"fresh" 80% ethanol for Ampure XP

[mapplebe]

So I've read several different protocols that all suggest making "fresh" 80% ethanol for the wash steps because the 80% ethanol quickly absorbs water from the air, making it not-80% ethanol

My only question is whether it is really better to open your stock of 100% ethanol over and over every time you run a prep - wouldn't the 100% ethanol absorb water even more quickly, essentially causing the same problem? Do people buy smaller volumes of stock ethanol so the turnover rate is just faster?

I do believe this is a problem - I have noticed even with PCR cleanup kits that older bottles of wash buffer tend to wash most of my product away (grrr!) - but it doesn't make sense to me why opening the stock bottle more frequently would work better making lots of small, full aliquots in containers that are air/water vapor tight - am I missing something?

[rnaeye]

Ampure XP protocol by manufacturer suggest 70% ethanol, not 80% (at least that was the case last time I read). I make 80% in a 15 mL tube, and use it. If I am not going to finish it on the same day, I label the level by marker, if it looks the same after days. I still use it. I a not worried about ethanol absorbing water. I am more worried, ethanol is evaporating and water percentage is relatively increasing.

[pmiguel]

So I've read several different protocols that all suggest making "fresh" 80% ethanol for the wash steps because the 80% ethanol quickly absorbs water from the air, making it not-80% ethanol

My only question is whether it is really better to open your stock of 100% ethanol over and over every time you run a prep - wouldn't the 100% ethanol absorb water even more quickly, essentially causing the same problem? Do people buy smaller volumes of stock ethanol so the turnover rate is just faster?

I do believe this is a problem - I have noticed even with PCR cleanup kits that older bottles of wash buffer tend to wash most of my product away (grrr!) - but it doesn't make sense to me why opening the stock bottle more frequently would work better making lots of small, full aliquots in containers that are air/water vapor tight - am I missing something?

In principle 80% ethanol should not pull water from the air. Even if it did, how much water is in the air sitting just above your wash solution in a capped container? Not much.

The benefit of making a fresh batch of wash solution is that, presuming you made no mistakes, it will be in a known good state at that point. Not so the bottle of wash solution opened 10 months ago and used by everyone in the lab, including the (bitter post-doc|clueless undergrad) who dumped column washes back into the bottle after doing their last purification.

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Phillip

[microgirl123]

Why wouldn't 80% EtOH absorb water from the air? EtOH is hygroscopic. And I guess that the original poster's question about 100% EtOH absorbing water also makes sense, although I'd never thought about it. For non-expensive, less-important things, I keep using old EtOH mixtures, but I always make fresh for library prep and clean-up.

[pmiguel]

Why wouldn't 80% EtOH absorb water from the air? EtOH is hygroscopic. And I guess that the original poster's question about 100% EtOH absorbing water also makes sense, although I'd never thought about it. For non-expensive, less-important things, I keep using old EtOH mixtures, but I always make fresh for library prep and clean-up.

100% EtOH is hygroscopic. I don't think 80% EtOH is hygroscopic.

There could be a preferential evaporation of EtOH molecules over H2O molecules that would amount to nearly the same thing. But unless you leave your wash buffers perpetually uncapped, I doubt you would experience much change in composition.

Nevertheless your approach sounds like a prudent one to me.

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Phillip

[GenoMax]

100% EtOH is hygroscopic. I don't think 80% EtOH is hygroscopic.

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Phillip

Some interesting facts about ethanol/water: http://en.wikipedia.org/wiki/Azeotrope

http://en.wikipedia.org/wiki/Ethanol_purification#Purification

I suppose 80% EtOH would still technically be hygroscopic just not as much as 100%.

[kcchan]

I think the handling of the beads has a bigger impact on recovery rates than the concentration of ethanol. The beads should be washed very gently, making sure none of the beads get knocked off. You should dispense the ethanol as a slow drip off the tip of the pipette. Also make sure not to suck up any beads while disposing of the supernatant. If you do, dispense everything back and wait a minute or two to let the beads drift back to the edge of the tube. Where I work we use very strong magnets that lift the beads off of the bottom of the tubes. This makes it much easier to avoid disturbing the beads.

[Phlya]

The beads should be washed very gently, making sure none of the beads get knocked off.

Why would you say that? I routinely wash them pretty harshly (resuspending them in ethanol by pipetting), and the recovery is always close to 100%.

[pmiguel]

Some interesting facts about ethanol/water: http://en.wikipedia.org/wiki/Azeotrope

http://en.wikipedia.org/wiki/Ethanol_purification#Purification

I suppose 80% EtOH would still technically be hygroscopic just not as much as 100%.

"technically"? If you open a tube of 80% EtOH, to be hygroscopic it would need to increase in mass due to absorption of water vapor from the air. Do you think that would happen?

I agree that the % of EtOH would decrease, but that would be through preferential evaporation of EtOH.

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Phillip

[kerplunk412]

I think below 70% EtOH may be where you might lose some product during washing, so I think that many protocols suggest 80% EtOH so even if it is old and has absorbed water or evaporated it will still be over 70%. I do not believe there is any problem with using older 80% ethanol, but as pmiguel mentioned it is good practice to make it fresh every time to avoid cross-contamination and such.

Another interesting fact about water/ethanol mixture- the volume of the mixture is slightly lower than the volume of the individual constituents. So 80% EtOH should be made by adding 40 mL 100% EtOH to 10 mL water, not by adding 40 mL EtOH and bringing to 50 mL with water. The difference in volume isn't much, but it is noticeable, and will result in a slightly lower % ethanol than intended if done the second way.