Investigation of Sulphur Containing Organic Compounds in Groundwater
Using Differential Ion Mobility and Mass Spectrometry
by
Jadwiga Lyczko
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Master of Science
in
Chemistry
Guelph, Ontario, Canada
©Jadwiga Lyczko, August, 2013
ABSTRACT
INVESTIGATION OF SULPHUR CONTAINING ORGANIC COMPOUNDS IN
GROUNDWATER USING DIFFERENTIAL ION MOBILITY AND MASS
SPECTROMETRY
Jadwiga Lyczko
Advisor:
University of Guelph, 2013
Dr. Wojciech Gabryelski
Groundwater aquifers are the largest source of drinking water for human population.
Current available information of the quality of groundwater is quite limited mainly due to the
lack of comprehensive analysis of groundwater and the challenging task of applying any
analytical method in its investigation. In this thesis, a new method based on “soft” mass
spectrometry and differential ion mobility (FAIMS) was developed to discover previously
unknown sulphur-containing contaminants in groundwater in Ontario. Following this discovery,
de novo identification of these contaminants was accomplished by determining their elemental
composition based on mass measurements and their chemical structures from unique dissociation
patterns. The compounds characterized in this study were found to be thiotetronic acids which
are structurally related to synthetic and natural antibacterial agents such as the natural antibiotics
thiolactomycin and thiotetramycin, allowing for speculation as to their potential beneficial
properties.
Acknowledgements
First and foremost I would like to express my sincere gratitude to my supervisor Dr.
Wojciech Gabryelski for his tremendous help in my research. Without Dr. Gabryelski’s input,
much of this work would not be possible. Thank you for your words of encouragement, your
patience and understanding. I would also like to thank my fellow colleague Dr. Daniel Beach for
taking the time and showing me the ropes as well as for his support and advice. Finally I would
like to thank Dr. Richard Manderville and Dr. Mario Monteiro, who served on my advisory
committee, for their guidance and generous time.
iii
Table of Contents
ABSTRACT………………………………………………………………………………………ii
Acknowledgements ........................................................................................................................ iii
Table of Contents…………………………………………………………………………………iv
List of Tables ................................................................................................................................ vii
List of Figures ............................................................................................................................... vii
List of Schemes .............................................................................................................................. xi
Appendix Content .......................................................................................................................... xi
List of Abbreviations .................................................................................................................... xii
1.0
INTRODUCTION ............................................................................................................... 1
1.1
Drinking water resources ................................................................................................. 1
1.2
Groundwater quality......................................................................................................... 1
1.3
Groundwater contamination sources ................................................................................ 3
1.4
Current methods in drinking water analysis ..................................................................... 3
1.5
Novel analytical methods in drinking water analysis....................................................... 6
1.5.1
Electrospray Ionization ............................................................................................. 6
1.5.1.1 Nano – Electrospray.................................................................................................. 8
1.5.1.2 Electrochemical processes in ESI ............................................................................. 9
1.5.2
Differential Ion Mobility Spectrometry (DIMS) .................................................... 11
1.5.3
Time of flight mass spectrometry principles of operation ...................................... 14
1.6
2.0
ESI-FAIMS-MS method in drinking water analysis ...................................................... 18
DEVELOPMENT OF THE ESI-FAIMS-Q-TOF METHOD FOR THE
GROUNDWATER ANALYSIS .................................................................................................. 19
2.1
Groundwater analysis by ESI-FAIMS-MS .................................................................... 20
2.1.1
Sampling ................................................................................................................. 21
2.1.2
Sample preparation ................................................................................................. 21
iv
2.1.3
Nanospray Ionization .............................................................................................. 22
2.1.4
Differential ion mobility separation (FAIMS) ........................................................ 23
2.1.5
Mass spectrometry analysis .................................................................................... 24
2.2
Optimization of mass spectrometry detection in the analysis of groundwater by ESI-
FAIMS-MS ............................................................................................................................... 28
2.2.1
Optimization of the sampling interface of the QTOF mass spectrometer .............. 29
2.2.2
Optimization of ion transmission parameters of the QTOF mass spectrometer ..... 34
2.3
The ESI-FAIMS-MS analysis of water samples in positive mode ................................ 37
2.3.1
Analysis of groundwater samples from different locations .................................... 40
2.4
The ESI-FAIMS-MS analysis of groundwater samples in negative mode .................... 42
2.5
Analysis of groundwater samples by other laboratories. ............................................... 44
2.6
The ESI-FAIMS-MS/MS analysis of groundwater samples in positive mode .............. 44
3.0
CALIBRATION OF Q-TOF MASS ANALYZER ........................................................... 47
3.1
Significance of accurate mass measurements ................................................................ 47
3.2
Calibration methods in mass spectrometry .................................................................... 47
3.3
Standards for calibration ................................................................................................ 48
3.4
A new calibration method using formaldehyde ............................................................. 49
3.5
Characterization of formaldehyde calibration solution in the positive ionization
mode………………… .............................................................................................................. 50
3.5.1
3.6
Formation of methylene glycol polymers ............................................................... 51
Characterization of formaldehyde calibration solution in the negative ionization mode
………………………………………………………………………………………….54
3.6.1
Formation of formaldehyde polymers in the negative ionization mode ................. 56
3.7
Tuning and Calibration of the Q-TOF mass analyzer .................................................... 58
3.8
Optimization operational parameters of Q-TOF mass analyzer (tuning) ....................... 60
3.9
Stability of the Q-TOF mass analyzer and corrections for instrument drift................... 63
v
3.10
4.0
Summary ........................................................................................................................ 69
IDENTIFICATION OF THE DETECTED SPECIES ...................................................... 69
4.1
Spectral data in the positive ion mode ........................................................................... 77
4.2
Spectral data in the negative ion mode........................................................................... 84
5.0
CONCLUSIONS AND FUTURE WORK ........................................................................ 85
APPENDIX ................................................................................................................................... 88
REFERENCES ............................................................................................................................. 96
vi
List of Tables
Table 2.1: Measured masses of the molecular ion at m/z 203 and its dissociation in MS and
MS/MS……………………………………………………………………..…………………….46
Table 3.1: Elemental compositions and calculated accurate masses of ammoniated ions of
polymethylene glycol with one terminal methyl group………….……..………………………..51
Table 3.2: Elemental compositions and calculated accurate masses of ions of polymethylene
glycol detected in the negative ion mode…………………………….………………………….55
Table 3.3: Short and long term drift in the accuracy of mass measurements of formaldehyde
solution in positive ionization mode………………………….………………………………….64
Table 3.4: Elemental composition, calculated masses and relative errors (ppm) in mass
measurements for a mixture of nitrosamines…………………………...………………………..66
Table 3.5: Elemental composition, calculated masses and ppm errors after data correction from
the analysis of a mixture of carboxylic acids………………...…….…………………………….68
Table 4.1: Molecular formulas and measured masses of groundwater contaminants investigated
in the positive ion mode………………………………………………………………………….70
Table 4.2: Molecular formulas and measured masses of deprotonated thiotetronic acid and its
dissociation products in the negative ion mode………………...………………………………..74
List of Figures
Figure 1.1: Breakdown of the world’s freshwater resources…………………………………….1
Figure 1.2: Schematic diagram of ESI process…………………………………………………..7
Figure 1.3: Relative ions mobility at high and low electric field conditions as a function of the
electric field strength………………………………………………………………………….….11
vii
Figure 1.4: Ion motion between two parallel plates during application of an asymmetric
waveform in FAIMS……………………………………………………………….…………….12
Figure 1.5: Schematic diagram of the ESI-FAIMS-MS instrument that was employed in my
research…………………………………………………………………………………………..13
Figure 1.6: Schematic diagram of a TOF mass analyzer………………………………………..15
Figure 1.7: Schematic diagram of reflectron device…………………………………………….17
Figure 2.1: The analysis of a groundwater sample by ESI-FAIMS-QTOF-MS in the positive ion
mode. (A) The total ion compensation voltage (TICV) spectrum; (B) the mass spectrum
acquired at the compensation voltage (CV) of -10.0 V; (C) the mass spectrum acquired at the CV
of 3.5 V; (D) the mass spectrum acquired at the CV of 12.5 V; (E) the mass spectrum acquired at
the CV of 12.5 V…………………………………………………………………………...…….25
Figure 2.2: A schematic diagram of the QTOF mass spectrometer used in my research. The
sampling interface (source) of the instrument is shown in the inset…………………………..…30
Figure 2.3: Tuning page of the QTOF mass spectrometer for instrumental parameters controlling
ion transmission from the sampling interface to the TOF mass analyzer………………………..31
Figure 2.4: Results of the ESI-FAIMS-QTOF-MS analysis of the same groundwater sample at
the same experimental conditions except using different sample cone voltages at the sampling
interface. (A) The sample cone voltage of 50 V; (B) the sample cone voltage 30 V; (C) the
sample cone voltage of 14 V. All spectra were acquired in the positive ionization mode
following the separation of ions in FAIMS at the CV of 12.5 V. The extraction cone voltage of 1
V and the collision energy of 10 V were used in the experiment……………….…….…………33
Figure 2.5: Tuning page of the QTOF mass spectrometer with optimized parameters for the ion
transmission in the quadrupole section of the instrument……………………….……………….35
Figure 2.6: Results of the ESI-FAIMS-QTOF-MS analysis of the same groundwater sample at
the same experimental conditions except using different collision energies conditions. (A) The
collision energy of 15 V; (B) the collision of energy 10 V; (C) the collision energy of 4 V. All
spectra were acquired in the positive ionization mode following the separation of ions in FAIMS
viii
at the CV of 12.5 V. The extraction cone voltage of 1 V and the sample cone voltage of 14 V
were used in the experiment…………………………………………………..…………………36
Figure 2.7: Analysis of groundwater, blank and tap water in positive ion mode by ESI-FAIMSMS with Q-TOF detection. (A) The TICV spectrum of groundwater; (B) the mass spectrum of
groundwater at CV 12.5 V; (C) the TICV spectrum from blank analysis; (D) the mass spectrum
of the blank at CV 12.5 V; (E) the TICV spectrum of tap water; (F) the mass spectrum of tap
water at CV 12.5 V………………………………………………………………………………39
Figure 2.8: Analysis of groundwater samples by ESI-FAIMS-MS with Q-TOF detection. (A)
The TICV spectrum of groundwater from location 1; (B) the mass spectrum (CV 12.5 V) of
groundwater from location 1; (C) the TICV spectrum of groundwater from location 2; (D) the
mass spectrum (CV 12.5 V) of groundwater from location 2; (E) the TICV spectrum of
groundwater from location 3; (F) the mass spectrum (CV 12.5 V) of groundwater from location
3………………………………………………………………………………..…………………41
Figure 2.9: Results of the analysis of a groundwater sample in negative ion mode by ESIFAIMS-MS with Q-TOF detection. (A) the TICV spectrum; (B) the mass spectrum at the CV of
-10.0 V; (C) the mass spectrum at the CV of 13.0 V…………………………………………….43
Figure 2.10: The ESI-FAIMS-MS/MS spectrum of the m/z 203 parent ion that was transmitted
in FAIMS at the CV of 12.5 V in the positive ion mode………..……………………….………45
Figure 3.1: The mass spectrum of the formaldehyde calibration solution in the positive
ionization mode…………………………………………………………………………..………50
Figure 3.2: The mass spectrum of the formaldehyde calibration solution in the negative
ionization mode……………………………………………………..………………..…………..54
Figure 3.3: A schematic diagram of the Q-TOF MS instrument…………..………...…...……..58
Figure 3.4: TOF instrument tune page with optimized settings in positive ion mode…….…….60
Figure 3.5: Influence of different pusher-puller voltages on peak shape in negative ionization
mode (elemental composition of selected peak C5H11O7-; calculated accurate mass 183.0510).
Peak fronting (A); peak tailing (B); optimized symmetrical peak (C)…..……………..………..62
ix
Figure 3.6: Mass spectrum of nitrosamines mixture in positive ionization mode….…….……..66
Figure 3.7: The mass spectrum of a mixture of carboxylic acid in the negative ionization
mode…………………………………………………………………………………….………..68
Figure 4.1: Chemical structures of (A) thiotetronic acids; (B) thiolactomycin;
(C) thiotetramycin…...…………………………………………...………………………………77
Figure 4.2: MS/MS spectrum of m/z 203 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 10 V; (C) collision
energy of 20 V…………………………………………………………….……………………..78
Figure 4.3: MS/MS spectrum of m/z 221 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 10 V; (C) collision
energy of 20 V………………………………………………………………………..………….79
Figure 4.4: MS/MS spectrum of m/z 179 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V……….…………..80
Figure 4.5: MS/MS spectrum of m/z 161 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V; (C) collision energy
of 16 V…………………..……………………………………………………………………….81
Figure 4.6: MS/MS spectrum of m/z 143 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 12 V………………….82
Figure 4.7: MS/MS spectrum of m/z 101 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V………….………..83
Figure 4.8: MS/MS spectrum of m/z 201 ion that was transmitted in FAIMS at CV = 13.0 V in
negative ion mode. (A) Collision energy of 10 V; (B) collision energy of 20 V……..…………84
Figure 4.9: MS/MS spectrum of m/z 157 ion that was transmitted in FAIMS at CV = 13.0 V in
negative ion mode. (A) Collision energy of 10 V; (B) collision energy of 20 V…..……………85
x
List of Schemes
Scheme 3.1: Proposed mechanisms for the formation of polymethylene glycols which were
detected in the positive ion mode (Figure 3.1, marked as “*” and “v”). (A) The initiation step;
(B) the propagation step; (C) Termination of polymerization by methanol and formic acid……53
Scheme 3.2: Proposed reactions for the formation of negative ions of polymethylene glycol
without the terminal methyl group (marked with “*” and “v” in Fig. 3.2) during electrospray
ionization. (A) The initiation step; (B) the propagation step; (C) the termination step involving
formic and acidic acids…...….……………..……………………………………………………57
Scheme 4.1: Dissociation pathways of protonated thiotetronic acid which was detected in
groundwater samples at the CV of 12.5 V…………………………….…………………………73
Scheme 4.2: Fragmentation tree of investigated groundwater contaminantsin the negative ion
mode………………………………………………………………………………...……………76
Appendix Content
Scheme 1: Proposed reactions of m/z 203 and m/z 221 ions…..……..………………………….88
Scheme 2: Proposed reactions of m/z 221 ion. (A) Elimination of water; (B) elimination of
ketene…………………………………………………………………………………………….88
Scheme 3: Proposed reactions of m/z 203 ion. (A) Water pick up; (B) elimination of ketene; (C)
elimination of ethylene diol; (D) elimination of ethylenediol……..………….…………………89
Scheme 4: Proposed reactions of m/z 179 ion. (A) Elimination of two water molecules followed
by ring opening and water pick up; (B) elimination of ketene and water…..……...……………90
Scheme 5: Proposed reactions of m/z 161 ion. (A) Water pick up; (B) elimination of water; (C)
elimination of ketene and water; (D) elimination of ethylene diol…....…………………………91
Scheme 6: Proposed reactions of m/z 143 ion. (A) Water pick up; B) pick up of two water
molecules; (C) elimination of carbon dioxide; (D) elimination of water; (E) tautomerization
followed by ring opening; (F) elimination of hydroxyethyne……...……………………………92
xi
Scheme 7: Proposed reactions of m/z 101 and m/z 83 ions…...…..…..…………………………93
Scheme 8: Proposed reactions of m/z 201 ion at low collision energies. (A) Formation of
ethylene diolate at m/z 59; (B) elimination of carbon dioxide; (C) elimination of carbon sulphide;
(D) elimination of ketene….……………………………………………………………..………93
Scheme 9: Proposed reactions of m/z 201 ion at higher collision energies. (A) Tautomerization
of the negative ion of the thiotetronic acid; (B) elimination of ketene and carbon dioxide; (C)
elimination of two ketene groups; (D) elimination of carbonyl sulphide…....…………………..94
Scheme 10: Proposed reactions of m/z 157 ion in the negative mode. (A) Elimination of carbon
dioxide; (B) elimination of carbon sulphide; (C) formation of ethylene diolate at m/z 59
ion……………………………………………………………………………………………......95
List of Abbreviations
APCI
atmospheric pressure chemical ionization
CE
collision energy
CI
chemical ionization
CID
collision induced dissociation
CV
compensation voltage
DBP
disinfection byproducts
DC
direct current
DIMS
differential ion mobility spectrometry
DV
dispersion voltage
EI
electron ionization
xii
EPA
Environmental Protection Agency
ESI
electrospray ionization
FAIMS
high-field asymmetric waveform ion mobility spectrometry
FT-ICR
fourier-transform ion cyclotron resonance
GC
gas chromatography
GCxGC
two-dimensional gas chromatography
HAAs
haloacetic acids
HPLC
high performance liquid chromatography
IMS
ion mobility spectrometry
LC
liquid chromatography
m/z
mass to charge ratio
MCP
multichannel plate detector
MS
mass spectrometry
MS/MS
tandem mass spectrometry
PT
proton transfer
Q1
the first quadrupole cell in a Q-Tof
Q2
the hexapole collision cell in a Q-Tof
Q3
the third quadrupole cell in a Q-Tof
Q-Tof
quadrupole-time-of flight
RF
radio frequency
T
tautomerization
xiii
TIC
total ion chromatogram
TICV
total ion compensation voltage spectrum
TOF
time of flight
UPLC
ultra performance liquid chromatography
xiv
1.0 INTRODUCTION
1.1 Drinking water resources
Water is one of the essential resources for life on earth. It has been estimated that there is
about 1.4 billion km3 of water on the earth surface. However, in reality only 2.5% (35 km3) of
this volume consists of drinking water. The remaining 97.5% is saltwater stored in the oceans1.
