Fluorescent reagents, derivatization with

The aromatic nucleus adsorbs in the UV and thus, the derivative can be detected by a UV detector. This is the most common type of chemical derivatization but the derivative may be chosen to be appropriate for different types of detector. For example, the solute can be reacted with a fluorescing reagent, producing a fluorescent derivative and thus be detectable by the fluorescence detector. Alternatively, a derivative can be made that is easily oxidized and, consequently, would be detectable by an electrochemical detector. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

The microspheres—synthesised via a two-step process (acid-catalysed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPS) in aqueous solution, followed by condensation catalysed by triethanolamine)—have a narrow size distribution (Figure 5.16) and are considerably more stable than polystyrene divinylbenzene microspheres as shown in phosphoramidite oligonucleotide synthesis by the excellent retention of fluorescence intensity in each of the reagent steps involved in phosphoramidite DNA synthesis (Figure 5.17, in which the organo-silica microsphere free thiol groups are derivatized with ATTO 550 maleimide coupled to the entrapped dye). 

There are potentially viable reagents available that may be employed for the derivatization of compounds either for enhancing UV/visible radiation (called chromatags) or for reaction of non-fluorescent reagent molecules (called fluorotags) with solutes to yield fluorescent derivatives. 

Fluorescence detection was selected to increase sensitivity and selectivity. Histamine has no natural fluorescence and a post-column derivatization with OPT was found to be facile. The OPT reaction with histamine or any primary amine will only occur in an alkaline medium. The derivatization reagent, pumped into the system after the mixture has been separated on the column, must be strongly basic to neutralize the acid in the mobile phase. The structure of the OPT adduct has been found to be dependent upon the pH at which the reaction is carried out as wel1 as the sol vent system (15). 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

In their study on the automation of the derivatization reaction of carboxylic acids, Wolf and Korf (34) chose bromomethylmethoxycoumarin (Br-MMC) as a label because of its commercial availability. The automation of this reaction is problematic, because elevated temperatures are required in the manual procedures and the reaction had to take place in an aprotic environment. The addition of a solid salt also complicates the procedure. They describe a solution to this problem using a suspension of potassium carbonate and appropriate reagent concentrations. The samples were detected by a fluorescence detector equipped with a 5-ju.l cell, using an excitation wavelength of 325 nm and a cutoff filter of 398 nm. 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

Hsieh et al. [46] described an HPLC method for simultaneous determination of the R-(—) and (S)-(+)-enantiomers of vigabatrin in human serum after precolumn derivatization with a fluorescent chiral reagent (naproxen acyl chloride) and detection at 350 nm with excitation at 230 nm. Separation was achieved on an Agilent Eclipse XDB-C8 (5 /an) column (15 cm x 4.6 mm) using a mixed solvent of acetate buffer (pH 7)-methanol (60 40) as a mobile phase at a flow rate of 1.2 ml/min. The calibration graphs for each enantiomer were linear over the... 

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