CHROMATOGRAPHIC SYSTEMS HAROLD H. STRAIN Argonne National Laboratory, Chicago, I l l .
W
I T H I N the brief period following last year’s reviews (6, 8, 50, 67, 74, 86, 156, 179, 203, 220, 236, 236, 164, 2Y7, g8.2, 885,287, 298,916,518,3,%’9,333,373, 418) several novel modifications and numerous new applications of the chromatographic technique have been reported. Indeed, the current progress haa been so extensive and so diverse that many of the developments seem remotely related to the paper absorption procedure originated by Schonbein or to the columnar adsorption method perfected by Tswett. I n the light of this diversity of technique and application, Ypecial effort has now been spent in a search for those features of new publications that serve as a basis for correlation with earlier investigations. I n this way, the evolutionary aspects of the subject may be followed, and the advances of use to others may be hrought to light. Even in one year, several Significant trends are apparent in the development of chromatography. There has been a gradual realization t h a t chromatographic methods themselves serve as interpretative aids as well as observational and analytical techniques. The chromatographic technique has been amplified and combined with other analytical procedures such as electromigration. There has been continued advance in the use of neF absorptive phases and new solvents. This advance has, in turn, facilitated the application of chromatographic methods to a great variety of scientific investigations. Chromatographic methods have now found extensive use for studies of all kinds of soluble compounds ranging in amounts from a few hundred atoms or molecules (radioactive tracers) to tons (as in petroleum refining) (lor, 147). As more and more workers have utilized chromatographic methods from diverse but specialized points of view, a complex and conflicting terminology has been developed. This has resulted, in part, from the very large number of variable conditions in chromatographic systems, from the emphasis placed upon particular components of these systems, and from the manifold objectives of the workers. Several symposia concerning chromatographic analysis have provided numerous papers that serve as useful guides to this rapidly expanding subject. One group of papers pertaining to the properties of adsorbents appeared in the Journal de Chimie Physique et de Physico-chimie Biologique. Another group, pertaining to many aspects of chromatography, forms a single number of the Discussions of the Faraday Society. A third group, restricted to partition chromatography, comprises a monograph of the Biochemical Society (405). Yet another large group, selected by the Division of Petroleum Chemistry of the AMERICAN CHEMICAL SOCIETY,has been published in Industrial and Engineering Chemistry and in ANALYTICAL CHEMISTRY.Much of the chromatographic literature of the last decade has been carefully reviewed in a monograph by Zechmeister (418).
color, molecular architecture, biological activity, a r d radioctctivity. I n this way the origin, the function, and the transformations of many substances have been traced before knowledge of their chemical composition has been obtained. Chromatographic observations have provided new clues to the mechanism of many obscure phenomena. For example, in photcsynthesis wherein the carotenoid and chlorophyll pigments utilize incident light for the production of oxygen and organic matter, chromatographic investigations indicate that energy utilization occurs without detectable chemical change of the pigments (375). Similarly, combination of chromatographic methods with the remarkably sensitive radioactive methods has confirmed this view ( 7 5 ) and has revealed a great variety of organic substances that are formed rapidly by this utilization of energy in plants (IO,2Y, 60, Y5). Numerous similar examples have now been reported in many other aspects of biological investigations, such as the isolation of various radioactive drugs (270) and foods (391) that then serve as indicators of chemical change in plant and animal metabolism. I n this application of chromatographic technique, all the radioactive substances appear as discrete radioactive zones in the absorption medium. This comhination of high resolving power with great sensitivity for all the resolved substances is a remarkable advance in the perfection of analytical and observational technique. I t provides a convenient and rapid approach to the interpretation of many natural processes in advance of the usual, less sensitive chemical analysis. From this standpoint, chromatography serves not only as an observational tool of great discriminating power but also as an interpretative aid that is remarkably adaptable and reliable. COMMON PROPERTIES OF CHRO.MATOGRAPHIC SYSTEMS
All chromatographic methods are based upon the differential migration of solutes through polyphase systems in which the phases have preferential affinities for the solutes. Two-phase systems commonly employed are composed of solid plus liquid or dispersed liquid and support plus liquid. Other two-phase systems as solid or liquid plus gas are sometimes utilized (147,152,318). Solutes to be examined are forced to migrate through the polyphase system by flow of the fresh liquid or gas phme (51, 318). Recently, flow of electrical current (electrochromatography) has been utilized on an increasing scale. For these investigations, the solute mixture is contained initially in a narrow zone. Under these conditions, differential migration of the solutes results in their separation from one another. Continuous flow of solution through an adsorptive phase yields only a partial separation of the solutes, but it provides much useful information about the properties of the chromatcgraphic systems ( l d Y , 243). Examination of the variation of the composition of the advancing solution forms the basis of frontal analysis (195), of displacement analysis combined with frontal analysis (71, 160, 184, 382), and of carrier displacement analysis (383) in which a series of adsorbed solutes are displaced in succession by a mixture of slightly more adsorbed substances. From a theoretical point of view, a remarkably large number of different solid and liquid substances may be employed as the phases in chromatographic systems. For the separation of particular substances, however, the phases are limited by the properties of the solutes. All the solutes must be in a reversible or dynamic equilibrium between the phases. They must be retained in significant amounts by each phase. They must not react irreversibly with either phase, and the polyphase system itself must be a stable one.
CHROMATOGRAPHY A S AN INTERPRETATIVE AID
Owing to their high resolving power, chromatographic methods often reveal new and unsuspected constituents of complex mixtures. As first observed by Tswett, bhese new substances may occur in such small amounts that they cannot be examined readily by the conventional methods of chemical analysis. For this reason it is often impossible or unprofitable to define them in terms of their chemical composition. It is possible, however, to describe them in relation to their chromatographic behavior such as relative migration rates or adsorption sequences. These properties can usually be correlated with other properties such as 25
26
ANALYTICAL CHEMISTRY
For effective separations of mixtures in chromatographic eystems, the relative amounts of each solute in the two phases must be different. This requirement also limits the number of substances employed as mobile and nonmobile phases. It is one of the most difficult conditions to establish for various kinds of solutes. When mixtures of unknown substances are being investigated, it can be determined only empirically. It is surveyed in greater detail in subsequent sections concerning the phases and the specificity of chromatographic systems. CHROMATOGRAPHlC APPARATUS
For the utilization of these chromatographic systems, many adsorption techniques have been devised. Some of these modifications, which have resulted in an enormous variation of a p paratus, are indicated by Table I and Figures 1 to 5. All this
.
