Table I. Distribution Coefficients of Fatty Acids in Various Solvent Systems
-
Acid Caoric Lahric
~
Myristic Palmitic Stearic Table 11.
(Upper layer, petroleum ether) Lower Layer, 9 to 1 Lower Layer, Dimethyl Dimethyl SulfoxideSulfoxide HzO Settling time K - Settling time K 30 sec. 0.07 30 sec. 0.26 ~. .~ . 1 min. 0.17 8min. 0.77 2 min. 0.31 >1 hr. 2.56 10 sec. 0.72 1 hr. 6.73 21 min. 1.44 1 hr. ~
Lower Layer, 1 t o 1Dimethyl SulfoxideHz0 Settling time K 2 min. 0.44 9 min. 0.80 15 min. 2.18 45 sec. 1.17 15 sec. 2.45
Distribution Coefficients of Fatty Acids in Various Solvent Systems
(Upper layer, petroleum ether) Lower Layer 9 to 1 Lower Layer, 18:2:3 Dimethyl SuboxideDimethyl Sulfoxide1-Octanol H20-1-Octanol Settling Settling time, time, Acid sec. K B Bec. K B 30 0.28 20 0.09 Capric 1.8 6.6 30 1.86 30 0.16 Lauric 2.0 3.1 Myristic 40 5.80 25 0.32 1.7 1.2 50 6.69 Palmitic 40 0.55 1.8 1.3 45 8.63 30 0.99 Stearic Oleic 0.52 Linoleic 0.25 current distribution apparatus is actually used, predictions can be made as to the separability of substances if their distribution coefficients in the chosen solvent system are known. Distribution coefficients ( K values) for the acids were determined by taking a 0.5-gram sample and equilibrating i t in a 250-ml. separatory funnel (supplied with a Teflon stopcock) with equal volumes of upper and lower layers. An aliquot of the upper layer was then titrated. From these data, the concentration of fatty acid in each layer was calculated and thus the distribution coefficient. The solvents were chosen for their immiscibility in each other and their ability t o dissolve fatty acids. The
Lower Layer, 19:1:3 Dimethyl SulfoxideH20-1-Octanol Settling time, sec. K B 30
0.35
20
0.73
30
1.86
35
2.70
40
5.24
2.1
2.5 1.5
1.9
following tables give K values for fatty acids in various solvent systems. A solvent system was considered satisfactory if the settling time for the two layers was not greater than 5 minutes and if two adjacent R values had a ratio (beta value, 8) of the larger to the smaller in the range of 1.1to 4. Tables I and I1 list the distribution coefficients for several solvent systems. For the first solvent system in Table 11, which was found to be the optimum, oleic acid would not be separated from palmitic acid since their K values are practically the same. Choice of Solvent System. The solvent system using a petroleum ether upper layer and a 9 to 1 ratio of dimethyl sulfoxide to 1-octanol as a
lower layer (Table 11) was chosen as optimum because the beta values were reasonable, the settling time was less than 1 minute, and no third solvent constituent had to be added to the lower layer. Because of foaming of the fatty acids, necessitating long settling times, 1-octanol was used as a defoaming agent. Separation of Fatty Acids Clo-C18. Using the above-mentioned solvent system, a mixture of fatty acids Cla to Cl* totaling 13 grams (synthetic mixture) was added to the first five tubes of the apparatus. After 150 transfers of upper layer, an aliquot of every third lower layer was titrated for fatty acids. Figure 1 shows the distribution of the fatty acids in the tubes of the countercurrent distribution apparatus. A qualitative infrared analysis was made on the solutions from each peak tube. The results coniirm the presence of the individual fatty acids in each section of the apparatus. The R values shown in Figure 1 were calculated from the formula K = T,,, where ,,T is the tube number n - rmsr of maximum concentration and n is the number of transfers. The R values in Figure 1 are higher than those found in Table I1 for the respective acids because of the large amount of sample introduced initially. LITERATURE CITED
(1) Ahrens, E. H., Jr., Craig, L. C., J. Biol. Chem. 195,299 (1952). (2) Craig, L. C., Hausmann, W., Ahrene,
E. H., Jr., Harfenist, E. J., ANAL. CHEM.23, 1236 (1951). FRITZWILL,I11
Alcoa Research Laboratories Aluminum Company of America New Kensington, Pa. Presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1958.
