Labline Award Symposium
Some of the papers presented in the Labline Award Symposinm Honoring Harold E. Strain, Division of Analytical Chemistry, 139th Meeting, -4merican Chemical Society, St. Lonis, Mo., March 1961.
The Basis of Selectivity in Chromatography, Electrochromatography, and Continuous Electrochromatography HAROLD H. STRAIN Argonne National laborafory, Argonne, 111.
The selectivity of separations by chromatography, electrochromatography, and continuous electrochromatography depends upon the substances being separated and upon the migration conditions. The number of these differential migration conditions is enormous and indeterminate. There is no common basis for comparing the selectivity of the separations, and there is no widely applicable basis for predicting the selectivity of particular systems. No systematic relationship between the separability and the molecular structure of various substances has been demonstrated.
C
electrochromatography, and continuous electrochromatography have many features in common. They are differential migration methods of analysis. They serve for the resolution of multicomponent mixtures of molecularly dispersed, chemical substances. They facilitate the isolation, the comparison, and the partial description of all kinds of substances. They rank among the most widely applicable and the most sensitive and selective tools for the investigation of chemical species and their reactions (1, 2, 6, 9-11, 18-25, HROMATOGRAPHY,
SO, 31).
These differential migration methods of analysis are easily defined. Many of their modifications have been clearly described. Innumerable results obtained by their use are a matter of record (1, 2, 6, 9-11, 13, 18, 19, 22, 24,25,SO). In many respects, these differential migration methods of analysis are well understood. Basic concepts for their
operation have been developed. Explanations for their resolving power, based upon the mechanism or “theory” of the separations, have been propounded. The various modifications and applications have been correlated in relation to the migration conditions (1, 2, 6, 10,11,13,19,22, 24, SO). Many fruitful relationships between the sorbability of organic substances and their molecular structure have been established. There is hope that molecular structure may be derived from differential migration behavior (2, 11, 25, 26). I n some aspects, however, the differential migration methods exhibit complex, unrelated properties. The possibilities for variation are enormous. These intricacies stem from the great variability of the techniques. The separations and the selectivity often depend upon different phenomena. There is frequently no common basis for correlating the applications or for expressing or comparing the selectivity. The number and properties of the differential migration methods are indeterminate (24). There is a limited systematic basis for deriving the molecular structure from differential migration behavior. Highly selective with respect to separations, the methods are nonspecific with respect to structure (22,24). This report indicates some of the critical conditions required for the separations. It shows some of the ramifications of these conditions. It reveals the current limitations of our understanding of the essential conditions that determine separability and selectivity. It demonstrates the complexity of the relationships between sorbability
and the molecular structure of various organic substances. The separations by each of the differential migration methods depend upon particular conditions typical of the procedures themselves ( 1 , 2, 6, 9-11, 18, 19, 22, 24, SO). For simplification of the considerations, the methods are examined separately. CHROMATOGRAPHY
Definition. All the modifications of chromatography are included in the following definition. Chromatography is a method of analysis in which flow of liquid or gas promotes the separation of substances by differential migration from a narrow zone in a porous sorptive medium (22, 24, SO). This concept of chromatography stems directly from Tswett’s concepts. I t includes the two major variations based on the nature of the wash medium-namely, gas and solution chromatography. It also includes the variations based upon the dynamic sorptive properties of the porous systemnamely, adsorption on solids and *on fixed liquids (16, 28), sorption (partition) by fixed liquids, and sorption by ion exchangers such as zeolites and ionic resins. It embraces the modifications based upon reversal of the phases as employed with partition chromatography and also the various arrangements for flow of the wash medium as lineal or one-way flow, transverse lineal or two-way flow, radial flow, and segmental flow (22, 24,30,SS). Chromatographic Systems. For all chromatographic separations, particular combinations of the mixtures, the solvent, and the sorbent must be VOl. 33, NO. 12, NOVEMBER 1961
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SOLVENTS
SOLUTES SORBENTS
,'
Q
Starch
0 Acids
