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- Posts: 2
- Joined: Wed Jul 20, 2011 7:20 pm
The mobile phase specified in the method is 100mM KH2PO4, 500mM NaCl, and 0.1% Tween-80 at a pH of 6.0. The column is a Tosoh TSKgel G3000SW XL 7.8x300mm, 5µm. I keep the column at 25°C and the flow rate is 1.0 mL/min. I detect using UV-Vis at 280nm if that matters at all.
I have done some experimenting with the mobile phase. Using 0.1% Tween-80 in PBS (which is about pH 7.6), BSA and our enzyme separate pretty well, however BSA elutes before our enzyme which seems backwards. As I understand SEC, the larger molecule should elute first, right?
Does anyone have any suggestions for me to try? Is there a different column or mobile phase I should try? I almost feel like I need to start over with this method, but I don't know where to start.
Thanks in advance!