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Purdue UniversityPurdue e-Pubs
Open Access Dissertations Theses and Dissertations
January 2015
MASS SPECTROMETRIC STUDIES ONPETROLEUM ASPHALTENES ANDORGANOSULFUR COMPOUNDS, ONFUNCTIONAL-GROUP SELECTIVE ION-MOLECULE REACTIONS AND ON GAS-PHASE REACTIVITY OF META-BENZYNESTOWARD AMINO ACIDSWeijuan TangPurdue University
Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations
This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] foradditional information.
Recommended CitationTang, Weijuan, "MASS SPECTROMETRIC STUDIES ON PETROLEUM ASPHALTENES AND ORGANOSULFURCOMPOUNDS, ON FUNCTIONAL-GROUP SELECTIVE ION-MOLECULE REACTIONS AND ON GAS-PHASEREACTIVITY OF META-BENZYNES TOWARD AMINO ACIDS" (2015). Open Access Dissertations. 1199.https://docs.lib.purdue.edu/open_access_dissertations/1199
PURDUE UNIVERSITY GRADUATE SCHOOL
Thesis/Dissertation Acceptance
To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
Weijuan Tang
MASS SPECTROMETRIC STUDIES ON PETROLEUM ASPHALTENES ANDORGANOSULFUR COMPOUNDS, ON FUNCTIONAL-GROUP SELECTIVE ION-MOLECULEREACTIONS AND ON GAS-PHASE REACTIVITY OF META-BENZYNES TOWARD AMINO ACIDS
Doctor of Philosophy
Hilkka I. Kenttämaa
Scott A. McLuckey
Mahdi Abu-Omar
Hilkka I. Kenttämaa
Chengde Mao
R. E. Wild 04/30/2015
MASS SPECTROMETRIC STUDIES ON PETROLEUM ASPHALTENES AND
ORGANOSULFUR COMPOUNDS, ON FUNCTIONAL-GROUP SELECTIVE ION-
MOLECULE REACTIONS AND ON GAS-PHASE REACTIVITY OF META-
BENZYNES TOWARD AMINO ACIDS
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Weijuan Tang
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
May 2015
Purdue University
West Lafayette, Indiana
ii
To my husband, Huaming Sheng
To my parents, Yi Tang and Yali Gan
To my parents in law, Tiansheng Sheng and Tanni Wang
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to express my deepest gratitude to my advisor,
professor Hilkka I. Kenttämaa. She not only provided me a wonderful opportunity to join
her research group, but also gave me guidance, inspiration, encouragement and support
throughout my PhD study. I was able to follow my passion and explore a wide variety of
exciting research projects in mass spectrometry. More importantly, I learned how to think,
how to solve problems, and how to better communicate and collaborate with different
people. She helped me grow into an independent scientist who is ready to tackle
challenges ahead. I am forever grateful to be her student, and I truly appreciate all her
help along the way. Without her guidance and support, I would have never become who I
am today.
Special thanks go to Dr. John Nash for his help in quantum chemical calculations
on the mono- and biradicals studied in my thesis and all the great discussions. I would
also like to thank Mark Carlsen, Dr. Hartmut Hedderich, and members of the Jonathan
Amy Facility, for helping me repair an FT-ICR instrument (NEL) that was down for two
years. Many thanks go to my thesis committee members, Dr. Scott A. McLuckey, Dr.
Mahdi Abu-Omar and Dr. Chengde Mao, for valuable discussions. I also want to thank
Dr. McLuckey for writing me recommendations for a PhD internship.
iv
I would like to express my gratitude to the past and present group members in Dr.
Kenttämaa’s group. Special thanks go to Dr. Jinshan Gao and Dr. Benjamin Owen who
trained me on ICR and LQIT instruments when I first joined the group; to Dr. Matthew
Hurt and Dr. David Borton who mentored me on petroleum projects; to Dr. Huaming
Sheng and Dr. James Riedeman who provided lots of great suggestions to my research
projects and helped with mechanistic studies. I also want to thank Dr. Ashley Wittrig, Dr.
Tiffany Jarrell, Dr. Peggy Williams, Dr. Fanny Widjaja, Dr. Enada Archibold, Dr. Linan
Yang, Dr. Nelson Vinueza, Dr. Vanessa Gallardo, Alex Dow, Chris Marcum, Guannan Li,
Priya Murria, John Degenstein, Chunfen Jin, Hanyu Zhu, Joann Max, John Kong,
Xueming Dong, Xin Ma, Mingzhe Li, Ravikiran Yerabolu, Mark Romanczyk, Laurance
Cain, Raghu Kotha, Babu Mistry, Rashmi Kumar, and Yuyang Zhang. Without their
feedback, friendship and generous help, my graduate study would not have been so
enjoyable.
I would like to thank Dr. Daniel Raftery for introducing me to the interesting area
of metabolomics. I am grateful for all the mentorship and help that I received from him
and the group members. I also want to extend my thanks to the people whom I have
worked with during my internship, including Dr. Vincent Asiago, Dr. Jan Hazebroek, Dr.
Cathy Zhong, Dr. Bruce Orman, Chris Vlahakis and Teresa Harp.
Lastly, but most importantly, I would like to thank my loving family for always
being there to support me. My wonderful husband, Huaming Sheng, has given me endless
support, understanding and encouragement. I am very grateful to have met him in China
because of our common goal to pursue a graduate degree at Purdue. It has been enjoyable
and fruitful five years in graduate school. The most sincere thanks go to my dear parents,
v
Yi Tang and Yali Gan, for all the years of unconditional love, caring, guidance and
encouragement. I also wish to thank my parents-in-law, Tiansheng Sheng and Tanni
Wang, who constantly encourage me and treat me as their own daughter.
vi
TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ........................................................................................................... xi
LIST OF SCHEMES........................................................................................................ xiv
ABSTRACT ................................................................................................................... xvi
LIST OF PUBLICATIONS ............................................................................................. xix
CHAPTER 1. INTRODUCTION AND OVERVIEW ....................................................... 1
1.1 Introduction .............................................................................................................. 1 1.2 Overview .................................................................................................................. 3 1.3 References ................................................................................................................ 4
CHAPTER 2. THEORY, INSTRUMENTATION AND EXPERIMENTAL ASPECTS OF LINEAR QUADRUPOLE ION TRAP (LQIT) AND FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) MASS SPECTROMETRY ................................................................. 6
2.1 Introduction .............................................................................................................. 6 2.2 Ionization Methods .................................................................................................. 7
2.2.1 Electron Ionization (EI) .................................................................................. 8 2.2.2 Chemical Ionization (CI) ................................................................................ 8 2.2.3 Electrospray Ionization (ESI) ......................................................................... 9 2.2.4 Atmospheric Pressure Chemical Ionization(APCI) ...................................... 11
2.3 Linear Quadrupole Ion Trap (LQIT) Mass Spectrometry ...................................... 14 2.3.1 Introduction ................................................................................................... 14 2.3.2 Instrument Overview .................................................................................... 14 2.3.3 Ion Motions in LQIT ..................................................................................... 18
2.3.3.1 Radial Motion ..................................................................................... 19 2.3.3.2 Axial Motion ....................................................................................... 23
2.3.4 Ion Excitation and Detection ........................................................................ 24
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Page
2.3.5 Tandem Mass Spectrometry ......................................................................... 27 2.3.5.1 Ion Isolation ........................................................................................ 27 2.3.5.2 Collision-activated Dissociation (CAD) ............................................. 29
2.3.6 Ion-molecule reactions in LQIT .................................................................... 30 2.4 Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry ........ 32
2.4.1 Introduction ................................................................................................... 32 2.4.2 Instrument Overview .................................................................................... 32 2.4.3 Ion Motions in FT-ICR ................................................................................. 36
2.4.3.1 Cyclotron Motion ................................................................................ 36 2.4.3.2 Trapping Motion ................................................................................. 38 2.4.3.3 Magnetron Motion .............................................................................. 40
2.4.4 Ion Manipulations in FT-ICR ....................................................................... 43 2.4.4.1 Ion Transfer ......................................................................................... 45 2.4.4.2 QuadrupolarAxialization (QA) ........................................................... 46 2.4.4.3 Ion Excitation and Detection .............................................................. 47 2.4.4.4 Ion Isolation ........................................................................................ 50 2.4.4.5 Collision-activated dissociation (CAD) in FT-ICR ............................ 52
2.5 Fundamental Aspects of Gas-phase Ion-Molecule Reactions ............................... 53 2.5.1 Brauman’s Double-Well Potential Energy Surface ...................................... 53 2.5.2 Kinetics of Ion-Molecule Reactions ............................................................. 56
2.6 References .............................................................................................................. 60
CHAPTER 3. STRUCTURAL COMPARISON OF ASPHALTENES OF DIFFERENT ORIGINS BY USING MULTIPLE-STAGE TANDEM MASS SPECTROMETRY ...................................................... 65
3.1 Introduction ............................................................................................................ 65 3.2 Experimental Section ............................................................................................. 67 3.3 Results and Discussion .......................................................................................... 68 3.4 Conclusions ............................................................................................................ 77 3.5 References .............................................................................................................. 78
CHAPTER 4. CHARACTERIZATION OF ORGANOSULFUR MODEL COMPOUNDS RELEVANT TO FOSSIL FUELS BY USING HIGH-RESOLUTION TANDEM MASS SPECTROMETRY ................. 82
4.1 Introduction ............................................................................................................ 82 4.2 Experimental Section ............................................................................................. 85 4.3 Results and Discussions ......................................................................................... 86 4.4 Conclusions .......................................................................................................... 106 4.5 References ............................................................................................................ 107
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Page
CHAPTER 5. GAS-PHASE ION/MOLECULE REACTIONS FOR THE IDENTIFICATION OF SULFONE FUNCTIONALITIES IN PROTONATED ANALYTES IN A LINEAR QUADRUPOLE ION TRAP MASS SPECTROMETER ........................................................... 110
5.1 Introduction .......................................................................................................... 110 5.2 Experimental Section ........................................................................................... 112 5.3 Results and Discussions ....................................................................................... 114 5.4 Conclusion ........................................................................................................... 123 5.5 Reference ............................................................................................................. 124
CHAPTER 6. GAS-PHASE REACTIVITY OF META-BENZYNES TOWARD AMINO ACIDS ....................................................................................... 126
6.1 Introduction .......................................................................................................... 126 6.2 Experimental Section ........................................................................................... 130 6.3 Results and Discussion ........................................................................................ 134 6.4 Conclusions .......................................................................................................... 155 6.5 References ............................................................................................................ 157
VITA ............................................................................................................................... 161
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LIST OF TABLES
Table .............................................................................................................................. Page
Table 3.1 MWD and AVG MW of Molecules in the Six Asphaltenes Samples, and Structural Information for the Eight Selected Ions .......................................... 75
Table 4.1 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Thiophenes ....................................................................................................... 98
Table 4.2 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Thiols ............................................................................................................... 99
Table 4.3 MS2 and MS3 CAD Product Ions (with Relative Abundances) for ................ 100
Table 4.4 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Sulfides ........................................................................................................... 101
Table 4.5 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Disulfides ....................................................................................................... 102
Table 4.6 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 CAD Product Ions for Ionized Thiols ........................................... 103 Table 4.7 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm)
of MS2 and MS3 CAD Product Ions for Ionized Polyaromatic Sulfur Compounds .. 103 Table 4.8 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 and MS3 CAD Product Ions for Ionized Sulfides ................................................ 104 Table 4.9 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 and MS3 CAD Product Ions for Ionized Disulfides ............................................ 105
Table 5.1 Reaction products (m/z values and branching ratios) and efficiencies for reactions of protonated sulfones and sulfoxides with TMP (PA = 222.2 kcal/mola). ...................................................................................................... 118
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Table Page
Table 5.2 Reaction products (m/z values and branching ratios) and efficiencies for reactions between protonated N-oxides, ketones, hydroxylamines, carboxylic acids, aliphatic and aromatic amines with TMP (PA = 222.2 kcal/mola). ...................................................................................................... 120
Table 5.3 Reaction products (m/z values and branching ratios) and efficiencies for reactions of protonated sulindac and sulindac sulfone with TMP (PA = 222.2 kcal/mola). ............................................................................................ 122
Table 6.1 Reaction efficiencies (Eff.) and product branching ratios for biradiucals a – d upon reaction with tetrahydrofuran, allyl iodide, dimethyl disulfide, tert-butyl isocyanide, and cyclohexane; secondary products are noted as (2o) and are listed after the primary products that produce them ................... 148
Table 6.2 Reaction efficiencies (Eff.) and product branching ratios for monoradicals upon reaction with L-glycine, L-leucine, L-proline, L-lysine, DL-lysine-ε-15N. ................................................................................. 150
Table 6.3 Reaction efficiencies (Eff.) and product branching ratios for biradiucals a – d upon reaction with glycine, L-leucine, L-lysine, and DL-lysine-ε-15N. .. 151
Table 6.4 Reaction efficiencies (Eff.) and product branching ratios for biradiucals a – d upon reaction with L-methionine and L-cysteine. ................................... 153
Table 6.5 Reaction efficiencies (Eff.) and product branching ratios for biradiucals a – d upon reaction with L-proline and L-phenylalanine. ................................ 154
xi
LIST OF FIGURES
Figure ............................................................................................................................. Page
Figure 2.1 Examples of two chemical reactions used to ionize a radical precursor (R). ..................................................................................................................... 9
Figure 2.2 Depiction of the ESI process in positive ion mode. ........................................ 11
Figure 2.3 Depiction of the APCI process in positive ion mode. ..................................... 12
Figure 2.4 Schematic of the Thermo Scientific LQIT mass spectrometer with operating pressure indicated for each region of the instrument. ...................... 15
Figure 2.5 Components of the API stack and ion guides to which a downhill ................. 17
Figure 2.6 Schematic of the Thermo Scientific LQIT mass analyzer. .............................. 18
Figure 2.7 The oscillating RF potentials applied to the x- and y-axis pairs of rods ......... 19
Figure 2.8 The most well-defined stability region of Mathieu stability diagram for LQIT. The circles of different sizes represent ions of different masses. Ions that fall outside of this region have unstable motions in x and/or y directions as indicated. ..................................................................................... 22
Figure 2.9 Diagram of DC potential well for trapping ions in the center along the z-axis. .................................................................................................................. 23
Figure 2.10 Illustration of ion ejection from LQIT at (a) = 0.908 during mass selective instability scan, and (b) = 0.880 during resonance ejection. ........ 25
Figure 2.11 Depiction of the ion detection system in Thermo Scientific LQIT. .............. 26
Figure 2.12 The sequence of events for ion isolation and fragmentation by collision-activated dissociation (CAD). The ion of interest is represented by the green circle. ........................................................................................... 28
Figure 2.13 Tailored RF waveform for ion isolation. ....................................................... 29
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Figure ............................................................................................................................. Page
Figure 2.14 Schematic of an LQIT with a manifold set-up to introduce neutral reagent for ion-molecule reactions. .................................................................. 31
Figure 2.15 Schematic of the 3-Tesla dual-cell FT-ICR mass spectrometer utilized in this research. ................................................................................................ 33
Figure 2.16 Details of the dual-cell in FT-ICR. Reproduced with permission from Nicolet FT-MS 2000 instruction manual. Copyright 1985 Thermo Fisher Scientific Inc. ................................................................................................... 35
Figure 2.17 Cyclotron motion of a positively charged ion in the FT-ICR. ...................... 38
Figure 2.18 Depiction of the cyclotron motion and trapping motion of a positively charged ion in an ICR cell ................................................................................ 40
Figure 2.19 Depiction of an ion's cyclotron motion (little circles) and magnetron motion (large circles). It should be noted that the magnetron radius depicted here is exaggerated. ........................................................................... 42
Figure 2.20 The sequence of events for ionization of a radical precursor, generation of radical sites and ion-molecule reaction in a dual-cell FT-ICR. ................... 44
Figure 2.21 Illustration of (a) trapping a positively charged ion; (b) transfer of the ion into the other side of a dual-cell FT-ICR ................................................... 46
Figure 2.22 Depiction of (a) quadrupolar excitation for quadrupolar axialization (QA) and (b) dipolar excitation for ion detection. ........................................ 47
Figure 2.23 Illustration of ion excitation and detection in an FT-ICR cell. (a) Ions are kinetically excited to move coherently as ion packets. (b) An ion packet passes by the detection plates and induces an image current, which is converted to frequency domain spectrum and finally mass spectrum. .......................................................................................................... 49
Figure 2.24 Comparison of chirp excitation (left) and SWIFT excitations (right). .......... 51
Figure 2.25 Comparison of potential energy surfaces for ion-molecule reactions in the gas phase (top) and in solution (bottom). ................................................... 55
Figure 2.26 Brauman double-well potential energy surface illustrating the entropy constraints for a gas-phase ion-molecule reaction. .......................................... 56
Figure 2.27 A semi-logarithmic plot of the relative abundances of a reactant ion and its products versus time ........................................................................................ 59
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Figure ............................................................................................................................. Page
Figure 3.1 APCI mass spectrum showing the MWD and AVG MW of the Maya asphaltene sample. ........................................................................................... 69
Figure 3.2 Fragmentation pattern of molecular ions of m/z 634 ± 1 of Surmont asphaltene sample. ........................................................................................... 70
Figure 3.3 MS2 CAD mass spectrum of ions of m/z 500 ± 1derived from the Maya asphaltene sample, with the maximum total number of carbons in alkyl chains and the estimated aromatic core size indicated. .................................... 71
Figure 3.4 MS3 CAD mass spectrum of [M-CH3]+ fragment ions of m/z 485 ± 1
that were formed from molecular ions of m/z 500 ± 1derived from the same Maya asphaltenes sample (Figure 3.3).................................................... 72
Figure 3.5 General trend for the approximate maximum total number of carbons in alkyl chains as a function of MW of the molecules derived from the six asphaltene samples. .......................................................................................... 76
Figure 3.6 General trend for the approximate aromatic core size as a function of MW of the molecules derived from the six asphaltene samples. ..................... 76
Figure 4.1 Two forms of the molecular ion of benzenethiol ........................................... 89
Figure 4.2 MS2 spectrum measured for the molecular ion of 2,2’-bithiophene, and MS3 spectra measured for its three fragment ions ................................................... 90
Figure 5.1 A mass spectrum measured after 100 ms reaction of protonated dibenzothiophene sulfone with TMP in LQIT (*secondary products of protonated TMP). ........................................................................................... 116
Figure 5.2 Mass spectra measured after 300 ms reaction of protonated sulindac (top) and sulindac sulfone (bottom) with TMP in LQIT (*secondary products of TMP adducts; ** secondary products of protonated TMP) ......... 117
Figure 6.1 Structures of ortho- (1), meta- (2), and para-benzyne (3) ............................ 128
Figure 6.2 Structures of the meta-benzyne analogues (a-d) and related monoradicals (e-g) studied ........................................................................... 135 Figure 6.3 Relative energy versus dehydrocarbon atom separation for
meta-benzyne analogues a-d .......................................................................... 136
xiv
LIST OF SCHEMES
Scheme Page
Scheme 4.1 Fragmentation pathways for the molecular ions of (a) benzenethiol, and (b) benzyl mercaptan upon multiple-stage CAD. ................................... 88 Scheme 4.2 Fragmentation pathways for the molecular ion of diphenyl sulfide upon multiple-stage CAD. ............................................................................. 92 Scheme 4.3 Fragmentation pathways for the molecular ion of benzyl sulfide upon multiple-stage CAD. ...................................................................................... 93 Scheme 4.4 Fragmentation pathways for the molecular ion of phenyl disulfide upon multiple-stage CAD. ............................................................................. 95 Scheme 4.5 Fragmentation pathways for the molecular ion of dicyclohexyl disulfide upon multiple-stage CAD.. ............................................................................ 96 Scheme 4.6 Fragmentation pathways for the molecular ion of dibenzyl disulfide upon multiple-stage CAD.. ............................................................................ 97 Scheme 4.7 Fragmentation pathways for the molecular ion of butyl disulfide upon multiple-stage CAD.. ............................................................................ 98 Scheme 5.1 The proposed mechanism for the formation of a stable [TMP adduct- MeOH] product ion when a protonated sulfone reacts with TMP. ...............117 Scheme 6.1 Proposed mechanism for the formation of adduct – COOH for biradical d upon reaction with lysine .......................................................................... 143 Scheme 6.2 Proposed mechanism for HSCH3 abstraction from methionine by biradical c.. ....................................................................................................144
Scheme 6.3 Proposed radical mechanism for H2O abstraction from proline by biradical c. .....................................................................................................145
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Scheme Page
Scheme 6.4 Proposed nonradical mechanism for H2O abstraction from proline by biradical c.. .................................................................................................. 146
Scheme 6.5 Proposed mechanism for H2O abstraction from proline by biradical d. ..... 147
Scheme 6.6 Proposed mechanism for formation of adduct and adduct-CO2 for biradical d upon reaction of with proline. .....................................................147
xvi
ABSTRACT
Weijuan Tang. Ph.D., Purdue University, May 2015. Mass Spectrometric Studies on Petroleum Asphaltenes and Organosulfur Compounds, on Functional-Group Selective Ion-Molecule Reactions and on Gas-phase Reactivity of meta-Benzynes toward Amino Acids. Major Professor: Hilkka I.Kenttämaa.
Mass spectrometry has found a wide variety of applications in many fields of
study, such as fundamental chemistry, biological science, food and fuels, advanced
materials, etc. Due to its high sensitivity, selectivity and speed, mass spectrometry
provides an invaluable tool for direct mixture analysis. When coupled with separation
methods, such as gas chromatography or high performance liquid chromatography,
analysis of minor components in complex mixtures is possible. In addition to the
molecular weight information, mass spectrometers can provide structural information for
the ionized analyte molecules. However, mass spectrometric analysis of complex
mixtures is not without challenges, such as suitable evaporation/ionization methods are
not readily available for different types of samples. For example, because of such
limitations, little is known about the molecular weight or structural information of
asphaltenes, which are the heaviest components of crude oil and one of the most complex
mixtures in nature. Characterization of asphaltenes at the molecular level can alleviate
some of the problems they cause to petroleum industry and facilitate the discovery of
beneficial uses for asphaltenes.
xvii
Multiple-stage tandem mass spectrometry (MSn) based on collision-activated
dissociation (CAD) is usually a method of choice for structural elucidation of unknown
compounds. However, this method alone does not always unambiguously identify the
functional groups in an unknown analyte. Therefore, tandem mass spectrometry (MS/MS)
based on ion-molecule reactions was developed and implemented in a linear quadrupole
ion trap (LQIT) mass spectrometer for functional group identification. This method has
great potential for rapid identification of unknown drug metabolites in the pharmaceutical
industry.
Gas-phase ion-molecule reactions are also very useful in study of reaction kinetics
and mechanisms. The intrinsic chemical properties of such highly reactive molecules as
radicals can be studied in the gas phase, which are otherwise difficult to access by other
experimental approaches. Knowledge on the reactivity of aromatic carbon centered σ,σ-
type biradical intermediates is desirable as they are associated with the biological activity
of a naturally occurring enediyne antitumor agents. Of particular interest is the reactivity
of 1,3-biradical species (meta-benzynes) because of its therapeutic importance. In this
thesis, the reactivity of four meta-benzyne analogues towards eight amino acids was
examined by using “distonic ion approach” in a Fourier-transform ion cyclotron
resonance (FT-ICR) mass spectrometer.
The experiments described in this thesis were aimed to provide more detailed
structural information of mixture components by using different mass spectrometry based
methods. Chapter 2 briefly describes the theory, instrumentation, and experimental
aspects of the two instruments used for these studies. Chapter 3 focuses on structural
comparisons of asphaltenes of different origins by using multiple-stage tandem mass
xviii
spectrometry. Chapter 4 describes structural characterization of organosulfur model
compounds related to fossil fuels by using high-resolution tandem mass spectrometry.
Chapter 5 focuses on development of gas-phase ion-molecule reactions for the
identification of the sulfone functionality in drug metabolites. Chapter 6 is devoted to the
study of gas-phase reactivity of pyridine, quinoline, and isoquinoline based meta-
benzynes towards various amino acids.
PUBLICATIONS
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1
CHAPTER 1. INTRODUCTION AND OVERVIEW
1.1 Introduction
With more than a century of development, mass spectrometry (MS) has
undergone tremendous technological improvements. It has become one of the most
powerful, sensitive, selective, and versatile analytical techniques.1,2 Mass spectrometric
analysis includes three key steps: sample evaporation and ionization, separation of ions
by their mass-to-charge (m/z) ratios, and detection of the ions.3After sample evaporation,
the neutral analyte molecules are converted to ions. Numerous efforts have been
dedicated to the development of methods that allow ionization of different analytes
effectively, from small organic molecules to large biomolecules.3 The resulting ions are
then separated based on their m/z ratios, which can be done using various mass analyzers,
including those that utilize a combination of electric and magnetic fields under vacuum
conditions. After the ions are detected, mass spectra are generated, which show a plot of
the relative abundances of the ions as a function of their m/z ratios. If a molecular ion or
pseudo-molecular ion is formed for each molecule, the molecular weight information of
the analyte can be obtained. Different isotopes of a given element can also be easily
distinguished.4 It should be noted that high-resolution mass spectrometers can measure
accurate masses of the ions, which provides elemental composition for an unknown ion.
2
In addition to molecular weight information, mass spectrometers are capable of
providing structural information for the ionized analyte molecules. Multiple-stage tandem
mass spectrometry (MSn) is an important approach to achieve this.5 By isolating and
subjecting the ion of interest to collision-activated dissociation (CAD), it often generates
characteristic fragmentation products that provide structural information for the analyte.6
As another alternative to probe the structure of the ionic analytes, ion-molecule reactions
have been explored extensively.7-11 The ion of interest can be allowed to react with
selected neutral reagents, producing diagnostic products that facilitate identification of
different functional groups in the analyte molecules. This is particularly useful when
CAD alone does not provide enough structural information for the analytes. Isomer
differentiation also often gets easier when using ion-molecule reactions in mass
spectrometers.12,13
Because of the uniquely valuable information MS can provide, it has found a wide
range of applications in qualitative and quantitative analysis of both small molecules and
big polymers.14 MS has become an indispensable analytical tool in such areas as drug
discovery, clinical analysis, biological science, environmental chemistry, geological
study, and many others.15 MS has been successfully coupled with chromatographic
separations for the analysis of minor components in complex mixtures, which are
difficult to analyze by other techniques, such as nuclear magnetic resonance.16-18
3
1.2 Overview
This dissertation focuses on the structural characterization of petroleum
asphaltenes and organosulfur compounds in them, development of methods for drug
metabolite identification based on functional group selective ion-molecule reactions, and
exploring gas-phase reactivity of meta-benzynes towards amino acids. Chapter 2 briefly
describes the theory, instrumentation, and experimental aspects of the two instruments
used for these studies. They are linear quadrupole ion trap (LQIT) and Fourier transform
ion cyclotron resonance (FT-ICR) mass spectrometers. Chapter 3 focuses on structural
comparisons of asphaltenes of different origins by using multiple-stage tandem mass
spectrometry. Chapter 4 describes structural characterization of organosulfur model
compounds related to fossil fuels by using high-resolution tandem mass spectrometry.
