Quantitative characterization of physico-chemical properties of the active layers of reverse osmosis and nanofiltration membranes, and their relation to membrane performance

The main objectives of this dissertation were: (i) to develop experimental and analytical procedures to quantify different physico-chemical properties of the ultra-thin (~ 100 nm) active layers of reverse osmosis (RO) and nanofiltration (NF) membranes and their interactions with contaminants; (ii) to use such procedures to evaluate the similarities and differences between the active layers of different RO/NF membranes; and (iii) to relate characterization results to membrane performance. Such objectives were motivated by the current limited understanding of the physico-chemical properties of active layers as a result of traditional characterization techniques having limitations associated with the nanometer-scale spatial resolution required to study these ultra-thin films. Functional groups were chosen as the main active layer property of interest. Specific accomplishments of this study include the development of procedures to quantify in active layers as a function of pH: (1) the concentration of both negatively and positively ionized functional groups; (2) the stoichiometry of association between ions (i.e., barium) and ionized functional groups (i.e., carboxylate and sulfonate); and (3) the steric effects experienced by ions (i.e., barium). Conceptual and mathematical models were developed to describe experimental results. The depth heterogeneity of the active layer physico-chemical properties and interactions with contaminants studied in this dissertation was also characterized. Additionally, measured concentrations of ionized functional groups in the polyamide active layers of several commercial RO/NF membranes were used as input in a simplified RO/NF transport model to predict the rejection of a strong electrolyte (i.e., potassium iodide) and a weak acid (i.e., arsenious acid) at different pH values based on rejection results at one pH condition. The good agreement between predicted and experimental results showed that the characterization procedures developed in this study serve as useful tools in the advancement of the understanding of the properties and structure of the active layers of RO/NF membranes, and the mechanisms of contaminant transport through them.

Growth and Characterization of III-V Semiconductor Materials for MOCVD Reactor Qualification and Process Control

Metalorganic chemical vapor deposition is examined as a technique for growing compound semiconductor structures. Material analysis techniques for characterizing the quality and properties of compound semiconductor material are explained and data from recent commissioning work on a newly installed reactor at the University of Illinois is presented.

Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Low temperature magneto-photoluminescence characterization of high purity gallium arsenide and indium phosphide

Low-temperature magneto-photoluminescence is a very powerful technique to characterize high purity GaAs and InP grown by various epitaxial techniques. These III-V compound semiconductor materials are used in a wide variety of electronic, optoelectronic and microwave devices. The large binding energy differences of acceptors in GaAs and InP make possible the identification of those impurities by low-temperature photoluminescence without the use of any magnetic field. However, the sensitivity and resolution provided by this technique rema1ns inadequate to resolve the minute binding energy differences of donors in GaAs and InP. To achieve higher sensitivity and resolution needed for the identification of donors, a magneto-photoluminescence system 1s installed along with a tunable dye laser, which provides resonant excitation. Donors 1n high purity GaAs are identified from the magnetic splittings of "two-electron" satellites of donor bound exciton transitions 1n a high magnetic field and at liquid helium temperature. This technique 1s successfully used to identify donors 1n n-type GaAs as well as 1n p-type GaAs in which donors cannot be identified by any other technique. The technique is also employed to identify donors in high purity InP. The amphoteric incorporation of Si and Ge impurities as donors and acceptors in (100), (311)A and (3ll)B GaAs grown by molecular beam epitaxy is studied spectroscopically. The hydrogen passivation of C acceptors in high purity GaAs grown by molecular beam epitaxy (MBE) and metalorganic chemical vapor deposition (MOCVD) 1s investigated using photoluminescence. Si acceptors ~n MBE GaAs are also found to be passivated by hydrogenation. The instabilities in the passivation of acceptor impurities are observed for the exposure of those samples to light. Very high purity MOCVD InP samples with extremely high mobility are characterized by both electrical and optical techniques. It is determined that C is not typically incorporated as a residual acceptor ~n high purity MOCVD InP. Finally, GaAs on Si, single quantum well, and multiple quantum well heterostructures, which are fabricated from III-V semiconductors, are also measured by low-temperature photoluminescence.

In situ characterization of sediment oxygen demand across the spectrum of non-resuspension to full resuspension with varying bed shear stress

Sediment oxygen demand (SOD) can be a significant oxygen sink in various types of water bodies, particularly slow-moving waters with substantial organic sediment accumulation. In most settings where SOD is a concern, the prevailing hydraulic conditions are such that the impact of sediment resuspension on SOD is not considered. However, in the case of Bubbly Creek in Chicago, Illinois, the prevailing slack water conditions are interrupted by infrequent intervals of very high flow rates associated with pumped combined sewer overflow (CSO) during intense hydrologic events. These events can cause resuspension of the highly organic, nutrient-rich bottom sediments, resulting in precipitous drawdown of dissolved oxygen (DO) in the water column. While many past studies have addressed the dependence of SOD on near-bed velocity and bed shear stress prior to the point of sediment resuspension, there has been limited research that has attempted to characterize the complex and dynamic phenomenon of resuspended-sediment oxygen demand. To address this issue, a new in situ experimental apparatus referred to as the U of I Hydrodynamic SOD Sampler was designed to achieve a broad range of velocities and associated bed shear stresses. This allowed SOD to be analyzed across the spectrum of no sediment resuspension associated with low velocity/ bed shear stress through full sediment resuspension associated with high velocity / bed shear stress. The current study split SOD into two separate components: (1) SODNR is the sediment oxygen demand associated with non-resuspension conditions and is a surface sink calculated using traditional methods to yield a value with units (g/m2/day); and (2) SODR is the oxygen demand associated with resuspension conditions, which is a volumetric sink most accurately characterized using non-traditional methods and units that reflect suspension in the water column (mg/L/day). In the case of resuspension, the suspended sediment concentration was analyzed as a function of bed shear stress, and a formulation was developed to characterize SODR as a function of suspended sediment concentration in a form similar to first-order biochemical oxygen demand (BOD) kinetics with Monod DO term. The results obtained are intended to be implemented into a numerical model containing hydrodynamic, sediment transport, and water quality components to yield oxygen demand varying in both space and time for specific flow events. Such implementation will allow evaluation of proposed Bubbly Creek water quality improvement alternatives which take into account the impact of SOD under various flow conditions. Although the findings were based on experiments specific to the conditions in Bubbly Creek, the techniques and formulations developed in this study should be applicable to similar sites.