Fluorescent reagents, derivatization with chloride

The most common fluorotags (fluorescent reagents) are dansyl chloride and o-phthalaldehyde (OPA). Chromotags include p-bromophenacyl bromide (PBPB) for derivatization of carboxylic acids (K-salts) with a crown ether catalyst, ninhydiin for primary amines forming complexes that have their adsorption maxima at about 570 nm, and dansyl chloride for primary and secondary... 

An excellent and detailed discussion of pre-column and post column derivatization has been given by Lawrence (11) and includes a comprehensive list of available reagents, procedures and application examples. One of the more popular procedures is fluorescence derivatization and probably the most popular fluorescent reagent is dansyl chloride (12). 5-Dimethylamino-naphthalene-l-sulphonyl chloride (dansyl chloride, DNS-chloride or DNS-Cl) reacts with phenols and primary and secondary amines under slightly basic conditions to form a fluorescent sulphonate ester or sulphonamide. The quantum efficiency of dansyl derivatives is high whereas the reagent itself does not fluoresce. 

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. 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

Hsieh et al. [46] described an HPLC method for simultaneous determination of the R-(—) and (S)-(+)-enantiomers of vigabatrin in human serum after precolumn derivatization with a fluorescent chiral reagent (naproxen acyl chloride) and detection at 350 nm with excitation at 230 nm. Separation was achieved on an Agilent Eclipse XDB-C8 (5 /an) column (15 cm x 4.6 mm) using a mixed solvent of acetate buffer (pH 7)-methanol (60 40) as a mobile phase at a flow rate of 1.2 ml/min. The calibration graphs for each enantiomer were linear over the... 

A high degree of sensitivity and selectivity can be obtained with certain biomolecules by the chemical attachment of fluorophores. The most common fluorescent derivatization reagents include fluorescamine, dansyl chloride, pyridoxal, pyridoxal 5-phosphate, dansyl hydrazine, and pyr-idoxamine. Such derivatization procedures can be used to enhance the fluorescence of compounds with low quantum yields as well as impart fluorescent properties to compounds that do not fluoresce naturally. 

When the extracted analytes provide no response on passing through a flow-cell located in a detector connected on-line to the continuous extractor via a dynamic manifold, the extractor outlet can also be connected to a manifold where the extract is merged with a stream of an appropriate reagent to derivatize the analytes as they are extracted, thereby enabling their subsequent on-line determination. This approach has been used to extract selenium from sand and sludge [57], and selenium, arsenic and mercury from coal. In the latter application, on-line derivatization with sodium tetrahydroborate (for selenium and arsenic) and tin chloride (for mercury) allowed the analytes to be determined in a direct manner using the atomic fluorescence technique [46]. 

Chemical derivatization has become a very popular technique for increasing the sensitivity of a specific type of detector to compounds for which it normally exhibits little or no response. An examples of this procedure would be the reaction of an aliphatic alcohol, which contains no UV chromophore, with benzoyl chloride to form the benzyl ester which could then be detected by the UV detector. Derivatization can also be used to permit the use of an alternative type of detector to increase sensitivity. For example an amino acid may exhibit only weak absorption in the UV, but when reacted with a suitable fluorescing reagent, could be detected by means of the fluorescence detector at concentration levels one or two orders of magnitude lower than with the UV detector. It is clear that derivatization procedures can increase significantly the versatility of many detectors. 

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. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

Dansyl chloride was, for a long time, the most widely used fluorogenic derivatization reagent for amino acid determination. However, there are certain problems connected with the dansyl reaction at low ammo acid concentrahons (Neadle and Pollitt, 1965). Since dansyl chloride and its byproducts are fluorescent, serious interference can occur at high sensitivity levels... 

Peroxyoxalate chemiluminescence reactions are analytically important (particularly in HPLC). The most commonly used reagents are bis-(2,4,6-trichlorophenyl)oxalate (TCPO) and bis-(2.4-din-itrophenyl)oxalate (DNPO). Fluorescent compounds (including anthracene, perylene, aminoanthracenes, and aminopyrenes) and suitably derivatized analytes (such as amines, steroids with dansyl chloride thiols with N-[4-(6-dimethylamino-2-benzofuranyl)-phe-nyljmaleimide and catecholamines with fluoresca-mine) can be sensitively detected. 

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