Application bandwise

Figure 4 Linomat for sample application bandwise by spray-on technique. The dynamic range is 1-490 pL, the practical range for quantitative analysis is 2 to about 50 pL. With a typical band length of 7 mm with a distance of 3 mm to the next, a 20 x 10 cm HPTLC plate accommodates 18 sample tracks per side.

Band-shaped starting zones give the best possible separation efficiency attainable with the selected TLC system. As an example, Fig. 28 shows the effect of the application method on the separation result. The difference between spotwise and bandwise application can be clearly seen. 

If you have to perform assays by quantitative TLC, you will find fully automatic sample application indispensable. This will at least require the Automatic TLC Sampler (ATS 3) of CAMAG (Fig. 34), in which a PC controls the application and then, in combination with a TLC scanner, the evaluation. The PC also processes the measured data. The samples are applied from a steel capillary, which is connected to a dosing syringe driven by a stepping motor. The samples are applied to precoated layers (on glass or foil), up to the 20 x 20 cm format, either spotwise or bandwise as required. Applica-... 

The measurement consists of a direct photometric evaluation. The TLC plate is scanned lane by lane by monochromatic light of the appropriate wavelength. The measuring slit is adjusted to give a light beam whose dimensions suit the size of the spot. Spots obtained by pointwise application are scaimed over their whole diameter. With bandwise application, which is preferable for quantitative evaluation, an aliquot of 50-75 % is taken from the homogeneous central part of the band. 

Top line and bottom line concentrated plant extract solution applied using a Linomat IV Top line 10- il amounts (a) 4 s/pl pointwise, (b) 15 s/pl pointwise, (c) 4 s/pJ bandwise (8 mm) Bottom line 25-pl amounts bandwise (10 mm) (a) 4 s/pl, (b) 9 s/pl, (c) 15 s/pl Middle line 5-pi amounts of chlorophyll-containing solutions water, acetone, toluene, -hexane, chloroform (hand application)... 

The Automatic TLC Sampler III is the latest version of this kind. It applies samples automatically from a rack of sample vials. The samples are dispensed from a steel capillary, spotwise by contact transfer or bandwise using the spray-on technique. The capillary which delivers the samples moves according to the plotter principle at a lateral speed of 25 cm/s. The sample dispensing speed is selectable between 10 and 1(X)0 nL/s. The application module is controlled by an IBM-compatible PC, normally the same computer that controls densitometric chromatogram evaluation. 

Bandwise application of samples using spray-on technique and modem instrumental applicators, enables application of relatively large volumes of solutions (up to 99 (il). In some instances, it is even necessary to dilute solutions. The opposite situation is seen in the case of spotwise application on HPTLC plates. Recommended volumes for achieving the optimum results should not exceed 0.5 l. To obtain sufficient concentrations, samples may have to be concentrated, but concentrated aqueous solutions usually have relatively high viscosity and surface tension. This prevents the filling and emptying of capillaries caused by capillary forces. Therefore, it is advisable, if possible, to dilute concentrated solutions with methanol. Solid samples should be dissolved in distilled water (pH 5.5), dilute acid or alcohol and water mixtures, and treated as solutions. 

CAMAG Linomat IV (Figure 17) Microprocessor controlled bandwise applicator which optimizes conditions for subsequent densitometric measurement. It has a range of 5-490 pi for preparative separations and 1-99 pi for quantitative application. 

---