The addition of an ionic liquid into the mobile phase appeared to be useful in optimization of chromatographic separation of peptides. Different behavior of peptides in thin-layer chromatography (TLC) was observed after addition of 1-ethyl-3-methylimidazolium tetraﬂuoroborate to the eluent in comparison to the system without the ionic liquid. Nonlinear dependence of the retention coefﬁcient, RM, of peptides on the volume percentage of acetonitrile in the eluent was found in normal-phase TLC with and without immidazolium tetraﬂuoroborate in the mobile phase. In general, RM increased with increasing concentration of acetonitrile. In TLC systems without the ionic liquid, RM can be described well with a quadratic function. On the other hand, in a TLC system with an ionic liquid as the additive to the mobile phase, the retention behavior is better described with a third-degree polynomial function. The potential usefulness of ionic liquids for optimization of separation of peptides was demonstrated. Optimization of the separation conditions was supported by a commercially available computer program. Copyright © 2004 John Wiley & Sons, Ltd.