Chromatography Forum

I need a little help here with a fairly basic issue, that for some reason I am getting stuck on.

We have a method that monitors at a wavelength of 205 nm. We just switched from a Methanol mobile phase to Acetonitrile, and the absorbance increased. I know that this is due to the lower UV cutoff of ACN, but I would like to understand exactly how this works.

When the mobile phase has strong absorption you are essentially working on a high baseline (though you don't see it since absorbance is, by defenition, a subtraction of incident and transmitted light). But still we are in a high absorbance range, which means we are in the range where there would be significant non-linearity. I think this may be the reason for the reduced response. But is this the only reason for the reduced response. Or am I missing something.

Hi Adam,

The more absorbance, the mobile phase exhibits, the less is the effective absorbance range left for the analyte to operate in.
If you have 2 AU absorbance range (in a perfect world i.e. no absorbance from the mobile phase), the analyte has the hole range for it self. Now if you introduce a mobile phase that absorbs 1 AU, the analyte has to live with the remaining 1 AU, even though you don’t see the range reduction due to the autozeroing of the signal at the beginning of the chromatogram.

Best Regards

Hi Adam,

Bit confused by your post. Are you saying that the ACN absorbs more than MEOH at 205? If that is the case then you must have some contaminated ACN. UV cutoff for MeOH is 205 and for ACN it is 190 nm. UV cutoff means the wavelength at which the solvent has an absorbance of 1 AU for 1 cm path.

Yeah... at 205 the absorbance of MeOH is about 1 AU and for ACN it should be probably less than 0.1 or near zero. Do you mean your analyte peaks show higher absorption with ACN? That would make some sense.

No, I am saying that because of the lower UV cuttoff of ACN, the analyte has a higher area count (vs with methanol mobile phase).

adam, besides the nonlinearity you mention there is the possibility that the baseline is so noisy that the peak integration doesn´t work correctly with the MeOH. With a large peak I would bet on nonlinearity for a cause.
In any case, the real cause is easily determined.

...err doesn't extinction coefficient depend on solvent anyway?

It seems that the primary effect in changing solvent is a change of wavelength, which can cause a change of absorption at a given wavelength. That is why one does not use absorbance coefficients determined in different media. If Adam didn´t recalibrate after changing solvents he needs to learn a bit yet.

For a moment there I thought we might get through the discussion without any hot gas.

Yes the absorptivity is a function of the solvent, but this is a moderate effect. What we were seeing was far more significant and - I'm sure - due to the fact that we are at a low wavelength, where methanol absorbs significantly but where acetonitrile does not absorb much.

So far it seems most agree with my suggestion that the reason for the lesser sensitivity with methanol is that when the mobile phase absorbs significantly it puts us in a range where the response curve becomes non-linear and (more to the point) flatter - i.e. there is less absorbance for a given concentration.

But I am still open to any other suggestions.

Adam- I agree that there should be very little change in lambda max hence absorptivity, particularly in an aqueous environment.

I guess I wouldn't be surprised to see a fairly drastic effect. Try injecting analyte such that it gives an absorbance of 2.0 in ACN. That is essentially what you are doing when you measure an analyte absorbance of 1.0 in methanol at 205. I expect that it will take significantly more than twice the concentration (or volume injected) to get an absorbance of 2.0 than it does to get 1.0 with your ACN.

What is the analyte? Maybe there is some chemistry going on too if you have something like and enol or acid.

In my opinion there is lots of hot air in discussing this without knowing what the wavelength shift is, or discussing this without a recalibration.
When you get significant nonlinearity due to high total absorbance you are close to blacking out the detector. It is very easy and quick to check that by increasing the amount of analyte and noting whether the top of the peak flattens (if it wasn´t flat already). If that happens you can add as much more analyte as you want without much, if any, change in absorbance.