Quantitative Analysis by TLC

TLC is not the best chromatographic technique for quantitative analysis and, although it can provide quantitative results, the procedure is cumbersome then other chromatographic methods. Furthermore, for accurate work, expensive scanning equipment is required, which, virtually eliminates the cost advantage of the technique.

There are three approaches to quantitative TLC: extraction of the spot with subsequent measurement by spectroscopic or other techniques, comparative separations with visual assessment and finally optical scanning. The first is rarely used due to the difficulties involved in extracting the material from the spot, which often gives poor accuracy.

Comparative Spot Assessment by Visual Estimation

The quantity of material in a TLC spot cannot be estimated visually, as the retinic response of the human eye is not linear and varies widely with the wavelength of the reflected light. Visual estimation is further complicated by the variation of the iris with light intensity. The eye, however, is very sensitive to slight differences in light intensity and therefore can be used very effectively for comparing light intensities. In fact, in the early twentieth century, the majority of trace analyses (e.g. oxygen in boiler water, heavy metal contaminants such as chromium, manganese and cadmium, alkaloids, etc.) were carried out by forming colored derivatives and visually comparing the color with that of standards.

In TLC, a similar procedure is employed. The sample is run in parallel with a series of calibration solutions each containing the solute of interest at different concentrations. The first set of calibration standards can have relatively large concentration intervals, which allows the approximate concentration of the solute of interest to be identified. A second set of standards is then made up that embraces the suspected concentration in smaller intervals and the separation is repeated. By matching the intensity of the spots, the concentration of the sample can be estimated.

The TLC spots cannot be rendered visible by a destructive process (e.g., concentrated sulfuric acid) as such processes are not quantitative. If the solute of interest is colorless, a suitable colored derivative must be formed either before separation or more commonly after the separation by treating the plate with an appropriate reagent. Providing the separation is clean and a suitable colored derivative is employed, the visual comparative procedure can give quantitative results with an accuracy of about +/-10 %. Today, such accuracy is generally considered inadequate and as a result, the visual comparative technique has been largely replaced by instrumental methods of spot density measurement.