Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University

A STUDY- OP THE TRANS PATTY ACID CONTENT

OP MARGARINES AND SHORTENINGS

DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in The Graduate School of The Ohio State University

Ahmed Fahmy Mabrouk, B,Sc,,M,»Sc, The Ohio State University

1951*

Approved by:

\J ._____ ft 'AcUriser To My Parents

This dissertation is respectfully dedicated.

A. 50471. AC KNOWLEDGMENT

The author wishes to express his sincere gratitude to his preceptor Professor J. B. Brown, Director of The Insti tute of Nutrition and Pood Technology, for suggesting the problem and for his invaluable guidance throughout the suc cessful completion of the investigation and for correcting the manuscript, and to Professor Fred E, Deatherage, Chair man of the Department of Agricultural Biochemistry, who merits thankful considerations for his help during my study. My heartful gratitude is extended to Professors C. L. Wilson and J. I. Watters for the use of the Beckman IR2 infrared spectrophotometer; to Dr. Walter Frajola for the use of the DU ultraviolet spectrophotometer; and to Mr. A. Salant and his Associates of the Health Center Library for their help during my study. The author is grateful to the personnel of the Physical Medicine and Rehabilitation Department of The University Hospital for their encouragement and sincere help during recovery and convalescence from poliomyelitis which, I believe, contributed greatly to the successful culmination of this work. Acknowledgment should also be made to Dean Hussein Aref, Faculty of Agriculture, Giza, Egypt, whose sincere encouragement has been a continued source of stimulation to the author over recent years. • * • 1 11 iv The author is aware of the extremely valuable as sistance and. cooperation of* the Egyptian Education Bureau Personnel, Washington, D.C., throughout his study. This work was supported by a study leave grant from Cairo University, Egypt. TABLE OP CONTENTS Page I. INTRODUCTION AND STATEMENT OP PROBLEM ...... 1 II. HISTORICAL REVIEW OP HYDROGENATION ...... 3 A. Early Development and I m p o r t a n c e...... 3 B. General Chemical Changes Occurring During Hydrogenation ...... 6 C. Hydrogenation of Triglycerides...... 9 D. Hydrogenation of Unsaturated Patty Acids.*. 1 q. E. Nutritive Value of Hydrogenated Pats. . . . 29 III. HISTORICAL REVIEW AND DISCUSSION OP METHODS OP SEPARATION AND IDENTIFICATION OP PATTY ACIDS. . 3k

A. Methods of Separation of Patty Acids . . . . 3^4-

1. Fractional Distillation ...... 3^4- 2. Separation of Patty Acids and Their Compounds by Methods Based on . Solubility Differences ...... 37 3* Separation of Fatty Acids by Chromatography ...... k7 B. Methods of Identification of the Patty Acids 5>2 1. The Determination of the Degree of Unsaturation ...... 52 2. Procedures for Determining the Position of the Double Bonds In Unsaturated Acids ...... 55 3* Examination of Fatty Acids by Absorption Spectrophotometry ..... 62 (a) Ultraviolet Spectrophotometry . • 62 (b) Infrared Spectrophotometry . . . 67 (c) Raman Spectrophotometry .... 71 IV. SOME OBSERVATIONS ON OLEIC, LINOLEIC AND LINOLENIC ACIDS AND SOME OP THEIR ISOMERS CONCEIVABLY PRESENT IN HYDROGENATED PATS . . 73 V. OBJECTIVES AND GENERAL RESULTS OP THE PRESENT INVESTIGATION ...... 8l

VI. EXPERIMENTAL PART ...... 8 3

A. Analytical Methods ...... 8 3 vi

Page B. Reevaluation of Reference Standards on Pure Fatty Acids, Methyl Eaters and Triglycerides ...... 93 C. Ultraviolet Infrared Spectrophotometric Examination of Six Typical Margarines and Five S h o r t e n i n g s ...... • • 99

D. Spectrophotometrie Examination of Miscellaneous F a t s ...... lOij. E. Preparation and Examination of Mixed Methyl Esters of Selected Specimens of Margarine and Shortening ...... 1 0 8 F* Fractional Distillation of the Methyl Esters of M g ...... 113 G-. Further Separation of the CpQ Esters of Mg by Low Temperature Crystallization. The Isolation of C-,o Trans Ester Concen trates ...... • • • • 1 1 8 H. Fractional Distillation of Methyl Esters of S £ ...... 129

VII. DISCUSSION ...... 131

VIII. SUMMARY AND CONCLUSIONS ...... 137

BIBLIOGRAPHY...... 1J4.I I INTRODUCTION AND STATEMENT OP PROBLEM

Fata are one of the three main classes of foodstuffs in the diet of humans and animals. For human foods, they are used In the natural state, possibly after refining, or they may be profoundly altered as for example by hydrogena tion to give improved physical qualities. Assimilation of fats by man depends to some extent on their melting points, liquid oils and low melting fats being almost completely assimilated while fats which have been hydrogenated to a melting point of $0°C or above are appreciably less readily digested. A present trend in the American diet is the use of large amounts of shortenings and margarines. For example, In 1953 the consumption of margarines In the United States has exceeded that of butter for the first time. Simul taneously, lard consumption has declined along with the increased use of shortenings. The unsaturated fatty acids which comprise a major portion of the acids commonly found in food fats have always been assumed to be of the cis-confIguration with the exception of vaccenic acid, trans,11-octadecenoic a c i d ^ ) . Recent work in this laboratory^) has shown that summer butterfat contains 9*5-9*7 per cent trans acids while winter butterfat contains 5-4- Per cent. Vaccenic acid has been reported as a minor component In butterfat. It has been alleged t-o be a growth promoting factor, but 2 later work in this country has not confirmed this finding. In the literature, hydrogenated fats have been criticized for edible use for alleged lack of the nutritive value attaching to the so-called essential fatty acids hav ing more than one double bond In the molecular formula* Such statements, although they are regularly unaccompanied by the actual evidence, are cited In the scientific litera ture. With a view to avoiding this misunderstanding, the present investigation attempts to supply systematic In formation on the nature of the unsaturated fatty acids In shortenings and margarines with emphasis on the trans com ponents. Although hydrogenation of the glycerides is of greater commercial importance than hydrogenation of the fatty acids themselves or their simple alkyl esters, much of our knowledge of the reaction mechanism and the result ing products has been derived from studies upon the latter compounds. During the past two decades, there has been a rapid acceleration In the progress of fat research. The develop ment of new procedures, i.e., the Infrared spectrophotometric method for the determination of trans compounds, chromatographic analysis of fatty acids, the standardiza tion of spectrophotometric methods for the determination of polyunsaturated fatty acids using pure natural fats, and the application of the low temperature crystallization technique In preparing concentrates of trans components 3 have made the quantitative determination of the trans-

fatty acids in hydrogenated fats a relatively simple

operation. The present investigation is concerned only with composition, with no implications as to "biological values. It is hoped that these results will not only fill one of the obvious gaps in our knowledge of these im portant foods but will also help in future studies of their nutritional value.

II HISTORICAL REVIEW OF HYDROGENATION

A. Early Development and Importance. In 18639 Debus^3) showed that if a mixture of hy drocyanic acid vapor and hydrogen Is passed over platinum black methylamine is formed. It Is quite obvious that this reaction involves the addition of hydrogen to hydrocyanic acid, i.e., It Is hydrogenation. Saytzeff^^ In 1 8 7 3 found that when hydrogen and nitrobenzene vapors are passed over finely divided palladium nitrobenzene Is con verted into aniline. Sabatier and his associates in 1 9 2 2 ^^ demonstrated that many ethylenic compounds combined with hydrogen, yielding the corresponding saturated derivatives, If exposed in the vapor state to the action of various catalysts. Oleic acid vapor could thus be converted into stearic acid. The practical applications of the dis- k coveries of Sabatier and his associates lie in their demonstration that the expensive noble metals, palladium and platinum, are not essential catalysts for the reaction, and that certain cheap base metals (notably nickel, co balt, copper, and iron), when finely divided, are good catalysts at temperatures of 1 5 0 - 3 0 0 ° . However, it was not feasible to use the process in its original form for the hydrogenation of fatty acids and oils, because these are not readily vaporized without decomposition, except under very special conditions. At the beginning of the twentieth century, chemists succeeded in hydrogenating oils in the liquid state with nickel as catalyst; in doing so, the oils, liquid triglycerides, were usually converted to fats, solid triglycerides.

The hardening of vegetable and marine oils has be come today an industry of greatest commercial importance.

Before 191)4-* this industry was mostly confined to the manufacture of fats of technical qualities suitable for candles and soap making. The First World War, by stimu lating the demands for butter and fat substitutes, was primarily responsible for the fact that huge quantities of oils were hardened to products suitable for edible purposes. It is quite understandable that the higher price of fat compared with that of oil on the one hand, and the low cost of hydrogenation on the other hand, has been the reason for the rise of the fat-hardening industry. If the price of certain unsaturated compounds becomes greater than that or the corresponding saturated ones, the need for a dehydrogenation technique would develop automatically. Vegetable and animal oils are rich In glycerides of oleic, linoleic and linolenic acids, and these on complete hydrogenation yield tristearin. This process Is accompanied by a rise In the melting point, a decrease In the Iodine value, finally to zero, and a corresponding decrease In refractive index, Hie course of hydrogenation can be readily followed by determining the refractive Index from time to time. As a rule, complete hydrogenation produces too hard a fat, also one which is indigestible, but by partial hydrogenation It is possible to obtain fats of any desired consistency, suitable for use as a substitute for butter, lard or tallow. Soybean and cottonseed oils are the main oils used for hydrogenation In the United States while peanut, coco nut, and cottonseed oils are the principal oils used in the United Kingdom for this purpose. The principal unsaturated fatty acids in these oils are t Trivial Name Chemical Name Palmitoleic acid cis,9-hexadecenoic acid Oleic acid cis,9~octadecenolc acid Linoleic acid cis,9-iCis,12-octadecadienoic acid Linolenic acid cis ,9“ jds, 12-,cis, 15-octadecatrienoic acid 6 In addition to the above, there are present palmitic and stearic acids, and minor amounts of several other acids, both saturated and unsaturated. Hydrogenation is employed on a vast scale in both the soap and edible fat industries, for converting liquid oils to hard or plastic fats, for converting soft fats to firmer products, and for improving the resistance of fats and oils to deterioration through oxidation or flavor re version.

B. General Chemical Changes Occurring During Hydrogenation.

The main reaction talcing place during hydrogenation is the addition of two hydrogen atoms to each double bond, i.e., the conversion of unsaturated fatty acids in the triglyceride molecule to saturated ones. The result of this reaction Is the production of solid fats from liquid oils. As in most chemical reactions, side reactions occur during hydrogenation, the most important being isomeriza tion and interesteriflcation. These will be reviewed briefly below. Isomerization Hydrogenation of fatty acids in fats Is accompanied by the formation of Isomeric unsaturated fatty acids with properties different from the natural acids. The formation of these Isomers may be due to: (l) partial hydrogenation of polyethenoic acids to form Iso-unsaturated acids; (2) migration of* the double bond; or (3 ) the conversion of natural cis to trans acids*

(l) Partial Hydrogenation of Polyethenoic Acid3 To Form Iso-Unsaturated Acids. Van der Veen in 1931^^ demonstrated the formation of 9 ,1 5 - and 1 0 ,l4--octadecadienoic acids in hydrogenated linolenic acid. He thereby showed that the 12:13- or the middle double bond saturated preferentially to the 9 *1 0 - and l^:l6 -double bonds, which underwent a partial shift to the 1 0 :1 1 - and II4.: l^-positions respectively (see (2 ) below). Lemon In 1944-^^ likewise reported that in the hydrogenation of linolenic acid a very considerable amount of hydrogenation occurs at the middle bond to yield an isolinoleic acid or 9*l5-octadeeadienoic acid. Bailey and Fisher (194-6)^^ found that the hydrogen going to the middle double bond of linolenic acid may amount to 6 5 per cent of the total; hence in the hydrogenation of an oil originally rich in linolenic acid, such as linseed oil, this isomer may Increase to as much as 2 5 - 3 0 per cent of the total fatty acid. Smith in 194-8^*^ observed that under selective conditions of hydrogenation of linseed oil abcut 31 P©r cent of the hydrogenated linolenic acid Is transformed Into 9»l5-octadecadienoic acid while the remainder of the linolenic acid goes to oleic acid In either one or tv/0 steps. Rebello and Daubert In 1951^°^ demonstrated that the methyl Isolinoleate fraction ob tained by the low temperature crystallization technique was a mixture of at least tiiree isomers, the 8,1^-, 9»l5- and 1 0 ,114--isolinoleie acids. (2) Migration of the Double Bond. During hydrogenation, double bonds may migrate to new positions in the unsaturated fatty acids. The result ing isomers are of interest because of their relatively high melting points and the consequent effect on the con sistency of the hydrogenated products. In industry, the term "iso-oleic acid" refers simply to the unsaturated fatty acids of high melting point which appear with the saturated acids In the conventional lead soap separation of the latter. The Identity of these acids Is still un certain. Probably, they consist chiefly of positional Isomers of octadecenoic acids, but they may also be the trans Isomers, noted in (3 ) below. Minor amounts of con jugated fatty acids have also been observed in hydrogenated vegetable oils initially devoid of conjugation (11)•

(3 ) The Conversion of Natural Cis to Trans Isomers. The transformation of oleic acid Into elaldic takes place quite readily In the presence of certain reagents, the process being known as elaldlnization. During com mercial hydrogenation a partial elaldlnization may occur, which contributes to the increase In melting point. Feuge and co-workers in 1951 demonstrated that In cis-trans Isomerization during hydrogenation, as In elaldlnization brought about by other means, an equilibrium 9 is eventually reached in the later stages of hydrogenation of the monoethenoic acids resulting in a mixture of approxi mately two-thirds of the trans isomer and one-third cis isomer. Xnteresterification The phenomenon of the migration of fatty acids on the glycerol radical of fats during hydrogenation, i.e*, interesterification> may considerably alter the physical properties of the final product. Steger and Van L o o n ^ ^ found that on heating a mixture of triolein and tristearin in the presence of nickel at about 200° the quantity of tristearin in the product was considerably reduced. Ather ton and Hilditch in 1 9 lj-l^'1'^^ reported that interesterifIca tion takes place relatively slowly In normal hydrogenation especially at low temperatures. They found that at 1 8 0 ° about 5 cent of the glycerides present are involved in this reaction each hour and no reesterifIcation was observed at 60-70°, (Cf. Naudet and Desneulle^) . The lack of reliable analytical methods for the examination of the glycerides make the study of this phenomenon very difficult•

C. Hydrogenation of Triglycerides*

Studies on hydrogenation of triglycerides have been largely confined to changes in the component fatty acids without reference necessarily to their pattern of combination 10 in the triglyceride molecules themselves® Alterations in the component glycerides during hydrogenation of a fat are the resultant of a number of factors, such as: a) the structure of the glycerides in the original fat, b) the manner in which the fatty acid radicals per se are affected during hydrogenation (i.e., preferential hydrogenation of linoleic to oleic acid), and c) the manner in which mixed glycerides of different configurations behave differently toward catalytic hydrogenation, i.e., the apparent reluc tance with which an unsaturated beta-acyl group is hy drogenated in comparison with an unsaturated alpha-acyl group. Dhingra, Hilditch and Rhead in 1932 reported^ that the proportion of isooleic acids produced during se lective hydrogenation of cottonseed oil depends mainly on the temperature of hydrogenation and to a smaller extent on the catalyst concentration; it increases with rise of temperature and also with increasing concentration of catalyst at any given temperature. In 1932 Hilditch and Jones^^ studied the relative amounts of solid and liquid oleic acids present at different stages of the hydrogenation of olive and cottonseed oils. They noticed that selective hydrogenation of polyunsaturated glycerides generally leads to the production of more Isooleic than ordinary oleic acid. They also found that hydrogenation of glycerides of monoethenoic acids is usually accompanied by an Increase in the ratio of solid to liquid oleic acids. 11 In 1932 Hilditch. and J o n e s ^ ^ investigated the alterations in glyceride structure produced during pro gressive hydrogenation of olive and cottonseed oils* Be cause of the selective action whereby linoleic groups are very largely converted Into oleic or isooleic groups before the latter are fully saturated, little increase in the total amount of saturated fatty acids and consequently no appreciable increase in fully saturated glycerides take place until linoleo-glycerides have almost disappeared. After the linoleo-glycerides have disappeared, steady in crease in the total saturated acids commences, but develop ment of fully saturated glycerides Is relatively slow until towards the final stages of hydrogenation. Considering the sequence of actions involved in the conversion of a molecule of triolein Into tristearin, they concluded that: a) one oleic group only undergoes hydrogenation at each effective contact with the catalyst, the semi-hydrogenated glyceride then leaving the catalyst and requiring fresh adsorption before further addition of hydrogen takes place, and b) the hydrogenation of the different classes of unsaturated glycerides Is definitely selective, trioleins being more readily attacked than dioleo-glycerides, and the latter more so than the mono-oleo compounds. They Indicated also that mixed glycerides containing palmitic groups are prefer entially attacked to those containing only stearic residues. An unsaturated beta-acyl group is reluctant to hydrogenation 12 In comparison with an unsaturated alpha-acyl group in the glyceride molecule, Hilditch and Paul, 1935^^. When a mixture of natural fats consisting largely of mixtures of palmito-di-unsaturated glycerides and tri unsaturated glycerides (oleolinoleins), such as cottonseed oil or pig depot fat, are hydrogenated, the palmito-di- unsaturated glycerides pass into palmitostearin to a very large degree before any tristearin is produced, according to Bushell and Hilditch^. In 19^6, Hilditch^0^ suggested a mechanism for the selective hydrogenation of polyethenoic fatty compounds* He noticed that when a mixture of glycerides of oleic and linoleic acids is hydrogenated, the linoleic groups are reduced to the monounsaturated ones before any appreciable amount of saturated glycerides is formed* Ease of hydrogena tion is attributed to the presence of a -CHg- group between two unsaturated groups as in linoleic acid (-CH=CH-CH2 “CH=CH-) and In linolenates (-CH2 -CH=CH-CH2 -CH=CH-CH2 “cH=CH-); and the mechanism of selective addition of hydrogen is almost certainly connected with the ready detachment of a hydrogen atom from the central -QH2“ group between two ethenoid groups. The role of the active -CRg- group in selective hydrogenation Is supported by the following facts: (a) the 9,12-octadecadienoic group Is almost completely

hydrogenated selectively, and 9 *1 5 - as well as 9,12- and 12,15-octadecadienoates are formed from linolenates; 13 (b) linolenates with two active methylene groups are hy drogenated directly to monounsaturated groups; (c) the polyethenoid glycerides of marine animal oils

contain the -CH=CH-(CH2 )2 ~CH=CH- group as well as

-CH-GH-CH2 -CH=CH- group and do not exhibit the same selective hydrogenation; (d) the much lessened selective hydrogenation of the free acids as compared to the esters is explained by the

competitive action of the strong polar group, -C0 OH0 Several reports are very directly related to the objectives of the present investigation* Bailey, Feuge and Smith, 1 9 studied the various factors affect ing the yield of oleic acid and iso-oleic acid in the hydrogenation of cottonseed and peanut oils* The factors influencing the formation of iso-oleic acid include tem perature, pressure of hydrogen, degree of agitation, and the concentration and nature of the nickel catalyst* They reported that the formation of stearic acid is repressed and formation of iso-oleic acid is simultaneously favored by increasing the temperature, increasing the catalyst concentration, decreasing the pressure and decreasing the agitation. They also noticed that the nature of the nickel catalyst, as influenced by its method of preparation, may have a large effect on the composition of the hydrogenated products• On partial hydrogenation of trioloin under conditions which give rise to a relatively large amount of iso-oleic (2 2 ) acids, Benedict and Daubert, 1950* isolated trans-

8 - and trans-9-octadecenoic acids and identified them

physically and chemically. At least six different iso-oleic acids are to be found in partially hydrogenated cottonseed or soybean oils, with melting points ranging from 1jlj.-530, Jackson and Callen, 1951*. Feuge at al*^*^ found that the formation of trans isomers was accelerated by increasing the temperature, and concentration of the catalyst, and by decreasing the degree of dispersion of hydrogen. They noticed that trans isomers are formed at a slightly lower rate during hydrogenation of triolein than with methyl oleate. They also found that hydrogenation of methyl oleate always results in the es tablishment of equilibrium between cis and trans isomers; the concentration of the latter was always 67 per cent, while in the case of triolein the equilibrium concentration of trans was 62 per cent at 200° and 57 P©** cent at 175°.

