This thesis examines naphthenic acids and petroleum components using ultrahigh resolving power tandem mass spectrometry. Naphthenic acids are naturally occurring carboxylic acids in crude oil that cause problems for the petroleum industry such as pipeline corrosion and formation of deposits. The author uses Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to selectively ionize naphthenic acids and nitrogen-containing compounds in crude oil samples. Several tandem MS techniques including collision-induced dissociation and infrared multiphoton dissociation are employed to characterize naphthenic acid dimers and provide structural information on crude oil components. The high mass accuracy and resolving power of FT-ICR MS allows accurate mass assignments and

1.
Department of Chemistry
Thesis submitted for the degree of MSc in
Analytical Science: Methods and Instrumental
Techniques
August 2014
Ultrahigh resolving power tandem mass spectrometry of
petroleum components
By: Simona Gherghel
Supervisor: Dr Mark P. Barrow

2.
Table
of
Contents
1
Introduction
................................................................................................................
1
1.1
Crude
oil
..................................................................................................................................................................
1
1.2
Naphthenic
acid
chemistry
..............................................................................................................................
2
1.3
Formation
of
naphthenic
acid
dimers
........................................................................................................
5
1.4
Analytical
techniques
for
looking
at
crude
oils
......................................................................................
6
1.5
Fourier
transform
ion
cyclotron
resonance
mass
spectrometry
....................................................
7
1.6
High
mass
accuracy
.........................................................................................................................................
10
1.7
Kendrick
mass
defects:
the
key
to
unlocking
chemical
formulas
................................................
10
1.8
Double-­‐bond
equivalent
versus
carbon
number
plot
.......................................................................
12
1.9
Main
aims
.............................................................................................................................................................
13
2
Experimental
.............................................................................................................
14
2.1
Sample
preparation
.........................................................................................................................................
14
2.2
Fourier
Transform-­‐Infrared
Spectroscopy
(FT-­‐IR)
...........................................................................
15
2.3
Mass
Spectrometry
Analysis
........................................................................................................................
15
2.4
Calibration
...........................................................................................................................................................
17
2.5
Data
analysis
.......................................................................................................................................................
17
3
Results
and
discussion
...............................................................................................
19
3.1
Proof
of
naphthenic
acid
dimers
existence
in
solution-­‐phase
......................................................
19
3.2
2
year
old
Kodak
naphthenic
acid
sample
.............................................................................................
20
3.2.1
ISD
experiment
0
V
...........................................................................................................................................
20
3.2.2
ISD
experiment
(60
V)
....................................................................................................................................
25
3.3
Fresh
sample
......................................................................................................................................................
29
3.3.1
ISD
(0
V)
of
serial
dilution
............................................................................................................................
29
3.3.2
Importance
of
ion
accumulation
time
(IAT)
.........................................................................................
35
3.4
CID
on
the
2
year
old
Kodak
NA
sample
.................................................................................................
37
3.5
IRMPD
experiment
of
the
fresh
Kodak
NA
sample
............................................................................
40
3.6
Fresh
Kodak
NA
sample
doped
with
salts
.............................................................................................
44
3.7
EID
experiment
on
a
NIST
crude
oil
sample
.........................................................................................
47
4
Conclusion
................................................................................................................
52
5
References
................................................................................................................
54
6
Acknowledgments
....................................................................................................
58
7
Appendix
..................................................................................................................
59
7.1
Known
ions
mass
list
......................................................................................................................................
59
7.2
Mass
error
distribution
histograms
.........................................................................................................
65
7.2.1
2
year
old
doped
Kodak
NA
sample-­‐ISD
0
V
.........................................................................................
65
7.2.2
2
year
old
doped
Kodak
NA
sample-­‐ISD
60
V
.......................................................................................
66
7.2.3
Fresh
sample:
serial
dilution
(ISD
0
V
.....................................................................................................
67
7.2.4
CID
on
2
year
old
Kodak
NA
.........................................................................................................................
71
7.2.5
IRMPD
on
fresh
Kodak
NA
sample
............................................................................................................
73

3.
Abbreviations
APCI Atmospheric Pressure Chemical Ionization
CID Collision Induced Dissociation
DBE Double Bond Equivalent
EI Electron Ionization
EID Electron Induced Dissociation
ESI- Electrospray ionization
FAB Fast Atom Bombardment
FT-ICR MS Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
FT-IR Fourier Transform- Infrared Spectroscopy
GC-MS Gas Chromatography Mass Spectrometry
IAT Ion Accumulation Time
ICR Ion Cyclotron Resonance
IRMPD Infrared Multiphoton Dissociation
ISD In Source Dissociation
KMD Kendrick Mass Defect
MS/MS Tandem mass spectrometry
NA Naphthenic Acid
SARA Saturate, aromatic, resin and asphaltene
SORI Sustained Off-Resonance Irradiation

4.
Summary
The selective ionization of acidic compounds from a commercial naphthenic acid mixture and
of nitrogen-containing compounds from a crude oil sample was carried out using Fourier
transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in negative-ion and
positive-ion electrospray ionization (ESI), respectively. Naphthenic acids are of great concern
to the petroleum industry as they are responsible for various unwanted phenomena such as
pipeline corrosion, formation of sodium naphthenate deposits and they also are toxic to
aquatic organisms. Naphthenic acids have been previously demonstrated to form O4 class
naphthenic acid dimers. During this research, the formation of sodium bound naphthenic acids
(NaO4 class) has been observed. The ultrahigh resolving power and mass accuracy of FT-ICR
MS offers accurate mass assignments and information on compositional differences of
naphthenic acid species. Several tandem MS methods such as collision-induced dissociation
(CID) and infrared multiphoton dissociation (IRMPD) were further employed for structural
characterization of the naphthenic acid dimers. CID and IRMPD of the same precursor ions
produced very similar fragmentation patterns it has been shown that dimers reflect the most
abundant monomer species present. Electron-induced dissociation (EID) was used for
structural characterization of crude oil and it provided valuable fragmentation information
showing successive losses of alkyl chains leading back to the stable cores made of fused
rings.

5. Introduction
1
1 Introduction
1.1 Crude
oil
Crude oil is a fundamental energy source that has been playing a key role in the progress of
modern human society. Even under very optimistic estimates about the development of
alternative energy sources, it will remain one of the most heavily used energy sources for
many decades to come.1
Global dependence on crude oil originates from its high versatility; it
can be used as fuel for vehicles and airplanes, as a home heating source and almost all
chemical products, such as plastics, pesticides, detergents, dyes and even medicines begin
with oil-derived feedstock.2
Nevertheless crude oil is a limited resource and while its production is decreasing, its
consumption only keeps increasing. Therefore, lately there has been an increased focus on the
use of heavier, lower quality sources.3
Heavy oils are one of the most complex mixtures in
nature and they can contain thousands to millions of various components.4
Typically crude
oils have a high proportion of light hydrocarbons (CcHh) and other minor ingredients. The
hydrocarbons can be divided into four types: paraffin based (15 to 60%), naphthenes based
(30 to 60%), aromatic based (3 to 30%) and asphaltic based (remainder). Carbon accounts for
about 85% (83-87%) of the crude oil mass, hydrogen for about 12% (10-14%) and the H/C
ratio is about 1.8.5
The minor ingredients of crude oil include nitrogen (0.1-2%), oxygen (0.05-1.5%), sulfur
(0.005-6%) and traces of metal (0.1%) such as are sodium (Na), calcium (Ca), magnesium
(Mg), aluminium (Al), iron (Fe), vanadium (V), and nickel (Ni). Even though petroleum
contains more than 90% hydrocarbons, it is the heteroatom-containing compounds (NnOoSs)
the ones that cause most problems regarding pollution, poisoning of catalysts, corrosion and
formation of emulsions.6
Low quality crude oils contain higher amounts of sulfur, acidic
components and other hydrocarbons. Sulfur-containing compounds naturally occurring in
crude oils act as poisons to the catalysts used in the conversion of feedstock to useable
intermediate and end products.7
Sweet and sour are terms used to refer to a crude oil’s
approximate sulfur content. As a rule, when the sulfur content is in excess of 0.5% the crude
oil is considered sour, and when it is 0.5% or less it is considered sweet.8
Nitrogen-containing

6. Introduction
2
compounds also represent a concern as they too can play a role in the catalyst deactivation.9
Oxygen-containing components can include phenols, ketones and carboxylic acids. The
presence of acidic substances in crude oil was first observed in a Romanian oil in 1874 by
Hell and Medinger10
. Later on, in 1890 Aschan11
named them naphthenic acids. Nowadays, it
is well known that naphthenic acids cause corrosion in pipes and equipment used in the
processing plants and that they are toxic to aquatic environments.12
1.2 Naphthenic
acid
chemistry
Naphthenic acids (NAs) comprise of a large group of saturated monocyclic, acyclic and
aromatic carboxylic acids. They are naturally occurring compounds in most petroleum
sources, including crude oil and bitumen from the oil sands.13
The classical formula for NAs
is CnH2n+ZO2, where n indicates the carbon number and Z is referred to as the “hydrogen
deficiency” and is zero or a negative even integer number. More negative Z values represent
an increase in the number of hydrogen atoms lost as the structure gets more compact and as
rings are formed14
. The Z value is equal to 0 for saturated linear hydrocarbon chains and it
becomes -2 for monocyclic NAs, -4 for bicyclic, -6 for tricyclic and so forth.15
Sample
structures of naphthenic acids are shown in Figure 1.
Figure 1. Representative structures of naphthenic acids where Z is the hydrogen deficiency, R
represents an alkyl chain and n indicates the number of CH2 groups

7. Introduction
3
The chemical, physical and toxicological characteristics of different NA mixtures depend on
their differences in molecular structure, composition, volatility and polarity. NAs dissolve in
organic solvents and their pH influence their water solubility. Generally, their pKa values are
between 5 and 6.15
Because NAs are water soluble to some degree, their release to
wastewaters must be monitored.16
Naphthenic acid make up to 4 wt% of the crude oil composition17
and their presence in crude
oils is of great concern to the petroleum industry. NAs are responsible for corrosion in
refineries, pollution in refinery wastewaters and in oil sands extraction waters, formation of
calcium and sodium naphthenate deposits during production and processing, and formation of
emulsions.18
Naphthenate deposition can obstruct pipelines (Figure 2), causing production
irregularities and in some cases production shutdowns that can be very expensive for the oil
companies. Formation of emulsions and naphthenate deposits results in millions of dollars
expenses for the petroleum industry.
Figure 2. Naphthenate deposit causing obstruction of a hydrocyclone19

