ON-LINE PRE-CONCENTRATION TECHNIQUES IN CAPILLARY ELECTROPHORESIS FOR ENVIRONMENTAL ANALYSIS CHUANHONG TU NATIONAL UNIVERSITY OF SINGAPORE 2004 ON-LINE PRE-CONCENTRATION TECHNIQUES IN CAPILLARY ELECTROPHORESIS FOR ENVIRONMENTAL ANALYSIS BY CHUANHONG TU (M.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgement During my PhD study, I have been assisted by many wonderful individuals who have generously contributed their knowledge, experience and talents. To all these people, I express my deepest gratitude and heartfelt appreciation. First of all, I would like to express my sincere thanks to my supervisor, Professor Lee Hian Kee, for his invaluable suggestions, guidance and encouragement during the course of my study. Special thanks go to Ms. Frances Lim and Ms. Tang Chui Ngoh for their technical assistance. I am thankful to the staff in the Chemical Store, and the General Office of the Department of Chemistry. I also thank my colleagues Dr. Zhu Lingyan, Dr. Gong Yinhan, Ms. Zhao Limian, Ms. Sun Lei, Ms. Wen Xiujuan, Ms. Shu Yan, Dr. Hou Li and Mr. Zhu Liang, Mr. Shen Gang, Mr. Zhu Xuerong, Mr. Jiang Xianmin, Mr. Zhang Jie, Mr. Chanbasha Basheer for their advice and discussions. I thank the National University of Singapore for awarding me a research scholarship which supportd my study. Last but not least, I am grateful to my family, my wife and my parents for their endless understanding and support. i Table of Contents Acknowledgements i Table of Contents ii Publications v Summary vii List of Tables X List of Figures X CHAPETER ONE INTRODUCTION & LITERATURE REVIEW 1.1 Brief History of Capillary Electrophoresis 1.2 Basic Principles of CE 1.2.1 Electrical migration of charged species 1.2.2 The electroosmotic flow (EOF) 4 1.3 INSTRUMENTAL SETUP OF CE 1.3.1 Capillary 1.3.2 Sample Introduction 1.3.3 Detection 10 1.4 OPERATING MODES OF CE 11 1.5 ONLINE PRECONCENTRATION TECHNIQUES IN CE 1.5.1 On-line preconcentration using physical barrier 1.5.2 On-line chromatographic preconcentration techniques 1.5.2.1 On-line solid phase extraction 1.5.2.2 Pseudo-stationary phase partition-based techniques 1.5.3 Online electrophoretic pre-concentration techniques 1.5.3.1 On-line pre-concentration based on electric field enhancement 1.5.3.2 On-line pre-concentration based on varying mobility 13 14 16 16 18 21 22 31 1.6 THE OBJECTIVES OF THIS PROJECT 32 CHAPTER TWO EXPERIMENTAL 34 2.1 INSTRUMENTATION 34 2.2 GENERAL CE PROCEDURES 35 2.3 MOBILITY MEASUREMENT 35 2.4 REAGENTS AND MATERIALS 36 ii CHAPTER THREE SAMPLE SELF-STACKING IN CZE FOR THE DETERMINATION OF NITRATE IN SEAWATER 38 3.1 INTRODUCTION 38 3.2 EXPERIMENT 3.2.1 Chemicals 3.2.2 Apparatus and procedures 3.2.3 Sample collection and pretreatment 40 40 41 41 3.3 RESULTS AND DISCUSSION 3.3.1 Chloride-induced leading-type sample self-stacking 3.3.2 Optimization of sample self-stacking 3.3.2.1 Effect of DDAPS concentration on mobility and sample selfstacking 3.3.2.2 Effect of BGE concentration. 3.3.2.3 Influence of chloride concentration 3.3.3 Current change during the sample self-stacking 3.3.4 Determination of nitrate in seawater sample 41 41 44 44 45 46 50 52 3.4 CONCLUSION 54 CHAPTER FOUR DUAL TRANSIENT ISOTACHOPHORESIS IN CZE FOR THE DETERMINATION OF HALOACETIC ACIDS 55 4.1 INTRODUCTION 4.1.1 Occurrence, toxicity and analysis of haloacetic acids 4.1.2 CE for HAAs analysis 55 55 56 4.2 EXPERIMENTAL 4.2.1 Instrument and procedures 4.2.2 Chemicals 4.2.3 Off-line sample pretreatment 58 58 59 59 4.3 RESULTS AND DISCUSSION 4.3.1 Separation of HAAs 4.3.2 Large volume sample stacking 4.3.3 NaOH Effects in Large Volume Sample Stacking 4.3.3.1 BGE of pH 2.9 4.3.3.2 BGE of pH 5.8 in the presence of DDAPS 4.3.3.3 Optimization of the BGE for sample stacking. 4.3.4 Effect of injection volume 4.3.5 Sample stacking mechanism 4.3.5.1 Current changes during sample stacking 4.3.5.2 Sample stacking mechanism 4.3.6 Method Validation 4.3.7 Real Sample Analysis 60 60 67 68 68 70 73 74 75 75 79 84 84 4.4 CONCLUSION 88 iii CHAPTER FIVE BASE-AIDED LARGE VOLUME SAMPLE STACKING IN CZE FOR PHENOXY ACIDS WITH MEDIUM PH BUFFER 89 5.1 INTRODUCTION 89 5.2 EXPERIMENTAL 5.2.1 Instrument and procedures 5.2.2 Chemicals 90 90 90 5.3 RESULTS AND DISCUSSION 92 5.3.1 Large volume sample stacking vs. base-aided large volume sample stacking 92 5.3.2 The effect of BGE composition on BA-LVSS 94 5.3.2.1 Effect of pH of the BGE 94 5.3.2.2 Effect of DETA concentration 95 5.3.3 The effect of injection volume 96 5.3.4 Tolerance of salt 99 5.3.5 