(Polar)
(Polar)
(Activated)
Figure 1. Combinations of solvents and sorbents utilized for separation of the polyene hydrocarbons, the carotenes, and the hydroxy and oxy derivatives, the xanthophylls ( I 7,25-28)
employed. -4s s h o w ~ iby nmny adsorption experiments with carotenoid pigments, various specific combinations of solvznts and sorbelits may be utilized ( I f , 2.5, 26). As illustrated in Figure 1, the solvents should be less polar than the sorbed carotenoids, whereas the adsorbents are more polar. The weakly polar petroleum ether must be employed for sorption of the weakly polar carotenes, whereas more polar solvents may be utilized for the xanthophylls, ivhich contain polar hydroxy grorips. l'he sorption phenomena utilized in chromatography have frequently been oversimplified. For example, adsorption, either at a liquid-liquid (28) or a t a liquid-solid interface (6, l l ) , and partition, as between two immiscible solvrnts (Z4), are commonly regarded as acting independently and singly in the respective chromatographic systems. There are, however, circumstances when all these effects may be operative simultaneously in one system. as indicated schematically in Figure 2 . In the early days of chromatography, the effectiveness of the separations was attributed largely to the selective properties of the sorbents, many of which were employed in the sorption columns. Later i t was found that the solvent has a pronounced effect upon the separations. Certain chloroplast pigments, for example, were readily separable with some solvents but not with others. With traces of 1-propanol in petroleum ether, chlorophyll a is more sorbed than lutein in columns of powdered sugar. With increasing quantities of I-propanol, the chlorophyll becomes relatively less and less sorbed so that a t one concentration it is not separated from the lutein. At concentrations above this point, it is less sorbed than the lutein, and the chro1734
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ANALYTICAL CHEMISTRY
matographic sequence is reversed (158r). When paper chromatography was widely adopted, variation of the solvent was necessary to improve the effectiveness of many separations, particularly in the two-way development with two different solvents in succession (2, 6, 10, 11). With the introduction of the ion exchange resins as chromatographic sorbents, the selectivity of the separations was improved by adding various complevforming solutes to the wash liquids ( 2 . 1 0 , l l ) Variation of separability and selectivity with variation of solvent may result from the effect of the solvent on the sorbent, from the effect of the solvent upon the dissolved substances, or from both these rffects. The solvent may displace the sorbed substances in some preferential way. It may also dissolve or alter the sorbed substances so that their affinity for the sorbent is changed. Examples of each effect have been found by adsorption of the chloroplast pigments (25-98). The effective combinations of solvent and sorbent can be described only in relation to the separation of specific substances. At the present stage of knowledge, the chromatographic system is a function of the nature of the substances to be separated as well as a function of solvent and sorbent (62, 64). By way of illustration, two substanccs, A and B, may be separated effectively by a particular solventsorbent combination. If now a third substance, C. is mixed with A and 13, the mixture may or may not be resolved, because C may contaminate either A or B. The effectivencss of the system must then be defined in terms of the separability of A, B. and C. The number of possible chromatographic systems is enormous. Various mixtures of diverse kinds of substances I
Figure 2. Schematic representation of substances sorbed on a solid (center circle), in a fixed liquid (between the two circles), and on the surface of the fixed liquid (outer circle) ( 7 6)
may be investigated by chromatographic methods. A very large number of liquids, liquid mixtures, and solutions serve as the chromatographic solvents and mash media. hIany more are constantly being introduced as new substances become available. S u merous solids, fixed liquids, resins, and polymers serve as the sorptive media. Many new sorbents are being devised to serve for special separations. Thus far, no widely applicable methods have been devised for selecting new and effective chromatographic systems (22, 24) The significance of the chromatographic system cannot be overemphasized. This system proi-ides separability and selectivity. The combination of mixture, solvent, and sorbent is a critical triad. It is the trinity of the chromatographer (22,24,25). Determination of Selectivity. The selectivity of chromatographic systems is indicatcd by the separability of substances. Conseqiiently, t h e selectivity may be determincd most directly by observation of the separations. The selectivity may also be estimated by comparison of the socalled R values, provided