Optimum support properties for protein separations by high-performance size exclusion chromatography

Optimum chromatographic properties of high performance size exclusion chromatography (HPSEC) of proteins, such as resolution, molecular weight accuracy and recovery, are obtained on packings and columns with tailor-made physical and chemical structures, employed at properly adjusted eluent compositions and operation conditions. SEC-theory suggests that a broad molecular weight fractionation range and high linearity of the log—linear calibration plot can be achieved by the use of two packings (10- and 80-nm pore size, characterized by a pore-size distribution (psd) equal to or less than 1 decade and by equal internal column porosity ϵ p), rather than a single 30- to 50-nm pore-size packing with a wide psd. Favourably high-phase ratios of ϵ p/ϵ o⪢/ 1.0 for HPSEC columns were accomplished with a minimum interstitial column porosity ϵ o and a high value for the internal column porosity ϵ p (the specific pore volume, ν p, multiplied by the packing density, ϱ p.) Ligands such as diol, N-acetoxyamino and oligomeric ether with a propyl- or propoxy-spacer bonded to the silica at the highest density appear to provide high mass recovery and bioactivity as well as chemical stability. Such packings, available in 3–5 μm particle size ranges of narrow distribution, packed into columns ⪢6 mm i.d. and ⪢500 mm in length, offer the best compromise with respect to resolution, speed and pressure drop. More careful studies are required to explain the effects of protein conformational changes and interconversions during elution on HPSEC columns.