Linearity, precision and detection limits 99 5.3.6 Investigation of stacking mechanism 103 5.3.7 Sample analysis 111 5.4 CONCLUSION CHAPTER SIX FIELD-AMPLIFIED SAMPLE INJECTION IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY FOR ENRICHMENT OF PHENOLS 111 113 6.1 INTRODUCTION 113 6.2 EXPERIMENT 6.2.1 Instrument 6.2.2 Reagents and solutions 114 114 114 6.3 RESULTS AND DISCUSSION 6.3.1 Field amplification sample injection in MEKC 6.3.2 Effect of sample matrix 6.3.2.1 Water as sample matrix 6.3.2.2 NaOH solution as sample matrix 6.3.3 Method Validation 6.3.4 Sample analysis 115 115 116 116 121 124 125 6.4 CONCLUSION 126 CHAPTER SEVEN CONCLUDING REMARKS 128 REFERENCES 132 iv Publications and Conference Presentations 1. Chuanhong Tu, Lingyan Zhu, Chay Hoon Ang, Hian Kee Lee, Effect of NaOH on large volume sample stacking of haloacetic acids in capillary zone electrophoresis with a low pH buffer, Electrophoresis, 24 (2003) 21882192. 2. Lingyan Zhu, Chuanhong Tu, Hian Kee Lee, On-line concentration of acidic compounds by anion-selective exhaustive injection-sweeping-micellar electrokinetic chromatography. Anal. Chem. 74 (2002) 5820-5825. 3. Chuanhong Tu, Hian Kee Lee, Determination of nitrate in seawater with capillary zone electrophoresis with chloride-induced sample self stacking. J. Chromatogr. A, 966 (2002) 205-212 4. Lingyan Zhu, Chuanhong Tu, Hian Kee Lee, Liquid-Phase Microextraction of Phenolic Compounds Combined with On-Line Preconcentration by FieldAmplified Sample Injection at Low pH in Micellar Electrokinetic Chromatography, Anal. Chem. 73 (2001) 5655-5660 5. Li Hou, Xiujuan Wen, Chuanhong Tu, Hian Kee Lee, Combination of liquidphase microextraction and on-column stacking for trace analysis of amino alcohols by capillary electrophoresis, J. Chromatogr. A, 979 (2002) 163– 169 6. Xiujuan Wen, Chuanhong Tu, Hian Kee Lee, Two-step Liquid-liquid-Liquid microextraction of nonsteroidal anti-inflammatory Drugs in Wastewater, Anal. Chem. 76 (2004) 228-232. 7. Chuanhong Tu, Lingyan Zhu, Hian Kee Lee, The effect of counter-ions of the background electrolyte on large volume sample stacking of phenoxy v acids in capillary electrophoresis with phosphate background at pH 6.0. Manuscript under preparation. 8. Chuanhong Tu, Lingyan Zhu, Hian Kee Lee, Base-aided large volume sample stacking of haloacetic acids with a low pH buffer, presented at HPCE 2002, April 13-18, 2002, Stockholm, Sweden. 9. Chuanhong Tu, Lingyan Zhu, Hian Kee Lee, Approaches to liquid-phase microextraction/capillary electrophoresis for environmental analysis, presented at EnviroAnalysis 2002, May 27-30, Toronto, Canada. vi SUMMARY This work focused on the development of on-line sample pre-concentration techniques to improve detection sensitivity in capillary electrophoresis (CE). Several on-line enrichment methods were established for acidic compounds with various pKa values, including strong acid (nitrate), weak acids (haloacetic acids, phenoxy acids, pKa 0.6-4.8) and very weak acids (phenols, pKa 7.510.6), in different sample matrices. For nitrate in seawater sample, a zwitterionic surfactant was added into the background electrolyte (BGE) to increase the mobility difference between chloride and nitrate, so that a leading-type sample self-stacking could be employed to pre-concentrate low concentration nitrate in seawater using native chloride in the sample as the leading ion, and the co-ion in the BGE as terminating ion. Thus, a highly conductive sample could be injected in a large volume with about 4-fold sensitivity enhancement compared to large volume sample stacking in which nitrate was dissolved in pure water. A detection limit of nitrate of 35µg/L was achievable for seawater with relatively low concentration BGE. At an analyte concentration near the limit of detection (LOD), the mole ratio between the matrix and the analyte was around 106:1. Organic solvent is often used for sample extraction during off-line sample pretreatment. Unfortunately, samples in common organic solvents, such as hexane, cannot be analyzed directly by CE. Aqueous alkaline solutions are usually employed to back-extract organic weakly acidic compounds from organic solvent in sample pretreatment. We developed three on-line preconcentration methods for acidic compounds dissolved in NaOH solution, vii are still inadequate for practical world analysis. Thus, off-line sample pretreatment to preconcentrate analytes may still be necessary. In sample pretreatment, organic solvent is generally