Methods for the Estimation of Glycerol in Neutral Glycerides, Phospholipids, and Cardiolipins by ^ Gas-Liquid Chromatography

This dissertation has been 65—3865 microfilmed exactly as received

HOLLA, Kadambar Seetharama, 1934- METHODS FOR THE ESTIMATION OF GLYCEROL IN NEUTRAL GLYCERIDES, PHOSPHOLIPIDS, AND CARDIOLIPINS BY ^ GAS-LIQUID CHROMATOGRAPHY.

The Ohio State University, Ph.D., 1964 Chemistry, biological

* University Microfilms, Inc., Ann Arbor, Michigan METHODS FOR THE ESTIMATION OF GLYCEROL IK

• NEUTRAL GLYCERINES,‘PHOSPHOLIPIDS, AND f ■ CARDIOLIPIijs BY GAS-LIQUID

/ CHROMATOGRAPHY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate pl.,/ School of The Ohio State University /

\ Kadambar Sejptharama Holla, B.Sc., B.Sc . (Tech.) , M.Sc.(Tech.)

# * * * *

The Ohio State University 1961*

Approved by

Adviser Department of Physiological 'Chemistry \ I f

ACKNOWLEDGMENTS

r ' > I am deeply indebted to my adviser, Dr. David G.

Cornwell, for his guidance, timely suggestions, keen interest, criticisms and the encouragement that he gave throughout my work. his vast scientific knowledge and experiences helped me to progress and complete my work.

I thank Dr. J, B. Brown for his parental advice that he gave during the tenure of my studies. I thank all my friends in this laboratory, and Dr. L. A. Horrocks ,

Cleveland Psychiatric Institute, for their co-operation.

I am indebted to the College of Medicine for support through a Research Fellowship in Physiological Chemistry.

I appreciate the study leave generously granted to me by

The Tata Oil Mills Co., Ltd. (India).

ii J X VITA

May 16, 193b. . . . Born - Kadambar, Manjeshwar, Madras, India

19 5 5...... B.Sc., St. Aloysius College, Mangalore, Madras University

19 57...... Technology, University of Bombay, India

1957-1958 . .

1958-1959 . .

19 59...... Technology, University of Bombay, India.

1959-1961 r 3. Ltd.

1961...... Tata Oil Mills Co., Ltd.)

1961-1963 ...... T eaching Assistant and .Research Assistant The Ohio State University

196k D epartmental Fellow, Department of Physiological Chemistry, The Ohio State- University, Columbus, Ohio

PUBLICATIONS

"Detection of Adulteration of Butterfat." Holla, K. S. Bombay Technologist 9:16, 1958-1959*

"Fat Splitting, and Fatty Acid Distillation." Gopalan, A. K . , K. S. Holla, N. Desikachar, and M. V. A-. Iyengar. Chem. Age India 10:577, 1959. ~ VITA - Continued

"Improved Determination of Glycerol and F atty Acids in Glycerides and Ethanolamine Phosphatide s by Gas-Liquid Chromatography." Holla, K. S., L. A. Horrocks , and D. G. Corn-well. J. Lipid Res . 5:263 , 1964.

i v CONTHNTS

Page

ACKNOWLEDGMENTS...... '. ii

VITA ...... iii

LIST OF T A B L E S ...... viii

Chapter

I INTRODUCTION ANDSTATEMENT OF THE PROBLEM . . 1

II HISTORICALREVIEW ...... 5

A. Methods for the estimation of glycerol. . 5 1. Oximetric methods...... „ . . . 6 2. Colorimetric m e t h o d s ...... 11 3. Physical methods ...... 12 k. Enzymatic methods...... 13 5. Assay of glycerolderivatives. .... 13 B. Glycerol in phospholipids ...... l6

III EXPERIMENTAL EQUIPMENT, MATERIALS, AND METHODS...... 18

A. Equipment ...... &l8 B. .Materials ...... 18 C. M e t h o d s ...... 20 1. Preparation of egg yolk triglycerides- and ethanolamine phosphatides. . . . 20 2. Preparation of phospholipids (lecithin and cephalin) from egg yolk...... 21 3. Hydrogenolysis-acetylation ...... 2h 1*. Saponif icat ion-acetylat ion ...... 25

4- CONTENTS - Continued.

Chapter - - Page

5. Acetolysis;...... 25 a. Acetolysis of cephalin. . . . 25 b. Acetolysis of cephalin and l e c i t h i n ...... 26 6 . Gas-liquid chromatography .... 26

IV R E S U L T S ...... 28

A. Composition of reference compounds . 28 B. Relative molar response determina tions ...... 29 C. liydrogenolysis-acetylation...... 31 D. Improved hydrogenolysis-acetylation. 31 E. The use of internal standards with hydrogenolysis-acetylation...... 3^ 1. Docosenyl acetate...... 3^+ 2 . Eicosanyl acetate...... 36 F. Saponification-acetylation ...... 36 G . Acetolys is-saponification-acetylation...... ^1 H. Modified acetolysis...... k6 I. Estimation of glycerol content in cardiolipins...... 58 J. Effect of modified acetolysis on fatty acid components...... 6l

V DISCUSSION...... 62

A. Sample purity and relative molar response...... 62 B. Hydrogenolysis-acetylation ..... 63 C. The use of internal standards. . . . 6h D. Saponification-acetylation ...... 65 E. Acetolysis ..... 66

vi CONTENTS - Continued.

Chapter Page

F. Modified acetolysis...... 67 G....Cardiolipin structure...... 69 H. Effect of modified acetolysis on fatty acid components...... 73

VI S U M M A R Y ...... 75

REFERENCES...... 7 8

vi i LIST OF TABLES

Table | Page.

1 . C^foposition of reference compounds...... -28

2. Relative molar response of triacetin, eicosanyl acetate and docosenyl acetate...... 30

3. Hydrogenolysis-acetylation and GLC. [Horrocks and^Cornvell (1) ]...... 32

k. Hydrogenolysis-acetylation and GLC...... 33 r

5 .* -diydrogenolysis-acetylation of a standard methyl ester mixture and prolonged vacuum evaporation...... 3^ /\ '

6 . The fatty acid and glycerol content of glycer ide samples estimated by hydrogenolysis- acetylation and GLC...... 37

7. The fatty acid and glycerol content of glycer ide samples estimated by hydrogenolysis- acetylation and GLC...... 3 8

8. Duplicate_analyses of egg yolk triglyceride by hydrogenolysis-acetylation and GLC. . . . ^0

9. Glycerol content of glyceride samples estima ted by saponif i c at ion-ac e ty lat ion and GL-Q. , k2

10. Ester-to-phosphorus ratios of phospholipids . k3

11. Glycerol content of cephalin estimated by acetolysis-saponification-acetylat ion and GLC...... hh

vi i i TABLES - Continued.

Table ^ Page

12. Glycerol and fatty acid contents of cephalin by acetolysis-hydrogenolysis-acetylation and GLC...... U 5

13. Glycerol content of lecithin by acetolysis- saponification-acetylation and GLC ...... 1*7

ll+. Studies on the hydrolysis of phospholipids. . 1*8

15. Glycerol content of lecithin estimated by modified acetolysis-saponification-acetylation and G L C ...... 55

16. Glycerol content of lecithin estimated by incomplete saponification-acetylation and GLC...... 5 6

17. Glycerol content of cephalin estimated by modified acetolysis-saponification-acetylation and GLC...... 57

18. Glycerol content of cardiolipin estimated by modified acetolysis-saponification-acetylation and GLC . . 60 .»

19. Comparison of glycerol recovery from cephalin estimated by both methods...... 70

20. Comparison of glycerol recovery from lecithin estimated by b^oth methods...... 71

i x CHAPTER I

INTRODUCTION AND STATEMENT OF

THE PROBLEM

A number of analytical methods have been proposed for the estimation of glycerol since the nineteenth century.

These methods were applied especially for the determination of glycerol in commercial processes developed in the paint, cosmetic, and wine industries. The early methods were based on oxidation, and since they were non-specific re quired special attention in the purification of glycerol.

Large samples were necessary. Hydrolytic procedures caused significant losses. While large samples are available for the analytical methods employed in industry, only small samples are generally available for biochemical investi gations. Thus it is difficult to apply these routine analy tical methods in biochemical researcb-r

In 1952, with the advent of gas-liquid chromato graphy (GLC), the quantitative analysis of lipids and other volatile compounds became more rapid and dependable. GLC

1 2

can also be used to isolate compounds in pure form and re places, in part, distillation techniques. The fatty acid moieties of neutral lipids, phospholipids, polyphospholipids and waxes can be estimated and identified by GLC.

The low volatility of. glycerol made its estimation by

GLC very difficult. Hence derivatives of glycerol which are more volatile than glycerol itself are prepared for GLC.

The nethods applied to prepare glycerol derivatives are acetylation and the synthesis of isopropylidene glycerol.

The first report on the application of GLC for the determination of glycerol was by Horrocks and Cornwell (l).

Glycerides were reduced with lithium aluminum hydride (LAH)

•i> yielding lithium aluminum alcoholate of fatty alcohols and glycerol. The lithium aluminum alcoholates were then acetylated by the direct addition of acetic anhydride.

(■ This method permits the simultaneous determination of the relative composition of fatty acids and glycerol in neutral lipids. Reproducible results are possible with this pro cedure. However, alcohol refluxing to remove excess acetic anhydride sometimes resulted in low yields which may be explained by triacetin solubility in wash solvents or partial hydrolysis of triacetin. A modified procedure was needed to obtain quantitative and consistent data. The glycerol content of phospholipids nd cardiolipins has been determined by several authors who subjected these

“'""I compounds to hydrolysis and reported the different consti tuents by paper chromatography. The components isolated were estimated either by oxidation or colorimetric methods.

The possibility for degradation and low glycerol recoveries existed at several steps in these procedures. However, the hydrogenolysis procedure mentioned above was not quantitative with phospholipids. Thus a new method was needed for the estimation of glycerol in phospholipids and cardiolipin.s.

This work was undertaken to find consistent and (I quantitative procedures for the estimation of glycerol in • neutral lipids and phospholipids. The following areas were investigated:

(a) Modifications in the hydrogenolysis-acetylation

method

(b) Evaluation of internal standards for estimating

absolute glycerol content

mating glycerol alone in glycerides with a complex

fatty acid pattern u

(d) Hydrogenolysis-acetylation and saponification- ace-

tylation of phospholipids

(e) Methods for the acetolysis of phospholipids

(f) Quantitative estimation of glycerol in phospho

lipids (acetolysis-saponificat ion-acetyl ation) II. HISTORICAL REVIEW

A . Methods for the estimation x of glycerol _ - -

The history of glycerol since its isolation by Scheele in 1779 (2 ) to the present day is an interesting example of the development of modern methods of science and technology.

