Liquid chromatography with alkylammonium formate ionic liquid mobile phases and fluorescence detection

Fluorescence detection of various pharmaceuticals and the amino acid tryptophan (low molecular weight organic compounds) as well as the enzyme lactate dehydrogenase LDH (high molar mass compound) has been studied in aqueous solutions using alkyl ammonium formate ionic liquids as the primary solvent component. It was expected that the high viscosity of such ionic liquid-water mixtures would enhance fluorescence. Pharmaceuticals such as riboflavin and naproxen showed no such enhancement in the presence of ethylammonium formate (EAF) or isopropylammonium formate (IPAF) but the fluorescence of warfarin was substantially enhanced by a factor of 4 with 80% EAF and a factor of 7 with 70% IPAF. However, this improved fluorescence using alkylammonium formates did not seem to be general for other coumarin compounds except for bromadiolone which showed a similar fluorescence enhancement using EAF. Enhancement of tryptophan fluorescence was also seen for both EAF and IPAF. During the reversed phase elution of LDH on a polymeric HPLC column, remarkable enhancements in LDH peak intensity and activity were observed by incorporating 6% PEG 8000 in the organic mobile phase that contained either 20% acetonitrile or IPAF. Using higher concentrations of PEG 8000 is not recommended, not only because of the high viscosity, but also because the stabilizing effect of PEG 8000 is gradually reduced at higher concentrations.