apparatus is subject to variation in dimensions and in many cases it can be employed with various absorptive and liquid phases. Kotable among the recent innovations of chromatographic apparatus have been those designed for use of paper as the absorptive medium (Table I), a subject recently reviewed by several workers (7'4, 86, i5,5, 373). This use of paper is not due to any unique or specific absorptive property but to its dimensions, uniformity, stability, permeability, and translucency. The latter property permits the use of color-producing reagents for detection of absorbed solutes, and it facilitates the use of various optical scanning devices (299). Paper lends itself to use with the ascending or descending flow of the wash liquid (Figures 1 and 2 ) . I t may also be supported between glass plates which permit use a t any angle and which prevent evaporation of the wash liquid (Figure 3). It may be utilized as a support for more active adsorbents (58, 91, 127, 118, 268, 337). Substances resolved on paper are readily recoverable (238). Apparatus employing paper as the nonmobile phase has proved exceptionally useful in separations based upon the principle of electromigration (373) or upon electromigration plus chromatography (Figure 4)(376). This use of paper permits simultaneous separation of cations and anions, whereas the older use of electromigration in columns permitted separation either of cations or of anions during a single operation (Figure 5 ) (34, 35, 83, 96, 99, 106, ,827, 268, 376, 382, 388). For these separations with electrical current, t'he paper may be placed in a stoppered tube (ass),in a closed jar ( I O S ) , between plastic sheets on a metal block (227'), or between glass plates that may be cooled (34, 376). An apparatus permitting the continuous separation of solutes has been designed and applied t,o the resolution of mixtures of various inorganic and organic ions. Separations may depend upon electromigration alone (106, 268) or upon electromigration plus chromatography (376). I n these uses of electromigration plus chromatography, electrolytic reactions a t the electrodes may yield acidic and basic zones that migrate toward each other and may interfere with the separations of the solute mixture. This deleterious effect may be reduced by the use of a long apparatus, large volumes of buffer solution between the electrodes and the paper strip (106, ass),
Figure 1. Formation of Chromatograms by Upward Migration of Wash Liquid Solute mixture applied as band (or spot)
Table I.
Adsorption Apparatus
Tubes with nonmobile solids: adsorption or chromatographic column,
partition column, ion exchange column, etc. Tubes with nonmobile solids plus electrodes: electrochromatographio column (Figure 4)
Porous cohesive solids: chromatobar (889) Thin layers of powdered solids: chromatostrip (219), surface chromatography (286) Single strips of paper: paper chromatography Single strips of paper plus electrodes: electrochromatography ($76) Flat sheets of paper: paper Chromatography, two-way chromatography (260) Flat sheets of paper plus electrodes: electrochromatography Circular sheets of paper: paper chromatography, radial chromatography (556) Cylindrical beds: radial chromatography (265) Circular sheets of various fabrics between embroidery rings (326) Tubular sheets of paper: paper chromatography Stacks of filter paper: chromatopile (292) Paper impregnated with various absorbents: paper adsorption chromatography, paper partition chromatography, etc. (68, 91, 127, 218, 228, 357)
Figure 2. Formation of hromatogram by Downward Migration of Wash Liquid Solute mixture applied as band (or spot)
V O L U M E 23, N O . 1, J A N U A R Y 1 9 5 1
27 There have been iniprovements in the interferometric (189)and potentiometric ( 2 0 8 , 209) methods for esamination of effluent. Further refinements of the use of thin layers of adsorbents h a v e been made (286, 399). Macro (298,424) and micro (286, 299) separations have been obtained rapidly in paper. A proportional divider, t h e P a r t o g r i d , aids m e a s u r e m e n t of RF values (39%’).
Table 11. Ways of Forming a Chromatogram Flow of solvent in one direction: unidimensional chromatography, columnar chromatography, one-way paper chromatography, fractional elution (829), progress chromatography (17 7 ) , carrier displacement (383) Flow of solvent in one direction followed by flow of solvent at right angles: two-way chromatography in paper or fabric sheets (326)
Flow of electrical current: electrochromatography (376),ionography ($68) Simultaneous flow of solvent at right angles t o flow of electrical current: electrographic analysis (376) Flow of electrical current in one direction with one solvent followed by flow of current a t right angles but with another solvent: two-way electrochromatography l.’low of electrical current opposed to flow of solvent (273) (not tested with an absorptive phase) Various additional combinations of flow of solvent, flow of electrical current and other forces (see text and Table 111) Flow of solvent containing a mixture of carefully selected displacers: carrier displacement chromatography (583)
FORMATION OF CHROMATOGRARIS I I
I
I
I I I I I
I I
I I
I I I
I I I I I
I
I I I I
I I
I I I I I I I I
I
I I I
I I 1 I
I
I
I I
I
I
I
I
I
I
I
I
I
I I I I I I
I
I
I I I
I
L
4
A
Figure 3. Formation of Chromatogram in Paper Strip, P , between Glass Plates (Dashed Line)
The differential migration of solutes in a polyphase system requires the use of a driving force. Flow of solFigure 4. Arrangement for vent, t h e common Separation of Cations or Anions method of forming a by E l e c t r o c h r o m a t o g r a p h y chromatograni (Table (without Flow- of Solvent) 11), e v r t s a uniform force on all the substances in solution. The differential migration of the solutes depends upon the amount of each in solution relative to the total amount present in the polyphase system. Flow of electrical current for formation of chroinatograms e+ erts a preferential driving force on the components of the solute mixture. Here the differential migration is a function of the sign of the electrical charge and of the mobility in the polyphase system. As with flow of solvent, migration of an ion in a polyphase system also depends upon the amount of solute in solution relative to the total amount in the adsorption zone (376). Other forces, such as magnetic fields, have been suggested as thc driving mechanism for chromatographic separations (268). Two different forres may be applied successivply or siniultaneously a t right angles to each other. An example of the former is the cross-capillary analysis or two-way chrnmatoyraphy with different solvents (260). An example of the latter is the combination of electromigration with flow of solvent (373, 376‘). This latter principle can be adapted to continuous separation processes (Ci76).
For upward or downward flow of wash liquid
(376) L
nonpolarizable electrodes ( 2 - 8), or weak organic acids or ammonia as the electrolyte (376). A number of new laboratory devices have recently been described. These include a fooboperated pressure and vacuum regulator (bo),an ophthalmoscope scanning or spot-locating light (@), a dispenser for powdered adsorbents (84), a rack of Duralunlin for holding many paper sheets during chromatographic development (89), polarographic detection of proteins containing cysteine or cystine ( I O I ) , photography of bacterial spot tests with polarized light ( 1 0 2 ) , weighted paper strips for use in milk bottles ( I 3 7 ) , in cylinders (262), and in tubes (Sib),an electronic image converter for location of substances that, absorb infrared light ( 166), and an ultraviolet-sensitive photomultiplier tube for location of substances that absorb ultraviolet light (944).
I I
-
, ,
P
El
Figure 5. Arrangement for Formation of Electrochromatogram in Paper Strip, P , between Glass Plates (Dashed Line) (376) Two different forces, as flow of solvent and electromigration, may be allowed to act in opposite directions. This application has been tested thoroughly for the separation of isotopes in systems with little or no absorption capacity (87.9).
28
ANALYTICAL CHEMISTRY
With so many forces applicable to chromatographic systems, there is now opportunity for further advances in the formation of chromatograms. Three different forces may be utilized simultaneously to effect a continuous resolution of mixtures. This could be accomplished in a rectangular column with flow of solution into the bed near one corner, with simultaneous flow of solvent through the remainder of the bed, and with electromigration and magnetic forces applied horizontally and a t right angles to each other. A three-way discontinuous development has now following two-way developbeen achieved by electromigration ment in a sheet of paper.
r--l
C'
1
0L
oc
a;
P
'8
Figure 6.
