SOLUBILITIES CT FATTX ACIDS IX f f i » S D OROAMIC SQLVKWT8 AT 209 W r B ITW M
DISSERTATION Pr«i«at«d In Partial Polflllatnt of tho Rofolromato Per the Degree Doctor of Philosophy in the Gtradaate Seheel of the Ohio State University
By-
Dor la KolLhe B.So • 0 M»Se* It The Ohio State Baieerelty 1953
Approved hyt ACEHOVLEDGMEHT
To Dr* J, B» Brown go my sincerest thank# for his helpful counsel and his constant smile of encouragement* 1 also wish to thank Dr* M* S. Bowman, who kindly consented to act as my co-adviser. I am grateful to the University for the fellowship which for the past three years has been granted to me from funds allocated by the Research Foundation for fundamental research*
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A 16487 TABLE OP CONTENTS Fag* I* STATEMENT CEF PROBIEM 1 XI • HISTORICAL 2 A. Review of Methods for Separating Fatty AcI da and Their Compound* 2 B. Development of the Low Temperature Crystallization Technique 19 C. Review of Previous Work on Fatty A d d Solubilities 23 III. EXPERIMENTAL 38 A. Plan of Investigation 36 B. Preparation of Fatty Aolds 1^2 C. Analyses Used for Criteria of Purity 59 D. Purification of Solvents 62 E. Procedure for Measuring Solubilities 62 IV. SOLUBILITY DATA ?0 V. DISCUSSION 85 VI. SUGGESTIONS FOR FUTURE WORK 100 SUMMARY 101 BIBLIOGRAPHY 101*.
111 INDEX TO TABLES P*g» 1. Fatty Aold Solubilities as Complied by Brown In 1941 29 2. Solubilities of the Saturated Acids 31 3* Solubilities of the Unsaturated Acids 32 4* Solubility Ratios of Fatty Acids Under Various Conditions 33 5» Typical Data of Singleton 34 6* Solubilities of Hoerr and Harwood for Oleic Acid 36 7. Solubilities of Hoerr and Harwood for Llnolele Aold 37 8* Fractional Distillation of the Methyl Eaters of OU ts Oil kS 9* Fractional Distillation of the Methyl Esters from Rapeseed Oil 52 10* Analytical Constants of the Fatty Acids 61 11. Sample Data Showing Change of Solubility with Time 69 12* Solubility Data on Fatty Acids In Various Solvents 71 13. Fatty Acid Solubilities In Various Hydro carbon Solvents % 14. Comparison of Solubilities with Data Previously Reported by Other Investigators 97
iv XVXXSX TO ILLtJSTRATIOKS
Page 1 , Low Temperature Crystallisation Apparatus J+7 2 * Preparation of Methyl Oleate by Crystallization of the C^0 Methyl Esters of Olive Oil lj.8 3 * Preparation of Methyl Erucate by Crystallization of the C2 2 Methyl Esters of Rapeseed Oil 53 l\.m Preparation of Methyl Eleosenoate by Crystal* 11sation of the Con Methyl Esters of Rapeseed Oil 55 5* Crystallisation of Hormel Llnoleie Aold 58 6 . Constant Low Temperature Apparatus 6l(. 7* Apparatus for Removing Samples of Saturated Solution 6? 8. Solubility of Fatty Acids in Methanol 7l|. 9. Solubility of Fatty Aelds in Ethyl Aoetate 75 1 0 * Solubility of Fatty Acids In n*Heptane 76 11* Solubility of Fatty Aelds in Acetone 77 1 2 * Solubility of Fatty Aelds in Diethyl Ether 76 13* Solubility of Fatty Acids in Toluene 79 lit* Solubility of Stearle Aold In Various Solvents 80 15* Solubility of Olele Acid in Various Solvents 61 1 6 . Solubility of Llnoleie Acid in Various Solvents 82 I* STATEMENT OF PROBLEM
In 1937 there appeared the first papers of a series by Brown and coworkers (l, 2) dealing with the technique of fractional crystallization at low temperatures as a means for separating fatty acid mixtures* These Investi gators worked with dilute solutions of fatty acids in organic solvents and within the range of temperatures obtainable with dry ice. Low temperature crystallization has since found wide application as a means for separating and purifying fatty acids, particularly the unsaturated acids. It may well be considered one of the most useful yet simplest tools at the disposal of the fat chemist. The utility of the method has been somewhat limited, however, by the fact that so little information is avail able relating to the solubility behavior of the fatty acids at such low temperatures. It has been the purpose of this investigation to carry out an extended study of the solubilities of a number of highly purified fatty acids in various organic solvents at low temperatures. This endeavor has first necessitated the preparation of the fatty acids themselves, since materials of the degree of purity required for such a study are not commercially available. Solubility measurements have then been taken with the aid of an 2 apparatus designed to maintain constant temperatures down to -7f>° C. It is hoped that these data will not only Till one of the very obvious gaps in our knowledge of the physical behavior of the fatty acids but will also serve as a guide for more advantageous utilization of the method of low temperature crystallization*
II. HISTORICAL
A. Review of Methods for Separating Fatty Acids and Their Compounds*
The separation of the various component fatty acids occurring in a natural fat or oil is one of the most im portant problems in fat chemistry* The process is usually a difficult one* especially if the separation must be carried out at all quantitatively* The mixed fatty acids from a given source often Include some acids of widely different chemical properties as well as others which have almost identical chemical characteristics and differ only slightly in their physical behavior* There are a number of methods available for resolv ing fatty acid mixtures* The methods include both chemical and physical means of separation* and they vary in degree of efficiency* convenience* and applicability to a given separation problem* Usually a combination of several methods comprises the best procedure* particularly if the . 3 mixture Is at all complex* The techniques which are available can be roughly divided into processes Involving (l) distillation, (2) adsorption and extraction, and (3 ) methods dependent upon solubility.
1 . Distillation Procedures. As applied to the separation of fatty acids and their derivatives there are three distinct distillation techniques, each useful in performing a particular type of separation* These are the methods of steam distillation, vacuum distillation, and molecular distillation. Steam distillation is used to separate the fatty acids of such materials as milk fats, containing acids such as butyric and caproic, which volatilize readily with steam* The method may serve as a means for partial separation of the shorter chain from the longer chain acids; however, in order to obtain any kind of efficient separation it is necessary to employ some type of frac tionating column. The history of vacuum distillation, with respect to fats, dates back to the work of Chevreul (3 ) during the early part of the nineteenth century* A review of the extensive work that has appeared on the subject since that time, and particularly within recent years, is quite beyond the scope of this paper* Since there are numerous texts (I4-, 5 , 6 ) dealing with both theoretical and practical aspects of fractional distillation under vacuum, the technique need be discussed only briefly here* In general the distillation unit consists of the following essential parts: (l) a boiler or still pot equipped with a source of heat, (2 ) a fractionating column, including packing and Insulation, (3) a still head, equipped with a condenser and a device for reflux control, (4.) a fraction cutter of some sort, with a set of receiving vessels, and (5) a means for producing and controlling the vacuum in the system* Much Ingenuity has been exercised In designing modifications of these various parts. Probably the most attention has been directed toward the column Itself and the development of various types of packing materials. These have Included various types of rings, tubes, Jack chains, and BB shot, straight and bent carding teeth, Vigreux Indentations, wire and glass helices, spiral coils, crimped wire, and the conical type of wire gauze packing developed by Stedman* Industrial columns are usually of the bubble-cap type, while a modified spinning band column, with minimum pressure drop, has been used in the laboratory by Murray (7 ) with excellent results. Distillation was first applied to fat materials by direct distillation of the oils themselves, then later by attempted fractionation of their mixed fatty acids.. Some investigators were able to distill certain glycerides, but they had little success in achieving fractionation by this method. This Is not surprising in view of the present picture of the heterogeneous nature of the glycerides comprising natural fats and oils. Work with the free acids was largely unsuccessful because of their tendency to decompose on prolonged heating. In addition, since the acids tend to associate even in the vapor phase, such separations as were obtained were poor. The use of methyl ester distillation was first reported In 1906 (8 ). This method permitted distillation at lower temperatures and largely eliminated the dangers of association and decomposition. Ester distillation has since been used In the investigation of a vast number of fatty substances, and, although other esters have some times been used, the methyl esters are the ones most commonly employed. Fractional ester distillation, carried out under vacuum with an efficient column, Is probably the best technique available for separating fatty acids according to chain length. Ordinarily, however, there Is little or no separation of saturated from unsaturated components accomplished by distillation. For this reason ester distillation is generally used in conjunction with some other means of separation when more complete resolution 6 • • Is desired. Molecular distillation differs from ordinary vacuum distillation In that the system must be much more highly evacuated (ca. 0 .0 0 1 mm.) and the distillation path ex tremely short, with only 1 -2 cm. distance between evapora tion surface and condensation surface* By using this method It is possible to distill many normally non- di3tillable substances. There Is a great deal of litera ture devoted to this subject, much of It being incorporated in a number of review articles (9 # 1 0# H ) • Molecular distillation has been used with varying degrees of success for the separation of glycerides and such non-glyceride lipids as sterols, hydrocarbons, and vitamins. It has not been extensively applied to the separation of fatty acids, since ordinary methods of dis tillation permit more convenient and efficient separation of most mixtures. The technique has been valuable, how ever, as a means for separating mixtures of highly un saturated and thermally unstable acids, such as occur in fish oils and brain phosphatides, and for separating monomeric and polymeric fatty acids and esters.
2 . Adsorption and Extraction Methods. (Notes Since discussions of these two topics would tend to overlap, they are considered here under a common heading.) The use of chromatographic adsorption as a means for separating fatty acids is currently receiving a * great deal of attention* The method is.based upon the fact that the active surfaces of certain.finely divided solids have great adsorptive power, different substances exhibiting different affinities for a given adsorbing surface. Adsorption behavior differs considerably even among very closely related substances* The process of chromatographic separation consists, in general, of passing a solution of the mixture under investigation through a column of some suitable finely divided solid* Activated carbon, clay, and metal oxides are typical adsorbents* As the solution flows through the column, the more highly adsorbed substances become concentrated at the top, while the less strongly adsorbed materials are displaced downward along the column* The technique was first employed in 1906 by a botanist named Tswett (12), but the potentialities of his method were not realised by chemists until about 1 9 3 0* In 1939 Cassidy (13) discussed the possibilities of Tswett 9s work with respect to the field of fatty acid separation* He also made a study of the adsorption iso therms of a number of saturated fatty acids (1J4.) and 1 * pointed out (1 5 ) that even very complex fatty acid mixtures could be separated by chromatographic means If the proper adsorbing material were selected* Since then adsorption has been widely used in the field of fatty acid separation* It has been employed in the purification » • 4 • of such compounds*as oleic a'eid, methyl llnoleate, and methyl llnolenate and has been* applied to the separation of numerous types of fatty mixtures•• One of the problems which arise when colorless compounds are subjected to chromatographic treatment Is that of determining where the bands of material are lo cated along the column* Graff and Skau (l6) followed the progress of fatty acids through the column by having the adsorbent (magnesium oxide) impregnated with phenol red* Dutton (17) has described a method for following colorless compounds through a continuous flow column by measuring changes in the refractive index of the eluate with a highly sensitive refractometer* Ion exchange chromatography» so successful in the separation of many Icnizable substances, has not found application In the field of fats; however, its use has been reported In connection with the volatile .fatty acids obtained from grape Juice wastes (18)• One chromatographic technique very useful in fat chemistry is that of carrier displacement (1 9) • In this method the mixture to be separated is combined with* some "carrier11 which can later be easily removed* The carrier usually contains a number of substances whose adsorption Isotherms lie intermediate between those of the components of the mixture* The components* * are thus .spaced out * by
"* * the carrier, and the zones, even when quite small, are * relatively easy to separate. In the case of the saturated • * , fatty acids, the esters of the acids themselves have been •found to be the most satisfactory system of carriers, (20) Martin and Synge (21) in 19^1 introduced a variation of the chromatographic process in which two liquid phases are used. This method Is known as "partition chromato graphy". The column form Is retained, but the adsorbent (e.g* silica gel) functions mainly as a support for one liquid phase (water), while a second liquid, immiscible with the first, travels down the column. A relatively recent development Is that of paper partition chromato graphy, In which paper constitutes the solid or stationary phase. This technique has been applied with success to the separation of both fatty acids (22, 23, 21}.) and their salts (25# 26). Boidingh (27) has prepared filter paper strips Impregnated with vulcanized rubber latex In such a way that the porous structure of the paper Is retained and has been able to achieve quite efficient fatty acid separations. Partition chromatography is actually just a special • * technique for carrying out liquid-liquid extractions. Al~‘ though extraction has long been a standard laboratory method for carrying out separations of compounds with distinctly different properties,, its use as -a means for separating closely related substances is relatively new* * • Separation by extraction of materials With similar parti-, tion coefficients requires some sort* of fractionating device in order to keep the process from becoming un bearably tedious, since an extremely large number of indi vidual extractions is necessary for each separation. Several types of apparatus have been developed for carry ing out separations by countercurrent extraction (28). These are said to permit several thousand almost quanti tative extractions in just a few hours of operation. Mixtures both of fatty acids (29) and o f their oxidation products (30) have been separated by countercurrent dis tribution methods. An extensive treatment of counter- current distribution, and of extraction in general, is given in a recent discussion by Craig and Craig (31)* Many excellent separations of fatty acids and re lated substances have been reported using the methods of adsorption and extraction. Both methods, however, are best suited to cases where small samples are involved. Moreover, the equipment is rather complicated in some cases, and a good deal of preliminary research is usually necessary in order to determine just ^xat con ditions should be used for the resolution of a particular mixture. 3* Methods Dependent on Solubilities of Fatty Acid Derivatives 9 •
* There are a number of methods available for the separation of fatty acid mixtures which depend upon the relative solubilities of the acids or their deriva tives In various solvents* Most of these methods find their chief use In separating mixtures Into saturated and unsaturated fractions, or further separating unsaturated acids into fractions of varying degrees of unsaturation* None of the methods is completely quantitative be cause of the mutual solubility effects which the compounds In fat mixtures exert upon one another* However, they are able to resolve rather complex mixtures Into much simpler ones, some separations being much more efficient than others# Several of these solubility methods also serve as very effective tools for purifying individual fatty acids. Methods Involving various fatty acid derivatives will be discussed briefly below, while the separation .of the free acids will be taken up in more detail In the next section*
Metal Salts* When fatty acids, are reacted with metal ions, they form metallic salts, or soaps, whose properties depend both on the nature of the metallic ion and on the chain length
* and degree of unsaturation of the fatty aold* The differ- enCe in solubilities or these metal soaps has been the* basis for a number of procedures for fatty acid separation. One of the first and most efficient methods of this type was the lead salt-ether method proposed by Gqsserow (32) in 1828. This means of separation, sometimes referred to as the Varrentrapp (33) method, involves conversion of the acids to their lead salts and then treatment of the mixture with ether, in which the saturated salts are in soluble. The saturated components can then be removed by filtration. The lead salt-ether technique has been often investigated and has been modified a number of times to improve its general applicability* One important modifica tion was that of Twitchell (3I4-)# who suggested the use of 95% ethanol in place of ether as the partitioning solvent. In actual practice complete and sharp separations are not generally obtainable with lead soap procedures. Certain unsaturated acids, such as erucic, eleostearlc, and elaidic, yield salts which are only sparingly soluble in ether or alcohol. On the other hand, some of the saturated acids found in such materials as butterfat and coconut oil have appreciable solubility and remain in part or entirely with the unsaturated fraction. There are also considerable mutual solubility effects, and it has been observed that the solubilities of saturated lead soaps are much greater when there are large quantities of ' . ' 13 unsaturated soaps In solution. (3$) In addition. It has been suggested that lead soaps may cause isomerization of oleic acid. (3&) A number of other metal Ions have also been used with some success. Barium salts, for example, have been separated with benzene (37) or with methanol (30). sy these methods barium oleate remains Insoluble and Is fil tered out with the saturated acids. Another widely used soap separation Is the lithium salt-acetone method of Tsujimoto (39)• for the separation of monoethylenic from polyethylenic unsaturated acids* This technique has been of particular value in connection with the fish oils. Lithium soaps may also be treated with other solvents, such as ethanol or water, to effect separa tion of closely related saturated acids (e.g. palmitic and stearic)• Various other separation procedures have employed the salt3 of sodium, potassium, calcium, magnesium, zinc, strontium, and thallium, or such heavy metals as Iron, nickel, cobalt, silver, gold, and mercury. Certain of these methods have been shown to permit efficacious separa tion when applied to particular types of mixtures, but their usefulness tends to be rather specialized. *' * Bromo Derivatives. Btfominatlon has been used for many years as a means ' for examining the unsaturated fatty acids present in fats and oils. When bromine is added to unsaturated fatty acids dissolved in some cold organic solvent, such as • ether or chloroform, bromo derivatives of the acid3 are formed by addition at the double bonds of the molecules. Monoethylenlc acids (oleic, erucic, ricinoleic, etc.) form dibromides which are readily soluble in most organic sol vents. Linoleic acid forms a tetrabromide which is soluble in ethyl ether but nearly insoluble in cold petroleum ether. The hexabromo derivative obtained from linolenic acid is insoluble in ethyl ether and in many other solvents. The octabromide from arachidonic acid is still more insoluble, and the polybromide from clupanodonic acid is insoluble in practically all organic solvents. Advantage has been taken of the differences in solubility of these bromo derivatives to separate unsaturated fatty acid mixtures into fractions of varying degrees of unsaturation. The bromination method has been a highly important preparative tool. After the desired polybromides have been separated and carefully recrystallized, the free acids can be regenerated by treatment with zinc in some suitable solvent. Bromination has even been utilized as a quantitative technique for estimating the polyunsaturate content of fatty acl£ mixtures, although the actual yields of poly- bromides ape far from quantitative. Linoleic acid yields only about one hal-f the theoretical amount .of insoluble bromo derivative, while linolenic gives about one third and arachidonic approximately one fourth of the quantita tive yield. This results from the fact that isomeric bromides with different solubilities are produced as a consequence of the formation of two asymmetric centers as each double bond is brominated. Nevertheless, reasonably quantitative results have been possible for the estimation of arachi donic (J4.0 >, linolenic (I4.I) , and linoleic (Ip2) acids using the empirical constants worked out by Brown and coworkers In conjunction with polybromide numbers. More recent work from that same laboratory (i+3, I4J4., I4.5) suggests that under carefully controlled conditions the method can be used very quantitatively, employing the polybromide yield in conjunction with an empirical standard curve. A noteworthy disadvantage of the bramination- debromination procedure is the danger of isomerism during the process* By low temperature crystallization Matthews, Brode, and Brown (I4.6) showed that debromination linoleic and linolenic acids contain at least 12-15 P®** cent of isomeric acids.
