NOVEL APPLICATIONS OF ELEVATED TEMPERATURE HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY: ANALYSES OF

TRIAZOLE FUNGICIDES AND VITAMIN E ISOMERS

SEE HONG HENG

UNIVERSITI TEKNOLOGI MALAYSIA

NOVEL APPLICATIONS OF ELEVATED TEMPERATURE HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY: ANALYSES OF

TRIAZOLE FUNGICIDES AND VITAMIN E ISOMERS

SEE HONG HENG

A thesis submitted in fulfilment of the

requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia

APRIL 2005

iii

Specially dedicated to my beloved mother, sister and brother.

iv

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisor, Professor Dr.

Mohd Marsin Sanagi, for his constant ideas and enthusiasm. His excellent scientific

guidance, infinite patience and unconditional help have enabled me to approach work

positively.

I also would like to thank my co-supervisors, Assoc. Prof. Dr. Wan Aini Wan

Ibrahim and Assoc. Prof. Dr. Ahmedy Abu Naim for their helpful comments and

advices. Thanks to the laboratory assistants of the Department of Chemistry, U.T.M.,

particulary to En. A. Kadir A. Rahman for the technical assistance.

My sincerest gratitude also goes to my post-graduate friends and all the staff

in chromatography team. I would like to take this opportunity to thank everyone who

has contributed whether directly or indirectly to this project.

I would also like to thank the Ministry of Science, Technology and

Innovation (MOSTI) for the financial support through the IRPA program and a

studentship for myself.

To my family, I cannot thank you enough for your support and faith in me.

v

ABSTRACT

Control of column temperature has become increasingly accepted as a

separation parameter in high performance liquid chromatography (HPLC). In this

study, high-temperature reversed-phase (RP) HPLC using water-rich and superheated

water eluents is evaluated as a new approach for the separation of selected triazole

fungicides. Using a polybutadiene-coated zirconia column at temperatures of 100°C

to 150°C, clear separations were achieved when 100% purified water was utilized as

organic-free eluent. Excellent limits of detection down to pg level were obtained

under optimum conditions. Nevertheless, poorer separation efficiency was observed

when the triazole fungicides were separated on carbon-clad zirconia column using

water-rich eluents. Novel separation of eight vitamin E isomers (α-, β-, γ-, δ-

tocopherol, and α-, β-, γ-, δ-tocotrienol) and α-tocopherol acetate on both normal-

phase (NP) and reversed-phase (RP) HPLC were also examined. All the isomers

were successfully separated using NP-HPLC on amino and silica columns. By

simply increasing the temperature for silica column, excellent separation efficiencies

and shorter analysis times were achieved. Seven vitamin E isomers were successfully

separated using RP-HPLC at high temperatures. Both developed separation methods

are rapid, showed excellent repeatability, and suitable to be used as a quantitative

method in analyzing vitamin E isomers. Pressurized liquid extraction (PLE) along

with elevated temperature NP-HPLC is evaluated as a new approach for the

determination of β-carotene and vitamin E isomers in residue oil obtained from palm

pressed fiber (PPF). The new developed method demonstrated an outstanding

performance with excellent efficiency in terms of total extraction time, total solvent

usage, total carotene and vitamin E isomers contents as well as the exceptional

method repeatability.

vi

ABSTRAK

Pengawalan suhu turus telah semakin diterima sebagai parameter pemisahan

dalam kromatografi cecair prestasi tinggi (HPLC). Dalam kajian ini, HPLC fasa

terbalik bersuhu tinggi dengan turus zirkonia tersalut polibutadiena menggunakan

pengelusi kaya-air dan air lampau panas telah dikaji sebagai suatu pendekatan baru

bagi pemisahan fungisid triazola terpilih. Dengan menggunakan turus zirkonia

tersalut polibutadiena pada suhu 100°C hingga 150°C, pemisahan lengkap telah

tercapai apabila menggunakan 100% air tulen sebagai pengelusi bebas organik. Had

pengesanan yang rendah sehingga tahap pg telah tercapai pada keadaan optimum.

Walau bagaimanapun, kecekapan pemisahan yang lemah telah dicerap apabila racun

rumpai fungisid triazola dipisahkan dengan turus zirkonia tersalut karbon

menggunakan pengelusi kaya-air. Pemisahan baru lapan jenis isomer vitamin E (α-,

β-, γ-, δ-tokoferol, and α-, β-, γ-, δ-tokotrienol) and α-tokoferol asetat dengan HPLC

fasa normal (NP) dan fasa terbalik (RP) juga telah dikaji. Semua isomer telah berjaya

dipisahkan menggunakan NP-HPLC dengan turus amino dan silika. Dengan hanya

meningkatkan suhu bagi turus slika, kecekapan pemisahan yang baik dan masa

analisis yang lebih pendek telah tercapai. Tujuh jenis isomer vitamin E telah berjaya

dipisahkan menggunakan RP-HPLC pada suhu tinggi. Kedua-dua kaedah pemisahan

yang dibangunkan adalah cepat, menunjukkan kebolehulangan yang tinggi, dan

sesuai digunakan sebagai kaedah kuantitatif dalam menganalisis isomer vitamin E.

pengekstrakan cecair bertekanan (PLE) bersama dengan NP-HPLC pada suhu

tertingkat telah dikaji sebagai pendekatan baru dalam menentukan β-karotin dan

isomer vitamin E dalam sisa minyak diperoleh daripada sabut kelapa sawit (PPF).