Relative Positions of Lutein, L, and of Chlorophyll, C
After washing with benzene B , and petroleum ether plus 0.5% propanol, P (upper s q u k e s ) , and after washing with P and B (lower squares)
Two-way development in sheets of paper, usually effected by flow of two different solvents ( 6 7 ) ,can also be achieved by flow of solvent in one direction and with electromigration in the other, and by electromigration with two different solvents. With these two-way developments the final position occupied by a solute depends upon the order in which the forces are applied (Figure 6). With three-way development achieved by electromigration and by solvent flow, eight sequences of application are possible (Table 111). As the total number of solvents and mixtures of solvents that can be used is very great, the total number of variable conditions is enormous.
Figure 7. Random Distribution of Polar Molecules between Droplet of Stationary Liquid Phase and Mobile Liquid Phase Partition diagram. C, concentration. S stationary liquid phase; L, mobile liquid phkse
fication is arbitrary, however, because some substances may act by several processes, depending upon the nature of the solutes and the solvent. In this classification, paper is often a surface-active aolid like other polysaccharides such as starch and powdered sugar. Under other conditions, the paper may retain water or glycols that hold solutes by solution. Cnder still other conditions, as with ammoniacal copper solutions, the paper may retain the solute by ion exchange or by chemical combination (916). Similarly, ion exchange Chromatography might be regarded as a subdivision of cheniichroniatography. h more complex classification ot absorbents based on the principal state of the solutes in each phase (685) is subject to these same ambiguities. In vieaof these complexities, a detailed classificativn of absorptive substances does not seem to be helpful. Likewise, the classification of the subject of chromatography into categories based upon t h e properties oi a single absorptive substance (paper chromatography) is certain to produce conflicting concepts concerning identical phenomena in various systems.
A
T3
I
NONMOBILE PHASES IN CHROMATOGRAPHIC SYSTEMS
Most substances employed as the solid or nonmobile phases in chromatographic systems fall within four groups as indicated by Table IV. There the absorptive substances are classified with respect to their common reaction with the solutes. This classi-
Table 111. Sequences in Which Electromigration in Different Solvent8 (E) and Solvent Flow (S) May Re Combined in Three-way Development XO.
1 2 3
p.
4
7
Direction 1 E E E E S S S S
Direction 2
E E S
8
s
Direction 3 E S
E S S
6
E
E
E
E
S
Figure 8. Random Distribution of Ionized Molecules between Ion Exchanger, S , and Mobile Liquid Phase, L S s has been pointed out many times (151),sorption botherms give much useful information regarding the kind of forces that are effective in the nonmobile phases of chromatographic systems. A graphic illustration of the condition of a solute in various absorptive phases is provided by Figures 7 to 9. I n the simplest case, the solutes are in solution in the nonmobile phase; hence there is a random distribution of solute molecules in both phases. With ion exchange or other chemically reactive solids, there is a random, slow neutralization of charge in the solid phase. With surface-active absorbents there is a rapid concentration and often a specific orientation of solute molecules a t the active surface of the solid phase (Figure 9).
29
V O L U M E 2 3 , N O . 1, J A N U A R Y 1 9 5 1 Table IV. Solid or Nonmobile Chromatographic Phases Surface-active solids: chromatography by adsorption Ion exchange minerals and resins: chromatography by ion exchange (376) (ionography, radioionography) (198) Bound liquids or gels: chromatography by partition (281,406) Chemically reactive solids: chemichromatography (373)
I n many uses of paper, however, water and water-miscible solvents yield separations equivalent to those in the prwence o€ water-saturated solvents (28, 150). The relative roles played by paper and by a separate nonmobile phase in these three-phase systems remain to be established. As is discussed in another section, the improved separations may be attributed t o eptablishment of a. sorption gradient in the system. ,MOBILE PHASES 1% CHROM4TOGRAPHIC SYSTEMS
Mobile Liquid Phases Employed in Chromatography Nonioniamg, nonnolar solvents: essential to chromatography of nonpolar solutes ionizing polar solvents: essential to chromatography of polar solutes by ion exohange and by electromigration Solvent8 with preferential affinity for absorptive phase: essential to displacement analysis and carrier displacement analysis (160, Table V.
184, 386)
Solvents that react with solutes, useful for variation of distribution coefficients and of electrical charge by complex formation (376)
The liquid mobile phase plays a critical role in the formation of chromatograms. Thus far, however, i t has been difficult to classify liquids adequately in relation t o their part in chromatographic system. A preliminary, tentative classification based upon usefulness is shown by Table V, but owing to the possibility for variation of the absorptive phase, numerous exceptions might be cited. Classification based upon physical state of the solute in the liquid phase is subject to similar complications (286). One reason for the difficulty in classification of the chromatographic solvents lies in the complexity of their reactions with the nonmobile phase and with the solutes themselves. The simplified drawings shown in Figures 7 to 10 do not indicate the role of the solvent, yet in many two-phase systems the effect of the solvent may be very great. Small quantities of some solvents may displace large quantities of other solvents and of solutes (374). Some solvents may combine tenaciously with various solutes and thereby affect their adsorbability and their migration. From these points of view the solvent may exert various specific influences in chromatographic systems (39,160, 184, 190,283, SlS, $76, 406). I n the recent extensive investigations with paper as an absorptive phase, the solvents range from polar substances such as water, mineral acids (245), organic acids, and organic bases to nonpolar hydrocarbons (23, 28). Sometimes just the correct amount of “impurity” or acid or salt must be present in the solvent (39, 31, 45, 4 7 ) . The solvent may be miscible with water, immiscible with water and dry, or immiscible with water and saturated with water.
Figure 9. Oriented Distribution of Polar Molecules between Impervious Polar Surface, S, and Mobile Liquid Phase, L The sorption or distribution isotherms indicate the average distribution behavior of a solute when in equilibrium between the two phases. They do not indicate the rate of adsorption and deeorption. Neither do they reveal the actual distribution mechanism. For example, small molecules may penetrate the narrow pores or interplanar spaces of a porous adsorbent as indicated by Figure 10. Large molecules would be unable to penetrate these pores, yet on the outer surface they too would be adsorbed. In this way surface-actlte adsorbents possess a specificity in relation to space and to the size and molerular architecture of solutes that is not shared by solutions, by gels, or by resinous ion exchangers. For many solute mixtures, surfaceactive adsorbents should, therefore, exhibit more extensive resolving powers than partition or ion exchange absorbents. .4n indication of the validity of this conclusion is the effectiveness of surface-active solids for resolution of various complex mixtures, such as petroleum hydrocarbons that are not readily resolved by other sorptive systems (76,97, 207,124,125,173). When solutes adsorbed in paper (155, 179) or in columns of cellulose ( f a y , 188) are irrigated with organic solvents zaturated with mater containing polar substances, the separability of the mixtures is often enhanced, and the trailing portions of the adsorption zones are usually reduced. This effect, which has long been known with other adsorbents, has often been attributed to the formation of a separate liquid phase in the paper, so that the separations result from partition between two liquid phases.