Figure 1.1 presents further breakdown of freshwater resources.
World’s freshwater resources
LAKES, RIVERS, SWAMPS
1%
30%
GROUNDWATER
GLACIERS AND
PERMANENT SNOW
69%
Figure 1.1: Breakdown of the world’s freshwater resources1,2.
As presented in Figure 1.1, 7% of freshwater resources are stored in the form of glaciers
and permanent snow, only 1% of drinking water comes from lakes, rivers and swamp. Thus,
groundwater aquifers provide the biggest source of freshwater.
1.2 Groundwater quality
Groundwater is found beneath the Earth’s surface in soil and rock pores. Its composition
may vary significantly in different regions. Groundwater is usually free from bacterial pollution
however, it contains dissolved minerals and organic matter at various concentrations. Those
1
chemical components of groundwater may be harmless or even beneficial to consumers but
certain compounds can be harmful or toxic. Most minerals in groundwater derive from rocks as
they dissolve when the flowing water comes in contact with the rock. These minerals contain:
sodium, calcium, magnesium, potassium, chloride, bicarbonate, and sulfate. The dissolved
minerals are usually responsible for the tangy taste of groundwater. When the quantity of the
minerals exceeds 1,000 mg/L, water typically is not considered desirable for drinking3.
Moreover, groundwater contains humic and fulvic acids which appear to be released from
organic carbon found in the soil/subsurface matrix4,5. Furthermore, monomers and polymers of
neutral sugars and amino acids have also been identified in groundwater6,7.
In recent studies, Longnecker et al. employed Electrospray Ionization coupled with FourierTransform Ion Cyclotron Resonance Mass Spectrometry (ESI FT-ICR MS) to examine a
complex mixture of natural organic compounds in groundwater8. Their research, implementing a
very sophisticated mass spectrometry detection system, has revealed that a large proportion of
dissolved organic matter in groundwater represent compounds containing nitrogen and sulphur.
Based on measurements of mass to charge (m/z) values of detected groundwater contaminants,
they established elemental compositions of 292 chemical compounds which are unique
components of groundwater. Moreover, their studies indicate that groundwater contaminants
may represent a very complex mixture of organic compounds including a large group of
structural isomers. Longnecker et al. could not establish or propose chemical structures to the
detected groundwater constituents due to the utilized in the study analytical method. Moreover,
current known techniques in water research are not capable of providing such information. It is
important to mention that the structural elucidation of such unique chemical components of
groundwater is the most challenging task for any analytical method used in water analysis.
2
1.3 Groundwater contamination sources
Groundwater is commonly recognized as a relatively safe source of drinking water as it gets
filtered by soil and rocks which make it less prone to bacterial and chemical contamination3.
However, groundwater quality has been significantly influenced in recent years by the urban
development and increased industrial waste. There are two main categories of groundwater
contamination sources: point sources and distributed (non-point) sources. The first category
includes: municipal landfills, industrial waste disposal sites, leaks or spills of industrial
chemicals at manufacturing facilities, leaking tanks or pipelines containing petroleum products,
livestock wastes, on-site septic systems, leaking sewer lines, sludge disposal areas at petroleum
refineries, wells for disposal of liquid wastes, runoff of salt and other chemicals from roads and
highways, spills related to highway or railway accidents. The second category includes
contamination from fertilizers and pesticides on agricultural lands and forests, contaminants in
rain, snow, and dry atmospheric fallouts9.
1.4 Current methods in drinking water analysis
Drinking water is a very complex mixture of thousands of different compounds which are
present at a relatively low concentration10. A list of new water contaminants is released every
year by the Environmental Protection Agency (EPA), however, a large fraction of organic
compounds in water in not known or poorly characterized. For that reason there is a great need
for analytical methods which will be able to detect not only currently known water contaminants
but also discover new pollutants that are present in water but remain unknown at this time.
Gas chromatography (GC) and Liquid chromatography (LC) coupled to Mass
Spectrometry (MS) still continue to be the most popular techniques in water analysis11. Gas
3
Chromatography-Mass Spectrometry (GC-MS) is suitable for monitoring low polarity
contaminants which are volatile and thermally stable. More polar compounds, often non-volatile
or thermally labile are not amenable to GC-MS analysis without chemical derivatization.
Universal ionization methods used in GC-MS: electron impact (EI) and chemical ionization (CI),
allow for ionization and subsequent detection of thousands of less polar organic compounds in
water12. Over the last few years, there is an expanding trend of using two-dimensional GC
(GCxGC) which can address enormous complexity of water samples11. Two GC capillary
columns with different stationary phases are connected in this technique through a special
interface (modulator). The role of the modulator is not only to connect two GC columns but also
to preserve the separation achieved in the first separation dimension, which provides superior
separation power relative to the traditional one-dimensional GC. Time-of Flight (TOF) mass
analyzers are usually used in this technique as detectors, due to their rapid data acquisition
capabilities13.
Liquid Chromatography-Mass Spectrometry (LC-MS) is also a very popular analytical
technique in water analysis. LC-MS is suitable for separation and detection of non-volatile and
thermally labile compounds which cannot be analyzed by GC-MS. For that reason many
pesticides, pharmaceuticals and other highly polar compounds have been analyzed using this
method14. Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI)
are most widely employed ionization techniques to couple LC with a variety of mass
spectrometers12. Over the last few years, Ultra Performance Liquid Chromatography (UPLC) has
been successfully used in water analysis11. In contrast to the traditional High Performance Liquid
Chromatography (HPLC), UPLC separation columns are shorter and packed with much smaller
4
particles of stationary phase. The advantage of UPLC is the reduced time of analysis with
superior resolution and sensitivity15.
The major missing gap in our knowledge about water composition is related to highly
polar contaminants that pose analytical challenges for their characterization using conventional
methods in water analysis. Hyphenated analytical methods such as LC-MS and GC-MS can
deliver accurate results, however, there are certain limitations with these techniques because they
face enormous challenges in the analysis of water samples containing a complex mixture of
many thousands of organic contaminants occurring mainly at very low concentrations. HPLCMS methods have successfully been employed for monitoring and studying water pollution with
respect to a wide range of polar organic contaminants. Each traditional LC-MS method,
however, is usually restricted to a few pre-selected, “suspected”, target compounds (usually less
than 30), for which the particular analytical method has been developed, optimized and
validated. This approach (target analysis) is not sufficient to fully assess the quality of water
because many currently unknown contaminants, previously not considered as potential
pollutants, may be present in water and pose a threat to the environment and human health. The
most important limitation of current analytical techniques in water analysis is that they can
mainly be employed for the detection of target analytes which have previously been identified in
water samples.
In this case, the presence or absence of a compound in a sample can be
determined by using a commercially available standard for the target analyte. These methods do
not provide sufficient information about non-target components of the sample. There is growing
interest in the detection and determination of chemical structures of such non-target abundant
contaminants, but their identification in water involves usually new analytical techniques and
methods which are developed for this purpose.
5
1.5 Novel analytical methods in drinking water analysis
Electrospray Ionization-High Field Asymmetric Waveform Ion Mobility SpectrometryMass Spectrometry (ESI-FAIMS-MS) is an analytical method with great potential in the area of
drinking water analysis. This emerging technique, developed in Canada, can detect target water
contaminants in a convenient fashion with no need for sample preparation, pre-concentration, or
liquid separation in a matter of a few minutes. ESI-FAIMS-MS also has unique capabilities of
detecting thousands of highly polar contaminants in drinking water and providing sufficient
spectral information for the structural identification of non-target pollutants present in water even
at a sub-parts-per-billion concentration level10. This difficult task cannot be completed by any
other analytical method.
Such unique analytical capabilities of ESI-FAIMS-MS can be
accomplished by combining three critical techniques: Electrospray Ionization (ESI) for ionizing
components of a water sample, High Field Asymmetric Waveform Ion Mobility Spectrometry
(FAIMS) for separating electrospray-generated ions, and Mass Spectrometry (MS) for detecting
ions after their separation.
1.5.1
Electrospray Ionization
The discovery of electrospray ionization (ESI) had a great impact on the mass
spectrometry field. In the 1960s Malcolm Dole and his co workers reported that gas-phase ions
can be generated by passing solution through a conductive capillary, which tip was electrically
charged16,17. Based on that discovery John B. Fenn from Virginia Commonwealth University and
his co workers developed an improved ESI method which could be interfaced to a mass
spectrometer18. Initially, their work was dedicated to the protein analysis, and then further
extended to polymers, biopolymers and eventually to the analysis of small polar molecules19,20.
6
In 2002 John B. Fenn was awarded the Nobel Prize in Chemistry for the development and
application of electrospray ionization21.
Figure 1.2: Schematic diagram of ESI process.
Figure 1.2 presents a schematic diagram of an ESI source. The ESI process takes place at
room temperature and atmospheric pressure. A sample, dissolved in a polar solvent, flows
through a fused silica capillary which is located inside of a slightly larger metal (conductive)
capillary. The tip of the capillary is maintained at a high potential (~4000 V) relative to the
grounded counter electrode. As a result an electric field is created between the tip of the capillary
and the counter electrode19,20. When the sample emerges from the silica capillary, applied field
penetrates the solution, causing a partial separation of positively and negatively charged species.
When positive potential is applied to the capillary an enrichment of positive ions will take place
at the capillary tip and a drop of charged analytes will be formed22. Initially formed drop will
have a spherical shape. However, when charge accumulation will increase on its surface the drop
will elongate forming a ‘Taylor cone’ (Fig. 1.2). At the point when the charge density is high
7
enough to overcome the surface tension which holds the droplet together, a coulombic explosion
will occur and a spray of highly charged offspring droplets will be formed19,20.
Currently there are two mechanisms which have been proposed for the formation of
charge molecules from the offspring droplets in ESI. The first theory, known as a coulomb
fission states that due to solvent evaporation a coulombic explosion process is repeated many
times until desolvated gas-phase ions of analytes are formed. The second mechanism (ion
evaporation mechanism) assumes that with solvent evaporation and increasing charge density,
ions are released from the charged droplet23. Electrospray-generated ions are then attracted to the
entrance of the mass spectrometer. The ESI process takes place at room temperature and
atmospheric pressure. Thus, before ions can be analyzed they have to enter to the vacuum region
of the mass spectrometer. This transition can be accomplished by high efficiency pumping
systems of mass spectrometers. Furthermore, coaxially injected gas (usually nitrogen) or heated
capillary aid the formation of gas-phase ions by the evaporation of solvent molecules19,20.
ESI is an excellent ionization technique for highly polar and non volatile compounds.
Furthermore, ESI is a soft ionization technique where very fragile ions can be analyzed
intact19,21. Thanks to this method, ionization of neutral compounds and the transfer of ions
directly from the solution to the gas phase can be accomplished. ESI is commonly used in LCMS methods in which the ESI source is located at the entrance of a mass spectrometer to produce
gas phase ions from compounds separated in LC20.
1.5.1.1 Nano – Electrospray
Nano-electrospray or nanospray ionization is practically electrospray ionization at sub
µL/min flow rates. It was developed by Wiliam and Mann in late 90s as a solution to limited
8
quantities of sample available for analaysis17. However, smaller consumption of analyte is not
the only advantage of nanospray when compared to the traditional electrospray. This nanoflow
technique also involves smaller quantities of solvent used in sample preparation. With higher
flow rate, electrospray droplets have a larger diameter. Thus, the distance between the ESI
capillary and counter electrode has to be large enough for sufficient solvent evaporation and
generation of gas phase ions. Nanospray droplets are much smaller and more charge is available
for the analyte for ionization. Thus, the distance between the capillary and counter electrode can
be reduced, resulting in better sensitivity and more efficient formation of gas phase ions24. In
addition to those advantages, nanospray is also a soft ionization technique which preserves the
original structure of ions allowing them to be analyzed intact25. For these reasons, ESI in a
nanoflow regime will be employed in my research.
1.5.1.2 Electrochemical processes in ESI
It is very important to note that analytes in ESI can be greatly influenced by
electrochemical processes occurring at the tip of the charged capillary20. We can refer to the ESI
source as a special type electrochemical cell. A special character of this cell comes from the fact
that the transport of ions between electrodes does not occur in solution, like in a traditional
electrolysis, but in the gas phase17. During the analysis, when the capillary is held at positive
potential the charged capillary is an anode and the counter electrode is a cathode. When the
positive ions are attracted towards the mass analyzer, the buildup of negatively charged species
in the capillary will take place. Therefore, in order to counter balance the excess of negatively
charged ions, the oxidation reactions will occur in the electrospray capillary (anode). The type of
reaction that will dominate depends on the oxidation potential of analytes, solvent and material
of the metal capillary. Oxidation of different species takes place starting from the species with
9
the highest oxidation potential until the charge balance will be established17,26. When the
capillary is held at negative potential, it becomes a cathode and the counter electrode is an anode.
Reduction reactions have to take place in the electrospray capillary (cathode) when the negative
ions are attracted towards the mass analyzer to counter balance an excess of positively charged
species26. In addition to oxidation and reduction reactions at the capillary tip, the reduction and
oxidation reactions have to take place at the counter electrode. However, these processes are of
less significance as they do not have an influence on the ions observed in mass spectra.
The total number of ions generated in ESI is dependent on electric current which is
produced by red-ox reaction occurring at the capillary tip20. Based on that phenomenon several
researchers have been conducting studies to investigate the effect of red-ox reactions on analytes
in sprayed samples. Van Berkel et al. performed series of experiments to study pH changes in the
solution as a result of electrochemical processes occurring at the capillary tip27.
Their
investigation revealed that the composition of electrospray solution can change significantly
during the electrospray process. In the positive ion mode they observed a decrease in the solution
pH due to oxidation of water which was used as a solvent. Furthermore, the decrease in pH was
greater when the flow rate was reduced27. This fact is not surprising as when the flow rate is
reduced analytes have more contact with the surface of the conductive capillary and there is
more time for red-ox reactions to take place. Thus, the effect of the electrochemical processes
occurring at the capillary tip in more pronounced in nanospray than in electrospray with higher
flow rates28. Analogically, in the negative ion mode, the increase pH was observed due to
formation of hydroxide ions, generated during water reduction at the capillary tip27. Another
study conducted by Van Berkel et al. revealed that when the capillary tip is made of Fe, Fe2+ ions
are generated as a result of oxidation of the capillary tip. Similar phenomenon can be observed
10
when the electrospray emitter is made of other metals such as Zn, Ag, Cu for example29. The
described above research findings prove the importance of red-ox reactions occurring at the
capillary tip therefore they cannot be ignored when performing ESI experiments.
1.5.2
Differential Ion Mobility Spectrometry (DIMS)
In traditional drift time ion mobility spectrometry ions travel through the buffer gas in the
presence of a low electric field. The mobility of an ion depends on its size, mass and charge and
it is characteristic for this particular ion30. At the low electric fields (< 10000 V/cm), the ion
mobility (K) is independent on the applied electric field. At higher electric fields, the ion
mobility (Kh) is no longer constant and depends on the strength of the electric field. The
phenomenon is illustrated in Figure 1.3. Three different ions (A, B, and C) can experience
different high electric field mobilities (Kh) relative to their mobilities (K) at low electric fields.
These unique properties of ions are used in differential ion mobility techniques to separate ions
based on their relative mobilities at high and low electric field conditions31. The same principle
has been used in developing High-Field Asymmetric Waveform Ion Mobility Spectrometry
(FAIMS) which is implemented in my research as a primary separation method for ions of water
contaminants generated in ESI.
Figure 1.3: Relative ions mobility at high and low electric field conditions as a function of the
electric field strength (adapted from32).
11
Figure 1.4 illustrates operational principles of High-Field Asymmetric Waveform Ion
Mobility Spectrometry (FAIMS) which is an ion separation technique based on the difference in
ion mobility at high electric field relative to low electric field10. Ions are introduced between two
parallel electrodes and driven by a stream of buffer gas (e.g., nitrogen). One electrode is kept at
the ground potential (bottom electrode) while an asymmetric waveform V(t) is applied to the
second electrode (top electrode). During one cycle of the asymmetric waveform ions are exposed
alternately to strong and weak electric fields. Since the ion mobility is different during the
application of high electric field and low electric field portion of the waveform, the ion will drift
towards one of the electrodes. A few asymmetric waveform cycles eventually will cause
collision of the ion with the electrode. To overcome this drift, a small DC voltage called
compensation voltage (CV) can be applied to one of the electrodes. With the appropriate CV, the
ion will remain between the plates and will find its way to the mass analyzer, while other ions
will hit one of the electrodes and will be lost31.
