"Do not use 70% ethanol to sterilize the wand, it does not burn completely. Better yet, 70% becomes 50%, becomes 30%... WATER DOESN'T BURN!!"More recently in a comment, Kevin Z muses, "I don't really see how it matters to make fresh ethanol dilution or keep a 85% stock around." Being graduate students, and having totally forgotten basic high school science, this became a question: Is this true, does ethanol in solution evaporate preferentially over time? (Of course, being graduate students, it took 2 years to come up with and test the question).
There were two schools of thought on the possible answer:
1) A mixture of water and ethanol will form a perfect solution with its own unique properties (different percentages will have different evaporation rates but the ethanol percentage will remain constant over time)
2) Ethanol, being more volatile than water, will preferentially leave solution resulting in a decrease in ethanol content over time.
To answer the question, a series of ethanol dilutions ranging from 0 to 100% were prepared and left open on the benchtop in 15 mL tubes (10mL of each dilution). The volume was recorded every day for a period of 1 week and plotted.
Figure 1: Change in volume of open ethanol solutions over a period of 1 week. Slopes represent evaporation rate (mL/hour).
As Figure 1 demonstrates, each solution has a unique evaporation rate and slopes appear roughly linear. However, closer examination reveals that the graphs are not linear but rather the slope of each curve changes subtly over time. That is, for example, the calculated slope of the line over the first 3 days differs from that over the last 3 days (data not shown). This change in evaporation rate over time suggests that the ethanol content of each solution is changing, in line with view (2).
Because the changes in slope were subtle, a second metric was chosen to verify that the ethanol content of each solution was changing over time. For this, the mass of one mL of each solution was measured at the end of the week and compared to the density of solutions of known composition.
Figure 2: Density of various ethanol solutions. Diamonds indicate the density of freshly prepared solutions at the indicated ethanol percentages. Bars indicate the measured density after one week on the benchtop.
As can be seen in Figure 2, the density of ethanol solutions does, in fact, change over time - indicated by the difference between the points and the height of each bar. The difference is most noticeable at mid-range mixtures (~30 - 50% EtOH). Final ethanol percentages after one week were calculated based on the known standards. (I realize the x-axis doesn't scale correctly [eg. the space between 0 and 10 is the same as between 10 and 30], this was a quirk of Excel that I couldn't figure out in a short amount of time. The standard curve and R2 were determined on a separate graph with proper scaling [not shown] using the same data) The summary can be seen in the table below.
Table 1: Summary of Ethanol Densities
|% EtOH in solution||Density (g/mL)||Density (after 1 week)||Estimated final % EtOH|
Long story short, the postdoc in question was correct. After a week on the bench, 70% ethanol used to sterilize bacteria spreading wands will be a shadow of its former self. Regarding Kevin Z's comment, if you're in the habit of leaving 85% EtOH open on the bench, then by all means you should be making it fresh - but I suspect that's not the case. This carries over to other areas in the lab as well. Oft times, the people filling TC spray bottles with 70% ethanol for sterilizing work surfaces (maybe this is where the confusion at the bacterial bench comes from?) will prepare too much and leave the excess in a graduated cylindar on the bench - sometimes covered, sometimes not - to fill bottles at a later time. This, again, will lead to evaporation of ethanol and spray bottles filled with less than 70%.
This could have been figured out in a much simpler way. Water and ethanol form an azeotrope - a mixture whose composition can't be changed by distillation - at a ratio of 95.6 : 4.4 ethanol : water. In this mixture, the vapour has the same constituent ratio as the solution. Other ratios will have one component evaporating off at a different rate than the other. Which is why, of course, we're able to distill spirits to increase their alcohol content. Check out the above link to read more about azeotropes, phase diagrams and Raoult's Law.
The real lesson, though, is to put the cap back on your Jameson's when you're not drinking it.