HPLC Troubleshooting

[Mr Bates88]

I currently work for a quality lab for a sanitizer/soap manufacturing facility. I am experiencing a problem with my Varian HPLC. The system is composed of two ProStar 210 pumps and a 325 detector. I am specifically testing PCMX. The eluents are Methanol and 0.25M Sodium perchlorate adjusted to a pH of 2.5. The method is to be ran at 85% Methanol and 15% Sodium perchlorate at 1mL/min. The detector uses a deuterium lamp.

I first experienced the problem a week ago. The pressure on the instrument when flowing 1mL/min of Methanol is typically around 1100-1200. When the problem originally occurred, I noticed the pressure of flowing 1mL/min of Methanol was down to ~850psi. I checked all connections of the HPLC and have not found any leaks. I then removed the column and tested the pump at 2mL/min for 20 minutes. I collected the eluent and at 20 minutes the amount was almost spot on at 40mL. I then reconnected the column. After injecting a few samples the retention time of my PCMX peak was extending further and further out. Initially the retention time was around 4.8 minutes and continued to extend out (5.1min, 5.25min, 5.75min, 17min).

Even though the pump test without the column was successful, I figured there might still be an issue with the pump head. I removed the pump head and replaced the consumables within it (seals, back-up seals, and o-ring). I reconnected the pump head and attempted to flow 1mL/min of methanol through the system. The pressure was back up to 1200psi and was holding consistent. I figured the system might be fixed and started to shoot samples again. I tried to calibrate the instrument 4 or 5 times across 2 days. The calibration never came out great; however, the retention time for the PCMX peak was very consistent. The next calibration I tried, was not as successful. The PCMX peak began to shift later once again. I pushed 1mL/min of methanol through the system again and the pressure was back down to 860psi.

I figured I had deduced that the problem was the pump head, since removing it and rreplacing the consumables seemed to have resolved the issues for ~12 hours of operation; however, I was a bit baffled when the problem reemerged.

I have replaced the consumables in the mixer and I have replaced the inline filter that is after the mixer, but before the injection valve. Nothing has seemed to have helped.

Yesterday I took off the cover on the detector and did notice a bit of salt buildup surrounding the connection to the outlet port of the flowcell. My next thought is to try to clean/replace the flowcell. I have not gone down this path yet, because I'm not quite sure how a flowcell would cause the issues that I'm experiencing.

Does anyone have an idea as to what might be wrong with my instrument?

[Consumer Products Guy]

Wow, Master Bates, that mobile phase is way too complex for the analysis of PCMX. Unfortunately, if your company cGMP-validated such procedure, you may be stuck with it.

As to that unit: my only hands-on experience is with Agilent stuff over 4 decades. But I will volunteer that we used 0.5% acetic acid in water with either methanol or ACN as the organic, and detection at 280nm. With acetic acid as the modifier/acid component, there's no chance of any precipitation in the system causing higher pressures.

So keep the mobile phase simple, like acidify with acetic acid or phosphoric acid and keep anything with sodium away if you can, just potential for issues. That way you can eliminate pH meter steps and filtration of your buffer.

Try washing out your flowcell with water, and your system with 10% methanol in water. Start with low flow rates.

[Mr Bates88]

Try washing out your flowcell with water, and your system with 10% methanol in water. Start with low flow rates.

Would a contaminated flowcell cause such issues?

Thanks

[Mr Bates88]

I was trying to purge the system with water. Does it make any sense that when pumping 0.5mL/min of water through my pump A (Typically methanol) that my pressure is around 350psi, but when i pump 0.5mL/min of water through my pump B (Typically the Sodium perchlorate) my pressure is around 1100psi?

The pressure module is installed on the side of pump A. So I have a SS line running from pump A to the pressure module, and then a SS line running from the pressure module to the mixer which is on the side of pump B. Pump B line goes straight to the mixer.

[James_Ball]

I was trying to purge the system with water. Does it make any sense that when pumping 0.5mL/min of water through my pump A (Typically methanol) that my pressure is around 350psi, but when i pump 0.5mL/min of water through my pump B (Typically the Sodium perchlorate) my pressure is around 1100psi?

The pressure module is installed on the side of pump A. So I have a SS line running from pump A to the pressure module, and then a SS line running from the pressure module to the mixer which is on the side of pump B. Pump B line goes straight to the mixer.

When you replaced the components in the pump head, did you also replace the pistons? If not, did you examine the pistons under a microscope or a good magnifying glass? If there are scratches on the piston, it can quickly degrade the new pump seals and cause the low pressure to return.