| Analytical separations in conjunction with powerful detection methods make possible the quantitation of large and small molecules important in environmental science, food science, pharmaceutical, and medical matrices. Many of these separations utilize instrumental methods such as capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) to alleviate the complexity of these various matrices. Fluorescence, laser-induced fluorescence (LIF), evaporative light scattering detection (ELSD), and mass spectrometry (MS) are detection methods commonly employed in conjunction with these separation strategies. Three distinct projects to develop and exploit such analytical technologies are described. First, few biological molecules are natively fluorescent, so they often must be labeled with a fluorescent tag in order to be detected via LIF. Furthermore, protein separations using CE-LIF may be complicated by protein interactions with the negatively charged surface of the inner capillary wall, arising from the ionization of surface silanol groups. Presented here is a method for quantitation of protein-dye complexes using a self assembled, surfactant-coated capillary and on-column protein labeling with a non-covalent squarylium dye, Red-1c. Second, seasonal sugar levels are relevant to the relationship between winter anthocyanin production and drought stress in angiosperm evergreen species, and so the development of a method for rapid analysis of sugars in seasonal tree leaf tissue is needed. An HPLC-ELSD method was developed for the quantitation of three sugars, glucose, fructose, and sucrose. The method was subjected to standard addition validation and was shown to provide precise and accurate measurement of all three analytes in real samples.;Third, in addition to their many uses as solvents, ionic liquids (IL) have been utilized as highly effective substrates for complexation and subsequent quantitation of anions by electrospray ionization mass spectrometry (ESI-MS) and high performance liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The present work provides an alternative method utilizing capillary electrophoresis coupled with electrospray ionization mass spectrometry (CE-ESI-MS). This method offers advantages over LC-MS in that CE, in general, provides shorter separation times, requires less reagent and solvent, and needs less sample volume. Mono- and divalent anionic analytes, were complexed with di- and trivalent cationic liquids. The complexation reaction was undertaken in a variety of modes, including pre-column, on-column and post-column, and the resulting complexes, which were positively charged and of higher mass than the native analytes, were subsequently analyzed by MS. This method allowed for greater sensitivity than could be achieved by direct detection. |
