Baseline rise with temperature

[Kevin Kelly]

I am running a gradient UPLC method (flowrate 0.5 mL/min) that starts out 53:47 (pH 6 NH4Acetate) : ((50:50) ACN:MeOH). The gradient starts 10 minutes after isocratic to 47:53 at 20 minutes, then isocratic for 10 minutes. At column temp of 10C the baseline goes down during gradient to about -0.006 AUFS (and stays down during the second isocratic step); at column temp of 20C or a column temp of 30C the baseline goes up during the gradient to about 0.4 AUFS (and stays up during the second isocratic step). The column temperature is monitored and is constant. The baseline change is repeatable over 10 injections of 30 minutes each. Does anyone have an explanantion why the baseline behaves like this?

[Uwe Neue]

Changes in the refractive index can influence this behaviour. The changes in RI will depend on the temperature.

[Phycal]

What wavelength are you monitoring at? If you are using 210 - 220 nm you may have to look at the grade of solvents being used (I am assuming it is absorbance based on the fact you are using AUFS).

I would check all the chemicals to make sure you using the highest grade possible - HPLC grade at least (especially for the ammonium acetate). Also, you do know that you are not in the buffering range of ammonium acetate at pH 6.0? pKa is 4.8 - so you need to be between 3.8 and 5.8 to be in the buffering capacity of ammonium acetate (also what concentration?)

[HW Mueller]

Phycal, how do you explain the temp. effect with impurities?
I would have expected a downtrent of the baseline at all temps, as acetate/acetic acid appears to be the strongest light absorber here. I also associate an increase in temp. with a decrease in RI for probaly most substances, but there seem to be some surprises here.
Another possibility I see, especially since this pH is not buffered, that mixing in organic and increasing the temp. lowers the pH such that an esterification occurs. The methyl ester absorbs at higher wavelength.

[Phycal]

Phycal, how do you explain the temp. effect with impurities?
I would have expected a downtrent of the baseline at all temps, as acetate/acetic acid appears to be the strongest light absorber here. I also associate an increase in temp. with a decrease in RI for probaly most substances, but there seem to be some surprises here.
Another possibility I see, especially since this pH is not buffered, that mixing in organic and increasing the temp. lowers the pH such that an esterification occurs. The methyl ester absorbs at higher wavelength.

Well - if there is impurities - they are washing them off. As I said also, they are NOT buffering anything at that pH with ammonium acetate.

It is not RI - due to them saying AUFS (Absorbance Units Full Scale) - RIU would be what is normally used for RI. That is why I asked what wavelength they are detecting at.

We basically need more information (what column and phase) as well as detection method and wavelength if UV-Vis.

[HW Mueller]

Phycal, you didn´t explain the temp. effect.

We are talking about an RI effect in UV/VIS absorption detectors.

[Phycal]

My take is that there may be some contaminant on the column that at one temp will elute and at other temp will not - may due to a pH shift but it has been a long time since I have used ammonium acetate in LC. Temp can affect pH in major ways - particularly with ammonium acetate out of the buffering range.

[tom jupille]

In my experience, most temperature-related baseline effects with UV detectors are due to refractive index changes. All UV detectors have some sensitivity to refractive index, because it is impossible to perfectly collimate a light beam (lasers excepted!) so that it is perfectly parallel. That means that at least *some* of the light will enter the flow cell off-axis and deflect at the interface between the window and the mobile phase. How strongly the detector responds will depend on the design of the flow cell and optics (some detectors are better than others), and also on the alignment of the flow cell with the optical axis of the detector (if it's out of whack, the effect will be worse).

While we *can* speculate about temperature-induced pH shifts, "Occam's razor" (http://en.wikipedia.org/wiki/Occam%27s_razor) makes the RI explanation the more likely one (to my mind, anyway!).

[Kevin Kelly]

Thank you everyone for your comments and ideas.

To answer some good (and important!! I should have included this info in my original topic) questions asked by the Forum members, this analysis is performed with 0.05M NH4Acetate (at the non-ideal pH=6) and measuring the UV absorbance at 235nm on a Kinetex C18 100 x 3.0 mm x 3.6 um. All organics are HPLC grade, water is MilliQ, and NH4Acetate is ACS reagent.

Phycal, we don't think this is column contaminate problem as we are only injecting standard solutions of pure compounds made up in 75:25 methnaol:water. Additionally, injections of diluent (75:25 MeOH:Water) give the same temperature dependence of the baseline disturbance.

We too are leaning toward some sort of RI interference of the baseline absorbance (ala Uwe Neue, HW Mueller, and Tom Jupille).

Thanks again to everyone for your help.
Kevin