Optimization of indirect ultraviolet detection in high-performance liquid chromatography and capillary electrophoresis

Abstract

The similarities and differences in indirect UV detection in ion-exchange chromatography (IEC) and capillary zone electrophoresis (CZE) are discussed. In IEC the UV absorption of the buffer is the limiting factor, because of the relative long detection path length of usually 10 mm. The highest signal-to-noise ratios are obtained at low buffer concentrations. As the retention time is also determined by the buffer concentration, the exchange capacity of the stationary phase additionally has to be optimized. In practice, it is optimal to work close to the cut-off of the buffer solution to achieve the highest detection sensitivity. In CZE, the UV absorption of the buffer usually does not cause any problems because of the far shorter optical path length, which is determined by the inside diameter of the capillary. Here, the optimum detection sensitivity can be achieved when working at the absorption maximum of the buffer. Optimization of buffer concentration can follow the CZE requirements, i.e., low heat generation and low electrodispersion. Indirect UV detection in CZE gives surprisingly high detection sensitivity in the ppb range. The independence of signal-to-noise ratio of optical path length derived for IEC, when commonly used eluent concentrations and molar absorptivities are applied, is not valid for CZE. It is borderline case for optical path lengths above 4 mm. For a fast understanding of the influence of all variables in indirect UV detection, a simplified mathematical model was derived. This may be applied to both IEC and CZE measurements.