c’L Figure 10. Oriented Distribution of Polar Molecules between Interstitial Polar Surfaces, S, and Mobile Liquid Phase, L I n practice, the elution of strongly absorbed solutes is often difficult. For partition absorbents this elution may be facilitated by use of the liquid contained in the nonmobile phase as eluant (251). Presumably, use of the specific groups of ion exchangers in soluble form would likewise facilitate elution of solutes retained tenaciously in ion exchange columns. REVERSAL OF PHASES IN CHROMATOGRAPHIC SYSTEMS
Owing to the great density and polarity of solid substances, the polar phase in chromatographic systems has usually been the fmed, nonmobile component. With the use of suitable supporting
ANALYTICAL CHEMISTRY
30
material such as colloicial vulcanized rubber latex, chlorinated hydrocarbons, or surface-treated Celite that can be impregnated wit,h liquid hydrocarbons, the less polar phase can be made the nonniobile phase. Under these conditions the most polar solutes flow through the column first, the reverse of the usual chromatographic separation (39, 190, 313, 405), and excellent resolutions are ohtained. SPECIFICITY OF CHRORIATOGRAPHIC SYSTEMS
Studies of the sorption of various conipounds have providcd inforination about the selective action of many chromatographic systems. -4s indicated, most attention has been centered on the preferential attraction between the solutes and the adsorbent. Investigators were attracted by the variability of adsorbents, by their preference for polar substances, and by the opportunity to study the adsorption of gases in the absence of the solvent phase. At a recent symposium, the specificity of chromatographic systenis was shown to be a func.tion of solvent, solute, and adsorbmt. For most systems, both the solvent and the adsorbcnt may exhibit preferential affinities for similar solutes. For many systems, particularly those involving chemical and surface reactions with the solutes, this specificity can be determined and expressed only as a property of the system itself. For other systems, especially liquid-liquid systems, the specificity is related to the ratio of the solubility of the solute in each phase. A4ccordingly, solubility measurements as well as the usual partition or distribution determinations (Figure i ) should serve as a basis for estimation of specificity and chromatographic behavior. SPECIFICITY OF ELECTROCHRORl.IT0GRAPHIC SYSTEMS
As an aid to the separability of solutes, various complex-forming reagents may be added to mixtures so that neutral solutes are converted into anions or cations, and charged species may be converted into undissociated species or into ions of opposite charge. These effects become of great practical value, particularly in ion exchange chromatographic systems and in electrochromatographic analysis (3?6). For ions of identical sign, chromatographic sequences are ubually identical with electrochromatographic sequences. But for ions of opposite sign, electrochroniatography is a more selective and efficient method of analysis than conventional chromatography (376). As isotopes are poorly separable in chroniatographic systems and in electromigration systems (156, 279), isotopes should be poorly separable by the electrochromatographic method. 5l'ECIFICITY IN RELATION TO TIOLECULAR STRUCTURE 111 the
course of tn-enty years, numerous relationships between the structure of organic niolecules and their adsorbability have been elucidated. \Vith the rapid development of paper chromatography many of these relationships have been encountered 108, 293, 300, 329, 374, 404, 417) and many again (43, 44,50,66, new ones have been reported (23, 43, 47, 169, 186-188, 261, 272, 275, 283, 230, 307, 317, 321, 325, 327, 3.51, 366, 369). In most of this work, the nuclear skeleton of the molecule has been held constant and various polar groups varied. I n this way, the adsorbability of the molecule can be attributed to the effect of particular groups, and a definite value, designated as AR,w, may he assigned to the effect of the group upon the chromatographic behavior of the molecule (23, 49). R.%f = log
(A -
1).
Although AR,w is nearly constant for a structural group in similar molecules, many eweptions were observed. Even with the simplest molecules, the addition of a polar group may have various anomalous effects upon chromatographic behavior. I n certain aromatic quinones the position of the hydroxyl groups is of greater significance than the number of hydioxyl groups (191). Likewise, the closer the amino group to the
carboxyl group of itn aliphatic anilno acid, the rreuker the attraction ot the molecule to the polar phase ( 3 2 7 ) . I n organic acids, the addition of double bond^ increases affinity for the nonpolar phase (hod), whereas the reverse effert is observed with hydrocarbons (420) and carotenoids. Surprisingly, amides are more absorbe i than the corresponding acids ( 4 7 ) . A hydroxyl group in an anthraquinone nucleus is more adsorbed than a hydroxyl group in a side chain (Mt9). PERCENT SOLUTE IN SOLUTION 0
100
\
\
\
\ 100
PERCENT SOLUTE ADSORBED
Figure 11. Hypothetical Sorption Gradient in Chromatographic Column The role of solvent in chromatographic relationships may be illustrated with the aliphatic carbosylic acids. With these substances, the polar carboxyl group remains constant and the nonpolar carbon chain becomes the structural variant. Lower members of the series are more soluble in polar solvents such as aqueous alcohol than the higher members, and with this solvent the lower members are less absorbed on charcoal than the higher members. Here preferential solubility in the mobile phase has apparently outneighed the attraction of charcoal for the polar groups, and a reversal of sequence analogous to t'hat obtained by reversal of phase has been obtained (160, 181, 303). "lie elution of these acids is in homologous sequence, not precisely in the order of decreasing ionization constants. With columns of silica gel and with chloroform plus butanol as solvent, the acids of lowest molecular weight are t,hen the least soluble and the most absorbed (283). With this system, too, separability of the acids and their hydroxy derivatives is more closely related to relative Jolubilities in the two phases than to polarity or degree of ionization. I n paper with butanol plus amnionia as solvent, the acids of lowest molecular weight migrate a t the slowest rates ( 5 3 ) . Likewise in paper, but with butanol plus formic acid as solvent, keto acids and aliphatic hydroxamates of lowest molecular weight migrate a t the slowest rate (275, 366). For substances of similar kind and dimenaions, chromatographic separations segregate the molecules in relation to functional groups. Distillation segregates the molecules in relation to size (420). A combination oi the two processes provides a new approach to petroleum fractionation ( 7 6 , 97, 107, 124, 195, 277, 353,357, 397).
