The separation of color pigments of chestnut sawdust was carried out on various direct (silica, alumina, and diatomaceous earth) and reversed-phase (RP) (silica, alumina and diatomaceous earth impregnated with paraffin oil) TLC layers, and the plates were evaluated by a dual wavelength TLC scanner. The mean fraction was tentatively identified by on-line and off-line TLC-FTIR. The titration curve and strong binding of pigments to alumina indicated that each pigment fraction is of acidic character. Pigments showed anomalous retention behavior under RP-TLC conditions; retention decreased with increasing concentration of organic modifier in the mobile phase.The anomaly was tentatively explained by the suppression of dissociation of the highly polar groups of pigments, resulting in enhanced apparent lipophilicity. Multi-step gradient elution sepa-rated pigments on both silica and impregnated silica layers, the separation being better under RP conditions. Only the presence of a carbonyl group in the main fraction was verified by on-line FTIR, because the method requires a higher concentration of solutes than the TLC scanner did. Off-line FTIR spectra suggested that each pigment fraction contains -OH and -COO groups, and that they are probably tannic acid like compounds.