used to extract organic compounds. The final extract is therefore not suitable for direct analysis by CE. For some applications, it is possible to back-extract analytes into an aqueous solution, making it possible to use CE for analysis. Following this approach, here, we developed methods for on-line concentration of acidic compounds involving NaOH solution as the matrix for back extracting the analytes into sample matrix. For haloacetic acids (pKa 0.6-2.9) dissolved in NaOH solution, a phosphate background electrolyte (BGE) at pH 3.0 was used for separation. It was found that the peak heights of analytes were increased by the presence of NaOH in the sample with large injection volume. The mechanism underlying this sample stacking was proposed to be hydroxide-induced dual transient isotachophoresis. In the first stage of isotachophoresis, hydroxide played the role of a leading ion. Since the hydroxide was reactive with the low-pH BGE, the reaction product, HPO42- was the leading ion in the second stage of isotachophoresis. In both stages, the co-ion of the BGE (H2PO4-) was the terminating ion. Finally, the analytes were destacked and separated in CZE mode when the leading ion, HPO42-, was consumed due to continuous reaction with H+ in the low pH BGE. For the CZE separation of phenoxy acids (pKa 1.9-4.8), a higher pH BGE had to be used due to higher pKa values of these analytes. When diethylenetriamine (DETA) was used as EOF suppressor with phosphate BGE at pH 6.0, the sample dissolved in the NaOH solution could be injected into the 129 capillary in a large volume, and the analytes were concentrated on-line. Further study indicated that the presence of a protonated amine in the BGE as counter-ion was critical for this sample stacking. The EOF was suppressed by by DETA, and the analytes migrated against the EOF. After sample stacking, the sample matrix was removed from the separation capillary by the EOF pumping from the inlet; thus had no influence on the CZE separation that followed. The above methods cannot be applied for the online pre-concentration of phenols due to their high pKa values and lack of a suitable EOF modifier in a high-pH BGE. Micellar electrokinetic chromatography (MEKC) was therefore employed for the separation of these analytes with a low pH BGE to suppress the EOF. The pre-concentration was carried out by field amplified sample injection (FASI) of the sample dissolved in NaOH solution. FASI was implemented by pre-injecting a long water plug to produce a field amplification effect. As a result, the phenols accumulated at the interface between the water plug and the BGE since they were neutralized due to the dynamic pH junction. Since the EOF was from the anode to the cathode, the water plug would be pumped out of the capillary by the EOF from the inlet. MEKC separation was initiated by transferring the inlet from the sample vial to one containing the BGE after a period of injection. In summary, in developing on-line pre-concentration techniques, the sample matrix, together with the EOF are important factors to be considered. The EOF can be used for the removal of the sample matrix after sample stacking if the migration direction of EOF is against the analytes. 130 Finally, further research may be suggested. The on-line preconcentration techniques described in this work are mainly based on the manipulation of the velocity through the electric field strength that the ions experience. In fact, the velocity of electrophoretic migration is a product of mobility and electric field strength. Therefore mobility manipulation is another possibility to consider in sample stacking. Theoretically, the mobility is influenced by the charge, molecular volume and the viscosity of the medium. Possibly, a high viscosity BGE can be used to retain the analytes along their migration path. Since the effect of viscosity on the mobility is not selective to the analytes, a universal method may be developed. In another future research possibility, the methods developed in this work may be transferred to microchip-based CE since electrophoresis in the fabricated micro-channels also suffers from the same low sensitivity problems as conventional CE. 131 References [1] F. Kohlrausch, Ann. Phys. 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A 788 (1997) 185. 144 [...]