Fluorescence Detectors

 

Many compounds fluoresce when exposed to light of a specific wavelength and the light emitted can then be sensed and used for detection and quantitative measurement of the substances concerned. The intensity of the fluorescent light is proportional to the intensity of the excitation light and the concentration of the material being excited. Fluorescence detection is more sensitive than light absorption and in addition, can be more selective. As the sensitivity is directly proportional to the intensity of the exciting light, the laser might appear (and sometimes is) the best type of light source to use. Common light sources are the mercury lamps emitting at 365 nm or laser lamps. However, the mercury lamp and the laser can provide no wavelength selectivity and allow the wavelength of the excitation light to be selected such that the maximum light emission is obtained. As a consequence, xenon, and deuterium lamps that give a broad spectrum of light are preferable and if a diode array sensor is also employed a very narrow ranges of wavelengths can be selected for optimum sensitivity Unfortunately there are limited number of compounds that are fluorescent and so derivatization is often necessary to provide fluorescent derivatives to improve the detection limits of a specific substance. There a number of reagents that link specific chemical groups to the solute molecule, e.g. UV chromophores or fluorophores. Each reagent class comprises groups based on the chemical nature of the solutes with which they react (e.g. fluorescent reagents that derivatize acids, alcohols, amino acids, etc.).

 

Two UV absorbing reagents used to derivatize amino groups are benzoyl chloride and m–toluoyl chloride. Both reagents add a benzene ring to the molecule that contributes a strong UV absorbing chromaphore. The p–nitrobenzoyl chloride reagent imparts an even stronger UV chromaphore than the simple aromatic ring and may be preferred for this reason. The reaction is fairly rapid and can take place at room temperature. Pyridine is usually employed to scavenge the hydrochloric acid that is released. Similar reagents are the toluene-sulphonyl chloride and the benzene sulphonyl chloride, which both produce sulfonamides with the amines. As an example, gentamicin, a poly-functional amino compound, can be analyzed after labelling with benzene sulphonyl chloride.  The reaction is completed in 10 minutes at 75˚C. 1-Fluoro-2,4-dinitrobenzene is also used for derivatizing amino compounds (e.g. the amino glycosides such as neomycin, fortimicin, amikacin, tobramicin, gentamicin and sissomicin).

 

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 reagent 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 high quantum efficiencies; 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.

 

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.