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Effects of Freezing Point by Organic Buffers - (May/18/2014 )



I was just wondering if someone could help me understand the change in freezing temperature that has seemingly occurred in a 50mM Tris-HCl, pH 8.0 - as compared to distilled water.  I went to get a Nickle Column that I had stored in the fridge (it's 4C - confirmed), and liquid over the top of the Nickle resin had frozen. 


What's the basic science behind the lowering of freezing temp by Organic Buffers?  Theorizing: Tris somehow bound strongly enough (more strongly than H-bonds alone) to some water molecules, allowing the molecules to stay still at a higher temperature than what is observed by water alone...


Is this what is happening? Any additives to prevent this freezing that are compatible with Nickle resin (I don't know if glycerol is compatible?)


I enjoy this trips back to basic science!


Thanks for any help.




Hmmm - it shouldn't freeze at 4 C.  Usually freezing point depression means that you lower the point of freezing relative to the concentration of the salt added.  For example (numbers made up!) a 100 mM NaCl solution might freeze at -1 and a 1 M NaCl might freeze at -8.  This should also happen for organic buffers such as Tris as these are usually in the salt form (you added HCl to get the pH correct).


Are you sure that it is only the 50 mM tris that is over the column?  None of the agarose beads to which the Nickel is attached are in there or could have been solubilized?  The fridge doesn't have an ice-box (or display any ice anywhere) at the top does it?  If so, the temperature of the fridge will vary quite a bit depending on proximity to the ice-box and the circulation system of the fridge. 


Hi There!


I put a thermometer right where the column was sitting to confirm the temp.  In addition, DNA samples in 10 mM Tris were right next to the column, unfrozen!  I don't think any beads were suspended, as I spun the column before storage, and stored in the upright position.


So, what you're saying is that salt concentration is inversely proportional to freezing temp (as in, salt concentration increases, freezing temp decreases?)  Can you explain the chemistry behind this?  Are ions competing ionically with H/O, preventing effective hydrogen bonding?  Thus, the water molecules would have to be brought down to a lower temperature to "slow down" to a point of freezing?


I've no idea of the physics behind it - but i do know that salty water freezes at a lower temperature than fresh water - this is one of the bases for the Fahrenheit scale, and its also the reason you might add ethylene glycol (anti-freeze) to the radiator of your car.  Trust me, it is nothing to do with the Tris!


Note that you should immerse the thermometer in water in the fridge to get an accurate reading.   You could also take some 50 mM tris and check if it freezes in the fridge, which it shouldn't.


Could there be SDS in the buffer? - this will precipitate and freeze at relatively high temperatures.


as bob1 points out, you may not be seeing ice. you may be seeing crystals of one (or more) of the medium's components.


Apart from this, fridges don't have a homogeneous temperature distribution but there are warmer and colder zones...Especially the lower compartments and near the cooling plate (evaporator) at the rear are cooler zones (if you have a commonly built one) and if stuff is too close to this parts it might freeze.
For the chemistry of freezing-point depression, check wikipedia, even there it's complicated enough (that it works you see in colder countries where in winter streets are de-iced with different types of salt).