Phenol fluorescent derivatives

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. 

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. 

This fluorescent acid chloride can be used to form derivatives of alcohols, amines, and phenols. Using these fluorescent derivatives, an analysis of a series of n-alcohols from Ci to C4 was developed. A chromatogram produced by this technique is shown in Figure 3. Derivatives were also formed from ammonia, dimethylamine, and phenol. A derivative was formed from pentachlorophenol but was not fully characterized. The quantum yields of fluorescence of the alcohol derivatives of V were lower than those of the alkyl halide derivatives of III. 

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. 

Dansyl Chloride (afluorotag) is invariably used to obtain fluorescent derivatives of proteins, amines and phenolic compounds, the excitation and emission wavelengths being 335 to 365 nm and 520 nm respectively. 

Chloro-7-nitrobenz-2,l,3-oxadiazole (NBD-C1) reacts with primary and secondary aliphatic amines to produce intensely fluorescent derivatives. Although anilines, phenols and thiols react with NBD-C1, they produce derivatives which do not fluoresce or which are only weakly fluorescent. This limits the types of compounds which can be analyzed with this reagent and thus makes the technique more selective. This selectivity is desirable in trace analysis because of a lower degree of interference from co-extractives. The general reaction scheme for formation of the fluorescent amine derivatives is shown in Fig.4.50. 

Several alkaloids of the phenylethylamine, morphine and Ipecacuanha classes can be detected directly with DNS-C1 [121-124]. This reagent reacts with the primary and secondary amino groups or phenolic hydroxyl groups of the alkaloids to form highly fluorescent derivatives, nanogram amounts of which may be detected after chromatographic separation. 

Chlorpromazine and many of its metabolites react with DNS-C1 to form fluorescent derivatives [135,136]. The reaction involves dansylation of the amino and/or the phenol groups of the molecules. The metabolites which form DNS derivatives are listed in Table 4.24. 

Used for the precolumn preparation of fluorescent derivatives of primary and secondary amines, phenols, amino acids, and imidazoles Reference 10... 

N-Methylcarbamate insecticides have been labelled with dansyl chloride (28). The procedure involves hydrolysis with aqueous base to form a phenol and methylamine. The reagent reacts rapidly with both compounds and the newly formed fluorescent derivatives can then be applied and separated by tic (29, 30). A typical reaction scheme is shown in Figure 7. Detection limits are good ( 1 ng per spot) and dansylation of pesticides can be accomplished with extracts from water and soil samples (31J. ... 

In a study of the analysis of organophosphorus pesticides, fenchlorphos, cruformate and fenthion were hydrolyzed to the corresponding phenols which were reacted with dansyl chloride to form fluorescent derivatives. Separation of the three derivaties was achieved by adsorption chromatography on silica. Separation of the cis and trans isomers of three organophosphorus pesticides, dimethyl-... 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

Reaction with 7-Nitrobenzofurazan 9 Fluorescent derivative (phenols)... 

Phenols, thiols 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) Formation of fluorescent derivatives... 

Analytes containing hydroxyl groups, such as phenols, glycols, and alcohols, can be converted with 3.5-dinitrobenzoyl chloride [276] or dabsyl chloride [277] - [279] into compounds that ab.sorb UV or visible light. Fluorescent derivatives can be obtained with 7-(chlorocarbonyl) methoxy-4-methylcoumarin [280]. 

Fluorescence detectors are based on filter-fluorimeters or spectrofluori-meters. They are more selective and can be up to three orders of magnitude more sensitive than UV absorbance detectors. The detector responds selectively to naturally fluorescing solutes such as polynuclear aromatics, quinolines, steroids and alkaloids, and to fluorescing derivatives of amines, amino acids and phenols with fluorogenic reagents such as dansyl chloride (5-(dimethylamino)-l-naphthalene sulfonic acid). 

We have also investigated other oxalate esters as a potential means to improve the efficiency. The most commonly used oxalates are the 2,4,6-trichlorophenyl (TCPO) and 2,4-dinitrophenyl (DNPO) oxalates. Both have severe drawbacks namely, their low solubility in aqueous and mixed aqueous solvents and quenching of the acceptor fluorescence. To achieve better solubility and avoid the quenching features of the esters and their phenolic products, we turned to difluorophenyl oxalate (DFPO) derivatives 5 and 6 (Figure 14). Both the 2,4- and the 2,6-difluoro esters were readily synthesized and were shown to be active precursors to DPA chemiluminescence. In fact, the overall efficiency of the 2,6-difluorophenyl oxalate 5 is higher than for TCPO in the chemical excitation of DPA under the conditions outlined earlier. Several other symmetrical and unsymmet-rical esters were also synthesized, but all were less efficient than either TCPO or 2,6-DFPO (Figure 14). 

The fluorescent signal will change with variation in quantum yield of fluorescence and with molar absorptivity. Not only do fluorescence quantum yields vary with the different dansyl derivatives formed, but so do the molar absorptivities (12). Another problem is exemplified by the 30-nm difference in the emission maxima of the dansyl derivatives of phenol and 2,4,5-trichlorophenol (13). 

Dansyl chloride that exhibits a blue intrinsic fluorescence, reacts with many amines and phenols to yield derivatives with fluorescence of another color. 

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