Thin Layer Chromatography - Thin Layer Chromatography Applications
Thin Layer Chromatography Applications
Quantitative analysis by TLC is most frequently carried out by the visual comparison of the intensity of the spots of interest to those of a standard. For example, such a procedure would allow the sample concentration to be compared with that of a legal standard to ensure compliance with regulatory limits. However, for more accurate work, spot scanning techniques must be used. In general, it is the optical properties of the separated material that are used for the quantitative assessment of each thin layer spot. If the spot adsorbs in the visible range of wavelengths, then, either or both reflected and transmitted light can be measured. If, however, the substance in the spot only adsorbs in the UV range of wavelengths, then only reflected light can be measured, as the silica base of the plate itself will adsorb UV light. If the substance fluoresces, then the fluorescent light can be directly measured as emitted light, or, alternatively, if a fluorescent plate is used, the quenching due to presence of the spot can be measured. Unfortunately, there is no simple linear relationship between solute mass and spot transmittance or reflectance, and calibration curves must be obtained using synthetic standards.
The relationship (with the exception of fluorescence) between the measured property and the mass of solute in the spot is obtained by arbitrarily fitting an appropriate function (usually a second or third order polynomial) to a set of calibration data. The calibration curve is then used for subsequent analysis. The calibration data are usually fitted to a polynomial function as these are the simplest and easiest to calculate. However, care must be taken not to use too high an order of polynomial as the improved correlation will usually include fine structure in the curve fit which does not exist in practice and which will often lead to serious errors. To avoid this situation the order of the polynomial should never be greater than one-fifth of the number of data points. For example, to fit a second order polynomial there should be at least 10 calibration points that scan the total range of concentrations that are likely to be analyzed. If a third order polynomial is needed, then there should be at least 15 data points spanning the concentration range. Fluorescence measurements have a number of advantages over transmittance and reflectance as the relationship between emitted light and solute mass is more closely linear.