Change in UV baseline during injections

Hello,

I am running an Agilent 1100 series with a quat pump. The column is Agilent Poroshell 120 EC-C18 3.0x50mmx2.7um. Column compartment is at 50C. UV signal is 228nm. The mobile phases are 0.1% formic acid in water and 0.05% formic acid in methanol with a flow rate of 1.0mL/min. The gradient looks like this: 

Time Mobile Phase A Mobile Phase B

0             40                     60

1.0         40                      60

7.0         23                      77

8.2         5                        95

9.5         5                        95

10         40                       60

13         40                       60

I have been getting a consistent UV baseline during injections during the few weeks I've been running this system. Then I had to replace MPB and started getting a different baseline profile. I remade the mobile phase using newly opened methanol and cleaned all glassware used prior, with the same result. I tried making new MPA as well without any success. Nothing on the system changed in between these events so I assume it is related to the mobile phase but I cannot get the baseline to look how it did before. Here is an example below where the blue trace was how it looked before and the green trace is now it is looking now. The baseline is declining as the organic phase ramps in the gradient.

Any thoughts on what could be attributing to this?

Thank you!

Top Replies

  • Hi

    There is clearly a difference between the fresh mobile phases that you have just prepared and the mobile phases you used before.  When you first started, did you prepare fresh mobile phases…

  • Hello,

    if you look at the baseline for multiple injections, is it the same for every injections now? Also is the pressure alike to what you had before you changed mobile phase?

  • Hi

    There is clearly a difference between the fresh mobile phases that you have just prepared and the mobile phases you used before.  When you first started, did you prepare fresh mobile phases?  That may sound like an odd question but, for what it's worth I actually prefer the green chromatogram to the blue one.  The green chromatogram has a cleaner, "flatter" baseline than the blue one.

    Was the older mobile phase prepared with the same formic acid?

    When troubleshooting problems like this, I think about what the chromatograms are showing me.   

    The green chromatogram suggests that mobile phase B absorbs less at 228nm than mobile phase A.  Is that reasonable?  Mobile phase B contains methanol which has some absorbance at 228nm, and formic acid also absorbs at this same wavelength.  Does the lower percentage of formic acid in mobile phase B compensate for the absorbance of methanol?  In both cases, maybe the answer is no.

    The blue chromatogram suggests that either mobile phase B absorbs more at 228nm than mobile phase B.  Is that reasonable?  I could repeat the arguments above, but what I can also see in the blue chromatogram are lumps and bumps in the middle of the chromatogram.  To me, these look like some contamination being eluted slowly from the column.  These contaminants could come from many places, but I would suspect that they come from the mobile phase.

    I don't want to guess at the problem, but I think the probable cause of the difference lies with the mobile phases.

    I would always recommend preparing fresh mobile phases daily, if that is practical.  Make what you need and always use clean bottles; avoid the temptation to just top-up the bottles that are on the instrument.  Using this approach, you avoid the possibilities of contamination due ageing mobile phases.

    Hope that long answer gives you some help to understand what you're seeing.

    /Andy

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