Troubleshooting Reversed Phase Chromatography (RPC)

Ghosting

Poor-quality eluent components can cause a phenomenon referred to as “ghosting”. Trace levels of organic impurities bind to the medium, concentrating during equilibration and sample application. When elution begins, these contaminants appear in the chromatogram as unknown, or “ghost” peaks. The size of a ghost peak will usually depend on the equilibration time and the level of organic impurities in the eluent.

Ghosting may also be caused by incomplete elution of molecules in a previous run. Run a blank gradient, with no sample, as a check, especially if subsequent runs are to be performed with highsensitivity detection.

Baseline drift: balancing eluents

During a typical run the baseline can progressively increase or decrease in an approximately linear fashion as the proportion of eluent B increases. This phenomenon may originate from an ion-pairing agent (or strong acid component) or an organic modifier that absorbs significantly at the detection wavelength. The background absorbance caused by eluent components is corrected for during column equilibration. As the proportion of organic component increases so the absorbance properties change.

Compensate for a drifting baseline by using different concentrations of UV-absorbing ion-pairing agents (or buffer acids) in eluent A and B and thereby balancing the “concentrations” with respect to UV-absorption properties to give an approximately straight baseline. Because of batch-to-batch variations in the absorption properties of eluent components and other differences between the conditions in different runs, it is not practical to give specific recommendations. The following example can assist to illustrate the principle: gradients from TFA in water to TFA in acetonitrile will usually require that the concentration of TFA in acetonitrile is 10–30% lower than in water. The balanced concentrations of UV-absorbing components should then be determined empirically. The difference in concentration of ion-pairing agent between the two eluents is generally not large enough to adversely affect the separation. A typical example would be to use 0.065% TFA in eluent A and 0.05% TFA in eluent B.