Hello, 

I am afraid that you cannot use fucose to identify your fucoidan using HPLC, unless you hydrolyse your fucoidan and test its fucose content, using the sigma fucose as your standard. Bare in mind that the definition of fucoidan is it a polysaccharide, contains mainly of fucose and it is sulfated. Therefore, if you can quantify the percentage of fucose in it, and it shows that it has high fucose content, then you can say that it is fucoidan. In order to check the sulfate content, you can either use the spectrophotometric method, or CHNS elemental analyser. Alternatively, you can use FTIR to determine if the sulfate functional groups are present or not. 

I would recommend you to use a screening method to screen the percentage of fucoidan available from your seaweed sample. This method utilises a colourimetric reaction to identify the presence of fucose. Next, get a fucoidan standard, either from sigma or food grade fucoidan (from Marinova, Australia; YSK, Japan; etc) to identify the extracted fucoidan using HPLC. Please find attached the article that mention the methods above:

Article Isolation and antioxidant capacity of fucoidan from selected...

Will it be a flawed concept to use fucose as standard, and thereby saying that fucoidan is present by the appearance of the same peak as that of the fucose in the sample? (But I think i get your point that fucose is a monosaccharide while my sample is expected to have multiple chains of monosaccharides wherein possibly there are unique substituents in between linkages) Nevertheless, will the conceptual method be entirely flawed?

For the colorimetric reaction, are microplate readers appropriate?

I noticed that you used Chloroform:Methanol:Water for LMW extraction and defatting. However I have read articles (Michailovna et al., 2009) saying that CHCl3 is quite not desired to be used for seaweed fucoidan extraction. In my prior phytochemical screening practice, hexane was used to defat.

I'm attaching here my propsed fucoidan extraction process. Please let me know what you think. I also intend to insert somewhere the addition of hexane to defat. Would you recommend that I insert defatting process at the start: that is, ethanol:hexane proportions instead of ethanol alone?

I have downloaded your article and will talk it out with my team. Your knowledge and input is very much appreciated.

Thanks!

Both fucoidan and fucose have different properties (even though fucoidan is made from fucose). Fucoidan is a polysaccharide, while fucose is the monosaccharide, making the big differences in molecular size and polarity, thus you will get different retention time for both fucose and fucoidan. Therefore, your conceptual method is not workable if you inject fucoidan without prior hydrolysing the fucoidan into its monosaccharides. 

As for the colorimetric reaction, yes, use microplate reader. More efficient that way.

We are actually working on a modified extraction method right now, which is similar to yours. In our method, we use only 80% ethanol to defat, without hexane. I would think the mixture of solvents enables the extraction of different compounds with different polarity, thus, more efficient. 

Hi,

I have just found out that hexane treatment for defatting is actually after initial maceration with 80% ethanol, wherein hexane is mixed with the residue via separatory funnel (of course after rotavap to remove ethanol). After letting it settle, the fatty layer is discarded and the residue is once again introduced to rotavap (or steam bath), thereby removing hexane.

For your previous research, did you try to employ any test to identify and quantify any possible protein content remaining after intensive purification? (The same is true for sulfate and sugar/monosaccharide content)

Lastly, what purification method would you suggest us, undergraduate students, to do when we cannot afford agarose for gel filtration or anion exchange process? What is available to me as of the moment is Ultrafiltration, Liquid Chromatography-Mass Spectrometry and perhaps ion-exchange (for desalting, I think).

PS: Do you happen to have come across full copies of the articles linked below? Unfortunately subscribing to them would be a hardship for me and I humbly ask that if you have access or copies of them, I would greatly appreciate it.

All in all, thank you for continually sharing your knowledge and experience!

http://pubs.acs.org/doi/abs/10.1021/ac60111a017

http://onlinelibrary.wiley.com/doi/10.1002/0471142913.fae0302s00/full

http://www.sciencedirect.com/science/article/pii/S0008621502000538

http://www.sciencedirect.com/science/article/pii/S0144861707000495

http://www.sciencedirect.com/science/article/pii/0003269776905273

Hello,

I didn't test for protein content, however, I tested for the sulfate content via CHNS elemental analyser. While doing that, I get the results for nitrogen content as well, thus showing the presence/absence of protein. 

I would say that ultrafiltration is good for purification, but you must look into the type of membrane used for the ultrafiltration. LC-MS is more of testing the purity as well as the impurities present. 

As for the publications, yes, I have some of them. Probably you can give me your email, and I will forward it to you. Glad that I could help.

Hi,

My email is [email protected]

I am really glad for your effort in sharing indispensable information. I hope our Q&A's will help other researhgate members who are, like me, still budding researchers.

Thank you!

PS: I still have questions in my mind but I have to attend to class today. Have a nice day!