Capillary scale light emitting diode based multi-reflection absorbance detector

Abstract

We describe a light emitting diode (LED) based multi-reflection capillary scale absorbance detector based on both square and round capillaries and compare their performance with a conventional single-pass on-tube detector. The optical path length is extended by silver coating, the external surface of the capillary. The reflective geometry has been reported to be less prone to artifacts induced by refractive index changes; we do find this to be true. Although the detection volume/illuminated volume is increased some, a multi-reflection cell based on a 180 μm bore capillary with a ∼2-cm long illuminated volume shows over a 50-fold gain in signal-to-noise (S/N) compared to a single-pass on-tube configuration with the same capillary. The limit of detection (LOD) is 4.4 fmol (2.6 pg, 1 μL of 22.0 nM injected dye) BTB under pulseless (pneumatic) flow conditions. The cells behave as multipath devices where the effective path lengths are greater at low absorbance values. In our experiments, where non-coherent light is launched through optical fibers that are large compared to capillary bore dimensions, increase in the effective path length of the cell do not occur in a predictable fashion with the angle of incidence of the light beam. Although the effective path length almost linearly increases with increasing distance between the light entry and exit windows, the absolute values of the effective path lengths are always lower than this physical distance, suggesting that after some passage through the solution, light largely travels through or along the glass wall. Square capillaries have better light transmission and offer some performance advantages. Multi-reflection cells can indeed be of value for sensitive detection in microflow systems.