I have a problem with my HPLC instrument that the pressure increases when it is turned off and restarted the next day and I wonder what I can do about it. I usually analyze nucleotides on a C18 column using a mobile phase of a mixture of solution A (0.7 g/l tetrabutylammonium bromide, 5.8% acetonitrile, 23 g/l KH2PO4 pH adjusted to pH 5.6 with KOH) and solution B (same as A but without phosphate) at 1 ml/min. In the end of the day, I usually equilibrate the column for 10 min with 7% acetonitrile before turning it off. When I change pump solutions, I usually suck in 15 ml of the new solution before starting the pump with the new solution (in this case 7% acetoitrile).
Nowadays when I come back in the morning and turn the HPLC on it is always much higher pressure than the day before regardless if I use solution A, B or just 7% acetonitrile. The normal pressure is usually around 300 bar, but it can be up to 50 bar higher when I start the HPLC the next morning. To normalize the pressure, I have to run the guard column backwards at a high speed and force whatever is blocking the column out. It does not matter what samples I run, this happens also if I just run standards. I have no idea what is accumulating over night and have been thinking if this could be air, bacteria or trapped salts but it is definitely something that seems to increase over time when the pumps are still. The higher pressure is generally built up on the guard column but I do not know if it starts there or if it is particles that originate somewhere else but follow the stream and end up there. It does not seem to help if I equilibrate the system including the guard column with 70% methanol or isopropanol before turning the pumps off. I have also tried to change precolumn numerous times without success.
To address the increasing pressure in your HPLC system, check for blockages in the guard and main columns, thoroughly purge the system to remove air or residues, and inspect pump components for wear. Regular maintenance and proper mobile phase preparation can also help prevent pressure fluctuations.
Thanks a lot for your suggestions. I think I have done all of that, including numerous washes/purges, and changed the pistons and packings in the pump. The situation has become better though and now the pressure will normalize after running the pumps at low speed for 20 min when I start up the HPLC in the morning before using the regular speed. I am still curious what it is though that is causing this problem, and what is the most efficient type of washes in this case. So far, the most efficient wash for me has been 0.4 M H3PO4 (containing 7% ACN just to protect the precolumn). There is no meaning to wash the column the same way since the high pressure develops over the precolumn and not the column.
Hi Anders Hofer,
The concentration of phosphate buffer (23 g/L KH2PO4) in the mobile phase is too high, which could be a significant factor contributing to the elevated pressure. I strongly recommend the following procedure:
This should help remove any buffer deposits, if present.
Thanks a lot for the answer. However, do you really think that phosphate precipitation could be the main problem although the organic content in the buffers is so low? Both solution A and B contain only 5.6% acetonitrile. Nevertheless, it is a good idea to try to wash with high temperature water and I will try it.
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