Separation, Quantification and Identification

These various methods of analysis as applied to the analysis of organic acids in physiological fluids are described below in the succeeding sections. Electrophoresis (Nordmann and Nordmann, 1961) and isotachophoresis (Bocek et aL, 1976) of organic acids have not received wide application, particularly in the clinical and metabolic fields, and are not dealt with in this section. 

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The interfacing of CE with ESI mass spectrometry has progressed substantially in recent years. On-line CE/MS has been widely used for both qualitative and quantitative analysis of many chemically diverse molecules. This method becomes a useful and sensitive analytical tool for the separation, quantification and identification of biological, therapeutic, environmental and other important classes of chemical analytes. The developments of CE/MS have been reviewed [22-26]. 

The methods discussed for separation of alkanes and cycloalkanes from alkenes, aromatics, resins and other more polar constituents of petroleum can be employed also for synthetic mixtures, asphalts, bitumens, etc. However, for the quantification and identification of such homologous families as n-alkanes, branched alkanes, etc., further analysis is needed. Whereas the GTA relates to functionality and differences in polarity, the separation within the alkane family cannot be based on these characteristics. 

Whereas the power of resolution depends on the quality of the column, quantification and identification of the separated compounds is a function of the detector. The oldest GC detector, still used for hydrocarbon gases, is the TCD (Thermal Conductivity Detector). The TCD response is proportional to the molar fraction and the specific heat of the compound detected. Its sensitivity is in the jug (10" g) range, but even for the same homologous family, this changes with molecular weight ... 

The use of mass spectroscopy and HPLC offers great potential for the quantification and identification of polymer additives. However, its adoption has been fraught with practical problems, created by the very nature of the eluent from the chromatograph. One possible solution to this problem is the moving-belt interface, in which the eluent is sprayed on to a circulating belt and the solvent evaporated in stages, so that finally only the analyte is carried into the mass spectrometer. This approach has been successful in the separation and identification of some polymer additives [60]. However, it can only be used for Cl and El mass spectroscopy, and the interface can only be operated with mobile phases containing less than 50% water. Newer interfaces such as thermospray and particle beam offer potential in this area. 

Analytical Approaches. Different analytical techniques have been appHed to each fraction to determine its molecular composition. As the molecular weight increases, complexity increasingly shifts the level of analytical detail from quantification of most individual species in the naphtha to average molecular descriptions in the vacuum residuum. For the naphtha, classical techniques allow the isolation and identification of individual compounds by physical properties. Gas chromatographic (gc) resolution allows almost every compound having less than eight carbon atoms to be measured separately. The combination of gc with mass spectrometry (gc/ms) can be used for quantitation purposes when compounds are not well-resolved by gc. 

The mass spectrometer (ms) is a common adjunct to a chromatographic system (see Mass spectrometry). The combination of a gas chromatograph for component separation and a mass spectrometer (gc/ms) for detection and identification of the separated components is a powerful tool, particularly when the data are collected usiag an on-line data-handling system. QuaUtative information inherent ia the separation can be coupled with the identification of stmcture and relatively straightforward quantification of a mixture s components. 

Chandra, A., Rana, J., and Li, Y., Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS, J. Agric. Food Chem., 49, 3515, 2001. 

Khachik, F. and Beecher, G.R., Separation and identification of carotenoids and carotenol fatty acid esters in some squash products by liquid chromatography. 1. Quantification of carotenoids and related esters by HPLC, J. Agric. Food Chem., 36, 929, 1988. 

Rohrer, J. S., Cooper, G. A., and Townsend, R. R., Identification, quantification, and characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein proteolytic digests, Anal. Biodiem., 212, 7, 1993. 

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