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 OF AMINO 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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ates were examined by infrared spectroscopy, ethoxy-group determination, and elemental analysis. Figure 1 shows the infrared spectra of the four amino acid derivatives isolated by the above procedure. These spectra were obtained from about 1 to 2 mg. of the concentrated eluate by the potassium bromidepellet technique. The spectrum of an authentic sample of y-ethyl ester of glutamic acid wm identical with that of the unknown coneentrate, derived from glutamic acid, giving further evidence about the identity of the derived compound. No authentic samples of the ethyl esters of the other amino acids were available, and their infrared spectra could thus not he compared. However, two strong bands in the regions of 5.7 and 8.2 microns indicated the presence of an ester linkage: these two bands were absent from the parent amino acids (3). Ethoxy-group determination by the method of Pregl (19) was positive, but not quantitative. Carbon-hydrogen d e terminations, likewise, were not quantitative, indicating a mixture of amino acids and their ethyl esters. This latter point was demonstrated by paper chromatography of the purified esters: the concentrates were shown to be a mixture of the parent amino acids and their derivatives. It seemed difficult to prevent slight hydrolysis of the esters during chromatographic development. Heyns has also observed artifact spots when chromatographing glutamic acid with butanol-acetic acid (IO). Although he did not isolate these artifacts, he reasoned that one of them probably was identical with the y-butyl ester of glutamic acid. Other artifacts, thought to be due to the interaction of glutamic acid and glycine, have been shown actually to he due to the interaction of glntamic acid and phenol (2,9). To minimize the formation of the ethyl esters of these four amino acids, the samples should not be stored over ethanol for any length of time. This is especially applicable to plant extracts having a high acid content. 2-Propanol is the recommended solvent for the amino acids from protein hydrolysates, because, according to Block, this solvent is an effective preservative and yet does not cause esterification (6). A sample of glutamic acid may be kept indefinitely in a solution of 10% aqueous2-propanol, acidified with 0.1N hydrochloric acid. Figure 2 shows a chromatogram of glutamic acid dissolved in acidified 2-propanol and ethanol. No Zpropanol ester is formed, whereas the ethyl ester may be clearly seen. Figure 2 also illnstrates another phenomenon of the dicarboxylic amino acids, the formation of “multiple spots” with an unbuffered solvent like phenolwater (spots 5 and 6). When Dent published his map of amino acids in 1947 (67,
Table I.
RJ Values of Amino Acids, Dissolved in Ethanol-Hydrochloric Acid
[Solvent. 1-Butanol-acetic acid-water (4: 1:5, v./v.)] Freah 36-Hour-Old Color Amino Acids Solution Solution Ninhydrin Glutamic acid 0.35 0.35 0.60 y-Ethyl ester glutamic acid 0.35 (minor) 0.35 (minor) 0.60 0.60 Aspartic acid 0.28 0.28 y-Aminohutpic acid
0.45
&Alanine
0.37
he correctly showed that aspartic and glutamic acids split into two spots each with phenol as solvent. How these multiple spots may l e d to a false interpretation of an experiment is illustrated in Figure 3. This is an aukradiogram of an alcohol extract of an excised bean leaf, treated with the herbicide Simazine (Geigy Product) and exposed to C’% in the light. The effect of the herbicide seemed to lie in the formation of compounds A and B (see Figure 3). Cochromatography with aspartic acid showed that both spots were due to aspartic acid, and that no new compound has been formed (20). DeVay, Weinhold, and Zweig (7) have studied the chromatography of aspartic and glutamic acids in phenol, saturated with buffers at pH 2.5 (below the isoelectric point) and pH 7.2 (above the isoelectric point). Figure 4 shows that each amino acid produced single spots at each pH, but that the R, values were lower at the higher pH. This finding seemed to indicate that the anion of each amino acid gave a lower RJ value than the corresponding cation. At an intermediate pH, as may be expected from unbuffered phenol, both ionic species exist in equilibrium, thus producing two spots or a streak, (Figures 2 and 3). The formation of multiple spots of the dicarboxylic amino acids may be prevented by the use of buffered phenol or buffered paper (4, 14, 16). Recause commercially available phenol has a high acid content, i t is usually distilled. A recent innovation is the availability of neutral “liquid phenol-without preservative’’ which bears on its label the inscription “suitability for amino acid paper chromatography-to pass test.” Similar difficulties, like streaking or multiple spots, have been observed with the basic amino acids. Aronoff (1) has attributed multiple spots of lysine, when phenol-water was used as solvent, to the formation of association complexes with the solvent. ARTIFACTS OF ORGANIC ACIDS AND BASES
Multiple spots or long trails, known as “comets,” have been observed fre-. quently during the chromatography of
0.50 0.45 0.73 0.37 0.68
Blu; Green-blue
Figure 2. One-dimensional chromatogroms
paper
A.
y-Elhyl ester glutamic acid Glutomicocid in 10% 2-propanol C. Glutamic ocid in 80% ethonoi, trace HCI Solvent, phenol-woter
B.
organic acids and bases. These artifacts are again due to the ionization of these compounds into more than one form. In the case of the acids, this may be overcome by the addition of a “swamp” acid to the solvent, like formic acid, in order to repress the ionization of the organic acid to be chromatographed (16). For weak bases, like alkaloids, a solvent should contain a mineral acid with a common anion (17). Thus, a single species. the salt. results in a sinele. ._ nell-defined spot. Another ohenomenon. that of “masking,” has been observed when chromatographing organic acids. This artifact I
VOL. 31, N O . 5, MAY 1 9 5 9
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Figure 3. Two-dimensional autoradiogram of extroct from C’40z-exposed bean leaf 1, 2.
