Chromatography Forum

Hallo fellow chromatographers,

Has anybody encountered problems related with the purity of Tween 80. We use this product in our diluent solvent ( 0.02 to 0.01% ), in order to prevent adsorption of our biomolecules and by-products.
We are doing SEC chromatography and measure at 214 nm
While running blancs ( diluent solvent with the tween 80 at concentration of 0.02% and 0.01%) we observe peaks eluting at the retention where high molecular weigth products elute, in casu our molecules of interest and their aggregates. It contributes to the (im) purity calculation of our monomer...
Tween itself has an MW of 250 and should elute at the total permeation volume, so what could be the product ?

If we inject a 1% solution of Tween a clear peak is integrated at the retention time of high molecular weigth product.
We do not have a slightest idea what the productcould be : oligomers, impurities?

Looking forward to receive comments

Regards

Philippe

tween-80 is a mixure of fatty acid esters of sorbitan polyethoxylates and it can give you several peaks in LC . only solution to move away any tween-80 peak from your compound of interest.
since it is a natural product mixure and chromatographically its profile may change from source to source and on storage.

sticky stuff!!!

JM

Phillipe,

These types of heterogeneous surfactants are a chromatographic nightmare for both LV/UV and LC/MS. In addition, they may often contain significant levels of formaldehyde that may react with some molecules.

Try to find a very pure, crystalline homogeneous surfactant (SDS ??) instead.

JMB

What is the column and mobile phase that you use for SEC?

Yes, what is your mobile phase and column. If separation cannot be achieved with adjustment to your gradient then try different column stationary phase.

Thanks for the comments

The column we use is a BioRad Bio Sil 250 column and the mobile phase is 0.2 m Na phosphate pH 6.8 and we run the isocratic mode
It hardly impossible to change this method..

Regards

Philippe

Could have some of the dimer of the reduced sugar in it which is then ethoxylated and esterified or more than one ester on the end group. Tween 80 should have a much higher MW than 250.

see

http://images.google.com/imgres?imgurl= ... D%26sa%3DN

And also do a search of tween 80 with google and look at images instead of Web tab to see some other structures/chromatograms.

Could analyze your sample by LC-MS if got rid of the phosphate..