D. Hydrogenation of Unsaturated Fatty Acids®#

#In this section the subject of hydrogenation has been dis cussed from the point of view of acids of varying degrees of unsaturation. Many of the data, however, were obtained as a result of hydrogenation of natural glycerides® 13 General Concepts. In general# hydrogen tends to add first to the most unsaturated fatty acid present^^ . Upon contact of a glyceride molecule containing oleic, linoleic, and linolenic acids with hydrogen and a catalyst, the most probable re action is the addition of hydrogen to one of the double bonds of linolenic acid to produce, for example, oleo- dillnolein. There are three positions on the linolenic acid molecule at which hydrogen may add, - at 9>10“ or 12,13- or 15,16- yielding 32,15-, 3 , 15- or 9,12-octadecadienoic acids respectively. As a consequence three different glycerides are obtained. This is the most probable reaction, but the follow ing reactions are also possible:

1) hydrogenation of bonds l5*l6- and 1 2 ,1 3 - in the lino lenic group, simultaneously to give normal dioleo- linolein; 2) hydrogenation of bonds 9,1°“ and 12,13- In the linolenic radical to give an Isomeric dioleo-linolein; 3) hydrogenation of bond 12,13- of linoleic acid, to give normal dioleo-linolenin; if) hydrogenation of bond 9,1°- In the linoleic radical to give an isomeric dioleo-linolenin; 5) hydrogenation of the oleic acid to give stearo-linoleo- linolenin. There is a multiplicity of possibilities for the addition l6 of the second mole of hydrogen, and so on until the glyceride molecule Is completely saturated* r The different reactions which occur simultaneously and consecutively with the fatty acids of an oil such as cottonseed, which contains no linolenic acid, are shown dlagrammatically in the following figure: Oleic acid

Linoleic acid Stearic acid X, Iso-oleic acid ‘DIAGRAM (I)

The overall reaction is termed "selective” when the velocity constants for reactions 1 and 2 are large in relation to those for reactions 3 and !{.* High selectivity Is invariably accompanied by a relatively large production of Iso-oleic acids from reactions 2 and Bailey 191^9#

The following diagram represents the different re actions which occur simultaneously and consecutively in the hydrogenation of an oil containing linolenic, linoleic, and oleic acids (the most unsaturated acid is linolenic)•

Oleic

Linolenic Stearic

Isolinoleic Iso-oleic DIAGRAM (II) 17 Conditions leading to high selectivity in Diagram (l) have the effect principally of making reactions 1,2,3, and l\. proceed relatively rapidly in comparison to reactions 5,6, and 7. On increasing selectivity, there is a slight increase in the rate of reactions 1, 2, and 3 at the expense of If, and of 5 at the expense of 6 and 7* As in Diagram (I), selectivity favors the production of iso-oleic acids in Diagram (II)* ( ocZ\ Bailey, 19^4-9 , presented equations for estimating from the composition of oil samples the relative reaction rates of the different unsaturated fatty acids In an oil subjected to catalytic hydrogenation. He reported that the ratio of reaction rates, linoleic acid to oleic acid varies from about if to 1 In very non-selective to about 50 to 1 In very selective hydrogenation of cottonseed oil. On hydrogenating linseed oil, selectivity and non-selectivity showed the following relative reaction rates for oleic, isolinoleic (9#l£-oetadecadienoic acid), linoleic, and linolenic acids, respectively: non-selective 1 2.5 7*£ 12.5

selective 1 3.85 3 1 . 0 77.0 The behavior of soybean oil hydrogenated selectively was quite similar to that of linseed oil. Under selective hydrogenation, both oleic and iso oleic acids have the same reactivity toward hydrogenation according to Bailey and Fisher^^ . Thompson In 1 9 ^ 1 ^ ^ 18 hydrogenated a 5?0-£0 mixture of tung oil and linseed oil and analyzed samples at Iodine value Intervals of ten for fatty acid composition. He found that during the G initial stages of hydrogenation, eda^os tearin was many times more reactive than normal linolenin at equivalent c? concentrations but, as Ql^ostearin decreased, Its re activity lessened until at the 1-2 per cent level it was only slightly more reactive than normal linolenin at an equivalent concentration. He noticed also that the con jugated linolein formed from alaeostearin was many times as reactive as the non-conjugated linolein when present In con siderable amounts. He reported that normal linolenin and normal linolein have similar reactivities. In order to account for the presence of Iso-acids, several theories have been presented^^ and 28) ^ nameiy; a) hydrogenation occurs randomly at all of the double bonds of a polyunsaturated fatty acid; b) oils containing polyunsaturated fatty acids are hy drogenated progressively; c) during hydrogenation, not only hydrogen is fixed at the double bonds of the unsaturated fatty acids, but

at high temperatures In heterogenous media dehydrogenation also occurs; d) the formation of Iso-acids Is due to migration of the double bonds; e) any one or all of these reactions may occur simul- 19 taneously or successively depending on the conditions of* hydrogenation- Hydrogenation of* Linolenic (Octadecatrienoie) Acid, As noted previously, if* one mole of hydrogen is added at random to a mole of linolenic acid (9>12,15>-octadecatrienoic acid), three isomeric forms of octadecadienoic

acid are produced, namely, 9>12-, 9 *1 5 -j and 12,15>-octadecadienoic acids. Since each positional isomer can exist as four geometrical isomers, the number of the formed isomers would be correspondingly increased. The addition of a second mole of hydrogen to the three above mentioned isomers, yield three positional isomeric forms of octadecenoic acid, namely, 9~> 12- and l^-octadecenoic acids. Since each one of these isomers can exist in two geometrical forms, the total number of the resulting isomers would be six. On partial hydrogena tion of an oil containing linolenic acid, it is possible to have twelve octadecadienoic acids and six octadecenoic acids, assuming that hydrogen adds at random. As hydrogen adds preferentially to a particular double bond, this specific bond is completely saturated in a large number of molecules before a second bond is at tacked. This preferential addition to a specific double bond reduces the number of the resulting isomers formed from linolenic acid to one specific positional isomer; if geometric isomerization also occurs, to two geometrically 20 isomeric octadecadienoic acids. On further preferential ad dition of hydrogen, tv/o geometrically isomeric octadecenoic acids are formed.

Xhe preferential addition of hydrogen to specific bonds may likewise be accompanied by a shift in the position of the double bond. By definition fifteen positionally iso meric octadecadienoic acids are possible on the saturation of one double bond of linolenic acid molecule, assuming a shift by one carbon atom of each of the remaining two unsaturated bonds. Xn the above discussion, three assumptions of hydroge nation of linolenic acid were discussed. Experimental evi dence indicates that the double bonds in polyunsaturated fatty acids are dissimilar with respect to their energy of activa tion; consequently, they do not add hydrogen at the same rate. Sims and Hllfman, 19^3^^* noted that conditions which favor the selective hydrogenation of oils also favor the development of trans isomers. Bauer and Erman,1930 reported that the 9*10“ double bond of linolenic acid molecule saturates more readily than the l5:l6-double bond, which in turn saturates more readily than the 1 2 :1 3 -double bond, resulting mainly in the formation of 12,lf>-octadecadienoic acid. Van der Veen, 1931^^ > noted that on hydrogenation of methyl linolenate with one mole of hydrogen, the 12,13-double bond adds hydrogen first pro ducing 9,15-octadecadienoate which partially isomerizes to 21

form. 10 , li^-octadecadlenoate. On farther hydrogenation

with one mole of hydrogen 8- and 1 0 -octadecenoate, trans,

10 -octadecenoate and stearate were formed. Lemon^) con

firmed the above mentioned conclusion and showed that the

acid formed on partial hydrogenation of linolenic acid

contains double bonds which are non-eonjugatable on

alkali Isomerization with ethylene glycol potassium hy

droxide solution and therefore are not capable of determina

tion by the spectrophotometric method of Mitchell, Kraybill and Zschiele^O) . The observations of van der Veen and

Lemon were substantiated by Bailey and Fisher, 19^4-6 , who hydrogenated cottonseed, soybean, and linseed oils under selective conditions and isolated 9 *15 >~octadecenoIc acid from the products of partial hydrogenation. Subse- (81) (82) quently Mattil J and Filer at al ascertained the presence of 9*l£”Ocfcadecadienoic acid In hydrogenated soybean oil. The acid is named iso-linoleic acid*

Fisher, O'Connor and Dollear, 19^4-7^^^, stated that Iso-linoleic acid is not produced during the hy drogenation of cottonseed and peanut oils.

Bailey and Fisher^®) reported the relative reac tivities toward hydrogenation of oleic, iso-linoleic, linoleic, and linolenic acids. They found iso-linoleic acid to hydrogenate three times as fast as oleic acid whereas linoleic and linolenic acid are hydrogenated 20 and ij.0 times as fast as oleic acid respectively. 22

Gunstone and Hilditch, 194-5^^, and Hilditch,

I9J4-6 , reported that the relative rates of oxidation of

methyl oleate, linoleate and linolenate were approximately

in the ratio of 1:12:2 5 - both hydrogenation and oxida

tion the effect of the -Ch=CH-Ch2 “^H=Ch- group is to in

crease the rate of reaction 12 - 20 time3 over that for

the monoethenoid system, and that the introduction of a

second reactive -CH2 - group doubled the rate again^ 0 ).

Lemon and Cross, 1914.9 ^ ^ , concluded from a study

on hydrogenated linseed oil that hydrogenation is ac

companied by a els-to-trans change in some of the double

bonds of the fatty acids, and that methyl iso-linoleate

has at least one double bond with a trans configuration*

Rebello and Daubert, 19 ^ 1 * hydrogenated methyl

linolenate and isolated a fraction rich in methyl iso-

linoleate which was found to be a mixture of at least three

isomers, the 8,lij.-, 9,1$- 10,lij.-octadecadienoates• It

is quite obvious that the 9,10- and l5 *l6 -double bonds of

linolenic acid underwent a shift during the hydrogenation

of this acid at the 1 2 ,1 3 -double bond, to the 8,9 “ # 1 0 ,1 1 -

and lif-, 15- positions respectively. Infrared examination

indicated a trans-configuration for at least one of the

double bonds of iso-linoleic acid.

During hydrogenation of linseed oil, comparatively

large amounts of conjugated linoleic acid are formed, Lie and Spillum, 19.^2 . Their data show that: 23

a) At 2 3 5 ° conjugation of the double bonds is very large

in comparison with that at l6o°.

b) The hydrogen seems to serve as an additional activator

for the isomerization,,

c) By hydrogenation at 23 5 ° II4. per cent conjugated linoleic

acid appears in the hydrogenated fat; at l60 °, only

3 ,6 per cent,

d) At 2 3 5 ° the reaction, linolenic ---^ conjugated linolenic

- >- conjugated linoleic ------oleic acid, predomi

nates *

At l6 o° the reaction

Iso-linoleic

Linolenic Oleic, predominates.

Linoleic

DIAGRAM (III) e) It is well known that high temperature promotes the

selectivity of the nickel-hydrogenation catalyst. High,

temperature during hydrogenation causes high isomeriza

tion and increased reactivity toward hydrogenation, f) The hydrogenation of linoleic acid Is more likely to

proceed as in the following diagrams

Conjugated Linolenic Conjugated Linoleic 4 _ V ' / s Linolenic ------Linoleic : Oleic

Iso-linoleic DIAGRAM (IV) 21*. On partial hydrogenation of methyl linolenate,

U e n o ^ ^ found that the l5 *l6 - position hydrogenates

first, then the 9 *I0 -Position, while the 1 2 ,1 3 -position

is the least susceptible one.

In considering the problems of investigating the

nature of the unsaturated fatty acids in a fat manufactured

by hydrogenating soybean oil, it should be borne in mind

that oxidative cleavage provides no information regarding

the geometric configuration of the acid, and that all four

geometric Isomers of ordinary linoleic acid would give the

same oxidation products on complete fission. The difficul

ties of distinguishing between the various predictable

geometrical Isomers of octadecadienoic acid which result

from the partial hydrogenation of linolenic acid are almost

Insuperable in the present state of development of ana

lytical methods. All of the above mentioned Isomers must be considered as possibly present in the shortening or margarine being studied.

Hydrogenation of Linoleic (Octadecadienoic) Acid.

Hydrogenation of the 12,13-double bond In linoleic acid to form 9 *1 0 -octadecenoic (oleic) acid appears to be always accompanied by a minor amount of hydrogenation of the 9 ,1 0 -double bond to form 1 2 ,13 -octadecenoic acid, van der Veen, 1931^^ • Moore In 1919^^ and Hilditch and Vidyarthi in 1929^^ found 8-, 10-, and 11-octadece noic acids In partially hydrogenated linoleates. In 19^4-5* 25

Mattil^1^and Daubert and Filerproved that during the hydrogenation of linoleic acid esters there is some dis placement of the position of the two double bonds to relatively widely separated positions. Hilditch and

Vidyarthl^39) noticed that, on hydrogenation of methyl linoleate, saturated esters are not formed until about

90 per cent of the polyunsaturated fatty acid esters are reduced to octadecenoates. In a study of hydrogenated cottonseed oil, Bailey^^ found that under very selective operating conditions the ratio of hydrogenation rates for linoleic and oleic acids Is about 50 to 1 while under very non-selective conditions the ratio is lj. to 1. It Is generally believed that in the hydrogenation of linoleic esters the bond farthest removed from the ester linkage tends to be reduced first. Suzuki and Inoue^^-^ noticed that on hydrogenating one mole of linoleate with one mole of hydrogen, cis-9 -octadeeenoate is produced. Related to this Is the finding^ that in the hydrogenation of position isomers of octadecenoic acids, the highest rate of hydrogenation was shown by the double bond farthest removed from the carboxyl group. If these were the only reactions taking place during the partial hydrogenation of linoleic acid, iso-oleic acids would not be formed. However, partial hydrogenation of the natural linoleic acid found In oils produces relatively large proportions of high melting Iso-oleic acidsand These Iso-oleic acids appear early in the course of the

reaction* Possibly some are positional isomers of cls-9-

octadecenoic acid produced either by the saturation of

9 il0 -double bond of linoleic acid or the displacement of

the position of the double in the resulting oleic acid*

The high-melting iso-oleic acids are geometrical isomers

of cis-octadecenoic acids* This assumption is supported

by the fact that, as a group, the trans isomers of 6 -,

8-* 9 -, 10 -, 1 1 -, and 1 2 -octadecenoic acids possess high

melting points appreciably above those of the corresponding

cis-isomers, Huber, 1 9 5 1 ^ ^ -

O'Connor et al, 1 9 5 3 (^6 ), investigated the mechanism

of hydrogenation of methyl linoleate prepared from refined

and bleached cottonseed oil. They reported the following

findings: 1 ) The amount of diene conjugation increased as

the temperature and catalyst concentration increased and as the degree of hydrogen dispersion decreased, 2 ) The relative rate of hydrogenation for conjugated linoleate was 3 .7 to 8 ,7 times as great as that for nonconjugated linoleates. 3 ) ^be maximum amount of nonconjugated trans isomers formed during hydrogenation increased as the tem perature increased and as the catalyst concentraticn and degree of hydrogen dispersion decreased, ij.) After linoleate disappeared, the percentage of trans isomers on the basis of the total amount of oleate tended to remain well above the equilibrium concentration of 67 per cent 27 for elaidinized methyl oleate.

Hydrogenation of Qleic and Other Qctadecenoic Acids.

Many of the comments in the preceding sections above have covered general aspects of the hydrogenation of oleic acid. Addition of hydrogen to the octadecenoic acids is accompanied by other reactions which may involve shift of the double bond in the carbon chain. In all cases the final product Is stearic acid, so by studying the inter mediate products of partial hydrogenation, a true picture of the accompanying reactions can be deduced. Upon partial hydrogenation of oleic acid and Its esters, substantial quantities of Iso-oleic acids are formed In addition to stearic acid. These iso-oleic acids, formed by a shift of the double bond to adjacent positions, are a mixture of both cis and trans acids. The formation of Iso-acids occurs largely during the Initial period of hydrogenation, and, as hydrogenation proceeds, formation of Iso-acids decreases, Mazume^?) . Conversely, oleic acid may also be formed during the hydrogenation of Iso-oleic acids, but the ratio of iso-oleic acid to oleic is much greater than in the hydrogenation of oleic acid.

Piguleviski and Artamonov, 1 9 P found In com paring the hydrogenation of a number of positional isomers of octadecenoic acid that the location of the double bond affects the ease of hydrogenation; the 2 -octadecenoic acid was the least reactive, the 3 “°ctadecenoIc was hydrogenated 28

more rapidly, witli 6- and 9-octadecenoic acids showing

progressively greater hydrogenation rates. Recently, Ar-

1 9 ^ 3 ( 1| . 8 ) ^ tamonov, confirmed the above observations by ex

tending these studies to the monounsaturated fatty acids

of the C-^q , and ser^es* He concluded that the

rate of hydrogenation generally declines with an increase

in molecular weight.

On partial hydrogenation of methyl oleate, Hilditch and Vidyarthi, 1 9 2 9 ^ ^ * Indicated a displacement of the

double bond with the formation of 8 ,9 - and 10,11-octadecenoate. This conclusion was confirmed by Bauer and Krallls,

193^ ^ 9 ), w j10 studied the migration of the double bond in oleic acid on heating In the presence or absence of metals and by Steger and Scheffers^^ who studied the Intra molecular rearrangements on hydrogenating the methyl esters of oleic, elaidic, petroselenic and petroselaidlc acids. In all cases migration of the double bond In the direction opposite to the ester group is strongly pronounced. More recently, Waterman et al, 1 9 5 3 ^ ^ , investigated the partial hydrogenation of the methyl esters of oleic, elaidic, petroselenic and linoleic acids. They noticed that migra tion of the double bond In both directions takes place, but migration in the direction opposite to the ester group is strongly pronounced. They found that the place and con figuration of the double bond in the original ester seem to be of little importance. On the other hand, according to 29

Piguleviski and Artamonov, 1 9 £ 3 ^ ^ , cis-6 -oetadecenoic

acid (petroselenic) hydrogenates more rapidly than its

geometrical isomer, trans-6 -octadecenoic acid (petrose»

laidic)o

Nutritive Value of Hydrogenated Fats

Hydrogenation of fats serves the double purpose of

increasing resistance to rancidity and of increasing the

proportion of solid fatty acids. The Increased resistance

to rancidity depends largely upon the selective hydrogena

tion of polyunsaturated fatty acids to those containing

one double bond. This situation partly accounts for the widespread but mistaken impression that margarines and

shortenings manufactured from hydrogenated fats contain no glycerides derived from fatty acids more unsaturated

than oleic acid.

The vegetable oils used in the manufacture of shortening and margarine are mixed triglycerides primarily of palmitic, stearic, oleic, linoleic and linolenic acids in different proportions. For the manufacture of margarine, vegetable oils are usually hydrogenated under selective conditions, while for shortening non-selective conditions are employed. Under selective hydrogenation, the preferen tial conversion of linoleic acid to oleic and iso-oleic acids rather than oleic to stearic acid takes place,

Bailey^-^ . It is quite obvious from Table 1 that butter and lard contain lower amounts of linoleic and linolenic 30 acids in comparison with vegetable fats commonly used for hydrogenation,

TABLE I

FATTY ACID COMPOSITION OF SEVERAL IMPORTANT FOOD FATS

Butter- Cotton Soykeai Fatty Acid fat Lard Crisco Margarine seed oil oil (5.3 (53) (55) <54>.... (56) (56)

Butyric 3.1 -- _ „

Caprolc 1.9 — — -- — Caprylic 0 .8 — — -- --

Capric 2 .0 — — -- —

Laurie 3-9 — -- -- —

Myristic 1 0 .6 2.9 0 .5 6 0 *lt 2 0.3 m m mm

Palmitic 2 8 .1 2 ^ . 9 1 4 .1 1 7 .0 1 9. 1 6.5

Stearic 8.5 H + . 5 6.3 2 . ll|. 1 . 9 4 -.2

Arachidic 1 .0 -- 1 .6 2 1 - 4 4 0 .6 0.7 Oleic 3 6 .4 lj.2.7 6 1 .1 8 4 2 . 4 7 3 3 . 1 5 3 2 .0

Octadecadie- 3*7 1 3 . 9 1 1 .614. 1 3 . 5 7 39-35 4 9 . 3 noic(Linole ic)

Octadeca- trienoic -- 0 .0 2 0 . 0 5 2 .2

Arachidonic — 1 .1 — — —— —

The vegetable fats, in general, have much higher proportions of the so-called "essential fatty acids" than 31 do tlie animal fats (Table 1). Linoleic and linolenic acids are the only biologically active unsaturated fatty acids in vegetable fats, while animal fats Include also

small amounts of arachidonic acid, which exhibits the greatest biopotency of the three acids.

The distinctive value of linoleic acid and of other unsaturated fatty acids, commonly classed as es sential for the rat, has not yet been clearly established in human nutrition, but is commonly accepted. Andrews and

Richardson, 19^3 ^ ^ , found several brands of shortening to contain 17 to 37 P©*1 cent of glycerides derived from linoleic acid. They analyzed 227 samples of edible fats collected from widely scattered cities in the United States and found that the average percentage of glyceride derived from linoleic acid increases in the order: butterfat, margarine, lard, hydrogenated shortening and blended shortening containing some hydrogenated fat or animal fat as a stiffening agent.

Trans isomers formed during hydrogenation of glycerides are high melting and less digestible, Sims^®^.

Animal and vegetable fats are equally effective in meeting the nutritional requirements and both fats have the same ranges of digestibility, Galamini^^) and Deuel^0^ .

Euler and Euler, 195>1^*^* compared butter and margarine as to their dietary Influence on rats over a period of

18 months and covering l\. generations. They could find 32

no significant differences relative to growth rate,

longevity, number of females bred, number of litters

born, average number of young per litter, number of young

of the subsequent generation, and cause of death*

Phatak and Patwadhen, 1952^^, Investigated the

nutritive value of certain iso-oleic acid fractions

isolated from partially hydrogenated oils by chemical and

physical methods. The growth of young rats fed these

iso-oleic acids was significantly better than on an oleic

acid diet. These unexpected data were confirmed by re

peating the same experiment. They are of the opinion that

the chemical separation of the iso-oleic acids (lead soap

method) may have caused certain chemical changes such as

shift In the position of the double bond and/or decom

position of the acid. When they applied a physical separa

tion procedure (mercury salt) to obtain iso-oleic acid,

there was practically no difference in growth rates of

rats fed on this iso-oleic acid fraction and on pure oleic

acid. The nature of the growth promoting components in

the fraction obtained by the chemical separation method

is not yet known. Rats were capable of assimilating iso-

oleic acids to about 91 pep cent and oleic acid to 9 3 —9^4- per cent; the difference Is not significant.

