Miami University – the Graduate School

MIAMI UNIVERSITY – THE GRADUATE SCHOOL

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation

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 photoreactor 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.

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

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

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

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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

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

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

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 the stabilization of LO enzyme activity when 61 PEG-LO samples stored in an ice-bath in the refrigerator

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2.8 Effect of CL signal as a function of storage time for the lactate- 63 PEG-LO sample at low temperature 2.9 Calibration curve for the FI-CL determination of L-lactate using 64 the spiral flow cell 3.1 Instrument design for (A) FI and (B) LC with on-line photo- 74 oxidation and quenched CL detection 3.2 Effect of flow rate on the sensitized photogeneration and CL 79 detection of oxygen species 3.3 Effect of solvent on the sensitized photogeneration of oxygen 80 species 3.4 Effect of pH on the (A) CL intensity and (B) CL signal profile 82 obtained from the photochemical-luminol reaction sequence 3.5 Effect of photosensitizer concentration on the photogeneration of 85 oxygen species 3.6 CL-inhibition and UV detection chromatograms for the 88 separation of phenols 4.1 Structures of PBSA and thirteen aromatic compounds used in this 99 study 4.2 Instrumental setup for the indirect FL determination of aromatic 102 compounds by (A) FI and (B) HPLC 4.3 Molecular spectra showing the effect of pH and photolysis time on 105 the off-line photolysis of PBSA 4.4 Shielding effect on the off-line photolysis of PBSA 108 4.5 Analysis of photolyzed PBSA (MW=274.3) after photolysis for (A) 111 0 min, (B) 30 min and (C) 60 min by reversed phase ion-pair LC- ESI-MS using tert-octylamine as an ion-pairing agent 4.6 The photolysis effect on ion intensity and ion distribution in MS 113 spectra of PBSA derived from the total ion current chromatograms obtained by LC-MS 4.7 Concentration effect on the on-line photolysis of PBSA as 116 measured by the FL intensity ratio

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4.8 Flow rate effect on the on-line photolysis of PBSA as measured by 117 the FL intensity ratio 4.9 Organic solvent effect on on-line photolysis of PBSA as measured 118 by the FL intensity ratio 4.10 The molar absorption efficient (ε) at 217 nm and sensitivity of 121 determination of 13 aromatic compounds by the shielding effect- based FL detection method 4.11 PBSA concentration effect on FL intensity of nitrofurantoin, 4- 123 nitroaniline, and 4-nitrophenol separated by HPLC 4.12 Flow rate effect on the FL intensity of nitrofurantoin, 4- 125 nitroaniline, and 4-nitrophenol separated by HPLC 4.13 pH effect on FL intensity of nitrofurantoin, 4-nitroaniline, and 4- 126 nitrophenol separated by HPLC 4.14 Chromatogram of 5 aromatic compounds taken by HPLC with 127 indirect FL detection via the shielding effect on photolysis of PBSA 5.1 Chemical structures of volatile amines (A), aromatic sulfonates 137 (B), and phospholipids (C) 5.2 Chromatograms of aromatic sulfonates by reversed phase ion- 143 pair LC with MS (A) and UV detection (B) 5.3 The effect of trap drive level in ion trap MS on the peak height (A) 146 and the pattern of ions comprised in BSA (B), 1,5-NSA (C), 1,3,6- NSA (D), and 2-NSA (E) sample peaks in the ion chromatograms 5.4 Representative MS spectra of aromatic sulfonate sample peaks in 151 the ion chromatogram obtained with trap drive level set at 32.5 (A) and 55 (B) using MS 5.5 The chromatograms (total ion, extracted ion, and UV) obtained 154 from separation of aromatic sulfonates by reversed phase LC

using four different volatile ion-pairing agents (NH4OH (A, E), TOA (B, F), DHA (C, G) and TBA (D, H)

5.6 MS spectra of four phospholipids obtained from ESI-MS in both 161 positive (top) and negative (bottom) modes by direct infusion analysis 5.7 Chromatograms (A) and average MS spectra (B) of sample peaks 165 generated from reversed phase LC separation of phospholipids in the presence of TOA as the ion-pairing agent in mobile phase. 5.8 Chromatograms generated from the reversed phase LC 170

separation of phospholipids in the presence of NH4OH as the ion- pairing agent in mobile phase 5.9 Reconstructed base peak chromatograms produced from LC 173 separation of phospholipids in the presence of 5 mM TOA (A) and

NH4OH (B)

TABLE OF ABBREVIATIONS

Acetonitrile ACN Atmospheric pressure chemical ionization APCI Atropine AT Brompheniramine Br-Phe Benzenesulfonic acid BSA Cinnamic acid CA Capillary electrophoresis CE Chemiluminescence CL Chlorpheniramine Cl-Phe Dihexylamine DHA Detection limit DL Dimethylsulfoxide DMSO 2,4-Dinitrophenyl oxalate DNPO Electrochemical EC Electrospray ionization ESI Fast atom bombardment FAB Fluoroethylene propylene copolymer FEP Flow injection FI Fluorescence FL Fourier transform ion cyclotron resonance FT-ICR Gallic acid GA Gas chromatography GC Height equivalent to one theoretical plate HETP High performance liquid chromatography HPLC Ion-pair IP Liquid chromatography LC Liquid chromatography-mass spectrometry LC-MS Lactate dehydrogenase LDH

Lactate oxidase LO Mass spectrometry MS Nitroaniline NA 4-Nitrobenzoic acid 4-NBA Nitrofurantoin NF Nitronaphthalene NN 2-Nitrophenol 2-NP 3-Nitrophenol 3-NP 4-Nitrophenol 4-NP 1,3,6-Naphthalenetrisulfonic acid 1,3,6-NSA 1,5-Naphthalenedisulfonic acid 1,5-NSA 2-Naphthalenesulfonic acid 2-NSA Octadecyl silica ODS Phosphatidic acid PA Polycyclic aromatic hydrocarbons PAHs 2-Phenylbenzimidazole-5-sulfonic acid PBSA L-α-Phosphatidylethanolamine PE Polyethylene glycol PEG L-α-Phosphatidylinositol PI Photochemically initiated chemiluminescence PICL Photochemically induced fluorescence PIF Peroxyoxalate PO L-α-phosphatidylserine PS Photosensitizer PS Quinine sulfate QS Radio frequency RF Reactive oxygen species ROS Relative standard deviation RSD 2+ Tris (2,2’-bipyridyl) ruthenium (II) Ru(bpy)3 Supercritical fluid chromatography SFC Sodium salicylate SS

xii tri-n-Butylamine TBA 2,4,6-Trichlorophenyl oxalate TCPO 2-(3,6,9-Trioxadecyloxcarbonyl)-4-nitrophenyl oxalate TDPO tert-Octylamine TOA

xiii

DEDICATION

To my parents, Zhang Jincang and Fu Ping, To my aunt, Fu Xiamei To my sisters, Zhang Yina and Zhang Yilan To my wife, Zheng Ying

xiv

ACKNOWLEDGEMENTS

First of all, I would like to thank my research advisor, Dr. Neil Danielson, for his guidance and patient assistance during my graduate study in Miami University. I also thank him for encouraging me to try some new research ideas with his full support. It is my real honor to work with such a nice professor. Secondly, I would thank my graduate committee members, Dr. John Sebastian, Dr. André Sommer, Dr. Shouzhong Zou, and Dr. Catherine Almquist, for their help and reviewing my dissertation. Thirdly, I would liked to thank my colleagues and friends in Dr. Danielson’s research group, Lining Qi, Yaqian Liu, Martin Waichigo, Beata Musial, and Abdul Niewman for their help. I also would like to thank Yumin Chen, my best friend with whom I have had many interesting dialogues on research and many other academic areas, for his friendship and help. Furthermore, I would like to thank my parents for their support in everything I have attempted. Without their encouragement, I could not reach this stage in my life. I also want to thank my wife for her love and spiritual support. She has provided her assistance to type several parts of my dissertation. Finally, I would like to thank Miami University and Chemistry Department for awarding me a chance to pursue the Ph.D. degree.

Chapter I Introduction

This dissertation describes primary research on the development of photochemically initiated luminescence detection methods for flow injection and/or liquid chromatography (LC):

a direct chemiluminescence (CL) detection method for on-line determination of H2O2 and lactate using a 5.5 µL micro spiral flow cell (Chapter II), an on-line quinine-sensitized photooxidation and quenched luminol CL detection method for phenolic compounds (Chapter III), and an indirect fluorescence detection method for nonfluorescent or weakly fluorescent compounds that exhibit a special shielding effect on the UV photolysis of 2-phenylbenzimidazole-5-sulfonic acid (PBSA) (Chapter IV). Furthermore, the research on the separation of ionic aromatic sulfonates and phospholipids by LC-mass spectrometry (MS) using the more volatile ion-pairing agent tert- octylamine is also included in this dissertation (Chapter V). Chapter I is an introduction chapter that serves the purpose to briefly depict the background, theory, and development associated with luminescence, liquid chromatography, and mass spectrometry techniques. It consists of six sections. Section A will cover the chemiluminescence theory, chemiluminescence reactions, and their application as detection methods for flow injection (FI) and LC. Section B will discuss the molecular fluorescence theory and its applications in analytical chemistry. Section C will introduce the luminescence detection methods based on photochemically initiated fluorescence and chemiluminescence. Section D will focus on the introduction of fundamental chromatography theory, retention mechanisms in reversed-phase liquid chromatography and ion-pair chromatography, stationary phase selection, and LC instrumentation. Background information about LC-MS and its applications, interfaces, electrospray ionization process, and LC-MS instrumentation will be included in section E. The purpose of this research is described in section F of this chapter.

Section A: Chemiluminescence

Chemiluminescence (CL) phenomena have been known for a long time in nature in association with light emission from organisms such as the fireflies, luminous bacteria and protozoa, the sea pansy, the marine fireworm, and certain jellyfish [1,2]. In 1877, Bronislaus 1

Radziszewski synthesized lophine (2,4,5-triphenylimidazole) and observed the first CL phenomenon from a synthetic organic compound when it was shaken in air with an alkaline alcoholic solution [3]. However, the definition of CL was not established until Wiedemann classified six types of luminescence in 1888 [4], including CL as a result of a chemical reaction. The modern definition of CL is the emission of electromagnetic radiation (in UV, visible and near-infrared regions) as a result of the chemical reaction that yields an electronically excited species, which as it returns to its ground state, or transfers its energy to another species, generates light emission [5]. For a chemical reaction to be a CL reaction by producing light, the precursor(s) of the light-emitting species must participate in a reaction that releases a large amount of energy. It requires around 40-70 kcal/mol for visible light emission (400-750 nm). Figure 1.1 illustrates the energy pathway for a CL reaction. ∆HA is the activation energy for a dark reaction competing

with the CL pathway whose activation energy is ∆HA*. The reaction toward the CL pathway will

be favored when ∆HA* < ∆HA. Because of its exothermic energy requirement, CL reactions are largely limited to those redox reactions using oxygen and hydrogen peroxide or similar potential oxidants [6]. Two reaction pathways, type I and type II, represent the direct and indirect CL reaction mechanisms as shown in Figure 1.2. In the type I direct CL reaction, a CL reagent and an oxidant, in the presence of some cofactors, react to yield an intermediate or product that is in an electronically excited state, sometimes in the presence of a catalyst. When the excited species relaxes to its ground state, light emission is produced. Although the type II reaction also involves the formation of an excited species in its initial step, this intermediate is not the actual light emitter, but rather transfers its energy to a fluorophore, an energy acceptor. As the excited fluorophore returns to the ground state, light emission is produced. The type II CL reaction is also called a sensitized or energy transfer CL reaction. The efficiency of the CL reaction is an important parameter in determining the CL intensity, which is the ratio of total number of photons emitted to the number of molecules

reacting and is also represented by the quantum yield. This quantum yield (ΦCL) can be defined for a type I CL reaction as:

ΦCL = ΦR × ΦES × ΦF

C* + D ∆HA

∆HA* A + B

Potential energy level ∆H

C + D

Reaction coordinate

Figure 1.1. Energy pathway for chemiluminescence.

A + B + ( Cofactors) (CL precursor) (Oxidant) r) o catalyst h p ro o lu P* (f Energy Transfer CL Direct CL F (Type I) (Type II) P + F* F + hν P + hν

Figure 1.2. Types of chemiluminescence.

where ΦR is the chemical yield of molecules that are going through the CL pathway, ΦES is the yield of excited state molecules, and ΦF is the fluorescence quantum yield of excited molecules that actually produce a photon. The quantum yield for a type II CL reaction can be written as:

ΦCL = ΦR × ΦES ×ΦET × ΦF where ΦET is the efficiency of energy transfer from the initially excited species to the

fluorophore. If there are no dark reactions competing with the CL pathway, ΦR is practically taken as 1. The CL efficiency is primarily dependent on the factors that influence the formation of species in the excited state and the pathways for such species to lose energy, e.g. luminescence, molecular dissociation, intra- and intermolecular energy transfer, and quenching by solvent or other molecules [5]. Therefore, experimental factors, such as the CL precursors, chemical structure, catalysts, metal ions, temperature, pH and ionic strength, hydrophobicity of the solvent, and the nature and concentration of other substrates can all affect the CL pathways and favor other nonradiative pathways, Energy transfer acceptors or quenching molecules will also have an effect on the CL quantum yield and the CL intensity. The CL intensity, depending on the quantum yield and the rate of CL reaction, can be expressed as:

ICL = ΦCL(-dA/dt)

where ΦCL is the CL quantum yield and (-dA/dt) is the rate at which the CL precursor A is consumed. In the type I CL reaction, the formation of the excited product is usually a rate-

limiting step. If the CL precursor A or oxidizing agent B is in excess, the ICL can be rewritten as:

ICL = ΦCL kr [A] or ΦCL kr [B]

where kr is a rate constant of the oxidation reaction which produces an excited product. If the reaction conditions are well controlled, there is a linear quantitative relationship between the CL intensity and concentration of the CL reagent or substrate participating in the CL reaction. Among many CL reactions discovered so far, only a few of them have become practical

detection techniques used in FI and LC due to low CL efficiency with ΦCL below 0.001, and/or CL emission wavelengths beyond the UV-Visible region needed for most common PMT detectors [6]. The CL reactions commonly used for CL detection in FI and LC are shown in Figure 1.3. Oxidation of luminol in basic solution as shown in Figure 1.3 (a) is one of the most

studied and versatile Type I CL reaction, having a ΦCL value of about 0.01 in water and 0.05 in 5 (a) NH NH 2 O NH 2 O 2 O * NH O base N + Oxidant + N2 NH O metal ion N

O HO OO O

NH2 O

O + hν O

O (b) O O O O Fluorophore C C Ar O C C O Ar + H2O2 OO

O O

C C Fluorophor Fluorophore * + 2CO2 OO

Fluorophore * Fluorophore + hν

O N CH3

CH3 N

+ hν

(d)

CH3 CH3 N N * oxidant or reductant hν base

C O O OR

Figure 1.3. (a) Luminol CL reaction. (b) Peroxyoxalate CL reaction. (c) Lucigenin CL reaction. (d) Acridinium ester CL reaction.

6 dimethylsulfoxide (DMSO). By controlling certain experimental conditions, a linear response between CL intensity and the concentration of an oxidant or catalyst or enhancer or inhibitor can − − be established. The primary oxidant for the luminol-CL reaction can be H2O2, MnO4 , OCL , I2, 2− 1 NO2, and S2O8 [7-12]. H2O2 and reactive oxygen species such as singlet oxygen (O2 ), and superoxide (O2•-) are useful oxidants because those species are generated through metabolism in biosystems and certain photochemical reactions permitting the indirect determination of certain biomolecules and other organic compounds [13-16]. Oxidation of luminol in aqueous solution requires the use of a catalyst, such as a transition metal ion (e.g. Co(II), Cu(II), Cr(III) and Fe(II)), ferricyanide, an organometallic complex (e.g. hemin), or a peroxidase (e.g. horseradish peroxidase) [17-23]. Some metal ions that are able to quench luminol-CL, such as Th(IV), Tl(III), Sb(III), Zn(II), and Cd(II), can be determined by this detection method [24-27]. Quenched luminol-CL can also be used to indirectly determine ascorbic acid due to its antioxidative ability [28]. Many other compounds can be determined by this luminol-CL method

if they have an ability to produce a reactive oxidizing species. Glucose and lactate produce H2O2 when the oxidation reactions are catalyzed by glucose oxidase and lactate oxidase [29], respectively. Another peroxyoxalate (PO) reaction-based methods produce CL light by oxidation of aryl oxalate esters by hydrogen peroxide in the presence of a fluorophore as shown in Figure 1.3(b). PO chemistry provides the most efficient non-enzymatically catalyzed CL reaction, reaching a quantum yield of 0.34 in the best case. The PO reaction mechanism follows the type II pathway. A high-energy intermediate, 1,2-dioxetanedione, generated from the oxidation of oxalate by H2O2, forms a charged complex with the fluorophore, donating one electron to the intermediate. As this electron is transferred back to the fluorophore, an electronically excited

state of the fluorophore is reached with liberation of CO2. CL emission is actually produced from the fluorophore. The most commonly used PO reagent includes 2,4,6-trichlorophenyl (TCPO), 2,4-dinitrophenyl (DNPO), and 2-(3,6,9-trioxadecyloxcarbonylyl)-4-nitrophenyl (TDPO) oxalates [30-32]. Because the CL intensity is proportional to the concentration of substrates, such as oxalates, hydrogen peroxide, and fluorophores, a variety of compounds can be directly or indirectly determined by PO CL-based detection methods. Fluorescent polycyclic aromatic hydrocarbons and nonfluorescent or weakly fluorescent compounds amino acids, carboxylic acids, aliphatic amines, catecholamines, nitrophenols and unsaturated disaccharides that can be

7 pre-derivatized to a fluorescent species, as well as compounds such as glucose, uric acid, and cholesterol, whose enzyme-catalyzed oxidation reactions usually generate H2O2 as a byproduct are examples [30, 33-42]. Both lucigenin and acridinum ester react with hydrogen peroxide in the presence of a base to generate CL light (Figures 1.3(c) and 1.3(d)). The relaxation of N-methylacridone that is generated either from the decomposition of a dioxetane intermediate formed in the lucigenin oxidation step or from a dioxetanone formed in the acridinium ester oxidation step is believed to be the result of CL emission at 440 nm. Lucigenine-CL can occur without a catalyst, but a relatively intense CL emission can occur in the presence of a transition metal ion as a catalyst. CL emission is also observed from the reactions of lucigenin with various reducing compounds. Lucigenin-CL is very similar to luminol-CL. Both can be used to measure the reactive oxygen ·- species; lucigenin-CL is selective toward the superoxide (O2 ) species [43]. The application of this type of CL reaction has been reported for the determination of transition metal ions (e.g. Co2+, Cr3+, Fe3+, Mn2+, and Pb2+, and Ag+), reducing compounds (e.g. Cr2+, Fe2+, Mo5+), ascorbic

acid, tetracyclines, glucose, oxidants (e.g. H2O2 and reactive oxygen species), and compounds

generating H2O2 [44-55]. 2+ Tris (2,2’-bipyridyl) ruthenium (II) (Ru(bpy)3 ) electrochemically reacts with itself to 3+ +. generate the oxidized and reduced species, Ru(bpy)3 and Ru(bpy)3 These species react with 2+ certain reductants and oxidants, respectively, to form the excited [Ru(bpy)3 ]*, which emits light as it relaxes to the ground state. This elctrogenerated CL process can be expressed by the following equations:

2+ 3+ − Ru(bpy)3 → Ru(bpy)3 + e electrochemical oxidation 2+ − + Ru(bpy)3 + e → Ru(bpy)3 electrochemical reduction 3+ 2+ Ru(bpy)3 + reductant (analyte) → Ru(bpy)3 * + product + 2+ Ru(bpy)3 + oxidant (analyte) → Ru(bpy)3 * + product 2+ 2+ Ru(bpy)3 * → Ru(bpy)3 + hν

2+ This Ru(bpy)3 -CL reaction has been applied to determine organic acids (e.g. oxalate, pruvate, and ascorbic acid), aliphatic amines, amino acids, alkaloids, pharmaceutical compounds containing the amine functionality (e.g. antihistamines and antibiotics), and biochemical 8 compounds (e.g. NADH and DNA PCR products) [56-64]. The decreasing sensitivity order for aliphatic amines is tertiary, secondary, and primary, opposite to fluorescence derivatization methods [65a, 65b]. The instrumentation for CL detection is remarkably simple and may be composed of a reaction cell (e.g. a quartz flow cell and a Teflon coil reactor), a light detector (e.g. a photomultiplier tube (PMT)), a light-tight chamber that holds the reaction cell and light detector inside, a device for introducing and pre-mixing CL reagents and/or the sample, and a data processor or recorder. Because the CL measurement is taken against a dark background, the background noise is extremely low usually resulting in excellent detection limits. Besides their extensive use in FI and LC, CL detection techniques have been coupled to capillary electrophoresis (CE) or immunoassay.

Section B: Fluorescence

Fluorescence (FL) is luminescence from a species in the singlet excited state, previously generated by absorbing electromagnetic radiation, when it returns to the ground state (Figure 1.4). It returns to its lowest excited state by a series of rapid vibrational relaxations and internal conversion with no emission of radiation produced. FL arises from a transition from the lowest vibrational level of the first excited electronic state to one of its vibrational levels of the electronic ground state. Due to the radiationless vibrational relaxation step involved in most cases, molecular FL emission bands occur at longer wavelengths than that for excitation light. The occurrence of FL is associated with an energy transition, such as σ → σ*, n → π* and π → π*. FL resulting from σ → σ* is seldom observed because it requires excitation of the molecule by UV light below 250 nm, which will cause deactivation of the excited states by predissociation or dissociation. Although FL is produced most effectively when the lowest electronically excited state is reached by a n → π* transition from the ground state, it is more likely to be reached by a π → π* transition to a higher energy excited state followed by internal conversion. The inherent life-time associated with a n → π* transition is much longer than the π → π* transition; as a result, there is a much greater chance of intersystem crossing (singlet-to- triplet transition). Therefore, FL is more commonly produced by a π → π* transition for which the deactivation processes that compete with FL are less likely [66].

Vibrational relaxation Intersystem conversion

s n

e o

r i

o t

p r

p o

s s

P A Fluorescence .

Ground

state λ1 λ2 λ3

Figure 1.4. Energy diagram showing fluorescence transitions

The quantum yield (Φf) of FL is calculated from the ratio of the number of molecules that

luminesce to the total number of excited molecules. Values of Φf are in the range of 0 to 1, which

are largely determined by structure. A high value of Φf is generally associated with molecules possessing a highly delocalized system of conjugated double bonds that result in a relatively rigid structure [66,67]. Compounds such as fluorescein, 2-phenylbenzimidazole, anthracene, perylene, and other polycyclic aromatic hydrocarbons (PAHs) with condensed aromatic ring structures, usually exhibit intensive FL due to the low energy π → π* transition levels. Besides the structural effect, temperature, solvent, pH and dissolved oxygen will affect the FL quantum yield to some degree.

The FL intensity (If) is given by: −εbc If = K’I0 (1 − 10 )

where K’ is a constant related to the quantum yield, I0 is the intensity of the excitation light, ε is the molar absorption coefficient, c is the concentration of the fluorescent compound, and b is the path length of the cell. If εbc < 0.05 as is the case for quite diluted solutions, the equation above can be simplified to:

If = 2.3K’I0 εbc = Kc FL intensity is largely dependent on the power of the light source as well as the concentration of the fluorescent compound. When the power of the excitation source is constant, there is a linear relationship between the concentration of the compound and its FL intensity, which allows for the quantitative determination of compounds. The basic instrumentation for FL detection as shown in Figure 1.5 consists of a source of UV radiation, an excitation wavelength selector or monochromator, sample holder (e.g. a quartz curvette for the static FL measurement or a quartz flow cell for continuous FL measurement in flow injection or HPLC), an emission monochromator, a PMT detector to respond to the light emission, and a read-out system to record the FL signals. Because the second monochromator is placed perpendicular to the incident light beam and in most cases the FL emission with a longer wavelength than the excitation light is detected by the PMT, the background signal from the excitation light is minimized. As a consequence, the FL signal is measured against a virtually black background and thus has a high signal-to-noise ratio. Furthermore, the FL signal intensity can be enhanced simply by using a powerful excitation source, e.g. a laser source, without bringing in background noise to the signal. In contrast, the use of a powerful excitation source

sample P λex 0 source excitation monochromator emission

monochromator

λem

PMT

Figure 1.5. Simplified diagram of a FL detector

12 will not change the absorbance but greatly raise the background noise level because the UV absorbance measurement is taken for the incident and emitting light at the same wavelength including any background noise from the excitation source. Therefore, FL detection is much more sensitive than UV absorbance. Direct FL detection is limited to a small number of compounds exhibiting sufficiently intensive FL, e.g. quinine, hydroquinone, and PAHs. However, chemical derivatization techniques can make FL detection methods suitable for determination of a variety of non- fluorescent or weakly fluorescent compounds by FI and LC. For instance, 5- dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) has been used as a pre- or postcolumn fluorescence labeling agent that introduces a fluorophore into the structure of primary amines, secondary amines, and compounds carrying phenolic, alcoholic hydroxyl, or thiol groups [68-73]. The detection limits of those compounds with FL detection are in the sub- ng-to-pg range. There are quite a few chemical reagents avaliable for off-line or on-line FL derivatization of organic compounds, e.g. o-phthaldialdehyde for amines and amino acids, 4- bromomethyl-7-methoxycoumarin for carboxylic acids, and 5-dimethylaminonaphthalene-1- sulfonylhydrazine for compounds with carbonyl groups [74-76]. FL derivatization via a complex formed between a metal ion and an organic compound or chelating agent has been also applied for the FL determination of either metal ions or organic species [77]. Besides the chemical derivatization techniques, photochemical derivatization techniques have been used recently for the FL determination of non-fluorescent or weakly fluorescent compounds, such as thiamine and the insecticide imidacloprid [78,79]. Furthermore, indirect FL methods based on the formation of a stable or temporary complex between a fluorophore and a quencher (analyte) that results in the decrease of FL from the fluorophore have been reported for the indirect determination of nitro compounds [80,81].

