Hydrophobic nonspecific

In any of its different modes, LC behaves as a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the LC mode, different types of adsorption forces may be included in the retention process. Hydrophobic (nonspecific) interactions are the main ones in reversed-phase separations. Polar interactions including dipole-dipole, and dipole-induced dipole forces dominate in the normal-phase mode, whereas ionic interactions are responsible for the retention... 

Despite the intrinsically nonspecific nature of ion-exchange and reversed-phase/hydrophobic interactions, it is often found that chromatographic techniques based on these interactions can exhibit remarkable resolution this is attributed to the dynamics of multisite interactions being different for proteins having differing surface distributions of hydrophobic and/or ionizable groups. 

Figure 9.11 Amino acid sequences of homeodomains from four differenf franscription factors Anfp is from fhe Antennapedia gene in the fruitfly Drosophila, a2 is from the yeast Mat o2 gene, eng is from fhe engrailed gene in Drosophila and POU is from fhe POU homeodomain in the mammalian gene Oct-1. Residues colored green form the hydrophobic core of the homeodomain, blue form nonspecific interactions with the DNA backbone and red form contacts with the edges of the DNA bases.

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Membranes of extreme halophilic (Kushwaha et al. 1975, Anwar et al. 1977, Anton et al. 2002, Lutnaes et al. 2002, Oren 2002) and thermophilic bacteria (Alfredsson et al. 1988, Yokoyama et al. 1995) contain a large concentration of polar carotenoids. Membranes of these bacteria, which live in extreme conditions, should provide a high barrier to block nonspecific permeation of polar and nonpolar molecules. Incorporation of dipolar carotenoids into these membranes at a high concentration serves this purpose well because dipolar carotenoids increase the hydrophobic barrier for polar molecules (Wisniewska and Subczynski 1998, Wisniewska et al. 2006) and increase the rigidity barrier... 

Biotin modification reagents are widely used to attach a biotin group to proteins or other molecules for subsequent use in avidin, streptavidin, or NeutrAvidin separations or assays. Traditional biotin compounds containing aliphatic or other hydrophobic linker arms are discussed in detail in Chapter 11. In this section, the biotin-PEG compounds exclusively are discussed due to their unique hydrophilic properties, which include low nonspecific binding character and low immunogenicity. 

Adsorbents for biomacromolecules such as proteins have special properties. First, they need to have large pore sizes. A ratio of pore radius to molecule radius larger than 5 is desirable to prevent excessive diffusional hindrance (see Intraparticle Mass Transfer in this section). Thus, for typical proteins, pore radii need to be in excess of 10-15 nm. Second, functional groups for interactions with the protein are usually attached to the adsorbent backbone via a spacer arm to provide accessibility. Third, adsorbents based on hydrophilic structures are preferred to limit nonspecific interactions with the adsorbent backbone and prevent global unfolding or denaturation of the protein. Thus, if hydrophobic supports are used, their surfaces are usually rendered hydrophilic by incorporating hydrophilic coatings such as dextran or polyvinyl alcohol. Finally, materials stable in sodium hydroxide solutions (used for clean-in-place) are... 

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