My current thesis is regarding the application of Gel Permeation Chromatography (GPC) equipment and principles to the study of polyelectrolyte configuration in solution. The main focus of this study is the effect of salt on the hydrodynamic volume/solution properties of polyelectrolytes and the ability of GPC to effectively determine the degree of variation. This involves the comparison of different salt types and concentrations in aqueous solution. The specific polyelectrolyte examined is poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC), a zwitterionic polymer (i.e. one having both positive and negative charges on the monomer). PMPC is a fairly new and promising polyelectrolyte for use in protein drug delivery and biomaterial surface applications. Understanding the effects of salt on this polyelectrolyte will aid in the development of technologies involving this polymer as well as other zwitterionic polymers. Different salts were utilized to formulate an effect of salt ions so that a systematic analysis could be performed. Using a control as reference it was determined that the salt does have an effect on the solution properties of the polyelectrolytes, as expected. The specific properties examined were characterized into two categories; solution properties and ion properties. Solution properties involved the investigation of salt concentration, solution ionic strength and solution pH effects. For ion properties, salts were selected on the basis of ion charge, charge type and ion size, so that the effects of both the cation and anion components could be analyzed. Two parameters, namely, ion size/type and ion valency were examined for both the cation and the anion. In addition the configuration of the anion was also investigated. vSpecific conclusions found in this study were as follows: 1) The salt concentration has an effect up to a certain "saturation" point. 2) The ionic strength has no visible effect (any effect is related to the concentration component). 3) There is no observable solution pH effect. 4) There is no observable cation effect. This includes no cation type/size or cation valency effect. 5) There is a significant anion effect. 6) A smaller anion has a greater effect than a larger anion. 7) A divalent anion has a greater effect than a monovalent anion. 8) A monatomic anion has a greater effect than a polyatomic anion. It was found that the effect of ion properties is related to mechanisms associated with the geometry of the polyelectrolyte. The negative charge group of the polyelectrolyte which is situated closer to the backbone (inside) is less important to the change in hydrodynamic volume resulting from ionic interactions with the salts since it is shielded by the positive charge group situated at the end of the side chain (outside). The observed phenomena were also explained by other chemical and physical properties such as charge density and ionic potential. In addition to the original plan of study, other phenomena were observed and later explained, such as the presence of four distinct regions associated with salt concentration and the variation m the degree of hydrodynamic volume change with different molecular weight samples. The application of aqueous GPC equipment (including a differential refractive index detector (DRI)) and principles to the study of polyelectrolyte solution effects minimizes the detection equipment required and, provides sufficient resolution and accuracy for examination of solution properties while remaining time and cost effective. The project discoveries have shown that size exclusion chromatography provides an excellent means of obtaining a complete and accurate set of correlations between polyelectrolyte charge and salt effects. / Thesis / Master of Applied Science (MASc)

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The detection and identification of particles within fluid samples is key in the prevention of the spreading of disease. This has created a growing need for devices able to successfully separate and identify multiple particles for this purpose while operating at a high enough throughput to be applicable in the field. A well investigated method of manipulating particles in this way is Dielectrophoresis (DEP), which is the use of varied electric fields gradients to generate a force on small particles. The strength of DEP depends of the properties of the particle medium, the signal generating the electric field, and the properties of the particles themselves. This method and its interaction with all small particles, including biological particles such as blood and cancer cells, has allowed devices utilizing this idea to be investigated for various biological purposes. This thesis investigates methods to increase the throughput of these types of devices in order to increase their ability to process large amounts of samples in reasonable amounts of time. This is done in primarily two methods. One approach uses the application of chromatographic methods to DEP devices to separate particles by altering their individual transit time through a device, allowing identification during constant flow. Another method is through mass parallel channels which each individually operate as a standard DEP particle trapping device. This allows for the summation of the maximum flow through the device due to its design layout. / Master of Science / Micrometer scale devices are popular for the identification, separation, and characterization of micron scale particles. This includes uses in biological fields for the manipulation of particles such as blood cells, cancer cells, and bacteria. A common method of manipulating these particles is Dielectrophoresis, a force that causes particles to be repelled or attracted to geometric designs within the device generated by an applied electric field. The strength and direction of this force on the particles is dependent on the properties of the electrical signal applied to the device, the physical properties of the particles, such as size and shape, and the properties of the medium the particles are suspended in within the device. Biological devices utilizing this force have been tested before, allowing for particles to be separated out of mixed particle solutions. Most of these devices operate by moving through very little material at one time, somewhere in the microliter per hour range. This thesis explores attempts to increase the rate at which samples can be processed by these devices in multiple ways. Chapter 2 explores methods of DEP by applying Chromatography principles, which is to constantly move samples through the device at a high rate and slow the target particles, so they exit the device at a different time than other particles. Chapter 3 investigates increasing device throughput by replicating a standard DEP channel multiple times on one device so that several may operate all at once.

