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Question:
“I am running a gradient method of 20 to 45% B over 55 minutes, where B is acetonitrile and A is acetate buffer at pH 4.8. There is a large peak at the end of the gradient as shown below. This peak is observed for a blank injection of the sample diluent so I think it must be due to a contaminant in the mobile phase. I have tried preparing fresh mobile phase from different reagent batches but it is still observed. Can you suggest what could be causing it?”
Answer:“Contamination of the mobile phase is usually one of the primary suspects when unexpected peaks occur in gradient analysis. However, it is commonly observed that a peak such as that shown in the chromatogram occurs when the strong solvent conditions at the end of the gradient are rapidly changed back to the original conditions. This only occurs with acetonitrile and is not yet fully explained to everyone’s satisfaction. It occurs after the separation is complete and thus is not normally a problem. Simply ignore the peak or set your CDS to stop collecting data after the last peak of interest.”
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