The Development of In-situ NMR Photoreactors and Analysis of
Photochemical Processes in the Environment
by
Liora Bliumkin
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Chemistry
University of Toronto
© Copyright by Liora Bliumkin (2016)
ii
The Development of In-situ NMR Photoreactors and Analysis of
Photochemical Processes in the Environment
Liora Bliumkin
Master of Science
Department of Chemistry
University of Toronto
2016
Abstract
Photolysis is a major abiotic process in the environment. Current understanding of
environmental photolytic processes is limited due to restricted information offered by
conventional analytical techniques and lack of in-situ studies. In-situ nuclear magnetic resonance
(NMR) photoreactors were developed to directly integrate light sources with NMR spectroscopy
to probe into environmental photochemistry in a non-invasive manner. They were applied to a
series of environmental systems including an atmospheric pollutant, crude oil extracts,
groundwater (at natural abundance), and dissolve organic matter (DOM). Intermediates and
degradation products were identified along with kinetic profiles of specific compounds in
complex environmental mixtures. It was also shown to be a great non-invasive chromatographic
tool to investigate the phototransformation of DOM. Also, two dimensional (2D) NMR
experiments were used to characterize and quantify components in DOM. Overall, the work
demonstrates that in-situ NMR spectroscopy is an important analytical tool in unraveling
complex environmental photolytic processes.
iii
Acknowledgements
First and foremost I would like to thank my supervisor, Professor André J. Simpson, for
the opportunity to work on this project as well as his guidance and insight which made this
research possible. I also would like to thank my supervisory committee members Professor
Myrna Simpson and Professor Jon P. D. Abbatt for their helpful advice and suggestions. In
particular, I would like to thank Professor Myrna Simpson for being my second reader and
Professor Jon P. D. Abbatt for sharing his wisdom on the photochemistry of atmospheric
pollutants and providing samples.
I would like to thank Dr. Ronald Soong for his assistance with technical help involving
setting up NMR experiments and his advice along the way. I want to acknowledge Dr. Ran Zhao
for his valuable input and sharing his knowledge on the photochemistry of atmospheric
pollutants. Also, I want to thank Dr. Eric Reiner for providing samples. A special gratitude to
both Simpson groups (past and present members) for their encouragements and assistance as
well as the welcoming and enjoyable atmosphere in the lab. Daniel Lane-Coplen thank you very
much for your help fixing the Suntest system and the HPLC pump. I would not been able to
finish my research without your help. I would also like to thank the Krembil Foundation for
providing funding.
Lastly, I am grateful for my family’s and friends’ continued support and encouragement
throughout my MSc program.
iv
Table of Contents
Acknowledgments………………………………………………………………………………..iii
List of Figures…………………………………………………………………………………...viii
List of Abbreviations………………………………………………………………………..........xi
List of Appendices………………………………………………………………………….…...xiv
Preface………………………………………………………………………………………........xv
1 Chapter 1 - Introduction………………………………………………………………………...1
1.1 Overview……………………………………………………………………………….......1
1.2 Environmental Photochemistry…………………………………………………………….2
1.3 Common analytical techniques used to study environmental photochemistry……………4
1.3.1 Optical Spectroscopy…………………………………………………………..........5
1.3.2 Fluorescence Spectroscopy…………………………………………………............5
1.3.3 Mass spectrometry (MS)……………………………………………………………6
1.4 Nuclear magnetic resonance (NMR) spectroscopy: a novel technique in environmental
studies……………………………………………………………………………………...7
1.4.1 Basics of NMR spectroscopy……………………………………………………….8
1.4.2 Solution-state Proton NMR spectroscopy…………………………………………..9
1.4.3 Analysis of complex samples using diffusion-editing techniques…………...........11
1.4.4 Multidimensional NMR spectroscopy…………………………………………….13
1.4.4.1 1H-1H Correlation Spectroscopy (COSY)………………...………………........14
1.4.4.2 1H-13
C Heteronuclear Single Quantum Coherence (HSQC) and
edited-HSQC…………………………………………………………………...14
1.4.4.3 1H-1H 2D Total Coherence Spectroscopy (TOCSY) and
v
selective 1D TOCSY…………………………………………………………...15
1.5 The importance of analyzing photochemical reactions of natural
organic compounds and pollutants in the environment…………………………………...16
1.5.1 Photolysis of atmospheric pollutants……………………………………………....16
1.5.2 Photolysis of soil contaminants and groundwater………………………………....17
1.5.3 Photolysis of surface water pollutants……………………………………………..19
1.5.4 Dissolved organic matter (DOM)………………………………………………….20
1.6 Research Objectives…………………………………………………………………........21
1.7 References………………………………………………………………………………...23
2 Chapter 2 – The development of an in-situ NMR photoreactor to
study environmental photochemistry……………………………………………………...…35
2.1 Abstract…………………………………………………………………………...…........35
2.2 Introduction……………………………………………………………………………….36
2.2.1 Current techniques used in photochemical analysis………………………………36
2.2.2 Why NMR Spectroscopy? An important tool in environmental research………..37
2.3 Experimental Section……………………………………………………………………..39
2.3.1 Light Sources and Optical Fiber……………………………………………...……39
2.3.2 Chemical Actinometry and Calibration of the Suntest…………………………….41
2.3.3 Sample Preparation………………………………………………...………………41
2.3.4 In-situ photolysis analysis on NMR Spectroscopy using
OceanOptics HPX-2000 and PX-2………………………………………………...42
2.3.5 In-situ photolysis analysis on NMR Spectroscopy using
vi
Original Hanau Suntest model…………………………………………………….43
2.4 Results and Discussion……………………………………………………………………44
2.4.1 Comparison of different light sources……………………………………………..44
2.4.2 Photooxidation and mineralization of an atmospheric pollutant………………….48
2.4.3 Oil spills: the fate of water soluble fraction (WSF) of crude oil upon
exposure to light…………………………………………………………...………50
2.4.4 Monitoring photochemical changes of groundwater at natural abundance………55
2.5 References…………………………………………………………………………….......59
3 Chapter 3 – Analysis of DOM phototransformation using a looped NMR system
integrated with a sunlight simulator…………………………………...…………..66
3.1 Abstract…………………………………………………………………………………...66
3.2 Introduction……………………………………………………………………………….67
3.3 Experimental Section……………………………………………………………………..70
3.3.1 Light source………………………………………………………………………..70
3.3.2 Sample Preparation………………………………………………………………..70
3.3.3 In-situ analysis of DOM photolysis using diffusion-editing NMR experiments....70
3.3.4 DOM photolysis analysis using 2D NMR experiments……………………….......72
3.4 Results and Discussion…………………………………………………………………...73
3.4.1 Monitoring the phototransformation of DOM using in-situ NMR spectroscopy...73
3.4.2 Using diffusion-editing NMR as a chromatographic tool to study DOM………....77
3.4.3 Monitoring the photolytic fate of specific compounds using in-situ
1H NMR spectroscopy…………………………………………………...………...78
vii
3.4.4 2D NMR identification of biochemical classes and specific metabolites
in DOM…………………………………………………………………...………..80
3.5 References…………………………………………………………………………….......86
4 Chapter 4 – Conclusion and Future Directions………………………………………………93
4.1 Light sources and system design: potential and limitations……………………………....93
4.2 Evaluating aqueous photochemical processes using in-situ NMR spectroscopy………...94
4.3 Monitoring the photolytic fate of dissolved organic matter (DOM) using an in-situ NMR
photoreactor……………………………………………………………………………….95
4.4 Future Directions………………………………………………………………………….96
4.4.1 Parallel acquisition and dual receivers………………………………………….....97
4.4.2 Influence of DOM on the photodegradation of organic contaminants…………....97
4.4.3 Combining in-vivo NMR with in-situ NMR photoreactors……………………….98
4.4.4 Combining MS with in-situ NMR spectroscopy…………………………………100
4.4.5 In-situ photoirradiation of pesticides using solid-state and
comprehensive multiphase (CMP) NMR spectroscopy…………………...……..101
4.5 References……………………………………………………………………………….103
5 Appendix A – Supporting information for Chapter 2………………………………………107
6 Appendix B – Supporting information for Chapter 3………………………………...…….139
7 Appendix C – Copyrights and Permissions……………………………………………..….162
viii
List of Figures
Figure 1-1. Proposed photodegradation pathway of chloroacetanilides, such as metolachlor, in
the absence (A) and presence (B) of DOM. Reprinted with permission from Wilson, R. I.;
Mabury, S. A. Photodegradation of metolachlor: isolation, identification, and quantification of
monochloroacetic acid. J. Agric. Food. Chem. 2000, 48(3), 944-950. Copyright 2015 American
Chemical Society.
Figure 1-2. A) depicts the SPR-W5-WATERGATE sequence. Selective pulses are depicted by
an open ‘shape’, whereas hard pulses are indicated by solid blocks. B) shows 2mM sucrose in
90%/10% H2O/D2O without any solvent suppression. C) using a basic pre-saturation as a
comparison, and D) using the SPR-W5-WATERGATE sequence with the parameters optimized
for natural water samples. Reproduced from Lam, B.; Simpson, A. J. Direct 1H NMR
spectroscopy of dissolved organic matter in natural waters. Analyst. 2008, 133(2), 263-269 with
permission from The Royal Society of Chemistry.
Figure 1-3. Pulse sequences for a diffusion-editing NMR experiment used for demonstrative
purposes. Pulse sequences for editing (a) 1H NMR spectra using a combination of T1 and T2
relaxation times with solvent suppression using field gradients and (b) 1H NMR spectra based on
differences in diffusion coefficients and T2 relaxation times (DIRE). Sequence b incorporates the
WATERGATE solvent elimination sequence. The narrow bars are 90° pulses, the open
rectangles are 180° pulses, G¢ is a rectangular z-direction magnetic field gradient, the vertical
hatched rectangles comprise the “3-9-19-19-9-3” 180° pulse sequence used in the
ix
WATERGATE solvent suppression sequence, and G are sineshaped z-direction magnetic field
gradients. The figure is adapted with permission from Liu, M.; Nicholson, J. K.; Lindon, J. C.
High-resolution diffusion and relaxation edited one- and two-dimensional 1H NMR spectroscopy
of biological fluids. Anal. Chem. 1996, 68(19), 3370-3376. Copyright 2015 American Chemical
Society.
Figure 1-4. A) Proposed photodegradation pathway of florasulam in soil. B) Proposed
photodegradation pathway of florasulam in water. The figure is adapted with permission from
Balmer, M. E.; Goss, K. U.; Schwarzenbach, R. P. Photolytic transformation of organic
pollutants on soil surfaces - an experimental approach. Environ. Sci. Technol. 2000, 34(7), 1240-
1245. Copyright 2015 American Chemical Society.
Figure 2-1. Schematics, NMR data, and kinetic information for the photodegradation of the
reference sample, 34.52 mM riboflavin solution, using three different light sources.
Figure 2-2. 1H spectra of p-nitrophenol (7.74 mM) and its photoproducts at three different time
points during the light exposure inside the Suntest.
Figure 2-3. Phototransformation of WSF of crude oil with HPX-2000 (right) and Suntest (left)
light sources. A and C are prior to light exposure and B and D are following light exposure.
x
Figure 2-4. A: final 1H spectrum of groundwater sample (TOC: 1.96 ppm) after 6 hours in the
dark (0-4.5 ppm region). B: 1H spectrum of groundwater after the sample was exposed to light
for 1 day and 12 hours inside the Suntest solar simulator.
Figure 3-1. The % photomineralization of different DOM fractions, at each day relative to day 0
(=”light-off”, prior to light exposure), over the course of 5 days using in-situ NMR photoreactor.
Figure 3-2. Kinetic plot for acetone and carboxylic acid products from three DOM sources over
five days of photoirradiation.
Figure 3-3. A: 1H-
13C HSQC NMR spectra of Suwannee River NOM prior to light exposure. B:
1H-
13C HSQC NMR spectra of Suwannee River NOM following light exposure.
Figure 3-4. 2D COSY NMR spectrum of Pony Lake Fulvic Acid prior to light exposure.
Metabolites were matched with Woods, G. C. et al., 2011 and with AMIX against Bruker
Biofluid Reference Compound Database.
Figure 3-5. 2D COSY NMR spectrum of Pony Lake Fulvic Acid following a month of
photoirradiation. Metabolites were matched with Woods, G. C. et al., 2011 and with AMIX
against Bruker Biofluid Reference Compound Database.
xi
List of Abbreviations
1D One Dimensional
2D Two Dimensional
3D Three Dimensional
AMIX Analysis of MIXtures
BTEX Benzene, Toluene, Ethylbenzene, Xylene
CDOM Chromophoric Dissolved Organic Matter
CMP-NMR Comprehensive MultiPhase Nuclear Magnetic
Resonance
COSY COrrelation SpectroscopY
CRAM Carboxyl-Rich Alicyclic Molecules
DE Diffusing Editing
DEPT-HSQC 2D Distortionless Enhancement by Polarization
Transfer - Heteronuclear Single Quantum Coherence
Spectroscopy
DIPPMPO 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-
oxide
DOM Dissolved Organic Matter
DSR Downward Surface Radiation
EPR spectroscopy Electron Paramagnetic Resonance spectroscopy
FA Fulvic acid
FID Free Induction Decay
xii
GC Gas Chromatography
HPLC High Performance Liquid Chromatography
HSQC Hetero-nuclear Single-Quantum Coherence
IHSS International Humic Substances Society
INEPT Insensitive Nuclei Enhanced by Polarization Transfer
MAS Magic Angle Spinning
MDLT Material Derived from Linear Terpenoids
MS Mass Spectrometry
NMR Nuclear Magnetic Resonance
NOSEY Nuclear Overhauser effect spectroscopy
PAHs Polycyclic Aromatic Hydrocarbons
PPCP Pharmaceuticals and Personal Care Products
ROS Reactive Oxygenated Species
S/N Signal-to-Noise
SCA Short Chain organic Acids
SEI SElective Inverse
SOA Secondary Organic Aerosol
SPE Solid Phase Extraction
SPR-W5-WATERGATE Shaped PResaturation-WATER suppression by
GrAdient Tailored Excitation with W5 pulse trains
SW ShortWave
TCI Triple resonance Carbon Inverse
TOC Total Organic Carbon
xiii
TOCSY Total Correlation SpectroscopY
TXI Triple resonance Inverse
WHO World Health Organization
WSF Water Soluble Fraction
xiv
List of Appendices
Appendix A Supporting information for Chapter 2………………………………………108
Appendix B Supporting information for Chapter 3…………………………………….140
Appendix C Copyrights and Permissions………………………………………………162
xv
Preface
Chapter 2 of this thesis was submitted to a peer-reviewed journal while Chapter 3 is a
manuscript that is being prepared for submission. Therefore, this thesis may contain unavoidable
repetition. Contributions from authors are as follows:
CHAPTER 1:
Introduction
Written by Liora Bliumkin with critical comments from André J. Simpson and Myrna J.
Simpson.
CHAPTER 2:
The development of an in-situ NMR photoreactor to study environmental photochemistry
Authors: Liora Bliumkin, Ronald Soong, Jon P.D. Abbatt, Ran Zhao, Eric Reiner, and André J.
Simpson. Submitted for publication.
The experimental design was developed by Liora Bliumkin and André J. Simpson. The
experiments were conducted by Liora Bliumkin with help from Ronald Soong and André
J. Simpson. Data interpretation was performed by Liora Bliumkin and André J. Simpson.
The manuscript was written by Liora Bliumkin with critical comments from André J.
Simpson, Jon P.D. Abbatt, Ran Zhao, and Eric Reiner.
CHAPTER 3:
Analysis of DOM phototransformation using a looped NMR system integrated with a
sunlight simulator
The experimental design was developed by Liora Bliumkin and André J. Simpson. The
experiments were conducted by Liora Bliumkin with help from Daniel Lane-Coplen and
André J. Simpson. Data interpretation was performed by Liora Bliumkin and André J.
Simpson. The manuscript was written by Liora Bliumkin with critical comments from
André J. Simpson.
CHAPTER 4:
Conclusion and future directions
Written by Liora Bliumkin with critical comments from André J. Simpson.
1
1 Chapter 1 – Introduction
1.1 Overview
This thesis focuses on the development and application of in-situ NMR-based
photochemical reactors to investigate the photolytic fate of natural and anthropogenic pollutants,
groundwater, and dissolved organic matter (DOM) in the environment with high temporal
resolution. The environment is a highly complex and dynamic system with chemical, biological,
and physical processes all playing critical roles. Compounds are constantly broken down and
new ones are formed. Photochemistry is the most important abiotic process determining the fate
of compounds in the atmosphere, soil and plants, and aquatic ecosystems. However, much of the
chemistry is still poorly understood. A better understanding of environmental photolytic
processes can provide a deeper understanding as to the fate and transformation of compounds in
the environment. Moreover, humans can leverage this knowledge to their advantage by utilizing
solar radiation for the production of chemical goods and breakdown of anthropogenic pollutants.
Multiple analytical techniques have been previously utilized to study environmental
photochemistry, including mass spectrometry (MS)1, optical spectroscopy
2,3, and nuclear
magnetic resonance (NMR) spectroscopy1. However, MS and optical spectroscopy may require
chemical derivatization to analyze complex environmental samples and often offer limited
structural information. The ability of NMR spectroscopy to analyze a sample in a non-invasive
manner, along with its ability to solve de-novo chemical structure, makes it an important
technique for environmental research. NMR has also been shown to be an excellent tool to
follow the progress of chemical reactions.4 However, previous studies were performed ex-situ
with low temporal resolution which could be problematic when elucidating a reaction’s
2
mechanism especially if reactive and short-lived intermediates are formed or rapid reactions take
place.
This thesis first compares three different designs of in-situ NMR photoreactors from
relatively cheap xenon arc lamps with optical fibers (light is fed directly into the NMR) to a
simple looped flow NMR system integrated with a sunlight simulator (light source outside of the
spectrometer). The in-situ NMR photoreactors are then applied to a series of environmental
systems, including individual compounds, crude oil extracts, and groundwater to assess their
applicability to a wider range of environmental systems followed by the investigation of the
phototransformation processes of DOM. The research presented in this thesis demonstrates that
environmental photolytic reactions can be monitored in-situ and in real time using NMR
spectroscopy. The in-situ NMR photoreactor systems developed offer high temporal resolution in
an automated fashion providing a range of valuable information such as structural
characterization of intermediates and products as well as kinetic information. The work
presented here demonstrates that an in-situ NMR photoreactor is an important tool in unraveling
environmental photolytic processes providing complimentary information to more conventional
analytical approaches.
