Derivatizing Reagents for Detection of Organic Compounds By HPLC

Derivatization is the process of chemically modifying a compound to develope a new compound which has properties that are suitable for analysis using HPLC. Derivatization improves the detectability of a target analyte by reaction with suitable derivatizing agent. Derivatization reactions are simple chemical modification of substance that make it compatible with the selected separation method or transforms substance with a low UV- absorption into highly sensitive product. Derivatization reactions in liquid chromatography modify the solutes adding a chomophore for easy UV detection or a fluorophore for sensitive fluorescent detection. The chemical structure of the compound remains same and just modifies the specific functional group for reacting compounds to derivative of deviating chemical and physical properties in order to make them detectable. Introduction of certain elements or groups through chemical derivatization may enhance the detector’s response helpful for the elucidation of structure of analytes. In conclusion, the present review describe various derivatization reagents for pre-column and post column derivatization process in HPLC by UV-visible and fluorescence detection are summarized along with reactions and some practical aspects. The commonly used derivatizing reagents in HPLC are 1-fluoro-2, 4-dinitrobenzene, ninhydrine, 4-N-N-dimethylaminoazobenzene-4’-sulfonyl chloride, benzoyl chloride, phenyl isocyanate for UV-visible detection and o-phthalaldehyde, fluorescamine, 1-dimethylaminonapthalene-5-sulfonyl chloride (DNS-Cl), 9-fluorenylmethyl chloroformate (Fmoc-Cl), benzofurans for Fluorescence detection.