In order to separate and identify functional groups of molecules responsible for fluorescence compounds present in biodiesel, a column chromatography coupled with infrared spectroscopy and multivariate analysis was performed. A biodiesel sample was packed in a chromatographic column and the fractions obtained were used to perform the analyses. Before undergoing the separation process, the biodiesel sample was analyzed by light emitting diode (LED)-induced fluorescence and compared its spectrum with β-carotene and soybean oil patterns. The low cost and speed of analysis suggest that this technique can be used in the separation of biodiesel substances. The fluorescence emission spectra allowed identifying molecules such as β-carotene, in which the spectrum of its pattern exhibited fluorescence within a region ranging from 500 to 700 nm and chlorophyll molecules. When soybean oil is excited at around 405 nm, it features a fluorescent emission band within the region of 670 nm, which reveals the presence of chlorophyll. Infrared spectroscopy coupled with principal component analysis allowed to discriminate the fractions and to identify the functional groups of compounds present in the sample.