question about making phenol/TE and

[Tim Fitzwater]

Phenol may contain 0.15% H3PO2 as a preservative which may liberate spontaneously flammable phosphine when distilled.  Distill only under an inert atmosphere.  Phenol becomes explosive when heated above 79°C.

Oxidized phenol is pink to red.  The oxidation products are quinones, a wide variety of phenolic coupling compounds and diacids.  Quinones are a, b-unsaturated ketones that are capable of reacting with primary amines.  The reaction of quinones with multiple amines can form stable crosslinked products.  Quinones can crosslink nucleic acids and may break down phosphodiester bonds.  The diacids arise from further oxidation of orthoquinone, while phenolic coupling products result from phenoxide radicals.  Quinones are faintly yellow.  Phenolic coupling reaction compounds are intense yellow or red.     
0.1% 8-Hydroxyquinoline can be used as an additional preservative; this also turns the phenol layer yellow, making separation of phases easier.  
Some protocols add 70 mL m-cresol/500 g phenol as antifreeze and as an additional deproteinizing agent.  The m-cresol should be colorless.

Phenol is most stable as crystals at -20°C, but is most practically stored in a liquid-saturated form at 4°C.  The aqueous phase of water-saturated phenol is typically pH 3.0-4.5.  The low pH is due to partitioning of the diacids into the aqueous phase.  Partitioning will proceed more rapidly in a buffered solution of a higher pH, however, phenol has a pKa = 10.0 and is much more susceptible to oxidation at high pH.  Buffers used to equilibrate phenol should not exceed pH 8.0.  

There is a wide variation in the amount of NaOH or Tris buffer required to adjust the pH of phenol, which makes purchase of commercial versions very attractive and highly recommended.

To measure the pH of phenol:
Organics such as phenol and chloroform have much lower dielectric constants than water.  The very large liquid junction potential can cause problems such as pH drift, long stabilization times and pH electrode damage.  The indicator chemical in pH paper is destroyed by phenol resulting in inaccurate pH measurement.  In order to accurately measure the pH of saturated phenol it is necessary to solubolize the phenol in an aqueous medium. 

For saturated phenol (no chloroform), mix 2 mL organic phase with 5 mL methanol and 13 mL water and pH entire sample.  pH will drift down over the course of about 30 seconds until it stabilizes.  (This method is from the Ambion 1994 catalog.)  

For phenol:chloroform or acid phenol:chloroform, mix 2 mL of the organic phase with 8 mL methanol and 10 mL water.  Mix and measure pH of the entire sample with the pH electrode.  pH will drift down over the course of about 30 seconds until it stabilizes. 

Silver/silver chloride type electrodes are not recommended because they do not accurately measure the pH of Tris.  Since the electrode dehydrates during phenol testing, it should be soaked in Type I water to rejuvenate the glass bulb.