Hydrogenated fats compare favorably with a natural fat of comparable consistency in serving as a source of essential fatty acids, Deuel and Melnick, 19 5 ^ - ^ ' ^ . 33 Linoleic acid isomers, which are formed during the hy drogenation of vegetable fats and cannot be detected by the ultraviolet spectrophotometric method, exhibit essential fatty acid activity. Using a microbiological assay tech nique, these investigators demonstrated that the iso-oleic acids are not antimetabolites for natural oleic acid and that they are used as nutrients. Likewise, conjugated unsaturated fatty acids are not antimetabolites of the essential fatty acids and are readily metabolized to carbon dioxide and water. Prom the previous literature survey, we can conclude that margarines and shortenings contain lower percentage of linoleic and linolenic acids in comparison with cottonseed and soybean oils from which they are manufactured. On the other hand, both products are deficient in arachidonic acid, the essential fatty acid that exhibits the greatest biopotency. The percentage of octadecadienoic acids in shorten ing is less than that in lard* The essential fatty acids occur naturally in both plant and animal fats in the cis- form. Because of the fact that the octadecadienoic acids found in hydrogenated fats consist of positional and geo metrical isomers, and because of the lack of information about the precise amounts of linoleic acid present in them, the biopotency of margarines and shortenings with respect to their content of essential fatty acids is questionable. Ill HISTORICAL REVIEW AND DISCUSSION OP METHODS OF SEPARATION AND IDENTIFICATION OF FATTY ACIDS

(A). Methods of Separation of Fatty Acids. In general, the separation of the various fatty acids in natural fats or oils is a difficult process, es pecially if It Is to be carried out quantitatively. The methods that are applicable nowadays include both chemical and physical means of separation. The most satisfactory means for obtaining pure fatty acids Is not confined to a single method of separation but combines two or more pro cedures. The choice of the method of separation depends quite largely upon the starting material available and upon the object to be attained. The available procedures may be roughly divided Into three general types involving separa tion by: (l) fractional distillation; (2) solubility; and (3) chromtography. (l) Fractional Distillation. Three types of distillation can be applied to separate mixtures of fatty acids: (a) steam distillation; (b) fractional distillation; and (c) molecular distillation. None of these methods Is adapted to the separation of cis- trans pairs of acids. (a) Steam Distillation. Distillation with steam is applied to separate the so-called volatile from the non-volatile acids. This 35 method is used mainly to separate short chain Tatty acids such as butyric, caproic, caprylic, etc*, Trom milk Tat.

It is also applicable to coconut and palm kernel oils. (b). Fractional Distillation.

Fractional distillation is probably the best tech nique available Tor separating Tatty acids according to

their chain length. The boiling points oT the Tatty acids

Increase with increasing length oT carbon chain. The boil

ing points oT saturated and unsaturated acids with the same number oT carbon atoms do not diTTer suTTiciently to permit Tor their separation, except with very eTTicient columns. Methyl esters boil at lower temperatures (ca 15°) than their corresponding acids and are usually employed in order to avoid association and decomposition.

Norris and Terry, 1 9 discussed the important

Tactors in the construction and operation oT laboratory

Tractionating columns such as column diameter, Insulation, packing, pressure drop, eTTect oT reTlux and molecular weight on Tractionating eTTiciency, reTlux regulation, choice oT operating pressure, constant pressure mainte nance, and accurate measurement oT pressure.

Several types oT column have been used Tor Trac- tional distillation, including the simple pot stills typITIed by the WiHstatter pattern, and, more recently, columns packed with aluminum rings^ 5 ) # or single-turn glass hellcs^^ which are heated externally by an 36 electrically heated sleeve or jacket. Other types of ef ficient fractionating column now available are the spiral- gauge screen and the Bower gauze spiral^^' and fibrous glass packed columns^^ .

The development of efficient packed columns has made it possible to almost completely separate carbon series as found in natural fatty acid mixtures in one through-put#

It is even possible to separate methyl oleate and methyl stearate, the boiling points of which differ by only 3 ° at 1 mm. pressure. Edwards and Robb, 195>1^^ , stated that fractional distillation of the methyl esters of fatty acids would not give a separation of methyl linoleate, linolenate and oleate and that chemical changes may take place in the process. Within recent years, fractional dis tillation on a commercial scale has been developed^O) #

The effect of heat on unsaturated fatty acid esters during the fractionation process has been considered by Norris et al, 19^-3. They found that esters of acids with three or fewer unconjugated double bonds are substantially unaffected, while those of more unsaturated acids are also little altered, although slight rearrangement may occur to conjugated isomers, followed by some dimerisation.

Molecular distillation, known as short-path dis tillation, is carried out under extremely high vacuum.

It differs from ordinary distillation by virtue of the 37 fact that evaporation and condensation occur in a highly

evacuated atmosphere (0 .0 0 1 mm. Hg or less), between sur

faces separated by a short distance, usually of the order

of 1 -2 cm., and by the further fact that evaporation of the

distilland occurs from the surface of thin films (0 .0 1 to

0 .2 mm.). Molecular distillation has the disadvantage of

providing very poor fractionation unless the boiling

points of the compounds to be separated are widely spaced.

By means of this method vitamins and hydrocarbons

are now being distilled from fish and fish liver oils on a

commercial scale. It is not at present used commercially

to any great extent for the fractionation of fatty acids.

From time to time reviews covering various phases

of developments in molecular distillation have appeared and many types of distillation equipments have been described together with their application to the separation of a variety of substances^7 2, 73 and 7^)#

(2 ) Separation of Fatty Acids and Their Compounds By Methods Based on Solubility Differences.

The relative solubilities of the free acids in mixtures and of their various derivatives in different solvents or in the same solvent at different temperatures have been employed as a means of obtaining Individual acids from mixtures and of purifying specific acids. Owing to the mutual solubility effects exerted by one component of the mixture on the solubility of the other components or vice versa, none of the methods is strictly quantitative. 38 Nevertheless, they afford a means of resolving relatively complex mixtures into simpler ones which can in turn be separated further by other means. The oldest of these methods depends on the relative solubility of the metallic salts of a mixture of fatty acids, while more recent ones depend on the relative solu bility of the free fatty acids or their esters in solvents cooled as low as -75° or on certain of their derivatives. (a) Separation of Patty Acids as Metallic Salts. The solubility characteristics of a large number of fatty acid salts have been investigated, and many methods have been proposed for the separation of fatty acids, based on the salt solubility. Generally, these methods are con fined to the separation of unsaturated from saturated acids. The most widely used metal soap method is the Official lead soap-ether method^-^ or the Twitchell lead soap-alcohol modification^^>,^^ • This method is based on the fact that the lead salts of saturated acids are practically insoluble whereas those of unsaturated acids are appreciably soluble in ether or in alcohol. In this method, the lead soaps of trans acids usually behave as saturated acids. Other salts have been proposed as a means of separating liquid from solid acids--lithium, ammonium, zinc, barium, thallium, mercury, sodium, potassium, calcium, magnesium, strontium, Iron, nickel, cobalt, silver and gold. However, no method has been developed which 39

gives quantitative separation of each, component acid of

fats and oils, but only leads to fractions which are

relatively homogeneous with respect to certain types of

acids* Regardless of the method used, complete and sharp

separation of the saturated and unsaturated fatty acids is

generally not possible.

(b) Low Temperature Crystallization*

The technique of crystallization from appropriate

organic solvents at temperatures as low as -75>° has been

developed especially for the separation of the fatty acids

and their esters by J. B, Brown and his colleagues. The

procedure may be applied advantageously to the mixed fatty

acids of most fats and oils without any preliminary treat

ment. Thus, in most cases, the acids may be divided into

three or four solubility groupsi saturated, monoethenoid,

diethenoid, and triethenoid. In this general classifica

tion, each solubility group may include one or more minor

component acid. Brown and co-workers have applied this

method to the preparation of oleic, linoleic, linolenic

and arachidonic acids from the mixed unsaturated acids of a variety of fats and oils and animal lipids.

In 1937 9 Shinowara and Brown^?^ reported the preparation of oleic acid by direct crystallization of the

fatty acids of olive oil. A unique application of the low

temperature crystallization method was made by Brown and

Green, 19^0 ^7 9) t jn the preparation of methyl ricinoleate ko and ricinoleic acid from castor oil* Frankel and Brown,

19^-P-^ f described a crystallization procedure for the preparation of pure linoleic acid. This method was later (fii ) used by Frankel, Stoneburner and Brown, 19^3 * bo pre pare linoleic acid from sesame, cottonseed, grapeseed and poppyseed oils. The acid isolated from these oils by crystallization is essentially Identical with corn oil linoleic acid prepared by this method and with recrystal lized alpha-linoleic acid, prepared by reduction of tetra- bromostearic acid* Hilditch and Riley in 19l}-6^®^ modified this method to meet the case of fatty oils in which con jugated elaeostearic acid is present in quantity. The crystals formed at -60 ° are rich in elaeostearic acid*

Cramer and Brown, 1 9 ^ - 3 ^ ^ » applied this method of low temperature crystallization from methanol, acetone or petroleum ether to the separation of the methyl esters of C^, C^£, and acids obtained from human body fat.

Methyl esters are more soluble in a given solvent than their respective acids. Foreman and Brown, 1 9 i | i ^ ® ^ , reported solubility data on the saturated acids from lauric to behenic and on oleic, linoleic, linolenic, eicosenoic and erucic acids in acetone, methanol and petroleum ether at temperatures down to -^0 ° and lower.

Kolb, 19i>3^-^ , reported further determinations of the solubility of several of the common higher fatty acids as well as of several cis-trans pairs of unsaturated acids l+l and of on© acetylenic acid in methanol, acetone, diethyl ether, ethyl acetate, toluene and n-heptane. Cornwell, (2 ) Backderf, Wilson and Brown, 1 9 5 3 , described low tempera ture crystallization procedures for the preparation of concentrates of C^q trans-octadecenolc acids, containing

8 7 -8 8 per cent of their octadecenoate as the trans ester isomer.

It has been emphasized by Bailey, 1 9 5 0 ^ ° ^ , and others that the solubility of any given acid is closely related to its melting point, and to a certain degree to the nature of the solvent. In general, the even carbon, normal saturated and unsaturated acids become less soluble with increase in molecular weight. It may be further stated for the unsaturated acids that solubility increases with the number of double bonds. Also, the trans acid

(high melting point acid) of a cis-trans pair is less soluble than the els acid (lower melting point acid).

Crystallization procedures for separating the fatty acids from one another and for actually preparing them in the pure state constitute the simplest and most direct method so far available.

Many other application of the low temperature crystallization procedure have been recently reviewed by

Kolb and Brown, 1 9 5 ^ *

Counter-current extraction Is the name given to a particular type of multiple-batch extraction which, is carried out in laboratory devices originally designed by

Craig, * Later, in 19^4-9* Craig and Post^®®^ con structed an apparatus by means of which several thousand extractions can be made in a few hours. Barry, Sato and

Craig^^* 90) studied the separation of normal C2 to C^q fatty acids with the use of aqueous buffers as hypophases and isopropyl ether, n-heptane, and iso-octane as hyper phases. The method has been shown to be convenient for the separation and identification of these compounds.

Ahrens and Craig, 1 9 5 2 ^ " ^ , reinvestigated the separation of the higher fatty acids with the use of improved solvent systems and increased number of transfers. Their studies were extended to the unsaturated octadecenoic acids. Com plete separations were achieved in the unsaturated series by the application of 6£0 transfers and in the homologous series of the saturated acids by the application of lj.00 transfers. Cannon, Zilch and Dutton, 19^2 ^ 2 ) ^ showed that partition coefficients increased with chain length and decreased with increasing unsaturation, with conjuga tion of double bonds, and with introduction of hydroxyl and hydroperoxide groups.

The separation of linseed oil fatty acids by counter- current distribution between pairs of solvents was investi gated and a linolenic acid concentrate, containing linoleic acid only as impurity, was obtained, Davies and Edwards, it-3 195>1^*^ « The applicability of the counter-current ex

traction technique to a diversity of problems In lipid

chemistry is increasingly apparent* Up to date no Informa

tion is available on the distribution behavior of the

acids a n d / o r methyl esters of octadecenoic Isomers and

octadecadienoic Isomers* Consequently, this technique was

not used in this investigation, but is suggested for

future work*

By means of counter-current extraction procedures

applied to the dicarboxylic acids resulting from the

degradative oxidation of the methyl ester of Isollnoleic

acid, Rebello and Daubert, 19^1^°^, concluded that It Is

a mixture of at least three Isomers* This method Is sug

gested for use in identifying the double bond position In

unsaturated acids, and Is being further adapted to this

use by R. H. Backderf In this laboratory*

(d) Separation of Patty Acids as Their Bromo- derlvatives.

Bromine reacts with the double bonds of unsaturated

fatty acids to form 1,2-dibromides which differ greatly In

solubility in different organic solvents, such as, pe

troleum ether, ethyl ether and chloroform* Based on this

fact are methods for separation and Identification of some

of the common unsaturated fatty acids* Table 2 gives a

general idea of the solubilities of these typical bromides*

Bromination and separation of the resulting bromides on the basis of solubility have been applied for the TABLE 2 SOLUBILITIES OP BROMIDES OF SEVERAL COMMON UNSATURATED PATTY ACIDS Solubility In Fatty acid Bromide Petrol. Ethyl Chloro M.P. Forme d ether ether form °C Oleic Dibromide Sol. Sol • Sol. Liquid Linoleic Te trabromi de Sol. - Sol. Sol. 115 warm Insol.- cold

Linolenic Hexabromi de Insol. Almost Sol. 1 8 6 Insol. Arachidonic Octabromide Insol. Practically Insol. 228-30 Insol. Pentaenoic Deca-and Ins ol• Insol• Insol. Decomposi and higher higher at 2 5 0 bromides without melting

qualitative and quantitative examination of the unsaturated fatty acids present in fats and oils. This method has been subjected to several modifications to improve Its utility. Prom Table 2, it is quite obvious that if a mixture of various fatty acids is brominated in cold petroleum ether, those bromides which are insoluble (tetra-, hexa-, octa-, and deca-) will precipitate and can be filtered off, washed, dried and weighed. Similarly, If brominated in ethyl ether, the hexa-, octa-, and deca- bromides are pre cipitated, while In chloroform the octa- and deca- bromides separate out. Therefore, the precipitation of the insoluble bromides forms the basis for rough estimation of the

different proportions of the various unsaturated fatty acids

present. However, the yields of Insoluble bromo-derivatives

from polyethylenic acids are not quantitative, e.g., arachi-

donlc acid gives only about one-fourth, linolenic approxi mately one-third of the theoretical yield of bromides, the remainder of the material being liquid, soluble iso-bromides.

Brown and co-workers^94*9£*96*97) proposed various empirical constants for calculating the composition of mixtures of unsaturated fatty acids on the basis of their polybromide numbers. Recently, Brown and co-workers (98*99*300) have suggested that, under controlled conditions, the method

can be used quantitatively, employing the polybromide yield in conjunction with an empirical standard curve.

(e) Urea Inclusion Compounds of Patty Acids.

Bengen, I9I4.O ^^ , found that urea forms well-defined and easily handled crystalline compounds with a wide variety of straight chain aliphatic compounds but not with branched chain ones. These novel compounds have provided a multitude of research applications in the separation of mixtures, in the purification and identification of com pounds and as a means of analysis. The remarkable differ ence is also observed among carboxylic esters. A number of branched chain esters failed to form crystalline inclusion compounds under conditions which yielded stable adducts from normal esters with a total chain length of about ten ij.6

atoms. Slightly shorter normal chains gave less stable

compounds and very simple esters none at all*

Urea adducts have been used to separate mixtures of

esters by Linstead and Whalley, 1 9 5 0 ^ ° ^ . Newy et al,

19J?0^10^ t employed the method for fractionating mixtures

of fatty acids on the basis of their degree of unsaturation

by extractive crystallization with urea. Schlenk and Hol

man, 1 9 5 0 ^ ° ^ , studied the preparation of urea inclusion

compounds from many pure fatty acids and pointed out that

saturated and unsaturated fatty acids or their methyl esters

could be separated at room temperature, taking advantage of

their preferential formation of urea compounds by the sat

urated components of a mixture. They also isolated fairly

pure methyl oleate and methyl linoleate from olive oil and

corn oil respectively.

Swern et al, 1952^10^ , found that u r e a compounds

meet almost all the criteria for an ideal derivative. The

dissociation temperatures of adjacent or closely adjacent

members of the four homologous series studied (fatty acids,

methyl esters, alcohols and vinyl esters) are sufficiently

far apart to permit this new characteristic to be employed

for identification purposes. They also reported that the

technique can be used on a semimicro or micro scale and

the original compound can be readily recovered. Swern et al, 1 9 5 2 ^ ^ ^ , employed urea inclusion compounds in the preparation of pure oleic acid (oleic acid content 80-95 14-7 per cent) from various grades of inedible animal fats and red oils while Swern et al, 1952^^*^, used the urea inclusion compounds in determining the configurations of the 9»10-dihydroxystearic acids prepared from elaidic and oleic acids by hydroxylation with potassium permanganate. They confirmed the fact that hydroxylation with potassium permanganate proceeds by cis or normal addition, and that opening of the oxirane ring of the isomeric 9>10-©poxy- stearic acids involves an inversion. Swern et al, 1 9 5 3 ^ ° ^ » by the use of urea adducts, prepared concentrates of natural linoleic acid (linoleic acid content, 85-95 per cent) from corn oil fatty acids, and concentrates of linolenic acid (linolenic acid, 8 7 -8 9 per cent) from perilla oil fatty acids.

(3 ) Separation of Patty Acids by Chromatography. The chromatographic technique which is now commonly used in lipid separations falls into the five general classes described briefly below. (a). Elution Analysis Chromatography* This method, introduced by the Botanist Tswett for the separation of plant pigments, involves the passing of a solution through an adsorbent column, and development of the adsorbed zones by elution or washing of the column with additional solvent, either the same or different from the original solvent. The least adsorbed substance lies in the lower zone and is the first substance to emerge from 4 8 the column* The most adsorbed substance lies in the upper zone and is the last one to emerge* (b) Frontal Analysis. When a solution containing a mixture of solutes is passed continuously through a column of adsorbent, the solutes are adsorbed from the solution and are retarded to different degrees. The components appear in the effluent in order of their adsorbability. When successive small increments of the effluent are subjected to physical or chemical analysis, a front will be ovserved for each component in the mixture. When the last front appears, the composition of the percolate is the same as that of solution pressed in. The only component which is separa ted pure is that portion of the least adsorbed compound v/hich appears ahead of the second front. (c) Displacement Analysis. If a sample to be analyzed is dissolved in a solvent and added to a chromatographic column, and a solution of a more strongly adsorbed substance is pressed in after it, the solutes arrange themselves in zones on the column, in order of their adsorbability. As the displacer compound advances down the column, the substances in the sample arrange themselves in zones ahead of it, each in turn dis placing the next more weakly adsorbed substance. Each dis placed substance emerges from the column in a characteristic concentration, and in the more general case, if concentra ^9 tion of solute is plotted against volume of effluent, a stepwise curve is obtained. The height of the step is characteristic of the substance in a given chromatographic system, and the area under the step is proportional to the quantity of the component. (d) Partition Chromatography. This modification of chromatographic procedure util izes the differences in concentration of solutes in two liquid phases. One liquid phase is held immobile by a solid carrier, the other is allowed to flow past the immo bile phase. In practice, partition chromatography is very similar to elution chromatography. The basic difference is that in partition chromatography the Immobile phase is liquid, and separation depends upon the partition coeffic ients of solutes in the two liquid phases. (e) Paper Chromatography. It is in reality one dimensional or two dimensional partition chromatography on a paper support. Its applica tion In the lipid field is limited. By chromatographic analysis with appropriate ad sorbents, It is possible to separate fatty acids or their esters according to carbon chain length, or to degree of unsaturation and also by virtue of other factors in the chemical structure of the acids in question. Some of the more important literature citations follow. Cassidy, 19^4-1^^^^, reported the separation of lauric, myristic, palmitic, t.nd stearic acids by elution analysis. Monostearin is more highly adsorbed by adsorbing agents than tristearin; distearin is intermediate, Kaufmann, 191+0^1101 Swift, Rose and Jamieson, 191+3^ , applied elution analysis to the preparation of pure methyl linoleate from cottonseed oil using alumina as an adsorbent and petroleum ether as an eluent. Riemenschneider, Herb and Nichols, 19l+9^ , described an elution analysis procedure for the isolation of pure natural linoleic and linolenic acids as their methyl esters. Methyl isolinoleate was isolated from partially hydrogenated linseed oil using silica gel as adsorbent and chloroform as an eluent, Lemon, 19i+9(113). Displacement chromtography has been developed in this country largely by Holman and co-workers. Saturated fatty acids can displace their lower homologues from char coal columns, Holman and Hagdahl, 19 l+8 ^ ^ ^ . The best displacer for a fatty acid is its nearest homolog of lower solubility in a solvent which just dissolves the desired concentration of displacer. Depressed solubility caused by addition of water increases separability of fatty acids as shown in frontal and displacement analysis,

Holman and Hagdahl, 1 9 S>0 ^ . Holman in 1 9 5 1 ^ ^ ^ summarized as follows: increas ing the number of isolated double bonds decreases the ad sorption; the change saturated -- 5#- unsaturated ------>- 51 acetylenic acid decreases adsorption; differences in ad sorbability of cis and trans isomers are slight; one isolated double bond in the molecule decreases adsorption roughly equivalent to two fewer carbon atoms; conjugation of unsaturation increases adsorbability. Recent adaptations of paper chromatography are the following: Fatty acids can be separated and identified by paper chromatography of their sodium salts using butanol saturated with 1*5 H 1'JHj^OH as a solvent, Brown and Hall,

1 9 ^0 ( . Bromocresol green and bromothymol blue re vealed the anions as yello spots and the cations as blue spots, Mixtures of stearic and oleic acids, stearic and linoleic acids, and oleic and linoleic acids were separa ted by this method, Kaufmann, 1950^^*^^, Kaufmann and (119) Budwig, 1951 , investigated paper chromatography of propionic, butyric, valeric, caproic, enanthic, caprylic, octenoic, pelargonic, decanoic, undecylenic, stearic, oleic, elaidic, linoleic and erucic acids. Later in 1952, they applied the following fluorescent indicators: quinine, acridine orange, Rhodamine B, Fluorol G, chlorophyll, anthracene, anthranol, and combinations of them in separ ating and identifying the previous acids^120), Kaufmann, Budwig and Schmidt, 1952 ^ Investigated the technique as applied to 9 * H “ 9 *1 2 -octadecadienoie acids, and to B-eleostearic and B-licanic acids by using reactions with organic salts and dying with organic dyes, Inoue and 52 (122) Noda, 1953 , applied paper partition chromatography to the separation and identification of saturated fatty acids, unsaturated acids, hydroxy acids and polybasic acids; and previously, 19^-9^'“^^ > to the separation and Identification of fatty acids by means of color reaction with the hydroxamic acids, which were obtained from fatty acid esters by reac tion with hydroxylamine. They have also described the paper partition chromatography of hydroxamic acids using ferric chloride as a coloring reagent which can be applied to the separation and Identification not only of saturated acids, but also of unsaturated and hydroxy-monocarboxyllc acids as well as di- and tricarboxylic acids, Inoue and Noda, 195l(12^ . In view of the above citations, it seems unlikely that chromatography will be useful In separating the mix tures of C-^g Iso-acids such as are likely to be found in hydrogenated fats.

