Yes, Diana brings up really important point. ÊNot only is acetic acid volatile, certain salt mixtures (like NH4 salts) can make the matter worse. ÊDrifting pH, particularly in poorly designed vapor diffusion experiments, can yield irreproducible results if the volatility of the buffer components is not recognized. ÊI agree with Nadir that common practice need not slavishly followÊappropriate 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. Ê

I am reminded of a horror story that Dino Moras told me about his group in Strasbourg. ÊCrystals could not be reproduced after a technician left, even though the conditions were seemingly repeated exactly. ÊThe 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. ÊSo whatever method one chooses to use, write it down explicitly so others can follow it.

Michael

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R. Michael Garavito, Ph.D.

Professor ofÊBiochemistry & Molecular Biology

513 Biochemistry Bldg.ÊÊÊ

Michigan State UniversityÊ ÊÊ Ê

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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
Ê Cellular & Molecular Biochemistry
Ê INSERM U-724
Ê Nancy University, School of Medicine
Ê 9, Avenue de la Foret de Haye, BP 184
Ê 54505 Vandoeuvre-les-Nancy Cedex
Ê France
Ê Phone: +33 (0)3.83.68.32.73
Ê Fax: Ê +33 (0)3.83.68.32.79
Ê E-mail: [log in to unmask]



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 (@25ûC), 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/

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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




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 <

This is a job for the trusty 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







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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: [log in to unmask]
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