Frequent measurements with HPLC columns often result in a decreased quality of the chromatographic results in HPLC laboratories, after a method has yielded stable and good results over a long period of time. Possible effects could be the formation of double peaks or the appearance of tailing. As a cause, the abrasion of the column can be considered. To test this, many manufacturers provide a test chromatogram that serves as a comparison. This measurement is carried out under the indicated conditions and the results are compared with the test chromatogram. In the case of deviations of up to 10% in the separation step number or symmetry, the column is still in order and the poor results must have a different cause.
Regarding the separation step number, however, it is important to note that the only considerating this number is not very conclusive, since this parameter is dependent on the other chromatographic parameters. It must therefore be ensured that the column was properly equilibrated before the first use, before the quality of the column is questioned in case of large deviations of the number of separation stages.
In the case of reversed-phase columns, there is the possibility that the stationary phase may have changed after use in another mode, such as, for example, ion pair chromatography. This can lead to other results. For normal-phase columns, the water content of the eluent and water portions on the columns must be checked as this may also lead to other separation results.
An incorrect storage of RP columns is an extremely high risk for a decreasing quality. RP columns should be stored in methanol or the like, thus preventing drying out. When reused, step-by-step conditioning to the required water content of the column is then important.
So before exchanging the columns with decreasing results, other causes should be examined. In the case of RP columns, storage is crucial for the quality of the column, whereas for NP columns the water content decisively influences the quality.