The peak formed is therefore the end point. ACKNOWLEDGMENT
The aid given this work by C. P. Krula, Central Research Department, who designed the electronic circuitry, is gratefully acknowledged. LITERATURE CITED
(1) Bricker, C. E., Sweetser, P. B., ANAL.CHEM.24,409 (1952).
(2) De Ford, D. D., Miller, J. K., Ibid., 29,475 (1957). (3) Goddu, R. F., Hume, D. S . , Zbid., 26, 1679 (1954). (4) Zbid., p. 1740. (5) Headridge, J. B., Talanta 1, 293 (1958). ( 6 ) Malmstadt, H. v.1 Roberte, '2. B., ANAL.CHEM.28,1408 (1956).
(7) Malmstadt, H. V., Vassallo, D. A,, Zbid., 31,862 (1959). (8) Marple, T. L., Hume, D. S . , Ibid., 28, 1116 (1966). (9) Muller, R. H., J . Opt. icoc .h25, . 342 (1935).
(10) Reilley, C. M., Schmid, R. W., ANAL.CHERI.31,887 (1959). (11) Robinson, H. A., Trans. Electrochem. SOC.92,445 (1947). (12) Underwood, A. L., J. C h m . Educ. 31, 394 (1954). (13) Weilley, C. A Chalmers, R. A., Analyst 82,329 (19k7).
RECEIVEDfor review June 10, 1959. Accepted October 20, 1959. Division of Analytical Chemistry, Beckman Award Symposium on Chemical Instrumentation Honoring Howard Cary, 135th Meeting, ACS, Boston, Mass., April 1959.
PoIa rogra p hic Dete rminati o n of AI pha-Methy I- ~ ~ - ctiynes R. J. THIBERT and R. M. OTTENBRITE Department of Chemistry, Essex College, Assumption University of Windsor, Windsor, Ontario, Canada
b Because of a recent synthesis of a substituted amino acid, a - m e t h y h cystine, it became necessary to have a method of determining this compound. The polarographic determination of the substance in 0.1N hydrochloric acid, using thymol as a maximum suppressor, was investigated. The relationships of concentration of the compound and temperature to the diffusion current were studied. The influences of thymol concentration and pH on the apparent half-wave potential were determined. A linear relationship of the diffusion current to the concentration of a-methyl-DL-cystine was observed in the range of 5 X to 2 X 1 O - W . The system is not reversible.
A
synthesis O f a-methyl-DLcystine by Arnstein ( I ) , which has been confirmed in this laboratory ( 8 ) , made it desirable to establish a method of analysis for this compound. Cystine can be determined colorimetrically by reduction to cysteine and b y using reagents which form colors in the presence of free sulfhydryl groups ( 2 , 9). Cystine can also be determined polarographically, and studies of this type have been reported ( 7 ) . Because the compound under investigation has a structure similar to cystine, it was thought feasible to attempt a polarographic estimation. RECEST
EXPERIMENTAL
Apparatus and Materials. T h e Sar-
gent ( E . H. Sargent & Co.) Model XXI Polarograph was employed for this study. A Heyrovsk3 polarographic cell was used during most of this work; a n H-cell with S.C.E. was used t o determine Eliz. The capillary
106
ANALYTICAL CHEMISTRY
Table I. Effect of Concentration on Diffusion Current
2 x 15 1x 7 5
5
x
10-3 X 10-8 X 10-4
8.66
8 _ ._ M
6 42 4 19 3.64 2 28
6 i8
4 60 3 53 2 36
8.03 6 03 3 98 3 06 2 01
Average of three determinatious. Table II.
O
c.
0.0 11.5 19.0 22.5 25.0 30.0 35.0 40.0 45.0
Diffusion Current, 4.28 5.28 5.66 6.00 6.22 E.54 l.06
7.3ti 8.00
*
RESULTS AND DISCUSSION
Effect of Temperature on Diffusion Current
Temperature,
DL-cystine and thymol, and diluting to volume with 0.1N hydrochloric acid or buffer. The solutions were transferred to a polarographic cell and nitrogen (purified by assing through ammoniacal cuprous ciloride) was bubbled through for 5 minutes. Polarograms were run through the range of 0.0 to -1.0 volt. The drop rate was adjusted to 3 seconds and the temperature was controlled to 0.1" c.
pa.