8. Introduction
4
Naphthenic acids are also responsible for the corrosion of pipeline carbon steel alloys that are
otherwise resistant to corrosion from compounds containing sulfides.20
An example of
pipeline affected by NA corrosion is shown in Figure 3. Corrosion by naphthenic acid is not
fully understood but it is known that it involves the chelation of the metal ion by the
carboxylate group with the release of hydrogen gas.21
Qu and his colleagues showed that
corrosivity of NAs depends greatly on the molecular mass and structure; NAs with higher
averaged molecular weight and higher averaged number of ring structures were less
corrosive.22
Temperatures between 220 and 400° C facilitate corrosion whereas at
temperatures above 400 °C the NAs
decompose forming a film that protects the alloy.23
Figure 3. Naphthenic acid corrosion in crude oil unit piping 24
With the increase for oil mining, transportation and application, more and more soils are
susceptible to crude oil contamination. At the same time, more soils contaminated with crude
oil enter the aquatic environment through surface runoff.25
Naphthenic acids are
environmentally significant because they are known to be toxic to a variety of aquatic
organisms, algae and mammals by acting as endocrine disruptors.26
The extraction processes
of bitumen, an extra heavy oil subgroup, from oil sands produce tailing waters that contain
naphthenic acids. As a consequence, oil sands companies need to monitor and report the
concentration of NAs in the waters on and near their leases.27

9. Introduction
5
Nonetheless, NAs also represent a resource. They and their metal carboxylates are used as
dryers in dyes, as rubber plasticizer and as fungicides for wood preservatives.28
By separating
naphthenic acids and other acidic species present in crude oils, the quality of the crude oil will
improve, and on the other hand the NAs can be used as feedstock.
1.3 Formation
of
naphthenic
acid
dimers
Naphthenic acids are both hydrogen-bond acceptors because of the –C=O carbonyl group and
hydrogen bond donors because of the –OH hydroxyl group; therefore they can participate in
dimer formation by hydrogen bonding. Smith and his colleagues29
used Fourier
transform
ion
cyclotron
resonance
mass
spectrometry
(FT-­‐ICR
MS) to show that naphthenic acids
self-associate in the gas-phase at high enough concentration (1 to 10 mg/mL), forming
naphthenic acids dimers (general formula CnH2n+zO4). Moreover, they were able to
demonstrate that the higher the concentration of naphthenic acids, the higher their tendency to
form aggregates.
Although dimer formation is an accepted phenomenon in the world of petroleomics, there
have not been many studies focused on this topic. Da Campo et al.30
have taken a further look
at CnH2n+zO4 aggregates. Fourier transform infrared spectroscopy of these dimers revealed
that they are also present in the solution-phase. Using an FT-ICR MS instrument, they
demonstrated that these compounds are bound by noncovalent bonds and they reported a
possible general structure as the one shown in Figure 4, where a proton is shared between two
oxygen atoms. The formation of these species is dependent on the accumulation time of the
ions in the hexapole of the ion source. It was noticed that by increasing the accumulation
time, and thus increasing the number of collisions taking place, there was a decrease in the
signal intensity of these species, which was related to the weak noncovalent bonds that hold
together the dimers.

10. Introduction
6
Figure 4. Possible structure for the singly charged noncovalent naphthenic acids dimers30
Mapolelo et al31
studied calcium naphthenate deposits and sodium naphthenate emulsion
sample using FT-ICR MS, only after preparation in the laboratory to first remove the metals
from the naphthenic acids and thus creating free acids.
Naphthenic acids dimers will affect the behaviour of the naphthenic acids in the solution-
phase and so the problems associated with them. In this paper, a better understanding of
naphthenic acid dimers was pursued through the use of ultrahigh resolving power mass
spectrometry.
1.4 Analytical
techniques
for
looking
at
crude
oils
Petroleomics is referred to as the detailed study for elucidating the chemical composition of
the components naturally occurring in petroleum and crude oil samples. High resolving power
mass spectrometry, especially FT-ICR MS is nowadays the central petroleomics-grade
technique.
How effective the oil recovery methods are depends on the composition of the oil of interest.
Analysis of the composition of crude oil can be highly complex and separation of the major
crude oil components can facilitate their characterization. Saturate, aromatic, resin and
asphaltene (SARA) analysis is a separation technique that has been widely used in
petroleomics. It involves the separation of crude oil components based on their solubility and
polarity into four main classes: saturate, aromatic, resin and asphaltene fractions. The first
step is the precipitation of asphaltenes in n-alkane solvents, followed by liquid

11. Introduction
7
chromatography using silica or alumina columns to separate the three remaining fractions by
their polarity.32
Elucidating the chemical structure of heavy components in crude oil can allow a better
understanding and prediction of the petroleum behaviour during processing. Because the
organic composition of heavy crude oil is so complex, its characterization has been in the past
limited to bulk properties (such as viscosity, density, electric conductivity, light scattering,
UV-visible and infrared spectroscopy, 13
C nuclear magnetic resonance NMR, X-ray
diffraction)33
and various chemical separation methods based on solubility (like SARA
analysis), boiling point, gas and liquid chromatography.34
NMR and X-ray diffraction have
been employed in determining the molecular structure of crude oil compounds, but both
techniques are limited in the sense that they can only provide an average molecular structure
of components. 35
At present, Fourier transform ion cyclotron resonance mass spectrometry is an indispensable
method of choice for the analysis of complex samples such as petroleum. FT-ICR MS
dominates the petroleomics field because of its high mass accuracy and ultrahigh resolving
power, making it possible to calculate the elemental composition of the compounds with great
accuracy.
1.5 Fourier
transform
ion
cyclotron
resonance
mass
spectrometry
All types of mass spectrometer allow compounds to be identified by the production of gas-
phase ions from a neutral sample and their subsequent sorting and detection based on their
mass-to-charge ratio (m/z). The sample can be solid, liquid or gas, depending upon the type of
ion source used. The ions are transferred into the mass analyser (ICR cell) and the differences
in masses of the fragments enable the mass analyser to separate the ions according to their
mass to charge (m/z) ratio.36
FT-ICR MS instruments determine the m/z of an ion by measuring the cyclotron frequency of
the ion trapped in a fixed magnetic field. The magnetic field is usually generated using a
superconducting magnet. The equation that relates frequency to the m/z of the ions is the
following:

12. Introduction
8
𝑓 =
!"
!!"
(eq. 1),
where f is the cyclotron frequency (Hz), m is the mass of the ion (kg), q is the charge on the
charged particle (coulombs, C) and B is the magnetic field (tesla, T).37
Due to the inherently ultrahigh resolving power (for example 400 000 (full width at half
maximum) at m/z 400) and accurate mass measurement, Fourier transform ion cyclotron
resonance mass spectrometry has enabled analysts to study heavy crude oil at a molecular
level.38
FT-ICR MS has been playing a key role for petroleomics ever since its first
application to petroleum distillates39
as it has allowed a better understanding of the structure
of the components that make up this highly complex mixture and thus an insight into their
behaviour.
Most of the characterization of naphthenic acids has been performed using different mass
spectrometric (MS) techniques, including gas chromatography mass spectrometry (GC-MS)40
,
two-dimensional GC-MS41
and liquid chromatography (LC) MS.27
Also, different ionization
approaches such as electron ionization (EI)42
, fast atom bombardment (FAB)43
, atmospheric
pressure chemical ionization (APCI)44
, and lately electrospray ionization (ESI)17
have been
successfully used for the study of naphthenic acids.
ESI is a soft ionization technique that has gained great popularity in the past few years and it
has been shown that is it able to selectively ionize acidic and basic components in petroleum
samples.45
The analyte dissolved in a suited solvent is sprayed from a small capillary tube into
a strong electric field in the presence of a drying gas (typically nitrogen) and thus, generating
a fine “mist” of highly charged droplets as illustrated in Figure 5.46
Under negative-ion ESI
acids are deprotonated generating negative ions and under positive mode ESI (basic) neutral
are protonated forming positive ions.34
Although crude oil contains less than 10% heteroatom-
containing compounds, they represent the main concern for the petroleum industry. Because
of their high polarity, ESI is specific and especially efficient in generating their gas-phase
ions. Moreover, negative-ion ESI is a “soft” ionization techniques that generates [M − H]-
without extensive ion fragmentation or matrix interference.

13. Introduction
9
Figure 5. Schematic representation of ion formation by electrospray ionization (ESI)
Tandem mass spectrometry (MS/MS) techniques allow insight on structure of naphthenic
acids by offering further information about specific ions. The ions of interest from the ion
source are selected and then subjected to fragmentation through different dissociation
techniques such as collision induced dissociation, infrared multiphoton dissociation and
electron induced dissociation.
Collision-induced dissociation (CID), also known as collisionally activated dissociation
(CAD), is by far the most common MS/MS technique and it involves the acceleration of the
ions by electrical potential to high kinetic energy followed by the collision of the ions with
neutral gas molecules, resulting in further dissociation of the ions.
Central to infrared multiphoton dissociation (IRMPD), is the absorption of multiple infrared
photons, leading to a continuous build-up of internal energy by the ions. When the internal
energy overcomes the barrier against dissociation, fragmentation of the ions occurs.47
The
irradiation of ions is typically carried out using a pulsed CO2 laser.
Electron induced dissociation (EID) involves the fragmentation of singly-charged ions
succeeding their interaction with high-energy electrons (>10 eV).48
EID has been used in the
past for the analysis of peptide ions, metabolites and sodium clusters cations49
but as to date,
there are no publications testing the viability of EID for petroleum samples. EID would allow

14. Introduction
10
going beyond the analysis of petroleum using elemental composition alone by obtaining
greater structural insight.
1.6 High
mass
accuracy
In mass spectrometry, mass accuracy is defined as the ratio of the m/z measurement error to
the true m/z, generally expressed in parts per million (ppm) and mass resolving power
represents the ability to distinguish between two peaks varying slightly in m/z.50
Each isotope of each chemical element has a different mass defect and they are known to a
great accuracy. By using the power of the inherent high mass accuracy and ultrahigh
resolving power of Fourier transform mass spectrometry, it is possible to compare petroleum
samples on a molecular level with high accuracy. The importance of ultrahigh resolving
power can be illustrated in the case of CH4 and O as they both have a nominal mass of 16, but
their exact mass is 16.0313 Da and 15.9949 Da respectively, resulting in a mass difference of
36.4 mDa. Similarly SH4 and C3 have a nominal mass of 36, but their exact masses (36.0034
Da and 36.0000 Da, respectively) differ by 3.4m Da.4
But even with a high mass accuracy and knowledge of the ion’s charge state, the chemical
complexity of naphthenic acids increases greatly with molecular weight and the assignment of
the elemental composition can be unambiguously done only to about 500 Da.51
Nevertheless,
by using Kendrick mass defect plot the mass range can be extended up to three times.52
1.7 Kendrick
mass
defects:
the
key
to
unlocking
chemical
formulas
Kendrick mass defect (KMD) plots have been successfully applied for the study of petroleum
samples using ultrahigh resolving power mass spectrometry as they have allowed data
interpretation and visualization of complex data sets. KMD plots are particularly useful for
indicating the characteristics and behaviour of the species of interest.53
Kendrick mass defect calculation transforms the IUPAC mass scale (12
C=12.000 00 Da) to
the Kendrick mass scale (CH2=14.00000 Da instead of 14.01565 Da) using the following
equation:

