Buffering Phenol: The Light Dawns

Jim Owens jow at helix.nih.gov
Fri Jun 10 09:26:48 EST 1994


In article <2t4u1u$pe5 at mserv1.dl.ac.uk> Dr. C.J. Chan, cchan at crc.ac.uk
writes:
>I have the same problem when I tried to buffer some phenol myself. I
just 
>cannot get the pH to 8 and when I add NaOH, the whole thing goes into a
single
>phase. If someone knows why and how to solve this problem, please let 
>me know.

OK, phenol phans, here is the definitive answer:

I learned from the original poster of this question that pH of the phenol
phase was being "measured".  Non, non, mes amis!  (Hercule Poirot)

All the methods for measuring pH only give the negative log of hydrogen
ions at ionic strengths below 0.01M, although they are good enough
approximations up to 0.1M ionic strength.   The theory that underpins
measurement of pH, on which use of both pH meters and pH papers depend,
is only good for such dilute aqueous conditions.

Therefore,  pH of the aqueous phase with which it has been equilibrated
is used to determine the "pH" of the phenol.  I have heard that it is
possible to measure the pH of an organic solution, but a pH meter or the
pH papers will not give an accurate measure.

I was taught by a former PhD student (Leona Fitzmaurice) from Alan
Williamson's lab in Scotland.  She said that he had done extensive
empirical testing of how to prepare phenol for extraction of DNA and RNA.
 Best results for DNA came from phenol prepared the way she taught me. 
That is: equilibrate (shake) the phenol against 0.1M TrisHCl, pH8, and
let the phases separate.  Then determine the pH by putting a drop of the
AQUEOUS phase, against which the phenol had been equilibrated, on pH
paper.

It makes sense both theoretically (for the reason given above) and by
common sense.  If phenol is equilibrated with aqueous pH 8 buffer, the
phenol will not change the pH of DNA dissolved in TE at pH8.  The DNA
will remain in the aqueous phase if the pH of the aqueous phase is over
7.5.  

I think it would be natural to "measure" the pH of the phenol if you have
not taken a course in physical chemistry.  In such a course, especially
one for biochemists, the theory of pH measurement is discussed.

According to the Merck Manual (9th ed), the pK of phenol is 10.0.  So if
enough sodium hydroxide is added, you will have the sodium salt of
phenol.  The ammonium salt of phenol seems to be completely miscible with
water.  (Or is it the other way around?)  Sodium salts are generally less
soluble than ammonium salts, but only a little less.  Thus it is quite
possible that this would explain the lack of two phases when one adds
NaOH to raise the pH of the phenol phase, if it is measured directly.

Sorry to be such a didact, but I hope it will be helpful.  Perhaps this
could be added to the FAQ without all the history, theory?

Good luck,

Jim Owens



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