Sample application
Drugs are applied to TLC plates as spots or bands with a homogeneous distribution of material within the starting zone. For high-performance layers, starting spot diameters of about 1.0–2.0 mm are desirable, corresponding to a sample volume of 100–200 nL if applied by a dosimeter (micropipette). For conventional TLC plates, sample volumes 5- to 10-fold greater are accept able. Attention should be paid to the solvent used for sample dissolution and application. Desirable properties of the sample solution are summarised in Table 13.6. If scanning densitometry is used for detection, manual sample application with hand-held devices is inadequate. For densitometry, the starting position of each spot must be known accurately, which is achieved easily with mechanical devices that operate to a precise grid mechanism. Also, the sample must be applied to the layer without disturbing the surface, something that is nearly impossible to achieve using manual application.
Sample application devices for TLC encompass a wide range of sophistication and automation. The most popular devices for quantitative TLC use the spray-on technique. A controlled nitrogen-atomiser sprays the sample from a syringe or capillary, to form narrow, homogeneous bands on the plate surface. The plate is moved back and forth under the atomiser on a translational stage to apply bands of any length between zero (spots) and the maximum transit length of the spray head. Bands are typically 0.5 or 1.0 cm in length, with the longer bands used primarily for preparative-scale separations. The rate of sample deposition can be adjusted to accommodate sample solutions of different volatility and viscosity. An advantage of spray on devices is that different volumes of a single standard solution can be applied for calibration purposes and the standard addition method of quantification is carried out easily by over spraying the sample already applied to the layer with a solution of the standard. Fully automated sample applicators can be programmed to select samples from a rack of vials and deposit fixed volumes of the sample, at a controlled rate, to selected positions on the plate. The applicator automatically rinses itself between sample applications and can spot or band a whole plate with different samples and standards without operator intervention.
capillary tip is brought into contact with the plate surface using a mechanical device to discharge its volume. A click-stop grid mechanism is used to provide an even spacing of the samples on the layer and a frame of reference for sample location during scanning densitometry. Layers with a preadsorbent zone (a narrow zone prepared from a silica gel of low surface area with weak retention) simplify some aspects of sample application. This allows relatively large sample volumes or dirty samples to be applied to the preadsorption zone and their reconcentration to a narrow band at the inter face between the preadsorbent and separation zones by a short development prior to chromatography. However, since the distribution of the sample may not be even within the band, the quantitative accuracy of densitometric measurements may be impaired using this approach.
Although manual sample application is not advised for quantification, it is acceptable for qualitative purposes. A variety of glass microcapillaries are available for sample application. In some of these, the microcapillary is calibrated and the use of a thin wire inside the capillary enables positive displacement of the sample to enable quantitative application.