15. Introduction
11
Kendrick Mass=IUPAC Mass×
!"
!".!"#$#
(eq. 2).
Then the Kendrick mass defect is defined by:
Kendrick Mass Defect (KMD) = Nominal Kendrick Mass-Kendrick Mass (eq. 3),
where Kendrick mass is calculated as shown above and nominal Kendrick Mass is the
Kendrick mass rounded to the nearest integer.
For samples containing a high number of hydrocarbons, such as petroleum, KMD plots
simplify the display by showing peak patterns lined up in the horizontal direction. The
effectiveness of KMD plots relies on the fact that members of a homologous series from
crude oils, i.e. compounds that have the same constitution of heteroatoms and number of rings
plus double bonds but different alkyl chain length, will have an identical Kendrick mass
defect. In a KMD plot, a homologous series will appear on a horizontal row, making it easy to
distinguish from species of other class and type. Another great advantage is that “outlier” data
can be immediately determined if they fall outside the main patterns54
.
Furthermore, unambiguous assignment of a series of related compounds at low m/z serves to
determine all other higher mass members of that species. As a result, KMD scaling extends
elemental composition assignment to masses up to three times higher than would be possible
using mass-measurement accuracy only.
For illustrative purposes, a KMD plot of naphthenic acids is shown in Figure 6. The primary
trend horizontally is a constant difference in Kendrick nominal mass of 14.01565 (indicating
an increase with a CH2 group) and vertically there is a constant Kendrick mass defect
difference of 0.013455
(indicating a difference of two hydrogen atoms and thus an increase in
the degree of unsaturation).

16. Introduction
12
Figure 6. Kendrick Mass Defect plot of monomeric naphthenic acids
1.8 Double-­‐bond
equivalent
versus
carbon
number
plot
The first step to evaluate the structure of a compound is to determine its elemental
composition. The exact mass measured with MS can be used to calculate the elemental
formulae (CcHhNnOoSs). Once the elemental composition is known, double-bond equivalent
(DBE), also known as degree of unsaturation, plays a key role in predicting the chemical
structure of the compounds present in crude oil samples. DBE represents the number of
double bonds and rings in a given molecule. DBE and Z values are different ways of thinking
about the same thing, i.e. the hydrogen deficiency (rings and double bonds and degree of
unsaturation).
The DBE values calculated from the elemental composition can be plotted against carbon
number, resulting in a “DBE plot”. Species of the same DBE, i.e. from the same homologous
series, will be plotted on a horizontal line. The DBE plots may be rendered more informative

17. Introduction
13
by colour-coding each m/z ion depending on its measured relative abundance. Herein, the
gradient goes from red for the least abundant ions to yellow for the most abundant ions.
Additionally, the size of each plot dot can differ with the abundance of that ion. Such a plot of
naphthenic acids is shown in Figure 7.
Figure 7. Example of DBE plot for O2 class species, where the most abundant species have a
DBE value of 3.5
1.9 Main
aims
The aim of the following study is to use ultrahigh resolving power FT-ICR MS and tandem
MS techniques for characterization of petroleum components.
The initial focus is on aggregation of naphthenic acids in the solution phase: understanding
their composition and whether these aggregates form selectively or depending on the
available monomers. Then electron-induced ionization will be applied for the first time to
petroleum in order to obtain preliminary data for proof-of-concept of a new approach for
structural insights into petroleum. The interaction of singly-charged ions with high-energy
electrons (>10 eV) is expected to result in mass spectra with more extended ion fragmentation
than slow-heating dissociation techniques such as CID or IRMPD.48

18. Experimental
14
2 Experimental
2.1 Sample
preparation
A commercial Kodak naphthenic acid mixture (The Eastman Kodak Company, Rochester,
NY) was used for all the analysis carried out for the characterization of naphthenic acids
using negative-ion ESI.
The water used in the experiments was purified using a Direct-Q 3 Ultrapure Water System
from Millipore (Billerica, MA, USA). An already prepared two years old sample containing 4
mg of Kodak NA mixture dissolved in 5 mL of acetonitrile (WVR, Leuven, Belgium) and 5
mL of Milli-Q water was now doped with 50µL Agilent #1969 ESI-L Low Concentration HP
calibrating mix (Agilent, Palo Alto, CA). The concentration of the sample is 0.4 mg/mL and
is expressed in mass/volume instead of molarity as naphthenic acid is a mixture. This sample
was then subjected to in-source dissociation (ISD) at 0 and 60 V and to CID fragmentation at
2, 6, 8 and 12 V. ISD is a variation of CID, where an increase in the difference in potential
between the two ion funnels gives ions higher kinetic energy at an early stage of the
instrument. ISD is a non-selective technique useful for dissociation of noncovalent species.
To compare the results obtained from the 2 year old Kodak NA sample, a fresh solution of
Kodak NA sample was prepared. This fresh solution containing 19.9 mg of Kodak NA
mixture was dissolved in 5 mL of acetonitrile and 5 mL of Milli-Q water (~2 mg/mL). Using
this stock solution a serial dilution with the following concentrations: 0.5, 0.1, 0.05, 0.001,
0.005 and 0.001 mg/mL was created and subjected to ISD (0 V) experiments. In the previous
CID experiments, the quadrupole was used to isolate the m/z 503.4 ion (with a 1 Da wide
isolation window). The ions in the isolation window pass though the collision cell, where CID
experiments were performed and then the ions were passed to the ICR cell. To improve the
quality of the isolation and the resolving power of the resulting spectra, the quadrupole was
used to isolate the species of interest then a correlated sweep (in the cell) plus correlated shots
(1% clean up shots power, pulse 0.15846 sec) in the cell was used. The fragmentation was
carried out using IRMPD rather than SORI-CID as the later will raise the pressure in the cell
and decrease performance.

19. Experimental
15
Doping of a fresh Kodak NA sample with sodium chloride and analysing the effects salt has
on NAs was the next step. A fresh 0.05 mg/mL stock solution Kodak NA mixture was
prepared in 10 mL of acetonitrile and 10 mL of Milli-Q water. A 600µM stock solution of
sodium chloride was prepared in 20 mL of Milli-Q water (0.7 mg NaCl). Three samples
containing the same naphthenic acid mass concentration (0.05 mg/mL) and varying salt
concentration values were prepared. The concentration of salt in the three samples was as
follows: 0.6 µM, 54.55 µM and 300 µM, respectively. The blank stock solution of Kodak NA
sample, as well as the three samples of Kodak NA doped with salt was subjected to MS
analysis. A 0.05 mg/mL solution of Kodak NA was also doped with significant higher
amounts of NaCl to mirror the seawater salinity, i.e. approximately 600 mM, but it was not
run thought the FT-ICR MS.
A light-sour crude oil sample, which is a standard reference material from the National
Institute of Standards and Technology (NIST, Gaithersburg, Maryland, U.S.A.) was used to
obtain a positive-ion ESI spectrum. Then isolation was performed using a window with the
width of 60 Da so that the isolation window spans ions of two higher/lower carbon numbers
each side of the central ion. EID experiments were carried out and a single EID spectrum was
obtained. Because of the difficulty faced by tuning with low signal, the spectrum collected
was the average of 500 scans and it took about an hour to acquire.
2.2 Fourier
Transform-­‐Infrared
Spectroscopy
(FT-­‐IR)
FT-IR analysis was carried out on a JASCO FT/IR-470 plus. The Kodak NA mixture was
analysed directly, with no prior dilution in solvents. The baseline correction was produced in
a nitrogen flow atmosphere, followed by the acquisition of the sample FT-IR spectrum as an
average of 200 scans.
2.3 Mass
Spectrometry
Analysis
All mass spectrometry experiments were carried out on a Bruker solariX Fourier transform
ion cyclotron resonance mass spectrometer fitted with an Apollo II ion source and a 12 tesla
superconducting, refrigerated and ultrashield magnet (Bruker Daltonik GmbH, Bremen). An
instrument schematic of the instrument used is shown Figure 8.

20. Experimental
16
Figure 8. Schematic of 12 T Bruker Solarix FT-ICR MS (courtesy of Bruker Daltonik,
GmbH, Bremen)
For the Kodak NA samples, the instrument was run in negative-ion conditions as negative
ESI-MS favours the ionization and detection of acidic compounds such as naphthenic acids.
These acidic sites give up a proton and thus creating negatively charged molecules. For the
crude oil sample, the instrument was run in positive-ion conditions due to the high abundance
of basic nitrogen species, which can be readily protonated and thus, providing a strong signal
for MS/MS experiments.
The conditions of ESI were the following: syringe flow of 120-300µL/hour, drying gas
(nitrogen) temperature of 220° C and a flow rate of 4L/min. The nebulizer gas (also nitrogen)
was kept at a pressure of 1.2 bar. The ion accumulation time (IAT) was tuned while
monitoring the transient lifetime. For all the CID experiments the IAT was set to 0.5 sec, for
IRMPD experiments it was 2 sec and for the ISD experiments of the old and fresh samples it
was 0.001s. A set of ISD experiments where the IAT varied between 0.1 and 0.4 was carried
out in order to obtain an understanding how the spectrum is affected by accumulating ions for
longer period of times.
The mass range was set between m/z 147.41 and 3000. Mass spectra consisted of 4M Word
data points the signal-to-noise ratio was enhanced by summing 100 time domain transients.
Transient length was approximately 1.67 s with a resolving power (m/Δm50%) of about 400

21. Experimental
17
000 at m/z 400 (Δm50% is the mass spectral full peak width at half height). After acquisition,
data were zero-filled56
once and sine-bell apodization57
was applied.
2.4 Calibration
Mass lists were obtained using the processing software Data Analysis 4.2 (Bruker Daltonik
GmbH, Bremen) where the S/N threshold was set to be higher than 4. Calibration was carried
out externally for the old Kodak NA sample doped with Agilent #G1969 Low Concentration
HP calibrating mix. In this way, accurate m/z values were obtained over a wide mass range
that bracketed the monomer and dimer region of interest and thus improving the mass
accuracy and confidence in assignments.
For subsequent spectra, the calibrating material was not used in order to eliminate possible
peak contributions. The calibration was carried out internally using the O2, O4 and NaO4
homologous peaks (the list of known mass ion used for calibration is provided in the
Appendix).
2.5 Data
analysis
Spectral interpretation was carried out using the Data Analysis 4.2 software. For the
calculation of the elemental formulas, the usual CcHhNnOoSs formula for petroleum was
considered, where c and h are unlimited, n and o are between 0 and 5 and s is between 0 and
4. Up to two 13
C atoms were also included in the calculation because of the high content of
carbon, along with 1 atom of Na. Other constraints included even electron species, maximum
H/C ratio of 3 and a minimum of -0.5 and a maximum of 40 rings plus double bonds.
Several approaches for data analysis and visualization of the large amount of data sets
acquired were used in this paper, including Kendrick mass defect plots, double bond
equivalent plots, bar charts of contributions from different type of species and distribution of
the intensity of various Z series versus carbon number. Data were processed with Excel 14
(Microsoft Corporation, Redmond, Washington, U.S.A.), Origin 9.1 (OriginLab,
Northampton, MA, USA) and GraphPad Prism 6 (GraphPad Software, LA Jolla California,
USA).