Section C: Photochemically initiated luminescence detection

Although luminescence detection techniques based on CL and FL have achieved low detection limits and high sensitivity in LC and FI analysis, their use is largely confined to compounds that either participate in the CL reactions through an enhancement or inhibition effect or exhibit strong FL themselves or strongly quench the FL of fluorophores. Chemical

13 derivatization techniques have been most commonly applied for the determination of those compounds lacking CL and FL functionalities. However, on-line chemical derivatization has encountered some problems: low derivatization yield, excess reagent and byproduct interferences toward detection, long reaction times, and required sample cleanup and preconcentration [82]. Photochemical reactions have been demonstrated to be an efficient pre-or post-column option. The analytical potential of photochemical reactions arises from the inherent advantages of this type of reaction over reactions with chemical reagents. Because light is the only “reagent” that is used to induce on-line photochemical reactions, no other reagents except the photolyzed sample reach the detector and the selectivity is greatly enhanced due to the photochemical reaction that is easily controlled by using a certain type of light source, its power, and irradiation time. Furthermore, on-line photochemical reaction systems are easy to be implemented. The photoreactor can be as simple as PTFE tubing helically coiled around the lamp, which is easily coupled between a column and a detector. Photochemically initated (PI) CL is a type of luminescence detection technique. The main feature of the PICL detection is a combination of the simple and achievable photochemical reactions that produce certain species with their sensitive CL detection. For instance, photolysis of photosensitizers (PS) can generate superoxide, singlet oxygen and other reactive oxygen species via two possibly pathways:

1 3 PS + O2 O2 hν ISC (1) PS → 1PS* → 3PS* 3 O2 •+ •− PS + O2

PS•− + R•+ R hν ISC (2) PS → 1PS* → 3PS*

RH PSH• + R•

•− • 3 •− • PS or PSH + O2 → O2 or HO2 + PS

where 3PS* is the photosensitizer in the triplet excited state that is produced from 1PS* in the excited singlet state via intersystem crossing, and R or RH is a reducing agent. A number of

compounds, such as methylene blue, rose bengal, riboflavin, perylenequinone derivatives, TiO2 and even luminol can be used as photosensitizers [83-86]. Those oxygen species are very sensitively detected by reacting with luminol and lucigenin to produce CL. Besides photosensitizers and reducing agents in the second pathway acting as analytes for PICL, theoretically, compounds that are subject to the photooxidation consuming those reactive oxygen species can also be determined via the quenching effect on CL intensity. As far as I know, no one has reported the quantitative determination of those compounds by this type of PICL detection method with FI and LC, but it has been used to determine the superoxide dismutase activity, antioxidant activity in biological systems, and the antioxidant properties of wine and tea [87-89]. In addition to the PICL method mentioned above, other PICL techniques based on photogeneration of CL species or intermediates, oxidants, and catalysts for CL reactions have been applied successfully for FI and LC determination of compounds that do not directly participate in the CL reaction process. The determination of lactate been carried out by 3+ 2+ photodecomposition of lactate in the presence of Fe and UO2 and then coupled with luminol- CL detection of generated Fe2+ as a catalyst [90]. Besides its ability to make compounds detectable by CL techniques, PICL can greatly improve the sensitivity of the conventional CL 3+ detection, such as [Ru(bpy)3] -based CL detection of aromatic amines [91]. Polyols have been 3+ determined by [Ru(III)(bpy)3] -based CL detection of oxalate that is generated from on-line photooxidation of polyols [92]. PICL determination of sparfloxacin takes advantage of a complex formed between Tb3+ and a photoproduct from on-line photolysis of sparfloxacin, 2− which sensitizes the CL reaction of Ce (IV)-SO3 [93]. Inorganic nitrate has also been determined by PICL techniques because photogenerated peroxonitrite can undergo a CL reaction with luminol [94]. Furthermore, CL from photolyzed azaaromatics (e.g. acridines, quinoline, isoquinolines, and phenanthrolines) in amide solvents has been observed upon addition of base, indicating potential as an analytical PICL detection method [95]. Another type of luminescence detection technique is photochemically induced fluorescence (PIF) detection. On-line PIF detection instrumentation is simply made up of a photoreactor in which several types of non-fluorescent or weakly fluorescent compounds

15 undergo photochemical reactions generating strongly fluorescent photoproducts. Because most photochemical reactions take place via free radicals, the PIF conversion efficiency is quite high. PIF methods have been successfully coupled with FI and LC for determination of quite a few compounds, such as aflatoxins, phenothiazines, chlorophenoyacid herbcides, benzoylurea insecticides, and folic acid [96-100]. Those photoreactive compounds have been detected by PIF conversion to their fluorescent derivatives without any sensitizer or oxidizing reagent present. For organic compounds not so photoreactive, a photosensitizer or oxidizing reagent is generally needed to improve the PIF conversion rate. For instance, PIF conversion of methotrexate and its metabolite, nicotinic acid and nicotinamide, kynurenines, and pyridinecarboxylic acids has been achieved by irradiation in the presence of hydrogen peroxide [101-103]. The use of acetone, sodium sulfite, and TiO2 as photosensitizers for the photochemical reactions of menadione, sodium bisulfite, sulfamethazine, and 2,6-dimethylphenol, respectively, has enabled PIF conversion of those compounds for FL detection [104-106]. Some organized media, such as cyclodextrin and surfactant-based micelles have been proven useful to enhance the PIF detection of nonfluorescent herbicides and pesticides [107,108]. However, those PIF methods are limited to compounds that are easily and efficiently converted to fluorophores by UV irradiation, sometimes in the presence of a photosensitizer and an oxidizing reagent. Therefore, it is necessary to develop new methods taking advantage of an on-line photochemical reactions and the high sensitivity of FL detection for FI and LC determination of those compounds that are not easily amenable for PIF conversion.

Section D: Liquid chromatography

The development of chromatography is dated back to a century ago. A Russian botanist Michael Tswett used a glass column packed with a solid adsorbent under liquid flow to carry out research work on chlorophyll extracts and published the report in 1903, which became the first systematic work of what is recognized now as chromatography. He also first introduced the definition of chromatography, “a method in which the components of a mixture are separated on a adsorbent column in a flow condition”, in another early paper in 1906 [109]. However, establishment of modern chromatography became possible after Martin and Synge’s work on partition chromatography of amino acids using silica gel particles wetted with water in 1941 and

16 the plate theory of chromatography that lead to their Nobel Prize in 1952 [110,111]. Gas chromatography was developed by Martin and his co-worker James in 1950s [112]. Because thermally unstable and non-volatile compounds cannot be determined by GC, high performance LC (HPLC) was developed and improved by Huber, Kirkland, Knox, Horváth, Snyder and Scott between the 1960s and 70s. Separation of the components with high resolution was achieved by using a column packed with usually 5 or 10µm µm-size solid particles and a high-pressure pump to transport a liquid mobile phase through the column at a sufficient flow rate. Although the acronym HPLC is still used, it is now often assumed that just LC means HPLC. LC will be used in that context in this dissertation. Among chromatography-based separation techniques, LC has become at least as popular as GC with numerous applications in quality control, drug discovery and formulation, synthetic chemistry, biomedical research and other disciplines associated with chemical and biological systems. The general instrumentation of LC is made up of the mobile phase reservoirs, the high pressure pumps that are used to transport the mobile phase either under isocratic or gradient conditions, a sample injector that is used to introduce a certain amount of sample into a high- pressure stream of the mobile phase, a column in which the chromatography process is occurring, a detector with a flow cell (e.g. UV, refractive index, FL, electrochemical, and mass spectrometric detectors), and data acquisition and read-out. A classical way to describe the chromatography process in LC is as follows: the distribution of solutes between a stationary phase and a liquid mobile phase is continuously taking place while solutes are migrating in the flowing direction of the mobile phase. Those solutes, distributed preferentially in the mobile phase, will move more rapidly than those distributed preferentially in the stationary phase. As a consequence, the solutes will elute in order of their increasing distribution coefficients with respect to the stationary phase. The distribution coefficient (K) of each solute can be expressed as:

K = CS / CM where CS is the concentration of a component in the stationary phase and CM is the concentration of a component in the mobile phase.

Typical chromatographic peaks are shown in Figure 1.6. The retention time (tR) is measured from the time when the sample is injected and the time when a component is eluting

tR2

tR1 Component 1 Component 2

Signal

w1 w2

Time

Figure 1.6.The chromatogram and its characteristic parameters

from the column and t0 is the elution time of an unretained component. The fundamental dimensionless measurement of retention of components in LC is the retention factor (k’) that can

be calculated by substituting tR and to into the equation below:

k’ = (tR − to) / to The retention factor is more practical than the distribution coefficient for describing the ability of a stationary phase to retain components because the difference in the retention property of each component in LC results in a difference of the elution time of each component from the LC column and it is easy to measure time than the concentrations of a solute distributed in a stationary phase and a mobile phase. Another advantage is that k’ is independent of flow rate and column dimensions. The result of separation of two adjacent peaks can be evaluated by two

parameters, selectivity (α) and resolution (Rs). The selectivity is defined by,

α = k2’ / k1’

where k2’ and k1’ are the retention factors of the first and second peaks to elute, respectively. When α is around 1.1, the baseline resolution of two adjacent peaks can be achieved if k’ is controlled between 2 and 10. Resolution is calculated from the following equation:

RS = (tR2 − tR1) / (0.5 (w1 + w2))

where tR1, w1 and tR2, w2 are retention time and peak width of the first peak and second peak,

respectively. When Rs is 1.5, the two adjacent peaks are basically baseline resolved. The number of theoretical plates (N) used to represent the column efficiency, is expressed by the following two equations: 2 2 N = 16 (tR / wb) = 5.54 (tR / w0.5)

where wb and w0.5 are the peak widths at the baseline and half height, respectively. N is also associated with band broadening of peaks in the chromatogram. The higher the column efficiency, the less band broadening of the peaks in a given segment of the column. Such a relationship can be expressed in the equation below, H = L / N where H is a band broadening parameter that is defined as the height equivalent to one theoretical plate (HETP) and L is the column length. Because separation is taking place on the column, the retention properties of components, the ability of the stationary phase to selectively retain the components, and most importantly the

19 column efficiency will all impact peak resolution. Thus, resolution can be rewritten by an integration of k’, α and N parameters into the following expression: 1/2 RS = (1/4) (N) (k’1 / (k’1 + 1)) ((α − 1) / α) In order to achieve a fast separation with high resolution, the k’ (2-10) and α (around 1.1) need to be controlled by optimizing experimental conditions, e.g. mobile phase solvent ratio, and flow rate, using a highly efficient column. LC can be categorized into at least four separation modes: normal-phase, reversed-phase, ion-exchange, and size-exclusion. Normal-phase LC is limited to the separation of fairly nonpolar compounds with a relatively small polarity difference between the non-polar mobile phase (e.g. hexane (δ = 7.0) and the polar silica stationary phase (δ = 17). Reversed-phase LC with a large polarity difference between the polar mobile phase (e.g. water (solubility parameter (δ) = 23.4) and the non-polar stationary phase (e.g. octadecyl silica (δ = 7.0)) will allow separation of compounds with a wider range of polarity as compared to the normal-phase LC [113]. Inorganic ionic species are best separated by ion-exchange LC while large molecules, e.g. polymers, are best suited for separation by size-exclusion LC. However, reversed-phase LC, because of its compatibility with aqueous sample solutions, is the most commonly used mode of LC for the analytical and preparative separation of compounds in the chemical, biological, pharmaceutical and biomedical areas. Even some ionizable organic compounds can also be separated using an ion-pairing agent in the mobile phase by reversed-phase LC. The remaining discussion of LC is focused on the introduction of reversed-phase LC. The most commonly used stationary phase in reversed-phase LC is silica covalently bonded with octadecylsilane (ODS or C18) via a siloxane linkage. Other analogous hydrocarbon

type of stationary phases include octylsilyl silica (C8), butylsilyl silica (C4), and methylsilyl silica

(C1). Cyano-bonded and phenyl-bonded phases are also used in reversed-phase LC because those phases provide a difference in selectivity to ODS. Furthermore, polymer-based stationary phases, e.g. cross-linked poly(divinylbenzyl)styrene, have been developed for reversed-phase LC due to its applicability for mobile phases with a wide pH range (pH 1 to 13). The choice of stationary phases is based not only on solvophobic interactions but also on other mechanisms, e.g. polar interactions with free silanol groups, dipole-dipole interactions on cyano-bonded phases, and π-π stacking interactions on phenyl-bonded phases. Practically, ODS is suitable for separation of nonpolar to moderately polar solutes while the short-chain hydrocarbon stationary phases are 20 better for more polar compounds. Cyano-bonded phases are good for highly polar compounds that have a little retention on ODS. Phenyl-bonded phases are quite effective for the retention of polycyclic aromatic hydrocarbons. The retention mechanism of reversed-phased LC is associated with the hydrophobic effect. Retention of solutes by such a nonpolar stationary phase is due to the repulsion of the hydrophobic solutes by water molecules in the mobile phase. A more detailed description of retention mechanism based on the solvophobic theory can be obtained from Horváth and his coworkers’ work in 1976 [114]. Although the hydrophobic interactions are determined by the specific solute-solvent interactions in the mobile and stationary phases, retention in reversed-phase LC is very much controlled by interactions in the mobile phase because the composition of the stationary phase remains constant with the change in composition of the mobile phase. The effects of solvent ratio and solvent strength on the retention in reversed-phase LC have been proposed by Synder and his coworkers [115] in the following equation:

log k’ = log k’w – S Φ where S is the solvent strength parameter, Φ is the organic solvent composition (v/v) in the

mobile phase that is prepared from mixing of water and organic solvent, and k’w is the retention factor of solute in a completely aqueous mobile phase (Φ = 0). In reversed-phase LC, usually, the mobile phase is composed of water, the weakest solvent in terms of solvent strength (S = 0), and an organic solvent used to modify the polarity of the mobile phase. Unlike normal-phase LC in which a variety of solvents can be chosen to prepare a relatively non-polar mobile phase, only a few organic solvents that are not only miscible with water but also have a low UV cutoff can be used to prepare the mobile phase with water. These include methanol (S = 2.6), acetonitrile (S = 3.2), 2-propanol (S = 4.2) and tetrahydrofuran (S = 4.5) [116]. When a certain type of organic solvent is chosen to prepare the mobile phase with water, the retention factor of the solute

decreases with increasing organic solvent composition in the mobile phase because k’w and S values are constant. Based on such an effect of the mobile phase composition on retention properties of solutes, gradient elution by changing the solvent ratio in the mobile phase is commonly applied in reversed-phase LC to achieve a fast separation. Figure 1.7 exemplifies the effects of varied organic solvent contents in mobile phase on the results of LC separations of two molecules with a large difference in hydrophobicity.

Log k’ Biomolecule (B)

Small molecule (S)

A B C % Organic solvent S

B A B S B

B S

Time

Figure 1.7. The effects of varied organic solvent content in the mobile phase on the elution behavior of a large biomolecule and a small organic molecule separated by reversed phase HPLC.

Reversed-phase LC applied to the separation of ionic compounds in the presence of an ion-pairing agent in the mobile phase is termed reversed-phase ion-pair LC, an alternative to ion- exchange LC. The aim for using a chemical agent (ion-pairing agent) with an opposite charge to the ionic analytes in the mobile phase is to form neutral ion-pairs with analyte ions to facilitate retention by reversed-phased LC. Quarternary amines (e.g. tetrabutylammonium) have been used as ion-pairing agents for the separation of anionic analytes, such as sulphonated aromatic compounds and carboxylic acids [117-119]. Perchloric acid and alkylsulphates (e.g. dodecyl (lauryl) sulfate) have been used as ion-pair agents for many basic samples [120,121]. There are two possible mechanisms for the retention of solutes by reversed-phase ion-pair LC [122,123]. The first model is formation of the ion-pair in the mobile phase followed by distribution of the ion-pair between the mobile phase and stationary phase. The other is a dynamic ion-exchange process in which the analyte interacts with the ion-pairing agent adsorbed on the surface of the stationary phase. According to the complete thermodynamic equilibria as shown in Figure 1.8, Harváth et al. and Foley [122,124] have obtained the mathematical expression of retention factors for both mechanisms:

k’ = ( (KA + K1 KIPA [IP]m) VS) / ((1 + K1 [IP]m) Vm) (first mechanism)

k’ = ( (KA + K2 KIP [IP]m) VS) / ((1 + K2 [IP]m) Vm)) (second mechanism) − where KA, KIP and KIPA are the distribution coefficients of the anionic analyte, A , cationic ion- + + − pair agent, IP , and the formed ion-pair, IP A , respectively, and K1 and K2 are the ion-pair formation constants in the mobile and stationary phases, respectively. Both expressions are equivalent to each other, which means that two mechanisms are possible and indistinguishable. Furthermore, both equations predict that retention increases with either increasing adsorption of the formed ion-pair (IP+A−) or ion-pair agent (IP) onto the stationary phase. In general, for ion- pairing agents with short alkyl chains, the first ion-pairing formation mechanism appears to govern retention whereas for ion-pairing agents with long chains, the ion-exchange mechanism dominates [123,125]. Therefore, it is important to control the concentration and the type of ion- pair agent, temperature, and other experimental parameters that contribute to the change of adsorption of either the ion-pair reagent or the formed ion-pair onto the stationary phase.

KA − − Am AS

+ + KIP + + IP m IP S

K1 K2 KIPA + − + − IP A m IP A S

Mobile phase Stationary phase

Figure 1.8. Thermodynamic equilibria involved in reversed phase ion-pair LC.

Section E: Liquid chromatography-mass spectrometry (LC-MS)

Mass spectrometry (MS) detection has revolutionized LC-based analytical techniques in many ways. First of all, MS provides the molecular weight and structural information of compounds of interest and makes on-line identification of chemical species eluting from the LC column more positive. LC with a conventional detector, e.g. UV, FL and refractive index, only provides information of the relative retention factors of sample component peaks as compared to standards. Sometimes, it is hard to know what standards to run for unknown samples. Secondly, MS is essentially a universal detector by which many compounds lacking either a chromophore or fluorophore for UV and FL detection can be detected without any chemical derivatization. Thirdly, MS detection can be more sensitive than some conventional detection techniques. Although the current detection limits of MS may not be as good as those by fluorescence or electrochemical detection, it has the potential to improve if sample introduction and ionization efficiency can continue to be improved. Furthermore, LC-MS can allow a mobile phase gradient program to be run during the separation with little effect on the ion chromatogram, but running a gradient elution with an electrochemical or refractive index detector can be problematic. Finally, LC coupled with MS has become a more user-friendly technique that can provide solutions to analytical problems. The history of LC-MS started in the early 1970s. In order to couple LC with MS that requires the detection of ions under high vacuum conditions, the interface must take a flowing liquid stream to acceptable MS pressure (<10-3 Pa) while transferring as much solute as possible with little band broadening. At least seven interfaces have been developed since the 70s, including direct liquid introduction (DLI), moving-belt, monodisperse aerosol generation (MAGIC) or particle beam, thermospray, continuous-flow fast atom bombardment (FAB), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) [126-132]. Among those interfaces, ESI has become the most common interface in commercial LC-MS. Development of the ESI interface for MS had a tremendous impact on modern MS methodology culminating in the 2002 Nobel Prize in chemistry. Before ESI was available, interfaces used for continuous introduction and ionization of solution samples in commercial MS were mostly the thermospray and APCI interfaces. Thermospray is not suitable for introduction and ionization of nonvolatile and thermally unstable samples because ionization of the sample

25 molecules results from the thermal vaporization process in the presence of aqueous ammonium acetate in a heated capillary. APCI is an efficient technique in which the vaporized mobile phase reacting with high energy electrons generated from a 63Ni β− emitter or a corona discharge generates various adduct ions for chemical ionization of sample molecules, but it is only applied for ionization of small molecules. Those limitations, which prevented MS from analyzing nonvolatile compounds, e.g. metal complexes, phospholipids, peptides, proteins and synthetic polymers, have been overcome by the ESI technique based on ionization of molecules by spraying a solution from the tip of an electrically charged capillary. This is a “soft” ionization technique by which those large biomolecular ions may retain their solution-phase three- dimensional conformation [133]. Multiple charging from ESI allows for determination of high- mass molecules (e.g. polypeptides and proteins) with a relatively low m/z range MS instrument [134,135]. The general ESI process starts when solution passes through a metal capillary with a high potential, e.g. 4000-4500 V, held at the tip that is surrounded by a concentric tube with a nebulizing gas flowing through it. Under the effect of an electrostatic field and the shear force generated by the nebulizing gas from a pneumatic nebulizer, a mist of highly charged droplets is generated, passing down to the MS inlet. During that transition, evaporation of solvent is occurring as a countercurrent dry gas and heat are applied to the droplets before they enter the vacuum of MS. A decrease in the droplet size occurs and the charge density on the surface is raised. When the force of the Coulombic repulsion exceeds the surface tension, the droplet explosion (Columb explosion) generates much smaller droplets containing a single ion, eventually leading to the formation of bare analyte ions that are directed into an orifice through electrostatic lenses leading to the mass analyzer. Figure 1.9 shows the ion formation from a droplet generated by the ESI process. By switching the polarity of the potential applied to the capillary for ESI, either positive or negative ions can be generated. Furthermore, selection of appropriate solvents to prepare sample solutions is critical in enhancing the sensitivity for both positive and negative ion formation through ESI. The ESI interface as a continuous sample introduction and ionization device has been coupled with nearly all major mass analyzers, including quadrupole, quadrupole ion

+•_+_•+_+ +_•+ _+•_+ _•+_ +_•+_•+_+•_+_+ Droplet +_ •+_+•_+•_+ +_•+ _+_

Dry gas and heat

Solvent evaporation + +• +•+ + _+•_+ +•+ +• •+_•+_ + +•+ Coulomb + +•++ explosion Further droplet

fission and /or Ion evaporation

Figure 1.9. Diagram for electrospray ionization process

27 trap, magnetic sector, Fourier transform ion cyclotron resonance (FT-ICR), and time of flight. Figure 1.10 shows the instrumentation of the Bruker Esquire-HP LC-MS with an ESI interface coupled with an ion trap mass analyzer. An ESI source in which a pneumatic nebulizing needle is orthogonally positioned relative to the sampling orifice is used to introduce and ionize the sample solution from the LC column. Analyte ions are generated by solvent evaporation followed by Coulombic explosion before entering the sample orifice. Under the influence of pressure and electrostatic gradients, ions pass through the glass capillary into the transport and focusing region. Two skimmers are used to remove the bulk of the dry N2 gas and some remaining solvent vapor. Ions pass into the first octopole ion guide that focuses and transport the ions to the second octopole with a relatively low pressure. After going through the second octopole ion guide and lenses, ions reach the ion trap mass analyzer. The ion trap is made up of a ring electrode between two endcap electrodes. A high radio frequency (RF) voltage from the primary RF generator is applied to the ring, while the endcap electrodes are held at ground. A pseudo-potential well is formed inside the quadrupole field. The ion trapped in the ion trap will move in an oscillating harmonic motion with a secular frequency that is determined by the m/z ratio of the ion and the RF drive level. The exit endcap opposite to the detector is also connected to the auxiliary RF generator that produces the dipole field. If the frequency of this dipolar field is the same as the secular frequency of the ions, ions takeup the energy very quickly and leave the ion trap. By ramp scanning the RF voltage in both the primary and auxiliary RF generators, ions with the characteristic secular frequency will be ejected from the ion trap and detected by a conversion dynode based detector (Daly detector). The ion current produced from the detector will lead to the generation of a mass spectrum (plot of ion current versus m/z ratio).

Section F: Purpose of research

CL detection methods have been applied for determination of a variety of compounds by FI and LC because the sensitive CL measurement by the PMT is carried out against a dark background. If the CL reaction and reaction conditions are selected, the sensitivity and detection limits are largely dependent on the mixing efficiency of analyte and CL reagent in the flow cell. The problem associated with most FI-CL systems using a conventional design is, CL reagents

and analyte H2O2 are mixed first in the mixing tee before entering the flow cell of the detector.

Instrument Scheme

Figure 1.10. The instrumentation of the Bruker Esquire-HP LC-MS with an ESI interface. Reprinted by permission from Bruker.

