Development of sample pretreatment and liquid chromatographic techniques for antioxidative ...

86 Pages·2009·1.25 MB·English

by  Maarit Kivilompolo

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University of Helsinki
Department of Chemistry
Laboratory of Analytical Chemistry
Finland
D
EVELOPMENT OF SAMPLE PRETREATMENT AND
LIQUID CHROMATOGRAPHIC TECHNIQUES FOR
ANTIOXIDATIVE COMPOUNDS
MAARITKIVILOMPOLO
Academic Dissertation
To be presented, with permission of
the Faculty of Science of the University of Helsinki
for public criticism in Chemicum, Auditorium A 129,
(A.I.Virtasen aukio 1, Helsinki)
on June 5th, at 12 o’clock noon.
Helsinki 2009
Author
Maarit Kivilompolo (née Ollanketo)
Laboratory of Analytical Chemistry
Department of Chemistry
University of Helsinki
Finland
Custos
Prof. Marja-Liisa Riekkola
Laboratory of Analytical Chemistry
Department of Chemistry
University of Helsinki
Finland
Reviewers
Prof. Heli Sirén Prof. Pavel Jandera
Laboratory of Applied Chemistry Department of Analytical Chemistry
Department of Chemical Technology Faculty of Chemical Technology
Lappeenranta University of Technology University of Pardubice
Finland Czech Republic
Opponent
Prof. Paola Dugo
Department of Food and Environmental Science
Faculty of Science
University of Messina
Italy
ISBN 978-952-92-5574-0 (paperback)
ISBN 978-952-10-5584-3 (pdf)
http://ethesis.helsinki.fi/
Cover picture: Tuulia Hyötyläinen
Yliopistopaino, Helsinki, 2009
TABLE OF CONTENTS
TABLE OF CONTENTS 1
PREFACE 3
ABSTRACT 5
ABBREVIATIONS AND SYMBOLS 6
LIST OF ORIGINAL PAPERS 8
1 INTRODUCTION 9
2 AIMS OF THE STUDY 11
3 ANTIOXIDANTS 13
3.1 Lycopene 14
3.1.1 Structure, occurrence and concentrations 14
3.1.2 Health claims 15
3.2 Phenolic compounds 16
3.2.1 Structures, occurrence and concentrations 16
3.2.2 Antioxidativity and health claims 20
4 SAMPLE PRETREATMENT TECHNIQUES 21
4.1 Supercritical fluid extraction 21
4.1.1 Factors affecting the extraction 22
4.1.1.1 Choice of a fluid 23
4.1.1.2 Modifiers 23
4.1.1.3 Matrix 23
4.1.1.4 Pressure, temperature, flow rate and extraction time 23
4.1.1.5 Collection of extract 24
4.1.2 Applications 24
4.2 Pressurised hot water extraction 25
4.2.1 Factors affecting the extraction 26
4.2.1.1 Temperature 26
4.2.1.2 Pressure, flow rate and extraction time 27
4.2.1.3 Collection of extract 27
4.2.2 Applications 27
4.3 Sonication-assisted extraction 27
4.3.1 Factors affecting the extraction 28
4.3.2 Applications 28
4.4 Comparison of techniques 29
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5 LIQUID CHROMATOGRAPHIC TECHNIQUES 31
5.1 Comprehensive two-dimensional liquid chromatography 33
5.1.1 Comprehensiveness and orthogonality 35
5.1.2 Peak capacity 36
5.1.3 Modes of comprehensive two-dimensional liquid chromatography 37
5.2 Coupling of extraction on-line to liquid chromatography 38
5.2.1 Factors affecting the coupling 38
5.3 Applications 39
5.3.1 High and ultra performance liquid chromatography 39
5.3.2 Comprehensive two-dimensional liquid chromatography 40
5.3.3 On-line coupled extraction and liquid chromatography 41
6 EXPERIMENTAL 42
6.1 Chemicals and materials 42
6.2 Equipment and procedures 43
6.2.1 Pressurised hot water extraction 45
6.2.2 Dynamic sonication-assisted extraction 46
6.2.3 Comprehensive two-dimensional liquid chromatography 46
6.2.4 Coupling dynamic sonication-assisted extraction on-line to liquid chromatography 48
7 RESULTS AND DISCUSSION 50
7.1 Sample pretreatment techniques 50
7.1.1 Supercritical fluid extraction 51
7.1.2 Pressurised hot water extraction 52
7.1.3 Dynamic sonication-assisted extraction 55
7.2 Experiments on storage and stabilisation 56
7.3 Liquid chromatographic techniques 57
7.3.1 One-dimensional liquid chromatography 57
7.3.2 Comprehensive two-dimensional liquid chromatography 58
7.3.2.1 Column selection, modulation time and selectivity correlation 59
7.3.2.2 Comparison of different second dimension separation systems 62
7.3.2.3 Peak capacity 65
7.4 On-line coupled dynamic sonication-assisted extraction and liquid chromatography 66
7.4.1 Optimisation of the solid-phase trapping 67
7.4.2 Comparison of the on-line and off-line systems 67
7.5 Quantitative and qualitative results of herbs and beverages 70
7.5.1 Herbs 70
7.5.2 Beverages 73
8 CONCLUSIONS 75
9 REFERENCE 78
APPENDICES: PAPERS I - VI
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PREFACE
This thesis is based on research carried out in the Laboratory of Analytical Chemistry of the
Department of Chemistry, University of Helsinki, during the years 1997-2000, 2002 and 2006-
2008. Financial support from the University of Helsinki, the Alfred Kordelin Foundation, the
Academy of Finland, the Oskar Öflund Foundation and the Finnish Concordia Fund is gratefully
acknowledged.