(B) Methods of Identification of the Fatty Acids.

(1 ) The Determination of the Degree of Unsaturation. Determination of the Iodine value by a standard method (WIjs) Is a direct measure of the degree of unsat- uratlon of a fatty acid or its ester* Table 3 gives a general Idea of the iodine values of some of the unsat urated fatty acids and their methyl esters. 53 TABLE 3 IODINE VALUES OP SEVERAL FATTY ACIDS AND THEIR METHYL ESTERS iodine Value Patty Acid (Theore tical) Methyl Trivial Name Geneva Name Acid ester

Myristoleic 9 -tetradecenoic 1 1 2 * 1 5 1 0 5 .6 0 Palmitolelc 9 -hexadecenoic 99*78 94*57 Oleic 9 -octadecenoic 89 #87 8 5 * 6 2

Linoleic 9 ,1 2 -octadecadienoic 1 8 1 * 0 3 1 7 2 .4 1 Linolenic 9 ,1 2 ,l5 -octadecatrienolc 273.51 2 6 0 .4 0 Eleostearic 9 ,ll,1 3 -octadecatrIenoic 273*51 2 6 0 * 4 0 Arachidonic 5 ,8 ,1 1 , l/^-eicosatetraenoic 333*50 3 1 8 .8 1

In the case of* conjugated acids, iodine values are mislead ing because the values obtained by standard methods are much lower than the theoretical values# The theoretical iodine value or eleostearic acid is 273*5* By the Wljs method It is about 1 8 0 , a reaction for two double bonds# Consequently, It was regarded for a long time as a diethenoic acid, but by molecular refractivlty of the acid, the amount of hy drogen absorbed in order to effect complete conversion into stearic acid and by modified methods of iodine absorption, It was later demonstrated that it contains three ethenoic c on jugate d linka.ge s.

It Is also to be noted that IC1 (Wljs) and iBr (Hanus) react incompletely with an unsaturated acid, if the double 514- bond Is closer to the carboxyl group than the 5 - 6 position, as noted in Table i\.9 Bull, 1937 ,

TABLE 1|. IODINE VALDES OF SEVERAL UNIQUE UNSATURATED ACIDS Iodine Value Acid Formula Theoretical Found

Crotonic c h 3 -c h =c h -c o o h 2914- 25 Maleic cis-H00C-CH=CH-C00H 2 1 9 6 . 0

Fumaric trans -HOOC-CH=CH-COOH 2 1 9 6 .0

2 ,3 -oleic CH 3 -(CH2)^-CH=CH-C 00H 89 «87 9 . 0

3 ,l|.-oleIc ch3 -(ch2 )1 3 -ch=ch-ch2-cooh 8 9 .8 7 1 6 .3

1{.,5 - oleic CH3-(CH2 )1 2 -CH=Ch-(CH2 )2 -C00H 8 9 .8 7 2 6 . 9

The polyunsaturated fatty acids may be examined for conjugated unsaturation by determination of the diene number which is Kaufmann1s^12^ analytical adaptation of the Diels- Alder reaction* From this value the percentage of the double bonds which are conjugated can be calculated* A second and more informative method Is by examination of the ultraviolet absorption spectrum of the compound* This is more fully described later*

Kaufmann 128) ,ji3 c o y e r e 4 that unsaturated fatty acids react with thiocyanogen in a reproducible manner, if the reaction conditions are adequately controlled. Thiocyanogen adds quantitatively to the ethylenic bond of oleic acid, but with only one of the two double bonds In 55 linoleic, and with, two of* the three linolenic acid, double bonds* The thiocyanogen value is analogous to the iodine value, being calculated on the same basis, in terms of iodine* It is used frequently in conjunction with the iodine number for the determination of the composition of mixtures of unsaturated fatty acids or their esters^75)„

(2 )• Procedures for Determining the Position of the Double Bonds in Unsaturated Acids. In order to ascertain the position of the double bond In unsaturated fatty acids these acids, in the pure state If possible, are usually oxidized in such a way that mainly a mixture of monocarboxylic and dicarboxylic acids results:

Ch 3 -R -C h =CH-R 1 -COOH + Ij. 0 ------>- CH3 -R-COOH -+ HOOC-R-j-COOH

By identification of the resulting acids ..and even in many cases only one of them, the position of the double bond In the starting compound can be deduced. Practically, the oxidation reaction never proceeds so simply as Is indicated by the above equation, because lower decomposition products are always formed. These disturb more or less severely the exact estimation. The monocarboxylic acids seem especially subject to such undesirable secondary decomposition. The dibasic acids are more stable and therefore the identifica tion of these acids is preferred In determining unsaturated acid structure. Separation of monocarboxylic and dicar boxylic acids can be readily performed by steam distilla- 56 tion, the lower monocarboxylic acids (lower tlian being volatile. In addition, advantage of the different solubilities of earboxylic acids In light petroleum ether may be used in separating mono- and di-carboxylic acids, the former being readily soluble, while the latter are practically insoluble in this solvent. In general, the products of oxidation of unsaturated fatty acids vary with the reagent used, and the conditions of oxidation, i.e. temperature, concentration of reagent and sample, solvent in which oxidation is taking place, catalyst, etc. The oxidizing agents used in the oxidation of fatty acids are: potassium permanganate, hydrogen perox ide, peracids, ozone and lead tetracetate. Since oleic acid has been the most thoroughly Investigated of all un saturated acids, the determination of its structure will be discussed to Illustrate the several methods available. (a) Oxidation with Potassium Permanganate. Potassium permanganate is the oxidizing agent most often employed in Investigating the structure of unsaturated fatty acids. Long chain saturated fatty acids are generally unaffected by this reagent except at elevated temperatures and after long action. On the contrary, mono- and polyethenoid acids react rapidly at relatively low temperatures. Two types of potassium permanganate oxidation are used: oxidation In aqueous alkaline medium at low temperatures,

0 -3 0 °, and oxidation in nonaqueous or acidic media at more 57 elevated temperatures. Oxidation with Aqueous Alkaline Potassium Permanganate, The products of oxidation in the cold are predomi nantly hydroxy acids of the glycol type. Thus, oleic acid in ice-cold alkaline potassium permanganate solution is rapidly oxidized to dihydroxystearic acid. ch3 -(ch2 )7 -ch=ch-(ch2 )7-cooh mrxDk a ch3 -(ch2 )7 -ch(oh)

-ch(oh)-(ch2 )7-cooh

Lapworth and Mottram, 1 9 2 5 ^ ^ ^ * investigated the various factors affecting the oxidation reaction of oleic acid. They reported the following conditions for obtaining the maximum yield of dihydroxystearic acids: low temperature

(0 -1 0 °); a concentration of oleic acid not to exceed 0 . 1 per cent; reaction to be carried out on the sodium or potassium salt; concentration of permanganate not to ex ceed 1 per cent; very short (5 minutes) reaction time; and slight excess of alkali at the outset to avoid the production of ketohydroxy stearic acid. Under these conditions, they were able to obtain dihydroxystear!c acid equivalent to 9 & P®*1 cent of the original pure oleic acid. Oleic acid yields the high melting dihydroxystear!c acid

(1 3 2 °) whereas peracid oxidation yields the low melting acid (9 5 °)• On the other hand, alkaline permanganate oxidation of elaidic acid produces the low melting di hydroxys tearic acid (95°) 9 Markley, 19^-7^ . Green and 58 Hilditch, 1935>^-^^, applied the Lapworth and Mottram method to linoleic acid and isolated two tetrahydroxy- stearic acids, melting at 1 7 3° and 1 5 5 ° respectively. On oxidation of alpha-linolenic acid, with alkaline per manganate they isolated two hexahydroxystearic acids melt ing at 2 0 3 ° and 1 6 9 °, respectively. Oxidation with Neutral Aqueous Potassium Permanganate, King, 1 9 3 6 ^ ^ ^ , found that oxidation of oleic and elaidic acids with neutral aqueous potassium permanganate yielded mixtures of 9 “*iydroxy-1 0 -ketostearic and 1 0 -hydroxy

9.-ln3'kostearic acids together with varying amounts of dihydroxystearic acids. Doree and Pepper, 1914.2 ^-^^, sub jected erucic (cis-1 3 -docosenoic) and brassidic (trans-1 3 - docosenoic) acids to both alkaline and neutral permanga nate oxidation and found that the principal products of oxidation were dihydroxybehenic acids under alkaline condi tions and hydroxyketobehenlc acids under neutral conditions Honaqueous Oxidation with Potassium Permanganate. When unsaturated fatty acids are subjected to oxida tion with potassium permanganate in acetic acid or acetone solutions, fission occurs at the double bonds. Theoreti cally, disruptive oxidation of monoethenoid acids produces a dibasic and a monobasic acid. For example, disruptive oxidation of oleic acid should produce only azelaic acid and nonanoic (pelargonic) acid in equimolar quantities in accordance with the following equation: 59

CH^~(CH2 )-CH=CH-(CH2 )7“C00H GH3 -(CH2 )7-COOH Oleic acid Honanoic acid

HOOC-(CH2 )7 -COOH Azelaic acid

Practically, the two acids are not produced in the same proportions. This is due to the fact that intermediate products, such as hydroxy acids, are formed prior to fission and that the fission products are themselves sus ceptible to further oxidation. The monobasic acids undergo additional oxidation much more rapidly than the dibasic acids, which often results in appreciably lower yields of the former as compared with the latter acid, Armstrong and Hilditch, 1925 > ^ ^ ) » described a method for the dis ruptive oxidation of methyl oleate in dry acetone or in glacial acetic acid. They stated that by using one of these solvents no additional decomposition occurs; the use of the acids in the form of their methyl or ethyl esters is stressed. Methyl oleate under these conditions yields 8 0 - 9 0 per cent of the theoretical amount of nonanoic acid. On disruptive oxidation of ethyl oleate, the yields of azelaic and nonanoic acids are equivalent to 95 P©r cent and 5 9 Pe** cent, respectively, of theory. Mowry, Brode and Brown, 19i^2^^^* applied disruptive permanganate oxidation to establish the position of the double bonds in arachidonic acid. 6o (b) Oxidation with Ozone. In general, oxidation with alkaline permanganate Is preferable with monoethenoid acids, while ozonation yields more satisfactory results with polyethenoid acids, Markiey, 19^7^^30)^ Ozonation of polyunsaturated acids occurs smoothly and with the formation of much fewer secondary reactions than in the case with the more drastic permanga nate oxidation. Ozone reacts with unsaturated fatty acids, the reaction being the addition of a molecule of ozone at the double bond to produce an ozonide. These are oily, unstable substances which readily break down on treatment with water or dilute acids to yield aldehyde and acidic decomposition products. Ozonation of oleic acid and sub sequent hydrolysis in the presence of air occurs according to the following scheme: 0 — 0 CH3-(CH2)7-CH=CH-(CH2 )7-COOH-tO^ — ^ CH3 -(Ch2 '7 -CH CH-(CH2 }f-C00H v o/ Oleic Acid Oleic Ozonide

CH3-(CH2 )7 -C00H + CHO (CH^^-COOH n-nonanoic acid Azelaic semialde' hyde Oleic ozonide h 2o or l-^ch3-*(ch2 )7-cho + H00C-(CH2>7-C00H n-nonanal Azelaic acid

When the preceding hydrolysis Is carried out in the presence 6i of an excess of hydrogen peroxide, nonanoic and azelaic acids are formedo (c) Oxidation with Lead Tetraacetate. This method involves preparation of the dihydroxy acid followed by splitting the glycol grouping by lead tetraacetate into two aldehydes, Criegee, 1 9 3 1 ^ ^ ^ . R-CHOH-CHOH-Rf-t- 0 — >-R-CH0 -+ R»-CH0+ H20 Further oxidation of the resulting aldehydes to carboxylic acids is practiced in most cases. (d) Oxidation with Peracids. The peracids commonly used for the investigation of the fatty acids are peracetic, perbenzoic, periodic, and performic acids. The first step of oxidation with peracetic acid is, presumably, the addition of peracetic acid at the double bond, followed by a hydrolysis to the dihydroxy acid. On treatment with dilute alkali the hydrolysis is completed as shown by Hilditch and Lea, 1 9 2 8 ^ ^ ^ , and

Boeseken and Belinfante, 1 9 2 6 ^ ^ ^ . Perbenzoic acid re acts with unsaturated fatty acids in a somewhat different manner, the first product being usually a crystalline oxido-acid which can be converted quantitatively into the dihydroxy compound by hydrolysis.

Bauer and Bg.hr, 1 9 2 9 ^ ^ ^ * investigated the reaction of perbenzoic acid with mono- and polyethenoid acids. They found that in all cases an oxido acid is the principal pro duct which on hydrolysis is converted to the corresponding 62 hydroxy acid. Swern, Findley and Scanlaa^ ^ 0 anci investigated the mechanism of peracetic and perbenzoic acid oxidations of unsaturated fatty acids and concluded that in both cases the primary reaction product is an epoxy compound, but under the conditions generally em ployed with peracetic acid the oxirane ring, -CH-CH-, is ^c/ opened to form hydroxyacetoxy derivatives* Either a short reaction time at high temperature or a longer time at lower temperatures resulted in opening of the oxirane ring* (3)- Examination of Fatty Acids by Absorption Spectrophotometry* When organic compounds are exposed to energy of ap propriate wave length, absorption occurs with accompanying conversion of energy into one of three kinds of molecular or atomic motion, depending upon the wave length of the light. This results in three types of spectra, rotational, electronic, and vibrational. Rotational spectra occur in the far infrared and microwave regions and have had little application to organic chemistry. Electronic spectra form the basis for visible and ultraviolet spectroscopy* The third type occurs when the absorption of radiant energy gives rise to changes in the vibration of the molecule, and it is the measurement of this absorption which is applied in infrared spectroscopy as well as in the use of Raman spec tra for the examination of fatty acids. (a) Ultraviolet Spectrophotometry. 63 Saturated aliphatic compounds are transparent except in the far ultraviolet region. Polyunsaturated fatty acids with conjugated double bonds have absorption bands at 2 3i}->

2 6 8 , and 3 1 6 nu^ for 2 , 3 » and Ij. double bonds respectively. Moore in 1937^*^^ and later Burr and co-workers,

1 9 3 8 found that if fatty acids containing more than one double bond were subjected to prolonged saponification there was a marked increase in ultraviolet absorption due to conjugation of the double bonds. The absorption with vegetable and land mammal fats^-^^ was at 2 3 0 and in marine oils 2 7 0 mi^. lie found that tung oil fatty acids showed intense absorption at 2 7 0 mi^ even before saponifica tion. Later, he observed^ that when saponified linseed oils were subjected to prolonged heating an increased ab sorption at 2 3 0 took place first and later a development of an absorption maximum at 270 mi^. The latter change was accompanied^by the formation of a solid unsaturated acid which resembles elaeostearic acid. Later work resulted in the observation that the absorption observed in polyethylenic acids at 2 7 0 im^ was caused by three conjugated linkages, while that at 2 3 0 mi^ was due to two such linkages. Burr and Miller, 1 9 ^ 1 ^ ^ ^ , found that Moore’s reaction can be made reproducible and suggested an empirical quantitative procedure for estimating linoleic and linolenic acids in vegetable oils. In their work, the natural, non-conjugated acids are converted to conjugates by heating with alkali In ethylene glycol at l8 0°. Mitchell, Kraybill and Zscheile, 19^3^^, established a precise quantitative method for the determination of linoleic and linolenic acids in fats and oils in which alkalinity and time of isomerization are carefully controlled. In 19^-fy- Beadle and Kraybill publi shed reference values for arachidonic acid, as well as for linoleic and linolenic acids. Thus, they extended the spectrophotometric method to include estimation of arachidonic acid. To effect Improvements In sensitivity, Hilditch and co

workers, 1 9^5 ^^^^ recommended 60 minutes as time reaction

to conjugate diene unsaturated acids at 1 8 0° and 1 5 minutes

at 1 7 0° to conjugate triene unsaturated acids. Hilditch, Patel and Riley, 1 9 5 1 ^ reinvestigated the values of

the extinction coefficients (E^/0 ) for ultraviolet ab- Iom^

sorption bands at 2 3 k mH an<^ ^ 68 which are developed when linoleic and linolenic acids are subjected to alkali isomerization* They found that acids obtained by debroml- natlon of tetrabromldes and hexabromides gave different values than fatty acids obtained by physical methods, I.e. by the Brown et al technique ^ followed by adsorption on silicic acid. They found no appreciable difference under their conditions of isomerization for the two types of linoleic acid. Nichols and Riemenschneider, 19^9 9 showed that ultraviolet absorption intensities of methyl esters of polyunsaturated fatty acids obtained by the 65 partition chromatographic method differed significantly from, those obtained by debromination. The spectrophotornetrie method can be used only with reservations for the determination of unsaturated fatty acids in hydrogenated fats and oils because in the process of hydrogenation diene acids are produced which fail to conjugate upon alkali isomerization* Lemon, 19M4-^ ^ * Daubert and Filler, 19^4-5^^ and Popov et al, 1 9 5 0 ^ - ^ ^ . From a study of the kinetics of alkali isomerization of linoleic, linolenic and arachidonic acids, Riemenschneider

et al, 1 9 5 0 ^ ^ ^ , found that 9 °i3 »1 2 cis-octadecadienoic

acid isomerized twenty times faster than the 9 ^rans 9 I2 trans- octadecadienoic acid and produced a mixture of dienoic con jugated isomers having a specific extinction coefficient at the maximum more than five times that of the latter. Jackson et al, 1952^^^, investigated the alkali isomerization of the geometrical isomers of octadecadienoic acid- They found that the intensity of the absorption of isomeric octadecadienoic acids was dependent on the duration of alkali isomerization. They used their results in estab lishing equations for the determination of these isomers in mixtures• The procedures were restandardized, with pure speci mens of the methyl esters of linoleic, linolenic, and arachi donic acids, prepared by physical methods, as standards by

Brice and co-workers, 1 9 5 2 ^1^3 ), They recommended an iso 66 merization time of i|_5 minutes rather than 2 5 or 30 minutes, and preferred a glycerol-air technique for general use because of its simplicity and high precision. Ethylene glycol was a second choice because of the greater trans parency of reagent blanks. In very recent studies of the technique by Riemen- schneider and Herb, 1952^*^^, optimum conditions for the production of maximum conjugation of methyl arachidonate comprised heating the sample in 21 per cent KOH-ethylene glycol for 15 minutes at 180°. These conditions when ap plied to methyl linoleate, linolenate, eicosapentaenate and docosapentaenate prepared entirely by physical methods increased the sensitivity of the spectrophotometric method for all except linoleic acid for which the sensitivity was unchanged. In 195>3* these authors (-*-3>5) described a micro method for determining fatty acids containing from two to five double bonds. They compared the results of analyses of fats and oils by the micromethod with those from a standard macromethod, and the results were In good agree ment. Lundberg and Hammond, 1953^*^^, studied especially the alkali isomerization of the methyl docosahexaenoate and the spectral properties of conjugated hexaene fatty acids. The present status of ultraviolet spectrophoto metric methods as applied to fats and oils may be sum marized as follows: they are highly precise for the common polyunsaturated acids of 2-6 double bonds; they provide a Rao and Daubert, 19l).8^ ^ ^ and Swern et al, 1950 ^ ^ , means of determining both conjugated and non-conjugated noted that all trans-monounsaturated compounds show a

fatty acids in mixtures; they are far more sensitive than strong absorption band at 10.36l(,which is absent in the

the thiocyanometric and other methods in detecting small corresponding cis-monounsaturated and saturated compounds,

proportions of non-conjugated acids and, in the range be lio marked differences exist between corresponding trans-,

low about per cent, far more accurate; and they are cis- and saturated compounds in other regions of the infra

capable of high reproducibility in experienced hands. red spectrum. They concluded that this maximum absorption

Finally, as noted previously, they must be applied with at I O . 3 6 i s characteristic of a trans configuration. reservations to hydrogenated fats. Properly applied, they In 19l;9 Lemon and C r o s s ^ indicated the possibility offer important information concerning their structure. of utilizing infrared spectral properties for analytical

(b) Infrared Spectrophotometry. purposes, They concluded that the intensity of absorption

