Fluorescamine emission

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. 

Glycine was analyzed by the fluorescence of its fluorescamine derivative with excitation at 366 nm and emission at 480 nm (7). A standard working curve prepared simultaneously with the analyte permitted quantitation. 

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. 

In order to improve detection sensitivity, aspartame can also be separated by HPLC as a fluorescent derivative (41). Fluorescamine derivatives are separated on LiChrosorb RP-8 using acetonitrile in 50 mM acetate, buffer, pH 6, 22 78, v/v (53), or on Spherisorb S5 ODS-2 RP using 0.2 M phosphate buffer (pH 9) acetonitrile methanol, 2 1 1, v/v (54). Detection is performed at 397 nm excitation and 482 nm emission (54). 

Method 1 (manual column chromatography). An aliquot portion (20-50 pi) of the column effluent, containing less than 10 nmoles of the peptide or amino acid, is placed in a glass tube (13 X 100 mm) and mixed with 1.85 ml of 0.5 M sodium borate buffer (pH 8.5). A 0.15-ml volume of a fluorescamine solution in acetone (0.03%) is added to the tube which is held on a vortex mixer. Vigorous mixing is continued for a few seconds, and the fluorescence is then measured directly in the tube at 390 nm (excitation) and 475 nm (emission). 

Formation of carbonyl groups causes a decrease in amino group number in protein molecules. Therfore the loss in amino group content is also used as a measure of oxidative protein modification marker. The protein amino group quantitation is most commonly performed employing the fluorescamine assay. Fluorescamine reacts with primary amines to form a fluorescent product (excitation wavelength 390 nm emission at 440-500 nm) (R9). Older methods, such as employing trini-trobenzenesulfonate (H2), are less sensitive and more troublesome. 

Fluorescamine (4-phenylspiro(furan-2-(3H),r-phthalan)3,3 -dione) is also a commonly used fluorescence reagent. It reacts almost instantly and selectively with primary amines, while the excess of the reagent is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction is carried out in aqueous acetone at a pH of about 8-9 and the derivatives can be chromatographed directly. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Two disadvantages of the reagent are its cost and the fact the products are less stable, cannot be stored and should be injected onto the column immediately after formation. Fluorescamine has been employed in the analysis of polyamines, catecholamines and amino acids. 

An older method employing fluorescamine may be used if improved detection limits are required. In this method, fluorescamine (Fig. 1.6) reacts with the primary amine groups of the protein, generating flourophores that can be excited at 390 nm to generate emission at 475 nm. Figure 1.6 also shows calibration curves for a variety of proteins this method is capable of detecting as little as 10-ng total protein.10... 

The original reported use of lasers in CE with fluorescence detection was described by Gassmann et in which laser-induced fluorescence (LIE) was used to distinguish and measure the chiral enantiomers in mixtures of dansylated amino acids. Since that time, lasers have become commonplace in CE systems with fluorescence detection. The most common types of lasers used have been the He-Cd, Ar-ion, and He-Ne lasers, as they are relatively inexpensive and have emission lines that match commonly used fluorescent reagents, such as o-phthalaldehyde (OPA) (325 nm) and fluorescamine (354 nm). 

Methoxy-2-diphenyl-3(2//)-furanone, which is structurally related to fluorescamine, has the same unique properties. The reaction of MDPF with primary amines proceeds rapidly only above pH 9 (Weigele et al, 1973). Much higher reagent concentrations are often required, e.g., 10 mg/ml, in order to obtain nearly quantitative fluorophor formation (Wideman et al, 1978). This requirement is due to the more rapid hydrolysis of MDPF. In contrast with fluorescamine, MDPF dissolved in methanol is as stable and as reactive as when it is dissolved in acetone. The MDPF fluorophors also have excitation and emission maxima at 390 and 475 nm, respectively. Various peptide fluorophors have been found to be stable between pH 2 and 11, and it has been possible to lyophilize the fluorophors without any decomposition. The fluorescence intensity also varies with solvent composition, but not with pH. 

Other fluorogenic reagents are available. o-Phthalaldehyde reacts rapidly with primary amino groups in the presence of 2-mercaptoethanol at alkaline pH and room temperature (Roth, 1971). The excitation and emission maxima are at 340 and 450 nm, respectively. As with fluorescamine and MDPF, o-phthalaldehyde is nonfluorescent. The reagent is water soluble and is dissolved and stored in the aqueous buffer (pH 10.0) used for the reaction. The fluorophors are less stable than those formed with fluorescamine (Mendez and Gavilanes, 1976). However, the use of ethanethiol, rather than 2-mercaptoethanol, for the fluorogenic reaction has been reported to yield fluorophors with a half-life of 2 days in aqueous solution at pH 9.1 (Simons and Johnson, 1977). 

Fluorescamine is employed for more sensitive determination of amino acids by fluorescence spectroscopy. Since it undergoes rapid hydrolysis under aqueous conditions, the reagent is normally dissolved in acetone. It is commonly employed for the detection of amino acids on surfaces, such as thin-layer chromatography (TLC) plates. Amino acids are detected based on emission of light at 470 nm following excitation at 390 nm. Detection limits for amino acids are in the low picomole range. 

Fluorescamine (A) triethylamine (10%, v/v) in dichloromethane (B) fluorescamine (4-phenyl-spi ro[f u ran-2-(3H), 1 -phthalan]-3,3 -dione) (0.05%, m/v) in acetone Plate sprayed with A, air-dried, sprayed with B, air-dried, then sprayed again with A spraying with base is necessary to stabilize and intensify fluorescence Spedlic for amino- and acetamidodeoxy sugars fluorimetric scanning (excitation 390 nm, emission 475 nm) 50-100pmol... 

Residual monomers of epoxy resins (i.e., m-xylylenediamine and bisphenol A diglycidyl ether) were extracted from cured epoxy resins and analyzed on a C g column (A = 275 nm, ex 300 nm, em for m-xylylenediamine) using a complex 20-min 30/70 -> 75/25 acetonitrile/water gradient [1019]. Excellent resolution of bisphenol F, bisphenol A, three bisphenol F diglycidyl ether isomers, and bisphenol A diglycidyl ether resulted. Peak sh s were also excellent. Linear concentration curves from 20 to 1000 pg/L were obtained. The m-xylylenediamine was derivatized with fluorescamine and analyzed with the same column and gradient as for bisphenol (but with different excitation/emission wavelengths) and linear curves from 10-800 pg/L were obtained. 

A simple, rapid fluorimetric assay for reducing sugars involved condensation with hydrazine then fluorescamine, and measurement of fluorescence intensity (excitation at 4(X) nm, emission at 490 nm). ° Glucuronic, galacturonic and gluconic adds, as well as glucono-l,S-lactone and mannono-1,4-lactone were separately determined by measurement of fluorescence intensity of the... 

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