Chapter 5 focuses on the development of gas-phase ion-molecule reactions for the
identification of the sulfone functionality in drug metabolites. Chapter 6 is devoted to the
study of gas-phase reactivity of pyridine, quinoline, and isoquinoline based meta-
benzynes towards various amino acids.
4
1.3 References
1. McLuckey, S.; Wells, J. Chem. Rev.2001,101, 571.
2. Perry, R. H.; Cooks, R. G.; Knoll, R. J. Mass Spectrom. Rev. 2008, 27, 661.
3. de Hoffman, E.; Stroobant, V. Mass Spectrometry: Principles and Applications, 2nd ed.; John Wiley and Sons, Ltd.: New York, 2002.
4. McLafferty, F. W. Interpretation of Mass Spectra, 4th ed.; University Science Books: Sausalito, CA, 1993.
5. Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry:
Techniques and Applications of Tandem Mass Spectrometry; VCH Publishers: New York, 1988.
6. McLuckey, S.J. Am. Soc. Mass Spectrom. 1992, 3, 599.
7. Brodbelt, J. S. Mass Spectrom. Rev. 1997, 16, 91.
8. Eberlin, M. N. J. Mass Spectrom. 2006, 41, 141.
9. Watkins, M. A.; Price, J. M.; Winger, B. E.; Kenttämaa, H. I. Anal. Chem.2004, 76, 964.
10. Habicht, S.; Vinueza, N.; Archibold, E.; Duan, P.; Kenttämaa, H. I. Anal. Chem. 2008, 80, 3416.
11. Sheng, H.; Williams, P. E.; Tang, W.; Riedeman, J. S.; Zhang, M.; Kenttämaa, H. I. J. Org. Chem. 2014, 79, 2883.
12. Schwartz, J.; Wade, A.; Enke, C.; Cooks, R., Anal. Chem.1990,62, 1809.
13. Fu, M.; Duan,P.; Li,S.; Habicht, S. C.; Pinkston, D. S.; Vinueza, N. R.; Kenttämaa, H. I. Analyst, 2008,133, 452.
14. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse,C. M. Science1989, 246, 64.
15. Gross, J. H., Mass Spectrometry, a textbook. Springer: New York, 2004.
5
16. McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. 3rd. Anal Chem. 1997, 69, 767.
17. Ermer, J.; Vogel, M. Biomed. Chromatogr.2000,14, 373.
18. Loegel, T. N.; Danielson, N. D.;Borton, D. J.; Hurt, M. R.;Kenttämaa, H. I. Energy Fuels, 2012, 26, 2850.
6
CHAPTER 2. THEORY, INSTRUMENTATION AND EXPERIMENTAL ASPECTS OF LINEAR QUADRUPOLE ION TRAP (LQIT) AND FOURIER TRANSFORM
ION CYCLOTRON RESONANCE (FT-ICR) MASS SPECTROMETRY
2.1 Introduction
With its unique attributes in sensitivity, selectivity and versatility, mass
spectrometry (MS) has become an invaluable analytical tool across a broad range of
applications.1-4 Mass spectrometric analysis involves three key steps: sample evaporation
and ionization, separation of ions by their mass-to-charge (m/z) ratios, and detection of
the ions. In the ion source, the analyte molecules are evaporated and ionized by addition
or loss of proton(s), cation(s), anion(s) or electron(s).1 Mass analyzers, which separate the
ions based on their m/z ratios, are the core components of mass spectrometers. Generally,
they can be categorized into two types, trapping and scanning.2 In trapping mass
analyzers, the ions are manipulated at different points of time while confined in the same
space. In scanning mass analyzers, the selected ions fly to different regions of the
instrument for mass spectrometric manipulations. Finally, the separated ions reach the
detector where they are detected.
In this thesis, two mass spectrometers with trapping mass analyzers were
employed: linear quadrupole ion trap (LQIT) mass spectrometer and Fourier-transform
ion cyclotron resonance (FT-ICR) mass spectrometer. The theory, instrumentation, and
7
experimental aspects of these two mass spectrometers are discussed in the following
sections.
2.2 Ionization Methods
There are different ways to ionize the analyte molecules in mass spectrometry,
such as electron ionization (EI),5 chemical ionization (CI),6 electrospray ionization
(ESI),7,8 atmospheric pressure chemical ionization (APCI),9,10 atmospheric pressure
photoionization (APPI),11 desorption electrospray ionization (DESI),12 direct analysis in
real time (DART),13 matrix-assisted laser desorption ionization (MALDI),14 inductively
coupled plasma (ICP)ionization,15 fast atom bombardment (FAB),16 field desorption /
field ionization (FD/FI),17 and laser-induced acoustic desorption (LIAD) / ionization,18,19
among others. Each ionization method has its own advantages and disadvantages, and a
choice is often made depending on the nature of the molecules to be analyzed. Four
ionization methods, EI, CI, ESI, and APCI, were employed for the work in this
dissertation and will be discussed in detail in the following sections.
8
2.2.1 Electron Ionization (EI)
Electron ionization (EI), introduced by Dempster in 1918, is the oldest ionization
method in mass spectrometry.5 Ionization is achieved by bombarding the analyte
molecule with a beam of energetic electrons (typically ~70 eV) in the gas phase.20
Molecular ion of the analyte molecule can be generated if the kinetic energy of the
electrons is greater than the ionization energy of the analyte. However, EI can deposit
more energy than needed for ionizing the molecule, thus fragmentation may occur.
Although molecular weight information is not retained in this case, it can be useful for
structural elucidation of the analyte based on reproducible fragmentation patterns
produced by EI.
2.2.2 Chemical Ionization (CI)
Chemical ionization (CI) is a soft ionization method, which can generate pseudo-
molecular ion of the analyte molecule with minimum fragmentation.6 Therefore,
molecular weight information can be obtained. The analyte of interest is ionized through
chemical reactions with reagent ions, such as electron and/or group transfer.21,22 Figure
2.1 gives two examples of chemical reactions to ionize a radical precursor (R). Figure 2.1
(a) shows how a protonated radical precursor [R+H]+ is generated via self-chemical
ionization (self-CI) and CI. First, an acetone molecule undergoes self-CI with an acylium
ion that was generated by EI of another acetone molecule, forming protonated acetone
ion as the final CI reagent ion. Then the neutral radical precursor is allowed to react with
the CI reagent ion to generate protonated radical precursor. This proton transfer reaction
has to be exothermic to occur. Typically, a CI reagent with PA slightly lower than that of
th
sh
un
ca
an
an
ca
co
ra
d
ot
m
he analyte m
hows how
ndergoes EI
ation to the n
Figure 2.1
Electr
nalysis.7,8 It
nalytes, such
an form mu
orresponding
anges. Proto
eprotonation
ther ionizati
most basic or
molecule is c
a methylate
I and self-CI
neutral radic
Examples o
rospray ioniz
has been su
h as proteins
ultiply charg
g analytes, w
onation typic
n generally
ion methods
r acidic analy
chosen, such
ed radical
I to form dim
cal precursor
f two chemi
2.2.3 Elec
zation (ESI)
uccessfully a
s, oligonucle
ged gas-pha
which enabl
cally occurs
occurs for m
, ESI has lim
ytes in a com
h as acetone
precursor [
methyl iodid
r, subsequen
cal reactions
ctrospray Ion
is a soft ion
applied to ge
eotides, lipid
ase ions, th
les their det
s for molecu
molecules in
mitation in te
mplex mixtur
e or methan
[R+CH3]+ is
de cation, w
ntly generatin
s used to ion
nization (ES
nization tech
enerate gase
ds, etc.23,24 U
herefore redu
tection in in
ules in posi
n negative i
erms of ioni
re are ionize
nol. Similarly
s generated
which then tr
ng a methyla
nize a radica
I)
hnique for m
eous ions for
Upon ESI, la
ucing the m
nstruments w
itive ionizat
onization m
ization bias,
ed.
y, Figure 2.
d. Methyl io
ransfers a m
ated molecul
l precursor (
ass spectrom
r thermally l
arge biomole
m/z ratios o
with limited
tion mode, w
mode. As wit
because onl
9
.1 (b)
odide
methyl
le.
(R).
metric
labile
ecules
of the
mass
while
th all
ly the
10
In electrospray ionization, ions are preformed in solution, and they are transferred
from solution to gas phase through a stepwise process.26 As shown in Figure 2.2, the
solution containing the analyte of interest first passes through a high voltage needle (±3-5
kV), from which a fine mist of charged droplets is ejected.24 With the assistance of
nebulizing gas (typically nitrogen), the solvent molecules keep evaporating and the
droplets shrink in size, which causes the charges to move towards each other. Once the
charges are concentrated to a critical point, known as the Rayleigh stability limit, the
droplet explodes to produce smaller droplets. This process occurs as the Coulombic
repulsion overcomes the surface tension of the droplet, and it repeats until singly or
multiply charged gas-phase ions are released and reach the mass spectrometer inlet.26
io
a
g
ca
re
so
to
co
Atmo
onization tec
method of
enerate ionic
an be obtain
elies on a ser
olution conta
o several hu
ommonly us
Figure 2.2
2.2.4 At
spheric pres
chnique.9,10 A
choice for
c molecules
ned, althoug
ries of reacti
aining the an
undred degr
sed as sheat
Depiction of
mospheric P
ssure chemi
APCI is often
analytes wi
with no or l
gh this is not
ions to ioniz
nalyte of int
rees of Cels
th and auxili
f the ESI pro
Pressure Che
cal ionizatio
n used to ion
ith low to m
little fragmen
t always the
ze the analyt
terest flows i
sius (300 °C
iary gas, als
ocess in posi
emical Ioniza
on (APCI)
nize thermal
medium pola
ntation, thus
e case.27 AP
te of interest
into the ion
C - 500 °C
so assists wi
itive ion mo
ation(APCI)
is also cons
lly stable mo
arity. APCI
s molecular m
PCI is simila
t. As shown
source, whe
C) and vapo
ith the solut
de.
)
sidered as a
olecules. It is
can be tun
mass inform
ar to CI in t
in Figure 2.3
ere it is heate
orized. Nitro
tion nebuliza
11
a soft
s also
ned to
mation
that it
3, the
ed up
ogen,
ation,
cr
h
b
so
m
to
re
pr
reating a fin
igh-voltage
elow) occur
A typ
olvent.28 Firs
molecules) to
o form reage
eagent ions,
rotonated m
ne mist of dr
corona disch
in the zoom
Figure 2.3 D
pical APCI r
st, the coron
o produce the
ent ions H2O
H3O+. The
molecules in p
oplets. Whe
harge needle
med plasma r
Depiction of
reaction seri
na discharge
e primary io
O+, which th
final reagen
positive ion
en the mist le
e, where a se
egion.
the APCI pr
ies is illustr
needle ioniz
ons. These pr
hen undergo
nt ions then
mode. It sh
eaves the va
equence of c
rocess in pos
rated below,
zes the carri
rimary ions
ion-molecul
ionize the a
hould be note
aporizer heat
chemical rea
sitive ion mo
where wat
er gas N2 (th
react with s
le reactions
analyte mole
ed that the s
ter, it passes
actions (discu
ode.
er is used a
he most abun
solvent mole
to form the
ecules, gener
specific choi
12
s by a
ussed
as the
ndant
ecules
final
rating
ice of
so
th
olvent, such
he types of io
as carbon d
ons formed i
disulfide, tolu
in APCI.29,30
uene, or chlo
0
oroform, as well as sheaath gas can a
13
affect
14
2.3 Linear Quadrupole Ion Trap (LQIT) Mass Spectrometry
2.3.1 Introduction
Linear quadrupole ion trap (LQIT) mass spectrometers have found a wide range
of applications since they were first introduced in 2002.31,32 These 2-dimensional (2-D)
ion traps operate by trapping the ions in a confined space while performing ion
manipulations based on time. The underlying principles of 2-D LQIT are similar to those
of 3-D quadrupole ion traps (QIT).33 However, LQITs have improved ion trapping
capacity and efficiency, which results in higher sensitivity.31 Compared to the traditional
QIT, LQIT has five-fold lower detection limit.31 In order to combine tandem mass
spectrometry capabilities of LQIT with high-resolution measurements, several hybrid
instruments emerged not long after the stand-alone LQIT was introduced, such as LQIT-
TOF,34 LQIT-Orbitrap,35 and LQIT-FT-ICR.36 The latter two hybrid instruments were
used to perform accurate mass measurements for part of the work discussed here.
2.3.2 Instrument Overview
LQIT experiments were performed using a Thermo Scientific LTQ linear
quadrupole ion trap mass spectrometer.31,37 A general schematic is shown in Figure 2.4.
The LQIT is equipped with an atmospheric pressure ionization source, such as ESI or
APCI. In these instruments, the ionized molecules are drawn into the API stack region,
whose pressure is maintained at approximately 1 Torr by two Edwards E2M30 rotary-
vane mechanical pumps (10.8 L/s). The ions then travel through a series of ion optics and
reach the mass analyzer. The pressure of the ion trap is maintained at approximately 0.5-
1
tu
pu
ef
m
st
g
(7
.0 x 10-5 T
urbomolecul
umps (10.8
fficiencies a
multipole Q0
Figure 2.4 S
Comp
tack compri
enerated, a n
760 Torr, 3-
Torr by on
lar pump, wh
L/s). Anot
are 25 L/s an
region (1 m
Schematic ofpressu
ponents of th
ises a trans
negative pre
5 kV) to the
ne inlet (40
hich is back
ther two inl
nd 300 L/s, p
mTorr) respec
f the Thermoure indicated
he API stack
sfer capillar
essure gradie
e heated tran
00 L/s) of
ked by two E
lets of the
pump down t
ctively.
o Scientific Lfor each reg
k and ion gu
ry, tube len
ent and large
nsfer capillar
a Leybold
Edwards E2M
turbomolecu
the multiple
LQIT mass sgion of the in
uides are sh
ns, and skim
e potential d
ry (1 Torr, ±
d TW220/15
M30 rotary-
ular pump,
Q00 region
spectrometernstrument.
hown in Figu
mmer cone.
decrease fro
±20 V) draw
50/15 triple
-vane mecha
whose pum
(500 mTorr
r with opera
ure 2.5. The
. After ion
m the ion so
w the ions int
15
e-inlet
anical
mping
r) and
ating
e API
s are
ource
to the
16
API stack. A potential of 0 to ±10 V (positive for positive ions and negative for negative
ions) is applied to the transfer capillary to aid in focusing ions into a concentrated beam.
Additionally, the transfer capillary is normally heated to 250-300 °C to aid in ion
desolvation. A mass-dependent potential is applied to the tube lens to focus the ions into
a tighter packet for transmission. It should be noted that the voltage on tube lens can
increase the kinetic energy of the ions, which may induce fragmentation. The slightly off-
axis skimmer is a grounded lens (0 V) used to remove neutral molecules and act as a
vacuum baffle to the lower pressure ion guide region.
After exiting the API stack, ions are guided through a series of lenses and
multipoles into the mass analyzer. Q00 and Q0 are quadrupole assemblies with square
rods; multipole Q1 is an octupole assembly with round rods. RF potentials are applied to
the rods of these multipoles so that the ions are confined in the x-y plane. A downhill DC
potential gradient is applied to the lenses and multipoles so that the ions gain kinetic
energy that facilitates their transmission further into the ion trap.
ro
m
sl
an
FigureDC p
The m
ods. Each of
mm) sections
lits (0.25 mm
nd an electro
e 2.5 Compopotential grad
mass analyze
f the four ro
s, as shown i
m × 30 mm
on multiplier
nents of the dient was ap
er of the LQ
ds is divided
in Figure 2.6
dimension)
r is on each
API stack applied to aid
QIT mass sp
d into front
6. At the x-a
which allow
side of the tr
and ion guidein ion transf
pectrometer
(12 mm), ce
axis of the c
w for ion eje
rap for ion d
es to which afer in the z-d
r consists of
enter (37 mm
enter two ro
ection. A co
detection.
a downhill direction.
f four hyper
m), and bac
ods, there are
onversion dy
17
rbolic
k (12
e two
ynode
cr
p
ro
to
tr
Fig
Ions a
reates an o
otential (±5
ods so that th
o all three se
rapped axiall
gure 2.6 Sch
are trapped
scillatory io
kV, 1.2 MH
he ions are t
ections of th
ly (z-directio
hematic of th
2.3.3
in LQIT b
on motion.33
Hz) with opp
trapped radia
he mass ana
on).
he Thermo S
Ion Motions
y a combin
3,38-41 As sh
posite polarit
ally (x-y pla
alyzer to cre
Scientific LQ
s in LQIT
nation of RF
hown in Fig
ty is applied
ane). A statio
ate a potent
QIT mass ana
F and DC p
gure 2.7, an
d to the x- an
onary DC po
tial well, so
alyzer.
potentials, w
n oscillating
nd y-axis pa
otential is ap
that the ion
18
which
g RF
airs of
pplied
ns are
2
p
ca
w
th
el
ar
th
Figure 2.
.3.3.1 Radia
In ord
otentials is a
an be describ
wherein is
he angular fr
lectric field
re subject to
hat trap the i
.7 The oscill
al Motion
der to trap
applied to th
bed as show
the DC pot
requency of
between the
o a potential
ons in the x
lating RF pofor trapping
the ions ra
he four hyper
wn in Equatio
Ф
ential, is t
the RF field
e four rods. W
in the x-y p
and y direct
tentials applg ions radial
adially (x-y
rbolic rods o
on 2.1.
the zero-to-p
d, and is tim
When the io
lane, which
tions are sho
lied to the x-lly (x-y plan
plane), a c
of the LQIT
cos Ω
peak amplitu
me. These po
ons are trans
is described
own in Equat
- and y-axis ne).
combination
mass analyz
ude of the R
otentials crea
sferred into t
d by Equatio
tions 2.3 and
pairs of rods
n of RF and
zer. The pote
Equatio
RF potential
ate a quadru
the ion trap,
on 2.2. The f
d 2.4.
19
s
d DC
ential
on 2.1
l,Ω is
upolar
, they
forces
20
Ф , Ф Equation 2.2
Ф Equation 2.3
Ф Equation 2.4
wherein is the mass of the ion, is the elementary charge, is the number of charges
of the ion, and is the radius of the circle inscribed within the ion trap. Rearrangements
of the above equations result in Equations 2.5 and 2.6 respectively.
cosΩ 0Equation 2.5
cosΩ 0Equation 2.6
These two equations are similar to the general form of Mathieu equation (Equation 2.7).
2 cos 2ξ 0Equation 2.7
The Mathieu equation can be rearranged into Equation 2.8 by defining as ,
cos Ω 0 Equation 2.8
21
Therefore, the radial motion of an ion can be described as shown in Equations 2.9 and
2.10.
Equation 2.9
Equation 2.10
wherein and are Mathieu stability parameters. An ion will have a stable trajectory
in the LQIT only when its and values fall within the stability region. The most
well-defined stability region is shown as the overlap area in Figure 2.8. Ions that fall
outside of this region have unstable motions in x and/or y directions as indicated.
Generally, the LQIT operates with 0 so that a broad range of ions can be trapped
simultaneously. Since value is inversely proportional to the ion's m/z ratio, larger ions
have lower values while smaller ions have higher values. This is illustrated by the
circles of different sizes in Figure 2.8, which represent ions of different masses.
F
se
w
fr
Figure 2.8 ThThe circles o
this
At a g
ecular (reso
wherein
requency
he most wellof different s
s region have
given RF pot
nant) freque
of an ion is
l-defined stasizes represee unstable m
tential, each
ency, which
. Since th
s one half of
ability regionent ions of d
motions in x a
h ion with a d
is described
e maximum
f the RF frequ
n of Mathieudifferent masand/or y dire
different m/z
d in Equation
m value of
uency.38
u stability disses. Ions thaections as ind
z ratio oscill
n 2.11,
is 1, the m
agram for Lat fall outsiddicated.
lates at a spe
Equation
maximum se
22
QIT. de of
ecific
n 2.11
ecular
2
p
H
th
T
in
es
d
en
F
.3.3.2 Axial
In ord
otentials are
Higher poten
hat a potenti
Trapping ions
n the x-rods
Addit
ssential for i
irection and
nergy.
Figure 2.9 D
Motion
der to trap
e applied to t
ntials are app
al well is cre
s in the cent
, but also re
ionally, the
ion trapping
d collisions w
Diagram of D
ions axially
the front, ce
plied to the
eated, which
ter not only
educes fring
helium buf
g.31 Ions gain
with the buff
DC potential
y (z-directio
enter and bac
front and ba
h traps the io
facilitates ef
e field effec
ffer gas, wh
n kinetic ene
fer gas help
well for trap
on) in the m
ck sections o
ack sections
ons in the ce
fficient ion e
cts created b
hich is prese
ergy while b
to cool the
pping ions in
mass analyz
of the four h
s than the m
enter, as show
ejection thro
by the front
ent in LQIT
being transfe
ions and red
n the center a
zer, differing
hyperbolic ro
middle sectio
wn in Figure
ough the two
and back le
at ~3 mTo
erred along t
duce their ki
along the z-a
23
g DC
ods.31
on, so
e 2.9.
o slits
enses.
orr, is
the z-
inetic
axis.
24
2.3.4 Ion Excitation and Detection
An ion's radial motion in quadrupole fields can be expressed mathematically in
terms of the Mathieu equations. As shown in equations 2.9 and 2.10, is proportional to
the DC voltage while is proportional to the amplitude of the applied RF voltage. The
LQIT normally operates at = 0. By scanning the amplitude of the RF voltage linearly,
ions of increasing m/z ratios will be pushed to the instability boundary at = 0.908. At
this point, ions’ motion becomes unstable and the ions are ejected through the slits in the
x-rod for detection. This process is known as the “mass selective instability scan”.42 This
technique suffers from low mass spectral resolution because not all ions of a specific m/z
ratio are ejected from the trap simultaneously.
In order to increase mass resolution, another technique known as "resonance
ejection" is often employed.43 Using this technique, ions are scanned out at = 0.880, as
shown in Figure 2.10. This is achieved by applying a small supplemental RF voltage of
fixed frequency during the ramp of the main RF voltage. When an ion is about to be
ejected from the trap by the main RF voltage, it is brought to resonance with the
supplemental RF voltage. This facilitates ion ejection from the trap and improves mass
resolution and sensitivity.
F
d
si
co
ap
g
el
T
Figure 2.10 I
After
etection syst
ide of the i
onversion dy
pplied to it.
enerated. Po
lectrons, and
These second
Illustration oinstability s
ions are ejec
tem, which
on trap.31 A
ynode with
When an ion
ositively cha
d negatively
dary particle
of ion ejectioscan, and (b)
cted through
consists of a
As shown in
either -15 k
n hits the su
arged ions g
y charged io
es are focus
on from LQI) = 0.880
h the slits in
a conversion
n Figure 2.1
kV (for posi
urface of the
generate one
ons generate
sed by the c
IT at (a) =during reson
the x-rods,
n dynode and
11, the eject
itive ions) o
dynode, mu
e or more n
e one or m
concave surf
= 0.908 durinnance ejectio
they are dire
d electron m
ted ions are
or +15 kV (f
ultiple secon
negatively c
more positive
face of the
ng mass seleon.
ected toward
multiplier on
e attracted t
for positive
dary particle
charged ions
ely charged
dynode, an
25
ctive
ds the
n each
o the
ions)
es are
s and
ions.
d are
ac
th
m
th
th
fr
ccelerated to
he dynode a
multiplier, wh
he electron m
he number o
rom the trap.
Figure
oward an ele
and the elec
hich generat
multiplier. F
of secondary
.
2.11 Depicti
ectron multip
tron multipl
tes a cascad
Finally, a me
y particles, a
ion of the ion
plier becaus
lier.31 Each
de of electro
easurable cur
and thus pro
n detection s
e of a large
secondary p
ons that cont
rrent is crea
oportional to
system in Th
potential di
particle will
tinue to strik
ated, which i
o the numbe
hermo Scien
fference bet
l hit the ele
ke the surfa
is proportion
er of ions ej
ntific LQIT.
26
tween
ectron
ace of
nal to
ected
27
2.3.5 Tandem Mass Spectrometry
Multiple-stage tandem mass spectrometry (MSn) is a powerful technique to probe
the structure of an ionized unknown compound.44 After the ion of interest is isolated, it
can be subjected to collision-activated dissociation (CAD) to generate characteristic
fragment ions; or it can be allowed to undergo ion-molecule reactions to form diagnostic
product ions, both of which shed light on the structure of the ionic parent molecule. Since
LQIT is a trapping instrument, tandem mass spectrometry events occur in the same space
but sequentially in time.
2.3.5.1 Ion Isolation
As shown in Figure 2.12 I-IV, a series of events occur before an ion of interest
(represented by the green circle) is isolated and subjected to CAD. The ion of interest is
first placed at = 0.880 by ramping the RF voltage, during which process ions of lower
masses are ejected from the trap (I-II). Next, a tailored broadband RF waveform is
employed to eject all remaining unwanted ions, except the ion of interest (III). This
isolation waveform is similar to what is employed for ion excitation and detection as
described above. It consists of a 5–500 kHz multi-frequency waveform with sine
components spaced every 0.5 kHz, with a notch at q = 0.83 so that the ion of interest
remains in the trap (Figure 2.13). Finally, the RF voltage is decreased to move the ion of
interest to q = 0.25 (IV). Upon CAD, the parent ion dissociates into fragment ions (IV),
which can be efficiently trapped for detection or further dissociation reactions.
Figure 2.12activated d
2 The sequedissociation
nce of event(CAD). The
ts for ion isoe ion of inter
olation and frrest is repres
fragmentationsented by the
n by collisioe green circl
28
on-le.