V O L U M E 23, NO. 1, J A N U A R Y 1 9 5 1 For inorganic substances, chromatographic behavior is related
to the ionic charge, the degree of hydration, and the atomic dimensions and structure (33, 186, 361-363, 377, 380). These effects are attributed largely to the attraction between the ionic species and the absorptive ion exchange phase (226, 877, 380). In the presence of complex-forming reagents, the chromatographic behavior of inorganic ions is subject to great variation. I n all considerations of the relationship between molecular structure and chromatographic behavior, two principles must always be kept in mind. One is the tremendous number of attractive forces in the simplest chromatographic systems. The other is the great adaptability or variability of molecules in the different chromatographic systems. These variations may include reaction with either mobile or nonmobile phase, ionization, association, and distribution of energy (200) or polarity within the molecule itself (417). L)ISTRIBUTlON O F SOLUTES IN SORPTION ZONES
The distribution of a solute in a sorption zone has been found to vary a great deal, depending upon the conditions in the chrcmatographic system. When a solution is permitted to flow through a chromatographic system, a uniform sorption zone is usually formed. If several solutes are present, these likewise form uniform, superimposed zones which migrate through the column a t different rates (160, 284). When an initially narrow sorption zone is washed with fresh solvent, as in the formation of the chromatogram, the uniform zone becomes diffuse a t the trailing boundary and sometimes it becomes diffuse a t the leading boundary. These effects have been correlated with the rate of change of the distribution coefficient and are represented by the sorption isotherms (Figures 7 to 10) (150, 161). They are now reconfirmed by careful &dies of the adsorption of nitroaniline upon silica gel (587). In practice diffuseness of the sorption zones may be reduced by sorption of the solutes a t low concentration (585) and by the addition of strongly sorbed substances to the solutions (275, 317). This diffusenes may also be reduced by the use of systems with a11 increasing sorption capacity in the direction of solvent flow. The addition of strongly sorbed substances to a solvent sometimes causes unexpected variation of the distribution of other 8oIutes, such as xanthophylls, in the sorption zones. I t may cause tllic zones to become very narrow; it may induce variation of the sequence of the bands; i t may result in the elution and crystallization 01 the sorbed solutes; and it may even cause the formation of two distinct zones from a sinzle homogeneous solute. These anomalous effects have been observed most frequently when slightly soluble, strongly sorbed, polar substances are added to a nonpolar solvent (374). Analogous results have also been observed in paper chroinatogranis (a,275, 517). In the light of this experience, the formation of diffuse zones or even of multiple zones in a chromatographic system is not necessarily proof of heterogeneity or of isomeric forms OF a solute (9). An unusual distribution of pigments in their adsorption zones has frequent,ly heen observed when a petroleum ether extract of plant material? is percolated through a column of magnesia. Some of the minor carotenoid constituents form very narrow zones a t some diet'ance from the top of the column. The rate of migration of these zones may he very slow, so that additional pigment carried with the solution accumulates. This effect, which is of great help in the isolation of the minor pigments, may now be attributed to reaction b e h e e n colorless substances and the chromatographic system, resulting in the formation of n sorption gradient. Similar evidence indicates that sorption gradients may be formed in paper-for example, the migration rate of solutes in paper with mixed solvents as wash liquids varies with the distance t>he solutes move ( 4 7 ) . Similarly, the trailing portions of the
31 sorption zones are often reduced by the use of mixed solvents and of water-saturated solvents (28,29,47,275,317). SORPTION GRADIENTS IN CHROMATOGRAPHIC SY STEhIS
As the effect of additives and of many mixed solvents may be considered the resultant of the formation of an adsorption gradient, it has seemed desirable to examine, qualitatively, the behavior of a solute in a column having greatest adsorption capacity a t the base and least a t the top. For purposes of illustration, a column may be assumed to have a nearly tenfold variation of adsorption capacity, as illustrated in Figure 11. If a solute with a nearly linear sorption isotherm (Figure 7 ) is adsorbed in such a column, the sorption zone will not migrate a t a uniform rate as is usually the case (71,150,161). Instead, the lower portions of the zone, having a greater proportion of solute adsorbed than the upper portion, will migrate a t a slower rate. The initial zone will be compressed and the solute will be Concentrated as indicated by Figure 12 (left).
d
TOTAL SOLUTE IN ZONE
Figure 12. Estimated Distribution of Solute in Chromatographic System with Sorption Gradient of Figure 11
For an initial zone occupying about one tenth of the column (Figure 12), the solute a t the upper boundary will be moving about 10% faster than the solute a t the lower boundary. As the solute is washed along with fresh solvent, it should form a narrower and narrower zone, the frontal and trailing boundaries remaining well defined. The greatest amount of narrowing or concentration should occur during the initial washing, but the percentage of narrowing should remain nearly constant, Figure 12 (center and right). For solutes with the usual curved adsorption isotherms (Figures 9 and lo), the distribution behavior in a column with an adsorption gradient would be more complex, especially if the gradient were not sufficient to offset the natural trailing tendency of the adsorption zone. THEORIES OF CHROMATOGRAPHY
There has been continued progress in the development and testing of theories of chromatography, which have been based on the assumption of equilibrium and of nonequilibrium conditions (30, 71, 150, 151, 213, 2Y8, 300, 30&306, 361, 387). These studies have contributed to an understanding of the kinetic processes involved in separations, and they have given clear concepts
ANALYTICAL CHEMISTRY
32 of the variability of adsorbents (200, 295). They have also revealed many variable conditions in the operation of columns (90, 966, 587). I n a recent study, some two dozen terms were required in order to encompass these variable factors, yet these conditions were not critical in the resolution of mixtures of similar solutes (lithium isotopes) (152). The separability of mixtures depends primarily upon differences among the partition or distribution ratios of the solutes in the nonmobile and the mobile phases (66). For distribution between liquid-liquid phases, the critical conditions are primarily solubility relationships (978, 9O4, 352). For liquid-ion exchange systems, the critical conditions are less clear, and for liquidadsorbent systems the critical conditions are obscure, for they depend upon the inherent selectivity of the solid phase itself (863-256). I n most chromatographic systems with a surface-active sorbent, the proportion of solute adsorbed increases a t low concentration (199, ,953-256). With nitroanilines adsorbed on silica gel, however, this proportional distribution is constant. Consequently, the theoretical treatment of these chromatographic systems is much simpler than that for systems with variable proportional distribution relationships (387). SORPTIVE PROPERTIES OF VARIOUS SUBSTANCES
Much effort has been spent in an investigation of the surface properties of various solids (108, 221, 996) such as precipitates (346), silicates (393), carbon (22, 55, 953-256, 267, S26, S97), silica (199, 253-256), titania (217), alumina (324, 325, 334, 335, 969, 378, SrQ), and manganese oxide (395). Attempts to characterize chromatographic adsorbents have not been concerned with specificity (295). Rlonolayers have been found to be weakly adsorptive (94,100,200). An interpretation of the slow penetration of solutes into resins has been based on the Donnan equilibrium ( 6 5 ) , a phenomenon involved in the penetration of ions through membranes and into living cells (32, 92). Surprisingly, ionic movement a t a phase boundary is nearly a million times slower than in homogeneous solutions (92). Adsorption has been investigated with radioactive indicators (131), and there have been additional studies of the adsorption isotherms ( 5 , 123, 506, 333). These results have demonstrated a remarkable difference between the selectivity of surface-active sorbents, as silica gel and charcoal, for the same mixtures of hydrocarbons (124,125, i z ) . TERMINOLOGY
