Anion Pairing Method Development

[has1705]

I need some help developing an experimental anion pairing chromatography method for my research. I'd like to use a C18 column and preferably a methanol-based mobile phase, if possible. I'm not entirely sure what substances I'm looking for, only that they may be similar to sulfated steroids, and our starting samples are fairly crude and complex since they are derived from raw urine. Our previous separations have used XAD4-acetone extraction from raw urine followed by two separate RP-HPLC separations: the first on a C18 column using a methanol gradient (30-80% MeOH, 0.1% TFA), and the second separation on an analytical HyperCarb column with a THF gradient (10-50% THF, 1% TFA); all with post-separation screening for activity and MS analysis. I'd like to utilize a anion pair method compatible with our other separations. Any suggestions on where to start would be fantastic. Thank you all!

[mattmullaney]

Hi has1705,

In a general way, Sigma-Aldrich sells quite a large variety of ion-pairing agents, including those used for MS work. Probably the Chapter 7, and in particular, Section 7.4, of the text:

Practical HPLC Method Development, 2nd Edition, Lloyd R. Snyder, Joseph J. Kirkland, Joseph L. Glajch
ISBN: 978-0-471-00703-6, 800 pages, March 1997.

can help more than most other sources. Also, here's another text;

Ion-Pair Chromatography and Related Techniques, Teresa Cecchi, October 15, 2009 by CRC Press
Reference - 215 Pages - 24 B/W Illustrations
ISBN 9781439800966

and another link:

http://www.chromatographyonline.com/ion ... &&pageID=1

Best Wishes!