Hi Everyone,
I was wondering if you guys had any ideas/suggestions that I can work with to try to fix my HPLC. I have attached a picture of one of our Blanks (100% ACN). This shape is unusual, however it is consistent.
It is usually smooth along 0 mAU for the majority of the blank with a slight upward slope up to about 1 mAU by 10min.
We are running c18 Raptor-Ark from Reztek with ACN, H2O and Phosphoric acid.
Gradient: 70% ACN + 30% H20 ---> 95% ACN + 5% H2O over the course of 13min. Both phases are acidified with 0.085% H3PO4
We tried changing to another column we had on site (Not in the greatest shape) and the chromatogram looked nearly identical to the one in this image.
What is going on here, how should I go about systematically troubleshooting this?
Also I apologize for the low quality image, I took it on my phone as I didn't have my flash drive with me.
Thank you all for the help.
You most likely have contamination of your mobile phase (or of the column). You also may not have a proper column wash step/time after each analysis to allow for proper removal of any retained matrix and allow for re-equilibration (which should never be part of the analysis method, but separate). ACN is poor at dissolving and removing many types of material, esp at high concentrations. MeOH is often better for this purpose. However, the "best" wash solution to use is always the one (or group of solutions) that FULLY dissolves all of the material to wash it off the column.
PS: You referred to your injection of ACN as a "blank". A true blank would be the mobile phase solution, not pure ACN. If you were running isocratically, a blank would be the acidified 70/30 solution, but since you are running a gradient, no true blank exists (so you should use the acidified solution,70/30 as a T0 blank). HPLC takes many years to achieve even a basic level of proficiency so seek out more training in the fundamentals as well as specific training on the hardware and software used.
Have you done multiple blank injections to verify that you do not have contamination in the system? If you have is the base line constant or does it drop off?
You most likely have contamination of your mobile phase (or of the column). You also may not have a proper column wash step/time after each analysis to allow for proper removal of any retained matrix and allow for re-equilibration (which should never be part of the analysis method, but separate). ACN is poor at dissolving and removing many types of material, esp at high concentrations. MeOH is often better for this purpose. However, the "best" wash solution to use is always the one (or group of solutions) that FULLY dissolves all of the material to wash it off the column.
PS: You referred to your injection of ACN as a "blank". A true blank would be the mobile phase solution, not pure ACN. If you were running isocratically, a blank would be the acidified 70/30 solution, but since you are running a gradient, no true blank exists (so you should use the acidified solution,70/30 as a T0 blank). HPLC takes many years to achieve even a basic level of proficiency so seek out more training in the fundamentals as well as specific training on the hardware and software used.
Hi !
I agree with most of the comments you received so far:
- First replace all your mobile phases with fresh ones it is most likely a contamination (I guess you filter your phase before entering the system) and check if that helps.
- you can try running the mobile phase through a zero-dead volume fittings (avoid passing eluents through the column) and check if you still have this weird baseline. (if so, the column is likely not an issue)
- check if you see this fluctuation when you simply monitor it (without injection) in that case maybe the pump my be the problem (unlikely but HPLCs are capricious sometimes)
- check if any of those bumps corresponds to big pressure variations in your gradient program
That should already help figuring which part of the machine to blame !
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