Urea Complexes. In 19lt-0 it was observed by Bengen (ip7 ) in Germany that urea has the property of forming complexes with straight chain organic compounds, but not with their branched chain and cyclic analogs. This fact was not given much attention until about ten years later when Zimmerscheid (1^.8 ) confirmed Bengen*s observation and in vestigated the urea complex formation as a research tool* The complex is believed to be formed by the incorporation of the hydrocarbon chain in tunnel-like spaces in the urea crystal lattice* Whereas distillation sorts compounds ac cording to size, and adsorption according to type or class, this method separates them according to shape.
U r e a complex formation has been very successfully applied to certain phases of fatty acid separation. The complexes are prepared by the simple addition of the fatty acid (with warming) to a saturated solution of urea in methanol (16 g./lOO ml.). Crystallization takes place at room temperature. Complexes have been prepared for a con siderable number of fatty acids (l^), and in experiments with mixtures, some separation has been achieved in cases where the solubilities of the complexes are sufficiently different. Unsaturated fatty acids form complexes with urea as well as do the saturated straight chain acids. The degree of unsaturation, position of the double bond, and geometrical configuration of the acid apparently have little effect on the composition of the complex. (5 0) Urea complexes of unsaturated .fatty acids are remarkable in their resistance to autoxidation. Although, both, saturated * + and unsaturated acids form urea complexes, the saturated chains combine preferentially in a mixture. Acids with shorter hydrocarbon chains tend to give less stable adducts, those of low molecular weight yielding none at all. (5l) Swern, et al., have applied the urea technique to the preparation of oleic acid (52), obtaining a product with 97“ 99 pe** cent purity. The method has also been used to prepare concentrates of linoleic and linolenic acids (53)• Extractive crystallization with urea has been con sidered commercially, and a type of unit process has been described (5U) utilizing the urea reaction in practical large-scale separation^. Thiourea (5 5) ha3 also been used with success for hydrocarbon complex formation.
Hydroxamlc Acids Inoue and his coworkera in Japan have developed a method for separating fatty acids.by means of their hydroxamic acids. (5 6 ) The hydroxaraic acids are crystal line substances with melting points considerably higher than those of the corresponding qarboxylic acids. The hydroxamic acid derived from oleic acid, fof* example, melts at 6 l ° . Patty acids are converted quantitatively to * hydroxamic a.cids by reaction with hydroxylamlne under anhydrous conditions, and they can b© later regenerated by refluxing with dilute alcoholic sulfuric acid solution* Treatment of hydroxamic acid3 at 0° with organic, solvents, such as alcohol, ether, petroleum ether, or carbon tetrachloride, is recommended for separating fatty acid mixtures into saturated and unsaturated fractions. The hydroxamic acid method is also reported to be superior to steam distillation as a means for separating volatile and non-volatile fatty acids. (57) The technique has been suggested as a means for obtaining pure oleic acid from olive oil (58) and pure erucic acid from rapeseed oil (5 9)* Moreover, it is claimed that repeated crystallization of hydroxamic acids from or ganic solvents facilitates the preparation of highly purified linoleic acid from cottonseed oil and linolenic acid of high purity from soya bean oil (60)• . The use of hydroxamic acids has been applied by these same Japanese investigators (6l) to fatty acid separa tions by paper partition chromatography. Ethyl acetate, n-butanol, and butyrone were found to be suitable solvents, and a 10 per cent alcoholic solution of ferric chloride was employed -as the chromogenic agent. An extensive series of Bp values was reported for hydroxamic acids derived from both saturated and unsaturated fatty acids, from certain common substituted acids, and from a number • ■ * of polycarboxylic acids* . * * 19 B. Development or the Low Temperature Crystallization Technique
One or the simplest methods ror separating ratty acids is that or direct crystallization or the mixed acids from some suitable solvent. Although solvent crystallization is a common purirication tool in organic chemistry, its role as an important process ror separating Tatty acid mixtures is relatively recent. This is because earlier investigators usually attempted separations from rather concentrated solu tions and at only slightly reduced temperatures, and the separations were generally inerricient. For example, in 1927 Bertram (62 ) used crystallization from acetone at -15° - as a method Tor purifying oleic acid, and several years later Raymond (63) reported a similar process using ethanol as solvent. In 1937 Brown and coworkers Introduced a technique or crystallization at very low temperatures as a means Tor separating Tatty acids. They used dilute solutions, usually in acetone or methanol, and worked In the range of tempera tures obtainable with dry Ice. Fractionation was achieved by carrying out crystallizations at successively lower temporat tires. In general, the separation scheme involved an Initial crystallization from a 10. per cent solution at -20 to - 2 5 ° , to separate the saturated from the unsaturated acids, then further crystallizations at -50 to - 7 0 ° for separating the individual unsaturated components. 20 The introductory paper of a series was that of Brown and Stoner (l), who described the low temperature solvent crystallization of linoleic acid from the fatty acids of corn and cottonseed oils. By using the procedure described above, they obtained linoleic acid preparations with purity up to 93 P©** cent. Brown and Franks 1 (6)4.) did further work in order to improve the crystallization tech nique as a method for preparing linoleic acid and were suc cessful in obtaining the acid in a pure state by this com pletely physical method. Brown and Shinowara (2 ) had shown that pure oleic acid could be readily prepared by crystallization of the mixed acids of olive oil, and they were also able to obtain concentrates of linolenic acid (6f>) from linseed and perilla oils. The linolenic acid content of their best product, however, was only 88 per cent. Cramer and Brown (66) employed the method of lew tem perature crystallization from acetone, methanol, and petro leum ether in order to separate the methyl esters of human fat. The esters were first fractionated by vacuum distilla tion, and then the esters of the C^g, and C^g acids were each subjected to crystallization* Brown and coworkers applied the same method to analyses of the fatty acids of corn oil (67), menhaden oil (68), and human milk fat (69)*
* A combination' of crystallization and fractional die- 21 tlllatlon was also used to prepare methyl arachidonate or 95 per cent purity from the methyl eaters of suprarenal phosphatides (70, 71 )* Brown and Green (72 ) were able to prepare very pure methyl riclnoleate from castor oil, and by saponification and further low temperature crystallisa tion they prepared its difficultly isolatable acid in 95 pax* cent purity. Erucic (73 ) osid. brassidic (7 ^-) acids had been earlier purified by crystallization at moderately reduced temperatures, and several Investigators had used the method to prepare eleostearic acid from tung oil (75* 76, 77)• Earle and Milner (78 ) showed that low temperature crystallization was suitable for the quantitative determina tion of the saturated fatty acids In soybean oil. Their procedure involved repeated crystallization from a 10 per cent acetone solution at -i|.0o and calculations based on the weight and iodine value of the solid acids. Mi11lean and Brown (7 9 ) used low temperature crystal lization to isolate oleic acid from olive, peanut, c o m , cottonseed, and linseed oils and from chicken fat. The oleic acid preparations from all these materials were identical. In addition, from lard, beef tallow, beef adrenal phosphatides, pork liver lipids, human fat, soy bean oil, and rapeseed oil were Isolated octadecenoic acids which’contained isomers other than oleic. Idnoleic acid (60 ) was prepared by low temperature crystallization of the fatty acids of sesame, cottonseed. . *
22 grapeseed,. and poppy sedd oils and shown to be identical with the material obtained from c o m oil. An attempt to prepare the acid from olive oil was not very successful. Isollnoleic acid (8l) was separated from partially hydrogenated linseed oil by a combination of the methods of low temperature crystallization and chromatographic adsorption. A combination of crystallization and molecular distillation was used to obtain 9 0 -9 5 per cent methyl peroxido oleate (82). Matthews, Brode, and Brown (I4.6 ) were able to demon* strate that the linoleic and linolenlc acids prepared by debromlnation procedures contained 12 to 1*> per cent of isomeric acids. By employing low temperature crystalliza tion to remove the isomeric material, they were able to raise the melting points of both acids by several degrees. Holman and coworkers (8 3) utilized this purification tech nique for preparing linolenlc acid for spectrophotometrie studies. Hildltch and coworkers (814.) have incorporated the low temperature crystallization technique into their pro cedure for studying the fatty acid composition of fats and oils. It has replaced the lead salt method for separating saturated from unsaturated components. Fractional crystal lization at low temperatures, and then analysis of the • * various fractions, has also been applied to the glycerides themselves for determining the glyceride structures of fat ‘ materials (65)• Low temperature solvent fractionation has even proved to be practical on a commercial scale* In 19^-2 Emory Industries, Inc. (86) put into operation their first com mercial unit for the fractionation of fatty acid mixtures by low temperature crystallization. Using methanol both as solvent and as the heat exchange medium in the multi- tubular crystal11zer, they have developed a continuous crystallization process, which has been applied to the production of stearic and oleic acids at a rate of several million pounds each month. Hie advantage of low temperature crystallization lies in the fact that it is such a simple technique and yet such an effective means of separation. The operation re quires no complicated equipment, and the solvent can be recovered and reused. The procedure is comparatively rapid and without the hazards of isomerization and thermal decomposition. It is probably one of the best methods available both for separating saturated from unsaturated fatty acids and for purifying individual unsaturated acids.
C. A Review of Previous Work on Fatty Acid Solubilities.
1. Solubilities at temperatures above 0°. Studies of fatty acid solubilities at room tempera- turo.and above have been limited to Investigation, of the 21* normal' saturated acids, since the unsaturated compounds ar9| for the most part, infinitely soluble in most solvents at such elevated temperatures* A fairly extensive amount of data has been collected with respect to the saturated fatty acid series* Humorous investigators have made solubility determinations for the lower molecular weight acids (formic to caproic) in a variety of solvents, and many of their results have been compiled by Seidell (87)• This discussion, however, has been limited to the longer chain fatty acids and reviews briefly the work that has been done pertaining to their solubilities both In water and in organic solvents. Fatty acids exhibit appreciable solubilities in water, as compared with the corresponding hydrocarbons, because of the presence of the hydrophilic carboxyl group* The solubilities of the lower members are quite high, but they decrease rapidly with increase in the length of the hydrocarbon chain* The solubility of butyric acid, for example, is Infinite at room temperature, while that of caproic is about one per cent and that of palmitic less than 0*001 per cent* Halston and Hoerr (88) made a systematic investiga- tlon of the aqueous solubilities of the normal saturated fatty acids from caproic to stearic between 0 ° a n d 60°,
These results tend to be a little high, since the interfer ence of .carbon dioxide was neglected* Solubility curves were later redetermined at the same laboratory (89) for the aclda from Cg to and at temperatures between 30° and 6o°. The solubility of water in fatty acids was also studied (90). In addition, John and MeBain (9 1) have carried out solubility studies of the even carbon acids from caprlc to stearic at 2f>° and 50°.In water, using conductivity measurements. The solubilities were found to oscillate with odd and even carbon acids; e.g., a given even carbon fatty acid would tend to be more soluble than its next higher even carbon homolog, but less soluble than the next higher odd carbon acid. A zigzag arrangement of the acids has been used to account for this phenomenon (92). The most extensive and probably the moat reliable solubility data on the saturated acids in non-aqueous solvents have been supplied by Ralston and coworkers (93# 9I4-) at Armour and Company. These investigators worked at temperatures between 0° and 60® using the so-called **syn— the tic1* method of solubility measurement. Known amounts of acid and solvent were placed in sealed tubes and set in a bath, the temperature of which was raised and lowered until the saturation temperature (i.e., the temperature at which crystals Just began to form) was determined. Curves were then constructed from this data.
* Solubilities of the normal saturated acids, both odd and even, from Cg to C^g were determined in ethanol, acetone, 2-butanone, benzene, glacial acetic acid,*cyclo- hexane, chloroform, carbon tectrachlorlde, ethyl acetate, butyl acetate, methanol, 1aopropanol, n-butanol, nitro- ethane, and acetonltrlle• In general, the acids tended to be more soluble In chloroform and less soluble In nitro- ethane and acetonltrlle. The same alternation of solubility between the even- and odd-carbon acids was observed as was noted with aqueous solutions. Solubilities of the even- carbon acids were also determined In toluene, o-xylene, chlorobenzene, nitrobenzene, 1,4-dioxane, furfural, 1,2- di chi or oe thane, and nltrome thane. In addition, there was some work carried out on three-component systems (95) • Hixon and coworkers (9 6* 97.» 9$) Have investigated the solubilities of certain fatty acids and derivatives in liquid propane, working at temperatures in the region of 100°. They studied binary and some ternary mixtures of propane with the following materialsi oleic acid, stearic acid, palmitic acid, cetyl stearate, trlcaprylin, trl- palmitin, tristearin, and refinedcottonseed oil. There have been several minor studies on fatty acid solubility undertaken as part of specialized research pro jects. For example, solubilities of caprylie and higher acids were detemlned in sodium glyoocholate solution at pH 6.2 (99). The solubilities of lauric, myrlstic, pal mitic, and stearic acids, as well as sever?! hydroxy acids, triglycerides, and other lipids, were detemlned at room temperature In tetrahydrofuran and in tetrahydrofuran con- taining 8 per cent water* (100) Curves have also been de termined Tor solubilities of the mixed fatty acids of copra, arachls, and castor oils as a function of the alcohol strength of the solvent (101)• In addition to work with the free fatty acids, the Armour group has reported solubility data on the methyl esters (102) of acids from capryllc to stearic in a number of organic solvents* Other work at the same laboratory has Included solubility studies of fatty alcohols (103)» ketones (IOI4.) , ,primary (105), secondary (106), and tertiary (107) amines, long chain amides, anllldes, and N,N-diphenyl amides (108), primary amine hydrochlorides (109), alkyl quarternary ammonium salts (110), dialkyl dimethyl ammonium chlorides (111), nltriles (112), and hydrocarbons (113)* Other solubility studies of fatty acid derivatives Include the work of Loskit (III4.) on the simple saturated triglycerides from tricaprln to trlstearln, that of King and coworkers (115, 116) on synthetic diacid triglycerides, that of Chen and Daubert (117) on a number of synthetic trlaeid triglycerides, and that of 3wern (ll8) on the ascorbic esters of palmitic and laurlc acids* There has been a vast quantity of solubility data recorded for numerous metal derivatives of the fatty acids, but since the soaps constitute a rather special type of fatty acid derivative, they have not been included here* Solubilities at Low Temperatures
Although considerable solubility work has been done with saturated fatty acids, data on the solubilities of the unsaturated acids are much less extensive* This Is partly because unsaturated acids are so difficult to ob tain in sufficient purity for solubility studies, but mainly because the unsaturated acids are almost infinitely soluble in most solvents at ordinary temperatures. Work on the solubilities of these acids must therefore be carried out at rather low temperatures* The only quanti tative studies which have included the unsaturated fatty acids have been the low temperature investigations carried out by Foreman and Brown (119 ) * Singleton (120, 121), and Hoerr and Harwood (122)* Tables containing much of the pertinent data collected by these investigators are repro duced on the following pages* Foreman and Brown (119) carried out the first im portant solubility study to be undertaken with fatty acids at low temperatures* What little data there had been accumulated up until that time (all of it on saturated acids) was listed by Brown (123) in a review in 19^1 (of* Table 1). The materials used by Foreman and Brown were of high purity, and the scope of their investigation was rather wide* They studied the even saturated acids from laurlc to behenic and five unsaturated acids, including 0 H\ * o d * 0 0
Table 2. Solubilities of the Saturated Acids (1 1 9).
