by
Zoraku » Wed May 09, 2018 2:38 pm
Rndirk wrote:
I think it was worth the try. You're ruled out quite a bit already:
- Column issue
- Instrument issue
- Standard/sample degradation
- Mismatch between injection solvent/mobile phase
- Temperature should be stable
In my opinion, the next thing worth taking a look at is the mobile phase. Is the MeOH/water mixture prepared in a single bottle or do you use the pump to mix? Is it prepared freshly? Did you by any chance change vendors for MeOH/water recently? Could you take a different source of MeOH and water to prepare the mobile phase and try?
If prepared in a single bottle, note that making a MeOH/water mixture is trickier than it sounds. Taking 100mL MeOH and adding to the 500mL mark with water will NOT give the same mixture as taking 400mL water and adding to the 500mL mark with MeOH. The way you prepare it should be written down in an SOP and always be the same.
Vendor for methanol has not changed, we use Thermofisher. Our mobile phase is mixed online, with each individual component being degassed as well as possible prior to placing the solvents on the HPLC.
I would have to double check the actual lot number of the HPLC water and Methanol I am using, it could be an issue with the lot itself - great thinking.
I have changed the methanol and water out, but as I just mentioned earlier, I may have replaced it with the same lot - just different physical bottle.
Unfortunately my predecessors in this position were not very thorough with writing SOP's or spelling out how they prepared their mobile phase. I am very familiar with solvent expansion and contraction when mixing organics and aqueous liquids together and I usually practice measuring them separately in separate graduated cylinders prior to mixing together but unfortunately the way it was prepared for this method specifically was never written down (whether the method was developed by online mixing or mixing together prior to putting on the instrument).
Because it was never written down I always just mixed online and had results as expected.
I'll be having a PM performed on the instrument soon and I will be sure to have a very thorough maintenance replacing rotor seals, pump seals, etc...
Until then I will look at the solvents and see if I can find out anything more. As I had mentioned earlier, even though the peak looks to be poorly shaped - the phenomena occurs in both standard and formulations and isn't adversely impacting my quantification. Even so, I want to figure out what's going on.