Differential chromatographic retentions

As already mentioned, we chose three different physicochemical properties for studying the influence of the surface area and fractal dimension in the ability of dendritic macromolecules to interact with neighboring solvent molecules. These properties are (a) the differential chromatographic retention of the diastereoisom-ers of 5 (G = 1) and 6 (G = 1), (b) the dependence on the nature of solvents of the equilibrium constant between the two diastereoisomers of 5 (G = 1), and (c) the tumbling process occurring in solution of the two isomers of 5 (G = 1), as observed by electron spin resonance (ESR) spectroscopy. The most relevant results and conclusions obtained with these three different studies are summarized as follows. 

Case 2. The chromatographic retention factors and the M/c of X and Y in this case are identical. Since their electrophoretic mobilities and MCe are different, there MCec will be different too and the two components can be separated in CEC, e.g., components B and C have the same chromatographic retention factors, but different electrophoretic mobilities. Hence, they are separated by virtue of differential electrophoretic migration as illustrated in Fig. 1.12. 

The k" measures the magnitude of retention in CEC due to reversible binding of the analyte to the stationary phase. Inspection of Eq. (11) shows that for all components (neutral or charged), k" is always positive, as a chromatographic retention factor should be. Further, while the retention factor in HPLC and the velocity factor in CZE are able to characterize the respective differential migration processes alone, both of them are required to characterize CEC. 

Separation of solutes injected into the system arises from differential retention of the solutes by the stationary phase. The net retention of a particular solute depends upon all the solute-solute, solute-mobile phase, solute-stationary phase and stationary phase-mobile phase interactions that contribute to retention. The t3q3es of solute-stationary phase interactions involved in chromatographic retention include hydrogen bonding, van der Waal s forces, electrostatic forces or hydrophobic forces. 

It follows from the discussion in this paragraph that only standard differential thermodynamic functions can be calculated from any chromatographic distribution constant defined in whatever way. Also, it is necessary to always specify the choice of the standard states for the solute in both phases of the system. Without specifying the standard states the data on the thermodynamic functions calculated from chromatographic retention data lack any sense. When choosing certain standard states it may happen that the standard differential Gibbs function is identical with another form of the differential Gibbs function, or includes such a form situations described by equations 46 and 49 may serve as examples. The same also holds true for standard differential volumes, entropies and enthalpies (compare Section 1.8.3). However, every particular situation requires a special treatment. 

The use of mass spectrometry in the structural analysis of carbohydrates, first reported in 1958 (114), was developed in detail by Kochetkov and Chizhov (115). They showed that, under electron impact, the acetylated and methyl ether derivatives of monosaccharides provided a wealth of structural information through analysis of typical fragmentation pathways of the initial molecular ion. This has proved of enormous utility in the structural elucidation of polysaccharides and complex oligosaccharides sequential permethylation, hydrolysis, reduction to the alditol, and acetylation, affords mixtures of peracetylated, partially methylated alditol acetates that can be separated and analyzed by use of a gas chromatograph coupled directly to a mass spectrometer (25). The mass spectra of stereoisomers are normally identical, while the gas chromatographic retention times readily permit differentiation of stereoisomers. 

Often, size exclusion chromatograms (SEC) (compare section 11.7, Size Exclusion Chromatography) of polymers under study are expressed as differential representations of molar mass dispersity. The chromatographic retention volumes are directly transformed into the molar masses. This approach renders useful immediate information about tendencies of molar mass evolution in the course of building or decomposition polyreactions but the absolute values of molar mass can be only rarely extracted from it. As a rale, polystyrene calibrations are applied for molar mass calculation so that one deals with the polystyrene equivalent molar masses, not with the absolute values. The resulting dispersity (distribution) functions may be heavily skewed because the linear part of the calibration dependence for the polymer under study may exhibit well different slope compared with the polystyrene calibration, which was employed for the transformation of retention volumes into molar masses. 

From the Van Deemter plot, it is clear that there exists an optimum velocity of mobile phase at which the HETP function attains its minimum. The low HETP value is favorable because it will imply a large number of theoretical plates in the column of a given length (cf. Equation 6.1). Thus, interactions between the sample and the stationary phase will favor differentiation of retention times of various analytes traversing the chromatographic column. 

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

The silanophilic character of 16 reversed-phase high-performance liquid chromatographic columns was evaluated with dimethyl diphenycyclam, a cyclic tetraza macrocycle [101]. The method is rapid, does not require the removal of packing material, and uses a water-miscible solvent. The results demonstrate two points first, cyclic tetraza macrocycles offer substantial benefits over currently used silanophilic agents second, the method can easily differentiate the performance of various columns in terms of their relative hydrophobic and silanophilic contributions to absolute retention. 

An advantage of the mass spectrometer as a detector is that it may allow differentiation of compounds with similar retention characteristics or may allow the identification and/or quantitative determination of components that are only partially resolved chromatographically, or even those that are totally unresolved. This may reduce the time required for method development and is discussed in more detail in Chapter 3. 

Mass spectra of the cis and trans isomers of the two substituents are virtually indistinguishable, but the IR spectra allow easy differentiation in the region near 1320 cm . The relative stereochemistry of the natural alkaloids can be determined by comparison of gas chromatographic behavior with synthetic material of known stereochemistry, since the two isomers have different retention times. 

The thermodynamic dead volume includes those static fractions of the mobile phase that have the same composition as the moving phase, and thus do not contribute to solute retention by differential interaction in a similar manner to those with the stationary phase. It is seen that, in contrast to the kinetic dead volume, which by definition can contain no static mobile phase, and as a consequence is independent of the solute chromatographed, the thermodynamic dead volume will vary from solute to solute depending on the size of the solute molecule (i.e. is dependent on both ( i )and (n). Moreover, the amount of the stationary phase accessible to the solute will also vary with the size of the molecule (i.e. is dependent on (%)). It follows, that for a given stationary phase, it is not possible to compare the retentive properties of one solute with those of another in thermodynamic terms, unless ( ), (n) and (fc) are known accurately for each solute. This is particularly important if the two solutes differ significantly in molecular volume. The experimental determination of ( ), (n) and( ) would be extremely difficult, if not impossible In practice, as it would be necessary to carry out a separate series of exclusion measurements for each solute which, at best, would be lengthy and tedious. 

Alhedai et a also examined the effect of exclusion on dead volume measurement. A mobile phase consisting of n-octane, the same chain length as the bonded phase, was employed to ensure no differential interaction between the solute and the two phases. A range of aliphatic hydrocarbons from, n-hexane to n-hexaiiiacohtane were chromatographed at two temperature 30°C and 50°C. The two temperatures were used to ensure that the retention mechanism was solely exclusion and not partition. If partition was the mechanism promoting retention, then different retention volumes... 

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