Kaedah baru yang dibangunkan menunjukkan prestasi cemerlang dengan kecekapan

yang baik dari segi masa pengekstrakan, jumlah penggunaan pelarut, jumlah

kandungan karotin dan isomer vitamin E dan juga kebolehulangan kaedah yang

tinggi.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE PAGE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENTS iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xiii

LIST OF FIGURES xv

LIST OF SYMBOLS/ABBREVIATIONS/

NOTATION/TERMINOLOGY xx

LIST OF APPENDICES xxii

1 INTRODUCTION 1

1.1 Preamble 1

1.2 Principles of High Temperature Operation in

Reversed-Phase High Performance Liquid

Chromatography (RP-HPLC) 1

1.3 Instrumental Consideration and Performance in

High Temperature RP-HPLC System 4

1.4 Water-Rich and Superheated Water Eluents on

High Temperature RP-HPLC 6

1.5 High Thermal Stability Stationary Phase in the

RP-HPLC 8

1.5.1 Zirconia based stationary phase 10

viii

1.5.2 Types and classification of

zirconia based stationary phases 11

1.5.2.1 Polybutadiene-coated zirconia

stationary phase 13

1.5.2.2 Carbon-clad zirconia stationary

phase 15

1.6 Influence of Narrow-Bore Column in HPLC

Separation 17

1.7 Classification of Triazole Fungicides 18

1.7.1 Chromatographic techniques in

analyzing triazole fungicides 18

1.8 Analysis of Tocopherol and Tocotrienol Isomers 19

1.8.1 Chromatographic techniques in

analyzing tocopherols and tocotrienols. 20

1.8.1.1 Normal-phase HPLC 21

1.8.1.2 Reversed-phase HPLC 22

1.9 Tocopherols and Tocotrienols in Palm Oil and

Palm Pressed-Fiber (PPF) 22

1.9.1 Extraction of residue oil obtained from

PPF 23

1..9.1.1 Soxhlet extraction 24

1.9.1.2 Pressurized liquid extraction 24

1.10 Problems Encountered in HPLC Technique 25

1.11 Objectives and Scope of Work 26

1.12 Division of Chapters in Thesis 26

2 EXPERIMENTAL 27

2.1 Introduction 27

2.2 Reagents 27

2.3 Chromatographic Conditions 29

2.4 System Conditioning Procedure 31

ix

2.5 Procedures for High Temperature RP-HPLC

of Triazole Fungicides On Zirconia Based

Stationary Phase 31

2.5.1 Separation of triazole fungicides by high

temperature RP-HPLC on polybutadiene-

coated zirconia stationary phase 31

2.5.2 Separation of triazole fungicides by high

temperature RP-HPLC on carbon-clad

zirconia stationary phase 32

2.6 Enhanced Separation of Tocopherols and

Tocotrienols By NP and RP-HPLC at Elevated

Temperature 33

2.6.1 Separation of tocopherols and

tocotrienols by high-temperature

RP-HPLC on PBD-coated zirconia

column 33

2.6.2 NP-HPLC of tocopherol and

tocotrienol isomers on amino and silica

columns at ambient temperature 33

2.6.3 NP-HPLC of tocopherol and

tocotrienol isomers on silica column at

elevated temperature 34

2.7 Determination of Carotene, Tocopherols and

Tocotrienols in Palm Pressed-Fiber 35

2.7.1 Palm pressed fiber samples 35

2.7.2 Extraction of residue oil from palm

pressed fiber 35

2.7.2.1 Soxhlet extraction 35

2.7.2.2 Pressurized liquid extraction 35

2.7.3 Determination of tocopherols and

tocotrienols content in residue oil 36

2.7.4 Determination of carotene content 37

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3 HIGH TEMPERATURE RP-HPLC OF TRIAZOLE

FUNGICIDES ON ZIRCONIA BASED

STATIONARY PHASE 38

3.1 Introduction 38

3.2 Separation of Triazole Fungicides on a

Polybutadiene-Coated Zirconia Stationary Phase 38

3.2.1 Elution behavior on a PBD-coated

zirconia column 38

3.2.2 Selectivity and resolution on a PBD-

coated zirconia column 42

3.2.3 Influence of temperature on retention

factor on a PBD-coated zirconia column 45

3.2.4 Influence of temperature on column

efficiency on a PBD-coated zirconia

column 46

3.2.5 Influence of temperature on PBD-coated

zirconia column separation mechanism 48

3.2.6 Limit of detection 51

3.3 Separation of Triazole Fungicides on a Carbon-

Clad Zirconia Stationary Phase 53

3.3.1 Elution behaviour of triazole fungicides

on a carbon-clad zirconia column 53

3.3.2 Selectivity and resolution on carbon-

clad zirconia column 57

3.3.3 Influence of temperature on retention

factor 56

3.3.4 Influence of temperature on column

efficiency 60

3.3.5 Influence of temperature on separation

mechanism 61

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4 ENHANCED SEPARATION OF TOCOPHEROLS