(All solubilities expressed in p. /1000c;. solution)
t±emp A . M „ . Ace t one Methyl Alcohol Skellysolvo L °c.
LA’JRIC ACID Ou - — - - — 15.1 -10° _ - _ 23.3 10.7 -2 0 ° 17-4- 17-0 3.79 -30° 1 2 .3 8.23 — — — -50° 0.25 --- MYRISTIC ACID 0 U 2 2 .7 lb.Ip 9.4-3 -10° 10.7 8 .2 6 3.25 -2 0 ° 1+.33 3*44 1.31 -30° 1.74- 1.53 0.20 -50° ---- PALMITIC ACID 10° 17.7 13.1 6 .3 8 0° 7.15 3.96 1.25 -10° 2 . 30 1.4.6 0 .2 6 -20° 1 • 34. 0 .6 3 0 .1 2 -30° 0.4.5 0.2 0 0 .0 9 STa-AHIC ACID — JAU...... 2.59 1.31 0° 2 .1 9 0 .9 2 0 .24. -10 0 0.38 0 .3 2 0 .1 0 -20 0 0 .1 0 A2AC:-IDIC ACID 10u 1.33 0 .9 8 0.58 0 ° 0.75 0 .6 5 0.4-8
E E H E N I C ACID 10u 0.51 0 .4.2 0 .2 9 0° 0 .1 0 0 .1 0 0 .1 0 32 Table 3* Solubilities of the Un3aturated Acids (119).-
(All solubilities expressed in £*/lOOOp. solution)
Temp • Ac e tone Methyl Alcohol Skellysolve £ °c.
(a) EICCS3NGIC ACID -25^ ...... 1 6 .1 io76 _ -30° ii—59 3.5$ 2.95 -4.0° 1 *57 1.29 1 .0 7 (b) ERUCIC ACID -29'u 3.52 1 .7 6 - _ — -30° - — 0.87 o.59 (c) OLEIC ACID _ ^0 ° 1 5 .2 7 .0 3 1 1 .6 -50° 5 .1 6 3.29 5.33 -3o° 1.39 C . ;:9 1 .0 5 -So° o .61 0.51 0 .52 - ! 0 o.5 0.32 0.25 (d) LIEOLEIC ACID q O 5 3.2 25.2 17-0 _A 7° l/f.2 9.25 3.27 _ - - > C‘ ” i 5.15 . 3.95 0.09 ( e) LIITCLELIC ACID -6;^ ■-tO * 17.6 5 *0-3 Table if. Solubility Ratios cf’ Patty Acids Under Various Conditions (119). (All solubilities expressed in p. /1000 y. solution)
H a t i 0 Solve nt Temp . C1 e 1 c Pals’, 1 tic Oleic: Palmitic Tethyl acetate ™or c 1 0 .0 " . 7l|- Uf.7:l A ctlone jvon O 1 \ O 30 .0:1 _ Of. c O.afi diethyl alcohol 7-03 0 .20 35 -if: 1 Eutyl alcohol _2 rjO 6 2 .5 1.32 :i7r / • A * -*-1 EthylIdene dichloride -25° 2 6 .3 3 -2if 82.7 si Skellysolve b -30° 1 1 .8 0 .0 9 1 3 0 .0 :1 ■ Carbon disulfide -30° 15.7 0 .1 157.0:1 Toluene -30° 5 0 .2 0 .1 5 0 0 .0 :1 Ethyl ether -4.0 ° 4-3 -7 0 .1 If 50 .0 :1
Ratio Solvent Temp. LInoleic Ole ic Lino!eic; Oleic
Skellysolve B -70° 0 .6 0 0 .2if 2.5:1 Carbon disulfide -62 ° lf.1? 0 .308 1 0 .3 :1 1-ethyl alcohol -70° 3.91)- 0-32 12.3:1 Ace t one -70° 5.19 O.IjO 1 3 -0 :1 Table 5 » Typical Data of Singleton (121). Compositions of the Original Oleic Acid-Palmitic Acid-Acetone Mixtures and Phases at Equilibrium (Compositions are expressed in weight (gram) per cent of total solution).
SOLID PHASE ORIGINAL MIXTURE LIQUID PHASE By difference Py calculation Temp, Palmitic Oleic Solvent Palmitic DTeTc’ Solvent PaTmiTTc""$leic T O n l H c ' TOs'i'c °C. aclA acii. acid acid acid acid acid___
0 ♦ • f • • • • + • • • « • • t 0.650 ♦ + • « • 99.350 100.00 ..... 10 0.00 2.560 2.560 94.880 0.825 2.583 96.592 100.00 0.0 ..... 1.608 7 .1)82 Q0.820 1.103 7 4 3 6 91461 100.00 0.0 .... 1.686 l54o6 82.908 15.876 100.00 0.0 9 • • t I * 9 I • • T O T f • « * • • -wm ..... w ISO T ■ O ’ 0.670 2459 96.880 O.379 2.377 97*244 100.00 0.0 0.779 7.736 9 1 .ij.e5 O.ko2 7 .71)5 100.00 0.0 2.381 1L.6116 0.621 Ilu298 65.081 100.00 0.0 • 9 9 • « « I * • I 6.319 f • « 9 # m O.094 « • • • • 0.6 **14 + « • 9 I t 100.00 0.0 0.590 1 .5 4 s 97.865 0.134 1.51)3 96.323 98.00 2.0 1.182 3.945 9^.873 0.153 3.822 96.025 85.93 14.07 * • t • * * • 9 « • 1.062 9.382 89-556 0.156 5.212 9^.632 17.06 82.94 17 .61) 82.36 0.871 8.000 91.129 0.16 5.356 94.480 18.61 Bl.39 « * * * 9 9 9*99 0.035 9.612 J0.31 0.0 5.600 0.0 0.0 0.0 0.0 1 0 0 6 *•4*+ 100*00 9 9 9 9 1 7733T 9 9 9 • • m o.o3« • * t • • rss 9 9 9 9 9 100.00 ..... 100.00 0.307 * * 9 * * 99.693 0.032 99.968 1.879 1.253 96.868 0.065 1.2IU 98.691) 97.91 2.09 ..... 0.244 2410 97.346 0.060 1.967 97.973 28.87 71.13 ... 0.039 2. 0.039 1.978 7.983 0.0 100.00 ..... 0.232 2716w o.bij 0 .6 4 93IT 12774 ■55775 66.10 1 -3 3 1 i £ § 2 97.872 0.009 0.658 99.333- ,^»21 35.67 61^3 commercial hexane, the solubilities being carried out at temperatures from 0 ° to -30°. Typical data appear in Table 5. From Singleton's work it is apparent that the presence of oleic acid has a considerable solubilizing ef fect upon each of the saturated acids, but the saturated acids seem to lend relatively little enhancement to the solubility of oleic acid. Moreover, the solubilizing effect of oleic acid is more marked in hexane than in acetone. The most recent solubility study on fatty acids at reduced temperatures is that of Hoerr and Harwood (122). These authors made an intensive study of the solubilities of oleic and llnoleic acids in a number of organic solvents. The acids were very carefully prepared, and solubilities were determined by the so-called "synthetic*1 method, cover ing the temperature range from about 10° to -ip0 ° for oleic
(Table 6 ) and from about -10° to -5>0 ° for llnoleic (Table 7) Since this article was not published until the present investigation was nearing completion, the two studies tend to overlap to some extent. In general, however Hoerr and Harwood have worked within a somewhat higher tem perature range, so that in reality the two Investigations are complementary, the few values which do overlap only serving to confirm the accuracy of the data. Table 6. Solubility Data of Hoerr and Harwood (122) for Oleic Acid
g. oieie acid per iUU g. solvent 5 O > H O O H 0 c 1 • G Solvent -ij.0 ,0* •
' -30.0' 0 0 -2 0 . » n-Hexane 0 .1 1 .2 9.1 44*4 160 720 Benzene8, Eutectic system • • ♦ • 253 910 C yc1ohexane D Eutectic system • * 80 233 870 Carbon tetrachloride0 Eutectic ,. 2k. 6 68 160 590 Chloroform 11.5 2 3 .3 *4.6 .0 92 205 760 o-Xylened Eutectic • • 30.5 88 250 1100 Diethyl ether 1 .2 4*4 17.9 60 195 870 Chlorobenzene 2.5 6 .2 2 7.0 85 220 900 1 ,2 -Dichloro- e thane • <# < 0 .1 1.3 2 6 .1 130 670 Nitrobenzene® Eutectic system • • • • 220 1100 Dioxane** Eutectic system • ♦ • • 237 1120 Furfural® • # 0 .1 0.3 1.3 4*7 1 4 . 5 Ethyl acetate 1 ,6 12.2 185 750 n-Butyl acetate 2,3 6.4 IJ4..3 ifco 200 770 Acetone o,5 l.k 5.1 2 7 .4 159 870 2-Butanone 1 .0 2 .6 3.6 33.5 170 880 Methanol 0.3 0.9 4 .0 3 1 .6 250 1820 9 5.0^ ethanol 0.7 2 .2 9.5 47.5 235 1470 2-Propanol 1 .1 ? * 2 1 1 .5 55.0 226 1160 n-Butanol 1.3 4.0 15.2 56.5 100 950 Hi tromethane“ 0.1 0.2 0 .4 0 .6 0.3 1.0 Nitroe thane1 .2 .3 1 .3 2.2 3.4 8.7 AcetonltrlleJ .1 .3 0 .7 1.1 1.3 7.7
®Eutectic at 5 wt. % oleic acid, -9*2 Eutectic at 31:19 wt. % oleic acid, -12.1* °Eutectic at 9 * 4 wt• % oleic acid, -25.6°. Eutectic at 6,0 wt, % oleic acid, -31*0° ®Eutectic at 58.5 wt. % oleic acid, 0,0°. 'Eutectic at 61. 6 wt, % oleic acid, -3*3° SMiscible above 26,2° hMiscible above 94*5° iMiscible above 31*7° JMiacible above ol.0° 37 Table 7* Solubility Data of Hoerr and Harwood (122) for iiinoleic Acid
a.'T XSSrelg Acid t>er "10 ? g.’ Tblve’nt— Solvent -5u.u8— — -3 8.au— -iovs° n-Hexane 3.0 It. 3 53 170 990 Benzene® system e * 320 1250 C yc lohexane^ system • e 275 1210 Carbon tetra chloride0 Eutectic • * 70 160 6oo Chloroform 19-0 4.0 .0 88 210 770
Ethyl acetate 5.6 4 - 1 58 200 1300 Acetone 0 .0 27*2 lk7 1200 2-Butanone 1 0 .6 37*0 185 1220 Methanol 9-9 L 8 .1 233 1850 9 5.0# ethanol 1 1 .1 k2.5 208 1150 2 -Propanol 11.7 203 1080 n-Butanol 1 8 .9 56 180 870 Nltroethane® < 0 .1 0 .J+ 2 .1 8 Acetonltrlle® < 0 .1 0 .2 o.t k aEutectic at 7t 6 wt- % llnoleic acid, -2 1 .2°. ^Eutectlc at 51 8 wt* % llnoleic acid, -2 8*3°. cEutectlc at 31 9 wt* % llnoleic acid, -3 5 .3°. ^Mlcclble above 1*5°. eMisclble above 39-5. 38 III. EXPER IMENTAL
The experimental work Included in this study has been considered under the following headings: plan of in vestigation, preparation of the pure fatty acids, analytical procedures for estimating their purity, methods for purifying solvents, and techniques used in the actual solubility n e a s u remen t s.
A. Plan of Investigation
This investigation was patterned, in general, after that of Foreman and brown (119) except that in the present study more emphasis was given to the solubilities of the unsaturated acids, a larger number of solvents was used, and special attention waa paid to the problem of attaining equilibrium. The following acids were selected as subjects for study: palmitic, oleic, and llnoleic were chosen because of their general Importance in fat chemistry and their abundant natural occurrence.* It was also hoped that linolenlc acid might be included; however, difficulty was encountered In purifying the acid, and time did not permit Its inclusion in this paper. Eicosenoic and erucic acids
^It has been postulated that palmitic and oleic acids are probably present to some extent in all fats and oils • 39 represent relatively common monoethylenic acids with chain lengths different from oleic, Petroselinic acid was in teresting as a positional isomer of oleic acid. Previous work from this laboratory by Millican and i-rcwn (79 ) in dicated that a number of natural fats contained octadecenoic acids other than oleic. It was postulated that these fats contained isomeric acids, seme perhaps simple positional 3somers. Relative solubility data for clelc and petroselinic acids might Indicate v/hether cr not such Isomers wore separable by crystallization. Stearic, arachlciic, and be- henic acid solubilities were determined in order to round cut tho saturated series and to compare their solubilities with those of the corresponding unsaturated compounds, dlaidic, petroselaldie, and brassidic acids represented typical acids with trans configurations. do data on acids of this type have been published. Recent work from this laboratory (12lj_) has shown that the acids of buttorfat contain as much as lb per cent trans acids* Aork now In progress Indicates that the fractions from vegetable shortenings and oleomargarines contain about IpO-pO per cent trans acids (1 2 5 )- was felt that data on the solubili ties of typical trans acids might help in applying the crystallization method to the separation of such naturally occurring trans components. Solubility measurements were also made with stearolic acid, a synthetic acetylenic acid, since the material was available, having been prepared in 1^0 • this laboratory by Khan (126), and represented a type of unsaturated fatty acid whose solubility behavior had not been investigated. Solvents were chosen so as to include representa tives of a number of different solvent types. It was also necessary In such low temperature work to choose solvents which would remain liquid at -75°. Because a rather large number of acids was to be studied, the number of solvents was limited to six. Included were an alcohol, an ester, an ether, a ketone, and both an aliphatic and an aromatic hydrocarbon. Methanol was selected as the simplest member of the alcohol family, diethyl ether as the most readily available of the ethers, ethyl acetate as a common member of the ester group, and acetone as a typical ketone. In the aromatic hydrocarbon series benzene has a very high freez ing point, but toluene remains liquid at -95>° and is therefore suitable as a solvent for low temperature work. Petroleum ether is the solvent generally employed when an aliphatic hydrocarbon is desired. In Foreman's work Skellysolve B was used. Although much more uniform In quality than petroleum ether, Skellysolve B is nevertheless a mixtxire of hexanes and not a pure substance. The pure compound, n-hexane, Is a rather expensive material; how ever, Its closest homclogs, n-pentane and n-heptane, are both relatively Inexpensive. Since the high volatility 1+1 of n-pentane makes it less convenient to handle, n-heptane was selected as a model aliphatic hydrocarbon. A very limited study was also carried out using a series of other hydrocarbons in order to note possible effects of hydrocarbon structure on solubility. The procedure used for measuring solubility was a modification of that used by Foreman and Brov/n (119)* alternative method would have been the "synthetic" tech nique used by the group at the Armour Company. This latter method involves placing known amounts of acid and solvent in a tube and raising and lowering its temperature to determine the point at which turbidity begins. To use this procedure would have required the construction of a special bath, whereas the apparatus of Foreman and Brown was already available. Moreover, the heavy frost which forms on exposed surfaces at low temperatures would have made it difficult to observe the appearance of turbidity, wnile the analytical method of Foreman and Brown depends on the analysis of saturated samples withdrawn from the test solutions and does not require visual observation. The work of Foreman and Brown has been criticised (127) on the grounds that equilibrium conditions were not always attained, so a special effort was made in this investiga tion to reach equilibrium as completely as possible. When a solute is dissolved in a solvent, the curve of concen tration vs. time for the solution usually starts out with 14-2 a rather, steep slope and then gradually levels off at-the concentration of the saturated solution, Samples were with drawn at intervals at least twenty-four hours apart until two consecutive samples yielded the same analytical results* It v/as then assumed that the level portion cf the curve had Lccn reached and hence that equilibrium had been estab lished. Several experiments in which the test temperature was approached both from the warm side and from the cold side indicated that this assumption was valid.
