Delhaye, M., J . Am. Chem. SOC.74, 5768 (1952). (16) Dibeler, V. H., J. Research Natl. Bur. Standards 49, 235 (1952). (17) Dibeler, V. H., Mohler, F. L., Reese, R. M., J . Chem. Phys. 21, 180 (1953). (18) Eaborn, C., J. Chem. SOC. 1953, 3148. (19) Fishl, W., Young, I. G., Appl. Spectroscopy 10,213 (1956). (20) Gilman, H., ed., “Organic Chemistry,” Vol. 111, pp. 143-51, Wiley, New York, 1953. (21) Gilman, H., Dunn, G. E., J. Am. Chem. SOC.72, 2178 (1950). (22) Goodman, L., Silverstein, R. M., Shoolery, J. X., Jr., Ibid., 78, 4493 (1956). (23) Goubeau, J., Siebert, H., Winterwerb, bl., 2. anorg. Chem. 259, 240 (1949). (24) Goubeau, J., Warneke, R., Ibid., 259,233 (1949). 125) Harms. D. L., ANAL. CHEM. 25. . 1140 (1953). (26) Hawkins, J. A., Wilson, M. K., J . Chem. Phys. 21,360 (1953). (27) Holzman, G. R., Lauterbur, P. C., Anderson, J. H., Koth, W., Ibid., 25, 172 (1956). 128) Hummel, D.. Farbe u. Lack 62. 529 . (1956). (29) Hummel, D., Eunststoffe 46, 442 (1956). (30) Ingebrigtson, D. N., Smith, A. L., ANAL.CHEY.26, 1765 (1954). .
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(31) Kagarise, R. E., Weinberger, L. A., U. S. Naval Research Laboratory, Office of Technical Services, Rept. PB 111438 (May 11, 1954). (32) Kline, G. M., ed., “Chemical Analysis of High Polymers,” Chap. 14, Interscience, New York, 1959. (33) Kreshkov, A. P., Mikhailenko, Yu. Ya., Yakimovich, G. F., Zhur. Anal. Khim. 9,208 (1954). (34) McLafferty, F. W., ANAL. CHEM. 28,306 (1956). (35) Murata, H., J . Chem. Phys. 19, 659 (1951). (36) Murata, H., Science & I d . (Osaka) 30. 164 (19561. (37) ’Murata, H., Hayashi, S., J. Chem. Phys. 19,1217 (1951). (38) hfurata, H., Okawara, R., Watase, T., Ibid., 18,1308 (1950). (39) Pozefsky, A., Grenoble, M. E., Drug & Cosmetic Ind. 80,752 (1957). (40) Radell, J., Hunt, P. D., Murray, E. C., Burrows, W. D., ANAL.CmM. 30, 1280 (1958). (41) Richards, R. E., Thompson, H. W., J . Chem. SOC.1949. 124. (12) Rochow, E. G.; LcClair, H. G., J . Inorg. & Nuclear Chem. 1, 92 (1955). (43) Savidan, L., Bull. SOC.chim. France 1953, 411. (44) Scheidt, U., Appl. Spectroscopy 7, 75 (1953). (45) Schnell, E., Rochow, E. G., J. Am. Chem. SOC.78, 4178 (1956). (46) Schnell, E., Rochow, E. G., J . Inorg. &Nuclear Chem. 6 , 303 (1958).
(47) Sharkey, A. G., Jr., Friedel, R. A., Langer, S. H., ANAL. CHEW 29, 770 (1957). (48) Slobodin, Ya. M., Shmulyakovskif, Ya. E., Rzhedzinskaya, K. A., Doklady Akad. Nauk S.S.S.R. 105, 958 (1955). 149) Smith. A. L.. J . Chem. Phvs. 21. 1997 (1953). (50) Smith, A. L., Angelotti, N. C., Spectrochim. Acta, in press. (51) Smith, A. L., Brown, L. H., Tyler, L. J., Hunter, M. J., Ind. Eng. Chem. 49, 1903 (1957). (52) Smith, D. C., French, J. hf., O’Neill, J. J., U. S. Naval Research Laboratory, Office of Technical Services, Rept. P-2746 (January 1946). (53) Sokolov, N. N., Andrianov, K. A., Akimova, S. M.,Zhur. Obshchei Khim. 25,675 (1955); J.Gen. Chem.(U.S.S.R.) 25. 647. (54) ’Ulbrich, R., 2. Naturforsch. 9b, 380 (1954). (55) Wright, X., Hunter, M. J., J . Am. Chem. SOC.69, 803 (1947). (56) Yegorov, Y . P., Bazhulin, P. A,, Doklady Akad. Nauk S.S.S.R. 88, 647 (1953). (57) Yonemoto, T., Senzok, K., Bull. Electrotech. Lab. (Tokyo) 18,428 (1954). (58) Youn C. TV., Servais, P. C., Currie, C., Hunter, M. J., J . Am. Chem. SOC.70, 3758 (1948). .
