If you are not seeing an expected peak in an HPLC assay - Tips & Suggestions
September 27, 2013
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If you don’t see a peak, the first thing to do is detach the column and replace with a column union. Inject the solution again and see if there is a peak. If not, then you may have a sample preparation problem where the analyte is not extracting or otherwise not ending up in the sample.
Check your sample to make sure the level is high enough for the autosampler injection syringe. If it is too low, the needle may be aspirating air instead of sample.
Also, consider the possibility that your detector has an issue and may be a cause.
Another common cause of peaks not being detected is with systems in which flow can be directed to a variety of columns. Check that flow is being directed to your column. You will notice this easily if there is no pressure with your column connected. If you do see a peak with the column union, it may be not retaining or not eluting with your column.
Check your sample to make sure the level is high enough for the autosampler injection syringe. If it is too low, the needle may be aspirating air instead of sample.
Also, consider the possibility that your detector has an issue and may be a cause.
Another common cause of peaks not being detected is with systems in which flow can be directed to a variety of columns. Check that flow is being directed to your column. You will notice this easily if there is no pressure with your column connected. If you do see a peak with the column union, it may be not retaining or not eluting with your column.