Rasmussen and co-workers in 19lf7^ ^ found that at the peak of the 968 cm"1 band is a measure of the extent a strong absorption band at 10.36 microns is common to of the cis-to-trans change that is associated with the monounsaturated hydrocarbons having the trans configura hydrogenation of unsaturated fatty acids, Swern and co tion at the double bond. In the case of unsaturated fatty workers, 1 9 5 0 ^ ^ , presented infrared absorption data on acids, the absorption band at 968 cm"1* is apparently a number of pure long chain saturated and monounsaturated associated with a trans-configuration, since the same band fatty acids, methyl esters, triglycerides and alcohols, is found in the spectra of unsaturated fatty acids, or Within each class, trans compounds were distinguished their esters, after heating for three hours in the presence readily from cis- and/or saturated compounds. Terminally of 0,3 per cent selenium, which is a well-known method^1* ^ unsaturated compounds were distinguished readily from the of synthesizing an equilibrium mixture cis and trans-isomers. internally unsaturated and/or the saturated types, Their

data constitute the most complete study to date of the

* 968 cm"^ is the wave number; it is the value of infrared spectral properties of fatty acids and esters, 1 ,.. 1- ci-1 or 10.3311 =0 = 968 cm*1. lO.jj x 10=4 /V Their work was extended by Singleton et al, 1951 ^ ^ , to

the absorption maxima of the lower saturated fatty acids,

As a result of these observations, Swern and co-workers, 69 1950^ » se'k UP an infrared analytical method, for the quantitative determination of trans isomers of monoun saturated acids and esters in the presence of cis-isomers and of saturated compounds. Swern et al, 195>0^"^^, con cluded that the infrared method is more rapid, specific and accurate than the lead salt method; it is directly ap plicable to the determination of trans isomers in acid or ester mixtures; and only small samples are required. Simi lar conclusions were made by Jackson and Gallen in 1 9 ^ 1 ^ ^ ^ . Infrared spectrophotometry in relation to fatty acid chemistry was reviewed in 195>0 by Binkerd and Harwood^^^ . Brown et al, 1 9 5 1 ^ ^ * ^ » Investigated the effect of posi tional isomerism on the absorption maxima of several natural and synthetic cis- and trans-octadecenoic acids. They found no difference for the two naturally occurring cis-acids, i.e., petroselenlc and oleic acids. The syn thetic cis-acids showed slightly greater extinction coef ficients, which may indicate either than these compounds absorb more strongly or that the extra absorption at 10.36 Is due to the presence of very small quantities of the corresponding trans-isomers, due to failure to achieve complete stereochemical specificity in Raney-NI reductions. All the positional isomers of the trans-acids had similar maxima except for the trans,-8-octadecenoic acid. Infrared spectra for unsaturated fatty acids lower 70 than the C-^g series have not been reported and little has been published concerning the spectra of the more highly unsaturated f&tty acids. No quantitative absorption data are reported for the polyethenoic acids with more than two double bonds. Jean Lecomte in 1950 and 1951^^ * 1&9* 170) presented an excellent description and discussion of the infrared spectra of the constituents of fats, Wheeler and co-workers, 1952(l52)^ pound that methyl octadecadienoates with isolated trans, conjugated trans- trans, or conjugated cis-trans double bonds were character

ized by different bands in the spectral region from 9.18 to

11.0 < • The 9 8 8 cm”'*’ band characteristic of isolated trans double bonds is approximately additive since linolelaidate has about twice the absorption of elaidate and a cis-trans-linoleate has about the same absorption as elaidate. Trans-trans conjugated linoleate is character

ized by a band at 9 8 8 cm"'*', while cis-trans and trans-cis conjugated linoleates are characterized by two bands at

9J4.8 and 9 8 2 cm”’*', both of them strong and sharp. They noticed that the absorption coefficient of linolelaidate was very nearly twice that of the elaidate and that

cis- 9 9 trans-1 2 -linoleate showed only the 9 8 8 cm"’*’ band and had an absorption coefficient quite close to that of elaidate and very nearly one-half that of linolelaidate. Lemon and Cross, 19^4-9^"^ > found that the isolinoleic acid produced by hydrogenation of linoleic acid 71 has at least one double bond with a trans-configuration.

However, since no increase in the absorption at 9 6 8 cm"'*' results on the isomerization of methyl isolinoleate, they concluded that both double bonds have a trans-configuration* The infrared method is superior to the Twitchell method for the determination of trans components* It handles simple and complex isomers of octadecenoic mix tures with equal ease in view of identical infrared ab sorption for trans isomers of different double bond posi tion. This method does not differentiate between the trans monounsaturated acids and the trans-polyunsaturated fatty acids. The only available method for the determination of the geometrical isomers of octadecadienoic acid is that of Wheeler et a l ^ ^ ^ , in which alkali isomerization is con ducted for three intervals followed by ultraviolet measurement* The values for trans-octadecadienoic acids recorded in this research are empirical due to the fact that no ac curate value for the absorption of linolelaidate is available• Raman Spectrophotometry By means of Raman spectra, it is possible to de termine the strength of bonds between atoms in their normal state and to calculate the frequency and amplitude of atomic vibrations. In some cases it is possible to de termine the spatial configuration. Despite the wide 72 utility of the Raman spectra in th© field of organic chemistry, it has found only limited application in the field of fat and oils. The Raman spectra of only a few of the normal, straight chain fatty acids have been examined. Many investigations have been made with respect to the effect of ethylenic bonds on Raman spectra of organic compounds, and especially the relation of frequency shifts to cis-trans-isomerism, Blanche Gredy, 1 9 3 5 ^ 7*^ , pointed out that cis- and trans-ethylenic isomers could be clearly

distinguished by difference of 1 2 -l£ units in frequency in

the neighborhood of 1 6 0 0 cm“^ characteristic of the double linkage, the cis-compounds having a lower frequency than the corresponding trans-isomer. In a study of the Raman spectrum of some ethylenic compounds of the general formula Et-CH=CH—R and comparison with compounds of the formulas

H-Ch=Ch-R and Ch3 -Ch=Ch-R, she, 1 9 3 7 ^172^, found that the decrease in ethylenic Raman frequency in general depends more on the position of a substituent group in reference to the double bond than on its nature, McCutcheon et al, 1914-1 , applied Raman spectra to an Investigation of the cis- and trans- isomers of the ^18 unsa^ui>a‘^e^ acids. They found that the characteristic ethylenic frequency in ethyl oleate was llf units lower than In ethyl elaidate, and that the Intensity of the line at

3 0 0 9 was approximately twice as strong for ethyl oleate as for ethyl elaidate. Prom the study of the Raman data 73 obtained with alpha- and beta-linoleates, they concluded that these two substances were identical. This conclusion

is in agreement with those of McCutcheon, 1 9 3 8 ^ ^ ^ » of Riemenschneider et al, 1939^^^* and of Kass and Burr,

1 9 3 9 C^76). Elaldinlzation of linoleic acid produced a re versal of both double bonds from the cis- to the trans configuration. On the basis of the Raman and infrared spectra of ethyl llnolenate, they concluded that the double bond configuration was cis-cis-cis. No applications of Raman spectra have been made in the present Investigation.

IV. SOME OBSERVATIONS ON OLEIC» LINOLEIC AND LINOLENIC ACIDS AND SOME OF THEIR ISOMERS CONCEIVABLY PRESENT IN HYDROGENATED PATS

In 19^4^4- Millican and Brown^^^ reported the isola tion of oleic acid from a number of animal and vegetable fats by a combination of fractional distillation and low temperature crystallization techniques. The acid obtained from fats of vegetable origin showed a melting point very

close to that reported for pure oleic acid, 1 3 «0 -1 3 *lf°. Oleic acid prepared from beef tallow and from several other animal lipids melted 3° lower than pure oleic acid. Re- / -1 ry O \ cently, Orians and Brown' have described similar re sults with human milk fat, and Orians^ f £n unpublished work, obtained comparable data on oleic acid from butterfat and was unable to separate and identify the isomers. The 7k abnormal molting points of these preparations were at tributed to the presence of small amounts of geometrical and/or positional isomers of oleic acid. Sixteen positional isomers of oleic acid exist, as follows:

2:3 6:7 1 0 :1 1 34:15

3 =ii- 7:8 1 1 :1 2 15:16

Ip :5 8:9 1 2 :1 3 1 6 :1 7

5**6 9 :1 0 13:34 1 7 :1 8

Each of the above mentioned isomers except the 1 7 :1 8 may exist as two geometrical isomers, cis and trans. Conse quently, there are fifteen trans-isomers possible. Only three of the possible octadecenoic acids have been found in nature: oleic (cis-9 -octadecenoic), petroselenic (cis-6 - octadecenoic) and vaccenic (trans-ll-octadecenoic) acids. Oleic acid is the most widely distributed of all fatty acids, and has been alleged to occur in all natural lipid mixtures. Because many of the theoretically possible octadecenoic acids may be found in hydrogenated fats, the physical and chemical constants of both the natural and synthetic octadecenoic acids are comprehensively reviewed in Tables 5 and 6 . TABLE (5) PHYSICAL AND CHEMICAL CONSTANTS.OF CIS-OCTADECENOIC ACIDS m.p.,of. Setting Method m.p. of diketo- Acid m.p. Neutral,» Iod. Point of dihydroxy stearic Reference °C Equiv. Val. °C Prepn. derivative acid Theory a m mm m 282.5 39.9 .... * —— ...... 2- S9.o -- .... -- Koczy et al.(l83)

50.5-51.0 -- -- ...» Synthesis 106 .0-107.0 -- Myers (184) 126.04m 3- 56,0-57,0 -- mm m mm m Synthesis ...... Eckert et al»{l85) 4- 52.0-53*0 ------Synthesis -- .... Eckert et al.(l85) 5- 1)2,0-1^3.0 .... -- -- Synthesis -- Egorov(a) (186)

6- 30.0-30.2 281.7 88.73 -- Parsley 113.5- -- Brown et al,(l67) seed oil 114*5 * 30.0 ...... Parsley 114.0- .... Hilditch et al.(l87) seed oil 115 .0** 33. 0 - 3 M ...... Ralston (188)

28.5-29.2 ---- Synthesis 121.5 *** -- Lumb & Smith(l89^

2 9 4 --- 89.9 --- Parsley 122.2*** Van Loon (190) seed oil 7- 12.5-13.1 282. £ 89.79 Synthesis 94.0-94*5* 87*0 Brown et al.(l67) 11.8-12.5 282.1 87.70 11.3 Synthesis 95®o-96.o* Huber (45)

8- 22.8-2l),.2 282.9 89.90 .... Synthesis 94.0-94*3* Brown et al*(l67)

22.7-23.3 280.2 88.80 23.1 Synthesis 96 .0-97.0* ---- Huber (45) TABLE (5) (continued) PHYSICAL AND CHEMICAL CONSTANTS OP CIS-OCTADECENOIC ACIDS m.p, of Setting Method m.p. of diketo- Acid m.p, Neutral,1 lod. Point of dihydroxy stearic Reference °C Equiv. Val. °C Prepn. derivative acid 9- 13*3-15.5 "285. £" 89.95 Olive oil 94.0* Brown et al,(lb?T 10.9-11.5 282.9 8 8 4 10*9 Synthesis 93.0-9 4-0* Huber (45)

13.0 ------mm mm mm ^ Olive oil ------Ralston (188)

13.0 --- Olive oil ---- Brown & Shinowara (78) 10- 22*2-22.8 264.8 87.2 21.5 Synthesis 98.0-99.5* Huber (45)

11- 10.5-12.0 88.1 Synthesis 93.o-9t.o* Ahmad, Pumpus 127-128*** & Strong(lQl) 4.5-15.5 282.7 88.5 Synthesis 92-93* Brown et al,(l67)

13.o-ll|-0 281)..3 89.1 12.9 Synthesis 9I4..5 -980O* --- Huber 45)

12- 26 .8-27.6 282.3 87.3 27-2 Synthesis 98.5-97.0 --- Huber 45) 9.8 -1 0 4 ------—------— Grun et al« (192)

------17- 55.5-56.1 282.6 88.7 Synthesis Huber (45) -.■i {&. t -■ Oxidation by Peracetic Acid {*?*) Oxidation by Alkaline KMnO|^ (■£&#) Configuration not proved (a) TABLE (6) PHYSICAL AND CHEMICAL CONSTANTS. OF.. TRANS-OCTADECENOIC ACIDS m.p* of Setting Method m.p. of diketo- Acid m.p. Heutral. Iod. Point of dihydroxy stearic Reference °C Equiv. Val. °C derivative acid Theory 89-9 ------

2- 57.0-57-5 — --- Synthesis 125.0-125.5** — — Myers (181|.) 101.0-102. 0a*a 3- — - — — ———— v n «■«! — k ------— —— “— — ------

5 - 47.5 ------— ------Posternak(l93)

6— 53.0 — — Isomerization 121.0-122.0*--- Hilditch & Grif fith (19!^) 52.7 Isomerization 117.2*** --- Steger & Van Loon (195) 52.S-53.0 281.9 89.22 Isomerization 121.0-121.5* --- Brown et al.(l67) 52.0-53.0 ...... Isomerization 116.5*** --- Lumb & Smith(l89) 7- 44«2-44«5 281.6 88.72 Isomerization 130*5-131.0 87.0 Brown et al.(l67)

45.5 ------Synthesis 86.5 Posternak (193)

51 .0-52.0 ------— ------Isomerization ------Vanin et al0(l9o)

52.0 ------Synthesis - - ■— PIgulevskii et al. 1197)

43.5-44.5 282.8 87.9 43.6 Isomerization 132-133* — — Huber (45) TABLE (6) (continued) PHYSICAL AND CHEMICAL CONSTANTS OP TRANS-OCTADECENOIC ACIDS m.p. of Setting Method m.p. of diketo- Acid m.p. Neutral* Iod. Point of dihydroxy stearic Reference °C Equiv. Val. C Prepn. derivative acid 8-^ 53.0 synthesis — -- — - Arnaud & i^oster- nak( 198) 50.5-50.7 282.1 90*0 isomerization 118-119 92.5 -93*.0 Brown et al.(l67)

51.5-52*3 282.3 88.3 51.3 Isomerization 118-119 --- Huber (1+5)

5 2 4 - 5 3 4 Prom partially -— - --- Benedict & Daubert hydrogenated (22) triolein 9- 4 . 5 4 5 . 5 279.2 88.6 lj3.ii- Isomerization 129.0-131.0* — — Huber 45)

14*5 ------— ------Ralston (188) 14*0 ------I3O.O-130.5 * --- Brown et al.(l67) ii5*o45*5 ------Synthesis — — — - Thomas et al.(199) 10- 1*2.0 ------Boeselenet al.(200)

52 .0-52.6 279.7 86.2. 51.2 Isomerication 120.0-121.0*— Huber (1+5) li- lj.3.5-14*5 28i|..0 88.7 1+3.3 Isomerization 129^130* --- Huber (1^5) lj.3.0-14.0 --- 87.3 --- 93.0-91+.0***--- Ahmad et al.(2Cl.)

I1.3.6 -I4 .0 282.5 88.33 --- Isomerization 127.5-128.0* Brown et al.(l67) TABLE (6) PHYSICAL AND CHEMICAL CONSTANTS OP TRANS-OCTADECENOIC ACIDS m.p. of Setting Method of m.p. of diketo- Acid m.p. Neutral. Iod. Point . Prepn. dihydroxy stearic Reference °C Equiv. Val. °C derivative acid & l|2.5 --- —- --- Bertram method------Rao 2c Daubert(l59 ) 45<>o-ij.5»5 — - — -- --- Synthesis Thomas et al•{199) l^.^-l^.o ------——- Synthesis 126.5-128.0# --- Walter & Thomas . (202) . 12- Ij2 .5-I1.3.0 279.2 86.9 51.5 Isomerization 119-120# ---- Huber (i(5) 39®7-i}.0.1 ------Thomas et al.(199i

Oxidation by Performic acid (#) Oxidation by Peracetic acid (##) Oxidation by Alkaline Permanganate (««*) 80 Linoleic acid has been unequivocally identified as the principal octadecadienoic acid present in plant and animal fats and possesses the cis,cis structure. When two

or more double bonds are present in an unsaturated C^q fatty acid the opportunity for the occurrence of positional and geometrical isomers is greatly increased. Although no such isomer has been isolated from plant fats, the exist ence of such isomers has been inferred, Frankel, Stone- / p-| \ burner and Brown, 19^4-3 isolated linoleic acid from olive oil by low temperature crystallization and stated that it contains 19 per cent to i^O per cent of these isomers, Cornwell^®^ inferred the presence of 1 .2 per cent of trans,trans-octadecadienoic acid in summer butterfat^c^* also (2))^ Isolinoleie acids, both positional and geometrical, may be found in hydrogenated soybean and cottonseed oil as a result of rearrangement of the double bonds in the original linoleic acid therein and also as a result of partial hydrogenation and rearrangement of the double bonds in linolenic acid* 81 V. OBJECTIVES AND GENERAL RESULTS OF THE PRESENT INVESTIGATION

The objectives of this investigation are: 1. To investigate the nature of the unsaturated fatty acids in typical margarines and shortenings as manufactured In the United States.

2. To determine the content of trans-acids in these products•

3. To study the distribution of trans-acids In the several carbon series of hydrogenated fats.

J^. To study In detail the nature of the trans-acids In the

Ciq fraction of a typical margarine.

£. To identify some of these trans-acids, If time permits.

With these objectives in mind, we purchased on the market six representative margarine samples and five shortenings. These were examined by the usual analytical procedures. Unsaturated acids were determined spectrophotometrically In shortenings and margarine fats. The trans acids v/ere estimated both as triglycerides and as methyl esters by the infrared spectrophotometric method. It was necessary to develop new Infrared reference values for several pure acids and esters to standardize the procedure and the IR spectrophotometer used In the present 82 work; these standards are described later. The triglycerides of a shortening and of a margarine were converted to their methyl esters by methanolysis; the methyl esters were fractionally distilled, and the fractions analyzed chemically. Each of these fractions was examined by the infrared spectrophotometrie method. The C^g fraction of the methyl esters of one margarine fat was resolved by low temperature crystallization by two schemes. The use of the ultraviolet spectrophotometric method was evaluated by comparing its results on the low temperature crystallization fractions with those calculated from the iodine values and weight fractions. As a result of this work, we have shown that margarines and shortenings with one exception contain from 2 2 .6 9 "to

14-1*73 per cent ti>ans glycerides calculated as trielaidin. The exception was a blended shortening which was slightly hydrogenated and was found to contain only a trace of de tectable trans glycerides. The data obtained on the fractions resulting from fractional distillation of the methyl esters of one of the margarines, M£, and the resolution of the C^q esters by low temperature crystallization indicate that hydrogenated fats contain highly complicated mixtures of fatty acids. In addition to the usual naturally occurring fatty acids, several positional and geometrical octadecenoic and octa decadienoic acids are present. The positional Isomers of 63 the octadecadienoic acids probably consist of three types according to the diene system present in the carbon chain: a) conjugated acids, b) acids with diene systems in which the two double bonds are separated by one methylene group, and c) acids with diene systems in which the two double bonds are separated by more than one methylene group. Each one of these acids may occur in one or more of four geo metrical forms; cis,cis-, cis,trans-, trans, cis- and trans, trans-. In addition, evidence is presented for the presence of trans hexadecenoic acids in one or more forms. Time has not permitted further resolution of the complex mixture of acids which occur in these important foods•

VI. EXPERIMENTAL PART

A. Analytical Methods. Saponification Number Saponification numbers were determined by refluxing 3-5 gm of margarine or shortening with 50 ml. of 0.5 N alcoholic potash solution for 1 /2 hour, then titrating back with 0*5 N HC1 solution to a phenolphthalein end point. In all cases duplicate determinations and two blank titrations were made. Saponification Equivalent (Mean Molecular Weight) The saponification equivalent of methyl esters was % determined by the same procedure as the saponification number but with the appropriate calculation* The mean molecular weight of the constituent acids of methyl esters was calculated by subtracting lij. from the saponification equivalent. Iodine Value Iodine values were determined according to the Official and Tentative Methods of The American Oil Chemistfs Society- 0*2N Wij's solution was used In these determina

tions* The weight of samples ranged from 0 .2 -0 *I{. gm. The reaction time was limited to one hour. Ultraviolet Spectrophotometric Examination The ultraviolet spectrophotometric method of the American Oil Chemist's Society was used for the determina tion of polyunsaturated acids with certain modifications of the official equations as suggested by Brice et al, 19^5^^* Because of its Importance in the present work, a full de scription of the method follows. Apparatus and Reagents Measurements were made with a model D U Beckman photoelectric quartz spectrophotometer equipped with a hydrogen lamp. The fused silica absorption cells were

I4.8 mm high and had 10 mm light paths and ground glass stop pers • Isomerization was carried out in a constant tempera ture bath similar to the one recommended by the Official 85 (75) Method. The bath temperature was maintained at 180* 0 .1 °. A glass manometer was used to regulate the nitrogen pressure during the isomerization. The nitrogen gas was purified by passing it over copper turnings heated to

3 0 0 -3 2 5 and then through concentrated sulphuric acid to get rid of oxygen and moisture respectively* Isooctane ,?Spectro Grade" (Philips Petroleum Company) was used as solvent for determination of conjugated con stituents originally present in the sample and methanol (Baker and Adamson, reagent Grade) for determinations of conjugated acids in the isomerized products* Whenever possible, the dilutions were adjusted so that the observed absorptions were between 0*1 and 0 *8 * The specimens were isomerized in a 6.5 per cent solution of KOH in ethylene glycol. This reagent was pre pared by adding 60 gms. of KOH, A.C.S. grade, to ethylene glycol, Eastman No* 133* and heating the mixture under dry-oxygen free nitrogen to a temperature of 190o . The solution was allowed to cool under nitrogen and its nor mality was checked by titration. The solution, kept in the refrigerator (7°) under nitrogen, remained without sig nificant change for 7 - 8 weeks. Small Pyrex glass cups

10 mm in diameter and lij. mm high were used for weighing and introducing samples into the reaction tubes. Procedure a) For Conjugated Polyunsaturated Acids Originally Present in Sample. 86

100 mgms« of sample was accurately weighed and the cup and sample Introduced into a 100 ml glass-stoppered volumetric flask® The sample was dissolved In isooctane and the solution wa3 made up to the mark. Spectral density readings v/ere taken at 233, 262,

2 6 8 , 2 7 l\., 3 1 0 , 3 1 6 , and 322 nn^, the original solution being diluted if necessary so that the observed densities lie between 0.2 and 0.8. The observed optical densities were recorded against the Isooctane blank. Values obtained thus before alkali isomerization measure the conjugation present in the original sample. With natural lipids these values are very low. b) For Non-conjugated Polyunsaturated Acids. 11.0-0.1 gm. of the KOfi-ethylene glycol solution was weighed into a 10,f x l” Pyrex test tube and main tained at 1 8 0° for 20 minutes under an atmosphere of nitrogen. The glass cup containing the 100 mgm sample was then dropped Into the test tube and swirled vigorously for a few seconds, after which the tube was returned back to the bath. After the addition of the samples, heating was continued for 25> minutes. The resulting material was then quantitatively transferred to a 100 ml. volumetric flask and made up to the mark with methanol. Two blank determinations v/ere run with each series of Isomerlzations. Spectral density measurements were made at the wave lengths specified In the analysis for conjugated constituents. 87 Calculations The specific extinction coefficient, K, is calcu lated for each, wave length according to the formula: K - D where "b x c D = observed spectral density b = cell length in cm, c = concentration in gms/liter

The resulting K-values are designated Kggg* ^ 268 and

Kg^.6 * etc . (a) For Conjugated Constituents Originally Present* The specific extinction coefficient at 233 mi^, Kggg, was corrected for absorption by COOR and -C=C- groups, as suggested by Brice et a l ^ ® ^ .

