A new general method is developed for the optimization of HPLC ternary or pseudo-quaternary mobile phases which are represented by the trilinear coordinate system. This method can predict the global optimum of the mobile phase composition. The global optimum composition along each edge of the triangle and the corresponding selectivity factor of the worst-separated peak pair(s) are used in this method. This method is named the weighted pattern comparison optimization method (WPCO) and is applicable for both known and unknown samples. The WPCO method is simpler than those currently in use. The WPCO method was tested by using 68 literature data sets whose separation response surfaces are different. Results of the WPCO method agree with the results obtained by the minimum α plot method and by the grid search method, and do so with substantially fewer experimental measurements. Compared with the 5% (in eluent composition) step size grid-search procedure, the WPCO method using the same step size reduces the experimental work by 75%. For further reducing the experimental work, the original WPCO method is simplified. In an ordinary HPLC separation, the separation factor and resolution are approximately proportional to the logarithm of the selectivity factor. Based on this, the separation factor replaces the logarithm of selectivity factor in the original WPCO method. This further reduces the experimental work and avoids the error introduced in the measurement of the column dead volume. The simplified WPCO method has been tested in the normal-phase and reversed-phase chromatography separation cases. The simplified WPCO method has been tested by using 27 literature data sets whose separation response surfaces are different. Results of the simplified and original WPCO methods are nearly identical when the capacity factors of the solutes of the worst-separated peak pairs are greater than 5. When the capacity factors are less than 5, the simplified WPCO method is satisfactory in less complex, less critical applications. Two new metabolites of nefopam have been separated from greyhound urine. In the separation process, flash chromatography is used for cleaning up and preseparating the samples in a single step. Compared with other techniques, experimental work is reduced. The structure of one of the newly discovered metabolites is determined using MS and NMR. The most probable structure of the other metabolite is determined using MS. The main metabolic pathways at different doses in greyhounds are studied. / Graduation date: 1991

A reverse-phase high pressure liquid chromatographic method for the quantitative analysis of unchanged ascorbic acid in human urine is described. Selection of an appropriate mobile phase and discussion of some of the analysis problems are presented. Twenty-four hour ascorbate excretion profiles from two subjects were determined. Standard redox titration procedures were used to corroborate the chromatographic method. When compared to classical titration or colorimetric redox procedures, the new assay features straightforward sample preparation and improved sensitivity.Ball State UniversityMuncie, IN 47306

Oil from flaxseed (Linum usitatisssimum L.) contains hydrophobic cyclic peptides or cyclolinopeptides (CLs) comprising eight or nine amino acids. These bioactive compounds have potential therapeutic applications and may be used as scaffolds for increased utility. Two steps were undertaken to increase the potential utility of these compounds. Initially multigram quantities of flax CLs were highly enriched from flax oil. Subsequently new synthetic procedures were developed for modification of the CLs through the methionine group (Met). Finally, the utility of the modified CLs was tested in a number of applications. CLs were recovered from a crude oil extract that contain five CLs (CLA, CLC, CLE, CLJ and CLK). Oxidation of this mixture reduced the complexity of the mix to just three CLA, CLJ and CLK. CLJ and CLK were enriched then characterized by NMR and MS-MS methods. CLs containing methionine sulfoxide groups (Mso), CLC and CLE were isolated from crude mixture then selectively reduced to afford Met containing analogs: CLB and CLE'. The Met of modified CLs was used as a point for attachment of tags and couplers for various applications. Cyclic peptide modification through Met groups has not been reported previously. Synthetic methods were devised to introduce activating functional groups such as -CN, -COOH, -OH and -NH2 to the sulfur atom of Met. The modified CL conjugates were characterized using spectrometric techniques including 1D and 2D NMR spectrometry, as well as mass spectrometry. After activation the CLs were covalently linked to molecules or materials of interest including fluorescence tags (coumarin), affinity chromatography media and bovine serum albumin (BSA) for production of polyclonal antibodies. Fluorescence studies were performed in methanol, ethanol, dimethylformamide and acetonitrile to study the solvent effect. CLs attached to solid affinity matrix showed specific binding to apolipoprotein A1 after incubation with chicken serum. These CLs also act as hapten and have been used to couple BSA to produce polyclonal antibodies. Met modification was a satisfactory approach to produce a range of useful peptide products where more conventional methods of molecule attachment are not available.

