Liquid Chromatography of Oligomers

Liquid Chromatography of Oligomers Constantin V. Uglea Institute of Biological Research Ministry of Research and Technology and the Medical and Pharmaceutical University lasi, Romania

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Preface

Liquid chromatography has become of late an efficient and fast tool for identifying the qualitative and quantitative composition of complex mixtures. Having been diversified by numerousprocedures, liquid chromatography is applied not only to separate mixtures madeup of small molecules, but also to determine molecular weightdistribution in oligomers and polymers. This was an important gain for specialists, since oligomersand polymers are noteasy to access by classical methodsof fractionation. Liquid chromatography is also an efficient means to control synthesis processes and to recognize the kinetics and mechanisms of oligomerization and polymerization reactions. As a result, developing liquid chromatography “homo polymericus” created the possibility of obtaining information on complex functionality in oligomers and polymers. This information is extremely useful in the formulationof new materials that provide features required by their various applications. This book focuses on two major objectives: (1) the description of the procedures used by liquid chromatography for the characterization of oligomeric mixturesand (2) the presentation of the equipment and optimal iii

iv

Preface

working conditions used to perform the separation of both well-defined oligomeric speciesand of fractions with narrow molecular weight distribution. The reader will become conversant witha complete description ofthe applications and possible performance of liquid chromatography in oligomerseparation.Moreover, Liquid Chromatography of Oligomers willbea valuableguideforthereaderonthenecessaryinformationtoestablishan optimal strategy for the practical application of liquid chromatography in the field o f oligomer characterization. Chapter 1 deals with the definition and history of chromatography and the nomenclature of oligomers. Chapter 2 discussesmolecularnonhomogeneityofsyntheticoligomers. Oligomers as well as polymers are characterized by both molecular weight distribution and nonhomogeneity of chemical composition. Functionalitydistributionisalsoincludedin the molecularheterogeneityof oligomer mixtures. Liquid chromatography of oligomers is presented in Chapter Although liquid chromatography may be performed by various procedures (thin-layer chromatography, partition chromatography, adsorption chromatography, etc.) we consider that liquid-solid chromatography represents the most important means for the study of the molecular weight distribution of oligomers. Gel permeation chromatography becamea part of liquid chromatography primarily because of its equipment characteristics. Its mechanism and performance make gel permeation chromatography a unique method with valuable applications in the chemistry of oligomers and polymers. Chapter 4 is dedicated to this method covering the theoretical basis and mechanisms; column packings (gels), calibration procedures, and applications and equipment. Liquid Chromatography of Oligomers is intended both for experts with experience in the field of oligomer and polymer synthesis and characterization and for students interested in becoming acquainted with the fascinating world of oligomers and macromolecules.

Constantin V. Uglea

Contents

Preface 1. Definition, History, and Nomenclature

iii 1

Definition I. 11. History 111. Classification of Chromatographic Methods IV.Oligomers:Nomenclature and Classification References

2. Molecular Nonhomogeneity of Synthetic Oligomers Introduction I. 11. Experimental Determination of Functionality References

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Contents

vi

3. Liquid Chromatography I. Theoretical Basis 11. ColumnPackings 111, Column Performances IV. Choice and Optimization of Variables in Liquid Chromatography References 4. GelPermeation Chromatography

I. II . 111. IV. V. VI, VII. VIII. IX.

Index

Introduction Theoretical Basis Packings Calibration Band Broadening Non-Size ExclusionEffects Aspects ofGPC Analysis of Copolymers Selected Applications Recycle GPC and High-Performance GPC References

42 43 82 118

129 213

258 258

264 269 275 28 1 288

297 301 306 313

341

l Definition, History, and Nomenclature

Chromatography ranks among the most prominent discoveriesof our century that have greatly influenced the progress of science and technology. At present it is one of the most important analytical methods, a tool for investigating surface phenomena and solutions, well as diverse physicochemical properties of substances; itrepresents a means for thepurification and separation of substances, the basis of the automatizationof technological processes, and has several fields of application in chemistry, biology, medicine, agriculture, etc. This chapter deals with the definition of chromatographic processes, history of chromatography development, and classification of chromatographic methods. The explanation of some characteristic terms involved in the nomenclature of oligomer science is an important topic of this chapter.

DEFINITION Chromatography is a general term applied to a wide range of separation techniques based upon the sample partitioning between a moving phase, I

Chapter I

2

which can be a gas or liquid, and a stationary phase, which may be a liquid or a solid. Wide application of chromatography for theanalysis and preparative scale separation ofcomplexmixturesresulted in confusion about themeaning of the chromatographic process as such and itsapplication. Thus, in the last 50-60 years [l-61, chromatography was determined as a physicochemical process for the separation of the constituents of a mixture. From such a definition it follows that if a simple substance is injected into a chromatographic column, no chromatographic process occurs in spite of the movement of the solute through the adsorptive bed accompanied by sorption-desorption and by formation of a chromatographic band with the corresponding distribution of solute concentrations. In other words, if the aim of the process isnot theseparation of mixtures in the case of nonanalytical applications, then according to thewell-known definitions such a process couldnot be considered chromatography. For that reason a new definition of chromatography appears necessary independent of its applications. The following definition is proposed

A chromatographic process is a process based on the movement of the restricted band the substance through the adsorptive bed inthe moving phase flow and one linked with repeated elemental sorption-desorption acts. The chromatographic process occurs as a result of partitioning of the solute between two phases one of which [is] relativeto the other. Since the velocity of the movement of the solute and the position of its band on the sorbent bed in any instant is due to the velocity of the moving phase and the value of the partition coefficient, the mixture of substances having different values of the partition coefficient will be separated in the course of their movement through the adsorptive bed. The reference to a restricted band eliminates rectification and countercurrent extraction. The term Chromatography is applied both to the process and to the scientific subject matter that studies and employs this process.

HISTORY Since time immemorial humanity has competed with nature. As we have often felt powerless before overwhelming forces of nature, humanity has tried, at the beginning shyly, then quite perseveringly, to appropriate the means of nature and then to achieve its performances. Prometheus and Icarus are the first examples that come to mind. Learning from thefailures, humanity realized that ouronly chance in this encounter comes from having a profound knowledge of the mecha-

Definition, Nomenclature History, and

3

nisms on which natural phenomena are based. Their reproduction was the immediate purpose of scientific endeavors. This brings us to the inception of biomimetics, a science whose main aim isto reproduce phenomena and natural products by means of some systems able to decompose complex natural processes into simple and measurable steps. We consider that thechromatographic separations, .and especially the preparative ones, are biomimetic tentatives aimed to mimic the naturally occurring phenomena. In spite of the fact that the concept of chromatography was introduced only in the first years of this century, a convincing amount of evidence is available on the “naturaloccurrence’’ of chromatographic processes and their essential contribution to various natural phenomena. This idea is, naturally, true only on the assumption that the term chromatography is applied to the process of the migration of substance bands along a sorbent bed. In this connection it is very useful to consider the natural processes on the basis of the formalism that has been worked out foranalytical and preparative chromatography, because inthis way, we can achieve a profound understanding of these processes. On the other hand, if fully understood, some natural phenomena may also serve as an example or as a prototype forthe development of efficient methods. The migration of petroleum through rocks accompanied with the alteration of its composition is the most clearcut evidence of the “natural occurrence” ofpreparative chromatography. As has been pointedout by V. A. Sokolov [g], “the long way of petroleum in the rocks to some degree compensates for the low separating properties of such a chromatographic column; therefore, under favorable conditions, a certain separating effect will result.” Day [ g ] represents the pioneering work in this field. When proving the inorganic origin of Pennsylvania petroleum hestated that, provided the limestone is saturated with oiland some pressureis produced at the bottom, the oil migrates, the emerging product being lighter in composition and more clear than the original petroleum. Thus having percolated the heavy oil of Western Ohio (Silurgian age), a lighter product was obtained with a composition similarto thatof Pennsylvania petroleum (Devonian age). A few years later, Day [lo] proposed to use this principle for the laboratory fractionationof petroleum and its products. Day’s method can be interpretated as the frontal version of liquid-solid chromatography. However, his explanation of the results was incorrect: he assumed that the different diffusion rates of the petroleum components through the small bores between the particles of lime or clay are the main reason for the separation effect and did not mention the contributionof adsorption. Russian petroleum engineers who lived in Baku at that time thoroughly studied the problems related to the migration of petroleum and its

4

Chapter I

laboratory fractionation [ll]. As a result of this work, the Surakhamy deposit near Baku is well known to everybody active in petroleum engineering. A colorless (“white”) petroleum consisting mainlyof gasoline fractions is emerging from that deposit. A hypothesis was advanced that the petroleum from the deep levels rises to the surface through the porous medium which retards theheavy fractions. To support thistheory, one of the drilled holes was deepenedand a dark fountain of heavy oil emerged[ 121. Different authors offered various explanations of this and similar phenomena in the light of the hypothesis concerning the origin of petroleum. Nevertheless the ability of clays and other rocks to separate the oil into various fractions was accepted as an immutable fact. As was pointed out by Herr in 1908 [ 1 l ] , “nature working with an unlimited quantity of material can attain a fractionationresulting in a high purity filtrate, as can be seenin the case ofSurakhamy petroleum.” Detailed investigations ofthe percolation of oil through Fuller’s earth carried out by Herr [l11 showed that in this case the clay mainly retains aromatic compounds and compounds containing heteroatoms. On the other hand,the high-boiling paraffins andnaphthalenes pass along with the higher gasoline fractions. Thus, the overall result of the processis the bleaching of petroleum. From the modern point ofview this result can easily be explained as due to frontal liquid chromatography on the polar active solid.The composition of petroleumcan change during its migration in various ways depending on the nature of the sorbtion media: either by the preferential sorption of aromatics and substances containing sulfur, oxygen, and nitrogen, or by fractionation according to the molecular weight. The water present in the pores of the rocks acts as the modifier of the sorption properties. Various bituminous substances may also serve as modifiers. Chromatographic processes are certainly notthe onlyreason for changes in the composition of petroleum oilsduring their migration; gravitation processes, sublimation and condensation due to temperature fluctuations, etc., must also be taken into account. Nevertheless the contribution of chromatographic processes israther significant. Petroleum gases consist only of a few components, and here, itis very easy to detect the effect of separation. A number of examples are given in the literaturepointing out thechromatographic distribution of gases in the porous layers surrounding the gas and petroleum deposits [ 13-16]. Lighter hydrocarbons remain in the layeras the distance from the gas deposit increases, and only methane occurs at a distance of 175 km. Thus, the so-called halochromatographic distribution of hydrocarbons occurs around the gas (and petroleum) deposits. This is due to the combination of diffusion and sorption phenomena. The adsorbing rock slows down the diffu-

Definition, Nomenclature History, and

5

sion of gases and naturally; this slowing-down process is weakerfor lighter hydrocarbons. Sokolov [8] called this process diffusion chromatography. It is of interest to note that in 1971 a new method was proposed for gas analysis that in principle is similar to diffusion chromatography 1171. This method utilizesthe diffusion of gasesthrough the chromatographic column rather than elution. one additional factshould benoted. The high pressuresand temperatures characteristic of the depth of the earth may be considerably higher than the critical parameters. Under suchconditions the substance is a supercritical fluid with a much smaller viscosity and greater mobility than a liquid. Thus, here is a process similar to the well-known method of fluid chromatography [ 181. The differences in the amount metals in crude oils are also due to chromatographic phenomena. Thus the concentrations of vanadium, copper, and nickel in petroleum in one of the Oklahoma deposits decreases from West to East and this was accepted as one of the evidences for the migration of petroleum from theWest [ 191. Besides this, it has beennoted [ 191 that theincrease of the distance of migration is followed bya decrease in the concentration of the I3Cisotope. These regularitiesin combination with the dataconcerning the hydrocarbon composition of petroleum make it possible to describe their “history,” the direction of their migration, and-depending on the place and depth of the deposition and onthe type of the rocks servingas the medium for themigration -to make certain predictions. Chromatographic processes must also be taken into account when choosing underground gas deposition. Either exhausted gas deposits or water strata should be selected. However,it is necessary in the firstcase to removehydrogen sulfide (to avoid deterioration in the quality of the pumped gas) and in the second case to dry the stratum. These processes may be covered with the same formalism as chromatographic separation [20,21]. Scientists investigatingthe formationof ores and distribution of various metals in them, concluded that in these cases chromatographic processes are very significant [22]. The ions of iron, copper, nickel, cobalt, and other metals were stated to move in silica and alumina gels due to diffusion. Thus, there is a combination of diffusion and ion exchange. Chromatographic phenomena are importantin natural biological processes. This particularly refers to the process of breathing. For example, the absorption of oxygen by blood hemoglobin in the lung as well as the absorption (bothreversible and irreversible) ofvarious harmful impurities are to a certain degree chromatographic processes. The utilization of hemoglobin in analytical gas chromatography is a logical extension of this natural

6

Chapter 1

biologicalprocess and indeed, this hasbeendescribed in two papers [23,24]. In these investigations,blood, coated on a solid support, served as the stationaryphase for the separation of oxygen from the otherinorganic gases and its determination. The examples outlined in these paragraphs indicate that chromatography is a natural process and is always realized in nature. Thus, just as in other fields of knowledge, we must regard nature as our teacher, perceive its laws, refine and further develop the natural processes, and apply them to the tasks we are facing. The discovery of “synthetic chromatography” is generally creditedto Tswett, a Russian scientist. Brief information concerning his life has been published in some papers [25-271. He was born on May 14,1872, in Asti, a little town in Italy. His mother, Maria De Dorozza, was an Italian born in Turkey and educated in Russia. His father, Semen Tswett, was a Russian from Chernigov (Ukraine), where the family name of Tswett had been known for a longtime. M. S. Tswettwas educated in Switzerland (in Lausanne and Geneva). In 1891 he entered the University of Genevaand in 1893 obtained his baccalaureate. In 1896 he presented his doctoral thesis in botany entitled “Investigations on the Physiology of Cells.” In the same year he moved to Russia where he lived first in Simferopol, then worked for a very short time in the Botanic Garden of Odessa University, and in late 1896 he left it forSt. Petersburg. Here Tswett worked at theBiological Laboratory, became a reader in botany at a school for women attached to it, and began to work on the preparation ofhisMaster’s thesis at the Botanical Laboratory of the Russian Academy of Sciences. In 1900, upon the recommendation of several prominent scientists, Tswett was given a membership of the St. Petersburg Society ofNatural Scientists. In 1901 Tswett prepared his Russian Master’s thesis entitled “A Physico-Chemical Study of the Chlorophyll Grain. Experiments and Analysis” which he dedicatedto thememory of his father. In January 1902 Tswett moved to Warsaw, where at first he held a modest positionat theuniversity, that of a supernumerary laboratory assistant, and later,of an instructor atthe Chair of plant anatomy and physiology. In the same year he was promoted to an assistant professorship and qualified to lecture. Here at the library of Warsaw University worked a Czech, Helena Trusevich, who Tswett married in 1907. In 1908 he was accepted on the staff of the Polytechnic Institute andcompleted his Doctorate thesis, published two years later as Chromophyllsin Plants and Animals W .

During his sojourn in Warsaw he traveledaround Europeto acquaint himself with the status of scientific research work and the methods of teaching botany in higher educational institutions, particularly in the Bo-

Definition, Nomenclature History, and

7

tanical Institutes of Berlin, Kiel, Amsterdam, Leyden, Delft, Brussels, and Paris. In 1915 when the German Army approached Warsaw, Tswett moved with Polytechnic Institute to Moscow and in 1916, to Nizhny Novgorod. Finally, in 1917, he was appointed Ordinary Professor and Director of the Botanic Garden at theUniversity of Yuriev (now Tartu, Estonia).After the town had been occupied by the Germans in February 1918, he left Yuriev for Voronezh where hewas professor of Botany at the University until his death resulting from heart disease on June26, 1919. While still workingin Petersburg on his Master’s thesis, Tswett persistently sought a physical method that would permit the separation of a mixture of chlorophyll pigments beingsure of the complexity ofits composition. In Warsaw hecontinued his experimentsand finally madehis discovery. His paper “On a New Category of Adsorbtion Phenomena and Their Application to Biochemical Analysis” was presented at the meeting of the Biological Department of the WarsawSocietyof Natural Scientists on March 8-21,1903 [29]. Although the principles of the method had been already laid down in his Master’s thesis, it is this date thatshould be considered the birthday of chromatography, rather than theyear 1906 mentioned by manyauthors. Tswett started with static experiments on the absorption of the pigment on a strip of filter paper from a solution in an alcohol-petroleum ether mixture. Upon drying, the paper strips were dark green in color; treatment withpetroleum ether produced a yellow solution, and when treated with petroleum ether to which alcohol has been added the solution became dark green, while the strip of paper was discolored. Later he used particles ofa sorbent instead of paper, also under static conditions, and the next stage wasfiltering through a bed of the sorbent placed at the top of a filtering funnel, i.e., under dynamic conditions. It was then easy to observe a difference in color according to the height. Later he usedtubes (columns) filled with a sorbent. Tswett began the experiments on the separation of pigments by using the frontal mode; later he discovered the development (elution) version of chromatography and at once appreciated its advantages. The only question that is not mentioned in his first communication is the term chromatography, whichby no means prevents this work from being consideredthe firstfundamental research inchromatography. In connection with the new term chromatography, the dataavailable on thepeculiar features of Tswett’s character make quite plausible the suggestion made by Purnell “It would be nice to think, that Tswett, whose name in Russian means color, took advantage of the opportunity to indulge his sense of humor.”

8

Chapter I

In his two subsequent papers published in 1906, three years later than his first report, Tswett already introduced the term chromatography. In the first of these two papers he writes about his discovery in a vivid, picturesque language: Like light rays in the spectrum the different components of a pigment mixture are separated on the calcium carbonate column and then can be qualitatively and quantitatively determined. I call such a preparation a chromatogram, and the corresponding method chromatographic method. It is very essential that in his publication Tswett has pointed out that the method is equally suited for theseparation of colorless substances. Tswett widely used the chromatographic method not only to separate a mixture and to prove that it consistsofmany components, but also for quantitative analysis; he contemplated the possibility of introducing reference (marker) components into themixture of facilitate identification. He was the first toemploy chromatography as a preparative method to produce individual compounds; he was the first to use a change in the properties of the mobile phase (gradient elution) during the chromatographic process. He also pointed out the need for a spectral study of the compounds in the adsorbed state. Tswett developed the apparatus for theprocess of liquid chromatography, realized for the first time the possibility of carrying out chromatographic processes in a vacuum or under pressure, summarized the recommendations for the preparation of effective columns, and was the first to use both micropacked and preparative columns and theinverse flow ofthe mobile phase in the column; he drew attention to the necessity of accounting for the simultaneous occurrence of adsorption processes and purely diffusion phenomena in the column. He carried out separation of substances both by their partial elution and by complete elution from the column. Apart from developing the fundamentals of the method and extensively using it to solve many problems that are complicated even at the modem level of science, he also introduced many of the basic conceptsand terms of the new method, including the name of the method, chromatography, development, displacement, chromatogram, etc. Tswett imparted a new, deeper meaning to the adsorption method than was known before. In particular, thelater development ofpaper chromatography is to a considerable degree the result of extending the general relationships discovered by Tswett to theparticular case of adsorbtive separation of a mixture on paper strips. He has to a certain measure also foreseen the advent of thin-layer chromatography (TLC) by showing the

Definition, History, and Nomenclature

9

analogy between the properties of a paper strip and those of a calcium carbonate layer used for thechromatographic analysis ofsolutions. This detailedexamination of Tswett’s discoveries makes us fully agree with Zechmeister [33], who wrote, “M. Tswett isthe trueinventor of chromatography in all ofits fundamental aspects.” One of the characteristic features in the history of development of science and techniques, chemistryand biology in particular, is the fact that the trend and rate of development dependto alarge extent on theutilization of certain methods of research and their efficiency. This has been stressed among others by Tswett himself: “Every scientific advance is an advance in method” [28]. In the 18th century it was crystalyzation that wasbeing developed for nonvolatile substances and then turned into multistage crystalization; in the 19th century, for volatile substances, evaporation that had been known for centuries, was transformed into multistage distillation; in the 20th century sorbtion and extraction of volatile and nonvolatile compounds was converted into multistage and continuously highly efficient processes. Chromatography is one of them. The importance of many outstanding discoveries is not always appreciated at first. Further developments often begin only after a considerable period of time has elapsed. The duration of such a latent period is a measure of the degree to which the discoverer has advanced ahead of his contemporaries. The latent periodof chromatography lasted about 25 years, only a small number of various applications of the method being reported during this time. Thus after 1911, DhCrC [34], in Switzerland, and Stoklassa, in Czechoslovakia [28] systematically used chromatography; in the United States this method was used somewhat later by Palmer [35]. Other scientists usingchromatography in this latent period include Goward [36] and Lipman [37] who were concerned withthe study of extracts from flowers and the separation of rhodoxanthine and xanthophyll, and Bird and others were close to the development of gas chromatography (GC) [38]. And yet chromatography did not advance significantly during this period. This is due partly to the negative comments of Wilstatter who warned against chemical reactions of the pigments with the adsorbents. However, the mainreason was the absence of a genuineneed for the method. The latentperiod lasted until 1931 when Lederer, Kuhn, and Winterstein [39,40] demonstrated the usefulness of the method. Since this time, the growth of chromatography has been continuous; newer and newer versions have been developed and applications in newer and newer fields demonstrated. However, based on the results of studies of old literature [41-461 some experimentsperformed in the pre-Tswett period should bementioned.

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Chapter I

Thus it would seem that the earliest results in this field are obtained by Runge who used in 1834 a radial method for conducting the process of separating salts and organic substances [47,48]. Then, in 1851, Schonbein and Goppelsroeder [49-511 started aseries of investigationson the so-called capillary analysis which consists of the separation of zones on the paper strips. Tswett was well acquainted with these works and wrote: “Goppelsroeder devoted himself since 1861 to the practical development of Schonbein’s idea with an astounding patience and accumulated an enormous amount of observations” [28]. He also noted the work done in 1851 by Cohn who obtained a radial chromatogram similar to Runge, with three rings whenevaporating drops of solution of “phycocyan”[52]. Another group of studies was devoted to separation on asorbent bed. It would seem that in this case it is bestto begin with the words of Tswett, whocitingSachsse,wrote [28], “The adsorptive properties ofsoilwith respect to salts were known already to Aristoteles since he pointed out the conversion of sea waterto potable water when makingit to pass through a layer of earth.” Williams [45] referred to the work of Thomson and Williams who were approaching separation on a column with an adsorbent long before 1850. Subsequently, the results of Read (1893) [53] should be mentioned, who observed some changes in the composition of a solution of salts when filtered through powdered kaolin. The results of Day in 1897 also belong here [54]. He observed the clarification of oil upon being filtered through Fuller’s earth. In 1900 S. K. Kvotka received a patent for a method of fractionating petroleum by filtration through porous media. Similar results were obtained by Englerand Albrecht [S]. In all of these cases, the separation by adsorption have been carried out by the simplest versions of frontal analysis. However, in spite of their unquestionable value,usefulness, and interest, none oftheseproposed methods are able to evolve into thechromatography that we extensively use today and is basedon theworks of Tswett. Then in the late 1930s and early 1940s chromatography began to evolve. The foundation of thin-layer chromatography (TLC) was laid in 1938 by Izmailov and Schreiber [56] and later refined by Stahl [57,58] in 1958. The remarkable work of Martin and Synge in 1941 [59], not only revolutionized chromatography but in general setthe stage for thedevelopment of gas chromatography and paper chromatography [60]. In the late 1960s more and more emphasiswas placedupon developing liquid chromatography as a complementary techniqueto gas chromatography. High performance liquid chromatography (HPLC) or high pressure, high speed, and modern liquid chromatography has evolved from this effort. Advances in both instrumentation and column packings occurred rapidly that it was difficult to maintain a state-of-the-art expertise. Indeed,

Definition, Nomenclature History, and

I1

even today the technique is rapidly maturing and is rapidly attaining an equal stature with other investigation techniques. During the early 1970s the advantages of liquidchromatography were really just beginning to draw attention to a wide audience. Revolutionary improvements in equipment, materials, techniques, and application of theory have brought liquid chromatography to what Snyder and Kirkland [61] have aptly called “modern liquid chromatography.” Resulting from the phenomenal interest and continued growthinhigh performance liquid chromatography, an analyst during the last decade has seen value added to the already existing advantages of convenience, accuracy, speed, and the ability to successfully deal with very difficult separation problems. It will hardly come as a surprise to the reader that the large majority of the references cited inthis book about HPLC of oligomers will come from the past 10 or 15 years. To effect most of these difficult separations has required the resources of modern liquid chromatography. Both classical LC and TLC have long histories. In the late 1950s TLC started to emerge as a simple, low-cost, relatively efficient means of separating less volatile components of organic mixtures. HPLC surplanted GC in the late 1960s and early 1970s for chromatographing nonvolatile compounds. Just as chromatography (an efficient means of fractionation) has widely replaced fractional crystallization, has HPLC increasingly replaced classicalliquid column chromatography. Applications of the latter have not grown nearly as fast as HPLC in recent years simply because HPLC is a more advantageous way to accomplish the same objectives.The goals of maximum efficiencyin a minimum time haveshifted the emphasis from traditional liquid chromatography techniques into modern HPLC and GC, including small-bore column technology. Even TLC, while preserved in muchits traditional form, hasprogressedsuch that manyhigh-performance TLC (HPTLC) applications have appeared [all. It is interesting and useful to contrast and compare liquid chromatographic techniques. Modern HPLC is fast, very efficient, relatively expensive,easily automated, and useful for analytical aswell as preparative needs.Classicalcolumn chromatography islessexpensive, but ismuch slower, less efficient, and not as easily automated. Itis also useful for either preparative or analytical work. Both paper and TLC are closely related in technical aspects: the details of development, sampleapplication and detection are much the same. They differ in the nature of the stationary phase, with paper having that phase anchored to the carrier while TLC has the sorbent loosely spread or fixed on a rigid (glass) support. The traditional borderlines between these two techniques have blurred, but TLC has appeared to be more widely retained for general laboratory applications. TLC is often indispensable

Chapter I

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tool in many laboratories. It is easy to learn, comparatively inexpensive, fast, convenient, versatile, uses standardized stationary phases (prepared plates), and is reasonably efficient. HPTLC can offer equivalent resolution in reduced time or improved resolution in equivalent time (compared to normal TLC). TLC is attractive especially to those laboratories having low capital budgets. It is especially useful wherea large number of samples are to be examined and where the components of interest are relatively easily separated. TLC bridges a gap between traditional column liquid chromatography andmodern HPLC.

CLASSIFICATION OF CHROMATOGRAPHIC METHODS The state of aggregation of the two phases, the mechanism of separation process, the shape of the adsorptionisotherm that characterizes the elementary separation process and some specific workingconditions are thebasic criteria that areapplied in the classification ofchromatographic methods. Taking into account the characteristics of the two phases,chromatography may be dividedinto liquid chromatography (LC) and gas chromatography (GC). According to this classification paper chromatography (PC), thin layer chromatography (TLC), liquid-liquid chromatography (LLC), steric exclusion chromatography or gel permeation chromatography (SEC or GPC), affinity chromatography (AC), and ion-exchange chromatography are the most important procedures of LC. According to the mechanism and drive forces that act during the elementary process of separation, chromatography maybedivided into adsorption chromatography, partition chromatography, ion-exchange chromatography, and SEC or GPC. The shape of the chromatographic peak is determined by the shape of the isotherm specific to the elementary separation process. From this point of view, chromatography may be a linear or nonlinear process. In the case of a linear isotherm, the chromatographic peak (elution curve) is represented by a normal distribution curve while the nonlinear isotherm determines an asymmetric elution curve. This asymmetry is proportional to the degree ofnonlinearity of the isotherm. Some experimental details determine the characteristics of the chromatographic process. Dependingon thepressure, the chromatographic separation may bea high pressureor a low pressure process. Depending on the structural characteristics of the oligomers, among the chromatographic methods LSC and SEC are preferably used for the characterization of molecular nonhomogeneity of synthetic oligomers.

Definition, History, and Nomenclature

13

IV. OLIGOMERS:NOMENCLATUREAND CLASSIFICATION IUPAC defines an oligomer asa substance composed of molecules containing a few of one or more species of atoms or groups of atoms (constitutional units) repetitively linked to each other. This definition does not specify an absolute degree of polymerizationor molecular weight that distinguished an oligomer from a polymer, but it does further state that the physical properties of an oligomer vary with the addition or removal of one or a few of the constitutionalunits from molecule. This structure-property definition is perhaps the most meaningful definition of oligomer. The term oligomer originates from Greek words oligo = few, and meros = part andwas first used by Burckard et al. [62]. Burckardalso was originator of the term oligopeptide [63]. Telechelic oligomers, macromonomers, and prepolymers are three other terms used in oligomer science. The term telechelic polymer was proposed in 1960 by Uraneck et al. [64] to designate relatively low molecular weight macromolecules possessing two reactive functional groups situated at both chain ends. The term originates from the Greek words telos = far and chelos = claw, thus describing the molecule as having two clawsfar away from each other, i.e., at theextremities of the chain, able to grip something else. An oligomer can be consideredto be a telechelic if it contains at least one reactive end group which can react selectively to give a bond with another molecule. Dependingon functionality (which must be distinguished from the functionality of the end group itself), telechelics can be classified as mono-, di-, tri-, or polytelechelics [65]. The original definition of telechelic needs some additional comments in connection with end groups reactivityand the influence of experimental conditions upon this reactivity. This means that apolymer may be telechelic under certain experimental conditions, but nottelechelic underothers. Theoretically all oligomerscan be considered to be telechelic provided one can find a reaction that is selectivefor thechain ends ofthe polymers, provided a given reagent is able to distinguish the end groups from the main chain. This interpretation leads to a more generalized interpretation. Thus atelechelic may be any oligomeric species that contains at least one “informational site” capable of a selective replyagainst an external stimuli in a given experimental system. A second problem that is encountered when looking for a general definition of the telechelic is the “functionality” of the telechelic. We have to make a distinction between “functionality” of oligomeric speciesand the functionality of the end group itself. Not only the functionality of the end

14

Chapter I

group itself is of considerable importance. A telechelic oligomer may have more than two reactive end groups because they are branched or have a star-shaped structure. These polymers can be designated as tritelechelic, tetratelechelic, or polytelechelic. Of course, the oligomers that possess only one reactive functional end group have been called monotelechelics or semitelechelics. In defining the nature of a telechelic oligomer or polymer, an important question is if such a compound having different functionalend groups can be regardedas telechelic. Supposewe can make a linear polymer having one hydroxyl endgroup andone amino end group. Undoubtedly, this polymer would give a number of possibilities for constructing interesting welldefined polymer structures. The condition is, however, that all molecules have indeed one hydroxyl and one amino end group. If only the average amount of hydroxyl and amino end groups is one for each polymer molecule, it will not be possible to use this compound to produce a well-defined structure. Keeping this in mind, the classical polycondensation polymers, obtained from two bifunctional monomers, cannot be regarded as telechelic, although they possess two end groups, unless their synthesis has been conducted in such a way that the end groups are the same for each individual molecule presentin the mixture. For example, a linear polyester having two hydroxyl endgroups or two carboxyl endgroups is a telechelic polymer, but a linear polyester witha distribution of molecules having two hydroxyls, one hydroxyl and one carboxyl, and two carboxyls is not a telechelic polymer. Onthe other hand, linear a polyesterin which all macromolecules have one hydroxyl and one carboxyl end group may again be considered as a telechelic. It will be monotelechelic for reactions involving either the hydroxyl or the carboxyl; it will be ditelechelic if it reacts at both extremities. Thus, thetelechelic concept hasa pragmatic sense. This statement has no reference about chemical structure oftelechelic, but itrepresents the behavior of this “material expression’’ in a given experimental system and especially its capacity to create previsible and welldefined macromolecules. More precisely,this concept contains the possibility of a given oligomeric species to be involved in the transport of an amount of information as precursor of a di- or tridimensional macromolecule with regulatedstructure. A very important requirement of telechelic oligomers is their perfect terminal functionality, i.e., the average number functionality of a ditelechelic oligomer must be 2.0, that of a tritelechelic must be etc. Most research to date concentrated on defining and developing clean systems, i.e., systemsthat reproduce and conveniently yield perfect average over a desirable molecular weight terminal functionality (1.O or 2.0 or range and molecular weightdistribution.

15

Definition, Nomenclature History, and

A large amount of data has been generated with terminally functionalized polyisobutylenes (PIB) prepared by the inifer method [66-681. Quantitative derivatization of these new telechelic oligomers gave riseto a whole new family of unique materials. A most promisingnew development concernsthe synthesis of phenoltelechelic PIBs. These materials are structurally closely relatedto bisphenol A and therefore the linear product is called bisphenol-PIB. The versatile terminal phenol function allows further derivatizations. The chlorine telechelic products can be quantitatively dehydrochlorinated and in this manner olefin-telechelic oligomerscan be obtained [69]. The latter materials can be quantitatively further derivatized to other telechelics. For example, sulfonation of the terminal olefin groups by acetyl sulfate withsubsequent neutralization yielded new “endless”ionomers [70,71].

