SUMMARY
This is a rapid method with sufficient precision t o give reliable results on large numbers of research and control samples of condensed phosphates and phosphate detergents. The modified solvents and use of the densitometer each contribute to the saving of considerable time over previously published methods. One operator can evaluate 20 samples per day with time during running, drying, etc., for other work. If i t is desired to use an elution tech-
nique for evaluation (1, 4-7), use of the modified solvents will decrease the time needed for separation and eliminate the necessity for a two-solvent, two-dimensional separation. ACKNOWLEDGMENT
The authors express their gratitude to P\larie LTndermood for assisting in development of the method. LITERATURE CITED
(1) Crowther, Joan, ANAL. CHEW 26, 1383 (1954).
(2) Ebel, J. P., Bull. SOC.chim. France 20,991,998 (1953). (3) Hanes, C. S., Isherwood, F. .4,, Nature 164, 1107 (1949). (4) Huht,i, A. Id., Gartaganis, P. A.., Can. J . Chem. 34,785-97 (1956). 15) Karl-Krout~a. Editha. ANAL. CHEM. ' 28,1091-7 (i956). ' (6) Smith, M. J., Ibid., 31, 1023 (1959). ( 7 ) Westman, A. E. R., CronTther, J., J . -4m Ceram. s O C * 37, 420 (1954).
RECEIVEDfor review May 9, 1958. Accepted November 28, 1958. Division of Analytical Chemistrv, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
Purification of Plant Amino Acids for Paper Chromatography JOHN F. THOMPSON, CLAYTON 1. MORRIS, and ROSE K. GERING U. S. Plant, Soil and Nutrition laboratory, Agricultural Research Service, U. S. Departmenf o f Agriculfure, Ifhaca, N. Y.
b In a method for the purification of amino acids by use of ion exchange resins, basic amino acids are retained on Dowex 50 on the ammonium form and other amino acids are retained on the hydrogen form. All amino acids are eluted with ammonia. The hydrolysis of labile substances is avoided by carrying out procedures at 0" to 6" C. Some amines may also b e obtained in a separate fraction, and satisfactory recoveries were obtained with standards, protein hydrolyzates, and plant extracts.
S
rater-soluble materials such as salts and sugars interfere n i t h the satisfactory separation of amino acids by paper chromatography (4)(Figure 1). Purification procedures which have been utilized include solvent extraction ( I ) , electrolytic ( 5 ) , and ion exchange resin methods (3, 13, 15, 16). Solvent extraction methods cause a breakdown of acid-labile compounds, losses of amino acids in the removal of lipides, and incomplete recovery of amino acids through the multiplicity of operations ( g ) . Electrolytic methods result in losses of amino acids (18) and the breakdown of arginine (18, 19) and glutamine (9). They also are cumbersome ( I ) . and do not successfully remove organic acids and nonionic substances. Disadvantages of published procedures for the purification of amino acids with ion exchange resins include the incomplete recovery of amino acids (3, 15, I7), incomplete separation of salts ( I S ) , use of reagents which cause hydrolysis of amides ( I S ) , loss of basic OME
1028
ANALYTICAL CHEMISTRY
amino acids ( 6 ) , and esterification of glutamic acid (16). The procedures described utilize a sulfonic acid ion exchange resin t o remore interfering substances from amino acids so as to avoid the breakdown of labile substances and accomplish the separation of the basic amino acids and some amines from the other amino acids. EXPERIMENTAL
Materials and Reagents. Deionized SS7ater. Water previously passed through a commercial water purifier is p u t through a column made u p of 2 Darts of Amberlite IRA400 a n d 1 part of IR-120. Ion Exchange Resin. Dowex 50-X4 1200 to 400 m&h) (The Dow Chemical Co.) is soaked in water overnight and stirred \vith a n equal volume of water in a cylinder. The fines are removed by decanting the supernatant after 30 minutes and repeating the process twice. Hydrogen Form. Donex 50-X4 is heated for 16 hours a t 100" C. n-ith 2 volumes of 1N sodium hydroxide, then poured into a column, and drained. (A convenient column is made by attaching a coarse sintered-glass funnel, 5 em. in diameter, to a glass tube a t least 40 cm. long.) After the column of resin is washed with deionized Tvater to remove the excess sodium hydroxide, it i.: treated with five column volumes of 6LV hydrochloric acid. The hydrochloric acid is removed with deionized n-ater until the effluent is free of chloride ions. Smmonium Form. Doxex 50-X4 in the hydrogen form is treated, in a column, with 10 volumes of 2N ammonium hvdroxide made with deionized water. The resin column is mashed with boiled deionized r a t e r (10 to 20 column
volumes) until the effluent reaches a p H of 8 to 9. If not used within a week, i t should be thoroughly washed just before use to remove dissolved resin. This resin should be preserved n-ith chloroform to prevent the growth of microorganisms. Glass Columns for Dowex 50. Columns are made of glass tubes (20 X 0.9 em.) with capillary tubes (10 X 0.2 em.) attached at the bottom. The top is flared to approximately a 60" taper and 3 to 4 em. in diameter. -4small plug of glass wool is placed in the column and the hydrogen or ammonium form of resin is added to a depth of 7 em. The t x o columns are arranged so that the column of ammonium resin is directly over that of the acid resin. ,4n aqueous solution having approximately 500 y of amino nitrogen, by ninhydrin analysis ( I $ ) , is adjusted to pH 7.0 =t0.1 with ammonium hydroxide or hydrochloric acid and cooled to 0" to 6" C. The resin columns are similarly cooled and subsequent operations are performed a t this temperature. The sample solution is poured on the column of ammonium resin and the effluent passes directly on the column of acid resin. The resins are then rinsed n i t h four successive 10-ml. portions of cold deionized water. If the samples contain sufficient amounts of polyphenolic substances t o interfere with separation of amino acids, the columns are further rashed with 80% alcohol (about 100 ml.) until the effluent gives no blue color with a 0.1JIsolution of ferric chloride. The effluent solution contains anions and nonionic materials. The two columns are separated and eluted individually. The column of Dowex 50 in ammonium form retains the basic amino acids and the amines. The basic amino acids and some amines are eluted with 80 ml. of 2N ammonium hydroxide.
The eluate is dried don-n under carbon dioxide-free conditions a t moderate temperatures (
