simona gherghel-msc thesis

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

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

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

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.

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

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

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

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

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.

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

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, 13C 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:

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.

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

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 (12C=12.000 00 Da) to

the Kendrick mass scale (CH2=14.00000 Da instead of 14.01565 Da) using the following

equation:

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).

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

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

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.

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.

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

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 13C 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).

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.

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

1600165017001750180018500.0

0.1

0.2

0.3

0.4

0.5

Abs

orpt

ion

(a.u

.)

Wavenumber (cm-1)

1742(monomer)

1703(dimer)

C=O stretch(COOH)


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.


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)


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.


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.


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


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.


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


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


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 40 420

1

2

3

4

5

6

7

8

9

10

11

12

13

14 Old Kodak NA sample (ISD 0 V)O2 class DBE plot

DB

E

Carbon number10 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

DB

E

Carbon number

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 420

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 40 420

2

4

6

8

10

12

14Old Kodak NA sample (ISD 0 V)

NaO4 class DBE plot

DB

E

Carbon number

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 420

2

4

6

8

10

12

14Old Kodak NA sample (ISD 60 V)

NaO4 class DBE plot

DB

E

Carbon number


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%.


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


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.


32

Figure 20. DBE plots of O2 and O4 species from spectra of a fresh Kodak NA sample at

various concentrations


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


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


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.


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


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.


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)


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)


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


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


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


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


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)


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


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


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.


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)


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 70 800

5

10

15

20

25 NIST crude oilN1 class DBE plot

DBE

Carbon number

0 5 10 15 20 25 30 350

2

4

6

8

10

12

14

16

18

20

22 Isolation N1 Class DBE plot

DBE


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.


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 30 352

4

6

8

10

12

14

16

18

20

22

DBE

Carbon number

EID-30 V cathode biasN1 class DBE plot

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.

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.

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.

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.