Hydroboration followed byoxidation of olefin-telechelicPIBs yielded hydroxy telechelic oligomers [68], which have been used in a great variety of subsequent transformations. Indeed, one most promising lead is the synthesis of PIB-based polyurethanes by reacting HO-telechelic liquids with various isocyanates inview of the synthesis of “model” or “perfect” network (i.e., a network in which the molecular weights between crosslink points are the same). Finally, here are few thoughts about nomenclature. The nomenclature” can beused for ditelechelic oligomers. It uses the usual prefix substituent names for the functional end groups, followed by the name of oligomer. For example:

-NH2

H0 H 0 -OH H0

: a-hydroxy-polyethylene : a,o-hydroxy-polyethylene : a-hydroxy-w-amino-polyethylene

The oligomer withthe following structure H0

H0 has been termed byGoethals [72] trihydroxytelechelic polyethylene. The prefix terminated nomenclature or prefix telechelic such as “carboxy-terminated polyethylene”or “carboxy-telechelic polyethylene”can be used to designate a class of polyethylene having carboxylic end groups, but the description is in fact incomplete becauseit does not say if it is a mono-, di-, or polytelechelic.

16

Chapter I

For complex endgroups, the prefix name isput between brackets and preceded bymono-, bis-, or tris-; examples:a,o-bis(phydroxypheny1)polyethylene. The concept of prepolymers, which can be transformed into final products, with well-specified properties by reaction of end-standing functional groups with multifunctional coupling agents was not new. early as 1937, Otto Bayer [73] used this concept in polyurethane chemistry based on hydroxy-terminated oligomers. The functionality of the reactive end groups itself is another important parameter that plays an important andprimordial role in the potential uses of the telechelic oligomers. The usual telechelic oligomers havemonofunctional end groups, i.e., end groups that can form one bond with another functional group. Other end groups may be unequivocally bifunctionalor multifunctional. Such groups, if they can participate in a polymerization reaction, are of special importance because copolymerization of oligomers containing such end groups with low molecular weight monomers leads to graft copolymers (for monotelechelics) or to polymer networks (for di- or polytelechelics). Such telechelic oligomersare “macromolecular monomers’’and are commonly calledmacromonomers or macromers. Therefore, macromers have to be considered as a special class of telechelics, the classical monomers being bifunctional monotelechelic. The term macromonomer or macromer was introduced by Milkovich in 1974 [74]. However, Bamford et al. [75,76] had demonstrated in 1958 that graft copolymers could beprepared by copolymerizinga small monomer with the terminal double bond of poly(methy1methacrylate) prepared by radical polymerization under conditions in which the termination reaction is mainly disproportionation [771. The great interest in telechelic oligomers resides in the fact that such molecules can be used, generally together with suitable linking agents, to carry out three important operations: Chain extension ofshort chains to long ones by means of bifunctional linking agents Formation of networks by use ofmultifunctional linking agents Formation of block copolymers by combination of telechelics with different backbones These conceptsare of great industrial importance since they form the basis of the so-called liquid polymer technology exemplified by “reaction injection molding” (RIM). Great interest has also been shown bythe rubber industry because the formationof a rubber is based on network formation. In classical rubber technology, this is achieved by the crosslinking of long chains that show high viscosity. The classical rubber technology, therefore,

Definition, Nomenclature History, and

17

requires an energy-intensive mixingoperation. The use of liquidprecursors, which can be end-linkedto the desired network, offers notonly processing advantages, but in some cases, also better properties of the end product. Finally, the development of “thermoplasticrubbers” has furthermore stimulated the industrial interest in telechelic polymersthat arepotential starting materials for such thermoplastic rubbers. The use of telechelics in epoxy resins, polyurethane, and polyesteracrylates production represent another example the great interest in the field of synthesis and characterization of oligomers.

REFERENCES 1. Keulemans, A. I. M., Gas Chromatography, Reinhold Publ. Corp., New York, 1957. 2. Bayer, E., Gas-Chromatographie,Springer-Verlag,Berlin-Gotingen-Heidelberg, 1959. 3. Schay, G., Theoretische Grundlagen der Gas-Chromatographie, VEB Deutscher Verlag der Wissenschsften, Berlin, 1961. 4. Dal Nogare, S. and Juvet, R. S., Gas-Liquid Chromatography. Theory and Practice, Interscience Publishers, New York, London, 1962. 5. Golbert, K. A. and Vigdergauz,M. S., Treatise of Gas-Chromatography, Khimija, Moscow, 1967 (in Russian). 6. Vigdergauz, M. S., On thedefinition of a chromatographic process, Chromatographia, 5, 107, 1972. 7. Kholkin, Yu. I., Remarks, Prikl. Khim., 2,9, 1970 (in Russian). 8. Sokolov, V. A., Processes of formation and migrationof petroleum and gas, Nedra, Moscow, 1965, pp. 178-186 (in Russian). 9. Day, D. T.,Proc. Amer. Phil. Soc., 112, 1897 (cited in ref. 8). 10. Day, D. T.,La variation des caractkes des huiles brutesde Pennsylvanie et de

11. 12. 13. 14. 15. 16.

YOhio, Intern. Congr. Petroleum, Notes, Memoires et Documents, Paris, 1900, pp. 53-60. Herr, V. F., Trudy Bakinskogo OtdeleniyaRusskogo Tekhnicheskoy Obshchestva, 22,21, 1908; 22,39 1908; 25,81, 1911 (cited in ref. 8). Kalitzki, K., Proc. ofthe Geol. Committee, St. Petersburg, 30,585,1911; cited in ref. 8 (in Russian). Nagy, B., The sedimentary strata as a chromatographic column, General Petroleum ChemistrySymposium, New York, 1959. Nagy, B., Review of chromatographic “plate” theory with reference to fluid flow in rocks and sediments, Geochim. Cosmochim. Acta, 19, 1, 1960. Nagy, B. and Wourms, J. P., Chromatographic separations and concentration of organic compounds in sediments, Bull. Geol. Soc. Am., 35, 1662, 1958. Roper, W. A., Doscher, T., and Kobayashi, T., Evidence of chromatographic effect during flow of gasesthrough oil-field cores, J. Petroleum Tech., March 1958, p. 15.

18

Chapter I

17. Khokhlov, V. N., Okhotnikov, B. P., and Zhukhovitskii, A. A., Diffusion chromatography, Zavod. Lab., 37,533 1971 (in Russian). 18. Klesper, K., Corvin, A. H., and Turner, D. A., The influence of migration upon the composition of petroleum, J. Org. Chem., 27,700, 1962. 19. Silverman, S. R., Migration and separation of oil and gas fluids in substractive environments, Amer. Assoc.of Petroleum Geologists, Geology of Fluid Symposium, Midland, January 30, 1964. 20. Shtof, M. D., Trudy Kuibyshev N.I.I.N.P., 40,242,1968(in Russian). 21. Karimov, M.F. and Odegov, A. I., Petroleum Refinerand Petroleum Chemistry Synthesis, Petroleum Inst. Ufa, 1974,p. 293 (in Russian). 22. Ritchie, A. S., Chromatography in Geology, Elsevier, Amsterdam, 1964. 23. Gil-AV, E. and Herzberg-Minzky, Y., Chromatographic phenomena in biological processes, J. Amer. Chem. Soc., 81,4749, 1959.

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Zhuknovitskii, A. A.,Anew stationary phase for liquid chromatography, Khim. Tekhnol. Topliv. iMassel, 1972,No. 6,p. 7 (in Russian). Richter, A. A. and Krasnopolskaja, T. A., M. Tswett-A short Biography in Adsorbtion Chromatography Analysis, Izd. Akad. Nauk, Moscow, 1946, p. 229 (in Russian). Hesse, G. and Weil, H., Michael Tswett’sfirst paper on chromatography,M. Woelm-Eschwege, 1954. Sakodynskii, K. and Solujanov, P., Gas Chromatography, Niitechim, Moscow, 1967,p. 116 (in Russian). Tswett, M., Chromophyll in plants and animals, Izd. Karbasnikov, 1910 (in Russian); cited in ref. 27. Tswett, M., Trudi Varshavskovo Obshestva Estetsvoi spitalei otd. biologii, 14, 20, 1903 (in Russian); citedin ref. 27. Parnell, H., Gas Chromatography,J. Wiley BE Sons, New York, 1962. Tswett, M., Ber. deutsch. Bot. Ges., 24,316, 1906; cited in ref. 41. Tswett, M., Ber. deutsch. Bot. Ges., 24,384, 1906; cited in ref. 41. Zechmeister, L. and Cholonsky, L.,Principles and Practiceof Chromatography, New York, 1943;cited in ref. 42. Dhtrt, C. and Vegezzi, G., C. R.Acad. Sci., 2,399, 1916; cited in ref. 42. Palmer, L. S., Carotinoids and related Pigments, New York, 1922;cited in ref. 42. Goward, K., Biochem. J., 18,11 14, 1924; cited in ref. 42. Lipman, Th., Compt. Rend.,182,867, 1926; cited in ref. 42. Berl, E. and Wacherdorf, E. Z.,Angew. Chem., 37,298,1930;cited in ref. 42. Kuhn, R. and Lederer, E.,Ber. deutsch.Bot. Gm.,64,218,1931;cited in ref.41. Kuhn, R., Winterstein, A., and Lederer, E., 2 . Phys. Chem., 197, 147, 1931; cited in ref. 41. Weil, H. and Williams, T., History of chromatography, Nature, 166, 1O00,

1950. 42. Faradane, J., History of chromatography, Nature, 167, 120, 1951. 43. Weil, H. and Williams, T., Early history of chromatography, Nature, 167, 906,1951. 44. Zechmeister, L.,Early history of chromatography, Nature, 167,405, 1951.

Definition, Nomenclature History, and 45.

19

Williams, T., History of chromatography, Anal. Chem., 25,531, 1953.

46. Heines, S. V., Evolution of chromatography, J. Chem. Educ., 46,315, 1969. XWZ, 65,1834; cited in ref.44. 47. Runge, F.F.,Annalen der Physik und Chemie, 48. Runge, F. F.,Farbenchemie, 111, 1850; cited in ref. 44. Schonbein, C.V., Naturforsch. Verh. Basel, 3,240, 1861; cited in ref. 42. Schonbein, C., J. Chem. Soc., 33,304, 1878; cited in ref. 42. Goppelsroeder, F., Verh. Naturforsch. Basel, 3,268, 1861; cited in ref. 42. Kohn, A., 2.Wiss. Zool., 3,264, 1851; cited in ref. 42. Read, L., Proc. Chem. Soc., 9,123,1893; cited in ref. 42. Day, D.,Znd. Techn. Petr. Rev., 3,9, 1900; cited in ref. 46. Engler, C. and Albrecht, E. Angew. Chem., 14, 865, 1904; cited in ref. 46.Izmailov, N. A. and Schreiber, M. S., A new method of separation for the organic compounds,Farmatsiya, 3,15,1938 (in Russian). 57. Stahl, E., A new version of adsorbtion chromatography, Chemiker Ztg., 82,

49. 50. 51. 52. 53. 54. 55. 56.

323,1938. 58. Stahl, E., Theoretical aspects of thin layer chromatography, Pharmazie, 11, 633, 1956. 59. Martin, A. J. P. and Synge, R. L. M., Theory of chromatography, Biochem. J., 35, 91, 1941.

60. Consden, R., Gordon, A. H., and Martin, A. J. P., Equilibrium theory of

61. 62. 63. 64. 65. 66. 67.

chromatography, Biochem. J., 38, 224, 1944; for refs. 56-60 see Liteanu, C., Gocan, I., Hodisan, T., and Nascu, A., Liquid Chromatography, Academic Editorial House,Bucharest, 1974, p. 26. Snyder, L. R. and Kirkland, J. J., Introduction to Modern Liquid Chromatography, 2nd ed., J. Wiley & Sons, New York, 1979, Ch. 1. Burckard, H., Bohn, E., and Winkler, S., Ungesatige Derivate von Gentiobiose und Cellobiose, Berichte, 63B, 989, 1930. Burckard, H. and Griinert, H., N-methansulfonyl derivate von amino-sauren und oligopeptiden, Ann., 545, 178, 1940. Uraneck, C. A., Hsien, H. L., and Buck, 0. G., Telechelic polymers, J. Polymer Sci., 46, 535, 1960. Goethals, E. J., Telechelic polymers: synthesis and applications, CRC Press, Boca Raton, 1987. Kennedy, J.P.and Marbchal, E., Cationic Polymerization, John WileyInterscience Publishers, New York, 1982. Fehervary, A., Kennedy, J. P., and Tiidos, F., New telechelic polymers and sequential copolymers by polyfunctional initiator-transferagents (inifers). IV. Chain transfer, molecular weight distribution and NMR study of telechelic a,w-Di(tert)chloropropylisobutylene, J. Macromol. Sci., Chem., AIS, 215, 1980.

Ivan, B., Kennedy, J. P., and Chang, V. S. C., New telechelic polymers and sequential copolymers by polyfunctional initiator-transferagents (inifer). VII. Synthesis and characterization ofa,w(dihydroxy)polyisobutylene, J. Polymer Sci., Polym. Chem. Ed., 18,3177, 1980. 69. Kennedy, J. P., Chang, V. S. C., Smith, R. A., and Ivan, B., New telechelic polymers and sequential copolymers by polyfunctionalinitiator-transfer

68.

Chapter I

20

70.

71.

72.

73.

74. 75. 76. 77.

agents (inifer). V. Synthesis of a,o-Di(isopropeny1)polyisobutylene and a-tertbutyl-w-isopropylpolyisobutylene,Polymer Bull., I , 375, 1979. Kennedy, J. P., Storey, R. F., Mohajer, Y.,and Wilkes, G. L., New polyisobutylene based model ionomers. I. Synthesis and characterization, Abstracts, IUPAC MACR0’82, p. 905, Bucharest. Kennedy, J. P., Storey, R. P., Mohajer. Y.,and Wilkes, G. L., Mew polyisobutylene based model ionomers. 11. Structural-property relationship, Abstracts, IUPACMACR0’82, Bucharest, p. Nuyken, 0. and Pask, S. D., Telechelics by carbocationic reactions in Telechelic Polymers: synthesis and applications,Goethals, E. J., ed., CRC Press, Boca Raton. 1987, Ch. 5. Bayer, O., Die neue methode gestatted Zum ersten male kunstoffe mit praktisch beliebigen eigenschaffen und eindentich klarem chemischen aufbau herzustellen, Angew. Chem., 76,553, 1947. Milkovich, R. and Chiang, M. T.,U.S. Patent, 3,786,116, 1974. Bamford, C. H.and White, E. F. T., Synthesis of a new copolymers based on poly(methy1methacrylate), Trans. FaradaySoc., 54,268, 1958. Bamford, C. H.,Jenkins, A. D., and White, E. F. T., The termination reaction in the copolymerization process, J. Polymer Sci., 34,271,1959. Bamford, C. H. and Jenkins, A. D., Disproportionation reactions in the copolymerization of methyl methacrylate with other vinyl monomers, Nature, 176, 78, 1955.

Molecular Nonhomogeneity of Synthetic Oligomers

INTRODUCTION Molecular species forming oligomeric mixtures differ through their molecular weight, chemical composition, and functionality. If molecular weight and chemical compositionare two characteristics equally involvedin polymer and oligomer chemistry,the functionality is a more specific parameter for the monomer and oligomer chemistry. The concept of functionality was introduced by Kienle [ l ] and today is understoodto mean the positions per oligomeric species capable of reacting under specific conditions. Let us define functionality F by

F = -N/2 l/Mn

- NM,,

”

2

where N is the total number of functional groups per gram of sampleand M,, is the number average molecular weight the of sample [2]. Functionality can assume all values from zero upward, including fractions, but chain molecules are formed onlyif the value is at least two. 21

Chapter 2

22

The above interpretation represents only a pragmatic understanding as long as the functionality is not an absolute property of a functional group, but always has to be considered in relation to the reaction partner and, sometimes, withreaction conditions. The following examplesillustrate the influence of monomer or oligomer structure as well as of the reaction conditions upon the value of functionality. A lone isocyanate group, -NCO, in a monomer is, for example, monofunctional with respect to a lone -OH group if both groups are present at about thesame concentration. Therefore, to form polyurethanes with the urethane group -NH-CO-0-, diisocyanates must react with diols. With an excess of isocyanate groups, however, the urethane groups can convert to allophanates:

+ -NH-CO-0-

-NCO

"NH"C0

I

-N-CO-0-

(2)

Since two isocyanategroups react withone hydroxyl group to form allophanates, the -NCO group is semifunctional. With polymerization initiators (chain growth kinetics), however, the isocyanate group is always bifunctional:

n N=CO

-(N-CO-)"-

I

I

R

R

The chemical structure of the resulting macromolecule, moreover, will be decided not only bythe functionality of the groupcapable of encatenation, but also by the functionality of the molecule. The strained heterocycle of lactams is bifunctional with respectto cationic initiators. However, neutral and protonatedpolymer amide groups can also take part in the disproportion, viz.:

+

I " C O

-

CO

+

1

-

+

-CO

CO +

-

I-

Molecular Nonhomogeneity

Oligomers Synthetic

23

The new polymer cation can, in turn, initiatelactam polymerization again. These side reactions increasethe mean functionality of these base units to more than two. The mean functionality of the monomer, however, can also be smaller than the sum of the functionalities of the groups contained in its molecule. For example, in the Diels-Alder condensation of a bisdiene with benzoquinone (where no byproduct is formed, yet the polymer is formed through a stepwise kinetic process), the total functionality of the bisdiene in chain formation is two perunbranched macromolecule:

Carothers derived a simple equation relating the degree of polymerization (DP) to the extent of reaction p , where p is defined as the fractionof functional groups that have reacted at time t. Thus 1 - p is the fractionof groups unreacted. If it is assumed that there are No number of moleculesat the start,then the number of remaining unreacted moleculesis

N = No(1

-p)

(7)

The average number of repeatingunits in all molecules at any stage in the reaction is the original number of molecules dividedby the remaining number of molecules:

-

o DP = NN The expression for W in terms of Carothers equation:

reaction conversion is known as the

According to this limiting equation, oligomers of M = 20 to 50 are formed for conversions of 95 to 98%. Very few reactions in organic chemistry can be forcedto andbeyond 98%, yet this requirement is the first characteristic that should be considered when itis proposed to apply a new reaction to a step-growth polymerization.In order to obtain step-grown oligomers ofM equal to 200, the fractional conversionpshould be 0.995. The general equation for the formation of a linear polymer by the step reaction of bifunctional reactants A and Bmay be writtenas follows: nA

+ nB

A(BA),-IB

(10)

Chapter 2

24

The probability of finding a repeating unit AB in the oligomer chain is p and the probability of finding n - 1 of the repeating units is p'"'. Since the probability of finding an unreacted molecule of A or B is p - 1 , the probability (P,,)of finding a chain with n repeating units (BA),, is

P,, = ( 1

- p)pI-'

(11)

Hence, the probability of the totalnumber of repeating units (BA),, is N,, = N ( 1

- p)p'"l

(12)

where N is the number of molecules in the mixture after the reaction has occurred to the extent p . Since N = No(1 - p). Equation 12 becomes N,, = No( 1

- P)~P'"

(13)

The corresponding weight-average molecular weight distribution W,, may be calculated from therelationship W,, = nN,,/No:

The number-average molecular weight weight M, are as follows:

a,,and weight-average molecular

- mN0 M,, = N and

where m is the molecular weight ofthe monomer. The index of polydispersityM J M , for the most probable molecular weight distribution is then "

Thus, as mentioned above, when p = 1, i.e., for a total conversion, the index of polydispersityfor themost probable distribution for step-reaction polymers is 2. The limitation on is usedto advantage when it is desired to obtain polymers with lower molecular weight by step-growth polymerizations. It is obvious that the value of p may be reduced by using an excess of one ofthe reactants or by adding a calculated amount of a monofunctional reactant (quenching the reaction before completion or adding a stoichiometric excess of one reactant is not economical).

m

Molecular Nonhomogeneity

of Oligomers Synthetic

25

If the number of monofunctional molecules present in the reaction mixture is Ni in addition toNo difunctional molecules A-B, the relationship (eq. 9 ) between the fractionalconversion ofeither A or B groups in the must be modified to take into account the effect of this monofunctional, polymer-chain termination:

DP =

+ Ni/No 1 - p + NJN0 1

For example, if the monofunctional derivative is present to the extent of 1%, eq. 18 predicts that a reaction conversion 98% will yield a polymer with a DP approximately 34 instead 50 as seen above. Assuming that the step-growth polymerization involves AA and BB monomers and that the B groups are present in excess, the stoichiometric imbalance defined bythe ratior

determines the number of unreacted functional groups after the reactions reaches the extent p , as shown below:

and

T N B = ( 1 - p r ) N : = ( 1 - p r )N:

(21)

where N t and NE are the number of A and B functional groups, respectively, in the initial reaction mixture, and N A and N B are the number of these groups at various stages of reaction. The total number of chain ends is the sum of N A and N B , and the total number of chains, N,, half the number of chain ends, is

= A2(

1

+ -r1 - 2 p ) N t

The totalnumber of repeating units distributed among these chains,N,, is the number of monomer molecules present initially: N,

= -1A T :+ -1N : 2

2

=

2

(23)

Chapter 2

26

Dividing the number of repeat units by the number of chains, one obtains the number average degree of polymerization:

DP =

l + r 1+r-2p

Considering eq. 23, for a 1 Yo excess of BB, i.e., r = a of 50 is attained at reaction a conversion of98.5%. The Carothers [ 3 ] equation as originally derived contained a factor, to account for the effect of monomers having more than two functional groups on the degree of polymerization.In this case, is the averagefunctionality, i.e., the number of functional groups per monomer moleculefor all types of monomer present:

where Ni and& are the number of molecules and the functionality of the ith component in the reaction mixture, respectively. The original number of functional groups in the reaction mixture is then No? and the number of functional groups that have reactedis 2(N0 + N being the total number of molecules present in the reaction mixture at an extent of reaction p . Therefore

Elimination of Nfrom eqs. 26 and 8 gives the modified Carothers equation:

When the average degree offunctionality of 2, this equation reverts to the previous form given by eq. 9. The modified Carothers equation demonstrates how sensitive step-growth polymerization for is thepresence of small amounts of polyfunctional monomers which can act as branching or crosslinking sitesin the growing chain. For a fixed extent ofreaction, the presence of multifunctional monomers in an equimolecular mixture of reactivegroups increases the degree of polymerization. Conversely, for the same mixture, a lesser extent of reaction isneeded to reach a specified with multifunctional reactants than without them. If the number of functional groups is unequal, this effect works in opposition to the multifunctional groups. For example, in eq. 10, if there is one trifunctional monomer in every ten original A monomer molecules, then f = 2.1 and a conversion of 95% will result in a polymer

Molecular Nonhomogeneity

of Oligomers Synthetic

27

having a of 200 instead of 20, as mentioned above for a stoichiometric mixture of bifunctional partners. Carothers attempted to correlate the concept of functionality with the structure resulting polymers. He concluded that linear polymers are formed only when the monomers are bifunctional, and if the functionality is higher than 2, gelation takes place and a three-dimensional structure is formed. Korshak [4]showed, however, that many tri-, tetra-, andhigher functional monomers can form linear polymers. Korshak consideredthat there are three types of factors which determine the relation between monomer functionality and polymer structure. The first factor, illustrated by the reaction of glycerol (f = with phthalic anhydride (f = 2), is determined by the monomer structure. The reactivity of functional groups is variable. Only at temperatures above 18OOC are glyphthals formed. At lower temperatures, linear polyesters are formed since the polycondensation involves primarily the -CH20H groups of glycerol, which are much more reactive than secondary CHOH hydroxyls. The second type of factor refers to the influence of experimental conditions. The reactivity of functional groups might dependon the nature the catalyst used, the proportionof monomers,the nature of the solvent, and thetemperature. For example, in the reaction of formaldehyde (f = 2) with phenol (f = not all of the active hydrogens are equally active. Under acid conditions, the quinoid structure

>

is stabilized as a reaction intermediate favoring the para additions of the protonated formaldehyde molecule. There are also indications that the reaction of one position alters the reactivity of others, the reactivity depends on the extent of reaction as well. Low molecular weight polymers with chain capped by phenol repeat units (known as A-stage resins, novolacs, or resole prepolymers)are formed when the ratioof CHzO toC6H50H is less than unity. The reactionis either acid or base catalyzed, and branching is uncommon at this stage. When the reaction is carried out under basecatalysed conditions and with a formaldehyde/phenol ratio greater than unity, a final crosslinkedpolymer(called a C-state resin or resite) is formed. The influence of solvents is illustrated by the reaction of "diamino-4,4'-dihydroxy-diphenylmethane with carborane dicarboxylic acid: in common solvents, a three-dimensional product is obtained, while the addition of tributyl amine favors the formationof linear polymers[4]. The third type refers to some favorable arrangements of functional

Chapter 2

28

groups in monomer molecules, and can be illustrated by the so-called ortho effect [5]. This phenomenon explainsthe formationof linear polymers with heterocycles in the main chain from tri- and tetrafunctionalmonomers. A typical example is the two-step preparation of an aromatic polyimide bythe reaction of pyromellitic anhydride with aromatic diamines. In the first stage, polyamic acid is formed in aprotic solvents (dimethylformamide, dimethyl-sulfoxide):

150%

Polyamic acid

Bonding occurspredominantly at thepara position; there is relatively little attack at the meta positions. In order to avoid crosslinking reactions, the solid content of the solution is restricted to 10-15070 and the yield to 50%. In the second stage, water is eliminatedat 300OC. The step-growth reactions in the early stages of the process yield oligomers which react to form polymers. The course of further conversion depends not only on the amount of functional groups in monomers, but also on thearrangement and activity of functional groups in oligomer molecules. It is therefore necessary to extend the concept of functionality from monomers to oligomers. Moreover, side reactions may alter the original functionality of monomersor thatof oligomers. Consequently, the functionality should be defined as possible functionality, q5po, or theoretical functionality, i.e., the highest number of reactive functional groups derived from themolecular structure, oras practical functionality, &, i.e., the number of functional groups capable of reacting under given conditions. The latter can change with reaction conditions (temperature, concentration of monomers, nature of catalyst, type of solvents, etc.). Based on these definitions, it is possible to express the functionality of a given monomer in step-growth polymerizations in terms of relativefunctionality, qiR, which is the ratio of to This new quantity characterizes the reactivity of both parent monomers and resulting oligomers. Usually, &,rand 4R2 1. When = 2, the resulting oligomersor polymers are linear and if4h 3, three-dimensionalstructures are formed.

&.

Molecular Nonhomogeneity

Oligomers Synthetic

29

Other types of functionality include the number-average functionality, &,and the weight-average functionality,&, which are defined by the following relations:

+ +Po2 + +P03 +

+Pol

"=

*

*

-

cZN, +poi

"

Nl+N2+N3+---

and

-

+Eo1

=

6PolNl

+ 4kO2 + + + +Po82 + +P083 +

' *

c c

&oi

(31)

+PO&

where and Ni represent the possible functionality and the numberof moles of species i , respectively. The oligomers used in industrial processes as starting materials are characterized both by molecular weightand functionality distributions. In other words each oligomeric sample is, in fact, a mixture of oligomeric species characterized by different molecular weights and functionalities. The concept of functionality distribution was introduced by Entelis et al. [5a]. This concept may be used in the characterization of the behavior of oligomers during the processing as well as for the determination of final properties of the products. The functionality distribution may becharacterized by number-average functionality, which can be determined bythe following relation:

an

where is the number average molecular weight the of oligomeric sample and M, represents the equivalent molecular weight of the repeatable unit. In turnM, may be defined by the following relation:

M, = M,

1m/s

(33)

where M, and S represent the molecular weightand concentration (in weight percent) of an oligomeric speciesin a given oligomeric mixture. Since the physical properties of polymer networks dependon the crosslink density, and thecrosslink densityis largely determined bythe oligomer reactive-groups,equivalentweight, and the averagenumberofreactive groups per molecule, the functionality is of greatest importance to those working with these materials. Functionality is usually calculated by dividing molecular weight by equivalentweight (relation The determination of molecular weight in the range lo3 - lo4 is, however, subject to an uncertainity of at least * W O even when sure that one is working with a pure material. A need exists for amore precise method.

Chapter 2

30

The preceding relations contain no information specific for the evaluation of the functionality distribution. For example, = 2 represents an ideal value for a difunctional oligomeric mixture but this value is also characteristic to a oligomeric mixture which contain equal amounts of monoand trifunctional species. We can avoid uncertainty using 4,. These two averages allow usto approximate the width ofthe functionality distribution curve of the analysed oligomeric mixture. The value of $,, is influenced bythe existance ofthe unfunctionalized or cyclicspecies in the oligomericsample.This fact determines a high degree of uncertainity in the experimental determination ofthe 4,J4,, ratio. In this case the functionality distribution curve is determined by chromatographic methods. Functionalized oligomersform multicomponent systems within which the molecular species are distinguished through their molecular mass and functionality. In turn, functionality distribution maybedivided in two components: quantitative and qualitative. Quantitative functionality distribution represents the amounts of functional groups per molecular species. This distribution gives no information about the position and the repartition of the functional groups along the molecular species. These details are revealed by the qualitative functionality distribution. Unfortunately, the experimental determination ofthe qualitative functionality distribution represents eventoday a debated problem of oligomer chemistry. given oligomeric species can be affected simultaneouslythe by molecularweight and functionality distributions. Only in a fewcasesfine organic synthesis give rise to well-defined functionalized oligomers. In practice, the experimental determination of molecular weight distribution in oligomeric samples is directly influencedthebychemical composition variation and functionality distribution ofthe given sample[a-1 l]. Let us consider one oligomeric species i which is characterized by a given functionality4. In this case

+,,

"

and

Molecular Nonhomogeneity

of Oligomers Synthetic

31

where and represent number and weight average molecular weight of an oligomeric species withfunctionality$ Mm is molecular weightof a functional group. From the relations 34-37 result (MW/M,,),= M J M , whenM,/M,, = 1 and M,, B 2flM,. "

"

"

II. EXPERIMENTALDETERMINATION OF FUNCTIONALITY Besides chromatographic methods, other physical and physicochemical methods may be usedin order to determine the value of averagefunctionality?,, (relation 32). Since @,, and Recan be determinedby direct methods, it is obvious thatf,, may be also determined by direct methods. Osmometry, ebulioscopy, vapor pressure osmometryand other methods may be usedfor the determination of G,,.On the other hand, equivalent molecular weight ae(relation 33) is determined by the known analytical methods [ 12,131. Thus esterification with aceticor phthalic anhydride is used for thedetermination of the concentration of hydroxylic groups while acidictitration and reaction with HBr are used for the determination of the content amidic and, respectively, of epoxy groups the analyzed samples. The content of double bonds is determined bythe bromine index method. Indirect determination of the functionality is based on gel point and crosslinking densitymethods.

A. The Gel Point Method The Flory-Stockmayerpolycondensation theory [ 14-19] for complex, branched reactants predicts the extent of reaction at which weight average properties of the product, such as the weight average molecular weight, become infinitely large. This critical reaction extent is defined as the gel point. The determination of the gel point may be achieved by using equations connecting the extent ofreaction at thegel point with the functionality of the reactants. This method could be reversed and the functionality calculated from the extent of the reaction at the gel point. In order to do this, the equation connecting the various factors must bein terms of two average functionalities, one for each type of reactant, each functionality capable of taking on any positive value. Flory limited his theory to systems in which only one of the reactants had a functionality greater than 2 [ 15-18], whereas Stockmayer extended the theory of three-dimensional condensation polymers to reactions between molecules with two different functional groups but no restricted functionality distribution. The so-called Stockmayer gel equation resulting from this theory is

Chapter2

32

shown belowfor anAB-type polycondensation, a reaction where groups of type A can react only withgroups of typeB. =

- 1)-'(g, -

l)-' where PAand PErefer to the respective fractions of groups A andB reacted and f w and g, are weighted average functionalities (referred to as effective functionalities) of react A andB, respectively, defined as (PApB)wl

(fw

and

Here Ai is the number of moles of reactant A bearing i functional groups and Bi is the number of moles ofreactant B bearing j functional groups. Stockmayer's gel equation is useful for the prediction of gel point or for the determination of effective functionality, provided three basic assumptions of the gel equation are met, equal reactivity of like groups, absence of cyclization, and of side reactions. For certain systems, such as carboxy-terminated polybutadiene-polyol reactions, these assumptions appear to be fulfilled, as has been shown by Strecker and French [20,21]. In their application, Strecker and Frenchconsider that the A groups (e.g., carboxyl groups) can react only withB groups (e.g., hydroxyl groups) andvice versa. At every time ofreaction equal amounts of reactive groups of type A and B must havereacted. By transformation ofeq. 38 one can write

and

where r is the ratio of the total number of B groups initially present to the total number of A group initially present. According to eq. 42, in a test for functionality it is necessaryto know only the functionality of the crosslinking agent, the ratio of reactants, and the extent of reaction of either of the reactants at the gel point. The value

Molecular Nonhomogeneity

of Oligomers Synthetic

33

Tw.

obtained is a weighted averagefunctionality, If the spread in functionality is small,fW will be closeto the?". In thecase of carboxy-terminated polybutadiene-polyol reactions, the determination of unreacted carboxyl groups at the point was carried out principally byinfrared methods. The titration methods may be equally used in this respect. Some work was done to see whether titration of carboxyl groups with standard base was sufficiently accurate for functionality measurements. Onedifficulty with the titrationmethod liesin the time required to withdraw a viscous aliquot from the reaction mixture. The mixture is viscous when approaching the gel point that a minute or two may be required to remove a sample large enoughto titrate. In that time the reaction has proceeded sufficientlyto introduce a significant error. Phenolic antioxidants are often present in commercial oligomers. These react with a base and may or may not react with epoxidesor aziridine crosslinking agents. To remove this uncertainity the oligomers must be purified. At best the titration method cannot be considered as accurate as the infrared method. The last method needs a correct calibration in order to determine the true content of carboxylic end group during the condensation reaction with phenols. In the case of polyisocyanated, the basic requirements of Stockmayer's gel equation cannot usually be meet, mainly because of the frequent occurrence of like groups of unequal reactivity and the need for relatively dilute systems to detect the gel point [22]. However, a useful form of Stockmayer equations can be retained for the calculation of the true effective functionality of such reactants. 1.

