estimated by analyzing a series of samples in sealed tubes. As shown in Table I, the average RuO4 recovery was 97.9%, with a standard deviation of *1.5%, The greatest loss of RuOc occurred in transferring a weighed amount of RuOl to the Carius tube and subsequent sealing. For this reason, the accuracy of the analytical method is probably greater than that shown in the table. This was demonstrated by recoveries of 99.0 and 99.6y0 when the RuO4 mas dissolved directly in carbon tetrachloride instead of being sealed in a Carius tube. The method was applied to 300 to 400 samples and found to be satisfactory. Typical types of samples are shown in Table I. These contained hod, RuOz, Anhydrone, ruthenium metal, and/or silica gel. The procedure was modified for the latter, since the CC14 extraction of RuOl from the silica gel was very slow and required several extractions. The RuOc and silica gcl were dissolved in 2N KOH (CCll m-as used to wash the silica gel from the tube into the KOH). When the silica gel mas added to the KOH, there was some gas evolution, but a t no time was there an odor of RuOl. Tests with RuOz and silica gel indicated that there was no significant dissolution of RuOz during this treatment. The dissolved silica gel did not interfere with the optical measurements. If any RuOz n a s carried over with the silica gel, it was removed by centrifuging. Marshall and Rickard reported that
Table I. Summary of Results Sample Composition, Mg. Ru RuOz metal Anhydrone ... ... ... ... ... ... ... ... ... 1000 ... ... ... 1000 ... 1000 ... 1000 ... ...
RuO, 52.1 84.5 48.0 39.7 58.7 64.1 40.9 65.5
150
61.1
...
io0
...
... ... ...
...
42.7 36.0 51.9 46.6
‘50
...
1000
1000 ... ... ... ...
% RuOa
Silica gel
Recovered 98.3
...
96.1 96.6 97.1 97.7 96.3
lbbb
1000 1000
1000 Av.
100.0 97.9
c?
potassium ruthenate solutions are unstable but can be stabilized for a t least l/z hour by making the solution 2N in KOH. Rithenate-solutions, prepared using the present procedure, exhibited exceptional stability. For example, the absorbance of a freshly made sample was 0.527, after 1 day 0.528, and after 5 days 0.530. Initially the stability was ve&_ Door. but reneated use of the-same glassware apparently had a beneficial conditioning action. As a result, all new glassware was soaked for several days in KOH-ruthenate solution prior to use. I
ACKNOWLEDGMENT
The authors acknowledge with thanks
1.5
the guidance provided by C. M.Slansky of Phillips during the course of the -program. LITERATURE CITED
(1) Marshall, E. D., Rickard, R. R.,
ANAL.CHEM.22,795-7 (1950). (2) Sandell, E. B., “Colorimetric Determination of Traces of Metals,” 3rd ed.,
Interscience, New York, 1958. CHARLES J. ANDERSON RICHARD DEL GROSSO
gl”bJl)MARTIN ratoriesH. ORTNER
~ ~ $ - ~ & ~ ~ ~ f
Vitro Laboratories West Orange, N. J. WORKsupported by a subcontract from Phillips Petroleum Co., prime contractor to the Atomic Energy Commission under contract AT(10-1)-205.
N e w Solvent System for Separation of Fatty Acids C,,-C,, by Countercurrent Distribution n-heptane as the upper layer and acetonitrile, methanol, and acetic acid as the lower layer, appears to be equivalent in performance to the new solvent
SIR: Ahrens and Craig ( I ) used the countercurrent distribution principle for the separation of fatty acids using several solvent systems. One system, 3.6 3.2
PALMITIC ACID
2.8
K.0.70
STEARIC ACID K: 1.2
Figure 1 . Countercurrent distribution of fatty acids
2.4
Upper layer. Petroleum ether Lower layer. 9 to 1 dimethyl rulfoxide-octa-
2.o
1.6
no I
system presented here. However, no third solvent is required in the new system. Fatty acids CIO to C18 have been separated from each other by the countercurrent distribution technique in the new solvent system using petroleum ether as an upper layer and a 9 to 1 ratio of dimethyl sulfoxide to 1-octanol as a lower layer. This communication describes the determination of the distribution coefficients of fatty acids in various solvent systems and the separation of the acids in the new solvent system. EXPERIMENTAL
1.2
0.8 0.4
0.0
0
10
20
30
40 5 0 60 TUBE NUMBER
70
80
90
100
Apparatus. Craig-type countercurrent distribution apparatus (,!?), 100 tubes, 40-ml. lower layer capacity, 40ml. upper layer capacity with automatic filling device (H. 0. Post Scientific Instrument Co., Maspeth, N. Y.). Determination of Distribution Coefficients. Before the counterVOL. 33, NO. 4, APRIL 1961
* 647
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.. 2 6 ~. .~ 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