22. Experimental
18
The mass spectra gathered varied from 200 to 300 entries in the peak list for the CID and
IRMPD experiments and between 1000-3000 data points for the ISD experiments. The m/z,
intensity and resolving power values were the parameters imported from Data Analysis
software into Microsoft Excel.
Using eq. 2 and eq. 3, a method was created for the calculation of the Kendrick mass defects.
An example of the calculation is shown next.
For an ion with the composition C15H25O2
-
(the negatively singly charged ion of the C15H26O2
naphthenic acid with Z=-4) the IUPAC mass is 237.186004, but using the Kendrick mass
scale, the ion has a Kendrick mass of 237.186004*14/14.01565=236.921160. The Nominal
Kendrick mass is 237 and the Kendrick mass defect is 237-236.92116=0.07884.
Each data entry was manually assigned on the basis of Kendrick mass defects and of m/z
values within an error range typically of 1 ppm or less. Then each formula was used to
calculate the Z and DBE values, followed by various visualization methods. For all mass
spectra, mass error distribution histograms of the main class species present were created and
they can be found in the Appendix. The low mass errors obtained using FT-ICR MS allow
more confident assignments than by using other type of mass analyser.
Double bond equivalent was calculated using the following equation58
:
DBE= 𝑐 −
!
!
+
!
!
+ 1, for the elemental formulae CcHhNnOoSsNa1.
To ensure persistence in data analysis, all DBE values were calculated for the molecular ions.
So, for example, for the molecular ion C15H29O2
-
, DBE=15-29/2+1=1.5 (corresponding to a
saturated naphthenic acid compound, Z=0). Equivalently, all deprotonated ions of monomeric
naphthenic acids from the ESI experiments will have an odd DBE value.
For convenience purposes, assignments were categorized in terms of their carbon number,
number of double bonds and rings, and their constituent heteroatoms (O, N, S, Na). The
heteroatom(s) determines the compound class; for example, O or O1 denotes a composition of
CcHhO1 and O2 denotes a composition of CcHhNnO2.

23. Results
and
discussion
19
3 Results
and
discussion
3.1 Proof
of
naphthenic
acid
dimers
existence
in
solution-­‐phase
Carboxylic acids are well known to have a characteristic twin-peak infrared absorption band,
with a small peak between 1740 and 1750 cm-1
(monomer form) and a strong peak between
1700 and 1715 cm-1
(dimer form). The pure, undiluted in organic solvents, Kodak naphthenic
acid mixture was analysed using a Fourier Transform Infrared Spectroscopy. Its FT-IR
spectrum is shown in Figure 9 and it displays this pattern with an apex at 1703, associated
with the presence of NA dimers and a smaller apex at 1742 cm-1
, associated with the
monomeric NA structures. This readily proves that the components from the Kodak mixture
associate to form dimers in the solution-phase at a sufficiently high concentration.
Figure 9. Expanded region of the FT-IR spectrum of the Kodak NA mixture showing the
absorption band characteristic to the C=O stretching in the carbonyl group. The maxima at
1703 cm-1
corresponds to dimeric species and the maxima at 1742 to the monomeric species.
Figure based on data provided from previous study20
using the same Kodak NA mixture as in
this work
160016501700175018001850
0.0
0.1
0.2
0.3
0.4
0.5
Absorption(a.u.)
Wavenumber (cm-1
)
1742
(monomer)
1703
(dimer)
C=O stretch
(COOH)

24. Results
and
discussion
20
3.2 2
year
old
Kodak
naphthenic
acid
sample
3.2.1 ISD
experiment
0
V
During an ISD experiment (0 V), using the 2 year old Kodak naphthenic acid sample doped
with Agilent #G1969 ESI-L Low Concentration HP calibrating mix, it was observed that
besides the monomer distribution in the m/z 170-390 region (attributed to the naphthenic acid
species, i.e. O2 class species), there are three other distinct distributions present in the m/z
365-675, 670-920 and 950-1200 m/z region along with clusters in the m/z 1080-1230, 1370-
1520 and 1700-1850 regions (Figure 10).
Figure 10. Mass spectrum of a 2 year old Kodak NA sample showing several distributions
(negative-ion ESI, ISD 0 V). The peaks marked with a star originate from the Agilent #G1969
ESI-L Low Concentration HP calibrating mix
For convenience purposes, the four distributions present were named using roman numerals
as shown in Table 1.

25. Results
and
discussion
21
Table 1. Assigned names for the four distributions in the mass spectrum of a 2 year old Kodak
NA sample (negative-ion ESI, ISD 0 V)
m/z
region
Distribution
name
170-­‐390
I
365-­‐675
II
670-­‐920
III
950-­‐1200
IV
A very effective visual method to illustrate features and behaviour of the species of interest is
the use of Kendrick plots. The m/z for all the peaks in the spectrum were rescaled to the
Kendrick Mass scale and the nominal Kendrick Mass was plotted against KMD to reveal the
four distributions present (Figure 11). Even though two-dimensional KMD plots do not
typically show the relative intensity of the compounds, they are very useful in illustrating the
degree of unsaturation. It can be clearly noticed that distribution II has the highest Kendrick
mass defect variation and thus its components extend over a wider range of degrees of
unsaturation when compared to the other distribution present such as distribution I, i.e. the O2
class species. Also an overlap of species can be observed in distribution II.
Figure 11. Kendrick Mass plot of the four distributions present in the 2 year old Kodak NA
sample (negative-ion ESI, ISD 0V)

26. Results
and
discussion
22
Carrying out data analysis, the majority of the peaks in distribution II were assigned to O4 and
NaO4 class species. Distribution III and IV have tentative assignments of NaOS and NaO3,
respectively. Within acceptable mass errors, the compositions were not naphthenic acid
multimers, and so further work would be necessary to comprehensively characterize these
distributions.
The 2 year old Kodak NA sample exhibits mainly oxygen-containing species such as: O2,
O3S, O4, NaO4, O4S and O5S. The OxS species are believed to be contaminants from sulfur-
containing compounds, such as sodium dodecyl sulfate, a common surfactant in many
cleaning and hygiene products with the general formula CH3(CH2)11OSO3Na.
The inherent ultrahigh resolving power and mass accuracy of the FT-ICR MS used in this
study for determining the elemental composition of naphthenic acids is readily evident, even
when operating in broadband (m/z 147.41 and 3000) as shown in Figure 12, which represents
a 20 Da and a 16 mDa mass scale-expended segment. An increase in the mass spectra
resolving power can be achieved by lengthening the transient acquisition (tacq). Longer tacq are
obtained by increasing the number of data points collected and/or by increasing the lowest
m/z value.
The m/z 468-501 zoom in spectrum, reveals single charged molecules, separated by
approximately 14 Da for members of the same homologous series (m/z=473.36, m/z=487.38
and m/z=501.4) and by approximately 2 Da for compounds that have a DBE difference of 1
(m/z=473.36 and m/z=475.38). The 16 mDa zoom-in region (m/z
469.324-­‐469.340
window) reveals a resolved doublet of molecules: a NaO4 class species on the left hand side
and an O4 class species on the right side. The
two
peaks
are
being
separated
by
about
2.4
mDa,
i.e.
1
mDa
less
than
between
SH4 and C3. This pair of peaks repeats throughout the
dimer distribution, pointing out the importance of ultrahigh resolving power and mass
accuracy.

27. Results
and
discussion
23
Figure 12. Mass scale-expanded segments (20 Da and 16 mDa wide) of negative ESI FT-ICR
mass spectra of the 2 year old Kodak NA mixture. The bottom spectrum is a zoom in on a
NaO4/O4 pair.

28. Results
and
discussion
24
The abundance values of the species present in distribution I and II have been calculated as
the sum of the intensities of all species from the same class (Table 2). O4 class dominates
distribution II, fitting with previous work reported by Da Campo30
. At a smaller contribution,
it is shown for the first time the formation of sodium bound naphthenic acid dimers.
Table 2. Summed absolute intensity and normalized relative intensity of the species present in
the monomer and dimer distributions for the old 0.4 mg/mL Kodak NA sample (ISD 0 V)
Species Intensity Relative intensity (%)
Monomer O2 1701126842 23.60
O3S 7415251 0.10
Dimer O4 4783158046 66.34
NaO4 612119306 8.49
O4S 11957646 0.17
O5S 93864521 1.30
The relative intensity of the compounds present in the monomer and dimer distributions are
illustrated as graph in Figure 13. O4 class species represent approximately 66% of the species
present in the dimer distribution and NaO4 represent approximately 8%, respectively.
Figure 13. Relative intensity of the species observed in the monomer and dimer distribution in
the 2 year old Kodak naphthenic acid mixture (negative-ion ESI, ISD 0 V)
0
10
20
30
40
50
60
70
O2
O3S
O4
NaO4
O4S
O5S
Relative
Intensity
(%)
Species
present
Old
Kodak
naphthenic
acid
sample
composition
(ISD
0
V)
monomer
dimer

29. Results
and
discussion
25
3.2.2 ISD
experiment
(60
V)
By increasing the in source fragmentation energy to 60 V, most of the dimeric species
dissociate back to monomeric species, and thus a decrease in intensity of the dimer
distribution was noticed while the intensity of the monomer increases as shown in Figure 14.
Figure 14. Mass spectrum of 2 year old Kodak naphthenic acid sample showing several
distributions (negative-ion ESI, ISD 60 V)
The main event noticed when increasing the fragmentation power to 60 V was that no O4, O4S
and O5S class species were present anymore and the dimer distribution consisted of only
NaO4 class species. The summed absolute and relative intensities of the species present in the
old Kodak NA sample are gathered in Table 3.

