Elimination of Electrochemical Oxidation During Sample Ionization

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Elimination of Electrochemical Oxidation during Sample Ionization

Using Liquid Sample Desorption Electrospray Ionization (DESI)

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree of

Master of Science

Najah K. Almowalad

August 2016

This thesis titled

Elimination of Electrochemical Oxidation during Sample Ionization

Using Liquid Sample Desorption Electrospray Ionization (DESI)

NAJAH K. ALMOWALAD

has been approved for

the Department of Chemistry and Biochemistry

and the College of Art and Science by

Hao Chen

Associate Professor of Chemistry and Biochemistry

Robert Frank

Dean, Collage of Art and Science

ABSTRACT

ALMOWALAD, NAJAH K., M.S., August 2016, Chemistry

Elimination of Electrochemical Oxidation during Sample Ionization Using Liquid

Sample Desorption Electrospray Ionization (DESI)

Director of Thesis: Hao Chen

This thesis introduces a method to eliminate the electrochemical oxidation that takes place during sample ionization by using liquid sample desorption electrospray ionization (DESI) method.

Several organic compounds including N, N, N', N'-tetramethyl-p- phenylenediamine (TMPD), N-phenyl-p-phenylenediamine (PPD), phenothiazine (PT), andtriphenylamine (TPA)were observed to produce significant amount of oxidized product ions when electrospray ionization (ESI) in the positive mode ion was used for their ionization due to the inherent oxidation by ESI. Interestingly, the oxidation of these organic compounds can be avoided by using liquid sample desorption electrospray ionization (DESI). This phenomenon could be explained by the fact that no high voltage is directly applied to the sample solution during DESI analysis.

Master of Analytical Chemistry. Elimination of Electrochemical Oxidation during

Sample Ionization Using Liquid Sample Desorption Electrospray Ionization (DESI).

DEDICATION

I dedicate this thesis to my parents, my king, the US and brothers.

ACKNOWLEDGMENTS

First, I would like to express my thankfulness and appreciation to my advisor, Dr.

Hao Chen, for his great support in science and wonderful advice for three years. He gave me all the encouragements and eager to do experiments and conduct research. He also guided me to work hard and to enjoy research at Ohio University. Second, I would like to thank Dr. Peter Harrington and Dr. Mark McMills for cooperation as committee members to give me suggestions about my thesis. Third, I would like to thank Dr. Hao

Chen‘s group members, Dr. Mei Lu, Dr. Pengyuan Liu, Dr. Qiuhua Wu, Dr. Zhi Li, Dr.

Hetong Qi, Si Cheng, Dr. Qiuling Zheng, Yi Cai, Li Xiyang, Chang Xu, and Yuexiang

Zhang for their support, cooperation and friendship.

Fourth, I really want to thank my father for his support and for being with me in the U.S for the period of my study. Also, I want to thank my mother for her emotional support and encouragements from my home country (Saudi Arabia).

