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THE PHYSICAL PROPERTIES OF

TRIACYLGLYCEROLS

IN RELATION TO HILKFAT

A thesis presented for the degree of

Doc t or of Philosophy

in Chemistry

at Massey University

by

Robert Norris

1977

ii

ABSTRACT

Enantiome ric and racemic triacylglyce rols (TGs) repre sentative

of the major structural classe s in milkfat we re synthe sise d and the ir

polymorphism was characte rise d by diffe rential scanning calorime try (DSC)

and infrare d (IR) spe ctroscopy.

With butyric (B) , ole ic (0), palmitic (P) and stearic (S) acids

as starting mate rials, 22 racemic TGs were prepare d. The main TG classe s

were : 1) palmitoyl-stearoyl TGs (e . g. PSS ) , 2) 1-butyryl TGs (BPS) ,

3) 1-ole oyl TGs (OPS) , 4) 2-ole oyl TGs ( POS ), 5) 1-butyryl-2-oleoyl

TGs (BOS) and 6) 1, 2-dioleoyl TGs (OOS) . Three TGs containing e la idic

acid (E) were also synthe sise d (BES, ESS and SES) . In a ddition, enan­

tiome rs of three of the racem ic TGs be longing to the 1-butyryl and

1-ole oyl classe s we re prepare d (�-SSB, -SSO an d -PPO) .

The polymorphic forms of each TG we re classifie d as a, �· or�

by comparison of the ir IR spe ctra with the spe ctra of the polymorphic

forms of monoacid TGs. Solvent crystallise d forms we re also chara cte rise d

by X-ray powde r diffraction . Ne lting poin ts of all polymorphs and heats

of fusion of the least stable (a) and most stable forms we re de te rmine d

by DSC . Howe ve r, the de termination of the p olymorphic assignment and

heat of fusion of the inte rme diate forms was often unce rtain be cause of

the difficulty in obtaining a pure phase.

The principal findings we re :-

1) C orre spondin g enantiome ric and racemic TGs e xhibite d similar poly­

m orphic be haviour e xcept that the a forms of the enan tiome rs trans­

forme d more rapidly than those of the ir racemate s.

2) For TGs in which one fatty acid was ve ry diffe rent from the othe r two

(e . g. BSS, OOS) , the position of the unusual acid de te rmine d the chain

pack ing of the stable form . If the acid was in a primary position,

the TG was �'-stable (e . g. BSS, OSS, OOS) , while if it was in the

se condary position, the TG wa s �-stable (e . g. SOS, SBS) .

3) The re we re close paralle ls be tween the stable forms of corre sponding

butyryl and ole oyl TGs (e . g. BSS, OSS; SBS, SOS; BOS, OOS) , although

in othe r re spe cts the ir p olymorph ism ha d little in common . The stable

forms of BSP and OSP showe d anomalous the rmal, diffra ction and spe ctral

data compare d with the rema ining 1-butyryl and 1-ole oyl TGs.

4) The re sults obtaine d for the 1-ole oyl, 2-ole oyl and 1, 2-diole oyl TGs

we re in gene ral a greement with earlie r reports, a lthough some

diffe rences we re note d in the transforma tion of OSP, OPS, SOS and POP.

Furthermore, previously undetected tran sitions were observed for a ll

the oleoyl TGs, a lthough these were minor. A new polymorph of OPP

was a lso characterised. With the exception of POS, all monooleoyl

TGs showed anomalous crysta llisation beha viour.

iii

5) The results for the polymorphism of the palmitoyl-stearoyl and elaidoyl­

stearoyl TGs were a lso in accord with p revious reports. The presence

of a � ·2 form was confirmed for all TGs except SPS and PSP. The heats

of fusion of the � forms of SPS an d PSP were comparable with those of

their unsymmetrica l counterparts, PSS an d PPS, but the heats of fusion

of their stable� · forms were much higher than those of� · SSS, PSS,

PPS and PPP.

These findings are discussed in rela tion to the prin ciples of

close packing and their relevan ce to the phase behaviour of milk fat.

