UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

NONPOLAR MATRICES FOR MATRIX-ASSISTED LASER DESORPTION/IONIZATION – TIME OF FLIGHT – MASS SPECTROMETRY

A dissertation submitted to the

Division of Research and Advanced Studies of the University Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTORATE OF PHILOSOPHY

in the Department of Chemistry of the College of Arts and Sciences

April 2005

by

Chad LaJuan Robins

B.S., Southern University and Agricultural & Mechanical College Baton Rouge, Louisiana – Campus, 1999

Committee Chair: Dr. Patrick Alan Limbach

© Copyright 2005 Chad LaJuan Robins All Rights Reserved

ABSTRACT

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)

has been used extensively for the characterization of large biomolecules, synthetic

polymers and small molecules. Typically, low molecular weight acidic matrices have

been used in MALDI-MS for such analyses. Here, the use of low molecular weight

nonpolar matrices for MALDI-MS is investigated. In particular, the analytical and

physical properties of this class of matrices are studied.

The effect of instrumental factors on nonpolar matrix behavior was

investigated. Two different MALDI target substrates, stainless steel and

poly(methylmethacrylate) (PMMA), were used. The radical molecular ion abundance

of nonpolar analyte ions analyzed in the presence of various nonpolar matrices, such

as anthracene and 9-cyanoanthracene, were determined as a function of the MALDI

target substrate. It was found that PMMA MALDI target substrates yielded higher

radical molecular ion abundances for nonpolar analyte ions than stainless steel

MALDI target substrates. This difference is attributed to the absence of photoelectrons which reduce charged species in the plume.

To determine whether the desorption process for nonpolar matrices is different than the desorption process for polar, acidic matrices, matrix initial ion velocities were measured in MALDI-MS. The relative initial ion velocities of the nonpolar

matrices, anthracene, 9-cyanoanthracene, pyrene, and acenapthene and were

investigated and compared to the relative initial ion velocities for 2,5-

dihydroxybenzoic acid (DHB), sinapinic acid (SA), and all-trans retinoic acid (RTA),

polar matrices. It was found that DHB has a greater relative initial ion velocity than

I the nonpolar matrices. It does not appear that the desorption process is different between nonpolar and polar matrices based upon these results. In addition, when using any matrix, polar or nonpolar, to analyze nonpolar polymers, the addition of a metal salt increased the relative initial ion velocity of the matrix, suggesting a different desorption mechanism in these cases.

To illustrate an application of nonpolar matrices for analytical MALDI-MS, an atmospheric resid crude oil fraction was characterized. The MALDI mass spectral data for this sample was difficult to interpret when a polar matrix, 3-indole acrylic acid, was used, due to fragmentation and matrix clustering in the analyte molecular weight distribution range. The nonpolar matrices anthracene and 9-cyanoanthracene were found to be very suitable for the analysis of this sample in either linear or reflectron mode time-of-flight mass spectrometry. These matrices did not lead to fragmentation or clustering, and identification and characterization of the analyte peaks were straightforward.

II

In Memory of my Father

“Son, I just want you to do better than I did.”

- Charley Robins -

For I know the plans I have for you," declares the LORD,” plans to prosper you and not to harm you, plans to give you hope and a future.

Jeremiah 29:11

III ACKNOWLEDGMENTS

I would like to begin by acknowledging God as Lord and Savior of my life. I

thank God for the knowledge, wisdom, direction, and patience He has given me to go

through on this journey. I could not have accomplished what I have without him.

What I’ve learned He has taught me, where I’ve come from, He has brought me, and

where I’m going, He is bringing me to. So, I owe all that I’ve accomplished to God.

I also, thank God for placing me within Dr. Patrick A. Limbach’s research group where he has been very, very patient with me as well as guiding and directing

me through graduate school and the research process. Dr. Limbach, I’m grateful to

you for the time that you have spent molding and shaping me into the graduate

student that I am today. Where ever I go, I will take with me the principles that you

have set before all the students that you have mentored for they are very valuable, not

only in the scientific community but outside the scientific community as well. I’m

also grateful to Mrs. Kay Limbach, who has given of her husband unselfishly as he

sought to prepare his graduate students for life after graduate school. May God richly

bless the Limbach family.

I would like to thank my advising committee, Dr. Thomas Ridgway and Dr.

Bill Connick for their helpful discussions as it supported and related to my research.

I’m also grateful to them because they have also prepared me for life in the scientific

community. I thank Louisiana State University (LSU) and The University of

Cincinnati and the entire Chemistry Department and staff at both Universities for

allowing me to pursue graduate studies at such prestigious institutions. I would also

IV like to acknowledge Dr. James Mack (Assistant Professor, The University of

Cincinnati) for your words of wisdom and encouragement.

To my colleagues in the Limbach Group (past and present), I thank you for all

of your support and I want you all to know that I value very dearly the family type

atmosphere that is apart of the Limbach group. That family type of atmosphere is the

staple of the Limbach Research Group; never loose that. Special thanks to Dr.

Beniam Berhane for being there for me. Your dedication and desire for achieving

goals in life helped to inspire me while here at The University of Cincinnati and I’ve

looked up to you as my “2nd Big Brother”. Thanks for your prayers and words of

encouragement.

To my mother, Ardene Robins, and late father, Charley Robins, thank you for

always being there for me as parents. Your teaching me in the way of the Lord

(Proverbs 22:6) has prepared me for life’s journey. For that and as well as your

words of wisdom, encouragement, and prayers, I’m grateful. To my siblings,

Reginald and Kendra, thank you too, for your encouragement and prayers, I love you all dearly. To my mother and father-in-law, Pastor & Mrs. Allen E. Middleton, I thank you also for your words of wisdom, prayers, and encouragement.

To my wife, Kalilah L. Robins, without you, I don’t believe I would be at this point in my life now. I thank you for always being there for me. Thank you for your encouragement, praying for me and with me. I thank you for putting your life on hold that I may accomplish the career goals that I desired to achieve. You have truly been to me as the Lord desires a wife to be to her husband, for that I’m very grateful. I can honestly say that I would never want to experience life without you. I

V praise God for joining our lives together. One chapter of our life is now complete and now it is time to move onto the next chapter in our life and fulfill the purpose for which God united us together in marriage to accomplish. I love you.

VI TABLE OF CONTENTS

TABLE OF CONTENTS...... 1

LIST OF TABLES AND FIGURES...... 3

LIST OF ABBREVIATIONS AND SYMBOLS ...... 7

CHAPTER ONE: Matrix-Assisted Laser Desorption Ionization – Mass Spectrometry………...... 10 1.1 Purpose of Work ...... 10 1.2 Mass Spectrometry and Basic Principles of MALDI Analysis of Nonpolar Analytes ...... 10 1.3 MALDI Analysis of Nonpolar Analytes...... 14 1.4 Role of matrix ...... 17 1.5 Polar vs. Nonpolar Matrices ...... 18 1.6 MALDI Ion Formation ...... 21 1.7 Mass Analyzer...... 23 1.8 Significance of Work ...... 26

CHAPTER TWO: CHARACTERIZAITON OF STAINLESS STEEL AND POLYMER MALDI TARGETS AND THE EFFECT OF THE SUBSTRATE ON NONPOLAR MATRIX IONIZATION EFFICIENCY...... 29 2.1 Introduction...... 29 2.2 Experimental...... 30 2.2.1 Materials ...... 30 2.2.2 Methods...... 30 2.2.2.1 Polymeric MALDI Sample Target Disc Fabrication...... 30 2.2.2.2 Imaging ...... 31 2.2.2.3 MALDI Analysis...... 31 2.3 Results and Discussions...... 33 2.3.1 LDI of MALDI Substrates...... 33 2.3.2 Solvent Effects on Polymeric Substrates ...... 34 2.3.3 Charge Transfer Analysis on MALDI Substrates...... 35 2.4 Conclusions...... 39

CHAPTER THREE: DETERMING THE INITIAL ION VELOCITY FOR NONPOLAR MATRICES...... 41 3.1 Introduction...... 41 3.2 Experimental...... 43 3.2.1 Materials ...... 43 3.2.2 Methods...... 43 3.2.3 MALDI Analysis...... 44 3.3 Results and Discussion...... 44 3.3.1 Measurement of the Initial Ion Velocity...... 44

1 3.3.2 Initial Ion Velocities of Polar Matrices with Polar Analytes (Proton- Transfer Ionization) ...... 47 3.3.3 Initial Ion Velocities of Nonpolar Matrices with Nonpolar Analytes. 48 3.3.4 G-Value Determination...... 49 3.3.5 Correlation of Nonpolar Matrix and Initial Ion Velocity for Aromatic Nonpolar Polymer, Polar Polymer, and Linear Chain Nonpolar Polymer ...... 51 3.3.6 Correlation of Polar Matrix and Initial Ion Velocity for Aromatic Nonpolar Polymer, Polar Polymer, and Linear Chain Nonpolar Polymer ...... 55 3.4 Conclusion ...... 58

CHAPTER FOUR: The Use of Nonpolar Matrices for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Analysis of High Boiling Crude Oil Fractions……………...... 60 4.1 Introduction...... 60 4.2 Experimental...... 63 4.2.1 Materials ...... 63 4.2.2 Methods...... 63 4.2.2.1 MALDI Analysis...... 63 4.2.2.2 FI Analysis...... 64 4.3 Results and Discussion...... 64 4.3.1 Candidate MALDI Matrices...... 64 4.3.2 Linear vs. reflectron mode MALDI-MS...... 66 4.3.3 Solvent effects...... 69 4.3.4 Comparison with LDI- and FI-MS ...... 72 4.3.5 Evaluation of nonpolar MALDI-MS ...... 74 4.4 Evaluation of BTLP Crude Oil Fraction on PEEK Target ...... 78 4.5 Conclusions...... 80

CHAPTER FIVE: CONCLUSIONS AND FUTURE DIRECTIONS ...... 81 5.1 Summary...... 81 5.2 Future Directions...... 83

BIBLIOGRAPHY...... 86

2 LIST OF TABLES AND FIGURES

List of Tables

Table 3.1 Values of β from eq. 3.3 for the specified geometry...... 47

Table 3.2 Relative initial ion velocity of synthetic polymers desorbed from different nonpolar matrices at 337 nm with the calculated mass resolution, R (G=91%, U=20 kV) ...... 53

Table 3.3 Relative initial ion velocity of synthetic polymers desorbed from different polar matrices at 337 nm with the calculated mass resolution, R (G=91%, U=20 kV).... 56

List of Figures

Figure 1.1 Block Diagram of a mass spectrometer...... 11

Figure 1.2 MALDI sample preparation of matrix and analyte...... 13

Figure 1.3 Schematic of Desorption process in MALDI...... 14

Figure 1.4 Common polar matrices and nonpolar matrices used for MALDI – MS analysis...... 20

Figure 1.5 Schematic diagram of linear time-of-flight (L-TOF) instrument...... 24

Figure 1.6 Schematic diagram of a reflectron time-of-flight instrument (re-TOF)...... 25

Figure 2.1 Structures of chemical compounds used as analytes in this chapter...... 32

Figure 2.2 Mass spectrum of a LDI analysis of a PMMA MALDI substrate. The mass spectrum of the other polymers (PC, PS, PEEK) were the same. LDI experiments were investigated using a laser power of 20 – 30 %...... 33

Figure 2.3 CCD images of unspotted (left) and spotted (right) polystyrene MALDI substrates...... 34

Figure 2.4 MALDI – MS of ferrocene (MW = 186.03 Da) analyzed using anthracene (MW = 178.23 Da) as the matrix carried out on a stainless steel (left) and PMMA (right) MALDI target substrate...... 35

Figure 2.5 MALDI – MS of triphenylphosphine (MW = 262.29 Da) analyzed using 9- cyanoanthracene (MW = 203.24 Da) as the matrix carried out on a stainless steel (left) and PMMA substrate (right)...... 36

3 Figure 2.6 MALDI – MS of mixture of three heteroatom compounds, Xanthene (MW = 182.22 Da), Thianthrene (MW = 216. 32 Da), and triphenylphosphine (MW = 262.29 Da) analyzed using anthracene (a,b) (MW = 178.23 Da) and 9- cyanoanthracene (c,d) (MW = 203.24) carried out on a stainless steel (left) and PMMA (right) MALDI target substrate...... 37

Figure 3.1 Schematic diagram of the linear TOF mass analyzer setup to measure the relative initial ion veloicities...... 44

Figure 3.2 Plot of delay time (τ) vs. flight time (t) for Na+ directly desorbed off MALDI sample target. The non-zero slope denotes field penetration during pulsed ion extraction...... 45

Figure 3.3 Initial ion velocities for DHB and polar analytes. The field-independent velocity is extrapolated from the high-mass velocity data (dashed line)...... 48

Figure 3.4 Chemical structures of the synthetic polymers investigated in this study...... 49

Figure 3.5 Initial ion velocities measured at various G-values. The optimal G-value for this mass spectrometer was determined to be 91%...... 50

