Fluorescent derivation reagents

In long-wavelength UV light (2 = 365 nm) carbohydrates, e.g. glucose, fructose and lactose, yield pale blue fluorescent derivatives on a weakly fluorescent background. In situ quantitation can be performed at = 365 nm and 2fi = 546 nm (monochromatic filter M 546) [19]. Further differentiation can be achieved by spraying afterwards with p-anisidine-phosphoric acid reagent [8]. 

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

Another reagent that readily forms fluorescent derivatives with primary amines is o-phthalaldehyde (trade name "Fluoropa"). The reaction proceeds in aqueous solution in the presence of a mercaptan at a pH of 9-11 producing an isoindole. 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Direct use of pyridone sodium salts as fluorescent deriv atizing reagents. 

A third possibility of converting nonfluorescent substances to fluorescent derivatives, without the use of additional reagents apart from the stationary phase on which the separation has been carried out, is to place the plate in a plasma chamber for some time. 

In the first step tin(Il) chloride in acetic acid solution reduces the aromatic nitro groups to amino groups. The aromatic amines produced then react with fluorescamine in weakly basic medium to yield fluorescent derivatives (cf. reagent monograph Fluorescamine Reagent , Volume la). 

Note Primary amines yield fluorescent chromatogram zones even before the application of reagent 3. Secondary amines do not yield fluorescent derivatives until they have been treated with reagent 3. Hence, the reagent sequence allows the stepwise detection of primary and secondary amines. Taurine is preferred as the essential component of reagent 3 over the multiplicity of other possibilities because it produces intense fluorescence it is also not very volatile and is readily available. Amides and substances with peptide linkages, eg. hippuric acid, are not detected, neither are secondary amines that are volatile at high temperatures. 

Only theophylline yields an intensely fluorescent derivative under long-wavelength UV light when treated with chloramine T — sodium hydroxide reagent. The purine derivatives caffeine and theobromine investigated at the same time fluoresce very weakly or not at all. 

The reaction course has not been elucidated (cf. also sodium hydroxide reagent). Hydrolyzation reactions and aromatizations are probably primarily responsible for the formation of colored and fluorescent derivatives. Substituted nitrophenols - e.g. the thiophosphate insecticides — can probably be hydrolyzed to yellow-colored nitro-phenolate anions by sodium hydroxide or possibly react to yield yellow Meisenheimer complexes. Naphthol derivatives with a tendency to form radicals, e.g. 2-naphthyl benzoate, react with hydrolysis to yield violet-colored mesomerically stabilized 1,2-naph-thalenediol radicals. 

REAGENTS FOR THE PRECOLOMN IN LIQUID CHROMTOGRAPHY PREPARATION OF FLUORESCENT DERIVATIVES... 

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

However, most molecules and ions show no fluorescence and so it is necessary to resort to other methods in these cases. Derivatisation methods can be used where the substance of interest is not fluorescent but may be converted into a fluorescent derivative by reaction with a suitable (nonfluorescent) reagent. 

There are potentially viable reagents available that may be employed for the derivatization of compounds either for enhancing UV/visible radiation (called chromatags) or for reaction of non-fluorescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives. 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

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