As a consequence, not all of the emitted light is detected. A large quantity of sample up to the hundred-µL level must be introduced into the system to achieve low detection limits. The research goal for this project (Chapter II) is to characterize a micro-flow cell for the rapid mixing of reagents and analyte and simultaneous detection of CL emission. Application for the on-line determination H2O2 and lactate by FI with CL detection was also shown. Phenol, chloro- and nitro-phenols are considered priority pollutants by the EPA due to their toxicity and persistence in the environment. Quite a few analytical techniques have been used for phenol analysis. The direct determination of some chloro- and nitrophenols by gas chromatography at low concentrations without chemical derivatization can result in peak tailing and compound discrimination in the injector or capillary column. Fluorescent methods for non- fluorescent chloro- and nitro-phenols also require a time-consuming prederivatization step. Amperometric EC detection is extremely sensitive but requires the use of quite high potentials, generating high background currents and chromatographic interferences as matrix components are oxidized. The research goal in this project (Chapter III) is to develop an on-line quinine- sensitized photo-oxidation with quenched CL detection method that is based on the decrease of light emission from the luminol CL reaction due to the photo-oxidation of phenols that scavenge 1 ·− the photogenerated reactive oxygen species (e.g. singlet oxygen ( O2) and superoxide (O2 )). Application of this detection method will be tested for various functionalized phenols by FI and LC. Aromatic compounds are often determined by FI and LC with FL instead of UV detection to gain selectivity and detection limit advantages. FL determination of those nonfluorescent or weakly fluorescent aromatic compounds (e.g. nitro or halogen functional group-substituted aromatics) by FI and LC generally requires either chemical derivatization or photochemical derivatization. Off-line derivatization procedures are most commonly used, but sometimes extreme conditions (e.g. long reaction times and time-consuming sample purification and preconcentration procedures) are required. Nitroaromatic compounds such as nitrophenols via on-line photolysis seldom convert to significant fluorescent products unless an oxidizing reagent 2+ (e.g. H2O2) and a photocatalyst (e.g. TiO2 or Fe ) are present in the analyte solution. The primary research goal in this project (Chapter IV) is to develop an indirect fluorescence (FL) detection method for nonfluorescent or weakly fluorescenct compounds that exhibit an effective shielding effect on the UV photolysis of 2-phenylbenzimidazole-5-sulfonic acid (PBSA). The

30 second goal here is to study the fundamental aspects of the shielding effect on the UV-photolysis of PBSA off-line using UV, FL spectroscopy, and reversed phase ion-pair LC-MS. For determination of polar ionic organic compounds by LC-MS, reversed-phase ion-pair LC has been often coupled with ESI-MS. A mobile phase containing a nonvolatile ion-pair agent (e.g. tetraalkyl ammonium) can provides sufficient resolution for the separation of ionic species by LC equipped with an UV or FL detector, but can cause severe contamination of the interface, capillary, and skimmers when it is used in LC-MS. The primary research goal here (Chapter V) is to investigate the use of volatile tert-octylamine as a new ion-pairing agent for separation of highly polar ionic aromatic sulfonates and polar phospholipids by reversed phase LC coupled with ESI-MS. The second goal is to study the effect of trap drive level on the ion intensity and ion patterns that appear in the ion chromatograms and MS spectra.

References

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Chapter II Characterization of a micro spiral flow cell for chemiluminescence detection

Section A: Introduction

Chemiluminescence (CL) is the emission of light derived from a chemical reaction when a species in the excited state drops down to the ground state. Besides the often very good sensitivity and selectivity of CL, instrumentally it is a simple technique. Introduction of the sample and CL reagent, often by flow injection (FI) or after liquid chromatography (LC), into a flow cell mounted in front of a photomultiplier tube (PMT) is a common approach. Analytical applications of CL have been reviewed for FI, LC, and immunoassay [1-4]. Biosensors based on immobilized oxidase enzymes for the determination of peroxide by luminol CL are quite common examples [5,6]. Reviews of luminol type CL derivatization reagents for LC and capillary electrophoresis (CE) and CL detectors for CE are recently available [7,8]. The commercial flow cell design for CL detectors is often based on at least two inlet ports, one for the sample and one or two for the CL reagents which connect to a 0.3-0.5 mm i.d. x 50-100 cm long Teflon tube arranged in a spiral pattern in front of the PMT. The advantage of the spiral flow cell design is the reaction kinetics for optimum light emission is less dependent on the flow rate of the FI carrier stream or the LC mobile phase flow rate. Because manufacturers of these CL detectors indicate flow cell volumes of 60 – 120 µL, these detectors are really only suited for conventional analytical LC using flow rates near 1 mL/min and often greater. An application for the determination of peroxide in microalagae by luminol CL chemistry used such a large volume flow cell [9]. Luminol, the Cr(III) catalyst, and the basic pH sample carrier stream were set at 2.5, 2.5, and 11 mL/min, respectively, and the injection volume was 400 µL. Peroxide (50 µL sample size) has been determined by FI using immobilized horse radish peroxidase at micro flow carrier flow rates of 0.01 – 0.1 mL/min with a spiral flow cell [10]. Although the dimensions of the flow cell were 0.96 mm i.d. x 6 cm which calculates to a volume of 44 µL, the actual volume was smaller since the cell was packed with immobilized enzyme glass beads. A similar FI method for H2O2 but using immobilized luminol and Co(II) on ion exchange columns involved instrumentally a 200 µL sample loop and a 200 µL spiral flow cell

[11]. Subnanomolar levels of H2O2 in sea water were determined by the same luminol reaction using an FI instrument equipped with a 60 µL sample loop and a 660 µL spiral flow cell [12]. A comparison of flow cell designs including the standard quartz flow cell, a helix flow cell, the spiral flow cell, and a novel bundle flow cell was made by FI for the determination of Cr(III)

using luminol and H2O2 [13]. The helix, spiral flow cell, and the bundle flow cell all had dimensions of 0.8 mm x 50 cm which calculates to a volume of 251 µL. Injection sizes were 200 µL with a flow rate of 1.5 mL/min. The sensitivity of the bundle flow cell was found to be 50% better than the spiral flow cell under these experimental conditions. Recently, the FI determination of decylamine using a commercial CL detector equipped with a 120 µL flow cell with a 12 cm pathlength has been reported [14]. A sample size of 50 µL with a carrier flow rate of 0.75 mL/min was used. Although not commercially available, CL flow cells for microbore and capillary LC have been developed. A study of spiral flow cell tubing dimensions for detection after semi-micro LC using conditions of a 20 µL injection size, 0.1 mL/min flow rate, and 1.5 mm i.d. x 250 mm reversed phase column has been made [15]. A tubing i.d. of 0.25 mm as compared to 0.5 mm was important to suppress band broadening and the optimum tubing length was found to be 60 cm. The separation of 3-aminoperylene derivatized carboxylic acids was done using a 0.5 µL injection on a 1mm x 250 mm reversed phase column at 0.2 mL/min with peroxyoxalate CL detection in a 100 µL flow cell [16]. Dansylated amino acids were separated by reversed phase LC using similar conditions as just described except a flow rate of 0.6 mL/min was used [17]. A comparison of 5 and 50 µL standard straight flow cells using the peroxyoxalate CL detection chemistry showed because of band broadening, the 50 µL flow cell could be recommended for analytical scale but not microbore LC [18]. A similar straight 0.3 mm i.d. Teflon tube flow celI was investigated for capillary LC [19]. Because the peroxyoxalate CL chemistry has fast kinetics permitting the use of a 200 µL/min combined flow rate for CL reagents as compared to a 10 µL/min mobile phase flow rate, band broadening effects could be reduced. Peroxyoxalate CL detection for capillary LC was demonstrated using a zero dead volume mixing tee to permit introduction of the CL reagents and then light detection further downstream in a 0.25 mm x 4 cm straight PTFE tube [20]. The effective cell volume was estimated to be only 2.1 –2.5 µL due to the sheath flow of the CL reagent which served to reduce extra column band broadening. To the

45 best of our knowledge, a low volume spiral flow cell with tubing less than 0.25 mm i.d. has not been previously reported. Use of small diameter tubing is important since band broadening is dependent on the square of the tubing radius but only proportional to the tubing length. In this paper, a newly designed spiral micro-flow cell (5.5µL) using 100 µm i.d. capillary Teflon tubing for the rapid mixing of reagents and analyte and simultaneous detection of CL emission was made and characterized using the luminol-H2O2-hemin CL reaction. The flow rate effect on CL signal intensity and profile were studied during the flow cell characterization. Comparative studies were carried out with a standard FI instrument with a mixing tee and conventional 12 µL flow cell in a HPLC fluorometer with the source lamp off. Polymer stabilized enzyme lactate oxidase (LO) was injected with the sample for the FI-CL determination of L-lactate using the spiral micro flow cell instrument. Factors that affect the CL signal intensity such as flow rate, temperature, activity units (U) of LO, and sample stability were also investigated. Finally this enzymatic FI-CL method was applied to the determination of L-lactate in two beer samples.

Section B: Experimental

B.1. Apparatus and instruments

The determination of H2O2 and lactate was carried out with the micro-flow FI-CL instrument diagrammed in Figure 2.1(A). Two micro-flow pumps (P#1 and P#2) with a computer interfaced controller unit composing the Ultra-plus Micro LC system (Micro-Tech Scientific, Mountain View, CA) were used to pump the filtered CL reagent and carrier buffer streams, respectively. Samples of interest (1µL) were injected with a Rheodyne model 7410 injection valve into the buffer stream. A Model 900 Isotemp refrigerated circulator water bath (Fisher Scientific, Pittsburgh, PA) was used to control the reaction temperature of a mixing coil made from 100 cm x 100 µm ID Teflon capillary (Cole-Parmer, Ann Arbor, MI) required for the enzyme-catalyzed oxidation of L-lactate. PEEK tubing of 175 µm i.d. x 30 cm (Upchurch

Scientific, Oak Harbor, WA) was used to connect the injector to the detector for the H2O2 study or two pieces of 175 µm i.d. x 40 cm PEEK tubing connected the coil reactor to the injector and detector for the lactate study. The CL signal was continuously measured by a Lumi-Tec photometer (St. John Associates, Inc., Rockville, MD) with a custom-designed 5.5 µL spiral flow

A Lumi-Tec photometer Micro-flow pump detector with a 5.5µL spiral flow cell

CL reagent P#1 P M Sample T

Carrier buffer # P 2 Waste Filter Injector (1µL)

Coil reactor

& isotemp unit

Figure 2.1(A). A schematic diagram of the FI-CL instrument for the determination of lactate.

The same instrument but without the coil reactor was used for the H2O2 study. P#1, P#2 = microflow pumps, PMT = photomultiplier. Each of the five small rectangles in the solution stream represent a filter. See experimental Section B.1 for more details.

B Teflon capillary 70cm × 100µm ID

(Top View)

(Side view)

Waste outlet

Inlet 1 Inlet 2

(Bottom view)

Figure 2.1(B). Design diagram for the 5.5 µL spiral flow cell. Top view: spiral flow cell tubing held in place on an Al disk by a clear acrylic retaining disk with two screws (1), waste outlet (2) also shown. A 5 mm long channel formed in the center of the underside of the acrylic disk is where the inlet tubing (7,8) end and mixing can take place. An outlet from the center of this channel (not shown) is where the tubing to form the spiral shape is connected. Side view: spiral tubing flow cell held between the acrylic disk (3) and the aluminum disk (4) mounted to the bottom Teflon disk (5) all with a diameter of 2.5 cm. Bottom view: waste outlet (6) and inlets (7,8) shown as well as four screws holding the Al and Teflon disks together.

48 cell. The design diagram of this flow cell is shown in Figure 2.1(B). The key part of this flow cell is a spiral 70 cm × 100 µm ID Teflon capillary (Cole-Parmer) that is mounted between two disks of diameter 2.5 cm in front of the PMT with two inlets for the mixing of CL reagents and buffer carrier stream carrying the analyte, and a waste outlet. A more detailed description is given in the figure legend. Initially, a nylon capillary 75 µm x 80 cm was tried for the spiral flow cell but reliability was poor due to kinking and pressure instability of the tubing. Besides solvent filters, an in-line filter (Upchurch) after the injection valve also protected the reaction coil from particles. Fittings with built-in 0.5 µm PEEK microfilters (Part # M-560, Upchurch) were used to connect the PEEK tubing to the detector inlets (Figure 2.1(A)) For comparison to the spiral FI-CL detector, a standard FI-CL detector was constructed as follows. The same pump configuration as shown in Figure 2.1(A) was used. No enzyme coil was needed since only the H2O2-luminol reaction was studied for comparison. The connection between the injector and mixing tee was made by 0.25 mm i.d. x 35 cm stainless steel tubing. The CL reagent and carrier buffer with analyte were combined in a low dead volume stainless steel tee (Valco, Houston, TX) before they entered through a 0.30 mm i.d. x 30 cm stainless steel tube into the HPLC flow cell for CL measurement. The CL signal intensity was measured by a RF-551 spectrofluorometric detector with a 12 µL quartz square flow cell (Shimadzu, Columbia, MD). The detected emission wavelength was set at 431 nm as recommended [21] and the excitation light source was switched off. All CL signals were recorded by a Chromatopac C-R6A integrator (Shimadzu). B.2. Reagents and solutions Luminol (3-aminophthahydrazide) was from Aldrich (Milwaukee, WI). Hemin (chloroferriprotoporphyin) from bovine, L-(+)-lactic acid (sodium salt), lactate oxidase (LO) from Pediococcus species (L-0638, 50 units, EC 232-841-6), and polyethylene glycol (PEG) with a molecular weight of 3350 were purchased from Sigma (St. Louis, MO). Hydrogen peroxide (30% solution) was obtained from Fisher Scientific. All other chemicals used were of analytical reagent grade. Distilled and doubly deionized water was obtained from the E-Pure deionization system (Barnstead, Dubuque, IA). B.3. Procedure

The CL reagent solution delivered by P#1 contained 0.4 mM luminol, 8 µM hemin, and 0.1 M sodium phosphate dibasic buffer solution (pH 11.6). The composition of this CL reagent is based on previous work [22]. The carrier buffers delivered by P#2 to propel the injected analyte were either 0.1M sodium phosphate diabasic buffer solution (pH 11.6) for the FI-CL determination of H2O2 or 0.1 mM sodium phosphate monobasic buffer solution (pH 7.5) for the determination of lactate. Both buffer solutions were prepared from 0.5 M stock solutions by

appropriate dilution with water. H2O2 stock solutions of 20 mM and 1 mM were prepared fresh

daily. Serial dilution of the stock solutions with water provided the required H2O2 standard solutions that were prepared immediately before analysis. For the flow rate optimization work,

either a 50 µM H2O2 standard or a 50 µM lactate standard was used. For the calibration curve, each lactate standard solution, made up of 7.5% PEG (v/v), 5 U/ml LO, lactate and buffer, was prepared in a 1mL vial immediately before FI-CL analysis. The 1000 µM lactate stock solution was prepared fresh daily.

For the H2O2 study, no coil reactor was needed as part of the instrument however for the lactate work, the temperature controlled coil reactor set at 37.5 oC was important. In order to study the stability of the lactate samples, a 0.5 mL volume of 100 µM lactate containing 7.5% PEG and 5 U/mL LO was prepared in each test tube. Those solutions were placed in the ice-bath and stored in the refrigerator before analysis at various times. In order to investigate the stabilization of LO by PEG, a 250 µL volume of 15% PEG aqueous solution was mixed with 50 µL of LO solution with an activity value of 50 U/mL and 150 µL of pH 7.5 phosphate buffer solution in each test tube. Those test tubes were placed in the ice-bath and stored in the refrigerator. Lactate needed for a final concentration of 50 µM was added just prior to analysis. Under the optimal temperature and flow rate conditions, the calibration curve for the determination of L-lactate was obtained. Under the same analytical conditions as before, a 50 µL volume of beer was taken with 50 µL of LO to make each 0.5 mL sample solution with pH 7.5 buffer. The results of L-lactate in beer were calculated from the calibration equation. Both the sample analysis as well as the calibration work should be done using the same enzyme solution to minimize signal response variation from different LO reagent bottles.

Section C: Results and discussion

C.1. Flowrate studies for the spiral and conventional HPLC flowcells Characterization of the 5.5 µL spiral flow cell flow cell–CL detector was based on

injected H2O2 with luminol CL detection using the instrument diagrammed in Figure 1A but without the reaction coil. The effect of flow rate on the CL signal intensity measured as peak height was studied at both 15 and 25 µL/min for P#1 over a range of 40, 50, 60, and 70 µL/min for P#2 (Figure 2.2). As the P#1 flowrate was held constant at either value, the peak became sharper with less time needed to reach the peak height, with the highest peak observed at 50 µL/min. The CL intensity profile with P#1 at 15 µL/min increased from about 15 to a maximum of 17 mV at 50 µL/min and then back down to 14 mV as a function of the P#2 flowrate. The CL intensity profile with P#1 at 25 µL/min varied slightly from 19 to a maximum of 21 mV at 50 µL/min and then back to 18 mV as a function of P#2 flowrate. Using 25 µL/min for P#1, the baseline peak width decreased from 2.5 min to 1.1 min and the peak asymmetry improved by a factor of 2 as the P#2 flow rate increased from 40 – 70 µL/min (Figure 2.3). Previous work [17] indicated baseline peak widths estimated at 1-2 min for early eluting peaks even at a high flow rate of 0.6 mL/min. The optimal flow rates for P#1 and P#2 were 25 µL/min and 50 µL/min, respectively. There was no further optimization of flow rate of CL reagents at P#1 due to the concern about the problem of pressure buildup inside the Teflon spiral flow cell tubing. Comparative studies were also done on the same CL system as diagrammed in Figure 2.1(A) but without the reaction coil and instead a low volume mixing tee leading to a HPLC detector with a 12 µL square flow cell. Flow rate optimization was studied at 15, 25, and 35 µL/min for P#1 over a six point range from 90 – 140 µL/min for P#2 (data not shown). The RSD for these data range from 0.5-3% for n=4. The improved RSD is likely due to the difference in the stability of the electronic readout of the two detectors. The flow rate responses for P#2 increased slightly by about 4 –6 units starting from 11, 17, and 24 mV for the respective increasing P#1 flowrates before falling off at about 130 µL/min. The optimal flow rate of 120 µL/min for P#2 was found in these gradual profiles for all three P#1 flowrates. In search of the optimal flow rate of CL reagent for P#1, the flow rate at P#2 was set constant 120 µL/min and the flow rate for P#1 was varied over a seven point range from 15 to 75 µL/min. The CL intensity increased steadily from 14 to 35 mV before leveling off at about 50 µL/min. The optimal flow rate for P#1 was considered to be 55 µL/min.

15 µL/min at pump#1 25 25 µL/min at pump#1

CL intensity (peak height), mV 10 35 45 55 65 75

Flow rate at pump#2, µL/min

Figure 2.2. Effect of the flow rate on the CL intensity for the FI-CL system with a 5.5 µL spiral flow cell. Analytical conditions: 0.4 mM-luminol-8 µM hemin solution buffered at pH 11.6 # # (P 1); pH 11.6 carrier buffer (P 2), 1µL of 50 µM H2O2 for each injection, room temperature. The relative standard deviation (RSD) of these data ranged from 3-10% for n=6.

Flow rate: 25/40 25/50 25/60 25/70 µL/min (P#1/P#2)

Figure 2.3. Effect of the flow rate on the CL signal profile

C.2. Calibration curves and detection limits for H2O2 Under the optimal flow conditions, the relationship between the concentration of each

standard H2O2 solution and its CL signal intensity for both the spiral flow cell and the conventional square flow cell was found. For the spiral flow cell (Figure 2.4(A)), an exponential profile is noted. The equation of the best fit calibration curve was Y = 0.32X1.41 with a regression 2 coefficient r = 0.9978. A detection limit for H2O2 of 1.5 µM (1.5 pmol for 1 µl sample) was determined from the measurement with three times the signal-to-noise ratio. The reproducibility

in terms of relative standard deviation (RSD) was 8.8% (n=5) for a 10 µM H2O2 sample. This absolute detection limit for the spiral flow cell in terms of pmol was also 6 times lower than that of the similar flow cell made from 6 cm × 0.96 mm ID Teflon tubing [10]. For the 12 µL square flow cell (Figure 2.4(B)), a slight exponential profile is again noted from 50 – 1000 µM (n=6). The best fit calibration curve from 50 to 1000 µM was Y = 1.16 2 0.0081X and r =0.9987. The detection limit of H2O2 was 37 µM (37 pmol for 1 µL sample), which was again calculated from three times the standard deviation of the blank measurement.

Reproducibility (RSD) was 1.8% (n=7) for a 300 µM H2O2 sample. Although some band broadening in the connecting tubing could have been a factor despite the higher flowrate for the

12 µL fluorescence flowcell FI system, the direct mixing of the CL reagent and H2O2 at the start of the spiral tubing design and the relatively longer residence time in front of the PMT likely contributed to the lower detection limit and higher sensitivity for the FL-CL determination of

H2O2 with the 5.5 µL spiral flow cell. C.3. L-lactate assay

The L-lactate assay was based on the luminol FI-CL measurement of H2O2 that was generated from the oxidation of lactate in the presence of lactate oxidase (LO) using the instrument diagrammed in Figure 2.1(A). Factors that affected the CL intensity, such as flow rate, temperature, enzyme concentration, and mixing time were considered. Polyethylene glycol (PEG) stabilization of the enzyme activity was also shown. Due to the narrow pH range (pH 6- 7.5) for the optimal enzyme activity [23], the pH 7.5 phosphate buffer replaced the pH 11.6 phosphate as the carrier buffer delivering the injected lactate samples.

Figure 2.4. Calibration plots for the FI-CL determination of H2O2. (A) 5.5 µL spiral flow cell. (B) 12 µL square flow cell. P#1 and P#2 were set at 25 and 50 µL/min for (A) and 55 and 120 µL/min for (B), respectively. See experimental Section B.3 for more details.

45 A: a 5.5 µL spiral flow cell

35 30 25 20 15 10 5

mV height), (peak CL intensity 0 0 5 10 15 20 25 30

Hydrogen peroxide, µM 30 B: a 12 µL square quartz flow cell 25

CL intensity (peak height), mV 0

0 200 400 600 800 1000

Hydrogen peroxide, µM

C.3.1 Flow rate optimization The flow rate optimization for the lactate assay is shown in Figure 2.5. The initial optimization step shown in Figure 2.5(A) was to determine the optimal flow rate of the CL # reagent at P 1 by using 50 µM H2O2 as a sample. As expected, the CL response tended to be higher as the flow rate for P#2 decreased due to a longer residence time for the optimum CL emission intensity. If the flow rate of the CL reagent was below 45 µL/min, any change in flow rate of the carrier buffer at P#2 had a minimal impact on the CL signal intensity. In the second optimization step using lactate shown in Figure 2.5(B), the highest CL signal intensity was obtained when the flow rate of carrier buffer was increased to 60 µL/min. This is likely due to mixing in the flow coil was more efficient between the still narrow nondiffused lactate-PEG-LO injection plug and the CL reagent. There was no further attempt to increase the flow rate at P#2 in case of damage to the Teflon-made flow cell caused by the high pressure buildup inside the flow cell. Therefore, 45 µL/min at P#1 and 60 µL/min at P#2 were set as the operational flow rates throughout the remaining lactate studies. C.3.2. Temperature and enzyme concentration Temperature-CL intensity curves obtained with different LO activities clearly indicated maximum catalytic activity as the reaction temperature reached 37 to 38°C (Figure 2.6). Surprisingly, strong CL signals were still evident at 60 oC. During the study of the effect of LO concentration on CL intensity with the temperature set at 38°C, it was observed that the highest average CL signal intensity reached 51 mV with 5 U/mL LO. The average CL signal intensity obtained at 1 and 10 U/mL LO was 26 and 44 mV respectively. For the lactate assay, it was decided the optimal reaction temperature was 37.5 oC and the optimal concentration of LOD was 5 U/mL. C.3.3. Stability of lactate samples The stabilization of lactate dehydrogenase (LDH) by PEG has been used previously in FI and CE studies to ensure reproducibility of the analytical response over a time period of at least 8 hr at 30 oC [24,25]. A comparison of the CL signal using lactate samples with and without 7.5 % PEG as a function of low temperature storage time is shown in Figure 2.7. The variation of CL intensity values was less than 5mV for the enzyme solution containing PEG while it reached up

Figure 2.5. Flow rate optimization for the determination of lactate. (A) Determination of the # optimal flow rate of CL reagent at P 1 using a 50 µM H2O2 sample. (B) Determination of the optimal flow rate of the carrier buffer at P#2 using 50 µM lactate with 7.5% PEG and 10 U/mL LO. CL reagent of 0.4 mM luminol, 8 µM hemin, and 0.1 M phosphate buffer (pH 11.6) delivered by P#1 but the pH 7.5 buffer solution was now the carrier buffer (P#2). Temperature was set at 38 oC. RSD for these data points ranged from 7-18% for n=4.

A 15 µL/min at pump#1 14 25 µL/min at pump#1 35 µL/min at pump#1 12 45 µL/min at pump#1 10 8

4 2 0 CL intensity (peak height), mV height), (peak CL intensity -2 25 30 35 40 45 50 55 60 65

Flow rate at pump#2, µL/min

B: 45 µL/min at pump#1 55

CL intensity (peak height), mV height), (peak intensity CL 30 25 30 35 40 45 50 55 60

Flow rate at pump#2, µL/min

250 230 1U/mL LOD 210 5U/mL LOD 190

170

150

130 110 90 70 CL intensity (peak height), mV 50 10 20 30 40 50 60 70

Temperature, deg

Figure 2.6. Effect of temperature (oC) on the LO enzyme activity. Optimal flow rates of 45 and 60 µL/min for P#1 and P#2, respectively. Sample: 50 µM lactate in 7.5% PEG with either 1 or 5 U/mL LO. Other conditions as in Figure 2.5(B). RSD values for 6 injections were in the range between 7-15%.

40 without 7.5% PEG with 7.5%PEG 35

CL intensity (peak height), 10mV 0246810121416182022 Mixing time, hour

Figure 2.7. Effect of PEG on the stabilization of LO enzyme activity when PEG-LO samples stored in an ice-bath in the refrigerator. Diamond points – no PEG, RSD 12-20% for n= 6-8. Square points – 7.5 % PEG. RSD 8-16% for n =6-8. Conditions as in Figure 2.5(B) but 5 U/mL LO and 37.5 oC.