AC KNOWLEDGE1ENTS

I would particularly like to thank my two supe rvisors, Dr J.C .

Hawke and Prof. G. N. Malcolm, for the opportunity to work at Massey

Unive rsity. I am also inde bte d to the Ne w Zealand Dairy Re search

Institute for financial support during the course of this work.

In the preparation of the the sis, thanks are due to Mrs . A.

Stre tton for drawing m ost of the figure s, to M iss V.L. Be the ll and

Dr D . A. D . Parry for proof-reading and to Mrs J. R. Parry for typ ing.

I am similarly obligate d to Me ssrs P. T. Tuttiet t and D. S. M unro, who

wrote the compute r p rograms for graphical pre sentation of the thermal

analysis data. Dr M .W. Taylor made helpful sugge stions of a gene ral

nature .

iv

Finally, I am grate ful to m y wife an d family for the ir continue d

inte re st and encouragement ove r what must have seeme d an in te rminable

pe riod.

C hapte r 1.

1. 1.

1. 1. 1

1. 1. 2

1. 1. 3

1. 1. 4

1. 1. 5

1.1. 6

1. 2

1. 2. 1

1. 2. 2

1. 3

1. 3. 1

1. 4

1. 4. 1

C hapter 2.

A

2. 1

2. 1. 1

2. 1. 2

2. 1. 3

2. 1. 4

2. 2

2. 2. 1

2.2. 2

2. 2. 3

2. 2. 4

TABLE OF CONTENTS

INTRODUCTION

Polymorphism of Triacylglyce rols

Historical Background

Structural Fe ature s of Lipids in the Solid State

Monoacid Saturate d Triacylglycerols

Unsaturate d Triacylglyce rols

Diacid Saturate d Triacylglycerols

Enantiome ric Triacylglyce rols

Infrare d Spe ctroscopy of Triacylglyce rols in the Solid State

Assignments of C haracte ristic Absorption Bands

Spe ctral Diffe rence s be tween Polymorphic Forms

Me lting and Solidification of Fats

Te chnical Imp ortance of the Phase Behaviour of Fats

Polymorphism of Fats

Solid Miscibility of Fats

He lting and Solidification of Hilkfat and Milkfat Fraction s

Aim of the Pre sent Work

Se le ction of Triacylglyce rols for Synthe sis

HATERIALS AND HE.rHODS

Analytical and Synthe tic Me thods

Mate rials

Solvents

Re age nts

Fatty Acids

Storage of Glyce ride s and Synthe tic Inte rme diate s

Analytical Me thods

Thin-Layer C hrom atography

Column Chromatography

Gas-Liquid C hromatography

Structural Analysis of Triacylglycerols

V

1

1

1

2

5

13

18

21

22

22

24

27

27

27

28

29

33

33

36

36

36

36

36

37

37

37

37

39

40 40

2. 3

2. 3. 1

2. 3. 2

2. 3. 3

2. 3. 4

2. 3. 5

2. 4

2. 4. 1

2. 4. 2

2. 4. 3

B

2. 5

2. 6

2. 6. 1

2. 6. 2

2. 6. 3

2. 7

2. 7. 1

2. 7. 2

2. 7. 3

2. 8

2. 8. 1

2. 8. 2

2. 8. 3

2. 8. 4

2. 8. 5

2. 9

C hapter 3.

