NOVEL APPLICATIONS OF ELEVATED TEMPERATURE HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY: ANALYSES OF
TRIAZOLE FUNGICIDES AND VITAMIN E ISOMERS
SEE HONG HENG
UNIVERSITI TEKNOLOGI MALAYSIA
BAHAGIAN A  Pengesahan Kerjasama*
Adalah disahkan bahawa projek penyelidikan tesis ini telah dilaksanakan melalui
kerjasama antara _______________________ dengan ________________________
Disahkan oleh:
Tandatangan : ………………………………………….. Tarikh: ………………...
Nama
: …………………………………………..
Jawatan
: …………………………………………..
(Cop rasmi)
* Jika penyediaan tesis/projek melibatkan kerjasama.
BAHAGIAN B  Untuk Kegunaan Pejabat Sekolah Pengajian Siswajah
Tesis ini telah diperiksa dan diakui oleh:
Nama dan Alamat Pemeriksa Luar
: Assoc. Prof. Dr. Md Jelas Bin Haron
Chemistry Department,
Faculty of Science and Environmental Studies,
Universiti Putra Malaysia,
43400 Serdang, Selangor.
Nama dan Alamat Pemeriksa Dalam : Assoc. Prof. Dr. Mohamed Noor Bin Hassan
Chemistry Department,
Faculty of Science,
Universiti Teknologi Malaysia
81310 Skudai, Johor.
Disahkan oleh Penolong Pendaftar di SPS:
Tandatangan : ………………………………………….. Tarikh: ………………...
Nama
: Ganesan A/L Andimuthu
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
ii
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-, δtocopherol, and α-, β-, γ-, δ-tocotrienol) and α-tocopherol acetate on both normalphase (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/
1
NOTATION/TERMINOLOGY
xx
LIST OF APPENDICES
xxii
INTRODUCTION
1
1.1
Preamble
1
1.2
Principles of High Temperature Operation in
Reversed-Phase High Performance Liquid
Chromatography (RP-HPLC)
1.3
Instrumental Consideration and Performance in
High Temperature RP-HPLC System
1.4
4
Water-Rich and Superheated Water Eluents on
High Temperature RP-HPLC
1.5
1
6
High Thermal Stability Stationary Phase in the
RP-HPLC
1.5.1
8
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
1.6
1.7
Influence of Narrow-Bore Column in HPLC
Separation
17
Classification of Triazole Fungicides
18
1.7.1
Chromatographic techniques in
analyzing triazole fungicides
1.8
Analysis of Tocopherol and Tocotrienol Isomers
1.8.1
1.9
18
19
Chromatographic techniques in
analyzing tocopherols and tocotrienols.
20
1.8.1.1 Normal-phase HPLC
21
1.8.1.2 Reversed-phase HPLC
22
Tocopherols and Tocotrienols in Palm Oil and
Palm Pressed-Fiber (PPF)
1.9.1
2
15
22
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
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
2.5.1
31
Separation of triazole fungicides by high
temperature RP-HPLC on polybutadienecoated zirconia stationary phase
2.5.2
31
Separation of triazole fungicides by high
temperature RP-HPLC on carbon-clad
zirconia stationary phase
2.6
32
Enhanced Separation of Tocopherols and
Tocotrienols By NP and RP-HPLC at Elevated
Temperature
2.6.1
33
Separation of tocopherols and
tocotrienols by high-temperature
RP-HPLC on PBD-coated zirconia
column
2.6.2
33
NP-HPLC of tocopherol and
tocotrienol isomers on amino and silica
columns at ambient temperature
2.6.3
33
NP-HPLC of tocopherol and
tocotrienol isomers on silica column at
elevated temperature
2.7
34
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
2.7.3
2.7.4
pressed fiber
35
2.7.2.1 Soxhlet extraction
35
2.7.2.2 Pressurized liquid extraction
35
Determination of tocopherols and
tocotrienols content in residue oil
36
Determination of carotene content
37
x
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
3.2.1
Elution behavior on a PBD-coated
zirconia column
3.2.2
42
Influence of temperature on retention
factor on a PBD-coated zirconia column
3.2.4
38
Selectivity and resolution on a PBDcoated zirconia column
3.2.3
38
45
Influence of temperature on column
efficiency on a PBD-coated zirconia
column
3.2.5
3.2.6
3.3
46
Influence of temperature on PBD-coated
zirconia column separation mechanism
48
Limit of detection
51
Separation of Triazole Fungicides on a CarbonClad Zirconia Stationary Phase
3.3.1
Elution behaviour of triazole fungicides
on a carbon-clad zirconia column
3.3.2
56
Influence of temperature on column
efficiency
3.3.5
57
Influence of temperature on retention
factor
3.3.4
53
Selectivity and resolution on carbonclad zirconia column
3.3.3
53
60
Influence of temperature on separation
mechanism
61
xi
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
4.2.1
Influence of column temperature on
retention factor
4.2.2
4.2.4
68
Influence of column temperature on
separation resolution
4.2.3
64
70
Influence of column temperature on
column efficiency
71
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
4.3.2.2 silica based stationary phase
4.4
76
81
Separation of Tocopherols and Tocotrienols
by NP-HPLC on Silica Columns at Elevated
Temperature
4.4.1
4.4.2
82
Separation repeatability, reproducibity
and linearity test
85
Limits of detection
86
xii
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
5.3
89
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
5.3.2.4 Choice of the extraction pressure
5.4
97
Comparison of Soxhlet Extraction with PLE
Extraction
6
95
Quantification of Tocopherols and Tocotrienols
using Optimized PLE Method
5.5
94
97
CONCLUSIONS AND FUTURE DIRECTIONS
101
6.1
Conclusions
101
6.2
Future Directions
103
REFERENCES
105
Appendix A
115
xiii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
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
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
Retention factor of four triazole fungicides as a function
of temperature using different proportion of organic
modifier on PBD-coated zirconia column
46
Enthalpy data for the tebuconazole and hexaconazole at
high column temperatures on PBD-coated zirconia
column.
