Due to the wide K range of molecules they cannot be easily separated using isocratic methods, gradient elution is suggested in such a case. The most important reason for gradient elution is to rapidly move the strongly retained analytes of the mixture, but at least resolve the retaining component well. Several problems related to isocratic elution in reversed-phase chromatography can be conquered with the use of gradient elution. In this form of analysis, the composition of the mobile phase is changed throughout the analysis by increasing the number of organic modifiers.
The final composition of the mobile phase is selected to make sure all molecules are separated from the HPLC column within the desired time. It is feasible to increase the concentration of organic modifiers to wash away potentially contaminated and strongly retained compounds from the column. The gradient method is the best, for analysis performed using reversed-phase chromatography, normal phase chromatography, and ion-exchange chromatography. Special HPLC pumps are needed to perform gradient analysis, allowing the on-line mixing of the mobile phase composition.
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