PEG Separation Problem?

[rick1112]

Hi
I am currently working on Developing a SEC method for analysis of PEG(12kd) on analysis i have got the following profile

http://i36.tinypic.com/a2d8w4.jpg

its seems as multiple species are Co-eluting with the main peak (R.t of around 12.0 min)..the zoom view is shown below...

http://i36.tinypic.com/29l23xl.jpg

is there any way i could seperate these species??
My run conditions are
Column:- Shodex Kw 803(8.0 X 300mm)
Mobile phase :- Water only
Run:- Isocratic (30 min)
Flow rate:- .5 ml/min

Thanks a lot

[bhuvfe]

Which detector are you using? RI?
From your snapshots looks like you are overloading the column.

If you inject less do you get the same pattern for the main peak?

Normally, a concentration between 2000 and 4000 ppm should be enough to give a decent chromatogram.

Best wishes,

bhuvfe

[rick1112]

hi bhuvfe

yes i am using RI detector

regarding overloading..i also thought the same and even tried a lower injection amount..but then my impurites(high molecular weight) disappers...which is quite a problem..as they are visible in my electrophoresis analysis (SDS-PAGE)...

any way i will look into injection amount in detail...but what is gets my attention is that i have rub this same samples in Shodex oHpak, Tosho GMPW, ProteinPaK 120A(waters, TSK G2000SWxl (others condition remains same) but i they all doesnt show such mutiple peak co-eluting although either they show a high peak tailing or skewness of peak in the post peak reagion..which i think is also an indication of co-eluting species...
so can anyone suggest how can i separate these co-eluting sepcies

thanks