HPLC of Human serum albumin, peak bifurcating?

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weilanstudent
weilanstudent's picture
HPLC of Human serum albumin, peak bifurcating?

Hello,
         I want to seprate the human serum albumin and Apo-2L from lyophilized powder, and i can do it very well 4 years ago. But the human serum albumin appeared peak bifurcating in the same  chromatographic conditions in recent years.  
        
         And then i tested the titerof my column, and the plates is 17000, it is showed that the state of  my column is good. Then i contact factory of my column,they said it is the problem of my samples, 
         
          but the sigma product (pure albumin of human serum) and the baxter albumin product also appeared the peak bifurcating. i don't know if it is because the albumin have the isomers in the two products.

          my  chromatographic conditons : tempreture 40℃, Mobile phase:acetonitrile and H2O(0.01%TFA).
           someone please help me why the peak bifurcate? Thank you very much!

Sami Tuomivaara
Sami Tuomivaara's picture
weilanstudent,

weilanstudent,

Please tell what columns you are using and what test did you do to get the theoretical plates. From your experiment, looks like we can eliminate the protein as the source of this behaviour. Just to confirm, you got two peaks 4 years ago (pure albumin and pure Apo-2L peaks), and now you have three?

Cheers,

weilanstudent
weilanstudent's picture
HI, Suola,

HI, Suola,
        Thanks a lot for your reply. My column is C4 of Grace. In order to describe what i said clearly, i have upload two images. The first one is recent chromatogram, the previous peak is the human serum albumin, and we can see that it is bifurcatied, The second image is the human serum albumin chromatogram 4 years ago, and it is not bifurcatied. so i want to see whether my column is in good conditon. and later, i eliminate the protein amount, but it is useness, it is also bifurcatied. the column is good, i don't know if it is the problem of the samples.  
              and the standard  albumin chromatogram is the third image. it is bifurcatied too. Did the albumin have the isomers in the solution? Thank you very much!