Figure 3.6 Pulsed ion extraction linear mode MALDI mass spectrum of Insulin. Matrix: DHB, wavelength 337nm. Determination of G-Values was done by varying the ion extraction voltage (IS2) to the total ion voltage (IS1). Resolution determined to be (a) 62 and (b) 649...... 51

Figure 3.7 Linear – positive ion mode MALDI mass spectra of synthetic nonpolar polymer, Polystyrene 5000 (spectra on left) and polar polymer, PEG 4600 (spectrum on right) analyzed in four different nonpolar matrices anthracene (a,b), pyrene, (c,d), acenapthene (e,f) and 9-cyanoanthracene (g,h). All matrices and polymers were dissolved in THF...... 52

Figure 3.8 Linear – positive ion mode MALDI mass spectra of PBD 5000 analyzed in two nonpolar matrices in (a) 9-cyanoanthracene and (b) anthracene. All matrices and polymers were dissolved in THF...... 53

Figure 3.9 Linear – positive ion mode MALDI mass spectrum of PBD 5000 analyzed retinoic acid. Both matrix and polymer was dissolved in THF...... 55

Figure 3.10 Linear – positive ion mode MALDI mass spectra of synthetic nonpolar polymer, Polystyrene 5000 (spectra on left) and polar polymer, PEG 4600 (spectrum on right) analyzed in three different polar matrices DHB (a,b), RTA (c,d) and SA (e,f). All matrices and polymers were dissolved in THF...... 57

4 Figure 4.1 Laser desorption/ionization mass spectra of neat solutions of (a) trans-3- indoleacrylic acid (3-IAA), (b) 9-cyanoanthracene (9-CA) and (c) anthracene in THF. 3-IAA yields many fragment and cluster ions in addition to the molecular ion at m/z 187. In contrast, 9-CA and anthracene yields only a single radical molecular cation upon laser desorption...... 65

Figure 4.2 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of trans-3-indoleacrylic acid (matrix) in linear (a) and reflectron mode (b)...... 66

Figure 4.3 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of anthracene (matrix) in linear (a) and reflectron mode (b)...... 67

Figure 4.4 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of 9-cyanoanthracene (matrix) in linear (a) and reflectron mode (b) ...... 68

Figure 4.5 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. THF was used as a common solvent...... 69

Figure 4.6 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. Pyridine was used as a common solvent...... 70

Figure 4.7 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. 50% aqueous acetonitrile was used as a common solvent...... 71

Figure 4.8 (a) LDI mass spectrum of BTLP in THF. (b) FI mass spectrum of BTLP in THF. Data were obtained in reflectron ion mode...... 71

Figure 4.9 LDI mass spectrum of BTLP in DCM (a), methanol (b), ACN (c), and 50% ACN/H20 (d). Data was obtained in the reflectron mode...... 72

Figure 4.10 FI mass spectrum of BTLP in DCM (a), methanol (b), ACN (c), and 50% ACN/H20 (d)...... 73

Figure 4.11 Reflecton mode MALDI-TOF mass spectra of BTLP with 9- cyanoanthracene as the matrix with both matrix and crude oil fraction dissolved in like solvents, (a) DCM, (b) methanol, (c) ACN, and (d) 50% ACN. For all spectra, 1000 µL of matrix were mixed with 2 µL of the crude oil fraction resulting in an approximate mole ratio of 250:1 (matrix:analyte). Even at higher matrix/analyte mole ratios, the molecular mass distribution and mass spectral features for the BTLP sample do not change...... 76

5

Figure 4.12 Linear mode MALDI-TOF mass spectra of BTLP with (a) anthracene and (b) 9-cyanoanthracene as the matrix. For both spectra, 1000 µL of matrix were mixed with 2 µL of the crude oil fraction resulting in an approximate mole ratio of 250:1 (matrix:analyte). Even at higher matrix/analyte mole ratios, the molecular mass distribution and mass spectral features for the BTLP sample do not change...... 77

Figure 4.13 Reflectron mode MALDI-TOF mass spectra of BTLP on PEEK Polymer Target...... 78

Figure 4.14 Reflectron mode MALDI TOF-MS mass spectra of BTLP with (a) anthracene and (b) 9-cyanoanthracene as the matrix on PEEK polymer target...... 79

6 List of Abbreviations and Symbols

λ – wavelength.

µJ – microjoules.

3 – IAA – 3-Indoleacrylic Acid.

9-CA – 9-Cyanoanthracene.

ACN – Acetonitrile.

ATW – Acetonitrile/Trifluoroacetic acid/Water.

BTLP – Bottom Total Liquid Product.

C60 – Buckminsterfullerene.

CCD – Charge Coupled Device.

CHCA – α – cyano-4-hydroxycinnamic acid.

Da – Dalton.

DCM – Dichloromethane.

DHB – 2,5-dihydroxybenzoic Acid.

ESI – Electrospray Ionization. eV – electron Volt.

FAB – Fast Atom Bombardment.

FI – Field Ionization.

FD – Field Desorption.

FT-ICR – Fourier Transform-Ion Cyclotron Resonance.

FWI – Formic Acid/Water/Isopropanol

GPC – Gel Permeation Chromatography.

IP – Ionization Potential.

7 kV – kilovolts.

LDI – Laser Desorption Ionization.

LDI-MS – Laser Desorption Ionization – Mass Spectrometry. m – Mass. mM – milliMolar.

MALDI – Matrix Assisted Laser Desorption/Ionization.

MALDI–MS – Matrix Assisted Laser Desorption/Ionization – Mass Spectrometry.

MS – Mass Spectrometry.

MW – Molecular Weight. ns – nanoseconds.

PA – Proton Affinity.

PBD – Polybutadiene.

PC – Polycarbonate.

PEEK – Polyetheretherketone.

PEG – Polyethylene Glycol.

PMMA – Polymethylmethacrylate.

PS – Polystyrene.

RA – all-trans Retinoic Acid.

SA – Sinapinic Acid.

SIMS – Secondary Ion Mass Spectrometry. t – Time.

TFA – Trifluoroacetic Acid.

THF – Tetrahydrofuran.

8 TOF – Time-of-Flight.

TS – Thermospray Ionization.

µL – microliter.

VPO – Vapor Phase Osmometry. vo – Initial Ion Velocity.

9 CHAPTER ONE: Matrix-Assisted Laser Desorption Ionization – Mass

Spectrometry

1.1 Purpose of Work

From the very beginnings of matrix-assisted laser desorption/ionization mass

spectrometry (MALDI-MS), polar matrices have been used to analyze all types of compounds such as peptides, proteins, oligosaccharides, polymers, crude oil fractions, etc. However, not until recently have nonpolar matrices been used for polymers

(Macha 2000) and crude oil fractions (Robins 2003). The aim of this work is to investigate more thoroughly the behavior and applicability of nonpolar matrices for

MALDI-MS.

1.2 Mass Spectrometry and Basic Principles of MALDI Analysis of Nonpolar

Analytes

Mass spectrometry (MS) is an indispensable analytical technique used in

biochemistry, inorganic chemistry, environmental chemistry, forensics, pharmacy,

and medicine. This analytical method is applicable to all types of analytes: volatile or

nonvolatile, polar or nonpolar, and solid, liquid, and gas materials. MS is used to

characterize and identify unknown compounds as well as to reveal structural and

chemical properties of molecules. A mass spectrometer is an instrument that

measures the masses and relative concentrations of individual atoms and molecules

that have been converted into ions. As these atoms and molecules are so small, stating their mass in the standard unit (i.e.; kilograms) is cumbersome. For instance,

the mass of a single carbon atom is approximately 1.99266 x 10-26 grams.

10 Consequently, the Dalton (Da), which is equal to (1/12) of the mass of a single atom of the isotope of carbon-12 (12C) is the most common unit for denoting molecular

weights (Dass 2001).

A mass spectrometer is used to measure the mass of analytes by determining

the mass-to-charge (m/z) ratio of individual molecules that have been converted into gas-phase ions under vacuum. This is normally accomplished by introducing the analyte of interest (solid, liquid, or gas) into a sample inlet system, then ionizing the sample in the ion source, followed by separating the ions in the mass analyzer region, and finally detecting the ions at the detector. As a result, a mass spectrometer consists of mainly four basic components as shown in Figure 1.1.

Figure 1.1 Block Diagram of a mass spectrometer.

The mass spectrometer has been revolutionized to a great extent over the past

60 years. It has seen major innovative developments as it is now applicable to biological, petroleum, and environmental analysis, just to name a few. However, it was the field of biological analysis that really led to an explosion in using this method

11 because historically there was no suitable form of ionization for large compounds that did not lead to the degradation or decomposing of compounds being exposed to high temperatures. The search for improved ionization methods led to the discovery of field desorption (FD) (Becky 1977), fast-atom bombardment (FAB) (Barber 1981), secondary ion mass spectrometry (SIMS), thermospray ionization (TS) (Blakley

1983), electrospray ionization (ESI) (Yamashita 1984; Whitehouse 1985), laser desorption ionization (LDI) (Cotter 1987), and matrix assisted laser

desorption/ionization (MALDI) (Karas 1987; Tanaka 1988). Of the above mentioned

ionization techniques, ESI and MALDI are the most recent developments in the field that have significantly advanced the application of mass spectrometry to high

molecular weight, nonvolatile, and thermally labile samples.

LDI was introduced in the late 1960’s (Fenner 1966; Vastola 1968; Vastola

1970) for the analysis of biological molecules. Practitioners have employed the use

of lasers with different wavelengths and of various pulse widths, e.g. nitrogen lasers

(337 nm), excimer lasers (193, 248, 308, and 351 nm), Q-switched, frequency-tripled

and quadrupled Nd:Yag lasers (355 and 266 nm, respectively) (Nordhoff 1992),

Er:Yag (2.94 µm) (Overberg 1990; Nordhoff 1992) and TEA-CO2 laser (10.6 µm)

(Hillenkamp 1991; Overberg 1991). Two universal observations were acquired from

these different studies; one is that for efficient energy transfer to the sample, a

resonant absorption is required at the laser wavelength, and the second is that to

circumvent thermal decomposition of the thermally labile molecules, the energy

transfer must transpire within a short window of time. However, in the LDI

12 experiments, the compounds that were accessible were no more than 103 Da due to a

lack of resonant absorption of the laser energy by large biomolecules.

To overcome this deficiency, researchers combined light-absorbing

compounds that were admixed with the analyte for laser desorption mass

spectrometry. Two research groups investigated different pathways that lead to

analyzing proteins of about 105 Da. Tanaka and co-workers used ultrafine cobalt

powder (particle size 30 nm) added to analyte solutions in glycerol (Tanaka 1988;

Tanaka 2003), while Karas et. al co-crystallized the analyte with an organic matrix

(Karas 1985; Karas 1987; Karas 1988; Karas 1989).

Figure 1.2 MALDI sample preparation of matrix and analyte.

These approaches produced intact gas-phase molecular ions without fragmentation at

a much lower laser power than used in the LDI approach. This great achievement in

the field opened the door for many other types of nonvolatile compounds to be

analyzed that were not previously accessible by the LDI technique.

13 In MALDI, a very small amount of a minute concentration of analyte solution is thoroughly mixed with a large amount and concentration of an aromatic organic

matrix solution. This matrix-analyte mixture is then spotted on a MALDI target to

dry and solidify into a crystalline solid. A pulsed laser of a specific wavelength is

absorbed by the matrix and is used to facilitate both desorption and ionization of the

analyte of interest.

Figure 1.3 Schematic of Desorption process in MALDI.

1.3 MALDI Analysis of Nonpolar Analytes

Since the introduction of MALDI-MS, the primary utilization of this

analytical technique has been the characterization of polar organic and bio-organic

molecules. The matrices used for analysis of such compounds are acidic organic molecules such as 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid, (α-CHCA), and 3,5-dimethoxy-4-hydroxycinnamic acid (better known as

Sinapinic Acid (SA)). For these types of matrices and analytes, the predominant mechanism for ionization appears to be either proton transfer reactions between the

14 analyte and matrix or cation adduction. Although not nearly as common as reports on

the production of protonated or cation-adducted molecular ions, there are several

reports in the literature on the formation of analyte molecular radical cations during

MALDI-MS experiments (Juhasz 1993; Michalak 1994; Lidgard 1995). In addition, laser desorption studies of matrix materials reveal that many compounds that have

. been used as matrices form M+ ions upon ionization (Ehring 1992; Gimon-Kinsel

1997).

The first and most complete description on the production of radical cations

using MALDI was presented by Juhasz and Costello (Juhasz 1993). They reported

the successful use of four different matrices (2-(4-hydroxyphenylazo)benzoic acid

(HABA), quinizarin, dithranol, and 9-nitroanthracene) for the characterization of

ferrocene and ruthenocene oligomers and polymers up to m/z 10,000. These authors

noted that the role of the matrix in the ionization process could not be established:

either charge transfer from the matrix to the analyte or photoionization of the analyte

directly could account for their results. Further, the chemical stability of these

matrices was not reported in their work (although in another paper they noted that

HABA is characterized by a strong (M+H)+ ion, (Juhasz 1993)) and it is unclear

whether it was a matrix molecular radical cation or some other feature that yielded the

reported mass spectral results.

