Difference between mobile phase on SEC column TSKgel Polymer

[kurian]

Hello,

We have analyzed various samples where the mobile phase is the solution of each sample (different solutions) on TSK3000SWxl in RI. Each solution have different salinities and pH.
With here, we visualize large differences between samples, a difference equal to another analysis (assay Free PS)
Then we analyzed these same samples in their initial solution but with a single e same mobile phase. Here we can see very little difference, and is no longer identical to the Free PS assay.
I have difficulty understanding and explanation of this phenomenon.
The mobile phase used for the second test is of 200 mM NaCl, 50 mM PO4 pH 7 for all samples.
What happens when the samples in the presence of the mobile phase?
Is there a smoothing of the differences caused by the same buffer?
Decreased hyrdrophobe interactions ?

[kurian]

Erratum : Polymer stationnary phase - TSKGMPWxl and SEC on conjugates Carrier-Polysaccharrides

[Uwe Neue]

If I understood you correctly, you initially tried to do SEC of your samples in mobile phases of different salinity to match the salinity of your samples. The peak profiles that you got were different from the peak profiles that you are getting now when you are analyzing all samples in the same mobile phase. Furthermore, the profiles that you are getting now are very similar to each other.

SEC works on the principle that there is no interaction between your samples and the stationary phase, and no interaction of the sample with itself in the mobile phase. Furthermore, it will give you an elution based on the size of the molecule in the mobile phase. I would think that changes in the salt concentration will affect the molecular size of your analytes, and that this is the primary reason for the differences observed, but it is also possible that you have some interaction with the surface of the packing. One can possibly sort this out by doing some experiments around the salt concentration etc. However, for the question of comparing the different samples to each other, a single mobile phase is required. Based on this requirement, you latest analysis (all analytes in the same mobile phase) is valid.

[kurian]

OK,

It's probably an interaction of the sample protein/PS with the stationnary phase, where the mobile phase induced more affinities like hydrophobicity, electrostatic interaction and hydrogen bonding.

For more details, you will see a very good newest article where i fell over.
"The Critical Role of Mobile Phase Composition in Size
Exclusion Chromatography of Protein Pharmaceuticals"

http://www3.interscience.wiley.com/cgi- ... /HTMLSTART

[Uwe Neue]

The mobile phase that you are describing is a rational mobile phase for proteins on this column. There is no reason for changing this, and your SEC data on this column are valid.

[kurian]

For the mobile phase describe above, i think also there is no problem, but for the different mobile phase equal to the sample solution, i'm not very certain.
Compositions are with Arginine 10mM - maleic acid 10mM, Propylene glycol 200mM - maleic acid 10mM, etc.

[Uwe Neue]

Right... The mobile phase that you just described is just junk for this application and useless. As I said before, your reliable results are obtained with the mobile phase used for the second test, which is 200 mM NaCl, 50 mM PO4 pH 7 for all samples