Publications

Micellar electrokinetic chromatography (MEKC) was successfully coupled to Fourier transform infrared (FTIR) detection, using a micromachined IR-transparent flow cell with an optical path length of 15 μm for the on-line detection of five neutral analytes. Tight connections between the flow cell and the capillaries were achieved by creating a small O-ring of UV-curing epoxy adhesive on the sharply cut capillary ends. The background electrolyte consisted of 15 mM phosphate buffer at pH 7 and 40 mM sodium dodecyl sulfate (SDS). Five analytes (paracetamol, caffeine, p-nitro benzyl alcohol, m-nitrophenol and p-nitrophenol) were successfully separated, yielding detailed IR stack plots that could be used for quantification and identification. Linear calibration graphs were obtained for each individual analyte present in mixtures at concentrations up to 10 mM. The limit of detection (3 S/N) ranged between 1.1 and 1.5 mM (1.2–1.8 ng). Analytes were identified by comparing spectra obtained during the MEKC separation with those resulting from completely filling the capillary with each individual analyte dissolved in the micelle-containing electrolyte. Information on the specific functional groups of all analytes could be elucidated from the spectra. Since FTIR is a nondestructive detection technique, a conventional on-line UV detector was introduced directly after the developed IR flow cell to test the system's performance and to demonstrate that tandem FTIR and UV detection is feasible.

A capillary electrophoresis method for the enantiomeric separation of the tetrapeptide H–Tyr–(d)Ala–Phe–Phe–NH2 (TAPP), has been developed and validated. The preferred background electrolyte (BGE) consisted of 0.1 M aqueous phosphoric acid adjusted to pH 3.0 with triethanolamine. The chiral selectors 18-crown-6-tetracarboxylic acid (18C6H4) and heptakis(2,6-di-O-methyl)-β-cyclodextrin (2,6-DM-β-CD) were compared and the crown ether 18C6H4 was found to be superior. The separation of TAPP and its enantiomer was accomplished within 30 min with a resolution greater than 3.5. The method was then investigated with respect to selectivity, linearity, accuracy, range, precision, detection limit (DL), quantitation limit (QL) and robustness, essentially following International Conference of Harmonisation (ICH) guidelines for the validation of analytical methods. The DL and QL for the TAPP enantiomer were found to be 0.3 and 0.8%, respectively, at the target TAPP concentration of 1 mg/ml. Robustness was tested using a full factorial design for the following four experimental variables varied at two levels: pH of the BGE, chiral selector concentration in the BGE, phosphoric acid concentration in the BGE, and temperature. The method showed good performance with respect to all of the validation parameters, and proved to be robust to changes in the experimental parameters within the tested domain.