Difference Between Isocratic and Gradient Elution

The key difference between isocratic and gradient elution is that isocratic elution refers to maintaining a constant concentration in the mobile phase, whereas gradient elution refers to maintaining a varying concentration in the mobile phase.

The terms isocratic and gradient elution are used in chromatography. During a chromatographic run, we use a stationary phase, which is a non-moving substance, along with a mobile phase, the moving substance. The isocratic and gradient elution describes the properties of the mobile phase.

1. Overview and Key Difference
2. What is Isocratic Elution
3. What is Gradient Elution
4. Side by Side Comparison – Isocratic vs Gradient Elution in Tabular Form
6. Summary

What is Isocratic Elution?

Isocratic elution is a term used in chromatography when the mobile phase has a constant concentration. Here, the concentration of the mobile phase is constant throughout the chromatographic process. In this process, we can observe the peak width increasing with retention time linearly in the chromatogram. However, this leads to a disadvantage – the late-eluting peaks for late elution get very flat and broad. Therefore, these broad peaks become difficult to be recognized as peaks.

Moreover, in isocratic elution, the selectivity does not change according to the column dimensions. This means the selectivity does not depend on the changes in column dimensions. Here, the length and diameter are considered as column dimensions. Therefore, the peaks elute in the same order.

What is Gradient Elution?

Gradient elution is a term used in chromatography when here the mobile phase has a varying concentration. In other words, the concentration of the mobile phase does not have to remain constant. For example, in HPLC, a common separation method uses methanol 10% initially and ends at 90%, by increasing the concentration gradually. The mobile phase has two components: a weak solvent and a strong solvent. Weak solvent allows the solute to elute slowly while strong solvent causes the rapid elution of the solute. In reverse phase chromatography, we use water as the weak solvent and organic solvent as the strong solvent.

Difference Between Isocratic and Gradient Elution

Figure 01: HPLC

Moreover, the gradient elution method decreases the later eluting components to make them elute faster, giving a narrow peak in the chromatogram. This method improves the peak shape and the peak height as well. Furthermore, in gradient elution technique, the elution order changes with the changes in column dimensions.

What is the Difference Between Isocratic and Gradient Elution?

The terms isocratic and gradient elution are used in chromatography. Isocratic and gradient elution describe the properties of the mobile phase. The key difference between isocratic and gradient elution is that isocratic elution refers to the maintenance of a constant concentration in the mobile phase whereas gradient elution refers to the maintenance of a varying concentration in the mobile phase.

In the isocratic elution technique, the peak width increases with the retention time linearly. However, in the gradient elution technique, the retention of the later-eluting components is decreased, so that the elution become faster and gives narrow peaks. Apart from that, in the isocratic elution, selectivity does not depend on the column dimensions, but in gradient elution, the selectivity changes with changing column dimensions.

The following table summarizes the difference between isocratic and gradient elution.

Summary – Isocratic vs Gradient Elution

Isocratic elution and gradient elution describe the properties of the mobile phase. The key difference between isocratic and gradient elution is that isocratic elution refers to the maintenance of a constant concentration in the mobile phase whereas gradient elution refers to the maintenance of a varying concentration in the mobile phase.