B. Preparation of Fatty Acids
Most of the fatty acids used In this investigation were prepared from natural oils. When long chain compounds occur so abundantly in natural products, It is usually not feasible to synthesize them. On the following pages the methods used to prepare the individual acids are described. The preparation of oleic acid has been considered In great detail in order tc Illustrate the various techniques and types of apparatus used.
1. Oleic Acid Oleic acid was prepared from olive oil essentia H y by the method of Foreman and Brown (119)* a. Preparation of Methyl Bsters One kilogram of olive oil was refluxed for about *4-3 twenty-four hours with 1200 ml. methyl alcohol containing 2 per cent dry hydrogen chloride. The resulting esters were then washed several times with 3-lp 1. water. The water layer was removed with a syphon.and finally with a separatory funnel. The last traces of water and alcohol were removed by warming the material under reduced pressure.
The esters were then distilled under vacuum (ca. 1 -2 mm, Hg.) in an all-glass Claisen distilling apparatus, b. fractional Distillation of Methyl asters of Olive Oil.
The esters were fractionally distilled through a
Fenske column, 90 cm. long and 2 .3 cm. in diameter and packed with l/8w glass helices. The top and bottom halves of the column were individually heated with nichrome coils, regulated by variable voltage transformers. The distilling pot and head were both connected to the column with glass ball joints. The still pot was heated by a Glascol heating mantle which covered the entire flask. A variable reflux distilling head was 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 capillary was a Newman stopcock, by which the reflux ratio was regulated. A glass thermometer well extended down through the condenser, and a glass tube connecting the top of the condenser to the output tube kept the system open during total reflux. Connected to the output tube was a modified k k Pauli or cow-udder type receiver, so constructed that eight Tractions could be collected without disturbing the vacuum simply by turning the receiver. Round bottom 250 ml, pyrex Tlasks were used to collect the various fractions, A take-off pressure of about 0,1 mm, was main tained by means of a Cenco Idegavac pump, a Dubrovin vacuum guage being used for pressure measurement. Volatile ma terials from the system were caught in a dry ice trap. This apparatus afforded excellent separation of mixed methyl esters according to carbon chain length. An initial charge of 605> g- olive oil methyl esters yielded 505 g- of C^q esters (cf. Table c) .
c. Crystallization of Esters,
The ^2.8 e3ters of olive oil contain methyl oleate with small amounts of stearate and linoleate. The latter were removed from the oleate by crystallization at low temperatures. A solution of 23>0 g. of the above esters in 7 1*
acetone was prepared in a If? x lj.5 cm. pyrex crystallizing cylinder and placed in the low temperature bath pictured
in Pig* 1. The bath consisted of a wooden box i^.8 x I4.Q x 55 cm., heavily lined with rock wool insulation and containing
an inner cylindrical chamber 26 cm* in diameter and I4.5 cm. deep. This copper chamber was partially filled with acetone and copied to the desired temperature with dry ice. k s Table 8. Fractional Distillation of the Methyl asters of Olive Oil
Fraction Boiling range Weight Sap. equiv. Iodine 0.1 mm. in g. value
I 105-110° i-i-7 • 3 1.14-385 275.0 9.4 II 110-125° 22.9 1 .[{J4.38 2 6 6 .0 49.7 III 126 -12?° 60.7 1.4495 " IV 127 -128° 7 2 .1 1.4497 V 128-128.5° 1 6 1 .9 1.4495 > 301.4 92.9 VI 1 2 8.5 -1 2 9.5 ° 168.5 1.4494 VII 129.5-130° 314-.S 1.4494 ^
Orig. esters 605 -- — 84.2 A Weston low temperature thermometer with a bimetallic stem and a round dial permitted easy temperature reading. The cylinder was equipped with a special monel paddle type stirrer, and the solution was stirred slowly for about an hour or two, the temperature being held as constant as possible by small additions of dry ice. The 3tirrer was then removed, and the cylinder was left undisturbed for several minutes to allow most of the crystals to settle to the bottom of the vessel, thus facilitating easier and more rapid filtration. The liquid portion was remove d by gentle suction through an inverted porcelain filter into a br 1, suction flask (cf. Pig, 1). Four lead weights, previously hung on the cylinder, served to anchor the vessel as its contents were removed. The filtration was thus carried out at the temperature of crystallization. As shown in Pig* 2, the purification of methyl oleate required seven successive crystallizations. The first four, at -6 3 to -65°, removed the linoleate, the next two, at -35°, precipitated most of the stearate, and the final crystallization, at -65°, recrystallized the methyl oleate, leaving behind in the filtrate the small traces of stearate (and possibly linoleate) still present. Yield of methyl oleate, 136 g.; I.V., 85>.6. d. Preparation of Oleic Acid.
The purified methyl oleate was next converted to V 7 k Q
--M X -r . O — Preparation of Methyl Oleate by Crystallization of the C^g Esters of Olive Oil
25>0 g. C-^g Esters (Olive Oil) dissolved In cooled to 7 1 . acetone -65° C-l F- 1 47.6 g. dissolved. In cooled to 1.7. 136.7 7 1 . acetone -6^° C-T~ F-2 13*3 dissolved in cooled to i.v. 99.6 7 1 . acetone -63° c-T P-3 12.3 g . dissolved in cooled to I.V. 35.3 7 1 . acetone -63° c-ir T -h dissolved In cooled to I.V 7 1 . acetone -35° c-5 0.3 g. 1 65.4- s* dissolved in cooled to I4- 1 . acetone -35° c-6 “F -6 21.1 g. I4 4 . dissolved In cooled to i4. 1 . acetone -65° 0-7 “ F-7 136 g. 8 .2 g. I.V. 85.6 ^9 oleic acid. The ester was first refluxed with potassium hydroxide in alcohol, the alcohol was distilled off, water was added to the saponified material, and the mixture was acidified with excess hydrochloric acid. The oleic acid was transferred with ether to a Claisen flask and distilled ac a pressure of about 1 mm. The distillate was a col^rlvjs liquid with the following constants: m.p*, 13*3°; I.V.,
59-9* neut • equiv., 282.5; and np , l.lf6 0 0 . The acid was placed in sealed ampules under vacuum and stored at -20° until ready for use.
2. Elaidic Acid
Elaidic acid was prepared by isomerizing purified oleic acid. Sixty-seven grams of oleic acid was mixed with 670 mg. of powdered selenium and heated to 175-*l3p0 for about twelve minutes with nitrogen bubbling through the system. The mixture was then allowed to cool under nitrogen. A very small amount of Darco powdered charcoal was added for decolonization and the mixture filtered
through a sintered glass filter. Acetone (670 ml.) was used for transfer and solution of the oleic-elaidic equili brium mixture.
The acetone solution was kept at -2 0 ° overnight. The precipitated crystals were then removed by filtration, the yield of crystals amounting to 33 grams, although, the removal of solvent was not complete. The crystals were redissolved in 330 ml. of acetone and again kept overnight at -20°. The crystal crop was filtered off and dried under vacuum, yielding 2 0 .Ij. grams of flaky white crystals i m.p., !g3.6°; I.V. , 89*85; neut. equiv., 282.5*
3. Stearic Acid
Stearic acid was prepared by purification of the commercial product Hystrene 9 7 Stearic Acid, a gift from the Industrial Chemicals Department of Atlas Powder Company* The material was first esterified, and about 500 grams of the methyl esters were fractionally distilled. About 60 grams of low boiling esters came over between IOI4.0 and 12if° at 0.1 mm. pressure. Then a 365 g* fraction of methyl stearate was collected at I2I4.-1250 * The methyl stearate was saponified and reconverted to the acid, which was washed three times with hot water and then recrystallized several times from petroleum ether (3O-600 ) at -20°. The purified stearic acid came out as white flaky crystals: m.p., 69*50; I.V., 0.01; neut. equiv., 28q.5*
!4 . Palmitic Acid
The palmitic acid used had been prepared by Foreman (1 1 9) by fractional distillation of the methyl esters of palm oil. The acid was recrystallized from petroleum ether before use and had the following constants: m.p., 6 2 .8°; I.V., 0.0; neut. equiv., 256.3* 51 ‘ 5. Erucic Acid
Erucic acid was prepared from rapeseed oil, in which erucic constitutes the principal acid, being present to the extent of over 4.O per cent of the total fatty acids, Hapeseed oil methyl esters (14-75 6*) were fractionally dis tilled as shown in Table 9 yielded 193• U- £• of ^22 esters with a mean molecular weight of 3 5 2 .2 and an iodine value of 71*8.
The C2p esters were crystallized from acetone first at -2 0 ° to precipitate the behenate present. The solution was then cooled to -60 °, at which temperature methyl erucate is practically insoluble but more highly unsaturated esters tend to remain in solution (cf. Pig. 3). After two crystal lizations at -60 ° the iodine value of the recovered methyl erucate was only 71 *7 5 (theor. 7 1 *9 9)* product was redissolved in acetone and cooled slowly until crystal formation was observed (ca. -30°)• The solution was held for an hour at that temperature and the precipitated crystals removed by filtration. The filtrate contained 70.0 g. of methyl erucate, I.V., 72.0. The ester was converted to erucic acid and distilled: yield, 65*i|- g*J m.p.» 33*5°; I.V., 75*0; and neut. equiv., 338.5* 52 Table 9- Fractional Distillation of Methyl Esters from Rapeseed Oil fraction Boiling range Weight Sap. equiv. Iodine 0.1 mm. in g. value
I 95-115° 4-3 256.1*. 32.4 II 115-117° 7.9 2 7 0 .7 18.5
III 117-120° 6 .0 2 8 0 .3 62.3 IV 120-1 24 ° 1 6 6 .9 2 9 7 .0 148.5 V 12l4.-ll4.0 o 9-7 313.6 92.7 VI 114.0 -114.10 43-7 3 2 2 .0 7 6 .0 H O H VII i4l-i54° • 3 3 3 .6 72.2 VIII 1514.-156° 1 9 3 .4 352.2 71.8 IX 1 56 - 160 ° 7.5 3 6 1 .1). 7 0 .6
Residue -- 21.8 - —
Qrig. esters 53
Fig. 3. Preparation of Methyl Erucate by Crystallization of the C^2 Methyl Esters of Hapeseed Oil.
Cop Eaters (R hapeseed Oil) 80 g.; I.V. 71.8 dissolved in cooled to If 1 . acetone -2 0 ° C-l i 5*3 g. 79*1 g*; 1 .v. 73.7 i.v. gi.o dissolved in cooled to 2 1 . acetone -60 0
C-2: f-2 73*9 i.v. 7 2 .3 5 .*+ g. i.v. 9 3 .1 dissolved In cooled to 2 1 . acetone -6 0 0 C-■3 *-3 71 .^4- 6 » i I.V. 71.8 2.3 g. I.v. 8 9 .2 dissolved In cooled to 2 1 . acetone -30°
1*3 s* 7 0 .0 g. I.v. 67*3 I.V. 72.0 5 h 6 . Eicosenolc Acid
Eicosenoic acid was also obtained from rapeseed oil. The methyl ester fractionation shown in Table 9 yielded one fraction of mean molecular weight 3 2 2 , the correct value for the CgQ series. The fractions from this and a similar distillation were combined and refractionated through a I4.5 cm. packed column, yielding 1^9 *4- grams of esters. These were further separated by low temperature crystallization according to the scheme in Fig. J4.. The final crystal fraction, C-3, was saponified and the free acid distilled under vacuum. A colorless oil was obtained which gave the following constants: m.p., 22.0°; I.V.,
8 1 .7 ; neut. equiv., 3 1 1 *0 .
The arachidic acid used had been prepared by Foreman (119) by the catalytic reduction of eicosenoic acid. It was recrystallized twice before use to give colorless plates: m.p., 74-*7°; I.V., 0.0; neut. equiv., 313.0.
8. Bohenic Acid
The behenic acid used in this study was the purified material prepared by Foreman (119)* It was made by the hydrogenation of erucic acid and had the following constants: m.p., 80.0°; I.V., 0.0; and neut. equiv., 3i|-0*3* 55
Fig i^. Preparation of Methyl Eicosenoate by Crystallization of the C20 Esters of Rapeseed Oil.
14.5.5 g- c20 Me Asters (I.V., 7 6 .0) dissolved in cooled to 2 1 . acetone -2 0 ° F-l 3.0 g. 1^2.5 g.; I.V., 82.1 dissolved in cooled to 2 1 . acetone - 60 ° C-2 F-2 37.5 g.; I.V. 77.5 5.0 g. dissolved in I cooled to 2 1 . acetone I -5 5 to 60 C-3 F-3 32 g.; I.V. 7 8 .1 5.5 g. 56 9. Brassidic Acid
Brassidic acid was prepared by elaidinization of purified erucic acid, using the procedure described for the preparation of elaidic acid. The crystals were colorless and had the following constants: m.p., 60.0°; I.V., 7if*97* and neut. equiv., 338.6.
10. Petroselinic Acid
The petroselinic acid used in this investigation was prepared by Fusari (128). His method included frac tional distillation of the methyl esters of parsley seed oil followed by low temperature crystallization of the fraction. The material had the following analytical con stants: m.p., 30-30.2°; I.V., 88.73; neut. equiv., 28l.7«
11. Petroselaidic Acid
Petroselaidic acid was prepared from petroselinic by isomerization with powdered selenium (cf. preparation of elaidic acid)• The material crystallized in flaky colorless crystals which had the following constants: m.p., 53.0°; I.V,, 89.25; and neut. equiv., 282.0.
12. Stearolic Acid
The sample of stearolic acid used in this study was prepared by N. A. Khan (126). His procedure involved 57 bromination of oleic acid to the dibromo derivative, followed by dehydrohalogenation with sodamide in liquid ammonia to the acetylenic compound, Khan*s material was recrystallized twice from petroleum ether at 0 °C. The recrystallized material consisted of white needles which melted at .2-lj_9.50 and pave the following; constants: I.V., 39.5; neut. equiv., 280.0*
13- Linoleic Acid
Pure linoleic acid was prepared by a modification of the method of Matthews, Brode, and Brown (Ij.6 ) . Fifty grams of material, obtained by debromination of tetra- bromostearic acid, was purchased from the Kormel Institute and repeatedly crystallized from petroleum ether at -6 2 ° (cf. Fig. 5). Seven crystallizations yielded a product which melted at -5 *2 ° and gave the following constants: I.V., lol.O; neut. equiv., 280.^; , 1.1+701; per cent purity as determined by bromination (I4.3 ) » 1017°. 58 Fig. 5* Crystallization of Hormel Linoleic Acid
I4.2 .7 g. Hormel linoleic acid (m.p., -10.6°) in cooled to ether -62 ° C-l F-l 8-9 g* dissolved in cooled to 1 1 . pet.ether -62 ° C-2" F-2 2 .8 g. dissolved in cooled to 1 1 . pet.ether -6 2 °
C-3 F-3 2.3 g. dissolved in c o d e d to 1 1 . pet.ether -62 ° e n F—1+. 1 .2 g, dissolved in cooled to 1 1 .pet.ether -6 2 ° F-5 2 .1 g • dissolved in cooled to 1 l.pet. - 62° ether Q 1 “F- 6 2 3 *8 g.; m.p., -5*8° 1 .6 g. dissolved cooled to in 1 l.pet. -6 2 to -61*.° ether C-7 F-7 2 1 .7 g.; I.V., 1 8 1 .0 . 2 .0 g. ra.p. -5 .2 ° C. Analyses Used for Criteria of Purity
1 . Neutral Equivalent A 1-3 g. sample of acid was dissolved In about £0 ml. of neutral ethanol and titrated against 0.1 N aqueous sodium hydroxide, using phenolphthalein as indicator. A 10 ml. microburet was used to measure the quantity of alkali required.
