Silica compounds protein interactions

The eluent is rather acidic in most protein separations. With silica-based packings, the reason for the selection of this condition is related to the surface silanols which are weakly acidic groups. Above pH 3.5-4, these groups are dissociated and ionic interactions will occur with basic sample compounds. These interactions can be reduced simply using acidic mobile phases that prevent ionization of the silanols. Another reason is the stability of the silica which is higher in acidic media72). 

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

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... 

The most important point during sample preparation is to prevent oxidation of ascorbic acid. Indeed, it is easily oxidized by an alkaline pH, heavy metal ions (Cu and Fe ), the presence of halogens compounds, and hydrogen peroxide. The most suitable solvent for this purpose is metaphosphoric acid, which inhibits L-ascorbic oxidase and metal catalysis, and it causes the precipitation of proteins. However, it can cause serious analytical interactions with silica-based column, e.g., C18 or amino bonded-phases [542] and it could co-elute with AA. 

The wall of a fused silica capillary, containing silanol groups, has been identified as a potential site of interaction for solutes in electrophoretic measurements. In particular, adsorption of proteins, peptides and other basic compounds has been more specifically addressed [1-3]. In the ideal situation, the interaction is strictly chromatographic so that the process is reversible with the net result being a loss in the observed efficiency for CE [2,4]. A more serious problem can result when there is irreversible adsorption of the analyte on the capillary wall leading to a lack of repro-... 

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. 

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