lodometric Assay of Natural and Synthetic Penicillins, 6-Aminopenicillanic Acid and Cephalosporin C SIR: Since the publication of the original (8) work on the iodometric method for the assay of penicillin a host of other penicillins have appeared. These include the natural penicillins, penicillin V (phenoxymethyl penicillin acid) and a newly discovered cephalosporin C as well as a synthetic penicilli (a - phenoxyethylpenicillin). Also, 6-aminopenicillanic acid is the key intermediate (3, 6) through which new penicillins may be synthesized. 648
ANALYTICAL CHEMISTRY
This study shows that the relationship between the iodine consumption of these various compounds under the normal conditions used for the assay is stoichiometric and that a chemical equivalent for the various compounds can be derived experimentally. Moreover, baminopenicillanic acid has little or no bioactivity when measured against the usual test organisms even though it possesses the P-lactam structure common to the natural peni-
cillins. Therefore, it may when present, lead to false values in the iodometric assay for penicillin. Because the iodometric method corrects for the presence of other iodineconsuming substances by an initial titration it has been used successfully over many years on diverse formulated products, on many derivatives, and even on properly treated broth samples (1,4). In the production of synthetic penicillin, however, there does exist the possibility
that 6-aminopenicillanic acid may appear along with penicillin. Acidification of the mixture and extraction with amyl acetate will separate the two compounds and these may then be assayed independently. EXPERIMENTAL
Millimolar solutions of potassium benzylpenicillin, phenoxymethyl penicillin acid, potassium a-phenoxyethylpenicillin, 6-aniinopenicillanic acid, and cephalosporin C were made up and assayed under the conditions shown in Table I. Ten-milliliter aliquots representing one hundredth of a millimole were used in each experiment so that the number of iodine equivalents can be readily observed in the last column. In each case there was no demonstrable consumption of iodine before alkali treatment. The volume of 0.01N iodine solution used in all cases was 15 ml. which represents an approximate 50% excess of this reagent. RESULTS AND DISCUSSION
Because 6-aminopenicillanic acid and cephalosporin C were new compounds the effect of a higher concentration of alkali was studied. The results indicate that the increased alkali concentration has no effect on the iodine equivalent. An inactivation time of 10 minutes was adequate except for 6-aminopenicillanic acid which required 15 minutes. Those compounds having the configuration,
(CH&C-
CH-COOH
I
I
HC,
,N\
C,O CH
I
- -
HN CO R consume nine equivalents of iodine. 6-Aminopenicillanic acid,
(CH3)2 CI
s
CH-COOH I
N
llc’
‘co
\ /
CH
I
“2
consumes only eight equivalents of iodine. Cephalosporin C consumes approximately four equivalents. This value, it must be stressed, is tentative because
Table 1.
Iodine Equivalents
Compound
M.W.
Wt., Mg.
Potassium benzylpenicillin
372
3.72
2N
NaOH, M1.
Inactivation Time, Minutes
1
15 30
12
Consumed, M1.
60 a-Phenoxyethylpenicillin
350
acid
3.50
1
15 30
8.95 9.02 8.98
15 30
60
7.95 8.03 7.97
3
15 30 60 15 30 60
8.02 8.04 7.98 7.96 8.05 8.02
1
15 30
8.96 9.00 9.03
15 30 60 15 30 60 15 30
4.05 4.10 4.07 4.12 4.10 4.15 4.10 4.17 4.20
60
6-Aminopenicillanic acid
216
6-Aminopenicillanic acid
216
Potassium phenoxyethylpenicillin
402
2.16
1
2.16
2
4.02
60
Cephalosporin C
437
4.37
1 2
3
60
other data indicate a purity of approximately 90% on the particular sample used for investigation. The purity of penicillin preparations is expressed in terms of biopotency. For example, 1 mg. of sodium benzylpenicillin is equivalent to 1670 International or U.S.P. units. One milligram of potassium benzylpenicillin, on the other hand, is equivalent to 1595 units; thus the bioactivity is a function of its molecular weight. When using the iodometric method in purification processes the purity can be calculated in terms of percentage or in terms of micrograms per milligram. (V,
- Vl) M.W. Nof R.pen Sample wt. in mg.
fig. R.pen/mg.
where V z = volume of exactly 0.01N iodine consumed after inactivation with alkali VI = iodine consumption before inactivation R.pen = the particular species of penicillin to be determined
N
8.97 9.00 8.97
= number of iodine equiv-
alents. Any new species of penicillin can be chemically assayed by substituting the correct molecular weight in the equation. ACKNOWLEDGMENT
The author thanks M. R. Siino for carrying out many of the experiments involved and H. A. Friediani of Bristol Laboratories for the supply of purified 6-aminopenicillanic acid. LITERATURE CITED
(1) Abraham, E. P., Chain, E. B., Nature 146,837 (1940). (2) Alicino, J. F., IND.ENQ. CHEM., ANAL.ED. 18. 619 11946). --, (3) Batchelor, ’Fa-R., Doyle, F. P., Nayler, J. H. C., Rolinson, G. N., Nature 183,257 (1959). (4) Ferrari, A., Russo-Alesi, F. M., Kelly, J. M., ANAL.CHEM.31, 1710 (1959). (5) . . Sheehan, J. C., Henerv-Logan. K. R.. Johnson, ‘D. A:, J. A h . chem. Soc: 75, 3292 (1953); 81, 5838 (1959). \--
JOSEPH F. ALICINO Squibb Institute for Medical Research New Brunswick, N. J. VOL. 33, NO. 4, APRIL 1961
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