Figure 1.4: Ion motion between two parallel plates during application of an asymmetric
waveform in FAIMS (adapted from32).
12
Figure 1.5 shows a FAIMS device with two parallel plates. This simple configuration has
been used in the past but a number of new FAIMS analyzers have been developed in the last
decade. New FAIMS devices operate on the same principle but they are designed to improve ion
transmission and ion separation efficiency. Figure 1.5 shows a schematic diagram of a FAIMS
device with cylindrical geometry that was employed in water analysis by ESI-FAIMS-MS.
In
developed ESI-FAIMS-MS method (Fig. 1.5), a water sample is infused to the ESI source and
electrospray-generated gas-phase ions enter the annular space of the FAIMS analyzer. FAIMS
operates at atmospheric pressure and at room temperature and acts as an ion filter which, from a
mixture of ions at the inlet of the FAIMS analyzer, transmits only selected ions into the
Quadrupole-Time-of-Flight- Mass Spectrometer (QTOF-MS) in a continuous fashion.
Figure 1.5: Schematic diagram of the ESI-FAIMS-MS instrument that was employed in my
research (adapted from10).
Experimentally, the difference in high and low electric field mobility of ions oscillating
between two concentric electrodes is reflected in the value of a compensation voltage (CV) at
13
which only one type of ions is selectively transmitted through the device. The CV can be
scanned, sequentially passing a bandwidth of ions to the fast QTOF mass detector. QTOF-MS
permits the acquisition of full scan (MS) and product ion spectra (MS/MS), with the accurate
mass of the parent and its daughter ions which are generated through collision induced
dissociation (CID) in the collision cell.
This information will be used for the structural
identification of non-target contaminants. The key advantage of ESI-FAIMS-QTOF-MS, over
LC-MS, is the continuous mode of gas-phase ion mobility separation, which not only eliminates
interferences from the ionization of liquid sample matrix in ESI but also improves the quality of
spectral data by supplying investigated ions for the MS and MS/MS detection for unlimited
amount of time when FAIMS is operated in non-scanning mode.
The FAIMS separation technique is orthogonal to both liquid chromatography and mass
spectrometry, which means that FAIMS can separate species that would not be separated by LC
or distinguished by MS. This technique is especially effective in separating isomers ions, which
is extremely helpful in the determination of their structures. A major advantage of FAIMS in
water analysis is its ability to reduce chemical background, which allows for identification of
low abundance compounds that cannot be detected by other methods10.
1.5.3
Time of flight mass spectrometry principles of operation
Ion separation and mass measurements in a time of flight mass spectrometry (TOF-MS)
are based on very simple principles. A schematic diagram of a TOF mass analyzer is shown in
Figure 1.6. Investigated ions are accelerated by electric field voltage (V) and then travel down
the field free drift region (l) before reaching the detector20.
14
Figure 1.6: Schematic diagram of a TOF mass analyzer.
In the field free region, ions are separated and their m/z are measured based on the flying
time from the ion source to the detector (small ions travel faster than heavier). To determine
relationship between m/z and flying time (t) we have to introduce few simple equations20.
The kinetic energy of accelerated ions can be represented by equation (1):
KE = 1/2mv2 = zeV
(1)
Where:
KE – ion kinetic energy
m – mass of an ion
v – ion velocity in the field free region
z – charge state of an ion
V – accelerating potential
e – electron charge
From this equation we can conclude that all ions at the same state (z) after acceleration, will
acquire the same kinetic energy.
The flying time through the drift free region is given by equation 2 and depends on the length of
the field free drift region (flying tube) and ions velocity:
15
t = l/v
(2)
Where:
t – time of flight
l – travel distance/ length of the flying tube
v – ion velocity
When we will combine equation 1 and 2 and rearrange them we can determine factors
influencing time of flight (equation 3):
t2 = m/z (l2*2eV)
(3)
Since the acceleration voltage (V) and length of the tube (l) are constant, we can conclude from
equation 3 that the time of flight is proportional to square root of m/z20.
Despite these simple principles, TOF mass analyzers exhibit a limited resolution and
significant errors in mass measurements. The first factor contributing to limited resolution is the
length of the flight tube (usually 1-2m) which is not sufficient to resolve ions with similar m/z20.
Secondly, not all the ions with the same masses are arriving at the detector at the same time,
mainly due to their spatial and energy distribution. During the acceleration event these
distribution problems arise from different location and velocity component with respect to the
direction of their acceleration. Ions initially located further from the detector will spend more
time in the acceleration region; they will be exposed to the higher acceleration potential and will
acquire higher kinetic energy and drift velocity. As a result their flying times will be shorter. To
resolve this issue current TOF mass analyzers have been equipped with a reflectron33. A
schematic diagram of a reflectron is shown in Figure 1.7. The device consists of few lenses to
which a high voltage is applied. In the most common configuration, the difference in voltage
16
between each consecutive lens is the same, which creates a linear potential gradient across the
mirror assembly. Ions with the same m/z but a higher velocity will not penetrate to the reflectron
region to the same extend. Ions with more kinetic energy (ion 1 and 2 in Fig. 1.7) will spend
more time in the reflectron region than ions with less kinetic energy (ion 3 in Fig. 1.7). As a
result all three ions will arrive at the detector at the same time.
Figure 1.7: Schematic diagram of reflectron device.
Furthermore, a reflectron not only improves resolution by compensating for the energy
distribution but also by extending the flight path. The time of flight is directly proportional to
the length of the flight tube (equation 3). Thus, longer tube will provide longer flight times and
better resolution. The TOF instrument, employed in my study, operates on the same principles
described above and is also equipped with a reflectron device for improved performance.
17
1.6 ESI-FAIMS-MS method in drinking water analysis
Electrospray Ionization-High Field Asymmetric Waveform Ion Mobility SpectrometryMass Spectrometry (ESI-FAIMS-MS) is a new analytical method which can address some
important needs in water analysis. In the past, FAIMS was successfully applied in the analysis of
target and nontarget water contaminants.
Liu and al. developed a method for the analysis of nitrosamines in drinking water by
using FAIMS as an attractive alternative to chromatographic separation. Using available
analytical standards, the authors investigated separation of four nitrosamines by FAIMS in order
to use this technique in the analysis of drinking water samples spiked with previously
investigated nitrosamines34. Ells at al. reported detection of 9 disinfection byproducts (DBPs)
present in drinking water at a part-per-trillion concentration level using ESI-FAIMS-MS method.
Investigated DBPs represent various chlorinated and brominated haloacetic acids. Haloacetic
acids (HAAs) are the major byproducts of water chlorination and have been recognized as
potential carcinogens. Elimination of interferences form the sample matrix by FAIMS
significantly improves detection limits of these compounds in water35,36. Another study,
employed FAIMS technique in separation of water samples. It is difficult to determine tracelevel quantities of perchlorate in water using conventional ESI-MS because of the presence of
other more abundant interfering ions such as bisulfate and dihydrogen phosphate. The ESIFAIMS-MS method could separate perchlorate from the interfering ions and provide its rapid,
selective and sensitive detection37.
In addition to these examples of target analysis of water, FAIMS separation was also
applied to the nontarget analysis of highly polar contaminants in the drinking water. In this
18
approach, contaminants in chlorine-treated drinking water were indentified based solely on
spectral data from ESI-FAIMS-MS. Accurate mass measurements and dissociation reactions of
investigated species in tandem mass spectrometry allowed for the identification of the most
abundant water contaminants such as glycolic acid which was never considered to be present in
drinking water before the application of FAIMS. In addition, 25 carboxylic acids present at subparts-per-billion concentration levels have been also identified in the study10. The application of
ESI-FAIMS-MS is not limited to analysis of water samples. Other environmental and biological
samples have been analyzed by this technique with great success38.
In this thesis, I will present my work devoted to the development of the ESI-FAIMS-MS
technique for nontarget analysis of groundwater samples. Furthermore, I will apply this method
to the detection and chemical (structural) identification of abundant, but previously unknown,
sulfur-containing groundwater contaminants, which have been present beyond the scope of
conventional analytical methods in water analysis for the last 30 years. The work described in
this thesis has led to the development of a new analytical approach in water analysis. The key
feature of this approach is “soft” mass spectrometry which is capable of detecting and
investigating new classes of contaminants that normally would have not been detected by
“classical” mass spectrometry techniques in water analysis.
2.0 DEVELOPMENT OF THE ESI-FAIMS-Q-TOF METHOD FOR THE
GROUNDWATER ANALYSIS
Groundwater is commonly recognized as a relatively pure and safe source of drinking
water, however, it is speculated that the growth of population, industry and new mining
technologies in recent years significantly influenced groundwater quality. Although hidden
19
beneath the earth surface, groundwater is still vulnerable to environmental pollution. In the past,
the analysis of groundwater has been focused on selected trace contaminants which are usually
detected at very low concentrations. The major problem with the target analysis is its limited
scope which may not take into account currently unknown and potentially abundant groundwater
contaminants. With recent advancements in analytical methods in water research, it is possible
to investigate chemical composition of drinking water with respect to many thousand compounds
that can be detected in a quick and convenient fashion. More importantly, new analytical
techniques are not only limited to the target analysis, they also provide unique capabilities to
discover novel, previously not reported groundwater contaminants which could affect water
quality.
2.1 Groundwater analysis by ESI-FAIMS-MS
Mass Spectrometry combined with Liquid Chromatography is the most commonly used
technique in water analysis. New mass spectrometry methods, supported by new analytical
separation techniques provide unique capabilities for investigating extremely complex mixture of
chemical components in drinking water. In this thesis, I will present comprehensive studies on
the development of a new analytical strategy in water analysis based on combining the mass
spectrometry detection (QTOF-MS) with the differential ion mobility separation (FAIMS) for
groundwater samples subjected to nano-electrospray ionization. The ESI-FAIMS-QTOF-MS
technique has never been applied to groundwater analysis. Consequently, new and challenging
aspects of the analysis has to be addressed at each stage of the analytical process including
sampling, sample preparation, nanospray ionization, ion mobility separation and particularly
mass spectrometry detection. In this chapter, I will describe the optimization of analytical steps
of the ESI-FAIMS-QTOF-MS method, which lead to the discovery of new contaminants in
20
groundwater.
2.1.1
Sampling
Sampling and sample storage are initial steps in the analytical process and their
importance should not be ignored. Careless sampling and sample handling can greatly influence
further stages of the analysis. In my study, groundwater samples were collected from different
wells in rural areas around Guelph, Ontario, Canada into specially prewashed glass jars with
“Teflon” lids (VRW, Radnar, PA, USA). Teflon lids have been chosen over soft plastic lids
because compounds (plasticizers) from a plastic material can leak into a water sample and be a
source of high background noise in the analysis. The samples were analyzed immediately after
arrival to our laboratory or stored in the refrigerator at 5°C to avoid decomposition of analytes.
2.1.2
Sample preparation
Sample preparation is a very important and usually time-consuming step in the analytical
process. Without proper sample preparation even the most sophisticated techniques will not
deliver satisfactory results. Commonly used separation method in water analysis, such as LC
and GC, require extensive sample preparation. The ESI-FAIMS-MS technique can analyze water
samples without fractionation, extraction or pre-concentration. For example, simple dilution of a
tap water sample with methanol, is the only sample preparation step required for the FAIMS
analysis. Groundwater, unlike tap water, may have higher content of different inorganic salts
which perturb the operation of electrospray ionization. Therefore, the series of experiments were
performed to determine the optimal dilution factor for groundwater samples. It was established
that 10 times dilution of a sample with the methanol buffer containing 0.1mM ammonium
acetate provides the optimal signal intensity for analytes and stable ionization conditions.
21
Methanol and ammonium acetate are added in order to aid the ionization process. Methanol
lowers surface tension of water samples whereas small quantities of ammonium increase the
electrospray current17,39.
2.1.3
Nanospray Ionization
Prepared/diluted water samples were introduced to a nanospray ionization source
(Waters, UK) using the injection manifold of a capillary Ultra Performance Liquid
Chromatography system (Waters, UK). The capillary format of UPLC is specifically designed to
provide stable low flow rates of pumped liquids. Each diluted water samples were injected into
the 50 μL injection loop. The capillary UPLC pumping system was pushing the samples solution
from the loop to the nanospray at the optimal flow rate of 400 nL /min., providing continuous
delivery of each injected sample for over 2 hours. Operating electrospray at reduced flow rates
(nanospray) offers many advantages which are described in more details in section 1.5.1.1.
For the purpose of the ESI-FAIMS-MS analysis, the position of the nanospray probe
(relative to the sampling plate of the FAIMS device) was optimized during the series of
experiments. When the probe was located perpendicular to the sampling plate, large droplets and
nonvolatile salt particles from electrospray could enter the FAIMS analyzer. For that reasons,
the electrospray probe was configured at a 20 degrees angle with respect to the FAIMS entrance.
This off-axis geometry eliminates potential contamination problems because only desolvated
ions can enter the annular space of the FAIMS analyzer. The nanospray source was operated in
the positive and negative ionization mode. The optimal electrical potential at the electrospray
capillary, for obtaining a steady and uniform spray, ranges between 3 to 4 kV (positive mode)
and -2 to -3 kV (negative mode) depending on the surface tension of ionized samples39. Certain
22
samples with higher surface tension are more difficult to be ‘stretched’ into a Taylor cone;
therefore, higher electric potentials need to be applied. However, when the applied potential is
too high, an electric discharge can occur and lead to poor ionization efficiency of analytes
17
.
Consequently, the spray voltage was adjusted for each groundwater sample to ensure steady and
uniform electrospray.
2.1.4
Differential ion mobility separation (FAIMS)
The FAIMS technique was employed in my research as a separation tool for ions
originating from chemical components of groundwater samples. The principles of ion separation
in FAIMS are described in section 1.5.2.
An Ionalytics Selectra FAIMS analyzer with cylindrical geometry was operated at the
maximal dispersion voltage of 4000 V. The separation of ions was carried in a carrier buffer gas
(20% of CO2 in N2) at its total flow rate of 1.3 L/min. These conditions were found to be
optimal for providing efficient and reproducible differential ion mobility separation results and
maintaining high spectral intensities of separated and detected ions. The addition of a relatively
large proportion of CO2 to the carrier gas has a very important impact on the separation process
and overall analysis. A differential ion mobility of ions separated in FAIMS can be greatly
influenced by the presence of traces of water vapor, CO2 and other volatile compounds in the
carrier gas.
This phenomenon results from interactions between analyte ions and volatile
molecules during the separation process. Formation of an ion cluster with volatile molecules can
significantly change its differential ion mobility relative to the differential ion mobility of the
naked ion. An excess of CO2 in the carrier gas can “out-compete” the potential formation of ion
clusters with other volatile molecules which are present in the buffer gas at trace level. As a
23
result the reproducibility of the FAIMS separation is improved38. In addition, CO2 cluster ions of
different analytes are often transmitted through the FAIMS device at a much wider range of
compensation voltage than their corresponding naked ions, which improves the separation
capacity and the separation efficiency of the method.
The addition of CO2 to the carrier gas also creates a very unique environment for
electrospray-generated ions which are introduced to the mass spectrometer following the FAIMS
separation. CO2 can “preserve” the integrity of labile molecular ions which normally (with the
absence of CO2) would dissociate through collisions with N2 gas molecules at the source of the
mass spectrometer.
2.1.5
Mass spectrometry analysis
The mass spectral detection in ESI-FAIMS-MS was carried out using a quadrupole time
of flight mass spectrometer (QTOF micro) (Waters, UK). The instrumental parameters of this
mass analyzer were set to the standard values optimized and recommended by the instrument
manufacturer. These setting were suitable for the analysis of water samples and metabolites in
urine in our laboratory in the past.
Figure 2.1 presents spectral data from the ESI-FAIMS-MS analysis of a groundwater
sample in the positive ionization mode. Figure 2.1 A represents the total ion compensation
voltage (TICV) spectrum which was acquired by scanning the compensation voltage (CV) from
0 to 50 V (with a 0.5 V interval) and detecting the total intensity of ions that are transmitted
(separated) in FAIMS at each CV value.
24
Normalized Ion Intensity
100
(A)
B
(NOM)
D
80
60
40
C
20
0
Normalized Ion Intensity
-20
100
0
20
40
CV [V]
60
80
100
(B)
80
60
40
20
0
500
1000
1500
2000
2500
Normalized Ion Intensity
m/z
100
(C)
80
60
40
20
0
100
100
150
200
250
m/z
101
(D)
Normalized Ion Intensity
Normalized Ion Intensity
50
80
60
119
40
127
20
83
300
100
350
(E)
400
450
500
101
34 S
50
102
103
102
103
0
100
101
104
m/z
86
0
50
100
150
m/z
200
250
Figure 2.1: The analysis of a groundwater sample by ESI-FAIMS-QTOF-MS in the positive ion
mode. (A) The total ion compensation voltage (TICV) spectrum; (B) the mass spectrum acquired
at the compensation voltage (CV) of -10.0 V; (C) the mass spectrum acquired at the CV of 3.5 V;
(D) the mass spectrum acquired at the CV of 12.5 V; (E) the mass spectrum acquired at the CV
of 12.5 V.
