David Chen

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Coupling the capillary electrophoresis (CE) to mass spectrometry (MS) provides an attractive analytical platform with minimal sample consumption, short analysis time, and high separation efficiency. Small-molecule biomarkers are metabolites associated with biological or pathogenic processes. The comprehensive study of small molecules found within the biological system is known as metabolomics. The work presented herein aimed to fulfill the advantage of CE-MS in analytical efficiency and fill the gaps of current methods by achieving the nonaqueous-CE-MS analysis of isomeric prostaglandins and enabling the peak alignment in CE-based metabolomics with inconsistent flow velocities.Urinary creatinine is commonly measured to normalize urinary metabolite concentrations or to assess renal function. In Chapter 2, multisegment injection-CE-MS was combined with a dilute-and-shoot strategy to analyze urinary creatinine. The diluted urines can be directly analyzed with a total analysis time of
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Glycoproteins are of interest as therapeutic agents. Characterization of the glycan component of glycoproteins is challenging due microheterogeneity, but necessary as glycans can cause negative immune system reactions. Erythropoietin is a glycoprotein hormone that stimulates the production of red blood cells and is used as a therapy for anemic patients. However, erythropoietin is also used as a doping agent in sports to boost performance by increasing the oxygen-carrying capacity of blood. Techniques for studying the glycans of erythropoietin and for differentiating between endogenous and exogenous erythropoietin are of interest in the scientific community. This thesis presents work on the development of techniques for the study of erythropoietin and its glycans for differentiation between endogenous and exogenous erythropoietin. Chapter 2 presents a capillary electrophoresis-mass spectrometry method that separates the terminal residue found on glycans, sialic acid. The presence of one type of sialic acid indicates an exogenous origin since humans cannot produce that sialic acid. By manipulating the post-column chemical conditions of the separation, the sensitivity of the method was increased. The most common sialic acid and the sialic acid that cannot be produced by humans were separated, and the feasibility of the method to detect low abundance levels of sialic acids from erythropoietin was shown. A key factor in lowering the LOD was the ability to inject reproducibly from a 1 µL sample volume. In chapter 3, different sialic acids from the producing conditions of erythropoietin (e.g. in the body or in cell culture) change the isoelectric point. A capillary isoelectric focusing method was developed with ultraviolet and laser induced fluorescence detection to analyze erythropoietin produced both endogenously and exogenously. The method was able to separate erythropoietins produced from different sources, and the isoelectric points of the various glycoforms of recombinant erythropoietin were determined. Chapter 4 focuses on simulations of an electro-fluid-dynamic microfluidic device for the feasibility of desalting glycoproteins with the device. Simulations concerning the ability of the device to selectively separate and purify analytes using a single power supply by manipulating the dimensions of the lengths and widths of the collection channels of the device.

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Qualitative and quantitative determination of trace compounds in complex samples is critically important in all fields of scientific research. The direct coupling of suitable sample preparation methods and direct analysis in real time mass spectrometry (DART-MS) has been shown to be a promising solution to trace analysis of compounds in complex matrices. In Chapter 1, a technical introduction of DART-MS and a review of some of the most common sample preparation techniques in trace analysis are presented. The coupling of sample preparation and DART-MS are also briefly reviewed in this chapter. Chapter 2 presents the direct coupling of dispersive magnetic solid-phase extraction (DMSPE) to DART-MS using a metal iron probe and its application in the trace analysis of six triazine herbicides in aqueous environmental samples. The ratio of magnetic core to graphene oxide in the sorbents and other key parameters in both DMSPE and DART-MS were investigated and optimized. This developed method combined the advantages of these two analytical techniques, and good analytical performance was achieved. In Chapter 3, a sorbent and solvent co-enhanced DART-MS method (SSE-DART-MS) was demonstrated for the determination of trace phthalic acid esters (PAEs) in water. Six common organic solvents were added on to two graphitic carbon nitride-based materials (g-C₃N₄) with different morphologies to study the enhancement effect.In Chapter 4, nine silica-supported ionic liquids (ILs) with different hydrophobicity were synthesized. They were then used separately as coatings for the solid-phase microextraction of five PAEs followed by DART-MS analysis to study how the hydrophobicity of sorbents influence the signals of analytes in DART-MS, and further investigation is needed to verify the general trend. In Chapter 5, the principle of deep eutectic solvent-based microwave-assisted extraction (DES-MAE) was systematically studied. The study also showed that in most cases, adding water will reduce the heating efficiency of DES under microwave irradiation, and DES containing carboxylic acid may react with cellulose, hemicellulose, and lignin to promote cell wall destruction. Chapter 6 summarizes the work presented and provides a brief outlook for future directions.

