High-performance liquid chromatography for reversed-phase separation has both isocratic elution and gradient elution robust and convenient in its nature, particularly when working with compounds whose polarity varies widely. Some of the common issues with isocratic separations consist of poor resolution of initial eluted peaks, peak broadening, peak width for later eluted molecules because of peak dispersion and peak tailing, this often outcome in an unwanted lengthy analysis and columns contamination by strongly retained analytes. Gradually mode of HPLC can help to defeat some of these problems, usually offering better peak shape and the ability to eliminate molecules with a wider range of hydrophobicity values at a reasonable desired time.
Here are mentioned some advantages of gradient elution over isocratic elution.
- The initial mobile phase composition is selected in order that the strength is suitable to retain and resolve the initial eluting components.
- It is possible to increase or decrease the polarity of the mobile phase according to our requirements.
- The separation strength is then increased in a predetermined manner to separate the molecules with good resolution.
- We can select the ending composition of the mobile phase to confirm the separation of all analytes within a reasonable time; this is not possible in isocratic elution.
- Complex mixtures of samples can be separated without taking the time.
- It is possible in the gradient method to increase the concentration of organic modifier for column washing.
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