Fluorescamine

Udenfriend and co-workers showed that phenylacelal-dehyde, which is formed by oxidative decarboxylation of phenylalanine by ninhydrin, reacts with excess of ninhydrin and with a primary amino group to give a fluorescent product [228]. The elucidation of this mechanism and of the structure of the fluorescent reaction product [229] led to the synthesis of fluorescamine [230]. 

4-Dimethylamino-l-naphthylisothiocyanate [224], 4-(benzyloxycarbonylaminomethyl)phenylisothiocyanate [225] and 4-(dimethylaminonaphthalene-l-sulphonyl-amino)phenylisothiocyanate [226, 227] have been introduced in order to improve the sensitivity of the Edman method. These reagents are used in the same way as the other isothiocyanates, and allow end-group determinations in the picomole-range. 

Fluorescamine (4-phenylspiro[furan-2(3H), I -phthalan]-3,3 -dione) is a non-fluorescent compound, with m.p. 

Compounds with nucleophilic functional groups (primary and secondary amines, alcohols, water, etc.) are capable of reacting with fluorescamine, but only primary amines form fluorescent products. Fluorescamine is, therefore, a specific reagent for compounds with primary amino groups. 

The estimation of amines, amino acids, peptides and proteins is carried out by essentially the same procedure 2S2, 233]. Three volumes of a solution of a primary amine, peptide or protein in 0.05 M phosphate buffer, pH ft-9.5 (or a 0.05 M sodium borate buffer, pH 8-9.5) are mixed under vigorous stirring with 1 volume of fluorescamine solution (0.28 mg ml in acetone or 0.56 mg mlin dioxane). Fluorescence measurement is carried out 5—30 min after mixing. Yields of the fluorophore are 80—90%. There is a linear relationship betv/een the concentration of the amine and the observed fluorescence up to 25—30 nmol ml 

---

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). 

Reactions can also occur during chromatographic development. These can either be undesired reactions or planned derivatizations. Thus, Weicker and Brossmer [11] have reported, for example, that hexoses, pentoses and disaccharides can be ammated when ammonia-containing mobile phases are employed on silica gel G layers. On the other hand, fluorescamine or ninhydrin have been added to the... 

Apply indole derivatives dissolved in sodium bo- [105] rate buffer solution (c = 0 2 mol/1, pH 9 0) — ethanol (1 -I-1) Dip TLC plate in fluorescamine solution to just above starting zone (15 s) Then dry at room temperature and develop In case of indole amines followed by spraying with 40% perchloric acid... 

The following are amongst the reagents that have been reported as being added to the mobile phase acids for quinine alkaloids [184], ninhydnn for amino acids [185 — 187], fluorescamine for biogenic amines [188] Fluorescein sodium [189], dichlorofluorescein [190], rhodamine 6G [191], ANS reagent [192] and bromine [193] have all been descnbed as additives to mobile phases... 

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. 

Fluorescamine reacts directly with primary amines to form fluorescent products. Secondary amines yield nonfluorescent derivatives which can be transformed into fluorescent products by a further reaction with primary amines. 

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

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. 

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

The specific detection of aromatic nitro compounds is a second example. These can be converted by reduction to primary amines, which are then diazotized and coupled to yield azo dyes (cf. reagent sequence Titanium(III) chloride — Bratton-Marshall reagent ). Sodium nitrite —naphthol reagent, diazotized sulfanilic acid and other reagents specific for amino groups (e.g. ninhydrin, fluorescamine, DOOB, NBD chloride [9]) can also be used in the second stage of the reaction (Fig. 21). 

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). 

Reagent 2 Spray solution 2 Dissolve 20 mg fluorescamine in 100 ml acetone. 

Fluorescamine reacts directly with primary amines to yield fluorescent derivatives of the general formula 1. On the other hand, secondary amines react in weakly basic medium to yield nonfluorescent derivatives of type II after the hydrolysis of excess fluorescarrtine, these are converted to fluorescent products of type I by reaction with a primary amine, e.g. taurine. 

Note The aromatic amines produced by reduction with SnCl2 in acidic medium can be detected with fluorescamine (after neutralization of the layer by spraying with sodium carbonate) instead of 4-(dimethylamino)-benzaldehyde [5]. 

---