Problem with IEX-HPLC (Anion-exchange)
Posted: Mon Apr 06, 2009 9:12 pm
Hi folks, anyone here familiar with IEX- HPLC ?
I've tried IEX-HPLC for my first time, more precisely i tried to separate 4 small proteins with theoretical pI's = 5.5, 6.2, 7.7 and 9.0 .
The column i'm using is MiniQ (strong anion-exchanger). Eluents i've used were 20mM Tris/HCL pH 8.0 and 20mM Tris/HCL + 1.0M NaCl pH 8.0.
--> i couldn't find any protein peak... so i used the same Buffers with 30% ACN and pH. 9.0 --> i could find one small broad peak which already eluted at ~ 14% Eluent B (Buffer with 1.0M NaCl). My impression is that the proteins don't proper bind to the column ?
Now i'm confused --> i should have at least 3 peaks because at pH 9.0 the proteins with pI = 5.5, 6.2 and 7.7 should be negative charged and bind to the column.
Can anyone make suggestions what to change/try out ?
thx
Diablo
I've tried IEX-HPLC for my first time, more precisely i tried to separate 4 small proteins with theoretical pI's = 5.5, 6.2, 7.7 and 9.0 .
The column i'm using is MiniQ (strong anion-exchanger). Eluents i've used were 20mM Tris/HCL pH 8.0 and 20mM Tris/HCL + 1.0M NaCl pH 8.0.
--> i couldn't find any protein peak... so i used the same Buffers with 30% ACN and pH. 9.0 --> i could find one small broad peak which already eluted at ~ 14% Eluent B (Buffer with 1.0M NaCl). My impression is that the proteins don't proper bind to the column ?
Now i'm confused --> i should have at least 3 peaks because at pH 9.0 the proteins with pI = 5.5, 6.2 and 7.7 should be negative charged and bind to the column.
Can anyone make suggestions what to change/try out ?
thx
Diablo