Monoclonal antibodies are large, complex biomolecules that can be difficult to characterize. Characterization is important because of the various post translational modifications that can occur during manufacturing, processing, and storage. Some modifications can lead to efficacy and safety issues and therefore are heavily monitored. A leading way to monitor various modifications is by using liquid chromatography. The high sensitivity, reproducibility, and ability to quantitate analytes makes it very attractive for monoclonal antibody characterization. The large molecular size of monoclonal antibodies (150 kDa) makes them challenging to separate efficiently and with high enough resolution to be helpful. New column technologies that would help improve protein separation efficiencies and slectivities would greatly help in this challenging process. In this thesis, three novel bonded phases are developed for the separation of monoclonal antibodies including a weak anion and cation exchanger (WAX, CEX) for the separation of charged species as well as a novel hydrophilic interaction chromatography (HILIC) for the separation of glycoforms. Column develop is achieved by optimizing selectivity and improving efficiency of separations by altering particle surface chemistry.

This thesis focuses on my endeavors to prepare new porous polymeric monoliths that are viable to use as supports in flow-through processes. Polymer monoliths of various porous properties and different chemical properties have been prepared utilizing the thermally induced phase separation (TIPS) phenomenon and step-growth polymerization reactions. The aim has been to find appropriate synthesis routes to produce separation supports with fully controlled chemical, physical and surface properties. This thesis includes preparation of porous monolithic materials from several non-cross-linked commodity polymers and engineering plastics by dissolution/precipitation process (i.e. TIPS). Elevated temperatures, above the upper critical solution temperature (UCST), were used to dissolve the polymers in appropriate solvents that only dissolve the polymers above this critical temperature. After dissolution, the homogeneous and clear polymer-solvent solution is thermally quenched by cooling. A porous material, of three dimensional structure, is then obtained as the temperature crosses the UCST. More than 20 organic solvents were tested to find the most compatible one that can dissolve the polymer above the UCST and precipitate it back when the temperature is lowered. The effect of using a mixture of two solvents or additives (co-porogenic polymer or surfactant) in the polymer dissolution/precipitation process have been studied more in depth for poly(vinylidine difluoride) (PVDF) polymers of two different molecular weight grades. Monolithic materials showing different pore characteristics could be obtained by varying the composition of the PVDF-solvent mixture during the dissolute­ion/precipitation process. Step-growth polymerization (often called polycondensat­ion reaction) combined with sol-gel process with the aid of porogenic polymer and block copolymer surfactant have also been used as a new route of synthesis for production of porous melamine-formaldehyde (MF) monoliths. In general, the meso- and macro-porous support materials, for which the synthesis/preparation is discussed in this thesis, are useful to a wide variety of applications in separation science and heterogeneous reactions (catalysis).

No description available.

Continuous chromatography is an attractive alternative to traditional batch chromatography because it can have higher productivity, solvent efficiency, and product concentrations. However, several barriers prevent further use of continuous chromatography. There are many operating parameters that must be determined when designing continuous systems making it difficult to achieve high purity, yield, and productivity. Through the identification and strategic combination of the key dimensionless groups that control a continuous separation, it is possible to design highly productive systems that produce products with high yield and high purity. In this dissertation, three examples were selected to demonstrate the significance of a model-based method when designing continuous chromatography systems. (1) The Speedy Standing Wave Design and simulated moving bed splitting strategies for the separation of ternary mixtures with linear isotherms. (2) The Standing-wave Design of Three-Zone open-loop non-isocratic SMB for purification. (3) The Continuous Ligand-Assisted Displacement for the separation of Rare Earth Elements.<div>In the first example, the Speedy Standing Wave Design equations were developed for multicomponent separations with linear isotherms and a systematic splitting strategy was developed for the design of multiple sequential Simulated Moving Beds (SMBs). By performing the easiest split first, the overall productivity and solvent efficiency can be significantly improved. Rate model simulations were used to verify that the SSWD equations achieved target yields and purities. In systems where only one component is desired, the sorbent should be selected such that this component is the most or least retained so that it can be separated in a single SMB.</div><div>In the second example, the Standing Wave Design method was extended to non-isocratic three zone open loop SMBs. The standing wave design equations were derived and then verified using rate model simulations. In two case studies it was shown that non-isocratic SMBs designed using the standing wave design method show an order of magnitude higher productivity than a comparable batch system when the impurities are weakly adsorbing. When the impurities are competitive, the SWD method produces SMB systems with 2 orders of magnitude higher productivity than comparable batch systems. Because the design is based on dimensionless groups, the resulting designs are easily scalable and no rate model simulations are required to design high yield, high purity, and high productivity SMBs.</div><div>In the third example, the constant pattern design method was extended to continuous LAD systems. A continuous operation mode was developed that reduced the cycle time of LAD systems to further increase the productivity. In cases where the feed was equimolar, the continuous configuration increased the productivity between 20-50%. A multizone continuous LAD configuration was developed for the separation of a complex mixture of Dy, ND, and Pr that simulated a crude magnet feed. The resulting overall productivity for this system was 190 kg/m³day which was two orders of magnitude higher than a single column batch system and 70% higher than a multizone batch system. The robustness of the constant pattern design method was demonstrated through a simulated case study and it was determined that adding a safety factor through the reduction of the flowrate was more effective than reducing the design length.</div><div>Using a model-based design allows for the consistent design of continuous chromatography systems. The effects of a change in a feed or operating condition can be more easily understood through the lens of the model. This means that adjustments can be made pre-emptively when necessary and the new designs can be tested with virtual experiments before being implemented. The understanding of key dimensionless groups allows for designs that meet key design criteria at all scales of operation and thus allows for the easy transition from one scale to another.</div>