AND TOCOTRIENOLS BY HPLC AT ELEVATED

TEMPERATURE 64

4.1 Introduction 64

4.2 Separation of Tocopherols and Tocotrienols by

High-Temperature RP-HPLC on PBD-Coated

Zirconia Column 64

4.2.1 Influence of column temperature on

retention factor 68

4.2.2 Influence of column temperature on

separation resolution 70

4.2.3 Influence of column temperature on

column efficiency 71

4.2.4 Influence of column temperature on 72

separation mechanism

4.3 Separation of Tocopherols and Tocotrienols

by NP-HPLC on Amino and Silica Columns

at Ambient Temperature 75

4.3.1 Elution behavior in NP-HPLC 76

4.3.2 Optimization study of normal phase

system 76

4.3.2.1 Amino-bonded silica stationary

phase 76

4.3.2.2 silica based stationary phase 81

4.4 Separation of Tocopherols and Tocotrienols

by NP-HPLC on Silica Columns at Elevated

Temperature 82

4.4.1 Separation repeatability, reproducibity

and linearity test 85

4.4.2 Limits of detection 86

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5 DETERMINATION OF CAROTENE,

TOCOPHEROLS AND TOCOTRIENOLS IN

PALM PRESSED-FIBER 89

5.1 Introduction 89

5.2 Enhanced Separation of Tocopherols and

Tocotrienols by NP-HPLC at Elevated

Temperature 89

5.3 Determination of Tocopherols and Tocotrienols

in Residue Oil from Palm Pressed Fiber 91

5.3.1 Soxhlet extraction 91

5.3.2 Pressurized liquid extraction 93

5.3.2.1 Optimization and validation of

the PLE extraction technique 93

5.3.2.2 Choice of the sample amount 93

5.3.2.3 Choice of the extraction

temperature 94

5.3.2.4 Choice of the extraction pressure 95

5.4 Quantification of Tocopherols and Tocotrienols

using Optimized PLE Method 97

5.5 Comparison of Soxhlet Extraction with PLE

Extraction 97

6 CONCLUSIONS AND FUTURE DIRECTIONS 101

6.1 Conclusions 101

6.2 Future Directions 103

REFERENCES 105

Appendix A 115

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LIST OF TABLES

TABLE NO. TITLE PAGE

1.1 General characteristics of the four reversed-phase LC test columns 9

1.2 HPLC phases based on porous zirconia 13 2.1 Properties of four triazole fungicides 28 2.2 Properties of tocopherols and tocotrienols 29 2.3 Separation conditions used for NP-HPLC at ambient

temperature 34 3.1 Column efficiency (N/m) and resolution (Rs) of four

triazole fungicides as a function of temperature using 100% pure water as eluent on PBD-coated zirconia

column 43 3.2 Retention factor of four triazole fungicides as a function

of temperature using different proportion of organic modifier on PBD-coated zirconia column 46

3.3 Enthalpy data for the tebuconazole and hexaconazole at

high column temperatures on PBD-coated zirconia column. 49

3.4 Retention factors of four triazole fungicides on carbon- clad zirconia column as a function of temperature using different proportions of organic modifier 59

3.5 Enthalpy data for the hexaconazole and tebuconazole at

high column temperatures on carbon-clad zirconia column 63

4.1 Retention factors of tocopherols and tocotrienols as a

function of temperature using different proportion of organic modifier on PBD-coated zirconia column 69

4.2 Resolution values as a function of temperature ranging from 80οC to 140οC using different mobile phase compositions 71

xiv

4.3 Enthalpy data for the α-tocopherol acetate at high column temperatures on PBD-coated zirconia column 73 4.4 Resolution (Rs) of adjacent peaks and efficiencies (N/m)

of the amino and silica columns in separating eight natural vitamin E isomers at ambient temperature 80

4.5 Retention factors (k’), theoretical plates/column length (N/m), and resolution (Rs) of eight vitamin E isomers as a function of column temperature using hexane-1,4- dioxane 96.0:4.0 (v/v) as the eluent on silica column 84

4.6 Repeatability and reproducibity of the optimum separation condition based on the retention factors and the peak area respond factor on the same day and different days 85

4.7 Regression analysis of the plot of peak area versus concentration for each isomer 86

5.1 Determination of tocopherols and tocotrienols in residue oil from PPF using Soxhlet extraction 93

5.2 Comparison of results obtained on the determination of residue oil contents using Soxhlet extraction and PLE methods 100

xv

LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 Control of solvent polarity (dielectric constant) by changing temperature (at 50 bar) with pure water compared to methanol- or acetonitrile-water mixtures at 25οC and ambient pressure 7

1.2 Strong Lewis acid sites that can undergo ligand- exchange interactions with Lewis bases 12

1.3 Structure of cross-linked polybutadiene-coated zirconia stationary phase 14

1.4 Structure of carbon-clad zirconia stationary phase 16 1.5 Chemical structures of tocopherols and tocotrienols 20 2.1 Experimental set-up: A, mobile phase reservoir; B,

HPLC pump; C, injection valve; D, column; E, oven; F, cooling system (ice water); G, UV detector; H, integrator; I, backpressure regulator; J, mobile phase pre-heating coils 30

2.2 Schematic diagram of home-made pressurized liquid

extraction (PLE) system: A, HPLC pump; B, three-way switching valve; C1 & C2, static valves; D, pressure gauge; E, oven; F, preheating coil; G, extraction vessel; H, pressurized nitrogen gas; I, collection vial. 36

