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 theComposition 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 Butterfat ...... 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 the total fatty acids) was made up of geometrical Isomers of this acid. Hilditch and Jasperson (15) detected traces of triethenolc acids and of conjugated dlethenoic C-^g acids. White and Brown (16), 1939, on the basis of tetrabromide studies, suggested that about one-third of the octadecadienoic acids of butter were isoadds, probably of the trans type. By using spectroscopic methods Schaffer and Holm (17) claimed that summer butters contained 2.6-

2.7$ llnoleic a d d and 0.8-1.2% linolenic acid but their method was not specific for these acids. Recently it has been shown that the ultraviolet method for estimating these polyunsaturated adds is un­ reliable when the fats contain isollnoleic acids (18, 19). Cornwell et.al. (20) fractionated the C^g esters of butterfat by low temperature crystallisation and found that at the lower temperatures filtrate fractions were obtained which contained a higher proportion of trans acids than the starting material. These more soluble trans acids were presumed to be isollnoleic acids of the c is-trans or trans-cis type.

There is also present in butterfat a very small percentage of acids of higher molecular weight. Hilditch and Jones (13) reported the presence of small amounts of arachidic acid while Bosworth and Brown

(7) proposed that arachidoni-, tetracosanoic, behenic, and cerotic acids were present in butterfat.

In addition to the above mentioned components of butterfat, one other unusual acid is known to be present. This is vaccenic, or trans-ll-octadecenoic acid. In 1928 Bertram (21) isolated this acid 5 from butterfat using a lead soap-mercury soap crystallization procedure, and estimated that 0.01* of the fatty acids of butter was vaccenic.

Subsequent investigators have reported this a d d to be present in butterfat as follows s Qrossfeld and Sinner (22), 1.1-1*. 7*; Qeyer

et.al. (23), 0.5-0.7*; Cornwell et.al. (20), 2.3-8*. The presence of elaidic acid in butterfat has not been

established. This acid has, however, been reported as a constituent

of beef fat by Swern and co-workers (21*). In any event, trans-acids

have repeatedly been reported as minor constituents of various fats of

animal origin. The diets of these animals, mostly herbivorous, do

not contain trans-acids. One is left with the problem of trying to explain the origin of these iso-acids within the body of the animal.

Swern (21*) has proposed that trans-octadecenolc adds may have been produced endogenously from a cls-aonoethenoic acid by the action of oxidases. Shorland (25) on the other hand, has found trans-acids in

the fat of ruminants but not in the fat of non-ruminants and believes

that their presence could better be explained on the basis of bacterial action in the rumen. He also found that about nine percent of the fatty adds isolated Aram the rumen contents of pasture-fed sheep were

trans, while no appreciable amount of trans-adds was found in the pasture lipids. Reiser (26) has shown that rumen bacteria hydrogenate

linolenic acid to linoleic acid, and since hydrogenation is usually

accompanied by cla, trans isomerization, this could explain the presence

of trans-acids in the rumen. Another observation which may bear some

direct relation to this problem was stated by Hofmann and Tausig (27)

in 195U* These investigators have found a wide-spread distribution of 6 da-vaccenic a d d in bacterial lipids, it being a major lipid con­ stituent of L, arabinosus, L, easel, a Strephtococcus species, and A. tumefaciens. Cia-vaccenic acid accounts for approximately 68$ of the total fatty a d d s of the latter organism. These observations have prompted the above authors to suggest that intestinal bacteria are a likely source for the trans-vaccenic a d d in the lipids of higher animals.

The fatty a d d composition of butterfat is subject to considerable seasonal variation. Among the many factors which have been suggested as being responsible for these variations are: a) diet, b) plane of nutrition, c) stage of lactation, d) temperature of environment, e) degree of freedom of movement, f) age of cow and, g) breed of cow. Of these various factors Hansen and Shorland (28) consider plane of nutrition and stage of lactation the most important causes. The effect of diet, according to these investigators, is of little importance since the diet fats of pasture and stall fed cows total only 2-U$ of the dry weight in either case, A comparison of

the seasonal variations observed by two different investigators is

given in Table U * 7 Table U

Seasonal Variations In the Fatty Acid Composition of Butterfat

New Zealand Butter(Shorland) (28) English Butter (Hilditch)(15)

Length of carbon chain Mole % sat. Mole % unsat. Mole % sat. Mole % unsat. % 10.2-10.9 — 9.5-9*9 — C6 — 3.5-U.l —

C8 1.2-2.2 — 0.8-1.6 —

°10 2.7-lw3 0.3 2.6-3.2 0.1 °12 3 0.3 2.3-3.U 0.1-0.2

ciU 9.1t-12 .2 1.0-1.2 8.2-11.5 0.6-0. 9 °16 21.6-23. h 1.6-1.9 2U.0-26.7 2.3-U.3 cl8 10.3-11.7 22.1-29.6 7.6-10.7 25.5-32.0

C 20 0 .5-0.7 1.5-2.1 0.9-1.6 0.8-1.3

H I A Review of the Methods of Separation and Identification of Fatty Acids and Their Esters.

A. Methods for Separating the Fatty Acids,

Very often the most formidable barrier that separates an organic chemist from achievement is the problem of isolating pure

compounds from mixtures* He is often forced to settle for much less.

This situation is perhaps never more apparent than in the field of fatty acid chemistry where one deals with complex mixtures of high

molecular weight compounds, all of which possess similar chemical

and physical properties. The following is a brief review of the

various methods that have been employed to effect resolution of such fatty acid mixtures. 1, fractional Eater Distillation*

This method is primarily useful for separating mixtures of fatty acids or esters whose components hare different molecular weights* It is usually found expedient to fractionally distill the methyl esters of fatty acids rather than the acids themselves, since by so doing the distillation temperatures may be reduced considerably*

In addition the column efficiency may be appreciably better for esters than for free acids, due to the better wetting properties that the esters display toward the column packing material* Since simple distillation of a mixture of these high boiling fatty esters, the boiling points of which differ by only a few degrees, will effect very little resolution, it is necessary to magnify such slight separations by distilling over and over again in series, or by employing a packed distillation column. The important factors involved in the construction and operation of such a column have been reviewed by Norris and Terry

(29) and by Hurray (30)* Under proper conditions these columns may easily display fractionating efficiencies of from ten to one hundred theoretical plates* One factor which determines the efficiency of the fractionation is the height of the column* This factor is especially important when distilling high boiling fatty esters since extremely low pressures must be employed in order that the distillation temperature may be low enough to prevent excessive polymerisation and isomerisation of the esters* A packed distillation column offers a certain resistance to vapor flow, and at the same time supports a column of vapor above the still pot* Both of these factors increase the pressure on the distilling liquid and increase the tenqperature* The height of the colunn le therefore U n i t e d by the maximum temperature which nay be safely used*

The rather high pressure drop encountered with the use of conventionally packed columns can be greatly reduced by the use of a spinning band column* The use of this type apparatus was first described by Lesesne and Lochte (31) In 1938* The essential feature of such a column Is that rapidly spinning bands or rotors are substituted for the usual packing* In 19$1 Murray (32) described a

Ut5 cm* column with an efficiency in the region of 70 theoretical plates* The low pressure drop of this column (O.bmm at 0.5mm head pressure) made possible the fractionation of compounds of high molecular weight which would have suffered some decomposition In a packed column* Straight chain alcohols, esters and acids of from 30 to 3U carbon atoms could be safely distilled* The disadvantages of such a column were the high cost of construction, and the tediousness of operation* 2* Separations of Fatty Acids by the 0se of Metal Soaps*

The fatty acids and their esters are extremely soluble in organic solvents at room temperatures • It Is therefore Impossible to employ ordinary fractional crystallisation techniques with mixtures of these compounds* Metal salts of fatty acids, however, often possess useful solubility characteristics in these solvents. The most widely used metal salt methods are the Official lead soap-ether method (33) and the Twitchell lead soap-alcohol method (3U, 35)* These methods are based upon the fact that the lead salts of saturated acids are practically Insoluble in ether or in alcohol whereas those of the 10 unsaturated acids are appreciably soluble* Such procedures are exceedingly cumbersome and sharp separation of the saturated from

the unsaturated fatty acids is generally not possible.

3. Low Temperature Crystallisation. Although the fatty adds and their esters may not be crystallised from organic solvents at ordinary temperatures, their

solubilities at lower temperatures are so reduced that fractional crystallization may be employed. In 1937 Shinowara and Brown (36) reported the preparation of pure oleic acid by the direct crystalli­

zation of the fatty acids of olive oil* These investigators found

that the fatty acids may be divided into three or four solubility groups; le. saturated, monounsaturated, diunsaturated, and poly­

unsaturated. Brown and co-workers have applied this technique to the

isolation of pure specimens of oleic, linoleic, linolenic, and arachidonic acids from the mixed acids obtained from a variety of

natural sources (37, 38)• It has been pointed out by Bailey (39) and others, that the solubility of a fatty acid is closely related to its melting point*

In general, the solubilities of fatty acids and esters decrease with increasing molecular weight, and increase with increasing unsaturation.

U. Urea Inclusion Compounds. In 19U0 Bengen (UO) reported that he had prepared crystalline

urea adducts of hydrocarbons, alcohols, aldehydes, ketones, acids and ester s. He found that the formation of these urea compounds with

straight chain but not with branched chain molecules is a general

principle and can be used for their separation. It has subsequently 11 been found that nearly all of the naturally occurring fatty adds form these adducts. All urea Inclusion compounds crystallize In hexagonal prisms, and analysis of the crystal structure reveals that the regular hexagonal prisms are built up exclusively of urea (1*1, 1*2). This honeycomb-like structure suggests that the aliphatic molecules occupy the free space Inside of the hexagonal channels. This assumption would neatly explain why branched-chaln compounds refuse to form these compounds by reasoning that the effective diameters of

the extended molecules are too large. Knight et.al. (1*3) found that the urea compounds of the fatty acids display many of the characteristics of a perfect derivative. The crystals are easily formed or decomposed, and have a temperature of dissociation characteristic for the particular included fatty acid or

ester. The main interest in the urea inclusion phenomenum has been focused on the fractionation of fatty acids according to unsaturation. In the series stearic, oleic, linoleic, and linolenic acids, the ease of adduct formation decreases with increasing unsaturation and the efficiency of separation is best between oleic and linoleic acids (1*1*). The shorter chain length saturated fatty acids display the same general tendency to form the urea adduct as oleic and linoleic acids, so that these components must first be removed by fractional distillation. Usually a preliminary solvent crystallization is performed to eliminate saturated acids. Using these methods in conjunction with the adduct

method, Swern and Parker (1*5) prepared 99% pure methyl oleate from olive 12 oil* Schlenk and Holman (U6) were able to prepare methyl linoleate from corn oil methyl esters which possessed the theoretical iodine number* These investigators also found that the trans configuration forms the adduct more readily than the corresponding cis configuration.

There are as yet, however, no reported instances where this difference has been used for the separation of trans fatty acids from natural mixtures*

$• Chromatography.

The chromatographic procedures which are commonly used in lipid separations fall into four general classes and are described briefly below (U7) •

Elution analysis chromatography is the original procedure introduced by Tswett for the separation of plant pigments* It involves passing a solution through an adsorbent column, and developing the adsorbed zones by washing the column with additional solvent. The least adsorbed solute lies in the lower sons of the column and is the first to emerge upon washing. The most adsorbed solute lies in the upper zone and is the last to emerge. Several of the more coomonly used adsorbent materials used for the construction of such columns are charcoal, silica gel and alumina.

Displacement analysis differs from elution chromatography only in that a solution of a more strongly adsorbed substance is used as the developing solvent. The sample to be analyzed is dissolved in a solvent and added to the chromatographic column. A solution containing the displacer compound is pressed in and the components of the sample arrange themselves in zones ahead of this displacer. Each 13 solute will In turn displace the next more weakly adsorbed solute.

This method offers the advantage that nearly quantitative recoveries of solutes may be obtained whereas ordinary elution chromatography often results In large solute losses due to permanent adsorption.

Partition chromatography was Introduced by Martin and

Synge (U8) in 19U1 for the micro determination of the water soluble higher monoamino-acids in protein hydrolysates and was later extended to the separation of fatty acids by Ramsey and Patterson (U9). In practice, partition chromatography is most like elution chromatography.

The basic difference is that the immobile phase is a liquid supported by a solid carrier whereas in elution chromatography the inmobile phase is a solid. The mobile phase is allowed to flow past the immobile one and separation of a solute mixture depends upon the partition co­ efficients of the solutes between the two liquid phases.

Paper chromatography is one or two dimensional partition chromatography using paper as the support for the immobile phase. It is useful when extremely small quantities are to be analyzed.

It has been found possible to separate fatty acids chromato- graphically according to their chain length or their degree of un­ saturation. In I9I4I Cassidy (50) reported the separation of lauric, rayristic, palmitic and stearic acids by elution chromatography. Swift,

Rose and Jamieson (51), 19ll3 separated pure methyl linoleate from cottonseed oil methyl esters using elution chromatography. White and

Brown (52) prepared a very pure specimen of methyl arachidonate from the fatty a d d s of beef adrenal phosphatides by using a combination of 1U low temperature crystallization and elution chromatography*

Siemenschneider et.al. ($3), 19U9, described an elution procedure for preparing pure linoleic and linolenic adds from natural sources.

Fairbaim and Harper (5U), 1950, reported the separation of the saturated acids from acetic to caprylic using a silica gel column and eluting with a chloroform-butyl alcohol mixture. In 1951 Nijkamp (55) published a simimicro analytical procedure for the chromatographic determination of the volatile fatty acids from acetic to capric. Howard and Martin (56), 1950, separated the saturated acids from lauric to stearic by a reversed phase partition chromato­ graphy. Displacement chromatography was developed in this country largely by Holman (57, 58) and co-workers. Using charcoal as adsorbent and aq. ethanol as the solvent he was able to separate saturated acids from unsaturated acids of the same chain length. He was also able to separate all of the saturated even carbon atom acids from k to 16 carbon atoms. Linoleic and oleic acids are separable, but they both lie between myristic and palmitic in adsorbability. Cia and trans Isomers are not separable by this technique, but by using carrier displacement linoleic acid was separated from its conjugated isomer. Holman found that one Isolated double bond In a molecule decreases adsorption roughly equivalent to two fewer carbon acids in the fatty acid chain.

