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gaurd column binding.. rp-hplc - (Apr/03/2008 )

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.



yes, it will bind to the guard column. but it will also interact with the matrix in the column.