Date of Award
Doctor of Philosophy
Kelsey D. Cook
David C. Baker
The utility of mass spectrometry as a method of analysis for polydisperse polymer samples is assessed. Emphasis is placed on characterization by electrospray ionization mass spectrometry (ESI-MS); therefore a fundamental study of aspects affecting the invasiveness of the ESI process was included. The fundamental study used laser-induced fluorescence spectrometry to profile pH changes as droplets evaporate in an electrospray plume by measuring emission spectra of 2( or4 )-[10-( dimethylamino )-3-oxo-3H-benzo[ c ]xanthene-7-yl]benzenedicarboxylic acid (carboxy SNARF®-1 ), a pH-sensitive fluorescent dye. The observed pH changes depend on initial droplet pH and polarity. In some instances, small or negligible changes of pH are· observed, consistent with expected buffering. The pH of initially acidic droplets decreases along the spray axis in both positive- and negative-ion modes, to an extent larger than expected from solvent evaporation. This phenomenon may be a manifestation of droplet cooling, droplet subdivision, and/or heterogeneous charge distribution within the spray plume or within individual ESI droplets. Next, an investigation of the use of Fourier transform methods applied to synthetic polymer ESI-MS spectra was conducted. Fourier transform methods were used to obtain information about the monomer mass, charge distribution, combined end-group mass, and polydispersity in cases where the convolution of molecular weight, charge, and isotope distributions renders spectra otherwise uninterpretable. Charge state information can be used with the original mass spectrum to estimate the sample average molecular weight. In cases where multiple end groups or adducting species are present, the method returns an average end group mass, rather than individual values. The method is tested using simulated and experimental spectra. Successful application requires a reasonable signal-to-noise ratio and spectral digitization rate, but does not require full resolution of contributing peaks. In addition, Fourier transform analysis of simulated ESI-MS spectra of homopolymer mixtures and synthetic copolymers can also be used to obtain information about the monomer mass and monomer ratios from unresolved spectra. Collision-induced dissociation (CID) in the source of an ESI mass spectrometer was employed to characterize putative samples of the ionic terpolymer poly(styrene sulfonate-co-acrylic acid-co-2-acrylamido-2- methylpropane sulfonic acid). Qualitative and semi-quantitative information about the monomer content was quickly obtainable from ESI-MS spectra, and indicated that some samples contained little or none of one or two expected comonomers. For two representative samples, confirmatory nuclear magnetic resonance (NMR) data were acquired. The NMR experiments required sample clean-up (to remove additives) and long acquisition times (up to 720 min) for 13C NMR. Cleanup also improved the ESI-MS results, providing better agreement with the NMR data. However, qualitative and semi-quantitative information was obtainable by ESI-MS (but not by NMR) without the cleanup step. Full quantitation of monomer ratios would require suitable standards, but even without such standards the ESI-MS measurements provide a rapid ( < 1 min) means for differentiating these samples (e.g., for process or quality control). Additionally, CI D was employed for quantitation of monomer ratios in copolymers using monomer and polymer standards. It was observed that differences in fragmentation of samples with different monomer ratios compromise the use of one polymer sample as a standard for assessing the monomer ratio in another at high cone voltage (CV). Use of monomer mixtures as standards is also precluded at high CV due to extensive fragmentation when sampling free MA monomers. Use of a lower CV and consideration of fragments as well as monomer ions leads to better performance. Using monomer mixtures as calibrants at low CV also provides accurate quantitation of the copolymer monomer ratios, provided that the total intensity ratios are used. Comparison of monomer and polymer breakdown diagrams provides the insight needed to determine the appropriate CV for quantitation with either polymer or monomer standards. Similar results are obtained for a second ionic copolymer system (2-acrylamido- 2-methylpropane sulfonic acid -co-acrylic acid). The studies involving in-source depolymerization for co-polymer analysis required assessment of the limit of detection (LOO) for monomers in the absence and presence of polymer. Differences in fragmentation were observed when sampling pure monomer relative to monomer derived from depolymerization. In light of this observation, the dependence of monomer:fragment ratios on the level of monomer contamination from CID in an ESI-MS source were used for assessing monomer contamination. Although the LOO was 2 orders of magnitude worse than that obtained by direct monomer analysis at low-CV, the method may by of diagnostic value when CID is used to monitor monomer ratios. Evaluation of ESI-MS methods was also carried out on more delicate molecules: oligosaccharides derived from hyaluronic acid by action of bovine testicular hyaluronidase (hyaluronate 4-glycanolyrolase, E.C. 184.108.40.206). The sugars were characterized by ESI-MS and matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS). Oligomers with both an odd number and even number of sugar units with molecular masses up to eight kDa were observed in the ESI-MS spectra. However, the generation of odd-numbered oligomers is not consistent with the regiospecificity of the enzyme and with the MALDI-MS results, which indicated even-numbered oligomers exclusively. In addition, a third method of characterization, high-performance anion-exchange chromatography, showed only even-numbered oligomers. Relative intensities of the odd-numbered oligomers in ESI-MS demonstrated a CV dependence, suggesting that the odd-numbered oligomers resulted from collisional activation. In order to achieve results by ESI-MS that mirror results from the other techniques, the CV must be kept low and precisely controlled. This study displays the relative usefulness and possible vulnerabilities of ESI-MS and MALDI-MS when utilized for carbohydrate analysis without corroborating data from other methods of analysis. Finally, the Thauera strain MZ1T exopolysaccharide abundance in an industrial wastewater treatment facility was positively correlated to episodes of poor dewaterability of the activated sludge. In order to begin to understand the potential role of Thauera strain MZ1T exopolysaccharide in viscous bulking episodes and poor dewaterability of activated sludge wastes, its glycosyl composition was determined. GC-MS analysis of the alditol acetate and trimethylsilyl methyl glycoside derivatives of an approximately 260KDa exopolysaccharide (EPS) extracted from cell-free culture supernatants of Thauera strain MZ1T grown on minimal medium revealed that the extracted EPS was composed of five monosaccharides including: rhamnose, galacturonic acid, glucose, N-acetyl-glucosamine, and N-acetyl-fucosamine. Spectroscopic analyses by FTIR and NMR further revealed the presence of esterified component groups on the polymer. This study-confirms that the EPS contains a rare combination of monosaccharides, which have been hypothesized to exist in Zoog/oea-like organisms but never shown.
Prebyl, Benjamin Scott, "Characterization of polydisperse polymer systems via mass spectrometry. " PhD diss., University of Tennessee, 2003.