Home > Community > Why is saponification needed before HPLC analysis?
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+ Seaweed
+ Hplc analysis
+ Carotenoids
+ Purification
+ High-performance liquid chromatography
+ Chemistry
+ Extraction
+ Hplc-ms
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Kim Aaron

Why is saponification needed before HPLC analysis?

Anil Saraf  Follow

Bruce Neagle and Narong Chamkasem thank you for your response.

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Benson Wangombe  Follow

A quick search on the internet, I found two papers mentioned about the extraction with solvent, no need to saponify it . It the natural antioxidant and is not in the ester form, so you don't need to saponify it like vitamin A-acetate. You may need to show us the method you have that recommend to saponify the sample.

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David Paul  Follow

Mahmood M. Barbooti thanks for your response. It was exactly what I need to know.


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Carol Campbell  Follow

I looked at the molecular structure and it has ester at the end. If you saponify and break that bond, you would not have the molecule of Fucoxanthin anymore. It will be something else (acid form of acetic derivative) which I am not sure if you can call it as Fucoxanthin. Purification is one thing to separate the analyte from interference. You have asked about how to extract it from the sample. That is two different things. It is different than the vitamin A case. Vitamin A is sensitive to light and oxidation. The food maker added vitamin A -acetate or palmitate so it is stable in the final product. To determine vitamin A content, you need to saponify to get acetate out of vitamin A and determine vitamin A after that and the result is vitamin A concentraton.

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Damian Sanchez-Marin  Follow

@Amirreza, because you need to remove the ester if you want estimate free Fucoxanthin. more over, even you can try estimate without saponification too by suitable method. but you should know that fucoxanthin is mono ester or diester also the molecular weight of the particular ester. Now mathematically you can derive the free fucoxanthin content from ester values. unless other xanthophylls, fucoxanthin may not saponify because it is epoxicarotenoid, so during the saponification it may converted to fucoxanthinol.



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Brian Tucker  Follow

Amirreza... HPLC is a well certified and reliable method for the separation and analysis of components in mixtures. The components sometimes have some common properties that will cause overlapping of the chromatographic signals. OR... the matrix is so complicated that affects the analysis. Chemical modification is necessary in such cases to release the components or remove some interfering compounds. Thus, pretreatment of samples by saponification as in your case or esterfication for the analysis of fatty acid mixtures. The success of your analysis highly depend on the success of the pretreatment... Good luck...

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Bob Fishell  Follow

Interested information dear Mahmood

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Barry F  Follow

Thank you Dear @Narong Chamkasem , I have studied both of article but both of them used a silica column for purification of Fucoxanthin. Most of article used a purification or saponification stags. I want to know what is the importance of this stage and what will happen if we don't do it? Thanks

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Daniel Silvers  Follow

Take a sample and homogenize it in buffered water. Perform a 'light' (0.01 N NaOH) saponification to ensure complete release of fucoxanthin from the cells. Take an aliquot and dissolve it in mobile phase, ultrafilter a portion, and inject into the HPLC.

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Angel Wallace  Follow

Amirreza.... you are welcome.. Regards

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