61 to 18 mV for the enzyme solution in the absence of PEG. Although the absolute CL response was lower for the first 6 hr, PEG did have a positive effect on the stabilization of LO enzyme activity. However, the effect was not as dramatic as that for LDH that has 4 subunits. The enzyme LO with 7.5% PEG was mixed directly with the lactate sample solutions prior to FI injection in order to minimize consumption of the expensive enzyme. These samples were kept in an ice-bath stored in the refrigerator. The FI-CL analyses of those sample solutions were carried out at different times and the results are as shown in Figure 2.8. The CL intensity continuously decreased with the mixing time with a slope during the first 2 hr of about 0.6 mV/min. From 4-17 hr, the slope was only about 0.1 mV/min. The CL signal decay was somewhat surprising considering the low storage temperature. Based on the finding from these experiments, the time spent for lactate, LO, and PEG in solution should be as short as possible. C.3.4. Calibration, detection limit, and real sample data for L-lactate The relationship between lactate concentration and its CL signal intensity in terms of peak height under optimum analysis conditions is shown in Figure 2.9. The calibration range for the lactate assay from 0 to 50 µM was fitted to the equation Y = 0.39X + 6.90 with r2 = 0.9923. The detection limit of lactate calculated from 3 times the standard deviation of the blank measurements was 2.9 µM (2.9 pmol for 1 µl sample). The reproducibility (RSD) was 12.2% (n = 4) for 10 µM lactate and 8.8% (n = 4) for 25 µM lactate. Compared to other methods for lactate assays, such as the enzymatic FI-CL method based on luminol-H2O2-horseradish CL reaction with a detection limit of 0.08 µM (8 pmol for 100µL sample injection) [26], the FI method based on UV detection of NADH generated from the oxidation of lactate catalyzed by lactate dehydrogenase enzyme (LDH) with a detection limit of 0.13 mM (65 pmol for 0.5 µL sample injection) [24], the FIA method based on photochemical reaction with the detection limit of 18 nM (1.8 pmol for 100 µL injection) [27], the enzymatic

FI-CL method based on the luminol-H2O2-K3Fe(CN)6 CL reaction with the detection limit of 5 mg/L (44.5 µM) [28], this FI-CL detection limit of 2.9 pmol for lactate was better or comparative. The proposed method using the external standard calibration method was applied to the determination of L-lactate in two beer samples. The determination of lactate in beer is important since excessive lactate will cause an off-taste and the level of lactate is related to the fermentation process in general for the control of bacterial contamination [29]. For n = 5, a 62

300

250

200

150

100

CL intensity (peak height), mV height), (peak intensity CL 0 0 2 4 6 8 10 12 14 16 18

Mixing time, hour

Figure 2.8. Effect of CL signal as a function of storage time for the lactate-PEG-LO sample at low temperature. Conditions as in Figure 2.5(B) but 5 U/mL LO and 37.5 oC. RSD for these data were 7-15% for n=6.

45 40 35 30 25 20 15 10 5

CL intensity (peak height), mV 0 0 102030405060708090100

L-lactate, µM

Figure 2.9. Calibration curve for the FI-CL determination of L-lactate using the spiral flow cell. Conditions as in Figure 2.5(B) but 5 U/mL LO and 37.5 oC. RSD for these data ranged from 7.8- 15.2% for n=4.

64 sample of George Killian’s Irish Red was found to have 14.5 + 1.3 mg/L lactate and a sample of Michael Shea’s Black & Tan was analyzed for 8.1 + 1 mg/L lactate. Both of these values are somewhat low as compared to a previous study for L-lactate in Czech and Italian beers in which levels were on the order of 20 – 50 mg/L [29].

Section D: Conclusions

Mixing of the luminol reagent and H2O2 analyte inside the spiral cell with simultaneous detection of CL emission resulted in an improvement in sensitivity and detection limit as compared to a tee mixer and a standard fluorescence flow cell. Application to oxidase enzyme chemistry was shown. Although the enzyme (LO) when combined with PEG was quite stable when stored at low temperature, the LO-PEG-substrate (L-lactate) mixture was not for any significant length of time. However the determination of L-lactate was possible if the LO-PEG- lactate mixture was injected just after preparation. Addition of LO-PEG in the carrier stream should still be cost effective using rapid sample injections spaced about 2 min apart. The sample carrier flowrate range of 50-60 µL/min for the spiral flowcell FI system is considered more compatible with small microbore HPLC. It is expected this low volume spiral flow cell would minimize band broadening for microbore HPLC applications such as the separation and detection of glucosides [22]. Application to wide bore capillary LC should also be feasible.

References

[1] W. R. G. Baeyens, S. G. Schulman, A. C. Calokerinos, Y. Zhao, A. M. Garcia Campana, K. Nakashima, D. De Keukeleire, Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays, J. Pharm. Biomed. Anal. 17 (1998) 941. [2] M. G. Sanders, K. N. Andrew, P. J. Worsfold, Trends in chemiluminescence detection for liquid separations, Anal. Commun. 34 (1997) 13H. [3] A. Roda, P. Pasini, M. Guardigli, M. Baraldini, M. Musiani, Bio- and chemiluminescence in bioanalysis, Fresenius J. Anal. Chem. 366 (2000) 752. [4] A. M. Garcia Campana, W. R. Baeyens, X. R. Zhang, E. Smet, G. VanDer Weken, K. Nakashima, A.C. Calokerinos, Detection in the liquid phase applying chemiluminescence, Biomed. Chromatogr. 14 (2000) 166. [5] K. Hayashi, S. Sasaki, K. Ikebukuro, I. Karube, Highly sensitive chemiluminescence flow injection analysis system using microbial peroxidase and a photodiode detector, Anal. Chim. Acta, 329 (1996) 127. [6] U. Spohn, F. Preuschoff, G. Blankenstein, D. Janasek, M. R. Kula, A. Hacker, Chemiluminometric enzyme sensors for flow-injection analysis, Anal. Chim. Acta 303 (1995) 109. [7] M. Yamaguchi, H. Yoshida, H. Nohta, Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis, J. Chromatogr. A 950 (2002) 1. [8] Y-M. Liu, J-K. Cheng, Ultrasensitive chemiluminescence detection in capillary electrophoresis, J. Chromatogr. A, 959 (2002) 1. [9] R. Escobar, S. García-Domínguez, A. Guiraúm, O. Montes, F. Galván, F.F. de la Rose, A flow injection chemiluminescence method using Cr(III) as a catalyst for determining hydrogen

peroxide. Application to H2O2 determination in cultures of microalgae, Luminescence 15 (2000) 131. [10] O. Nozaki, H. Kawamoto, Determination of hydrogen peroxide by micro-flow injection- chemiluminescence using a coupled flow cell reactor chemiluminometer, Luminescence 15 (2000) 137.

[11] W. Qin, Z. Zhang, B. Lin, S. Liu, Chemiluminescence flow-sensing system for hydrogen peroxide with immobilized reagents, Anal. Chim. Acta 372 (1998) 357. [12] J. Yuan, A. M. Shiller, Determination of subnanomolar levels of hydrogen peroxide in seawater by reagent-injection chemiluminescence detection, Anal. Chem. 71 (1999) 1975. [13] P. Campins-Falco, L. A. Tortajada-Genaro, F. Bosch-Reig, A new flow cell design for chemiluminescence analysis, Talanta 55 (2001) 403. [14] P. J. Fletcher, K. N. Andrew, S. Forbes, P. J. Worsfold, Automated flow injection analyzer with on-line solid-phase extraction and chemiluminescence detection for the determination of dodecylamine in diesel fuels, Anal. Chem. 75 (2003) 2618. [15] K. Takezawa, M. Tsunoda, K. Murayama, T. Santa, K. Imai, Study on the fluorescent 'on- off' properties of benzofurazan compounds bearing an aromatic substituent group and design of fluorescent 'on-off' derivatization reagents, Analyst 125 (2000) 293. [16] K. Honda, K. Miyaguchi, K. Imai, Evaluation of fluorescent compounds for peroxyoxalate chemiluminescence detection, Anal. Chim. Acta 177 (1985) 111. [17] K. Miyaguchi, K. Honda, T. Toyo’oka, K. Imai, Application of a microbore high- performance liquid-chromatography chemiluminescence detection system to the n-terminal amino-acid-analysis of bradykinin, J. Chromatogr. 352 (1986) 255. [18] G. J. de Jong, N. Lammers, F. J. Spruit, R. W. Frei, U. A. Th. Brinkman, Analytical implications of the half-life of the chemiluminescence signal in the peroxyoxalate detection system for liquid-chromatography, J. Chromatogr. 353 (1986) 249. [19] G. J. de Jong, N. Lammers, F. J. Spruit, C. Derwaele, M. Verzele, Low-dispersion chemiluminescence detection for packed capillary liquid-chromatography, Anal. Chem. 59 (1987) 1458. [20] A. J. Weber, M. L. Grayeski, Peroxyoxalate chemiluminescence detection with capillary liquid-chromatography, Anal. Chem. 59 (1987) 1452. [21] J. Lee, H. H. Seliger, Quantum yields of the luminol chemiluminescence reaction in aqueous and aprotic solvents, Photochem. Photobiol. 15 (1972) 567. [22] P.J. Koerner, Jr., T.A. Nieman, High-performance liquid-chromatographic determination of glucosides (glucose conjugates) with post-column reaction detection combining immobilized enzyme reactors and luminol chemiluminescence, J. Chromatogr. 449 (1988) 217.

[23] F. Mizutani, K. Sasaki, Y. Shimura, Sequential determination of l-lactate and lactate- dehydrogenase with immobilized enzyme electrode, Anal. Chem. 55 (1983) 35. [24] J.R. Marsh, N.D. Danielson, Determination of substrates using poly(ethylene glycol)- stabilized dehydrogenase enzymes by microliter per minute flow-injection, Analyst 120 (1995) 1091. [25] J.M. Fujima, N.D. Danielson, Online lactate dehydrogenase substrate and activity determinations by capillary electrophoresis, J. Cap. Elec. 3 (1996) 281. [26] A. Hemmi, K. Yagiuda, N. Funazaki, S. Ito, Y. Asano, T. Imato, K. Hayashi, I. Karube, Development of a chemiluminescence detector with photodiode detection for flow-injection analysis and its application to l-lactate analysis, Anal. Chim. Acta 316 (1995) 323. [27] T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, J. Martín, Flow injection determination of lactate based on a photochemical reaction using photometric and chemiluminescence detection, Analyst 124 (1999) 1517. [28] R.W. Min, J. Nielsen, J. Villadsen, Simultaneous monitoring of glucose, lactic-acid and penicillin by sequential injection-analysis, Anal. Chim. Acta 312 (1995) 149. [29] S. Girotti, M. Muratori, F. Fini, E. N. Ferri, G. Carrea, M. Koran, P. Rauch, Luminescent enzymatic flow sensor for D- and L-lactate assay in beer, European Food Res. Technol. 210 (2000) 216.

Chapter III Determination of phenols by flow injection and liquid chromatography with on-line quinine-sensitized photo-oxidation and quenched luminol chemiluminescence detection

Section A: Introduction

Phenol and substituted phenols are used extensively in the manufacture of a wide variety of products, such as polymers, fertilizers, adhesives, paints, insecticides, herbicides, antiseptics, and explosives [1,2]. Phenols are also produced as by-products in many industrial processes (e.g. petroleum, paper, tanning dye and soap industries) [3]. Furthermore, phenolic compounds are also formed during the natural decomposition of humic substances, tannins, and lignins, and photolytic or metabolic degradation of herbicides and insecticides [4,5]. Due to their toxicity and persistence in the environment, phenol, chloro- and nitro-phenols are considered priority pollutants. Methods based on separation techniques such as high performance liquid chromatography (HPLC) [6,7], gas chromatography (GC) [8,9], capillary electrophoresis (CE) [10,11], and supercritical fluid chromatography (SFC) [12], have been successfully applied to the determination of phenols. Although GC equipped with various detectors such as flame ionization, electron capture, or mass spectrometry (MS), has achieved high sensitivity and selectivity, the direct analysis of some chloro- and nitrophenols at low concentrations without chemical derivatization can result in peak tailing and compound discrimination in the injector or capillary column [13]. Although pentafluorobenzyl derivatives of phenols have been determined by GC with either electron capture [14] or mass spectrometry (MS) detection [15], methylation using the hazardous reagent diazomethane has been instead recommended for nitrophenols [16,17]. Recently, phenols after methylation using methyl iodide in a phase transfer catalysis reaction were determined by GC with flame ionization and MS detection [18]. There has been a definite trend to use HPLC as an alternative technique for the determination of phenols in aqueous solutions [19]. In order to achieve low detection limits and high sensitivity, two types of approaches, enrichment (e.g. solid-phase micro-extraction, liquid-

69 liquid extraction, and solid-liquid extraction) [20-22], and special detection techniques (electrochemical (EC), fluorescence, and MS) have been applied for phenols [20, 23-25]. However, amperometric EC detection requires the use of quite high potentials (>1V for nitrophenols) [26] and high background currents and chromatographic interferences increase as matrix components are oxidized at such a potential. To minimize the necessity of cleaning the amperometric working electrode, coulometric detection using a higher surface area flow through graphite electrode has been proposed [27]. Fluorescent methods for chloro- and nitro-phenols have required prederivatization chemistry. Dansyl derivatives of chlorophenols upon post- column photochemical reaction can be detected fluorometrically at the 200 pg level [24]. After reaction with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride at 60 oC for 30 min, chlorophenols as well as phenol could be determined after HPLC separation at the low pmol range [28]. Photo-oxidation of phenols using three approaches, direct UV photolysis [29], photocatalysis on semiconductor particles [30-32], and photosensitized oxidation [32-34], have been researched for wastewater treatment. Although photolysis of phenols by photosensitizers may not be an economical way for water treatment, photosensitizers can dramatically increase 1 the quantum yield of singlet oxygen ( O2), a major oxidizing species that is believed to be responsible for the photo-oxidation of the phenols [33]. Photosensitizers can react with 1 dissolved oxygen in solution to generate singlet oxygen ( O2) by energy transfer or superoxide − radical anion (O2 ) via electron transfer between the photoexcited sensitizer and O2 [33-36].

Photo-oxidation of phenols has been done in organic solvents such as CH3OH, CCl4, CH2Cl2, and benzene, using different photosensitizers, e.g. eosin, rose bengal, methylene blue, riboflavin and the Zn (II) complex of tetraphenyl porphyrin [33]. Quinine sulfate is used as a 1 photosensitizer because its major component, quinine, sensitizes the photogeneration of O2 in the aqueous solution with a quantum yield of 0.36 [37]. Quinine has been used to sensitize the photo-oxidation of a variety of compounds, including iodide ion, 2,5-dimethylfuran, indoleactic acid, histidine, tryptophan, theophyline, and uric acid [38]. In analytical FI applications, quinine has been reported to sensitize the CL reactions involving the drug tiopronin (N-2- mercaptopropionylglycine) upon oxidation with Ce (IV) [39] and pyruvate upon oxidation with

KMnO4 [40].

Although high sensitivity and selectivity of analysis can be achieved simply by measuring the CL light using a photomultiplier, the use of CL detection methods for FI and LC of phenolic compounds is uncommon. Rhodamine labeled chlorophenols were determined by peroxyoxalate CL after HPLC separation [41]. 10-Methyl-9-acridinium carboxylate has been used for CL labeling of chlorophenols before HPLC separation [42]. The photochemical degradation of dansyl derivatives of alkyl-, chloro-, and nitro-phenols as described in [24] was adapted for use with peroxyoxalate CL [43]. Recently dansyl derivatives of alkyl phenols were detected by the bis(2,4,6-trichlorophenyl)oxalate – hydrogen peroxide reaction [44]. The FI determination of volatile phenols based on a quenching method by which the CL signal from the

H2O2-p-chlorobenzenediazonium fluoroborate (p-CBDA) reaction was suppressed by a carrier stream of added phenols as they reacted with p-CBDA [45]. However this method cannot be adapted to LC because the phenols when injected as discrete samples generate only weak CL. The luminol-based CL reaction is a well-known method for the detection of reactive •− 1 oxygen species (ROS), such as O2 , O2, and H2O2, because those species quickly react with luminol in alkaline solution to emit light, as the hydroperoxide intermediate of luminol decomposes into aminophthalate [46-48]. This approach has been used for several biological applications such as the assessment of ROS in sperm [49] and ROS produced from redox-cycling drugs in cultured heptatocytes [50]. The antioxidative effects of hydroxybenzoic acids were studied by following the decrease of luminol CL in the presence of ROS [51]. Photo-oxidation of the phenols in the presence of quinine coupled with luminol-based CL reaction should be a promising detection method for phenol analysis. The CL signal will be suppressed as the concentration of phenols increases because more oxygen species are consumed in solution. In general, post-column luminescence detection of underivatized phenols seems to be an unusual approach. In this paper, a quinine-sensitized on-line photo-oxidation and quenched CL detection method was developed for the determination of ten phenolic compounds by FI and LC. This detection method was most effective for phenol, 4-chlorophenol, and 4-nitrophenol. The effects of several important experimental parameters such as flow rate, pH, solvent, and photosensitizer concentration, on the generation and detection of oxygen species were studied in the FI system. Separation of phenol, 4-nitrophenol and 4-chlorophenol in an aqueous mixture was carried out by LC with this detection method.

Section B: Experimental

B.1. Chemicals and solutions 2-Nitrophenol, 3-nitrophenol, 4-nitrophenol, 4-chlorophenol and luminol (3- aminophthahydrazide) were provided by Aldrich (Milwaukee, WI). Phenol was from MCB (Cincinnati, OH). Quinine sulfate was obtained from Fisher Scientific (Fair Lawn, NJ). Hemin (chloroferriprotoporphyin) from bovine was purchased from Sigma (St. Louis, MO). Acetonitrile and methanol were obtained from Burdick & Jackson (Muskegon, MI). All other chemicals are at least of analytical reagent grade as well. Distilled-deionized water used for preparation of solutions was taken from an E-Pure deionization system (Barnsted). One liter of 500 µM quinine sulfate (QS) stock solution was prepared by dissolving 0.3915 g QS in 1 L of 0.1 M potassium phosphate monobasic buffer solution (pH 7). A 350 µM QS solution used in FI or LC was obtained from the appropriate dilution of this stock solution. For pH studies, HCl-KCl, acetate-acetic acid, and phosphate buffer solutions described in the literature [52] were used to adjust the pH of 500 µM QS in the range of 1-8.5. The CL reagent solution contained 0.4 mM luminol, 8 µM hemin and 0.1 M sodium phosphate dibasic buffer solution (pH 11.6). A stock CL solution was prepared by dissolving 0.0708 g of luminol in 200 mL of 0.5 M sodium phosphate diabasic buffer solution (pH 11.4) and diluting it to 1 L after adding 40 ml of 0.2 mM hemin solution. Each phenol standard solution for FI was prepared by mixing an appropriate volume of an aqueous stock solution (0.5 or 10 mM for the nitrophenols, 1mM chlorophenol and 1 mM phenol) and 35 mL of 500 mM quinine sulfate solution (pH 7.0) in a 50-ml volumetric flask. Phenol mixtures used for LC were prepared by proportionally mixing each phenol sample and 1.6 mL of acetonitrile together and diluting to 4 mL with water in 15 × 45 mm vials. B.2. FI and LC instrument design for photo-oxidation and quenched CL detection The FI instrument with on-line photo-oxidation and quenched CL detection is shown in Figure 3.1(A). Two Ultra-plus micro LC pumps (pump #1 and #2) (Micro-Tech Scientific, Sunnyvale, CA) were used to transport the luminol solution and water (sample carrier). Samples

72 with added QS (20 µL) were injected with a Rheodyne model 7125 injection valve into the carrier stream from pump #2. The QS-sensitized oxidation of the phenolic compounds took place in a coil photo-reactor made from a 3048 mm (l) × 1.6 mm (od) × 0.25 mm (id) fluoroethylene- propylene copolymer (FEP) tubing (Upchurch Scientific, Oak Harbor, WA) circling around a Nippo GL 4 mercury UV lamp powered by a LDC UV III LC detector. After the photolyzed phenol sample stream left the reactor and was mixed with the luminol stream in a Valco mixing tee, CL emission at 431 nm [53] was generated and its intensity was measured by a Shimadzu RF-551 spectrofluorometric detector (Shimadzu, Columbia, MD) with the excitation light source switched off. CL signals were processed and recorded by a Shimadzu Chromatopac C-R6A integrator. The FI instrument diagramed in Figure 3.1(A) was modified for LC as shown in Figure 3.1(B). Pump #3, a Beckman 110A LC pump, was used to transport the acetonitrile-water mobile phase while the two Ultra-plus micro LC pumps (#1 and #2), were used to transport the luminol CL reagent and photosensitzer QS solution, respectively, as for FI. The phenol sample was injected into a MetaChem Polaris C18-A 3µm HPLC column (50 × 4.6 mm id) (ANSYS Technologies, Lake Forest, CA) using the Rheodyne model 7125 injection valve (20 µL). The UV absorbance of the column eluent was continuously measured at 254nm using a Shimadzu SPD-6AV UV-VIS spectrophotometric detector and recorded using a Linear 1201 chart recorder. Unlike for FI, the LC eluent was mixed with the QS solution from pump #2 through tee 2 first before entrance into two tandem coil reactors, each having the same dimensions (3048 x 0.25 mm ID) coiled around the Hg lamp as above for the FI instrument.

B.3. Procedures

B.3.1. Photo-oxidation and quenched CL detection of phenols by FI Under the optimum flow conditions, aqueous phenol samples containing 350 µM photosensitizer QS and buffered at pH 7 were injected into the carrier stream of deionized water propelled by pump #2. A series of 0, 10, 25, 50, 100, 150, 200, and 250 µM standard solutions was injected five times to generate calibration curves for each monosubstituted nitrophenol. The calibration data for phenol and 4-chlorophenol resulted from five repetitive injections, each of

Figure 3.1. Instrument design for (A) FI and (B) LC with on-line photo-oxidation and quenched CL detection.

Micro-flow pump Fluorometer

CL reagent P#1

T-piece

P Sample M T

Water P#2

Injector Photoreactor (20 µL) (3048 × 0.25 mm FEP Waste B + Hg lamp)

Pump Sample HPLC column

P#3 UV detector QS Mobile phase T-piece P#2

T-piece

P FEP photoreactors + Hg lamps Fluorometer M T P#1 CL reagent

Waste

75 the standard series of 0, 1, 5, 10, 25, 50, 75, 100 and 150 µM solutions. A similar range of concentrations to these but up to 400 µM were tried for the other compounds. B.3.2. LC separation of phenols The LC separation of the phenol mixture was carried out with an isocratic flow rate of 100 µL/min using an acetonitrile-water (40:60, v/v) mobile phase at ambient temperature. The aqueous phenol sample mixture could be injected without being pre-mixed with photosensitizer solution. With optimum flow rates of 100 µL/min for 350 µM QS and 370 µL/min for 0.4 mM luminol, the quenched CL chromatogram was obtained after UV detection at 254 nm.

Section C: Results and discussion

C.1. Photo-oxidation chemistry of phenols and the luminol CL reaction with oxygen species Photo-oxidation of phenols is occuring via the oxidation reaction between phenols and oxygen species that are generated from photolysis. The primary step for the photosensitized oxidation is the absorption of UV light by quinine sulfate (QS). After excitation to the singlet excited state (1QS*), most of the singlet QS molecules decay quickly to the triplet excited state (3QS*) via intersystem crossing (ISC). Energy transfer between 3QS* and triplet ground state 3 1 oxygen ( O2) leads to the generation of singlet oxygen ( O2) followed by oxidation of the phenolic compound (PC). Photo-oxidation of phenol in the presence of photosensitizer QS through the so-called type II mechanism is expressed as follows [33,54]: hν ISC QS → 1QS* → 3QS* (1)

3 3 1 QS* + O2 → QS + O2 (2)

1 O2 + PC → PCox (3)

where PCox stands for the oxidized phenolic compound. 1 Although O2 is a dominating oxidizing species in the photo-oxidation, the formation of ⋅− 3 superoxide radical anion (O2 ) via the electron-transfer between QS* and molecular oxygen

76 make photo-oxidation possible through a superoxide pathway. This type I mechanism is depicted in the following equations [33, 55]: 3 3 ⋅+ ⋅− QS* + O2 → QS + O2 (4)

⋅+ QS + PC → QS + PCox (5)

⋅− − ⋅ O2 + PC−H → HO2 + PC (6)

− ⋅ PCox + HO2 + PC → further oxidation reactions (7)

In this work, hemin, an organic iron complex, is used as the primary oxidant to generate the luminol radical at basic solution (pH 11.6). The sample stream containing unconsumed 1 3 ⋅− oxygen species ( O2, O2, and O2 ) after photo-oxidation is mixed with the luminol stream in a ⋅− flow cell. The O2 can readily oxidize the luminol radical to form hydroperoxide [56,57] while 3 ⋅− O2 reacts with luminol radical to produce O2 and diazaquinone (L) first and then newly- ⋅− generated O2 further reacts with luminol radical to form hydroperoxide [56, 58]. In contrast to 3 1 − O2, O2 can oxidize luminol (LH ) directly into hydroperoxide [59]. The CL reactions between luminol and oxygen species are summerized in the following equations where APA is 3- aminophthalate.

