基于分析方法优化制备方法的流程

Abstract 

In analytical HPLC good resolution is required over the entire gradient; in preparative HPLC it is only important to achieve good resolution around the target compound, which should be collected in highest purity. With a small set of optimized gradient methods a much better resolution can be achieved than with a single generic method. The generation of a small method set based on retention time windows in the pre-preparative analytical run is described in this Application Note. Using Agilent standards with known elution properties makes this an easy four-step process, which can be accomplished in less than one hour. An application example demonstrating how the resolution of the target compound can be improved is also shown.

Introduction 

Baseline separation of peaks is required for accurate quantification of compounds in analytical HPLC. Therefore, a resolution of about 1.5 is needed if the peak areas are not too dissimilar. A good resolution is only required for the target compound in preparative HPLC1, and that compound is required with high purity. The separation of the remainder of compounds in the chromatogram is not of interest. Generic gradients of 5 – 95 % organic mobile phase are frequently used for purification runs, which do not always lead to sufficient resolution for the target compound. To increase the resolution an optimized method with a shallower gradient around the elution composition of the target compound could be used. Since the preparative method cannot be optimized for each compound this is achieved in practice by using a small set of methods. The preparative method is selected according to a retention time window, in which the compound elutes in the pre-preparative analytical run. The process of generating an optimized preparative method set for retention time windows in the prepreparative analytical run is described in an article by Blom et al. In this Application Note the process described in the article is simplified by using Agilent standards but it can also be performed with any other standard compounds.

Conclusion 

In this Application Note the generation of a small set of optimized preparative methods based on retention time windows in the pre-preparative run was explained. By using the Isocratic Test Sample and the Electrospray LC Demo Sample the complete process was done in four easy steps taking less than one hour to accomplish. The improved resolution of or near the target compound using optimized gradients was presented in an application example.