First solvent, phenol-woter (right to left1 Second solvent, butonol-propionic a c i d w a t e r Idown-up)
occurs when the developed chromatcgram is sprayed with an acid-base indicator, like hromophenol-blue, and when sodium or similar ions are present in the original sample. The spot due to the interfering cation turns dark-blue and may thus mask an acid (yellow) (present as the anion) which may have the same R , value. When the R , values are similar, a crescent-shaped spot results, as illustrated in Figure 5 . This artifact may be prevented by first purifying the sample by ion exchange or ether extraction. ARTIFACTS OF 2,4-DINITROPHENYLHYDRAZONES
Multiple spots occur when the 2,4-dinitrophenylhydrasones of pyruvic and oxalacetic acids are chromatographed. This has been shown to he due to the differential migration of the cis and trans isomers of the phenylhydrasones (fd). The two structures are: CHs
Aspartic ocid 2. Glutomis acid Solrent% A. Phenol-buffered p H 2.5 8. Phenol-buffered p H 7.2 1.
tography of inorganic ions have been reported by Erdem (8). He found that cadmium (11) acetate resulted in three spots when chromatographed with a solvent containing ammonium hydroxide. Sprayed with quercetin, these three spots have been tentatively identified as cadmium-ammonia complexes. Erdem showed this by comparing their color with that of synthetic cadmiumammonia mixtures on spot tests. He observed similar colors ranging from yellow, to brown, to purple. Erdem (8) observed that disodium hydrogen phosphate and disodium hydrogen arsenate gave two spots with benzene-ethanol-water solvent, p r y sumably due to the mono- and dibasic ions. When phosphorus and arnenic NO.
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“.H/ cis ARTIFACTS DUE T O PURIFICATION
In the purification of sugars from plant extracts by passage through a strong alkaline ion exchange resin (Dowex 2 OH-), lactic acid is formed. On spraying the chromatogram with silver nitrate, a false spot, due to lactic acid, may appear. The remedy is purification of the sugar solution by a basic ion exchange resin in the carbonate cycle (it). ARTIFACTS O F INORGANIC IONS
Multiple spots in the paper chroma-
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ANALYTICAL CHEMISTRY
Figure 4. One-dimensional chromatograms
a
trans
acids were used, only single spots resulted.
Figure 5. One. dimensional paper chromotogram of alcohol extract of excised bean leaf 1. Dark-blue spot duetoNa+ions 2. Unidentified orgonlc acids-yellow spots. Solvent, e t h e r formic acid-water,
5:2:1.
K/V.
(4) Berry, H. K., Siltton, H. E., Cain, L., Berry, J. S., University of Teras Puhl. 5109, 25 (1951). (5) Blyk, R. J., Durrum, E. L., Zn-eig, G., Paper Chromatography and Papet‘ Electrophoresis,” 2nd ed., p. 117, Academic Press, Kew York, 1958. (6) Dent, C. E., Biochem. J . 41, 210-53 (1947).
(7) DeVay, J. E., Weinhold, A. R., Zreig, G., ANAL.CHEM.31, 815 (1959). (8)Erdem, B., Reo. fac. scz. zmii,. Istanbul ZOC, 332-48 (1955). (9) Hackman, R. H., Lazarus, M., Bioehem. Biomhvs. . “ Acta 17. 147-8 (1955). (IO) Heyns, K., Koch, K., KBnigsdorf, W., Naturwissaschnfla 39, 381 (11152). (11) Hulme, A. C., Nature 171, Gl0-11 (1953). (12) Isherwood, F. A., Jones, R,. L., Ibid., 175,419-21 (1955). (13) Koch, R., Hsnson, H., Z . physiol. Chem. 292, 180-3 (1953). (14) Levy, A. J,,, Chung, D., .ISAL, CHEM.25,396-9 (1953). (15) I.ugg, J. 15’. H., Overell, B. T., Nature 164, 87-8 (1947). 116) MoFarren. E. F.. . ~ N A L .CHEM.23, 168-74 (1950. (17) Munier, R., Machehoenf, hl., Cherrier, N., Bull. mc. ehim. b i d . 34, 204-14 (1952). (18) Plaisted, P. H., Contribs. Boyca Thompson Insl. 19, 23144 (1958). ( l e ) Pregl, F.,“Quantitative Orgmische Mikrosnalyse,” 4th ecl., pp. 210 ff, Julius Springer, Berlin, 1035. (20) Zwelg, G.,Ashton, F. AI., University of California, Davis, unpuhliehed results. ~
ACKNOWLEDGMENT
The author is indebted to Paul 0. Amrhein, Perkin-Elmer Corp., for ohtaining the infrared spectra. LITERATURE CITED
(1) Aranoff, S., Science 110, 590 (1949).
(2) Beck, M. T., Ebrey, P., Acta Chim. Sei. Hung. 4, 231 (1954). (3) Bellamy, L. J., “ I n f y e d Spectra of Complex Molecules, Methuen & Co., London, Wiley, New York, 1954.
RECEIVED for review November 26, 1058. Accepted January 30, 1959.