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Capillary electrophoresis (CE) is known for its low sample consumption, high-resolution efficiency as a separation technique, while mass spectrometry (MS) provides unprecedented selectivity and sensitivity as a detector, while adding another dimension in separation. The development of a robust CE-MS interface makes it possible to combine these two technologies for the analysis of many types of compounds. However, the coupling of CE to MS reduces its usable electrolyte ingredients substantially, and many of the popular buffer systems based on phosphate or borate, the MEKC methods dependent on the use of non-volatile surfactants, have to be excluded. As a result, formic acid and acetate buffers are the most widely used background electrolytes (BGEs) in CE-MS. In this work, we used mainly organic solvent systems to expand the choices of BGEs for CE-MS. In chapters 2 and 3, we presented two quantitative 100 % nonaqueous CE-MS methods for the separation and determination of small hydrophobic molecules. Two different BGEs were developed in this part: a basic buffer system for the analysis of negatively charged compounds and an acidic buffer system for the analysis of positively charged compounds. The compounds studied are three anthraquinones extracted from traditional Chinese medicine (TCM), Rhubarb, and six synthesized hydrophobic peptides, respectively. A high-organic-content CE-MS (HOCE) method was developed for the proteomics analysis of envelope proteins. A field amplified sample stacking technique was optimized to improve the concentration sensitivity of CE-MS for samples containing a large number of different analytes. The introduction of methanol into the buffer increased the performance of CE-MS for the detection of hydrophobic peptides in complex proteins digest. A half organic CE-MS method was used for the sequencing of novel mAbs. It was demonstrated that CE-MS/MS provided highly complementary information to LC-MS/MS with much less sample consumption. The last part of this thesis describes the application of a new generation mass spectrometry, timsTOF Pro connected with LC for the site-specific O-glycomics. In TIMS, charged compounds were separated based on their differential sizes and charges, similar to CE. The results suggest that combining CE-MS for PTMs analysis can be even more productive in the future.

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Capillary electrophoresis−mass spectrometry (CE−MS) is complementary to liquid chromatography−mass spectrometry (LC−MS) because of the orthogonal separation mechanism. An electrospray ionization (ESI) interface is the pivotal device in CE-MS and its emitter location relative to the MS inlet orifice has a significant influence on both ESI stability and detection sensitivity. A flow-through microvial interface provides a path for ion transmission between the CE and the MS systems. Based on the microvial interface with its small dilution volume and asymmetric emitter geometry, CE−MS concentration sensitivity can arrive at the nanomolar level with often-used mass spectrometers.To further improve the concentration sensitivity in CE–MS, dynamic pH junction focusing has been studied. By a focusing mechanism similar to isoelectric focusing (IEF), pH junction method can achieve a significant on-line preconcentration effect on weakly alkaline and zwitterionic analytes prior to the CE separation. Dynamic pH junction and its technical variant, dynamic pH barrage focusing have been investigated both theoretically and experimentally regarding peak symmetry, capturing efficiency, quantitative performance and compatibility to MS detection. Simulation-based optimization provided theoretical evidences that both configurations can capture targeted analyte with negligible or no loss during analyte focusing, giving adequate calibration curve linearity. Most theoretical predictions, alongside the capability of selective focusing in complex matrices, were verified by experimental evaluations with optical or MS detection.High resolution capillary isoelectric focusing mass spectrometry (cIEF−MS) enabled by the microvial interface is an attractive method for the charge heterogeneity characterization of biopolymers. Peptide and protein were used to demonstrate the feasibility of two online electrolyte configurations. pI resolution up to 0.02 pH in neutrally-coated capillaries demonstrated the high separation power. Isotopically resolved mass spectra collected by an Orbitrap MS instrument provided protein structural information. Analyses of intact monoclonal antibodies (mAb), including infliximab and adalimumab, an unidentified mAb and a reference mAb, showed the potential of cIEF–MS for charge variant separation and online identification in the biopharmaceutical industry. Automated cIEF–MS analysis shortened separation and identification process of mAb charge variants from days to approximately 2 h.

