Selection of Chromatographic Mode

Total theoretical peak capacity for the ID and 2D LC/MS analyses of the yeast ribosomal protein sample was calculated as 240 and 700, respectively. Individual separation peak capacities were calculated by dividing the total separation time by the average peak width at baseline, and the 2D peak capacity determined as the product of the peak capacity of the two dimensions. These theoretical calculations rely on optimal use of the two-dimensional separation space, which in turn is dependent upon the lack of correlation between the component retention times in the two separation modes. Thus, the maximum use of the theoretical peak capacity is not only dependent on the selection of chromatographic modes based on different physicochemical... 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

It is important for the analyst to be able to select the best stationary phase to use for a particular chromatographic analysis. Silica gel can be used in two modes of chromatographic separations as a stationary phase in normal elution development or as a stationary phase in exclusion chromatography. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

The aim of this chapter is to evaluate the orthogonality of selected 2DLC systems for the separation of peptides. The orthogonality of different chromatographic modes was quantitatively characterized using a novel geometric approach. Practical peak capacity was calculated from the theoretical peak capacity and the knowledge of... 

TABLE 12.1 Chromatographic Modes and Conditions Used for LC-MS Study of Tryptic Peptides Separation Selectivity... 

On the basis of the preceding discussion, it should be obvious that ultratrace elemental analysis can be performed without any major problems by atomic spectroscopy. A major disadvantage with elemental analysis is that it does not provide information on element speciation. Speciation has major significance since it can define whether the element can become bioavailable. For example, complexed iron will be metabolized more readily than unbound iron and the measure of total iron in the sample will not discriminate between the available and nonavailable forms. There are many other similar examples and analytical procedures that must be developed which will enable elemental speciation to be performed. Liquid chromatographic procedures (either ion-exchange, ion-pair, liquid-solid, or liquid-liquid chromatography) are the best methods to speciate samples since they can separate solutes on the basis of a number of parameters. Chromatographic separation can be used as part of the sample preparation step and the column effluent can be monitored with atomic spectroscopy. This mode of operation combines the excellent separation characteristics with the element selectivity of atomic spectroscopy. AAS with a flame as the atom reservoir or AES with an inductively coupled plasma have been used successfully to speciate various ultratrace elements. 

The three main modes of chromatographic operation are elution chromatography, selective adsorption/desorption, and simulated countercurrent chromatography. Of these, elution chromatography, used as a cyclic batch process, was the first to be developed for large-scale separations. 

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