Michalak et al. have presented MALDI mass spectra of several proteins

(insulin, cytochrome C, and serum albumin) using C60 as a matrix (Michalak 1994).

The positive ion mass spectrum of C60 shows production of the molecular radical cation and a large number of fragment ions while the negative ion mass spectrum is

15 composed primarily of the molecular radical anion. As C60 has no proton sources for proton transfer reactions to occur, these authors assign the molecular ions of each

. . protein analyzed as the M+ or M- species. However, due to the high molecular weight of these proteins (Mr > 5,000 Da in all cases) and the low resolution of the time-of-flight mass spectrometer used, these assignments are tentative. Formation of radical molecular ions from these proteins would be unique and in contrast to other studies of radical molecular ion formation. For example, Juhasz and Costello noted that insulin could not be detected when 9-nitroanthracene, which has no labile proton for proton transfer reactions, was used as a matrix, but the (M+H)+ ion of insulin was detected when other matrices that have labile protons, such as dithranol, were used

(Juhasz 1993).

Recently, McCarley and co-workers and Macha and co-workers have investigated a new class of nonpolar compounds having suitable properties to function as charge transfer matrices for MALDI-MS analysis of nonpolar analytes

(McCarley 1998; Macha 1999). Their initial studies focused on examining the

MALDI properties of such matrices. The compounds investigated were anthracene and terthiophene. Both of these compounds have strong molar coefficients at the 337 nm, the wavelength of the nitrogen laser used for these studies. The radical molecular cation for each compound was readily observed upon direct LDI.

Next they utilized those compounds as matrices for the analysis of low molecular weight, nonpolar analytes. Macha and co-workers investigated nonpolar analytes such as decamethylferrocene, 1,2-diferrocenylethane, ferrocene, and 2,2’- methylenebis(6-tert-butyl-4-methylphenol) with anthracene and other potential charge

16 transfer matrices (Macha 1999). For all analytes investigated, an abundant radical molecular cation was detected only in the presence of the nonpolar matrix. To establish that direct photoionization of the analyte was not responsible for the production of radical molecular cations, matrices having molar extinction coefficients five orders of magnitude larger than the analytes were used and the matrix-to-analyte ratio was varied from 100:1 to 10,000:1. Direct laser desorption ionization experiments of the analytes in the absence of matrix did not result in the production of a radical molecular cation.

In the majority of the cases studied, the presence of an analyte radical molecular cation can be predicted from the thermodynamics of the charge transfer process: analytes with ionization energies less than the ionization energy of the matrix are detected while analytes with ionization energies greater than the ionization energy of the matrix cannot be detected. That work lends further evidence of the mechanism of ionization using this relatively new approach to MALDI-MS. This technique should prove valuable for the analysis of electro-active and nonpolar molecules, such as those found in crude oil fractions.

1.4 Role of matrix

The most intrinsic property that characterizes MALDI-MS is the matrix. The matrix is typically a small acidic organic compound, in the mass range of 100 – 300

Da, which has a relatively high molar absorptivity (ε) at the wavelength of the laser.

The matrix must have an appropriate chromophore because energy transfer is based on the strong absorption and ensuing electronic excitation of the matrices. Frequently used MALDI matrices have molar absorptivities on the order of 103 to 105 M-1 cm-1

17 (Limbach 1998). A large number of potential MALDI matrices have been

investigated over the years with a relatively small number becoming standards for

MALDI-MS characterization of polar and nonpolar compounds.

The matrix is crucial in promoting the formation of intact gas-phase molecular ions from the analyte being analyzed. Analyte isolation by the matrix physically separates the analyte molecules one from the other. Normal molar ratios of matrix to analyte range from 100:1 to 10,000:1. The matrix absorbs the laser radiation. The analyte is not ionized directly upon laser irradiation as the laser radiation is preferentially absorbed by the matrix. The preferential absorption of the laser irradiation by the matrix reduces subsequent fragmentation of the analyte, improving the production of intact molecular ions of the analyte.

Analyte desorption is another function that the matrix serves. It is presumed that the laser radiation absorbed by the matrix allows desorption of the first few layers of the sample; after desorption occurs, the MALDI plume is formed containing all charged and neutral species within the source region of the instrument. However, the exact matrix characteristics that facilitate analyte desorption are being investigated.

The matrix has an active role in ion production either directly or indirectly (Zenobi

1998; Karas 2000; Knochenmuss 2000).

1.5 Polar vs. Nonpolar Matrices

The selection of a matrix is crucial for success in MALDI experiments

(Zenobi 1998). Most often, an analyte is co-crystallized with an excess of a solid

matrix material; and the capacity of matrix molecules to separate analyte molecules is

made possible by the chemical nature of the intermingling molecules (Macha 2002).

18 Due to their similar chemical properties, polar matrix molecules interact well with

polar analyte molecules while nonpolar matrices interact well with nonpolar analytes.

Many polar matrices are derivatives of benzoic acid, cinnamic acid, and

related aromatic compounds which were identified early on as good MALDI matrices

for proteins (Beavis 1989). 2-(4-hydroxyphenlazo)-benzoic acid (HABA) was

introduced as a matrix by Juhasz and co-workers for peptides, proteins and glycoproteins up to 250 kDa (Juhasz 1993). 3-hydroxypicolinic acid (3-HPA) (Wu

1993; Wu 1994), glycerol (Berkenkamp 1998), and succinic acid (Nordhoff 1993) are

matrices that have been used with UV and infrared lasers for the analysis of

oligonucleotides.

However, biological molecules are not the only type of compounds that have

been analyzed using polar matrices. Hydrocarbon polymers and crude oil fractions

have been analyzed using 2,5-dihydroxybenzoic acid (DHB), HABA and all-trans

retinoic acid (RTA). HABA and all-trans RTA have been effectively used with

polystyrene (Juhasz 1993; Schriemer 1997; Whittal 1997; Yalcin 1997; Zhu 1998;

Guo 2000). all-trans RTA has also been effectively used with polybutadiene and

polyisoprene (Yalcin 1997). DHB and trans-indole acrylic acid have also been used

for hydrocarbon polymers (Belu 1996; Whittal 1997).

Because the use of polar matrices has been so successful with the analysis of

various kinds of analyte compounds, the use of nonpolar matrices has not received

much attention. However, the application of nonpolar matrices has been very

successful with small molecules as well as hydrocarbon nonpolar polymers. A

majority of research with nonpolar matrices has been done by the Limbach research

19 group. McCarley and co-workers showed that aromatic compounds having no

functional groups, anthracene and terthiophene, generated radical molecular cations

upon laser desorption (McCarley 1998). Macha and co-workers not only used the

matrices in the analysis of small molecule compounds but extended the use of these matrices to the analysis of nonpolar polymers (Macha 2000).

Figure 1.4 Common polar matrices and nonpolar matrices used for MALDI – MS analysis.

20 1.6 MALDI Ion Formation

In a MALDI experiment, only a few types of ions are commonly observed in positive ion mode in the final MALDI mass spectrum. These ions are radical molecular cations, protonated molecular ions and cationized molecular ions in the form of metal adducts. Formation of gas phase molecular ions fall into two general categories. Primary ionization refers to generation of ions from neutral molecules in the sample. This process begins with laser photons absorb by the matrix in multiple steps until the ionization energy of the matrix is exceeded and the excited matrix ejects an electron to produce matrix molecular cations (M+.) or anions (M-.).

Secondary ionization refers to ions that are not directly generated by primary processes. In this ionization process, it involves ion – molecule reactions taking place within the MALDI plume.

Zenobi and Knochenmeuss addressed primary ionization mechanisms in a prior review (Zenobi 1998). In this same review, many secondary ionization mechanisms were considered as well and only a couple will be given attention. The two secondary ionization mechanisms that have been under scrutiny in these recent years have been ion-molecule reactions with proton and electron-transfer cationization reactions. The appearance of radical molecular ions and how they come about have been discussed in great detail (Karas 2000; Knochenmuss 2000).

Ion-molecule reactions involving proton-transfer reactions lead to the appearance of singly charged protonated molecular cations which are the most commonly observed ions in MALDI. Karas and co-workers debate that in the

MALDI plume, there are many multiply charged species that are being neutralized by

21 electrons and reduced to singly charged protonated molecular ions that are the lucky survivors of a cluster desorbed gas phase ion plume (Karas 2000). Knochenmuss and co-workers argument is that these ions in the mass spectrum are the result of secondary ion molecule reactions that can be predicted by thermodynamics

(Knochenmuss 2000). The protonated molecular ion is observed in the mass spectrum when the proton affinity (PA)1 of the analyte is greater than the proton affinity of the

matrix (Eq 1.1, 1.2).

+ + M . + A → ( M − H ) . + ( A + H ) (1.1)

+ + ( M + H ) + A → M + ( A + H ) (1.2)

Ion-molecule reactions involving charge (electron) – transfer reactions and the

appearance of singly charged radical molecular cations are rare. The appearance of

these ions in the MALDI mass spectrum is also said to be the result of secondary ion

molecule reactions (Knochenmuss 2000). The radical molecular ion is observed in

the mass spectrum when the ionization potential (IP)2 of the matrix is greater than the

ionization potential of the analyte (Eq 1.3).

+ . + . M + A → M + A (1.3)

Macha and co-workers, working with nonpolar matrices such as terthiophene and

anthracene, addressed this issue and suggested that ion-molecule reaction

thermodynamics influence MALDI spectra in a systematic way (Macha 1999).

1 Proton affinity is defined as the energy associated with the heterolytic cleavage of the element – hydrogen bond in the gas phase. 2 Ionization potential is the amount of energy required to break up a chemical into electrically charged particles.

22 1.7 Mass Analyzer

After the desorption/ionization event and ions are formed, these ions are then

sent to a mass analyzer and separated based on their mass to charge (m/z) ratio. There

are many different mass analyzers, such as the magnetic sector (B), linear quadrupole

(Q), linear quadrupole ion trap (LIT), quadrupole ion trap (QIT) , Fourier transform

ion cyclotron resonance (FT-ICR), and time of flight (TOF) that are used with the

various types of ion sources. However, for MALDI, two mass analyzers are used

more often, the TOF and FT-ICR. Of these two, the TOF is the most universally used

due to reasons such as cost efficiency, no difficulty in design and use, (theoretically) unlimited mass range, ability to detect all charged species simultaneously, rapid analysis time, and well-suited for the MALDI pulsed ionization source (Muddiman

1997).

In MALDI, charged species are created by a laser pulse that ablates the solid- crystalline matrix-analyte sample located on the surface of a MALDI target which is located in the source region containing an electric field (figure 1.5). Charged species of different masses are then accelerated to the same final kinetic energy of a few kV.

The electric field accelerates the charged species into the “field-free” drift region resulting in the separation of the charged species in space and time and detected at a detector at the end of the ion flight tube. Depending on instrumental design, the length (L) of the flight tube ranges between 1 to 3 meters (m). The time (t = seconds) that is required for charged species to pass through the “field-free” region is dependent on their m/z ratio as shown in the equation 1.4 in which e is the

23 fundamental charge (1.6 x 10-19 C), z is the number of charges, and V is the

accelerating voltage of the charged species in the source region.

1  m  2 t = L (1.4)  2 zeV 

Hence, the lightest charged species arrives at the detector first followed by other

charged species of succeeding heavier masses. Figure 1.5 shows a schematic of a

linear TOF mass analyzer.

Figure 1.5 Schematic diagram of linear time-of-flight (L-TOF) instrument.

Though the TOF mass analyzer has its advantages, a disadvantage is its limited mass resolving power. Mass resolving power is the ability of the mass analyzer to separate ions of different masses. Resolution, R, is defined as the ratio of the mass of interest, m, to the difference in mass, ∆m. For a TOF mass analyzer resolution is proportional to the flight time t of the ion divided by twice the time interval (∆t) of the ion arrival at the detector as shown in equation 1.5.

m t R = = (1.5) ∆ m 2 ∆ t

24 To overcome limited resolving power, two instrumental modifications have been

developed. These are time-lag-focusing (also referred to as delayed extraction, or pulsed ion extraction) and the reflectron. Figure 1.6 shows a schematic diagram of

the reflectron mode TOF mass analyzer.

Figure 1.6 Schematic diagram of a reflectron time-of-flight instrument (re-TOF).

The principle of time-lag-focusing (Cotter 1997; Gross 2004) is to produce

ions in a “hypothetically” field free environment, in so doing allowing them to

separate in space according to their different initial ion velocities. Then, after a short

delay of some hundreds of nanoseconds (ns) the accelerating voltage is switched on

with a fast pulse. This method also guarantees that laser-induced reactions have been

completed before the ions are pulsed out of the extraction region. At the time of

extraction, ions with high initial ion velocities will have traveled farther than slower

ions, and therefore experience only a fraction of the extraction voltage between the

sample target plate and the extraction grid. Time-lag-focusing compensates for the

initial ion velocity distribution of the MALDI generated ion packet such that same

m/z ions arrive simultaneously at the detector.