3. 1

3. 2

3. 2. 1

Preparation of Racemic Triacylglycerols

Acyl Chlorides

1-Acylglycerols

1,3-Diacylglycerols

1,2-Distearoylglycerol

Triacylglycerols

Preparation of Enantiomeric Triacylglycerols

1,2-Isopropylidene-�-glycerol

1,2-Diacyl-�-glycerols

Triacylglycerols

Physical M ethods

M elting Point Determination

X-Ray Powder Diffraction

Apparatus

Preparation of Samples

Identification of Polymorphs

Infrared Spectroscopy

Apparatus

Investigation of Polymorphism

Identification of Polymorphs

Thermal Analysis

Apparatus

Calibration

Presentation of Data

Investigation of Polymorphism

Identification of Polymorphs

Preparation of� PSP and�· SPS

RESULTS

Structural Analysis of Triacylglycerols

Polymorphism of Racemic Triacylglycerols

Palmitoyl-Stearoyl and Elaidoyl-Stearoyl Triacylglycerols

1-Butyryl-2-0leoyl and 1,2-Dioleoyl Triacylglycerols

1,2-Dibutyryl-3-palmitoylglycerol

vi

40 42

42

44

47

49

50

50

52

53

54

54

54

54

54

54

55

55

55

56

56

56

57

57

59

61

61

62

62

62

62

64

66

C hapte r

3.2.4

3.2.5

3.2.6

3.2.7

3.2.8

4. 4.1

4.1.1

4.1.2

4.1.3

4.1.4

4.1.5

4.1.6

4.1.7

4.1.8

4.2

4.2.1

4.3

2-0le oyl Triacylglycerols

2-Butyryl-1,3-distearoylglyce rol

1-Butyryl Triacylglyce rols

1-Butyryl-2-e laidoyl-3-ste aroylglycerol

1-0le oyl Triacylglycerols

Polymorphism of Enantiome ric Triacylglyce rols

Comparison of the Polymorphism of Corre sponding Enantiome ric an d Race mic Triacylglycerols

DISC USSION

Polymorphism of Race mic Triacylglyce rols

Palmitoyl-Ste aroyl and Elaidoyl-Ste aroyl Triacylglyce rols

1-Butyryl-2-0le oyl and 1,2-Diole oyl Triacylglyce rols

1,2-Dibutyryl-3-palmitoylglyce rol

2-0le oyl Triacylglycerols

2-Butyryl-1,3-distearoylglyce rol

1-Butyryl Triacylglyce rols

1-Butyryl-2-elaidoyl-3-ste aroylglyce rol

1-0le oyl Triacylglyce rols

Polymorphism of Enantiome ric Triacylglyce rols

Comparison of the Polymorphism of Corre sponding Enan tiome ric and Race mic Triacylglyce rols

Con clusion

APPENDIX

The rmal and Spe ctral Data for the Polymorphic Forms of the Individual Triacylglycerols

BIBLIOGRAPHY

vii

67

72

73

75

76

81

81

120

120

120

121

123

124

127

128

132

133

136

136

138

140

19 3

Table

1.1

1.2

1.4

1.6

3.4

3-5

3.6

3-7

3.8

3-9

3.10

3.11

3.12

1

2

3

LIST OF TABLES

C lassification of the Polymorphic Forms of Glyceride s

The Polymorphs of 1-0le oyl and 2-0le oyl Disaturate d Triacylglyce rols

C haracteristic Bands in the Solid State Spe ctra of Triacylglyce rols

viii

8

15

23

Me thylene Waggin g Band Distributions for the Alpha Forms 25 of the Monoacid Triacylglyce rols of Elaidic, Ole ic, Palmitic and Ste aric Acids

C omparison of the Spe ctra of a, � · and� Polymorphs of 26

M on oacid Saturate d Triacylglycerols

Selection of Triacylglyce rols for Synthe sis 34

Positional Analysis of Synthe tic Triacylglycerols 83

Stereospe cific Analysis of �-SSO and �-SSB 83

X-Ray Short Spacin gs of the Solvent C rystallise d Forms of 84

Palmitoyl-Ste aroyl and Elaidoyl-Ste aroyl Triacylglycerols

Me lting Points and He ats of Fusion of the Polymorphic 85 Forms of Palmitoyl-Ste aroyl and Elaidoyl-Stearoyl Triacylglycerols

Me lting Points and He ats of Fusion of the Polymorphic 86

Forms of OOS, OOP, BOS, BOP and BBP.