25
The TICV spectrum can be compared to a total ion chromatogram (TIC) in Liquid
Chromatography-Mass Spectrometry (LC-MS), however, the distributions of detected ions in the
CV spectrum and the total ion chromatogram are related to completely different separation
principles. The TICV spectrum represents the separation of ions based on their high and low
electric field mobility in a gas-phase. The TIC on the other hand represents the distribution of
species with respect to their elution times from a chromatographic column. The chromatographic
separation can be controlled by changing the solvent strength of the mobile phase, whereas the
FAIMS separation relies on scanning the compensation voltage at different rates. The most
significant difference between FAIMS and LC is that the CV scan in FAIMS can be stopped at
any CV for an extended period of time (up to 2 hours) to acquire high quality spectral data for
ions that are separated by at this CV. In LC, the spectral data acquisition has to be accomplished
in a short period of time during the elution of the chromatographic peak of analyte. Figure 2.1 B,
C and D present mass spectra that have been acquired by “parking” the CV at -10.0 V, 3.5 V and
12.5 V, respectively for a longer period of time (1 minute) to detect ions transmitted at these
particular conditions.
The mass spectrum acquired at the CV of -10.0 V (Fig. 2.1 B) shows only a small
fraction of the extremely complex mixture of chemical components of natural organic matter
(NOM) in groundwater, detected as positively charged ions. Dissolved natural organic matter is
known to contain humic and fulvic acids which are released from organic mass found in the
soil/subsurface matrix4,5, but little is known about the huge numbers of components of NOM
detected by ESI-FAIMS-MS in the CV range between -1 V and -15V. Mass spectra of NOM
(e.g., Fig. 2.1 B) show multiple species present at each nominal mass in the low and high mass
range. No current analytical method has the capabilities of identifying these polar components
26
of NOM, which are present in groundwater at a wide, but usually low range of concentrations.
Electrospray Ionization coupled directly to Fourier-Transform Ion Cyclotron Resonance Mass
Spectrometry (ESI FT-ICR MS) has recently been implemented to examine a complex mixture
of organic compounds of NOM in groundwater8. This research established elemental
compositions of 292 chemical components of NOM including a large proportion of compounds
containing nitrogen and sulphur. It is evident that the identification of even the most abundant
components of NOM is an extremely challenging task due to the enormous complexity of NOM.
It is important to mention that the FAIMS technique shows some ability to separate components
of NOM based on their size because positive ions of larger components of NOM are transmitted
by FAIMS at progressively more negative compensation voltages. Positive ions of NOM display
negative differential mobility properties because these ions experience larger ion mobilities at
high electric fields and smaller ion mobilities at low electric fields. Consequently, ionized
components of NOM are transmitted in FAIMS at negative compensation voltages.
The FAIMS analyzer is capable of separating ions of NOM from ions of small polar
groundwater contaminants which are transmitted in FAIMS at positive compensation voltages
due to their positive differential mobility properties.
The elimination of the chemical
background from numerous components of NOM is critical for the detection and further
identification of other compounds in groundwater, especially those present at a trace level. For
example, the mass spectrum in Figure 2.1 C, shows a hundred of less abundant groundwater
contaminants which were detected as positive ions following the FAIMS separation at the CV of
-3.5 V. Without FAIMS, the detection of these ions would be problematic because of a high
level background from NOM.
It is important to realize that large ions of relative polar
components of NOM are quite labile and can dissociate to smaller fragment ions before the
27
detection. For this reason, the chemical background from NOM in mass spectrometry also
includes small fragment ions at practically each nominal mass as observed in the mass spectrum
in Figure 2.1 B. NOM is a source of problems for LC-MS methods in which an extensive
preparation of water samples is required to eliminate or reduce the content of NOM in purified
samples subjected to the water analysis.
The mass spectrum (Fig. 2.1 D) represents the most abundant ions which have been
detected in the groundwater sample. All major ions (m/z 101, 119, and 127) at the CV of 12.5 V
exhibit isotopic patterns indicating the presence of sulphur atoms in their structures. Sulphurcontaining ions, such as the m/z 101, can be recognized by considering the relative intensity of M
(101) and M+2 (103) spectral peaks. Due to the natural abundance of isotopes of 32S (95.0 %)
and 34S (4.2 %), the spectral intensity of M+2 ions for sulphur-containing species is significantly
larger than the spectral intensity of M+2 ions for non-sulphur species. The most abundant ions at
m/z 101, 119 and 127 (Fig. 2.1 D) show the isotopic patterns in which their corresponding M+2
ions at 103, 121 and 129 (Fig. 2.1 D) have been detected at spectral intensities which are
characteristic for sulphur compounds.
2.2 Optimization of mass spectrometry detection in the analysis of groundwater by
ESI-FAIMS-MS
The discovery of sulphur-containing species in groundwater was quite intriguing at first,
considering that these compounds appeared to be the most abundant contaminants in
groundwater samples analyzed by ESI-FAIMS-MS in our laboratory. My further work focused
on the optimization of mass spectrometry conditions for the analysis of these compounds. This
part of my research revealed that extremely labile ions of sulphur-containing contaminants have
28
to be analyzed at completely different conditions than other compounds. For this reason, these
abundant sulphur contaminants could not be detected in groundwater by conventional mass
spectrometry techniques used in the past.
2.2.1
Optimization of the sampling interface of the QTOF mass spectrometer
Figure 2.2 illustrates most essential elements of ion optics of the QTOF mass
spectrometer used in my study. Molecular ions of groundwater contaminants from nanospray are
transported through multiple devices during their “journey” to the detector. If ions have a fragile
nature, certain instrumental parameters (settings) can induce their dissociation. The dissociation
of labile ions can occur during ionization, at the vacuum interface (source) and inside the mass
spectrometer.
The operation of the QTOF is recommended under a set of instrumental
parameters which have been determined by the manufacturer to provide its optimal performance
with respect to ion transmission (sensitivity), resolution and the accuracy of mass measurement.
These recommended conditions are optimal for a wide range of instrument applications but not
appropriate for the analysis of groundwater. Due to significant complexity of ion optics in
QTOF-MS, changing instrumental parameters beyond the recommended values is seldom
attempted, but it was necessary in my research.
Electrospray ionization and FAIMS separation take place at atmospheric pressure. The
QTOF mass analyzer on the other hand is kept under high vacuum conditions therefore, ions
entering the mass analyzer region will experience a significant pressure change. The pressure at
the vacuum (sampling) interface is reduced in multiple stages to avoid fragmentation of ions40.
The inset in Figure 2.2 illustrates the sampling interface of the QTOF mass spectrometer. It
29
consists of two plates: sample cone and extraction cone and the region between the plates is kept
under the vacuum conditions by the differential pumping system.
Figure 2.2: A schematic diagram of the QTOF mass spectrometer used in my research. The
sampling interface (source) of the instrument is shown in the inset.
During the ESI-FAIMS-MS analysis, a potential is applied to the sample cone and
extraction cone. The potential between these two electrodes can be controlled by changing the
voltage at each electrode. Figure 2.3 shows the tuning page where parameters controlling the
nanospray source and the sampling interface can be accessed. During the ESI-FAIMS-MS
analysis, certain settings such as: desolvation temperature, source temperature, cone gas flow,
desolvation gas flow and syringe pump flow (Fig. 2.3) do not apply to the analysis. The only
parameters that can be adjusted using this software page include: the capillary voltage
30
(nanospray potential), sample cone voltage
and extraction cone voltage. The extraction
cone voltage is normally set between 0-2 V.
The sample cone voltage is recommended
by the manufacturer to be between 30 and
70 V. It was determined that these last two
parameters play a crucial role in the
dissociation of fragile molecular ions. The
extraction voltage of 1 V and the sample
cone voltage of 14 V were determined to be
optimal for sampling intact molecular ions
from the FAIMS analyzer into the QTOF
Figure 2.3: Tuning page of the QTOF mass
spectrometer for instrumental parameters
controlling ion transmission from the sampling
interface to the TOF mass analyzer.
mass spectrometer during the ESI-FAIMSMS analysis of groundwater samples.
Figure 2.4 shows results of experiments to determine the optimal conditions at the
sampling interface during the ESI-FAIMS-QTOF-MS analysis. All mass spectra in Figure 2.4
have been acquired for the same groundwater sample and using the same experimental
conditions in ESI-FAIMS-QTOF-MS except that the sample cone voltages were varied. Because
the spectra were acquired in the positive ionization mode following the separation of ions in
FAIMS at the CV of 12.5 V, sulphur-containing ions can be detected in the groundwater sample.
When the sample cone was set at 50 V (Fig. 2.4 A), only small ions are observed. However,
31
when the cone voltage was decreased to 30 V (Fig. 2.4 B) ions with higher masses started to
appear in the spectrum. With the sample cone at 14 V (Fig. 2.4 C), further increase in the
spectral intensity of higher mass ions was evident.
The observed spectral features clearly show that the dissociation of fragile ions occurs at
the interface of the mass spectrometer. The kinetic energy of ions entering the vacuum region of
the mass spectrometer is determined by the potential difference between the sample cone and
extraction cone. At recommended potentials of the sample cone, the kinetic energy of labile
molecular ions is sufficient to cause their collision induced fragmentation41. This phenomenon
also known as the source or nozzle-skimmer collision induced dissociation, can significantly
influence appearance of detected in the spectrum ions40,41. With lowering the sample cone
voltage, the dissociation of ions is reduced and the transmission of ions also decreases. Thus, we
have chosen the optimal voltages for the sampling cone (14 V) and the extraction plate (1 V) to
minimize ion fragmentation without compromising ion transmission and sensitivity.
32
101
Normalized Ion Intensity
100
Sample cone 50 V
(A)
80
60
119
40
127
20
83
86
0
50
150
100
200
250
m/z
161
Normalized Ion Intensity
100
Sample cone 30 V
(B)
80
60
143
40
101
203
119
20
0
250
200
150
100
50
m/z
161
Normalized Ion Intensity
100
203
(C)
Sample cone 14 V
80
60
175
138
40
143
20
133
221
184
0
50
100
150
200
250
m/z
Figure 2.4: Results of the ESI-FAIMS-QTOF-MS analysis of the same groundwater sample at
the same experimental conditions except using different sample cone voltages at the sampling
interface. (A) The sample cone voltage of 50 V; (B) the sample cone voltage of 30 V; (C) the
sample cone voltage of 14 V. All spectra were acquired in the positive ionization mode
following the separation of ions in FAIMS at the CV of 12.5 V. The extraction cone voltage of 1
V and the collision energy of 10 V were used in the experiment.
33
2.2.2
Optimization of ion transmission parameters of the QTOF mass
spectrometer
The dissociation of labile ions can occur not only at the sampling interface but also inside
the mass spectrometer.
Figure 2.2 shows the quadrupole section of the QTOF mass
spectrometer. This part of the instrument consists of three main transmitting devices called
multipoles (Q0, Q1 and Q2) which are responsible for transmitting ions from the sampling
interface (source) to the TOF mass analyzer. During MS experiments when ions are detected, all
multipoles act as ion transmitting/focusing devices. A small DC voltage is applied across each
multipole to move ions from its entrance to its exit. In this way, Q0 (quadrupole) can transmit
ions from the sampling interface to Q1 (quadrupole), Q1 can transmit ions to Q2 (hexapole) and
Q2 can transmit ions to the TOF mass analyzer for detection. Each multipole also acts as an ion
focusing device because transmitted ions are tightly focused to the center of the device by the
applied RF radio frequency voltage and through collisions of ions with residual gas molecules.
As a result, the diameter of the ion beam is decreased and ion energy spread is reduced. This is
extremely important for the performance of TOF mass analyzer as its sensitivity and resolution
significantly improves with tightly focused ion beam. In MS/MS (tandem) experiments, Q1 acts
as a mass filter which transmits only a selected parent ion to Q2 which serves as a collision cell
where the dissociation of parent ions takes place as a result of their collisions with gas molecules
(argon) that have been introduced to the cell42. The CID process can be controlled by applying a
DC voltage across the collision cell. Following CID, ions are transmitted to the TOF mass
analyzer for detection33.
34
Figure 2.5 shows tuning page for the quadrupole section of the instrument, where
different sets of parameters related to the transmission of ions through instrument multiples (Q1,
Q2, Q3) can be adjusted. During the series of
experiments, all these parameters have been
investigated and their optimal values are
presented the in tuning page in Figure 2.5. It
was established that a DC voltage applied
across the hexapole (Q2) plays the most
important role in the dissociation of labile ions.
This DC voltage corresponds to the parameter
called Collision Energy in the tuning page in
Figure 2.5.
In the MS operation mode, the
collision energy is usually set to 10 V in order
to maximize ion transmission and minimize ion
fragmentation. In the case of fragile ions, the
collision energy CE should be even lower to
Figure 2.5: Tuning page of the QTOF mass
spectrometer with optimized parameters for the avoid their fragmentation. In the MS/MS
ion transmission in the quadrupole section of
the instrument.
operation mode, the collision energy is adjusted
in order to obtain the desired fragmentation of a parent ion39.
Figure 2.6 presents spectral data from the ESI-FAIMS-MS analysis of the groundwater
sample at three different collision energy settings. These experiments have been performed to
determine the optimal collision energy voltage for groundwater contaminants that are separated
in FAIMS at CV of 12.5 V.
35
Normalized Ion Intensity
100
101
(A)
CE = 15 V
143
80
60
40
83
20
203
0
50
150
100
200
250
m/z
143
Normalized Ion Intensity
100
CE = 10 V
(B)
80
203
60
101
40
73
161
20
0
250
200
150
100
50
m/z
161
Normalized Ion Intensity
100
(C)
175
203
CE = 4 V
138
143
80
60
184
40
133
221
20
73
101
119
0
50
100
150
200
250
m/z
Figure 2.6: Results of the ESI-FAIMS-QTOF-MS analysis of the same groundwater sample at
the same experimental conditions except using different collision energies conditions. (A) The
collision energy of 15 V; (B) the collision energy of 10 V; (C) the collision energy of 4 V. All
spectra were acquired in the positive ionization mode following the separation of ions in FAIMS
at the CV of 12.5 V. The extraction cone voltage of 1 V and the sample cone voltage of 14 V
were used in the experiment.
36
Based on mass spectra acquired at different collision energies, it is evident that “gentle”
conditions during ion transmission through the hexapole are important to avoid fragmentation of
extremely labile ions. At relatively harsh conditions (the collision energy of 15V in Fig. 2.6 A),
only a small number of ions could be detected at the CV of 12.5 V. When the collision energy
was decreased to 10 V (Fig. 2.6 B) one new ion at m/z 161 appeared in the spectrum. With
further decrease in collision energy (4 V in Fig. 2.6 C), many new ions were detected in the
groundwater at the CV of 12.5 V. Consequently, the collision energy of 4 V was determined to
be sufficient to provide the detection of most fragile ions.
2.3 The ESI-FAIMS-MS analysis of water samples in positive mode
The optimization of ESI-FAIMS-MS has led to establishing unconventional mass
spectrometry conditions for the detection of very labile ions originating from groundwater. The
new ESI-FAIMS-MS method, implementing this “soft” mass spectrometry approach, has a
potential to detect new contaminants in drinking water. For this reason, the analysis of tap water
and groundwater samples from different locations was performed. These experiments were
conducted to determine if the presence of sulphur-containing compounds in groundwater is an
isolated case or a common contamination problem.
Figure 2.7 shows spectral data from the ESI-FAIMS-MS analysis of groundwater, blank
(HPLC-grade water) and Guelph tap water. The total ion compensation voltage (TICV) spectra
were acquired in the positive ionization mode by scanning the CV from 0 V to 50 V and
detecting ions which were transmitted through FAIMS during the scan. Soft mass spectrometry
conditions were used for ion detection. The TICV spectra for each water sample show distinct
distributions.
The TICV spectrum for groundwater (Fig. 2.7 A) shows the maximal total
37
intensity of detected ions at the CV of 12.5. The mass spectrum (Fig. 2.7 B), acquired at this
CV, contains sulphur-containing contaminants which were not previously detected in
groundwater. The TICV spectrum for tap water (Fig. 2.7 E) shows a different ion intensity
distribution in FAIMS separation than groundwater, which indicates different compositions of
tap water and groundwater. The mass spectrum of tap water at the CV of 12.5 (Fig. 2.7 F) does
not contain sulphur-containing contaminants detected in groundwater but displays other
abundant unknown species. The blank analysis was performed using HPLC-grade water which
was sampled, stored and prepared in the same way as all authentic water samples in the ESIFAIMS-MS method. The TICV spectrum from blank analysis (Fig. 2.7 C) shows a very low
background level. In addition, the mass spectrum, acquired at the CV of 12.5 V (Fig. 2.7 D),
shows the absence of intense background ions. Sulphur-containing contaminants are not present
in tap water and they were not detected in the blank.