TABLE OF CONTENTS

Abstract……………………………………………………………..………….………….3

Dedication…………………………..…………………………………..…………………4

Acknowledgments………………………………………………………..………………..5

List of Figures………………………………………………………………..…….……...8

Chapter 1: Introduction………………………………………………..…………………10

1.1 Mass Spectrometry………………………………………………..…………..10

1.2 Ionization Methods……………………………………………..…………….10

1.2.1 Chemical Ionization (CI) and Electron Ionization (EI)…..………..10

1.2.2 Electrospray Ionization (ESI)………….…………………..………11

1.2.3 Ambient Desorption Mass Spectrometry……………….……...... 12

1.2.4 Desorption Electrospray Ionization (DESI)…………….…..……..15

1.2.5 Inherent Oxidation by ESI………..…………………….…………17

1.3 Mass Analyzers………………………………………………….……………18

1.3.1 Quadrupole Mass Analyzer……………...……...... ……...…..….18

1.3.2 Ion Trap Mass Analyzer………………...………...……..….……..19

1.3.3 Time of Flight Mass Analyzer……………...... ……………….…20

1.4 Tandem Mass Spectrometry…………………...…………..………………….21

Chapter 2: Comparison of ESI and DESI for the Ionization of Several Organic

Compounds…...…...... 23

2.1 Chemicals and Materials …………………………………….………………23

2.2 Experimental Conditions …………....…………………………………..…..24

2.3 Results and Discussion…………………………………………………...….25

2.3.1 N,N,N',N'-tetramethyl-p-phenylenediamine(TMPD)………...…....25

2.3.2 N-phenyl-p-phenylenediamine(PPD)………………...... …….…...28

2.3.3 Phenothiazine (PT)………...………..……………...…………..…31

2.3.4 Triphenylamine(TPA)………...….....…….………..…...…...... ….34

Chapter 3: Summary and Future Work…………….………...……..……………..……..36

References.…………...... ……..……………..………………..……………….…37

LIST OF FIGURES

Figure 1-1: Schematic showing the process of ESI[1]…………..…..….....…..….………11

Figure 1-2: Some direct ionization techniques [8]…………………………...... ………….14

Figure 1-3: Some direct desorption/ionization techniques[8]……………….………...... 14

Figure 1-4: Some two-step ionization techniques[8]…………….………………...... ….15

Figure 1-5: DESI for analyzing solid sample on surface[7]……………….……...... ….…16

Figure 1-6: DESI for analyzing liquid sample[9] …………………………………....…...17

Figure 1-7: Schematic showing quadrupole mass analyzer[1]….…....……...... …....…..19

Figure 1-8: Schematic showing ion trap mass analyzer………..……...... ………….….20

Figure 1-9: Schematic showing TOF mass analyzer[10]……………..…...... …...……..21

Figure 1-10: Diagram of a triple quadrupole mass spectrometer[13]…………...... …...22

Figure 2-1: Structures of organic compounds TMPD, PPD, PT and TPA...…...... …….24

Figure 2-2: a) ESI-MS spectrum of 30 µM of TMPD dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1). b) DESI-MS spectrum of 30 µM of TMPD dissolved in solvent ACN/H2O/HOAc (v/v/v, 50:50:1)…………………...... ………....25

Figure 2-3: a) MS/MS spectrum of the protonated TMPD [M+H]+ (m/z 165)generated by

DESI. b) MS/MS of the protonated TMPD [M+H]+ (m/z 165) generated by DESI; c)

MS/MS of the radical cation TMPD (m/z164) [M+.] generated by ESI….…...... ……..26

Figure 2-4: a) ESI-MS spectrum of 50 µM of PPD dissolved in solvent ACN/H2O/HOAc

(v/v/v, 50:50:1). b) DESI spectrum of 50 µM of PPD dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1)……………………………...... ……………...28

Figure 2-5: a) MS/MS spectrum of the protonated PPD [M+H]+(m/z 185) generated by

ESI. b) MS/MS spectrum of the protonated PPD[M+H]+of (m/z 185) generated by

DESI………………………………………...………...…………...... ………..………….29

Figure 2-6: a) MS/MS spectrumof the radical cation PPD M+. (m/z 184) generated by ESI; b) MS/MS of [M-H]+(m/z 183) generated by ESI………………...... ….……...…..30

Figure 2-7: a) ESI-MS spectrum of 50 µM of PT dissolved in solvent ACN/H2O/HOAc

(v/v/v, 50:50:1). b) DESI-MSspectrum of 50 µM of PT dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1)………………………...... …….………..…31

Figure 2-8: a) MS/MS spectrum of the protonated PT (m/z 200) generated by ESI. b)

MS/MS spectrum of the protonated PT (m/z200) generated by ESI. c)MS/MS spectrum of the PT radical cation (m /z 199) generated by ESI………...... ………...……32

Figure 2-9: a) ESI-MS spectrum of 50 µM of TPA dissolved in solvent ACN/H2O/HOAc

(v/v/v, 50:50:1). b) DESI-MS spectrum of 50 µM of TPA dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1)………….…………...... …....…..34

Figure 2-10: a) MS/MS spectrum of the protonated TPA (m/z 246) generated by ESI. b)