K2 = Kggg " 0 o07 ...... «(For fats and esters)

The specific extinction coefficient at 268 ^2 6 8 * was corrected for background absorption as follows: K 3 = 2.8 [ kz66 - 1/2(K262 -t- K27k)J

The specific extinction coefficient at 316 Kg]_£, corrected for background absorption is:

|/^K3l6 “ 1//2^K310 K 3 2 2 ^J Conjugated diene, % - Cg = 0*87 Kg

Conjugated triene, % = Cg = 0*lj.7 Kg

Conjugated tetraene, % = = o.k-9 Kh

(b) For Non-conjugated Constituents* The specific extinction coefficient after isomeriza- 88

tion, at 233 rm^, Kl2 3 3 ’ was co^rected. for the amount of diene acids originally present by this relationship, re presenting non-conjugated diene material in the sample:

K ,2 “ K * 233 “* K233

The specific extinction coefficient at 268 mi^, K*2 68 * non-conjugated material in the sample was corrected for background absorption and for undestroyed conjugated triene:

K '3 = I4..I [ > > 2 6 6 .- 1/2 (K> 2 6 2 + K'27l^)3 -K3

The specific extinction coefficient at 3 l6 mi^, k '3 i6 ’

tetraene, was corrected for background absorption and for undestroyed conjugated tetraene by the following calculation: K'^ = 2.5 / k > 3i6 - 1/2(K»310 -+ K'322) J -K^

From the above:

Linoleic acid, ^,(X) = l.l6 k 1 - 1.33K' ^ + 0.09

Linolenic acid, - 1 .8 8 K'^ - i|..i|.3 K'^

Arachidonic acid, %,(Z) - .1^3 K

I.V. of Sample - 1.8ll(C2 -tX) + 2 . 7 3 7(0-3 + Y) +3.337(C^^Z) 0.899 Saturated acids, % - % total fatty acids - oleic acid + % conjugated acids + % non-conjugated acids) The total fatty acids in most natural oils is 95*6 per cent; for methyl esters 95*2 per cent. 89 To calculate to a Tatty acid basis, multiply the percentage value obtained by 100 / % total fatty acids. Examination by Infrared Spectrophotometry. The infrared technique is so important to the present investigation that it is discussed below more fully than the other analytical methods. (1). Introductory Comments. In fat chemistry, Infrared spectrophotometry has al ready become an Indispensible tool. Swern and co-workers^ adopted the method to the quantitative determination of trans fatty acids and compared briefly their results with the Twitchell lead salt method, with striking confirmation of the previously recognized failure of the Twitchell method to detect all of the trans-acids present. The Twitchell method was shown^ to have fair accuracy for simple trans-acid mixtures but gave very low values (error of 35 to I|.5 per cent) with hydrogenated fat3 . (2) Procedure. In the present work, Infrared examination of the hydrogenated fats and esters prepared from them was carried out according to the method of Swern et al, 1950^^^ . A Beckman IR-2 spectrophotometer was employed. This instrument, housed in an air-conditioned room, was maintained at 2 5 -1 ° by a constant temperature circulating bath supplied with the instrument. All measurements were made in a liquid cell, which consisted of two rock salt 90 windows separated by an amalgamated lead spacer* The cell thickness was 0 ,1 0 6 cm. An approximately £0 mg. sample was weighed accur ately into a 10 ml. glass stoppered volumetric flask and diluted to the mark with pure CS2 * 3n general, the total concentration was adjusted to give a solute optical density between 0 .2 and 0 .6 . (The CS2 used was chemically and optically pure, I.e., it should have the highest trans mission to the infrared radiation obtainable.) The solutions were allowed to stand In the tightly stoppered flasks for at least one hour at 25>-l°. All measurements were made under these conditions. A portion of the solution was Introduced Into the absorption cell by a hypodermic syringe, and the optical density of the solution plus cell at exactly 1 0 .3 6 • was measured by the null method. The optical density at

1 0 .3 6 ^. of the cell filled with CSg was then measured under Identical conditions, and the difference was taken as the absorption due to the solute. The optical density difference was then used to calculate the extinction coef ficient, E, by the formula E = d , where d Is the c 1 optical density, c is the concentration in grams per liter, and 1 is the cell thickness in centimeters. All readings were made with a slit width of O.lj.00 mm.

(3 ) Calculation of Results. The following equations were used in calculating the weight percentage of trans components In mixtures: 91 a) Mixtures containing cis and trans octadecenoic compo nents only. Trans component, weight^ = ^ ^ K T - K c ! where K0^s observed extinction coefficient for the mixture. K extinction coefficient of pure cis compound, o extinction coefficient of pure trans compound. b). Mixtures containing trans-octadecenoic and saturated (mainly) components only. Trans component, weight^ “ 100 - Kg) (2) kT - Ks where is the extinction coefficient of the pure saturated compound. c) Mixtures containing cis- and trans-octadecenoic and saturated components.

Trans component,weight^ = IOOCKq-^q -KcY - KSZ) ...... (3) IKT - K c} ! where Y is the total weight fraction of octadecenoic components (cis plus trans), and 2 is the weight fraction of saturated components. Y is calculated from the iodine value of the mixture and Z is obtained by difference. (Ij.) • Discussion of Factors Affecting Values in Preceding Calculation.

Swern et al, 195 >0 ^ ^ ) , and Brown et al, 1951^ -^7), pointed out that a shift in the position of the double bond in either the cis or the trans-octadecenoic acid pro duced little or no change in the extinction coefficient. 92 Therefore, even though the exact structures of the trans- and cis-octadecenolc acids in a mixture are uncertain, the measurements made in this investigation were valid* In the present work, it was assumed that the specific extinction coefficients of elaidic acid and methyl elaidate were sub stantially the same as those of any of the isomeric trans- octadecenoic acids or esters, respectively which seem likely to occur in the mixtures under investigation* It may be stated further that extinction coefficients for the various cis-octadecenolc and saturated acids studied are all approximately the same. This relation also holds for the cis and saturated glycerides and to a somewhat lesser degree for the cis and saturated methyl esters. Some of the crystallization fractions described later possessed iodine values Indicating the presence of polyunsaturated components, mainly diene (linoleic) acid, Wheeler and co-workers, 1 9 5 2 ^ ^ ^ , showed that linolelaidate has about twice the absorption of elaidate and a cis-trans-linoleate has about the same absorption as elaidate. By assuming that all polyunsaturated compounds in these high Iodine value fractions were cis-trans linoleate or cis-cis-linoleate and by further assuming that this cis-trans isomer had the same extinction coef ficient as methyl elaidate, it was possible to make a very loose estimation of the total percentage of trans compo nents in these highly unsaturated fractions. In these 93 Tractions the weight per cent trans component reported consists of the trans-octadecenoate and the cis-trans- octadecadienoate* The precision of the infrared method is to a degree dependent on the magnitude of the extinction coefficient obtained, Cornv/ell^®^^ • Materials with extinctions less

than 0*100 can be analyzed with a precision of -I4..O per

cent; material with extinctions of above 0 *3 0 0 , with a precision of -0*7 rcr cent. In this investigation, the total concentration of the solution was adjusted where

possible to give solute optical densities between 0*300

and 0 .6 0 0 . Due to variations In such factors as scattered radiation, wavelength calibration, slit width settings and accuracy of cell-thickness measurements, it is necessary to determine reference extinction coefficients on the In strument being used under the exact conditions to be em ployed In the analysis.

B. Reevaluation of Reference Standards on Pure Fatty Acids, Methyl Esters and Triglycerides.

The extinction coefficients of fatty acids and their esters, reported in the literature^* 1 6 2 , 163 and 1 6 7 )^ cannot be employed directly In this investigation due to variable factors In the spectrophotometer to be used, as noted above* The effect of these factors is quite obvious 9^ when one compares the reference values cited in the litera ture, Table 7* where these values were determined in several laboratories. It was necessary, therefore, to es tablish reference values of pure compounds with the in strument used in the present Investigation. The Infrared instrument was standardized by using it to determine the k-values of pure fatty acids, methyl esters of pure acids and pure triglycerides. Values for the acids are Included, although they were not actually used later to analyze mixtures, mainly because pure speci mens were available at the time, and it was thought that the values would be useful in later investigations. Description of Pure Reference Materials The reference compounds employed, some of their characteristics, and methods of preparation are shown in

Table 8 . The K values for palmitic and stearic acids are slightly higher than those reported by O'Connor et al^*^^. This may be attributed to their use of chloroform as a solvent which does not give good results In comparison with carbon disulphide used In this vvork^®^^ , TABLE (7) 95 EXTINCTION COEFFICIENT AT 10,36 MICRONS FOR PURE CIS AND TRAMS MONOUNSATURATED AND SATURATED ACIDS, METHYL ESTERS AND TRIGLYCERIDES Extinction Coefficient C ompound Published Determined in ______this laboratory A) FATTY ACIDS

Caproic acid 0 .2 3 (1 6 2 ) Caprylie acid o.i6 (1 6 2 ) Capric acid O.llj. (1 6 2 ) Laurie acid 0.13 (1 6 2 ) 37332 AFM Myristic acid 0.12 (1 6 2 ) 0 .1 1 7 AFM Palmitic acid 0 . 1 1 (1 6 2 ) 0.135 AFM 0 .1 2 9 (1 6 3 ) Hexadecenoic acid 0 .1 7 8 AFM Stearic acid (1 6 2 ) 0 .1 3 6 AFM 0*123 (1 6 3 ) 0.136 (2) Cis-6 -octadecenoic O.llJ. (167) 0 .1 2 9 (163) Cis-7-octadecenoic 0 .1 5 (167) Cis-8-octadecenoic 0 .1 7 (167) Cis-9 -octadecenoic 0 .1 3 3 (163) RHB o . i k 3 (2 ) 0 . 1 k (3.67) Trans-6 -octadecenoic 3.67) O.ij.6 (167) o.56o (163) Trans-7-octadecenoic o.ij.8 (3.67) Trans-8-octadecenolc 0 4 3 Trans-9 -octadecenoic 0.552 i t S 6 .3 6 0 AFM 0.552 (2) 0.582 RHB 0.k7 Trans-11-octadecenoic o.ii-9 w

Linoleic acid 0 *175 * (2 )

B) METHYL ESTERS Caproate 0 .0 8 (1 6 2 ) Caprylate 0.05 Caprate 0.07 m i Laurate 0* 0 4 (1 6 2 ) THTJiPi. AFM Myristate o.oli (1 6 2 ) 0 .045 . AFM Palmitate 0 .0 3 (1 6 2 ) 0 .0 2 9 AFM Stearate 0.028 (1 6 3 ) 0 .034. AFM 0.03 (1 6 2 ) TABLE (7) (continued) 96 EXTINCTION COEFFICIENT AT 10.3 6 MICRONS FOR PURE CIS AND TRANS MONOUNSATURATED AND SATURATED ACIDS, METHYL ESTERS AND . TRIGLYCERIDES Extinction Coefficient Compound Published Determined in ______this laboratory

Cis-9 -octadecenoic 0 .0l*.l (1 6 3 ) o.olji*. RHB 0.01+9 (2 ) Trans-9-octadecenoate 0 .1ji+2 (1 6 3 ) 0 .9 2 3 RHB 0.911 (2 ) Trans-6 -octadecenoate 0 .1+91*. (163) Linoleate 0 .0 6 6 (2 )

C) T R I G L Y C E R I D E S Trimyristin 0.087 (163) Palmitostearin 0.087 (163) Tristearin 0 .0 8 8 (167) 0 .0 6 2 AFM Triolein 0 .081+ (163) 0 .1 0 6 AFM 0 .0 1 0 6 (167) Trielaidin 0.1+79 (163) 0.963 AFM 0 .9 2 8 (167)

Reference (162) reported the extinction coefficient at 1 0 .Ij.0 microns, using chloroform as a solvent. Triolein, trielaidin, and tristearin were kindly supplied by Dr. Daniel S w e r n of The Eastern Regional Research Laboratory. Linoleic acid prepared by debromination was supplied from Horrael Institute and was purified by repeated crystalliza tion from petroleum ether by Mrs. Doris Kolb. The extinction coefficient values for oleic and elaidic acids and their methyl esters were determined by Mr. R. H. Backderf* TABLE (8) 97 CHARACTERISTICS OP REFERENCE COMPOUNDS Reference 171 o *p * I.V. Method of preparation Compound °C Wi,1s M.M.W, and purification Fatty Acids Laurie 44*0 0.00 250.3 Prepared by Foreman and then crystallized twice from acetone at 0° Myristic 54*3 0.0 o 228.4. Prepared by Sheer and crystallized tv/ice from acetone at 0° Palmitic 6 2 .8 0 .0 0 2 5 6 .3 Prepared by Foreman, purified by crystallization from acetone twice Hexadecenoic _____ 99*80 254*^ Prepared by F.A.SUii^h

Stearic 6 9 .5 0.0Q 284*5 Purified by low tempera ture crystallization from acetone at -20°G

Oleic 13.3 8 9 .9 8 282.5 Prepared by Clemmons from olive oil Elaidic 43.6 89.85 282.5 Prepared by isomerizing oleic acid by Backderf

Methyl Esters Laurate 0.50 214.4 Prepared by Foreman. Purified by fractional distillation Myristate 0.01 242.4 Prepared by Foreman Palmitate 0 . 0 0 270.0 Prepared by Foreman Stearate 0.00 298.5 Prepared from human milk fat and purified by crystallization at 0

Oleate 85.60 2 9 6 .5 Methanolysls of oleic acid, Backderf Elaidate 85.60 2 9 6 . 5 Isomerization of methyl oleate,Backderf 98 C. Ultraviolet Infrared Spectrophotometric Examination of Six Typical Margarines and Five Shortenings. The present investigation attempts to supply sys tematic information on the distribution of the unsaturated fatty acids in hydrogenated shortening and margarines. These products are manufactured in America mainly from cottonseed and soybean oils. Their composition has been studied by a number of investigators but little reliable information was obtained due to the analytical methods used. Andrews and Richardson^^ reported that the per centage of glycerides derived from linoleic acid decreases in the following order• shortening, lard, margarine and butterfat. Recently, Richards et al*^- and examined spectrophotometrically a sample of shortening and of

margarine and found II.6 I4. per cent linoleic acid in the former and 13.^7 par cent in the latter. In previous in vestigations no differentiation between the conjugated and non-conjugated dienes were reported. No specific data are available on the amounts of trans acids present In products of this type. (l) Description of Specimens of Margarines and Shortenings. The specimens used In the present investigation were purchased at random on the market. A n analyses of shorten ings were made on samples as received. Each margarine sample was melted and the water layer was siphoned off. Then the fat layer was dehydrated under vacuum, filtered to remove the salt and curd and stored in an atmosphere nitrogen at -2 0 °. General information on the several specimens of margarines and shortenings is given on Table 9«

(2)• Eesults of Infrared and Ultraviolet Analysis, The eleven specimens described in Table 9 were analyzed for saponification number and Iodine value and for trans glycerides by the infrared method described

previously. The results are shown in Table 1 0 , TABLE (9) 100 DESCRIPTION OF SHORTENINGS AND MARGARINES

Sample Brand Company Origin of Fat

Shortenings si Crisco Proctor & Gamble Vegetable oils S2 Spry Lever Brother Co* Vegetable oils Swift’ning Swift & Company Meat fats & vegetabli s 3 fats* Durkee * s Durkee Famous Foods Vegetable fats % Food Club Topco Assoc. Inc. Vegetable fats sS

Margarines

Ml All Sweet Swift & Company Cottonseed 3c Soybean oils m 2 Parkay** Kraft Foods Co. Soybean Sc cottonseed oils Good Luck Lever Brother Co. Soybean & cottonseed M3 oils Dixie The Capitol City Cottonseed & soybean Products Co. oils Mg Nu-Mad The Miami Cottonseed and soy Margarine Co. bean oils m 6 ParkayJKs- Kraft Foods Co. Soybean 3c cottonseed oils

* The unique composition of this material Is due to the fats from which It Is manufactured. ■JHfTwo specimens purchased at different times. 101 TABLE (10) EXAMINATION, INCLUDING INFRARED, OF SHORTENINGS AND MARGARINES

Sample Sap. Iod. K Trans Components No. Val. as Trielaidin .%____ Shortenings

S1 191.2 7 6 .7 9 0 *2014. 22.69

s2 1 91.1*. 7 5 .4 o 0.258 3 3 .4 1 196.1 0.097 00.43 S3 5 9 *4° 192.9 73*67 0.272 36.62 S4 0.264 s 5 196.3 7 2 .5 5 34*90

Mx 19 3*1 7 5 .5 3 0.268 3 5 .5 1

m 2 193.2 81.36 0.272 3 7 .1 1 m 3 192.3 7 8 .6 7 0.260 34.77

M4 192.2 7 4-1*9 0.251 32.93 1 9 1 .1 8 4 .7 7 0.293 4 1 .7 3

m 6 192.3 80.26 0 .2 5 4 3 3 .1 5 102

From Table 1 0 , it is noted that the saponification numbers of margarines and shortenings range from 191.1 to 1 9 6 .1 * These data support the view that the basic oils used in the manufacture of these two products are cotton seed oil (Sap. No. 191-196 ) and soybean (Sap. No. 189-19^-) oils. If any of the coconut products (Sap. No. 2^1-265 ) is used as one of the ingredients in the manufacture of hy drogenated fats, an increase in the saponification number will be observed. Margarines and shortenings have high iodine values (7 2.55 ~Ql|-«7 7) hut are solid at room temperature. This is due to the presence of high-melting trans-monounsaturated acids (m.p. . With one exception, the shortenings and margarines contain high percentages of trans acids (2 2 .69 -^-l«73) ♦ These trans acids are responsible for the plasticity, solid texture and keeping quality of these pro ducts. The exception, S3, is manufactured by blending animal and vegetable fats with a minimum of hydrogenation* Its composition more nearly resembles a natural fat than any of the products described in Table 1 0 . Results of ultraviolet spectrophotometrlc examination are given in Table 11, the absorption values before and after alkali Isomerization having been obtained by the procedures described previously. Before isomerization, absorption Is due to conjugated systems present In the original specimen. After alkali isomerization, absorption TABLE (11) ULTRAVIOLET SPECTRAL ANALYSIS OF SAMPLES OF SHORTENINGS AND MARGARINES

Fatty Acids > Conjugated Acids /o Sample Iod* Satura Oleic Linoleic Linolenic Arachi - Liene Trione Tetraene Val. ted * * donic

S1 76.79 23.696 63.895 11.802 0.437 0 . 0 0 0 0.168 0.004 0 . 0 0 0 s2 7 5 4 0 16.562 79.238 3.937 0.029 0 . 0 0 0 0.234 0 . 0 0 0 0 . 0 0 0 s3 5 9 4 0 44.943 42.513 11.339 0.539 0.450 0.210 0.004 0.002

73.67 19.970 74.639 0.198 0 . 0 0 0 0.806 0.012 0 . 0 0 0 % 4.375

21486 72.880 0 . 0 0 0 0 . 0 0 0 s5 72.55 4.701 0.165 0.755 0.013

H i 75.53 17.618 76.942 4.332 0.173 0 . 0 0 0 0.421 0.04 0 . 0 0 0 m2 81,36 15.211 75.628 8.105 0.561 0 . 0 0 0 0.488 0.007 0 . 0 0 0 -rf" O C O O 78.67 20.550 ©. 10.636 O.I46 0 . 0 0 0 0.351 0.013 0 . 0 0 0

% 7449 23.036 66.326 10.084 0.069 0 . 0 0 0 O .476 0.009 0 . 0 0 0

84.77 15.592 71.303 10.753 0.367 0 . 0 0 0 1.471 0.016 0 . 0 0 0 h 6 80.26 15.196 75.560 8.207 0.479 0 . 0 0 0 0.547 0.009 0 . 0 0 0 103