1.2 Environmental Photochemistry
Solar radiation is a primary source of energy on Earth. As such, photolysis is a major
abiotic process that plays an important role in the biogeochemistry of the environment.5,6
Photochemical processes at the Earth’s surface are dominated by the absorption of photons
between 290-600 nm by chromophores (eg. conjugated double bonds) that can undergo π→π∗ or
n→π∗ transitions to become photochemically active.7 Energy transfer within the system may
3
induce chemical changes such as mineralization (conversion to CO2 and H2O) and
photogeneration of new compounds via bond cleavage, isomerization, rearrangement or
intermolecular reactions.8 Photochemical processes are commonly divided into direct and
indirect photolysis. In direct photolysis, a chromophore directly absorbs photons that trigger a
chemical change.7,9
In contrast, indirect photolysis involves a reactive transient such as ·OH,
nitrate, and singlet oxygen that initiates a chemical reaction.7,10
Both direct and indirect photolysis can have either a beneficial or harmful impact on the
environment. Amino acids and DOM are both susceptible to photodegradation which can
influence nutrition availability in aquatic ecosystems and result in oxidative damage of
extracellular proteins of living organisms.11
Also, certain water pollutants, such as
pharmaceuticals and personal care products (PPCPs) and pesticides, have been shown to produce
toxic products upon irradiation. For instance, metolachlor, a widespread herbicide in the
environment, produces phytotoxic products such as monochloracetic acid in the presence of
DOM (Figure 1-1).12
A) B)
Figure 1-1. Proposed photodegradation pathway of chloroacetanilides, such as metolachlor, in
the absence (A) and presence (B) of DOM. Reprinted with permission from Wilson, R. I.;
Mabury, S. A. Photodegradation of metolachlor: isolation, identification, and quantification of
monochloroacetic acid. J. Agric. Food. Chem. 2000, 48(3), 944-950. Copyright 2015 American
Chemical Society.12
4
Sunlight also holds a great importance as a renewable and sustainable energy source.13
Solar
radiation can be utilized as a green remediation alternative to current wastewater treatment
methods. Incorporation of photooxidative techniques can help reduce sludge generation from
biological treatment as well as toxic products following chlorination.14,15
Furthermore, many
plants and organisms depend on sunlight to carry out cellular metabolic processes.
Photochemical reactions are also an important part of atmospheric chemistry that can have an
enormous impact on the composition of the atmosphere, air quality, and human health.16
1.3 Common analytical techniques used to study environmental photochemistry
Fluorescence spectroscopy, optical spectroscopy, and MS are commonly used to study
photolysis as they offer high time resolution and sensitivity.6 Information on chemical properties
such as quantum yields2 can be obtained using optical spectroscopy while fluorescence
spectroscopy and MS can provide structural information for relatively simple mixtures17
.
However, such approaches may require extensive sample extraction, isolation and potentially
derivatization for analysis.18
Such treatments can remove important information on conformation
and layering as well as lead to chemical fractionation which has the potential to drastically
change the photoreactivity of the sample over the true natural state. The selective nature of these
analytical approaches is also a limiting factor, providing limited insight into environmental
processes at a molecular level.19,20
The limitations of these conventional analytical techniques are
discussed below. Special considerations must be taken into account in order to monitor the
progress of environmental photolytic reactions in a non-invasive and indiscriminate fashion.
5
1.3.1 Optical spectroscopy
Optical properties such as refractive index and absorption characteristics can be used for
structural elucidation of photodegradation products. UV-Vis spectroscopy can be further used to
measure photolytic rates, estimating lifetimes, and to elucidate a reaction’s mechanism for
relatively simple compounds.2 However, spectral overlap is a common issue with environmental
samples due to their complexity.21
UV-Vis spectra are broad and thus it becomes challenging to
unambiguously differentiate absorption peaks between different structures, even in a simple
system such as Cl2 and ClOOCl.3 Another issue that must be taken into account is the frequent
discrepancies between different research groups of absorption cross sections that are used for
kinetic measurements such as rate constants.2,3
1.3.2 Fluorescence spectroscopy
Fluorescence spectroscopy is a more selective, highly sensitive, and non-destructive
alternative to UV-Vis spectroscopy in the study of photolytic processes in the environment.19,20
Only a few chemicals exhibit fluorescence, but they do have their own unique excitation
wavelength which reduces spectral overlap and matrix effect.20
The fluorescence signal is
strongly influenced by temperature, pH, polarity, and concentration of the sample which
provides detailed conformation and structural information. For example, two tryptophan
molecules located at different sites of a protein will display a different fluorescence spectra due
to their different local environment.22
However, certain compounds, such as naphthenic acids,
may florescence in acidic pH but not in a basic environment.23
Also, the detection of various
chemicals, especially high molecular-weight compounds, in a complex sample is limited by
6
fluorescence.20
Analysis of photochemical reactions by fluorescence is highly dependent on the
structural features of a compound than it is with UV-Vis spctroscopy.20
1.3.3 Mass spectrometry (MS)
MS is commonly used in environmental studies for exposure analysis and risk
assessments as the coupling of high performance liquid chromatography (HPLC)/gas
chromatography (GC) with MS is considered to be one of the most useful techniques for the
determination, identification, and quantification of natural and anthropogenic contaminants in
complex environmental systems (eg. air, water, soil).24,25
MS is a great analytical technique for
targeted analysis. It requires careful selection of solvents, column type, ionization technique, MS
detector, and sample preparation technique which can differ from sample to sample. It becomes
challenging to use HPLC/GC-MS for non-targeted analysis and specifically when analyzing an
unknown environmental sample because: 1) MS can only detect those ionized molecules that
have reached the detector26
, 2) the sample may contain reactive species; thus leading to the
detection of the decomposition products and not the precursors by HPLC/GC-MS26
, 3)
compounds, and/or their photoproducts, can irreversibly bind to the stationary phase or interact
with the mobile phase,26
4) many environmental systems cannot be easily analyzed in their
natural state. For instance, the site of each carbohydrate in glycoproteins could not be
differentiated from mass spectra following the removal of protein from the carbohydrates during
sample preparation.27
On the other hand, NMR is a great tool for non-targeted analysis of
environmental processes in their natural state.6 The higher resolution (MS: ~8,000,000,
1H NMR:
~2500)28
and low detection limit (~pM with the ability to detect molecules of low zeptomoles)29
offered by MS makes it a great complementary tool to NMR.
7
GC-MS commonly uses electron impact ionization which results in very reproducible
fragmentation patterns. Libraries have been developed that contain fragmentation patterns for
millions of compounds. As such, GC-MS is a very effective tool for identifying unknowns as
long as the compounds are in the database and are thermally stable. HPLC-MS however uses a
wide array of ionization approaches which lead to different fragmentation and isotope patterns
from laboratory to laboratory.30
Therefore, HPLC-MS databases are less developed making
identification of unknowns more challenging. Also, various MS detectors are coupled with
HPLC which makes it difficult to have a library like with GC.30
1.4 Nuclear magnetic resonance (NMR) spectroscopy: a novel technique in
environmental studies
The non-invasive nature of NMR spectroscopy makes it a powerful tool in environmental
studies as it can be used for structural elucidation and investigation of complex environmental
processes in their natural state.6 For instance, partitioning, equilibrium, and micelle formation
can all be analyzed using NMR spectroscopy.6,31,32
NMR spectroscopy can be used to analyze
gaseous, solution, solid, gel-like, and even heterogeneous samples such as soil demonstrating its
wide applicability in environmental chemistry.33
Moreover, as a non-selective tool, it offers
efficient and indiscriminate information that can be missed by conventional techniques.34
It is a
versatile, robust, and highly reproducible technique that requires little to no sample preparation
and can be used for both qualitative and quantitative analysis.35,36
For example, radical adducts
of 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DIPPMPO) can be identified and
quantified using NMR but are left undetected by electron paramagnetic resonance (EPR)
spectroscopy and GC-MS.37
Furthermore, the use of multidimensional NMR does not only
8
reduce spectral overlap but also provides a variety of molecular information from structural
composition, dynamics, and isomeric discrimination to intermolecular and intramolecular
interactions.28
For example, enantiomers can be easily differentiated by two dimensional (2D)
NMR but not by MS.38
As such, NMR spectroscopy has become an important analytical tool for
the investigation of environmental photolytic processes.
1.4.1 Basics of NMR spectroscopy
NMR spectroscopy is a key analytical tool in understanding environmental samples and
processes at a molecular level. It centers around the interaction of electromagnetic radiation with
matter. A sample is immersed in an external magnetic field (B0) where the atomic nuclei align
with or against B0 based on their nuclear spin. The nuclei are simultaneously excited as the
sample is subjected to brief pulses of radio-frequency (RF) radiation which rotate the magnetic
vectors 90o such that they are parallel to the main field and induce a sinusoidal current in the
detector coil. The Free Induction Decay (FID), is detected by the receiver coil surrounding the
sample as the nuclei relax back to eventually re-aligning with the main Bo field. The time domain
data is then converted to a frequency domain data by Fourier Transformation. Multiple “scans”
are often accumulated in each experiment to improve the signal to noise.39,40
The chemical shifts
in an NMR spectrum correspond to the frequencies at which the atomic nuclei resonate at due to
the different local environment that they experience. Interactions between bonded nuclear spins
give rise to spin-spin coupling effect that can be utilized for structural elucidation and for a
deeper understanding of the chemical dynamics of a sample.40
Consequently, photochemical
reactions can be analyzed using NMR spectroscopy by monitoring changes in chemical shifts
and splitting patterns over time as even subtle changes in a nuclei’s chemical environment will
9
result in a different shielding effect.41
Additionally, integration of peak areas allows for
quantitative analysis of chemical reactions. For fully quantitative data the recycle delay needs to
be >5xT1 to ensure complete relaxation between scans.42
1.4.2 Solution-state Proton NMR spectroscopy
High spectral resolution can be obtained with solution-state NMR due to the random and
rapid tumbling of nuclei spins that average out anisotropic NMR interactions. Proton’s natural
abundance of 99.8% and the higher spectral dispersion (~2500 in 1H NMR in comparison to ~20
for UV-Vis spectroscopy) , spin-spin coupling information, and quantitative reliability of 1H
NMR are key for targeted and non-targeted analysis of complex environmental samples.28
A re-occurring issue with environmental samples is the water resonance that saturates the
NMR receiver and dominates the NMR spectrum. This becomes a significant problem in NMR
spectroscopy as many aqueous environmental samples have a low abundance of natural organic
matter (1-2 ppm in groundwater)43
, thus, hindering the detection of signals from the sample. In
this thesis, the problem was overcome with the use of a water suppression technique called SPR-
W5-WATERGATE with a perfect echo (Figure 1-2).44-46
The SPR saturates the water proton
signal and W5 inverts all signals except for water. A pair of gradients refocus the inverted signal
magnetization from the solute while de-phasing the water signal that was not flipped (i.e
water).42
At the same time, it reduces baseline distortions and maximizes signal detection by
permitting the NMR receiver to be optimally set. As a result, the high sensitivity obtained using
water suppression techniques and the high resolution with solution-state NMR permits structural
elucidation of biological molecules present in low concentrations.43
10
Figure 1-2. A) depicts the SPR-W5-WATERGATE sequence. Selective pulses are depicted by
an open ‘shape’, whereas hard pulses are indicated by solid blocks. B) shows 2mM sucrose in
90%/10% H2O/D2O without any solvent suppression. C) using a basic pre-saturation as a
comparison, and D) using the SPR-W5-WATERGATE sequence with the parameters optimized
for natural water samples. Reproduced from Lam, B.; Simpson, A. J. Direct 1H NMR
spectroscopy of dissolved organic matter in natural waters. Analyst. 2008, 133(2), 263-269 with
permission from The Royal Society of Chemistry.46
11
Another challenge with 1H NMR experiments is the peak overlap in complex environmental
samples.47-49
A series of multidimensional experiments have been previously developed to
overcome this challenge and provide additional information on molecular composition and
molecular interactions. This will be discussed later in section 1.4.4.
1.4.3 Analysis of complex samples using diffusion-editing techniques
Diffusion-editing (DE) builds upon a spin-echo sequence with the addition of two
gradient pulses that are identical in amplitude and width. These gradient pulses are able to
suppress resonances from small molecules while highlighting signals from rigid components by
de-phasing the resonances using the first gradient pulse and then re-phasing them at the end with
the second gradient pulse (Figure 1-3).50,51
The magnetization of molecules that have diffused
within the NMR tube during the diffusion delay (Δ) are not re-phased and essentially their signal
decreases and cannot be observed in the spectrum. The observed signals originate from
molecules that experienced little to no self-diffusion.
12
Figure 1-3. Pulse sequences for a diffusion-editing NMR experiment used for demonstrative
purposes. Pulse sequences for editing (a) 1H NMR spectra using a combination of T1 and T2
relaxation times with solvent suppression using field gradients and (b) 1H NMR spectra based on
differences in diffusion coefficients and T2 relaxation times (DIRE). Sequence b incorporates the
WATERGATE solvent elimination sequence. The narrow bars are 90° pulses, the open
rectangles are 180° pulses, G¢ is a rectangular z-direction magnetic field gradient, the vertical
hatched rectangles comprise the “3-9-19-19-9-3” 180° pulse sequence used in the
WATERGATE solvent suppression sequence, and G are sineshaped z-direction magnetic field
gradients. The figure is adapted with permission from Liu, M.; Nicholson, J. K.; Lindon, J. C.
High-resolution diffusion and relaxation edited one- and two-dimensional 1H NMR spectroscopy
of biological fluids. Anal. Chem. 1996, 68(19), 3370-3376. Copyright 2015 American Chemical
Society.52
When applied to an environmental sample such as rainwater this approach helps
discriminate between different fractions (small metabolites and macromolecules) without
altering the sample.53
A “diffusion-editing” NMR spectrum only contains 1H NMR signals from
rigid and large molecular weight structures while a NMR spectrum with signals from small
13
molecules and soluble components is referred to as “inverse diffusion-editing” NMR spectrum.
This becomes important when analyzing complex environmental samples such as DOM where
certain peaks may be masked by the overlapping peaks in a simple 1H spectrum.
54 Also, it aids in
differentiating between resonances that originate from macromolecular and small metabolites
and monitoring the changes of these fractions over the course of a chemical reaction.54
1.4.4 Multidimensional NMR spectroscopy
A common issue in environmental research is a sample’s complexity that results in
overlapping signals in the one dimensional (1D) NMR spectra. NMR spectroscopy can overcome
this obstacle by dispersing the signal into multiple dimensions. Three dimensional (3D) NMR
experiments for example approach a peak capacity of 100,000,000.28
Multidimensional NMR
experiments not only provide additional dispersion but also additional information that varies
depending on the experiment but could include, couplings through bonds, interactions through
space, diffusion coefficients, and dynamics. Multiple dimensional experiments allow for analysis
of complex and heterogeneous samples such as lignin, soil and dissolved organic matter.35,55,56
For instance, 2D NMR experiments have been shown to be very useful in the characterization of
soil organic matter.57,58
3D NMR data have also been used to identify the major components in
dissolved organic, itself reported as the most complex mixture known.59
Multidimensional NMR experiments provide unequivocal structural information,
including isomeric and stereoisomeric discrimination, as well insight into molecular interactions,
diffusion, dynamics and conformation. For instance, 1H-
1H correlation spectroscopy (COSY) and
nuclear Overhauser effect spectroscopy (NOESY) experiments provided valuable information on
14
the molecular structure, mechanism, and regiospecificity of the photochemical cyclization
reaction of N-(9-Oxothioxanthenyl)benzothiophene carboxamides.60
1.4.4.1 1H-
1H Correlation Spectroscopy (COSY)
COSY experiments have been often utilized to identify small metabolites in a complex
systems such as DOM and to characterize biomolecular effects of environmental stressors on
organisms such as Haliotis rufescens.47,55,61
This NMR technique correlates between adjacent
proton nuclei via magnetization transfer from one proton nuclei to the next on adjacent carbon
atoms within a single molecule.62
The data is then plotted in a space defined by F1 (ppm) and F2
(ppm) dimensions. The cross peaks provide information on spin-spin coupling between hydrogen
atoms in a molecule that can then be used for structural elucidation of unknown structures.63
For
instance, COSY experiments have been shown to be useful in the structural elucidation of
organic pollutants’ photoproducts, such as alloxydim herbicide, that is key in determining their
toxicity level in the environment.64
Advanced NMR software (AMIX, version 3.8.14, Bruker
BioSpin) can also be utilized to identify specific compounds by matching a 1D or 2D spectrum
against standards from Bruker Biofluid Reference Compound Database (v 2-0-0 to v 2-0-3).
1.4.4.2 1H-
13C Heteronuclear Single Quantum Coherence (HSQC) and edited-
HSQC
HSQC is another powerful NMR technique for structural elucidation. It correlates proton
atoms with their directly attached carbon atoms through magnetization transfer from a 1H nuclei
to a 13
C nuclei and back to the proton nuclei.62
The data is plotted in a similar manner as a COSY
spectrum where the proton chemical shifts are along the F2 dimension while the carbon chemical
15
shifts are plotted along the F1 axis. However, unlike COSY, it does not display a diagonal.
HSQC NMR techniques have been previously applied with DOM for compositional elucidation
and identification of carboxyl-rich alicyclic molecules (CRAM) components.65
Edited-HSQC
builds up on a simple HSQC experiment where -CH2- correlations can be distinguished from –
CH3 and -CH- because they are oppositely phased. This can help simplify a complex HSQC
spectrum of DOM and provide multiplicity information.
1.4.4.3 1H-
1H 2D Total Coherence Spectroscopy (TOCSY) and selective 1D
TOCSY
TOCSY experiments are very similar to COSY but display higher sensitivity, especially
for macromolecules.62
While COSY provides information on one bond correlations, a TOCSY
experiment also identifies long range correlations within a 1H-
1H spin system via spin-spin
coupling.62
The magnetization can be transferred over up to six bonds as long as coupling exists
between neighbouring protons. The observed crossed peaks in a 2D TOCSY spectrum are
dependent on the mixing time.62
Increasing mixing time provides longer range connectivity
information. Therefore, a TOCSY experiment with short mixing times may resemble a COSY
spectrum while longer mixing time identifies long range correlations. A selective 1D TOCSY
experiment is a useful technique in structural elucidation of a specific molecule in a complex
system. Like 2D TOCSY, a selective 1D TOCSY identifies long range correlations within a spin
system. However, in 1D TOCSY a specific resonance is selected to be excited from which the
magnetization is then transferred to all its J-coupled protons. J-coupled protons close to the
selected peak will display a higher multiplet intensity in comparison to protons that are further
16
away from the selected resonance.63
Thus, selective TOCSY can simplify a spectrum with
overlapping peaks and allow the investigator to focus on a specific molecule of interest.
1.5 The importance of analyzing photochemical reactions of natural organic
compounds and pollutants in the environment
Solar radiation is an important abiotic force that drives the chemistry in the atmosphere
(troposphere and stratosphere) and at the Earth’s surface (aqueous ecosystems, plant, and soil
surfaces). Chapter 2 monitors various aqueous photochemical processes that may take place in
the atmosphere and in natural waters while Chapter 3 focuses solely on surface water
photochemistry. The significance of atmospheric, aqueous, and terrestrial environmental
photochemistry are discussed here.
