UNITED STATES DEPARTMENT OF THE INTERIOR GEOLOGICAL. SURVEY AN INVESTIGATION OF THE UNSATURATED FATTY ACIDS OF BUTTERFAT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By RICHARD H. BACKDERF, B.S., M.A. The Ohio State University 1956 Approved by* I, -- £ adviser Department of Chemistry DEDICATION To Mary Anne ii ACKNOWLEDGMENT The author wishes to express his sincere gratitude to his adviser Professor J. B. Brown for the suggestion of this problem and for his invaluable guidance and encouragement through­ out its successful completion. Appreciation is also felt for the use of an infrared instrument furnished by Dr. Urone and Mr. Anders of the Ohio Department of Health. The summer butter used in this investigation was kindly supplied by the Pickerington Creamery Inc., Pickerington, Ohio. This work was supported in part by a grant from the Development Fund of The Ohio State University through the Institute of Nutrition and Food Technology. iii TABLE OF CONTENTS Page I Introduction ....... .................. • .1 II Historical Review on the Composition of the Fatty Acids of Butterfat............... 2 III A Review of the Methods of Separation and Identification of Fatty Acids and Their Esters................ .......... ..... ..... ...... ..... 7 A. Methods for Separating the Fatty Acids.............................................. 7 1. Fractional Ester Distillation...... • • • •..... 2. Separations of Fatty Acids by the Use of Metal Soaps.............................. 9 3• Low Temperature Crystallization.10 U. Urea Inclusion Conpounds......................... 10 5. Chromatography......... ..... .................... .12 6. Fractionation of Fatty Acids by Countercurrent Extraction ........ 15 B* Methods of Identification of Fatty Acids and Esters. .... 16 1. Determination of the Degree of Unsaturation of Fatty Materials ................. .16 2. Determination of the Positions of Double Bonds in Fatty Acids and Their Esters............. 16 a) Ozonation of Uhsaturated Fatty A c i d s . .16 b) Permanganate Oxidations of Unsaturated Fatty Acids................. 18 c) Peracid Oxidations of Uncaturated Fatty Acids..................... *............. .19 d) Separation and Identification of the Products Resulting from Oxidative Double Bond Cleavage of Unsaturated Fatty Acids ..... .21 iv V Page 3* Optical Methods Used for the Identification of Fatty Acids•.••.••••••••*••••••. *2U a) Ultraviolet Spectrophotometry........... 21* b) Infrared Spectrophotometry.. ................... 26 c) X-ray Spectroscopy*. .............. *.......... 26 IV Objective of this Investigation........................ 29 V Experimental............. *30 A. Analytical Methods................................ 30 1. Saponification Equivalent and Iodine Value...... *................. 30 2. Neutralization Equivalent........... 31 3. Ultraviolet Spectrophotometric Examination............... 31 U. Infrared Examination ................ 31 B. Preliminary Analyses of Several Butterfats..................................... .*35 C* Isolation of the Ci6 and 0^8 Methyl Esters from Summer Butter by Fractional Distillation.......... 3? D. Fractional Crystallization of the Ci8 Methyl Esters of Low Temperatures........ ........................ ••.•.U0 E. Development of the Chromatographic Procedure for the Resolution of Mixtures of Bicarboxylic Acids.................... .U3 1. Preparation of the Chromatographic Column............................ ........... * *lUi 2. Preparation and Synthesis of the Various Acids Used to Standardize the Chromatographic Procedure.................... U6 3. Standardization of the Chromatographic Procedure....................................... 5>3 vi Page F. Cleavage Reactions of Pure Acids and Mixtures of Acids and Chromatographic Analysis of the Cleavage Products*............... *............... 56 1. The Oxidative Cleavage of Pure Acids.......................................... 56 a) Oxidation of Oleic Acid via the Method of Begemann**......*............... 56 b) Modification of the Begemann Oxidation Procedure .............. *....... 57 c) Oxidation of Oleic Acid with Perforate and Periodic Acids and KMnOlj in Acetic Acid.............. .........58 d) Oxidation of Oleic Acid with Perforate and Periodic Acids and Ag20«................ *59 e) Ozonation in Acetic Acid...... 59 f) Discussion of the Cleavage Reactions a to ................ 60 2. Oxidation of a Known Mixture of Monoethenoic Fatty Acids ..... 63 0. Oxidative Analysis of the Various Fractions Obtained from Summer Butterf at ...... 66 1. Investigation of the Ci8 Series................. 66 2. Investigation of the C^5 Series................. 8l VT Dis cuss ion........................................... 83 VII Summary.......... 