4.26 5.28 5.66 6.00 6.21 6.64 i.04 7.36 8 00
constant, m z W 6 ( m = 2.57 nig. per second; t = 3.00 seconds). was 2.253 at 25' C. and -0.555 volt. All pH measurements were made with a Beckman Model G p H meter. Buffer solutions employed were prepared according to Clark and Lubs ( 3 ) . The a-methyl-DL-cystine was recrystallized three times from absolute ethyl alcohol prior to use in this study. A stock solution of 1 X lO-*Jf was prepared in 0.1i17 hydrochloric acid. A 1.2 x lO-3M thymol solution in 0.1N hydrochloric acid was prepared for use as a maximum suppressor. Procedure. T h e solutions used for analysis were prepared in a 100-ml. volumetric flask by adding t h e appropriate concentrations of a-niethyl-
Effect of Concentration on Diffusion Current. T h e effect of concentration of a-methyl-nL-cystine (using 1.2 x 10-4M thymol as t h e maximum suppressor a t 25O, 30°, and 37.5' C.. respectively) on t h e diffusion current results in a linear relationship (Table 1)* The diffusion current was measured from the top of the first wave to the top of the second wave as there occurs a prewave with thymol. This type of prewave also occurs with cystine and has been studied by Kalousek, Grubner, and Tochstein (6). Figure 1, curve 2, is a typical example of a polarogram obtained under the above conditions. Except for the study of diffusion current dependence on temperature, where the height of the diffusion current represents the prewave plus the top wave, the diffusion currents and half-wave potentials are based on the top wave only wherever a prewave occurred. The half-wave potential ( E M ) m s measured us. S.C.E. at 20' C. using 1 X 10-3M a-methyl-DL-cystine. E1 2 was observed to be -0.555 and -0.718 volt for thymol concentrations of 1.2 X and 4.8 X 10-4M, respectively.
CZ
CI
I I 06 07 VOLTAGE VERSUS M E M U R Y PCCL
03
I
I
I
04
05
08
09
Figure 2. potential
Figure 1. Effect of thymol concentration on shape of polarogram 1. 2.
2.4 1.2
x x
10-5~ 10-4~
3. 4.
Diffusion Current Dependence on Temperature. Polarograms of 1 X 10-3Af a-methyl-DL-cystine using 1.2 X 10-4M thymol were run in the range of 0" to 45' C. A linear relationship between temperature and total diffusion current occurred (Table 11). Effect of pH on Apparent HalfWave Potential. Solutions of aniethyl-DL-cystine (1 X 10-3M) s-ere prepared using media of various p H lwels (3) with 1.2 >: 10-4JI thymol. The solutions were analyzed a t 21.1" C. nnd the apparent half-ware potential as determined (Table 111). The polarograms had different shapes (Figure 2) and the apparent half-wave potential \ras observed to increase negatively with increase in pH. The higher the pH, the more nearly S-shaped the polarogranis became (Figure 2 ) . Effect of Thymol on Apparent HalfWave Potential. T h e effect of thymol concentration on the shape of t h e polarographic wave was studied. Solutions of 1 X 10-,jAlf a-methyl-DLcystine were analyzed at different thymol concentrations and the apparent Eli%was determined. The value of the apparent half-wave potential changes with thymol concentration (Table IV) because of a change in the shape of the polarographic wave (Figure 1). A well defined Sshaped curve occurred a t a thymol concentration of 4.8 >: 10-4Jf. Effectof Digestion on Polarographic Wave of oc-Methyl-Dr,-Cystine. Duplicate 10-nil. samples of 1 x l O - * X a-methyl-DL-cystine were digested according to t h e method of Koch and Jlchfeekin (4). A distilled 1%-aterblank vas treated similarly. Polarographic analysis of the digests in 0.1s hydrochloric acid a t 19.4" C. using 1.2 X 10-431 thymol revealed that the characteristic wave was absent in both the blank and the samples. This as taken as evidence that no
2.4 4.0
x x
Table 111. PHa 0 12
1 10 1 97 3 00 3.48 3 96
I I I I 04 05 06 07 VOLTAGE VERSUS MERCURY POOL
03
I
IO
(See Table 111 for buffers) pH 0.12 3. pH 1.97 pH1.10 4. pH 3.00 5. pH 3.96
Effect of pH on Apparent Half-Wave Potential Apparent Half-Wave Medium Used* Potential, Volt
S HCl 2,c k C 1 64.5 ml. 0 . 2 5 HCle 2d1 KC1 10.6 ml. 0.2N HClc 2.11 potassium acid phthalate 20.3 ml. 0.21V HClc 2-11potassium acid phthalate 6.0ml. of 0.2-YHClC 0 231 potassium acid phthalate 0.40 ml. 0.2AVNaOHc 1 0 0 0 0
I
Os
Effect of pH on apparent half-wave 1. 2.
