Chromatography Forum

There are different reports on ideal pore size for molecules of some MW.

If injected 3 peptide standards with molecular weights- 1.5, 8, and 20 kDa on PLRP columns with 60, 100, 300, 1500 Angstrom pore sizes, (and used a MeCN grad with 0.1% TFA) what could I expect as far as

Loading capacity
resolution, peak shape
recovery

If using a smaller pore (100 Angstrom) PLRP column to purify a 7,000 Da peptide, how would the presence of larger (50 kDa) proteins, such as BSA, affect separation (assuming BSA elutes much later in the gradient than 7 kDa peptide)? [/list][/code]

I dont know if this answers your question, but in one of our work (J. Chromatogr. A, 1167 (2007) 63.) we have compared gradient elution of three proteins with different molar mass (insulin, trypsin, BSA) on five comercial C18 columns with various internal structure (porous, superficially porous, nonporous and monolithic). Three of them had pore size 300 A. The (model) separation was better on the superficially porous and monolithic columns than on the fully porous materials.

So I think that internal structure and type of your stationary phase can be more important than the pore size.

BSA is ~ 66kDa. Under the right condtions, it can be excluded from 10nm pore columns.
Any interaction would be (in theory) with the surface and not inside the pores.

loading capacity (related to alpha, surface area, column i.d., ect.)
resolution (k', peak shape, efficiency, selectivity of column)
peak shape (purity of silica, endcapping, ect.)
recovery (column temp, pore size, pore size distribution, purity of silica, ect.)

If you have 20k Da protein - you should stick with widepore column (30nm).
On occassion, 12nm can work for 20kDa (depending on actual size of polymer).