Chem 152: Biochemistry laboratory
Department of Chemistry
School of Science and Engineering
Loyola Schools, Ateneo de Manila University
BUFFERS
Required reading before the lab: pH meter manual (available at the stock room)
Introduction:
A buffer is a solution which consist of a definite proportion of conjugate base [A-] to weak acid [HA] with
a pH near the pKa of the weak acid. Since the ratio [A-]/[HA] ranges from 10-1 to 101, the two species are
always present in considerable amounts. Together they resist large change in pH by partially absorbing the
H+ and OH- ions added to the system, as shown by the following equations:

H+ + AOH- + HA

HA
(1)
A- + H2O
(2)
Buffer solutions do change in pH but the change is much less than that which would occur if no buffer were
present.
In the Henderson-Hasselbalch’s equation (eqn. 3), the most effective buffering system contains equal
concentration of [HA] and [A-]. When [A-] is equal to [HA], pH equals pKa (eqn. 4). The effective range
of a buffer system is generally two pH units, centered at the pKa value.
pH = pka + log [A-]/[HA]
(3)
pH = pKa + log [0.10/0.10]
pH = pKa + log 1
pH = pKa
(4)
Procedure:
Part 1: Determining the Ka
You need to determine the pH under conditions of concentration and temperature that are as close as
possible to the conditions you will be using in the laboratory.
The acetic acid solutions in the laboratory are about 3M. You will be diluting the stock solution of acetic
acid to concentrations of around 0.12M. You have to make 50 mL samples of the buffer. Determine
roughly how much stock solution of acetic acid is required to make a 50mL solution of about 0.12M
concentration. Round up or down to get a volume of stock solution that can be measured using the pipets
in the laboratory.
Pour a small amount of acetic acid stock solution into a clean, dry beaker. The amount you put into the
beaker should be only slightly more than you need for your pipet so that there will be little wasted
chemical. Never pipet directly from the stock bottles. Transfer the desired amount of acid to a 50mL
volumetric flask. Do NOT dilute yet.
Based on the concentration of acid you will have after diluting to 50mL, determine how many grams of
sodium acetate you will need to add to the dilute acid so that the concentrations of both the weak acid and
the salt are identical. Weigh out the amount of sodium acetate at the balance and carefully transfer the
entire amount of salt to the volumetric flask. Add water to the 50 mL mark and mix well.
Determine the pH of the buffer. From the pH, determine the actual Ka for acetic acid solutions. Use the
derived Ka throughout the entire experiment.
Part II: Making the Buffer
In part 1, the ratio of salt to acid was 1:1. For the second part of the experiment, you will use the same
concentration of dilute acetic acid used in part 1 (0.12M), but alter the amount of salt to obtain the pH that
will be assigned to your group.
Using the Ka you derived from part 1, determine how many grams of sodium acetate you need to add to the
acetic to obtain your assigned pH. Make a 50.0 mL sample of the buffer and mix it well.
Check the pH of the buffer.
Part III: Adding Acid or Base to a Buffer
Pour half of the buffer solution into a clean and dry beaker, and the other half into another beaker. Note the
exact amount of buffer.
Add 5mL of dilute HCl to the buffer.
Calculate the expected pH and check.
Use a dry stirring rod to thoroughly mix the acid into the buffer.
Add 5mL of dilute NaOH to the other half. Use a dry stirring rod to thoroughly mix the acid into the
buffer. Calculate the expected pH and check.
Part IV: Making a Tris-HCl Buffer (Preparation for the next experiment)
For the previous part of this experiment, you prepared a buffer using precise volumetric apparatus. You
will now prepare a buffer via a more practical method in preparation for the next experiment. You are to
make a Tris-HCl Buffer of pH 8.00. An important organic base, tris is the abbreviated name for
trishydroxymethylaminomethane. The molar mass of Tris is121.14 g/mol.
Weigh out enough of the solid Tris base to create a 500mL of a 0.1M solution. Quantitatively transfer this
to a 1L beaker. Add a small amount of distilled, deionized water to dissolve the base. Measure the pH of
this solution. Slowly, add small amounts of dilute HCl to bring the pH down to about 8.00. Once this is
reached, fill the beaker until it reaches the 1L mark. Measure the pH one last time. Add 2-3 drops of 10%
sodium azide solution.
References:
Boyer, R. 1993. Modern Experimental Biochemistry, 2nd ed. The Benjamine/Cummings Publishing
Company, Inc.
NAME_______________________________
YEAR & COURSE______________________
DATE______________________
CH152.1 Biochemistry Lab
BUFFERS
I.
Determining the Ka
Initial Acetic Acid Concentration_______________
Target Acetic Acid Concentration________________
Target Acetic Acid Solution Vol._______________
TRIAL 1
TRIAL 2
TRIAL 3
Vol. Acid pipetted
Mass sodium acetate added
pH reading
pKa
Average Ka____________________________
Notes and Calculations:
II.
Creating a buffer
Target pH____________________
Actual pH___________________
Target mass of sodium acetate _________________
Acual mass of sodium acetate________________
Notes and Calculations:
III.
Adding acid//base to the buffer
HCl Concentration____________________
NaOH Concentration____________________
Amount HCl added____________________
Amount NaOH added____________________
Computed final pH____________________
Computed final pH______________________
Actual final pH_______________________
Actual final pH_________________________
Notes and Computations:
IV.
Making a Tris-HCl Buffer
Mass of Tris needed_____________________
Actual mass of Tris used__________________
Final pH______________________________
Calculations:
Draw the structure of Tris
1. Compare your experimental Ka to the theoretical Ka of acetic acid published in acid/base tables. Are they the
same? If not, explain your results.
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2. Why will adding extra water in part IV not affect the pH of the buffer?
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