References

56

31. Mapolelo, M. M.; Stanford, L. A.; Rodgers, R. P.; Yen, A. T.; Debord, J. D.; Asomaning, S.; Marshall, A. G., Chemical Speciation of Calcium and Sodium Naphthenate Deposits by Electrospray Ionization FT-‐ICR Mass Spectrometry. Energy Fuels 2009 23 (1), 349-‐355. 32. Fan, T. W., Jianxin; Buckley, Jill S., Evaluating Crude Oils by SARA Analysis. Soc. Petrol. Eng. 2002, Conference Paper (SPE/DOE Improved Oil Recovery Symposium, 13-‐17 April 2012, Tulsa, Oklahoma ). 33. Merdrignac, I.; Espinat, D., Physicochemical characterization of petroleum fractions: The state of the art. Oil Gas Sci Technol 2007, 62 (1), 7-‐32. 34. Marshall, A. G.; Rodgers, R. P., Petroleomics: chemistry of the underworld. Proc Natl Acad Sci U S A 2008, 105 (47), 18090-‐5. 35. Michael, G.; Al-‐Siri, M.; Khan, Z. H.; Ali, F. A., Differences in Average Chemical Structures of Asphaltene Fractions Separated from Feed and Product Oils of a Mild Thermal Processing Reaction. Energy Fuels 2005, 19 (4), 1598-‐1605. 36. Barrow, M. P.; Headley, J. V.; Peru, K. M.; Derrick, P. J., Fourier transform ion cyclotron resonance mass spectrometry of principal components in oilsands naphthenic acids. Journal Chromatogr. A 2004, 1058 (1–2), 51-‐59. 37. Marshall, A. G.; Chen, T., 40 years of Fourier transform ion cyclotron resonance mass spectrometry. Int. J. Mass. Spectrom. 2014, In press. 38. Hsu, C. S.; Lobodin, V. V.; Rodgers, R. P.; McKenna, A. M.; Marshall, A. G., Energy Fuels 2011, 25 (5), 2174. 39. Hsu, C. S.; Liang, Z.; Campana, J. E., Hydrocarbon characterization by ultrahigh resolution fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 1994, 66 (6), 850-‐855. 40. Merlin, M.; Guigard, S. E.; Fedorak, P. M., Detecting naphthenic acids in waters by gas chromatography-‐mass spectrometry. J Chromatogr A 2007, 1140 (1-‐2), 225-‐9. 41. Hao, C.; Headley, J. V.; Peru, K. M.; Frank, R.; Yang, P.; Solomon, K. R., Characterization and pattern recognition of oil-‐sand naphthenic acids using comprehensive two-‐dimensional gas chromatography/time-‐of-‐flight mass spectrometry. J Chromatogr A 2005, 1067 (1-‐2), 277-‐284. 42. St. John, W. P.; Rughani, J.; Green, S. A.; McGinnis, G. D., Analysis and characterization of naphthenic acids by gas chromatography-‐ electron impact mass spectrometry of tert.-‐butyldimethylsilyl derivatives. J Chromatogr A 1998, 807 (2), 241-‐251. 43. Fan, T. P., Characterization of naphthenic acids in petroleum by fast atom bombardment mass spectrometry. Energy Fuels 1991, 5 (3), 371-‐375. 44. Mohammed, M. A.; Sorbie, K. S., Naphthenic acid extraction and characterization from naphthenate field deposits and crude oils using ESMS and APCI-‐MS. Colloids Surf., A 2009, 349 (1-‐3), 1-‐18. 45. Qian, K.; Edwards, K. E.; Dechert, G. J.; Jaffe, S. B.; Green, L. A.; Olmstead, W. N., Measurement of Total Acid Number (TAN) and TAN Boiling Point Distribution in Petroleum Products by Electrospray Ionization Mass Spectrometry. Anal. Chem. 2008, 80 (3), 849-‐855. 46. Ho, C. S.; Lam, C. W.; Chan, M. H.; Cheung, R. C.; Law, L. K.; Lit, L. C.; Ng, K. F.; Suen, M. W.; Tai, H. L., Electrospray ionisation mass spectrometry: principles and clinical applications. Clin Biochem Rev 2003, 24 (1), 3-‐12. 47. Sleno, L.; Volmer, D. A., Ion activation methods for tandem mass spectrometry. J Mass Spectrom 2004, 39 (10), 1091-‐1112.