LH− + hemin → L⋅− (8)

⋅− 3 ⋅− L + O2 → L + O2 (9)

⋅− ⋅− + − L + O2 + H → LHOO (10)

− 1 − LH + O2 → LHOO (11)

− LHOO → (APA)* + N2 (12)

(APA)* → APA + hν (13)

C.2. Effects of experimental parameters on the on-line photo-generation and CL detection of oxygen species

Simpler CL reactions were considered for the determination of phenol at a range of concentrations (25, 50, and 100 µM) under UV photolysis. Using either water, 0.4 µM luminol (pH 11.6), or 0.4 µM luminol with 8 µM hemin (pH 11.6) as the carrier stream, no signal different from the blank was observed upon injection of any of the three phenol solutions. When the same concentrations of phenol with 350 µM quinine sulfate were injected into the same three carrier streams, no signal was seen for water, weak signals from 10 – 20 units were found for luminol, and strong signals from 200 – 400 units were observed for luminol plus hemin. Therefore the importance of the strategy outlined above was confirmed and is summarized below. This detection method consists of three chemical processes: (1) photo-generation of oxygen species sensitized by QS; (2) oxidation of phenols by these oxidizing agents; (3) luminol- CL reaction of the remaining oxygen species. To characterize the on-line QS-sensitized process, several important experimental parameters and their effects on the generation and detection of oxygen species were studied by FI. C.2.1 Flow rate With the flow rates of carrier stream from pump #2 set at either 40 or 60 µL/min, the CL intensity was measured as a function of pump #1 flow rate (Figure 3.2). A lower flow rate of 40 µL/min for pump #2 lead to a somewhat higher yield of oxygen species generated due to a longer photolysis time in the coil reactor. However a 30% faster analysis time is possible when pump #2 is set at 60 µL/min. The flow rate at pump #1 influenced mixing efficiency of luminol and sample streams, and residence time of the CL reaction mixture in the flow cell. The CL intensity increased with increasing flow rate at pump #1 until a plateau was reached as the flow rate exceeded 75 µL/min. The flow rates of 95 µL/min at pump #1 and 60 µL/min at pump #2 (only a 10% loss in response for the higher pump #2 flowrate) were used throughout the remaining FI work. C.2.2 Solvent Two types of dual-component solvents, acetonitrile-water and methanol-water, were used to prepare 350 µM QS sample solutions. Their effects on CL intensity are shown in Figure 3.3.

600

500

400

300

200

100 CL Intensity (peak height), mV

0 45 55 65 75 85 95 105 115

Flow rate at pump #1, µL/min

Figure 3.2. Effect of flow rate on the sensitized photogeneration and CL detection of oxygen species. Analytical conditions: 0.4 mM-luminol-8 µM hemin CL solution buffered at pH 11.6 (P#1); Water as a sample carrier (P#2) at flow rates: (●) 40 µL/min and (▲)60 µL/min; 500 µM QS aqueous sample.

600

500

400

300

200

100 CL Intensity (peak height), mV

0 0 102030405060708090100 Organic solvent content, %

Figure 3.3. Effect of solvent on the sensitized photogeneration of oxygen species. Flow conditions: 95 µL/min (P#1) and 60 µL/min (P#2); 350 µM QS samples prepared using solvents (▲) acetonitrile-water and (○) methanol-water; other conditions as in Figure 3.2.

Acetonitrile seemed to act as an enhancer because the CL intensity showed a 27% increase for the QS sample prepared in 10% acetonitrile and 7% in 20% acetonitrile as compared to that obtained in an aqueous QS solution. In contrast, the CL intensity was nearly the same in 10% methanol but indicated a 36% decrease in 20% methanol from that obtained in aqueous solution. The CL intensity started to decrease with increasing organic solvent content after the organic solvent content exceeded 10% in both acetonitrile and methanol. However, the declining CL intensity reached a lower level earlier in methanol solutions (50-90%) than that in acetonitrile solutions (70-90%). QS was insoluble in an aprotic solvent such as 100% acetonitrile. As a result, its CL intensity rapidly fell down to a very low value near the baseline. In contrast, the CL intensity in 100% methanol had just a 24% decrease from the 60 – 90% in the methanol plateau region. The mechanism of CL enhancement by acetonitrile at low concentration is not clear, but it has been reported as a type of photosensitizer in the photolysis and CL determination of chloramphenicol [60]. CL enhancement could be also attributed to the increased lifetime of the excited species or radical in the low percentage acetonitrile medium. The CL intensity declined

with increasing organic solvent content possibly because of the decrease of O2 solubility and the change of solvent polarity in the dual-component solvents. The polarity parameter for water is 10.3 but only about half this for acetonitrile or methanol. C.2.3. pH The pH effect on the intensity of CL emission obtained from the QS-sensitized photochemical and luminol-CL reactions is shown in Figure 3.4(A). The CL intensity was quite steady between pH 1-5, but it dramatically increased until pH 7 where it plateued at pH 8.5. The pH effect study did not go beyond pH 8.5 because of the formation at pH 9 of a white precipitate believed to be quinine in the neutral form in the 500 µM QS solution. Three different ionization forms of quinine species can exist in aqueous solution, depending on pH: the dication, monocation and neutral form. The dissociation constants (pKa) of those species are 4.3 for the dication and 8.4 for the monocation [61]. The presence of quinine as monocation likely plays some role in its photosensitizing ability. The pH effect on the CL signal profile is shown in Figure 3.4(B). The CL signal at pH 1 consisted of two adjacent peaks; the left one started to grow and the right one started to shrink as pH increased. The maximum peak response appeared at pH 7. The peak profile was smoother but

Figure 3.4. Effect of pH on the (A) CL intensity and (B) CL signal profile obtained from the photochemical-luminol reaction sequence. The 500 µM QS aqueous samples are buffered at pH in the range 1-8.5; other conditions as in Figure 3.3.

450 400 A 350

300

250 200 150

100

CL intensity (peak height), mV 0 0123456789

83 not higher at pH 8. The efficiency of the luminol-CL reaction with oxygen species possibly depended on pH because the rate constant of decomposition of luminol hydroperoxide to aminophthalate in the monoanion form (Eq. 12) was observed to monotonically increase with pH approaching 10.5 [56]. Therefore, the shape of the CL signal could be related to the pattern of pH variation when the photolyzed sample stream from the photoreactor was mixing with the luminol solution (pH 11.6) from pump #1. The mixing of two streams with a large pH difference could result in a pH shift from its buffered pH value for the CL reaction. However, two streams with a small pH difference could easily maintain a buffered pH value, thus generating a CL peak. Furthermore, the influence of pH on the photosensitizing ability of quinine species mentioned as above might contribute to the change of CL signal profile. Aqueous sample solutions prepared at pH 7 were used for the remaining FI experiments. C.2.4. Concentration of photosensitizer QS as a photosensitizer played a very important role in the photogeneration of oxygen species. Such an effect was quantified by the CL measurement of those species in aqueous QS solution as shown in Figure 3.5. The increase in CL intensity (y) with QS concentration (x) displayed a linear relationship of y=1.4944x − 60.943 with a linear range of 50-350 µM and a regression coefficient (r2) of 0.9963. As the QS concentration increased beyond 350 µM, a leveling of CL response appeared. It was appropriate to choose 350 µM as a working concentration of QS for the subsequent phenol analysis because the background CL at a higher concentration of QS might decrease the quenched CL sensitivity. C.3. FI determination of phenols FI with this dual reaction detection method was applied to ten phenolic compounds and their calibration curves over 1-2 orders of magnitude are summarized in Table 3.1. Analytical results for each compound were obtained on the same day because the variation of dissolved oxygen in all reagents day-to-day could lead to the 2-4% enhancement of background CL of 350 µM QS. Chlorophenol and phenol presented similar but the strongest inhibition to the CL reaction and 4-nitrophenol quenched the CL signal more effectively than 2- and 3-nitrophenols. Precision data for 25 and 100 µM samples expressed by relative standard deviation (RSD) were in the range 0.7-1.7% (n=5). Disubstituted phenols such as 2,4-dichlorophenol and 2,4- dinitrophenol showed weaker responses than analogous monosubstituted compounds. Catechol

700

600

500

400

300

200

100

CL Intensity (peak height), mV 0

0 100 200 300 400 500 Quinine Sulfate, µM

Figure 3.5. Effect of photosensitizer concentration on the photogeneration of oxygen species. Aqueous QS samples buffered at pH 7; other conditions as in Figure 3.4.

Table 3.1. Calibration equations for ten phenolic compounds

Sample Calibration equation a Linear range (µM) r2

phenol y = -5.14x + 670 5-50 (n=4) 0.9924

4-chlorophenol y = -5.51x + 715 5-50 (n=4) 0.9947

4-nitrophenol y = -3.06x + 771 0−100 (n=5) 0.9891

3-nitrophenol y = -1.67x + 759 0-200 (n=7) 0.9997

2-nitrophenol y = -2.38x + 761 0-200 (n=7) 0.9991

2,4- y = -0.38x + 356 5 – 400 (n=6) 0.9861 dichlorophenol 2,4-dinitrophenol y = -0.96x + 354 5 – 250 (n=9) 0.9895 catechol y = -1.48x + 740 0 – 100 (n=3) 0.9972 salicylate y = -1.99x + 391 0 –100 (n=4) 0.9869

3-hydroxy-L- y = -2.53x + 459 0 –150 (n=5) 0.9936 knurenine a y, CL intensity (peak height); x, concentration of sample. N = data points defining the linear range.

86 and salicylate showed response factors similar to 3-nitrophenol. The biological compound 3- hydroxy-L-kynurenine, produced enzymatically by monooxygenase in the catabolism pathway for tryptophan, showed a response in the middle of the range. Some phenols such as gallic acid (3,4,5-trihydroxybenzoic acid) were not detectable due to their instability at high pH. Tannins such as pentagalloyl glucose also showed no response. Non phenolic compounds such as benzoate and acetylsalicylate were detectable with a sensitivity similar to slope values of 3-nitrophenol and salicylate, respectively, in Table 3.1. Kynurenine, the substrate for the enzyme monooxygenase, responded with a sensitivity two times less than 3-hydroxy-L- knurenine. An interference study involving common aromatic compounds with oxygen containing substituents was also made. The concentration (µM) of the interfering compound which caused a 5% increase in the response of 50 µM phenol with no interfering compound present is summarized as: acetophenone (100), anisole (200), benzaldehyde (400), and phenethanol (750). Good selectivity of this CL reaction for phenols as compared to other hydroxylated alkyl aromatics seems likely. Detection limits calculated from 3 times the standard deviation of the blank measurement were 4.1 and 4.4 µM (82 and 88 pmol) for phenol and 4-chlorophenol and 7.4 – 13.5 µM (148 – 270 pmol) for the nitrophenol isomers. Using FI with fluorescence detection, phenol could be detected as low as 0.002 µM (0.04 pmol for 20 µL injection), but none of the other chloro or nitro phenolic compounds showed any fluorescence response. This is not surprising since electron-withdrawing substituents such as -CL and -NO2 quench fluorescence. C.4. Determination of phenols by HPLC Chromatographic separation conditions were optimized using the instrument diagramed in Figure 3.1(B). Because of the time effect on the sensitized photochemical reaction, a flow rate of 100 µL/min for the mobile phase using pump #3 was chosen after comparison of the separation results from their UV and CL-inhibition chromatograms obtained at different flow rates. The mobile phase solvent ratio was 40:60 (v/v) acetonitrile/water optimized according to the retention time and resolution of each peak on the UV chromatograms. Both UV and quenched CL chromatograms for the aqueous sample mixture containing phenol, 4-nitrophenol, and 4-chlorophenol are presented in Figure 3.6. The retention factors (k’) for these compounds were 0.9, 1.5, and 2.4, respectively. 2-Nitrophenol with a k’ of 2.3 could also be separated but there was overlap with the 4-chlorophenol peak. The presence of two coil 87

UV 1

05 10 Time, min

CL-quench 1 3

Figure 3.6. CL-inhibition and UV detection chromatograms for the separation of phenols. Aqueous phenol mixture (50 µM each compound). 1 = phenol, 2 = 4-nitrophenol, and 3 = 4- chlorophenol. Polaris C18-A 3 µm HPLC column (50 × 4.6 mm id), 100 µL/min of acetonitrile- water mobile phase (40:60, v/v) (P#3), UV detection at 254 nm, 100 µL/min of 350 µM QS aqueous solution of pH 7 (P#2), and 370 µL/min of 0.4 mM luminol CL solution (P#1).

88 photoreactors in tandem after UV detection of phenols caused an approximately 1.4 min delay for CL detection. In the CL chromatogram, the average inhibited CL intensity values (negative peak heights) for the phenol, 4-nitrophenol, and 4-chlorophenol peaks were 77.5%, 34.9%, and 75.4% of the background CL intensity value with RSD in the range of 2.2 − 6.8% (n=3). Phenol and 4-chlorophenol displayed the strong inhibition to CL, almost twice that for 4-nitrophenol. In contrast, in the UV chromatogram, the weak UV absorption of 4-chlorophenol resulted in its peak height only 25% and 14% that of the peak heights for phenol and 4-nitrophenol, respectively. This photochemical quenched CL method did not achieve LC detection limits for phenols in the nM range as reported for EC methods or in the low pmol range as reported for prederivatization FL or CL methods. The LC detection limits were about 1 µM (20 pmol) for phenol and 4-chlorophenol and 3.5 µM (70 pmol) for 4-nitrophenol; these were about 2-4 times better than FI because of the higher LC flowrate giving sharper peaks. The longer LC photolysis reactor also ensured that the reaction time was only somewhat shorter. The major problem that limited the detection limit was variation of background CL up to 6% due in part to pump noise. Using LC with UV absorbance at 254 nm, detection limits of 0.4 µM (8 pmol), 0.7 µM (14 pmol), and 3.5 µM (70 pmol) were found for 4-nitrophenol, phenol, and 4-chlorophenol, respectively. Although the LC quenched CL detection limits are at best comparable to those by UV detection, this CL reaction when coupled in series to a UV detector could aid in the qualitative identification of oxygenated aromatics. Such an application could be the photocatalytic degradation of toluene in aqueous solution using a TiO2 catalyst and a surfactant which generates 4-methylphenol, benzaldehyde, and other oxygenated aromatics [62].

Section D: Conclusions

The use of quinine sulfate as a photosensitizer in a FI or LC mode for the production of reactive oxygen species (ROS) that could react with aromatic compounds, particularly phenol, 4- chlorophenol and 4-nitrophenol, causing an analytical decrease in the luminol CL signal was demonstrated. LC detection limits for phenols with this quenched post-column CL method were higher than those found for prederivatization based luminescence methods. If adapted for CE, this CL detection method may be more competitive than UV detection which is limited by the

89 short capillary pathlength. Because nonphenolic compounds with reactive oxygen species- scavenging activity such as acetophenone, anisole, benzoic acid, and knurenine were detectable, the potential reactivity of this CL method still needs to be explored.

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Chapter IV Indirect fluorescent determination of aromatic compounds via a shielding effect on the UV-photolysis of 2-phenylbenzimidazole-5-sulfonic acid

Section A: Introduction

At present, aromatic compounds are often determined by flow injection (FI) and liquid chromatography (LC) with FL instead of UV detection to gain selectivity and detection limit advantages. For those non-fluorescent or weakly fluorescent aromatic compounds, e.g. nitro or halogen functional group-substituted aromatics, chemical derivatization that introduces fluorophores to those molecules is generally required for FL detection [1-4]. Off-line derivatization procedures are most commonly used because of reaction versatility, but sometimes extreme conditions, e.g. long reaction times and time-consuming sample purification and preconcentration procedures are required [5-7]. Therefore, development of on-line derivatization techniques for fluorescence detection of non-fluorescent compounds has been a research area in analytical chemistry for the last two decades [8-14]. For example, nitro-substituted polynuclear aromatic hydrocarbons can be determined by FL detection method via post-column

electrochemical derivatization approach by which the NO2 group is reduced to the NH2 group using an electrochemical detector. Photochemical derivatization, applied to aromatic compounds that react under UV irradiation, can lead to the formation of strongly fluorescent photoproducts [10, 15-18]. Because light is used instead of the chemical reagent, on-line photochemical derivatization is simple and flexible as compared to on-line chemical derivatization approaches. However, application of photochemical derivatization is restricted to certain types of photoreactive compounds or drugs that can efficiently convert into fluorophores under UV irradiation. Nitroaromatic compounds such as nitrophenols via on-line photolysis seldom convert to significant fluorescent products 2+ unless an oxidizing reagent (e.g. H2O2) and a photocatalyst (e.g. TiO2 or Fe ) are present in the analyte solution [19]. Even under those conditions, a powerful UV light source and a long photolysis time were still required to achieve efficient conversion. Because of those

97 disadvantages, on-line photochemical derivatization may not be a practical approach for FL determination of some nitro compounds by FI and LC. 2-Phenylbenzimidazole-5-sulfonic acid (PBSA) is a UV-B sunscreen agent with characteristic UV absorbance at 302 nm and strong fluorescence around 350 nm. PBSA is also a photoreactive compound that readily undergoes photodecomposition in alkaline solution under UV irradiation, resulting in a fast decrease in its fluorescence. It has been reported that PBSA 1 •– acts as a sensitizer for photogeneration of singlet oxygen ( O2) and superoxide (O2 ) that are capable of damaging DNA [20, 21]. Those oxygen species could be the oxidizing agents for further oxidation of PBSA. Therefore, compounds that can have strong UV absorbance at certain wavelengths or/and are reactive toward those oxygen species can have a shielding effect on the UV-photolysis of PBSA. An indirect FL detection method via this shielding effect on the UV-photolysis of PBSA has been developed in our laboratory. In this work, thirteen aromatic compounds were studied on their shielding effect, including 4-nitroaniline, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, α- nitronaphthalene, nitrofurantoin, atropine, brompheniramine, chlorpheniramine, 4-nitrobenzoic acid, gallic acid, cinnamic acid and sodium salicylate. The molecular structures of PBSA and these thirteen aromatic analytes are shown in Figure 4.1. The aromatic compounds with electron withdrawing groups are non-fluorescent. Nitrofurantoin is an antibacterial agent used for the treatment and prevention of urinary tract infections [22]. Chlorpheniramine and brompheniramine are representative antihistamine drugs [23, 24]. Atropine is widely used as a parasympatolytic, anticholinergic and antiemetic drug [25]. Nitrophenols are phenol polutants widely present in the environment where they appear as intermediates in the synthesis of many drugs, dyes, various pesticides as well as major metabolic or photolytic degradation products of these compounds [26]. Nitroaniline is widely produced as dye and pesticide intermediates and also used as a photoinitiator of polymerization processes [27]. To study the fundamental aspect of this indirect FL detection method, off-line photolysis of PBSA and the shielding effect of several aromatic compounds were investigated using UV and FL spectroscopy. Reverse phase ion-pair LC-MS in the presence of a volatile amine ion-pairing reagent in the mobile phase was effective for the separation of PBSA photolysis products. The optimization of experimental conditions for photolysis and FL detection was also investigated. Finally, application of this

OH OH OH NH COOH 2 O NO2 ONa

OH NO2 NO2 NO2 NO2

4-Nitrophenol 3-Nitrophenol 2-Nitrophenol 4-Nitroaniline Sodium salicylate 4-Nitrobenzoic acid

COOH NO2 COOH O N O 2 O N N NH HO OH O OH N Gallic acid Nitronaphthalene trans-Cinnamic acid Nitrofurantoin

Cl Br H HO3S N O N N N O OH

N N

Chloropheniramine Brompheniramine Atropine 2-Phenylbenzimidazole-5- sulfonic acid (PBSA)

Figure 4.1. Structures of PBSA and thirteen aromatic compounds used in this study.

99 detection method for the on-line determination of aromatic compounds was carried out by FI and LC.

Section B: Experimental

B.1. Chemicals and solutions 2-Phenylbenzimidazole-5-sulfonic acid (PBSA), 4-nitroaniline (4-NA), 2-nitrophenol (2- NP), 3-nitrophenol (3-NP), 4-nitrophenol (4-NP), 4-nitrobenzoic acid (4-NBA), α- nitronaphthalene (NN), and tert-octylamine were purchased from Aldrich (Milwaukee, WI). Nitrofurantoin (NF), atropine (AT), brompheniramine (Br-Phe), chlorpheniramine (Cl-Phe), gallic acid (GA), and sodium salicylate (SS) were provided from Sigma (St. Louis, MO). trans- Cinnamic acid (CA) was purchased from Tokyo TCI (Tokyo, Japan). HPLC grade methanol (MeOH) and acetonitrile (ACN) were obtained from Burdick & Jackson (Muskegon, MI) and Pharmaco (Brookfield, CT), respectively. All other chemicals used are at least of analytical reagent grade as well. Distilled-deionized water used for preparation of solutions was taken from the E-Pure deionization system (Barnstead, Dubuque, IA). A 1 mM PBSA stock solution was prepared daily by dissolving 0.028 g of PBSA in 1 mL of 0.2 M NaOH solution and then diluting to 100 mL with water. PBSA solutions of 100 µM (pH 7 and pH 12) for off-line photolysis studies were prepared by a 10-fold dilution of PBSA stock solution with 0.1 M potassium phosphate monobasic buffer solution (pH 7) and NaOH/KCl buffer solution (pH 12). PBSA solutions of 1 µM for off-line shielding effect studies were obtained from serial dilution of the PBSA stock solution with NaOH/KCl buffer (pH 12). For the study of the pH effect on FL detection in HPLC, phosphate dibasic and NaOH/KCl buffer solutions described in the literature [28] were used to prepare 2 µM PBSA solutions with the pH values in the range between 11.6 to 12.6. All PBSA solutions were stored in flasks or vials that were covered with aluminum foil to avoid light and kept in the dark when not in use. Stock solutions of 1 mM for eleven aromatic samples were prepared in aqueous solution except gallic acid and α-nitronaphthalene which were made in acetonitrile. Benzoic acid, trans- cinnamic acid and nitrofunrantoin stock solutions were prepared by dissolving samples in 2 mL of 0.2 M NaOH solution first and then diluting with water to 100 mL. For HPLC analysis, a 1 mM nitrofurantoin stock solution was prepared in acetonitrile. A 10 mM tert-octylamine stock

100 solution was prepared by mixing of equimolar amount of 0.877 mL tert-octyamine (95%) and 0.287 mL acetic acid (100%) in 10 mL methanol and then diluting to 500 mL with water. All light sensitive sample solutions were stored in flasks and vials covered with aluminum foil to keep from light exposure. B.2. Apparatus The instrumental set up for FI and HPLC determination of aromatic compounds with on- line FL detection via the shielding effect of the photolysis of PBSA is given in Figure 4.2. Ultra- plus micro LC pumps (pump #1 and #2) (Micro-Tech Scientific, Sunnyvale, CA) were used to transport the sample carrier buffer in the FI system as shown in Figure 4.2(A) and the mobile phase and PBSA solutions in the HPLC instrument as shown in Figure 4.2(B). Samples of interest were injected using a Rheodyne model 7125 injection valve with a 20-µL loop. A MetaChem Polaris C18-A 3 µm HPLC column (50 × 4.6 mm id) (ANSYS Technologies, Lake Forest, CA) was used to separate aromatic analytes in the sample mixture. Two tandem photoreactors are the key part for on-line UV photolysis of PBSA and analyte mixture solutions that were premixed before injection for FI or combined on-line using a low dead volume mixing tee for HPLC. Each one was made from 3048 mm (l) × 1.6 mm (od) ×0.25 mm (id) fluoroethylene polymer (FEP) tubing (Upchurch Scientific, Oak Harbor, WA) circling around the NIPPO GL4 Hg lamp. The FL emission of photolyzed samples was measured at 350 nm using a Shimadzu RF-551 spectrofluorometric detector (Shimadzu, Columbia, MD) with an excitation wavelength set at 302 nm. FL signals were processed and recorded by a Shimadzu Chromatopac C-R6A integrator. The off-line photolysis experiments were carried out in a round 5-mL quartz cell placed against a NIPPO GL4 Hg lamp. FL spectra of photolyzed sample solutions were measured using a Perkin Elmer LS 55 luminescence spectrometer (Perkin Elmer, Boston, MA) with an excitation wavelength of 302 nm. Absorption spectra of PBSA and analyte samples were recorded using a Hewlett-Packard 8453 UV-VIS spectrophotometer (Agilent, Palo Alto, CA). MS spectra of photolyzed PBSA samples were obtained using an Esquire LC-electrospray ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). A MetaChem Polaris C18-A 3 µm HPLC column (50 × 4.6 mm id) was used to separate PBSA and its associated photolyzed species from the sample matrix.

101

A Sample

NaOH/KCl P#1 FL detector Pump Injector Coil photoreactor Waste

B Sample

ACN/H20 # (40/60) P 1 LC column Coil photoreactor

2 µM PBSA P#2 FL detector

Waste

Figure 4.2. Instrumental set up for the indirect FL determination of aromatic compounds by (A) FI and (B) HPLC.

102

B.3. Procedures B.3.1. Off-line photolysis of PBSA and PBSA with aromatic compounds A 4 mL volume of 100 µM PBSA prepared in pH 7 and pH 12 solution were photolyzed for 0, 15, 45 minutes. A 100-fold dilution of the PBSA samples after photolysis was required to get the FL spectra. UV absorption spectra were obtained directly from the photolyzed samples without further dilution. PBSA aqueous solutions of 100 µM (pH 12) were photolyzed for 0, 30 and 60 min. A 250 µL volume of each photolyzed sample was diluted with 750 µL methanol before a 20-µL injection into the LC-MS instrument. The mobile phase for the separation of anionic PBSA and its associated species by HPLC was 40/60 methanol/H2O (v/v) in the presence of 2.5 mM tert- octylamine as an ion-pairing agent. The flow rate of the mobile phase was set at 320 µL/min and negative ionization mode was chosen for ESI-MS. Nitrogen dry gas, temperature, and nebulizer pressure were set at 9 L/min, 365 ˚C, and 40 psi, respectively. The capillary high voltage was set at 3500 V. Total-ion current chromatograms and MS spectra of photolyzed PBSA samples were obtained and the number of spectra averaged was 4. Aqueous solutions containing 1 µM PBSA and 4-nitroaniline with concentrations of 0, 0.1, 0.5, 1.5, 2.5, 5, 10, and 15 µM were prepared in pH 12 buffer solution. The 4 mL sample solution was photolyzed for 30 minutes before the FL spectrum was taken. To study the shielding effect of other aromatic compounds, 1 µM PBSA aqueous solutions each containing 5 µM 4-nitrobenzoic acid, sodium salicylate, 3-nitrophenol, 2-nitrophenol, 4-nitrophenol, and 4- nitroaniline were prepared in pH 12 buffer solution. Their FL spectra were taken after 30-minute UV photolysis of each 4 mL sample solution. B.3.2. Flow injection and HPLC of aromatic compounds After experimental factors, including PBSA concentration, flow rate, and solvent were investigated by FI, 13 aromatic samples containing 2 µM PBSA with concentrations of 0, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 40, 50, 60, 75, and 100 µM were prepared from appropriate dilution of their stock solutions with pH 12 NaOH/KCl buffer solution. The pH 12 NaOH/KCl buffer solution was used as a FI sample carrier. With a flow rate of 175 µL/min, five injections for each sample solution generated a calibration curve for each aromatic compound. Analytical figures of merit for this FI indirect FL detection method were determined.

