A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic

Abstract

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This ‘off-column’ flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10³ (reserpine, λ = 220 nm), and a concentration detection limit of 5.1 × 10⁻⁸ mol l⁻¹ were observed. Baseline noise was measured at 3.5 × 10⁻⁵ absorbance units (AU), with a standard deviation of 1.7 × 10⁻⁵ AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50–100 μm, and flow rates from 100 nl min⁻¹ to 1 μl min⁻¹. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.