BIOCHEMISTRY LABORATORY MANUAL
EXPERIMENT NO.1
Buffers
I. BACKGROUND
Maintaining the pH of biological system is of great importance in preserving their functions.
Buffers are substances that resist pH changes that occur when an acid or a base is added to
the system and consist of a weak acid and its conjugate base (or a weak base or its conjugate
acid).
The pH of a buffer can be determined by the Henderson-Hasselbalch equation as shown
below:
pH = pka + log
[base]
[acid]
This equation is derived from the dissociation of the weak acid:
HA → H+ (aq) + A− (aq)
Upon addition of a strong acid, the base part of the buffer reacts with the added acid.
Alternatively, when a strong base is added, the acid part of the buffer reacts with the added
base. Hence, no change in pH is observed.
II. OBJECTIVES
At the end of this activity, the students should be able to:
1. Prepare a buffer of a given concentration and volume.
2. Compare the buffering capacity of buffer solution of different concentrations.
BIOCHEMISTRY LABORATORY MANUAL
III. MATERIALS
The student needs to bring the following:
One (1) empty 250 mL water bottle
Reagents
0.1 M HCl
0.1 M NaOH
1. 0 M acetic acid
sodium acetate trihydrate
sodium dihydrogen phosphate
sodium hydrogen phosphate
sodium bicarbonate
sodium carbonate
Materials (per group)
(2) 50-mL buret
(1) 10-mL graduated cyclinder
(1) 50-mL graduated cyclinder
(2) 100-mL volumetric flask
(1) 250-mL volumetric flask
(1) 125-mL Erlenmeyer
(1) serological pipette
pH paper (optional)
Equipment
pH meter
IV. SAFETY
• Wear lab gown and protective goggles at all times
• Place highly volatile samples under fume hood.
• Wash hands thoroughly with soap and water before leaving the laboratory.
BIOCHEMISTRY LABORATORY MANUAL
V.
PROCEDURE
A. Preparation of pH 4, 0.2 M Acetate Buffer
1. Based on your prelab answers, weigh the amounts needed and place in a
250-mL volumetric flask. Add the volume of 1 M acetic acid that you have
calculated in Step 1.
2. Fil the volumetric flask to the mark with distilled water.
3. Transfer the buffer you have prepared to a pre-labeled empty water bottle.
Label the buffer with its contents (0.2 M buffer pH 4), your name(s), and date.
4. Check the pH of your buffer using a pH meter or (pH paper)
B. Determination of the Buffering Capacity
1. Get 10-mL of your prepared pH 4 buffer solution and add 40 mL of water in an
Erlenmeyer flask.
2. Add HCl to the buffer until the pH reaches a value of 2.
3. Titrate the buffer with NaOH. Record the pH value every 1mL of NaOH added.
4. Keep titrating until you reach pH Value of 11.
5. Prepare a 1:10 dilution of your buffer with a total volume of 50 mL. Calculate
the volume of the buffer that you will need to make this dilution.
6. Have your instructor check your work before proceeding to the next step.
7. Plot the volume of NaOH added (mL) vs. pH.
WASTE DISPOSAL: Dispose all wastes in the Inorganic Waste Bottle
BIOCHEMISTRY LABORATORY MANUAL
PRELAB QUESTIONS – EXPERIMENT NO.1
Buffers
Group no.: _________
Score: __________
NAME
1. Complete the table below.
Chemical
sodium acetate trihydrate
sodium dihydrogen phosphate
sodium hydrogen phosphate
sodium bicarbonate
sodium carbonate
CLASS NO.
Chemical Formula
Molar Mass
2. Show by calculation how you would prepare 250 mL of a 0.2 M acetate buffer from 1 M
acetic acid and solid sodium acetate trihydrate.
a) Calculate the total moles of the buffer.
b) By using the Henderson-Hasselbalch equation, calculate the ratio of mol
base/mol acid.
c) By using the ratio calculated in Part b, calculate the mL of 1M acetic acid to be
used.
d) By using the ratio calculated in part b, calculate the grams of solid sodium
acetate trihydrate to be used.
3. Show by calculation how you would prepare a 1:10 dilution of a 50-mL 0.2 M buffer. In
your description, be sure to indicate the volume of reagents and glassware to be used.
BIOCHEMISTRY LABORATORY MANUAL
DATA REPORT SHEET – EXPERIMENT NO. 1
Buffers
Group no.: _________
Section: _________
NAME
CLASS NO.
Instructors: _______________________________________
A. Preparation of pH 4, 0.2 M Acetate Buffer
Mass of sodium acetate trihydrate weighed:
pH of 0.2 M acetate buffer:
Score: _____________
_____________ g
_____________
B. Buffering Capacity of 0.2 M Buffer: ____________
Buffering Capacity of 0.2 M buffer
Vol of NaOH added
(mL)
pH reading
Vol of NaOH added
(mL)
pH reading