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- Posts: 22
- Joined: Tue Oct 18, 2011 12:01 pm
Hope everyone is well.
I've encountered a curious pressure issue with my Waters Acquity Arc Bio HPLC. These HPLC's have the ability to switch between HPLC and UHPLC capabilities.
On Friday last week (8th April) I finished some work on a method I regularly use in the lab to monitor micro-fungal fermentation. Samples are prepped for analysis by centrifugation at 11000 rpm and then the supernatant is filtered through a 0.2um filter. This filtrate is what is injected into the system. The method in question uses an isocratic 5mM sulphuric acid mobile phase, flow rate 0.7 ml/min, USP designation L17 column (Waters Ion-Pak in this case) 300mm x 7.8mm, 7um particle, column oven 40degC, RID detection 55degC, inj vol 20ul. The operating pressure for this is normally 1150 psi and this is what was recorded at the end of operations on Friday. This is all done through the HPLC path of the instrument.
Skip forward to Monday (11th April) and I switched the system to the UHPLC side for a method I'm using to monitor mycotoxins in the same micro-fungal fermentations. Samples are prepped in the same way as above. The method uses a C18, 150mm x 3mm, 2.5um particle column. Mobile phase is a HPLC water:MeCN gradient, flow rate 0.6 ml/min. It starts at 17% B rises to 45% B over 20 minutes. There is then a 5 minute clean at 95% B before returning to 17%B for 5 minutes equilibration. So 30 minutes in total. Column oven 30degC, detection DAD 192nm to 800nm inj vol 50ul. The operating pressure for this is normally around 6500 psi (with the starting mobile phase) which isn't particularly unusual for a UHPLC application.
However after I'd set everything up on Monday I recorded a pressure of nearly 8500 psi! I did a few basic checks but nothing seemed untoward and so, perhaps foolishly given I wanted to get some data that morning, I ran the samples I had given the pressure, whilst high, was safely within a) the pump limit and b) the column limit. After concluding the analysis I allowed the LC to tick over at 0.1ml/min overnight with 100% MeCN (the storage solvent for the column is 100% MeCN in line with what it was shipped in).
The next morning I removed this column and set up my system for the aforementioned ion exclusion method. The pressure recorded was 1750 psi, a huge increase on what I'd seen only a few days ago. I ran a SST sample and the chromatography was horrendous. Preparation of fresh mobile phase and then SST (sequentially) did not improve the situation.
I then tried switching back to column 2. On this occasion the pump started to trip out due to the pressure being above the limit of the pump (9500 psi). I therefore ran the system at 100% MeCN with all other parameters the same. This gave me a pressure of 3149 psi. I left the instrument to tick over at 0.1 ml/min 100% MeCN again overnight. When I came back in the morning the pressure was nearly 3000 psi!
I've used a small 5cm C18 column in between switching my main two columns and a consistent pressure of 880 psi is recorded each time. I've ruled out there being any sort of blockage before you get to the column by running the system without a column and pressures are negligible for the HPLC component and what you'd expect for the UHPLC. Therefore it would appear that the issue lies with both columns given the system pressure for both seems to be increasing with time.
However I'm keen to try and find out what has caused this, particularly given column 1 and my routine HPLC method were fine on Friday. This issue appears to have started on Monday (or at least has been picked up on Monday). I've got some new columns on the way but, of course, without understanding why I'm possibly doomed to experience the same fate again further down the line.
Possible information overload here folks, but then the opening post from Tom does say provide as much as possible, lol. Any help would be much appreciated as I venture forward.
Cheers, James