90 VIII Bibliography.................. 91 I Introduction. The Tatty acid composition of butterfat is one of the most complex of all naturally occnrlng lipids. Like other fats it is now assumed to be primarily a mixture of mixed triglycerides, but but ter fat glycerides contain a larger number of different fatty acid species than any other common natural fat. More than twenty five different fatty acids have been reported to be present, but, as with most other lipids, oleic and palmitic acids are the major components. This thesis represents primarily an investigation of the octadecenolc or "oleic" acid fraction obtained from a summer butter but, in addition, describes the "linolelc" and "palmltoleic" component acids of this fat. The problem is based upon the observation that oleic acid prepared from butterfat by the usual means displays a melting point that is several degrees lower than that of oleic acid prepared from olive oil. Since we have taken olive oil oleic acid as a standard of purity it is the natural conclusion that the lower melting octadecenolc acid from butterfat is in fact a mixture of acids. Millican and Brown (1) have studied the melting points of the octadecenolc acids, and their dihydroxy derivatives, obtained from several natural sources and have concluded that there is a rather general occurrence of octadecenolc acids other than oleic acid in fats and other lipids of animal origin. Although thirty geometrical and positional isomers of oleic acid are theoretically possible, only vaccenic (trans-ll-octadecenolc) acid has been reported to be present in butterfat. Indeed, the structure assigned to natural vaccenic acid is not above doubt, for while the natural a d d and synthetic trana-ll-octadecsnoic a d d possess very similar physical end choiical properties, the z>riy diffraction patterns of the two acids are distinctly different (2). This fact led Bumpus, Taylor and Strong (3) to conclude that natural vaccenic acid preparations consisted essentially of trans-ll-octa­ ds cenoic acid, but were contaminated with small amounts of isomeric acids sufficient to produce an altered crystal structure* The natural vaccenic acid was prepared from beef fat by the Bertram metal soap method (U) whereas the a d d mixtures described in this paper were prepared from butter by low temperature crystallisation techniques exclusively* nevertheless, on the basis of the conclusions of Bumpus et*al*, it may be suspected that natural vaccenic add, regardless of the source or method of preparation, may be a mixture* II Historical Review of the Composition of the Fatty Acids of Butterfat* The fatty acid composition of milk fats differs significantly from the depot or organ fats of the same animal and from the plant fats* This difference is especially marked with the milk fats of ruminants and is due to the presence in these fats of m i l but definite proportions of butyric, caproic, caprylic, capric and lauric acids* The fatty acid composition of cow*s milk fat has naturally received the most detailed investigation* In addition to the lower saturated acids, it has been established that small quantities of mono—ethenoic a d d a of 10, 12, lit and 16 carbon atoms are also present* The presence of a decenoic acid in butterfat was first postulated by Saedley (5) in 1912. In 1922 Orun and Wirth (6) isolated and identified this a d d as well as the 3 Ci)(> and acids* In these cases the doable bond was found to be In the 9-position. In 1933 Bosworth and Brown (7) confirmed the presence of decenolc and tetradscenoic acids in butterfat to the extent of 0.18JC and 0.875t respectively* In addition to palmitic, stearic, oleic and llnoleic acids there are trace amounts of a number of unusual fatty acids* Shorland and co-workers hare recently reported the presence of a surprising number of branced-chain fatty acids derived from New Zealand butterfat* These acids are listed below in Table It Table I The Saturated Branched-chain Fatty Acids of Butterfat Acid Wt« % of Butterfat Reference Isoheptadeoanoic — 8 Anteisoheptadecanoic — 8 A multibranched C20 a d d 0*006 9 Iso-pentadecanolc 0.37 10,12 Anteiso-pentade canolc 0*!i3 10,12 Iso-tetradecanolc 0*05 U N-pentadecanolc 0.82 12 The chemical identity of the polyunsaturated acids of butter­ fat has been the subject of considerable discussion* In 1929 Hilditch and Jones (13) found that llnoleic acid from butterfat failed to give the usual yields of the tetrahydroxystearic acids given with llnoleic acid from vegetable sources* Bosworth and Brown (7), 1933, failed to detect any llnoleic acid in butterfat. Hilditch (lit) concluded in 1937 that minute quantities of els, cle—linoleic aeid occur in butterfat, but that a larger percentage (3-5# of