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The chemical characterization of biological and environmental samples are areas of research which involve the analysis of highly complex chemical mixtures. While the samples from these two fields differ greatly in composition, they present similar challenges. Complex mixtures provide a challenge to the analytical chemist as compounds in the mixture can have matrix effects which interfere with the analysis. Indeed, these interfering compounds may even be analytes themselves. High resolution mass spectrometry, which separates and detects ions based on their mass-to-charge ratio, is a powerful tool in the analysis of such mixtures. The amount of data resulting from such analyses, however, can be intractable to manual analysis, necessitating the use of computational tools. Furthermore, for the data to be reliable it is important that the performance of the mass spectrometer is optimal and consistent, but the complexity of the data again makes manual interpretation of the quality difficult. Thus, there is a need for computational assistance in analysis as well as method optimization and quality control.In Chapter 2:, we present a review of considerations toward the design of a standard mass spectrometry-based method for the quantification of naphthenic acids. The study provides recommendations for how these considerations can be addressed. In Chapter 3:, we describe a computational method of resolving dicarboxylic acids in high resolution mass spectrometry data of mixtures of derivatized naphthenic acid fraction compounds. The study is a proof-of-concept and demonstrates that derivatization-based methods of analyzing these diacid components is feasible but requires further investigation.In Chapter 4: and Chapter 5:, we present two computational tools which assist in method optimization and quality control of Thermo Orbitrap mass spectrometer systems. Chapter 4: presents RawQuant, a software tool which extracts scan quantification and meta data from data-dependent analysis data files from Orbitrap mass spectrometer systems. The tool is designed to inform the user toward method optimization. Chapter 5: presents RawTools, which builds upon RawQuant by adding the ability to track important measures of mass spectrometer performance longitudinally across a multi-run experiment. The tool is demonstrated using a 140-file dataset and provides easy visual monitoring of instrument performance.

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Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) combines the superior separation efficiency of CE with the detection capability of MS to provide information-rich data on samples with complex matrices. CE-ESI-MS is applied to a range of complex mixtures to semi-quantify unknown components and find patterns among groups of samples. The first part of this thesis tells how the industrial generation of oil sands process-affected waters (OSPW) poses an environmental threat, and a review is given on the state-of-the-art of analytical chemistry in the semi-quantification of toxic naphthenic acid fraction compounds (NAFC) in OSPW. The chapter ends with a technical description of the CE-ESI-MS system which was utilized.CE-ESI-MS was demonstrated to produce effective analyses of a well-known complex mixture: human urine. CE-ESI-MS was used to quantify and “fingerprint” components in human urine via targeted and untargeted analyses of the sub-5 kDa urine metabolome of patients with prostate and/or bladder cancer. For targeted analysis, endogenous levels of sarcosine and 5 other metabolites were quantified in four patients and in a pooled healthy urine sample. An untargeted analysis of patient urine was also performed identifying over 400 distinct molecular features per patient.Next, a CE-ESI-MS method was developed for the analysis of a relatively unknown complex mixture: NAFCs in OSPW. A standard mixture of amine-derivatized naphthenic acids was analyzed in under 15 min, detecting NAFCs between m/z 250 and 800. Derivatization of NAFCs consisted of two-step amidation reactions mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide in three different solvents. The optimum BGE composition was determined to be 30% (V/V) methanol in water and 2% (V/V) formic acid.The optimized CE-ESI-MS method was then used to find patterns in NAFCs across porewater samples impacted by oil sands waste. CE-ESI-MS, Orbitrap MS, and a standard GC-FID method were used to characterize porewater samples. Differences in measured amounts of total petroleum hydrocarbon by GC-FID and NAFC by HRMS indicate that the two methods provide complementary information about dissolved organic species in water leachate samples. CE-ESI-MS also provides complementary information and is a feasible and practical option for source evaluation of NAFCs in water.

