found in this solvent system as compared with analytical runs with 1 gram of material (2). This was thought to be possibly due to incomplete draining during the decantation and transfer stages. When the fractions were flash-evaporated and lyophilized, the combined dry weight of the LH-RH active area was 2.7 grams. Thus a purification of over 60fold resulted in this run, with an excellent recovery of biological activity which was estimated to be essentially quantitative. The proportionate increase in the specific biological activity was evident from bioassays for LH-RH shown in Table I. The pressor activity was found in fractions 60-99 (mean K = 0.76). Since LH-RH contains an inherent activity of FSH-releasing hormone (FSH-RH) (7, a), this activity was also found in tubes No. 130-230. The fractions which remained in the CCD train contained the bulk of the dry weight and were devoid of LH-RH and FSH-RH activity at doses of 20 kg. This experiment is an example of the successful application of the technique of countercurrent distribution for the purification of a biologically active material from the hypothalamus on a preparative scale. The versatility of the apparatus is also apparent as the approach can be conveniently modified to suit one’s purpose.
Similar CCD techniques could be used for the purification of other biologically active substances as stated many times by Craig and associates (6, 10, 11, 13, 18). The advantages and suitability of the CCD method for this type of separation are once more confirmed. It may be pertinent also to mention that a modification of CCD called the “counter double countercurrent distribution” (CDCD) has a still much larger capacity (19). ACKNOWLEDGMENT We are deeply grateful to Professor Lyman C. Craig, Rockefeller University, New York, for generous advice on the techniques of CCD. We wish to thank Mr. K. Schuerger, H. 0. Post Scientific Instrument Co., Inc., for his cooperation in constructing the adjustable metal flap for the timing disk. RECEIVED for review April 14, 1971. Accepted May 28, 1971. Supported in part by USPHS Grant AM-07467 and The Population Council, New York, N. Y . (19) 0. Post and L. C. Craig, ANAL.CHEM., 35, 641 (1963).
Sclvent Isotope Effects on Decomposition of N, N- DiaIky Idithiocarbamic Acids K. I. Aspila, S. J. Joris,‘ and C. L. Chakrabarti* Department of Chemistry, Carleton University, Ottawa, Ontario, KIS 586, Canada
STUDIES ON THE DECOMPOSITION of dithiocarbamates in DzO have appeared recently in the literature ( I , 2). It was shown that the rate of decomposition of C-N,N-tetramethylenedithiocarbamic acid at pH 1.0 is 2.6 times faster (1) in DzO than in H 2 0 ; and that the p D dependence of the decomposition of N,N-diethyldithiocarbamic acid is similar to its pH dependence (I, 2). In this paper, solvent isotope effects on decomposition rates of several dialkyldithiocarbamates are compared. The synthesis of the dithiocarbamates used in this study was described in an earlier paper (3). D 2 0was 97 % pure. Rate measurements were done spectrophotometrically (3) at 15 i 0.1 “C and at pH (or pD) values of 1 =k 0.2. In this range of acidity the rates of decomposition of dithiocarbamates are independent of pH (pD) since nearly 99% of the dithiocarbamate anions are converted to the unstable acid form ( I , 3). It appears from Table I that the magnitude of the solvent isotope effect is related to the substituent in the dithiocarbaPostdoctoral research fellow. Present address, Noranda Research Centre, 240 Hymus Boulevard, Pointe Claire, Quebec,
Canada. Correspondence should be addressed to this author. (1) K. I. Aspila, V . S. Sastri, and C. L. Chakrabarti, Tularrta, 16, 1099 ( 1969). (2) S. W. Dale and F. Fishbein, J . Agr. Food Chem., 18, 713 (1970). (3) S. J. Joris, K. I. Aspila, and C . L. Chakrabarti, J . Phys. Chem., 74, 860 (1970).
Table I. Solvent Isotope Effects on the Decomposition of N,N-DialkyldithiocarbamicAcids (Temp = 15.0 f 0.1 “C, pH = pD = 1) Ratio of decomposition Decomposition rate rate Dithiocarbamic constants, set+ constants KD,O Kn2o KD,o/KH20 acid Dimethyl 0.0506 0.0180 2.80 0.0922 0.0363 2.54 Diethyl 0.0150 1.81 Dibutyl 0.0272 0,0352 0.0827 0.42 Diisopropyl
mate molecule. An inversion of isotope effect is observed in the case of diisopropyl-dithiocarbamate for which the rate of decomposition is slower in DzOthan in H20. It was shown (4) that dithiocarbamic (DTC) acids are formed by protonation of a sulfur atom of the dithiocarbamate anion (models I and 11). Through subsequent redistribution of electron densities in the acid molecule (5). the nitrogen atom accepts (3) a hydrogen bond. As the dithiocarbamate anion picks up only one proton (4,the hydrogen bond formed at the nitrogen atom is either intramolecular (4) S. J. Joris, K. I. Aspila, and C. L. Chakrabarti, ANAL.CHEM., 41, 1441 (1969). ( 5 ) D. M. Miller and R. A. Latimer, Can. J . Chern., 40,246 (1962).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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(model 111) or is the result of solvent participation in the transfer of the proton from the sulfur atom to the nitrogen atom of the DTC acid (model IV). Solvent participation is likely to occur in the case of DTC acids with small alkyl substituents (e.g., dimethyl- or diethyl-DTC acid) as the studies with mixed solvents indicated ( 3 ) that for these DTC acids the solvent is able to approach closely the N-C bond (See Scheme 1).
