Phenol

[microBloom]

I ordered the wrong type of Phenol for my lab, we needed Phenol saturated with Tris and I just ordered just regular liquid phenol, so I have now brought it upon myself to sature the Phenol with Tris. I have a protocol which I will write here, after which I have a question or two.

 

The protocol tells me to add equal volumes of 0.5 M TrisHCl pH8, mix, and take off the aqueous phase. Then, add equal volumes of 0.1 M TrisHCl pH8, mix, and take off aqueous phase. Then, add equal volumes of 0.1 M (or 0.05 M) TrisHCl pH8, mix, take off aqueous phase, and messure the pH to make sure it's around 7.6-8.

 

My questions:

1. Should I do this in a beaker that has a stir bar in it, or do it in a large cylinder, cover it with parafilm and mix by flipping it upside down and right side up a couple times? The first way seems safer (since Phenol is so dangerous) but I was told that it really needs to be mixed vigourously and a stir bar might not do the trick. The cylinder seems more dangerous, but if I put enough parafilm on there, would it really hold the liquid? (My total volume will be 1 L)

 

2. When taking of the aquous phase, whether in a beaker or cylinder, is it ok if I pour of at least the beginning of it so I don't have to pipette off the whole 500 mL, or will the bottom layer pour out as soon as I tip over the beaker/cylinder. I was told that if it's in a cylinder the phases will have better separation than a beaker... opinion on this?

 

3. At the end, does it matter if for the last saturation step I add 0.1 M or 0.05 M TrisHCl pH8? Should I maybe measure the pH before this and determine from that whether I need more or less Tris?

 

4. How do I know how much mixing is enough?

 

5. Should I let it stand for any significant period of time beyond letting the phases separate?

 

Does anyone have any other tips?

[hypervalent_iodine]

My questions:

1. Should I do this in a beaker that has a stir bar in it, or do it in a large cylinder, cover it with parafilm and mix by flipping it upside down and right side up a couple times? The first way seems safer (since Phenol is so dangerous) but I was told that it really needs to be mixed vigourously and a stir bar might not do the trick. The cylinder seems more dangerous, but if I put enough parafilm on there, would it really hold the liquid? (My total volume will be 1 L)

 

The second option would be a bad idea. From the sounds of it (from an organic chemist POV), you would ideally want to use a separatory funnel and do a liquid-liquid extraction (there are plenty of YouTube videos on this). It will save you a lot of time.

 

2. When taking of the aquous phase, whether in a beaker or cylinder, is it ok if I pour of at least the beginning of it so I don't have to pipette off the whole 500 mL, or will the bottom layer pour out as soon as I tip over the beaker/cylinder. I was told that if it's in a cylinder the phases will have better separation than a beaker... opinion on this?

 

See above. Get your hands on a sep funnel, probably a 1000mL one.

 

3. At the end, does it matter if for the last saturation step I add 0.1 M or 0.05 M TrisHCl pH8? Should I maybe measure the pH before this and determine from that whether I need more or less Tris?

 

If the protocol says either or will work, then it shouldn't matter too much (though I'd maybe stick with 0.1 M if you're trying to saturate it).

 

4. How do I know how much mixing is enough?

 

If you're using a sep funnel, you would invert it and swirl it around a little or shake it (being sure to relieve the pressure every few seconds) a couple of times. I'm unsure how easy it is to saturate phenol with tris, but if it doesn't get to be the pH you need straight away, you can just keep washing fresh Tris through the phenol (i.e. separate the aqueous and organic layers, place the phenol layer back in the sep funnel and add some more Tris) until it does. Alternatively, you could leave it to stir for a while in a beaker (you shouldn't have a problem with using a stirrer bar) and then separate it with a sep funnel.

 

 

5. Should I let it stand for any significant period of time beyond letting the phases separate?

 

You should let it stand for as long as it take to separate. If it looks like it's taking a long time and you have a lot of emulsion at the interface, you can try and get rid of it by eluting the bottom phase off very slowly.

 

 

Does anyone have any other tips?

 

My tip is to get a sep funnel. Maybe biochemists have different ways of doing this, but a separatory funnel should work fine. If you don't have one in your lab, try borrow one from somewhere else. You could potentially use a beaker for this and leave it to stir with a stirrer bar and then decant the top layer off carefully, but this can be arduous and it will mean you'll have to pipette the last however much off.

 

Also, make sure you know which layer is the aqueous and which is the organic.

[CharonY]

The funnel is an elegant way to do it. Parafilm is a really bad idea. If you do not have access to anything, I would take bottle that is sealed tightly before shaking or vortexing phenol. For the most part, a stirring bar works also well, but I would disrupt the strring a little bit every now and then and restart, to ensure homogenous mixing. In the end it should look like a nice emulsion, without any obvious separation.

The problem with decanting is that either you will waste quite a bit (if you want to avoid getting the aqueous phase) or you will have to pipette the last bit rather carefully. Certainly not ideal, but doable.