References

57

48. Zhurov, K. O.; Fornelli, L.; Wodrich, M. D.; Laskay, U. A.; Tsybin, Y. O., Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chem. Soc. Rev. 2013, 42 (12), 5014-‐5030. 49. Kaczorowska, M. A.; Cooper, H. J., Electron induced dissociation (EID) tandem mass spectrometry of octaethylporphyrin and its iron(iii) complex. Chem. Commun. 2011, 47 (1), 418-‐420. 50. Scigelova, M.; Hornshaw, M.; Giannakopulos, A.; Makarov, A., Fourier transform mass spectrometry. Mol Cell Proteomics 2011, 10 (7), M111 009431. 51. Kim, S.; Rodgers, R. P.; Marshall, A. G., Truly "exact" mass: Elemental composition can be determined uniquely from molecular mass measurement at ∼0.1 mDa accuracy for molecules up to ∼500 Da. Int. J. Mass Spectrom. 2006, 251 (2-‐3 SPEC. ISS.), 260-‐265. 52. Rodgers, R. P.; Schaub, T. M.; Marshall, A. G., PETROLEOMICS: MS returns to its roots. Anal. Chem. 2005, 77 (1), 20 A-‐27 A. 53. Hur, M.; Yeo, I.; Kim, E.; No, M. H.; Koh, J.; Cho, Y. J.; Lee, J. W.; Kim, S., Energy Fuels 2010, 24 (10), 5524. 54. Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Qian, K., Kendrick mass defect spectrum: A compact visual analysis for ultrahigh-‐resolution broadband mass spectra. Acc. Chem. Res. 2001, 73 (19), 4676-‐4681. 55. Kendrick, E., A mass scale based on CH2 = 14.0000 for high resolution mass spectrometry of organic compounds. Anal. Chem. 1963, 35 (13), 2146-‐2154. 56. Comisarow, M. B., Error estimates for finite zero-‐filling in fourier transform spectrometry. Anal. Chem. 1979, 51 (13), 2198-‐2203. 57. Aarstol, M.; Comisarow, M. B., Apodization of FT-‐ICR spectra. Int. J. Mass Spectrom. Ion Processes 1987, 76 (3), 287-‐297. 58. Islam, A.; Cho, Y.; Yim, U. H.; Shim, W. J.; Kim, Y. H.; Kim, S., The comparison of naturally weathered oil and artificially photo-‐degraded oil at the molecular level by a combination of SARA fractionation and FT-‐ICR MS. J Hazard Mater 2013, 263 Pt 2, 404-‐11. 59. Sannes-‐Lowery, K. A.; Hofstadler, S. A., Characterization of multipole storage assisted dissociation: implications for electrospray ionization mass spectrometry characterization of biomolecules. J. Am. Soc. Mass. Spectrom. 2000, 11 (1), 1-‐9. 60. Bushey, J. M.; Danell, R. M.; Glish, G. L., Iterative Accumulation Multiplexing Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 2009, 81 (14), 5623-‐5628. 61. Pan, C.; Hettich, R. L., Multipole-‐storage-‐assisted dissociation for the characterization of large proteins and simple protein mixtures by ESI-‐FTICR-‐MS. Anal. Chem. 2005, 77 (10), 3072-‐3082. 62. de Koning, L. J.; Nibbering, N. M. M.; van Orden, S. L.; Laukien, F. H., Mass selection of ions in a Fourier transform ion cyclotron resonance trap using correlated harmonic excitation fields (CHEF). Int. J. Mass Spectrom. Ion Processes 1997, 165–166 (0), 209-‐219.

Acknowledgments

58

6 Acknowledgments

This thesis would not have been possible without the help and advice of several people.

I am very grateful and indebted to my supervisor Dr Mark P. Barrow for excellent guidance,

patience and for giving his valuable time, advice and opinion to this thesis from the very

beginning of the research up to the end of the writing. His painstaking effort in proof reading

the drafts is greatly appreciated. I have been very lucky to have a supervisor who took so

much interest in this project and who was always willing to help me.

I would also like to thank the PhD students from the Ion Cyclotron Resonance Laboratory

Chris Wootton, Maria van Agthoven, Andrew Soulby, Juan Wei and Federico Floris as well

as to my AS:MIT colleagues Lying Huang and Haytham Hussein who have been a source of

friendship, laughs as well as good advice.

