Let's start with the obvious first: The two acids are completely different, available in different purity grades, different pH / pKa's, with different absorption and ionization properties. Ask yourself, why is the acid being used? Is it there to improve separation on the column OR is it there only to improve ion formation in the MS? If it is being used to improve the chromatography, then you will need to adjust the pH to the original value (failure to do so may result in a change in retention time and separation). If it was chosen to enhance ion formation, then the response may be different.

• What, if any effect each acid will have on your method will entirely depend on YOUR actual chromatography method (which you did not provide) and why the acid was initially used/chosen. If you substitute one for the other, please be sure and document this change and also any related changes made in the concentration as you are no longer using the original developed method.

Additional critical info to consider: Acetic acid is very weak (pKa ~ 4.8) compared to Formic acid (pKa ~ 3.8).

• "Common pKa Values for ACIDS & BASES used in HPLC and LC/MS Method Development"; https://hplctips.blogspot.com/2015/12/common-pka-values-for-acids-bases-used.html

**For LC-MS use, I have been teaching that we prefer to use fresh formic acid not acetic acid for most ESI applications. It is available in far higher purity grades and since it is a stronger acid (but not too strong), you can use less of it to obtain similar ionization effects. When developing LC-MS methods, the use of high-purity, low concentration additives is always wise. Formic acid is also well documented to improve ion formation too. By contrast, acetic acid is not available in high purity forms and often is quickly contaminated or degraded. It often results in additional noise and higher baselines absorbance for both MS and UV/VIS detectors. Now, if you are not concerned with high sensitivity, then this may be of no concern and in those cases you can use acetic acid as an additive, but only if it is there to promote ionization and not to improve the chromatography.

Hi Shazeli.

I am assuming you are using ESI ionization source in analysis, you didn't write about this. If you to change formic acid for acetic acid could afect retion time in chromatography, peak shape and signal intensity, becuase you are modify ionizantion condiction in your system. If this should be impact your analyis in more or less degree will be dependent of your analytical conditions and target compound. You could test this modification and consider this with a robustness test for your analytical method. I hope this help you.

You should see similar chromatography. Formic acid is a stronger acid than acetic, so you will be running at a lower pH. I prefer formic for ESI work over acetic acid, especially for unbuffered runs like yours. You might be slightly more sensitive for the flavanoids with formic acid but in general I would expect similar performance.

Let's start with the obvious first: The two acids are completely different, available in different purity grades, different pH / pKa's, with different absorption and ionization properties. Ask yourself, why is the acid being used? Is it there to improve separation on the column OR is it there only to improve ion formation in the MS? If it is being used to improve the chromatography, then you will need to adjust the pH to the original value (failure to do so may result in a change in retention time and separation). If it was chosen to enhance ion formation, then the response may be different.

• What, if any effect each acid will have on your method will entirely depend on YOUR actual chromatography method (which you did not provide) and why the acid was initially used/chosen. If you substitute one for the other, please be sure and document this change and also any related changes made in the concentration as you are no longer using the original developed method.

Additional critical info to consider: Acetic acid is very weak (pKa ~ 4.8) compared to Formic acid (pKa ~ 3.8).

• "Common pKa Values for ACIDS & BASES used in HPLC and LC/MS Method Development"; https://hplctips.blogspot.com/2015/12/common-pka-values-for-acids-bases-used.html

**For LC-MS use, I have been teaching that we prefer to use fresh formic acid not acetic acid for most ESI applications. It is available in far higher purity grades and since it is a stronger acid (but not too strong), you can use less of it to obtain similar ionization effects. When developing LC-MS methods, the use of high-purity, low concentration additives is always wise. Formic acid is also well documented to improve ion formation too. By contrast, acetic acid is not available in high purity forms and often is quickly contaminated or degraded. It often results in additional noise and higher baselines absorbance for both MS and UV/VIS detectors. Now, if you are not concerned with high sensitivity, then this may be of no concern and in those cases you can use acetic acid as an additive, but only if it is there to promote ionization and not to improve the chromatography.

I think it will maintain a similar chromatographic separation, but will change the response in MS. The molar mass of acetic acid is higher.

I think William Letter has given a very precise answer to your question and it should help you in any eventualities.