I express special thanks to Professor Marja-Liisa Riekkola for giving me the opportunity to carry
out this work in the Laboratory of Analytical Chemistry and for her patience when this project
went on and on for such a long time!
I am most grateful to my supervisor Docent Tuulia Hyötyläinen for her never-ending
encouragement, fresh ideas, help and positive attitude even in situations where the research
seemed to go nowhere. I really appreciate it that you allowed me to grow towards my Ph.D with
my own time schedule! Sincere thanks to Docent Kari Hartonen, who introduced me to the
interesting world of extraction and helped me to find “what’s under the surface”. I owe my thanks
to Dr. Kathleen Ahonen for improving the language of most of the original papers. Professors
Heli Sirén and Pavel Jandera are thanked for their careful pre-examination of the thesis. I also
want to thank my co-authors Vít Ob(cid:292)rka and Anna Nurmi (née Peltoketo).
I am also indebted to our laboratory engineer, Matti Jussila, who always had the time and will to
fix all problems dealing with instruments and computers. Merit Hortling, Liisa Heino, Pentti
Jyske and Erja Pyykkö are sincerely thanked for helping with various practical things over the
years.
My former and present colleagues in the lab (Antti, Arvind, Géraldine, Heidi, Jaakko, Jaana,
Jana, Jari, Jarno, Jaroslav, Jevgeni, Jie, Joanna, Johan, Johanna, Laura, Lucia, Maria, Pasi, Pertti,
Piia, Ruth, Sami P., Sami V., Simo, Stella, Susanne, Tapio, Totti and Yohannes) deserve warm
thanks for making my work easier during these years, for giving me a hand in solving problems,
and for providing a pleasant working atmosphere. I have enjoyed solving scientific problems with
you, but even more importantly, our discussions outside the scope of science! I still enjoy the
special humour of our “special lab family”; thank you Joonas, Juhani, Kati L., Kati V., Masahiko,
Minna and Terhi. I also want to thank Juhani and Joonas, for their excellent team work and help
in the field of teaching and pedagogy. My foreign co-workers are especially thanked for patiently
answering my questions about cultural differences and bringing a fresh perspective to our
discussions. My dear friends, some of them all the way from the childhood, some appearing in
my life during my studies and work, are very precious. Thank you for your friendship!
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My warmest thanks go to my parents, Vieno and Väinö, for their confidence in me and for being
supportive and interested in my work and well-being and, above all, allowing me to make my
own choices. My brother Harri and his family are thanked especially for taking my mind to other
issues, e.g. like relaxing at the cottage by lake Saimaa.
Finally, my warmest thanks are addressed to my family, my husband Mika for his love and
support and for handling the world of computers so well, and to our three active and wonderful
children, Aada, Saana and Teemu, for being the sunshine of our lives.
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ABSTRACT
In this study, novel methodologies for the determination of antioxidative compounds in herbs and
beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit
oxidative events. They are a part of the human defense system and are obtained through the diet.
Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables
and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid
oxidation and formation of free radicals under conditions of cooking or storage and to reduce the
concentration of free radicals in vivo after food ingestion. There is growing interest in natural
antioxidants, and effective compounds have already been identified from antioxidant classes such
as carotenoids, essential oils, flavonoids and phenolic acids.
The wide variety of sample matrices and analytes presents quite a challenge for the development
of analytical techniques. Growing demands have been placed on sample pretreatment. In this
study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised
hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied.
SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the
extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic
acids from Lamiaceae herbs. In the development of extraction methodologies, the main
parameters of the extraction were studied and the recoveries were compared to those achieved by
conventional extraction techniques. In addition, the stability of lycopene was also followed under
different storage conditions.
For the separation of the antioxidative compounds in the extracts, liquid chromatographic
methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid
chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LC(cid:117)LC)
were studied and compared with conventional high performance liquid chromatography (HPLC)
for the separation of antioxidants in beverages and Lamiaceae herbs. In LC(cid:117)LC, the selection of
LC mode, column dimensions and flow rates were studied and optimised to obtain efficient
separation of the target compounds. In addition, the separation powers of HPLC, UPLC,
HPLC(cid:117)HPLC and HPLC(cid:117)UPLC were compared.
