Choice and optimization of the mobile phase

The best mobile phase strength for a specific separation problem can be determined by thin-layer chromatography (TLC). It is represented by that solvent or solvent mixture which gives an value of about 0.3 in the thin-layer chromatogram. TLC results are readily transferable to HPLC and k values can be predicted if the stationary phase in both methods is the same product, albeit with a difference in particle size. A developing distance of 5 cm is sufficient for this test, so it can be carried out on small TLC plates in a very short time. Small sealable jam-jars make good developing tanks. 

By this TLC test the correct strength (c ) can be found but the selectivity may need optimization as individual peaks may remain only partly resolved. 

In order to obtain selectivity changes it is necessary to choose solvents which differ in their localization and basicity. In many cases the mobile phase consists of two solvents, A and B. The usual A solvent is hexane which has no strength, localization or basicity. For B it is best to use either a non-localizing, a non-basic localizing, or a basic localizing solvent. For systematic selectivity tests and maximum changes in elution pattern, the separation should be tried with all these types of B solvents. 

Typical non-basic localizing B solvents are acetonitrile and ethyl acetate. Acetonitrile is only slightly miscible with hexane ethyl acetate has a high UV cutoff of 260 nm. 

A typical basic localizing B solvent is tert. butylmethyl ether. 

Thin-Layer Chromatography, Hiithig, Heidelberg, 1986, chapter X Transfer of TLC Separations to 

---