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Taylor dispersion analysis (TDA) can be used for characterizing size of particles over a wide range. This method is performed on a capillary column and its simplicity and reproducibility offer many advantages over other more commonly used techniques for sizing. In the first part of the thesis, different sizing techniques are reviewed and their advantages and disadvantages are discussed.In the second part of this thesis, numerical modeling is used to optimize the operating conditions for performing TDA. In addition, the validity of TDA conditions is verified and the optimum conditions for performing TDA are discussed. Also, the effect of electric field on the validity of TDA and its application for a mixture of molecules with different sizes is studied. It is concluded that care must be taken to choose parameters in a way that lead to optimal TDA results. For the mixtures, using electric field is advantageous, because it not only leads to separation of molecules but also gives information on the charge of the molecules.In addition to numerical modeling, the equations for TDA to be used in the presence of electrophoretic mobility are modified and compared to the diffusion coefficients obtained using classical TDA. In this part of thesis, in addition to pressure, voltage is also applied to separate the components of mixtures and characterize the size of individual species. The results indicate that using the modified equations make the resulting values more consistent with the ones obtained from classical TDA. Our method can be used for mixtures, and in this thesis, a preliminary attempt has been made to characterize the size and charge of a mixture of proteins and peptides and amino acids.The final part of this thesis discusses the dispersion of proteins and small peptides during electrokinetic migration. Like conventional Taylor dispersion, peak broadening is moreprronounced for particles with smaller diffusion coefficients. The theoretical description of band broadening caused by electroosmotic flow dispersion (EOFD) and the experimental verification of this phenomenon are presented.

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Binding analysis has been a very important topic of many scientific research works for years. In Chapter 1, some most common binding analysis techniques were briefly reviewed. Six interesting variants of the CE-based methods are discussed including capillary electrophoresis frontal analysis (CE-FA). In Chapter 2, we demonstrated the development of a CE-FA method and its application in the affinity determination between a DNA binding domain and a drug candidate. The current data processing method of CE-FA mandates specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. Chapter 3 showed how significant errors occur when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real world applications becomes questionable. A mobility-based correction method was developed based on the flux of the ligand molecules. Conventionally, adding external pressure during the process of capillary electrophoresis separation has not been a common approach. While accelerating the separation, drawbacks including peak broadening and reduced resolving power are almost inevitable in pressure-assisted capillary electrophoresis (PACE). Frontal analysis (FA), with its unique characteristics, could potentially benefit from using external pressure while avoiding related drawbacks. In Chapter 4, the possible impact from pressure was studied mathematically and the physical process was simulated with COMSOL Multiphysics®. PACE-FA is applied to study the binding interactions between hydroxypropyl β-cyclodextrin (HP-β-CD) and small ligand molecules. In Chapter 5, we compared the binding constants obtained with electrospray ionization mass spectrometry (ESI-MS) and ACE. Two simulation programs were deployed to estimate the solvent impact on the binding interactions. In Chapter 6, a free solution method was developed for evaluating specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of non-specific binding without the need of reference compounds was proposed and tested. ESI-MS was first used to rapidly screen the small molecule candidates, then the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA).

