Chromatography Forum

how can we guarantee there is no interaction between stationary phase and solute? and how can we know there is no interaction?

That is a good question and difficult to answer. The easiest way to recognize adsorption is when parts of the polymer are eluting behind the volume of the dead volume marker.
Another possible case when you would notice adsorption is when the reproducibility is very poor. The reason of it can be numerous. I would contact your column supplier for tips and tricks.

If money is not a problem:
- GPC-MALLS will shed some light on the separation mechanism.
- If your polymer is very low molecular weight (< 5000 Da) LC-MS
might help.
- MALDI after GPC fractionation could help too

what can we do to minimize adsorption? for example, choice of mobile phase? any thought?

Cleaning up, I just got Tosoh`s "The Customer Magazine", #01, 2009 in my hand. There was an article in there talking about
E. Daisuke, et. al., J Chrom, vol. 1094, #1-2, 49-55 (2005). It was suggested that arginine in the mobile phase might be the best way to go in SEC of some proteins. They mentioned that high concentrations of salts can eliminate ionic interactions, but may cause an increase in hydrophobic interaction. Organic modifyers and urea, while increasing solubility, can be expected to also increase ionic interactions.
They did not mention pH, as a pH of 7 was apparently considered mandatory. If this is not the case one can also vary pH to eliminate absorption.

On the more generic side, i.e. for SEC in general, protocols have been worked out for any polymer that you can think of. By "protocol" i mean conditions of mobile phase composition and temperature that not only solubilize any polymer but also minimize interaction with the stationary phase. You'll find a good list in my book on pages 147-149.

I am new here. where can i find your book?

"Uwe Neue"]On the more generic side, i.e. for SEC in general, protocols have been worked out for any polymer that you can think of. By "protocol" i mean conditions of mobile phase composition and temperature that not only solubilize any polymer but also minimize interaction with the stationary phase. You'll find a good list in my book on pages 147-149.

why pH 7 is mandatory?

Cleaning up, I just got Tosoh`s "The Customer Magazine", #01, 2009 in my hand. There was an article in there talking about
E. Daisuke, et. al., J Chrom, vol. 1094, #1-2, 49-55 (2005). It was suggested that arginine in the mobile phase might be the best way to go in SEC of some proteins. They mentioned that high concentrations of salts can eliminate ionic interactions, but may cause an increase in hydrophobic interaction. Organic modifyers and urea, while increasing solubility, can be expected to also increase ionic interactions.
They did not mention pH, as a pH of 7 was apparently considered mandatory. If this is not the case one can also vary pH to eliminate absorption.

Please read my statement again. I didn´t say that pH = 7 is mandatory. The statement is merely an attempt to explain why pH was not mentioned in the advice by Tosoh. I have gotten some interesting and weird results by going away from pH = 7 with antibodies. Part of this was apparently due to drastically changing the configuration of the protein. The following statement is probably true for most proteins: If you want to keep them in their natural state you have to keep them near pH = 7, among other things.

You can buy my book (Uwe D. Neue, HPLC Columns, Wiley-VCH) from Amazon.com or from Waters Corp part number WAT038216.

Thanks for your reply, i am more concerned about adsorption of small molecules on sephadex G15. do you think 2-mecaptoethanol is an organic modifier?

Cleaning up, I just got Tosoh`s "The Customer Magazine", #01, 2009 in my hand. There was an article in there talking about
E. Daisuke, et. al., J Chrom, vol. 1094, #1-2, 49-55 (2005). It was suggested that arginine in the mobile phase might be the best way to go in SEC of some proteins. They mentioned that high concentrations of salts can eliminate ionic interactions, but may cause an increase in hydrophobic interaction. Organic modifyers and urea, while increasing solubility, can be expected to also increase ionic interactions.
They did not mention pH, as a pH of 7 was apparently considered mandatory. If this is not the case one can also vary pH to eliminate absorption.

do you mean you recommend use offline MS to check GPC fraction after Ve.

If money is not a problem:
- GPC-MALLS will shed some light on the separation mechanism.
- If your polymer is very low molecular weight (< 5000 Da) LC-MS
might help.
- MALDI after GPC fractionation could help too

Mercaptoethanol? For what? To prevent oxidation? I wouldn´t use that stinking stuff, unless it did some miracle for me.
OK, I was thinking of proteins when replying. I have never trusted SEC for small molecules, so have not used it, but preventing these from sticking would be just like getting molecules to elute at tm (to) in other methods. If you think the molecules react with the Sephadex you should give us more info.

Maybe slight off-topic - but I have had some problems with hydrophobic interactions between one of our peptides and Superdex SEC columns. In one case, the largest and most hydrophobic peptide eluted last - even though it should have eluted first.

The only way to control that was to add acetonitrile to the mobile phase and adjust the elution of that compound. Some may see that as an advantage, to have one more parameter to use for separation.

The problem is that no SEC columns are checked for hydrophobicity when manufactured, so don't count of the same behaviour of the next batch of columns... We had to send back one batch that was just too different - but they still passed all tests at Amersham.

Sephadex actively adsorbs aromatic compounds. See the references I posted today in the other string on this subject. It's a function of the crosslinking agent used to form dextrans into the bead shape, so there's no way to avoid it with carbohydrate-based SEC media. A good silica-based SEC material does not have this problem, and one can separate molecules as small as amino acids with the right combination of pore diameter and mobile phase. If you want to read about all the variables involved in tedious detail, then read my book chapter from 1999 on SEC of Small Solutes. It's available in the Literature section of the PolyLC web site.