Fluorescamine reagent Fluorescence

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). 

Sulfonamides are used for the prevention and treatment of human infectious diseases and also as growth promoters in swine. It is necessary to monitor pork products for drug residues of sulfonamides. Various sulfonamides were separated on Whatman LKCigF layers in a methanol-0.5 M NaCl (50 50) mobile phase spots were detected with UV light (366 nm) after spraying with fluorescamine reagent followed by 0.5% triethanolamine in chloroform to stabilize fluorescence. The Rp values of the sulfonamides were as follows sulfabromomethazine, 0.19 sulfadimethoxine, 0.39 sulfamethazine, 0.59 sulfathiazole, 0.76 sulfaguanidine, 0.90 (Whatman TLC Technical Series, Volume 3). 

Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),l -phthalan]-3,3 -dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compoimds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm. 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Note If netilmicin is to be chromatographed alone it is recommended that the methanol content of the mobile phase be increased (e.g. to 23 -I- 7), in order to increase the value of the hRf. The detection limit for the substances in the application tested was more sensitive using DOOB reagent on RP layers than when NBD chloride, fluorescamine or o-phthalaldehyde were employed. The derivatives so formed were stable and still fluoresced after several weeks if they were stored in the dark. 

Another commercially available fluorescing reagent, "Fluorescamine", (4-phenylspiro(furan-2-(3H),T-phthalan)3,3 -dione) reacts directly with a primary amine in aqueous acetone at a pH 8-9. 

In some situations, the direct attachment of a large group (such as fluorescamine) to a biologically active substrate can reduce activity. This is due to steric hindrance which can cause a change in conformation or physically block an active site. This condition can be obviated in many cases by attaching the bulky moiety to a spacer arm composed of two or more methylene groups. To this end, a variant of F-D was also synthesized in which a spacer arm of beta-alanine was inserted between the fluorescent and chemically reactive moieties of the reagent. 

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. 

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Latent fingerprints on paper have been revealed by combining the amino acids present with reagents such as ninhydrin (see 37), dansyl chloride (92), fluorescamine (154), 4-chloro-7-nitrobenzofurazan (127a) and o-phthalaldehyde (see reaction 7). To avoid some problems encountered with these reagents it was proposed to use 1,8-diazafluorenone (155), leading to the formation of highly fluorescent ylides (156)349. 

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. 

Whenever only primary amines need to be derivatized, fluorescamine often constitutes the reagent of choice. Fluorescamine, although nonfluorescent itself, can react with primary amines forming highly fluorescent pyrrolinones (139-144). Aliphatic primary amines favor derivatization reaction at pH 8-9, whereas primary aromatic amines exhibit optimal reactivity at pH 3-4. Secondary amines are also fully reactive with fluorescamine but their products do not fluoresce. However, secondary amines can be detected with fluorescamine if they are converted to primary amines by oxidation with N-chlorosuccinimide prior to their fluorescamine derivatization (145, 146). Alcohols can also interact with fluorescamine but this reaction is reversible as a result, alcohols just slow down the reaction rate of fluorescamine with primary amines. On the other hand, tertiary amines and guanidines are not reactive at all with fluorescamine. 

Fluorometric detection has also been employed for the determination of sulfonamides in edible animal products, because it confers the advantages of selectivity and sensitivity. Although sulfonamides possess weak native fluorescence, their sensitive lluorometric detection necessitates use of precolumn or postcolumn derivatization producing the corresponding fluorescent derivatives. The most commonly used derivatizing reagent for precolumn derivatization is fluorescamine (217, 228, 230, 238, 239), while for postcolumn derivatization fluorescamine (231), and o-phthalaldehyde (OPA), and -mercaptoethanol (219) are most often used. 

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

Fluorescamine reacts instantaneously with the primary amino groups of peptides, yielding a fluorescent product with an excitation peak at 390 nm and an emission band at 475 nm. It is insoluble in water and is usually prepared in acetone. Neither the reagent nor the degradation products of the excess reagent in an aqueous medium are fluorescent, which is a great advantage, particularly when postcolumn derivatization is used. 

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). 

The use of chemiluminescence reactions for the detection of metal ions by liquid chromatography was recently reported [59,60]. The detectors made use of the chemiluminescence produced in the reaction between luminol and hydrogen peroxide which is catalyzed by transition metals. The column effluent was mixed with the reagents in order to yield the chemiluminescence. The reaction was fast and was carried out at room temperature. By varying the pH of the buffer, selectivity towards certain metals was also achieved. For example, at pH 10-11 nickel could be analyzed but lead and aluminium were inactive at pH 13-14, the converse was true [59]. Aminco-Bowman has marketed a liquid chromatographic system in which amino acids and amines are analyzed by means of the fluorescence produced on reaction with the reagent fluorescamine. Fluorescamine does not fluoresce, but it does react with primary amino groups to produce fluorescent derivatives. The reaction is instantaneous and may be carried out at room temperature, usually at pH 9. This detection system promises to be far more sensitive than the ninhydrin detection system and is much more easily adapted to HPLC. 

Fig.4.41. The reaction of fluorescamine (Fluram ) with primary amines 1 = reagent II - amine III = fluorescent product.

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. 

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