... analytes in different zones to accomplish the concentration 1.5.1 On- line preconcentration using physical barrier The principle of on- line pre- concentration using a physical barrier is the same as in the classical technique of ultrafiltration The leading species are stopped by the physical barrier, allowing the molecules following behind to 14 eventually reach the same physical space, thus increasing concentration. .. multiple -capillary system to separate the enrichment capillary from the separation capillary These include the use of a double -capillary system [69], and an on- line switching valve [85] In the double -capillary system, two capillaries were connected with a T connector The pre- concentration by SPE was carried out in one capillary; the separation was performed in the other capillary In the on- line switching... for stacking neutral analytes in high-salt sample matrix with electrokinetic injection The analytes were injected into the capillary by EOF and was stacked at the inlet due to their partitioning into negatively charged micelles This scheme could be performed on conventional CE or CE on a microchip [105,106] In summary, the on- line pre- concentration methods based on partition into pseudo-stationary also... capillary tubing as in conventional CE CE -on- a-chip has the potential to be multiplexed for highthroughput applications 1.3.2 Sample Introduction Sample can be introduced into the capillary in two common ways in CE One is the hydrodynamic injection, and the other is electrokinetic injection In hydrodynamic injection, a pressure difference between the inlet and outlet is applied to move the sample into... 1.5.3.1 On- line pre- concentration based on electric field enhancement 1.5.3.1.1 Field-amplified sample stacking Field-amplified sample stacking (FASS) is the simplest method for on- line pre- concentration It can be induced by injecting a large volume of sample dissolved in a low conductivity sample matrix The effects of injecting samples in a low-conductivity matrix were first reported by Mikkers in 1979... switching valve design, the analytes were retained on a stationary phase within the valve The retained analytes were transferred to the separation capillary by valve switching The on- line coupling of LC with CE 17 provides a possible technique for on- line pre- concentration in addition to a twodimensional separation [86-89] In the multiple -capillary scheme, detection enhancements of 400- to 500-fold [90,91]... pseudo-stationary phase, temperature, organic modifier, etc 1.5 ONLINE PRECONCENTRATION TECHNIQUES IN CE Although the separation efficiency of CE is higher than that of HPLC, the limits of detection (LOD) for capillary electrophoresis are constrained by the dimensions of the capillary For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary In. .. electrophoretic pre- concentration techniques To concentrate the analytes in a large sample plug, the velocities of the analytes in the direction of their movement should be reduced The analytes in the leading part slow down, those in the tailing part will catch up Thus, the analytes are accumulated into a small volume This principle is applicable to all on- line sample pre- concentration techniques 21 In electrophoresis, ... The interface for online CE-MS can be electrospray ionization (ESI) [32] or continuous-flow fast atom bombardment (CF-FAB) [33] For off -line CE-MS, matrix-assisted laser desorption/ionization (MALDI) is commonly used [34] 1.4 OPERATING MODES OF CE Different modes of capillary electrophoresis can be performed using a standard CE instrumental set-up with different electrophoretic media In the continuous... thoroughly They found that, theoretically, the peak width in sample stacking was proportional to the ratio, γ, of buffer concentration in the original sample solution to that in the BGE This difference in the concentrations inside the capillary tubing generated an electroosmotic pressure originating at the concentration boundary The laminar flow resulting from the electroosmotic 22 . partition-based techniques 18 1.5.3 Online electrophoretic pre- concentration techniques 21 1.5.3.1 On- line pre- concentration based on electric field enhancement 22 1.5.3.2 On- line pre- concentration based. 1.3 INSTRUMENTAL SETUP OF CE 7 1.3.1 Capillary 8 1.3.2 Sample Introduction 9 1.3.3 Detection 10 1.4 OPERATING MODES OF CE 11 1.5 ONLINE PRECONCENTRATION TECHNIQUES IN CE 13 1.5.1 On- line preconcentration. preconcentration using physical barrier 14 1.5.2 On- line chromatographic preconcentration techniques 16 1.5.2.1 On- line solid phase extraction 16 1.5.2.2 Pseudo-stationary phase partition-based