Structural Characterization of Freshwater Dissolved Organic Matter from Arctic and Temperate ...

234 Pages·2012·5.46 MB·English

Structural Characterization of Freshwater Dissolved
Organic Matter from Arctic and Temperate Climates
Using Novel Analytical Approaches

by

Gwen C. Woods

A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Chemistry
University of Toronto

© Copyright Gwen C. Woods (2012)
Structural characterization of freshwater dissolved organic matter from
polar and temperate climates using novel analytical approaches
Doctor of Philosophy Degree, 2012
Gwen C. Woods
Department of Chemistry
University of Toronto

ABSTRACT
Dissolved organic matter (DOM) is comprised of a complex array of molecular
constituents that are linked to many globally-relevant processes and yet this material is still
largely molecularly uncharacterized. Research presented here attempted to probe the
molecular complexity of this material from both Arctic and temperate climates via
multifaceted and novel approaches. DOM collected from remote Arctic watersheds provided
evidence to suggest that permafrost-disturbed systems contain more photochemically- and
biologically-labile material than undisturbed systems. These results have large implications
for predicted increasing temperatures where widespread permafrost melt would significantly
impact stores of organic carbon in polar environments. In attempting to address the
complexities and reactivity of DOM within global environments, more information at the
molecular-level is necessary. Further research sought to unravel the molecularly
uncharacterized fraction via use of nuclear magnetic resonance (NMR) spectroscopy in
conjunction with hyphenated and varied analytical techniques. Directly hyphenated high
performance size exclusion chromatography (HPSEC) with NMR was explored. This
hyphenation was found to separate DOM into structurally distinct fractions but proved limited
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at reducing DOM heterogeneity. Of the many high performance liquid chromatography
(HPLC) techniques tested, hydrophilic interaction chromatography (HILIC) was found the
most effective at simplifying DOM. HILIC separations utilizing a sample from Florida
resulted in fractions with highly resolved NMR signals and substantial reduction in
heterogeneity. Further development with a 2D-HILIC/HILIC system to achieve additional
fractionation was employed. This method produced fractions of DOM that were homogenous
enough to produce excellent resolution and spectral dispersion, permitting 2D and 3D NMR
experiments to be performed. Extensive NMR analyses of these fractions demonstrated
strong evidence for the presence of highly oxidized sterols. All fractions, however, provided
2D NMR spectra consistent with oxidized polycyclic structures and support emerging data
and hypotheses suggesting that cyclic structures, likely derived from terpenoids, are an
abundant, refractory and major component of DOM. Research presented within this thesis
demonstrates that HILIC and NMR are excellent co-techniques for the analysis of DOM as
well as that oxidized sterols and other cyclic components with significant hydroxyl and
carboxyl substituents are major constituents in DOM.
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ACKNOWLEDGEMENTS
There are a number of individuals for whom I would like to extend my gratitude. First
and foremost I would like to thank my supervisor, Prof. André Simpson, who has made this
dissertation possible. I would particularly like to thank André for his wonderful insight and
assistance with NMR as well as for a continuously positive attitude. I would also like to
thank Prof. Myrna Simpson for her help in analyzing data from the High Arctic, rides to
Kingston to visit colleagues at Queen’s University and general helpfulness in many aspects of
the research presented. Further gratitude is extended to the chair of my committee, Prof.
Jonathan Abbatt as well as examination committee member Prof. Jennifer Murphy both of
whom inspired and encouraged thoughts of my future career. I am additionally thankful to
Prof. Lihini Aluwihare for taking the time and effort to be the external examiner.
A special thank you to all the wonderful members of both Simpson groups, past and
present. You have been a tremendous source of support and entertainment. I have had
invaluable conversations with many of you with regards to scientific inquiries, future careers
and life in general. Best of luck to all of you!
I would like to acknowledge funding sources for the projects contained within this
thesis: the Natural Sciences and Engineering Research Council (NSERC) of Canada (AJS)
and the Government of Canada International Polar Year program (MJS) for funding provided
as well as the Polar Continental Shelf Project (Natural Resources Canada) for logistical
support; the government of Ontario for an Early Researcher Award and the Natural Sciences
and Engineering Research Council (NSERC) of Canada Strategic Grant (AJS); a University
of Toronto Fellowship, a Teaching Reduction Fellowship, a UTF Conference Travel Grant
and a Doctoral Thesis Completion Grant (GCW).
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A special thank you is extended to all of the wonderful people I have met while living
in Toronto for graduate studies. Many of you have become good friends over the years, kept
me entertained and always been supportive; I am lucky to have you. I would especially like
to thank the Chabane family for greeting me into their home, welcoming me on holidays and
treating me as family.
This short acknowledgement cannot begin to convey the gratitude I feel toward my
family. To my parents, you have given so much. I know that you are and always will be my
biggest supporters. Thank you for everything. Galen, Alyn and Ellenore, you have taught me
some of life’s most interesting and not always easiest lessons! I have missed you so much the
last few years but look forward to seeing so much more of you!
Lastly I need to thank my personal chef, best friend and love of my life. Stefan, I
could not have done this without you.

