SEC impurity assay with a "sticky" enzyme
Posted: Mon May 14, 2012 6:33 pm
Hi, I inherited an SEC HPLC method when I began working here a few years back. Recently, I have been trying to get ready to validate this method. Since we have no available impurities to spike with, I was going to use BSA as an impurity to spike our enzyme with. I immediately noticed that BSA and our enzyme co-elute in this system. However, our enzyme is ~150 kDa and I believe BSA is generally about 66.5 kDa which tells me that something is wrong. Our enzyme is known to be pretty "sticky" so I'm thinking that it is interacting with the SEC column in more than just a physical size exclusion sort of manner. Unfortunately, my previous HPLC experience has been mostly in reversed phase and small molecules, so I'm at a disadvantage trying to troubleshoot this method.
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!
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!