Methodology of Sample Application:
The amount of sample applied in HPTLC is much smaller, classical volumes being 100-200 nL along with a compact diameter of 1.0-1.5 mm. Number of samples might be increased because of its compact size. After developing the plate up to a distance of 3 to 6 cm, compact separated spots are obtained which may have 10 times better detection limits than the conventional TLC.
Application of the sample on the plate is a critical process in HPTLC. A convenient spotting device used is platinum-iridium capillary of 100-200 nL whose tip is polished to gives a smooth, planar surface of area 0.05 mm2. It is used with a mechanical applicator which minimizes damage to the plate surface. Quantitative analysis requires scrapping of the spot followed by dissolution or direct scanning. In order to shorten the process, solid phase extraction column is used to concentrate the sample, followed through automatic sampling device so that multiple samples may be processed in a short period and highly reproducible results are obtained.