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TABLE OF CONTENTS

ABSTRACT……………………………………………………………………….…….… ii
ACKNOWLEDGEMENTS……………………………………………………….…….… iv
TABLE OF CONTENTS………………………………………………………….…….... vi
LIST OF TABLES..……………………………………………………………….…….… xi
LIST OF FIGURES...……………………………………………………………….….…. xii
LIST OF ABBREVIATIONS……….………………………………………………..…… xvii
PREFACE…………………………….……………………………………………….…... xix

CHAPTER 1: Introduction: Structural characterization of dissolved….… 1
organic matter, global implications and analytical approaches

1.1 Introduction…………………………………………………………………………. 2
1.1.1 Global significance and motivation for DOM research…………………..…. 3
1.1.2 Global significance and motivation for Arctic DOM research…………..….. 4

1.2 Analytical approaches to DOM……………………………………………….… 6
1.2.1 The application of high performance liquid chromatography with DOM………... 8
1.2.2 The application of nuclear magnetic resonance spectroscopy with DOM………… 9
1.2.3 The application of multidimensional NMR spectroscopy with DOM………….…. 12
1.2.3.1 Two dimensional correlation spectroscopy………………………... 14
1.2.3.2 Two total dimensional correlation spectroscopy…………………... 14
1.2.3.3 Heteronuclear single quantum correlation………………………… 15
1.2.3.4 Heteronuclear multiple bond connectivity…………………………. 16
1.2.4 Analysis of lignin-derived phenol biomarkers in DOM…………………….. 16
1.2.5 Analysis of DOM with 3D EEMs and parallel factor analysis……………… 17

1.3 Study objectives…………………………………………………………………… 20

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1.4 Thesis summary…………………………………………………………………… 21

1.5 References………………………………………………………………………….. 24

CHAPTER 2: Evidence for the enhanced lability of dissolved organic…... 39
matter following permafrost slope disturbance in the Canadian
High Arctic

2.1 Abstract……………………………………………………………………………… 40

2.2 Introduction………………………………………………………………………… 42

2.3 Experimental………………………………………………………………………. 45
2.3.1 Study area and sampling……………………………………………………. 45
2.3.2 Solution-state 1H NMR analysis…………………………………………… 49
2.3.3 Organic carbon content and lignin-derived phenol extraction…………...... 50
2.3.4 EEMs and PARAFAC analyses……………………………………………. 51
2.3.5 Photolytic degradation experiments………………………………………... 53

2.4 Results and discussion…………………………………………………………… 55
2.4.1 Solution-state 1H NMR…………………………………………………….. 55
2.4.2 Characteristics of lignin-derived phenols in Arctic DOM………………..... 58
2.4.3 PARAFAC components derived from fluorescence EEMs………………… 66
2.4.4 Effects of simulated solar radiation on Arctic samples…………………….. 71

