P i w 4 84e New England Association of C h e m B c h e r s
David Racusen University of Vermont
Burlington, Vermont
The Investigation of Unknowns by Paper Chromatography
T h e usual means of introducing paper chromat,ography in the teaching laboratory is the examination of "known" compounds. For this purpose the student is given a solution containing, let us say, an amino acid mixture which he proceeds to analyze by spotting on paper, running in a given solvent, and detecting with ninhydrin. While this is a useful introduction, it does reduce the student to the familiar level of cook-book user. A subsequent, and more stimulatr ing exercise, is the examination of natural mixtures, such as orange juice, blood, urine, etc., for easily identifiable metabolites as amino acids, sugars, etc. There is an even more creative approach which involves tentative identification of an unknown by paper chromatography. The only limitations necessary are that any unknown chosen must be amenable to analysis by chromatography: that is, it should be a stable, nonvolatile, organic compound, somewhat soluble in H20, and of moderately low molecular weight. None of these criteria offer a serious limitation. (In a real research situation, the means of crude isolation might well be sufficient evidence. For example, extraction with aqueous solvents, evaporation, and dialysis of the material would indicate, respectively, water-solubility, nonvolat,ility, and low molecular weight.) Also, the choice of unknowns could be extended to include not only true metabolites but synthetic compounds such as drugs, etc. There is obviously a limit of molecular complexity beyond which even the most talented student cannot penetrate in several laboratory sessions. However, experience leads me to believe that students can go much further than a bald demonstration of amino acid chromatography. In doing so, they gain a valuable insight into the relationship of molecular structure to partition and chromagenic properties. The General Problem
At present there is no coherent scheme for the systematic study of an unknown by paper chromatography. However, there is a logical approach. The first and most insistent problem is that of detection. Having found a means of visualizing the compound(s), the next step is finding a solvent system. Finally, one would study the behavior of the compound by use of other color reactions and by modifying the solvent system. This combined data should make possible a tentative 484
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identification based on information in such compilations as Block, Durrum, and Zweig, "Paper Chromatography and Electrophoresis," Academic Press (1960) and Lederer and Lederer, "Chromatography," Elsevier (1957). Methods of Detection
Methods of detection can vary from intrinsic color (as in dyes) to radioactivity (as in '4C-labeled metaholites). Most often some sort of color reaction must be used. Fortunately, there are a few general color tests which apply to almost any compound regardless of functionality (i.e., alkaline KMnO4 or ammoniacal AgNOd. In practice the initial color reaction is performed on n dried sample of the unknown on filter paper. For example: lop1 (0.01 ml) of a ly0 solution will give a spot about the size of a penny on Whatman KO. 1 filter paper. The dried spot will contain lOOpg (0.1 mg), an amount more than adequate for almost any color test. A testing order of roughly increasing complexity and specificity is as follows: (1) Use short (253 mp) and long (370 mp) wavelength ultraviolet light, then observe fluorescence or absorption. These phenomena are shown by many aromatic or other highly conjugated compounds. (2) Spray with 1% KMnOa in 2% NanCOa. Many organics will reduce the KMn04, leaving a yellow area in a purple background. The immediacy with which this happens is an indication of how good a reducing agent is present. Unsaturated, phenolic, and aldehydic compounds usually give fast color changes. (3) Treat with a pH indicator to demonstrate acid or basic groups. One such indicator is 0.04% bromphenol blue in ethanol. (4) Ehrlich's reagent (p-dimethylaminobenzaldehyde in HC1 and ethanol) gives yellow colors with free amines and purple colors with indoles (tryptophane, etc.) and pyrroles. ( 5 ) Ninhydrin gives purple colors with aliphatic amines including some amino sugars and most amino acids. Yellow and brown colors are obtained with proline and asparagine. (6) Pauly reagent (diazotized sulfanilic acid) gives orange colors with imidazoles (histidine, etc.) and phenols.