1.5.1 Photolysis of atmospheric pollutants
Solar energy is the driving force of many atmospheric processes. Thus, understanding
atmospheric photochemistry is important for several reasons: 1) modeling the photolytic fate of
atmospheric pollutants, 2) reducing uncertainties in atmospheric models, and 3) understanding
the impact of atmospheric photochemical reactions on climate and human health. Organic
aerosols have a significant impact on atmospheric pollution and atmospheric chemistry due to
their ability to absorb and scatter solar radiation.16
Also, water vapours, H2O2, NO3-, and NO2
-
are a major source of hydroxyl radicals that participate in atmospheric photochemical
processes.16
The lack of detailed knowledge on the photochemistry of atmospheric pollutants and
secondary organic aerosols (SOAs), in aqueous and heterogeneous phase, add to the lack of
correlation between field measurements of SOAs and atmospheric models.67
For example, it has
17
been shown that the mechanism of direct photolysis of pyruvic acid is dependent on its physical
state and its environment. When in gas phase, decarboxylation is the main degradation pathway
of pyruvic acid upon exposure to UV light.68
However, in aqueous phase, the mechanism as well
as the photoproducts formed are quite different.16
The ability to monitor chemical reactions in-
situ and in real time can deepen our understanding on the mechanism of aqueous-phase
photooxidation of atmospheric pollutants and provide insight into the atmospheric chemistry of
relevant photosensitive compounds.16
1.5.2 Photolysis of soil contaminants and groundwater
The application of agrochemicals and improper waste disposal are common
anthropogenic sources of toxic chemicals released into the environment that lead to soil and
groundwater contamination.69,70
In 1999, approximately 1.2 million tons of herbicides were
applied to crops in America alone.71
As the photodegradation of these chemicals are quite
different on soil surface than in an aqueous system, there is an increasing need to study the
photolytic mechanism and fate of these pollutants on soil before they are approved for use.71,72
For instance, the proposed photodegradation pathway and rate of florasulam, an herbicide used
in Canada and Europe, is different in soil due to the contribution of indirect photochemical
processes in water (Figure 1-4).72
18
A) B)
Figure 1-4. A) Proposed photodegradation pathway of florasulam in soil. B) Proposed
photodegradation pathway of florasulam in water. The figure is adapted with permission from
Balmer, M. E.; Goss, K. U.; Schwarzenbach, R. P. Photolytic transformation of organic
pollutants on soil surfaces - an experimental approach. Environ. Sci. Technol. 2000, 34(7), 1240-
1245. Copyright 2015 American Chemical Society.72
It has been a common practice to study the photodegradation of herbicides and pesticides in soil
by measuring the total loss of compound from spiked soil layers as a function of time.73
However, it is extremely important to also identify the intermediates and products formed in the
photodegradation of the parent compound for a more accurate assessment of a contaminant’s
toxicity and persistence in the environment. For instance, the photodegradation products of
carbamazepine were determined to be more toxic than the parent compound, demonstrating the
need for a better understanding of the photolytic fate of contaminants in the environment.74
Contaminated soil can also result in groundwater contamination, a main source of
drinking water; making it unsafe for human use.75
Some of the main sources of groundwater
contamination are leeching of industrial chemical waste discharge, landfills, and pesticides
through the soil.24
Groundwater contamination is not just a regional problem, but rather a
19
widespread problem since, along with water, contaminants, pollutants, sediment, and debris also
move through the rocks.24
In recent years, there has been a lot of interest in the application of
sunlight and advanced oxidation processes in water treatment as an efficient and cheap
remediation alternative.24,34
Analyzing the photolytic fate of molecules in groundwater can also
provide insight into the origin of chemicals found down the stream.
1.5.3 Photolysis of surface water pollutants
Many anthropogenic pollutants, such as PPCPs, can be detected in aqueous systems
worldwide as less than 50-90% of anthropogenic pollutants are broken down in wastewater
treatment plants.76
For example, PPCPs have been detected in 80% of tested rivers and lakes
throughout the United States.77
The release of these pollutants into the environment is a major
concern as accumulation of toxic chemicals and prolonged exposure can harm aquatic systems
and human health.78,79
Many of these anthropogenic pollutants that have persisted through
biological treatment in wastewater treatment plants will likely show no further bio-degradation in
wastewater waterways, but are known to be susceptible to photodegradation.80-82
For instance,
pharmaceutical drugs paracetamol and diclofenac that resisted bio-degradation have been shown
to be rapidly degraded by advanced photooxidative processes.83
Similarly, oil spills are a major
environmental issue in which toxic chemicals are continuously introduced into the environment.
Since the industrial revolution, there has been a widespread introduction of crude oil and
petroleum into aquatic environments. Oil spills are largely due to anthropogenic activities such
as oil spills from oil extraction, exploration, and transportation, and with a small percentage due
to natural causes.84
According to World Health Organization (WHO), BTEX (benzene, toluene,
ethylbenzene, and xylene), components found in petroleum and crude oil, are of main concern in
20
relation to drinking water standards.85
As such, it is important to investigate the photolytic fate of
these pollutants in the aquatic environment for an accurate evaluation of the environmental risks
that these anthropogenic pollutants pose. The photodegradation products may possess a higher
degree of toxicity than its parent compound.
1.5.4 Dissolved organic matter (DOM)
Photosensitizing species in natural waters such as chromophoric dissolved organic matter
(CDOM) have a large influence on the biogeochemistry of aquatic systems and on the
photodegradation of organic contaminants.80,86-88
DOM photolysis in itself signifies a large
carbon photomineralization process with increasing importance due to the melting of sea ice
cover as a result of climate change.89,90
DOM is a product of allochthonous (eg. terrestrial plant
material) and autochthonous (algal-derived material and microbial processed material in lakes)
sources and, therefore, its composition varies depending on the local environment.59,89,91
Consequently, its photosensitizing properties are highly dependent on its molecular composition.
The absorption of photons initiates structural changes in DOM by the generation of singlet
excited state 1DOM*.
1DOM* may relax back to ground state or form the triplet excited state
3DOM* by the de-activation of
1DOM* through intersystem crossing.
92,93 DOM can undergo
direct phototransformation from its triplet excited-state (3DOM*) and also generate reactive
oxygenated species (ROS) such as ∙OH, singlet oxygen, and superoxide radicals for the indirect
photolysis of organic pollutants.87,89,94,95
Indirect photolysis of DOM is the main abiotic process
in which aquatic and anthropogenic pollutants, such as pesticides and pharmaceuticals, are
breakdown in natural waters.80,88
For instance, the photodegradation rate of the antibiotic
amoxicillin is enhanced in the presence of DOM.9
Yet, there is a lack of knowledge on the
21
structural composition of DOM and, therefore, its chemistry. With that in mind, the
photodegradation of DOM will be investigated in this dissertation.
1.6 Research objectives
Environmental photochemistry is an important abiotic process that initiates the
breakdown and generation of compounds in the environment. Photolysis has been often studied
ex-situ using conventional techniques that frequently required chemical derivatization. These
studies generally lacked high temporal resolution and provided limited information on reactive
and short-lived intermediates. The ability of NMR spectroscopy to analyze a sample in its natural
state and in a non-invasive manner is a very important factor in environmental studies. NMR has
been shown to be an excellent tool to follow the progress of chemical reactions. The focus of this
dissertation was to develop and explore various approaches to performing in-situ photochemical
NMR. As such, chapter 2 is focused on the design and comparison of three in-situ NMR
photoreactor systems, from relatively cheap xenon arc lamps to more realistic “sunlight
simulators” as-well as comparing flow systems (light source outside the spectrometer) to optical
fiber (light directly into the NMR). The systems were then tested on a range of environmental
samples to test their applicability to a wide range of environmental systems. Chapter 3 probes
molecular changes of DOM from three different sources (Suwannee River, Nordic reservoir, and
Pony Lake) upon light exposure. The photolytic fate of DOM was monitored in-situ and in real
time over a course of 5 days using 1H NMR and
1H diffusion-edited NMR experiments.
Subsequently, the DOM samples were analyzed using a series of 2D NMR experiments (1H-
13C
HSQC, edited-HSQC, and COSY) prior and following a monthly exposure to light. The specific
objectives of this dissertation are as follows:
22
1. Developing in-situ NMR photoreactors to study environmental photochemistry in real
time.
2. Compare the advantages and disadvantages of each system. Specifically, identify the
system that permits the most environmentally relevant photodegradation.
3. Demonstrate the applicability of in-situ NMR photoreactors to a wide range of
environmental samples, including atmospheric pollutants, simple compounds, extracts of
crude oil, and groundwater.
4. Apply various NMR techniques, such as diffusion-editing and a series of 2D NMR
experiments, to study the photolytic fate of DOM at the molecular level.
Objectives 1-3 are discussed in chapter 2 while objective 4 will be presented in chapter 3.
Finally, chapter 4 provides the overall conclusions of the research presented in this
dissertation as well as recommendations for future experiments.
23
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55. Woods, G. C.; Simpson, M. J.; Simpson, A. J. Oxidized sterols as a significant
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59. Woods, G. C.; Simpson, M. J.; Simpson, A. J. Oxidized sterols as a significant
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35
2 Chapter 2 – The development of an in-situ NMR photoreactor to
study environmental photochemistry1
2.1 Abstract
Photochemistry is a key environmental process directly linked to the fate, source, and
toxicity of pollutants in the environment. In this study various approaches for directly integrating
light sources with nuclear magnetic resonance (NMR) spectroscopy are explored. To assess their
applicability, the in-situ NMR photoreactors were applied to a series of environmental systems
ranging from an atmospheric pollutant, crude oil extracts, and groundwater. The study
successfully illustrates that environmentally relevant photochemical processes in the aqueous
phase can be monitored in-situ and in real time using NMR spectroscopy. A range of
intermediates and degradation products were identified and matched to the literature.
Preliminarily measurements of half-lives were also obtained from kinetic curves. The Suntest
system was shown to be the most suitable model to explore environmental photolytic processes
using in-situ NMR spectroscopy. Other light sources with more intense UV output hold potential
to evaluate the full range of possible reactions, useful when evaluating UV as a remediation
alternative in areas such as wastewater treatment plants. Finally, the ability to analyze the
photolytic fate of trace chemicals in groundwater, at natural abundance, using a cryogenic probe
demonstrates NMR spectroscopy’s viability as a powerful and complimentary technique for
environmental applications in general.
1 Authors: Liora Bliumkin, Ronald Soong, Jon P.D. Abbatt, Ran Zhao, Eric Reiner, and André J. Simpson.
Submitted for publication. Liora Bliumkin performed the experiments with the help of Ronald Soong and André J.
Simpson. Data was analyzed by Liora Bliumkin and André J. Simpson. The manuscript was written by Liora
Bliumkin with critical comments from André J. Simpson, Jon P.D. Abbatt, Ran Zhao, and Eric Reiner.
36
2.2 Introduction
Environmental photochemistry looks at the transformation of compounds, found on
Earth’s surface and in the atmosphere, due to the absorption of photons between 290-600nm.1
The absorption of photons (electromagnetic energy) provides sufficient energy for electrons to be
excited from ground state into an excited state (commonly π→π∗ or n→π∗).1 In order for the
excited electrons to return to ground (stable) state, they must release the excess energy. One way
this can be achieved is through the initialization of a chemical reaction that requires an input of
energy.1 These photochemical reactions can then result in mineralization (conversion to CO2 and
H2O) or generation of new compounds via bond cleavage, isomerization, rearrangement or
intermolecular reactions and can take place in both the aqueous (atmospheric aerosols or surface
water) and solid phases (plant and soil surface).2 Generally, photochemical reactions can be
broken into two subcategories: direct and indirect photolysis. In direct photolysis, a chromophore
directly absorbs a photon and becomes excited.1 Conversely, indirect photolysis involves a
photosensitizer, that upon absorption of photons, transfers the energy to initiate a chemical
reaction in nearby compounds.1 Not only does solar radiation play a pivotal role in the
composition and fate of both natural and anthropogenic chemicals in the environment but much
of the life on Earth also relies on it as its source of energy.3
2.2.1 Current techniques used in photochemical analysis
Photochemistry is commonly studied using fluorescence, optical spectroscopy, and mass
spectrometry (MS) due to their high temporal resolution and sensitivity.4 Chemical properties,
such as quantum yields, can be obtained using optical spectroscopy. However, it becomes
37
challenging to elucidate such information from complex samples due to spectral overlap, as in
the case of polycyclic aromatic hydrocarbons (PAHs) in cosmic water ice.5 Other studies have
demonstrated that UV-Vis spectrometry provides more ambiguous information relative to higher
resolution techniques such as NMR.6 Florescence is a highly sensitive alternative, but is
unfortunately restricted to only a small fraction of molecules that fluoresce and provides only
limited information as to chemical structure. MS is arguably one of the most efficient and
informative techniques and can characterize photoproducts in a complex mixture based on their
fragmentation patterns.7 High performance liquid chromatography (HPLC) and gas
chromatography (GC) are generally coupled with MS to enhance selectivity and reduce spectral
overlap.8 Nonetheless, MS may require extensive sample preparation that can potentially lead to
the introduction of variability and artifacts.9 For example, many free radicals (such as nitrous
oxides) can be detected in solution using florescence while much of this information is lost with
MS if the preparation time is too long.10
Furthermore, while MS provides critically needed
molecular formulae information, identification of exact structures may not be possible if novel
structures are formed (i.e. library fragmentation not available). As such there is need for
complimentary techniques, especially those that can provide high resolution isomeric
information required to solve de-novo molecular structure.
2.2.2 Why NMR Spectroscopy? An important tool in environmental research
In recent years, NMR spectroscopy is emerging as an important complimentary tool as it
can provide unprecedented information regarding molecular structures, mechanisms, and kinetics
that are key in the elucidation of photochemical reactions.11
Moreover, it is a non-selective,
versatile, robust, and highly reproducible technique that offers efficient and indiscriminant
38
information that can be missed by conventional methods.12,13
NMR has the ability to identify and
quantify reactive species such as radical adducts of 5-diisopropoxy-phosphoryl-5-methyl-1-
pyrroline-N-oxide (DIPPMPO) that become undetectable with electron paramagnetic resonance
(EPR) spectroscopy and GC-MS.14
The ability to observe subtle changes in molecular
composition with NMR has increased its popularity in other fields such as nutrition, medicine,
pharmacology, and toxicology.15
A common issue in environmental research is the sample’s complexity. However, NMR
spectroscopy can overcome this obstacle by employing multidimensional experiments, with three
dimensional (3D) experiments approaching a peak capacity of 100,000,000.16
Woods et al. have
demonstrated that increasing the number of dimensions leads to the reduction in spectral overlap
and provides additional connectivity information permitting spectral assignment even in samples
such as dissolved organic matter (DOM), which are amongst the most complex mixtures
known.13,17
Multidimensional NMR experiments provide unequivocal structural information,
including isomeric and stereoisomeric discrimination, as well as insight into molecular
interactions, diffusion, dynamics and conformation. For instance, correlation spectroscopy
(COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments provided valuable
information on the molecular structure, mechanism, and regiospecificity of the photochemical
cyclization reaction of N-(9- Oxothioxanthenyl)benzothiophene carboxamides.18
The ability of NMR spectroscopy to analyze a sample in its natural state and in a non-
invasive manner is a very important factor in environmental studies. NMR has been shown to be
a useful tool to follow the progress of chemical reactions. An excellent example is the study of
trifluralin degradation by 19
F NMR where samples in NMR tubes were placed outside in direct
sunlight and then periodically brought in for NMR analysis.19
The study identified a range of
39
degradation products and reaction mechanisms. However, while such studies are accessible and
easy to perform they lack high temporal resolution which could be problematic if reactive short-
lived intermediates or rapid reactions occur. In this manuscript we explore and develop various
approaches to performing in-situ photochemical NMR. These include comparing light sources,
from relatively cheap xenon arc lamps to more realistic “sunlight simulators” as-well as
comparing flow systems (light source outside the spectrometer) to optical fiber (light directly
into the NMR). The various light setups are tested on a range of media including individual
compounds, crude oil extracts, and groundwater to test the applicability to a wide range of
environmental systems. The study demonstrates that environmentally relevant photochemical
processes in aqueous phase can be monitored in-situ and in real time using NMR spectroscopy.
Once constructed the photochemical NMR systems are relatively easy to operate permitting
studies with high temporal resolution in an automated fashion without user intervention.
Considering the highly complementary nature of NMR to MS, especially in terms of structural
elucidation, in-situ photochemical NMR will likely play an important role unraveling
photochemical processes especially in more complex system where MS alone is insufficient.
2. 3 Experimental Section
2.3.1 Light Sources and Optical Fiber
OceanOptics HPX-2000: 35W continuous xenon light source with wavelength coverage
between 185-2000 nm (main output is 290-800 nm. Figure A.1a) and equipped with INLINE-
TTL-S Inline TTL shutter (powered by a 12 VDC signal with maximum operating frequency of
5Hz. Manufactured by OceanOptics, Ostfildern, Germany) (Table A.1).
40
OceanOptics PX-2: a pulsed xenon lamp with adjustable flash rate (it was set to 500
pulses per second) and a wavelength range from 220-750 nm (Figure A.1b). The light source is
equipped with INLINE-TTL-S Inline TTL shutter (powered by a 12 VDC signal with maximum
operating frequency of 220 Hz. Manufactured by OceanOptics, Ostfildern, Germany) (Table
A.1).
Original Hanau Suntest: a xenon burner with daylight filter specifically designed to
mimic the spectrum of sunlight received at the Earth’s surface (Figure A.2). The average global
shortwave (SW) downward surface radiation (DSR) reported in the literature is ~17.16mW/cm2
while the average global net absorbed surface shortwave radiation flux is ~14.94mW/cm2.20
The
Suntest spectral output was confirmed by chemical actinometry as described in the main
manuscript.
Optical Fiber
In some applications an optical fiber was used to direct the light from the source into the
sample inside the NMR. A fiber was custom made by polymicro technologies of 6.7m length
with a standard SMA905 connector on one end. The core was 600 uM ID made from of high -
OH silica specially manufactured to transmit deep UV-VIS down to 190nm (FVP series,
Polymicrotechnologies, Phoenix, AZ). This fiber represents the largest diameter deep UV
transparent fiber available and was selected in order to transmit as much light as possible from
the source. The polyamide coating was removed from a ~ 10 cm section at the end of the fiber
that entered the NMR tube using a Microsolve CE Window MakerTM
(Microsolv, Eatontown,
NJ) which further increase light transmittance into the sample.23
41
2.3.2 Chemical Actinometry and Calibration of the Suntest
The average global shortwave (SW) downward surface radiation (DSR) reported in the
literature is ~17mW/cm2 while the average global net absorbed surface shortwave radiation flux
is ~15mW/cm2.20
NMR based chemical actinometry using a 2mM solution of 2-
nitrobenzaldehyde in 70% D2O and 30% H2O was used to measure the average radiation flux
between 290-380nm reaching the reaction vessel inside the Suntest. The radiation flux exposed
to the reaction vessel inside the Suntest was calculated to be ~8.53mW/cm2 with an average
photon flux of 2.46∙1014
(s-1
∙cm-2
∙nm-1
) which was consistent with Zhao et al..21
This is ~2 times
lower than the average global SW DSR and ~1.75 times lower than the net absorbed radiation
flux reported in the literature and is consistent with the net absorbed shortwave radiation in New
Orleans, Casablanca, and Beijing in January and Paris and Berlin in October.20
No attempts were
made to attempt to calibrate the HPX-2000 or PX-2 in relation to natural sunlight as their spectral
output differs greatly from sunlight and numerous disadvantages observed in this paper make
them less suitable for photochemical studies that aim to mimic the natural environment.
2.3.3 Sample Preparation
34.52mM riboflavin (Sigma-Aldrich, Canada, CAS#:83-88-5) solution (pH 11.43) was
prepared in 70% D2O and 30% H2O (Cambridge Isotope Laboratories (CIL) Inc., USA).
