Microbore columns were investigated for their application to High Performance Liquid Chromatography. Microbore columns consist of stainless steel tubing, 1/16¹¹ OD x 1mm ID, packed with silica gel or other suitable material. These columns were evaluated in terms of extra-column variance effects, mass sensitivity, and solvent consumption. Three methods of extracting the extra-column variance effects were evaluated. When used with an instrument whose contribution to total peak variance is small compared to the column, microbore columns provide the best performance for analyses where sample is limited or where reduction of solvent usage is desirable. A method from the literature for extracting extra column variance effects was found to be easy and reliable. It is proposed as a useful way of allowing column performance to be compared from instrument to instrument and for determining whether a particular instrument is suitable for use with microbore columns. / M.S.

The extraction of fifteen (15) polymer additives with supercritical carbon dioxide which are used as antioxidants, uv stabilizers, process lubes, flame retardants and antistats from eight formulations of polystyrene is demonstrated and compared to traditional dissolution/precipitation extractions. The purpose of the study was twofold: 1) the development of a high performance liquid chromatography (HPLC) method(s) for the additives and 2) the determination of the viability of supercritical fluid extraction (SFE) for the additives from polystyrene. Separation of some of the additives was achieved using reversed phase liquid chromatography. Nine of the additives were assayed in this manner while, the remaining six additives could not be assayed using reversed phase liquid chromatography. In order to develop an extraction method for the additives, the effects of static extraction time, CO2 density, and temperature were first investigated. These preliminary extractions revealed that a static extraction period which afforded an opportunity for the polymer to swell combined with a high CO2 density and extraction temperature above the glass transition (Tg) yielded quantitative recoveries of the additives. Triplicate extractions of the various polystyrene formulations matched additive recoveries obtained by the traditional dissolution/precipitation method. / Master of Science

Tobacco has been studied as a host for producing recombinant therapeutic proteins on a large-scale, commercial basis. However, the proteins expressed in tobacco usually need to be purified to high yield and purity from large amounts of biomass in order for their production to be commercially viable. The methods needed to purify proteins from tobacco are very challenging and not well studied. The objective of this research was to develop a process for the purification of the acidic model protein, recombinant β-glucuronidase (rGUS), from transgenic tobacco leaf tissue to high yield and purity. Polyelectrolyte precipitation with polyethyleneimine (PEI) was identified as an initial purification step for purifying acidic recombinant proteins from tobacco. Polyethyleneimine precipitation allowed for high recovery and concentration of the target protein while removing large amounts of impurities from the initial extract. At dosages of 700-800 mg PEI/g total protein, nearly 100% of the rGUS activity was precipitated with generally more than 90% recovered from the pellet. In addition, more than 60% of the native tobacco proteins were removed in the process, resulting in a purification factor near 4. Recombinant GUS was further purified by a step of hydrophobic interaction chromatography (HIC) followed by a step of hydroxyapatite chromatography (HAC). The HIC step served to remove PEI and other contaminants such as nucleic acids that were accumulated during the precipitation step, while the HAC step served to separate rGUS from the remaining native tobacco proteins, most notably ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Nearly 40% of the initial rGUS activity was recovered as a near homogeneous fraction based on SDS-PAGE analysis after the three step process. The main steps used in this process are anticipated to be scalable and do not rely on affinity separations, making the process potentially applicable to a wide variety of acidic recombinant proteins expressed in tobacco as well as other leafy crops. / Master of Science

Yes / Microemulsion is a stable, isotropic clear solution consisting of oil based substance, water surfactant and cosurfactant. There are two types of microemulsion which are used as a mobile phase; water in oil (w/o) and oil in water (o/w).Microemulsion has a strong ability to solubilize both hydrophobic and hydrophilic analytes, therefore reducing the pre-treatment of the sample which is needed for the complex sample. Recent reports found that separating the analytes by using microemulsion high performance liquid chromatography can be achieved with superior speed and efficiency compared to conventional HPLC modes. In this work, Oil in water (o/w) microemulsion has been used for the determination of nifedipine in pharmaceutical preparation. The effect of each parameter on the separation process was examined. The samples were injected into C18, analytical columns maintained at 30°C with a flow rate 1 ml/min. The mobile phase was 87.1% aqueous orthophosphate buffer 15 mM (adjusted to pH 3 with orthophosphoric acid), 0.8% of octane as oil, 4.5 SDS, and 7.6% 1-butanol, all w/w. The nifedipine and internal standard peaks were detected by UV detection at λ max 237 nm The calibration curve was linear (r2=0.9995) over nifedipine concentrations ranging from 1 to 60 μg/ml (n=6). The method has good sensitivity with limit of detection (LOD) of 0.33 μg/ml and limit of quantitation (LOQ) of 1.005 μg/ ml. Also it has an excellent accuracy ranging from 99.11 to 101.64%. The intra-day and inter-day precisions (RSD %) were <0.45% and <0.9%, respectively.