Influence of Functional Groups of Unequal Reactivity

When a polycondensation reaction isallowed to proceed to completion under nonstoichiometricconditions, for example groups of type A being in excess, PAis equal to the initial mole ratio of B to A groups, and PB= 1. Thus, there will be a critical initial ratio of the reactive groups for which gelation will coincide with the completion of the reaction. In this case, Stockmayer's gel eq. 38. simplifiesthen to the following form

where (PA)gcl is the critical initial ratio of B to Agroups. The corresponding equation for theconverse case( B groups in excess) is

The eqs. 43 and 44 have been determined by Fogiel [22] usingthe criterion of gelation given by Case [23].

Chapter2

34

2. Effect of Cyclization The problemofring formation in polycondensation reaction hasbeen treated theoretically by Jacobson and Stockmayer [24]taking intoaccount an original mixture of reactants consisting of A,, A,, . . , Ai moles of reactants of type A bearing, respectively, 1, . . , i functional groups and B,, B,, . . . , Bj of reactant of type B bearing, respectively, 1,2, ., j functional groups. All unreacted groups ofthe same kind are assumed to be equally reactive. Let rnbe the probability of forming an n-unit ring, where a single unit is defined to contain two groups of each type along the backbone forming the ring. Consider a bond -[AB]- picked at random from thereacted system. The objective is to compute the probability for the recurrence of the bond without cyclization (partial propagation expectation P;). After summing up over all monomers and ring sizes (total propagation expectation), gelation will occur when P = 1. The probability that B of the -[AB] - bond belongs to Bj monomer is pi; the probability that any of the (j 1) groups of type B has reacted with a group A belonging to Ai, which isnot a part of the molecule chosen at random,is

.

.

..

-

n'

p p B ( j- 1)P ( 1

- nC rn) =l

(45)

where n' is the number of units present in the chain selectedat random. The probability of any of the ( i - 1) groups reacting with any B group other thanthose derived from the same molecule isthe partial propagation expectationpb, viz. n'

P;J

=ppB(j

2

- I)Pi( - n=l r n ) P A ( i

- l)

(46)

Summing piJ over all species and ring sizes and equating the sum of unity yields the gel point

or

For a small extent of cyclization, the gel equation can be approximated to

35

MolecularNonhomogeneityof Synthetic Oligomers -1

m

(pApB)gel

=

(1 - c

rn)

(fw

-

l ) -'(gW

-

l ) -I

(49)

l

The problem remains to compute the value of Cz=lrnof the re1ation 49. According to Jacobson and Stockmayer [ M ] the ratio of the number of rings n units (R,,)to the number of chains of n units (C,) is

where B=(%) is a parameter the reaction conditions, V denotes the total volume of the system, v is the number of chain atom per unit, and b is the effective bond length. Assuming a small amount of cyclization

where c is the concentration of reactive groups one of the reactants in moles perliter. Summation of over all ring sizes yields

The infinite series m

is a well-known Reimann I function which has the value of 1.341. For a given polycondensation system, allparameters may be combinedto give 2 5 r n = k-

c Substituting this value into eq. 49 yields Stockmayer's gel equation [l41 modified to account for ring formation, viz. n=l

The method of gelpoint has been verified using various oligomeric systems. Thus, the value o f f wwas determined for hydroxyl or carboxyl terminated oligobutadienes [25,26], polypropylene glycols [27], and tiokols [28].

Chapter 2 Table 1 Average Functionality o f Liquid Tiokols (solidifier: manganese dioxide) Functionality of linear part of oligomer

ii?, 1

- = B”

2470 2170 2350 2500 2600 3500 2060 2300 2050

S3

2.38 2.40 2.45 2.58 2.53 2.57 2.33 2.30 2.40

2.40 2.41 2.44 2.50 2.50 2.55 2.20 2.20 2.20

2.20 2.35

2.45

-

2.37 2.23 2.30

‘Calculated from -SH determination data, bCalculated from fractionation data. Source: Ref. 25.

The variation of functionality in the case of industrial tiokols is presented in Table 1. Table 2 gives some results on functionality of carboxyl terminated oligobutadienes obtained by using Epon X-801with a functionality of as curing agent. The value of functionality of the resin is approximately the same, whether calculated from the epoxide reaction or

Table 2 Weight Average Functionality of Carboxyl-Terminated Oligobutadienes Reaction Glycerol Reaction Epoxide

6

COOH react. time COOH Gel react. time Gel (mm 290 242 152 136 152 178 Source: Ref. 20.

(min)

(qo)

2.40 2.37 2.95 2.38

24.0 26.5 24.5 19.0

59.8 60.2 50.3 60.2

2.40 2.38 2.97 2.38

2.24

18.5

64.2

2.22

04

Molecular Nonhomogeneity

Oligomers Synthetic

37

from the glycerol reaction. Other characteristics concerning various oligomeric systemsare presented in Tables In theory, the number of average functionality, g,, can be determined from theaverage molecularweight M,, (corrected for water) and theequivalent weight E,, according to the equation

The determination of functionality of high molecularweight polyols bythe molecular weight method is rather unsatisfactory due to a relatively high experimental error ( *5%), but particularly due to a very strong effect of potentially present low molecularweight inert materials. The gel point method, on the other hand,is insensitiveto the presence of inert materials and it is precise to about1070. Moreover weight average and number average functionalities of conventional polyols usedfor flexible foams will differ by only 4% in the most unfavorable case (50/50 equivalent ratio of diol and triol). Therefore, the gel point method appears to be suitable for thedetermination of number, as well as weight average functionalities of conventional polyols. In Table 5 functionalities of the polyols are compared with respectto the method used. Even though the water content has been excluded in

Table 3 Number and Weight Average Functionalityof Oligoesters (curing agent:hexamethylenediisocyanate) Glycerol To

content, Oligomer 2.26Oligoesterb 2.17

M,, j/j,,

0.75 1S O 1280 1.so 2.241490 1.21 3.22 2.65 1890 3.00 Oligobutadienpolyol 1.63 3.43 - 2.10 2300 1.43 3.96 - 2.77 2275 2.86 1730 1.51 5.15 3.41 2000 2.44 5.61 - 2.30 2400 8.10 - 2.80 2800

j~j,,

1680

-

2.22 1.042.31 2.40

1.07

1.61 4.61

2.89

'Determined by gel point method. bCopolyester based on adipic acid, diethylenglycol, glycerol, and hydroxy terminated oligobutadiene. Source: Ref. 25.

02

Chapter 2

38

Table 4 Number and Weight AverageFunctionality of Carboxyl Terminated Oligobutadiene and Copolymers of 1,3-Butadienewith Acrylonitrile (curing agent: epoxyline-5) Gel Oligomer Oligobutadiene 2.09 220 2.05 0.99 2.06 280 1.00 2.07 260 0.98 2.04 240 1.03 2.00 210 1.02 1.97 320 Copolymer 1,3-butadieneacrylonitrile Copolymer 1,3-butadienemetacrylic acid

2.08 2.07 2.08 1.94 1.93

3690 3230 3110 3120 2570 1760 3130 430 3280 3230 360 2520 2310 2310

386 1.08 0.98 1.03 - 2.26 - 2.95 - 2.95

1.90 2.13 1.98 2.10 2.01 2.01

2.06 2.08 2.04 4.74 5.94 5.93

Source: Ref. 25.

the calculation of molecular weight, the functionality of these materials is appreciably lower by the molecular weight method than by the gel point method, the lattergiving results closerto the expected functionalities.

B. CrosslinkingDensity Method The accuracy with whichthe functionality of a given oligomer can be measured dependson the accuracy with whichnumber or weight average molecular weight can be measured. Table 5 Functionality ofSelected Polyols Used for Flexible Foams Material Polyester “triol” (“Voranol” CP-3OOO) Polyester ‘Yriol” (Witco “Fomrez” 50) Polyester (Witco “Fomrez” 52)

g,’

a,,

E,“

2073 2.672.08 996 2810 2.672.561087 2265 2.312.02 1120

‘Equivalent weight. bDeterminedby the following relation: g, = M,/,!?,. aetermined by gel pointmethod. Source: Ref. 22. ”

g,”

Molecular Nonhomogeneity

Oligomers Synthetic

39

From an examination of available test methods and their limitations for number average molecular weight measurements, vapor pressure mometry was selected as the most suitable method for the low molecular weight range (1000-5000). Vapor pressure osmometry(VPO) is a thermoelectric differential vapor pressure technique for determining M,, of oligomers. The thermoelectric methods has significant advantages in speed and in small sample size. The basis of the thermoelectric method is the measurement of the small temperature difference resulting from a differential mass transfer between droplets of pure solvent and oligomer solution maintained in an atmosphere of solvent vapor. It is true that molecular weight measurements can be seriously lowered by the presence of low molecular weight impurities, notablyresidualsolvent and antioxidants. There are other factors, though, which must be considered:the possibility of oligomer-oligomerassociation which leads to anapparently higher molecular weight, and the phenomenon of oligomer-solvent interaction. If a polymer is formulated at a one-to-one ratio of reactive groups and complete reaction is obtained, the crosslinking density, X,, as moles of branch points per gram can be obtained and is givenby the following relation:

where n = number of different reactants; i = the individual reactants, values 1 to n; f = functionality of the reactants known for all ingredients except oligomer and not to exceed in value; W = weight fraction of ingredients; E = equivalent of ingredients. The possibility of using the critical conditions of gel formation and concentration of internodal chains of a network to calculate the?,, and?, has been analyzed by Entelis l] using as an example the system involving the copolymer of tetrahydrofuran andpropylene oxide, 1,4-butanediol, 1,1,1-trimethylol propane and 2,4-toluylenediisocyanate. The effecthas been shown ofthe reactivity ofinitial reagents, temperature and concentration of a catalyst on the value of the $,, and f, determined by indirect methods. In this instance, the change of relative capacity of reaction of reactants under the influence of temperature has been determined. The value of determined functionality was also influenced bythe mechanism of oligomerization reaction. Both influencesare materialized by a twenty percent variation of the $,, and ?, values. The differences between ?,, and f, values obtained by indirect methods or by determination of chemical composition may determined also by the other three additional factors: the

40

Chapter2

uncertainity of functionality determination, the difference between chemical and effective functionalities and by the incertitude of molecular weight determinations. The differences between chemical and effective functionalities appears mainly when the value of monomer functionality is higher than Another shortcoming of indirect methodsis the fact that these methods do not take intoaccount the unfunctionalized species. The application of indirect methods is limited to the monomers with functionality lower than However, these methods are suitable for the determination of effective functionality of various oligomeric systems. In this manner some predictions are possible concerning the structure and physicochemical properties of the end products.

REFERENCES 1. Kienle, R. N., Kinetics polycondensationprocesses, Znd. Eng. Chem.,22, 590,1930. 2. Min, T. L., Miyamoto, T., and Inagaki, H., Determination of functionality distribution in telechelic prepolymers by TLC, Rubber Chem. Technol., 50, 63, 1976. 3. Carothers, W. H.,Polymers and polyfunctionality, Trans. Faraday Soc., 32, 39, 1936. 4. Korshak, V.

V., Dependence of polymerstructure on monomer functionality, Acta Polym., 34,603, 1983. 5. Korshak, V. V., Thermoplastic Polymers, Nauka, Moscow, 1972, 211(in Russian); a) Entelis, S. G., Functionality distribution in synthetic oligomers systems, J. prakt. chem., 313,484, 1971. 6. Uglea,C. V., Hemicellulose fractionation, Makromol. Chem., 175, 1535,

1974. 7. Simionescu, N., Uglea, C. V., and Feldman, D., Uber die polydispersitat ‘der methylcellulose, Cell. Chem. Technol., I, 199, 1967. 8. Uglea, C. V., Simionescu, N., and Feldman, D., Contribution au fractionnement des copolymbres. I. Copolymbres acrylonitrile-acetate de vinyle, Rev. Roum. Chim., 13,949, 1968. 9. Feldman, D., Uglea, C. V., and Simionescu, N., Contributions au fractionnement des copolymbres. 11. Copolymbres ternaires acrylonitrile-acbtate de nyle-alphamethyl styrene, J. Polymer Sci., A-l, 7,439, 1969. 10. Uglea, C. V., Lupu, V., and Sgndescu, F., Contribution B la caracterisation des copolmbres.V. Influence des conditions experimentalessur les resultats du fractionnement, Rev. Roum. Chim., 16,1399, 1971. 11. Adams, H.E., Ahad, E., Chang, M. S., Davis, D. B., Franch, D. M., Ager, H. J., Law, R. D., and Simkins, J. J. P., A cooperative molecular weight distribution test,J. Appl. Polymer Sci., 17,269, 1973. 12. Baczek, S. K.,Anderson, J. N., and Adams, H. E., Methods of functionality determination intelechelics, J. Appl. Polymer Sci., 19,2269, 1975.

Molecular Nonhomogeneity

ofOligomers Synthetic

41

13. Schneko, H., Degler, H., Dongowski, H., Caspary, R., Angerer, G., and Ng, T. S., Synthesis and characterizationof functional diene oligomers in view of their practical application, Angew. Makromol. Chem., 70,9, 1978. 14. Stockmayer, W. H., Molecular weight distribution in synthetic polymers, J. Polymer Sci., 9,69, 1962. 15. Flory, P. J., Molecular size distribution in three dimensional polymers. I., J. Amer. Chem. Soc., 63,3083, 1941. 16. Flory, P. J., Molecular sizedistribution in three dimensional polymers. II., J. Amer. Chem. Soc., 63., 3091, 1941. 17. Flory, P. J., Molecular size distribution in three dimensional polymers. III., J. Amer. Chem. Soc., 69,30, 1947; IV., ibid., 63,3096, 1941. 18. Flory, P. J., Molecular size distribution in three dimensional polymer units, V., J. Phys. Chem., 46, 132, 1942; Flory, P. J., Molecular size distribution in three dimensional polymer units. VI. Branched polymers containing A-R-Bf., type units, J. Amer. Chem. Soc., 74,2718, 1952. 19. Stockmayer, W. H., Molecular distribution in condensation polymers, J. Polymer Sci., 11,424, 1953. 20. Strecker,R.A. H. and French, D.M., The determination of reactive-group functionality from gel point measurement,J. Appl. Polymer Sci., 12, 1697, 1%8. 21. French, D. M. and Strecker, R.A. H., Functionality and observed versus predicted gel points, J. Macromol. Sci. Chem., AS, 893, 1971. reactions of polyiso22. Fogiel, A. W., Effective functionality and intramolecular cyanates and polyols, Macromolecules, 2, 581, 1969. various 23. Case, L. C., Theoretical interpretation of functionality distribution in oligomeric systems, J. Polymer Sci., 26,333, 1957. 24. Jacobson, H. and Stockmayer, W. H., Intramolecular reaction in polycondensation. I. The theoryof linear systems, J. Chem. Phys., 18, 160, 1950. 25. Valuev, V.I., Shlyakhter, E. G., Erenburg, E. G . , and Poddubnyi, I. Ia., Study of the functionality distribution of various oligomers, Vysokomol. Soedin., AI4,2291, 1972. 26. Consaga, J. P., The determination of functionality distribution in carboxyl terminated oligobutadienes, J. Appl. Polymer Sci., 14,2157, 1970. 27. Evreinov, V. V., Ol’kov, Yu. A., Baturin, S. M., and Entelis, S. G . , Indirect methods for determination thefunctionalities of oligomers, Vysokomol. Soedin., A20,2146, 1978. 28. Prom. SK, 1976, No. 3, p. 15 (Russian). 29. Entelis, S. G . , Evreinov, V. V., and Kuzaev, A.I., Reactive oligomers, Khimia, Moscow, 1985, Ch. 3. 30. Ol’kov, Yu. A., Lugovoi, V. B., Baturin, S. M., and Entelis, S. G . , Kinetics of three-dimensional polymerization and the effect of monofunctional molecules on the properties of cross-linked poly(etherurethane) elastomers, Vysokomol. Soedin., A14,2662,1972. 31. Ol’kov, Yu. A., Baturin, S. M., and Entelis, S. G., Reactivities of functional groups ofcomponents and theirinfluence on thethree-dimensional polymerization. Kinetics and the properties of cross-linked poly(ether-urethane)s, Vysokomol. Soedin., A15,2758, 1973.

Liquid Chromatography

During the early 1970s the advantages of liquid chromatography(LC) were really just beginning to draw the attention of a wide audience. Revolutionary improvements in equipments, materials, techniques,and the application of theory have brought LC to what Snyder and Kirkland have aptly called Modern Liquid Chromatography [l]. Liquid chromatography is now a mature analytical technique that is based on a well-developed theoretical background and as a consequence is carried out with fairly efficient LC equipment. The high level of performance achievable by LC today, however, is the direct result of over two decades of careful study into the basic theory of chromatographic separations. Liquid-solid chromatography (LSC) is the oldest form of the four basic modes ofLC (ion-exchange, gel permeation,partition, and LSC). The first person who was able to explain correctly the phenomena taking place during the movement of substances along a sorbent bed was Michael S. Tswett [2]. Using these phenomena, he created a remarkable analytical method, showed its wide scope, and named not only the tech42

Liquid Chromatography

43

nique itself but also the process and the branch of science dealing with it. On March M. S. Tswett presented at a meeting of the Biological Section ofthe Warsaw Society ofNatural Sciences a preliminary communication “On a New Category of Adsorbtion Phenomena and their Application to Biochemical Analysis’’ In this lecture, he reported on the first part of his experiments aimed to separate plant pigments. Tswett systematically tested a very large number of solids to check the possibility of using them as adsorbents; he definitely understoodthe nature of adsorbtion and already took an important step forward by being ableto have spatial separation of several zones on the adsorbent. Although Tswett didnot describe in hismethod(whichwas not yetfinalized)as“chromatography” (this name was first used in in his two well-known papers his investigations were already in a well advanced stage. Thus, we can safely characterize his communication as his first paper on chromatography. When Michael Tswett began to perform chromatography, he used a polar support as a stationary phase and a nonpolar solvent as the developing fluid. This form of chromatography is known today as normal phase liquid chromatography(NPLC). The term reverse-phase liquid chromatography(RPLC) means a procedure that is a logical extension of NPLC. In this form of chromatography the mobile phase is more polarthan the stationary phase. It is unfortunate that many chromatographers tend to consider this a unique technique distinct from so-called NPLC since the technique is becoming very popular and is usedto separate a wide variety of mixtures.

THEORETICAL BASIS Chemists are more familiar withthe behavior of small moleculesthan that of macromolecules (oligomersand polymers), while chromatographers are accustomed to working with isocratic elution (constant composition of the mobile phase) rather than with gradient elution (variable composition of the mobile phase). It is not surprising then that there is some confusion when it comes to the principles of chromatographic separation of macromolecules. Practitioners often are puzzled by the outcome of some change in separation conditions. The apparent absence of predictability and of guidelines for method development also preventsthe full potential of these separations from being achieved. Such apparent anomalies in the separation of macromolecules have led workersto make new proposals for thebasis of such separations. Many chromatographers assume that macromolecules are retained at the column top, until at somepointthey are desorbedcompletely and then move through the column without further interaction with the stationary phase.

44

Chapter

Other workers have suggestedthat chromatographic migration in these systems occurs bya precipitation-redisolution process rather than by interaction of solute molecules with the stationaryphase; that is, solute solubility determines separation. Another view is that the multipoint attachment of macromolecules to the surface of the stationary phase leads to a retention process that differs fundamentally from that forsmall molecules. The general problem of chromatography is, in a practical sense, not solvable. For ordinary column chromatography unknowable variables exist involving everythingfrom the natureof the complex multisitesurface to the details of the method of packing the column. Assumptions can be, and have been made that allow for an approximate solution to the theoretical problem. The approximate results have been shownto confirm experience to a fairdegree. The principal problemof chromatography is separation of substances. In a typical procedure the material of interest is started from a band near the topof the column and is washed downthe column with pure solvent. As the washing proceeds,there is a separation of the original band into component substances. The value of the method is enhanced by any factor that can further the separation of the component bands, and that can minimize eachband dispersion. The mechanism of separation in LC is a subject of much debate [591. Snyder [8] describes and compares each of the proposed mechanisms. Presently there is no “best” model for all packings, solutes, and solvent strengths. Snyder’s model [5,8] provides a good understanding of alumina and is fairly good for silica with weak solvents. Other models [7,9] best handle silica with strong solvents and are useful for selecting solvents for gradient elution works. An important goal in separation science is to develop a quantitative understanding of the molecular mechanism of retention in LC. This would not only permit prediction of retention and separation behavior from molecular structures, but also permit the development of chromatographic methods for the purpose of exact physicochemical measurements of properties of solutions and forthe purpose of better understanding of interphases of chain molecules, including Langmuir-Blodgett films, micelles, and bilayer membranes. The statistical mechanical theories of polymer chain adsorption at a solution surface have been studied extensively by Rubinand Dimarzio [ 10121. These studies represent one of the first todevelop tentativelya theoretical basis of LC. The central problem of such a study is to enumerate all possible configurations of a flexible chain polymer in the vicinity of a solution surface and keep track of the number of monomer units in each chain configuration which lie (or are adsorbed) in the surface layer. With

Liquid Chromatography

45

this information, it is possible to assign a probability to each configuration which takes into account the energy gained with 7 monomer units are adsorbed in the surface layer and thus tocalculate the average properties of a polymer chain. Rubin [ 101 states that the polymer chain configurations are based on a lattice model in which there is a one-to-one correspondence between random walk paths on the lattice and polymer chain configurations. An isolated polymer chainat a solution surface is investigated. Onedimensional characteristics of the monomer unit distribution are determined analytically in the limitof a longpolymer chain, neglecting the self-excluded volume.The mean number of monomerunits adsorbed in the surface layer, is determined, assuming that one end of the polymer chain lies in the surface layer. The parameter N is the number of monomer units in the chain, and 8 is the adsorption energy of each monomer unit in the surface layer measured in unitsof kT. In addition, themean distance of the free end of the chain from the surface z(8,N) is determined. The lattice model considered include the simple-cubic, hexangonal-closepacked, facecentered-cubic, and body-centered-cubic lattices.For N + both and z(8,N) exhibit a very interesting discontinuity at a lattice-dependent adsorbtion energy 8,. For example, for 8 > e,, (which is also proportional to the average adsorption energy of a polymer chain) is proportional to N. For 8 8,, is proportionaltoa constant of order unity; and for 8 = e,, is proportional to NI'*. It is shown that the probability distribution of the end ofthe chain decreases exponentially with increasing distancefrom the surface layer for 8 > 8,. In addition, themean number of monomer units in the kth layer from the surface is determined for N 1 and 8 > 8, and is found to decrease exponentially with increasing k. In effect, for 8 > 8, the polymer chain exists inan adsorbed state. The adsorption of a long flexible-chain moleculeon a long rigid-rod molecule wasalso studied by Rubin [ 11 The principal difference between the rod-and surface-adsorption model is in the form of the function

for 8 > ln(6/5), where f ( 8 ) is the limiting average function of monomer units in adsorbing sites. In particular, as 8 decreases toward e,, the slope of thef(8) vs. 8 curve approaches 25 in the surface adsorption model whereas the slope and all higher derivatives ofthe f ( 8 ) vs. 8 curve approach zero in the rod-adsorption model. The rodlike molecule is represented by the lattice sites on the of a single-cubic lattice; sites which are nearest neighbors to the are adsorbing sites. The dimensionless adsorbtion energy per monomer unit is 8 = d k T . The problem of enumerating polymer chain configurations tak-

46

Chapter 3

ing into account the increased probability of occupying adsorbing sites and the zero probability of occupyingZ sites is formulatedand solved as a random-walk problem. This model aisnatural generalization ofa randomwalk model ofadsorption on a plane solution surface.The average function of monomer unitsin adsorbing sitesfR(0) is computed as the limit in which the number of monomer unitsin the polymer chain approaches infinity. On the other hand, DiMarzio and Rubin [ 121 apply the lattice model a of adsorbtion of an isolated chain polymer between two plates using matrix formalism and a grand canonical ensemble formalism. The matrix formalism is particularly convenieni for calculating the polymer segment density as a function of the distance from one of the plates for different fixed plate separations. The grand canonical ensemble formalism consists of loops (sequences of polymer segments whose ends are in contact with one plate and whose intermediate segments lie between the two plates), bridges (sequences of polymer segments whose ends are in contact with different plates and whose intermediate segments lie betweentwo plates), and trains (sequences of polymer segments which are wholly incontact with one plateor the other). All of the foregoing quantities have been calculated in the limit of infinite molecular weight as a function of the distance of separation between the plates and the energy of adsorption of a polymer segment on a plate. The self-excluded volume of the polymer chain is ignored. In addition, the average size loops, bridges, and trains, and the effective force of attraction between the plates is calculated. The above statistical-mechanical theory was later developed by Grosberg [l31 and Entelis [l41 and applied in the case of functionalized oligomers. Successful fractionation of high molecular weight homopolymers using gradient elution high-performance liquid chromatography (HPLC) has been reported [ 15-24]. For example, polystyrene homopolymers or oligomers in a molecular weight range 102-107 have been separated efficiently and rapidly by means of gradient elution HPLC utilizing either C-l8 orC-8 chemically bonded phaseand a methylene chloride-methanol mixed mobile phase [19]. Other mixed mobile phases such as THF-H20 has also been employed [20,24]. Separation of styrene oligomers also been achieved by supercritical fluid chromatography with a density-programmed supercritical n-pentane mobile phase Based on the above mentioned experimentaldata, a statistical thermodynamic theory of polymer fractionation and its application to gradient elution HPLC has been developed by Boehm, Martire, Armstrong, and Bui (BMAB-1) to describe the retention behavior of an isolated flexible homopolymer molecule distributed between a binary mixed mobile phase and an idealized stationary phase consisting of sorbed solvent on a homoge-

Liquid Chromatography

47

neous planar surface [27]. In this analysis the Flory-Huggins lattice model approach is applied to an isolated polymer moleculeand entrained solvent molecules in each chromatographic phaseand nearest neighbor interactions are included in the Bragg-Williams random-mixing approximation [28,29]. The BMAB-I theory successfully accounts for the observed experimental trends of an increasingly abrupt transition from very high to very slow retention asthe degree of polymerization,DP, of the polymer increasesand as themobile phase becomes sufficiently enriched to a critical composition of the better solvent for the polymer. The critical composition represents that mobile phase composition where the capacity factor becomes unity. The critical composition is predicted to be a monotonically increasing function of DP for flexible homopolymers of sufficiently largeDP (> 15) and this dependence emanates primarily from the molecular flexibility of the polymeric solute which allows sizeand shape alterations by solventuptake or exclusion in responseto its solvent and/or surface environment [28,29]. The dependence ofthe critical compositionon DPalso generatethe opportunity for polymer fractionation by gradientHPLC. The BMAB-l theory was subsequently extended to include intermolecular polymer segment interactions and thus be applicable to semidilute polymer solutions. This extension is hereafter referred to as the BMAB-2 theory [30]. The onset of the semidilute regime where different flexible polymer molecules begin to interpenetrate appreciably occurs in good solvents for a volume fraction of chain monomers given byd, = DP -'" [31]. Hence departures from infinite dilution can be anticipatedfor small values of d,, when DP islarge(e.g.,when IO3 DP 4 2 d, 2 1 X lo-').Whensolutesampleconcentrationduringelutionfallsin the semidilute solution range, concentrationeffects on chromatographic retention behavior must be included for meaningful comparisons between theory and experiment. The BMAB-2 theory predicts that the transition region between high and low polymeric solute retention becomes more gradual as the concentration increases. Later, Boehm and Martire[32],byapplicationof the McMillanMayer procedure, developa more general derivation ofthe BMAB theory. This is achieved by the derivation of a more general expression for the capacity factor k' for a flexible chainlike polymer molecule distributed between mobile and stationary chromatographic phases. This generalization of the original BMAB-l theory is more reliable for lower molecular weight oligomers since more general expressions for the solvent entrained polymer coil expansionfactors are introduced that apply for smaller values of DP. Theoretical predictions of partition coefficients of oligomers and polymers between imiscible solvent phases as well as explicit expressions are obtained for the chromatographic capacity factors and corresponding

Chapter

48

retention timesin gradient elution for flexible, chainlike oligomers and polymers. Furthermore, the authors also investigate the effects of including nonlinear (i.e., quadratic) contributions to the mobile phase composition dependence on In k’ on chromatographic retention timesin a linear gradient elution. The nonlinear contribution are found to be more important forlower DP solutes. In the infinite dilution limit, the BMAB theory applies statistical thermodynamics to investigate the equilibrium distribution of an isolated, flexible chainlike macromolecule between a binary solvent mobilephase and an idealized planar stationary phase surface with sorbed solvent(s). The relative phase preference ofthe macromolecule dependson DP, 6, all possible types of nearest neighbor interactions such as solvent molecule-polymer segment,solventmolecule-solventmolecule,polymersegment-polymer segment, solvent molecule and/or polymer segment-stationary phase surface site, and also the configurational entropy of the flexible polymer molecule in each chromatographic phase. The flexible polymer coil plus the entrained solvent molecules is assumed to behave as a swollen spherical (thin cylindrical)gel in a favorable mobile (stationary) phase environment. In its simplest linearform, theBMAB theory predicts that In k’ = S(4c - 4 ) = IAIIDP(4c

- 4)

or

where

and represents the slope of -In k with mobile phase composition in the case of nonzero solute concentration, c, and c = 0, respectively; k’ ,capacity factor (theoretical) for a flexible oligomer or homopolymer when the “exact” and asymptotic, large DP solute expansion factors areutilized; &, asymptotic limiting value of the critical composition as DP + m;4, phase ratio. A I asymptotically approaches a constant value independent of DP for large values which is usually negative for reasonable solutions of the mixed solvent mobile phase. In the simplest version of the BMAB theory which applies when only sorbtion of the better polymer solvent onto the stationary phase occurs, the following predictions for 4cand A I result:

4c = 4: and

-

lalDP-In

+ ~ I D P - ~+” c,DP” + c,DP“ln

DP

(5)

Liquid Chromatography

A1 =

x12

+

x13

- x23

49

+ dDP-4’5 + cDP

(6)

Here, 4:(0 &, 4c 1) is the asymptotic limiting value of the critical composition as M + and xu (ij= 1-3) represents the reduced interchange energy required to form an i-jnearest neighbor pair. The indices 1 and 2 respectively refer to the better and poorer mobile phase solventsand 3 corresponds to amonomeric segment ofthe polymer. The quantities a, b, cl, c,, d, and c introduced in eqs. 5 and 6 are independent of DP and depend on the interchange energies and weakly upon the mobile phase composition [321.

Eqs. 2 and 3 indicate that for large DP homopolymers, k’ = exp [ [ A lI M(& - 4)] undergoes an abrupt transition from a very large to a very small value within a very narrow mobile phase composition range centered at = &. Thus, a very slight changein mobile phase composition from &(l - e) to &(l + e), where e is a small positive infinitesimal such that lim e + 0 as DP + leads to an increasingly abrupt change from very large to very small retention of solute as DP increases. Eq. 5 predicts that & becomes a monotonically increasingfunction of DP for sufficiently large DP which asymptotically approaches r#~ as DP + Consistent with the above considerations, the BMAB theory also predicts that fractionationof homopolymersand oligomers with respect to DP is possible providedcertain requirements are satisfied:

:

The macromolecules are flexibles. A combination of a good solvent (1) and a poor solvent (2) for the polymer is employed in the mixed mobile phase such that xlz x13 - x23 0. The good solventis preferentially sorbed onto the stationary phase.