7.74mM p-nitrophenol (Sigma Aldrich, Canada, CAS#: 100-02-7) with 38mM H2O2 (Pure
Standard Products (PSP), 3% w/w in water) in 70% D2O and 30% H2O solution was prepared. A
mixture containing 4mL crude oil (Baar, Pennsylvania) : 1mL of 17.51 mM SDS solution (Fisher
Scientific, USA,CAS#: 151-21-3; dissolved in 70% D2O and 30% H2O) was vortexed for 15
minutes and left to settle in the dark for 24 hours. The added SDS is a surfactant that helps
42
disperse the oil into the water phase as previously described.24
The water soluble fraction (WSF)
was extracted and H2O2, an oxidant, was then added to the WSF for a final concentration of 68
mM. Photooxidation in the presence of H2O2 has been previously proven as an important
remediation technique involving the generation of reactive hydroxyl radicals that are capable of
degrading a wide range of organic pollutants.7 Groundwater was collected from a spring
emerging from the valley wall above Highland Creek, Scarborough, Ontario, Canada (grid
reference: 43.781069, -79.193631). Upon collection, 0.125% sodium azide was added to prevent
microbial activity and 0.08% D2O (99.99% D2O from Sigma Aldrich, Canada) to minimize
dilution while providing a sufficient signal for the spectrometer lock.25
2.3.4 In-situ photolysis analysis on NMR Spectroscopy using OceanOptics HPX-
2000 and PX-2
Light was transferred using an optical fiber that was connected to OceanOptics HPX-
2000 or PX-2 at one end with the other end placed approximately 3 cm (~0.5 cm above the NMR
coil) from the bottom of a 5mm NMR tube (Norell Inc., NJ, USA). The photolytic reactions were
continuously observed inside a Bruker BioSpinAvance-III-HD 500 mHz NMR fitted with a 2H-
1H-
13C-
15N TCI prodigy
TM cryoprobe with actively shielded z-gradients. The water signals were
suppressed using SPR-W5-WATERGATE water suppression sequence, with a 125µs binomial
delay, integrated with a perfect echo to reduce J-coupling modulation and a relaxation delay
corresponding to 5xT1.25,26
The numbers of scans for photolytic reactions using HPX-2000 and
PX-2 were adjusted based on the sample heterogeneity (128 scans for p-nitrophenol, 256 scans
for riboflavin, and 512 scans for crude oil). The 16,384 time domain points were multiplied by
an exponential function corresponding to 0.3-1Hz line broadening in the transformed spectrum
43
and a zero filling factor of 2. “Light-off” experiments were performed prior and following light
exposure to demonstrate that no factor, other than light, influenced the observed spectral
changes.
2.3.5 In-situ photolysis analysis on NMR Spectroscopy using Original Hanau
Suntest model
Experiments were performed using a simple closed-flow system. Samples were placed
inside a glass beaker with a quartz cover inside Suntest light system. The flow rate was set to
1.5mL/min. All experiments were acquired using Bruker BioSpin Avance III 500 mHz NMR
fitted with a 1H,
13C,
15N, TXI (Triple resonance Inverse) Z-gradient 250µL injection NMR flow
probe with the exception of groundwater. Due to lower concentration the groundwater was
analyzed using Bruker BioSpin AvanceIII 500 mHz NMR fitted with a 1H,
13C,
15N, cryo TCI
(Triple resonance carbon inverse) probe and a custom flow cell.27
The experimental parameters
were repeated as above with the exception of groundwater that was acquired using 4096 scans.
“Light-off” experiments were performed prior and following light exposure. Dark control
experiments were also conducted. 500μL of each sample that was to be analyzed using the
Suntest model was transferred into a 5mm NMR tube and covered in aluminum foil. The NMR
tube was placed inside Suntest for the duration of the light experiment and was later analyzed.
The controls ensure changes are from the light alone and not for example heating within the
Suntest.
Kinetic plots were created using ACD Labs NMR Workbook Suite 2015 (ACD Labs, Toronto,
Canada). Due to limited spectrometer time, the experiments could not be run in triplicate, instead
44
the data reprocessed, rephrased and reintegrated three times to generate error bars for the kinetic
curves.
2.4 Results and Discussion
2.4.1 Comparison of different light sources
Riboflavin represents a simple, cheap and well characterized photosensitive compound
ideal for investigating the basic performance of the in-situ NMR photoreactors before application
to more environmentally relevant systems later in this paper. Riboflavin is highly sensitive to UV
and visible light and forms reactive oxygenated species (ROS) upon light exposure.28
Absorption
maxima have been reported around 224, 268, 373, and 445nm.29,30
The photodegradation of
riboflavin has been extensively studied, and is known to form 2 main photoproducts: lumichrome
and lumiflavin.28
HPX-2000, PX-2, and Suntest light systems were selected for comparison. These are
discussed more in the supporting information. Light from both the HPX-2000 and PX-2 were fed
directly into the NMR via optical fibers. For the Suntest, solution was flowed through the NMR
into the Suntest in a looped system. Figure 2-1 provides schematics, NMR data and kinetic
curves for the degradation of riboflavin using the 3 different light sources. Expanded NMR
spectra including various controls are provided in the supporting section (A.4 through A.12). The
controls included light-off before exposure (to ensure no change prior to analysis), light-off after
exposure (to ensure changes halt when light is removed) and dark controls (demonstrate changes
are caused by the light). In all controls no changes were observed confirming all reactions to be
photolytic.
45
On light exposure using both PX-2 and HPX-2000 the two singlet (methyl) peaks at 2.05
and 2.17ppm and aromatic peaks at 7.09 and 7.19ppm decreased in intensity. Eight new peaks
appeared between 6-8.5ppm along with two large signals at 3.61 and 3.77ppm (Figure 2-
1,S4,S6). The solution changed from light orange to a dark orange solution and a dark orange-
brown precipitate was formed inside the NMR tube (more visible with HPX-2000 (see figure
A.4)). The NMR products were not consistent with products expected to be formed from
riboflavin in sunlight22,28
and showed some similarities to spectra of riboflavin polymers
previously reported in the literature.31
However, in-depth identification, which is beyond the
scope of this paper, were not attempted.
Figure 2-1. Schematics, NMR data, and kinetic information for the photodegradation of the
reference sample, 34.52 mM riboflavin solution, using three different light sources.
46
Interestingly, the results with the Suntest were very different. The peaks at 7.09 and
7.19ppm were replaced by five new singlet peaks between 6.7-8.5ppm. Also, new peaks between
3.4–3.8ppm were observed. These are all consistent with the main photoproducts of riboflavin
reported in the literature for >254nm light, namely lumichrome, lumiflavin, erythrose, and 1-
deoxy-xylulose.22,28
Assignments are provided in Figure A.8. Different photoproducts between
the light systems are likely due to the different spectral output. While not clear from Figure A.1
the manufacturer reports wavelengths down to 185 nm for the HPX-2000 and 220nm for the PX-
2. Riboflavin is known to contain several chromophores ranging between 200-500nm, including
a chromophore at 224nm.29,30
The kinetics also highlighted differences between the light systems. The PX-2 and HPX-
2000 show unusual sigmoidal curves (Figure 2-1). These likely arise due to the placement of the
optical fiber within the NMR tube. To avoid shimming problems, the end of the stripped fiber is
placed in the reaction solution but above the detection coil. It appears that the light induces
photolytic reactions but the products take time to diffuse into the detection coil region. The lack
of an immediate and prominent reaction suggests that the light cannot penetrate directly into the
coil region due to self-absorption from the sample itself. This clearly highlights the difficulties in
distributing light uniformly through any sample based on an optical fiber which in turn would
complicate calculating kinetic parameters such as half-lives. While the fiber could be completely
stripped of polyamide coating and submerged to the bottom of the tube the NMR tube, the
capillary may perturb the magnetic homogeneity of the sample leading to line shape distortions
and care would have to be taken to make sure no extraneous light interferes NMR detection
circuitry, potentially problematic in cryoprobes that have cryogenically cooled electronics in
very close proximity to the sample.
47
Conversely, the Suntest, which is based on a flow design produces a logical decay profile
indicating the reaction starts immediately after light exposure and continues in a two-step first-
order mechanism21,22
, initial rapid degradation followed by slower degradation. The half-life can
be calculated as ~1.88 hours. Based on chemical actinometry (see experimental section) the
Suntest system produces light that is consistent with that measured in New Orleans, Casablanca,
and Beijing in January and Paris and Berlin in October which is ~ 1/2 of the global solar
average.20
The average half-life of riboflavin in the environment can be measured by accounting
for the difference in radiation flux (a factor of two) between the Suntest and the environment.
Previous kinetic studies suggest a linear relationship between photon flux and the rate of
riboflavin photodegradation.21,22
Once the reduced light output from the Suntest (~50%
compared to average global net) is accounted for, the global average half-life of riboflavin is
~0.94hours. This is consistent with reported previous reports of riboflavin photodegradation in
milk.32
Considering this along with the fact that Suntest produced the expected degradation
products22,28
and is fundamentally designed to simulate sunlight, it is clear this approach is best
suited to investigate and monitor environmental photochemistry. This said, the drawbacks of
such a flow system include the requirement for larger volumes of sample, the rigorous cleaning
required between samples to prevent carry over, the cost of a Suntest simulator, and the need to
design a NMR flow cell or have access to an NMR flow probe. Arguably the HPX-2000 and PX-
2 may have use if reactions in the deep UV are of interest for example the photoremediation of
contaminants.
48
2.4.2 Photooxidation and mineralization of an atmospheric pollutant
Solar radiation is known to be the driving force of many processes in the atmosphere and
the generation of atmospheric radicals which are considered the “cleanser” of the
atmosphere.33,34,35
Hence, the mineralization of p-nitrophenol was monitored here to demonstrate
the phototransformation process of a simple environmentally relevant compounds using in-situ
NMR photoreactors. Nitrophenols are introduced to the atmosphere via biomass burning
emissions, as well as atmospheric oxidation of aromatic pollutants.36
Nitrophenols are
phytotoxic37
; and therefore, their photochemistry and decay products are of great interest to the
atmospheric chemistry community. In particular, p-nitrophenol is sufficiently water-soluble that
it is subject to aqueous-phase photooxidation and mineralization in cloud and fog waters.38
In-situ and real time information from NMR spectroscopy enabled the identification of
the reaction’s intermediates (Figure A.13). Figure 2-2 provides an overview of the NMR data
and kinetic curve for the mineralization of p-nitrophenol using the Suntest as a light source.
Detailed NMR spectra for both HPX-2000 and Suntest light sources, including various controls,
are provided in the supporting section (Figures A.11-A.15). All spectral changes were confirmed
to be photolytic.
49
Figure 2-2.
1H spectra of p-nitrophenol (7.74 mM) and its photoproducts at three different time
points during the light exposure inside the Suntest.
The signals from the parent compound and photoproducts decrease over the course of
light exposure, suggesting either photomineralization to CO2 or release of small volatile organic
products (Figure 2-2). As stated in the literature, the major photoproducts of p-nitrophenol are
hydroquinone and 4-nitrocatechol.39
These primary intermediates reacted further with hydroxyl
radicals leading to ring-opening products and formation of oxygenated aliphatic compounds such
as 2-butenedioic acid.39,40
Additionally, benzoquinone was formed from hydroquinone while
formic acid, p-phthalic acid, and 1,2,3-benzenetriol were detected based on their chemical shifts
(Figure A.13).26,41
The ability to monitor a reaction’s progress with high temporal resolution
using in-situ NMR spectroscopy and NMR’s highly complementary nature to MS should prove
50
useful in the elucidation of reaction mechanisms in general. The half-life of p-nitrophenol was
determined to be 2.47 hours (Figure 2-2). Unlike with riboflavin, estimating the half-life of p-
nitrophenol in the environment has proven to be more challenging with a wide range of half-lives
reported since the photooxidative degradation of p-nitrophenol is highly dependent on the
concentration of ·OH and substrate concentrations as well as the light source used.39
In contrast, only slight photomineralization were observed with HPX-2000 as the light
source (Figure A.15). In large part, this is likely related to the spectral output of the two sources.
The Suntest produces 90uW/cm2/nm (converted from ~0.9W/m
2/nm, figure A.2) which is ~9
times more intense than the ~10uW/cm2/nm for the HPX-2000. Furthermore, loss in the optical
fibers and low surface area exposure would render an external optical fiber solution, such as the
HPX-2000, less appealing for most environmental applications.
2.4.3 Oil spills: the fate of water soluble fraction (WSF) of crude oil upon exposure
light
To demonstrate the application of photochemical NMR to a more complex environmental
mixture the WSF of crude oil was studied as an example. Oil spills are a significant
environmental problem in which toxic chemicals are released into the environment.42,43
Certain
gasoline components are resistant to biodegradation, but are photoliable.41
Here, the fate of water
soluble oil components upon light exposure using the HPX-2000 and the Suntest light sources
was investigated.
SDS was added to simulate the use of surfactants which are often used to help disrupt
large oils spills and disperse oil components into the aqueous phase.24
An SDS control
experiment confirmed that SDS is not photolabile (Figure A.16). Other controls, including light-
51
off prior and following photoirradiation and dark controls, confirmed all reactions to be
photolytic (Figures A.17,A.20,A.25). With both models, new peaks were observed between 0.7-
1.5ppm, suggesting the formation of aliphatic compounds (Figures 2-3B (yellow), S18, S21,
S23).45
Furthermore, new photoproducts between 3.5-3.8ppm hint at hydroxylation of crude oil
precursors (Figures 2-3B (orange), A.18, A.21, A.23).45
52
Figure 2-3. Phototransformation of WSF of crude oil with HPX-2000 (right) and Suntest (left)
light sources. A and C are prior to light exposure and B and D are following light exposure.
53
Two dimensional (2D) Distortionless Enhancement by Polarization Transfer -
Heteronuclear Single Quantum Coherence Spectroscopy (DEPT-HSQC) provides additional
spectral dispersion and H-C connectivity over one bond (1H-
13C JCH). The DEPT component
encodes CH3/CH and CH2 with different phases based on the different evolution of these units
during the experiment. In simple terms, DEPT-HSQC provide a high dispersion map of the H-C
units in the mixture with the CH2 units coloured green and the CH/CH3 units coloured blue.
Region 6 in the DEPT-HSQC data (Figure A.21) supports the production of a small quantity of
hydroxylated products. A notable reduction in aromaticity between 6.5-8ppm following light
exposure (Figures 2-3C&D, A.19,A.24) was also observed. The aromaticity decreased by ~13%
with HPX-2000 and by ~35% with the Suntest model (Figure 2-3). Based solely on the spectral
output of the lamps the Suntest is ~9 times more intense than HPX-2000 between 300-800nm
and likely explains the increased breakdown down of more aromatic structures (Figures 2-
3C&D). It is proposed that photooxidation of aromatic compounds was initiated by ring
oxidation followed by ring-opening reactions that yielded a range of oxygenated, mainly
aldehydes and acids, and unsaturated products (Figures 2-3B&D, A.18, A.19, A.22, A.23,
A.24).46
The clearest indicators are the new signals between 5.0-6.0ppm (HC=C) region
following irradiation with HPX-2000 further which are consistent with the formation of double
bonds (Figure 2-3D (green) and HSQC region 5, Figure A.21).
Other protons in the same 1H-
1H spin system can be isolated using selective TOCSY. In
this experiment the double bond signals are selectively excited and then a homonuclear spinlock
transfers magnetization down the chain to other protons within the same 1H-
1H spin system. The
result is a sub-spectrum of the structural motif that contains the double bonds (Figure A.22). The
54
units are consistent with linear aliphatic constructs and are most likely the result from aromatic
ring opening reactions. Interestingly, these products are more intense with the HPX-2000. One
argument is that the deeper UV offered by this lamp leads for enhanced degradation of the
aromatics. This is partially supported by Figures A.18 and A.23 which show the HPX-2000 more
efficient degradation of selective BTEX components. However, this is inconsistent with the
overall aromatic region that decreases more with the Suntest (Figure 2-3D). Double bonds are
seen to form with the Suntest system (see Figure 2-3D) but they do not appear to accumulate.
These unsaturated products are known to be photolabile, and hence, can further react via
oxidative cleavage of double bonds to form aldehydes, ketones and acids.47
In Figure 2-3D, two
singlet peaks at ~8.2 (purple, likely formic acid) and ~9.6ppm (brown, an aldehyde) were formed
following photoirradiation, the signals are present after photodegradation with both the Suntest
and HPX-2000. However, signals corresponding to -CH2-/-CH- signals adjacent to carboxylic
groups at ~2.5ppm (red) following irradiation were larger with the Suntest model (Figure 2-3B).
The kinetic profile of this region shows that carboxylic groups accumulated over time with the
Suntest while it remained relatively the same with HPX-2000 (Figure A.26). This suggests that
ring-opening products continue to react with the Suntest model due to greater light intensity
forming acidic end products.
In summary, NMR is a useful tool to help explain the overall changes occurring during
photochemical process, for example, the formation of new structural categories (double bonds)
and the degradation of aromatics. With additional assignments from a list of specific compounds
of interest (for example BTEX) it should be possible to combine both non-targeted and targeted
analysis and extract a wealth of process information in a relatively short amount of time and in a
non-invasive fashion.
55
In this study 1H NMR data of WSF using HPX-2000 as the light source was acquired
with a cryogenically cooled probe while the 1H NMR data using the Suntest system was acquired
with a room temperature flow injection probe, as the latter is much easier to integrate into a flow
system. However, as can be seen from Figure 2-3C, the sensitivity of the cryoprobe (HPX-2000,
right) is ~2 times that of the flow probe (left). While it is possible to integrate flow into
cryoprobe systems it involves developing custom flow cells as previously reported by Soong et
al..48
To demonstrate a flow-application taking advantage of additional sensitivity of the
cryoprobe the next section deals with the flow analysis of a low concentration environmental
sample, at natural abundance.
2.4.4 Monitoring photochemical changes of groundwater at natural abundance
The final example demonstrates the application of cryogenically cooled NMR probe for
analysis of complex environmental samples. For centuries, groundwater has been used as a main
source of drinking water. Today, it is still a favorable source of drinking and agricultural water,
yet little is known about its composition and phototransformation.49
Additionally, there has been
a growing interest in evaluating the application of sunlight in water treatment as a result of
groundwater contamination originating from leeching of industrial chemical waste discharge and
pesticides through the soil.50
Groundwater was analyzed at its natural state and in a non-invasive
manner, a key factor in environmental studies, using a sunlight simulator. Dissolved organic
carbon (DOC) concentration in groundwater in North America can be as low as 1-2ppm
depending on the season.51
The total organic carbon (TOC) of the groundwater discussed in this
paper was 1.96ppm. Considering the total volume of the NMR is ~300uL, there is only ~588ng
of total organic matter in the coil with many species in the low ng range.
56
Control experiments confirmed all reactions to be photolytic (Figures A.27, A.29, A.30).
The chemical fingerprints of groundwater prior to light exposure were relatively easy to identify
as it consisted of mostly biological molecules that are well represented in bio-reference NMR
databases (Figure 2-4). The molecular composition of groundwater was shown to contain many
similarities to the spectral composition of DOM in glacial ice.25
It was found to consist of: acetic
acid, alanine, glycerol, glycine, lactic acid, pyruvic acid, and short chain organic acids (SCA)
(Figure 2-4). The degradation products were more challenging to identify using the NMR
database alone (Figure 2-3B). Acetone is a likely mineralization intermediate of an oxygenated
precursor while formic acid, at ~8.1ppm, is a general breakdown product found in many DOM
samples. Figure A.31 provides a kinetic profile of the photodegradation of lactic acid and dual
photogeneration and consumption of acetone over the course of light exposure, demonstrating
the viability of in-situ NMR analysis in understanding photochemical processes of environmental
samples taken directly from the environment and analyzed at natural abundance.
57
Figure 2-4. A: final
1H spectrum of groundwater sample (TOC: 1.96 ppm) after 6 hours in the
dark (0-4.5 ppm region). B: 1H spectrum of groundwater after the sample was exposed to light
for 1 day and 12 hours inside the Suntest solar simulator.
58
Additional information such as diffusion, connectivity information, dynamics, and
conformation, could not be obtained as the trace amounts of organic material in groundwater
prevent 2D NMR analysis at natural abundance. This stated it should be possible to concentrate
(freeze dry, speed vacuum) the sample and run 2D NMR at high concentration to elucidate
structures. 1H NMR can then be used to follow these assigned molecules at natural abundance.
Interestingly, the flow system employed using the Suntest could theoretically permit a small flow
to split to MS. The direct combination of NMR and MS is proving very powerful in metabolic
research, where the co-variance between signals over time in the two instruments can be used to
statistically correlate peaks in NMR and MS.52
Such applications in environmental research
could be very powerful and should directly relate molecular formulae (MS) and isomeric
information (NMR) to provide an unrivalled combination in terms of identifying new species.
