Gradient program containing mobile phase A with 0.1 % Trifluoroacetic acid in water and B with 0.1% TFA with Acetonitrile.
Unknown peak elute at same retention time at main component in blank solution.
Tried almost every factor but unknown peak elution remain same.
Kindly update if any.
Hello Dilip,
The unknown peak elutes at the same retention time as the main component in the blank solution, suggests that there might be a co-eluent or interference molecule bound in your column. I can suggest few adjustments to address this issue:
1. Try column conditioning by a blank run with acetonitrile for an extended period of time at a low flow rate. It will be helpful to purify the column and remove contamination.
2. Change the mobile phase composition: Adjust the ratio of mobile phase A (0.1% TFA in water) and mobile phase B (0.1% TFA in acetonitrile). Try different gradient profiles, such as increasing the proportion of mobile phase B or altering the gradient slope. This will help to separate the unknown peak from the main component.
3. Modify pH of mobile phase: In addition to trifluoroacetic acid (TFA), consider using alternative acidic additives like formic acid or phosphoric acid. These acids may provide different selectivity and improve peak separation.
4. Change the column: Try using a different column with the alternative stationary phase. Different columns can exhibit varied selectivity, which may help resolve the unknown peak from the main component.
5. Adjust the column temperature: Altering the column temperature can affect the separation efficiency and selectivity. Lowering or increasing the column temperature within a reasonable range might help separate the unknown peak from the main component.
BE SURE to validate any changes made to the method by conducting appropriate method validation experiments to ensure the result's accuracy, precision, and reliability.
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