulsed l a s e r s i n s t e a d o f t h e n o r m a l l y a p p l i e d pulsed Xenon sources i s a promising development ( r e f s . 167-169). Another instrumental technique t o increase t h e s e l e c t i v i t y o f phosphori m e t r y i s synchronous scanning as proposed by Vo-Dinh

(ref.

170). The

e x c i t a t i o n and emission monochromators o f a phosphorimeter a r e s e t w i t h a constant wavelength difference

of ~h = hem

- hexc and

b o t h monochromators

are scanned a t t h e same r a t e , A phosphorescence peak o n l y occurs when b o t h

302 Axc and Aem correspond simultaneously t o wavelengths a t which e x c i t a t i o n and emission o f a p a r t i c u l a r compound occurs. I n t h i s way, sharper peaks

a r e obtained. Second d e r i v a t i v e phosphorimetry has a l s o been used by Vo-Dinh and workers ( r e f .

CO-

171). By means o f t a k i n g t h e second d e r i v a t i v e o f an RTP

emission spectrum, o v e r l a p between phosphorescence bands c o u l d be reduced and

the

phosphorescence

background

decreased.

Both

the

synchronous

scanning and t h e second d e r i v a t i v e technique have been a p p l i e d t o t h e anal y s i s o f PAH m i x t u r e s ( r e f s . 172, 173). 3.2.2

MICELLE-STABILIZED RTP

The use o f organic media such as m i c e l l a r s o l u t i o n s and c y c l o d e x t r i n s t o induce RTP i n c e r t a i n compounds has been i n t r o d u c e d by C l i n e Love e t al.

(refs.

174, 175). Reviews of t h e a n a l y t i c a l i m p l i c a t i o n o f m i c e l l e

chemistry i n phosphorimetry have appeared r e c e n t l y ( r e f s . 176, 177). The advantages o f t h i s method are: 1) an increase i n s e n s i t i v i t y because t h e organized environment reduces i n t r a m o l e c u l a r processes competing w i t h photoemission;

2) b e t t e r s o l u b i l i t y o f non-polar compounds w i t h respect t o

aqueous s o l u t i o n s ;

3) t h e p o s s i b i l i t y t o b r i n g a n a l y t e s and heavy atoms

together very e f f i c i e n t l y t o c r e a t e a heavy atom e f f e c t . A disadvantage i s t h a t oxygen s t i l l has t o be removed from m i c e l l a r s o l u t i o n s because t h e m i c e l l e s do n o t p r o t e c t t h e phosphorophores a g a i n s t quenching species. The a p p l i c a t i o n o f t h e p r i n c i p l e o f m i c e l l e enhanced phosphorescence as a d e t e c t i o n method i n 1 i q u i d chromatography has been proposed by Weinberger e t a l . ( r e f . 178) i n two ways. F i r s t by u s i n g a m i c e l l a r s o l u t i o n as t h e mobile phase and secondly by post-column a d d i t i o n o f t h e m i c e l l a r s o l u t i o n t o t h e column e f f l u e n t . not

always

easily

U n f o r t u n a t e l y , t h e use o f m i c e l l a r s o l u t i o n s i s

compatible

with

liquid

chromatography

conditions.

DeLuccia and C l i n e Love s t u d i e d t h e s e n s i t i z e d phosphorescence o f b i a c e t y l i n organized media ( r e f s . 179, 180). The p o t e n t i a l o f synchronous scan and second d e r i v a t i v e techniques i n m i c e l l a r RTP was examined by Femia and C l i n e Love ( r e f . 181).

3.2.3

RTP I N NORMAL FLUIDS It i s g e n e r a l l y accepted t h a t i n normal f l u i d s o l u t i o n s , where f a c t o r s

suppressing

the

phosphorescence

diffusion o f

triplet

i n t e n s i t i e s are

too

quenchers low

to

be

a r e commonly used

for

absent,

analytical

purposes. Several fundamental s t u d i e s i n t h i s f i e l d have been p u b l i s h e d d u r i n g t h e l a s t 20 years (refs,

182

-

185). The h i g h e s t phosphorescence

emission i n t e n s i t i e s a t room temperature have

been

reported

by

Almgren

303 ( r e f . 183) f o r b i a c e t y l i n benzene w i t h a phosphorescence quantum y i e l d o f 0.08

and by Parker and Joyce ( r e f .

fluorormethylcyclohexane w i t h a

%

%

184) f o r acetophenone i n per-

o f 0.0581.

For benzophenon, w i t h a

quantum y i e l d o f 1.0 and a t r i p l e t l i f e t i m e o f around 7 msec a t 77 K no RTP could be observed i n hexane; Turro ( r e f . 186) reported a 9.1 x

lom3

i n water, and Joyce ( r e f .

184) a

t

o f 0.097

$

o f only

i n perfluoro-

methylcyclohexane. Turro e t a l . ( r e f . 185) showed t h a t i n a c e t o n i t r i l e , a solvent widely used i n reversed phase l i q u i d chromatography, a phosporescence emission could be achieved f o r 1,4-dibromonaphthalene, quantum y i e l d

i n the order of

Table

V

includes

with a

a number of

exceptional compounds t h a t emit "strong" phosphorescence i n normal f l u i d solutions. From an a n a l y t i c a l p o i n t o f view, t h i s phenomenon can o n l y be u t i l i z e d i n an i n d i r e c t way. The phosphorophore i s present as a s o l u t e and the analyte acts e i t h e r as a s e n s i t i z e r o r as a quencher o f phosphorescence. Both techniques methods i n HPLC.

have been successfully

applied as d e t e c t i o n

Phosphorescence data f o r naphthalene (N) , 1-bromonaphthalene (1-BrN) , 2-bromonaphthalene (2-BrN), 1,4-bromonaphthalene (1,4-BrN), 4,4'-dibromobiphenyl ( 4 , 4 ' - B r B), 2-bromobiphenyl (2-BrB) and 4-bromobiphenyl (4-BrB) i n 2 % e t h y l t e t r a h y d r o f u r a n a t 77 K and i n n-hexane a t room temperature (295 K); from ( r e f . 187).

TABLE V

77 K

295

K

Compound

N

t

T

0.03

2.1

P

t

,msec

T

P

,msec

lo3

1-BrN

0.27

15.0

0.10

1.9

2-BrN

0.38

16.8

0.14

2.8

1, 4-Br2N

0.27

5.3

0.18

1.7

4,4'-Br2B

0.49

12.5

0.08

0.86

0 0.012

-

2-BrB

0.15

-

4-BrB

0.65

22.5

The s t a t e o f t h e a r t i n 1983 has n i c e l y been overviewed by Hurtubise (ref.

166)

and by Vo-Dinh

(ref.

188).

At

t h a t date t h e a n a l y t i c a l

p o t e n t i a l o f s o l u t i o n quenched phosphorescence was n o t y e t known; t h i s method has been introduced q u i t e r e c e n t l y and i t s p o t e n t i a l i s subject of current research.

304 3.2.4

WHY RTP?

The reason t h a t much e f f o r t has been devoted t o t h e extension of phosphorimetry undoubtly i s t h a t phosphorescence, as explained above, can be considered as complementary t o fluorescence.

Furthermore f o r

many

purposes no a d d i t i o n a l instrumentation i s required. O f course, compounds detectable by d i r e c t o r by s e n s i t i z e d phosphorescence are also measurable by U V - V I S absorption spectroscopy. Nevertheless, i n many a p p l i c a t i o n s luminescence measurements are e s s e n t i a l n o t o n l y because they are more selective,

but e s p e c i a l l y because f o r t r a c e analysis o f r e a l samples

frequently lower d e t e c t i o n l i m i t s are required than a t t a i n a b l e by absorpt i o n measurements. Solution quenched phosphorescence

i s applicable t o compounds t h a t

r a p i d l y react w i t h the e x c i t e d phosphorophore, t h e i r own absorption c h a r a c t e r i s t i c s are not relevant. Therefore t h i s technique i s e s p e c i a l l y of i n t e r e s t f o r analytes badly detectable by d i r e c t U V - V I S absorption spectroscopy, as f o r instance inorganic ions ( r e f s . 35-37). 3.3

EXPERIMENTAL ASPECTS

3.3.1 REMOVAL OF OXYGEN Essential f o r RTPL detection i s t h e long t r i p l e t s t a t e l i f e t i m e o f the phosphorophore under consideration. This imp1 i e s t h a t special experimental requirements have t o be met t o make phosphorimetry i n f l u i d s o l u t i o n s a useful a n a l y t i c a l method. To t h i s end t h e s o l u t i o n s have t o be deoxygenated as much as possible since oxygen acts as a very e f f i c i e n t quencher and the solvents have t o be p u r i f i e d c a r e f u l l y t o avoid i m p u r i t y quenching. Moreover, the experimental set-up has t o be cleaned thoroughly and d i r e c t contact between s o l u t i o n s and s y n t h e t i c m a t e r i a l s as t e f l o n should be minimized. I n p r a c t i c e , these conditions can be f u l f i l l e d r e l a t i v e l y easy.

For

a l l types o f experiments, i n batch, i n f l o w i n j e c t i o n analysis and i n liquid chromatography purging o f solvents w i t h n i t r o g e n gas reveals a sufficient

reduction

in

oxygen

a v a i l a b l e n i t r o g e n gas

concentration

(ref.

39).

Commercially

(containing about 5 ppm o f oxygen)

i s passed

through a column f i l l e d w i t h a heterogeneous reduction c a t a l y s t (i.e., pyrophorous copper) and kept a t a constant temperature of 100 OC. I n t h i s way the oxygen content o f t h e N2 gas i s reduced t o l e s s than 0.2 ppm. The p u r i f i e d N2 f l o w i s l e d through a washing b o t t l e and ( i n batch experiments) subsequently through t h e sample s o l u t i o n . A f t e r 5-10 minutes of purging the deoxygenation i s completed and a s t a b l e phosphorescence signal

i s obtained.

During t h e measurements a constant

N2

flow

maintained over t h e sample i n order t o prevent re-entrance o f oxygen.

is

305

In f l o w i n j e c t i o n a n a l y s i s and l i q u i d chromatography t h e deoxygenation of t h e s o l u t i o n s occurs i n t h e e l u e n t v e s s e l.

In F ig. 16 a s p e c i a l l y

c o n s t r u c t e d vessel as d e s c r i b e d i n r e f . 43 i s depict ed; t h e c r u c i a l p o i n t i s t h a t t h e use o f s y n t h e t i c m a t e r i a l s has been avoided so t h a t o n l y glas s , q u a r t z and/or s t a i n l e s s - s t e e l

have been a p p l i e d . A schematic d i a -

gram o f a HPLC system i s g i v e n i n Fi g . eluent

v es s el,

pump,

i n j e c t i o n valve,

17. The i n t e r c o n n e c t i o n s between column and d e t e c t i o n a r e

all

s t a i n l e s s s t e e l c a p i l l a r i e s . The o v e r a l l system i s c l o s e d by l e a d i n g t h e o u t p u t c a p i l l a r y back t o t h e e l u e n t v e s s e l , whereas d u r i n g e l u t i o n ,

the

v a l v e t o waste i s opened t o a v o i d c o n t a m i n a t i o n o f t h e e l u e n t . I n t h i s way t h e e l u e n t can b e used c o n t i n u o u s l y phosphorescence s e m i t i v i t y

F ig. 16

.

o v e r weeks w i t h o u t any l o s s o f

D e t a i l s o f t h e eluent vessel, c o n s i s t i n g o f a 3 1 glass b o t t l e and g l a s s s t o p p e r 855 which f i t s w e l l i n a ground g l a s s j o i n t . The n i t r o g e n gas used f o r deoxygenation t h e e l u e n t e n t e r s v i a a g l a s s Lube ( l ) , w i t h s p e c i a l g l a s s j o i n t (cup s i z e 13/5, R ot u lex ) , v i a an opening (2) and a g l a s s f i l t e r (3); t h e o u t l e t i s v i a a g l a s s t u b e ( 7 ) . The deoxygenated e l u e n t i s pumped i n t o t h e f l o w system v i a a s t a i n l e s s - s t e e l c a p i l l a r y ( 4 ) , which forms one u n i t w i t h a s t a i n l e s s - s t e e l b a l l p a r t (5) f o r t h e o u t l e t o f deoxygenated e l u e n t ; (6) r e p r e s e n t s a c o n s t r u c t i o n i d e n t i c a l t o 4 and 5 f o r t h e i n l e t o f e l u e n t ( r e f . 43).

In o r d e r t o b e s u r e t h a t t h e q u a l i t y o f t h e s o l u t i o n i s c o n s t a n t o v e r a l o n g e r p e r i o d o f t i m e , t h i s has t o be checked. I f b i a c e t y l i s a p p l i e d as phosphorophore, t o d a t e by f a r t h e most i n d i r e c t phosphorescence d e t e c t i o n measurements a r e based on t h i s compound, t h e phosphorescence t o f l u o r escence s i g n a l r a t i o (see F i g . 18) i s an i n d i c a t i o n f o r t h e q u a l i t y o f t h e

306 system regarding O2 and impurities and thus for the sensitivity that can be obtained in the measurements (ref. 38).

Fig. 17

Schematic representation of the dynamic system for liquid chromatography with phosphorescence detection. The broken 1 ines represent stainless-steel capillaries for the nitrogen gas stream that after being washed in a washing bottle containin some eluent is led into the eluent vessel (depicted in Fig. 167 and goes eventually to waste or is used to deoxygenate the sample solution. The solid 1 ines represent stainless-steel capillaries for the eluent stream, connecting eluent vessel, injection valve, analytical column and luminescence detector. The system is closed under normal conditions, to prevent entrance of oxygen or impurities; during the recording of the chromatograms the valve to waste is open. The inverter is not strictly necessary; it serves to record the inverted phosphorescence signal, which is useful in quenched phosphorescence (ref. 35)

I 'I

L .-

U

k-4

Fig. 18

biacetyl nm. The fluorescence

M

307 3.3.2

INSTRUMENTATION

The d e t e c t i o n devices t h a t

can be used f o r t h e measurements

of

phosphorescence i n f l u i d s o l u t i o n s a r e standard commercially a v a i l a b l e fluorescence d e t e c t o r s , though some simp1 i f i c a t i o n s a r e p o s s i b l e .

First,

use can be made o f a l e s s expensive d e t e c t o r , as o n l y a r e s t r i c t e d number of wavelengths are important. I n s e n s i t i z e d phosphorescence t h e choice of t h e e x c i t a t i o n wavelength A exc depends on t h e absorption c h a r a c t e r i s t i c s of t h e analyte, whereas t h e emission wavelength A e m can be fixed; i n quenched RTPL both A e x c and ,,,A, t h e d i f f e r e n c e between A e x c

a r e f i x e d . Secondly, f o r phosphorescence

and Aem i s l a r g e r than f o r fluorescence w i t h

t h e r e s u l t t h a t background r a d i a t i o n due t o s c a t t e r i n g and Raman e f f e c t s i s more r e a d i l y reduced.

For t h a t reason t h e emission g r a t i n g mono-

chromator can be replaced by simple c u t - o f f f i l t e r s , which a r e n o t o n l y l e s s expensive b u t a l s o a l l o w a h i g h e r l i g h t throughput t h u s r e v e a l i n g higher s e n s i t i v i t i e s . As already mentioned a g a i n i n s e n s i t i v i t y can be obtained w i t h t h e pulsed source-time resolved d e t e c t i o n technique. With a s h o r t e x c i t a t i o n source f l a s h t h e molecules a r e e x c i t e d . I n o r d e r t o e l i m i n a t e background due t o r a p i d l y decaying emission ( i m p u r i t y f l u o r escence and s c a t t e r ) t h e phosphorescence s i g n a l i s recorded d u r i n g a t i m e i n t e r v a l t ( g a t i n g time) which s t a r t s a t i m e i n t e r v a l td (delay time) 9 a f t e r t h e f l a s h (see Fig. 19). By choosing t h e s u i t a b l e l i g h t pulse, d e l a y

-

and g a t i n g time,

background luminescence and s c a t t e r i n g o f t h e l i g h t

source can be suppressed considerably. sowcepulse

sowcepulse

4

i

-

id t'3 I

0

Fig. 19

Time

-

E x c i t a t i o n and emission s i g n a l dependence o f t i m e a f t e r source delay f l a s h a t t = 0 o p e r a t i n g i n t h e phosphorescence mode: t gated time; t , gate w i d t h o f d e t e c t o r . The emission s i g n a l By choice o f an apa f t e r tffe source f l a s h by a delay t i m e t p r o p r i a t e value f o r t background emissign caused by s c a t t e r i n g anclSfluorescence i m p g i t i e s , which have l i f e t i m e s s h o r t e r than 10 s , i s n o t detected. This r e s u l t s i n a r e d u c t i o n i n t h e n o i s e o f t h e system.

.

fi

308 For l i q u i d s t a t e phosphorescence w i t h a l i f e t i m e o f 1 additional

instrumental

requirements

needed

for

/.LS

t o 10 ins t h e

time

resolution

measurements can be met more e a s i l y than f o r fluorescence w i t h a l i f t i m e o f 1-100 ns (refs. 40, 4 8 ) . Nowadays luminescence detectors w i t h a pulsed Xe-lamp (pulses i n the 50 Hz range, w i t h a width o f about 50 /.L S ) and a gated photomul t i p 1 i e r are commerci a1 l y avai 1able.

3.4

INDIRECT PHOSPHORESCENCE DETECTION

3.4.1

SENSITIZED PHOSPHORESCENCE

3.4.1.1 INTRODUCTION I n sensitized phosphorescence a f t e r e x c i t i n g a donor molecule energy t r a n s f e r t o an acceptor molecule takes place and t h e phosphorescence of the acceptor i s monitored. I n general t h i s i n d i r e c t method i s applied f o r non-fluorescent

analytes w i t h a high

phosphorescence i n l i q u i d s o l u t i o n s

(e

BiSc

which do n o t emit d i r e c t

i s n e g l i g i b l e ) . This means t h a t

P f o r these compounds the r a d i a t i v e phosphorescence t r a n s i t i o n i s t o o slow t o compete sucessfully w i t h non-radiative

decay.

It

i s the

aim

of

sensitized phosphorescence t o circumvent t h i s decay and t o r e a l i z e energy t r a n s f e r t o an acceptor, a compound w i t h an exceptional high phosphorescence e f f i c i e n c y i n l i q u i d s o l u t i o n s . This method has been applied both i n homogeneous and i n m i c e l l a r s o l u t i o n s . I n most a p p l i c a t i o n s t h e analyte i s the donor compound, however, a l s o an i n t e r e s t i n g example has been r e ported i n which t h e analyte acts as t h e acceptor.

3.4.1.2

THEORETICAL ASPECTS

The s e n s i t i z e d RTPL pathway i s depicted i n t h e energy diagram o f Fig. 20 and the s i m p l i f i e d r e a c t i o n scheme i n Table V I . The donor (0) i s excited by means o f l i g h t absorption and reaches eventually t h e lowest s i n g l e t excited s t a t e denoted as 'D*. Subsequently t h e molecule crosses over t o t h e t r i p l e t s t a t e 3D*. In absence o f t h e acceptor, the f o l l o w i n g step i s t h e r e t u r n t o t h e ground s t a t e . However, i n t h e presence o f an acceptor (A), energy t r a n s f e r t o t h e t r i p l e t s t a t e o f the acceptor may occur, so t h a t an acceptor molecule i n i t s lowest t r i p l e t e x c i t e d s t a t e 3A* i s produced:

3 D*

k

+

'A

-b 'D + 'A*

kt i s the bimolecular r a t e constant o f t h i s energy t r a n s f e r r e a c t i o n , expressed i n M-l s-'. 3A*.

The f i n a l step i s t h e phosphorescence emission from

309 I t i s r e a d i l y seen t h a t t h e i n t e n s i t i e s o f s e n s i t i z e d phosphorescence I (sens) can be expressed as a product o f f o u r independent f a c t o r s ( r e f . P 39), i.e., D D D A A $, (sens) = Iabs (33) ' 'isc ' ' t ' 'P

BiscD

i s t h e intersystem IabsD i s t h e r a t e o f l i g h t a b s o r p t i o n by 0, c r o s s i n g e f f i c i e n c y o f D and thus t h e e f f i c i e n c y o f t r i p l e t f o r m a t i o n o f A t h e donor, i s t h e e f f i c i e n c y o f energy t r a n s f e r from D t o A and 0 P i s t h e phosphorescence e f f i c i e n c y o f A.

aDA

I n absence o f i n n e r f i l t e r e f f e c t s I:bs i s p r o p o r t i o n a l t o t h e concent r a t i o n o f D:

wherein

D t h e i n t e n s i t y o f t h e l i g h t source a t hexc, {xc

absorptivity o f D a t Aexc

and 1 t h e o p t i c a l pathlength.

the molar

I f the analyte

I (sens) i s P l i n e a r l y dependent on t h e a n a l y t e c o n c e n t r a t i o n , as i s obvious from Eqs. (33) and (34). Obviously, t h e c r u c i a l f a c t o r i n s e n s i t i z e d phosphorescence d e t e c t i o n i s BDA.This e f f i c i e n c y depends on t h e r a t e constant kt, t h e acceptor c o n c e n t r a t i o n [A] and t h e l i f e t i m e of 3D* i n absence o f t h e acceptor, denoted as T~D : a c t s as a donor t h e s e n s i t i z e d phosphorescence s i g n a l

To approach a t r a n s f e r o f 100 p e r cent, t h e c o n d i t o n

D

k, [A] >> 1

TO

should be f u l f i l l e d . This means t h a t energy t r a n s f e r should be much f a s t e r than t h e o v e r a l l decay r a t e of t h e t r i p l e t s t a t e o f t h e donor. I n general, provided t h a t t h e energy t r a n s f e r r e a c t i o n i s exothermic, kt approximates t h e d i f f u s i o n a l - c o n t r o l l e d r a t e constant. Nevertheless some c a u t i o n should be taken. The t r a n s f e r r e a c t i o n i n v o l v e s a change o f s p i n s t a t e , which i s

310 only allowed in the electron exchange mechanism, requiring collisional interaction between 3D* and 1A. In this mechanism kt is proportional to the overlap between the normalized emission spectrum of D, i.e., 3D* +'0, and the normalized singlet-triplet absorption spectrum of A, 'A+ 3A*. In other words the intensities of the emission and the absorption band play no rule but nevertheless their shapes are important (ref.189). The problem is that S j T absorption spectra are not simply accessible. SO it might be possible that for exothermic energy transfer kt is much smaller than expected due to unfavourable overlap. Unidirectional energy transfer from 3D* to 1A takes place if the energy of 3D* is at least 20 J/mol higher than the energy of 3A* (ref. 190); if the difference is smaller back transfer of energy plays a role (ref. 191).

SO

I Fig. 20

Decay pathways in sensitizedlphosphorescence. kD kD and ko are the rate constants in s of the intramo1ecfiiarn6eactivhlFon of the donor via fluorescence, intynal cony,ersion and intersystem crossing, respectively. k and k are the phosphotescence Arate constants of the d&or and tfe acceptor, while k and k are the overall rate constqts of intra- and intermof&ular #&radiative deactivation in s of the T state of the donor and acceptor. k [A] is_Fhe apparent rate cbnstant of the energy transfer reactidn in s (ref. 39).

Furtheron it is obvious from Eq. (36) that for a high sensitized RTPL signal the concentration of the acceptor should be taken as high as possible. It should be realized, however, that direct excitation of the acceptor must be avoided, because it would lead to a background phosphorescence signal. Fortunately, biacetyl, a compound with a high 8 in a P variety of liquid solutions, has very low molar absorption coefficients over a wide range of excitation wavelegths. Nevertheless, its concentration must not be chosen too high, i.e., about 10-4M. Finally, it is emphasized that obviously the phosporescence efficiency of the acceptor is unfavourable influenced by impurity quenching. This

means

that

quenchers,

including

oxygen

should

be

removed

from

the

s o l u t i o n , as much as p o s s i b l e . TABLE V I

Reaction scheme o f s e n s i t i z e d RTPL by t r i p l e t - t r i p l e t energy transfer.

1. E x i t a t i o n o f t h e donor (D) ' 0 t hyex D +'D*

2. Intersystem c r o s s i n g t o t h e t r i p l e t s t a t e 'D*

3D*

3. Energy t r a n s f e r t o t h e acceptor (A) kt 3D* t 'A 0' '

+

3A*

4. Phosphorescence o f t h e acceptor 3A* + l A

3.4.1.3

+ h A

yP

APPLICATIONS

Various organic compounds w i t h low n a t i v e fluorescence can be sens i t i v e l y detected by s e n s i t i z e d RTPL; e s p e c i a l l y t h o s e compounds t h a t undergo an e f f i c i e n t n o n - r a d i a t i v e decay v i a i n t e r s y s t e m crossing. I n p r i n c i p l e s e n s i t i z e d RTPL i s g e n e r a l l y a p p l i c a b l e t o a l l compounds meeting t h e f o l l o w i n g two requirements: t h e i r T1 s t a t e energy must be h i g h e r than t h a t o f t h e acceptor and they must show phosphorescence i n r i g i d s o l u t i o n s a t 77 K, s i n c e t h e main c o n d i t i o n t o be f u l f i l l e d i s an e f f i c i e n t t r i p l e t formation. As s e n s i t i z e d RTPL i s an i n d i r e c t emission method, o n l y t h e e x c i t a t i o n p r o p e r t i e s o f t h e a n a l y t e ( a c t i n g as donor) p l a y a r o l e . A l l compounds a r e detected a t a s i n g l e wavelength, i.e., t h e emission wavel e n g t h o f t h e acceptor which i s r e l a t i v e l y long. M o n i t o r i n g o f t h e same phosphorescence s i g n a l f o r a l l a n a l y t e s imp1 i e s t h a t u s u a l l y chromatographic separations a r e necessary if complex matrices, c o n t a i n i n g s e v e r a l s e n s i t i z i n g and even quenching compounds have t o be analyzed. The commonly used phosphorophore i n HPLC i s b i a c e t y l ,

a compound w i t h favourable

phosphorescence e f f i c i e n c i e s i n v a r i o u s s o l v e n t s , used as e l u e n t i n HPLC. M i n order The c o n c e n t r a t i o n o f b i a c e t y l i s u s u a l l y n o t h i g h e r than t o avoid background emission due t o d i r e c t phosphorescence.

312 Compounds measured by s e n s i t i z e d phosphorescence are t h e we1 1 known polychlorinated biphenyls (PCBs) and naphthalenes (PCNs) (ref. detection l i m i t s are i n t h e low nanogram region.

43).

The

Furtheron, ortho-sub-

s t i t u t e d PCBs do n o t produce any RTPL signal. Therefore t h e combination of sensitized RTPL and UV absorption d e t e c t i o n provides perspectives f o r t h e identification

of

complex

PCB mixtures

as

frequently

enountered

in

i n d u s t r i a l samples. In Fig. 21 t h e UV and s e n s i t i z e d RTPL detected chromatograms o f Aroclor 1221 are depicted. A l l PCBs present in the sample are detected i n t h e UV-chromatogram. As expected t h e s e n s i t i z e d RTPL chromatogram i s more simple since the peaks o f t h e o r t h o - s u b s t i t u t e d biphenyls are missing. O f course t h e e x c i t a t i o n wavelength i s an a d d i t i o n a l select i v e l y parameter i n s e n s i t i z e d RTPL.

6

Fig. 21

4

2

0

tlme (min)

6

4

2

0

Reversed-phase chromatograms o f Aroclor 1221 obtained w i t h UV detection ( A= 224 nm), concentration 50 ppm and s e n s i t i z e d RTPL detection ( h = 265 nm, h = 520 nm), concentration 10 ppm. Peaks: 1 = biflgnyl I 2 = 2-ch%robiphenylI 3 = 4-chlorobiphenyl , 4 2,2'-dichlorobiphenyl and 6 = 4,4'-dichlorobiphenyl ( r e f . 43).

A number o f PCNs e x h i b i t n a t i v e fluorescence.

I n v e s t i g a t i o n s on mix-

tures o f PCNs reveal f o r most of t h e compounds a s e n s i t i z e d RTPL d e t e c t i o n

l i m i t comparable w i t h those obtained w i t h fluorescence d e t e c t i o n ( r e f . 191). Furtheron t h e l i n e a r range i s the same as u s u a l l y found by f l u o r escence, i.e.,

3 t o 4 decades. However, t h e t r i - and t e t r a - s u b s t i t u t e d

compounds have a d e v i a t i n g behaviour.