30. Results
and
discussion
26
Table 3. Summed absolute intensity and the normalized relative intensity of the species
present in the monomer and dimer distributions for the old Kodak NA sample (ISD 60 V)
Species Intensity Relative intensity (%)
Monomer O2 4786823693 80.36
O2S 5123729 0.09
O3S 24629987 0.41
Dimer NaO4 1139835910 19.14
The relative intensity of the compounds present in the monomer and dimer distributions are
illustrated as bar chart graph in Figure 15.
Figure 15. Relative intensity of the species observed in the monomer and dimer distribution in
the 2 year old Kodak naphthenic acid mixture (negative-ion ESI, ISD 60 V)
For a visual comparison, the content of O2, O4 and NaO4 class species in the Kodak NA
sample at the two different ISD experiments (0 and 60 V) is illustrated as stacked bar chart in
Figure 16. If at 0 V, O4 naphthenic acid dimers predominate the spectrum, at 60 V there are no
O4 species present, while the monomeric naphthenic acids dominate the spectrum. The
explanation behind it is that at higher voltages, O4 dimers dissociate back to the monomeric
form of NAs. The NaO4 dissociate less easily than the O4 dimers as they are tightly bound due
to the sodium atom. This result, determining the role of sodium, will later lead on to be
looked at using a fresh Kodak NA sample.
0
10
20
30
40
50
60
70
80
90
O2
O2S
O3S
NaO4
Relative
Intensity
(%)
Species
present
Old
Kodak
NA
sample
composition
(ISD
60
V)
dimer
monomer

31. Results
and
discussion
27
Figure 16. The intensity of main class species present in the monomer and dimer region for
the 2 year old Kodak NA mixture during ISD experiments at 0 and 60 V. At 60 V ISD, there
are no O4 dimers present
The DBE plots of the three main components from the ISD 0 V and ISD 60 V spectra of the 2
year old Kodak NA sample are shown in Figure 17. The DBE plots at ISD 0 V and at ISD 60
V are very similar for O2 and NaO4 class species, respectively. The carbon number varies
between 11 and 26 for O2 class compounds, between 22 and 42 for O4 class compounds and
between 24 and 41 for NaO4 class compounds. The DBE values observed in negative-ion ESI
are half integers due to the change in electron availability attributed to loss of a proton. The
saturate species (Z=0) will have a DBE of 1.5 due to the carboxylic acid group. All higher
DBE values are associated with rings (or double bonds). The DBE ranges from 1.5 to 8.5 for
O2 species, from 2.5 to 12.5 for O4 species and from 4 to 12 for NaO4 class species. The most
abundant species have a DBE value of 3.5 for O2 class, of 5.5 for O4 class and of 6 for NaO4
class.
0.E+00
1.E+09
2.E+09
3.E+09
4.E+09
5.E+09
6.E+09
7.E+09
8.E+09
0V
60V
Contributions
of
the
O2,O4
and
NaO4
class
species
(ISD
experiments)
NaO4
O4
O2

32. Results
and
discussion
28
Figure
17.
DBE
plots
of
O2,
O4
and
NaO4
species
(from
top
to
bottom)
at
0
V
(right
side)
and
60
V
(left
side)
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14 Old Kodak NA sample (ISD 0 V)
O2 class DBE plotDBE
Carbon number
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14 Old Kodak NA sample (ISD 60 V)
O2 class DBE plot
DBE
Carbon number
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
2
4
6
8
10
12
14
Old Kodak NA sample (ISD 0 V)
O4 class DBE plot
DBE
Carbon number
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
2
4
6
8
10
12
14
Old Kodak NA sample (ISD 0 V)
NaO4 class DBE plot
DBE
Carbon number
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
2
4
6
8
10
12
14
Old Kodak NA sample (ISD 60 V)
NaO4 class DBE plot
DBE
Carbon number

33. Results
and
discussion
29
3.3 Fresh
sample
The existence of sodium naphthenates in the two year old Kodak NA sample was believed to
be caused by the presence of sodium from glassware. In order to investigate this hypothesis a
serial dilution of fresh Kodak NA was prepared and analysed using ISD at 0 V to mirror
previous experimental conditions.
3.3.1 ISD
(0
V)
of
serial
dilution
The data analysis revealed that the main compounds in the dimer region of the fresh Kodak
NA sample are mainly O4 class species with a very small contribution from NaO4 class
species. Because the interaction timescale is minutes rather than years, there is less time to
interact with sodium from glassware and so much less of the NaO4 class species are observed
when compared with the 2 year old Kodak NA sample.
The work carried by Smith et al29
using negative ESI FT-ICR MS on a 375-400° C distillation
cut from Athabasca bitumen showed that high sample concentration must be used in order to
observe multimers aggregation. At 1 mg/mL, multimers formation was evident and as
concentration increased to 5 mg/mL and 10 mg/mL the relative abundance of multimers was
higher than that of monomers. In this paper, the dimer formation is shown to occur at lower
concentration of Kodak NA sample (0.001 to 0.5 mg/mL).
It was noticed that by increasing the concentration of the naphthenic acid sample, there was a
large increase in the relative abundance of the O4 class species, whereas the O2 monomers
decrease significantly (highlighted data in Table 4). Increasing the concentration from 0.001
mg/mL to 0.5 mg/mL, the abundance of monomeric naphthenic acids drops from ~82% to
~4.5%, while the abundance of O4 dimers increases from ~10% to ~82%.

34. Results
and
discussion
30
Table 4. Relative abundance of the species present in the fresh Kodak NA sample as a factor
of concentration (ISD 0 V).
Comparing the abundance of NaO4 class species from the old fresh Kodak NA sample with
that from the fresh sample, a decrease in the latter was noticed. Where the relative abundance
of NaO4 class species in the 2 year old 0.4 mg/mL Kodak NA sample was ~8.5% (Table 2), in
the fresh 0.5 mg/mL Kodak NA sample it is ~2.8%.
The relative abundance of naphthenic acid monomers and dimers as a factor of concentration
(on a logarithmical scale) is shown in Figure 18. The crossover between the O2 and O4 class
species occurs between 0.005 mg/mL to 0.01 mg/mL.
Figure 18. The relative abundance of O2, O4 and NaO4 class species present in the fresh
Kodak NA sample depending on the sample concentration (ISD, 0 V)
Species/Concentration
(mg/mL)
0.001 0.005 0.01 0.05 0.1 0.5
Monomer O2 81.84734 48.54421 41.65119 17.33382 9.2603 4.42647
O3 0 0.06375 0.10108 0.04236 0 0.14678
O4 1.61367 1.2567 1.17202 0.34764 0.03462 0.02243
O2S 1.55263 1.70097 1.46321 0.84632 0.24235 0.40252
O3S 4.86567 0.1608 0.1229 0.01219 0.1459 0
Dimer NaO4 0 0.44595 0.68072 0.95765 1.90841 2.8396
O4 9.74035 45.27648 52.36459 74.5034 84.42081 82.30006
O5 0 0 0.1012 0.4483 0.16802 0.76221
O4S 0.30642 1.99654 2.34308 5.46864 3.81959 8.82549
O5S 0.07392 0 0 0.03969 0 0.27444

35. Results
and
discussion
31
Plotting out the data from Table 4 into a bar chart (Figure 19) can provide useful overview on
how concentration affects the overall distribution trend in the naphthenic acid sample. By
increasing concentration, naphthenic acid monomers decrease in abundance, whereas
noncovalent O4 dimers and sodium bound naphthenic acid dimers increase in abundance.
Figure 19. The relative abundance of the species present in the fresh Kodak NA sample at
various concentrations (ISD, 0 V)
Further information can be provided by using DBE plot of different compound classes at
various concentrations. DBE plots of the O2 and O4 species at 0.001mg/mL, 0.01mg/mL and
0.1 mg/mL dilution samples are illustrated in Figure 20. Although DBE plots are usually
created for a single class, for this set of data it was decided to include the O2 and O4 classes in
the same DBE plot. The resulting DBE plots help visualise how an increase in concentration
by an order of magnitude changes a shift from high content of monomeric species to high
content of dimeric aggregates. In the 0.001mg/mL sample, the naphthenic acids with a DBE
number of 3.5 are predominant, while in the 0.1mg/mL sample, the naphthenic acid dimers
with a DBE of 5.5 are most abundant.

36. Results
and
discussion
32
Figure 20. DBE plots of O2 and O4 species from spectra of a fresh Kodak NA sample at
various concentrations

37. Results
and
discussion
33
The effect concentration has on the ratio of NaO4/O4 species from the fresh Kodak NA sample
was explored next (Table 5). With increasing concentration the NaO4/O4 ratio increases.
Table 5. The absolute abundance of NaO4 and O4 class species and their ratio in the fresh
Kodak NA sample at various concentrations (ISD, 0 V)
Class/
Concentration
(mg/mL)
0.001
0.005
0.01
0.05
0.1
0.5
NaO4
0
1.77E+07
3.31E+07
9.71E+07
6.58E+08
8.07E+08
O4
1.79E+08
1.79E+09
2.55E+09
7.56E+09
2.91E+10
2.34E+10
NaO4/O4
0
0.010
0.013
0.013
0.023
0.035
By plotting the absolute abundance of the NaO4 and O4 class species from Table 5 into a bar
chart (Figure 21), the change in overall intensity is displayed.
Figure 21. The absolute abundance of the O4 and NaO4 compounds in a fresh Kodak NA
sample at different concentrations. The lowest concentration analysed, i.e. 0.001mg/mL
contains no NaO4 class species
0.0E+00
5.0E+09
1.0E+10
1.5E+10
2.0E+10
2.5E+10
3.0E+10
3.5E+10
0.001
0.005
0.01
0.05
0.1
0.5
Absolute
intensity
Concentration
(mg/mL)
O4
and
NaO4
absolute
intensity
as
a
factor
of
concentration
NaO4
O4

38. Results
and
discussion
34
For a better visualization of the NaO4/O4 ratio at different Kodak NA concentrations, a
stacked bar chart normalized to 100% was created (Figure 22). It can be observed that an
increase in concentration leads to the presence of more NaO4 class species relative to the
abundance of O4 species. Where there was no NaO4 class species at 0.001 mg/ml, at 0.5
mg/mL, they make up to ~3.3% of the summed relative abundance of NaO4 and O4 class
species. A possible explanation is that O4 species dissociate more readily, while NaO4 species
are more tightly bound.
Figure 22. The summed relative intensity of the O4 and NaO4 compounds in a fresh Kodak
NA sample at different concentrations. The y scale shows the 0-10% segment out of 0-100%.
0%
1%
2%
3%
4%
5%
6%
7%
8%
9%
10%
0.001
0.005
0.01
0.05
0.1
0.5
Relative
intensity
(%)
Concentration
(mg/mL)
O4
and
NaO4
normalized
to
100%
contribution
as
a
factor
of
concentration
O4
NaO4

39. Results
and
discussion
35
3.3.2 Importance
of
ion
accumulation
time
(IAT)
Accumulating ions in an rf-only multipole for longer period of times before the mass analysis,
also referred to as multipole storage assisted dissociation (MSAD), is a well-known
phenomenon to affect the degree of fragmentation.59
The space-charge limit is defined as the largest amount of charge that can be stored within a
trap.60
It is hypothesised that once the ion density exceeds the space-charge limit, the
coulombic-repulsive forces between the charges will push the ion to larger radii. As a
consequence of the expansion, the ions are subject to higher amplitude oscillations and
therefore they reach higher kinetic energy. Collisions with the background gas molecules in
the hexapole at these higher energies results in fragmentation.61
As illustrated in Figure 23, increasing ion accumulation time from 0.1 to 0.4 s facilitates the
number of undergoing collisions, and thus causing a decrease in the relative intensity of the
naphthenic acid dimers, as well as a slight shift towards higher m/z.
Therefore, ion accumulation time is an important parameter that needs to be carefully
controlled throughout the same set of experiments, otherwise there is a significant change in
monomer/dimer distribution.

