Question

A polystyrene resin SEC column has a diameter of 8.8 mm and a length of 30 cm. The particle skeleton (solid portion) occupies 20% of the volume, the intraparticle pore space occupies 40% of the volume, and the interstitial volume (interparticle space) constitutes the rest. The column is operated at a flow rate of 1 mL/min

What would the elution time of a very large protein (MW > 600 kDa) that is completely excluded from the pores be?

What would the elution time of a small molecule marker (like uracil) be

A hydrophobic protein elutes at 18.2 min. What does this imply about its retention mechanism?

Answer 1

For proteins that elute later than expected I either assume that either (a) the protein is interacting with the resin, or (b) the AKTA is pumping slower than it thinks it is (check for air in the pumps). For example I have a 14 kDa protein that elutes after 130 ml on a Superdex 75 16/60 (i.e. after the column volume)!

I wouldn't worry too much about your apparent MW being 20-30 kDa and not 40. The column calibration isn't that reliable. SEC MALS is much better. Don't forget, SEC is called "size-exclusion" and not "mass-exclusion" for a reason. The hydrodynamic size is the key thing. In your case it seems you may have some self-interaction and transient binding to the superdex matrix. Do you have enough NaCl in the buffer (~150 mM is advised)? You could try a different column e.g. Sephacryl