3.1 Separation of four triazole fungicides on ZirChrom-

PBD column (100 × 2.1 mm I.D.). Chromatographic conditions: mobile phase: acetonitrile-water 10:90 (v/v); flow rate: 0.5 mL/min; temperature: 100οC-120οC; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.1 mg/mL. Peaks: 1 – solvent; 2 – tebuconazole, 3 – hexaconazole, 4 – propiconazole, 5 – difenoconazole 39

xvi

3.2 Separation of four triazole fungicides on ZirChrom- PBD column (100 × 2.1 mm I.D.). Chromatographic conditions: mobile phase: acetonitrile-water 5:95 (v/v); flow rate: 0.5 mL/min; temperature: 120οC-140οC; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.1 mg/mL. Peaks: 1 – solvent; 2 – tebuconazole, 3 – hexaconazole, 4 – propiconazole, 5 – difenoconazole 40

3.3 Separation of four triazole fungicides on ZirChrom-

PBD column (100 × 2.1 mm I.D.). Chromatographic conditions: mobile phase: 100% pure water; flow rate: 0.5 mL/min; temperature: 130°C-150°C; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.1 mg/mL. Peaks: 1 – solvent; 2 – tebuconazole, 3 – hexaconazole, 4 – propiconazole, 5 – difenoconazole 41

3.4 Relationship of separation factor (α) for propiconazole

(a), difenoconazole (b) as a function of mobile phase composition at different column temperature on PBD- coated zirconia column 44

3.5 Variation of column efficiency as a function of

temperature using low proportion of organic modifier on PBD-coated zirconia column. (Solute: tebuconazole) 48

3.6 Van’t Hoff plots for tebuconazole (a) and hexaconazole (b) using different proportions of acetonitrile in the eluent at high column temperatures on PBD-coated zirconia column 50 3.7 Limit of detection of four triazole fungicides separation

on ZirChrom-PBD column (100 × 2.1 mm I.D.). Chromatographic condition: mobile phase: 100% pure water; flow rate: 0.5 mL/min; temperature: 140οC; detection: UV absorbance at 195 nm; injection volume: 1 µL 52

3.8 Separation of four triazole fungicides on ZirChrom- CARB column (100 × 2.1 mm I.D.). Chromatographic condition: mobile phase: acetonitrile-water 10:90 (v/v); flow rate: 0.5 mL/min; temperature: 100οC-120οC; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.1 mg/mL. Peaks: 1 – solvent; 2 – hexaconazole, 3 – propiconazole, 4 – tebuconazole, 5 – difenoconazole 54

xvii

3.9 Separation of four triazole fungicides on ZirChrom- CARB column (100 × 2.1 mm I.D.). Chromatographic condition: mobile phase: acetonitrile-water 5:95 (v/v); flow rate: 0.5 mL/min; temperature: 120οC-140οC; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.1 mg/mL. Peaks: 1 – solvent; 2 – hexaconazole, 3 – propiconazole, 4 – tebuconazole, 5 – difenoconazole 55

3.10 Separation of four triazole fungicides on ZirChrom- CARB column (100 × 2.1 mm I.D.). Chromatographic condition: mobile phase: acetonitrile-water 1:99 (v/v); flow rate: 0.5 mL/min; temperature: 120οC-150οC; detection: UV absorbance at 220 nm; injection volume: 1 µL; solute concentration: 0.05-0.1 mg/mL. Peaks: 1 – solvent; 2 – hexaconazole, 3 – propiconazole, 4 – tebuconazole, 5 – difenoconazole 56

3.11 Relationship of separation factor as a function of

mobile phase composition at different column temperature on carbon-clad zirconia column for propiconazole 59

3.12 Variation of column efficiency as a function of temperature using low proportion of organic modifier on carbon-clad zirconia column for hexaconazole 60

3.13 Van’t Hoff plots for tebuconazole (a) and hexaconazole (b) using different proportions of acetonitrile in the eluent at high column temperature on carbon-clad zirconia column 62 4.1 RP-HPLC separations of a balanced mixture of

tocopherols and tocotrienols on polybutadiene-coated zirconia column at different column temperature. Chromatographic conditions: mobile phase: acetonitrile- water (50:50 v/v); flow rate: 0.4 mL/min. Peaks: 1 – solvent, 2 – δ-tocotrienol; 3 – γ-tocotrienol, 4 – α-tocotrienol, 5 – δ-tocopherol, 6 –γ-tocopherol, 7 - α-tocopherol, 8 -α-tocopherol acetate 65

4.2 RP-HPLC separations of a balanced mixture of tocopherols and tocotrienols on polybutadiene-coated zirconia column at different column temperature. Chromatographic conditions: mobile phase: acetonitrile- water (45:55 v/v); flow rate: 0.4 mL/min. Peaks: 1 – solvent, 2 – δ-tocotrienol; 3 – γ-tocotrienol, 4 – α-tocotrienol, 5 – δ-tocopherol, 6 –γ-tocopherol, 7 - α-tocopherol, 8 -α-tocopherol acetate 66

xviii

4.3 RP-HPLC separations of a balanced mixture of tocopherols and tocotrienols on polybutadiene-coated zirconia column at different column temperature. Chromatographic conditions: mobile phase: acetonitrile- water (40:60 v/v); flow rate: 0.4 mL/min. Peaks: 1 – solvent, 2 – δ-tocotrienol; 3 – γ-tocotrienol, 4 – α-tocotrienol, 5 – δ-tocopherol, 6 –γ-tocopherol, 7 - α-tocopherol, 8 -α-tocopherol acetate 67