+

Furthermore, fractionation of higher DP homopolymers requiresgradient elution which commencesat aninitial mobile phasecomposition that is less than the critical composition of the lowest molecular weight homopolymer in the sample and themobile phasecomposition is then temporally enriched withthe better solvent to a final value, 4, that atleast exceeds the criticial composition of the highest DP homopolymer present. This can be achievedif 1 1 4, > For veryhighvaluesof DP, 4c increasesonly slightly withDP rendering fractionation more difficult. In a linear gradient, the temporal variation of the mobile phase composition is

4(?)= 4 ( 0 ,

+ Bt

t 0 (7) where q+?) and is the mobile phase composition entering the column inlet at time t and t = 0, respectively, and B is the rateof increase of thevolume fraction of the good solvent.The solute retention time, tR,in linear gradient elution if eqs. 2 and 3 apply for k is

Chapter

50

t~ =

to

+

(IAIIDPB)”ln[l

- 4CO))ll

+

[All DPBtoexp[IAII DP (4, (8)

where torepresents the column dead time.When

l 4 DPBto

expt 1

4 (+c - +(O))I

1

eq. 8 reduces to tR

= to

+ (4, - + ( o ) ) / ~ + ( S B ) “ln(SBt,,)

(9)

where S = IDP. This result can be utilized to determine the critical composition, 4,, and S = -a In k/a 4, by gradient elution measurements provided the linear approximation to In k given by eq. 2 is valid over the entire mobile phase composition range where chromatographically significant values of k are encountered (10 2 k 2 0.1). A linear relationship between In k and over the relevant composition range is anticipated to become moreaccurate as DP increases. In practice, B(tR - to) ismeasured for variousselectionsof the gradient rate, B, with +(o) and t maintained at fixedvalues.Linear regressionisemployed to obtain the best linear fit of B(tR - to) to ln(Bt,) and the gradient determined value of the slope, S,, and critical composition C$ lo5 daltons) values of S can be 30- to 100fold greater than for small molecules, meaning that retention is then extremely sensitive to change in 4. This behavior of large solute molecule may appear strange to chromatographers who are more familiar with the separation of smaller molecules (lower values of S). Because of the dependence k on S in gradient elution (eq. 4a, Table l), the gradient separation of macromolecular samples requires different experimental conditions to achieve optimum values ofk’ during the separation. Chromatographers who have comparedthe resolving power of different columns for both large and smallmoleculeshave often drawn two conclusions: first, that the plate numbers measured for large-solute molecules are generally much smaller than those measured for small molecules (same column)and, second, that “good” columnsfor small molecules separation (large M values) are often “poor” when applied to macromolecular samples and vice versa. This again has been interpreted as evidence of a nonchromatographicprocessin the separationofmacromoleculesby HPLC. On the other hand, chromatographic theory holds that eq. 15 applies to all samples for a given column. These contradictory observations will be analyzed inthe following paragraphs. The reduced velocity y of eq. 20 is proportional to VD,,,.The solute diffusioncoefficient D, isroughly proportional to (solutemolecular weight) “0.4 which meansthat will be proportional to (molecular other factors being equal [l]. Thus present separations of small molecules with 3-5 pm particles usually involve y values in the range < y < 15, whereas macromolecule separations typically involvey values greater than 50. This meansthat eq. 1 is approximately givenas

h =A

1.5 for the first band). Throughput can be increased in some(but not all cases) by charging larger samplesthan correspond to the case of two bands of interest just touching ( R , = 1). The effect of particle size on throughput iscomplex and there is currently no single “rule of thumb” to guide its choice. Because of this complexity, the optimum particle size for any given case can best be determined only from a knowledge of the exact circumstances surroundingthe separation.

E. Equipment Figs. 14 and 15 are a schematic of the instrumentation required for highperformance liquid chromatography and, respectively, a general view of a liquid chromatograph produced by Knauer company (Germany). The instrumentation comprises several components: A solvent reservoirthat contains the mobile phase A high pressure pump used to push the mobile phase through the tightly packed column A pressure gaugefor monitoring the pump pressure An injection device, usually containinga sample loop, used to introduce the sample into the moving mobile phase A column, typically tube a of stainless steel10 to 30 cm in length, 3 or 4 mm i.d., which has been tightly packed with small particles (10 pm) of the material usedto effect the separation A detector, frequently a variable wavelength UV spectrophotometer, used to measure the concentration of the sample components as they elute fromthe column A potentiometer recorderthat produces the chromatogram An optional data handling module to facilitate quantitative analysis and report writing The solvent reservoirfor HPLC is determined bythe type of pumping used (e.g., syringe pumps containa limited reservoirequal to the volume of the fullydisplacedpiston). Here the solventisdegassedexternally and

166

Chapter

Figure 14 Schematic of HPLC instiumentation: 1, filter; 2, solvent reservoir; 3, pump; 4, pulse dampener;5, sample valve;6, guard column;7, column; 8, detector; 9, waste reservoir; 10, data system (optional);11, recorder.

i

Figure 15 General viewof a liquid chromatograph produced by Knauer Company (Germany).

Liquid Chromatography

167

added to the pump chamber. However, other typesofpumps,suchas reciprocating piston types, containa separate solvent reservoir. Ideally,the reservoir must meet several requirements: Contain a volume adequate for repetitive analysis Provide solvent degassing either by heating or applying vacuum or allowing sparging with heliumand Be inert with respectto the solvent Frequently,glass or stainlesssteelcontainers of 0.5 to 2 Lmake suitable solvent reservoirs. Where space allows,the use of the glass bottle in which the solvents are purchased makes an excellent solvent reservoir. These should be carefully cappedto avoid contamination from the laboratory atmosphere. The solvent is the mobile phase; its purpose is to carry the sample through the column and produce a reasonable distribution (capacityfactor) between the mobile and stationary phases. The solvent must dissolve the sample. In addition the solvent must be of high purity, preferably HPLC quality. Other desirablefactors include low cost, low viscosity, low toxicity, and low boiling point. Depending the type of column employed, the solvent will vary. Table 16 illustrates the most commonly used solvents in liquid chromatography. One of the most important components in HPLC is the pumping system. By producing reproducible high pressures, the pump is a major factor in obtaining high resolution, high speed analyses, and reproducible quantitative analyses.The requirements ofa good pump include: A stable flow without pulsations to minimize detector noise A range of low rates for solvent delivery suitable for the various HPLC modes (usually0.5 to 10 mL/min) A constant volume delivery to facilitate qualitative and quantitative analysis A tolerancefor high pressure (6000 psi) and An easy adaptabilityto gradient operation

Table 16 Commonly Used HPLC Solvents

HPLC ~

NormalphaseHexane

or iso-octane, methylenechloride, chloroform, ethyl acetate Reversed phase Water, methanol, acetonitrile

168

Chapter

The pump with reciprocating piston utilizesa piston in direct contact with the solvent. The piston is driven either mechanically with motors and gears or by solid-state pulsing circuits. A simple versionof the reciprocating pistonpump is shownin Fig. 16 and Fig. 17. The last figure represents the analytical version of Knauer (Germany) HPLC pump Type A motor-driven piston moves rapidly back and forth in a hydraulic chamber. By means of check valves on the backward stroke, the piston sucks in solvent from a reservoir. At this time the outlet to the column is closed to preserve the operating pressure insidethe column. On theforward stroke the pump pushes solvent out to the column and the inlet from the reservoir is closed. eccentric cam drives the piston in three steps: (1) rapid reset; (2) rapid displacement until operating pressureis reached; and a smooth constant volume displacement to provide a uniform flow rate. This is an inexpensive pump that allows a wide range of flow rates. Unfortunately, it does produce flow pulses,and a pulse dampening system must be employed. One approach to eliminate pulsations is to use multihead pumping systems. Twoor three pumping assemblies offsetin time can produce essentially pulseless flow. Other models electronically sense the pressure between

2-

Figure 16 Schematic of reciprocating pistonpump: 1, column; 2, piston; reservoir;6, check values;7 , hydraulic chamber.

4, motor; 5 , external

cam;

Liquid Chromatography

I69

Figure 17 Analytical versionof Knauer HPLC pump type 64.

pump pulses and automatically change the speed of the stepping motors to minimize flow pulses. Gradient operation is achieved either by premixing the solvents at low pressureprior to pumping or by the addition of a second pump and mixing at operating pressures. The syringe pumps operate by a screw gear placing the plunger through the solvent reservoir. They are expensive, but usually produce stable flow rates and highpressures.This is sometimes overshadowed by the inconvenience of the several washings required when changing solvent systems. Syringe pumps are particularly well suited to gradient operation because each solvent is contained in separate reservoirs and the speed of each syringe plunger can be electronically controlled to produce the desired gradient. Of course, two syringe pumps must be usedfor gradient operation.

l . InjectionSystems Samples are introduced by one of three systems: (1) syringe injection; sample valve;and automated sample valves. Sample valvesare themost

I 70

Chapter

widely used because they providea more reproducible volume injectedand better quantitative analysis. They can be easily automated for unattended operation, andthey are not expensive. Syringe Injection. Low pressuresyringes for gas chromatography (GC) can be successfully used up to 1500 psi;however, precautions must be taken. At higher pressures, specially designed syringes are available. Requirements for septum materials are more rigorous in HPLC operation than in GC. The elastomer must have sufficient strength to enable successive needlepenetrations without total ruptureof polymer extrusion through the needle hole. This requirement is problematic with elastomers which swell in solvents. Generally, silicone polymers such as those used in GC are the most popular for LC systemsoperating with aqueous, aqueous-alcohol, or other polar mobile phases. However, with nonpolar organic solvents, swelling of the silicone elastomer precludestheir usage. Data are available from LC manufacturers concerning compatibility of elastomers with nonpolar solvents. Perfluoroelastomers are ideal with hydrocarbons and other nonpolar mobilephases.Septum material coveredwith a thin sheet of Teflon or otherinert polymer is also available. SampleValve. A fiied-volume loop isfilledwith the sample and, by turning a valve, this loop is placed into the solvent stream before the column. In Fig. 18, on the left,is a sample loop of stainless steelthat has been filled with sample from a syringe. For analytical work, the sample loop volume is usually 10 or 20 pL. Note that in this filling position the mobile

B

Figure HPLC sample valve. A, sample loading position. B, sample introduction position; 1, sample; 2, syringe; sample loop.

Liquid Chromatography

I71

phase at high pressure is passing directlythrough this sample valve to the column. On theright in Fig. 16, the valve has been rotated that the high pressure mobile phase now sweeps through the sample loop carrying the sample with it onto the column. This is a very reproducible techniqueand sample valves can be easily automated. Sample sizes can be changed by changing the sample loop volume. Unlike syringes, they do not suffer lack of repeatability due to operator variability or downtime due to septum deterioration. Sample valves must be constructed with minimum dead volumes and be of suitable materials to eliminate reaction between sampleor solvent. Some valves contain a large volume sample loop and allow “variable volume” injection by syringes. This isa convenience in method development, but it does not provide as good quantitative results as with fixed volume loops whichare completely filled with sample. The samplevalve approach hasbeen automated for unattended HPLC operation. In several versions, the sample is placed in small glass vials. The vials are indexed to a position where an excess volume of sample is pumped through a high pressure injection valve. The excess volume is necessary to thoroughly clean the sample loop. The sample is automatically injected and the sampler indexed to a new vial. Microprocessors can be used to repeat the injection of the same sample,to rinse the sample loop and connecting tubing between each sample,or simply to inject in sequencethe samples as loaded in the sampler tray.

AutomatedSample Valves.

2. Column Oven Many HPLC separations can be performed at ambient temperatures without the aid of a column oven. However, elevated temperatures are useful either to reduce retention volumesor to decrease the mobile phase viscosity and thereby increase the column efficiency. Lower mobile phase viscosity also enablesoperation at lower pumping pressuresor high flow ratesat the same pumping pressure. Three approaches are currently used in temperature control: (1) an external heat exchanger enclosingthe column through which water from a thermostatically controlled waterbath is circulated;(2) submerging the column ina water bath; using a hot air circulation oven as inGC. Useful temperature rangesare from 30 to 15OOC. Recent work has emphasizedthe change in column selectively caused by changes intemperature (see Table 17)[550]. This istrue even for adsorbents suchas silica gel. Thermostats were always required for ion exchange, gelpermeation, and liquid partition chromatography;theynow appear highly advantageous even for adsorption and bonded phase chromatography.

172

Chapter

Table 17 Effect of Column Temperature

Temperature ("C) 25 50 75

Platedm R 2.4

11,m 12,400 12,500

1.20 1.14 1.11

Source: Ref. 550.

3. Columns Columns for HPLC vary depending primarily on the type of separation desired and the physical characteristics ofthe packing materials. The most popular column length ranges from 10 to cm. Analytical columns have internaldiametersof 3 or 4 mm and are precision-borestainlesssteel. Column packings of small diameters(3 and 5 pm) use shorter columns (10 to 15 cm) due to the higher efficiency and larger pressure drops. The column packing is usually retained by inserting nickel or stainless steel frits into the fittings at the end of the column. The frit is effective in reducing dead volumeas well as producing uniform flow profiles. Columns for preparative separations are usually wider. Preparative scale columns have internal diameters ranging from 1 to 10 cm. They are useful for l-mg to l-g quantities of sample. Larger flow rates are required and most often automatic sample collectors are used in preparative scale work. Microbore columns are l-mm i.d. A 50-pL flow rate would correspond to 1mL/minwith normal analyticalcolumns.Thesesmallflow rates show increased sensitivity if the same sample size is applied to both microbore and analytical columns. Microbore columns with their very small flow rates have a definite advantage where expensive or exotic solventsare used. In addition, they provide a good interface for LC/MS, LC/FTIR, and LC/NMR. They do not, however, show any better resolution or faster analysis than regular analytical columns because chromatographic parameters do not depend on column diameter.

4.

Detectors

Although time has singled out some HPLC detectors as preferred devices for quantitation in the majority of separations, there is still a need for more sensitiveand selective detectors. Present research concentrateson the improvement of existing detection technology to perform more sensitive

Liquid Chromatography

I73

measurements, and onthe development of novel devices that impart greater selectivity or specificity. Detectors have been classified according to the principle ofoperation by which solutesare monitored selectively. Although the majority of detectors fall under the broad classification of spectroscopic or electrochemical techniques, due regard has been given to the expanding field of research concerned with postcolumn reactors based on immobilized reagents. The purposes of the detector are to measure the sample concentration in the mobile phase to produce an electrical signal proportional to the sample concentration. Detectors operating on the principle of monitoring ultraviolet (UV) absorption at a specific wavelength stilldominate HPLC as the most widely used detector type. The basic design has changed little in recent years, and the regular replacement of UV sources still represents the major maintenance consideration. Shown schematically in Fig. 19 is a simple fixed wavelength doublebeam UV photometer. On the left is the source, a low pressure mercury lamp that emits a sharp (essentially monochromatic) line spectrum with a strong line at nm. A quartz lens focuses the UV radiation on the sample and reference cells. The sample cell usuallycontains about 10 to 20 pL of the continuously flowing column effluent.The reference cell is usually filled with air. A UV filter removes unwanted radiation. The radiation passing through the reference and sample cells fallson two photodetectors. The output of these two detectors are passed through a preamplifier to a log comparator that produces the electric signal for the recorder. The log comparator is necessaryto convert the photons measured at the photodetector (transmittance) into absorbance units that are directly proportional to concentration.

a

Figure 19 Schematic of UV photometer, 254 nm; 1, mercury source 254 nm; 2, UV filter; mask; 4, sample cell; 5, electronics; 6, photodetector; 7 , reference cell; 8, quartz lens.

Chapter3

I74

The UV detector is inexpensive, sensitive, insensitive to normal flow and temperature fluctuations, and well suited to gradient elution. It is, however, a selective detector. Only sample moleculesthat absorb at254 nm can be detected. It cannotbe used for lipids, hydrocarbons, most polymers and oligomers, carbohydrates, and fattyacids. Because many compounds do not absorb at 254 nm, a variety of W / v i s detectors offering other wavelengths have become available. With the medium pressure mercury lamp and appropriate filters, wavelengths available include254,280,312,365,436, and 546 nm [551]. Spectrophotometers adapted for HPLC arepreferably the most common HPLC detectors today. Some are true recording spectrophotometers that allow a UV/vis spectrumto be generatedon an eluting peaktrapped in the flow cell. Others have onlymanual selection of wavelength (usually200 to 800 nm). This allows selection ofa wavelength to maximize sensitivityor minimize interferences, but does not allow a spectrum to be recorded. Figure 20 shows a schematic spectrophotometer used as an HPLC detector. This instrument uses a grating, thus allowing selection of any wavelength from 200 to 800 Light from a continuous source is focused on the entrance slit of a grating monochromater. By the appropriateoptics, this “white light” is focused on the grating where it is dispersed into the various wavelengths. By varying the position of the grating, the desired wavelength is focused on the exit slit and then passed through sample and referencecells by means of a beam splitter. The detector measures the

nm.

9 Figure 20 Schematic of spectrophotometerfor HPLC detector; 1, source; 2, grating; 3, exit slit;4, entrance slit;5, monochromator;6, beam splitter;7, reference cell; 8, detector; 9, sample cell; 10, rotating chopper.

Liquid Chromatography

I75

difference in lightintensity passing between sampleand reference cells. The detector signal is converted into absorbance by a logarithmic comparator. Spectrophotometers allow the selection of any useful wavelength in the UV and visible regions. The primary benefit is the increased sensitivity for the compounds of interest. Many compounds would not absorbwell at 254 nm, and thereforegreater sensitivity is possible with a spectrophotometer. In some cases, noise or interference from the sample matrix is also reduced by usinga spectrophotometer. UV detection in capillary liquid chromatography has been studied to determine the relationship betweenmaximumachievablesensitivity and minimum extracolumn dispersion. There is an apparent trade-off between best sensitivity and minimum dispersion, which is achieved through oncolumn detection. Diode array detection (DAD), as an extension of conventional UV absorption detection, has found wide application in HPLC method development for peak identification and tracking. Although the optical hardware hasnot changed significantly,software development revealsthat there is a lot of ongoing research. Information retrieval and processing is a very important aspect that has contributed to anincreasing range ofDAD applications. few manufacturers of liquid chromatographic detection equipment have introduced low cost devices that monitor a limited number of wavelengths by rapid scanning and offer some of the facilities available on the DAD systems. These instruments allow the entire UV/vis spectra to be recorded rapidly and stored digitally in a microprocessor where it can be recalled or handled in several ways. large number of detector elements (32 to 512 inpresent designs)can store and read out theimpinging radiation in milliseconds. Major uses include peak identification by having a UV/vis spectra for each liquidchromatographic peak, determination of peakpurity by absorbance ratioing at two wavelengths, and multiple chromatographic recordings at avariety of wavelength,that can be displayed after the runto maximize sensitivityor minimize interferences. Limitations of the photoiodide array detector include the high cost (15,OOO-25,000 USD), depending on the microprocessor capability, limited linear range ofthe present diode arrays, and, finally, limited signal/noise primarily due to both optical and electrical noise. Keller and Massart [552] described a method for peak purity control based on evolving factor analysis. The factor analysis is performed on a moving window with a fiied, rather than increasing, number of spectra. Impurities with similar spectra to the analyte could be detected below19'0. Bunger et al. [553] applied the Meister method of curve resolution to resolve overlapping peaks of complex hydrocarbon mixtures. The specific resolution of polyaromatic hydrocarbons was found to depend on thenum-

I76

Chapter 3

ber of principal components used in the curve resolution method. Results wereshown for up to five principal components. A novel approach to enhance peak recognition has combined postcolumn continuous-flow analysis with DAD [554-5561. Fell et al. [557] described further work where KOH solution is addedto the carrier stream to generate pH-shifted differa ence spectra. There were obtained a number of dipeptides containing tyrosyl residue by subtractingthe normalized spectrum under alkaline conditions (pH 12.4) from the spectrum obtained at pH 4.4 (reversed phase conditions). Strasters et al. [558] reviewedthreemultivariatetechniques (multicomponent analysis, targetfactor analysis, and iterative target transformation-factor analysis) for peak deconvolutionas a means ofautomated peak recognition for computer-guided strategies in HPLC. They conclude that the final result of the optimization is substantially dependent upon the technique selected and spectral information. Fell and Berridge [559-561 J have applied the multichannel detection capability of DAD to assist automated optimization strategiesin HPLC. Verzele et al. [562] reported procedures to enable commercial DAD systems to be adapted for microliquid chromatography. A piece of fusedsilica capillary, stripped of polyimide coating, was mounted in a detector block. The paper discussed the control of a number of parameters that affectdetectorperformance,includingrefractiveindexchanges,optical dispersion, and optical alignment. Refractive index is a universal detectorthat will respond to all sample types. It is more expensivethan the simple UV detector, but less expensive than a spectrophotometer. It requires very goodtemperature and good flow control, and is not amenable to gradient elution. Sensitivity is limited to about 1 pg of sample. The refractive index (RI) detector is useful in gel permeationchromatographywork,preparativeseparations, and routine quality control where trace analysis or gradient elution is not required. It is usually neither sensitive nor stable enough for routine analytical work

5. Fluorescence Conventional fluorescence has been applied for highly selective and sensitive detection ofboth naturally fluorescent analytesand analytes that have been derivatized. The proportional response of fluorescent emission intensity to exitation intensity has inevitably led to the development of laserinduced fluorescence(LIF). Modern lasers are more robust and stable and offer an increasing selection of emission wavelength. Two complementary reviews on LIF are reported. One discusses the development of LIF, its advantages over conventional fluorescence detec-

Liquid Chromatography

I77

tion, and the use of different flow cell designs The other places an emphasis on trace bioanalysis of drugs, with a focus on techniques such as frequency doubling, variable wavelength dye lasers, and derivatization procedures The detection of glucuronic acid conjugatesafter derivatization with the fluorescein fluorophore was reported A LIF system constructed around a continuous-wave argon-ion laser enabled a limit of detection of ca. mol.Thisrepresentsagain in sensitivityoverconventional fluorescence detection ofan order of magnitude greater than 4. Gooijer et al. applied LIF toconventional HPLC of a standard mixture of polyaromatic hydrocarbons.A frequency-doubled argon-ion laser was used to excite at nm. Short-wavelength excitation reduced Raman scattering of the eluent, which can interfere with analyte fluorescent emission, while increasingthe application of LIF to a wider range of analytes. Improvements in limits of detection of 4 to times were reported, with further improvements limited by background luminiscence ofthe solvents and quartz cell walls. Further work by Gooijer reported the addition of an intensified linear diode array (ILDA) detector to their frequency-doubled LIF. This enabled identification through real-time monitoring of the fluorescence emission spectrum. Delorme developed a dual-mode laser-based detection system for HPLC. LIFwas combined with thermal lensing spectroscopy in a single flow cuvette.The detection of separated NDand U was reported. Fell et al. examined conventional fluorescence detection with an ILDA detector for its potentialcontribution in pharmaceutical and biomedical analysis. The data were presented as an isomeric plot (emission intensity, emission wavelength, time)and techniques to assess peak homogeneity were applied. Goijer et al. described a detection systemfor liquid chromatography based on the measurement of long-lived luminiscence in an attempt to discriminateagainstshort-livedbackgroundfluorescence. A pulsed Xe lamp and a gated photomultiplier were used.The lanthanide ion Tb(II1) was used as a luminophoric label with thiol-containing analytes. Luminescence sensitizationand derivatization through the thiol functionality were achieved in the reagent 4-maleimidylsalicilic acid. 6. Chemiluminescence

The sensitivity of fluorescence detection is frequently limited by the presence of impurities that give rise to interfering luminescence background signals. Oneapproach to overcome this problemis to applychemical, rather than spectroscopic, excitation. Chemiluminescence involves postcol-

I78

Chapter

umn reaction with the HPLC eluent and collection of emitted light at a specific wavelength. In a comprehensive review,Jong and Kwakman [574] address recent developments in the chemiluminescence detection of biomedicalsamplesseparated byHPLC.Details arediscussedonperoxyoxalate, luminol, and lucigenin based chemiluminescence, as well as a brief account of bioluminescence reactions. Kwakman et al. [575] described peroxyoxalate chemiluminescence detection of primary aminesafter precolumn derivatization with naphthalene2,3-dialdehyde (NDA), or anthracene-2,3-dialdehyde(ADA). A modified commercial fluorescence detector with excitation source turned off was utilized to obtain enhancement in limits of detection of25 and 50 times for NDA and ADA derivatives, respectively. ADA derivatives suffered from rapid decomposition, especially in the presence of hydrogen peroxide. Although LIF could give further improvements in detection limit enhancement, the chemiluminescence technique was considered preferable, due to its lowcost,asameans gainingsuperiorsensitivitytoconventional fluorescence detection. Jones et al. [576] described a similar chemiluminescence detection system based on a commercial fluorescence detector with the excitation lamp shutter in the closed position. A postcolumn reagent based on luminol was usedto detect cobalt at picogram levels after separation on a cation-exchange column.

7. OpticalActivity Polarimetric measurement of optical activity and the related technique of circular dichroism have played increasingly important roles as HPLC detectors. Chiral phases for the separation of enantiomers coupled to detectors that exploit the measurement of optical activity provide an important and growing research field within HPLC technology. Lloyd and Goodall have recently reviewed polarimetric detection in HPLC [577], with an emphasis on the selective detection of optically active molecules. Meihard and Bruneau [578] employed tandem UV absorptionpolarimetric detection in studies to develop a chiral phase that would resolve the deltamethrin racemate. The UV detector gave the sum of the signals due to the two optical antipodes and thepolarimeter the difference. These data were usedto quantify the two optical antipodesin a mixture and assist in the development of a new HPLC chiral support. Goodall et al. 15791 applied a dual optical rotation-UV absorbance detector system for the determination of the enantiomeric purity of ephedrine and pseudoephedrine separatedby HPLC. Goodall et al. [580] described the construction and application of a diode-laser-based optical rotation detector. The detector components are

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summarized in Fig. 21. A collimated laser diode source emitting at 820 nm was focused onto an HPLC flow cell ( 8 pL) via a polarizer. A Faraday effect modulator oscillating at 1.45 kHz was arranged to give a modulation angle of up to 1 The cell beam, after passing an analyzer (similar to the polarizer) was detected by a silicon photodiode. Due to the inherently low flicker noise characteristics of diode lasers, the design could be simplified. The determination of D- and L-tryptophan mixtureswas demonstrated. A circular dichroismdetector, with simultaneous measurement of absorbance, was usedto evaluate the anisotropy factor andhence the enantiometric excess of the eluates [581]. This approach was applied to preparative chiral HPLC to enable optimized collectionof enantiomeric fractions.

8. AtomicSpectroscopy Element-specific detection in liquid chromatography still accounts for only a small proportion of detection principles employed. The perceived complexity of atomic spectroscopic techniques and the challenges presented in the interface design have prevented the development of commercial detectors based on atomic spectroscopy. However, the inherent benefits of element-specific detectionare demonstrated for the analysis of trace organics in complex samples. Furthermore, effort continues in the research associated with linking liquid chromatography to atomic spectroscopic systems for specialized applications. Kientz et al. [582,583] have designed and evaluated an interface for the on-line coupling of microcolumn liquid chromatography with a flame photometric (FPD) detector. In this work, a commercially available FPD designed for gas chromatography was modified to permit direct introduca

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l80

Chapter

air

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Figure 22 Schematic diagram of LC flame photometric detector. (a) Burner assembly, (b) stainless steel tube (0.50 mm i.d.), (c) fused-silica capillary (0.32 mm i.d.), (d) fused-silica capillary interface (0.10 mm i.d.). tion ofthe column effluentinto a hydrogen-air flame. A simple design (Fig. 22) in which a fused-silica capillary (0.1 mm i.d.) is inserted into the base of the flame was assessed accordingto a number of operating parameters. The position of the capillary, gas flow rates, and quenching effects of the eluent were considered, and the detection a number of organophospho20 pg rus compounds was demonstrated. A detection limit of phosphorus per second was claimed. Graphite furnace atomic absorption spectroscopy (GFAAS) has the advantage of high sensitivityand selectivity for small sample volumes.The discontinuous nature GFAAS sampling presents an obvious disadvantage. Astruc et al. [584] investigated the theoretical limits of GFAAS as a discontinuous detection procedurefor liquid chromatography. Their theory shows that optimum detection may be achieved when the liquid chromatographic peak width is the same order of magnitude as the cycle time of the GFAAS. Their theory was verified by the speciation of butyltin compounds at sub-mg/L concentrations. Haswell et al. [585] applied reversed phase HPLC interfaced to electrothermal atomization atomic absorption spectrometric detection.The specificity ofthe detection system toward cad-

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miumwasused to monitor the separation and speciation of cadmiummetallothionein type complexes.

9. Mass SpectrometricDetectors It is beyond the scope of this chapter to review the rapidly increasing applications of liquidchromatography-massspectrometry(LC-MS). It is important, however, to highlight a few significant developments that have demonstrated novelty in the interface-ionization mechanism. It is the technology by which liquid chromatography is coupled to mass spectrometry that ultimately limitsthe performance and application of LC-MS as a sensitive and selective LC-detectorcombination [586,587]. Dynamic fast atom bombardment(FAB)LC-MS,which can give molecularweight information and structure-characteristic fragmentation data, has been associated mainly with low-resolution MS. Kostianen et al. [588] demonstrated LC-MS usinga frit-FAB interface coupled to a high-resolution MS. This was applied to the identification of several trichothecenes. The reversedphase separation consisted of a methanol-water gradient witha postcolumn addition of glycerol. Polyethylene glycol was added as a calibrant for an MS scan range m/z 150 to 500, with resolution of8000. An alternative to the frit-FAB interface is the continuous-flow FAB (CF-FAB), that is basedon setting up a film on a nickel, silver,or stainlesssteel target. Niessen et al.[589]studiedsomeof the important factors affecting the performance of a CF-FAB interface based on a target fed from a fused-silica capillary (Fig. A range of conditions were studied for the fourtarget materials, with the conclusion that thewettability of the target was a key factor in determining stable operation of the FAB intera

Figure 23 Schematic diagram of a continuous-flow FAB-MS probe interface.(a) LC-capillary, (b) FABtarget,(c)compressedpaperwick, (d) exchangeable ion volume, (e) homogeneous film of LC mobile phase on target surface.

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Chapter 3

face. The effect of glycerol concentration in the mobile phase was also investigated, with optimum performance obtained with 7% (w/w) of glycerol, representing a supply rate of 0.3 pL/min. Pullen et al. [ 5901 described the design and construction of a microcolumn LC-MS interface based on corona discharge ionization. The system was designed for the detection of known target compounds at trace levels in pharmaceutical formulations and biological fluids. High sensitivity and minimum sample workup were essential prerequisites. The ionization chamber was based on a precisely controlled point-to-point discharge that enabled successful focusing on the region of maximum ion generation with the ion slit and quadrupole analyzer. Stenhagen and Alborn [591] have utilized an electron input ion source that permits direct connection with microcolumn liquid chromatograph based on a packed fused-silica column (0.22 mm i.d.). The nebulization of the eluent was controlled by the electrostatic field set up between the column end and an extraction-focusing plate. The main advantage claimed for this interface arrangement was reduced thermal decomposition of labile compounds. Operation of the interface was demonstrated for a number of separated plant allelochemicals. Speciation of tributylin in water samples was presented by Ebdon et al. [ 5921 who demonstrated HPLC coupled to inductively coupled plasmamass spectrometry (ICP-MS). With a standard ICP torch, a limit of detection of 25 ng/mL was achieved.

10. Electrochemical Detectors Electrochemical methods of detection in HPLC have failed to achieve widespread popularity. For a number of applications, the selectivity of electrochemical measurements has ensured their adoption over detectors employing other measurement principles. The complexity of operation of some electrochemical detectors and the need for regular maintenance of electrodes and flow cells are possible reasons for their confinement to special applications. The conductivity detector has been successful when applied to ion-chromatography monitoring followed by the pulsed amperometric detector that maintains electrode surface integrity through electrochemical cleaning. The potential for the application of electrochemical measurement systems to HPLC detection is increasing due to research in the areas of flow-cell design, new electrode materials, and advances in the associated instrumentation. In a recent review, Buchberger [593] highlighted advances in the coupling of electrochemical detectors with HPLC. Voltametric detectors were emphasized, with discussion of new electrode materials, new excitation functions, and new application resulting from these developments [ 594-5961.

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A convenient means of classifying electrochemical methods of detection is to distinguish between principles of operation, grouping detectors that use equilibrium as opposed to dynamic processes. Detection systems that do not promote oxidation or reduction of the analyte are termed “equilibrium,” whereas all other systems that involve electron transfer (amperometric, voltametric, and coulometric) may be termed “dynamic.”