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8.
RECEIVED for review December 22, 1958. Accepted March 16, 1959.
Spectrophotometric Determination of Amino Acids Alkaline Copper Salt Method Using Cuprizone, Biscyclohexanoneoxalyldihydrazone RAYMOND BORCHERS Department of Biochemisfry and Nutrition, University of Nebraska, Lincoln 3, Neb.
b The sensitivity of the alkaline copper salt method for amino acids has been increased b y the use of cuprizone, biscyclohexanoneoxalyldihydrazone, for the determination of solubilized copper. Amino acids may b e determined on a 1-ml. sample a t 0.1 to l.OmM concentration. Prior treatment of samples with nitrous acid provides a procedure for checking on nonamino acid interfering compounds. The method has been applied in following the course of proteolytic activity and to amino acid determinations on 0.2ml. blood sample.
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alkaline copper salt method for the determination of amino acids of Pope and Stevens (8) as modified by Schroeder, Kay, and Mills (9) has been further modified. Modifications have been concerned with the estimation of copper brought into solution by the amino acids from a n alkaline suspension of washed copper phosphate. HE
The copper has been estimated by iodometric titration ( 9 ) , spectrophotometry of the copper-amino acid complex (3, 4, 10, l l ) , spectrophotometry involving diethyldithiocarbamate ( l a , I S ) , polarography ( 5 ) , and flame photometry ( 2 ) . A procedure for the application of cuprizone, biscyclohexanoneoxalyldihydrazone, to the determination of copper in the alkaline copper salt method for amino acids is presented. Cuprizone has been used as a chromogenic reagent for copper (6) and applied in the determination of serum copper (7). The comparative sensitivity of cuprizone for copper is attested by its molar absorbancy of 16,000 (7); 12,700 for diethyldithiocarbamate (7) ; and 6200 and 56 for the copper-amino acid complex a t 235 and 620 mp, respectively (11). REAGENTS
WASHED COPPER PHOSPHATE Sus-
Prepare as directed (9). Age 2 days before using. Prepare weekly. BORAXBUFFER. oH 9.1. Preoare as directed (9). CUPRIZONE, 0.27,. Dissolve 200 mg. of cuprizone, biscyclohexanoneoxalyldihydrazone (G. Frederick Smith Chemical Co.) in 100 ml. of 50% ethyl alcohol with gentle heating. It is stable indefinitely. AMINO ACIDS AXD RELATEDCOMPOUNDS. Make freshly prepared solutions in distilled water. These compounds were used as purchased. PEXSION.
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PROCEDURE
Add one volume of sample (0.1 t o l.OmM amino acid concentration) &to one volume of copper phosphate suspension. Neutralize samples when necessary by adding one drop of phenolphthalein plus sodium hydroxide to a faint pink. Prepare a blank with one volume. of water plus one volume of suspension. Mix and let stand for 5 minutes. Centrifuge a t 2000 r.p.m. for 5 minutes. To 1 ml. of the superVOL. 31, NO. 7, JULY 1959
* 1179
natant solution add 5 ml. of 0.1M boric acid and 0.2 ml. of cuprizone. Read after 5 minutes in a spectrophotometer a t 600 mp. Calculate amino acid concentration by reference to a curve prepared from standard copper solutions. Ordinarily, 2 mmoles of amino acid or 28 mg. of amino nitrogen were calculated equivalent t o 1 mmole of copper. EXPERIMENTAL
COLORREACTION.The optimum pH for maximum color development was reported (7) to occur between pH 7.0 and 9.0. One milliliter of supernatant solution or a half-strength borax buffer solution plus 5 ml. of water gave a pH of 9.0 to 9.1. Under these conditions color development was slow and irregular. Replacing the water by 5 ml. of 0.1M boric acid gave a pH of 7.9 to 8.0, where color development was immediate and a maximum. I n the absence of interfering compounds no fading was observed after 12 hours a t room temperature in the dark.
Table I. Compounds Found to Cause Interference Maximum Effect of Concn. in Sample Excess Giving Cpd. on Color
