It had been ascertained that in the use of microbore liquid chromatography (LC) and on-column focusing (peak compression), there were advantages to be had in using the strongest eluting injection solvent that would still bring about on-column focusing. In such cases, it was found that the scope for the use of on-column focusing with microbore LC was greater than previously imagined. It has been possible to explain these results by considering on-column focusing to take place in two distinct phases, i.e. focusing of the sample band due to the injection solvent, followed by further focusing of the sample band by the sample solvent/mobile phase step gradient. In this content, an equation has been developed which provides a much more accurate estimate of the maximum allowable injection volume for a given injection solvent and mobile phase than has been possible until now.