Hello,
I'm somewhat new to gradient HPLC method development. I've been trying to separate synthetic impurities of an amino acid by RP-HPLC with UV detection at 205nm. My gradient starts at 98% KH2PO4, pH 3.0, 2% ACN and goes to 70% ACN. I observe, very reproducibly, 2 very large peaks that originate from the buffer solution. I must operate at 205 nm for sensitivity. How do I alleviate these two artifact peaks which elute late in the gradient. I've tried better grades of reagents (KH2PO4 and H3PO4). Has anybody else seen this phenomenon?

Thanks for your help

Water quality could be the issue, including plasticisers in the tubing of your water lines or even your HPLC instrument. Verify if the peaks are getting larger when you equilibrate longer with the starting composition of your gradient.