Selection of derivatization reagents

Table 5 Some examples of the form of presentation of the recommendations on the selection of derivatization reagents for different classes of organic compounds.

Finally, Chapter 7 discusses the use of selected fluorescence and pH indicator detection reagents, nniversal charring methods, and selective derivatization reagents that produce colored zones. 

Prechromatographic derivatization reactions re usually favored when it is desired to modify the properties of the sample to enhance stability during measurement (i.e., minimize oxidative and catalytic degradation, etc.), to improve the extraction efficiency of the substance during sample cleanup, to improve the chromatographic resolution, or to simplify the optimization of the reaction conditions [698-702]. As both pre- and postchromatographic methods enhance the sensitivity and selectivity of the detection process a choice between the two methods will usually depend on the chemistry involved, ease of optimization, and which method best overcomes matrix and reagent interferences. 

Solvent removal.. Lzurge amounts of solvents and excess derivatizing reagents, etc., can be excluded from the main separation column. Prevents column deterioration and improves the performance of sensitive, selective detectors. 

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. 

The use of organomagnesium reagents as terminal reductants in zirconocene-catalyzed diene reductive cyclization permits derivatization of the resulting bis(magnesiomethyl)cycloalkanes. However, the use of other stoichiometric reductants is likely to afford catalytic systems that exhibit complementary selectivity profiles. Molander reports the... 

FIA has also found wide application in pharmaceutical analysis.214,215 Direct UV detection of active ingredients is the most popular pharmaceutical analysis application of FIA. For single component analysis of samples with little matrix interference such as dissolution and content uniformity of conventional dosage forms, many pharmaceutical chemists simply replace a column with suitable tubing between the injector and the detector to run FIA on standard HPLC instrumentation. When direct UV detection offers inadequate selectivity, simple online reaction schemes with more specific reagents including chemical, photochemical, and enzymatic reactions of derivatization are applied for flow injection determination of pharmaceuticals.216... 

The indirect approach has been widely applied since many functional groups can be derivatized with various chiral reagents and the covalent diastereoisomers can be separated with inexpensive non-chiral systems. Other advantages of the indirect approach are that method development is rather straightforward and that the detection sensitivity of the enantiomers can be improved by the selection of an appropriate CDR having a strong chromophor or fluorophor. 

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). 

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