The looped photochemical NMR reactor described here paves the way to make such future
studies possible.
The study has successfully demonstrated that photolytic reactions in aqueous phase can
be explored in-situ and in real time using NMR spectroscopy. It provides unambiguous
information on kinetics and structural identification of intermediates and degradation products
which can be further used to elucidate reaction mechanisms. It was determined that Suntest in
combination with a loop flow system is the most suitable model to explore environmental
photolytic processes using in-situ NMR spectroscopy. Its application to a range of environmental
systems have illustrated that NMR is a powerful complimentary tool that can be used to study
simple chemical reactions down to groundwater at natural abundance. The isomeric information
provided by NMR spectroscopy is extremely complimentary to MS and has an important role in
unraveling photochemical processes in complex environmental systems.
59
2.5 References
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24. Simpson, A. J.; Mitchell, P. J.; Masoon, H.; Liaghati, Y.; Adamo, A.; Dicks, A. P. An oil
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25. Pautler, B. G.; Woods, W. C.; Dubnick, A.; Simpson, A. J.; Sharp, M. J.; Fitzsimons, S.
J.; Simpson, M. J. Molecular characterization of dissolved organic matter in glacial ice:
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26. Adams, R. W.; Holroyd, C. M.; Aquilar, J. A.; Nilsson, M.; Morris, G. A. “Perfecting”
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27. Soong, R.; Nagato, E.; Sutrisno, A.; Fortier-McGill, B.; Akhter, M.; Schmidt, S.;
Heumann, H.; Simpson, A. J. In vivo NMR spectroscopy: toward real time monitoring of
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28. Jung, M. Y.; Oh, Y. S.; Kim, D. K.; Kim, H. J.; Min, D. B. Photoinduced generation of
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29. Cheng, C. W.; Chen, L. Y.; Chou, C. W.; Liang, J. Y. Investigations of riboflavin
photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase
activity. J. Photochem Photobiol B. 2015. doi: 10.1016/j.jphotobiol.2015.04.028
30. Drossler, P.; Holzer, W.; Penzkofer, A.; Hagamann, P. pH dependence of the absorption
and emission behaviour of riboflavin in aqueous solution. Chem Phys. 2002, 282 (3),
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31. Iida, H.; Iwahana, S.; Mizoguchi, T.; Yashima, E. Main-chain optically active riboflavin
polymer for asymmetric catalysis and its vapochromic behavior. J. Am. Chem. Soc. 2012,
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32. Wishner, L. A. Light-induced oxidations in milk. J. Dairy Sci. 1964, 47(2), 216-221.
33. Zhang, W.; Xiao, X.; An, T.; Song, Z.; Fu, J.; Sheng, G.; Cui, M. Kinetics, degradation
pathway and reaction mechanism of advanced oxidation of 4-nitrophenol in water by a
UV/H2O2 process. J Chem. Technol. Biotechnol. 2003, 78 (7), 788-794.
34. Finlayson-Pitts, B. J.; Pitts, J. N. Jr.. Chemistry of the upper and lower atmosphere:
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39. Daneshvar, N.; Behnajady, M. A.; Asghar, Y. Z. Photooxidative degradation of 4-
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mechanism. J. Hazard Mater. B. 2007, 139 (2), 275-279.
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66
3 Chapter 3 – Analysis of DOM phototransformation using a looped
NMR system integrated with a sunlight simulator
3.1 Abstract
Aquatic dissolved organic matter represents the largest pool of mobile organic carbon on
Earth and its production, transformation, and fate are intimately tied to the global carbon cycle.
Photochemical transformation plays an important role functionalizing and degrading dissolved
organic matter (DOM), producing one of the most complex mixtures known. Nuclear magnetic
resonance (NMR) spectroscopy is a powerful non-invasive approach that provides detailed
molecular information that can be used to elucidate structural change in complex mixtures. In
this study, using a flow-based design, NMR is directly interfaced with a sunlight simulator
affording the possibility to study DOM photochemical transformation with high temporal
resolution. Sample from Suwannee River (Florida), Nordic Reservoir (Norway), and Pony Lake
(Antarctic) are studied. DOM photolysis is dominated by the photodegradation of aromatics and
unsaturated structures (many arising from lignin) into carboxylated and hydroxylated products.
Using complimentary off-line two dimensional (2D) NMR a range of metabolites are assigned.
The research demonstrates the applicability of the looped system to follow degradation in a non-
targeted fashion (the mixture as a whole) and target analysis (tracing specific metabolites). The
later holds great potential to study the fate and transformation of contaminants and nutrients in
the presence of DOM. In summary, the photolytic fate of DOM is successfully monitored using
in-situ NMR, providing preliminary measurements of reaction rates and insight into the
photochemical properties of DOM.
67
3.2 Introduction
DOM is a very complex mixture of organic molecules that plays a key role in the
biogeochemistry of aquatic ecosystems.1 It is a product of plants and microbial decomposition
and, therefore, its composition varies depending on its source.2,3
It plays an active role in the
global carbon cycle, transport of organic pollutants, and it is both biodegradable and
photoreactive.1 It is estimated that the total carbon pool in DOM is as large as 700 Pg C, which is
approximately equivalent to the carbon content in atmospheric CO2.3 Therefore, DOM photolysis
represents a huge carbon reserve that could potentially be converted to CO2 and significantly
impacting future climate shifts.4
DOM photolysis is an important abiotic reaction in natural water where photons (300-
800nm) are absorbed by chromophoric dissolved organic matter (CDOM).1 The energy is passed
on to unsaturated bonds, preceded by the generation and attack from reactive oxygenated species
(ROS) such as ∙OH, singlet oxygen, and superoxide radicals.1,2
DOM can undergo
phototransformation from its triplet excited-state (3DOM*) and also act as a photosensitizer in
the indirect photolysis of organic pollutants.1,2
For instance, cationic antibiotic norfloxacin have
been shown to be photosensitized by DOM.5 It is hypothesized that DOM accounts for up to
75% of aquatic photochemical processes.2 Yet, the photolytic fate of DOM is still far from
understood.1
DOM has been previously analyzed by high resolution NMR spectroscopy, mass
spectrometry (MS), and optical spectroscopy.6 As DOM is amongst the most complex mixtures
known, many conventional analytical techniques lack high temporal resolution to effectively
study the photolysis of DOM without altering its chemistry.1,3,7
So far studies of the
68
phototransformation of DOM have focused on the bulk, fractions or isolates, or focused on
specific smaller molecules in the matrix.1,3,8
MS has been most frequently employed to study
DOM on a molecular level due to its outstanding sensitivity. However, MS’s selective nature of
extraction methods, such as solid phase extraction (SPE), limits the understanding of the
photolytic fate of DOM as a whole.1 Moreover, MS generally lacks isomeric discrimination and
ability to solve structure de-novo a considerable limitation when studying mixture where
multitudes of new molecules may form.1
NMR spectroscopy is a highly complementary analytical tool to MS, that excels at
isomeric discrimination and has been central in unraveling many of the structural components in
DOM.3,9,10-12
NMR spectroscopy is a powerful non-selective and non-invasive analytical tool that
can provide detailed structural information and connectivity information on complex
samples.3,13-15
NMR spectroscopy can be used spectroscopically to separate components based
on self-diffusion which permits the analysis of different fractions (small metabolites and more
rigid large molecular weight molecules), as in the case of rainwater, without altering the
sample.16
Multidimensional NMR experiments provide additional connectivity information and
dispersion via the second dimension which reduces spectral overlap and permits spectral
assignment of complex samples such as DOM.3,7,13
Multidimensional experiments offer more
detailed structural information, isomeric discrimination, information on distances and
interactions between different nuclei, as-well as insight into the dynamics, exchange and
conformation all critical for the elucidation of unknown structures and reaction mechanisms. A
range of simple molecules in Suwannee River DOM have been previously identified using
Correlation Spectroscopy (COSY) and Heteronuclear Single Quantum Coherence (HSQC)6 as
69
well as hyphenated 2D and three dimensional (3D) NMR.3,12
Furthermore, environmental
processes can be monitored in-situ and, sensitivity permitting, in real time using NMR
spectroscopy. These afford the potential to identify a range of reactive intermediates, follow their
transformation, and calculate half-lives of either individual species (acetone for example) or bulk
components (for example, aromatics in general). The viability of in-situ photoirradiation using
NMR spectroscopy has been previously demonstrated by analyzing phototactic reactions of
Pharanois phoborhodopsin in Natronomonas pharaonis using solid-state NMR spectroscopy
under magic angle spinning (MAS).17
This study aims to apply solution-state NMR to study DOM from three different sources
(Suwannee River, Nordic reservoir, and Pony Lake) upon light exposure. The samples selected
represent DOM samples from different latitudes, from Antarctica (Pony Lake), Norway (Nordic)
and Florida (Suwannee River), which receive differing degrees of natural light which in turn may
influence the photochemical reactivity of the organic material in the environment. In the first
section, the photolytic fate of DOM is monitored in-situ and in real time over a course of 5 days.
The samples flow through the NMR probe into a solar simulated light source and monitored
using both 1H NMR and diffusion based-editing. In addition, DOM samples are analyzed off-line
using a range of 2D NMR experiments (1H-
13C HSQC, edited-HSQC, and
1H-
1H COSY) prior
and following a monthly exposure to simulated light to help with spectra assignment. The study
successfully demonstrates that the photolytic fate of specific metabolites in DOM can be
monitored in-situ and in real time to obtain a better understanding of the photochemical
properties of DOM. Furthermore, in-situ analysis of different fractions of DOM using diffusion-
edited NMR experiments provides insight into the underling processes involved in the
phototransformation of DOM.
70
3.3 Experimental Section
3.3.1 Light Source
Original Hanau Suntest was utilized in this study as it is specifically designed to simulate
the spectral output of sunlight (Figure B.1.A). The radiation flux measured by NMR chemical
actinometry using 2 mM solution of 2-nitrobenzaldehyde18
in 70% D2O and 30% H2O
(Cambridge Isotope Laboratories (CIL) Inc., USA) for this model was ~8.53 mW/cm2. This is
approximately twice as low as the average global shortwave (SW) downward surface radiation
(DSR) reported in the literature. It is however in agreement with the net absorbed shortwave
radiation in New Orleans, Casablanca, and Beijing in January and Paris and Berlin in October.19
3.3.2 Sample Preparation
Pony Lake Fulvic Acid (FA) Reference material, Suwannee River NOM and Nordic
Reservoir NOM were obtained from the International Humic Substances Society (IHSS). All
samples were prepared in 99% Milli-Q water and 1% D2O solutions (99.9% D2O, Sigma-
Aldrich, Canada, CAS#:7789-20-0) and the pH were adjusted to ~7 with NaOD (99.5%
deuterated and 30% in D2O, Cambridge Isotope Laboratories (CIL) Inc., USA, CAS#: 14014-06-
3).2,10
DOM samples for in-situ NMR analysis were prepared at a concentration of ~2.6mg/mL.
DOM samples for 2D NMR analysis were prepared at a concentration of ~57mg/mL. All
samples were vortexed and sonicated for 15 minutes prior to analysis.
3.3.3 In-situ analysis of DOM photolysis using diffusion-editing NMR experiments
All in-situ and diffusion-editing NMR experiments were acquired using Bruker BioSpin
AvanceIII
500 mHz NMR fitted with a 1H,
13C SEI (Selective Inverse) Z-gradient 120µL
71
injection NMR flow probe. Water signals were suppressed using SPR-W5-WATERGATE water
suppression sequence, with a 125µs binomial delay, incorporated with a perfect echo and a 5xT1
relaxation delay.20-22
“Light-off” 1H NMR experiments were acquired prior and post photoirradiation to
illustrate that light was the only variable influencing the observed spectral changes (Figures B.2-
B.7).
Experiments were performed using a simple closed-flow system (Figure B.1.A) with a
flow rate of 0.50mL/min where 1H NMR spectra were continuously collected every hour for 20
hours. Subsequently, the Suntest and the flow were turned off for 4 hours to allow the acquisition
of diffusion-editing NMR data. This cycle was repeated over the course of 5 days. The
experiments were repeated again under the same conditions using a 5 mm Wilmad® quartz
NMR tube (Sigma-Aldrich, USA) (Figure B.1.B) as the reaction vessel instead of the beaker in
figure B.1.A to act as a control and account for any evaporation inside the beaker. The flow had
to be turned off for the diffusion-editing experiments as the approach measures self-diffusion
(i.e. movement from one physical location within the flow cell to another). In the presence of a
flow such measurements are not possible. Diffusion-edited (DE) NMR experiments were
performed with a bipolar pulse pair longitudinal encode-decode sequence.23,24
2048 scans and 32
dummy scans were collected using a 1.2 ms, sine shaped gradient pulse at 40 gauss/cm, a
diffusion time of 100 ms, and 32,768 time domain points at 298K.23,25
The 32,768 time domain
points were multiplied by an exponential function corresponding to 10 Hz line-broadening and a
zero filling factor of 2. Inverse Diffusion-edited (soluble and low molecular weight structures)
were generated via difference from reference NMR spectra (collected identically to the diffusion-
editing spectrum but with the power of the diffusion gradients set to zero).26
In simple terms the
72
reference spectra contain signals from all molecules and the diffusion-edited NMR spectra
contain only signals from molecules with restricted diffusion. A weighted subtraction leads to a
sub-spectrum that contains only rapidly tumbling small molecules.26,27
In this study an approach
should highlight the small molecules formed from the photochemical breakdown of DOM.
1H NMR spectra were divided into 5 regions: material derived from linear terpenoids
(MDLT), 0-1.6 ppm; carboxyl-rich alicyclic molecules (CRAM), 1.6-3.2 ppm; carbohydrates
(and lignin methoxy), 3.2-4 ppm; unsaturated components, 5.5-6.5 ppm; and aromatics, 6.5-7.8
ppm; (Figures B.8-B.10).3,9,10
Kinetic plots were created using ACD Labs NMR Workbook Suite
2015 (ACD Labs, Toronto, Canada). Due to limited spectrometer time and duration of photolysis
experiments, each experiment was acquired once. Error bars were generated after reprocessing,
rephrasing, and reintegrating in triplicate each dataset.
3.3.4 DOM photolysis analysis using 2D NMR experiments
To support the one dimensional (1D) spectra collected via the looped NMR system, 2D
NMR spectra were also collected on samples that were photodegraded over a course of one
month within quartz NMR tubes. DOM samples (at ~57 mg/mL) were placed in 5 mm Wilmad®
quartz NMR tubes (Sigma-Aldrich, USA) and exposed to simulated solar radiation inside the
Suntest for a month. Samples were analyzed both before and after light exposure using 1H-
1H
COSY, 1H-
13C HSQC, and edited
1H-
13C HSQC NMR experiments with a
2H-
1H-
13C-
15N TCI
prodigyTM
cryoprobe fitted with an actively shielded z-gradient. The water signals were
suppressed using SPR-W5-WATERGATE as described above. COSY was acquired using 4096
time domain points, 16 dummy scans and 256 scans for each of the 128 increments. COSY
spectra were processed using an unshifted sine-squared function and a zero filling factor of 2.
1H-
13C HSQC was performed following the method outlined by Koskela et al. using echo/anti-
73
echo gradient selection with 64 scans, 2048 data points in the F2 dimension and 96 increments in
the F1.28
Identical to that described by Koskela et al. the 1H-
13C coupling was transferred using
constant time adiabatic CPMG trains using 4 x 1JCH values optimized to make the transfer
constant and independent of varying 1JCH. Spectra were processed with an exponential function
in F2 corresponding to a 15Hz line broadening and an sine-squared function shifted by π/2 in F1.
DEPT-HSQC was acquired and processed identically with the exception that a standard non-
quantitative sequence was employed and editing was achieved via 1/(2J(XH)) evolution delays
such that XH and XH3 are positive and XH2 negative in the final spectrum. Metabolites were
identified using Analysis of Mixtures (AMIX, version 3.9.14, Bruker BioSpin) in combination
with the Bruker Biofluid Reference Compound Databases, version 2-0-0 through version 2-0-4).
Identification was performed using a procedure developed for complex mixtures.12
Compounds
with a greater than 80% match (automated search) were selected for manual inspection. The
chemical shifts of the identified compounds were compared with database values (r2
= 0.99, σ =
0.01) to confirm matching and any compounds not meeting these requirements were removed.
3.4 Results and Discussion
3.4.1 Monitoring the phototransformation of DOM using in-situ NMR
spectroscopy
Similar spectral changes were observed across all DOM samples following 5 days of
light exposure (Figure 3-1). All 5 regions in the 1H NMR spectra (MDLT, CRAM,
carbohydrates, olefinic, and aromaticity) have photomineralized to a certain degree which was
evident by the overall decrease in spectral intensity (Figures B.8-B.10).
74
Figure 3-1. The % photomineralization of different DOM fractions, at each day relative to day 0
(=”light-off”, prior to light exposure), over the course of 5 days using in-situ NMR photoreactor.
75
MDLT, CRAM, and carbohydrates fractions had a lower degree of mineralization in comparison
to olefinic and aromatic structures due to relatively lower concentration of reactive
chromophores (Figure 3-1). This fundamental property in combination with the fact that
terpenoids are general poor biological food sources may explain why such a high proportion of
these type of material accumulate in the DOM and can persist for many 100’s of years in the
ocean (Table B.1).29,30
On the other hand, conjugated unsaturated molecules are highly
susceptible to direct photolysis1,31
explaining their higher degree of photodegradation (Figure 3-
1). DOM can also generate ROS (eg. superoxides, singlet oxygen, and hydroxyl radicals) for
indirect photolysis and photooxidation of the highly reactive double bonds.2
Carbohydrates content have decreased across all samples (Figure 3-1). The change was
greater with Nordic Reservoir NOM in comparison to Suwannee River NOM and Pony Lake FA
(Figure 3-1). This may be due to the contribution of indirect photolysis. Studies have shown that
terrestrial humic substances enhance the photodegradation of carbohydrates via indirect
photolysis while carbohydrates alone have low susceptibility to photodegradation due to
differences in the absorbance spectrum with that of sunlight and also due to the presence of
nonchromophoric carbohydrate fraction in DOM.32,33
Nordic Reservoir NOM and Suwannee
River NOM contain terrestrial plant-derived inputs (allochthonous carbon sources)31
while Pony
Lake FA is algal- and microbial-derived (containing photosynthetic algae and mixotrophic algae
autochthonous sources)34
providing a possible explanation for the observed differences. The
higher degree of photomineralization of carbohydrates in Pony Lake FA in comparison to
Suwannee River NOM is likely due to differing natural light intensities where Suwannee River
NOM (Florida) and Pony Lake FA (Antarctica) were collected, thus influence the photoreactivity
of DOM in the environment.
76
Conjugated unsaturated structures and aromatic constituents (eg. lignin, lignin-like
species, and proteins) have also experienced photomineralization.1,31
Photoirradiation of these
groups initiated ring opening reactions and photogeneration of oxidized aliphatic intermediates
which will resonate in the MDLT and CRAM spectral regions (Figure 3-1).1 One possible
reaction mechanism is the addition of oxygenated species, such as H addition, to unsaturated
carbon bonds. This leads to upfield spectral shift of proton signals originating from protons
bound to unsaturated carbons or from -CH2- next to an unsaturated bond into the MDLT and
CRAM spectral regions.35
Ring opening products of aromatic constituents may also lead to the
observed change. As such, the lower degree of photomineralization of MDLT and CRAM
relative to olefinic and aromatic structures is also attributed to photogeneration of products that
will resonate in these regions.
