We are analyzing amino acids using a C18 column (150 mm × 3 µm) with PITC:Triethylamine (1:1) derivatization, but this method is resulting in broad peaks and poor separation of the amino acids mixture.
We attempted various modifications to our protocol, including adjustments to the gradient, column temperature, injection volume, and other parameters. However, these changes did not yield the desired results.
For better PITC pre-column derivatization of amino acids on a C18 column, ensure complete drying after reaction to remove excess PITC, use a buffered mobile phase (pH ~6.5) with gradient elution, and control column temperature for sharper peaks and improved separation. Optimizing these steps typically resolves broad peaks and poor resolution issues.
Sir, thank you for your response. We will try the drying process, as we are currently using hexane to remove excess PITC reagent. Could you please provide guidance regarding the appropriate reaction time and the required amounts of reagents for derivatization? We already tried the other modifications you suggested but they did not resolve the resolution issue. Abdelhak Maghchiche
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