Hanahan, in his book Lipide Chemistry, states "the determi nation and estimation of glycerol is one of the more dif ficult and less rewarding analytical problems in lipide * chemis try."(3 )

Different procedures are used in the estimation of glycerol. These procedures may be grouped in the following areas: (l) oximetric methods; (2 ) colorimetric methods;

(3) physical methods; (U) enzymatic methods; (5 ) assay of glycerol derivatives. Each procedure has its own advantages and limitations. The principal limitations of many proce dures are mentioned in the Introduction. These include lack of specificity and necessity for purification, lack of sensitivity, and finally hydrolytic degradation and other losses encountered during isolation and purification. The 6

analytical procedures in this study were developed to elimi nate these problems in the determination of9 glycerol.

1. Oximetric methods. Lead peroxide oxidation, an early method (H) was applied to crude glycerol. A precipi tate with the formula C^HgO^ Pb wa£ obtained. The percent age of glycerol was obtained from the weight of lead perox ide consumed multiplied by an appropriate factor. Benedikt and Zsigmondy (5) oxidized crude glycerol with alkaline permanganate to oxalate, carbon dioxide and water. The oxalate was precipitated as the calcium salt. The oxalate was then incinerated to give either carbonate or calcium oxide which was then titrated against a standard acid solu tion. Later workers either titrated the oxalate or trapped the carbon dioxide and weighed it as barium or calcium car bonate. This method was later modified by several workers,

(6-18). Freeman and Friedemann (19) oxidized glycerol with potassium permanganate after removal of fatty acids and determined the excess potassium permanganate iodometrically.

This method was applied in several laboratories but inconsis tent results were reported (7,20-22). Each of these methods has its own disadvantages. For example, erroneous results will be obtained if oxidizable organic compounds are present, thus leading to nonquantitative recovery of oxalate.

Cerate oxidimetry for the determination of glyc erol was used by Cuthill and Atkins (23). They used eerie sul fate and excess eerie sulfate was back titrated with stand-

o ard ferrous sulfate. Glycerol was oxidized to carbon dioxide and water by eerie sulfate. Fulmer et al. (2*0 applied this method to fermentation products for the esti mation of glycerol and dextrose. Fairly good results were obtained. Later Smith and Duke (25) improved this method by using perchlorato-ceric acid for oxidizing glycerol and the excess unreacted reagent was back titrated with stand ard oxalate using nitroferroin as an internal indicator.

This method required only a short time. The stoichiometry is expressed in the following equation:

C3 H 8O 3 + 8 H 2 Ce(C10j+)6 + 3 II20 > 3 HCOOH + 8 Ce(C10^ ) 3 +

2k H C 1 0 "

Silverman (26) used this method and found it suitable only after removing chloride, fatty acids and proteins. In 1955

Sharma and Mehrotra (27) conducted controlled o'xidation studies on the mixture of glycerol using eerie sulfate and

standardized the method. 8

Quinquavalent vanadium oxidation of glycerol was car ried out by West and Skoog (28). The excess vanadium was titrated against ferrous ammonium sulfate. The results agree fairly well with those obtained by the periodate, cerate and dichromate methods. Both cerat.e and vanadium will oxidize polyalcohols and hence the material for the analysis must be pure. These methods are employed in industry, but they are not used commonly.

In one dichromate method, purified glycerol was oxi dized and the carbon dioxide liberated was estimated either gravimetrically or manometrically (29-32). Richardson and

Jaffe (33) gave a table relating the amount of dichromate consumed to the glycerol content. This method was later adopted and improved by several authors (3^-37). A dichromate-iodine-sodium thiosulfate method was used by various workers (38-H3) with good results. The reaction is given below:

3 C3H3O3 + 7 K2Cr20T + 28 H2S0^ ---^ 9 C02 + 7 Cr2(SO^)3+

7 KgSO^ + UO H20

K2Cr20T + 6 KI + 7 H2S O ^ > Cr2 (SOi+)3 + U KgSO^ +

3 I2 + 7 H20 The advantages and disadvantages of this method compared to other methods have been reviewed (25,UU-U8). Electrometric titration of excess dichromate was used at the Procter and

Gamble Co. (U9,50). Diphenylamine was used as an internal indicator by Randa (51) • The kinetics of thermal and photo ) chemical oxidation of glycerol by potassium dichromate were studied by Rohatgi (52).

Since dichromate oxidizes many organic compounds, gly cerol must be free of impurities. Very dilute solutions of glycerol could be determined by this method, while other methods frequently require a concentrated glycerol sample.

Glyceryl phosphate, glycol and trimethylene glycol must be separated before subjecting glycerol to oxidation. This method is still in use in the soap industry.

Periodic acid selectively oxidizes aliphatic compounds containing hydroxyl groups on adjacent carbon atoms while other hydroxy compounds remain unchanged. Trimethylene glycol is not oxidized. Glycerol and glycol are oxidized to formic acid and formaldehyde, and formaldehyde respect ively ( 53) • CHOH + 2 H5I O g ^ 2 HCHO + HCOOH + 2 HIO3 + 5 H20 I c h 2oh c h 2oh ■ | + H j I O g -----> 2 HCHO + HIO3 + 3 H20 c h 2oh

Several ways of analyzing glycerol after the oxidation are:

(a) Estimating the excess periodate by the iodine liberated when potassium iodide is added, (b) Neutralizing the excess

9 periodate with glycol and titrating the formic acid derived from glycerol, (c) Colorimetric determination of formalde hyde formed.

Chaumeille in 1902 reported a method of analysis of glycerol by iodic acid (5*0 and potassium iodate. The iodine liberated was titrated with standard sodium thiosulfate

c h 2oh I T I205 + 5 CHOH --- 15 C02 + 20 H20 + T I2

c h 2oh

Iodometric methods have been used by several workers (55-62) with good and consistent results comparable to other methods.

Periodic acid in neutral solution yields formic acid from glycerol and this can be estimated volumetrically by 11

titrating with standard alkali. These methods have "been em ployed by several authors (h6 ,kQ , 63-8l ). Many of these /

V studies are cited in reviews by Reese and Williams ( ) , and Miner and Dalton (82).*

Periodic acid oxidation of glycerol and glyceryl phosphate and estimation of them by colorimetric methods will be discussed later. Glycerol must be free of sugars.

Bromine oxidation, a new and rapid method for the determination of glycerol in aqueous solution, was developed by Juhlin (83). Glycerol reacts with excess bromine water.

The remaining bromine water i:s titrated iodometr i cally .

Ilogai Ka used bromine water to oxidize glycerol (8U) and estimated excess bromine colorimetrically with a Lovibond

Tintometer. Comparable results were obtained. This method was later improved by Mikkelsen ( 85).

2. Colorimetric methods. A spectrophotometric method can be employed to estimate''glycerol if there are no impur ities in the sample. Advantage is taken of the°color for mation of glycerol with sodium and copper. Sodium-cupri- glycerol-complex is measured as a blue color spectrto,photo- metrically at 630-635 mA. This~~method was employed by several authors (86-89). Glycerol-copper-coraplexing was 12

satisfactorily applied in two studies (90,91). Other spectrophotometric or titration methods have also been employed

(73,86,92-98).

The colorimetric method of estimation of glycerol is carried out after oxidizing glycerol with periodate to formic acid and formaldehyde. The excess iodate and periodate are reduced with sodium arsenite. The formaldehyde produced is then determined spectrophotometrically after reaction with

1,8 dihydroxy-naphthalene-3-6 disulfonic acid (9 6 ).

3. Physical methods. In a number of studies, glycerol was first isolated and purified by chromatography (3,99).

Specific physical and chemical analytical procedures were then^applied. Papers have been published on either paper or column chromatographic separation of glycerol and its subsequent estimation iodometrically and colorimetrically

(78,96,100-107). Specific gravity and boiling point (108,

109), refractive index and distillation (110,111), extrac tion (112-117) and viscometric method (118) were employed early but only when pure glycerol was available. They may be used after chromatographic purification, however a number of other procedures are much more sensitive than the physical methods described here. 13

b, Enzymatic■methods. The oxidation and reduction of

NADH and NAD coupled with dehydrogenase enzymes and spectrophotometric analysis was used by several people (119-123).

A general mechanism is given below (122):

Glycero-kinase Glycerol + ATP ^ ------^ L-Glycerol-1 POJ4 + ADP

Pyruvate kinase ADP + PEP >ATP + Pyruvate

Lactic acid dehydrogenase Pyruvate + NADH ------Lactate + NAD

Correction for the possible presence of other interfering substances or inhibitors are usually made. Also these enzymes might react nonspecifically. Free glycerol was determined by enzymatic methods. hydrolytic methods are ? necessary for the estimation of glycerol in glycerides.

5. Assay of glycerol derivatives. The preparation of 1 crystalline glycerol derivati.v-es.r , " "especially ' glycerol tribenzoate provides for the identification and estimation of glycerol (10 8,12^-127). The aqueous glycerol was made alkaline with sodium hydroxide and treated with benzoyl chloride. The resulting tribenzoate was purified by three recrystallization from ligroin and had a, melting point of

72° .■

Hydriodic acid was used by Zeisel and Panto (128) to •convert glycerol to isopropyl iodide. The advantage of the

method is that organic compounds which interfere with the «• usual analytical methods do not yield isopropyl iodide,

C3H 5(OH)3 + 5HI -■----- ^ C3HtI + 3 1I2 0 + 2. I2

Isopropyl iodide which is volatile, is carried into an

alcoholic solution of silver nitrate by means of a stream

of nitrogen or carbon dioxide. Phosphorus used in the re

action flask absorbs liberated iodine. From the weight of

silver iodide formed, the weight of glycerol is calculated.

The method cannot be used if glycols are present. Crudes

from soap lye cannot be used. The method is tedious, time

consuming, and requires large .amount of glycerol. Several

authors have used this method in the determination of

•glycerol (129-137).

The American Oil Chemists' Society Approved Official

Method (A.O.C.S.) (53) determines glycerol by acetylation.

This method is nonspecific and will estimate glycols and

polyglycerols. Two general procedures, acetylation and

saponification of glycerol triacetate or acetylation fol

lowed by titration of the unreacted acetic anhydride, have

been developed and improved since 1888 (130 ,136,13 8-11+9 ).

