Chromatography Forum

Hello All,

For HPLC seperations, such as size and ion exchange, why does it typically take a few injections (we usually do 3) for the chromatogram to look normal?

What is happening during the "column conditioning" stage?

Thank You.

This may be related to your column storage conditions. Especially in "size", which I assume means size exclusion chromatography, there is no good reason for a change in the chromatogram.

Also, what is "not normal" in your chromatogram?

when the storage condition is differnt from running condition, you have to replace the former by the latter in the column. For example, you replace 50/50 ACN/water as stored to 80/20 Buffer/ACN as run. How long you need to equilibrqte the colmn depends how different between the two conditions, and how quick the mobile phase can fully interact with the stationary surface(column chemitry). Sometimes it takes more than 20 column volume. I do not think you need inject the sample, which can use as monitor though.
If the conditions were the same, I expect much faster equilibrate.

Uwe put it very nicely: "there is no good reason....."
Here is another bad reason for nonequilibrium: The analyte needs to cover some active sites (or whatever).

With ion exchange, the column may have a large capacity compared to the mobile phase, and so it takes longer to remove the storage ions and replace them with the running ions. Gel-type resins also swell and shrink in response to ionic strength, and that process can be a bit slow; you often don't start at complete equilibrium, but some fixed time after the previous gradient.