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Capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) combines the superior separation capability of CE and detection and characterization ability of MS. Different CE separation modes can be coupled to ESI-MS, employing an interface with a flow-through microvial. In the first part of the thesis, recent development of CE and CE-MS applications in the analysis of complex samples are reviewed. Capillary isoelectric focusing (cIEF) is an important tool for the separation and characterization of amphoteric molecules based on isoelectric points. Minute structural changes on a large protein can result in changes in isoelectric point, and the changes can be detected by slab gel isoelectric focusing or capillary isoelectric focusing. A systematic study on the interactions among carrier ampholytes, sample media and capillary inner coatings was carried out to provide guidelines for choosing feasible combinations that can achieve isoelectric focusing and successful chemical mobilizations. Within the 0.1%-1% (w/v) carrier ampholytes concentration range, small forward EOFs will ensure a higher chance of good focusing and successful electrophoretic mobilization, while a negative EOF will hinder these processes. Feasible combinations of experimental conditions are summarized. Using the optimized conditions, we reported the direct observation of the shape of focused ampholyte bands in the cIEF process by online cIEF- ESI-MS. The ampholyte bands directly detected by MS have the potential to enable a more accurate pI determination for unknown amphoteric molecules. Immunoglobulin G from rabbit serum is used to demonstrate this possibility. In Chapter 6, a CE-MS method was developed to monitor the concentration variations of major nutrients and/or metabolites in human embryonic stem cell CA1S culture medium over a culturing cycle. Concentration changes for nutrients and/or metabolites in the culturing media provided information on the cell growth behavior without destructing living cells. In the last part of the thesis, an atmospheric ion lens was applied to the flow-through microvial CE-ESI-MS interface to improve the electrospray ionization and sampling efficiency. A mixture of amino acids was tested to show the increased signal-to-noise ratios. The atmospheric ion lens also gives more flexibility when choosing the EOF and chemical modifier flow rates.

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Glycosylation of proteins is ubiquitous and has the ability to significantly alter the biological and biophysical properties of proteins. The need to study structure-function relationships of glycans in a living organism requires the continuous development of rapid and sensitive technologies for the characterization of glycan components. In recent years, a broad range of technologies have evolved to provide new developments and emerging glycomics techniques. In this thesis we present the development of new methodologies based on capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the study of protein N-glycosylation in complex biological systems and therapeutic recombinant drugs. Our approach involves the use of a flow-through microvial, a novel technology that provides a robust and easy-to use strategy for interfacing CE separation with MS detection. In chapter 2, we report a simple and robust CE-ESI-MS methodology for comprehensive characterization of glycosylated proteins at the level of intact protein, enzymatically released glycopeptide and glycans. In chapter 3, we characterize a complex set of enzymatically released N-glycans from a recombinant therapeutic drug that revealed extensive glycan heterogeneity. The study demonstrated the potential of our approach to complement established techniques for glycan characterization of therapeutic glycoproteins in the pharmaceutical industry. Chapters 4 and 5 of this thesis are devoted study protein glycosylation of relevant biological systems. In chapter 4, O-acetylated N-glycans from fish serum were characterized in their native state and the structural variations of their isomeric species were investigated by tandem MS approaches. The developed CE-MS methods may be useful not only for the characterization of acetylation of complex glycans but also to study other types of glycan modifications in different contexts. In chapter 5, we present CE-MS methodologies for characterizing protein N-glycosylation in human serum associated with prostate cancer and asthma. Comparison of glycan compositions and relative abundances revealed abnormal glycosylation in prostate cancer and asthma serum. The capability of our CE-ESI-MS method to perform global glycan profiling of human serum demonstrates its potential for comprehensive glycan profiling in the context of malignancies and for the discovery of glycan disease markers with high selectivity and specificity.

