Chromatography Forum

hi..
i have a very basic question..
i work with protein rp-hplc..
i use a say for example, c18 gaurd column, followed by c18 column, same brand.. similar matrix. i have a gradient of say 25-75%acetonitrile with TFA.. at 25% the protein binds and at say around 60% it is seen to elute. now i have a doubt! at 25%.. does my protein bind only to the gaurd column.. cos the amount i load can be carried by the gaurd column. if yes.. wil it elute only at say 55-60%, and then if it is coming with this high % acn.. will it be able to bind the column.. or wud it just travel thru the column? what is the difference in binding at the gaurd column and analytical column.. except for the length and hence the resolution. the protection part of gaurd column i do understand.

thankyou!

Proteins behave just like any other molecule in reversed-phase. At low % organic, the equilibrium distribution strongly favors the stationary phase, and the protein binds. As the %B is increased during a gradient, the equilibrium gradually shifts increasingly toward the stationary phase, and the protein begins to migrate down the column, moving slowly at first, then accelerating as it moves down the column.

The relationship between %B and retention (k') for proteins is very steep, so they accelerate quickly compared to small molecules, but the difference is quantitative not qualitative (i.e., same mechanism, just faster).

So the answer is that the protein does spend proportionally more time interacting with the guard cartridge (because that's where it's migrating more slowly), but it is incorrect to say that it interacts only with the guard cartridge.