tone solvent minimizes streaking or mashing out of the shorter chain saturated esters, while in the chloroform system it decreases smearing and gives more discrete spots. Still, the separation of the whole spectrum of esters from laurate to linolenate in the presence of formic acid is not ideal, because laurate and, to a lesser degree. myristate streak slightly from the starting point without migrating as definite spots when the acetone solvent is used to separate the unsaturated esters. This interference, however, does not prevent subsequent development of the chromatograms in the chloroform system for the separation of the saturated esters from each other. I n this manner, complete resolution of all compounds can be achiered on the same paper. The R j values of the esters in the two solvent systems are such that after chromatography in both solvents, the laurate and myristate esters still may overlap the unsaturated compounds to some extent-it., laurate is sometimes found between oleate and linoleate-but the use of indicators differentiating between saturated and unsaturated acid esters permits unambiguous interpretation of such chromatograms. The unsaturated esters are detected specifically as dark b r o m spots, by staining with iodine vapors, while the saturated esters are not visible and cholesterol is barely noticeable under these conditions. The saturated esters are detected together with the unsaturated ones and free cholesterol by phosphotungstic acid, as described by Michalec (19). The two indicators can be applied successively to the same paper after final chromatography in the chloroform solvent. but it is necessary to discharge iodine thoroughly from the paper to obtain well defined spots with the phosphotungstic acid indicator. The phosphotungstic acid reagent is more sensitive than the iodine indicator and can be used to detect as little as 5 y of esters. About three times this amount of cholesteryl oleate is needed with the iodine indicator, although lesser amounts of the more unsaturated esters are required. Separation is best nith about 10 to 20 y of each component. These lipides tend to streak when overloaded, perhaps because of their high molecular weights, and it is necessary to chromatograph different amounts of unknown mixtures to Separate satisfactorily the major components and to detect the compounds present in minor amounts. Triglycerides move approximately the same distance as the cholesteryl
1470
ANALYTICAL CHEMISTRY
esters. IT-hile fatty acids, their methyl or ethyl esters, monoglycerides, and tliglycerides migrate farther and can be separated from cholesteryl esters. I n general, however, triglycerides do not interfere with the identification of the cholesteryl esters, as they do not react with phosphotungstic acid. Autoxidation products of the unsaturated cholesteryl esters migrate very rapidly and are found close to the solvent front. The R j values of the esters are influenced by the amount of silicone on the paper, the type of paper used, and the composition of the solvent. A high silicone content. represses migration. particularly of the long-chain saturated fatty acid esters. Whatman T o . 1 filter paper was used for most of the experiments, but Whatman Yo. 3 paper is satisfactory also, especially when only unsaturated esters are present. The S o . 1 paper is preferred when the four saturated esters are present too. Increasing the amount of formic acid in the solvent systems decreases R j values. The solvent compositions given earlier and in Table I1 were used for most of the work. However, for any batch of prepared filter paper and set of conditions, it is advantageous to adjust the composition of the solvents. by varying the formic acid content, to give the least smearing of the saturated esters and to retain good separation of the unsaturated compounds. ACKNOWLEDGMENT
The authors are grateful to H. K. Mangold for suggestions concerning the paper chromatographic studies, and to George 1Iizuno for his technical assistance in obtaining the infrared spectra. They are indebted to Dennis Chapman and D. E. C. Corbridge of 1-nilever, Port Sunlight. for the x-ray studies, and to Page R. Edmondson, Department of Medicine, University of l h n e s o t a . for the near-infrared .spectra. REFERENCES
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RECEIVED for revier December 21, 1957. Acrepted April 17, 1958. Supported in part by a grant-in-aid from Oscar AIayer & Co. and by The Hormel Foundation. Hormel Institute Publication KO.165.
Determination of Water in Liquid Bromine-Correction In the infrared quantitative analp i s data [R. B. Duvall and L. R. Kiley, -%SAL. CHEX 30, 549 (195S)l the range presented should read 0-100 p.p.ni., not 0-100 %, and the accuracy 1' + 0 3 p.p.m., not i 0 . 5 %. The final note in the method should read: ,.Relative absorbance is given as the slope of the Beer's law concentration curve used expressed in terms of absorbance per 100 p.p.ni. of constituent."
