CHROMATOGRAPHIC PROCEDURES FCR THE ISOLATION OF TOE ORIGINAL CONSTITUENTS OF NATURAL WAXES, WITH SPECIAL REFERENCE TO THE STUDY OF OURICURI WAY

DISSERTATIONPresented In Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in the Graduate School of Ihe Ohio State

University

By

LESLIE JOHN NORMAN COLE, B.Sc. (Agr.), M.Sc.

The Ctiio State University 1956

Approved by:

Adviser ~rtn^nt n*' A-t IcuI tural 73 ioohcml rt ry

ACKNOWLEDGMENTS

The author is indebted to, and wishes to express his gratitude for the kind help and guidance given by Professor J. B, Brown, Fh»P,, throughout the execution of this research, and in the preparation of this dissertation; also to Professor F, E, Deather&ge, Fh,D,, whose expressions of kindness to the author during this period is greatly appreciated. Thanks are also due to him for allowing the author to be registered in the Department of Agricultural Biochemistry,

This investigation was made possible through a fellowship en­dowment by S, C, Johnson & Son, Inc*, in the Department of Physiological Chemistry, The chairman of this department, Professor C, S, Smith,K.D., Ph.D,, merits thanks for his genuine interest in the research program.

The author is indeed grateful to Mr, E, S, HcLoud, Director of Basic Research, S, C, Johnson & Son, Inc., far his continued in* terest, and for supplying the ouricuri wax used in this investigation.

To hi8 fellow workers Dr, B, Sreenivasan and Mr, R, H, Backderf, for their cooperative spirit, which helped to minimize the pressures of post-graduate study, the author wishes to say a sincere thank you.

ii

TABLE OF CONTENTSPage

I INTRODUCTION . . . . . . ............................... * 1TT HISTORICAL R E V I E W ....................................... 3

Review of Recent Wool W ^ Research 3Review of Recent Research on Plant Waxes . . . . . . . 6Review of the 6enGral Principles of Adsorption

Chromatography and Its Applications in the Analysis of Wax Constituents........ . * ........ 8

III OBJECTIVES................................................ HiIV EXPERIMENTAL.............................................. 16

A. Methods of Analysis of Functional ^Toups Foundin Waxes . . . . . . . . . 16

Carbonyl Value . . . . . . . . . . . . . . . 16Hydroxyl Function ............... 38Acid V a l u e ................................. 20Ester Value.............................. . 21Molecular Weight Determination . . . . . . . . 22

B. The Chromatographi c Separation of Synthetic Compounds 2li1. description of Reference Compounds and

Materials . . • • • • • • • • • . • • . . 21;2. Preliminary Considerations on the Hse of

Alumina as an Adsorbent . . . . . . . . . 253. Chromatographic Adsorption Fxperiments

with Reference Compounds .......... 27a. Chromatographic experiments on

small columns involving ternary mixtures ..................... 28

b. Cliroma togra phi c experiments on small columns involvingquatenapy mixtures . ........ * 32

c. Ciiromatographi c experiments on large columns involvingquaternary mixtures . . . . . . . 2h

C. Separation of the Constituents of Spermaceti Wax . . 37D. Attempted Separation of the Constituents of

Candelilla Wax by Chroma to graphic Adsorption . . . Id;

iii

E# The Application of Adsorption Chromatography to the Separation and Isolation of the Constituents of Ouricuri Wax . . . . . .........................

a. Initial attempts at a chromatographicseparation of ouricurl w a x .............. .

b. Experiments to solve the problem ofpreparing alumina suitable for separating ouricuri u a x ...................

c. Final experiments loading to the separationof ouricuri wax into major chromatographic entities.................................

d. The separation of some of the fractionsobtained in (c) into less heterogeneous mixtures with silica gel . . . . . . . . .

V DISCUSSION OF Ri'SULTS AND IMPLICATIONS OF THE INVESTIGATION; WITH SUGGESTIONS fOR FUTURE RESEARCH....................

VI SUMMARY....................................................EIBL3OGRAPHY

I. INTRODUCTION

The early workers in the field thought of waxes as being simple esters of normal acids and alcohols, with email percentages of these components in the flree state. This conclusion was arrived at mainly on the basis of the study of the products of saponification. Re* cently, doubts have bean expressed as to the validity of this assump­tion, and in some cases even the experimental data have been questioned. Waxes are unusual in their chemical complexity and in the resulting physical nature. Methods suitable for the isolation and consequent elucidation of wax constituents have not until recently become available. As a result there is little exact information about the nature of this group of compounds. The present work is designed to develop methods which will make possible a study of the true nature of the waxes.

Chemical investigation in this field l.as as its goal the isolation and elucidation of the various fragments obtained from saponification of the raw material and, second, determination of the nature of the constituents as they occur in the natural product.The present investigation falls into the latter category.

Waxes are important articles of world commerce. They are used extensively in the manufacturing and maintenance industries, such as in the making of electric cables, polishes far shoes metal and wood; and in cosnetics, adhesives, food packaging and carbon paper, to name only a few, Ouricuri wax is derived, according to Ivanovsky (l), from the under surface of the leaves of a tall palm Attalea excelsa,

1

2Martin, which is indigenous to tropical America, Kew Royal botanical Gardens classifies Syagrus coronata, Becc, as the palm from ■which ouricuri wax is obtained, Ouricuri wax is of particular Importance to the carbon paper industry, because it exhibits certain charac­teristics in relation to dye oleed not shown by other waxes. The widespread use of this wax stands in striking contrast to the paucity of si gnl f L- uit * nformation concerning its ch*wii stry. It is because of tills condition that ouricuri wax was selected as the nrinclpal subject of this investigation.

II. HISTORICAL REVIEW Historically, the chemistry of the vaxes may be divided for con­

venience into two periods. The classic period, up to about 19U0, Is marked by an accumulation of a large amount of empirical data of a semi quantitative nature. The modern period is characterized by the application of new and precise techniques and the availability of de­pendable results. The history of the former period has been covered adequately by previous workers in this laboratory (2, 3 ) • This review will deal with the advances made since 19U0, and will relate the sig­nificant contributions in the study of wool wax, some plant waxes, and the application of chromatography to wax research.Review of Recent Wool Wax Research

Current major Interest in the wax field centers around the chemistry of wool wax. The adds and alcohols occur in this wax in about equal amounts, each group constituting about 5066. Bertram (U) was among the first to express doubts concerning the validity of the assumption that these two entities were joined in a simple ester linkage. From experimental determinations he found the molecular weight of the wax to be about 800, whereas, were the above assumption true, this value would have been 600, or if based on the saponification value it would be 550. Wool wax is a uniquely complex mixture, perhaps even more so than any of the plant waxes. This is attested to by the fact that prior to the advent of more precise and accurate methods of measurement much confusion dominated the literature* In 19U5 Weitkamp (£) isolated 32 addle components from saponified wool wax using extended or amplified distillation, a procedure originally

3

u

suggested by Axe and Bratton (6). The free wax adds were removed from the starting material, and the remaining esters saponified. The adds which were subjected to distillation were derived fi*cm the original esters. These adds fell into four classes: 1) normal;2) iso-, in which the terminal group is isopropyl; 3) ante-iso, in which the terminal group is isobutyl, i.e. the branch occurs on the ante-penultimate carbon atom; U) 2-hydraay-acids. The members of the last group with a chain length greater than C16 are thermolabile.Horn et al. (7, 8)have quantitatively improved the isolation of the 2-hydraoy-acids. first, they separated them from the total acidic components by solvent fractionation and then partially separated the resul tant mixture of 2-hydraxy acids by distillation in a spinning band column* Pure components were isolated from the various fractions by crystallization, and,where necessary by solvent fractionation.Four pure 2-hydroxy-acids were identified, and found to be 2-hydroxy-n- dodecanoic,n-tetradecanoic, n-octadecanoic, and -l6-methylheptadeca- noic adds.

Only recently has a corresponding investigation of the alcohols been carried out. Murray and Schoenfeld (9) by systematic research es­tablished the unequivocal identification of several alcohols. They distilled them as their acetates in a highly effldent spinning band column and also applied the amplified distillation technique. The sterols and triterpene alcohols were first removed from the unsaponifi- able. They Isolated 10 branch-chain components, six of which belonged to the ante-i so dextro-rotatory group; four were of the iso-variety.Von Rudloff (10) treated wool wax alcohols with urea in an attempt to

$

separate the n-aliphatic alcohols from the cyclic components* Heobtained a mixture of monotaydric alcohols contaminated with a frnial.l

alcoholsamount of the dihydric. The separation between mono- and dihydric/ was achieved by chromatography on alumina. This separation was later improved by Tiedt and Truter (ll), who acetylated the mixture of mono- and dihydric alcohols, and again subjected them to urea adduct forma­tion* The primary alcohol acetates form the adducts readily, but the secondary do not* Arom a 26-stage fractional crystallization scheme in constant boiling ethanol-banzene they Isolated five n-aliphatic alcohols; viz, octadecanol, eicosanol, docosanol, tetracosanol, and hexacosanol* Murray (12) claimed to have identified seven n-alcohols:

Gao* C22» c2*> Cae> Cze* C30. However, 2% of the wool wax alcohols were still unidentified and awaited elucidation (13). The presence of diols, long suspected by Kuwata and Katuno (lU), was con­firmed by Horn and Hougen (15)* They characterized five such entities, four of which are branch chain diols, viz, n-hexadecane-, 16 methyl- heptadecane-lB-ajethylnonadecano-, 20-methylheneicosane-, 22-methyl- tricosane-l:2-diol. There is therefore representation among the alcohols of each of the configurational types found among the acids.

The cyclic alcohols of wool wax have received more attention, and much is known about their composition and structure* They fall into two classes: a) the sterols, and b) the isocholesterols, long thought to be triterpenes, but now definitely established as having the basic cyclopentanoperhydrophenanthrene skeleton of the sterols with side chain attached at the same point* The three methyl groups, however, appear to be located like those of the triterpenes (16,17,18).

6The first successful attempt to Isolate an original pure ester

component of wool wax was carried out by Tiedt and Truter (19)* The material precipitated by methanol from an ether solution of wool wax was extracted with hot acetone and the residue from this subjected to adduct formation with urea* None was forthcoming* The esters were carried through an 8^-stage crystallization in methyl ethyl ketone*The Tma-Sn components isolated were cholesteryl 26-methyloctacosanoate, cholesteryl 28-methyltriacosanoate, and a diester of an hydroxy-acid* Prom -the latter fraction both alcohols and acids formed urea adducts* The acidic fraction was readily separated into two components by crystallization firom benzene* The predominant component of the more soluble portion was 26-«ethyloctacosanoic acid; the other was found to be an hydroxy-acid* This evidence was consistent with Bertram1 s (U) premise that diesters of hydroxy-acids do occur in wool wax* It also provided a simple explanation of the mechanian by which branch ehaln esters can give rise to straight chain saponification products*Review of Recent Research on Plant Waxes

Since 19U0, investigations of plant waxes have been sporadic.The literature contains few reports of systematic and accurate studies of the acids and alcohols of this group, Caraauba wax appears to have been studied more than the rest (20). Some of the information currently accepted could profitably be reviewed, and some of the recently developed techniques applied* In partial answer to this need, the findings of Koonce and Brown (21, 22), Murray and Schoenfeld (23), and also Chib nail and his associates (2b* 25) are outstanding. The nature of the alcohols from caraauba wax was elegantly elucidated by

7Murray and Schoenfeld (23), They first acetylated the alcohols and then subjected the mixture to amplified distillation in a spinning hand column* They obtained confirmatory evidence far the presence of the erven numbered alcohols from C24 to C34, and also for three closely related glycols. These results confirm the earlier findings of Eoonce and Broun (21) that the bulk of the alcohols consisted of the C30, C32 and C34 homologues. The recent investigations of Murray and Schoenfeld resulted in their claim to have elucidated the total composition of saponified camauba uax« They were able to find four CO-diols in addition to the normal and iso- alcohols. The fraction of acidic com­ponents unidentified by Kbonce and Broun, but predicted by them to be different in nature tram n-aliphatic, proved to be 6*3 -iiydroxy acids, the members of which included the Cie, C^of C22, C24, C2e, C3Q homo­logues (26, 27, 28).

Schuette (29, 30, 31) and his co-workers have investigated saponified candelilla and ouricuri waxes. From candelilla wax they isolated normal C^e acids and alcohols, which had not been hitherto found, in addition to the already established claim for the presence of C3Q, C32, and C34 homologues of both acids and alcohols* The hydrocarbons, which were isolated by chromatography on alumina and purified on silica gel amounted to 52.2$. In a study of ouricuri wax these workers claim to have obtained by means of solvent fractionation a fraction composed mostly of melissyl cerotate. The paraffinic hydrocarbons were obtained by extraction of the wax with acetone and this extract in turn with petroleum ether. This mixture was distilled in a molecular still, and the distillate chromatographed on alumina.

8

They concluded that the and C33 homologues were undoubtedly1 present and that C26, Ca6 and C30 were strongly indicated. In the only other significant investigation into the properties of ouricuri wax, Lodecke (32) observed that the wax has the following constants: m.p. 82-83*1 acid no. 16-22$ ester no. 62-70* saponification no. 78-92$ iodine value 6.3-8; hydroxyl no. 130$ ash 2.1j6$ moisture 1.2^* He found his samples to contain $.8-6*255 of resins.A Review of the General Principles of Adsorption Chromatography and Its Applications in the Analysis of Wax Constituents.

The name chromatography, implying restriction to colored sub­stances, is of historical significance, but bears little relationship to the way in >dilcb the method is applied today. Tswett (33) is generally credited with having invented chromatography when he used it for a separation of leaf pigments. However, this same technique had been used previously by Day (3b) in the separation of petroleum products.

Chromatographic behavior depends on a great many factors and as a consequence is highly empirical. Because of this, the method is quite versatile. Despite the fact that theories of the mechanism are more difficult to formulate than are those of a relatively simple process like distillation, three generally accepted types of analysis are recognized: 1) frontal $ 2) elution; and 3) displacement analysis. In the first type the mixture is introduced and allowed to pass through the column. In this case the developing solvent is not changed fi*om the one originally used for solution of the mixture.The point of emergence and the concentration of each front is then

9observed in the percolate. This type of analysis results in a partial separation only, but is of value when studying the homogeneity of a sample. The second type is essentially the same as that used by Tswett (33)* and involves the removal of the components ty applying suitable eluting solvents. Ideally all concentrations of a given substance should travel at the same rate, and the zones should move undistorted through the column. In displacement analysis a solution of a substance is added which is adsorbed more strongly than any of the components to be separated. Owing to the competition for the ad­sorbent* the components arrange themselves in a succession of adjoin­ing zones* each of a constant characteristic composition. In this investigation all three general types are represented.

A few instances were cited above where chromatography has bean used in conjunction with some other major operation. This section deals with those investigations in which chromatography was the major device used to effect a separation of wax components. In the first application of the technique to the fractionation of wax constituents, Tischer and Ulnar (35) investigated beeswax in the following manner. Yellow honey beeswax was dissolved in petroleum ether at 70-80#C. Overnight* 55.6 of the wax precipitated out as colorless solids, m.p. 67.5-68.5*. Further crystallization from acetic acid, petroleum ether and ethanol raised the melting point to 73-75*. The product containing difficultly soluble acids and unsaturated esters was sapord fled to give a mixture of acids and alcohols* The petroleum ether decantate was adsorbed on alumina producing eight distinctly colored zones; these were displaced into

10the filtrate by washing with petroleum ether, after prior removal of the hydrocarbon* The first zone contained tree wax acids almost exclusively; these were divisible into three portions when eluted by alcoholic petroleum ether* Zones II to VXI were found to contain esters, while the majority of the material was found in Zone VTII,The solids from the latter were again chromatographed on alumina to give a mixture of esters, alcohols and a small amount of hydro­carbon, Later chromatographic investigation of the pigments only shoved them to contain carotenoids, lutein and carotene (36).Trappe (37* 38* 39), seeking new methods for the separation of fats from their natural sources, systematically investigated the possi­bility of applying the adsorptive process to the separation of these mixtures. He worked out an elutropic series of solvents. This was an arrangement of solvents in increasing order of their eluting power on a given adsorbent, or conversely, the ease with which a given compound will, be adsorbed from a given solvent. This series depends largely on the quantity and activity of the adsorbent. In order to standardize the procedure he coined the term "adsorption value" to define the percentage of material adsorbed on one volume weight of adsorbent from three volume parts of 100 mg, per cent solution. The volume weight is defined as that weight of substance which occupies 1 cc. after centrifugation for a half hour at 3000 r.p.rc. and 1!? cm. radius. He judiciously worked out a simple method for the separation of free from estcrified cholesterol present in blood serum, without the usual recourse to digitonin precipitation (Uo). Daniel, Lederer and Velluz (hi, U2) subjected the unsaponifiable fraction of wool wax

11to chromatography on alumina, and separated out six distinct fractions using a series of solvents, With petroleum ether they obtained hydro­carbons, Successive fractions obtained with varying proportions of benzene and petroleum ether were: 1) an unidentified dextro-rotatory steroid ketone; 2) cholesta-3-5> diene-7-one, considered to be an artifact of saponification* 3) isocholesterols, viz., lanosterol, dihydrolanostero1, and agnosterol; U) cholesterol; 5) a mixture of unidentified dextro-rotatory substances.

