Change in UV baseline during injections

Hi Rossf011

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

Hi Rossf011

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