natural antioxidants and high performance liquid chromatography

194 Pages·2013·2.24 MB·English

NATURAL ANTIOXIDANTS AND HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC) HYPHENATED SCREENING TECHNIQUES

A thesis submitted in accord with the requisites of the Degree of Doctor of
Philosophy (Science)

By

Mariam Mnatsakanyan

School of Natural Sciences
University of Western Sydney
New South Wales, Australia

May 2010
“Happiness lies in the joy of achievement and the thrill of creative effort”

F. D. Roosevelt
TABLE OF CONTENTS

Table of Contents .......................................................................................................... i
Statement of Authentication ................................................................................ vi
Acknowledgements ................................................................................................... vii
Publications Arising From This Thesis ....................................................... viii
List of Abbreviations ................................................................................................. ix
List of Tables ................................................................................................................... xi
List of Figures ................................................................................................................ xii
Abstract .......................................................................................................................... xviii
Preface .................................................................................................................................1
Chapter 1
Introduction .......................................................................................................................5
1.1 Defence Against Oxidants: Amtioxidants .....................................................................6
1.2 Antioxidants and Their Mechanisms of Action .............................................................7
1.3 Natural Products and Antioxidants ...............................................................................8
1.4 Methodologies in Total Antioxidant Assessment .........................................................8
1.4.1 2,2´-Diphenyl-1-Picrylhydrazyl (DPPH) Assay .........................................................9
1.4.2 Chemiluminescence (CL) Methods ...........................................................................9
1.5 High-Resolution Antioxidant Screening Techniques .................................................. 10
1.6 High Performance Liquid Chromatography (HPLC) .................................................... 12
1.6.1 Resolution ............................................................................................................. 12
1.6.2 Peak Capacity ........................................................................................................ 13
1.6.2.1 Isocratic Elution .................................................................................................. 14
1.6.2.2 Gradient Elution ................................................................................................. 16
1.6.2.3 Multidimensional HPLC (MDLC) ......................................................................... 17
1.7 Two-Dimensional Liquid Chromatography ................................................................. 17
1.7.1 Orthogonality ........................................................................................................ 18
1.7.1.1 Geometric Approach to Factor Analysis (GAFA) .............................................. 19
1.7.2 Sample Dimensionality.......................................................................................... 20
1.8 The Practical Criteria of 2D HPLC Applications (Natural Products) ........................... 22
1.8.1 Sample Dimensionality Selection .......................................................................... 23
1.8.2 Selectivity Studies-Stationary Phase ..................................................................... 24
1.8.3 Selectivity Studies-Solvent Selectivity ................................................................... 26
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1.8.4 Modes of 2D HPLC Separations ............................................................................ 27
1.8.5 Two-Dimensional System Designs ........................................................................ 29
1.8.6 Data Collection and Analysis ................................................................................. 30
1.9 Coffee Espresso: A Complex Sample of Natural Origin .............................................. 32
1.10 The Research Problems ............................................................................................. 33
1.11 Project Aim ................................................................................................................ 34
1.12 Project Objectives ..................................................................................................... 34
Chapter 2
General Experimental ............................................................................................... 35
2.1 Chemicals, Reagents and Samples .............................................................................. 36
2.2 Sample and Reagent Preparation ............................................................................... 36
2.2.1 Sample Preparation .............................................................................................. 36
2.2.2 Reagent and Standard Preparation ...................................................................... 37
2.3 Equipment ................................................................................................................... 37
2.3.1 Chromatographic Instrumentation ....................................................................... 37
2.3.2 Mass Spectrometer Analysis ................................................................................. 37
2.3.3 Chromatographic Columns ................................................................................... 38
2.3.4 Development of On-Line Post-Column DPPH Assay Technique .......................... 38
2.3.4.1 Instrumental Set-up .......................................................................................... 38
2.3.4.2 Results and Discussion ...................................................................................... 39
2.3.5 Chemiluminescence (CL) Detector......................................................................... 42
2.4 Chromatographic Separation Methods ...................................................................... 42
2.5 Data Analysis ............................................................................................................... 42
Chapter 3
High performance liquid chromatography with two simultaneous
on-line antioxidant assays: Evaluation and comparison of espresso
coffees ................................................................................................................................. 44
3.1 Introduction ................................................................................................................. 45
3.2 Experimental ............................................................................................................... 47
3.2.1 Chemicals, Reagents and Samples ........................................................................ 47
3.2.2 Sample and Reagent Preparation ......................................................................... 47
3.2.3 Chromatographic Instrumentation and Columns ................................................. 47
3.2.3.1 Chromatographic Instrumentation ................................................................... 47
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3.2.3.2 Chemiluminescence (CL) Detector..................................................................... 48
3.2.4 Chromatographic Separation and On-Line Antioxidant Assays ............................ 48
3.2.4.1 On-Line DPPH• Assay......................................................................................... 48
3.2.4.2 On-Lline Chemiluminescence (CL) Assay ........................................................... 48
3.3 Results and Discussion ................................................................................................ 49
3.3.1 Separation and Detection Conditions ................................................................... 49