Paper chromatography has only recently found extensive application in the field of fatty acids. The anions of these acids may be separated on paper using such solvents as ammoniacal butanol. 15 Kaufmann and Budirlg (59) found that different metallic soaps gave characteristic colors with basic dyes, particularly Rhodaaine B and Nile Blue. They Investigated the paper chromatography of propionic, butyric, valeric, caproic, enanthic, caprylic, octenoic, pelargonlc, decanolc, undecylenic, stearic, oleic, elaldic, llnoleic and eruclc acids. In 1952 (60) they applied certain other fluorescent indicators such and quinine, chlorophyll and anthracene to the identification of these acids. 6. Fractionation of Fatty Acids by Countercurrent Extraction.

This method of separating fatty acid mixtures Is based on the differential solubility of Individual acids in two immiscible solvents. "Countercurrent distribution" is the name given to a particular type of multiple-batch extraction which Is carried out in ingenious laboratory devices designed by Craig (61). Whereas manual extractions may be used only when solute partition coefficients are widely divergent, these devices permit the separation of solutes whose partition coefficients are quite similar. Countercurrent distribution is roost useful for separating the isologous series of

C^3 adds, for which other methods are generally ineffective. For example, oleic, llnoleic and linolenic acids may be separated using a 650 stage extraction. The respective partition coefficients of these adds are h*9t 2.0 and 1.6 in a heptane-formamlde-methyl alcohol-acetic acid solvent system (62).

This procedure offers the advantages of an extremely gentle, low temperature fractionation and almost complete recovery of starting materials* It suffers the disadvantage of being an extremely tedious 16 manipulation with which only small quantities may be fractionated.

According to Craig (6 3 ), the method would not be applicable to the separation of mixtures of geometrical Isomers such as oleic from elaidic acid. B. Methods of Identification of Fatty Acids and Esters.

1. The Determination of the Degree of Unsaturation of Fatty Materials. The usual procedures for determining the degree of unsaturation involve adding halogen or hydrogen to the double bond in such a way that the addition may be quantitatively measured. Conditions must be arranged so that quantitative addition takas place without the substitution of carbon-bound hydrogen. A summary of the various chemical procedures used for these determinations has recently been given by D. S. Bolly (6U).

2. The Determination of the Positions of Double Bonds in Fatty Acids and Their Esters. Due to the importance of double-bond cleavage reactions in the present investigation, there will follow first a brief resume of the reactions most widely used with unsaturated fatty acids, followed by a description of the methods used for separating and identifying the cleavage fragments. Any method which depends upon the cleavage of and ethylenic bond for establishing its position in an alkyl chain must assume that there has been no migration of the double bond either before or during the cleavage reaction.

a) Ozonation of Unsaturated Fatty Acids.

The classical procedure for determining the position of a double bond in an unsaturated compound involves dissolving this material 17 in some inert solvent and then passing a stream of ozonized air or oxygen through this solution until no further absorption of ozone can be detected. The resulting solution of ozonide can subsequently be decomposed in either a reducing or an oxidising medium with the production of the corresponding aldehydes or carboxylic acids.

Isolation and identification of these fragments will determine the original position of the double bond.

The action of ozone on oleic acid was first investigated by Molinari in 1903 (6$). At about the same time Harries (66) began a series of 96 papers on the reaction of ozone with unsaturated organic compounds. A number of solvents have been found useful by various workers. F. G. Fischer (67) states that dry pure ethyl acetate is the best solvent for straight-chain unsaturated compounds.

Acetic acid, hydrocarbons, carbon tetrachloride and methyl aid ethyl chlorides have been used frequently and successfully as solvents. Dull (68) has made a comprehensive investigation of a number of different procedures for the oxidative cleavage of ozonidss. He

concluded that decomposition of the ozonldes with alkaline perman­ ganate or with alkaline hydrogen peroxide were the most useful methods.

Until recently no accurate study had been made of the cleanness of the double bond cleavage obtainable by the various techniques used.

In 195>0 Begemann (69) et.al. used a chromatographic technique to separate the cleavage products obtained from several pure fatty acids and esters by quantitatively determining the mole percentages of the dicarboxylic acids produced. These authors concluded that ozonation in chloroform at -10° to -20° and permanganate oxidation in acetic acid 18 according to the method of Armstrong and Hilditch (70) gave equally satisfactory results, with the latter method being favored because of its sinqplicity. Ozonation of methyl oleate or methyl elaidate produced a mixture of dicarboxylic acids which indicated a slight secondary disintegration of the primary cleavage products. This was small, however, as only y - h % of suberic acid could be identified in addition to the main product of azelaic add. Later Allen and

Kiess (71), 1955* claimed that ozone in methyl acetate was superior to permanganate in acetone for the oxidation of oleic acid and its isomers•

b) Permanganate Oxidations of Unsaturated Fatty Acids.

Prior to 1925 the oxidation of oleic acid with some of the more common oxidizing agents such as nitric or chromic acid or permanganate had been extensively studied by numerous workers. In all cases a variety of products resulted. Mild oxidation would produce mainly the glycol while more vigorous oxidation would split the carbon-carbon double bond into mono- and dicarboxylic acids.

These oxidation products were then themselves oxidized to lower homologues so that a spectrum of mono carboxylic acids from pelargonic to acetic and di carboxylic acids from azelaic to oxalic might result (72). In 1925 Armstrong and Hilditch (70) reported a perman­ ganate oxidation of oleic acid using acetone or acetic acid as a solvent. The dibasic acid could be recovered in at least 80£ of the theoretical yield with very little secondary degradation. A typical oxidation was reported to have produced azelaic acid in 95.3 % yield and nonanoic acid in 59*13& yield. Iodine absorption of the oxidation 19 mixture indicated that 9 5 -9* of the original unsaturation had been

oxidised by this procedure. Recently, as noted previously, a quanti­ tative determination of the products obtained by the Armstrong -

Hilditch oxidation was reported by Begamann et.al. (69). These inves­

tigators found that acetic acid was far superior to acetone as sol­ vent for permanganate oxidations. The dicarboxylic acids obtained

from fatty acids and esters were separated and analyzed by partition

chromatography. Table III illustrates the results published by

these authors.

Table III

The Oxidation Cleavage of Methyl Oleate and Elaidate with Potassium Permanganate (69).

Dicarboxylic Acids Methyl Oleate Methyl El&idate from the Oxidation Products in acetone in acetic in acetone in acetic acid acid

Total yield 8 0 * 87* 75* 85*

Sebadc acid 0* 0 * 0 * 0 *

Azelaic acid 63* 95* 72* 91.5*

Suberic acid 28* 5* 23* 8*

Pimelic acid 8* 0 * 5* 0 *

These authors did not investigate the accompanying mono-

carboxylic acids which were produced.

c) Peracid Oxidations of Unsaturated Fatty Acids.

In 1928 Malaprade (7 3 ) reported the use of periodic acid for

the oxidation of glycol, glycerine, mannitol and other polyhydroxy 20 compounds. These were reported to have been oxidised according to the equation: (CH20H) 2(CHQH)n + (n + 1) H K ^----- > 2HCH0 ^n H C O ^ + (n + 1)HI0^ HgO.

Ten years later King (7U) used this method to prepare azelaic semi-aldehyde from 9,10-dlhydr oxys tearic acid. The yield of semi­ aldehyde was never theoretical but in the neighborhood of 30-l*0£. A similar reaction utilizing lead tetraacetate as oxidising agent was reported by Criegee (75) in 1930. Criegee's reaction has had very wide application in determining the structures of sugars and it has also been applied to the demonstration of the positions of the hydroxyl groups in di- and tetrahydroxystearic a d d s and in certain cork acids

(76). Monoethenolc fatty acids may be oonverted to the corres­ ponding oc-glycol acids in good yields by the action of hydrogen peroxide in glacial acetic acid (77, 78)* It was known that when the sodium salt of oleic acid was oxidized in cold dilute at lrai in** solution by potassium permanganate a 9,10-dihydroxystearic acid was produced which melted at 132° (79). Elaidic acid under these conditions produced an isomeric dihydroxystearic acid melting at 95° • When these same acids were hydroxylated with hydrogen peroxide in acetic acid the melting points of the resulting dihydroxystearic acids were reversed (77) • In 19U0 Scanlan and Swern (7 8 ) published a procedure for cleaving oleic acid by employing the peracetic acid hydroxylation reaction followed by the Criegee lead tetraacetate reaction. The resulting aldehydes and aldehyde—acids were obtained in yields ranging from 12 to 85% of theoretical. Swern inproved this method in 19U5 by 21 hydroxylating with hydrogen peroxide in formic acid (8 0 ), and reported yielda of 80-100% from oleic acid* Another procedure which has been used for the cleavage of oc -glycols la the use of a solution of potassium periodate in dilute sulfuric acid (7U)* As is the case with other cleavage reactions, this method when used in conjunction with one of the hydroxylating procedures does not usually produce quantitative yields of the cleavage products* However, Ross et.al. (81), 19US, reported the cleavage of

erucic acid by this method with subsequent recovery of the thirteen-

carbon-atom dicarboxylic acid In theoretical yield* The method suffers from the difficulty that the products formed are aldehydes and are

subject to polymerization and oxidation reactions. Since the most

effective chromatographic procedures have been developed for the

separation of homologous dicarboxylic acids, the aldehyde-acids

produced by the peracid-perlodate scheme must be further oxidized* In

order to employ this technique for the cleavage of fatty acid double

bonds one must therefore employ three separate oxidation procedures*

This is to be compared with the one-step oxidations possible with

permanganate in acetic acid or the two step oxidations with ozone. The

peracid-periodate oxidations are time-consuming and tedious and may be

used only if they axe capable of producing increased yields with fewer side products*

d) The Separation and Identification of the Products Resulting from the Oxidative Double Bond Cleavage of Unsaturated Fatty Acids.

Unsaturated fatty acids when oxidized by the Armstrong -

Hilditch method or by the decomposition of fatty acid ozonides with 22

1*2^2* produce mixtures of mono- and dicarboxylic adds* In order to analyze a given monoethenoic fatty acid preparation for positional isomerism of the double bond, it is desirable to have at ones disposal an effective and quantitative means for separating homologous series of both the mono- and dibasic acids. The technique of chromatography has been used most effectively toward this end. The chromatographic procedures which have been developed for the separation of homologous saturated fatty acids should be applicable for the analysis of these same acids produced as a result of bond cleavage. In practice, however, quantitative analysis of mixtures of lower molecular weight mono carboxylic acids is usually unsatisfactory because these acids are more subject to secondary degradation than are the corresponding dicarboxylic acids, and the chromatographic separation methods are not sufficiently quantitative.

However, several procedures have been recently developed which permit a quantitative determination of mixtures of monocarboxylic acids. Boldingh (82), 1950, applied the principle of reversed phase partition chromatography to a quantitative micro determination of the saturated n-fatty acids from C6 to C^g. He used systems containing benzene absorbed in vulcanized natural Hevea rubber as the immobile solvent and mixtures of methanol, acetone and water as the mobile solvent.

The quantitative estimation of the component dicarboxylic acids in a mixture by chromatographic means has only recently received widespread attention. As noted previously, Begemann et.al.

(69) in 1950 published a very elegant method for the analysis of mixtures of dicarboxylic acids obtained from the oxidation of unsaturated 23 fatty acids. She used partition chromatography with alcohol-water on silica gel as the immobile phase, and benzene as the mobile phase. Such a system permitted the separation and quantitative determination of the dicarboxylic acids from C£ to C^. Marvel (8 3 ), 1950, developed a similar but less versatile system using water as the stationary phase. This method was not suitable for convenient and quantitative analysis of mixtures of widely differing composition, especially those containing large proportions of the higher dicarboxylic acids. In 1952

Higuchi (8U) et.al. used a column having a citrate buffer as the internal phase for separating dicarboxylic acids with from six to ten carbon atoms. Since oxidative double bond cleavage of unsaturated fatty acids produces both mono- and dicarboxylic acids, the ideal chromato­ graphic procedure for analyzing such oxidation products would be one that could separate the homologues of both species of acids. Vandenheuvel and Hayes (85), 1952, developed a multicolunm technique which allowed the separation of monocarboxylic acids with from two to twelve carbon atoms, and,with a modification of the column mixture, of dicarboxylic acids with from four to ten carbon atoms. In 1955 Zblnovsky (86) published an ultramicro method with which he was able to separate a mixture of seven monocarboxylic acids with eight dicar­ boxylic acids on a single column. He used an internal phase of cellosolve and water supported on silicic acid. When both mono- and dicarboxylic acids were placed on the column, the monocarboxylic acids could be eluted with Skellysolve while the dicarboxylic acids remained stationary. The dicarboxylic acids were subsequently eluted with di-n- butyl ether. 21* 3* Optical Methods Used for the Identification of Fatty Acids* a) Ultraviolet Spectrophotometry* Polyunsaturated fatty acids which contain conjugated double bond systems have characteristic absorption bands at 231*, 266 and

3l6nys for two, three and four conjugated bonds respectively. Satxzrated and monoethenoic acids are transparent in this region* In

1937 Moore (87) noted that when polyunsaturated fatty acids were subjected to prolonged saponification there was a marked increase in ultraviolet absorption due to conjugation* Burr and Miller (88), 191*1, found that Moo re* s reaction could be made reproducible and suggested an empirical quantitative procedure for estimating llnoleic and llnolenic acids in vegetable oils. They conjugated natural acids by heating with alkali in ethylene glycol at 180°• Mitchell, Kraybill and Zscheile (8 9 ), 191*3, published an exact empirical method for the determination of llnoleic and llnolenic acids in natural oils in which the time, temperature, and alkalinity of the isomerization was carefully controlled. In 191*1* the method was extended to the estimation of arachidonic acid (90)* The following year Brice et.al*

(9 1 , 9 2 ) published a modification of the existing ultraviolet spectrophotometric methods for application primarily to the detection and estimation of low proportions of conjugated and nonconjugated unsaturated constituents in fats, oils, and soaps. Brice estimated that the errors of the method were within 10 percent of the quantity present when that quantity was near 10 percent, 25 percent near 1 per­ cent, and that the results were at least correct in order of magnitude when the quantity present was 0.1 percent or less* However, when this 2$ method was applied to a number of seed oils and concentrates having high contents of llnoleic and llnolenic acids, serious discrepancies between spectrophotometric and thiocyanometric analysis were en­ countered. It was established that the spectrophotometric method was at fault* The chemical standards used heretofore had been prepared by bromlnation-debromination procedures. Such preparations contain substantial proportions of geometric isomers other than the natural all-d a variety* These configurational differences have been shown to produce significantly different ultraviolet absorption

intensities following alkali isomerization (93)* Nichols, et.al.