The objective of this research is to develop and apply a HILIC UHPLC stationary phase that allows for separation of intact glycoproteins. In Chapter 1 I give an overview of the problems of current glycosylation profiling with regards to biotherapeutics, and my strategy to separate the intact glycoprotein with HILIC. Chapter 2 describes the methods used to produce the nonporous packing material and stationary phase. In Chapter 3 I describe previous work in developing a HILIC polyacrylamide stationary phase, and further improvements I have made. Chapter 4 describes development of an assay in collaboration with Genentech of therapeutic mAb glycosylation. In Chapter 5, I show HILIC-MS of digested ribonuclease B as a beginning step to analyze glycosylated biomarkers.

In the area of forensic chemistry, improved or new analysis methods are continually being investigated. One common and powerful technique used in forensic chemistry is wall-coated open-tubular column (WCOT) gas chromatography with electron ionization single quadrupole mass spectrometry (GC-MS). Improvements to and effectiveness of alternatives to this instrumental platform were explored in an array of parallel inquiries. The areas studied included the column for the chromatographic separation, the universal detection method employed, and the fragmentation method used to enhance molecular identification. <br><br>Superfine-micropacked capillary (SFµPC) columns may provide an alternative to commercial packed GC columns and WCOT GC columns that combines the benefits of the larger sample capacity of packed columns and the benefits of the excellent separation capabilities and mass spectrometry (MS) flow rate compatibility of WCOT columns. SFµPC columns suffer from high inlet pressure requirements and prior reported work has required specialized instrumentation for their use. Fabrication of and chromatography with SFµPC GC columns was successfully achieved with typical GC-MS instrumentation and within the flow rate limit of a MS. Additionally, the use of higher viscosity carrier gasses was demonstrated to reduce the required inlet pressure for SFµPC GC columns.<br><br>Recently, a new vacuum ultraviolet spectrometer (VUV) universal detector has been commercialized for GC. The ability of VUV detectors to acquire absorbance spectra from 125 nm to 430 nm poses a potential alternative to MS. As such, GC-VUV provides an exciting potential alternative approach to achieving excellent quantitative and qualitative analysis across a wide range of analytes. The performance of VUV and MS detectors for forensic analysis in terms of quantitative and qualitative analysis was compared. Analysis of alkylbenzenes in ignitable liquids was explored, which can be important evidence from suspected arson fires and are difficult to differentiate with MS. The VUV detector was found to have superior specificity and comparable sensitivity to the MS detector in scan mode.<br><br>Addition of thermolysis (Th) as an orthogonal fragmentation pathway provides the opportunity to increase the differences between MS fragmentation patterns. Fragmentation has been widely established to aid in identification of molecules with MS by providing characteristic fragments at characteristic relative abundances. However, molecules with very similar structures do not result in sizable spectral differences in all cases with typical MS fragmentation techniques. A series of Th units were fabricated and integrated into GC-Th-MS instruments. Th-MS was conducted with the thermally labile nitrate esters across a range of instrumentation and thermal conditions.<br>