Findley and Brown (U3) used molecular distillation in conjunc­tion with functional analysis to investigate the nature of the original constituents of plant waxes. By this means it was possible to obtain a true picture of the constituents of seme of the waxes. However, with ouricuri wax considerable chemical changes were en­countered, These workers subsequently applied the chromatographic technique with varying results (3)* As a consequence of their in­vestigation and the work of Broadhead et al, (UU), who studied the chromatographic behavior of some synthetic compounds related to the waxes, the groundwork for the present investigation was provided.A method whi ch accurately measures the hydrocarbon content of waxes has recently been reported by Wilder (US) in which the wax is chromatographed on activated alumina at $0* and the hydrocarbon eluted with heptane.

It has been known for some time that hydrocarbon mixtures could be separated chromatographically, and this procedure was used by a number of workers (U6, U7, US). Schuette et al. (h9) claim that this method can be used as a test of absolute purity of an

12hydrocarbon. They Isolated n-tetracosane fi*om both oat oil and

ouricuri wax. This is in contrast to the current belief that only odd numbered hydrocarbons occur in waxes, fly chromatography on silicic acid columns Fillerup and Head (50) separated artificial lipid mixtures and also plasma lipids into the following groups: sterols, sterol esters, triglycerides, fatty acids and phospholipids. By this tech­nique eleostearic acid was found exclusively in the triglyceride fraction of plasma lipids of rats after being fed as the methyl ester.

The first successful application of the chromatography process to the separation of a whole wax without the aid of additional methods

was accomplished by Fuchs and deJong (51). They dissolved beeswax in carbon tetrachloride and passed the mixture on to a column of

activated alumina using ascending chromatography under pressure. With the following eluant schedule, they separated the wax into the compo­nents indicated below:

Eluant Components Removed;Carbon tetrachloride Hydrocarbon and simple estersToluene Un saturated estersChloroform Hydroxy estersChloroform acetic acid 5-8 Acids

A more definitive separation was obtained on silica gel by use of a slightly altered eluant schedule. There was a wider gap between the appearances of hydrocarbon and simple esters. A second crop of simple esters was removed with toluene, while the unsaturated esters wore re­moved with chloroform. Chloroform-ethanol, 2:1, was found suitable

for elution of hydroxy esters, and the acids required a mixture of

13chloroform-ethanol-ncotic acid in the ratio of 50:2 5:1* The com­position of yellow Deeswax as determined from their results canpnares

favorably with tfiat calculated from the saponification products on a

theoretical basis of simple combination of acids and alcohols (£2),

This wax then would constitute a typical example of the classical con­cept of the structure of waxes, Kauftnann and Kholmeyer (53) quite

recently have devised a method for the chromatographic partition on

paper between even numbered alcohols from C10 to C10, oleyl al­

cohol, cholesterol and fatty acids. The alcohols were identified by

staining the curomatogram with the dye, Khodamin B, or alternately by

oxidizing them to the acids and treating with acidic permanganate,

with the consfjquent production of brown spots.

In summary, the chief methods used to Isolate wax constituents are saT'onification followed by fractional distillation, solvent frac­tionation, urea adduct formation and c!rronatography. These methods which usually complement saoon.' fication have been U3ed individually or jointly. Fractional distillation in spinning band columns has been shown to be ideally suited for separation of the different moieties resulting from saponification. Only chromatograpliy, however, has so far provided an orderly ini tial separation of the main ori ginal con­st! t lontu.

IH, OBJECTIVES This investigation is an integral part of a program in this

laboratory designed to develop new procedures for the separation of compounds occurring in natural waxes, and to apply these procedures to the separation and identification of such constituents. In this under­taking the technique employed was chromatography.

The classical method of investigating natural waxes involves disruptive attack by saponification, followed by an examination of the fragments so obtained, Frcm these results it has been concluded that waxes are largely mixtures of normal esters of the high molecular weight acids and alcohols. Recently doubt has been cast on the validity of this assumption, especially with reference to wool wax (U). By applying less severe procedures it was proposed to isolate the constitu­ent compounds in their native form, and thus obtain exact information as to their nature and distribution.

Prior to the study the waxes themselves, a study was to be made of the chromatographic behavior of specific synthetic mixtures, similar to those alleged to be present in waxes. The information ob­tained from this operation would serve first as an index of the probable behavior of natural material, and second as a standard of reference. Suitable representatives of the following chemical classes were selected: l) a hydrocarbon; 2) a ketone; 3) an ester;U) an alcohol; 5) a fatty acid. When the conditions for separation were obtained they were to be applied to the resolution of a simple wax, a classic example of which would be spermaceti. Next it was in­tended to progress to a more complex mixture such as is found in theplant waxes, candelilla and ouricuri. The greater part of the research

Hi

15•was to be conducted on the latter material. It was expected to achieve a separation based primarily on the increasing polarity of the func­tional group. The highest degree of separation which could be hoped for at this stage would be that of homogeneous functional groups. However where there were di-functional molecules, it would be expected that these would be included with those molecules containing the stronger polar function. If waxes could be resolved into less complex mixtures, than further elucidation of the naturally occurring con­stituents will be greatly facilitated.

IV. EXPERIMENTAL A. Methods of Analysis of the Functional Groups Found in Waxes

The analytical detern!nations which are the basis for following the separations described in this investigation were carried out by use of the methods originally developed in this laboratory by Koonce (2) and by Findley (3> U3)* Certain modifications were introduced in same of the determinations, but basically the methods are those of the previous workers. The procedures used in these determinations, along with a critique of their accuracy and precision, are given below. Carbonyl Values. This is defined as the number of milligrams of potassium hydroxide required to neutralize the hydrochloric acid liberated from hydroxylamine hydrochloride in its reaction with the carbonyl groups present in 1 g, of substance. The reagent used was an 0.5 N. solution of hy dr oxylami ne hydrochloride prepared fresh on each occasion by dissolving 3*65 g. in a minimal quantity of water and adding absolute alcohol to a volume of 100 ml* The sample (0.1- 0.5 g.) was first dissolved in 5“10 ml. benzene and 10 ml. of the reagent was added* The mixture was than heated under reflux for an initial period of 2 hours and then titrated against 0.5 N. alcoholic potassium hydroxide with thymol blue as indicator. After titration, the flask was returned to reflux for an hour, and again titrated if the color had reverted from yellow to red. This procedure was re­peated until the yellow color was permanent. The determination is based on the fact that an amount of hydrochloric acid is liberated, equivalent to the amount of ketone entering into reaction to form the oxime* A 10 ml. aliquot of the reagent was taken as a blank;

16

17this usually required no more than 1 or 2 drops of alkali*

The precision and accuracy of the method was checked with known compounds, the results of which are given below, along with a sample calculation* The carbonyl value for the purposes of this investiga­tion is expressed in terms of ml111-molea per gram* This is arrived at in the following way* The number of nrilli-cquivalents of base consumed in neutralizing the liberated hydrochloric acid, is divided by the weight of the sample taken for the determination* This figure is readily converted to the more formal expression, equatable to the saponification value, by multiplying by the gram equivalent weight of potassium hydroxide, i*s* $6.1, This calculation is shown in the following example:

Hie Justification for using this mode of expression is that it provides an easy means of comparison; further when pure compounds are involved, the molecular weight is obtainable simply by dividing the m Ifcl./g, into a thousand. All this holds true if the compound under investiga­tion is mono functional with respect to the function being determined. With di-functional compounds the value determined will no longer represent the number of naili -moles per gram, but will represent twice that figure, and is correctly expressed as the number of milli- equivalents per gram* The results obtained with pure samples are given in Table I,

weight of sample ml, of base normality of base

0.S701 g.2.6UO.U223

no. of ra Mol./g. - x O.U223 - 1.9$$

TABLE IDetermination of Carbonyl Groups inKnoun Compounds

Sample"" m MolCar-

bonyl/g. Added

m fool. Car- bony l/g. Found (Mean)

Mo.'o'fDetermi­nations

Range of Values (m Mol*/ . £..) _

Stearone 1.96 1.95 u 1.9UO-1.955Stearone in the presence of octa- decyl stearate 1.05 1.07 3 1.07-1.08Stearone mixed with octadecanol 0.92 0.905 3 0.900-0.915Octadecyl stearate 0.00 0.02 1 0.0212-keto-steari c acid* 3.26 3.05 3 3.03-3.06

♦Neutralization Equivalent (N.E.) 307 (found) j theory, 298; purity about 91%•

Pram the above results it was concluded that the method is capable of estimating ketones, both as a pure compound and in the presence of esters and alcohols. Esters interfere to a limited extent, ■while alcohols do not.Hydroxyl Function. This is expressed, customarily, either as hydroxyl value or as acetyl value. The fomer is obtained by titrating the excess acetic acid present after acetylation of a given sample, and is defined as the number of milligrams of alkali equivalent to the hydroxyl content of 1 gram of substance. In the acetyl value the number of milligrams of alkali required to neutralize the acetic acid obtained by saponifying 1 gram of an acetylated substance are measured. In this study the method used followed the principle of the hydroxyl value determination. The determination was carried out in the following manner. The sample (0.1 - C.5 g,) was placed in

19a 2^0 ml. Erlenmeyer flask, fitted with a standard taper neck, and U ml, of reagent ware added. A 75 era* air condenser was inserted into the flask, both fitted with V joints, and the two surfaces wet with pyridine so as to ensure a better seal* The flask was then heated for 90 minutes on a hot plate, cooled, and 10 ml* of 855 aqueous pyridine added* The flask was again warmed for 2 minutes to redissolve the mixture, and 35 ml. of benzene or chloroform was added. When cooled the solution was titrated against 0.5 N alcoholic potassium hydroxide with phanolphthalein as an indicator. The acetylating reagent was a mixture of one part acetic anhydride in three parts pyridine, with OjjjG water added. A blank determination was carried out in the same manner* To obtain the alkali equivalent of acidic material, another sample was dissolved in 10 ml. pyridine and 35 ml. of benzene and then titrated against the standard alkali. As was the case with the carbonyl determination, the hydroxyl value was also expressed in terms of milli-moles per gram. The principle of the calculation is the same with one small modification. The difference between the amount of alkali required for the blank and for the sample was first obtained. This is the number of ml. of aTJcn_H equivalent to the acetic add required to acetylate the sample. The rest of the calcu­lation follows as with the carbonyl value. In this procedure the use of chloroform was more appropriate than was butanol as recommended by Ogg et al. (5U). The addition of O.L$ of water to the acetylating reagent was recommended by Hawke (55). This had the effect of mini­mising the discoloration caused by the prolonged heating. The 1:7 ratio of acetic anhydride to pyridine recommended by West (56) has

20been shown by Ogg et al, (5U) to be less efficient than the 1*3 solu­tion, The results obtained from analysis of known compounds are given In Table II,

TABLE II

Samplem Mol, Hydroxyl/g. (Found)

m Mol,Hydroxyl/g, (added) Theory

Octadecanol ) 3.67 ) 3.65 ) 3.66 ) 3.67

mean 3.61? 3.71Mono-hydr oxy- stearic acid ) 3.28

) 3.29 ) 3.29

mean 3.2? 3.3UOctadecylstearate 0,00 0,00

From the results In Table II it can be seen that the reaction with acetic anhydride did not go quite to completion in the cases of octa— decanol and mono-hydroxy-stearic acid. However the precision was satisfactory for use in the present investigation.Acid Value, This is usually defined as the number of milligrams ofpotassium hydrojd.de required to neutralize the acid in 1 g, of sample. However, in this work it is expressed as the number of mi 111-moles per gram of sample, the principle of calculation being the same as in thecase of the carbonyl value. The determination was carried out in thefollowing way: A 0,1-0,5 g, sample was weighed out into a 125 ml, Erlenmeyer flask and 10 ml, benzene added. The flask was wanned and

21about 5 wl. of alcohol (neutralized to the pink color of phenolphthalein) added, the flask was then cooled to roc® temperature and the contents titrated against 0,1 N aqueous alkali, to an end point which remains pink far 3 minutes. The method was found to give accurate and precise results. Three determinations on a known sample of stearic acid gave acid values of 197,1; 197.0; 197.0; theory (197.2).Ester Value. This is the difference between the saponification value and the acid value. In this investigation, following the reasoning given previously, the saponification number was expressed in mi 111- moles per gram. Consequently, there were no changes introduced in the calculation of ester value in milll-moles per gram, A comparison of this procedure with the approved macro-procedure was carried out, using a sample of known saponification value, and the comparison found to be good, Koonce (2) has found the optimum conditions for wax saponification to be: 1) reaction time 90 minutes; 2) ml nitron effective concentration 0,25 N alkali; 3) minimum excess of 192JC. The method used throughout this study was as follows* A sample weighing 0,1-0,5 g, was dissolved In 5 ml, benzene and 10 ml. of an approximately 0,5 N solution of potassium hydroxide were added. The flask was heated under reflux for 90 minutes and then titrated against 0.5 N hydrochloric acid (micro-burette). Table III shows the compari­son between the method employed for saponification value determination in this investigation and the approved macro-procedure. The sample used was from the C18 cut of a fractionation of the methyl esters of sheep liver fatty acids. This sample had a saponification value of 191,7 or 3.1*1 mi Hi-moles per gram of ester.

TABLE inComparison of Saponification Value Procedure with Approved

Macro-MethodSend.-mi cro Methodm Mol. Ester/g.

Macro-method m Mol, Estar/g,

3,la 3.U13.U2 3.1*23.1*2

mean 3.1*2 T.H5

Molecular Weight, The following modification of the Rest method (57) for the determination of molecular weight by the depression of the melting point of camphor was adopted* A 12 x 75 son* pyrex glass tube was blown out at the closed end to form a bulb, and tared. From this point the tube was untouched by the naked hand* About 20 mgms, of the sample was accurately weighed into the bulb, followed by an amount of camphor equal to 19 times the weight of sample. The open end of the tube was then drawn out and sealed off. The tube was then placed in the melting point bath and maintained at 178* until the contents had melted completely. The molten material was swirled to obtain thorough mixing, and the tube removed and cooled. The melting point of the mixture was then determined. The melting point was that temperature at which the last crystal disappeared. The ther­mometer used was graduated in hundredths of a degree, A satisfactory sample of camphor was prepared in the following manner, Camphor, supplied by the Jean Vivaudau Co,, Inc,, was first mixed with a num'll amount of ether in a mortar, and as the ether evaporated the solid was ground to a powder. The resultant powder was exposed to the air for two minutes and placed in a glass stoppered bottle. The melting

22

23point of an acceptable sample varied between 177-178*0* After the preparation of each batch of camphor the molecular depression constant was determined, using an analytically pure sample of benzoic add and a sample of octadecyl stearate (saponification value 101**8), For molecular weights of the order tested the method was found to be accur­ate within if care was exercised in the weighing of the sample and in the reading of the melting points* It was found that temperatures had to be read to the nearest 0*05*» and that the rate of Increase of temperature around the melting point should not be greater than 0*3 * per minute, Trial determinations were carried out on samples of do- triacontane and stearone. These results are given in Table IV*

TABLE IVMolecular Weight Determinations of Known Compounds

Substance" Mbl'. vt.

