A group of papers presented
Separation through Differential Migration Analysis
before the Division of Analyti-
cal Chemistry at the 134th Meeting
of
the
American
Chemical Society,
Chicago,
Ill., September 1958
Requirements for Analysis by Differential Migration HAROLD H. STRAIN Argonne National laboratory, lemont, 111. ,Differential migration is the basis of several widely applicable techniques for the resolution of mixtures of chemical substances. It also serves for the description and comparison of the substances themselves. For examination b y differential migration, substances must be molecularly dispersed as in a gas, vapor, or solution. Permeable migration media, ranging from a vacuum to stabilized solutions, are required. For the most extensive separations, the mixture must be added to the migration medium as a narrow zone. To promote migration, a driving force is required, and the resultant migration is usually opposed by a resistive action. The driving force, the resistive action, or the resultant of these effects must produce selective or differential migration of the components. Two driving forces may be applied transversely, providing two-way, discontinuous separations from spots of the mixture or continuous separations from a narrow stream. Substances separated by differential migration may be located and estimated by methods that vary with the substances and the properties of the medium. Substances may be described, compared, and identified b y their rates of migration or migration relative to other substances. Migration methods may be varied in so many ways that there is no systematic basis for classification of all variations.
T
Symposium on Differential Migration Methods of Analysis was designed to illustrate the importance of differential migration effects in analytical chemistry. Although these techniques are of great usefulness to analytical chemists, many of the methods were developed in other fields. For example, chromatography was devised and refined HE
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ANALYTICAL CHEMISTRY
in botany and plant physiology. Two of its important modifications,.paper chromatography and gas partition chromatography, were first demonstrated in organic chemistry and biochemistry. Electrochromatography (zone electrophoresis) was worked out in biochemistry and inorganic and clinical chemistry. Continuous electrochromatography was a product of biochemical and inorganic investigations. Liquid-liquid partition, partition chromatography, countercurrent distribution, and density-gradient centrifugation were developed in biochemical research and mass spectrometry was developed in physics, particularly nuclear physics. Many of these techniques have only recently been adopted in conventional analytical chemistry, and few have been included in formal courses of instruction in analytical chemistry. Differential migration methods of analysis serve primarily for the resolution of mixtures of chemical substances. They permit comparison, description, and isolation of the constituents of multicomponent mixtures. They are applicable to the investigation of virtually all kinds of chemical substances, including unknown or undescribed compounds. They are useful in all branches of science concerned with chemical compounds and their reactions. Because of their usefulness as exploratory or research tools, they have found their greatest application in the hands of specialists in various areas of research concerning chemical substances and their reactions. These differential migration methods are very simple in principle, but they are endlessly variable with respect to modification and application. Consequently there have been few attempts to systematize the basic principles and the requisite conditions (1-8). This paper describes the important features that are common to all differential mi-
gration methods of analysis. Special modifications and applications of the technique are illustrated by the other papers of the symposium. REQUISITES FOR
DIFFERENTIAL MIGRATION
Differential migration methods of analysis and some of their principal requirements are summarized in Table I. All these methods require the use of molecularly dispersed mixtures, in the form of a gas, a vapor, or a solution. Mixtures of solids or of liquids should be vaporizable or soluble without chemical change or alteration. As most substances may be dissolved, whereas fewer substances may be vaporized, migration methods that utilize solutions (solution chromatography, electrochromatography, batchwise countercurrent partition, sedimentation, and diffusion) are more widely applicable than those that utilize gases and vapors (gas chromatography, mass spectrometry, and thermal gravitational diffusion) (2). All differential migration methods of analysis require the use of special, permeable, migration media. These media include a vacuum, as in mass spectrometry, gases, as in gas chromatography, and solutions, as in solution chromatography, electrochromatography countercurrent partition, differential sedimentation, and diffusion. With many differential migration methods of analysis, the migration media must be stabilized to prevent mixing of the migrating substances. In electrochromatography and diffusion, for example, paper and gels serve as stabilization agents. I n electrochromatography and differential sedimentation, density gradients also serve as stabilization agents. With many of these methods, the stabilization agent has little or no effect upon the migrating substances. With chromatographic methods, how-