Antibodies represent the largest group of biopharmaceuticals. Due to the nature of their clinical applications, they often need to be produced in large quantities. Plants have distinct advantages of producing large quantities of recombinant proteins, and tobacco is arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high biomass yields and robust transformation technology. However, to produce proteins using transgenic tobacco for human applications, purification of the proteins is challenging. On the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and purification of antibodies. An affinity chromatography purification step utilizing Protein A resin introduced early in the purification process can reduce successive unit operations, thereby reducing the overall process cost. However, directly applying tobacco extract to Protein A chromatography columns may be problematic due to the non-specific binding of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were studied to provide valuable information for future downstream processes for antibody purification from transgenic tobacco. The efficiency of the post load wash buffer to reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post load wash preformed best at reducing the non-specific binding of NTP to the ProSep A resins, while higher salt concentrations were more effective at reducing the amount of NTP contaminants present during elution of the columns. Using a post load wash buffer with an intermediate pH between the binding buffer and the elution buffer was more efficient at eluting our model antibody, human IgG. However, lowering the ionic strength and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract samples were loaded onto the column. Nevertheless, cleaning the columns with denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was effective in regenerating the DBC of the resins and prolonging the life cycle of the resins. This is important to evaluating the economic feasibility of directly using Protein A chromatography to recover antibodies from tobacco extract. Of the three Protein A resins studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of 5. / Master of Science

Gas Chromatography (GC) is a technique for separating volatile compounds by relying on adherence differences in the chemical components of the compound. As conditions within the GC are changed, components of the mixture elute at different times. Sensors measure the elution and produce data which becomes chromatograms. By analyzing the chromatogram, the presence and quantity of the mixture's constituent components can be determined. Machine Learning (ML) is a field consisting of techniques by which machines can independently analyze data to derive their own procedures for processing it. Additionally, there are techniques for enhancing the performance of ML algorithms. Feature Selection is a technique for improving performance by using a specific subset of the data. Feature Engineering is a technique to transform the data to make processing more effective. Data Fusion is a technique which combines multiple sources of data so as to produce more useful data. This thesis applies machine learning algorithms to chromatograms. Five common machine learning algorithms are analyzed and compared, including K-Nearest Neighbour (KNN), Support Vector Machines (SVM), Convolutional Neural Network (CNN), Decision Tree, and Random Forest (RF). Feature Selection is tested by applying window sweeps with the KNN algorithm. Feature Engineering is applied via the Principal Component Analysis (PCA) algorithm. Data Fusion is also tested. It was found that KNN and RF performed best overall. Feature Selection was very beneficial overall. PCA was helpful for some algorithms, but less so for others. Data Fusion was moderately beneficial. / Master of Science / Gas Chromatography is a method for separating a mixture into its constituent components. A chromatogram is a time series showing the detection of gas in the gas chromatography machine over time. With a properly set up gas chromatographer, different mixtures will produce different chromatograms. These differences allow researchers to determine the components or differentiate compounds from each other. Machine Learning (ML) is a field encompassing a set of methods by which machines can independently analyze data to derive the exact algorithms for processing it. There are many different machine learning algorithms which can accomplish this. There are also techniques which can process the data to make it more effective for use with machine learning. Feature Engineering is one such technique which transforms the data. Feature Selection is another technique which reduces the data to a subset. Data Fusion is a technique which combines different sources of data. Each of these processing techniques have many different implementations. This thesis applies machine learning to gas chromatography. ML systems are developed to classify mixtures based on their chromatograms. Five common machine learning algorithms are developed and compared. Some common Feature Engineering, Feature Selection, and Data Fusion techniques are also evaluated. Two of the algorithms were found to be more effective overall than the other algorithms. Feature Selection was found to be very beneficial. Feature Engineering was beneficial for some algorithms but less so for others. Data Fusion was moderately beneficial.

<p><em>Size exclusion chromatography</em> (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance.</p><p>Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work.</p><p>The method developed was tested on cannabis, coffee and 'Spice' with good results.</p>

Size exclusion chromatography (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance. Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work. The method developed was tested on cannabis, coffee and 'Spice' with good results.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

This research contributes to worldwide efforts to miniaturize one of the most powerful and versatile analytical tools, gas chromatography (GC). If a rapid, sensitive and selective hand-held GC system is realized, it would have a wide range of applications in many industries and research areas. As a part of developing a hand-held GC system, this research focuses on the separation column, which is the most important component of a GC system. This thesis describes the development of a miniature separation column that has low thermal mass and an embedded heating element for rapid thermal cycling. The worlds first thin polymer film (parylene) GC column has been successfully developed. This thesis includes: first, a study of theoretical column performance of rectangular GC column; second, the design optimization of parylene column and embedded heating element; third, the development of new processes such as parylene micromolding and stationary phase coating technique for parylene column; fourth, the fabrication of parylene GC column with an embedded heating element; and lastly, the testing and evaluation of parylene GC column through GC analysis.

The protic acid-catalyzed polymerization of 1,6-anhydro-6-D-glucopyranose (I) was first reported one-half century ago; however, the mechanism of this reaction has not been resolved and is the topic under investigation in this thesis. In an attempt to resolve this mechanism, a number of 1,6-anhydrides structurally related to 1,6-anhydro-B-D-glucopyranose (I) were prepared and polymerized. The C-2, C-3, or C-4 hydroxyl group was either specifically blocked, replaced by a hydrogen atom or positioned different sterically. The relative rates of disappearance of monomer in the polymerization reaction were measured and this information used to propose a reaction mechanism.