Ligand density

In this work the state-of-the-art and perspectives of column characterization and compai ison have been presented and discussed. All information about physico-chemical properties of RP HPLC Cl8 and C8 columns as porosity, average surface area, free silanol concentration, binding ligand density and others, were summarized. The points of views about column classifications, its advantages and disadvantages were discussed. It was shown that Cl8 and C8 HPLC column classification processes do not allow selecting the column with the same or preai range selectivity. 

The elution of [60]- and [70]fullerenes was measured in water-methanol as a function of temperature on a poly(octadecylsiloxane) phase.67 The retention was shown to be dependent on the surface tension of the stationary phase through a simple geometrical model in which the solute formed a cavity in the stationary phase. In affinity chromatography, it was demonstrated that low ligand density may be a requirement for specificity of binding.68... 

Clarke, S. J., Hollmann, C. A., Aldaye, F. A. and Nadeau, J. L. (2008). Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots. Bioconjug. Chem. 19, 562-8. 

Ligand density is a primary determinant of selectivity in HIC.3-5 This has two important ramifications (1) minor variations in ligand density can significantly alter selectivity and (2) reproducibility of column selectivity is more difficult for manufacturers to achieve than it is for nonhydrophobic retention mechanisms. This makes it necessary for assay developers to document adequate lot-to-lot reproducibility of a given column medium before investing major resources in assay development. 

Fujimoto et al. [77] synthesized a novel phase by coupling a dodecylamino-substituted P-cyclodextrin (P-CD) to 3-glycidoxypropyl-derivatized silica gel. The surface coverage of this phase was reported as 0.37 xmol/m, which amounts to a surface coverage of 2.6 xmol/m for C12 chains (seven chains per fi-CD). An increase in shape selectivity was observed when compared with a conventional Cis monomeric phase as determined by selectivity differences between j9/m-terphenyl, j9/o-terphenyl, and coronene/phenanthro[3,4-c]phenanthrene solute pairs and was attributed to the localized high ligand density as constrained by the fi-CD platform structure. 

Besides the packings mentioned above, a range of other stationary phases exists as well, bnt they are rarely used in pharmaceutical analysis. Even with this more limited selection of ligands, packings with quite different selectivities can be created, as in shown in Section III. F. The diversity stems from the choices in the properties of the silica, such as pore volume, pore size, surface area and purity as well as differences in ligand density. Nevertheless, general statements about different phases can be derived, and these are covered in the next section. 

Particle Size, Pore Diameter, and Ligand Density Effects in the Reversed-Phase Chromatography Separation of Peptides... 

Figure 13 Plots of the Capacity Factors, K, of the Paracelsin A-D as a Function of the n-Alkyl Length of Different RPC Silica Sorbents of Constant Ligand Density in H20/MeOH/MeCN (22 39 39) 1201ab...

Typical n-alkyl ligand densities that can be achieved with -alkylchlorosi lanes are within the range of 2.5-3.2pmolm-2, whilst with disilazanes, ligand densities approaching the limited values can be reached under optimized conditions, i.e. between 3.50 and 4.20 pmolm-2. Surface-modified zirconia or other metal oxide based RPC sorbents can be similarly prepared by either of the above two strategies. Compared to n-alkylsilicas, these ceramic RPC sorbents show different selectivities with synthetic peptides, as well as different chemical stability profiles. Consequently, RPC sorbents based on these types on surface-modified, porous metal oxide materials fulfill useful and complementary roles, but at this point in time, have achieved a more limited range of applications for the resolution of synthetic peptides due to their limited availability. 

Fig. 10. Typical adsorbent surfaces can be considered to have very high binding site ( ligand ) densities, resulting in multivalent interactions with adsorbed protein. If the multivalent interactions are of sufficient number and energy, the adsorptive interactions is irreversible ...

Modern HPLC techniques have resulted in a spectacular improvement in speed, resolution, and sensitivity. Separation times in HPLC are usually short, mostly in the interval of 10 - 20 min, with simpler separation problems being carried out in less than 10 min. This is due generally to the use of stationary phase that consists of very small and uniform porous particles with high ligand density. These conditions lead to better mass transfer and higher column efficiency. Consequently, high-performance separations can be obtained in short times (5). 

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