reversephase hplc of proteins..mechanism of binding/elution

[alicee!]

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?

[tom jupille]

There is good discussion of that entire topic in the book "Basic HPLC and CE of Biomolecules" by Cunico, Gooding, and Wehr. To a first approximation, retention in reversed-phase is described by a log-normal relationship between k' and the fraction strong solvent in the mobile phase (i.e., log(k') is a linear function of %B). The slope is typically varies approximately with the square root of molecular weight, so that a small molecule shows a shallow slope, something like a peptide a higher slope, and a protein even higher. For large proteins, the slope is steep enough that it effectively looks like an "on/off" mechanism, but it's actually just an extension of general behavior.