Thin Layer Chromatography - Scanning Densitometry 5


CAMAG provides three light sources from which to choose: a deuterium lamp generating light having wavelengths from 190 to 400 nm, a tungsten filament lamp providing light having wavelengths from 350 to 800 nm and a low pressure mercury vapor lamp, which provides high intensity line emissions at wavelengths 254 nm and 578 nm. Light from the lamp light source passes through a lens that focuses it through a slit and onto a diffraction grating. Light from the diffraction grating of the selected wavelength is reflected by a plane mirror through a selectable slit, and thence to another plane mirror and then onto a half silvered mirror. Half the light intensity is taken from the half silvered mirror to a reference photocell and the other half passes to the TLC plate. The light reflected from the plate is monitored by another photocell, aligned at 30˚ to the normal of the plate and the transmitted light is monitored by yet another photocell placed directly under the plate. The stage is driven by stepping motors in both the (x) and (y) directions and the reproducibility of positioning the monitor is claimed to be ±50 mm in the (y) direction and ±100 mm in the (x) direction at a maximum scanning speed of 150 mm/sec. An example of the results from a scan of a thin layer plate employing light of 220 nm is depicted in figure 21. The double beam scanner is basically two such optical systems that allow two parts of the plate to be scanned simultaneously.


Courtesy of CAMAG Inc.

Figure 21. The Analog Calibration Curve for a Series of Sulfonamides Produced by Scanning a TLC Plate with the CAMAG Scanner


The scanning time is seen to be over 80 minutes and the measuring points were averages from consecutive sample pairs. Nevertheless, a very respectable chromatogram is achieved from which quantitative data can be readily obtained. An example of the flexibility of multi-wavelength scanning is shown clearly in figure 22, where the separation of some sulfonamides and antibiotics is depicted, having been scanned over a series of different wavelengths. The wavelengths had been previously identified as optimum for certain of the components (i.e. each component gave a maximum response to light of the chosen wavelength). The optimum wavelengths proved to be in the UV range, viz., 254 nm, 365 nm, 302 nm, 313 nm and 366 nm. This presentation is similar to that produced by a diode array UV-LC detector.