Acetylation requires high concentrations of glycerol. 15

With the discovery of chromatographic techniques, es pecially gas-liquid chromatography (150), new glycerol pro

cedures were introduced. Clifford (151) measured relative

responses for glycerol, glycol and trimethylene glycol by

gas chromatography. Murray and Williams (152) determined

glycerol directly by GLC. They used diethylene glycol as

an internal standard. A simultaneous method for the esti

mation of glycerol and fatty acids by GLC was developed by

Horrocks and Cornwell (l). The limitations of this study

are discussed in the Introduction. An improvement of this

method is described in this dissertation and has already

been published (153). At the same time Mason et al. (15*0

developed a different simultaneous method for glycerol and

fatty acids. In this procedure

CHoOCOR CHo 1. NaOCHo, Benzene, CHoOh' 1 I CHOCOR + C(OCH3 )2 ^

ch2ocor ch3 2. CH3OH, HC1

3 RCOOCH3 (Fatty acid methyl esters) +

CH2OH

Isopropylidene glycerol neutral lipid is treated with methanol, 2,2 dimethoxypro-

pane,benzene and sodium methoxide. Fat is hydrolysed and

fatty acid methyl esters are formed. On neutralizing the

solution with methanolic hydrochloric acid further esteri-

fication occurs and isopropylidene derivatives are formed.

The mixture is then used for GLC. Quantitative conversion

of glycerol to the isopropylidene derivatives required

many manipulations. Also this method was not extended to

the analysis of phospholipids.

B. Glycerol in phospholipids

It is difficult to estimate the glycerol content of

phospholipids since these compounds are difficult to hydro

lyze. Phospholipids are not hydrolyzed easily by acids.

Hanahan (3 ,9 9 ) states that glycerol is destroyed by 6 N

hydrochloric acid. He claims that little glycerol is

destroyed with 2 N hydrochloric acid. Nevertheless, the con

centration of aqueous hydrolysates results in a low yield of

glycerol due to its volatility (9,^8,155*156). Acetolysis

was introduced by Bevan et al. (157) in 1953. According tp'

-the procedure 1 :3-dipalmitoyl phosphatidic acid yielded 1-a-

acetyl-2 :3-dipalmitin on refluxing with acetic acid-acetic

anhydride (U:l, v/v). They suggested a Walden inversion

mechanism. This was confirmed by using active (optically) IT

C3 / 1-methyl heptyl diphenyl phosphate which upon acetolysis yielded 1-methyl heptyl acetate. ho percentage recoveries were reported for any compound, except for octadecyl phos- » phate which was 100 per cent. ^

The reaction is: \

( AcpOrAcOH ( CH0P0 ( NHPh )g ------C H O C O C 1 5 H 31 + PhHllC0CIi3 I I CH2OCOC15K31 c h 2o c o c h 3

Hoefnagel et al. (158 ) used this method for finding acyl migration in diphenyl distearoyl-a-phosphate and obtained

90-93 percentage conversion to distearoyl-a-acetyl glycerol.

Hanahan and coworkers (159,l6o) employed this method routinely in their laboratory with phospholipids. They saponified the lipid and prepared the isopropylidene derivatives of glycerol for GLC analysis. Kenkonen (l6l) in his recent paper, a proposal for a general scheme for phospholipid analysis, stated that very nearly quantitative yields of diglyceride acetates were obtained by preparative acetolysis of purified egg lecithins. Percentage recoveries were not reported in this paper. It is evident that detailed anal ysis of acetolysis reactions are not available in the literature. III. EXPERIMENTAL EQUIPMENT,

MATERIALS, AND METHODS

A , Equipment

The Aerograph A-350-B gas chromatograph (Wilkins

Instrument and Research, Inc., Walnut Creek, Calif.) was used in the present Investigation. This is a dual column

instrument capable of temperature program procedures.

Columns were purchased from Applied Science Laboratories,

Inc., State College, Pa.

A Beckman D. U. Model No. 21+00 Spectrophotometer

was used in spectrophotometric determinations.

A Wheelco model 8000-1+600-S266 one millivolt electronic

recorder and A-2 electronic integrator were purchased from

Barber Colman Co., Rockford, 111.

B. Materials

Monopalmitin (M.P.), tripalmitin (T.P.), monostearin

(M.S.), distearin (D.S.), tristearin (T.S.), dipalmitin

(D.P.), monomyristin (M.M.), dimyristin (D.M.), and 19

trimyristin (T.M.) were kindly supplied by Dr. F. Baur

(Procter and Gamble Co., Cincinnati, Ohio),

Batyl distearate (B.S.), A grade, and batyl dipalmi.tate (B.D.), A grade, were obtained from California Cor poration for Biochemical Research, Los Angeles, Calif.

Glyceryl triacetate was obtained from Eastman Organic

Chemicals, Rochester 3, N.Y.

Standard methyl esters of fatty acids were obtained

from Applied Science Laboratories.

Ethylene glycol dimethyl ether (glyme) was purchased

from Ansul Chemical Co., Marinette, Wis.

Dioxane was purchased from Carbide and Carbon Chemical

Corporation, New York, N.Y.

L-a-Cephalin, and L-a-Lecithin (chromatographically

pure) were obtained from General Biochemicals, Chagrin Falls,

Ohio.

Cardiolipin and beef heart lecithin were purchased from

Sylvana Chemicals Co., Orange, N.J. (distributed by Arnel

Products Co., New York, N.Y.).

Double distilled TO per cent perchloric acid was ob

tained from The Frederick Smith Chemical Co., Columbus, Ohio. 20

Helium and Nitrogen Hi grade were obtained from General

Dynamics, San Carlos, Calif.

Potassium tertiary butyl alcoholate was obtained from

M.S.A. Research Corp., Callery, Pa.

Lithium aluminum hydride was obtained from Metal

Hydride Inc., Beverly, Mass.

Unisil silicic acid was obtained from Clarkson Chemi

cal Co., Williamsport, Pa.

Methyl eicosanoate was obtained from Lachat Chemicals

Inc., Chicago, 111.

Methyl erucate was kindly supplied by Dr. Fred Kruger.

Xylene (B.P. 138.5-lHo°) was obtained from J. T. Baker

Chemical Co., Phillipsburg, N.J.

Trifluoroacetic acid and trifluoroacetic anhydride

were purchased from Eastman Organic Chemicals, Rochester,

N.J.

Hamilton syringes were purchased from The Hamilton

Company Inc., Whittier, Calif.

C. Methods

1• Preparation of egg yolk triglycerides.and ethanol-

amine phosphatides. Egg yolk triglycerides and ethanolamine

phosphatides were prepared from a lipid extract (162) of 21

two egg yolks by the method of Horning et al. (163). A

U cm column packed with 85 g Unisil silicic acid was eluted with increasing amounts of benzene in hexane. The benzene:hexane (3 :2 , v/v) fraction was rechromatographed to give triglycerides which were homogeneous as judged by thin layer chromatography (TLC) (l6U,l65). Elution was contin ued with benzene and chloroform and then a chloroform:methanol ( U : 1, v/v) fraction was isolated. This fraction was rechromatographed on Unisil and diethylaminoethy1 cellu lose in the acetate form (1 6 6 ) to give ethanolamine phos phatides, which were identified and judged homogeneous by

TLC ( \61*, 16 5 ) . Thin layer plates were prepared as described by Stahl (167 ) using 30.0 g Silica Gel G and 60 ml of water.

The lipid samples were spotted on the thin layer plates and developed in chlor of orm: me thanol :water ( 65 :2 5 :**, v/v)

as the solvent system. The spots were identified by spray ing the plate with 50 per cent sulfuric acid followed by

charring on a hot plate. These compounds were prepared in

collaboration with Dr. Lloyd A. Horrocks of the Cleveland

Psychiatric Institute.

2.' Preparation of phospholipids (lecithin and cenhalin)

from egg yolk. Yolks from 20 eggs were combined (361 g wet 22

weight), lipids were extracted, and the neutral lipids were separated from the phospholipids according to the procedure of Rhodes and Lea (l6 8 ). Most of the water from the com bined wet egg yolks was removed with acetone (500 ml). The residue obtained after filtering the blended egg yolks, was blended further with 500 ml of acetone and filtered.

This process was repeated once more. Thus most of the neutral lipids were removed. The residue was extracted with 350 ml of chloroform:methanol (1 :1 , v/v) and filtered.

This process was repeated twi.ce. The filtrate contained most of the phospholipids. The solvent was removed by evaporation and the residue was dissolved in 80 ml of petroleum ether (b.p. i+0-60°). Most of the phospholipids (k were purified by precipitation four times at 0 ° from a 15

* per cent solution of petroleum ether by addition of 8 vol umes of acetone. The phospholipid fraction was then^dissolved in 100 ml of petroleum ether and kept at 0 ° for l6 hr and centrifuged. This step removed most of the sphingomyelins. The crude phospholipids obtained, 19 g, were dissolved in 250 ml of chloroform and stored under nitrogen in a deep freeze. Aliquots of 35 ml* containing 23

about 2 g of crude phospholipid were used for the separation

and purification of cephalin and lecithin. Chromatography was performed on Uo g alumina (Woelm, basic) packed into

a chromatographic column (2.5 cm by 1+5 cm), constricted at the base. Lecithin was eluted with chloroform:methanol

(1:1, v/v) according to the method of Rhodes and Lea (l68 ).

A flow rate of 2-2.5 ml/min was maintained with positive nitrogen pressure. Fifteen milliliter fractions were col lected. The fractions were-^nonitored by TLC using chloro form: methanol:water ( 65 : 2 5 :1+, v/v) as the solvent system.

Tube numbers 1-9 contained almost all the lecithin and some presumed lyso compounds but were free of cephalin. Before pooling, each fraction was tested on TLC. The solvent

system was changed after 9 tubes to ethanol:chloroform: water (5:2:2, v/v) to elute cephalin. Tube numbers 10-17

contained almost all the cephalin as judged by TLC.