3.3.2 Comparison of Espresso Coffees ........................................................................... 51
3.4 Conclusions .................................................................................................................. 56
Chapter 4
A Discussion on the Process of Defining Two-Dimensional
Separation Selectivity ................................................................................................ 57
4.1 Introduction ................................................................................................................. 58
4.1.1 Statistical Metrics ................................................................................................. 59
4.2 Experimental ............................................................................................................... 60
4.2.1 Chemicals and Samples ......................................................................................... 60
4.2.2 Chromatographic Instruments and Columns ........................................................ 60
4.2.3 Chromatographic Separation ................................................................................ 61
4.2.4 Data Analysis ........................................................................................................ 62
4.3 Results .......................................................................................................................... 62
4.3.1 Sample Set Selection ............................................................................................. 64
4.3.2 System 1. 2D HPLC System Performance Measured Using the Entire Ristretto
Espresso Sample ............................................................................................................. 65
4.3.3 System 2. 2D HPLC System Performance Measured Using Selected Regions of the
2D Separation Space ...................................................................................................... 69
4.4 Discussion .................................................................................................................... 74
4.5 Conclusions .................................................................................................................. 74
Chapter 5
The Assessment of  Selective Stationary Phases For Two-
Dimensional HPLC Analysis of Foods: Application to the Analysis of
Coffee ................................................................................................................................. 76
5.1 Introduction ................................................................................................................. 77
5.2 Experimental ............................................................................................................... 79
5.2.1 Chemicals and Samples ......................................................................................... 79
5.2.2 Chromatographic Instrumentation and Columns ................................................. 79
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5.2.2.1 Chromatographic Instrumentation ................................................................... 79
5.2.2.2 Chromatographic Columns ............................................................................... 79
5.2.3 Chromatographic Separations .............................................................................. 79
5.2.3.1 First Dimensional Separations .......................................................................... 79
5.2.3.2 Second Dimensional Separations ...................................................................... 80
5.2.3.3 Operation .......................................................................................................... 80
5.2.4 Mass Spectra Analysis ........................................................................................... 80
5.2.5 Data Processing .................................................................................................... 81
5.3 Results and Discussion ................................................................................................ 81
5.3.1 Preliminary Studies: Solvent Selectivity ................................................................ 82
5.3.2 Stationary Phase Selectivity .................................................................................. 83
5.3.2.1 Qualitative Assessment of the Selectivity Changes ........................................... 89
5.3.2.2 Quantitative Assessment of the Selectivity Changes ........................................ 98
5.3.2.3 Localised System Performance ....................................................................... 100
5.4 Overview .................................................................................................................... 101
5.5 Conclusions ................................................................................................................ 103
Chapter 6
The Analysis of Café Espresso using Two-Dimensional Reversed
Phase-Reversed Phase High Performance Liquid Chromatography
with UV-Absorbance and Chemiluminescence Detection .................... 104
6.1 Introduction ............................................................................................................... 105
6.2 Experimental ............................................................................................................. 106
6.2.1 Chemicals, Reagents and Samples ...................................................................... 106
6.2.2 Chromatographic Instrumentation and Columns ............................................... 107
6.2.2.1 Chromatographic Instrumentation ................................................................. 107
6.2.2.2 Chemiluminescence (CL) Detector................................................................... 107
6.2.2.3 One-Dimensional On-Line HPLC-DPPH Instrumentation ............................... 107
6.2.2.4 Chromatographic Columns ............................................................................. 107
6.2.3 Chromatographic Separations ............................................................................ 107
6.2.3.1 2D Chromatographic Separations and On-Line Chemiluminescence (CL)
Assay .......................................................................................................................... 107
6.2.4 Data Analysis and Plotting .................................................................................. 108
6.3 Results and Discussion .............................................................................................. 108
6.4 Conclusions ................................................................................................................ 117
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Chapter 7
2D HPLC Fingerprinting Technique:
Applications To The Analysis of Wine and Apple Samples ................ 118
7.1 Introduction ............................................................................................................... 119
7.2 Experimental ............................................................................................................. 120
7.2.1 Chemicals, Reagents and Samples ..................................................................... 120
7.2.2 Chromatographic Instrumentation and Columns ............................................... 121
7.2.2.1 Chromatographic Instrumentation ................................................................. 121
7.2.2.2 Chemiluminescence (CL) Detector................................................................... 121
7.2.2.3 2D Chromatographic Columns ........................................................................ 121
7.2.3 2D Chromatographic Separations ....................................................................... 121
7.2.4 2D HPLC-CL Analysis ............................................................................................ 122
7.2.5 Data Analysis and Plotting .................................................................................. 122
7.3 Results and Discussion .............................................................................................. 122
7.4 Conclusions ................................................................................................................ 132
Chapter 8
General Conclusion .................................................................................................. 133