38
(A) Groundwater
Ion Intensity
3000
CV 12.5
2000
Normalized Ion Intensity
4000
100
203.0
(B)
80
221.0
60
40
161.0
20
143.0 179.0
0
1000
200
150
100
50
250
300
m/z
0
4000
10
(C) Blank
Ion Intensity
3000
CV 12.5
2000
20
30
Compensation Voltage CV [V]
Normalized Ion Intensity
0
100
40
50
(D)
80
60
40
20
208.0
0
1000
50
200
150
100
250
300
m/z
0
4000
10
(E) Tap
Water
Ion Intensity
3000
CV12.5
2000
20
30
Compensation Voltage CV [V]
Normalized Ion Intensity
0
100
40
50
186.9
(F)
80
60
40
142.9
162.9
20
195.0
0
1000
50
100
150
200
250
300
m/z
0
0
10
20
30
Compensation Voltage CV [V]
40
50
Figure 2.7: Analysis of groundwater, blank and tap water in positive ion mode by ESI-FAIMSMS with Q-TOF detection. (A) The TICV spectrum of groundwater; (B) the mass spectrum of
groundwater at CV 12.5 V; (C) the TICV spectrum from blank analysis; (D) the mass spectrum
of the blank at CV 12.5 V; (E) the TICV spectrum of tap water; (F) the mass spectrum of tap
water at CV 12.5 V.
39
2.3.1
Analysis of groundwater samples from different locations
Figure 2.8 shows results of the analysis of groundwater samples from three different wells
located in rural areas around Guelph using the developed and optimized ESI-FAIMS-MS method
in the positive ionization mode. All TICV spectra, presented in Figure 2.8, were obtained by
scanning the CV from 0 V to 50 V. Mass spectra of groundwater samples were acquired at the
CV of 12.5 V to determine the presence or absence of ions containing sulphur. For all three
groundwater samples, the ion at m/z 203 was the most abundant sample’s component
representing characteristic isotopic pattern for a sulphur-containing compound. The analysis of
the samples from the different locations clearly shows that it would be important to identify the
chemical structure of the most abundant contaminant in groundwater and determine the potential
source of this contamination.
40
Normalized Ion Intensity
Normalized Ion Intensity
100
(A)
80
60
100
203.0286
(B)
221.0322
80
60
161.0160
40
179.0257
20
0
40
100
150
200
m/z
250
300
20
0
0
20
30
Compensation Voltage CV [V]
Normalized Ion Intensity
Normalized Ion Intensity
100
10
(C)
80
60
100
40
50
203.0050
(D)
80
60
175.0147
40
161.0022
20
221.0154
0
40
50
100
150
250
200
300
m/z
20
0
Normalized Ion Intensity
100
(E)
80
60
40
40
30
20
Compensation VoltageCV [V]
Normalized Ion Intensity
10
0
100
50
203.0281
(F)
80
161.0183
60
221.0371
175.0358
40
143.0097
20
0
50
100
150
200
250
300
m/z
20
0
0
10
20
30
Compensation Voltage CV [V]
40
50
Figure 2.8: Analysis of groundwater samples by ESI-FAIMS-MS with Q-TOF detection. (A)
The TICV spectrum of groundwater from location 1; (B) the mass spectrum (CV 12.5 V) of
groundwater from location 1; (C) the TICV spectrum of groundwater from location 2; (D) the
mass spectrum (CV 12.5 V) of groundwater from location 2; (E) the TICV spectrum of
groundwater from location 3; (F) the mass spectrum (CV 12.5 V) of groundwater from location
3.
41
2.4 The ESI-FAIMS-MS analysis of groundwater samples in negative mode
Groundwater samples were analyzed by ESI-FAIMS-MS in negative mode of nanospray
ionization. All analytical steps of the ESI-FAIMS-MS method were optimized in the same way
as presented for positive ions. Figure 2.9 shows spectral data from the analysis of a groundwater
sample in the negative ionization mode. The TICV spectrum (Fig. 2.9 A) was obtained by
scanning the CV from -20 to 100 V and detecting ions which were transmitted through FAIMS
within this CV range. Thousands of negative ions were observed in the TICV spectrum including
a complex mixture of negative ions of NOM. Negative ions of NOM, separated at the CV -10.0
V, are shown in the spectrum in Figure 2.9 B. Similarly to the positive ion mode, negative ions
of large components of NOM were separated from smaller ions of polar contaminants present in
the sample. Most importantly, the m/z 201 ion with characteristic “sulphur fingerprint” was
separated at the CV of 13.0 V in FAIMS (Fig. 2.9 A) and detected in mass spectrometry as one
of the most abundant organic components of the sample. This ion represents deprotonated form
of the m/z 203 ion which was detected in the positive ion mode. The TICV spectrum (Fig. 2.9 A)
illustrates the detection of inorganic anions (HSO4-, HSO3- and HCO3-) in groundwater, which
have been previously reported to be transmitted in FAIMS at the high CV values10. It is
important to note that the spectral intensity of the m/z 201 ion is comparable with the spectral
intensity of sulphate anion (detected as HSO4-) which is usually present in groundwater at low
mg(s)/L. This means that sulfur contaminants in groundwater are quite abundant.
42
Normalized Ion Intensity
100
(B)
(A)
80
HSO3
HSO4
60
(C)
40
HCO3
20
0
Normalized Ion Intensity
100
20
0
-20
100
80
60
40
Compensation Voltage CV [V]
(B)
80
60
40
20
0
500
Normalized Ion Intensity
100
1000
1500
2000
2500
m/z
201.0103
(C)
80
60
40
20
0
160
180
200
m/z
220
240
Figure 2.9: Results of the analysis of a groundwater sample in negative ion mode by ESIFAIMS-MS with Q-TOF detection. (A) the TICV spectrum; (B) the mass spectrum at the CV of
-10.0 V; (C) the mass spectrum at the CV of 13.0 V.
43
2.5 Analysis of groundwater samples by other laboratories.
A groundwater sample with abundant sulphur contaminants was submitted for the mass
spectrometry analysis to the University of Guelph Mass Spectrometry Facility and Prof.
Pawliszyn’s research group at the University of Waterloo.
The groundwater sample was
analyzed by various analytical methods including LC-MS with three different mass
spectrometers (Ion Trap, Orbitral and triple Quadrupole) as well GC-MS with Ion Trap. All these
methods failed to detect sulphur-containing compounds which were successfully analyzed by the
new ESI-FAIMS-MS method in our laboratory. This confirmed the fragile nature of the detected
ions and the requirement for a special analytical technique capable of detecting them.
2.6 The ESI-FAIMS-MS/MS analysis of groundwater samples in positive mode
Figure 2.10 presents the tandem mass spectrum (MS/MS) of the parent ion at m/z 203 that
was transmitted in FAIMS at the CV of 12.5 V in the positive ionization mode. The spectrum in
Figure 2.10 shows that the investigated ion dissociated in collision induced dissociation
producing a large number of fragment ions, which will facilitate structural identification of this
most abundant contaminant in drinking water.
44
100
101.0058
Normalized Ion Intensity
CID 20 V
80
60
40
119.0163
20
143.0172
161.0221
82.9985
179.0386
203.0355
0
60
80
100
120
140
160
180
200
220
240
m/z
Figure 2.10: The ESI-FAIMS-MS/MS spectrum of the m/z 203 parent ion that was transmitted
in FAIMS at the CV of 12.5 V in the positive ion mode.
The spectral information that was acquired in the analysis of the groundwater samples by
ESI-FAIMS-MS (MS/MS) may be sufficient to determine the structure of the most abundant
contaminants in groundwater. Their identification will be based on analyzing spectral data to
determine elemental composition of intact molecular ions and all fragments ions from tandem
mass spectrometry. With known elemental composition of ions, further study of their gas phase
chemistry can be performed to establish their chemical structures based on their reactivity. The
key requirement for such de novo identification approach is the quality of spectral data in mass
and tandem mass spectrometry. However, when performing multiple MS and MS/MS analysis
of investigated contaminants in groundwater in positive and negative ion mode, the lack of
precision in accurate mass measurements of ions was observed. Table 1 shows data from mass
measurements for six different MS and MS/MS experiments in positive ion mode for the most
abundant contaminant detected at the nominal m/z of 203.
45
Table 2.1: Measured masses of the molecular ion at m/z 203 and its dissociation in MS and
MS/MS.
MS experiments CV 12.5 V
Nominal
mass of ion
MS/MS experiments (CID) of m/z 203 ion
Measured accurate mass of ion
Run 1
Run 2
Nominal
mass of ion
Run 3
Measured accurate mass of ion
Run1
Run 2
Run 3
221
221.0322
221.0578 221.0506
221
221.0466
221.0291 221.0650
203
203.0286
203.0461 203.0392
203
203.0424
203.0254 203.0461
179
179.0257
179.0417 179.0352
179
179.0386
179.0158 179.0417
161
161.0160
161.0307 161.0307
161
161.0221
161.0122 161.0368
143
143.0172
143.0071 143.0302
119
119.0163
119.0058 119.0269
101
101.0058
101.0002 101.0148
83
82.9985
82.9932
83.0021
Based on this data it is not possible to determine elemental composition of investigated ions
with high confidence. Therefore, the aim of the next chapter of this thesis is the development of
a suitable calibration method of the Q-TOF mass spectrometer to improve its performance with
respect to the accuracy of mass measurements, which in addition to the detection of new
contaminants would provide high quality spectral date for their chemical identification.
46
3.0 CALIBRATION OF Q-TOF MASS ANALYZER
3.1 Significance of accurate mass measurements
The accurate mass (exact mass) of a molecule is defined as its monoisotopic mass
calculated with respect to the most abundant isotopes. John Beynon realized 60 years ago that
the accurate mass measurement of an ion can be used to determine its elemental composition43.
His statement has significantly changed directions in the mass spectrometry field and today
Time-of-flight (TOF), Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass
analyzers offer accurate mass measurements in mass spectrometry. With proper tuning and
calibration, these instruments are capable of delivering mass measurements with relative errors
below 5 ppm and absolute errors smaller than the mass of an electron44. This performance is
sufficient to determine empirical formulae of ions from small molecules (up to 300 Daltons).
However, when the mass of an ion gets larger, even higher mass accuracy is required to establish
its correct empirical formulae among significantly increased number of potential elemental
compositions of ions at this particular mass43.
3.2 Calibration methods in mass spectrometry
Accurate mass measurements cannot be accomplished without appropriate instrument
calibration which involves the spectral analysis of reference compounds producing ions at known
mass to charge (m/z).
Calibration is used to tune a mass analyzer to analysis therefore,
calibration and analysis should be performed at the same experimental conditions (instrumental
parameters). There are two types of calibration methods which can improve the accuracy of
mass measurements. External calibration is performed prior to analysis and is effective only
when operation of mass analyzer is very stable43. The second approach is the internal calibration
47
in which reference compounds are introduced to the mass spectrometer along with the analyte.
Internal calibration can compensate for a short instrument drift, which can improve performance
of all mass analyzers43.
3.3 Standards for calibration
[Glu1]-Fibrinopeptide B has been used in our laboratory as a reference compound for the
calibration of the Q-TOF mass spectrometer. To calibrate the low mass range of the instrument,
collision induced dissociation of the peptide was performed to generate small ammonium ions of
amino acids with known compositions and masses. The dissociation of [Glu1]-Fibrinopeptide B
provides only few fragment ions at m/z below 400 and can be only accomplished at the sample
cone voltage of 50 V and the collision energies of 10 V. These instrumental conditions are quite
different from those (cone voltage (14 V), collision energy (4 V)) required for the detection of
extremely labile molecular ions
of sulfur-containing contaminants
in
groundwater.
Consequently, the calibration method is not compatible with the analysis. As shown in Chapter
2, the performance the Q-TOF is compromised for this calibration approach. For this reason, a
new calibration method is required in “soft” mass spectrometry in order to carry on the
calibration step and the analysis at the same experimental conditions to improve the quality of
spectral data. Polyethylene glycols (PEGs) and polypropylene glycols (PPGs) have been used
for calibration because their ions can cover a wide mass range in positive and negative ionization
mode39,45. PEGs and PPGs, however, can easily adsorb to any surface (e.g., fused silica tubing)
and deposit on elements of ion optics (e.g., transmission quadrupole) causing instrument
contamination (memory effect). This problem in some cases (frequent calibrations) can be very
severe and require a week of extensive instrument cleaning prior to the analysis, which is not an
attractive option from a point of view of the user46. Aqueous solutions of different salts (cesium
48
iodide, sodium iodide, tetraethylammonium iodide) are used in electrospray to generate cluster
ions which can also cover a wide mass range for positive and negative ions. However, alkali
metal ions are a source of many problems in mass spectrometry including not only the
instrument contamination but also ionization suppression and formation of different salt adducts
with the analyzed compounds, which can be observed in mass spectra for months46. Therefore, it
is reasonable to avoid any instrument contamination by salts.
3.4 A new calibration method using formaldehyde
The calibration of a Q-TOF micro mass spectrometer (Waters, UK) has been
accomplished by using a new approach in which a diluted solution of formaldehyde serves as the
calibration standard in positive and negative ionization modes. Formaldehyde is an inexpensive
and commonly used solvent which is susceptible to nucleophilic addition and has tendency to
undergo spontaneous polymerization47. Formaldehyde polymers are excellent calibration
standards. The calibration solution can be prepared simply by diluting 7µL of formaldehyde
(37.3% by weight) (Fisher Scientific, Fair Lawn, NJ, USA) in 1 mL of methanol/water (9/1 v/v)
containing 0.1 mM ammonium acetate. The solution was infused to the electrospray source at
the 5µL/min flow rate using a syringe pump built into the Q-TOF instrument. The electrospray
source was operating at 80°C with the cone N2 gas flow at 20 L/h and desolvation N2 gas flow at
380 L/h. The electrospray capillary voltage was set at 2 kV and -2.3 kV in the positive and
negative ionization mode, respectively. This calibration solution can be analyzed at “gentle”
conditions of ion sampling at the interface of the mass spectrometer. The sample cone voltage
can be adjusted as low as 2 and 3 V in the positive and negative ion modes. At the same time,
optimal spectral intensities (just below 200 counts/sec) can be obtained for ions used in
49
calibration. The formaldehyde calibration solution does not contaminate the instrument and can
be easily removed from the system after few minutes of flashing with a solvent.
3.5 Characterization of formaldehyde calibration solution in the positive ionization
mode
Figure 3.1 shows the mass spectrum of the formaldehyde calibration solution in the
positive ionization mode. To ensure that low intensity peaks are well defined, the spectrum was
acquired for 10 minutes with a scan time of 1 second to obtain at least 1000 counts continuum
data for the lowest spectral intensity ion used for calibration. After acquisition, the data were
summed and smoothed two times using Savitzky-Golay method and then centered.
230
*
100
200
*
260
*
* HO
C
O
CH3
+
NH4+
+
NH4+
n
80
Normalized Ion Intensity
v HO
170
*
60
290
*
216
v
276
v
40
140
*
110
* 126
v
50
100
H
n
320
*
306
v
350
336 *
v
156
v
380
366
*
v
396 410
v *
80
*
0
O
246
v
186
v
20
C
150
200
250
m/z
300
350
400
440
*
450
Figure 3.1: The mass spectrum of the formaldehyde calibration solution in the positive
ionization mode.
50
The calibration spectrum shows few series of evenly spaced peaks corresponding to ions
of polymethylene glycol. The most intense series of peaks (marked with “*” in Fig. 3.1) is
associated with ammoniated ions of polymethylene glycol with one terminal methyl group. The
structure of these ions is presented in Figure 3.1 and their elemental composition and calculated
accurate masses are listed in Table 3.1. The second most intense series of ions (marked as “v” in
Fig. 3.1) corresponds to ammoniated polymethylene glycol without the terminal methyl group, as
shown in Figure 3.1.
Table 3.1: Elemental compositions and calculated accurate masses of ammoniated ions of
polymethylene glycol with one terminal methyl group.
Elemental
Composition
Calculated mass
C2H10O2N
C3H12O3N
C4H14O4N
C5H16O5N
C6H18O6N
C7H20O7N
C8H22O8N
80.07061
110.08117
140.09174
170.10230
200.11287
230.12343
260.13400
Elemental
composition
Calculated mass
C9H24O9N
C10H26O10N
C11H28O11N
C12H30O12N
C13H32O13N
C14H34O14N
290.14456
320.15513
350.16569
380.17626
410.18682
440.19739
In principle, all ions of methylene glycol polymers can be employed as reference ions for
the calibration. In my calibration procedure, only the most intense ammoniated ions of
methylene glycol polymers with the terminal methyl were selected as reference ions.
3.5.1
Formation of methylene glycol polymers
Due to the tendency of undergoing spontaneous polymerization, formaldehyde is usually
sold as an aqueous solution (30-50% by weight). The rate of polymerization of formaldehyde in
51
water decreases because of formation of formaldehyde hydration product – methylene glycol48.
Aqueous formaldehyde contains methanol (12% in my study), but its concentration can be as
high as 15%49.
Methanol also decreases the rate of polymerization of formaldehyde. In
addition, formaldehyde contains other stabilizers in milligram per liter concentration levels
including formic acid which also was found to decrease the rate of base catalyzed formaldehyde
polymerization process49-50. The presence of these stabilizing agents can explain the origin of
different polymers detected in the calibration spectrum (Fig. 3.1).