2 . Iodine Value The official Wijs one hour method was used. (129)
3 . Melting Point Melting points were determined using two different procedures. One was the capillary method, In which a small sample of acid was placed in a fine capillary and placed in a suitable bath. For those melting points lying above room temperature a well-stirred oil bath heated with a micro burner was used. As the melting point of the sample was approached, the rate of rise in temperature was slowed down
to about 1° per minute. Temperature was measured with a
thermometer graduated in 0 .1 ° divisions and calibrated against a Bureau of Standards thermometer. For those sub stances melting below rpom temperature the oil bath was replaced either by a water bath cooled with Ice or by an alcohol bath cooled with dry ice. The bath was cooled to
about 5>° below the freezing point of the sample and the 6 0 temperature allowed to rise very slowly by thermal trans fer from the atmosphere. For very low melting acids a special thermometer was used covering the temperature range -3 0 to 0 ° and graduated in 0 ,1° divisions. A second method involved the construction of time vs. temperature heating and cooling curves. About 5 g* of acid was placed in a 1,5 x 15 cm. test tube. This was then set inside a 2 , 5 x 15 cm. tube, the tubes being held together with a rubber stopper. A cork carrying a ther mometer graduated in 0 .1° divisions was placed in the smaller tube. This outfit was then placed in a suitable bath liquid contained in a 3-4 1» vessel. As the bath temperature was very slowly raised or lowered, the tem perature of the fatty acid was plotted against time. The temperature at which the curve levelled off (i.e., where the temperature became momentarily constant) was taken as the melting point of the compound. The flatness of this region of the curve was also an indication of purity.
A summary of the analytical constants of the
fatty acids used in this investigation appears in Table 1 0 . Reference values are also included. Theoretical iodine values and neutral equivalents were obtained by calculation. The melting point reference values represent the most reliable of previously reported melting points for these acids• 6i Table 10. Analytical Constants of the Patty Acids.
Acid Iodine Value Neut • Equiv• M.P. Ref. obs. the or . obs. theor• obs • value o o Palmitic « - 256.3 256 .lf2 6 2 .8° 6 2.9° a
Stearic 0 .0 - 284.5 281+.*4.7 69.5° 6 9.6° a o o Arachidic • - 313.0 312.52 74*7° 75.35° a o o Behenic • - 314.0.3 340.57 8 0.0° 79.95° a
Oleic 89-9 89*87 282.5 282.^5 13.3° 13.36° b n n Elaidic 89.85 282.5 43.6° 14.3.68° ° O 0 N 0 Petroselinic 88.73 281.7 w 30--3 0 .2 ° It n Petroselaidic 89.25 2 8 2 .0 53.0° 53° c
Eicosenoic 81.7 81.75 3 1 0 .8 3 1 0 .5 2 2 .0° 22® 8.
Erucic 75.0 Ik- 98 338.5 338.56 33.5° 3 3 .1*° ° n M Brassidic 74* 97 338.6 6 0 .0° 60° ° Linoleic 1 8 1 .0 1 8 1.0 ^* 2 8 0 .4 2 8 0.1*4 -5.2° -5.2-5.0° •Stearolic 89.5 181.03* 2 8 0 .0 " 14.6 .2-14.6 .5 ° I4.80 1
a. Francis. P., and Piper, S. H.f J. Am. Chem. Soc., 6l, $77 (1939). t , — b. Smith, J. C., J. Chem. Soc., 97H- (1939)* c. Griffiths, H. N., and Hilditch, T. P., J.Chem. Soc., 2315 (193^). d. Hopkins, C. Y . , Chisholm, M. J., and Harris, J., Can. J. Res., B27, 35 (19*4-9). e. Matthews, ». L. , Brode, W. R., and Brown, J. B., J. Am* Chem. Soc., 6^, 1064 (1941)* f. Postemak, M • 7., Compt• rend., 162, 944 (19X6) •
«The practical iodine value is actually one-half the theoretical value, since only one molecule of iodine Is added to the triple bond. 62 D. Purification of Solvents
All solvents used were reagent grade materials and were redistilled before use. Prior to distillation some of the solvents were treated with suitable drying agents: toluene and diethyl ether were dried with metallic sodium; acetone and ethyl acetate were dried over potassium car bonate; and anhydrous methanol was prepared by treating C.P. methanol with magnesium and iodine. The hydrocarbon solvents were pure grade chemicals (99 mol % minimum) ob tained from Phillips Petroleum Company. The solvents were usually distilled through a small Vigreux column, the first and last portions of distillate being discarded.
E. Procedure for Measuring Solubilities
1. Description of Constant Low Temperature Apparatus
An apparatus for providing constant low temperatures has been described previously by Foreman and Brown (119). So successful was this apparatus in maintaining constant low temperatures. It has been used in this investigation without change of design* Pictured in Fig. 6, the bath consists of two indi vidual monel metal compartments, one a working chamber and the other a cooling unit. The bath measures 95 x 55 cm. at its top surface and is 1^0 Qm* deep. The dimensions of the two monel compartments are 26 x 29 x 27 cm. each. 63 and they are surrounded on all aides with 12.5 cm. of rock wool insulation. Alcohol is the medium of thermal transfer in both compartments, a mixture of ethanol and methanol being used. The working chamber of the bath is equipped with an electric stirrer, a bimetallic thermostat, and an iron-constantan thermocouple, through which the temperature is measured and recorded by a Leeds and Northrup Model R ■»'icromax Recorder. She thermostat operates by controlling a pump in the cooling unit of the bath, starting and stop ping it by a time-delay relay. The pump forces the bath liquid from the working compartment through a copper coil in the cooling chamber and then back again. Thu3 dry ice can be added to the colder reservoir without disturbing the
flasks in the working chamber (e.g. with excessive foaming) *e and only a small portion of the working bath liquid is circulated through the cooling coll at a time, so that the bath temperature is not lowered too rapidly. By means of this apparatus it has been possible to maintain a constant temperature below room temperature and as low as -75° with a maximum deviation of 0.5°* although the limits have actually been more narrow in moat cases.
2. Equilibration of Patty Acid Solutions.
Into the working chamber of the bath described above were placed the flasks whose contents were to be equili brated. Wide-mouthed 250 ml. Erlenmeyer flasks were used Fig. 6. Constant Low Temperature Apparatus 65 for this purpose. The diameter of the flask openings measured 3*5 cm. so that they would permit insertion of a 3.0 cm. sintered glass filter tube. Six of these flasks could be conveniently kept in the bath at one time. To each flask was added about 150 ml. of solvent and an excess of the fatty acid whose solubility was being studied. The flasks were then stoppered and placed in the bath on metal racks which served to steady them. After they had been allowed to reach approximately the same tem perature as the bath, the stoppers were carefully replaced by neoprene stoppers fitted with mercury sealed stirrers. The flask contents were stirred rapidly for about ten hours, after which time the stirring was discontinued and a sample of liquid drawn off. The flask was usually allowed to remain in the bath overnight, and then stirring was resumed for ten or twelve hours and a second sample drawn. This procedure was repeated until two successive samples yielded the same results. It was then assumed that equilibrium had been established as nearly as possible. The temperature of the bath was then lowered 10° and a second set of solubility data taken on the same acid- solvent mixtures. This process was repeated until the desired temperature range was covered. An improvement on the equilibration technique was realized by the Installation of a mechanical rocker to 66 replace the Individual mercury-sealed stirrers. The rocker consisted of a perforated copper plate, equipped with holders for six ml. flasks, joined by means of a connecting rod to a motor located outside the bath. A continuous duty speed reducer motor was used to move the rocker at a rate of 30 cycles per minute. The rocking device is pictured In operation In Pig. 6.
3, Withdrawal of Samples from the Saturated Solutions.
After about ten hours of stirring or rocking, agita tion was stopped and the solutions allowed to stand quietly for about ten minutes. Then the stoppers were removed, each being replaced immediately by another neoprene stopper fitted with a 3*0 cm. sintered glass inversion filter. The filter sticks had 7/2f> inner joints at their open ends, covered by rubber caps to keep out moisture. Each filter disc was pushed down sufficiently far so that it was close to, but not touching, the surface of the solution. The filters were allowed to remain in that position for about an hour in order that they might be cooled to the tempera ture of the bath* In drawing a sample the filter disc was pushed down into the solution and the rubber cap replaced by a special withdrawal pipette (of* Pig* 7) equipped with the outer member of the joint on the filter* A sample of saturated liquid was then drawn up into the bulb of the pipette by
68 gentle auction and, by proper manipulation of the two-way stopcocks, allowed to flow into a tared 125 ml* glass- 3toppered Erlenmeyer flask, Ihe flask was then refitted with its glass stopper, allowed to reach room temperature, and weighed to the nearest 0,01 gram.
Calculation of Solubilities
After a sample of saturated liquid had been drawn and weighed, the solvent was removed under vacuum and the flask reweighed. The acid residue was then dissolved in 25 ml, of neutral ethanol containing phenolphthalein as indicator. The alcohol solution was titrated to the phenolphthalein end point against 0.01 N or 0.1 N sodium hydroxide in a 10 ml. microburet. The titration values thus obtained were converted to gram per cent solubility, i.e. grams/100 grams solution, by the following equation! t r ml. x If x mol. wt. g T T 5 ------in which X s solubility in grams per 100 grams solution N & normality of alkali ml. • milliliters of alkali mol.wt. * molecular weight of fatty acid g m grams of solution
The solubility values calculated in this way were checked against those obtained gravimetrically, using the weights of the acid residues in the equation! 69 Solubility - wt. acid x 100 wt• solution
5. Problem of Attaining Equilibrium Sample data for several fatty acid-solvent systems taken at tine interval's during the approach to equilibrium appear In the following table.
Table 11. Sample Data Showing Change of Solubility with Tine*
Aoid-Uoivent Temperature, - 30u sample so. Mixture encroached from 1 2 3 it Oleic aeld- warm side I.7 0 1 .6 8 1.68 Acetone cold side i:8 1.68 1 .6 8 1 .68 Erucic aeld- warm side 0 .2 0 3 0.160 0.121 0 .11 2 Ethyl acetate cold side 0 .0 9 5 0.110 0 .11 3 0 .111 Petroselinic aeld- warm side o .0625 0 .051 O.Olfl O.OifO n-Heptane cold side 0.037 0 .039 O.OijjO O.OlfO Elaidic aeid- warm side 0.077 0 .0 6 5 0 .0 6 k 0 .0613 Methanol cold side 0 .06 1 0 .0 6 k 0.065 o .o 6t Stearic aeld- warm side 0 .0 5 5 0 .0 5 3 0.050 0.051 Diethyl ether cold side 0 .05 1 0.051 0.052 0.051
*The test temperature was approached first from room tempera ture and then from the temperature of a dry loe-cooled bath. The flasks were plaeed in a -30° bath and stirred ran idly for about five hours, then the first sample was taken. A second sample was removed after five more hours of stirring. The flasks were then allowed to remain (unstirred) at -3 0 ° over night. On the following day stirring was resumed and samples 3 and if were withdrawn at successive five-hour intervals.)
These data indicate that equilibrium was attained for these representative systems within a two-day period. Thus, in most subsequent work an Initial sample was removed 70 from, each solution after 1 1/2-2 days in the bath, and then a second sample was taken on the following day. Agreement of results for the two samples was accepted as evidence that equilibrium had been attained. Table 11 also demonstrates that equilibrium can be more rapidly attained when the test temperature is ap proached from the cold side. The reason for this undoubtedly lies in the fact that fatty acids tend to form supersaturated solutions which would impede progress toward equilibrium when the test temperature is approached from the warm side.