Equilibrium Electrochemical Detection. A novel detector for simultaneous conductivity and pH measurement was described by Slais [597]. The cell volume, estimated at 150 nL, was suitable for detection of microcolumn liquid chromatographic effluent. Measurement of the pH, based on an antimony microelectrode, enabled the detection of changes as low as f 0.004 pH. A miniaturized lead-lead sulfate reference electrode was used to avoid noble metal contamination of the antimony surface and any consequent degradation in the performance of pH measurement. Conductivity, with a sensitivity of f 50 ns/cm, was detected between the antimony microelectrode and a third auxiliary electrode constructed from a stainless-steel capillary. Slais and Janecek [ 5981 reported a simultaneous photometric and conductivity detector in an earlier paper. This detection system, also designed for microcolumn liquid chromatography, claimed a volume of 100 nL with an optical path length of 1 mm. Optical fibers were used as an optical link between the flow cell, UV source, and photomultiplier. Examples of indirect UV-conductivity detection of separated anions and carboxylic acids were presented. Slais and Oscik-Mendyk [ 5991 demonstrated the application of a simultaneous conductivity and amperometric detector for the determination of dicarboxylic acids separated by microcolumn liquid chromatography. The detector, based on a manganin working electrode, has a reported cell volume of 20 nL. Electrokinetic detection is based on the measurement of the streaming current generated by the movement of a liquid along a solid surface. This phenomenon can be exploited for the measurement of liquid chromatographic effluent composition. The streaming current arises from the fact that part of the spatial charge of the electric double layer is carried by the streaming liquid. In practice, the charge carried may be measured by an isolated collector electrode. The generation of a streaming current, I,, is described by the Helmholtz-Smoluchovsky equation, where

r, = A

~ P

( 106)

A is a constant dependent upon the composition of the streaming liquid

and measurement cell geometry, P is the pressure gradient on the streaming current generation cell, and is the zeta potential, which defines the edge of the outer Helholtz layer. For a capillary, the zeta potential is given by:

184

Chapter

where 11 is the viscous force per unit area, 4 is the volume flow per unit time, q is the capillary cross-sectional area, e, is the permittivity of free space, and e is the relative permittivity of the streaming liquid, such that is also a function of column effluent composition. Vespalec and Neca [600,601] have exploitedthis electrochemical phenomenon for liquid chromatographic detection. They studied the effect of detector construction with respectto the magnitude and polarity of streaming currents for both normal and reversed phase separation modes. Detection limits for several nitrophenols were achieved in the range 10-9-10”1 mol. A detection limit of1.8 molwasclaimed for dipicrylaminein a mobile phase ofn-heptane-acetone 10) with10 ppm of nitric acid. Dynamic Electrochemical Detection. Amperometric detection is the most

widely used example of dynamic electrochemical measurement for liquid chromatography detection. Gratzfeld-Huesgen and Haccker[602]discussed the optimization ofsensitivity through selection of appropriate working potentials in thin-layer cell amterometric detectors for HPLC. The stability of operation is also discussed, with reference to electrochemical cleaning to promote electrode life. The cell designis very important to the operation voltametric detectors. Wall-jet geometries can offer considerable advantages over thin film electrodes both in terms of simplicity of construction and in terms of reduced band broadening. Horvai et al. [603] studied band broadening in large volume wall-jetcells used for electrochemical detection inliquid chromatography. The necessity for smooth flow patterns and the conditions that promote stable operationwere discussed. Nagels etal. [604] investigated large volume wall-jetdetectors and studied rapid-scan staircase voltammetry under convection-diffusion and diffusion-controlled conditions. The importance of the parameters of flow rate, electrode diameter, and scan rate was demonstrated for microbore and microliquid chromatography as well asfor conventional liquid chromatographic conditions. Electrode materials. New electrode materials and chemically modified electrodes continue to be reported for applications in electrochemical measurements.Amperometric detection hasbeenused in conjunction with some novel electrode types and with electrodes that have chemically modified to impart selectivity or improve performance. Hart et al. [605] demonstrated the determination glutathione in human plasma samples using HPLC coupled amperometric detection. A carbon-epoxy resin composite working electrode, which has been chemi-

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cally modified withcobalt phthalocyanine, was reported. The cobalt phthalocyanine actedas an electrone mediator to assist the kinetics of the electron transfer process of glutathione electro-oxidation. A detection limit of 55 ng/mL for glutathione was claimed. Nagels et al. [606] and Schuddinck et al. [607] reported a novel carbon-polymer chip electrode for microliquid chromatography detection. The working electrode was constructed from 20% (w/w) carbon-loaded ethylene-vinyl actetate copolymer (containing 9% vinyl acetate) that was bonded around two polyamide-coated fusedsilica quartz capillaries (100-pm i.d.). A stainless-steel wire, previously inserted into the spaced capillaries, was withdrawn after two polymer chips were compressed on a hot plate to form a conducting tubular electrode between the fused-silica capillaries. The detection of catecholamines, separated by microliquid chromatography after solid phase extraction from urine samples, was demonstrated. Stulik et al. [608] utilized a passivated copper electrode for selective amperometric detection of ethylenethiourea in beverage samples separated by HPLC. The selectivity was attributed to the complex reaction between ethylene thiourea and copper I1 ions. A detection limit of 3 ng/mL was reported for a200 pL injection volume. Smythet al. [609]compared amperometric detectionemploying a static mercury drop electrode (SMDE) with a glassy carbon working electrode for the determination of cisplatin following HPLC separation. A detection limit of 5 pg/mL for the SMDE compared with ng/mL for the glassy carbon was reported. It was found, however, that regular polishing was required for the glassy carbon electrode to maintain performance. alternative cell arrangement, where a dual-in-parallel amperometric detection mode was employed, enabled a decrease in the detection limit to 25 ng/mL, due to enhanced signal-to-noiseconditions.

Electrode Arrays. Electrode arrays, where a numberofindependently controllable working electrodesmonitor HPLC effluent either seriallyor in parallel, could be to electrochemical liquidchromatographic detectors what the diode array spectrometer is to UV absorbtion detection. To date, the only commercially available electrochemical array detector is based on serial measurementofliquid chromatographic effluent through interconnected coulometric cell compartments. Onlyrecentlyhave the potential applications of such a detection system been explored. Rizzo et al. [610] reported the determination of neurochemicals in biological fluids using a 16-electrode coulometric array detector. Of molecules searched for, 21 could be measured after direct injection of a plasma sample,illustrating the potential selectivity of electrode array detectors. An alternative to the costly and complex arrangement required for

Chapter

186

coulometric arrays is to control a single cell comprisingan array of miniature disk working electrodesand anassociated auxiliaryand reference pair. Fielden et al. [61 l ] demonstrated preliminary results ofan array of miniature electrodes mounted ina single plane.A wall-jet cell geometry was used to minimize band broadening (Fig. 24) and maintain a rapid response. The electrochemical detection of a mixture of phenols was reported with simultaneous monitoring of eight different oxidation potentials.

1 l . Postcolumn Electrochemical Derivatization A few specific examples of electrochemical reagent generationfor postcolumn derivatization have been reported. Many examples of enzyme reactors linked to amperometric detection have also been described (see postcolumn reactors, following). Electrochemical generation of reagents can potentially be finely controlled with microcell volume compatibility. Mascher and Kikuta [612] describe an HPLC separation couple to a fluorescence detector via a coulometric cell. The determination of amoxicilin was studied with electrochemical postcolumn oxidation of the separated analyte leadingto the formation of a fluorescent byproduct.An absolute detection limit of 10 pg injected was claimedfor the direct injection of plasma samples without prior treatment. Palmisano et al.[613 J utilized a postcolumn coulometric cellto gener-

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Figure 24 Schematic diagramto show constructionof the wall-jet electrode array for LC-voltametric detection. (A) Section through the cell: (a) platinum auxilliary electrode, (b) electrode array assembly, (c) working electrode cell half, (d) O-ring seal, (e) outlet, (f) maincellbody, (g) inlet jet (0.3 mm i.d.), (h) silver-silver chloride reference electrode with sintered glass junction. (B) Close-up of working (1 electrode array, comprisedof high-regularly spaced glassy carbon-disk electrodes mm diameter).

Liquid Chromatography

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ate bromine on-line. The microtoxins altenuene and isoaltennene were derivatized by the electrochemically generated bromine after reversed phase liquid chromatographic separation. A detection limit of 8 ng on column was reported. Malcolme-Lawes etal. [614] describeda computer-controlled electrochemiluminiscence monitor for liquid chromatography. The chemiluminiscence intensity could becontrolled by electrochemical parameters, such that computer-optimized chemiluminiscencewas realized.

7 2 . Detection by immobilized Enzyme Reactors A survey ofpostcolumn reaction technology has been included in this chapter, as column reactors have become more widely used for enhanced selectivity in the detection process for liquid chromatography. It is convenient to classify such column reactors as those based on enzymes and those that empldy other reagent chemistries. Marko-Varga et al. [615,616] have comprehensively reviewedthe application of immobilized enzyme reactors for selective detection in liquid chromatography. The incorporation of immobilized enzyme in liquidchromatographic systems, selectivity toward interferences, and many examples are discussed. An emphasis is place on systems that combine immobilized enzyme reactors with electrochemicaldetection, particularly amperometric detection. The application of chemically modified electrodes to this task is also surveyed. Tagliaro et al. [617-6191 reported the development of postcolumn immobilized enzyme reactors based on alcohol oxidase. Details of the immobilization procedure were given, witha consideration of band spreading due to reactor void volume. The reaction product, hydrogen peroxide, was monitored amperometrically at a platinum working electrode polarized to +500 mVvs.Ag-AgC1. Detection limits of 2.5 nmol and 0.6 nmol for ethanol and methanol, respectively, were determined. Marko-Varga [620] has utilized two serial immobilized enzyme reactors for the determination of mono- and oligosaccharides in fermentation broths afterliquid chromatographic separation. The detection principle was based on a sequence of enzymatic reactions. Free glucose monomers were first generated bythe hydrolysis of poly-and oligoglucans due to amyloglucosidase (AMG) maltodextrins

AMG

+

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(108)

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Chapter

188

NAD+

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D-gluconate

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that resulted inthe oxidation ofthe 0-D-glucose. Mutarotase was added to the second reactor column to transform the a-D-glucose formed in the first step to the 0-anomeric form. A phenoxazine-modified electrode was used in an amperometric detector to monitor the NADH produced by the second reactor column. Variations on this approach werealsoreported for the enzymatic production and analysis of D-xylulose [621] and the determination of sugars in spent sulfite liquor [622]. Marko-Varga et al. [623] reported stereoselective detection ofL- and D-amino acids using a postcolumn immobilized enzyme reactor containing either L-or D-amino acid oxidase. A second reactor containing immobilized horseradish peroxidase was used to promote the formation of a red-colored complex bythe reaction of hydrogen peroxide generated the by first reactor with 4-aminophenazoneand dichloropheno-sulfonyl chloride. Townshend and Leon-Gonzales [624] employedthe inhibition of immobilized acetylcholinesterase to selectively detect organophosphorus and carbamate pesticides separated by liquid chromatography. a-Naphthyl acetate was hydrolyzed by the immobilized enzyme to an a-naphthol, which was reacted withp-nitrobenzene-diazonium fluoroborate to give a continuous absorbance at 50 nm. Inhibition of the cholinesterase ledto a decrease in baseline absorbance. An extensive review of Brinkman et al. [625] considered the design, construction, and application of postcolumn reactors. This included discussion on open-tubular,packed-bed,segmented-stream, and hollow-fiber membrane reactors. Irth et al. [626] describeda postcolumn ligand exchange reactor coupled to selectivespectrophotometricdetection for the determinationof PbII, CdII, and BiIIIseparatedastheirdiethyldithiocarbamate (DTC) complexes by reversed phase liquid chromatography. Ligand exchange with copper(I1) phosphate led to selective detection of Cu(DTC), at 435 nm. In a subsequent paper, Irth et al. [627] applieda postcolumn ligand-exchange reaction for the selectivedetectionofD-myo-1,2,6-inositol triphosphate (1,2,6-IP,). A reagentstreamof the Fe(II1)-methylcalceinblue(MCB) complex, which is weakly fluorescent, gave a low fluorescent background. Strongly fluorescent MCB, released the by ligand-exchange reaction, gave a detection limitof 3-10 ng of 1,2,6-IP3.

13. Recorders Standard strip chart recorders (1 and 10mV)such as are used in GC are also employed in HPLC. Chart speeds should be reproducible that

Liquid Chromatography

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retention times (chart distance) can be used to identify peaks. Quantitative analysis is performed as in GC (i.e., peak areas or peak heights are measured and compared with standards). Disc integrators, electronic integrators, and computers are also usedto measure peak areas.

14. Data Systems Data systems are divided into two classes; the microprocessor-based electronic integrator and the computer workstation (Fig. 25). Electronic integrators are relatively easy to use requiring little or no setup. Most provide default settings that can be used for typical analyses. Electronicintegrators are limited in that they employ hardware programs that may not be suited for a given analysis, theydo not provide for storage or archiving ofchromatograms, and most cannot redo a chromatogram using different integration parameters without repeating the entire analysis.

Figure 25 Eurochrom software/hardware data system and monitor chamber HPLC system (Knauer Company, Germany).

a dual-

190

Chapter3

Although costing morethan electronic integrators, computer workstations provide more flexibilityand power. Mostcomputer workstations provide means for finer control over integration functions as well as archiving chromatograms, redrawing base lines, and aiding in data analysis and report generation. Disadvantages of computer data stations are their complexity, longer setup times, a lack of mobility, and higher initial cost than electronic integrators. As the electronic integrators gain both features and power and computer workstations become more user friendly, the line separating these classes of data systems becomes less distinct. The type of system neededcan only be determined by carefully evaluating whatisavailable (hardware and software) and matching the features available with the needs of the analysis.

75. Factors in Choosing HPLC Instrumentation Several major factors must be considered when purchasing an HPLC system. There are usually, in addition, several factors unique to each laboratory. The comments concerning each factor are the subjective opinion of the author. It is essential to knowif HPLC will solve the analytical problem. Fortunately, most manufacturers have well-equipped applications laboratories that can run samples. Prospective buyers are urged to take the time to run thesample before making a purchase. Most samples can be run isocratically; however, for an initial purchase, gradient elution is a strongly recommended capability. It is essential in research and method development and lessusefulin routine quality control. A Tied wavelength (254 nm) UV photometer is recommended as a standard component for everything exceptgel permeation chromatography work. Most sample types show someabsorbance at 254, and it is a robust, inexpensive, and sensitive detector. Sample typesthat might require a variable wavelength UV or refractive index detector include sugars, fatty acids, triglycerides, most polymers,and nonaromatichydrocarbons. However, in view of chromatographic separation of oligomers, the author’s first choice is a refractive index detectorthat can be used for both sensitivity or selectivity. Optionally, this detection system maybe combined withUV detector. Modular systems are more flexible and usually less expensive than integrated systems. They allow start-up with a modest investment and can be upgraded later. Modular systems usuallyrequire some experienceon the part of the chemist and may require more “playingin the lab”initially to set up and optimize the modules. Integrated systems use matchedcomponents

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in a fully assembled unit; start-up time is minimal and the manufacturer often provides installation, start-up assistance, and some training. Integrated systems are usually more expensiveand easier to automate, butthey frequently cannot be taken apart forswapping parts or troubleshooting. The buyer must finally choosea specific instrument manufacturer. In many cases the previous considerations will have narrowed the field to two or three candidates. Table presents the major suppliers and models of HPLC equipment (in alphabetical order). The most important factors are the manufacturer’s reputation in HPLC and the after-sales service. A good picture can be seen by talking to several local LC customers who have purchased equipment in the last few years. Try to avoid “good” or “bad” opinions created years ago, often in a different product line or associated with a particular person. A minor factor (in most cases) should be the relative cost, provided there is not more than variation. The important criterion is the price/ value; frequently, highly pricedunits offer more value.

F. Applications of Analytical and Preparative Liquid Chromatography

The were a very exciting period in the area of applications. Few separation problems could not be solved with either HPLC alone or in combination with other techniques. HPLC is used not only to separate and purify products of synthetic and naturally occurring reactions, but also to obtain intermediates of reactions, purify starting materials, and obtainenough of a purified compound for futuretesting or for structuraldeterminations. HPLC has expanded to include ion chromatography, affinity, immunoaffinity, and chiral chromatography in addition to the more common modes of adsorption, ion-exchange, normal phase, reversed phase, and size exclusion chromatography. Supports, besides the commonlyusedsilica,include other oxides carbon polymeric resins hydroxyapatite beads and agarose Packings come ina wide range of particle and pore sizes. There are specialty packings for affinity and immunoaffinity separations oligomers and macromolecules, and especially for biologically active macromolecules Mixed-mode and mixed-bed packings have been investigated as have been coupled columns for compounds with multiple functional groups Columns come in various lengths and internal diameters to solve any problems at hand. In addition to the commonly used stainless steel columns, glass-lined stainless steel and glass columns are now available as are plastic cartridges. The problem now is

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Liquid Chromatography

197

deciding which of the plethora of columns is the right one to buy, given that column prices are high and many academic laboratories have limited funds forequipment Several forms of preparative liquid chromatography have been developed according to various requirements for sample size and difficulty of the separation. Analytical or preparative liquid chromatography involves slightly enlarged column volumes obtained by either lengthening the column or enlarging itsinternal diameter. Larger columns allow difficult separations, but the back pressureand separation time soon become prohibitive. Enlarging the column diameter is thus the most practical means to achieve increased capacity. High-performance laboratory preparative liquid chromatography is currently performed primarily on columns cm long withan i.d. of 710 mm. Such columns hold g of silica gel and can handle sample loads from to mg, depending on the difficulty of the separation. A larger column cm long with an i.d. of cm) that holds approximately g of packing material is often used in the research laboratory. The optimum flow rate of such a column is mL/min, that can easily be accommodated by mostanalytical liquid chromatographic instrumentation. This size column is the largest that can be packed with conventional packing methodsand thatwill show reasonable column stability when handled carefully. Even larger columnsare needed for production-scale preparative liquid chromatography, and two solutions to this problem have been developed. The first employs enlarged systems similar to those used in analytical liquid chromatography with stainless steel columns filled with silica gelbased packing materials and high-pressure solvent delivery systems. The second, used primarily in biotechnology, uses soft gels in glass or plastic columns undermoderate pressure. Analytical columns are usually packed by pumping a slurry of the packing material at pressures of up to400-1000 bar. Packing fluid is forced at very high velocity through the column bed as it forms. This forces the stationary phase particlesinto amore or less stable bed with a porosity that is still far from what is theoretically possible, making the stability of these columns uncertain. Under prolonged use, further settling of the stationary phase may occur, a void will appear at the top of the column, and the efficiency ofthe system will be reduced. It is difficult, however, to pack large i.d. columns in this manner. Stainless steel columns of cm i.d. can accommodate the necessary pressures only whenthe wall is prohibitively thick. Pumps that can generate that required pressures while delivering the necessary volume of solvent

198

Chapter

simply do not exist. Early preparative liquid chromatographic systems avoided this problem by using coarser stationary-phase particles; particles with an average size of25 pm can indeed be packed simply bypouring them evenly into the column. Adding solvent to the particles without wetting them can improve dry packingresults. Glass or plastic columnscan be used for such large-particle columns, that have good stability because of the mild pressure drop and general chromatographic requirements of coarser particles. The recent tendency in preparative liquid chromatography, however, is toward high-efficiency columns usingsmall particles with a diameter of 10-25 pm. Packing problems are avoided by using compressed bed techniques in which dry packing material or a slurry is placed in the column that is then compressed. Very little liquid is forced through thebed during packing. Although bed compression was developed as a packing technique for large-scale columns, recent experience has shownthat it is also important in maintaining columns in an efficient state during the chromatographic run. Friction heat is generated in the column as the solventis forced through thepacked bed. Thisappears to be a problem for the small-particle columns used in modern preparative liquid chromatography. Such friction heat is a function of the linear flow velocityand viscosity of the solvent as well as the particle size and pressure drop over the column. With the larger particles and low viscosity solvents usedin normal phase liquidchromatography, friction heat is negligible. With smaller particle columns and the viscous solvents used in reversed phase liquid chromatography however, friction heat becomesa serious problem. opening appears at the column wall because of the difference in thermal expansion, coefficients between the packed bed and column wall material. In a 20-cm i.d. column, packed efficiently withWpm reversed phase stationary phase, a visible opening of several tenths of a millimeter appears during the firstchromatographic run. All solvent pumpingthrough thecolumn then passes along the wall and not through the packed bed, resulting in the “thermal wall effect” [649,650]. This problem can be solved by continuous bed compression during the chromatographic run such that the volume of the space available to the packed bed is continuously corrected. Use of columns that have been removed from the compressed packed station can result in thermal wall effects for reversed phase small-particle separations. Bed compression can, in principle, be achieved axially, radially, or by a combination of the two. Axial compression can be exerted from the top of the column, the bottom of the column, or both together, and the piston can be actuated either mechanically or hydrolytically. Because mechanical

Liquid Chromatography

199

pistons require a lot of operating space, hydraulic pistons are preferable from an installation point ofview. Radial compression can be obtained by gas or liquid pressure on a flexible sheet againstthe column wall insidethe metal column. The flexible wall obviously must be inert to the solvents used and must withstand frequentbending.Combinedradial and axialcompression is obtainedby pressing a wedge-shaped shoft in the column. The term dynamic axial compression was recently introduced[649]; such a term is useful in differentiating more clearly between “dynamic preparative liquid chromatography” (continuous bed compression) and “static preparative liquid chromatography” (without bed compression). With the advent of commercially available large-scale compressed column instrumentation [649],this technique has become widely used the in pharmaceutical industry. For large-scale industrial preparative liquidchromatography, the economics of operation are important. The cost per kilogram of processed compound is between 50 and 500 US$, which is not prohibitive for compounds of high commercial value, dependingon the difficulty of the chromatographic separation. Solvent handling and recovery account for much of the operating costs ofthis type of large scale system; other costs include instrumentation,installation,maintenance, and labor. Stationary-phase costs depend greatlyon whether the silica gel must be derivatized. Although a large number of octadecylated silica gels are available commercially for large-scale preparative liquid chromatography, all are different and will produce different alpha values for a given separation. It is thus extremely important to compare such materials on an analytical liquid chromatographic systemto find the one that gives the most efficient separation. Practically all analytical chromatography today is performed with mixtures of solvents,but it is almost impossibleto recover large volumes of a ternary solvent without changing its composition in the distillate. This problem can be avoided by usinga single solvent,but a single solvent does not often provide the desiredselectivity. The stationaryphaseis thus adapted to a particular separation problem by changingthe degree of derivatization, changing the derivatization reagent, mixingdifferent stationary phases, derivatizing with mixed reagents, or sequentially using different derivatization reagents on the same phase. Such special stationary phases may be expensiveat first, but in the final economic picture they may prove to be worthwhile. Because stationary phase packing material is expensive, particularly in the amounts required for large preparative liquid chromatographic columns, it is economically advantageous to regenerate used columns. Used packing materials can be dirtied by samples, lose bonded chains, or exhibit

200

Chapter

increased back pressure aasresult of fine particles that are generated during packing or by continuous compression. Although some researchers believe that removal of fine particles by reclassification introduces unwanted changes in selectivity because new active surfaces are being generated, this does not appear to be the case. The increase insurface area from compression is extremely small,and even if all the particles were halved, the surface area of a 20-pm packing material would increase less than 1 m2/g, compared with a total specific surface area of 200 m2/g. Regeneration of used packing materials isthus a viable proposition. The feasibility of a production-scale liquid chromatographic process is, ofcourse, determined by running the separation on analytical instrumentation first. Itis, however, practically impossible to reproduce exact analytical liquid chromatographic data on a larger scale system. Even when the two columns are packed with the same stationary phase using the same packing material (which is normally not possible), wall effects will result in the columns having different packing densities, which in turn cause variations in retention data. Nevertheless, analytical results can be a great help in optimizing a preparative liquid chromatographic separation. In spite of the fact that the retention mechanism in reversed phase liquid chromatography (RPLC) still remains controversial, many applications liquid chromatographic separation of oligomers have been reported (see Table 19). RPLC yielded separations of lower oligomers far superior to those achieved bygel permeation chromatography (GPC). Styrene oligomers having polymerization degrees N, up to 11 were separated on octadecylsilica columnselutedwith pure acetonitrile [750].Better separations can be achieved usinggradient elution with an increasing concentration tetrahydrofuran (THF) in water [751-7541 or of THF in methanol [755]. The influence of various factors on the separation of styrene oligomers has been investigated, such as the nature, pore size and surface area of chemically bonded nonpolar phases and solvents used as the mobile phase [756,757]. Some combinations ofbondedphases and solvents used as the mobile phases allow both the separation of styrene oligomers, according to their degree of polymerizationand theresolution of some stereoisomers of polystyrene oligomers [756,757].Nonaqueous reversed phase liquidchromatography on octadecyl-silica columnseluted with a gradient of dichloromethane in methanol separated styrene oligomers having values Nof of up to 20 and some higher polymers of low polydispersity [758]. The separation is believed to occur by a combination of hydrophobic and precipitation mechanism and a theory describing this process was developed [759]. Aqueous and nonaqueous reversed phase liquid chromatography on phenyl-bonded

Liquid Chromatography

201

phases were used for isocratic separations of styrene oligomers with THFwater, acetonitrile-water or THF-n-hexane as mobile phases, and an equation was derived to describe the relationships between the logarithm of capacity factors, k', of the oligomers and both the degree of polymerization and the concentration of the organic solvent in water as the eluent [760,761]. Normal phase liquid chromatography (NPLC) on nitrile-bonded phases eluted witha gradient of dichloromethane in isooctane separated the first twelve styrene oligomers, and a linear relationship between log k' of the oligomers and thedegree of polymerization, N , was reported [762]. Similar results were achieved on silica gels with a convex gradient of dichloromethane in n-hexane, which allowedthe partial separation of 8-10 low molecular weight styrene oligomers [763]. Whenthe mobile phase was comprised of THF and n-hexane or of ethyl acetate and n-hexane, the styrene oligomers were separated only accordingto the number of oligomer units, whereas withdichloromethane-n-hexane,separation of the stereoisomers of the styrene oligomersalso occurs [764]. The retention behavior was explained on the basis of additive contributions to the adsorption from identical oligomer unitsand from end groups [765]. A calibration method using alkylbenzene calibration series and two indices to characterize the retention of each solute over a wide range of mobile phase compositions has been introduced [766]. One index, nE, accounts for thehydrophobicity of a solute and the other, qi, for thepolarity of its functional group(s) interacting with mobile phase components. This approach makes it possible to predict the selectivity changes induced by changes in the mobile phase compositions and may to used to predict relative and absolute retentions under isocratic or gradient elution conditions in binary and ternary mobile phases [767,768]. The lipophilic and polar indicesmaybecalculated from the additive contributions of structural elements [767]. with homologous series [769],a regular increase inthe logarithms of the capacity factors with an increasing number ofrepeat structural units was observed for some oligomeric series studied systematically in reversed phase systems, such as for oligoethylene glycol phenyl ethers, in agreement with the Martin rule [769].

G. Unsolved Problems and Future Trends of Liquid Chromatography In liquid chromatography (LC) there is a lack of a reliable modelto predict the capacity factor fora wide range of solutes, the retention mechanism on some phases isnot fully understood, and data on diffusion coefficients are scarce.However, approximations havebeenroutinelyappliedover the

b

8

Ghro ~ aogr t a p h for ~

ligo~er

n

2~000 2,0~-6,000

Stationary Phase

luent

Silica gel Silica gel ben~~ne-eth~nol

3,100 Silica gel ~ Q 0 - 4 Silica ~ ~ gel ~ , ~ ~ligobuta~i~ne 80Q-1,900 Uligobutadiene 2 ~ 5 ~ligoisobutylene 2,200 ~ l i g o i s o ~ylene ut 900 ~ligoisop~ene 3,100 Gopoly(1,3-butadieneisoprene) Copoly(1,3-b~tadieneisoprene) Chpoly( 1 ~ ~ - ~ u t ~ ~ i e n e - I ,~Q0-3 iso~rene~ 294 Gopoly(d ,3-~utadien~isoprene) Co~oly( 1,3-~utadieneisoprene) Copoly( 1~ ~ - ~ ~ t ~ ~ i e n e isQprene)

Silica 0 gel Silica gel Silica ~ gel Silica gel Silica gel Silica gel

CCt,, toluene, CKCl,, dichloroethane CGl~-~~Gl~-e~hanol

~bservations

ef.

G~adientelution Gradient elutiQn Gradient elution Gradient elution Gradient elution lsocratic elution

[651,652] [652-6541

~ r a d i e nelution t Gradient elution Gradient elution Gradient elution ~ r a d i e nelution ~ ~ r a d i e nelution t

[6601 [661] [662] 16581 f658,663f 16621

I6551 1656,6571 [658] [659]

lsocratic elu~ion

f

Silica gel

~ r a d i e nelution t

j16651

Silica gel

~ r a d i e n~lution t

16663

Isocratic elution

I6671

Gradient ~ l ~ t i o n 16583

C o ~ o l1y, ~ - ~ u t a d ~ e n e isoprene) Copoly~ 1,3-butadie~ep~perylene)

Silica gel

eel4-

Gradient elution

[6~~1

Gradient elution

[6693

G ~ ~ ~ ieel n~t t ~ ~ [ 6n 7 ~f ~ ~ 7 ~ Copoly~tetra~ydro~uran~~Q~ylen~glycol~ Oligotetrahydrofuran Copoly~tetrahydr~fu~anepichlorohydrin) ~ ~ ~ o l y ~ e toxidehy~en~ 500-5,000 Silica gel ~ r o ~ y l e glycol) ne Oligo~r ~pyleneglycol 5 , ~ OSilica gel ~ligoox~ropyl~~e ,OOO Silica gel ~ligotetr a~ethyl~ne500-3,000 Silica gel glycol ~ligodi~thyl~neglycol ~ 0 0 - 2 , Silica ~ gel adipate ~ l ~ g Q ~ b dycol) ut~le~ 1~ ~ O - ~Silica , Ogel~ Tiokol 2 , ~ 0 0 - 3 , 0 Silica ~ gel ~ligosulfide Glass Epoxy resins Silica gel ~ ~ h a - ~ y d r o ~ ~ - o ~ e g ~ -2,000 Silicagel ~enzoligo~ 1,3,6-trioxane) ~ycloole€in oli~o~ers ligoacrylate ~ligoca~r~l~ctone h,

t2

ethyl e t ~ketone ~ l (

Isocratic elution

[6721

I ~ u ~ r a telution ic Isocratic elution

[673] 16741

EK -et~anol(6:2:1)

MEK

en~ene-ethanol(75/25) CC14-6 ~en~e~e-hexane ~etra~~~r~~uran THF

~socratic~ l u t ~ o n[ ~ ~ 5 , 6 7 7 , 6 ~ $ f ~socraticelution [67~-6$6] Isocratic elution [6 $ 7 - ~ ~ 0 1 Isocratic elution

[691-6961

Isocratic elution Gra~ientelution Gradient elution Isocratic elution Isocratic elution

~6~7~6~$1 ~69~,700] [701,702] ~703-~07~ 170~~70~1

socrati~el~tion [7 G r a ~ i e nelution ~ [710-7 121 ~ ~ aelution ~ i ~[713'f ~ t

Table 19 Continued -

Oligomer

!

M"

Polycarbonate

9,000

Maleic anhydride oligomers

3,000

Oligoesters Oligoacetilene Oligostyrene Oligo(phenylene sulfone) Epichlorohydrine oligomers Oligo(4-vinylpyridine) P henol-formaldehyde oligomers Oligo(viny1naphthalene) Polynuclear aromatic hydrocarbons Oligo(styrene-acrylate) Peptides Oligo( 1,3,6-trioxane) Oligonucleotides Oligosaccharides

1,500 1,000 3,000 1,000 5,000 1 300

Stationary Phase Organic packings Organic packings Silica gel Silica gel Silica gel Silica gel Silica gel Bondapak

1,500 2,000

LiChrosorb

4,500 1,500 1,800 2,000 4,000

LiChrosorb Lichrosorb

Eluent

Observations

Ref.

Gradient elution Gradient elution Hexane-MEK( 8:92) -

Tetrahydrofuran CHCl,-CCl, CHC1,-CC1, Pentane-CH,OH Methanol( 80% )-water(20Vo )

Tet r ah ydro furan

Isocratic elution Isocratic elution Isocratic elution Gradient elution Gradient elution Gradient elution Isocratic elution

17181 [ 7 19-7241 17251 17261 [ 727,7281 16861

Gradient elution Gradient elution

[730-7341

Gradient elution Gradient elution Isocratic elution Gradient elution Gradient elution

[735-7431 [ 744,7451 17461 17471 [ 748,749J

[ 716,7 171

t7291

Liquid Chromatography

205

years and some questions still remain which are not very clear to users. The questions are more numerous in liquid chromatography than in gas chromatography. These questions may be classified in two categories: those regarding the solvent effects and those which are involved in retention problems. In normal phase liquid chromatography (NPLC) the SnyderSoczewinski displacement model states that retention is given by eq. 110 [7701 : logk' = log(

%) + a(S" - €'AS) +

&as

Vm

where a! is the adsorbent activity, V, and V, are the volumes of stationary and mobile phase, respectively, E' is solvent strength parameter, and A, s are constants. There is no adsorbent of standard activity to refer to and a! is determined from chromatographic measurements. In the model (eq. 110) the solute retention in a binary mixture of solvents (apolar diluent polar modifier) is given by

+

l o g k ' = a - blog(X,) where X, is the mole fraction of the polar modifier. When using mixtures of the same eluting strength the same retention should be observed. This is not the case as indicated by data in Matyska and Soczewinski [771]. The different retention data obtained in the experiments with heptane and chloroform are normal since solvents exhibit their own different characteristics. Differences in selectivity are the result of displacement and solvation equilibria in the mobile phase [ 7721. Identical retention is observed with solutes of identical properties. T o our knowledge there is no predictive theory of selectivity to account for these differences. This problem is more acute in reverse phase mode liquid chromatography (RPLC) and very troublesome when dealing with optimization procedures. Observed retention at the three selected solvent compositions of the triangle apices is often a matter of perplexity [773]. This can be overcome but the problem is complex when 3 or 4 solvents are involved. Peak crossover occurs when the whole surface of the mixture design is considered and one cannot predict the relative solute behavior. Solvent effects are weak and cannot be measured accurately; they are the consequence of individual effects which can be synergistically reinforced or diminished. In RPLC the retention mechanism is still not fully understood. Retention modeling is required for any computer-assisted optimization procedure and description of the retention behavior of the solutes under isocratic conditions is a key factor to gradient elution. The prediction of retention time and optimization of separation conditions from physicochemical properties is not com-

Chapter

206

pletely successful, the correlation between AHand k’ is only fair. One very serious drawback is the stability of the packing; only few manufacturers can provide guaranteed reproducible packings. Moreover, it is common practice to use columns withvery different mobile phases and this is detrimental to the reproducibility of retention. The simplest relationship is kw Ink’ = In

- SI$

(1 12)

where I$ is the volume percent of organic modifier in a binary mixture: waterlorganic modifier. Thislinear relationship onlyholds true over a limited range of composition (Fig. The linear part is smallest whenthe solute is capable of hydrogen bonding. To account for deviations from linearity, second order or more sophisticated relationships have been derived. The problem is that the range of linearity is not known. To enhance the domain Johnson [774] used the solvatochromic theory of Kamlet and Taft and compared the linearity In k’ = f ( 4 ) and In k’ = f(so1va-

log k’

/

thylbcnzene

1.5

Nllrobcnzcnc

1.c

c

.