2
w
bu
in
k
su
ap
m
en
R
am
"n
.3.5.2 Collis
In the
wherein ions
uffer gas ov
nterest is iso
inetically e
upplemental
pproximately
multiple colli
nergy and ev
RF voltage ap
In The
mount of k
normalized
Figure
sion-activate
e LQIT, col
undergo fr
ver a relative
olated, the q
excited by
l RF voltage
y 10-100 m
isions with t
ventually cau
pplied to the
ermo Scient
kinetic energ
collision en
e 2.13 Tailor
ed Dissociati
lision-activa
agmentation
ely long activ
value of 0.2
resonance
e (~ 1 V) ma
ms. During
the buffer g
uses the ion
e x-rods is re
tific LQIT, t
gy an excit
ergy" on a
red RF wave
ion (CAD)
ated dissocia
n after many
vation time.
25 is genera
excitation.
atching the s
this process
as, which co
s’ dissociati
ferred as "tic
wo paramet
ted ion gain
scale from
eform for ion
ation (CAD)
y low-energy
45,46 As disc
ally chosen.
This is ac
secular frequ
s, the kinet
onverts their
ion into frag
ckle voltage
ters are usua
ns during C
0% to 100%
n isolation.
) is a slow
y collisions
cussed above
The isolated
ccomplished
quency of the
tically excit
r kinetic ene
gment ions. T
e".
ally adjusted
CAD. One p
%. The high
heating me
with the he
e, once the i
d ion can the
by applyi
e isolated io
ed ions und
ergy into int
The supplem
d to determin
parameter i
her the value
29
ethod,
elium
on of
en be
ing a
on for
dergo
ternal
mental
ne the
s the
e, the
30
more energy is deposited into the parent ion. The exact collision energies are unknown.
Another factor is the q value. At higher q values, ions oscillate at a higher frequency and
have higher kinetic energies, which may result in more fragmentation upon collisions
with helium. Typically, a q value of 0.25 is chosen to activate the ion of interest. This
value allows most of the fragment ions to be efficiently trapped during CAD.31 For ions
that require higher energy to dissociate, a q value that is greater than 0.25 can be used.
However, this limits the mass range of fragment ions that can be trapped.
2.3.6 Ion-molecule reactions in LQIT
In addition to performing CAD experiments on the ion of interest, tandem mass
spectrometry experiments based on ion-molecule reactions are also of great use for
structural investigations.47 This is particularly true for isomer differentiation because
CAD may yield the same fragmentation patterns while ion-molecule reactions often show
distinct products for isomeric ions.48 Figure 2.14 shows a schematic of an LQIT with a
manifold set-up that can be used to introduce neutral reagents for ion-molecule reactions.
In this example, a neutral reagent, trimethyl phosphite, is introduced into the ion trap. The
neutral reagent is then allowed to react with the analyte ion of interest for various periods
of time, ranging from 30 ms to 10 s.
FFigure 2.14 S
Schematic off an LQIT wion-
with a manifo-molecule re
old set-up to actions.
introduce neeutral reagen
31
nt for
32
2.4 Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry
2.4.1 Introduction
In 1932, Ernest Lawrence and Stanley M. Livingston first discovered the principle
of ion cyclotron resonance (ICR).49,50 It was later introduced to mass spectrometry,
followed by the commercialization of the first ion cyclotron resonance (ICR) mass
spectrometer.51,52 Afterwards, the successful combination of Fourier transformation with
nuclear magnetic resonance (NMR) spectroscopy inspired Comisarow and Marshall to
couple Fourier transformation (FT) with ion cyclotron resonance (ICR). 53,54 In 1974, the
FT-ICR mass spectrometry was created. FT-ICR is a very powerful mass spectrometric
technique, offering ultra high resolution and high mass accuracy.55,56 Its many
applications include complex mixture analysis and accurate mass measurement, as
demonstrated in Chapters 3 and 4. Moreover, the high vacuum condition of FT-ICR
instrument allows the ions to be trapped for relatively long periods of time. Therefore,
gas-phase ion-molecule reactions and multiple stage MS/MS experiments can be
performed, as demonstrated in Chapter 6.
2.4.2 Instrument Overview
The gas-phase reactivity study of radicals described in this dissertation was
carried out in a ~3 Tesla dual-cell Extrel model FTMS 2001 mass spectrometer (Figure
2.15). The source side and analyzer side of the instrument are two differentially pumped
regions, the purpose of which is to maintain different pressures in each cell. The
instrument is equipped with an IonSpec data station running on IonSpec Omega 8
s
b
m
i
a
c
n
t
l
software. A
by two Pfeif
mechanical p
is measured
above the ce
convert the
neutral reage
Figure 2.1
Differ
to liquids an
liquids, such
high vacuum
ffer HiPace7
pump serves
by a Bayar
ell. Accordin
ion gauge re
ent concentr
15 Schemati
rent methods
nd gases. G
h as methyl
m with a nom
700 turbo pu
s as a back p
rd-Alpert ion
ngly, ion gau
eadings to r
ation in the c
ic of the 3-T
s can be use
aseous samp
iodide, can
minal baselin
umps, one on
pump for eac
nization gau
uge correcti
real pressure
cell (describ
esla dual-celin this resea
ed to introdu
ples, such a
be introduc
ne pressure o
n each side o
ch turbo pum
uge, which s
ion factors n
e, which allo
bed in section
ll FT-ICR march.
uce various
as helium an
ced via pulse
of ~1x10-9 T
of the instrum
mp. The pres
sits approxim
need to be m
ows for dete
n 2.5.2).
mass spectrom
samples ran
nd argon, as
ed valves. T
Torr is maint
ment. An A
ssure of each
mately one m
measured so
ermination o
meter utilize
nging from s
s well as vo
This instrume
33
ained
lcatel
h cell
meter
as to
of the
ed
solids
olatile
ent is
34
equipped with two sets of pulsed valves on the source side and one set on the analyzer
side. If a constant pressure rather than a pulse of the reagent is desired, batch inlets can
be employed for introduction of volatile liquids. For volatile solids and highly volatile
liquids that do not require heating, they can be introduced into the instrument by using
Varian leak valves. For nonvolatile and thermally stable solids, they are introduced into
the instrument by using solids probes, which can be heated up to ~250˚C to facilitate
sample desorption. Laser-induced acoustic desorption (LIAD) is an alternative method
of introducing nonvolatile and thermally labile samples.57 On either side of the
instrument, it is equipped with a heated solids probe, batch inlet and Varian leak valve.
Electron ionization filament, which emits electrons to induce ionization of the analytes,
is located in the analyzer side of the instrument.
The dual-cell consists of two cubic cells that are aligned collinearly within the
homogenous magnetic field. The two cells consist of a total of eleven stainless steel
plates, as shown in Figure 2.16. Three of them are perpendicular to the magnetic field.
They are trapping plates, which are used for trapping ions along the z-axis (discussed in
section 2.4.3.2). The trapping plate in the center, called the conductance limit, is shared
between the two cells.58 The three plates all have 2 mm diameter holes in the center,
which allow ions to be transferred from one cell into another, as well as allow the
electron beam to travel from the analyzer side into the source side for sample ionization.
The remaining eight stainless steel plates are parallel to the magnetic field. For each cell,
one pair of plates is used for ion excitation and another pair of plates for ion detection.
F
Figure 2.16 DFT-MS
Details of the2000 instruc
e dual-cell inction manual
n FT-ICR. Rl. Copyright
Reproduced wt 1985 Therm
with permissmo Fisher Sc
sion from Nicientific Inc.
35
icolet
36
2.4.3 Ion Motions in FT-ICR
In FT-ICR mass spectrometers, ions undergo three types of motion, cyclotron,
trapping, and magnetron motions.52,53 The electric fields applied to the trapping plates
confine the ions along the z-direction, whereas the homogeneous magnetic field confines
the ions in the x-y plane. The sum of the forces (F) an ion experience arises from the
electric and magnetic fields, and it can be described by Equation 2.12.53
Equation 2.12
wherein q is the ion's charge, E is the electric field, B is the strength of the magnetic field,
and is the velocity of the ion.
2.4.3.1 Cyclotron Motion
The most important ion motion, cyclotron motion, arises from the interaction of a
moving ion with the homogeneous magnetic field. The magnetic field exerts an inward-
directed Lorentz force, FLorentz, on the ion, as shown in Equation 2.13
FLorentz qvxyB Equation 2.13
wherein q is the ion's charge, vxy is the velocity of the ion in the x-y plane, and B is the
strength of the magnetic field. Furthermore, the ion also experiences an outward-directed
centrifugal force, FCentrifugal, as shown in Equation 2.14,
Equation 2.14
37
wherein m is the mass of the ion, vxy is the velocity of the ion in the x-y plane, and r is the
radius of its cyclotron motion.
As an ion moves circularly in a unidirectional magnetic field, the Lorentz force
and centrifugal force counter balance each other (Figure 2.17). Therefore Equations 2.13
and 2.14 are equal, resulting in Equation 2.15, which can also be rearranged to Equation
2.16. This equation shows that an ion's cyclotron radius ) is proportional to the mass-
to-charge ratio of the ion and its velocity in the xy-plane while inversely proportional to
the strength of the magnetic field.
Equation 2.15
Equation 2.16
Since frequency ѡ can be expressed as shown in Equation 2.17, the ion's
cyclotron frequency (ѡ ) in FT-ICR can be described as shown in Equation2.18,
ѡ Equation2.17
ѡ Equation2.18
An ion's cyclotron frequency is independent of its velocity or kinetic energy, and
proportional to the strength of the magnetic field and inversely proportional to the ion’s
mass to charge ratio. FT-ICR detects ions based on their cyclotron frequencies, which are
unique to the m/z ratios of the ions. Therefore, FT-ICR has higher mass accuracy and
re
sp
2
o
p
tr
is
esolution co
pectrometer
Figu
.4.3.2 Trapp
The m
f the cell if
otential (+2
rapping plate
s shown in F
ompared to
which meas
ure 2.17 Cycl
ping Motion
magnetic fiel
f no constra
V for posi
es to constra
Figure 2.18. T
o other m
sure ions' ma
lotron motio
d constrains
aint is impos
itive ions an
ain the ions
The trapping
, ,
ass spectro
asses based o
on of a positi
s ions in the
sed in the z
nd -2 V for
along the z-
g potential is
ometers, su
on their velo
ively charge
xy-plane, ye
z-direction. T
r negative i
-axis. Trappi
s shown in E
2
uch as tim
ocities.53
d ion in the
et the ions ca
Therefore, a
ions) is app
ing motion i
Equation 2.19
e-of-flight
FT-ICR.
an freely dri
a small repu
plied to the
in the z-dire
9.
Equation
38
mass
ft out
ulsive
three
ection
n 2.19
39
wherein is the voltage applied to the trapping plates, and and are constants related
to the geometry of the cell. For a cubic cell, = 0.3333 and = 2.77373. For a trapping
voltage of 2 V, the trapping plates have the maximum value of 2 V, whereas the center of
the cell has a minimum value of 0.67 V ( ). The frequency at which the ions oscillate
harmonically between the trapping plates (along z-direction) can be shown in Equation
2.20.
Equation 2.20
wherein is the charge of the ion, is the voltage applied to the trapping plates, is a
constant related to the cell's geometry, is the mass of the ion, and is the distance
between the trapping plates.
2
tr
m
th
un
in
ce
Fig
.4.3.3 Magn
Ions a
rapping moti
magnetron m
he electric fi
niform elect
n equation 2
enter of the c
gure 2.18 Dep
netron Motio
are constrain
ion, respecti
motion, canno
eld along the
tric field gen
.21. It oppos
cell, causing
piction of tha positively
on
ned in the x
ively. Howe
ot be neglect
e z-axis is no
nerates an ou
ses the Lore
g them to orb
F
he cyclotron charged ion
xy-plane an
ever, the pre
ted (Figure 2
ot uniform d
utward-direc
enz force and
bit at larger r
F r
motion and n in an ICR c
nd z-axis by
esence of an
2.19). This m
due to the fin
cted radial fo
d pushes the
radii in x-y p
trapping mocell.
y their cyclo
n undesirable
motion is du
nite size of th
orce (F , wh
e ions radiall
plane.
otion of
otron motion
e ion motion
ue to the fac
he cell. The
hich is illust
ly away from
Equation
40
n and
n, the
ct that
e non-
trated
m the
n 2.21
41
wherein is a constant related to the cell's geometry ( = 1.39 cm for a cubic cell), is
the charge of the ion, is the voltage applied to the trapping plates, is the distance
between the trapping plates, and is the radius of the magnetron motion.
The frequency of the magnetron motion ) is defined by Equation 2.22,
wherein is the cell geometry factor, is the voltage applied to the trapping plates, is
the distance between the trapping plates, and is the strength of the magnetic field. It
should be noted that the magnetron frequency is independent of an ion's m/z ratio, hence
all ions will have the same magnetron frequency. Generally, the magnetron frequencies
are not detected, because they are much smaller than the cyclotron frequencies.53 It
should be noted that magnetron motion can adversely affect ion transfer, sensitivity,
resolution, and mass accuracy.58 Quadrupolar axialization (described in section 2.4.4.2)
can be used to counter these effects.
Equation 2.22
F(
Figure 2.19 D(large circles
Depiction ofs). It should b
f an ion's cycbe noted tha
clotron motiat the magnet
ion (little cirtron radius d
rcles) and madepicted her
agnetron moe is exagger
42
otion ated.
43
2.4.4 Ion Manipulations in FT-ICR
Similar to the LQIT mass spectrometer, FT-ICR is a trapping instrument. A series
of events occurs in the same space but at different times. The high vacuum of FT-ICR
enables the ions to be trapped for a relatively long period of time. Therefore, FT-ICR
provides an ideal environment for studying ion-molecule reactions in the gas phase. The
event sequence used for studying radical reactions in a dual-cell FT-ICR is shown in
Figure 2.20. It includes desorption and ionization of the radical precursor, transfer of the
ions into a clean cell, generation of radical ions by sustained off-resonance irradiation
collision-activated dissociation (SORI-CAD), isolation of the desired radical ions, and
allowing them to react with a neutral reagent, followed by excitation and detection of the
reaction products.
Figure 2.200 The sequenradical site
nce of eventss and ion-mo
s for ionizatiolecule react
ion of a radiction in a dua
cal precursoal-cell FT-IC
r, generationCR.
44
n of
45
2.4.4.1 Ion Transfer
In the dual-cell FT-ICR, ion transfer is an essential step needed to perform ion-
molecule reactions in a clean environment. Protonated or methylated radical precursors
are generated in the source cell and transferred into the clean analyzer cell for reactions.
Ion transfer is achieved by grounding the conductance limit plate for a certain period of
time, as shown in Figure 2.21. The optimal transfer time is dependent on the m/z ratio of
the ion, and can be calculated using Equation 2.23.
⁄ 10μ Equation 2.23
In preparation for ion transfer, the voltage for the remote trapping plate is changed
from +2.0 V to -3.5 V for 15 ms, so as to eject all ions from the analyzer cell. This step
ensures that the charged radical precursors will enter a clean analyzer cell for reactions.
During transfer, ions gain kinetic energy. Thus, they need to be cooled before performing
further experiments.59,60 This is accomplished by allowing the ions to undergo energy
dissipation through collisions with neutral molecules or atoms present in the analyzer cell
for 1 – 5 s. After transfer, the voltage on the conductance limit plate is brought back to +2
V, so that the transferred ions remain trapped in the analyzer cell. In the work described
in this dissertation, argon was pulsed in as cooling gas. Collisions with argon gas convert
the ion's kinetic energy to internal energy which can be released by IR emission.
F
2
th
p
T
em
m
an
co
m
F
tr
p
igure 2.21 Il
.4.4.2 Quadr
Due t
he radius of
late, the tran
This results in
mployed to o
Quadr
motion. It wa
nd detection
ollisions wit
motion increa
inally, the i
ransfer.
Quadr
otential mat
llustration of
rupolarAxial
o the presen
the magnetr
nsferring ion
n loss of ion
overcome th
rupolar excit
as achieved b
n plates of th
th a collision
ases slowly,
ions are kin
rupolar axia
tched the cy
f (a) trappingthe other s
lization (QA
nce of magn
ron motion i
ns will collid
n signal. Qua
his disadvant
tation works
by applying
e source cel
n gas, usuall
, while the
etically coo
alization is
yclotron freq
g a positivelide of a dual
A)
netron motio
s larger than
de with the co
adrupolar axi
tage.61-63
s by convert
an RF poten
l, which gen
ly helium (~
radius of th
led and axia
mass select
quency of t
ly charged iol-cell FT-IC
n, ion transf
n the 2-mm h
onductance
ialization (Q
ting ions' ma
ntial of oppo
nerated a qua
~10-5 Torr), t
heir cyclotro
alized into t
tive. The fr
the ions of
on; (b) transfCR.
fer is not alw
hole in the c
limit and be
QA) is a tech
agnetron mo
osite phases
adrupolar ele
the radius of
on motion d
the center o
requency of
interest. On
fer of the ion
ways efficie
conductance
ecome neutra
hnique that c
otion to cycl
s to the excit
ectric field. U
f ions' magn
decreases rap
of the cell b
f the applied
nly the ions
46
n into
ent. If
limit
alized.
an be
lotron
tation
Upon
netron
pidly.
before
d RF
with
cy
ax
ex
se
2
in
Io
m
yclotron freq
xialized into
xcitation and
ection 2.4.4.
Figure 2.22
.4.4.3 Ion Ex
Ion ex
n Section 2.4
on detection
move on orbi
quency that i
o the center
d dipolar ex
3) respective
2 Depiction oan
xcitation and
xcitation is a
4.4.4) and co
is discussed
its of small r
is in resonan
r of the cell
xcitation for
ely.
of (a) quadrund (b) dipola
d Detection
an essential
ollision-activ
d here. The i
radii in the c
nce with the
l. Figure 2.2
the purpose
upolar excitaar excitation
step for ion
vated dissoc
ions' kinetic
cell. In order
applied qua
22 shows a
es of QA and
ation for quafor ion dete
detection as
ciation (discu
energies are
r for them to
adrupolar RF
a comparison
d ion detect
adrupolar axection.
s well as iso
ussed in Sec
e typically ve
o be detected
F potential w
n of quadru
tion (discuss
ialization (Q
olation (discu
ction 2.4.4.5
ery low, and
d, they need
47
will be
upolar
sed in
QA)
ussed
).53,64
d they
to be
48
kinetically excited to larger radii so that they move closer to the detection plates.
Moreover, ions move in random phases in FT-ICR. They need to be excited to move
coherently as ion packets to yield detectable signal. Therefore, a dipolar RF voltage is
applied to the excitation plates to kinetically excite the ions. When the cyclotron
frequency of an ion is in resonance with the RF frequency, it will absorb energy from the
field, accelerate, and move to a larger orbit. An ion's radius after excitation is given by
Equation 2.24,
Equation 2.24
wherein is the peak-to-peak RF voltage, is the excitation time, is the distance
between the two excitation plates, and is the strength of the magnetic field. It should be
noted that this radius is independent of an ion's m/z ratio, thus all ions will be excited to
the same radius with the same RF amplitude. Typically, a frequency sweep, known as
"chirp", is employed to excite ions of different m/z ratios to larger orbits. In the
experiments discussed here, a chirp excitation with a bandwidth of 2.7 MHz and sweep
rate of 3200 Hz/μs was used to excite ions for detection.
When the excited ion packet passes by the detection plates, a small current called
image current is induced.64-66 The image current contains frequency and amplitude
information, which correspond to ion's cyclotron frequency and relative abundance,
respectively. The image current is converted to voltage, digitalized, and recorded as a
function of time (transient). Fourier transformation generates a frequency domain
spectrum, which can be subsequently converted to a mass spectrum by using Equation
2.18. The process of ion excitation and detection is described in Figure 2.23. Image
cu
in
a
re
m
urrent detect
n the FT-ICR
mass spect
esolution, o
molecules pre
Figure 2.23kinetically
detection pl
tion is a non
R cell after d
trum with h
therwise ion
esent in the c
3 Illustrationexcited to mates and ind
n-destructive
detection. A
igher resolu
n signal wi
cell.
n of ion excitmove coherenduces an imag
spectrum a
e technique,
transient obt
ution. High
ill decay du
tation and dently as ion page current, wand finally m
which mean
tained for lo
vacuum is
ue to collis
etection in aackets. (b) A
which is conmass spectrum
ns that ions c
onger period
also critical
sions with n
an FT-ICR ceAn ion packenverted to frem.
can still be s
of time resu
l to achieve
neutral atom
ell. (a) Ions aet passes by equency dom
49
stored
ults in
high
ms or
are the
main
50
2.4.4.4 Ion Isolation
In the process of generating desired radical ions, unwanted ions are always also
generated. As mentioned in the previous section, ion's kinetic excitation can also be used
for ion isolation. Different from ion detection, where all ions are kinetically excited to
radii that are slightly smaller than the dimensions of the cell, ion isolation is achieved by
exciting unwanted ions to radii that are larger than the cell dimensions. The unwanted
ions can be ejected when colliding with the cell plates.
The broadband frequency sweep, known as chirp excitation, can be employed to
excite and eject ions with a broad mass range.53,67 However, this method suffers from two
major limitation. First, the RF amplitude varies throughout the frequency domain,
resulting in non-uniform ion excitation. Therefore, not all unwanted ions will be ejected
from the cell. Second, tailing in both ends of the RF chirp may result in ejection of
desired ions and hence poor ion signal. These drawbacks can be avoided by the use of
stored-waveform inverse Fourier transform (SWIFT) excitation. 67
SWIFT is a tailored excitation method.68-70 A frequency-domain waveform is pre-
defined. The frequencies are determined by the mass-to-charge ratios of the selected ions
that are to be ejected. An inverse Fourier-transformation is applied to this waveform to
generate a time-domain excitation waveform, which is then used to excite all the selected
ions simultaneously. The processes of chirp and SWIFT excitation are shown in Figure
2.24. In the work described in this dissertation, chirp was used for ion excitation and
detection, while SWIFT was used for isolation of charged radicals.
Figure 2.24 Compariison of chirpp excitation ((left) and SWWIFT excitations (right)
51
.
52
2.4.4.5 Collision-activated dissociation (CAD) in FT-ICR
As discussed above, kinetic excitation is not only necessary for ion detection and
isolation, but is also used for ion dissociation. In FT-ICR, a single-frequency RF voltage
corresponding to the cyclotron frequency of the ion of interest can be applied to the
excitation plates to kinetically excite the desired ion. In the presence of an inert gas, the
kinetic energy of the excited ion is converted into internal energy through multiple
collisions. When sufficient amount of internal energy is accumulated by the ion,
fragmentation occurs. This process is called on-resonance collision-activated dissociation
(CAD).67 The characteristic fragment ions formed through CAD can provide structural
information for the parent ion, which is particularly useful in structural elucidation of an
unknown compound.
In the case of generating radical ions, off-resonance irradiation collision-activated
dissociation (SORI-CAD) is often used.71,72 As its name suggests, an off-resonance RF
voltage is applied to the excitation plates to excite the ion of interest. Typically, an RF
frequency 1000 Hz higher or lower than the ion's cyclotron frequency is used, which
causes the ion's cyclotron radius to increase and decrease repeatedly. In the course of
SORI-CAD, the ion of interest gains less kinetic energy as compared to on-resonance
CAD. Through collisions with inert gas, the ion's internal energy increases slowly,
resulting in its dissociation via the lowest energy pathway.
In this dissertation, SORI-CAD was employed to generate radical ions for gas-
phase reactivity studies. Argon (typically at the pressure of ~10-5 Torr) was used as the
inert gas for collisions. The radical precursors selected have weakly bound nitro- or iodo-
substituents, which can be cleaved off via homolytic cleavages to generate radical sites
53
upon off-resonance CAD. The number of SORI-CAD events needed is dependent on the
number of radical sites desired, being one for monoradicals and one or two for biradicals.
After the radical ions were successfully generated, they were cooled through collisions
with neutral molecules or atoms before their ion-molecule reactions were examined.
2.5 Fundamental Aspects of Gas-phase Ion-Molecule Reactions
Reactions between ions and neutral molecules, which are most often studied in
mass spectrometers, have received considerable interest for a long time.70,73-76 They can
be used as an alternative method to probe the structure of an ion of interest, because
sometimes dissociation reactions do not give much useful structural information.
Moreover, ion-molecule reactions are proven to be a powerful tool for studying
thermodynamics and kinetics of reactions. Compared to solution, gas phase provides an
ideal environment to investigate the intrinsic chemical properties of highly reactive
intermediates without interference of solvent. For the work discussed in this dissertation,
gas-phase ion-molecule reactions were used to study the chemical properties of carbon-
centered ơ-type mono- and biradicals that cannot be easily generated in solution. This
section introduces the fundamental and experimental aspects of gas-phase ion-molecule
reactions in FT-ICR and LQIT.
2.5.1 Brauman’s Double-Well Potential Energy Surface
The potential energy surface for gas-phase ion-molecule reactions is different
from that in solution (Figure 2.25). In solution, both endothermic and exothermic
54
reactions can occur. In the case of endothermic reactions, the reactants acquire energy
from outside of the system to overcome the reaction barrier.77 In gas phase in high
vacuum, however, energy is conserved, and the overall reaction must be exothermic to
occur. In order to explain the rates of gas-phase ion-molecule reactions, Brauman
proposed a double-well potential energy surface model.78-80 Based on this model,
reactions in the gas phase proceed through formation of a reactant complex and product
complex. The reactant complex is formed between the ion and a neutral reagent molecule
due to long-range ion-dipole and/or ion-induced dipole forces.81 These attractive forces
lower the potential energy of the reactant complex, which is known as solvation energy.
The magnitude of this energy is dependent on the dipole moment and polarizability of the
neutral molecule. The solvation energy is available for the reactant complex to overcome
energy barriers along the reaction pathways. Ultimately, whether the reactant complex
can proceed to form products or dissociate back to separated reactants is determined by
the height of the reaction barrier. If this barrier is greater than the total energy of the
reactants, the reaction cannot occur. The rate at which the reaction occurs is largely
controlled by the energy difference (∆E) between the transition state and the separated
reactants.
Gas-phase ion-molecule reactions do not always occur, even when the net
reaction is exothermic and the system has enough energy to overcome the reaction barrier.
