Accessible surface area of common silica particle types
In reverse-phase liquid chromatography, the surface area stationary phase is proportional to analyte retention. Typically, surface area is measured by the nitrogen adsorption isotherm. However, many analytes being characterized, such as therapeutic proteins, are much larger than nitrogen molecules. As the analyte size increases, the accessibility into the small pores of conventional columns decreases, lowering the accessible surface area. The accessible surface area of three column materials (fully porous, superficially porous, and nonporous particles) were studied as a function of analyte size, using two different techniques, frontal analysis and free particle extraction. Frontal analysis involved continuously loading analyte onto the different columns under retention conditions until a breakthrough signal was detected. The time needed to achieve breakthrough was used to compare the accessible surface area. Free particle extraction used fluorescently labeled analytes to compare the accessible surface of the three particle types. Analytes were extracted under retention conditions then released from the particles by decreasing the polarity of the solvent. Fluorescence measurements of the extracted supernatant were used to compare the accessible surface area of the three particle types. It was found that for small molecules (DiI, 934 Da), the accessible surface area of the fully porous particles was an order of magnitude higher than the other two materials, which agrees with the results from nitrogen adsorption isotherm. For large proteins (BSA, 65 kDa and IgG, 150 kDa), the three particle types provided similar surface areas. This demonstrated that large analytes are greatly excluded in the small pores of fully porous and superficially porous particles.
Wirth, Purdue University.
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