these values have been determined under similar conditions (12 ) . The migration of a sorbed substance relative to the migration of the solvent or wash medium has long been employed to compare the sorbability of rarious substances (9, 5, 11, 12, 1 4 ) . For many conditions, this ratio, the R value, increases with concentration. The R value for lineal flow in a paper strip or column is not directly proportional to radial flow from a spot in a sheet of paper (5, 33). It is proposed, therefore, that the values for lineal flow and radial flow be designated lin-R and rad-R, respectively. With surface active adsorbents and with ion exchange sorbents, the R value usually increases with concentration of the solute.. K i t h fixed liquids as sorbents, the R ralue s h o w
little variation with concentration (2, io). Tables of R values are frequently recorded, however, without reference to the variation of R with concentration. In the true liquid-liquid partition systems, the R value is related t o the relative solubility of the substance in the two phases. This conclusion is indicated by the distribution ratio when the two phases are saturated and bj. the constancy of the ratio at lower concentrations (7,8). Basis of Selectivity. If two substances, A and B, are separable with a particular combination of solvent a n d sorbent, then t h e R values must be different. The primary requisite for separability is R.4 # RB. The greatest selectivity of various systems for the separation of A and B is indicated by the greatest difference between RA and R g . From the standpoint of the distribution mechanism, the R value is the ratio of the amount of solute in solution in a chromatographic zone relative t o the total amount of solute in that zone. I1p.
=
d (in solution) 4 (in solution) h (sorbed) A (in solution)
+
rZ (total)
Because separations are tested in a particular migration arrangement, the selectivity is primarily a function of the distribution ratio in that arrangement. All other factors such as porosity, density, activity of the sorbent, and rate of flow apply equally to all the substances being separated ( 2 2 ) . Where the R value varies with concentration. as in many ion exchange and adsorption systems, the distribution ratio and the selectivity are also functions of the concentration ( 2 , 1 0 ) . However complex the sorption process, however varied the solution composition, and whatever the mechanism of the distribution phenomena. the R values for the particular conditions prokide an all-inclusive measure of the relative rates of migration. These values provide a numerical basis for comparing the selectivity of each chromatographic system. Molecular Structure and Chromatographic Selectivity. Chroniatographic methods are useful for separating all kinds of closely related organic substances including homologs and spatial isomers. Some of the earliest experience, especially t h a t with carotenoid pigments, revealed increasing sorbahility with incrcasing numbers of double bonds, Kith increasing conjugation of the double bonds, and rrith increasing numbers of polar hydroxyl and keto groups ( 2 , IO, 11, 25-27). From this kind of experience, one might expect to find systematic rela-
tionships that would permit the estimation of molecular structure from the chromatographic behavior of organic substances. In practice, however, these hopes have not been fulfilled. The sorbability of organic molecules is not specific with respect to composition or t o structure. Nor is i t specific with respect t o the molecular skeleton nor in relation t o particular functional or polar groups, which usually increase the sorbability (2, IO, 11,22, 24,26). The concept of polarity employed by the chromatographer differs from that often employed by the physical chemist. From certain physical chemical viewpoints, polarity is related t o the dipole moment. It is greatest with reactive functional groups in unsymmetrical arrangements as in nitrobenzene and o-dinitrobenzene. It is least, or zero, with the groups in symmetrical arrangements as in p-dinitrobenzene. From the chromatographic standpoint, the polarity of functional groups varies little with their arrangement in the molecule. The thrce dinitrobenzenes, for example, are much more sorbed than nitrobenzene. Nevertheless, the sorbability due to the functional groups varies sufficiently with their location in a particular structure so that various isomers, such as the three dinitrobenzenes, may be separated (Ii). Similarly, the sorbability attributed to functional groups in a homologous series also varies enough so that the members may be scparated. The sorbability attributed to any group in a molecule varies x i t h the structure of the rest of the molceule just as the solubility attributed to a particular group also varies with the structure of the rest of the inolecule (8). There are other limitations to the use of chromatography for the elucidation of organic structure. There is no single chromatographic system for the