Introduction

Aqueous Normal Phase (ANP) chromatography is a powerful retention mode that bridges characteristics of both traditional Normal Phase (ONP) and Reversed Phase (RP) separations.

It uses reversed‑phase solvents—primarily water and acetonitrile—along with small amounts of acid or base, yet produces a retention pattern aligned with classic Normal Phase trends. This article defines ANP within the broader chromatographic framework and clarifies how its retention behavior differs from ONP and RP. 

Understanding Normal Phase (ONP)

Normal Phase chromatography increases retention as the mobile phase becomes less polar, typically by decreasing water or another polar solvent. Highly non‑polar mobile phases (e.g., 100% hexane) produce the longest retention times for polar analytes because analyte–stationary‑phase interactions dominate when mobile‑phase polarity is minimal. 

Understanding Reversed Phase (RP)

In Reversed Phase chromatography, the opposite trend occurs: retention increases as the mobile phase becomes more polar. As water concentration rises, hydrophobic analytes interact more strongly with the non‑polar stationary phase, generating longer retention times. This makes RP the most widely used mode for moderately non‑polar or hydrophobic analytes.

What Defines Aqueous Normal Phase (ANP)

ANP chromatography creates a Normal Phase‑like retention pattern using reversed‑phase solvents such as acetonitrile and water, modified with small acid or base concentrations. Maximum retention typically occurs at very high organic levels, up to ~98% acetonitrile, with only ~2% aqueous content. As the aqueous portion increases, retention decreases, eventually reaching a minimum under low‑ACN, high‑water conditions. This behavior distinguishes ANP from both ONP and RP even though the solvent system resembles RP mobile phases.

Practical Interpretation

• In ANP, increased water reduces retention, mimicking Normal Phase behavior.
• ANP allows use of RP solvents, making it far easier to integrate with MS‑compatible mobile phases compared to classical ONP.
• Because retention is strongest at high ACN, ANP is particularly effective for polar and ionic compounds that otherwise elute at or near void volume in standard RP methods.

Conclusion

Aqueous Normal Phase chromatography delivers Normal Phase retention behavior while maintaining the solvent compatibility, flexibility, and practicality of reversed‑phase systems. By understanding the relationship between organic content and analyte retention, chromatographers can leverage ANP to retain polar analytes efficiently without reverting to traditional non‑polar Normal Phase solvents.