4.4 Variation of column efficiency as a function of temperature using different proportions of organic modifier for α-tocopherol acetate 72

4.5 Van’t Hoff plots for α-tocopherol acetate using different proportions of acetonitrile in the eluent at high column temperatures on PBD-coated zirconia column. (a) Linear extrapolation on all temperature studied. (b) Linear extrapolation with a temperature range from 80οC to 120οC. Data points in brackets ( ) represent the point that are excluded from the linearity calculations for the plots. 74 4.6 NP-HPLC separations of a balanced mixture of tocopherols and tocotrienols on amino-bonded silica column at ambient temperature with different mobile phases. Chromatographic conditions: flow rate: 0.2 mL/min. Peaks: 1 – α-tocopherol acetate, 2 – α-tocopherol, 3 – α-tocotrienol, 4 – β-tocopherol, 5 –γ-tocopherol, 6 - β-tocotrienol, 7 - γ-tocotrienol, 8 - δ-tocopherol, 9 - δ-tocotrienol 77 4.7 NP-HPLC separations of a balanced mixture of

tocopherols and tocotrienols on Hypersil silica column at ambient temperature with different mobile phases. Chromatographic conditions: flow rate: 1 mL/min. Peaks: 1 – α-tocopherol acetate, 2 – α-tocopherol, 3 – α-tocotrienol, 4 – β-tocopherol, 5 –γ-tocopherol, 6 - β-tocotrienol, 7 - γ-tocotrienol, 8 - δ-tocopherol, 9 - δ-tocotrienol 78

4.8 NP-HPLC separations of a balanced mixture of

tocopherols and tocotrienols on Hypersil silica column at different column temperatures. Chromatographic conditions: mobile phase: hexane-1,4-dioxane (96.0:4.0 v/v); flow rate: 1 mL/min. Peaks: 1 – solvent, 2 – α-tocopherol acetate; 3 – α-tocopherol, 4 – α-tocotrienol, 5 – β-tocopherol, 6 –γ-tocopherol, 7 - β-tocotrienol, 8 - γ-tocotrienol, 9 - δ-tocopherol, 10 - δ-tocotrienol 83

xix

4.9 Limit of detection of a balanced mixture of tocopherols and tocotrienols on Hypersil silica column. Chromatographic condition: mobile phase: hexane-1,4-dioxane (96.0:4.0 v/v); flow rate: 1 mL/min; temperature: 40οC; detection: UV absorbance at 295 nm.; injection volume: 5 µL 87

5.1 NP-HPLC separations of crude palm oil on a Hypersil silica column. Chromatographic conditions: mobile phase: n-hexane-1,4-dioxane (96.0:4.0 v/v); flow rate: 1 mL/min; temperature: 40οC. Peaks: 1 – α-tocopherol, 2– α-tocotrienol, 4 – γ-tocotrienol, 5 - δ-tocotrienol, 3 & 6– unknown 90

5.2 NP-HPLC separations of residue oil extracts

obtained by n-hexane (A) and chloroform (B) on a Hypersil silica column. Chromatographic conditions: mobile phase: n-hexane-1,4-dioxane (96.0:4.0 v/v); flow rate: 1 mL/min; temperature: 40οC. Peaks: 1 – α-tocopherol, 2 – α-tocotrienol, 4–β-tocopherol (internal standard), 5–γ-tocotrienol, 6 - δ-tocotrienol, 3 & 7 – unknown 92

5.2 Effect of (a) sample amount, (b) temperature, and (c) pressure on pressurized liquid extraction (PLE) efficiency 96

5.4 NP-HPLC separations of residue oil extracts obtained by pressurized liquid extraction on a Hypersil silica column. Chromatographic conditions: mobile phase: n-hexane-1,4-dioxane (96.0:4.0 v/v); flow rate: 1 mL/min; temperature: 40οC. Peaks: 1 – α-tocopherol, 2 – α-tocotrienol, 4– γ-tocotrienol, 5-δ-tocotrienol, 3 & 6–unknown 98

5.5 Comparison of vitamin E isomers and β-carotene

concentration of optimized PLE and Soxhlet extraction. (Error bars represents standard deviation of results, n=4) 99

xx

LIST OF SYMBOLS/ABBREVIATIONS/NOTATION/TERMINOLOGY

IUPAC - International Union of Pure and Applied Chemistry

RP-HPLC - Reversed-phase High Performance Liquid Chromatography

HPLC - High Performance Liquid Chromatography

η - Mobile phase viscosity

T - Absolute Temperature

∆P - Pressure drop

φ - Flow resistant factor

L - Column length

u - Linear flow rate

dP - Particle diameter

Dm,25 - Molecular diffusivity at 25oC

H - Theoretical plate height

I.D. - Internal diameter

UV - Ultra violet

PS-DVB - Poly(styrene-divinylbenzene)