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Temporary interception and then re-infusion of oxygenated blood into heart during on-pumpCoronary Artery Bypass Grafting (CABG) surgery causes ischemia-reperfusion injury (IRI).Propofol is an intravenous anesthetic agent that maybe potentially cardioprotective against IRI. Thisthesis presents the development, validation and application of analytical methods for monitoringpropofol and a series of prognostic biomarkers in hope of identifying contributory factors to IRI andpropofol cardioprotection.I developed a Capillary Electrophoresis (CE) method to quantify propofol concentrations inblood. Propofol concentrations in the μg/mL range were measured from 400 μL samples. A dosefindingstudy using this method determined a practical infusion rate of 120 μg•kg•min⁻¹ to achievethe target blood concentration of 5 μg•mL⁻¹.To measure the oxidative stress biomarker, 15-F₂t₋isoprostane, the nitrosative stressbiomarker, 3-nitrotyrosine, the myocardial protective factor, adenosine, and the cardiovascular riskfactor, asymmetric dimethylarginine (ADMA), I developed simple, sensitive and robust LiquidChromatography-Mass Spectrometry (LC-MS) or Liquid Chromatography Tandem MassSpectrometry (LC-MS/MS) methods. Briefly, a basic mobile phase and base-resistant column wereused for the LC-MS analysis of 15-F₂t-isoprostane. A one-step solid phase extraction andpentafluorophenyl (PFP) core-shell column were employed for the LC-MS/MS analysis of 3-nitrotyrosine. A Strong Cation Exchange (SCX) solid phase extraction and modifier-free mobilephase were implemented for the LC-MS/MS analysis of adenosine. A new derivatization method toenable baseline separation of ADMA and its regio-isomer symmetric dimethylarginine (SDMA) wasdeveloped for the ADMA LC-MS/MS quantitation. Performance parameters for these methods,including linearity, precision, accuracy, Limit of Detection (LOD), Limit of Quantitation (LOQ),Lower Limit of Quantitation (LLOQ) and stability were found satisfactory.Concentrations of 15-F₂t₋isoprostane, 3-nitrotyrosine and adenosine were found to rise afteron-pump CABG surgery. However, these changes were not able to explain the cardioprotectiveeffect of propofol. Nonetheless, the correlations of 15-F₂t₋isoprostane with diabetes, glucoseconcentration and PTEN level were significant. Patients with low cardiac output syndromeexperienced more 3-nitrotyrosine increase than patients without this syndrome. The basal adenosinelevel was found to increase more in patients with low left ventricular ejection fraction. Thesefindings and the underlying methodologies are important for identifying new prognostic biomarkers.

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The development of separation science is one of the most important contributions inanalytical chemistry, and current separation systems can analyze less than femtomoles ofanalyte. However, the need for such ultrasensitive technology is partly driven by thedifficulties in obtaining appreciable quantities of pure substances. Therefore, a platformenabling the purification of chemicals in a preparative fashion from complex mixtures isrequired.In this thesis, a new continuous chemical purification platform is introduced,based on the interactions of analyte with multiple types of driving forces in a twodimensionalelectro-fluid-dynamic (2-D EFD) system, in which both electric field andhydrodynamic pressure are simultaneously utilized in 2-D microfluidic channelnetworks.Mass conservation is the guiding principle for analyte distribution in channelintersections. However, in EFD devices, the mass distribution is more complex. In orderto understand the analytes’ transportation behaviour in multiple force fields, we studiedthe mass transfer in EFD devices, and discovered the effective volumetric flow rateconservation principle. It can be used to predict the analyte concentration in a channel,and provides a theoretical basis for investigating the mass distribution in EFD devices.Y-shaped microfluidic devices have been extensively used for mixingcomponents. With the strategically applied electric potential and hydrodynamic pressure,such spontaneous mixing process can be reversed in the same device. A continuoussolution stream containing a mixture of two analytes can be separated into two channels,each containing a pure compound. By increasing the geometry complexity, more complex samples can be processed. Amultiple-branched device is introduced forcontinuously purifying multiple analytes. Each component in the introduced mixture canbe directed to enter its specific collection channel, without any contamination.In the study of sample injection methods, the hydrodynamic injection is superiorto the electrokinetic injection in the purification process by providing faster sampleprocessing and being more resistant to the fluctuating electroosmotic flow. In addition,the buffer depletion problem can be fully resolved. The stringent control and ease ofoperation of this technique could lead to a new generation of purification devices to servethe needs of academic research and commercial activities.

