Monolith và cột đóng gói hạt nhỏ cho sắc ký lỏng - Xin Zhang

Monoliths and Small Particle-Packed Columns for Liquid Chromatography By Xin Zhang A dissertation submitted to the Faculty of the Graduate School of the University at Buffalo The State of New York in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry January 1, 2007 UMI Number: 3244236 UMI Microform 3244236 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.

Box 1346 Ann Arbor, MI 48106-1346 Dedication This thesis is dedicated to my husband, Aihua Xu and my daughter Julia Xu. To my husband, it was him who brought me to the U.A and encouraged me to pursue my dream. He gives me advice and support whenever I need. To my seven month old daughter, she is my angel and her smile is everything to me.

Thank you both for everything. ii Abstract Advances in chromatography have led to two recent active areas of study: the reduction of particle size for column packing and development of monolithic materials. Reducing the size of the packing material leads to an increase in chromatographic efficiency and a decrease in the analysis time. The monolithic column is composed of one single piece of material with through-pores that offers very high permeability, thereby, allowing the column can offer a variable external porosity and operation at high linear velocity with low pressure requirements.

The performance of commercially available columns, a monolith and a sub-2 μm particle packed column was examined. Higher efficiencies were obtained with the sub-2 μm particle packed column. Very high pressures 14,000 psi were required to operated the sub-2 μm packed column to obtain a linear velocity of 6.4 mm/sec, while a pressure of 1,600 psi was required to achieve a linear velocity of 12 mm/sec with the monolithic column. Examination by the van Deemter model shows that the monolithic column behaves like a 3 μm packed column.

A new approach to synthesize monolithic columns for capillary LC and capillary electrochromatography (CEC) was also examined. A silica based hybrid monolithic material was synthesized and characterized. The material fabricated inside capillary columns consisted of an allyl-functionalized monolith, which was prepared in a one-pot reaction. The material was characterized by SEM, nitrogen absorption (BET method), and chromatographically by CEC and CLC.

The chromatographically tested column with best performance had a surface area of about 100 m2/g with through pores 7 μm and 6 nm mesopores. iii A stationary phase for CEC was synthesized by polymerizing n- isopropylacrylamide on aminated silica particles. The poly-n-isopropylacrylamide was characterized by IR spectroscopy and thermogravimetric analysis. Both IR spectroscopy and thermogravimetric thermograms showed the grafting of the poly-n- isopropylacrylamide on the aminated silica.

Tested under CEC conditions, using a series of neutral and acidic compounds, the stationary phase showed a mixed mode separation mechanism with both hydrophobicity and hydrophilicity contributing to the separation. iv Acknowledgement My thanks and appreciation go to Dr. Colón for persevering with me as my advisor through out the time it took me to complete this research and write the dissertation. Through his guidance and wisdom, I was able to grow as a scientist.

I would also like to thank him for being a friend. The experiences that he has provided me with are something that I will carry with me forever, and I am truly grateful. The members of my dissertation committee, Dr. Diana Aga, Dr.

Troy Wood and Dr. Bing Gong, have generously given their time and expertise to better my work. I thank them for their contribution and their good-natured support. I would like to thank the past and present members of the ColónLab with whom I have had the distinct pleasure of working with, either here or via collaborations: Yong Guo, Jose Cintrón, Hector Colón, Jason Anspach, Carl Adams, Murali Seenappa, Jennifer Herrmann, David Hoth, Li Li, Qian Wang, Melissa Dunkle, Glorimar Vicente, Jose Rivera, and Richard Brice.

Thanks for the discussions, both scientific and not, lunches, and good times, as well as a lot of great memories. I am grateful to many persons who shared their memories and experiences, especially Honghai Jiang, Xing Dai, Hsien-Po (Paul) Chiu, Chi-Tung (Thomas) Chiang, Ying Shao, Zhengzheng Jiang and Minfeng Li for their friendship, help, and good times through this process. I would like to thank my cousin Zhiming Tan, my aunt Guifang Ma and my uncle Meiqi Tan for taking care of my parents for the past five years while I am studying in the U. v I would like to thank my parents-in-law, Hailian Cao and Baosheng Xu, for taking care of my daughter during my thesis writing.