Further purification of lecithin and cephalin frac

tions was carried out on Unisil columns. The solvent sys

tem was chloroform:methanol (6 8 :3 2 , v/v), and procedure was

identical to that of Lea et al. (l68,l69)» The flow rate was 5-6 ml/min. Fifteen milliliter fractions were collected

and the homogeneity of the sample was tested by TLC. In lecithin purification, tube numbers U-28 were pooled. In cephalin purification, tubes 15-25 were pooled. The ester value was determined by the method of Stern and Shapiro

{170). Methyl palmitate., was used as standard. The phos phorus content was determined according to the method of

Lowry et al. (1T1)•

3. Hydrogenolysis-acetylation. From 30 to 100 mg of glyceride and a known amount of methyl eicosanoate or methyl erucate was dissolved in 20 ml of dry ether and placed in a

100 ml round-bottomed flask. About 200 mg of lithium aluminum hydride was dissolved in 30 ml of ether in a- separ

ate flask. The residue was allowed to settle and the solu tion was transferred by pipette, 1 ml at a time, to the

glyceride solution until boiling stopped. A volume excess of this LAH solution was added and the mixture refluxed for from 60 to 90 min (l). Acetic anhydride was added dropwise to decompose excess lithium hydride, followed by

25 ml of acetic anhydride and 30'ml of xylene (Baker, A.R., b.p. 138.5-1^0°). Ether was removed by boiling with the

flask open or could be removed before addition of excess

acetic anhydride and xylene by evaporation with a water

pump. When the vapor temperature rose to.110-115°» the 25

flask was closed and the contents were refluxed for 6 hr.

The mixture was filtered and the filtrate evaporated at

55° for 60 min first with a water-pump, and then high vacuum. The residue was dissolved in dry ether for GLC.

If early extraneous peaks were found in GLC, xylene was added, and vacuum evaporation repeated.

U. Saponification-acetylation. From 30 to 100 mg of

glyceride and.a known amount of hexadecanyl acetate (pre

pared from the pure methyl ester by hydr ogenoly s i s, acety.- lation and Claisen distillation) were dissolved in 30 ml

of absolute methanol and placed in a round bottomed flask.

About 25 mg of sodium was dissolved in 10 ml methanol and

added to the solution. The mixture was refluxed for 2 hr,

and methanol was evaporated with a water-pump. Water

(5 ml) was added and the mixture /y.as- refluxed for 60 min.

Acetic anhydride (35 ml) and 35 ml of xylene were added and

the acetates prepared and isolated as described above.

5. Acetolysis. a. Acetolysis of cephalin: From 50 to

100 mg of cephalin and a known amount (30 mg) of hexadecanyl

acetate were weighed into a flask. Ten milliliter of a

mixture of acetic acid and acetic anhydride (1*:1 , v/v) was

added (157,158). The sample was refluxed for 10 hr and the acetic acid and acetic anhydride were removed with a water- pump, then high vacuum. The flask was flushed with nitro gen. Twenty milliliter of methanol was added to dissolve the sample. Methanol, 10 ml containing 50 mg of sodium, was added and the mixture refluxed for 2 hr. Methanol was removed using a water-pump. Water, 5 ml, was then added and the mixture refluxed for 2.5 hr. Acetic anhydride

(35 ml) and 35 ml xylene were added and the mixture re fluxed for 6 hr. Acetates were then analyzed by GLC.

b. Acetolysis of cephalin and lecithin: From 50 to

100 mg of phospholipid and 30 mg of hexadecanyl acetate were weighed into a flask. Ten milliliter of a solution containing trifluoroacetic acid and acetic anhydride

(1:4, v/v) was added and the mixture refluxed for 10 hr.

Saponification and isolation were then accomplished by the procedure described above. however, the amount of sodium added for the saponification was increased from 50 to JO mg and 2 ml of 1 h sodium hydroxide and 3 ml of water were used in place of 5 ml of water used previously.

6 . Gas-liquid chromatography. The Aerograph (A-350-B) dual column temperature programmer gas chromatograph was used in the analysis of lipid components. The instrument 27

contained dual columns, dual H-filament detector cells, and dual collectors. The temperature of the column oven, in jector block, cell oven, and collectors was measured by accurate pyrometers. A sample was injected in either in jector with Hamilton syringes of 10 or 25 microliter capacity and analysed by the corresponding column. The detector signal was amplified and recorded by a Wheelco model 8000-

H600-S26 6 •one millivolt electronic recorder and the peak area was recorded by a type A-2 electronic integrator.

When identical columns are used bleeding is nullified and a steady base line results. The flow rate was adjusted by using an in-line flow-restricture.

A 10 ft stainless column, 0.25 inch i.d., containing from 10 to 13 per cent ethylene glycol succinate polyester

(EGS) on 60-80 mesh Gas Chrom P was used. Helium was used as the carrier gas. The flow rate (60-100 ml/min) and temperature (170-200°) of the column were varied for the separation of lipid components. Peak areas were corrected for molar response relative to hexadecanyl acetate taken as 100 (172). Columns were conditioned before use for 35 to U0 hr at 205° and an approximate flow rate of 30 ml/min to eliminate bleed off. Programming was used for compounds with a very high retention time. IV. RESULTS

A , Composition of reference Compounds

Reference compounds were converted to alcohol acetates and their purity was determined by GLC. Minor components were found in several compounds. The composition of these materials is reported in Table 1.

Table 1.--Composition of reference compounds.

Number of Impurity in Minor Sample Tracings Mole % Components

Distearin 6 k.2 Palmitate

Methyl Eicosanoate T 2.2 Stearate (Lachat Chemical Corp. )

CThimyl Dipalmi- 5 T.3 Stearate t at e

Batyl Distearate 6 12.2 Palmitate

28 29

B . Relative molar response determinations

Relative molar response factors (RMR) for glyceryl triacetate, eicosanyl acetate and docosenyl acetate in the thermal conductivity detector were calculated as follows:

Astd x RMR x 100

Mstd = moles of standard (hexadecanyl acetate)

Mx = moles of compound

■^std = integrated area for standard

Ax = integrated area for compound

The RMR of glyceryl triacetate was determined with a mixture of hexadecanyl acetate and triacetin. The RMR of triacetin was also obtained from different glycerides which were sub jected to hydrogenolysis and acetylation. The results are given in Table 2. Docosenyl acetate and eicosanyl acetate were synthesized from.their methyl esters and RMR estimated with a known amount of hexadecanyl acetate or a pure refer ence glyceride (Table 2).

The two eicosanyl acetate samples differ in their RMR.

The RMR of the pure sample agrees with a previous study (IT2) •

The commercial sample has a lower RMR and it is evident that the 18:0 minor component is not sufficient to account for 30

Table 2.— Relative molar response of triacetin, eicosanyl acetate and docosenyl acetate.

Number of Relative molar Sample Trac ings Respons e^

+ Triacetin 9 69.8 3e 7 73.3 + 3 h 70.3 + 2 6 70.7 + 2 + • 11 73.0 5 + 6 ■ 69.9 3 6 70. U + 2 6 71.3 + 1 7 71.7 + 1 • 5 70.9 + 0 • h 71.0 + 3

Mean 71.0

+ Eic osanyl acetate8. 8 101. 5 2 5 103 .0 + 3 + 5 10U .6 3 8 99.2 + 5 Mean 10 2.0

Eicosanyl acetate^5 12 111.0 + 3

+ Docos enyl acetate2 6 97. 5 k + 5 100 .0 5 6 101.0 + 2 + 5 103.0 3 + 3 101.0 5 1 99.0

1 . 1OU.0

- Mean 101.0

a Prepared from methyl eicosanoate purchased from Lachat Chemical Corporation. b Pure sample kindly supplied by Dr. J. B. Brown, c Programmed from 170° to 200° with a 3° per min gradient. d RMR for hexadecanyl acetate was 100. e Mean t standard deviation the variation. The RMR of triacetin with the Aerograph

A-35O-B was 71# whereas the RMR was 92 with the Aerograph

A-90-C (172). The RMR for compounds within a homologous

series such as alcohol acetates did not vary beiween

''fts instruments.

C . Hydrogenolys is-acetylat ion

The procedure described by Horrocks and Cornwell (l)

was repeated in a preliminary study. Glyceride samples

were reduced with lithium aluminum hydride (LAH) and ace-

tylated. Excess acetic anhydride was removed by refluxing

with absolute ethanol. Ethyl acetate and ethanol were f removed under water-pump. The contents of the flasks were

extracted with ether and washed. This ether solution was

filtered through sodium sulfate and reduced in volume for

GLC. The results are given in Table 3. Wide variations

in ester-to-glycerol ratios were obtained (Table 3).

D . Improved hydrogenolysis- ac etylation

A number of modifications in hydrogenolysis-acetyla-

tion were attempted to improve ester-to-glycerol ratios.

The modification that was finally selected eliminated the

ethanol refluxing and the aqueous wash steps. Xylene was 32

Table 3. — lly dr ogenolys is-acetylation and GLC. [Horrocks and Cornwell (l)]

Humber of Ester/Glycerol Sample Tracings Calculated Found

Tripalmitin 2 3 .00 b .90 1 3.00 3.30 Monopalmi tin 1 1.00 1. 5U 3 1.00 0.91 1 1.00 1.07 3 1.00- 0.93

added and evaporated, first with a water-pump and then high vacuum to remove excess acetic anhydride and acetic acid.

The results are sumniar-i zed in Table 1+.

Since the excess reagents were removed under vacuum it .was necessary to determine if fatty alcohol acetates were lost in this procedure. Hence a standard fatty acid methyl ester mixture was subjected to hydrogenolysis-acetylation.

This was followed by prolonged vacuum evaporation at a water bath temperature of 55° and a vacuum of one millimeter of mercury. The results are' given in Table 5. Hydrogen- olysis-acetylation of a Cg through Cgo methyl ester test mixture gave quantitative results with Cqo through C20 acetates even after prolonged vacuum evaporation while octyl acetate was recovered in low yield. Table U .--Hydrogenolysis-acetylation and GLC.

Number of Ester/Glycerol Sample Trac ings Calculated Found

Tripalmitin 5 3.0 3 .00 k 3.0 3.11 5 3.0 , 3.17 5 3.0 3 .20 b 3.0 3.08 2 3.0 2.85 6 3.0 3.27 6 3.0 2.98 8 3.0 * 2.98 1+ 3.0 3.03 Mean 3 .07

Monopalmitin U 1.0 ' 1 .0 U 6 1.0 1.08 5 1.0 1.00 7 1.0 i.o6 It 1.0 0.99. 5 1.0 1.03 It 1.0 1.08 6 1.0 1.07 • 5 1.0 1.05 7 1.0 o .96 Mean 1 .0U

Dipalmit in 5 2.0 2.20 9 2.0 2.18 6 2.0 2.05 Mean . 2 .lit

Distearin 5 2.0 2 .lit 2 2.0 2.05 Mean 2 .09

Monoste arin 5 1.0 1.03 Monomyristin 5 1.0 0.98 3 1*

Table 5.— Hydrogenolysis-acetylation of a standard methyl ester mixture and prolonged vacuum evaporation.

