Amines fluorescamine

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... 

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. 

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. 

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). 

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]. 

For fast reactions (i.e., < 1 min.), open tubular reactors are commonly used. They simply consist of a mixing device and a coiled stainless steel or Teflon capillary tube of narrow bore enclosed in a thermostat. The length of the capillary tube and the flow rate through it control the reaction time. Reagents such as fluorescamine and o-phthalaldehyde are frequently used in this type of system to determine primary amines, amino acids, indoles, hydrazines, etc., in biological and environmental samples. 

Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M., Fluorescamine a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range, Science, 178, 871, 1972. 

Benson, J. R. and Hare, P. E., o-Phthalaldehyde fluorogenic detection of primary amines in the picomole range comparison with fluorescamine and ninhydrin, Proc. Natl. Acad. Sci. U.S.A., 72, 619, 1975. 

Determination of Number of PEG Chains Linked to a Protein Molecule. This parameter was calculated by multiplying total number of amino groups on a particular protein by the fraction of amines that were consumed by the modification, which was measured by TNBS assay (28) or in some instances by fluorescamine assay (29). 

Figure 2 Structure of fluorescamine (FR) and its fluorogenic reaction with primary amines.

The concentration of neomycin in urine has been determined with a fluorimetric method 15. Interfering amines were separated by chromatography on SE Sephadex C-25 before reacting the isolated neomycin with fluorescamine. 

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. 

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. 

Fluorescamine reacts with primary amines to form fluorophores (see Fig. B2.2.4) that are excited at 390 nm and fluoresce at 475 nm. Peptides react with fluorescamine at pH 7.0, giving higher fluorescence than amino acids, which have maximum fluorescence at pH 9. The reaction proceeds rapidly with primary amines at 25°C. The resulting fluorescence is proportional to the amine concentration. The fluorophores are stable for several hours. A negligible interference is produced with ammonia. 

Fluorescamine, or 4-phenylspiro[furan-2(3//),l/-phthalan]-3,3,-dione, is used to introduce a fluorescent label on electroblotted proteins via reaction with free amines. Transferred proteins are visualized on blot transfer membranes with UV light. This stain can be very sensitive and can be used in conjunction with a second detection method such as immunoblotting (also see Basic Protocol 3). However, the protein is irreversibly modified because fluorescamine reacts with available amino groups (i.e., lysines and the protein N terminus if it was not previously blocked). 

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