HPLC Technique for seperating derivatives of Cholic acid ?

[vanjas]

I'm trying to set up the method which quantitates cholic acid derivatives such as ursodeoxycholic acid, chenodeoxycholic acid, cholic acid, lithocholic acid, deoxycholic acid and hydrodeoxycholic acid at a time.
I'm using Capcellpak C18 column and UV-Detector. As my trial, these substances have very low peak responses at UV-Detector. My question is that is there any way that I can increase the peak sensitivity of them?
I'm using 0.1M phosphate buffer(pH 3.0) and acetonitrile as mobile phase with gradient method. Is my choice is right? Also, is there any way that I can reduce baseline noise ?
It would be really helpful if someone who has a experience dealt with this matter gives me advice.
Thanks for your help in advance.

[yaoguocan]

in my opinion ,you would better check the condition of your uv lamp .because low energy lamp will cut dowm the peak sensitivity of them.
what's more ,you would better clean detector cell .unclean detector cell will rasie baseline noise .make the lod higer than normal . i think if you just want to seperate them .lower flow rate but more polarity mobile phase should be applicable.

[yaoguocan]

in my opinion ,you would better check the condition of your uv lamp .because low energy lamp will cut dowm the peak sensitivity of them.
what's more ,you would better clean detector cell .unclean detector cell will rasie baseline noise .make the lod higer than normal . i think if you just want to seperate them .lower flow rate but more polarity mobile phase should be applicable.

[Ary]

Vanjas

These compounds have very poor UV response. You will need to derivatise them with a UV absorbing compound to improve your detection. I did look at these compounds a long long time ago but I'm afraid I cant recall what the derivatising agent used was. However there are texts which will give you plenty of options. Unfortunately I dont have any to hand.

Regards

Steve

[Mark Tracy]

When I used to work at Pickering Laboratories (www.pickeringlabs.com), we sold a custom post-column reaction system for that. Perhaps someone there can find the procedure.

Another alternative is to use evaporative light scattering detection. You will need to switch to a volatile buffer (ammonium acetate or formate).