Did you ever feel like someone was out to get you? I remember the story one of my Master Class students told about leaving on a vacation. She left instructions with her lab mate to shut down her HPLC system the following morning after all the samples had run but when she returned from vacation two weeks later, she found that all her friend had done was turn off the power. She could hardly see through the bottle of buffer as there was so much junk growing in it.
How do you recover from such an episode? My approach would be to do a major system clean and replace some of the parts that are not worth cleaning. First, I would remove the column and replace it with a piece of connecting tubing. Next, remove all the frits from the solvent inlet lines and place the lines in a bottle of HPLC-grade water. Purge the system so that 10–20 mL of water flows through each line. Hopefully this will flush any buffers and solvents from the system.
The primary system cleaning procedure is to flush it with 30% phosphoric acid. Just take a bottle of concentrated phosphoric acid and dilute 2 parts with 1 part of water. Then pump ≈20 mL of this through each solvent channel. This should remove any microbial contamination from inside the tubing, degasser, pump, autosamplers and detector. After flushing with acid, flush with HPLC-grade water. You should be able to tell when the acid is washed out by checking the pH of the waste leaving the detector.
Now replace the frits in the solvent reservoirs with new ones — I prefer large-porosity frits, such as 10–15 µm. These are not worth trying to clean. Install a known good column and run a system test or set up a simple and reliable method and run a system suitability test. Hopefully you’ll be back in business at this point. If there is trouble maintaining pressure, you may need to clean or replace the check valves and replace the pump seals.
Once the system is working well, you can check the original column and see if it is still serviceable. It may have survived unscathed or sitting in buffered mobile phase for a couple weeks may have ruined it.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com