At last, I would like to thank my parents Xiaoming Zhang and Jufang Ma, for the endless support and love. Especially my Mom, she barely speaks English but flew to U. by herself to take care of me and my new born daughter. vi Table of Content Dedication ii Abstract iv Acknowledgements v Table of Content vii List of Figures xi List of Tables xvii List of Abbreviations xviii Chapter 1 Fundamentals of Monolithic and Small-Particle Packed Columns in HPLC 1 1.1 Silica-Based Monolithic Columns in Liquid Chromatography 1 1.2 Silica Based Monoliths 2 1.1 Sol-gel Process 2 1.3 Rinsing and Aging 5 1.3 Monolith in a Tube or in a Capillary 9 1.4 Advantages and Disadvantages of Monolithic Silica Columns 10 1.5 Chromatographic Properties of Monolithic Silica Columns 11 1.6 Increase in Separation Speed by Using Monolithic Silica Columns 14 1.2 Ultrahigh Pressure Liquid Chromatography (UHPLC) 15 vii 1.2 Advantages of Sub-2-μm Particles 16 1.3 Instrumental requirement for small particles 18 1.4 Heat dissipation in UHPLC 20 1.3 Scope of Dissertation 22 1.4 References of Chapter 1 23 Chapter 2 Monolithic and Sub 2 μm-packed Columns for HPLC 27 2.1 Material and Chemicals 28 2.3 Results and Discussions 33 2.1 Separations with Monolithic and 1.2 van Deemter Plots 41 2.3 Pressure Drop and Mobile Phase Consumption 49 2.4 Separation of Basic Compounds at Different pH Buffered Mobile Phase 54 2.5 Separation of Base Drugs 59 2.5 References for Chapter 2 64 Chapter 3 Chromatographic Characterization of Allyl-functionalized Monolithic Column 66 3.2 Material and Chemicals 68 3.4 Electroosmotic Mobility and Permeability 71 3.3 Results and Discussion 72 3.1 Effect of the Amount of PEG as Porogen 72 3.3 Column Aging Time 82 3.4 Alkaline Stability Test and Surface Coverage 87 3.5 References for Chapter 3 92 Chapter 4 Evaluation of poly(-N-isopropylacrylamide-co-(3-(methacryloylamino) propyl) trimethylammonium) as a stationary phase for capillary electrochromatography 94 4.2 Material and Methods 96 4.2 Material and Chemicals 96 4.4 Preparation of Poly-NIPAAm-co-MAPTA Modified Silica 99 4.3 Results and Discussion 102 4.1 TGA Analysis 102 ix 4.3 Chromatographic and Electrophoretic Characteristics 106 4.1 Electroosmosis and Mobile Phase pH 106 4.2 Organic Modifier in the Mobile Phase 111 4.4 Separation of Basic Compounds 123 4.5 References for Chapter 4 126 Chapter 5 Conclusions and future directions 128 5.2 Future Research Directions 129 5.3 References for Chapter 5 134 x List of Figures Figure1-1: Schematic representation of the silica reactions involved in the sol-gel process.

Figure1-2: Formation of menisci during: concave or convex meniscus on wetting angle Figure 2-1: Chromatograms obtained on the ONYX monolithic column: A) provided by manufacture, B) collected in our laboratory. Peak 1: thiourea, peak 3: anthracene, mobile phase: Acetonitrile/Water (60:40), flow rate: 2.0mL/min, UV 254 nm detection, separation at room temperature. Injection volumn for A: 1 μL, B: 600 nL Figure 2-2: Separating of test mixture using the monolithic column at different linear velocity. The components in order of elution were thiourea, aniline, benzene, naphthalene and 1-methyl naphthalene.

The mobile phase was 60%:40% acetonitrile:water used as mobile phase. Injection volumn: 600 nL; Detection UV: 214 nm. Figure 2-3: Chromatograms showing the separation of test compounds using a 1.7 μm packed column under different linear velocity. The components in order of elution were thiourea, benzene, naphthalene and 1-methyl naphthalene.