Alcohol acetate. Mole %

Known Found3

+ 8:0 5.08 1.5^ o .0Tb j- 10:.0 9-58 9.21 1 .37 12:0 10.09 11.10 + 0.1+7 lU: 0 13 .25 ll+.QO + 0 .6 0 . 16 :0 15. 62 16.30 + 0.52 + 18 :0 20.02 20 .60 0.51* 20 :0 26.30 26.00 + 1.00

a Average of seven tracings,

b Mean t standard deviation.

E . The use of internal standards with hydrogenolysis-acetylation

(l) Docosenyl acetate. The selection of an internal standard is important. It should be one that is inert towards all sample components and is"not present in the samples analyzed. The internal standard should have a different retention volume than the components of the sample under consideration. Docosenoic acid is not a common com ponent fatty acid in natural lipids; however, docosenyl-M acetate has an extremely high retention volume and hence requires programming. On the other hand, eicosanyl acetate 35

has a reasonable retention volume, but eicosanoic acid is

found in many natural lipids.

Jis ter-to-glycerol ratios of reference compounds and

their fatty acid components and the absolute amounts of

these components were determined using docosenyl acetate

as the internal standard. A sample calculation is given

below:

A mixture of methyl docosenoate (53^+ Vmoles ) and monopal-

mitin (267 ymoles) was subjected to hydrogenolysis-acety-

lation and GLC. The area under each component was read

•from the integrated area and substituted in the equation

given below.

M A x RMR s t d s td x Mx

(a) Area of docosenyl acetate (RMRs-t^, 101) is 6o.9

(b) Area of hexadecanyl acetate (RMRX , 100) is 31.1+

Substituting these values in the above equation,

60.9 x 71 or Mx = 267 pmoles 21.k 101

The theoretical glycerol value was 267 pmoles and the 36

experimental value was 267 y moles. The sample was programmed

\ after the fatty alcohol acetates were eluted. This enhanced

the elution of docosenyl acetate. During programming the

column temperature was increased from 170° to 200°. The .

rate of temperature increase was 3C per minute. The results

are given in Table 6.

(2) Eicosanyl acetate. Eicosanyl acetate was used

with reference compounds of a known fatty acid composition.

The results of hydrogenolysis-acetylation and GLC of a

number of samples are given in Table 7• Consistent and

reproducible data were obtained. The internal standard was

used for the analysis of egg yolk triglycerides. These

results are shown in Table 8. Minor peaks with longer

retention times, presumably polyunsaturated fatty alcohol

acetates, were observed in this study; however, they were

not identified or included in the calculations, The total

ester content obtained with GLC agreed with the total ester

estimated by a colorimetric method.

F . Saponificat ion-acetylation

Glycerides were saponified and the fatty acids were

methylated with sodium methoxide. Methyl esters were

completely saponified by refluxing the mixture with water. Table 6.— The fatty acid and glycerol content of glyceride samples estimated by hydrogenolysis-acetylation and GLC.a

I V moles

Number of 16 : 0 18:0 Glycerol Sample Tracings Ester/glycerol Known Found Known Found Known Found

Monopalmi tin 7 1*88 k9l!6h U88 k6brt±2 1.06

6 267 273±8 . - — 267 257+12 1.06 +1 1 0 0 —i

Distearin 5 - 265 250+8 133 I2U+I1 2.10

Batyl distearate k - 3oj;5 187 167^9 - - - Hexad ecanyl 1 331 3 ^2+11* ac etate /

a Methyl docosenoate was the internal standard,

b Mean * standard deviation. Table 7.— The fatty acid and glycerol content of glyceride samples estimated by hydrogenolysis-acetylation and GLCa

{' y moles }

Humber of ll*:0 16:0 18:0 Glycerol Ester/ Sample^* Tracings Glycerolc Known Found Known Found Known Found Known Found

Hexadecanyl 8 392 39116 d not measured ac etate Monopalmitin 6 2 5 1+ 26618 8, 10l2 25I* 256110 1 .0U Monopalmitin 6 162 152i2 not measured 162 ll*2l3 1.07 Monopalmitin 5 161 155—1+ not measured l6l . 11*313 1.08 Monostearin 10 271+ 276116 268 263120 1.05 Dimyristin 9 268 292ll8 not measured 131* 13018 2.20 Dimyristin 6 212 211*16 15 12 + 2 106 10315 2.08 Dipalmitin 9 377 388119 not measured 188 17718 2.20 Dipalmitin 6 203 20316 101 99+7 2.05 Dipalmitin + 6 3^0 356111* 322 33l*ll8 not measured 331 32lllU 2.15 Dimyristin Trimyristin 10 2 U6 26615 12 11+2 ■ 82 8218 3 .20 Tripalmitin 5 1*01 389110 11 15±3 131* 122+6 3.20 Chimyl Dipalmi - 6 1-1*6 1321U 20 20l3 - - - tate

Batyl Distear 6 \ 18 18+1 131* 13315 - - - ate a Methyl eicosanoate was the internal standard. 1 h Sample and internal standard impurities were allowed for in the calculation of ^ "known lipid content."

c 18:0 impurity from internal standard omitted.

d Mean t standard deviation

UJ VO Uo

Table 8.— Duplicate analyses of egg yolk triglyceride by hydrogenolysis-acetylation and GLCa

Component Sample A Sample B

y moles/ml mol e % y moles/ml mole %

16':0 3.3 t 0 .2b 26.0 3.3 t 0.1 25.6

16:1 0.3 ± 0.1 2 . b o.u i 0.1 2.9

18:0 1.3 ± 0.2 10.0 1.3 i 0.1 10.2

18:1 6.9 i 0.3 5b.1 6.8 i 0.2 52.6

18:2 0.9 i 0.1 7.5 1.1 J 0.1 8.7

Glycerol b.3 t 0.2 b .2 + 0.2

Total esterc 12.7 12 .9

Ester/Glycerol 3.0 3.1

Total ester 13.1 + 0 .8 13.1 t 0.8

a Methyl eicosanoate was the internal standard,

b Mean 1 standard deviation,

c Colorimetric -analysis (170). 1+1

Water produces sodium hydroxide from sodium methoxide. A large excess of acetic anhydride (35 ml) was added to re move the water and to acetylate the glycerol. Xylene (35 ml) was added along with acetic anhydride and refluxed.

Solvents were removed with a water-pump and then with high vacuum. The sample was then used for GLC analysis. Hexa- decanyl acetate was used as an internal standard. The results are shown in Table 9.

This is an important extension of the method not only for the analysis of glycerides that contain eicosanoic acid hut also for analysis of glycerides that may contain com pounds which elute with glyceryl triacetate. Thus saponification-acetylation increases the flexibility of the p r o c e d u r e .

G . Acetolysis-saponification- ac etylat ion

Karrer and Jucker (173) described a hydrogenolysis procedure which resulted in the dephosphorylation of lecithin.

When their conditions were used only 65 per cent of the cal culated glycerol content was obtained from cephalin. Hydro- genolysis followed by acetolysis of cephalin gave an 88 per cent glycerol yield. Because the hydrogenolysis-acetylation 42

Table 9•--Glycerol content of glyceride samples estimated by saponification- acetylation and GLC.a

Glyc erol dumber of Sample Trac ings Known Found % Recovery 1J mole

Monopalmitin 5 303 3l4ll6b 103 .6 1+ 2 41 232+3 96.3 6 189 184+2 97.4 7 303 32015 10 5.0 6 207 20 8+4 100.5 Monostearin 5 266 277 +9 104.0 Distearin 6 140 139±1 99 .3 Dimyristin 7 146 147+3 100.7 Monopalmit in + 7 313 316 + 7 101.0 Monoste arin Trimyristin 5 165 168+3 101.8 Tripalmitin 5 125 129±4 103.2 6 142 l43±3 100.7 10 162 159+3 98.1 7 158 15411 97.5 Tripalmitin + 9 l4l 13814 97.9 Trimyristin

a Hexadecanyl acetate was the internal standard,

b Mean 1 standard deviation.

procedure did not work with the phospholipids, several at tempts were made to hydrolyze the phosphate group as a preliminary step in the analytical procedure. The phospho lipids used in this study were tested by TLC for their purity and their ester-to-phosphorus ratios determined

(Table 10). U3

Table 10.— Ester-to-phosphorus ratios of phospholipids

S a m p l e dumber of Determination Ester/Phosphorus

Lecithin ( egg) 10 2.01

L e c i t h i n ( GBI) k 1.99

C e p h a l i n (egg) 8 2.00

C ep h a l i n (GBI) 1 2.0 9

The methods of Bevan etal. (157) were applied to phospha tidyl ethanolamine prepared from egg yolk. According to this procedure, a known amount of the lipid was mixed with hexadecanyl acetate as an internal standard and then re fluxed with a mixture of acetic acid and acetic anhydride

( : 1, v/v) for 10 hr. After the completion of the reaction, excess reagents were removed first with a water-pump and then with high vacuum. The flask was flushed with nitrogen for about a minute. Saponification-acetylation was carried out as described in Section F. The sample was analyzed by

GLC. The results are given in Table 11, A good conversion

of phospholipid glycerol to glyceryl triacetate was obtained.

The glycerol content was calculated from the phosphorus

analysis. kk

Table 11.— Glycerol content of cephalin estimated by acetolysis-saponification-acetylation and GLC.a

Glycerol Knownb Found Number of Sample Tracings y moles % Recovery

i Cephalin (egg) k 96 . 8 90 .ii2c 93.1 k 96.8 92 .3±2 95.U Ethanolami ne 5 69.0 6 5.5!t2 95.0 Phosphatide. 5 69.0 65 .0±3 9^.2 Cephalin (GBI) 5 95.9 9^.5±1 98.5 6 95.9 9**.1±1 98.1 9 77.3 7 7 .2±k 99 .9 8 107. 8 10 8 .8+U 100.9 5 95.9 93.5ll 97.5 7 78.9 80.312 101.7 8 85 . 5 86 .7^3 101 . k 7 87.2 * 88 .8+2 101.8 8 95.6 106.5±2 111.0

a Hexadecanyl acetate was the internal standard,

b Estimated from weight or phosphorus content,

c Mean + standard deviation.