The observed increase in spectral intensity between 2.3-2.5ppm across all samples
(Figure B.11 was used as an example) suggests the photogeneration of carboxylic acid
photoproducts. This also suggests photoinduced bond cleavage of precursor into a carbonyl
molecule and peroxy radical that later generates an aldehyde and a carboxylic acid product.35
Proton signals from -CH2 next to these functional groups will also resonate in the MDLT and
CRAM regions.36
These observations were also in agreement with Gonsior, M. et. al.’s work.1
The photomineralization of each DOM sample was calculated based on the total proton
integration before and after light exposure. It was calculated that ~32% of Pony Lake FA, ~28%
of Nordic Reservoir NOM, and ~16% of Suwannee River NOM was mineralized in 5 days of
light exposure. The higher degree of photomineralization of Nordic Reservoir NOM relative to
Suwannee River NOM may be attributed to the original higher content of conjugated unsaturated
and aromatic structures that are photolabile (Table B.1). Furthermore, Suwannee River NOM
77
originates from Okefenokee Swamp in South Georgia, US37
, an area that experiences higher
daily light intensity relative to Vallsjøen, Skarnes, Norway where Nordic Reservoir NOM was
collected.31
Thus, most of the reactive chromophores in Suwannee River NOM has likely reacted
and degraded prior to sample collection, leaving behind relatively stable photoproducts and
chromophores. Pony Lake FA, an autochthonous-derived DOM38
, has different photosensitizing
properties from Suwannee River NOM and Nordic Reservoir NOM, containing allochthonous-
derived carbon sources such as terrestrial plants38
, and thus its photomineralization cannot be
directly compared. Additionally, Pony Lake FA has been isolated differently than Suwannee
River NOM and Nordic Reservoir NOM with certain components removed that would be
commonly present in the environment.
3.4.2 Using diffusion-editing NMR as a chromatographic tool to study DOM
Diffusion-editing (DE) techniques are great NMR chromatographic tools to study the
photosensitizing properties of different DOM fractions in a non-invasive manner.39
DE expands
on a simple spin-echo sequence with the addition of two identical gradient pulses. The first
gradient pulse de-phases the resonances while the second gradient pulse re-phases them at the
end of the experiment. As a result, magnetization from small molecules that have diffused within
the detection coil region during the diffusion delay (Δ) are not re-focused efficiently.24,25
Consequently, a diffusion-edited NMR spectrum suppresses these resonances and highlights
signals from molecules that experience little to no self-diffusion (eg. large and rigid structures
such as lignin). The decrease in signal to noise (S/N) (~64% in Pony Lake FA, ~34% for Nordic
Reservoir NOM, and ~29% in Suwannee River NOM) supports the hypothesis of photoinduced
78
bond cleavage (eg. dealkylation) into lower molecular weight products that experience higher
degree of self-diffusion (figures B.12-B.14).1,3,40
On the other hand, an inverse diffusion-editing NMR spectrum displays signals only from
small metabolites and soluble components. The appearance of NMR signals over time supports
the hypothesis of photogeneration of low molecular weight product with high degree of self-
diffusion (Figure B.15 is used as an example). Signal loss is a limiting factor of inverse-
diffusion-editing NMR since the spectrum is obtained by subtracting a diffusion-edited spectrum
from a reference spectrum. The use of a cryogenic probe or longer NMR experiments may be
required to compensate for loss in signal to noise.
As mentioned in section 3.4.1, the decrease in S/N was greater in the olefinic and
aromatic region in comparison to MDLT and CRAM (Figures B.12-B.14). Previous studies have
provided strong evidence that MDLT and CRAM are by-products and derivatives of terpenoid
structures.3 Terpenoids are known to be stable structures, especially as functionalized cyclic
structures with refractory properties.3,10,41
They have lower content of reactive chromophores that
absorb light with the exception of some MDLT components that contain conjugated double
bonds.9 Furthermore, CRAM are highly branched, interlinked acyclic molecules that can form
aggregates and limit its photoreactivity by trapping the photolabile regions inside its structure
and away from reach of photons.2,10
3.4.3 Monitoring the photolytic fate of specific compounds using in-situ 1H NMR
spectroscopy
The use of looped flow system for directly integrating NMR and a sunlight simulator
offers high temporal resolution that can provide insightful information on the photochemistry of
79
DOM. So far, DOM photolysis was defined by the overall changes of assigned regions in the 1H
NMR spectra. However, due to its complexity, a number of photochemical reactions can take
place simultaneously and at different rates. If specific compounds within a mixture are of an
interest, as long as their chemical shifts are resolved, it should be possible to follow their kinetics
in close to real-time. This is somewhat challenging to perform for DOM because of the spectral
overlap in such a complex mixture.1 This stated, the fate of 2 components, acetone and
carboxylic acid products (2.2-2.5ppm), which are present in all the DOM samples are used as an
example to demonstrate monitoring target kinetics of species within an mixture (Figure 3-2).
Figure 3-2. Kinetic plot for acetone and carboxylic acid products from three DOM sources over
five days of photoirradiation.
80
Acetone was identified using Bruker’s AMIX software (AMIX, version 3.8.14, Bruker BioSpin)
by matching 1H NMR and COSY spectra against standards from Bruker Biofluid Reference
Compound Database (v 2-0-0 to v 2-0-3). Acetone is continuously produced upon exposure of
DOM to light suggesting that it is a product of the photodegradation of other precursors in DOM
(Figure 3-2). Similarly, carboxylic acid photoproducts are continuously produced which is in
agreement with Gonsior, M. et. al.’s work and section 3.4.1.1 The photogeneration of acetone
and carboxylic acid photoproducts was higher in Nordic Reservoir NOM followed by Suwannee
River NOM and Pony Lake FA (Figure 3-2). The ability to monitor the photolytic changes of
individual molecules in DOM over time, with high temporal resolution, can potentially aid in the
elucidation of the photochemical processes and photosensitizing properties of DOM. In addition,
this approach could be very informative if used to monitor the degradation of contaminants that
contain sensitive NMR nuclei such as 19
F. As 19
F is not naturally abundant in DOM then great
specificity should be afforded regarding the photochemistry of the contaminant itself even in the
presence of other complex matrices such as DOM.
3.4.4 2D NMR identification of biochemical classes and specific metabolites in
DOM
1D NMR spectra alone are insufficient for detailed molecular characterization due to high
degree of spectral overlap. 1H-
13C HSQC NMR spectra provided additional spectral dispersion
and connectivity information for a more detailed classifications of DOM. Figures 3-3, B.16, and
B.17 identify a range of structural groups such as anomeric units in carbohydrates (1) ,
unsaturations (2), aromatics (3), N-acetyl and/or O-acetyl, S-CH3 (4), MDLT and aliphatics (5),
CRAM (6), methoxy group from lignin (7), methylene (-CH2-) units in carbohydrates (8),
81
methine (-CH-) units in carbohydrates (9), protein alphas (10) in accordance with edited-HSQC
(not shown) and Lam, B. et al.9,42
Figure 3-3. A:
1H-
13C HSQC NMR spectra of Suwannee River NOM prior to light exposure. B:
1H-
13C HSQC NMR spectra of Suwannee River NOM following light exposure.
82
The results in figures 3-3, B.16, and B.17 are in agreement with section 3.1 and 3.2,
demonstrating that aromatic and conjugated unsaturated structures are highly susceptible to
photodegradation. The aromatic region in Pony Lake FA originates from proteins while region 7
illustrates that lignin-like species are present in Pony Lake FA (Figures B.17 and B.18).43
Region
7 in B.17 and B.18 does not correspond to lignin from terrestrial sources as the landscape
surrounding Pony Lake is free from terrestrial plants.44
Instead, region 7 corresponds to lignin-
like species found in algae in Pony Lake FA that contain similar molecular formulae to lignin
found in Suwannee River.44
2D COSY NMR spectra were also acquired for more detailed and unambiguous
characterization of metabolites in DOM through bond connectivity of protons up to 3-4 bonds
away. A wide range of acids and alcohols, as well as alanine, were identified by matching the
spectra with Woods, G. C. et al., 2011 and with AMIX against the Bruker Biofluid Reference
Compound Database. 2D COSY NMR spectra of Pony Lake FA prior and following
photoirradiation are shown as an example (Figures 3-4 & 3-5).
83
Figure 3-4. 2D COSY NMR spectrum of Pony Lake FA prior to light exposure. Metabolites
were matched with Woods, G. C. et al., 2011 and with AMIX against Bruker Biofluid Reference
Compound Database.
84
Figure 3-5. 2D COSY NMR spectrum of Pony Lake FA following a month of photoirradiation.
Metabolites were matched with Woods, G. C. et al., 2011 and with AMIX against Bruker
Biofluid Reference Compound Database.
There was a strong correlation (r2 > 0.99) for all chemical shifts between the assigned
metabolites in figures 3-4 and 3-5 and the reference chemical shifts from Bruker Biofluid
Reference Compound Database (v 2-0-0 to v 2-0-3) (Figure B.18, Table B.2). Acetone, ethanol,
and formic acid are photochemically produced from CDOM while amino acids, such as alanine,
and carboxylic acids (eg. hydroxybenzoic acids) can be naturally found in DOM.8,45-47
Saturated
long chain aliphatic acids are also present in DOM and in rainwater.25,48
The main limitation of
85
assigning metabolites in this manner is that it is restricted to known structures in the database and
hinders the structural elucidation of unknown metabolites in DOM.
A variety of NMR techniques were used to characterize and study the photochemistry of
DOM. It was evident that DOM photolysis was highly dependent on its source and molecular
composition. DOM rich in conjugated unsaturated and aromatic structures were highly
susceptible to both direct and indirect photolysis. Pony Lake FA’s high degree of
phototransformation in comparison to Suwannee River NOM and Nordic Reservoir NOM may
be due to differing light intensities between Florida, Norway, and Antarctica as well as the
presence of lower molecular weight structures that have higher photolysis quantum yields and
are more accessible to photons. From in-situ and diffusion-edited NMR it is apparent that
photoirradiation induces bond cleavage of large DOM structures into oxygenated lower
molecular weight products. Furthermore, photochemical changes of specific metabolites in DOM
were successfully monitored using in-situ NMR spectroscopy. In the future, real time analysis
can provide a deeper understanding on the photosensitizing properties of DOM and its impact on
aquatic ecosystems.
86
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(10), L50-L52.
18. Zhao, R.; Lee, A. K. Y.; Huang, L.; Li, X.; Yang, F.; Abbatt, J. P. D. Photochemical
processing of aqueous atmospheric brown carbon. Atmos. Chem. Phys. 2015, 15 (2),
2957-2996.
19. Hatzianastassiou, N.; Matsoukas, C.; Fotiadi, A.; Pavlakis, K. G.; Drakakis, E.,
Hatzidimitriou, D.; Vardavas, I. Global distribution of Earth's surface shortwave radiation
budget. Atmos. Chem. and Phys. 2005, 5 (10), 2847-2867.
20. Pautler, B. G.; Woods, W. C.; Dubnick, A.; Simpson, A. J.; Sharp, M. J.; Fitzsimons, S.
J.; Simpson, M. J. Molecular characterization of dissolved organic matter in glacial ice:
coupling natural abundance 1H NMR and fluorescence spectroscopy. Environ. Sci.
Technol. 2012, 46 (7), 3753-3761.
21. Adams, R. W.; Holroyd, C. M.; Aquilar, J. A.; Nilsson, M.; Morris, G. A. “Perfecting”
WATERGATE: clean proton NMR spectra from aqueous solution. Chem. Comm. 2013,
49 (4), 358-360.
22. Lam, B.; Simpson A. J. Direct 1H NMR spectroscopy of dissolved organic matter in
natural waters. Analyst. 2008, 133, 263-269.
89
23. Simpson, A. J.; Song, G.; Smith, E.; Lam, B.; Novotny, E. H.; Hayes, M. H. B.
Unraveling the structural components of soil humin by use of solution-state nuclear
magnetic resonance spectroscopy. Environ. Sci. Technol. 2007, 41 (3), 876-883.
24. Wu, D.; Chen, A.; Johnson, C. S. Jr. An improved diffusion-ordered spectroscopy
experiment incorporating bipolar-gradient pulses. J. Magn. Reson. A. 1995, 115, 260-264.
25. Simpson, A. J. Determining the molecular weight, aggregation, structures and
interactions of natural organic matter using diffusion ordered spectroscopy. Magn. Reson.
Chem. 2002, 40, S72-S82.
26. Courtier-Murias, D.; Farooq, H.; Masoom , H.; Botana, A.; Soong , R.; Longstaffe, J. G.;
Simpson, M. J.; Maas, W. E.; Fey, M.; Andrew, B.; Struppe, J.; Hutchins, H.;
Krishnamurthy, S.; Kumar, R.; Monette, M.; Stronks, H. J.; Hume, A.; Simpson A. J.
Comprehensive multiphase NMR spectroscopy: basic experimental approaches to
differentiate phases in heterogeneous samples. J. Magn. Reson. 2012, 217, 61-76.
27. Lam, L.; Soong, R.; Sutrisno, A.; de Visser, R.; Simpson, M. J.; Wheeler, H. L.;
Campbell, M.; Maas, W. E.; Fey, M.; Gorissen, A.; Hutchins, H.; Andrew, B.; Struppe,
J.; Krishnamurthy, S.; Kumar, R.; Monette, M.; Stronks, H. J.; Hume, A.; Simpson, A. J.
Comprehensive multiphase NMR spectroscopy of intact 13C-labeled seeds. J. Agric. and
Food Chem. 2014, 62 (1). 107-115.
28. Koskela, H.; Heikkilä, O.; Kilpeläinen, I.; Heikkinen, S. Quantitative two-dimensional
HSQC experiment for high magnetic field NMR spectrometers. J. Magn. Reson. 2010,
202 (1), 24-33.
29. Eppley, R. W.; Peterson, B. J. Particulate organic matter flux and planktonic new
production in the deep ocean. Nature. 1979, 282, 677–680.
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30. Follett, C. L.; Repeta, D. J.; Rothman, D. H.; Xu, L.; Santinelli, C. Hidden cycle of
dissolved organic carbon in the deep ocean. PNAS. 2014, 111 (47), 16706-16711.
31. Yan, M.; Fu, Q.; Li, D.; Gao, G.; Wang, D. Study of the pH influence on the optical
properties of dissolved organic matter using fluorescence excitation-emission matrix and
parallel factor analysis. J. Lumin. 2013, 142, 103-109.
32. Cawley, K. M.; Hakala, J. A.; Chin, Y. P. Evaluating the triplet state photoreactivity of
dissolved organic matter isolated by chromatography and ultrafiltration using an
alkylphenol probe molecule. Limnol. Oceanogr. Methods. 2009, 7 (6), 391-398.
33. Grzybowski, W. Terrestrial humic substances induce photodegradation of
polysaccharides in the aquatic environment. Photochem. Photobiol. Sci. 2009, 8 (10),
1361-1363.
34. Brown, A.; McKnight, D. M.; Chin, Y. P.; Roberts, E. C.; Uhle, M. Chemical
characterization of dissolved organic material in Pony Lake, a saline coastal pond in
Antarctica. Mar. Chem. 2004, 89 (1-4), 327-337.
35. Graedel, T. E. Terpenoids in the atmosphere. Rev. Geophys. Space Phys. 1979, 17 (5),
937-947.
36. Xie, H.; Zafiriou, O. C.; Cai, W. J.; Zepp, R. G.; Wang, Y. Photooxidation and its effects
on the carboxyl content of dissolved organic matter in two coastal rivers in the
southeastern United States. Environ. Sci. Technol. 2004, 38 (15), 4113-4119.
37. Green, N. W.; McInnis, D.; Hertkorn, N.; Maurice, P. A.; Perdue, E. M. Suwannee River
natural organic matter: isolation of the 2R101N reference sample by reverse osmosis.
Environ. Eng. Sci. 2015, 32 (1), 38-44.
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38. Nason, J. A.; McDowell, S. A.; Callahan, T. W. Effects of natural organic matter type
and concentration on the aggregation of citrate-stabilized gold nanoparticles. J. Environ.
Monit. 2012, 17 (7), 1885-1892.
39. Goldberg, S. J.; Ball, G. I.; Allen, B. C.; Schladow, S. G.; Simpson, A. J.; Massom, H.;
Soong, R.; Graven, H. D.; Aluwihare, L. I. Refractory dissolved organic nitrogen
accumulation in high-elevation lakes. Nat. Commun. 2015, 6, 6347, DOI:
10.1038/ncomms7347.
40. Bindman, N.; Merkx, R.; Koehler, R.; Herrman, N.; van der Donk, W. A. Photochemical
cleavage of leader peptides. Chem. Comm. 2010, 46 (47), 8935-8937.
41. Hertkorn, N.; Harir, M.; Koch, B. P.; Michalke, B.; Schmitt-Kopplin, P. High-field NMR
spectroscopy and FTICR mass spectrometry: powerful discovery tools for the molecular
level characterization of marine dissolved organic matter. Biogeosciences. 2013, 10 (3),
1583-1624.
42. Mitchell, P. J.; Simpson, A. J.; Soong, R.; Oren, A.; Chefetz, B.; Simpson, M. J.
Solution-state NMR investigation of the sorptive fractionation of dissolved organic
matter by alkaline mineral soils. Environ. Chem. 2013, 10 (4), 333-340.
43. Cawley, K. M.; McKnight, D. M.; Miller, P.; Cory, R.; Fimmen, R. L.; Guerard, J.;
Dieser, M.; Jaros, C.; Chin, Y. P.; Foreman, C. Characterization of fulvic acid fractions
of dissolved organic matter during ice-out in a hyper-eutrophic, coastal pond in
Antarctica. Environ. Res. Lett. 2013, 8 (4), 045015.
44. D’Andrilli, J.; Foreman, C. M.; Marshall, A. G.; McKnight, D. M. Characterization of
IHSS Pony Lake fulvic acid dissolved organic matter by electrospray ionization Fourier
92
transform ion cyclotron resonance mass spectrometry and fluorescence spectroscopy.
Org. Geochem. 2013, 65, 19-28.
45. de Bruyn, W. J.; Clark, C. D.; Pagel, L.; Takehara, C. Photochemical production of
formaldehyde, acetaldehyde and acetone from chromophoric dissolved organic matter in
coastal waters. J. Photochem. Photobiol. A Chem. 2011, 226 (1), 16-22.
46. Fischer, H.; Meyer, A.; Fischer, K.; Kuzyakov, Y. Carbohydrate and amino acid
composition of dissolved matter leached from soil. Soil Biol. Biochem. 2007, 39 (11),
2926-2935.
47. Page, S. E.; Arnold, W. A.; McNeill, K. Assessing the contribution of free hydroxyl
radical in organic matter-sensitized photohydroxylation reactions. Environ. Sci. Technol.
2011, 45 (7), 2818-2825.
48. Cottrell, B. A.; Gonsior, M.; Timko, S. A.; Simpson, A. J.; Cooper, W. J.; van der Veer,
W. Photochemistry of marine and fresh waters: a role for copper-dissolved organic matter
ligands. Mar. Chem. 2014, 162, 77-88.
93
4 Chapter 4 - Conclusion and Future Directions
4.1 Light sources and system design: potential and limitations
Chapter 2 concludes that the Suntest model in combination with a loop flow system is the
most suitable system to investigate environmental photolytic processes using in-situ nuclear
magnetic resonance (NMR) spectroscopy. HPX-2000 and PX-2 models can be used to elucidate
photochemical reactions under harsher environmental conditions with the focus on evaluating
UV and UV/H2O2 as a remediation alternative in areas such as wastewater treatment plants. All
three in-situ NMR photoreactors are relatively easy to operate and allow for investigations of
photochemical processes with high temporal resolution in an automated fashion without user
intervention.
