What is the Difference Between Reverse Phase and Normal Phase HPLC?

The main difference between reverse phase and normal phase HPLC lies in the polarity of the stationary phase and the mobile phase. In reverse phase HPLC, the stationary phase is non-polar, while the mobile phase is polar. In contrast, in normal phase HPLC, the stationary phase is polar, and the mobile phase is non-polar.

In reverse phase HPLC, a non-polar stationary phase, such as silica with C18 chains, is used. Common mobile phases include polar solvents or solvent mixtures like acetonitrile-water or methanol-water. Polar molecules elute quickly in reverse phase HPLC, while non-polar molecules elute slowly.

On the other hand, in normal phase HPLC, a polar stationary phase, such as silica, is used with non-polar mobile phases like hexane or non-polar solvent mixtures like 2-propanol in hexanes. In normal phase HPLC, polar molecules elute slowly, and non-polar molecules elute quickly.

In summary, the key differences between reverse phase and normal phase HPLC are:

• Reverse phase HPLC uses a non-polar stationary phase and a polar mobile phase.
• Normal phase HPLC uses a polar stationary phase and a non-polar mobile phase.
• Polar molecules elute quickly in reverse phase HPLC and slowly in normal phase HPLC.
• Non-polar molecules elute slowly in reverse phase HPLC and quickly in normal phase HPLC.

Comparative Table: Reverse Phase vs Normal Phase HPLC

The main difference between reverse phase and normal phase HPLC lies in the polarity of the stationary and mobile phases. Here is a comparison table highlighting the differences:

In reverse phase HPLC, the stationary phase is nonpolar and the mobile phase is polar, while the opposite is true for normal phase HPLC. In reverse phase HPLC, more polar solutes elute first, and in normal phase HPLC, less polar solutes elute first. Reverse phase HPLC is more commonly used, accounting for approximately 70% of HPLC methods, due to its broad applicability and reproducibility.