Method Development – the Key to Success in HPTLC

Method development is a common task for laboratories performing chromatography. Generally methods are sought, which are rapid, fit for purpose, inexpensive, and reliable. Precision and accuracy are not just important during validation for compliance with cGMP but also of concern for the daily analytical routine. Method development, optimization, and validation must be considered as an integrated process.

It starts with suitable sample preparation. Here HPTLC offers an important advantage due to the fact that chromatographic plates are disposable. Matrix does not have to be eluted as in column chromatography. This may simplify the separation. A selective solvent for the extraction of target compounds may be desirable. For qualitative analysis sonication with methanol for 10 min is a good starting point. Different extraction solvents and extraction methods or times can be compared on the same HPTLC plate.

As stationary phase silica gel is most widely used because it can separate many substances classes based on type und number of functional groups. Alternatively reversed and polar bonded phases are also available. Why not try out an amino phase for separation of carbohydrates or phenolic compounds of different conjugation?

One of the most challenging steps in method development is the selection of the appropriate mobile phase. The great choice of solvents and almost unlimited possibilities for combinations offer extreme flexibility but can also be overwhelming. The first step should always be a thorough literature search i.e. in the comprehensive database CCBS (Click on article webpage link).

For method development from scratch a guided trial and error approach can be recommended. More information on this can be found in our brochure “TLC/HPTLC method development“ (CAMAG website > Literature > Catalogue & Brochures) or in textbooks such as (p.182 ff). Whether optimization or method development is performed the HPTLC Vario-System can make finding a suitable mobile phase easier because 6 runs with different solvents can be performed in parallel on a 10x 10 cm plate (picture below).

The possibility of convenient chemical derivatization is another strength of HPTLC. Numerous reagents, specific or non-specific are known. Not only for visualization but also to improve detection limits of quantitative determinations and to introduce chromophoric groups into a molecule, derivatization may be suitable. For a wide range of popular reagents and their use please consult the literature.

Method development is a complex subject, but it is worth taking it seriously. Your routine analytical laboratory will thank you!