ever, the powdered or fibrous sorptive media not only stabilize the flow of gas or solution but also effect the separations through their selective sorption of the migrating substances. For the migration of the constituents of mixtures, a driving force is required. It may be nonselective, as flow of gas in gas chromatography and as flow of solution in solution chromatography (produced by gravity, suction, pressure, or centrifugal force). It may be selective, as direct current electrical potential in mass spectrometry and in electrochromatography, as centrifugal force in differential sedimentation, and as thermal diffusion in thermal gravitational diffusion. The migration of the molecules usually encounters a resistive action in the migration medium. This resistive effect may be negligible in a vacuum, as in mass spectrometry. But under other conditions, it may be very great (nearly large enough t o stop the migration), as in some modifications of chromatography. It is nonselective in some circumstances, as in certain modifications of electrochromatography, yet in other circumstances it is the basis of the selective migrations, as in chromatography and some modifications of electrochromatography. The driving force, the resistive action, or the resultant of these two effects must produce differential migration of the components of mixtures. Driving forces may be utilized in various geometric arrangements to improve the separations. For example, two different driving forces may be applied transversely and in succession, as in mass spectrometry, or two resistive forces may be utilized transversely and in succession, as in tn-0-way paper chromatography. Two different forces may also be applied transversely and simultaneously as in batchnise separations by chromatography plus electrochromatography and in continuous separations by continuous electrochromatography . In all differential migration methods of analysis, the dimensions of the initial zone of the mixture determine, to a large degree, the extent of the separations. The over-all separation depends upon the relative rates of migration, the dimensions of the initial zone of the mixture, and the distance of migration. If the initial zone of the mixture is w r y wide or continuous, the components form overlapping zones with only partial separations a t the leading regions of the zones. Examples of these partial separations are found in capillary analysis, break-through or frontal analysis, the moving boundary method of electrical migration or electrophoresis, and most diffusion methods. But if the initial zone of the mixture is narrow, the components will yield separate zones each
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containing a single constituent of the mixture, as in chromatography, electrochromatography, etc. (see Table I). An exception is diffusion. The definition of the zones of migrating substances varies with the migration systems. K i t h some systems, as in partition and ion exchange chromatography, the zones are usually diffuse a t both the leading and trailing boundaries. With other systems, as in adsorption chromatography, the zones are very diffuse a t the trailing boundaries but very sharp a t the leading boundaries. The rate of migration of a substance serves as a basis for its description and its comparison with other substances. I n many systems, the rate of migration varies with many different conditions, such as the intensity of the driving force and the resistive action, the concentration of the migrating substance, the temperature, and the permeability of the migration medium. All these conditions must be carefully standardized and specified, if the migration rate is to represent reproducible characteristics of the migrating substance. In solution chromatography, the rate of migration of the solute relative to the migration of the wash liquid, the R or R F value, is widely employed for the description of all kinds of soluble substances. The rate of migration relative to an authentic reference substance and the migration sequences of various substances (which may vary with the migration conditions) (3) are widely employed for the description of unknown substances separated by chromatography. The quantity and the concentration of the mixtures that may be separated by differential migration methods are limited by the migration systems themselves. As a rule, the upper limits of quantity and concentration are small compared to chemical methods of separation. But the lower limits are usually much lower than those encountered in chemical separations. I n fact, most of the differential migration methods permit separations of the smallest quantities that can be detected by the most sensitive tests. For this reason, differential migration methods are remarkably effective with substances that are found in minute quantities-for example, the synthetic transuranium elements, fission products, neutron activation products, vitamins, hormones, and enzymes. Most differential migration methods must be supplemented with detection techniques for the location, detection, and estimation of the separated substances. With separatory methods based upon the use of solutions, wherein the substances are diluted with many molecules of the solvent, these detection techniques may depend upon various characteristics of the separated substances, such as their nuclear, physical, chemical, 820
ANALYTICAL CHEMISTRY
3Solvent
or biological properties (2). With gas chromatography, wherein the substances are diluted with relatively few molecules of the wash gas, several detection techniques, such as the gas density balance and the ionization of the gas fractions, serve as nigh universal procedures for the detection of all kinds of gaseous substances. With mass spectrometry, the detection of the charge carried by the separated substances is an extremely sensitive and universally applicable technique for the detection of the separated substances. For this reason, mass spectrometry itself is employed for the detection and estimation of volatile substances separated by other differential migration techniques.
