Chromatography Forum

I have a particular protein that binds nicely to all RP columns, but does not elute completely, even on the weakest RP phases. Avoiding the modes of HIC and IEX, if we stay with RP, does anyone have any experience with this problem and can recommend a stronger buffer system or different type of RP column that can hopefully elute this problem child.

Thanks

Which stationary phases have you tried? Have you tried C4 (300A)? Have you tried IPA instead of acetonitrile?

Can you tell us more about the protein mwt?

Bryan,

thanks for getting back to me.
I have tried C4-C18 phases, with varying carbon loads.
I have also tried acetonitrile with low levels of IPA.

ok - please send me an email when you get a moment

Will your protein remain in solution in 70% organic solvent? If so, then consider HILIC instead of RPC. Works nicely for integral membrane proteins, for example. It looks at the polar residues in a protein and simply ignores the nonpolar ones. Try a 1:1 blend of PrOH and ACN, with 50 mM formic acid as well, and run a decreasing PrOH/ACN gradient. Also be sure to use a HILIC column with a thick-well-hydrated coating; uncoated silica will not do.

For proteins it does not matter whether it is C3 or C18, but pore size does. use 300A as suggested. But the most important is to raise temperature to at least 60C. I am using Zorbax SB 300 (any carbon loading) with excellent results. I may be able to help you further if you provide MW of your protein, and it's origin.

I've observed similar recovery issues when working with larger, relatively hydrophobic peptides.

It has already been stated that use of shorter alkyl chain RP columns and elevated temperature is generally thought improve recovery. From my experience, recovery of hydrophobic peptides was much better on HILIC vs RP columns, as Andy Alpert mentioned.

You might look into using a polymeric (PLRP) column. Hydrophobicity is similar to a C4, however, PLRP is not silica-based. This permits use of more aggressive elution conditions (higher temp, pH). Additionally, the lack of silanol interaction with basic residues is thought to contribute to increased recovery compared to an alkyl chain column.

You can try the Bio-C18 column...it is 200, 300 Angstrom: http://www.sepax-tech.com/Bio-C18.php
Or possibly size exclusion?

Also, try HILIC, just thought of that: http://www.sepax-tech.com/HILIC.php

Here's two references on the use of HILIC for separation of intact membrane proteins in a volatile solvent:
1) J. Carroll et al., PNAS 103 (2006) 16170;
2) J. Carroll et al., PNAS 104 (2007) 14330.

Hi people,

I have the same kind of problem.... Tried all sorts of reversephase column, but yet to try polymeric column. My proteins mW is similar to antibody but its a fusion protein, with lot of glycosylation. It elutes at 50% 0f ACN. Do you have any more suggetions.

thnkyou,
jith

There's a lot of potential issues with porous materials for protein separation, including:
- poor peak shape, poor recovery, and poor efficiency (high mass transfer resistance).

To help solve these issues, Imtakt developed Presto FF-C18 (2um non porous ODS).
Below is data for Lectin and Polyglutamic acid (up to 6MDa):

http://www.imtaktusa.com/site_media/fil ... TI582E.pdf
http://www.imtaktusa.com/site_media/fil ... TI560E.pdf

I have a particular protein that binds nicely to all RP columns, but does not elute completely, even on the weakest RP phases. Avoiding the modes of HIC and IEX, if we stay with RP, does anyone have any experience with this problem and can recommend a stronger buffer system or different type of RP column that can hopefully elute this problem child.

Thanks

If it never elutes completely, what's your reference then for complete elution? Did you check peak area without any column? (Assuming you dialyzed the sample extensively against your loading buffer.) And how much exactly are you missing? (Are you sure there are absolutely no aggregates in your sample? How did you determine concentration of the sample?

If recovery is problematic, I wouldn't even think about using a C18, or a C8. A cyano column is one of the least retentive RP supports. On the other hand, Uwe Neue has mentioned these supports have a propensity for undergoing hydrolysis, and exposed silanols are believed to negatively impact recovery.

Is your column new? Sometimes column manufacturers recommend repeated injections of an inexpensive protein to "condition" the support (column presumably has active sites where protein irreversibly binds).

By the way-- How soluble is your protein in 50% MeCN? How about higher MeCN? Could it be that your protein precipitates somewhere between the injector and column outlet?