Acetolysis-hydrogenolysis-acetylation was carried out on cephalin in order to determine both the glycerol and fatty acid composition. The fatty acid and glycerol con tents are reported in Table 12. Methyl eicosanoate was the internal standard. Table 12.— Glycerol and fatty acid contents of cephalin by acetolysis-hydrogenolysis-acetylation and GLC.a

Glycerol 16:0 alcohol Acetate

Number of Sample Tracings Known^ Found % Recovery 1t Known Found % Recovery

V moles p moles *

Cephalin (GBI) T 77.9 75.213° 96.6 156 i 5 9 n 102.0 -

7 55.8 57.8+3 103 .6 111 116+3 10h .5

6 55.2 5U.Uil 98.5 110 10913 99.1

‘5 70.3 6*1.O 92.0 lUl ll*5l!* , 103.0

a Methyl eicosanoate wals the internal standard

b Estimated from weight or phosphorus content

c Mean 1 standard deviation U6

Acetolysis-saponification-acetylation was applied to both

commercial and egg yolk lecithins. The results are reported

in Table 13. Yields were not satisfactory, hence an im

proved method was needed for the analysis of* glycerol. A >, f summary of the different modifications attempted is given

in Table lU. When tr if luoroac.et ic acid and t r i f luor oa cet i c

anhydride (8:2, v/v) was used for acetolysis, the glycerol

recovery was 89 per cent (Method 16, Table l M . When

trifluoroacetic acid and acetic anhydride (1:9, v/v) was used, the yield of glycerol was 99 per cent (Method 23,

T a b l e ih). However, low values were sometimes obtained. \ If the tnfluoroacetic acid content was lowered from 1 ml

to 0.3 ml the yield of glycerol dropped from 99 per cent

to 83 per cent (Method 2U, Table 1^). An intermediate

trifluoroacetic acid concentration was chosen. This reflux

procedure, trifluoroacetic acid and acetic anhydride

(1:U , v/v) is called modified acetolysis.

H . Modified acetolysis

In the modified acetolysis procedure lecithin was

mixed with a known amount of hexadecanyl acetate and re

fluxed with trifluoroacetic acid and acetic anhydride

(l:U, v/v) for 10 hr. Extensive charring occurred. The Table 13»--Glycerol c on t e n t of lecithin by acetolysis. saponification-acetylation . and GLC •a

Gly ce r o l

K n o w n 13 F o u n d ' /£ R e c o v e r y d u m b e r of Sample Tracings U m o l e s

Lecithin (egg) 5 157.1+ 115 .0 l 3 d 7 3 . l c 6 157.!+ 99.1+13 6 3 . 2 5 9 2 . 5 1+0 .611 1+3.9 5 9 2 . 5 1+0 .9+1 44. 2 3 9 2. 5 . 1+7. ll3 5 0 . 9 7 8 . 7 3 8 . 7 + 2 1+9.2 5 7 8 .7 3 8 . 3 1 3 1+8.7 5 7 8 . 7 3 9 . 6 1 2 50.3 5 171.0 7 1 . 9 1 2 4 2. 0 5 1 7 1 . 0 8 4 . 7 1 2 4 9 . 5 4 171.0 5 9 . ill 3 4 . 6 Lecithin (GBI) 11 58.8 3 9 . 2 1 3 6 6 .7 C 12 58.8 3 7 . 5 1 2 6 3 . 8 C L 6 58.8 2 7 . 2 + 1 4 6 . 3

a Hexadecanyl acetate was the internal standard

b Kstimated from phosphorus content

c Additional acetolysis reagent and increased reflux t i m e

d Mean 1 standard deviation

c. Table lU.— Studies on the hydrolysis of phospholipids

Glycerol Recovery

Of Known Found /0

Methods / Sample Condition of hydrolysis U moles

1 DL-Cephalin (GBI) 100 mg of cephalin dissolved in Mo conversion 10 ml of ethanol containing 100 mg sodium and stirred for 6 hr, re fluxed with 30 ml of acetic an hydride and 30 ml of xylene and flash evaporated. 2 DL-Cephalin (GBI) 100 mg of cephalin dissolved in \ No conversion 10 ml of ethanol containing 100 mg sodium, stirred for 6 hr, 5 ml acetic acid added, reduced with 1 g LAH for 1 h r , acetylated and flash evaporated. 3 Monopalmitin About 100 mg of monopalmitin dis- No reduction, good solved in 10 ml ethanol containing conversion to tri- 100 mg sodium, stirred for 6 hr, acetin 5 ml of acetic acid and 1 g LAH added, reduced for 1 hr, acetylated and flash evaporated. -t=- OD Table lH.— Continued.

Glycerol Recovery

Known Found %

Methods Sample Condition of Hydrolysis \i moles

h DL-Cephalin 25 ml of 5% methanolic hydrochlor- Ho conversion ide added to 100 mg of cephalin* refluxed for 5-6 hr, solvent re moved, reduced with 200 mg LAH for 1 hr, acetylated and flash evapor abed. 5. Monopalmitin 100 mg of monopalmitin dissolved in Ester/Glycerol 10 ml of methanol containing 50 mg ratio of 0.96 sodium, stirred for 5-6 hr, reduced with 200 mg LAH, acetylated and evaporated. 6 DL-Cephalin 75 mg of cephalin dissolved in 20 Ester/Glycerol ml of methanol containing 2 mg ratio of 3.7 sodium, stirred for 5-6 hr, 5 drops of acetic anhydride added, reduced with 200 mg LAII, flash evaporated, acetylated and flash evaporated. Sodium glycero- 100 mg sodium glycerophosphate slur- Ho conversion phosphate ried with 20 ml of methanol contain ing 50 mg sodium, refluxed for 10 hr, 5 drops of acetic anhydride added, ' flash evaporated, acetylated and flash evaporated. Table lU.— Continued.

Glycerol Recovery

Known Found tocl

Methods Sample Condition of Hydrolysis y moles

Sodium glycero- 100 mg of sodium glycerophosphate dis poor conversion phosphatea solved in 20 ml tetrahydrofuran, 3 ml of 2N hydrochloric acid added, refluxed for 6 hr, acetylated and flash evapor ated . Sodium glycero 100 mg of sodium glycerophosphate dis poor conversion phosphate3 solved in 2 ml water, H ml of trifluoroacetic acid added, refluxed for 6 hr, flash evaporated, acetylated and flash evaporated. 10 Sodium glycero- 100 mg sodium glycerophosphate refluxed 100 60 60 pho sphatea with 10 ml of acetic acid and acetic anhydride (U:l, v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 11 Sodium glycero- About 100 mg of the sample refluxed 100 70 70 pho'sphate3. with 10 ml of acetolysis mixture for 10 hr, flash evaporated, saponified, ace tylated and flash evaporated. 12 Cephalin (GBI)a 100 mg of cephalin dissolved 7 ral t-bu- no conversion tanol containing 50 mg of potassium t- VJI O butoxide, refluxed for 7 hr, flash evap orated, acetylated and flash evaporated Table 15.— Continued.

Glycerol Recovery

Known Found Methods Sample Condition of Hydrolysis Vi moles

13 Sodium glycero- 30 mg of the sample dissolved in 10 ml poor conversion phosphatea of trifluoroacetic acid, refluxed for 10 hr, evaporated, saponified, acetyl ated and flash evaporated. it Cephalin (GBI) About 100 mg of cephalin dissolved in 66 b3 65 n-butyl ether, reduced with too mg of LAH, acetylated and flash evaporated 15 Lecithin (egg): Sample refluxed with 10 ml of a mixture t6 38 82 of trifluoroacetic acid and trifluoroacetic anhydride (8:2, v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 16 Lecithin (egg) Sample refluxed with 10 ml of a mixture k6 89 of trifluoroacetic acid and trifluoroacetic anhydride (8:2, v/v) for 10 hr, flash evaporated,, saponified, acetylated and flash evaporated. 17 Cephalin (GBI)*3 Reduced with 200 mg of LAH n-butyl ether, 8l k2 acetylated, and flash evaporated.

VJ1 !— 1 f

Table lU.— Continued.

Glycerol Recovery

Known Found %

Methods . Sample Condition of Hydrolysis y moles

18 Cephalin (GBI) Sample refluxed with 10 ml of a 5^ k8 89 mixture of acetic acid and acetic anhydride (U:l, v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 19 Cephalin (GBI)a Sample refluxed with 10 ml of a 85 77 90 mixture of trifluoroacetic acid and acetic anhydride (1 :1 0 , v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 20 Cephalin* Sample refluxed with a mixture of 5U 8H trifluoroacetic acid and acetic anhydride (0 .2 :9 .8 , v/v) for 10 hr, flash evaporated, saponified, acetyl ated and flash evaporated. 21 Cephalin (GBI)a Sample refluxed with 10 ml of a 87 81 93 mixture of trifluoroacetic acid and acetic anhydride (1 :1 0 , v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated.

VI ro Table Ik.— Continued.

Glycerol Recovery

Known Found % Methods Sample Condition of Hydrolysis u moles

22 Sodium glycero- Sample refluxed with 10 ml of a mixture 100 73 73 pho sphatea of trifluoroacetic acid and trifluoroacetic anhydride (it: 1, v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 23 Lecithin (GBI)a Sample refluxed with 10 ml of a mixture 80 79 99 of trifluoroacetic acid and acetic an hydride (l:9, v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 2k Lecithin (GBI)a Sample refluxed with 10 ml of a mixture 80 66 83 of trifluoroacetic acid and acetic an hydride (0.3:9.7* v/v) for 10 hr, flash evaporated, saponified, acetylated and flash evaporated. 25 Lecithin (GBI)a Sample refluxed with 1 ml of trifluoro- 80 9 11 acetic acid in 10 ml of methanol for 10 hr, flash evaporated, saponified, acetyl ated and flash evaporated. 26 Lecithin (GBI)a Sample refluxed with 0.2 ml of trifluoro- 80 3 it acetic acid in 10 ml of methanol for 10 hr, f/Lash evaporated, saponified, acetyl ated and flash evaporated.

a Hexadecanyl acetate was the internal standard, 00 b Methyl eicosanoate was the internal standard. trifluoroacetic acid and acetic anhydride were removed at

first with water-pump and then under high vacuum. It was

necessary to remove trifluoroacetic acid and acetic anhy dride before subjecting the product to saponification.

Flushing with nitrogen was also necessary. The sample was

then dissolved in absolute methanol (20 ml) and TO mg of

sodium in methanol (10 ml) was added and refluxed for 2 hr.

The solvent was removed and 2 ml of IN sodium hydroxide

and 3 ml of water were added. The mixture was refluxed

for 2.5 hr. The rest of the procedure was carried out

as described for saponification-acetylation. The results

of the glycerol recovery from commercial and egg yolk

lecithins are given in Table 15. Glycerol recoveries of

92 to 102 per cent were obtained. Low recoveries occurred

when saponification was not complete. These low recoveries

were obtained before the introduction of an extra step, the

addition of sodium hydroxide. These low recoveries were

caused in part by methyl esters elating with triacetin and

* hexadecanyl acetate. Typical results for incomplete sapon

ification are reported in Table 16.

