Chromatography Forum

Dear all,

Here is my question. When you do the compound tuning as well as FIA, has the solvent used dissolving your target compounds to be the same as your mobile phase? If yes, why and how would this affect my later method development?

At the very beginning, I used isopropanol to dissolve my standard because I will use it as my mobile phase for chiral separation using a chiral column. But it turns out that the separation is not good. And I decided to separate the achiral analytes first using a C18 column (MP: ACN) by using the same values of DP, EP, etc. tuned at the beginning. And later I found my repeatability is so poor with intensities jumping up and down without any rules. So now I am doing the compound tuning and FIA again using the same solvent as my MP.

Thanks for your attention.

Best regards,
Kenny

For compound tuning, I prefer where possible to tee the analyte into a flow from the chromatography system, in which case the solvent in which the analyte is dissolved doesn't matter too much, because it's mostly diluted out by the flow from the pump.
My understanding is that the adducts formed will depend to some extent on the solvent in which the material enters the spray chamber (assuming you're doing ESI). Once you've formed a particular adduct ion, its behaviour in fragmentation doesn't depend on the solvent (because the solvent is long since gone by the time you reach the collision cell or ion trap or wherever else fragmentation is happening), but if your sample hits the spray chamber in a different solvent to the solvent you will eventually use in a chromatographic method, then you may see different adducts, and find, for example, that you've developed a method based around the sodium adduct while the chromatography runs have 95% as the hydrogen adduct.

I agree with lmh and recommend using a T-piece. Make sure to use the same flow rate as you will in the method. Personally I take the column out of the flow path to make sure nothing is eluting from the column while infusing.

I would like to add the following for gradient methods.

If you have a gradient method for the compound, you know the retention time, and therefore the composition of mobile phase at that retention time.

For example, your method starts at 5% MeOH and ends at 70% MeOH over 5 minutes. Compound comes out at 2min. From your gradient setup you can read that ideally you want to tune your compound using a T-piece at, let's say, 25% MeOH.

I think it's more important when MP 1 and MP 2 have different (amounts of) additives. But even without additives, there is a desolvation efficiency difference between water, methanol, acetonitrile,... As desolvation is part of the ionization efficiency, you can expect differences in signal intensity in different MP compositions.

However, if you have a gradient method with compounds eluting at different retention times, you'll have to find a consensus setting for tune parameters that can't be switched during the run.

I normally do initial setup without the Tee, putting the analytes in the syringe mixed in what should be approximately the mobile phase as what they elute in, this allows for adducts and other influences to be accounted for, and you can find your masses without needing a super high concentration mixture. After all of that is setup, I then infuse into the Tee connection at full flow rate to adjust my temperature, disolvation gas flow and the spray needle axis until I max out the response.

kennywurb wrote:
Dear all,

Here is my question. When you do the compound tuning as well as FIA, has the solvent used dissolving your target compounds to be the same as your mobile phase? If yes, why and how would this affect my later method development?

At the very beginning, I used isopropanol to dissolve my standard because I will use it as my mobile phase for chiral separation using a chiral column. But it turns out that the separation is not good. And I decided to separate the achiral analytes first using a C18 column (MP: ACN) by using the same values of DP, EP, etc. tuned at the beginning. And later I found my repeatability is so poor with intensities jumping up and down without any rules. So now I am doing the compound tuning and FIA again using the same solvent as my MP.

Thanks for your attention.

Best regards,
Kenny

No, not really. I infuse compounds for method development regularly and invariably put them in 50:50:0.1 MeOH:H2O:Formic while the mobile phase that is eventually used for the method ends up being CH3CN.