* Values for 2-, 3- and [{.-double acids are stated as linoleic, linolenic and arachidonic acids, respectively, but the method is not specific for these acids. 10i|. at 233 and at 268 is increased, the increase being calculated as linoleic and linolenic acids respectively. The calculations shown in Table 11, including results for oleic and saturated acids, are not highly accurate, being offered only as an approximation indicating the order of magnitude of the values for fatty acids more unsaturated than oleic acid. Apparently this is the first time this method has been applied in detail to the determination of the composition of shortenings and margarine. Values for triene conjugated acids in Table 11 are negligible; those for diene conjugation range from 0,l68- l,!f71 per cent. With one or two exceptions these values are no higher than those for cottonseed and soybean oils, described later in Table 1 3 * Apparently, therefore, very little or no conjugation is introduced into these products during hydrogenation. Values for linolenic acid range from 0 ,029-0.8 6 7 per cent. Linoleic acid values are 3 *937-11 • 802, avgs, 7,321 per cent for shortenings; and J4..832 to 10.753» avg., 8*769 per cent for margarines. It is to be noted that the linoleic acid values are actually for total octadecadienolc acids of the type which will isomerlze to diene conjugation under the conditions of the analysis. D) Spectrophotometric Examination of Miscellaneous Eats. Shortenings and margarines contain high percentages of trans fatty acids as well as small amounts of conjugated unsaturated fatty acids. For purposes of comparison, a further study of the spectral absorption of some vegetable fats used in the manufacture of shortenings and margarines (cottonseed, soybean, peanut, coconut, corn and palm oils), and of lard, olive, and lettuce seed oils seemed likely to yield useful information. Data on human fat and human milk fat are also included because of the writer’s interest in the procedure and because specimens were available. Many of these fats have not been previously examined by these methods. The data are given in Tables 12 and 1 3 * io6 TABLE (12)

INFRARED EXAMINATION OF MISCELLANEOUS OILS AND FATS

Tpans Component Sample Iod. K Calculated as Val. Trielaidin & ______Cottonseed Oil 112.£ 3 O.lOlj. 0. 6)4.2 Corn Oil 12i|_* 32 0.107 0.000 Coconut Oil 68.90 0.113 2.570 Lettuce Oil 127.18 0.102 0.000 Palm Oil Neutralized I4.5.60 0.1014. 2.398 Palm 011^ African 5 2 .5 6 0.088 2.114.1

Peanut Oil 9 3 .5 8 0 .0 9 3 0.000 Olive Oil 62.90 O.lli-3 9.14-22 Soybean Oil 128.14.6 0.101 0.000

Lard 67.38 O.90 0.000 Human Milk Fat 61.29 0.129 7.281 Human Depot Pat 69.114. 0.112 2 .9 9 8 TABLE (13) ULTRAVIOLET ANALYSIS OF MISCELLANEOUS OILS AND FATS

T a t W Acids jo . . " . . ^ Sample iod* Satura "Oleic" Linoleic Linolenic Araciii- - Biene Triene I’etraei Val* ted Acid Acid Acid Acid donic Acid Cottonseed oil 112.53 '20.118 " ' 29*945" w .143 0.309 0.000 0.414 0.071 676oo'

Corn oil 124.32 6.588 44*017 itf.733 O.909 0.000 0.566 0.187 0.000 Coconut oil 68.90 20.531 78.780 O.460 0.000 0.000 0.229 0.000 0.000

Lettuce seed 127*18 4*549 32.229 62.568 0.0,15 0.000 0,631 0.008 0.000 oil Palm oil, 45*6o 54*771 37*797 6.429 0.278 0.000 0.724 0.001 0.000 Neutralized Palm oil, 52*56 51*111 37.162 11.068 0.354 0.000 0.303 0.002 0.000 African Peanut oil 93*58 21.950 47*915 29.613 0.253 0.000 0.266 0.003 0.000

Olive oil 62.90 30.167 67.147 1.003 0.597 0.000 1.075 0.011 0.000

Soybean oil 128.46 17.927 23.801 49.399 7.842 0.000 0.834 0.197 0.000

Lard 67.38 34*560 53.034 11.882 0.116 0.196 0.170 0,002 0.000 Human milk fat 61.29 43*272 44*886 9.460 0.761 1.052 0.567 0.001 0.002

Human depot fat 69*14 33.073 56.799 6.974 0.912 1.358 0.884 0.000 0.000 108

It is quite clear that the vegetable fats in Tables 12 and 13 with the exception of coconut, palm and olive oils are practically devoid of trans acids. The 0 *61j.2 per cent of trans glycerides'in cottonseed oil is considered to be questionable, perhaps within the experimental error* The trans component values for palm, coconut and olive oils are significant. Previous work in this laboratory on olive oil has Inferred the presence of isollnolelc acids of the cis,trans-type in this oil. Lard contains no trans components while human depot fat contains 3 per cent and human milk fat 7*3 pe** cent. The high content of trans acids in the latter two fats may be the result of the dietary Ingestion of margarines and shortenings. E) Preparation and Examination of Mixed Methyl Esters of Selected Specimens of Margarine and Shortening. One specimen each of margarine and shortening was converted to methyl esters which were to be examined di rectly and later fractionally distilled. Methyl esters were prepared directly from the fats by alcoholysis. The specimen, about 1000 gm., was re fluxed for forty-eight hours with 2000 ml. methanol con taining 2 per cent dry hydrogen chloride. The resulting esters were then washed several times with distilled water. The water layer was siphoned off and the final separation was carried out in a separatory funnel. The last traces of water and alcohol were removed by warming 109 in a 5-liter round bottom flask under reduced pressure. The crude methyl esters were then distilled under reduced pressure (ca. 1-2 mm. Hg) in an all-glass Clalsen distilling apparatus. By this method a 1015 gm. specimen of (sap, no., 1 92.3 ; I.V., 8 0.2 6 ; content of trans material calculated as trielaidin 33.2 per cent) gave 1000 gm. crude methyl esters: sap. equiv., 290*5; iod. val., 78*50; and K, 0.2l6* The crude methyl esters were distilled at 2 -3 mm. Hg pressure, the greater part coming over between 1926-216°; yield, 975*0 gm.; sap. equiv. 2 9 0 .8 , I.V. 7 8 .91 and K, 0 .2lij.. Similarly, 1 0 .13*0 gms. of S2 yielded 985*0 gm. crude methyl esters which upon distillation gave 9&5 ©n- the pure mixed methyl esters; I.V. 7^*2 5 ; and K, 0 .2 1 7; and sap equiv. 290.3; K, 0.217. Table 1J4- summarizes the preceding data and comprises the K values of the crude and distilled methyl esters with those of the original fat for specimens and Sg. These data were obtained primarily to compare infrared results when applied to glycerides and to mixed esters, also, to observe changes, if any, on transesterlfication and vacuum distillation. For the same reasons, the specimens were also analyzed by ultraviolet before and after alkali Isomerization, the results being given In Table 1 5 ® 110 TABLE ll+

INFRARED ANALYSIS OF ORIGINAL FATS, CRUDE ESTERS AND DISTILLED ESTERS

M6______m— — S2 Sample ' "lod~I Trans lod* Trans ______Valo_____ k______jo____ Val._____ k % Original Fat 80*26 0*2$!+ 33.15 75.^0 0 .2 5 8 3 3 .1+1 Crude Methyl Esters 78.50 0.216 36*12 7^.32 0.220 36*93

Distilled Esters 7 8 . 9 1 0.21^ 36.85 71+.25 0.217 36*33 TABLE 15

ULTRAVIOLET ANALYSIS OP ORIGINAL FATS, CRUDE ESTERS AND DISTILLED ESTERS

i'atty Acids % Conjugated Acids % Sample Iod. Saturated Oleic Linoleic Linolenic Diene Triene Val. acids acid acid acid

Margarine 6

Original fat 80.26 15.196 75.560 8.207 0.1+79 0.547 0.009 Crude methyl esters 78.50 19.189 70.820 9.073 0.1+13 0.486 0.019 Distilled esters 78.91 19.017 70.865 9.077 0.535 0.4.89 0.017 Shortening 2

Original fat 7540 13.011 77.910 3.993 0.023 0.262 0.001 Crude methyl esters 7 k - 3 2 13.669 81.837 4 .19J4- 0.02I4. 0.275 0.001 Distilled esters 7 k 13.667 81*900 14..150 0.022 0.260 0.001 112 Purification of the methyl esters by Claisen dis tillation, Table llj., has little effect on the extinction coefficient, the difference being 0*002 and 0*003 and S2 respectively which may be within the experimental error* On the contrary the difference in extinction coefficient between the triglycerides and the methyl esters is quite appreciable, 0*0lj.0 and 0 *3 1 * This is attributed mainly to the higher extinction coefficient of triglycerides in comparison with methyl esters. For example, the extinction coefficient of trielaidin is 0.f?63 while that of methyl elaidate is 0.323? triolein has 0.106 and methyl oleate 0 . Oiplp. Methanolysis as used in this investigation is un doubtedly the mildest possible method for the preparation of methyl esters. It has no effect on the components of margarines or shortenings* The data in Table If? shows that Claisen distillation lasting 3 hours at I96-2160 produced no significant increase in the conjugated un saturated fatty acids In the resulting methyl esters. This is in agreement with a previous study on the effect of heat on fatty acid esters^®^ . Kummerow and Potter, 1950^^3) reported that the presence of nonsaponifiable matter in lipids can cause appreciable differences in calculated mixed fatty acids and that the error can be appreciable in lipids extracted from liver. The results In Table If? indicate that the 113 unsaponiflable matter has no effect on the absorption spectra in the ultraviolet region. Differences In mono- ethenolc and saturated acids may be due to two factors: higher value of the triglycerides and errors In the calcu lation due to unsaponiflable In the original fat. This Is not present In the esters. Difference In saturated acids In the fat and the esters is due to the fact that saturated acids are calculated by difference. Also It should be pointed out that monoethenoic acids are calculated by difference between the ultraviolet values and total iodine value• F) Fractional Distillation of the Methyl Esters of M^. In this laboratory, fractional distillation is carried out by use of the following apparatus: a). a 2000 ml. Pyrex distilling pot equipped with a thermometer well and ground glass female ball joint. The distilling pot Is heated by an electric heating mantle which covers the entire pot. b)* The pot is set under a modified Penske column consist ing of three concentric Pyrex glass tubes. The Innermost glass tube Is packed with 1/8" glass helices for a height of 90 cm. and is equipped with a male ground glass ball joint at the bottom and a female ball joint at the top. The intermediate glass tube Is wound In two sections with a #20 nichrome wire heater. The heating is adjusted with two Variac controls to prevent excessive condensation on the wall of the column. The outer glass tube is used as an air jacket insulation to control heat loss* c) « A variable reflux distilling head is used, equipped with a water-cooled condenser and a narrow trough to catch the condensate and lead it to the output tube* Sealed into the output tube is a Newman stopcock, by which the reflux ratio is regulated. A glass thermometer well extends down through the condenser. d)• Attached to the output tube is a inodified Pauly or cow-udder receiver. It is constructed so that eight fractions can be collected v/ithout disturbing the vacuum simply by turning the receiver, Brown, 1 9 2 9 ^ ^ ^ . Eight round-bottom 2$0 ml. flasks were used to collect the fractions* From the center of the bottom of the Pauley receiver the system is attached to a Cenco-Megavac pump through a dry ice trap. Pouring operation, pressures are maintained at the minimum obtainable by the pump (0 .1-0 .2 mm.Hg), a Dubrovin vacuum guage being used for pressure measurements. The rate of distillation is controlled by the stopcock in the stillhead. A charge of 935*8 gm. of the methyl esters of was fractionally distilled as described in Table 16. Infrared results are included In this table, and ultra violet analysis of the fraction In Table 1 7* TABLE (16 ) 115 RESULTS OF FRACTIONAL DISTILLATION OF METHYL ESTERS OF MARGARINE AND ESTIMATION OF TRANS COMPONENTS OF FRACTIONS

Trans Frac 0 b,p* range wt. Iod. Mean Mol. K Esters No* at 1mm Hg. s* Val* Wt.,acids % 1 102-111 6 .1 5 5-13 23I4..2 O.OI4.8 3 -1^8

2 111-118 123J4.0 10*99 237.60 0.058 5 J+5 3 118-132 73.50 7 3 .8 5 27^.50 0.168 3 0 .3 9

k- 132** 151.00 95.37 279.50 0.208 3 5 .1 3 5 132-13!).-::- 1I4.6 ,ij.o 9 8 .0 7 281.8 0.219 3 7.1|-2 6 131)--* 112.90 9 7 .1 5 281.9 0.221 3 7 .8 1 7 131)-** 112.90 9 3 .6 5 2 8 1 .5 0.23ij. li-0 .9 9 8° 131).** 8 7 .[{.0 88.014. 282.8 O.2I4.O I4.2.15

9 13t).-135 * 15.60 85-19 278.8 0.2^5 U-3-37

10 136** 72.80 75.30 2 8 2 .1 0 .2 3 3 14-0 .914- 11 136-138 6 .3 5 6 8 .8 5 281.2 0 .2 0 5 35.30

-----

O *■» *• «■ O O Held in column H • 0.114.8 Total 918.^0

Pot residue 17.^0 ------0.130 -----

Original esters 935.80 7 8 .9 1 276.8 O.21I4. 3 6 .8 5

The raise of temperature followed the change in reflux ratio from 1 S10 to 1 :3 ® o Distillation stopped after 38 hours. TABLE (17) 116 ULTRAVIOLET ANALYSIS OP ESTER FRACTIONS OP

Prac. Saturated Octadecenolc Octadecadienoic Octadecatrlenoic Acid Acid Acid Acid % % Oon j • Nonconl. Conj. ^ohconjo % % % % 1 5.084 4 .7 4 7 0.129 00.040 00.000 00.000 2 90.199 8.301 0.062 1*475 00.000 00.063 3 20.816 68.634 0.169 10.048 00.000 00.332 4 3.276 81.718 0.221 14-307 00.000 00.478 5 2.819 81.032 0.243 15.376 00.000 00.530 6 0. 8I4.6 86.073 0.177 12.789 00.000 00.451 7 2.386 86.948 0.223 9.934 00.000 00.511 8 7 .5 io 83.226 0 .3 3 9 8.281 00.000 00.644 9 14.841 71.680 0.557 12.380 00.005 00.407 10 19-912 72.680 1*895 5 .5 0 0 00.001 00.012 -d- 00 CO 0 0 11 32.006 56.542 7*727 3.231 00.002 »

Original esters 19*017 70.865 0.489 9*077 00.017 00.535

F-j_ is calculated as except the unsaturated acids were calculated as ^2.6* P2 Is calculated as 117 Fraction 1 (Table l6) (M.M#W. of acid 23^.2 ) con sists mainly of methyl myristate (228.J4.) contaminated with small amounts of C-j^ unsaturated esters* From the iodine value it is assumed the unsaturated esters of this fraction consist of 3*Jl-8 per cent tran3-hexadecenoate and 1.27 per cent cis-hexadecenoate• Fraction 2 (M.M.W. of acid 257 *6 ) is composed mainly of palmitic acid and trans- and cis- hexadecenoic acid esters 5J+5 per cent and 2.85 per cent respectively. Fraction 3 (M.M*W. of acid 27lj-.5o) Is a mixture of and C^g methyl esters. Fraction ij. (M.M.W. acid 279*5) is mainly material but contains some C^g esters, the amount of which is small, no doubt, because of the large intermediate fraction (3)• Fractions 5 * 6 > 7, 8, 9, 10 and 11 are all C-^g methyl esters. However for the preparation of concentrates of trans acids later, methyl esters fractions If. to 10 were combined. From Table 17s it Is quite obvious that as dis tillation proceeds, the amount of non-conjugated polyun saturated esters decrease, as these constitute 15*38 P©n cent and 3*23 per cent of fractions 5 and 11 respectively. On the contrary, the amount of conjugated octadecadienoate rises as the distillation proceeds and shows Its highest percentage in fraction 11, 7*73 per cent of the total fraction. The percentage of methyl stearate increases gradually In the successive C-^g distillates and reaches Its maximum 118 In fraction 11 (32 .0 per cent of the fatty acids of this fraction). This finding supports the known fact that methyl stearate is the highest boiling component of the C-j^q mixture. Likewise, trans-unsaturated esters behave In a similar way, in that they show gradual increase in the several fractions; from 35*13 per cent in fraction Ij. to J4.0.9I4- per cent In fraction 10 with a maximum of J_p3 • 37 per cent in fraction 9 * From the preceding data one may con clude that methyl stearate and conjugated dienoate are somewhat concentrated in the higher boiling C-^g fractions; also, methyl esters of trans acids* It is seen, further, that trans components occur in all fractions. Therefore, both and C^g trans fatty acids are found in this margarine. G-) Further Separation of the C-^g Esters of Mg by Low Temperature Crystallization. The Isolation of C^g Trans Ester Concentrates. The methyl ester solutions were placed in a l5 xl|-5 cm. Pyrex crystallizing cylinder and placed in the low tempera ture bath which is standard equipment in this laboratory. The bath consists of a wooden box lj-8 x 1^.8 x 55 cm., heavily lined with rock wool Insulation and containing an inner cylindrical copper chamber 26 cm* in diameter and I4.5 cm* deep. This chamber was partially filled with acetone and cooled to the desired temperature with dry ice. A Weston bimetallic low temperature thermometer permitted easy temperature readings. The solution was stirred slowly 119 for two hours by means of a monel paddle type stirrer. The temperature was held constant by the addition of small pieces of dry Ice. After the desired temperature Is main tained for two hours, the stirrer was removed and the solu tion was left undisturbed for a few minutes to allow the formed crystals to settle to the bottom of the cylinder, thus facilitating the filtration. The filtrate was drawn off -under auction through an Inverted porcelain filter. The apparatus described above will handle Ij. liters of solu tion conveniently. A second crystallization apparatus is available which is deeper and which will accommodate 7 liters of solution. It was used when these volumes were crystallized as noted in Chart 2 below. One hundred grams of the combined ester fractions (iodine value 9 2 .5>8, K, 0 .2^6 ; N.E. 2 8 1.2 ), composed of combined fractions i{.-1 0 , Table IJ4., was dissolved in I4.OOO ml. methanol. The solution was cooled to -65 ° and maintained at this temperature for two hours, and was then filtered with suction. The crystals were washed once with cooled (-65 °) methanol. The filtrate and the washings were col lected and designated as P*^- The crystals were dissolved in six liters methanol and again cooled to -65°, the re sulting filtrate was designated as F»2* The crystals were dissolved in methanol and cooled to -20°. The resulting filtrate was cooled to -6o°, designated as F* g and the crystals as C»2* The resulting crystals at -20° were 120 dissolved in four liters methanol and cooled to 0°, the filtrate designated as and the crystal fraction as C» The methyl ester3 in the several filtrates were re covered by removing the methanol under reduced pressure, and the crystalline fractions were recovered by simply drying under vacuum to constant weight. A full description of the crystallization operation and analytical data on the resultant fractions are given in Chart 1 and Tables 18 and

1 9 . In another experiment a similar crystallization was carried out, except that the resulting crystals at -65° were dissolved and cooled to 0°, -20°, and then to -60° respectively, as described in Chart 2. Analysis of the fractions is likewise found in Tables 18 and 1 9 * TABUS (18) 121 ULTRAVIOLET ANALYSIS OP CRYSTALLIZED FRACTIONS Octa- Frac Satur dece- Octadecadienoic Octadecatrienoic Total tion ated noic Acid Acid . .% Acid Acid Conj. Noneonj. Conj. Nonconj.