(Found)Mol* wt* (Calcd,)

Dotriacontane 10*71*5510*0 )| *30

mean T & - l*5oStearone h9$

1*901*8 31*92

mean l*£o 506

It is generally recognized that the error inherent in this method increases with increasing molecular weight. Consequently for values of this order the precision achieved here is satisfactory al­though it is agreed that it is not good. However the method does furnish the operator with a reasonably good index, within 5% of the

2k

mean molecular size of the material under investigation,5, The Chromatographic Separation of Synthetic Compounds,1, Description of reference compounds and materials.

The following materials were used in this investigation. Activated alumina, 80*200 mesh, was obtained from the Fisher Scientific Co, and from M, Wo elm, supplied by the Standard Scientific Supply Corp. Heptane, supplied by Hill lips Petroleum Co,, was claimed to be 99 moles per cent pure. This solvent was dried over drier!te and redis­tilled before use. Highest grade Propanol was distilled over calcium oxide before use. Glacial acetic acid was dehydrated by refluxing one liter of the acid with 50 ml, of acetic anhydride for 3 hours.The acid was then redistilled. All other chemicals used were chemically pure grade.

Do triacontane had been prepared previously by Katchen (58), by reacting cetyl bromide with metallic sodium in anhydrous ether; m,p. 69,5* (reported, 70*)• C, 8$,?0fC; H, lU,69^ (theory C, 85,21:,H, Ui,75).

Octadecyl stearate was prepared according to the method of Hilditch ($9), in which pure octadecanol, Sh g*, was reacted with 55,6 g, of stearic add in the presence of 0,b g, of -naphthalene sulfonic acid and 500 ml, benzene. The mixture was refluxed in a manner such that the moisture in the benzene-water azeotrope formed could be removed from the reaction mixture. At the end of 1* hours the reaction was stopped and the mixture recrystallized from toluene at -20*, The recovered crystals were washed on a Bftchner filter with cold acetone to remove residual toluene, and dried in air (sap, value:

25found, 10U.8; calcd. 10U.5).

Octadecanol had been previously prepared by Katchen (58) from crude octadecanol supplied by Eastman-White, by recrystallization three times from 95% ethanol and once from methanols ».p#, 57*8* (reported 58.0*); hydroxyl value, 208*8 (theory 207*?)*

Stearic acid was obtained by purification of Hystrene (T-97), supplied by Atlas Powder Co., by twice crystallizing frees acetone at 0* (N.F. 285.0, theory 28U.U6; I.V. 0.30, theory 0.00).

Stearone was supplied by Armour. The crude product was twice crystallized from 50{£ heptane-ace tone; ketone equivalent 505, theory 5o6.9.2. Preliminary Considerations on Use of Alumina as an Adsorbent.

Findley (3) reported varying degrees of molecular disruption in chromatographic experiments with sugar cane and other waxes.Cahn and Fhipers (60) have also described the hydrolysis of an alkali labile substance (diacetyl taxicarol) by alumina, but separa­tion of esters has also been carried out successfully with this adsorbent (61, 62, 63). Broadhead et al. (UI4) found that the detri­mental effects of alumina could be satisfactorily rectified by pre­washing the adsorbent with 1% HC1. Initially the objective in this section of the investigation was to attain the highest degree of resolution of a synthetic mixture, while keeping side reactions at a minimum. Previous reports showed that alumina which had the greatest resolving power also caused greatest disruption, but silica gel while giving lower overall resolution produced no side reactions. Because of the greater potential in resolving power, alumina was chosen as the

26adsorbent upon which the major separation was to be developed*It was found that when alumina prepared according to roadhead et al. (UU) was subsequently subjected to temperatures of UOO* or above the undesirable characteristics returned. This was illustrated by the following preliminary experiments. In the first experiment the pretreated alumina was oven dried at 105* for 12 hours. To a column (10 x 3*3 cm,) prepared from this batch of alumina, 1,1192 g, of octadecyl stearate was added in approximately 1 solution in heptane. Elution with 1500 ml, of 2% acetone-heptane, gave a 97,6/6 recovery of ester. However when the ester was chromatographed on alumina which had been subjected to muffling at UOO* for 5 hours, only U9,1# of the ester could be recovered, the remainder, having presumably been saponified,

Brockman and Schodder (6h) claimed to have prepared five grades of alumina distinguishable by their varying adsorptive capacities.This was measured by observing the separation of various dyes in columns 10 cm, long and 1*5 cm, in diameter. In the test 20 mg, each, of two dyes were dissolved in 10 ml, of pure benzene, the solution diluted to 50 ml, with petroleum ether, and a 10 ml, aliquot passed throu i the column, followed by 20 ml, of the same solvent, free of dye. Alumina (I) possessed the desired activity when p-methoxy-azobenzene (upper) and azobenzene were separated before the latter dye had passed through the column, With alumina (II), of lower capacity, azobenzene washed completely through the column, but sudan yellow and p-methojey-azobenzene should separate. Alumina (III) was characterized by the fact that sudan red could be separated

27as the upper layer ft*can sudan yellow, which was still adsorbed as the lower band. Alumina (IV) should fail to hold sudan yellow but should be capable of separating amino-azobenzene and sudan red into distinct upper and lower bands respectively. Finally the weakest product (V) was of suitable activity Wien p-tydroxy-azobenzene formed a distinct zone above a band of p-amino-azobenzene. Ibis series depends essentially on the increasing moisture content of the alumina*

Although the Brockman and Schodder procedure does provide a method by which activity of alumina may be standardized, it does not prevent a certain amount of variability from one sample to another, i.e. separation of the two zones nay occur at any position within the 10 cm. length of the adsorbent. In order therefore partially to lessen the degree of variability, the following procedure was adopted throughout for the grading of alumina. Instead of a 10 cm* column of the above dimensions, £.£ g. of alumina were used to make a column $ cm* length and 1.3 cm. diameter. Hie rest of the procedure followed the one given above. All the chromatographic separations reported were carried out on alumina which would fall between Brockman activity grades II and III. The only pair of dyes used was p-methoxy-azobenzene and sudan yellow. Activities determined by the latter method are designated in arable numerals.3. Chromatographic Adsorption Experiments with Reference Compounds.

In this section, the work involving the chromatographic separa­tion of ternary and quartemary mixtures of the reference compounds will be described. At first the experiments were carried out on smaller columns, and from the information obtained in these experiments

28larger columns von used in an attempt, to increase the quantity of starting material*

a* Chromatographic experiments on small columns involving ternary mixtures,

MimH nn (300 g,) was washed with Uoo ml* of l£ HC1 and finally with distilled water to bring the adsorbent to a neutral pH* It was then oven dried at 105* for 10 hours* This produced alumina an which p-methoxy-asobenaene (lower) would just separate from sudan yellow on a column containing 5 g* of adsorbent* Adsorbent which displayed these characteristics was designated grade 2*

The chromatography apparatus used in this investigation con­sisted of three parts, rig, the reservoir, the column, and the outlet device* For the small column, a glass tube (3*U cm* diameter) was fitted at one end with a 3U/28 V and at the other with a 3U/U5 S, both joints being female. Hie reservoir consisted of a 1 liter round bottom flask, to the base of which was sealed a 9*0 cm* length of glass tubing* The end of this tubing was fitted with a 3h/hS V (male). Another tube (0*5 cm* diam*) was joined to the base of the flask, being inserted inside the larger tubing* An opening from the flask into the snail tube was then made* The outlet device consisted of a 3U/28 S (male), tapered so that a 2 mm* stopcock could be fitted to it. The large column measured 5*5 cm* in diameter and was also fitted with a 3U/U5 I (female) at one end, the other end being 55/35 ■ (female). The packing of the column was carried out in the following manner* With the stopcock closed the entire column and half the reservoir was filled with heptane. Alumina (120 g*) was then poured

29Into the reservoir, and sucked slowly into the column upon opening the stopcock to allow passage of the liquid through the system. The filling procedure was accompanied by gentle tapping of the column, so that the alumina settled evenly. This was the procedure followed for the preparation of all columns used in this investigation, irrespective of size. A ternary mixture consisting of the following compounds was transferred to the column as a 1!£ solution in heptane.

Dotriacontane 0.5057 g. “ 1*123 milliHaolesOctadecanol 1.0282 g. • 3.800 milliHaolesOctadecyl Stearate 1.U159 g* * 2.6U0 milli-aoles

After the solution had been passed through the column, elution with the solvent schedule shown in Table V was begun. The elution rate for the removal of hydrocarbon was 700 ml. per hour. This rate was increased to 1 liter per hour for the subsequent material. These were the rates used by Findlay (3). Table V shows the results ob­tained in this experiment. The figures reported for total milllHUoles of ester and alcohol in the recovered solids are derived ty multiply* ing the mill i -mole3 per gram by the total weight of the fraction.In this experiment and for all succeeding ones, the solvent was removed from the solids by distillation under reduced pressure. The last traces of solvent were removed by heating to 100* in a vacuum oven at about 5 an* pressure.

TABLE V

Frac­tion

Eluant vol. (ml.)

MaterialRecovered

g.

---------------TCsar * “

Ester (m Mol.)

fatalAlcohol (m Mol,)

1000 heptane 0.U8U0 (0.5057) o.ooo 0.000

f2 2500 2£ acetone in heptane 1.9386 2.620 1.9U0

f3 2000 1$ propanol in heptane 0.U780 0.020 1*760

2.9666* 2.62 (2.6U) 3.700 (3.80)*h

VTotal yield 98.1$ by weight*Values given in parenthesis are the amounts of each constituent present in the original mixture.

The results of this experiment showed that, 1) degradation of ester material had not occurred; 2) that with alumina of reduced ac­tivity, acetone solution was ineffective in rendering a separation of ester from alcohol as was pointed out by the fact that slightly less than half of the octadecanol was found in the ester fraction (Fs)>3) in the solids of fraction Fx there wore no detectable amounts of either ester or alcohol. It must therefore have consisted solely of hydrocarbon.

Since acetone was not effective in separating esters and alco­hols a solvent of weaker polarity was indicated. For this reason anhydrous, peroxide-free ethyl ether was tried. A ternary mixture comprised of octadepyl stearate 0.8U83 g* (1.581 m Mol.), stearone0.53U6 g. (1.0 3 ® Mol.), octadecanol 0.6200 g. (2*292 m Mol.) was dissolved in 1 per cent solution in heptane. Hydrocarbon was

30

31omit tod from this experiment because major emphasis was placed on a separation of ester from octadecanol* This solution was added to an alumina column of similar dimensions and activity to the one used In the last experiment* The results of this separation are given in Table VI.

TABLE VISeparation of a Ternary Mixture of Octadecyl Stearate, Stearone, and Octadecanol on AluminaFrac­tion

Eluant vol.MaterialRecovered(g.)

TFotaT' " Ester (m Mol.)

"Total--Alcohol (m Mol.)

Totkl"' ' Ketone (m Mol.)

l£ ether in heptane, 15>00 1.3717 1.97 0.00 1.050

Fa propanol in heptane:2000 0.6165

(1.98)**2.28 0.0000

1.9952* 2.2B(2.29)**

“W 5 0(1.053)**

* Total yield 99.9& by weight***Values given in parenthesis are the amounts of each constituent present in the original mixture.

The results shown in Table VI clearly indicate that a l£ solution of ether in heptane is effective in permitting a separation of ester from octadecanol under the conditions of the experiment. It will not, however, allow the separation of ester from stearone. This conclusion was made on the basis of the fact that all the octadecyl stearate and none of the octadecanol was present in Fraction F1# However, Fx also contained all of the stearone* These results brought out the fact that the ketones of which stearone is a representative, and the high molecular weight esters have similar polar characteristics. It was decided not to try, for the present, to separate these two groups

of substances.32

b. Chromatograph!c experiments on small columns involving a quarternary mixture.

Having established the conditions which appeared to give a satisfactory separation of synthetic mixtures into hydrocarbons, esters and ketones, and alcohols, the next experiment was designed to determine the suitable conditions for a quaternary mixture. In this separation the following compounds in l£ solution in heptane were passed through a column of alumina of the same dimensions and ac­tivity as in the previous experiments,

Dotriacontane 0,7871 g, 1,71*5 milli-equivalentsOctadecyl stearate 1,2036 g, 2,21*5 mi lli'-equivalentsOctadecanol 0,9325 g, 3*1*00 mi lli-equivalentsStearic acid 0,71*26 g, 2*610 nri. lli-equivalents

The solvent schedule required foiy and the analytical results obtained from this separation are given in Table VII,

TABLE VIISeparation of a Quaternary Mixture of Synthetic Cporpounda on -lumlaaFrac­tion

Eluant vol,(«1.) ____.

VeigjitRecovered(g.)

Total Ester (m Hoi,)

total Alcohol (m Mol,)

Totar------Acid (m Mol.)

heptane 1500 1**4126 1.160 0.00 0.00

Fa 1% ether in heptane:2000 0.5830 1.085 0.00 0.00

f3 1% propanol in heptane: 1500 0.9257 0.000 3.U3 0.00

F4 acetic add in heptane: 1500 0.737U 0.000 0.00 2.59

3.6?87* "srsw(2.2H3 )-■:-»

W "(3.U5)** (2.61)*-*

*Total yield 99.7%.**Values given in parenthesis are the amounts of each constituent present in the original mixture*

The results obtained from the last experiment show that 1*16 m Hoi* or 0.6230 g, octadecyl stearate had been eluted by heptane, along with all of the hydrocarbon. This could have been caused by small differences in activity between the alumina used in this experiment and in the preceding ones; even though they were of the same grade. However clear-cut separations of octadecanol and stearic acid were obtained.

The next objectives were: first, to find the conditions which would permit a suitable separation of hydrocarbon and ester, and second, the quantity of adsorbent required for a charge of 1$ to 20 g. There are three interrelated factors which have direct bearing on these objectives, viz: 1) the activity of the adsorbent; 2) the quantity of adsorbent; 3) the quantity of solvent required, An in-

33

31*crease in the height of the column was definitely indicated. This in turn would mean that more solvent would be required. If the activity of the alumina were decreased, so that less solvent would be required, it would impair the effectiveness of the attainable separation. Con­sequently a critical point, i.e., in terms of weight of alumina had to be found, idxich would: l) give a satisfactory separation of hydro­carbon from ester; 2) keep the solvent requirement at a reasonable level*

c. Chromatographic experiments on large columns involving quartemary mixtures.

The conditions under which an adequate separation of a quaternary mixture could be obtained are illustrated in the next experiment* The compounds present in the mixture were: dotriacontane, octadecyl stearate, stearone, and octadecanol. Stearic acid was omitted from the mixture because it had been established in pre­liminary experiments that neither its inclusion or omission had direct bearing on the effectiveness of separation of the other com­ponents. In addition its omission would mean a saving in solvent and in time*

Alumina of lesser activity than that used in previous ex­periments was prepared in the following way. Fisher alumina (600 g.) was washed with 800 ml. of a solution of 2$ HC1 accompanied by vigorous stirring for one hour. After allowing the particles to settle, the supernatant was decanted and the residue washed with sufficient water to bring the alumina to a neutral pH. Three hundred grams of the adsorbent in a 1 liter beaker were then placed

35in a drying oven at 105* and heated for 10 hours* Alumina of de­sired activity was that which, under the test conditions, permitted the complete removal of p- nethoxy-azobenzane and left sudan yellow as a well defined band beginning 0*5-0,7 cm* from the top of the ad­sorbent* This alumina was called grade 2a* The following mixture dissolved in 800 ml. heptane was transferred to the column* (5*5 x 30 can.) Do triacontane 2*800.9 octadecyl stearate 2,6200 g., stearone1*1562 g*, octadecanol 2,6226 g* The heptane solution was poured into a 3 liter reservoir and passed through the column at 30° ml, per hour* After the solution had drained to within an inch of the top of the adsorbent, three liters of heptane were then poured into the reser­voir and percolation continued at the same rate. The passage of the final 500 ml* of heptane failed to remove further solids from the column. The same procedure was continued using a 1$ solution of ethyl ether in heptane, to remove ester and ketone. After U liters had been passed only traces of solid material were recovered. The flow rate was increased to 500 ml* per hour, during elution of F2 and F3. For the removal of octadecanol five liters of a solution of 1% propanol in heptane were required. The passage of this component down the column could be followed with the aid of ultraviolet light, as it gave a characteristic fluorescence. Solids appeared in the first liter of eluant, indicating that there had been incomplete removal of the preceding fraction. Since in previous experiments it had been observed that alcohols failed to appear before three liters of 1 propanolic heptane had been passed, these solids were combined with fraction F2.

