Silica-based supports proteins

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants. 

Rapid Separation of Proteins and Peptides using Conventional Silica-Based Supports Identification of 2-D Gel Proteins Following In-Gel Proteolysis... 

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... 

Silica-based supports whose surfaces are chemically bonded with hydrophilic compounds like glycerol propylsilane have been developed, and have been mainly used for the separation of proteins. One example of a commercially available materials is shown in Table 7.2. They are generally very rigid, and particles of 5-10 fim in diameter can be used. However, they are chemically unstable and hence they must be operated in rather limited pH range, usually 2-8. In addition, many silanol groups which are negatively charged at pH above c. 5 remain on their surfaces [5], and therefore ionic interactions with samples readily occur. 

The pH of the buffers used in HIC separations is important because of the adsorption of proteins to the chromatographic support. Increase in the pH value (up to 9—10) of the mobile phase also decreases the hydrophobic interactions between proteins and the hydrophobic ligands, due to the change in charge of the protein. At high pH, silica-based supports are unstable and inadequate for protein purification. Generally, lowering the temperature promotes protein elution. Therefore, labile proteins should be separated at low temperatures [40]. 

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... 

---