The use of ¹⁹F observation using MLEV ¹H decoupling in LC/¹⁹F {¹H} NMR was investigated as an alternative to LC/¹H NMR for fluorine containing mixtures and in order to avoid the solvent background problems associated with LC/¹H NMR. P-fluorobenzoate derivatives of various alcohols were analyzed by both LC/¹⁹F {¹H} and LC/¹H NMR. Another alternative exists in supercritical fluid chromatography. A delivery system was assembled and an NMR flow probe was developed and demonstrated practical for directly coupled SFC/¹H NM. The alkane substituents of a model fuel mixture were identified using SFC/¹H NMR in contrast to using the normal phase LC/¹H NMR approach. The relaxation behavior and molecular motion of dilute solutions of benzene and acetonitrile in sub- and supercritical CO₂ were determined using stopped flow ¹H and ¹⁴N NM. The nuclear spin-lattice relaxation times (T₁) for ¹H and ¹⁴N were measured through inversion recovery and linewidth, respectively. Relaxation was found to be dominated by spin-rotation interactions with molecular correlation times (r_sr and _rc) being determined directly from the ¹H and ¹⁴N T₁ over a wide range of viscosities and temperatures. Line-narrowing improvements of ¹⁴N averaged 3-fold as a result of the increased molecular motion. The increased molecular motion as a result of supercritical CO₂ resulted in improved signal enhancement using flow dynamic nuclear polarization. The observed enhancements were two times greater than that typically achieved for the same system and configuration using normal liquid solutions. Through observed NMR enhancements, relative microwave magnetic field values in the vicinity of the NMR coils were measured for typical flow and static DNP-NMR configurations. The advantages of the former were noted. / Ph. D.

A method has been developed whereby various physical properties of middle distillate fuels may be rapidly and accurately calculated by a group property approach from data obtained from a directly coupled Liquid Chromatograph -⁻¹H Nuclear Magnetic Resonance Spectrometer (LC-⁻¹H NMR). The physical properties include cetane number, cetane index, density, specific gravity, pour point, flash point, viscosity, filterability, heat of combustion, cloud point, volume percent aromatics, residual carbon content, and initial, 10%, 50%, 90%, and end boiling points. These property predictions have accuracies approaching the error for measurement of the experimental physical property and require less than two hours analysis time per fuel. An interface was developed between the NMR spectrometer and a personal computer to aid in automation of the LC-⁻¹H NMR data collection and to perform off-line analysis of the LC·⁻¹H NMR data. This interface and all associated software is described. Also presented is a series of model compound studies in which the physical properties of pure hydrocarbons (i.e., alkanes, monocyclic and dicyclic aromatics) were predicted by a similar group property approach. / Ph. D.

Small diameter LC columns provide rapid thermal equilibration and are ideal candidates for temperature programmed LC. Special instrumentation requirements are presented and details of column assembly are given to permit the preparation of highly efficient, stable microbore columns. Three LC temperature control systems are described and their individual strengths and weaknesses are discussed. Problems encountered in raising the temperature of an LC column are addressed and solutions are described. Experimental results of column and instrumentation evaluation are given and the effects of temperature on speed, efficiency, stability and retention of a broad range of samples is reported. Temperature and solvent programming are compared directly. / Ph. D.

