Difference Between Reverse Phase and Normal Phase HPLC

The key difference between reverse phase and normal phase HPLC is that the reverse phase HPLC uses a nonpolar stationary phase and a polar mobile phase whereas the normal phase HPLC uses a polar stationary phase and a less polar mobile phase.

The normal phase HPLC is the oldest technique of HPLC that Tswett used in his separations of plant extracts; he used chalk in a glass column. This was the classical mode of chromatography that led to naming it as the “normal” technique. Reversed-phase HPLC, on the other hand, is the opposite of the normal technique, which scientists developed lately.

1. Overview and Key Difference
2. What is Reverse Phase HPLC
3. What is Normal Phase HPLC
4. Side by Side Comparison – Reverse Phase vs Normal Phase HPLC in Tabular Form
5. Summary

What is Reverse Phase HPLC?

Reverse phase HPLC is a chromatographic technique in which we use a hydrophobic stationary phase. Among all HPLC methods, we use this method for approximately 70% because of its broad applicability, and reproducibility. The stationary phase is nonpolar and the mobile phase is polar.

Most of the times, scientists use an aqueous blend of water with a miscible, polar organic solvent, such as acetonitrile or methanol as the mobile phase. Therefore, the analytes interact with the nonpolar stationary phase.

What is Normal Phase HPLC?

Normal phase HPLC is a chromatographic technique in which we use a hydrophilic stationary phase. It is the traditional method of HPLC, though we do not use it that much. The stationary phase is polar, and the mobile phase is nonpolar. More importantly, the mobile phase of this technique is 100% organic. It means that no water is used for this.

Difference Between Reverse Phase and Normal Phase HPLC

Figure 01: A sample profile for normal phase and reverse phase HPLC chromatograms according to the polarity of components in the analyte.

Typically, the stationary phase contains silica, cyano, diol, amino bonded phases, etc. Mobile phases include organic solvents such as hexane, ethyl acetate, etc. This technique bases on the retention of solvent or analyte molecules onto the polar stationary phase.

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

Reverse phase HPLC is a chromatographic technique in which we use a hydrophobic stationary phase. The stationary phase of this technique is nonpolar while the mobile phase is polar. Moreover, scientists use an aqueous blend of water with a miscible, polar organic solvent, such as acetonitrile or methanol as the mobile phase in reverse phase HPLC. In contrast, the normal phase HPLC is a chromatographic technique in which we use a hydrophilic stationary phase. The stationary phase of this technique is polar while the mobile phase is nonpolar. In addition to that, scientists use organic solvents such as hexane, ethyl acetate, etc. as the mobile phase of normal phase HPLC. The below infographic presents the difference between reverse phase and normal phase HPLC in a tabular form.

Summary – Reverse Phase vs Normal Phase HPLC

Reverse phase and normal phase HPLC techniques are two liquid chromatographic techniques. The key difference between reverse phase and normal phase HPLC is that the reverse phase HPLC uses a nonpolar stationary phase and a polar mobile phase whereas the normal phase HPLC uses a polar stationary phase and a less polar mobile phase.