As the name implies, multiple separation mechanisms are purposely blended to enable unique HPLC column selectivity. They are tremendously powerful for separations of ionizable and neutral compound mixture. There is no limitation on the amount of organic from 100% water and 100% ACN while maintaining column stability and reproducibility...etc.
Most of mixed-mode columns have polar embedded groups which are ionizable, so when you use high organic, for example, you might easily go from reversed-phase ion-exchange mode to HILIC/ion-exchange mode. They are typically less hydrophobic than C18 reversed-phase columns, i.e., having C12 carbon chain. Polar ionizable groups on the surface further reduce hydrophobicity of the HPLC column.
When running gradients, you need to consider both reversed-phase and ion-exchange retention mechanisms. A suggested gradient runs from 0% to 60% ACN while increasing the amount of ions from 5 mmol to 50 mmol. The separation mechanisms may be complex, but method development is usually straight forward.
Tips on using mixed-mode HPLC columns
To confirm reproducibility, it is recommended that you inject the sample multiple times. If the results are not reproducible, you can further modify the mobile phase composition or the gradient
Precise salt concentration and pH must be prepared by weights or volumes. Do not use pH meters or titration methods since they are imprecise.
Optimal buffer concentration most likely differs among methods. Thus, it is recommended that you adjust the mobile phase with salt, acid, or alkali to improve peak shape.
Typical applications are pharmaceutical counter ions, surfactants, and ionizable/neutral compound mixtures.