I have a task to conduct shake-flask fermentation and purify the biosurfactant produced by Streptomyces sp. From the papers that I have gone through so far, many of them suggested using the silica gel column chromatography but with different kinds of solvent system. But I was suggested to conduct silica gel column chromatography with solvent system of chloroform: methanol: n-hexane, with ratio of 1:1:1. However, I still couldn't find any papers that conduct this method and I am having a hard time to understand the procedure for this method. I truly appreciate your willingness to help me with my understanding in regards to this method. Thank you.
Check https://www.researchgate.net/publication/290122979_Strategies_of_Solvent_System_Selection_for_the_Isolation_of_Natural_Products_by_Countercurrent_Chromatography
Also check https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/silica-gel
Dear Syafiqah Nadhirah Mukhtar thanks for sharing this interesting chemical problem with the RG community. There are very many relevant papers available in which the purification of natural product using column chromatography is described in detail. In your case you need to preparae a 1:1:1 (by volume) mixture of the three solvents. Then suspend the desired amount of silica gel in this solvent and fill a column of suitable size with the suspension. Let the silica gel settle and let the supernatant solvent run though the column. Then load the concentrated biosurfactant solution onto the column and let it run into the silica gel. Then elute the product with fresh solvent mixture and get the purified product as eluent. Finally evaporate the solvent to get your product.
You should also check the literature for related work describing the preparation and purification of biosurfactants produced by Streptomyces sp. For example, the authors of the following paper use only methanol for the column chromatography:
Production and characterization of biosurfactant from marine Streptomyces species B3
Article Production and characterization of biosurfactant from marine...
Please see if you can access the full text of this article through your institution. Alternatively you can request the full text directly from one of the authors. Two of them have RG profiles and can be contacted directly via RG.
Also please have a look at closely related technical questions which have been posted earlier on RG such as the following:
Regarding column chromatography for extraction of flavonoids, can silica gel be used as absorbent and hexane as eluent?
https://www.researchgate.net/post/Regarding-column-chromatography-for-extraction-of-flavonoids-can-silica-gel-be-used-as-absorbent-and-hexane-as-eluent
I hope this helps. Good luck with your work and best wishes, Frank Edelmann
Dear Syafiqah Nadhirah Mukhtar ,
The thing is that it depends on what you are aiming for. Is it a protein/peptide, is it a phospholipid, is it a mainly hydrophobic or hydrophilic substance (for example some sugar) etc. The overall process can be divided in several steps: production, extraction, separation, and purification. Each steps determines the direction towards certain types of substances.
If for example you extract your biosurfactant with the well-known Blight & Dyer extraction method then you end up with a selection of (phospho-)lipids, if you use some sort of acid precipitation method you end up with (primarily) proteins (and possibly glycolipids etc.).
The same is true for the separation and purification step. Every combination of solvents and every ratio used will result in different yields and end-products. See for example Table II in this paper:
Jiang, W., Shan, H., Song, J., & Lü, H. (2016). Separation and purification of ombuoside from Gynostemma pentaphyllum by microwave-assisted extraction coupled with high-speed counter-current chromatography. Journal of chromatographic science, 55(1), 69-74.
where you can see that the Partition coefficient (K) for their substance of interest varies dramatically for the different solvent combinations.
Finally, the choice in different types of chromatography is seemingly endless and is another determining steps.
Like you I was unable to find an example where they used the combination and ratio you indicated. I think that you should ask the one that suggested you this particular choice of solvents and ratio (and preferably a literature suggestion). All I can think of is that the biosurfactant you are being asked to go after is more hydrophobic than average and the one who came up with this suggestion expects a higher yield of purified product.
Otherwise, I suggest you go for suggestions like indicated by Frank T. Edelmann (see enclosed file for the first paper).
Best regards.
Hi Syafiqah Nadhirah Mukhtar . I suggest for you the following link: https://lab-training.com/column-chromatography/
See also the following link: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/column-chromatography
Also see the following link: https://microbenotes.com/column-chromatography/
Kindly see also the following link: https://chemistry.miamioh.edu/gung/CHM244/pdfs/column.pdf
Check on this paper.
https://www.degruyter.com/document/doi/10.2478/s11532-012-0037-y/pdf
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