References ...................................................................................................................... 138

Appendix I

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STATEMENT OF AUTHENTICATION

The work presented in this thesis is, to the best of my knowledge and belief, original
except as acknowledged in the text. I hereby declare that I have not submitted this
material, either in full or in part, for a degree at this or any other institution.

Mariam Mnatsakanyan

May 2010

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ACKNOWLEDGEMENTS

I take this opportunity to express my gratitude to many people for their support and
friendship during the time I spent at the University of Western Sydney.

First and foremost, I wish to sincerely thank my principal supervisor, A/Prof.
Andrew Shalliker, who generously and tirelessly offered his advice, assistance and
inspiration through the period of this research. This work could not been succeeded
without Andrews encouragement and help. I have learned a lot from him.

I want to appreciate Prof. N.W Barnett and Dr X.A. Conlan from Deakin University
(School of Life and Environmental Sciences) for their expertise provided during this
work.

I appreciate the support of my panel supervisors, Dr Rosalie Durham and A/Prof.
Kaila Kailasapathy. I want to thank late A/Prof. Geoff Skurray for the interesting
conversations. Special regards to Dr Michael Phillips for his assistance with any
questions that I might have had.

I am grateful to Paul Stevenson for his friendship and for writing an algorithm that
enhanced the data analysis during this research.

My deepest appreciation goes to the International Postgraduate Research
Scholarship (IPRS) scheme of University of Western Sydney, for providing me with
the necessary financial assistance. Special regards to Tracy Mills, for the given
opportunity.

I would like to express my gratitude to all my colleagues and friends; Arianne, Kirsty,
Coleen, David for their friendship and support throughout the period of my studies.

I’m grateful to Mr Steven MacJohn for always being there for me. I am indebted to
my parents for their endless love.

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PUBLICATIONS ARISING FROM THIS THESIS
Refereed Journal Publications
1. Mnatsakanyan M., Goodie T.A., Conlan X.A., Francis P.S., McDermott G.P.,
Barnett N., Shock D., Gritti F., Guiochon G., Shalliker R.A., High
performance liquid chromatography with two simultaneous on-line
antioxidant assays: evaluation and comparison of espresso coffees. Talanta
81(3) (2010); 837.

2. Mnatsakanyan M.; Stevenson P.G.; Shock D.; Shalliker R.A.: The
Assessment of  Stationary Phases for Natural Products. Talanta 82(4)
(2010); 1349.

3. Mnatsakanyan M., Stevenson P.G., Conlan X.A., Francis P.S., Goodie T.A.,
McDermott G.P., Barnett N.W., Shalliker R.A., The Analysis of Café
Espresso using Two-Dimensional Reversed-Phase High Performance Liquid
Chromatography with UV-Absorbance and Chemiluminescence Detection.
Talanta 82(4) (2010); 1358.

4. Stevenson P.G., Mnatsakanyan M., Francis A.R., Shalliker R.A., A
Discussion on the Process of Defining Two-Dimensional Separation
Selectivity. Journal of Separation Science 33 (2010); 1.

5. Stevenson P.G., Mnatsakanyan M., Shalliker R.A., Peak Picking from 2D-
HPLC data. Analyst 135 (2010); 1541.

6. Shalliker R.A.; Stevenson P.G.; Mnatsakanyan M.; Dasgupt P.K.; Guiochon
G.: Application of Power Functions to Chromatographic Data for the
Enhancement of Signal to Noise Ratios and Separation Resolution. Journal of
Chromatography A 1217(36) (2010); 5693.

7. McDermott G.P., Noonan L.K., Mnatsakanyan M., Shalliker R.A., Conlan
X.A., Barnett N.W., Francis P.S., The on-line HPLC-DPPH• antioxidant
assay: methodological considerations and application to highly complex
samples. Analytica Chimica Acta 675 (2010); 76.

Book Chapters
1. Milroy C., Stevenson P.G., Mnatsakanyan M. and Shalliker R.A.,
Multidimensional High Performance Liquid Chromatography in Hyphenated
and Alternative Methods of Detection in Chromatography. Editor R.A.
Shalliker, Publishers Taylor and Francis, (2010); (In submission).

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