HPLC-grade methanol and distilled or de-ionised water are used to prepare the eluting solvent,which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with minimumwater content of 25% should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory foreluting substances with a log P of 6 within an hour, at a flow rate of 1 ml/min. For substances with a log Pabove 6 it may be necessary to shorten the elution time (and those of the reference compounds) bydecreasing the polarity of the mobile phase or the column length.