In high-performance liquid chromatography, the isocratic and gradient are separation methods they are very useful for the separation of analytes. The separation mode in which the mobile phase composition remains constant during the process is called an isocratic elution. Generally, it is widely used in reversed-phase chromatography. The isocratic method is usually successful in the separation of sample molecules that unusual in their affinity for the stationary phase. Many chromatographers suggest that when an isocratic elution will work for you, avoid using the gradient.
Advantages of isocratic elution in HPLC:
- No specific HPLC pump is necessary to flow the solvents/mobile phase.
- The method transfer is easily possible.
- There is no need to re-equilibrate with the initial mobile phase composition between consecutive injections.
- It is a cost-effective method as compared to gradient elution.
- It is a simple way of separation as compared to gradient elution.
Disadvantages of isocratic elution in HPLC:
- This is a time-consuming method.
- The major disadvantage is that the delayed eluting peaks become very broad and flat.
- Poor resolution of initial eluting peaks.
- This will separate a limited number of molecules.
- This method is not suitable for clean HPLC columns.
- The selectivity doesn’t change if column dimensions change.
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