Chromatography Forum

Hi,

I recently configured a new Agilent system to run size exclusion on various antibodies varying in size from roughly 50 kDa to 200 kDa. All works well; however I have never had to determine K and Alpha values before. I can find plenty of literature but am still struggling to understand how I may identify the correct values to allow calibration of the system and estimation of molecular weights.

Will one value suit all my samples or will each require alterations?

Any help would be appreciated.

Alpha is to be determined for a critical separation of a peak pair.
If more than 2 components are to be separated, Alpha could be determined for all components using the nearest peaks for reference. Alternatively one can identify the most critical separation – i.e. the closest 2 peaks and argue that if those peaks are separated all the others (the less critical ones) should be OK as well.
K is determined for the peak/s of interest – typically the one that is to be isolated or reported, using the exclusion volume as a dead time/volume. The inclusion volume could be used for reference as well, but it should be noted and used consequently.

Best Regards