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Membrane technology has been actively used as a separation tool in the chemical, environmental, and biopharmaceutical industries for several decades. As membrane quality requirement in the industry has increased, efforts have been directed towards enhancement in mechanical strength, chemical durability and functionality of membranes. One of the approaches for membrane quality enhancement is based on the combination of hydrogel technology with membrane technology. This thesis focused on the application and development of hydrogel based membranes, notably hydrophilized PVDF (polyvinylidene fluoride) membrane for hydrophobic interaction membrane chromatography; the fabrication of paper-hydrogel composite membranes for membrane chromatography; development of a technique for coating alginate (a natural hydrogel) on the outer surface of a hollow fiber membrane for potential application in bioreactors and the use of hollow fiber membranes as mold for fabrication calcium alginate fibers for biomedical and tissue engineering applications. A membrane chromatography-based polishing technique was developed for removing leached protein-A and aggregates from monoclonal antibody (mAb). A commercial synthetic membrane that is known to be hydrophilized by hydrogel grafting was employed to develop this polishing process that resulted in highly pure mAb, free from aggregates and protein-A. This mAb polishing technique could easily be integrated with a hydrophobic interaction membrane chromatography based mAb purification process. A paper-hydrogel composite membrane was developed as an inexpensive alternative to commercial synthetic membranes used for carrying hydrophobic interaction membrane chromatography. Poly(N-vinylcaprolactam) or PVCL hydrogel was coated on Whatman filter paper to prepare these membranes. These environment responsive membrane which responded to changes in salt concentration, gave excellent fractionation of multi-component protein mixtures. As case study, a mixture of immunoglobulin G, human serum albumin and insulin was fractionated. A technique for modifying the surface of synthetic hollow fiber membranes with alginate (a natural hydrogel) was developed. This manner of surface modification led to the improvement in membrane mass transport. The alginate was cross-linked on the outer surface of the membrane by diffusion of the cross-linker (calcium ions) through the membrane pores. The calcium alginate coating layer was characterized by optical and transmission electron microscopy, contact angle measurement, hydraulic permeability measurement and by examining solute transport. Hollow and solid calcium alginate fibers were fabricated using a novel hollow fiber membrane based moulding technique. The pore present on the hollow fiber membrane served as the reservoir for the calcium chloride solution with cross-linked the alginate within the lumen. The calcium alginate fibers produced were characterized by optical, transmission electron, and scanning electron microscopy. Cell immobilization experiments were carried out to demonstrate biocompatibility and potential for tissue engineering applications. / Thesis / Doctor of Philosophy (PhD)

During conditions of osmotic stress, some plants accumulate compatible osmolytes such as glycine betaine or choline-0-sulphate. Choline is required as a precursor for synthesis of both osmolytes and choline is also required by all plants as a component of phospholipids. In the betaine accumulator spinach, choline synthesis requires three sequential N-methylations of phosphoethanolamine (PEA) to generate phosphocholine (PCho), with the first N-methylation being catalyzed by S-adenosyi-L-methionine: PEA Nmethyltransferase (PEAMeT). Choline synthesis and, more particularly the activity of PEAMeT, are up-regulated by salinity (Summers and Weretilnyk, 1993). This thesis reports on the partial purification and preliminary characterization of PEAMeT from spinach. A variety of column chromatography matrices including DEAE Sepharose, phenyl Sepharose, w-aminohexyl agarose, hydroxylapatite, phenyl Superose, Mono Q and adenosine agarose, have been used to purify PEAMeT. A 5403- fold purified preparation yielded a specific activity of 189 nmol· min-1 • mg-1 protein. SDS-PAGE analysis of this preparation revealed a number of polypeptide bands but only one which photoaffinity cross-linked to [3H]SAM. The estimated native molecular weight (MW) of PEAMeT was found to be 77 kDa by gel filtration chromatography and an estimated MW of 54 kDa was determined by SDS-PAGE. SDS-PAGE analysis of samples photoaffinity crosslinked to [3H]SAM gave a slightly higher estimated MW of 57 kDa. Effects of various factors on PEAMeT assay conditions were evaluated using partially purified PEAMeT preparations. PEAMeT activity as a function of pH gave a unimodal curve with an apparent pH optimum at 7.8 with 1 00 mM HEPES-KOH buffer. In vitro PEAMeT activity was inhibited by phosphate, PCho, S-adenosyi-L-homocysteine, ca+ 2, Mn+2 and co+2 but not by choline, betaine, ethanolamine, mono- and dimethylethanolamine or Mg+2 • Phosphobase N-methyltransferase activities present in preparations enriched for PEAMeT activity can catalyse the reaction sequence PEA- PMEA - PDEA - PCho. Under optimized assay conditions using PEA as the sole substrate, PMEA, PDEA and PCho were quantified and were detected in the order: PMEA (77%) > PDEA (17%) > PCho (6%). Thus a single enzyme, PEAMeT, is capable of converting PEA to PCho in leaves of spinach. The existence of a second enzyme which converts PMEA to PCho has also been reported for leaves and roots of spinach (Weretilnyk and Summers, 1992). The presence of two enzymes with overlapping activities raises questions regarding the roles of these two enzymes in choline metabolism. For example, do these enzymes also have overlapping functions in choline synthesis, particularly under conditions of osmotic stress? / Thesis / Master of Science (MSc)

