Yes, Diana brings up really important point. Not only is acetic acid
volatile, certain salt mixtures (like NH4 salts) can make the matter
worse. Drifting pH, particularly in poorly designed vapor diffusion
experiments, can yield irreproducible results if the volatility of
the buffer components is not recognized. I agree with Nadir that
common practice need not slavishly follow appropriate chemistry
theory, but with any method of buffer making, good note-taking and/or
defined lab protocols are needed to ensure that any experiment can be
reproduced.
I am reminded of a horror story that Dino Moras told me about his
group in Strasbourg. Crystals could not be reproduced after a
technician left, even though the conditions were seemingly repeated
exactly. The problem was that the former technician prepared the
buffer for the reservoir differently than did the other lab members,
and it was pH drift which led to crystallization. So whatever method
one chooses to use, write it down explicitly so others can follow it.
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]
****************************************************************
On Jul 23, 2008, at 1:03 PM, Diana Tomchick wrote:
One important consideration with acetate buffer is volatility. All
the care in the world to painstakingly reproduce the pH of the
buffer will not help you if store your acetate buffer for long
periods of time in a container that doesn't minimize evaporation
(so all of you out there trying to reproduce an acetate buffered
condition from a commercial crystallization screen that was stored
in plastic 15-ml tubes, beware!).
Diana
On Jul 23, 2008, at 10:26 AM, Nadir T. Mrabet wrote:
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
* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B
Dallas, TX 75390-8816, U.S.A.
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)