When modified acetolysis was applied to either com

mercial cephalin or cephalin prepared in the laboratory 55

Table 15.--GlyceroT content of lecithin estimated by modified acetolysis-saponification-acetylation . and GLC.a

Glycerol

Known** Found Number of Sample Tracings y moles % Recovery

Lecithin (egg) 5 114. 0 103.013° 90 .4 6 157.4 156.4+3 99 .4 12 157.4 150.0+4 95.3 7 157.4 155.2+5 9 8.6 7 7 8 . 7 7 1 .4+1 90.7 6 78.7 7 2 .2+2 91.8 5 114. 0 116.014 101. 8 6 114.0 113.0+1 99.1 4 114.0 107.718 94.5 Lecithin (GBI) 5 79.5 78.913 99 .3 8 117.5 116.415 99.1 5 58.8 54.412 92.5 5 117.5 109.314 93.0

a Hexadecanyl acetate was used as the internal standard,

b Estimated from phosphorus content,

c Mean ± standard deviation. 56 from egg yolk, consistent and reproducible results were obtained. The results are shown in Table 17. Percentage recoveries were from 91 to 100.

Table 16.--Glycerol content of lecithin estimated by incomplete saponification-acetylation and GLC.a

Glycerol

Humber of Known^ Found Samples Tracings y moles % Recovery

Lee ithin ( egg) 8 7 8 . 7 6 6 . 5±2C 8 U . 5 1+ 111}. o 9 6 . 5±0 8I4.6 CO CO tr\ Lecithin (GBI) 5 • 5 0 . 5 1 2 8 5 . 9

a liexadecanyl acetate was the internal standard

b Estimated from phosphorus content

c Mean i standard deviation

oince charring occurred during the modified acetolysis, the acetolys,ls products obtained with dipalmitin, monopalmitin,

glycerol and phospholipids were studied further-. Acetyl diglycerides, synthesized from egg yolk phospholipids by acetolysis with acetic acid and acetic anhydride ( h : 1, v/v), have nearly the same as tripalmitin with hexanerether

(1:1, v/v) solvents. The products obtained with egg yolk 57

Table 17 .--Gly cerol content of cephalin estimated by- modified acetolysis-saponification-acetylation and GLC.a

Glycerol

Number of Known13 Found Sample Trac ings V mol es % Recovery

C ephalin (GBI) 6 95.9 9 2 .3±lc 9 6.3 " k 83.9 8 2 .9^0 98.8 5 ■ 95.9 93.5ll 97.5 6 95.9 9 6 .2+2 100.3 C ephalin (egg) h 127. k * 117.7+2 92. h 5 127. u 116.2±k 91. 2

a Hexadecanyl acetate was the internal standard

b Estimated from phosphorus content

c Mean i standard deviation

phospholipids by acetolysis with trifluoroacetic acid and acetic anhydride (1:b , v/v) streaked from the origin and showed no distinct glyceride sppts. A streak was also found with the charred acetolysis mixture after it was refluxed without lipid. Model compounds such as dipalmitin, monopalmitin and glycerol were, therefore, refluxed with trifluoroacetic acid and acetic anhydride. Products corres ponding to the monoacetyl dipalmitin, diacetyl monopalmitin

Iv ti and triacetin were visible on TLC. 58

I Estimation of glycerol content in cardiolipins

The glycerol content of cardiolipin (Sylvana Chemical

Company) was determined. TLC of this sample with a solvent system containing chloroform:methanol:water (65:25:^* v/v)

gave one major spot which was near the solvent front.

When hexane:ether (1:1, v/v) was used as a solvent, there was no movement of the cardiolipin spot and there was no

spot at the solvent front wher e triglycerides would mi grat e

in this solvent system. This showed that there was no

triglyceride present.

Attempts to chromatograph and isolate different

cardiolipin components, if any, with Unisil column chroma

tography were tried. About 300 mg of cardiolipin was

chromatographed on 25 g Unisil and 50 ml fractions were

collected. Each fraction was analyzed for phosphorus con

tent. Tube numbers 1-lU were eluted with chloroform:methanol

(9:1, v/v). The solvent system was changed to methanol at

this point and tube numbers 15-18 were collected. Only two

peaks were obtained. These were found in tubes 2 and 15.

When these fractions were analyzed for ester value and

phosphorus content, they had the same composition. The 59

two initial peaks were obtained when individual peaks were rechromatographed. Several runs were carried out with similar results. Thus cardiolipin was not fractionated by this procedure.

Cardiolipin samples containing from 75 to 100 micro moles of phosphorus were mixed with a known amount of hexadecanyl acetate (30 mg) and a mixture of trifluoroacetic acid and acetic anhydride (10 ml, It**, v/v) was added.

This mixture was refluxed for 10 hr. Saponification-acety1- ation was then carried out as described previously. The glycerol content, phosphorus content, and ester content are tabulated in Table 18.

To determine whether plasmalogens were present, 200 mg of cardiolipin was hydrogenated completely with platinum dioxide (50 mg) as the catalyst. The pressure was 35 psi and the time of hydrogenation was 7 hr. The glycerol con tent and phosphorus content were then determined (Table 18).

Glycerol-to-phosphorus ratios of 1.15 to 1.20 were obtained.

Since hydrogenation did not decrease the yield of glycerol,

it was apparent that plasmalogens were not present. Table 18.--Glycerol content of cardiolipin estimated by modified acetolysis- saponification-acetylation and GLC.a

Number of Ester / Glycerol / Sample Trac ings Phosphorus Ester Glyc erol Phosphorus Phosphorus

y moles

Cardiolipin 5 78.6 169.8 94.6±3c 2.16 1.20 4 56.2 120.3 65.3l2 2.14 1.16 5 86.it l88.lt 95.013 2.18 1.10 7 93.9 204.7 112.2+3 2.18 1.20 5 93.9 204 .7 112.412 2.18 1.20 Cardiolipin 4 it6 .3 102.8 56.5±2 2 .22 1.22 (hydrogenated) 3 it 6 .3 102.8 52.Oil 2.22 1.13 Cardiolipin13 4 3^.2 74.6 19.7l2b 2.18 0.58

a Hexadecanyl acetate was the internal standard,

b Acetolysis with acetic acid and acetic anhydride (4:1, v/v).

c Mean 1 standard deviation. 61

J . Effect of modified acetolysis on fatty acid components

Since a black color was produced when the modified acetolysis procedure was applied, further work was under taken to show the nature of the degradation products.

When the acetolysis mixture was refluxed alone, a black color was produced. About 100 mg of tripalmitin was re fluxed with 10 ml of trifluoroacetic acid and acetic anhydride (1:U, v/v). The reagents were removed under water-pump and then with high vacuum. The mixture was flushed with nitrogen. The product was subjected to hydrogenolysis and acetylation. Components of lipids were then analyzed by GLC. An ester-to-glycerol ratio agreed with t'heOry (3.0). Egg yolk lecithin was subjected to the same procedure. It was observed that unsaturated fatty acids were almost completely destroyed whereas saturated fatty acids and glycerol were not affected. V. DISCUSSION

A . Sample purity and relative molar response

The purity of the components of the neutral glycerides and standard fatty acid methyl esters used in this study was tested by the synthesis and GLC analysis of alcohol acetates.

The results in Table 1 show that reference compounds had small percentages of impurities. If the saponification number was taken as the criterion of purity, as was done by earlier workers, it would be hard to detect these impurities.

Methyl eicosanoate (Lachat Chemical Corporation) was stated to be more than 99 per cent pure but it was found to have

2 per cent stearate. The RMR of pure eicosanyl acetate was

111 (Table 2) but the RMR of the commercial sample was 102.

The lower RMR of the commercial sample could not be attri buted only to 18:0 alcohol acetate, a minor component.

Other factors .such as the incomplete removal of solvent might contribute to the lower RMR. Thus reference compounds should not be used without determining both their fatty acid composition and an experimental RMR, The RMR of

62 63

compounds in a homologous series was found to be the same with two different chromatographs, but the RMR for other compounds varied. Thus triacetin had an RMR of 92 in the

Aerograph A-90-C (172) and had an RMR of 71 in the Aero graph A-350-B (Table 2). Hence RMR values should be determined experimentally when different instruments are used.

B . Hydrogenolysis-acetylation

The results in Table 3 were obtained with the pro cedure of llorrocks and Cornwell (l). on different samples.

Inconsistent ester-to-glycerol ratios were- obtained. Some values were good, but others were very high, such as 3.3 to U.9 for triglycerides. This indicated that glyceryl triacetate could be lost either by interester ifi cation with ethanol or solution in water during the aqueous wash procedures. Glyceryl triacetate solubility in water is

7 parts per 100. If some ethanol, ethyl acetate or acetic acid were present in the sample when water was added, the glyceryl triacetate partition between ether and water could be altered. It is difficult to control these conditions.

Some unnoticed variation in procedure could lead to appre

ciable losses. The lithium and aluminum acetate formed 6k

in situ is a good catalyst for quantitative acetylation of fatty alcohols and glycerol.

Hence the procedures described above.were modified,

eliminating refluxing with ethanol and an aqueous wash which were used previously to remove excess acetic anhydride

and acetic acid. Glyceryl triacetate and fatty alcohol

acetates were isolated directly as the residue after evap

oration of a xylene solution under high vacuum. Consistent

and theoretical ester-to-glycerol ratios were obtained.

Table 5 shows that prolonged vacuum evaporation of

a standard mixture of 8:0 to 20:0 alcohol acetates at a

water bath temperature of 55° resulted in the loss of about

TO per cent of the octyl acetate, but 10:0 to 20:0 alcohol

acetates were recovered quantitatively.

C . The use of internal standards

Internal standards are necessary for the estimation

of absolute amounts of fatty acids and glycerol in lipid

s amples.

There is no need to know the exact amount of unknown

glyceride as it is possible to calculate this from the

known amount of internal standard. In some studies aliquots

of a standard mixture were injected and a graph was prepared 65

for the calculation of unknown mixtures. It is difficult to inject exact aliquots. When the internal standard is injected with the sample, this difficulty is overcome.

Equations for the calculation of an unknown lipid composition with an internal standard are given in Section E*

) The results obtained by this procedure were consistent and compared with the theoretical data within the experimental error of the procedure (Tables 6 and T)• When this method was applied to a triglyceride obtained from egg yolk (Table 8) the results were similar to data for methyl esters of egg yolk triglycerides (17U). Minor peaks with longer retention times, presumably polyunsaturated fatty alcohol acetates, were observed in the study; however, they were not identi fied or included in the calculation. The total ester content obtained with GLG- agreed with the total ester content estimated by a colorimetric method.

