Chromatography Forum

Hi, I ran the same sample twice using the same analytical conditions, but in one instance i used methanol as the organic modifier and in the other I used acetonitrile. In acetonitrile the active response is markedly decreased compared with the response in methanol. Has anyone seen this before.
Can anyone explain what is happening.
Hope someone can help
Thanks
Liv

Hi Olivia,

what detector are you using?

Liv,

Your problem interests me but I have to admit I need more information to help think of reasons.

If you are using UV detection, then one possiblility is that the solvents shift the wavelength of absorbtion for your analyte. This is uncommon and would be more likely if your peak came off very fast, before the sample was thoroughly mixed with the mobile phase. If your analyte has strong hydrogen-bonding character, this might be a possibility, especially if you are using a narrow band-pass UV detector and you are monoitoring off-peak or off the maxima a bit.

If you are using ELSD, then I might expect that the droplet size would be impacted by the sovation of the analyte. Again, this would require that the sample did not fully mix with the mobile phase or fully equilibrate before eluting.

These ideas may be off base, but the more you can share about how you are running your assay, the more we can generate ideas and see if they make sense. I would like to know what your column phase, mobile phase and detection are for a start. If you can provide a general idea of the analyte, then we can be more definite in our ideas.

Thanks for your patience as we work through this.

I hope that some others may have seen this change and may be able to help.

Hi, thank you so much for the responses
I am using an Agilent vwd detector
Many thanks
Liv

UV, hmm...

Does your analyte have a pK near the pH of the mobile phase?

Methanol has a higher absorbance than acetonitrile, especially below 230nm, so that would not explain your observation.

Some remote possibilities are that your sample is not completely soluble in the acetonitrile mobile phase, or that any change in retention with the different solvents was not handled well by the integrator.

Good luck,

Bruce Hamilton

Hi Liv,

Did you use the same aqueous/organic ratio in both cases? How about the retention time – did the analyte elute at the same time both with ACN and methanol as a modifier? Also, what is the detection wavelength?
Finally, when you refer to response, do you mean peak area or peak height?

Best Regards

Are you injecting exactly the same sample when using the different mobile phases?

Have you tried a linearity check with both mobile phases.

You could try a UV scan in the 2 phases.

I think the most likely problem is solubility as anumber of substances are less soluble in acetonirile compared to methanol solutions.

Olivia,

I am curious what you have found as the source of your detection differences. Can you update us on what your response is to the various suggestions and what you now think is the source of the difference?

Thanks for whatever you can share.

olivia, acetonitrile has tendencies to attarct the proton so if u use the acetonitrile as a organic mobile phase then ur compound gets affinity with actonitrile and thus its responce gets lower . On the other hand methanol has proton donating tendencies so it may turns your actual compound in the methayl ester and it is highly lypophylic so it is possible that your compound gets better responce in methanol

This may help:
http://en.wikipedia.org/wiki/Solvatochromism

We should really know more about your separation. Do you use peak areas or peak heights (already asked but no answer so far)? Do you use the same percentage of B solvent for both the water/MeOH and water/ACN separation? Or did you adjust the %ACN in order to get identical retention times?

What is your sample solvent? The sample solvent may influence the response, see J. Kirschbaum et al., J. Chromatogr. 507 (1990) 165.

Hi sorry for the lack of response. I used the same % of organic in both cases, and the sample solvent was methanol.
I am referring to peak areas.
Many thanks for all your help
Liv

Hi sorry for the lack of response. Please note that there is an error in the previous post, the sample solvent was 50% methanol-water.

Many thanks for all your help
Liv

We still need more info. What is the nature of the analyte? Does it contain functional groups that can ionize? Are you using a buffer?