Derivatization fluorescence reagents

Fluorescent derivatizing reagents with benzofuran skeleton for HPLC with spec-trometric detection 98YZ483. 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Pyridone Acid Chlorides as Fluorescent Derivatizing Reagents. A second derivatizing reagent, a fluorescent acid chloride, was synthesized from the sodium salt of 3-phenyl-2(lH)pyridone (IV). 

Mitsui, A., Nohta, H., and Ohkura, Y., High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent, /. Chromatogr., 344, 61, 1985. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Mukherjee, P. S. and Karnes, H. T., Ultraviolet and fluorescence derivatization reagents for carboxylic acids suitable for high performance liquid chromatography A review, Biomed. Chromatogr., 10, 193, 1996. 

Prados P, Fukushima T, Santa T et al (1997) 4-/V,/V-Dimethylam inosu 1 lbnyl-7 -N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography. Anal Chim Acta 344 227-232... 

Table 2 Application of Fluorescence Derivatization Reagents to F1PLC with PO-CL Detection ... 

Figure 14 Representative fluorescence derivatization reagents used for PO-CL detection. DNS-CI, dansyl chloride DBD-F, 4-(A,A-dimethylaminosulphonyl-7-fluoro-2,l, 3-benzoxadiazole NDA, naphthalene-2,3-dicarboxaldehyde DNS-H, dansyl hydrazine DBD-H, 4-(iV,iV-dimethylaminosulphonyl-7-hydrazino-2,l,3-benzoxadiazole).

SA Cohen, DP Michaud. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-A -hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211 279-287, 1993. 

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. 

H. Nohta, T. Yukizawa, Y. Ohkura, M. Yoshimura, J. Ishida and M. Yamaguchi, Aromatic glycinonitriles and methylamines as pre-column fluorescence derivatization reagents for catecholamines, Anal.Chim. Acta, 344, 233-240 (1997). 

Chiral and fluorescent derivatization reagent (Prof. H. Ohrui)... 

Gifford, L.A. Owusu-Daaku, F.T.K. Stevens, A.J. Acenaphthene fluorescence derivatization reagents for use in high-performance liquid chromatography. J.Chromatogr.A, 1995, 715, 201-212... 

Kondo, J. Suzuki, N. Naganuma, H. Imaoka, T. Kawasaki, T. Nakanishi, A. Kawahara, Y. Enantios-pecific determination of ibuprofen in rat plasma using chiral fluorescence derivatization reagent, (—)-... 

Wang, H., Li, J., Liu, X., and Zhang, H.-S., A -hydroxysuccinimidyl fluorescein-O-acetate as a highly fluorescent derivatizing reagent for aliphatic amines in hquid chromatography. Anal. Chim. Acta, 423, 77-83, 2000. 

Among the more sensitive methods for the determination of A-nitrosamines are the ones based on the denitrosation of A-nitroso compounds by hydrobromic-acetic acid, and the subsequent detection of the liberated secondary amines via fluorescence derivatization. Precolumn or postcolumn derivatization has been used for the determination of A-nitrosamines. The most commonly used fluorescent derivatization reagents are listed in Table 12.4. 

Fluorescent derivatizing reagents withbenzofiiran skeleton forHPLC with spec-... 

The fluorescent derivatizing reagents most commonly used in this context are primary or secondary amines that react with isocyanates to form ureas according to the reaction given below ... 

A wide range of fluorescent derivatization reagents and techniques have been developed to utilize the analytical advantages that fluorescence offers. These derivatization reagents are usually specific to functional groups (e.g., amine, hydroxy, thiol), and their specificity offers yet another opportunity for discriminating against interferents. For excellent reviews, the reader is referred to references [38] and [39]. 

If a fluorescent derivatizing reagent that is non-fluorescent when unreacted (and therefore detector-transparent) but fluoresces when it becomes the tag (i.e., the part of the derivatizing reagent that is chemically bonded to the analyte) cannot be found. 

Gamoh K, Takatsuto S (1989) A new horonic acid derivative as a highly sensitive fluorescence derivatization reagent for brassinosteroids in liquid chromatography. Anal Chim Acta 222 201-204... 

Takechi H, Goto Y, Machida M. 4-(7-diethylaminocoumarin-3-yl)benzoyl cyanide (DBCB-CN) a highly sensitive fluorescent derivatization reagent for alcohols in high performance liquid chromatography. Chem Pharm Bull 1998 46 159-62. 

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