In mixed-mode HILIC chromatography, selectivity of separation can be adjusted by amount of acetonitrile, amount of buffer and buffer pH. Buffer concentration and pH will affect retention of ionizable compounds to a different degree. Retention of neutral compounds can be adjusted by the amount of acetonitrile. Carboxylic acid, three amino acids and two sugars are separated by combination of HILIC and ion-exchange mechanisms. Compounds can be monitored by ELSD, Corona (CAD), LC/MS or low UV. UV-transparent mobile phase /buffer is required for UV monitoring of this mixed-mode separation. This HPLC method can be adopted as general approach for analysis of sugars, amino acids and carboxylic acids.

Two amino acids, aspartic acid and asparagine, are separated on an Obelisc R column by combination of reverse-phase and ion-exchange mechanism. Method can be used for analysis of these and other underivatized amino acids by HPLC with UV, ELSD, CAD and LC/MS detection.