I am using C18 column for analyzing urine and my mobile phases include water, methanol and 250 mM phosphate buffer, pH 6.2 in HPLC UV. The flow rate is 0.300 ml/minute. Everything looks normal except yesterday because pump pressure is increased. I have used 100 % water but it did not work. Can any fellow suggest me the possible solution for removal of phosphate buffer and to reduce the prossure.
Dear Shehwaz,
When buffer salts are used in mobile phase/s, be careful that the buffer salts are not allowed to precipitate out of solution and cause the pump parts to stick. Hence when the system is not used for injections, ensure that the buffer solution is always circulated through the pumps at a low flow rate, such as 0.2 ml/minute. If necessary, re-circulate the mobile phase/s. Never let the pump lie unused for more than around ten minutes, if there are buffer salts within the mobile phase/s.
When you have finished using the buffer, flush the system with 15%/85% v/v methanol /water for two hours. When buffers with a very high salt content are used, it is useful to back-flush the pump pistons, please see the pump user manual.
Using 100% water will cause the buffer salts to precipitate out of solution.
It is possible that phosphate buffer has precipitated out and blocked parts.
If there is a blockage, from the precipitation of phosphate buffer, then this would explain a pressure build up. Cleaning blockages is tricky. Disconnect the pump from the injection valve, and try, even for a few moments at a time, to pump warm 15%/85 % methanol/water (HPLC grade) through the pump, into waste. Try to clean the check valves with acetone (HPLC grade).
If/when the pump starts to pump, clean the injection ports and flow cell by pumping warm 15%/85% methanol/water (HPLC grade), through the injection ports and flow cell, without any column being in place.
Kind regards,
Ade Kujore
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Follow the washing procedure carefully (use many solvents aq...organic...aq).
Thanks a lot Ade Kujore and Abdallah Hussien Fathy,
I did backflush of column according to manual, and passed 5% methanol : 95 % water for two hours at low flow rate (0.1 ml/minute). Now it looks working and pressure is reduced from 9.9 to 6.0.
Let us monitor for full night.
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