V O L U M E 2 2 , NO. 1 2 , D E C E M B E R 1 9 5 0
1561
Schulek, E., and It62sa, l’.,Z. nrtnl. Chent., 117, 400 (1939). Shapiro. .J,, .4m.J. Clin. Path. Tech. Sect., 12, 66 (1942). Smith, L., Sceiisk Kem. Tid., 56, 153 (1944). Snell. F. D.. and Snell, C. T., “Colorimetric Methods of .-\nalpsis.” Vol. 11, K e w Tork, D. Van Sostrand Co., 1937. (26) Thouvrnin. R., Ann. chim. nnnl., 26, 72 (1944).
(22) 123) (24) (25)
(13) hlehlig, J. P.. and Hulett, H. It., Ibid., 14, 869 (1942). (14) Mehlig, J. P., and Shepherd, hl. *J., Jr.. ANAL. CHEM..21, 641 (1949). 115) (16) (17)
Mellon, M. G., IND.ENG.CHEM.,*\SAL. ED., 17, 81 (1945). Murray, W. M., and .ishley, S.E. Q., 10, 1 (1938). Newman, H. W., and Abramson, h l . , J . Pharmncol. Ezptl. Therap., 74, 369 (1942).
Sicloux, M., Ann. fermentalions, 1, 513 (1936). Pudovik, A. N., and sinaiskii, G. M.,J , ~ ~ ~ l i (U.S.S.R.), 21, 862 (1948). (20) Rowland, G. P., IND. E r o . CHEW,ANAL.ED.,11, 442 (1939). (21) Sandell, E. B.. “Colorimetric Determinations of Traces of Metals,” pp. 190-4. Sew York. Intersrienre Publishel S, 1944. (18) (19)
April 17. 1950. Presented beforr t h e Section of Analytical ~ RECEIVED d
Chemistry a t the Pacific Northwest Regional Meeting of t h e AMERICAN C H E r i c a L SOCIETY,Richland, Wash.. J u n e ed for the estimation of individual amino :icids or their mixtures in chromatographic, Ytudies. The original procedure of Folin ( 5 ) i n d the subsequently modified form of the procedure by Englis and Fieis ( 4 ) were the hasis of the present worh. This color]metric process offers advantages for routine k\ork and i t is based upon the reaction between an amino w i d and sodium-2-naphthoquinone-4-sulfonate, the product being in w c h case a highly c-oloiet1 cwniporintl.
Mqo( N
E-0042 D-0.028 c -0.02 I
ri, 0
-OH
w\/ SI’
Figure I.
R--VH
(’OOIl
Wavelength, m p Extinction-Concentration Study for Gl? cine from $00 to 600 nip Volurnea in all raws 50 rnl.
Seither of the papers referred to gave a comparative survey of the behavior of the individual amino arids through the clesiretl c~trnc.cntrationrange, 0 to 0.05 mg. of amino nitrogen in ;iO-nil. final volume of the solution after color development. Tlif method was further studied by Frame et al. (6)withafilterphotom(Iter with special referrnc,c to the cletermination of amino nitro~ c nin blood. A comparative study of the method with thc ninhydrin manometric. method has been made by Chinard and \.a11 Slyke ( 3 ) . In the course of the studies here reported the wide variation in the slopes of the extinction-concentration curves of the various
’ Present address. U. S. Atomic Energy Commission, New Brunswick, N. J.
amino avida was again noted. This fact \vould not militate against the use of the method in connection with efficient chromatographic separations, but it does preclude its use for an accurate estimation of a-amino nitrogen in mixtures of several amino acids. The wide variations in color are apparently due to the varying inductive effect of the group R of the amino acid. This hypothesis is considered further under discussion of results. Block ( I ) , Bull et nl. (Z), and Moore and Stein ( 7 ) found that various amino acids gave different extinction values a t the same molar concentration by the photometric ninhydrin procedure according t o their iniproved technique. In t.his case the same colored product. t l i k e t o h \ d r i n t ~ ~ l i d e n e ~ i k e t o h y d r i n d a misi
ANALYTICAL CHEMISTRY
1562 formed in all cases, but the color yield varies relative to leucine taken as 1.00 from 0.03 for hydroxyproline up to 1.12 for lysine. The "neutral" amino acids show a yield that is very close to 1.00.
/A .300
EXPERIMENTAL
Materials. The amino acids were obtained from Merck & Company, Inc., Rahway, N. ,J. Through the courtesyof H. A. Frediani, a number of reference samples that had been analyzed both by formol titration and by the phase purity technique were obtained. Stock solutions were prepared by dissolving the calculated amounts of the pure preparations in distilled water that had been sterilized by boiling. The addition of a trace of solid mercuric iodide prevented bacterial action and was without apparent influence upon analytical procedures that were used.
.200
s
.-
+ U
.-C f; W
I
-
.loo.
L
.01
.200 Figure 3.