49
Retention factors of four triazole fungicides on carbonclad zirconia column as a function of temperature using
different proportions of organic modifier
59
Enthalpy data for the hexaconazole and tebuconazole at
high column temperatures on carbon-clad zirconia
column
63
Retention factors of tocopherols and tocotrienols as a
function of temperature using different proportion of
organic modifier on PBD-coated zirconia column
69
Resolution values as a function of temperature ranging
from 80οC to 140οC using different mobile phase
compositions
71
1.1
3.1
3.2
3.3
3.4
3.5
4.1
4.2
xiv
4.3
4.4
4.5
4.6
4.7
5.1
5.2
Enthalpy data for the α-tocopherol acetate at high
column temperatures on PBD-coated zirconia column
73
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
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-1,4dioxane 96.0:4.0 (v/v) as the eluent on silica column
84
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
Regression analysis of the plot of peak area versus
concentration for each isomer
86
Determination of tocopherols and tocotrienols in residue
oil from PPF using Soxhlet extraction
93
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
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
Strong Lewis acid sites that can undergo ligandexchange interactions with Lewis bases
12
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
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
Separation of four triazole fungicides on ZirChromPBD 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
1.1
1.2
1.3
2.2
3.1
xvi
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Separation of four triazole fungicides on ZirChromPBD 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
Separation of four triazole fungicides on ZirChromPBD 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
Relationship of separation factor (α) for propiconazole
(a), difenoconazole (b) as a function of mobile phase
composition at different column temperature on PBDcoated zirconia column
44
Variation of column efficiency as a function of
temperature using low proportion of organic modifier
on PBD-coated zirconia column. (Solute: tebuconazole)
48
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
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
Separation of four triazole fungicides on ZirChromCARB 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
3.10
3.11
3.12
3.13
4.1
4.2
Separation of four triazole fungicides on ZirChromCARB 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
Separation of four triazole fungicides on ZirChromCARB 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
Relationship of separation factor as a function of
mobile phase composition at different column
temperature on carbon-clad zirconia column for
propiconazole
59
Variation of column efficiency as a function of
temperature using low proportion of organic modifier
on carbon-clad zirconia column for hexaconazole
60
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
RP-HPLC separations of a balanced mixture of
tocopherols and tocotrienols on polybutadiene-coated
zirconia column at different column temperature.
Chromatographic conditions: mobile phase: acetonitrilewater (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
RP-HPLC separations of a balanced mixture of
tocopherols and tocotrienols on polybutadiene-coated
zirconia column at different column temperature.
Chromatographic conditions: mobile phase: acetonitrilewater (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
4.4
4.5
4.6
4.7
4.8
RP-HPLC separations of a balanced mixture of
tocopherols and tocotrienols on polybutadiene-coated
zirconia column at different column temperature.
Chromatographic conditions: mobile phase: acetonitrilewater (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
Variation of column efficiency as a function of
temperature using different proportions of organic
modifier for α-tocopherol acetate
72
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
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
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
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
5.1
5.2
5.2
5.4
5.5
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
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
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
Effect of (a) sample amount, (b) temperature, and
(c) pressure on pressurized liquid extraction (PLE)
efficiency
96
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
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
A
TITLE
PAGE
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]:
ln η = a +
b
T
(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
∆P = φLηu
dp
2
(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]:
 T  η 25 

Dm = Dm, 25 

 298  η 
(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 RPHPLC 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 hightemperature RP-HPLC.
Schrenker [18] has implemented the study on the effect of mobile phase preheating on HPLC column performance. The results showed that control of constant
column temperature using conventional temperature-controlled devices such as “airbath” 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 “waterbath” 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 RPHPLC
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).
% Methanol or Acetonitrile in Water
Dielectric Constant, ε
Methanol/Water (25οC)
Acetonitrile/Water (25οC)
Pure Water
Pure Water Temperature / οC
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(styrenedivinylbenzene) (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].
Description
Types of Stationary Phases
Graphitized CarbonClad Zirconia
3
C18 Silica
3.5
Polymeric
5
PolybutadieneCoated Zirconia
3
300
300
100
300
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
Particle size (µm)
Pore size (Å)
Column size (mm x mm)
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