Appendix

59

7 Appendix

7.1 Known ions mass list # TuneMixNeg m/z charge Z HPmix2 301.998139 1-‐ HPmix3 601.978977 1-‐ HPmix4 1033.988109 1-‐ HPmix5 1333.968947 1-‐ HPmix6 1633.9498786 1-‐ HPmix7 1933.930624 1-‐ HPmix8 2233.911463 1-‐ C10H20O2 171.139053 1-‐ 0 C12H24O2 199.170353 1-‐ 0 C14H28O2 227.201653 1-‐ 0 C15H30O2 241.217303 1-‐ 0 C16H32O2 255.232953 1-‐ 0 C18H36O2 283.264253 1-‐ 0 C20H40O2 311.295553 1-‐ 0 C22H44O2 339.326853 1-‐ 0 C24H48O2 367.358153 1-‐ 0 C12H22O2 197.154703 1-‐ -‐2 C15H28O2 239.201653 1-‐ -‐2 C16H30O2 253.217303 1-‐ -‐2 C17H32O2 267.232953 1-‐ -‐2 C20H38O2 309.279903 1-‐ -‐2 C22H42O2 337.311203 1-‐ -‐2 C24H46O2 365.342503 1-‐ -‐2 C12H20O2 195.139053 1-‐ -‐4 C15H26O2 237.186003 1-‐ -‐4 C16H28O2 251.201653 1-‐ -‐4 C17H30O2 265.217303 1-‐ -‐4 C18H32O2 279.232953 1-‐ -‐4 C19H34O2 293.248603 1-‐ -‐4 C20H36O2 307.264253 1-‐ -‐4 C22H40O2 335.295553 1-‐ -‐4 C24H44O2 363.326853 1-‐ -‐4 C26H48O2 391.358153 1-‐ -‐4 C28H50O2 417.373803 1-‐ -‐4 C12H18O2 193.123403 1-‐ -‐6 C15H24O2 235.170353 1-‐ -‐6 C17H28O2 263.201653 1-‐ -‐6 C20H34O2 305.248603 1-‐ -‐6 C22H38O2 333.279903 1-‐ -‐6 C24H42O2 361.311203 1-‐ -‐6 C26H46O2 389.342503 1-‐ -‐6 C12H16O2 191.107753 1-‐ -‐8 C15H22O2 233.154703 1-‐ -‐8 C17H26O2 261.186003 1-‐ -‐8

Appendix

60

C20H32O2 303.232953 1-‐ -‐8 C22H36O2 331.264253 1-‐ -‐8 C24H40O2 359.295553 1-‐ -‐8 C26H44O2 387.326853 1-‐ -‐8 C12H14O2 189.092103 1-‐ -‐10 C15H20O2 231.139053 1-‐ -‐10 C20H30O2 301.217303 1-‐ -‐10 C22H34O2 329.248603 1-‐ -‐10 C15H18O2 229.123403 1-‐ -‐12 C20H28O2 299.201653 1-‐ -‐12 C22H32O2 327.232953 1-‐ -‐12 C21H41O4Na 379.282978 1-‐ 0 C22H43O4Na 393.298628 1-‐ 0 C23H45O4Na 407.314278 1-‐ 0 C24H47O4Na 421.329928 1-‐ 0 C26H51O4Na 449.361228 1-‐ 0 C28H55O4Na 477.392528 1-‐ 0 C30H59O4Na 505.423828 1-‐ 0 C31H61O4Na 519.439478 1-‐ 0 C32H63O4Na 533.455128 1-‐ 0 C33H65O4Na 547.470778 1-‐ 0 C34H67O4Na 561.486428 1-‐ 0 C36H71O4Na 589.517728 1-‐ 0 C38H75O4Na 617.549028 1-‐ 0 C40H79O4Na 645.580328 1-‐ 0 C22H41O4Na 391.282978 1-‐ -‐2 C23H43O4Na 405.298628 1-‐ -‐2 C24H45O4Na 419.314278 1-‐ -‐2 C26H49O4Na 447.345578 1-‐ -‐2 C28H53O4Na 475.376878 1-‐ -‐2 C29H55O4Na 489.392528 1-‐ -‐2 C30H57O4Na 503.408178 1-‐ -‐2 C31H59O4Na 517.423828 1-‐ -‐2 C32H61O4Na 531.439478 1-‐ -‐2 C33H63O4Na 545.455128 1-‐ -‐2 C34H65O4Na 559.470778 1-‐ -‐2 C35H67O4Na 573.486428 1-‐ -‐2 C36H69O4Na 587.502078 1-‐ -‐2 C37H71O4Na 601.517728 1-‐ -‐2 C38H73O4Na 615.533378 1-‐ -‐2 C39H75O4Na 629.549028 1-‐ -‐2 C22H39O4Na 389.267328 1-‐ -‐4 C23H41O4Na 403.282978 1-‐ -‐4 C24H43O4Na 417.298628 1-‐ -‐4 C26H47O4Na 445.329928 1-‐ -‐4 C28H51O4Na 473.361228 1-‐ -‐4 C30H55O4Na 501.392528 1-‐ -‐4 C32H59O4Na 529.423828 1-‐ -‐4 C33H61O4Na 543.439478 1-‐ -‐4