T h e i r t r i p l e t s t a t e energies are

lower than f o r b i a c e t y l , so t h a t these compounds do n o t a c t as s e n s i t i z e r s

313 but as quenchers o f the b i a c e t y l phosphorescence ( r e f . 43).

This aspect

w i l l be discussed thoroughly i n t h e next section.

Another i n t e r e s t i n g group o f compounds which can be detected w i t h RTPL are t h e parent compound dibenzofuran and i t s c h l o r i n a t e d d e r i v a t i v e s (refs. 32, 192). These compounds, known as environmetal hazards have a high t o x i c i t y depending on t h e numbers and p o s i t i o n s o f t h e c h l o r o subs t i t u e n t s . Sensitized RTPL d e t e c t i o n seems appropriate as t h e t r i p l e t formation e f f i c i e n c i e s are considerable and t h e t r i p l e t energies are h i g h enough t o guarantee an e f f i c i e n t energy t r a n s f e r

t o biacetyl.

As

an

example the chromatogram o f a m i x t u r e o f dibenzofuran (DBF) and 2,8-dichlorodibenzofuran (2,8-C12DBF)

i s given i n Fig. 22. Various c h l o r i n a t e d

dibenzofurans also e x h i b i t a n a t i v e fluorescence, a property t h a t has n o t received a l o t o f a t t e n t i o n i n t h e l i t e r a t u r e . Hence, i t i s i n t e r e s t i n g t o compare the s e n s i t i v i t i e s o f HPLC combined w i t h fluorescence, s e n s i t i z e d phosphorescence and UV-absorbance detection. I t became c l e a r t h a t t h e r e i s a need t o increase the s e n s i t i v i t y o f the HPLC method by applying a preconcentration procedure.

Furtheron,

the data revealed t h a t t h e f l u o r -

escence detection o f c h l o r i n a t e d dibenzofurans i s more s e n s i t i v e than t h e sensitized RTPL mode, i f a g r a t i n g instrument i s used, whereas w i t h a f i l t e r instrument the reverse i s t r u e ( r e f . 192). This may be a t t r i b u t e d t o t h e f a c t t h a t i n s e n s i t i z e d RTPL s c a t t e r i n g background plays a l e s s important r o l e . 3

2

I

1

10

8

6

4

2

0

t (min 1

Fig. 22

L i q u i d chromatogram o f a mixture o f dibenzofuran (DBF) and 2,8C1 DBF, separated on LiChrosotb RP-18 column, l e n g t h 11 cm; f l o w ra?e 1 ml/min; eluent; 10- M b i a c e t y l i n a c e t o n i t r i l e / w a t e r 83.7/ 16.3 ( v / v ) ; s e n s i t i z e d RTPL d e t e c t i o n ( hexc = 290 nm, h = 522 nm); 1 = solvent peak; 2 = DBF (34 ng); 3 = 2,8-C12DBF ($7 ng) ; 4 = unknown ( r e f . 39).

Another i n t e r e s t i n g a p p l i c a t i o n of s e n s i t i z e d RTPL i s t h e a n a l y s i s of biacetyl

i t s e l f . This

determination i s

important because

of

t h e great

314 influence o f b i a c e t y l on the f l a v o u r o f beer,

wine and several d a i r y

products. I n these samples b i a c e t y l concentrations i n t h e order of 1 ppb t o 1 ppm are relevant. Though b i a c e t y l i s a good phosphorophore in f l u i d solutions, i t i s very i n e f f i c i e n t l y e x c i t e d , because o f i t s extremely low molar a b s o r p t i v i t y (above 220 nm E < 20 M - l cm").

This implies t h a t t h e

detection l i m i t s f o r b i a c e t y l i n beer, achieved by UV/Vis absorption and by d i r e c t phosphorescence measurements are both very unfavourable. However, s e n s i t i z e d phosphorescence d e t e c t i o n can be invoked t o improve the e x c i t a t i o n o f b i a c e t y l i n an i n d i r e c t way ( r e f . 40, 41). Now b i a c e t y l , a c t i n g as acceptor i s the analyte and a s u i t a b l e donor compound has t o be found. The appropriate donor should have a high a b s o r p t i v i t y , i t should guarantee a high energy t r a n s f e r e f f i c i e n c y t o b i a c e t y l , i t should be nonphosphorescent i t s e l f , but i t s t r i p l e t s t a t e TI

should have a l a r g e l i f e -

time. For extremely low b i a c e t y l concentrations i t i s obvious t h a t

so t h a t the e f f i c i e n c y o f energy t r a n s f e r (see Eq. 22) can be approximated as

620

-

560

SO0

440

emirslon wsvelength(nm)

Fig. 23

Sensitized phosphorescence emission spectrum o f a mixture o f NDSA and b i a c e t y l i n a deoxygenated s o l u t i o n o f w a t e r / a c e t o n i t r i l e 70/ 30 ( v / v ) a t room temperature. The background fluorescence o f NDSA i s recorded without deoxygenation; keXC = 302 nm (a favourable wavelength t o e x c i t e NDSA) ( r e f . 35).

315 Under these c o n d i t i o n s t h e s e n s i t i z e d phosphorescence s i g n a l I (sens) i s P l i n e a r l y dependent on t h e b i a c e t y l c o n c e n t r a t i o n . A number o f p o t e n t i a l donors has been tested;

1,5-naphthalenedisulfonic a c i d disodium s a l t

(NDSA) i s an a p p r o p r i a t e donor. This compound has a f a v o u r a b l e s o l u b i l i t y i n p o l a r s o l v e n t m i x t u r e s and g i v e s no r e t e n t i o n on a reversed-phase column.

Fig.

23

shows

the

emission

spectrum

of

a

deoxygenated

4 w a t e r / a c e t o n i t r i l e 70/30 (v/v) s o l u t i o n o f 1.5 x 10- M NDSA and about 5 x M b i a c e t y l . A chromatogram f o r a beer sample c o n t a i n i n g 14 ppb b i a c e t y l i s given i n Fig. 24; t h e corresponding l i m i t o f d e t e c t i o n i s 0.5 PPb

. biacety I

I

1

6

4

1

2

0

t-tR(min)

F i g . 24

HPLC chromatogram o f a beer sample c o n t a i n i n g 14 ppb b i a c e t y l w i t h s e n s i t i z e d phosphorescence d e t e c t i o n ( h = 310 nm; h = 516 nm). Column: 25 cm x 4.6 mm I . D . 5 p f#kerisorl);l elu&!t: w a t e r l a c e t o n i t r i l e , 70/30 (v/v), pH = 6.5, w i t h 2 x 10 M NDSA. 1 = s o l v e n t f r o n t ; 2 = unknown ( r e f . 35).

QUENCHED PHOSPHORESCENCE INTRODUCTION

3.4.2 3.4.2.1

Another i n d i r e c t d e t e c t i o n method based on a dynamic p r i n c i p l e concerns t h e quenched phosphorescence i n 1 i q u i d s o l u t i o n s

at

room tem-

perature. Analytes a b l e t o quench t h e phosphorescence o f a compound, f o r example b i a c e t y l , cause a decrease of t h e monitored s i g n a l . As t h e amount o f quenching i s determined by t h e r a t e constant o f t h e r e a c t i o n , between a n a l y t e and e x c i t e d phosphorophore, t h e quenched phosphorescence method has an i n h e r e n t s e l e c t i v i t y . I t i s e s p e c i a l l y u s e f u l when one o r a few analytes have t o be determined i n presence o f a number o f o t h e r compounds, f o r example i n complex m a t r i c e s such as body f l u i d s o r environmental samples

.

THEORETICAL ASPECTS

3.4.2.2

The r e a c t i o n scheme showing t h e r e a c t i o n pathways f o r quenched RTPL i s given

in

Table V I I . For convenience

i t i s based on b i a c e t y l (B) as t h e

316 phosphorophore. F i r s t b i a c e t y l i s e x c i t e d by means o f l i g h t absorption d i r e c t l y i n an excited s i n g l e t s t a t e . Subsequently b i a c e t y l f a l l s down t o i t s t r i p l e t s t a t e T1. I n absence o f a quencher, t h e f o l l o w i n g step i s t h e r e t u r n t o t h e ground s t a t e by emission of phosphorescence. I n presence of a quencher which i s able t o r e a c t r a p i d l y w i t h 3B* t h e l i f e t i m e of b i acetyl i n t h e t r i p l e t s t a t e w i l l decrease, r e s u l t i n g i n a reduction of t h e monitored b i a c e t y l phosphorescence signal ( r e f . 42). TABLE V I I

Reaction scheme f o r quenched RTPL o f b i a c e t y l by tri p l e t - t r i p l e t energy t r a n s f e r . 1. E x c i t a t i o n of b i a c e t y l (B) 'B

+ hyB exc

+ 'B*

2. Intersystem crossing t o t h e t r i p l e t s t a t e 'B*

43B*

3. Phosphorescence o f b i a c e t y l 3B*+1B

+ hy

P

4 . Quenching o f b i a c e t y l phosphorescence by a quencher Q (the analyte)

I n absence o f quencher, t h e phosphorescence

1, o f b i a c e t y l can be ex-

pressed as

I =IB 0

eB eB

(39)

abs isc P

B where Iabs i s the r a t e o f l i g h t absorption by b i a c e t y l and QSc

represent

the

respective

efficiencies

of

intersystem

and e B P crossing and

phosphorescence (since o n l y the phosphorescence p r o p e r t i e s o f B p l a y a r o l e , i n the f o l l o w i n g t h e superscript e f f i c i e n c y i s given by

B w i l l be deleted).

The l a s t

317 i s t h e t r i p l e t s t a t e l i f e t i m e o f b i a c e t y l ( 5 ) . I f , however, a quencher i s present t h e l i f e t i m e w i l l be reduced, and t h e f o l l o w i n g r e l a t i o n holds

wherin

T~

kQ i s t h e b i m o l e c u l a r r a t e constant o f t h e quenching r e a c t i o n (M

-1 -1

s ) and

[ Q ] t h e c o n c e n t r a t i o n o f t h e quencher (M). As a r e s u l t t h e phosphorescence s i g n a l i n t e n s i t y w i l l decrease from 1, t o I . For t h e r a t i o Io/I a relation s i m i l a r t o t h e well-known Stern-Volmer derived

equation i n fluorescence can be

As t h e q u o t i e n t kQTo/Io f o r a chosen phosphorophore and a n a l y t e ( t h e

quencher) has a constant value i t i s c l e a r from Eq. (42) t h a t I - l - I o - ' d e pends l i n e a r l y from

[Q]. This i m p l i e s t h a t p l o t t i n g o f 1 - l versus [Q]

d e l i v e r s a s t r a i g h t l i n e w i t h a slope p r o p o r t i o n a l t o k

Q

and an i n t e r c e p t

equal t o t h e i n v e r t e d i n t e n s i t y o f t h e unquenched s i g n a l . I n o r d e r t o r e a l i z e a l i n e a r response between t h e c o n c e n t r a t i o n and t h e s i g n a l h e i g h t i n several experiments an e l e c t r o n i c s i g n a l i n v e r t e r has been i n t r o d u c e d between t h e photomul t i p 1 i e r t u b e and t h e recorder. Furtheron i t i s obvious from Eq.

(42)

t h a t t h e s e n s i t i v i t y of t h e

quenched RTPL method f o r a p a r t i c u l a r phosphorophore and quencher i s determined by t h e quenching r a t e constant k I f t h e l i f e t i m e T~ i s known t h e r a t e constant k

Q'

Q

can be c a l c u l a t e d from t h e s l o p e o f t h e Stern-Volmer

p l o t . I n s t a t i c experiments, t h e value o f

T~

i n d i f f e r e n t s o l v e n t s can be

r e a d i l y estimated i n an i n d i r e c t way from t h e r a t i o o f t h e phosphorescence and fluorscence i n t e n s i t i e s of b i a c e t y l , measured a t t h e i r r e s p e c t i v e maxima. O f course, i n time-resolved experiments

T~

can be d e r i v e d d i r e c t l y

from t h e decay o f t h e phosphorescence s i g n a l . Subsequently an e s t i m a t i o n o f t h e l i m i t s o f d e t e c t i o n f o r t h e a n a l y t e s can be obtained, s i n c e i t can be d e r i v e d t h a t f o r batch experiments approximately holds

3 18

10 (M1 s-1) 1.o.d. (M)=-

(43)

k

Q

This implies t h a t the quenched RTPL method would be o f i n t e r e s t f o r analytes quenching the b i a c e t y l phosphorescence w i t h r a t e constants lo7 t o

l o 9 M-l s-', depending on the a v a i l a b i l i t y o f a l t e r n a t i v e d e t e c t i o n methods. O f course the s e n s i t i v i t y depends a l s o on T~ and thus on t h e amount o f oxygen and i m p u r i t i e s present i n the s o l u t i o n . Furthermore, t h e achievable d e t e c t i o n l i m i t s improve w i t h increasing I,,

since a t higher I.

the signal t o noise r a t i o becomes more favourably. The quenched RTPL i s also a s e l e c t i v e method, because o n l y analytes w i t h s u f f i c i e n t l y high k

Q

values can be detected. Otherwise t h i s i s t h e o n l y requirement f o r d e t e c t -

a b i l i t y . The absorption spectra o f the analytes do not p l a y a r o l e . The same holds f o r the quenching mechanism. Quenched RTPL i s not l i m i t e d t o analytes w i t h t r i p l e t states l y i n g lower than t h e T1 s t a t e o f t h e phosphorophore. Other mechanisms, such as e l e c t r o n t r a n s f e r and proton a b s t r a c t i o n may be also involved i n t h e quenched RTPL. Especially t h e former seems t o be important.

This explains why f o r instance some inorganic ions are

s e n s i t i v e l y detectable w i t h quenched RTPL. F i n a l l y i t i s emphasized t h a t a l s o fluorescence quenching has been introduced

as

a detection

method

in

HPLC,

i.e.,

by

the

group

of

Winefordner i n 1981 (refs. 193, 194). However, i t should be r e a l i z e d t h a t t h i s detection p r i n c i p l e

i s essentially

phosphorescence method described

different

from the

i n t h e present chapter.

quenched

Fluorescence

quenching i s based on the s t a t i c quenching as a r e s u l t o f the i n t e r a c t i o n between the fluorophore and the analyte i n t h e i r e l e c t r o n i c ground s t a t e s and/or on the absorption o f fluorescence r a d i a t i o n by t h e analyte. Dynamic quenching o f fluorescence i s n o t e f f e c t i v e since the l i f e t i m e o f t h e lowest excited s i n g l e t states i s t o o short. 3.4.2.3

APPLICATIONS

The p o t e n t i a l o f quenched RTPL f o r t h e d e t e c t i o n of a l a r g e number o f both organic and inorganic compounds has been examined. The relevance o f quenched RTPL i s obvious since i t i s applicable f o r t h e s e n s i t i v e detection o f various groups of compounds w i t h otherwise poor d e t e c t i o n properties. The chromophoric p r o p e r t i e s of

t h e analyte do n o t play a r o l e i n

quenched RTPL; the analyte needs n o t t o be excited. Also t h e mechanism o f the quenching process i s n o t important. Besides energy t r a n s f e r reactions, a l s o electron t r a n s f e r reactions o r even proton a b s t r a c t i o n reactions may

319 be o p e r a t i v e . The o n l y c o n d i t i o n i s t h a t t h e quencher r e a c t s r a p i d l y w i t h biacetyl

i n t h e t r i p l e t state;

t h e r a t e constant

k

determines

Q

the

s e n s i t i v i t y o f quenched RTPL f o r a p a r t i c u l a r a n a l y t e . G e n e r a l l y , quenched RTPL d e t e c t i o n i s r e l e v a n t f o r a n a l y t e s w i t h r a t e c o n s t a n t s r a n g i n g f r o m

lo7 t o

lo9

M-l s - l ( r e f . 42).

A screening t e s t o f various types o f

compounds has re v e a l e d t h a t quenched RTPL can be used among o t h e r s f o r h i g h e r c h l o r i n a t e d naphthalenes, a r o m a t i c and a l i p h a t i c amines, s u l p h u r c o n t a i n i n g o r g a n i c s such as t h i o u r e a s and p henot hioazines and s e v e r a l i n o r g a n i c i o n s (e.g., NO2-, SCN-, S2032- , Sn2+ , Cr042-) and complexes (e.g., some a n t i t u m o r agents based on P t ( I 1 ) ) . It i s emphasized t h a t quenched RTPL has a l s o an i n h e r e n t s e l e c t i v i t y . T h i s i s i n t e r e s t i n g as i n t h e f i e l d o f i o n chromatography m o b i l e phases w i t h h i g h i o n i c s t r e n g t h s are

used.

In

ge n e r a l

only

those

ions

are

observed

by

quenched

phosphorescence t h a t e i t h e r have a l o w l y i n g t r i p l e t s t a t e energy o r a l o w o x i d a t i o n p o t e n t i a l . The o t h e r s can b e used as e l u e n t c o n s t i t u e n t s w i t h o u t i n d u c i n g any background s i g n a l . I n t h e f o r e g o i n g s e c t i o n we have seen t h a t PCNs can be d e t e c t e d by s e n s i t i z e d b i a c e t y l phosphorescence though t h e s e n s i t i v i t y f o r some h i g h e r c h l o r i n a t e d PCNs i s low. T h i s has t o be a s c r i b e d t o t h e t r i p l e t energy values of t h es e compounds, which a r e about t h e same as, o r even l o w e r , than t h e t r i p l e t energy o f b i a c e t y l . I n t h i s case a reversed energy t r a n s f e r r e a c t i o n f rom b i a c e t y l t o t h e PCN must b e t aken i n t o account, which leads t o a decrease i n t h e s e n s i t i z e d (and t h e d i r e c t ) RTPL s i g n a l o f b i a c e t y l . A re v erse d energy t r a n s f e r i m p l i e s t h a t i n a d d i t i o n t o UV abs o r p t i o n and s e n s i t i z e d phosphorescence a l s o quenched phosphorescence can be invoked f o r t h e analyses and i d e n t i f i c a t i o n o f i n d u s t r i a l PCN m i x t u r e s ( r e f . 43). I t i s emphasized t h a t t h e a p p l i c a t i o n o f quenched RTPL i n s t e a d s e n s i t i z e d RTPL r e q u i r e s o n l y an a d j u st ment o f t h e e x c i t a t i o n wavelength. I n F i g . 25 t h e r e v e r s e d phase chromatograms o f Halowax 1099 of

obta ined w i t h represented.

UV,

sensitized

RTPL

and

quenched

RTPL

detection

are

The s e n s i t i v i t y o f t h e quenched phosphorescence d e t e c t i o n i s demonstrated f o r thiourea derivatives.

I n Fi g . 26 a chromatogram d e t e c t e d by

quenched RTPL i s d e p i c t e d , u s i n g a s i g n a l i n v e r t e r ( r e f .

42).

Quenched

RTPL has been used t o d e t e c t some P t ( I 1 ) c o o r d i n a t i o n complexes, which a r e w e l l known agents w i t h a n t i t u m o r a c t i v i t y ( r e f . 46). The k values f o r t h e

8

‘ 8

quenching o f b i a c e t y l phosphorescence a r e 7 x 10 and 4 x 10 M-l s - l f o r CDOP ( c i s p l a t i n ) and CBDCA ( c a r b o p l a t i n ) , r e s p e c t i v e l y ; t h e s e values g i v e r i s e t o i n t e r e s t i n g LOD values. On t h e c o n t r a r y CHIP, a P t ( I V ) d e r i v a t i v e , does h a r d l y show any quenching (k c 106M- 1s- 1 ). T his r e s u l t may be an i n dication

that

the

quenching

Q

is

based on

electron

t r a n s f e r from t h e

320 platinum i o n t o t h e e x c i t e d b i a c e t y l molecule. I n Fig. 27 a chromatogram of a standard s o l u t i o n o f CDDP and CBDCA i s presented using quenched phosphorescence as d e t e c t i o n (

= 415

m, , ,A

= 520 nm). The l i m i t s of

detection c a l c u l a t e d from t h i s chromatogram are 3.0 x

M f o r CDDP and

M f o r CBDCA. These data compare favourably w i t h LODs obtained

3.3 x

v i a other detection methods (see Table V I I I ) . Experiments on u r i n e .and plasma samples showed i n t e r f e r i n g compounds c o - e l u t i n g w i t h the platinum species, so t h a t a clean-up procedure i s necessary ( r e f . 47). I n Fig. 28 a chromatogram f o r a blank plasma sample and a sample spiked w i t h CDDP i s depicted, obtained a f t e r applying such a sample hand1 i n g procedure; t h e chromatography i s based on a solvent generated anion exchanger system. I t

i s c l e a r t h a t i n t h i s way CDDP can be determined q u a n t i t a t i v e l y i n plasma. The same holds f o r CDDP i n u r i n e . The s e n s i t i v i t y o f t h e method i s s u f f i c i e n t f o r the monitoring o f therapeutic CDDP l e v e l s i n c l i n i c a l samples.

l

uv

SENSITIZED

9

14

18

16

14

12

l0

B

6

4

2

0

18

16

14

10 B timelrninl

12

6

L 4

2

0

OUE NCHED

Fig. 25

Reversed-phase chromatograms o f Halowax 1099 obtained w i t h UV detection ( A = 233 nm; 0.32 a.u.f.s.), s e n s i t i z e d RTPL detection ( hexc = 300 nm; hem = 520 nm) and quenched RTPL detection ( = 415 nm; A = 520 nm); i n j e c t e d amount o f sample i n a l l t%romatograms: qm @. The corresponding peaks i n t h e three chromatograms are i n d i c a t e d by the f i g s . 1 t o 15; i t i s obvious t h a t combination o f these t h r e e d e t e c t i o n modes i s h e l p f u l f o r i d e n t i f i c a t i o n purposes ( r e f . 43).

F i n a l l y , we w i l l consider t h e determination o f chromate as an example of quenched RTPL d e t e c t i o n i n ion-chromatography ( r e f . 48). The t r i v a l e n t c a t i o n Cr(II1) which i s an e s s e n t i a l t r a c e element t o man, hardly does quench b i a c e t y l phosphorescence.

The chromate ion Cr(V1) , an enzymatic

poison leading t o h e p a t i t i c and renal damage by exposure, can be determined s e l e c t i v e l y a f t e r separation on a paired-ion reversed phase HPLC system w i t h quenched phosphorescence detection.

In these measurements

321

the possibility o f time resolution in quenched phosphorescence detection has been invoked. The signal reduction caused by the quencher Cr04'depends on both the delay time td and the gating time t experimental pa9' rameters in a pulsed source detection system. This leads to a modified Stern-Volmer equation :

wherein the subscription "pulse" is used to denote that the pulsed phosphorescence detection mode i s applied. This equation applies provided that t is chosen long enough to guarantee that after t seconds less than 9 9 1 per cent o f the signal is recorded. It is obvious that is not linearly dependent on [Q]. The influence o f the exponential term decreases if td is shortened. Experiments on the biacetyl system have shown that for t is 0.9-1.0 ms the signal to noise ratio is the most 9 favourable and that the optimum value for td is about 0.10 ms. In Fig. 29 a chromatogram for a 1.7 x lom6 M chromate standard solution is given. The detection limit achieved is 1.4 x M, corresponding to 16 ppb, which underlines the relevance of the method as the maximum concentration allowed in drinking water is 50 ppb. The linearity of the method is three orders of magnitude provided that an electronic signal inverter is applied. From the data in Table I X it is clear that the sensitivity of the quenched RTPL method compares reasonably with other detection methods.

1

-

10 0 6

Fig. 26

4 2

6

tR(min)

Quenched -8TPL chromatogram o f some thiourea derivatives; eluent: 1.0 x 10 M biacetyl in water; column RP-18, 12 cm, d = 5 K flow rate = 1 ml/min; observed t (= t ) for NaN02 = 48 s; 1 = thourea (41 ng), 2 = thhhydanqoine (63 ng), 3 = ethylenethiourea (55 ng), 4 = methimazole (62 ng) (ref. 42).

322 TABLE V I I I

Comparison o f 1.o.d. values (ng/ml) f o r CDDP and CBDCA derived from l i q u i d chromatography w i t h d i f f e r e n t d e t e c t i o n methods.

Detection method

uv UV a f t e r d e r i v a t i z a t i o n

E l ectrochemi c i a1

CDDP

CBDCA

20,000 40 15

Chl o r i de-assi sted electrochemical

50

QRTPL

90

Ref.

20,000 1200 15 A f t e r precolumn d e r i v a t i z a t ion

195 195 196

Not measured

197

122

46

TABLE I X Comparison o f 1.o.d. values (ng/ml) f o r chromate obtained w i t h d i f f e r e n t HPLC methods. Detection method

1 .o.d.

Ref.

E 1e c t rochemi ca 1

4

198

100

199

7

200

16

48

(as diethyldithiocarbamate complex) Col orimet r ic (as l15-diphenylcarbazide comp 1ex) D i r e c t current plasma (DCP) emission spectroscopy Quenched RTPL

The chromatogram o f tapwater (Fig. 30) and surface water samples spiked w i t h chromate show the appl i c a b i 1 it y o f t h e quenched phosphorescence method f o r r e a l samples.

323

Fig 27

Quenched phosphvescence chromatogram of CDDP (6.1 . M) and CBDCA (8.3 , 10- M) in 0.15 M aqueous NaC1. 1 = chloride, 2 = unknown. Column: ODS Hypersil column 5 f l (10 cm x 4.6 mm) prepared with hexadecyltrimethylammonium bromide (HTAB). Mobile pha5e: water/methano$, 99/1 ( v / v ) , pH 5.0 (citrate buffer), 2 x 10 M HTAB and 10- M biacetyl. Aex = 415 nm, A, = 520 nm (ref. 46).

Fig. 28

Chromatogram of a blank N a s m a sample (A), and o f a plasma sample spiked with 5 x 10 M CDDP ( B ) . For chromatographic conditions, see Fig. 27 (ref. 47).

324

t

h

3 L

v

x

e C

01

r

c_

z 01

In J!

8

a 0

8 P

3

-

6

4

0

2

tR(min)

Fig. 29

Chromatogram o f a standard s o l u t i o n o f chromate (1.7 x 10'6M). 1 and 2 are quenching i m p u r i t i e s , 3 = chromate. Chromatographic cond i t i o n s : column 12 cm x 4.6 mm I.D. 5 IQI ODS Spherjsorb; mobile phase: w a t e r / a c e t o n i t r i l q 95/5 ( v / v ) , 2 x3 10- M phosphate b u f f e r , pH = 7.1,_{ x 10- M TBACl and 5 x 10- M b i a c e t y l ; flow Time-resolved phosphorescence detection: td r a t e = 1.9 m l min = 0.01 msec, t = 1.00 msec, lexc = 400 nm (broad band f i l t e r ) , A = 515 nm (Pef. 48). em

.

r 4

0

4

2

0

Chromatogram o f a blank tat water sample (A) and o f a t a p water sample spiked w i t h 1 x 10 M chromate ( B ) . The p o s i t i v e peaks are a t t r i b u t e d t o i m p u r i t i e s present i n t h e samples e x h i b i t i n g n a t i v e fluorescence o r s e n s i t i z e d phosphorescence o f b i a c e t y l . The negative peaks are caused by quenching compounds. Chromatographic and detection c o n d i t i o n s as i n Fig. 29 ( r e f . 48).