40. Results
and
discussion
36
Figure
23.FT-­‐ICR
mass
spectra
(negative-­‐ion
ESI,
ISD
0
V)
of
a
1
mg/mL
NA
Kodak
mix
in
1:1
acetonitrile
and
water
at
different
ion
accumulation
times

41. Results
and
discussion
37
3.4 CID
on
the
2
year
old
Kodak
NA
sample
To obtain an initial insight into the naphthenic acid dimers and how they dissociate back to
naphthenic acids, a collision induced dissociation experiment was carried out. The molecular
ion at m/z=503.410574, corresponding to C32H55O4
-
(Z=-8, DBE=5.5), was chosen as the
precursor ion because of its high intensity. The isolation window was set to be 1 m/z wide.
Within the 1 m/z window, besides the O4 class compound, there was also present a NaO4 class
compound (C31H44NaO4
-
, z=-16, DBE=10), as well as a small number of other very low
intensity ions.
The intensity of the two species during the 2, 6, 8 and 12 V CID experiments are shown in
Table 6. At 2 V, the intensity of the NaO4 compound was significantly less than the intensity
of the O4 compound. It was noticed that by increasing the collision cell energy, there was a
decrease in the intensity of the O4 species, while the intensity of the NaO4 species was slightly
increasing. The significant decrease in intensity of the O4 species occurs from going from 2 V
to 6 V collision cell energy. By 12 V, C31H44NaO4
-
is more intense than C32H55O4
-
.
Table 6. The intensity of the precursor ions during the 2, 6, 8 and 12 V CID experiments
Precursor ion / Collision
cell energy (V)
2 6 8 12
C32H55O4
-
2.05E+09 4.01E+08 7.63E+07 2.09E+06
C31H44NaO4
-
2.72E+07 3.53E+07 3.51E+07 4.00E+07
The absolute intensity of the precursor ions were normalized to relative intensity and then
plotted against the collision cell energy for a better visualization (Figure 24). Higher collision
cell voltages, i.e. higher kinetic energy collisions, result in an increased chance for the weakly
bound O4 class species to undergo fragmentation. The crossover between the two species
happens between 8 V and 12 V.

42. Results
and
discussion
38
Figure 24. The relative intensity of the O4 and NaO4 precursor ion as a function of the
collision cell voltage. By increasing the collision cell voltage, the intensity of the O4 class
species decreases
An example of a CID spectrum is illustrated in figure 25, where the parent ion (m/z=503.4)
and the fragmented species can be observed.
Figure 25. FT-ICR MS spectrum (CID 8 V)

43. Results
and
discussion
39
The resulting spectra for the four different CID experiments, gave rise to monomeric
naphthenic acid species with very similar characteristics in regards to the number of carbon
atoms, the degree on unsaturation and the relative intensity distribution of the NAs with
different z series (Figure 26).
Figure 26. Distribution of the NA homologous series fragments produced from the
dissociation of C32H55O4
-
(Z=-8) during different CID experiments (2, 6, 8 and 12 V)
The main difference between the four resulting spectra was an increase in signal intensity of
the NA monomers with the increase in collision cell energy. Increasing collision energy
increases abundance of fragments, but does not change fragments produced. The most intense
NA homologous series produced during CID experiments has a Z number of -4, as a result of
the dissociation in half of the O4 dimer precursor ion with a Z number of -8. NA monomers
with Z=-6 and Z=-2 are also very intense. The low intensity monomeric NAs with a Z value
between -10 and -18 are believed to originate from the NaO4 dimer precursor ion.
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
12
14
16
18
20
22
Intensity
Carbon
number
Distribution
O2-­‐6
V
(CID)
0.E+00
1.E+07
2.E+07
3.E+07
4.E+07
5.E+07
6.E+07
12
14
16
18
20
22
Intensity
Carbon
number
Distribution
O2-­‐
2
V
(CID)
0
-­‐2
-­‐4
-­‐6
-­‐8
-­‐10
-­‐12
-­‐16
-­‐18
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
3.5E+08
4.0E+08
12
14
16
18
20
22
Intensity
Carbon
number
Distribution
O2-­‐
12
V
(CID)
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
12
14
16
18
20
22
Intensity
Carbon
number
Distribution
O2-­‐
8
V
(CID)

44. Results
and
discussion
40
3.5 IRMPD
experiment
of
the
fresh
Kodak
NA
sample
During CID experiments, the precursor ions are selected using the quadrupole and then the
ions are passed to collision cell for fragmentation with argon gas (pressure in collision cell is
~7×10-6
mbar). As seen before leakage can happen through the isolation window.
To improve the quality of the isolation, besides isolation in the quadrupole, a correlated sweep
and correlated shots in the ICR cell were used.62
Zoom-in insets on the precursor regions are
shown in Figure 27, where it can be easily observed that carrying out just the isolation in the
quadrupole allows leakage of other ions into the isolation window, and when the correlated
sweep and correlated shots are added the isolation window is cleaner.
Figure 27. Enlarged mass spectrum of the precursor region for the quadrupole isolation (upper
spectrum) and for the quadrupole isolation plus correlated sweep plus correlated shots
(bottom spectrum)
After performing higher resolving power isolation, the options for dissociation in the ICR cell
are sustained off-resonance irradiation - collision induced dissociation (SORI-CID) or
infrared multiphoton dissociation (IRMPD). IRMPD has a great advantage over SORI-CID
because it does not require the use of collision gas. In this way, the pressure in the analyser

45. Results
and
discussion
41
cell is maintained at an optimal level while the pumpdown time is eliminated and thus there is
no diminish in the performance of the instrument (peak shape, resolving power).
IRMPD on the fresh solution was carried out on a series of ions of same carbon number and
class, but differing DBE. This allows a study of fragmentation patterns as a function of DBE.
A difference in Z number by 2 can mean a double bond but also the formation of a new ring.
Firstly the dissociation of the m/z 503.4 precursor ion (C32H55O4
-
, Z=-8) was carried out to
compare with the CID data of the old solution. The resulted spectrum is shown in Figure 28.
Figure 28. FT-ICR MS spectrum (IRMPD) showing the precursor ion at m/z 503.4 and the
fragmented ions
From Figure 29, it can be observed that the monomer distribution obtained from the
dissociation of the m/z 503.4 precursor using IRMPD is very similar with the dissociation of
the same ion using CID (Figure 26). From the fragmentation of the molecular ion with Z=-8,
once again Z=-4 is the most intense fragment series, followed by Z=-2 and Z=-6. While for
the CID data, the apex for Z=-6 (at carbon number 17) is more intense than the apex for Z=-2
(at C16), for the IRMPD data, the apex for Z=-6 (at C17) is less intense than the apex for Z=-
2 (at C16). Also, to be noticed the apex for the series with z=-4 has a maxima at carbon
number 16, i.e. half of the number of carbons the precursor ion has. The fragments obtained

46. Results
and
discussion
42
from both CID and IRMPD data show that the dimers reflect the most abundant monomer
species present and there is no preferential selectivity during dimer formation.
Figure 29. Distribution of the naphthenic acids homologous series fragments produced from
the dissociation of the molecular ion C32H55O4
-
(z=-8) using IRMPD
Next on, IRMPD was carried out on two less saturated precursor ions: C32H53O4
-
(m/z=501.4,
Z=-10) and C32H51O4
-
(m/z=499.4, Z=-12) (Figure 30). By increasing the Z number (index of
hydrogen deficiency), there are more possible combinations for the fragments to form. It
appears to be a trend with respect to the relative intensities of the different homologous series
as a function of the Z value of the selected dimer. A more negative Z value leads to Z series
of monomers being more evenly spread, rather than one or two Z series predominating.
0.E+00
1.E+08
2.E+08
3.E+08
4.E+08
5.E+08
6.E+08
7.E+08
8.E+08
10
12
14
16
18
20
22
24
Intensity
Carbon
number
O2
distribution-­‐framentation
of
m/z
503.4,
Z=-­‐8
(IRMPD)
0
-­‐2
-­‐4
-­‐6
-­‐8

47. Results
and
discussion
43
Figure 30. Distribution of the naphthenic acids homologous series fragments produced from
the dissociation of the molecular ion C32H53O4
-
(z=-10) on the left graph and of the molecular
ion C32H51O4
-
(z=-12) on the right graph (IRMPD)
The most negative Z value for the monomer region does not exceed the Z value of the dimer
which proves that there is no additional fragmentation is occurring.
0.0E+00
2.0E+07
4.0E+07
6.0E+07
8.0E+07
1.0E+08
1.2E+08
1.4E+08
1.6E+08
1.8E+08
2.0E+08
10
12
14
16
18
20
22
Intensity
Carbon
number
O2
distribution
-­‐framentation
of
m/z
501.4,
Z=-­‐10
(
IRMPD)
0
-­‐2
-­‐4
-­‐6
-­‐8
-­‐10
0.0E+00
2.0E+07
4.0E+07
6.0E+07
8.0E+07
1.0E+08
1.2E+08
10
12
14
16
18
20
22
Intensity
Carbon
number
O2
distribution
-­‐framentation
of
m/z
499.4,
Z=-­‐12
(
IRMPD)
0
-­‐2
-­‐4
-­‐6
-­‐8
-­‐10
-­‐12

