What caused the shift of my peak? urgent !

[wxmwqr]

My peak always shift.
Conditions:
waters HPLC system with autosampler and online degaser, but no oven of the column that was exposed to the air.
Column: phenomenex Luna
Mobile Phase: Acetonitrile and Phosphrat buffer (pH=6.98, phosphric acid to adjust) gradient elution. Actonitrile from 27% to 37% at 0-15min, from 37% to 80% at 15-30min.
I do the same method with the same bottle of mobile phase. But at the morning and noon the peak change, that cause the bad peak resolution of peak 2 and 3. pleas see the Image:

I want to know what cause the peak shift.
I think the resone :
1. the temperature is changing. but I test it, the chang range in our lab is +/- 3 centigrade. It is a problem?
2. the pump is not good. I suspect the the ratio is not stable. ( the pump vaile has been changed)

Could you give me the other advise!
Thanks![/img]

[lcrandall]

Hi
I can't see the image, but...
Is this happening only today? or have you seen it in the past?
How long do you equilibrate your column before making the first injection? Do you run a blank gradient before running sample?
Do the retention times hold steady in the PM?
Lisa

[wxmwqr]

It happened in the past. I test the temperature is 25 centigrade.
I change two different columns. It is the same.
I equilibrate for about 20min before the injection. And run the blank gradient mobile phase, it is ok!
The retention time is no regulations.
By the way, you can copy that
http://tinypic.com/usermedia.php?uo=GVq ... GtNg%3D%3D
into the IE address.

Maybe you can see the image.

Thanks!

[tom jupille]

I copied the correct "IMG" code from tinypic for you:



It could be either a temperature problem or a pump proportioning problem.

change range in our lab is +/- 3 centigrade. It is a problem?

Yes, a 6 degree shift in temperature could well cause the kind of retention shift you are seeing (retention decreases in the afternoon as the lab warms up). Tape a thermocouple to your column and monitor the actual temperature at the column over the course of a day and see if your retention times correlate with temperature.

You can check the proportioning accuracy of your pump using some variant on the step-test (http://www.lcresources.com/resources/TSWiz/hs450.htm).

[wxmwqr]

Thanks TOm.
Now I coated the column with some cotton , and the shift is weak.
I think the temperature is the problem.