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Glycerol is behaving strangely

glycerol biochemistry proteinase k

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#1 W.T. Lagarde

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Posted 13 March 2014 - 06:28 AM

I am currently performing genotyping in mouse tails using Proteinase K. In order to prolong the shelf life of our Proteinase K we wanted to create a storage buffer. One important part of this buffer is 50% (v/v) glycerol. According to all the literature I could find, the pH of glycerol should be about 7-7.5. However, after discovering our Proteinase K still would not keep, I decided to check the pH of all my ingredients. It turned out, our glycerol (99+%) was actually around 3.5. A different batch of glycerol, with a slightly lower concentration of glycerol (87%, diluted with Milli-Q) was even slightly lower at a pH of 3.2. Even stranger, diluting both batches of glycerol to the 50% required by our buffer brought the pH down even further. To me, and many of my colleagues, this made no sence at all.

All possible explanations I am able to come up with seem very unlikely. Are both batches of glycerol somehow tainted? Is there some reaction going on? Can some of the trace elements (Heavy metals or magnesium <5ppm) have such an extreme effect on pH?

Since I am not able to figure this thing out, I hope someone out there might have the answer for me. In any case, all help will be greatly appreciated.

#2 hobglobin


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Posted 13 March 2014 - 08:58 AM

Glycerol has a pKa of 14.2, therefore it's not unusual for glycerol to have this pH. Additional water might include more dissolved carbon dioxide or more solvent to dissociate (not sure). Anyway have you any buffering substance in your storage buffer? Usually I'd expect Tris-HCl as ingredient  for this.

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#3 El Crazy Xabi

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Posted 13 March 2014 - 04:37 PM

Always check the MSDS first. The ones I checked at Sigma say that glycerol pH (99% and 86%) should be 5.5-8 and characteristics to be checked after 3 years. I'm not sure about its reactivity but it may be aged and have formed some oxidation byproducts such glyceric acid (pKa 3.55) either by chemical or biological reactions. I would order new glycerol.


And yes, for enzymes 50% glycerol in water and storage in freezer is the common way.... and for bugs too

Edited by El Crazy Xabi, 13 March 2014 - 04:38 PM.

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