IV. SOLUBILITY DATA A compilation of the data obtained during the course of this investigation appears in Table 12. The same data are represented graphically In Figs• 8 to 13* In these graphs solubility (in grams/100 grams solution) has been plotted against temperature in °C. In order to consider the relative solubilities In the various solvents on a more equivalent basis, the solu bilities have also been plotted as the logarithm of the mole fraction of acid vs. the reciprocal of the absolute temperature. This method has been applied to a number of solubility studies carried out by Hildebrand (130) and has been used by Bailey (127) for correlating fatty acid solubilities. Typical seta of curves plotted in this manner appear in Pigs. Ilf to l6. Ideal solubility curves 71 Table 12. Solubility Data on Fatty Acids in Various Solutions (All values In grains acid per 100 grains solution)
Solvent Temp• taethanol Ethyl Diethyl Acetone 'i'oluene formal Acetate Ether______Heptane Stearic Acid 10° 0 .2 6 0.58 2.k0 o .5i^ 0 . 0 .0 8 0 0 .0 9 0 0 .1 3 0.95 0 .1 1 0 .0 8 0 0 .0 1 8 -10° 0.031 0.027 O .3 8 0 .0 2 3 0 .0 1 5 0.00^ -20° 0.011 0 .0 0 6 0.15 0 .0 0 5 0 .0 0 3 -30° 0.051 Oleic Acid -20? I*.*02 5*95 5 .2 0 2.25 -30° 0.66 1.90 1 .6 8 3.12 0 .6 6 4 o ° 0.29 0.62 5.15 0.53 0 .9 6 0 .1 9 -5o° 0.10 0.20 1.80 0 .1 7 0.28 0 .0 5 0 -6o° 0 .0 3 0.057 0 .6 1 0.055 0.075 0 .0 1 1 -70° 0.21 Elaidic Acid 0° 0.59 -10° 0.£q 0.86 0 .1 9 -20° 0 .1 8 0.29 l.kO 0 .2 6 0.20 0.060 -30° 0.0614. 0.10 0.60 0 .0 9 2 0.056 0 .0 1 9 4 o° 0 .0 2 0 0 .0 2 7 0.23 0 .0 2 9 0.013 0 .0 0 7 -50° 0 .0 1 0 0 .0 0 8 0.10 0 .0 0 9 Iilnoleic Acid
- 50° 3 .1 0 1*.. 1 * 0 * it.io e» 0 .9 8 -60 ° 0 .9 0 1 .3 8 1 .2 0 S t , 0.20 -70° 0 .2 5 0.39 0.35 0.01*2 Araehldlc Acid
1 0 o 0 .0 8 0 0 . 11*. 0 .9 0 0 .1 3 0.12 0.028 0° 0 .0 2 8 0 .0 3 6 0 .3 8 0.035 0 .0 2 6 0.005 72 Table 12. Solubility Data (cont.) (All values In grams acid per 100 grams solution)
------" /^oTrenfc Temp• Methanol ithyl Dletbyl Acetone Toluene formal Acetate Etber Heptane Eicosenolc Acid
"20o 0 .8 0 1 .3 0 - 1 .1 0 *,3°o 0.35 0 .6 0 3.90 0.54- 1 .1 0 0.45 0 .1 5 0 .2 6 1.70 0.27 0 .3 0 0 .1 5 -50 0 .0 6 0.11 0.68 0.12 0 .0 7 0 .0 4 8 -6o° 0.02 0 .0 4 0.22 0.05 - 0 .0 1 Petroselinlc .Acid -10° . o.5o -20° \ 0.48 0.73 3*52 0.78 0 .9 8 0 .1 3 -30° \ 0.18 0 .3 0 1 .6 8 0.31 0 .2 3 0 .0 4 0 -^°o » 0 .0 6 0 o.ll 0 .7 6 0.11 0 .0 ^6 0 .0 0 9 0.018 0 .0I4.0 0 .3 1 0 .0 3 5 0 .0 0 8 - -60 ° — - - - Petroselaldic Acid
0° 0 .5 0 *» V 1 .4 0 0 .2 2 -10° 0.20 0 .3 6 0 .3 2 0 .1 9 0 .0 3 0 -20° 0 .0 8 2 0 .3 4 0.13 0 .0 6 0 0 .0 0 8 -30° 0.028 0 .0 5 0 0 .5 1 0 .0 5 0 0 .0 2 0 0.002 0.010 0.018 0.19 0.019 0 .0 0 8 • -5o° - 0 .0 7 0 -- - Stearolic Acid 0° -10° 0*65 1.15 4*85 1 .2 6 1 .7 0 0.050 -20° 0 .2 5 0.45 2 .0 0 0.52 0 .4 0 0.010 0 .1 0 3 0.17 0.78 0.19 0 .0 8 3 0 .0 0 6 -£0° O.OifcO 0.065 0.32 0.070 0 .0 1 8 - Palmitic Acid
10° 1 .3 0 1 .6 0 1 .4 1 0 .3 0 0° 0.L6 0.52 2 . 9 5 0.66 0 .3 6 0 .0 8 -10° 0 .1 6 0.18 1.35 0.27 0 .0 8 6 0.02 -20° 0 .0 5 0 0 .0 6 0 0.56 0 .1 0 0 .0 1 8 0.005 -30° - 0 .0 1 8 0.21 0 .0 3 8 -- 73 Table 12. Solubility Data (cont*) (All values In grams acid per 100 grams solution)
' ' Solvcni/." ... "" Temp* Heinanol aibyl i>l ethyl Acetone toluene formal ______Acetate Ether______Heptane Behenlc Acid 0 0 O H H H o oo 0 .0 1 9 0.055 O .4 8 0 .0 5 0 0 .0 4 0 0 .0 1 2 0 .0 0 7 0 .0 1 6 0 .1 8 0 .0 1 4 0 .0 1 0 0 .0 0 2 1 0 .0 0 2 0 .0 0 4 0 .0 6 8 0 .0 0 4 0 .0 0 2
Hruclc Acid o o o 0 0 0 o o o o 1 1 1 1 1 o . li-9 0. 3 5 0.19 0.31 0 .2 8 0 .6 8 0 .1 1 0 .0 6 6 0 .1 1 1 .2 0 0 .1 0 0 .1 6 0 .0 3 0 0 .0 2 4 0 .0 4 0 0.49 0.037 0 .0 4 4 0 .0 0 6 0.007 0 .1 8 Brassldlc Acid O O O 0 o H CvJ H o oooo 0.27 0.70 0. 6 8 0.62 0.20 0 . 094 0 .2 6 0 .2 4 0 . 1 8 0 .0 5 8 I 1 1 0.035 O .0 9 6 O .7 8 0.0 6 5 0.050 0 .0 1 6 0 .0 1 0 0.028 0.28 0 . 024 0 .0 1 3 0 .0 0 5 0 .0 0 3 0.01 0.10 . . ,00 Gro*» *>'u,i0n Groms fcc'd a s -
Oleic
Steorolic
Polmitic
HO. ™ ______Eicownoic
Ftetroselinic
______Steorolic
Brossidic Grams Acid in 100 Grams Solution 1.0 0.2 0.0 6 0 0.4 i. 0 Slblt o Fty cd i n —Heptane. in Acids Fatty of Solubility 10. Fig. 60 -6 o 40 -4 Uj o 20 O tu o lit Ui Eicoseooic Petroselenic Steoroiic £'\co$en°'c 100 Groms Solution Acid *n
Petrosehnic Erucic Stearate Eloidic o—
Petroseloidic
Polmitic
Brossidic Stearic X lO4
Fig. 14. Solubility of Stearic Acid in Various Solvents. Il lll»lal«lal 50 48 46 44 42 40 38 4 - x IO *
Fig. IS Solubility of Oleic Acid in Various Solvents. Log Mole Fraction of Linoleic Acid i. 6 Slblt o Lnli Ai i Vros Solvents Various in Acid Linoleic of Solubility 16. Fig. x s 83 have been drawn in order to indicate the deviation of the actual solubility curves from ideality. Hie ideal curves have been constructed according to the equation: log N2 = - ag 1 - 1 H*- T
in which 1*2 = mole fraction of acid AH - heat of fusion H - gas constant T * absolute temperature Tm * melting point (in °K.) of acid
In Table 13 ***e listed a limited number of solubilities of oleic, linoleic* and stearic acids in a series of hydro carbon solvents. The solubility of stearic acid was also studied in dimethyl formamlde. The data obtained were as follows: Temp. Solubility (in g./lOO g. solution) 10® 1.15 0° 0.38 -10° 0.102 -20° 0 .02*4. The solubilities are higher than those for most of the other solvents. Dimethyl formamlde was not further studied be cause of its high toxicity and its low volatility* which made solvent removal difficult. It was not considered a convenient medium for use in the low temperature crystallisa tion procedure. 84 Table 13. Patty Acid Solubilities In Various Hydrocarbon Sol vents (All values expressed In grains acld/1 0 0 grams solution)
Stearic Acid Methyl Iso- Temp. n-Heptane Cyclohexane n-Pentane pentane
10° 0.080 0.20 O.O89 O.096 0° 0.018 0.06 0.015 O.OI4.O -10° O.OOI4. 0.021 0.003 0.017 -20° - 0.005
Dllso- 2 -Methyl Iso- Temp. propyl Neohexane pentane octane 10° 0 .07 0 0 .0k 0 .0 8 6 0.051 0° 0.015 0 .0 1 0 0.03 0 .02 0 -100 o.ook 0.003 0.01 0.008 -20° - - 0.003 0.003
Oleic Acid Methyl Dllso- Iso- Neo- 2-Methyl Temp. n-Heptane Cyclohexane propyl octane hexane pentane -I4.O0 0.19 0.34 0.212 0.162 0.13 0.19 -50° 0.050 0.11 0.112 0.070 0.050 0.08
Llnolelc Acid Methyl Di iso Temp. n-Heptane cyclohexane propyl -50° 0 .9 8 2 . 0 6 o .9k -6o° 0 .20 0.38 0 .1 7 -70° 0.0^2 0 .0 7 2 0 .0 3 2 85 V. DISCUSSION The data presented above are Important primarily as physical constants which serve to extend our knowledge of the physico-chemical properties of an important class of long chain organic compounds. However, their practical value, from the standpoint of fatty acid separation, lies In the fact that they Indicate relative solubilities, in six different type solvents, for such series of acids as: stearlc-olelc-linoleic (C1g acids with increasing unsatura tion) ; palmitic-stearic-arachidic-behenic (saturated acids with Increasing chain length ); stearic-oleic-elaidic, or stearic-petroselinic-petroselaidic acids, saturated and els and trans unsaturated); arachidlc-eicosenoic (Cgo acids, saturated and uns&turated); behenic-erucic- brasaidic (C22 acids, saturated and cis and trans unsaturated) stearic-oleic-stearollc (C^g acids, saturated, olefinic, and acetylenic); and oleic-petrosellnic unsaturated acids with double bonds in different locations)• Of course the data listed here indicate only what the simple solubilities of these pure fatty acids are with re spect to one another. If each fatty acid in a mixture went into solution Independently and there were no mutual solubility effects, It would be possible to predict and to carry out rather remarkable separations of these com pounds by crystallisation. However, no information Is given by these data as to how these same acids behave when ■ 86 they are present in mixtures. Actually It Is known that fatty acids do show mutual solubility effects, but the degree to which they Influence one another depends both upon the solvent and upon the nature of the particular mixture under consideration. Waentig and Pescheck (131) investigated the inter- solubillzation of saturated fatty acids in a series of organic solvents. They reported relatively small solubiliza tion effects in ethyl alcohol, ethyl ether, ethyl acetate, and benzaldehyde, but rather large effects in chloroform, carbon tetrachloride, benzene, toluene, and nitrobenzene. The effects were especially marked in the case of the chlorinated solvents. For example, the solubility of palmitic acid in carbon tetrachloride was found to be in creased 250 per cent by the addition of lauric acid. It was assumed that there was some sort of compound formation between the acids in such solvents. The results of Kalston and Hoerr (132)* however, were not in agreement with those of Waentig and Pescheck. In fact molecular association seemed to have little influence on the solubility behavior of the binary fatty acid mixtures which they studied. They found instead that solubility appears to be closely linked with melting point, the most soluble binary mixture being that of the same composition as the eutectic mixture. Solubility studies carried out by Singleton (120, 121) with ternary systems, consisting of two fatty acids 87 plus an organic solvent, Indicate that the presence or oleic acid has a rather large solubilizing effect upon palmitic or stearic acid, but that the saturated acids have relatively little Influence on the solubility of oleic acid* It was also shown that the nature of the solvent is Important, the solubilizing effect of oleic acid being considerably larger in hexane than In acetone. Although It must be kept In mind that such mutual solubility effects do exist, the solubility curves for the pure acids are nevertheless valuable as guides for determin ing what conditions to use in attempting separation of given mixtures by the crystallization method, or for deciding whether or not low temperature crystallization is feasible for the separation of a particular fatty acid mixture. An additional phenomenon which sometimes prevents clear-cut separations by the crystallization method is that of mixed crystal formation. The close similarities in size and chemical properties of certain fatty acid combinations make it possible for the molecules in the crystal lattice of one acid to be partially replaced by those of another* The physical difficulty encountered in removing fil trate from crystals might also be mentioned* When large volumes of fatty acid crystals are precipitated, certain quantities of mother liquor tend.to become incorporated with the crystal fraction, thus preventing complete 88 separation of crystals and filtrate. This difficulty may be remedied either by recrystallization or simply by wash** ing the crystals with precooled solvent. An Inspection of the solubility curves shown on preceding pages reveals a number of general trends in fatty acid solubility behavior. Solubility is shown to be enhanced, of course, both by decrease in chain length and by unsaturation. The introduction of a double bond greatly increases fatty acid solubility, the increase being greater the farther the point of unsaturation is removed from the carboxyl group. Changing an acid with a cis double bond to its trans isomer reduces its solubility considerably, as does converting its olefinic function to a triple bond. It Is easy to understand why unsaturated acids can be so successfully removed from their corresponding satur ated compounds by low temperature crystallization; and it is obvious from these curves why elaldlc acid can be so easily crystallized from its isomerization mixture with oleic acid by crystallization from alcohol at about -25°, and why petroselaidic and brassldic acids can be similarly crystallized from their respective cis isomers. The solubility curves provide a guide by which condi tions may be chosen for attempting the separation of a given mixture of fatty acids. Stearic acid might be separated from elaldlc, for example, by crystallization from methanol or n-heptane at 0 ^, from ethyl acetate, acetone, or toluene at -10°, or from diethyl ether at -20°. Or, In the series, behenlc and brassidic acids might be separated with ether at 0°. Brassidle and stearic acids, on the other hand, would appear to be almost impossible to separate by crystalli zatlon procedures, A mixture of oleic and stearolic acids would be expected to be relatively easy to separate by crystallization from heptane at -2 0 °, and oleic and petrose- linic acids should be readily separable from a toluene solution at It has been shown that although the location of the double bond along the chain has a definite effect on the melting points of cis unsaturated acids, the trans compounds are not so affected. These data are insufficient to show whether or not the same relationships hold for the solubili ties of the cis and trans acids, but It is likely that they do • The postulation by Ralston and Hoerr (132) and by Bailey (127) that solubility is closely linked with melting point Is supported by these data, at least in a qualitative sense. In general, the order of the solubility curves for the various acids is the same for all six of the solvents studied; however, one peculiar reversal was noted* The solubility of stearolic acid appears to change relative to eruclc and elaldlc acids in the following manner: In acetone, methanol, and ethyl acetate, stearolic > eruclc> * 90 elaldlc; In ether and toluene, eruclc > stearolic > elaldlc; and In n-heptane, eruclc > elaldlc > stearolic. On the basis of comparative melting points alone, stearolic would be expected to behave In all solvents as It does In n-hep- tane; that Is, stearolic acid (m.p. 6 .5 °) should be less soluble than elaldlc acid (m.p. 1|_3»7°) , which Is In turn less soluble than eruclc acid (m.p. 3 3 -5°)• The propor tionately greater enhancement of solubility of stearolic acid by polar solvents as compared with eruclc and elaldlc acids might indicate that stearolic acid is somewhat more polar than the other two compounds. In Table 13 are listed the results of a limited study on the solubilities of stearic, oleic, and llnolelc acids In a series of hydrocarbon solvents. There appears to be not too much difference In the solvent properties of the various hydrocarbons studied. However, several effects of solvent structure may be noted. The cyclic heptane, methyl cyclohexane, Is a better fatty acid solvent than is the corresponding straight chain compound. n-Heptane ap pears to be a slightly poorer solvent than its five-carbon homolog, n-pentane. Similarly lsooctane appears to be a poorer solvent than the homologous isopentane. Hydrocarbons containing the lsopropyl group seem to have solvent proper ties not too different from the normal compounds, while the neopentyl group appears to decrease fatty acid solubility to a small degree. A comparison of the curves In Pigs. II4.