Fig. 30

3.5

2

ALTERNATIVE PHOSPHOROPHORES/LUMINOPHORES Though t h e i n d i r e c t phosphorescence detection,

quenched,

both s e n s i t i z e d and

has proved t o g i v e promising r e s u l t s i n HPLC experiments w i t h

325 biacetyl

as

phosphorophore

this

combination

has

some

inherent

dis-

advantages. F i r s t b i a c e t y l i s consumed d u r i n g t h e experiments as i t i s p a r t o f t h e e l u e n t . Secondly i t i s n o t s t a b l e a t pH values h i g h e r than 7. T h i r d l y , t h e measurements have t o be done under oxygen f r e e c o n d i t i o n s . The f i r s t drawback can be r a i s e d by u s i n g immobilized phosphorophores ( r e f . 49). I n o r d e r t o e l i m i n a t e t h e oxygen removal s t e p r a r e e a r t h metal ions, which a r e w e l l known luminophores have been s t u d i e d i n quenched luminescence d e t e c t i o n ( r e f . 50). I n t h i s case t h e t r a n s i t i o n r e s p o n s i b l e f o r t h e l i g h t emission does n o t i n v o l v e a t r i p l e t and s i n g l e t s t a t e . Therefore t h e more general term luminophore i s used. I n t h i s s e c t i o n a t t e n t i o n w i l l be p a i d t o two a l t e r n a t i v e phosphorophores/luminophores as d e t e c t i o n method i n f l o w systems as HPLC. 3.5.1

IMMOBILIZED PHOSPHOROPHORES There i s a number o f advantages i n employing immobilized phosphoro-

phores i n i n d i r e c t phosphorescence d e t e c t i o n . F i r s t t h e r e i s an e x t e n s i o n of

the solvent

compatability,

so t h a t

a p o l a r phosphorophores may be

combined w i t h aqueous mobile phases and reversed phase HPLC columns. Furtheron, because o f i m m o b i l i z a t i o n no losses o f phosphorophore occur and f i n a l l y , t h e l o c a l i z a t i o n o f t h e phosphorophore i n t h e c e l l makes i t poss i b l e t o apply o t h e r s i m p l e r oxygen removal procedures. Disadvantages a r e t h e background s c a t t e r i n g caused by t h e support and t h e l i m i t e d t r a n s parancy

of

the

cell

which

limits

the

excitation

range

of

the

phosphorophore down t o about 340 nm. F o r t u n a t e l y , t h e e f f e c t o f s c a t t e r i n g can be e f f i c i e n t l y

suppressed by u s i n g a pulsed source-time

resolved

luminescence d e t e c t i o n system. Results obtained w i t h 1-bromonaphthalene,

a compound known from i t s

RTPL p r o p e r t i e s (see Table V) have been reported. The phosphorophore i s c o v a l e n t l y bonded t o a s o l i d support v i a a spacer t o c r e a t e pseudo solution

conditions.

The

immobilization

reaction

(ref.

49)

is

schematically represented i n F i g . 31. The synthesized batches, which a r e very s t a b l e ,

c o u l d be used under f l o w c o n d i t i o n s a t l e a s t d u r i n g s i x

months. They c o u l d be a p p l i e d w i t h o u t problems between pH 2 and 8. The immobi 1 iz a t ion h a r d l y affects t h e spectroscopic emission c h a r a c t e r i s t i c s o f t h e phosphorophore. Although e x c i t a t i o n o f 1-bromonaphthalene i t s e l f i s almost impossible a t wavelengths h i g h e r than 340 nm, i n t r o d u c t i o n of t h e

C=O group on t h e 4- p o s i t i o n r e s u l t s i n a low a b s o r p t i v i t y i n t h e 340-375 nm region. The p o s s i b i l i t i e s o f t h i s system f o r quenched phosphorescence M n i t r i t e solution d e t e c t i o n have been t e s t e d by i n j e c t i o n of a 7 x on a HPLC column. The a n a l y t i c a l d a t a a r e comparable w i t h those o b t a i n e d M. w i t h t h e homogeneous b i a c e t y l system; t h e d e t e c t i o n l i m i t i s 2 x

326 For I - a lower signal was observed. According to the Stern-Volmer relation (see Eq. 42) the sensitivity of the quenched phosphorescence detector is determined by the product kQTo. As the phosphorescence 1 ifetime T~ depends mainly on the phosphorophore used ( T~ is about 0.9 ms) the lower signal for I- has to be described to the relatively low quenching constant k (k = 1 x 10'M-I s-l for I- and kQ = Q Q 3.3 x lo9 M - l s-l for NO2-). From the data represented, the potential of an immobilized phosphorophore in quenching phosphorescence detection i s clear. Until now an example, wherein the immobilized phosphorophore acts in the sensitized mode has not been reported. IJr

Fig. 31

a. Synthesis of 10-(4-bromo-l-naphthoyl) decylamine, intermediate I. b. Immobilization of intermediate I to silanized silica gel or CPG (ref. 49).

3.5.2 RARE EARTH METAL IONS AS LUMINOPHORES The spectroscopic properties of lanthanide ions have been subject of research since many years, but the application of the luminescence of these ions as detection method in liquid chromatography (LC) is still rather restricted. Wenzel at al., have developed an LC method for the determination of polynucleotides and nucleic acids with xanthine, guanine and thioridine unities (ref. 201). After chromatographic separation, a postcolumn complexation of the analytes with Eu(II1) or Tb(II1) leads to products that can be sensitively detected by lanthanide luminescence. The use of Eu(II1) and Tb(II1) as luminophores in LC in a sensitized detection mode has been described by Dibella et al. (ref. 202). The analytes

327 ( o r g a n i c compounds) a r e e x c i t e d by UV r a d i a t i o n , energy t r a n s f e r f rom t h e t r i p l e t s t a t e o f t h e a n a l y t e t o t h e l a n t h a n i d e i o n occurs and luminescence of t h e l a n t h a n i d e i s detected. I n t h i s s e t -up energy t r a n s f e r i s t h e c r u c i a l s t e p s t a r t i n g a t t h e donor i n t h e t r i p l e t s t a t e , which e x p l a i n s t h a t deoxygenation o f t h e s o l u t i o n i s r e q u i r e d . Baumann e t a l . have examined t h e use o f r a r e metal i o n s E u ( I I 1 ) and T b ( I I 1 ) as luminophores f o r quenched d e t e c t i o n i n LC ( r e f . 50).

from (for b idden) t r a n s i t i o n s between l e v e l s b e l o n g i n g t o t h e 4 fn e l e c t r o n c o n f i g u r a t i o n . As t h e 4 f e l e c t r o n s a r e s h i e l d e d by 5 s and 5 p e l e c t r o n s , t h e observed t r a n s i t i o n s a r e v e r y sharp, even f o r s p e c t r a o f l i q u i d sol u t i o n s . N o n - r a d i a t i v e decay u s u a l l y competes s t r o n g l y w i t h r a d i a t i v e r e l a x a t i o n , e s p e c i a l l y i n H20. The

long

living

lanthanide

luminescence

(9.1

ms)

arises

From a p r a c t i c a l p o i n t o f v i e w compared t o b i a c e t y l phosphorescence, E u ( I I 1 ) and T b ( I I 1 ) luminescence have t h e s t r o n g advantage t h a t oxygen quenching h a r d l y p l a y s any r o l e .

T h i s i m p l i e s t h a t j u s t a common HPLC

set- up i s c omp at i b l e w i t h t h e quenched l a n t h a n i d e luminescence d e t e c t i o n mode;

no s p e c i a l e x p e r i m e n t a l r e q u i r e m e n t s need t o be f u l f i l l e d . On t h e

o t h e r hand t h e f a c t t h a t oxygen does n o t i nduce s i g n i f i c a n t quenching i m p l i e s t h a t , i m comparison w i t h b i a c e t y l , o t h e r quenching mechanisms may be o p e r a t i v e so t h a t another s e l e c t i v i t y s h o u l d be expected. i n s e c t i o n 3.4.2.2 the detection l i m i t s achiev able i n dy n a m i c a l l y quenched luminescence d e t e c t i o n depend on t h e As has been p o i n t e d out

n o i s e on t h e luminescence s i g n a l and t h u s on t h e i n t e n s i t y o f t h i s s i g n a l . U n f o r t u n a t e l y f o r E u ( I I 1 ) i n p r a c t i c e a r e l a t i v e l y l o w luminescence l e v e l i s reached because o f i t s l o w a b s o r p t i v i t y ( € < 10 M - l cm-l and t h e ab-

s o r p t i o n peaks a r e narrow). For T b ( I I 1 ) t h e s i t u a t i o n i s more f avourable: f 75d 1 l y i n g a t 220 nm i s a l l o w e d ( € 300 M - l

t h e t r a n s i t i o n 4 f8 - 4

so t h a t r a t h e r e f f i c i e n t e x c i t a t i o n i s p o s s i b l e p r o v i d e d t h a t a lamp i s a v a i l a b l e w i t h a f a v o u r a b l e o u t p u t a t t h a t wavelength.

cm-l

i n a r a t h e r broad band)

P r e l i m i n a r y HPLC experiments have been r e p o r t e d f o r n i t r i t e s o l u t i o n s . The EuC13 c o n t a i n i n g m o b i l e w a t e r phase was pumped d i r e c t l y t hrough a L i Chrosorb RP-18 a n a l y t i c a l column. The t i m e r e s o l v e d measurement ( t d = 30 ps, tg = 2.0 ms) r e s u l t s i n a l i m i t o f d e t e c t i o n o f o n l y 2 x

M. As

expected f o r T b ( I I 1 ) t h e 1.o.d. i s more f a v o u r a ble. I t was found t o be 5 x 10- 8M, a q u i t e p ro m i s i n g r e s u l t ( r e f . 50). O b v ious ly t h e r e a r e s t i l l some aspects t o be examined. F i r s t of a l l , i t has been shown t h a t t h e E u ( I I 1 )

about

a

factor

o f 30

luminescence l i f e t i m e i s increased

upon g o i n g f r o m H20 t o D20; hence such an e l u e n t

328

might be very interesting assuming that microbore HPLC i s feasible. Secondly, the choice of buffers, frequently necessary in ion-chromatographic separation seems to be limited; some ions form stable complexes with the lanthanides thus reducing dynamic quenching of luminescence. Thirdly, the excitation efficiency of the lanthanides might be improved via cornplexation; on the other hand the ligands used in these complexes should not inhibit dynamic quenching processes. Sumnarizing this section, we do expect in the near future the publication of some interesting applications of lanthanide luminescence detection in LC especially for the measurements of inorganic ions. CONCLUDING REMARKS From the results described above it is obvious that phosphorescence detection has its particular applicability field in HPLC, despite of the fact that the phenomenon of phosphorescence in normal fluid solutions is quite exceptional. It is important to emphasize that the experimental requirements to be met are not difficult both to realize and to maintain and that the same equipment as applied in fluorescence detection can be utilized. The main requirement is to apply a closed chromatographic system based on stainless steel capillaries and to deoxygenate continuously the eluent by leading nitrogen gas through the eluent vessel. In sensitized phosphorescence detection, generally the analyte acts as a donor. Hence this detection mode is only appropriate for compounds with an absorption spectrum in the near UV and visible wavelength region. Therefore the results should be considered as additive to those obtained with absorption detection. As has been shown above, the combination of both detection techniques, and if relevant further combintion with fluorescence and / or quenched phosphorescence detection, is quite adequate for the analysis and identification of complex mixtures. The applicability of sensitized phosphorescence wherein the analyte is the acceptor, as for instance utilized for the determination of biacetyl in beer samples, i s limited since it requires a high phosphorescence efficiency for the analyte itself. The quenched phosphorescence detection mode presumably has a wider applicability range than the sensitized one. This indirect method is essentially different from indirect UV absorption- and fluorescence detection known in i o n chromatoraphy which both are based on displacement effects. In phosphorescence detection, a dynamic quenching effect i s monitored, i .e. , a decrease of the phosphorescence efficiency induced by the analytes. This explains the impressive detection limits obtained for 3.6

efficient

quenching

analytes.

Especially

in

i o n chromatography

this

d e t e c t i o n t ec hniqu e has a good p o t e n t i a l . Ne v ert heles s phosphorescence d e t e c t i o n i s n o t w i d e l y a p p l i e d y e t . Obviously, t h e need t o deoxygenate t h e e l u e n t i s a s e r i o u s hindrance f o r i t s genera l acceptance. describ ed i n s e c t i o n 3.5, and r a r e e a r t h

From t h i s p o i n t o f view t h e new developments namely t h e use o f immobilized phosphorophores

luminophores,

o f f e r new p e r s p e c t i v e s .

p r e l i m i n a r y r e s u l t s o b t a i n e d f o r E u ( I I I ) and T b ( I I 1 )

Especially

the

luminophores a r e

q u i t e pro mis in g s i n c e i n t h e s e systems oxygen removal i s n o t necessary i n t h e quenched mode.

It

i s emphasized t h a t ,

compared

to

fluorescence

d e t e c t i o n , phosphorescence has t h e g r e a t advantage t h a t background r a d i a t i o n due t o f l u o r e s c e n t i m p u r i t i e s and t o s c a t t e r i n g can be e l i m i n a t e d q u i t e e a s i l y w i t h o u t t h e need of expensive equipment; a p u l s e d Xe-lamp i n strument s u f f i c e s .

REFERENCES 1 H. Lingeman, W.J.M. Underberg, A. Takedate and H u l s h o f f , J. L i q . Chromatogr., 8, 789 (1985). 2 R.W. F r e i , N.H. V e l t h o r s t and C. G o o i j e r , Pure 81 Appl. Chem., 57, 483 (1985). 3 U.A.Th. Brinkman, G.J. de Jong and C. G o o i j e r , Pure 8I Appl. Chem., 59, 625 (1987). 4 U.A.Th. Brinkman, Chromatographia, 24, 190 (1987). 5 P. L i n d r o t h and K. Mopper, Anal. Chem., 51, 1667 (1979). 6 J. D'Souza and R.J. O g i l v i e , J. Chromatogr., 232, 212 (1982). 7 P. Kucera and H. Umagat, J. Chromatogr., 255, 563 (1983). 8 P. Boehlen and H. M e l l e t , Anal. Biochem., 94, 313 (1979). 9 J.F. Lawrence and R.W. F r e i , Chemical D e r i v a t i z a t i o n i n L i q u i d Chromatography, E l s e v i e r , Amsterdam (1976). 10 J. F. Lawrence, Prechromatographic Chemical D e r i v a t i z a t i o n i n L i q u i d Chromatography, i n Chemical D e r i v a t i z a t i o n i n A n a l y t i c a l Chemistry, Vol. 2: S epar a t i o n and Continuous Flow Techniques, R.W. F r e i and J.F. Lawrence Eds., Plenum Press, New York (1982). 11 R.W. F r e i and A.H.M.T. Scholten, J. Chromatogr. Sci., 17, 152 (1979). 12 R.W. F r e i , J. Chromatogr., 165, 75 (1979). 13 J.T. Stewart, Trends Anal. Chem., 1, 170 (1982) 14 G. Schwedt and E. Reh, Chromatographia, 14, 249 (1981). 15 G. Schwedt and E. Reh, Chromatographia, 14, 317 (1981). 16 E.C. Toren Jr. and D.N. Vacik, Anal. Chim. Acta, 152, 1 (1983). 17 R.W. F r e i , Chromatographia, 15, 161 (1982). 18 J.F. Lawrence, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr., 185, 473 (1979). 19 R.J. Reddingius, G.J. de Jon , U.A.Th. Brinkman and R.W. F r e i , J.Chromatogr., 205, 77 (19817. 20 J.H. Wolf, C. de R u i t e r , U.A.Th. Brinkman and R.W. F r e i , J. Pharm. Biomed. Anal., 4, 523 (1986). 21 C. de R u i t e r , J. Hefkens, U.A.Th. Brinkman, R.W. F r e i , M. Evers, E. M a t t h i j s and J.A. M e i j e r , I n t . J. E n v i r o n . Anal. Chem., 31, 325 (19871. 22 F. Smedes, J.C. Kraak, C.E. Werkhoven-Goewie, U.A.Th. Brinkman and R.W. F r e i , J. Chromatogr., 247, 123 (1982). 23 A.H.M.T. Scholten, U.A.Th. Brinkman and R.W. F r e i , Anal. Chim. Acta, 114, 137 (1980).

330 24 25 26 27 28 29 30 31 32 33 34 35

M. U i h l e i n and E. Schwab, Chromatographia, 15, 140 (1982). M.L. Grayeski, Anal. Chem., 59, 1243A (1987). P. van Zoonen, Thesis, Free U n i v e r s i t y Amsterdam, 1987. W.R. S e i t z and M.P. Neary i n Contemporary Topics i n A n a l y t i c a l Chemistry, Volume I . D.M. Hercules (Ed.), Plenum, New York (1977). G. Mellbin, J. Liq. Chromatogr., 6, 1603 (1983). S. Kobayashi and K. Imai, Anal. Chem., 52, 424 (1980). P. van Zoonen, D.A. Kamminga, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chim. Acta, 167, 249 (1985). P. van Zoonen, D.A. Kamminga, C. Gooijer, N.H. V e l t h o r s t , R.W.Frei and G.Gubitz,Anal .Chim.Acta, 174, 151 (1985) G. Gubitz, P. van Zoonen, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chem., 57, 2071 (1985). P. van Zoonen, I . de Herder, C. Gooijer, N.H. V e l t h o r s t , R.W. F r e i , E. Kuntzberg and G. Gubitz, Anal. Letters., 19, 1949 (1986). P. van Zoonen, C. Gooijer, N.H. Velthorst, R.W. F r e i , J. H. Wolf, J. G e r r i t s and F. Flengte, J. Pharm. & Biomed. Analysis, 5 , 485 (1987). C. Gooijer, R.A. Baumann and N.H. Velthorst, Progr. Anal. Spectrosc.,

10, 573 (1987). 36 37 38 39 40

J.J. Donkerbroek, Thesis, Free U n i v e r s i t y Amsterdam, 1983. R.A. Baumann, Thesis, Free U n i v e r s i t y Amsterdam, 1987. J.J. Donkerbroek, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chem., 54, 891 (1982). J.J. Donkerbroek, N.J.R. van Eikema Homes, C. Gooijer, N.H. Velthorst and R.W. F r e i , Chromatographia, 15, 218 (1982). R.A. Baumann, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chem.,

57, 1815 (1985). 41 42 43 44

R.A. Baumann, C. Gooijer, N.H. Velthorst. R.W. F r e i , J. S t r a t i n g , L.C. Verhagen and R.C. Veldhuyzen-Doorduin, I n t e r n . J. Environ. Anal. Chem., 25, 195 (1986). J.J. Donkerbroek, A.C. Veltkamp, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chem., 5 5 , 1886 (1983). J.J. Donkerbroek, N.J.R. van Eikema Hommes, C. Gooijer, N.H. Velthorst and R.W. F r e i , J. Chromatogr., 255, 581 (1983). C. Gooijer, N.H. Velthorst and R.W. F r e i , Trends i n Anal. Chem., 3,

259 (1984). 45

52 53 54 55

C. Gooijer, P.R. Markies, J.J. Donkerbroek, N. H. V e l t h o r s t and R.W. Frei, J. Chromatogr., 289, 347 (1984). C. Gooijer, A.C. Veltkamp, R.A. Baumann, N.H. V e l t h o r s t , R.W. F r e i and W.J.H. van der Vijgh, J. Chromatogr., 312, 337 (1984). R.A. Baumann, C. Gooijer, N.H. V e l t h o r s t , R.W. F r e i , I . K l e i n and W.J.F. van der Vijgh, J . Pharm. Biomed. Anal., 5, 2, 165 (1987). R.A. Baumann, M. Schreurs, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Can. J. Chem., 65, 965 (1987). R.A. Baumann, C. Gooijer, N.H. V e l t h o r s t , R.W. F r e i , I . Aichinger and G. Gubitz, Anal. Chem., 60, 1237 (1988). R.A. Baumann, D.A. Kamminga, H. Derlagen, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , J. Chromatogr., 439, 165 (1988). W.P. Bostick, M.S. Denton and S.R. Dinsmore i n Bioluminescence and Chemiluminescence, Instruments and Applications, K. van D i j k e (Ed.), CRC, Boca Raton, (1985) Vol. 11. J.W. B i r k s and M.C. Kuge, Anal. Chem., 52, 897 (1980). B. Shoemaker and J.W. B i r k s , J. Chromatogr., 209, 251 (1981). H. Schaper, J. Electroanal. Chem., 129, 335 (1981). R.C. Massey, C. Crews, D.J. McWeeny and M.E. Knowles, J. Chromatogr.,

56 57 58 59

W.C. Yu and E.U. Golf, Anal. Chem., 55, 29 (1983). A.V.Hartkopf and R. Delumya, Anal. Lett., 7, 79 (1974). M.P. Neary, W.R. Seitz and D.M. Hercules, Anal. Lett., 7, 583 (1974). R. Delumya and A.V. Hartkopf, Anal. Chem., 18, 1402 (1976).

46 47 48 49 50 51

236, 527 (1982).

33 1 60 61 62

63 64 65 66 67 68 69 70 71 72 73 74 75 76

77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101

J.L. Burguera, M. Burguera and A. Townshend, Anal. Chim. Acta, 127, 199 (1981). T.Hara, M. Toriyama and K. Tsukagoshi, B u l l . Chem. SOC. Japan, 56, 1382 (1983). T. Hara, M. Toriyama and T. Ebuchi, B u l l . Chem. SOC. Japan, 58, 109 (1985). A. MacOonald and T.A. Nieman, Anal. Chem. 57, 936 (1985). J.P. Auses, S.L. Cook and J.T. Maloy. Anal. Chem., 47, 244 (1975). O.T. B o s t i c k and D.M. Hercules, Anal. Chem., 47, 447 (1975). C. Ridder, E.H. Hansen and J. Ruzicka, Anal. L e t t . , 15, 1751 (1982). C.A. Koerner and T.A. Nieman, Anal. Chem., 58, 116 (1986). R. Fagerstroem, P. Seppanen and J. Janne, C l i n . Chim. Acta, 143, 45 .. (1984). U. Bachrach and Y.M. Plesser, Anal. Biochem., 152, 423 (1986) M.P. Neary, R.W. S e i t z and D.M. Hercules, Anal. L e t t . , 7, 583 (1974) M.L. Gandelman and J.W. B i r k s , J. Chromatogr., 242, 21 (1982) T. Kawasaki, M. Maeda and A. T s u j i , J. Chromatogr., 328, 121 1985). R.L. Veazey and T.A. Nieman, J. Chromatogr., 200, 153 (1980). R.A. Steen and T.A. Nieman, Anal. Chim. Acta, 155, 123 (1983) M. Maeda and A. Tsu.ji. J. ChromatoQr., 352, 213 (1986). C.L.R. C a t h e r a l l , T;F; Palmer and E.Bi Cundall, J . Chem. SOC. Far ad ay Trans. 2, 80, 823 (1984); 80, 837 (1984). F.J. Alvarez, N. J. Parekh, B. Matuszweski, R.S. Givens, T. Higuchi and R.L. Showen, J. Am. Chem. SOC. , 108, 6435 (1986). P. van Zoonen, D.A. Kamminga, C. G o o i j e r , N.H. V e l t h o r s t , R.W. F r e i and G. Gubitz, Anal. Chem., 58 1245 (1986). P. van Zoonen, H. Bock, C. G o o i j e r , N.H. V e l t h o r s t and R.W. F r e i , Anal. Chim. Acta, 200, 131 (1987). K.W. Sigvardson and J.W. B i r k s , Anal. Chem., 55, 432 (1983). K.W. Sigvardson, J.M. Kennish and J.W. B i r k s , Anal. Chem., 56 1096 (1984). M.L. Grayeski and A.J. Weber, Anal. L e t t . , 17, 1539 (1984). K. Miyaguchi, K. Honda and K. Imai, J. Chromatogr., 316, 501 (1984). K. Miyaguchi, K. Honda and K. Imai, J. Chromatogr., 303, 173 (1984). K. Miyaguchi, K. Honda, T. Toyo'oka and K. Imai, J. Chromatogr., 352, 255 (1986). G. M e l l b i n and B.E.F. Smith, J. Chromatogr., 312, 203 (1984). T. K o z i o l , M.L. Grayeski and R. Weinberger, J. Chromatogr., 317, 355 (1984). K. Kobayashi, J. Sekino, K. Honda and K. Imai, Anal. Biochem., 112, 99 (1981). K. Honda, K. Miyaguchi and K. Imai, Anal. Chim. Acta, 177, 111 (1985). 6. Mann and M.L. Grayeski, J. Chromatogr., 386, 149 (1987). L.O. Bowers, Anal. Chem., 58, 513A (1986). L. Dalgaard, Trends i n Anal. Chem., 5 , 185 (1986). K. Honda, K. Miyaguchi, H. N i s h i r o , H. Tanaka, T. Yao and K. Imai, Anal. Biochem., 153, 50 (1986). F.M. Freeman and W. R. S e i t z , Anal. Chem., 50, 1242 (1978). K. Hool and T.A. Nieman, Anal. Chem., 59, 869 (1987). R.O. Lippman, Anal. Chim. Acta, 116, 181 (1980). B.R. B r a n c h i n i , F.G. S a l i t u r o , J.D. Hermes and N.G. Post, Biochem. Biohpys. Res. Comm., 97, 334 (1980). J.R. Poulson, J.W. B i r k s , G. Gubitz, P. van Zoonen, C. G o o i j e r , N.H. V e l t h o r s t and R.W. F r e i , J. Chromatogr., 360, 371 (1986). J.R. Poulson, J.W. B i r k s , P. van Zoonen, C. G o o i j e r , N.H. V e l t h o r s t and R.W. F r e i , Chromatographia, 21, 587 (1986). C.E. Goewie, M.W.F. Nielen, R.W. F r e i and U.A.Th. Brinkman, J .Chromatogr., 301, 325 (1984). H. Jansen, R.W. F r e i , U.A. Th. Brinkman, R.S. Deelder and R.P.J. S n e l l i n g s , J. Chromatogr., 325, 255 (1985).

.

332 102 103 104 105 106 107 108 109 110 111

112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139

D. P i l o s o f and T.A. Nieman, Anal. Chem., 54, 1698 (1982). G. Scott, W.R. Seitz and J. Ambrose, Anal. Chim. Acta, 115, 221 (1980). J.L. Meek and C. Eva, J. Chromatogr., 317, 343 (1984). H.H. Weetall i n "Immobilized Enzymes" (U. Moshbach Ed.) , Academic Press, New York (1976). G. Damsma, B.H.C. Westerink and A.S. Horn, J. o f Neurochem., 45, 1649 (1985) P. van Zoonen, D.A. Kamminga, C. Gooijer, N.H. V e l t h o r s t and R.W. Frei, J. Liq. Chromatogr. 10, 819 (1987). K. Honda, J. Sekino and K. Imai, Anal. Chem., 53, 940 (1983). P. van Zoonen, D.A. Kamminga, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chem., 58, 1245 (1986). P. van Zoonen, H. Bock, C. Gooijer, N.H. V e l t h o r s t and R.W. F r e i , Anal. Chim. Acta, 200, 131 (1987). K. Honda, K. Miyaguchi and K. Imai, Anal. Chim. Acta, 177, 103 (1985). C. Gooijer, P. van Zoonen, N.H. V e l t h o r s t and R.W. F r e i , J. of Bioluminescence and Chemiluminescence, i n press (1989). G.N. Lewis and M. Kasha, J. Am. Chem. SOC., 66, 2100 (1944). N.J. Turro "Modern Molecular Photochemistry", The Benjamin/Cummings Publishing Co., CAI (1978). J.B.F. Lloyd and J.N. M i l l e r , Talanta, 26, 180 (1980). J.D. Brown, J. Chem. SOC., (1958). M. Roth, J. Chromatogr., 30, 276 (1967). E.M. Schulman and C. Walling, Science, 178, 53 (1972). E.M. Schulman and C. Walling, J. Phys. Chem., 77, 902 (1973). R.T. Parker, R.S. Freedlander and R.B. Dunlap, Anal. Chim. Acta, 119, 189 (1980). R.T. 'Parker, R.S. Freedlander and R.B. Dunlap, Anal. Chim. Acta, 120, 1 (1980). S.L. Wellons, R.A. Paynter and J.D. Winefordner, Spectrochim. Acta, 30A, 2133 (1974). T. Vo-Dinh, E. Lue Yen and J. D. Winefordner, Talanta, 24, 146 f 1977). jiS. McHale and P.G. Seybold, J. Chem. Ed., 53, 654 (1976). E. Lue Yen Bower and J.D. Winefordner, Anal. Chim. Acta, 101, 319 (1978) G.J. Niday and P.G. Seybold, Anal. Chem., 50, 1577 (1978). C.G. de Lima and E.M. de Nicola, Anal. Chem. , 50, 1658 (1978). R.T. Parker, R.S. Freedlander, E.M. Schulman and R.B. Dunlap, Anal. Chem., 51. 1921 119791. M.L. Meyers, R. Zellmer, R.K. Sorrel1 and P.G. Seybold, J. Lum., 20, 215 (1979). C.D. Ford and R.J Hurtubise, Anal. Chem., 51, 659 (1979). D.L. McAleese and R.B. Dunlap, Anal. Chem., 56, 836 (1984). D.W. Abbott and T Vo-Dinh, Anal. Chem., 57, 41 (1985). M.M. Andino and J D. Winefordner, J. Pharm. Biomed. Anal., 4, 317 (1986). C.D. Ford and R.J Hurtubise, Anal. Chem. 50, 610 (1978). C.D. Ford and R.J Hurtubise. Anal. Chem.. 52. 656 (1980). W. Homer, G. Krabichler, S.'Uhl and D. Oelkrug, J.'Phys; Chem., 87, 4872 (1983). R.M.A. von Wandruszka and R.J. Hurtubise, Anal. Chem., 48, 1784 f.~~ 1976). ~, R.M.A. von Wandruszka and R.J. Hurtubise, Anal. Chem., 49, 2164 (1977). R.M.A. von Wandruszka and R.J. Hurtubise, Anal. Chim. Acta, 93, 331 (1977).