This is due to entropy constraints.79 As shown in Figure 2.26, the transition state leading
to product formation can be "tight" (low entropy) as the orientation of the complex is
specific, which has few rotational modes to it. However, the transition state leading to
separated reactants is "loose" (high entropy) as there are many rotational modes available.
T
b
F
Therefore, so
ack to separ
igure 2.25 C
ometimes it
ated reactan
Comparison o
is entropica
ts than to pr
of potential phase (top)
ally favored
oceed to form
energy surfa) and in solu
d for the rea
rm products.
aces for ion-ution (bottom
actant comp
-molecule rem).
plex to disso
actions in th
55
ociate
he gas
w
o
re
co
on
ra
Figure 2.2
In the
were investig
f interest. Th
eactions. It
onstant, and
n the param
ate constant
26 Braumanconst
2
e work desc
gated, becaus
he reaction
can be calcu
d is the
meterized traj
measuremen
n double-weltraints for a g
2.5.2 Kinetic
ribed in this
se they shed
efficiency is
ulated by E
theoretical c
ajectory theo
nts is estima
ll potential egas-phase io
cs of Ion-Mo
s thesis, rea
d light on the
s defined as
quation 2.25
collision rate
ory proposed
ated to be 5
energy surfacon-molecule
olecule Reac
action efficie
e intrinsic ch
the percent
5, wherein
e constant w
d by Su.82 T
50%, and the
ce illustratinreaction.
tions
encies and r
hemical prop
tage of collis
is the e
which can be
The accuracy
e precision i
ng the entrop
reaction pro
perties of th
sions that le
experimenta
calculated b
y of the rea
s less than
56
py
oducts
he ion
ead to
l rate
based
action
10%.
57
100Equation 2.25
The gas-phase ion molecule reactions studied here follow second-order kinetics as
shown in Equation 2.26, where indicates the reaction rate, is the second-order
experimental reaction rate constant, and are the concentrations of the neutral
reagent and the ion, respectively.
Equation 2.26
Under the experimental conditions used here, the neutral reagent is in great excess
as compared to the radical ions. Hence, the concentration of the neutral reagent is
presumed to be constant throughout the reaction. Therefore, the reactions can be assumed
to follow pseudo-first order kinetics as shown in Equation 2.27, where equals
. can be derived by using Equation 2.28.
Equation 2.27
Equation 2.28
In order to calculate , and need to be determined. As shown in
Figure 2.27, plotting ln versus time generates a line with a slope equal to - ,
wherein and are the relative abundances of the reactant ion at time t and time
zero. The concentration of the neutral reagent [N] is derived from its nominal pressure (P)
read by an ion gauge. A conversion factor (1 Torr = 3.239 × 1016 molecules/cm3) is used
58
to convert the pressure of the neutral reagent into its concentration. An ion gauge
correction factor (IGCF) is also necessary to correct for the location of the ion gauges,
because they are located about one meter above the cell where the reactions take place.
The ion gauge correction factor is obtained by measuring the reaction rate of an
exothermic reaction that is assumed to occur at collision rate, such as electron transfer or
proton transfer reaction. For instance, carbon disulfide radical cation is used to abstract
an electron from the neutral reagents, or protonated acetone or methanol is used to
transfer a proton to the neutral molecules. Additionally, the ion gauge's sensitivity
towards different neutral reagents (B/I) is corrected.83 In summary, the experimental
reaction rate constant can be experimentally determined by using Equation 2.29.
. /
Equation 2.29
m
ab
re
fr
th
F
pr
Figure 2.27
Besid
measured. Th
bundance by
elative abun
rom seconda
he primary p
igure 2.27,
roduct of pro
A semi-loga
es reaction
he branching
y the sum o
ndances with
ary products
products and
products 1
oduct 1.
arithmic plotpro
rates, the re
g ratio for a p
f all primary
hin short rea
. Secondary
d further mo
and 2 are
t of the relatoducts versu
eaction prod
primary prod
y products' a
action times,
products ca
onitoring the
primary pro
tive abundanus time.
ducts and th
duct is determ
abundances.
, primary pr
an also be co
eir reactions
oducts, whil
nces of a reac
eir branchin
mined by div
. By measur
roducts can
onfirmed by
s. For the ex
le product 3
ctant ion and
ng ratios are
viding its rel
ring the prod
be distingu
isolating ea
xample show
3 is a secon
59
d its
e also
lative
ducts'
uished
ach of
wn in
ndary
60
2.6 References
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65
CHAPTER 3. STRUCTURAL COMPARISON OF ASPHALTENES OF DIFFERENT
ORIGINS BY USING MULTIPLE-STAGE TANDEM MASS SPECTROMETRY
3.1 Introduction
Asphaltenes, the heaviest components in crude oil, are generally defined by its
solubility regime: insoluble in n-alkanes and soluble in aromatic solvents such as toluene,
benzene, or pyridine.1,2 They are extremely complex mixtures containing molecules with
multiple fused aromatic rings, alkyl chains, heteroatoms and metals.1Asphaltenes are
problematic to the petroleum industry since they result in reduced oil recovery,
precipitation in transport pipelines, adsorption on refinery equipments, fouling of
catalysts used in crude oil conversion2-4 Moreover, the depletion of conventional lighter
crude oils necessitates exploration of heavy crudes that have a high concentration of
asphaltenes.5 In order to address these emerging problems and potentially convert
asphaltenes to useful chemicals, an in-depth understanding of the molecules that
comprise asphaltenes is necessary.6
A wide range of analytical methods have been used to interrogate the bulk
properties of asphaltenes. Nuclear magnetic resonance (NMR) spectroscopy sheds light
on the molecular parameters such as carbon aromaticity and average size of asphaltenes;7
X-ray absorption near edge structure (XANES) measures the sulfur and nitrogen
functionalities.8,9
66
The molecular weight distribution of asphaltene remains controversial.10-12 Vapor
pressure osmometry, size-exclusion chromatography yielded molecular weight of several
thousand amu or even larger values.13-15 However, fluorescence depolarization indicated
an average molecular weight (MW) of 450-850 Da.16-20 Other literatures using different
techniques such as NMR and mass spectrometry summarized a range of 500-2000 Da.1,19-
27 The disparity in measurements was suggested as a result of asphaltene aggregation.27,28
In the past decades, two structural models are debated about the asphaltene
molecules: the island model and the archipelago model.16,19-22,29The island model
(sometimes referred as continental model30) has only one aromatic core with peripheral
alkyl chains, whereas the archipelago model has multiple aromatic cores that are bridged
by alkyl chains and may also containperipheral alkyl chains.20 Multiple experimental
methods, such as time-resolved fluorescence depolarization, Taylor diffusion, and NMR
spectroscopy provide strong support to the island model.16,18-23,31,32 However, the
presence of archipelago structures has also been demonstrated by NMR spectroscopy and
average structural parameter calculations,33 mass spectrometry,34 and thermal cracking of
asphaltenes.35,36 Which of these two motifs predominates asphaltene structure has not
reached a general consensus yet.
Mass spectrometry is an important analytical tool to characterize asphaltenes at
the molecular level, yet it faces many challenges. Asphaltenes have a tendency to degrade
and aggregate upon introduction into the gas phase.28,37-39 Compatible solvent system
needs to be carefully chosen for asphaltene desolvation.28,40 Ionization bias is another
concern because of the highly complex composition of asphaltene mixture. 41
Electrospray ionization (ESI) is especially suitable for ionizing polar constituents with
67
high heteroatom content; atmospheric pressure chemical ionization (APCI) and
atmospheric pressure photo ionization (APPI) are more suitable for ionizing nonpolar
hydrocarbons.37,42-43 In addition, a variety of desorption/ionization methods have been
used for asphaltene analysis, such as matrix-assisted laser desorption/ionization,14 field
desorption/field ionization,44,45 and laser-induced acoustic desorption/electron
ionization.39
In this work, positive ion mode APCI doped with carbon disulfide (CS2) was used
to study six petroleum asphaltenes samples of different geographical origins in a linear
quadrupole ion trap (LQIT) mass spectrometer. CS2 reagent has been demonstrated to
generate stable molecular ions for the asphaltenes based on previous experiments.25,46-47
Furthermore, multi-stage tandem mass spectrometry was employed to examine the
structures of asphaltene molecules by subjecting the selected molecular ions to
collisionally activated dissociation (CAD). In addition to the molecular weight
distribution (MWD) and average MW, structural information, including maximum
number of carbons in alkyl chains and minimal sizes of the aromatic cores, was obtained.
3.2 Experimental Section
Chemicals. The petroleum asphaltene samples from Bohai (China), Maya
(Mexico), Claire (UK), Surmont (Canada), Montana (US), and McKittrick (US) were
provided by ConocoPhillips. Carbon disulfide (>99.9 %) and heptane (>99.9 %) were
purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification.
The samples were dissolved in heptane and sonicated for 1 hour, followed by filtration
and drying under inert gas flow to remove the maltene content.
68
Instrumentation. A Thermo Scientific linear quadrupole ion trap (LQIT) was
used for mass spectrometric analysis. The asphaltenes were dissolved in CS2 at a
concentration of 0.5 mg/mL. The sample solutions were introduced into the APCI source
via direct infusion from a Hamilton 500 μL syringe through the instrument’s syringe
pump at a flow rate of 20 µL/min and ionized via positive ion mode APCI (at 300°C) by
using CS2 as a dopant so that only molecular ions were generated. Molecular ions with
eight randomly selected mass-to-charge (m/z) ratios ranging from m/z 500 up to m/z
808were isolated using an isolation window of 2 Da (±1 Da), and subjected to CAD at an
energy of 35 arbitrary units. The use of an isolation window of 2 Da (±1 Da) results in
isolated ion populations that may contain isomeric and isobaric ions. This large window
was used due to the relatively low ion signals measured for these complex mixtures. This
is justified as it was previously demonstrated that the fragmentation patterns and main
fragment ions of ionized asphaltenes are independent of the size of the isolation window
as long as it is equal or less than 2 Da.25 The data were processed by using Thermo
Xcalibur software. All measurements were repeated four times. The averaged results or
ranges are reported in Table 3.1.
3.3 Results and Discussion
In this study, APCI doped with CS2 was used to ionize asphaltenes in the positive
ion mode so that only stable molecular ions were generated. 25,46-47 Based on the measured
mass spectra, the molecular weight distributions (MWD) were determined for six
petroleum asphaltene samples originating from Bohai, Maya, Claire, Surmont, Montana
and McKittrick. All of the studied asphaltenes from the American continent, Europe and
C
w
sa
w
an
(r
8
n
d
sh
China have s
weight (AVG
amples with
with an AVG
Fi
Struct
nd MS3 exp
randomly se
08) were iso
ominal colli
erived from
how a simila
similar MW
G MW), wh
h different g
G MW of ~61
igure 3.1 AP
tural features
periments of
elected) mas
olated and s
ision energy
the differen
ar decay patt
WD, ranging
hich was cal
geographical
15 Da is show
PCI mass speof the M
s of the asph
f the isolated
s-to-charge
subjected to
y. The fragm
nt asphaltene
tern (for exa
from 200 u
lculated usin
l origins (Ta
wn Figure 3
ectrum showMaya asphalt
haltenemolec
d ions. For
ratios (m/z
collision-ac
mentation pat
e samples w
ample, see Fi
up to 1400
ng Equation
able 3.1). A
.1 for Maya
wing the MWtene sample.
cules were e
each sampl
500, 515, 6
ctivated diss
tterns of mo
were compar
igure 3.2 for
Da. The av
n 1,25 varies
A typical mo
a asphaltenes
WD and AVG
examined by
e, molecular
606, 626, 63
sociation (C
olecular ions
red. All the
r the fragmen
verage mole
s slightly am
onomodal M
s.
G MW
y performing
r ions with
34, 704, 736
AD) at the
of the same
ions consist
ntation patte
69
ecular
mong
MWD
g MS2
eight
6, and
same
e m/z
tently
ern of
th
ra
on
T
F
±
[M
ar
fr
m
fr
du
he molecula
adical loss, l
n, with the
The reason fo
Figure 3.2 Fr
For co
± 1 derived f
M-CH3]+ fra
re presented
ragment ion
molecular ion
ragment ion
ue to the [M
ar ions of m
less favored
larger alkyl
or such aspha
ragmentation
omparison p
from the Ma
agment ions
d below. Int
ns derived f
ns themselv
s have great
M-CH3]+ ion
m/z 634 + 1
ethyl radica
radical loss
altene behav
n pattern of m
purpose, MS
aya asphalten
of m/z 485
terestingly, M
from the m
es (Figure 3
ter abundanc
n population
of Surmon
al loss, even
ses always b
viors is still u
molecular iosample.
2 CAD mass
nes sample,
± 1 that wer
MS3 mass s
molecular ion
3.3) although
ces than slig
n being a som
nt asphaltene
n less favore
being less fa
under invest
ons of m/z 63
s spectrum o
as well as M
re formed fr
spectrum (F
ns show sim
h not quite
ghtly larger
mewhat sim
es), with a d
ed propyl rad
avored than t
tigation.
34 ± 1 of Sur
of molecular
MS3 CAD m
rom the abov
Figure 3.4) o
milar decay
as smooth,
fragment io
mpler mixtur
dominant m
dical loss, an
the smaller
rmont aspha
r ions of m/z
mass spectru
ve molecular
of the [M-C
y patterns a
as some sm
ons. This ma
e of isobaric
70
methyl
nd so
ones.
altene
z 500
um of
r ions
CH3]+
as the
maller
ay be
c and
is
io
le
co
io
su
someric ions
ons formed
engths. Hen
ommonly ob
ons fragmen
urprising as
Figure 3.3asphaltene
s than the m
from the [M
nce, these fr
beyed by fra
nt to yield e
aromatic ion
3 MS2 CAD sample, with
molecular ion
M-CH3]+ions
ragmentation
agmentations
even-electro
ns are the mo
mass spectrh the maximestimated ar
ns. Similar to
s correspond
ns do not o
s observed i
on and not o
ost common
rum of ions omum total num
romatic core
o molecular
d to losses o
obey the Ev
in mass spec
odd-electron
n species sho
of m/z 500 ±mber of carbe size indicat
ions, all the
of alkyl rad
ven Electro
ctrometry (i.
n ions). Thi
owing this ty
± 1derived frbons in alkylted.
e major frag
dicals of diff
on Rule48 th
.e., even-ele
is is not en
ype of behavi
rom the Mayl chains and
71
gment
ferent
hat is
ectron
ntirely
ior.48
ya the
F
m
ex
io
th
4
ob
m
io
fr
d
m
igure 3.4 Mformed fro
Comp
molecular io
xperiment) r
ons of m/z 5
he isolated io
71 and m/z
btained for
molecular ion
ons of m/z
ragment ion
emonstrated
molecules.24
S3 CAD masom molecula
parison of th
ns (MS2 ex
reveals that
00 yield maj
ons of m/z 4
457 (Figur
the molecu
ns and not v
457 are for
ns of m/z 4
d that this is
ss spectrum ar ions of m/z
sam
he m/z valu
xperiment) a
they differ b
jor fragment
485 forms fr
re 3.4). Thes
ular ions in
via further f
rmed directl
485 or m/z
also true for
of [M-CH3]z 500 ± 1dermple (Figure
es of the fr
and those o
by one mass
t ions of m/z
ragment ions
se observati
n MS2 expe
fragmentatio
ly from mo
471, as sh
r losses of a
+ fragment irived from the 3.3).
ragment ions
obtained fro
s unit. For e
z 485, m/z 4
s of m/z 470
ions demons
eriments are
on of larger
lecular ions
hown in Fig
alkyl radicals
ions of m/z 4he same May
s obtained d
om the [M-
example, wh
471 and m/z
0 and m/z 45
strate that th
e formed d
fragment io
s of m/z 50
gure 3.3. A
s from proto
485 ± 1 that ya asphalten
directly from
CH3]+ions
hile the mole
457 (Figure
56 instead o
he fragment
directly from
ns. For exam
00 instead o
A previous
onated aspha
72
were nes
m the
(MS3
ecular
e 3.3),
f m/z
t ions
m the
mple,
of the
study
altene
73
The MS2 and MS3 results discussed above indicate that the fragmenting ion
populations in all cases contain many isomeric and possibly isobaric ions with alkyl
chains of differing lengths since no single structure can undergo losses of so many
different alkyl radicals. Further, the results support the island model more than the
archipelago structural model due to the absence of facile losses of large aromatic moieties
that would be expected for archipelago structures. These findings are in agreement with
earlier results obtained by tandem mass spectrometry for both protonated and molecular
ions of asphaltenes.24,25
Based on MS2 CAD mass spectrum of the selected asphaltene molecular ions, the
maximum total number of carbons in alkyl chains was determined by counting the
number of carbons in the largest eliminated alkyl radical that formed the smallest
detectable fragment ion (considered to be the ion with approximately 1% relative
abundance from the most abundant ion in the mass spectrum). The aromatic core size was
estimated by counting the maximum number of fused aromatic rings possible for an ion
to reach the m/z value of this fragment ion (methylene functional groups possibly left on
the aromatic core after loss of the alkyl chains). For molecular ions of m/z 500 ±1 derived
from the Maya asphaltene sample as shown in Figure 3.3, the smallest detectable
fragment ion has an m/z value of 233, therefore it is determined that there are 4 fused
aromatic rings after cleavage of alkyl chains with at least 19 carbons. It should be noted
that the above determination is based on the assumption that all non-aliphatic carbons are
aromatic carbons, and all aromatic rings are fused. The accuracy of this determination
method is under investigation as the heteroatoms and metals are not taken into account,
yet the
74
above values would not be significantly changed. Overall, it provides insight into the
qualitative structural comparisons of asphaltene samples of different geographic origins.
The molecular weight and structural information for the eight selected molecular
ions derived from the six asphaltenes samples were summarized in Table 1. Since the
experiments were repeated for four times, a slightly different ion population may be
isolated and fragmented each time. Therefore, the maximum total number of carbons in
alkyl chains and the estimated aromatic core size may differ, which are represented by
ranges respectively. For visualization of the general trends, the number of aliphatic
carbons, as well as estimated aromatic core size, was plotted against a series of MWs
studied for the six asphaltene samples respectively, as shown in Figure 3.5 and Figure
3.6. The results that fall within a range are represented by the average value and standard
error.
For asphaltene molecules with MWs ranging from 500 to 808 Da, the maximum
total number of carbons in the alkyl chains ranges from 17 to 41, while the approximate
number of aromatic rings ranges from 3 to 7. These results are consistent with other
reports suggesting that asphaltene aromatic moieties contain 4-10 fused rings, and the
length of aliphatic chains covers a wide range up to 30-40 carbon atoms.49-50 Generally,
molecules of greater MWs were found to have more carbons in alkyl chains, yet they do
not always have more aromatic rings in the cores. For instance, molecules of smaller
MWs (from 500 to 626 Da) derived from Montana, Maya and Surmont asphaltenes have
fewer carbons in alkyl chains and larger aromatic cores compared to those molecules of
75
larger MWs. Additionally, molecules of MWs ranging from 634 to 808 Da have about the
same number of carbons in alkyl chains and aromatic cores regardless of their geographic
origins.
Table 3.1 MWD and AVG MW of Molecules in the Six Asphaltenes Samples, and Structural Information for the Eight Selected Ions
Bohai Maya Claire Surmont Montana McKittrick
MWD 250-1450 200-1400 300-1500 200-1350 300-1400 200-1360 AVG MW 702 615 681 575 664 571
Ion of m/z 500 Maximum number of
Carbons in chains 22 17-21 21-22 17-21 17 21-22
Estimated Core Size 3 4 3-4 4-5 5 3-4 Ion of m/z 515
Maximum number of Carbons in chains
23-24 18-19 23-24 16-18 13-14 22-23
Estimated Core Size 3 5 3 5 7 3-4 Ion of m/z 606
Maximum number of Carbons in chains
29-30 28-30 28-30 27-28 25-28 28
Estimated Core Size 3 3-4 3-4 4 4-5 4 Ion of m/z 626
Maximum number of Carbons inchains
30-31 31 31 30 28-31 30-31
Estimated Core Size 3-4 3 3 4 3-5 3-4 Ion of m/z 634
Maximum number of Carbons inchains
30-32 30-32 31-32 31-32 30 30
Estimated Core Size 3-4 3-4 3-4 3-4 4 4 Ion of m/z 704
Maximum number of Carbons inchains
35 34-35 35 34-35 35 34
Estimated Core Size 4 4 4 4 4 4 Ion of m/z 736
Maximum number of Carbons inchains
36-37 35-36 37 36 36 36-37
Estimated Core Size 4 4-5 4 4 4 4 Ion of m/z 808
Maximum number of Carbons inchains
39-40 40 40 40-41 40 39-41
Estimated Core Size 5 5 5 5 5 5
76
Figure 3.5 General trend for the approximate maximum total number of carbons in alkyl chains as a function of MW of the molecules derived from the six asphaltene samples.
Figure 3.6 General trend for the approximate aromatic core size as a function of MW of the molecules derived from the six asphaltene samples.
0
5
10
15
20
25
30
35
40
45
500 515 606 626 634 704 736 808
MW
Num
ber
of A
liph
atic
Car
bons
Bohai
Montana
Maya
Claire
Surmont
McKittrick
0
1
2
3
4
5
6
7
8
500 515 606 626 634 704 736 808
MW
Aro
mat
ic C
ore
Siz
e Bohai
Montana
Maya
Claire
Surmont
McKittrick
77
3.4 Conclusions
Examination of six petroleum asphaltene samples of different geographical
origins by using positive ion mode APCI doped with CS2 and multi-stage tandem mass
spectrometry allowed the determination of the molecular weight distribution (MWD),
average molecular weight (MW) as well as structural information for these samples. The
asphaltene samples studied have similar MWDs, ranging from 200up to 1450 Da. The
average MWs range from 570 up to 700 Da, and are dependent on the origin of the
samples. Eight randomly selected molecular ions with m/z values ranging from m/z 500
up to m/z 808, derived from the different asphaltenes, all show a similar fragmentation
pattern, providing support to the island structural model of asphaltenes. The fragmenting
ion populations in all cases contain many isomeric and possibly isobaric ions with alkyl
chains of differing lengths since no single structure can undergo losses of so many
different alkyl radicals. The maximum total number of carbons (ranging from 17 to 41) in
all alkyl chains generally increase with the increase of MWs of the asphaltene molecules
ranging from 500 to 808 Da. However, the number of aromatic rings (ranging from 3 to
7) in the cores does not reveal an obvious correlation with MWs of the asphaltene
molecules.
78
3.5 References
1. Mullins, O. C.; Sheu, E. Y.; Hammami, A.; Marshall, A. G. Asphaltenes, Heavy Oils, and Petroleomics; Springer: New York, 2007.
2. Adams, J. J. Energy Fuels 2014, 28, 2831.
3. Ancheyta, J.; Betancourt, G.; Centeno, G.; Marroquin, G.; Alonso, F.; Garciafigueroa, E. Energy Fuels 2002, 16, 1438.
4. Trejo, F.; Centeno, G.; Ancheyta, J. Fuel 2004, 83, 2169.
5. Rana, M. S.; Samano, V.; Ancheyta, J.; Diaz, J. A. I. Fuel 2007, 86,1216.
6. Akbarzadeh, K.; Hamami, A.; Kharrat A.; Zhang, D.; Stephan Allenson; Creek, J.; Kabir, S.; Jamaluddin, A.; Marshall, A. G.; Rodgers, R. P.; Mullins, O. C.; Solbakken, T. Oilfield Rev. 2007, 19, 22.
7. Ostlund, J. A.; Wattana, P.; Nydén, M.; Fogler, H. S. J. Colloid Interface Sci. 2004, 271, 372.
8. Zhang, L.; Wang, C.; Zhao, Y.; Yang, G.; Su, M.; Yang, C. J. Fuel Chem.Tech. 41 (11), 2013, 1328–1335.
9. Pomerantz, A. E.; Seifert, D. J.; Bake, K. D.; Craddock, P. R.; Mullins, O. C.; Kodalen, B. G.; Mitra-Kirtley, S.; Bolin, T. B. Energy Fuels 2013, 27, 4604.
10. Mullins, O. C.; Martinez-Haya, B.; Marshall, A. G. Energy Fuels 2008, 22, 1765.
11. Herod, A. A.; Bartle, K. D.; Kandiyoti, R. Energy Fuels 2008, 22, 4312.
12. Strausz, O. P.; Safarik, I.; Lown, E. M.; Morales-Izquierdo, A. Energy Fuels 2008, 22, 1156.
13. Harvey W. Yarranton, H. A., Jakher, R. Ind. Eng. Chem. Res. 2000, 39, 2916.
14. Trejo, F.; Ancheyta, J.;Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2007, 21, 2121.
15. Anderson, S. I.; Speight, J. G. Fuel 1993, 72, 1343.
16. Groenzin, H.; Mullins, O. C. J. Phys. Chem. A 1999, 103, 11237.
17. Buch, L.; Groenzin, H.; Buenrostro-Gonzalez, E.; Anderson, S. I.; Lira-Galeana, C.; Mullins, O. C. Fuel 2003, 82, 1075.
79
18. Badre, S.; Goncalves, C. C.; Norinaga, K.; Gustavson, G.; Mullins, O. C. Fuel 2006, 85, 1.
19. Mullins, O. C. SPE J. 2008, 13, 48.
20. Mullins, O. C. Annu. Rev. Anal. Chem. 2011, 4, 393.
21. Mullins, O. C. Energy Fuels 2010, 24, 2179.
22. Mullins, O. C.; Sabbah, H.; Eyssautier, J.; Pomerantz, A. E.; Barre, L.; Andrews, A. B.; Ruiz-Morales, Y.; Mostowfi, F.; McFarlane, R.; Goual, L.; Lepkowicz, R.; Cooper, T.; Orbulescu, J.; Leblanc, R. M.; Edwards, J.; Zare, R. N. Energy Fuels 2012, 26, 3986.
23. Sabbah, H.; Morrow, A. L.; Pomerantz, A. E.; Zare, R. N. Energy Fuels 2011, 25, 1597.
24. Borton, D.; Pinkston, D. S.; Hurt, M. R.; Tan, X. L.; Azyat, K.; Scherer, A.; Tykwinski, R.; Gray, M.; Qian, K. N.; Kenttämaa, H. I., Energy Fuels 2010, 24, 5548.