D . Saponification-acetylation

Saponification-acetylation is a modification of the method for the analysis of glycerol in glycerides which contain eicosanoic acid or other fatty acids which are also used as internal standards. Saponification-acetylation may also be used for the analysis of glycerol in 66

glycerides that contain fatty acids" which are converted to alcohol acetates which elute with glyceryl triacetate.

This method yields glyceryl triacetate and long chain fatty acids. The latter remain on the GLC column. Typical mean recoveries of glyceryl triacetate were from 97 to

105 per cent (Table 9). A large excess of acetic anhy dride was necessary to remove water and acetylate glycerol.

Sodium acetate is a good catalyst for acetylation. The results were similar to hydrogenolysis-acetylation.

E . Ac etolysis

Karrer and Jucker (173) state that hydrogenolysis of lecithin with LAH resulted in quantitative yield of fatty alcohols and glycerol. When the hydrogenolysis-acetylation procedure was used for the dephosphorylation of phospho lipids in this study, phosphate was not cleaved quantita tively. When cephalin (GBI) was subjected to hydrogenolysis- acetylation, glycerol recoveries from U5 to 65 per cent were obtained (Methods lH and 17, Table lU). When hydrogenolysis was followed by acetolysis of cephalin the glycerol yield was 88 per cent. Bevan et al. (157) and Hoefnagel et al.

(158) employed acetic acid and acetic anhydride (^:1 , v/v) for acetolysis of phospholipids. The recovery of glycerol when acetolysis-saponification-acetylation was applied either

to cephalin (GBI) or egg yolk cephalin was from 93 to 1(A per cent (Table 11). Acetolysis followed by hydrogenolysis-

acetylation on cephalin (GBI) resulted in good recovery of both glycerol and alcohol acetates (Table 12). However, a very poor recovery of glycerol was obtained when either

commercial or egg yolk lecithin was subjected to this ace

tolysis method followed by saponification-acetylation

(Table 13). Additional work was done in order to see whether additional acetolysis reagent and increased reflux

time could result in complete recovery of glycer'ol. The

results in Table 13 show that a 73 per cent recovery of

glycerol from lecithin was possible.

F . Modified acetolysis

Because acetic acid is not a strong organic acid, it

appeared that trifluoroacetic acid could be substituted for

acetic acid. The advantage of trifluoroacetic acid is that

it boils around 70°, hence can be removed with a water-pump. .

The mechanism for dephosphorylation of phospholipids can

be postulated from early work on the acid catalyzed acetyl-

ation of alkoxy groups bonded to the silicon atom (175,176). 68

Ac20 A c 20H.

<£> Ac 2OH A c® + AcOH

PC “ Pho sphat idy-1 choline

AcOH = Acetic acid

Ac20 = Acetic anhydride

E OH = Diglyceride

Trifluoroacetic acid exists in the presence of acetic

anhydride as a mixed anhydride (ITT). Modified acetolysis

derivatives of dipalmitin, monopalmitin, and glycerol were

found to have Rp values similar to the monoacety1 ,^diacetyl

and triacetyl derivatives of dipalmitin, monopalmitin and

glycerol respectively. 69

Modified acetolysis with trifluoroacetic acid and acetic anhydride (1 :U , v/v) on egg yolk and commercial lecithins yielded a good recovery of glycerol (Table 15).

The validity of the method for glycerol estimation in cephalin was carried out and a good recovery of glycerol was obtained (Table IT).

Tables 19 and 20 show the recovery of glycerol by acetolysis and modified acetolysis on egg yolk and commer cial cephalins, and on egg yolk and commercial lecithins respectively.

G . Cardiolipin structure

Pangborn first isolated cardiolipin in 19^2 (178).

She analyzed cardiolipin in 19^7 and obtained a phosphorus: glycerol:ester molar ratio of 1:1.3:2 (179). A structure was proposed in which four glycerol residues were connected by three phosphate groups in diester linkages. The re maining hydroxyl groups were esterified with fatty acids.

In 1952, McKibbin and Taylor (180) questioned this structure. TO

They found a molar ratio for phosphorus:glycerol:ester of

2:3:3. Faure and Morelec-Coulon (l8l) obtained a molar ratio for phosphorus:glycerol:ester of 2:3:4. This work was later confirmed by MacFarlane and Gray (182). Recently

Marinetti (183) obtained a molar ratio for phosphorus: glycerol:ester of 1:1.5:2 which agreed with MacFarlane and Gray. Chang and Sweeley (18U) isolated the deacylated polyglycerol phosphate from cardiolipin and supported

Pangborn's .structure on the basis of the acidity of this compound.

Table 19.— Comparison of glycerol recovery from cephalin estimated by both methods.

% Recovery

Sample TFA:AcgO Ac OH;Ac 20

Cephalin (egg) 92. 4 93.1 91.2 95.4 “ — 95.0 94.2 Cephalin (GBI) 97.5 98.5 100.3 98.1 96.3 100.9 98.8 96.6 97.5 99.9 97. 5 101.8 101.4 101.7

TFA trifluoroacetic acid AcOH acetic acid AC2O acetic anhydride 71

Table 20.— Comparison of glycerol recovery from lecithin estimated by both methods-.

% Recove'ry

Sample TFA:Ac pO AcOH:AC2O

Lecithin (egg) 90 . h 73.la 0 99. U 63.2a 95.3 U3.9 98.6 hk.2 90.7 50.9 91. 8 k9.2 99 .1 50.3 101.8 U8.7 9^.5 ^9.5 3^.6 Lecithin (GBI) 99 .3 66. 7a 93.0 63. 8a 99 .1 i+6.3 92.5 1+6.3

a Additional acetolysis reagent and increased reflux time. 72

These structures are written below:

0 T CH2OCOR CH2-0-P-0-CH2 CHgOCORg

CHOCOR2 3 CHOCOR^ CHOCORCHOCOR Pangborn (179) I ^ 0 and CH2-O-P-O Chang and Sweeley OH (l8i+)

0 -I* ch2ocor1 ch2-o-p-o-ch2 chocor2 choh CHOCOR3

CHgOCORl* OH MacFarlane and Gray (182) and Marinetti (183)

LeCocq and Ballou (185) recently established the overall absolute configuration of the cardiolipin molecule. They assumed the structure proposed by MacFarlane and Gray in their study. It is interesting that LeCocq. and Ballou (185) did not even determine ester-to-phosphorus molar ratios.

The molar phosphorus:glycerol:ester ratios obtained in this study (Table 18) are 1:1.2:2.2. The glycerol-to- phosphorus molar ratios obtained were similar to that of

Pangborn; but ester-to-phosphorus ratios were slightly higher. Phosphatidic acid has a molar ratio of phosphorus: glycerol:ester 1:1:2 and migrates along with cardiolipin in the solvent systems used for its identification. It would be difficult to assign a definite structure for cardiolipin from these observations. However, the ratio could result from a mixture containing a completely acylated cardiolipin with the MacFarlane and Gray struc ture (1:1.5:2.5) and phosphatidic acid (1:1:2). Equal molar 'quantities of these compounds would have a molar ratio of 1:1.25:2.2 5. Further work on the isolation of cardiolipins is necessary before a structure is verified.

H . Effect of modified acetolysis on fatty acid components

The trifluoroacetic acid and acetic anhydride used

'for the acetolysis of phospholipids did not affect the glycerol content. When tripalmitin was refluxed with a mixture of trifluoroacetic acid and acetic anhydride, fol lowed by hydrogenolysis-acetylation, an ester-to-glycerol ratio of three was obtained. When this procedure was applied to egg yolk lecithin almost all the unsaturated fatty acids were lost. Meade (186) observed that acetic acid was added to double bonds of the fatty acids in gly cerides yielding acetoxy saturated glycerides. Renkonen

(l6l) did not observe this type of addition on acetolysis. Meade employed a perchloric acid catalyst in his acetolysis mixture. This reaction may occur when stronger acids such as trifluoroacetic acid are used for modified acetolysis. VI. SUMMARY

In the present study gas-liq_uid chromatography (GLC) was used for the analysis of glycerol and fatty acids in lipids. Several preliminary studies were undertaken. Com mercial methyl ester preparations were examined and the presence of impurities noted. The relative molar response

(RMR) factors for triacetin, eicosanyl acetate and doco-

senyl acetate were determined. Published RMR values could not be used when commercial ester preparations were used

as GLC standards. The RMR factors in a homologous series of fatty alcohol acetates or methyl esters were found to be the same with different GLC instruments, whereas the

RMR for other compounds varied with the instrument employed.

The ester-to-glycerol ratios of neutral glycerides were determined by the method of riorrocks and Cornwell (l).

An improvement of this method was obtained by acetylating

the hydrogenolysis sample with acetic anhydride in xylene.

Losses due to vacuum evaporation were found with octyl

75 acetate. Wo losses were found with higher homologous com pounds. Eicosanyl acetate was prepared from commercial methyl eicosanoate and used as the internal standard.

Different internal standards were compared for the quan titative estimation of lipid components.

A saponification-acetylation procedure was developed for the analysis of glycerol alone in glycerides which contained the same fatty acid as the internal standard.

This procedure was also used for the analysis of glycerides that contained fatty acids whose alcohol acetates eluted

* w.it'n triacetin.

Egg yolk triglycerides and phospholipids were iso- G lated and purified by column chromatography and their purity was determined by thin layer chromatography.

Hydrogenolysis of phospholipids was not successful as a dephosphorylating procedure. Acetolysis with a mix ture of acetic acid and acetic anhydride (U:l, v/v) followed by saponification-acetylation resulted in a good recovery of glycerol from cephalin. The acetolysis method was not applicable to lecithin. Low glycerol recoveries were obtained. A modified acetolysis using trifluoroacetic acid and acetic anhydride (l:U, v/v) was developed for the quantitative recovery fo glycerol from egg yolk and com mercial lecithins.- This method was found suitable for the estimation of glycerol in cephalin.

^ Attempts to purify and isolate different components in commercial cardiolipin were not successful. The glycerol content of cardiolipin was estimated by the modi fied acetolysis-saponification-acetylation procedure.

Molar phosphorus:glycerol:ester ratios of 1:1.2:2.2 were obtained.

Modified acetolysis derivatives of dipalmitin, monopalmitin and glycerol were found to have lower Rf values

than triglycerides. The Rf values were similar to acetyl derivatives of dipalmitin, monopalmitin 'and glycerol

respectively. The trifluoroacetic acid and acetrc

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