What Happens to Beet Root Pigments When Cells are Disrupted with Chloroform?

[Artermis]

Quote:
Originally Posted by Artermis
Hello Moonbear,

I don't know if you know me but I'm a relatively new member but I know that you are very knowledgeable and helpful, hopefully you'll be willing to help me with this.

Hi, I've seen you in the biology forum recently. Glad to have you on-board.


Quote:
But alas! Betanin and chloroform do not mix together, so the betanin should stay inside the cell and may not diffuse out. There should not be a visible change and the test tube should remain clear.

I don't know this specific example, so can't be entirely sure, but if you're disrupting the cells with chloroform, anything in the cell has to come out. If what comes out (betanin in your example) doesn't mix with the solvent, then it should just separate into two phases, an aqueous phase and an organic phase. The aqueous phase would contain all your water-soluble components, and the organic phase would contain all the hydrophobic compounds.

This is a perfectly good question for the bio forum, so why don't you post it there and see if anyone there has tried this, perhaps as a lab exercise in a class. They might be able to verify if what I think would happen is really what happens.

Cheers,
Moonbear
________________________________________________________
some interesting and helpful sites:
http://www.fed.cuhk.edu.hk/~johnson/tas/investigation/membrane_teacher.htm

 

[Moonbear]

For folks who might be confused, Artermis asked me this question via PM and I suggested she post the question out here to get more input.

Here is her full question:

A few slices of beet root (containing the red pigment betanin, which is very common in high school biology experiments) are submerged into a test tube of 10 mL of chloroform. What happens? Do you observe any red pigment release?

I know you don't help people unless they've done some work and thought about it, so here is my rudimentary analysis:

chloroform is not soluble in water, whereas betanin is. chloroform, because it is an organic solvent, should denature the proteins in the cell membrane of the beet roots and "destroy" the cell membrane (I'm not sure if the cell membrane is actually destroyed but is still there but now easily permeable - the extent of the damage to the cell membrane is eluding me), which should allow the betanin molecules to diffuse (again, not sure about the specific damage done to the cell membrane) out into the chloroform. But alas! Betanin and chloroform do not mix together, so the betanin should stay inside the cell and may not diffuse out. There should not be a visible change and the test tube should remain clear.

That is my thinking, I would really appreciate it if you could clarify. Thank you!

 

[Ouabache]

I don't know this specific example, so can't be entirely sure, but if you're disrupting the cells with chloroform, anything in the cell has to come out. If what comes out (betanin in your example) doesn't mix with the solvent, then it should just separate into two phases, an aqueous phase and an organic phase. The aqueous phase would contain all your water-soluble components, and the organic phase would contain all the hydrophobic compounds.

I have not done this experiment, but speculating on what was given and reading the same http://www.fed.cuhk.edu.hk/~johnson/tas/investigation/membrane_teacher.htm you had found (a lot of good insight there), I agree. Once the cell membrane has been damaged, the contents would leak out. Even if the pigment was not soluble in the surrounding medium it would at least be soluable in the aqueous phase. The organic phase should remain clear unless there were some additional unaccounted pigments which dissolved there.

Another thought. If you were to shake the contents of the tube, the two phases would likely mix and form a suspension. Then it would appear that the pigment has mixed with the chloroform until you wait some time and allowed the two phases to re-separate. But from Artermis' description, I don't think this scenerio will happen.

synonyms of betanin: in literature you may find betalain and anthocyanin

 

[Artermis]

Does the phospholipid bilayer reverse when it comes into contact with the chloroform solution? Normally in an aqueous solution like water, heads would be out and tails in, (polar heads, apolar tails) but in chloroform which is a non-polar substance, would the bilayer reverse and have tails out and heads in? Interested in exactly what happens to the bilayer and the extent of change/damage done to it.

 

[Ouabache]

Does the phospholipid bilayer reverse when it comes into contact with the chloroform solution?

I am no biochemist, however my speculation is that this configuration is not mobile. As a bilayer, the hydrophobic edges face inward towards one another, and both hydrophilic edges face outward as in this http://courses.washington.edu/conj/membrane/plbispac.gif. Looking at this configuration, I suspect they are chemically bound in position and cannot flip around.

 

[Moonbear]

I really don't know how chloroform disrupts the membrane...I've just known since I don't know when that it does, and never gave much thought to how. I always just thought of it as "poking holes" in the membrane by dissolving the phospholipid bilayer, but I just never gave it enough thought to confirm if that was the mechanism.

As for the rest of the beet root lab, I found explanations of the rest of the lab and expected results on this site:
http://www.fed.cuhk.edu.hk/~johnson/tas/investigation/membrane_teacher.htm

 

[EnumaElish]

Beet root has to be available in your neighborhood grocer, and you should be able to buy chloroform from a good pharmacy. Should you not?

 

[Moonbear]

Beet root has to be available in your neighborhood grocer, and you should be able to buy chloroform from a good pharmacy. Should you not?

I don't think chloroform would be available to the general public. I don't think artermis was asking about where to get the supplies anyway, she was asking the mechanism by which chloroform disrupts the cell membrane, and then what happens to the pigments within the cells that are not soluble in chloroform.