Carboxylic acids are common in naturally occurring compounds (e.g. fatty acids, prostaglandins, bile acids etc.) all of which have weak UV and visible absorption. Reagents introducing UV chromaphores into such compounds are phenacyl bromide and naphthacyl bromide. Benzoyl chloride, m-toluoyl chloride and p–nitrobenzoyl chloride are also used to derivatize compounds with hydroxy groups but, 3,5-dinitrobenzoyl chloride introduces a really strong UV chromaphore. The two common reagents for derivatizing carbonyl groups are 2,4-dinitropheny-lhydrazine and p–nitrobenzyl-hydoxylamine. These reagents are commonly used to derivatize aldehydes, ketones, ketosteroids and sugars. p–Nitrobenzyl-hydoxylamine has been used successfully to insert UV chromaphores into prostaglandins extracted from biological samples.
A popular fluorescent reagents is 5-dimethyl aminonaphthalene-1-sulphonyl chloride (dansyl chloride, DNS-chloride or DNS-Cl). Dansyl chloride reacts with phenols and primary and secondary amines, under slightly basic conditions, to form a fluorescent sulfonate ester or sulfonamide. Dansyl derivatives have a high quantum efficiency; whereas the reagent itself does not fluoresce. Unfortunately, the hydrolysis product, dansylic acid, is also very fluorescent causing interference with water-soluble derivatives. The detection limits of the dansyl derivatives are about 1 x 10-9 g/ml and the excitation and emission maxima vary between 350-370 nm and 490-540 respectively. This reagent has been used successfully in the analysis of amino acids, alkaloids, barbiturates and pesticides.
4-Chloro-7-nitrobenz-2,1,3-oxadiazole (NBD chloride) reacts with aliphatic primary and secondary amines to form highly fluorescent derivatives. Aromatic amines, phenols and thiols yield weakly or non-fluorescent derivatives; consequently, the reagent is somewhat specific to aliphatic amines. The reaction proceeds under basic conditions and the products are extractable from aqueous mixtures with solvents such as benzene or ethyl acetate. The fluorescence can be significantly reduced by the presence of water, thus, the solution should be dry and so the reagent can not be used to form derivatives for reversed phase chromatography. Detection limits are 2–5 x 10-10 g/ml. The advantage of this reagent over dansyl chloride is that both the reagent and its hydrolysis products are not fluorescent. The excitation and emission wavelengths are also higher (480 nm excitation and 530 nm emission). NBD chloride derivatives have been used for the analysis of amino acids, amphetamines, alkaloids and nitrosamines.
Fluorescamine (4-phenylspiro(furan-2-(3H),1'-phthalan)3,3'-dione) is another fluorescence reagent that reacts almost instantly and selectively with primary amines, while the reagent excess is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction proceeds in aqueous acetone at a pH of about 8–9 and the derivatives can be chromatographed directly. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Fluorescamine has been employed in the analysis of polyamines, catecholamines and amino acids.
An alternative to fluorescamine is o-phthaldehyde (OPT) and the derivatives are more stable and can be stored overnight. It is used in a similar manner to fluorescamine the detection limits being 4 x 10-10 g/ml. OPT has been used in the analysis of dopamine, catecholamines and histamines. Other fluorescence reagents include 4-bromoethyl-7-methoxycoumarin, diphenylindene, sulphonyl chloride, dansyl-hydrazine and a number of fluorescent isocyanates. For further information on GC derivatizing reagents the reader is strongly recommended to refer to the Handbook of Derivatives for Chromatography edited by Blau and Halket [16].
