A method is described for acquiring identifiable Fourier transform infrared (FTIR) spectra of well resolved chromatographic components. The technique involves the combination of high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), each utilizing a different separation mode. In this way, the full identification power of FTIR can be realized for mixture analysis. In this particular work, microcolumn size-exclusion HPLC was followed by conventional silica gel TLC. Subsequently, the infrared spectra of each spot immobilized on the plate were measured by diffuse reflectance spectrometry. Spectral features of the eluates could be readily distinguished when 8 μg of each component were injected into the column. The feasibility of measuring submicrogram amounts of materials directly on a TLC plate was demonstrated.