Silica-based supports recovery

There are many ligands used for RPC, but the most popular for protein analysis are butyl (C4) and octyl (Cg). Little difference in selectivity for proteins is observed with ligand-chain-length variation, but mass recovery is often enhanced on the shorter chains. Due to their higher efficiencies and wettability, silica-based supports are generally used for protein analysis. The... 

The materials were tested as supports for SPE techniques. Excellent recoveries are observed, exceeding silica-based SPE materials (40). The supports were also investigated for their retention behavior for phenols, alcohols, carboxylic acids, aldehydes, ketones, esters, chloroalkenes, and polycyclic aromatic hydrocarbons (41). 

Hydrophilic size separation columns for use with aqueous samples are very popular choices for purifying proteins and carbohydrates. Protein separation columns are available on both silica and polymeric supports. It is surprising that the best of these protein purification columns in terms of resolution and in recovery of native protein are silica-based columns. One would expect that protein release from silica would be a real problem. It certainly is in many other silica columns. These columns, however, especially the TSK family of columns, give excellent recovery of enzymatic activity. I have talked to other column manufacturers who have investigated the problem. They say that when you remove the bonded phases from these columns they appear to be identical to bonded phases from a number of other, less successful, columns designed for protein purification. All of these bonded phases are primarily diol ether polymers, very hydrophilic, but of intermediate polarity. Some modification of... 

The attributes of a SEC column packing material are listed in Table 2. As indicated, the support must be optimized with respect to specific resolution, efficiency, column pressure, and mechanical, chemical, and thermal stability. Recovery of mass and activity is particularly important in the analysis and purification of biopolymers. It also plays a role in the analysis of nonbiochemical synthetic polymers on silica-based SEC columns. In addition to recovery losses by adsorption, the recovery for both groups of polymers can also be reduced by polymer degradation as a result of, for instance, mechanical shear. 

In any case, for such compounds, silica gel-based column material should be avoided as much as possible. Fortunately, various type of non-silica gel-based C-18 and other reverse-phase columns are now widely available. They include matrices such as polystyrenedivinylbenzene and hydroxyethyl meth-acrylate/dimethylacrylate copolymers with various functional groups. Recovery rates are generally much better on these polymeric materials, and are accompanied by a much longer column life. These alternative supports are also more tolerant to extreme pH and high concentrations of buffers such as ammonium acetate. 

Different resins, including acid alumina, silica gel, C-18 reverse phase, and florisil, as well as different solvents, were evaluated for their ability to clean-up the heptachlor-spiked samples for an immunoassay. The data obtained using acid alumina and C-18 reverse phase resin as the solid support and elution with 20 mL of the solvent indicated are siunmarized in Table n. It may be noticed that many of the resin/elution solvent combinations were able to give >9S% recoveries of the heptachlor (based on 1 C-heptachlor recovery), but when non-spiked beef fat samples were analyzed in the cELISA inhibition values of from 18 to 7S% were observed. The inhibition, observed in nonspiked samples represents unknown interfering compounds that coeluted with heptachlor. In the case of chromatography on an acid alumina column followed by elution with 20 mL of 75% acetonitrile in water, only 18% inhibition in nonspiked samples was observed. Material from identical experiments using nonspiked and spiked beef fat were collected as 3 mL fractions. Each fraction was then analyzed... 

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