Fluorescent derivatization agents

Similar dicholoro precursor 205 was utilized by Katoh and co-workers in the discovery of a new fluorescence derivatizing agent for fatty acids <99H299>. The conversion of 205 to fatty acid-coupled 206 proceeded through the displacement of both chloro groups with morpholine in several high-yielding steps. 

For those without native fluorescence, two common approaches have been employed, namely, derivatization and indirect fluorescence. Derivatizing agents should be pure, low fluorescent, and stable, as well as react quickly and uniquely with the analytes and, thus, formed compounds should be strongly fluorescent and stable. These include dansyl chloride, fluorescamine, 4-clair-7-nitro-benz-2-oxa-l,3-diazole (NBD), o-phthaldialdehyde (OPA), fluorescein isothiocyanate (FITC), and naphthalene-2,3-dicarboxaldehyde (NDA), which have been used for the analyses of amino acids, peptides, proteins, thiols, and sugars with LOD in 1-100 nM range. Compared to LINF, approaches based on derivatization provide the advantages of relatively low cost and versatility in instrumentation, but they may suffer from contamination and loss of temporal information. 

Most BAs do not show fluorescence or UV adsorption and a labelling procedure is indispensable to allow their detection. One of the most frequently studied fluorescent derivatizing agents in both the pre- and post-column methods is o-phthalaldehyde (OPA). Despite the limited stability of derivatives, the main advantages of OPA are a very fast... 

The second beneht in the earlier example illustrates the primary application of derivatization in HPLC. Inspection of Table 13.2 suggests that if we want to derivatize for improved selectivity and sensitivity, introduction of fluorescence should be our first choice. Table 13.3 lists a number of fluorescence derivatization agents and their target functionalities. Generally we wish to derivatize only one group at a time. 

Fluorescence analysis has been extended to many nonfluorescent species by the development of a wide range of derivatizing agents that form a fluorescent product. This approach has been especially useful with biochemical molecules, many of which are not natural fluorophores. 

At this point, the anaiyte may not be amenabie to UV, FL, or EC detection. In this case, the best course of action may be to choose LC/MS (see Section 4.2). However, one other option is to use a pre- " or post-coiumn derivatization step to increase the detectabiiity of the anaiyte with respect to FL or UV. Fluorescent or UV labels are available for carboxylic acids," amines, phenols, and thiols. The decision to use pre- or post-column derivatization is predicated upon the functionality of the analyte available for derivatization and the rate and extent of the reaction between each derivatizing agent and the analyte. 

Although tetracyclines possess the inherent ability to fluoresce, few methods exploiting this property have been reported (300, 306, 309). Instead, fluorometric methods based on the reaction of tetracyclines with suitable derivatizing agents have been developed. The use, for example, of zirconyl chloride as a fluorescence label in the postcolumn derivation of tetracyclines, has allowed highly selective and sensitive detection of these antibiotics in animal tissues (294, 295). 

Fig. 2 Postcolumn derivatization scheme for aflatoxin analysis 1, mobile phase 2, HPLC pump 3, injection valve 4, precolumn 5. analytical column 6, derivatizing agent solution 7, auxiliary HPLC pump 8, T-valve 9, oil or water bath 10, reaction coil 11, fluorescence detector 12, waste 13, chromatographic data handling system.

Derivatization is a technique that is most commonly performed prior to UV absorption or fluorescence detection. It is not restricted to these detection modes, however, and postcolumn electrochemical31 and postcolumn chemiluminescence32 detection have also been reported. Table 3.6 provides a list of some of the more common derivatizing agents and the compounds with which they react.33 Derivatization has also been used to aid in the detection of compounds such as amino acids,34 amines,35 saccharides,36 thiols,37 carboxylic acids,38 steroids,39 alcohols,40 fatty acids 41 and several inorganic species.42... 

The two forms of capillary array electrophoresis are emerging as powerful methods for the determination of enantiomeric purity of chiral compounds in a truly high-through-put manner. Various modifications are possible, for example, detection systems based on UV/Vis, MS, or electrical conductivity. Moreover, chiral selectors in the CE electrolyte are not even necessary if the mixture of enantiomers is first converted into diastereo-mers, for example, using chiral fluorescent-active derivatization agents [51,57]. 

Peptides are commonly detected by absorbance at 200-220 nm. However, most of the compounds present in wine may interfere in the ultraviolet detection of peptides when low wavelengths are used. Thus, for the analysis of these compounds it is useful to apply sensitive and selective detection methods. To this end, it is possible to form derivates of the peptides that can be detected at higher and more specific wavelengths. Detection by fluorescence can also be used to detect peptides containing fluorescence amino acids (tyrosine and tryptophan). For peptides without this property, the formation of derivates with derivatizing agents have been proved to be very useful (Moreno-Arribas et al. 1998a). 

Figure 11.14 A derivatizing reaction (it couid be either pre-or postcoiumn) between the derivatization agent, ortho-phthaiaidehyde (OPA), a primary amine (R2NH2), and a reduced sulfhydryi. The product is intenseiy fiuorescent and is readily detected by using a fluorescence detector.

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