CHROMATOGRAPHIC PROPERTIES OF SILICA-BASED MONOLITHIC HPLC COLUMNS By Jennifer Houston Smith Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Chemistry APPROVED Harold M. McNair, Chairman Mark R. Anderson James O. Glanville Larry T. Taylor Jimmy W. Viers September 2002 Blacksburg, VA Keywords: HPLC, Monolith, Chromatography Copyright 2002, Jennifer H. Smith CHROMATOGRAPHIC PROPERTIES OF SILICA-BASED MONOLITHIC COLUMNS Jennifer Houston Smith ABSTRACT Silica-based monolithic HPLC columns contain a novel chromatographic support in which the traditional particulate packing has been replaced with a single, continuous network (monolith) of porous silica. The main advantage of such a network is decreased backpressure due to macropores (2 µm) throughout the network. This allows high flow rates, and hence fast analyses that are unattainable with traditional particulate columns. The Chromolith SpeedROD (EM Science, Gibbstown NJ) is a commercially available silica-based monolithic column. This work investigated the chromatographic properties of the 50x4.60 mm (ODS) SpeedROD. Data fit to the van Deemter equation (mean square error=0.834) indicated that the van Deemter model was valid for monolithic columns. An effective particle size of 4 µm for the SpeedROD column was assigned by comparing the minimum of van Deemter curves with a series of particulate columns having various particle diameters. Separation Impedance (E), an empirically derived measure of column performance, was calculated as an alternate method of evaluating column efficiency. Data collected using this model confirmed monolithic columns behaves as a (more efficient) 3 µm column. A series of experiments were designed to compare the effects of mobile phase strength and mobile phase viscosity between the SpeedROD column and a particulate column. The results indicated both solvent strength and viscosity have effects on the monolithic column at the optimum linear velocity. A fast (90 s) HPLC method was developed using the SpeedROD column and a seven-component test mixture with a large range of hydrophobicities. The precision for both retention time and peak area was measured at high linear velocities (8 mL/min) and the percent relative standard deviation (RSD) calculated. Column to column reproducibility (n=6) was measured. The overall percent RSD ranged from 0.25% to 4.56% for retention time and from 1.08% to 6.77% for peak area. Run to run reproducibility (n=15) was measured for all six columns. Averages ranged for retention time from 0.89% to 5.09% RSD and for peak area from 4.65% to 6.18% RSD. Applications for the SpeedROD column with various sample types were developed and discussed. These methods demonstrated the effectiveness of the SpeedROD at fast flow rates. ACKNOWLEDGEMENTS Many people are responsible for my completion of this project, far to many to name, but a few of you deserve singling out. First and foremost, many thanks to Dr. Harold McNair for the opportunity to work and learn in a supportive environment where I was given the time and space to develop at my own pace (and allowed to make plenty of mistakes!). You were always understanding of my special needs as both mom and student, and I am deeply appreciative of your thoughtfulness in this matter. Thanks also to my committee members, Dr. Mark Anderson, Dr. Jim Glanville, Dr. Larry Taylor and Dr. Jimmy Viers, many of whom I had the opportunity to have as professors. I also must thank the chemistry department at Emory & Henry College for their constant encouragement and support throughout my stay here at Virginia Tech. Many thanks go to the members, past and present, of the McNair group, but especially to the current members, Amy Kinkennon, Kevin Schug, Kari Urias and Laura Nakovich; my partners in crime and life-long friends. It’s been wonderful working with such a great group of people. Last but not least, a special thanks to my family for their patience and support over the last four years, in particular, to my parents, John and Pat Houston; my husband, Sean, and my children, Jessica and Kelly. An acknowledgement is extended to the following companies who graciously donated chromatographic columns used in this study: EM Science, MetaChem, Optimize Technologies, Phenomenex, Varian, and Waters. Scynexis (Research Triangle Park, NC) provided funding for this research. iv DEDICATION "Challenges are what make life interesting; overcoming them is what makes life meaningful." -Joshua J. Marine Dedicated to my children, Jessica and Kelly Brown, so that they might know that anything is possible with the right attitude and enough hard work. v TABLE OF CONTENTS ACKNOWLEDGEMENTS........................................................................................................ IV DEDICATION...............................................................................................................................V TABLE OF CONTENTS............................................................................................................ VI TABLE OF FIGURES .............................................................................................................VIII TABLE OF TABLES ....................................................................................................................X CHAPTER 1: INTRODUCTION AND HISTORICAL BACKGROUND.............................. 1 INTRODUCTION TO HPLC............................................................................................................ 1 FAST HPLC ................................................................................................................................. 4 Factors affecting fast HPLC................................................................................................... 7 MONOLITHIC COLUMNS .............................................................................................................. 9 Monolithic Columns in CEC................................................................................................. 11 Organic-Based Monoliths in CEC........................................................................ 13 Silica-Based Monoliths in CEC ............................................................................ 16 Monolithic Columns in HPLC .............................................................................................. 19 Organic-Based Monoliths ................................................................................. 19 Inorganic-Based Monoliths............................................................................... 21 CHAPTER 2: EXPERIMENTAL.............................................................................................. 24 INSTRUMENTATION ................................................................................................................... 24 CHEMICALS ............................................................................................................................... 24 HPLC COLUMNS ....................................................................................................................... 26 VAN DEEMTER PLOTS................................................................................................................ 27 SELECTIVITY ............................................................................................................................. 29 FAST HPLC ............................................................................................................................... 29 Method Development............................................................................................................ 29 Column-to-Column and Run-to-Run Precision .................................................................... 29 APPLICATIONS........................................................................................................................... 29 CHAPTER 3: PRODUCTION OF MONOLITHIC COLUMNS........................................... 31 INTRODUCTION.......................................................................................................................... 31 SOL-GEL CHEMISTRY................................................................................................................ 31 Sol-gel processing (Nakanishi Method)................................................................................ 33 Mixing, Casting and Gelation............................................................................... 33 Aging..................................................................................................................... 39 Solvent Exchange and Drying .............................................................................. 41 Heat Treatment...................................................................................................... 43 Column Packaging and Surface Modifications..................................................... 43 CHAPTER 4: BAND BROADENING IN MONOLITHIC COLUMNS ............................... 45 INTRODUCTION.......................................................................................................................... 45 VARYING ORGANIC CONCENTRATION...................................................................................... 47 SOLVENT STUDIES..................................................................................................................... 51