103

Concentration, flow rate, and pH of the PBSA solution delivered from pump #2 were optimized for indirect FL detection with HPLC by separation of a three-component sample mixture that contained 25 µM nitrofurantoin, 5 µM 4-nitrophenol and 4-nitroaniline. Under the flow conditions of 150 µL/min for 40/60 ACN/H2O mobile phase from pump #1 and 125 µL/min for 2 µM PBSA solution (pH 12), from pump #2, a five-component sample mixture that was made up of salicylate, nitrofurantoin, 4-nitroaniline, 4-nitrophenol, and 2-nitrophenol was separated by HPLC. Five-component sample solutions with concentrations of 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µM for nitrofurantoin and concentrations of 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30 and 40 µM for other four components were injected four times for each sample mixture to generate the calibration curves for this HPLC method. Analytical figures of merit were also determined.

Section C: Results and Discussion

C.1. Off-line photolysis of PBSA Before the development of the on-line indirect FL detection method based on the shielding effect of analytes on the photolysis of PBSA, experimental effects on off-line photolysis of PBSA were studied, including pH, photolysis time, and reaction conditions. Reverse phase ion-pair LC-MS was also used to study the photolyzed PBSA sample. C.1.1. pH and photolysis time FL spectra of the photolyzed PBSA sample are shown in Figure 4.3(A). Photolysis of PBSA aqueous solutions proceeded slowly at pH 7 as compared to that at pH 12. A decrease of FL intensity by approximately one third of the original value was observed after UV photolysis of 100 µM PBSA solution at pH 12 for 15 min. The UV absorption spectra of a PBSA sample photolyzed at pH 7 and 12, which are shown in Figure 4.3(B), exhibited similar results to the fluorescence data with little change at pH 7 and a matched absorbance decrease at pH 12. The UV absorbance displayed a large change at 217, 247 and 302 nm before and after UV photolysis of PBSA at pH 12 for 45 minutes, which can be observed in the inserted diagram in Figure 4.3(B). The existence of different absorbance and florescence spectra for aqueous PBSA under neutral and alkaline pH is the result of the difference in the electronic distribution in the ground state and excited states because the singly charged PBSA anion (pKa1 = 5.23) and doubly

104

Figure 4.3. Molecular spectra showing the effect of pH and photolysis time on the off-line photolysis of PBSA. (A) FL spectra obtained after A 100-fold dilution of the 100 µM PBSA solutions; (B) UV spectra obtained from 100 µM PBSA solutions with the inserted plot of UV absorbance difference before and after photolysis for 45 minutes vs. wavelength. Photolysis of 100 µM PBSA (pH 7): (a) 0 min; (b) 15 min; (c) 45 min. Photolysis of 100 µM PBSA (pH 12): (d) 0 min; (e) 15 min; (f) 45 min.

105

a A b d c

e e t

c s

r o u f

4.5 B 1.6 4 1.4 1.2 3.5 1 0.8 3 0.6 0.4 2.5 difference Abs. 0.2 a 0 2 -0.2 b d 200 250 300 350 400 450 1.5 c Wavelength, nm

Absorbance, AU Absorbance, e 1

0.5 f

0 200 300 400 500 600 Wavelength, nm

106

charged PBSA anion (pKa2 = 11.91) are formed at pH 7 and pH 12, respectively. The reasons for a fast photodecomposition of PBSA in alkaline solution were not clear. Besides the excited states 1 of PBSA, pH might affect the photogeneration of oxygen species such as singlet oxygen ( O2) •– and superoxide (O2 ) [21] that were believed to be involved in the self-oxidation of PBSA during the UV photolysis. C.1.2. Chemical shielding effect The chemical effect on the photolysis of PBSA that results in the recovery of FL intensity is called a shielding effect. Figure 4.4(A) showed such an effect from 4-nitroaniline. A linear increase in FL intensity with concentration of 4-nitroaniline in the range from 0 to 5 µM was observed for the off-line photolyzed PBSA samples. When the concentrations of 4-nitroaniline in PBSA solution were above 5 µM, the shielding effect reached the upper limit. Like 4- nitroaniline, other nitro compounds and salicyate exhibited the shielding effect as shown in Figure 4.4 (B). The shielding effect order of those compounds was 4-nitroaniline > 4-nitrophenol > 2-nitrophenol > 3-nitrophenol > sodium salicylate > 4-nitrobenzoic acid. According to the molar absorption coefficients (ε) determined from 50 µΜ aqueous samples in 1 cm quartz cuvette at 217, 247, and 302 nm, nearly all those aromatic compounds had the highest ε values at 217 nm. Compounds in order of decreasing ε values were 2-nitrophenol (12538), 3-nitrophenol (11730), sodium salicylate (6058), 4-nitroaniline (6011), 4-nitrobenzoic acid (5585), and 4- nitrophenol (5582). The ε values for 2-nitrophenol, 3-nitrophenol, sodium salicylate, 4- nitrobenzoic acid seemed to confirm that the shielding effect on photolysis of PBSA was attributed to the UV absorption capacities of those compounds, but the large shielding effects caused by 4-nitrophenol and 4-nitroaniline was surprisingly considering their relatively small ε values, 5582 and 6011, respectively. Therefore, another mechanism besides the shielding effect from UV absorbance by aromatic compound is involved. Because 4-nitroaniline and phenolic compounds are subject to photochemical reactions, shielding by the photooxidation of aromatic 1 •– samples that compete with photooxidation of PBSA for oxygen species (e.g. O2 and O2 ) during the UV photolysis is a possibility. C.1.3 Study of photolyzed PBSA samples by reverse phase ion pair LC-MS It was of interest to determine the photolysis products of PBSA by LC/MS. Tetraalkyammonium compounds have been used commonly as ion-pairing agents for acidic aromatic sulfonates but can cause severe contamination of the interface and decreased sensitivity 107

Figure 4.4. Shielding effect on the off-line photolysis of PBSA. (A) FL spectra of 1 µM PBSA solutions (pH 12) containing 4-nitroaniline with concentrations of 0, 0.1, 0.5, 1, 1.5, 2.5, 5, 10 and 15 µM (in order of increasing peak height) after 30-min UV photolysis; (B) FL spectra of 1 µM PBSA solutions (pH 12) containing 5 µM 4-nitrobenzoic acid, sodium salicylate, 3- nitrophenol, 2-nitrophenol, 4-nitrophenol and 4-nitroaniline (in order of increasing peak height) after 30-min photolysis.

108

s n

n I

e c

e r

u l

Intensity Fluorescence

109 of electrospray ionization (ESI) MS due to their extremely poor volatility [29]. Volatile tert- octyamine (b.p. 137-143˚C) was used as an ion-pairing agent for the LC-MS analysis of photolyzed PBSA samples as shown in Figure 4.5. The total ion current (TIC) chromatograms consisted of two major peaks, the sample matrix peak eluted around 2 minutes, and the PBSA peak eluted around 7.5 minutes. Apparently, no one has studied the photolysis products of PBSA. The structures listed for those ions below are just the ones with molecular weights close to the corresponding m/z values without the confirmation by other analytical techniques. Ions in the sample matrix peak were mostly cluster ions that were made up of K, Na, Cl and sulfonate

(SO3) ions. In the MS spectra of photolyzed PBSA samples, the m/z 273.3 ion was a depronated - - molecular ion of PBSA ([M-H] ). The m/z 193.7 ion was a desulfonated ion [M-SO3-H] ). The m/z 222.8 ion is possibly an impurity present in PBSA sample. The possible ion structure was - [(M-SO3-H)+CHO] according to its m/z value. Ions with m/z 315.3, 340.7, and 359.4 were most possibly generated from the oxidative breakage of the imidazole ring of PBSA. According to - - their m/z values, the possible structures were [Ø(NHCOph)2-H] or [Ø(NHCOph)(NH2)(COph)] - for the m/z 315.3 ion, [Ø(NHCOph)(NHCOH)(SO3Na)-H] for the m/z 340.7 ion, and - [Ø(NHCOph)2(COOH)-H] for the m/z 359.4 ion. Ion with m/z 547.2 was the PBSA dimer anion ([2M-H]-). Both m/z 569.4 and 585.0 ions were the sodium-adducted ([2M-2H+Na]-) and potassium-adducted PBSA dimer ions ([2M-2H+K]-), respectively. The ion with m/z 821.0 was the PBSA trimer ion ([3M-H]-). Both m/z 842.9 and 858.9 ions are the sodium-adducted ([3M- 2H+Na]-) and potassium-adducted PBSA trimer ions ([3M-2H+K]-). Photolysis had a great effect on the intensity and chemical composition of ions comprised in the PBSA sample peak as shown in the TIC chromatograms. As compared to the ion intensity of PBSA and its associated species before photolysis, a decrease of ion intensity (all ratios less than 1) by a range of 31% to 79% for 30-minute photolysis and 54% to 98% for 60-minute photolysis was observed for ions with m/z values at 193.7, 222.8, 273.3, 547.2 and 821 (Figure 4.6). However, a remarkable increase in ion intensity from the original values obtained with intact PBSA (all ratios greater than 1) by 35% to 65% for 30-minute photolysis and 105% to 139% for 60-minute photolysis was generated for ions with m/z values at 315.4, 340.7 and 359.3. The presence of those ions that had increased ion intensity with photolysis time indicated that the opening of the benzimidazole ring in the PBSA molecular structure was a major step leading to the formation of products that exhibited weak or non-fluorescence. The reaction mechanism was not very clear, but

110

Figure 4.5. Analysis of photolyzed PBSA (MW=274.3) sample after photolysis for (A) 0 min, (B) 30 min and (C) 60 min by reversed phase ion-pair LC-ESI-MS using tert-octylamine as an ion-pairing agent. Negative mode. See ion-pair LC-MS operation parameters in Experimental.

111

112

3.000

30-min photolysis 2.500 60-min photolysis

2.000

1.500

1.000

0.500

Ion intensity ratio (I(t)/I(0)) 0.000

193.7 222.8 273.3 315.3 340.7 359.3 547.2 569.1 585 821 842.9 858.9

m/z

Figure 4.6. The photolysis effect on ion intensity and ion distribution in MS spectra of PBSA derived from the total ion current chromatograms obtained by LC-MS.

113 photogenerated active oxygen species were the most possible agents to attack the benzimidazole ring. C.2. Indirect FL detection via the shielding effect on photolysis of PBSA Based on the findings from the studies on the off-line photolysis of PBSA, this indirect FL detection method could be described by the following processes:

hυ ISC PBSA 1PBSA* 3PBSA* (1)

3 1 PBSA* + O2 PBSA + O2 (2)

3 •+ •– PBSA* + O2 PBSA + O2 (3)

1 •– PBSA + O2 or O2 photolytic products (4)

Analyte + hυ (UV) partially shielding UV light from PBSA (5)

1 •– Analyte + O2 or O2 oxidized products (6)

Under UV irradiation, PBSA was excited to the singlet state. Through intersystem crossing (ISC), a triplet PBSA* species is formed. Singlet oxygen and superoxide are generated via the energy transfer and electron transfer between triplet PBSA* and an oxygen molecule at ground state, respectively. The reactions between PBSA and those oxygen species will decompose PBSA*, resulting in the decrease of FL intensity of PBSA measured at 350 nm. In the presence of aromatic analytes that partially shield UV light and/or compete with PBSA for oxygen species in the photochemical reactions, the number of photo-excited PBSA molecules decreases and less PBSA molecules are photodecomposed. As a result, the FL signal of PBSA will go up. The recovery of FL signal of PBSA is dependant on the shielding effect and the concentration of analytes. Because of that, this indirect FL detection method is suitable for on- line determination of non-fluorescent aromatic compounds by flow injection (FI) and HPLC. C.3 Application of the indirect FL detection method for on-line determination of aromatic compounds by FI The effects of experimental conditions on the on-line photolysis of PBSA were investigated by measuring the FL intensity ratios of PBSA samples without and with UV

114 photolysis. Aromatic compounds were determined by this on-line FL detection method under appropriate experimental conditions. C.3.1. PBSA concentration Figure 4.7 shows the PBSA concentration effect on the photodecompsition of PBSA. The ratio of FL intensity of PBSA without and with UV irradiation was used to evaluate the photolysis efficiency. A large ratio of FL(UV-off)/FL(UV-on) (peak area/peak area) represented a high photolysis efficiency. The FL intensity ratio increased rapidly with PBSA concentrations from 0.05 to 0.4 µM but just had a little change from 0.4 to 2 µM. A large decrease in the FL intensity ratios occurred when PBSA concentrations were increased in the range of 2 to 16 µM. Although the photolysis efficiencies for 0.4 and 2 µM PBSA samples were close to each other, FL signal intensity (peak height) for 2 µM PBSA samples with and without photolysis was l17.4 and 890.8, respectively, as compared to 22.8 and 180.6 for 0.4 µM PBSA. To make this detection method sensitive to the shielding effect of analytes, 2 µM PBSA was used for the remaining FI work. C.3.2. Flow rate Flow rate controlled the photolysis time and analysis time as well. Figure 4.8 shows the flow rate effect as measured by FL (UV-off) / FL (UV-on) on the photolysis of 2 µM PBSA. Low flow rate lead to high photodecomposition yields. As flow rates were 175 µL/min or below, more than 90% of the PBSA was photo-decomposed. However, at 500 µL/min, approximately 50% of the PBSA sample was decomposed by UV photolysis. Because this detection method took advantage of the shielding effect of analytes on the photolysis of PBSA, on-line photolysis at the flow rate of 175 µL/min would provide a good response to the shielding effect of analytes. C.3.3. Solvent Solvent effect on photolysis of 2 µM PBSA as measured by FL (UV-off) / FL (UV-on) is shown in Figure 4.9. On-line photolysis of 2 µM PBSA samples prepared in ACN-H2O and

MeOH-H2O was much slower than those in aqueous solution. The influence of increasing organic solvent content from 5-30% on photolysis showed a slightly downward trend for the FL intensity ratio. A considerable decrease of the FL intensity ratio occurred for increasing MeOH content in the range from 1.25% to 5%. The reason that photolysis of PBSA in ACN-H2O was a little more efficient than in MeOH-H2O was not clear. The solvent polarity parameters P’ for

115

2 FL(uv-off)/FL(uv-on) 0 0 2 4 6 8 10 12 14 16

PBSA, uM

Figure 4.7. Concentration effect on the on-line photolysis of PBSA as measured by the FL intensity ratio. A 20 µL volume of each PBSA sample (pH 12) with concentration in the range from 0.04 - 16 µM was injected into the FI system. Flow rate of sample carrier buffer (pH 12) was set at 200 µL/min. The ratio of FL intensity without and with UV photolysis was used to represent the photolysis efficiency. RSD for these data ranged from 0.2 to 2.6% (n = 4).

116

16 14 12 10 8 6

4 FL(uv-off)/FL(uv-on) 2 0 150 200 250 300 350 400 450 500

Flow rate, uL/min

Figure 4.8. Flow rate effect on the on-line photolysis of PBSA as measured by the FL intensity ratio. Sample solutions of 2 µM PBSA were injected into the FI system at flow rates in the range between 150 and 500 µL/min. RSD for these data ranged from 0.3 to 4.2% (n = 4).

117

12 ACN 10 MeOH

FL(uv-off) /FL(uv-on) 2

0 5 10 15 20 25 30 Organic solvent content, %

Figure 4.9. Organic solvent effect on on-line photolysis of PBSA as measured by the FL intensity ratio. 2 µM PBSA samples were prepared in solution buffered at pH 12 with organic solvent in the range from 0 to 30% (v/v). Flow rate by FI was set at 175 µL/min. RSD for these data ranged from 1.0 to 3.4% (n = 4) for ACN and 0.1 to 2.6% (n = 4) for MeOH.

118

MeOH and ACN are similar (5.1 and 5.8) but there could be some effect on the excited states of PBSA, which are important for the photogeneration of active oxygen species. C.3.4. Flow injection determination of 13 aromatic compounds Under the optimal conditions, thirteen aromatic compounds as shown in Figure 4.1 were determined by this indirect FL detection method based on the shielding effect of analytes on the photolysis of PBSA. Among these thirteen aromatic compounds, there are nine non-fluorescent compounds (4-NP, 4-NA, 2-NP, 3-NP, NN, NF, 4-NBA, AT, Cl-Phe, and Br-Phe) and three modestly fluorescent compounds (SS, CA and GA). Tables 4.1(A) and (B) list calibration curve data and analytical figures of merits for those aromatic compounds determined by this proposed detection method. Detection limits were determined from the three times the standard deviation of five replicate FL measurements of a 2 µM PBSA solution. The relative standard deviation (RSD) values for determination of thirteen compounds with concentration of 5 µM are in the range between 0.66% and 3.42% (n=5). The sensitivity of this detection method was largely influenced by the shielding effect of analytes. As mentioned before, the shielding effect is associated with the ability of analytes to either absorb UV light at certain wavelengths that is strongly absorbed by PBSA during the photolysis or compete with PBSA molecules for active oxygen species from their own photochemical reactions or a combination of both. The capability of UV shielding by the analyte was best represented by the molar absorption coefficient (ε). UV-photolysis of PBSA exhibited a large decrease of UV absorbance at 217, 247 and 302 nm as mentioned before. The molar absorption coefficient (ε) values used for this shielding effect study were those highest values obtained at 217 nm because the low molar absorption coefficent values of analytes measured at 247 and 302 nm could not give any better correlation with the sensitivity data of analytes. The molar absorption efficient (ε) at 217 nm and the sensitivity (slope of the calibration curve by this detection method) for 13 aromatic compounds are compared in Figure 4.10. A fair correlation observed in compounds with the detection sensitivity below 13 (NN to 4-NBA) suggested that the UV absorbance of those compounds at 217 nm may be a dominant factor responsible for the shielding effect on the photolysis of PBSA. However, for compounds with detection sensitivity above 18 (4-NP to AT), the shielding effect was not merely coming from the UV-absorbance at 217 nm because their ε values were not as high as expected. From our previous work on photooxidation of phenolic compounds, phenol, nitrophenols and chlorophenol were reactive

119

Table 4.1A. Analytical figures of merit of the proposed FL detection method for FI determination of nitroaromatic compounds

Molecules studied Regression Linear Detection Correlation equation range, µM limit, µM coefficient(r2) 4-Nitrophenol y = 34.0x + 44.3 0.5-10 0.18 0.9991 4-Nitroaniline y = 28.9x + 76.9 0.5-5 0.21 0.9999 2-Nitrophenol y = 29.6x + 73.3 0.5-10 0.21 0.9976 3-Nitrophenol y = 25.5x + 54.9 0.5-10 0.24 0.9979 α-Nitronaphthalene y = 12.5x + 1-15 1.0 0.997 226.3 Nitrofurantoin y = 5.0x + 80.0 5-50 1.24 0.9953 4-Nitrobenzoic acid y = 1.4x + 42.7 5-75 4.29 0.9951

Table 4.1B. Analytical figures of merit of proposed FL detection method for FI determination of aromatic compounds. Molecules studied Regression Linear Detection Correlation equation range, µM limit, µM coefficient(r2)

Sodium salicylate y = 20.5x + 54.1 0.5-10 0.30 0.9981

Atropine y = 18.2x + 64.0 0.5-7.5 0.34 0.9929

Chlorpheniramine y = 9.1x + 54.4 1-15 0.68 0.9988 Brompheniramine y = 8.4x + 33.6 1-15 0.74 0.99 trans-Cinnamic y = 3.8x + 52.3 5-40 1.63 0.9948 acid

Gallic acid y = 2.7x + 224.3 5-50 4.68 0.9987

120

40 40000.0

35 Sensitivity 35000.0

30 molar abs. coeff. 30000.0

25 25000.0 20 20000.0

Sensitivity 15 15000.0

10 10000.0 Molar abs. coeff.(217 nm) 5 5000.0

0 0.0

E E F A -NP SS AT NN H N CA GA 4 2- NP 4-NA 3-NP -P -NB L 4 C BR-PH Ana lyte

Figure 4.10. The molar absorption efficient (ε) at 217 nm and sensitivity of determination of 13 aromatic compounds by the shielding effect-based FL detection method. The molar absorption coefficient of each analyte was determined from UV measurement of a 50 µM sample solution using a 10-mm quartz cuvette.

121 toward oxygen species generated from photolysis [30]. The reactivity order of nitrophenols was 4-NP> 2-NP> 3-NP, which is in accordance with sensitivity results from the current study. 4-NA is structurally similar to 4-NP. AT is not a phenolic compound, but the hydroxymethyl group in the alpha position on the benzene-ester structure could possibly still make AT subject to photooxidation. Therefore, the photoreactivity of analytes that compete with PBSA for oxygen species may be the most important factor in determining the shielding effect of those aromatic compounds with an amino or hydroxyl group on or nearby the benzene ring although the UV absorption coefficient values of 2-NP, 3-NP and AT are larger than those of 4-NP, 4-NA and SS. Photoreactive GA, an antioxidant, with a detection sensitivity value below 3 is an exception because it is very unstable and oxidized rapidly in the alkaline solution [31]. It seems that only optical chemical sensor-based FL quenching detection techniques have been reported on the determination of nitro aromatic compounds without prederivatization being involved [32-35]. The sensor-based detection limits for 4-nitrophenol [32], 2-nitrophenol [33], nitrofurantoin [34], 2-nitroaniline [35] are 10, 80, 0.48, and 0.1 µM, respectively, but detection of 4-nitroaniline is not as sensitive as 2-nitroaniline. Atropine also can be detected at 4.3 µM by this optical sensor method [36]. As compared to our FL detection method based on shielding effect, those sensor methods are about 12 to 380 times higher in terms of detection limit although sensor detection of NF is better than ours. Furthermore, sensor response times from 10 sec to min are generally required to get stable quenched FL signals and this response recovery time may prevent continuously monitoring of analytes in a flow stream, such as FI and HPLC. C.4. Application of this FL detection method for on-line determination of aromatic compounds by HPLC C.4.1. Optimization of experimental conditions for FL detection It has been previously shown that photolysis of PBSA in solutions containing an organic solvent is quite different from that in aqueous solutions because pH and solvent polarity affect the excited states of PBSA. The optimization of experimental conditions for FL detection, including concentration, flow rate, and pH of the PBSA solution that was on-line mixed with the eluent from the HPLC column, is provided here. Figure 4.11 shows the influence of PBSA concentration on FL detection of nitrofurantoin, 4-nitroanailine and 4-nitrophenol after HPLC separation. When the PBSA concentration increased beyond 1 µM, FL detection of nitrofurantoin, 4-nitrophenol, and 122

160 140

120 100 80

60 40 FL Intensity (mV) Intensity FL 20

0.5 0.75 1 1.25 1.5 1.75 2

PBSA, uM

Figure 4.11. PBSA concentration effect on FL intensity of nitrofurantoin (), 4-nitroaniline () and 4-nitrophenol (▲) separated by HPLC. Flow rates: 150 µL/min of 40/60 ACN/H2O mobile phase from P#1; 150 µL/min of 2 µM PBSA stream from P#2. A 20 µL injection of sample containing 25 µM nitrofurantoin, 5 µM 4-nitrophenol and 4-nitroaniline. RSD for these data ranged from 4.9 to 7.8% (n = 4) for NF, 0.4 to 2.2% (n = 4) for NA, and 0.6 to 2.8% (n = 4) for 4-NP.

123 nitrophenol reached a plateau. A further increase in PBSA concentration above 2 µM generated a FL background too high to be subtracted. Therefore, 2 µM was considered the optimal PBSA concentration. Figure 4.12 shows the flow rate effect on FL detection. Flow rate change seemed to have little effect on FL intensity of nitrofurantoin. FL detection of 4-nitrophenol was more intense as the flow rate decreased to 100 µL/min or below, but FL intensity of 4-nitroaniline was increased in the flow rate range of 50 to 150 µL/min. The PBSA flow rate of 125 µL/min was chosen as an optimal compromise for indirect FL detection with HPLC. Figure 4.13 shows the influence of pH on the FL detection with HPLC. FL intensity of nitrofurantoin was affected very little by a pH change between 11.6 and 12.6, but FL intensity started to decline at pH > 12 for 4-nitrophenol and pH > 12.2 for 4-nitroaniline. The optimal pH for the PBSA solution is pH 12. C.4.2. Calibration curves and analytical figures of merits Under the optimal experimental conditions for FL detection, five aromatic compounds in a sample mixture were separated by HPLC as shown in Figure 4.14. All sample peaks were well resolved with the retention factor (k’) of 1.28, 2.89, 4.74, 5.66, and 7.55 for SS, NF, 4-NA, 4-NP and 2-NP, respectively. Because of instability in light [37], NF was most likely to be photodecomposed early before its shielding effect could apply to the photolysis of PBSA. The NF peak is the lowest among the five sample peaks and also has a split top. The SS peak in the chromatogram is much smaller than expected according to the FI results. The organic solvent used in the mobile phase might cause the change of photoreactivity of SS to be much larger than that for the other nitro compounds. All calibration data and detection limits (S/N = 3) of the five aromatic compounds studied by indirect PBSA FL detection after HPLC are listed in Table 2. The relative standard deviation (RSD) (n=4) values for the HPLC determination of 7.5 µM 4-NP, 4-NA, 2-NP, SS and 30 µM NF in a sample mixture are 2.2%, 4.0%, 4.2%, 5.0%, and 13.3%, respectively.