MS/MS spectrum of the protonated TPA (m/z 246) generated by DESI……...... …....35

CHAPTER 1: INTRODUCTION

1.1 Mass Spectrometry

Mass spectrometry (MS) is a sensitive tool for chemical analysis. Since 1912,

MS was used to differentiate the isotopes of Neon (masses: 20,22).[2] After that, MS was widely used for isotopic and elemental measurements especially for the analysis of nuclear weapons such as Uranium (U235, U238).[2] Also, MS was used for analyzing complexes mixture such as petroleum products and organic compounds. In the last 20 years, MS has been established as a method for the analysis for large and bio-molecules such as proteins and nucleic acids.[3] Nowadays, MS has become a useful analytical tool that provides information about qualitative and quantitative analysis.[1]

1.2 Ionization Methods

1.2.1 Chemical Ionization (CI) and Electron Ionization (EI)

There are many ionization methods that produce ions during the mass spectrometric analysis such as electron impact (EI) and chemical ionization (CI). EI is commonly used for organic mass spectrometry.[4] By using an electron with kinetic energy of 70 eV to ionize a molecule in the gas phase, an electron could be expelled from the target molecule to generate the corresponding radical cation (i.e., the molecular ion).

In this EI process, several extensive fragments could be produced. The fragmentation could prevent the molecular ion from being observed. Despite EI, the ionization by CI for a reagent gas such as hydrocarbons, amines or alcohols by the electron impact occurs

+. first. For example, methane can be used as a reagent gas, which leads to form [CH4]

+ that reacts subsequently with another methane molecule. A mix of ions such as [C2H5] ,

+ + [C3H5] , and [CH5] are subsequently yielded. In the end, a proton transfers from these reagentions to the analyte M and produces the protonated analyte [M+H]+.[4]

1.2.2 Electrospray Ionization (ESI)

Nowadays, ESI is the most commonly used ionization technique. Because ESI can be used to produce ions from both small and large chemical substances such as peptides, proteins, and non-covalent complexes by using microliters of the sample, it can be used for both identification and quantification purposes.

The first invention of ESI was reported in the end of 1980s by John Fenn and his co-workers.[5] They described how large chemical molecules in solutions could be converted to ions in the gas phase without fragmentation. This discovery led to further progress in biomolecule analysis.[5]It is also called a "soft ionization technique" because it provides few fragments. This discovery led to many applications in bioanalysis.

Figure 1-1: Schematic showing the process of ESI[1]

As shown in (Figure 1-1), for ESI-MS analysis, the analyte solution is injected to the ion source chamber, which contains a quartz silica or a stainless steel capillary emitter. A high voltage (2.5-6 kV) is applied to the analyte solution to produce charged droplets with the assistance of a nebulizating gas such as N2. These charged droplets are created at the tip of the capillary (Taylor cone) and then travel to the analyzer. During that, the solvent in these droplets starts to evaporate, leaving these droplets smaller in size. When the size of the droplet decreases, the charge density increases on its surface.

After this step, the electrostatic repulsion of the droplet becomes stronger than its surface tension; thus, the charged droplets break down into smaller droplets. This process repeats and eventually produces dry gaseous ions for MS analysis.[1][6][7]

There are many factors that affect ESI signal including surface tension and the concentration of the electrolyte. By using diluted solutions with organic solvents such as methanol, acetonitrile, or isopropanol, the surface tension could be decreased to facilitate solvent evaporation. The solution should have enough electric conductivity around 10-9-

10-8Ω-1cm-1. After adding some additional chemicals such as acetic acid or formic acid into the analyte solution, the electric conductivity of the solution is increased.

Furthermore, heating the spray chamber is useful to enhance evaporation of the solvent from the droplets.[7]

1.2.3 Ambient Desorption Mass Spectrometry

Ambient mass spectrometry is a widely used technique. It is a developed analysis method that can be used to ionize nonvolatile polar and volatile nonpolar samples in open atmospheric circumstances with little or no sample preparation. There are several techniques of ambient desorption mass spectrometry. According to the paper,[8]there are

13 three main types of ambient desorption mass spectrometry: direct ionization, direct desorption ionization and two-step ionization.