(7) The Rydon-Smith reaction (Ch followed by starch-iodide) yields dark blue colors with any compound containing the -XH- grouping. (With the exception of UV examination, each reagent should he tested on a fresh spot.) Positive test results, as obtained ahove, should be accepted with some reservation. Spots directly applied to paper sometimes yield a spurious color reaction more attributable to pH difference or degree of wetting than to specific functionality. The intensity of reaction is useful of course, but a more convincing demonstration is that obtained after chromatography. Negative results are also suspect when examining complex mixtures; for example, UV absorbing and fluorescing materials may cancel each other when viewed in a mixture. Thus, it is well to repeat the series of tests following chromatography. I t is possible that the performance of all these tests would not guarantee success. Yet, if one had nothing further to go on, it would be best to use these simple and fairly general reactions. In addition to the ahove, there are numerous tests of a more specialized nature. Some of the common ones are: (1) 0.4Y0 22,-dinitrophenylhydradne in 2 N HC1 for aldehydes and ketones. (2) 5% aniline 5% xylose in 50% ethanol, heated a t 125', for organic aids. (3) Diazotization and coupling (nitrous acid followed by a phenol) for aryl amines. diphenylamine phosphate for sugars. (4) Aniline perchloric acid UV for phos(5) Molyhdate phate esters. (6) Cyanogen bromide for alkaloids. If, a t this point, the compound has been sufficiently characterized, it may he possible to find a ready-made solvent system in the chromatographic literature. The general practice has been to report solvent systems useful in separating all the likely members of a particular chemical family. For example, if one obtained a strong ninhydrin reaction, then one should try a solvent system which is known to give separation of non-volatile, aliphatic amines such as amino acids. With luck, one may find known R, values and color reactions which agree with the unknown compound. Co-chromatography of the unknown with the corresponding known compound should make identification possihle. But what of situations in which, (a) satisfactory chemical characterization by color tests fails or, (b) there is insufficient literature to allow direct comparison of R*values? The first case implies that one must invent a solvent system without benefit of the literature or arbitrarily choose a described solvent. The other case implies that one must somehow guess the structure of the unknown from the properties of known members of the same family. The following may clarify prohlems and offer a rational approach to their solution. Essentially paper chromatographic migration depends on the partition of the solute between a HzOcellulose complex (the stationary phase) and an organic solvent plus H20 (the mobile phase). Materials that are highly lipophillic tend to dissolve in the organic solvent and migrate rapidly. Very hydrophillic materials would rather partition into the H20-cellulose complex and migrate slowly, if a t all. Between these extremes there are many compounds having both lipophillic and
+
+
+
+
hydrophillic character. Thus, the common process in paper chromatography is the battle between these opposing properties. The hydrophillic part of the molecule attempts to cling to the stationary phase, whereas the lipophillic part tries to dissolve in the mobile phase. In a successful chromatogram a compromise is usually reached for the solute moves part way along the paper. Solutes that differ in lipophillic-hydrophillicbalance are usually separable in a single chromatographic pass. The kinds of functionality which lead to partition preference are well known to the student of organic chemistry. They are briefly indicated in Table 1. Toble 1. Partition Preference of Some Functional Groups, in lncreosing Order of Hydrophillic Character, Reading Down
-CHs
-CHz-
-CONH:
-CONR?
-3H
-COzH
-0-
etc.
\
0
,, -NH2
ete.
S
H
ete.
From such simple information it is possible to make predictions regarding solvent composition and relative rates of migration. These are considered separately. Solvent Composition
The mobile phase contains an organic solvent plus some H20. By increasing this H,O-content one can encourage a solute molecule to pass into the mobile phase. Increased H,O-content tends to attract the hydrophillic part of the molecule away from the H20-cellulose complex. The lipophillic part is already disposed toward the organic solvent so that the net result is increased mobility. The practical consequences are quite useful because there are many instances when one would like to increase the mobility of a particular solute. Information concerning the nature of the functional groups of an unknown can he obtained by comparing migration rates in two solvents which differ only in pH. The effect of pH is to change the migration rateof compounds having ionizable groups. At alkaline pH's carboxylic acids will he retarded while a t acid pH's amines will be retarded. In an alkaline environment the acid will dissociate to the carboxylate form which has more hydrophillic character (Table 1). This will tend to bind the molecule more firmly to the stationary phase and lower the mobility rate. Low pH, on the other hand, protonates the amino group and thus produces a lower mobility due to the more hydrophillic ammonium form. Designing N e w Solvent Systems
The previous discussion leads to a simple experimental approach as follows: Two large test tubes or small hydrometer jars may be set up as in the drawing. Each vessel contains 10 ml of 70y0 ethanol plus 0.5 ml of either concentrated acetic acid or concentrated NH40H. The unknown material is spotted onto a strip of Whatman 1 paper, about 1 in. from the bottom. The strip should he tapered as shown to prevent its clinging to the vessel walls. The glass hook should be adjusted so that the origin is at least 0.5 in. from the solvent level. Allow the spot to migrate several inches past the origin. If both solvents Volume 39, Number 9, September 1962
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give the same result in migration value, separation of materials, spot size and shape, one may conclude that the material contains neither a free acid nor basic group.' Subsequent chromatography may be c nducted with neutral solvent systems. If no migration occurs, or the migration occurs a t the solvent front, the ethanol-H10 ratios must be changed. A higher HzO content will increase migration, or a lower H 2 0 content will decrease migration. After a number of trials, it should be possible to decide if pH is a factor and which ethanol-H?O ratio gives reasonable results. At this stage it might be useful to try an organic solvent other than ethanol, especially if the spots seem to spread and become diffuse and elongated. Another alcohol may be tried such as isopropahole
This has value because it allows one to tentatively identify a compound by comparison with compounds of known Rr. For example, it would be possible to predict the order of migration of the following compounds by reference to Table 1: 7OZH RCH8 R-H RC02H RNtH2 R \
In this example the order of migration reads from left CO?H to right. That is, R-CH3 moves fastest, and R