25 A reflectron time-of-flight mass analyzer uses an ion mirror that focuses ions

of different kinetic energies in time. Reflectors which are used to focus the ions are

located at the rear of the field free drift region. In application, a reflector is made up

of a series of grids at increasing potential. The reflector voltage is set to about 1.05 –

1.10 times the acceleration voltage which assures that all ions are reflected within the

homogenous section of the electric field of the device. The ions infiltrate the

reflectron until they reach zero kinetic energy and are then expelled from the reflector

in the reverse direction. The kinetic energy of the leaving ions remain unaffected,

however their flight paths differ according to their differences in kinetic energy. Ions

possessing a high kinetic energy will travel further into the decelerating field, and

thus spend more time within the reflector than ions with low kinetic energy. Thereby,

the reflector achieves a correction in time-of-flight that substantially improves the

resolving power of the TOF analyzer (Cotter 1992; Guilhaus 1995; Ioanoviciu 1995).

1.8 Significance of Work

This research will lead to an improved understanding of the behavior and applicability of nonpolar matrices in MALDI – MS. To date, fundamental investigations of the MALDI process have been based on the behavior of polar matrices, such as DHB. Such experiments have included a quantitative model for

UV-MALDI ion generation for both primary and secondary ionization processes

(Knochenmuss 2002; Knochenmuss 2003), plume velocities (Kinsel 1997;

Gluckmann 1999; Puretzky 1999), and the magnitude of ion yield (Dreisewerd 1995) among others. In contrast, only a handful of investigations have focused on nonpolar

26 matrices, and this research will provide significant information relating to nonpolar

matrices that is currently unavailable.

Chapter 2 describes studies performed using nonpolar matrices on different

sample target substrates including a conventional stainless steel substrate and polymer

– based substrates. Because of the interaction of the matrix with a metal substrate,

photoelectrons produced in the MALDI plume have been found to reduce the analyte

ion signal (Frankevich 2002; Frankevich 2003; Frankevich 2003). The use of a polymer substrate reduces photoelectron production. The hypothesis being tested is that a reduction in photoelectron production should lead to an increase in radical molecular cations detected in the mass spectrum.

Chapter 3 examines the desorption process for nonpolar matrices in MALDI.

A parameter that has been used to elucidate the desorption process in MALDI is the

initial ion velocity (Juhasz 1997; Gluckmann 1999; Karas 2003). It is known that the

initial ion velocity of ions is matrix – dependent. Further studies suggest that the

initial ion velocity is also dependent upon analyte incorporation into the matrix. The

primary hypothesis to be tested is that the initial ion velocity for nonpolar analytes

will be higher than the initial ion velocity for polar analytes measured for any

particular nonpolar matrix. In addition, a correlation between the initial ion velocity

and mass spectral resolution will be established for these studies.

Chapter 4 will describe the application of nonpolar matrices to a crude oil

sample. The hypothesis to be tested in this work is that nonpolar matrices, which

exhibit minimal background ions in MALDI, will be more favorable for the analysis

of a low molecular weight crude oil sample than polar matrices, which exhibit a

27 complex background in MALDI due to matrix fragmentation and clustering. These results will also be compared to data obtained using LDI and FI, which have been used historically to investigate this class of samples.

28 CHAPTER TWO: CHARACTERIZAITON OF STAINLESS STEEL AND

POLYMER MALDI TARGETS AND THE EFFECT OF THE SUBSTRATE

ON NONPOLAR MATRIX IONIZATION EFFICIENCY

2.1 Introduction

Although MALDI – MS is a popular analytical method, there are still areas

this method can be improved. One area to be improved is sensitivity. It has been

found that the production of charged analyte species in MALDI is relatively low.

One of the reasons for this was the discovery of electrons in the MALDI plume which

were produced as a result of the interaction of a thin matrix layer and the metal

MALDI target substrate upon laser irradiation (Frankevich 2002). It was found that

these electrons contribute to the neutralization of charged species within the MALDI

plume as well as the reduction of positive ion intensity in the final mass spectrum.

Those findings then led Frankevich and co-workers to investigate how to

reduce or eliminate the formation of electrons in the MALDI plume (Frankevich

2003; Frankevich 2003). They found that the use of a polymeric MALDI substrate

during experimental analysis can have an extraordinary effect on the MALDI mass spectra (Frankevich 2003). This was evident as the appearance of multiply charged ion species of higher ion intensity during MALDI analysis (Frankevich 2003).

Our research group has been investigating nonpolar matrices used for the

formation of analyte radical molecular ions via a charge – transfer ionization

mechanism (McCarley 1998; Macha 1999). The appearance of the analyte radical

molecular ion is observed when the ionization potential of the matrix is greater than

the ionization potential of the analyte. Unlike other MALDI secondary ionization

29 processes, such as proton – transfer ionization mechanisms, where the appearance of singly charged protonated molecular ions as well as possible multiply charged protonated molecular ion are observed, the electron – transfer ionization mechanism

only yields an analyte radical molecular ion and it is often low in intensity.

Therefore, it is the aim of this investigation to examine the effect that the MALDI

substrate has on the ion abundance of the analyte radical molecular cation via a

charge transfer ionization mechanism using nonpolar matrices. Also, I will

investigate how the difference in ionization potential between the nonpolar matrix

and analyte relates to the abundance of the analyte ion in the mass spectrum.

2.2 Experimental

2.2.1 Materials

Samples were used as supplied by the manufactor, 9-cyanoanthracene (9-CA),

C60, thianthrene, triphenylphosphine, and xanthene were obtained from Sigma-

Aldrich Chemical (Milwaukee, WI, USA). Anthracene and perylene were obtained

from Fluka Chemicals (Milwaukee, WI, USA). Tetrahydrofuran (THF) and methylene chloride were obtained from Fischer Scientific (Fairlawn, NJ USA).

Polycarbonate (PC), Polyetheretherketone (PEEK), Polymethylmethacrylate

(PMMA) and Polystyrene (PS) sheets were purchased from Goodfellow (Devon, PA).

2.2.2 Methods

2.2.2.1 Polymeric MALDI Sample Target Disc Fabrication

Polymer sheets (Goodfellow, Berwyn, PA) were cut into two-inch by four-

inch rectangular sections using a vertical band saw (Cincinnati Machinery,

Cincinnati, OH). An RB (I) bed type mill (Taichung Hsien, Taiwan) with vertical

30 spindle was tramed3 to the mounting table. This milling machine, fitted with a TRAC

CNC controller (Southwestern Industries, Rancho Dominguez, CA), is specified to position with an accuracy of ±0.001 inch over the X, Y horizontal plane and repeatability of ±0.0005 inch over the X, Y horizontal plane. A vice (Kurt Mfg. Co,

Minneapolis, MN) was mounted and tramed to 90° of the mill spindle. A two-inch by four-inch piece of wood was mounted in the vice. The rectangular polymer piece was mounted to the wood using double-sided sticky tape. A one-inch hole saw (Sears and

Roebuck, Hoffman Estates, IL), operated at 310 RPM, was employed to fabricate the polymer MALDI target probes. A steady stream of Kool Mist cutting fluid (Santa Fe

Springs, CA) and compressed air were used for chip removal and to lubricate the cutting edge. Polymer disks were removed from the mounting device and cleaned.

2.2.2.2 Imaging

Electron microscope images were taken using a XL30 environmental scanning electron microscope (FEI Company, Hillsboro, Oregon). Optical images were taken using an Eastman MDS 100 digital camera (Kodak Digital Science, USA) outfitted with a macro 10x lens (Computar, Japan).

2.2.2.3 MALDI Analysis

All MALDI experiments were performed using a Bruker Daltonics Reflex IV

(Billerica, MA, USA) reflectron time-of-flight (TOF) mass spectrometer equipped with pulsed ion extraction. A nitrogen laser (λ = 337 nm), with a maximum radiant pulse energy of 300 µJ, was operated at a laser power between 20 and 30%. An

3 Tramed in engineering machinist terms means aligning the z axis (spindle) with the x-y axis (mounting table).

31 acceleration voltage of 20 kV was used for all LDI and MALDI analyses. Anthracene

(m/z 178) and C60 (m/z 720) were used for external calibration. On average, 100

laser shots were taken to obtain each mass spectrum.

Nonpolar matrices were prepared as 100 – 200 mM solutions. The nonpolar

analytes, Figure 2.1, were prepared at a concentration of 25 – 50 mM. Typically, 100

– 125 µL of nonpolar matrix solution was combined with 1 or 2 µL of the analyte.

Then a 1 µL aliquot mixture was spotted on the MALDI target and allowed to air dry.

Figure 2.1 Structures of chemical compounds used as analytes in this chapter.

32 2.3 Results and Discussions

2.3.1 LDI of MALDI Substrates

LDI analysis of MALDI polymer targets was carried out at the normal laser

power (20 – 30 %) used for the analysis of nonpolar matrices and analytes. For all

polymers investigated, no peaks related to background ions were detected (Figure

2.2). Ions representative of the polymer are not detected until a laser power of 50% is reached.

Figure 2.2 Mass spectrum of a LDI analysis of a PMMA MALDI substrate. The mass spectrum of the other polymers (PC, PS, PEEK) were the same. LDI experiments were investigated using a laser power of 20 – 30 %.

33 2.3.2 Solvent Effects on Polymeric Substrates

The second criterion in choosing the most suitable polymer substrate was to

examine the polymer’s resistance to solvents. If the solvent used dissolves the

polymer when the aliquot mixture is spotted onto the polymer substrate, the

interaction of the polymer with the matrix and analyte may result in additional

unwanted peaks in the mass range of interest. These unwanted peaks might possibly

arise from the polymer or from low molecular weight additives that are used to make

the polymer. A normal 1 µL matrix – analyte mixture was spotted on the polymer

substrates and allowed to air dry. As can be seen in Figure 2.3, when the matrix –

analyte mixture is spotted on the polystyrene substrate, the solvent readily dissolves

the polymer. This same trend was observed when the matrix – analyte mixture was

spotted on polycarbonate. However, when the matrix – analyte mixture was spotted

on PEEK or PMMA, the polymer was not seen to be dissolving. As a result, all

studies were performed on the PMMA MALDI substrate, which gave optimum

results and the best visibility of matrix-analyte mixture on the MALDI spot monitor.

Figure 2.3 CCD images of unspotted (left) and spotted (right) polystyrene MALDI substrates.

34 2.3.3 Charge Transfer Analysis on MALDI Substrates

It is known that formation of a radical molecular analyte ion via an electron –

transfer ionization mechanism is rare; however, it still useful for the analysis of

compounds with low ionization potentials (Zenobi 1998). Therefore, throughout

these investigations, matrix:analyte pairs were chosen whereby it is known that an electron – transfer ionization mechanism would take place. The purpose for this set of experiments was to determine the analyte ion abundance observed in the mass spectrum when using a stainless steel versus a PMMA MALDI substrate.

From previous analyses, ferrocene is known to undergo charge – transfer ionization when admixed with nonpolar matrices (Macha 1999). As can be seen in

Figure 2.4, the radical molecular cation for ferrocene is detected on both substrates.

The analyte ion abundance for ferrocene on the stainless steel MALDI target substrate is ~ 300 ion counts, while the analyte ion abundance is nearly 5000 ion counts on the

PMMA MALDI target substrate.

Figure 2.4 MALDI – MS of ferrocene (MW = 186.03 Da) analyzed using anthracene (MW = 178.23 Da) as the matrix carried out on a stainless steel (left) and PMMA (right) MALDI target substrate.

35 Figure 2.5 shows the analysis with 9-cyanoanthracene and

triphenylphosphine. The radical molecular cation was detected for

triphenylphosphine on both stainless steel and PMMA MALDI target substrates.

The ion abundance of the radical molecular cation for triphenylphosphine on stainless steel is ~ 400 ion counts, while when analyzed on PMMA, the ion abundance is nearly 1200 ion counts.

Figure 2.5 MALDI – MS of triphenylphosphine (MW = 262.29 Da) analyzed using 9-cyanoanthracene (MW = 203.24 Da) as the matrix carried out on a stainless steel (left) and PMMA substrate (right).

The data shown in Figures 2.4 and 2.5 correlate well with my hypothesis that the radical molecular ion abundance will be higher on a polymer substrate than it would be on a stainless steel substrate. Though photoelectrons produced from a thin layer of matrix on a stainless steel (metal) surface (Frankevich 2002) are not detected in the experiment, their secondary effects are evident in Figures 2.4 and 2.5.

36

Figure 2.6 MALDI – MS of mixture of three heteroatom compounds, Xanthene (MW = 182.22 Da), Thianthrene (MW = 216. 32 Da), and triphenylphosphine (MW = 262.29 Da) analyzed using anthracene (a,b) (MW = 178.23 Da) and 9- cyanoanthracene (c,d) (MW = 203.24) carried out on a stainless steel (left) and PMMA (right) MALDI target substrate.