Scheme 3.1 shows chemical reactions in the aqueous formaldehyde solution, leading to
formation of methylene glycol polymers. The process can be divided into three steps: initiation,
propagation and termination. In the first step, the polymerization is initiated by the nucleophilic
attack of hydroxide anion on the carbonyl carbon of formaldehyde (Scheme 3.1 A). The product
of initial nucleophilic addition (ion not detected in the spectrum) is then involved in the
nucleophilic attack on another formaldehyde molecule (Scheme 3.1 B). In this way the process is
propagated and multiple polymers with different chains lengths are produced.
52
A) INITIATION
H
H
OH-
O
HO
O
H
H
B) PROPAGATION
H
O
HO
H
H
O
O
O
HO
O
H
H
H
H
H
H
H
n
C) TERMINATION
O
O
HO
OH
H
H
n
n
O
O
HO
H
H
n
H
H
H
H
OH-
OCH3 +
O
HO
H3C
H
H
H
H
H
H
OH
O
HO
O
+
O
H
H
H
H
HO
O
n
Scheme 3.1: Proposed mechanisms for the formation of polymethylene glycols which were
detected in the positive ion mode (Figure 3.1, marked as “*” and “v”). (A) The initiation step;
(B) the propagation step; (C) termination of polymerization by methanol and formic acid.
Eventually, the process will be terminated by either methanol or formic acid (Scheme 3.1
C). The nuclephilic substitution on the carbon of methanol molecule will result in methylene
glycol polymers with the terminal methyl group. Termination by formic acid will occur when a
53
polymer will obstruct the proton from the acid to form methylene glycol polymers without the
terminal methyl group. Polymethylene glycols form ammonium ion adducts in electrospray and
these ions can be detected in MS (Fig. 3.1).
3.6 Characterization of formaldehyde calibration solution in the negative ionization
mode
Figure 3.2 presents the mass spectrum of the formaldehyde calibration solution in
negative ionization mode. Similarly to the positive ion mode, data acquisition was performed for
10 minutes, so the low intensity peaks can be well defined.
O
O
H
100
a)
H
O
80
b)
O
c)
183
*
O
213
*
CH3
O
153
*
O
*
Normalized Ion Intensity
H
O
H
H
H
n
OH
v
123
*
O
O
HO
O
H
H
O
60
O
O
HO
O
H
H
CH3
O
H
243
*
93
*
40
H
H
n
273
*
303
*
20
45
a 59 75
b c
107
v
137
v
167
v
197
v
227
v
257
v
287
v
333
317 * 347 363
v
v *
393
*
0
50
100
150
200
m/z
250
300
350
400
Figure 3.2: The mass spectrum of the formaldehyde calibration solution in the negative
ionization mode.
54
The most abundant series of negatively charged ions (marked with “*” in Fig. 3.2) in the
calibration spectrum corresponds to polymethylene glycol without the terminal methyl group.
These polymers were detected as adducts with formate anion as illustrated in Figure 3.2.
Elemental compositions and calculated masses of these ions are presented in Table 3.
Table 3.2: Elemental compositions and calculated accurate masses of ions of polymethylene
glycol detected in the negative ion mode.
Elemental
Composition
CHO2
C2H3O2
C2H3O3
C2H5O4
C3H7O5
C4H9O6
C5H11O7
Calculated mass
Elemental
composition
Calculated mass
44.99820
59.01385
75.00877
93.01933
123.02990
153.04046
183.05103
C6H13O8
C7H15O9
C8H17O10
C9H19O11
C10H21O12
C11H23O13
C12H25O14
213.06159
243.07216
273.08272
303.09329
333.10385
363.11442
393.12498
In addition, second series of peaks (marked as “v” in Fig. 3.2) are also associated with
polymethylene glycol without the terminal methyl group. These polymers were detected as
adducts with acetate anion as shown in Figure 3.2. In addition formate anion (m/z 45), acetate
anaion (m/z 59) and anion of glycolic acid (m/z 75) were detected in the low mass region of the
calibration range (Fig. 3.2). The most abundant series of ions representing adducts of
polymethylene glycol with formate anion (“*” in Fig. 3.2) along with formate, acetate and
glycolic acid anion (a, b, c in Fig. 3.2) have been employed as reference ions for calibration.
Their calculated accurate masses are showed in Table 3.2.
55
3.6.1
Formation of formaldehyde polymers in the negative ionization mode
Based on the detection of polymethylene glycol ions in the positive mode (Fig. 3.1),
polymethylene glycol with the terminal methyl group are the major products of the reactions
presented in Scheme 3.1. However, negative ions of these polymers are barely detected. This
observation suggests that the detection of abundant polymer ions in the negative ionization mode
must involve and additional process of formation of polymethylene glycol without the terminal
methyl group.
This process takes place during electrospray ionization but only when the
electrospray is operated in the negative ion mode due to reduction reactions occurring at the tip
of the electrospray capillary (cathode).
Consequently, the reduction of water present in the
calibration solution can occur, generating hydroxide ions which can trigger polymerization of
formaldehyde.
Scheme 3.2 illustrates the polymerization process in which hydroxide ion initiates
polymerization of formaldehyde (Scheme 3.2 A). Polymerization propagates through a series of
nucleophilic addition reactions (Scheme 3.2 B).
The polymerization is terminated by the
formation of adduct ions in which the proton is shared between the polymer moiety and an anion
(formate or acetate). These adduct ions are detected in MS. The described reactions most likely
occur inside electrospray droplets on their way to the entrance of the mass spectrometer. When
the solvent evaporates and droplets are becoming smaller, the amount of formaldehyde
molecules available for the polymerization also decreases. In highly condensed and charged
droplets, the polymer ions will eventually encounter formic acid or an acetic acid molecule and
form the adduct ions to stabilize its charge.
56
A) INITIATION
H
H
HO
O
OH
-
O
H
H
B) PROPAGATION
H
HO
O
H
H
O
O
O
HO
O
H
H
H
H
H
H
H
n
C) TERMINATION
O
H
H
H
H
O
O
O
HO
H
O
H
H
H
H
n
n
O
H
H
H
H
O
O
HO
HO
H
H
+
O
O
O
HO
O
O
HO
+
O
H
H
H
HO
CH3
CH3
H
H
n
n
Scheme 3.2: Proposed reactions for the formation of negative ions of polymethylene glycol
without the terminal methyl group (marked with “*” and “v” in Fig. 3.2) during electrospray
ionization. (A) The initiation step; (B) the propagation step; (C) the termination step involving
formic and acidic acids.
57
3.7 Tuning and Calibration of the Q-TOF mass analyzer
The formaldehyde solution was used to tune the Q-TOF mass analyzer and perform its
calibration. The tuning process involves optimization of all instrumental parameters that affect
the performance of TOF with respect to sensitivity, resolution and peak symmetry. Figure 3.3
illustrate the main components of the Q-TOF micro mass spectrometer including its analyzed
TOF mass.
Figure 3.3: A schematic diagram of the Q-TOF MS instrument (adapted from the Q-TOF micro
Mass Spectrometer Operator’s Guide, Waters Corporation).
Before the entrance to the acceleration region TOF mass analyzer, the ion beam is
focused by a series of lenses showed in the inset of Figure 3.3. The acceleration (pusher-puller)
region consists of three parallel plates: back plate (pusher) and two grids (Fig. 3.3)39. Initially,
58
acceleration region is field free so ions enter the gap between the pusher plate and the puller
(first grid) and accommodate their positions according to their initial velocity in y-direction. A
pulse is then applied to the pusher and puller to eject ions in the direction orthogonal to their
initial velocity. The frequency of ejection pulses is set up in a way that the fill up time of
acceleration region is the same as it takes for the ion with the largest m/z to reach the detector.
The push-out pulses are very sharp so all ions are ejected essentially at the same time. Otherwise,
the spatial focusing in the extraction region (accelerator) will be compromized33.
After leaving the first acceleration region (pusher-puller) ions are accelerated to their
final energy of ∼6 kV. The whole acceleration process is split into two phases to reduce the
amplitude of the push-out pulse as even with the current technology it is difficult to achieve
pulses with good reproducibility and short rising times for amplitude higher than 1 or 2 kV.
Furthermore, to avoid penetration of high field from the main acceleration region into the
pusher-puller region, the second grid is placed between these two regions. A relatively small bias
potential applied to the second grid sufficiently protects the pusher–puller region from a
penetrating field from the main acceleration region51.
After the orthogonal acceleration, ions enter the field-free drift space where TOF mass
separation occurs according to the principles described in Chapter 1. In the field free drift tube,
ions travel to the reflectron device where the ion beam is reflected at nearly 180 degrees and then
travel along the second field free drift region towards the multichannel plate (MCP) ion detector.
The time it takes for an ion to travel through the two TOF regions is still proportional to square
root of m/z of the ion as presented in Chapter 142.
59
3.8 Optimization operational parameters of Q-TOF mass analyzer (tuning)
Figure 3.4 represents the time of flight tune page which can be accessed from the
MassLynx software to control instrumental
parameters of the TOF mass analyzer. All the
settings presented in this figure have been
optimized in my studies during the series of
experiments. The tuning page (Fig. 3.4) shows
optimal settings for the TOF mass analyzer for
soft conditions of ion probing at the sampling
interface and during ion transmission through
the quadrpole section of the instrument. Three
different groups of instrument parameters can
be controlled using this software page:
transfer lens, TOF flight tube and MCP
detector.
Figure 3.4: TOF instrument tune page with
optimized settings in positive ion mode.
Transfer lens parameters are critical
in QTOF for the maximal sensitivity and resolution as ion beam entering the flight tube should
be tightly focused by action of three different lenses shown in Figure 3.3.
Second set of parameters (TOF flight tube parameters) controls the acceleration of ions
and their drift in the flight tube. Therefore, these settings have the biggest influence on
resolution, peak shape and accuracy of mass measurements. The particular elements of this part
60
of the instrument are showed in Figure 3.3. Using the software page (Fig. 3.4) there are few
different settings which can be adjusted in this section:
Pusher – is a back plate in the TOF acceleration region. The pulse voltage applied to the
plate causes orthogonal acceleration of ions. The pulse voltage applied to the pusher plate
normally has a height of 830 V39.
Puller – is a grid placed in parallel position to the pusher. Both pusher and puller create
the first acceleration region of TOF. The pulse voltage applied to this grid normally has a height
of 645 V39.
The pusher-puller voltages have profound influence on the resolution, peak shape and
accuracy of mass measurements. Repetition frequency of their pulses (cycle time) should be
adjusted in the way that the fill up time of acceleration region is the same as it takes for the ion
with the largest m/z to reach the detector. If pulses frequency of pusher-puller is shorter than the
time needed for the ions with the largest selected m/z to reach the detector, it will result in poor
resolution and peak shapes41. These frequencies can be adjusted either manually or
automatically. Manual option can be selected from the tune page of TOF (Fig. 3.4) and requires
entry of a particular cycle time, i.e. for ions below 1000 m/z, this time was established to be
33µsec. When manual pusher option is not employed, the instrument automatically will adjust
the pulse frequency according to the highest m/z requested in the acquisition range39.
Even more critical is to determine pulse voltages of the pusher-puller. During my
investigation and optimization of instrumental parameters these two settings proved to have an
essential role in the instrument performance. Figure 3.5 illustrates the influence of different
pusher-puller voltages on the peak shape in the negative ionization mode. Overcompensation of
61
the puller voltage (pusher -815 V; puller -705 V) results in peak fronting and absolute positive
mass error (+96 ppm) as shown in Figure 3.5 A. Overcompensation of pusher voltage (pusher 830 V; puller -660 V), on the other hand, results in peak tailing and absolute negative mass error
(-473 ppm) as shown in Figure 3.5 B. Properly chosen pusher-puller voltages (pusher -835 V;
puller -630 V) should provide perfectly symmetrical peaks and small mass error (-5 ppm).
Correct are shown in Figure 3.5 C.
Normalized Ion Intensity
100
(A)
183.0680
+96ppm
80
60
40
20
0
182.90
Normalized Ion Intensity
100
182.95
183.00
183.05
m/z
183.10
183.15
183.20
183.05
m/z
183.10
183.15
183.20
183.15
183.20
(B)
182.9644
-473ppm
80
60
40
20
0
182.90
Normalized Ion Intensity
100
182.95
183.00
(C)
183.0502
-5ppm
80
60
40
20
0
182.90
182.95
183.00
183.05
m/z
183.10
Figure 3.5: Influence of different pusher-puller voltages on peak shape in negative ionization
mode (elemental composition of selected peak C5H11O7-; calculated accurate mass 183.0510).
Peak fronting (A); peak tailing (B); optimized symmetrical peak (C).
62
After the optimization process of our instrument for positive and negative ionization
modes, the values are as follows:
Pusher (V)
Puller (V)
Positive mode
825
660
Negative mode
-835
-630
Other parameters from the TOF tune page (Fig 3.4) have been optimized in the same
way. After establishing optimal instrument conditions, the instrument was ready for the external
calibration. After the calibration, the instrument does not require any adjustments for months.
However, if operational parameters of TOF, sampling pate voltage or collision energy are
changed, the instrument should be recalibrated.
3.9 Stability of the Q-TOF mass analyzer and corrections for instrument drift
Following tuning and the external calibration, the stability of the instrument with respect
to mass accuracy measurement was investigated using the same formaldehyde calibration
solution as used for its calibration. Table 4 shows results for mass measurements on Q-TOF in
the positive mode at different time intervals following the external calibration. Run 1 and run 2
were performed on the day of calibration with ~50 ppm and ~ -90 ppm error, respectively. Run
3 was performed one day after the calibration with ~ +60 ppm error. Run 4 (a week after
calibration) and run 5 (a month after calibration) were completed with ~ +10 ppm and ~ +35
ppm errors, respectively. The determined accuracy of mass measurements clearly indicates that
the external calibration alone is not sufficient to obtain correct masses which in return would
provide correct elemental compositions. The observed drift of the Q-TOF mass analyzer can be
caused by temperature fluctuations. The changes in temperature can influence the performance
of Q-TOF instrument by changing the length of the flight tube and applied accelerating
potentials52.
63
64
350.1778
380.1894
410.2007
C11H28O11N
C12H30O12N
C13H32O13N
-49
-49
-48
-48
-49
-48
-48
-47
-46
-49
-47
ppm
error
-89
-11.1
-20.2
-18.6
-16.9
-15.5
-14.3
-90
-91
-90
-90
-91
-90
-90
-9.5
-12.6
-88
-91
-7.1
-7.7
-88
ppm
error
-36.9
-34.5
-31.5
-28.8
-26.3
-23.4
-20.4
-18.1
-14.9
-12.8
-9.7
error in
mDa
Run 2 (12-07-12)
-5.2
error in
mDa
Run 1(12-07-12)
63
63
63
64
63
63
63
63
65
64
64
ppm
error
25.8
24.0
22.2
20.6
18.1
16.4
14.6
12.6
11.1
8.6
7.0
error in
mDa
Run 3 (13-07-12)
-10
-10
-8
-9
-10
-9
-9
-8
-9
-10
-8
ppm
error
-4.1
-3.7
-2.9
-2.7
-2.8
-2.4
-2.1
-1.7
-1.4
-1.2
-0.9
error in
mDa
Run 4 (17-07-12)
34
35
35
37
35
35
34
37
38
36
37
ppm
error
13.9
13.1
12.1
11.8
10.2
9.1
7.9
7.3
6.4
4.6
4.0
error in
mDa
Run 5 (15-08-12)
Table 3.3: Short and long term drift in the accuracy of mass measurements of formaldehyde solution in positive ionization mode.
320.1313
230.1313
C7H20O7N
C10H26O10N
200.1202
C6H18O6N
290.1548
170.1087
C5H16O5N
C9H24O9N
140.0917
C4H14O4N
260.1431
110.0812
C3H12O3N
C8H22O8N
Calculated
mass
Elemental
composition
An important trend can be observed in the results presented in the Table 3.3. For each particular
run, the mass of all ions was determined with the same relative mass error. This discovery can be
used to correct spectral data and compensate for the instrument drift. In this case, if the mass of
one ion present in the spectrum is known, we can calculate the relative error of its mass
measurement. Consequently, we can correct the mass of any ion in the spectrum by this constant
relative error.
To compensate for the instrument drift, the internal calibration is commonly performed. For
this purpose, the new generation of Q-TOF instruments is equipped with the dual-sprayer ion
source. In this configuration the ion source is used to introduce a sample, whereas the second
sprayer introduces a calibration (reference) standard. The sample and reference sprayer are
switched frequently, which allows determining an error in mass measurement of reference ions at
any time. However, the absolute mass error for the reference ion is used to correct analyte ion by
the same factor using the instrument software53. Essentially, the entire mass spectrum is ‘moved’
by the same factor corresponding to the absolute error of the reference ion. Software algorithms
do not take into account that the absolute error in mass measurements is proportional to the
measured mass. Therefore, “tight” calibration is required in Q-TOF to obtain satisfactory results.
When the mass difference between a reference ion and analyte ion becomes larger, the mass
error for the corrected mass of analyte ion also increases.
My alternative correction method was used in the analysis of a mixture of nitrosamines in the
positive ion mode and a mixture of carboxylic acid in the negative ion mode. The purpose of
these experiments was to demonstrate the effectiveness of the new method which can
compensate for the instrument drift.