(9U), 1950, showed that natural all-cis llnoleic acid isomerized

twenty times faster than the all-trana isomer and produced a maximum specific extinction coefficient of more than five times that of the

latter. These considerations prompted Brice, et.al* (95>) to re-

standardize the spectrophotometric method, using the methyl esters of natural llnoleic and llnolenic acids as standards* The method at

this juncture is presumed accurate for natural fats and oils regardless of the percentage of polyunsaturation, but may not be accurately applied to fats and oils which contain trans-bonds or whose double bonds are separated by more than one methylene group. Such isomers

are produced during hydrogenation. In addition, small quantities of these isomeric llnoleic acids have been detected in butterfat as has been previously discussed.

In an attempt to overcome this failure of the spectrophoto­

metric method to give reliable results with fats and oils containing

cis,trans and trans,trans isomers of llnoleic acid, Jackson et*al. (18) 26 synthesized these various isomers and used them for standards. They found that the intensity of the absorption of isomeric octadeca- dlenoic acids vas dependent upon the duration of alkali isomerization, and suggested that three isomerization times be used. The specific extinction coefficients so obtained could be used with a set of three simultaneous equations in order to calculate the various percentages of els,els- cis,trans- and trans,trans-llnoleic acids in a given fat.

Unfortunately this modification apparently may not be used with specimans containing llnolenic add. Cornwell et.al. (20) found that when conjugation time was increased the estimated llnolenic acid content decreased seriously. There is at present, therfore, no completely satisfactory procedure for determining the llnoleic acid content of fats and oils which contain appreciable quantities of the cis,trans- and trans,trans-llnolelc acids. b) Infrared Spectrophotometry.

McCutcheon, Crawford and Welsh (96), 19U1, were the first to demonstrate that the cis-trans configuration of the ethylenlc linkage modifies the infrared spectra of unsaturated fatty acids in a characteristic manner. The most prominent difference between the spectra of trans-fatty acids and the saturated and all-cia acids occurs in the region from 10.27-10.38fA . Rasmussen and Brattain (97) first associated a prominent bond in this region with the trans-substituted ethylene structure. This bond is presumably a result of the out-of- plane bending vibrations of C=C-H carbon-hydrogen bonds when the ethylenic bond has the trans configuration (98). L band near lU.5 0 f4 has been associated with the analogous vibration of the cis-3ubstituted 27 ethylenic group (97). In 19h9 Lemon and Cross (99) suggested that the Intensity of the absorption peak at 10.33p was a measure of the extent of the cis-to-trans change associated with the hydrogenation of unsaturated fatty adds. Swern and co-workers (100) established that this absorption peak obeyed Beer's law in that for any given concentration of mixed fatty acids, esters or glycerides, the extinction at 10.36^4 was directly proportional to the percent trans- ethylenic linkages present. As a result, they were able to set up an Infrared analytical method for the determination of trans-octa- decenoic acids, esters (Including glycerides), and alcohols In the presence of the corresponding cis and saturated compounds. Pusari et.al. (101) examined the extinction coefficients of a series of synthetic cis- and trans-octadecenoic acids at this wavelength and found that little or no difference in absorption maxima occurred as a result of positional isomerism. Jackson, Wheeler and co-workers

(18) found that the band at 10*33 f* was approximately additive since methyl linolelaidate had about twice the absorption of elaldate, and a cis,trans-linole&te had about the same absorption as elaldate. The effect of conjugation was also investigated by these workers. They found that the absorption band of conjugated trans, trans-llnoleate was shifted to 10.12^ , while conjugated cis,trans-llnoleate was characterized by a doublet at 10.56 and 1 0 .19^1 .

This infrared method was compared with the classical lead salt-alcohol methods for determination of trans-octadecenoic acids by Swern and co-workers (102) in 1950. These authors concluded that the spectroscopic method was more rapid, specific and accurate than 28 the chemical method* One obvious limitation of this method is that

It cannot differentiate between trans-monoethenoic and trans-poly* ethenoic fatty acids. In addition, it Is difficult to separate mixtures of these two types of acids by crystallization techiques, since elaidic, linolelaidic and stearic acids all have similar solubilities in organic solvents* One possible solution to this problem has been suggested by Jackson et.al* (18) who used three different alkali isomerization times for each of the three different types of geometrical isomers of llnoleic acid, followed by ultra­ violet measurement*

c) X-ray Spectroscopy,

By means of x-ray diffraction it is possible to determine crystalline structure and the molecular dimensions of fatty acids.

Diffraction patterns obtained on a photographic plate by this method are Interpreted by using the Bragg (103, 101*) equation*

The x-ray diffraction pattern of synthetic trans-ll-octa- decenoic acid (105>) has recently been found to differ significantly from that of natural vaccenlc acid (2). Therefore while mixed melting point, infrared, and cleavage studies indicated that these two acids were identical, Burapus et.al. (3) concluded that they were not* He concluded that it was possible that the natural vaccenlc acid preparations submitted to x-ray examination consisted essentially of trans-ll-octadecenoic acid, but were contaminated with small amounts of isomeric acids sufficient to produce an altered crystal structure. Swern et.al. (106), 1955, investigated the x-ray diffraction patterns of some binary mixtures of trans-6 through 29 12-octadecenoic acids. They found that mixtures of even-even or odd-odd acids form a one phase crystal system, and show similar x-ray diffraction patterns. Mixtures of odd-even adds form a two phase crystal system and the diffraction patterns of these mixtures are distinctly different from the odd-odd or erven—even mixtures.

IV Objectives of this Investigation.

The object of this investigation is to stu

If a satisfactory cleavage reaction can be established, it would next be necessary to adopt a procedure which would permit a qualitative and a quantitative analysis of the cleavage fragments.

Used in conjunction with the infrared methods for determining the percentage of trans double bonds, and with the ultraviolet methods 30 for estimating the percentages of llnoleic and llnolenic adds, this cleavage analysis would make possible a more rigorous evaluation of the unsaturated fatty acids of butterfat. For example, it would enable a direct comparison of the percentage of 11-octadecenoic a d d with the percentage of trans-octadecenoic acid. Used on suitable crystal fractions of butterfat fatty esters, the method could be used to prove or disprove the presence of isomers of oleic and vaccenlc acids in this fat, and the relative percentages of all of these

isomers could be determined. While the presence of 9-hexadecenoic acid in butterfat has been definitely established, the possibility of small percentages of isomeric acids has not been eliminated. The

investigation will therefore be extended to include the sixteen-

carbon-atom unsaturated acids. Iso-linoleic acid is known to be present in butter, but the chemical identity of this acid, or mixture of acids, has not been established. Therefore it is finally hoped that cleavage analysis used with the refinements of the infrared and ultraviolet methods will permit a more rigorous evaluation of these polyunsaturated components•

V Experimental A. Analytical Methods.

1. Saponification Equivalent and Iodine Value. Saponification equivalents were determined by refluxing two to five grams of methyl esters (or glyceride) in alcoholic KOH for thirty minutes. The amount of KOH required for saponification 3i was determined lay titrating the excess with 0.5 N HC1 using phenol- phthalein indicator and subtracting the titration volume from the volume obtained from blank titrations* Iodine values were determined by the Wijs method according to the Official and Tentative Methods of

The American Oil Chemists1 Society, Official Method Cd 1 -25.

2. Neutralization Equivalent•

a) Fatty Acids. The equivalent weights of free fatty acids were determined by the direct titration of solutions of 2 - 5 g. of the acid in 50 ml. ethanol with 0.5 N NaOH, using phenolphthalein as the indicator.

b) Dicarboxylic Acids. These determinations were made by the direct titration of solutions of 20 - 50 mg. of the acid in 10 ml. ethanol with 0.05 N aqueous NaOH using phenolphthalein. The solutions were both agitated and protected from atmospheric co2 by bubbling a stream of nitrogen through them during the titrations.

3. Ultraviolet Spectrophotometric Examination.

Determinations of the percentages of polyunsaturated fatty acids were made according to the Official and Tentative Methods of

The American Oil Chemists* Society, method Cd 7-U8 (1953).

ii. Infrared Examination.

The percentages of fatty acids with trans double bonds in a mixture of saturated and unsaturated acids, esters or glycerides were determined through the use of the infrared analytical procedure described by Swern and co-workers (100). This method had previously been used in this laboratory with a satisfactory degree of precision 32 and accuracy and was apparently quite reliable. It haa been stressed by the authors of this method that universal values for the standard extinction coefficients may not be established.

Variations in such factors as scattered radiation, wave-length calibration, slit-wldth settings, and accuracy of cell-thickness measurements make it necessary to determine the required extinction coefficients on the instrument being used under the exact conditions to be employed in the analysis. In this laboratory it has been found that there is appreciable variation in the calculated extinction coefficients of pure fatty acids from instrument to instrument and from year to year using the same instrument. It was therefore necessary to redetermine standard extinction coefficients from time to time during the progress of the investigations described by this thesis. Table IV lists typical extinction coefficients of pure fatty materials as determined here and in other laboratories. The sources of the various compounds used as reference standards along with their physical and chemical constants are listed in Table V, 33

Table 17

Standard Extinction Coefficients of Pore Fatty Acids, Esters, and Glycerides at 10.36 microns.

Extinction Coefficients

Coapound Published Ref. Determined in this lab

Tristearin 0.068 100 0.088 Triolein 0.081* 99 0.106 Trielaidin 0.1*75 99 0.562

Stearic acid 0.123 99 0.136 Oleic a d d 0.133 99 0.11*3 Elaidlc acid 0.552 99 0.582

Methyl stearate 0.028 99 0.029 Methyl oleate O.OJ4I 99 0.0U2 Methyl elaldate 0.1*1*2 99 0.509

Palmitic acid O .129 99 0.158 Cis-9-hexadecenoic acid 0.178 106 0.182 Trans-9-hexadecenoic acid — - 0.61*2

Methyl myristate o.ol* 107 0.01*1** Methyl laurate o.ol* 107 0.01*1** Methyl caprate 0,07 107

* Determined by Hr. A. F. Mabrouk. Table V

Characteristics of the Reference Compounds Used for the Infrared Analyses

1.7. Mol. Ut. Source and Method of Compound M.P. I.V.WiJs Theory Hoi. Wt. Theory______Preparation_____

Tristearin 0.0 891.5 Kindly supplied by Dr.Daniel Triolein 85.1 86.1 - 885.11 Swern of the Eastern Regional Research Laboratory. Trielaidin I41.3-U1.5 85.1 86.1 885.11

Stearic acid 69.$ 0.01 0.0 28U.5 28U.5 Prepared by Doris Kolb from Hystrene 97-6* a gift from the 13.3 89.9 282.5 282.5 Atlas Powder Company. Oleic acid 89.9 Prepared from olive oil by Doris Kolb. ELaidlc acid U3.5-U3.7 89.7 89.9 282.5 282.5 Prepared from oleic a d d by selenium isomerization. Methyl stearate o.cto ' O.U” ' ' Z98'.5— '798:5“ Obtained from human milk fat by Betty Qnians. •» 85.6 296.0 296.5 Obtained from olive oil methyl Methyl oleate 85.14 esters. Prepared by isomerization of Methyl elaldate 85.6 85.6 296.5 296.5 methyl oleate. Prepared by H. Foreman from Palmitic acid 62.8 0.00 0.0 256.3 256.1i palm oil. Prepared by Doris Kolb from cod Cis-hexadecenoic acid —10.2—0.0 98.8 99.8 253.6 25U.U liver oil. Prepared from ^almitoleic4* acid Trans-hexadecenoic acid 31.3-31.5 98.7 99.8 253.6 25U.li by Doris Kolb. Methyl myristate - 0.01 0.0 2^2,U 21*2.U

Methyl laurate - 0.50 0.0 2ll*.li 21U.3 Prepared by H. Foreman

Methyl caprate - - 0.0 - 186.3 It must be emphasized that the several groups of specific extinction coefficients which have been listed in Table IV were determined at widely different times* It was found necessary to determine the coefficients of some groups using a different infrared Instrument than was used for the other groups. These groups may not, therefore, be related to one another for purposes of establishing standard values in another laboratory from redeterralnations of one trans and one cis compound. B. Preliminary Analysis of Several Butterfats. Several sanqples of commercial butters were obtained in

March, 1953. These butters were dehydrated and filtered and the various analytical constants determined. In addition, a sample of human nrfilc fat was examined in order to compare the percentage of

trans -glycerides present with that found in cow's milk fat. These

data are given in Table VI. Table VI Analytical Constants of Comercial Butterfats and Human Milk Fat with Special Reference to Their Content of Trans-Glycerides.