.

.

333 140 141 142 143 144

S.M. Ramasamy and R.J. H u r t u b i s e , Anal. Chem., 59 432 (1987). V.P. S e n t h i l n a t h a n and R.J. H u r t u b i s e , Anal. Chem., 57, 1227 (1985). R.A. D a l t e r i o and R.J. H u r t u b i s e , Anal. Chem., 54, 224 (1982). R.A. D a l t e r i o and R.J. H u r t u b i s e , Anal. Chem. 5 5 , 1084 (1983).

147

V.P. S ent h iln a t h a n . S.M. Ramasamv and R.J. Hurt ubise, Anal. Chim. Acta, 157, 203 (1984). R.P. Bateh and J.D. Winefordner, Ta l a n t a , 29, 713 (1982). J.J. Dekkers. G.Ph. Hoornwea. K.J. T e r o s t r a . C. MacLean and N.H. V e l t h o r s t , Chem. Phys., 34,-253 (1978): J.L. Ward, E. Lue Yen-Bower and J.D. Winefordner, T alant a, 28, 119

148 149 150 151 152 153

D.L. McAleese and R.B. Dunlap, Anal. Chem., 56, 600 (1984). E.M. Schulman and R.T. P a r k e r , J. Phys. Chem., 81, 1932 (1977). D.L. McAleese and R.B. Dunlap, Anal. Chim. Acta, 162, 431 (1984). P.G. Seybold and W. White, Anal. Chem., 47, 1199 (1975). W. White and P.G. Seybold, J. Phys. Chem., 81, 2035 (1977). E. Lue Yen-Bower and J.D. Winefordner, Anal. Chim. Act a, 102, 1

154 155 156 157 158 159 160 161 162 163 164

M.L. Meyers and P.G. Seybold, Anal. Chem., 51, 1609 (1979). T. Vo-Dinh and J.R. Hooyman, Anal. Chem., 51, 1915 (1979). R.J. Hu rt ubis e , Talanta, 28, 145 (1981). R.J. H u r t u b i s e and G.A. Smith, Anal. Chim. Acta, 139, 315 (1982). D.L. McAleese and R.B. Dunlap, Anal. Chem., 56, 2244 (1984). S.M. Ramasamy and R.J. H u r t u b i s e , Anal. Chem., 54 2477 (1982). S.M. Ramasamy and R.J. H u r t u b i s e , Anal. Chim. Acta, 152, 83 (1983). C.M. O'Donnell and J.D. Winefordner, Clin. Chem. 21, 285 (1975). R.P. Bateh and J.D. Winefordner, Anal. L e t t . , 15, 373 (1982). J.N. M i l l e r , Trends Anal. Chem., 1, 31 (1981). T. Vo-Dinh, G.L. Walden and J.D. Winefordner, Anal. Chem., 49, 1126

165 166 167 168 169 170 171 172 173

J.B.F. L loy d, A n a l y s t , 103, 775 (1978). R.J. H u r t u b i s e , Anal. Chem., 5 5 , 669A (1983). G.D. B o u t i l i e r and J.D. Winefordner, Anal. Chem., 51, 1384 (1979). G.D. B o u t i l i e r and J.D. Winefordner, Anal. Chem., 51, 1391 (1979). R.M. Wilson and T.L. M i l l e r , Anal. Chem., 47, 256 (1975). T. Vo-Dinh, Anal. Chem., 50, 396 (1978). T. Vo-Dinh and R.B. Gammage, Anal. Chim. Acta, 107, 261 (1979). T. Vo-Dinh and P.R. M a r t i n e z , Anal. Chim. Acta, 125, 13 (1981). T. Vo-Dinh, R.B. Gammage and P.R. M a r t i n e z , Anal. Chim. Acta, 118,

145 146

(1981).

(1978).

(1977).

313 (1980). 174

L.J.

C l i n e Love, M. S k r i l e c and J.G.

Habarta, Anal. Chem., 52, 754

(1980).

175 176

M. S k r i l e c and L.J. C l i n e Love, Anal. Chem., 52, 1559 (1980). L.J. C l i n e Love and R. Weinberger, Spectrochim Acta, 38B, 1421

177

L.J. C l i n e Love, J.G.

(1983).

.

Habarta and J.G.

Dorsey, Anal. Chem., 56, 1132A

(1984)

C l i n e Love, Anal. Chem., 54, 1552

178

R. Weinberger, P. Yarmchuk and L.J.

179

F.J. DeLuccia and L.J. C l i n e Love, Anal. Chem., 56, 2811 (1984). F.J. DeLuccia and L.J. C l i n e Love, Ta l a n t a, 32, 665 (1985). R.A.Femia and L . J . C l i n e Love, Anal.Chem., 56, 327 (1984). H.L.J. Backstrom and K. Sandros, A c t a Chem. Scand., 12, 823 (1958). M. Algrem, Photochem. and P h o t o b i o l . , 6, 829 (1967). C.A. Parker and T.A. Joyce, Trans. Faraday SOC., 65, 2823 (1969). N.J. T u rro, L. Kou Chiang, C. Ming-Fea and P. Lee, Photochem. and P hot o biol. , 27, 523 (1978). N.J. T urro, Mol. Photochem., 4, 213 (1972). J.J. Donkerbroek, J.J. E l z a s , C. G o o i j e r , R.W. F r e i and N.H. V e l t h o r s t , T a l a n t a , 28, 717 (1981).

iao

181 182 183 184 185 186 187

(1982).

334 188

189 190 191 192 193 194 195 196 197 198 199 200 201 202

T. Vo-Dinh, "Room Temperature Phos horimetry for Chemical Analysis", Wiley-Interscience, New York (1984p. See e.g., J.A. Barltrop and J.D. Coyle, "Principles of Photochemistry", Wiley Interscience, New York, Chapter 4 (1978). N.J. Turro, Modern Molecular Photochemistry, Chapter 9, Benjamin/Cummings, CA (1978). J.J. Donkerbroek, N.J.R. van Eikema Hommes, C. Gooijer, N.H. Velthorst and R.W. Frei, Applied Spectr., 37, 188 (1983). E. Blanco-Gonzalez, R.A. Baumann, C. Gooijer, N.H. Velthorst and R.W. Frei, Chemosphere, 16, 1123 (1987). S.Y. Su, A. Jur ensen, D. Bolton and J.D. Winefordner, Anal. Lett., 14, A l l 1 (198lp. S.Y. Su, E.P.C. Lai and J.D. Winefordner, Anal. Lett., 15, A 5 , 439 (1982). K.C. Marsh, L.A. Sternson and A.J. Repta, Anal. Chem., 56, 491 (1984). 1,s. Krull, X . 4 . Ding, S. Braverman, C. Selevka, F. Hochberg and L.A. Sternson, J. Chromatogr. Sci., 21, 166 (1983). W.N. Richmond and R.P. Baldwin, Anal. Chim. Acta, 154, 133 (1983). A.M. Bond and G.G. Wallace, Anal. Chem., 54, 1706 (1985). J. Ruter, U.P. Fislage and B. Neidhart, Chromatographia, 19, 62 (1985). I.S. Krull, D. Bushee, R.N. Savage, R.G. Schleicher and S.B. Smith, Anal. Lett., 15 ( A 3 ) , 267 (1982). R.J.Wenze1 and L.M. Collette, J. Chromatogr., 436, 299 (1988). E.E. Dibella, J.B. Weissman, M.J. Jose h, J.R. Schultz and T.J. Wenzel, J. Chromatogr., 328, 101 (1985p.

335 CHAPTER V I I

CONTINUOUS SEPARATION TECHNIQUES I N FLOW- INJECTION ANALYSIS M. VALCARCEL and M. 0. LUQUE DE CASTRO

1. Introduction 2. Gas-1 i q u i d i n t e r f a c e s 2.1 Gas-diffusion 2.2 D i s t i 11a t i o n 2.3 Hydride generation 3. Gas-sol i d i n t e r f a c e s 4. L i q u i d-1 iqu id i n t e r f a c e s 4.1 Extraction 4.2 Dialysis 5. Solid-liquid interfaces I o n exchange 5.1 Adsorptive preconcentrat i o n 5.2 P r e c i p i t a t i o n and d i s s o l u t i o n 5.3 HPLC-FIA a s s o c i a t i o n 6. Pre-column assemblies 6.1 6.2 Post-column assemblies F i n a l remarks 7. Acknowl edqernent 8. References 1.

INTRODUCTION

Chromatographic

and non-chromatographic

continuous

separation pro-

cesses are c u r r e n t l y among t h e most r e l e v a n t aspects o f a n a l y t i c a l chemis t r y . Both a r e c h a r a c t e r i z e d by t h e continuous motion o f one o r b o t h liq u i d o r gas phases involved, accomplished by means o f a p r o p e l l i n g system (e.g.

a p e r i s t a l t i c o r p i s t o n pump, p r e s s u r i z e d gas, e t c . ) .

The l i q u i d

(gas) sample can be introduced i n t o t h e system e i t h e r by i n j e c t i o n o r i n s e r t i o n , o r by continuous a s p i r a t i o n , These systems g e n e r a l l y accommodate a continuous d e t e c t o r a l l o w i n g i d e n t i f i c a t i o n and/or q u a n t i t a t i o n o f t h e analytes

concerned.

One o f

their

most

interesting features

is

the

p o s s i b i l i t y t o decrease t o a g r e a t e r o r l e s s e r e x t e n t human p a r t i c i p a t i o n i n t h e a n a l y t i c a l process ( a u t o m a t i z a t i o n ) thanks t o t h e i r i n h e r e n t dynamism. This t r e n d towards a u t o m a t i z a t i o n has been r e i n f o r c e d w i t h t h e i n c r e a s i n g use o f t h e c u r r e n t l y i r r e p l a c e a b l e microprocessors, used b o t h t o c o n t r o l t h e process and f o r d a t a a c q u i s i t i o n and treatment purposes ( r e f . 1). Gas chromatography i s t h e obvious continuous s e p a r a t i o n technique t o be chosen whenever t h e c a r r i e r o r t h e sample i t s e l f i s a gas. There a r e three

analytical

hydrodynamic techniques o f widespread use, namely h i g h

336 performance l i q u i d chromatography (HPLC) ( r e f . Z), f i e l d f l o w f r a c t i o nation (FFF) (refs. 3, 4) and continuous f l o w analysis (CFA) i n i t s two c h i e f variants: segmented flow analysis (SFA) ( r e f . 5) and f l o w i n j e c t i o n analysis (FIA) (refs. 6, 7). The f i r s t two methodologies are based on t h e continuous separation of t h e analytes and i n t e r f e r e n t s present i n a chromatographic column o r i n t h e a p p l i c a t i o n o f a c e n t r i f u g a l f o r c e o r a thermal o r e l e c t r i c gradient. On t h e o t h e r hand, continuous f l o w analysis methods are n o t p r i m a r i l y intended f o r separation purposes, but f o r automatization o f a n a l y t i c a l determinations o f one o r more analytes; y e t , they do a1 low f o r r e a l i z a t i o n of continuous separations, although these are markedly l e s s e f f i c i e n t

-

p a r t i c u l a r l y as regards d i s c r i m i n a t i o n

between several analytes. FIA

is

a CFA

mode developed

along t h e

characterized by a number o f features, segmented by a i r bubbles;

past

namely:

twelve years

and

(a) t h e f l o w i s n o t

(b) t h e l i q u i d sample i s d i r e c t l y irljected o r

inserted i n t o a f l u i d stream; (c) t h e sample-reaction zone i s dispersed i n a p a r t i a l , c o n t r o l l e d fashion i n t h e f l o w and (d) n e i t h e r physical (homogenization) nor chemical e q u i l i b r i u m has been reached by t h e t i m e detection i s performed. FIA recordings resemble t y p i c a l chromatograms. On comparing t h e instrumental schemes i n Fig. 1 one may conclude t h a t HPLC and F I A are r e l a t i v e l y s i m i l a r : i n f a c t , both techniques use l i q u i d reservoirs, pumps, i n j e c t o r s and continuous systems. However, t h e r e are also a number o f differences between both, c h i e f l y as regards working pressure, use o f an i n t e r f a c e , v e r s a t i l i t y , type o f a n a l y t i c a l problems d e a l t with, cost, etc. I n any case, t h e greatest d i f f e r e n c e l i e s i n t h e continuous separation c a r r i e d out i n t h e chromatographic column, which i s essential t o HPLC and o n l y occasionally employed i n F I A ( r e f . 8). Interfaces are used i n F I A f o r one o f two c h i e f purposes. On t h e one hand, they can be employed t o develop continuous separation processes improving

on

others

formerly

carried

e x t r a c t i o n , ion-exchange, adsorption, etc.).

out

manually

(1 i q u i d - 1 i q u i d

On t h e other hand, they can

be used f o r non-separative purposes (e.g. t o improve o r f a c i l i t a t e t h e a n a l y t i c a l determination). I n t h i s case, advantage i s taken o f t h e chemical r e a c t i o n t a k i n g place between a s o l i d phase and a l i q u i d f l o w i n g through it. Thus,

redox columns have been used t o handle reagents

s e n s i t i v e t o atmospheric agents ( r e f .

9) and t o perform multidetermi-

nations (e.g. o f n i t r i t e and n i t r a t e ) . The use o f enzymes immobilized on packed columns o r tube w a l l s i s a l s o a very i n t e r e s t i n g a l t e r n a t i v e ( r e f . 11). Voltammetric and potentiometric s t r i p p i n g techniques performed i n a continuous fashion can a l s o be included here i n s o f a r as they pursue t h e

337 two aforesaid objectives in their two essential steps: preconcentration and determination (refs. 12 14).

-

H I G H PERFORMANCE LIQUID CHROMATOGRAPHY LIOUIO RESERVOIRS

H

PROPULSION SYSTEM

H

I NTROOUCTIO

H

CONTINUOUS

COLUMN

DETECTOR

F L O W INJECTION ANALYSIS

PROPULSION

R E SERVOIRS

REACTOR INJECTION

SEPARATOR

OET ECTOR i

Fig. 1 Basic components of HPLC and FIA. The different types of interfaces used in FIA and the corresponding continuous separation techniques employed are summarized in Table I. The second phase, which acts as an analyte or interference collector, can be continually introduced into the separation system (liquid-liquid extraction, dialysis and gas diffusion). Alternatively, it can be a permanent part of the FIA system (e.9. an adsorption or ion-exchange minicolumn) - in this case, FIA and HPLC are nearly identical and only differ appreciably in that retention-elution processes in the former are one-stage rather than multi-step. The second phase can also be created in the separation system itself through a physical change (distillation) or a chemical reaction (precipitation). Whenever an analytical reaction is required, this can be developed in the course of the separation process (e.g. metal chelate or ion-pair extraction) or at a later stage - by merging the line carrying the isolated analyte(s) with one or several reagent lines prior to the detector, in much the same way as in postcolumn HPLC reaction detectors. This chapter deals with the most relevant features and applications of continuous separation techniques used in FIA configurations. Especial emphasis is placed on the coupling o f FIA systems to liquid chromatographs. The objectives pursued and advantages offered by the incorporation of non-chromatograph i c separation techniques into unsegmented flow configurations are also critically discussed.

I

338 TABLE I

Continuous separation techniques used i n F I A Interface

Technique

gas-liquid

d i s t i 1l a t i o n

gas-di f f usion hydride generation gas-sol i d

extraction 1i q u i d - l i q u i d dialysis

ion-exchange adsorption liquid-solid precipitation others

2.

GAS-LIQUID INTERFACES The g a s - l i q u i d

separation systems

typically

used

c l a s s i f i e d i n t o t h r e e groups, namely (a) gas-diffusion,

i n FIA

can

be

i n which a gas

present i n the donor phase o r generated i n a chemical r e a c t i o n d i f f u s e s t o the other phase

-

also l i q u i d

-,

a c t i n g as acceptor;

(b) d i s t i l l a t i o n ,

where the gas phase i s formed by heating, condensation a t a s u i t a b l e temperature and c o l l e c t i o n i n t o a second l i q u i d phase and (c) hydride generation, i n which the gas phase i s t h e r e s u l t o f a chemical r e a c t i o n and the second one i s a gas d r i v i n g t h e sample t o t h e d e t e c t i o n system. 2.1

GAS-DIFFUSION

The t r a n s f e r o f a gas between two donor streams, s t i l l n o t t o o widely applied i n F I A , can be used w i t h a l a r g e v a r i e t y o f analytes, matrices and detection systems (Table 11). The analyte making the gas phase can be as a gas i n t h e donor (02, 03, C12) o r a l t e r n a t i v e l y be generated i n i t by a s t r a i g h t f o r w a r d chemical reaction induced by an a c i d (formation o f

SO2, C02 o r HCN) o r a base

(formation o f NH3), o r through t h e a c t i o n o f r e l a t i v e l y high temperatures (formation o f acetone from oxidized ketone bodies). Photometry has been so f a r

the

detection technique most f r e q u e n t l y used i n conjunction w i t h

339 these systems on account of its suitability for analytes with acid-base properties, which usually diffuse to a solution containing an indicator whose colour change is a function of the amount of analyte diffused. As a rule, phase separation is isotermal and is effected by passage through a suitable membrane - usually PTFE. Occasionally, there is no separation membrane, but this is produced between two parallel rubber sheets supported by PerspexR plates, The stream containing the sample spreads throughout the lower sheet and yields a film which traverses the entire length of the rubber before going to waste. During the transport, if the partial pressure of the gas in the liquid is higher than that in the surrounding atmosphere, it volatilizes and diffuses to and is absorbed by the stream where its partial pressure is lower (ref. 15). The chief analytical purpose of the FIA/gas-diffusion association is interference removal from complex matrices (biological fluids, foods, vegetable tissues, environmental samples, etc.); yet, enhanced selectivity can also be obtained - as demonstrated by Pacey et al. (ref. 16) - by incorporating kinetic discrimination in the system timing or the reagent concentrations and experimental conditions of a given method and using a gas diffusion unit. These authors describe two examples of kinetic discrimination, namely the determination of ozone with Indigo Blue and that of chlorine dioxide based on luminol chemiluminescence. Both compounds react with chlorine. In the batch determination of ozone with Indigo Blue, equilibrium measurements do not allow discrimination between ozone, chlorine and manganese (VII) (ref. 17); yet, the differences between the rates of reaction of these analytes with the reagent permit their discrimination by using a single-channel FIA system which increases the selectivity for ozone over chlorine by a factor of about 3 , and that over Mn (VII) by a factor of about 2 - these factors can be further increased by incorporating a gas-diffusion unit into the manifold (Fig. 2). The diffusion membrane completely overcomes the interference by Mn (VII), while that posed by chlorine is significantly decreased from 1 mg chlorine (corresponding to an apparent ozone concentration of 0.36 mg/ml) in the manual method to an apparent 0.008 mg 03/ml in the gas-diffusion method. Selectivity is enhanced by a factor of 2.5 as a result of the kinetic discrimination introduced by the reagent, and by a further factor of 45 resulting from the incorporation of the gas-diffusion unit, so that the overall selectivity enhancement factor amounts to 112. Such a selectivity level i s adequate to safely determine residual ozone in the presence of chlorine in disinfected water samples.

TABLE I 1

W P

Features of FIA methods involving gas-diffusion processes

Analyte NH3

Matrix whole blood b 1 ood plant

NH3, urea

Phase separation

pH change ,,

membrane

,I

none membrane

I,

,I

chemical reaction pH change

NOx. NO2so2

Gas-phase formation

wine, beer, fruit juice

0,

I,

COP c102

p,

P

I,

,I

11

,I

waste water,

Oxidized ketone bodies

milk

P: photometry

pot: potentiometry opt: optosensor

heat

11

Pot Pot opt Pot

I,

I1

amp.: amperometry

P

membrane

,I

pH change

Detection

,I

clop 02

CN-

Analytical purpose

I,

chemi ca 1 reaction wine, food, p 1 asma

0

use of an indicator

20 23

integrated microconduits ISE

15 22 19

24 28

amp. P

29 21

di scrimination- chem. separation P I .R. Pot I,

chem.: chemiluminescence I.R. interference removal

Ref.

chem.

I,

I,

Special features

P

25 16

26 30 27

341 The d i f f e r e n c e s i n t h e s i g n a l produced by C12 and C102 i n t h e chemiluminescence determination o f c h l o r i n e d i o x i d e w i t h l u m i n o l a r e t i m e and pH-dependent.

C h l o r i n e d i o x i d e r e a c t s extremely q u i c k l y w i t h l u m i n o l ,

w h i l e c h l o r i n e r e a c t s more s l o w l y and w i t h l o n g e r l i f e t i m e s . By use of a flow-injection

system,

both

the

timing

and

the

chemistry

can

be

c o n t r o l l e d i n such a way as t o minimize t h e s i g n a l y i e l d e d by c h l o r i n e . The added use o f pH 13 makes t h e reagent even more s e l e c t i v e c o n d i t i o n s t y p i c a l l y used i n F I A overall

selectivity

factor

-

is

-

under t h e

towards c h l o r i n e d i o x i d e . The observed over

500.

The

incorporation

of

a

g a s - d i f f u s i o n u n i t i n t o t h e system overcomes t h e i n t e r f e r e n c e posed by b o t h i o n i c and organic m a t e r i a l s absorbing

light i n the ultraviolet

r e g i o n ( r e f . 18). The s e l e c t i v i t y enhancement between C102 and C12 r e s u l t i n g from t h e use o f t h e membrane i s 3.1; t h i s , m u l t i p l i e d by t h e enhancement f a c t o r r e s u l t i n g from t h e d i f f e r e n c e i n molar a b s o r p t i v i t y between both species a t t h e wavelength used,

175, y i e l d s t h e o v e r a l l

f a c t o r o f 500, which can be f u r t h e r increased t o over 5,000 thanks t o t h e masking e f f e c t o f o x a l i c a c i d on c h l o r i n e ( r e f . 18).

s te

Fig. 2

M a n i f o l d f o r t h e d e t e r m i n a t i o n o f c h l o r i n e d i o x i d e based on luminol chemi luminescence. Both t h e donor and t h e acceptor stream a r e wa_ger a t pH 2, adjusted w i t h s u l p h u r i c a c i d . The reagent, 1 10 M l u m i n o l , i s merged a t t h e T - c e l l p r i o r t o t h e p h o t o m u l p l i e r tube (PMT). The membrane c o n s i s t s o f 0.45 pm PTFE and t h e t u b i n g (0.5 mm i.d.) i s a l s o PTFE. A l l f l o w - r a t e s a r e 1 ml/min. (Reproduced from ( r e f . 16) w i t h permission o f E l s e v i e r Science Pub1 i s h e r s ) .

M a r t i n and Meyerhoff ( r e f . 19) have developed a general procedure f o r enhancing t h e s e l e c t i v i t y of anion-responsive 1 iquid-membrane e l e c t r o d e s based on t h e use o f an acceptor channel r e c e i v i n g t h e f l o w through a gasd i f f u s i o n u n i t as t h e i n j e c t i o n membrane prevents

ionic

loop o f an F I A system.

interferents

from

reaching

A

suitable

an i o n - s e l e c t i v e

342 electrode in the final flow stream. The authors apply their highly selective semi-automated method to the determination of dissolved nitrogen oxides or nitrite at levels above 5 f l . Nitrogen dioxide is trapped across a PTFE membrane in the separation unit and converted to nitrate by a buffered peroxide recipient solution that is injected and carried to a tubular nitrate electrode. As noted by its proponents, the method excels the selectivity and detection capabilities of the nitrate electrode alone and of conventional sensing systems based on pH electrodes. Gas-liquid interfaces have been used in a number of interesting clinical determinations such as that of ammonia in whole blood and plasma (ref. 20) or that of carbon dioxide in plasma (ref. 21). The former, proposed by Evensson and Anfalt, uses the configuration depicted in Fig. 3, where the sample is injected into a distilled water stream later merged with a 0.5N NaOH solution converting ammonium ion to ammonia, which in turn diffuses across the PTFE membrane to a stream of Phenol Red in NaOH, the resultant process being monitored at 540 nm. The method is quite convenient, uses small sample volumes (90 pl) and features a relatively high sampling frecuency (60 h- 1) . According to its proponents, it yielded excellent results upon application to samples from 17 individuals. The use of plasma instead of whole blood makes coils A and B in Fig. 3 dispensable (refs. 15-30). holder - -Module -- - -1 I Perspex modules I

,-, -.--...- .......

5 od i u rn hydroxide

9

Sample Sample

0.35 mL /min I

III

I I

~

~

I

I

117 - 1

1.12 rnL lrnin

I I W

Gas-diffusion cell

---

'-

---

1 -I ' I

L - -

Fig. 3

I In1

+?I

I

Membran+k'-

Phenol Red

I

I

----

Photometer

Manifold for determination of ammonia in blood accommodating a gas-diffusion system. (Reproduced from (ref. 20) with permission of Elsevier Biomed. Press).

343 A s i m i l a r c o n f i g u r a t i o n i s used f o r t h e d e t e r m i n a t i o n o f C02 i n plasma, an a c i d stream a c t i n g as donor and one o f Red Cresol as acceptor.

Ruzicka and Hansen ( r e f . 2 2 ) have a p p l i e d r e f l e c t a n c e spectrophotometry t o f l o w - i n j e c t i o n measurements o f pH and ammonia and urea assays w i t h t h e aim o f demonstrating t h e p r i n c i p l e behind and t e s t i n g t h e p e r formance o f optosensors i n t e g r a t e d i n t o microconduits.

With pH measure-

ments, d e t e c t i o n i s based on commercial non-bleeding acid-base i n d i c a t o r papers placed i n t h e f l o w i n g stream a t t h e t i p o f t h e f i b r e o p t i c . The determination o f ammonia and urea ( v i a urease) i n v o l v e s t h e use of

a

Bromothymol Blue stream and a m i n i a t u r e g a s - d i f f u s i o n d e v i c e ( r e f . 8). 2.2

DISTILLATION Continuous m i c r o d i s t i l l a t i o n systems have l o n g been used i n c l a s s i c a l

continuous air-segmented

configurations f o r determination o f v o l a t i l e

v o l a t i l e a c i d i t y i n wine), and a r e commercially a v a i l a b l e

analytes (e.9.

from a number o f companies such as Technicon o r Skalar. On t h e o t h e r hand, t h e r e i s o n l y one FIA method u s i n g d i s t i l l a t i o n w i t h t h e c h i e f purpose of i n t e r f e r e n c e removal from such a complex m a t r i x as waste water u s u a l l y i s f o r t h e determination o f cyanide. proposed by P i h l a r and Kosta ( r e f . consisting

of

a

distillation

b o r o s i l i c a t e g l a s s half-packed heating wire,

and

The method i n q u e s t i o n ,

31), uses a d i s t i l l a t i o n system an

absorption

unit.

The

former,

w i t h g l a s s h e l i c e s and wrapped

in a

i s entered by t h e n i t r o g e n stream a t t h e bottom o f t h e

d i s t i l l a t i o n column and c a r r i e s hydrogen cyanide through t h e condenser i n t o t h e absorption u n i t . An 0.1M s o l u t i o n o f sodium hydroxide i s pumped to

the

of

top

the

absorption

column.

