Abdul Bari Shah I didn't see that any modifiers were added. This means the pH of your mobile phase is floating anywhere from pH 6.5 to 7.5. That's very close to the pKa of at least one of those acidic phenolic groups, which means it is 50% ionized and 50% unionized. The ionized form is much more polar and would elute first. This is not the most robust chromatography because a slight change in pH (different water supplies, for example, or water that has been sitting for a while) can cause the compounds to elute differently.

I suggest lowering the pH to at least below 6, which will keep the phenolic groups as "free acids" using 0.1% trifluoroacetic acid, formic acid, or acetic acid added to both the water and acetonitrile.

After your experiment (end of the day), wash the column with acetonitrile with no acid. I didn't see which ODS you are using. Most ODS have the C18 bonded as a silyl ether and this slowly hydrolyzes over time at low pH; washing the acid out will extend your column life. Some columns have a different bonding that is more resistant to hydrolysis.

Another note about the type of ODS- some columns have an end-capping of some sort of polar group to prevent phase collapse when running at very low organic solvent concentrations- these usually have the term "AQ" or something similar. It is possible that the AQ end-capping is allowing retention of the more polar compound better than the less polar material, assuming that the isoflavones are not ionized.

Hi Abdul,

I think you need to provide more information about your HPLC process so we can better understand exactly what may be happening. What kind of chromatography are you performing? What are the elution conditions?

As mentioned above, we need more information

• Normal phase or reverse phase?
• Mobile phase?
• What modifiers are being used?
• What pH?

Depending on the pKa of the various phenolic groups, daidzein may elute faster

As mentioned before, it is important to know the experimental conditions to look for a possible cause. If you are working in C18 reversed phase, it could be possible that one compound was protonated and the other not at the pH at which you work, however the pKa are very similar (see DOI: 10.1021 / je4010905) and are in a pH zone where normally you do not work in reversed phase

Dear Tomás Monteiro Fernandes ,we have a sample containing these two compounds, for confirmation we do HPLC with ACN and Water, starting from 95% water and then decreasing the water and increasing the ACN, in short solvent system elute POLAR first then nonpolar (column ODS).

Abdul Bari Shah I didn't see that any modifiers were added. This means the pH of your mobile phase is floating anywhere from pH 6.5 to 7.5. That's very close to the pKa of at least one of those acidic phenolic groups, which means it is 50% ionized and 50% unionized. The ionized form is much more polar and would elute first. This is not the most robust chromatography because a slight change in pH (different water supplies, for example, or water that has been sitting for a while) can cause the compounds to elute differently.

I suggest lowering the pH to at least below 6, which will keep the phenolic groups as "free acids" using 0.1% trifluoroacetic acid, formic acid, or acetic acid added to both the water and acetonitrile.

After your experiment (end of the day), wash the column with acetonitrile with no acid. I didn't see which ODS you are using. Most ODS have the C18 bonded as a silyl ether and this slowly hydrolyzes over time at low pH; washing the acid out will extend your column life. Some columns have a different bonding that is more resistant to hydrolysis.

Another note about the type of ODS- some columns have an end-capping of some sort of polar group to prevent phase collapse when running at very low organic solvent concentrations- these usually have the term "AQ" or something similar. It is possible that the AQ end-capping is allowing retention of the more polar compound better than the less polar material, assuming that the isoflavones are not ionized.

Dear Abdul,

I recommend the answer from Jack Silver. Best regards.

thank you very much Jack Silver and Tomás Monteiro Fernandes