Compact optical detector utilizing light emitting diodes, 50 nL L-shaped silica capillary cell and CCD spectrometer for simultaneous multi-wavelength monitoring of absorbance and fluorescence in microcolumn liquid chromatography

Abstract

Optical detection setup utilizing light emitting diodes (LEDs), 50 nL L-shaped silica capillary detection cell (L-cell), and low-cost CCD spectrometer is described in this work. Experimental configuration can be equipped with two different LEDs for absorbance measurement and other two LEDs for fluorescence excitation. This setup is capable of simultaneous multi-wavelength monitoring of absorbance and fluorescence when light produced by the individual LEDs and light emitted by the fluorescent analytes is resolved in the spectrum outputted by the CCD spectrometer. Effective optical path of the 0.25 μm I. D. L-cell is 1 mm. Absorbance baseline noise is 1 mAU due to use of low-cost and relatively noisy CCD spectrometer and LED drivers. Nevertheless, the setup can detect adenosine 5'-monophosphate down to micromolar concentration. Performance of fluorescence monitoring allows detection of 5·10^-10 M fluorescein when 23 mW 470 nm LED is used for excitation. The dynamic range of absorbance and fluorescence measurement is 867:1 and 1622:1, respectively. Separation of test mixture (alkylbenzenes and polyaromatic hydrocarbons) demonstrate the effective use of the detector for simultaneous absorbance and fluorescence detection with 0.2 × 150 mm packed capillary column. The benefits of the setup are relative simplicity, compact design and the fact that it can be operated without any optical filters, slits, and extremely precise positioning of the optical elements.

Keywords: CCD spectrometer; Light emitting diodes; Microcolumn liquid chromatography; Miniaturised detection cell; Simultaneous absorbance-fluorescence detection.