C
.3 c
.02
.03
.04
.05
.06
Mq.of d-Nitrogen per %ml, Extinction 1;s. Concentration for Some Basic Amino Acids
Lysine. B . Histidine. C. Arginine Extinctions a t 470 mp relative t o reagent blank
A.
U
C
: ,100 w
A- Isoleucme
B- Valine
C -Glycine
D
~
Leucine
I
.01
.02
.03
.04
.05
.06
Mq.of d,-Nitroqen per 5 0 m l . Figure 2. Extinction vs. Concentration for Several Amino Acids of Neutral Group Extinctions a t 470 m p relative to reagent blank
A fresh 0.5% (0.5 gram per 100 ml.) aqueous solution of sodium2-naphthoquinone-4sulfonate was prepared immediately before each series of determinations. Solutions of 0.05 A: hydrochloric acid and 0.114 N sodium carbonate were prepared. For 4% sodium thiosulfate the proper weight of the pentahydrate was dissolved in water that had been sterilized by boiling. One milliliter per liter of 0.1 N sodium hydroxide was added to stabilize the solution. The sodium acetate-acetic acid buffer had 12.5 grams of reagent grade crystallized sodium acetate and 125 ml. of glacial acetic acid per 500 ml. Apparatus. The microburets and 50.00-ml. volumetric flasks were tested for correctness of calibration. Extinction values were determined mTith a Beckman Model DU spectrophotometer at 470 mp with a slit width of 0.045 mm. The c e h were calibrated relative to a designated cell. Procedure (3). An aliquot of the stock solution, containing a maximum of 0.003 millimole of alpha-nitrogen, was run into the 50.00-ml. volumetric flask and diluted to approximately 20 ml. with water, and 3 ml. each of 0.05 X hydrochloric acid and 0.114 r\ sodium carbonate were added. Two milliliters of freshly prepared 0.5% sodium-2-naphthoquinone-4-sulfonate solution were added and the solutions were allowed t o stand in the dark for 2.5 hours. At the end of this period 1ml. of the acetic acidsodium acetate buffer and 3 ml. of a 4% sodium thiosulfate solution were added, the latter to bleach the excess reagent. The solutions were made homogeneous a t 50.00 ml. and the extinction values were read relative to a reagent blank a t 470 mp with an 0.045-mm. slit width. At 470 mp there is a favorable development of extinction relative to the blank. RESULTS AND DISCUSSION
A typical wave length-concentration study for glycine is shown in Figure 1. Extinction-concentration graphs for the neutral amino acids that were studied are presented in Figure 2. These measurements gave straight-line calibration curves up to 0.04 to 0.05 mg.
of a-amino nitrogen per 50.00-ml. final volume after color development. It was noted that the depth of color of the amino acid-paraquinoid color complex was in increasing order: norleucine, leucine, glycine, valine, isoleucine. Except for glycine, the order of increasing depth of color is in line with the increasing inductive effects ( 8 ) of the radical R, which leads to the hypothesis that the radical affects the bond strength of the Schiff's base that is formed in the color reaction. There is no adequate explanation of the anomalous position of glycine in the series. I t is very frequently found that the first member of a homologous series differs widely in various properties from other members of the series and this has been noted in various reactions of glycine. Results for three basic amino acids are shown in Figure 3. In the case of lysine the unusually large extinction values are probably caused by participation of amino groups other than the aamino group in the color formation. The fact that amino groups other than alpha may participate in the formation of a colored complex absorbing in the region of 470 mp was evident in a stud>of @-alanine. Although the beta compound gave a nonlinear extinction-concentration curve, the extinctions of the two (nomplexes a t like concentrations were of the same order of magnitude. The depth of color is not in the same order as the inductive effects for the basic amino acids, and it appears that the divergence may be attributed to a combination of the inductive effect and the participation of a part of the other amino or imino nitrogen in the color development. Glutamic and aspartic acids were studied a s representatives of the acid amino acids; unusually low extinction values resulted and there was no linear extinction-concentration relation over the range studied. This conclusion is in accord with the findings of Folin. LITERATURE CITED
( 1 ) Block, Proc. SOC.Ezptl. Biol. Med., 72, 357 (1949). (2) Bull et al., J . Am. Chem. Soc., 71, 550 (1949).
(3) Chinard, F. P., and Van Slyke, D. D., J . Biol. Clbern., 169,571 (1947). (4) Englis and Fiess, Ind. Eng. Chem., 36,604 (1944). (5) Folin, O., J . B i d . Chem., 51, 377 (1922). (6) Frame, E. G., Russell, J. A , , and Wilhelmi, A. E., Ibid., 149, 255 (1943). (7) Moore, S.,and Stein, W. H., Ibid., 176,367 (1948). (8) Watson, H. B., "Modern Theories of Organic Chemistry," 2nd ed., pp. 31, 57, London, Oxford Vniversity Press, 1941. RECEIVED h l s y 3, 1950.