X-Ray Short Sp acings of the Solvent C rystallise d Forms 87 of SOS, POS, POP and SBS

Me lting Points and He ats of Fusion of the Polymorphic 88

Forms of SOS, POS, POP and SBS

X-Ray Short Spacings of the Solvent Crystallise d Forms of 89 BSS, BSP, BPS, BPP and BES

Me lting Points and He ats of Fusion of the Polymorphic 89 Forms of BSS, BSP, BPS 1 BPP and BES

X-Ray Short Spacings of the Solvent C rystallise d Forms 90 of OSS, OSP, OPS and OPP

Melting Points and He ats of Fusion of the Polymorphic 91 Forms of OSS, OSP, OPS and OPP

X-Ray Short Spacings of the Solvent C rystallise d Forms 92 of C orre spondin g Racemic an d Enantiome ric Triacylglyce rols

Me lting Points and He ats of Fusion of the Polymorphic 92 Forms of C orre spondin g Racemic and Enantiome ric Triacylglyce rols

APPENDIX

Me lting Points of the Stable Forms of Triacylglycerole

Some Common Bands in the Infrare d Spe ctra of Re late d Stable Forms

Some Common Bands in the Infrare d Spe ctra of Re late d [3 and � 1 Form s

141

142

143

ix

LIST OF FIGURES

Figure

1-1 Triclinic parallel and orthorhombic p erp endicular 3 subcells

1-2 The main polym orphic forms of monoacid triacylglycerols 6

1-3 Melting points of monoacid triacylglycerols 9

1-4 Molecular arrangement of trilaurin, b-axis projection 10

1-5 Triacylglycerols in the liquid state, p roposed lam ellar 12 structure

1-6 Triple chain structure of the � stable form of 2-oleoyl- 16

distearoylglycerol

1�7 Layer stacking of methyl end groups in the � forms of 20 C /C 2

triacylglycerols showing the two terrace structures n n±

1-8 Triacylglycerols of milkfat 31

2-1 Scheme for the p reparation of oleic acid 38

2-2

2-3

2-4

2-5

2-6

3-1

3-2

3-3

3-4

3-5

3-6

3-7

Gen eral schem e for the synthesis of racemic triacylglycerols 41

Synthesis of racemic triacylglycerols 43

Synthesis of racemic 1,2-diacylglycerol 48

Synthesis of enantiomeric triacylglycerols 51

C onventions for the presentation of thermal analysis data 58

X-Ray diffraction patterns of the � stable forms of p almitoyl-stearoyl and elaidoyl-stearoyl triacylglycerols

X-Ray diffraction p atterns of the� and�· forms of PSP and SPS

Melting thermograms of � SSS, SPS, PPP and SES

Melting thermograms of � PSP, PSS, PPS and ESS

Melting therm ograms of �· sss, ppp and SES

Melting thermograms of �I PSS, PPS and ESS

Melting thermograms of the solvent crystallised �I and � forms of SPS and PSP

93

94

9 5

96

9 7

9 8

9 9

3-8 IR spectra of the �1 stable forms of OOS, OOP, BOS and BOP 100

3-9 Melting therm ograms of � OOS, OOP, BOS and BOP 101

3-10 X-Ray diffraction p atterns of the � stable forms of SOS, 102 POS , POP and SBS

3-11 IR spectra of the � stable forms of SOS, POS, POP, SBS, 103 SES and ESS

3-12 IR spectra of comparative �2 and�� polymorphs of SOS, 104

POS and POP

3-13

3-14

3-15

Melting thermograms of � SOS, POS and POP

Melting thermograms of the intermediate forms of SOS, POS and POP

Polymorphism of SOS, POS and POP

105

106

107

Figure

3-16

3-17

3-18

3-19

3-20

3-21

3-22

3-23

3-24

3-25

3-26

3-27

4-1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Page

IR spectra of the�· stable forms of OXY and BXY 108

triacylglycerols

X-Ray diffraction patterns of the�' stable forms of OXY 109 and BXY triacylglycerols

Melting thermograms of a BSS, BSP, BPS and BPP 110

Melting thermograms of the intermediate forms of BSS, BSP, 111 BPS and BPP

Melting thermograms of a OSS, OSP, OPS and OPP showing 112 the presence of the a2 form at high heating rates

