If you're new to HPLC (High Performance Liquid Chromatography) or somewhat experienced, understanding the differences between Normal Phase (NP), Reversed Phase (RP), and Aqueous Normal Phase (ANP) chromatography can help you choose the right method for your analysis.

🔹 Normal Phase (NP) Chromatography

• How it works: The column is polar, and the mobile phase is non-polar.
• Retention behavior: Polar compounds stay on the column longer (more retention) when the mobile phase is less polar (e.g., 100% hexane).
• Use case: Best for separating polar compounds using non-polar solvents.

🔹 Reversed Phase (RP) Chromatography

• How it works: The column is non-polar, and the mobile phase is polar (usually water mixed with an organic solvent like acetonitrile or methanol).
• Retention behavior: Non-Polar compounds are retained longer as the mobile phase becomes more polar (e.g., increasing water content).
• Use case: Most common HPLC method, ideal for a wide range of compounds.

🔹 Aqueous Normal Phase (ANP) Chromatography

• How it works: ANP uses reversed phase solvents (water and acetonitrile) but behaves like normal phase chromatography.
• Mobile phase: Typically high in acetonitrile (up to 98%) with a small amount of water (2%) and sometimes an acid or base.
• Retention behavior: Polar Compounds are retained longer in high acetonitrile (low water) conditions. As you increase the water content, retention decreases.
• Use case: Great for polar compounds that don’t retain well in reversed phase but are difficult to work with in traditional normal phase.

🧪 Summary

🔗 Want to learn more?
Check out these resources for deeper explanations:

• Why Aqueous Normal Phase Methods are better than HILIC
• Wikipedia: Aqueous Normal Phase Chromatography