% Trans-Glyceride Specimen Sap. No. I.V. as Trielaidin

Blue Valley 229.8 3U.5 5.3 Mayflower winter 229.U 33.6 5.0 Mayflower summer 228.2 36.2 8.8

Borden 231.8 33.2 5.5 Fairmont 230.U 32.1 5.0

Armour 231.0 33.6 6.1 Human milk fat* 61.8 U.5 * Prepared by Betty Orians. 36 The constant* obtained for the specimens of winter butter­ fat showed little or no variation. The summer butterfat possessed a slightly higher 1.7. than the winter specimens, but a much higher percentage of trans-glycerides. On the other hand human milk fat contained a smaller trans-glyceride concentration than cow*a milk fat even through it had nearly twice the degree of unsaturation. In view of the findings of Hartman et.al. it was rather surprising to find any trans-glycerides in human milk fat, since it was his opinion that these glycerides were found in ruminating animals only. These trans -glyceride s may have originated exogenously however, as a result of the use of hydrogenated fats (margarines or shortenings) or oow*s milk fat in the mothers1 diet.

The percentages of "trielaidin" found to be present in the specimens listed in Table 71 are probably high. This error is brought about by the presence of lower molecular weight fatty acid residues in the glyceride structures of butterfat. Since the methyl esters of the lower saturated fatty acids have a higher specific extinction co­ efficient at 1 0 .36^4 than does methyl stearate (1 0 8 ) (see Table 17), it seems reasonable to assume that glycerides containing these lower acids would in like manner have slightly higher specific extinction co­ efficients than would tristearin. However, tristearin was used as the reference standard for all of the saturated components of these butter­ fats, and calculations based thereon might be expected to be as much as

3% too high in trielaidln.

Two of the winter butterfat specimens and "S" butterfat were analyzed by alkali isomerization and subsequent ultraviolet examination 37 In order to note any seasonal variation. The results of these analyses are shown in Table VII. Table VII Analyses of Three Butterfats by Ultraviolet Spectrophotometry. Fatty acid type Bordens Blue Valley Mayflower (summer) ( all values in percent)

Conj. diene 0.8 0.9 1.2 ConJ. triene 0.0 0.0 0.0 ConJ. tetraene 0.0 0.0 0.0

Llnoleic 1.9 1.8 1.6

Llnolenic o.U 0.5 0.9 Arachidonic 0.5 o.U o.U

Oleic 29.7 3 1 .U 30.9 Saturated 66.7 65.0 65.0

The data recorded in Table VII show that no significant seasonal variation in the composition of butterfat could be detected by this method of analyses.

C Isolation of the 0^5 and C18 Methyl Esters from Summer Butter by Fractional Distillation. The Mayflower summer butter described previously was 90 score butter, churned July U, 1952, by a continuous churn process. This butter was obtained from the Pickerington dreamery through the Department of Dairy Science of The Ohio State University. It is this butter which has been analysed by the various techniques to be described in this thesis and it will hereafter be referred to as "S" butter. 38 A 3 kg* sample of "S" batter was dried and filtered. Eleven hundred grama of the resulting fat were converted to methyl eaters by the usual methods. After washing repeatedly with water, drying and vacuum distilling, 1037 g* of the crude methyl esters were obtained. One kilogram of these crude esters was charged into a 2 1. stillpot and fractionated by distillation through a Fenske column, 90 cm. long and 2.5 cm. in diameter. This column was packed with single turn 1/8" glass helices and was heated with two nichrome coils, regulated by variable voltage transformers. A variable reflux still-head was used, equipped with a water condenser and a thermometer well. The reflux ratio was regulated with a Newman stopcock which was sealed into the take-off capillary. Distillate was collected by means of a Pauly type receiver so constructed that eight fractions could be collected without distrubing the vacuum. A still-head pressure of 0.05 to 0.10 mm. was maintained with a Duoseal pump, a Dubrovin vacuum gauge being used for pressure measurement. The main fractions obtained from "S" butterfat methyl esters were collected using a reflux ratio to 5*1, while intermediate fractions were collected at a 15*1 ratio. Analytical data on these fractions are described in Tables V H I and IX. The sap. equiv. of the fraction (297*2) would indicate that this fraction is a mixture of methyl stearate and oleate (theoretical sap. equiv. of methyl oleate, 296.5)* The sap. equiv. of this fraction calculated from Table IX is 297*1 and may be offered in evidence that a complete separation of carbon series was achieved. 39 Table VIII Fractional Distillation of the Methyl Eater from Butterfat.

Carbon B.p., % Trane as Fraction series 0.1 mm. Wt. in g. Sap.equiv. I.V. elaldate

1 Below C^g $2 - 108 92.5 220.8 7 .01* m

2 Below C^5 75 - 116 1U9.9 21*6.3 8.36 -

3 °16 116 - 118 21*3.2 271.6 6.67 3* 1* Ci6 - C18 118 - 128 30. U 290.2 55.16 10* 5 ClB 128 - 135 1*16.1* 297.2 61*. 98 15.5

- Holdup - 20.1* 302.9 61*.13 ee

Residue _ 1*0.9 319.7 80.11* -

* These values are rough approximations only, due to the lack of appropriate infrared standards.

Table IX Composition of the Tractions from MS" Butterfat, as Determined by Alkali Isomerization and Ultraviolet Spectroscopy

Fatty acid type Total esters C16 fraction Cig - C^g C^g fraction

( all values in percent)

ConJ. diene 1.0 0.1 0.5 1.2 Diene 1.8 0.1 1*.5 3.1*

Triene 0.8 0.2 1.6 1.8

Tetraene 0.5 0.0 0.0 0.0

Monoene 30.9 6.2 62.8 60.9

Saturated 65.0 93.1* 30.6 32.7 1*0 D Fractional Crystallisation of the Methyl Eaters at Low Temperatures.

These crystallizations were performed from 3-k% solutions in methanol. The solutions were prepared in a 1$ x U5 cm. pyrex crystallizing cylinder and placed into an acetone/dry ice bath of the desired temperature. The bath consisted of a heavily insulated wooden box containing a copper can 26 cm. in diameter and Uf> cm. deep. During the crystallizations the solutions were agitated slowly with a paddle-type monel metal stirrer upon which was mounted a Weston low temperature thermometer. The temperature was held as constant as possible by the addition of small portions of dry ice, and after the desired temperature had been maintained for two hours or more, the agitation was discontinued and the crystals allowed to settle. While still in the cooling bath the supernatant liquid was drawn into a U 1. suction flask through an inverted Buchner filter (30). These filtrations were thus carried out at the temperature of crystallization. The crystals were washed with several hundred ml. of cold methanol and filtered again, then dissolved in methanol at room temperature. The methanol was distilled from the solutions of filtrate and crystal fractions, and both fractions were freed from the last traces of solvent by distilling them under vacuum.

One hundred grams of the esters were dissolved in 3000 ml. of methanol and crystallized at low temperatures according to Chart I in an attempt to obtain a concentrate of trans-methyl octa-

decenoates. The various crystal fractions were examined by the standard ultraviolet and infrared procedures and these analyses to­ gether with other analytical data are tabulated in Table X. Chart I

Low Temperature Crystallization of the C^g Fraction from Sumer Butter.

lOOg.j I.V. 6iu98 1 % S % trans 30.8g.j I.V. U.72 Cooled U.2Jf trans to O'- 2 hrs. Cl 76.3% "oleic" as trans Cooled in 3250 ml. Cooled in 1000 ml. to - 20° methanol to - 20° ■ethanol overnight overnight Cooled in 3500 ml. U.Og.; I.V. 21 to - 38° methanol 23. trans overnight 97£*oleic"as trans Filtrate *L 33«2g.; I.V. 106.U 28.6g.j I.V. 8U.U 18.1% trans 2$.2% trans I 2J>.6#,oleic"as trans dissolved in 3000 ml. methanol Ci - F Cx - C Cooled 2 hrs. s 1 to - 60° 1.6g.j I.V. 79.k 25.7g.; I.V. 1*3.6^ trans 1.U2 - F It------F1L- - k7% "oleic" as 1,0$ trans c------trans 5856 "oleic" .v. 1U5.U 16.ljg.; I.V. 87.6 trans ns 9.9% trans Table Z

Analytical Data for the Various Crystal Fractions Obtained from the Total C1g Methyl Esters of "S" Butterfat.

Fraction Total Cj_8 o i esters Cl C1 - F cx - c c2 c3 *1 Fx - F Weight, grams 100.0 30.8 1.6 25.7 U.o 28.6 33.2 10.0 16.1, I.V. 65.0 li.7 79.1* 1.1* 21.0 81*.!, 106.1, 11*5.1, 87.6 Percent trans 15.5 U.2 U3.6 1.0 23.6 25.2 18.1 28.6 9.9 Percent "oleic" as trans 20.1. 16.3 1*7.0 58.8 97.0 25.6 - 9.9 Percent of total trans 100.0 8.1, (6.2) (2.2) 6.1 JU6.6 38.9 (2l*.8) (Ui.l)

Conj. diene, % 1.2 - - - 0.6 - 7.0 1.7 Llnoleic, % 3.1* --- - 0.0 - 18.6 0.9

Linolenic, % 1.8 - mm -- 0.0 - li,.2 0.3

Tetraene, % 0.0 mm -- - 0.0 - 1.2 0.0

Oleic, % 60.9 - - -- 97.0 - 70.3 95.6

Stearic, % 31.7 - - - - 2.U - -10.9* 1.5

* This anomaly is caused by the presence in this fraction of nonconjugatable polyunsaturated acids. U3 It was suggested in a previous report from this laboratory

(20) that a complete separation of the trans-fatty esters of butterfat cannot be made by low temperature crystallization techniques (l) •

This premise has been born out by the results of the above crystallization where it is seen that the best concentration of trans-octadecenoate obtained was only U3*6£ in fraction - F.

Fraction C2 while being composed of only 23*8^ trans-esters, had nearly all (97£) of its octadecenoate component In the trans configuration* This being the case, it is obvious that oxidative cleavage of the unsaturated adds present in this fraction and sub­ sequent identification of the fragments would enable us to establish the structure of a trans-octadecenoic acid present in summer butter. It may not be presumed, however, that the trans-acid present in fraction C2 is the only trans-acid found in butter, nor may it be presumed that C2 it­ self is composed of only one trans-acid. Therefore in the work that follows a method of oxidation must be used which will permit separation and identification of the whole spectrum of oxidation products which may arise from any "iso-oleic" esters possibly present* As noted previously, such a method has been developed by Begemann et.al* (69) who used the potassium permanganate oxidation technique of Armstrong and Hilditch (70), followed by a chromatographic separation of the resulting dicarboxylic acids.

£ The Development of the Chromatographic Procedure for the Resolution of Mixtures of Dicarboxylic Acids*

The adoption of a suitable chromatographic technique was a major factor toward the progress of this investigation, and a detailed description of the exact procedure is given in the following section. Uh

1. Preparation of the Column, The chromatographic column as originally described by Begemann et.al, (69) was constructed in the following mannert Silica gel was prepared according to Oordon et,al, (109)* Ten grams of this gel were mixed with 12 to 1U ml* of the aqueous phase of a two phase system obtained when 3 parts of ethanol, U parts of methanol, and 3 parts of water are shaken with 10 parts of benzene and allowed to separate into layers* The powder-like silica gel mixture thus formed was dispersed In the benzene phase and transferred in small volumes to a 10 mu* diameter glass tube and slightly pressed together to form a column of desired length* About 20 mg. of a dicarboxylic acid mixture to be analyzed was dissolved in 2 to 5 ml* benzene phase and poured on the top of the column. This was then washed continuously with more benzene phase* The percolate was collected in 1 ml. portions and titrated with 0.0$ N alcoholic sodium hydroxide solutions using phenolphthalein as indicator* This procedure was modified first by enlarging the column to 20 mm* in diameter and to a length of $0 cm.. It was found that the preparation of the chromatographic column could be greatly simplified* A 60 cm* length of glass tubing 20 mu. in diameter was fitted with a 2lt/U0 female ground glass joint at the top, and a slight constriction was placed in the tube about $ cm. from the other end* This end was then tapered below the constriction and a 5 cm. length of 6 mm* tubing attached* A Oooch crucible plate was placed in the tube on the constriction and a tightly fitting blotting paper disc was placed on u$ top of It* Silica gel'1' was poured into the column and the outalde of the glass tube was tapped gently to allow the entrapped air to escape.

Another tightly fitting blotting paper disc was placed on top of the column and 300 to 5>00 ml. of aqueous phase was forced through the column of dry silica gel. Gravity was used until the liquid had passed through about half of the oolumn, and then a pressure of about

20 cm. mercury was applied. After the water phase had passed through 2 the column, 200 ml. of benzene phase was forced through and the column was ready for we . The silica gel may be dried at 110° for several hours but gave satisfactory columns when used directly from the freshly opened can. The resolutions obtainable with such columns, for mixtures of dicarboxylic acids with from 8 to 13 carbon atoms, were as good or better than those prepared according to Begemann et.al., and the columns could be used as often as five times before discarding.

A sample weight of $0 to 100 mg. of a mixture of dicarboxylic acids to be analyzed was dissolved in 5 ml* benzene phase and pressed into the column* This was followed by £ ml. of additional solvent after which the column m s developed by washing continuously with 300 to U00 ml. of the benzene phase* The eLuate was collected in 5 ml* portions using a mechanical fraction collector which was activated by an automatic siphoning device* The direct titration of the 5 ml*

samples with alcoholic KOH was not found to be a satisfactory procedure,

1* Davison Silica Gel (mesh size thru 200) obtained from The Davison Chemical Corporation, Baltimore 3, Maryland, was found to be satis­ factory for the preparation of these columns.