2.5 DOM biogeochemistry in paired Arctic watersheds……………………….. 74

2.6 Acknowledgements……………………………………………………………….. 76

2.7 References………………………………………………………………………….. 78
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CHAPTER 3: Online high-performance size exclusion chromatography-… 89
nuclear magnetic resonance for the characterization of dissolved
organic matter

3.1 Abstract……………………………………………………………………………… 90

3.2 Introduction………………………………………………………………………… 91

3.3 Materials and methods…………………………………………………………… 93
3.3.1 Sample collection and preparation………………………………………….. 93
3.3.2 HPSEC separation…………………………………………………………… 94
3.3.3 Fraction collection…………………………………………………………… 95
3.3.4 Solution 1H NMR……………………………………………………………. 95

3.4 Results and discussion…………………………………………………………… 96
3.4.1 Online techniques…………………………………………………………… 96
3.4.2 Effects of concentration……………………………………………………... 102
3.4.3 Structural information……………………………………………………….. 104
3.4.4 Variability of environmental samples……………………………………….. 106

3.5 Acknowledgements……………………………………………………………….. 109

3.6 References…………………………………………………………………………... 110

CHAPTER 4: HILIC-NMR: toward the identification of individual…….... 114
molecular components in dissolved organic matter

4.1 Abstract……………………………………………………………………………… 115

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4.2 Introduction………………………………………………………………………… 116

4.3 Experimental …………………………………………………………………….. 119
4.3.1 HILIC separation…………………………………………………………..... 119
4.3.2 NMR analysis……………………………………………………………….. 120
4.3.3 Fluorescence analysis……………………………………………………….. 121
4.3.4 Statistical Analyses…………………………………………………………. 121

4.4 Results and discussion……………………………………………………………. 122
4.4.1 HILIC separation and NMR analyses………………………………………. 122
4.4.2 Fluorescence analysis……………………………………………………….. 129
4.4.3 Statistical analysis of NMR and PARAFAC……………………………...... 130

4.5 Acknowledgements……………………………………………………………….. 133

4.6 References…………………………………………………………………………... 134

CHAPTER 5: Oxidized sterols as a significant component of dissolved….. 138
organic matter: evidence from 2D HPLC in combination with
2D and 3D NMR spectroscopy

5.1 Abstract……………………………………………………………………………… 139

5.2 Introduction………………………………………………………………………… 140

5.3 Experimental ……………………………………………………………………... 142
5.3.1 2D-HILIC/HILIC separation………………………………………………… 142
5.3.2 NMR analysis...……………………………………………………………… 148

5.4 Results and discussion………………………………………………………….... 151
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5.4.1 One dimensional 1H NMR………………………………………………….. 151
5.4.2 Identification of small acids………………………………………………… 154
5.4.3 Evidence of sterols from DIPSI, DOSY and selective TOCSY
experiments……………………………………………………………….. 154
5.4.4 Evidence of sterols with three dimensional NMR…………………….…..... 162
5.4.5 NMR spectral predictions for sterols………………………………….…….. 163
5.4.6 Variability in structures……………………………………………….…….. 169
5.4.7 Further considerations………………………………………………….….... 171

5.5 Acknowledgements………………………………………………………….……. 173

5.6 References………………………………………………………………………….. 174

CHAPTER 6: Conclusions and future directions……………………….……… 185

6.1 Conclusions and future directions………………………………….…….…….. 186

6.2 Future Arctic research……………………………………………………….…… 187

6.3 Theoretical approaches…………………………………………………………... 188

6.4 Sterol oxidation to identify possible end-members………………………… 189

6.5 Targeted approaches………………………………………………….…………… 190

6.6 References………………………………………………………………….……….. 192

APPENDIX……………………………………………………………………….……….. 194

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