Differential retention

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. 

Greenhalgh, J.R., A. Birchall, A.C. James, H. Smith and A. Hodgson, Differential Retention of Pb-212 Ions and Insoluble Particles in Nasal Mucosa of the Rat, Phys. Med. Biol. 27 837-851 (1982). 

For separation, analytes must be retained and have differential migration in the column Separation of components cannot occur without retention and interaction with the stationary phase. In addition, analytes must show differential retention vs. other components. 

As mentioned earlier as an axiom, separation between two components is only possible if they have different migration rates through the column. Selectivity or separation factor (a) is a measure of differential retention of two analytes. It is defined as the ratio of the capacity factors k ) of two peaks as shown in Figure 4. Selectivity must be >1.0 for peak separation. 

Fischer, H., A. Sachse, C. E. W. Steinberg, and M. Pusch. 2002. Differential retention of dissolved organic carbon by bacteria in river sediments. Limnology and Oceanography, in press. 

Although it is well understood that molecules must be able to enter the cavity of the cyclodextrin molecule for complexation to occur, and therefore, under chromatographic conditions, for retention to result, the differential binding of two stereoisomers within the cyclodextrin that allows for their differential retention is not always apparent. An understanding of this can be obtained through the use of three dimensional computer graphic imaging of the crystal structure of the inclusion complex. 

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. 

According to Eq. (1), the retention of an analyte component is governed by the two transport coefficients D and Dj. The coefficient D scales directly with hydrodynamic volume and is the same parameter that differentiates retention in SEC. Thus, thermal FFF, like SEC, separates material according to differences in their hydrodynamic volume, which is related to molecular weight. However, V, increases with D in SEC and decreases with D in thermal FFF, so the elution orders are opposite in the two techniques low-molecular-weight components elute ahead of higher-molecular-weight components in thermal FFF. 

On-line SPE methods rely on the differential retention of small organic... 

Actually, size exclusion should not be the only possible separation mechanism resulting in a spontaneous chromatographic salt resolution. Any type of differentiated retention of the cation or anion must also generate acidic and basic effluent zones. Thus, in the case of ammonium acetate, simple dispersive (hydrophobic) interactions of the acetate anions overbalance their partial exclusion from the Styrosorb 2 polymer phase. Here, the ammonia-enriched alkafrne firactions elute first, while the acetic acid-smelling acidic fractions elute from the column with a pronounced retention (Fig. 12.17). It should be noted that contrary to the bulky tetrabutylammonium cations, the acetate anions are barely excluded from the polymer phase and can experience hydrophobic retention in fine pores. 

Some of the most relevant methods of enzyme purification by protein fractionation applicable at large scale will be reviewed. Such methods can be roughly divided into those based on fractional precipitation and those based on differential retention in a solid matrix. 

Micellar electrokinetic chromatography (MEKC) is a particular EKC mode where the secondary phase is composed by micellized surfactant (MEKC is discussed in detail in Chapter 3 by Terabe). Solute differential retention occurs as a result of a partition mechanism between a dispersed phase defined by the total volume of micelles and the remaining aqueous phase. MEKC modes of elution comprise normal, restricted, and reversed MEKC, based on the relative migration of the analyte and secondary phase apparent velocity. 

In liquid chromatography, chiral additives are frequently used with the mobile phase, and their direct effect on the differential retention of a pair of enantiomers can be very complex and difficult to calculate (although not impossible if sufficient data is available). The addition of an additive to the mobile phase changes both the interactive character of the mobile phase and the stationary phase, as the additive is distributed between the two phases in exactly the same way as a solute. The additive, however, is present at much higher concentrations than the solute and thus the isotherm is not linear. In order to understand the effect of an additive to the interactive nature of the stationary phase in LC, it is necessary to develop the Langmuir Isotherm which will provide an equation that describes the surface coverage by the chiral agent, in terms of its concentration in the mobile phase. 

The differing levels of cytokinins in tobacco leaves of varying age could be due to one or a combination of the following factors (1) differential translocation of xylem cytokinins (2) differential metabolism of xylem cytokinins (3) differential retention of xylem cytokinins and (4) differential biosynthesis of cytokinins in situ in the leaves. These four factors were assessed. By RIA, the principal cytokinins in tobacco xylem exudate were identified as Z, (diH)Z, [9R]Z and (diH)[9R]Z. The distribution and metabolism of the two ribosides in the tobacco shoot were determined after supply via the xylem. The major metabolites of [9R]Z in tobacco leaf laminae were adenine, adenosine and AMP, while the principal metabolite of (diH)[9R]Z was the 7-glucoside of (diH)Z. However, expanded pre-senescent and early senescent laminae did not differ in their cytokinin metabolism, while small expanding laminae showed a higher rate of metabolism. There was no differential distribution of the ribosides to these laminae, and no differential retention in leaves of differing maturity. 

NI, J., PANG, S. T. YEH, S. 2007. Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhihition efficacy in prostate cancer cells. Prostate, 67, 463-71. 

Boron-11 MRS has been used to assess the delivery of boron neutron capture therapy (BNCT) agents to a target lesion. Uptake and retention of these compounds was determined in vivo in humans and animals and the results show that there is differential retention between normal and abnormal tissue. Furthermore, H-observed B-edited MRS has been shown to provide much higher sensitivity and could be utilized to monitor the distribution and excretion of boron agents noninvasively in patients about to undergo BNCT. 

The most commonly used approach to chromatographic resolution of enantiomers has been the formation of diastereomers either transiently or covalently. As diaste-reomers have different physicochemical properties, there is a differential retention in a chromatographic system. 

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