ODS - Octadecylsilyl

PAHs - Polycyclic aromatic hydrocarbons

UV-Vis - Ultra-violet visible

LC - Liquid chromatography

MS - Mass Spectrometry

PBD - Polybutadiene

CARB - Carbon

Zr - Zirconia

GC - Gas chromatography

CE - Capillary electrophoresis

C - Carbon number

HT-HPLC - High temperature high performance liquid chromatography

MEKC - Micellar electrokinetic chromatography

xxi

TLC - Thin layer chromatography

NP-HPLC - Normal phase high performance liquid chromatography

PFPS - Pentafluorophenylsilica

ODPVA - Octadecanoyl polyvinyl alcohol

PPF - Palm pressed fiber

CPO - Crude palm oil

PFAD - Palm fatty acid distillate

PLE - Pressurized liquid extraction

MAE - Microwave assisted extraction

Log P - Logarithm of partition coefficient n-octanol/water

as - Absorbance of the sample

ab - Cuvette error

W - Weight of sample in gram

Rs - Resolution

w - Peak width

wA1/2 - Peak width at half height

t - Retention time

N/m - Theoretical number of separation plates per column length

RSD - Relative standard deviation

α - Separation factor

k - Retention factor

to - Column void volume values

MeCN - Acetonitrile

R - Gas constant

∆Ho - Standard enthalpy

∆So - Standard entropy

Φ - Column phase ratio

THF - Tetrahydrofuran

xxii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Presentations and Publications 115

CHAPTER 1

INTRODUCTION

1.1 Preamble

The originator of chromatography as it is practiced today was Michael Tswett

(1872-1919) [1]. In 1906 Tswett, a Russian botanist used the term chromatography

to describe his work on the separation of colored plant pigments into bands on a

column of chalk and other materials and stated “ Chromatography is a method in

which the components of a mixture are separated on an adsorbent column in a

flowing system” [2].

Chromatography is a separation method in which a mixture is applied initially

as a narrow zone to a stationary, porous sorbent and the components are caused to

undergo differential migration by the flow of the mobile phase, a liquid or a gas.

According to IUPAC, chromatography can be defined as a physical method of

separation in which the components to be separated are distributed between two

phases, one of which is stationary while the other moves in a definite direction [2,3].

1.2 Principles of High Temperature Operation in Reversed-Phase High

Performance Liquid Chromatography (RP-HPLC)

Control of column temperature has become increasingly accepted as a

separation parameter in RP-HPLC. Besides, high temperature as an optimization

parameter in the separation process of the RP-HPLC system has been widely studied.

2

This is due to the recent findings of the alternative stationary phase, which has high

thermal stability at high temperatures. Recently, the use of higher operating

temperatures in RP-HPLC has been demonstrated to be viable and useful for both

polymer and pharmaceutical application [4-9].

The advantages of doing RP-HPLC at high temperatures (>100οC) are

obvious. High temperature operation in RP-HPLC provides the opportunity to reduce

the quantity of organic solvent used in mixed organic-water mobile phase, decreased

total analysis time and column back pressure significantly. Elevated temperatures can

also increase analyte mass transfer rates and thereby decrease peak width [10-14].

The use of ambient operating temperature in conventional RP-HPLC system

with higher flow-rates to reduce analysis time is not recommended. The applicability

of high flow rates is limited by the back pressure that different parts of the

chromatographic system (pump, injector, and column) can withstand [10]. High flow

rates in conventional RP-HPLC system might cause a significant loss in resolution,

sacrifice the ruggedness of the separation, shorten the column lifetime and damaged

the pump seal [15].

Elevated column temperature operation in RP-HPLC can be used as a tool to

overcome the flow rate problem associated with high back pressure, allowing the use

of higher flow rates that otherwise could not be applied. The pressure reduction is

due to a decrease in eluent viscosity with increasing temperature. The relationship

between viscosity, η, and absolute temperature, T, is given by the empirical

expression [10]:

Tba +=ηln (1.1)

Where a and b are empirically determined constants. The lower viscosity decreases

the pressure drop across the column and allows higher linear velocities as the limit of

pump pressure is approached [16]. The pressure drop across a packed column can be

approximated by equation 1.2 [10]:

3

2pd

uLP ηφ=∆ (1.2)

where ∆P is the pressure drop, φ is the flow resistance factor, L is the column length,

η is the mobile phase viscosity, u is the linear flow rate, and dp is the particle

diameter. The viscosity (η) is proportional to the inverse of the temperature (T),

therefore, higher temperature can significantly decrease mobile phase viscosity (η)

and at the same time decrease the pressure drop (∆P) across a packed column where

all others parameter remain constant.

The advantage of lower pressure drop across the system is that it allows

higher flow rates to be applied without decreasing the efficiency of the separation.

Increase in the mobile phase flow rate can assist in stabilizing the pressure across the

column. Pressure stabilization across the column is extremely important in avoiding

the thermal mismatch and temperature gradient that might occur.

Carr and Li [17] in their paper described the rapid analyses of polycyclic

aromatic hydrocarbons and typical reversed-phase test mixtures at elevated

temperatures and high flow rates. The results showed that analysis time could be

decreased about 18-fold at high temperatures and flow rates without any significant

loss in resolution relative to that at the conventional temperatures and normal flow

rates.

An increase in temperature also increases the diffusion coefficients of the

mobile phase and the analytes. According to the Stokes–Einstein relationship, the

diffusion coefficient is directly proportional to the absolute temperature and

inversely proportional to the viscosity [13]:

=

ηη25

25, 298TDD mm (1.3)

where Dm,25 and η25 are the molecular diffusivity and the viscosity of the eluent at

25οC, respectively. High-temperature separation has been shown to improve analyte

4

resolution by decreasing mobile phase viscosity and by increasing the diffusion rate

of the sample species, thus increasing mass transfer of the analyte to the stationary

phase and thereby decreasing the peak width [13].