Appendix

61

C34H63O4Na 557.455128 1-‐ -‐4 C36H67O4Na 585.486428 1-‐ -‐4 C38H71O4Na 613.517728 1-‐ -‐4 C39H73O4Na 627.533378 1-‐ -‐4 C32H57O4Na 527.408178 1-‐ -‐6 C34H61O4Na 555.439478 1-‐ -‐6 C36H65O4Na 583.470778 1-‐ -‐6 C38H69O4Na 611.502078 1-‐ -‐6 C40H73O4Na 639.533378 1-‐ -‐6 C24H39O4Na 413.267328 1-‐ -‐8 C26H43O4Na 441.298628 1-‐ -‐8 C28H47O4Na 469.329928 1-‐ -‐8 C30H51O4Na 497.361228 1-‐ -‐8 C32H55O4Na 525.392528 1-‐ -‐8 C28H45O4Na 467.314278 1-‐ -‐10 C30H49O4Na 495.345578 1-‐ -‐10 C32H53O4Na 523.376878 1-‐ -‐10 C30H47O4Na 493.329928 1-‐ -‐12 C32H51O4Na 521.361228 1-‐ -‐12 C34H55O4Na 549.392528 1-‐ -‐12 C30H45O4Na 491.314278 1-‐ -‐14 C32H49O4Na 519.345578 1-‐ -‐14 C34H53O4Na 547.376878 1-‐ -‐14 C35H55O4Na 561.392528 1-‐ -‐14 C36H57O4Na 575.408178 1-‐ -‐14 C30H43O4Na 489.298628 1-‐ -‐16 C32H47O4Na 517.329928 1-‐ -‐16 C34H51O4Na 545.361228 1-‐ -‐16 C30H41O4Na 487.282978 1-‐ -‐18 C32H45O4Na 515.314278 1-‐ -‐18 C34H49O4Na 543.345578 1-‐ -‐18 C30H39O4Na 485.267328 1-‐ -‐20 C32H43O4Na 513.298628 1-‐ -‐20 C34H47O4Na 541.329928 1-‐ -‐20 C30H37O4Na 483.251678 1-‐ -‐22 C32H41O4Na 511.282978 1-‐ -‐22 C34H45O4Na 539.314278 1-‐ -‐22 C24H48O4 399.347984 1-‐ 0 C26H52O4 427.379284 1-‐ 0 C28H56O4 455.410584 1-‐ 0 C30H60O4 483.441884 1-‐ 0 C32H64O4 511.473184 1-‐ 0 C34H68O4 539.504484 1-‐ 0 C36H72O4 567.535784 1-‐ 0 C38H74O4 595.567084 1-‐ 0 C23H44O4 383.316684 1-‐ -‐2 C24H46O4 397.332334 1-‐ -‐2 C26H50O4 425.363634 1-‐ -‐2 C28H54O4 453.394934 1-‐ -‐2

