From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: buffers
Date: 23 Jul 92 12:59:21 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>>I usually assume that any dilution of a 0.2M solution 
[referring to phosphate buffer]
>>will not change the pH provided that nanopure, or equivalent, water is used.

>The pH would actually change on dilution because the ionic strength
>would change and the pKa of phosphate changes as a function of pH.
_____________________________________________________________________________
The following generalizations can help when trying to select a buffer:

Anionic buffers (phosphate, borate, acetate, etc) tend to change little with
temperature, but do change with ionic strength.  (Their ionization requires
charge separation [HA --> H+ + A-] which is made easier as ionic strength 
increases.  When the ionic strength is decreased, charge separation 
(= ionization) is more difficult, so the pH rises.)

Cationic buffers (ammonia, tris, etc) are little affected by ionic strength
(no charge separation: eg NH4+ --> NH3 + H+), but are quite sensitive to 
temperature.  Thus, caution needs to be used for pH-sensitive reactions if
a Tris buffer adjusted at room T is going to be used at 0 or 37 deg.

Zwitterionic buffers (such as "Good's buffers" like HEPES) are not much 
effected by ionic strength and also tend be less sensitive to temperature
than tris.

The Calbiochem buffer booklet says that diluting 0.5 M pH 6.6 phosphate to 
0.05 M will raise the pH to 6.9, while a 10-fold dilution of a tris buffer 
raises the pH only about 0.1 unit.  But tris buffer of pH 7 at 4 deg will
have a pH of 5.95 at 37 deg.  (This booklet also has other useful information 
to help when selecting and using buffers.)

Fortunately, many reactions work well over a range of pH, but if there are
problems, unthinking use of the wrong buffer may be one of the reasons.

(The preceding is posted in response to requests for information on why we
do some of things we do.)
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators