Specificity - Gradient method and run time

[danor285]

in the analysis of specificity, it is necessary to run 3 times the retention time in a gradient method,? What is your opinion?

[dap]

Hello.
Usually as long as possible gradient should be run for stress testing samples than gradient end time can be set to the elution time of the latest peak * 1,15. The latest peak should be ≥ 0,05 % imho.

[tom jupille]

For *specificity* (as opposed to stability testing), you need to establish that your compound is separated from any other compound likely to be present in the sample. With any single set of running conditions, there is always the possibility that another compound may coelute with your peak of interest. In an isocratic method, you might change the aqueous/organic ration by 10% or so and confirm that you still see a single peak under both conditions. Changing the steepness of a gradient (i.e., keeping the initial and final %B constant but changing the time by a factor of 3) is chromatographically equivalent to changing the isocratic composition by 10% (assuming reversed-phase chromatography and small-molecule sample here).