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Abstract
The HPTLC bioluminescence coupling is a method, which combines the separation ability of HPTLC with biodetection, applying the luminescent bacterium Vibrio fischeri that is also used in a cuvette assay for wastewater analysis according to a European norm method. As a result, a black and white image of the HPTLC plate is obtained, on which bioactive substances appear as dark spots on a brightly luminizing background.
The evaluation of the image was usually done visually and, hence, qualitatively. Currently, available image evaluation programs showed to be unsuitable for quantitative evaluation because of insufficient background correction and/or tedious procedures. Furthermore, special corrections like a horizontal background correction and the recalculation of the sigmoid dose response relationship of the bacteria's reaction are needed. Available programs could not fulfill these requirements.
Therefore, a method was developed by using existing common or freeware programs with which the proper corrections could be accomplished. Steps were the selection of the regions of interest from the HPTLC image, the conversion of the image file into a text file, followed by the main calculation in the spreadsheet program Microsoft Excel. For calculation, adapted versions of the cuvette test calculations were used.
As Excel does not contain the necessary integration tools, two export methods were included. The first method allowed for a routine evaluation of chromatograms giving peak height and area as parameters. The second method enables an in-depth evaluation of chromatograms using an HPLC software leading to parameters like, e.g., signal-to-noise ratio, peak asymmetry, or peak width.
Results obtained with the method were convincing. With a macro bundle, the calculations were not very time consuming and could be applied for routine use.