E. P. Heimer1 Montcloir State College Montclair, N e w Jersey 07043
Quick Paper Chromatography of Amino Acids
The use of paper chromatography in the identification of amino acids as well as their separation is of paramount importance. Since its introduction by Martin in 1944 (I), the technique has been widely used and is an essential experiment in the undergraduate and graduate laboratory courses in organic and biochemistry. The solvent systems generally described in laboratory manuals (2-6) have been based on n-butano1:acetic acid: water and phenol-water. Although these systems are satisfactory, they have the major drawback that development requires 2-3 hr. Since this is about the length of a usual laboratory period, the student cannot satisfactorily complete the experiment in the allotted time. Gabriel (7) described the use of acetonitrilebuffer systems for the separation of nucleic acid components. The outstanding features are that the developing time is less than 40 min and the papers do not have to be pre-tquilibrated. The present study applies these systems t,o some amino acids and overcomes the difficulty cited above. Many useful sources for lecture material are available (8-11). Experimental Systems and Reagents All the optically active amino acids were of the lrconfiguration in 0.1 M stock solution. Five-microliter spots were applied on Whetman 3 MM paper sheets, 20 X 20 cm (this is the paper of choice because of its rapid solvent migration rate, relatively high solute cs~acitv. .,and eood wet strenethi. - . The chromato~rams were suspended in an appropriate frame and developed in the ascending fashion without pre-equilibration. The developing chambers were 10 x 10 x 3-in. in glass thin layer chromatogmphy tanks such as the ones a,vailahle from Brinkmann Instruments Inc., Westbury, N. Y. When the solvent front reached a height of 12-14 cm, usudly in less than 40 min, the paper was withdrawn and dried. Bands were detected by heating at llODC after spraying with 0.2% ninhydrin in 95 ml of n-butanol and 5 ml of 10% aqueous acetic acid. The bufferp~ was adjusted by the addition of acetic acid or concentrated ammonium hydroxide.
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Table 1. R, Values Obsewed with the Solvent System Consisting of 7 0 Parts Acetonitrile to 3 0 Park 0.1 M Ammonium Acetate (V/V)
Amino acid
pH 4.0
pH 7.2
pH 9
Leucine Tryptophan Valine Proline Alanine Hydroxy proline Glycine Histidine Glutamic acid Lysine Aspartic acid
0.53 0.48 0.40 0.36 0.27 0.27 0.22 0.16 0.15 0.14 0.10
0.48 0.41 0.36 0.37 0.23 0.23 0.24 0.17 0.11 0.14 0.09
0.50 0.47 0.38 0.35 0.27 0.27 0.20 0.18 0.12 0.15 0.11
Table 2. R, Values Observed with the Solvent System Consisting of 6 0 Parts Acetonitrile to 4 0 Parts 0.1 M Ammonium Acetate (V/V)
Amino acid
aH 4.0
pH 7.2
aH 9.2
Leucine Tryptophan Vdiline Proline Alanine Hydroxy proline Glutamic acid Glycine Histidine Lvsine ~ i ~ a r t ,acid ic
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Results
Refer to Tables 1 and 2. The R, values presented here are an average of various determinations. Discussion
The results of this investigation show that the acetonitrile-buffer systems can be used as a tool for the separation of certain amino acids. The relatively fast developing time makes it feasible to use this as a teaching tool in the general biochemistry laboratory. The papers should not be pre-equilibrated and can be developed immediately after mixing the solvents. Certain amino acids can be resolved from mixtures; thus the system, with the proper choice of pH, can be used to determine unknowns. Care should be taken,
' .Mailing address: Chemical Resemch Dept., Hoffmann-LaRoche Inc., Nutley, N . J. 07110.
as with any other cyanide, in the handling of acetonitrile (12). Further applications of this system in the separation of oligopcptides, carbohydrates, and amines are current,lybeinginvestigated. Many thanks are due for helpful discussions with Dr. Joseph Becker, Montclair State College and Drs. A. L. Nussbanm, A. XI. Felix, and A. H. Ramel of Hoffmann-La Roche Inc. Literature Cited (1) MARTIN.A . J. P.. CONSDEN. R., AND GORDON. A. H.. Biochem. 5.. 38, 224 (1944). (2) CTARK,JOHNM.. JR., "Experimental Bioohemistyy:' The W. H. F r e e man 61 Co., Sen Franeiaco. 1964, p. 110. (3) CHATKIN, S., "Biochemistry Laboratory Techniques:' John Wiley 61 Sons. Inn..New York. 1966. (4) P n r ~ m n r ,G. H.. "Anderson's Laborstow Experimenta in Bioohemistry," The C V. Moshy Company. St. Louis. 1968, p. 69. (5) LmuAca. 0.. Experimental Bioohemistry." John Wiiey & Sona, Inc., New Yark, 1960, p. 141. (6) F r ~ a ~ L. n . F.. "Organic Experimenta:' D . C. Heath and Co.. Boston, 1964, D. 148. (7) G*nnmG, T. F.. J . Chromotaci., 36,518 (1968). (8) HEFTMANN.E.. ' l C h r o m ~ t ~ g m p h y(2nd " ed.). Reinhold Publishing Co.. New York, 1963. (9) H u s . I. M.. AND MACE%X.. "Pwer Chrom~togr~phy.'' Academia Press, New Vork, 1963. (10) Shn~x.I . , ''Chromatographic and Electrophoretic Techniques" (3rd ed.). Interaoienoe Publishers (Division of John Wiiey & Sons, Ino.). New York, 1969, Vol. I. (n)Zuerc. 0. A N D WAITAJER.J. R..''PBPBTChromatography and Elmtrophoreais." Aeademio Press. New York. 1971, Vol. 11. (12) "The Merek Index" (8th ed.), Merek and Co., Ine.. Rahway. N. J.. 1968, p. 8.
Volume 49, Number 8, August 1972
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