Thin-Layer Chromatography, TLC, HPTLC Error analysis for quantitation chromatographic and sampling error often largest
SummaryThe paper describes the measurement and calculation of the primary statistical errors in the evaluation of TLC or HPTLC using in situ reflectance measurements. The primary errors are the errors of the sample volume spotted onto the plate, errors caused by the chromatographic process itself, the positioning error of the plate with respect to the centre of the light beam and the error in the light measurement. By scanning the same spot, the same track or the same plate under different conditions, the various errors can be determined.When scanning a TLC-plate using a spectrophotometer for quantitative in situ analysis, there will be some errors caused by different parameters. The main sources of errors in scanning are:0" the error in the sample volume spotted to the plate, (sc the error due to chromatographic causes, for example deviation of the plate from the vertical during development and/or the error in migration distance.the error of positioning the spot in the centre of the light beam, and the error in the measurement of the sample spot, for example the error in the peak area using an integrator.Since the error propagation law is only valid for statistical errors, all parameters causing systematic errors e. g. errors due to spots near the solvent front when using a single beam spectrophotometer, and spots near a so-called pfront, have to be omitted from consideration here. For the same reason measurements have been confined to spots which are sufficiently far from the edge of the plate to prevent systematic errors caused by differences in the layer thickness near the edge. The error in the measurement, a' , , can be determined experimentally by repeatedly scanning one track of the developed TLC-plate without changing any of the other relevant parameters. Typical results are shown in Table 1. In this case the peak heights of the recorded peaks were used. From measurements of a large number of different scans and plates, a mean standard deviation of 0.315 mm has been found and all further calculations will be done with this value. The standard deviation must be the same for all peaks, whereas the relative standard deviation depends on the real peak height. It will be noted that in all the tables the calculated approximate standard deviations are given to one more digit than necessary; this is only done to avoid rounding off errors when subtracting the sauares.The error of positioning the spot in the centre of the light beam may now be evaluated. In this case the same track of the TLC-plate has to be repeatedly measured and the light beam has to be repositioned with respect to the first spot each time. While in the first experiment, in which the position of the plate was held constant, the error in the measurement of the spot size was determined, in this case the error in measurement and in positioning will infkence the result, i.e. the error is given by equation (2), and the positioning e...