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A novel capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) interface has been developed to provide a robust, user-friendly and more sensitive alternative interface strategy. The new interface uses a flow-through microvial design and a bevelled sprayer tip geometry. The capillary column terminus is surrounded by a tapered stainless steel hollow needle, and the interior of the needle tip acts as the CE outlet while its exterior tip surface provides the electrode surface for electrospray ionization. A chemical modifier is supplied to the open-ended microvial at the CE outlet through a standard tee union, serving the purpose of maintaining electrical continuity, and supporting a stable electrospray. The chemical modifier supplied through the flow-through microvial can also be used to improve the compatibility of CE effluent with electrospray ionization. The bevelled sprayer tip design extends the optimal flow rate range for ESI and requires lower flow rate compared to conventional blunt tips or symmetrically tapered sprayer tips. This feature leads to reduced dilution effect caused by the chemical modifier solution and improves the detection sensitivity. The mass transport process in the flow-through microvial was investigated by numerical simulation and experimental comparison of on-column and post-column detection. Both approaches demonstrated that the laminar flow profile inside the microvial does not significantly distort the peak shape and the major characteristics of the eluted peaks are maintained when the modifier flow rate is properly adjusted. The chemical modifier solution in the flow-through microvial enables CE separation without electroosmotic flow (EOF). One useful application of this feature is interfacing online capillary isoelectric focusing (cIEF) with ESI-MS detection, which could be a potential replacement in many applications of two-dimensional gel electrophoresis (2DE) for protein analysis in the future. The final part of the thesis elucidates the electric field distribution in the ESI source with an atmospheric ion lens, which could be incorporated in the CE-ESI-MS interface to improve the ionization and sampling efficiency in the future.

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Coronary artery disease is the leading cause of death in North America. The invasiveness of its treatment depends on its severity; less severe disease can be treated pharmacologically or surgically without significantly different outcomes, but coronary artery bypass grafting (CABG) clearly reduces mortality among medium- and high-risk patients compared to percutaneous and non-surgical intervention. Although the majority of patients undergoing surgical revascularization emerge without severe postoperative complications, a significant portion of patients encounter a postoperative complication known as low cardiac output syndrome which can quadruple the overall mortality rate for CABG. Intraoperative ischemia reperfusion injury is a major factor in the development of low cardiac output syndrome; so effective intraoperative myocardial protection is central to reducing its incidence, and represents an opportunity to considerably improve patient outcomes.The introductory chapter of this thesis describes the origin and role of reactive oxygen species (ROS) in myocardial ischemia-reperfusion injury. In addition, it introduces key strategies targeted to reduce ROS-mediated myocardial ischemia-reperfusion injury, highlighting key clinical studies that translated these strategies to reduce the severity of ischemia-reperfusion injury during CABG.The central hypothesis of the clinical project on which this thesis is based states that propofol reduces the incidence of low cardiac output syndrome subsequent to CABG with CPB by decreasing the magnitude of 15-F₂t-isoprostane generation during ischemia-reperfusion. The second chapter introduces propofol, and will review previous studies that explore its cardioprotective potential. The experimental section of this thesis describes the development of a quantitative technique for propofol analysis in whole blood, and its application in a dose finding study that define the parameters for achieving experimentally relevant concentrations of propofol during cardiopulmonary bypass. These two studies were fundamental to the development of a clinical study evaluating ROS generation and the incidence low cardiac output syndrome in patients undergoing CABG surgery. Preliminary results that address the central hypothesis are subsequently presented, along with an alternative proposed mechanism for propofol-mediated cardioprotection. This thesis will conclude with a summary of findings and a description of several future studies aimed at testing, generating, and evaluating new hypotheses.

