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Comparing silica and agarose resins for analysis of proteins with SEC

Silica- and agarose-based resins are the two most used types of size exclusion chromatography (SEC) resins available for protein analysis purposes. But how do they compare and which should you use?

Agarose is obtained from natural sources and contains a very low amount of ionic and hydrophobic groups that could interact with the compound of interest. In contrast, silica-based resins have silanol groups that require coating before use by size exclusion chromatography (SEC). Let's take a look at some of the differences between them:

Chemical composition: silica beads are composed of SiO2, whereas agarose beads are composed of Polysaccharide (D-galactose-3,6-Anhydro-L-galactose).

Surface coating: on silica beads the silanols are blocked to minimize adsorption, whereas surface coating is not required on agarose beads.

pH stability: for silica beads this is typically from pH 2-8 for operational use - for agarose beads this is 3-11 for operational use, but can be extended to 1-12 for cleaning-in-place.

Mechanical stability: this is rigid, high-pressure stability for silica beads, whereas for agarose beads the stability is dependent on chemical cross-linking.

Porosity: in both silica and agarose beads this can be controlled to achieve the desired separation range.

Higher alkali stability of agarose resins enables efficient cleaning
Blog2Figure2_v4_smallSuperdexTM Increase and SuperoseTM Increase are agarose-based resins, which tolerate high pH and can therefore withstand NaOH cleaning. This means that they can be efficiently cleaned and there is little if any need to use a guard column. The recommended process for cleaning the column is to inject NaOH after 10 to 20 separation cycles. As shown in the Figure, sample injections 1 and 200 on a Superdex 75 Increase column gave consistent results for efficiency and resolution when the NaOH cleaning regime had been applied.

Silica-based resins are mechanically very stable below a pH of 7 but do not tolerate pH > 8. Silica columns typically do not tolerate the common cleaning regimes for protein chromatography columns since those regimes involve NaOH, which gives high pH. Between pH 7 and 8, performance of the columns might be affected with a change in resolution as the outcome. An advantage with agarose-based resins is that the same SEC column can be used for different samples, with minimal risk for cross contamination. This is not the case with silica-based resins as they do not tolerate cleaning in NaOH.

 

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