Fluorescent labeling, derivatizing

See also. Analytical Reagents Specification. Derivat-ization of Analytes. Fluorescence Derivatization Fluorescence Labeling Quantitative Anaiysis. Lipids Fatty Acids. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Pharmaceuticai Applications. Mass Spectrometry Forensic Appiications. Spectrophotometry Derivative Techniques. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Since a relatively small number of analytes of interest have native fluorescent properties, derivatization reactions are frequently employed to enable this detection technique to be extended to a broader range of compounds. This is an excellent means of increasing the detectability for a whole range of molecules, but it is important to realize that there are certain limitations. First, it is difficult to obtain quantitative yields at low analyte concentrations. This implies that in some cases, the obtainable detection limit are not limited by the detector sensitivity, but instead by low yields in the derivatization reaction. Furthermore, to shift the equilibrium toward the product side at low analyte concentrations, as much as 104 times excess of fluorescent label may be necessary. Tow concentrations of impurities in the label can be present at levels greater than the analytes of interest and as a result, numerous interfering peaks in the chromatograms may be observed. These problems are discussed in detail in Ref. 181. 

Although various fluorescence-labeling derivatization reagents have been used in drug residues analysis, those replacing active hydrogens of hydroxyl, sulfydryl, or amino groups account for most applications. 

Fluorometric detection has been mainly employed for the determination of aminopenicillins such as amoxicillin and ampicillin in edible animal products because it confers the advantages of selectivity and sensitivity. Fluorometric detection of penicillins, however, necessitates their precolumn derivatization to produce the corresponding fluorescent derivatives. The most commonly used derivatizing reagents are formaldehyde (100, 117, 118), salicylaldehyde (83), and mercury dichloride (91). 4-Bromomethyl-7-methoxycoumarin has also been employed as a fluorescence label for the selective and sensitive detection of seven penicillins in milk (96). 

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). 

Zhuang et al. [152] derivatized glycan samples with 8-aminopyrene-l,3,6-trisulfonic acid to get a charge for electrophoresis and a fluorescent label for detection. They analyzed glycan derivatives in the blood of cancer patients... 

A variety of pre- and postcolumn sample derivatization schemes has been developed in CE, mainly for the attachment of fluorescent labels for detection purposes. Precolumn sample modification is usually less demanding than postcolumn reactions for reasons discussed in Sect. 3.2.4. 

Although on-column fluorescence detection can provide excellent detection limits, the technique is less versatile than UV detection because many solutes of interest do not exhibit native fluorescence and must be derivatized with some type of fluorophore. Consequently, the literature contains many examples of capillary electrophoresis separations of fluorescence-labeled solutes such as dansylated amino acids.50,51... 

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