-I6 indicates that deviations from ideality in polar solvents are greater for oleic than for stearic or linoleic acids and suggests that oleic acid is the least polar of the three. A similar finding was reported by Hoerr and Harwood (122). The following dipole moments have been reported for these acids (133): stearic e .3 .u. oleic n linoleic n Although these values have been criticised (I3I4.) with re spect to their absolute accuracy, the comparative magnitudes would appear to verify the solubility results. All of the systems Included in this study exhibit positive deviations from ideality. Such deviations result from the combination of intermolecular forces existing be tween the fatty acid molecules and those of the various solvents. The effects are magnified by the great differ ences in size and shape which exist between the organic solvent molecules and the long chain acids. The inter- molecular forces are of several different types. They include forces of dipole attraction, due to the polarity, or electrical dissymmetry, of the various molecules; hy drogen bonding forces, resulting from certain peculiarities In the structure of the hydrogen atom, making it capable of forming linkages e.g. between two atoms of oxygen; and the attractive forces referred to as van der Waals or London forces, which, operate between all molecules and which are best explained in terms of quantum mechanics. Many authors use the term nInternal pressure1* to refer to the total attractive forces between the molecules In a liquid. It would be theoretically possible to calcu late the solubilities of compounds knowing only the Internal pressures of the solutes and solvents. For nonpolar sub stances of similar structure and equivalent molecular volume the calculated values agree quite closely with actual solu bilities. This Is not the case, however, with solutions of fatty acids In organic solvents, where the disparity In size, shape, and structure between the solute and solvent molecules and the large hydrogen bonding forces present In the pure liquid fatty acids make the calculations In valid. Actually there is no consistent relationship be tween either the internal pressures or the dipole moments of the solvents and fatty acid solubilities. The solubilities listed for oleic acid refer in each case to the most stable fora of the acid in that system. The dimorphic character of oleic acid causes it to exhibit two solubility curves in some solvents. With the "synthetic* technique of solubility measurement used by the Armour group of investigators, it has been possible to observe portions of the unstable curves in same solvents• However, the • • analytical method followed in the present study Involved 93 a long waiting period to permit the establishment of equilibrium, and, since the transformation from the un- stable to the stable form occurs rather rapidly, the oleic acid data listed here may be assumed to refer to the stable modification of the acid. The sources of error inherent in the procedure used for determining solubilities may be summarized as follows: (1) The purity of the compounds studied is always an im portant factor controlling the accuracy of solubility data. However, the fatty acids used In this study were of suf ficient purity that error due to solute Impurities should be of minimum significance, (2) The use of pure solvents is necessary in order to obtain reliable solubility information. In this Investigation all the solvents used were reagent grade materials and were re distilled before use. Host of the solvents were also treated with suitable drying agents before distillation, (3 ) If true solubilities are to be measured, the system under consideration should be allowed to reach equilibrium. It is difficult to know whether or not complete equilibrium was achieved in all the systems studied here, but a definite effort was made to equilibrate the solutions as nearly as possible. Samples were drawn about twenty-four hours apart until two consecutive samples gave results which agreed within experimental error. It was then assumed that equilibrium conditions had been reached* Actually it Is 9^ possible that certain of these fatty acid-organic solvent mixtures might approach equilibrium at rates which are al most imperceptibly slow. However, several experiments in which the test temperature was approached both from the cold side and from the warm side indicated that several days was usually adequate for the establishment of equili brium. (I4.) Water has been shown to have a marked effect on the solubility behavior of fatty acids, so it was important that moisture be excluded from the solutions. At the low temperatures used in this work condensation of atmospheric moisture was very troublesome. Precautions were taken wherever possible to minimize contamination which might arise from this source. For example, mercury-sealed stirrers were used during the early part of the work, and later a rocking device was installed so that the flasks could remain tightly stoppered during the agitation period. Each stopper was kept covered with waterproof plastic in order to prevent ice crystals from forming Inside the flask lip and falling into the solution when the stopper was removed. When it was necessary to unstopper the flasks Ce. g. for insertion of filter sticks for sample removal), the process was carried out very rapidly, so that the flask contents were exposed to the atmosphere only for about a second. (5) In the ease of the unsaturated fatty acids there is some danger of impurity formation through air oxidation of the acids at double bonds, For this reason the purified unsaturated aclda were stored at -20° C. either under nitrogen or in sealed evacuated tubes. During solubility measurement the flasks were filled with nitrogen before being placed in the constant-temperature bath, although there was really not too much danger of oxidation at the very low temperatures used in most of this work, (6) Probably the largest source of error to be considered is that due to temperature, since solubility varies greatly with temperature In most of these systems. Extreme care was exercised to see that the bath temperature was set as precisely as possible, and the Micromax temperature recorder was checked frequently for proper adjustment, The apparatus used was capable of maintaining a constant temperature to within at least 0 ,5 °, and the limits were sometimes within 0 .1°, Nevertheless, in those regions of the solubility vs, temperature curves where the slope Is very steep, even a small discrepancy in temperature may produce significant error in the solubility results, (7) The taring of the 125 ml* glass-stoppered flasks and the weighing of samples were carried out on a sensitive analytical balance, so that the weights were accurate to 0 ,1 mg. When samples of solution were drawn, they were stoppered and allowed to reach room temperature before being weighed, so as not to Introduce error from thermal effects, Actually the solution weights had to be known only to an accuracy of 0 ,0 1 g. Since It was possible that the weights of the acid residues might be a little high (e.g. because of Incomplete solvent removal or slight oxida tion of the acid during evaporation), the gravimetric re sults were used only to check the magnitude of the results obtained volumetrlcally. (8) Titrations were carried out with standardized sodium hydroxide solution using a 10 ml* microburet, by means of which volume could be easily read to 0*01 ml* A well- lighted white enameled titration stand afforded excellent conditions for observing the end point indicator change* The titration values, for the most part, were considered accurate to within several parts per thousand* For purposes of comparison Table li*. has been pre pared* This table compares certain solubility determinations made during this study with values which have been reported by other investigators* In general, the agreement Is very good* However, several discrepancies may be noted* The solubilities in ethyl acetate, for example, tend to be considerably lower than those of Hoerr and Harwood. The value listed for diethyl ether, on the other hand, is sig nificantly higher than that of these investigators* It Is interesting to note that in a number of cases the results of this study agree quite closely with those of Foreman and Brown, indicating that their ten-hour waiting period was adequate in these Instances for attainment of equilibrium* o
97 . 1 • Table li+. Comparison of Solubilities with Data Previously Reported by Other Investigators (All solubilities expressed In grams acid per 100 grams solution) 0 0 0 H o OJ H 0 0 Stearic Acid 0 ° 1 1 Methanol 0 *2 6 „ 0 .0 9 0 0.031 0.011 0 . 259^ 0 .0 9 2 F 0.032P 0.010F
Acetone 0 .1 1 p. 0.023™ o.oo5a 0 . 219* 0.038^ o.ool*3 0 . 79* 0 .2 1 R 0 . 021S 0 .1 0 3 3
Olele Acid - 2 0 ° - 30° -l*o ° - 5o ° - 6 o° Methanol 1*.02 0 *8 6 0 .2 9 0 .1 0 0 .0 3 3 . 9s 0 . 708* o .329P 0.089* 0.051* 0 . 9s 0 . 3s Ethyl 5 .9 5 1 .9 0 0 .6 2 Acetate 1 0 . 9s 1*. ^ i . 6h
Acetone 5 . 2° 1 .6 8 0 .5 3 0 .1 7 0 .0 5 5 5.6o3 1 . 1*2* 0 .5 1 6 * 0.189* 0.061* 2 .0SS 0 .6 6 s 1.1+H 0 . # Diethyl Ether 1**37 1 . 2«
Eloosenole Acid - 2 0 ° - 30° - 1*0 °
Methanol 0.80 0 .3 5 0 .1 5 l . o 6F 0 . 3l*£* 0 .1 2 9 * Acetone . 1.10 0 . 51* 0 .2 7 l.6l* 0.1+58* 0.11*7*
F. Data of Foreman and Brown (119) • S. Data of Singleton (120, 121). H. Data of Hoerr and Harwood (122). R. Data of Ralston and Hoerr (93)* Table 14« Comparison o f Solubilities (cont.) * . (All solubilities expressed In grams acid per 100 grams solution)
J — — — - --- 0 H 0 O 0 0 1 1 1 H O ro Palmitic Acid 0° 0 0
Methanol 1.30 0 .I4.6 0 *1 6 0 .0 5 0 1.31 p 0.396f o.U*6p 0.o63P • 1.3“ o.8r Ethyl 0.52 Acetate 0 .8 Acetone 1 .6 0 0 .6 6 0 .2 7 0 .1 0 0..038 1.77F o.7i5p 0 .280P 0.13l+.P 0 .01^ 1.90R o.6or 0.289S 0.09U.8 0 .035s 0.65s
Linoleic Acid -5o° -60° - -70° Methanol 3 .1 0 °.9o 1 0.25 2 .5 2 0 • 925 o.39l*-p 3 .2H
Ethyl Acetate 5.3s ■
Acetone l^.io 1.20 0.35 ' * 4*82 l.lf2 0.519 • 3.2® . • Eruelc Acid -20° --30° m
. Methanol 0 .1 9 « 0 .068- « • » • • * * 0.176 0 .0 8 7 * •
Acetone • 0.28 * 9 * 0.352
F. Data of Foreman and Brown (119) • S. Data or Singleton (120, 121)* H. Data or Hoerr and Harwood (122)* R. Data or Ralston and Hoerr (93)* • • « * • • m * 1► * • • * * • 4 • 99 * . • • * Table li+.* Comparison ot Solubllltl.es (cont*) (All solubilities .expressed In -grams acid per 100 .grams solution) • « p • • * • - « » • * 0 H 'Arachidic Acid o 0 ° Methanoi 0 ,0 8 0 ’ 0 .0 2 8 o.096p o .o 6 5p Acetone 0 .1 3 0 .0 3 5 * 0 . 183P o .075p 0 H BehSnlc Acid o 0 ° 4 .* Methanol '■ 0.019 0.007- • . * ( 0 . 0l+2p 0.010P Acetone . - ■ 0 .050' 0 .0 1 4 * * ‘ ■ * , o . o5 i p 0.010P P. Data of-Fbremsn And Brown (119) S..‘Diti of Singleton (120* 121)• H. . JData of H<«rf .'tnd H*rwood (122) fi* .-Ditt of Ralston - and Hoerr (93)*
• 1 ; ‘ « * * '*- VI. SUGGESTIONS FOR FUTURE WORK
1* Since association phenomena tend to interfere with certain separations involving free fatty acids, a solubility study should be carried out with unsaturated fatty acid esters. Up to the present time the only ester solubilities
reported have been those of the saturated acids (1 0 2 ). 2. The triethylenic acid, linolenic, is rather conspicuously absent from the data presented here. Attempts were made to
prepare pure linolenic acid, but up to the time of this writing a product of sufficient purity for solubility measure ment has not been obtained. Any discussion of unsaturated
fatty acids Is Incomplete without this important unsaturated
fatty acid. Efforts to prepare this acid should be con tinued, and Its solubility in common organic solvents should
be studied. The only data now available on this acid are
the few isolated values of Foreman and Brown ( 1 1 9 )• 3. Solubility Information on all the various octadecenoic acids, Including both cis and trans Isomers, would be very
valuable. It would be Interesting from a theoretical stand
point to correlate the solubilities with the corresponding melt.Irig point data. The data would also have practical
•application in the separation and Identification of naturally • • * 1 ‘ • * * * * * .* ' 1 . • , t ’ occurring*Isgmeric-*oct^decernoic* acids*. ■ • * • . * * ~ - •; * . ■ • • ■ • * ■ . • ■ ■ •*•• .■ * ‘ .* . . * $ecauser, of * the solubijizatidp effePtSand mlied .'crystal:. * formatien’exhibited* py fajb'ty acids-,’ th^' sol-ubilitfes of the . *. acids when they are present in mixtures are different from those of the pure acids. It would be desirable to carry out an extensive solubility study with three-component systems, made up of two fatty acids and an organic solvent-.
Several investigations of this type have been reported by ‘
Singleton (120, 121), but oleic acid was the only unsaturated acid which was studied. Our work has indicated that linoleic and linolenic acids, for example, exhibit marked mutual solubility effects* Solubility measurements taken with mixtures of these acids would thus have great practical v a lue•
5. The solvents used In this investigation were limited to six common organic solvents. The work might be extended to include such solvents as acetonitrile, dioxane, carbon disulfide, nitroalkanes, and chlorinated hydrocarbons.
SUMMARY
1. The following fatty acids have been prepared.In high' purity by a combination of the methods of fractional methyl, ester distillation and low temperature crystallizations’-
oleic acid, .from- olive o i l ; stearic- acid*,; from Hyfctrpne-.^J-S'. (commercial ^stearic acidi.Jf*‘and’*erucic and eicosehdio..acidsNV from rape seed' oil.' - ‘•Linble.ic ac id ’* was obtained hy\ repeats dV crystallization, of. mateMfel ^rbpar’a&htiy ttte* Hormpi**Iiis tit fete . . V - . **. - . : . . *V ; / •• • '• (by .debr,omina1U*on>. *’Add!tiondl'hlgnly purified .ejomfiounds.V .* *. • •,* * •*•• * *• • ' already available included b’&li<ic, aracnidic, behenicj • and pet*roseliplc acids and t h e * synthetic• acetylenic*acid. o «
* • *% ;• * 102. •* * ■ * •• • . h * . stearolic. Elaidic, petroselaidic> and brassidic • acids.* * • . * * . ‘ have been prepared by isomerization of oleic, petroselinic., * • • and eruclc acids respectively.
2. Solubility measurements were made according to the pro- cedure of Foreman and Brown (119)* modified so as to assure attainment of equilibrium.
3. A special withdrawal pipette was designed for removing samples of saturated solution. It consisted essentially of a 50 ml. glass bulb equipped at both ends with two-way stopcocks and connected by a standard taper joint to a sintered glass filter tube.
if. Although mercury-sealed stirrers were used during the early part of this work, they were later replaced by a mechanical rocker which afforded simultaneous agitation of six solutions and. allowed the.flasks to remain tightly stoppered* ' - ■. - .
* 0 " . 1 - ' * 4 , ’ * * * * • '* 5* • 3 ©lubi^^ty ^terminations have be eh made in methanol, ethyl/ja£e$a^^ diethyl, .etjiei*, .and . • ,v tAfferent'. isolveht:’;types °
• large, but* several geheraf^Izat JLons^c’oncdhdlng. the iijfjJ. lienee * • • • • • • • •* • • • • of -solvent structure have been drawn from.the data.
* * s 7* Solubility data have been presented both in tabular 'and In graphic form.
3. The data have been compared with those of other in vestigators in those cases where previously reported values were available.
9. The solubility data have been discussed with respect to their application in separating various fatty acid mixtures.
10. Factors tending to limit the separation of fatty acids by the method of low temperature crystallization have also been mentioned.
11. A number of suggestions for future work have been included. • •
v - - • • 1 0 1 4 . * ■ BIBLIOGRAPHY V •'* . . ■ * * • * » *,• * t* *i * . . ■ * * 1. Brown, J. B. , and Stoner,. G.S-,., J. Am. ‘Chem* Soc..k‘ . . £ 2. 3 (1937). . / ' . ’ 2. Brown, J. B., and Shinowara, G,Y., ibid., ff9v .6 (1-937X*' 3. Chevreul, M. E. ,*Recherches sur lea Corps Gras"i.l823. If. Robinson, C. S., and Gilliland, E. R» , "Elements of Fractional Distillation," 3rd ed., McGraw-Hill,- N.Y., 1939. . 5. Morton, A. A*, "Laboratory Technique in Organic Chemistry," McGraw-Hill, N.Y., 1938.