25. Hurt, M. R.; Borton, D. J.; Choi, H. J.; Kenttämaa, H. I. Energy Fuels 2013, 27, 3653.
26. Loegel, T. N.; Danielson, N. D.; Borton, D. J.; Hurt, M. R.; Kenttämaa, H. I. Energy Fuels 2012, 26, 2850.
27. Tanaka, R.; Hunt, J. E.; Winans, R. E.; Thiyagarajan, P.; Sato, S.;Takanohashi, T. Energy Fuels 2003, 17, 127.
28. McKenna, A. M.; Donald, L. J.; Fitzsimmons, J. E.; Juyal, P.; Spicer, V.; Standing, K. G.; Marshall,A. G.; Rodgers, R. P. Energy Fuels 2013, 27, 1246.
29. Gray, M. R.; Tykwinski, R. R.; Stryker, J. M.; Tan, X. Energy Fuels 2011, 25, 3125.
30. Tukhvatullina, A. Z.; Barskaya, E. E.; Kouryakov, V. N.; Ganeeva, Y. M.; Yusupova, T. N.; Romanov, G. V. J Pet Environ Biotechnol 2013, 4, 152.
31. Andrews, A. B.; Edwards, J. C.; Pomerantz, A. E.; Mullins, O. C.; Nordlund, D.; Norinaga, K. Energy Fuels 2011, 25, 3068.
80
32. Majumdar, R. D.; Gerken, M.; Mikula, R.; Hazendonk, P. Energy Fuels 2013, 27, 6528.
33. Morgan, T.; Alvarez-Rodriguez, P.; George, A.; Herod, A.; Kandiyoti, R. Energy Fuels 2010, 24, 3977.
34. Podgorski, D. C.; Corilo, Y. E.; Nyadong, L.; Lobodin V. V.; Bythell B. J.; Robbins
W. K.; McKenna, A. M.; Marshall, A. G.; Rodgers, R. P. Energy Fuels 2013, 27, 1268.
35. Rueda-Velasquez, R. I.; Freund, H.; Qian, K.; Olmstead, W. N.; Gray, M. R. Energy Fuels 2013, 27, 1817.
36. Karimi, A.; Qian, K.; Olmstead, W. N.; Freund, H.; Yung, C.; Gray, M. R. Energy Fuels 2011, 25, 3581.
37. Gaspar, A.; Zellermann, E.; Lababidi, S.; Reece, J.; Schrader, W. Anal Chem. 2012, 84, 5257.
38. Wu, Q.; Pomerantz, A. E.; Mullins, O. C.; Zare, R. N. J Am Soc Mass Spectrom. 2013, 24, 1116.
39. Pinkston, D. S.; Duan, P.; Gallardo, V. A.; Habicht, S. C.; Tan,X.; Qian, K.; Gray, M.; Müllen, K.; Kenttämaa, H. I. Energy Fuels 2009, 23, 5564.
40. Kim, Y.; Kim, S. J Am Soc Mass Spectrom. 2010, 21, 386.
41. Rodgers, R. P.; McKenna, A. M. Anal. Chem. 2011, 83, 4665.
42. Hughey, C. A.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2002, 74, 4145.
43. Cunico, R. L.; Sheu, E. Y.; Mullins, O. C. Pet. Sci. Technol. 2004, 22, 787.
44. Qian, K. N.; Edwards, K. E.; Siskin, M.; Olmstead, W. N.;Mennito, A. S.; Dechert, G. J.; Hoosain, N. E. Energy Fuels 2007, 21, 1042.
45. Nyadong, L.; McKenna, A. M.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2011, 83, 1616.
46. Owen, B. C.; Gao, J.; Amundson, L.; Archibold, E.; Tan, X.; Azyat, K.; Tykwinski, R. R.; Gray, M. R.; Kenttämaa, H. I. Rapid Commun. Mass Spectrom. 2011, 25, 1924.
47. Jarrell, T. M.; Jin, C.; Riedeman, J. S.; Owen, B. C.; Tan, X.; Scherer, A.; Tykwinski, R. R.; Gray, M. R.; Slater, P.; Kenttämaa, H. I. Fuel 2014, 133, 106.
81
48. Karni, M.; Mandelbaum, A. Org. Mass Spectrom. 1980, 15, 53.
49. Mullins, O. C., Sheu, E. Y. Structures and Dynamics of Asphaltenes; Plenum Press: New York, 1998.
50. Calemma, V.; Rausa, R.; D’Antona, P.; Montanari, L. Energy Fuels 1998, 12, 422.
82
CHAPTER 4. CHARACTERIZATION OF ORGANOSULFUR MODEL COMPOUNDS RELEVANT TO FOSSIL FUELS BY USING HIGH-RESOLUTION
TANDEM MASS SPECTROMETRY
4.1 Introduction
Sulfur-containing compounds in fossil fuels are of big concern to environment, as
they cause pollution by releasing toxic gases, such as H2S and SO2, upon combustion.1-3
Hydrodesulfurization is usually required to lower the sulfur content of petrochemical
products and vacuum residues below the legal limits in many countries.4,5 The chemistry
of desulfurization involved in processing crude oil is greatly dependent on the forms of
sulfur in the oil.6,7 Since sulfur exists in different chemical bonding environments in
crude oil, such as in thiophenes, thiols, sulfides, disulfides, and polyaromatic sulfur
compounds, they require different conditions to be removed from hydrocarbons.6-8
Therefore, better understanding of the molecular structures of organosulfur compounds in
crude oil is highly desirable to aid in rational improvement of the desulfurization
processes.
Many attempts have been made to determine the chemical bonding environments
of sulfur in complex geological samples. X-ray absorption near-edge structure
spectroscopy (XANES) and X-ray photoelectron spectroscopy (XPS) have been used for
detection and quantitation of sulfur compounds in petroleum and coal samples.9-11
Nuclear magnetic resonance (NMR) coupled with sulfur functionality derivatization has
83
been employed to identify nonvolatile sulfur compounds in crude oil.12 Mass
spectrometry (MS), coupled with separation methods, such as gas chromatography (GC)
and liquid chromatography (LC), has become an increasingly important analytical tool to
identify organosulfur components in complex hydrocarbon mixtures.13-16
Different ionization methods have been employed to ionize organosulfur
compounds in mass spectrometry, such as electron ionization (EI), chemical ionization
(CI), electrospray ionization (ESI), and atmospheric pressure chemical ionization
(APCI).17 Several studies focused on EI mass spectra of organosulfur compounds.18-20
However, EI is not an ideal ionization method for complex mixture analysis because it
tends to induce fragmentation.21 For CI experiments of sulfur-containing heterocycles,
more than one ion type is generated for each analyte, such as protonated molecule
[M+H]+ and nitric oxide complex [M+NO]+.22
Traditional ESI is not optimal for nonpolar compounds’ analysis because it is
biased towards polar compounds.23 Therefore, the influence of various dopants on ESI of
sulfur-containing heterocycles has been studied.24-26 For instance, addition of Pd2+ ions
has been used to enhance molecular ion formation for selected sulfur-containing
heterocycles in ESI.27,28 However, this method may have problems with samples of
unknown sulfur content since the concentration ratio of Pd2+ and organosulfur
compounds is crucial for successful molecular ion generation. Another approach used to
ionize sulfur-containing heterocycles in ESI is to convert them to S-methyl sulfonium or
S-phenyl sulfonium salts in solution.29 This method, however, leaves open the question as
to whether all forms of organosulfur compounds are derivatized equally efficiently.
84
Compared to the ionization methods mentioned above, APCI is a more generally
applicable approach. The chemistry of APCI ionization can be tuned to achieve
molecular ion generation by choosing a proper reagent with desired thermochemical
properties.30,31 Recent research has demonstrated the production of stable molecular ions
for non-polar and polar aromatic compounds by using APCI with carbon disulfide (CS2)
as the solvent.31 In this work, the APCI/CS2 method was explored for the ionization of
various organosulfur compounds. Stable molecular ions were generated for all the
compounds studied except for a few sulfides and disulfides that showed minor
fragmentation upon ionization.
In order to probe the structures of the ionized organosulfur compounds, multiple-
stage tandem mass spectrometry (MSn) based on collision-activated dissociation (CAD)
can be used.32-34 In this work, the fragmentation patterns of the molecular ions of
organosulfur compounds with various sulphur-containing functionalities, including
thiophenes, thiols, polyaromatic sulfur compounds, sulfides and disulfides, were
systematically studied, whereas many other studies have mainly focused on molecular
ions of protonated ions of polyaromatic sulfur compounds.35-40 The characteristic
fragment ions generated in MS2 and MS3 experiments provide clues for the chemical
bonding environment of sulfur atoms in the examined compounds.
85
4.2 Experimental Section
All organosulfur compounds and carbon disulfide (>99.9 %) were purchased from
Sigma-Aldrich and used without further purification. The analytes were dissolved in CS2
at a concentration of approximately 1 mg/mL and sonicated for 20 min if dissolution did
not occur instantaneously. These sample solutions were directly infused into the ion
source at a flow rate of 20 μL/min by using a syringe pump.
Multiple-stage tandem mass spectrometry experiments were carried out using a
Thermo-Scientific linear quadrupole ion trap (LQIT) equipped with an atmospheric
pressure chemical ionization (APCI) source. The instrument parameters were as follows:
ion mode, positive; vaporizer temperature, 300 °C; source voltage and current, 4 kV and
4 μA, respectively; capillary temperature, 275 °C; nitrogen sheath gas and auxiliary gas
flow rates, 40 μL/min and 10 μL/min, respectively; tube lens voltage, 20-65 V. The
voltages for the ion optics were optimized for each individual analyte by using the tune
feature of the LTQ Tune Plus interface.
In MS2 experiments, molecular ions of the organosulfur compounds were isolated
using an isolation window of 1 Da (±0.5 Da), and subjected to collision-activated
dissociation (CAD) at a collision energy of 20-35 arbitrary units. In MS3 experiments,
fragment ions generated in MS2 experiments were isolated and subjected to CAD. A q
value of 0.25 was generally used for CAD, as small fragment ions can be efficiently
trapped when using this value. For those molecular ions requiring higher collision energy
to fragment, a higher q value of 0.4 was used so that more structural information on the
ions could be obtained. The relative abundances provided for the product ions in Tables
1-5 are the average values measured over 50 scans. A threshold of 1% was used, i.e., the
86
MSn products with a relative abundance lower than 1% relative to the base peak were
ignored. The data were processed by using Thermo Xcalibur software.
High-resolution experiments were performed using a Thermo-Scientific LQIT
coupled with a Fourier transform ion cyclotron resonance (FT-ICR; 7-T magnet) mass
spectrometer. The accurate masses of some ionic fragment ions were measured, based on
which molecular formulae can be assigned. This information helps to confirm the
identities of the neutral molecules lost during CAD process. The measured accurate
masses were mostly within 10 ppm from the expected values, as shown in Figure 4.6-4.9.
4.3 Results and Discussions
APCI(+)/CS2 generated stable molecular ions for all 19 organosulfur compounds
studied, with no or little fragmentation. Tables 4.1 – 4.5 summarize the CAD products of
ionized organosulfur compounds obtained in MS2 experiments and further CAD products
of those fragment ions obtained in MS3 experiments. Relative abundances of the ions are
listed. The sulfur containing neutral molecules that were lost during fragmentation are
highlighted in red color, including S (32 Da), HS● (33 Da), H2S (34 Da), CS (44 Da),
●CHS (45 Da), etc. Generally, losses of HS● and H2S were found to be associated with
aliphatic sulfur moieties, while losses of S, CS and ●CHS were more common for
polyaromatic sulfur compounds. Accurate mass measurements were performed for some
of the MS2 and MS3 CAD product ions, with their elemental compositions and mass
accuracy (ppm) shown in Tables 4.6 – 4.9. The proposed fragmentation pathways for
molecular ions of the selected organosulfur compounds are shown in Schemes 4.1 – 4.7,
with the major fragment ions highlighted by boxes.
87
Thiophenes. Three thiophene compounds with or without alkyl chains were
studied (Table 4.1). They form abundant molecular ions upon ionization via APCI(+).
The molecular ion of thiophene readily loses H and C2H2 upon CAD. No fragmentation
products were observed in MS3 experiments. For 2-methylthiophene molecular ion, H
loss is the most facile fragmentation, followed by C2H4 loss. Isolation of the [M-H]+
fragment ion followed by CAD results in C2H4 loss as well. The molecular ion of 2-
ethylthiophene fragments by alpha-cleavage, producing [M-CH3]+ ion that further
fragments by the loss of C2H4. None of these CAD products of the ionized thiophenes
provide much structural information of sulfur.
Thiols. Two thiol compounds were studied (Table 4.2). They form abundant
molecular ions upon ionization via APCI(+), except that benzyl mercaptan has minor [M-
HS]+ fragments. Their fragmentation pathways are proposed in Scheme 4.1. It was
noticed that benzenethiol molecular ion requires higher energy collisions for
fragmentation, thus a q value of 0.4 was used. It fragments by loss of H, C2H2 and CS.
Since isolation and further CAD of the [M-C2H2]+. fragment ion (m/z 84) results in
another acetylene loss, which is also observed for thiophene molecular ion, m/z 84 is
proposed to be thiophene radical cation. Elimination of CS possibly forms a five-
membered carbocyclic radical cation (m/z 66). This mass spectrometric fragmentation
product was also reported by Earnshaw et al. by using isotopic labeling and energetics
considerations.41 The molecular ion of benzenethiol was suggested to consist two forms,
as shown in Figure 4.1, and m/z 66 must only arise from a seven-membered ring
88
(expanded ring) structure.41 When m/z 66 is isolated and subject to further CAD, it loses
hydrogen atom and acetylene.
As for benzyl mercaptan molecular ion, the major fragmentation pathway is the
elimination of HS, producing a stable tropylium cation (m/z 91). Upon further isolation
and CAD, tropylium ion loses acetylene. In summary, losses of sulfur containing neutral
molecules, including CS and HS, were observed for molecular ions of thiols. This could
provide information about the presence of sulfur in the ionized molecules. Specifically,
when the molecular ions are subject to CAD, CS elimination appears to be associated
with aromatic compounds, while HS● loss is associated with aliphatic sulfur moieties.
Scheme 4.1 Fragmentation pathways for the molecular ions of (a) benzenethiol, and (b) benzyl mercaptan upon multiple-stage CAD.
89
Figure 4.1 Two forms of the molecular ion of benzenethiol
Polyaromatic Sulfur Compounds. Five polyaromatic sulfur compounds were
studied (Table 4.3). Abundant molecular ions are generated upon ionization via (+)APCI.
The molecular ions of benzothiophene and dibenzothiophene fragment by losses of S, CS
and CHS, apart from acetylene loss. In MS3 experiments, isolation and further CAD of
the above fragment ions readily loses H and C2H2 (Table 4.3). As for the molecular ion of
4,6-dimethyldibenzothiophene, in addition to H and CH3 losses upon CAD, HS loss was
observed as well (Table 4.3). The [M-HS]+ fragment ion (m/z 179) can be explained as a
facile hydrogen atom loss followed by sulfur atom elimination.39
For molecular ion of thianthrene, the major fragmentation pathway is the
elimination of S, while CHS and CS2 losses ware also observed (Table 4.3). Upon further
isolation and CAD of the predominant fragment [M-S] + ion (m/z 184), it readily loses
another S and CHS, along with H and C2H2 losses. This MS3 fragmentation behavior is
very similar to that of benzothiophene molecular ion, which indicates that the [M-S]+ ion
(m/z 184) generated during MS2 of ionized thianthrene has the same structure as that of
benzothiophene.
For another two sulfur atoms containing analyte, 2,2'-bithiophene molecular ion
fragments predominantly by losses of S, CS, CHS, and 2S (Table 4.3). Upon further
CAD of the fragment [M-S]+ ion (m/z 134), additional S, CS and CHS were lost besides
ac
fu
fo
fr
ob
w
b
b
cetylene los
urther CAD.
or its three fr
In sum
rom the m
bservations
which carbon
ecause carb
enzene, and
Figure 4.2 M
s. Moreover
The MS2 sp
ragment ions
mmary, sulfu
olecular ion
are consiste
n or hydrog
bon and hyd
polyaromati
MS2 spectrumspe
r, the [M-CH
pectra measu
s were show
fur containin
ns of poly
ent with sev
gen atoms a
drogen atom
ic sulfur com
m measured ectra measur
HS]+ fragme
ured for the m
wn in Figure 4
ng molecules
aromatic su
veral literatu
are eliminat
m scrambling
mpounds. 42,4
for the molered for its th
ent ion (m/z
molecular io
4.2 as an exa
s, including
ulfur compo
ure reports.27
ted during f
g readily oc
43
ecular ion ofhree fragmen
z 121) readil
on and MS3
ample.
S, CS and C
ounds durin
7,28,39 It shou
fragmentatio
ccurs for io
f 2,2’-bithiopnt ions.
ly loses CS
spectra mea
CHS, can be
ng CAD. T
uld be noted
on is not kn
onized thiop
phene, and M
90
upon
sured
e lost
These
d that
nown
hene,
MS3
91
Sulfides. Six sulfide compounds were studied (Table 4.4). Abundant molecular
ions were generated for most of the sulfide compounds via APCI. Upon ionization,
methyl phenyl sulfide is accompanied by minimal fragmentation while benzyl methyl
sulfide and benzyl sulfide form [M-H]+ fragment ions and/or other fragment ions.
The molecular ions of all sulfide compounds studied lose HS or H2S or both upon
CAD, with the exception of ionic benzyl phenyl sulfide. For example, methyl phenyl
sulfide molecular ion predominantly loses HS to form tropylium cation (m/z 91). CH3
and CH2S losses were also observed upon CAD. Ethyl phenyl sulfide molecular ion
fragments by cleavage of the alkyl chain, producing benzenethiol radical cation (m/z 110)
and methyl phenyl sulfide cation (m/z 123). At the meantime, minor [M-HS]+ products
(m/z 105) were observed. Further isolation and CAD of [M-C2H4]+. fragment ion (m/z
110) results in similar fragmentation behavior as that observed in benzenethiol molecular
ion. Further isolation and CAD of [M-CH3]+ fragment ion (m/z 123) results in loss of CS.
For molecular ion of benzyl methyl sulfide, the primary fragmentation pathway is -
cleavage, producing [M-CH3S]+ ion. Upon CAD, CH3, H2S and CH2S losses also
occurred. In MS3 experiments, only a hydrogen atom is lost upon CAD of [M-CH3]+
fragment ion (m/z 123).
For the molecular ion of diphenyl sulfide, two hydrogen atoms loss is the
dominant fragmentation pathway, as it drives the formation of conjugated aromatic rings,
producing m/z 184 (Scheme 4.2). Further isolation and CAD of this [M-2H]+. product ion
generates similar fragmentation behavior as that of dibenzothiophene molecular ion,
which supports the structure proposed for the fragment ion of m/z 184. Additionally,
diphenyl sulfide molecular ion fragments by the loss of H, CH3, HS and H2S. Further
92
isolation and CAD of [M-CH3]+ ion (m/z 171) results in loss of CS, CHS, H and C2H2,
suggesting that sulfur is present in the ring system for the fragment ion of m/z 171.
Further isolation and CAD of [M-H2S]+ ion (m/z 152) results in consecutive losses of
hydrogen atoms and acetylene, which is indicative of the aromatic ring structure
proposed for the fragment ion of m/z 152.
Scheme 4.2 Fragmentation pathways for the molecular ion of diphenyl sulfide upon multiple-stage CAD.
For the molecular ion benzyl sulfide, the primary fragmentation pathway is shown
in Scheme 4.3. H2S loss is observed upon CAD of the molecular ion, which can result in
the formation of a conjugated ring system for the fragment ion of m/z 180. Cleavage of
C-S bond yields two fragment ions, m/z 92 and m/z 91. Benzylic cleavage yields two
fragment ions, m/z 123 and m/z 122. Isolation and further CAD of [M-C7H8]+ ion (m/z
122) results in losses of H, CS and CHS, which suggests that sulfur is incorporated in the
93
ring system for m/z 122. Additionally, a benzene loss is observed upon CAD of ionic
benzyl sulfide parent ion (m/z 214). Isolation and further CAD of [M-C6H6] + ion (m/z
136) results in losses of H, 2xH, CS and CHS, suggesting that m/z 136 is likely to be a
sulfur-containing heterocyclic fragment ion.
Scheme 4.3 Fragmentation pathways for the molecular ion of benzyl sulfide upon multiple-stage CAD.
94
Based on the above observations, HS and H2S appear to be the characteristic
neutral molecules lost upon CAD of the sulfide molecular ions. However, benzyl phenyl
sulfide is an exception. Its molecular ion fragments predominantly by -cleavage and
produces tropylium cation (m/z 91).
Disulfides. Four disulfide compounds were studied (Table 4.5). Upon CAD,
phenyl disulfide molecular ion not only undergoes -cleavage, but also fragments by
losses of HS, H2S and 2 S atoms (Scheme 4.4). MS3 CAD experiments were conducted
for the two most abundant fragment ions generated in MS2 CAD. Isolation and further
CAD of [M-HS]+ fragment ion (m/z 185) results in consecutive hydrogen losses, as well
as HS, H2S and CH2S losses. It is reasonable that the fragmentation behavior of
protonated dibenzothiophene (m/z 185) is different than that of dibenzothiophene
molecular ion (m/z 184), which shows S, CS, and CHS losses upon MS2 CAD (Table
4.3). Isolation and further CAD of the [M-2S]+ ion (m/z 154) results in consecutive
hydrogen atom losses and acetylene loss.
95
Scheme 4.4 Fragmentation pathways for the molecular ion of phenyl disulfide upon multiple-stage CAD.
For dicyclohexyl disulfide molecular ion, however, no HS or H2S losses were
observed. The primary fragmentation pathway is α-cleavage, which generates two
product ions, m/z 148 and m/z 83 (Scheme 4.5). Isolation and MS3 CAD of [M-C6H10] +
fragment ion (m/z 148) results in losses of S2H, the product ion (m/z 83) of which has the
same structure as that of MS2 CAD product (m/z 83). This is because that isolation and
further CAD of both m/z 83 fragment ions results in the same fragmentation pattern
(acetylene loss). The difference in MS2 CAD behaviors between molecular ions of phenyl
disulfide and dicyclohexyl disulfide is due to the fact that ionized phenyl disulfide can
form a stabilized 3-membered aromatic ring upon HS and H2S loss (Scheme 4.4), while
ionized dicyclohexyl disulfide cannot.
96
Scheme 4.5 Fragmentation pathways for the molecular ion of dicyclohexyl disulfide upon multiple-stage CAD.
For dibenzyl disulfide molecular ion, the primary fragmentation pathway is the
loss of S2H besides -cleavages (Scheme 4.6). Further isolation and CAD of the [M-
S2H]+ ion (m/z 181) results in losses of methyl radical, acetylene and two hydrogen
atoms. Isolation and MS4 CAD of [M-S2H-CH3]+ ion (m/z 166) results in consecutive
hydrogen atom losses at higher collision energy (data not shown), which is typical
fragmentation behavior observed for conjugated aromatic rings. Such information
supports the resonance stabilized structures proposed for the fragmentation products of
m/z 166.
97
Scheme 4.6 Fragmentation pathways for the molecular ion of dibenzyl disulfide upon multiple-stage CAD.
Butyl disulfide molecular ion fragments by -cleavage of a C-S bond, generating
product ions of [M-C4H8]+ (m/z 122) and [M-C4H9S2]
+ (m/z 57). Meanwhile, S-S bond
cleavage products, ions of m/z 89 and m/z 88, were also observed, as shown in Scheme
4.7. Isolation and further CAD of the [M-C4H8]+ (m/z 122) fragment ion results in losses
of HS (m/z 89) and S2H (m/z 57). Further CAD of the fragment ions of m/z 89 results in
loss of H2S (m/z 55). This is consistent with what was observed for sulfide molecular
ions in previous section of this work. Upon CAD, HS and/or H2S losses are characteristic
for ionized organosulfur compounds with sulfur in an alkyl chain.
98
Scheme 4.7 Fragmentation pathways for the molecular ion of butyl disulfide upon multiple-stage CAD.
Table 4.1 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Thiophenes
Analyte (MW) MS (m/z)
MS2 CAD product ions (m/z) and their relative
abundance
MS3 CAD product ions (m/z) and their relative
abundance
Thiophene (84)
M+● (84) 84 – H (83) 2% 84 – C2H2 (58) 100%
No further fragmentation
2-Methylthiophene
(98)
M+● (98) 98 – H (97) 100% 98 – H – C2H4 (69) 11%
97– C2H4 (69) 100%
2-Ethylthiophene
(112)
M+● (112) 112 – CH3 (97) 100% 97 – C2H4 (69) 100% 97 – C2H2 (71) 2%
99
Table 4.2 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Thiols
Analyte (MW) MS (m/z) MS2 CAD product ions (m/z) and their relative
abundance
MS3 CAD product ions (m/z) and their relative
abundance
Benzenethiol
(110)
M+● (110) 110 – H (109) 10% 110 – C2H2 (84) 62% 110 – CS (66) 100%
84 – C2H2 (58) 100% 66 – H (65) 100% 66 – H –C2H2 (39) 67%
SH
Benzyl mercaptan
(124)
M+● (124) 100% [M-HS]+ (91) 11%
124 – HS (91) 100% 91 – C2H2 (65) 100%
Note: The sulfur containing neutral molecules that were lost during CAD are highlighted in red color.
100
Table 4.3 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Polyaromatic Sulfur Compounds
Analyte (MW) MS (m/z) MS2 CAD product ions (m/z) and their relative
abundance
MS3 CAD product ions (m/z) and their relative
abundance
Benzothiophene
(134)
M+● (134)
134 – C2H2 (108) 100% 134 – S (102) 43% 134 – CS (90) 75% 134 – CHS (89) 60%
108 – C2H2 (82) 100% 102 – C2H2 (76) 100% 90 – H (89) 100% 90 – H – C2H2 (63) 27%
Dibenzothiophene
(184)
M+● (184)
184 – H (183) 3% 184 – C2H2 (158) 3% 184 – S (152) 100% 184 – CS (140) 6% 184 – CHS (139) 30%
152 – H (151) 30% 152 – 2 H (150) 100% 152 – C2H2 (126) 4% 139 – 2 H (137) 43% 139 – C2H2 (113) 100%
4,6-
Dimethyldibenzo thiophene (212)
M+● (212)
212 – H (211) 2% 212 – CH3 (197) 100% 212 – HS (179)a 14%
197 – 2H (195) 2% 197 – C2H2 (171) 5% 197 – S (165) 100% 197– C3H6 (155) 3% 197 – CS (153) 39% 197 – CHS (152) 11% 179 – H (178) 100% 179 – 2 H (177) 3% 179 – 3 H (176) 14% 179 – C2H2 (153) 2% 179 – H – C2H2 (152) 25%
Thianthrene (216)
M+● (216) 216 – S (184) 100% 216 – CHS (171) 5% 216 – CS2 (140) 3%
184 – H (183) 2% 184 – C2H2 (158) 3% 184 – S (152) 100% 184 – HS (151) 2% 184 – CHS (139) 33%
2,2’-Bithiophene
(166)
M+● (166)
166 – S (134) 96% 166 – CS (122) 16% 166 – CHS (121) 100% 166 – 2 S (102) 7%
134 – S (102) 40% 134 – CS (90) 61% 134 – CHS (89) 55% 134 – C2H2 (108) 100% 121 – CS (77) 100%
a Refer to explanation on the formation of this m/z 179 fragment ion (212 – HS) in the text.