This dynamic reversible system is requisite for the migrations in all chromatographic separations. But for effective separations, an additional condition must be operative. The distribution of the components of the mixture between the solvent and the sorbent must be different. Except in homologous series, this preferential distribution, or selective sorption as it is commonly called, varies seemingly unpredictably with variation of the sorbed substances, solvent, and sorbent ( 3 , t ) . Because chromatographic separations may be made with mixtures of gases, vapors, and solutes, chromatographic methods fall into two principal categories: gas chromatography, for use with gases and vapors, and solution chromatography, for use with solutions. The principal interactions in gas chromatography are shown below. The principal interactions in solution chromatography (S) are shown below. These sorptive systems may be modified in many ways. Both the solvent and the sorbent may be varied with respect to their chemical and physical properties. Solvents ranging from the nonpolar hydrocarbons to the polar acids, bases, and aqueous solutions have been employed. Sorbents have ranged from the surface active powders (ad-
VARIABILITY O F DIFFERENTIAL MIGRATION METHODS
Differential migration methods are variable in so many ways that it is impossible to consider all the ramifications in a brief report. Chromatography is the most variable, and in many respects, the most adaptable, because it may be used for the examination of both vapors and solutions of all kinds of chemical substances ( 2 ) . Some illustrations of the complexity of differential migration may be gained by a brief consideration of chromatographic methods. I n all chromatographic techniques, there is a dynamic interaction among the solvent, the sorbent, and the components of the mixture-e.g.,
I
SOLVENT
[$'
/ SORBENT-
+ vapor]-----MIXTURE
+
c
Interface
1
fixedOrliquid
Principal interactions in gas chromatography
Liquid,
Soluble substances -MIXTURE
+ -
SORBEST-
Interface Liquid-solid Li uid liquid Fixel liqiid Ion exchanger Zeolite Resin
Principal interactions in solution chromatography
sorption chromatography) through the fixed liquids immiscible in the wash liquid (partition chromatography) to the ion exchange resins and zeolites (ion exchange chromatography). As yet there is no precise, comprehensive basis for the correlation of the numerous combinations of solutes, solvents, and sorbents that provide the dynamic and selective interactions essential to all chromatographic separations. The geometry of the sorbent may also be varied. Examples of this kind of variation are the dimensions of the tubes and columns in gas and solution chromatography, and the variation of paper from strips to sheets in paper chromatography, two-way paper chromatography, and radial paper chromatography (1, 4-r), No simple relationship between the chemical composition or molecular structure of unrelated substances and their
sorbability in the chromatographic columns has been established (3). For this reason, it is impossible to deduce composition or structure from chromatographic behavior alone. From this standpoint, chromatographic methods (and virtually all differential migration methods) cannot replace the conventional chemical methods for the determination of composition and structure. These differential migration methods do not even serve for the classification and identification of substances unless authentic preparations are available for comparison. Differential migration methods are most effective for the resolution of mixtures, the partial description of various substances, and the identification of substances by comparison with authentic preparations. I n all these respects, these methods augment and improve the armamentarium of the analyst.
LITERATURE CITED
(1) Strain, H. H., h A L . CHEW 23, 25 (1951). (2) Zbid., 30,620 (1958). (3) Strain, H. H., “Chloroplast Pigments
and Chromatographic Analysis,” 32nd Annual Priestley Lectures, Department of Chemistrv. Pennsvlvania State University, Unibersity P“ark, Pa., 1958. (4) Strain, H. H., in Yoe, J. H., and Koch, H. J., Jr., “Trace Analysis,” pp. 7-33, Wiley, Kew York, 1957. (5) Strain, H. H., Murphy, G. W.,ANAL. CHEM.24, 50 (1952). (6) Strain, H. H., Sato, T. R., Zbid., 28, 687 (1956). (7) Strain, H. H., Sato, T. R., Engelke, J., Zbid., 26, 90 (1954). (8) Tiselius, A., Endeavour 11, 5 (1952).
