Chromatography Forum

I have a need to analyze bile acids - cholic acid, deoxycholic acid, chenodeoxycholic acid in aqueous solution.

Problem number one is that these acids are only very slightly soluble in water unless at a pH of around 10 or so, pKa's are around 5. I have an RI and a UV detector, the low concentrations in neutral pH I don't think I am going to be able to detect.

We haven't designed the experiment yet, we may be able to run at high pH. If so, can someone suggest a column? Would a "ruggedized" silica column work ok or would I have better luck with a organic resin based column?

Supposing we cannot work up the experiment at high pH. I am thinking maybe I can dry down the supernatant from the experiment, dissolve in MeOH and run in reversed phase conditions.

Any thoughts? I know there are some sharp people on this board.

Thanks,

Mike

Why don't you try HILIC at alkaline conditions using a polymeric column...

Such a column is available and you find more information at www.sequant.com

I would run this under reversed-phase conditions with acetonitrile as the mobile phase modifier. These compounds are fairly hydrophobic, so you will need a large fraction of acetonitrile, and they will be easily soluble under such conditions.

I have a need to analyze bile acids - cholic acid, deoxycholic acid, chenodeoxycholic acid in aqueous solution.

Problem number one is that these acids are only very slightly soluble in water unless at a pH of around 10 or so, pKa's are around 5. I have an RI and a UV detector, the low concentrations in neutral pH I don't think I am going to be able to detect.

We haven't designed the experiment yet, we may be able to run at high pH. If so, can someone suggest a column? Would a "ruggedized" silica column work ok or would I have better luck with a organic resin based column?

Supposing we cannot work up the experiment at high pH. I am thinking maybe I can dry down the supernatant from the experiment, dissolve in MeOH and run in reversed phase conditions.

Any thoughts? I know there are some sharp people on this board.

Thanks,

Mike

I used ion pairing agent, keystone C18 5U, 25cm with UV detector. ACN as organic. Buffer should be around 7. You may try different columns which works well in relatively high pH. I developed the method long time ago. Quite roboust.

I agree with Uwe, the molecule lends itself well to reverse-phase. I use a 60/40/0.1 ACN/H2O/H3PO4 mixture for the analysis of deoxycholic acid on a Luna C18 column. Worked pretty well for me.

An acetonitrile - phosphate buffer (30:70), pH 7.5, containing 5*10-3 M tetrabutylammonium hydrogen sufphate (TBA) or B) acetonitrile - phosphate buffer (65:35), pH 7.5, containing 5*10-3 M TBA;
works very well on a C18 150 x 4.6 mm ID column the link is included
https://www.macherey-nagel.ch/web/MN-WE ... =HPLC00902