The easiest type of ambient desorption ionization is direct ionization. It is sufficient for both small organic compounds and large biomolecules. It requires high electric energy to ionize the analyte from solutions by using an electrosonic spray ionization (ESSI) sprayer. Some conditions are important to maintain during experiment such as filtering sample's solution to avoid any clogging of the ESSI capillary, and using small flow rate for the sample.

The interest of direct desorption/ionization (DI) method for analyzing biological and chemical substances increased in the beginning of 1980s. The main concept of direct desorption/ionization DI is to ionize the sample by using the impact of some charged particles or photons to the sample surface.

The two-step ionization system contains three parts: a unit that produces and transfers the analyte, a source of ambient ionization methods to produce charged ions and a zone or area where the interaction between the analyte and charged ions. The following charts (Figures1-2,1-3, and 1-4) show some of these three types of techniques.[8]

Figure 1-2: Some direct ionization techniques[8]

Figure 1-3: Some direct desorption/ionization techniques[8]

Figure 1-4: Some two-step ionization techniques[8]

1.2.4Desorption Electrospray Ionization (DESI)

Desorption electrospray ionization mass spectrometry (DESI-MS) is a kind of ambient desorption/ ionization mass spectrometry.[7]It is a useful type of direct desorption/ionization technique for quick analysis of variable samples such as abusive drugs, pharmaceuticals, metabolites, and chemical warfare agents. By using a pneumatically supported ESSI sprayer that produces charged solvent droplets, the interaction occurs between these charged droplets and samples on the surface to produce sample ions (Figure 1-5). DESI-MS can be used for either liquid or solid samples. For solid samples, it can be directly desorbed and ionized while liquid samples are often dried

16 before ionization because the liquid could be blown immediately from the surface with the use of a high velocity nebulizing gas; as a result, the signal could disappear.

Figure 1-5: DESI for analyzing solid sample on surface [7]

One new technique of DESI was presented by Dr. Hao Chen's lab group at Ohio

University OU for direct analysis of liquid samples.[9] In this method, sample solutions are introduced progressively for ionization via a fused silica sample's transfer capillary as driven by a syringe pump (Figure 1-6). This method provides two advantages. First, biological analytes such as urine could be directly analyzed from their natural environments without the need of drying the sample. Second, the capability of DESI for continuous analyzing is that the flowing liquid samples allows coupling MS with other devices such as chromatography and electrochemical cells. This method is quite

17 effective and powerful for analyzing a wide range of analytes such as peptides, proteins, and protein complexes.[9]

Figure 1-6: DESI for analyzing liquid sample [9]

1.2.5 Inherent Oxidation by ESI

Some organic compounds can be oxidized during the ionization step of ESI where there is a high voltage potential (3-5 kV) applied to the sample solution. This phenomenon is called inherent oxidation in ESI. This kind of electrochemical oxidation could lead to form a stable radical cation M+. or oxygenated products. The extent of oxidation depends on the electrolyte concentration and electrochemical redox potential for analytes in solution. The formation of the radical cation often happens to organic compounds that contain hetero atoms such as (N or O) or a highly conjugated

18 system.[10]N, N, N’, N'-Tetramethyl-p-phenylenediamine (TMPD), N-phenyl-p- phenylenediamine (PPD), phenothiazine (PT), and triphenylamine (TPA) are examples of this kind of inherent oxidation as found in this study.