RECEIVED for review December 16, 1958. Accepted February 27, 1959. Work performed under the auspices of the U. S. Atomic Energy Commission.
Some Artifacts in Paper Chromatography GUNTER ZWEIG University of California, Davis, Calif.
b Artifact spots have been observed on paper chromatograms of amino acids, organic acids-bases, and inorganic ions. Some of these artifacts have now been identified as the ethyl esters of amino acids and separate ionic species of dicarboxylic amino acids, by study of infrared spectra of isolated artifacts. Other artifacts discussed include “comet” and “masking” effects of the organic acids, geometric isomers of keto-acid derivatives, and multiple ionic species of inorganic anions. Caution must be exercised in interpreting “unknown” chromatographic spots. Corrective measures are recommended to minimize the occurrence of artifacts.
T
technique of paper chromatography has become an almost indispensable tool to the researcher who is dealing with micro amounts of a multicomponent mixture. Were it not for paper chromatography, the separation of the amino acids from protein hydrolyzates would be a time-consuming task, The metabolic fate of biological intermediates has been frequently elucidated by the combination of paper chromatography and autoradiography. The observation of an “unknown spot” on a paper cliromatogram has led to the eventual discovery of hitherto unknown comHE
pounds. These “unknown chromatographic spots” must be scrutinized very carefully, however, before deciding that they represent undiscovered compounds. Some spots may represent “multiple spots’’ or artifacts due to different ionic species of polar compounds or experimental manipulations. ARTIFACTS O F A M I N O ACIDS
Koch and Hanson (IS) observed the formation of the monoethyl esters of aspartic and glutamic acids by storing these amino acids in ethanol-N hydrochloric acid (75:26 v./v.) for several days. Plaisted (18) has also reported that glutamic acid in the presence of 80% ethanol and a strong cationic ion exchange resin (Dowex 50 H+) was converted to the y-ethyl ester of glutamic acid. This compound gives a ninhydrinpositive reaction and occupies a position near the leucines on a two-dimensional chromatogram, when phenol and butanol-acetic acid-water are used as solvents. This ninhydrin spot may be mistaken for an unknown amino acid when plant extracts are chromatographed. Furthermore, for quantitative analysis of the amino acids by paper chromatography, the value for glutamic acid would be low if the 7-ethyl ester were ignored. DeVay, Weinhold, and Zweig (Y)have studied the reaction between ethanol and the commonly occurring amino
acids in the presence of hydrochloric acid, and found that the follolTing amino acids formed ethyl esters with ease: glutamic acid, aspartic acid, y-aminobutyric acid, and p-alanine. Glutamic and aspartic acids chromatographed in phenol produced multiple spots due to two ionic species of the dicarboxylic amino acids. With butanol-acetic acidwater as solvent only one additional artifact was observed, because the ionization was repressed (see Table I). The R, value for the fast moving artifact of “old” glutamic acid was the same as that for authentic ?-ethyl ester glutamic acid. (The authentic sample of the ester contained a trace of glutamic acid.) The artifact spot of the four amino acids under investigation was faster moving in the two solvent systems than the parent amino acids. In this respect the chromatographic behavior was the same as that of ?-ethyl ester glutamic acid. The compounds, formed by the action of ethanol and hydrochloric acid with the amino acids, were isolated in milligram quantities. Thirty-six-hour-old solutions of each amino acid were streaked on Whatman 3hIM paper (18 X 22 inches) and chromatographed with phenol-water or butanol-acetic acidwater as solvents by the descending technique. Guide strips were developed with ninhydrin, and the faster moving, major band mas eluted with dilute hydrochloric acid. The concentrated eluVOL. 31, NO. 5 , M A Y 1959
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