TABLE VIIISeparation of a Quaternary fixture of Known Compounds on a Large Column of Alumina

COMPOSITION OF MIXTUREKetone nicohorRECOVERIES

frrac- hydrocarbon Sster tion Total wt. Total vt. Total vtT Total vt.

m Mol. g, m Mol* g. m Mol, g. m Mol. g.

none none

Compounds Weight Total Frac- Eluant vol. wt._________ (g.) m Mol, tion ( m l . ) __________________Dotria-contane 2.8019 6.23 Fx heptane: 2.7807 6.37 2.7807 none

3600Octadecyl ethylStearate 2.6290 L.89 F2 ether in 3.73UO none - L.R5 2*60 2,2h 1.33bO none

heptane:booo

Stearone 1,1562 2.28 On

Octadeca- 2.6228 9.72 F3 156 propanol 2,5965 none 0.0 none 9.62 2.5965nol in heptane:

5000Total 9.2009 9.1112* 6.37 2.7807 L.8L 2.60 2.2b 1.131*0 9.62 2.5965

♦Yield 98.956 by weight.Flow Rates: F1 removed at 300 ml. per hour.

F2 removed at 500 ml. per hour.F3 removed at 600 ml. per hour.

37Table VIII shove the analytical results obtained from this

separation, and indicate clearly that the conditions for a satis­factory resolution of the mixture bad been achieved* Fraction F1 consisted entirely of hydrocarbon, while Fa was a mixture ofoctadecyl stearate and stearone* Octadecanol was found only in F3*There was a loss of 1.3# of the original mixture in the operation*It is doubtful whether this represents the amount retained on the column, as some of the material may have been lost during the transfers involved in recovering the solids*

C. Separation of the Constituents of Spermaceti Wax.Frcm the results obtained with synthetic material it was con­

cluded that the conditions which gave an effective separation of the mixture into four groups, vis: hydrocarbon, ester and ketone, alcohol, and acid, could be successfully applied to waxes* If waxes were truly what they have been alleged to be, then difficulty should not be encountered in obtaining a separation of these waxes into similar groups.

Spermaceti wax is reported by Warth (52) to be composed chiefly of cetyl palmitate, which is present to the extent of 90{6. The ap­parent simple composition suggested that this wax would provide a suitable medium for bridging the gap between synthetic compounds, and the more complex plant waxes* The constituents of the latter are of higher molecular weight, and generally exhibit different solubility characteristics* Two experiments with spermaceti wax are reported in this section along with the conclusions which were drawn from the results* The sample used in this investigation was obtained from

38Qtt, Brown and Price (a pharmaceutical house) and claimed by then to be authenticated spermaceti.

In the first experiment ip,72 g. of the wax were dissolved in 1 liter of warm heptane and passed on to a column(5.0 cm* diameter, 30 cm* length) of alumina, grade 2a,(600 g*). Table H shows the elution schedule applied and the analytical data obtained in this separation. With this wax, in addition to the alcoholic material, the passage of the acidic components could be followed with ultra­violet light. They both exhibited a characteristic white fluorescence.

TABLE IXThe Chrcaaatographlc Separation of Spermaceti Wax (Experiment I) ____

Fraction K.eq. M.eq. M.eq. M.eq. M.eq. M.eq.Frac- Eluant vol. Weight Ester/ Ester —OH/g* -OH Acid/g. Acidtion (ml.)_______ (g.)_____g.________ Total____________ Total___________________ Total

heptane:11.500 11.6798 2.280 26.600 0.0 - 0.0

Fa 1$ etherin heptane:2.500 1.96U7 2.078 U.30 0.0 - 0.0

F3 l£ propanolin heptane:5000 1.0680 0.0 - 3.680 3.930 0.0

F4 1% aceticacid in heptane:5000 0.7928 0.0 - 0.0 - 3.8O 3.010

Fe 1$ aceticadd in heptane:1000 0.2133 0.0 - 0.0 - h.UO 0.870

Total 15.7186* 30.800 3.930 3.880Spermaceti Wax 15.7252 2.130_____ 33.500 0.10______ 1.570 O.O37 0.690*Tield 99.9? by weight. Ketones were not found in this wax*

aoSeveral features of this experiment are significant* 1) So lids

were not found in the first 1500 ml* of heptane percolate* This is important because in previous experiments where hydrocarbon was present, it began to appear after about $00 ml* of percolate had been collected. Hie inference is that hydrocarbons are not present in this sample of spermaceti. 2) The bulk of the material in Fraction Fx was obtained after 1500 ml* and before 3 liters of heptane had passed throujfo the column* The appearance of esters at this stage is probably due to overloading, i.e., a greater quantity of esters was present than could be adsorbed by the alumina. Further some esters are clearly held more strongly than others. This is shown by the fact that nearly 2 g* were recovered in the 2% ether percolate. In order to record the analytical data correctly, the values are ex­pressed in milli-equivalents and not in mi lli-moles, since the use of the latter term does imply a knowledge of the molecular weight,Tt was not intended to determine the mean molecular weights of the different fractions of this wax by physical methods. The effective- nexx of the separation could be obtained without this knowledge*Table IX shows that there is a deficiency of 2.70 milli-equivalents of ester in the recovered material, when compared to the starting wax* This loss of ester is partially compensated for ty the increase of 2,36 milli-equivalents of detectable alcohols. There was an in­crease of acids amount!ng to 3*19 milli-equivalents* If the decrease of esters was due to hydrolytic disruption, then there should be similar increases in the recovered alcohols and acids* This pre­supposes that the material undergoing disruption was simple e art jr.

Ul

Nevertheless* the wax did undergo some disruption. This was not ex­pected since the conditions of the experiment were those which did not produce any chemical changes when applied to synthetic compounds.

For the second experiment the alumina used was washed ex­haustively with methanol and then with water* and the regenerated adsorbent returned to activity grade 2a Jn the previously described manner* Eluant was withdrawn from the column at the rate of 300 ml* per hour until 1 cm* of solvent remained above the adsorbent* when the following elution schedule was introduced. Percolates were divided into approximately S00 ml. batches for the first five frac­tions * thereafter cuts were made whenever expedient. One per cent propanolic solution was introduced before all the ester had been eluted* This was only a device to avoid the unnecessary use of solvent. Alcohols which are eluted by l£ propanol run so far behind ester material that the first l$0O ml. of this eluant was used for the removal of residual material which could have been eluted by 1% ether. In this case propanol elution was resorted to only after 85 of the ester had been removed. Similarly is the case with the use of acetic acid. It has been found repeatedly that with alumina of this grade acids required 6^00 ml. for elution, and that most of the acids were collected in the last 1$00 ml. of percolate. Conse­quently acetic acid was added to the column after passage of U300 ml. propanolic heptane and before complete removal of the alcohol fraction. As in the previous experiment with spermaceti both the alcoholic and acid fractions gave a blue white fluorescence in ultraviolet light. The free alcohols and free acids are not completely

U2

solid at room temperature. This would indicate the presence of tin- saturated material since the analytical values rule out the possi­bility that shorter chained compounds were present, Warth (52) observes that the presence of any appreciable iodine value in sperma­ceti is probably due to contamination with small amounts of sperm oil. The fractions were insufficient for iodine value determination. The data obtained in this separation are given in Table 1 which shows the eluant schedule, and also serves to illustrate the balance obtained between original material and recovered solids.

TABLE XThe Chromatographic Separation of Spermaceti Wax (Scpariment II)

Frac­tion

Eluant vol# (ml*)

Fractionwt.(g .)

Ester Alcohol Acidm.eq,/g.

Totalm.eq.

Wean*mol.wt.

m.ea./g.

Totalm.eq.

Wean*mol,wt.

m,eq./g.

Total®.®q.

Wean*mol.wt.

*1 Heptane:$00 l.#32 1.9$ 3.Oil $10 0.00 0.00f2 Heptane:$00 $.8073 2.10 12.U2 U76 0.00 0.00f3 Heptane:>30 3.0578 2.20 6.80 U$$ 0.00 0.00F* Heptane: $00 1.8009 2.19 3.93 li 51 0.00 0.00Fe Heptane:$00 1.8723 2.19 li.U U57 0.00 0.00Fs Heptane:$00 0.77 $6 2.23 1.7U hli9 0.00 0.00f7 1$ ether in

heptane:1$00 1.833U 2.20 U.0U ii$$ 0.00 0.00Fe 15& propanol in

heptane :ll|00 1.2892 2.2h 2.89 liii$ 0.00 0.00f9 lS> propanol in

heptane :3000 0.>i919 0.00 3.69 1.816 2711% acetic inheptane:$00

*10 l£ acetic inheptane:6000 0.2282 0.00 0.00 3.36 0.768 297.$

Total 18.7U98 38.97 1.816 0.768Spermaceti Wax 18.9357 Uo.37 1.89 0.702field 98*9% on a weight basisT. — — — — — — — — — — — — — — —— — — — — — —*The values reported for mean mol. wt* are derived from the ester, hydroxyl, and acid values respectively, and assume the molecules to be mono functional.

hh

The results of this experiment may be summarized as follows,1) Spermaceti wax was successfully separated into its main functional groups without significant disruption of the molecules. This is evi­denced by the fact that there is no increase in the amount of alcohol recovered as compared to the amount originally present, and there is only a very small increase in acids. 2) The solids appearing in the first six fractions do so in order of decreasing molecular weight.This fact could bear further investigation and might conceivably form the basis of a method for the separation of high molecular weight esters, using elution analysis, 3) The molecular weights of the ester fractions calculated on a basis of the ester values indicate that cetyl paljritate (mol, wt, U8l) is probably present to a lesser extent than generally believed, U) The alcohols recovered appear to contain unsaturated material, as suggested by the melting point and molecular weight data. The mean molecular weight derived from the hydroxyl value is 271 (octadecenol, 268) and the melting point range was 31-36*. While this fraction is undoubtedly a mixture, it is conceivable that it does contain some octadecenol, £) The mean molecular weight of the acid fraction calculated from the acid value was 297,£ and the melting range was found to be 31-35** This suggests that the fraction contains a mixture of acids some of which are probably unsaturated,

D, Attempted Separation of the Constituents of Candeli11a Wax by Chromatographic Adsorption,

Candelilla wax is obtained from a weed Pedilanthus pavonis, Boisser, or from P, aphyllius which grows in the semi-arid regions

h$

of northern Mexico, and southern Texas* Arizona and southern California* It is a yellow brown solid, hard, brittle and easily pulverized* The melting point of samples derived from various sources have been re­ported by several workers (65, 66, 67 * 68), the temperatures varying from 6U-71"* This leads to the conclusion that standardized samples are difficult to obtain* Another species, Euphorbia antisyphilitica, Zucc,, produces a wax closely resembling the one derived from P* p3Vonis. The specimen used in this investigation was obtained from P, pavonis, and was supplied by Innis Speiden & Co. as refined candelilla wax* The analytical constants obtained for this wax ex­pressed in conventional units were found to be: acid value 16*3, saponification value 5l,l> ester value 3U*8* The reported mean values (52) for a number of different samples are: acid value 16,6, saponification value 58*1, ester value ljl*5*

Candelilla wax was not intended to be the subject of main interest, principally because it is unique in that it is reported to contain over 50% of hydrocarbons. However, its chromatographic characteristics would serve as an index of the probable behavior of the more complex plant waxes. Refined candelilla wax (20.0 g.) wasadded to 1 liter of hot heptane. Most of the wax dissolved onshaking, but a portion was insoluble. The bulk of the solution wastransferred to a column of regenerated alumina of activity grade 3,i.e., the sudan yellow band began 1,5 cm. below the top of the column.To avoid transferring the insoluble portion some of the solution was withheld along with the insolubles. The portion withheld, on evapora­tion to dryness, weighed 5.032 g* After the solution was passed on

U6to the column, elution was begun with heptane at room temperature#The first two fractions of heptane percolate contained 8#1$88 g. of a white solid which had neither ester, carbonyl, hydroxyl or acidic functional groups# This was presumed to be hydrocarbon# One per cent ether in heptane (l$00 ml#) removed 0#U$U2 g, of a white shiny plastic but tough solid. One per cent propanolic heptane (1000 ml#) eluted 0#1*880 g# (f4) of a white solid, similar in appearance to the solids in the previous fraction, but also possessing an aromatic odor# The fluorescent band characteristic of the alcohols iwierged after the passage of a further 2000 ml# of 1# propanol. The first $00 ml# of liquid collected after elution of the solids of Fraction F4 contained no solute# The alcoholic band (Fe) and the material eluted with it contained 2#92l*0 g# of greenish-yellow greasy substance with a characteristically pleasant odor# Fraction F6 contained more of similar material# The first 2000 ml# of l£ acetic acid in heptane yielded 0#2$Ul g. of a brittle, varnish-like, light brown solid (F7)# Fractions F6, F6, and F7 in addition to showing an appreciable hydroxyl value, also had a significant ester value. The acidic frac­tion was eluted in three stages, Fe-7l0# with l£ acetic-heptane.In each case they exhibited a significant hydroxyl value. The solvent schedule and fraction weights are given in Table XI, along with the analytical data on the fractions.

TABLE XI

Fraction m.«q. M.eq.* m.eq. ®.®q. m.eq. m.eq. m.eq. m.eq.Frac­ Eluant vol# vt. ester ester -OH -OH carbonyl carbonyl acid acidtion (ml*) - ~ Vs. total _ /£* total h . .

total __ /g. _ totalFi lieptane:750 7.1908 O .X 0.00 0.00 •» 0.00 - 0.00 -F2 heptane*300 0*6680 0.00 0.00 0.00 - 0.00 - 0.00 -?3 l£ ether in

heptane :1^00 0.1512 0.70 0.318 0.00 - 0.00 0.00 0.00 -F< 1$ propanol

in heptane:1000 0.1560 0.80 0.390 0,00 - 0.21 0.118 0.00 -

Fe 2$ propanol in heptane:1000 2.921:0 0.535 1.560 1.21 3.621 0.00 0.00 0.00 0.00

Fe 1% propanol in heptane:2000 0.8121 0.628 0.510 1.01 0.321 0.00 0.00 0.00 0.00

f7 15S acetic acid in heptane:1500 0.29a 0.580 0.117 1.135 0.365 0.00 0.00

fb 1 $ acetic acid in heptane:3000 0.6176 M . * N.p. 2.108 1.300 . 1.860 1.318

Fb 15b acetic acidin heptane:50C 0.3093 N.n. N.I>. 1.12 1.278 - 1.510 0.170

Fl0 2% acetic acidin heptane:1300 O.23O0 N.D, N.F. 1.1? 0.918 - 1.500 0.315Total 11.2181** 2.925 8.336 0.118 1.963Fraction of wax

chroma tographedw^rk m nn+. Heforrr

11.9700rl noH

9.330 wji. V* oH J 8.11 v a r ter . v «4 /.V 4- 0.181 2.830

U8This exploratory separation of candellUa wax led to the follow­

ing conclusions* 1) The portion of the wax which was actually chromatographed appeared to have been separated Into hydrocarbons, esters and ketones, alcohols and hydroxy esters, and acids, including hydroxy acids* 2) There was a considerable loss of esters, as shown by the imbalance between the quantity originally present in the original and recovered materials* Unfortunately ester values were not obtained far the acid fractions* It was found later, in the first ex­periment with ouricurl wax, that the acidic material did possess a significant ester value* It is questionable whether sufficient amounts were present In these fractions to offset the Imbalance* 3) There was an appreciable amount of acidic material unaccounted for* U) Ho specific conclusions could be arrived at concerning the composition of candelilla wax as a result of this experiment* However, it is to be noted that extensive chemical changes in the wax occurred on alumina which was less active than that lhich gave satisfactory results with the synthetic material, and with spermaceti wax*

Although the effects of the chemical changes ihich were taking place on the column were not understood, it was decided to carry out a second trial in order to observe more closely the significant features of the experiment, and to gain more experience with plant wax material*

The adsorbent used in this experiment was a fresh sample of Fisher alumina* It was washed in the usual manner with l£ HCl and water and brought to grade 2, similar to the activity used in the separation of synthetic mixtures* A column of similar dimension as

U9had obtained in the last experiment was used.

