Ethanol is super useful. It’s great for killing bugs, setting things on fire, and forcing nucleic acids out of solution. But not all ethanol is created equal, and not all kinds of ethanol are suitable for every task. To help you make sense of your flammables cabinet, here’s the rundown on the grades of ethanols typically used in molecular biology, as well as some important rules for how to use them correctly.
1) 95% (95.6%) ethanol
This is the highest concentration of ethanol you can get by distillation, because 95.6% ethanol is an azeotrope, which means the vapor state has the same ethanol:water ratio as the liquid state.
2) Absolute (99-100%) ethanol
Some procedures that are sensitive to the presence of water require absolute ethanol. A common method to produce ethanol with a higher concentration than 95% is to use additives that disrupt the azeotrope composition and allow further distillation. For this reason, absolute ethanol sometimes contains trace amounts of these additives (such as benzene). Absolute ethanol is hygroscopic (it attracts water), so don’t expect it to remain ‘100%’ ethanol for long if it’s left uncapped.
3) Denatured ethanol
Denatured ethanol (either 95% or absolute) contains additives (such as methanol and isopropanol) that render it unsafe to drink, and therefore exempt from certain beverage taxes. This makes it cheaper than pure ethanol.
Ethanol for molecular biology
You should not use denatured ethanol when precipitating nucleic acids, since some of the additives can interfere with downstream applications. This is a bit tricky to predict, because the additives vary between producers, but it is safest to avoid denatured ethanol altogether. Non-denatured ethanol at either 95% or 100% can be used, although when working with fluorescently-labelled nucleic acids, I don’t use absolute ethanol, in order to avoid background fluorescence from residual benzene.
Ethanol for disinfection
Aside from substantial differences in cost, it doesn’t really matter which ethanol grade you use for disinfection, so most labs use a denatured stock. What does matter is the final concentration of ethanol. Ethanol is an effective disinfectant at concentrations between 70% and 90%. Strangely, no-one has established experimentally exactly why undiluted ethanol doesn’t kill some kinds of bacteria as rapidly as diluted ethanol, although the most likely reason is just that aqueous ethanol is a more effective protein denaturant than absolute ethanol1.
However, there are two things to watch out for when diluting ethanol. One is that diluted ethanol is no longer an azeotrope, so evaporative loss will result in a gradual decrease in ethanol concentration. People who keep unsealed beakers of 70% ethanol on their bench should not be surprised when the ethanol ‘stops working’.
The second thing to watch out for is that ethanol/water mixtures don’t have additive volumes. That means that if you want to make 1 L of 70% ethanol from absolute ethanol, you can’t do it by mixing 700 mls of ethanol and 300 mls of water. The correct method is to measure 700 mls of ethanol and then bring it to a volume of 1 L with water.
Of course, you also shouldn’t forget that ethanol of any grade is highly flammable when undiluted. Watch that flame!
1 Ali, Y, Dolan, MJ, Fendler, EJ and Larson, EJ (2001) Alcohols in Disinfection, Sterilization, and Preservation, ed. Block, SS, 5th ed.