A

debubbler

voltammetric d e t e c t o r removes a l l gas from t h e system.

prior

to

the

Differentiation

between t o t a l and s t r o n g l y bound metal cyanide complexes i s achieved by UV photodecomposition o f t h e complexes. Cyanide i s thereby

recovered

quantitatively,

except when i t s c o n c e n t r a t i o n i s beyond t h e o p e r a t i n g

range

distillation

of

the

and/or

the

absorption

unit.

Either

a

c a l i b r a t i o n graph o r t h e s t a n d a r d - a d d i t i o n method can be used, though t h e l a t t e r i s t o be p r e f e r r e d when t h e sample v i s c o s i t y i s markedly d i f f e r e n t from t h a t o f t h e standards. Up t o 60 samples p e r hour can be thus assayed by a l t e r n a t i n g samples and washing s o l u t i o n s every 30 sec.

2.3

HYDRIDE GENERATION This i s a s u i generis example o f a g a s - l i q u i d s e p a r a t i o n process

c a l l i n g f o r a chemical agent

-

u s u a l l y sodium borohydride

-

t o yield a

v o l a t i l e compound which i s separated from t h e s o l u t i o n by a gas a c t i n g as the

second

phase and

transporting

the

a n a l y t e t o an atomic

optical

344 d e t e c t o r . Hydride generation i s u s u a l l y aimed a t i n t e r f e r e n c e removal ( r e f s . 32 38) and, secondarily, t o s p e c i a t i o n based on t h e d i f f e r e n t

-

r a t e o f formation of t h e h y d r i d e s o f t h e d i f f e r e n t chemical forms i n which a given a n a l y t e occurs ( r e f . 37, Table 3 ) . The F I A l h y d r i d e generation a s s o c i a t i o n has m a t e r i a l i z e d i n two types o f generic c o n f i g u r a t i o n d i f f e r i n g i n t h e way gas-separation i s e f f e c t e d , namely by means of

a conventional

debubler from which t h e generated

h y d r i d e i s d r i v e n t o t h e spectrophometer q u a r t z c e l l by a gas stream (Fig. 4) o r by use of one of t h e above-mentioned g a s - d i f f u s i o n u n i t s where t h e stream r e c e i v i n g t h e gas (hydride) t r a n s f e r r e d across t h e membrane i s another gas which f u n c t i o n s t o d r i v e i t t o t h e d e t e c t o r (Fig. 4 ) . The former t y p e o f c o n f i g u r a t i o n was used i n 1982 f o r d e t e r m i a n t i o n of bismuth by Astrom ( r e f . 32), who emphasized t h e promising advantages of F I A i n c o n t r o l l i n g i n t e r f e r e n c e e f f e c t s i n h y d r i d e g e n e r a t i o n systems. The c o n f i g u r a t i o n (Fig. 4 ) ,

designed t o avoid v o i d volumes as f a r as

p o s s i b l e , i n v o l v e s an a c i d stream i n t o which t h e sample (700 p l ) i s i n j e c t e d and l a t e r mixed w i t h sodium borohydride and sprayed w i t h n i t r o g e n

or argon i n t o t h e g a s - l i q u i d separator. The gas h y d r i d e i s swept i n t o t h e e l e c t r i c a l l y heated tube furnace and t h e c o n c e n t r a t i o n i s then measured a t 223.1 nm on an atomic a b s o r p t i o n spectrophotometer. Standard bismuth s o l u t i o n s a r e always i n j e c t e d before and a f t e r each i n t e r f e r i n g t e s t s o l u t i o n t o check f o r p o t e n t i a l changes i n s e n s i t i v i t y . C o n s u l t a t i o n of the l i t e r a t u r e , prompted t h e a u t h o r t o use t h e i n t e g r a l r a t h e r than t h e peak h e i g h t as a measure o f t h e h y d r i d e c o n c e n t r a t i o n

-

t h e peak h e i g h t

is dependent upon t h e o x i d a t i o n s t a t e o f t h e element i n q u e s t i o n because of

kinetic effects

involved,

while

the

integral

i s not

(ref.

34).

However, as bismuth does n o t pose t h a t o x i d a t i o n s t a t e problem, and on account o f t h e ease w i t h which a n a l y s i s ,

d e t e c t i o n and r e c o r d i n g a r e

performed i n FIA, t h e authors opted f o r making peak h e i g h t measurements, which i n a d d i t i o n made t h e method more w i d e l y a p p l i c a b l e i n s o f a r as many instruments are n o t equipped w i t h i n t e g r a t i n g f a c i l i t i e s .

The r e s u l t s

obtained show t h e e f f i c i e n c y of t h e F I A system i n m i n i m i z i n g t h e severe i n t e r f e r e n c e from copper and n i c k e l

(refs.

35, 36)

-

the interferent

concentrations t o l e r a t e d a r e 100 t o 1000 h i g h e r than those a f f o r d e d by conventional h y d r i d e generation systems f o r bismuth. Except f o r t h e i d e a l reagent concentrations used, t h e improvement i s t h e r e s u l t o f keeping t h e r e a c t i o n t i m e as s h o r t as p o s s i b l e i n o r d e r t o f a v o u r t h e main r e a c t i o n . The l a t t e r type o f c o n f i g u r a t i o n i s represented by a m a n i f o l d designed by Pacey e t a l . ( r e f . 33) f o r d e t e r m i n a t i o n o r a r s e n i c , which i s i n j e c t e d intended t o i n t o a water stream merging w i t h an a c i d and an I K stream

-

remove i n t e r f e r e n t s formed i n t h e h y d r i d e g e n e r a t i o n and t o

improve

the

345 arsenic s i g n a l

-

p r i o r t o m i x i n g w i t h t h e sodium borohydride stream. The

gas passing across t h e membrane o f t h e g a s - d i f f u s i o n u n i t i s d r i v e n t o t h e d e t e c t o r by a hydrogen stream. This dual-phase g a s - d i f f u s i o n system provides remarkably b e t t e r r e s u l t s than a conventional ( r e f s . 37

-

configuration

41).

GAS-SOLID INTERFACES

3.

There a r e few methodologies i n v o l v i n g gas-sol i d i n t e r f a c e s i n general and o n l y one d i r e c t determination o f c h l o r i n e and bromine i n F I A ( r e f . 4 2 ) . This i s based on t h e m o n i t o r i n g o f t h e t r a n s i e n t s i g n a l r e s u l t i n g from two consecutive r e a c t i o n s t a k i n g p l a c e a t a g a s - s o l i d i n t e r f a c e . The r e a c t i o n g i v i n g r i s e t o t h e increased s i g n a l i s t h a t o f t h e halogen and a-naphthoflavone, y i e l d i n g a red-brown complex. The b a s e l i n e r e s t o r a t i o n i s due t o a slower, simultaneous combination o f two processes, namely t h e

spontaneous decomposition o f t h e t r a n s i e n t complex and t h e r e a c t i o n of the

coloured

complex

with

As(III),

which

in

a-naphthoflavone

is

regenerated and t h e halogens are reduced t o t h e i r corresponding ha1 i d e s . A l l these r e a c t i o n s take p l a c e a t t h e s u r f a c e o f a p i e c e o f f i l t e r paper on which a l a y e r o f d r i e d r e a c t i n g m i x t u r e has been deposited. The course o f t h e r e a c t i o n i s monitored by t r a n s m i t t a n c e spectrophotometry. method i n v o l v e s no separation process indeed.

TABLE 111 Features o f FIA methods i n v o l v i n g h y d r i d e g e n e r a t i o n

Analyte

Hatrix

As ( I I I ) , A s W )

h a l y t ical prrpose I.R.

Detection A.A.S

Special features

Ref.

use of gas-

33

d i f f u s i o n membrane As,

Sb, 81, Se,

32 37

1.R

A.A.S

1.R

A.A.S

standard reference

I.R.

A.A.S

NBS orchard Leaves

I.R.

MECA

38 39

I.R.

ICP-AES

41

I.R.