comparison and classification of all organic substances. The several distribution phenomena t h a t serve as a basis for the separations are remotely related. Chromatographic sorbability. particularly as indicated by sequences (25-28), varies unpredictably with various solvents and with various sorbents. The adsorption systems, based upon sorption at interfaces, show enormous and unpredictable variation with increasing complexity of organic molecules (25-28). The partition systems, dependent upon the distribution betrveen tn-o liquids, also exhibit complex variations, Ivhich, as noted alrrady, are related to the relative solubility in the tn-o phases. Systematic correlation of sorbability and solubility with molecular structure has not progressed far enough to provide a basis for the estimation of molecular structure (8). Chromatography may be utilized in
conjunction \\ ith specific organic chemical reagents to determine whether or not particular functional groups are present. If an unknown substance is converted into a less sorbed product when treated with acylating agents, an alcohol group, or its analog, is probably present. I n conjunction with physical properties, such as visible and infrared absorption spectra, chromatographic behavior often indicates the degree of unsaturation and confirms the presence of functional groups. Chromatography is a valuable adjunct t o conventional organic chemistry, but i t is no substitute for the systematic specific reactions, degradative procedures, and synthetic techniques usually required for the determination of molecular structure (22,24). ELECTROCHROMATOGRAPHY
Definition. Like chromatography, electrochromatograpliy may also be defined as a n analytical procedure. Electrochromatography is a method of analysis in which direct current electrical potential promotes t h e separation of substances by differential migration from a narion zone in a stabilized, electrically conducting solution. Numerous synonyms are applied to this separation procedurenamely, differential electrical migration, ionography, zone electrophoresis, electropherography, etc. (1,22,24,SO). This concept of electrochromatography includes all the modifications without consideration of the migration conditions. I t emphasizes the significance of the narrow initial zone of the mixture. It includes all modifications of the migration system such as variation of the solvent, the background eleetrolj te, coinplexing reagents, and the nature and form of the stabilization medium. It includes systems in which there is little or no sorption of the migrating substances and also those in m-hich sorption plays a critical role in the separations ( I , 19, 20, 23, 2 s ) . It embrace. tn-o-n ay or transverse electrical migrations and certain aspects of separatory procedures based upon electrical migration preceded or followed by transverse flow of solvent. It comprises one component or vector of batchnisc methods based upon electrical migration transverse to .imultaneous f l o ~of solvent ( 1 8 , S l ) . Electrochromatographic Systems. For all electrochromatographic separations, special combinations of t h e mixture, the solvent, t h c backgiound electrolj-te, and t h e stabilization niedium must be employed. Aqueous solutions, oning to their large electrical conductivity, have usually been used, although effective separations have also been made in nonaqueous solutions (1, 19, 32). The stabilization media include paper, powders packed in VOL. 33, NO. 12, NOVEMBER 1961
1735
same way that R values are a function of the sorbability. Presumably the fraction of the species remaining nonsorbed in solution is free to migrate. The fraction fixed by the sorbent is not. Various factors such as the porosity, the average pathway of migration, and osmotic effects (8) apply equally to all the substances being separated in a particular migration system and should not affect the selectivity. As with the R values in chromatography, the mobilities are a function of so many variable conditions that they cannot be regarded as physical constants unless special pains are taken to define the system itself. Molecular Structure and Electrochromatographic Selectivity. There are some important relationships between the electrochromatographic behavior and the molecular structure of organic and inorganic molecules. The direction of migration reveals the sign of the electrical charge. Through the formation of complexes, such as the borates of polyhydroxy compounds, clues to the presence of particular molecular structures may be obtained. From variation of mobility with changes of pH, isoelectric points due to the presence both of acidic and basic groups may be indicated. By their effect upon the polyvalent cations, molecules with structures that form complexes may be detected. As in chromatography, these electrochromatographic methods are largely nonspecific with respect to structure. They serve as adjuncts to organic chemical methods but not as substitutes for them (24).
