What principle does ion exchange chromatography operate on?

• A. Size differences of molecules
• B. Net surface charge of molecules
• C. Mass of molecules
• D. Heat stability of molecules

Answer: (B)

Ion exchange chromatography operates on the principle of the net surface charge of molecules. In this technique, a column is packed with charged stationary phase materials, which can either be positively charged (anion exchange) or negatively charged (cation exchange). As a mixture of molecules passes through the column, those with a charge opposite to that of the stationary phase will be attracted and bind to the column, while those with the same charge will be repelled and elute more quickly.

The separation occurs based on the net charge of the molecules in relation to the conditions of the mobile phase (like pH and ionic strength), allowing for the effective separation of proteins, nucleic acids, or other charged biomolecules. This makes ion exchange chromatography a powerful tool for purifying and characterizing biomolecules based on their surface charge properties, rather than size, mass, or thermal stability.