I am currently working on bile acids separation. I started with literature study and found researchers have tried on 3.0 pH and 6.9 pH buffers. I 've tried both, but I feel that I am not getting the desired output due to improper peak tailing issues and co elution problems. I have designed a gradient program that gave good separation. But there are few issues.
Please help me with them.
1. Concentration of aqueous buffer salt and pH
2. Choosing the diluent. All bile acids are water soluble. Is water fine?
3. Plate count of the first peak is around 3000 - 4000. Whereas the rest are above 9000.
4. Co elution no effect on gradient. Temperature of column. I 've tried everything. Will organic modifier like TEA TFA help.
5. My method target is under 15 minutes. Three bile acids under my study. Glycocholate glycochenodeoxy chocolate and taurodeoxy cholate.
6. What is maximum bile acid concentration I can go without peak saturation.
7. Since all are having spectra at 200-220 nm. Can I go for ultra pure solvents.
See the websites of Agilent, Waters, and Phenomenex for HPLC methods for bile acids. Water alone will not help, nor will TEA.
Please be aware that pH 9, which is recommended in the link suggested by Sabah might damage most HPLC columns in the long term. Please find links to some other methods and a review.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/hplc-separation-of-bile-acids-and-their-conjugates-with-ascentis-express-c18.pdf
https://www.ncbi.nlm.nih.gov/pubmed/24627129
http://www.ingenieria-analitica.com/downloads/dl/file/id/2116/product/320/quantification_of_bile_acid_by_reversed_phase_hplc_with_evaporative_light_scattering_detection.pdf
http://www.jlr.org/content/49/12/2690.full
http://article.sciencepublishinggroup.com/pdf/10.11648.j.ajac.20140206.11.pdf
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