So I was doing some live heptane extraction and LLE and came across something very strange…
My initial heptane extract has some green tones; and then I mix in 3 parts methanol to pull cannabinoids out, while letting the fats hang out in heptane.
I was expecting all the greens to go into the methanol, as we’ve been generally assuming alcohols like chlorophyll…
My methanol layer is coming out orange/red and my heptane remains dark green. After multiple washes the green all stays in the tane…
Have we been wrong about chlorophyll solubility?
What’s the mechanism at play? (Other than the obvious straightforward answer of affinities).
Chlorophyll is often enmeshed in the thylakoid membrane which is non polar.
Guess that’s a wrap!
Oddly enough though… If I start with a methanol extract and stir some heptane on top to pull some fats etc. My resulting methanol layer is still dark…
Is there another part to that riddle?
Maybe methanol breaks up the membrane?
I don’t think the answer is that it’s encased in cell parts. If that’s the case, any ultrafiltration membrane would immediately remove all the chlorophyll entirely. And that does not occur in most situations.
Are you mixing water with your methanol? Or is it 100% organic? Chlorophyll is insoluble in water so the addition of water to alcohol phase could make it prefer heptane.
In general during a solvent extraction the chlorophyll will be released from the chloroplast
Methanol must be more polar than chlorophyll prefers relative to heptane. Chlorophyll is very non polar. Chlorophyll is likely a lot more soluble in ethanol than methanol
Definitely some water in everything
I had forgotten the structures, just remembered it was more of an issue with EtOH pulls.
Mainly nonpolar indeed.
Every day I realize how little I know
I just remembered the similarity to heme but forgot about the long ass tail. I wonder if it self assembles into micelles with its polar head and nonpolar tail. I guess the head is too planar tho.
Throw some sodium sulfate in there while it’s in heptane and watch the sodium swell up with the chlorophyll and sink to the bottom.
The head is much more nonpolar than polar as well. Although there is an ionic group it is not too accessible to solvent.
Interesting… I also would have expected the chlorophyll to move to the methanol phase.
I’d be interested to see what happens with other immiscible polar solvents. Acetonitrile, DMF, DMSO, etc.
Thanks for sharing your results!
Google always has the answer
Meaning you will pull more with tanes and less with etho or iso.
But thats not the case in practice. Hmmm
My best guess is you are forming a micelle structure. Looking at the structure of chlorophyll A & B, it very well could arrange into micelles. Thy hydrophobic tail isn’t that long (C16) many food emulsifiers are C12-C18; as for the head group, while the porphyrin ring is rather hydrophobic, the ionic character increases polarity.
Saturated fatty acids/triglycerides become insoluble/immiscible in ethanol/methanol at room temp around 12-14 carbons (the tail is pseudo-saturated because of the trans double bond). However, I would guess the head group has some alcohol solubility (large aromatic compounds typically have low solubility in most solvents).
This seems to suggest some kind of micelle structure in methanol and heptane but the original phase in which you start will determine the orientation of the micelle. With heptane, the tails are out, with methanol the heads are out, and you have various other plant compounds that are distributed between the interstitial spaces of the tails or heads.
Something you could try is a temperature swing where you gently heat the sep funnel with water (on the outside) to try and disrupt the supposed micelles. Another option would be to sonicate it briefly to see if the helps or hurts (I find ultrasound has many unintended consequences). I’m guessing you have small droplet sizes, which means the droplets have kinetic stability, so it may be difficult to break the emulsion.
All this said, it seems like this is a good problem. Since the cannabinoids are going into the methanol and the waxes stay in the heptane, then the chlorophyll staying there seems like a win! Thanks for posting this, this may illuminate a bit on color remediation and a way to remove the crap!
This was a far more accurate description by what I meant by ‘enmeshed’.
My grad group made solar cells out of PSI extracted from spinach leaves. They had to sonicate the extract in methanol to get the chlorophyll free of the thylakoid membrane. Then used chromatography to separate and then linked it to a long chain thiol so they could form self assembled mono layers of it. Cool stuff.
I don’t think it could be micelles for the reason I already stated – they’d be removable by pure filtration very easily if that was the case.