Twenty grams of wax were dissolved in 1000 ml* of hot heptane and the solution was transferred to the standard column; the insoluble

portion (resins) was not put on the column (0*5000 g*)* After heptane

and 1% ether in heptane had removed most of the hydrocarbon and simple

ester, 1.5 liters of 1% propanolic heptane eluted 0.2367 g. (F3) of a white plastic substance similar in appearance to Fraction F2 but possess­

ing a pleasant odor. No more solids were recovered from a further

500 ml. of this solution. The passage of an additional 1.5 liters of

eluant resulted in the removal of 2.998 g. (F4) of greenish-yellow

material with a soft spongy consistency* This preceded the main

fluorescent alcoholic band whereas in the previous run this portion was

mixed with the main band* This band (F0) was removed after a further

500 ml* of propanol and 1000 ml. of 1% acetic acid solution was col­

lected*

One and a half liters of 1# acetic acid in heptane removed

O.blUO g. (Fx) of a white powdery solid which was adulterated with a

small amount of a brown gummy material. This material was non-fluor­

escent. The acid band which exhibits blue white fluorescence was

taken off in 3 sections* Part 1 (Fe) was the portion immediately

preceding, and including some of the 1 cm. wide band, and part 2

the trailing edge (F9). Part 3 consisted of the remainder of

material eluted by 1 per cent acetic acid (Fl0)*

At the completion of elution with l£ acetic acid the

column was washed with two liters of a 50^ mixture of petroleum

ether and methanol* The residue after distillation of the solvent

5oconsisted of 2 g. of a solid which was only partially soluble in all of the wax solvents tried* Analytical determinations made on this fraction are not valid because of the partial insolubility, and the values are not reported*

This experiment exhibited, in most respects, the features shown

in the first separation of candelilla wax* The slight increase in

activity of the alumina affected the quantity of eluant required to

remove acids from the column* Whereas 6 liters were required in the

first case, here, 9.5 liters of 1% acetic acid in heptane were re­

quired* The analytical data given in Table show that even

greater disruption had occurred in this separation. This is illus­

trated by the greater discrepancy existing between the amounts of

original and recovered esters. In both experiments with candelilla

it was found that the wax consisted for the most part of hydrocarbons.

In the last separation over HJ> was isolated. This contrasts with the

52,2^ isolated by Schuette from a specimen of candelilla wax. Only

8<# of the wax was recovered after applying the customary eluant

schedule. The most unusual feature was that an appreciable quantity

of material could be removed with 50^ methanol-petroleum ether,

erven after the more powerful eluant, (i.e.) l£ acetic acid in

heptane, had been used. This indicated that the material remaining

on the column consisted of substances whi ch were different from any

hitherto encountered. These facts suggest that candelilla and

probably other plant waxes, were not solely comprised of the simple

ester compounds represented by octadecyl stearate.

TABLE XnResults

Frac- Eluant vol. tion (ml,)

factionwt.

m.eq,ester/g.

m.eq.estertotal

m.eq.-CH/g._ .

m*eq.-OKtotal

ffl.eq.carbonyl/ft

m.eq.carbonyltotal

m.eq.acid/g.

B*.eq.acidtotal

*1 heptane:1000 0.00 0.00 - 0.55 -Fa heptane:2,300 0^160 0.80 0.571* 0.00 - 0,3 83 0.131 **T?L3 1% propanol in

heptane:1^00 0.2367 0.85 0.201 0.00 - 0.915 0.215 m -1$ propanol inheptane: 2000 2.9980 0.127 1.277 1.30 3.90 0.00 0.00

f8 propanol inheptane: 500

acetic acidin heptane: 200 1.1793 0.60 0.706 1.59 1.87

f6 acetic acid inheptane:800 0.3269 0.715 0.21*2 1.65 0.5U0

F7 1% acetic acidin heptane: 1500 0.1*11*0 1.09 O.L.52 1.85 0.766 0.00 0.00

Fe acetic acid inheptane:2500 0.5295 N.D.* - 0 .00 0.00 1.61- 0.87

F9 1$ acetic acid inheptane: 3000 0.9197 M . - 1.16 1.06 2.20 2.02

F10 1# acetic acid inheptane: 1500 0.^555 H.P. - 0.558 0.27 0.00 1.62 0.71*5Total 15.9206** 3.1*52 8.366 0.31*6 3.635

Candelilla waxN.iJ.-fr ■ not detflrminod

20,0000 " W

0.620 12.1*0 ri Pld R r

0.532 10.6U 0.170 0.31*0 0.29 5.80

52E, The Application of Adsorption Chromatography to the Separation and Isolation of the Constituents of Ouricuri Wax*

a* Initial attempts at a chromatographic separation of ouricuri

wax*Although the problems arising out of the experiments with cande­

lilla wax were far from solved it was decided to begin a study of

ouricuri wax in preference to continuing with candelilla because as

stated previously the fonner is a more important article of commerce,

hence asjy information about its constitution would have greater

practical value.

The only available information on the composition of ouricuri

wax is contained in the reports of Schuette (31) and Ludecke (32),

The latter worker has published a list of some analytical and physical

constants of a sample of this wax and has claimed the presence therein

of hydrocarbons (17 ) and esters of myristic and cerotic acids,

Schuette has claimed isolation of a number of hydrocarbon fractions

and also the presence of melissyl cerotate.

Ouricuri wax used in this investigation was supplied and

authenticated by Mr, E, McLoud of S, C, Johnson & SQn, Inc,

The wax was dark brown in color and melted at 79-81°, The analytical

constants, expressed in conventional units, obtained for this sample

of ouricuri wax were: acid value 22,8; saponification value 106,9:

ester value 88,1; hydroxyl value 60,8, The values reported by

Warth (92) for the wax of A, cxcelsa, are: acid value 23,8; saponi­

fication value 89,3: ester value 61,9; m,p, 8U,3*,

In the first experiment, hereafter referred to as 01, 20*0 g« of the wax dissolved in one liter of hot heptane was transferred to a column (5*0 cm* x 30«0 cm) of fresh grade 3 alumina (600 g,). In the grading test the sudan yellow band began 1*5 cm* from the top of the column* Elution of the wax was begun with pure heptane, but as the system cooled upon addition of cold heptane, the flow rate de­creased, Even when pressure was applied the rate steadily fell off. This was an unusual result, and had not been encountered in any of the previous experiments. The effect was probably due to precipita­tion of wax material preventing the continuous flow of liquid through the system. However, when the column was heated by infra­red lamps the flow rate returned to the desired level. The solids collected from 1600 ml* of heptane eluate were designated as OI-Fjl.This was the small amount of material which preceded the sudden ap­pearance of large quantities of solids in the cool percolate, frac­tions 0I-F2, 01-F3, and 0I-F4, eluted with heptane, all shared the same general characteristics, in that they did not contain functional groups of greater polarity than carbonyl. When the quantity of material appearing in the heptane percolate showed signs of decreasing, the eluant was changed to 1# ether in heptane. This, however, did not affect the elution rate. Consequently, after one liter of the latter had been added, the eluant was changed to 1% propanol in heptane, Fraction 0I-Fe consisted of the solids obtained from one liter of 1$ ether and 2*5 liters of 1# propanol. The introduction of the latter eluant at this stage did not interfere with the sharp­ness of the separation of esters and alcohols as shown by the fact

&that 200 ml* of solid-free percolate were collected immediately pre­

ceding the emergence of the fluorescent alcohol band* Fraction 0I-F6

was composed of the solids present in the first liter of percolate

collected after making the ester cut. It consisted of all the ma­

terial present in the white fluorescent band. The solids were

greenish-brown in color. The bulk of 01-F6, however, was white

crystalline 3olids, of a chalky consistency. The eluant was changed

to IjC acetic acid in heptane just preceding the emergence of the

alcohol band* Fraction 0I-F7 therefore was removed using the latter

eluate, and bore similar analytical characteristics to solids of

oi-f6.VB.th the addition of acetic acid a new ultraviolet

fluorescent band developed at the head of the column* This band

emerged from the column after 3750 ml. of 1$ acetic acid were col­

lected* Fraction 0I-Fe consisted of the solids recovered from the

fluorescent band of acidic material, and 0I-Fg, those solids which

move at a slower rate than the preceding ones* Fractions 0I-FlO and

OI-Fj^ contained more brown coloring matter than did either fractions 0I-Fe or 0I-F9.

The material removed in this experiment (10.6507 g.) ac­

counted for only 53 .3 of the original charge. When the column was

washed with 2.5 liters of a $0-j0 mixture of methanol-petroleum

ether, a fur tiler 8.016 g. were recovered. This was not all derived

from the wax, but contained large amounts of a white crystalline

solid, mixed with brown wax material. The former was insoluble in

the usual wax solvents. Consequently the analytical determinations

55are not valid and are not reported# Table XIII is the balance sheet

of this first experiment with ouricuri. It shows the amounts of

material recovered in terras of railli-equivalents of esters, alcohols

and acids in comparison with the quantity present in the original

charge.

TABLE XIII

Showing the Amount of Recovered Material Compared to the Original ______________ Charge in Ouricuri (Experiment 01)______________

WeightW.eq.EstersTotal

M.eq.-OHTotal

M.eq.AcidTotal

Total fromrecoveredfractions 10.6^07 6.861 8.190 5.830

Ouricuri wax 20.000 30.000 21.150 8.000

The analytical data for the several fractions obtained in 01

are not presented because the overall recovery was very low (53.3^)

and consequently the recovered fractions were not representative of

the true nature of the total wax constituents#

A general appraisal of the results obtained in the investiga­

tion up to this point showed that less alteration of the waxes oc­

curred in those separations conducted on regenerated alumina, viz.

in the second experiment with spermaceti wax, and in the first with

candelilla. Apparently, exposure of the alumina to the eluant

schedule brought a trout unexplainable changes which decreased the

tendency of the adsorbent to alter the wax during chromatography.

In order to carry this reasoning to its logical conclusion, a

second experiment, Oil, was carried out on the same sample of

56alwwrlnft used in Experiment I, but which was regenerated by washing exhaustively with methanol, then water, and finally drying at 10*?* to produce the required grade 3. For this run 15 g» of ouricuri wax were dissolved in 1500 ml. of hot heptane, and the solution trans­ferred to the column. The column was wrapped with a coil of nichrome wire, and then enveloped by a suitably sized glass tube. The wire was heated by means of a variable resistance coil, and kept at 60* throughout the experiment. This type of heated column was used in

later work with alumina. The progress of the experiment followed the same pattern set in the previous run, and the salient features are represented in Table 17.

TABLE XIVPie Chroma to granhi c Separation of Oaricnri Wax on luinina (Experiment Oil)

ft*action Eluant vol. (mi.)

tractionWeight(g.)

K.eq.Ester/g.

W.'SqV "EsterTotal

“TT.Tq.-OB/g#

M.aq.-OHTotal

M.eq.Acid/g.

fc.eq.AcidTotal

OII-Fl heptane :1300 o.i55o 0.00 0 .00 n.B.* N.D. ft. - TTJ5T "oii-f2 heptane : $00 0.5370 1.08 0.905 0.00 0 .00 0 .00 0.000H-F3 heptane:2,100 2.3220 1.02 2.370 0.00 0 .00 0 .00 0.00oii-f* l£ propanol in

heptane:3550 3.8070 1.10 U.393 1.26 h.80 0 .00 0 .00011-Fg 1$ propanol in+ heptane: 1500 0.7700 1.75 1.3U8 0.66 0.508

oii-f6 1 acetic acid inheptane:1500

oii-f7 l£ acetic acid inheptane*3000 1.8871 0 .71 1.338 1.310 2.L70 1.650 3.1150II-Fe 1 acetic add inheptane: 800 1.6L78 0.595 0.980 0.820 1.350 1.6U5 2.710

oii-f9 1% acetic add inheptane12000 0.9020 1.00 0.902 2.156 2.215 1.2U 1.120

0II-FlO toluene: 800 1.660 1.862 3.091 1.00 1.660 0.61:5 1.071Total 13.9979*-* 15.130 13.003 8.016Ouricuri wax 15.0000 1*50 22.500 1.075 16.120 0.L00 6.000#N.D. ■ rot determined

**Yield 93 mJ% by weight.

58Fraction OII-Fx (Table IIV) contains the small amount of material

eluted by heptane before the sudden appearance of large amounts of solid.

This is probably hydrocarbon, but does not represent the total amount

in the charge, the remainder being found with the ensuing ester fraction.

Fractions OII-F2 and OII-Fa possessed the same characteristically

sweet odor of their counterpart in 01. Similarly 0I-F4 et seq. all

behaved in like manner to the corresponding fractions in the previous

run. When no more solids were eluted by 1% acetic acid, the column

8till retained material. This was shown by the characteristic brown

area at the head of the column, and which extended down for six or seven centimeters* In an attempt to remove this residue, 800 ml. of

toluene were used, resulting in the recovery of 1.660 gms. of a light

brown mass of chalky consistency} not all of the color was removed

from the column however.In this experiment 93*3% recovery was attained, exceeding by

U0£ the yield in 01. This result supports the hypothesis that prior

subjection of the alumina to the eluant schedule brought about in­

trinsic changes in the alumina, thereby affecting its behavior. In

fractions 0II-Fx to Fe, the amount of material recovered was $2,6%

of the original charge as compared with 3U.03^ for the same materials

in 01. The recovery of total ester equivalents, though increased to

67*3% of the original charge was still too low to be considered satis­

factory. The increase in recovered acidic material without corre­sponding increases in alcohol equivalents pointed to the nature of

the labile material as being other than simple ester.

There was an increase in acids compared to the amount originally

59present, indicating that acids had been set free during the experiment. However this was only an increase of 2.016 railli-equivalents, and therefore would not account for all, or even the greater portion of the unrecovered ester material. This assumes that the increase in acids was derived fro* esters. In addition there was a deficit of 3.117 inilli-equivalents of alcoholic material* This suggests that the remaining alcoholic function was to be found in the unrecovered 7%m It may be concluded therefore that although the use of re­generated alumina was instrumental in improving the yield, it did not prevent alteration of the wax during chromatography.

It seemed most likely at this point that of the eluants used acetic acid would be most likely to affect the future behavior of the adsorbent. Therefore, for the next run, a batch of alumina (6£0 g«) was washed with two successive 5>00 ml. portions of a 2% aqueous solu­tion of acetic acid instead of HC1. The mixture was stirred vigorously for 30 minutes on each occasion. After washing, the supernatant was decanted and the alumina next washed four times with distilled water.It was then brought to grade 3 in the usual way* Ten grams of ouricuri wax dissolved in 2 liters of heptane were chromatographed at 55** A comparison of the fraction weights (Table IV) with Oil showed that changes in the wax had also occurred in this experiment* Frac­tion 01135^ was apparently a fairly pure sample of hydrocarbon and probably represented the total amount present in the charge, thus showing that alumina of this grade was still active enough to give a complete separation of this constituent* Only 3U*7U£ was recovered prior to the removal of acidic material as compared to 52.6$ in Oil.

TABLE XVEluant Schedule and fraction ^eights of the Chromatographic Separation

Fraction Eluant Volume (ml.)FractionWeight(g.)

QIII-FX heptane:2750 0.1359

0III-Fa heptane:20Q0 1 propanol*3000 1.6810

OIII-F3 1 propanol in heptane:2^00 1.6570

oiii-f4 1$> acetic acid in heptane:6000 2.5370

0III-F5 toluene:!,100 1.DL30

Total recoveiy* 7.1539Charge 10.0000

Further analytical work was not done nor thought desirable, because knowledge of these analytical constants would not help in the solution

of the problem. It was clear that this type of pre-treatment was not

satisfactory.The crux of the problem at this point was to find the conditions

required to produce alumina incapable of effecting chemical change in this wax, and still be active enough to separate it into its major functional groups*

The following summarizes the experimental results obtained up to tliis stage, l)Unused alumina (Fisher) caused severe disruptive ef­

fects on the mixtures which were chromatographed on it, 2) Alumina, pre-treated by washing vith l£ HCl, and standardized to give grade 2, effected a satisfactory separation of a synthetic mixture of hydro­

carbon, n-ester, alcohol and fatty acid. 3) Alumina preparod according60

61

to the specifications above was not suitable for the separation of

spermaceti wax. However, when this wax was chromatographed on re­

generated adsorbent, a separation free of disruption resulted. U) When

candelilla and ouricuri waxes were chromatographed on regenerated

alumina (grade 3)# the yields were improved; however, the ability to

alter the natural constituents of the wax was still retained, although

to a lesser degree. 5) The unrecovered material on the column con­

sisting of either original or altered material, was not eluted by 1^

acetic acid in heptane, either because it was more polar than 1$

acetic, or it was insoluble in this solution.

b* Experiments to solve the problem of preparing alumina

suitable for separating ouricuri wax.