Human pathogenic yeasts of the genus Candida and 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 are responsible for about 10% of hospital-acquired infections. In addition, drug-resistant yeasts are rapidly emerging with the use of anti-fungal drugs. Common drugs such as Fluconazole and Amphotericin B target the ergosterol pathway in yeast. Learning about other metabolic differences in yeasts may also give a new understanding to their role as pathogens. Metabolomics is a field of study about the large spectrum of metabolites necessary for the growth and survival of an organism. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the metabolome of different yeasts in three different studies. The first study was a targeted analysis of the ergosterol pathway in 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 and a double drug-resistant mutant was found to have several changes in its sterol composition while Fluconazole-resistant strains were similar to the wild-type. In the second study, a comprehensive analysis of the polar and lipid metabolite profiles of six pathogenic yeasts revealed that lipid profiles were more conserved than polar profiles, thereby better reflecting their taxonomical relationship according to 265 rRNA sequences. However, there were several potential species-specific metabolites and short regions in the metabolite profiles with enough peak differentiation that could be used to rapidly distinguish between these yeasts by visual inspection. In the third study, the metabolic phenotypes of three strains of 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 were analysed to determine the extent of contribution of the metabolite phenotypes from two parents to their hybrid offspring. While the lipid metabolite phenotypes of all strains resembled each other, the polar metabolite phenotype of the hybrid offspring strongly resembled one parent but not the other. / Thesis / Master of Science (MS)

Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters. The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface. PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC. The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media. For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values. In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments. / Ph. D.

Alkylphenol ethoxylates (APEOs), alcohol ethoxylates (AEOs), and alcohol propoxylates (APOs) are non-ionic surfactants used in daily care products and detergents. They are formed as an oligomeric series with a varying distribution, which determines their commercial application. The goal of the research performed was the development of sample characterization methods for non-ionic surfactants utilizing supercritical fluid chromatography (SFC) under mild instrument operating conditions. The aryl group present in APEOs allowed ultraviolet (UV) detection, with an equal molar response for oligomers, allowing average molar oligomer values to be calculated. APEOs were separated by ethoxylate unit via SFC-UV as well as normal phase HPLC-UV employing packed columns. Stationary phase and column length were varied in the SFC setup to produce the most favorable separation conditions. Fractions from SFC runs of APEOs were collected and analyzed by flow injection analysis electrospray ionization mass spectrometry (FIA-ESI-MS) to identify fraction composition. SFC provided shorter retention times with similar resolution as HPLC for separation of APEOs and consumed a smaller amount of organic solvent. AEOs and APOs lack functionality capable of absorbing UV light outside the UV cut-off of normal organic solvents. SFC was able to separate AEOs and APOs derivatized as trimethylsilyl ethers (TMS) with pure CO2 with detection at 195 nm. The instrumental conditions, however, needed for separation necessitated high temperature and high CO2 pressure. Derivatization of alcohol polyether samples with an UV absorbing agent was achieved with phenylated disilazane-chlorosilane mixtures forming phenylsilylethers detected at 215 nm. Use of an organic solvent-modified CO2 mobile phase afforded lower pressure and temperature conditions for oligomer separation. The use of polar embedded alkyl phases combined with use of organic modified CO2 produced good resolution between oligomers. Better peak shape and shorter retention times were realized with methanol-modified CO2 than acetonitrile-modified CO2. Peak assignments were made via SFC coupled with ESI-MS detection in the positive ion mode. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values. Proton nuclear magnetic resonance (1H-NMR) analysis of non-derivatized samples was performed to determine average molar oligomer values and was used for comparison with values calculated from SFC-UV data. / Ph. D.