The column dimension;s were 100 x 2. Others conditions as in Figure 2-2 Figure 2-4: van Deemter equations of 3 different columns for ethylbenzene. xi The compounds were in order of elution, thiourea, benzene, N,N’- dimethylaniline, toluene and ethylbenzene, the mobile Phase was 70:30 acetonitrile:water; Other conditions were as in Figure 2-2. (A): 3 µm ODS HPLC column (100 x 4.) mm), (B): Monolithic column (100 x 4.) mm), (C): Column packed with 1.

Figure 2-5: Chromatograms of test compounds obtained in three different columns at optimal linear velocity. All conditions are as in Figure 2-4 Figure 2-6: Pressure drop as a function of linear velocity for three different columns. The mobile phase used was 60:40 acetonitril/water. Linear velocity was measure using thiourea as an unretained marker.

Figure 2-7: Mobile phase consumption as a function of linear velocity for three different columns. (A) Absolute flow rate vs. (B) Flow rate normalized to the column cross section area vs. All the conditions are as in Figure 2-6 Figure 2-8: Separation of basic compounds at different pH using the monolithic column (100 x 4.

The compounds were (in order of elution) thiourea, aniline, N-ethyl aniline, N,N-dimethyl aniline, N-ethyl, N-methyl benzenamine. The injection volumn was 600 nL, detected byUV at 254 nm. The pressure drop is at 400 psi.1, 5 mM phosphate, 60% acetonitrile as xii organic additive and (B) pH=7.0, 5 mM phosphate, 60% acetonitrile as organic additive Figure 2-9: Separation of base compounds at different pH using a 1.1, 5 mM phosphate, 70% Acetonitrile as organic additive, (B) pH=7.0, 5 mM phosphate, 70% Acetonitrile as organic additive, (C) pH=10.2, 5 mM phosphate, 70% Acetonitrile as organic additive. Operation pressure is 5000 psi.

Other conditions are as in Figure 2-8. Figure 2-10: Separation of (1) nortriptyline and (2) amitriptyline in a 1.7 μm packed colum using a high pH mobile phase. Condition: mobile phase, pH=10.2, 5 mM phosphate with 70% acetonitrile as organic modifer; UV detection, 254 nm; Flow rate, 250 μL/min.1 mm packed with 1.7 μm C18 material Figure 3-1: Schematic representation of the synthetic procedure to fabricate allyl-functionalized monolithic capillaries Figure 3-2: SEM images and electochromatograms obtained from three different monolithic columns prepared with different amounts of PEG as porogenic agent; (A) 69 mg PEG, (B) 90 mg PEG and (C) 108 mg PEG. Other conditions are shown in Figure 3-1.

The separation conditions were mobile phase: 4 mM borate (pH=9. 660 V/cm electrifield, effective capillary length: 20 cm with 50 μm inner diameter; injection at 10 kV for 6 seconds, UV detection at 214 nm. Peaks (in the order of elution) xiii are: 5mM DMSO, 15 mM benzene, 12.5 mM ethyl benzenes. Figure 3-3: SEM images and electochromatograms obtained from the monolithic columns prepared using different sol-gel reaction times, (A) 30 min, (B) 60 min, (C) 90 min, (D) 120 min, (E) 180 min.

Other conditions as in Figure 3-2. Figure 3-4: SEM images and electochromatograms obtained in monolithic columns prepared using different aging times. Separation conditions as Figure 3-2 Figure 4-1: Preparation scheme to modify aminated silica surface with poly- NIPAAm-co-MAPTA stationary phase Figure 4-2: Thermogravimetric analysis of (A) initial aminated silica particle (5 µm), (B) initiator activated particle, and (C) poly-NIPAAm-co- MAPTA modified particle Figure 4-3: FT-IR spectra of (A) poly-NIPAAm-co-MAPTA-modified and (B) aminated particles Figure 4-4: Electroosmotic mobility as a function of pH. Column length: 30 cm total, 20 cm effective length。 Mobile phase is made of a total of 5 mM phosphate at different pH with 30% acetonitrile as modifier.

The column is 30 cm total with 20 cm effective length; 5 kV, 8 sec injection, reversed polarity; Separation voltage is at 25 kV with reversed polarity, 20 psi applied to both ends of the xiv capillary; Detected at UV 214 nm. Figure 4-5: CEC separations of five model compounds with different organic additives. Mobile phase: 5 mM phosphate at pH = 3.