The separation of basic nitrogen-containing compounds has been investigated via supercritical CO₂ and 1% methanol modified CO₂. Packed columns with the following stationary phases were employed: silica, octadecyl (C₁₈), propylamino (NH₂), and polystyrene-divinyl benzene (PRP). Without modifier the range of basicities which could be eluted increased in the order of silica < PRP < C₁₈ = NH₂. Chromatographic peak shapes and selectivity were much better with propylamino column. Different aromatic amines and azaarenes were successfully separated on both analytical scale and microbore propylamino bonded phase packed columns with 100% supercritical CO₂. Separation is compared with both reversed phase and normal phase high performance liquid chromatography (HPLC). The retention mechanism study for these aromatic amines and azaarenes shows that the elution order not only depends on basicity and steric hindrance, but also on the solubility of the solute in CO₂. New cross-linked cyanopropyl and phenyl bonded phases are studied as stationary phases for packed column SFC, as well as for separation of nitrogen-containing compounds. The cross-linked bonded phase impedes access to uncapped silanol sites, thereby giving rise to better peak shapes, and more rapid elution without the necessity of a polar modifier in the mobile phase. Experiments both at elevated temperature and in the presence of methanol modifier revealed that there is no short or long term deleterious effect on the column. The separation of model mixtures of nitrated diphenylamine and nitrated anilines via SFC employing cyanopropyl packed and capillary columns is described. Peak identification and peak purity were performed by on-line Fourier transform infrared spectrometric detection. Supercritical CO₂ is employed with cyanopropyl packed columns for separation of non-polymeric components in double-base rocket propellants. Both supercritical CO₂ and CH₂Cl₂ were compared as a solvent for extraction of nonpolymeric components in "good" and "bad" double-base propellant. Finally, twenty-four phenylthiohydantion amino acids (PTH-AA) have been rapidly and efficiently separated on a cyanopropyl packed column by gradient elution of supercritical CO₂ and tetramethyl ammonium hydroxide-modified methanol. Complete or partial resolution of 22 derivatives is observed with only valine co-eluting with norleucine and lysine co-eluting with asparagine. No modifier was required for elution of neutral PTH-AA's from the cross·linked stationary phase. The addition of base plays a major role in elution of acidic and basic PTH-AA's. / Ph. D. / incomplete_metadata

Cannabis sativa L. is a species of flowering plant from the Cannabaceae family which contains over 100 different phytocannabinoids and terpenes. The therapeutic effectiveness of Cannabis sativa L. depends on the cannabinoid content, and the unique aroma in this plant is produced by the terpenes. The two most widely known cannabinoids are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). The legalization of hemp (defined as Cannabis sativa L. < 0.3% Δ9-THC by dry mass) has flooded the market with various hemp and hemp-derived consumer products. These studies focus on using Liquid Chromatography and Gas Chromatography to survey the cannabinoid and terpene content observed for commercially popular Cherry Wine hemp cultivars. Twelve samples of Cherry Wine hemp were obtained representing three field grown hemp plants, a hemp plant grown in a controlled indoor environment, and eight cloned hemp plants also grown in a controlled indoor environment. The analyses revealed variability in the cannabinoid and terpenes contents that reflect plant genetics, daylight exposure duration, and hemp processing and storage conditions. Nitrogen was examined as a substitute carrier gas for increasingly expensive helium for the analysis of terpenes. Finally, Liquid Chromatography was also applied to evaluate the cannabinoid content versus label claims of ten Δ8-Tetrahydrocannabinol (Δ8-THC) hemp-derived consumer products. Examples of under and over reporting of cannabinoids were observed indicating potential risks to consumer safety and need for improved product regulation.

In chemical analyses, it is crucial to distinguish between chemical species. This is often accomplished via chromatographic separations. These separations are often pushed to their limits in terms of the number of analytes that can be sufficiently resolved from one another, particularly when a quantitative analysis of these compounds is needed. Very often, complicated methods or new technology is required to provide adequate separation of samples arising from a variety of fields such as metabolomics, environmental science, food analysis, etc. An often overlooked means for improving analysis is the use of chemometric data analysis techniques. Particularly, the use of chemometric curve resolution techniques can mathematically resolve analyte signals that may be overlapped in the instrumental data. The use of chemometric techniques facilitates quantitation, pattern recognition, or any other desired analyses. Unfortunately, these methods have seen little use outside of traditionally chemometrics focused research groups. In this dissertation, we attempt to show the utility of one of these methods, multivariate curve resolution-alternating least squares (MCR-ALS), to liquid chromatography as well as its application to more advanced separation techniques. First, a general characterization of the performance of MCR-ALS for the analysis of liquid chromatography-diode array detection (LC-DAD) data is accomplished. It is shown that under a wide range of conditions (low chromatographic resolution, low signal-to-noise, and high similarity between analyte spectra), MCR-ALS is able to increase the number of quantitatively analyzable peaks. This increase is up to five-fold in many cases. Second, a novel methodology for MCR-ALS analysis of comprehensive two-dimensional liquid chromatography (LC x LC) is described. This method, called two dimensional assisted liquid chromatography (2DALC), aims to improve quantitation in LC x LC by combining the advantages of both one-dimensional and two dimensional chromatographic data. We show that 2DALC can provide superior quantitation to both LC x LC and one dimensional LC under certain conditions. Finally, we apply MCR-ALS to an LC x LC analysis of fourteen furanocoumarins in three apiaceous vegetables. The optimal implementation of MCR-ALS and subsequent integration was determined. For these data, simply performing MCR-ALS on the two dimensional chromatogram and manually integrating the results proved to be the superior method. These results demonstrate the usefulness of these curve resolution techniques as a compliment to advanced chromatographic techniques.