Gas Chromatography (GC) is one of the most important and widely used tools in analytical chemistry. However, they are bulky, have a longer measurement cycle, and consume a high amount of power. Micro-Gas Chromatography (µGC) is portable and energy-efficient, which allows onsite, real-time biological, forensic, and environmental analyses. This thesis presents a ready-to-deploy implementation of microfabricated gas chromatography (µGC) system capable of separating complex samples. We describe robust, modular, and scalable hardware and software architecture based on Real-Time Operating System (RTOS) and Python Graphical User Interface (GUI) integrated with various microfabricated devices to realize a fully functional µGC system. A sample heater for headspace injection, microfabricated separation column (µSC), a Photoionization Detector (PI-D), and a flow controller unit are integrated with the modular hardware and software to realize a fully functional Vacuum Outlet µGC system. We have designed a novel auto-calibration method for temperature calibration of the microfabricated devices which does not require changing the electronic circuitry or reprogramming the device. The vacuum outlet µGC setup is tested with various mixture of analytes. For these experiments, an average relative standard deviation (RSD) for retention time repeatability of 2.5% is achieved. Data processing techniques for raw chromatograms, including baseline correction and peak detection, are implemented on a microcontroller board and tested extensively as a part of this work. A novel algorithm for multidimensional analysis for the identification of co-eluting compounds in complex samples is implemented with a prediction accuracy of 94%. / Master of Science / Toxic volatile organic compounds (VOCs) such as benzene and toluene found in gasoline and xylene used in ink, rubber, and leather industries are of concern as they are present at elevated concentrations due to their higher vapor pressure. Sufficient exposure to these toxicants, even at lower concentrations like 100 parts-per-billion-volume (ppbv), may cause adverse health effects. Gas Chromatography (GC) has been the established method for assessing the presence and concentration of VOCs in the environment. Traditional GC systems are bulky, power-hungry, expensive, and require expert supervision for analysis. Recent research in microelectromechanical systems (MEMS) has reduced the size of the GC components, also called micro-GC (µGC), while improving the performance. The majority of the research and development of µGC is aimed at advancing microfabricated components such as preconcentrators, separation columns, and gas detectors. However, the integration of these different components is an important topic that requires more investigation. In this thesis, we present a robust and scalable software and hardware architecture that can be used to develop a portable and modular µGC system. The thesis discusses different experiments to calibrate various microfabricated devices, which are then used to build a fully modular µGC system. We show the separation capacity of the modular µGC system by passing complex compounds like kerosene and diesel. As the chromatogram from the µGC system has noise, the second part of the thesis explores data analysis techniques such as baseline correction, peak detection. These data analysis tools are used to filter the noise, detect relevant peaks in the chromatograms, and identify the compounds in a complex sample.

If the gas chromatographic retention behavior of a compound is known under isothermal conditions at several temperatures, then it is possible to predict accurately the retention time under temperature programmed conditions. This work details the calculations required to perform such predictions. A discussion of isothermal and temperature programmed retention is presented, along with a discussion of carrier gas flow relationships. These theories are then applied to the problem of predicting temperature programmed retention times from isothermal data. It is shown that accurate and precise predictions are possible for many situations. These predicted retention times are then applied to qualitative analysis by the use of retention indices. An architecture for a retention time or index database is proposed. Finally, the use of gas chromatography for the determination of thermodynamic quantities and gas viscosities is demonstrated. / Ph. D.