Appendix

62

C28H54O4 481.426234 1-‐ -‐2 C32H62O4 509.457534 1-‐ -‐2 C34H66O4 537.488834 1-‐ -‐2 C36H70O4 565.520134 1-‐ -‐2 C38H74O4 593.551434 1-‐ -‐2 C39H76O4 607.567084 1-‐ -‐2 C23H42O4 381.301034 1-‐ -‐4 C24H44O4 395.316684 1-‐ -‐4 C26H48O4 423.347984 1-‐ -‐4 C28H52O4 451.379284 1-‐ -‐4 C30H56O4 479.410584 1-‐ -‐4 C31H58O4 493.426234 1-‐ -‐4 C32H60O4 507.441884 1-‐ -‐4 C33H62O4 521.457534 1-‐ -‐4 C34H64O4 535.473184 1-‐ -‐4 C35H66O4 549.488834 1-‐ -‐4 C36H68O4 563.504484 1-‐ -‐4 C37H70O4 577.520134 1-‐ -‐4 C38H72O4 591.535784 1-‐ -‐4 C39H74O4 605.551434 1-‐ -‐4 C40H76O4 619.567084 1-‐ -‐4 C41H78O4 633.582734 1-‐ -‐4 C23H40O4 379.285384 1-‐ -‐6 C24H42O4 393.301034 1-‐ -‐6 C26H46O4 421.332334 1-‐ -‐6 C28H50O4 449.363634 1-‐ -‐6 C30H54O4 477.394934 1-‐ -‐6 C32H58O4 505.426234 1-‐ -‐6 C33H60O4 519.441884 1-‐ -‐6 C34H62O4 533.457534 1-‐ -‐6 C35H64O4 547.473184 1-‐ -‐6 C36H66O4 561.488834 1-‐ -‐6 C37H68O4 575.504484 1-‐ -‐6 C38H70O4 589.520134 1-‐ -‐6 C39H72O4 603.535784 1-‐ -‐6 C40H74O4 617.551434 1-‐ -‐6 C41H76O4 631.567084 1-‐ -‐6 C24H40O4 391.285384 1-‐ -‐8 C26H44O4 419.316684 1-‐ -‐8 C28H48O4 447.347984 1-‐ -‐8 C30H52O4 475.379284 1-‐ -‐8 C32H56O4 503.410584 1-‐ -‐8 C34H60O4 531.441884 1-‐ -‐8 C35H62O4 545.457534 1-‐ -‐8 C36H64O4 559.473184 1-‐ -‐8 C38H68O4 587.504484 1-‐ -‐8 C40H72O4 615.535784 1-‐ -‐8 C26H42O4 417.301034 1-‐ -‐10 C28H46O4 445.332334 1-‐ -‐10

Appendix

63

C30H50O4 473.363634 1-‐ -‐10 C32H54O4 501.394934 1-‐ -‐10 C34H58O4 529.426234 1-‐ -‐10 C35H60O4 543.441884 1-‐ -‐10 C36H62O4 557.457534 1-‐ -‐10 C38H66O4 585.488834 1-‐ -‐10 C39H68O4 599.504484 1-‐ -‐10 C40H70O4 613.520134 1-‐ -‐10 C41H72O4 627.535784 1-‐ -‐10 C29H46O4 457.332334 1-‐ -‐12 C30H48O4 471.347984 1-‐ -‐12 C32H52O4 499.379284 1-‐ -‐12 C34H56O4 527.410584 1-‐ -‐12 C36H60O4 555.441884 1-‐ -‐12 C37H62O4 569.457534 1-‐ -‐12 C38H64O4 583.473184 1-‐ -‐12 C39H66O4 597.488834 1-‐ -‐12 C41H70O4 625.520134 1-‐ -‐12 C30H46O4 469.332334 1-‐ -‐14 C32H50O4 497.363634 1-‐ -‐14 C34H54O4 525.394934 1-‐ -‐14 C36H58O4 553.426234 1-‐ -‐14 C37H60O4 567.441884 1-‐ -‐14 C38H62O4 581.457534 1-‐ -‐14 C40H66O4 609.488834 1-‐ -‐14 C42H70O4 637.520134 1-‐ -‐14 C30H44O4 467.316684 1-‐ -‐16 C32H48O4 495.347984 1-‐ -‐16 C34H52O4 523.379284 1-‐ -‐16 C36H56O4 551.410584 1-‐ -‐16 C38H60O4 579.441884 1-‐ -‐16 C40H64O4 607.473184 1-‐ -‐16 C42H68O4 635.504484 1-‐ -‐16 C30H42O4 465.301034 1-‐ -‐18 C32H46O4 493.332334 1-‐ -‐18 C34H50O4 521.363634 1-‐ -‐18 C35H52O4 535.379284 1-‐ -‐18 C36H54O4 549.394934 1-‐ -‐18 C38H58O4 577.426234 1-‐ -‐18 C40H62O4 605.457534 1-‐ -‐18 C42H66O4 633.488834 1-‐ -‐18 C30H40O4 463.285384 1-‐ -‐20 C32H44O4 491.316684 1-‐ -‐20 C34H48O4 519.347984 1-‐ -‐20 C35H50O4 533.363634 1-‐ -‐20 C36H52O4 547.379284 1-‐ -‐20 C37H54O4 561.394934 1-‐ -‐20 C38H56O4 575.410584 1-‐ -‐20 C39H58O4 589.426234 1-‐ -‐20

