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Miami University – the Graduate School MIAMI UNIVERSITY – THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Wei Zhang Candidate for the Degree: Doctor of Philosophy Dr. Neil D. Danielson, Director Dr. John F. Sebastian, Reader Dr. André J. Sommer, Reader Dr. Shouzhong Zou, Reader Dr. Catherine Almquist, Reader Graduate School Representative ABSTRACT DEVELOPMENT OF PHOTOCHEMICALLY INITIATED DIRECT AND INDIRECT LUMINESCENCE DETECTION METHODS FOR LIQUID CHROMATOGRAPHY (LC) AND STUDY OF AROMATIC SULFONATES AND PHOSPHOLIPIDS USING REVERSED PHASE ION-PAIR LC-MASS SPECTROMETRY by Wei Zhang The first project is development of a 5.5 µL spiral micro-flow chemiluminescence (CL) cell that allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light for flow injection (FI) using a 25 µL/min flow rate for luminol and a 50 µL/min buffer carrier flow rate. The detection limit of 1.5 µM achieved by using a spiral flow cell is 24 times lower than that obtained from a conventional FI system with a premixing tee and a straight 12 µL flow cell. This luminol FI method is applied to the enzymatic determination of L-lactate from 5–50 µM using polyethyleneglycol-stablized lactate oxidase. The second project is development of a quinine-sensitized photo-oxidation and quenched CL detection method for phenols using FI and LC. This method is based on the decrease of light emission from the luminol CL reaction due to the photo-oxidation of phenols that scavenge the photogenerated reactive oxygen species. On-line photo-oxidation is achieved using a coil photo- reactor made from FEP tubing coiled around a mercury UV lamp. This method is applied for the FI determination of ten phenolic compounds, mostly nitro- and chloro-phenols, and the LC determination of phenol, 4-nitrophenol and 4-chlorophenol with detection limits of about 1 µM. The third project is development of an indirect fluorescence (FL) detection method via the shielding effect on the UV-photolysis of 2-phenylbenzimidazole-5-sulfonic acid (PBSA). Compounds that have a strong UV absorbance at 217 nm and/or are reactive toward the photogenerated oxygen species can possess such a shielding effect. This method is applied for both the FI determination of thirteen aromatic compounds, mostly non-fluorescent nitro compounds, and the LC determination of salicylate, nitrofurantoin, 4-nitroaniline, 2-nitrophenol, and 4-nitrophenol with detection limits of about 0.2 µM. The fourth project is separation of ionic organic compounds by reversed phase ion-pair LC with MS detection. tert-Octylamine (TOA) is studied as a new more volatile ion-pairing agent for separation of aromatic sulfonates. The separation results are compared with those obtained from NH4OH, dihexylamine, and tri-n-butylamine. TOA is the preferred ion-pairing agent as compared to NH4OH for the gradient separation of phospholipids. DEVELOPMENT OF PHOTOCHEMICALLY INITIATED DIRECT AND INDIRECT LUMINESCENCE DETECTION METHODS FOR LIQUID CHROMATOGRAPHY (LC) AND STUDY OF AROMATIC SULFONATES AND PHOSPHOLIPIDS USING REVERSED PHASE ION-PAIR LC-MASS SPECTROMETRY A DISSERTATION Submitted to the Faculty of Miami University in partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Wei Zhang Miami University Oxford, Ohio 2003 Dissertation Director: Professor Neil D. Danielson TABLE OF CONTENTS Page Chapter I Introduction 1 Section A Chemiluminescence 1 Section B Fluorescence 9 Section C Photochemically initiated luminescence detection 13 Section D Liquid chromatography 16 Section E Liquid chromatography-mass spectrometry (LC-MS) 25 Section F Purpose of research 28 References 32 Chapter II Characterization of a micro spiral flow cell for 44 chemiluminescence detection Section A Introduction 44 Section B Experimental 46 1. Apparatus and instruments 46 2. Reagents and solutions 49 3. Procedure 49 Section C Results and Discussion 50 1. Flowrate studies for the spiral and conventional HPLC 51 flowcells 2. Calibration curves and detection limits for H2O2 54 3. L-lactate assay 54 3.1 Flow rate optimization 57 3.2. Temperature and enzyme concentration 57 3.3. Stability of lactate samples 57 3.4. Calibration, detection limit, and real sample data for 62 L-lactate Section D Conclusions 65 References 66 ii Chapter III Determination of phenols by flow injection and liquid 69 chromatography with on-line quinine-sensitized photo- oxidation and quenched luminol chemiluminescence detection Section A Introduction 69 Section B Experimental 72 1. Chemicals and solutions 72 2. FI and LC instrument design for photo-oxidation and 72 quenched CL detection 3. Procedures 73 3.1. Photo-oxidation and quenched CL detection of phenols 73 by FI 3.2. LC separation of phenols 76 Section C Results and Discussion 76 