Fluorescamine fluorescence detection

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). 

Bis-derivitisation with fluorescamine and HPLC with fluorescence detection Aqueous simulants — HPLC with fluorescence detection. Olive oil extracted with methanol /water after addition of hexane Aqueous simulants — HPLC with UV detection. Olive oil extracted with 80% aqueous acetonitrile Headspace GC with FID detection and pentane internal standard Aqueous simulants GC. Olive oil extracted with water/ethanol and GC detection... 

The HPLC methods mainly use UV detectors, but one uses ampero-metric (18) and one uses fluorescent detection (25), Fluorescent detection after derivatization with fluorescamine is the method most commonly used for detection on TLC plates, Vilim (24) used TLC to... 

A less costly alternative to fluorescamine is o-phthaldehyde (OPT), the derivatives of which are more stable and consequently can be stored overnight if necessary. It is used in a similar manner to fluorescamine the detection limits being about 0.1 ng ca. 4 x g/ml). OPT has been used in the analysis of dopamine, catecholamines and histamines. Other fluorescence reagents that are sometimes used include 4-bromoethyl-7-methoxycoumarin, diphenylindene, sulphonyl chloride, dansyl-hydrazine and a number of fluorescent isocyanates. 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

Fig. 11.2.10. HPLC of human leucocyte interferon hydrolysate using post-column fluorescence detection with fluorescamine. Chromatographic conditions column, DC-4A (Dumim) (500 x 4.6 mm I.D.) mobile phase, 0.175 N sodium citrate, 2.5% isopropanol, pH 3.5 (Buffer A), 0.175 N sodium citrate, pH 3.45 (Buffer B), 0.2 N sodium citrate, 0.05% thiodiglycol, pH 4.1 (Buffer C), 0.52 N sodium citrate, 1 N sodium chloride, 0.05% thiodiglycol, pH 7.9 (Buffer D), elution was achieved with the following gradient Buffer A (15 min). Buffer B (17 min). Buffer C (24 min). Buffer D (41 min), flow rate, 18 ml/h temperature, 57 ° C. Peaks 1, aspartic acid 2, threonine 3, serine 4, glutamic acid 5, cysteine 6, proline 7, glycine 8, alanine 10, valine 11, methionine 12, isoleucine 13, leucine 14, norleucine 15, tyrosine 16, phenylalanine 17, histidine 18, lysine 19, ammonia 21, arginine. Reproduced from Stein and Brink (1981), with...

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). 

Many reported methods for BA and AA involve pre- or postcolumn (or capillary) derivatization of these compounds before detection using 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde," 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, l,2-naphthoquinone-4-sulfonate," < -Phthalaldehyde (OPA), fluorescamine, and many other procedures reported in Table 30.2. OPA is disadvantageous in that it reacts only with primary amines, and the fluorescent derivatives are associated with significant instability. Dabsyl- and dansylchloride are better in this respect as they react with both primary and secondary amino groups, and provide stable derivatives. Indirect UV, indirect fluorescence detection, conductivity," and electrochemical detection have been utilized after CE separation as well as mass spectrometry Kvasnicka et al. " developed a direct,... 

Fluorescamine has also been employed for postcolumn derivatization with fluorescence detection. As discussed previously, a fluorescent product is generated in the presence of primary amines within a few seconds. Fluorescamine undergoes hydrolysis, but the hydrolysis reaction is much slower than the reaction with primary amines and the product of that reaction does not fluoresce. For postcolumn addition, the reagent is added as a solution in acetone, acetonitrile, or methanol. As with OPA, proline can be detected through conversion to aminobutyraldehyde by oxidation with N-chlorosuccinimide. Unlike OPA, fluorescamine reacts poorly with ammonia. Therefore, problems with baseline drift are not as prominent. Since the reaction of fluorescamine with primary amines produces an equilibrated mixture of two products, it is used most frequently as a postcolumn reagent. 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making derivatization a prerequisite for fluorescence detection. Derivatization has been described with naphthalene-2,3-dicarboxaldehyde-(S-mercaptoethanol for determination of substance P and its metabolites, fluorescamine, and 5-carboxyfluorescein succinimidyl ester for luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, and 3-endorphin. Kostel and Lunte compared various postcolumn reactor designs, whereas Advis et al. employed precolumn derivatization, among others. In order to improve sensitivity, laser light is frequently employed for exciting the fluorescent molecules referred to as laserinduced fluorescence (LIE) and provides a 500-1000 times improved sensitivity compared to UV detection. 

Amino-3 -deoxythymidine, a catabolite of AZT, was determined in plasma by derivatization with fluorescamine and reversed-phase h.p.lx. with fluorescence detection. ... 

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. 

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. 

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. 

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