Figure 2.6 shows results from analysis of three heteroatom compounds to see if selective ionization could be achieve from the nonpolar matrices. Xanthene (MW =

182.22 Da), thianthrene (MW = 216.32 Da), and triphenlyphosphine (MW = 262.29

Da) analytes were investigated by MALDI. Anthracene and 9-cyanoanthracene were the matrices selected for this investigation. The ionization potentials of xanthene and triphenylphosphine are reported to be 7.65 (Bouchoux 1981) and 7.80 eV (Ikuta

37 1982), respectively, while the ionization potential of thianthrene has been evaluated as 7.8 eV (Mallard 2003). The ionization potential for anthracene has been evaluated as 7.439 (Mallard 2003) and 9-cyanoanthracene has been reported to be 7.80 eV

(Klasinc 1983).

Figure 2.6 a and b show the anlaysis of the mixture with anthracene. The radical molecular cation for triphenylphosphine is detected on both stainless steel and

PMMA MALDI substrates. Although the ionization potential for triphenylphosphine is greater than the ionization potential of anthracene similar results for triphenylphosphine has been reported previously (Macha 1999). Thianthrene was detected at low abundance on the PMMA MALDI target substrate.

Figure 2.6 c and d shows the analysis of the mixture with 9-cyanoanthracene.

9-cyanoanthracene has an ionization potential that is greater than or equal to all three analytes within the mixture, so the analyte radical molecular cations for all three analytes are expected to be detected. The radical molecular cation for both triphenylphosphine and thianthrene are detected on both stainless steel and PMMA

MALDI substrates, while the radical molecular cation for xanthene is only detected on the PMMA substrate. When the analysis of the mixture was performed on the stainless steel MALDI substrate, the analyte ion intensity for triphenylphosphine and thianthrene is less than 500 ion counts. However, when the analysis was performed on the PMMA MALDI substrate, the analyte ion intensity for both analytes is well above 1000 ion counts.

The PMMA MALDI substrate demonstrates that a reduction in photoelectrons from a polymer substrate leads to increases in the radical molecular cation

38 abundances. The analyte ion abundance for the analysis of both matrices increased by a factor of two to three for some analytes. Also, noted in this mixture analysis was a clear dependence on the difference between the ionization potential of the matrix and analyte, and the analyte ion abundance. Anthracene has an ionization potential of

7.439 eV, while triphenylphosphine has a recently reported ionization potential of

7.80 eV and other literature values has been reported as low as 7.37 eV, (Debies

1974). Under those same conditions communicated in a previous article (Macha

1999), the 7.37 eV ionization potential for triphenylphosphine fits this situation, and therefore the difference in the ionization potential is 0.069 eV. With this difference, the analyte ion intensity for triphenylphosphine was ~ 1000 ion counts. Likewise, 9- cyanoanthracene has reported ionization potential 7.80 eV. Using the 7.37 eV ionization potential value for triphenylphosphine, the difference in ionization potential for the matrix and analyte is 0.43 eV. With this difference, the analyte ion intensity is ~ 2000 ion counts. Though only two matrices were used for this analysis, the results are consistent with an increase in the analyte ion intensity with increasing ionization potential between the matrix and analyte.

2.4 Conclusions

In this investigation, I have demonstrated that nonpolar matrices used for charge – transfer MALDI provide increased analyte radical molecular cation abundance on a polymer substrate. Single component analytes and mixtures were admixed with nonpolar matrices and investigated on stainless steel and polymer

MALDI target substrates. An increase in analyte ion abundance is clearly seen in the analysis that took place on a polymer substrate, while low analyte ion abundance was

39 observed in the analysis taken place on the stainless steel substrate. Presumably, photoelectrons produced from the laser and interaction of a thin matrix layer and metal target plate neutralize the charged species resulting in a reduced number of charged species being detected. Also, increased analyte ion abundance was detected when the ionization potential difference between the matrix and analyte was large, while decreased analyte ion abundance was observed when there was a small difference in ionization potential of the matrix and analyte.

40 CHAPTER THREE: DETERMING THE INITIAL ION VELOCITY FOR

NONPOLAR MATRICES

3.1 Introduction

One of the many issues in MALDI that has been addressed within the last few

years has been the study of the initial ion velocity of MALDI ions (Beavis 1991;

Dominic Chan 1994; Juhasz 1997; Gluckmann 1999; Karas 2003). The initial ion

velocity is the velocity ions possess when they leave the target surface and enter the

gas phase prior to an electrostatic acceleration (Spengler 2003). The initial ion velocity of MALDI ions is matrix dependent, analyte mass independent, charge independent (singly, doubly, or multiply charged), and ion polarity (positive – negative mode) independent. Typical initial ion velocities of analyte ions vary between 300 and 1000 m/s, whereas those of matrix ions vary between 300 and 1700 m/s (Juhasz 1997).

While the initial ion velocity is matrix dependent, there are other factors that have an effect on the initial ion velocity of MALDI ions. These factors include analyte class (peptide/proteins vs. oligosaccharides), sample preparation (single crystal, dried droplet, solvents), and additives (salts or co-matrices). Initial ion velocity discrepancies because of the class of analyte were first observed by

Glückmann and Karas (Gluckmann 1999). They found that slower initial ion velocities were found for oligosaccharides ( average ~ 194 m/s) than for proteins

(average ~ 543 m/s) when 2,5-dihydroxybenzoic acid was used as the matrix. They found that neutral oligosaccharides are not truly incorporated into the matrix and therefore a lower initial ion velocity is observed relating to a reduced role of the

41 matrix. When the authors placed a charged functional group onto a small neutral oligosaccharide an increase in the initial ion velocity was detected.

It has been found that in an analysis of carbonic anhydrase with α-CHCA

(matrix) dissolved in both formic acid/water/isopropanol (FWI) and acetonitrile/trifluoroacetic acid/water (ATW), an initial ion velocity of ~ 270 – 290 m/s was observed. Whereas the same analyte admixed with DHB in FWI had an initial ion velocity of 420 m/s, while when dissolved in ATW an initial ion velocity of

550 m/s was observed.

The initial ion velocity has proven to be a useful parameter for the characterization of the desorption process, but most of the prior experiments have focused on biomolecules (Juhasz 1997; Gluckmann 1999). The initial ion velocity of nonpolar matrices has not been reported, thus in this chapter initial ion velocities will be measured for synthetic polymers using polar and nonpolar matrices.

Initial ion velocities of nonpolar matrices with nonpolar aromatic polymers will be compared to that of nonpolar matrices with polar polymers as well as nonpolar linear chain polymers. This analysis shows that for analysis of a synthetic nonpolar aromatic polymer with a nonpolar matrix, a high initial ion velocity is observed, while in the analysis of a polar polymer with a nonpolar matrix, a low initial ion velocity is observed. The higher initial ion velocity for the nonpolar matrix/nonpolar aromatic polymer combination suggests that the nonpolar polymer is more soluble with a nonpolar matrix, while the lower initial ion velocity of nonpolar matrix/polar polymer is indicative of polar polymer not being soluble in a nonpolar matrix.

Improved peak resolution will confirm these predictions.

42 3.2 Experimental

3.2.1 Materials

Samples were used as supplied by the manufacturer. 9-cyanoanthracene (9-

CA), acenaphthene, pyrene, C60 (fullerene), thianthrene, triphenylphosphine, silver trifluroacetate (AgTFA), adrenocorticotropic hormone (18 – 39) (ACTH 18-39),

Isulin chain b oxidized, and insulin were obtained from Sigma-Aldrich Chemical

(Milwaukee, WI, USA). 2,5-dihydroxybenzoic acid (DHB), α-cyano-4- hydroxycinnamic acid (CHCA), all-trans retinoic acid (RA), anthracene, polystyrene

5000 (PS 5000), polyethylene glycol 4600 (PEG 4600), polybutadiene 5000 (PBD

5000), and sinapinic acid (SA) were obtained from Fluka Chemicals (Milwaukee, WI,

USA). Tetrahydrofuran (THF), methylene chloride and acetonitrile were obtained from Fischer Scientific (Fairlawn, NJ USA).

3.2.2 Methods

Polar and nonpolar matrices were prepared as 100 – 200 mM solutions, except for 9-cyanoanthracene, which was prepared as a 50 mM solution. The nonpolar analytes were prepared at a concentration of 100 mM, while the polar analytes were prepared at a concentration of 0.1 mM. The polymer standards, both polar and nonpolar, were prepared at concentrations of ~ 4.5 – 5.0 mM. Typically, 500 µL of nonpolar matrix solution was combined with 1 or 2 µL of analyte, except for 9- cyanoathracene where only 200 µL of matrix was used. A 1 µL aliquot mixture was then spotted on the MALDI target and allowed to air dry.

43 3.2.3 MALDI Analysis

Measurements were performed using a single-stage MALDI-TOF mass

spectrometer (Bruker Daltonics Reflex IV, Billerica, MA) in the linear mode. Pulsed

ion extraction was limited by software to delay times of 200, 400 or 600 ns. The

distance between the sample plate and grid is 0.35 cm. The flight tube length is

1.4965 m, which is the length between the extraction grid and ion detector.

Figure 3.1 Schematic diagram of the linear TOF mass analyzer setup to measure the relative initial ion veloicities.

3.3 Results and Discussion

3.3.1 Measurement of the Initial Ion Velocity

Because these experiments were conducted using a single-stage acceleration source, penetration of the acceleration field during the pulsed ion extraction delay could lead to significant increases in the initial ion velocities of lower mass ions

(Gluckmann 1999). To evaluate this effect, sodium and potassium salts were analyzed by LDI-MS. The plot of delay time (τ) versus flight time (t) for Na+ is

44 shown in Figure 3.2. As the flight time is not independent of delay time, field penetration is assumed. From the values in Figure 3.2, a residual field of ~70 V/cm is present during pulsed ion extraction. This field can have a significant effect on low mass (150 to 400 Da) ions such as those commonly ionized by charge-transfer ionization. Therefore two approaches were investigated to differentiate the behavior of nonpolar and polar matrices.

4160 y = 0.1974x + 4014.5 4140 2 4120 R = 0.9357 4100 4080

Ion Flight Time (ns) 4060 4040 100 200 300 400 500 600 700 Delay Time (ns)

Figure 3.2 Plot of delay time (τ) vs. flight time (t) for Na+ directly desorbed off MALDI sample target. The non-zero slope denotes field penetration during pulsed ion extraction.

Approach #1 – Numerical Fitting of the Data

The first approach involved generating the best fit between the experimentally

measured flight times and the time-of-flight equation, Equation 3.1.

− = (v1 v0 ) × m  + L ttotal (3.1)  E1 z v1

45 where v1 is the velocity of the ions leaving the acceleration region, v0 is the initial ion

velocity, E1 is the field strength in the acceleration region during pulsed-ion

extraction and L is the field-free drift length. The reduction in accelerating energy

due to the initial drift of the ions during pulsed-ion extraction was accounted for as

before (Gluckmann 1999).

Approach #2 – Extrapolating From High Mass Ions

The second approach involved calculating initial ion velocities for only

higher-mass analyte ions, and then extrapolating this data to a steady-state, initial ion

velocity for the matrix. Because field-penetration effects are most significant for

lower mass ions, we reasoned that by performing these measurements with higher

mass analytes, the linearity of Equation 3.2 would still be applicable for relative

comparisons of initial ion velocities (Juhasz 1997).

−1 v ≈ t × dt  (3.2) 0  β dτ 

where,

h β = (3.3) 2 d 1 L e where β is a coefficient. d1 is the length between the target plate and extraction grid. h is defined by

2d = − − 1 h (1 G)L 1 (3.4) (1− G) 2

while Le is defined

2d = + 1 Le L 1 (3.5) (1− G) 2

46 When these values are obtained and placed back into equation 3.3, the following β values are determined for the specified geometry (G-Value) in table 3.1.

Table 3.1 Values of β from eq. 3.3 for the specified geometry.

G-Value β (cm-1)

80 0.268

85 0.1947

88 0.150119

89 0.13509

90 0.12

91 0.10498

92 0.08918

94 0.0614

3.3.2 Initial Ion Velocities of Polar Matrices with Polar Analytes (Proton-

Transfer Ionization)

The matrices used in this study can be found in chapter 1 (figure 1.4). Prior experimental determination of the initial ion velocities of DHB using two-stage

MALDI-TOF instruments yielded 543 ± 40 m/s under no field penetration conditions

(Karas 2003) and 658 ± 27 m/s under minimal field penetration conditions (Juhasz

1997). Using the value of Juhasz as a lower limit, a numerical fit of delay time vs. flight time data for insulin desorbed from DHB was done. A best fit value of 992 ±

81 m/s for two grid voltages was obtained. As the value obtained here for DHB with a single-stage system is significantly higher than those obtained from two-stage

47 systems indicating substantial field effects during pulsed ion extraction for even higher mass ions, all further evaluations were conducted by Approach #2 outlined above. Further, only relative comparisons between the various matrices are possible from these data.

Velocity (m/s) DHB 3000

2000

1000

0 0 3000 6000 9000 12000 m/z

Figure 3.3 Initial ion velocities for DHB and polar analytes. The field- independent velocity is extrapolated from the high-mass velocity data (dashed line).

