HPLC Products

Integrating a very large peak, and some associated impurities at very low concentration can give many errors. This situation can arise when analysing a product prepared to 98-99% purity, so the total impurity concentration is around 1% of the main peak.

Analysis using a single chromatogram assumes the detector response is linear over a very wide concentration range, and the analysis of the smaller peaks is more difficult because of the lower signal to noise ratio. It is also difficult to find an appropriate Peak Width setting, unless the chromatography is such that it can be changed during the chromatogram.

The solution is to analyse the sample twice. Make up the sample at a concentration calculated to give the smaller peaks at a respectable level, (eg 0.2-0.3 Volts.) Then make up a dilution of say 1:100. The dilution ratio should approximate to the concentration ratio of the sample and impurities. The aim is to achieve a dilution which gives a sample peak of similar height to that of the impurities in the undiluted sample. The diluted sample should be run first, to minimise the possibility of sample carryover, followed immediately by the stronger sample, and under identical conditions. In this way the sample and impurity peaks will be of comparable size and their integration will be of equivalent quality. Integration conditions can be identical, and the main peak concentration can be calculated automatically by introducing a dilution factor of 100 for the first chromatogram.

It is important that where possible the samples are diluted in the mobile phase to avoid any errors from solvent effects. The only possibility of introducing error is from the dilution step, and so this should be done very carefully. However all the other errors arising from a single injection analysis (non-linear detector response, difficulties in identifying the peak start and end of small peaks, peak width problems) are eliminated.