1.3 Instrumental Consideration and Performance in High Temperature RP-

HPLC System

Elevated temperature as an optimization parameter in the separation process

of the RP-HPLC system is less popular among the researchers. This is due to few

reasons. Firstly, alternative stationary phases that have high thermal stability force at

high temperature are generally inadequate. The traditional silica-based stationary

phases are less stable at high temperatures. Secondly, the design of a

chromatographic system could not minimize thermal mismatch broadening and

balance heat transfer in the heater effectively. Thirdly, not all the analytes are

thermally stable on the time scale of the chromatographic run. Therefore, analyte

stability at high temperatures should be well considered [16].

To avoid the problems during the operation of the RP-HPLC system at high

temperature, a few modifications should be considered. Carr and Thompson [16]

suggested that one of the methods to solve the main problem was to minimize the

thermal mismatch broadening in high-temperature RP-HPLC. The temperature

mismatch between incoming mobile phase and the column must be minimized

because such a mismatch is a very serious cause of peak broadening of high-

temperature RP-HPLC.

Schrenker [18] has implemented the study on the effect of mobile phase pre-

heating on HPLC column performance. The results showed that control of constant

column temperature using conventional temperature-controlled devices such as “air-

bath” would lead to significantly axial and radial temperature gradients at

temperature different from ambient if the mobile phase enters the column at ambient

temperature. The use of conventional temperature-controller always lead to

5

insufficient heat transfer from air to the column wall and through the column wall

into the mobile phase.

Foong [19] carried out a study on the column efficiency differences of

separation process by comparing the high temperature RP-HPLC system with a

mobile phase pre-heating coil and one without it. The results showed that thermal

mismatch problem between incoming mobile phase and the column can be overcome

by simply modifying the conventional RP-HPLC system with an additional mobile

phase pre-heating coils. The column efficiency of the RP-HPLC system with a

mobile phase pre-heating coils showed higher values of plate number compared with

the system without the modification.

Several journals have described the instrumentation structure of RP-HPLC

system at high temperatures. Generally, instrumentation system for RP-HPLC at high

temperatures is almost the same with a commercial RP-HPLC system in the market.

The most obvious difference is that for RP-HPLC system at high temperature, both

mobile phase and column will be placed inside the temperature controller or simply

inside a heater.

Commercial temperature controller or column thermostat such as “water-

bath” and “block heater” are less popular among the researchers. New type of

column thermostat such as gas oven is well accepted by most of the researchers

because of it low heat capacity which allows sufficient heat transfer from air to the

column wall and through the column into the mobile phase. According to Schrenker

[18], a good column thermostat can save analysis time and improve detection limits,

because at higher column temperatures lower plate height (H) are usually observed

and the optimum of the Van-Deemter curve shifts to higher mobile phase velocities

which allowed the use of a higher flow rate in the separation.

The use of high operating temperature in conventional RP-HPLC system is

strongly dependent on maintaining the mobile phase in the liquid state. As we know,

the boiling point for mobile phases (water~100οC and acetonitrile~81.6οC) are

usually lower than the instrument operating temperature [20]. Therefore, an extra

piece of equipment is needed to overcome this problem. A pressure regulator is

6

usually attached to the detector outlet to provide back pressure to eliminate the

formation of bubbles in the mobile phase and thereby stabilize the baseline. Besides

pressure regulator, small I.D. restriction tubing (~ 0.10 mm) also can be used to

maintain a constant back pressure (~20 bar) at the outlet of the detector. Because the

pressure regulator is attached behind the detector, it would not cause extra column

broadening [2,10,19,21].

In our studies, the mobile phase is preheated to the same temperature as the

column oven temperature and then passes through injection valve and reaches the

column. The hot mobile phase exiting the column will be immediately cooled by ice

water before it reaches the detector.

1.4 Water-Rich and Superheated water Eluents on High Temperature RP-

HPLC

The mobile phase is one of the important parameters that need to be

considered in RP-HPLC. Peak shape, specification of functional group, and other

operating system parameters are strongly dependent on the nature of the mobile

phases that are used [20].

Organic solvents such as acetonitrile, methanol, and tetrahydrofuran are

commonly mixed with water and used as mobile phase in conventional RP-HPLC

system. Acetonitrile is widely used because of its high elution strength compared

with methanol and low UV transparency value. However, it is highly toxic and

expensive. The control of organic material waste disposal and its implication towards

chemist’s health were the problems that should be overcome [22-25].

In order to reduce the usage of organic solvents in RP-HPLC system,

attention was paid to new substitutes, for instance pure water. Water is always

characterized as a unique solvent because of its highly hydrogen-bonded structure,

and at ambient temperature it has disproportionately high boiling point for its mass, a

high dielectric constant, and high polarity [26]. It is readily available, relatively

7

cheap, non-toxic, and causes no significant problems with disposal. However, water

at ambient condition is too polar to solvate most organic pollutants. The polarity of

liquid water can be controlled over a wide range by changing temperature under

moderate pressures to maintain water in the liquid state [24]. Increasing the water

temperature to 200οC–250οC causes a similar change in solvent polarity (measured

by dielectric constant), as achieved by the common HPLC method of mixing

methanol or acetonitrile with the water to a liquid concentration of 100% (Figure

1.1).

Die

lect

ric C

onst

ant,

ε

Pure Water Temperature / οC

% Methanol or Acetonitrile in Water

Pure Water

Acetonitrile/Water (25οC)

Methanol/Water (25οC)

Figure 1.1: Control of solvent polarity (dielectric constant) by changing temperature (at 50 bar) with pure water compared to methanol- or acetonitrile-water mixtures at 25οC and ambient pressure [27-28].