The biggest limitation to conventional gas chromatography (GC) is limited peak capacity, making the analysis of complex mixtures a difficult or even impossible task. Comprehensive two-dimensional gas chromatography (GC×GC) significantly increases peak capacity and resolution, improves sensitivity and generates structured 3D chromatograms. This is achieved by connecting two columns coated with different stationary phases through a special interface (modulator). The interface samples the first column effluent and periodically injects fractions of this material, as narrow injection pulses, onto the second column for further separation. Commercial instruments achieve this with cryogenic agents. Since this expensive approach permits only in-laboratory analysis, the development of simple, economical and field-capable GC×GC systems is in demand. This report summarizes the fundamentals governing GC×GC separations and a brief history of technological advances in the field. It also documents the construction of a simple interface, devoid of moving parts and cryogenic consumables, and hence highly suitable for field analysis and monitoring applications. Evaluation of the interface suggests on-par performance with more complicated cryogenic modulators. GC×GC separations of technical mixtures of fatty acid methyl esters (FAMEs), common environmental pollutants (EPA 8270), polychlorinated biphenyls (PCBs), pesticides (toxaphene), as well as selected essential oils and major distillation fractions of crude oil indicate very good performance. Most notably, the interface prototype was applied for the first ever time-resolved on-site analysis of the semivolatile organic fraction of urban air particulate matter (PM2.5).

The biggest limitation to conventional gas chromatography (GC) is limited peak capacity, making the analysis of complex mixtures a difficult or even impossible task. Comprehensive two-dimensional gas chromatography (GC×GC) significantly increases peak capacity and resolution, improves sensitivity and generates structured 3D chromatograms. This is achieved by connecting two columns coated with different stationary phases through a special interface (modulator). The interface samples the first column effluent and periodically injects fractions of this material, as narrow injection pulses, onto the second column for further separation. Commercial instruments achieve this with cryogenic agents. Since this expensive approach permits only in-laboratory analysis, the development of simple, economical and field-capable GC×GC systems is in demand. This report summarizes the fundamentals governing GC×GC separations and a brief history of technological advances in the field. It also documents the construction of a simple interface, devoid of moving parts and cryogenic consumables, and hence highly suitable for field analysis and monitoring applications. Evaluation of the interface suggests on-par performance with more complicated cryogenic modulators. GC×GC separations of technical mixtures of fatty acid methyl esters (FAMEs), common environmental pollutants (EPA 8270), polychlorinated biphenyls (PCBs), pesticides (toxaphene), as well as selected essential oils and major distillation fractions of crude oil indicate very good performance. Most notably, the interface prototype was applied for the first ever time-resolved on-site analysis of the semivolatile organic fraction of urban air particulate matter (PM2.5).

Air is one of the most important resources in the world and is essential for life. With the development of industry, air pollution is becoming a severe problem. Air Pollution not only affects the quality of the air we breathe; it also impacts the land and the water. Volatile organic compounds (VOCs), which can cause short-term and long-term health problems, are found as contaminants in both indoor air and the environment. Therefore, it is necessary to develop accurate and convenient sampling methods to determine VOCs at trace levels in both community and occupational environment. The focus of this project is to develop the needle trap devices (NTDs) with appropriate sorbents and employ them to do air samplings by diffusive or active sampling mode. For diffusive sampling, the NTD with sorbent Carboxen1000 was developed to monitor benzene, toluene, ethylbenzene and o-xylene (BTEX) in the air, coupled with GC-MS. The factors such as sorbent strength, response time, face velocity, temperature and pressure, relative humidity and sampling duration were investigated. Method validations were done both in the laboratory and in field. The results demonstrate that the NTD with Carboxen1000 is a successful diffusive sampler for monitoring Time-weighted average (TWA) concentrations of BTEX. On the other hand, the NTD with divinylbenzene (DVB) coupled with GC-MS by thermal desorption was developed for sampling and analysis of volatile thiols. The factors such as sorbent strength, desorption efficiency were investigated. The applications, such as vegetable analysis and field sampling analysis, indicate that the NTD with sorbent DVB is a successful active sampler for determine volatile thiols in food and air samples.

