Microchromatography. I. A Technique for Separation and Identification of Traces of Amino Acids, Sugars, etc. IL Lakshminarayanan From the University
Botany
Laboratory,
Madras-b,
India
Received November 30, 1953
Since the researchesof Consden et al. (l), paper chromatography has come to stay as a simple, rapid, and reliable analytical tool in the study of various problems in biological chemistry. In a recent communication the author (2) has reported a simple technique of irrigation of paper-disk chromatograms using fine capillaries wherein the importance of controlling the rate of irrigation for efficient separation of amino acids and also the reproducibility of their Rf values, has been indicated. In the present communication an adaptation of this technique and a microscopic method of examination of the chromatograms for accurate measurement of the Rf values are described for the separation and identification of traces of amino acids and sugars in the order of 0.25 pg. EXPERIMENTAL AND RESULTS The arrangement is largely similar to the one described earlier (2) for the larger chromatograms (7.5 cm. diam.) and consists of a flat-mouthed glass cylinder (12 X 4 cm.) in which is kept a small 2-ml. vial containing the solvent, supported on a glass base. (Ordinary glass tumblers are equally good enough.) The irrigator consists of a small capillary tube (5 X 0.005 cm.) inserted into a thin square card board (1 X 1 cm.). The irrigator is introduced into the vial containing the solvent (n-butanol-acetic acid-water 4:1:5 was found to be the best for both amino acids and sugars; ethyl acetate-pyridine-water 2.5:1:3.5 was found to be equally good for the sugars) and adjusted so as to be in level with the glass cylinder. Using a fine capillary, the mixture of amino acids or sugars (0.25 pg. of each of the substances in 0.25 ~1.) is spotted in the center of a square filter paper (Whatman No. 1,4 X 4 cm.) with a circle of 2.3 cm. diam. drawn in its center. The filter paper is air-dried, kept in position over the glass cylinder with its center just touching the capillary tube, and covered with a glass plate. A few milliliters of used solvent are introduced into the cylinder earlier, to saturate it inside with the solvent vapor. The whole arrangement is covered with a bell jar. The filter paper is steadily 367
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irrigated by the capillary flow of the solvent and it takes about 40-45 min. for irrigating the whole area. A battery of chromatograms like this could be simultaneously run. Preliminary trials are conducted to fix the dimensions of the irrigator so that the time of irrigation is maintained constant in each case for a given solvent mixture.
Development of the Chromatograms In the case of the amino acids the air-dried filter papers are developed with ninhydrin as described by the author earlier (2). In the case of sugars the following technique based on the procedure described by Trevelyan et al. (3) has been found to be very satisfactory. The air-dried chromatograms after irrigation are dipped for 1 min. in a Petri dish containing a solution of silver nitrate in acetone, treated with ether, airdried, sprayed with 0.5 M alcoholic solution of aqueous NaOH, and airdried for 5 min., whereupon a dark-brown deposit of AgzO forms on the filter paper with brownish black bands of silver indicating the position of the different sugars. The dried filter paper is dipped for 10 min. in 6 iV ammonia, whereupon silver oxide dissolves leaving the background white and the bands brownish or yellow. The chromatograms are washed for 10 min. with successive changes of distilled water and momentarily dipped in distilled water freshly saturated with HzS, whereupon the bands turn jet black due to the formation of Ag2S. The chromatograms are washed again and dried under the fan. Other spraying reagents as aniline hydrogen oxalate described by Partridge (4) for aldoses, and orcinol trichloroacetate described by Klevstrand and Nordel (5) for ketoses, have been found to be quite useful for the differential identification of sugars. Mounting
and Examination
of the Chromatograms
The dried papers are cut into square bits just enclosing the circular chromatograms inside and mounted on a microscopic slide under a thin cover slip (0.1 mm. X 2.4 cm. X 4 cm.) and sealed with paraffin wax on the sides. The sealed chromatograms wrapped in black paper retained the colors longer than others, but it has not been possible to make permanent specimens. The slides are examined on the microscope stage using the low-power objective and suitable color filters (green for the purple amino acid bands and red for the yellow sugar bands). No filters are used in the case of the black bands of AgzS. The horizontal and vertical stage readings from center to periphery for the positions of the different bands are recorded. The chromatogram is moved
MICROCHROMATOGRAPHY.
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FIG. 1. Chromatograms showing the separation of mixtures of amino acids and sugars. The bands represent the constituents in the order from center to periphery. I. Ornithine, histidine, glycine, alanine, methionine-valine, norleucine, 6 bands. II. Lactose, arabinose, galactose, and raffinose, 4 bands; 0.25 pg. of each of the substances in 0.25 pl. spotted. Filter paper: Whatman No. 1. Solvent: n-butanolacetic acid-water 4:1:5. Diameter of the solvent boundary 2.3 cm. Time of irrigation : 45 min.
about the center through +45’ and -45” successively, and the corresponding values recorded and Rf values calculated in each case. The average of the 12 values is taken as the mean Rf value. The accompanying plates (Fig. 1) show the separation of seven amino acids and four sugars by this technique. Employing the present technique, consistent and reproducible RI values have been obtained for the common amino acids and sugars comparing favorably with those obtained in the case of the larger size chromatograms by the technique described by the author earlier (2). It would, however, be necessary to have the irrigation repeated with different solvent mixtures, as in all cases, to resolve overlapping bands of substances having close Rf values in a given solvent mixture, and also to employ different spray reagents for confirmations. The use of the microscope greatly facilitates the accurate measurement of the distances traveled by the components, and in this way the present technique is claimed to be superior to others employing scales or planimeters for the measurements. The use of color filters aids in a better evaluation of the Rf values, since it clearly shows the small area of diffuse coloration running into the filter paper fibers even in sharply separated bands (not perceptible to the naked eye). This technique of mounting and microscopic examination of the chromatograms could also be used with facility even in the case of the larger chromatograms of 7.5 cm. diameter described by the author earlier (2) by cutting small sectors in the eight directions mentioned
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above. The present technique could be conveniently adapted for the identification and separation of traces of other groups of chemical substances. Work on the quantitative aspect of the present technique based on the area and intensity of the bands is under way and will be published in due course. ACKNOWLEDGMENTS I am deeply indebted to Professor T. S. Sadasivan for his guidance and constant encouragement. Grateful thanks are also due to Mr. M. R. Govinda Rajan for technical help and to my collegues Dr. C. V. Subramanian and Mr. M. Lakshmanan for going through the manuscript and to the Government of India for the award of a senior research scholarship.
SUMMARY
A simple micro technique is described for the separation and identification of traces of amino acids, sugars, etc. in the order of 0.25 pg. on filterpaper disks of 2.3 cm. diam. by employing fine capillary tubes for irrigation. The developed chromatograms are mounted on a microscopic slide under a cover slip and examined under the microscope using suitable color filters. Consistent and reproducible Rf values comparable to those of larger chromatograms have been obtained. REFERENCES 1. CONSDEN, R., GORDEN, A. H., AND MARTIN, A. J. P., Biochem. J. 38, 224 (1944). 2. LABSHMINARAYANAN, K., Arch. Biochem. and Biophys. 49, 396 (1954). 3. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S., Nature 166, 444 (1950). 4. PARTRIDGE, S. M., “Partition Chromatography.” Biochemical Society Symposia, No. 3, p. 57, 1951. 5. KLEVSTRAND, R., AND NORDEL, A., Acta Chsm. &and. 4,132O (1950).