1.3 Mass Analyzers

1.3.1QuadrupoleMass Analyzer

The purpose of using a mass analyzer is to allow the measurement of the mass to charge ratio of an analyte ion that reaches the detector. The quadrupole mass analyzer is powerful, small, efficient, and widely used. It is composed of four parallel metal rods separated from each other by the same space. Each two opposite rods are attached to each other by an electric potential. Both a radio frequency (RF) AC and a reversed DC voltages are applied to these rods. These AC and DC voltages resulting in an electric field that causes ions to move vibrationally over x-y plane toward z axis. By changing and controlling these AC and DC voltages, ions that have a particular value of m/z ratio can pass through the mass analyzer to reach the detector while other ions that have different values of m/z ratio cannot pass through the mass analyzer because they collide with the rods and lose their charges to be neutralized. Therefore, ions can be measured by scanning their m/z ratios using the mass analyzer (Figure 1-7). [1]

Figure 1-7: Schematic showing quadrupole mass analyzer [1]

1.3.2 Ion Trap Mass Analyzer

Ion trap mass analyzer is regarded as a three dimensional quadrupole mass analyzer. It consists of three electrodes: one ring cap electrode and two end cap electrodes (Figure 1-8). The radio frequency fields are applied to trap sample ions in the three dimensional trap.[1]

Figure 1-8: Schematic showing ion trap mass analyzer

1.3.3 Time of Flight Mass Analyzer

Time of flight mass analyzer (TOF) is widely used in many instruments (Figure 1-

9). The separation of ions depends on the flight time that ions spend before reaching the detector. Ions are discharged or generated accumulatively from an ion source. Because of the potential difference that is applied between the grid of extraction and ion source electrode, ions are accelerated to the flight tube depends on their masses and velocities as they have the same kinetic energy. Smaller ions reach the detector faster than the bigger ones. After passing the acceleration area, ions travel to the detector and separate depending on their velocities. After measuring the time that ions spend to travel from the source to reach the detector, the mass-to-charge ratio is obtained. TOF instruments are compatible for using matrix assisted laser desorption ionization (MALDI) as the ion source. There are some hybrid instruments that combine two of mass analyzers such as quadrupole time of flight (Q-TOF).[4]

Figure 1-9: Schematic showing TOF mass analyzer[4]

1.4 Tandem Mass Spectrometry

Tandem mass spectrometry (MS/MS) is a useful technique to obtain structural information about analyte molecules. One common MS/MS technique is also called collision induced dissociation (CID) in which the analyte ions dissociate via collision energy with N2 or Ar gas. In the MS/MS experiment, ions are mass selected by the first stage of qaudrupole and collide with the collision gas to produce fragment ions in the 2nd quadrupole. The fragment ions are scanned by the 3rd stage of thequadrupole. The triple quadrupole is an example of tandem mass spectrometry as shown in Figure(1-

10).[11]

Figure 1-10: Diagram of a triple quadrupole mass spectrometer[11]

CHAPTER 2: COMPARISON OF ESI AND DESI FOR THE IONIZATION OF

SEVERAL ORGANIC COMPOUNDS

2.1 Chemicals and Materials

N, N, N’, N'-Tetramethyl-p-phenylenediamine (TMPD) (MW: 164 g/mol), N- phenyl-p-phenylenediamine (PPD) (MW: 184 g/mol), phenothiazine (PT) (MW: 199.28 g/mol), and triphenylamine (TPA) (MW: 245.32 g/mol) were purchased from Sigma

Aldrich (Figure 2-1). Methanol in HPLC grade was purchased from Fisher Scientific and acetonitrile HPLC was purchased from Avantor Performance Materials Inc. The distilled water used for sample preparation was obtained by using a Nano-pure Diamond

Barnstead Purification System (Barnstead International, Dubuque, IA). Figure (2-1) shows the structures of organic compounds TMPD, PPD, PT, and TPA that were studied in this thesis.

Figure 2-1: Structures of organic compounds TMPD, PPD, PT and TPA

2.2 Experimental Conditions

Finnigan LCQ Deca XP Max (ThermoFinnigan) was used for applying ESI and

DESI for TMPD, PPD, PT, and TPA.

For ESI, N2 gas was used as a nebulizing gas. The high voltage (5000 V) was applied to the sample solution for spraying. The commercial ESI source contained a stainless steel capillary to introduce the sample solution. The flow rate of the sample used was 10 µL/min. Tandem mass spectrometry (MS/MS) was used with normalized collision energy of 35 (manufactory arbitrary unit).

For DESI, the same samples were used. A fused silica capillary was used for introducing samples for DESI ionization. Additionally, the flow rate for both spray solvent ACN/H2O/HOAc (v/v/v, 50:50:1) and sample solution was 5µL/min.