1. Photo-oxidation chemistry of phenols and the luminol 76 CL reaction with oxygen species 2. Effects of experimental parameters on the on-line photo- 77 generation and CL detection of oxygen species 2.1 Flow rate 78 2.2 Solvent 78 2.3. pH 81 2.4. Concentration of photosensitizer 84 3. FI determination of phenols 84 4. Determination of phenols by HPLC 87 Section D Conclusions 89 References 91 Chapter IV Indirect fluorescent determination of aromatic compounds 97 via a shielding effect on the UV-photolysis of 2- phenylbenzimidazole-5-sulfonic acid Section A Introduction 97 Section B Experimental 100 iii 1. Chemicals and solutions 100 2. Apparatus 101 3. Procedures 103 3.1. Off-line photolysis of PBSA and PBSA with aromatic 103 compounds 3.2. Flow injection and HPLC of aromatic compounds 103 Section C Results and Discussion 104 1. Off-line photolysis of PBSA 104 1.1. pH and photolysis time 104 1.2. Chemical shielding effect 107 1.3. Study of photolyzed PBSA samples by reverse phase 107 ion pair LC-MS 2. Indirect FL detection via the shielding effect on 114 photolysis of PBSA 3 Application of the indirect FL detection method for on- 114 line determination of aromatic compounds by FI 3.1. PBSA concentration 115 3.2. Flow rate 115 3.3. Solvent 115 3.4. Flow injection determination of 13 aromatic 119 compounds 4. Application of this FL detection method for on-line 122 determination of aromatic compounds by HPLC 4.1. Optimization of experimental conditions for FL 122 detection 4.2. Calibration curves and analytical figures of merits 124 Section D Conclusions 124 References 130 iv Chapter V Volatile amines as ion-pairing agents for separation of 134 aromatic sulfonates and phospholipids using reversed phase ion-pair liquid chromatography-mass spectrometry (LC-MS) Section A Introduction 134 Section B Experimental 136 1. Chemicals and solutions 136 2. Apparatus 140 3. Procedures 140 3.1. LC-MS of aromatic sulfonates 140 3.2. ESI-MS analysis of phospholipid samples by syringe 141 infusion 3.3. LC-MS analysis of phospholipids 141 Section C Results and Discussion 142 1. Analysis of aromatic sulfonates by reversed phase ion- 142 pair LC-MS 1.1. Optimization of solvent ratios in mobile phase 142 1.2. Trap drive level effects 145 1.3. Separation results using different ion-pairing agents 150 2. Determination of phospholipids by reversed phased ion- 160 pair LC-MS 2.1. ESI-MS analysis of phospholipid samples by direct 160 syringe infusion 2.2. Separation of four phospholipids by LC using TOA 160 and NH4OH as ion-pairing agents Section D Conclusions 175 References 176 Chapter VI Significance and Future work 180 v LIST OF TABLES Page 3.1 Calibration equations for ten phenolic compounds 86 4.1A Analytical figures of merit of the proposed FL detection method 120 for FI determination of nitroaromatic compounds 4.1B Analytical figures of merit of proposed FL detection method for 120 FI determination of aromatic compounds 4.2 Analytical figures of merit of proposed FL detection method for 128 HPLC determination of aromatic compounds 5.1 Comparison of the separation results obtained from reversed 159 phase ion-pair LC using four types of volatile ion-pairing agents 5.2 The chromatographic, chemical and structural data obtained 168 from LC separation of phospholipids using TOA as an ion-pairing agent 5.3 The chromatographic, chemical and structural data obtained 172 from LC separation of phospholipids using NH4OH as an ion- pairing agent vi LIST OF FIGURES Page 1.1 Energy pathway for chemiluminescence 3 1.2 Types of chemiluminescence 4 1.3 (a) Luminol CL reaction. (b) Peroxyoxalate CL reaction. (c) 6 Lucigenin CL reaction. (d) Acridinium ester CL reaction 1.4 Energy diagram showing fluorescence transitions 10 1.5 Simplified diagram of a FL detector 12 1.6 The chromatogram and its characteristic parameters 18 1.7 The effects of varied organic solvent content in the mobile phase 22 on the elution behavior of a large biomolecule and a small organic molecule separated by reversed phase HPLC 1.8 Thermodynamic equilibria involved in reversed phase ion-pair 24 HPLC 1.9 Diagram for electrospray ionization process 27 1.10 The instrumentation of the Bruker Esquire-HP LC-MS with an 29 ESI interface 2.1(A) A schematic diagram of the FI-CL instrument for the 47 determination of lactate 2.1(B) Design diagram for the 5.5 µL spiral flow cell 48 2.2 Effect of the flow rate on the CL intensity for the FI-CL system 52 with a 5.5 µL spiral flow cell 2.3 Effect of the flow rate on the CL signal profile 53 2.4 Calibration plots for the FI-CL determination of H2O2 55 2.5 Flow rate optimization for the determination of lactate 58 2.6 Effect of temperature (oC) on the LO enzyme activity 60 2.7 Effect of PEG on
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