Chromatography Forum

Hello,

for the quantification of amino acid, I am using a C18 column. Mobile phase is 40mM phosphate puffer pH 7.8 (channel A) and acetonitrile/ methanol/ water 45:45:10 (channel B). Furthermore, to quantify amino acids, OPA derivatization on- line prior to injection was performed. The method worked really well, but recently I have problems with too high pressure. I have disconnected my column from the system, and the pressure remained high. I would assume that I have salt precipitation in the system. Therefore, I have used warm water to flush the system without the coulumn. The pressure is still very high. I am wotking with Agilent binary pump.

Thanks

Disconnect each fitting working from the column back towards the pump, when you find the connection where the pressure drops then you know the blockage if from there forward.

It can be in the autosampler valve, or the outlet valve or the mixing area between the pumps, but you have to just work stepwise until you know where it could be. If it is the valve at the autosampler you may need to replace the rotor or simply sonicate the valve face to remove a blockage in one of the ports.

First thing I would do would be to replace the PTFE purge frit in the purge valve.

After that I would run the system with the column inlet disconnected using 100% water. Route the tubing into a waste beaker.