Melting thermograms of a OSS, OSP, OPS and OPP recorded 113 after tempering at the a2 peak temperature

Melting thermograms of a OSS, OSP, OPS and OPP recorded 114

at low heating rates

Melting thermograms of the intermediate forms of OSS, OSP, 115 OPS and OPP

Polymorphism of OSS, OSP, OPS and OPP 116

IR spectra of the�· stable forms of racemic and 117 enantiomeric triacylglycerols

X-Ray diffraction patterns of the stable forms of corres- 118

ponding racemic and enantiomeric triacylglycerols

Helting thermograms of the a forms of rac- and sn-SSB and 119 SSO showing the more rapid transformatiOn of the-antipodes

Comparison of glycerol conformations in "tuning-fork" and 130 "chair" structures

Thermal behaviour

Thermal behaviour Thermal behaviour

Thermal behaviour

Thermal behaviour

Thermal behaviour

Thermal behaviour

IR spectra of SES

Thermal behaviour

IR spectra of ESS

Thermal behaviour

IR spectra of OOS Thermal behaviour

IR spectra of OOP

Thermal behaviour

IR spectra of BOS

Thermal behavi·our

of sss of PSS

of PPS

of ppp of SPS

of PSP

of SES

of ESS

of OOS

of OOP

of BOS

of BOP

APPENDIX

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

X

xi

Figure Page

18 IR spectra of BOP 161

19 Thermal behaviour of BBP 162

20 IR spectra of BBP 163

21 Thermal behaviour of SOS 164

22 Crystallisation of SOS from the melt 165

23 IR spectra of SOS 166

24 Thermal behaviour of POS 167

25 IR spectra. of POS 168

26 Thermal behaviour of POP 169

27 IR spectra of POP 170

28 Thermal behaviour of SBS 171

29 IR spectra of SBS 172

30 Thermal behaviour of sn-SSB 173

31 Thermal behaviour of BSS 174

32 IR spectra of BSS 175

33 Thermal behaviour of BSP 176

34 IR spectra of BSP 177

35 Thermal behaviour of BPS 178

36 Thermal behaviour of BPP 179

37 IR spectra of BPP 180

38 Thermal behaviour of BES 181

39 IR spectra of BES 182

40 Thermal behaviour of sn-SSO 183

41 Thermal behaviour of OSS 184

42 IR spectra of OSS 185

43 Thermal behaviour of OSP 186

44 IR spectra of OSP 187

45 Thermal behaviour of OPS 188

46 IR spectra of OPS 189

47 Thermal behaviour of sn-PPO 190

48 Thermal behaviour of OPP 191

49 IR spectra of OPP 192

xii

NOMENCLATURE

In general , the recommendat ions of the IUPAC-IUB Commission on

Biochemical Nomenclature ( 1 976 ; Lipids � ( 1977 ) , 455 ) are followed for

the nomenclature of lipids containing glycerol . However , the abbreviations

MG , DG and TG are used for mono- , di- and triacylglycerol respectively .

For the sake of brevity , TGs are specified by a three let t er c ode in

which the lett ers indicat e the acyl group ( B , butyryl ; E, elaidoyl ;

L , lauroyl; O , oleoyl ; P , palmitoyl and S , st earoyl ) and the order o f

t he symbols represents t he position of the acyl group in the TG molecule.

Enantiomeric TGs are dist inguished by the prefix ·�-', while TGs without

a prefix are understood t o be racemic . Thus �-PPO represent s

1 , 2-dipalmitoyl-3-oleoyl-�-glycerol and POS ( or SOP ) represents

�-1 -palmitoyl-2-oleoyl-3-st earoylglycerol .

The polymorphic forms o f TGs are designat ed according t o

Larsson's modificat ion ( Larsson , 1 966a ) on Lut t on's syst em o f classificat ion

( Lutton , 1 950) . Where necessary , the chain mult iplicity of a part icular

form is indicat ed by a suffix. Thus the t erm · �-2 1 represents a � form

with a chain multiplic ity of 2.