The diversity and wide applicability of chromatographic methods have led to the development of numerous terms on which there is by no means universal agreement. Paper chromatography, for example, may refer to the use of cellulose in columns or to paper in sheets, strips, or stacks. A chromatogram in paper is a papergrani (317). A chromatogram from a column is an adsorptograni (97), a term that might well be condensed to sorptogram. Thjs would follow from sorptography, once considered but never proposed as a substitute for chromatography. Depending upon their use, liquids may be solvents, eluants, elutriants, wash liquids, developers (387), desorbents ( 9 7 ) , and displacents (76). Paper partition chromatography, normally applied to the use of a separate, fixed liquid phase in paper, has often been used for separations in the absence of such a phase ( 7 4 ) ,as with water or with water-miscible liquids for the mobile solvent phase. Even with water-immiscible liquids as solvent, a separate aqueous phase may not be present in the paper. This may sometimes be the case even if the wash liquid is saturated with water. When the paper is soaked in the aqueous phase before formation of the chromatogram (62,416) there is no doubt about the presence of a liquid-liquid system. Paper treated in this way is one component of a three-phase system, liquid-liquid-solid. These con-
siderations apply to all adsorbents and supports for liquid phasea as well as to paper (296, 974). Progress chromatogram has been introduced for fractional elution (177). Hot chromatography (395), suggested for chromatography a t elevated temperatures (380), is likely to be confused with the common term for intense radioactivity. In many instances, electrochromatography is more exact than electromigration, which includes electrophoresis and ionophoresis (106). Perhaps, by analogy with chromatography, it would be convenient to use electrochromatography for those separations in which a narrow zone of ions is caused to migrate through an absorptive, polyphase medium. Ionography, proposed for the separation of ions by electrical migration in paper (Mi?),had previously been applied to the chromatography of ions (198). The R value, a simple, precise, and useful term (373),is being modified in many ways. For paper chromatography, it is commonly represented by RF without change of meaning. The RF values of various substances such as sugars may be divided by the value for a standard substance such as glucose, thus giving the RQvalue (187). With the amino acids, leucine as reference gives analogous, modified R values (295). 4 more complicated , = log ( l / R - 1 ) . All these R values term is the R.V ( 2 3 ) . RM vary with the chromatographic systems employed for their determination. Because no single chromatographic system is available for determination of R values of many substances, comparison of R values is most useful among substances of similar chromatographic properties. CHROMATOGRAPHY BY ADSORPTION
The use of activated adsorbents provides one of the most selective methods for the resolution of mixtures of weakly polar substances. I n columns a few hundred grams of these adsorbents permit the resolution of several grams of most solute mixtures. Of the many available adsorbents, activated alumina (115, 166, 402) finds wide application, frequently in the fractional elution procedure. I t has been utilized for the analysis of mixtures of inorganic ions (338,343), particularly those in alloys of copper zinc and silver (564, 365). Sluniina finds use for determination of vitamin A and its ester (109, 110, 181), for isolation of anhydrovitamin A (345) and oxidation products of carotenes (400), for isolation of a carotenoid acid from a tubercle bacillus (389),and for preparation of carotenoids from the gonads of a limpet (154j. Other organic substances examined with alumina are: 2,4-dinitrophenylhydrazones of keto acids (90); radioactive phosphates of milk (368); the drug podophyllin (167); serum protein (402); the alkaloids colchicine (215), sparteine (246), and 1-methylpyrrolizidine ( 2 4 7 ) ; acylated uronides (163); amaroids of quassia (1); various glucosides ( 2 , 69, 116120, 194, 241, $70, 371); soybean sapogenols (288); biochemical oxidation products of ionone (334); dihydrolysergic acids (372); the desoxysugar, sarmentose (168); anthraquinone derivatives (369); various steroids (2.9,20,52,170,259,284) including cholestenone and testosterone labeled with isotopic carbon (390); amylase hydrolysis products of starch (396); various enzymes (419); black oils or asphalts ( 2 4 2 ) ; and fats and fatty acids (404). Activated charcoal has been utilized for isolation of dugars (4O3), for separation of amino acids by carrier displacement chromatography (383),for separation of aliphatic acids by displacement analysis (160, 184) and by the usual development (SO.??), and for investigation of high molecular polymers as nitrocellulose, dextran, and others ( 7 2 ) . Magnesium silicates have served for isolation of alkaloids (411 ) and various nitrogenous compounds from shale oil (307). Permutit has likewise been employed for the preparation of radio-active alkaloids from plants (270). Calcium carbonate has been utilized for the adsorption of enzymes (196), vitamin A ( S d O ) , naphthoquinone pigments from sea urchins (153), frangula extract (204), the phenylosazones of the common sugars ( 1 2 6 ) ,and urinary porphyrins (146).
33
V O L U M E 23, NO. 1, J A N U A R Y 1 9 5 1 Various salts have been employed for separation of amino acids @SU, .2s4), carotenes ( l S O ) , acylated carbohydrates (410), and peroxides (Iff). Lime (69, 211, 22 a), magnesia (75, 85, 223, S66), and zinc carbonate find extensive use for separation of carotenoids, whereas sugar is usudly employed for chlorophylls (273,S74). Talc has been employed with urinary porphyrins (146) and with abalone-shell pigment (82), and starch has proved useful for isolation of amino acids (41, 296,319,320,406). Silicic acid, owing to its preferential attraction of olefinic hydrocarbons, is finding wide use in the fractionation of petroleum (76, 97,1W,Ii?&1.25, 155, 172, 55S, S95). It also finds use with various substances such as proteins (950), organic acids (25, 281 ), nitroanilines ( S 8 7 ) , 2,Pdinitrophenylhydrazones (159), ethers and sulfides (195), various hydroxyanthraquinones (191). For latter, magnesium carbonate has been employed (189). CHROMATOGRAPHY BY ION EXCHANGE
Synthetic ion exchangers continue to find wide application for the resolution of mixtures of inorganic substances (as the rare earths and the heavy synthetic elements) and of ionizable organic substances. Progress has been made in the preparation of sintered colloidal resins, in the preparation of uniform membranes (605, 228), and in the definition of conditions under which the various groups in resins may be active (398). Other hydrated dissociated gels as the alginates (294) and the soil colloids (231, 23.2) exhibit exchange properties like those of t.he synthetic resins (gs, 104, 112,161, 232).
An increasing amount of evidence indicates that the sorptive forces in an exchanger may be very comples, involving van der Waals attractions (301, 398) and covalent bonds (231 ). Variation of the combining or exchange properties of resins with variation of the degree of cross linking (18, 95, 98) may be ascribed to the differences in hydration as well as to the different porosity. For most chromatographic separations particles of small dimensions are desirable (58). Ton exchange in resins has been investigated as an equilibrium or kinetic phenomenon (204, 140, 141, 161, 625, 229) and as a statistical phenomenon ( 9 3 ) . The hydrated resin may be treated as a solid solution (116, 186) exhibiting the Donnan equilibrium phenomenon (66). Equilibria with resins have yielded dissociat,ion constants of radium-organic acid complexes (S42) and have demonstrated the formation of colloids (radiocolloids) of zirconium and niobium (S41). Cation exchangers in columns have proved useful for separation of heteropoly acids and salts (16,262),for isolation of fission products without carriers (385), for separation of iron and titanium (415), and for the rapid separation of ferricyanide from ferrocyanide ( 7 7 ) . Cation exchangers have served for separation of rare earths (561-363) (on both an analytical and a preparative scale), for the analytical separation of sodium and potassium i n the presence of magnesium (33),for the microseparation of larithanum and yttrium (198),for investigation of the sodium-hydrogen exchange system (106), and for investigation of the absorptioii of vanadium (207). The heavy isotope of lithium (Li’) was slightly concentrated a t the leading boundary in a column of acidic exchange resin (152). An interesting and important development lies in the use of strong hydrochloric acid to vary the absorbability of the heavy elements on anion and on cation exchangers (192, 266, 377, 360). I n the latter case, there is a reversal of the absorption or elution sequence with strong acid, so that americium and curium were separated from the rare earths in a single column (1 mm. by 10 cm.) ( 3 7 4 ) . Berkelium was isolated by elution with concentrated hydrochIoric acid followed by readsorption and fractional elution with citrate a t p H 3.5 (980). Anion exchange resins have been useful for the isolation of ribonucleotides (64, 78-80, ass), their isomerization products (7B, 8631, adenosine polyphosphates (81),and leucine (349).
