Topics - Derivatization
Derivatization, prior to chromatographic analysis, can be used for a number of reasons in sample preparation. Volatile derivatives of involatile substances can be prepared to render them amenable to separation by gas chromatography. In liquid chromatography, fluorescent derivatives can be prepared to render the substances specifically detectable at high sensitivity. To render involatile substances volatile for GC analysis, organic acids can be esterified using boron trifluoride as a catalyst or directly with diazomethane. Sometimes, the polarity of a solute needs to be drastically reduced to improve its chromatographic behavior and reduce tailing. Polarity reduction can often be achieved for amino, hydroxyl and thiol groups by acylation. Acylation is simply achieved by direct reaction with acid anhydrides and acid chlorides (e.g. acetyl chloride and acetic anhydride). In LC analyses, UV chromaphores and fluorophores are often introduced into sample molecules to increase their sensitivity to UV absorption and fluorescence detection. Benzoyl chloride, m-toluol chloride and p-nitrobenzoyl chloride are reagents that can add a benzene ring to a solute molecule and render it UV absorbing. To introduce UV chromophores into a solute containing a carbonyl group, 3,5-dinitrophenylhydrazine and p-nitrobenzylhydroxylamine are probably the two most common and effective reagents. To prepare fluorescent derivatives of phenols, and primary and secondary amines, dansyl chloride (5-dimethyl aminonaphthalene-1-sulphonyl chloride) is strongly recommended. Another fluorescent derivative is 4-chloro-7-nitrobenz-2,1,3-oxadiazole (NBD chloride) which provides highly fluorescent derivatives of primary and secondary amines but aromatic amines, phenols and thiols only yield weakly or non fluorescent derivatives.