I use hydrochloric acid to release a steroidal sapogenin from its saponin, then neutralise it with sodium bicarbonate. I am confident the hydrolysis works because the mixture no longer produces a persistent foam.
I know, from the literature, that this sapogenin is poorly soluble in water but very soluble in chloroform, so I phase extract the aqueous mixture with chloroform. I send the chloroform layer to my collaborator who evaporates it and resuspends in deuterated chloroform to do NMR. I expect several milligrams of the sapogenin but he has been unable to detect any. I'm losing my sapogenin. What am I doing wrong?
Some details and observations:
1) I add 2mls of concentrated (10N) hydrochloric acid to 8mls of the saponin which is in 45% ethanol (it's a “tincture”) so gets diluted to 36% ethanol when the hydrochloric acid is it added.
2) This (10ml) 2N reaction is incubated for 4 hours at 80C in a sealed polypropylene tube.
3) After incubation, the entire 10mls is poured into a flask containing 3 grams of sodium biocarbionate and mixed. Once the foaming (caused by release of carbon dioxide) has stopped, I pour the material into a new polypropylene tube. A lot of salt (NaCl) is produced from the neutralisation step. This salt and unreacted sodium bicarbonate settle to the bottom. I assume the solution is saturated with sodium ions.
4) Copious amounts of a dark red precipitate forms – even under conditions using much less acid, such as 0.01N reactions. When the solution is spun in a table top centrifuge, it produces a pellet that is about 1.5ml (on top of the salt formed by neutralisation) from 10mls of solution. The supernatant (from this 0.01N reactions) still produces a persistent foam. The red pellet, suspended in water, does not.
Not surprisingly, reactions at 2N also produce this precipitation but it is much darker – like a dark purple approaching black – and there is no persistent foam.
I don't know what this stuff is but I'm guessing it's polytannins.
5) I add 4 mls of chloroform to the (hydrolysed and neutralised) solution. Seal the (polypropylene) tube and shake vigorously for 30 seconds. I then spin the tube in a table top centrifuge at 4000RPM for 5 minutes. Next I use a graduated plastic (polypropylene) transfer pipette to penetrate the aqueous layer, bubbled out the tip (which has a “plug” of “polytannins” and some aqueous) and then collect the chloroform from the bottom, while avoiding the salt pellet. I transfer this into a glass (scintillation) vial. Then I add another 4mls of chloroform and extract again. I end up with more than 8mls in the vial because some of the ethanol moves into the chloroform. (They're miscible.)
Attached is a pdf with some photos and experimental flow chart.
Thanking you in advance for your suggestions.
Best regards,
Jamie
I had a problem following one potentially crucial point. After hydrolysis and neutralization, did you add chloroform to the hydrolysis and neutralization flask? Or did you pour out the liquid to secondary tubes and then treat with chloroform? If the sapogenin is very insoluble in water or aqueous ethanol, it is possible that much of your sample is clinging to the walls of the hydrolysis flask.
Great point, Joe. You may be on to something.
I neutralised in the glass flask and swirled the solution around and up the inside of the flask to get it well mixed – the worst thing do to, if your hunch is correct – then poured the solution into a new polypropylene tube.
I'll repeat the experiment but this time, after I've poured out the solution, I'll add a few millilitres of chloroform into the flask, swirl it to collect any clinging sapogenin and transfer that “chloroform wash” to the tube containing the (neutralised) solution to complete the extraction. Then repeat.
I'll post a follow up here. Many thanks.
And, if anyone wants to weigh in with info that supports this idea or an alternative hypothesis, please feel free to do so.
Best regards.
Jamie
The washing of the reaction flask with organic solvent is a good suggestion in this case to recover the aglycone. In the aqueous phase you may find the saccharides previously linked to the saponin, but these are not soluble in deuterated chloroform. A more polar solvent should be used to solubilize them.
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