HPLC Troubleshooting - Peak Shape Problems & Ghost Peaks

1. Column

If every peak has a double peak in the sample analysis process  (the shorter the appearance time, the less likely it is to have a double peak), especially the analysis of the pure sample, there is a problem in the column (column head damaged, stationary phase in the column head dirty or lost).

If the sample injection volume is small and the column works well, the peak is a large peak with a small peak, it is not trailing. This may be caused by the column block. Wash the column with mobile phase solvent or acid cleaning or other solvents in a reverse direction, the block in the column will be washed away. Forward direction washing sometimes also can solve this problem.
If the peak shape is trailing with little difference between two peaks, it may be caused by the dirty or loss of the stationary phase in the column header.

2. Solvent polarity and injection volume

For reversed-phase chromatography, the common mobile phases are methanol, acetonitrile, and water. Other additives are added to improve the separation performance. The sample is generally dissolved in a soluble solvent with the mobile phase, and the best solution is to use the mobile phase. When the solvent is strong polarity reagent (such as pure methanol, acetonitrile, pure ethanol) and the analysis system is mainly water, the double peaks phenomenon will occur with a large injection volume of pure sample (such as quantitative tube for 20ul): a peak combine with a smaller peak (different every time) with a tail, the retention time (relative to less sample quantity) will be earlier. If the injection volume is reduced by more than half, the peak shape will return to normal. This is because the solvent polarity of the sample is too different from that of the mobile phase, the mobile phase has no time to dilute it to reach equilibrium.

Another reason is that the injection volume is not necessarily large, but the absolute amount is large, and the two peaks on the chromatogram are close together, basically at the same height, without trailing (if the peak appearance time is very short, it may also be a column problem). If it is caused by a large injection and overload column, dilute the sample before injection.

3. Sample characteristics

Some samples, due to the characteristics of their chemical structure, have the phenomenon of tautomerism, and the tautomerism can not be separated, but in a dynamic equilibrium existence. In the chromatographic analysis, under a specific condition, a substance will appear double peaks. These double peaks are very close with the same height and no tail. The double peak phenomenon will disappear when the condition is changed (especially pH).

4. Device parameters

The parameters of the record are generally set internally and do not need to be modified without special needs. The HPLC recording time is generally 5ms. If the recording interval is shortened, one peak will become two or more peaks.