HAROLD 8. CASSIDY Yale University, New Haven, Connecticut
CHROMA TOG RAP^ is a method by means of whi::h mixtures may be separated so that under favorable conditions the components are obtained pure. But since there are so many kinds of mixtures, and so many different processes that may be used in analyzing them, it is necessary to look a t the entire realm of analysis in order t o see clearly the special features that distinguish chromatography and the various chromatographies. There are three kinds of analysis: qualitative, quantitative, and relationship (or structural) analysis. These can be sufficiently clearly marked off from each other under normal conditions that they can be used for classification. (There are situations in which it may be difficult to make a clear distinction between any two, but these are not of particular importance here.) Chromatography is a form of qualitative analysis. As with all qualitative methods chromatography may he made quantitative, but the method, in its strictly chromatographic aspects, only separates mixtures and tells neither how much of each component is present nor the relationship of that component to the others in the original mixture. It is important to notice this because the apparatus and procedures of chromatography are essentially very simple. The quantifying accessories are usually of necessity complicated. They are important and necessary parts of the apparatus, but are only adjuncts to the chromatography. This has t o be emphasized because when this distinction is not made the chromatographic parts of analyses often disappear under the tremendous variety and complexity of the associated instrumentation, and what is essentially simple from a functional point of view becomes hidden in a mass of contrivances. Qualitative analysis (as well, also, as quantitative and relationship analysis) may be applied to all kinds of mixtures. These may be classed for convenience as mixtures of bulk-level matter, as molecular mixtures, and as submolecular level compositions. These classes are not rigidly defined, since some large molecules approach particulate bulk (colloidal) matter in size, and since separations of ions are much more conveniently classed as molecular than as submolecular Presented as part of the Symposium an Vapor Phase Chromatography before the Division of Analytical Chemistry a t the 129th Meeting of the American Chemical Soci~ty,Dallas, 1956, snd taken largely from the manuscript of a new book on chrome tagraphy now in preparation. 'Communication No. 1370 from the Sterling Chemistry Laboratory, Yale University.
species separations. This classification is for the purposes of utility rather than for the files of the formalist. Chromatography belongs among the methods applicable to molecular mixtures. For this reason it is wrong to speak of chromotography as a filtering process. Filtration applies to the separation of bulk particulate matter (see Table 1) and it is misleading to speak of filtering molecular mixtures apart in a chromatography apparatus. This means, also, that in order to investigate a substance chromatographically it must be reduced to a molecular mixture. This is sometimes not ensured, and may account for some of the nonmoving material occasionally found at the top of a column or a t the site of the initial zone on paper, whence the mention here. There are in general two types of qualitative analytical methods for separating n~olecular mixtures: the chemical and the physical. Through both of these methods the object is to convert the molecular mixture into new mixtures of more desirable compositions, or in the most favorable cases into pure const~ituents, which are separable by bulk procedures. For example, a chemical separation of a molecular mixture of silver as the nitrate from sodium nitrate might depend on plating out the silver, inducing it to deposit on a bulk electrode that may later be removed mechanically from the system. An example of a physical method is found in distillation. The mixture to be separated is partially volatilized under conditions of temperature (and reflux, perhaps) that produce a vapor of different composition from the liquid, then these two phases are mechanically separated, usually by condensiug the vapor separately. Put in general terms, the homogeneous molecular mixture (a phase) is converted to a heterogeneous system, the phases of which can then be separated by mechanical means. This is typical of most of these separations at the molecular and submolecular levels. The distinction between chemical and physical classes (Table 1) is convenient and not absolute. In the former, the interactions that lead to separation are chemical in nature; in thelatter, physical. Chromatography belongs among the physical methods, though as Tswett pointed out in the early 1900's there is no real reason why there may not be developed chromatographies that depend on chemical reactions. Some of these are, indeed, known. Among the qualitative methods that depend pri-
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TABLE 1 The Field of Analvsis Analysis