<p>Canines can detect different diseases simply by smelling different biological sample types, including  urine,  breath  and  sweat.  This  has  led  researchers  to  try  and  discovery  unique  volatile  organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using  miniaturized  biosensors.  However,  the  identity  of  the  specific  VOC  biomarkers  must  be  demonstrated before designing and fabricating sensing systems. Through  an  extensive  series  of  experiments,  VOCs  in  urine  are  profiled  by  solid  phase  microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that  unique  classes  of  urinary  VOCs,  primarily  terpene/terpenoids  and  carbonyls,  are  potential  biomarkers  of  breast  cancer.  Through  implementing  chemometric  approaches,  unique  panels  of  VOCs  were  identified  for  breast  cancer  detection,  identifying  tumor  location,  determining  the  efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples.  VOC biomarker identification is an important step toward developing portable gas sensors, but  another  hurdle  that  exists  is  that  current  sensors  lack  selectivity  toward  specific  VOCs  of  interest.  Furthermore,  testing  sensors  for  sensitivity  and  selectivity  is  an  extensive  process  as  VOCs  must  be  tested  individually  because  the  sensors  do  not  have  modes  of  chromatographic  separation or compound identification. Another set of experiments is presented to demonstrate that SPME  fibers  can  be  coated  with  materials,  used  to  extract  standard  solutions  of  VOCs,  and  analyzed  by  GC-MS  to  determine  the  performance  of  various  gas  sensing  layers.  In  the  first  of  these  experiments,  polyetherimide  (PEI)  was  coated  onto  a  SPME  fiber  and  compared  to  commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity  for  urinary  VOC  extraction  relative  to  a  polydimethylsiloxane  (PDMS)  SPME  fiber.  These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers. </p>

<p>  </p> <p>Atmospheric aerosols formed through primary emissions, secondary gas-particle formations, and multi-phase chemical processes are composed of solid, semi-solid, or liquid-like particles suspended in the air that have direct implications towards the global radiative balance and human health as air pollutants.  Direct emissions of primary organic aerosols (POA; e.g. soot, BrC) and multi-phase formation of secondary organic aerosols (SOA) from the oxidation of biogenic monoterpene isomers represent two important sources/classes of particulate matter in the atmosphere. Multi-phase chemical processes driving the atmospheric and environmental aging through the photochemistry of iron(III), FeIII in organic aerosol particles and aqueous media drives the multiphase chemistry leading to systematic aging of their chemical composition and modifications to resulting light-absorption properties. The molecular composition, organic structures, physical properties, and sources of emissions are complex requiring development of powerful multi-modal analytical metrology, such as high-resolution mass spectrometry (HRMS) hyphenated with liquid chromatography (LC), photodiode array optical detection, drift tube ion mobility (IM) spectrometry, and desorption and ambient ionization of multi-components mixtures in atmospheric particles using temperature programmed desorption Direct analysis in real time (TPD-DART). Disseminating the molecular-specific composition, chemical and physical properties of complex mixtures in atmospheric organic particles and mixed inorganic/organic systems will help improve our understanding of their formation mechanisms, transformative chemical ageing processes, as well as improved detection of individual components in complex mixtures. </p> <p>     </p> <p>Chapter 1 and 2 of dissertation introduces complexity of atmospheric organic, carbonaceous aerosols, and complex environmental mixtures and discusses analytical metrology, experiments, and data analysis procedures used for detailed molecular-level characterization of mixtures. Chapter 3 the development of a robust analytical method for untargeted screening and determination of the physical and chemical properties (e.g. vapor pressures, enthalpies of sublimation, and saturation mass concentrations) of single components out of complex SOA particles using temperature programmed desorption Direct analysis in real time ionization – high resolution mass spectrometry (TPD-DART-HRMS).  Chapter 4 introduces the use of ion mobility - mass spectrometry (IM-MS) separation and multidimensional characterization of structural isomers in complex SOA mixtures. The chapter discusses the advanced usage of IM-MS to investigate the molecular and structural properties of isomers of alpha-pinene and limonene derived SOA, use of advanced data analysis procedures to resolved complex conformational and structural isomers, and investigate single-molecule structural changes from atmospheric-like ageing in SOA particles using IM-MS.  Chapter 5 discusses the chemical characterization and analysis of individual brown carbon (BrC) chromophores out of mixture of colorless organic carbon constituents and insoluble soot particles generated from controlled flame combustion of ethane fuel, a surrogate system representing gasoline combustion of motor vehicles. The chapter focuses on the quantitative method development and use of state-of-the-art liquid chromatography coupled to photodiode array followed by dopant assisted atmospheric pressure photoionization and HRMS (LC-PDA-HRMS) analysis, followed by conversion to quantitative optical information for comparisons with retrieved literature reports. Chapter 6 examines the complex multiphase photochemical cycling of Fe(III)-citrate, a relevant proxy for [FeIII-carboxylate]2+ complexes in atmospheric water using complementary analytical metrology of optical spectroscopy, LC-PDA-HRMS, oil immersion flow microscopy. Multi-modal datasets from these complementary techniques provide a unique experimental description of various stages of FeIII-citrate photochemistry, elucidate individual components of this reacting system, determine mechanistic insights, and quantify environmental parameters affecting the photochemistry. </p>

