pH stable solid phase for SEC HPLC

[ginebra]

Hi,

does anybody have an idea what column I could use for SEC HPLC of large proteins (pore size >250 A) which is pH stable up to at least 9?
I need it for analytical purposes.

Silica is stable only up to pH 7.5.

Cheers,
Chris

[Gerhard Kratz]

Best would be a column based on polymetacrylate like the Tosoh PW column, called TSKgel G2500PWXL. Please visit www.tosoh.com and click through to get the specifications and applications, and ask for a Free of Charge Test column. Good luck.

[DaveM]

Also Shodex do a group of polymethacrylate columns, the SB400 series. Just got hold of one (had a look at Tosoh but they didn't do the dimensions I needed) so I'm hoping they do the job!

DM

[ScottHorn]

I looked into this issue a while back when trying to decide on an HPLC SEC column for antibodies and antibody conjugates. Tosoh's website seemed to recommend the SW series (silica based, supposedly stable up to pH 7.5) over the PW series for proteins. I'm not sure why this is, so I just went with the SW and adjusted my mobile phase to pH 6.9 to be absolutely safe. Has anyone tried the PW series with proteins? The supersw3000 works really well for my application, but I wonder how much the difference in pH between my mobile phase and the storage buffer changes my analysis, especially when quantifying aggregates.

[danko]

Ginebra,


You might like to check out these columns:

http://www.gelifesciences.com/aptrix/up ... rentId=549

Best Regards

[ginebra]

thanks for the replies,

The silica-based columns seem to have by far the highest resolution but are not pH stable higher than 7.5.
All other types of solid phases have significantly lower resolution.
As far as I know the hydrolysis of the OH-groups is the problem with high pH on silica based solid phases.

Do derivatized silica columns exist where the OH-groups are protected?

I already had a look on the ge-columns. unfortunately too low resolution.

cheers,
chris

[Andy Alpert]

Silica can be "zirconia-clad" by treatment with ZrOCl2. This is described in detail in the chromatography literature in the early 1980's. The resulting material resists pH as high as 12 and can be given the same coatings as untreated silica. Some manufacturers offered this material but I haven't seen it mentioned for a long time. I presume that the size of the market was so small that it wasn't in anyone's interest to keep it in stock. Consider buying silica with the appropriate pore diameter and treating it yourself per the old published methods.

[ginebra]

Hi Andy,

very interesting what you are writing about. Do you have certain papers/links at hand?
does this treatment have any effect on resolution?
is on-column treatment possible or do I have to prepare it myself?

chris

[Andy Alpert]

Initially, look up: R.W. Stout and J.J. DeStefano, J. Chromatogr. 326 (1985) 63-78. It concerns treatment of silica to make it suitable for SEC of proteins at high pH, per your stated interest. Thereafter, go to the archives of Journal of Chromatography and look up "zirconia cladding" for additional papers on the subject.
Effect on performance: No deleterious effects have been reported.
Treatment of a previously packed column: Since your SEC column presumably has a silane-based coating already, then it's not a candidate for any such treatment. The conditions for zirconia cladding are too stringent for treatment of a packed column anyway.

[Gerhard Kratz]

Unfortunately silica based SEC columns are only stable up to pH7,5. Resolution and number of theoretical plates on polymer resin based SEC columns is not as high as on silica based, but still better than with softgel packings.
On silica based SEC packings the hydrophilic endcapping will be stripped off at pH values above 7,5. Most manufacturers offer also other column dimensions than mentioned in their catalogues. Just ask them. For the polymer resin based SEC columns pH stability is mostly limited on the specifications up to pH 11, but they can be used up to pH 12. I only can recommend to ask the manufacturer to get a test column to see if you get a acceptable resolution.
Otherwise, if a silica based SEC column gives best results, please use a plain silica guard column as a kind of saturator column to increase lifetime of your SEC column when your mobile phase has pH above 7,5. Sometimes that will help.
So far I know there is no SEC column commercial available based on zircona treated silica. I cannot recommend to use a "home made" column if you want to compare your results with others in the world or if you want to do a validation. Anyhow, it will be nearly impossible to get unmodified silica 5µm in bulk, the same what is used to produce SEC columns. Good luck

[Andy Alpert]

Gerhard's suggestion to use a saturator column of uncoated cheap silica is a good one. SynChrom used to sell them, but you can easily make your own using any cheap silica that you can dry-pack quickly into a column.

If you want to perform zirconia cladding and can't find another source of 5-um silica, we'll sell you some. Pore diameters available are 60, 100, 200, 300, 500, and 1000 Ã…. For intact proteins, the 200, 300 and 500-Ã… materials are best, with the choice depending on the fractionation range you want. We won't do the zirconia cladding for you. Gerhard is also right about the difficulty of comparing the performance of the resulting material with any regular commercial product, but then there will be no results with any commercial product with which to compare performance at pH 9.