00

GO

.

.

40

,

. 20

.

0

Figure 26 Typical relationships between log k’ and volume fraction of CH,OH in water/methanol. Solid lines linear regions. (Adapted from Ref.776.)

Liquid Chromatography

207

tion parameter). They observedbetter linearity in the lattercase but did not go beyond 80% in acetonitrile. In eq. 112, kw is the extrapolated value of k’ when 6 = 0 (pure water). Starting with the two binary mixtures (watedacetonitrile and water/ methanol) the same valuefor kw should be observed. Thisis very often not true. The value of kw must be considered asthe intercept of the line witha given mixture. We have no prediction model for binary mixtures and, moreover, none for ternary or quaternary ones. Jandera [776] published a paper on ternary solvent optimization. The lack of reliable theory prevents the routine use of computerizedoptimization. An index system does not exist in LC. Smith and Burr [777] proposed alkylaryl ketones asstandard solutes. The retention index of a solute is the sum of the retention of a parent compound, of the increment for substitution groups, and interaction terms. With benzene derivatives the parent compound is obviously the benzene. Unfortunately the indices are not very reliable for different solvent compositions and their use in optimization theory looks difficult. The main problemis the accurate determination of k’ .Each molecule may have its own volume [778] and a review on this topic has been published by Malik and Jinno[779]. The questions involved in retentions problems are numerous. With polar bonded-phases the exact nature of the retention on a organophase is still a matter of dispute since some papers claim that residual silanols are only involved in retention [780]. No definitive explanation is given for the fact that cyano phases exhibit basic properties in chloroform and acidic properties in methyl t-butylethers [781]. Based on past experience and current needs, it is possible to predict trends in separation techniques. Unfortunately, I donot have a crystal ball to see preciselythe developments that will take place in the future.The only thing one can be sure of by daring to write sucha paragraph is that a good deal of what is said will come back to haunt the author. “Those who ignore the past are bound to repeat it,” but I propose to approach the topic of this chapter by first offering a view of how liquid chromatography development originates in the past and finally, I would like to answer the questions of what needs to be the view for both instrument development for the near future, and what techniques must be first researched and what discoveries one could hope for which would generate another direction for development in separation. It would be useful to have an independent measure of what stage of development a given separation method has reached. Such a ruler would help in predictions of the future in, or of, a given method. No certain

208

Chapter 3

method has been established as yet, but there are a number of approaches including market surveys and total sales. The most obvious need for HPLC instruments will be in biotechnology and the life sciences. As the biological sciences continue to migrate toward a molecular basis, chromatography will play an importantrole and the market will expand. Separation science needsto direct its efforts toward other futures and othermethodologies and there are ample challengesto do New challenges in environmental monitoring and forensic medicine will requires sophisticated and reliable separation techniques. HPLC will play an important role in molecular biology not only on the analytical scale, but also on thepreparative scale. Both fast andmicrobore column HPLC will be used for various phases of the work, especially in mapping the human genome. HPLC will also be a vital tool in cancer and AIDS research and diagnosis and in determining biochemicalmarkers for detecting and monitoring the course of many inherited genetic defects and otherdiseases. In biotechnology,high-speed separations willbeused for process monitoring of fermentation and growth media. Microbore columns with sensitivities in the molerange will be used routinely, and singlemolecule manipulation may be possible because extreme sensitivity -”) is within sight using either capillary HPLC orcapillary electrophoresis. Another potential use is the separation of cells and the rapid identification of viruses and bacteria. HPLC will also continue to be the mainstay of the pharmaceutical industry, not only in analytical laboratories, butalso for quality control of starting materials and final products; for on-line process monitoring, and, on the preparative and processscale, for the isolation and purification of drug products. The increased sensitivity of capillary HPLC and thevery rapid analysis that can be achieved withshort columns will make this technique indispensable in biochemistry, molecular biology, and themedical sciences. The great potential of HPLC-coupled techniques will be valuable in toxicology, pharmacology, and forensic medicine. Because greater emphasiswillbeplaced on the stringent requirements of regulatory agencies, HPLC/MS will become a necessity in many laboratories and will be used routinely in organic synthesis and medicinal chemistry to separate, purify, and determine molecular structures simultaneously in one sample. Emerging applications in the life sciences will includethe analysis of carbohydrates (especially glycoproteins), phospholipids, and surfactants. With miniaturization, instruments can be taken into the field for environmental and clinical work, and possibly even into outer space. HPLC will also be important in monitoring the purity of materials used by the electronics industry in manufacturing processes, especiallyfor computer chips.

Liquid Chromatography

209

Most ofthe advances in separation in oligomericand macromolecular systems has arisen outside of the traditional chromatography community. There are numerous reasonsfor this. Part of the difficulty lies inthe preference by separation scientists for small molecules and the easy relationships of directed forces, sample kinetic theories, and other qualities common to such systems. Future advances in large molecule (including oligomers), if they are tocome from thechromatography community, will needto include the abandonment of this comfortable area and the application of a good deal of chemical insight as it relates to oligomers and polymers, which occupy space, have many functional groups, differ in chemical structure and in molecular size; consequently, results of separation can change with changing separation conditions. “More than 90% of liquid chromatography is reversed phase” is a remark one currently hears quite often in some circles. This is true if one ignores all exclusion chromatography done by those who must deal with synthetic oligomers and polymers including biopolymers. A large number ofvery practical separation problems are amenable to solution by RPHPLC, and that is certainly why it remains popular. This should not be construed that NP-HPLCmethods have been eliminated because they have no value. Certainly some of the separations currently carried out on reversed-phase with additivesto improve resolution might be better done by normal phase. There are, however, many more gaps in the scientific basis for designing normal phase systems than are left in predicting reversedphase behavior. The problems with normal phase are the volatility and flammability of the solvents, their relative cost, and the lack of much research to determine the requirements for producing optimal, bonded, normal-phase packing materials. Unless commercial interest in pursuing normal phasecan be stimulated bya potentially large market need, itis unlikely that much will bedone in improving the stateof normal-phase methods. Liquid chromatography is still in a growth phase because of its adoption by scientificareas that can not ordinarily use gas chromatography as a routine tool. Included in these are those devoted to characterization of polymers and especially the knowledge of biologically active oligomersand polymers, ions and very water soluble compounds. The demand for very sophisticated, multisolvent, multiwavelengthinstrumentation is not seen as yet in much ofthis community. Manyof the “bio-problems” are solved with binary gradients and single-wavelength detectors. This will undoubtedly change as the potential of the technique is recognized and the need for selectivity control increases. There are many problems to be investigated and solved in this arena. The trend in the future willbe toward “bigger and bigger” and “smaller and smaller” instrumentation. To allow easy use of capillary col-

210

Chapter 3

umns, instrumentation properlyengineered for thesecolumnsmustbe made commercially available. Users will need microcolumn-LC to interface more easily with mass spectrometers, with other detectors such as FT-IR, or with other techniques such as capillary electrophoresisor gas chromatography. Miniaturization of instrumentswill also mean using less solvent and column packingand will conservelaboratory space. In this decade there will be great activity in the development and monitoring of specialized column packings. Although work in this area started in the the efforts will intensify. Better and more effective chiral packings will be needed to satisfy the pharmaceutical industry’s stringent requirements for enantiomeric purity of drugs. Affinity and immunoaffinity columns will be of the utmost importance for the production of highly purified biotechnology products because regulatory agencies both in United States and in Europe will demand ultrapurification to remove all debris from thehost cells. Columns that have a hydrophilic exterior and a hydrophobic interior willbe in demand for protein and peptideanalysis, as well as for the characterization of functionalized oligomers. These packings must be of small particle size and large pore size. Although they are currently available, more sophisticated versions of packings will be developed with solid support other than silica. Packingsthat arespecifically designedfor nucleic acids, oligonucleotides, nucleotides, nucleosides,and their bases, as well as adducts of these compounds, are need for biotechnology and for studying disease processes and environment. Chelate packings that are selective for various metal ions will be of interest for monitoring metal contamination in water. Another interesting type of packing is the cyclodextrins. Small molecular weight compounds are excluded because of their limited steric access; thus, these packings can be used as cleanup columns priorto analysis or for purification of small moieties. Eventually, for every compound there will bea packing. Silica wasthe solid support of the but other supports began appear in the latter part of the decade. Organic resins are making a comeback after declining in popularity in the and early These supportshaveadvantages,as do polymer-coatedsilicas,especiallyinbiotechnology. Porous graphitic carbons also have great potential as solid supports. They are chemically unreactive, can be used in a wide pH range, are highly reproducible and stereoselective, can be modified by adsorption of high molecular weight materials,and can be usedfor size exclusion.The major limitation of this support currently is the dearth of literature on publications and separation conditions for various compounds. Mixed-mode and mixed-bedcolumnswillbecommonlyused. The mixed-mode technique will be used for column switching or for various

Liquid Chromatography

21 I

types of columns in series(e.g.,cyclodextrin or exclusion, followed by reversed phase and/or ion exchange), or a packing material on which two or more functional groups are bonded. Mixed-bed columns contain two or more types of packing materialsfor example, anion and/or cation exchangers mixed with a reversed-phase packing. The mode of operation can be changed simply by switching the mobile-phase conditions.In fact,it may not even be necessaryto have mixed beds. Some ion exchangers can be operated in the hydrophobic interaction mode by changing the salt concentration of the mobile phase. The use of lasers as detectors will become increasinglyimportant. As a universal detector, the LSD will replace the RI detector for separations that require increased sensitivityor gradient elution. This detector can also be used to determine molecular weight. As a highly selective and sensitive mode of detection, LIF will play an important role, not only with HPLC but also with capillary electrophoresis. As capillary HPLC instruments become commercially available, it will be possible to adapt the best in GC detectors for usewith HPLC, such as the FID or the electron capture detector. However, as mass spectrometers decrease in size and price, and the interfaces improve, mass spectrometers will become the detectors of choice for obtaining structure information or for positive identification of an analyte, as in court cases or drug testing in athletes. The good old workhorse of the field, the UV-vis detector, will continue to hold its place ofhonor for routine analyses of compounds containing a UV chromophore. Its ruggedness, reliability, ease of operation, and reproducibility make this detector desirablefor laboratories where the ultimate in sensitivity is not necessary. In our laboratory, the UV-vis detectors purchased in the early 1970s have been used bya large number ofgraduate students and are still giving good results. Multiple detectorswill also play a major role in HPLC. Two or more detectors will beusedeitherinseries or via stream splitting. It can be helpful to have a universal detector determinethe number of solutes present in the effluent and one or more very selective detectors identify the compounds. The primary constraint is that the solvent system must be compatible with all detection systems. Computer optimization is here to stay even though some people are still reluctantto use these programs. As the younger chemists become more computer literate, resistance will diminish and a tremendous amount of time will be saved by computer optimization. The use of robots will also increase exponentially inthe next decade. Robots will be used for all routine tasks and will be especially necessaryin sample preparation and handling of potentially dangerous samples, suchas physiological fluidsfrom AIDS patients,other viral and bacterial materials,

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contaminated environmental samples,and radioisotopes. Robotics will improve laboratory safety and will help to fill the gap due to the diminishing pool of science graduates available for jobs in industry and academia. In addition, the use of robots can increasethe number of samples reliably and efficiently processed per day, which is clearly an advantage in high-volume analytical laboratories. Control programs that use artificial intelligence will be the next stepin robotics. In the future, multitasking instruments will be commonplace. For example, after preliminary sample preparation by specialization robots, several instruments will be interfaced that all the steps from rigorous sample preparation to separation, peak identification, structure determination, and quantitation will be done automatically. Not only willthe instrument have several columnsand/or detectors, but it will be interfaced with capillary electrophoresis, GC, or mass spectroscopy. This combination of instruments and techniques is often referred to as multidimensional chromatography. In protein analysis, peptide mapping may be included in the multitasking program as will the determination of the base sequence in nucleic acidsin genome investigation. HPLC, alone or in combination with one or more techniques, will continue to be the separation technique that is the backbone of the analytical laboratory, especially in the pharmaceutical and biotechnology industries, because of the ease of sample preparation, automation, speed, and sensitivity. In the future, fast HPLC may be used instead of flow injection analysis to monitor real-time fermentation processes that are used in biotechnology. Althoughthe dominant applications will bein the life sciences, HPLC will face new challenges in environmental testing and in forensic medicine. Better separation techniques are also needed in the chemical industries for the manufacture of adhesives, sealants, catalysts for petroleum processing, and materials used in the manufacture of microcircuits. Good preparative and process HPLC techniques are required to separate, isolate, and purify most consumer products such as food, drugs, vitamins, and cosmetics. Several separation techniques will be competitive with HPLC in the next decade: capillary electrophoresis, supercritical fluid chromatography, countercurrent chromatography (CCC), and field-flow fractionation (FFF). However, Ido not believe that these techniqueswill replace HPLC. Because of its ruggedness, versatility, and separating power-especially for watersoluble, nonvolatile, thermally labile compounds -HPLC will maintain its solid position. Although capillary electrophoresis isa strong competitor in the separation of large biopolymers and biologically active molecules, more research is needed in injection systems, detectors, and peak identification before it can achieve its full potential. HPLC willbeused for routine

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analysis of molecules with molecular weightsin the range of 200 to several thousand. GC will continue to be the method of choicefor thermally stable, volatile molecules that have a molecular weight below 200, whereas capillary electrophoresis will be the method of choice for biopolymers. Supercritical fluid chromatography will be the preferred preparative technique if moremobilephases are found inwhich analytes are soluble. major advantage of this technique is the case of removing the solvent from the product solution. CCC is also useful for preparative work and has been used recently in the purification of peptides and proteins. However, many researchers prefer HPLC because they are able to scale up their analytical separations easily. Instrumentation for FFF currently is available, and in the next decade will be used in laboratories for theseparation of macromolecular or particulate species. Although other separation techniques are available, HPLC will not be replaced as the mainstay of the analytical laboratory, where all types of medium-sized molecules must be separated. Instead, in the 1990s GC and HPLC will be joined in the laboratoryby capillary electrophoresisand, for specific needsor problems, bySFC, CCC, and FFF. growing recognition ofthe potential formation of artifacts on column can be found in the current literature. This has many implications for the continued spread of LC into areas such as molecular biology. Artifact formation in gas chromatography has always been a problem and can be expected when labile substances are examined. The occurrence of artifacts is less a problem in small-molecule liquid chromatography and has been ignored in some earlystudies of the application of HPLC to diagnosis, for example. Much research will be needed to identify the likely occurrenceand cause of artifact formationin the separation of labile biomolecules if both methods of determination and collection of pure materials is to be reliable achieved. The future of chromatography seems quite bright, as does the future of molecular science. The future in chromatography is less clear. It will be fascinating to watch what happensin the next few decades.

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4 Gel Permeation Chromatography

INTRODUCTION In the landscape of liquid chromatographic methods,gel permeation chromatography (GPC)occupies a special place. The arguments are numerous which cansupport the differences betweenGPC and many other procedures that form liquid chromatographic method. In GPC, macromolecules are separated according to their hydrodynamic volume. This separation results from a decrease in the entropy of macromolecules when they pass from the channels of the mobile phase of the chromatographic columninto the pores of a stationary phase. In the adsorption form of liquid chromatography (LC),additional retention is observed. The elution of macromolecules in this instance is determined not only by entropy changes but also by the enthalpy changes that they undergo inthe interphase transition. The reason for the enthalpy changes is the energetic interaction between the macromolecules and the stationary phase matrix. The magnitude of this interaction is proportional 258

omatography Gel Permeation

259

to the number of adsorbing units of the chain. For an adsorbing homopolymer it is a degree of polymerization. In the case of heterogeneous polymers, it is possible that only one kind of unit interacts with the packing. Therefore, the magnitude of this interaction is proportional to the number of such units in the macromolecule. Consequently, the chromatograms obtained by GPC show distributions of the macromolecules of the sample according to size, whereas in LC the chromatograms reflect both this size distribution and also the number of the adsorbing monomer residuesin the macromolecule. Since the general liquidchromatographic instrumentation and column techniques are becoming an integral part of GPC, it is useful that practitioners be acquainted with the equivalences and differences in GPC and general LC technology. There are four ways of reporting conventional LC peak retention: retention time (t,), retention volume ( V,), peak capacity (k‘ ) and solute distribution coefficient (KLc). The simple experimental value,t R , measured by the time required for a peak to elute from the column following injection, is useful only for comparing peaks that have appeared in the same chromatogram. The value of tRis sensitiveto changes in experimental conditions such as flow rate and the specific columns used; therefore, it is not very specific for defining sample components. The retention volume V, is a more fundamental quantity in that it accounts for flow rates differences. To calculate V,, the mobile-phase flowrate, F,must be known aswell as the t R values, since V, = Ft,. While peak retention reported as V, is not subject to flow change, it can still vary with differences in column size and instrumental dead volume. Such variations are inherently compensated for with the more basic retention parameter, k ’ . Physically, k’ represents the ratio of the weight of solute in the stationaryphase to that in the mobile phase. Thus the weight fraction of solute remaining in the mobile phase is 1/(1 k ’ ) . For an unretained peak, tR = to,k’ = 0, and the value for the solute weight fraction in the mobile phase equals unity, meaning that the solute resides only in the mobile phase. Since solute migrates only when in the mobile phase, the retention time should be inversely proportional to this weight fraction:

+

or

and

Chapter 4

260

assuming constant flow rate, where V , = Ft,, for the retention volume of the unretained solute. Although widely used for comparing conventional LC data, values of k' still do notcompensate for differences in the stationary-phase concentration caused by the difference in the surface area and porosity of the column packing. Peak retention, or value of k ' , increases with increasingstationary-phase loading. account for differences instationary-phase loading, the parameter KLc should be used to define retention. In fact KLcis the only parameter that can uniquely define the retention characteristics of different organic compounds in conventional LC experiments witha specified column packing, mobile phase, and temperature. Physically KLcis the ratio of solute concentration in the stationary phase to that in the mobile phase. For a given mobile phase and column packing, values ofK,, uniquely reflectthe basic thermodynamic balance of solute between the phases. Assuming that the equivalent liquid volume for a stationaryphase is V,, K L C is related to k' by

Inserting this relation into eq.

one can show that

Eq. 5 represents the equilibrium theory of conventional LC peak retention. It explains why the experimental value of V, is determined solely by the thermodynamic balance ofsolute distribution between phases. In GPC, solutes are partially excluded from the column packing and elute ahead of the solvent peak. As a solute band moves along with the solvent downthe column and around thepacking particles, the solute molecules repeatedlypermeate or diffuse in and out of the pores of the packing. The driving force for the process is the concentration gradient between the pores. While in gel filtration separation (GFS) of naturally occurring macromolecules, or in GPC separation of small molecules, several distinct peaks may be obtained; in synthetic polymer analysesthe GPC chromatogram or elution curve usuallyis just a broad, continuous elution pattern. To extract polymer molecular weight distribution (MWD) information from a GPC chromatogram, the exact MWD versus V, calibration relationship the GPC column is required. In discussing LC retention, the volumes of the mobile phase inside

Gel

261

and outside of column packing are grouped into one volume term, V,, the retention volume ofthe solvent peak(eq. 5). Since all peaksin the otherLC methods elute after V,, it is not important to distinguish between the stagnant versus the moving parts of the mobile phase volume,V,. Subdivision of V, is necessary for explaining GPC (where the term mobile phase simply means, in GPC,the carrier solvent), because the stagnant part of the “mobile” phase residing in the pores is, in effect, the “stationary” phase for GPC separation (Fig. 1). To avoid confusion with the stationary-phase volume V, in the other LC methods, the stagnant structure in GPC is designated as the internalpore volume. The remaining liquid volume in a GPC system is designated as the void volume V,, which is mainly the interstitial liquid volume between the packing particle.By definition, then V, = V,

+ V;.

(6)

Size separation in GPC is the result of differential solute distribution between the solvent spaces outside and inside the pores of the column packing. Thissolute distribution can be described bythe GPC distribution coefficient KG=, that represents the ratio of the average solute concentration in the pores to thatoutside the pores. Because ofthe size-exclusion effect, not all the pore volume & is accessible to large solute. Solute concentration inside the pore decreases with increasing solute size. In effect, then, the total accessible liquid volume for different-size solute is not (V, but (V, KcPC&).Substitution of this accessible liquid volumefor V, in eq. 6 can be minimized by using inert column packings to avoid interference of surface effects on GPC solute retention. With negligible surface effects, GPC retention can be approximated as

+

+

Figure 1 GPC retention mechanism: I, mobile phase; 11, stagnant mobile phase; 111, porous packing.

262

Chapter 4

V, = V0

+ KcpCK

(7)

The values of KcPCcompensate for column size variation. For any GPC column regardless of size, KGpc= 0 at exclusion and KGw = 1 at total permeation. Intrinsically, GPC is a low-resolutiontechnique.Unlike other LC methods, which can be developed to resolve up to hundreds of component peaks representing many column volumes and extended retention times, GPC separations are constrained to occur within the limits of the packing pore volume. Thus only few peaks can be fully resolved in GPC. The large difference in peak capacity between GPC and other forms of LC can also be explained in terms of basic retention parameters. The value of KGpcis constrained to be between0 and 1, which means that solute distribution favors the unrestricted space outside the pore. On the other hand, values of K,, are unlimited, which means that solute distribution favors the stationaryphase, as in the case for most LC peaks. This difference in peak capacity between GPC and other LC methods is indicative that different thermodynamic balances are involved in controlling solute distribution. As will be described in the following, GPC is uniquely different from the other LC methods in that it is a chromatographic process controlled by entropy, notenthalpy. Direct use of terminology traditional to LC in GPC applications can sometimes be awkward. In GPC (Fig. 1) the size separation occurs only within the mobile phase volume, V,, where different size solutes distribute differently between V, and K, that is, between the solvent moving outside the packing and the stagnant solvent inside the pores of the packing. The distribution favors V, more for larger solutes. According to LC terminology and eq. the GPC chromatogram would have to be interpreted with awkward negative values of k ’ , since V, V, in GPC as solute elutes before the solvent peak (i.e., tR < to and k’ 0). This is why the distribution coefficient KcPCis used in GPC as the peak retention index instead of k’ as in conventional LC. For the same reason, the separation factor a, defined in conventional LC the ratio of k’ for two solutes, is not used in GPC. According to eq. failure todistinguish the moving and the stagnant parts of the mobile phase doesnot affect the estimation of k ‘ . However, it does causean errorin the calculation of solvent velocity,v:

where L is the column length. It is a known fact that polymers are not unique substances but tures formed from molecular species that can distinguish themselves through

Gel

263

molecular weight and composition or chemical structure. The direct consequence of this situationis that in the polymer’s casethe molecular weight is expressed by average values generated by the existence of a distribution of the molecular weight and physical constants are replaced by transition states. GPC was born outof the need to have an efficient deviceto determine molecularweight distribution in naturaland synthetic polymers. This method succeededin transforming polymer fractionation from an academic activity into a routine work also accessible for unsophisticated labs. GPC has eliminated on thespot the main disadvantages of polymer fractionation by classical methods: the huge time consumption, uncertain and unreproducible results, as well as tiresome work. However, 20 years have been necessary for thegel permeation effect to turn intoworking a methoduseful for oligomer and polymer fractionation. Since the 1925-1935 period when the gel permeation effect was discovered [ 1,2], in whicha pioneer’s role was held by the Romanian scientist Cernescu until the 1 9 5 0 ~ no~application was found to this effect. The first real application of gel permeation effect was made by Porath [4] who used this effect for separation and characterization of mixtures in biological chemistry.The method thus created, to be only applied inan aqueous medium, has been calledgelfiltration. However,in my opinion, the true historyof GPC begins in 1964 when Moore published hispaper “Gel permeation chromatography. A new method for the determination of molecular weightdistribution in high polymers” ( J . Polym. Sci., Part A, 2, 835, 1964). Immediately after that GPC has continually widened its application range. Identification of auxiliaries from polymers and quantification of oligomer presence represent only two of the main developments ofGPC in the analytical chemistry of polymers. Oligomer presence in polymers represents a natural state.Often oligomer separation determines their restoration in the polymer mass via degradative processes. Oligomers are to be called macromolecules whose molecular weight varies between 10’ and lo4 daltons. Beginning with the dimers and ending with species with a molecular weight of several thousands, the oligomers represent a special, well-defined world, which has become extremely useful and particularly powerful. Many times the oligomers are considered to be the diplomats of the polymer country [5,6]. During the past ten years, there has been less emphasison GPCtheory and band-broadening studies and more focus on applications. However, to finish off the theoretical basis of GPC, three essential elementsare needed: to know the separation process, to create a convenient and realistic model for the solute particle, and, finally, to achieve a correct image of the packing.

264

Chapter 4

II. THEORETICAL BASIS Gel permeation chromatography is sometimes viewed as a liquid chromatographic separation technique, sometimes as a molecular weight and molecular weight distribution method specificfor high polymers and oligomers. It fulfills the latter role only when the column is calibrated with appropriate standards; itis not always clear whatconstitutes an appropriatecalibration. Unlike other modes of chromatography, GPC is an entropically controlled separation technique that depends on the relative size or hydrodynamic volume of a macromolecule with respectto the size and shape of the pores of the packing. In GPC solute-packinginteractions are absent. In GPC as solute molecules migrate through the chromatographic column, they transfer back and forth between the moving and stationary phases, constantly redistributing themselves between the phases to satisfy the thermodynamic equilibrium. Under normal chromatographic conditions, solute distribution approximating thermodynamic equilibrium is achieved. Thermodynamic equilibrium is defined as the condition in which the chemical potential of each solute component is the same in the two phases. For dilute solutions at equilibrium, solute distribution can be related to the standard free energy difference (AGO) between the phases at constant temperature and pressure: AGO = -RTln K

(9)

with AGO

=

- TASO

(10)

where K is the solute distribution coefficient, R the gas constant, T the absolute temperature, and Moand ASo standard enthalpy and entropy differences betweenthe phases, respectively. The theory of macromolecular GPC was essentially formulated by Cassassa and coworkers 17-91. Their theory is based on the calculation of changes in the entropy of a macromolecule as it penetrates from themobile phase into a sorbent pore, and make use of a model of a flexible-chain macromolecule in a thermodynamically idealsolvent, assuming a low polymer concentration in the solution and the quasi-equilibrium nature of the chromatographic process. The chief result of the theoretical studies performed by Casassa[7-9] was a universal relationship between the distribution coefficient, K, and the ratio of the radiusof gyration of the macromolecule, r, to pore radius, R. For a model of slitlike pore of width 2R, this relationship has the following form [7]:

Gel

265

where the summation is performed for added values of m. In the limiting wide-pore and narrow-pore cases, the K vs. r / R relationship takes simpler forms:

It follows from eq. 13 that with large macromoleculesand narrow pores, at r > R, the distribution coefficient isnot equal to zero. This signifiesthat a number of large macromolecules penetrate into narrow pores, assuming elongated conformations that are different from theequilibrium conformations of macromolecules in solution. Penetration of large molecules into narrow pores is a specific feature of polymer chromatography, which Casassa [7] was first to recognize and that was not accounted for in other GPC theories [ 10,l l]. The validity of explaining GPC retention only in terms of thermodynamic considerations requires that the solute distribution in a GPC experiment be close to thermodynamic equilibrium. Thus GPC separation is controlled by the differential extent of permeation rather than by the differential rate of permeation. The results of the temperature, flow rate, and static mixing experiments clearly show that GPC retention is an equilibrium-controlled, size exclusionprocess [12-141. Thismechanisticmodelindicates that solute diffusion in and out the pores is fast enough with respect to flow rate to maintain equilibrium solute distribution. Thermodynamic size exclusion is the fundamental basis commonto all the GPCtheories discussed inthe next paragraphs, when models for different-shaped solutesare considered. The linear dependence of on K r / R in wide pores is of a fairly general nature It reflects a decreasing effective volume availablefor accommodation of a macromolecule in a pore. In wide pores, macromolecules have approximately the same conformations in an unrestricted volume and behave in chromatography like spherical solid particles with an equivalent radius. In fact, eq. 11 is a definition of the effective chromatographic radius of the macromolecule. It can be seen from comparing eq. 12 with the expression for the distribution coefficient of a spherical particle of radius in a slitlike poreat P < R [lo],

Chapter.4

K = l - -P R that in theprocess of chromatography in wide pores the polymer chain is like a spherical particle of an equivalent radius

Comparing the results of GPC theory for various forms ofmolecules, Casassa [9] drew a general conclusion to the effect that the equivalent chromatographic radius of an arbitrary particle is to half its mean span (the mean span being the greatest projection of the molecule onto the selected, averaged for all possible molecular orientations and conformations). The effective chromatographic radius for molecules of all typesstudied is proportional to the radius of gyration, but the coefficient of proportionality has proved to be different both forvarious forms of rigid particles [ 1 l], viz., spherical, ellipsoidal and rod-like, and for polymer molecules of various topology, i.e., linear, branched, and ring [9]. It has thus been shown that the radius of gyration is not strictly a universal chromatographic characteristic for macromolecules. Other characteristic dimensions, e.g., the Stokes radius determinable from the friction coefficient in diffusion and sedimentation processes, and the hydrodynamic radius associated withBenoit’s “universal calibration” dependence parameter [15], have also been denied the status of universal chromatographic characteristic which they are notin fact [ 101. However, in a series of molecules of certain type, any of these dimensions may be used as a characteristic chromatographic radius. Despite decades of investigations, considerable debate still exists about the proper form of the theoretical relationship among equilibriumconstant of GPC (KGpc), pore size, and macromoleculardimensions. Therefore, conversion of GPC elution volumes to some well-defined dimension isnot possible inthe sense that say the measured diffusivity leads to an equivalent Stokes radius. It is clear that the retention time in GPC bearing nonsteric interactions between the solute and the stationary phase-must depend uniquely on the dimensions ofthe solute and the stationary-phase pore. If the objects possessed simple geometry,the dependence of KGpc on pore andsolute sizes would take avery simple form; i.e.,

-

Gel

267

where R is the radius of a spherical solute and rpthe pore radius, and where X = 1, or for slab, cylindrical, or spherical pores [ 161 and Kopc is the theoretical equilibrium constant equivalent to the relative solute concentration within the pore. It is important to note that KGpc is equivalent to what others have called K,, but different from K,, which early researchers defined as the fraction of the swollengelaccessible to the solute. The situation, however, is complicated by the ill-defined and possibly nonuniform geometries of both the cavities and the solute. Attempts to convert this problem to a tractableform have taken avariety of routes, in which, of necessity, the geometry of at least one of the two components is reduced to a simple form. Laurent and. coworkers [l71 used the treatment of Ogston [l81 to model the separation as the diffusion of hard spheres of radius R through a concentrated solution of rigid rods. The result of this treatment may be expressed as

or K, = exp[ --?r l ( R

+ r,)

where r, is the rod radius (cross-sectional radius of the fibers that constitute the gel) and l is the linear concentration of rods in the gel (stationary phase). K, is the probability that a spherical particle is included in the network of the gel and is related to KGpc by the expression K, =

KGPC

vi +

h V,

where 6 is the total pore volume and V, is the volume of the gel matrix (the nonporous portion of the gel) in the column. The sum V, V, V, is the total geometric volume of the column; when this is referred to as “V,,” then K, may unfortunately appear as (V, - V,)( V, - V, ) - l , with confusing similarity to the general equation of elution volume. Ackers [ 191 avoided any precise geometric description of the stationary phase, envisaging it as a collection of semi-accessible regions, eachone having a probability of being permeatedequal to unity for all solutes below critical size, or zero, for all larger solutes. Assuming that this distribution of “pores” couldbe described viaan error function,Ackers obtained

+

K = a, i.e.,

+ b,erf

(1

-K)

+

(20)

268

Chapter 4

K =1

- erf[ ( R - a,)/b,]

(21

In a gelwith identifiable pore geometry, a, wouldbe the average pore radius, which is designatedas FP [ 191, and b, would be the standarddeviation of the pore distribution, referred to later as Ar. Note that eq. 21 is incorrect in the limit R = 0 since ,,= 1 - erf( - a,/b,). In both of the preceding treatments, the solute is considered to be spherical. Such a description oversimplifies the shape of nearly all macromolecules ofinterest. Gidding [20] took a moregeneral view of solute dimensions and analyzed the permeation of capsule-shaped rigid solutes into a stationary phase modeled as a collection of randomly intersecting planes. This treatment leadsto the result

(mR

K = exp(

(22)

where is the half of the mean projection length of the solute and is the pore surface area per unit of free volumes, i.e.,a measure of the mean pore size. For flexible chain macromolecules one must recognize the statistical nature of the solute dimensions, as represented by, for example, the mean square radius of gyration (s2). A theoretical treatment for flexible chains in cylindrical cavitiesof radius rp leads to [21]

where p, are the rootsof the equation .l,(@) = 0, .l,(/3) denoting the Bessel function of the firstkind of order zero. It may be shownthat eq. 23 predicts that a wide variety of chain macromolecules, eluted on a single column, should yield a common dependence of KGpcon the product of intrinsic viscosity and the molecular weight, [VIM R:, where R,, is frequently referred to as the hydrodynamic radius [22]. The fact that this “universal calibration” [23J has been experimentally confirmedfor numerous synthetic macromolecules has persuaded many polymer chemists to the position that R,, (less ambiguously referred to as the viscosity radius) [24] is the fundamental GPC parameter, much as empirical correlations of Kopc with the Stokes radius have convinced manyprotein chemists that R, is the controlling dimension. All of the preceding expressions have some current support among experimentalists, yet we may hardly believe that all could be consideredto be verified by experiment. We may citeuncertainties about theexperimental values rp, I , and S in theforegoing expressions as major difficulties in

-

269

Gel

validation of these models [ a ] . To some extent, it may be possibleto attribute the current state of uncertainty to the ambiguity in the solute R size . In thedevelopment of basictheory for GPCmuch lesseffort has been devoted to improve the structuraldescription of intraparticle morphology. However, computer simulations showed that the topology of the pore volume plays a significant role in determining the mass transport process. The network model [25] proposedto describe the intraparticle porous structure of a GPC column shows significant differences when compared with the results obtained using a conventional model of a parallel bundle of capillaries. This fact demonstrate the importance of including morphologicalcharacteristics into themodel to account for themass transfer restrictions.

