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Experiment No. 1
Preparation of Buffers

Theory:

A buffer solution is one that is resistant to change in pH when small amounts of strong acid or
base are added. For example, when 0.01 mole of strong acid or base are added to distilled water, the pH
drops to 2 with the acid and rises to 12 with the base. If the same amount of acid or base is added to an
acetic acid – sodium acetate buffer, the pH may only change a fraction of a unit.
Many buffers are prepared by combining a weak acid and its conjugate (acetic acid and
sodium acetate) or a weak base and its conjugate (ammonia and ammonium chloride). In general, the pH
range in which a buffer solution is effective is +/- one pH unit on either side of the pKa. The Henderson–
Hasselbalch provides the information needed to prepare a buffer.

pH  pKa  log
conjugatebase
weakacid 
There is a limit to the amount of acid or base that can be added to a buffer solution before one of
the components is used up. This limit is called the buffer capacity and is defined as the moles of acid or
base necessary to change the pH of one liter of solution by one unit.

Buffer Capacity = (number of moles of OH- or H3O+ added)

(pH change)(volume of buffer in L)

Materials:

Graduated cylinder, 100 mL 0.10 M K2HPO4

Beaker, 400 mL 0.20 M KH2PO4
Volumetric Flask, 100 mL 0.10 M CH3COOH
Pipette 0.02 M NaOH
pH Meter Sodium Acetate
Bottle

Safety:

• Always wear an apron and goggles in the lab.

• Report any spills so they may be cleaned up.
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Sample Computation

How would you prepare 10mL of a 0.01M phosphate buffer, pH 7.40, fromstoc solutions of 0.10M
KH2PO4and 0.25M K2HPO4? pKa of KH2PO4 = 7.20. Prepare 10 mL of a 0.01 M phosphate buffer, pH 7.70,
from stock solutions of 0.1 M K2HPO4and 0.2 M KH2PO4. (pKa for the weak acid = 7.20).

1. Use the Henderson Hasselbalch equation to find the ratio of A to HA.

pH = pKa + log [A-] / [HA]
7.40 = 7.20 + log [A-] / [HA]
0.20 = log [A-] / [HA]
1.584893192 = [A-] / [HA]*
*Since [A-] / [HA] = 1.584893192, we can say that [A-] / [HA] = 1.584893192/1. In this case [A-] =
1.584893192; [HA] = 1.

2. Calculate the decimal fraction (part/whole) of each buffer component.

A-= 1.584893192 / (1.000 + 1.584893192)
= 1.584893192/ 2.584893192 = 0.61313682
HA = 1.000 / 2.584893192= 0.38686318

3. Find the molarity (M)of each component in the buffer by simply multiplying the
molarity of the buffer by the decimal fraction of each component.
MA- = 0.01M x 0.61313682 = 0.006131368M
MHA= 0.01M x 0.38686318 = 0.003868632M

4. Calculate the moles of each component in the buffer.

Moles = Molarity x Liters of buffer
molesA- = 0.006131368M x 0.01L = 6.131 x 10 -5 moles
molesHA= 0.003868632M x 0.01L = 3.869 x 10-5 moles

5. Calculate the volume of each stock solution required to make the buffer
Liters of stock = moles of the buffer component / Molarity of the stock
LA- = 6.131 x 10-5 moles / 0.25 M = 2.452 x 10-4 L = 245µL
LHA= 3.869 x 10-5 moles / 0.10 M = 3.86 9 x 10-4 L = 387µL

6. To prepare this buffer, one would use appropriately-sized pipets to measure and
transfer each component to a 10mL volumetric flask and bring the solution to
volume with dH2O.

Compilation of Biochemistry Experiments and Exercises

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Procedure:

A. Phosphate Buffer

1. Prepare 100 mLof a 0.01 M phosphate buffer, pH 7.70, from stock solutions of 0.1 M K2HPO4and
0.2 M KH2PO4. (pKa for the weak acid = 7.20).

a. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
b. Check your calculations with other students. See the instructor if there is uncertainty.
c. Make the solution and check the pH of a portion of your buffer solution using the pH meter.

B. Acetate Buffer

2. Prepare 100 mLof 0.01 M acetate buffer,pH 3.80, from stock solutions of 0.1 M acetic acid and
0.02 M sodium hydroxide. pKa acetic acid= 4.76.

1. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
2. Check your calculations with other students. See the instructor if there is uncertainty.
3. Make the solution and check the pH of a portion of your buffer solution using the pH meter.
4. Calculate the exact volume of the 0.01 M acetate buffer required to make 100 mLof a
0.0005 M acetate buffer.
1. Prepare this new buffer using the following equation to aid you in your calculations. C1V1=C2V2
2. Check the pH of this new buffer.

C. Bicarbonate Buffer

3. Prepare 100 mL of 0.200 bicarbonate buffer, pH 10 by calculating the mass of sodium

bicarbonate and sodium carbonate

a. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
b. Check your calculations with other students. See the instructor if there is uncertainty.
c. Make the solution and check the pH of a portion of your buffer solution using the pH meter.

Compilation of Biochemistry Experiments and Exercises

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Report Sheet
Experiment # 1
Preparation of Buffers

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Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA:
Show your calculation:
A. Preparation of Phosphate Buffer

Calculated Volume
K2HPO4
KH2PO4

Actual pH of the buffer: _____________________

B. Preparation of Acetate Buffer

Calculated Volume
CH3COOH
KH2PO4

Actual pH of the buffer: _____________________

C. Preparation of Bicarbonate Buffer

Calculated Volume
CH3COOH
KH2PO4
Actual pH of the buffer: _____________________

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GUIDE QUESTIONS:

1. How would you relate the importance of pH to the body fluids?

2. Calculate the pH if 0.30M acetic acid, with Ka = 1.8 x 10-5 is added to 0.20 M sodium
acetate.

3. On the laboratory shelf are 250mM solutions of both acetic acid and sodium
hydroxide. How would you make a 100 mL solution of 25mM acetate buffer of pH
5.50 using these stock solutions?

4. In the phosphate buffer system containing K2HPO4and KH2PO4, what is the weak
acid? What is its conjugate base?

Compilation of Biochemistry Experiments and Exercises