The following series of experiments was designed to determine

the characteristics which alumina must possess, to permit its use as

a satisfactory adsorbent. The series was to be carried out on a

smaller scale than had obtained for previous separations of waxes.

Six experiments OIV-QIX are described and in each the column was

one of inside diameter 3Ji cm. The weight of alumina was standardized

at 120 g., and the amount of wax at U g. All separations were con­

ducted at 55°.

In OIV and OV the fact that adsorbent should not be muffled

immediately prior to Its use in chromatography was re-established.

The alumina in OIV was a previously used specimen, subjected to calcina­

tion in a muffle furnace for 5-6 hours at 600#, and brought to grade ?

by exposure to air. Four grams of wax in UOO ml. of heptane were

transferred to the column, and elution begun with 1^ acetic acid in

62heptane. After 750 ml* of eluant had been collected, solids were no

longer forthcoming, so the eluant was changed to toluene (600 ml*).

The combined solids from both percolates were then analyzed, with the

results shown in Table XVI* In all the separations of this series

the facts of specific interest were: 1) total yield, 2) amounts of

ester and acid equivalents recoverable* These data were used as

indices of the efficacy of separation* The optimum conditions would

be those which permitted the maximum recovery of esters with the

minimum of acidic material, so long as the total yield remained relatively high.

TABLB XVI Results Obtained in Experiment OIV

SampleM.eq.Ester

_ /g.

M.eq.EsterTotal

M.eq.Acid/g.

M.eq.AcidTotal

Weight. . . <£•) .

Wax recovered 1.073 3.65 0.61 2.20 3.1130::

Wax, original 1.50 6.00 0.10 1.60 1.0000

*Yield 85.3^ by weight

P'ram the results given in Table XVI it was concluded that a

significant amount of alteration had occurred. This was shown by*

l) a deficit of 2.35 mi111equivalents of ester in the balance

between the original and recovered material; 2) an excess of 0.60

mi111equivalents of acid, even though only 85^ of the original

charge had been recovered.

In the next experiment^ 0^,a sample of unused alumina was pre­

washed with glacial acetic acid (l ml* per 5 gm* of adsorbent), and

water* After 30 minutes of continuous stirring the supernatant was

63decanted and the adsorbent washed with several portions of water*

The supernatant was again decanted and the adsorbent heated in a

muffle furnace for 5 hours, after first removing most of the moisture

by heating at 10fJ* for 12 hours, When this sample of alumina, reduced

to grade 2, was used for chromatography the resultant yield was only

50.6£, showing that the alumina was unsatisfactory* Analytical de­

terminations were not carried out.In the next experiment, OVI, the adsorbent was first subjected

to the same pre-treatment as in the previous case, except that it was

not muffled* The sample was heated in the drying oven for a suf­

ficient period to produce grade 3 (usually 9 hours for 3 ^ 0 g.). In OVI 3 eluants were used and 2 fractions collected, the solids from the

toluene percolate being Included with the acidic portion. The

analytical results are given in Table XVII*

TABLE XVIIThe Chromatographic Separation of Ouricuri Wax (Experiment OVI)

Fraction EluantFractionWeight

(g.)

iCeq.Ester/£•

m Mol Ester Total

M.eq*Acid/g.

M.eq.AcidTotal

0 1$ propanol 1.900 1.07 2.036 o.o5 O.09Sovi-f2 2 AcOH

toluene 1 .3 6 1 1.78 2.U20 0 .9 2 1 .2 5 0

Total 3.?6l* U.U56 1.3U5Ouricuri Wax L.ooo 6.00 1.600

* Yield - 8l*6£ by weight

These data show an improvement in the number of ester equiva­

lents recovered, 7U.25C of the original charge, and that U7»5% of tne

wax was recovered before elution with 1^ in acetic acid in heptane*

6UThe latter value is also important because it serves as an additional

index of the degree to which the wax may or may not have been altered.

For the next experiment (OVil), the following changes were

introduced. The grade was reduced to U; this was obtained by heating

the alumina * after the final wash in a drying oven for 8 hour# at

105>°. This heating time was found satisfactory for batches of 300 g.

placed in 1 liter beaker3. The concentration of the final acetic-

heptane eluant was increased to 10{£. This eluant removed added

material from the column, as shown by the reappearance of color in

the percolate* Propanol, 2% in heptane was used instead of l£. The

analytical results given in Table XVIII show that: 1) the percentage

of recovered esters had increased to 76 *1; 2) the amount of material removed before elution with l/£ acetic acid was increased to 56*71$.

The percentage of mill 1.-equivalents of recovered acids was reduced

to 76*3.TABLE XVIII

The Chromatographic Separation of Ouricuri ^ax (Experiment OVTl)

Fraction Eluantfraction

wt.(g.)

m.eq.ester

m*eq*estertotal

m.eq. acid

. /g.

m.eq.acidtotal

ovii-yx 2% propanol 6.27? " 1.366 2.97 CCT? a.i<w0VlI-?2 5-10? AcOH 0.808 l.aob 1.60 0.982 1.113

toluene 0.329Total 3.U1U* a. 57 1.220Ouricuri Wax h.ooo 6.00 1*600

-Yield 85.35$ by weight.

It appeared that decreasing the adsorptive strength of tie ad­

sorbent enhanced the chances of recovering the wax in an unaltered

65form.

In OVIII, the next run, alumina made by M. ^oelm, designated

as anj onotropic, and having pH b.O, was used. ^ie alumina had not

been previously used, and was not subjected to pre-treatment, except

exposure to the atmosphere long enough to produce grade U (time

dependent on humidity). The experiment was conducted in a similar

manner to the others of this series* Benzene was introduced after

propanol and before acetic acid to test the effect of this eluant

on removing difficultly soluble esters. The solids from this

eluant, after weighing, were combined with the acetic acid fraction.

TABLE XIIThe Chromatographic Separation of Ouricuri Wax (Experiment OVIII)

Fraction Eluantfractionweighta (g-)

m.eq.ester/g.

m.eq.estertotal

m.eq.acidA *

m.eq.acidtotal

2$ propanol £.2300 1.361 3.636 0.5U2 0.69bo v h i -f2 benzene

2-10$ acOH0.22080.9325

1.U72 1.695 0.898 1.035

Total 3.3833* U.730 1.1290Ouricuri Wax b.oooo 6.00 1.600

*Yield 8b.58$ by *»ight.

Table XXI shows "that: l) the yield of recovered ester had been in­creased to ?8.8 %: 2) the amount of material appearing previous to 1$

acetic acid elution was 55.75$. 3) The recovered acids had decreased

to 7 0 The results of this series up to this stage show that the effect of untreated alumina (Fisher) in altering wax constituents

car be traced, at least in part, to its alkalinity, or the alkaline

material adsorbed on the alumina surfaces* It is noteworthy that

66this effect is not successfully counteracted by merely washing with

hydrochloric acid until the supernatant attains a neutral pH. The

critical factors appear to be: l) adsorptive activity; the alumina

must be of grade b (by the test conditions previously stated);

2) the pH of the supernatant after the preparatory water wash should

be between h and 5.In the next experiment OH, a sample of alumina (Fisher),

previously used in this series was washed with acidulated water, ac­

companied by vigorous stirring for 30 minutes. The acidic wash was

considered satisfactory when the supernatant liquid produced a green

color with bromcresol green (pH 3.8-5.U, yellow to blue). The super­

natant was decanted, and the alumina brought to grade U in the

previously described manner.

In O U the following eluant schedule was used: 1) heptane;

2) 2$ propanolic heptane; 3 ) propanolic heptane; b) benzene;

5) 2-10$ acetic acid in heptane. The analytical results and salient

data are given in Table IT.TABLE XX

The Chromatographic Separation of Ouricuri Wax (Experiment OS)Fraction Eluant

Fractionweight(gj)

m.eq.ester

/

/ §•

m.eq.estertotal

m.eq. acid . /g.

m.eq.acidtotaldRZTi heptane 0.n?02 o.87o 0.715 -

OLX-Fa 2-10$ propanol 2.3bliO 1 J.'h? 3.395 0.205 0.1-020IX-F3 benzene

2-10$ AcOH0.080*-k0.2135**

1.513 0.1.81 1.01*7 0.368

Total 3.U577* b.591 0.850Ouricuri Wax b.000 6.00 1.600* Yield (36.l<b/£ by weight. -^Combined.

67The data in Table JX show that 10£ propanolic heptane was instrumental

in removing most of the material formerly eluted by benzene and in­

cluded some of the solids hitherto present in the acetic acid-heptane

percolate* The data further show that there was a slight decrease in

recovered ester to 76,5$: however, this was not considered significant.

The percentage of recovered acids had decreased to 53 »1 (a very

favorable feature), and the amount of solids appearing in fractions

OIX-Fi and F2 was 79,3^, The small quantity in the benzene percolate

was included with the acidic fraction, A signi ficant quantity of

acidic material was present in fraction OUC-Fa, This was probably

brought down by occlusion of some acidic material on elution with the

more polar 1($ propanolic heptane.

In each separation attempt in this series, there had been a

significant amount of the original charge which had resisted removal,

even by a powerful eluant such as 1CJS acetic acid, A part of this

was elutable with methanol, but at the same time was irretrievably

contaminated with an unknown inorganic solid, Dioxane proved to be a

useful agent in removing the residual wax and not the inorganic

matter. However, the solids from the dioxane percolate, which were

found later to be resins, are not included in the data reported for

OIX, In this series of experiments it was found that acetic acid

appeared to enter into reaction with alumina or material adsorbed

on its surfaces. The compound so formed when removed by methanol was

found to contain alumina in qualitative tests and also gave a pungent

acetic odor on hsating.

68c* Final experiments leading to the separation of ouricuri wax

into major chromatographic entities*

The results obtained in experiments OIV-OIX indicated that when

freBh adsorbent was to be employed VIoelm alumina should be the one of

choice. Large enough quantities of this adsorbent were not available

when the series to be described was begun, consequently for the first

attempt, on return to relatively large scale separations, the adsorbent

chosen was a specimen regenerated after previous use by washing with

methanol and water and then heating in a muffle furnace at 600* for

$ hours* Finally, the alumina, after muffling, was washed with

acidulated water (about 2 ml* hydrochloric acid per 100 g, alumina)

until the supernatant produced a green color with bromcresol green;

it was then brought to grade U* The separation Ctt was conducted on

li80 g* of this alumina* It was decided to elute the hydrocarbons

along with simple esters, and later resolve the mixture on a second

chromatogram using sili ca gel as adsorbent* As a consequence a

smaller quantity of adsorbent was required, resulting in a saving of

time and solvent. In the elution schedule propanolic heptane was

substituted for 1C#« The general trend shown In Oil was repeated

here. In an effort to remove as much of the residue (after toluene

elution) as possible both dioxane and methanol were employed and the

percolates were combined before evaporation of the solvent. This

was an ingenuous step, as methanol removed the typical adulterating

non-wax solids. The percolate from methanol elution is deceptively

clear and therefore gives no hint of the oresence of the non-wax

69material* The methanol percolate was in this case clear and brown in

color. On concentration of the solution, copious amounts of idiite

crystalline material precipitated from solution even at steam bath

temperature. The material so obtained was worthless for analytical

purposes, because of the insoluble material. Discounting this

portion of the eluted material, and considering the results obtained

from the preceding fractions it was found that 65,3^ of the original

charge was present in fractions aX-F1-OXF3, and that 68,?^ of the

esters had been recovered. However 96,5^ of acids had already been

recovered, with a total weight yield of 88,0{6,

This separation was not sufficiently free of disruptive

changes to be considered as a satisfactory basis for a true evalua­

tion of the nature of the original wax constituents. However, the

following general conclusions were drawn. Fraction OX-Fj probably

contained all the hydrocarbon as well as most, if not all, the simnle

ester units. These solids were white with a tinge of yellow.

Fractions CX-F2 and F3 appear to contain mixtures of simple and

hydroxy-esters of varying molecular weights, and free alcohols.

While fraction 0X-F2 is green in color, CK-F3 was a brown solid

which cracked on cooling, A comparison of the mean molecular weights

obtained from the melting point depression of camphor^ and those

calculated from the ester and hydroxyl value is not of great signifi­

cance in this experiment, since artifacts may be present. An unusual

feature of these analytical determinations was, that when the acid

values were determined in hot or warm solution the results were sig­

nificantly higher than when the solutions were cooled to room

70temperature. Warming 'was necessary to dissolve the sample. The difference could conceivably be due to the presence of lactones or lactone-like substances. Furthermore the determinations in hot solution were characterized by quickly fading end-points.

With addition of acetic acid in heptane the general character

of the effluent solids changed. Fraction QX-F* was a brown solid

composed predominantly of acids, some of which may have been

hydroxy-acid s. The possible identity of the nature of the constituents

of fraction 0X-Fc posed a greater problem, and at this stage of the

investigation was open to conjecture. The molecular weight calculated

from the ester value is $60, and from the acid value lk2$: this

constant obtained by physical means is 720. Thi s would suggest that

the fraction consists of a mixture of acids and hydroxy-diesters,

since the probability of simple esters being present is very small*

The toluene fraction 0X-F6, a very dark brown shiny solid,

gives evidence of containing a substance of relatively high molecular

weight, a portion of which may have appeared in the previous fraction.

The molecular weight was found to be 1080. The value predicted from

the ester number is UU9, and from the acid value 20^0. The indica­

tion is therefore, that this fraction consists almost entirely of

diesters or polyesters joined through secondary hydroxyl groups. The

results of thds run are set forth in Table XXI*

TABLE XXIThe Chromatographic Separation of Ouricuri Max (Experiment OX)

Fraction Eluant vol. (ml.)

Fractionweight(g.)

m.eq.ester

m.eq.estertotal

m.eq. m.eq, -OH -OH /g. total

m.eq.acid/g.

m.eq.acidtotal

mol.vt.(Rast)

tS-F,. heptane:22CClg propanol:1000 3.8200 1.03 3.9U0 0.203 0.776 0.00 0.000 N.P.**

ox-f2 1$ propanolicheptane:27^0 3.7 U 5 1.127 U.180 1.20 U.U50 o.oia 0.151 610

qx-f3 % propanolicheptane:2100benzene:1000 2.2700 1.515 3.UI4O 1.61 3.720 0.175 0.397 720

gx-f4 3% AcCH:3l5010* Ac0H:250 2.1630 0.57 1.238 0.565 1.220 2.02 U.U50 h96 S

QX-F„ lOg AcOH:1750 0.8510 1.851 1.575 1.15 0.979 0.902 0.668 720QX-FS toluene:1000 0.3950 2.1*0 0.9U7 1.56 0.616

COCO«0 0.192 1080Total 13.2135* 15.320 11.771 5.858

Ouricuri Wax 15.0000 1.50 22.500 1.075 16.150 O.ljOO 6.00

*Yield 88.0g (byieight)

s-K-N.r. ■ not determined.

72In separation 0X1, the conditions which obtained in the

previons experiment were duplicated except for differences in the

elution schedule* The adsorbent was the same specimen used in OX,

but regenerated in the usual manner, A larger amount of heptane

was used in eluting fraction QXl-Fx, Fraction 0Xl-F7 consisted of

the solids eluted by dioxane. The analytical results show that

in the first 6 fractions 7U.C$ of the total ester material was re­

covered, and that the percentage of material present in the first

3 fractions was 67,5, The amount of acids appearing in the first

6 fractions had decreased to 6l$ of the original charge. These

fi gures do not take the dloxane fraction into consideration and

are given so that a comparison may be made with the previous

separation. When the solids of OXl-F? are included the amount of

recovered esters increases to 92.05f* the acids however show an

excess of 1 ,2 5 mi 111equivalents in a total yield of the recovered

material of The salient features of this separation aregiven in Tabie XXII.

TABLE m i

Fraction Eluant vol. (ml.)