Note: The sulfur containing neutral molecules that were lost during CAD are highlighted in red color.
101
Table 4.4 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Sulfides
Analyte (MW) MS (m/z) MS2 CAD product ions (m/z) and their relative
abundance
MS3 CAD product ions (m/z) and their relative
abundance
Methyl phenyl sulfide (124)
M+● (124) 100% [M-HS]+ (91) 3% [M-CH2S]+ (78) 3%
124 – CH3 (109) 1% 124 – HS (91) 100% 124 – CH2S (78) 44%
91 – C2H2 (65) 100% 78 – H (77) 5% 78 – C2H2 (52) 100% 78 – H – C2H2 (51) 93% 78 – C2H4 (50) 15%
Ethyl phenyl sulfide (138)
M+● (138) 138 – CH3 (123) 50% 138 – C2H4 (110) 100% 138 – HS (105) 6%
123 – CS (79) 100% 110 – H (109) 8% 110– C2H2 (84) 63% 110 – CS (66) 100%
Benzyl methyl sulfide (138)
M+● (138) 100% [M-H]+ (137) 60% [M-CH3S]+ (91) 45%
138 – CH3 (123) 24% 138 – H2S (104) 9% 138 – CH2S (92) 12% 138 – CH3S (91) 100%
123 – H (122) 100% 104 – H (103) 26% 104 – C2H2 (78) 100% 104– H – C2H2 (77) 35% 91 – C2H2 (65) 100%
Diphenyl sulfide
(186)
M+● (186)
186 – H (185) 17% 186 – 2 H (184) 100% 186 – CH3 (171) 5% 186 – HS (153) 8% 186 – H2S (152) 11%
184 – C2H2 (158) 5% 184 – S (152) 100% 184 – CHS (139) 43% 171 – 2 H (169) 18% 171 –C2H2 (145) 8% 171 – CS (127) 100% 171 – CHS (126) 43% 152 – H (151) 90% 152 – 2 H (150) 100% 152 – 3 H (149) 80% 152 –C2H2 (126) 30%
Benzyl sulfide
(214)
M+● (214) 100% [M-H]+ (213) 40%
214 – H2S (180) 2% 214 – C6H6 (136) 15% 214 – C7H7 (123) 44% 214 – C7H8 (122) 100% 214 – C7H6S (92) 5% 214 – C7H7S (91) 4%
136 – H (135) 40% 136 – 2 H (134) 8% 136 – CS (92) 100% 136 – CHS (91) 62% 122 – H (121) 100% 122 – CS (78) 4% 122 – CHS (77) 3%
Benzyl phenyl sulfide (200)
M+● (200) 200 – C6H5S (91) 100% 91– C2H2 (65) 100%
Note: The sulfur containing neutral molecules that were lost during CAD are highlighted in red color.
102
Table 4.5 MS2 and MS3 CAD Product Ions (with Relative Abundances) for Ionized Disulfides
Analyte (MW) MS (m/z) MS2 CAD product ions (m/z) and their relative
abundance
MS3 CAD product ions (m/z) and their relative
abundance
Phenyl disulfide
(218)
M+● (218) 100% [M-C6H6]
+ (140) 10%
218 – HS (185) 87% 218 – H2S (184) 4% 218 – 2 S (154) 100% 218 - C6H6 (140) 2% 218 - C6H5S (109) 1%
185 – H (184) 100% 185 – 2 H (183) 30% 185 – HS (152) 91% 185 – H2S (151) 2% 185 – CH2S (139) 3% 154 – H (153) 35% 154 – 2 H (152) 100% 154 – 3 H (151) 12% 154 –C2H2 (128) 3%
Dicyclohexyl
disulfide (230)
M+● (230) 230 – C6H10 (148) 100% 230 – C6H11S2 (83) 5%
148 – S2H (83) 100% 83 – C2H4 (55) 100%
Dibenzyl disulfide
(246)
M+● (246) 246 – S2H (181) 100% 246 – C7H7S2 (91) 28%
181 – 2 H (179) 7% 181 – CH3 (166) 100% 181 – C2H4 (153) 14% 91 – C2H2 (65) 100%
Butyl disulfide (178)
M+● (178) 100% [M-HS]+ (145) 4%
178 – HS (145) 1% 178 – C4H8 (122) 100% 178 – C4H9S (89) 4% 178 – C4H10S (88) 8% 178 – C4H9S2 (57) 3%
122 – HS (89) 14% 122 – S2H (57) 100% 89 – H2S (55) 100% 88 – C2H4 (60) 100% 88 – H2S (54) 40%
Note: The sulfur containing neutral molecules that were lost during CAD are highlighted in red color.
103
Table 4.6 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 CAD Product Ions for Ionized Thiols
Analyte (MW) Measured Mass Elemental Composition Mass Accuracy (ppm)
Benzenethiol
(110)
MS2: 110.01863 66.04693
MS2: C6H6S C5H6
MS2: 0.158 0.528
Benzyl
mercaptan (124)
MS2: 124.02839 91.05461
MS2: C7H8S C7H7
MS2: -9.042 0.383
Table 4.7 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 and MS3 CAD Product Ions for Ionized Polyaromatic Sulfur Compounds
Analyte (MW) Measured Mass Elemental Composition Mass Accuracy (ppm)
Benzothiophene
(134)
MS2: 134.01792 108.00254 102.04611 90.04605 89.03808
MS2: C8H6S C6H4S
C8H6 C7H6 C7H5
MS2: -4.122 -2.615 -2.859 -3.907 -5.579
Dibenzothiophene
(184)
MS2: 184.03346 152.06154 140.06176 139.05408
MS2: C12H8S C12H8 C11H8 C11H7
MS2: -3.600 -3.366 -2.084 -1.056
4,6-
Dimethyldibenzo thiophene (212)
MS2: 212.06462 211.05697 197.04145 179.08488 MS3: 165.06920 153.06927 152.06144
MS2: C14H12S C14H11S C13H9S C14H11 MS3: C13H9 C12H9 C12H8
MS2: -3.785 -2.974 -2.525 -3.612 MS3: -4.101 -3.965 -4.024
Thianthrene (216)
MS2: 216.00548 184.03353 171.02595 140.06171 MS3: 152.06139 151.05383 139.05392
MS2: C12H8S2 C12H8S C11H7S
C11H8 MS3: C12H8 C12H7 C11H7
MS2: -3.302 -3.220 -2.032 -2.441 MS3: -4.352 -2.627 -2.206
104
Table 4.7, continued
Analyte (MW) Measured Mass Elemental Composition Mass Accuracy (ppm)
2,2’-Bithiophene
(166)
MS2: 165.98980 134.01809 122.01803 121.01020 102.04607 MS3: 108.00240 102.04599 90.04595 89.03814 77.03804
MS2: C8H6S2 C8H6S C7H6S C7H5S
C8H6 MS3: C6H4S
C8H6 C7H6 C7H5 C6H5
MS2: -4.477 -2.854 -3.626 -3.698 -3.251 MS3: -3.911 -4.035 -5.017 -4.905 -6.967
Table 4.8 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 and MS3 CAD Product Ions for Ionized Sulfides
Analyte (MW) Measured Mass Elemental Composition Mass Accuracy (ppm)
Methyl phenyl sulfide (124)
MS2: 124.03352 91.05379 78.04590
MS2: C7H8S C7H7 C6H6
MS2: -4.858 -4.797 -6.429
Ethyl phenyl sulfide (138)
MS2: 138.04920 123.02589 110.01810 105.06958 MS3: 79.05368
MS2: C8H10S C7H7S C6H6S
C8H9 MS3: C6H7
MS2: -4.148 -3.312 -3.386 -2.825 MS3: -6.917
Benzyl methyl sulfide (138)
MS2: 138.04476 123.02579 104.05714 92.05713 91.05379
MS2: C8H10S C7H7S
C8H8 C7H8 C7H7
MS2: -11.893 -4.125
-47.203 -53.465
-4.797
Diphenyl sulfide
(186)
MS2: 186.04843 171.02593 153.06502 152.06165 MS3: 152.06137 139.05390 127.05363 126.04580
MS2: C12H10S C11H7S
C12H9 C12H8
MS3: C12H8 C11H7 C10H7 C10H6
MS2: -7.216 -2.149
-31.731 -2.643 MS3: -4.484 -2.350 -4.697 -4.774
Benzyl sulfide
(214)
MS2: 214.07693 180.08818 92.05710 91.05377
MS2: C14H14S C14H12
C7H8 C7H7
MS2: -19.352 -28.719 -53.791
-5.017
Benzyl phenyl sulfide (200)
MS2: 200.06039 91.05378
MS2: C13H12S C7H7
MS2: -5.033 -0.447
105
Table 4.9 Measured Accurate Masses, Elemental Compositions, and Mass Accuracy (ppm) of MS2 and MS3 CAD Product Ions for Ionized Disulfides
Analyte (MW) Measured Mass Elemental Composition Mass Accuracy (ppm)
Phenyl disulfide
(218)
MS2: 218.02111 185.04145 184.03368 154.07718 109.01034 MS3: 152.06151 151.05373 139.05379
MS2: C12H10S2 C12H9S C12H8S C12H10 C6H5S
MS3: C12H8 C12H7 C11H7
MS2: -3.363 -2.689 -2.405 -3.387 -2.820 MS3: -3.563
-3.0289 -3.141
Dicyclohexyl
disulfide (230)
MS2: 230.11458 148.03681 83.08500 MS3: 83.08494
MS2: C12H22S2 C6H12S2
C6H11 MS3: C6H11
MS2: -3.884 -4.616 -6.342 MS3: -7.064
Dibenzyl disulfide
(246)
MS2: 246.05226 181.10050
91.05381
MS2: C14H14S2 C14H13
C7H7
MS2: -3.590 -3.738 -4.578
Butyl disulfide (178)
MS2: 178.08367 145.10394 122.02118 89.04151 88.03369 57.06937 MS3: 89.04143 57.06931
MS2: C8H18S2 C8H17S
C4H10S2 C4H9S C4H8S
C4H9 MS3: C4H9S
C4H9
MS2: -4.344 -4.189 -5.436 -4.914 -4.913 -8.882 MS3: -5.813 -9.933
106
4.4 Conclusions
APCI(+) with CS2 as reagent can be used to generate stable molecular ions for all
the organosulfur compounds studied, with the exception of minor fragmentation for some
ionized sulfides and disulfides. Upon CAD, characteristic product ions were observed in
MS2 and MS3 experiments, which correspond to losses of S (32 Da), HS● (33 Da), H2S
(34 Da), CS (44 Da) and ●CHS (45 Da). These fragmentations indicate the presence of
sulfur in the ionized molecules. Losses of HS● and H2S were found to be associated with
aliphatic sulfur moieties, while losses of S, CS and ●CHS were more common for
heteroaromatic compounds. However, the reverse of the above statement is not true, i.e.,
not all molecular ions of organosulfur compounds show such characteristic losses. For
instance, molecular ions of thiophenes do not lose sulfur-containing molecules upon
CAD. Molecular ions of benzyl phenyl sulfide and some disulfides lose a sulfur atom
along with other parts of the molecules in different ways upon CAD.
In summary, knowledge of the chemical bonding environments of sulfur is
beneficial to improving the desulfurization process of fossil fuels in the petroleum
industry. High-resolution tandem mass spectrometry holds promise to determining the
presence and types of organosulfur compounds (aliphatic vs. aromatic sulfur) in complex
mixture. However, caution should be taken when making generalized conclusions about
the components in a mixture because not all molecular ions of organosulfur compounds
show characteristic sulfur losses upon CAD.
107
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109
41. Earnshaw, D. G.; Cook , G. L.; Dinneen, G. U. J. Phys. Chem. 1964, 68, 296. 42. Cooks, R. G.; Howe, I.; Tam, S. W.; Williams, D. H. J. Amer. Chem. Soc. 1968, 90, 4064. 43. Cooks, R. G.; Bernasek, S. L. J. Am. Chem. Soc. 1970, 92, 2129.
110
CHAPTER 5. GAS-PHASE ION/MOLECULE REACTIONS FOR THE IDENTIFICATION OF SULFONE FUNCTIONALITIES IN PROTONATED
ANALYTES IN A LINEAR QUADRUPOLE ION TRAP MASS SPECTROMETER
5.1 Introduction
Rapid identification of drug metabolites, degradation products, and impurities is
crucial in the drug discovery and development process since some of them are toxic.1-3
Analytical techniques, such as NMR, FT-IR, and X-ray crystallography can be utilized to
obtain information on functional groups and elemental connectivity in the analyte.4-6
However, compounds that are present in only small quantities in complex mixtures are
difficult to identify using the above methods.
Tandem mass spectrometry (MSn) has evolved to be a powerful technique for
mixture analysis due to its high sensitivity, selectivity and speed.7,8 Single-stage mass
spectrometry (MS) can provide molecular mass and elemental composition of the ionized
analytes, while MSn utilizing collision-activated dissociation (CAD) can provide
structural information for the isolated, ionized unknown analytes.9 When the
functionalities of ionized analytes cannot be definitively determined by CAD alone, an
alternative MSn technique based on ion-molecule reactions can be utilized to obtain
structural information.10 Our group has successfully developed methods based on ion-
molecule reactions to identify different functional groups in ionized analytes.11-21 Several
neutral reagents have been investigated extensively, including boron- and carbon-
111
centered reagents with good leaving groups.11-21 However, phosphorous-centered neutral
reagents have been rarely studied. The only report appeared thus far focused on the
identification of the amino functionality as well as differentiation of primary, secondary
and tertiary protonated amino functionalities by using diethyl methylphosphonate and
hexamethylphosphoramide.22 In this case, proton transfer reaction and adduct formation
are the two major reaction pathways.22
Oxidized sulfur functionalities, such as sulfone and sulfoxide, are common in
drug metabolites.23 Only a few tandem mass spectrometry (MS/MS) experiments based
on CAD of ionized sulfones and sulfoxides have been published, yet none of them
showed sulfone or sulfoxide specific fragmentation patterns.24-27 In an effort to enable
identification of sulfur-containing functionalities in drug metabolites, we recently
reported a boron-centered reagent (trimethyl borate, TMB) that allows the identification
of protonated sulfone analytes19 and another carbon-centered reagent (2-methoxypropene,
MOP) for the identification of protonated sulfoxide analytes.20 TMB was found to yield a
diagnostic product ion, adduct-Me2O, upon reaction with protonated sulfone analytes.
MOP was found to yield the same MOP adducts for protonated sulfoxides and N-oxides,
yet the distinction of sulfoxides was based on their high reaction efficiencies. In order to
search for another reagent that would differentiate protonated sulfoxides from other
functionalities based on different reaction products, a phosphorous-containing reagent,
trimethyl phosphite (TMP), was examined. However, to our surprise, TMP was found to
allow for the differentiation of protonated sulfone functionality from many other
functional groups, including sulfoxide, hydroxylamino, N-oxide, aniline, amino, keto and
carboxylic acid functionalities. The reaction specificity was further demonstrated by
112
using a sulfoxide-containing anti-inflammatory drug, sulindac, as well as its metabolite
sulindac sulfone.
5.2 Experimental Section
Chemicals. All chemicals were purchased from Sigma-Aldrich. Their purities were ≥
98%. All chemicals were used without further purification.
Instrumentation. All mass spectrometry experiments were performed using a Thermo
Scientific LTQ linear quadrupole ion trap (LQIT) equipped with an APCI source. The
analytes were dissolved in methanol with a final concentration of 0.01-1 mg/mL. The
sample solutions were introduced into the mass spectrometer through direct infusion at a
flow rate of 20 μL/min by using a syringe drive. The APCI source was operated in
positive ion mode. The temperatures for the vaporizer and transfer capillary were set at
300 °C and 275 °C, respectively. Nitrogen was used as the sheath gas and auxiliary gas,
with the flow rate maintained at 30 and 10 arbitrary units respectively. The voltages for
the ion optics were optimized for each individual analyte by using the tune feature of the
LTQ Tune Plus interface. The normal mass range (m/z 50-500) was used for all the
experiments, while the low mass range (m/z 20-200) was used for examination of the
exothermic proton-transfer reaction between protonated methanol and the reagent (TMP).
The type of the manifold that was used to introduce the reagent was first described by
Gronert.28,29 A diagram of the exact manifold used in this research was published by
Habicht et al.[13] TMP was introduced into the manifold via a syringe pump at the rate of
10 μL/h. A known flow of helium gas (0.8 L/h) was used to carry TMP into the mass
113
spectrometer. The syringe port and surrounding area were heated to ~70 °C to ensure
evaporation of TMP. A Granville-Phillips leak valve was used to control the amount of
the reagent introduced into the instrument, while another leak valve controlled the
amount of helium diverted to waste.30 A typical nominal pressure of TMP in the ion trap
during the experiments was 0.6×10-5 Torr.
Kinetics. After the analytes were ionized by protonation in the APCI source, the
protonated analytes were isolated by using an isolation window of 2 Da. The isolated ions
were allowed to react with the reagent TMP for variable periods of time. In the course of
ion/molecule reactions, the concentration of the neutral reagent is in great excess of that
of the ion of interest. Therefore, the pressure of TMP can be considered as a constant, and
the reactions follow pseudo-first-order kinetics. The reaction efficiency corresponds to
the fraction of ion/molecule collisions that lead to the formation of products. The reaction
efficiency, kreaction/kcollision, was calculated by measuring the rate of each ion/molecule
reaction (IM) and the rate of the highly exothermic proton-transfer reaction (PT) between
protonated methanol and the reagent (TMP) under identical conditions. The above rates
were measured by determining the relative abundances of the reactant ion and product
ions as a function of reaction time. In a semilogarithmic plot of the ion abundances as a
function of time, the decay slope of the reactant ion corresponds to the rate constant k
multiplied by the neutral reagent's concentration. Assuming that the exothermic proton-
transfer reaction (PT) between protonated methanol and TMP proceeds at collision rate
(kcollision; this can be calculated by using a parameterized trajectory theory31), the
efficiencies of the ion/molecule reactions can be obtained by using equation 1. The
114
reaction efficiency is based on the ratio of the slopes of the two reactions studied, i.e.,
kreaction[TMP] = slope (IM), kcollision[TMP] = slope (PT), wherein [TMP] = TMP
concentration. It is obvious that there is no need to measure the concentration of TMP as
it cancels out in the calculation. Additionally, the reaction efficiency is dependent on the
masses of the ion (Mi), neutral reagent (Mn), and methanol (M(PT)), as well as the pressure
read by an ion gauge for the reagent during the ion/molecule reaction (Pn(IM)) and the
proton-transfer reaction (Pn(PT)).
5.3 Results and Discussions
In an effort to search for a reagent that would allow the mass spectrometric
distinction of protonated sulfoxides from protonated sulfones, trimethyl phosphite (TMP)
was examined because its proton affinity (PA) (~220 kcal/mol32) is close to that of
sulfoxides (~220 kcal/mol19), and higher than that of sulfones (~205 kcal/mol19).
According to the reactivity that has been reported for a similar boron-centered reagents
TMB,11-13 TMP was expected to react with a protonated sulfoxide via proton abstraction
followed by replacement of a methanol molecule in the adduct ion of protonated TMP
and the neutral analyte molecule. Meanwhile, TMP was expected to react with protonated
sulfones mostly via proton abstraction.
115
TMP was allowed to react with protonated analytes containing sulfone or
sulfoxide functionality. As shown in Table 5.1, proton transfer was the major reaction for
both protonated sulfones and sulfoxides. However, contrary to the expectations, only
protonated sulfones showed the [TMP adduct-MeOH] product ion, not protonated
sulfoxides. A mass spectrum measured after 100 ms reaction of protonated
dibenzothiophene sulfone with TMP is shown in Figure 5.1 as an example. The most
abundant product ion (m/z 125) corresponds to proton transfer. The other product ion
(m/z 309) corresponds to [TMP adduct-MeOH], which was only observed for protonated
sulfones.
In order to probe the selectivity of the above reaction for protonated sulfones,
TMP was allowed to react with other protonated analytes containing various functional
groups, such as N-oxide, hydroxylamino, keto, carboxylic acid, and aliphatic and
aromatic amino. The reaction products and efficiencies are summarized in Table 5.2. The
main reactions were proton transfer, and minor addition reactions were observed for
some protonated analytes, yet no [TMP adduct-MeOH] product ion was observed. When
protonated sulindac and its metabolite sulindac sulfone were allowed to react with TMP,
only sulindac sulfone showed [TMP adduct-MeOH] product ion (Figure 5.2). This result
demonstrates the utility of this method for the identification of the sulfone functionality
in drug metabolites.
A mechanism is proposed for the formation of [TMP adduct-MeOH] ion for
protonated sulfones as shown in Scheme 5.1. The unique selectivity of TMP toward
sulfones can be rationalized by the six-membered transition state of the ion-neutral
complex of protonated sulfone and TMP (Scheme 5.1). Through this transition state, the
pr
at
O
pr
fu
d
roton could
ttack of the s
On the other
roton would
unctional gro
Figurdibenzothiop
be transferr
sulfone to th
hand, for oth
d most likely
oups could n
re 5.1 A masphene sulfon
ed to the les
he phosphoro
her protonat
y be transferr
not form the
ss spectrum mne with TMP
ss basic meth
ous center to
ted functiona
red to the m
six-member
measured aft in LQIT (*s
hoxy group,
o form [TMP
alities such a
more basic ph
red transition
fter 100 ms rsecondary pr
, which initi
P adduct-Me
as sulfoxide
hosphorous c
n state as sul
reaction of prroducts of p
ated nucleop
eOH] produc
and N-oxid
center since
lfone did.
rotonated rotonated TM
116
philic
ct ion.
e, the
these
MP).
S
Fs
Scheme 5.1 T
Figure 5.2 Msulindac sulf
The proposeproduct i
Mass spectra fone (bottom
s
d mechanismion when a p
measured afm) with TMPsecondary pr
m for the forprotonated su
fter 300 ms rP in LQIT (*roducts of pr
rmation of a ulfone react
reaction of p*secondary protonated TM
stable [TMPs with TMP
protonated suproducts of TMP).
P adduct-Me.
ulindac (top)TMP adducts
117
eOH]
) and s; **
118
Table 5.1 Reaction products (m/z values and branching ratios) and efficiencies for reactions of protonated sulfones and sulfoxides with TMP (PA = 222.2 kcal/mol a).
Reagent
(m/z of [M+H]+) PA
(kcal/mol)Product ions (m/z) and
branching ratiosReaction efficiency
(119)
206.3b Proton Transfer (125) 72%
Adduct – MeOH(309) 28%
90%
(157)
201.4b Proton Transfer (125) 74%
Adduct – MeOH (249) 26%
76%
(179)
203.7b Proton Transfer (125) 98% Adduct –MeOH (271) 2%
94%
(95)
193.5b Proton Transfer (125) 75%
Adduct – MeOH (187) 25%
98%
(121)
198.3b
Proton Transfer (125) 71% Adduct – MeOH (213) 29%
90%
(217)
205.0b
Proton Transfer (125) 94% Adduct – MeOH (309) 6%
59%
(197)
200.9c
Proton Transfer (125) 29% Adduct – MeOH (289) 7% Adduct (321) 64%
58%
(158)
202.9c
Proton Transfer (125) 96% Adduct – MeOH (250) 3% Adduct (282) 1%
89%
S
O
O
S
O
O
SO O
119
Table 5.1, continued
Reagent (m/z of [M+H]+)
PA (kcal/mol)
Product ions (m/z) andbranching ratios
Reaction efficiency
(163)
220.1b
Proton Transfer (125) 96% Adduct (287) 4%
87%
(203)
222.5b Proton Transfer (125) 95%
Adduct (327) 5%
73%
(79)
211.3a
Proton Transfer (125) 98% Adduct (203) 2%
101%
(166)
227.9c Proton Transfer (125) 1%
Adduct (290) 99%
3%
aReference 32. b Reference 19. c Calculated at the B3LYP/6-31G++(d,p) level of theory.
H3CS
OH
OO
NH2
120
Table 5.2 Reaction products (m/z values and branching ratios) and efficiencies for reactions between protonated N-oxides, ketones, hydroxylamines, carboxylic acids, aliphatic and aromatic amines with TMP (PA = 222.2 kcal/mol a).
Reagent (m/z of [M+H]+)
PA (kcal/mol)
Product ions (m/z) and branching ratios
Reaction efficiency
(96)
219.2b Proton Transfer (125) 98%
Adduct (220) 2%
94%
(146)
225.5b Proton Transfer (125) 66% Adduct (270) 34%
10%
(99)
201a Proton Transfer (125) 100%
110%
(183)
210.8b Proton Transfer (125) 98% Adduct (307) 2%
91%
(90)
218.6b Proton Transfer(125) 97%
Adduct (214) 3% 87%
(116)
215.9b
Proton Transfer (125) 95% Adduct (240) 5%
94%
(138)
206.7a Proton Transfer (125) 100% 71%
N
O
N
O
O
121
Table 5.2, continued
Reagent (m/z of [M+H]+)
PA (kcal/mol)
Product ions (m/z) andbranching ratios
Reaction efficiency
(139)
200.7c Proton Transfer (125) 100% 78%
(180)
207.5c Proton Transfer (125) 98%
Adduct (304) 2% 95%
(74) 220.2b
Proton Transfer (125) 95% Adduct (198) 5%
30%
(100)
223.3a
Proton Transfer (125) 94%
Adduct (224) 6% 86%
(94)
210.9b Proton Transfer (125) 100% 53%
(170)
214.4c
Proton Transfer (125)
97%
Adduct (294) 3% 46%
aReference 32. bReference 20. cCalculated at the B3LYP/6-31G++(d,p) level of theory.