AAS

40

81

termal water

speciation

Te AS

glycerine

H4

~~~~~

1.R.:

interference r m v a l

MECA: m l e c u l a r emission c a v i t y analysis

~~

AAS: atomic absorption spectrometry

ICP-AES: i n d u c t i v e l y carpled plasm-atomic emission spectromtry

The

346

7

Water

-

-

Pumps WaterSample

-

Water

Carrier-Waste

or

Reaged

Organic solvent

Fig. 4

-

L

(a) Flow-injection manifold for determination of bismuth by atomic absorption with hydride generation (S, injection port sample loop --;a, b and c, coils of i.d. 0.7, 0.5 and 0.5 mm, respectively; d, gas-liquid separator; W , waste). (b) Manifold for determination of As(II1) and A s ( V ) using gas diffusion. (Reproduced from (refs. 19, 20) with permission of the American Chemical Society).

347 LIQUID-LIQUID INTERFACES

4.

The way i n which t h e t r a n s f e r o f m a t t e r between two l i q u i d phases takes p l a c e i n continuous s e p a r a t i o n systems depends e s s e n t i a l l y on t h e t y p e of c o n t a c t between t h e phases i n v o l v e d and on t h e i r m i s c i b i l i t y . L i q u i d - l i q u i d e x t r a c t i o n r e l i e s on t h e i m m i s c i b i l i t y o f t h e two phases and t h e establishment o f a dynamic c o n t a c t zone between b o t h f a c i l i t a t i n g t h e t r a n s f e r o f matter. concerned

-

On t h e o t h e r hand,

g e n e r a l l y aqueous

-

i n dialysis,

t h e phases

a r e m i s c i b l e and t h e t r a n s f e r of m a t t e r

takes p l a c e across a semi-permeable membrane s e p a r a t i n g b o t h l i q u i d streams and accommodated i n t h e s e p a r a t i o n u n i t ( d i a l y s e r )

.

These two separation techniques have been used t o a d i f f e r e n t e x t e n t i n FIA. Thus, w h i l e e x t r a c t i o n has been employed r e l a t i v e l y f r e q u e n t l y ( r e f s . 43, 44), d i a l y s i s has been a p p l i e d t o a somewhat l e s s e r e x t e n t . 4.1

EXTRACTION

The o n - l i n e c o u p l i n g o f a l i q u i d - l i q u i d e x t r a c t i o n system t o an FIA c o n f i g u r a t i o n was simultaneously r e p o r t e d by K a r l b e r g and Thelander ( r e f . 45), and Bergamin e t a l . ( r e f . 46). I n b o t h cases, t h e e x t r a c t i o n system was l o c a t e d behind t h e i n j e c t i o n valve. C u r r e n t l y , t h e s e p a r a t i o n u n i t i s a l s o o c c a s i o n a l l y placed p r i o r t o t h e i n j e c t i o n system, so t h a t t h e separation process r e s u l t s

i n a continuous

stream o f o r g a n i c

phase

c o n t a i n i n g t h e a n a l y t e and f i l l i n g t h e i n j e c t i o n v a l v e ( r e f s . 46, 47). Less common i n FIA i s t h e o f f - l i n e p o s i t i o n i n g o f t h e system ( r e f s . 48

-

50). As a r e s u l t o f t h i s c o u p l i n g , a number o f c o n f i g u r a t i o n s o f v a r i a b l e complexity, s u i t e d t o s p e c i f i c needs, a r e now a v a i l a b l e , namely: (a)

Without phase separation.

I n t h i s mode

-

t h e simplest

-,

the

aqueous sample i s i n j e c t e d i n t o a single-channel m a n i f o l d c a r r y i n g t h e organic stream e x t r a c t a n t , which flows through t h e e x t r a c t i o n c o i l . This i s where t h e f o r m a t i o n o f an e x t r a c t a b l e complex between t h e a n a l y t e and t h e reagent d i s s o l v e d i n t h e o r g a n i c phase

-

measured as i t passes through t h e f l u o r i m e t r i c f l o w - c e l l ( r e f . 51) takes place. The a p p l i c a t i o n

o f ultrasounds t o t h e e x t r a c t i o n c o i l r e s u l t s i n t h e f o r m a t i o n o f a microemulsion t h a t increases c o n t a c t between t h e phases and hence t h e process

yield

and measurement

reliability

(ref.

52).

Microemulsion

formation p r i o r t o i n t r o d u c t i o n i n t o an FIA system has been i n v e s t i g a t e d by Worsfold ( r e f .

53).

This a u t h o r uses a s p e c i a l p r o p e l l i n g system

capable o f r e v e r s i n g t h e d i r e c t i o n of t h e f l o w i n o r d e r t o i n t r o d u c e an immiscible o r g a n i c phase p l u g i n t o t h e sample stream; t h e p l u g i s passed a l t e r n a t e l y i n b o t h d i r e c t i o n s through t h e d e t e c t o r , which a l l o w s c o n t r o l over t h e y i e l d o f t h e e x t r a c t i o n process and i t s e x t r a c t i o n k i n e t i c s t o be s t u d i e d ( r e f . 54).

3 48 (b) S i n g l e e x t r a c t i o n w i t h t h e s e p a r a t i o n system l o c a t e d p r i o r t o or after the injection unit. (c) M u l t i - e x t r a c t i o n , where t h e s e p a r a t i o n process i s repeated several times by u s i n g t h e same o r a d i f f e r e n t e x t r a c t a n t a t each stage ( r e f s . 55, 56), thereby i n c r e a s i n g t h e e f f ic i ency o f t h e o v e r a l l process.

selectivity,

sensitivity

and

(d) Back-extraction. This i s a m u l t i - s t a g e e x t r a c t i o n mode i n which t h e aqueous sample i s f i r s t e x t r a c t e d i n t o t h e organic medium and then back-extracted performed ( r e f . 57).

into

an

aqueous

phase where

measurements

are

The presence o f an organic phase i n an FIA system r e q u i r e s a s e r i e s of cautions t o be taken on account o f i t s c o r r o s i v e p r o p e r t i e s . Thus, t h e transport

tubing,

connectors

and

extraction

system must

be

steel,

platinum, g l a s s o r PTFE. An organic stream can be s e t i n motion by: (a) a t h e f l e x i b l e PVC t u b i n g commonly employed i n o t h e r p e r i s t a l t i c pump systems i s completely useless h e r e and i s t o b e replaced w i t h i n e r t

-

m a t e r i a l s such as m o d i f i e d PVC, s i l i c o n rubber o r f l u o r o p l a s t s ; (b) by t h e displacement technique, which i n v o l v e s pumping an aqueous stream i n t o a closed c o n t a i n e r - t h i s can be achieved w i t h a p e r i s t a l t i c pump and ordinary tubing

-

constant f l o w - r a t e

t h a t i s f i l l e d w i t h t h e o r g a n i c s o l v e n t and f e d a t a i n t o t h e FIA system;

(c)

by s e t t i n g a constant

pressure w i t h t h e a i d o f an i n e r t gas f o r c i n g t h e e x t r a c t a n t t o c i r c u l a t e along t h e F I A manifold. The most serious shortcoming a r i s i n g from t h e use o f F I A / l i q u i d - l i q u i d e x t r a c t i o n i s c u r r e n t l y t h e l a c k o f an e l a b o r a t e theory.

The s t u d i e s

c a r r i e d o u t so f a r i n t h i s area have o n l y d e a l t w i t h s p e c i f i c aspects of y e t , t h e number o f papers r e p o r t i n g new c o n t r i b u t i o n s i s t h e subject

-

f o r t u n a t e l y i n c r e a s i n g ( r e f s . 58

- 63).

I n t h e i r work on t h e hydrodynamic and i n t e r f a c i a l o r i g i n o f phase segmentation

, Sweileh and Cantwell

(ref,

60)

developed

a

semi-

q u a n t i t a t i v e physicochemical model f o r t h e process whereby a l t e r n a t i n g segments o f aqueous and immiscible o r g a n i c phases a r e produced on merging o f both phases. A growing "drop" o f one phase i s d i s l o d g e d t o produce a segment when t h e hydrodynamic f o r c e exerted by i t as a r e s u l t o f t h e f l o w o f t h e o t h e r s o l v e n t equals t h e i n t e r f a c i a l f o r c e h o l d i n g i t i n place. Hydrodynamic forces a r e expressed by Poi seui 11e' s and Bernoui 11 i ' s 1aws , w h i l e t h e i n t e r f a c i a l f o r c e i s expressed by a form o f t h e Tate equation ( r e f s . 64, 65) i n terms o f l i q u i d - l i q u i d i n t e r f a c i a l t e n s i o n and s o l i d l i q u i d c o n t a c t angle. These authors a l s o d e r i v e d a s e r i e s o f equations f o r c a l c u l a t i o n o f t h e e x t r a c t e d a n a l y t e f r a c t i o n , t h e dependence of t h e peak area on t h e f l o w - r a t e , t h e d i s t r i b u t i o n r a t i o and

a proportionality

349 constant c h a r a c t e r i s t i c o f t h e chemical system,

t h e peak h e i g h t as a

f u n c t i o n of t h e a n a l y t e c o n c e n t r a t i o n i n t h e i n j e c t e d sample, t h e f l o w r a t e r a t i o , t h e e x t r a c t e d a n a l y t e f r a c t i o n , spectrophotometer s e n s i t i v i t y and a f a c t o r s i m i l a r t o t h a t o f d i s p e r s i o n r e p o r t e d by Ruzicka ( r e f . 5) and adapted t o systems accommodating l i q u i d - l i q u i d e x t r a c t i o n . The e f f i c i e n c y o f a separation process has been s t u d i e d i n depth by Rossi e t a l . ( r e f . 58), who determined t h e optimum c h a r a c t e r i s t i c s o f t h e e x t r a c t i o n c o i l f o r d i f f e r e n t types o f phases and t h e i n f l u e n c e o f FIA v a r i a b l e s on t h e e f f i c i e n c y .

58) have i n v e s t i g a t e d t h e mechanism o f e x t r a c t i o n systems and determined i t s i n f l u e n c e on t h e peak w i d t h i n terms o f r e l a t e d v a r i a b l e s . I n a d d i t i o n , Backstrom e t a l . ( r e f . 62) have evaluated t h e e f f i c i e n c y achieved and t h e d i s p e r s i o n i n v o l v e d i n v a r i o u s phase separators. Laser-induced e x c i t a t i o n ( r e f . 63) and t h e a l t e r n a t e passage through t h e d e t e c t o r i n both d i r e c t i o n s ( r e f . 54) w i l l foreseeably a i d i n e s t a b l i s h i n g t h e t h e o r e t i c a l background o f t h i s technique. Every automatic s o l v e n t e x t r a c t i o n FIA system has t h r e e e s s e n t i a l components, namely: Nord and

dispersion

(a)

Karlberg

in

(ref.

flow-injection

Segmentor, i n which t h e streams o f t h e two phases i n v o l v e d

merge. I t s c h i e f aim i s t o o b t a i n i d e n t i c a l a l t e r n a t e segments o f b o t h immiscible l i q u i d s reaching t h e e x t r a c t i o n c o i l . (b)

Extraction c o i l ,

where t h e t r a n s f e r o f m a t t e r between t h e

segments o f b o t h phases i s e f f e c t e d . PTFE c o i l s r e p e l t h e aqueous phase, which i s c a r r i e d as bubbles; conversely, t h e w a l l s o f g l a s s c o i l s a r e wetted by t h e aqueous phase, so t h a t t h e o r g a n i c phase i s t r a n s p o r t e d by t h e former as bubbles. S e l l e y e t a l . ( r e f . 55) d e f i n e d some c r i t e r i a f o r c o i l s e l e c t i o n . Thus, c o i l s should i d e a l l y be made o f m a t e r i a l s a l l o w i n g t h e a n a l y t e t o pass i n t o t h e bubble phase. I n a d d i t i o n , t h e r a t i o between t h e i n t e r f a c i a l area and t h e i n i t i a l a n a l y t e volume should be as h i g h as p o s s i b l e and t h e a n a l y t e motion should be f a c i l i t a t e d t o achieve maximum efficiency. (c) Phase separator, which r e c e i v e s t h e segmented f l o w from t h e c o i l and c o n t i n u o u s l y s p l i t s i t i n t o two separate streams o f b o t h phases. O f a l l t h r e e elements, t h e most complex and i n t e r e s t i n g i s no doubt t h e phase separator, of which a v a r i e t y o f models have been designed w i t h

t h e aim t o improve on e a r l i e r ones ( r e f s . 44, 46, 63, 66 t h r e e c h i e f types o f continuous separator:

-

69). There a r e

(a) devices u s i n g a chamber

r e l y i n g on g r a v i t y t o separate t h e phases; (b) gravity-based devices w i t h a T-shaped separator and w i t h o r w i t h o u t a s o r t o f phase guide made of

m a t e r i a l wetted s e l e c t i v e l y

by

one

of

the

phases; (c) devices w i t h a

membrane separator based on the selective permeability of a microporous membrane towards the phase wetting it. This last type of separator features a number of advantages over the other two, namely: a smaller inner volume, which lessens the dispersion or dilution of the analyte or its reaction product and hence results in lesser band broadening and increased sensitivity; greater reliability in separating the phases at higher flow-rates, which redounds to shorter analysis times; flexibility for use with a variety of water-immiscible solvents as a result of no difference in density between the aqueous and the organic phase being required; greater separation efficiency. A recent publication (ref. 70a) has demonstrated that many of these above - mentioned advantages can also be achieved with a new generation of sandwich - type separators based on wetting . The joint use of FIA and liquid-liquid extraction has aided in solving a number of analytical problems in various areas - particularly environmental, clinical and pharmaceutical chemistry -, where this association has been chiefly applied for separation and occasionally - preconcentration o f the analyte. In Table IV are summarized the applications reported so far, classified according to the type of analyte determined (inorganic or organic) and, within each group, according to the detection system used. Fig. 5 shows the two basic types of FIA/liquid-liquid extraction assembly, namely with the extraction unit located prior to (a) or after (b) the injection system. The configuration including an extraction system prior to the injection valve is the most commonly used in the FIA/extraction/AAS association for analyte preconcentration and separation. The advantages offered by the joint use of this triad have been emphasized by several authors (refs. 6, 7, 70) (e.g. in the determination of copper, nickel, zinc and cadmium proposed by Nord and Karlberg (ref. 71), in that of zinc in biological and environmental samples (ref. 72) or iron matrices (ref. 591, or in that of perchlorate in serum and urine, based on the formation of an ion-pair between this anion and the Cu(I)/6-methylpicolinaldehyde azine complex (ref. 47). The sequential determination of nitrate and nitrite in foodstuff, also based on the formation of an ion-pair between nitrate and the Cu( II)/neocuproine complex which is extracted into MIBK is illustrative of the potential of this association for simultaneous analyses. In this determination, the samples are spiked with oxidant, Ce(IV), or inhibitor (amidosulphonic acid), for determination of the sum of both anions and nitrate alone, respectively (ref. 73).

-

351

To quartz cell 7

H CI

NaBHc

W

*

To quartz cell

I

Fig. 5

I

I

Generic types o f FIA/liquid-liquid extraction assemblies. (a) With the extraction system before the injection system ( 6 and B2, displacement bottles; S, segmentor; EC, extraction ioil; P, phase separator; I, injection system; AAS, atomic absorption photometer). (b) With the extraction system behind the injection system.

TABLE I V

Features o f FIA methods i n v o l v i n g l i q u i d - l i q u i d e x t r a c t i o n

w

vl

N

Ana 1y t e

Matrix

Mo

plant

Pb, Cd

water

Cd

urine

~

0

~

~

-

Anal y t e phase aqueous SCN/Fe system aqueous ,I

1,

Ga

water

aqueous/ lumogallion aqueous

NO2

,

NO;

cu Cd, Cu, Co, Pb, N i Ami nes

P

chlorofom/ d i t h izone

I,

tri b u t y l phosphate C C l 4/

soi 1

c104-

isoamyl alcohol chloroform

Special features

I,

biological, environmental ir o n urine, serum water food water diluted samples water

Ref. 46

calculation o f extraction constants new phase s e p a r a t o r

83 66

79

Zn

Zn, Cu, Pb, N i Zn

Detecti o n

biological I

U

Second phase

d ithizone isoamyl alcohol MIBK/APDC

m o d i f i e d T-piece I,

81

F

laser excitation

AAS

MIBK

59 I,

,I I,

,I

MIBK/APDC freon-113 H20 3 r d phase aqueous

47 sequential determination

FAAS back e x t r a c t i o n P

I

63 71 72

I,

aqueous/ SCNaqueous

80

calculation o f extraction constants aqueous/aqueous e x t r a c t i o n w i t h l i q u i d membrane

82 73 46 57 87 84

Caffeine

tablcts

Codeine, a c e t y l salicylic acid Anionic surfactants

,I

Cation i c surfactants Non-i o n i c surfactants Caf f e i ne, s u r f ac t a n t s 8-Dichlorotheop h y l 1ine, diphenyldramamine Procyclidine

industrial water sewage water waste water water

aqueous SCN-/ aqueous

ch 1oroform

,,

t o 1 uene

MIBK I1

tablets

cyclohexane ,I

biological tablets organic

Steroids

water

I,

chloroform

water

Drugs Vitamin B A1 kylaminh;

extraction membrane extraction membrane

increased s e n s i t i v i t y use o f v a r i o u s segmentors

85

86 95 48

a7 I,

chloroform

44

88 measurements i n b o t h phases

67

theoretical q u a n t i t a t i v i t y studies

89

iso-octane

74

n-heptane d i c h l oromethane

F

heptane

chloroform aqueous

aqueous/ 1u c i genin

1,Z-dichloroethane

chem

MBIK: methyl i s o b u t y l ketone

44

68

1,2-dichloroethane chloroform

beer, ma1t serum tablets

aqueous/aqueous with liquid aqueous/aqueous with liquid

,I

water

Bit t e r i n g compounds Terodiline Enal a p r i 1

P

gas chromatography

90 91

a d s o r p t i o n problems

92 93 53

microemulsions w i t h o u t phase s e p a r a t i o n

94

APDC: 1-pyrrolidinecarbodithioic a c i d

W Ln

W

354

The manifold depicted in Fig. 5b was developed and used by Ishibashi et al. (ref. 62) for the determination of gallium based on the formation of a fluorescent complex with lumogallion which is extracted into isoamyl alcohol. A similar configuration has been proposed by Karlberg et al. (ref. 70) for adaptation of the standard manual liquid-liquid extraction method for determination of bitterness by the FIA technique. The bittering compound is extracted into iso-octane and its absorption in the organic medium measured at 275 nm, thus making the separate solvent blank extraction, required in the batch procedure, unnecessary. The injected sample volume used in 100 pl and up to 60 samples can be assayed per hour with as little iso-octane consumption as 1 ml per sample. The method has been recently applied to the determination of this type of compounds in must as a means o f on-line monitoring of their evolution in the course of beer making (ref. 74). An interesting method for simultaneous determination of two organic compounds (diphenyldramamine and 8-chlorotheophylline) has been proposed by Fossey and Cantwell (ref. 67). These authors use a dual-membrane separator (1 ipophi 1 ic -PTFE- and hydrophi 1 ic -paper-) to obtain clear aqueous and organic phases, each being led to a different spectrophotometer. The aqueous portion, of pH 10, contains 8-~hlorotheophylline, while the organic phase (cyclohexane) contains dyphenyldramamine. The inherent versatility of FIA allows for adaptation of the configurations typically used to the particular characteristics of the system involved. Thus, the extraction efficiency can be improved through a salting-out effect by using further streams merging with the two-phase system (ref. 45). Also, the system can be adapted for changes in viscosity or pH scanning (ref. 78). The kinetic nature of this technique - measurements are made under non-equilibrium conditions, which allows FIA methods to be classed as fixed-time kinetic (ref. 72) is increased by joint use with liquid-liquid extraction, which further increases its selectivity (refs. 78, 80). This powerful association provides a number of valuable advantages such as lower sample, reagent and organic solvent consumption, higher determination rate, greater instrumental simp1 icity and reproducibility and less expensive instrumentation. The FIA/extraction association has also been applied for non-analytical purposes such as the calculation of the extracted analyte fraction (ref. 59) or that of the peak area (ref. 61) and height (ref. 59) -based on the use of a dual-membrane phase separator - as a function of other parameters typical of the chemical system such as acidity constants (ref. 78). The still small number of applications in this area

-

355 (Table IV) w i l l p r e d i c t a b l y be increased by t h e use o f laser-induced e x c i t a t i o n ( r e f . 62), m u l t i - e x t r a c t i o n systems ( r e f . 5 5 ) and f a s t - s c a n detectors (refs. 95). ( r e f s . 79

5 5 , 56) f o r s t u d y i n g r e a c t i o n mechanisms and k i n e t i c s

-

4.2

DIALYSIS Membrane-based F I A / l i q u i d separation systems have so f a r been almost

e x c l u s i v e l y used i n c l i n i c a l a n a l y s i s . T h e i r c h i e f use i s i n t e r f e r e n c e removal o n l y once have these been used f o r d i l u t i o n purpose ( r e f . 9 6 ) .

-

Table V l i s t s t h e most s i g n i f i c a n t achievements o f t h e F I A l d i a l y s i s assoc i a t i o n , o f which t h e work by Gorton and Ogren ( r e f . 97) i s a t y p i c a l example. These authors determine glucose and urea i n serum w i t h s u i t a b l e immobilized enzymes. I n t h e c o n f i g u r a t i o n used f o r d e t e r m i n a t i o n o f urea, depicted i n F i g , 6a, t h e sample i s i n j e c t e d i n t o a donor b u f f e r which i s d r i v e n t o waste once t h e a n a l y t e has passed through t h e membrane, wherefrom i t i s l e d by t h e acceptor stream (phosphate b u f f e r o f pH 6) t o t h e t u b i n g zone c o n t a i n i n g t h e r e a c t o r , packed w i t h urease immobilized on c o n t r o l l e d pore glass. The d e t e c t i o n system (ammonia-selective e l e c t r o d e ) c a l l s f o r t h e use o f a b a s i c stream merging w i t h t h e main l i n e a f t e r t h e enzymatic r e a c t o r i n order t o o b t a i n a sample p l u g o f a pH adequate f o r t h e r e l e a s e o f t h e monitored product ( r e f . 97). I n t h e i r r e p o r t , t h e s e authors evaluate t h e e f f e c t o f t h e d i a l y s e r , enzyme r e a c t o r and d e t e c t o r on t h e d i s p e r s i o n . Chang and Meyerhoff ( r e f . 98) used a membrane-dialyser i n j e c t i o n l o o p t o enhance t h e s e l e c t i v i t y o f anion-responsive 1 iquid-membrane e l e c t r o d e s i n FIA (Fig. 6b) and a p p l i e d t o t h e d e t e r m i n a t i o n o f s a l i c y l a t e . system c o n s i s t s

of

a tubular

polymer

membrane e l e c t r o d e

based

The on

manganese (111) t e t r a p h e n y l p o r p h y r i n c h l o r i d e t o sense s a l i c y l a t e i o n s formed i n a r e c i p i e n t b u f f e r s o l u t i o n h e l d w i t h i n t h e upper channel of t h e flow-through membrane d i a l y s e r assembly. Samples c o n t a i n i n g s a l i c y l i c a c i d a r e manually introduced i n t o t h e lower channel o f t h e d i a l y s i s u n i t , where a t h i n s i l i c o n e rubber membrane separates t h e two channels.

The

a n a l y t e i s trapped across t h e membrane as s a l i c y l a t e i o n s w i t h i n a s t a t i c layer o f a suitable recipient buffer. A f t e r a preselected trapping time, the

recipient

plug

is

flushed

to

the

electrode

in

the

typical

f l o w - i n j e c t i o n fashion. The peak p o t e n t i a l s obtained a r e l o g a r i t h m i c a l l y r e l a t e d t o t h e s a l i c y l i c a c i d c o n c e n t r a t i o n s i n t h e o r i g i n a l samples. A 2 near-Nernstian response i s obtained i n t h e range 10-4-10- M s a l i c y l a t e f o r a t r a p p i n g time o f 2 min. The d e t e c t i o n l i m i t s can be m o d i f i e d by changing t h i s t r a p p i n g time. The r e s u l t a n t system i s h i g h l y s e l e c t i v e towards s a l i c y l a t e (as s a l i c y l i c a c i d ) over most i n o r g a n i c and o r g a n i c anions commonly found i n blood.

TABLE V

Features o f FIA methods involving dialysis

W 0-l

m

Analyte

Matrix

Anal yt e phase

Li Various metal ions Zn

serum water

aqueous 1 i gand sol ut i on

0

borax 1 igand sol u t i on

I,

;;-

CI-, ~

Second phase

~

G1 ucose Glucose, urea Galactose Glucose

~

aqueous

serum -

acid solution

serum I,

Ref.

ISE

104 105

P

theoretical I

volt

milk urine

milk, waste water, fermentation broth urine, serum

Detection

Special features

reagent solution

buffer

103 106 107

P I,

96

basic medi um

It

new dialysis probe

99

p, Pot

enzyme reactor

97

8

I,

108 109 110 112 111

aqueous sample

sample

chem.

phosphate buffer aqueous

phosphate buffer aqueous

volt amp.

enzyme reactors

bi ol ogi cal fluid aqueous

aqueous buffer aqueous

Pot

ISE

F

reagent introduction by dialysis. speciation

100

aqueous

aqueous

101

aqueous

aqueous

bi ol ogi cal

aqueous

reagent introduction by dialysis. speciation liquid membrane for cleanup and preconcentrati on calculation of

P

plasma Galactose, lactose, dihydroxyl actose Salicylic acid

urine, milk serum

Sul phi te, sulphide, snow Ami nes Sulphonamides

serum

P

98

84

102

357

Pump

C)

Knotted line

n

Stab iI %1 Carrier

Passive Pressurizcd membrane membrane reaclor reactor NH3 or NAM Na O H

Waste

Fig. 6 (a) Configuration for determination of glucose in serum with sample dialysis. (Reproduced from (ref. 97) with permission of Elsevier Science Publishers). (b) Scheme of the dialyserlflowinjection set-up used for determination of salicylic acid (IS€, tubular PVC ion-selective membrane electrode: WE, working electrode for potentiometric measurements; SCE, saturated calomel electrode: V, pH/mV meter; DC, dialysis chamber; mem, silicor,e rubber membrane; V and V flow-injection valves). (Reproduced from (ref. 98) w i 8 permigion of Elsevier Science Publishers). (c) Membrane-based FIA system for determination of sulphite, sulphide and methanethiol in water. A KCN stream is used when formaldehyde is also present. a, b, c, and d are 24 cm, 15 CIT: reactor included -, 39 cm reactor included - and 148 cm i n length, respectively (Reproduced from (ref. 100) with permission of the American Chemical Society).

-

3 58 A d i a l y s i s probe has been developed f o r continuous sampling from

complex s o l u t i o n s such as f e r m e n t a t i o n b r o t h , m i l k and waste water, aimed a t rendering them s u i t a b l e f o r a n a l y s i s by l i q u i d chromatography, flowi n j e c t i o n a n a l y s i s , enzyme c a l o r i m e t r y , e t c . The a n a l y t e i s t r a n s f e r r e d t o a f l o w i n g stream separated from t h e sample by a d i a l y s i s membrane t h a t i s p r o t e c t e d from f o u l i n g by a s t r o n g t a n g e n t i a l l y f l o w i n g stream o f t h e sample e s t a b l i s h e d by p l a c i n g a magnetic s t i r r i n g b a r c l o s e t o t h e membrane surface. The device i s c o n s t r u c t e d from m a t e r i a l s a l l o w i n g t h e probe t o be s t e a m - s t e r i l i z e d when mounted i n s i d e a fermentor ( r e f . 99). Oasgupta e t a l . (ref. 100) c a r r i e d o u t a study on t h e p r e s e r v a t i o n of s u l p h i t e , sulphate and methanethiol i n b u f f e r e d formaldehyde and o x a l dihydroxamic

a c i d s t a b i l i z e r s aimed a t developing a method f o r f a s t

determination

o f these anions on t h e b a s i s o f t h e i r

reaction with

N-acridinylmaleimide (NAM) i n a water/#, N-dimethylformamide medium t o y i e l d a f l u o r e s c e n t product. I n t h e c o n f i g u r a t i o n used, d e p i c t e d i n F i g . 6c, t h e reagent i s introduced by d i a l y s i s and t h e c a r r i e r i s pumped a t a r a t e o f 440 PI min” through t h e i n j e c t i o n v a l v e and sample l o o p i n t o a passive membrane r e a c t o r immersed i n a concentrated ammonia s o l u t i o n t o r a i s e t h e pH t o about 10 and then through a p r e s s u r i z e d porous membrane r e a c t o r immersed i n an NAM s o l u t i o n . A superincumbent a i r pressure of 11.5

psi

i s adequate f o r

equivalent t o about 44

introduction min-’

o f NAM a t

a suitable

rate,

o f conventional a d d i t i o n . The t y p i c a l

r e a c t i o n t i m e i s 50 s and t h e d e t e c t i o n l i m i t s achieved

f o r the three

above-mentioned s u l p h u r - c o n t a i n i n g compounds a r e 0.04, 0.60 and 0.80 1-1 M, r e s p e c t i v e l y . The system a l l o w s f o r d i f f e r e n t i a l a n a l y s i s t o be i m plemented

(ref.

100).

s p e c i a t i o n determination

These

authors

f o r peroxides

have

also

developed

another

(H202 and CH30H2) by use of

a p r e s s u r i z e d PTFE membrane r e a c t o r c o n t a i n i n g t h e enzyme peroxidase. The pH of t h e flowing stream i s s e t by i n t r o d u c i n g ammonia through a nonporous cation-exchange r e a c t o r ( r e f . 101). A c o n f i g u r a t i o n f o r sample cleanup and amine enrichment

i n a flow

system r e c e n t l y r e p o r t e d i n v o l v e s passing t h e sample through a l i q u i d membrane whereupon t h e a n a l y t e i s released and subsequently trapped by a stagnant acceptor phase on t h e o t h e r side. The r e s u l t a n t a n a l y t e p l u g i s then swept‘from t h e membrane s e p a r a t o r t o t h e d e t e c t i o n system The proponents p r o v i d e a t h e o r e t i c a l d i s c u s s i o n of t h e mass t r a n s f e r across t h e membrane and t h e i n f l u e n c e o f t h e t r a n s p o r t on t h e acceptor c o n c e n t r a t i o n p r o f i l e . S t r i c t l y , t h i s i s an example of mixed e x t r a c t i o n - d i a l y s i s . The enrichment f a c t o r achieved w i t h t h i s c o n f i g u r a t i o n , whose r e s u l t s compare well

with

t h e o r e t i c a l p r e d i c t i o n s , i s dependent upon t h e sample volume,

359 supporting m a t r i x , type o f i m m o b i l i z i n g s o l v e n t used, donor f l o w - r a t e and c o e f f i c i e n t o f p a r t i t i o n o f t h e a n a l y t e between t h e donor and t h e membrane phase ( r e f . 84). Other non-determinative a p p l i c a t i o n s o f d i a l y s i s have been r e p o r t e d by Macheras and Koupparis ( r e f . 102) and by Bernhandsson e t a l . ( r e f . 103). The former authors used an automated f l o w - i n j e c t i o n a n a l y s e r i n t e r f a c e d t o a dialysis

u n i t t o study d r u g - p r o t e i n

between some sulphonamides

binding interactions

and bovine serum albumin),

with

(e.g. results

s i m i l a r t o those obtained by o t h e r procedures. A complete run, i n c l u d i n g calibration,

takes

about

100 min.

The d i a l y s a b l e

sulphonamides

are

q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y by a m o d i f i c a t i o n o f t h e B r a t t o n Marshall method. The system a l s o a l l o w s c a l c u l a t i o n o f d i a l y s i s r a t e constants. Berhandsson e t a l . ( r e f . 103) have discussed t h e t r a n s f e r of mass i n infinite parallel

plate dialysers

with

co-flow

between

d e t e c t o r streams by a p p l y i n g t h r e e t h e o r e t i c a l models.

sample

and

These authors

d e r i v e d a n a l y t i c a l expressions f o r t h e coupled d i f f u s i o n and t r a n s f e r phenomena o c c u r r i n g i n b o t h channels and obtained numerical s o l u t i o n s f o r a laminar f l o w regime by t h e f i n i t e - d i f f e r e n c e approximation method. They a l s o considered t h e r e s u l s obtained by a mixing-cup model a p p l i e d under

.

steady -s t a t e cond it ions With t h e dimensions t y p i c a l o f a n a l y t i c a l d i a l y s e r s t h e r e were o n l y small d i f f e r e n c e s between t h e r e s u l t s p r o v i d e d by t h e l a m i n a r - f l o w and p ug-flow models. The mixing-cup model p r e d i c t e d h i g h e r f l u x e s through t h e membrane than t h e o t h e r two, p a r t i c u l a r l y w i t h increased channel heights

The t h e o r e t i c a l r e s u l t s were c o n s i s t e n t w i t h

those e x p e r i m e n t a l l y obtained i n t h e d i a l y s i s o f Z n ( I 1 ) i o n s and t h e f l o w dependence a l s o agreed reasonably w e l l w i t h t h e o r y p r o v i d e d t h a t t h 2 hydrostatic

pressures

were

equal

on

both

sides

and

that

stresses

p o t e n t i a l l y r e s u l t i n g i n membrane b u l g i n g were kept low (see Table V ) ( r e f s . 104 - 112). 5.

SOLID-LIQUID INTERFACES Separation techniques i n v o l v i n g s o l i d and l i q u i d phases have been used

i n c o n j u n c t i o n w i t h F I A almost s i n c e t h i s technique was introduced. While i o n exchange was o r i g i n a l l y t h e separation technique most f r e q u e n t l y used with

