Basic Laboratory Techniques: Preparation of Buffer Solutions and Use of the pH Meter

Proper pH ranges and concentrations of solutions is critical for the satisfactory isolation and
characterization of biological molecules. In this lab you will be observing the effect of a buffer (acetate
buffer) when a strong acid or strong base is added.

Safety Considerations:

● Glacial acetic acid: Glacial (concentrated) acetic acid should be handled with the same
precautions used for other concentrated acids.

Part A: Preparation of Solutions

Do all calculations ahead of time and submit them to the instructor at least 24 hours BEFORE lab.

Prepare the following solutions.

0.2 M sodium acetate* (100 mL)
0.2 M acetic acid (100 mL)
0.2 M acetate buffer; pH 5.0 (from the weak acid and conjugate base solutions)

* the sodium acetate is a tri-hydrate, so be sure to use the correct molecular weight in your
calculations

Procedure:

1. You will need to prepare around 70-80 mL of acetate buffer. The buffer will be prepared from
the 0.2M sodium acetate and 0.2M acetic acid solutions that you prepare.

2. Prepare the 0.2 M sodium acetate solution from the sodium acetate trihydrate (use correct
MW).
a. A graduated cylinder is sufficiently accurate to measure the final volume of distilled
water needed to make your solution.

b. Prepare the solution in a clean beaker or flask and label it 0.2 M sodium acetate.

3. Prepare the 0.2 M acetic acid solution (MW = 60.1 g/mole).

a. For the calculations use the following information. Concentrated acetic acid, usually
called glacial acetic acid, has a density of 1.05 g/mL and is 99.7% pure.

To convert this information to a molarity (so you can use M1V1=M2V2 for your dilution)
you need to multiply the % (grams acetic acid over grams solution) by the density then
multiply by the molar mass and finally convert mL to L. Check with instructor on your
final answer before proceeding.
 b. Transfer using a micropipette the required volume of glacial acetic acid into distilled
water, then bring the volume to your final amount using a graduated cylinder.

c. Store the solution in a clean beaker or flask and label.

4. Prepare a solution of 0.2 M acetate buffer (200 mL) with a final pH=5.0 using the 0.2 M acetic
acid and 0.2 M sodium acetate solutions.
a. For the calculations use the Henderson-Hasselbalch equation to determine the ratio of
sodium acetate to acetic acid necessary to give a pH of 5.0. Note: The pKa of acetic acid
is 4.76.
pH = pKa+log ¿ ¿

(so you are solving for the RATIO—once you have this ratio, that tells you how much
volume you need of conjugate base and weak acid; as an example, if your ratio came
out to 2.5, then for every 2.5 volumes of conjugate base, you need 1 part volume of
weak acid)

b. From the ratio determined from the H-H equation combine the volume of 0.2 M sodium
acetate solution with 0.2 M acetic acid solution to make the final 0.2 M acetate buffer.
Remember that total volume should be around 70-80 mL.

c. Store the buffer solution in a clean beaker or flask labeled 0.2 M acetate buffer.

Part B: Measurement of pH & Titration with Strong acid and/or base

Procedure:

1. The proper use of the pH meter and the method of calibration will be re-enforced in the lab by
the instructor but you should have watched the associate video first. Be careful using the glass
electrodes.

2. Calibrate the pH meter as necessary with the given standard buffer solutions.

3. Determine the pH of the acetate buffer you prepared. Show the instructor your values before
proceeding to the titrations. pH values significantly away from the stated pH=5.0 will have to be
remade.

4. Once your solution is approved, begin titrating your buffer solution with either HCl or NaOH as
instructed by your professor.
a. Measure out 50 mL of your acetate buffer. Place into a clean 100-250 mL beaker

b. Using a plastic transfer pipet, drop in 5 drops of the strong acid/base. Stir (you may
CAREFULLY use the pH probe to stir the solution) and record the resulting pH.
 c. Continue adding 5 drops at a time, recording the pH after each 5 drop addition.

d. Your titration is over when you begin to get a significant jump in pH (greater than 1.0
units) upon addition of the next 5 drops of acid/base.

5. Rinse the pH probe well and repeat your titration (using the same titrant assigned to you) with
50 mL of distilled water.
a. Measure the pH of the water before you start (and no, it won’t be 7).

b. Using the plastic transfer pipet, add in 5 drops of acid or base, stir, and record the pH.

c. Continue adding 5 drops of acid or base until the pH has changed by one unit from the
starting pH of your water. (you shouldn’t have to add very much—think about this!)