Would someone please share their experiences on the observation of negative peaks in HPLC chromatograms. We are using a mobile phase that is 100% Phosphate Buffer. The column is a Supelco C-16 Amide and our diluent is water. The negative peak is observed in samples, standards, and diluent. The strange part is that the peak is shifting during the run but our analyte is not.

a little more info would be helpful. What kind of detector, what settings, where in the chromatogram are you seeing this peek?. And some more detail on the mobile phase and flow might provide some answers.
-Daren

We are using a Waters 2487 UV detector set at 215nm. The peak started out at 5.4 min and after approximately 18h it was eluting at 4.0 minutes. The peak width is approximately 1 minute. The mobile phase is simply 100% 50mM KH2PO4 pH 4.6 with a flow of 1.0 mL/min. Hope this helps.

First, phosphate is not a buffer at pH 4.6. Second, look at the t0 and calculate the Ks for that negative peak and your analyte.

Thanks for informing me that phosphate is not a buffer at pH 4.6, I didn't realize that. Perhaps you could give me a lesson in buffers, buffer capacity, fraction of dissociation, etc., since I apparently forgot. Also, if you read what I originally wrote, you would have noticed that I stated the retention time for the analyte did not change, but the negative peak did.

It can be a contamination in your buffer. You can check it by injecting your mobile phase. If the negative peak does not appear, most probably there is an absorbing impurity in your mobile phase. The impurity is equilibrated on the column and after injecting a sample deficient in it, you will observe a negative peak at its respective retention time. This phenomenon is used for indirect detection methods (mostly in ion chromatography)