3.3.3 Initial Ion Velocities of Nonpolar Matrices with Nonpolar Analytes

Accurate determination of initial ion velocities of the nonpolar matrices anthracene and 9-cyanoanthracene was significantly hindered by the presence of the accelerating field during pulsed-ion extraction. Common model analytes known to undergo charge-transfer ionization (e.g., ferrocene, triphenylphosphine, thianthrene) all have molecular weights less than 500 Da and so would gain a substantial velocity during the delay period due to the penetrating field. Measurements of initial ion velocity for these matrices, therefore, were limited to higher molecular weight analytes such as polystyrene (PS), polybutadiene (PBD), and polyethylene glycol

48 (PEG) (Figure 3.4) which are compatible with these matrices although these analytes are not ionized by a charge-transfer mechanism.

Figure 3.4 Chemical structures of the synthetic polymers investigated in this study.

3.3.4 G-Value Determination

One of the most important factors that need to be established for accurate measurement of the initial ion velocity is the difference between the voltages of the sample plate and the extraction plate, the G-value. As seen in previous experiments done by Juhasz (Juhasz 1997) and Karas (Karas 2003), this G-value is instrument dependent with an optimal G-value yielding better mass resolution and lower initial ion velocities. A graph of the G-value to the measured initial ion velocities is plotted in Figure 3.5 and was found to be optimal at 91%.

49 1110 1090 1070 1050 1030 1010 990 970 950 Relative Initial Ion Velocities (m/s) Velocities Ion Initial Relative 78 80 82 84 86 88 90 92 94 96 G-Values

Figure 3.5 Initial ion velocities measured at various G-values. The optimal G- value for this mass spectrometer was determined to be 91%

A pulsed ion extraction MALDI mass spectrum of insulin bovine was acquired using

2,5-dihydroxybenzoic acid as the matrix (Figure 3.6). The mass spectrum recorded in

Figure 3.6a yielded a resolution of 62 at a G-value of 94%. The mass spectrum recorded in Figure 3.6b, obtained at the optimum G-value, yielded a resolution of

649. Thus the optimum G-value results in the lowest initial ion velocity as well as the highest resolution. Because these studies were performed in a system where some field penetration is present, the 91% G-value represents the optimal setting for essentially as field free as our instrument can obtain.

50

Figure 3.6 Pulsed ion extraction linear mode MALDI mass spectrum of Insulin. Matrix: DHB, wavelength 337nm. Determination of G-Values was done by varying the ion extraction voltage (IS2) to the total ion voltage (IS1). Resolution determined to be (a) 62 and (b) 649.

3.3.5 Correlation of Nonpolar Matrix and Initial Ion Velocity for Aromatic

Nonpolar Polymer, Polar Polymer, and Linear Chain Nonpolar Polymer

PS 5000 (a synthetic nonpolar aromatic polymer), PEG 4600 (a synthetic polar polymer) and PBD 5000 (a synthetic linear chain nonpolar polymer) were analyzed using a variety of nonpolar matrices.

51

Figure 3.7 Linear – positive ion mode MALDI mass spectra of synthetic nonpolar polymer, Polystyrene 5000 (spectra on left) and polar polymer, PEG 4600 (spectrum on right) analyzed in four different nonpolar matrices anthracene (a,b), pyrene, (c,d), acenapthene (e,f) and 9-cyanoanthracene (g,h). All matrices and polymers were dissolved in THF.

52

Figure 3.8 Linear – positive ion mode MALDI mass spectra of PBD 5000 analyzed in two nonpolar matrices in (a) 9-cyanoanthracene and (b) anthracene. All matrices and polymers were dissolved in THF.

In the analysis of biomolecules with polar matrices, a high initial ion velocity is said to correlate with better mass resolution and little to no fragmentation of the analyte ions, while a low initial ion velocity is said to correlate with poorer mass resolution and increased fragmentation (Karas 2003). Although we are analyzing synthetic polymers, the same type of correlation is expected.

Table 3.2 Relative initial ion velocity of synthetic polymers desorbed from different nonpolar matrices at 337 nm with the calculated mass resolution. (G=91%, U=20 kV) Polymers PS 5000 PEG 4600 PBD 5000 Matrix IIV (m/s) R IIV (m/s) R IIV (m/s) R Anthracene 880 + 35 387 721 + 23 254 851 + 47 393 9- 777 + 89 372 833 + 29 496 888 + 22 350 cyanoanthracene Acenaphthene 805 + 27 402 740 + 52 156 N/A N/A Pyrene 808 + 45 521 679 + 15 373 N/A N/A

53 As seen in Table 3.2, higher resolution is consistently found to be correlated with higher initial ion velocity. For anthracene, PS 5000 yielded the highest initial ion velocity while PEG 4600 yielded the lowest. In the case of the 9-cyanoanthracene matrix, PBD 5000 was found to have yielded the highest and PS 5000 the lowest initial ion velocity. The relative initial ion velocities for pyrene and acenaphthene yielded trends similar to that of anthracene, with higher initial ion velocities found for

PS 5000 and the lowest values with PEG 4600.

The data presented in Table 3.2 and Figures 3.7 and 3.8 support our original hypothesis: the initial ion velocity for nonpolar analytes is higher than the initial ion velocity for polar analytes as measured for any particular nonpolar matrix.

Anthracene is a nonpolar aromatic matrix with no exocyclic functional groups. PS and PBD yielded statistically significant higher initial ion velocities than PEG when analyzed with this matrix. These higher initial ion velocities likely result because both the matrix and analytes are nonpolar, with PS yielding the highest velocity due to its aromaticity. Similarly, PS yielded higher initial ion velocities than PEG when acenaphthene and pyrene were used as matrices.

When 9-cyanoanthracene was used as the matrix, PBD yielded the highest initial ion velocity while the velocities for PS and PEG were found to be similar within the precision of measurement. Although I have classified 9-cyanoanthracene as a nonpolar matrix, it is very possible that the cyano functional group is the reason why this matrix promotes a higher initial ion velocity for PEG. It also seems likely that the double bond character of PBD promotes its interaction with both anthracene

54 based matrices. It remains unclear why PBD could not be analyzed with pyrene or acenaphthene.

3.3.6 Correlation of Polar Matrix and Initial Ion Velocity for Aromatic

Nonpolar Polymer, Polar Polymer, and Linear Chain Nonpolar Polymer

PS 5000 (a synthetic nonpolar aromatic polymer), PEG 4600 (a synthetic polar polymer) and PBD 5000 (a synthetic linear chain nonpolar polymer) were analyzed with a few polar matrices.

Figure 3.9 Linear – positive ion mode MALDI mass spectrum of PBD 5000 analyzed retinoic acid. Both matrix and polymer was dissolved in THF.

Again, because of what is already known about polar matrices and how initial ion velocities vary depending on the class of analyte, a prediction similar to what is already known of the polar matrices was made. We predict that a high initial ion velocity would be observed when a polar analyte is admixed with a polar matrix, while a low initial ion velocity would be observed when a nonpolar polymer is admixed with a polar matrix.

55 Table 3.3 Relative initial ion velocity of synthetic polymers desorbed from three different polar matrices at 337 nm with the calculated mass resolution, R. (G=91%, U=20 kV) Polymers PS 5000 PEG 4600 PBD 5000 Matrix IIV (m/s) R IIV (m/s) R IIV (m/s) R DHB 924 + 90 445 1006 + 75 494 N/A N/A RA 858 + 48 365 710 + 15 476 689 + 35 484 SA 700 + 60 468 697 + 52 285 N/A N/A

As can be seen in Table 3.3, higher resolution is not consistently found to correlate

with a higher initial ion velocity, except for DHB. For DHB, PEG 4600 yielded the

highest initial ion velocity, while PS 5000 yielded the lowest. However, in the case

of the retinoic acid matrix, PS 5000 yielded the highest and PBD 5000 yielded the

lowest. The relative initial ion velocity for sinapinic acid yielded trends similar to

retinoic acid, with higher initial ion velocities for PS 5000 and lower initial ion

velocities for PEG 4600, however, it did not produce any ion signal for PBD 5000.

The data presented in Table 3.3 and Figures 3.9 and 3.10 does not support my

original prediction: the initial ion velocity for polar analytes is higher than the initial

ion velocity for non polar analytes, as measured for any of the investigated polar

matrices, DHB. DHB was the only matrix that followed my prediction that the polar

analyte will have a higher initial ion velocity than the nonpolar analyte. DHB is a

polar matrix with a few exocyclic functional groups. PEG yielded a higher initial ion

velocity than PS when analyzed with this matrix. These initial ion velocities are

likely the result of both the matrix and analyte having hydroxyl (OH) functional

groups.

56

Figure 3.10 Linear – positive ion mode MALDI mass spectra of synthetic nonpolar polymer, Polystyrene 5000 (spectra on left) and polar polymer, PEG 4600 (spectrum on right) analyzed in three different polar matrices DHB (a,b), RTA (c,d) and SA (e,f). All matrices and polymers were dissolved in THF.

It is has been reported that a high initial ion velocity correlates with less fragmentation and better resolution. However, this was the opposite when retinoic acid was applied as the matrix, where the second highest resolution among the polar matrices was observed with the lowest initial ion velocity when analyzing PBD, a linear nonpolar polymer. Retinoic acid, a polar matrix, possesses an exocyclic linear

57 chain having double bond character throughout the linear chain. It is probably due to both matrix and polymer possessing double bond character that they interact or mix well with one another, which is represented by the high resolution in this analysis.

However, it remains unclear why PBD could not be analyzed by DHB or sinapinic acid.

3.4 Conclusion

The initial ion velocity of nonpolar matrices has been investigated with polar and nonpolar analytes. Our initial investigations were aimed at measuring the initial ion velocities of a variety of nonpolar matrices in the analysis of low molecular weight compounds. However, because the instrument was not essentially field free; the low molecular weight analytes experienced an increase in velocity during the delay period due to a retarding field. This field penetration would be expected to be significant for all analytes below molecular weight 3000 Da. Because of this, we turned our attention to synthetic polar and nonpolar polymers whose masses were beyond that which would experience field penetration effects.

It was found that the initial ion velocity of nonpolar analytes would be higher than the initial ion velocity of polar analytes when measured for any particular nonpolar matrix. Because the nonpolar matrices as well as the nonpolar polymer have an aromatic ring structure, it is believed that there is a better interaction between the matrix and analyte which results in such high initial ion velocities. It is also believed that within the same class of measurements of nonpolar matrix and nonpolar analyte that a matrix possessing exocyclic functional groups may lower the initial ion velocity of the matrix, which can be seen in the calculation of the resolution for the

58 particular combination. However, in order to confirm such a statement additional experiments with nonpolar matrices possessing exocyclic functional groups need to be performed.

59 CHAPTER FOUR: The Use of Nonpolar Matrices for Matrix-Assisted Laser

Desorption/Ionization Mass Spectrometric Analysis of High Boiling Crude Oil

Fractions

4.1 Introduction

Although high-boiling crude oil products are less amenable to mass spectrometric methods in general (Qian 2001), a variety of different crude oil related materials have been analyzed by MALDI-MS. Herod and coworkers have used

MALDI- or LDI-MS for molecular mass determinations of coal-tar pitches (Domin

1997; Lazaro 1997; Zhang 1997; Lazaro, Domin et al. 1999; Lazaro 2001; Menendez

2001), coal-derived liquids (Domin 1997; Deelchand 1999; Lazaro, Herod et al. 1999;

Herod 2000), kerogen extracts (Herod 1997), petroleum residues (Suelves 2001), bitumens (Domin 1999) and other petroleum samples (Zhang 1996; Pindoria 1997;

Johnson 1998; Johnson 1998; Johnson 1999; Menendez, Blanco et al. 2002).

In addition to those prior studies that used MALDI-MS for the characterization of crude oil related materials, Herod and coworkers have studied sample preparation and instrumental conditions appropriate for MALDI-MS of crude oil related materials (Domin 1997; Domin 1997; Herod 1997; Lazaro 1997). Domin found that increasing the extraction voltages increases the intensity of high mass ions

(Domin 1997; Domin 1997). Lazaro found that examining coal-derived liquids at low laser power revealed more structural information in the low mass range than when using a higher laser power (Lazaro 1997). Herod examined kerogen extracts in the absence and presence of a matrix. It was found that older kerogen extracts act as a

60 self-matrix while the younger kerogen extracts worked well with polar matrices

(Herod 1997).

MALDI- or LDI-MS has been used in combination with other analytical techniques (such as vapor phase osmometry, VPO, and gel permeation chromatography, GPC) to characterize the structural changes or reactivities of asphaltenes and resins (Miller 1998; Artok 1999; Seki 2000; Bartholdy 2001).

Typically, MALDI- and LDI-MS have been used to compare or confirm the molecular mass distributions found using VPO and/or GPC. Potential advantages of

LDI and MALDI-MS for such measurements include their minimal dependence on the solvent, temperature, or concentration, which all can be limiting factors when methods such as VPO and GPC are employed (Miller 1998). LDI-MS of asphaltenes typically yields molecular mass ranges from ~100 to 10 000 Da (Miller 1998; Seki

2000; Bartholdy 2001). A separate MALDI-MS study of asphaltenes yielded a much lower molecular mass, in the range ~200 to 3000 Da (Artok 1999). For those prior studies that used MALDI-MS, the most common and effective matrices employed were polar matrices such as sinapinic acid, a-cyano-3-hydroxycinnamic acid, 2,5- dihydroxybenzoic acid, 2-(4-hydroxyphenylazo)-benzoic acid, and 9- anthracenecarboxylic acid (Domin 1997; Herod 1997).