2.3 Results and Discussion

2.3.1 N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD)

Figure 2-2: a) ESI-MS spectrum of 30 µM of TMPD dissolved in solvent

ACN/H2O/HOAc (v/v/v,50:50:1). b) DESI-MS spectrum of 30µM of TMPD dissolved in solvent ACN/H2O/HOAc (v/v/v, 50:50:1).

Figure 2-3:a) MS/MS spectrum of the protonated TMPD [M+H]+ (m/z 165) generated by

ESI. b) MS/MS of the protonated TMPD [M+H]+(m/z 165) generated by DESI. c)

MS/MS of the TMPD radical cation M+.(m/z 164) generated by ESI.

When some selected organic compounds were ionized by ESI-MS, the in-source oxidation was observed for these compounds. For example, as shown in Figure (2-2a),

TMPD has a protonated peak [M+H] +(m/z165) and an oxidation peak for the radical cation M+.(m/z164). The radical cation M+. is produced as a result of the oxidation of

TMPD by one electron loss during ESI ionization. The oxidation can be avoided by using liquid sample DESI as the ionization method. As shown in Figure (2-2b), only the protonated peak [M+H]+ appeared at (m/z 165) while the radical cation (m/z 164) was not

27 observed. The reason for this difference is likely that, unlike in ESI, no high voltage is directly applied to the sample in the case of DESI; thus, it helps to avoid the oxidation of the sample. The lack of oxidation in the DESI analysis simplifies the acquired mass spectrum and would facilitate qualification. MS/MS was used to confirm the ion structures. As shown in (Figure 2-3), both(m/z 165) and(m/z 164) dissociate by the loss

. of methyl radical CH3 .

2.3.2 N-phenyl-p-phenylenediamine (PPD)

100 185 a) ESI of 50 µM PPD [M+H]+

183

50 + m/z 184 m/z 185 [M-H] 184 Relative Abundance Relative

[M]+.

0 120 140 160 180 200 220 240 260 280 300 m/z

100 185 b) DESI of 50 µM PPD [M+H]+

50 Relative Abundance Relative

0 120 140 160 180 200 220 240 260 280 300 m/z

Figure 2-4: a) ESI-MS spectrum of 50 µM of PPD dissolved in solvent ACN/H2O/HOAc

(v/v/v, 50:50:1). b) DESI-MS spectrum of 50 µM of PPD dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1).

168 100 108 185 a) MS/MS of m/z 185, ESI

50 Relative Abundance Relative -NH - 3

0 120 140 160 180 200 220 240 260 280 300 m/z 100 168

b) MS/MS of m/z 185, DESI

50 185 Relative Abundance Relative -NH3 108 -

0 120 140 160 180 200 220 240 260 280 300 m/z

Figure 2-5: a) MS/MS spectrum of the protonated PPD [M+H]+(m/z 185) generated by

ESI. b) MS/MS spectrum of the protonated PPD [M+H]+(m/z 185) generated by DESI.

184 100

a) MS/MS of m/z 184, ESI

50 Relative Abundance Relative 167

-NH3 0 120 140 160 180 200 220 240 260 280 300 166 m/z 100

b) MS/MS of m/z 183, ESI

183 50 Relative Abundance Relative

-NH3

0 120 140 160 180 200 220 240 260 280 300 m/z

Figure 2-6: a) MS/MS spectrum of the PPD radical cation M+.(m/z 184) generated by

ESI. b) MS/MS spectrum of [M-H]+(m/z183) generated by ESI.

PPD is the second example for comparison between ESI and DESI data. By using ESI, the protonated ion [M+H]+was observed at (m/z 185) as shown in Figure (2-

4a). PPD has two oxidation peaks:(m/z 184) for radical cation M+. and (m/z 183) for hydrogen loss. However, these two oxidation peaks were not seen with DESI analysis

(Figure 2-4b). This also shows the difference between ESI and DESI ionization. The fragmentation of these observed ions was obtained by MS/MS. MS/MS of(m/z 185) for

ESI and DESI shows in (Figure2-5). MS/MS shows that (m/z185) produces two fragment ions: (m/z 168) by the loss of ammonia NH3 and(m/z 108) by the loss of phenyl

. radical C6H5 . In contrast, both (m/z184) and (m/z183) dissociate via the loss of a neutral fragment NH3 (Figure 2-6).

