Peak Shapes in HPLC

Real-world chromatographic often deviate from the Gaussian model. This can be caused by a number of factors, including secondary analyte-stationary phase interactions and overload.

Secondary interactions are where the number of a secondary ‘active sites’ is insufficient to interact with all the molecules of a given analyte. Instead of affecting the retention of all analyte molecules, only the shape of the peak and modal retention average is affected. The most common type of secondary interaction is the ion-exchange interaction between acidic residual silanols on the silica surface and basic analytes. The presence of metals at the silica surface does contribute to silica acidity and ultra-pure silica such as Hypersil GOLD is especially recommended for the analysis of basic compounds.

Overload can occur when (i) the injected volume or elution strength of an injected solvent is too great, or (ii) the mass of injected compound(s) exceeds the capacity of the column packing to instantaneously retain them – see Scaling a Separation for recommended loading capacities.

Standard efficiency and resolution calculations assume that the shape of a chromatographic peak is Gaussian. There are two commonly used methods for determining the symmetry of a peak, both of which take into consideration the peak width and the distance between the peak apex and the start, or end of the peak. These are the tailing factor (USP, also called symmetry factor in other pharmacopoeias) and the asymmetry factor. See the Useful Equations section of this guide for additional information on how to calculate these.