Instrumentation

A number of different types of scanning spectroden-sitometers are available. Most are now either partially or fully computer-controlled. The parameters such as track length, number of tracks, distance between tracks, slit length and width, scanning wavelength and speed can all be programmed into the computer. Some spectrodensitometers can perform a pre-scanning run to determine the position of maximum absorption for the separated components on the track: this is particularly useful where spot application has been used. After scanning, the spectro-densitometer generates massive amounts of data from all the tracks, including peak height and area and position of zones (start, middle and end), for every component. Usually a chromatogram can be displayed for all tracks. This can be baseline-adjusted and excess noise from the background of the layer can be subtracted. All peaks can be integrated, ready for possible quantification. Although a number of scanning modes are available, such as linear, radial (scanning from the centre for circular chromatograms) and circular scanning around a ring (circular development), by far the most popular is the linear mode, as shown in Figure 1.

Normally, three light sources are used in scanning densitometry: a deuterium lamp (190-400 nm), a tungsten halogen lamp (350-800 nm), and a high pressure mercury vapour or xenon lamp for intense line spectra (254-578 nm), usually required for fluorescence determinations.

Three optical methods (Figure 2) have been used in the construction of scanning densitometers:

Figure 1 Linear scan of individual tracks using a scanning densitometer. Slit length and width, track length and speed of scan are all pre-selected.

1. single wavelength, single beam

2. single wavelength, double beam

3. dual wavelength, single beam

Construction 1 requires little explanation and is the type manufactured by most commercial TLC companies. Construction 2 divides the single beam into two by means of a beam splitter, so that one half scans over the chromatographic zone whilst the other scans over the background. Both beams are detected by matched photomultipliers and the difference in the signal measured. In construction 3, two wavelengths as close together as possible are chosen, such that fluctuations caused by light scattering at the light-absorbing wavelength are compensated for by subtracting the fluctuations at the different wavelength at which there is no absorption by the chromatographic zone.

In fixed-beam spectrodensitometers, the stage holding the TLC plate under the light beam moves at a constant rate, propelled by stepping motors. Where the light beam moves, it does so in a zigzag fashion over the surface of the stationary plate. Usually the zigzag scanners incorporate a curve linearization technique for absorption measurements. This uses the hyperbolic solution in eqn [2].

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

Get My Free Ebook


Post a comment