I'm trying to to purify protein sample using agilent HPLC system with C18 column. The moment I load sample and start program within few minutes the desired protein elute out along with solvent peaks. I tried changing mobile phase polarity (ACN- 40%, 20% and 15%) for 40 mins time to resolve but irrespective of polarity the sample elute out in initial few minutes.
How to tackle this problem?