48. Results
and
discussion
44
3.6 Fresh
Kodak
NA
sample
doped
with
salts
It was noticed in previous experiments that salt affects the dimer formation. For further
investigating of this matter, a fresh Kodak NA mixture was doped with sodium chloride. The
oil industry has long used a technique of pumping seawater into an oil reservoir to increase
the pressure and push oil toward producing wells. Understanding the ability of naphthenic
acid to form sodium bond dimers after coming in contact with seawater will help understand
deposition formation in industry.
The average concentration of salt in seawater is about 35 g/L or 599mM, but because such
high salt concentration would overwhelm the ESI process, the concentration of salt used in
this study for doping the naphthenic acid samples was much lower, in the µM range.
Nevertheless, for illustrative reasons, a 0.05 mg/mL Kodak NA sample was doped with
enough sodium chloride to imitate the actual seawater linearity (left hand side vial in Figure
31). At seawater salt concentration level, there is a dramatic change in the appearance of
naphthenic acid the sample when compared to a regular Kodak NA sample that has not been
doped with salt (right hand side vial).
Figure 31. Photography of a Kodak NA mixture (0.05 mg/mL) at seawater concentration of
salt (600 mM) (left hand side) and of a Kodak NA mixture (0.05 mg/mL) with no salt added
(right hand side)

49. Results
and
discussion
45
A fresh 0.05 mg/mL Kodak NA sample that contained no added salts was used as a blank
sample. Mass spectra zoom-ins on the NaO4/O4 pair of peaks from the blank Kodak NA
sample and the three Kodak NA samples doped with sodium chloride are shown in Figure 32.
Figure 32. Expanded mass range of the m/z 431.314 C25H44NaO4
-
and of the m/z 431.317
C27H43O4
-
. Increase in the concentration of salt, increases the relative intensity of NaO4 class
The data analysis carried showed that NaO4 class species make up to 7.23% of the dimeric
distribution in the NA blank sample (Table 7). As expected, by increasing the amount of salt
added to the NA sample, there is an increase in the NaO4 contribution at the expense of
dissociation of the weakly bound O4 species.
Table 7. The relative abundance of the species present in the dimer distribution at none or
different concentration of salt
Species/Concentration salt (µM) 0 0.6 54.55 300
O4 90.63 87.98 86.89 82.73
NaO4 7.23 11.11 11.94 16.20
O4S 2.14 0.92 1.17 1.07

50. Results
and
discussion
46
The relative abundance of the O4, NaO4 and O4S classes in the blank and in the salt doped
Kodak NA samples was plotted as line graph to show the general trend (Figure 33). By
increasing the salt concentration, there is a decrease in the contribution of the O4 class and an
increase in the contribution of the NaO4 class. Adding enough NaCl so the salt concentration
of the Kodak NA sample is 300 µM will increase the NaO4 class contribution to the dimer
from ~7% (the natural state, when no salt is added) to about 16%. A 300 µg/mL salt
concentration is 2000 times lower than the 600 mM seawater salinity.
Figure 33. Relative abundance of O4, NaO4 and O4S species in the dimer distribution of
Kodak NA mixture containing none or various concentration of salt

51. Results
and
discussion
47
3.7 EID
experiment
on
a
NIST
crude
oil
sample
As an additional line of work, further development of methods for understanding structures of
petroleum molecules was pursued. Determining the elemental composition for petroleum
samples has improved greatly the field of petroleomics, but understanding the structures of
the tens of thousands of components present represents the next challenge. In this work, EID
has been applied for the first time to crude oil samples.
Firstly, a positive-ion mode ESI spectrum of a NIST crude oil sample was obtained (Figure
34). The multitude of peaks in the mass spectrum proves the complexity of petroleum
samples.
Figure 34. Mass spectrum of the NIST crude oil (positive mode ESI)
The main contributor to the NIST crude oil mass spectrum is the N1 class (~66%), followed
by OS class (~26%) (Table 8). Other species such as NO, NO2, O4S and OS2 are present as
minor components.

52. Results
and
discussion
48
Table 8. The relative abundance of the species present in the positive-mode ESI spectrum of
NIST crude oil
Species
Relative
abundance
(%)
N
66.35
NO
3.11
NO2
0.35
O4S
0.34
OS
25.54
OS2
1.18
For a better visualization, the values from Table 8 are plotted as a bar chart in Figure 35.
Figure 35. The abundance of the species present in NIST crude oil (positive-ion conditions
ESI)
Because of the high content of N present in the petroleum sample and the known problems
associated with the N-containing compounds in crude oil, such as pollution and catalyst
poisoning, data analysis was focused on N class species. The DBE values of the components
of N1 class were plotted against their carbon number as shown in the DBE plot from Figure
36. DBE values extend from 3.5 to 22.5 with, DBE between 7.5 and 9.5 being most
abundant.
0
10
20
30
40
50
60
70
N
NO
NO2
O4S
OS
OS2
Relative
abundance
(%)
Species
present
NIST
crude
oil
composition
(positive
mode
ESI)

53. Results
and
discussion
49
Figure 36. DBE plot of the N1 class present in a NIST crude oil (positive-ion ESI)
The isolation window was chosen to be 60 Da wide in order to accommodate five data points
along homologues series. A DBE plot of the N1 class species from the isolation window is
shown in Figure 37. The DBE values extend from 3.5 to 19.5 while the most abundant species
have DBE values of 6.5 to 9.5. The carbon number ranged between 27 and 32.
Figure 37 DBE plot of the N1 class for the isolation window spectrum
A single EID spectrum (Figure 38) was obtained after one hour experimental time in order to
increase the signal to noise ratio. It was determined that a 30 V cathode bias produces the
0 10 20 30 40 50 60 70 80
0
5
10
15
20
25
NIST crude oil
N1 class DBE plot
DBE
Carbon number
0 5 10 15 20 25 30 35
0
2
4
6
8
10
12
14
16
18
20
22
Isolation
N1 Class DBE plot
DBE

54. Results
and
discussion
50
most extensive fragmentation for the NIST crude oil sample. The cathode bias controls the
energy of the electrons used for irradiation. Lower cathode biases were also tested, but it was
noticed that at values higher than 20 V, the degree of fragmentation improved.
Figure 38. EID spectrum of a NIST crude oil sample. The isolation window extends from m/z
375 to m/z 435
The DBE plot of the N1 class species from the EID spectrum (Figure 39) reveals that DBE
values extend from 3.5 to 20.5, showing a similar distribution as the N1 class species from the
isolation spectrum. When comparing the carbon number of N1 class species between the two
spectra, it is clear that additional fragmentation occurs during EID experiments. Where the
lowest carbon number during isolation was 27, the minimum carbon number after EID was
11. So, fragments smaller than the smallest intact components are seen.

55. Results
and
discussion
51
Figure
39. DBE plot of the N1 class for the EID spectrum
Fragmentation causes successive loss of alkyl chains from the core (fused rings) rather than
alkyl chains plus aromatic carbon, as proved by the horizontal lines to the left of the isolated
peaks in the DBE plot. However, fragmentation does not cause a reduction in aromaticity.
In this research, it was demonstrated that EID is a viable tandem mass spectrometry method
for petroleum analysis. Through EID significant fragmentation was produced with no increase
of pressure in the ICR cell. EID dissociates alkyl chain and thus it provides information about
the core of molecules. It could potentially be used to distinguish archipelago structures
(condensed aromatic cores connected by aliphatic chains) from island structures (one
aromatic core with aliphatic chains). For archipelago structures, DBE plot will show stable
structures favoured along the homologous series, while for island structures it will show loss
of alkyl chains evenly as seen with the NIST crude oil sample.
Therefore, it is possible to use EID on petroleum samples to remove alkyl chains and identify
the minimum number of carbons needed for just the stable core for a particular class species.
0 5 10 15 20 25 30 35
2
4
6
8
10
12
14
16
18
20
22
DBE
Carbon number
EID-30 V cathode bias
N1 class DBE plot

56. Conclusion
52
4 Conclusion
In this work, negative-ion ESI FT-ICR mass spectrometry reveals two forms of naphthenic
acid dimers to be present in a commercial naphthenic acid sample, instead of one as
previously believed: the O4 class species that have been formerly described by other groups
and the NaO4 class species. The power of the inherent ultrahigh resolving power and mass
accuracy associated with FTICR-MS made it possible to resolve NaO4/O4 pair of peaks,
separated by only 2.4 mDa.
Formation
of
naphthenic
acid
dimers
may
simply
be
the
route
that
naturally
occurs,
but
understanding
any
preferential
dimer
aggregation
would
help
understand
if
there
are
any
particular
culprits
or
whether
all
species
present
are
equally
guilty
for
corrosion
in
refineries
and
toxicity
to
aquatic
environment.
How easily naphthenic acids can form
dimers will impact the behaviour of these NAs and the availability of those protons to have
consequences for corrosion.
Comparing a 2 year old Kodak naphthenic acid solution with a freshly prepared one, it was
noticed that the dimers present depend on the solution age, and if a sample is preserved in
glassware for long enough time, the sodium bound naphthenic acid will be more abundant
(~8.5% contribution compared to ~2.8% contribution for the fresh sample).
The O4 class compounds have been demonstrated to dissociate readily as they are bound by a
weak noncovalent bond that breaks when increasing the collision power to higher voltages
such as 60 V for ISD experiments and 12 V for CID experiments. On the other hand, the
abundance of NaO4 class compounds remained relatively stable and it is believed that they are
bound by much stronger bonds.
A proposed structure is shown in Figure 40, where the sodium atom is shared between the
four oxygen atoms.

57. Conclusion
53
Figure 40. Possible molecular structure of the singly charged NaO4 class species present in
naphthenic acid sample, with delocalized electron density between the carbon atom and the
two oxygen atoms
A fresh Kodak naphthenic acid solution was doped with NaCl in order to monitor the effect of
sodium abundance upon the contribution of the NaO4 class species and to compare with the
aged naphthenic acid sample. This is highly relevant to the extraction of oil from reservoirs
process, where seawater is pumped down to help extract oil. Sodium bound dimers can form
precipitates when seawater comes in contact with naphthenic acids, leading to naphthenates
deposition inside pipelines. Using Kodak NA samples doped with sodium chloride, it was
shown that the more sodium is available, the higher the contribution of the NaO4 class is.
Doping the naphthenic acid sample with a salt concentration 2000 times lower than the actual
seawater salt concentration, increased the NaO4 contribution to the dimer from ~7% to ~16%.
Electron induced ionization was applied successfully for the first time to petroleum providing
proof of concept data. In petroleomics, fs. The EID experiment carried on a crude oil sample
demonstrated the great potential of EID MS/MS in structure characterization.
Further work can include possible tandem mass spectrometry methods for fragmentation of
species with tentative assignments such as the ones in distribution III and IV from the fresh
Kodak NA sample. Additional experiments can be carried out on doping naphthenic acid
samples with higher amounts of salt to mirror the actual salinity level of seawater and on
study of real world sodium naphthenate deposits. Further EID experiments can be carried out
using different cathode potentials on various class compounds from different petroleum
samples in order to obtain greater insight into molecular structures, including information
about favoured structures (archipelago versus island structures). EID MS/MS via FT-ICR MS
could be coupled with chromatography for structure information of isomeric compounds from
petroleum samples.

