Loading amount is a key parameter for preparative separation. In order to conduct efficient preparative separation, it is important to set proper loading depending on the purpose.
Injection volume and sample concentration are studied to select the proper loading amount. There are two methods to increase loading amount: “Concentration Load” (increasing concentration of the sample with fixed injection volume) and “Volume Load” (increasing injection volume with fixed concentration of the sample).
Loading condition studies are recommended to be conducted with Concentration Load because the peak shape is less influenced by Concentration Load than by Volume Load. Volume Load can be used in combination of Concentration Load. (Chart (2) and (3)).
Peak shapes can be affected by sample solvents (Chart (4)). It is preferable for samples to be dissolved in mobile phases or solutions with lower eluting power.
The optimum loading amount differs depending on the important criteria for the preparative purification, such as purity, recovery, and productivity. When purity and recovery are the priority, increasing loading can be difficult. On the other hand, when productivity is the priority, high loading separations are possible but purity and recovery tend to be low. It is important to set the optimum loading depending on the purpose.
Effect of sample loading on purification results
Loading studies may not be successful depending on a detection method. Proper loading study can be difficult with too high or too low detection sensitivity.
An appropriate detection method should be selected depending on the target substance.