-
- Posts: 2
- Joined: Wed Aug 24, 2022 9:55 pm
I am not sure if I am yelling into the ether, but I have been a lurker on these forums for a few months and they usually help a lot, so I thought I would give it a shot. If you couldn't tell from my username, I am a total newbie at HPLC (so please forgive me for my stupidity on certain things). I am also working on a slightly different software (Masshunter Data Acquisition to ChemStation for HPLC) than I have before. That being said, I am currently developing an assay that requires the determination of dwell volume/gradient delay volume/system delay volume (these have all been used interchangeably in the literature I have read, but correct me if I am wrong).
I attempted to do this by following a technique utilizing a mobile phase A of methanol and a mobile phase B of 0.1% acetone in meOH detailed on the HPLCtips blog (https://hplctips.blogspot.com/2017/02/d ... olume.html).
My mobile phases were as follows:
A: Water (HPLC-grade)
B: 0.1% acetone in water (also HPLC-grade)
I knew of similar tests run with 0.1 mM sodium nitrate in water as mobile phase B, so I thought this would be alright.
I removed the column and used a zero dead volume union. My gradient from 100% A to 100% B ran over the course of 10 minutes at a flow rate of 0.4 mL/min (very slow, I know, but we usually have issues with pressure** when we do have our column in and I wanted to keep the parameters similar enough) and a temperature of 40°C (again, just trying to keep parameters similar, though this may have been too warm for the pressurized acetone in retrospect). I flushed the system with 100% B for around half an hour, then 100% A for around half an hour. The baseline had stabilized at 0 with the water, and I ran my gradient.
I left for lunch and looked at my data when I returned. To my surprise, the signal looked more like a decreasing logarithmic function (it went WELL into the negatives). I ran the gradient again at ambient temperature to check, getting the same results.
I flushed with copious amounts of water at higher flow rates (1-2 mL/min) as I was worried a bubble had entered the system due to the acetone. Could this be the error? Should I remove the flow cell to sonicate it? Or is it a lost cause?
A couple of sources of error/factors which may be useful to the diagnosis of this issue:
Problem 1: Our lab did not have HPLC-grade acetone, but I was advised to use our anhydrous acetone (99.8%) in its place. It was not a new bottle, but it was septum-sealed and had to be removed using a cannula.
Problem 2: Adding even more to the gas being the issue, until very recently, the degasser was incorrectly installed (someone put the input lines into the output of the degasser and the output lines into the input). I reinstalled it correctly, but I am worried it may have done some damage (would this also explain the pressure issue?**).
**I am talking we are using a 2.1 mm x 50 mm column and it has a pressure ~ 100 bar with ACN alone
I am just at a total loss at the moment, so I would appreciate any help I can get greatly! Thank you so much.