As the general pharmacopoeial chapters on HPTLC were only recently published, most of the literature methods from before 2015/2017 are not harmonized with those chapters, and thus, may not be considered “standard HPTLC methods”.
Nevertheless, all TLC or HPTLC methods can be converted into standardized HPTLC methods. In a preliminary experiment, the standard and sample solutions of the original method are kept as well as developing solvent and derivatization reagent. All other parameters are set to “standard”:
- Plate material (HPTLC Silica gel 60 F254 20×10 cm);
- Plate layout (15 tracks, 8 mm bands, 8 mm from lower edge, 20 mm from left and right edges);
- Twin Trough chamber, saturated for 20 minutes with saturation pad;
- Plate activation (plate conditioned at a relative humidity of 33% for 10 minutes);
- Developing distance (7 cm from the lower edge);
- Drying step after development (5 min with cold air);
- Derivatization (dipping or automatic spray whenever possible);
- Digital documentation in short-wave UV light, long-wave UV light, and white light before derivatization, long-wave UV light, and white light after derivatization.
Other parameters can be adjusted: usually, application volumes of TLC methods are reduced to about one-fifth for HPTLC plates. Optimum application volumes for HPTLC are between 2 and 10 µL.
The preparation and use of derivatization reagents should follow the pharmacopoeias or the General SOPs of the HPTLC Association. Special derivatization reagents may be evaluated.
Sample preparation may be optimized if needed and simplified if possible. Cumbersome methods and harmful solvents should be avoided. A system suitability test (SST) (e.g., based on the Universal HPTLC Mix) must be introduced.
The following documents may be helpful do summarize all important parameters:
References: