Hi Hadeer

Can you give a little bit more details of your column(s)? Usually analytical SEC is not performed with only one Column, many systems are a mix of different sorbents or exclusion sizes to get a satisfying result. Anyway: the character of you stationary phase decides about effects like tailing.  What detector do you use?

iam using BIO-SEC column from silica gel i bought from agilent its dimensions :4 .6 I.D , 5 micrometer , 1000 A

and UV detector 

i usually got readings at 280 nm

Hi, Hadeer!

Firstly I think that your column have not an appropriate exclusion limit to separate this proteins. If I get you right, some of the whey proteins has sufficiently high MWs around 500-700 kDa, as your Bio-SEC column with 100A pores can separate proteins at best with 100 kDa masses. Try to use the same type column with larger pores (for example, 300 or 500A) or like Frank correctly wrote 2 columns with different porosity in series.

Secondly do you sure that CE purification fractionize your sample into individual compounds or a protein fraction with only small MWs? Maybe, you should try to check your whey protein sample without any additional purification steps at first and only after that perform SEC-analysis of cleaned-up fractions.

thanks maksim

regarding the first point my protein is 80 KDa so i decide that this column is suitable for its separation

but for purification i do gel electrophoresis  for purified fraction that show me only on band refer to 80 KDa protein which is not as the same results i found with SEC

iam really confused

i thought both column and mobile phases are not suitable do u think so ???