The limitation of the HPX-2000 and PX-2 setups lies in the use of optical fibers that feed
light directly into the NMR. Photolytic changes are not immediately observed using HPX-2000
and PX-2 but rather produce sigmoidal curves. This is likely due to the position of the optical
fiber within the NMR tube. The optical fiber must be positioned above the detection coil region
to avoid perturbation of magnetic homogeneity of the sample that can lead to line shape
distortions. Light is not transmitted uniformly across the sample resulting in a gradient of
decreasing photon concentration and energy that can initiate a chemical reaction from the upper
to the lower section of the detection coil region. Consequently, products take time to diffuse into
the detection coil region producing sigmoidal curves that complicate kinetic measurements such
as reaction rates and half-lives. This problem is avoided with the Suntest system as the
continuous flow mixes the solution in the reaction vessel placed inside the Suntest to generate a
logical decay profile of the parent compound. Furthermore, care is required when operating
94
HPX-2000 and PX-2 in-situ NMR photoreactors to prevent potential damage to the NMR
detection circuitry. Similarly, flow rate optimization is required while operating the looped-flow
system to prevent damage to the NMR flow probe and spectral distortion if the flow rate is too
high.
Although the spectral output of the Suntest model is specifically designed to simulate
solar radiation and the system is the best suited approach to investigating environmental
photolytic reactions, it has some drawbacks. The radiation flux measured inside the Suntest is
twice as less as the average global shortwave downward surface radiation reported in the
literature, but is consistent with the net absorbed shortwave radiation in New Orleans,
Casablanca, and Beijing in January and Paris and Berlin in October.1
Kinetic measurements can
be easily calibrated to account for the flow rate and differences in the radiation flux for first-
order photolytic reactions. This becomes more challenging when the photolytic reaction depends
on multiple variables such as substrate and reactive oxygenated species (eg. OH) concentrations.
The drawbacks of a flow system also includes the requirement for larger volumes of sample, the
rigorous cleaning between samples to prevent contamination, as well as designing a NMR flow
cell or have access to an NMR flow probe.
4.2 Evaluating aqueous photochemical processes using in-situ NMR spectroscopy
Aqueous photolytic reactions in the environment can be explored in-situ and in real time
using NMR spectroscopy by employing the Suntest model in combination with a loop flow
system. In-situ NMR spectroscopy offers high temporal resolution that allows the user to detect
and identify a range of reactive intermediates and degradation products for elucidation of
reaction mechanisms. Furthermore, generated kinetic profiles allow the user to assess the
95
stability of natural and anthropogenic compounds in the environment. For instance, the progress
of the photomineralization of p-nitrophenol in Chapter 2 was successfully followed using an in-
situ NMR photoreactor where a range of reactive intermediates such as 1,2,3-benzenetriol and
formic acid were detected, but could be missed by conventional techniques. Furthermore, the
high spectral dispersion offered by NMR spectroscopy permits both non-targeted and targeted
analysis of the photodegradation of crude oil’s water soluble fraction (WSF) and groundwater
over the course of light exposure in a non-invasive fashion. The ability to monitor the photolytic
fate of specific chemicals in crude oil and groundwater illustrates the rich information that can be
extracted on complex environmental processes using in-situ NMR spectroscopy. Additionally,
the ability to analyze samples taken directly from the environment, in their natural state and
natural abundance (eg. total organic carbon (TOC) of groundwater sample was ~2ppm), using
cryogenically cooled NMR probes demonstrates its viability in environmental studies.
4.3 Monitoring the photolytic fate of dissolved organic matter (DOM) using an in-
situ NMR photoreactor
DOM photolysis represents a large carbon photochemical process in natural waters where
DOM’s photosusceptibility to degradation is highly dependent on its source and molecular
composition. For instance, DOM rich in aromaticity, conjugated, unsaturated, and hydroxylated
structures are highly susceptible to phototransformation. Diffusion-editing NMR data
demonstrates that photoirradiation of these groups induces bond cleavage and ring-opening
reactions into oxygenated lower molecular-weight products such as acetone. In-situ and close to
real-time analysis, along with 1H NMR’s spectral dispersion capacity in relation to other
analytical tools such as UV-Vis spectroscopy, allows the user to follow the photolytic fate of
96
specific metabolites over time. 1H-
13C Heteronuclear Single Quantum Coherence Spectroscopy
(HSQC) and 1H-
1H Correlation Spectroscopy (COSY) NMR spectra provided additional
information on the molecular composition of DOM prior and following light exposure. A month
long light exposure also demonstrated the high photoreactivity of aromatic and conjugated
unsaturated structures. Overall, the work presented in this thesis demonstrates the potential of in-
situ NMR spectroscopy as a complementary analytical tool in unraveling complex environmental
photolytic processes.
4.4 Future Directions
A variety of 1H NMR techniques have been used to investigate the photolytic fate of
simple compounds, oil extracts, groundwater, and DOM in real time using in-situ NMR
photoreactors. However, valuable information for structural elucidation of reactive intermediates
and photoproducts can be missed by monitoring changes using a single nucleus. Furthermore,
information on the molecular composition of DOM and its effect on the photodegradation of
organic contaminants in aquatic ecosystems are limited. Given that, the use of dual receivers and
incorporation of mass spectroscopy (MS) with in-situ NMR photoreactors for a more detailed
structural elucidation are discussed in this section. The application of in-vivo NMR with in-situ
NMR photoreactors to study the phototactic reactions of organisms is also considered here.
Finally, an experimental design for investigating the photolytic fate of pesticides in soil using in-
situ solid-state NMR spectroscopy is discussed below.
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4.4.1 Parallel acquisition and dual receivers
The photodegradation of various environmental systems in this thesis were investigated
using a single receiver to demonstrate the viability of in-situ NMR photoreactors in
environmental studies. Recent advancement in NMR technology with dual receivers permits
simultaneous detection of two nuclei at the same time (for example 1H and
19F). Dual receivers
have already been implemented in protein NMR.2,3
In environmental research dual receivers
could be used to investigate the photolytic fate of halogenated (eg. –F and -Br) fire retardants
and phosphorylated molecules with one receiver following the DOM (for example 1H-
13C
HSQC) with the other the contaminant (eg. 19
F and 31
P NMR) in real time. One such case is
Mabury. S. A. et al. research on the photodegradation of trifluralin by 19
F NMR.4 In this study
various products containing both 1H and
19F were produced. A dual detection approach using
both 1H and
19F signals can provide important complementary information on the photolytic
mechanism of trifluralin and structural elucidation of reactive intermediates and products. This
idea can be also applied to other species such as 13
C, 13
P, and 15
N. The technology also allows
scientists to carry out multiple experiments more efficiently and reduce experimental time.
4.4.2 Influence of DOM on the photodegradation of organic contaminants
DOM plays a dual role in the transportation and sequestration of organic pollutants in
natural waters. Its photosensitizing properties also have a large impact on the photodegradation
of contaminants. Water pollutants, such as pharmaceuticals and personal care products (PPCPs)
and pesticides, produce toxic products in the presence of DOM. For instance, triclocarban
photodegrades into highly toxic chloroanilines and chloroiso-cyanatobenzene products in the
presence of DOM.5 Other studies have also demonstrated the dual action of DOM on the
98
photodegradation of antibiotics, by both increasing the photoreactivity via the photogeneration of
ROS and triplet excited state of DOM (3DOM*) as well as inhibition of phototransformation as a
result of inner filter effect.6 For these reasons, DOM plays a key role in the photochemical fate
and persistence of contaminants in aqueous environments. Unfortunately, there is still lack of
knowledge on the dual action of DOM in mediated photolysis of organic pollutants. Florescence
spectroscopy has been commonly used to investigate the photobleaching effects of DOM from
various sources. However, studies conducted using florescence spectroscopy alone are selective
and are limited to the DOM fraction that fluoresces. The non-selective nature of in-situ NMR
spectroscopy provides indiscriminate information that makes it an ideal tool for structural
elucidation of reactive intermediates and photoproducts in real time. Future studies should build
on the work presented in Chapter 3 and compare the phototransformation of aquatic pollutants in
the presence and absence of DOM to obtain a better understanding of the photosensitizing
properties of DOM and the photolytic fate of contaminants in aquatic ecosystems.
4.4.3 Combining in-vivo NMR with in-situ NMR photoreactors
Recent studies using Daphnia magna demonstrate the potential of studying living
organisms on a molecular scale using in-vivo NMR spectroscopy.7,8
Therefore, in-vivo NMR
spectroscopy could potentially be used to study the behavioural characteristics of living
organisms. For instance, Storz, et al. have indicated that ultraviolet light caused negative
phototaxis in D. magna while visible light led to positive phototaxis.9,10
In-vivo NMR in
combination with in-situ NMR photoreactors discussed in Chapter 2 can provide insight into the
phototactic reaction of D. magna to light. For this study, the Suntest is the most ideal light source
to mimic the environment as it is specifically designed to simulate solar radiation. An optical
99
fiber would have to be incorporated into the in-vivo flow system designed by Soong, R. et al. in
order to feed light from the Suntest into the NMR.8 NMR spectra of D. magna in the dark will
act as a control. Upon light exposure, biochemical changes can be monitored quantitatively using
quantitative HSQC NMR experiments, as previously demonstrated in chapter 3. Standards can be
acquired separately to obtain absolute measurements. Analyzing D. magna’s metabolic response
to light exposure in-vivo and in real time can provide a better understanding into the mode of
action governing these phototactic reactions.
This design poses a few obstacles that must be taken into account. The section of the
NMR tube in the detection coil region is filled with ~10 Daphnia for maximum signal.8 This
restricts D. magna’s movement in response to light exposure. On a similar note, light penetration
will be limited due to the large density of D. magna in the NMR tube. D. magna found at the
lower section might not experience a phototactic reaction. As a result, detection of biochemical
changes may become limited to the D. magna fraction that is exposed to light as well as the
detection limit of the NMR spectrometer. One possible solution is to reduce the number of D.
magna inside the NMR tube and compensate for the loss in signal by using a cryogenic probe for
higher sensitivity as well as increasing the number of scans per experiment. In addition, the
optical fiber must be positioned as to not cause harm to the organisms. D. magna’s transparent
outer shell protects them from being detected by predators. However, their transparency exposes
their internal components (eg. photolabile proteins and genes) to solar radiation that could
possibly introduce artifacts into the study.9,11
Conversely, the natural transparency of D. magna
provides an interesting opportunity to study their natural response to light in a biological context.
Once constructed, the in-vivo photochemical system can also be used to understand
metabolic stress responses of small organisms. D. magna is often used in aquatic ecotoxicology
100
studies due to its intermediate position in the food web, short life-span, and sensitivity to
toxins.12,13,14
As such, D. magna can be used to assess contaminant toxicity of various organic
pollutants and their photoproducts. D. magna inside the NMR tube will act as the reaction vessel.
A contaminant, such as triclocarban, will flow in a closed circuit from the Suntest into the NMR
tube. Over time the parent compound will photodegrade and D. magna will become exposed to
its photoproducts. Real time analysis of biochemical changes in D. magna upon exposure to a
contaminant (triclocarban) and its toxic photoproducts (chloroanilines and chloroiso-
cyanatobenzene products15
) can be used to re-assess the toxicity of anthropogenic contaminants
found in natural waters as the parent contaminant may display a low degree of toxicity while its
photoproducts may have an acute effect on D. magna. Further studies combining,
photochemistry, DOM, contaminants and their real-time impact on living organisms would be
challenging to perform using any other analytical approach but could provide a unique insight
into the complex synergism between environmental processes.
4.4.4 Combining MS with in-situ NMR spectroscopy
In-situ NMR spectroscopy is a versatile, non-selective, non-invasive, and robust
analytical tool that is rich in information for elucidation of structure and reaction mechanism of
complex systems.16,17
It provides structural information, such as chemical shifts and multiplicity,
and integrals for qualitative and quantitative analysis. It also provides insight into a wide range
of dynamic processes and intramolecular interactions, including diffusion in solution and ligand
binding such as endocrine disruptors with DOM.18
Yet, structural characterization using NMR
spectroscopy alone can be quite challenging in large part due to spectral overlap. While this can
be overcome through additional dispersion is afforded by multidimensional experiments, the
101
sensitivity of these experiments is much lower and takes longer to acquire making real-time
analysis challenging.
Conversely, MS offers higher sensitivity than NMR spectroscopy permitting the
detection of trace chemicals that can be missed by NMR, but cannot offer isomeric information.
Therefore, simultaneous detection by hyphenation of NMR and MS using a closed-flow system
will provide complementary information for accurate characterization of unknowns and reduce
ambiguity. For example in a complex system molecules could be assigned using two dimensional
(2D), three dimensional (3D) NMR spectroscopy which may take days, but after identification,
they could then be monitored using 1H NMR and even at trace concentrations using MS. A built-
in splitter system can be used to split a large portions of the flow to the NMR with a small
amount to MS. Calibration will be required for both quantitative and kinetic analysis of
photochemical processes to account for the continuous loss of sample to the MS. However, as all
molecules show the same response in NMR, the NMR data itself could be very important in
helping better calibrate MS responses which differ on a per molecule basis.
4.4.5 In-situ photoirradiation of pesticides using solid-state and comprehensive
multiphase (CMP) NMR spectroscopy
Many contaminants are introduced into the environment (eg. atmosphere, natural waters,
and soils) due to anthropogenic activities, leaving behind negative ecological consequences.19
This thesis has focused on understanding aqueous photochemical behaviour of natural and
anthropogenic contaminants in the environment. Soil is another major sink for environmental
contaminants such as pesticides. The heterogeneity of soil makes the phototransformation of
pesticides different than in aqueous solution and also much more difficult to understand.20,21
102
Thus, the photochemical degradation of contaminants in soil becomes of great interest as solar
radiation is one of the main destructive pathways.
Soil in its natural state is a complex heterogeneous multiphase sample that consists of
aqueous (pore-water and DOM), gel-like (swollen organic matter), and solid (microbial cell
walls, dry organic matter, and minerals) phases.22,23
With recent developments, photodegradation
of pesticides can be studied on soil in its natural and unaltered state using comprehensive
multiphase (CMP)-NMR spectroscopy.24
The ability of other analytical instruments to observe
changes in soil in its natural state is currently limited.22,23
Aqueous, gel-like, and solid
components can be analyzed simultaneously using CMP-NMR to provide detailed information
on all the bonds and interactions in all phases between a contaminant and soil.22
This is critical
for obtaining accurate information on an environmental system and kinetic measurements as
previous studies have shown that the photolytic half-lives of pesticides are highly dependent on
the presence of moisture in soil.25
Real time analysis can be achieved by feeding light from the
Suntest into the NMR via an optical fiber in a similar fashion to Tomonaga, Y. et al..26
In
Tomonaga, Y. et al. ‘s work, using solid-state NMR spectroscopy under magic angle spinning
(MAS), the optical fiber was fed into the NMR through a sealed cap that was attached to a
zirconia rotor and the light was illuminated from inside the rotor.26
In this manner the light is
concentrated on the center of the sample. In-situ photoirradiation using solid state NMR
spectroscopy have been demonstrated in various fields such as inorganic chemistry and medicine
(eg. trichloroethylene)27
, proving its viability and need in environmental studies. As CMP-NMR
can be applied to samples in their natural fully water-swollen state, photochemical studies
provide a unique window to permit the monitoring of photochemical degradation in soil and
sediment under close to environmental conditions.
103
4.5 References
1. Hatzianastassiou, N.; Matsoukas, C.; Fotiadi, A.; Pavlakis, K. G.; Drakakis, E.;
Hatzidimitriou, D.; Vardavas, I. Global distribution of Earth's surface shortwave radiation
budget. Atmos. Chem. Phys. 2005, 5(10), 2847-2867.
2. Kupče, E. NMR with multiple receivers. Top. Curr. Chem. 2013, 335, 71-96.
3. Kupče, E.; Kay, L. E. Parallel acquisition of multi-dimensional spectra in protein NMR.
J. Biomol. NMR. 2012, 54 (1), 1-7.
4. Mabury, S. A.; Crosby, D. G. 19
F NMR as an analytical tool for fluorinated agrochemical
research. J. Agric. Food Chem. 1995, 43 (7), 1845-1848.
5. Trouts, T. D.; Chin, Y. P. Direct and indirect photolysis of triclocarban in the presence of
dissolved organic matter. Elementa. 2015. DOI: 10.12952/journal.elementa.000050
6. Liang, C.; Zhao, H.; Deng, M.; Quan, X.; Chen, S.; Wang, H. Impact of dissolved
organic matter on the photolysis of the ionizable antibiotic norfloxacin. J. Environ. Sci.
(China). 2015, 27, 115-123.
7. Simpson, A. J.; Liaghati, Y.; Fortier-McGill, B.; Soong, R.; Akhter, M. Perspective: in-
vivo NMR – a potentially powerful tool for environmental research. Magn. Reson. Chem.
2015, 53 (9), 686-690.
8. Soong, R.; Nagato, E.; Sutrisno, A.; Fortier-McGill, B.; Akhter, M.; Schmidt, S.;
Heumann, H.; Simpson, A. J. In vivo NMR spectroscopy: toward real time monitoring of
environmental stress. Magn. Reson. Chem. 2015, doi: 10.1002/mrc.4154.
9. Storz, U. C.; Paul, R. J. Phototaxis in water fleas (Daphnia magna) is differently
influenced by visible and UV light. J. Comp. Physiol. A. 1998, 183, 709-717.
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10. Ebert, D. Ecology, epidemiology, and evolution of Parasitism in Daphnia; Bethesda
(MD) National Center for Biotechnology Information: US, 2005.
11. Janssen, E. M.; Erickson, P. R.; McNeill, K. Dual roles of dissolved organic matter as
sensitizer and quencher in the photooxidation of tryptophan. Environ. Sci. Technol. 2014,
48 (9), 4916-4924.
12. Lampert, W. Daphnia: model herbivore, predator and prey. Pol. J. Ecol. 2006, 54 (4),
607-620.
13. Soetaert, A.; van der Ven, K.; Moens, L. N.; Vandenbrouck, T.; van Remortel, P.; De
Coen, W. M. Dapnia magna and ecotoxicogenomics: gene expression profiles of the anti-
ecdysteroidal fungicide fenarimol using energy-, molting- and life stage-related cDNA
libraries. Chemosphere. 2007, 67 (1), 60-71.
14. Dang, Z.; Cheng Y.; Chen, H. M.; Cui, Y.; Yin, HH.; Traas, T.; Montforts, M.; Vermeire,
T. Evaluation of the Daphnia magna reproduction test for detecting endocrine disruptors.
Chemosphere. 2012, 88 (4), 514-523.
15. Ding, S. L.; Wang, X. K.; Jiang, W. Q.; Zhao, R. S.; Shen, T. T.; Wang, C.; Wang, X.
Influence of pH, inorganic anions, and dissolved organic matter on the photolysis of
antimicrobial triclocarban in aqueous systems under simulated sunlight irradiation.
Environ. Sci. Pollut. Res. Int. 2015, 88 (7), 5204-5211.
16. Comel, A.; Guiochon, G. The chemical composition of mixed wastes: analysis of the
photolysis products of organic ligands. J. Radioanal. Nucl. Chem. Art. 1994, 181 (2),
373-384.
105
17. Smith, M.E.; van Eck, E.R.H. Recent advances in experimental solid state NMR
methodology for half-integer spin quadrupolar nuclei. Prog Nucl Mag Res Sp, 1999, 34
(2), 159-201.