Acid exchangers have been used extensively for isolation of amino acids by flow of solvent (61, 73, 19S, $11, 401) and by displacement development (SO9, S I 4 ) . Basic exchangers aid the separation of acidic amino acids (308, 310). Other uses of exchange resins are: purification of glycerol (206). isolation of C14-glyceric acid ( 1 d d ) , and sorption of acids, bases, and alkaloids (301) of various milk constituents (142) and of eggwhite protein (558). CHROMATOGRAPHY BY PARTITIOS
Partition chromatography is applicable to the investigation of all substances that can be distributed between two immiscible liquids (14, 179, 951, 405). Already a number of liquids have been employed, and a great many more show promise for use with mixtures of various substances (251, 405). Many publications describing the use of water-immiscible solventg with papeiare summarized in the section devoted to paper chromatography (179). Organic acids have been separated by partition between silica gel plus water and chloroform (28s))by partition between Celite plus methanol and petroleum ether (56), by partition b e h e e n silica gel plus water and butanol plus chloroform (408),by partition between hydrocarbons on treated Hyflo Super-Gel and aqueous methanol and acetone (160), and by partition between hydrocarbons (benzene) plus vulcanized rubber and methanol plus acetone and water (39). The red eye pigment of Drosophila has been chromatographed in a column of silica gel with moist butanol as solvent ( 1 7 1 ) . Other substances sPparnted by partition are the methylated lructoses between water plus silica gel and toluene plus 0.33Tc ethyl alcohol ( 2 6 ) ,dinitrophenyl amino acids between chlorinated rubber and but,anol-saturated buffer ( S I S ) , anthocyanidins between 10% orthophosphoric acid plus silicic acid and butanol plus ether (360), tertiary amine salts of the penicillins between water plus silica gel and ethyl acetate (2&), alkylation producte of 3,5-dinitroaniline between strong aqueous acids and chloroform (SSO), and various amino acids (14) and biologically active substances from liver between moist silica and butanol and between moiet silica and propanol, the last eliminating the aqueous phase but still effecting separations (354). CHROMATOGR4PHY WITH LMPREGYATED PAPER
Paper has been impregnated with resin for separation of ions ( 2 2 8 ) ,with silicic acid for separation of 2,4-dinitrophenylhydrazones (618), with alumina for separation of ions by capillary analysis (1a?‘), for separation of vitamin A alcohol and its esters (91), and for separation of steroids ( 5 8 ) , with Quilon (stearato chromic chloride) for separation of tritiated sterols by reversal of phase (233), and with formaniide or propylene glycol for separation of sterols without reversal of phase. Addition of Burface-active substances changes the electrical charge of paper, thus facilitating the absorption of charged substances (337). PAPER CHROIM~TOGRAPHY
There has been a notable increase in the publications concerning paper chromatography (74,S?S), particularly in biochemiatry. Studies of the properties of paper have revealed most analytical papers suitable for chromatographic separations (R15). T h e kinetics of paper chromatography (300) are identical with those for other chromatographic systems. -4s reported long ago by Kolthoff, paper acts as an ion exchanger. I t absorbs radioactive calcium and lead (ThB tracer) reversibly and exhibits an equivalent Feight of about 20,000 (62, 239). I t s absorptive properties for ions are improved by boiling with dilute nitric acid ( 5 7 ) . I n the inorganic field, cellulose in columns ( 5 7 )and paper nerve for separation of nickel, cobalt, copper, and iron (5?’),for estimation of potassium (24),for separation of nickel and cobalt
34 (?237),for separation of aluminum, cadmium, arsenic, iron, and copper by two-way chromatography with collidine and butanol ( I l J ) , and for separation of elements of groups 1, 2, and 3 with butanol plus acetic acid (132). Xumerous organic subst,ances have been examined in papere.g., adrenaline (348) and noradrenaline ( I N ’ ) , various acyl hydroxamates in water-saturated butanol (366), various keto acids in butanol plus water and formic acid (275),and lower fatty acids in butanol in the presence of ammonia or ethylamine vapor (180) or in butanol saturated with 1.5 N ammonia ( 5 3 ) . Steroids examined in paper were: tritiated cholesterol and cholestenone in methanol and ethyl alcohol (233),adrenal cortical hormones in benzene or toluene with formamide or propylene in the paper (416), progesterone separated from fat with 80% ethyl alcohol ( l 6 7 ) ,separation of estrone, estradiol and estriol, etc., after coupling witn diazotized p-nitrobenzeneazodimethoxyaniline (169),and adrenocorticotropin (143, 144, 249). Soluble carbohydrat,es (38, 188, 307, 386), their methyl derivatives (188), and the sugar alcohols (186) are readily separable in paper with a variety of solvents. Carbohydrates of plants ( 4 , 4.4, 391) and of hydrolyzed polysaccharides (174, 178, 187, 202, 339) were readily separable in paper. Flavonol-3-glycosides, as rutin, quercitrin, and isoquercitrin were also separated (138). Organic products of photosynthesis consisting of carbohydrates, amino acids, and hydroxy acids with their phosphorylat,ed derivatives, all made detectable through assimilation of Cr402,have been resolved and identified by paper chromatography (10, 21, 27, 60, 891). Yarious antibiotics such as neomycin B (328), streptomycins ( S l y ) , and their alteration products (3.59, 407),penicillins (149, 210, 381 ), and Chloroinycetin (chloramphenicol) (148, 355) have been isolated in paper chromatograms. Nucleic acids from yeast, (62, 68, 276, 356), from muscle (68, 276), and from tobacco mosaic virus (280) were isolated in paper (S94). Development with 0.8 N hydrochloric acid in 70% watermiscible tert-butanol gave quantitative recovery (356). Paper has also served for isolation of adenosine triphosphate (3),for examination of synthetic purines (347), for following photodecomposition of the phosphorylated compounds in ultraviolet light ( 6 3 ) , and for observation of enzymatic exchange of purines and pyrimidines linked to the desoxyribosyl group (271). Phenolic substances including the tea catechins were readily separable in paper with water-immiscible solvents (23, 43, 45, 121, 261). Other substances adsorbed in paper were: basic and direct dyes from water and from mixed solvents (325); acridine dyes from water (243); anthraquinone pigments ( 3 5 1 ) ; groups of homologous compounds (321 ); curare