FIA,

adsorption

and

preconcentration

are

gradually

beco ing

commonplace i n t h i s context. 5.1

I O N EXCHANGE

has been p r e f e r e n t i a l l y used f o r preconcentration o f minor species from complex samples (e.g. i n d u s t r a1 I The FIA/ion exchange a s s o c i a t i o n

360 r a i n and sea water, s o l d e r i n g smokes, b i o l o g i c a l f l u i d s , e t c . ) , though i t has a l s o been employed f o r s e p a r a t i o n purposes and t o f a c i l i t a t e t h e determination o f d i f f e r e n t

a n a l y t e s i n t h e same sample by s e q u e n t i a l

e l u t i o n o f these, kept i n a s u i t a b l e a c t i v e agent ( r e f . 113). I n s o f a r as t h e analytes most f r e q u e n t l y determined a r e metal c a t i o n s , t h e commonest a c t i v e agents used a r e d i f f e r e n t types of c h e l a t i n g r e s i n . Table V I l i s t s t h e major species determined by methods i n v o l v i n g t h e F I A / i o n exchange association, c l a s s i f i e d according t o t h e t y p e o f a n a l y t e concerned i n t o c a t i o n i c species ( i n d i v i d u a l l y and i n m i x t u r e s ) , a n i o n i c species and conjugate acid-base p a i r s . A r e p r e s e n t a t i v e example o f t h e v e r s a t i l i t y of t h i s a s s o c i a t i o n and i t s ease of a d a p t a t i o n t o d i f f e r e n t problems l i e s i n t h e AAS determination o f heavy metals i n sea water proposed by Ruzicka e t al.

(ref.

114).

The metals a r e preconcentrated i n a c h e l a t i n g - r e s i n

microcolumn incorporated i n t o one of t h r e e c o n f i g u r a t i o n s o f d i f f e r i n g compexity t h a t these authors designed i n o r d e r t o overcome t h e problems successively encountered i n t h e i r experiments. A single-channel manifold f e a t u r i n g two s e r i e s o f i n j e c t i o n valves l o c a t e d p r i o r t o t h e column (Fig.

7a)

i s t h e simplest a l t e r n a t i v e f o r

concentration step.

implementation o f t h e pre-

The propel1 i n g system i s gas pressure-based.

The

c a r r i e r , ammonium acetate, d r i v e s t h e sample i n j e c t e d by means of v a l v e I1 t o t h e microcolumn through a c o i l and a by-pass o f v a l v e 12, where t h e

analytes a r e retained. The second s t e p i n v o l v e s i n j e c t i o n o f t h e e l u a n t through v a l v e I*. This c o n f i g u r a t i o n poses a s e r i e s o f problems such as t h e appearance o f a prepeak due t o the sample m a t r i x , a r i s i n g from changes

i n t h e r e s i n compactness

disturbances

i n changing from t h e

ammonium t o t h e p r o t o n from and t h e l a c k o f homogenization between sample and c a r r i e r i n t h e c e n t r a l zone o t t h e sample p l u g , which i s very a c i d i c and hinders r e t e n t i o n . These shortcomings were circumvented by u s i n g t h e c o n f i g u r a t i o n depicted i n Fig. 7b, w i t h a merging p o i n t f o r t h e ammonium acetate stream and e l u t i o n o f t h e a n a l y t e s i n t h e d i r e c t i o n opposing t h e retention

path

by

means

of

selecting

valves

whose

operation

is

i l l u s t r a t e d i n t h e f i g u r e . The a n a l y t i c a l procedure was automated by i t s proponents by u s i n g a m a n i f o l d w i t h a s i n g l e i n j e c t i o n v a l v e and a system c o n s i s t i n g o f two pumps and a t i m e r f o r s y n c h r o n i z a t i o n o f t h e o p e r a t i o n of t h e pumps (pumpl

acted d u r i n g t h e p r e c o n c e n t r a t i o n step, w h i l e pump2

worked d u r i n g t h e e l u t i o n ) . The sample m a t r i x never reached t h e d e t e c t o r i n e i t h e r case and t h e microcolumn was regenerated i n t h e e l u t i o n step. Townshend e t a l .

(refs.

115

-

116) have shown t h e v a s t p o t e n t i a l o f

a p p l i c a t i o n o f ion-exchange microcolumns i n F I A systems.

These authors

have developed a determination f o r Zn and Cd by u s i n g an exchange column

where both c a t i o n s , present i n t h e same sample, a r e r e t a i n e d t o be subsequently e l u t e d s e q u e n t i a l l y and t h e i r c o n c e n t r a t i o n determined i n d i r e c t l y through i n h i b i t i o n o f t h e c o b a l t - c a t a l y s e d

chemiluminescence

generation from luminol ( r e f . 115). These authors use t h e displacement o f t h i o c y a n a t e from a s t r o n g l y b a s i c ion-exchange r e s i n by o t h e r anions t o determine comon anions w i t h spectrophotometric d e t e c t i o n o f t h e i r o n (111)-thiocyanate

complex

formed.

N i t r a t e can be determined

in

the

presence o f c h l o r i d e and sulphate, which a r e removed by a precolumn packed w i t h a cation-exchange r e s i n i n s i l v e r form f o l l o w e d by a z i n c reductor. B i n a r y m i x t u r e s (e.g. c h l o r i d e and n i t r a t e ) can be determined simultaneously by s p l i t t i n g t h e sample i n t h e f l o w i n g system so t h a t p a r t of i t goes through t h e c h l o r i d e suppressor ( y i e l d i n g a response corresponding t o n i t r a t e alone) w h i l e t h e r e s t by-passes i t and g i v e s a response corresponding t o t h e sum o f c h l o r i d e and n i t r a t e . The j o i n t use o f i o n exchange and conversion techniques w i t h FIA has m a t e r i a l i z e d i n t h e o n - l i n e conversion o f s o l u b l e species t o i n s o l u b l e compounds by means o f a t a g m a t e r i a l which i s subsequently determined. This approach (Fig. atomic absorption

7) has been used t o determine s u l p h i d e by flame spectrometry w i t h t h e a i d o f

cadmium(I1)

as p r e -

c i p i t a t i o n t a g reagent. Excess Cd(I1) i s c o l l e c t e d on a c h e l a t i n g i o n exchanger and l a t e r eluted. The d e t e c t i o n l i m i t f o r s u l p h i d e i s 10 cg/l and t h e sampling r a t e achieved i s 100 samples hr-'

, t h e t y p i c a l standard

d e v i a t i o n being 1.2%. O f a l l p o t e n t i a l i n t e r f e r e n t s , o n l y phosphate has any e f f e c t on t h e determination (ref.117).

101, 118) use i o n exchange i n a r a t h e r - t h e r e q u i r e d pH change i s e f f e c t e d by i n t r o d u c i n g ammonia through a non-porous cation-exchange membrane r e a c t o r . Hwang and Dasgupta ( r e f s .

uncommon fashion i n t h e i r peroxide d e t e r m i n a t i o n

A novel, h i g h l y i n t e r e s t i n g c o n t r i b u t i o n t o t h i s area i s represented by t h e use o f i n t e g r a t e d microconduits ( r e f s . 119 - 137) (see Table V I ) .

5.2

ADSORPTIVE PRECONCENTRATION

The a d s o r p t i v e p r e c o n c e n t r a t i o n o f a n a l y t e s i n FIA has so f a r been t a c k l e d w i t h two c h i e f

a c t i v e agents,

namely a c t i v a t e d alumina and

e l e c t r o d e surfaces (carbon paste o r p l a t i n u m ) . Adsorption on a l u m i n i a has been used f o r preconcentration

o f chromic

i o n i n b i o l o g i c a l samples

( u r i n e ) p r i o r t o i t s d e t e r m i n a t i o n by ICP-AES ( r e f . 138) i n t h e s p e c i a t i o n o f chromium ( r e f . 139), as w e l l as f o r t h a t o f oxyanions such as

arsenate,

vanadate w i t h

borate, the

chromate,

molybdate,

phosphate,

selenate

and

same d e t e c t i o n system. Preconcentration on a carbon

362

paste electrode prior to the voltammetric determination of the analyte has been used in the analysis for drugs such as chlorpromazine (ref. 140) and doxorubicin (ref. 141) in urine. The pulsed amperometric determination of electroinactive adsorbates such as chloride and cyanide at platinum electodes (ref. 142) is but another proof of the FIA/adsorptive preconcentration association. An activated aluminia microcolumn has been used for separation and preconcentration of Cr(V1) from Cr(II1) in synthetic aqueous solutions prior to ICP detection at 267.72 nm, yielding a linear calibration graph between 0 and 1 000 ~ / lof Cr(V1) or Cr(III), with relative standard deviations at the 10 cg/l level of 2.2% and 1.1% for Cr(II1) and Cr(VI), respectively, for a 2-ml sample, the corresponding detection limits being 1.4 and 0.20 @/1, respectively. The procedure has been applied to the determination of both chromium forms at the cg/1 level in reference NBS water (ref. 139). Preconcentration and quantitation of doxorubicin, a cancer chemotherapy drug, are accomplished by a flow-injection approach involving adsorption of the drug onto a carbon paste electrode, medium exchange and differential voltammetry on the adsorbing surface (ref. 141). Linear response is obtained for concentrations from 10- 6M to the detection limit (loq9#). No preliminary steps are required for determination of the drug in urine by direct injection. Chlorpromazine can be determined under similar conditions in the presence of a tenfold excess of non-adsorbable species with similar redox potentials. The preconcentration step also results in increased sensitivity. A special type of adsorptive presoncentration is that of electroinactive species on Pt electrodes (ref. 142). Thus, chloride and cyanide modify the rate of surface oxide formation following a positive potential step. Hence, triple-step potential waveforms similar to those used successfully for pulsed amperometric detection of electroinactive adsorbates (e.g. alcohols, carbohydrates and amino-acids) can also be applied t o electroinactive adsorbates injected into an electrolyte stream. Depending o f the wave form, the overall anodic current at the detection peak will be greater or less than the baseline signal corresponding to oxide formation in the absence of the adsorbate. The sensitivy achieved i s very high indeed (ref. 143 and Table VII).

TABLE V I

Features of FIA methods involving ion-exchange processes

Anal yte

Matrix

Analytical purpose

NH3

rain water

seDaration

river water

Ni Ca cu soldering smokes

Zn, Cd

Detection

Amber1 i te- R120

Amberlite IRA-400 preconcentration chelating resin separation preconcentration 8-quinolinol separation preconcentration Dowex A-1 chelating resin chelating resin selectivity simultaneous Amberlite IRA-400 determination preconcentration Chelex-100 ,I

Mn, Pb, Cu

Pb, Cd, Ba, Be, Cu, Mn, Ni, Cu,

Active agent

Cd, Pb, Cu, Zn

I

tap, sea, polluted water sea water I,

Cd Ti, V , Al, Cr, F Cd, Co

,I

chelating resin

I1

Chelex-100

AAS

Ref.

column in samole 121 loop. Alternating h o w 122 123 multi-function valve 124 125

P

series injection valves 126 (sample-eluent) study of resin 49

chem.

sequential elution

115

AAS integrated microconduits 119 ICP-AES simultaneous 129 determination AAS two alternate columns 128

series injection and

114

selecting valves series injection and selecting valves

120

Chelex-100 and 8-quinolinol Chelex-100

AAS

129

,I

muromac A-1

ICP-AES

130

I1

tri PEN

FAA

131

8-quinolinol

AAS

TKS-gel SAX

P

,I

reference materi a1 s

F AAS

I,

,I

Cu Cd, Co Ni, Pb Pb, Cd

P

Speci a1 features

I,

indirect determination simultaneous detennination

continuous precipitation

117 132 W

m W

w m

P

Various anions sea water, serum, chlorinated reagents

Br-

Acid-base pairs Polyphosphates H202, CH30H2

rain water

H202

water

___ _ _

simultaneous determination separation

salt removal determination separation NH idroduct ion separation

bas i c resi n

,I

116

Amber1 i te XAD-2

,I

133

resin TXK-gel SAX Nafion 811X cation-exchange membrane Diaion SK-1

cond P F

P

.

hydrolytic catalysis enzyme membrane

134 135 136 118 137

____ __ cord.: condtictimelry ICP-AES: inductively coupled plasma-atomic emission spectroscopy

I__ _ _ _ _ I

F: fl:iorimct.j triPEN: N, N, N-tri(2-pyridylmethy1)ethylene

diamine

365

b) W

Fig. 7

q i m L Iminl

W

-

-

FIA/ion-exchan e c o n f i g u r a t i o n s o f v a r i a b l e increasing complexity. (a7 Single-channel manifold. (b) With e l u t i o n [a] i n t h e reverse d i r e c t i o n o f r e t e n t i o n [b]. (c) Automated m a n i f o l d . (Reproduced from ( r e f . 114) w i t h permission o f t h e Royal S o c i e t y o f Chemistry).

366

TABLE V I I Features of FIA methods involving adsorptive preconcentration Analytical Analyte

Matrix

pIrpose

Cr(II1)

urine

scpsrat im

Cr(III), Cr(VI1)

reference

Active

Detection

agent

Special

Ref.

features

138

ICP-AES

activated alumina

water Oxyanions

scparatim, prewncentrat ion prewncentrat ion,

u r i ne

speciatim

"

139 140

'I

atunina

sepsrat im Ch lorpromazi ne

activated alumina activated

preconcentrat i o n

carbon paste

volt

141

"

142

anp.

143

electrode Doxorubicin

carbon paste electrode

P t electrode

deterni M t in

Cl-

CN-,

TABLE V I I I

Analyte

Features o f FIA methods involving precipitation-dissolution

Matrix

Analytical wrpooe

Active agent ~

NH

3

, Cl-,

1042-

Detection

~~

theoretical

Special features

Ref.

~~

3 t +

Fe , Ag 2+ Ca

,

MS

with and w i t h w t

145

continuous prec i p i t a t e dissolution

c1-

waters

determination

~ g +

MS

normal and

foodstuff

siwltaneous

"

MS

mixture re-

147

reversed F I A c1-,

1-

solution

148

determinat ion Pb2+

uatars

preconcentratim NH

3

AAS

sample aspiration 149

367 5.3

PRECIPITATION AND DISSOLUTION

This separation technique, o f widespread use i n c l a s s i c a l a n a l y t i c a l chemistry applications, has been scarcely automatized owing t o t h e i n t r i n s i c d i f f i c u l t i e s involved. F i l t r a t i o n through a piece o f paper moving a t r i g h t angles t o t h e flow has been used i n air-segmented methods f o r cleanup o f samples p r i o r t o i n t r o d u c t i o n i n t o the system and i n some methods i n v o l v i n g t h e a n a l y t e p r e c i p i t a t i o n (e.9. tetraphenyl

t h e determination o f potassium i n f e r t i l i z e r s w i t h

borate and

photometric monitoring

of

the

precipitating

reagent ( r e f . 144)), though w i t h n o t t o o b r i l l i a n t r e s u l t s . Recently the authors have shown the p o s s i b i l i t y t o o b t a i n s a t i s f a c t o r y r e s u l t s from precipitation/dissolution processes implemented i n continuous unsegmented systems (Table V I I I ) . I n Fig. 9 i s shown t h e operational scheme o f an F I A c o n f i g u r a t i o n f o r i n d i r e c t AA determination o f anionic species incorporating a conventional HPLC cleanup f i l t e r . The system uses three valves; one f o r i n j e c t i o n ( I V ) o f t h e sample c o n t a i n i n g the anion; another f o r a l t e r n a t i n g i n t r o d u c t i o n o f washing and prec i p i t a t e solvent s o l u t i n s (SV1) and a t h i r d , four-way d i v e r t i n g one (SV2) f o r d i r e c t i n g streams t o t h e d e t e c t o r o r t o waste.

-

S,

Carrier

Eluent .-c

W

CdS lCd2*)

Carrier

Fig. 8 Configuration f o r p r e c i p i t a t i o n o f t h e analyte (sulphide) w i t h cadmium (11). The p r e c i p i t a t e c i r c u l a t e s f r e e l y along t h e system and i s detected by AAS. Excess Cd(I1) i s removed by t h e i o n exchange column ( I E C ) and subsequently eluted. (Reproduced from ( r e f . 117) w i t h permission o f E l s e v i e r Science Publishers).

In the p r e c i p i t a t i o n - f i l t r a t i o n step (Fig. 9a) , the sample i s i n j e c t e d i n t o the reagent-carrier.

A f t e r p r e c i p i t a t i o n , t h e f l o w i n g stream i s

passed by SV2 through the f i l t e r ,

where t h e p r e c i p i t a t e i s retained.

Since the cation-reagent stream continuously reaches t h e d e t e c t o r i n t h i s f i r s t stage, t h e signal (baseline) i s q u i t e high. On passage through t h e

368 detector, the precipitate fluid zone yields a negative signal (peak) proportional to the analyte concentration and corresponding to the reagent disappearance. Thus, no additional step is required - and valves SV1 and SV2 can be dispensed with - when the precipitate is relatively pure. A diluent stream later merged at the filter is occasionally needed when the reagent concentration in the carrier required to ensure precipitation is too high to be directly introduced into the atomic absorption spectrophotometer. In the wash step (Fig. 9b), valve SV1 introduces the washing solution, which is passed through the filter via SV2 and later led to the detector, where it gives a parasitic, nonanalytical signal corresponding to the adsorption (contamination) of the cation-reagent on the precipitate surface. Valve SV2 then sends the flow emerging from the precipitation coil to waste. In the dissolution step generally acidic (Fig. 9c), valve SV1 is switched to introduce a solvent. The remaining components act as in the previous operation. On passage of the stream through the filter, the freshly precipitated small mass is rapidly dissolved. A flow plug contains the stoichiometric amount of cation-reagent present in the precipitate. On passing through the detector, this zone yields a transient signal (peak) obviously proportional to the amount of precipitated anion-analyte. These continuous precipitation/filtration systems have been used with three types of precipitate: gelatinous (Fe203.xH20, with Fe3+ as carrier and NH3 samples), curdy (AgC1, with Ag+ as carrier and C1- samples) and crystalline (CaC204.2H20, using Ca2+ as carrier and C2042- samples). Recoveries close to 100% are achieved in every case, even at low analyte concentrations (ref. 145). Interferents are much less disturbing than in the classical precipitation-filtration procedure, probably as a result of the decreased precipitation and digestion times involved (ref. 146). By use of two FIA configurations (normal and reversed), continuous precipitation has been applied to the determination of chloride in different types of water with Ag(1) as reagent (ref. 147). Chloride and iodide have also been determined in various foods and drinks by using a configuration involving washing and sequential dissolution of the precipitates with ammonia and nitric acid for C1- and I-, respectively (ref. 148). These assemblies also allow the implementation of on-line preconcentration with the measuring device used. Such is the case with the determination of lead traces in various types of water (ref. 149). The direct aspiration of the sample into the flame of the atomic absorption spectrophotometer yields no signal. However, the set-up depicted in Fig. 10

-

369 a1 lows preconcentrating t o t h e extent required by continuously prec i p i t a t i n g the lead as a basic s a l t . concentration step. The sample stream

Only pump 2 works i n t h e pre-

-

aspirated r a t h e r than i n j e c t e d

-,

i s merged w i t h a p r e c i p i t a t i n g reagent stream (NH3) and t h e m i x t u r e i s l e d by SV2 t o the p r e c i p i t a t i o n c o i l and onto t h e f i l t e r , where t h e p r e c i p i t a t e formed i s r e t a i n e d and trough which t h e f l o w goes t o waste.

I n the d i s s o l u t i o n step, pump 2 i s stopped and a n i t r i c a c i d stream i s introduced i n t o the system by pump 1, valve SV2 leading t h e f l o w t o t h e detector. The n i t r i c acid r a p i d l y dissolves t h e p r e c i p i t a t e b u i l t up on the f i l t e r and a p o s i t i v e peak p r o p o r t i o n a l t o t h e amount o f a n a l y t e contained i n t h e aspirated sample volume i s yielded.

Lead can thus be

-

determined over a wide concentration range (1.2 1 500 ng/ml), w i t h a The p o t e n t i a l i n t e r f e r e n c e from

maximum preconcentration f a c t o r of 103

.

other t r a n s i t i o n metal ions a l s o p r e c i p i t a t e d and dissolved i n t h e process i s overcome by using t h e c h a r a c t e r i s t i c spectrum l i n e o f lead. HPLC-FIA ASSOCIATION

6.

Though a number o f papers i n t h e l i t e r a t u r e have HPLC and F I A among t h e i r keywords, few o f them a c t u a l l y r e p o r t on the o n - l i n e coupling o f both

techniques.

The

development

of

novel

continuous

hydrodynamic

detection systems o r the improvement o f those already e x i s t i n g involves

150). Also, p a r t s o f a l i q u i d chromatograph - separation column excluded - have been used i n F I A determinations, although t h i s a l t e r n a t i v e i s n o t r e commendable because o f t h e increased cost o f components designed t o withstand high pressures and o f t h e need f o r one high-pressure pump per channel ( r e f . 151). S t r i c t l y , o n l y when there i s a complementary p r e o r post-column i n j e c t i o n should t h e p o s s i b i l i t y o f an F I A system being coupled t o a l i q u i d chromatograph be considered. Thus, t h e basic components o f an on-1 i n e coupled HPLC-FIA system are two i n j e c t i o n valves, two pumps, a chromatographic column, a reactor and a continuous detector, i n a d d i t i o n t o t h e both techniques on account o f t h e i r common features ( r e f .

usual r e s e r v o i r s f o r the e l u e n t ( s ) , c a r r i e r ( s ) and reagent(s). R e s t r i c t o r c o i l s are also frequently used t o prevent t h e formation o f a i r bubbles.

I n Fig. 11 are i l l u s t r a t e d the t h r e e general manners i n which t h i s association can be experimentally implemented according t o t h e p o s i t i o n of

the i n j e c t i o n valve w i t h i n t h e F I A subsystem:

(a)

precolumn;

(b)

post-column w i t h i n j e c t i o n p r i o r t o merging o f t h e c a r r i e r o r reagent stream w i t h the chromatographic e f f l u e n t and (c) post-column w i t h t h e F I A valve placed a t the merging p o i n t i t s e l f .

3 70 a)

,

Sample

onion Reagent cation

Atomic

cbsorptlon

\

_ _ _ - ---'

5v1

-- -

- - -- -

- - - --

4 -1

- - - J

t

\

waste

4

S

l

V

"

Atomic a bsor p t i an

\

d)

Fig. 9 Continuous precipi tation/di ssol ut ion configurations for i ndi rect determination of anions by AAS. (a) Precipitation; (b) washing; (c) dissolution; (d) transient signal obtained in each step. For further details, see text.

371

PUMPS

\

0

-

NHI

Atomic absorption

-

Fig. 10 Continuous on-line system f o r preconcentration o f lead traces i n water p r i o r t o i t s determination by AAS w i t h continuous prec i p i t a t i o n (pump 2 i n operation) and p r e c i p i t a t e d i s s o l u t i o n (pump 1 i n operation). (Reproduced from ( r e f . 149) w i t h permission o f t h e Royal Society o f Chemistry).

6.1

PRE-COLUMN ASSEMBLIES Continuous pretreatment

(sample conditioning,

preconcentrat i o n and

interference removal) and d e r i v a t i z a t i o n systems applied p r i o r t o sample i n t r o d u c t i o n i n HPLC are o f special relevance whenever t h e p o s s i b i l i t y o f automatization i s involved. The most outstanding advances i n t h i s area have arisen from the use o f pre-columns packed w i t h an a c t i v e m a t e r i a l and coupled t o assemblies c o n s i s t i n g o f several r o t a r y valves f o r sample cleanup and t r a c e enrichment ( r e f s . 152, 153). L i q u i d - l i q u i d e x t r a c t i o n has occasionally been applied p r i o r t o 1i q u i d ( r e f . 154) o r gas chromatography ( r e f . 155). Indeed few FIA-HPLC systems use the F I A valve p r i o r t o t h e chromatographic column

(Fig.

lla).

A

7

t y p i c a l example i s the determination o f zinc i n t h e range 2 10-7-20 10- M based

on

its

activating

effect

on

metal-free

carboxypeptidase

A

immobilized i n a r e a c t o r ( r e f , 157) (Fig. 12). A d i v e r t i n g valve allows switching between water and regenerating s o l u t i o n (1,lO-phenanthrol ine) streams, where 500-pl samples containing t h e analyte and t h e substrate

(hippuryl-L-phenylalanine, t h e decomposition products o f which are sensed by reversed-phase 1 i q u i d chromatography) are injected. The two i n j e c t i o n valves used are connected o n - l i n e t o each o t h e r and a volume o f 10 pl i s i n j e c t e d i n t o t h e chromatograph 30 sec a f t e r sample i n j e c t i o n ( c a r r i e r flow-rate 1 ml/min).

372

S

bl

Reactor ----

VOlVQ

Confluence point

W

Cl S

R

W

Fig. 11 Generic types of HPLC-FIA assemblies. Precolumn flow injection (a); Post-column flow injection with valve located prior to (b) or at the point of merging (c) of the chromatographic eluate and the carrier or reagent. C, carrier; R, reagent; S, sample; D , continuous detector; W , waste.

373

P detector

F i g . 12 O p era t i o n a l scheme o f an FIA system used p r i o r t o a chromatog r a p h i c s e p a r a t i o n ( d e t e r m i n a t i o n o f z i n c t hrough i t s a c t i v a t i n g e f f e c t on an enzyme i m m o b i l i z e d i n t h e r e a c t o r . F o r d e t a i l s , see t e x t . (Reproduced f r o m ( r e f . 156) w i t h permisssion o f E l s e v i e r Science P u b l i s h e r s ) . 6.2

POST-COLUMN ASSEMBLIES

Post-column r e a c t i o n d e t e c t o r s a r e t h e commonest way o f implementing o n - l i n e d e r i v a t i z a t i o n i n HPLC aimed t o improve o r f a c i l i t a t e d e t e c t i o n ( r e f . 157). According t o F r e i e t a l . ( r e f . 158), one o f t h e c h i e f shortcomings o f t h e s e c o n f i g u r a t i o n s i s t h e need f o r post-column reagent addition.

The r e a g e n t can b e i n t r o d u c e d

namely (a)

i n t h r e e d i f f e r e n t manners,

i n a continuous stream merged w i t h t h e e f f l u e n t f rom t h e

chromatographic column;

(b) b y i n j e c t i o n i n t o a c a r r i e r l a t e r merging

w i t h t h e e f f l u e n t and ( c ) by means o f a s o l i d - p h a s e r e a c t o r where t h e reagent

-

generally a catalyst

-

i s i m m o b i l i zed. A f u r t h e r pump i s needed

i n t h e f i r s t two cases t o s e t t h e r e a g e nt o r c a r r i e r f l o w . post-column

pumpless

reaction

units

include

O t her

electrochemical,

photochemical and thermal sensing. Several FIA systems i n which i n j e c t i o n can be s u b s t i t u t e d by merging w i t h t h e chromatographic e f f l u e n t have been described overall

analyte concentration

th ro ugh t h e

FIA

( F i g 13).

The

i s determined by i n j e c t i n g t h e sample

v a l v e whereas d i s c r i m i n a t i o n between d i f f e r e n t a n a l y t e s

374 (multidetermination) is accomplished by acommodating the effluent in the post-column system, which uses no injection valve and thus acts as an open-tube reaction detector. Inorganic polyphosphates (ref. 159), pol)’phosphoric acids in phosphorous smokes (ref. 160), phosphate and phosphonate (with two parallel (ref. 161) or series (ref. 162) photometric detectors) and the complexing abilities of ligand for metal ions (refs. 163, 164) have been determined with these dual configurations. A real post-column on-line HPLC-FIA configuration is only justified when specific problems are involved. Such is the case with the determination of phosphinate, phosphonate and phosphate (ref. 165) , in which sodium bisulphite is previously required to oxidize P ( 1 ) and P ( I I 1 ) to P ( V ) , the species ultimately responsible for the analytical reaction with the chromogenic reagent - Mo(V)-Mo(1V). As the sulphite solution tends to corrode stainless steel and disturb the flow-rate of the reciprocating pump, it is introduced with a loop-valve injector to avoid contact with the pump.

\

/

CAREIER

I

3

I

‘2-

I W

REACTOR

REAGENT

-

W

Fig. 13 Alternate use of a flow-injection valve or the chromatographic effluent for individual and mu1 t i -determinations, respectively , wi th the same f 1 ow-i n ject i o n react i on/detect i on system. Another interesting way to couple an FIA system after a liquid chromatograph involves filling the flow-injection valve with the chromatographic effluent and introducing microvolumes of this into a reagent or carrier stream at regular intervals (Fig llc). The automatic functioning of the valve is obviously mandatory in this use. Mixtures of reducing sugars (ref. 116) and amino-acids have been resolved photometrically and amperometrical ly, respectively, with configurations involving the syn-

375 chronized operation of the two injection valves. In this respect it is worth noting the possibility to use an FIA assembly as the interface between a 1 iquid chromatograph and an atomic absorption spectrophotometer (ref. 168). In Fig 14 is shown the scheme o f the configuration developed for studying metal-ligand binding in clinical samples. It involves dual continuous detection; photometric of molecular species (citrate, albumin) and atomic absorption spectrophotometric of metal ions (Ca2+ and Mg2'). This configuration allows for individual optimization of both integrated processes (HPLC-photometric detection and FIA-atomic absorption detection). The serum samples used (250 p l ) are manually introduced into the HPLC injector, while the flow-injection valve sequentially and automatically introduces 11 pl of the effluent into the carrier with a delivery time of 5 sec (6 cycles per minute). The atomic spectroscopic detector gives a chromatographic 'peak' whose profile is formed by the maxima of the FIA peaks. Both chromatograms are recorded by a dualchannel recorder.

Gel per me a ti on

column

-

Fig. 14 Use o f a dual-detection - FIA system as interface between a 1 iquid chromatograph and an atomic spectroscopic detector in the study of metal-ligand binding interactions in clinical samples. For details, see text.

376 7.

FINAL REMARKS

Continuous separation techniques have so far been used Only occasionally in F I A - only in about 10% of all instances as can be seen from Fig. 15a. Liquid-liquid extraction is the separation technique most commonly used with this methodology. Roughly 20% of the work dealing with the joint use of F I A and separation techniques involved ion-exchange microcolumns. Gas-diffusion was used in a similar proportion, while dialysis was employed to a lesser extent. b)

0)

4

CWTINUOUS SEPARATION TECHNIWES

Fig. 15 Statistics on the use o f separation techniques with FIA. Membrane separation (Fig. 16) of molecules (dialysis), gases (gas diffusion) and immiscible liquids (extraction) was the foundation of over 60% of the continuous separation processes developed by FIA to date (ref. 169).

GAS- OlFFUSlON

0 1ALYS IS

LIQUID- LlQUlD EX TRACT ION

ANALYTICAL STREAM (L21 MEMBRANE SAMPLE STREAM I L I I

GAS

MOLECULES OR IONS

Fig. 16 Membranes used in continuous non-chromatographic separation techniques.

377

In addition to the use of solid-liquid interfaces for implementation of some analytical procedures, the chief purpose of the joint use of separation techniques and segmented flow systems is to improve sensitivity (preconcentration) and selectivity (sample cleanup, mu1 t i determinations), and in some cases - to improve or facilitate the analytical reaction and/or detection, otherwise unfeasible. Another advantage of this association over batch non-chromatograpic separation techniques lies in the higher sampling rates achieved, which is of great relevance to routine determinations. These separation processes carried out in a continuous fashion are intermediate, both kinetically and thermodynamically, between batch processes, where equilibrium is reached once or several times, and chromatographic processes, in which equilibrium is attained many times. It is interesting to note the decisive role played by kinetics in these continuous separation processes. As a rule, physico-chemical equilibrium is not reached by the time detection is performed, in contrast with batch and air-segmented continuous flow methods. This should result in decreased precision; yet, the relative standard deviations obtained by batch and air-segmented methods and by FIA are comparable. On the other hand, the kinetic discrimination afforded by the continuous methodology results in enhanced selectivity (ref. 1491, to the detriment of sensitivity - this should not be too much of a worry if the separation process involved is intended for preconcentration purposes. Despite their proven advantages, few FIA systems have been used in conjunction with continuous separation techniques so far. This can be attributed to the occurrence of a number of deterrent experimental factors influencing these dynamic systems. Nevertheless, it would suffice to test any of the above-described configurations to immediately realize the scarce technical and instrumental difficulties involved. Applications in this field will no doubt increase significantly in the years to come, particularly in clinical, food and environmental analysis, where the sample matrix and low analyte concentrations usually dealt with are decisive factors.