<p dir="ltr">Atmospheric organic aerosols (OA) have diverse compositions and undergo complex reactions and transformations within the atmosphere, leading to profound impacts on air quality, climate, and atmospheric chemistry. In particular, these aerosols play an important role in Earth's effective radiative forcing (ERF) through interactions with solar radiation, absorbing and scattering sunlight and terrestrial radiation. These interactions result in a warming and cooling effect on the climate, respectively. This dissertation seeks to unravel the intricate molecular characteristics of atmospheric OA, focusing specifically on its light-absorbing components, known as ‘Brown Carbon’ (BrC), and aims to comprehend its dynamic interplay within the atmosphere. The research employs state-of-the-art multi-modal mass spectrometry techniques to investigate atmospheric OA derived from the combustion of fossil fuels and biomass burning. Through a combination of controlled laboratory experiments and real-world sample analyses, these works provide molecular-level insights crucial for source apportionment and predictive modeling of OA fate. Chapter 2 details the instrumentation and data analysis methods, laying a robust foundation for subsequent chapters.</p><p dir="ltr">Chapter 3 delves into the investigation of smoldering-phase biomass burning organic aerosols (BBOA) and introduces an innovative fractionation method for high-level molecular characterization, targeted to streamline source apportionment of BBOA. This chapter also presents an extensive assessment of particle-to-gas partitioning of BBOA, providing valuable information for modeling atmospheric lifetimes and fate. In Chapter 4, a comparative analysis of BBOA from wild and agricultural fires is conducted, employing advanced molecular characterization techniques. Chapter 5 showcases the synergistic use of multi-modal mass spectrometry techniques to probe the chemical evolution of individual BBOA components. Finally, Chapter 6 examines the molecular analysis of secondary OA (SOA) generated from the photooxidation of a fossil-fuel proxy.</p><p dir="ltr">The comprehensive molecular-level studies presented contribute essential insights for climate modeling, aiding in resolving uncertainties associated with OA's impact on global ERF. The research not only challenges existing analytical methods but also introduces novel approaches for obtaining relevant information about atmospheric OA components. Overall, this work advances our understanding of the intricate dynamics of atmospheric aerosols, facilitating more accurate climate predictions and addressing uncertainties surrounding their net radiative impact.</p>

In forensic chemistry, a quicker and more accurate analysis of a sample is always being pursued. Speedy analyses allow the analyst to provide quick turn-around times and potentially decrease back-logs that are known to be a problem in the field. Accurate analyses are paramount with the futures and lives of the accused potentially on the line. One of the most common methods of analysis in forensic chemistry laboratories is gas chromatography, chosen for the relative speed and efficiency afforded by this method. Two major routes were attempted to further improve on gas chromatography applications in forensic chemistry.<br> The first route was to decrease separation times for analysis of ignitable liquid residues by using micro-bore wall coated open-tubular columns. Micro-bore columns are much shorter and have higher separation efficiencies than the standard columns used in forensic chemistry, allowing for faster analysis times while maintaining the expected peak separation. Typical separation times for fire debris samples are between thirty minutes and one hour, the micro-bore columns were able to achieve equivalent performance in three minutes. The reduction in analysis time was demonstrated by analysis of ignitable liquid residues from simulated fire debris exemplars.<br> The second route looked at a relatively new detector for gas chromatography known as a vacuum ultraviolet (VUV) spectrophotometer. The VUV detector uses traditional UV and far-ultraviolet light to probe the pi and sigma bonds of the gas phase analytes as well as Rydberg traditions to produce spectra that are nearly unique to a compound. Thus far, the only spectra that were not discernable were from enantiomers, otherwise even diastereomers have been differentiated. The specificity attained with the VUV detector has achieved differentiation of compounds that mass spectrometry, the most common detection method for chromatography in forensic chemistry labs, has difficulty distinguishing. This specificity has been demonstrated herein by analyzing various classes of drugs of abuse and applicability to “real world” samples has been demonstrated by analysis of de-identified seized samples.<br>