2. The solvents used in this laboratory were of commercial grade and were purified by a single simple distillation. 1*6 due to fading end points. This difficulty was presumably caused by the insolubility of the half soaps of the dicarboxyllc acids in benzene. The situation was rectified by allowing the benzene phase

solvent in these 5 ml. percolate samples to evaporate and replacing

it with 95% ethanol. The samples were then titrated against methyl

red indicator using 0.05 N aqueous NaOH. The above evaporation was

conveniently performed by placing the samples (which were collected

in 1* dram vials) in the gentle draft produced by a laboratory hood.

The evaporation was complete in 2U - 36 hours. Prolonged standing of

these samples is to be avoided as the various solutes are slowly

esterlfied by the alcohols present in the percolate. By the use of

this modification at least 95% of the starting wts. of dicarboxyllc

acids could be recovered from the column.

2. Preparation or Synthesis of the Various Acids Used to Standardize the Chromatographic Procedure.

In order to prepare standard chromatograms with which to compare chromatograms obtained from the oxidation products of butter-

fat fractions, it was necessary to have pure samples of the acids

expected from these oxidations. These pure acids were obtained as

follows:

a) Pimelic (Cy), suberic (Cg), azelaic (C9 ) and sebacic (C^q) acids

were obtained from the Eastman Kodak Company. These acids were

purified by crystallization from boiling water and from ether at -20°.

The resulting crystals were then Soxhlet extracted with petroleum

ether for 1*8 hours. The resulting purified acids were chromatographed

individually and found to be satisfactory for use as standards, ie.,

the chromatograms of these acids Indicated that no homologous acids U7 were present. b) Tetradscanedioic (C^^) acid was obtained from Sapon Laboratories-^ and purified by crystallisation from bensene and decolorisation with

Darco activated carbon-black. c) Tridecanedioic (Brassylic) a d d was mads by the disruptive oxidation of erucic acid. Thirty-five g. of erucic acid (neut. eqdv •, 339.2; theory, 338.6) were converted to the methyl ester. Twenty grams of this ester (I.V., 71«6; theory, 72.0) were oxidised in the usual

way with KHnOjj in acetic a d d according to the equations

CH3- ( CH2 ) 7-CH « CH- (CH2 ) n-C02We »CH3- ( CH2 ) 7-C0 2H ♦H02C-(CH2)ll-C°2*te The oxidation yielded 11.5> g* (83.2Jf) of crude brassylic acid* This crude acid was analysed chroma to graphically and the results are listed

in Table XI. Table XI The Composition of the Dicarboxyllc Acid Mixture Obtained by the Oxidation of Methyl Erucate.

Mole % Brassylic a d d "W

Azelaic a d d 6

Dihydroxy-acid 6

The acid was further purified by crystallisation from bensene and continuous extraction with petroleum ether and shown to be chromato­ graph! cally pure.

3. Sapon Laboratories, 101 E. Hawthorne Ave., Talley Stream, N. T. d) Hendecanedioic acid ( C n ) was synthesised by the following reaction scheme* LiAlH^ Et02C-CH2“(CH2)5'CH2“C02Et--- * HO-CH2 -(-CH2 )7-CH2-OH (110) ether HO-CH2-(CH2)7“CH2-OH S0°l2-» Ca-CH2-(CH2)7-CH2-C1 (105) pyridine

C1-CH2-(CH2)7-CH2-C1------ft CN-CH2-(CH2)7-CH2“CN 1 ethanol KOH CN-CH2-( ) 7-CH2-CN g g K02C-CH2- ( CH2 ) 7-CH2“C02k

The diethyl azelate was produced by placing 2 moles

(376.5 g.) aaelaic acid, 600 ml. absolute ethanol, 1000 ml. bensene and 50 g. conc. f^SO^ in a 3 1* flask. The mixture was refluxed

using a phase separating condenser until no further hydrophase was

formed; 1500 nil. H2O was added, and the resulting benzene layer was washed repeatedly with 2£0 ml. portions of water. The bensene was

distilled away and the residue vacuum distilled to yield k$2 g.

(93% yield) of diethylaselate. A solution of 0.855 mole (2l6.3g.) ester in 500 ml.

anhydrous ether was added to a stirred suspension of l.lli mole (l*3 «b g*)

lithium aluminum hydride in 1500 ml. anhydrous ether in a 3 1 . three

necked flask at a rate sufficient to maintain a gentle reflux. After

addition was completed, 130 ml. conc. H2S0^ in 1 1. H20 was added

slowly to hydrolyse the mixture. The resulting ether layer was washed,

filtered, dried, and the ether was removed by distillation. The

residue was vacuum distilled to yield 113 g. of an amorphous product

which was obviously contaminated with considerable quantities of 1*9 unreduced starting material* The product was purified by re crystal­ lisation from ether at -10° C to yield 96*U g* (6 8 j{ yield) 1,9- nonanediolt mp. (uncorr*), U2.9-U*.l°. The melting point has previously been reported to be U2 .5-U5 • $° (1 1 0 )*

The 1 ,9-nonanediol (0.575 mole or 92*3 g*) was placed in a 500 ml* three-necked flask equipped with stirrer, reflux condenser and dropping funnel* Pyridine (8 ml*) was added* The flask was

cooled in an ice bath and 250 g. thionyl chloride was added at such a rate that the temperature remained at about 10° • After addition, the ice bath was removed and the reaction mixture allowed to stand with stirring overnight, and then refluxed for three hours* Ice water was cautiously added to the reaction mixture* The organic layer was washed with conc* dil NaHCO^ and water, filtered through NagSOj^

and dried over CaCH^. The yield was 100 g. crude 1,9-nonanedichloride

yield)* The crude product was distilled through a packed column

to yield 90*3 g. (8 0 jC yield) pure nonanedichloridet b*p. o.lnna.> 73-

7?°.

Three grams of NaCN were dissolved in 60 ml* 9$% ethyl

alcohol and placed in a small r*b* flask with 0*012 mole (2*36 g.) of

the above dichloride. The mixture was refluxed for 90 hours* The

ethanol was removed by distillation and the dinitrile was hydrolyzed

without isolation by refluxing for 18 hours with a solution of 3 g*

KDH in 16 ml. HjO. The reaction mixture was washed with ether,

acidified with dilute H ^ O ^ and the free a d d extracted into ether*

The yield was 2*33 g. (90.0^) nonanedicarboxyllc acidt mp., 105.0-

106• 5° • The crude acid was purified by crystallizing it from a 105C 50 ethyl alcohol solution and by Soachlet extraction with petroleum ether to yield 1.95 g. of 1 ,9-nonanedicarbaxylic acid: top., 10 7 -108°.

e) Hexadecanedioic (Thapsic) a d d (C^) was prepared by a Kolbe electrolysis of the sodium salt of monoethylazelate according to the equation:

anode 2Et02C-CH2- (CH2 ) 5™OH2—C02Na 1 ■ >

Et02C-CH2-(CH2 )i2“0H2-C0 2Bt ♦ 2C0 2 ♦ 2NaOEt. (Ill). The monethylazelate was prepared by mixing 0.92 mole (173 g.) azelalc acid, 103 g. diethylazelate, 50 ml. di-n-butyl ether and 25 ml. conc. HC1 in a 1000 ml. Clalsen flask. The mixture was heated to

150°, then cooled to 120° and 60 ml. 95% ethanol added. The mixture was refluxed for 2 hours and 20 ml. additional ethanol were added. After two hours of further refluxing the mixture was vacuum distilled (112). Table X U describes the fractions obtained from this distillation. Table XII

Fractional Distillation of an Equilibrium Mixture of Mono- and Diethylazelate and Azelalc Acid.

Fraction Pressure mm. Temp, range °C Wt.,g. Conposition of fraction

I 20 - 82.6 butyl ether, alcohol, and water

IT 2 13U - 1 U1 119.2 diethylazelate

III 2 IliO - 16U 32.3 intermediate

17 3 170 - 195 1 3 0 .1* monoethylazelate 51 Fraction 17 was crystallised from 2000 ml. petroleum ether at -20° to yield 105 g* monoethylaaelate. Forty grama of this half ester were dissolved in 75 ml* methanol containing 0.30 g. (.013 mole) of sodium. This solution was poured into a 600 ml. tall type beaker which was equipped with a glass cooling coil and glass stirrer. The solution was electrolysed between two (2.5 x U.O cm.) platinum plates spaced 1-2 mm. apart. An e.m.f. of 2k volts was found to pass 1.2 amp. through this system. The acidity of the electrolyte decreased steadily and after 5 hours the current under 2k volts gradually fell off until less than 0.1 amp. was being passed through the electrolyte. An oily Insulating deposit had formed on the electrodes. The electrolyte did not become alkaline which indicated that the electrolysis reaction had not gone to completion. However, attempts to resume the electrolysis by cleaning the oily deposit from the electrodes and increasing the e.m.f. were unsuccessful. The methanol was distilled from the electrolyte and the residue was dissolved in 200 ml. ether* The ether solution was washed with 250 ml. water, then with two 250 ml. portions of cold 2% Ita^CO^. The solution was filtered through anhydrous Na^SO^ and dried over

CaCl2 * The ether was distilled away to leave 27.0 g. crude diethyl thapsate. This was purified by crystallizing twice from 300 ml. of

methanol at 0° and at -15°> to yield 15.U g. ester (lifljf yield). The ester (2.2 g.) was dissolved to 100 ml. 0.5 N ethanolic KOH and refluxed for 1 hour. The suspension was diluted with 56 ml. H£0 and again refluxed for one hour, after which the dear solution was acidified and extracted with ether. The ether extract was washed 52 thrice with water, dried, filtered and the ether evaporated to yield 1.82 g, of the free acid* m.p., 116-121.5°. The acid wae further purified by twice crystallising from 500 ml. diethyl ether at 5° to produce a final product* m.p., 1 2 2 .8-3 .6° and equiv. wt., ll*3 (equiv. vt., theory, 11*3 )•

This electrolysis had previously been attempted using a nickel anode and carbon rod cathodes. After 15 amp-houra of electrolysis under these conditions, the starting material was recovered unchanged. f) 9,10-Dihydroxystearic acid was obtained by the hydroxylation of oleic acid. Five grams of pure oleic acid obtained from olive oil were

converted to the 9,10-dihydroxystearic acid using cold dilute alkaline

KMnO^ according to the method of Lapworth and Mottram (79)* The crude product was Soxhlet extracted with 60-110° petroleum ether for

several hours, dissolved in acetone and filtered with ca. 50 mg. de­ colorising charcoal. The acetone was distilled away and the product

twice crystallised from U00 ml. ethyl acetate. The yield was 2.3 g.

dihydroxystearic acid* m.p., 129.5-130.5° and equiv. wt., 313 (theory,

316).

The stereoisomer of the above acid was prepared via the

hydrogen peroxide in formic acid method of Swern et.al. (80). Pure

oleic acid (llt.l g.) was converted to hydroxyformoxystearlc acid by

this method in $7% yield. This product was hydrolysed, and purified

by crystallization from ethyl acetate to yield 11.5 g. (72% overall

yield) of dihydroxystearic acid* m.p., 93»7-9U.3°.

g) Oleic acid was obtained from olive oil by crystallizing the C^g $3 distillation fraction from acetone at low temperatures. Two crystallisations at -60° followed by three at >20° were usually sufficient to separate oleic acid from the linoleic and stearic acids found in this fraction, to a purity of 9&% or more.

h) Elaidic acid was prepared by isomerisation of oleic acid with

1% selenium powder at 180° • The mixture was heated for one hour under agitation by a stream of purified nitrogen. The resulting equilibrium mixture was crystallised thrice from a methanolic solution at -20°•

The methyl esters of these acids were prepared by refluxing for forty-eight hours with excess methanol containing dry HC1 as a catalyst.

Chemical and physical constants for the standard acids described in this section are tabulated in Table XIII.

3. Standardization of the Chromatographic Procedure.

Known mixtures of the pure dicarboxylic adds described in the previous section were analysed chromatographically. Two chromato­ grams of such known mixtures are shown in Figures 1-a and 1-b. The percentage compositions of these mixtures were determined from the

chromatograms and are compared with the actual percentages in Table XIV.

The calculations for these analyses were made using the formula: Mole

% dicarboxylic acid *

Vol. 0.05 M MaOH represented by peak in chromatogram _ Total vol. 0.05 N NaOH required for total chromatogram 5U Table XEII

Chemical and Physical Constants for the Various Adds and Esters for use as Standards in the Chromatographic Procedure.

Neat, equiv. M.p . % °C (uncorr.)

Acid I.V. Found Theory Found Lit .value Ref.

Oleic acid 89.9 282.5 282.5 13.b 13.3 (101)

ELaldic a d d 89.8 282.5 282.5 U3.5 1*1*.5 (HI)

Methyl oleate 85.1* — — — — —

Ethyl oleate 80.8 — —— — —

Methyl elaidate 85.5 — — — — Suberic acid ■ ■ 87.6 87.1 139.5 11*5 (113)

Azelalc acid — 9U.7 91*.1 105.9 107 (113)

Sebadc acid — 101.9 101.1 132.7 133 (113)

Hendecanediolc acid — 108.8 108.1 107.0 113.5 (113)

Brassylic acid — 122.0 122.2 108.1 H3.5 (113) Tetradecanedioic acid — 130.8 129.2 12U.8 126.5 (113)

Thapsic acid — 11*3.1 11*3.2 122.8 125 (113)

Dihydroxystearlc acid — 315 316 93.7 91* (80)

Dihydroxystearic acid — 313 316 129.5 130 (101) 0,QE> N NaOH / eluate tube 0 . 1 1.0 1.5 0.5 0.5 Fig. 1 Chromatograms1Fig. of Artificialof Mixtures Pure •H CO 11 20 Cio Eluatetube number Dicarboxylic Acids Fig. Fig. 1-a 1 -b Table XIV Chromatographic Analyses of Known Mixture of Dicarboxylic Acids,

Dicarboxylic acid Mole % mixture A Mole % mixture B Found Actual Found Actual

c13 0 0 3.5 3.U Cll 0 0 20.7 22.U

c10 U 3 U.6 U.3 c9 5o 5U 67.U 65.6

c8 12 10 3.9 U.3

c7 3U 33 0 0

These results indicate that while not quantitative, the chromatographic procedure was of sufficient accuracyto merit its use for semi-quantitative analyses of unknown mixtures of dicarboxylic acids, F Cleavage Reactions of Pure Acids and Mixtures of Acids and Chromatographic Analysis of the Cleavage Products. 1, The Oxidative Cleavage of Pure Acids.

