If unknown substances are investigated, one can never be quite sure whether they are "pure" peaks, for example, if a peak can really only be assigned to one substance. However, some measures can be taken which significantly increase the purity of a peak. The coupling with MS or NMR devices is, of course, the first step here, but a high expenditure on equipment and high costs are involved for this. There are also alternative methods to be performed in smaller, less widely equipped laboratories.
At first, it should be ensured that all substances are detected at the selected wavelength. This is the simplest with a counter-sample to be determined: If one measures at the most common wavelength of 254 nm, one can measure as a counter-sample in the short-wave range, for example at 210 nm. However, the UV cut off of the eluent must also be taken into account. If you work with a diode array detector you can do without this test because all information is provided automatically.
An additional test may be the use of another detector to also identify aliphatic substances or sugars. For isocratic separations, a refractive index detector can be used. In the case of a gradient elution, however, a light-scattering detector is useful.
Changing the HPLC device and / or eluent can also further provide information about the purity of the peaks. Variables are here the composition of the mobile phase, the use of buffers and the pH. Also the exchange of the column can contribute to the assurance of purity.
If, despite changing the chromatographic conditions, the same results are obtained, the probability of obtaining pure peaks increases.