Chromatography Forum

Hey guys,

I'm trying to develop a method to quantify protame and tame ( tosyl-l-arginine methyl ester). Protame is only available diluted in DMSO. I first tried to inject my protame diluted in DMSO, but my peak for DMSO was enorm and it showed more retention than my protame. (I'm measuring around 235nm, because TAME absorbs very poorly on higher wavlengths) Is that possible I tought that the DMSO peak would elute around the void time. I'm working in HILIC conditions, with 95% ACN 5% H2o on a silica column. Does anyone ever experienced retention for dmso? I started diluting my protame with methanol but the dmso still elutes after protame.

Thanks in advance,

Hajar

There is nothing unusual in the retention of DMSO. Treat it like a normal polar molecule and it will no longer be surprising that it retains in the HILIC mode. Actually DMSO may be severely affecting your peak shape. Try to reduce the injection volume.

Thanks for answering. I actually have a very low absorption for my TAME, so I'm afraid that reducing the injection volume will affect the absorption.

if you use mixed-mode revered phase/cation-exchange column you can analyze your target in higher organic, when DMSO elutes very close to the void and retain your arginine-based molecule by cation-exchange mechanism. You can have elution of DMSO close to the void. Any inorganic acid of buffer will allow you to use any wavelength and see your target.

Something like this:
http://www.sielc.com/Compound-DMSO-Dime ... oxide.html

Also, in mixed-mode you can use much higher injections with mismatched solvents if you are exploring ion-exchange mechanism, so you can inject your compound in DMSO and the column will tolerate it and your peak shape will be okay.