8

Yang et al. [24] described that besides the polarity of water, two additional

mobile phase parameters which control liquid phase separation are the solvent

surface tension and viscosity. Low surface tension can significantly reduces the

retention for reversed-phase separation and low viscosity can results in better mass

transfer, thus achieving better chromatographic efficiencies. Water, heated at high

temperature has low viscosity and the relationship between the viscosity factor and

temperature has been explained previously (section 1.2).

A number of researches that use pure water as mobile phase at high

temperature liquid chromatography were recently reported. Mixtures of phenols,

parabens, barbiturates, and other analytes have been separated on poly(styrene-

divinylbenzene) (PS-DVB) and ODS-bonded silica columns at temperatures up to

210οC by Smith and Burgess [25] using superheated water as an eluent. Dasgupta

and Kephart [21] described the application of superheated water eluent in a capillary

scale reversed-phase liquid chromatography system.

The properties and characteristics of water at high temperatures had been

studied. The capability of the system at high temperature was demonstrated with the

separation of benzene derivatives on polybutadiene and elemental carbon modified

zirconia packing. Carr et al. [16,17,29] who introduced polymer coated zirconia

column performed a complete study. He succeeded in applying the superheated water

as mobile phase to the separation of polycyclic aromatic hydrocarbon (PAH)

compounds by developing a rapid separation at high temperature (200οC).

1.5 High Thermal Stability Stationary Phase in the RP-HPLC

Column plays a very important role in RP-HPLC system because all

separation process will occur in a column that is packed with specific stationary

phase. Reversed phase packing of alkyl silane-bonded phase is by far the most

popular stationary phase in RP-HPLC. However, these alkyl-bonded phases have

several shortcomings, the major one being their thermal and chemical instability

[30].

9

The typical conventional alkyl silane-bonded silica phase proved to be

unstable at temperatures 20οC-30οC higher than room temperature (>50οC) and

higher temperature accelerates the dissolution of silica in aqueous solution [31]. In

addition, degradation of the stationary phase occurs outside the pH range of 2.5-8.

Thus, the ion suppression method cannot be employed with sample solutes having

pKa value less than 2 or more than 7 [30-31].

To overcome the problem faced during the usage of alkyl silane-bonded silica

phase, a few alternative stationary phases that have higher thermal stability and

extreme acceptable pH range of 1-13 have been introduced (Table 1.1). Knox et al.

[32] introduced alternative stationary phase, which is called Porous Graphitized

Carbon (PGC). Meanwhile, Foong [19] have recently done a comprehensive research

about PGC column, which focused on the RP-HPLC operation system at high

temperature, using low organic solvent composition.

Table 1.1: General characteristics of the four reversed-phase LC test columns [32].

Types of Stationary Phases Description

Graphitized Carbon-

Clad Zirconia C18 Silica Polymeric Polybutadiene- Coated Zirconia

Particle size (µm) 3 3.5 5 3

Pore size (Å) 300 300 100 300

Column size (mm x mm) 150 x 4.6 150 x 4.6 150 x 4.6 150 x 4.6

Low pH limit 0.5 1.8 1 0.5

High pH limit 14 8 14 14

Temperature limit (oC) 200 80 150 200

Carbon loading (% carbon) 1.1 2.8 Not available 3.0

Polystyrene-divinylbenzene stationary phase can be regarded as one of the

earliest stationary phase introduced which is able to withstand extended exposure to

mobile phase at extreme pHs (1-14) and column temperatures as high as 200οC [30].

Separation mechanism and retention behavior on the PS-DVB stationary phase is

10

strongly dependent on the neutral non-polar polystyrene surface that function as the

active site for reversed phase separation with aqueous eluent [33]. Meanwhile, See

[34] has recently done a complete research about PS-DVB column, which focused on

the RP-HPLC operation system at high temperature, using water-rich and

superheated water as eluents.

1.5.1 Zirconia based stationary phase

Typically, the applicability of the novel stationary phase can be evaluated by

comparing to the Unger’s specification of the ideal phase. First, the particles must

have a narrow size distribution and high surface area. Second, the pores must have a

diameter appropriate to the size of the analyte and good connectivity to allow for fast

analyte mass transfer and third, the support material should resist thermal,

mechanical, and chemical degradation but have a surface that is both energetically

homogeneous yet chemically modifiable [35].

Zirconia based stationary phase from liquid chromatography has been

introduced by Carr and co-workers [16,17,29]. Zirconia based column have received

a great deal of attention recently because of their extraordinary stability under

extreme thermal and chemical conditions. The outstanding stability of the zirconia

can be explained in detail based on the physical properties of the zirconia structure.

In the monoclinic oxide, each zirconium atom has coordinate bonds to seven

neighbouring oxygen atom. In contrast, silica has only four bonds to oxygen atoms,

which largely accounts for zirconia’s superior resistance to chemical degradation,

especially by acid and base [36].

In order to examine the stability of the zirconium, it was dissolved in the pH

range from 1 to 14 and the results showed that there is no dissolution of zirconium in

this wide pH range using inductively coupled plasma MS as the detection method.

The ability to adjust the pH over a wide range can be quite critical in developing a

good separation. Use of high and low pH is often helpful in improving band spacing