In order to determine which of the several cleavage reactions was most suited to this study, the cleavage productsobtained fromthe oxidation of oleic ester or acid by the different oxidation procedures were examined chromatographically.

a) Oxidation of Oleic Acid via the Method of Begemann (69).

The KMnO^ in acetic acid oxidation according to the procedure of Begemann et.al. was the first to be attempted. By this method 2 g. of methyl oleate or oleic acid were dissolved in 20 ml. of glacial 57 acetic acid and placed in a 200 ml. r.b. flask. Four grains of finely ground KMnO}^ were added in small portions , with swirling, and at such a rate that the reaction temperature did not exceed 50°* The reaction mixture was then allowed to stand for one hour with occasional shaking. The acetic acid solvent was distilled away at 50° at reduced pressure and the residue decolorized with solution of 10% NaHS03 in an equal volume of 2 N l^SO^. The organic material was then extracted with ether. If the original material was an ester, a saponification step was necessary at this point. If the original material was the free acid, the above ether solution could be washed, dried, filtered and evaporated to yield a mixture of mono-and dicarboxylic acids. This mixture was washed twice with 10 ml. portions of petroleum ether, allowed to stand in a third 10 ml. portion of petroleum ether over­ night, and then washed again. The residue was then ready for chromato­ graphic analysis.

Two grams of oleic acid (I.V., 8 9 .9 ) were oxidized by this procedure. The resulting dicarboxylic acids were analyzed, and the results are tabulated in Table XV-a. Fig. 2-a is the chromatogram obtained from the analysis of this oxidation mixture. b) Modification of the Begemann Oxidation Procedure.

Because of the large percentage of secondary degradation resulting from the Begemann cleavage reaction, the procedure described above was modified by decolorizing the oxidation mixture with NaHS03/

H2S01* solution prior to the removal of acetic acid. The decolorized oxidation mixture was then diluted to 200 ml. with water and continuously extracted with ether. The ether extract was washed with 58

20 ml, distilled water and the ether distilled away to leave a residue of acetic acid and the oxidation products of oleic acid or ester* The acetic acid was removed by heating the mixture under vacuum on a steam cone* If the ester of oleic acid had been oxidised, a saponification step was necessary at this point; if the free acid had been oxidized, the residue from the above distillation was repeatedly washed with petroleum ether as before and chromatographed* The results of such a modification are shown in Table XV-b and by

Fig. 2-b. c) Oxidation of Oleic Acid with Performic and Periodic Acids and KMnO^ in Acetic Add* Oleic acid was oxidized by conversion to the dihydroaystearic acid via the performic acid oxidation described previously* Three

grams of KIO^ in 150 ml. of N H2S0^ were rapidly added to It g* of the

above dihydroxystearic acid (m.p., 93*7-9U*3°) in 200 ml. ethanol at U0°. After 10 minutes the clear solution was cooled to 15>° and diluted

with 200 ml* water and the resulting mixture was extracted with ether. An oil resulted on evaporation of the ether* Two grains of this oil were

dissolved in 1$ ml. glacial acetic acid and 1 g* finely powdered KMnOJi was added in small portions. The rate of addition was such that the temperature of the reaction did not exceed U0°. The 'reaction mixture was allowed to stand for four hours at room temperature* The acetic acid was distilled away on a steam cone using reduced pressure* The residue was decolorized as before, then extracted into ether and washed repeatedly with petroleum ether* Fig. 2-c and Table XV-c describe the results of this oxidation. d) Oxidation of Oleic Acid with Performic and Periodic Acids and Ag20. The aldehyde mixture obtained above via the periodate oxidation was oxidized with silver oxide (lilt) in hopes that this reaction would produce less secondary degradation.

A solution of 3*5 B* AgN03 in 100 ml. water was precipitated by the addition of a saturated solution of NaHC0 3 « The precipitate of

Ag20 was washed and added to a methanolic solution of one gram of the aldehyde mixture. The resulting suspension was kept alkaline by the dropwise addition of N NaOH using phenolphthalein as the indicator.

After thirty minutes the suspension was filtered. The filtrate was acidified with dilute H^Oj^ and extracted repeatedly with ether.

The oxidation products were washed with petroleum ether in the usual manner. Chromatographic analysis of the residue indicated incomplete oxidation of the aldehyde mixture by this method. A material having adsorption characteristics very similar to unde canedioic acid was separated from this Ag20 oxidation mixture. This material was not a dicarboxylic acid, however, as it was liquid at room temperature and it decolorized dilute alkaline KMnO^. On the basis of these observations it was presumed to be unoxidized starting material, viz. azelalc semialdehyde. For the analysis of this oxidation mixture see Fig. 2-d and Table XV-d.

e) Ozonation in Acetic Acid.

One gram of oleic ester or acid was dissolved in 20 ml. glacial acetic acid and a stream of ozonized ojqrgen was bubbled through this solution at room temperature. The ozonation was dis- 60 continued when no further ozone was absorbed by the solution as was evidenced by the reaction of the o zone —oxygen mixture with acidic aqueous KI according to the equations

O3 + 2KIH20 ------* l2 + 2K0H-*-O2. The ozonidt solution was decomposed by dropping It slowly and with stirring Into a solution of 8 ml. of 1 $% H202 In 25 ml* of acetic add. The resulting solution was heated on a steam bath for

2k hours to Insure complete decomposition of the ozonlde. The acetic

acid solvent was removed by vacuum distillation. If the original

sample had been and ester, a saponification was performed and the product worked up In the usual manner used In the KMnOjj oxidations. See Fig. 2-e and Table XV-e for an analysis of the products of this

oxidation. Table XV Interpretation of Fig. 2

Oxidation procedure Yield dicarboxylic acid, % Secondary degradation, £ a (Begemann) 61* 11-12

b (Begemann, mod.) 79 3 - U c (Peracid, KMnO^) 51 8

d (Peracid, Ag20) 30 k e (Ozone) 71 3 - 6

f) Discussion of the Cleavage Reactions a to e.

From the standpoint of completeness and cleanness of the cleavage reactions, the above data Indicate the oxidations with KMnO^

in acetic acid (method b) and with ozone in acetic acid (method e) are 0.05 N NaOH / eluate tube 1.0 1.0 1.0 1.0 2.0 1.0 2.0 2.0 via the oxidation of: a & b) methyl oleate with KMnO^ in KMnO^ oleate a with methyl b) of: & oxidation the via Fig. 2 Chromatograms of the dicarboxylic acids obtained acidsdicarboxylic ofthe Chromatograms 2Fig. acids and acidsAg and KMnOJj;and acids periodic and performic oleicacid with d) acetic acid; c) oleic acid with performic and periodic and performic c) oleicacid with acid; acetic o o o 0 2 w 20 ; e) oleic acid ;ozone. acid with oleic e) Eluate tube number tube Eluate 1 6

60 Fig. 2-d Fig. 2-b Fig. 2-a Fig.

62 the beat of the methods investigated. The small peak observed on the extreme left hand side of Fig. 2-b was assumed to be caused by dlhydroxystearic a d d since the material responsible for this peak melted in the range of 90-95° and had an equivalent weight greater than 300. This small amount of dihydroxystearic acid was not expected to interfere with subsequent analyses, and method (b) was adopted as the standard oxidation procedure. Ozonation apparently did not produce this side product, but it did produce slightly more secondary degradation and was not as conveniently performed. Since trans-octadecenoic acids were known to be present in butterfat, the standard oxidation procedure was used for the cleavage of pure elaidic add. This was done in order to compare the oxidation yields and percentages of secondary degradation obtainable by cleavage of geometrically isomeric unsaturated fatty acids. Two grams of elaidic acid (m.p., U3.5-U3.70; I.V., 69.9) were oxidized to produce azelalc acid in 8U£ yield. The secondary degradation amounted to about

2.5%, and about 2,5% of the elaidic acid was found to have been converted to dihydroxys tearic acid (m.p., 122-125°). It was interesting to note that the first stages of oxidation of elaidic acid with KMnOj^ in acetic acid were decidedly endothermic, while no temperature drop was ever observed during the oxidation of the cls-iaomer.

In order to test the KMnO^ in acetic acid oxidation on poly­ unsaturated acids, a sample of C^g fatty acids from c o m oil was oxidised by the standard procedure. The 1.7. of this fatty acid mixture was lU9 *0 ; this would correspond to a mixture of 27% oleic acid with 63% linoleic acid. The chromatogram of the oxidation products gave the 63 following results*

hendecandio1c acid 3 mole %

sebacic acid 1 mole %

azelalc acid 93 mole %

suberic acid 3 mole % From these data it was concluded that either a small per­ centage of positional isomers of llnoleic and oleic adds was present in the corn oil, or the oxidation medium caused some isomerization of the double bonds of llnoleic acid during the oxidation. At any rate, the oxidative cleavage reaction was judged to be applicable to mixtures containing polyunsaturated acids and the matter was pursued no further. 2. The Oxidation of a Known Mixture of Monoethenolc Fatty Acids. Table XVI describes the acids which were used to prepare an artificial mixture of known composition in order to evaluate the accuracy of the entire oxidation-chromatographic analysis. A sample of this mixture was oxidized by both the standard KMnO^ method and by ozonation in acetic acid. Table XVT Pure Monoethenolc Fatty Acids Used to Prepare the Artifical Mixture.

Acid I.V. Meut. equiv. M.p. Cls-11-octadecenoic-s 88.5 282.7 11*.5 - 15.5 Cis-13-do cos enoic-tt-fr 7U.5 339.2

Cis-9-octadecenoic 89.9 282.5 13.0 - 13.2

Cis-8-octadecenoic* 89.9 282.9 23.0 - 21*.0

* Prepared synthetically by Salvadore Fusari *-* Prepared from rape seed oil by Doris Kolb. 61* The dicarboxylic adds resulting from the above oxidations were analysed chromatographically and the chromatograms which were obtained are shown in Fig. 3-a and 3-b. Fig. 3-a was interpreted by assuming a 3% secondary breakdown of the primary oxidation products.

For example, this correction was made for aaelalc acid by subtract­ ing 3% of the total titration volume due to azelalc acid from the suberic acid volume and adding it to the azelaic acid volume. For

Fig. 3-b a h% correction was applied. The results of these in­ terpretations are listed in Table XVII. Table X7II Oxidative Analysis of an Artificial Mixture of Fatty Acids.

Mole %

of unsaturation Found(KMnOli) Found(Ozone) Actual

Cl3 12 8 9 cn 20 18 21 c9 1*1* 52 1*8

ce 21* 22 22

The high percentage of estimated from Fig. 3-a was presumably due to dihydroxystearic acid which is known to be produced by the permanganate oxidation. Mixtures of this acid with brassylic acid (tridecanedioic acid) are not completely resolved by the chromato­ graphic column and would therefore be calculated as one. Bond cleavage by ozone was therefore more suitable for mixtures containing a fatty acid whose unsaturation was beyond the eleventh carbon atom. At any rate, the results listed in Table XVII indicated that a satisfactory 0.05 N NaOH / eluate tube O.U 0.2 0.2 via theoxidation of an artificial mixture of erucic Pig. 3Chromatograms of thedicarboxylic acids obtained KMnO^ in aceticacid, and b) ozone. acid and cis- Cll 8 ’11 20 ,- 9 , and 10 Eluatetube number -11 65 octadecenoic acidswith: a) C,

6 6 method had been developed for the analysis of mixture a of positionally isomeric monounsaturated fatty acids. This method was next applied to the analysis of various fractions obtained from butterf at.

F. Oxidative Analysis of the Various Tractions Obtained from Sumner Butterfat.

Methyl ester samples obtained from summer butterfat by fractional distillation and crystallization were subjected to the cleavage analysis with the intention of determining the identitiee and percentages of the positional isomers of the monoethenoic fatty acids present. 1. Investigation of the C^g Series. The various crystal fractions described by Chart I were oxidized via the KMnO^ in acetic acid medium and analyzed chroma to- graphically. The results of these analyses have been tabulated in Table XVIII. Crystal fraction C^ - C was mostly methyl stearate and was not oxidized. Oxidation of fraction Ci - F indicated that 39^ of the octadecenoic acids present were positional isomers of oleic acid. Since

of the octadecenoic acids were trans, they could not have all been positional isomers of oleic acid, such as vaccenic add. One must conclude, therefore, either that elaidic acid was present in fraction Ci - F, or that geometrical isomers of llnoleic a d d were present. Analysis of the dicarboxylic acids obtained by the oxidation of crystal fraction C2 gave very surprising results. All of the un­ saturated acids in this fraction were trans, yet the chromatogram of the oxidation products showed the presence of little or no elaidic Table IVIII

Positional Isomers of Oleic Acid Present in the Various Crystal Fractions of Sumner Butterfat. Mole % positional isomers of oleic acid present "Oleic" as Percent of Oxidation Crystal fraction I,V. trans.i total trans Cy Cg C^ C C >C yield, %

Total esters 65.0 20.1* 100 - -- - - m Ml

Ci - C l.i* 1*7.0 2.2 -

Cx-F 79.1» 58.8 6.2 0 2 61 0 37 0 71*

C2 21.0 97.0 6.1 0 0 0 0 60 1*0 80

25.6 1*6.6 0 2 19 76 C3 3 73 3 Fi - C 87.6 9.9 ll*.l 0 1 91* 0 5 0 76

F, - F H6.h 28,6 2l*.8 3 9 71* 1 13 0 76 68 acid* A sharp curve was obtained indicating that vaccenic acid was a major component of the unsaturated acids present in C2 . In addition a sharp, heavy curve was formed by an acid material which passed rapidly through the column. Chroma ^graphically this acid behaved like stearic acid or dihydroxystearic acid, however it possessed an equivalent weight of about 11*9 and melted around 120° • The chromatogram of the

C2 oxidation products together with the chromatogram obtained from the oxidation of an artificial mixture of methyl elaidate and methyl stearate having the same I.V. as C2 are shown in Fig. U. The artificial mixture was oxidized in order to determine whether or not this un­ known curve from C2 had been produced from methyl stearate via an un­ expected oxidation of the saturated residue to some high molecular weight dicarboxyllc acid. The oxidation of methyl elaidate again produced a small amount of the dihydroxy-acid as was evidenced by the small hump on the chromatogram (Fig. U-b) between samples 9 and 12. The melting point range of this material was 130.1-130.6°. Chromatograms a and b (Fig. U) were completely different, however, so that the unknown curve described above must surely have been caused by higher dicarboxylic

acids produced from unsaturated fatty acids in which the double bond was more than 10 carbon atoms from the carboxyl group.

