Liquid-core waveguide technology for coupling column liquid chromatography and Raman spectroscopy

The on-line coupling of liquid chromatography (LC) and Raman spectroscopy (RS) via an entirely plastic liquid-core waveguide (LCW) was optimized in terms of excitation wavelength of the laser, especially in relation to the fluorescence background, and the length of the LCW. Excitation at 632.8 nm (He–Ne laser) was found to be a good compromise between a wavelength long enough to strongly reduce the fluorescence background and, on the other hand, short enough to avoid (re)-absorption of laser light and Raman signals by H 2O in LCWs of considerable length. This conclusion is supported by a theoretical discussion on the optimization of LCW lengths as function of the excitation wavelength for H 2O and 2H 2O. When using the He–Ne laser the optimum length is ∼50 cm for H 2O; this corresponds to a detection cell volume of 19 μl for an LCW of 220 μm I.D., which is fully compatible with conventional-size LC. The influence of an organic modifier, usually necessary for reversed-phase LC, on the free spectral window was evaluated. The potential applicability of LC–LCW-RS was shown for a mixture of adenosine 5′-monophosphate (AMP), guanosine 5′-monophosphate (GMP) and uridine 5′-monophosphate (UMP), utilizing an aqueous eluent without the addition of a modifier. Improved detectability was achieved by using the stopped-flow mode and applying a large-volume-injection procedure (injection volume: 200 μl). Under these conditions, the limit of identification for AMP, GMP and UMP was in the 0.1–0.5-mg/ml range.