Thin Layer Chromatography - Summary 1
The most popular and inexpensive method for quantitatively evaluating a thin layer plate is by simple 'spot matching' using the naked eye. The sample is separated together with a standard (or set of standards) alongside. After the separation is complete, the intensity of the spot of interest is compared with the intensity of the standard spot(s). A more accurate method is to scan the spot(s) by measuring the intensity of the reflected light, employing incident light of the appropriate wavelength or by fluorescence. If the substances of interest are not naturally fluorescent then fluorescent derivatives can be formed. Scanned chromatograms can be evaluated by using peak heights, peak areas or sliced areas. Sulfamethazine has been assayed in animal tissue using the visual comparative technique at concentration levels in the original sample as low as 50 ppb. Thin layer chromatography has also been employed to determine aflatoxins in palm oils and kernels at levels of 1-2 mg/kg. Although a somewhat lengthy procedure, an extraction efficiency in excess of 90% was easily achieved. In a similar analysis that involved the determination of phospholipids in human tears, the extracted phospholipids were hydrolyzed by acetylhydrolase to form 1-hexade-canoyl-sn-glycero-3-phosphocholine and then sprayed with the Mo-MG reagent to produce the intense blue-green spot of molybdo-phosphate malachite green. If the plate is scanned at the optimum wavelength, concentrations of 2-100 pmol were measurable with an RSD of about 2%. A hybrid thin layer plate (a plate half covered with silica gel and half with a laser desorption matrix) has been successfully employed as a transport medium for a laser desorption sampling system of a mass spectrometer. After separation in one direction, the spots on the plate are transferred to the section coated with the desorption matrix by transverse elution with another solvent The plate is then dried and then inserted into the mass spectrometer sampling system. The sensitivity that was attained ranged from 90 fmol to 6 pmol. Thin layer plates can also be scanned by a digital imaging camera and the resulting image passed to a computer and the analysis processed by suitable software. Calibration curves using this procedure are close to linear but there appears to be little difference between the precision and repeatability obtained by this method and that obtained by conventional scanning.