18. Bedard, M.; Giffear, K. A.; Ponton, L.; Sienerth, K. D.; Del Gaizo Moore, V.
Characterization of binding between 17β-estradiol and estriol with humic acid via NMR
and biochemical analysis. Biophys. Chem. 2014, 189, 1-7.
19. Siampiringue, M.; Chung, P. W. W.; Moursalou, K.; Tchangbedji, G.; Sarakha, M. Clay
and soil photolysis of the pesticides Mesotrione and Metsulfuron Methyl. App. Environ.
Soil Sci., 2014, 2014 (3), 1-8.
20. Garebing, P.; Frank, M. P.; Chib, J. S. Soil photolysis of herbicides in a moisture- and
temperature-controlled environment. J. Agric. Food Chem. 2003, 51 (15), 4331-4337.
21. Katagi, T. Photodegradation of pesticides on plant and soil surfaces. Rev. Environ.
Contam. Toxicol. 2004, 182, 1-189.
22. Lam, B.; Simpson, A. J. Direct 1H NMR spectroscopy of dissolved organic matter in
natural waters. Analyst. 2008, 133(2), 263-269.
23. Courtier-Murias, D.; Farooq, H.; Masoom, H.; Botana, A.; Soong, R.; Longstaffe, J. G.;
Simpson, M. J.; Maas, W. E.; Fey, M.; Andrew, B.; Struppe, J.; Hutchins, H.;
Krishnamurthy, S.; Kumar, R.; Monettte, M.; Stronks, H. J.; Hume, A.; Simpson, A. J.
Comprehensive multiphase NMR spectroscopy: basic experimental approaches to
differentiate phases in heterogeneous samples. J. Magn. Reson. 2012, 217, 61-76.
24. Simpson, A. J.; Courtier-Murias, D.; Longstaffe, J. G.; Masoom, H.; Soong, R.; Lam, L.;
Sutrisno, A.; Farooq, H.; Simpson, M. J.; Maas, W. E.; Fey, M.; Andrew, B.; Struppe, J.;
106
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Comprehensive Multiphase NMR. eMagRes. 2013, 2 (3), 399–414.
25. Garebing, P.; Chib, J. S. Soil photolysis in a moisture- and temperature-controlled
environment. 2. Insecticides. J. Agric. Food Chem. 2004, 52 (9), 2606-2614.
26. Tomonaga, Y.; Hidaka, T.; Kawamura, I.; Nishio, T.; Ohsawa, K.; Okitsu, T.; Wada, A.;
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revealed by in situ photoirradiated solid-state NMR spectroscopy. Biophys. J. 2011, 101
(10), L50-L52.
27. Hwang, S-J.; Petucci, C.; Raftery, D. In situ solid-state NMR studies of trichloroethylene
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Chem. Soc. 1998, 120 (18), 4388-4397.
107
Appendix A - Supporting information for Chapter 2
OceanOptics Spectral Output Graphs
OceanOptics HPX-2000
Figure A.1a. OceanOptics HPX-2000 pulsed xenon lamp spectral output. Spectrum was
obtained from OceanOptics.
108
OceanOptics PX-2
Figure A.1b. OceanOptics PX-2 pulsed xenon lamp spectral output. Spectrum was obtained
from OceanOptics.
Original Hanau Suntest Spectral Output graph
Figure A.2. Spectral output of Original Hanau Suntest. The spectrum was obtained from
ATLAS-Materials Testing Solutions Ltd.
109
Riboflavin
Table A.1. Advantages and disadvantages of three different light sources.
Light Source Characteristics Advantages Disadvantages 1. OceanOptics
HPX-2000 +
optical fiber
- 35W continuous xenon light
source
- wavelength coverage: 185-
2000 nm
- shutter is controlled by TTL
signal
- Easy handling
- Quick clean-up time
- Requires small amount
of sample and volume
of
deuterated solvent
- shutter is controlled by
TTL signal
- Can study atmospheric
reactions (photolysis)
- Requires careful
handling of the optical
fiber
- Intensity of light cannot
be controlled
- Harsh conditions
2. OceanOptics
PX-2 +
optical fiber
- pulsed xenon lamp
- adjustable flash rate
- wavelength range: 220-750
nm
- SMA 905 output connector
- controlled by a TTL signal
- adjustable flash rate
- Easy handling
- Quick clean-up time
- Requires small amount
of sample and volume
of
deuterated solvent
- shutter is controlled by
TTL signal
- Can study atmospheric
reactions (photolysis)
- Requires careful
handling of the optical
fiber
- Intensity of light cannot
be controlled
- Harsh conditions
3. Original
Hanau
Suntest –
closed-
circuit flow
system
- xenon burner
- removable UV filter
- wavelength coverage: 300-
830 nm
- radiation intensity: 830
W/m2
- Study photolytic
reactions at Earth’s
surface
- removable optical
filters (eg. UV) that
can be used to study
reactions with harsher
conditions
- IR mirrors for heat
reduction
- Long set up and clean-
up time
- Requires more sample
and solvent compared to
1 and 2.
- Intensity of light cannot
be controlled
110
OceanOptics HPX-2000
Figure A.3.
1H NMR spectra of a riboflavin solution prior to light exposure. A:
1H NMR
spectrum of a riboflavin solution at the start of the light off experiment. B: 1H NMR spectrum of
a riboflavin solution after 4 hours in the dark. This experiment simply demonstrates that the
sample is stable prior to light exposure.
111
Figure A.4.
1H spectrum of the phototransformed riboflavin sample after 12 hours and 20
minutes of light exposure using OceanOptics HPX-2000.
112
Figure A.5.
1H NMR spectra of the phototransformed riboflavin solution following light
exposure. A: 1H NMR spectrum of the phototransformed riboflavin sample immediately after
light exposure. B: 1H NMR spectrum of the phototransformed riboflavin sample after 4 hours in
the dark. These spectra are included to demonstrate that once the light is turned off the reactions
do not continue helping confirm that the light is responsible for the observed changes.
113
OceanOptics PX-2
Figure A.6.
1H NMR spectrum of the phototransformed riboflavin sample after 20 hours and 20
minutes of light exposure using the OceanOptics PX-2 system.
114
Original Hanau Suntest light system
Figure A.7.
1H NMR spectra of a riboflavin solution at three different time points during light
exposure (12 hours and 20 minutes) inside Original Hanau Suntest light system.
115
Figure A.8.
1H NMR spectrum of the phototransformed riboflavin sample after light exposure
inside Original Hanau Suntest light system.
116
Figure A.9.
1H NMR spectra of the phototransformed riboflavin solution following light
exposure. A: 1H NMR spectrum of the phototransformed riboflavin sample immediately after
light exposure. B: 1H NMR spectrum of the phototransformed riboflavin sample after 4 hours in
the dark. These spectra are included to demonstrate that once the light is turned off the reactions
do not continue helping confirm that the light is responsible for the observed changes.
117
Figure A.10.
1H NMR spectra of a riboflavin solution used in a dark control experiment. A:
1H
spectrum of the riboflavin sample prior to light exposure. B: 1H spectrum of the same riboflavin
sample after it was covered in aluminum foil and placed inside the Suntest system for the
duration of the light-on experiment (12 hours and 20 minutes).
118
p-nitrophenol
Original Hanau Suntest model
Figure A.11.
1H NMR spectra of the p-nitrophenol sample prior to light exposure. A:
1H NMR
spectrum of p-nitrophenol solution at the start of the light-off experiment. B: 1H NMR spectrum
of p-nitrophenol solution after 3 hour in the dark. This experiment simply demonstrates that the
sample is stable prior to light exposure.
119
Figure A.12.
1H NMR spectra of the p-nitrophenol sample at three different time points during
light exposure inside Original Hanau Suntest model.
120
Figure A.13.
1H NMR spectra of the phototransformed p-nitrophenol sample following light
exposure inside Original Hanau Suntest. A: 1H NMR spectrum of p-nitrophenol immediately
after light exposure. B: 1H NMR spectrum of p-nitrophenol after additional 3 hours in the dark.
These spectra are included to demonstrate that once the light is turned off the reactions do not
continue helping confirm that the light is responsible for the observed changes.
121
Figure A.14.
1H NMR spectra of p-nitrophenol sample from the dark control experiment. A:
1H
spectrum of the p-nitrophenol prior to light exposure. B: 1H spectrum of the same p-nitrophenol
sample after it was covered in aluminum foil and placed inside the Suntest for the duration of the
light-on experiment (12 hours and 40 minutes).
122
OceanOptics HPX-2000
Figure A.15.
1H NMR spectrum of p-nitrophenol sample after 12 hours and 40 minutes light
exposure using OceanOptics HPX-2000 light source.
123
Crude Oil
OceanOptics HPX-2000
Figure A.16.
1H spectra of 17.1mM SDS solution in 70% D2O and 30% H2O after it was
exposed to light from OceanOptics HPX-2000 for duration of 18 hours. A: SDS solution before
light exposure. B: SDS solution after light exposure. This experiment simply demonstrates that
the sample is stable prior to light exposure.
124
Figure A.17.
1H spectra of the water soluble fraction (WSF) of crude oil prior to light exposure.
A: initial 1H spectrum of WSF in the dark. B:
1H spectrum of WSF after 4 hours in the dark. This
experiment simply demonstrates that the sample is stable prior to light exposure.
125
Figure A.18. A:
1H spectrum of WSF of crude oil prior to light exposure (0.5-4.5 ppm region).
B: 1H spectrum of the photodecomposed WSF of crude oil after the light exposure for 18 hours
inside a NMR tube using OceanOptics HPX-2000 light box (0-4.5 ppm region). Spectral changes
from spectrum A are highlighted.
126
Figure A.19. A:
1H spectrum of WSF of crude oil prior to light exposure (5-10 ppm region). B:
1H spectrum of the photodecomposed WSF of crude oil after the light exposure for 18 hours
inside a NMR tube using OceanOptics HPX-2000 light box (5-10 ppm region). Spectral changes
from spectrum A are highlighted.
127
Figure A.20.
1H spectra of the photodecomposed WSF of crude oil after light exposure. A:
1H
directly after exposure. B: 1H spectrum after an additional 2 hours in the dark. These spectra are
included to demonstrate that once the light is turned off the reactions do not continue helping
confirm that the light is responsible for the observed changes.
128
Figure A.21. Edited-HSQC spectrum of crude oil following light exposure with HPX-2000. 1:
aliphatic compounds. 2: -CH2- adjacent to double bond (break down product from aromatics). 3:
-CH2- and -CH- adjacent to an aromatic ring. 4: -CH2- and -CH- adjacent to carboxylic group. 5:
-H bonded to unsaturated carbon (H-C=-) (ring opening product). 6: -CH- next to OH. 7: SDS
(overlapped with aliphatic compounds between 0.5-2 ppm).
129
Figure A.22. A:
1H spectrum of WSF of crude oil after light exposure using HPX-2000 light
source. B: 1H selective TOCSY spectrum of WSF of crude oil after light exposure.
130
Original Hanau Suntest Light system
Figure A.23. A:
1H spectrum of WSF of crude oil prior to light exposure (0-4.5 ppm region). B:
1H spectrum, in the 0-4.5 ppm region, of the photodecomposed WSF of crude oil after the
sample was exposed to light for 18 hours inside the Suntest model.
131
Figure A.24. A:
1H spectrum of WSF of crude oil prior to light exposure (5-10 ppm region). B:
1H spectrum, in the 5-10 ppm region, of the photodecomposed WSF of crude oil after the sample
was exposed to light for 18 hours inside the Suntest model.
132
Figure A.25.
1H spectra of WSF of crude oil from dark control experiment inside the Suntest
light system. A: 1H spectrum of WSF of crude oil prior to light exposure. B:
1H spectrum of the
same WSF sample after it was covered in aluminum foil and placed inside the Suntest system for
the duration of the light experiment.
133
Figure A.26. Kinetic profile of signals corresponding to -CH2-/-CH- signals adjacent to
carboxylic groups at
~2.5ppm from WSF of crude oil over the course of light exposure using
HPX-2000 and Suntest as light sources.
134
Groundwater
Original Hanau Suntest
Figure A.27.
1H NMR spectra of groundwater prior to light exposure. A:
1H NMR spectrum of
groundwater at the start of the light-off experiment. B: 1H NMR spectrum of groundwater after 6
hours in the dark. This experiment simply demonstrates that the sample is stable prior to light
exposure.
135
Figure A.28. A:
1H spectrum of groundwater prior to light exposure (0-4.5 ppm region). B:
1H
spectrum of the groundwater after the sample was exposed to solar radiation for 1 day and 12
hours inside the Suntest solar simulator.
136
Figure A.29.
1H NMR spectra of the phototransformed groundwater sample after light exposure.
A: 1H NMR spectrum of the phototransformed groundwater directly after light exposure. B:
1H
NMR spectrum of the phototransformed sample after additional 6 hours in the dark. These
spectra are included to demonstrate that once the light is turned off the reactions do not continue
helping confirm that the light is responsible for the observed changes.
137
Figure A.30. A:
1H spectrum of the groundwater sample prior to light exposure. B:
1H spectrum
of the groundwater sample after it was covered in aluminum foil and placed inside the Suntest
model for the duration of the light experiment (1 day and 12 hours).
138
Figure A.31. Kinetic profile of the photodegradation of lactic acid and dual photogeneration and
consumption of acetone in groundwater over the course of light exposure.
139
Appendix B - Supporting information for Chapter 3
Figure B.1.A. Design of the in-situ NMR photoreactor system used in this study as well as the
spectral output of Original Hanau Suntest light source which was obtained from ATLAS-
Materials Testing Solutions Ltd.
140
Figure B.1.B. Design of the in-situ NMR photoreactor system used in this study to account for
any evaporation in figure B.1.A.
141
“Light-off” 1H NMR spectra from in-situ NMR analysis
Figure B.2. A: Initial
1H NMR spectrum of Nordic Reservoir NOM prior to light exposure. B:
1H NMR spectrum of Nordic Reservoir NOM after 3 hours in the dark. This experiment simply
demonstrates that the sample is stable prior to light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
142
Figure B.3. A: Initial
1H NMR spectrum of Nordic Reservoir NOM following light exposure. B:
1H NMR spectrum of the phototransformed NOM after additional 3 hours in the dark. This
experiment simply demonstrates that the sample is stable following light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
143
Figure B.4. A: Initial
1H NMR spectrum of Pony Lake Fulvic Acid prior to light exposure. B:
1H
NMR spectrum of Pony Lake Fulvic acid after 3 hours in the dark. This experiment simply
demonstrates that the sample is stable prior to light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
144
Figure B.5. A: Initial
1H NMR spectrum of Pony Lake Fulvic Acid following light exposure. B:
1H NMR spectrum of the phototransformed DOM after additional 3 hours in the dark. This
experiment simply demonstrates that the sample is stable following light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
145
Figure B.6. A: Initial
1H NMR spectrum of Suwannee River NOM prior to light exposure. B:
1H
NMR spectrum of Suwannee River NOM after 3 hours in the dark. This experiment simply
demonstrates that the sample is stable prior to light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
146
Figure B.7. A: Initial
1H NMR spectrum of Suwannee River NOM following light exposure. B:
1H NMR spectrum of the phototransformed NOM after additional 3 hours in the dark. This
experiment simply demonstrates that the sample is stable following light exposure.
11 10 9 8 7 6 5 4 3 2 1 ppm
A
B
147
In-situ 1H NMR spectra before and after photoirradiation for 5 days
Figure B.8. A: 1H NMR spectra of Nordic Reservoir NOM prior to light exposure. B:
1H NMR
spectra of Nordic Reservoir NOM following to light exposure.
148
Figure B.9. A: 1H NMR spectra of Suwannee River NOM prior to light exposure. B:
1H NMR
spectra of Nordic Reservoir NOM following to light exposure.
149
Figure B.10. A: 1H NMR spectra of Pony Lake Fulvic Acid prior to light exposure. B:
1H NMR
spectra of Nordic Reservoir NOM following to light exposure.
Table B.1. % of total proton integration of different DOM fractions prior to light exposure.
MDLT CRAM Carbohydrates Olefinic Aromatics and lignin
Nordic
Reservoir
NOM
26.4 ± 0.5 48.9 ± 0.3 15.3 ± 0.4 3.0 ± 0.4 6.3 ± 0.2
Suwannee
River
NOM
23.5 ± 0.4 53.8 ± 0.2 15.1 ± 0.6 2.3 ± 0.4 5.2 ± 0.1
Pony Lake
fulvic acid
(FA)
reference
33.0 ± 0.2 53.2 ± 0.3 7.6 ± 0.9 1.7 ± 0.2 4.5 ± 0.1
150
Figure B.11. Overlaid 1H NMR spectra of Nordic Reservoir NOM (between 2-5ppm) every 20
hours during “light-off” portion of the experiment. Black: day 0, Red: day 1, Green: day 2,
Purple: day 3, Blue: day 4, Orange: day 5.The spectral intensity between 2.3-2.5ppm increases
over time.
151
In-situ diffusion-editing NMR spectra
Figure B.12. In-situ 1H diffusion-editing NMR spectra showing the change of the rigid and large
molecular weight structures of Nordic Reservoir NOM upon light exposure over the course of 5
days.
152
Figure B.13. In-situ
1H diffusion-editing NMR spectra showing the change of the rigid and large
molecular weight structures of Suwannee River NOM upon light exposure over the course of 5
days.
153
Figure B.14. In-situ
1H diffusion-editing NMR spectra showing the change of the rigid and large
molecular weight structures of Pony Lake Fulvic Acid upon light exposure over the course of 5
days. Note that CH2-COOH resonates in the CRAM region and forms at apex at 2.3ppm, this
signal clearly increases over time indicating the larger material is becoming carboxylated. While
this is visible to some extent in the Nordic and Suwannee samples the trend is clearest in this
figure (Pony Lake).
154
Figure B.15. In-situ
1H inverse diffusion-editing NMR spectra of Suwannee River NOM
showing the photogeneration of low molecular weight molecules over the course of 5 days.
155
1H-
13C HSQC NMR Spectra
Figure B.16. A:
1H-
13C HSQC NMR spectra of Nordic Reservoir NOM prior to light exposure.
B: 1H-
13C HSQC NMR spectra of Nordic Reservoir NOM following light exposure.
156
Figure B.17. A:
1H-
13C HSQC NMR spectra of Pony Lake Fulvic Acid prior to light exposure.
B: 1H-
13C HSQC NMR spectra of Pony Lake Fulvic Acid following light exposure.
157
Figure B.18. Zoomed region of
1H-
13C HSQC NMR spectra of three DOM samples before light
exposure, focusing on lignin methoxy group (fraction 7) and proteins (fraction 10).
158
Figure B.19. Strong correlation (r
2 > 0.99) for all chemical shifts between the assigned
metabolites in COSY and the reference chemical shifts from Bruker Biofluid Reference
Compound Database (v 2-0-0 to v 2-0-3).
159
Table B.2. Assignment of components in DOM from 1H-
1H COSY NMR experiments using
Bruker Database along with references of previous studies that have also identified these
components in DOM.
Compound References
Acetic acid 1,2
Fumaric acid 3,4
Levulinic acid 4,5
Methanol 2
Propionic acid 2,6,7
Pyruvic acid 8
Acetone 9
Formic acid 8
3-hydroxyprionic acid 10
Glutaric acid 3,11
Glycolic acid 3,7
Lactic acid 3,6,7
4-hydroxybenzoic acid 3,5
Ethanol
4-hydroxyhippuric acid
Alanine 12
C6-8 saturated carboxylic
acid and C6 dicarboxylic
acids (eg. Caprylic acid,
caproic acid, adipic acid)
13
160
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162
Appendix C – Copyrights and Permissions
163
164