* . * 6 . "Technique of Organic Chemistry," A. Weissberger, ed., Vol. if, Interscience, N.Y., 1951* .7. 7. Murray, K. E., J. Am. Oil Chemists1 Soc*, 28, 23-5 * ‘ • *- (1951). 8. Haller, A., Compt. rend., lq.3. 69k (1906). .. 9. Detwiler, S. B., Jr., and Markley, K. S., Oil-^c Soap, 1 6, 2 (1939). 10. Todd, S. S., ibid., 20, 20$ (I9I4.3). 11. Carney, T. P., "Laboratory Fractional Distillation*,* MacMillan, N.Y., 19k9* * . - * * * , , . . ♦ * * * 12. Tswett, M . , Ber. deut. botan. Ge.s., 2lfc, 3l6-, 3814- Xl906*‘)v. ■ • * * * ■ .' 13* Cassidy, H. G., J. Chem. Education,. 1'6’, 88 ..(-1939) * . : V* ■ * .*..*' -■' •:, v v ' - ;iif. Cassidy, H. G. , J. Am.-. Chem.’ Soc . , 8 2 3 0 7 3 i * 3076 X v 15. Cassidy, H. G. , ibid., 6^, 27 3 5 1 9 ^ 1 V. : • . ‘ f 7 *; . •. | • - - .• ° 0 *• T6 . Grat f , M. M., and Skau, E . L... Ind. Eng. .Chem. •••Anal.. V* '* ' Ed., is,- 3fo (X9ii3).. : .....•• *• ,1 • . ^ , > > i7. Dutton, .H. J.; . J. . Phys. Chem., Jfc§#.179; (J . 9 W U V : • •• * •*»* . . . * .. # • V • • *1*8. Matchett., J. R., Legault, R„*R., Nlmmo;.C. C ** and • ..*! Hot.ter, G. K., In 1 0 5 * « e .26. • Holman,**R. T., J. Am.. Chem. Soc./73# 126l (l*95l). • • « #* « « • ■ §• * * • * * m Hart In. *A. J.. P.,. and Synge, R. L. M., Biochem. J., * J & & < • >■/•*< 22/ -Broymj-PV* Biochem. J., k7, 599 (195>0)* * * - -*>**•“.•’** 4 # ^ • * • *• « * • • . • '"“231* * Kaufman,* H. P., Szakall, A., and Budwig, J., Fette u. ’ ; •• .SeEfen, 406 (l95l)*. ‘■ 2|^, ■ : Bi'sc'ojx.,^ * . *E* R., ' and Berridge, •* • N. J., Mature, ‘ 1 * 6 6. 522 \*(-l£50), • . . * • ■ 4 25i "Kaufjaan,* H'* P.*, Budwig* J., arid*Duddek, E., Fette u. ’ ’SelTen, *53', 2d5 (19510* ’ * ■ * ■ *■' "r' • , . • 26i.:’,'Br6wn^ P. and Hall, E. P. *,m Nature, 166. 66 (1950). 27." . “Bolding^,* J., Experlentia, i±, 270 (19^8) • ■ .. , • a • • . 2 $ . ; ^ CTraig,vL; C.f an *, . ? *■“" c * * • .‘3'£*' . Oysseijojr, C. A., Arch. Phamu, 2£$ 153 (1828). . ’ :„-'v : V^rrentrapp,. P., Ann.*, 35, 197 (18I4.O) • J # * ■ •3k*v Twltchell, J., Ind. Eng. Chem., 13» 806 (1921) . ;o”o‘ *. •*.* * * * * • ;35> *. I*e wk owl tech,* J., J. Soc. Chem. Ind., 9# 8Il2 (1890). • . * •Christopoulos, T., Praktika (Akad. A the non) , 9# 220 ** * (I93if). * • *37* • F*rnsteiner, K.. Z. Untersuch. Nahru. Oenuaam., 2, 1 (1899); k, 63 (1901); 6 , 161 (1903). ~ 38. EachSr, H. H., Helv. Chim. Acta.,12, 103 (1929). 39* Taujimoto, M., J. Chem. Ind. Japan, 2£, 1007 (1920). ko. Brown, J* B., J. Biol. Chem., 8 0, 1^55 (1928); 8 3. 777 (1929)* . 1 -• - *.* * • „ ' . -*■ **; - .. . % • •; • * 106 • .4 !*** * Sjninowara,’ G. Y., and Brown, J. B. J. Am* Chem. Soc., • • ■ r: §2., 273k .* «*»? . (1938). • * ’ U *■;; 1^.2°. Brown, J. B., and Frankel, J., ibid., 60 , 5k' (.193.6).* •* * *■° * • . '’•■'’,43. «hite, M. F., and Brown, J. B., J, Art* •C;,ii CUemis'ts.' ’ •Soc* , 2 6 /3 3 5 (19I+9) b 44. ibid., 26 , 133 (1949)• 45* White, M. F., 0 riar*3 , B. M.., and Brown, J. j--** , ibid., 2 6 , 53 (1949-). b.:.: 4 6 . Matthews, h . L., Brode, VP. R. , and Brown, . J, B., J. Am, Chem. Soc . 62, 3 06 .V (ly'.l) ■ 47* Bengen, F ., German patent application. 0, JL„ 1 2 .Y3 B - . , (March 1 > ) «: C^ch. Oil Mission neel l-i3, pp.’ 135-139)*-. ■ 0-//’ : J . j Biminerschied, W.* J , , L'i ner s t:e i h,' B . A * , .vett-Bamp, A. W . , and M^rschner/ H . . F , , Ind., -Bn p. Chem.,* 4 2 , 1300 (1950); J. Am. Chen. Soc., ?1 , 2 O4 ? 't- V9« Schlenk, H.f and Holman, 2. T., J* Ap. Chem. Soc. , 7 2 , 5001 (1 9 5 0 ). ;;'V4 '7 :;: v. v; * V5 7 . ibid., Science, 112, 1 9 (1950). 51., L instead, H. P., and Whalley , M-. ,.. J*, ChemA'Soc . , (1 9 5 0 ). ■ * 52. * Sw^rn, D. , and Parker. W . ^ , f J. Aru Gil Chem •Soc.. > 29, blk (1952). //\ v " • • A 53. * ibid.*:, 3£*, 5 (1953). •* ■»• «* * 5k* BaIJ.evy» »V. «A./“ J^., Bannerot, 'H. A., Fetterly, L. C, / • and Sn^lth, A. G,/ind, Bn£. Chem* 2125 (1951). * • J * i" 55. Fette^ly, L* C., y*S. Patents" .2 , 320 ,715 and 2,520,716 .' (1 9 50) t • • • * * • ‘ 56. Inoue ,*!.,• an^ Yukawa', Bull;. 1 cf. *C^iejp.e‘Soc . Japan, 16, 50i+ (194-0 )1 % • / • # *}'m** ** k : v • * * • «" 4 I, . . / V*„* 57. Icnoue, Y., Yukawa, H., and* Kfitsumata, ,H. ,*'ibi^ . 7 B 7 /•*/'•... * s‘ J4.91 (19^ 1 )• .*- • ; * * •; ...... • i y r . V « * ' • • 0,5 • »■ c /" *' (!« 58. Inoue, Y., and Yukawa,^., ibid. 5 ij • 1+11 *< i9l+l)^/; * O » * •• ■ •* . * 9• * a AO ® 10? 59- Yukawa, R . , and Inoue, Y . , ibid. ,. ,18 , 915 (l9q-2). Inoue, Y., and Yukawa, H. , Ibid. 1J_, 771 (I9 4 I) . Inoue, Y. , and $©'#a> i*U , J. Afcric. Chem. ^oc. Japan, i|, 29)+ 1193-0) . ~ 62. tertram, 3. H., Rec • trav. chim. , 6, 397 1 1 9 2 7 )* 63* Raynion'd, A., Chi-mie 9 Industrie, special Ko^ 523 (Feb. 1 9 2 9). J. and Brown,.J. 5., J. Am. Chem. 0 0 c., 6 3 * 65. Shd-nowara, G-. Y.., and Brown, J. 3. , ibid., 6 0 , 273*+ (193'S) • Cramer, B. 3., and Br.own, J. B. , J. Biol. Chem., 151« 4 ?7 (19^-3) - 6 7 . Baur, F. J., Jr., and brown, J. £■., J. Am. Chem* Sec. , 63. Smith. F. A., and Brown, J. B., Oil A Soap, 22, 277 ( l S f e K •own, J. - B. , and Brians, £. h., Arch. Biochem., 9, ... 2 01 (19ij.6 ) , , 70 • Shinow.ara, G . Y. , and B-rown, J. 'u. , J. Biol. Chem., 71. itfowry, i>. 9., Bro.de, A. R. andor own,' J. u. , ibid. , . . . 1^2,, 6 7 1 X 1 9 4 2 ) . B';-.-. 7 2 . brown, «I. U.„, and -^reen, R. 'B.., ; J. Am^ Chem. Boc. , ; . 6a,, ..... : 5v:'25'-v.;y.;:S; 7 '3B. Hod-d'e,, B. , and. ’tfllke., C., ange/h Che^m., 35>V 2'"89 ■d^5B :;Ay|g|2 2 -) . ' 74*- ’/Bef fie-r,;• •’Ii •.«1• -••••P-v> •'••• •Tv-.;Soc.•.Chem. Ind. , 55* 3311 (193^6 •) •’ 75* 3chumannC •\ £., ihdV Eric;. Chem., _8 , 5 (1 9 1 6 ) . 7 6 . Thomas, A. 7/., and Thomson, J. C.,J. Am. Chem. 3oc., 8 9 6 (193J+) • 7 7 .-* Ku, P.S., Ind. Eng. Chem., Anal. Ed., 9> 103 (19371-“’ i • * ’ v ‘‘ * * > • ♦ • * •• * • • • » • . * * • . %* * • 7 6 . Larle, F. R. , and Milner, R. T. , Oil b Soap, 17, 106 (19*4-0) . -- 79« ^illican, R. C., and Brown, J. S., J. biol. Che™. , 15k, J+3.7 (19U4) • 30. Frankel, J. S., Stoneburner, W., and nrown, J. S., J. Am. Chem. Soc., 6 g, . .2$9 (194-3)- 31. Lemon, H. >V., Can. J. Research, 27 By 6-Q5 (193.9) « 32. Swift, C. £., Oollear, F. C„, and O'Connor, R. T., Oil 3: Soap, 22, 353 (I9Z4.6 -) . 33* Holman, R • T. , Lundberp, W. 0 , , and Burr, 0. 0 . , J, Am. Chem. Soc., 6 7 , 1390 (194-5) • oip. Hilditch, T. P., and Riley, J. P., J. Soc. Chem. Ind., 6 Jt, 204. (1 9 4 5 ) - 35® Doershuk, A. P., and Daubert, b. 1'., J. Am. Oil Chemists1 Soc., 2J5, Lj.25 (19*4-8). 86. Kistler, 'R.-h.,, i^uckerheide, V. J., and Beyers, L. . D. , Gil & Soap, 21, 156 (194.6 ) . 8 7 * Seidell, A., "Solubilities of 0rr-a.nlc Compounds," Vol. II, 3rd ed., D. Van Hostrang, 194-1- 8 8 . Ralston, A. VV., and Hoerr, C . W"., «J. Crr. Chem. , 7 > 5 *4-6 (19+2). 8 9, Bgr,enber£er, D. H. , brobme, 2. K . , Ha 1::t o n , A %' 0 . Hperr, C. A., Pool, 3V.v ,6 y and. Ralston, -I. »*., Oil d . Soap , 19 , 126 (I9y8;) V-7 - /jl. John-, L. M., and i--chain, J. A’., J. Am, Oil Chemist's* ° * •• . .;Soc. , 2^, .NO (1938). • •' r . * • r . •'■V 0 ^ .*• . a * • * \ . *»• * * 92. -Kuthy, S., J. chim. phys. , ^2, 4.0 6 13-935? * • *•* i • -* .Ra’lsJtfbrw* ft-.-, WV y ail’d HperrC .-W. , ibid.* 1C*, 170 '•(19^ . : . • • • “ - - * ‘ •• °* . 0 © * •• •. •• • •-. ?v■ • * • . • . ,o . . . * 6 9, ; •• • :•* • •• •“ * * ■ . ' ‘ . • * ® ' © 96. Hixon, A. A'., and Ilixon, A. R., Trans, -am. Inst. Chem. Enrrs . , ?7, 92? (1941)• 97.- .rlixon, A. Vi,, 1 and be c Telman. -J • r . , ibid., 33, 891 (1 ? 4 ) . — OH Drew, L). A., and Hixon, A. H . , Ibid., 42, 675 (19^)* 99* Hoi we r da, A., biochem. A,, 294, 372 (1937). 100. Oremer, H. D. , ahd oe * uhlcr, H.., ibid., 3 2 0 , 112 (199-9). . . o ini. I.Iargaill an, L., and 0*0.3 tain? > 0* . , ll^'.ne Conp. . chim. Intl.., Paris, Oct. 193?-. (C.A. 2 9, 6>f; Ij_G) . 102-. Sedgwick,. R. 3., ; . Hoerr, C A . , and Harwood, II. J. , J. Ore. Chen., 17, 3 2 7 (1932). 103* ' Hoerr> C:. A., Harwood, IT. J.., and: 3'alstor, A. A1., .. ■ ibid.-, 2> 2 6 ? (19Vi) lOif. Garland, 3. IT., Hoerr, C . •/»., Pool, A. G.,_ and Ralston^ a. V/., ibid., jo, 3/424. (19+8/. 105. Ralston, A. A., K 00 r r, 0 . A . , Feel, V/. C . , and Harwood, H. J . , ibi d . , 102 (1944)• 106. Hoerr, C. w., Harwood. H, d. , and Hals ton, A'. V. . $ ibid., ot 201 (1 9 4 4 ). , 107. Ralston, A. W., Hoerr. G. A., and TuBrow, P. L., ibid., 2> 2 59 (1944)* 103. Ralston, A. W., Koerr, C. W., and Pool, 'A. C., ibid., 8, 473 (19431 * 109* 3ed£wick,-* R • Hperr, C. '.V., and Ralston, A. .7., *112. .Hoerr, C. W., Einkerd, E.'F., Pool., Y/.» G* , and Ralston, • *• A. W.,-ibirl.,*2 , 68 tL9!44). . f .. ° • ** * • * • o ** e 113. Jl^lston,0*A. *V., Hoerr, C. W., and Vro\r:s, L. T., ibid., w © • .“ 0^. 319 (19^/). ' ' • ®o * • ' . • • ’ ■ ' V' » O ... * • - - • • *• - * - •• * ’ • m hn Boot* Am. then, Monograph,. ed.fRe 2nd •.’ •• * 131. a . a * « * ^ " «| 'A • • * • flO O y 1 9 , , n ettv Mtos fte mr Oil Amer.the of Methods andTentative .1^9*, , : ^ 9 ■ llif. 115. 116. 1 . hn C. C Chen, F. E., and Daubert, 117. , 1 . oea, *D, n rw, . , Ol Sean, Oil B,, J. andBrown, H*D., Foreman, 119. 118. 2 . igeo, Vi, Singleton, .J, 3., 120. Am.- Oil. Che mi 2 . rw, B, hm Rev.123.. . B., (196-1). Chem. J 333 , 29,Brown, 1 2 1 2. or, C Hoerr, .V.,. . J 122. H. Harwood, and ., Phy's J . Chon.., t A ^ 04 • _ • 4 0 ^ A it. A © A 128. Fusari, S. A., Ph.D. Dissertation, Ohio State Ohio Dissertation, Ph.D. A., S. Fusari, 128. 126 7 Bie, . . "etn adSldfcto o Fats," of andSolidification "Melting E., A. 3 Bailey, 27. laboratory. this from Avlabrouk,work 125. unpublished F,, A. 130# 2., J.Brown, and F. B., Deatherage, A., K. Khan, 126. . onel D. C-. D Cornwell, _. R. ., Backderf, . L C , Ailson, , and o ibid,, . * * * “ Chamirs-b !s She.., ed., V^C 2nd Ed., . Mehlenbacher, akt K,E pyi. Chem.,E. physik. K., Laskit, clo, .E, n ig C 0, J. C. 0., and King, 0.E., McElroy, oisn H E, oh, «T Roche, E., H. Hobinson, • . K ,King, and wr, . Ibid. , D., Sworn, • h « **rt|• «■.*•*« * / ani, . adPshc, . E pyl. hm, " • • 9"3 • Chem., physlk. Ef G., Pescheck, and P., Waentig, * HlernV II .*, *HildebrandV •• ■ » •• • » O . ■ • • • ••• • •••••• • - •* . •* > p '• * / i . » • m “ * f * I ‘ . T ■ * i (• *» _ t % * . i / * • ' p >f . ,• *. • * . Bon J B• B J.' Brown, Biochem.,., Arch. inpress.' 6 19 (193^). 1191 £6, £6, nvriy 1950. University, og, . . OlBSa,|, 0 (196.65 201 |^,Soap, B Oil C., R.Fouge, . Ibid. , 529^(1919?. 153 J. Ani. 'Soc., OilChemists' (1951)* 27 £28, a s n i3 oorp, nesine 1552. Interscience, Monograph, 0il3 Fats and (19 l g S ) ; ‘ . ‘ • * * iU) ; ■ (i?U;). |f> 06 1 . " • .• t 6 2 £l±, Q 32 (196-9)*, 332 ( Bald'ey, 0 (52. ^^ •• : •• ^ •^ • (1532). 705 2 5 9 1 «C. «C. Ik/ A. •• ).; . . A. E , Singlet .on, A S. , and b « 9 «b ..•••'** ..•••'** Jl, # "S,olubili£y **of #"S,olubili£y •• 26 (196-9). 3256 *•* • * | f I f ^ ^ • § | ^ *• ^ ^ ®f• I # *«|tfH * ■.'■ 672-(1920)? 672-(1920)? ' ■ ■ - "•' ■** ■'. ‘'g ■ '* •**h 136-, % ib'i • o «, ■ o -• d. , 3 (1923). 135 J t f f i n e l e c ^ o l y t e s , " g,g , * y .'i':: : ' i ' . y 9m* 3 ts ■ 1 Soc. 15, 25» Am. 7 6 . , 1356 inherit!,• 19*36 ’* .« *.*• .. hm Soc., Chem. .• . *.-• C.G., 9 O : . y \ . • • . V 21., (196-5). 3 d • * • * * * > * % ; >•*“V- v . -h.;- • / f * 563 . ••• ... • * •• •*, ’• • • • * V ; • *:... * * ■ ^ ^ ■ v * ’ ■ -• . V . • ■ ’ • * .r v .• i n "■ d . «\ *.« . • 132. Ralston, A. W. , and Hoerr, 0. «V., J. 0r^, Chen.* - ■ !«*\* • 1 0 , 170 (19J4.5 ) • / '• ^ 133. Volarcvlch , M; P., and Stepanenko, N. H ., J. Exptl. Theoret. Pr.ys. U.S.S.R., 10, 61J (19^0). I3 J4.. Halston, A. Vvd ? ” F.atty' ACida and The 3 r Derivatives," . Johji Di ley and S on.s ," N . Y. , 1 vJ4-*'3. p . 31.-- • • AUTOBIOGRAPHY . t • *• . * • . • • • • • . I, Doris Kasey Kolb, was born In Louisville, Ky. , * • * * • • #on» August Jj-, 1927* I attended Louisville public primary* • * schools and graduated I» 19^5 from Shawnee* Girls1 High. * * School. I then entered the University. of Louisville with » , • .• • a major in chemistry and was graduated In 191+S* In * September* 19^4*8 ^ enrolled In the. Ohio State University # • • •• from which I received the degree Master of Science In • • 1950. Meanwhile*1 had also served as a teaching assistant #in th& Department of Chemistry. In July 1950 I was*. • # appointed* Research . Fellow Ip *the * Ohio State Department • • of Physiological Chemistry. * I have held this fellowship for the past three years, while completing requirements • ^ ► for the degree Doctor of Philosophy. • . • - *