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.) 4 # * * * * 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 AND STATEMENT OF THE PROBLEM . 1 II HISTORICAL REVIEW ................................... 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 glycerol derivatives. .... 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

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