2.3.3 Phenothiazine (PT)

199 100

[M]+. a) ESI of 50 µM PT

-e- [M+H]+ 50

MW 199 Da m/z 199

Relative Abundance Relative 198 200

[M-H]+

0 120 140 160 180 m/z 200 220 240 260 280 300

200 100 b) DESI of 50 µM PT [M+H]+

50 Relative Abundance Relative

0 120 140 160 180 200 220 240 260 280 300 m/z

Figure 2-7: a) ESI-MS spectrum of 50 µM of PT dissolved in solvent ACN/H2O/HOAc

(v/v/v, 50:50:1). b) DESI spectrum of 50 µM of PT dissolved in solvent

ACN/H2O/HOAc (v/v/v, 50:50:1).

Figure 2-8: a) MS/MS spectrum of the protonated PT (m/z200) generated by ESI. b)MS/MS spectrum of the protonated PT (m/z200) generated by DESI. c)MS/MS spectrum of the PT radical cation (m/z 199) generated by ESI.

PT is the third example for this study. Figure (2-7a) shows the spectrum generated by ESI for PT. In this spectrum, (m/z 200) corresponds to the protonated molecule [M+H]+(m/z 200). There is one oxidation product peak at(m/z 199) due to the formation of the radical cation M+.. On the other hand, after applying DESI as shown in

Figure (2-7b), only the protonated molecule [M+H]+ appeared, indicating that there is no oxidation. Figures(2-8a, 2-8b, and 2-8c) show MS/MS spectra of (m/z 200) generated by

ESI and DESI and MS/MS spectrum of(m/z199) generated by ESI. It is obvious that

(m/z200) generated by DESI and ESI loses one (-SH) group upon dissociation. In contrast, MS/MS spectrum of (m/z 199) generated by ESI loses one sulfur (S) atom upon dissociation.

2.3.4 Triphenylamine (TPA)

Figure 2-9: a) ESI-MS for 50 µM of TPA dissolved in solvent ACN/H2O/HOAc (v/v/v,

50: 50: 1). b) DESI-MS for 50µM of TPA dissolved in solvent ACN/H2O/HOAc (v/v/v,

50: 50: 1).

Figure 2-10: a) MS/MS spectrum of the protonated TPA [M+H]+(m/z 246) generated by

ESI. b) MS/MS spectrum of the protonated TPA (m/z 246) [M+H]+generated by DESI.

TPA is the fourth example for this study. Figure (2-9a) shows the acquired spectrum by using ESI for TPA. The (m/z246) corresponds to the protonated molecule

[M+H]+ and(m/z 245) presents the radical cation M+.. In contrast, when the sample was ionized by using DESI (Figure 2-9b), only the protonated molecule [M+H]+ appeared without oxidation. MS/MS of (m/z 246) shows the loss of (15 Da), which could be presumably the loss of (-NH) moiety group (Figure 2-10).

CHAPTER 3: SUMMARY AND FUTURE WORK

The oxidation of some organic compounds (aromatic amines) was explored in this thesis work. These compounds were N, N, N’, N'-tetramethyl-p-phenylenediamine

(TMPD), N-phenyl-p-phenylenediamine (PPD), phenothiazine (PT),and triphenylamine

(TPA). The oxidation of these compounds was observed after applying ESI-MS.

However, the oxidation was avoided by DESI-MS analysis of these species. The findings of this thesis suggests that the quantification of these compounds by using DESI-MS may provide more accurate results than ESI-MS. This quantification could be helpful because the compound signal would not be split into the protonated ion and radical cation in the case of DESI-MS analysis. In future, the quantification of these compounds by

ESI-MS could be beneficial from the finding in this thesis.

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