Air is one of the most important resources in the world and is essential for life. With the development of industry, air pollution is becoming a severe problem. Air Pollution not only affects the quality of the air we breathe; it also impacts the land and the water. Volatile organic compounds (VOCs), which can cause short-term and long-term health problems, are found as contaminants in both indoor air and the environment. Therefore, it is necessary to develop accurate and convenient sampling methods to determine VOCs at trace levels in both community and occupational environment. The focus of this project is to develop the needle trap devices (NTDs) with appropriate sorbents and employ them to do air samplings by diffusive or active sampling mode. For diffusive sampling, the NTD with sorbent Carboxen1000 was developed to monitor benzene, toluene, ethylbenzene and o-xylene (BTEX) in the air, coupled with GC-MS. The factors such as sorbent strength, response time, face velocity, temperature and pressure, relative humidity and sampling duration were investigated. Method validations were done both in the laboratory and in field. The results demonstrate that the NTD with Carboxen1000 is a successful diffusive sampler for monitoring Time-weighted average (TWA) concentrations of BTEX. On the other hand, the NTD with divinylbenzene (DVB) coupled with GC-MS by thermal desorption was developed for sampling and analysis of volatile thiols. The factors such as sorbent strength, desorption efficiency were investigated. The applications, such as vegetable analysis and field sampling analysis, indicate that the NTD with sorbent DVB is a successful active sampler for determine volatile thiols in food and air samples.

This research studies the chemical nature of zirconia and the complex surface chemistry of zirconia in order to better comprehend its behavior under chromatographic conditions. This research shows how the physical and chemical properties of zirconia depend strongly on the thermal treatment during synthesis. The morphology of the samples was also studied. The absorption capability of Adenosine Triphosphate (ATP) on zirconia was also monitored and spectrally characterized. The results of this research showed how the properties of zirconia vary with thermal treatment. It was observed that the zirconia prepared at a higher temperature had lower surface area, lower pore size and pore volume as compared to the zirconia prepared at a lower temperature. The morphology studies showed the porosity of the zirconia. The results from the absorption experiments showed that zirconia prepared at a higher temperature absorbed more ATP than the zirconia prepared at a lower temperature. Significant changes were also observed on the pellets of zirconia pre and post absorption experiments. I hope that this research sheds more light on the complex properties of zirconia’s surface chemistry and the results of this study could better help in the application and use of zirconia in chromatography to separate proteins.

The production and distribution of counterfeit drugs is a serious worldwide health problem. One recent example is the appearance of fake artesunate antimalarial tablets in Southeast Asia. Due to the malevolent circumstances in which these fakes are produced, concern over the presence of toxic tablet ingredients is very much a legitimate health issue. Therefore, quantification of the amount of active ingredient present in tablets marketed as artesunate, a drug used for the treatment of the multidrug-resistant Plasmodium falciparum malaria in Southeast Asia required liquid chromatography coupled to mass spectrometry (LC-MS). This quantification allows the classification of the tablets as genuine, sub-therapeutic or fake and the validation of field results using colorimetric tests. During the LC-MS experiments, there were the observations that several of the samples contained a “wrong” active ingredient (AI). This was identified via accurate mass measurements, chromatographic retention time and in-source collision-induced dissociation (CID)as erythromycin, a common antibiotic. Using multivariate unsupervised clustering algorithms, the LC-MS data was utilized for “chemically fingerprinting” the fake tablet samples and investigating the similarities between them. The results of this initial survey show a correlation between sample origin, the different types of fake authentication holograms found in the packaging and sample composition.

In order to discover marine bioactive compounds, we investigated the chemical constituents from the organic extracts of soft coral Klyxum molle. Repeated column chromatography on the ethyl acetate extract of soft coral Klyxum molle, resulted in the isolation of nine new eunicellin-type diterpenoids, klymollins V¡ÐZ (1¡Ð5) and klyxumollins A¡ÐD (6¡Ð9), and four known compounds (10¡Ð13). The structures of new compounds were determined on the basis of extensive spectroscopic data (IR, MS, 1D and 2D NMR) and by comparison of the spectral data with those of the related known compounds. The cytotoxicity of compounds 1¡Ð13 was evaluated against five tumor cell lines including CCRF-CEM (human T cell lymphoblast-like cell line), K562 (human erythromyeloblastoid leukemia cell line), Molt 4 (human acute lymphoblastic leukemia cell line), T47D (human ductal breast epithelial tumor cell line) and DLD-1 (human colorectal adenocarcinoma cell line). Compound 2 exhibited weak cytotoxicity toward three cancer cells (CCRF-CEM, Molt 4 and T47D) and compounds 4 and 9 showed weak cytotoxicity against CCRF-CEM cancer cells. Compound 11 was found to exhibit stronger cytotoxicity toward CCRF-CEM cancer cells (ED50 values of 4.2 £gg/mL). On the other hand, compound 3 showed weak activity against the accumulation of the pre-inflammatory cytokine IL-6 in LPS-induced macrophages, at a concentration of 25 £gM.