Reverse Phase HPLC And Normal Phase HPLC: Key Differences, Applications, and 7+ FAQs

Reverse Phase HPLC (RP-HPLC) is a chromatographic technique in which a non-polar stationary phase and a polar mobile phase-typically water mixed with organic solvents like methanol or acetonitrile used, whereas Normal Phase HPLC (NP-HPLC) employs a polar stationary phase and a non-polar mobile phase, usually consisting of organic solvents such as hexane or chloroform.

RP-HPLC and NP-HPLC are two primary modes of High-Performance Liquid Chromatography, used for separating, identifying, and quantifying the analytes.

Reverse Phase HPLC And Normal Phase HPLC

In reverse-phase high-performance Liquid Chromatography (RP-HPLC), the stationary phase is nonpolar (hydrophobic), and the mobile phase is polar. The mobile phase typically consists of a mixture of water and an organic solvent such as methanol or acetonitrile.

Normal Phase High-Performance Liquid Chromatography (NP-HPLC) is a chromatographic technique in which the stationary phase is polar and the mobile phase is non-polar. The mobile phase typically consists of organic solvents such as hexane, chloroform, or mixtures of non-polar solvents.

Reverse Phase HPLC (RP-HPLC)

Common nonpolar stationary phases include C18 (octadecylsilane), C8 (octylsilane), and C4 (butylsilane) bonded phases, which retain nonpolar compounds through hydrophobic interactions. For effective separation in reverse phase chromatography, the sample must be soluble in the mobile phase/ water/water-organic solvent mixture.

Stationary Phase: Non-polar (e.g., C18, C8 silica bonded with hydrocarbons)

Mobile Phase: Polar (e.g., water, methanol, acetonitrile)

Separation Principle:

• Polar compounds elute first.
• Non-polar compounds interact more with the non-polar stationary phase and elute later.

Applications: Widely used for pharmaceuticals, biomolecules, and polar to moderately non-polar compounds.

Advantages:

• Better reproducibility and stability.
• Broad applicability.
• Compatible with aqueous solvents

Normal Phase HPLC (NP-HPLC)

Common polar stationary phases used in normal phase chromatography include silica (SiO₂), diol, and cyano (CN) bonded phases. Separation is based on the differences in polarity between analytes, with more polar compounds interacting more strongly with the stationary phase and thus eluting later.

For effective separation in NP-HPLC, the sample preferably should be soluble in the mobile phase, which means it must dissolve in non-polar or moderately polar organic solvents. This technique is particularly useful for separating polar compounds that are not well retained in reverse-phase systems.

Stationary Phase: Polar (e.g., unmodified silica, amino, cyano)

Mobile Phase: Non-polar (e.g., hexane, chloroform, isopropanol)

Separation Principle:

• Non-polar compounds elute first.
• Polar compounds interact more with the polar stationary phase and elute later.

Typical Applications:

• Suitable for separating isomers, lipids, and non-polar organic compounds.

Advantages:

• Better for separating very non-polar or structurally similar polar compounds.
• Useful in chiral separations.

Reverse Phase HPLC and Normal Phase HPLC: Key differences

Case study: Elution of Benzoic acid and Toluene in Reverse Phase HPLC and Normal Phase HPLC

Benzoic acid is more polar than toluene, and hence the following will be the elution pattern:

Conclusion:

Reverse Phase HPLC (RP-HPLC) and Normal Phase HPLC (NP-HPLC) are two complementary chromatographic techniques, each suited for different types of compounds based on their polarity.

• RP-HPLC is the most widely used due to its versatility, compatibility with water-based solvents, and effectiveness in separating polar to moderately non-polar compounds, including drugs, peptides, and biological molecules.
• NP-HPLC is preferred for non-polar compounds, chiral separations, and isomeric mixtures, especially when RP-HPLC does not provide adequate separation.

Related:

• Relative Response Factor (RRF) in Pharmaceutical Analysis

FAQS: Interview Questions

Further Reading:

• EP