Previously, our group has investigated the use of nonpolar matrices

(McCarley 1998; Macha 1999) for MALDI-MS analysis of nonpolar analytes including hydrocarbon compounds (Macha 2000; Macha, Limbach et al. 2001). A characteristic of most nonpolar matrices is their minimal background at low mass

(Macha 2000). Because nonpolar matrices yield only a single radical molecular

61 cation, they are more compatible with lower molecular weight analytes due to the lack of interfering matrix-related ions in the mass spectrum.

Another unique property of nonpolar matrices is their ability to generate radical molecular cations of nonpolar analytes when the ionization potential of the matrix is greater than that of the analyte (Macha 1999; Knochenmuss 2000). Some typical compounds that can be found within a high boiling point crude oil fraction include poly- and mononuclear aromatics, and poly- and monocycloparaffins (Speight

1999).

Here I examine the use of nonpolar matrices for the characterization of an atmospheric resid, high boiling crude oil fraction. Previous electrospray ionization mass spectrometric characterization of similar resids yielded molecular mass distributions in the range 200–1000 Da (Qian 2001; Hughey 2002). Thus, one interest was to determine whether nonpolar matrices, which should yield a low background signal in the mass range of interest, offer any advantages over polar matrices for the MALDI-MS analysis of this sample type. As a point of comparison, matrices traditionally used for the analysis of hydrocarbon polymers (e.g., polystyrene) are also investigated. Although many analyte components (e.g., monocycloparaffins) found in a high boiling point crude oil fraction have ionization energies that are higher than typical nonpolar MALDI matrices (Speight 1999), another goal here was to determine whether these electron-transfer matrices introduce any discrimination effects into the MALDI-MS analysis of atmospheric resids. As part of these studies, the influence of mass spectrometer operational mode, matrix and sample preparation on the resulting mass spectral data will be presented.

62 4.2 Experimental

4.2.1 Materials

Anthracene, 9-cyanoanthracene (9-CA), buckministerfullerene (C60), and 3- indoleacrylic acid (3-IAA) were obtained from Aldrich Chemical (Milwaukee, WI,

USA) and used without further purification. The 650+ oF atmospheric resid crude oil fraction (bottom total liquid product—BTLP) was a gift from Exxon-Mobil

Corporation. HPLC-grade tetrahydrofuran (THF), methylene chloride

(dichloromethane), pyridine, acetonitrile, and benzene were obtained from Fischer

Scientific (Fairlawn, NJ, USA) and used as received.

4.2.2 Methods

4.2.2.1 MALDI Analysis

All MALDI experiments were performed using a Bruker Daltonics Reflex IV

(Billerica, MA, USA) reflectron time-of-flight (TOF) mass spectrometer equipped with pulsed ion extraction. A nitrogen laser (wavelength of 337 nm) operated at a laser power between 20 and 30% was used. Anthracene (m/z 178) and C60 (m/z 720) were used for external calibration. On average, 100 laser shots were taken to obtain each mass spectrum.

All matrices and calibrants used in the analysis were prepared to a concentration of 100 mM in each of the solvents investigated. The BTLP crude oil fraction was prepared by dissolving 0.075 g of the sample in approximately 1mL of the solvents used in the analysis. Next, the matrix solution was thoroughly mixed

63 with the BTLP crude oil fraction and typically 1 mL of this mixture was spotted on the sample plate and allowed to air dry before insertion into the sample chamber.

4.2.2.2 FI Analysis

Field ionization (FI) experiments were performed using a Waters GCT—TOF mass spectrometer with an FI emitter (Carbotec GmbH, Germany) under the following instrumental parameters. An emitter current of 8 mA, flash-off current of 8 mA, heater current of 1000 mA and an extraction voltage of 12 000 V were used for all analyses. Approximately 3 mL of sample were evaporated to dryness and placed onto the direct sample insertion probe. The temperature gradient for the analysis was

100 oC/min from 35 oC to 300 oC. Instrument calibration was done using heptacosabromotributylamine, methyltriazine, perfluorotrimethylcyclohexane, hexafluorobenzene, pentafluorobenzene, acetone, chloropentafluorobenzene and xylene.

4.3 Results and Discussion

4.3.1 Candidate MALDI Matrices

As the interest of this work was to investigate the use of MALDI- MS for crude oil fractions whose molecular weight distributions were in the range ~200 to

800 Da, it was necessary to characterize the behavior of the candidate MALDI matrices alone to ensure that the use of a matrix would not complicate the resulting mass spectral results. Matrices were chosen based on their solubility in common organic solvents (e.g., dichloromethane, acetonitrile, methanol), molar absorptivities at 337 nm, and their prior use in the analysis of hydrocarbon materials (Belu 1996;

64 Danis, Karr et al. 1996; Macha 2000; He, He et al. 2001; Macha, Limbach et al. 2001;

McCarley 2001).

Figure 4.1 Laser desorption/ionization mass spectra of neat solutions of (a) trans-3-indoleacrylic acid (3-IAA), (b) 9-cyanoanthracene (9-CA) and (c) anthracene in THF. 3-IAA yields many fragment and cluster ions in addition to the molecular ion at m/z 187. In contrast, 9-CA and anthracene yields only a single radical molecular cation upon laser desorption.

Figure 4.1 contains representative mass spectral results obtained from the laser desorption analysis of these compounds. In Fig. 4.1(a), laser desorption of 3-

IAA produces many fragment ions and cluster peaks that could interfere with MALDI analysis of the lower molecular weight crude oil fractions. In contrast, nonpolar matrices such as 9-cyanoanthracene (Fig. 4.1(b)) and anthracene (Fig. 4.1 (c))

65 produce only a single radical molecular ion that should not interfere with the lower molecular weight crude oil fractions.

4.3.2 Linear vs. reflectron mode MALDI-MS

The effect of the MALDI-TOF operating mode on the analysis of the heavy crude oil fraction was investigated next. Figures 4.2 – 4.4 display representative mass spectra generated from the MALDI-MS analysis of BTLP with various matrices.

Figure 4.2(a) shows the data obtained using 3-IAA, a traditional polar matrix used in the analysis of hydrocarbon polymers (Nielen 1999), in linear mode, and Figure

4.2(b) the data obtained from the same matrix in reflectron mode. Data obtained using

3-IAA in the linear mode is

Figure 4.2 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of trans-3-indoleacrylic acid (matrix) in linear (a) and reflectron mode (b).

66 characterized by a broad molecular mass distribution extending from about 200–3000

Da. Although a few features (which are due to the matrix) are superimposed on this broad distribution, this data is similar to that found by others when using polar matrices for crude oil related materials (Domin 1997; Herod 1997). In contrast, the data obtained in reflectron mode are much less informative. Many of the ions detected are due to the matrix and no clear molecular mass distribution that can be attributed to the BTLP sample is detected.

Figures 4.3 and 4.4 were acquired with the nonpolar matrices anthracene

(Figs. 4.3 (a) and 4.3 (b)) and 9-cyanoanthracene (Figs. 4.4 (a) and 4.4 (b)). In linear mode, both nonpolar matrices yield molecular mass distributions similar to that found with 3-IAA, however, without the complicating additional features due to the matrix.

9-cyanoanthracene yields a reproducibly more abundant distribution with signal above the baseline up to m/z 4000 like 3-IAA. In addition, the radical molecular ion for anthracene (178.2 Da) is the dominant feature in linear mode while a minimal matrix signal is detected for 9-cyanoanthracene.

Figure 4.3 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of anthracene (matrix) in linear (a) and reflectron mode (b).

67 In contrast to the data obtained using 3-IAA as the matrix, both anthracene and 9-cyanoanthracene yield mass spectra exhibiting broad molecular weight distributions in the range 200–600 Da in the reflectron mode. It was also found that 9- cyanoanthracene yielded additional ions above m/z 600 that were not detected when anthracene was used as the matrix. However, such signals are only detected in reflectron mode under particular sample preparation conditions. The difference in mass ranges found between the linear and reflectron mode data for any of the matrices investigated here may be complicated by the presence of fast neutrals that are desorbed and register at the linear detector. The Bruker instrument used in these studies does not allow for the deflection of fast neutrals. However, the use of delayed extraction as in this study should reduce the abundance of fast neutrals that reach the detector coincident with accelerated ions.

Figure 4.4 MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of 9-cyanoanthracene (matrix) in linear (a) and reflectron mode (b)

68 4.3.3 Solvent effects

In addition to the influence of the matrix on the mass spectral data obtained in these investigations, it is well known that the solvent can also play an important role in the MALDI process. The dissolution of the matrix and analyte in a common solvent is important to the success of a MALDI-MS analysis (Williams 1996). In addition, due to the heterogeneity of the BTLP sample, it is expected that the solvent polarity might also influence the resulting mass spectra (Yalcin, Dai et al. 1998;

Momcilovic, Wittgren et al. 2003).

Figure 4.5 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. THF was used as a common solvent.

MALDI-MS analysis of BTLP was done while varying the solvent used in sample and matrix preparation. Figures 4.5 – 4.7 summarize these results for a variety of solvents: tetrahydrofuran (Figs. 4.5(a) and (b)), pyridine (Figs. 4.6(a) and (b)), and

69 50% aqueous acetonitrile (Figs. 4.7(a) and (b)) presented in increasing order of solvent polarity. For the polar matrix 3-IAA, a dramatic difference in mass spectral results for each of the solvents was found. The least polar solvent, THF, yielded no discernable molecular mass distribution for the BTLP sample. Only the use of aqueous acetonitrile (Fig. 4.8 (a)) yielded a detectable molecular mass distribution.

However, the analyte signal is minor compared with the ions due to the matrix, and the distribution is only detectable around 300 Da.

The solvent used during sample and matrix preparation had much less of an effect on the mass spectral results when 9-cyanoanthracene was used. In general, a molecular mass distribution from 200–700 Da is detected, although the use of the most polar solvent, aqueous acetonitrile resulted in the narrowest molecular mass distribution.

Figure 4.6 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. Pyridine was used as a common solvent.

70

Figure 4.7 Reflectron mode MALDI mass spectra of 0.075 g/mL BTLP crude oil fraction with 100 mM of matrix ((a) 3-IAA and (b) 9-cyanoanthacene). 200 µL of matrix was mixed with 2 µL of crude oil fraction. 50% aqueous acetonitrile was used as a common solvent.

Figure 4.8 (a) LDI mass spectrum of BTLP in THF. (b) FI mass spectrum of BTLP in THF. Data were obtained in reflectron ion mode.

71 4.3.4 Comparison with LDI- and FI-MS

To determine the extent to which the added matrix influences the resulting mass spectral results, the BTLP sample was analyzed as a neat solution by LDI-MS

(Fig. 4.8(a)) and by FI-MS Fig. 4.8(b)). As seen in Fig. 4.8, this particular crude oil fraction yields a molecular weight distribution in the range 200–600 Da with either of these ionization methods.

Figure 4.9 LDI mass spectrum of BTLP in DCM (a), methanol (b), ACN (c), and 50% ACN/H20 (d). Data was obtained in the reflectron mode.

72 As can be seen in figures 4.9 analyzed by LDI and figures 4.10 analyzed by FI, different solvents used in the analysis had an effect on the mass spectra.

Figure 4.10 FI mass spectrum of BTLP in DCM (a), methanol (b), ACN (c), and 50% ACN/H20 (d).

For the LDI experiment, the nitrogen laser used in this study can allow for the direct photoionization of the sample for components whose ionization energies are less than

73 7.36 eV (2-photon ionization) or any highly absorbing lower molecular mass components of BTLP could serve as a ‘self-matrix’ for the subsequent desorption/ionization of other components (Domin 1997). For the FI experiment, typically those components whose ionization energies are less than 11 eV can be ionized if they can be vaporized into the gas phase. Because FI-MS requires analyte thermal vaporization for detection, the mass range for the samples investigated in this study are in the range expected for atmospheric resids. An interest in this work is whether the use of a nonpolar MALDI matrix would increase the mass range of analytes detectable by MALDI-MS.

4.3.5 Evaluation of nonpolar MALDI-MS

By comparison of the data in Figures 4.1 – 4.8 it is apparent that the choice of matrix and TOF configuration influence the mass spectral results obtained from the

BTLP sample. Taken together, these data demonstrate that a polar matrix, such as 3-

IAA, while appropriate for hydrocarbon polymers or much higher molecular weight crude oil components, is less effective at generating representative ions than the investigated nonpolar matrices. In addition, 3-IAA is more sensitive to sample preparation conditions with an aqueous solvent required to yield the most significant molecular mass distribution. Additional cationization salts (e.g., Ag+ or Cu2+) are typically used with polar matrices like 3-IAA during the MALDI-MS analysis of hydrocarbon polymers (Nielen 1999; Macha 2002). Because the objective of this work was to directly compare the effects of different classes of matrices on the mass spectral results, no such cationization salts were used in this investigation.