65
Figure 3.6 represent spectrum that was acquired for the mixture of nitrosamines in the
positive ion mode. N-Nitrosodi-n-buthylamine (m/z 159 in Fig. 3.6) was chosen as a reference
compound to correct masses of other nitrosamines detected in the spectrum.
100
C12H11ON2+
*
80
Normalized Ion Intensity
C8H19ON2+
*
60
C16H37O2N4+
*
40
C4H9ON2+
C5H11ON2+
*
* C6H15ON2+
*
20
C12H27O2N4+
C13H29O2N4+
*
*C14H33O2N4+
*
0
50
100
150
200
m/z
250
300
350
Figure 3.6: Mass spectrum of nitrosamines mixture in positive ionization mode.
Elemental composition, calculated masses of protonated nitrosamines along with obtained
ppm errors after proposed data correction are shown in Table 3.4.
Table 3.4: Elemental composition, calculated masses and relative errors (ppm) in mass
measurements for a mixture of nitrosamines.
N-nitrosamine compound
Elemental composition
Calculated
mass
Corrected mass
ppm error
N-nitrosopyrrolidine
N-nitrosomorpholine
N-nitrosodi-n-propylamine
N-nitrosodi-n-buthylamine
N-Nitrosodiphenylamine
C4H9ON2
C5H11ON2
C6H15ON2
C8H19ON2
C12H11ON2
101.07094
115.08659
131.01179
159.14919
199.08659
1
3
-1
0
0
66
N-nitrosopyrrolidine and Nnitrosodi-n-buthylamine dimer
N-nitrosomorpholine and Nnitrosodi-n-buthylamine dimer
N-nitrosodi-n-propylamine
and
N-nitrosodi-nbuthylamine dimer
N-nitrosodi-n-buthylamine
dimer
C12H27O2N4
259.21285
2
C13H29O2N4
273.22850
2
C14H33O2N4
289.25980
3
C16H37O2N4
317.29110
1
Figure 3.7 represent spectrum that was acquired for a mixture of carboxylic acids in the
negative ion mode. Similarly, to the positive ion mode, one carboxylic acid (2-hydroxypropanoic
acid; m/z 89 in Fig. 3.7) was randomly chosen as a reference compound to correct the masses of
the other carboxylic acids detected in the spectrum.
100
C9H9O3*
C15H15O2*
Normalized Ion Intensity
80
C3H5O3*
60
C5H9O3*
C6H11O3*
40
20
0
50
100
150
200
250
300
350
400
m/z
Figure 3.7: The mass spectrum of a mixture of carboxylic acid in the negative ionization mode.
Elemental composition, calculated masses and relative errors (ppm) from mass
measurements after corrections are shown in Table 3.5 for deprotonated carboxylic acids.
67
Table 3.5: Elemental composition, calculated masses and ppm errors after data correction from
the analysis of a mixture of carboxylic acids.
Carboxylic acids
Elemental
composition
Calculated mass
Corrected mass
ppm error
2-hydroxypropanoic acid
(Lactic acid)
2-hydroxy-2-methylbutyric
acid
2-hydroxycaproic acid
3-hydroxy-2phenylpropanoic acid
4,4’-(propane-2,2diyl)diphenol (Bisphenol A)
C3H5O3
89.02440
0
C5H9O3
117.0557
3
C6H11O3
C9H9O3
131.0713
165.0557
2
0
C15H15O2
227.1077
2
Results in Table 3.4 and 3.5 clearly demonstrate the effectiveness of the correction
method taking into account the same relative error in mass measurement of different ions at the
same time. This correction method provides accurate mass assignments with relative mass errors
below 5 ppm.
Furthermore, this correction method can be applied to all ions detected in a
spectrum when the accurate mass of just one ion in the spectrum is known. This particular ion
can be used as reference standard to correct masses of other ions for the instrument drift.
Finally, the correction method provides equally accurate masses for ions independent of a wide
m/z range spectrum and regardless of their spectral positions with respect to the spectral position
of the reference ion. For this reason, “tight” calibration required for other correction methods, is
not necessary in the correction method developed in my research. In the next chapter, this
simple approach will be used to determine elemental composition of compounds detected in
groundwater.
68
3.10
Summary
Several very important improvements have been introduced to the performance of the Q-TOF
mass analyzer through the work presented in this chapter. A novel and effective approach was
developed for calibrating the Q-TOF mass analyzer by discovering practically ideal calibration
standard for “soft mass spectrometry”, based on a simple dilution of 7 μL of formaldehyde in an
electrospray buffer solution. A detailed study of instrumental parameters and their influence on
quality of the spectral data was investigated. As a result the instrument performance was
optimized with respect to the peak symmetry and mass resolution. Following that, the external
calibration of the Q-TOF mass analyzer was performed. The most important discovery, during
the work presented in this chapter, comes from my studies on stability and instrument drift. The
new internal calibration method, developed in my study, can compensate for ion drift of the QTOF which now can provide high quality spectral so critical in non-target analysis.
4.0 IDENTIFICATION OF THE DETECTED SPECIES
The objective of my work that will be presented in this chapter is the structural identification
of the most abundant sulphur-containing groundwater contaminants which have been detected in
my study. Due to the improved performance of the ESI-FAIMS-QTOF MS technique, presented
in Chapter 3, it was possible to acquire high quality spectral data for groundwater samples.
Consequently, the chemical identification of a new class of sulphur-containing groundwater
contaminants could be attempted using the mass measurements along with the interpretation of
dissociation patterns of the investigated ions observed in tandem mass spectrometry (MS/MS)
under collision induced dissociation.
69
Table 4.1 lists measured masses of sulphur contaminants detected in groundwater. To
compensate for the instrument drift of TOF mass analyzer, the internal calibration method
(presented in section 3.9 in Chapter 3) was employed to improve accuracy of mass
measurements. For this purpose, ion at m/z 100.1120 (C6H14N+) was used as the internal
standard. This ion represents the protonated ion of cyclohexylamine, which is commonly
observed as a background ion in our instrument. By applying this approach, it was feasible to
determine the molecular formulas for the investigated ions with high confidence (Table 4.1).
Table 4.1: Molecular formulas and measured masses of groundwater contaminants investigated
in the positive ion mode.
Accurate Mass
221.0478
203.0373
179.0373
161.0267
143.0161
137.0267
125.0056
119.0161
101.0056
99.0263
82.9950
Molecular
Formula Found
C8H12O5S
C8H10O4S
C6H10O4S
C6H8O3S
C6H6O2S
C4H8O3S
C6H4OS
C4H6O2S
C4H4OS
C4H6S
C4H2S
Measured
Mass ([M+H]+)
221.00482
203.0375
179.0379
161.0271
143.0164
137.0272
125.0058
119.0165
101.0058
99.0265
82.9953
ppm error
2
1
4
3
2
4
2
3
2
2
4
The elemental compositions of investigated contaminants and the interpretation of
spectral data from MS/MS experiments were sufficient to determine the chemical structures of
the investigated species. During that process large amount of spectral data from MS and MS/MS
experiments was acquired and analyzed. Furthermore, in order to better understand the processes
occurring in the gas phase during collisional induced dissociation (CID), MS/MS experiments
were performed at different collision energies and different compensation voltages. This allowed
70
fragmentation reactions of ions to be investigated in a sequential “step by step” fashion, allowing
to establish their dissociation pathways.
Due to the extensive amount of spectral data which have to be presented for the
identification of these groundwater contaminants, all spectral data from MS/MS experiments are
presented at the end of this chapter. Moreover, the proposed mechanisms of CID reactions of
investigated species are presented in the appendix to this thesis.
Scheme 4.1 combines all dissociation reactions of the protonated m/z 203 ion (1) of
abundant sulphur contaminant which was detected in groundwater samples at the CV of 12.5 V.
Dissociation pathways (1) occur at extremely low activation conditions and involve formation of
20 fragment ions. Based on this information, the chemical structure of this contaminant was
elucidated. It belongs to a class of compounds called thiotetronic acids and its chemical structure
(1) is presented in Scheme 4.1.
The m/z 203 ion (1) and its fragmentation products exhibit complex and unique reactivity
which allowed determining its structural features. During CID, m/z 203 ion (1) undergoes ring
opening to form (2) with a very reactive ketene group which can react with a water molecule
(pick up a water molecule) and generate m/z 221 ion (3).
Tandem mass spectrometry
experiments at low collision energy shows that the ring opening process and water pick up are
reversible and as a result all three ions (1), (2) and (3) are present at equilibrium. At a higher
collision energy, each of three ions (1), (2) and (3) start to fragment in their own unique way.
The m/z 203 ion (1) can eliminate ethylene diol (loss of 60) and will form m/z 143 ion (7). The
m/z 203 ion (2) can lose ketene (loss of 42) generating m/z 161 (5) or ethylene diol (loss of 60)
producing m/z 143 ion (6). The m/z 221 ion (3) can eliminate ketene and generate m/z 179 ion
71
(4). Sequential dissociation reactions of ions described above, involve neutral loses,
tautomerization and rearrangements. Ions with reactive ketene group(s) can also react with water
molecule(s). The formation of m/z 143 ions involves more than one dissociation pathway. This
compound can be present in the form of different tautomers which can further eliminate carbon
dioxide, ketene, or water and form m/z 99 ion (14), m/z 101 ion (18) or m/z 125 (19) ion,
respectively. The m/z 143 ions in an open ring form with ketene group(s) can also pick up one or
two water molecules generating ions with m/z 161 (15) and m/z 179 (17) respectively.
Dissociation pathways of all ions (except three minor fragments (17), (18), (19)) lead to m/z 101
ion (14) and sequentially to m/z 83 ion (20). The m/z 83 ion (20) is the most stable ion among all
dissociation products of the thiotetronic acid. During collision induced dissociation, the m/z 83
ion (20) dissociates (disappears) at the collision energy of 30 V but the only ionic product of its
fragmentation is proton which is the final product of the dissociation of the thiotetronic acid in
the positive ion mode.
72
H
H H
H
OH
3
2
C
4
HO
5
S
HO
O
Ring Oppening
C
HO
Ring Closing
H
HO
m/z 203
(1)
O
HO
+H2O
O
1
SH
HO
SH
O
-H2O
m/z 203
(2)
O
C
m/z 221
(3)
O
-H2CCO
-H2CCO
-H2CC(OH)2
H
H
HO
H
C
O
HO
SH
m/z 143
(7)
S
m/z 161
(5)
-H2CC(OH)2
Ring Oppening
H
-2 x H2O
Tautomerization
C
O
O
O
-HCCOH
S
SH
m/z 143
(6)
HS
OH
H
C
C
C
O
-CO2
HO
O
C
O
SH
OH
C
HS
SH
O
m/z 143
(10)
m/z 99
(18)
O
C
HO
SH
m/z 161
(11)
-H2CC(OH)2
-HCCOH
O
H
+H2O
HO
m/z 179
(17)
m/z 161
(15)
H
H
O
S
H
OH
S
HO
H
-H2O
m/z 119
(13)
HO
C
H
m/z 161
(16)
m/z 137
(12)
OH
HO
SH
H
HS
-H2O
H
O
H
m/z 143
(9)
Ring Oppening
H
+H2O
OH
HO
+2 x H2O
+H2O
-H2O
-H2CCO
HO
C
HO
O
m/z 143
(8)
SH
H
Tautomerization
C
HO
O
m/z 179
(4)
-H2O
H
OH
C
+H2O
O
HO
C
HO
H
HO
S
O
C
S
S
m/z 125
(19)
C
H+
m/z 101
(14)
S
m/z 83
(20)
C
c
c
C
C
H
-H2O
H
Scheme 4.1: Dissociation pathways of protonated thiotetronic acid which was detected in
groundwater samples at the CV of 12.5 V.
73
In addition to the analysis in the positive ion mode, MS/MS experiments of the
deprotonated m/z 201 ion (21) was also performed in the negative ion mode. Table 4.2 presents
molecular formulas and measured masses of the anion of thiotetronic acid and its dissociation
products investigated in the negative ion mode. In order to determine the elemental composition
of these species, the acetate ion (C2H3O2-; m/z 59.0139) was used as an internal standard to
compensate for the QTOF drift. Acetate is always detected in the spectrum during analysis in
the negative ion mode because ammonium acetate is a component of the methanol buffer used
for diluting water samples. Thus, it is convenient to use acetate as the reference ion for the
internal calibration of spectral data.
Table 4.2: Molecular formulas and measured masses of deprotonated thiotetronic acid and its
dissociation products in the negative ion mode.
Accurate Mass
201.0227
159.0121
157.0506
157.0329
141.0557
117.0016
115.0223
113.0608
59.0139
Molecular
Formula Found
C8H10O4S
C6H8O3S
C7H10O4
C7H10O2S
C7H10O3
C4H6O2S
C5H8OS
C6H10O2
C2H4O2
Measured
Mass ([M+H]+)
201.0231
159.0125
157.0512
157.0334
141.0559
117.0019
115.0226
113.0611
59.0141
ppm error
2
2
4
3
1
3
3
3
4
The elemental composition of investigated ions in Table 4.2 along with the dissociation
patterns in MS/MS experiments, allowed for determining their chemical structures. In the
negative ion mode the dissociation of investigated compounds was less extensive. The MS/MS
spectral information used in the identification process are presented at the end of this chapter.
Furthermore, the appendix of this thesis contains reactions of all investigated ions with proposed
74
CID pathways for the investigated ions. Analogically to the positive ion mode, presented below
Scheme 4.2 will summarize the appendix pathways.
Based on spectral data from MS/MS experiments, it was established that m/z 201 (21) ion
can form two tautomers (ions (21) and (26) in Scheme 4.2). Moreover the dissociation of these
tautomers proved to be collision energy dependent. At low collision energy (10 V) the first m/z
201 tautomer ((21) ion in Scheme 4.2) dissociates and generates four fragments: m/z 59 ion
formed following the initial proton transfer reaction (ion (22) in Scheme 42), m/z 157 ion formed
by elimination of carbon sulphide (ion (24) in Scheme 4.2), m/z 157 ion formed by elimination of
carbon dioxide (ion (23) in Scheme 4.2) and m/z 159 ion formed by elimination of ketene (ion
(25) in Scheme 4.2). At higher collision energy (CE 20 V) the second m/z 201 tautomer (ion (26)
in Scheme 4.2) dissociates generating three ions: m/z 115 ion formed by elimination of carbon
dioxide and ketene (ion (27) in Scheme 4.2), m/z 117 ion formed by elimination of 2 ketenes (ion
(28) in Scheme 4.2) and m/z 141 ion formed by elimination of carbonyl sulphide (ion (29) in
Scheme 4.2).
75
CID at low
collision energy
CID at highier
collision energy
3
4
HO
5
O
S
H
H
O
2
C
1
O
O
Tautomerization
S
O
m/z 201
m/z 201
(21)
(26)
O
PT
O
HO
HO
O
PT
O
HO
O
m/z 59
(22)
O
O
O
m/z 157
(23)
-CO2
HO
m/z 159
(25)
O
S
S
S
m/z 157
(24)
-SCO
HO
O
O
HO
O
O
-2 x H2CCO
-CO2
-H2CCO
-H2CCO
-CO2
-CS
S
m/z 115
m/z 117
(27)
(28)
m/z 141 O
(29)
-CS
O
m/z 113
(30)
Scheme 4.2: Fragmentation tree of investigated groundwater contaminants in the negative ion
mode.
When the CID of m/z 157 ion (ion (23) in Scheme 4.2) was performed, m/z 113 ion (30)
was generated by the elimination of the carbon sulphide. Furthermore, by preselecting m/z 157
(ion (24) in Scheme 4.2) for MS/MS experiment two fragments were generated: (22) ion with
m/z 59 (formed following proton transfer) and ion (30) with m/z 113 (formed by elimination of
carbon dioxide).
The thiotetronic acid detected in groundwater and identified in the study, has not been
previously reported as a water contaminant. However, there are several available research papers
describing chemical structures possessing the thiotetronic acid ring (Fig. 4.1 A) analogues to the
detected species54,55. Moreover, the tiotetronic acid ring systems were found to be structurally
76
related to the natural antibiotics thiolactomycin (4.1 B) and thiotetramycin (4.1 C).
Thiolactomycine is one of the most important biologically active thiophene-based natural
products. It has been isolated from a soil bacterium Nocardia sp. and exhibits in vitro antibiotic
activity against many species of pathogens including gram-positive and gram-negative bacteria,
mycobacterium tuberculosis and malaria parasite Plasmodium falciparum. However, in vivo the
antibacterial activity of these compounds is reduced.
(A)
O
HO
O
O
R2
R1
Thiotetronic acids
O
HO
S
S
R1
(C)
(B)
R3
R3
S
O
HO
S
R2
Thiolactomycin
Thiotetramycin
Figure 4.1: Chemical structures of (A) thiotetronic acids; (B) thiolactomycin; (C) thiotetramycin
Based on this information we can speculate the potential source of the investigated
compounds in groundwater. Most likely they are the products of soil bacteria which penetrated to
the underground aquifers.