PACKING A great variety of gels has been used or tried for GPC. They are generally polymers of varying degrees of crosslinking and usually swell in the solvents for which they are made. Examples are polydextrans for aqueous solutions and polystyrenes for nonaqueous solutions. In contrastto the general views in this matter, the swelling was found not to be essential; the permeability (or porosity) is the important factor.Aside from permeability, the rigidity o f a gel determines its usefulness in practice. Soft particles do not pack well and tend to clog columns. This means high degrees of swellingare actually undesirable. High permeability and low swelling do not contradict each other necessarily sinceit has been possibleto prepare highly crosslinkedgels of high porosity. The permeability of a heavily crosslinkedgel is built into it by the diluent which must be presentduring crosslinking. If the diluent is well matched to the gel substance, increasing the amount of it is equivalent to increasing the swelling of the gel; the network becomes more tenuous. However, whenthe composition ofthe diluent is changed, it becomes possible to make the gel more rugged that it becomes more permeablebut not more tenuous. There is a variety of commercially available packings for GPC, and others are currently under development to improve the versatility, resolution, or speed of the GPC process. For a general review of GPC packing, see Refs. 26-34.

A. Semirigid Organic Gels Most GPC .analyses of synthetic organic polymers have been made using crosslinked semirigid polystyrene gel packings. Columns packed with spherical semirigid styrene-divinylbenzene copolymers are now available in a variety of pore sizes for 15 min analyses. These gels are applicable only

-

Chapter 4

270

in nonaqueous solvents. Advances in this area have focused on improving polymerization processes for increasing particle stability toward different mobile phases. Although most high-performance packings are in the range of 10 pm, there has been some development regarding smaller size packings for higher column efficiency. Mechan et [35] evaluated mixed-bed columns, whichcontain mixtures of individual pore-size crosslinked polystyrene packings. The authors suggest that for the analysisofpolymers above 1 lo6 g/mol, 10pm particles, rather than5 pm, should be used to avoid polymer sheardegradation. In addition, the larger size packing should be employed at high temperatures ( 1oO-15OoC) for increased column life. Kulin et al. [36] described the preparation of macroporous poly(styrene-divinylbenzene) particles usinga multistep swelling process. Packings of 5 , 10, and pmwere evaluated in terms of column efficiency and resolution. Finally, the theoretical prediction that the number of theoretical plates is proportional to the squareof the particle diameter was confirmed by these authors. Spherical particlesof controlled pore size vinylacetate copolymers are also suitable for use in organic solvents. Type OR-PVAgels (Fractogel, E. Merck, EM Laboratories) are used mainly for separating synthetic polymers. Pore sizes are available to separate materials with an exclusion limit up to about lo6 dalton, but the larger pore size particles are too soft for use in high performance GPC. These small-pore size, semirigid poly(viny1 acetate) gels are stable in organic solvents such as alcohols and acetone, even at elevated temperature ( 1oOOC) but limited to maximum column pressures ofabout 300-600 psi (20-40 bar). Porous organic gels that permit the separation of water-soluble solutes are yet not as well developed as packings for use in organic solvents. However, several useful packings are available and alarger selection can be anticipated. The hydrophilic organic gels used in gel filtration are made by the copolymerization of acrylamide with the crosslinking agent, N,N-methylenebisacrylamide (Bio-GelP). By regulating the concentration of these monomers, a series of covalently bonded gel products of various average pore size isproduced. Dextrans crosslinked with epichlorohydrine (Pharmacia Fine Chemicals) are also useful for aqueous phase separations. Because of high hydroxyl group concentration in the polysaccharide chains, Sephadex is strongly hydrophilic and swells in water and electrolyte solutions. While most of these materials are relatively soft, Sephadex G-25 has sufficient strength to be used at pressure at about 150 psi for molecular weights of up to about5,000 psi.

al.

Gel

271

A detailed study of Sepadex-75 was presented by Drevin et al. [37]. These authors studied the effect of mobile phasepH and ionic strength on the elution behavior of protein with different isoelectric point values. Sephadex LH-20 is a semirigid gel prepared by the propylation of Sephadex G-25. This gel is used with polar organic solvents (e.g., alcohols, tetrahydrofuran), but is also compatible with water.It is applied in a relativelywideparticlerange(25-100pm);however,narrowerparticle-size fractions (e.g., 25-30 pm) can be obtained by sizing to improve column performance. Hydrogel packings produced by Waters Associates also may be useful in GPC. These water-wettable beads are made from highly sulfonated, crosslinked polystyrene. Because ofthe surface sulfonic acid groups, ionic sorption effects may occur on either side of pH 7 in aqueous media, thus limiting mobile-phase solute compatibility. To overcome the poormechanical properties dextran gels, Tixier et al. [38] prepared mixed polyacrylamide-agarose gels in bead form. Four types of gel, developed by 1’Industrie Biologique Francaise, are available as Ultragel ACA-22, ACA-34, ACA-44, and ACA-54 from LKB. These gels differonlyincomponentconcentration, the firstdigitrepresenting the percentage of polyacrylamide and the second the percentage of agarose. Crosslinked poly(acryloy1 morpholine) (EnzacryP Gel) was prepared in bead form by copolymerization of acryloyl morpholine and N,N‘- methylene-diacrylamide in aqueous solution. This support contains few protons that are sufficiently labile to exchange with eluting solvent. In this respect it differs from other universal GPC matrices and from other water-compatible packings suchas crosslinked dextran and crosslinked polyacrylamide [39]. SpheronO is a macroporous, hydrophilic column packing originally developed at the Institute of Macromolecular Chemistry in Prague [40]. The packing is prepared in bead form by suspension copolymerization of hydroxyethyl methacrylateand ethylene glycol dimethacrylate.The pendant hydroxyl groups of Spheron gel permits chemical modification to be made. Consequently,Spheronderivativeswithspecificpropertiesmaybe obtained. These include: Spheron C, Spheron S, Spheron DEAE, Spheron ArA, and Spheron Oxine. Owing to the high content of the crosslinking agent, Spheron gel exhibits minimum swelling.Its rigidity is sufficiently highto permit its use at high flow ratesand high pressures( 3.5 pmole/m2), the residual silanolgroups remain unavailable for unwanted adsorptive interactions. Reactionof surface Si-OH groups with chlorosilane reagents to high yields is promoted by using a large excess of reactant, conducting the reaction in the neat liquid reactant or in a dry solvent, mechanically removing the volatile reaction product during the reaction (e.g., volatilization) [ M ] or by usingan appropriateacid acceptor such as pyridine [65]. If desired, untreated silica packing may be silanizedin situ in a reaction with chlorotrimethylsilane [66]. This approach is useful to resilanize a set of columns that have become somewhat adsorbing because of loss of deactivating bondedorganic groups. An improved mediumfor high pressuregel filtration was prepared by Lee and Jarrett [67], by derivatization of aminopropyl silica with glucose. The reaction involved was likely Schiff‘s base formation between the aldehyde of glucose and the amine of silica which was then reduced with NaCNBH,. This glucose-silica behaved as a nearly ideal molecular sieving support for thehigh pressuregel filtration chromatography of proteins and is superior in performance to many currently available supports. p-Bondage1 (Waters Associates) is a porous silica to which an ether functionality has been chemically bonded. This material is offered in four pore-size columns, plus an E-linear column that is a blend of pore sizes. The manufacturer states that these packings are compatible with solvents all polarity from n-heptane to buffered aqueous mobile phases; however, there is the usual pH limitations of 2-8.

-

Gel Permeation Chromatography

275

CALIBRATION In view of applying GPC to polymeric and oligomeric mixtureseparations, it is, nevertheless, necessary to know the mode of transformation the GPC elution curve in valuableinformation concerning the true composition of a certain mixture. This will enable us to offer a quantitative interpretation of the GPC results. However, one important problem is that GPC is a relative method and the quantitative interpretation is based on the validity calibration. The requirement for calibration arises in part from the absence of a complete theory for GPC, which would exactly define the relationship between the macromolecular dimensions of the solute and the experimentally measurable chromatographic partition coefficient K G p c .

where V, is the retention volume the solute, V, the interstitial volume (obtained experimentally as the elution volume of a solute too big to enter the pores), and V, the total volume of liquid in the column, equal to the sum of V, and the pore volume. K G p c thus represents the fraction of the pore volume accessible to the solute and ranges from zero to unity. However, one important problem is that the interpretation ofgel chromatograms and especially determination of the molecular weight the species separated needs a proper calibration curve. Since there are many solute-solvent systems involvedin GPC as well as many types of different pore sizes, the best way to obtain an accurate calibration is by experiment rather than by theoretical calculations. Errors in the calibration methods are minimized by obtaining a calibration curve under the same conditions as the sample separation. The calibration curve for a set of columns can be established experimentally by relating the peak elution volume to molecular weight for a seriesof narrow molecularweight distribution (MWD) standards. This is co-called direct calibration method with narrow MWD standards, The accuracy of this method depends on whether the reference compounds and analyzed sample all have a similar conformation and chemical structure. Gel chromatogram determination of each standard compound, plotting of the calibration curve, and their comparison represent the main steps of the GPC calibration by the direct method. The main disadvantage the direct calibration method is the lack of narrow standards. Polystyrene and a few other polymers are theonly polymers for which a series commercial standards of different molecular weight and narrow MWD is available. The calibration curve may be represented by the following equation

276

Chapter 4

where V,, is retention volume and A,B are constants. In the case of oligomers, the direct calibration method was applied using as standards well-defined substances with known molecular weight. Alkanes have been considered as potential standards in the calibration of GPC separation of oligomers. The absence of solute-solvent interactions and molecular associationssupport this idea. Universal calibration proceduresrepresent a tentative calibration associated with the determination of a rigorous way to transform the gel chromatogram into a true molecular weight distribution of the analyzed sample. The Q factor [68,69] and hydrodynamic volume [70,71] are the major parameters for universal calibration procedures. The Q factor is the ratio ofthe molecular weightto the extended chain length calculated from bond lengths and valenceangles. Its application assumes a very extended molecule along with the existence of a certain dependence between the "length of the molecular weight" and VEL.This typical length, expressed in angstroms ), yields the molecular weight of a linear polymer by means of an appropriatelinearity factor Q:

(A

M = QA

(28)

In this case, the universal calibration curve is based on therelationship VEL

=f(h3

A)

(29)

This dependenceis subsequently consideredto be independent of the nature and composition of the solvent-nonsolvent system. Applicable only to rigid polymers, the Q factor led to erroneous results in the fieldofflexible macromolecules [72]. The coil conformation adopted by macromolecules in solution suggested the idea of utilizingthe hydrodynamic volume asa universal calibration parameter in GPC. The universal calibration procedure utilizes the concept of the hydrodynamic volume of the solute molecule [73,74]. Hydrodynamic size of macromolecule R

=

(1M[rll)*"

is a universal characteristic of all macromolecules and solvents uniquely determining the retention time of macromolecules in a chromatographic experiment. The opinion of Russian scientists Vilenchik et al. [75] is that the all attempts to choose another universal characteristic have failed just because only the R value takes into account both the average size of the macromolecules and the density of the macromolecular coil as well as its partial draining determined by the value of the hydrodynamic interaction

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between the segments. It is precisely thesefactors that determine the behavior of macromolecules in the GPC process. In general, GPC calibration curves for solutesofdifferenttypesmerge into a singleplotwhen the calibration data are plotted as log [VIMvs elution volume instead of the usual logMvs. elution volume. The universal calibration method is conceptuallysound, but its use is still rather limited. However, with increased use GPC of viscometric detectors, therehasbeen a great deal of interest regarding the validity and application of universal calibration [76-841. Sanayei and O’Driscoll established universal calibration for polystyrene and poly(methy1 methacrylate) standards. Intrinsic viscosity of the oligomers of these polymers were estimated by extrapolation of the MarkHouwink equation, and these data were used to extend the universal calibration to the low molecular weight region. Using chromatographic analysis and viscometric investigations, it has been shown by Vilenchik et al. [75] that the linear relationship of the log [V]and log M of flexible chain macromolecules exhibits a break on passing from the oligomeric to the polymeric range. Simultaneously, the Huggins constant also changes [86]. These changes result from a decrease in the draining of macromolecules with increasing number of statistical segments. In particular, draining affects the hydrodynamic dimensions determiningthe GPC behavior of macromolecules. In themolecular weight range lower than l o 4 daltons, the Gauss statistic is no longer applicable [87-911. Actually, it is known that a viscosity theory, basedon the fact that the solute has molecular dimensions considerably higher than those of the solvent, cannot represent a valuable tool of viscosity determination for systems of particles having comparable dimensions. This has been observed since1958, when negative values of intrinsic viscosities werefound with certain oligomeric solutions[92]. Also with the same occasion, attention has been drawn [92] to the fact that, in the absence of irreversible adsorption phenomena, universal calibration may be employedwithsatisfactoryresults,even in the caseoflowmolecular weights, if the solutes do not have aromatic nuclei in its structure. Most failures of universal calibration are normally due to the failure of the size exclusion mechanism [93-971. In explaining the separation in GPC itis usually assumedthat thermodynamic equilibrium existsin solute distribution between the mobile and the gel phases in the column. On the other hand, the solute separation in GPC is mainly a result of steric exclusion from the gel pores. In fact the pure “size effect” is rarely encountered and thesolute separation is affected by other secondary mechanisms. These stem mainly from molecular interactions among solute, solvent, and gel in the column. Since 1981, Berek pointed out that both secondary effects,

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adsorption, and thermodynamic partition of the solute, usually represent only additional effects on thepredominating mechanism of steric exclusion [98]. However, theseeffects have to be taken into account, e.g., by employing the universal calibration concept for polymers in eluents with different thermodynamic quality and adsorptionstrength of the gel [99-1051. Under certain circumstances, the secondary effects may predominate over the basic mechanism of steric exclusion, as, e.g., in the case of the total retardationof the solute in the column as a consequence ofits adsorption on the gel. Anomalies in the application of the universal calibration method have also been noted [ 106,1071. Dubin and Principi [ 1081 have proposed that deviation from [q]-M correlations are only correct at infinite dilution. This approach appears to be marginally superior to the straightforward [TIM calibration for polymers havingdifferent conformations. The observed anomalies with universal calibration are of three types [109]: The unexpectedly large elution volumes determined by irreversible adsorption The deviation specific to systems oligomer-poor solvents The elution order in the domain of small molecules or oligomers not being governed only by the size of the solute particle GPC data in the domain ofoligomers and smallmoleculeshave pointed out the extra mechanisms involved inseparation [ 110-1 161. Besides the size exclusioneffect, in the GPC of oligomers, these secondary mechanisms are the solvation of solute species, the adsorption on thepore walls of the packing, and, for swelling packings, the partition between the gel and the whole phase. Tsitsilianis et al. [ 1171 tried to overcome the above problems by proposing another universal calibration procedure which can be applied to the low-molecular weight region.In order to obtain amore accurate value ofM at low molecular weights from the intrinsic viscosity, the following relation between [ T ] and M has been proposed[ 1181: [7)]-1

=

+ AIM-

(31)

where A, and are constants for a polymer-solvent system at a given temperature. This empirical linear relation for [v] versus M”” provides excellent fits of data in the molecular weight range 1,000-100,OOO[118, 1191. We now turn to theso-called universal calibration curve suggested by Benoit which is representedby the equation [71]

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where [v]is the intrinsic viscosity of the polymer. Eq. is valid for agreat numberof polymers withdifferent chemical composition and molecular structures. In aparticular chromatographic tem of a given set of columns with temperature and solvent specified, all the polymers with a given VELhave the same hydrodynamic volume[?]M, and consequently we may write

where the subscriptsp and S refer, respectively, to the polymer to be characterized and to a standardsample of another polymer. Obtaining [v]from thewell-known Mark-Houwink-Sakurada (MHS) empirical relation for bothpolymers

and rearranging we obtain [

K, log M, = -log K,

+

+ ++ a, log M,

Eq. can be used to obtain the calibrationcurve for the polymer p from the calibration curve ofstandards of another polymer if the MHS constants for bothpolymers are known [ Substituting [v]from eq. for thepolymer p and standardS into eq. and rearranging results in the following relation:

where

where AI,, Air, A,, and A, are the constants of eq. for the polymer standard andpolymer under analysis, respectively. Knowingthe calibration curve logM, = f(V,,) of the polymer standard it is possible, using eq. to obtain the molecular weight of the polymer p which corresponds to the molecular weight M, of the standard giving the same retention volume VEL.

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A. Calibration with Broad Molecular Weight Distribution Standards

Since narrow-molecular weight distribution standards are available only for polystyrene, broad-MWD polymer standards areused for GPCcalibration. There are two different ways of using these standards, the integral MWD method and the linear calibration method. The first method utilizes the complete MWD curve of polymer standards and the second method uses only the average molecularweight values of polymerstandards butassumes a linear approximation of the GPC calibration curve. Both methods are valid and useful at times, dependingon GPCconditions. The accuracy of the broad-standards calibrationmethods varies, depending on the accuracy of the available MWD information on the standards and theaccuracy ofthe linear-calibration approximation [121-1271. When the separation columns are purposely selected to assure linearity in calibration, theuse of linear calibration methods is definitely recommended overintegral MWD methods. Unlike those narrow-standards methods, the calibration curves obtained by broad-standards methods are affected by instrumental peak broadening. Without corrections, this calibration errorcan cause errors in molecular weight analyses of polymer samples. The integral MWD calibration method requires that the complete MWD of the broadpolymer sample intendedfor a calibration standard was fractionated by a classical method and themolecular weight determined for each of the fractions. The MWD information obtained in this way for the whole polymer, now the calibration standard, was then used to establish the GPC calibrationcurve. This experimental polymer fractionation approach has not generally been followed in practice because of the tedious nature and the questionable molecular weight precision of the experimental fractionation and the molecular weightsof the characterized fractions. A practical alternative approach suggested by Weiss[ 128 makes use of theoretical polymer MWD and average molecular weight values to provide the needed MWD information for theintegral-MWD calibration. This approach has been applied to both water-soluble polymers [l291 and organic soluble polymers[ 1301. Using this calibrationmethod, Bezdadea and Uglea [l221 analyzed poly(viny1 acetate) obtained by matrix polymerization. With this occasion, the formation of “replica” type macromolecules and “inclusion” type oligomers was evidenced. The presumed advantage of the integral method is that it makes no assumptions regarding the calibration curve shape and thus permits an accurate search for nonlinear calibration curves. However, this advantage

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often does not occur in practice, owing to the rather poor precision of the method. The linear calibration method using broad polymer standards are proposed by Hamielec [ 13 l] and developed by Yau [ 1321 and others [ 1331351. The Hamielec method [ 1311 consists of a search for an “effective” linear calibration and offers truly a practical way of obtaining GPC calibration curves that are specific to polymer type. The method needs only one broad-MWD standard of the same structure as the unknown samples. This can usually be providedeither by commercial standards or by converting of the samples into a working standard through independent determinations of its values of 2,and M,, using, for example, lightscattering and osmotic pressure techniques. Although a single broad standardis often used, the Hamielec method can be also used with two different molecular weightstandards, as long as there are two average molecular weight values known.The precision of the method increases with the difference between the two molecular weight values usedin calibration. In addition todependence on theaccuracy of the linear calibration approximation and on the experimental M, and M,,values of the standard, the Hamielec method has two other weaknesses: the physical significance of the effective calibration curve is not defined, and the calculated molecularweight values are accurate only for samples having a GPC elution curve similarto thatof the standard. In an improved version of the Hamielec method, GPC V2 [ 1321,compensation is provided for the symmetrical peak broadening caused by columndispersion effects. A more sophisticated versionof the Hamielec method, GPC V3 [135],includes a consideration of the skewness of the GPC column dispersion. In contrast to the integral MWD method, linear calibration methods pose no restrictions on the MWD shape of the standard. In practice, linear calibration methods provide a much better compromise than does the integral MWD method, because the linearity of the GPC calibration curve can be improved by the proper selection of GPC columns. For GPC columns with nonlinear calibration curves, a modified broad standardmethod must be used for which the linear calibration approximation is not assumed. In the last fewyearsmany attempts havebeenmadein order to develop new calibration methods using broad-MWDstandards [ 136-1461.

V. BANDBROADENING In spite of the efforts dedicated to the explanation of the GPC behavior of low molecular and macromolecular substances, there are still unanswered

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questions. One of themis the explanation of the coexistence of tendency of separating species when a mixture passes through the column, and that of broadening the peak, when the analyzed sample contains one molecular species. In column chromatography, a small volumeof the sample solution is injected to form a band at the top of the column. As this band migrates downstream, its width increases. Band broadening of a pure component can be used to measure the efficiency of the chromatographic system. The nomenclature used in reporting chromatographic band broadening in the literature is quite varied and sometimesconfusing.Readers should be aware of the many near synonyms for band broadening that appear in the literature, such as peak broadening, zone spreading, and instrumental, axial, longitudinal, or column dispersion. All forms of band broadening are detrimental to chromatographic separation. Basically, chromatographic separation is a demixing phenomenon and band broadening is a back-mixing or remixing phenomenon. Excessive peak broadening can result from poorly packed columns and can also result from excessive extracolumn volume that is present in the chromatographic instrument. An understanding of the basic column dispersion processes in wellplanned GPC equipment using a well-packed GPC column is needed for making efficient GPC separations with the best compromises among time, accuracy, effort, andconvenience. There are three fundamental processes leading to band broadening in GPC separation. The first is eddy diffusion that arisesbecausesample molecules take separateroutes through thepacked bed. The second contribution to band broadening occurs as a result of the resistance of solute to mobile phase masstransfer. This broadening process is caused bythe velocity gradient profile that exists in a single flow stream. Since liquid nearthe surface of the column packing particle moves relatively more slowly than the liquid at the center of the stream, solute molecules at the center migrate farther downstream than the others. Band broadening due to this dispersion process decreases with increasing lateral diffusion rate of the solute molecules between the fast- and the slow-moving liquid regions. At times this dispersion process is called “mobile-phase lateral diffusion” or “extra-particlemass transfer”. Longitudinal diffusion is the third basic band-broadening process in which the band is broadened along the column parallel to the flow direction by molecular diffusion of the solute. This form of band dispersion is important in gas chromatography but is insignificant in large-molecule GPC because of slowdiffusion. There are two ways of approaching the theoretical interpretation of

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chromatographic band broadening. In the kinetic or rate theory[ 1471 band broadening is explained in terms of realistic models involving molecular diffusion and flow mixing. In developing a rate theory, differential equations are derived to describe solute mass balance in a differential column section. The four classical, independent mass transfer process are considered in their partial differential form: longitudinal diffusion (D,&,/ax), eddy diffusion (DEaC,/ax), stationary mass transfer (D,dCJar) and mobile phase lateral diffusion (D,&3C,/ay),where is the column length variable along the column axis, y the distance variable in the lateral direction, and C, and CSthe solute concentrations in the mobile and the stationary phase, respectively. The quantity DEis the eddy-diffusion coefficient that is expectedto be proportional to dp(particle diameter of the packing) and v (flow velocity)[ 1471. A rate theory for GPCdispersion employsthe following partial differential equations. For stationary mass transfer (permeation)

where r is the radial distance from the center of the spherical porous particle. For mobile-phase masstransfer,

ac,

ac,

-++"DD,,~= at ax

a",

-uDsM

(x) acS r dp/2 =

+

where U = 6(1 - +)/dp+, with being the volume fraction of the extra particle solvent volume.For boundary conditions, at r = dJ2,

In these expressions, coupling of eddy diffusion to mobile-phase transfer and longitudinal diffusion effects are neglected.These assumptions are appropriate for GPC,since overall mobile-phase dispersion is usually small compared to permeation. Although the complete solution of these differential equations (eqs. 37 and 38) under the specific boundary conditions is not known, an approximate solution can be obtained for a limiting case where near-equilibrium solute distribution exists betweenthe phases. This limiting condition closely approximates most GPC experiments and predicts a near-Gaussian elution peak shape. The second theory on band broadening, which is a simplified, phenomenological approach, is the plate theory [ 1481. It explains band broadening by random fluctuations around the mean retention volume bya simulated partitioning model in a chromatographic column. The plate theory

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was first applied to liquid chromatographic studies by Martin and Synge [ 148 Because of its simplicity, the plate theorywill continue to be a useful, general model for studying chromatographic band broadening. The basic derivation of the general plate theory can be found in many GC and LC books [ 149,1501, and only a brief explanation is given below. In the plate model the chromatographic column is pictured as being divided into N number of adjoining separation zones, with each zone having such a length that there can be complete equilibrium the solute betwen the mobile and the stationary phases within the zone. Each zone is called a “theoretical plate,” and its length in the column is called “high equivalent to a theoretical plate,” HEPT, orsimply the “plate length,”H. To illustrate the plate concept, a primitive five-plate column( N = 5) is shown in Fig. 2, where the sequence the plates is indexed bythe serial number r. The figure shows values between q and p , which are thefractions of the total solute in mobile and stationary phases, respectively, with q p = 1. In this picture the flow of the carrier liquid is simulated by the sequential displacement ofthe entire top mobile phase sectionto the right, one plate at a time. The number of timesthat this volume displacement has taken place followingthe introduction of the sharp band into the first place is designated bythe index number n. With each volume displacement, only a fraction q of the solute in each plateis carried to the next plate, leavinga fraction p behind. The solute in each plate reequilibrates inthe new situation, and the displacement process repeats. This repetitive partition process leads to a solute distribution among many neighboring plates that follows the binomial distribution function. According to binomial statistics, the fraction of the original solute being in the rth plate following n displacements is

+

=

n! 4%”” r!(n- r)!

liquid

stationary

phase

0

1

2

4

Plate

Figure 2 Hypotheticalcolumn 151.)

fivetheoreticalplates.(AdaptedfromRef.

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In chromatography the solute concentration detector monitors the fraction q of the Nth (last) plate as a function of n. The elution curve is therefore described by q W(n,N),where n is proportional to elution volume. For the usual large number of plates in chromatographic columns ( N > 50),

the binomial solute distribution becomes indistinguishablefrom the Gaussian distribution curve [ 1511. With algebraic transformation, the Gaussian peak elution profile as predicted by the plate model can be expressed in terms ofthe experimental quantities of concentration (C), retention volume (V), peak retention volume ( V,), sample weight W , and p , the fraction of solute in the stationary phase:

By comparing eq. 41 with the general Gaussianfunction

where is the area of the peak, the standard deviation of the Gaussian peak in retention volume units, and h, V, and V, are as defined in the following equations:

i

where is the standarddeviation of a general statistical distribution, Vand the retention volume variables, V, the retention volume ofthe chromatographic peak, and the subscript i is the sequence index for the discrete equally spaceddata points used in the variance calculation:

p approaches unity

Chapter 4

286

Other results ofthe general plate theoryare

where L is column length. In summary, the predictions resulting from the general plate theory are 1. Gaussian peak shape (eq. 41). 2. Peak width increases linearly with retention volume (eq. 46).

Each peak in a chromatogram has approximately the same value of N and H (eq. 46). 4. Nincreases linearly with column length (eq.47). Items 2 and of these predictionsare notobserved inGPC. The GPC columns has many unique features. Whilethe volume of the solvent inside the porous packing does not affect solute selectivity in otherLC methods, it in fact serves as the stationary phase in GPC, in the sense that it causes the differential elution of solutes. Accordingly, while this liquid volume is described as thestagnant mobile phasein general LC discussions, it is called the stationary phase in GPC. This subtle difference in basic concept has caused much confusionand many inconsistencies betweenGPC and general LC terminology. Thus a classification of band broadening terminology is presented hereprior to the discussion ofthe GPC band broadening mechanism. The meaning of the phrase “stationary phase masstransfer” is different when used in GPC versus general LC discussions. Historically, all LC dispersion processes were considered as being independent of each other. Although the magnitude and the relative importance of each plate height contribution from various dispersion mechanisms vary from one form of LC to another, the overall plate height is given by

where HL, HsM, H,, and HMare the plate height contributionsdue to longitudinal-diffusion, stationary-mobile-phase, stationary phase, and interparticle-mobile phase transfer processes, respectively. The phrase “stationary phase mass transfer’’ means the H,,,,, term in GPC, but.the Hs term in other LC methods. In a classical sense the LC stationary term H, defines the dispersion effect of a distinct, separate LC stationary phase, but does not at all apply to GPC separations involving nonsorptive packings. In GPC the concern is the HsMterm, called the stagnant-mobile phase dispersion in LC discussions. Since the phrase “stagnant mobile phase” is somewhat

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confusing in GPCdiscussions, HSM was called simply the “stationary phase” by Yau [ 1521. With regardto band broadening in GPC, the plate height contribution due to longitudinal diffusion HL is insignificantly small because the large solute molecules commonly encounteredin GPC have very small diffusion coefficients. With HLand H, dropped from eq. 48, we have for GPC

H = HSM

+ HM

(49)

Besides the difficulties encountered when utilizing GPC for theseparation of small moleculesor oligomers, this method also has some intrinsic disadvantages, compared to theother chromatographic methods. Thus, mention must be made, among others, of the low value of peak capacity, n. This term is defined as the maximum number of peaks that can be resolved within a specified range of elution volume. In turn, the V, of a species is limited to the accessiblevolumeof the pores for the givenspecies. pointed out by Giddings[ 1531, GPC is uniquein that there isa well-defined limit to peak capacity. The concept of the theoretical plate is usedto compare the separation efficiency of columns. pointed out by Cazes[l541 The theoretical plate concept is borrowed from that area of chemical engineering involving fractional distillation. A theoretical plate, in the caseof distillation, refers to a discrete distillation stage constituting a simple distillation in which complete equilibrium is established between the liquid and vapor phases. In the caseGPC of where the two phasesare in constant motion, i.e., the solvent in the interstitial volume and the solvent within the gel pores, equilibrium is probably never achieved. The true significanceof the theoretical plate is lost. It must be realized, moreover, that the calculated theoretical plate in a chromatographic column represents a smaller separating ability than the theoretical plate in a distillation columnby a factorof twenty-fiveto fifty. Then, the determination of N in GPC is more a measure of how well a column is packed,that is, how much peak spreading it will cause, than how well it will resolve. Moreover,the method of determinationof N in the case of GPC is questionable. Whena solute of low molecular weight is used and its VELvalue, introduced in the relation N = 16

($)

2

where W represents the peak base width, is determined, the value of N thus obtained is meaningless, if referring to a GPC column, due to the fact that the gel is wholly accessible to the reference sample (employed for the determination of N) and partially accessible to the species forming the

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mixture subjectedto analysis. It is obviousthat in the case of an oligomeric or macromolecular mixture to be passed through the GPC columns, the support will offer a higher number of theoretical plates for smaller molecular species than forthose of higher molecular weight [ In addition, severalapproximations,althoughheuristicallyuseful, tend to propagate an ideal view which is longer validand which tend to induce overconfidencein the potential ofGPC. The assumption ofa perfect linearity of calibration curve, the use of the term “Gaussian”to qualify the shape of the gel chromatogram (this adjective implies morethan thesimple notion of symmetry), and the poor definition of the baseline (especially in the case of oligomer characterization)due to uncertain limitsof integration and thesignal-to-noise ratio are examples of excessive idealization or inadequate interpretation of GPC data. Although lacking a suitable theoretical base for a precise method of estimating its results, the GPC remains one of the most frequently used methods in the characterization of oligomeric mixtures, especially for a qualitative appreciation ofthe mixture composition[

VI.