A 0.12 g. sample of the C2 oxidation products was placed in a Soxhlet thimble and extracted with petroleum ether for forty-eight

hours. The insoluble residue (0.038 g.) was chromatographed. The size of the first peak relative to the second had decreased considerably and

the material which had been extracted was much more soluble in petroleum O.OJJ N NaOH/ eluate tube 1.0 2.0 2.0 1.0 via theKMnO^ oxidation ofia)fraction Co"Sn from butter- methylstearate having samethe I.V. as C Fig. U Chromatograms of the dicarboxylic acids obtained fat, and andb) artificial mixture of methylelaidate and 20 cl6 ■H 69 XI Eluatetube number 2 Fig.U-a * Fig. U-b

70 ether than any dicarboxylic acid heretofore examined* For example, a forty-eight hour Soxhlet extraction of tridecanedioic acid would extract less than one percent of the total starting weight, whereas over $0% of the oxidation products of Cg was extracted* The petroleum ether extract obtained above was washed once with pe t r o l e u m ether to yield 0.0725 g. of residue* This was passed through a 70 cm* silica gel column and a small fraction was obtained which melted sharply at 123*0-12U*0°. The sixteen-carbon-atora- d±carboxylic acid known as "thapsic acid” melts at 12U° and has an equivalent weight of 1U3* It would be expected to behave chromato- graphically like the 123-12U° melting material above* In order to test the premise that part of the material present in the first curve on Fig* U-a might be thapsic add, a sample of this acid was synthesized by methods which have been described previously* A mixed melting point was taken with the material isolated from the cleavage products of

fraction C2 :

m*p. of synthetic thapsic acid 1 2 2 .8-1 2 3 *6° m.p. of material from C2 123.0-121**0 m.p. of mixture 123*0-123*9 For comparison the melting point of a mixture of equal

quantities of thapsic acid and tetradecanedioic acid was determined. The results were as follows: m.p. of tetradecanedioic acid 121*.8-126.0° m.p. of thapsic acid 1 2 2 .8-123.6 m.p. of mixture 112.8-116.9 In an attempt to determine the nature of the other constituents responsible for the first curve of Fig. U-a, another trans-ester concentrate (Cg1) was obtained from summer butterfat as 71 before. One hundred grains of the total C^g methyl esters of "S" butterfat were fractionally crystallized at low temperatures and a small fraction (U.9 g.) was isolated which was 55.2JC trans-octa- decenoate. The I.V. of this fraction, 61.6, indicated that 80.5£ of the octadecenoate present in this fraction had the trans configuration.

This ester fraction was oxidized in the usual way and chromatographed using a 70 cm. column. Again a large amount of material was separated which behaved chromatographically like thapsic acid. Bottles #12 and

#13 obtained from this fractionation were combined and passed through the column a second time. The melting points of the various samples obtained from this second fractionation are shown in Table XIX. Table XIX Melting Points of the Various Eluate Samples Obtained from the Chromato­ graphic Separation of the Dicarboxylic Acids Produced via the Oxidation of a Trans-octadecenoate Concentrate from *S" Butterfat.

sample number Melting point range °C 11 llh.0-122.0

12 122.5-123.5 13 119.5-122.0

m 95.5-107.5 15 92.0-10H.0 16 105.0-112.0

17 1 0 5 .0 -108.0

A line joining the melting point maxima of the samples obtained from this second fractionation would show three dips* These minima would be presumed to be caused by mixtures of four compounds • 72

The first m i n i m u m would be caused by a mixture of stearic acid and thapsic acid* The melting point maximum and the sharp melting point of the material in bottle #12 indicated that the column had separated thapsic acid from stearic acid on the left of the chromatogram and from another compound on the right* This other compound was presumed to be dihydrcocystearic acid on the basis of its chromatographic behavior and equivalent weight, and because it is known to be produced by the oxidation procedure which was employed* Synthetic thapsic acid was found to pass through the silica gel column slower than stearic acid, but faster than dihydroxys tearic acid which was in agreement with the conclusions drawn from a consideration of Table XII. Finally, eluate sample number 17 (Table XIX) presumably contained a mixture of

dihydroxys tearic acids and other intermediate oxidation products such

as ketols and diketones* From this point it was desired to collect enough thapsic

acid from the butterfat oxidation products to enable the accurate determination of the equivalent weight and to run a carbon-hydrogen

analysis* This was accomplished by repeatedly passing the oxidation

material from C2 * through a 70 cm* silica gel column, removing after

each pass those samples whose melting points were greater than 120°•

About 20 mg* of the thapsic acid (m.p*, 122-121*) were collected in this way.

Analysis:

Calcd. for C ^ ^ O ^ ; C 67*09* H 10.96* equiv. wt. 1U3

Found C 67.01* H 10.96* equiv. wt. lUU 73 Five milligrams of the synthetic thapsic acid and 5 mg. of

the above acid were separately converted to the dlanilide. A mixed m.p. of the two anilides was taken as follows:

m.p. dlanilide of synthetic thapsic acid l61wU-l6f>.3° m.p. dlanilide of butterfat thapsic acid 163*0-I6iw8 m.p, mixture 16U.2-165*U m.p. dlanilide, literature 162 -163 (U5, 116)

The unsaturated acids in fraction analysed to be 26.6^

trans while 27Jt of these acids were positional isomers of oleic acid.

These results were especially significant since ultraviolet examination

of this fraction (see Table X) indicated that no polyunsaturated acids

were present. Here one is tempted to speculate that all of the positional

isomers of oleic acid had the trans configuration. However, it is

equally possible that elaidic acid was present in this fraction and that

a proportionate percentage of the positional isomers were els.

Fraction F^ - F was found to have 26£ positional isomers of

oleic acid while 28.6£ of the unsaturated acids were trans. Ultraviolet

examination of this fraction (Table X) indicated that ljl£ of the acids

present were polyunsaturated. This later percentage was undoubtedly too

low due to the anomalous minus percentage of stearic acid found by this

method. The error was caused by the presence of non-conjugatable

polyunsaturated acids. Because of the high solubility of this fraction

(filtrate @ -60°) it seems likely that all of the trans double bonds

were present in polyunsaturated acids, and not in elaidic or vaccenic

acids. The 13% of 11,12 unsaturated acids found, suggests the presence

of cis-vaccenic acid in butterfat, although it is possible that a

polyunsaturated acid such as 11,13-octadecandienoic might have been the 7U source of the 1356 dicarboxylic acid found In these cleavage products•

A quantitative estimation of the percent of 16-octadecanoic acid present in butterfat obviously was not possible by the analytical scheme which had been used* Dihydroxystearic acid, which the oxidative

cleavage reaction was known to produce, and monocarboxylic acids, which were not completely removed by the petroleum ether washes, were eluted with thapsic acid* It is not unlikely that other intermediate

oxidation products such as ketols and dike tones were present in the

oxidation mixture and were also eluted with thapsic acid* The various

analyses of crystal fractions reported in Table XVIII were all made by

ignoring small quantities of acid material which were eluted into

samples 8 to llu This material usually displayed a melting point range

of from fifteen to thirty degrees and was originally dismissed as being

a mixture of mono-carboxylic acids with dihydroxystearic acids* The conclusions drawn from the analysis of the oxidation products from

fraction C2 would indicate that higher dicarboxylic acids may also have

been present in the cleavage products of other crystal fractions.

It was hoped that double bond cleavage with ozone would not

produce these objectionable intermediate oxidation products and would

thus enable an accurate determination of the percentage of the per­

centage of higher dicarboxylic acids produced. A one gram sample of

the total C^g methyl esters of "S" butterfat was ozonized in acetic

acid, and the resulting ozonides were decomposed with hydrogen peroxide.

The chromatographic procedure of Zebinovsky (86) which had been recently published, looked like an elegant means of analyzing mixtures of dicarboxylic acids in the eight- to sixteen-carbon atom series, and an attempt wao made to evaluate the composition of the above mixture of ozonation products by this method# Fig* 5 is a chromatogram of the total ozonation products obtained from the C^g methyl esters of "S" butterfat using the Zbinovsky chromatographic procedure* Unfortu­ nately all efforts to make this chromatographic method quantitative and reproducible were unsuccessful and after many attempts it was abandoned. Fig. 5, considered only in a qualitative light indicated the presence of thapsic acid in the oxidation mixture, as well as its 11, 10, 9, 8 and 7 carbon-atom homologs* Heptanoic acid, which should have resulted from the cleavage of vaccenic acid, was not detected in the oxidation mixture, and was probably lost when the acetic acid solvent was vacuum distilled from the ozonation products. The most important advantage that was hoped to be gained through the use of the Zbinovsky procedure was that by this method it would not be necessary to separate the mono- and dicarboxylic acid cleavage products by washing with petroleum ether* The higher dicarboxylic acids are not as insoluble in this solvent as for example azelaic acid, and it was feared that these higher acids were being lost in the wash solvent. In another attempt to avoid this difficulty, the standard chromatographic method used previously (modification of Begemann method) was further modified by placing both the mono- and dicarboxylic acid cleavage products on the silica gel column. This was done by dissolving all of the ozonation products in benzene phase and pressing a suitable volume of this solution into the standard Begemann column. The column was then washed with ca. 2$0 ml. of a Skellysolve Microliters 0.1 N NaOH / eluate I 20 1*0 butterfat, using thechromatographic procedure of Zbinovsky ( Fig. $ Chromatogram of the ozonation products obtained Eluate tube number from t e tt l methylesters of "S" the total 86 ). 77 solution. This solution was formed when 10 parts Skellysolve B, U parts methanol, 3 parts water and 3 parts ethanol were shaken to­ gether and allowed to separate into two phases. Elution with the Skellysolve phase presumably washed the mono carboxyl ic acids from the column while the dicarboxylic acids remained stationary. This washing was followed by the usual development with benzene phase.

Fig. 6 shows the results obtained by this method for the ozonation products of the total methyl esters of butterfat and for pure ethyl oleate. The products from ethyl oleate were shown to contain

a spectrum of interfering acids which eluted in the thapsic acid range. However, a quantitative estimation of the various double bond positional isomers present in the unsaturated components of the C^g fraction was made by applying a correction for these acids.

This was done by subtracting h% of the total volume of standard NaOH

solution represented by Fig. 6-a from the first peak and 2% from the second peak. Such a correction was apparently valid since the

chromatogram shown in Fig. 6-b is reproducible in all respects. That is, ozonation of oleic ester repeatedly produced a small quantity of acid material which eluted in the thapsic acid range and the material

always represented 6% of the total chromatogram. The results obtained

from such an interpretation of Fig. 6-a are given in Table XX.

Since any attempt to quantitively analyze the total Ci8 esters of butterfat is complicated by the presence of isolinoleic esters, it was decided to remove most of these polyunsaturated esters by a single crystallization from methanol at -70°. Both the crystal and filtrate fractions so obtained were analyzed by infrared and ultraviolet . 0.05 N NaOH / eluate tube 1.0 0.5 1.0 Fig ozonationof* a) theC^gtotal esters "S" methyl of butterfat, and ethylb)pureoleate. 6 Chromatograms ofdicarboxylicthe acids obtained via the 20 Eluatetube number 78 °9 Fig*6-a Fig.6-b

79 spectrophotometry and by ozonation* The complete analyses of these fractions are also listed in Table XX*

Table XX

Analysis of the Total C^g Methyl Esters of "S" Butterfat.

Total esters Crystals, -70° Filtrate, -70° Wt. 20 g. 16.0 g. 3.8

I.V. 6U.98 £3.0 95.U

Trans, % 16 12 31*

"Oleic” trans, % 20 19 (31*)

Con;). diene, % 1.2 0.3 5.3 Con;), triene, % 0 0 0.1

Linolelc, % 3.1* 0.2 10.1

Linolenic, % 1.8 0 1*.3 Arachidonic, % 0 0 0.1

"Oleic", % 60.9 60.6 66.2

Saturated, % 32,7 36.9 13.9

A ^ mole %■> 7 7 0

£> ^ mole 13' 16 10

A mole 1 0 3

A ^ mole 7U 76 69

® mole 1** 3 1 18

* Percentages of various positional isomers based on total unsaturated components only. 80 The solubilities of the methyl esters of trans-octadecenoic acids are such that they would not be expected in the -70° filtrate (Table IX)* This being the case, all of the trans-acids detected in this filtrate are likely to be polyunsaturated. The fraction when analysed by infrared methods was found to be roughly 3h% trans, while analysis of this same material by alkali isomerization and ultraviolet

spectrophotometry indicated only 20% of the fraction was polyunsaturated. The ultraviolet analysis is known to be in error, however, since it may not be used for the quantitative estimation of the isolinoleic and isolinolenic acids present in butterfat. In view of the rather high

percentages of conjugated dlene material in the -70° filtrate, it is also very likely that the I.V. determined by the Wijs method was too low. In short, little quanitative significance may be attached to any of the data obtained for this fraction by ultraviolet methods. Cleavage analysis of the filtrate fraction again indicated the presence of cis-

vaccenic and cis-8-octadecenoic acids in butterfat. The ultraviolet analysis of the -70° crystal fraction (Table XX) was of significance, since practically no polyunsaturated material was present. Cleavage analysis again confirmed the presence

of a 16-octadecenoic acid in butterfat. By infrared measurement 20% of the unsaturated esters in this fraction were trans, but cleavage analysis

found 26% of these unsaturated acids to be positionally isomeric with oleic acid. Some of these positional isomers therefore must have been cis. Thapsic acid was not found in the oxidation products of the -70°

filtrate and one may conclude from this that no cis-16-octadecenoic acid was present in butterfat. It would appear then that about one- 81 third of the "vaccenic" acid detected by cleavage analysis was cis.