I . Quantitat~ve
Q~sl!~stive
Relatio!nship
May be a plied to
f
. Molecular1 m~xtnres
Mixtures of bud-level materials I
I
Methods of sepktion involve:
Methods of separation rely on
1
Sorting, filtering, mechanical I operations of all kinds physical interxtions
I
Distribution hetweenphases
ChemicalIresctions
II Thdmal analysis
Gravitat~onal 1. analysis
~ubmolecular-levelbornpositions Methods of sepmsdon involve breaking of chemical bonds
I
Electrophoretio analysis
(See ~ a 6 l 2) e
marily on physical interactions me can list a group that utilizes distribution between phases (Table 1). Chromatography falls in this .group. The group (Table 2 ) contains two classes of distribution systems: those that involve two bulk phases; and those that involve a bulk phase and a thin phase or film. The first kind includes distillation, absorption, extraction, crystallization, and sublimation. The second kind includes all the chromatographies. It should be pointed out that although only distributions between two phases are classified here, the classification may be extended t o more phases. Further, as with the previous categories, there are regions of overlap. For example it may be difficult to decide in certain cases how thick a film must grow to be before it is classed as a bulk phase. Also, the classification does not include combinations of two or more methods into one, as in extractive distillation. Such possible combinations can be derived from these tables. The nature of chromatography is not fully delineated until one further characteristic is brought out. The pairs of phases of all kinds, listed in Table 2 , can he brought into contact (so that the distribution can occur) in two basically different ways: in batchwise (which includes for our purposes cocurrent and cascade) and in differential countercurrent contacting. It is the second of these methods of contacting that characterizes chromatography. It can he understood, from Table 2, why it is that "chromatography" is such a tremendously extensive and varied field. Whereas among the systems that are produced by distribution between two bulk phases that are contacted in a differential countercurrent manner each system has a different name: distillation under reflux, extraction in a packed tower, etc., when a bulk phase and a thin phase are utilized all the systems are called chromatographic. The field of chromatograpy is therefore comparable in extent to that which includes all the others.
1
Electromagnetic analysis
Mass spectrographic analysis (some kinds)
DEFINITION OF CHROMATOGRAPHY
We are now in a position to define chromatography and to classify the different kinds of chromatography under the generic definition. Chromatography comprises a group of methods for separating molecular mixtures that depend on distribution between a bulk, usually mobile phase and a thin, usually stationary phase. These are brought into contact in a differential countercurrent manner. I n most chromatographic methods, the bulk mobile phase flom over the thin stationary phase. I n all cases the molecules of the mixture to he separated distribute between the two phases. Those that tend more to the stationary phase are retarded with respect to those that tend more to enter the mobile phase; the latter thus move away, in the stream of mobile phase, from the former and become separated. The mobile phase may be a solvent, developer, eluent, or displacer depending on its function. When the bulk mobile phase is a gas, or vapor, and the thin stationary phase is a liquid film held stationary on a support, the system comprises "gas-liquid partition chromatography" (GLPC) or "gas partition chromatography" (GPC). When the bulk mobile phase is a gas and the stationary phase is at the surface of an adsorbent, the system is "gas adsorption rhromatography" (GAC). The two may not always be distinguishable with absolute certainty, in which case a more noncommittal term "gas chromatography" may be used. When the hulk mobile phase is a liquid, and the thin stationary phase is a liquid film, a gel, or imbibed layer, the system is "liquid-liquid partition chromatography," or "partition chromatography," a special rase of which is "paper partition chromatography." If the stationary phase is furnished by the surface of a solid adsorbent, the system comprises classical "adsorption chromatography," or Tswett-column analysis. "Ionexchange chromatography" falls also in this group. These may not be clearly distinguishable under all
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TABLE 2 Cross-table of Phase-pair Distributions, and the Methods that They Generate --
Bulk phases Lipid (L)
Bulk phases
Solid ( S )
Gas or vapor ( G )
Liquid ( L ) Solid ( S ) T h i n phases Liquid flm or "mobile" inkrfaeial filni ( M )
! I !
Distillation, Absorption
L-L Extraction absorption
Sublimation
S-L Crystallization
Gas partition chromatography ( G P C )
M-L Liquid-liquidpartitionchromatog~shy, foamand emulsion chromstograpk
Gs
M-G
S-S (Enfleurage)
In this rase an artificial interface, a membrane or barrier, must be put between the phases.
