Electrophoresis microchip with integrated waveguides for simultaneous native UV fluorescence and absorbance detection

Abstract

Simultaneous label-free detection of UV absorbance and native UV-excited fluorescence in an electrophoresis microchip is presented. UV transparent integrated waveguides launch light at a wavelength of 254 nm from a mercury lamp along the length of a 1-mm long detection cell. Transmitted UV light is collected by another waveguide in the opposite end of the detection cell, while visible fluorescence is collected vertically through the lid of the chip. The background of scattered excitation light is suppressed by detection perpendicular to the excitation, the limited UV transparency of the borosilicate lid and by choosing a PMT insensitive to the excitation light. This way, the need for a fluorescence filter is eliminated. Calibration curves were measured for serotonin, tryptophan, propranolol and acetaminophen, and separations of the four compounds were demonstrated by electrophoresis and MEKC. All compounds could be detected in the micromolar range by absorbance detection, but fluorescence detection improved detection limits for compounds displaying native UV fluorescence up to ten times. The simultaneous detection also proved useful for the identification of compounds with similar retention times and even enables accurate quantification of co-eluting compounds.

Advances in Microfluidic Materials, Functions, Integration, and Applications

“…190 In an integrated approach, waveguides formed in a silicon microchip directed excitation light to the detection region and collected transmitted light (Figure 21), allowing analytes separated by μCE and MEKC to be detected by both UV absorbance and native UV-excited fluorescence. 188 …”

“…However, its sensitivity is reduced by the short optical pathlengths commonly encountered in microfluidic channels. 188 Both off- and on-chip UV absorbance formats have been shown in microdevices. A cross geometry fused-silica microchip was used for μCE of toxic alkaloids with UV-absorbance detection.…”

Label‐free analysis in chip electrophoresis applying deep UV fluorescence lifetime detection

“…However, sensitive detection of analytes is more challenging in microchips than in classical separation techniques due to the low sample amounts and technical constraints. UV absorbance detection, commonly applied in classical CE [4], is challenging in chip-based microfluidics [5,6] mainly due to the limited optical path length. Various detection methods have been implemented in MCE, the most common are electrochemical [7][8][9], mass spectrometric [10][11][12] and fluorescence-based [13,14] techniques.…”

An Integrated UV-Vis Microfluidic Device for the Study of Concentrated Solvent Extraction Reactions

“…Tunable thermal lens spectrometry which utilised a Xe lamp as an excitation source and optical cells 1-10 mm long were used. Ohlsson et al [23] used integrated waveguides to detect both UV-Vis absorbance and fluorescence signals in an electrophoretic microchip. Kuswandi et al [24] and Viskari et al [25] have reviewed studies on the use of UV-Vis spectroscopy in microfluidics.…”

“…Kuswandi et al [24] and Viskari et al [25] have reviewed studies on the use of UV-Vis spectroscopy in microfluidics. Generally, the integration of UV-Vis spectroscopy onto microfluidic platforms is difficult since the typical path length is in the order of 10 -4 cm and the intensity of the signal at the detector is weak [23,24]. Thus, methods that differ from traditional UV-Vis spectroscopy and/or strategies to increase the optical path length are often sought [22,[26][27][28][29][30].…”