Can anyone suggest the HPLC method to quantify spheroidenone carotenoid with a DAD detector and an Agilent zorbax SB-C18 column?
Hi. Probably, there will also be some impurities derived from red photosynthetic bacteria. In the following article, HPLC was performed on an ODS column (Zorbax Eclipse XDB-C18column (250 mm × 4.6 mm).). Please refer to it.
Article Assembly of functional photosystem complexes in Rhodobacter ...
Additional Notes. If you want accurate quantitation, you can purchase high purity standards from CaroteNature.
http://www.carotenature.com/wp-content/uploads/2022/03/PRICE_LIST_2022b.pdf
For a simplified approach, you can determine the total carotenoid content from the absorbance coefficients of the major known carotenoids in your sample by using absorbance spectrophotometry, and then perform HPLC at the same absorbance and estimate the concentration from peak purity by area normalization. However, this quantification method is not accurate.
As a side note, the only way to obtain the most accurate concentration is to use quantitative NMR. Even high purity standards are not strictly accurate in terms of purity, and the absorption coefficient is also only rough.
The bigger problem is perhaps the high price for the reference substance spheroidenone. Among others 10mg offered by Toronto Research Chemicals may become the best choice. You can test the purity the same time you prepare for your concentration-response correlation curve. The mass spectrum in the attached file has been made by GC-MS analysis. Isn't it to prefer before struggling with HPLC-MS?? It depends of course on your application and type of sample.
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