f
\
I
H
(W Scheme 1
The present authors propose that the solvent isotope effect observed in the decomposition of DTC acids containing small alkyl substituents is mainly due to the difference in position of either the deuteron or the proton in the hydrogen bond. Indeed, in agreement with zero point energy considerations, the probability of finding the deuteron close to the nitrogen atom is larger than the equivalent probability for a proton (6). As a result, the fractional charge on the nitrogen atom (6) R. E. Rundle, J . Phys., 25,487 (1964).
(model IV) will be larger when a deuteron rather than a proton occupies the “hydrogen bond.” We have shown previously how an increase of fractional charges in the DTC acid molecule enhances the decomposition rate (3). However, in the case of DTC acids containing large alkyl groups (e.g., diisopropyl-DTC acid), the solvent is less able to approach the N-C bond as is reflected in the great instability of these acids (3). The lower decomposition rate of diisopropyl-DTC acid in D?O suggests that the rate-determining step in the decomposition of that compound is the transfer of proton from the sulfur atom to the nitrogen atom (model 111). Indeed, the transfer of a deuteron from one base to another occurs at a slower rate than that of a proton. The opposite solvent isotope effects seen in Table I thus lead to the conclusion that, depending on the availability of a solvent molecule near the N-C bond, the rate-determining step in the decomposition of DTC acids is either the proton (or deuteron) transfer from the sulfur to the nitrogen atom or the decomposition of the intermediate IV. The solvent isotope effect in the decomposition of dibutyl-DTC acid (Table I) clearly illustrates the competition between these two possible rate-determining steps. If the above arguments are a true interpretation of the experimental results, the solvent isotope effect may be regarded as confirming the coilclusion reached by studies in mixed solvents (3); namely, that the rates of decomposition of DTC acids are primarily governed by the ease of approach of the solvent to the N-C bond. Solvent isotope effects have the peculiarity to throw light on the influence of the proton transfer on the overall reaction rate. ACKNOWLEDGMENT
The authors are grateful to Dr. P. M. Laughton and Dr. C. H. Langford, Carleton University, for helpful discussions. RECEIVED for review March 22, 1971. Accepted June 7, 1971. This paper constituted a part of the MSc. thesis of K. I. Aspila. The authors are grateful to the National Research Council of Canada for research grants.
Gas Chromatographic Determination of PeniciIIins Charles Hishta, David L. Mays, and Michael Garofalol Bristol Laboratories, Dicision‘of’Bristol-Myers Company , Syracuse, N . Y.
NUMEROUS CHEMICAL METHODS are available for the quantitative determination of penicillins; the identity of the penicillin must usually be determined by a different procedure, such as infrared spectrophotometry or thin-layer chromatography. Official microbiological methods for determining penicillins suffer from high variability and time-consuming procedures. A gas chromatographic procedure for the indirect identification of penicillins was reported by Kawai and Hashiba ( I ) . Organic acids produced by alkaline cleavage were Present address, Xerox Corporation, Rochester, N. Y .
converted to methyl esters and separated on a 3 . 5 % SE-30 column. Wolfe (2) and coworkers chromatographed the methyl ester of L-phenethicillin. A single peak without evidence of column decomposition was obtained from two columns coupled in series, 1% SE-30 on glass beads (70 cm) followed by 5 % NGS on Chromosorb (40 cm). The chromatography of methyl esters of several 6-amino penicillanic acid derivatives was reported by Evrard (3) in 1964. Some of these derivatives were separated on a 0.4% SE-52 column (2) S. Wolfe, Queens University, Kingston, Ontario, personal
communication, 1964. (1) S. Kawai and 1530
S. Hashiba, Burwcki Kugakir, 13. 1223 (1964).
(3) E. Evrard, M. Claesen, and H. Vanderhaeghe, Nature, 201, 1124 (1964).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
