ISDCSTRI.4 L AND LC’SGISEERISG CHEJIISTRY
July 15, 1930
can be deteriiiined the reagent blank error is negligible, as an inspectioii of Table ,Y will show. Literature Cited (1) Arnfeld, Dissertation, Berlin, pp. 1-60 (18991. (2) Asch, lv. and D . , “Silicates in Chemistry and Commerce,” translated by Searle, p. 16, London, 1913. (3) Atkins, J . .lIariiie B i d I s s o c n . , Cniied Kingdom, 14, 89 (1926). (4) Atkins, IhiLl , 15, 191 (1928).
(5) (6) (7) (8) (9) (10) (11) (12) (13) (14)
283
Atkins and Wilson, Biockem. J., 20, 1223 (1926). Bertrand. Bull. soc. chim. b i d , 6, 656 (1934). Dienert and Wandenbulcke, Compt. rend., 176, 1 4 7 8 (1923). Isaacs, B d l . soc. chim. bioi., 6, 157 (1924). King, J. B i d . Chem., 80, 25 (1928). King and Lucas, J . .Am. Chem. Soc., 50, 2395 (1928). S i m e c , Biochem. Z . , 190, 42 (1927). Travers and Nalaprade, Compt. r e n d . , 183, 292 (1926). Truog and I f e y e r , 1x0. ESG. C H E x , Anal. E d . , 1, 136 (1929‘1 Wu, J . B i d . Chem., 43 189 (1920).
A Source of Error in Polariscopic Measurements’ H. K. Miller a n d J a m e s C. Andrews DEP.4RTMEST
OF l”YSIOLOCIC.4L
CHEMISTRY, S C H O O L
w
OF
hfEDIClKE, UNIVERSITY O F PEVSSYLY.4SI.4, PHILADELPHIA, PA.
H I L E it is a matter of coniinon knowledge that accurate polariscopic readings require t h e use of clear solutions, there is little information available as to the extent and nature of the errors involved when polariscopic examinations are made of solutions which are very faintly turbid but not sufficiently so to prevent readings. The turbidity in solutions of amino acids is often of such nature that filtration is quite ineffective. Under these circuiiistances, if readings in short tubes are a t all possible, one is inclined to resort to this procedure rather than to other methods of clarification. The equivocal results of such a procedure are evident from the data shown below. While making polariscopic measurements on arginine and other ainino acids with comparatively lon. rotations, the writers observed rather large discrepancies for TThich there appeared to be no adequate explanation. This was first called to their attention by the values for specific rotation of d-cysteine published by 1-ickery and Leavenworth ( Z ) , who obtained figures considerably larger than those previously published by A n d r e w ( 1 ) . Repetition of these prepsrations under identical conditions still resulted in values which, for the present authors, duplicated the previously published figures of $9.0 to +10.0, while those obtained by Kckery and Leavenworth usually ranged from about 12.0 to 18.0. The polariscopes used. when checked with pure sucrose solutio’ns, gave practically identical results. A check solution of 1 per cent glutamic acid also gave identical results. However, the writers noted that in several cases the amino acid solutions shoned a slight turbidity which was very difficult to remove by filtration, and this sometimes obscured the field to the point of necessitating the use of a 1-dm. tube rather than the 4-dm. tube usually used by them. The observations of Vickery and Leavenv-orth were made with 2-dni. tubes. A series of measurements was then made of the optical activity of several amino acids and also of pure sucrose in 1-din. and 4-din. tubes. Table I shows the results obtained. It will be noted that the differences range from practically zero to a very considerable figure and that the greatest differences occur consistently with the more turbid solutions. I n the solutions of arginine (not too acid) and glutamic acid enough putrefaction can result from standing overnight in the laboratory to give a definite turbidity and, while this may not be so great as to prevent the use of the longer tube, one is far more prone to resort to shorter tubes in such cases. The cysteine samples were all prepared by reduction of 1cystine with tin and hydrochloric acid. The usual procedure
+
1
Rereived .4pril 26, 1930.
+
is to precipitate the excess tin as stannous sulfide and this is invariably followed by slow precipitation of free sulfur. When the solution with the stannous sulfide is filtered directly into the polariscope tube (the procedure used by T’ickery and Leavenworth and also by the authors in Experiments 8, 9, and lo), one is always on the verge of too much turbidity due to the sulfur, even with rapid manipulation. I n Experiment 7 the solution was filtered into the tubes without contact with hydrogen sulfide or removal of the tin; in Experiment 11 the solution was carefully protected from air for a day after the hydrogen sulfide treatment and then filtered into the tubes. Presumably the formation of free sulfur had ceased. T a b l e I-Specific
EXPP. 1 2 3 f a
6
k9
10 11
R o t a t i o n of V a r i o u s S o l u t i o n s i n T u b e s of Different Lengths
SOLS.
COSCS. Grams per
Sucrose Sucrose Glutamic acid hrginine Areinine
100 c c . 1 0470 1 0795 0 7213 8 264 1 030
Cystine Cysteine Cysteine Cysteine Cysteine Cysteine
1 000 1.250 1,000 0 995 0 825 0.902
[el?
[al?
LDM. TUBE
4-n~.
-C66.90 f67.00 f13.9 +26.7 -18.0 -213.0 f 8.8 f12.0 fl5.Z
+15.s f10.2
TUBE
REM.ARKS
f67.10 f67.00
Clear Clear Very faintly turbid Clear Lower aciditv than in Expt. 4 : faintly turbid Apparently clear Apparently clear Turbid Turbid Turbid Apparently clear
+11.4 +26.5 +14.5 -210.0 8.2 8.2 9.6 9.4 9.8
++ + ++
I t appears reasonable to conclude that in faintly turbid solutions of weakly rotatory substances identical specific rotations may not be obtained with tubes of different lengths. This is probably due to the effect of the turbidity in changing the character of the spot of light, and no doubt the possibilities of such a change are modified by the peculiarities of the polariscope and of the eye of the observer. If the error is caused by the interposition of so inuch turbidity as to change the appearance of the field, one would expect to obtain more correct values from shorter tubes, with, however, the disadvantage of a correspondingly greater percentage error in the final values obtained. Making duplicate determinations in tubes of two different lengths would appear to be a practicable means of insuring the absence of the turbidity factor. Literature Cited (1) Andrews, J. B i d . Chem., 69, 209 (1926). (2) Vickery and Leavenworth, I b i d . , 86, 129 (1930).
