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- Posts: 104
- Joined: Sun Sep 13, 2015 7:54 am
These are, as I mentioned, the exact same standard solution, utilizing the exact same mobile phase and column. The parameters are as follows:
40/40/20 - Acetonitrile/Methanol/2% Ammonium Acetate pH 5.3 buffer
1.0mL/min
30ºC column temperature
Column: EC 4/3 Nucleoshell Bluebird RP18 2.7um column (50x4.6mm)
I use a guard column that I replaced, changed nothing but pressure (decreased system pressure by about 10bar).
I have double checked the pH of the mobile phase...as this molecule is very sensitive to pH, and it came out to be 5.27, which is acceptable.
I used to run this sample on a longer 150x4.6mm column and the retention times I'm seeing now are more similar to what I would have seen running the longer column. I have been running this column with amazing success for the past 4 - 5 months with the reduced retention times...and all of a sudden...retention time shift. It is also consistent. Multiple injections of not only this standard, but other standard concentrations and sample preparations are giving now consistent longer retentions.
I am not convinced there's an issue with the standard solution. Furthermore, there is a set of isomers that is nearly identical to these...they elute slightly later. That standard solution is also showing a required 25min run time to elute both isomers when it should only be 6 minutes with this column.
I'm not seeing any pressure increase or pressure fluctuations between injections, no leaks, no air. I am at a loss as to what could be causing this change...literally nothing has changed between last week and this week in regards to running these samples yet I'm seeing significantly longer retention. The only thing I can think of is something happened that significantly altered the column chemistry significantly...but I've used many columns for many injections and never seen such a sudden change of retention time. My experience has always been a gradual shift if the column starts to get used more and more frequently.
Any ideas as to where I can start looking would be helpful.