Date of Award
Program or Major
Doctor of Philosophy
Sterling A Tomellini
Detection of some important amino group-containing compounds such as amino acids, aliphatic biogenic amines and aminoglycosides can present difficulties since these compounds do not possess strong fluorophores or chromophores. A novel indirect fluorescence detection method for these compounds has been developed, taking advantage of the fact they form complexes with Cu 2+ ions. The reaction between these compounds and a Cu(II)-L-tryptophan complex (Cu(L-Trp)2) in solution results in the displacement of L-Trp from the complex and an increase in L-Trp fluorescence, since the fluorescence of L-Trp is quenched in the Cu(II) complex. The increase in L-Trp fluorescence is taken as the measure of the presence of these amino group-containing compounds. This detection scheme has been interfaced to a HPLC system, by adding a buffered Cu(L-Trp)2 solution to the column effluent via a postcolumn mixing tee.
The detection system was optimized both theoretically and experimentally, taking into consideration that relevant equilibria existing in the solution that may affect the detection. The pH was found to be the most important detection parameter, affecting detection in several ways, including: (1) Cu(II)-L-Trp complexation, (2) Free L-TrP fluorescence efficiency; and (3) Cu(II)-analyte complexation. Chromatographic experiments were performed to determine the pH which provides the optimum S/N level. Other factors, such as the postcolumn reagent flow rate, and the effect of organic solvent modifier, were also studied.
This approach provides several advantages compared to other methods used to detect these analytes. Detection limits are reasonably good, generally on the order of low pmol injected. Linearity ranges to nmol injected. Our experience also indicates that small postcolumn mixing volumes which introduce minimal postcolumn bandbroadening can be used. Simplicity and robustness are among the other advantages of this approach to analyte detection.
Yang, Min, "A non-derivatization approach to HPLC fluorescence detection for some important amino group-containing compounds based on a ligand displacement reaction" (2000). Doctoral Dissertations. 2151.