Retention time shift on Cation Exchange HPLC

[Rocnex1]

Hi All,

I am running cation exchange for a monoclonal antibody. The column is Propac WCX-10, 4x250 mm. The MP is 50 mM MES pH 5.75 and plus 1M NaCl, gradient. The sample is not very clean and I usually see around 10 peaks.

I didn't use a column oven to maintain temperature. In a sequence of 60 injections (run O/N), the retention time kept shifting back and forth, even with the QC samples injected every 10 samples. My guess is the room temperature was not stable during the night. But the shift was as much as 1 min for any particular peak. The run time is 1 hour and all the peaks came out from 17 to 22 minutes.

Is there any particular reason why this shift could happen, except the room temperature? Does tween in the samples possibly cause this?

Thank you very much!

[tom jupille]

That kind of problem can also occur if you are running only a narrow concentration range in your gradient (e.g., 20 to 25% B). What happens is that the variation in pump proportioning is large relative to that narrow range.

For example, a proportioning accuracy of 0.25% becomes a 5% relative error when spread over a 5% range (0.0025/0.05 = 0.05 = 5%).

If you are running a narrow range, you can improve the situation by putting your starting NaCl concentration (plus the buffer, of course) in A and the ending NaCl concentration in B and then running a 0 - 100% gradient.

[Mark Tracy]

You may want to check the flow rate and gradient accuracy of your pump to make sure it is within spec.