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..............................................................................................