Hydrophobicity, bonded phases

The reverse-phase technique is used less, however, with the advent of hydrophobic bonded phases (Section 8.2(3)). 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

In general, there is a wide variety of chromatographic modes (types) that can be employed for the HPLC determination of food components, but only a few have been used for the determination of NOC. These include partition/adsorption on silica gel, liquid-liquid partition on polar-bonded phase (e.g., cyano, amino) or nonpolar hydrophobic-bonded phase (e.g., reversed-phase), and anion-exchange chromatography. Macrae (61) discussed the theories behind the various modes of chromatography. 

For acetonitrUe/water systems it was found that acetonitrile forms thick adsorbed layer on the surface of hydrophobic bonded phase, while methanol adsorption from water formed a classical monomolecular adsorbed layer [166]. The thick adsorbed layer of acetonitrile provides a suitable media for the adsorption of liophilic ions on the stationary phase adding an electrostatic component to the retention mechanism, while monomolecular adsorption of methanol should not significantly affect adsorption of ions. 

Since the introduction of commercial precoated plates in the mid-1960s, continual developments with regard to the increase of selectivity and improvement of separation efficiency were pursued [i.e., ready-to-use layers suitable for high-performance thin-layer chromatography (HPTLC), polar and hydrophobic bonded phases, plates with concentrating zones],... 

Polar inorganics (hydrophilic) Polar organics Polar bonded phases Hydrophobic bonded phases Ion exchangers Inorganic... 

The chemical structure of one of the more popular bonded phases that are used for this purpose is shown in Figure 9.5. Retention factors obtained from simple hydrophobic-bonded phases, like typical C-18 HPLC columns, are not predictive of absorption, but the immobilized artificial membrane (lAM) method has been shown to correlate well with other models and with in vivo absorption data. For example, one study found that it gave a correlation coefficient of 0.858 (taking into account MW) when plotted against rat s intestinal absorption data, for a set of 12 compounds. ... 

Many different phases can be designed using this technology. Later, other techniques were developed that accomplished the same goal. In one alternative technology, the hydrophobic bonded phase is covered with a crosslinked polyethyleneoxide, which prevents proteins from reaching the hydrophobic surface, but lets small molecules penetrate. 

As opposed to the usual polar stationary phase and a less polar or non-polar mobile phase, the stationary phase in reverse phase chromatography is hydrophobic (hydrophobic bonded phase usually possessing Cjg or Cg functional groups) and the mobile phase is polar (fully or partially aqueous). In this case, polar substances will interact more with the polar mobile phase and elute first. As the non-polarity of the solute components increases, their retention times will also increase since they will interact more with the non-polar stationary phase.The reverse phase system is therefore very useful for separation of non-polar solutes. Water, an extremely polar solvent becomes the weakest eluent here. Methemol and acetonitrile are stronger eluents than water. Solvents of intermediate eluting strength cem be obtained by mixing one of these solvents with water. 

Common applications for this bonded phase are separations of peptides and proteins because these less hydrophobic bonded phases lead to less denaturing/structural changes in the protein (in part because weaker mobile phases will cause elution). This type of support is typically wide pore (300A). 

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