my protein gives huge tailing.. - rp-hplc for a protein (Aug/18/2007 )
i m doing an rp-hplc for a protein.. on c4 column.. acetonitrile + tfa and water with tfa gradient. with any amounts of protein loaded, my protein gives huge tailing.. wat are the reasons. how do i overcome these?
It looks as though the protein is too sticky for the column. Check the column with other proteins, and see how they behave. Is the protein very hydrophobic?
Q1: How old is the column?
Q2: Are you confident the column is in good condition?
Q3: Do you have any other columns you cold use, say, a C8 or C18? If not, have you tried other solvents, such as isopropanol?
Do you have to use RP-HPLC? HIC can work well with hydrophobic proteins, and you can usually rely on ion exchange.
All the best.