10-4~ 10-4~
I 08
I
I
02
0'1
C
++
+++
-0.318 -0.537 -0 579 -0 663 - 0 727 -0 810
-0.320 -0 540 -0 575 -0 666 - 0 726 -0 810
Values represent final solution to be analyzed. Buffers vere prepared according to Clark and Lubs (5). -4mount of acid or base required to prepare 200 ml. of buffer solution.
Table IV. Effect of Thymol Concentration on Apparent Half-Wave Potential
Thymol Concn., 3Lole/ Liter
Apparent E1/2, \-o1t 2.4 X 10-5 -0.343 -0.343 1.2x 10-4 -0.656 -0.ki 2.4X -0.652 -0.652 4.8X lo-' -0.723 -0.723
Temperature, C. 21.7 . 21.9 21.9 18.2 ~~
substance present in the reagents was responsible for the wave usually obtained mlth a-methyl-Do-cS-stine. Diffusion Coefficient and Reversibility of the Reaction. By substitution in t h e IlkoriE equation, t h e diffusion coefficient for a 1 x 10-3N solution of a-methyl-~~-cystine at 25" c., using 1.2 X 10-4M thymol, was found to be 9.38 X lod6 sq. em. per second; the diffusion current was 4.19 ba. and the capillary constant 2.253. The value of the diffusion coefficient obtained is of the same order as that reported by Kolthoff and Bariiuni (6) for cystine (5.3 X 10 sq. cm. per second). The reversibility of the reaction way iD - i tested by plotting E os. log i2 for 1 X 10-3Llf Lu-niethyl-DL-cystine a t 20" C. a t tliyniol concentrations of
1.2 X and 4.8 X 10-4X, respectively. Straight lines were obtained for both thymol concentrations with .lopes of 0.0817 and 0.0633 for thymol and 4.8 concentrations of 1.2 x x l O - 4 J f , respectively. These values differ from the theoretical slope (0.0295). I n this respect the results are similar to Kolthoff and Barnum ( 6 ) , and Kalousek, Grubner, and Tochstein is), who concluded that the cystine reduction is not reversible. LITERATURE CITED
(1)Arnstein, H. R. V., Biochem. J. 6 8 , 333 (1958). ( 2 ) Chinard, F. P., Hellerman, L., dlethods of Biochem. Anal. 1, 1 (1954). (3) Hawk, P. B., Oser, B. L., Summerson, W. H., "Practical Physiological Chemistry," 13th ed., p. 35, ?\ZcGraP;Hill, K e v York, 1954. (4)Ibid., p. 547. (5) Kalousek, >!I Grubner, ,, O., Tochstein, A., Chem. listy 47, 1143 (1953). 1~, 6 ) Kolthoff. I. hI.. Barnum. D.. J . Am. Chem. Soc: 63, 520 (1941).' (7)Kolthoff, I. M., Lingane, J. J., L'Polarography," 2nd ed., Vol. 11, p. 779,Interscience, New York, 1952. (8) Xosicki, G. W., Ottenbrite, R. If., Thibert, R. J., unpublished studies. (9) Snell, F. D., Snell, C. T., "Colorimetric Methods of Analysis," 3rd ed., 5'01. 111, p. 478, Van Nostrand, New York, 1953. RECEIVED for review May 28, 1959. ilccepted November 6, 1959. M-ork. zupported by a grant from the National Research Council of Canada. VOL. 32,
NO. 1,
JANUARY 1960
107