. /° % % fa % % First !Batch (Chart 1)

00.000 47.283 8.143 1|.9 .£ 6 9 0.000 2.011 107.006 p2 00.000 58.892 I.676 4 0 .2 2 4 0.000 1.310 102.122 7-905 83.028 1.637 7 •>+0i4. 0.000 0.026 100.00 P3 (?2 1.341 9 8.6 ii-3 0.oij-5 0.168 0.000 0.000 100.00

Second Batch (Chart II)

*L 00.000 57.£ifO 8.628 llif .798 0.000 2.013 112.97 P 2 00.000 66.707 1.550 32.227 0.000 0 .3 3 0 100.314 2 .7 5 7 88.5 6 6 1 .5 1 1 7.020 0.000 0.146 100.00 4.987 d3 94*823 0.047 O.llj.3 0.000 0.000 100.00

Composition of combined fraction , subjected to Low Temperature Crystallization. 5.823 81.813 0.272 11.121 0.000 0.971 TABLE 19 122 METHYL STEARATE, METHYL OCTADECENOATE AND METHYL OCTADECA- DIENOATE VALUES CALCULATED PROM IODINE NUMBER

M e t h y 1 Octa- Octa deca- Frac Wto Temp. Iod* Stear dece die- Trans °C noate % * tion m * . Val* ate noate First Batch (Chart I)

p -i 13.50 -65-^ 1 5 2 .5 k 00.000 22.90 7 7 .1 0 k 3 .8 0 p .2 12*00 -65 -^ 132.^0 00.000 k6.io 53.90 kl.6o 3.90 -60-JHt- 87.65 00.000 97.20 2.80 19.00 f '3 C >2 ko.90 -60*HH* 8 k . 79 00.900 99.10 00.00 2 9 .5 0 P'^ 21.60 0 7k .53 12*90 87.10 00.00 90.60 C i 7.80 0 11*35 86.70 13.33 00.00 12.30

Second Batch (Chart II) F' »! 18.18 -65-^ 15 k *03 00.00 21.20 78.80 k 3 .k0 F» *2 5 .7 0 -6 5*^- 1 2 3 .kl 00.00 68.10 3 1 .9P kl. 80 pi it-.oo -60 -5W5- 95 .k7 00.00 88.60 11.ko ko.00 53.35 -60 85.59 00.00 100.00 00.00 kk.70 ° " 3 C» »2 12.10 -20 56.87 3 3 .6 0 66. ko 00.00 62.90 c . ' i 5 .9 0 0 17.00 80.00 20.00 00.00 lk.00

-:t- Trans % is calculated as elaidic acid ”methyl ester” In all Tractions* ■iKfrThese fractions are calculated here as binary mixtures of octadecenoate and octadecadienoate from the iodine values, on the assumption that no stearate is present In-6o.-65° filtrates. ^ -:BBKcCaleulated as a mixture of stearate and octadecenoate* CHART 1 123

Crystallization of C-^q Ester Fraction of to Produce

Trans Ester Concentrates

100 gm. of Ct_q fraction I.V. 92.58; K 0.256 Dissolved in I4.OOO m3, methanol Cool to “65° (2 hours)

I------1 Crystals Filtrate (F* ) Dissolved In 13.50 gm. 6000 ml methanol I.V , 152 .5 *4-, K 0 .2514- Cool to -65° (2 hours)

! ------Crystals Filtrate (F? ) Dissolved in 12.00 gm. 6000 ml methanol I.V. 132 .ip.; X 0 . 2 lj.3 Cool to -20° (2 hours)

1 Crystals Filtrate Dissolved in i|.000 ml methanol Cool to -60 ° Cool to 0° (2 hours) (2 hours)

1 \------I I------Crystals (C 1 -^) Filtrate(Ftl ) Crystals(C »2) Filtrate(F» 3 ) 7 *o0 gm. 21.60 gm. • ij.0.90 gm. 3 .9 0 gm. I.V. 11.35 I.V. 7^-53 I.V. 95.79 i.v. 87.65 K 0 .091}. K 0J|1}.2 K 0.185 K 0.135

CfQ fraction subjected to low temperature crystal lization Is a result of the combination of fractions Lj_—10 Chart 2 124 Second Crystallization of ClQ Esters of to Produce Trans Ester Concentration

100 gm. of C-]_q fraction I.V. 9 2 .5 8 ; K 0 .2 5 6 Dissolved in. 6000 ml methanol Cool to -65 °

I------Crystals Filtrate (F"x ) Dissolved in 18.60 gm. 6000 ml methanol I.V. l5 i|-.0 3 , K 0.252 Cool to -65 °

, ------Crystals Filtrate (P1^) Dissolved in 5.70 gm. 6000 ml. methanol I.V. 123.41, K 0.244 Cool to 0°

1------*------I Crystals (C * f Filtrate 5.90 gm. Cool to -20° I.V. 1 7.0 0 , K 0 .1 0 3

Crystals (C*^) Filtrate 12 .10 gm. Cool to -60° I.V. 5 6 .87, K 0 .3 4 2

1 Crystals(C"^) Fiitrate(F»^) £ 3 *35? gm. 4*00 gm« I.V. 8 5 .5 9 , K 0 .2 5 8 i.v. 95 .47,k 0.140 125 The C^g fraction of vegetable fats consists mainly of three solubility types of acids, stearic, oleic and linoleic acids which can be separated easily by cooling their solutions to -20° and -60 °. When thus crystallized stearic acid comes out at -20°, oleic acid at -60° and linoleic acid remains in solution at -60 °. The esters behave similarly. In hydrogenated fats, two more types of acids may exist: a) positional and geometrical isomers of octadecenoic acids, and b) positional and geometrical isomers of octadecadienoic acid. Changing an acid from its cis-form to its trans form reduces Its solubility consIderably. This fact Is used In separating elaidic acid from Its Isomerization mixture with, oleic acid by crystallization from methanol at -20°. The trans-isomers of oleic acid are expected to have Intermediate solubility between stearic and oleic acids. The position of the double bond In the carbon chain of octadecenoic acids has a definite effect on the melting points of both cis- and trans-acids (Tables 6 and

7) • Ralston and Hoerr^®^ and Bailey postulated that solubility is closely related to melting point. Recently, Kolb^®-^ showed that as the double bond is moved away from the carboxyl group of a cis-octadecenoic acid, the acid becomes somewhat more soluble. If this fact holds true, we may expect to find cis,12- and l5-octadecenoic acids, if present, to be more soluble than oleic acidj 126 and hence may appear In the linoleic acid fraction* The introduction of a second double bond further Increases the solubility; linoleic acid, for example, is about 30 times as soluble as oleic acid. The Increase in solubility is greater the farther the point of un saturation is removed from the carboxyl group. Rebello and Daubert^^ applied over thirty crystallizations to obtain an isolinoleic ester fraction, 95*8 per cent pure, consisting of at least three Isomers, the 8,11}.-, 9,l£- and 10,lif- octadecadienoic esters. Conjugated diethenoic esters are less soluble than nonconjugated ones. Rebello and Daubert succeeded In removing conjugated diethenoic esters from non-conjugated at -70°. Cis, trans- and trans,cis-octadecadienoic acids form an intermediate solubility class betv/een cis,cis-(the more soluble) and the trans,trans-(the less soluble) one. Fraction F»in Chart 1, calculated from the Iodine

value (Table 1 9 ) as a mixture of octadecenoate and octadecadienoate, contains 77*1 per cent of the latter. On the basis of ultraviolet analysis, F»i contains if.9.6 and 8.1 per cent of nonconjugated and conjugated dienoates, a total of 57*1 per cent. There is about 2 per cent error because ultraviolet also Indicates the presence of this amount of triene ester. The difference, about 20 per cent, may be accounted for by assuming it to be due in part to the presence of considerable amounts of octadecadienoates 127 which will not rearrange to conjugated diene systems with alkali, such as 9*15>“Octadecadienoate - addition the difference may be due to acids which require longer periods of alkali isomerization such as cis,trans- and trans,cis-

9 .1 2 - and 1 2 ,1 3 - octadecadienoic acids and the corresponding trans,trans acids. These latter types can not be determined under our experimental conditions. In order to estimate them even semi-quantitatively, it is necessary to Isomerize

the sample for 6 0 and 3 6 0 minutes, observing the absorptions at these times, as suggested by Wheeler et al^"^^ . This work was not done. However, on the basis of theoretical solubilities, we can assume that the types of diene acids found in F» ^ are, in part, cis,cis-, cis,trans-, and

trans,cis-9 *1 2 - and 1 2 ,13>-octadecedienoic acids and in part geometric isomers of octadecadienoic acids with a diene system which resists alkali isomerization such as

9 .1 3 -octadecadienoic acid. Also, as noted previously, this

fraction contains 8 per cent of conjugated diene acids. From the iodine value of this fraction is

calculated to contain 2 2 . 9 per cent of methyl octadecenoate. This value is over 2 per cent high because ultraviolet

analysis gave 2 per cent of triene ester in this fraction. This octadecenoate is largely methyl oleate, but it may also contain other soluble isomers of this ester such as methyl 15>-octadecenoate, etc. Trans octadecenoates are not likely to be present in appreciable amounts since 128 these would he sparingly soluble at -6 0 °. Most of the trans material in this fraction is likely diene in natures

Qctadecadienoate values for F»g in Tables 1 8 and 19 are J4.I.9 a n d 1|-6 . 1 per cent, respectively, the difference being due to acids of the types mentioned above, with per haps some emphasis on trans,trans types. Trans esters in this fraction, I4.I.6 per cent, are quite certainly of both the diene and octadecenoate classes. The trans component in fraction F*^ is mainly the trans octadecenoates, with the double bond beyond the 9 “ position. The crystals obtained at -60°, C»consist of

9 9 . 1 per cent octadecenoate, about one third of which is trans ester. The cis and trans octadecenoates in the fraction may include many of the predictable types, in cluding oleate and elaidate. The highest concentration of trans material is found in fraction F*j^. This fraction consists of stearate, trans-9 -octadecenoate and cis- octadecenoates of lesser solubility than oleic acid. Ninety per cent of the octadecenoate in this fraction is trans. The major component of is methyl stearate con taminated with trans- and cis-octadecenoates.

From the data of Tables 1 8 and 19* it is quite obvious that the scheme used in Chart 1 produces a fraction with the highest concentration of trans esters, in which 9 0 . 6 per cent of the unsaturated esters are of the trans form. The maximum obtained in Chart 2 was 6 2 . 9 129 per cent of the unsaturated esters in fraction CM2 . The two schemes of crystallization from methanol failed to produce the desired 100 per cent concentrate of trans com ponent . The advantages of low temperature crystallization in separating the C-^g esters into six simplified fractions are borne out by the data in Tables 1 8 and 19* Each of these simplified fractions still contains more than three components. Still it is suggested that the schemes de scribed in Charts 1 and 2 will be very useful in making simplified mixtures in preparation for applying chromatography, or countercurrent extraction methods. Because of the com plexity of margarines and shortenings in general it appears to be almost impossible to achieve the isolation of any single acid in a pure state by applying any single technic of separation.

H. Fractional Distillation of Methyl Esters of S2 *

A charge of 687*80 gm. of the methyl esters of S2 was fractionally distilled as described in Table 1 8 . The infrared analyses of the fractions are included in this table but time did not permit further examination. 130 TABLE 20 FRACTIONAL DISTILLATION OF METHYL ESTERS OF S2 AND INFRARED EXAMINATION OF FRACTIONS Me an Trans Frac. Boiling Range> wt. Iod. Mol. K C ompo- No. at 1 mm. gm. Val. wt. IO .36 nents °C Acids yom**cr/

1 93.0-10^.0 1.60 10.87 249.2 0.069 7.09

2 10]+. o-io 9.0 22.70 1.52 253-1 0 .01+6 2.50

3 109.0 -111.0 48.70 — 257.1 0.040 0.00

4 111.0 -123.5 18.20 76.88 270.7 0.177 27.77 CVI 0 O rn H - 5 123.5 -124.0 • 85.31 277.79 0.235 39-37 -=h OJ m 0 O 6 124.0-125.0 ♦ 85.28 261.9 0.250 43.01

7 125 .0 -127.5 47-95 76.30 282.5 0.260 45.30

8 127.5 -128.0 71.50 65.12 282,3 0.260 45.72

Pot residue -- - 28.20 61.37 — 0.194 31.73

Held In --- 10.75 46.81 — 0.201 33.32 column

Methyl esters Total 687.so 74*25 276.3 0.217 36.33

•^Calculated aa elaidate. In fractions 1 and 2, the material is likely to be actually trans hexadecenoate. 131 Since the fractional distillation of the methyl esters of S2 (Table 20) was similar to that of the data in Table 20 will be discussed only briefly. Prom the iodine and K values of Fraction 2 it is assumed that the unsaturated esters contain 2.5 P©r cent trans hexadecenoate. The percentage of trans esters in the fraction increases gradually and reaches its maximum in Fraction 8, 14.5*7 P©r cent. Due to time limitations, the ester fractions were not subjected to thorough, study like those of M^.

VII. DISCUSSION.

A great deal of the confusion that appears In the literature concerning the fatty acid composition of hy drogenated fats is probably due to limitations in the methods U3ed In their analysis. For example, Riemen- schneider^^ ^ found 3-5 per cent more linoleic acid in tobacco seed oil by the ultraviolet spectrophotometrie ( 7) method than by the iodine-thiocyanogen method. Lemon'' investigated the composition of hydrogenated linseed oil using both methods and found that the iodine-thiocyanogen method was unsatisfactory because of the presence of Isolinoleic acid which absorbs more thiocyanogen than normal linoleic acid. Subsequently, Fisher, 0*Connor and

Dollears( -)-}/3) reported that isolinoleic acid absorbs about two moles of thiocyanogen per mole of acid, while normal linoleic acid absorbs only one mole. Spectrophotometric 132 analysis (ultraviolet) may not be applied accurately to hydrogenated fats which contained triene or higher un saturation prior to hydrogenation since the isolinoleic acids formed during hydrogenation are not necessarily in cluded in the calculations and the resulting values for oleic and saturated acids will be erroneous, as stated earlier. In the process of hydrogenation diene acids are formed which fail to conjugate with alkali or do so more slowly than linoleic acid. The constants of the equations used In calculating the composition of fats by the Official Method^-^ are based on the assumption that the cls,cls- isoraer (linoleic) or the cis,cis,cis-isomer (llnolenic) are the ones found in normal vegetable fats. Likewise, if hydrogenation produces any Isomeric change to the trans configuration, the constants of the Official equations will not be strictly applicable* The Infrared spectrophotometric method is far superior to the Twitchell method for the determination of trans components In hydrogenated fats. It handles simple and complex mixtures of octadecenoic acids with reasonable accuracy in view of the almost Identical infra red absorption of various trans isomers of different double bond position. However, this method does not differentiate between the trans monounsaturated and the trans-polyun saturated fatty acids. The only available method which will give any information on the geometrical isomers of octa- 133 decadienoic acid is that of Wheeler et a l ^ ^ ^ , in which

alkali isomerization is conducted for three time intervals,

followed by ultraviolet measurement. This technique is not

adequately standardized and the resulting values must be

accepted with caution. For this reason this technique was not applied in the present work. The composition of margarines and shortenings in Table 11 was calculated by the use of the Official equa tions. Because of the limitations of the analytical methods, particularly the spectrophotometrie methods used for octadecadienoic and octadecatrienoic acids and the arbitrary procedures of arriving at corrected values for these constituents, the composition data in Table 11 must be regarded as only fair approximations. However, it seems safe to conclude that the amounts of linoleic acid (or perhaps better still octadecadienoic acid) in shortenings and margarines are higher than those found in butterfat. Shortenings contain from 3*9^- to 1 1 . 8 0 conjugated octadecadienoic acid and 0 . 1 7 to 0.9l conjugated diene acid. The total amount of octadecadienoic acid is lj..l7 to 11*97 per cent. The total amount of octa decadienoic acid in Crisco is 11*97 cent and this is in close agreement with the value reported by Richards et a l ^ ^ , 11.61^. per cent. Shortenings show a much wider range of values than margarines. The lower contents of octadecadienoic acids in shortenings is an indication of 13^ the probable presence of diene systems which, are not de tected by the methods used. The octadecadienoic acid con

tents of margarine fats range from 5 -2 5 -1 2 . 2 2 per cent. With the exception of one specimen (Swiftning) all of the margarines and shortenings are devoid of arachidonic acid. This sample contains 0.!{.5 per cent of this acid, most probably due to the lard (containing up to

0.6 per cent of this acid^2<"^ and beef tallow (0.1 per cent, Hilditch and Longenecker(209)j which are used to formulate this product. An important objective of this investigation was to determine the trans acid contents of six margarine samples and five shortenings by means of their infrared absorption at 10.36 The results, calculated as elaidin, ranged

from 3 2 .9 “4-l*7 per cent for margarines and 22.7-36.6 per cent for shortenings, except Swiftning, which again is unique and contains only traces of trans components. The infrared examination was extended to several vegetable oils with emphasis to those used in the manufacture of these products. Prom Table 12 it is quite obvious that most of the vegetable oils examined are devoid of trans components, the exceptions being coconut, palm oil (both belong to the Palmae Family) and olive oil (Oleaceae Family). These oils are extracted from fruit coat tissues. It is quite interest ing that both soybean and peanut oils (Leguminosae) do not contain trans acids. In 1952 Hilditch^^ reported that 135 the classification of species according to the constitu ent fatty acids in their seed fats leads to much the same result as that developed by the botanical classifications of Linnaeus and his successors. It seems that the trans components found within the limits of any given family are nearly the same, iDue to the limited number of samples tested, it is hard to make a general statement before making a thorough and intensive study. Olive oil contains

9 .il-2 per cent trans isomers. This accounts for the differ ences observed as found by Kass, Lundberg and Burr, 1914-0 ^^*^, who reported 1 3 * 9 P8*1 cent linoleic acid in olive oil by the thiocyanogen number-iodine number calculation, and only 3*0 per cent by the tetrabromide number determination. It Is likely that the Isomeric -acid is either the cis,trans- or the trans,cis-modification of 9>12-octadecadienoic acid and that it Is definitely not the trans,trans-form, Brown and co-workers, 1 9 ^4-3 ^ ^ * In further attempts to study the nature of the un saturated acids in margarines and shortenings, single specimens of each were converted to methyl esters and these esters fractionally distilled. The composition of the several ester fractions was previously discussed. It Is sufficient to state here that trans esters were found

In all fractions and comprise mainly and trans com ponents. The presence of trans hexadecenoic acid could only be the result of Isomerization and possible shift of 136 the double bond originally present. The C-^g esters of these fats contained IfO-jpO per cent of trans acids, both monoethenoic and diethenoic. In an effort to concentrate the trans acids in the C-lq fraction of Mg, low temperature crystallization of this fraction was carried out In methanol by two procedures, resulting In six simplified fractions. The C^g methyl acids of a typical margarine fat were thus shown to be a mixture of stearic acid, positional and geometrical isomers of octadecenoic, octadecadienoic and octadecatrienoic acids, a mixture so complex as to almost preclude Its com plete resolution. In commercial hydrogenation, the term "iso-oleic acid’1 commonly refers simply to the unsaturated fatty acids of high melting point which appear with the saturated acids in the conventional lead soap separation of the fatty acids of the product. Presumably, "iso-oleic" Is a mixture of the trans-isomers of oleic with melting points of [jlj. to 63° • Elaidic acid, long recognized as the most prevalent iso-oleic acid, melts at I4I}.0 • It has been identified in hydrogenated fats, but isolated only once In pure form^^^ . On the other hand, the cis-octadecenoic acids are comparatively low melting 10-30° (see Table 5>) • With the exception of oleic acid, they occur, but only in very limited quantities, in hydrogenated fats. In the hydrogenated vegetable fats, the conventional lead soap 137 method, used by various Investigators for the determination of iso-oleic acids, Is unsatisfactory. In a commercial

sample of hydrogenated vegetable fat containing 1 3 . 1 per cent Iso-oleic acids, Swern and co-v/orkers ^ found

3Il.2 per cent trans acids by the infrared method. Jackson and Callen^^5^ reported that the Twitchell method gives very low values with hydrogenated fats and that the error amounts to 35 to \\S per cent. The present work is a further development of the Infrared spectrophotometric method for the determination of the trans Isomers In fats and in ester mixtures and promises to make possible further investiga tions of the trans components in lipids generally.

VIII. SUMMARY AND CONCLUSIONS

(1). The chemical changes in the triglycerides and unsatura ted fatty acids which occur during hydrogenation of fats and the principal methods of fat and fatty acid investigation are reviewed. (2). A unique feature of the present investigation Is the use of the infrared spectrophotometric method for the quantita tive determination of the trans compounds in commercial margarines and shortenings and In methyl ester fractions prepared from same. This necessitated the evaluation of infrared reference standards in a considerable series of fatty acids and their compounds, a number of them for the first time. (3). Six margarines and five shortenings were examined by the usual methods and by infrared and ultraviolet pro cedures . (Ij.) . Ihe trans glycerides in the margarines ranged from

3 2 .9 “^4-l*7 per cent; in the shortenings, with one exception, from 22.7-36.6 per cent. These fats were shown to contain small amounts of conjugated diethenoic acids and traces of triethenoic acids. (5)• Swiftning, a blended shortening, contained only traces of trans glycerides. It was the only product examined which contained arachidonic acid (0.1j_5 per cent). (6). A series of fats of both animal and vegetable origin were examined by the infrared method. Cottonseed, soybean and peanut oils, commonly used to manufacture margarines and shortenings, and also corn and lettuce seed oils, con tained no trans glycerides. The trans glyceride contents of coconut, palm and olive oils were 2.6, 2.3 and 9°k- Per cent, respectively. This finding is discussed. The animal fats, lard, human milk fat and human body fat, contained

0 .0, 7*3 and 3.0 per cent trans glycerides* (7). Methyl esters were prepared from one specimen each of margarine and shortening. It was shown that no important changes in the constituent acids were produced as a result of the method of preparation. (8). The esters were fractionally distilled. The resulting fractions were shown to include esters of the C-^i , and 139 Ciq series. 'i'rans components were found in all fractions, but major amounts, ipO-50 per cent, occurred in tlie C^g esters.

(9)* Small amounts of trans hexadecenoic acids occur in margarines and shortenings.

(10)* In an attempt to produce concentrates of trans esters and to further study the nature of the trans unsaturated acids of the C^g series, the combined C^g esters of a specimen of margarine were fractionally crystallized at low temperature by two procedures. Six simplified frac tions were thus obtained and their composition studied from their iodine values and infrared and ultraviolet absorptions. One of these fractions contained 90 per cent of its unsaturated acids as trans acids.

(11). Evidence is presented that the unsaturated fatty acids of a typical margarine, and probably generally of margarines and shortenings, are actually an extremely complex mixture of many of the theoretically possible positional and geometric isomers of the octadecenoic and octadecadienoic acids. When the hydrogenated fat is made from an oil containing linolenic acid (soybean oil), the mixture will be even more complex than when made from cottonseed oil.

(12). The spectrophotometric method of analysis of hydrogenated fats is evaluated and shown to be subject to definite limitations* The ultraviolet procedure. especially, may tie in error due to the presence of diene acids which will not conjugate with alkali or do so more slowly than linoleic acid. Precision of the infrared method is also affected by the complex nature of the fatty acids in these products. 14-1 BIBLIOGRAPHY 1* Bertram, S. H., Biochem. J., 197» 4-33 (1928)* 2, Cornwell, D, G„, Backderf, R., Wilson, C. L. and Brown, <1. B„ Arch. of Biochem. and Biophys., J±6, 364. (1953).

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211. Kass, J. P., Lundberg, W. D. and Burr, G* 0 . Oil and Soap, 17, 55 (191+0). 152 AUTOBIOGRAPHY

I, Ahmed Fahmy Mabrouk, was born In Cairo, Egypt, September 30, 1923. 1 attended Cairo public secondary schools and graduated In 19^1• My undergraduate training v/as obtained at Cairo University, from which I received the Bachelor of Science degree in 19^5* After my gradua tion, I was appointed instructor in Cairo University Departments of General Chemistry and Food Technology. From the same University I received the Master of Science degree in 1950. A study leave v/as granted by Cairo University and I came to Ohio State University in the spring of 195>1* v/here I specialized in Agricultural Biochemistry. I have held this study leave for the last three and half years while completing the requirements for the degree Doctor of Philosophy,