Thin-layer chromatography (TLC) has, due to separation of samples in parallel on a two dimensional silica gel plate, the advantage of enabling an excellent visualisation of the obtained chromatogram. No other chromatographic technique can directly express the result as a colour image and make it available for visual evaluation.
This was one of the most important factors which made TLC a very valuable method in the past. The Direct visual evaluation in TLC requires trained experts for interpretation. Even though such interpretation proved to be effective and able to detect the finest detail, it is always subjective, difficult to report, and lacking reproducibility. So far no automated approach has been found and simple, low level digital imaging has not offered an adequate solution.
Camag, a specialist for instrumental TLC equipment, has now addressed this problem and is providing a solution especially dedicated to TLC image analysis, which delivers reproducible images with a dynamic sensitivity even surpassing the human eye.
A new Digital Imaging System (DigiStore2) is based on a widely proven multi wavelength illumination unit and taking advantage of a new scientific grade CCD camera. The optical setup is kept fully constant offering the advantage of being able to compensate spatial differences in sensitivity. Such compensation can only be performed if a reference is measured with exactly the same settings (focal length and opening of lens) as the actual image. This is not possible with automatic zoom lenses. Fig.1 compares an uncompensated image with a state-of-the-art compensated image, featuring a so called flat-field correction.
Apart from the spatial sensitivity the dynamic linearity is a second important factor. Consumer cameras are usually optimised to reduce the captured highly dynamic image for final best results when printed. Such algorithms act highly nonlinear and enhance the details in darker zones while weakening those in bright areas. This makes TLC images ‘look good’, but on images taken under white light or UV254 using fluorescence indicator, weak spots are completely lost and the detection limit is strongly reduced.
For the new system a scientific approach with linear image data acquisition and high dynamics has been taken. Instead of the commonly used 8bit
(255 non linear intensity levels) a 12bit system (4095 linear intensity levels) was selected. None of the currently available computer monitors can visualise such highly dynamic images, but the additional dynamics can be used to amplify weak zone. This allows dramatic enhancement of the sensitivity of detection. Therefore a Spot Amplification tool with discrete factors has been invented. It allows locating even the smallest fractions present on a TLC plate. Fig.2 shows a scientific 12bit image with weak TLC zones and its amplified version, on which all zones can easily be localised.
DigiStore2 improves scientific image documentation for TLC to a level comparable to the best evaluations performed with the human eye. Aspects of reproducibility and data storage, which were not addressed by pure visual evaluation, are now fully covered. Reproducibility independent of a dedicated expert has been achieved and is setting the base for future image analysis such as quantitative evaluation, pattern recognition and much more.
Dr Matthias Loppacher is Head of Research & Development, CAMAG, Muttenz, Switzerland. www.camag.com