Fractionweight

...(fit).. .

rn.9q.ester/g.

m,eq.estertotal

m.eq.-OH

. _ / * • _

m.eq.-OHtotal

m.eq.acid/fi*. ..

m,eq.acidtotal

mol,wt,(East)

dfc£-Px heptane :31|C0 14.7000 1.118 £.hoo 0.222 l.^i3 0.00 0.00 W.i;,**cki-f2 1$ propanolic

heptane:2$00$% propanolicheptane:500 3.1100 1.370 14.250 1.30 I4.O3O 0.029 0.090 750

axi-73 $% propanolicheptane:2L50 2,3100 1.690 3.910 1.65 3.520 0.275 0.635 633

oii-f4 1# acetic acid inheptane:3500 0.9700 0.630 0.610 0.20 0.X9U 2.03 1.970 h80

qki-f6 acetic acid inheptane:uOO1C$ acetic acid inheptane:2500 0.9220 1.820 1.680 1.15 1.060 0.702 0.6U6 750

oxi.f6 toluene:1300 0.359 2.227 0.797 1.56 0.560 0.503 0.180 1100OiI-F7 diaxane:l500 1.365 2.920 u.000 2.25 2.961 2.73 3.730 N.D.

Total 13.73^- 20.6!i7 13.668 7.251Ouri curi Wax 15.000 22.50 16.150 6.00

-"-Yield 91. U* by weight

“ not determined

The solids from QXI-F7 in Table XXII were light brown in color

and generally resinous In appearance* ^he difficulty encountered in

elutinr‘ this fraction was the main reason for the poor yields in

past separations* These solids gave no melting point, and tended to

char rather than melt, at hi-h temperatures, consequently molecular

we.i.cht determinations could not be made* As expected, a con­

siderable portion of the ester, alcohol and acidic functional groups

were found in QX-F7. The general conclusion drawn from the balance

obtained between the orirTinal and recovered material was that the wax

had been separated with the least amount of alteration so far ob­

tained*

So as to obtain bettor and more detailed information about

0XI-i?1-F3, it was proposed to chroma to.-raph these fractions on

silica gel using the basic scheme suggested by Fuchs and de Jong (51),

but with original modifications as appeared necessary, before this

could be done, however, more material had to be obtained, Conse­

quently another separation was carried out* Alumina (M, Woelm) had

been received in sufficient quantities to produce a column of the

desired dimensions. For OXII, U^O g* of alumina (Woelm) was exposed

to the atmosphere for a sufficiently lon:r enough period (time de­

pendent upon humidity) to produce rrade U* A no]umn was prepared in

the usual way and I S g* of wax in IS'00 ml* heptane was transferred

to it* Tne schedule and analytical data obtained in OXII are pre­

sented in Table XXIII at this time so that the description of the

course of the experiment can be followed*

TABLE IXIIIThe Chronatographic Separation of Ouricuri Wax on Alumina (Experiment XII)

Fraction Eluant vol. (ml.)

Fractionweight(g.)

m.eq.ester/jU,

m.eq.estertotal

m.eq,-OH/g.

m.eq.-Offtotal

m.eq.acid/g.

m.eq.acidtotal

Wt.tinwax

mol.vt.

(found)0KII-?X heptane: 3600 3.3l7^ 1.075 3.560 not - - 22.6$ li.b.-JHi-

detectableckii-f2 2$ propanolic

heptane:3000$% propanolicheptane: 00 li.2270 1.370 5.830 1.50 6.000 O.O63 0.255 28.19 H.D.

0XII-?3 propanolicheptane:2000 1.6130 1.65 2.992 1.68 3.0U5 0.161 0.292 12.07 730

cxn-?4 benzene:EtOH 2:1800 0.305 2.678 0.817 1,81 0.553 0.165 0.050 2.03 725

okii-f6 2% acetic acid in -v)Vj\heptane: 1500 1.3052 0.00 0,00 0.15 0.195 2.020 2.6bO 8.70

0XII-F6 0.5£ acetic acidin benzene 0.)i630 1.6U5 0.713 0,87 O.I425 0.960 O.Uib 3.06 AcyiW y W

otn-F7 $% acetic acidin benzene 0.80U7 2.80 2.252 1.57 1.261 O.I466 0.375 5.35 1950

o m - F e benzene:EtOH:AcCH:$0:25:l750 0.7210 3.78 2.722 2.13 1.536 h.550 3.2b2 U.80 N.D.

nxn-Fe benzene: EtOH:AcOH:50:50:52000 1.5021 2,66 3.985 2.27 3.b08 2.660 3.985 10.01 K.D.

Total m.ho85-> 22.870 16.003 11.283 96.30

Ouricuri Wax 15.000 ■U ■ - 1 22,500 16.150 6.00

** N.D. - not determined

76Fractions QXH-F1, F2 and F3 were removed in the customary

fashion, while O^II-F* was eluted with 800 ml* of benzene-ethanol,

2:1, instead of benzene alone* The acidic fraction, 0XII-Fs, was

eluted as usual with 1^ acetic acid in heptane. From this point the

elution schedule deviates from the pattern followed hitherto. The

material remaining on the column was removed in two stages* First

2 liters of a ^ solution of acetic acid in benzene-ethanol, 2:1,

removed solids which when taken to dryness were found to contain

large amounts of inorganic matter. This fraction per se is not

represented in Table XXIII, but was purified and rechromatographed

in a manner to be described later. The second fraction containing

the remainder of the colored matter on the column was eluted by

1 liter of a ^ solution of acetic acid in benzene-ethanol 1:1* This

percolate also contained a large amount of inorganic solids. To

purify those two fractions the following procedure was adopted. The

wax material from the first batch of solids was separated out by

washing with benzene and then decanting the solution into a separate

container. A final, wash with ethanol removed all the wax from the

white crystalline residue. The wax solution was then returned to a

column of alumina (120 g.) 3*U cm. diameter. A solution of

acetic acid in benzene removed the fraction referred to as 0XH-F6

in Table XXIII. This was followed by ^ acetic acid in benzene,

giving fraction 0XII-F7, Fraction 0XlI-7a was eluted with a ^

solution of acetic acid in benzene:ethanol (2:1). Again the perco­

late contained inorganic material. This was finally separated

from the wax material in the following way. The solids taken to

77dryness were redissolved in benzene end a little ethanol* This gave

a partial separation of the wax material* The solution was then

passed throu^i a column of silica gel and eluted with 5 acetic acid

in benzene* This procedure completely separated out the wax material

(resins) which is recovered in the percolate*The second batch of solids, i,e* those removed by 5% acetic

acid in benzene-ethanol 1:1, was purified by dissolving the material

in benzene and passing the solution through a column of silica gel

as with OXII-F0, The recovered material was free of inorganic matter

and is represented in Table XXIII as OXII-F9, The balance between

original material and the recovered fractions given in Table XXJII shows that there is an excess of acidic material in the recovered

fractions, contributed to mostly by resins* Since there is a small

increase of the amount of recovered ester, and only a slight de­

ficiency in alcoholic material, it can be presumed that these acidic

components are probably not derived fr*oro the rupture of ester

linkages*Because of the importance of the alumina used in QXn, details

of its preparation follows. Alumina, M* Toelm, pH U,0, is exposed

to moist air for a sufficient length of time to give a product, aof

standard 5,5 B* column (1,3 cm, diameter)/which behaves according

to the following specifications, A mixture of p-methoxy-azobenzene

and sudan yellow (20 mg, each) is dissolved in 10 ml. of benzene

and the solution made up to 00 ml, with petroleum ether, A ml,

aliquot of this solution is then passed on to the column of alumina, and eluted by 20 ml, of the same solution, free of dye. Alumina of

78grade U will permit the complete removal of p-methoxy-azobenzene, but

will leave a band of sudan yellow beginning about li cm. fr*om the top of the column.

d. Separation of some of the fractions obtained in (c) into

less heterogeneous mixtures with silica gel.

Having achieved a separation of the wax into its major

chromatographic entities on alumina, it was now desirable to resolve

some of these fractions into less heterogenous mixtures. The first

three fractions resulting from experiment OXII -were subjected to

chromatography on silica gel. A 2.7225 g. aliquot of fraction

QXII-F-l was dissolved in 300 ml. of heptane and the solution trans­

ferred to a column (3.U cm. inside diameter) containing 120 g. of

silica gel (Davidson). This column was used for all separations

described in this section. Elution with 750 ml. of heptane gave

0.1606 g. of a white plastic solid melting between 57-71*. This

material had no ester value and was therefore considered to be hydro­

carbon. The mean molecular weight of this fraction was 336. Heptane

elution was followed by a 50^ mixture of ethylene dichloride and

heptane (1000 ml.). The latter eluant removed 0.95UO g* of a white

crystalline solid tinged with yellow (O^ll-F^Pa), m.p. (82-87*).

This fraction had no hydroxyl value, and was therefore considered

to be simple esters, since this was the only functional group present.

The next eluant ethylene dichloride (800 ml.) removed 1.5Ul5 g. of

material similar in appearance to the previous fraction, and was

designated QXlI-F^-Pg, m. p. (82-118.?). This fraction possessed a

79higher ester value than the previous one, but had no hydroxyl

functional groups. It was therefore considered to be esters of lower molecular weight. Fraction CKII-F1-P4 was eluted by 2

propanolic ethylene dichloride, and consisted of 0.0650 g. of a

light brown finely divided crystalline substance. Enough material

for a single hydroxyl value determination only showed it to contain

alcoholic material. The impression is that this fraction consists

of free alcohols brought down by imperfect separation in OXII. The

analytical data obtained from these fractions are given in

Table XXIV.

TABLE XXIVThe Chromatographic Separation of Fraction OXII-F^ on Silica Gel

Sub- fraction Eluant volume (ml.)

Sub­fractionweight(g.)

Amount in original fraction (g.)

m.eq,ester/g.

estertotal

m.eq.-OH/g.

m.eo.-OH*total

Wt./f oforiginalwax

mol.vt.(Rast)

(ttll-Fi-tj, heptane: 7 00 0.1606 0.195& 0.0 — 0.0 - 1.30 336

QXII-Fi-Pa 50$ ethylene dichloride in hertane: 1000 0.95Uo 1.3600 1.03 1.395 0,00 0.00 7.75 920

oxii-f1-f3 ethylene dichloride: 800 U5°c 1.5M5 1,8760 1.265 2.375 0.00 0.00 12.1-9

cc780

CXIT-F^F* 2$ pronanol in ethylene di- chloriae 500 o.o65 0.078 N.D.* 3.02 0.196 0.52 N.D.

Total 3.309b 3.570 22.06Fraction CKII-?! 3.3175 1.075 3.56 not

detectable22.30

-*N.D. * not determined

81A 3.?77U g. aliquot of fraction OXII-F2 (Table XXIII) was

dissolved in 7? nil, of chloroform, and transferred to a column of

silica gel* This adsorbent was a specimen previously used and re­

generated by heating in a muffle furnace for ? hours at !?00*. ^h©

silica was subsequently washed with water and kept in a drying oven

(105°) for 2b hours. The first fraction (3XII-F2-P1 in this separation

(see Table XXV) was removed by 2£0 ml.of chloroform. Fuchs and deJong

($1) in their work with beeswax found that chloroform eluted simple

esters and not hydroxy-eeters. Consequently it was reasoned that if

there were residual simple esters in this fraction they should be

removed preferentially by chloroform. This proved to be correct, as

the solids m. p* (86-87°) in OXII-F^Pj. had only traces of hydroxyl

material. The rest consisted of simple esters. Fraction 0XIT-F2-P2

eluted by chloroform-ethanol (2:1), proved to be 1.8126 g. of a dark

green solid melting at 76-79°* This was considered to be probably a

mixture of free alcohols and hydroxy-esters. These conclusions were

based on the analytical data given in Table XXV, idiich showed the

hydroxyl value to be significantly in excess of the ester value;

the fraction could not therefore be composed solely of hydroxy-

esters, unless dihydroxy-mcno-esters were present.

Sub-fraction Eluant vol.(ml.)

TABLE XXVResults of the Chromatographic Separation of Fraction QXII-Fa on Silica QqJL________________

m.eq.m.eq.m.eq. m.eq, m.eq. m.eq. mol. WtJE ester ester

cDcii-f2«f1

QXII-F2-P2

axn-r2-r3

ch lor o form: 3 00

chloroform- ethane] :2:1300

chloroforro- etiianol-acetic acid: 50:50t2 15°0

Total

Fraction 0XII-F2

Sub- Amountfraction in weight original (g.) fraction0.3&10 b7l?60

1.0705 I.38U3,2771 U.P2U0

U.2270

-OH/g. total /g.

1.8110 0.598 oToo"

-OKtotal

acid acid vt. orig. /g. total wax

0.059 0.029 528 3.31

1.8126 2.3I1L.O 1.2la 2.905 1.725 h.OU 0.05 0.317 680 15.59

l.Mj8 2.018 1.1*0U.821

1.37 5.870 1.1*2

1.933 0.262 0.362 670 9.22

5.973 0.5086.000 0.063 0.266

28.12

28.19

COro

83The succeeding fraction QXII-F2-P3, (m.p. 86-87°), was eluted

by l^OO ml, of a 2 % solution of acetic acid in a 0C# mixture of chloroform and ethanol* The similarity of ester and hydroxyl values

as wall as the fair agreement between molecular weight determined ex­perimentally (670), and predicted from the ester value (690), sug­gests that this fraction consists mainly of hydroxy-mono-esters.

In continuance of the further separation of some of the fractions given in Table XXIII, an aliquot (I.O896) of 0XIJ-F3 was dissolved in chloroform and chromatographed on 120 g. of silica gel

using chloroform as developer. A wide diffuse band moved slowly down

the column as elution proceeded. The total soli ds recovered from this operation, 1.OU03 g*, (0XII-F3-P1) melted at 82-83“, and gave a higher ester value (2.336 m.eq./g.) than the original (1.600), and the hydroxyl value of this material was 1.66 m.eq./g. Hie remainder

of the charge, O.OI493 g«, resisted removal by more polar solvents*The molecular weight of the eluted fraction (83U) suggests that this material is hydroxy-di-ester, since the molecular weight predicted by the ester value is approximately half, i.e. U29.

Fraction 0Xll-F4 was not further chromatographed on silica gel. By the same reasoning it was considered to be hydroxy-di- eeters. Molecular weight found 720; predicted from ester value 37)j.

V. DISCUSSION OF RESULTS AND IMPLICATIONS OF THE INVESTIGATION} WITH

SUGGESTIONS FOR FUTURE RESEARCH

Hie underlying theme of this investigation was to find a method which would be suitable for use in isolating the original constituents of waxes in some orderly basis* This in turn would simplify the

problem of determining the true nature of plant waxes. Adsorption chromatography was considered to offer the greatest potential for

achieving the desired goal, in that it is milder than other available methods, it is sensitive and it is versatile. By using adsorption

chromatography one can take advantage of the differences In polarity between the different functional groups of the constituents of waxes, and thus separate them into fairly discrete groups.