Section D: Conclusions

The fundamental study of off-line photolysis of PBSA has revealed that the decrease of FL emission of PBSA at 350 nm was directly linked to the opening of benzeneimidazole ring

124

200

150

100

50 FL Intensity (mV) Intensity FL

0 50 75 100 125 150 Flow rate at P#2, uL/min

Figure 4.12. Flow rate effect on the FL intensity of nitrofurantoin (), 4-nitroaniline () and 4- nitrophenol (▲) separated by HPLC. Flow rates: 150 µL/min at P#1. Sample concentrations same as previous experiments. RSD for these data ranged from 1.7 to 7.0% (n = 4) for NF, 0.8 to 3.9% (n = 4) for NA, and 1.3 to 2.1% (n = 4) for 4-NP.

125

160 140 120

100 80 60

40 (mV) Intensity FL 20 0

11.6 11.8 12 12.2 12.4 12.6 pH

Figure 4.13. pH effect on FL intensity of nitrofurantoin (), 4-nitroaniline () and 4- nitrophenol (▲) separated by HPLC. 2 µM PBSA used; 125 µL/min at P#2; 150 µL/min at P#1. RSD for these data ranged from 1.9 to 7.4% (n = 4) for NF, 2.0 to 3.7% (n = 4) for NA, and 1.0 to 3.6% (n = 4) for 4-NP.

126

i 0.4

e 0.3

e 0.2

e 0.1

i t

R 0

2 4 6 8 10 12 14 16 18 20 Time, min

Figure 4.14. Chromatogram of 5 aromatic compounds taken by HPLC with indirect FL detection via the shielding effect on photolysis of PBSA. A 20 µL injection of sample containing 7.5 µM salicylate (1), 30 µM nitrofurantoin (2), 7.5 µM 4-nitroaniline (3), 7.5 µM 4-nitrophenol (4), and 7.5 µM 2-nitrophenol (5). See Experimental for chromatography conditions.

127

Table 4.2. Analytical figures of merit of proposed FL detection method for HPLC determination of aromatic compounds. Molecules studied Regression Linear Detection Correlation equation range, µM limit, µM coefficient(r2) 2-Nitrophenol y = 1.1×106x + 8.1 0.5-20 0.25 0.9969 ×105 4-Nitroaniline y = 3.7×105x + 2.0 1-20 0.29 0.9942 ×106 4-Nitrophenol y = 1.2×106x – 2.4 0.5-20 0.36 0.9965 ×105 Nitrofurantoin y = 2.5×104x + 2.6 5-50 2.17 0.9817 ×105 Sodium salicylate y = 3.7×105x – 1.5 5-30 3.58 0.9988 ×106

128 during the UV photolysis of PBSA in alkaline solution. Aromatic compounds that either have a strong UV absorbance at 217 nm and/or can undergo competitive photochemical reactions with active oxygen species have a shielding effect on the photolysis of PBSA, leading to the recovery of FL intensity. An indirect FL detection method based on the shielding effect of analytes on the photolysis of PBSA has been developed for the determination of aromatic compounds by FI and HPLC. The largest advantage of this FL detection method is on-line determination of non- fluorescent aromatic compounds and drugs, e.g. nitrophenols, nitrofurantoin, atropine, and chlorpheniramine, without going through time-consuming off-line fluorescent labeling procedures or weakly efficient, possibly catalyst-required on-line photochemical derivatization. Because of the high detection sensitivity of phenolic compounds, such as, salicylate, which undergoes photochemical reactions with superoxide and singlet oxygen, this indirect FL method has a potential application for the evaluation of antioxidant activity of related compounds. A potential limitation of this detection method is it may not be suitable for gradient HPLC because gradient elution will slow down the photodecomposition of PBSA, resulting in the increase of FL background and a decrease of sensitivity of this detection method.

129

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133

Chapter V Volatile amines as ion-pairing agents for separation of aromatic sulfonates and phospholipids using reversed phase ion-pair liquid chromatography-mass spectrometry (LC-MS)

Section A: Introduction

Liquid chromatography coupled with mass spectrometry (LC-MS) has quickly developed as a powerful analytical tool for pharmaceutical [1-3], biological [4,5], and environmental [6,7] applications. Unlike conventional UV, fluorescence (FL), and refractive index LC detectors, MS detection provides molecular weight and structural information of compounds of interest and makes positive on-line identification of chemical species eluting from the LC column possible. Chemical derivatization is usually not needed for MS detection of compounds lacking a chromophore or fluorophore. To couple LC with MS, at least six distinct interfacing techniques that convert the liquid- phase from the LC column to the gas-phase for introduction into the MS vacuum system have been developed [8-13]. The electrospray ionization (ESI) interface has become the most popular interface for commercial LC-MS [14-18]. As compared to other interfaces, ESI is able to ionize with little fragmentation not only a wide range of polar species from low-mass synthetic compounds to the high-mass polymers and biochemical compounds but also those nonvolatile and /or thermally labile analytes. The ESI interface is made up of a pneumatic nebulizer with an electrospray needle that is housed inside a concentric tube with a nebulizing gas flowing through it. The ESI process is dependent on many instrumental and experimental factors, but solvent composition play a role in various steps of the electrospray nebulization and ionization process [19,20]. Nebulization and ionization efficiency is largely affected by physiochemical properties of the solvent mixture, such as volatility, viscosity, acidity or basicity, ionic strength, polarity, and liquid conductivity. A too high ionic strength mobile phase results in discharge and poor spray performance of ESI [20,21]. Although the ESI process is compatible with many mobile phases commonly used in LC, the use of high concentrations of nonvolatile additives and pH buffers with high salt content in the mobile phase will easily cause ion suppression and

134 contamination of the interface [22]. Therefore, this limitation of ESI will hinder MS detection for ion exchange chromatography that uses buffers with high ionic strength unless post-column treatment of the mobile phase is involved. Recently, ion exchange chromatography with ESI-MS by using either an ion suppressor membrane to reduce the salt content in the mobile phase [23,24] or aqueous ammonium acetate solution [25] as the mobile phase has been shown. For the separation of polar ionic organic compounds by LC-ESI-MS, reversed-phase ion- pair LC has been often coupled with ESI-MS. Reversed-phased ion-pair LC is more versatile than ion exchange chromatography because both ionic and neutral compounds can be separated. In addition, by optimization with an appropriate ion-pairing agent and concentration, a great improvement in terms of resolution and selectivity can be achieved. There are quite a few of ion- pair agents commonly used in reversed phase LC, e.g. tetraalkylammonium salts for anionic analytes and alkylsulfates for cationic analytes. However, the use of those nonvolatile ion-pairing agents will deteriorate the performance of ESI and cause severe contamination of the MS interface, capillary, and skimmers [26]. On the other hand, if those ion-pair agents are used at low concentration to reduce contamination of the ion source, the lack of sufficient retention for highly polar analytes on the stationary phase will lead to poor resolution and selectivity [27]. On- line removal of nonvolatile ion-pairing agents using an ion-exchanger trap column or an ion suppressor cartridge has been carried out successfully for the determination of anionic sulfonated compounds [28] and cationic biopterin and guanidine [29] by LC-ESI-MS. This type of method requires the regeneration of cartridge at regular intervals. Another promising approach is the use of volatile ion-pairing agents with boiling points below the dry temperature (e.g. 365 °C) set for ESI interface, thus causing evaporation of the ion-pairing agent with the organic solvent. Triethylamine, N,N-dimethyl-n-butylamine, tri-n-butylamine, dihexylamine, and di-n- butylamine have been used as volatile ion-pair agents for the separation of anionic aromatic sulfonates [26,30 ]and anticoagulant rodenticides [31] by reversed-phase ion-pair LC. Trifluoroacetic acid, heptafluorobutanoic acid, and perfluoroheptanoic acid have been used as volatile ion-pairing agents for separation of basic amine analytes by reversed-phase LC [32]. In this work, tert-octylamine (bp 137-143°C) was studied as a new type of volatile ion- pairing agent for separation of four highly polar aromatic sulfonate salts (benzenesulfonate, 2- naphthalenesulfonate, 1,5-naphthalenedisulfonate, and 1,3,6-naphthalenetrisulfonate) by reversed-phase ion-pair chromatography coupled with ESI ion trap MS for detection in the

135 negative mode. The effect of trap drive level on the ion chromatogram and distribution of ions generated from the ESI process was studied. The LC-MS results using tert-octylamine were compared with those obtained from three other volatile ion-pairing agents (ammonium hydroxide, dihexylamine, and n-tributylamine). In addition, the use of tert-octylamine as an ion- pairing agent for separation of four polar phospholipids (phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and 1,2-dilauroyl-sn-glycero-3-phosphate) by reversed- phase LC with MS detection in the negative mode was investigated. The results were compared with that obtained using NH4OH as an ion-pairing agent.

Section B: Experimental

B.1. Chemicals and solutions Tert-octylamine (TOA) (95%, bp 137-143°C), tri-n-butylamine (TBA) (98.5+%, bp 216°C), and dihexylamine (DHA) (97%, bp 192-195°C) were purchased from Aldrich (Milwaukee, WI). Ammonium hydroxide and acetic acid were provided from Fisher (Fair Lawn, NJ). Benzenesulfonic acid (BSA) (98%, sodium salt), 2-naphthalenesulfonic acid (2-NSA) (90%, sodium salt), and 1,5-naphthalenedisulfonic acid (1,5-NSA) (95%, disodium salt) were obtained from Aldrich (Milwaukee, WI) and 1,3,6- naphthalenetrisulfonic acid (1,3,6-NSA) (trisodium salt) from TCI (Tokyo, Japan). All four phospholipids were purchased from Avanti Polar Lipids: L-α-phosphatidylethanolamine (PE) (soy, sodium salt, #830024P), L-α-phosphatidylserine (PS) (soy, sodium salt, #870336P), L-α-phosphatidylinositol (PI) (bovine, liver, sodium salt, #830042P), and 1,2-dilauroyl-sn-glycero-3-phosphate (PA) (sodium salt, #840635P). The molecular structures of the volatile amines, aromatic sulfonate salts, and phospholipids are shown in Figure 5.1(A)-(C). HPLC grade methanol and hexanes were obtained from Pharmaco (Brookfield, CT) and Fisher (Fair Lawn, NJ), respectively. All other chemicals used are at least of analytical reagent grade as well. Distilled-deionized water used for preparation of solutions and mobile phases was taken from the E-Pure deionization system (Barnsted). A 100 mL volume of 1 mM stock solution of each aromatic sulfonate was prepared in aqueous solution. A 10 mL volume of 200 µM stock solution of each phospholipid was prepared in hexane/isopropanol (3/2, v/v) and stored at –20 °C in a refrigerator. The 25 µM aromatic sulfonate and 20 µM phospholipid sample mixtures used for LC-MS analysis were made from

136

Figure 5.1. Chemical structures of volatile amines (A): tert-octylamine (TOA), dihexylamine (DHA), and tri-n-butylamine (TBA), aromatic sulfonates (B): benzenesulfonate (BSA), 2- naphthalenesulfonate (2-NSA), 1,5-naphthalenedisulfonate (1,5-NSA), and 1,3,6- naphthalenetrisulfonate (1,3,6-NSA), and phospholipids (C): L-α-phosphatidylethanolamine (PE), L-α-phosphatidylserine (PS), L-α-phosphatidylinositol (PI), and 1,2-dilauroyl-sn-glycero- 3-phosphate (PA) used in this study.

137

A . Volatile amines

H2N HN N

TOA, MW 129.25 DHA, MW 185.36 TBA, MW 185.36

B. Aromatic sulfonates

SO3 SO3

SO3

SO O3S 3

SO3 2-NSA, MW 207.23 1,5-NSA, MW 286.28 1,3,6-NSA, MW 365.34

SO3

BSA, MW 157.17

138

C. Phospholipids

O R2 H O R P 1 O O O R O 3

R2, R3 = Fatty acyl chain

R = -H Phosphatidic acid (PA) 1

-CH -CH -NH Phosphatidylethanolamine (PE) 2 2 3

NH3 -CH -CH 3 Phosphatidylserine (PS) CO2 OH

OH Phosphatidylinositol (PI) HO OH OH

139 appropriate dilution of mixed aromatic sulfonate stock solutions with water and phospholipid stock solutions with hexanes/isopropanol (3/2, v/v). For the separation of aromatic sulfonates by reversed phase ion-pair LC, mobile phase A and B both containing a 2.5 mM volatile ion-pairing agent, were prepared by mixing an equimolar amount of 42.2 µL of ammonium hydroxide or 109.6 µL of TOA or 150.2 µL of DHA or 151.2 µL of TBA with 35.9 µL of acetic acid in 20 mL water (A) and 20 mL methanol (B), and then diluting with water and methanol, respectively, to the mark in 250-mL volumetric flasks. For separation of phospholipids by reversed phase ion-pair LC, mobile phase A containing a 5 mM ion-pairing agent in methanol/water (88/12, v/v) was prepared from mixing an equimolar amount of 84.4 µL of ammonium hydroxide or 219 µL of TOA with 71.8 µL of acetic acid in 20 mL methanol in a 250-mL volumetric flask, and diluting with methanol to the mark after addition of 30 mL water. Mobile phase B containing a 5 mM ion-pairing agent in methanol/hexanes (88/12, v/v) was prepared in a similar way. B. 2. Apparatus The separation of aromatic sulfonates and phospholipids by reversed phase ion-pair LC was carried out on a Polaris C18 -A 3 µm LC column (50 × 4.6 mm id) (ANASYS Technologies, Torrance, CA) using an HP 1100 series LC instrument with with an auto-sampler plus a 7750- 044 Rheodyne injector as wellas a UV-VIS detector. ChemStation software was installed for instrument control, data acquisition and data analysis. A Bruker Esquire ion-trap mass spectrometer with an ESI interface was used to produce ion chromatograms and MS spectra for the eluates from the column. Reference spectra for each phospholipid were acquired in both positive and negative ion modes using a syringe pump (Cole-Parmer Instrument Co., Vernon Hills, IL) with a 250 µL syringe (Hamilton Co., Reno, NE) to introduce the sample solutions into the ESI-MS. UV absorbance spectra of aromatic sulfonates and phospholipids were recorded using an HP/Agilent 8453 UV-VIS spectrophotometer. B.3. Procedures B.3.1. LC-MS of aromatic sulfonates Optimization of the solvent ratio in the mobile phase for separation of aromatic sulfonates by reversed phase ion-pair LC was carried out under isocratic conditions with a flow

140 rate at 0.5 mL/min. The UV detection wavelength was set at 220 nm after obtaining a UV spectrum of each 25 µM aqueous aromatic sulfonate sample. The ESI-MS instrument, set in negative ion mode, was running all the time under the following conditions: nebulizer at 50 psi,

N2 dry gas at 9 L/min, dry temperature at 365 °C, high voltage (HV) of capillary at 3800 V, HV of end plate offset at –500 V, skimmer 1 at –28.9 V, capillary exit at –99.8 V and a spectral average of 4. The effect of the trap drive level on the ion chromatograms and distribution of ions generated from the ESI process was investigated using a water (A)/methanol (B) (33/67, v/v) mobile phase containing the ion-pairing agent TOA (2.5 mM). The trap drive level was changed from 27.5 to 55 at an interval of 2.5. The remaining three ion-pairing agents (NH4OH, DHA and TBA) were studied when the LC-MS trap drive level was set at both 32.5 and 55. Total-ion current, extracted ion current, UV chromatograms, and MS spectra of each aromatic sulfonate sample peak were produced for the comparison study. B.3.2. ESI-MS analysis of phospholipid samples by syringe infusion A 20 µM phospholipid solution was introduced into the ESI-MS instrument at a flow rate of 600 µL/hr by a syringe pump. Mass spectra for each phospholipid were acquired in both positive and negative modes. The ESI-MS instrument was run under the following conditions: nebulizer set at 15 psi, dry gas at 4 L/min, dry temperature at 300°C, HV of capillary at 4500 V for positive mode and 3500 V for negative mode, HV of end plate offset at –500 V, trap drive at 51.4 to 61.3 for positive mode and 56.4 to 61.4 for negative mode, skimmer 1 at 35.7 to 59.7 for positive mode and -33.7 to -43.7 V for negative mode, capillary exit at 120.3 to 139.3 V for positive mode and –112.2 to –134.8 V for negative mode, and a spectral average of 20. B.3.3. LC-MS analysis of phospholipids Separation of the phospholipids was achieved by a mobile phase containing a 5 mM ion- pairing agent under gradient conditions. Mobile phase A consisting of methanol/hexane/H2O (22/0/3, v/v/v) with a 5 mM volatile ion-pairing agent is changed linearly in 17 min to mobile phase B containing methanol/hexanes/H2O (22/3/0, v/v/v) with a 5 mM ion-pairing agent. Mobile phase B was maintained for 3 min and then linearly changed back to mobile phase A over the next 3 min. Column reequilibration took 10 min before the next sample was ready to be injected. UV detection was set at 205 nm after the UV absorption spectrum of each phospholipid sample was obtained. The flow rate of mobile phase was set at 0.5 mL/min. ESI-MS was run in

141

the negative mode with the following experimental conditions: nebulizer set at 50 psi, N2 dry gas at 9 L/min, dry temperature at 365 °C, high voltage (HV) of capillary at 3500 V, HV of end plate offset at –500 V, skimmer 1 –43.7 V, capillary exit -123.3 V, trap drive at 61.4, and a spectral average of 4.

Section C: Results and Discussion

C.1. Analysis of aromatic sulfonates by reversed phase ion-pair LC-MS C.1.1. Optimization of solvent ratios in mobile phase Mobile phases used for separation of 4 aromatic sulfonates (BSA, 2-NSA, 1,5-NSA and

1,3,6-NSA) were made up of methanol, water, a volatile ion-pairing agent (NH4OH, TOA, DHA and TBA) and an equimolar amount of acetic acid that was used to convert the amine into its positive ammonium ion. The ion-pairing agent concentrations for LC-MS are usually in the range between 2 and 10 mM [28]. All four ion-pairing agents studied in this work were prepared with a concentration of 2.5 mM. Further investigation of the concentration effect on sensitivity was not done because a strong reduction of sensitivity of MS detection when the amine concentrations are above 2.5 mM has been reported [26]. Under isocratic conditions with a flow rate at 0.5 mL/min, four methanol/water mobile phases containing 2.5 mM TOA with solvent ratios of 40/60, 35/65, 33/67 and 30/70 (v/v) were tested for the LC separation of the four aromatic sulfonates. The UV and ion chromatograms obtained using methanol/H2O (33/67) are shown in Figure 5.2. BSA and 1,5-NSA both appeared in two adjacent peaks (peak 1 and 2). Both 1,3,6-NSA (peak 3) and 2-NSA (peak 4) were two well-separated peaks. A decrease of methanol content in the mobile phase favored the separation of BSA and 1,5-NSA as shown in the resolution diagrams inserted in the upper right corners of Figures 5.2(A) and (B). Although the use of methanol/H2O (30/70, v/v) mobile phase resulted in at least baseline resolution (1.5) of the BSA and 1,5-NSA peaks in both the UV and ion chromatograms, its unfavorably long analysis time of 25.2 minutes made methanol/H2O (33/67) practically the optimal TOA- containing mobile phase for separation of aromaic sulfonates. The optimal solvent ratios for the methanol/H2O mobile phases containing NH4OH, TBA and DHA were 20/80, 35/65 and 50/50, respectively.

142

Figure 5.2. Chromatograms of aromatic sulfonates by reversed phase ion-pair LC with MS (A) and UV detection (B). Methanol/water (33/67, v/v) mobile phase containing 2.5 mM ion-pairing agent TOA. A 20-µL injection of 25 µM aromatic sulfonate aqueous solution. Trap drive level set at 54.5 in MS. See Experimental for chromatography and MS conditions. 1 = BSA, 2 = 1,5- NSA, 3 = 1,3,6-NSA, and 4 = 2-NSA. the peak at 1.3 minutes is due to the injection pulse.

143

A 1.90 A. Ion chromatogram 1.70 2 1.50 1.30

peaks 1.10 0.90 0.70 Resolution of BSA/1,5-NS of Resolution 0.50 40/60 35/65 33/67 30/70 Methanol/water 1 4 3

B. UV chromatogram A 2.10 1.90 1.70 2 1.50 1.30

peaks 1.10 0.90 0.70

BSA/1,5-NS of Resolution 0.50 40/60 35/65 33/67 30/70 Methanol/water 4

1 3

144

C.1.2. Trap drive level effect The mass analyzer in ion trap MS consists of a ring electrode between two endcap electrodes. A high voltage RF potential is applied to the ring, while the endcaps are held at ground. The oscillating potential difference established between the ring and endcap electrodes creates a strong quadrupolar field. Depending on the level of RF voltage, the field can trap ions of a particular mass range. Because trap drive values are associated with the RF voltage, it is important to investigate the effect of trap drive level on the type and quantity of ions that appear in the mass spectra. Figure 5.3(A) shows the effect of trap drive level on the peak height (ion intensity after subtraction of background) of four aromatic sulfonates in an ion chromatogram in the presence of TOA as the ion-pairing agent. The peak heights of both 1,5-NSA and 1,3,6-NSA increased with the increasing trap drive level, while the peak heights of BSA seemed to be oscillating around the middle values. An increase of trap drive had only a slight decreasing effect on the height of the 2-NSA peak. The trap drive affects the distribution of the characteristic ions associated with each aromatic sulfonate sample peak in the chromatograms. Plots of ion intensity versus trap level for various ions are displayed in Figure 5.3(B)-(E). For the BSA sample peak, such plots for characteristic ions with m/z 157.2 (M−), 315 ([2M+H]−), 337 ([2M+Na]−), and 516.9 ([3M+2Na]−) are shown in Figure 5.3(B). The most intense ion with m/z 157.2 is the molecular ion of BSA. Its intensity (peak height measured from the extracted ion chromtogram) strongly declined as the trap drive level rose from 27.5 to 37.5 and started to recover a little after 37.5 with oscillation between 40000 and 66000, making it still the most abundant ion at the high trap drive level. The second most intense ion (m/z 337) is a sodium-adducted BSA dimer ion whose intensity reached the highest value as the trap drive level approached values around 40-42.5 before declining. Other two less intense ions with m/z 315 and 516.9 have the highest intensity values with the trap drive level set at 35 and 47.5, respectively. For the 1,5-NSA sample peak with its characteristic ions as shown in Figure 5.3(C), the highest intensity values for ions with m/z 287.2 ([M+H]−), 309.2 ([M+Na]−) 331.4 (([M+2Na-H]−) 596.8 (([2M+Na+2H]−), 618.7 ([2M+2Na+H]−), and 640.8 ([2M+3Na]−) were reached with the trap drive level set at 35, 40, 50, 47.5, 50, and 52.5, respectively. When the trap drive level was set above 45, the m/z 331 ions became the two most abundant ions trapped inside the mass analyzer. For the 1,3,6-NSA sample peak, its characteristic ions with m/z 367.3 (([M+2H]−), 388.8 (([M+Na+H]−), 411.2 145

Figure 5.3. The effect of trap drive level in ion trap MS on the peak height (A) and the pattern of ions comprised in BSA (B), 1,5-NSA (C), 1,3,6-NSA (D), and 2-NSA (E) sample peaks in the ion chromatograms. Methanol/water mobile phase (33/67, v/v) containing 2.5 mM TOA. Other experimental conditions set same as in Figure 5.2. Points are connected for clarity.

146

350000 BSA A 1,5-NSA 300000 1,3,6-NSA 2-NSA 250000

200000

150000

100000

50000

intensity) (total-ion height Peak 0

27.5 32.5 37.5 42.5 47.5 52.5

Trap drive level

147

160000

B: BSA m/z 157.2 140000 m/z 315 120000 m/z 337 100000 m/z 516.9 80000

60000 Ion intensity 40000

20000

0 27.5 32.5 37.5 42.5 47.5 52.5

Trap drive level

90000 C: 1,5-NSA 80000 m/z 287.2 m/z 309.2 70000 m/z 331.4 m/z 596.8 60000 m/z 618.7 m/z 640.8 50000

40000

Ion intensity 30000

20000 10000 0 27.5 32.5 37.5 42.5 47.5 52.5 Trap drive level

148

16000 D: 1,3,6-NSA m/z 367.3 14000 m/z 388.8 m/z 411.2 12000 m/z 423.9 m/z 449.4 10000 8000 6000 Ion intensity 4000 2000

25 30 35 40 45 50 55

Trap drive level

160000 E: 2-NSA 140000 m/z 207.3 m/z 436.9 120000

100000

80000 60000 Ion intensity

40000

20000

0 27.5 32.5 37.5 42.5 47.5 52.5

Trap drive level

149

− − − (([M+2Na] ), 423.9 ([M-SO3+3K+Na-2H] ), and 449.4 (([M+2Na+K-H] ) as shown in Figure 5.3(D) have the highest intensity values with the trap drive level set at 35, 40, 42.5, 50, and 47.5, respectively. With the trap drive level rising from 35 to 50, the increase of ion intensity for m/z 423.9 apparently coordinated with the decrease of ion intensity of the 1,3,6-NSA molecular ion with m/z 367.3. For the 2-NSA sample peak as shown in Figure 5.3(E), its characteristic ions included a strong molecular ion with m/z 207.3 ([M−]) and its weak sodium-adducted dimer ion with m/z 437.9 ([2M+Na]−). The ion intensity of m/z 207.3 decreased with the increasing trap drive level, while the m/z 437.9 ion had a slight maximum intensity at a trap drive level around 42.5 to 45. The example of the change of ion patterns by trap drive level was best represented in the MS spectra obtained with the trap drive level set at both 32.5 and 55 as shown in Figures 5.4(A) and (B), respectively. Because of the strong effect of trap drive level on the ion intensity and ion pattern for the sample MS spectra, the most intense molecular ions of all four sulfonated aromatic compounds were only observed at low trap drive level, e.g. 27.5-35. Both molecular BSA and 2-NSA ions were still the most intense ions appearing in the MS spectra when the trap drive level was set above 42.5. However, the sodium-adducted dimer and monomer ions of 2,5- NSA would be best observed with highest intensity when the trap drive level was set above 45. There was a difference for 1,3,6-NSA from other sulfonate peaks when the trap drive level was − set above 40. The most intense ion with m/z 424 ([M-SO3+3K+Na-2H] ) present in the MS spectra was possibly associated with a decomposition product of 1,3,6-NSA. The cause of detachment of one of its three sulfonate (-SO3) groups from the naphthalene ring structure during the ESI process was not clear, which might be attributed to the possible difference of bonding energy among sulfonate groups substituted on different positions of the naphthalene ring. C.1.3. Separation results using different ion-pairing agents Under the above-mentioned optimal mobile phase composition, separation of four aromatic sulfonates (25 µM BSA, 2-NSA, 1,5-NSA, and 1,3,6-NSA) was carried out by reversed phase LC using four different types of volatile ion-pairing agents, 2.5 mM NH4OH, TOA, DHA, and TBA. UV, total ion current and extracted ion current chromatograms are shown in Figures 5.5(A)-(D) and 5(E)-(H) when the MS trap drive level was set at 32.5 and 55, respectively. The eluting order of four sulfonate compounds from the column were BSA, 1,5-NSA, 1,3,6-NSA and 2-NSA for mobile phases containing TOA and TBA, while it was BSA, 1,5-NSA, 2-NSA and 150

Figure 5.4. Representative MS spectra of aromatic sulfonate sample peaks in the ion chromatogram obtained with trap drive level set at 32.5 (A) and 55 (B) using MS. conditions as in Figure 5.3.

