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ANALYTICAL CHEMISTRY
Table I. Polarographic Characteristics at 25" C. of Anodic Vanadite Wave after Aging in -4lkaline Medium Electrolyte
PH 9.1 10.1
Eiiz ( m . S.C.E.) -0.25
10.5
10.7 10.9 12.1 12.4 12.6 12 8
13.7 (calcd.)
-0.34 - 0 34 -0.39 -0.42 -0.42 - 0 42 -0.37
-0.38 -0.42
1
-1.12 -1.06 -1.1 -1.02 -1.02 -0.83
-0.75 -1.01
-1.11 -1.11
O'Oi5 0.05 0 06
from 13.7 to 10 and are of the same order of magnitude as those reported by Lingane and Meites. For the diffusion current constants, values of -1.466 to -1.41 were reported in unaged solutions compared to values of -1.1 and less observed in aged solutions. The decrease in the diffusion current constant throughout the pH range in aged solutions, as well as the disappearance of the double waves, suggested that a slow association process takes place in alkaline solutions of vanadium( IV).
(0:Oi) 0.065
...
the reversibility of the wave ( 2 ) . The values of E314 - Elir given in the table indicate that these anodic waves may be considered reversible as far as the criterion is concerned.
ACKNOWLEDGMENT
The work was carried out in a test of the possible replacement of iron( 11) by vanadium( IV) in low temperature polymerization recipes under the sponsorship of the Reconstruction Finance Corp., Office of Synthetic Rubber, in connection with the synthetic rubber program of the United States Government. LITERATURE CITED
DISCUSSION
The half-wave potentials in the aged solutions vary only slightly between -0.42 and -0.34 volt when the p H is decreased
(1) Lingane, J. J., and Meites, L., J. Am. Chem. SOC., 69,1882 (1947). (2) Tomes, J., Collection Czechoslov. Chem. Communs., 9,26 (1937). RECBIYED for review March 18, 1954. Accepted May 5, 1954.
New Solvent System for Separation of Amino Acids By Paper Chromatography R. A. CLAYTON'
AND
F. M. STRONG
D e p a r t m e n t o f Biochemistry, University o f Wisconsin, M a d i s o n 6, W i s .
D
C R I S G purification studies on a plant growth factor from coconut ( 5 ) , fractionation progress was followed by paper chromatographic resolution and detection of the ninhydrinpositive components of the various concentrates. The more common solvent systems available for the separation of ninhydrinpositive materials were found to be unsatisfactory for this work. Consden at al. ( 3 ) and Block et al. ( 1 ) describe the use of various phenolic, organic base (both buffered and unbuffered), and butyl alcohol solvent systems. The phenolic systems were found to be undesirable for routine work because of their corrosive nature and because they are generally equilibrium systems, the composition of which is affected markedly by temperature fluctuations. The common n-butyl alcohol-acetic acid system (1) was found to be unsatisfactory because of the slow movement of the solvent front after several hours of development. Since the decrease in ~olventfront movement was accompanied by the development of the odor of ethyl acetate in the chromatographic chamber, tertbutyl alcohol was substituted for n-butyl alcohol in an attempt to reduce esterification. While this substitution did decrease esterification, and enhance the solvent front movement, the resolution of the ninhydrin-positive components was greatly impaired. The high cost of organic bases such as lutidine and collidine renders these solvents impractical for daily, routine investigations. The new system, which has been used successfully in this laboratory for the resolution of ninhydrin-positive compounds, consists of methyl ethyl ketone, propionic acid, and water in the volume ratios 75 :25 :30. The solvents are completely miscible and hence the composition of the system is not altered by temperature fluctuations. The solvent front moves about 40 cm. in 20 hours on ascending chromatograms, and can be air-dried in 30 minutes to allow spraying with a butyl alcohol solution of 1 Present address, Department of Biochemistry, George Washington University, Washington, D. C.
ninhydrin. The movement of 24 amino acids has been studied and the R, differences among the individual amino acids have been found to be as great as those reported with any of the existing unidirectional systems. Replirate R , determinations were found to agree within f 2 % . In application to natural produot work in this laboratory, this solvent system has allowed resolution of eight ninhydrin-positive spots in a unidirectional chromatogram of an acid hydrolyzate of a purified coconut meat fraction (2). In another experiment, this same hydrolyzate were resolved further into 12 spots by development of the chromatogram with the same solvent system in a second direction. This new solvent system can be used for routine, unidirectional paper chromatograms of amino acids, or in combination with one of the published systems for two-directional chromatograms where the separation of complex mixtures of amino acids or peptides is desirable. EXPERIMENTAL
Preparation of Samples. Solutions of 24 amino acids (Table I ) were prepared by dissolving a p rovimately 20 mg. of each amino acid in 1 ml. of L O N hydrocfloiic acid and diluting to 10 ml. with water. These solutions were kept frozen when not in use. Asparagine and glutamine gave two ninhydrin-positive spots 2 weeks after preparation of the solutions. This observation seemed to indicate partial conversion to the corresponding dibasic acids. Operating Procedure. Whatman No. 1 sheets (46 X 52 cm.) were used as supplied by the manufacturer throughout this work. Two to 4 Y of each amino acid were applied to the Paper on the base line at points 1.5 cm. apart. Ascending chromatograms were run with a solvent system which consisted of 75 ml. of methyl ethyl ketone, 25 ml. of propionic acid, and 30 ml. of water. The pa ers were run for 20 hours in a Chromatocab (Model A, Researci Equipment Corp., Oakland, Calif.) a t 22' to 24' C. The solvent front generally moved about 40 cm. during this period of time. After 20 hours, the papers were removed and air-dried for 30 minutes. The papers were sprayed with a butyl alcohol solution of ninhydrin (0.125%) which contained one crystal (5 mg.) of
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V O L U M E 2 6 , NO. 8, A U G U S T 1 9 5 4 phenol per 100 ml., and were air-dried for 20 minutes. A 10minute heating period in an 85" C. oven was usually sufficient to bring out all of the spots. Finally, the spots were sprayed with an ethyl alcohol solution of copper nitrate (4) to convert them to a permanent, salmon-pink color. A list of R, values is given in Table I.
Table I.
Rj Values of Amino Acids on Whatman No. 1 Paper at 2 2 " to 24" C."
Amino Acid Average R f b DL-Leucine 0.81 DL-Isoleucine 0.78 0.75 DL-Phenylalanine 0.69 DL-Tryptophan 7-Amino butyric acid 0.68 DL-,Methionine 0.65 0.62 DL-Valine 0.57 L-Tyrosine @-Alanine 0.55 0.51 L-Proline 0.47 alanine 0.44 L-Glutamic avi