58. References
54
5 References
1.
Cho,
Y.;
Ahmed,
A.;
Kim,
S.,
Application
of
Atmospheric
Pressure
Photo
Ionization
Hydrogen/Deuterium
Exchange
High-­‐Resolution
Mass
Spectrometry
for
the
Molecular
Level
Speciation
of
Nitrogen
Compounds
in
Heavy
Crude
Oils.
Anal.
Chem.
2013,
85
(20),
9758-­‐9763.
2.
Griffiths,
M.
T.;
Da
Campo,
R.;
O'Connor,
P.
B.;
Barrow,
M.
P.,
Throwing
light
on
petroleum:
simulated
exposure
of
crude
oil
to
sunlight
and
characterization
using
atmospheric
pressure
photoionization
fourier
transform
ion
cyclotron
resonance
mass
spectrometry.
Anal.
Chem.
2014,
86
(1),
527-­‐34.
3.
Colati,
K.
A.
P.;
Dalmaschio,
G.
P.;
De
Castro,
E.
V.
R.;
Gomes,
A.
O.;
Vaz,
B.
G.;
Romão,
W.,
Monitoring
the
liquid/liquid
extraction
of
naphthenic
acids
in
brazilian
crude
oil
using
electrospray
ionization
FT-­‐ICR
mass
spectrometry
(ESI
FT-­‐ICR
MS).
Fuel
2013,
108,
647-­‐655.
4.
Marshall,
A.
G.;
Rodgers,
R.
P.,
Petroleomics:
The
Next
Grand
Challenge
for
Chemical
Analysis.
Acc.
Chem.
Res.
2004,
37
(1),
53-­‐59.
5.
Boduszynski,
M.
M.,
Composition
of
heavy
petroleums.
1.
Molecular
weight,
hydrogen
deficiency,
and
heteroatom
concentration
as
a
function
of
atmospheric
equivalent
boiling
point
up
to
1400.degree.F
(760.degree.C).
Energy
Fuels
1987,
1
(1),
2-­‐
11.
6.
Mapolelo,
M.
M.
Extraction,
Chromatographic
Separation
and
Characterization
of
Polar
Acidic
Species
in
Crude
Oils
and
Naphthenate
Deposits
by
Ultrahigh
Resolution
Fourier
Transform
Ion
Cyclotron
Resonance
Mass
Spectrometry.
Florida
State
University,
2010.
7.
Liu,
P.;
Shi,
Q.;
Chung,
K.
H.;
Zhang,
Y.;
Pan,
N.;
Zhao,
S.;
Xu,
C.,
Molecular
Characterization
of
Sulfur
Compounds
in
Venezuela
Crude
Oil
and
Its
SARA
Fractions
by
Electrospray
Ionization
Fourier
Transform
Ion
Cyclotron
Resonance
Mass
Spectrometry.
Energy
Fuels
2010,
24
(9),
5089-­‐5096.
8.
McKetta,
J.
J.,
Encyclopedia
of
Chemical
Processing
and
Design.
Marcel
Dekker:
United
States
of
America,
1990;
Vol.
35.
9.
Rodgers,
R.
P.,
Hughey,
C.
A.;
Hendrickson,
C.
L.;
Marshall,
A.
G.,
Advanced
Characterization
of
Petroleum
Crude
and
Products
by
High
Field
Fourier
Transform
Ion
Cyclotron
Resonance
Mass
Spectrometry.
Fuel
Chemistry
Division
Reprints
2002,
47
(2).
10.
Hell,
C.;
Medinger,
E.,
Ueber
das
Vorkommen
und
die
Zusammensetzung
von
Säuren
im
Rohpetroleum.
Ber.
Dtsch.
Chem.
Ges.
1874,
7
(2),
1216-­‐1223.
11.
Aschan,
O.,
Ueber
die
in
dem
Erdöl
aus
Baku
vorkommenden
Säuren
mit
niedrigerem
Kohlenstoffgehalt.
Ber.
Dtsch.
Chem.
Ges.
1891,
24
(2),
2710-­‐2724.
12.
Kannel,
P.
R.;
Gan,
T.
Y.,
Naphthenic
acids
degradation
and
toxicity
mitigation
in
tailings
wastewater
systems
and
aquatic
environments:
a
review.
J
Environ
Sci
Health
A
Tox
Hazard
Subst
Environ
Eng
2012,
47
(1),
1-­‐21.
13.
Scott,
A.
C.;
Mackinnon,
M.
D.;
Fedorak,
P.
M.,
Naphthenic
Acids
in
Athabasca
Oil
Sands
Tailings
Waters
Are
Less
Biodegradable
than
Commercial
Naphthenic
Acids.
Environ.
Sci.
Technol.
2005,
39
(21),
8388-­‐8394.
14.
Dzidic,
I.;
Somerville,
A.
C.;
Raia,
J.
C.;
Hart,
H.
V.,
Determination
of
naphthenic
acids
in
California
crudes
and
refinery
wastewaters
by
fluoride
ion
chemical
ionization
mass
spectrometry.
Anal.
Chem.
1988,
60
(13),
1318-­‐1323.

59. References
55
15.
Headley,
J.
V.;
Peru,
K.
M.;
McMartin,
D.
W.;
Winkler,
M.,
Determination
of
dissolved
naphthenic
acids
in
natural
waters
by
using
negative-­‐ion
electrospray
mass
spectrometry.
J
AOAC
Int
2002,
85
(1),
182-­‐7.
16.
Qian,
K.;
Robbins,
W.
K.;
Hughey,
C.
A.;
Cooper,
H.
J.;
Rodgers,
R.
P.;
Marshall,
A.
G.,
Resolution
and
Identification
of
Elemental
Compositions
for
More
than
3000
Crude
Acids
in
Heavy
Petroleum
by
Negative-­‐Ion
Microelectrospray
High-­‐Field
Fourier
Transform
Ion
Cyclotron
Resonance
Mass
Spectrometry.
Energy
Fuels
2001,
15
(6),
1505-­‐1511.
17.
Barrow,
M.
P.;
McDonnell,
L.
A.;
Feng,
X.;
Walker,
J.;
Derrick,
P.
J.,
Determination
of
the
nature
of
naphthenic
acids
present
in
crude
oils
using
nanospray
Fourier
transform
ion
cyclotron
resonance
mass
spectrometry:
the
continued
battle
against
corrosion.
Anal.
Chem.
2003,
75
(4),
860-­‐6.
18.
Simon,
S.;
Reisen,
C.;
Bersås,
A.;
Sjöblom,
J.,
Reaction
between
tetrameric
acids
and
Ca
2+
in
oil/water
system.
Ind.
Eng.
Chem.
Res.
2012,
51
(16),
5669-­‐5676.
19.
Scaled
Solutions
LTD,
Organic
Deposits-­‐
Napthenates
http://www.scaledsolutions.com/Performance%20Tests/Organic%20Deposits
(accessed
18
Aug).
20.
El
Mahdy,
G.
A.;
Atta,
A.
M.;
Dyab,
A.
K.
F.;
Al-­‐Lohedan,
H.
A.,
Protection
of
Petroleum
Pipeline
Carbon
Steel
Alloys
with
New
Modified
Core-­‐Shell
Magnetite
Nanogel
against
Corrosion
in
Acidic
Medium.
J.
Chem.
2013,
2013,
9.
21.
Slavcheva,
E.;
Shone,
B.;
Turnbull,
A.,
Review
of
naphthenic
acid
corrosion
in
oil
refining.
Br.
Corrros.
J
1999,
34
(2),
125-­‐131.
22.
Qu,
D.
R.;
Zheng,
Y.
G.;
Jiang,
X.;
Ke,
W.,
Correlation
between
the
corrosivity
of
naphthenic
acids
and
their
chemical
structures.
Anti-­‐Corros
Method
M
2007,
54
(4),
211-­‐
218.
23.
Clemente,
J.
S.;
Fedorak,
P.
M.,
A
review
of
the
occurrence,
analyses,
toxicity,
and
biodegradation
of
naphthenic
acids.
Chemosphere
2005,
60
(5),
585-­‐600.
24.
GE-­‐Measurement
and
control,
Naphthenic
Acid
Corrosion.
http://www.ge-­‐
mcs.com/en/ndt-­‐corrosion-­‐inspection/naphthenic-­‐acid-­‐corrosion.html
(accessed
17
Aug).
25.
Wang,
Y.;
Zhou,
Q.;
Peng,
S.;
Ma
Lena,
Q.;
Niu,
X.,
Toxic
effects
of
crude-­‐oil-­‐
contaminated
soil
in
aquatic
environment
on
Carassius
auratus
and
their
hepatic
antioxidant
defense
system.
J
Environ
Sci
2009,
21
(5),
612-­‐617.
26.
Mohamed,
M.
H.;
Wilson,
L.
D.;
Peru,
K.
M.;
Headley,
J.
V.,
Colloidal
properties
of
single
component
naphthenic
acids
and
complex
naphthenic
acid
mixtures.
J.
Colloid
Interface
Sci.
2013,
395
(1),
104-­‐110.
27.
Yen,
T.
W.;
Marsh,
W.
P.;
MacKinnon,
M.
D.;
Fedorak,
P.
M.,
Measuring
naphthenic
acids
concentrations
in
aqueous
environmental
samples
by
liquid
chromatography.
Journal
Chromatogr.
A
2004,
1033
(1),
83-­‐90.
28.
Hemmingsen,
P.
V.;
Kim,
S.;
Pettersen,
H.
E.;
Rodgers,
R.
P.;
Sjöblom,
J.;
Marshall,
A.
G.,
Structural
characterization
and
interfacial
behavior
of
acidic
compounds
extracted
from
a
North
Sea
oil.
Energy
Fuels
2006,
20
(5),
1980-­‐1987.
29.
Smith,
D.
F.;
Schaub,
T.
M.;
Rahimi,
P.;
Teclemariam,
A.;
Rodgers,
R.
P.;
Marshall,
A.
G.,
Self-­‐association
of
organic
acids
in
petroleum
and
Canadian
bitumen
characterized
by
low-­‐
and
high-­‐resolution
mass
spectrometry.
Energy
Fuels
2007,
21
(3),
1309-­‐1316.
30.
Da
Campo,
R.;
Barrow,
M.
P.;
Shepherd,
A.
G.;
Salisbury,
M.;
Derrick,
P.
J.,
Characterization
of
Naphthenic
Acid
Singly
Charged
Noncovalent
Dimers
and
Their
Dependence
on
the
Accumulation
Time
within
a
Hexapole
in
Fourier
Transform
Ion
Cyclotron
Resonance
Mass
Spectrometry.
Energy
Fuels
2009,
23
(11),
5544-­‐5549.