circumstances, for, as Tiselius pointed out, if an adsorbed film becomes thick enough the conditions for partition chromatography may arise. Finally, if the hulk phase is liquid and the thin film is the interfacial region formed at the surface of bubbles of gas, or droplets of liquid passing through the hulk phase, we have foam or emulsion rhromatography, as the case may he. These six types comprise all the kinds of chromatography that differ because of the phases employed. There are, however, differences in technique that enable a further differentiation between chromatographic methods. I n principle, all of the above kinds of chromatography may be carried out by the techniques of frontal analysis, development and elution analysis, displacement and carrier displacement analysis, and gradient elution analysis. Frontal analysis is an old method of using a bed of adsorbent. The mixture to be treated is passed into the bed until break-through occurs, when a front of the least strongly retarded component of the mixture issues from the bed of stationary phase, followed by a front containing also the next least strongly held component, and so on, with, in favorable cases, one front for each component (including the solvent). The very old method of "capillary analysis" belongs in this category. In development analysis, an amount of the mixture to be analyzed that is small relative to the capacity of the bed of stationary phase is applied to the bed. The developer then separates the components of this mixture out, as described above, to form zones of the components in the bed. (The bed of stationary phase may, of course, he a strip or sheet of paper.) In elution analysis these zones are made to issue from the bed one after the other. Displacement and carrier displacement analyses employ more strongly adsorbed substances, added t o the mixture and already present in the mixture, t o displace less strongly adsorbed ones, so that the zones move along the bed and out of it in a compact array. Gradient elution analysis employs a
developer with gradually increasing eluting power to crowd the rear part of each zone toward the main body of the zone, thus decreasing the ill effects of tailing. These names were given to the methods by Tiselius and his co-workers, who invented the displacement techniques. I t has been pointed out that in all kinds of chromatography the distribution process that leads to a separation occurs between a bulk phase and a thin phase in differential countercurrent contact. A consequence of this method of contacting is that there is never any over-all equilibrium in the system (though a steady state may be achieved temporarily and locally). A further conseqnence is that as one phase is a thin film, and hence a region of somewhat limited freedom for the molecules that enter it, geometrical factors may become important in influencing the separation. This is particularly so when the thin film is a t the surface of a solid adsorbent, for here not oniy is the film thin, often of molecular thickness, hut also the surface of the adsorbent is practically always inhomogeneous in terms of interactive sites as well as of topology. This may explain the very subtle differentiations that occur between molecules admitted to the stationary phase and those largely excluded from it. GAS CHROMATOGRAPHY
A special feature of gas adsorption and gas partition chromatographies is that the distribution picture is greatly simplified, especially in the latter case, compared with the chromatographies that employ hulk liquid phases. Where the mobile phase is a hulk liquid, the separation depends on competitive interactions between the molecules of the mixture and those of the mobile and stationary phases. (The competitions involve, also, possible associative effects.) Further, there is an additional competition for the stationary phase by the molecules of the mobile phase. While these three competing interactions add a tremendous range of sensitivity and subtlety to these chromato-
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graphic methods, they do complicate prartice and theory. In the gas-liquid partition method two of thesc competing interactions virtually vanish. The carrier gas (the bulk mobile phase) is usually chosen to he virtually inert toward both the stationary phase and the mixture to he separated. Hence there are only the interactions between the molecules of the mixture and those of the stationary phase to be ronsidered in theory and practice, a t least to a good first approximation. (For this reason the mutual interartions of different kinds of molecules in the mixture may make their effects more noticeable.) It therefore is easier to see, in gas-liquid partition chromatography, the operation of dispersion, dipole-dipole, polarizability, and hydrogen bonding interactions as they operate in the separations of diffcrent mixtures on different stationary phases. The gas chromatographies are heing rapidly developed, and a great service would he done to workers in this field, as well as to newcomers, if suitable nomenrlature were agreed upon. At present, a t least
485
four different names are heing used for gas partition chromatography, and two for gas adsorption chromatography The inventors of the partition method, Drs. A. T. James and A. J. P. Martin, called it gasliquid partition chromatography, a name shortened by the Shell Development Group to GLPC. In the discussion during the last session of the Dallas symposium it seemed generally agreed that suitahle names for the t.echniques are "gas partition chromatography," and "gas adsorption chromatography," with the more general term "gas chromatography" to include To these names can he added the terms that qualify the terhniques of operation, if that should be necessary, namely frontal analysis, elution analysis, displacement analysis, and gradient analysis. In the last case the type of gradient, whether concentration, temperature, or whatever else, may have to be specified. General willingness to use these names would contribute to the rlarity of the growing literature on gas chromatography. Letter from tho chairman of the Symposium, Dr. Charlo8 R. Willinghnm.