To exploit the benefits of an integrated system, in which sample preparation and final separation
are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a
liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the
extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of
achieved by the LC(cid:117)LC system.
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ABBREVIATIONS AND SYMBOLS
1D one-dimensional
2D two-dimensional
AC affinity chromatography
ASE accelerated solvent extraction
BSTFA N,O-bis(trimethylsilyl)-trifluoroacetamide
DAD diode array detector
DMAE dynamic microwave-assisted extraction
DSAE dynamic sonication-assisted extraction
EIC extracted ion chromatogram
ESI electrospray ionisation
GC gas chromatography
GFC gel filtration chromatography
GPC gel permeation chromatography
HILIC hydrophilic interaction liquid chromatography
HPLC high performance liquid chromatography
i.d. inner diameter
IEC ion exchange chromatography
IPC ion pair chromatography
LC liquid chromatography
LC-LC heart-cutting liquid chromatography
LC(cid:117)LC comprehensive two-dimensional liquid chromatography
MAE microwave-assisted extraction
MMLLE microporous membrane liquid-liquid extraction
MS mass spectrometry
NPLC normal phase liquid chromatography
PAH polycyclic aromatic hydrocarbons
PEEK poly(etheretherketone)
PHWE pressurised hot water extraction
PLE pressurised liquid extraction
RPLC reversed-phase liquid chromatography
SAE sonication-assisted extraction
SAX strong anion exchange (solid-phase material)
SBSE stir bar sorptive extraction
SCF supercritical fluid
SEC size exclusion chromatography
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SFE supercritical fluid extraction
SLE solid-liquid extraction
SPE solid-phase extraction
SPME solid-phase microextraction
TBA tetrabutylammonium bromide
TFA trifluoroacetic acid
TOF time-of-flight
TPA tetrapentylammonium bromide
UPLC ultra performance liquid chromatography
UV ultraviolet
(cid:302) separation factor
d particle size of the column packing material [µm]
p
h relative plate height
H plate height [µm]
k retention factor
L column length [cm]
N plate number
n peak capacity of the first dimension separation
c,1
n peak capacity of the second dimension separation
c,2
n total peak capacity of the LC(cid:117)LC separation
n,tot
p critical pressure [bar, 1 bar = 105 Pa]
c
R resolution
s
t retention time of the first eluting peak [s or min]
R,1
t retention time of the last eluting peak [s or min]
R,n
T critical temperature [°C, 0°C = 273.15 K]
c
u linear velocity [mm/s]
W average 4(cid:305) peak width [s or min]
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LIST OF ORIGINAL PAPERS
This thesis is based on the following papers, hereafter referred to by their Roman numerals [I-VI]
I Ollanketo, M., Hartonen, K., Riekkola, M.-L., Holm, Y. and Hiltunen, R. Eur. Food Res.
Technol. 212 (2001) 561-565. “Supercritical carbon dioxide extraction of lycopene in tomato
skins”. Copyright Springer-Verlag 2001
II Ollanketo, M., Peltoketo, A., Hartonen, K., Hiltunen, R. and Riekkola, M.-L. Eur. Food Res.
Technol. 215 (2002) 158-163. “Extraction of sage (Salvia officinalis L.) by pressurized hot water
and conventional methods: antioxidant activity of the extracts”. Copyright Springer-Verlag 2002
III Kivilompolo, M. and Hyötyläinen, T. J. Chromatogr. A 1145 (2007) 155-164.
“Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs:
Characterisation and quantification of antioxidant phenolic acids”. Copyright Elsevier B.V. 2007
IVKivilompolo, M., Ob(cid:292)rka, V. and Hyötyläinen, T. Anal. Bioanal. Chem. 391 (2008) 373-380.
“Comprehensive two-dimensional chromatography in the analysis of antioxidant phenolic
compounds in wines and juices”. Copyright Springer-Verlag 2008
V Kivilompolo, M. and Hyötyläinen, T. J. Chromatogr. A 1216 (2009) 892-896. “On-line
coupled dynamic sonication-assisted extraction-liquid chromatography for the determination of
phenolic acids in Lamiaceae herbs” Copyright Elsevier B.V. 2009
VI Kivilompolo, M. and Hyötyläinen, T. J. Sep. Sci. 31 (2008) 3466-3472. “Comparison of
separation power of ultra performance liquid chromatography and comprehensive two-
dimensional liquid chromatography in the separation of phenolic compounds in beverages”
Copyright Wiley-VCH Verlag GmbH & Co. KGaA 2008
Paper I: Main responsibility for planning, carrying out the work and writing the article.
Paper II: Main responsibility for extractions done by pressurised hot water, liquid
chromatography analyses and shared main responsibility for writing the article.
Paper III: Main responsibility for planning, carrying out the work and writing the article.
Paper IV: Main responsibility for planning and writing of the article, also performed part of the
experiments.
Paper V: Main responsibility for planning, carrying out the work and writing the article.
Paper VI: Main responsibility for planning, carrying out the work and shared main responsibility
for writing the article.
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