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A novel interface for capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) has been developed with the goal of providing a robust and easy-to-use hyphenation strategy that also provides high sensitivity. The interface uses atapered stainless steel needle that surrounds the capillary terminus, so that the needleinterior acts as the CE outlet vial and the outside tip is the electrospray emitter. Achemical modifier solution, introduced via a tee junction, serves to improve thecompatibility of the background electrolyte with ESI and allows the interface to operateunder conditions where the bulk flow in the capillary is suppressed or reversed.A major novel feature of the interface is the use of a beveled conductive needle as the ESI emitter. Because electric field is highest at the sharpest edge of an electrode, liquid exiting the needle is drawn to the sharpest point of the bevel, whereas for aconventional symmetrically tapered emitter the field is equally distributed around thecircumference of the tip. Electrospray performance as a function of flow rate wasinvestigated for a variety of emitter geometries. The beveled emitters provided a morestable and efficient electrospray over a wider range of flow rates than traditional emitters.The second part of the thesis describes the application of the CE-ESI-MS interface with the beveled emitter to a variety of biomolecular species, includingpeptides, proteins and carbohydrates. These applications include the use of a variety ofcapillary coatings and separation conditions where the electroosmotic flow is suppressedor even reversed, so that the bulk flow in the CE capillary is away from the interface. Theinterface was also used to interface capillary isoelectric focusing (cIEF) with MS detection, enabling free solution and fully automated protein analysis that is analogous to 2D gel electrophoresis. The final part of the thesis examines the viability of a two-marker correction strategy for improving retention time reproducibility in reversed-phase liquidchromatography. Although the strategy provided up to a ten-fold improvement in reproducibility for repeated separations, the correction was not successful in relating retention times obtained under different gradient conditions.

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Capillary electrophoresis (CE) with complexation additives in the backgroundelectrolyte utilizes both equilibrium and electric field in the separation process. Therefore, acomprehensive understanding of the analyte migration behavior in CE processes becomesessential to effectively control the chemical separation process and to achieve the fullpotential of this powerful analytical technique.Computer simulation is one of the best ways to visualize the instantaneous behaviors ofphysicochemical systems. After the success of our first simulation program SimDCCE, aJAVA based computer simulation model of dynamic complexation capillary electrophoresis(CoSiDCCE) was developed based on the differential mass balance equation, the governingprinciple of analyte migration in all separation techniques. CoSiDCCE is highly efficient, andis capable of demonstrating various types of the affinity interactions between multiple speciesin CE in real time or faster.With the simulation program, a thorough study of the mechanism of vacancy affinity CEwas carried out. Thermodynamic binding constant was estimated with nonlinear regressionmethods. Thirteen scenarios in four different combinations of migration orders of free protein,free drug, and their complexes formed were studied.The specific protein-ligand interactions were determined using CE-frontal analysis, oneof the most effective CE modes. A new algorithm was derived to calculate the bindingparameters for higher order specific interaction in the presence of non-specific interactions.Computer simulation was used to study the migration behaviors of all species in the DCCEprocess.CoSiDCCE was used to elucidate the determining factors that result in CE separationsof amino acid enantiomers and predict the efficiency of the chiral selectors when used in otherseparation systems such as chromatography. With this program, the migration behavior ofdifferent species involved in the competitive dynamic complexation in chiral CE processeswas investigated, and the change in migration orders in some chiral separations was explained.The binding constants and complex mobilities were also determined.In addition, a micellar electrokinetic chromatography method was developed todetermine potentially anti-carcinogenic flavonoids in various wine samples. A systematicoptimization of the separation buffer and concentration of surfactant was carried out toimprove the reproducibility and sensitivity for the analysis of flavonoids.

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