The retention times of substances in a separation process could vary. A possible cause could be the incorrect setting of the temperature. Chromatography is temperature-dependent. Temperature changes has an effect in both the adsorption processes in normal phase chromatography and the distribution processes in reversed phase chromatography.
In non-airconditioned rooms, the temperature fluctuations over the year are very large, resulting in changes in retention times of substances during separation. In extreme case, it may also influence peak spacing or peak order. Column ovens help to keep the temperature in the device constant. Temperatures just above the maximum room temperature are often selected.
The injection of the eluent can also cause fluctuations in the retention times. These are produced and injected at room temperature. However, in the short time that the eluents have been introduced into the system, they cannot be heated to the appropriate temperature by the column oven. This results in a temperature gradient on the column – close to room temperature at the beginning of the column and rises to the selected system temperature close to the detector. Since temperature differences of as little as 1° C can be decisive, the eluents should therefore also be heated to the oven temperature.