How to choose between isocratic and gradient in liquid phase?
1??. Gradient elution is used for method establishment. When establishing HPLC methods for samples with unknown components, even if isocratic elution is planned for the final separation, gradient elution still needs to be used for method development first.
2??. The initial gradient elution separation can approximately estimate the retention value range of the sample, providing the necessary basis for selecting isocratic or gradient for subsequent experiments.
3??. If isocratic elution is the best choice, the initial gradient test can provide an estimate of the best %B for further testing.
4??. The initial gradient elution experiment can make the overall sample separation better and faster.
5??. The initial gradient elution is less likely to miss low-concentration components that elute early and late.
6??. The initial gradient is usually a linear gradient (full gradient), such as 5-100% B. According to the impurity situation, the test is carried out at 1.5 gradient/min or 5 gradient/min.
7??. During gradient elution, the absorption of the modifier in the aqueous phase is different from the absorption in the organic phase, resulting in baseline drift, which can be improved by mixing the mobile phase.
8??. Usually the isocratic method is often used for content. It is recommended to use the gradient method for relevant detection. When determining the content method, it is necessary to evaluate the impact of potential impurities on the main peak and the running time of the method.