NON-SIZEEXCLUSION EFFECTS

Flow Rate Effect If the equilibrium assumption in GPC is valid, then the elution volume should be independent of flow rate. The majority of earlier workers concluded that elution volumes were independent of flowrate within the usual flow rate ranges [ However, a number of studies have shown that elution volumes can either increase or decrease with flow rate, depending upon the experimental conditions [ Some of these observations have been rationalized, but others remain unexplained. Some ofthe possible explanations for these observations of flow rate dependent elution volumes include nonequilibrium effects, molecular structure changes with flow rate, stationary-phase changes with flowrate, and concentration/flow rate effects. Nonequilibrium effects in GPC can occur in two ways. The first is when equilibrium is not maintained at the micropore mouth between the solution in the interstitial volume and the solution within the micropore. The second is when equilibrium is maintained at the micropore mouth but the solution in the interior of the micropore is not at equilibrium with the solution in the interstitial volume. In this case diffusion of the solute into the micropore could beimportant [ At low flow ratesthe elution curve is Gaussian in shape, and therefore symmetrical, and the peak position is equalto the first moment. At higher flow rates, however, the elution curve can become skewed whenthe diffu-

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sion rate into micropores is sufficiently slow. Ifthe first moment is independent of flow rate, as predicted by Herman's theory, and the elution curve becomes more skewed with increasing flowrate, then the peak position of the elution curve should decrease as the flow rate increases. Hence, when observing flow rate dependent elution volumes, one must be careful to measure the position ofthe first moment,rather than the peak position. Molecular Degradation. A number of experimenters have demonstrated that high molecular weight molecules can be degraded in a chromatographic column at high flow rates[ 164-1661. These studies have involved a number of different polymers and a number of different instruments. Molecular degradation with increasing flow rate results in an apparent increase in elution volumes with increasing flow rates. Configurational Changes. The flowfield through packedcolumnsbelongs to a class of flows referred to as kinematically nonhomogeneous, meaning that the strength of the velocity gradient variesfrom point to point within the flow field. Macromoleculesare subject to gross configurational distortion depending on the strength of the local velocity gradientthat they experience. Severalauthors [ 167-1721 have speculatedthat, in nonhomogeneous flows, the lowest velocity gradient regions of the flow can become enriched in polymer concentration asthe macromolecules attempt to minimize configurationalentropy by escapingthe elongating effects the high velocity gradient regions.The quantitative aspects of this effect exist [ 1691, although many experiments indirectly support the idea. In any nonhomogeneous flow, macromolecules do not move with the local solvent velocity, leading againto the possibility ofa nonuniform spatial concentration distribution [ 1681. Purely hydrodynamic arguments are sufficient to show this, with no ad hoc thermodynamic assumptions as above. Cross-streamline migration is predicted towardthe concave side when the local velocity field is curvilinear and nonhomogenous. Clearly, the opportunity for both these modes of migration, thermodynamic and hydrodynamic, exists in principle in the flow through chromatographic columns. Maximum velocity gradient regions, of magnitude 100 to 1,OOO sec" under reasonable operating conditions, exist in close proximity to stagnant, low velocity gradient regions. Macromolecules travel curved paths in circumnavigating the packing particles. Thus it is likelythat nonuniform concentration profiles may arise from thefluid mechanicsin chromatographic columns. When macromoleculesare subject to large rates ofdeformation, their configuration can change by stretching and aligning with the flow streamlines [ 1701. Such a situation can occur at sufficiently high flow rates in a

290

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chromatographic column. Some workers have suggested that this could change the partition coefficient of the macromolecule and,. therefore, its elution volume could change with flow rate. These workers have concluded, however, that this effectis of no significance in GPC [ 171,1721. Flow Rate. A possibility exists that flow rate dependent elution volumes could arise from changes inthe pore volume or pore size with a change in flow rate and a corresponding change in the column’s pressure drop. Either of these effects would result in the elution volume decreasingas the flow rate increases. This isa possible mechanism of flow rate dependent elution volumes in columns packed with nonrigid packings [l731 although most authors have concluded that this is not a significant effect in the range of flow rates and pressure drops typically used in GPC [ 1741. Stationary Phase Changes

B. Concentration Effect Elution volumes have been observedto increase as thesolute concentration is increased [ 175-1771 and also as the volume injected is increased [ 1731. These observations suggest that, if equilibrium is maintained at the pore mouth, then the equilibrium partition coefficient must increase with increasingsoluteconcentration.Such an effecthasbeenexperimentally shown to be true for bothrigid and flexible macromolecules. Concentration effects, i.e., the dependence ofthe elution volumeand of the width of the elution curve on concentration and overall amount of injected polymer solution in GPC havebeenobserved in many works. Waters [ 1781 supposed the increase in elution volume with increasing concentration to be due to the higher viscosity ofthe injected solution. Boni et al. [179,180] observed that the change in elution volume with a change in concentration was a linear function of the logarithm of molecular weight or of intrinsic viscosity. In the latter case, they obtained a single linear dependence for variouspolymers.SimilarresultswereobtainedbyLambert [ 1811. The hypothesis of viscosity phenomena was supported by Goetze et al. [ 1821, who injecteda polymer solution ina solvent whose relative viscosity was higher than that of the solvent used asthe mobile phase. According to them, viscosity phenomena causea change in the elution volume, but the whole change cannot be assigned to these phenomena. Moore [l831 explained the viscosity phenomenaas “viscous fingering.” Ouanc [ 1841 stressed the effect of overloading of the column in the injection of solutions of mixtures ofstandard polymers having different molecular weights and high concentrations. Rudin [ 185-1871 showed that the effective hydrodynamic volume of macromolecules in solution decreased with increasing concentra-

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tion and that this effect mustbe taken intoaccount in constructing a universal calibration graph. The hypothesis concerningthe effect of concentration on the elution volume was supported also by other authors [188-1901, who observed that the effect of concentration on the elution volume in a thermodynamically poor solvent (under theta conditions, when the effective dimensions of the macromolecular coils do not vary with concentration) was weaker. However, this subject still remainsin debate taking into account the experimental data obtained by Dawkins [191-1931, Lecoutier [ 1941, and Garcia et al. [195]. When the organic packing material is crosslinked polystyrenegel and themobile phase is compatible with the polystyrene gel and with the polymer solute, the first condition being more important, solutes do not display preferential affinity or preferential adsorption for themobile or stationary phase. Hence,partition and adsorption mechanisms do not influence the solute size separation [191]. However, when the solute polymers are eluted in poor or thetasolvents for solute and gel (i.e., cyclohexane at with polystyrenesolute and polystyrene gel) solute-gel interactions become important and secondary mechanisms appear with the elution volumebeingdisplaced to highvalueswithrespect to universal calibration. When there is incompatibility between the solute polymer and cross-linked polystyrene gel, elution volumes are shifted toward smaller retention volumes [ 1941. The GPC behavior of synthetic polymers in good and theta solvents has been compared using polar inorganic packings (porous silica) [196, 1971. For these systems observed deviations with respect to the universal calibration are practically independent of the thermodynamic quality or compatibility of the eluent for thepolymeric solute or gel, respectively. The deviations appear when simultaneouslythe interactions between the solvent and gel and between solvent and polymer solute, are markedly modified by using a mixture of two solvents as eluent. Due to the preferential sorption (as a consequence of specificinteractions) of the gel by one of the components of the mixed eluent, a difference in the thermodynamic quality of the mobile and quasistationary phases appears and consequently, additional partition of the solute may occur [197]. Using the latter observations, it was suggested that the thermodynamic quality of the solvent should be estimated from concentration effects [99]. It was also observed that the mutual arrangement of the individual columns affected the concentration dependence of the elution volume [ 1981, and that, with increasing flow of the solvent, the concentration dependence of the elution volume decreased [ 199,2001. An increase in the width of the elution curve with increasingconcentration and volume of the injected polymer solution was observed by several authors [201-2031. Hazell et al. [204] assumed (but did not prove) an increase in concentration

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effects with decreasing efficiency of the column, Hellsing investigated the effect of concentration of the polymer present in the mobile phase on the elution volume of natural macromolecules. Bartick and Johnson outlined the possibility of using differential GPC in the study of concentration effects, while Bakos et al. utilized the same method in the study of incompatibility of various polymers and concentration effects under such conditions. Concentration effectswere interpreted by Belenkii and Vilenchik as a consequence of the osmotic pressure at the boundary of the mobile and stationary phase, leading to shrinkage of the gel in the eluting zoneand/or redistribution of macromolecules of various sizes in the polydisperse sample. Cantow et al. observed an exceptionally stronger effect of concentration with samples having a broad distribution compared to those with a narrow distribution. In a seriesof papers, Janca showed that the change in elution volume following a change in the concentration of injected solution in GPC is due to many contributing processes, i.e., the change in the effective size of permeating molecules, viscosity phenomena and secondary exclusion. The first two contributions lead to an increase in elution volumes, while the last, secondary exclusion, causes reduction in elution volumes with increasingconcentration. Song and coworkers presented a theory of concentration effects for polydisperse polymerson hydrodynamic volumereduction and viscosity phenomena. This model predicted the effects of concentration on elution volume, axialspreading, and peak skewing. Theseauthors also reported on a procedure for determining the second virial coefficient of polymersfrom concentration effects Moureyetal. studied the solution properties ofpoly [bis(trifluoroethoxy)phosphazene]and found thatconcentration-inducedchain compression was more severe in a good solvent as compared to a poor solvent. For eluents representing theta solvents for the given polymers, the concentration effect practically disappears; therefore the useof theta eluents is advantageous from this point It has been found that the concentration effects increase with both increasing relative molecular weight and decreasing polymolecularityof the polymer injected. They depend substantially also on the thermodynamic quality of the eluent Since the concentration effect complicates interpretation of GPC data,investigators usually try to eliminate or atleast minimize its influence. Berek et al. found that the magnitude of the concentration effect for polystyrene in some pure and mixed eluents on Si0,-based column material correlates with the product A,M, where is the osmotic second virial coefficient. This correlation was also supported by a theoretical analysis basedon the dependence ofconcentration induced shrinkage of polymer coils on the thermodynamic quality of the solvent

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293

[218]. These authors proposed a qualitative determination of from the concentration effect in GPC, as an alternative to the traditionalmethods of measurement by means of light scattering and osmometry. Ousalem and Busnel [223] discussedconcentration effects for several polymer typesand Hu andcoworkers [224-2261 published a series ofpapers on the concentration effects for polydisperse polymers and applied their theory to universal calibration [224], to radius of gyration calculations [225] and topolyisobutylene rubber characterization [226]. Cheng and Yan [227] studied the effect of polystyrene concentration on elution volume and were able to determine the critical concentration for coil shrinking. Their observations were in agreement with mean-field theory regarding the variation of polymer chain dimensions insolution. Czok and Guiochon [228] reported that viscous fingering is more pronounced with large-diameter columns that with microbore columns. Methods proposed to reduce this effect included increasingthe viscosity of the mobile phase to match that of the injected sample and following the injected sample witha large plug of eluent havinga viscosity slightly higher than thesample. Ye et al. [229] studiedthe effect of polymer concentration in vacancy GPC, a seldom used technique, in which the polymer solution is employed as the mobile phase withpure solvent injectedas the sample.

C. Solvent Effects Unlike other liquid chromatographic methods, the mobile phase in GPC usually is not varied to control resolution. Rather, the mobilephaseis limited to the solvents that can dissolve the sample macromolecule. If permitted, a solvent with lowviscosity at the temperature of separation is preferred to ensure high column plate count (due to improved mass transfer). To maintain high resolution, mobile phases that have boiling points only about 25-5OoC higher than the column temperature should be used. Of course, in the case of difficulty soluble samples, the solvent must be selected primarily to provide sufficient solubility, and viscosity considerations are then secondary. A particular mobile phase has also to be selected on the basis of its compatibility with the solute detector. Thus if a differential refractometer is to be used,the refractive index of the mobile phase should beas different as possible from that of the sample. Or the mobile phase must havea much lower absorption than thesolute at the wavelength of detection with a UV or IR photometric detector. An occasional problem in GPC is molecular association of the sample. For example, the behaviorofphenols and their derivatives during

Chapter 4

294

separation by GPC has been studied in order to explain some features of the mechanism of phenol-formaldehyderesin synthesis [230].The oligomer formed through phenol polycondensation with formic aldehyde, presents various structural forms. Calculations revealed that 13,203 linear isomeric structures are possible for a species made up of 10 phenolic units bound together in ortho or para positions by methylenic bridges [231]. On the other hand, resoles have molecular weight distrbution ranging between 18 dalton (themolecular weight of water) and dalton [232]. Very little is known about the conformation and existence molecular associations formed by phenol and its derivatives withtetrahydrofuran (THF). Hydroxymethylated derivatives of phenols dissolved in THF differ from mono- or polyphenols in that they also may form intramolecular hydrogen bonds in polar solvents. IR spectra of the solutions of these compounds in THF have shown that the peak characteristic the bond between the phenolic or alcoholic group and THF is found at 3280 and 3430 cm", respectively. Within the same field (3240 cm") there appears the bond characteristic of the intramolecular bond between the phenolic "OH group and the ortho-hydroxymethylene group. One has an adsorption in the same domain cm"). Consequently, the IR spectra of the omethylolphenol solution in THF cannot determine the nature the solutesolvent interactions. One should choose betweenthe two possiblestructures presented in Fig. 3. the intermolecular hydrogen bonds require the phenolic hydrogen, which has an acid character, conformation 1 (Fig. 3) has a reduced probability. Such a hypothesis is also supported by the behavior of this species during separation by GPC [230]. Further anomalies have been observed in the elution of styrene oligomers [233]. In a polar medium (e.g., dimethylformamide), the mixture of l-methyl-3-phenylindane and 1,3-diphenyl-l-butene (two dimers of styrene) behave like a monocomponent compound which, depending on the composition of the mixture, leave GPC columns at different values of VEL.

1

2

Figure 3 Two possible structures of hydrogen bonds in o-hydroxymethylated phenol-THF system.

hromatography Gel Permeation

295

All attempts to explain this behavior have, to date, been unsuccessful. However, controlled sample association can be advantageous for certain separations by GPC. The effect of the solvent on thevarious GPC packings must be considered. For example, the crosslinked polystyrenegels for GPC can tolerate a moderate range of organic solvents, but acetone, alcohols, and otherhighly polar solvents cannot be used. Aqueous systems outside the pH range of about 2-8 degrade siliceous packings. Strong bases, such as NaOH and tetramethylammonium hydroxide, should be avoided but organic amines (e.g., triethylamine) are well tolerated [234]. Salts ingeneral appear to hasten the degradation of silica packings,particuarly at temperatures above ambient, the effect of direct function of pH and ionic strength. The solvent that causes collapseof organic gels for GPC must be avoided. However, in GPC with certain very polar solvents such as DMF, a salt must often be added to the solvent to reduce solute adsorptive effects and maintain a constant ionic strength. However, large changes in salt concentration can cause an organic gel to collapse. Many commonly used solvents for GPC are listed in Table 1 together with most ofthe properties of interest [235].

D. Instrumental Anomalies Some instrumentalists have shown that apparent flow rate dependent elution volumes can result from instrumental anomalies, i.e., the performance of the instrument depending upon the flow rate of solvent through it. Two such anomalies were documented by Yau, Suchan, and Malone [236] on instruments that use a siphon for the purpose of monitoring the flow and elution volume. The first anomaly was due to the fact that solvent would continue to flow into the siphon as it was discharging. This anomaly results in apparent elution volumes appearing lower than they actually are and in apparent elution volumes decreasing with increasing flow rate. The second anomaly was due to the fact thatsolvent couldevaporate before the siphon discharged. At slow flow rates a significant portion of the solvent in the siphon could evaporate before the siphon discharged.This would also result in apparent elution volumes that were lowerthan the true elution volumes. However, since the evaporation effect decreases as the flow rate increases, the apparent elution volume would increase with increasing flow rate. When both of these anomalies are operative, the apparent elution volume increases with low flow rates and then decreases with high flow rates. A plot of apparent elution volume versus flow rate would show a maximum for a given molecular species. Yau, Suchan, and Malone demonstrated that these two anomalies were the major contribution their to observed flow rate dependent elution volumes.Withthese instrumental

Chapter4

296

Table

Properties of Solvents Commonly used in Gel Permeation Chromatography

Solvents Tetrahydrofuran 1,2,4-trichlorobenzene o-Dichlorobenzene Toluene N,N'-Dimethylformamide Methylene chloride Ethylene dichloride N-Methylpyrrolidone m-Cresol Benzene Dimethylsulfoxide Perchloroethylene oChloropheno1 Carbon tetrachloride Water Trifluoroethanol Chloroform Hexafluoro-isopropanol

Melting point ("C)

Boiling point ("C)

-65

66 213 180 110.6 153 40.1 84 202 202.8 80.1 189 121 175 76.8 100.0 73.6 61.7 58.2

17 - 19 -95 -61 -97 - 36 - 24 12 5.5 18 - 19 7 -23 0

-

-64 -34

uv cutoff (nm)

220 307 204 285 275 245 230 262 302 280 260 290

-

265

-

190 245

190

Refractive index at

1.4072 1.5717 1.5515 1.4969 1.4294 1.4237 1.4443 1.4700 1 S440 1 so11 1.4770 1.505 1.5473 1.4630 1.33 1.2910 1 .4457 1.2752

anomalies accounted for, Little et al. found no flow rate dependence of elution volumes on an instrument that was similar to that used by Yau, Suchan, and Malone Retentionof the solutebymechanisms other than sizeexclusion greatly complicatesthe process of obtaining sample molecular weight information from GPC data. Adsorption or "matrix" effects involvinga form of partition or adsorption can be superimposedon size exclusion, resulting in excessive retention that the desired relationship between retention volume and molecular size isnot obtained. Adsorption or matrix effects can often be minimized by utilizing the most polar mobile phase permitted by sample solubility.For example, water is an effective mobile phase with unmodified siliceous particles for some separations. At pH >4, the acidic Si-OH groups on silica are ionized and can function as ion-exchange sites. Aqueous phases containing buffers or salts can be used effectively to eliminate undesired ion-exchange interaction of certain solutes (e.g., proteins) with unmodified siliceous surfaces. Where permittedby the sampleproperties,working at pH 4. In some cases ionic interaction between the solute and the carboxylic groups in the matrix is strong that suppression of the ionization of the carboxylic groups is necessary. Other mechanisms of interactions between organic gels and aromatic and heterocyclic solvents suggest involvement of the ?r-electron systemof the solute and an electron-deficient or electronegative portion of the gel matrix. Hydrogen bonding between substitutents on aromatic solutes, or the heteroatom of heterocycles solutes, and certain functionalities on the organic gels is also possible. Non-size exclusion effects in GPC and mobile phase optimizationwere reported for cellulose nitrate [238], lignin [239,240], polyamic acids [241,242], poly(acrylic acid)[243],poly(amideimide) [ M ] poly(viny1 , pyrrolidone) 12451, asphalts [246], polycyclic aromatics [247], and alkyd resins [248]. The theoretical interpretation of the behavior of small molecules or oligomers during separation through GPC are based, partially or totally, on the theories proposed for the GPC of high polymers. Disagreements, observed mainly in the attempt to establish a suitable parameter for GPC calibration, have been caused, in our opinion, by the fact that structural characteristics and solvent-solute interactions dominate over the separation mechanism of exclusion. This observation is supported by early experimental data thatevidenced the very special behaviorof aromatic hydrocarbons [249-2571, as well as the influence of functional end groups on the GPC separation of oligomers[258-2651.

VII. ASPECTS OF GPC ANALYSIS OF COPOLYMERS Because the molecular size of dissolved molecules varies with composition and structurein the case of block and random copolymers, there has beena problem in establishing accurate molecular weight calibration for polymer or oligomer systems of varyingcomposition and for systems where composition is a function of molecular weight. In practice the use of GPC with selective detectors [266-2711 and the application of interactive liquid chromatographic modes are the procedures applied for measuring compositional heterogeneity in these types of polymers [272-2741.

298

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The fundamental existenceofso-calledcriticalconditionsiswellknown in polymer chemistry and in chromatography [275-2821. In these conditions the entropy losses of a macromolecule within a pore and the enthalpic effects due to adsorption of the chain units on thepore walls are exactly compensated. Underthe critical conditions, when passing from the solvent volumeinto thepores, the free energy changefor a macromolecule is found to be zero (the distribution coefficient K = 1). The first experimental works in connection with this subject were published by Tennikov and coworkers [283,284] and subsequently the results were confirmed for different polymersand various solvents [285,286]and a theoretical foundation was elucidated [287-3001. The Russian scientists use the expression that macromolecules become “invisible,” in order to emphasize that, under critical conditions, the retention volumes of homopolymer macromolecules of any molecular weight become equal to the retention volume of solvent molecules, in sucha way as to make them indistinguishible from each other and from the solvent. The idea of chromatographic “invisibility” has been used [288] with the aim of building up a theory of a two-block copolymer separation according to the lengths of one “visible” block only, provided that the copolymer was exposed underthe conditions criticalfor theother “invisible” block. The method of “invisible” is, in a way, analogous to the well-known procedure [289] of copolymer analysisby means of light scattering, where a solvent is chosen that has a refractive index identical withthat of one of the copolymer components. This results in the optical invisibility of this component and makes the measurement of the radius of gyration of the visible component possible. Critical conditions can be created experimentally by means of particular choices of mixed eluenttemperature or pH (when aqueous eluents were used) variations, etc. The results of this method of “invisible” providean efficient separation of macrocycles and linear polymers and theseparation of macromolecules accordingto the number of functional groups. The method of “invisibles” also makes it possibleto separate two-block copolymers accordingto the size of the “visible” block only, and to separate grafted polymers according to the backbone length [291-2941. The method of “invisible” in polymer chromatography is exactly rigorous for a number of polymer systems that contain a single continuous “visible” chain fragment. In particular, monofunctional macromolecules, two-block copolymers, three-block copolymers with the central block being investigated, partially cyclic macromolecules, and graft copolymers with “invisible” side-chains belongto such systems.The most convenient realiza-

hromatography Gel Permeation

299

tion of the method of “invisibles” in such systems would beobtained using wide-pore adsorbent. For other systems, e.g., copolymers with several “visible” fragments jointed together with “invisible” inserts, the method of “invisibles” becomes rigorous whenusing narrow-pore adsorbent only, the pore sizesbeing smaller than those of all the chain elements under critical conditions. For the realization of the method of “invisibles” in such systems, itis important to have narrow-pore adsorbents, with a uniform pore surface, and also to be able to select conditions that are both critical for one of the copolymer components and at the same time do not cause strong adsorption of the other “visible” component. This can be achieved by choosing a suitable eluent, variations of temperature and adsorbent surface modification. The theoretically predicted efficiency of the method of “invisible”for functionally active macromolecules has been demonstrated experimentally in detail for a large number of polymer systems,and areview waspublished by Entelis et al. [290]. Some other predictions of the theory, in particular the possibility of the application of the “invisibility”method to the analysis of both block and graftpolymers and to theseparation of linear and cyclic macromolecules, havenot received experimentalcorroboration to date. Molecular weight-sensitive and information-rich LC detectors have beenextremelyvaluable for characterizing complexpolymers. GarciaRubio [301,302]used simulation studies to investigate joint molecular weight-chemical composition distribution and secondary fractionation effects of linear copolymers. Compositional heterogeneity of methyl methacrylate-styrene copolymers was determined by using 3-phenylazobenzoyl peroxide as a initiator and labeling agent[303J . The use of a photoiodide array detector with GPC was described by Del Rios [304]. UV Spectrophotometric detection is the most widely used approach for determining chemical heterogeneity of complex polymers. UV detection was used to determine compositional heterogeneity of wood components [ poly(ethy1ene terephthalate)-poly(tetramethy1ene ether) multiblock copolymers [3061, and styrene-butadiene-styreneblock copolymers [3071. Using GPC coupled to UV and IR detectors Verenich et al. [308] determined the compositional heterogeneity of oliogmeric epoxy propiolates. On-line IR was used to determine functional group distribution of poly(dimethylsi1oxane)s [3091. Kilz [310,311] useda four detector system consistingof a multi-angle laser-light-scattering photometer, refractometer, UV detector, and an online viscometer for the characterization of block and statistical copolymers as well as for star- andcomb-shaped polymers. Grubisic-Gallot etal. [312]

300

Chapter 4

also used a fourdetector system for thecomplete characterization of several different types of block copolymers.In their system, a low-angle laser-lightscattering photometer was employed. Trothnigg [313,314] used an on-line density detector in series with a refractometer to characterize the composition ofpoly(ethy1eneglycol)poly(propy1ene glycol) blendsand copolymers of ethylene oxideand tetrahydrofuran. Mork and Priddy [315]determined the extentofphenolic end-capping in bisphenol A polycarbonates using GPC-UVdetection. Warner et al. [316] used GPC-UV detectionto determine the amount and location of functional groups in styrenic polymers. Pasch and coworkers [317] relied on UV detection for the analysis of polymers containing UV stabilizer units. UV-GPC was used in the characterization of polystyrene-poly(2,4dimethyl-l,Cphenylene ether) blends [3 181, and polyester-polycarbonate blends [3191. Podzinek et al. [320] used GPC-UVfor determining the composition of polyesters. The compositional heterogeneity of nonstoichiometric polyelectrolyte complexes between polymethacrylate and poly(N-ethyl-4-vinylpyridinium bromide) was investigated by Efremov et al. [321] using GPCUV. Styrene propylene oxide block copolymers [322]and linear and cyclic siloxane copolymers [323]also are characterized using this technique. An on-line interface for GPC/FT-IR to remove high-boiling mobile phases for subsequent FT-IR detection was described by Dekmezian and Morioka [324]. GPC/NMR was employed to characterize isotactic poly(methyl methacrylate) [325] and block and random copolymers of methyl and butyl methacrylates [326]. The use of multiple modes of liquid chromatography to determine the chemical composition of distribution ofcomplexpolymershasreceived considerable attention during the last ten years. Investigators are finding it convenient to first separate by molecular size, followed by adsorption chromatography. This approach has been used by Mori [327-3301, Glockner[331-3351, and others [336,337] for the characterization of various copolymers. Also, Jiang et al. [338] first fractionated copolymers blends by precipitation followed byGPC. Techniques involving the coupling of GPC columns to a reversedphase system have been reported for theseparation of proteins [339], glycoproteins [340], and additives in cellulose acetate [341]. A GPC/capillary electrophoresis apparatus has beenreported for thecharacterization of proteins [3421, and seeRefs.343-346 for other studies dealing with multiHPLC approaches for protein separation. An interesting multidimensional technique was reported by Cortes et al. [347], in which a GPC microcolumn system was coupledto a pyrolisis GC apparatus for the characterization of acrylonitrile-styrene copolymer.

Gel

301

Fujimoto andcoworkers [3481 described a technique utilizing microcolumn GPC followed by thin-layerchromatography (TLC). Detection was accomplished on the TLC plates withFT-IR. Orthogonal chromatography has also been used for the separation of complex polymers. With this approach, the sample is first separated by GPC in a given mobile phase. Through the use of a switching valve, the separated components are then eluted through a second column utilizinga poor solvent asthe mobile phasethat will either change the conformationof the polymer or encourage interaction with the packing [349-3511. Finally, Palladino andCohen [352] useda photoiodide array detector with GPC to measure secondorder derivative spectra of polypeptides. By comparison of these spectra to those of known amino acids and heterodipeptides, identification of the composition of unknown peptides was possible.

VIII. SELECTED APPLICATIONS Although lacking a suitable theoretical base for a precise method of estimating its results,the GPC remains one of the most frequently used methods in the characterization of macromolecules [353-3631. This section coversselected applications of GPC in the field of naturaland synthetic oligomers and polymers. In polymer dope dyeing, the dyestuff is introduced into thepolymerization system during synthesis. For this purpose, either reactive or inert dyes may be used [364-3661. In the first case, the dyestuff becomespart of the main chain by combination with suitable functional groups. In the second case, the dyestuff isdissolvedin a polymermoltenmass and a physical blend of macromolecular product and dyestuff is obtained. This process is applied in industry for poly(ethy1ene terephthalate)-PET-dope dyeing. The difference between dope-dyed polyesterand colorless polyester obtained under the same experimental conditions is shown by a lower intrinsic viscosity and higher amount of “COOH end groups and diethylene glycol (DEG) units. Efforst have been made to avoid these disadvantages by shortening the contact time between polymer and dyestuff during polycondensation. The results were not successful and polyester fiber and yarn producers now accept these specialfeatures of dope dyeing because of the economic characteristics of the dyed polymer obtained. The gel chromatograms of dope-dyed PET show the systematic influenceof dyestuff on the value and distribution of molecular weight in dope-dyed PET. In an experiment performed byus [367] polymer was obtained by batch reaction in a laboratoryplant. The polycondensation was completed in 270 min. To be able to follow the oligomer transformation during polycondensation, the process was interrupted after 90, 120, and 210 min. The

302

Chapter 4

samples obtained in this way will be called“interrupted samples.” Thus, we obtained 12 interrupted samples and 3 end products (items 1and 2 in Table 2 for colorless polymer, items6 to 10 for blue polymer, and items 10 to 15 for red-colored polymer). The dyestuff (1070 by weight for dimethylterephthalate, DMT) was introduced at the beginning of the polycondensation stage. The chemical and molecular characteristics of the resulting products are given in Table 2. The molecular weight distribution in the synthetized sampleswas determined by GPC. Theelution was carried out at 100°C by using a mixture of nitrobenzene-tetrachloroethane(NB-TCE) (5:95,v/v). The samples were dissolved in pheno1:TCE (3:2). Before use, TCE was neutralized and stabilized as follows: freshly distilled TCE was treated twice with 5% aqueous Table 2 SampleCharacteristics data

GPC

No.

-

dyestuff

M,’

data Analytical COOH [SI g-106) (dl/g)

Dmc (Vo)

1 77.0 0.075 3.45 1,010 0.205 2 22.0 1.29 4,270 0.293 1.96 3 CSd 35.0 6,750 0.611 1.72 4 49.6 16,500 0.740 1.09 5 57.0 21,000 0.080 6 1,400 200.0 6.40 2,800 0.120 1.50 7 0.105 168.0 1,950 0.180 8 ERLS‘ 98.0 3,700 2.55 96.09,6000.225 4,2700.210 2.50 9 . 4,550 10 78.0 38,500 16,500 0.6102.60 17,000 0.620 11 0.082 5.45 1,150 137.9 12 0.085 1.40 1,550 120.0 13 ESGFP‘ 0.255 3.00 47.0 5,200 0.365 3.01 14 58.2 11,OOO 58.7 0.545 3.20 15 17,200

(dl/g)

M,

-

-

0.080 0.140 0.240 0.690 0.760 0.110

1,480 4,000 6,800 13,400 24,700 2,300

2,200 7,500 10,300 20,100 58,300 3,100

0.140

3,700

4,600

0.75 0.71

21,500 19,500

69,000 55,000

[SI

Industrial samples

I I1

22,000 19,800

28.0 68.6

0.760 0.725

0.55 1.66

‘Determined by titration of end groups; bdetermined by potentiometric titration; ‘determined by gas chromatography; dcolorless samples;Tstofil Blue RLS (produced by Sandoz); ‘Estofil Red SGFP (produced bySandoz). Source: Ref. 367.

Gel

303

KzC03solution (10 m1 solution KzC03per loo0 mL TCE) with stirring for 30 min in each treatment. After separation of the aqueous phase, 1 mL of propylene oxide was added per 1,OOO mL and the TCE was then mixed in the required ratio with NB. The mixture was kept on silica gel until use (10 g silica per 1O ,OO mL), in brown, closed bottles. These experimental conditions were chosen for GPC to avoid the high viscosity the phenol-TCE mixture and because PET solutions in the TCE-NI3 mixture ( 9 5 5 v/v) are not stable. Table 2 shows that the colored samples are characterized by a higher content of “COOH end groups than the colorless samples. This finding suggests the occurrence ofa degradation reaction during the preparationof dope-dyed PET. Since the coloring matters used have a l-aminoanthraquinone structure in which hydrogen atoms of amine groups are partly substituted by different radicals, the higher content of “COOH end groups in colored PET may be explained easily.The basic character of the aromaticamines is decreased byp-?r conjugation which involves the unpaired electrons of the nitrogen atom and the n-electrons of the benzene ring. This process gives an extra positive charge at the nitrogen atom, that induces polarization of the ester link. See the following reaction: OkH2 --C

6+

, CH“O-C“C6H~” -H

a+

6-

4 +

0

+ CHz=CH-O-C-C$&-

“C \

OH

a

NH-R

1 NH-R

A

A

I

l

(51)

in which A = antraquinone rest and R = 1,3,5-trimethylphenyl or - C O - C ~ s . The GPC data confirm the systematic influence of the dyestuff on themolecular weight distribution of dope-dyedPET (Fig. 4) [367]. The analysis of product characteristics shows that in the firststage of the polycondensation process (after 90 min) the molecular weight distribution (MWD) of the dyed polymer shifted to the range of higher molecular weights. The same results wereobtained from the values of M,, and M, for samples 1 and 6 (Table In Table 3, the frequency of the species per range of molecular weight for samples 1 to 10 is given. From these data, it can be seen that sample 1 contains a higher amount of species with M,, < lo3than sample 6. Concerning the species with lo3 < M,, < lo4,sample 6 has a higher percentage. Asa result of this uneven distribution of species in the molecular weight range in samples 1 and 6, the dyed polymer has a higher M, and M , than thecolorless polymer. In the subsequent stages of polycondensation, the presence of dye-

Chapter 4

14

16

18

20 22 U ve,lcounts

-

Figure 4 Gel chromatogram of colorless samples (-) and dyed ones ( (A) After 90 min; (B) after 120 min;(C) after 180 min;(D)after 210 min; (E)after 270 min; (F) industrial sample. (From Ref. 367.) e).

Table Species Distribution (To) per Range of Molecular Weightin the Analyzed Samples

G

G,,of colorless samples

of colored samples

"

No. < l o 3< l o 4> l o 4 20.411 6.49 2 3 4 5

-

-

Source: Ref.

75.68 3.91 86.37 7.14 58.97 41.03 21.01 78.99 6.50 93.50

MJM,, 1.48 1.87 1.51 1.50 2.36

"

No. 6.27 6 7 8 0.71 9 10