Appendix

64

C40H60O4 603.441884 1-‐ -‐20 C42H64O4 631.473184 1-‐ -‐20 C44H80O4 659.504484 1-‐ -‐20 C30H38O4 461.269734 1-‐ -‐22 C32H42O4 489.301034 1-‐ -‐22 C34H46O4 517.332334 1-‐ -‐22 C35H48O4 531.347984 1-‐ -‐22 C36H50O4 545.363634 1-‐ -‐22 C37H52O4 559.379284 1-‐ -‐22 C39H56O4 587.410584 1-‐ -‐22 C41H60O4 615.441884 1-‐ -‐22 C43H64O4 643.473184 1-‐ -‐22 C45H68O4 671.504484 1-‐ -‐22

Appendix

65

7.2 Mass error distribution histograms

7.2.1 2 year old doped Kodak NA sample-‐ISD 0 V

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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6

8

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12

14

16

18

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22

24

Cou

nt

Error (ppm)

Old doped Kodak NA sample (ISD 0 V) O2 error histogram

Mean error: -0.064 ppmSTD error: 0.102 ppmRMS error: 0.120 ppm

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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40

50

60Old doped Kodak NA sample (ISD 0 V)

O4 error histogram

Cou

nt

Error (ppm)


-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Error (ppm)

Old doped Kodak NA sample (ISD 0 V) NaO4 error histogram


Appendix

66

7.2.2 2 year old doped Kodak NA sample-‐ISD 60 V

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

5

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25

30


O2 error histogram

C

ount

Error (ppm)

Mean error: 0.017 ppmSTD error: 0.062 ppmRMS error: 0.064 ppm

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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NaO4 error histogram

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Error (ppm)


Appendix

67

7.2.3 Fresh sample: serial dilution (ISD 0 V

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

5

10

15

20

C

ount

Error (ppm)

Fresh Kodak NA sample 0.5 mg/mL (ISD 0 V)O2 class error histogram

Mean error: -0.032STD error: 0.091RMS error: 0.095

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Cou

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Error (ppm)

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Appendix

68

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

10

20

30

40

50

Mean error: 0.014STD error: 0.287RMS error: 0.287


Cou

nt

Error (ppm)

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

10

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40



Cou

nt

Error (ppm)

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Appendix

69

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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15

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-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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60

80

100



Cou

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-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Error (ppm)

Appendix

70

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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60

70

80



Cou

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Error (ppm)

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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45



Cou

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Error (ppm)

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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20Fresh Kodak NA sample 0.001 mg/mL (ISD 0 V)O4 class error histogram

Cou

nt

Error (ppm)


Appendix

71

7.2.4 CID on 2 year old Kodak NA

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Error (ppm)

Old Kodak NA mixture (CID 2 V)O2 class error histogram


-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Error

Appendix

72

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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Cou

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Error

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Error (ppm)

Appendix

73

7.2.5 IRMPD on fresh Kodak NA sample

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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35


Fresh Kodak NA sample (IRMPD, m/z=499.4 precursor)O2 class error histogram

C

ount

Error (ppm)

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.00

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simona gherghel-msc thesis

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