4.1 Spectral data in the positive ion mode
Figures presented below show spectral data from the tandem mass spectrometry experiments
of the most abundant sulphur compounds detected in groundwater. In order to obtain tandem
mass spectrometry information for fragment ions with m/z 143 and m/z 101 in a source, CID was
performed by increasing the sample cone voltage to 30 V.
77
Normalized Ion Intensity
100
MSMS m/z 203
CID 4 V
203.0372
(A)
80
60
221.0468
40
+18
20
0
Normalized ion Intensity
100
250
200
150
m/z
100
50
143.0163
(B)
-60
203.0372
CID 10 V
80
-18
60
-42
161.0265
221.0463
40
179.0375
20
-42
-18
101.0047
0
50
Normalized Ion Intensity
100
100
150
m/z
101.0085
(C)
200
-42
250
CID 20 V
119.0173
80
-42
82.9983
-18
60
-18
-42
143.0163
40
-18
161.0265
20
137.0275
179.0378
0
50
100
150
m/z
200
250
Figure 4.2: MS/MS spectrum of m/z 203 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 10 V; (C) collision
energy of 20 V.
78
Normalized Ion Intensity
100
203.0372
(A)
-18
80
60
MSMS m/z 221
CID 4 V
221.0479
40
20
0
50
Normalized ion Intensity
100
150
m/z
100
143.0170
(B)
80
200
203.0372
-60
-18
-18
-42
250
CID 10 V
221.0474
60
161.0268
-42
40
179.0358
20
101.0094
0
50
Normalized Ion Intensity
100
(C)
80
150
m/z
100
200
101.0082
250
CID 20 V
-42
-42
-18
-42
60
-18
40
-18
119.0178
20
143.0167
161.0266
82.9986
137.0273
179.0358
0
50
100
150
m/z
200
250
Figure 4.3: MS/MS spectrum of m/z 221 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 10 V; (C) collision
energy of 20 V.
79
Normalized Ion Intensity
100
MSMS m/z 179
CID 4 V
(A)
161.0287
80
-18
60
40
179.0447
20
0
100
50
Normalized ion Intensity
100
(B)
150
m/z
143.0176
161.0285
200
250
CID 8 V
80
-18
60
-18
40
20
179.0447
101.0054
0
50
100
150
m/z
200
250
Figure 4.4: MS/MS spectrum of m/z 179 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V.
80
Normalized Ion Intensity
100
MSMS m/z 161
CID 4 V
(A)
143.0146
80
161.0269 179.0372
60
40
-18
+18
20
0
50
Normalized ion Intensity
100
100
150
m/z
200
(B)
CID 8 V
143.0175
80
250
101.0078
179.0372
161.0295
-42
60
-18
119.0128
40
-42
-18
20
0
50
Normalized Ion Intensity
100
100
150
m/z
200
(C)
250
CID 16 V
101.0044
80
143.0163
60
119.0173
82.9950
40
-18
20
0
50
100
150
m/z
200
250
Figure 4.5: MS/MS spectrum of m/z 161 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V; (C) collision energy
of 16 V.
81
Normalized Ion Intensity
100
(A)
MSMS m/z 143
CID 4 V
143.0161
-42
80
-44
60
+18
-18
40
101.0075
161.0260
20
99.0256
125.0052
0
50
Normalized Ion Intensity
100
100
150
m/z
200
(B)
250
CID 12 V
101.0058
80
-18
60
40
82.9971
20
99.0258
119.0118
143.0161
125.0056
0
50
100
150
m/z
200
250
Figure 4.6: MS/MS spectrum of m/z 143 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 12 V.
82
Normalized Ion Intensity
100
(A)
MSMS m/z 101
CID 4 V
101.0050
80
-18
60
40
82.9975
20
0
100
50
Normalized ion Intensity
100
150
m/z
200
(B)
250
CID 8 V
82.9965
80
60
-18
40
20
101.0050
0
50
100
150
m/z
200
250
Figure 4.7: MS/MS spectrum of m/z 101 ion that was transmitted in FAIMS at CV = 12.5 V in
positive ion mode. (A) Collision energy of 4 V; (B) collision energy of 8 V.
83
4.2 Spectral data in the negative ion mode
Figures presented below show spectral data from MS/MS experiments performed in the
negative ion mode.
Normalized Ion Intensity
100
(A)
201.0216
MSMS m/z 201
CID 10 V
-142
80
60
-44
59.0151
40
157.0321
20
0
50
100
59.0131
Normalized Ion Intensity
(B)
150
m/z
100
200
-88
-86
-84
-44
-42
-142
80
60
250
CID 20 V
157.0253
40
-60
115.0195
113.0443
159.0132
117.0051
20
141.0568
201.0261
0
50
100
150
m/z
200
250
Figure 4.8: MS/MS spectrum of m/z 201 ion that was transmitted in FAIMS at CV = 13.0 V in
negative ion mode. (A) Collision energy of 10 V; (B) collision energy of 20 V.
84
Normalized Ion Intensity
100
MSMS m/z 157
CID 8 V
157.0331
80
60
59.0172
40
113.0426
20
0
50
150
m/z
100
200
250
Figure 4.2-2: MS/MS spectrum of m/z 157 ion that was transmitted in FAIMS at CV = 13.0 V in
negative ion mode. (A) Collision energy of 10 V; (B) collision energy of 20 V.
5.0 CONCLUSIONS AND FUTURE WORK
The initial objective of my study was the application of the Electrospray Ionization - HighField Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry (ESI-FAIMS-MS)
technique for a non-target analysis of groundwater samples. Throughout the initial experiments I
faced numerous challenges with the detection of extremely labile groundwater contaminants.
This led me to the development of a new method with unique analytical capabilities. The method
was developed and optimized during a series of experiments which resulted in the detection of
previously unknown groundwater contaminants containing sulphur. The analysis of these
contaminants was only possible by “soft” mass spectrometry due to their extremely fragile
nature. In this technique, ions were introduced to the mass spectrometer at very gentle conditions
(sample cone 14 V). Additionally, ion transmission was carried out at gentle conditions (collision
energy 4 V) to further reduce the dissociation of ions in the hexapole collision cell.
85
The FAIMS device was used in the study as a primary separation tool combined with mass
spectrometry detection. This combination offers numerous advantages in water analysis but also
makes it more convenient for the user. Sample preparation is practically limited to a simple
dilution of groundwater samples with methanol-water buffer containing 0.1 mM ammonium
acetate. The most impressive capability of FAIMS in water analysis is the elimination of
chemical background through FAIMS separation, which results in high quality spectral data that
was presented in my thesis.
While conducting mass and tandem mass spectrometry experiments, the instability in mass
measurements on Q-TOF was observed and addressed. For the purpose of this study a novel
external calibration method using formaldehyde polymers as reference compounds was
developed. During the experiments absolute error in mass measurements was discovered to be
proportional to the m/z of an ion. Based on this principle a new approach was introduced to
compensate for the instrument drift during mass measurements. By employing this strategy, the
accuracy of mass measurement improved so significantly that elemental compositions of the
investigated groundwater contaminants could be determined with high confidence.
Based on the accurate mass measurements of investigated ions and their dissociation in
tandem mass spectrometry experiments, the structural elucidation of the most abundant sulphur
containing compounds detected in groundwater was achieved. The characterized compounds
(thiotetronic acids) have not been previously detected in water. These abundant groundwater
contaminants are structurally related to many natural and synthetic antibacterial agents such as
the natural antibiotics thiolactomycin and thiotetramycin. Thiolactomycine was originally
isolated from a soil bacterium Nocardia sp. and exhibits strong antibiotic properties in vitro. It is
86
reasonable to assume at this point that the detected in groundwater compounds come from soil
and perhaps are accumulated products of soil bacteria in underground aquifers.
A part of this thesis deals with one of the most difficult aspects of non target analysis
related to gas phase ion chemistry of compounds investigated in my study. The appendix to this
thesis contains a detailed illustration of gas phase chemistry of a new class of compounds which
previously have not been analyzed in mass spectrometry. It is also interesting and important to
note that one can predict the fate of ions in collision induced dissociation. In this study it was
established that protonated molecular ion of the investigated thiotetronic acid can generate 19
other ions and 17 out of 20 ions will end up as a proton during CID at high collision energy.
This can explain why thiotetronic acids have not been previously detected by different mass
spectrometry techniques.
The method developed in this thesis can be applied for the detection and identification of a
wide range of fragile contaminants in drinking water. With this new analytical capability, new
chemical components in water may be discovered considering limitations of conventional mass
spectrometry. The problem of the analysis of fragile ions is not limited to water research. The
same problem is observed in the analysis of polar metabolites, DNA adducts, modified
carbohydrate and proteins. Each sample submitted to a mass spectrometry facility for analysis
contains certain components which will survive at sampling conditions of conventional mass
spectrometry. The concept of “soft” mass spectrometry is especially attractive in combination
with differential ion mobility techniques because FAIMS plays an important role in the analysis
of labile ions generated in electrospray ionization.
87
APPENDIX
H
H
H
3
2
C
4
HO
5
S
HO
O
O
C
HO
1
SH
HO
Ring Closing
C
HO
Ring Oppening
O
HO
SH
H
m/z 203
(1)
O
-H2O
H
m/z 203
(2)
O
O
H
H H
H
H
OH
OH
OH
HO
+H2O
C
HO
C
O
O
SH
HO
m/z 221
(3)
O
O
SH
HO
SH
HO
C
HO
O
O
Scheme 1: Proposed reactions of m/z 203 and m/z 221 ions.
(A)
H
H
H
H
H
OH
OH
C
HO
C
HO
O
O
SH
HO
3
HO
-H2O
SH
HO
2
C
4
5
S
HO
O
1
H
m/z 221
(3)
O
(B)
H
O
H
H
OH
H
H
OH
HO
HO
C
C
HO
O
HO
O
SH
H
OH
HO
-H2CCO
O
SH
m/z 221
(3)
m/z 203
(1)
O
C
HO
O
SH
m/z 179
O
(4)
Scheme 2: Proposed reactions of m/z 221 ion. (A) Elimination of water; (B) elimination of
ketene.
88
(A)
H
H
H H
H
OH
OH
C
HO
O
HO
m/z 203
(2)
O
O
H
H
HO
HO
-H2CCO
SH
m/z 203
(2)
(C)
C
O
SH
O
H
HO
C
O
HO
m/z 221
(3)
O
C
HO
SH
HO
SH
H
(B)
O
HO
SH
m/z 161
(5)
O
H
H
C
O
PT
O
H
C
O
SH
O
C
HO
O
HO
SH
O
C
HO
+H2O
O
-H2CC(OH)2
C
O
O
SH
HO
SH
m/z 143
m/z 203
m/z 203
(2)
HO
(6)
HO
(D)
H
H
O
C
HO
PT
S
O
-H2CC(OH)2
O
S
OH
m/z 143
(7)
m/z 203
(1)
C
HO
RO
HO
C
O
H
OH
H
C
HO
S
H
O
OH
S
m/z 143
(8)
Scheme 3: Proposed reactions of m/z 203 ion. (A) Water pick up; (B) elimination of ketene; (C)
elimination of ethylene diol; (D) elimination of ethylenediol.
89
(A)
H
H
H
OH
HO
H
H
OH
HO
C
C
HO
HO
O
SH
O
-2xH2O
C
HO
O
SH
SH
H
(9)
143
m/z
m/z 179
(4)
H
H
H
H
OH
C
HO
O
+H2O
O
RO
C
HO
C
HO
O
SH
SH
SH
m/z 161
(11)
m/z 143
m/z 143
(10)
(B)
H
HO
H
HO
C
OH
HO
HS
H
H
O
C
m/z 179
(4)
HO
-H2CCO
OH
HO
OH
HO
HS
HS
m/z 119
m/z 137
(12)
O
SH
O
O
HO
H
-H2O
(13)
OH
HO
H
HS
Scheme 4: Proposed reactions of m/z 179 ion. (A) Elimination of two water molecules followed
by ring opening and water pick up; (B) elimination of ketene and water.
90
(A)
C
C
C
O
HO
OH
HO
OH
HO
HO
H
H
H H
H
+H2O
O
HO
O
HO
SH
SH
SH
m/z 161
m/z 179
(4)
m/z 161
(5)
(B)
H
H
HO
H
HO
C
O
HO
O
C
O
HO
SH
-H2O
C
HO
m/z 143
SH
H
SH
(9)
H
m/z 161
(5)
PT
(C)
H
H
C
HO
O
H
C
HO
O
O
S
-HCCOH
H
S
HO
S
HO
O
SH
T
H
HO
HO
m/z 161
(5)
m/z 119
H
(13)
-H2O
(D)
H
O
OH
HO
OH
OH
H
PT H
C
O
SH
O
C
H
HO
H
C
H
O
-H2CC(OH)2
SH
SH
HO
O
H
C
PT
C
S
SH
m/z 101
m/z 101
(14)
m/z 161
(11)
Scheme 5: Proposed reactions of m/z 161 ion. (A) Water pick up; (B) elimination of water; (C)
elimination of ketene and water; (D) elimination of ethylene diol.
91
(A)
H
H
H
H
H
H
OH
OH
C
O
O
SH
H2O
C
O
O
S
SH
m/z 143
(6)
(B)
C
HO
O
O
S
m/z 161
m/z 143
(15)
(8)
m/z 161
(16)
H
O
O
C
C
O
O
OH
HO
+2xH2O
H
C
H
C
C
SH
O
S
H
m/z 143
(6)
S
H
H
H
H
C
HO
C
C
O
S
S
m/z 143
(8)
O
-CO2
H
C
HO
O
PT
S
H
m/z 143
m/z 143
O
C
-H2O
C
H 2O
O
S
S
S
m/z 125
(19)
(7)
(8)
m/z 143
m/z 143
H
(8)
O
H
C
O
H
m/z 143
(9)
O
O
O
HO
C
HO
SH
H
S
SH
H
O
H
C
O
SH
m/z 143
(10)
PT
C
O
SH
RO
m/z 143
(10)
HS
HO
C
HO
H
C
O
T
(F)
H
(6)
C
HS
H
H
O
O
m/z 99
(18)
H
C
H
HS
O
HS
O
H
HO
C
PT
O
(E)
(17) O
m/z 179
H
(C)
(D)
C
HO
H 2O
C
H
O
H
O
HO
O
-HCCOH
C
PT
SH
SH
(14)
101
m/z
C
S
SH
Scheme 6: Proposed reactions of m/z 143 ion. (A) Water pick up; (B) pick up of two water
molecules; (C) elimination of carbon dioxide; (D) elimination of water; (E) tautomerization
followed by ring opening; (F) elimination of hydroxyethyne.
92
83
S m/z
H
HO
HO
C
PT
C
S
H
-H2O
S
m/z 101
H 2C
S
S
C
C
C
c
c
c
C
C
C
(20)
(14)
H
H
H
H+
Scheme 7: Proposed reactions of m/z 101 and m/z 83 ions.
(A)
3
HO
5
S
O
O
O
2
C
4
O
PT
1
HO
S
HO
m/z 201
(21)
O
m/z 59
(22)
O
O
(B)
O
HO
O
- CO2
HO
S
O
S
m/z 157
(23)
m/z 201
(21)
O
(C)
O
HO
S
HO
S
O
O
- CS
O
O
m/z 201
(21)
HO
C
HO
O
O
O
O
m/z 157
O
O
O
(24)
(D)
O
HO
S
O
m/z 201
(21)
O
O
O
HO
HO
S
m/z 201
O
-H2CCO
O
S
m/z 159 (25)
O
Scheme 8: Proposed reactions of m/z 201 ion at low collision energies. (A) Formation of
ethylene diolate at m/z 59; (B) elimination of carbon dioxide; (C) elimination of carbon sulphide;
(D) elimination of ketene.
93
H
H
(A)
O
HO
O
O
Tautomerization
S
O
S
O
m/z 201
(26)
m/z 201
(21)
O
O
(B)
O
S
O
O
O
O
-H2CCO
-CO2
O
O
S
S
m/z 159
m/z 201 O
(26)
(24)
m/z 115
(27)
(C)
O
O
O
O
O
S
-H2CCO
O
O
S
O
S
-H2CCO
m/z 117
m/z 159
m/z 201 O
(26)
(28)
(25)
(D)
O
S
O
O
HO
O
O
-SCO
O
O
H
O
m/z 201
(26)
m/z 141 O
(29)
Scheme 9: Proposed reactions of m/z 201 ion at higher collision energies. (A) Tautomerization
of the negative ion of the thiotetronic acid; (B) elimination of ketene and carbon dioxide; (C)
elimination of two ketene groups; (D) elimination of carbonyl sulphide.
94
(A)
HO
HO
O
O
- CO2
O
m/z 113
(30)
m/z 157
O
(24)
(B)
S
HO
C
O
HO
-CS
O
m/z 113
(30)
m/z 157
(23)
(C)
HO
O
O
PT
O
O
O
OH
HO
O
H
OH
m/z 157
O
(24)
O
O
O
m/z 59
(22)
Scheme 10: Proposed reactions of m/z 157 ion in the negative mode. (A) Elimination of carbon
dioxide; (B) elimination of carbon sulphide; (C) formation of ethylene diolate at m/z 59 ion.
95
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