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8. ACKNOWLEDGEMENT

The authors wish to acknowledge the support of the comision Interministerial de Ciencia y Technologia (Grant no. PA 86-0146) for research on this topic.

378 REFERENCES

1 2 3

8 9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

M. Valcercel and M.D. Luque de Castro, Automatic Methods o f Analysis, Elsevier, Amsterdam, 1988. C.F. Simpson, Ed., Techniques i n L i q u i d Chromatography, J. Wiley, New York, 1982. J.C. Giddings, K.A. G r a f f , K.D. Caldwell and M.N. Myers, Advances i n Chemistry Series, C.D. Craver, Ed., Amer. Chem. SOC., Waschington D.C., 1983. J.C. Giddings, Anal. Chem., 53 (1981), 1170A. W.B. Furman, Continuous Flow Analyis. Theory and Practice, Marcel Oekker, New York, 1976. J. Ruzicka and E.H. Hansen, Flow I n j e c t i o n Analysis, J. Wiley, New York, 1981. M. Valcarcel and M.D. Luque de Castro, Flow I n j e c t i o n Analysis: P r i n c i p l e s and Applications, E l l i s Horwood, Chichester, 1987. J. Ruzicka and E.H. Hansen, Anal. Chim. Acty, 179 (1986) 1. G. Den Boef and R.C. Schorthorst, Anal. Chim. Acta, 180 (1986) 1. L. Anderson, Anal. Chim. Acta, 110 (1979) 123. H.A. Mottola, Anal. Chim. Acta, 145 (1983) 27. D. Jagner, M. Josefson and K. Aren, Anal. Chim. Acta, 141 (1982) 147. J.A. Wise and W.R. Heineman, Anal. Chim. Acta, 172 (1985) 1. J. Wang and H.D. Dewald, Anal. Chim. Acts, 162 (1984) 189. E.A.G. Zagatto, B.F. Reis, H. Bergamin F and F.J. Krug, Anal. Chim. Acta, 109 (1979) 45. G.E. Pacey, D.A. H o l l o w e l l , K.G. M i l l e r , M.R. Straka and G. Gordon, Anal. Chim. Acta, 179 (1986) 259. H. Bader and J. Joigne, Water Res., 15 (1981) 71. D.H. Hollowell, Diss., Miami Univ., Oxford, OH, (1985). G.B. M a r t i n and M.E. Meyerhoff, Anal. Chim. Acta, 186 (1986) 71. G. Svensson and T. A n f i i l t , C l i n . Chim. Acta, 119 (1982) 7. H. Baadenhuijsen and H.E.H. Seuren-Jacobs, C l i n . Chem., 25 (1979) 443. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 173 (1985) 3. M.E. Meyerhoff, and Y.M. F r a t i c e l l i , Anal. L e t t . , 14 (1981) 415. J. Moeller and B. Winter, Fresenius Z. Anal. Chem., 320 (1985) 451. D.A. Hollowell, G.E. Pacey and G. Gordon, Anal. Chem., 57 (1985) 2851. M.R. Straka, 6. Gordon and G.E. Pacey, Anal. Chem., 57 (1985) 1799. P. Marstorp, T. A n f a l t and L. Anderson, Anal. Chim. Acta, 149 (1983) 281. D.A. Hollowell, J.R. Gord, G. Gordon and G.E. Pacey, Anal. Chem., 58 (1986) 1524. M. Granados, S. Maspoch and M. Blanco, Anal. Chim. Acta, 179 (1986) 445. C. Okumoto, M. Na ashima, S. M i z o i r i , M. Kazama and K. Akiyama, Eisei Kagaku, 30 $1984) 7 B. P i h l a r and L. Kosta, A i a l . Chim. Acta, 114 (1980) 276. 0. Astrom, Anal. Chem., 54 (1982) 190. G.E. Pacey, M.R. Straka and J.R. Gord, Anal. Chem., 58 (1986) 504. D.D. Siemer, P. Koteel and V. Jariwala, Anal. Chem., 48 (1976) 836. F.D. Pierce and H.R. Brown, Anal. Chem., 48 (1976) 693. F.D. Pierce and H.R. Brown, Anal. Chem., 49 (1977) 1417. M. Yamamoto, M. Yatsuda and Y Yamamoto, Anal. Chem., 57 (1985) 1382. R.R. Liversage and J.C. van Loon, Anal. Chim. Acta, 161 (1984) 275. M. Burguera and J.L. Burguera, Analyst, 111 (1986) 171. J.C. De Andrade, C. Pasquini, N. Baccan and J.C. van Loon, Spectrochim. Acta, Part B, Oct. 38 (1983) 1329. N.H. Tioh, Y. I s r a e l and R.M. Barnes, Anal. Chim. Acta, 184 (1986) 205.

379 42

S.M. Ramasamy, M.S.A. Jabbar and H.A. M o t t o l a , Anal. Chem., 52 (1980) 2062. 43 M.D. Luque de Castro, J . Autom. Chem., 8 (1986) 56. 44 B . K a r l b e r g , Anal. Chim. Acta, 180 (1986) 16. 45 B. K a r l b e r g aad S. Thelander, Anal. Chim. Acta, 98 (1978) 1. 46 H. Bergamin F , J.X. Medeiros, B.F. Reis and E.A.G. Z agat t o, Anal. Chim. A c t a , 101 (1978) 9. 47 M. G a l l e g o and M. V a l c B r c e l , Anal. Chim. Acta, 169 (1985) 161. 48 J . Kawase, Anal. Chem., 52 (1980) 2124. 49 A. D e r a t i n i and B. S e b i l l e , Anal. Chem., 53 (1981) 1742. 50 J.L. Burguera, M. Burguera, L. Cruz and O.R. Naranjo, Anal. Chim. Acta, 186 (1986) 273. 51 K. Kina, T. S h i r a i s h i and N. I s h i b a s h i , T alant a, 25 (1978) 295. 52 P. L i n a r e s , F. Ldzaro, M.D. Luque de C ast ro and M. V a l c a r c e l , Anal. Chim. A c t a , 200 (1987). 53 M.H. Memon and P.J. Worsfold, Anal. Chim. Acta, 183 (1986) 179. 54 A. R ios , M.D. Luque de C a s t r o and M. V a l c i i r c e l , Anal. Chem. ( i n pre s s ). 55 D.C. S h e l l y , T.M. Rossi and I.M. Warner, Anal. Chem., 54 (1982) 87. 56 T.M. R os s i , D.S. S h e l l y and I.M. Warner, Anal. Chem., 54 (1982) 2056. 57 M. Bengtsson and G. Johansson, Anal. Chim. Acta, 158 (1984) 147. 58 L. Nord and B. K a r l b e r g , Anal. Chim. Acta, 164 (1984), 233. 59 J.A. Sweileh and F.F. C a n t w e l l , Anal. Chem., 57 (1985) 420. 60 F.F. C ant w e l l and J.A. Sweileh, Anal. Chem., 57 (1985) 329. 61 K. Backstrom, L.G. D a n i e l s s o n and L. Nord, Anal. Chim. Acta, 169 (1985) 43. 62 L. Nord and B. K a r l b e r g , Anal. Chim. Acta, 125 (1981) 199. 63 T. Imasaka, T. Harada and N. I s h i b a s h i , Anal. Chim. Acta, 129 (1981) 195. 64 J.T. Davies and E.K. R i d e a l , I n t e r f a c i a l Phenomena, 2nd edn., Academic Press, New York, 1963, Chapter 1. 65 A.W. Adamson, P h y s i c a l Chemistry o f Surfaces, 2nd edn., I n t e r s c i e n c e , New York, 1967, Chapters 1 and 7. 66 J.L. Burguera and M. Burguera, Anal. Chim. Acta, 153 (1983) 207. 67 L. Fossey and F.F. C a n t w e l l , Anal. Chem., 55 (1983) 1882. 68 J. Kawase, S. Nakae and M. Yamanaka, Anal. Chem., 51 (1979) 1640. 69 K. Ogata, K. Taguchi and T. I m a n a r i , Anal. Chem., 54 (1982) 2127. 70 B. K a r l b e r g i n Chemical D e r i v a t i z a t i o n i n A n a l y t i c a l Chemistry Vol. 2, S e para t i o n and Continuous Flow Techniques, R.W. F r e i and J . F. Lawrence, Plenum Press, New York, 1982. 70a C. de R u i t e r , J.H. Wolf, K.A.Th. Brinkman and R.W. F r e i , Anal. Chim. A c t a 192 (1987) 267. 7 1 L. Nord and B. K a r l b e r g , Anal. Chim. Acta, 145 (1983) 151. 72 K. Ogata, S. Tanabe and T. I m a n a r i , Chem. Pharm. B u l l , 31 (1983) 1419. 73 M. G allego, M. S i l v a and M. V a l c i i r e l , Anal. Chim. Acta, 179 (1986) 439. 74 Y. Sahlestom, S. Twengstrom and B. K a r l b e r g , Anal. Chim. Acta, 187 (1986) 339. 7 5 0. K l i n g h o f e r , J . Ruzicka and E.H. Hansen, T alant a, 27 (1980) 169. 76 F. Ldzaro, M.D. Luque de C a s t r o and M. V a l c a r c e l , Anal. Chim. Act a, 169 (1985) 132. 77 P. L i n a r e s , M.D. Luque de C a s t r o and M. V a l c a r c e l , Anal. Chim. Acta, 161 (1984) 257. 78 L. Fossey and F.F. C a n t w e l l , Anal. Chem., 57 (1985) 992. 79 K. Ogata, K. Taguchi and T. I m a n a r i , Bunseki Kagaku, 31 (1982) 641. 80 K. Ogata, K. Taguchi and T. I m a n a r i , Bunseki Kagaku, 31 (1982) 89. 8 1 L. Sun, L. L i and Z. Fang, Fenxi Huaxue, 13 (1985) 447. 82 M. G a llego, M. S i l v a and M. V a l c a r e l , Fresenius Z. Anal. Chem. 323 (1986) 50.

380 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114

115 116 117 118 119 120 121 122 123 124 125 126 127

P. A. Johansson, B. Karlberg and S. Thelander, Anal. Chim. Acta, 114 (1980) 215. G. Audunsson, Anal. Chem., 58 (1986) 2714. B. Karlberg, P.A. Johansson and S. Thelander, Anal. Chim. Acta, 104 (1979 21. Y. H i r a r i and K. Tomokuni, Anal. Chim. Acta, 167 (1985) 409. M.J. Whitaker, Anal. Chim. Acta, 179 (1986) 459. Y. Sahlestom and B. Karlberg, Anal. Chim. Acta, 179 (1986) 315. L. Fossey and F.F. Cantwell, Anal. Chem., 54 (1982) 1693. L. Nord, S. Johansson and H. B r B t e l l , Anal. Chim. Acta, 175 (1985) 281. T. Kato, Anal. Chim. Acta, 175 (1985) 339. S. Johansson and H. B r o t e l l , Flow Analysis 11, Lund (Sweden) 1982. B. Karlberg and S. Thelander, Anal. Chim. Acta, 114 (1980) 129. M. Maeda and A. T s u j i , Analyst, 110 (1985) 665. M. Gallego, M. Silva'and M. Valcarcel, Anal. Chem., 58 (1986) 2265. E.H. Hansen and J , Ruzicka, Anal. Chim. Acta, 87 (1976) 363. L. Gorton and L. Ogren, Anal. Chim. Acta, 130 (1981) 45. 9. Chang and M.E. Meyerhoff, Anal. Chim. Acta, 186 (1986) 81. C.F. Mandenius, B. Danielsson and B. Mattiasson, Anal. Chim. Acta, 163 (1984) 135. P.K. Dasgupta and H.C. Yang, Anal. Chem., 58 (1986) 2839. H. Hwang and P.K. Dasgupta, Anal. Chem., 58 (1986) 1521. P.E. Macheras and M.A. Koupparis, Anal. Chim. Acta, 185 (1986) 65. B. Bernhardsson, E. Martins and G. Johansson, Anal. Chim. Acta, 167 (1985) 111. R.Y. Xie and G.D. C h r i s t i a n , Anal. Chem., 58 (1986) 1806. E. Martins, M. Bengtsson and G. Johansson, Anal. Chim. Acta, 169 (1986) 31. W.D. Basson and J.F. van Staden, Analyst, 104 (1979) 419. J.F. van Staden and W.D. Basson, Lab. Pract., 29 (1980) 1279. B. Olsson, H. Lundback and G. Johansson, Anal. Chim. Acta, 167 (1985) 123. D. P i l o s o f and T.A. Nieman, Anal. Chem., 54 (1982) 1698. P.J. Worsfold, J. F a r r e l l y and M.S. Matharu, Anal. Chim. Acta, 164 (1984) 103. H. Lundback and B. Olsson, Anal. Lett., 18(B7) (1985) 871. M. Masoom and A. Townshend, Anal. Chim. Acta, 166 (1984) 111. A. Townshend, Anal. Chim. Acta, 180 (1986) 49. S. Olsen, L.C.R. Pessenda, J. Ruzicka and E.H. Hansen, Analyst, 108 (1983) 905. J.L. Burguera, M. Burguera and A. Townshend, Anal. Chim. Acta, 127 (1981) 199. A.T. F a i z u l l a h and A. Townshend, Anal. Chim. Acta, 179 (1986) 233. B.A. Petersson, Z. Fang, J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 184 (1986) 165. H. Hwang and P.K. Dasgupta, Anal. Chem., 58 (1986) 1521. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 161 (1984) 1. Z. Fang, J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 164 (1984) 23. H. Bergamin Fo., B.F. Reis, A.O. Jacinth0 and E.A.G. Zagatto, Anal. Chim. Acta, 117 (1980) 81. H. Mikasa, S. Motomizu and K. Toei, Bunseki Kagaku, 34(8) (1985) 518. Z. Fang, S. Xu and S. Zhang, Fenxi Huaxue, 12 (1984) 997. O.F. Kamson and A. Townshend, Anal. Chim. Acta, 155 (1985) 253. L. Risinger, Anal. Chim. Acta, 179 (1986) 509. P. Hernandez, L. Hernhndez, J. Vicente and M.T. S e v i l l a , Anal. QuTm., 81 (1985) 117. S.D. Hartenstein, J. Ruzicka and G.D. C h r i s t i a n , Anal. Chem., 57 (1985) 21.

38 1 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166

2. Fang, S.Xu and S. Zhang, Anal. Chim. Acta, 164 (1984) 41. S. H i r a t a , Y. Umezaki and M. Ikeda, J. Flow I n j . Anal., 3 (1986) 8. S. H i r a t a , Y. Umezaki and M. Ikeda, Anal. Chem., 58 (1986) 2602. M. Bengtsson, F. Malamas, A. Torstensson, 0. Regnell and G. Johansson, Mikrochim. A c t a , 111 (1985) 209. H. Hirano, Y. Baba, N. Yoza and S. Ohashi, Anal. Chim. Acta, 179 (1986) 209. P . I . Anagnostopoulou and M.A. Koupparis, Anal. Chem., 58 (1986) 322. T.S. Stevens and T.E. M i l l e r , P a t e n t US 4290775. T. M i l l e r and T. Stevens, Adv. Instrum., 35 (1980) 21. N. Yoza, H. H i r a n o , Y. Baba and S. Ohashi, J. Chromat., 325 (1985) 385. T. Yamane, Bunseki Kagaku, 33 (1984) E203. A.G. Cox, and C.W. McLeod, Anal. Chim. Acta, 179 (1986) 487. A.G. Cox, I.G. Cook and C.W. McLeod, Analyst , 110 (1985) 331. I.G. Cook, C.W. McLeod and P.J. Wo r s f o ld, Anal. Proc., 23 (1986) 5 . J. Wang and B.A. Fr e i h a , Anal. Chem., 55 (1983) 1285. E.N. Cheney and R.P. Baldwin, Anal. Chim. Acta, 176 (1985) 105. J.A. P o l t a and D.C. Johnson, Anal. Chem., 57 (1985) 1373. J.M. S k in n e r and A.C. Docherty, T a l a n t a , 14 (1967) 1393. P. M a r t i n e z , M. G a l l e g o and M. V a l c i r c e l , Anal. Chem., 59 (1987) 69. M. V a l c i r c e l , A n a l y s t , 112 (1987), 729. P. M a r t i n e z , M. G a l l e q o and M. V a l c B r c e l , J. a n a l . Atom. Spec., 2 (1987) 211. P. Ma rt ine z , M. G a l l e g o and M. V a l c B r c e l , Anal. Chim. Acta, 193 (19871. 127. P. M a r t i n e z , M. G a l l e g o and M. V a l c B r c e l , Analyst , 112 (1987) 1233. C.E. Lunte, T.H. Ridgway and W.R. Heineman, Anal. Chem., 59 (1987) 761. F.P. B i g l e y , R.L. Grob and G.S. Brenner, Anal. Chim. Acta, 181 (1986) 241: R.W. F r e i , Swiss Chem., 6 (1984) 55. M.W.F. N i e l e n , R.W. F r e i and U.A.Th. Brinkman, S e l e c t i v e sample h a n d l i n g and d e t e c t i o n i n l i q u i d chromatography, Vol. I, ( E d i t o r s F r e i and Zech) E l s e v i e r , Amsterdam, 1987. J.N. Dolan, J.R. Grant, N. Tanaka, R.W. G ise and B.L. Karger, J. Chromat. Sci., 16 (1986) 616. E. Fo e l q u i s t , M. K r y s e l l and L.G. Danielsson, Anal. Chem., 58 (19867 1516. L. R i s i n g e r , L. Ogren and G. Johansson, Anal. Chim. Acta, 154 (1983) 251. I . S . K r u l l , Ed., R e a c t i o n D e t e c t i o n i n L i q u i d Chromatography, Marcel Dekker, New York, 1986. R.W. F r e i , J. Hansen and V.A.Th. Brinkman, Anal. Chem., 57 (1985) 1529A. Y. H i r a i , N. Yoza and S. Ohashi, Anal. Chim. Acta, 115 (1980) 269. R.S. B r a z e l l , R.W. Holmberg and J.H. Moneyhun, J. Chromatogr., 290 (1984) 163. Y. Baba, N. Yoza and S. Ohashi, J . Chromatogr., 295 (1984) 153. Y. Baba, N. Yoza and S. Ohashi, J. Chromatogr., 318 (1985) 319. N. Yoza, T. M i y a j i , Y. H y r a i and S. Ohashi, J. Chromatogr., 283 (1984) 89. N. Yoza, T. Shuto, Y . Baba, A. Tanaka and S. Ohashi, J. Chromatogr., 298 (1984) 419. Y. H i r a i , N. Yoza and S. Ohashi, J. Chromatogr., 206 (1981) 501. D. B e t t e r i d g e , N.G. Courney, T.J. Sly and D.G. P o r t e r , A n a l y s t , 109 (1984) 91.

382 167 168

J.B. K a f i l and C.O. Hubber, Anal. Chim. Acta, 139 (1982) 347. B.W. Renoe, C . E . Shideler and J . Savory, C l i n . Chem., 27 (1984)

169

M. Valcbrcel and M.D.

1546. 3.

Luque de Castro,

J. Chromatogr., 393 (1987)

383 SUBJECT INDEX A

barium ions 54 basic drugs 87 absorber solution 38,41 1,2-benzanthracene 285 acetylcholine 221 1,4-benzodiazepine 86 acetylcholine 262 benzodiazepines 95 acetylchol i nesterase 208 benz (a) pyrene 285 acetylsal icy1ic acid 353 biacetyl 263,306 bile acids acid digestion 39 220,248 bioluminescence acidic drugs 87 261 bismuth 84 acid-citrate dextrose (ACD) 344 acti vat i on analysi s 135 bittering compounds 353,354 blood active dialysis 45 82 actva ted charcoa1 94 blood level monitoring 96 adsorber tube 37 blood sample analysis 87 adsorption of sample constituents 54 blood sample clean-up 82 adsorptive preconcentration 361 blood sample extraction 85 215 blood sample preparation af1 atoxin 106 237 boric acid af1 atoxin determination 54 breakthrough air samples 52 70 aldehydes and ketones 215 1 bromonaph ta1 ene 303 1-bromonaphthalene aliphatic sulphonic acids 69 325 alkaline phosphatase 208 1,4-bromonaphtalene 303 a1 kylami nes 353 2-bromobiphenyl 303 a1 kylsulphates 261 2- bromonaph ta1ene 303 4-bromobiphenyl alumina cartridge columns 55 303 bromide a1 umi na columns 56 291 Amber1 i te XAD-2 94 bromine 345 3-aminofluoranthene 271,285 bromine solutions 39 amino acids 25,26,215,261 2-t-butyl-anthraquinone 277 amino acids 171 butene isomers 24 ammonia 247 ammonia in plasma 342 C ammonium sulfate 84 amobarbi tal 253 cadmi um 25 cadmium silicas amperometric detection 61 24 androstenedione 201 caffeine 187,353 calcium anion analysis 65 46,61 anionic surfactants 353 carbarnates 215 anthracene 285 carbary 1 140,187 anthraquinone 215,277 carbon dioxide in plasma 342 carboplatin anti coagul ants 84 319 arachidonic acid metabolites 190 carboxylic acids 26 Aroclor 1221 312 carbroma1 94 carotine aromatic carboxylic acids 69 201 aromatic monosulphonic acids 69 cartridge columns 50,52,55 catecholamines 19,197,215,246 aromatics 52 arsenic 344 cationic surfactants 353 ascorbic acid 265 cation-exchange cartridge column 60 atomic emi ss i on spectrometry cell clearance time 139 58 6 autoradiography 200 cellulose Cerencov counting 144 B charcoal 94 charge collectors 143 21 background count rate 165 chelating silicas back-extraction chemical derivatisation 53,261 86 chemical derivatization techniques 209 back-up adsorbent 37 barbiturates 94,215,237,248 chemical modification 44,54

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384 chemi 1 uminescence 144 I 261 chemi 1 uminescence detection 264 chemi luminescence detection 222.264 chenodeoxychol ic acid 248 t h 1 orami ne-T 183 chloride 35,39,55,71 chlorine 345 chlorine dioxide 341 chloroaniline 215 chlorpromazi ne 94,362 choice of supports 7 cholesterol 190 ,228 cholesterol oxidase 208 cholic acid 248 chol ine 221 I 262 choline oxidase 208 chromate 324 cisplatin 319 c 1 ean-up 209 codeine 353 coincidence circuit 144 coincidence time 162 column-switching 96 combustion accelerator 41 combustion flask 41 combustion methods 40 compl exat i on 5 complexation react ions 57,58 complexes surfaces 5 complexi ng functional groups 12 complexing metal sites 5 complexing s i 1 i ca 25 concentrations 125 concentrator column 63 35,62,69 conduct i v i ty detect i on conductometric detection 252 contamination 55 contaminat ion effects 53 contamination from stainless steel 60 continuos flow analysis 336 controlles pore glasses 7 copolymer resins 6 copper 19,23,344 copper-loaded si 1 icas 26 copper-si 1 icas 24 corrosion reactions 58 counting efficiency 138,162,164 counting efficiency 162 creati ne kinase 264 creat ini ne 265 cross-linked celluloses 7 cross-1 inking 9 cross-1 inking techn i ques 5 cyan i de 343 cyano complexes 49 cyanogeni c g 1 ycosi des 225 cyanopropyl column 107 cyclic undecapeptide 97 cycl odeytrins 302 cycl ospori ne 82

cyclosporine cyclosporine cyclosporine cyclosporine cycl ospori ne cyclosporine

adverse effects and 1 ipoproteins C conformers 0 metabolites

97 97 106 116 106 82,96

D

dansylated amino acids 250 data filtering techniques 160 dehydroascorbic acid 265 deltametrin 187 deoxychol i c acid 248 deoxygenat i on 305 9,lO-dephenylanthracene 285 deproteination 85 deproteination techniques 84 derivati zation of cyclosporine 106 detection in ion chromatography 35 detergents 26 1 di acetone si 1 ica 23 dialysis 355 dialysis-injection device 48 di a1 yt i c preconcentration 74 dialytic techniques 45 diamine silicas 23 diatomaceous earth 93 4,4'-di bromobiphenyl 303 dibenzofuran 313 2,8-di chl orodi benzofuran 313 8-dichlorotheo~hvlline 353 diethylenetri aminepentaacetic acid 183 diethylstilbestrol 243 digests 52 digital chromatography 109 di ketone si 1 icas 26 di pept ides 25 direct absorption 38 di rect detection 35 disposable cartridge columns 49 disposable fi 1 ters 55 di sulfides 234 disulfiram 237 dithiocarbamate silicas 26 divinylbenzene stationary phase 53 46 I 74 Donnan dialysis Donnan exclusion 35 dopami ne 77 doxl aminesuccinate 187 doxorubi ci n 362 doxy1 amine succi nate 138 drug analysis 186 dual ion-exchange 49 dual isotope detection 183 E

EDTA 54 electrochemical reagent production 208

385

eluent pH enalapri 1 enanti omer separation energy pulse height analysis enkephal in enkephalin metabolites enzymatic cleavage enzyme assay enzyme immobilization

68 353 27 144 154 190 183 135 208 187 321 291 347 38 6 93

ethyldipropylthiocarbamate

ethylenethiourea ethynyl thiourea extraction coil extraction methods extraction techniques Extrelut R

gl ucuronidase g 1 uthet imide glyceryltrinitrate g lycochenodeoxycholi c acid glycochol ic acid glycodeoxycholic acid glycolithocholic acid gl ycosi dase glycoursodeoxycholic acid goals of cleanup gold gold(1) cyanide guai acol guan i di ne guanine

208 94 187 248 248 248 248 208 248 44 77 77 38 215 326

H F

fast fourier transform fast-scan detectors fatty acid methyl esters fel odi pi ne fenoldopam ferritin F I A manifold field flow fractionation filtering algorithms fi 1 tration devices flow cell volume flow-injection analysis f1 ow-i nject i on manifold flow-through y -counting fluorescence detection fluorescence quenching f1 uori de f1 uori ne f1 uorocarbon fi lms fluorosilicic acid flux materials formaldehyde formaldehyde formaldehyde free bromine furnace combustion fusion methods fusion techniques

161 355 190 187 223 199 348 336 160 55 168 335,336 346 145 260 318 39 39 200 39 39 215 37 54 215 54 39 42 40 39 I

G

ga 1 actose gallium gas samples gas-diffusion gas-1 iqid interfaces gas-solid interfaces geological samples gl ucagon glucose glucose

356 354 37 338 338,342 345 39 183 265,356 356

ha1 f-1 i fe time 142 ha1 ides 40 ha1 onaphthol reaction 179 HBr 40 HC1 40 heparin 265 heterogeneous catalysis 5 heterogeneous scint. counting 153,153 hexacyanocobalt 77 hexobarb i ta 1 253 HF 40 HI 40 high pressure combustion bomb 42 hollow dialysis fibre 46 hol 1 ow fiber membranes 252 hollow fibers reactors 208 hol 1 ow fibre suppressor 77 hollow-fibre suppressor device 77 homovani 1 1 i c acid 227 3a-hydroxysteroid dehydrogenase 246 6-hydroxycortisol 197 6-hydroxyprednisolone 197 hydri de generation 343 hydrochloric acid 84 hydrogen peroxide 269 hyd rogenphtha 1 ate 45 hydroperoxides 215 hydrophobic interactions 35 hydroqui none 243 hydroxymethabe-su1 phonate 54 hydroxysteroid dehydrogenases 208 hypoxanthi ne 199 1

iminodiacetate silica immobi 1 i zati on immobilized acetylcholine esterase immobilized enzymes immobi 1 ized enzymes immobilized fluorophore immobi 1 ized phosphorophore

19 279 280 336 208 271 325

386 247 immobi 1 ized urease immobi 1 i zi ng enzymes 5 immuno assay 135 immunosuppressive agent 96 indirect detection 35 35 indirect UV absorption inorganic ions 33 inorganic polyphosphates 374 inorganic solutes 53 i nosi ne 226 internal standard 95 291 iodide i odi nated thyronines 235 iodine 54 ion chromatographic sample cleanup 49 ion chromatography 33,34,53,55 ion exchange resins 94 ion interaction 34 34 ion suppression ion-exchange chromatography 34 ion-exchange concentrator column 70 ion-exchanae functional i ti es 70 ion-excl usion chromatography 35 ion-i nteraction reagent 34 ,53 ion-pair 173 ion-pair formation 26 1 i ron 23 i somers 24 K

knitted reactors

211

1

labeled amino acids 1 actoperoxidase lactose lanthanide ions lanthanide luminescence laser induced fluorescence 1 aser-induced excitation 1 ead 1 igand exchange chromatography 1 iqid-sol id extraction 1 iquid-1 iquid clean-up liquid-liquid interfaces 1 iqoid-liquid sample clean-up lithocholic acid luciferase 1 ucigeni ne react ion 1 umi nescence detect i on luminescence detectors 1 umi no1 luminol chemi luminescence luminol reaction 1 umi nophores L-amono acid oxidase

152 183 356 326 328 260 355 55 23 6 87 347 86 248 264 264 260 308 341 341 264 326 208

M

magnesi um main adsorbent matrix elimination matrix elimination methods matrix interferences menadione meprobamate 2-met hy 1 ant h raqu i none 2-met hyl tetrahydrofuran metabolic pathways metabol ism metabolites metal chelating silicas metal cyanide complexes metal preconcentrati on metal separation metal si 1 icas methacrylate resin methane sulphonic acid methanethiol methanol methimazole methylamine methylene blue micellar solutions mice1 le-stabilized RTP mi c rocou 1 omet ri c ce 1 1 microdi st i 1 1 ation mi croemul s i on microsomal cytochrome P450 mobile phase reactions modified si 1 icas monoc 1 onal rad i oimmuno assay morphine

61 37 77 76 44 277 94 277 303 135 186 135 26 343 12 21 24,25 70 38 3 58 54 291,321 247 54 302 302 241 343 347 97 53 5,8 97 94

N a -naphtoflavone 345 1 ,5-naphtha1 enedi su 1 fonic acid 315 69 2-naphtyl ami ne-1-sul phonic acid 2-naphtyl selenylchloridf 106 naphtalene 303,312 narrow-bore columns 245 negatrons 141 neuronal tissue 22 1 neutron activation 136 nickel 23,344 nicotine 187 ninhydrin react ion 245 nitrate 35,55,71 nitrite 35,291 nitrogen dioxide 38 ni trophenols 253 non-i oni c surfactants353 353 nucleic acids 326 N-a1 kyl ami nes 246 N-a1 lylthiourea 29 1 N-ethyl -m-to1 ui di ne 291

387

N-methy 1 acri done N-methylcarbamate pesticides

N, N-dimethyl-phenylenediamine N, N-d imethylan i 1 i ne N,N-dipropylani1 ine

265 247,249 54 29 1 29 1

0

omeprazole on-line derivatization on-line reduction on-1 ine sample hand1 ing OPA reaction OPA reactor open tubular reactors ophthala1 dehyde (OPA) organ transplantation organic solutes organosulphur compounds oxalate oxine silicas ozone o-phtala1 dehyde

187 209 236 209 246 219 208 261 96 53 215 52,74 21 339 246

P

packed bed reactors 208,211 parathi on 140,187 parent compounds 135 passive dialysis 45 PCB mixtures 312 penici 1 1 in 215 pentanesulphoni c acid 53 peptide mapping 183 pept ide synthesi s 5 a4 perchlorid acid peroxyoxalate chemi luminescence 221 peroxyoxalate reaction 262,264 perylene 285 pestcide analysis 186 pesticides 140,171 phase separation 339 phase separator 349 phases for extraction 6 phenols 215 phenylbutazone 94 phosphate 41'74,374 phosphinate 374 phosphonate 374 phosphorescence 26 1 phosphorescence detection 296 30 1 phosphori met ry phosphorophores 30 1 phosphorus 40 photochemical deri vat ization 242 photochemical reactions 208 photochemical reactor 261,272 photolysis 245 phtalate eluents 60 phthalic acid 45

plant extract 52 pl at i num 19 polych 1 ori nated bi pheny 1 s 312 polycycl ic aromatic hydrocarbons 266 polymeric ani on-exchangers 60 polynucl eotides 326 polyphosphoric acids 374 polyvalent cations 61 positrons 141 post-column assemblies 373 post-column detector 208 post-column mixing system 247 post-col umn oxidat ion 236 post-col umn reaction 209 post-column reaction detection 208 post-col umn segmentation 170 preconcentration 5,6,71 preconcentration columns 62 preconcentration supports 7 predni solone 197 predni sone 197 pregnenolone 190 pre-column assemblies 371 pre-mixing system 247 primary amides 246 primary amines 261 procycl i di ne 353 prostaglandin 215 protective coating 57 protein precipitation 108 proteins 25 purine nucleotides 190 pyruvic acid 38 p-isopropylaniline 291 p-nitrophenacyl esters 266 p-to1 uenesulphoni c acid 69

Q quaternary ammonium type drugs 261 quenched detection 327 quenched phosphorescence 263,315 quenchers 163 quenching properties 163 quinidine 94 quinones 273 R

radio liquid chromatography 133 radioactivity detector 139 radi oanalysi s 135 radioassays 189 radioenzyme assay 135 radiohalogenides 179 183 radi oi odi nation radioiodination o f proteins 183 radioisotopes 133,135,141 radiopharmaceutical preparations 183 radio-chromatog rap hy 141

388 rare earth metal ions rare earth metals rat bile rat hepatocytes reaction detector reaction of solutes reactor types receptor assay red cresol reducing carbohydrate reflectance spectrophotometry reverse radiogram reversed energy transfer rhodi um rotating-mi rror phosphorescence

326 245 221 183 209 53 211 135 3 43 215 343 171 319 24 301

sulphide 356 sul phi te 291,356,358 sulphonami des 356 sulphonates 261 sulphonic acids 69 sulphur 40 116 supercri t. f luid chromatogr. supports 6,7 surface modification 9,lO surfactants 52 synchronous scanning 301 T

taurochenodeoxychol ic acid 248 taurocholic acid 248 S taurodeoxychol ic acid 248 taurolithocholic acid 248 sal icy1 ates tauroursodeoxychol ic acid 94 248 sampl e col 1 ec t i on temp.dependent distribution 37 98 sample digestion terodi 1 ine 39 353 sample filtration tertiary amine type drugs 44 261 sample aenerator tert.structure of cyclosporine 38 97 sample ireconcent rat ion 44,69 tetracene 285 sample treatment met hods 53 tetramethyl ammoni um hydroxide 53 Sandimmun R 82,96 thermolysis 245 sandwich type separator 350 thiamine 215 Savinsky-Go1 ay moving average 161 thiamine phosphat esters 215 Schoeniger flask 40 th iohydantoi ne 321 scinti 1 lation counters 143 thioridine 326 scinti 1 1 at ion counting 137 thiosulfate 52 seawater 39 thiourea 291 segmentation /storage system 172 thiourea derivatives 321 segmented flow analysis 336 three point rule 28 select ion of sorbents 209 to1 uenesulphonic acid 71 select i vi ty 68 trace enrichment 209 s e m i t i v i ty transition metals 68 5,23 sensitized phosphorescence 263,308 tri chloracet ic acid 84 she1 lfish 39 tri hydroxyndoles 236 triphenylphosphonium salts silicas 8 26 si 1 icates 6 trouble shootina 126 si 1 ica-based anion-exchangers 60 tryptophane 28 silver 24 tubular reactors 208,211,245 size exclusion 35 tyrosine 28 soil extract 52 solid scinti llator 144,153,164 U solid state reactors 264 solid-1 iqid extraction 93 ul traf i 1 tration 44,84 sol id-1 iquid purification 86 ultra-trace analysis 61 solution quenched phosphorescence 304 urapidi 1 173 station. phases for cartr.co1. 50 urea 247,356 steroid metabolites 190 urease 208 steroids 192,353 urease reactor 219 steroids cortisol 265 ursodeoxycholic acid 248 storage loop 171 streched polymer films 301 V s t rychn i ne 94 styrene-divinyl benzene resins 70 variable recoveries 109 sulfamethoxazole 125 vitamin 81 353 sulphate 41,55,71,74,358 vitamin D 192 ~

389

vitamin K 1 vitamin K3 vitamin K-3

243 215 276

X

XAD-2

xanthine xanthine oxidase xylenol orange

95 326 208 245

Y

yttrium silicate

144

z zinc zone compression effect

23 62

8

8-1 act am anti bi ot i c B-lactamase inhibitors

215

253

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39 1

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 Z. 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 . Turkova

Volume 13

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

Volume 11

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

Volume 15

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

392 Volume 16

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

Volume 17

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

Volume 18A

Electrophoresis. A Survey of Techniques and Applications. P a r t A: Techniques edited by Z. Deyl

Volume 18B

Electrophoresis. A Survey of Techniques and Applications. P a r t 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. P a r t A: Fundamentals edited by E. Heftmann

Volume 22B

Chromatography. Fundamentals and Applications of Chromatographic and Electrophoretic Methods. P a r t B: Applications edited by E. Heftmann

Volume 23A

Chromatography of Alkaloids. P a r t A: Thin-Layer Chromatography by A. Baerheim Svendsen and R. Verpoorte

Volume 23B

Chromatography of Alkaloids. P a r t 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

393 Volume 30

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

Volume 3 1

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

Volume 3 2

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

Volume 33

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

Volume 34

Polymer Characterization by Liquid Chromatography by G. Gliickner

Volume 35

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

Volume 36

Selective Gas 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

Volume 39A

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. P a r t A edited by R.W. Frei and K. Zech

Volume 39B

Selective Sample Handling and Detection in High-Performance Liquid Chromatography. P a r t B edited by K . Zech and R.W. Frei

Volume 40

Aqueous Size-Exclusion Chromatography edited by P.L. Dubin

Volume 41A

High-Performance Liquid Chromatography of Biopolymers and Biooligomers. P a r t A: Principles, Materials and Techniques by 0. Mike:

Volume 4 1B

High-Performance Liquid Chromatography of Biopolymers and Biooligomers. P a r t B: Separation of Individual Compound Classes by 0. Mikei

Volume 42

Quantitative Gas Chromatography for Laboratory Analyses and OnLine Process Control by G. Guiochon and C.L. Guillemin

Volume 43

Natural Products Isolation. Separation Methods for Antimicrobials, Antivirals and Enzyme Inhibitors edited by C.H. Wagman and R. Cooper

394

Volume 44

Analytical Artifacts. GC, MS, HPLC, TLC and P C by B.S. Middleditch

Volume 45A

Chromatography and Modification of Nucleosides. Part A: Analytical Methods for Major and Modified Nucleosides. HPLC, GC, MS, NMR, UV and FT-IR edited by C.W. Gehrke and K.C.T. Kuo

Volume 45B

Chromatography and Modification of Nucleosides. Part B: Biological Roles and Function of Modification edited by C.W. Gehrke and K.C.T. Kuo