HO
HO
O
HN
122
Table 5.3 Reaction products (m/z values and branching ratios) and efficiencies for reactions of protonated sulindac and sulindac sulfone with TMP (PA = 222.2 kcal/mol a).
Reagent (m/z of [M+H]+)
PAb (kcal/mol)
Product ions (m/z) and branching ratios
Reaction efficiency
Sulindac (373)
224 Proton Transfer (125) 89%
Adduct (481) 11%
53%
Sulindac sulfone (373)
203
Proton Transfer (125) 75% Adduct – MeOH (465) 9% Adduct (497) 16%
78%
aReference 32. bReference 33.
HOOC
F
SO
HOOC
F
S
O
O
123
5.4 Conclusion
A method based on a functional group-selective ion/molecule reaction in a linear
quadrupole ion trap mass spectrometer has been demonstrated for the identification of the
sulfone functionality in protonated analytes. A phosphorous-centered neutral reagent,
trimethyl phosphite (TMP), can form characteristic [TMP adduct- MeOH] product ions
only when allowed to react with protonated sulfone analytes. All other protonated
compounds investigated in this study, with functionalities such as sulfoxide, N-oxide,
hydroxylamino, keto, carboxylic acid, aliphatic and aromatic amino, react with TMP via
proton transfer and/or addition. The selectivity of TMP toward sulfones can be
rationalized by the six-membered transition state of the ion-neutral complex of
protonated sulfone and TMP, while other functionalities cannot form such transition state.
The results obtained for sulindac and sulindac sulfone suggest that this method allows the
identification of sulfone functional group in drug metabolites even in the presence of
other functionalities.
124
5.5 Reference
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2. X. Chen, S. Hussain, S. Parveen, S. Zhang, Y. Yang, C. Zhu, Curr. Med. Chem. 2012, 19, 3578-3604.
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5. D. P. Bhave, Muse, Carroll, Infect. Disord. Drug Targets 2007, 7, 140-158.
6. D. A. Dibbern, A. Montanaro, Ann. Allergy Asthma Immunol. 2008, 100, 91-100.
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8. R. H. Perry, R. G. Cooks, R. J. Knoll. Mass Spectrom. Rev. 2008, 27, 661-699.
9. R. G. Cooks, K. L. Busch, G. L. Glish, Science, 1983, 222, 273-291.
10. S. Osburn, V. Ryzhov, Anal. Chem. 2013, 85, 769-778.
11. M. A. Watkins, B. E. Winger, R. C. Shea, H. I. Kenttamaa. Anal. Chem. 2005, 77, 1385-1392.
12. K. M. Campbell, M. A. Watkins, S. Li, M. N. Fiddler, B. Winger, H. I. Kenttämaa, J. Org. Chem. 2007, 72, 3159-3165.
13. S. C. Habicht, N. R. Vinueza, E. F. Archibold, P. Duan, H. I. Kenttämaa, Anal. Chem. 2008, 80, 3416-3421.
14. M. Fu, P. Duan, S. Li, S. C. Habicht, D. S. Pinkston, N. R. Vinueza, H. I. Kenttämaa, Analyst 2008, 133, 452-454.
15. P. Duan, T. A. Gillespie, B. E. Winger, H. I. Kenttamaa. J. Org. Chem. 2008, 73, 4888-4894.
16. P. Duan, M. Fu, T. A. Gillespie, B. E. Winger, H. I. Kenttämaa, J. Org. Chem. 2009, 74, 1114-1123.
17. J. Somuramasami, P. Duan, L. Amundson, E. Archibold, B. Winger, H. I. Kenttämaa, J. Am. Soc. Mass Spectrom. 2011, 22, 1040-1051.
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18. R. J. Eismin, M. Fu, S. Yem, F. Widjaja, H. I. Kenttämaa, J. Am. Soc. Mass Spectrom. 2012, 23, 12-22.
19. H. Sheng, P. E. Williams, W. Tang, M. Zhang, H. I. Kenttämaa, J. Org. Chem. 2014, 79, 2883-2889.
20. H. Sheng, P. E. Williams, W. Tang, M. Zhang, H. I. Kenttämaa, Analyst 2014, 139, 4296-4302.
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25. W. Lam, R. Ramanathan, J. Am. Soc. Mass Spectrom. 2002, 13, 345-353.
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126
CHAPTER 6. GAS-PHASE REACTIVITY OF META-BENZYNES TOWARDS
AMINO ACIDS
6.1 Introduction
Radicals are known to attack proteins, resulting in their denaturation, oxidation,
fragmentation and degradation, which is associated with aging and many human
diseases.1-4 Therefore, it is important to understand the processes involved. Numerous
solution studies have been carried out on reactions of oxygen-containing radicals with
proteins, peptides, and amino acids,5-8 whereas carbon-centered radicals are rarely
investigated. However, carbon-centered biradicals are of great interest as they are the
biologically active intermediates of the naturally occurring anticancer antibiotics called
enediynes, which are known to attack DNA.9-11 Additionally, proteins can also be
damaged by such carbon-centered radicals.12-14 The one report that has appeared thus far
focused on reactions of phenyl radicals toward glycine in solution.15 Specifically, a para-
benzoic acid radical (4-dehydrobenzoic acid) was found to abstract a deuterium atom
from the α-position of α,α-dideuterioglycine in solution.15 The study of carbon-centered
radicals is challenging, which is largely due to the difficulty of generating pure radicals in
solution. Alternatively, gas-phase experiments can be performed to generate the radicals
relatively easily, which also allow their intrinsic chemical properties to be examined in a
solvent-free environment.
127
In order to investigate the gas-phase reactivity of carbon-centered radicals toward
various substrates, our laboratory has advanced the “distonic ion approach” by using
mass spectrometry.16-20 A distonic radical ion can be formed by attaching a chemically
inert charged moiety to the radical of interest, which allows for its mass spectrometric
manipulation and detection. This approach has been applied to gas-phase reactions of
phenyl radicals with amino acids and peptides, the results of which reveal similar
reactivity to that of neutral phenyl radicals in solution.21-24 For instance, both a gas-phase
positively charged phenyl radical (e.g., N-(3-dehydrophenyl)pyridinium) and a neutral
phenyl radical in solution (e.g., 4-dehydrobenzoic acid) abstract a deuterium atom from
the α-position of α,α-dideuterioglycine.15,21 In addition to the hydrogen atom abstraction
(radical mechanism), other reaction pathways were observed for more electrophilic
phenyl radicals (e.g., N-phenyl-3-dehydropyridinium) in the gas phase.21,22 These
reactions include NH2 abstraction, SH and SCH3 abstractions from cystein and
methionine, addition to the aromatic ring and side-chain abstraction from aromatic amino
acids, which were assumed to occur via addition-elimination mechanisms due to the
greater electrophilicity of these radicals.21,22 The electrophilicity of a radical can be
quantified by the calculated vertical electron affinity (EA) of its radical site, which is
defined as the energy released upon addition of an electron to the radical site with no
geometry change.25 The greater the EA, the more polar the transition state, thus the faster
are both radical and nonradical reactions. This can be rationalized by the ionic avoided
curve crossing model proposed by Anderson et al.26 EA has been demonstrated to be an
important reactivity controlling factor not only for carbon-centered σ-monoradicals but
also for related σ,σ-biradicals.25-27
128
Among carbon-centered biradicals, the three isomeric didehydrobenzenes, ortho-,
meta-, and para-benzynes (Figure 6.1), have attracted great attention in the scientific
community.28-41 They are important reactive intermediates and fundamentally intriguing
molecules. The ortho-benzyne 1 has been thoroughly studied both experimentally and
computationally.28-30 It has been generated from various commercially available
precursors in solution, and characterized by IR, UV-Vis, and NMR spectroscopy.31-33
Interest in para-benzynes (for example, 3) has grown rapidly since the discovery of para-
benzyne intermediates that are responsible for the antitumor activity of enediynes.9-14
This is due to the fact that para-benzyne analogues can abstract a hydrogen atom from
both stands of double-stranded DNA, thus causing irreversible DNA cleavage.35-38
However, the clinical use of these anticancer reagents is hindered by their high toxicity.34
The unwanted side effects of these radical intermediates can also be attributable to their
reactions with proteins.12-14 Compared to ortho- and para-benzynes, meta-benzynes (like
2) have not received the same degree of attention.
Figure 6.1 Structures of ortho- (1), meta- (2), and para-benzyne (3).
A key parameter that affects the reactivity of benzynes is the degree of interaction
between the two biradical electrons.39 This interaction can be described by the magnitude
of singlet-triplet (S-T) splitting, which is defined as the energy difference between the
129
singlet ground state and the lowest energy triplet state. The S-T splittings of the three
benzynes have been measured by negative ion photoelectron spectroscopy.41 The ortho-
benzyne has a large S-T splitting (-37.5 kcal/mol) due to strong through-space coupling,
therefore, a large amount of energy is required to uncouple the biradical electrons.41 This
explains why nonradical reactivity was observed for ortho-benzynes.42 The para-benzyne
has a much smaller S-T splitting (-3.8 kcal/mol) due to weak through-bond interaction
between the biradical electrons.41 Hence, it displays radical reactivity.43,44 It should be
noted that the radical reactivity of para-benzyne analogues is substantially lower than
that of related monoradicals because of the through-bond coupling of the biradical
electrons.39 The meta-benzyne has an intermediate S-T splitting (-21.0 kcal/mol),41 which
is thought to hinder radical reactivity and can potentially make a more selective warhead
for antitumor reagents than para-benzynes.39,40
Gas-phase studies have indicated that the reactivity of meta-benzynes is affected
by both EA and the extent of S-T splitting, as expected based on above discussion.
Additionally, another important reactivity controlling factor for meta-benzynes is
distortion energy (△E2.30), which is the energy required to distort the minimum energy
dehydrocarbon atom separation (DAS) to the separation of the transition state, which is
approximately at 2.30 Å.45-48 A small △E2.30 means that it is energetically easier to
uncouple the singlet biradical electrons in the transition state for radical reactions. This
explains why some meta-benzynes with very small △E2.30 undergo radical reactions
despite of their large S-T splitting.48 Moreover, the reactivity of meta-benzynes can be
"tuned" from electrophilic to radical-like by changing the substituent groups, which
influence S-T splitting, EA and distortion energy of the benzynes.47,48 Therefore, it is
130
critical to understand the factors that control the reactivity of meta-benzynes toward
organic substrates, and more importantly, biomolecules, including DNA and proteins.
Such knowledge could facilitate rational design of better antitumor drugs. This study
focuses on the understanding of meta-benzynes' reactivity toward proteins. Due to the
large size and complexity of proteins, the reactions of selected meta-benzynes with free
amino acids were examined. So far, only one report has been published on the reactions
of a meta-benzyne analogue (N-methyl-6,8-didehydroquinolinium cation) with free
amino acids and dipeptides.49 In this work, the reactivity of four meta-benzyne analogues
with varying EA, S-T splitting, and distortion energy was studied towards amino acids in
an FT-ICR mass spectrometer. To the best of our knowledge, this is the first study on the
effects of the above parameters on the gas-phase reactivity of meta-benzynes toward
different amino acids.
6.2 Experimental Section
All the experiments were carried out in a Finnigan FTMS 2001 dual-cell Fourier-
transform ion cyclotron resonance (FT-ICR) mass spectrometer. The amino acids were
introduced into the mass spectrometer by using a manual solids probe. The probe was
heated to 140 oC for all amino acids except lysine for which the probe was heated to
200°C. Observation of an abundant protonated amino acid and only minor amounts of
other ions upon reaction with protonated acetone (proton affinity = 194 kcal/mol50)
confirmed that the amino acids were introduced into the instrument without much thermal
decomposition. The proton affinities of the amino acids are 211.9 kcal/mol for glycine,
218.6 kcal/mol for leucine, 238 kcal/mol for lysine, 223.6 kcal/mol for methionine, 215.9
131
kcal/mol for cysteine, 220 kcal/mol for proline, and 220.6 kcal/mol for phenylalanine.50
All amino acids (purity ≥ 98.5 %)) except DL-lysine-ε-15N were obtained from Fluka
Biochemika. DL-Lysine-ε-15N (purity ≥ 98.5%) was obtained from Cambridge Isotope
Laboratories, Inc. All amino acids were used without further purification. The other
reagents, tetrahydrofuran, allyl iodide, dimethyl disulfide, and tert-butyl isocyanide, were
obtained from Sigma Aldrich Co. and were used as received.
The radicals’ precursor ions were generated by CH3I chemical ionization in one
side of the dual-cell mass spectrometer as described previously.27,49,51 For example, N-
methyl-6,8-didehydroquinolinium cation (radical b) was formed by introducing 6,8-
dinitroquinoline and methyl iodide into the same cell of the instrument through a solids
probe and a pulsed valve, respectively. An electron beam of 20-25 eV kinetic energy was
used; the filament current was 7 μA and the ionization time 1 s. Methyl iodide undergoes
electron ionization and self-chemical ionization to form dimethyl iodide cation, which
then transfers a methyl cation to the neutral radical precursor, subsequently generating N-
methyl-6,8-dinitroquinoline cation. The N-methylated precursor ion was transferred into
the other cell by grounding the conductance limit plate for about 154 μs. Quadrupolar
axialization (QA) was employed to increase ion transfer efficiency.52 Next, the radical
sites were generated by sustained off-resonance irradiated collision-activated dissociation
(SORI-CAD).53 This involved applying an off-resonance RF voltage to the excitation
plates of the cell and pulsing argon (at a nominal pressure about 10-5 torr) into the cell.
Collisions with argon for 0.5-1 s at an RF frequency 1000 Hz higher or lower than the
ions' cyclotron frequency resulted in homolytic cleavages of the two carbon-nitrogen
bonds. The other radicals were generated using a similar procedure except that different
132
precursors were used: 3,5-diiodopyridine for a, 5,7-dinitroisoquinoline for c, 5,7-
dinitroquinoline for d, 3-iodopyridine for e, 5-nitroisoquinoline for f, and 6-
nitroquinoline for g.
After the radicals were generated, they were isolated by ejecting all other ions
from the cell by applying a series of stored-waveform inverse Fourier transform (SWIFT)
excitation pulses to the plates of the cell.54 The isolated radical ions were allowed to react
with an amino acid for a variable period of time (typically 0.5-1000 s). Detection was
performed by using “chirp” excitation of 124 V amplitude, 2.7 MHz bandwidth, and 3.2
kHz/μs sweep rate to kinetically excite the ions so that they move coherently as ion
packets and closer to the detection plates which is required for their detection. All mass
spectra presented here are the average of five transients, which were recorded as 64k data
points and subjected to one zero fill prior to Fourier transformation. Each reaction
spectrum was background corrected by using a procedure described previously.55
All reactions were found to follow pseudo-first order kinetics, which allows for
the determination of the second-order reaction rate constant (kexp) from a semilogarithmic
plot of the relative abundance of the reactant ion versus reaction time and the
concentration of the amino acid. In the FT-ICR, the concentration of ions (charged
radicals) inside the cell is much smaller than the concentration of neutral molecules
(amino acids). Therefore, the concentration of the amino acid can be assumed to be
constant. The concentration of the amino acids was determined by measuring the pressure
inside the cell by an ionization gauge that is located on each side of the dual cell. The ion
gauge pressure readings were corrected for the sensitivity of the ion gauge toward each
amino acid and for the pressure gradient between the ion gauge and the cell. The
133
correction factors were obtained by measuring the reaction rate of an exothermic proton-
transfer reaction from protonated acetone or protonated methanol to the given amino acid.
Such reactions can be expected to occur at collision rate.56 The accuracy of the measured
rate constants is estimated to be around 50%, and the precision is estimated to be better
than 20%. The theoretical collision rate constants (kcoll) were obtained using a
parameterized trajectory theory.57 The efficiency of each reaction (the fraction of
collisions that leads to reaction) is given by kexp/kcoll. The primary products’ relative
abundances (branching ratios) are given as the ratio of a given primary product ions’
abundance divided by the sum of all primary product ions’ abundances.
Quantum chemical calculations were performed with the Gaussian 03 and Molpro
electronic structure program suites. Molecular geometries for the (bi)radicals were
calculated as described previously.48 For meta-benzyne analogues a-d, the S–T splitting
at the dehydrocarbon atom separation of the transition state (∆ES-T), the electron affinity
at the dehydrocarbon atom separation of the transition state (EA2.30), and the distortion
energy (∆E2.30) were calculated at the RHF-UCCSD(T)/cc-pVTZ//B3LYP/cc-pVTZ level
of theory. The potential energy surfaces for meta-benzynes a-d were calculated at the
UBLYP/cc-pVDZ//UBLYP/cc-pVDZ level of theory. The charge densities were
calculated at the UB3LYP/cc-pVTZ//UB3LYP/cc-pVTZ level of theory. The activation
enthalpies for hydrogen atom abstraction from methane and addition of water and
ammonia to different radical sites of the meta-benzyne analogues were calculated at the
MPW1K/6-31+G(d,p)//MPW1K/6-31+G(d,p) level of theory. For σ-monoradicals e-g,
the electron affinities were calculated at the RHF-UCCSD(T)/cc-pVTZ//B3LYP/cc-
pVTZ level of theory.
134
6.3 Results and Discussion
The four positively charged meta-benzyne analogues and three related σ-
monoradicals selected for this study are N-methyl-3,5-didehydropyridinium (a), N-
methyl-6,8-didehydroquinolinium (b), N-methyl-5,7-didehydroisoquinolinium (c), N-
methyl-5,7-didehydroquinolinium (d), N-methyl-3-dehydropyridinium (e), N-methyl-5-
dehydroisoquinolinium (f), and N-methyl-6-dehydroquinolinium (g) cations (Figure 6.2).
Before the reactivities of the meta-benzynes (a-d) toward amino acids were examined,
their reactivities towards simple organic molecules, such as tetrahydrofuran, allyl iodide,
dimethyl disulfide, and tert-butyl isocyanide, were investigated (Table 6.1). The reactions
of amino acids with related monoradicals were also examined (Table 6.2). Finally, meta-
benzyne analogues (a-d) were allowed to react with glycine, leucine, lysine, isotopically
labeled lysine (lysine-ε-15N), methionine, cysteine, proline and phenylalanine for variable
periods of time. Their reaction efficiencies and product branching ratios were determined,
as summarized in Tables 6.3-6.5. Three important reactivity-controlling parameters for
meta-benzynes were listed in the tables. They are S-T splitting (△ES-T), EA at the
transition state geometry (EA2.30), and distortion energy (△E2.30). The reason of
computing EA2.30 for meta-benzynes is because EA at the 2.3 Å transition state geometry
is a much more important reactivity controlling factor than the EA at the minimum
energy (ground state) geometry.48
135
Figure 6.2 Structures of the meta-benzyne analogues (a-d) and related monoradicals (e-g) studied.
In order to better understand the role of distortion energy as a reactivity
controlling parameter for meta-benzynes, potential energy surfaces for biradicals a-d are
shown in Figure 6.3. The distortion energies for N-methyl-3,5-didehydropyridinium (a)
and N-methyl-5,7-didehydroquinolinium (d) are similar, which are 7.6 and 8.6 kcal/mol
respectively. In contrast, the distortion energies for N-methyl-6,8-didehydroquinolinium
(b) and N-methyl-5,7-didehydroisoquinolinium (c) are much smaller, which are 4.2 and
3.5 kcal/mol respectively. Therefore, a small amount of energy is required to distort the
minimum energy dehydrocarbon atom separation (DAS) to the separation of the
transition state for biradical b and c. Hence, they are more likely to display radical
reactivity compared to biradical a and d. This finding was demonstrated in the present
study, as discussed below.
B
m
te
is
on
st
si
A
Fig
Biradicals’ r
The r
molecules ha
etrahydrofur
socyanide (tB
n the reactiv
tudied.
Biradi
imple organi
AI, a CN gro
gure 6.3 Rela
reactions wi
eactivity of
ave not be
an (THF),
BuNC) were
vity of the fo
ical b demo
ic substrates
up from tBu
ative energy meta-b
th simple or
the four N-
en examine
allyl iodide
e investigate
our meta-ben
onstrates mo
s. It abstracts
uNC (Table 6
versus dehy
benzyne anal
rganic mole
-methylated
ed previous
(AI), dime
ed. More im
nzynes befo
ostly radical
s an hydroge
6.1), just as
ydrocarbon alogues a-d.
ecules
biradicals a
sly. Therefo
ethyl disulfi
mportantly, th
ore their reac
l reactivity
en atom from
monoradica
atom separat
a – d towar
ore their re
fide (DMDS
his knowledg
ctions with a
when allow
m THF, an i
als do.58-60 A
tion for
d simple or
eactivity tow
S), and tert-
ge can shed
amino acids
wed to react
iodine atom
At the first gl
136
ganic
wards
-butyl
d light
were
with
from
lance,
137
this behavior appears to be in conflict with general expectations. A large S-T splitting,
such as that of b (-18.7 kcal/mol), is generally thought to hinder radical reactivity, as is
the case for many other meta-benzyne type biradicals.39,40 However, the key to the
behavior of biradical b is its small distortion energy (4.2 kcal/mol), an important
reactivity controlling parameter for meta-benzynes.48 Only a small amount of energy is
required to partially uncouple the biradical electrons in the transition state for radical
reactions. Some other reactions products, including 2H-atom abstraction, SCH3
abstraction, HCN abstraction, could be the results of nucleophilic addition reactions.
These results indicated that biradical b could react via both radical and nonradical
mechanisms.
For biradical c, fewer characteristic radical reactions were observed upon its
interaction with simple organic molecules (Table 6.1). Biradical c reacted with AI and
tBuNC by mainly I atom and minor CN group abstraction respectively, which are
characterized as radical reactions. However, no characteristic H-atom abstraction (radical
reaction) was observed for c upon reaction with THF. The other reactions products, such
as SCH3 abstraction from DMDS, HCN abstraction from tBuNC, could arise from
nucleophilic addition reactions. Generally, c was found to react with AI, DMDS, and
tBuNC slower than b did. These results indicated that biradical c could react with organic
substrates via both radical and nonradical mechanisms, but its radical reactivity is lower
than that of b. This can be rationalized as lower EA2.30 of c than that of b, although both
radicals have similar S-T splitting and distortion energy values.
Different from b and c, the only definite radical reaction observed for biradical a
was an iodide atom abstraction from AI (Table 6.1). It reacted with THF by 2H-atom
138
abstraction and C4H6 abstraction, while no H atom abstraction was observed. Addition
reactions were observed for a upon reaction with AI, and an exclusive HCN abstraction
was observed upon reaction with tBuNC. The predominant nonradical reactivity of a can
be rationalized by its relatively large distortion energy, i.e. an energy of 7.6 kcal/mol is
required to distort the biradical's minimum energy geometry to the separation of the
transition state. To avoid this energetically costly uncoupling of the biradical electrons,
biradical a tends to undergo nucleophilic addition reactions as opposed to radical
reactions. In terms of total reaction efficiencies, a reacted with most organic substrates
the fastest, except for THF. This is due to the fact that it has the largest EA2.30, which is
thought to enhance both radical and nonradical reactions. However, compared to
monoradicals, biradical a is less reactive.58-60 This is because of the biradical's relatively
large S-T splitting (-23.3 kcal/mol), which has been demonstrated to hinder radical
reactions.
Biradical d was found to be unreactive toward THF, AI and DMDS. It only
reacted with tBuNC by HCN abstraction at an efficiency 13-times lower than that of b.
The nonradical reactivity of d can be rationalized by its largest distortion energy (8.6
kcal/mol) among the four biradicals. The low reaction efficiency could be attributable to
its relatively small EA2.30 (5.14 eV). In agreement, a previous study reported that d is
unreactive toward dinucleoside phosphates whereas biradical b is not.51 In summary, the
radical reactivity of the four biradicals can be ranked as below based on their reactions
with organic substrates, b > c > a > d (none).
139
Monoradicals’ reactions with amino acids
The electorn affinity (EA) has been demonstrated as a major reactivity controlling
parameter for reactions of charged monoradicals with small organic substrates as well as
amino acids.21,22,25 The monoradicals studied here have decreasing EA values as follows:
radical e (5.75 eV) > radical f (4.74 eV) > radical g (4.57 eV). The ordering of EA's was
found to correlate with the monoradicals’ reaction efficiencies towards the amino acids
studied. In general, the greater the EA of the radical, the greater the total reaction
efficiency. Additionally, more NH2 abstraction and less hydrogen atom abstraction were
observed for radical e with larger EA than radical f and g with smaller EA's. This is in
agreement with the previous finding that, as the EA of a radical increases, its addition
reactions become faster than hydrogen atom abstraction.19,20 The details of the reactions
are discussed below.
Glycine and leucine react via two reaction pathways with radicals e – g, hydrogen
atom abstraction and NH2 abstraction (Table 6.2). The more electrophilic radical e reacts
by NH2 abstraction faster than the less electrophilic radicals f and g. The greater the EA
of the monoradical, the more the NH2 abstraction is favored. NH2 abstraction has been
suggested to occur via a nucleophilic addition-elimination reaction pathway that
progresses through the formation of an addition intermediate.21 In the case of glycine and
leucine, the formation of this addition intermediate is highly exothermic and fragments
due to the inability for gas-phase systems to transfer energy to the surroundings.
Additionally, leucine yields a higher branching ratio for hydrogen atom abstraction than
glycine (Table 6.2), suggesting that hydrogen atom abstraction preferentially occurs from
the side-chain of the amino acids. In fact, a previous study on the reactivity of partially
140
isotope labeled amino acids towards charged phenyl monoradicals confirms that most
hydrogen atoms are abstracted from the alkyl side chain and a small amount of hydrogen
atoms are abstracted from the α-carbon.24
Proline reacts with radicals e – g mainly via hydrogen atom abstraction. Proline
does not react by NH2 abstraction because its nitrogen atom is part of a five-membered
ring. However, a ring-opening product, formed by C2H4N abstraction, was also observed
for e – g. The C2H4N abstraction product was also observed for reactions of a charged
phenyl radical (N-phenyl-3-dehydropyridinium) with proline in an earlier study.24
C2H4N abstraction from proline likely starts by nucleophilic addition of the proline
nitrogen to the radical site, followed by elimination of CO2 as well as ethylene from the
ring in proline.24 Several additional reaction pathways were observed including
abstraction of OH group, addition and addition – OH, which could also be initiated by
similar nucleophilic addition of the proline to the radical.
