Effect of Both pH and Organic Content on a Separation of Sugars, Amino Acids, and Carboxylic Acids

In mixed-mode HILIC chromatography, separation selectivity can be finely tuned by adjusting the acetonitrile content, buffer concentration, and buffer pH. The retention of ionizable compounds is primarily influenced by buffer pH and concentration, while the retention of neutral compounds is governed mainly by the proportion of acetonitrile in the mobile phase.

Using the Zodiac HST N column, a combination of HILIC and ion-exchange mechanisms enables the effective separation of a carboxylic acid, three amino acids, and two sugars. Detection can be carried out using ELSD, Corona CAD, LC/MS, or low-wavelength UV detection. For UV detection, a UV-transparent mobile phase and buffer must be used.

This HPLC method offers a robust and flexible approach for the simultaneous analysis of sugars, amino acids, and carboxylic acids, and can be adopted as a general-purpose method in analytical laboratories.