A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic.
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 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter 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.
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
H
H
Wang
E C
EC
Yi
C A
CA
Ibarra
M
M
Hackett
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005336:Fiber Optic Technology; D055100:Optical Fibers; D012110:Reserpine / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet
D012110:Reserpine