For lysine and lysine-ε-15N, hydrogen atom abstraction and NH2 abstraction were
observed for reactions of radicals e – g. Both NH2 groups of L-lysine-ε-15N can react with
the radical sites, which agrees with the finding from a previous study.24 Additional
reaction pathways were observed including addition, addition – COOH, and abstraction
of CH2NH group. A possible mechanism for the formation of a stable adduct has been
proposed earlier for a charged phenyl radical (N-phenyl-3-dehydropyridinium) when it
reacts with lysine. 24
141
Biradicals’ reactions with amino acids
Glycine, Leucine, Lysine, and 15NH2-Lysine
The results for reactions of biradicals a – d toward the simple aliphatic amino
acids, glycine, leucine, lysine, and 15NH2-lysine are summarized in Table 6.3. A previous
study has demonstrated that the N-methyl-6,8-didehydroquinolinium cation b reacts with
amino acids via both radical reaction pathways and nucleophilic addition-elimination
pathways.49 The reaction products observed in this study are similar to those in the
literature report, including one (or two) hydrogen atoms abstraction, H2O abstraction,
addition, addition – CO2, addition – HCOOH and addition – COOH (Table 6.3). The
abstraction of two hydrogen atoms can be rationalized by radical reaction mechanisms,
which is likely initiated by H-atom abstraction from α-carbon or from the alkyl chain of
the amino acids.49 The other reactions observed are likely initiated by nucleophilic
addition of NH2 or OH group to the more electrophilic radical site at carbon 6.49
For biradical c which has similar S-T splitting (-17.6 kcal/mol) and distortion
energy (3.5 kcal/mol) compared to that of biradical b, it is reasonable to predict that it
may show some radical-type reactivity just as biradical b does. However, one hydrogen
atom abstraction (radical reaction) was not observed for biradical c upon interaction with
aliphatic amino acids. Only trace amount and small amount of 2H-atom abstraction were
observed when biradical c reacts with glycine and lysine respectively. Unlike H-atom
abstraction, 2H-atom abstraction can be either radical (consecutive hydrogen atom
abstraction) or nonradical reactions (hydride abstraction followed by proton transfer).
The majority of other reactions pathways observed for biradical c are addition, NH3
abstraction, H2O abstraction, addition – COOH and addition – H2O (Table 6.3), which
142
are likely initiated by nucleophilic addition-elimination reactions (nonradical reactions).
The absence of H-atom abstraction pathway for biradical c is likely due to its low EA2.30,
yet the radical reactivity of biradical c could not be ruled out based on these results.
For biradical a with a relatively large S-T splitting (-19.4 kcal/mol) and large
distortion energy (7.6 kcal/mol), only trace amount of 2H-atom abstraction was observed
when it reacted with glycine and leucine. Most of the other reactions were nucleophilic
addition reactions, which were similar to those observed for biradical b with the same
group of amino acids, such as H2O abstraction, addition, addition – CO2, addition –
HCOOH and addition – COOH (Table 6.3). This suggests that nonradical reaction
pathway dominates for the reactions of biradical a with aliphatic amino acids. It should
be noted that the branching ratio of 2H-atom abstraction increased for biradical a upon
interaction with lysine, which has a larger alkyl side chain. This finding agrees with what
has been observed for positively charged phenyl monoradicals, i.e. the larger the alkyl
side chain of an amino acid, the higher the branching ratio of H-atom abstraction.21 This
is due to the fact that hydrogen atoms can be abstracted from both α-carbon and the alkyl
side chain.24 Different from biradical b, biradical a displays an additional reaction
pathway, NH3 abstraction. Since both NH3 and 15NH3 groups were abstracted from lysine
labeled with 15N on the side chain, NH3 abstraction is likely initiated by NH2 abstraction
from either the amino terminus or the side chain of lysine followed by a hydrogen atom
abstraction by another unquenched radical site.
For biradical d with a relatively large S-T splitting (-24.6 kcal/mol) and large
distortion energy (8.6 kcal/mol), it is predicted to react mostly via nonradical instead of
radical pathways. Indeed, the only reaction pathways observed for d are H2O abstraction
143
and addition – COOH upon interaction with lysine, whereas no reaction products were
observed upon interaction with glycine or leucine. Moreover, d has the lowest reaction
efficiency, if any, among the four biradicals, albeit it has a slightly higher EA2.30 (5.14 eV)
than that of c (4.93 eV). This can be explained by the largest distortion energy of d
among the four biradicals. A possible mechanism for the formation of adduct – COOH
for biradical d upon reaction with lysine is shown in Scheme 6.1. Based on atomic charge
calculations, the radical site 7 of biradical d is the more electrophilic site, which is most
likely the radical site that initiates the reactions. Moreover, the calculated activation
enthalpies for the nucleophilic addition of ammonia and water to the radical site 7 of d
are 12.2 kcal/mol and 22.4 kcal/mol respectively, which indicates that the nucelophilic
addition of the amino acid's NH2 group to the radical site is kinetically favored.
N
CH3
N
CH3
N
CH3
HO
O
NH2
NH2
NHO
O
NN
CH3
N
NHCOOHLoss of
Addition-COOH
H
H
H H H
H
Scheme 6.1 Proposed mechanism for the formation of adduct – COOH for biradical d upon reaction with lysine
C
su
m
C
H
ac
co
sh
H
le
du
co
S
P
w
Cysteine and
The p
usceptible to
monoradicals
CH3).21 In co
HSR abstract
cids just lik
omplex diss
hown in Sch
H2O abstracti
ess prevalent
ue to the hi
ompeting rea
Scheme 6.2 P
Proline and P
The r
with biradica
d Methionin
presence of
o biradical a
s with this gr
ntrast, birad
tion (Table 6
ke for mono
sociates.49 A
heme 6.2. Co
ion, addition
t for reactio
igh reactivity
actions.
Proposed me
Phenylalani
eaction path
ls b are sim
ne
f the S-C
attack. Previ
roup of amin
dicals’ reactio
6.4), which
oradicals, fo
A possible m
ompared to H
n, addition –
ons of a – d
y of the SR
echanism for
ine
hways obser
ilar to those
bond in cy
ious studies
no acids are
ons with this
is likely init
llowed by H
mechanism f
HSR abstrac
CO2, additio
with this gr
R group in th
r HSCH3 abs
rved for the
e observed fo
ysteine and
have demon
e dominated
s group of a
tiated by SR
H atom abs
for HSCH3
ction, the oth
on – COOH
roup of ami
he amino ac
straction fro
reactions o
or glycine an
methionine
nstrated that
by SR abstr
amino acids a
R abstraction
straction bef
abstraction
her reaction p
H, and additio
ino acids. Th
cids, which h
m methionin
of proline an
nd leucine. T
e is particu
t the reactio
raction (R =
are dominate
n from the a
fore the coll
by biradical
pathways su
on – HCOOH
his is most l
hinders the
ne by biradic
nd phenylal
They include
144
ularly
ons of
H or
ed by
amino
lision
l c is
uch as
H are
likely
other
cal c.
anine
e one
(o
–
v
pr
ph
n
b
ra
b
S
or two) hydr
HCOOH an
ia both radi
roline mostl
henylalanine
onradical pa
e ruled out a
adical and n
iradical c, as
Scheme 6.3 P
rogen atoms
nd addition –
cal and non
ly by H2O a
e exclusively
athways. Ho
as discussed
nonradical m
s shown in S
Proposed rad
abstraction,
– COOH (Ta
nradical path
abstraction,
y by additio
wever, the r
d above for t
mechanisms w
Schemes 6.3
dical mechan
, H2O abstra
able 6.5), wh
hways, as di
addition and
on. These re
radical react
the reaction
were propos
and 6.4 resp
nism for H2O
action, additi
hich indicate
iscussed abo
d addition –
esults sugge
tion mechan
with organi
sed for H2O
pectively.
O abstraction
ion, addition
ed that biradi
ove. Biradica
– CO2, whil
ested that c
nism for bira
ic substrates
O abstraction
n from prolin
n – CO2, add
ical b could
al c reacted
le it reacted
tend to reac
adical c coul
s. Therefore,
n from prolin
ne by biradic
145
dition
react
d with
with
ct via
ld not
, both
ne by
cal c.
ph
to
re
p
ph
at
sm
d
ar
Scheme
Biradi
henylalanine
o react via n
eacted with
athways wer
henylalanine
t low efficie
mall EA2.30.
d upon reacti
re shown in
e 6.4 Propos
ical a reacte
e mainly by
nucleophilic
proline mo
re also obse
e. As discus
encies due t
Possible me
on with prol
Schemes 6.5
ed nonradica
ed with prol
H2O abstrac
addition-eli
ostly by add
erved. No re
sed above, d
to its large d
echanisms fo
line, i.e. H2O
5 and 6.6 res
al mechanismby biradica
line exclusiv
ction and ad
imination pa
dition, while
action produ
d is either u
distortion en
or all the no
O abstraction
spectively.
m for H2O aal c.
vely by H2O
ddition. Thes
athways as d
e H2O abstr
ucts were ob
unreactive to
nergy, large
onradical rea
n as well as
abstraction fr
O abstractio
se results ind
discussed ab
raction and
bserved whe
oward amino
e S-T splittin
actions obser
addition and
rom proline
on, and it re
dicate that a
bove. Biradi
addition –
en d reacted
o acids or re
ng and relat
rved for bira
d addition –
146
eacted
a tend
ical d
CO2
d with
eacted
tively
adical
CO2,
147
Scheme 6.5 Proposed mechanism for H2O abstraction from proline by biradical d
Scheme 6.6 Proposed mechanism for formation of adduct and adduct-CO2 for biradical d upon reaction of with proline.
Tab
le 6
.1
ES
-T,a k
cE
A2.
30,a
E2.
30,a k
c
MW
7 M
W 1
MW
9
Rea
ctio
n ef
fici
endi
met
hyl d
isul
fi
cal/m
ol
a eV
ca
l/mol
72
2 C E
168
A
94
S SS nc
ies
(Eff
.) a
nd p
de, t
ert-
buty
l iso
c
m
/z 9
2 a
-23.
3 6.
17
7.6
x H
abs
e 57%
C
4H6
abs
43%
E
ff. =
0.2
1%
I ab
s 5
5%
(2°)
I a
bs
Add
ition
45%
E
ff. =
3%
CH
3 ab
s 8
7%
(2°)
SC
H3
abs
(2°)
SS
CH
3 ab
s(2
°) C
H2
abs
SC
H3
abs
13%
(2
°) S
CH
3 ab
s E
ff. =
39%
N CH
3
rodu
ct b
ranc
hing
cyan
ide,
and
cyc
lth
e pr
imar
y
m/z
b-1
85.
2 4.H
abs
2 x
H a
bE
ff. =
Unr
eact
ive
I ab
s
Ally
l ab
All
yl-H
E
ff. =
U
nrea
ctiv
e i
SC
H3
ab (
2°)
SC
HS
CH
3 a
SS
CH
3 a
Eff
. =
Unr
eact
ive g
rati
os f
or b
irad
iulo
hexa
ne; s
econ
dpr
oduc
ts th
at p
ro
14
2 b 8.
7 25
2 7
0%
bs 3
0%
= 1
%
isom
er 7
%
83%
bs
15%
ab
s 2
%
2.5%
is
omer
40%
bs 9
6%
CH
3 ab
s ab
s 2
%
abs
2%
30
%
isom
er 7
%
N CH
3
ucal
s a
– d
upo
n r
dary
pro
duct
s ar
e od
uce
them
. m
/z 1
42
c-1
7.6
4.93
3.
5
No
Rea
ctio
n
I ab
s 8
3%A
ddit
ion
17 %
Eff
. = 0
.3%
SC
H3
abs 1
00(2
°) S
CH
3 a
E
ff. =
6%
reac
tion
wit
h te
trno
ted
as (
2o ) an
d
n %
% 0%
ab
s
rahy
drof
uran
, ally
d ar
e li
sted
aft
er
m/z
142
d
-2
4.6
5.14
8.
6
No
Rea
ctio
n
No
Rea
ctio
n
No
Rea
ctio
n
148
yl io
dide
,
148
ES
-T,a k
cE
A2.
30,a
E2.
30,a k
c M
W 8
a Cal
cula
ted
at th
cal/m
ol
a eV
ca
l/mol
83
H
he R
HF
-UC
CS
D(T
)/c
m
/z 9
2 a
-23.
3 6.
17
7.6
CN
abs
100
%
Eff
. = 6
9%
cc-p
VT
Z//B
3LY
P/cc
N CH
3
Ta
m/z
b-1
85.
2 4.
HC
N a
b (
2°)
C4
(2°
) H
C (
2°)
Ad
CN
ab
(
2°)
CE
ff. =
U
nrea
ctiv
e -p
VT
Z le
vel o
f th
eorab
le 6
.1, c
onti
nue
14
2 b 8.
7 25
2 bs
93%
4H
8 ab
s C
N a
bs
dditi
on
bs 7
%
H2+
. tran
sfer
52
%
isom
er 5
%
ry. N C
H3
ed
m
/z 1
42
c-1
7.6
4.93
3.
5
HC
N a
bs 8
5% (
2°)
HC
N a
b (
2°)
Add
itio
CN
abs
15 %
(2°)
CN
abs
Eff
. = 3
9%
%
bs
on
%
s %
m/z
142
d
-2
4.6
5.14
8.
6
HC
N a
bs 1
00%
(2
°) H
CN
abs
(2
°) A
dditi
on
Eff
. = 4
%
149
149
150
Table 6.2 Reaction efficiencies (Eff.) and product branching ratios for monoradicals upon reaction with L-glycine, L-leucine, L-proline, L-lysine, DL-lysine-ε-15N.
m/z 93 e
m/z 143
f
m/z 143
g
EA (eV)a 5.75 4.74 4.57
Glycine MW 75
H absb 35% NH2 abs 65%
Eff. = 19%
H abs 82% NH2 abs 18% Eff. = 2.5%
H abs 68% NH2 abs 32% Eff. = 2.6%
Leucine MW 131
H abs 62% NH2 abs 38%
Eff. = 71%
H abs 97% NH2 abs 3% Eff. = 12%
H abs 82% NH2 abs 18%
Eff. = 6%
Proline MW 115
H abs 62% C2H4N abs 16%
OH abs 16% Addition 3%
Addition-OH 3% Eff. = 26%
H abs 84% C2H4N abs 4% Addition 8%
Addition – OH 4% Eff. = 17%
H abs 34% C2H4N abs 8% Addition 37%
Addition– OH 21% Eff. = 6%
Lysine MW 146
H abs 43% NH2 abs 41% Addition 14%
Addition-COOH 2%
Eff. = 58%
H abs 92% NH2 abs 3%
CH2NH abs 3% Addition 2% Eff. = 31%
H abs 68% NH2 abs 20% Addition 12%
Eff. = 15%
DL-lysine-ε-15N MW 147
H abs 42% NH2 abs 29%
15NH2 abs 18% Addition 10%
Addition-COOH 1%
Eff. =65%
H abs 89% NH2 abs 3%
15NH2 abs 2% CH2NH abs 5%
Addition 1% Eff. =33%
H abs 49% NH2 abs 23%
15NH2 abs 16% Addition 12%
Eff. = 13%
aCalculated at the RHF-UCCSD(T)/cc-pVTZ//B3LYP/cc-pVTZ level of theory.
NH2
CH
C
CH2
OH
O
CH
CH3
CH3
N
CH3
151
Tab
le 6
.3 R
eact
ion
effi
cien
cies
(E
ff.)
and
pro
duct
bra
nchi
ng r
atio
s fo
r bi
radi
ucal
s a
– d
upo
n re
acti
on w
ith
gl
ycin
e, L
-leu
cine
, L-l
ysin
e, a
nd D
L-l
ysin
e-ε-
15N
.
m
/z 9
2 a
m
/z 1
42
b
m/z
142
c
m
/z 1
42
d
ES
-T,a k
cal/
mol
E
A2.
30,a e
V
E
2.30
,a kca
l/m
ol
-23.
3 6.
17
7.6
-18.
7 5.
25
4.2
-17.
6 4.
93
3.5
-24.
6 5.
14
8.6
G
lyci
ne
MW
75
Tra
ce 2
H a
bs
H2O
abs
57%
A
ddit
ion-
CO
2 4
%
Add
itio
n -
HC
OO
H 3
9%
Eff
. = 3
2%
H a
bs 7
%
2 H
abs
9%
H
2O a
bs 1
3%
Add
itio
n 2
4%
Add
itio
n-C
O2
38%
A
ddit
ion-
HC
OO
H 9
%
Unr
eact
ive
isom
er 2
4%
Eff
. = 7
.5%
Tra
ce 2
H a
bs
Add
itio
n 1
00%
E
ff. =
0.1
%
No
Rea
ctio
n
L
-leu
cine
M
W 1
31
Tra
ce 2
H a
bs
H2O
abs
42%
A
ddit
ion
32%
A
ddit
ion-
CO
2 1
%
Add
itio
n- H
CO
OH
20%
A
ddit
ion-
C4H
8 5
%
Eff
. = 7
0%
H a
bs 8
%
2 H
abs
9%
H
2O a
bs 2
7%
Add
itio
n 2
0%
Add
itio
n-C
O2
18%
A
ddit
ion-
HC
OO
H 1
8%
Eff
. = 2
3%
NH
3 ab
s 9
%
Add
itio
n 9
1%
Eff
. = 2
.2%
No
Rea
ctio
n
N CH
3
N CH
3
151
152
Tab
le 6
.3, c
onti
nued
m/z
92
a
m
/z 1
42
b
m/z
142
c
m
/z 1
42
d
ES
-T,a k
cal/
mol
E
A2.
30,a e
V
E
2.30
,a kca
l/m
ol
-23.
3 6.
17
7.6
-18.
7 5.
25
4.2
-17.
6 4.
93
3.5
-24.
6 5.
14
8.6
L
-lys
ine
M
W 1
46
2 H
abs
6%
N
H3
abs
23%
H
2O a
bs 5
%
Add
itio
n 1
%
Add
itio
n-C
O 3
%
Add
itio
n-C
OO
H 6
2%
Eff
. = 6
7%
2 H
abs
12%
H
2O a
bs 1
5%
Add
itio
n-C
OO
H 7
3%
Eff
. = 2
1%
2 H
abs
9%
H
2O a
bs 6
%
Add
itio
n-C
OO
H 7
5%
Add
itio
n-H
2O 1
0%
Eff
. = 1
9%
H2O
abs
12%
A
ddit
ion-
CO
OH
88%
E
ff. =
4%
D
L-l
ysin
e-ε-
15N
M
W 1
47
2 H
abs
10%
N
H3
abs
22%
15
NH
3 ab
s 1
2%
Add
itio
n-C
OO
H 5
6%
Eff
. = 8
1%
Not
rel
evan
t N
ot r
elev
ant
Not
rel
evan
t
a Cal
cula
ted
at th
e R
HF-
UC
CSD
(T)/
cc-p
VT
Z//B
3LY
P/c
c-pV
TZ
leve
l of
theo
ry.
N CH
3
N CH
3
152
153
Tab
le 6
.4 R
eact
ion
effi
cien
cies
(E
ff.)
and
pro
duct
bra
nchi
ng r
atio
s fo
r bi
radi
ucal
s a
– d
upo
n re
acti
on w
ith
L-m
ethi
onin
e an
d L
-cys
tein
e.
m
/z 9
2 a
m
/z 1
42
b
m/z
142
c
m
/z 1
42
d
ES
-T,a k
cal/
mol
E
A2.
30,a e
V
E
2.30
,a kca
l/m
ol
-23.
3 6.
17
7.6
-18.
7 5.
25
4.2
-17.
6 4.
93
3.5
-24.
6 5.
14
8.6
L
-met
hion
ine
MW
149
2 H
abs
d 33%
H
2O a
bs 3
0%
OH
abs
4%
SH
2 ab
s 6
%
HS
CH
3 ab
s 2
2%
Add
itio
n –
CO
2 5%
E
ff. =
74%
H a
bs 4
%
2 H
abs
4%
H
2O a
bs 3
9%
SH2
abs
1%
, S
CH
3 ab
s 4
%
HS
CH
3 ab
s 3
9%
SC2H
4 ab
s 4
%,
Add
itio
n 5
%
Unr
eact
ive
isom
er 1
2%
Eff
. = 5
5%
H2O
abs
12%
SH
2 ab
s 6
%
HS
CH
3 ab
s 8
2%
Eff
. = 4
7%
H2O
abs
88%
H
SC
H3
abs
12%
E
ff. =
5%
L
-cys
tein
e M
W 1
21
H2O
abs
22%
SH
2 ab
s 7
2%
Add
ition
– C
OO
H 6
%
Eff
. = 4
3%
H a
bs 4
%
2 H
abs
4%
N
H2
abs
2%
N
H3
abs
8%
H
2O a
bs 2
0%
SH
abs
1%
SH
2 ab
s 5
2%
Add
itio
n 5
%
Add
ition
– C
OO
H 3
%
Add
itio
n –
HC
OO
H 1
%
Unr
eact
ive
isom
er 2
5%
Eff
. = 2
2%
SH
abs
19%
SH
2 ab
s 7
1%
Add
itio
n 1
0%
Unr
eact
ive
isom
er 2
0%
Eff
. = 3
%
No
Rea
ctio
n
a Cal
cula
ted
at th
e R
HF-
UC
CSD
(T)/
cc-p
VT
Z//B
3LY
P/c
c-pV
TZ
leve
l of
theo
ry.
N CH
3
NH
2
CHC
CH
2
OH
O
CH
2S
CH
3
153
154
Tab
le 6
.5 R
eact
ion
effi
cien
cies
(E
ff.)
and
pro
duct
bra
nchi
ng r
atio
s fo
r bi
radi
ucal
s a
– d
upo
n re
acti
on w
ith
L-p
rolin
e an
d L
-phe
nyla
lani
ne.
m/z
92
a
m
/z 1
42
b
m/z
142
c
m
/z 1
42
d
ES
-T,a k
cal/
mol
E
A2.
30,a e
V
E
2.30
,a kca
l/m
ol
-23.
3 6.
17
7.6
-18.
7 5.
25
4.2
-17.
6 4.
93
3.5
-24.
6 5.
14
8.6
L
-pro
line
M
W 1
15
H2O
abs
d 100
%
Eff
. = 9
5%
H a
bs 2
%
2 H
abs
4%
H
2O a
bs 6
6%
Add
itio
n 6
%
Add
itio
n –
CO
2 7
%
Add
itio
n –
HC
OO
H 1
5%
Unr
eact
ive
isom
er 2
2%
Eff
. = 4
2%
2 H
abs
6%
H
2O a
bs 3
7%
Add
itio
n 3
8%
Add
itio
n –
CO
2 1
9%
Eff
. = 2
%
H2O
abs
17%
A
ddit
ion
70%
A
ddit
ion
– C
O2
13%
E
ff. =
0.7
%
L-p
heny
lala
nine
M
W 1
65
H2O
abs
50
%
OH
abs
13%
A
ddit
ion
30%
A
ddit
ion
– H
CO
OH
7%
E
ff. =
72
%
2 H
abs
5%
H
2O a
bs 3
4%
Add
itio
n 2
4%
Add
itio
n –
CO
2 1
2%
Add
ition
– C
OO
H 7
%
Add
itio
n –
HC
OO
H 1
3%
Add
itio
n –
OH
5%
U
nrea
ctiv
e is
omer
12%
E
ff. =
28%
Add
itio
n 1
00%
E
ff. =
3%
No
Rea
ctio
n
a Cal
cula
ted
at th
e R
HF-
UC
CSD
(T)/
cc-p
VT
Z//B
3LY
P/c
c-pV
TZ
leve
l of
theo
ry.
N CH
3
154
155
6.4 Conclusions
The reactivities of four meta-benzyne analogues, a – d, toward four organic
substrates and eight amino acids were examined in a dual-cell Fourier-transform ion
cyclotron resonance (FT-ICR) mass spectrometer. Based on the four biradicals' reactivity
toward THF, AI, DMDS, and tBuNC, their radical reactivity follows the order of b > c >
a > d (none). Similarly, biradicals b and c display more radical reactivity toward amino
acids than biradical a and d do. The radical or nonradical reactivity toward organic
substrates and amino acids is largely affected by the distortion energies. EA at the
transition state geometry affects the total reaction efficiency of meta-benzynes, with the
three biradicals following the order of a > b > c that is generally consistent with the
EA2.30 ordering.
Overall, three important parameters were found to affect the reactivity of meta-
benzynes toward amino acids as well as organic substrates. They are (1) the S-T splitting
at the separation of the transition state, ∆ES-T; (2) the electron affinity at the separation of
the transition state, EA2.30; (3) the energy required to distort the minimum energy
dehydrocarbon atom separation to the separation of the transition state, ∆E2.30. This is the
first study on how these distinct chemical properties can affect the gas-phase reactivities
of the selected meta-benzynes towards amino acids. Different from related monoradicals,
which reacted with amino acids mainly by H atom abstraction and NH2 group abstraction,
the biradicals reacted via multiple pathways, including one (or two) hydrogen atoms
abstraction, H2O abstraction, addition, addition – CO2, addition – HCOOH and addition –
COOH. The extent to which of these reaction pathways (radical or nonradical) dominate
156
is highly influenced by the three reactivity-controlling parameters of the meta-benzyne
analogues.
157
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VITA
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VITA
Weijuan Tang was born in Guiyang, Guizhou Province, China on April 9, 1988.
She graduated from Guiyang No.1 High School in the June 2006. Afterwards, she entered
China Pharmaceutical University in Nanjing, China, where she graduated with a bachelor
degree in pharmaceutical science in June 2010. Her undergraduate research focused on
synthesis and biological evaluation of promising drug molecules. In the summer of 2010,
she went to Purdue University for her Ph.D. degree. She joined Professor Hilkka I.
Kenttämaa’s group where she worked on a variety of analytical chemistry projects,
including structural characterization of petroleum asphaltenes and organosulfur model
compounds, functional group selective ion-molecule reactions for drug metabolite
identification and fundamental studies on the gas-phase reactivity of meta-benzyne
towards amino acids. She was granted the Doctor of Philosophy degree in Chemistry
from Purdue in May, 2015.