reverse phase hplc of proteins.. mechanism of binding and elution.. - your views.. ! (Mar/29/2008 )
hii alll...
there are many views on the binding and elution of proteins (or rather any hrydrophobic molecule) on an RP column.
to me.. the protein binds in the first minimal zone of the column and elutes and starts eluting at say 45-60% organic phase and then never rebinds just travels through the column..
do most of us agree? or is there a binding and elution.. rebinding reelution kindof partitioning?
and what is zone size 'as an idea' which the protein covers while binding.. i suppose it wud not be too large right?
any ideas?
it would be the bind-elute, rebind-re-elute... all the way through the column.
but once say 60% organic phase has reached the column.. and the protein elutes at 60% ACN.. how can it bind again? would it be allowed to bind again?
why r u saying what u r saying pls? thanks!
the protein that is eluted will be traveling along with (maybe even slightly ahead of) the mobile phase that is strong enough to elute it. not all interaction is cancelled. so some rebinds then re-elutes and so on. this is one of the reasons that you see sharp rises and tailing of the peaks.
i'm not sure if i'm explaining this well (i can see it in my head but may not be verbalizing well). you may be able to get more complete answers at the chromatography forum.