Although a column is often thought to be the cause of HPLC analyses failing to show the expected data trace, many cases can be attributed to other causes, including improper maintenance operations. This article discusses the case in which the grade of a solvent has impact on peak shapes. Here is a chromatogram of the analysis of a basic compound using acetonitrile/ water. Peak 2 represents the basic compound.

The figures on the right show the chromatograms obtained for two separations conducted under identical conditions except that the acetonitrile used was of different grades. One was HPLC grade (Figure 1); the other was reagent grade (Figure 2). Whilst the peak shape was broader with the HPLC grade acetoniteile, it was much sharper using the reagent grade. The peak shape differences can also be observed for acetonitrile from different suppliers, although they were of the same special grade. This may be because traces of impurities in the acetonitrile behave in the same way as modifiers added to an eluent. Replacing eluent with acetonitrile/ 5mM ammonium acetate produced a chromatogram similar to that in Figure 2 irrespective of the grade or supplier of the acetonitrile.

To avoid the influence of different grades, solvent specifically manufactured for HPLC should always be used. Compounds which have no dissociation groups can be analyzed with eluent containing no acid or salt, although eluents with additives such as salt should always be used when reproducibility is important.