Removal of Mobile Phase Components From Purified Peak

[Slammy1]

Due to time restrictions and problems in separating, we used the existing method to purify an observed impurity. The method is ion pair using a phosphate buffer and heptanesulfonic acid. The impurity has a molecular weight of around 250 and is positively charged. What's the best way to remove the phosphate and HSA from the sample to analyze on mass spec and run NMR for ID? TIA for any advice.

EDIT: The parent compound for the this material has a pKa of 7.28, and the HSA is about 1.35. The impurity appears to be always charged (quaternary amine) irrespective of pH, and takes a long time to elute from an SCX column. I'm thinking we might try a weak cation exchange resin in an SPE module to perform this. Sorry for the ambiguity, I'm coming in on the back end of this problem.

[tom jupille]

How about an anion exchanger in the formate or hydroxide form? That should pass your compound through unretained (if not excluded, even).

[Uwe Neue]

I second Tom. You can find methods of this type in the Oasis Applications booklet at www.waters.com.

[Slammy1]

Hey, thanks for the replies. After talking with tech support for Phenomenex, I ended up ordering some of the Strata weak cation exchange SPEs. I always get confused with the resin beds, which are defined from what they are meant to hold rather than the type of resin present. The sodium counter ions will be the limiting factor, but tech support indicated I'd be able to minimize their presence by flushing copious amounts of water through the bed. Still, better sodium clustering than phosphate clusters. I'm more used to approaching the problem from a protein perspective, where you can use MWCO filters. Now the challenging part, purification of the peak. We don't have an isolator, so instead we utilize the divert valve on the MS. Making use of what's available, it's grad school all over.

[Vlad Orlovsky]

You can avoid all extra work if you use column with ion-pairing reagent attached to the surface of silica. You can use almost any mobile phase and definetly use LC/MS compatible mobile phase composition (ammonium formate or acetate, formic or acetic acid). Check any of the applications for Primesep 100 or Primesep 200 columns:
http://www.sielc.com/Applications_By_Detection.html
http://www.sielc.com/Applications_By_Column.html
http://www.sielc.com/pdf/SIELC_January_2007.pdf

Regards,

Vlad

[Uwe Neue]

Well, you have elected to do the opposite of what Tom and I had suggested. How will you do the elution?

[Slammy1]

The reason I went with the cation exchange resin is that I talked with Phenomenex tech support before you had posted, and that was what they recommended. The sample will be eluted with 100% organic (probably ACN). Sorry, they were anxious to move forward. It's one of those hurry up and provide an answer now projects. The concern with the anion exchange resin would be the sodium. I really like the bonded ion pair idea. I've passed the data along to the project chemist.

Thanks
Sam

[pipettemonkey]

Could you not simply run the fraction through a solid-phase C18 extraction column, then elute with acetonitrile?

[reyoungs]

Remember that you can dilute the sample with water or an aqueus buffer to allow it to bind to an RP phase and run it through a C18, phenyl or similar SPE cartirdge, wash with water to flush ions and then elute with minimal high concentration of organic.

If you still have to do this for the next RUSH stage, try diluting it to the point where it is barely soluble and you will be surprised the capacity of an SPE. This will be faster than the cleanup for the ion exchange SPE.