tubes or troughs, and gels such as gelatin, agar, starch, and silicic acid 19J88J84)'
Electrochromatographic separations are a function of the migration system. Various electrolytes have been utilized with and without p H buffers. Various complexing and chelating agents, usually with particular p H buffers, have been added to the background solutions in order to improve the selectivity and the completeness of the separations (4, 19, 20, 29). As examples, cyanide in aqueous ammonia permits an excellent separation of thallium and silver ions (as), and borate makes possible the migration and separation of noncharged carbohydrates ( 1 , 19). All the ramifications of these systems have not yet been ascertained. Determination of Selectivity. As in chromatography, the selectivity of electrochromatographic systems depends upon the substances being separated, the composition of the background solution, and the nature and properties of the stabilization medium. Thus far, no widely applicable generalizations regarding the selective separation of various kinds of substances have been evolved (24). The selectivity of a particular system may be determined directly by migration of the mixture. I t may also be estimated by comparison of the mobilities ( p ) determined separately in the same system (29). 111 this respect, the mobility in the system is analogous to the R value. Basis of Selectivity. If substances are separable by electrochromatography, the mobilities in the migration system must be different, PA # p ~ The greatest selectivity of various systems for the separation of A and B is indicated by the greatest difference between p A and C(B. When A and B carry electrical charges of opposite sign so that they migrate in opposite directions, absolute separations n-ithout cross contamination are possible (20,29). The migration mechanism is usually complex. Migrations may be primarily through the background solution without sorption by the stabilization medium, through the background solution with concomitant sorption by the stabilization medium, or primarily through the stabilization medium alone (19). Sorption of the migrating species by the stabilization medium may enhance the separations, or it may reduce separations obtainable in the background solution alone ( 2 , 19, 20, IS).
From consideration of the migration mechanism, the selectivity is a function of the electrical mobility both in the background solution and in the stabilization medium. It is also a function of the sorbability in somewhat the
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ANALYTICAL CHEMISTRY
.
CONTINUOUS
ELECTROCHROMATOGRAPHY
Definition. Continuous differential electrical migration, as applied to the continuous resolution of mixtures, may be defined as a separatory procedure. Continuous electrochromatography is a method of analysis in which direct current electrical potential promotes the separation of substances by differential migration from a narrow stream of the dissolved mixture in a wide, stabilized stream of the background electrolytic solution flowing transverse to the electrical field. Synonyms include continuous zone electrophoresis, continuous electrophoresis, and continuous electropherography (1, 1719. 88, 84, SO, 31). This concept of continuous electrochromatography includes all the principal modifications-namely, beds of packed powders, fibrous media such as paper. and various electrolytic solutions with or without p H buffers and complex-forming reagents. It is based upon the procedure employed rather than upon the migration mechanism. Continuous Electrochromatographic Systems. I n all the continuous sepa-
rations, particular combinations of the mixture, the solvent, the background solution, and the stabilization medium are employed. The background solution is usually an aqueous solution of a salt, weak acid, or weak base, with or without pH buffers and complexing agents. The stabilization medium is usually soft paper, which may be supported between plates of glass or plastic, suspended in a closed vessel, or suspended against a support of solidified polystyrene foam (1, 9, 17-19, 21, 83,Sl). The mechanism of the separations involves two transverse migration effects: the lateral migration in the electrical field, and the cross migration due to the flow of the background solution. The migration may occur with or without sorption by the stabilization medium. In either case, the stabilization medium also presents a mechanical barrier to the migrating species. Determination and Basis of Selectivity. Because of the complexity and variability of the migration systems, the selectivity must be defined in relation to the composition of the mixture, the constituents of the background solution, and the nature of the stabilization medium. It is estimated most conveniently by the separation of particular substances. The selectivity may be compared in relation to the angular displacement of particular substances from the line of solvent flow. This displacement is a function of the electrical potential and the flow of the background solution. Comparison "; separations under different conditiolis must be corrected for these two variableq (9,17-19,31). The requisite f u i the separability of two substances is that the angle (