The design of existing beaded adsorbent materials for column-mode protein purification has emphasized the impact of diffusional transport phenomena upon adsorbent capacity. A design model is presented that optimizes molecular accessibility of proteins relative to the mechanical stability of the material by manipulation of size and solids content for uncross-linked cellulose beads. Cellulose beads of various sizes ranging from about 250 to 1000 pm diameter and having different solids contents were evaluated. Cellulose beads (1.2 mm diameter) gave pressure drops of less than 1 psi per cm of bed at superficial fluid velocities of 100 cm/min in a 1 5 cm bed. Solids content of greater than about 9% cellulose greatly reduced the permeability of large proteins such as thyroglobulin and p-Amylase into the beaded matrix at bead contacting times of 5 and 50 seconds. The amount of permeation in 3% cellulose beads by thyroglobulin at bead contacting times of 5 seconds was about tenfold larger than predicted by diffusion models using the diffusivity of the protein in water. The utility of a low solids content, large bead cellulose support was shown with immobilized IgG (Mr 155 kDa) capturing recombinant human Protein C (M, 62 kDa). The amount of immobilized antibody was varied and immunosorptive capacity of 1 mm cellulose beads was found to be equivalent to that of 0.1 mm cross~linked agarose beads. The immobilization of antibodies to these supports was studied by photomicroscopy of cross-sectioned beads containing immobilized fluorescent labeled antibodies. While 75% of the antibody was immobilized within 0.07 mm of the cellulose bead surface at an antibody density of 1 mg antibody per ml of beads, an appreciable amounts of antibody immobilized deeper into the bead may have been utilized in order to yield capacities equivalent to the smaller agarose beads. The beaded cellulose supports derivatized to form either immunoaffinity or anion exchange matrices exhibited very low non-specific binding. Thus, the particle size, solids content, and extent of derivatization of cellulose matrices can be engineered so as to create matrices that provide high flow rates with low pressure drops while also having desirable adsorptive capacity for proteins. / Ph. D.

A digital computer was used to measure accurate gas chromatographic peak symmetry, position and dispersion by central statistical moments. Benzene samples were chromatographed on squalane for columns of 3, 6 and 12 foot lengths and 1/8", 1/ 4" and 1/2" diameters as a function of sample size. Peak symmetry was monitored by measuring skew, γ₁ , and "excess," γ₂ two quantities derived from the higher central moments. Skew was found to increase in a positive manner for tailing peaks, pass through a maximum and approach a limiting step form for extremely overloaded columns. Skew could be used to indicate saturation of the liquid phase when it passes through a maximum. Negative skew for fronting peaks also approached a zero limiting form. Excess, γ₂ was found to decrease rapidly for all columns. A few microliters of sample were sufficient to cause significant negative values of excess. Excess provides a semi-quantitative measure of column capacity. Three general types of peak shapes were observed with increasing sample sizes: 1) gaussian behavior at very low sample sizes; 2) distorted peaks suitable characterized by central moments at normal analytical size samples; and 3) highly distorted peaks at larger sample sizes where central moments no longer reflect the step shapes observed. Moments can be used to set limits on sample sizes which will produce these highly distorted peaks. Two moment related measures of skewness were also calculated. Pearson’s skew, (Mean - Mode)/(variance)^½, along with Pearson’s β, γ measure of skewness, were found to qualitatively reflect peak shape behavior only in the region of analytical sample sizes. Pearson's skew is subject to difficult interpretation due to equivalent modal values for large samples and the β, γ measure was insensitive to fronting peaks. Third and fourth central moments were observed to have regular behavior as a function of sample size. A moment definition of resolution was derived R = 0.5 (M(1)₂ - M(1)₁) /( √M(2)₁ + √M(2)₂ ) where M(1) = mean and M(2) = variance. This definition was used to compare solvent efficiency for the separation of benzene and cyclohexane on three liquid phases, squalane, dinonalphalate and TRIS. In terms of equivalent throughput, defined as moment resolution per unit time per gram of sample, TRIS was found to be 175 times more selective than squalane. A preparative chromatograph was built with four thermal conductivity detectors at 50' intervals in a 200' x 3/8" column. Column efficiency was measured by comparison of moment parameters at the end of each 50' section. The column was operated both at normal, high pressure drop (ambient outlet pressure) and low differential pressure (constricted outlet). The condition of high pressure drop caused acceleration of samples through the last two column sections and resulted in much poorer column efficiency. The low differential pressure column, inlet 350 psi, outlet approximately 150 psi produced a more linear velocity and greater column efficiency. In fact 100 feet of the low differential pressure column generated the same resolution as 200 feet of the high pressure column. For most chromatographic peaks manual methods of peak evaluation are subject to significant operator errors due to the subjective nature of assigning base width and peak retention time. The use of moments greatly increases the accuracy of two important measurements; (1) retention time as measure from the first moment and (2) resolution for preparative scale samples as measured from the first and second moments. The method of moments provides an accurate means of measuring retention time, dispersion, resolution and preparative scale equivalent throughput. / Ph. D.