2. Investigation of the Series.

Butterfat is known to contain 9-hexadecenoic acid. The configuration of this acid is presumed to be cis. but it is possible that minor percentages of the trans isomer are also present. It is also possible that positional isomers of this acid are present. In order to explore these possibilities, the fraction obtained from

"S" butterfat was also analyzed using the same scheme used for the

series. The total methyl esters were fractionated once by crystallizing 100 g. from 3000 ml, of methanol at -20°. The results obtained from the analyses of both fractions are listed in Table XXI.

The data from Table XXI Indicate the presence of no positional isomers of 9-hexadecenoic acid in butterfat fatty acids, but about half of the hexadecenoic acids are transj hence, both cis- and trans-hexadecenoic acids are present. The latter has not been previously reported. 82

Table XXI

Analysis of the Ci6 Methyl Esters of "S" Butterfat.

Total Me esters Crystals, -20° Filtrate, -1

Wt. 100.0 g. 92.2 g. 8.7 g

I.v. 6.7 0.9 61.U

Trans, % 0 0 31*

Hexadecenoic as trans, % 0 0 53

Conj. diene, % 0.1 - 0.7

Nonconj. diene, % 0,1 - 0.6

Triene, % 0.2 - o.5

Hexadecenoic, % 6.2 - 67.0

Saturated, % 93.U 99 31.2 mole - - 0 A mole - - 0

^ mole - - 100 A ^ mole 0

* Percentages of various positional isomers based on total unsaturated components only. VI Discussion The general objectires of this investigation were to adopt some quantitative method of cleavage analysis for unsaturated fatty esters and to apply this method, along with other established analytical procedures, toward a more complete evaluation of the compo­ sition of butterfat. Toward this end, the quantitative chromatographic separation of mixtures of homologous dicarboxylic acids according to Begemann et.al. (69) was found to be most useful. In this laboratory the original procedure was modified in method of column construction and use, but the basic principles were the same. By the use of such a

column it was found that known mixtures of dicarboxylic acids could be

analyzed with a maximum error of±\x%, and column recoveries from such

analyses were usually greater than 9$%» Several methods of oxidative double bond cleavage were attempted on pure oleic acid or ester, and from them the KMnOj^ in acetic

acid oxidation of Armstrong and Hilditch (70) or ozonation in acetic acid was found to be most suitable. By these methods 3 - W secondary breakdown of the dicarboxylic acids resulting from double bond fission was observed and a suitable correction for this was applied to subse­

quent analyses. By far the mist disturbing feature of these oxidation

reactions was their failure to produce more than 80% of the theo­

retical yields of dicarboxylic acids. Cleavage analyses of mixtures of

positionally isomeric octadecenoic acids, based on such reactions, must

be made with the assumption that all of the components of such mixtures

were oxidized proportionately. This assumption was apparently a permissible one, since a known mixture of positionally isomeric un­ saturated fatty acids, oxidized by either method, gave results which were 83 8U within the experimental error of the chromatographic procedure. Armed with this method of cleavage analysis, a quantitative infrared method for the determination of $ trans fatty esters, and an ultraviolet procedure for the estimation of $ polyunsaturated fatty esters, an investigation of the methyl esters of a summer butterfat was begun.

The C]_8 methyl esters, obtained by the fractional distil­ lation of the total esters through a packed column, were crystallized from methanol at low temperatures. This was accomplished by lowering the temperature of a k% ester solution to 0°, then to -20°, -38° and

-60°, removing after each temperature reduction the resulting crystal fraction. The 0° crystal fraction was further fractionated by re- crystallizing at -20°. A discussion of the analytical data obtained for each of these crystal and filtrate fractions follows*

a) Fraction C^-C (crystal fraction obtained from recrystallization of 0° crystals) was of little interest since it was mostly methyl stearate (I.V., l.U). This fraction contained 2.2$ of the total trans esters present in the original Ci8 ester preparation and nearly 60$ of the unsaturation was trans.

b) Fraction Ci-F (filtrate fraction obtained from recrystallization of 0° crystals) was 93$ octadecenoate and 7$ stearate by I.V. calcu­ lation. This fraction accounted for 6.2$ of the total Ci8 trans acids and 1*7$ of the unsaturated esters present were trans. Cleavage analysis indicated that 37$ of the unsaturation was in the 11- position and was presumably due to vaccenic acid. There was also some un­ saturation in the 8- position (3$) and this may also have been trans.

However, even if all of the positional isomers had been trans, 7$ of 85 the trans esters would not be accounted for (ie., hl% - 37% - 3%=1%), and must have been present as methyl elaidate. This was the only fraction in which there was any evidence for the presence of elaidate in butterfat, and would correspond to about 0 .0UJ( methyl elaidate in the total butterfat methyl esters.

c) Fraction C2 (*20° crystals) was the most interesting of all the

specimens obtained via this crystallisation scheme because 97% of the unsaturated acids present was found to be trans. Cleavage analysis gave evidence for only a trace of methyl elaidate, and if this material

had been present in butterfat, it certainly would have been expected to

crystallize into this fraction. Instead, the unsaturation was found to

be in the 11- position (60%) and in the 16- position (Uo£). These

results were indeed surprising since a 16-octadecenoic acid had never

before been reported as a component of an animal fat. Its presence was

established by the isolation and identification of hexadecanedioic

(thapsic) acid from the cleavage products of C2j the configuration of

this unsaturated acid must surely have been trans. It was not possible to separate the trans-16-octadecenoic

ester from its mixture with methyl stearate and methyl vaccenate by

crystallisation techniques. A 16-octadecenoic acid has been prepared

synthetically by Kapp and Knoll (117) and it is assumed that this acid

was the trans isomer from a consideration of the method of synthesis

and purification. The melting point reported for this synthetic acid

was 62.8-63.5° and is to be compared with the melting points of 69°

and UU° (105) reported for stearic and vaccenic acids respectively.

Since solubility is closely related to melting point, it is not 86 surprising, therefore, that mixtures of the methyl esters of these three acids may not be completely separated by fractional crystal­ lization. At this point it was found that the determination of the percentages of acids whose unsaturation was beyond the 12- position was subject to considerable error due to the presence of dihydroxy­ stearic acids in the KMnO^ oxidation products. The dihydroxy-acids behaved chromatographically like the higher dicarboxylic adds and were eluted with them during the analysis. While this error would cause the analysis of 16-octadecenoic acid to be high, the greater solubility of thapsic acid in petroleum ether during the separation of the mono- from the dicarboxylic acid cleavage products would cause it to be low. An attempt was made later to eliminate these objection­ able features of the analytical procedure.

d) Fraction C3 (crystals at -3&°) was interesting in that it was a large fraction containing nearly half of the trans adds present

in the original ester mixture. This was not a good concentration of

trans-material, however, since only 25.6% of the octadecenoate present

had this configuration. Cleavage analysis found 26% of the octa­

decenoate to be positionally isomeric with oleate, and ultraviolet

examination indicated that less than l£ of this fraction was poly­

unsaturated. Because the percentage of positional isomers and the percentage of trans-isomers in this fraction were the same, it was

tempting to presume that all of the positional isomers were trans.

Such an assumption was especially attractive in view of the fact that little or no evidence for the presence of elaidate had been found in 87 fractions Cg and C^-F. Three percent of C3 was found to be 16- octadecenoate•

e) Fraction F^-C (crystals at -60°) contained at least 3% poly­ unsaturated esters but this small percentage was not expected to interfere seriously with cleavage analysis. The material was found to be lOjt trans, and 6% positional isomers of oleate. It had been hoped that some fraction could have been isolated which contained no trans-octadecenoate. Cleavage analysis of such a preparation would have enabled the positive proof or disproof of the presence of cis- vaccenic acid in butterfat. Unfortunately F^-C, the best concen­ tration of cis-octadecenoate obtained, was contaminated with 10% of a trans-isomer. Such being the case, it seems unlikely that an all- cis specimen could be obtained from butterfat C18 methyl esters by low temperature crystallization techniques, and this conclusion would clearly explain the failure of previous investigators to isolate pure oleic acid from butterfat.

f) Analysis of fraction F]_-F (filtrate at -60°) was so complicated by the presence of positional and geometrical isomers of the poly­ unsaturated fatty esters, that little significance could be attached to the ire suits obtained. The 1.7. of this material was found to be

1U5.U by the Wijs method, but due to the presence of conjugated diene material (118) the true I.V. was probably nearer to ll*9. Several approximations were made in an attempt to glean some meaning from the analysis of this fraction as listed in Table X. First of all stearic acid was assumed to be absent; the absurd -10.9£ value obtained spectrophotometrically for this acid being rejected. Next the values 88 obtained for linolenic and tetraenolc acids were assumed to be correct*

Then, on the basis of the ll*9 iodine value, the % oleic acid and % dienoic acid was recalculated* By such methods it was deduced that hXx% of the fatty acids were monoethenolc and hX% were diethenoic*

These figures are to be compared with the 10% and 26% recorded for

these components in Table X. Cleavage analysis indicated that considerable original un­

saturation had existed in the 8-, and 11 - positions, but not whether t these unsaturated components had been mono- or polyunsaturated. As a consequence the possibility of the occurrence of cis-vaccenic acid in this fraction has been neither proved nor disproved by this entire scheme of analysis* In another attempt to solve the cis-vaccenic acid problem,

the total C^g methyl esters were crystallized once at -70° to produce

a crystal fraction that displayed only 0.5% diene material when

analyzed by the standard ultraviolet procedure. Cleavage analysis

placed 1% of the unsaturation in the 16- position, 16% in the 11-

position, and 1% in the 8- position, while 19^ of the total un­

saturation was trans* Since 2U% of the unsaturation was in other

than the 9- position, but only 1 9 ^ of this unsaturation was trans,

at least 5% of the positional isomers must have been cis* Thapsic

acid was detected in the cleavage products of the -70° crystal fraction,

but not in the filtrate. If cis-16-octadecenoic acid were a com­ ponent of butterfat, the solubility of its methyl ester would be expected to be such that it would be found in both of these fractions. As it was not, it may be assumed that only the less soluble trans- 89 form occurs in butterfat and that cis-vaccenic acid was the cis- positional isomer of oleic acid present in this crystal fraction.

About 1% of the total fatty acids of butterfat would be cis-11-octa­ decenoic by this method of deduction. The total esters were subjected to cleavage analysis by ozonation, and by a modification of the chromatographic procedure that did not require washing of the oxidation products with petroleum ether prior to placing them on the column. Cleavage with ozone was used because this reagent apparently did not produce dihydroxystearic acids which interfered with the determination of mole# thapsic acid in the oxidation products. The analysis indicated that 1-2% of the total methyl esters of butterfat was methyl-16-octadecenoate, and

3-U5S was methyl-ll-octadecenoate. A similar investigation of the methyl esters indicated that all of the unsaturation in this fraction was in the 9- position, however half of this unsaturation was found to be trans. This would correspond to 0 .0U£ trans-9-hexadecenoate in the total methyl esters of butterfat. VII Summary 1. A method of cleavage analysis for unsaturated fatty acids and esters has been adopted which permits the determination of the mole % composition of a mixture of positionally isomeric unsaturated acids with a maximum error of±U£.

2. Used in conjunction with established optical methods of analyses, this method has been applied to an investigation of the composition of the and C^g fatty esters of butterfat.

3. The occurence of a unique acid (trans-16-octadecenoic) has been established, and this acid is believed to be present to the extent of of the total butterfat fatty acids.

U. Evidence has been presented for the occurence of trace amounts of elaidic acid in butterfat.

$, An 11-octadecenoic acid was found to be present to the extent of 3-h% of the total butterfat fatty acids. This acid was mostly trans, but evidence has been offered for the presence of 1% of cis-11-octadecenoic acid as well.

6 . Over 50% of the hexadecenoic acids of butterfat has been found to have the trans- configuration. These previously unreported trans- acids would account for 0 .0U£ of the total fatty acids in butterfat•

7. No positional isomers of 9-hexadecenoic acid were found to be present.

90 VTII Bibliography 1. Millican, R. C. and Brown, J. B., J. Biol. Chain., U37 (19l*U).

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I, Richard Harold Backderf, was born in Akron, Ohio,

July 3, 1927, and received my primary and secondary education there. After serving for one year as an enlisted man in The United States Navy, I entered the University of Akron, Akron, Ohio, and was granted the degree Bachelor of Science in June 1950. At this time I was employed by the General Tire and Rubber Company of Akron, Ohio, as a chemist in polymer research. In March 1951 I resigned this position to enroll in the Graduate School of The

Ohio State University where I specialized in Organic Chemistry. A research fellowship was granted to me in the autumn of that year by The Institute of Nutrition and Food Technology, and I received the degree Master of Arts in 1952. While completing the require­ ments for the degree Doctor of Philosophy at The Ohio State University, I have held either the above mentioned fellowship, or an assistantship in the Organic Division of the Department of Chemistry.

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