151

A BSA

1,5-NSA

1,3,6-NSA

2-NSA

B BSA

1,5-NSA

1,3,6-NSA

2-NSA

152

1,3,6-NSA for DHA. Because of the low MS ion intensity of the 1,3,6-NSA sample peak when the trap drive level was set at 32, it was seen clearly only in its extracted ion current chromatograms. Due to the complete overlap of the 1,3,6-NSA and 1,5-NSA peaks when

NH4OH was used as an ion pairing agent, only the 1,5-NSA component with m/z 324.7 ([M+K]−) was observed from its extracted ion current chromatograms with weak intensity and severe peak tailing (Figures 5.5(A) and 5(E)). Use of TOA (Figures 5.5(B) and 5(F)) showed a quite good resolution of all four compounds with clearly identifiable MS spectra. Unlike using TOA and TBA, the most intense characteristic ion associated with the 1,5-NSA ion peak with m/z 331.4 (([M+2Na-H]−) (Figure 5.5(G)) was observed using DHA when the trap drive level was set at 55. The lower shoulder peak (tR = 14.4 min), overlapping with the left major 2-NSA peak (tR = 13.6), as seen in the UV and ion chromatograms for TBA as shown in Figures 5.5(D) and 5(H), was actually an impurity composed mainly of 2-thionaphthalene ion with m/z 238.9. The comparison of separation results using four types of ion-pairing agents is summerized in Table 5.1. It was obvious that the use of NH4OH as an ion-pairing agent could not provide sufficient selectivity for separation of 1,5-NSA and 1,3,6-NSA with the multiple sulfonate functional groups on the naphthalene ring. Among three remaining amines, the mobile phase containing TBA exhibited the best selectivity for separation of BSA and 1,5-NSA. Amines in order of increasing resolution values calculated from the total-ion current chromatograms were TBA ≥ DHA> TOA while the order was TOA > TBA > DHA from the UV chromatograms. In general, the use of TOA for LC separation of four aromatic sulfonates achieved a comparable resolution as compared to the DHA and TBA. One of the advantages of using TOA over DHA and TBA was that the peak tailing occurring for the 1,3,6-NSA sample peak was not as severe as that in DHA and TBA. When the MS instrument set in a positive ion mode was checked using pure methanol for the possible memory effect or carry-over effect from amines, the positive ions of all three amines could be detected easily. The intensity of the TOA ion with m/z 130 in the range of 1-3 ×105 was nearly 10 times lower than TBA and DHA ions with m/z 186. The TBA and DHA memory effect remained in the MS instrument for a much longer period of time (6-7 days) than TOA (2-3 days). Therefore, the use of TOA should be a good alternative ion-pairing agent for the determination of highly polar ionic organic compounds by reversed-phase ion-pair LC-MS although its selectivity may not be as good as TBA.

153

Figure 5.5. The chromatograms (total ion, extracted ion, and UV) obtained from separation of aromatic sulfonates by reversed phase LC using four different volatile ion-pairing agents

(NH4OH (A, E), TOA (B, F), DHA (C, G) and TBA (D, H). Trap drive level in MS was set at both 32.5 to acquire A, B, C, D chromatograms and 55 to get E, F, G, and H chromatograms. A 20 µL volume of aqueous sample containing 25 µM BSA, 1,5-NSA, 1,3,6-NSA and 2-NSA was injected. Four mobile phases containing NH4OH, TOA, DHA, and TBA were prepared from methanol/water with ratios of 20/80, 33/67, 35/65, and 50/50 respectively. See Experimental for chromatography and MS detection conditions.

154

A: NH4OH, trap drive 32.5

B: TOA, trap drive 32.5

155

C: DHA, trap drive 32.5

D: TBA, trap drive 32.5

156

E: NH4OH, trap drive 55

F: TOA, trap drive 55

157

G: DHA, trap drive 55

H: TBA, trap drive 55

158

Table 5.1. Comparison of the separation results obtained from reversed phase ion-pair LC using four types of volatile ion-pairing agents. a Ion-pairing Retention time Retention Resolution Selectivity Peak Shape b c c agent (min) factor (k’) (RS) (α) (1,3,6-NSA)

NH4OH 2.3 (BSA) 0.44 3.6 (1,5-NSA) 1.25 NA NA Extremely NA (1,3,6-NSA) NA Severe 12.4 (2-NSA) 6.75 overlap and peak tailing TOA 3.9 (BSA) 2.00 1.26 (Ion) 1.23 Peak tailing 4.5 (1,5-NSA) 2.46 1.43 (UV) with a peak 9.3 (1,3,6-NSA) 6.15 asymmetry of 16.3 (2-NSA) 11.54 3.47

DHA 3.7 (BSA) 1.85 1.45 (Ion) 1.20 Severe peak 6.8 (1,5-NSA) 4.23 1.28 (UV) tailing 7.9 (2-NSA) 5.08 22.4 (1,3,6-NSA) 16.23

TBA 3.6 (BSA) 1.77 1.47 (Ion) 1.31 Severe peak 4.3 (1,5-NSA) 2.31 1.33 (UV) tailing 9.9 (1,3,6-NSA) 6.61 13.6 (2-NSA) 9.46 a. Experiments were carried out under the isocratic conditions with trap drive level set at 55 in MS. b. The retention time of each peak was measured from the total ion chromatogram except 1,5-NSA from its extract ion chromatogram When NH4OH was used as an ion-pairing agent. c. Both resolution and selectivity values were calculated from two adjacent peaks, BSA/1,5-NSA for separation in the presence of NH4OH, TOA and TBA, while 1,5- NSA/2-NSA in DHA case. Resolution was calculated from both total-ion and UV chromatograms.

159

C.2. Determination of phospholipids by reversed phased ion-pair LC-MS C.2.1. ESI-MS analysis of phospholipid samples by direct syringe infusion The MS spectra of all four phospholipids obtained by positive (top) and negative (bottom) ion modes are shown in Figure 5.6(A)-(D). In the positive mode, both protonated and sodium adducted molecular ions were present in the MS spectra as major peaks for PA (12:0), PS (16:0-18:2 and 16:0-18:1), PE (16:0-18:2 and 16:0-18:1), and PI (18:0-20:4 and 18:0-20:3). Furthermore, the substantial fragmentation of phospholipids as a result of breakage of the glycerol-phosphate ester bond was observed in PA, PS, and PE, leading to the formation of diacylglyceryl fragment ions and their sodium-adducted ions with m/z 575.6 ([M-140.01]+ for PE and [M-182.99]+ for PS), 599.5 ([(M-140.01)+Na-H]+ for PE and [(M-182.99)+Na-H]+ for PS), 439.5 ([M-95.96]+) and 461.4 ([(M-95.96)+Na-H]+) for PA), while PI showed only a little diacylglyceryl fragment ion peak with m/z 627.5 ([M-259.01]+) in its MS spectrum. The capillary exit voltages were believed to induce the fragmentation of phospholipids, thus generating diacylglyceryl ions [33]. As compared to the MS spectra obtained in the positive mode, MS spectra in negative mode were quite clean and composed of mainly negative molecular and/or sodium-adducted molecular ions, e.g. m/z 714.5 ([M-H]−) and 738.5 ([M+Na- 2H]−) for PE, m/z 535.5 ([M]−) for PA, m/z 886.9 ([M]−) for PI, m/z 760.5 ([M]−) and 783.2 ([M+Na−H]−) for PS. Therefore, MS detection of phospholipids in the negative mode is more appropriate.

C.2.2. Separation of four phospholipids by LC using TOA and NH4OH as ion- pairing agents The structures of phospholipids as shown in Figure 5.1(C) consist of three parts: a glycerol backbone, a polar head group (a phosphate diester with a negative charge on the phosphate group), and two fatty acid chains esterified at the sn-1 and sn-2 positions. Due to the hydrophobicity of phospholipids arising from the two long alkyl or alkenyl groups in the fatty acyl chains, normal-phase HPLC has been used extensively to separate phospholipids [34-38]. Separation of most phospholipids by reversed phase LC requires the use of ion-pairing agent that forms a neutral complex with the negatively charged phosphate group. PE will be a zwitterion in the middle of the pH range. UV detection of phospholipids is not very sensitive because underivatized phospholipids absorb near 205 nm with a low absorption coefficient. Therefore,

160

Figure 5.6. MS spectra of four phospholipids obtained from ESI-MS in both positive (top) and negative (bottom) modes by direct infusion analysis. PA, PS, PE, and PI samples prepared 20 µM in hexanes/isopropanol (3/2, v/v) were introduced separately by the syringe pump to the ESI- MS instrument generating spectra (A), (B), (C), and (D), respectively. See Experimental for MS conditions in direct syringe infusion analysis.

161

PA (12:0), MW= 535.35

PS (16:0-18:2), MW = 758.5 PS (16:0-18:1), MW = 760.52

162

PE (16:0-18:2), MW = 715.49 PE (16:0-18:1), MW = 717.51

PI (18:0-20:4), MW = 885.55 PI (18:0-20:3), MW = 887.56

163 reversed phase LC coupled with MS has provided some advantages over UV detection for the separation and structural analysis of intact phospholipids [38]. Because phospholipids contain extremely hydrophobic alkyl and alkenyl carbon chains, the mobile phase commonly used in reversed phase LC, such as methanol/water, acetonitrile/H2O, and even isopropanol/H2O, could not provide a sufficient solvent strength to elute the phospholipids that strongly interact with the hydrophobic C18 stationary phase. Therefore, an extremely non-polar solvent hexane(s) has been used to prepare the mobile phase for separation of phospholipids by reversed phase LC under gradient elution [33,39]. Figure 5.7(A) shows chromatograms for separation of four phospholipids (20 µM PE, PA, PI and PS) by reversed-phase LC in the presence of 5 mM TOA as the ion-pairing agent.

Four weak peaks (tR = 11.1, 12.9, 13.3 and 13.9 min) associated with PS, PE and PI sample components were barely visible in the UV chromatogram (second trace) while the PA peak was absent because PA had the lowest UV molar absorptivity among the four phospholipids (e.g. 12952 for PI, 12137 for PE, 7788 for PS, and 428 for PA, which was measured from the UV absorbance at 205 nm of each 20 µM phospholipid standard). In contrast, the PA ion peak (tR = 7.5 min) was clearly present in the total ion current chromatogram (first trace) with eight other peaks associated with the PE, PI and PS samples (tR = 10.2, 11.1, 12.2, 12.9, 13.3, 13.9, 15.0, and 15.4 min). To identify the chemical composition of each ion peak in the total ion chromatogram, the average MS spectra of all ion peaks present in the total ion chromatogram and subsequently the extracted ion current (EIC) chromatograms of those typical ions associated with each phospholipid component are shown in Figure 5.7(B). Both molecular ions and sodium adducted molecule ions associated with PA, PE, PS and PI were present in the corresponding MS spectra with sufficient ion intensity. Because each type of phospholipid sample might consist of a few analogous components that differentiate one another by a varied number of double bonds in their fatty acyl chains, the existence of multiple ion peaks with a mass difference of nearly 2 units between two adjacent peaks could be observed from the EIC 716-717.9, EIC 737.2-742.9, EIC 780.7-784.6, and EIC 883.5-889.6. PA studied in this work is a synthetic phospholipid with two saturated fatty acyl chains (PA (12:0)). Therefore, ions associated with its analogs were not observed in all MS spectra. The chromatographic, chemical, and structural information related to each ion peak in the ECI chromatograms are listed in Table 5.2.

164

Figure 5.7. Chromatograms (A) and average MS spectra (B) of sample peaks generated from reversed phase LC separation of phospholipids in the presence of TOA as the ion-pairing agent in mobile phase. Mobile phase A (methanol/hexanes/H2O (88/0/12)) and B

(methanol/hexanes/H2O (88/12/0)) both containing 5 mM TOA. Mobile phase running under a linear gradient program: 0-100% B in 17 min, 3 min hold, 100-0% B in next 3 minutes. A 20 µL injection of the sample mixture containing 20 µM PA, PS, PE and PI. See Experimental for chromatography and MS detection conditions.

165

166

167

Table 5.2. The chromatographic, chemical and structural data obtained from LC separation of phospholipids using TOA as an ion-pairing agent. a Sample Retention Peak height Ion pattern m/z Fatty acyl chains in time of (Ion intesnity) lipid component

ion peak (min)

− PA 7.5 357003 [M] 536 PA (12:0)

PE 14 1015572 [M-H]− 716 PE (16:0-18:2)

15.1 368710 [M-H]− 717.9 PE (16:0-18:1)

11.9 783250 [M+Na-2H]− 737.2 PE (16:0-18:2)

12.9 1684643 [M+Na-2H]− 738.8 PE (16:0-18:1)

14.1 374772 [M+Na-2H]− 740.9 PE (16:0-18:0)

15.5 494196 742.9 b

12.2 511802 [M+2Na-3H]− 759.4 PE (16:0-18:2)

PS 10.2 338310 [M+Na-H]− 780.7 PS (16:0-18:2)

11.1 1081522 [M+Na-H]− 782.7 PS (16:0-18:1)

12.3 406927 [M+Na-H]− 784.6 PS (16:0-18:0) PI 12.5 268784 [M]− 883.5 c 13.3 1626596 [M]− 885.8 PI (18:0-20:4) 13.9 954276 [M]− 887.7 PI (18:0-20:3) 14.8 269043 [M]− 889.6 PI (18:0-20:2) a. Data listed in this table were directly obtained from extract ion chromatograms and average MS spectra of ion peaks present in total ion chromatogram. b. Ion with m/z 742.9 was possibly a type of sodium adduct of reduced PE (H2PE (16:0- 18:0) that was produced from reduction of two carbonyl groups by proton from unknown source. Its ion pattern could be expressed by [(M+2H)+Na-2H]−. c. Ion with m/z 883.5 could be associated with PI (18:0-20:5) or PI (18:0-20:4)–2H although it was not found from Avanti’s lipid catalog about the lipid quality information.

168

For comparison of separation results obtained with TOA, 5 mM NH4OH was used as the ion-pairing agent for the LC separation of the same phospholipid sample (20 µM PE, PA, PI and PS) under the same experimental conditions. The total ion current, UV and ECI chromatograms are shown in Figure 5.8. Table 5.3 lists the chromatographic, chemical, and structural information corresponding to each ion peak present in the ECI chromatograms. The clear difference was that six ion peaks associated with lipid components were generated in the NH4OH total ion chromatogram as compared to eight ion peaks for that using TOA. Furthermore, as indicated in Table 5.3, there were five PS sample components eluting in order of 780.6, 783.3, 784.9, 782.6 and 784.5 instead of three PS sample components that eluted in order of 780.7, 782.7 and 784.6 when TOA was used as an ion-pairing agent. The cause of a strange elution order of PS components observed from using NH4OH was not clear, but it possibly resulted from the change of retention mechanism of reversed phase ion-pair LC from an ion-pair distribution model using TOA to a dynamic ion-exchange model using NH4OH [40,41]. Due to the complexity involved for the separation of phospholipid samples that actually are composed of multiple lipid components for each type of phospholipid sample, for a better comparison, two reconstructed base peak chromatograms (BPC) generated using the most intense ion present in the average MS spectrum of each ion peak are shown in Figure 5.9. Existence of better resolved separated ion peaks associated with PS (m/z 780.7, 782.7, and 784.6) and PE (m/z 738.8 and

716) was found in the chromatogram obtained with TOA as compared to that with NH4OH. In addition, severe tailing of the PA peak was observed with NH4OH as an ion-pair agent.

Therefore, TOA with good volatility would be a good replacement for NH4OH as the ion-pairing agent for separation of phospholipidsby reversed phase LC with MS detection. TBA and DHA were tested as ion-pairing agents for separation of phopholipids. Because of column problem, the results were inconclusive. However, by observation of ion chromatograms obtained from the same bad column using TOA, DHA, and TBA, the eluting order for PE, PI and PS components was similar although the PA peak with a major ion at m/z 536 was missing. When phospholipids are separated by reversed phase ion-pair LC, care needs to be taken. Because of the strong interaction between hydrophobic long fatty acyl carbon chains and the chemically bonded silica stationary phase, e.g. octadecylsilyl silica (C18) and octylsilyl silica

(C8), some phospholipid residues might be retained very strongly on the column. Furthermore,

169

Figure 5.8. Chromatograms generated from the reversed phase LC separation of phospholipids in the presence of NH4OH as the ion-pairing agent in mobile phase. Mobile phase A

(methanol/hexanes/H2O (88/0/12)) and B (methanol/hexanes/H2O (88/12/0)) both containing 5 mM NH4OH. Gradient and experimental conditions as in Figure 5.7.

170

171

Table 5.3. The chromatographic, chemical and structural data obtained from LC a separation of phospholipids using NH4OH as an ion-pairing agent. Sample Retention Peak height Ion pattern m/z a Fatty acyl chains in time of ion (Ion intensity) lipid component peak (min)

PA 6.9 21346 [M]− 536.5 PA (12:0)

PE 14.3 505847 [M-H]− 716 PE (16:0-18:2) 15.3 88900 [M-H]− 717.7 PE (16:0-18:1) 12.2 146584 [M+Na-2H]− 738 PE (16:0-18:2) 13.2 482833 [M+Na-2H]− 740 PE (16:0-18:1) 14.4 165117 [M+Na-2H]− 740.9 PE (16:0-18:1) 15.7 101316 742.8 b 11.9 53166 [M+2Na-3H]− 760 PE (16:0-18:2) PS 9.8 62462 [M+Na-H]− 780.6 PS (16:0-18:2) 10.7 297439 [M+Na-H]− 783.2 PS (16:0-18:1) 11.8 142581 [M+Na-H]− 784.9 PS (16:0-18:0) 14.3 101272 [M+Na-H]− 782.6 PS (16:0-18:1) 15.4 37316 [M+Na-H]− 784.5 PS (16:0-18:0) PI 11.7 120755 [M]− 883.4 c 13 820170 [M]− 885.8 PI (18:0-20:4) 13.6 459548 [M]− 887.7 PI (18:0-20:3) 14.4 79305 [M]− 889.5 PI (18:0-20:2) See a, b, and c items in Table 5.2.

172

Figure 5.9. Reconstructed base peak chromatograms produced from LC separation of phospholipids in the presence of 5 mM TOA (A) and NH4OH (B). In (A): 1 = PA (m/z 536.5), 2 = PS (m/z 780.7), 3 = PS (m/z 782.7), 4 = PS (m/z 784.6), 5 = PE (m/z 738.8), 6 = PI (m/z 885.8), 7 = PE (m/z 716), 8 = PI (m/z 889.6), and 9 = PE (m/z 742.9). In (B): 1 = PA (m/z 536.5), 2 = PS (m/z 780.6), 3 = PS (m/z 783.2), 4 = PI (m/z 883.4), 5 = PI (m/z 885.8), 6 = PE (m/z 716), and 7 = PE (m/z 742.8). see Tables 5.2 and 5.3 for structural information.

173

5 6 A. TOA

3 7

9 1 2 4 8

5 B. NH4OH

7 2 4 1

174 ion-pairing agents, like polar amines, are easily attached to the remaining silanol groups of the silica surface. Those factors will lead to the deterioration of column separation performance. Such an effect caused the decrease of peak height (intensity), disappearance of the PA peak, and change of eluting order for phospholipids gradually appeared when same experiments were repeated again. So far, the problem was not resolved yet. Carefully washing of the column with an appropriate solvent after each sample run or the use of polymer-based stationary phase might overcome this problem.

Section D: Conclusions

This work demonstrates that a volatile amine, TOA, is suitable to be an ion-pairing agent for separation of highly polar ionic compounds such as aromatic sulfonates by reversed phase LC coupled with ESI-MS. In a comparison study using four volatile ion-pairing agents for the LC separation of BSA, 1,5-NSA, 1,3,6-NSA and 2-NSA in an aqueous sample solution, NH4OH does not provide a sufficient resolution as those found for the three organic volatile amines. TOA achieves a comparable resolution and does not causes a severe peak tailing problem for 1,3,5- NSA as compared to those results using TBA and DHA. Furthermore, TOA generates less memory effect on the MS instrument than TBA and DHA whose characteristic m/z 186 positive ions are 10 times stronger than the m/z 130 ion for TOA and remain in the MS instrument for a longer period of time. Besides the study of ion-pair effect on separation, the study on trap drive effect on ion pattern appearing in MS spectra of sample peaks reveal that molecular ions of those sulfonates can be best observed at a low trap drive level, e.g. 27.5-32.5 for BSA, 32.5-37.5 for both 1,5-NSA and 1,3,6-NSA, and 27.5-37.5 for 2-NSA. This work also makes a first attempt to separate phospholipids (PA, PS, PE, and PI) by reversed phase LC with MS detection using TOA as the ion-pair agents. TOA achieves better results than NH4OH in terms of higher ion intensity and more lipid components resolved in the base peak chromatogram. However, the deterioration of column performance is a problem that needs to be resolved when reversed phase ion-pair LC-MS is applied for the separation of phospholipids.

175

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179

Chapter VI Significance and Future work

Development of three luminescence detection methods and a methodology for separation of ionic compounds by reversed phase ion-pair LC coupled with ESI-MS using a new type volatile amine as an ion-pairing agent provided the solutions to the problems as mentioned in the Purpose of Research (section F in chapter I). Although straight flow cells with µL level volumes were known for CL, a micro spiral flow cell on the order of 5.5 µL had not been previously developed or characterized. The cell was characterized using luminol for the on-line determination of sub pmol-level H2O2. This detection method was successfully applied for the determination of L-lactate that generated H2O2 from an on-line lactate oxidase-catalyzed oxidation reaction. Because the sample carrier flow rate was in the range of 50-60 µL/min, this detection method is considered compatible with small microbore HPLC or wide bore capillary LC. Therefore, application of this detection method for those two types of LC should be a continuation of this project in the future. The quinine-sensitized photo-oxidation and quenched CL detection method was developed originally for on-line determination of nonfluorescent nitrophenols and chlorophneols. Because detection is based on the ability of analytes to react with photogenerated reactive oxygen species, application of this detection method for those phenols did not need time- consuming chemical derivatization procedures that are essential for GC and LC-FL methods. Application of this method could be used to evaluate the activity of antioxidants of nonphenolic compounds in the future. Furthermore, coupling of this CL detection with CE should be advantageous because UV detection is limited by the short capillary pathlength. The indirect FL detection method was a unique approach based on the shielding effect of analytes on the photolysis of PBSA to permit the determination of non-fluorescent compounds without chemical and photochemical derivatization. It was suitable for those non-fluorescent compounds that are not strongly photoreactive but have strong UV absorbance around 217 nm, which was required by FL detection via photochemical derivatization. In the future, more compounds or drugs that have such strong UV absorbance around 217 nm could be studied for their shielding effect on PBSA, e.g. spiramycin, a macrolide antibiotics with two carbon-carbon

180 double bonds in its molecular structure. The influence of chemical structure of analytes and matrix compounds on the shielding effect will also be investigated. Application of this method for evaluation of antioxidative activity of related compounds could be investigated. High molecular weight biomolecules, such as proteins or DNA should also be good candidates for PBSA shielding. The use of the volatile tert-octylamine (TOA) as a volatile ion-pairing agent for separation of ionic organic compounds by reversed phase ion-pair LC-MS was successful due to its sufficient interaction with the stationary phase and a low MS memory effect. In the future, more sulfonated compounds with multiply substituted sulfonate functional groups could be tested. The minimum concentration of TOA as an efficient ion-pairing agent in the mobile phase needs to be determined. The pH effect on the reversed phase ion-pair separation of ionic compounds should be studied. Furthermore, the column deterioration problem associated with the reversed phase LC separation of phospholipids using NH4OH, TOA and other volatile amines needs to be addressed. Separation of phospholipids using either dihexylamine or tri-butylamine as the ion-pairing agent has not been compared to that using TOA. The research on the use of TOA as an ion-pairing agent for separation of phospholipid derivatives from same type of lipid should be conducted.

181