alkaloids from mixed solvents (340); vitamins Bl2 and B1U (422); xanthopterin (116); hydroxysulfapyridine and derivatives ( 4 6 ) ; porphyrins of acute porphyria ( 2 7 2 ) : glucose from hydrolysis products of liver glycogen after rats were fed KaHCl4O3( 1 4 5 ) ; various alkaloids (302); and sugars in the hydrolyzate of tomatin (266). Amino acids and proteins are two of the most important constituents of living cells, and paper chromatography has provided many new approaches to the investigations of these substances (157,203). Progress in t,he paper chromatography of these nitrogenous compounds can be att,ributed largely t,o their reaction with ninhydrin, which provides a rapid, convenient, and extremely sensitive method for their detection in the paper itself (11, 128, 139) or in extracts ot the paper (269). Amino acids dried in paper yield fluorescent spots that remain after extraction with water and serve for location and identification of the acids (139, 166, 316, 409). The .V-dinitrophenyl derivatives of amino acids are also resolvable in paper (36, 290, 295), and the amino acids with radioiodine (158, 245,384), sulfur (216), or both (116) are easily located by autographs. A novel method for detection and estimation of amino acids depends upon treatment of the unknown mixture with radioiodine-labeled p-iodobenzenesulfonyl chloride. The products are
ANALYTICAL CHEMISTRY then com-oared chromatographically with a similar mixture prepared from authentic amino acids with radiosulfur-labeled p iodobenzenesulfonyl chloride. Measurement of the radioactivity of the sulfur and iodine atoms provides an indication of the amino acids and of their relative amounts (216). Although most separations of amino acid mixtures in paper have been effected with mixtures of water-immiscible solvents ( l 2 8 ) , water-miscible solvents have also yielded excellent results (28, 29). Addition of various solutes often improves the definition of the zones (28, 29, 31, 158, 295). The tendency of lysine to form diffuse zones varies with the acidity before development (9). Paper chromatography has given information concerning the nitrogen metabolism of the thyroid (216, 245, 384), of enzyme of bacteria (88), of virussystems (17, I T ? ) , of plant stems (VI), infected eggs ( l l 4 ) , of nuclear sap (54),of liver mitochondria (297),in liver disease ( 9 5 ) ,and of alanine and taurine in the rat ( I S ) . It has revealed aminoethylphosphoric ester in rats and in human tumors ( 1 5 ) , various amino acids in rat tissues (fa),yaminobutyric acid in yeast (327), a-,e-diaminopimelic acid in bacteria (413), and various amino acids in silkworm eggs (103),in frog embryos (257),in urine ( S I , 6Z.4), and in fetal and maternal plasma ( 8 7 ) . I t has revealed biochemical differences among humans with respect to the urinary excretion of amino acids (31). Paper chromatography has facilitated the isolation of peptides as well as of amino acids (203) as in animal organs (134), enzyme and syntheses (49,162)analogous to solvolytic reactions ( 1 7 , Is%’), chemical preparations (164). It has aided the isolation of the hydrolysis products of proteins (251, 312) as ferritin (I%’), pituitary protein ( 3 7 ) , gramicidin (175), lipovidase (185), egg albumin and others (308, 318), horse-radish peroxidase ( 2 7 4 ) , tobacco mosaic virus protein (367), insulin (13S), the mitochondria of tissues (258), and the proteins of soil (48). It has been employed for isolation of proteins themselves (129, 201 ). CHEMICHROMATOGRAPHY
The use of %hydroxyquinoline for the resolution of inorganic ions provides a convenient method for estimation of various elements by observation of the length of the sorption zone (3’31). Selective reversible reactions, such as that between urea and hydrocarbons, show promise as the basis of new chromatographic systems (420). LOCATION OF RESOLVED SUBSTANCES
The chromatographic investigation of various substances has required a diversity of methods and reagents for detection and estimation of the resolved materials. These methods fall into two principal groups: those based upon physical properties and those based upon chemical properties. The former include fluorescence, radioactivity (21, 27, 42, 60, 75, 122, 145, 152, 198, 216,226,233,245,374,380, S84,385, 391), photography and photoelectric methods (102, 166, 344), indicators used with organic acids ( 7 ) , interferometry (102, 166, 344), and various visual methods including staining techniques (39, 201 ). Chemical methods for streak or brush methods include many colored and color-producing reagents (248, 419). I n paper chromatography there is a list of these chemical reagents nearly as long as the kinds of substances investigated, and these can easily be found by reference to the original papers. Reagents most widely used are ninhydrin, ammoniacal silver solutions (203), permanganate, iodine (279), and various colored derivatives. PROGNOSTICATIONS
Owing to its speed, selectivity, sensitivity, adaptability, and reliability, chromatography is certain to reveal numerous new phenomena that will challenge the analytical skill and the interpretative capacity of scientists. The value of chromatography
V O L U M E 2 3 , NO. 1, J A N U A R Y 1 9 5 1 lies in this promise of future usefulness as well as in its demonstrated aid t o scientific advance. LITERATURE CITED (1) Adanis, R., and JVhaley, TI-. RI., J . Am, C'hem. Soc., 72, 375
(1950). (2) Aebi, A , and Reichstein, T., Helu. Chim. A c t a , 33, 1013 (1950). (3) Albaum, H. G., Ogur, M.,and Hirshfeld, A,, Arch. Biochem., 27, 130 (1950). (4) Albon, N., and Gross, D., A n u l y s t , 75, 454 (1950). , 432 (1950). (5) Alexander. A . E . , and Posner, A. AI.,N a t 7 ~ r c 166, (6) hiion., Endeacorcr, 9, 53 (1950). (7) Anon., .J. ( ' h e m . Edztcutior~,27, 281 (1950). ( 8 ) Anon., Sufritiori Rea., 7, 195 (1949). (9) Aronoff, d.,Science, 110, 590 (1949). (10) AronoiT, S.,and Veriioii, L., Arch. Biochtm., 27, 239 (1950). (11) Atkinson, R. O., Stuart, R. G . , and Stuckey, R. E., A n a l y s t , 75,447 (1950). (12) Awapara, J . , .J. B i d . C ' h e n i . , 178, 113 (1949). , 76 (1950). (13) Awapara, J., S o t i ~ r r 165, (14) Awapara, ,J., Tecus R c p t s . Uiol. M e d . , 8, 117 (1950). (15) .-\napara J., Laiidua, A. J., and Fuerst, R., J . B i d . C'hcrn., 183, 545 (1950). (16) Baker, L. C'. It-., I,OEY. 13., and 3IcCutcheoii, T. P., J . Ana. Cliern. SOC.,72, 2Yi-4 (1950). (17) 13altaly, R., Berger, I. M.,and Rothstein, A. A , , I t i d . , 72, 4149 (1950). (18) Barrer, It. IT.,DiscussiorLs Faraday Soc., 7, 135 (1949) (19) Barton, D. H. I
