Hi, I am currently on a method development of GFC and I am very new on this technique. I use polystyrene sulfonate as standards and acetone as a maker of the exclusion volume of the column. However, the retention time of acetone seems different under different mobile phase. At a flow rate of 1ml/min, the retention time of acetone is 12.5 min in 100% water, 11.9min in 75% 10mM/20mM phosphate buffer + 25%ACN, 11.5min in 65%10mM/20mM phosphate buffer + 35%ACN, 13.8min in 80% (50mM phosphate buffer + 0.1M NaCl) + 20% MeOH. This is confusing me since there should be no secondary interactions between acetone and the column matrix, theoretically, the retention time should be the same, unless there are some fundamental that I missed since I never use SEC before.

Every time before changing the buffer, I flushed the column thoroughly with water and 30%ACN, it shouldn't be the retention problem (the retention times are very reproducible under each mobile phase). I also noticed that even the calibration curve made with polystyrene sulfonate are not linear at the low Mw, the slope became less steep, which also confuse me.

I wonder if acetone is a good marker for the exclusion volume, and how can I determine the exclusion volume at its optimal condition (all the secondary interactions are suppressed to the minimum)? More importantly, can anyone tell me why the calibration curve became less steep at the lower Mw?

Many thanks in advance!