Hiis investigation was carried out in three stages, first a satisfactory method was developed which provides an adequate separation of a known mixture, by virtue of the differences in polarity of the

functional groups in the mixture. The second stage was the application of this procedure to a separation of spermaceti wax. finally the com­plex plant waxes, candelilla and ouricuri, were investigated. The data given in Tahle V m ahow that dotriacontane was separated from octadecyl stearate and stearone, whj ch were in turn separated from

octadecanol, and although not included In the particular experiment presented in Taole VlII, the result of previous work left no doubt

that alcohols could be separated from high molecular weight acids,

fsters were not distinguishable from ketones by this procedure.This fact was not cause for great concern at this stage. With due

8 i

8$regard to the great complexity of the plant waxes* it was desirable

to separate them into the foregoing four major functional groups

first, Following this, more refined resolutions could be attempted

with probably greater success, than if too refined a resolution

were attempted on the crude wax itself*

Having obtained a separation of known compounds, the

standardized procedure was then applied to spermaceti wax. It was

found that about 8,0$ of the material originally present as esters

had been altered by the alumina. This loss of esters was compensated

for by increases in the amounts of alcoholic and acidic materials

recovered. The ability of alumina (Fisher) to alter or disrupt

esterified material was a feature first noticed by Findley (3 ) and

also in the work with known compounds, and lias been a constant

problem throughout this investigation. This effect had been

counteracted successfully in the case of synthetic material, by

washing with sufficient hydrochloric acid to neutralize the alkaline

material present, The detrimental effects on spermaceti wax caused

by this same adsorbent lead to the conclusion that there is present

in spermaceti, ester compounds other than those involved in a normal

ester linkage between n-acids and primary n-alcohols. When regenerated

alumina was used for the separation of spermaceti wax, then results

were obtained indicating that the separation had proceeded free of

disruption. In the second separation of spermaceti wax (Table X)

it was found that esters appeared in the percolate after 1500 ml, of

heptane had been collected. This effect showed that the activi-ty of

the alumina was not strong enou$i to permit retention of large

86quantities of this material. Some esters were retained; however,

these were removed by a solvent of greater polarity than heptane

viz: 1$ ether in heptane. Table X shows that fractions F^Fg were

eluted in order of decreasing molecular weight* This is a significant

fact, and can be interpreted to mean that a possible separation of

simple esters could be attained by elution chromatography under the

correct conditions. These conditions might include slightly less

active alumina, a narrower column, a decreased ratio of adsorbate

to adsorbent. The results also indicate that although cetyl palmi-

tate may be a major component it is probably not present to the

extent of 90^, as is currently believed (52).The mean molecular weight of the pure alcohols present in

spermaceti wax (271), calculated from the hydroxyl value, lies close to the molecular weight of octadecenol (268). The wide

melting range (31-36*) indicates strongly the presence of a

mixture of compounds, which would include unsaturated material. The

melting point (31-35°) of the acidic fraction also suggests that unsaturated material is present. The mean molecular weight of

these acids, 297.5> calculated from the acid value implies a mixture

of Cie and C20 acids.Although the conditions which were successful with spermaceti

wax failed when applied to candelilla wax, the two separations which

were described did supply valuable information which later led to

finding adequate condi tions for the successful separation of

ouricuri wax. The outstanding features associated with the two experiments with candelilla wax are as follows. 1) The over-

87whelmingly major constituent of this wax was found to be hydrocarbon*

2) The conditions which had been applied successfully in the separa-

ti on of known synthetic compounds and spermaceti wax produced con­

siderable disruption when applied to candelilla* 3) The unrecovered

ester material was not compensated for by corresponding increases in

recovered equivalents of alcohol and acid* It was found later that

the resinous material of candelilla wax contains much of the acidic

and ester units. Further, resinous material was not removed by the

elution schedule in operation at this time.

The main inference to be drawn from the investigation up to

this point was tlxat spermaceti and candelilla waxes do not consist

only of simple esters, alcohols and acids, otherwise these complicate

ing factors would not have occurred. Farther, there was no doubt

that once technical difficulties were overcame adsorption cliromato-

graphy lent itself particularly well to the resolution of waxes

into different functional groups. Next a number of small scale

chromatographic separations of ouricuri wax were conducted, under

conditions of different adsorbent characteristics and varying

elution schedules. The results of these experiments revealed that

the factors of critical importance are activity and pH of the

alumina, Thi s was convincingly demonstrated when alumi na (Woelm)

of pH !j*0 and grade U was used. This aasoroent gave satisfactory

results without being pre-treated. The strange behavior of Fisher

alumina, in that It required several washings with concentrated

acetic acid solution before use, was not entirely understood,

Generally, it was found that use of this adsorbent was to be

88avoided i&enever possible*

Originally it had been hoped to obtain in one chromatographic separation a resolution of the wax into functionally distinct groups. To achieve this using grade U alumina would require an impractical amount of adsorbent and eluants* In addition it mas questionable whether the highly complex mixtures present in plant maxes could be adequately separated in one pass through the column* Consequently* it mas decided to obtain a partial separation first on alumina* and later to separate the resultant fractions into less heterogenous mixtures an silica gel*

The initial resolution revealed that the constituents of ouri­curi max can be separated by this method into four distinct classes or groups* First there are the components ih1 ch are non-act die and mhlch vary in polarity from the non-polar hydrocarbons to the most polar hydroaty-esters and hydraxy-dl-esters* Secondly there are the acids* fairly soluble in heptane* and which are removed by 1% acetic acid In heptane* These appear to be normal acids*The third group comprises the most unusual class of compounds iso­lated from this wax in which the discriminatory functional group is carboxyl* However* this material is removed partially* by 10£ acetic acid in heptane* and parti ally by toluene* but readily with acetic add in benzene* Actually* toluene probably removes these components by mixing with acetic add already present in the system* and dissolving very high molecular wdght compounds* However* neither toluene nor 10$ acetic add in heptane are truly suitable eluants for this group* Toluene per se is too weak In

89polarity, and ace Lic-heptane is a poor solvent* The use of % acetic

acid in benzene was found to be well suited for the removal of thfs

material.The molecular weight of fraction QXTI-F6 (625) is fairly

close to that predi cted from the ester value. This would suggest

that each molecule contains one ester linkage. However an estimate

of the nature of this material is complicated by the presence of an

appreciable acid value (0,96 m,eq./g.). Consequently, evaluation of

this fraction is not possible at this stage. Fraction 0£n-F7 Has

all the earmarks of a high molecular weight poly-ester. The

molecular weight calculated from the acid value is 211*0, while the

experimentally determined figure (Rast) is 1950, It is clear thatfree

one can then assume that there is present 1 ^arboxyl group per

molecule. If this is correct then it follows that there are ap­proximately 6 ester linkages per molecule (2,d0-£- 0,1*66).

The fourth group of compounds, noted above, is composed of

resins. This material (QXll-Fe and Fg), contains a relatively high

concentration of ester, hydroxyl and acidic functional groups. The

resins of ouricuri wax, which are insoluble in heptane, are mainly

responsible for the low yields obtained in experiments 0I-0X, It is

conceivable that these resins undergo some change during the removal

of solvent from the percolate, because the resultant solids are not

as soluble in benzene. This alteration may account for the excess

of acidic function noted in the analytical data of Table XX.HI,

ihe first three fractions obtained in the initial separation

on alumina were further chromatographed on silica gel. The results

90of tiles© separations together -with that obtained in the initial

resolution form the basis upon which the composi tion of ouricuri waxwas

is calculated. The wax/found to contain 1*3,6 hydrocarbons (sub­

fraction OXlJ-yj-P^. Simple esters were considered to be the main

constituents of subfraction 0XTI-F1-P2 and P3 (20*2i$) and of sub­

fraction CKJ_I-F2-Pi (3.31^/. The estimated amount of simple esters

in this wax is therefore 23*656. In the sub-fraction eluted by

chloroform:ethanol, 0XIT-F2-P2> the hydroxyl value exceeds the

aster value by m,eq*/g* This excess is attributed to free

alcohols* The mixture therefore, was thought to contain O.UBU

millimole of alcohol per gram, and l,2Ul millimoles of hydrcocy-

ester per gram* If one assumes the moan molecular weight of the

alcohols to be equal to that found in 0Xxi-F1-P4, i.3* 331* then

the estimated amount of free alcohols in this wax including 0,5256

present in 0XlI-Pi-'P4 would be 3,0^, This percentage was arrived

at oy: l) multiplying 0,U8h by the sub-fraction weight (2,3U7 g*);

this gives the total number of millimoles of free alcohol* 2) the

weight of the alcohols (386 mg.) is obtained by assuming a mean

molecular weight of 331* Thus the percentage present in the wax

Is obtained* The total concentration of hydroxy-mono-esters is

obtained by summation of the amounts present In 0Xu-r2-P2 (l3.t£) and 0XH-F2-P3 (9.356) and QXII-Fe (3*lt). The latter fraction is

included in this group although some doubt surrounds its identity.

The total amount of hydroxy-mono-esters calculated in this way is

25,^, Fraction QXn-F3-P1 is considered to be composed of

hydroxy-di-estars by virtue of the fact that the molecular weight

91(Rast) la 83U» azid that predicted frost the ester number la U29«Since there is only a very m i l amount of non-ester material In the parent fraction GHI-F3, the entire fraction la calculated as di­ester j similarly CHI-F4. The estimated amount of hydroxy-di- esters by sunttation of these two fractions is lU.l£.

The total percentage of free acids represented in GIU-Fe is 8.7^ of the wax. In this connection It mnat be mentioned that the larger quantities of free acids in earlier experiments, Oil and OK, are considered to be due to disruption* Fraction QXII-F7 represents 5#35£ of hydroxy-poly-ester material and fraction OKII-Fe and F® 1U.8£ of resin.

In those experiments in ldxich disruption had occurred, e.g.Oil and CK, the amount of acidic material eluted by acetlc-heptane was greater than the amounts removed in satisfactory separations. Further the adds in O H and 01 gave significant hydroxyl values.This fact coupled with the behavior of fractions OXn-Fj., Fz and F3, which gave rapidly fading end points when titrated against 0.1 N alkali in warmed solution, suggests that the adds were derived from the opening of cyclic structures such as lactones.

A summation of the results shows that an estimate of the percentage composition can be made in terms of the functional entities. A comparison of the estimated composition of ouricuri wax based on this work, with that provided by Ludecke (32), of a sample of refined ouricuri is given below.

92

Ludecke (32) This Investigation

Constituent % Constituent %

hydrocarbons 17 hydrocarbons 1.3myristic and corotic simple esters 23.6acid ester 59.0 hydroxy-mono-es ters 25.U

saponifiable resins 11,6 hvdroxy-di-esters 1U.1free acids io. U hydroxy-,acidic poly­moi sture 2.0 esters $.uLnorgani c matter 1.0 resins lh.e

free acids 8.7free alcohols 3.0moi sture l.Uinorganic matter (ash) o,U

987T

The difference in the hydrocarbon content of the two reports indicate

that conceivably the starting materials were obtained from different

species of trees, The types of compounds covered under myristic and

cerotic acid esters is open to conjecture.

It can be stated that as a result of this investigation

proof has been established, based on the experience gained with

candelilla and ouricuri waxes, that waxes do not consist wholly orsimple

predominantly of the/esters of normal alcohols and aci'ds. It has

been shown that the behavior of ouricuri wax on alumina columns Is

in striking contrast to the behavior reported for beeswax by *uchs

and deJong (5l), The latter wax is uncomplicated by the presence

of resins and material other than simple esters, a result it is

readily separated into its different functional groups. On tho

other hand, ouricuri wax has been shown in the present work by means

of adsorption chromatography, functional group and molecular weight

analysis, uo consist of mixtures of hydrocarbons, simple n-osters,

hydroxy-mono-estars, hydroxy-di-esters, hydroxy-poly-esters, free

acids and free alcohols. In effecting the separation of this -wax into its various functional entitles, the overall objectives of the investigation have been accomplished, and for the first time a plant wax has been resolved into groups of less heterogenous mixtures, thus contributing to the understanding of the true nature of the original constituents of plant waxes. In addition, it appears that the general scheme as applied to ouricuri could be used successfully in the study of the original constituents of other plant waxes, al­though modifications may be required to fit each case.

Obviously, there is a considerable amount of work yet to be carried out on all of the above fractions, to establish unequivocally, the nature of each of the compounds present. However, because of the complexity of the total problem, only the initial objectives have been accomplished at this time. It is suggested that future research for the further isolation and identification of the constituents of ouricuri wax be conducted along the following lines. Each of the fractions could be saponified and the resulting acids and alcohols characterized, This would give further insight into the exact nature of the linkages by comparing the analytical data of the saponified material with the data of the original fraction. The alcohols might be separated from the hydroxy-esters by first acetylating the mixture and then subjecting it to urea adduct formation; the primary acetates formed with the free alcohols should give the crystalline adducts, but the branched acetates with secondary alcoholic groups of the parent hydroxy ester should not.If lactones are present, it would be expected that on saponification

9h

of the parent fraction* these lactones will also be saponified and

thus remain with the soaps, as salts of hydroxy-acids, These soaps

could then be divided into two portions, one part being converted to

the acids and the other esterified with methanol* Since on con­

version to the acids, lactones readily c"cliae, thus masking the

hydroxyl group, it is suggested that proof of the presence of these

compounds may be established on a basis of an expected increase in

hydroxyl value of the methyl esters, when compared to the acids,

Esters derived from lactones will have free hydroxyl groups, while

the free hydroxyl group concentration of the acid portion will be

considerably less. An attempt to separate out the various indi­

vidual esters could be made using partition chromatography and

solvent fractionation, either senarately or as complementary tech­

niques. Similarly the fraction consisting solely of acids might

be further resolved into individual components.

VI. SUMMART

1). A re-evaluation of send—micro methods for the determination of

acid, ester, hydroxyl and carbonyl values was made and certain

modifications instituted. Molecular weights were satisfactorily

determined by a modification of the depression of the melting

point of camphor method.

2), A method was developed for the chromatographic separation on

alumina (designated grade 2 for the purposes of this investiga­

tion) of a synthetic mixture of dotriacontane, octadecyl stearate,

stearone, octadecanol, and stearic acid. This separation was

based on the increasing polarity of the functional groups con­

cerned. Stearone and octadecyl stearate were indistinguishable

by this method.

3). This grade 2 alumina wid ch was suitable for the separation in

(2) when prepared fresh did not give a satisfactory separation

of spermaceti wax, but, on reuse after regeneration by ex­

haustive washing with methanol and water, spermaceti wax was

resolved into three functionally distinct groups. These were:

esters (96.2$), alcohols (2.6$), acids (1.2^).

Ii). The successful conditions for the separation of spermaceti wax

were found inapplicable to candelilla and ouricurL waxe3. The

results obtained from the separation of refined candelilla wax

showed that it had undergone changes during chroniatography,

ThJ s sample of wax was found to contain hl£ of hydrocarbons.

96Functional analyses of the rest of the recovered fractions indi­

cated that simple esters, hydroxy esters, free acids and free

alcohols were present in the wax,

?). Alumina (II, Woelm) of pH li,0 and grade U was found to be the best

starting adsorbent for the separation of ouricuri wax,

6 ), Ouricuri wax was separated by cliramatography on alumina into U

major classes (9 fractions) with a minimum of alteration of the

naturally occurring constituents,

7), The first fraction from the initial separation of the wax on

alumina, was rochromatographed on silica gel, found to

con tain, inter alia, hydrocarbons, simple n-csters, and a very

small amount of alcohols,

ft). The second fraction from the initial separation was further

resolvod on silica gel and found to contain, inter alia, simple

n-crters, a mixture of free alcohols and hydroxy-mono-esters,

and a fraction which probably contains only hydroxy-mono-esters,

9), The third and fourth fractions from the initial separation were

considered to consist predominantly of hydroxy-di-esters, The

fifth fraction consisted exclusively of acids, while the material

present in the succeeding fraction has been calculated as

hydroxy-ncno-esterc. This fraction is complicated by the

presence of a significant amount of acidic material. The follow­

ing fraction (seventh) consists of the most unique material

97isolated from this wax. It gives evidence of being a very high

molecular weight poly-^ster, The remaining material was found

to be resins,

10), The- estimated composition of ouricirri derived from these

separations is as follows: hydrocarbon 1,3^» simple esters 23,6$,

hydroxy-rcono-es t er s 25.1$, hydroxy diesters lli,3$> hydroxy- acidic-poly-esters 5*W»> resins lh,8$, free acids 8,70$, free

alcohols 3*C > moisture 1*1$, ash (inorganic matter) 0,1.'$,

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AUTOBIOGRAFHY

I, Leslie John Norman Cole, was bom in Jamaica, B.W.I., on

December 10, 1926, and received my primary and secondary education

there* In 19^2 I was successful in the School Cyo-tificate, and in

19U6, in the Higher School Certificate examination of the Cambridge

University Syndicate. From 19U5 to 19U8 I was a clerk in the Public

Service of Jamaica, which position I resigned to enter Macdonald

College, McGill University, Montreal, Canady j received the D«Sc.

(Agr.) second class honors in 1951, specializing in Animal Bio­

chemistry. In 1952 I received the M.Sc. in the Department of

Biochearn.ctry of the University of Saskatchewan. The research work

submitted for this degree was carried out in the Prairie Regional

Laboratory of the National Research Council of Canada, ^hilo at

the University of Saskatchewan I was appointed Demonstrator in the

Department of iiLochenistry in 1951 and 1952, and was a staff

member of the Prairie Regional laboratory during the summer of

1952* In 1953 I was awarded the S. C. Johnson & Son, Inc.,

fellowship at The Ohio State University. I have held this award

for the entire period during which this investigation has been

conducted.

102