How to apply a washing cycle when running an HPLC sequence

In most cases a guard column to protect the column and help maintain consistent drug retention time in biorelevant media. However, in some situations, a guard column is not as effective as introducing a washing cycle.

A washing cycle is recommended to be built into the analytical method of the drug in biorelevant media if significant change in system properties observed during specificity and precision. Typically, a washing cycle can be built into most analytical methods as a step gradient. Usually, 100% methanol is required for 2 minutes to clean the column. After cleaning with 100% methanol, saturation is required with mobile phase until the column, baseline and pressure are stable to the degree prior to analysis. A typical example of an analytical method for a diluted sample containing poorly soluble drug (e.g. rivaroxaban) in biorelevant medium is provided below.