HPLC Products

Normal

Problem

There are two problem scenarios in this case. One where all the flat top peaks are the same size, and one where they are different heights as shown here.

The above situation (flat top peaks, different peak heights) arises when the flow cell is too small. Peak volumes are then much bigger than the cell volume, so the cell reaches a point where it is filled with the sample peak, and as it continues to flow through the cell, the absorption remains constant, coming back down only once the tail of the peak elutes. The peaks are at different heights because the small volume cell has a short enough pathlength that even when filled with the sample peak, it does not take the detector offscale, and the peak heights represent the combination of sample concentration and extinction coefficient of each peak.

In another situation, flat top peaks can result with a normal flow cell when the sample concentration is high enough to take the detector offscale. In this situation, all flat top peaks are the same height, ie full scale.

Older detectors had a Range setting, which could be used to adjust the output scaling for a chart recorder. (ie if the peaks were small, the full voltage signal could be allocated to (for example) 0-0.005AUFS instead of 0-1AU. This allowed tiny peaks to be amplified and seen on a chart recorder. Normally this Range setting did not affect the Integrator output. However if the Range was set to 0.005AUFS (for example) any peak which gave a higher absorbance than this would be truncated, and give a flat top peak. So care should be taken if using the Chart Recorder Output on an older detector. On some more modern detectors, the Range setting has been re-introduced, this time affecting the Integrator output. So if for some reason the Range is set to a low value and not reset to full scale, it is likely that flat top peaks will result.