Chromatography Forum

how to minimize hydrophobic interaction between highly polar molecules and stationary phase from sephadex materials?

Generally one uses chaotropic substances rather than lyotropic additives. Look up "Hofmeister Series" for more info. This means you do the opposite of salting out.
Detergents can also be used, of course, also organic modifiers.
In the other chain I mentioned arginine, I have not used it, I imagine it is chaotropic.

Hydrophobic interaction of highly polar molecules? Hydrophobic interaction with Sephadex?

hydrophobic interaction between water soluble organic compounds and sephadex stationary phase

Hydrophobic interaction of highly polar molecules? Hydrophobic interaction with Sephadex?

I actually mean adsorption of organic compounds in sephadex packing material. did you use wrong term"hydrophobic interaction"?

Hydrophobic interaction of highly polar molecules? Hydrophobic interaction with Sephadex?

Sephadex is probably the most polar packing material that one can get. So I am not terribly concerned about hydrophobic interactions. Plus, if you think that you habe a problem, you can always add some organic solvent to your mobile phase...

Actually, Sephadex does adsorb compounds with aromatic residues. It's a function of the crosslinking agent used to link the dextran chains to form the spherical bead shape. The more highly crosslinked grades, such as G-10, G-15 and G-25, have the worst problem in this regard. Some of the early papers on SEC from the 1960's noted this with regard to a homologous series of phenylalanine oligomers, which were retained past the Vt time and which eluted in the opposite order from what you'd expect in SEC. Refs: 1) R.K. Bretthauer and A.M. Golichowski, BBA 155 (1967) 549; 2) J. Porath, J. Protein Chem. 16 (1997) 463.

Andy, what do you mean with "actively adsorps" and
"so there's no way to avoid it with carbohydrate-based SEC media."?
You don´t think it is possible, as I noted in my last statement above, that there should be a solvent which shifts the equilibrium completely away from the stationary phase to the mobile phase?

I just meant that all chromatography media consisting of crosslinked carbohydrate chains has this characteristic, judging from the literature. Sure, you can overcome this by including some organic solvent in the mobile phase, but that perturbs the elution order in SEC. If you add a lot of organic solvent and the solutes are particularly polar, then you'll start to superimpose hydrophilic interaction at ACN levels as low as 40%. Typically, elution orders in HILIC are the opposite of those in SEC. You can see examples of these effects on elution order in the book chapter I referred to in my previous posting. Since silica-based SEC materials don't have this problem, they are definitely preferable for SEC of small solutes that contain aromatic residues.

Pardon me; I see that my citing of the book chapter was in a different discussion string. It's in the Literature section of the PolyLC web site; look for the chapter from 1999 on SEC of small solutes.

Actually, Sephadex does adsorb compounds with aromatic residues. It's a function of the crosslinking agent used to link the dextran chains to form the spherical bead shape. The more highly crosslinked grades, such as G-10, G-15 and G-25, have the worst problem in this regard. Some of the early papers on SEC from the 1960's noted this with regard to a homologous series of phenylalanine oligomers, which were retained past the Vt time and which eluted in the opposite order from what you'd expect in SEC. Refs: 1) R.K. Bretthauer and A.M. Golichowski, BBA 155 (1967) 549; 2) J. Porath, J. Protein Chem. 16 (1997) 463.

Hi Andy,
Do you have any other info on this study involving phenylalanine oligomers?

Hi, Krab - I believe I cited two or three papers on the subject in my 1999 book chapter [available to download from the Literature section of the PolyLC.com Web site]. They all involved running Phe oligomers under SEC conditions and observing them eluting in order of increasing size. Anything with more than two or three subunits eluted after Vt, convincing evidence of active adsorption. What further info would you want?
I might note that the same phenomenon pertains to peptides, not just the homologous series of Phe oligomers. I've seen several posters on the use of Superdex Peptide where they noted that peptides with a lot of aromatic residues eluted later than one would expect.