Indirect chromatographic method

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

The advantage of the indirect chromatographic method consists in the fact that it can predetermine the elution order that is important for the determination of optical purities, but there are some limitations to this technique. The derivatization method is tedious and time-consuming due to the procedure that involves different chemical reaction rates of the individual enantiomers. The chiral derivatizing agent must be with high-optical purity and stable in solution or solid state, since optical impurity will result in two more diastereomeric products [53,54,56]. Moreover, this method cannot be used easily with the environmental samples [56]. 

The derivatization can be due in the different reaction rates of the individual enantiomers, and for a proper interpretation of the analytical result, it is necessary to have the reaction completed before analysis. The formation of undesired should be avoided and the diastereomeric mixture must be chemically and stereochemically stable. For preparative purposes, the indirect chromatographic method involves an additional synthesis step, because the derivatizing agent has to be cleaved off the separated diastereoisomers after their resolution in a nonchiral environment. Hereby, the impurities can be introduced or even the racemization of the just resolved enantiomers could be caused. Still, once a suitable chiral derivatizing agent and procedure have been found, the indirect method offers some advantages for preparative separations over the direct preparative separations, mainly due to the fact that, in large-scale resolutions, the nonchiral media are better handled than, for example, the stationary phases, and the conditions can be adjusted more easily to obtain the desired resolution [54]. 

Chai, X.S. and Zhu, J.Y. Indirect headspace gas chromatographic method for vapor-liqtrid phase equilibrium study, J. Chromatogr. A, 799(2) 207-214, 1998. 

Two ESI-MS approaches can be taken, namely, direct and indirect analysis of the complexes. Direct methods utilize exclusively ESI-MS to analyze the nature of the non-covalent complexes formed under native conditions in the condensed phase while analyzing the products in the gas phase. Indirect methods utilize biochemical and chromatographic methods for preparing and separating the complexes and ESI-MS as the ancillary detector for the individual products of the non-covalent complex, namely, the small molecules and the protein. 

On mixtures, except for one review [50], little information is available and measures are required both indirect (physical absorption) and direct (chromatographic) methods are possible. In UMR45, by high pressure adjustment of a chromatographic method, we proved that water in organic media, even in trace amount, has a dramatic negative influence on H2 solubility. 

In one study, a modified Monier-Williams method has been utilized as a preparative procedure to obtain both the free and bound sulfite fractions. The two fractions were analyzed by HPLC with indirect photometric detection using a 250 X 4.6-mm LC-SAX column eluted with potassium hydrogen phtalate (0.15 g/L, pH 5.7) and detected at 280 nm. Levels of 5-10 ppm of S02 in foods, corresponding to 30 - 60 ng injected, were reliably detected by this method. The results confirmed that the chromatographic method, unlike the Monier-Williams method, is able to avoid the potential interference of volatile substances derived from matrices or utilized chemicals (36). The HPLC conditions are summarized in Table 3. 

Titration The determination of assay values for reference standards, counter-ions, or impurities can often be independently determined via titration. While titration assays generally have less selectivity in comparison to chromatographic methods, the advantages of a broad spectrum of classical titration techniques that exist for organic functional groups is often overlooked. The methodologies include not only classical potentiometric acid/base titrations but also nonaqueous, redox, indirect, precipitation, and derivatization titrations.76-79... 

Carbon monoxide may be released from hemoglobin and then measured by GC, or it may be determined indirectly as carboxyhemoglobin by spectrophotometry. Gas chromatographic methods are accurate and precise even for very low concentrations of carbon monoxide. The spectrophotomet-ric methods are rapid, convenient, accurate, and precise, except at very low concentrations of carboxyhemoglobin C<2% to 3%). 

Enantiomers can be separated by traditional chromatographic methods, provided they have been previously derivatized with a chiral compound to produce diaste-reomers. This method of indirect separation of enantiomers is explained in Section 22.5. 

The kinetic characteristics are determined in GC methods both directly and indirectly. In direct pulse chromatographic methods the reaction rate can be established by the direct determination of the amount (concentration) of the reacting component, whereas in indirect methods this is done on the basis of the variations with time of the chromatographic properties of the reacting system, which are usually determined from the relationship between and the retention times of the non-reacting components and the composition of the reaction mixture used as the stationary phase [58]. Pulse chromatography... 

Polychlorinated biphenyls (PCB) are determined by photometric or gas chromatographic methods. In the former method, the polychlorinated biphenyls are determined by spectrophotometry indirectly, via mercury rho-danide after their mineralization with metallic sodium in ethanol. In the latter determination, a flame ionization detector is used [33]. 

A recent alternative to HPLC or GC in the analysis of organic acids is capillary zone electrophoresis. It provides completely different selectivities from chromatographic methods, shorter analysis times, and no derivatization is required. As a drawback, the lack of UV absorbance of aliphatic organic acids above 220 nm requires the addition of substances showing strong UV absorption for indirect UV detection. 

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