Sonoma State University 2001
Department of Biology Animal Physiology

A "buffer" is something that resists change. In common chemical usage, a pH buffer is a substance, or mixture of substances, that permits solutions to resist large changes in pH upon the addition of small amounts of H⁺ or OH^- ions. To put in other way, a buffer helps maintain a near constant pH upon the addition of small amounts of H⁺ and OH^- ions to a solution.

Common buffer mixtures contain two substances, a conjugate acid and a conjugate base (salt). Together the two species (conjugate acid and conjugate salt) resist large changes in pH by partially absorbing additions of H⁺ and OH^- ions to the system. If H⁺ ions are added to the buffered solution, they react partially with the conjugate base present to form the conjuagte acid. Thus, some H⁺ ions are taken out of circulation. If OH^- ions are added to the buffered solution, they react partially with the conjuagte acid present to form water and the conjugate base. Thus, some OH^- ions are taken out of circulation. Buffered solution do change in pH upon addition of H⁺ or OH^- ions. However, the change is much less than that would occur if no buffer were present.

In general, a buffer is used to maintain the pH relatively constant during the course of a reaction that produces or utilizes H⁺ ions. The ability of the buffer to maintain a near-constant pH increases as the strength of the buffer increases. However, it is not always possible to use a relatively concentrated buffer. The enzyme, tissue, or cells under investigation may be sensitive to high ionic strength, or the assay may require that the pH be adjusted easily to some higher ot lower value at the end of the reaction. Thus a compromise is necessary.

Purpose:

1) To become familiar with the terms and calculations involved in preparation of biological buffers.

2) To prepare a buffer of desired pH and ionic strength

3) To become familiar with the use of pH meter and analytical balance

Problem: Prepare 100 ml of a 0.2 M acetate buffer, pH 5.0, starting from solid sodium acetate (molecular weight: 136) and a 1 M solution of acetic acid. (pK_a of acetic acid: 4.77)

Before you start making up the buffer, do the calculations to determine the amount of sodium acetate, acetic acid and distilled water to be used/added. Get ok from the instructor or from one of the teaching assistants.

Before you use the pH meter, it should be calibrated. The following are the calibration steps:

1. Select the pH mode by pressing the mode key until pH is displayed on main display.
2. Place electrode into pH 7 buffer and stir moderately.
3. Press calibrate key to begin calibration.
4. Press 2 then yes to select 2 buffer calibration.
5. When "Ready" is displayed on prompt line, Press yes. The meter then automatically switches to buffer 2.
6. Remove electrode from first buffer. Rinse with deionized water and place electrode into second buffer.
7. When "ready" is displayed on the prompt line, press yes.
8. The electrode slope is calibrated and then the meter will automatically proceed to "MEASURE" mode. It is ready to read pH's.
   

Procedure #1:

1) Calculate the molarities of sodium acetate (salt) and acetic acid (acid) using the Henderson-Hasselbach's equation:

2) Weigh out the calculated sodium acetate and transfer it carefully to the flask (we will show you how to use the balance)

3) Measure the calculated volume of 1 M acetic acid and add it to the sodium acetate.

4) Calculate the volume of distilled water to be added in order to make 100 ml of the buffer. Add the distilled water and mix well using the stir bar and plate.

5) After you prepare the buffer, check the pH of the buffer and record it (we will show you how to use the pH meter).

How to do Procedure #1

molarity of acid + molarity of salt = molarity of buffer; we need 0.2 M acetate buffer

A 0.2 M acetate buffer contains a total of 0.2 mole of acetate per liter. Some of the total acetate is in the conjugate acid form (HOAc), and some is in the conjugate base form (OAc^-). The proportion(hence, the concentration) of each form may be solved by using Henderson-Hassalbach equation;

With this information, we can calculate the amount of HOAc and OAc^- needed to make 100 ml of 0.2 M buffer. Try it!


0.1 L x 0.2M = 0.02 mole total (HOAc plus OAc^-)

The total of 0.02 M is obtained from two sources

0.1 L x 0.126 M = 0.0126 mole OAc^-

0.1 L x 0.074 M = 0.0074 mole HOAc^-

The 0.0126 mole of OAc^- comes from solid sodium acetate

Now, since the molecular weight of sodium acetate is 136

The 0.0074 mole of HOAc comes from a 1 M stock solution of acetic acid.

Again,

number of moles = litres x M

0.0074 = litres x 1 M

litres = 0.0074

or 7.4 ml

Procedure #2, #3, #4

Take 50 ml of the prepared 1.0 g pancreatin/L physiological saline. This is protein taken from the liver. To show its buffering effect, place 50 ml of this protein solution in a 250 ml beaker, add a stir bar and the pH probe. Record the beginning pH. While stirring the solution add .04 M HCl one drop at a time recording the number of drops (up to 30 drops) and resultant pH's. Rinse the beaker and add fresh pancreatin and record resultant pH's when adding .04 M NaOH a drop at a time. Plot your results on a graph with drops vs. pH. Include the graph in your report. Do the same with 50 ml of physiological saline and plot on the same graph..

Thinkers

How does a buffer diminish the pH consequences of adding H+ or OH-?

At what pH is buffering the most effective?

Is protein a buffer? What are the reactions?

Pancreatin is most effective as an enzyme around a pH of 7.0. Is this the pH at which it is the best buffer?

If pancreatin buffers best on the acid side of pH 7, what does this tell you about its composition at physiological pH's around 7.0?

Chemicals, glass ware, and equipment needed:

Chemicals:

Sodium acetate (136 Mol Wt): solid, about 20-25 g
Sodium phosphate: about 30 g
Acetic acid: 100 ml, 1 M, labelled
.04 M NaOH: 6 X 50 ml, labelled
.04 M HCl: 6 X 50 ml, labelled
1 L 1.0 g pancreatin/L physiological saline
1 L physiological saline (8.5 g NaCl /L)

Glassware:

Beakers: 30 X 250 ml
Beakers: 12 X 50 ml
Graduated cylinder: 6 X 100 ml
Wash Bottles: 6
5 ml Pipette Tips
1 ml Pipette Tips

Equipment & Supplies:

Analytical balance: 2
pH meter: 6
Pipettes 5 ml: 6
Pipettes 1 ml: 6
Stir plate (and stir bars): 6
Spatulas for weighing: 2