HPLC Products

Problem

Negative peaks are a problem, because an HPLC Data handling program will have trouble integrating them. Negative peaks must be manually identified to the data system, or it will wrap a baseline round underneath them like a rubber band, and integrate all neighbouring peaks down to this artificial baseline.

With detectors which measure a difference in reading (eg Conductivity or Refractive Index) negative peaks are fairly normal. In the case of Ion Chromatography, for the analysis of cations, a strongly conducting acid eluent is used, and all peaks are detected as negative peaks. This presents no problem however, because by inverting the polarity, all peaks are treated as positive peaks and integrated normally. Using a refractive index detector, it is normal to use an eluent with as low a refractive index as possible, so that the majority of peaks come out as positive peaks. However, some peaks may be negative, and these must be handled separately.

The solvent front (void volume peak) usually also has a negative element, and hence it is recommended to turn integration off untill after the void volume peak has eluted. However if the flow rate is increased, the void volume elutes more quickly and care must be taken to update the 'Integration Off' timing so as not to miss the first peak of the chromatogram.

Where a negative peak may come as a surprise is when using a UV detector. Given that the baseline is normally at zero absorbance, it shouldn't be possible to absorb less than zero! However, some mobile phases absorb UV, especially when used near to their UV cut-off, or when buffer salts are used. In this case, it is normal to use the Autozero function to back off the eluent absorption, but it is then possible for a non-absorbing component to elute, which absorbs less light than the eluent, and in this case, a negative peak will result.

This can be used as a means to detect non-UV-absorbing species with a UV detector. An eluent is chosen which does absorb some UV light. It is important that the absorption is not so strong as to take the detector offscale! However when a non-absorbing component elutes, a negative peak is usually seen.