74 In contrast to 3-IAA, nonpolar matrices such as anthracene or 9- cyanoanthracene are effective at generating significant and reproducible molecular mass distributions from the BTLP sample. It was found that ion abundances from 9- cyanoanthracene are consistently higher than those from anthracene, which improves the ability to use reflectron mode TOF-MS for these analyses. Further, both 9- cyanoanthracene and anthracene are relatively insensitive to sample preparation conditions although aqueous solvent systems do result in a smaller molecular mass distribution for either matrix. Given the consistency of molecular mass distributions found using nonpolar MALDI-, LDI- and FI-MS and prior studies on atmospheric resids similar to the BTLP sample studied here (Boduszynski 1987; Boduszynski

1988; Qian 2001; Hughey 2002), it does appear that nonpolar MALDI-MS, at a minimum, does not adversely skew the detected molecular mass distribution.

Although it is clear from the data that the use of polar matrices, such as 3-

IAA, are ineffective for the characterization of BTLP samples, it is less obvious that the addition of a nonpolar matrix has any effect on the resulting mass spectral data.

For example, a comparison of the mass spectral results found using 9- cyanoanthracene prepared in various solvents (Fig. 4.11) appear to be similar to the

LDI results found with THF as the solvent (Fig. 4.8 (a)) or other solvents. Assuming that the average molecular mass of the BTLP sample is 400 g/mol, the data obtained in Fig. 4.8 (a) would be at a matrix-to-analyte mole ratio of ~50:1.

75

Figure 4.11 Reflecton mode MALDI-TOF mass spectra of BTLP with 9- cyanoanthracene as the matrix with both matrix and crude oil fraction dissolved in like solvents, (a) DCM, (b) methanol, (c) ACN, and (d) 50% ACN. For all spectra, 1000 µL of matrix were mixed with 2 µL of the crude oil fraction resulting in an approximate mole ratio of 250:1 (matrix:analyte). Even at higher matrix/analyte mole ratios, the molecular mass distribution and mass spectral features for the BTLP sample do not change.

Although it has been shown that nonpolar matrices (e.g., anthracene) are

involved in both the desorption and ionization steps in MALDI-MS (McCarley 1998;

Macha 1999), those studies were done using analytes having minimal molar

absorptivities at 337 nm. It could be possible that, even at the mole ratios used here,

components of the sample could be absorbing a significant fraction of the laser

76 irradiation, leading to the similar appearance between the MALDI- and LDI-MS results.

To investigate this possibility, studies were conducted at higher matrix/analyte mole ratios. For example, the data shown in Fig. 4.12 were obtained at ~250:1 matrix/analyte mole ratios for anthracene (Fig. 4.12 (a)) and 9-cyanoanthracene (Fig.

4.12 (b)). Results obtained at this mole ratio are similar to those found at lower mole ratios (e.g., Figs. 4.3 (a) and 4.4 (a)). Thus, it appears unlikely that, for the nonpolar

MALDI-MS analyses, the analyte is serving as a ‘self-matrix’.

Figure 4.12 Linear mode MALDI-TOF mass spectra of BTLP with (a) anthracene and (b) 9-cyanoanthracene as the matrix. For both spectra, 1000 µL of matrix were mixed with 2 µL of the crude oil fraction resulting in an approximate mole ratio of 250:1 (matrix:analyte). Even at higher matrix/analyte mole ratios, the molecular mass distribution and mass spectral features for the BTLP sample do not change.

In addition to the lack of interfering fragment and cluster ions, another unique property of nonpolar matrices is their ability to undergo electron-transfer reactions with analytes (McCarley 1998; Macha 1999). Even operating in reflectron mode,

77 only unit mass resolution of components in the BTLP sample could be obtained.

Thus, it is not possible to identify the components by their elemental composition and assign compound classes or types to confirm the generation of radical molecular cations solely by electron-transfer reactions with the matrix. However, it would be interesting to pursue such identifications using a higher resolution mass spectrometer, such as the latest high-field Fourier transform ion cyclotron resonance mass spectrometers (Qian 2001).

4.4 Evaluation of BTLP Crude Oil Fraction on PEEK Target

In chapter two, it was found that using a stainless steel metal target can cause the formation of photoelectrons leading to the reduction in the ion intensity of the analyte. Therefore it is of interest here to take the BTLP crude oil fraction and analyze it on a microfabricated polymer target. LDI – MS analysis of the BTLP crude oil fraction on a PEEK target yielded no ions (Figure 4.13). This result was opposite to that observed from the stainless steel target.

Figure 4.13 Reflectron mode MALDI-TOF mass spectra of BTLP on PEEK Polymer Target.

78

Figure 4.14 Reflectron mode MALDI TOF-MS mass spectra of BTLP with (a) anthracene and (b) 9-cyanoanthracene as the matrix on PEEK polymer target.

The analysis of the BTLP crude oil fraction incorporated with the nonpolar matrices, anthracene and 9-cyanoanthracene, on the PEEK polymer did not produce any peaks that were not observed on the stainless steel target (figure 4.14). However, the matrix and analyte ion abundances are greater from the PEEK target than the stainless steel.

This is evident as the most abundant peaks, the matrix ions, in figures 4.3 (b) and 4.4

(b) are less than 3000 ion counts. While the ion intensity of the matrix ion peaks on the PEEK target is approximately 10 000 ion counts. Peaks that are labeled in the inset in Figure 4.14 (b) are cluster and dimer peaks from the matrix.

79 4.5 Conclusions

The use of MALDI-MS, with nonpolar matrices in particular, has been demonstrated for the analysis of bottom total liquid products (BTLP) resulting from crude oil distillation. The nonpolar matrices investigated in this work, anthracene and

9-cyanoanthracene, are suitable for either linear or reflectron mode TOF-MS. These matrices are relatively insensitive to sample preparation conditions and yield molecular mass distributions ranging from 200–4000 Da (linear mode) and 200–600

Da (reflectron mode). Because nonpolar matrices do not fragment or cluster, they do not interfere with the analysis of the lower molecular components of the BTLP sample. Results obtained using nonpolar matrices and MALDI – MS compare favorably with those obtained by FI – MS or LDI – MS.

80 CHAPTER FIVE: CONCLUSIONS AND FUTURE DIRECTIONS

5.1 Summary

The overall objective of this dissertation is to gain an improved understanding of the behavior and applicability of nonpolar matrices in MALDI – MS. The use of these types of matrices would be very beneficial to the analysis of low molecular weight compounds because these nonpolar matrices form radical molecular cations, with no fragment or cluster peaks, making spectral interpretation effortless. Polar matrices, commonly used in MALDI have exocyclic functional groups which fragment and form clusters in the low molecular weight range making spectral interpretation more difficult.

Chapter 2 describes studies to determine the effect that conventional stainless steel and polymer MALDI sample target substrates have on nonpolar matrices used for charge transfer ionization analyses. Polymer sheets were milled to the size and shape of Bruker Daltonic’s microprobe plate (stainless steel 10 – spot round) MALDI substrate. LDI experiments were performed which demonstrated no background interference from the polymer targets. Charge – transfer ionization experiments were done using the stainless steel and polymer substrates and the analyte ion abundance was monitored. The analyte ion abundance was found to be highest when analyzed on the polymer substrates. A reasonable explanation for these results is that fewer photoelectrons are produced from the polymer substrate which could neutralize the charged species in the MALDI plume. These results also establish that the use of a polymer substrate simplifies the identification of the analyte ion of interest in the analysis.

81 In Chapter 3, I measured the initial ion velocity to examine the desorption process for nonpolar matrices in MALDI. The initial ion velocities of nonpolar matrices with nonpolar analytes were compared to the initial ion velocities of nonpolar matrices with polar analytes. Initially, nonpolar matrices were admixed with low molecular weight nonpolar analytes to determine their initial ion velocity.

However, because of the retarding extraction field that is experienced on our instruments, low molecular weight nonpolar analytes would gain an increase in internal kinetic energy leading to erroneous initial ion velocities for compounds up to

3000 Da. Therefore, synthetic polymers with a molecular weight average of ~ 5000

Da were investigated. High initial ion velocities were found for nonpolar matrices with nonpolar analytes, while low initial ion velocities were found for nonpolar matrices with polar analytes. Higher resolution was obtained with nonpolar matrices admixed with nonpolar analytes, while lower resolution was found for nonpolar matrices admixed with polar analytes.

In Chapter 4, I demonstrated the use of nonpolar matrices for the analysis of an atmospheric resid, high boiling crude oil fraction. The nonpolar matrices that were used in the analysis of a low molecular weight crude oil fraction made for easy spectral interpretation as they exhibited minimal background ions, as compared to polar matrices which exhibit a complicated background in MALDI from matrix fragmentation and clustering. Sample information obtained from the use of these nonpolar matrices in MALDI was comparable to the information obtained using conventional ionization techniques such as LDI and FI. The nonpolar matrices and crude oil fraction were further studied on a polymer MALDI sample target substrate.

82 An increase in the ion abundance of components of the crude oil fraction was found, as would be predicted from my results obtained in Chapter 2.

5.2 Future Directions

The application of nonpolar matrices for MALDI has been used for the analysis of low molecular weight analytes (Macha 1999), synthetic polymers (Macha

2000), and an atmospheric resid, high boiling crude oil fraction (Robins 2003). An advantage of a nonpolar matrix compared to a polar matrix is that a nonpolar matrix yields no background ions, which is very favorable when analyzing compounds of low molecular weight (< 1000 Da).

Two classes of analytes that would be worth characterizing using nonpolar matrices would be dendrimers and carbohydrates. A dendrimer is a highly branched polymer with a central core unit and a variable number of monomeric repeat units bearing a functional group at the branching point. Synthetically developed, dendrimers have found widespread application across many branches of chemistry.

Because of their poly – conjugated aromatic features, they would be ideal analytes for analysis with nonpolar matrices, whose chemical structure is also aromatic, suggesting good analyte incorporation into the analyte.

Carbohydrates are biological compounds that are traditionally analyzed using polar matrices; however, they are not ionized by a proton – transfer ionization mechanism as is usually the case for a polar matrix. The addition of an alkali cation is needed to promote ionization. Nevertheless, polar matrices have been used, and because of the ions that result from matrix fragmentation and clustering, there is

83 strong interference and sensitivity is very low (Gouw 2002). The use of nonpolar matrices could minimize matrix – related interferences.

Charge-transfer ionization mechanisms in MALDI are very rare, however, it is very useful and important in the analysis of low molecular weight analytes having low ionization potentials. In Chapter 2, it was found that there is an increase in the radical molecular cation of the analyte ion when there is an increase in the difference in ionization potential of the analyte and matrix. It also would be of interest to investigate the dependence of the analyte cation abundance on the analyte driving force for charge-transfer ionization of the analyte by the matrix cation. Results from these experiments will gauge if the radical molecular cation abundance of the analyte ion in the charge-transfer ionization process is under thermodynamic control.

Time – of – Flight Secondary Ion Mass Spectrometry (TOF – SIMS) is an ion desorption mass spectrometry technique developed specifically to examine the chemical composition of surfaces with high sensitivity (Gardella 1980; Hanton 1999).

In TOF –SIMS a pulsed high energy ion beam is used to probe the surface. The primary ion beam strikes the analyte surface, transferring energy to surface species.

Some of these secondary species are charged and have sufficient energy to escape the surface. These secondary ion beams are subsequently mass analyzed. The most significant advantage of the method is that it has the ability to obtain spatially resolved, chemically sensitive images of the surface. These images are produced by rastering the primary ion beam across the surface, recording the initial position and mass data for each ion.

84 Hanton and co workers used TOF – SIMS to investigate the sample preparation for MALDI analysis of synthetic polymers incorporated into polar matrices (Hanton 1999). Hanton and co workers not only collected molecular mass data, but they also obtained total ion images of the crystalline solid and individual ion images of the matrix, polymer and metal cationization reagent. The analyses showed that TOF-SIMS is a useful and viable tool to investigate the surfaces of MALDI sample preparations.

Based on the results that are seen within Chapter 3, investigating the initial ion velocity of nonpolar matrices, TOF – SIMS seems to be the most sophisticated analytical method to examine the interaction of the nonpolar matrix, polymer, as well as metal salt used in the analysis. Images obtained from TOF – SIMS investigations can show the surface interaction of the polymer and the host matrix crystal. From these images, one can examine how well nonpolar and polar polymers are incorporated into nonpolar matrices as well as nonpolar and polar polymers incorporated into polar matrices. These images can then be used to confirm that nonpolar polymers closely interacting with nonpolar matrices result in a high initial ion velocity, whereas polar polymers closely interacting with nonpolar matrices result in a low initial ion velocity.

Also, different metal cations have been found to work better with different classes of polymers (Montaudo 2002) providing higher resolution and mass accuracy.

As higher resolution correlates with a high initial ion velocity, it would be of interest to see how the identity of the metal cation affects the initial ion velocity measurements for nonpolar matrices.

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