I am currently preparing to isolate polyphenolics (Hydrolysable tannins) from aqueous extract of a plant. I want to set up a small sephadex column to guide the major isolation on a laerge column.
My column is 20cm high and 40ml. How do i pack it? Is it okay to dissolve my extract in 50% Ethanol, even though I am thinking of starting with 100% Ethanol, followed by 10%, 30, 50, 70% Methanol?
You have to try. I separate the tannins on HPLC. But not all of them. Unfortunately, sometimes you have to use trial and error. I would start with 100% ethanol.
Hello,
Sephadex LH20 should be suspended in a water-methanol mixture of 95-5 or 90-10 (V/V), then poured into the column. After the settling of the column and its Sephadex filling over night, your extract is put on the surface of the sephadex material. Stepwise (steps of only 5 %) the methanol content is slowly increased (based on a TLC check which compounds are eluted). I have only tried with methanol, but the same procedure (including the filling of the column) should also be possible with ethanol (instead of methanol). Perhaps it is the best way to start with a low ethanol content in your mobile phase (preferably 95-5 water-ethanol mixture) to be sure not to overlook any compound. I would take the mobile phase (e.g. 95-5 water-ethanol mixture) to solve your extract and to put this solution/suspension of your extract on the column.
Much luck!
Kind regards, Klaus Peter Latté, Berlin
Thank you Klaus Peter Latté and Agnieszka Barbara Najda for your responses. They are very helpful.
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