Michael's comments are correct and follow indeed appropriate chemistry
theory.
In practice however, as said earlier, it is less likely to read a
correct pH value with a pH-meter (unless extensive care and adjustments
are taken beforehand) than to predict amounts of acid and base to use
reliably based on the HH equation.
I take this opportunity to correct my mistyping in my previous mail: "In
the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - [NaOH])"
should be rewritten into "In the second case, the HH is written 4.5 =
4.76 + log([NaOH]/(25 - [NaOH]))".
Greetings,
Nadir
--
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED]
R.M. Garavito wrote:
Buffer making is very much an empirical process, but there is a
comment that needs to be made about the use of the H-H equation and
pKa values. I have to teach our department's biochemistry laboratory,
and I sadly would have to take off points from all the discussions as
the H-H equation and pKa won't give the right answer as pKa is for an
ideal (i.e., infinitely dilute) solution. A 25 mM Na acetate solution
is not dilute. You need to use the pKa' which sadly changes as the
concentration of the buffer increases or decreases: while the pKa of
acetic acid is 4.76 (@25˚C), at 100mM, the pKa' is 4.60. The National
Bureau of Standards (now NIST) has a detailed list of standard buffer
recipes and pKa' values for most of the common buffers (e.g., see
Bates, J. Natl. Bur. Stand. 66A, 179, 1962).
That said, Nadir's method is a fine way to make a buffer of a known pH
(using a well calibrated pH meter) at a known temperature, and it will
allow you to make a buffer with the same pH value almost every time
(depending on how your room temperature changes throughout the year).
Being able to consistently and reliably repeat the buffer formulation
is the most important point.
Cheers,
Michael
/****************************************************************/
/R. Michael Garavito, Ph.D./
/Professor of Biochemistry & Molecular Biology/
/513 Biochemistry Bldg. /
/Michigan State University /
/East Lansing, MI 48824-1319/
/Office:// //(517) 355-9724 Lab: (517) 353-9125/
/FAX: (517) 353-9334 Email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>/
/****************************************************************/
On Jul 22, 2008, at 11:20 AM, Nadir T. Mrabet wrote:
I bet it is more difficult to adjust a pH-meter than to use the
Henderson-Hasselbalch equation
and still get the expected pH with a pretty good accuracy especially
if your work near the pKa.
There are actually two ways to prepare this 25 mM buffer, pH 4.5.
The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so
don't worry too much about this).
Reference is "Buffers for pH and Metal Ion Control", Perrin &
Dempsey, Chapman & Hall, NY, ISBN 0 412 21890 9.
High-grade glacial acetic acid (99-100%) is 18 N.
Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final). Keep
is a dark, tightly closed bottle.
Make a stock solution of 250 mM sodium acetate (if you use FW, not
MW, to calculate mass to use, then no worry about anhydrous or not
since water is also taken into account if present)
or
make a stock solution of 5N NaOH. Keep is a dark, tightly closed bottle.
Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log
([A-]/[AH]).
In the first case, you write it : 4.5 = 4.76 + log ([sodium
acetate]/[acetic acid])
Second equation is [sodium acetate] + [acetic acid] = 25 mM
which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134 mM.
For 1.0 L buffer, mix adequate volumes of stock solutions of sodium
acetate and acetic acid and complete with water (add acid after un
first fill with water to ~ 800 mL).
In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 -
[NaOH]),
which gives [NaOH] = 8.886 mM (same result as above for sodium
acetate which was then the base).
The added advantage of using HH and stock solutions is that even if
your pH is not exactly 4.5, say 4.55, if you make a new buffer the
next day or even the next month,
your buffer will have the same pH value. I don't expect you can ever
achieve such a repeatability using a pH-meter.
HTH,
Nadir Mrabet
--
Pr. Nadir T. Mrabet
Cellular & Molecular Biochemistry
INSERM U-724
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: [EMAIL PROTECTED]
<mailto:[EMAIL PROTECTED]>
William G. Scott wrote:
So what, then, will be the concentration of the acetate ion in your
stock solution when you have finished?
(Disclaimer: I get to teach this stuff periodically in remedial
chemistry as a punishment for deployment of excessive sarcasm during
faculty meetings.)
On Jul 22, 2008, at 6:10 AM, Santosh wrote:
Hi,
Make a 1M Na-Acetate do not make up to the 1 Ltr volume. Leave some
extra
volume and now start adding Acetic acid till you get pH 4.5
(Glacial Acetic
Acid).
Now make up the volume to 1ltr or how much ever you are deciding to
make the
50X stock solution.
Best,
Santosh
On Mon, Jul 21, 2008 at 11:20 PM, William G. Scott <
[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>> wrote:
This is a job for the trusty Henderson-Hasselbalch equation:
http://en.wikipedia.org/wiki/Henderson-Hasselbalch_equation
On Jul 21, 2008, at 8:12 PM, Meg wrote:
Dear All,
I want to prepare 25 mM sodium acetate buffer pH 4.5. can anyone
give the
exact composition of how to prepare it. we prepare it using
sodium acetate
and acetic acid combination. i am not able to arrive at the
calculatation
correctly, so if anyone can explain me with the above buffer how to
calculate. and what sodium acetate [Anhydrous / trihydrate] and
acetic
acid
[glacial/ plain] to use.
thanks n regards
Meg goyal,
M.SC Biotechnology [Research]
Institute of science,
Fort
Mumbai, INDIA
