Medical Chemistry, First Year General Medicine 2005/2006

Practical Lesson No 2

BUFFERS AND BUFFER CAPACITY

Reagents:
1. NaH2PO4 0.1 mol/l
2. Na2HPO4 0.1 mol/l
3. NaCl 0.1 mol/l
4. HCl 0.1 mol/l
5. NaOH standard solution 0.1 mol/l

1 THEORY

1.1 Principle:
A buffer solution is a solution that resists changes in pH either when diluted or when
limited amounts of acid or base are added to it. Such a solution can be prepared by
combining a weak acid and its salt with a strong base (conjugated base) or, analogously, a
weak base and its salt with a strong acid (conjugated acid).

For example:
Acetate buffer: CH3COOH (the weak acid) + CH3COONa (the salt, conjugated base)

Phosphate buffer: NaH2PO4 (the weak acid) + Na2HPO4 (the salt, conjugated base)

Tris buffer:

(Tris: Tris [2-amino-2-(hydroxymethyl)-propan-1,3-diol)], an organic base)

The Henderson-Hasselbalch equation describes the behaviour of such a buffer and for the
mixture of a weak acid and its salt with a strong base (conjugated base) it has the form:
c
pH = pK a + log s
c ac
pKa negative logarithm of the dissociation constant for the weak acid
cs substance concentration of the salt (conjugated base)
cac substance concentration of the weak acid (conjugated acid)

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 Medical Chemistry, First Year General Medicine 2005/2006

Graphically the Henderson-Hasselbalch equation plotted as the acid : conjugated base

ratio vs. pH of buffer actually constitutes the titration curve of the weak acid (see figure
on this page). Note also that for the acid : base ratio 1:1 the pH of buffer just equals the
pKa.

The equation for a weak base and its salt with a strong acid (conjugated acid) has the
form:
c
pH = pK w − pK b + log b
cs
pKb negative logarithm of the dissociation constant for the weak base
cb substance concentration of the base
cs substance concentration of the salt (conjugated acid)
pKw = 14 = − log 10-14 (ionic product of water)

Buffer capacity (β) is defined as the amount of a strong acid or a strong base that has to be
added to 1 litre of a buffer to cause pH change of 1.0 pH unit:
∆c b ∆c
β= = − ac
∆pH ∆pH
The buffer capacity depends on the amounts of substance of the weak acid and its
conjugated base in the buffer. It is in fact directly related to the first derivative of the
buffer titration curve, or, in other words, the slope of the titration curve. As the slope of
the titration curve is smallest at the acid : base ratio 1:1, the buffer capacity is maximal at
the same point.

Titration curve of sodium phosphate buffer

10

9
pH of buffer

pH = pKa = 7.21
7

9:1 8:2 7:3 6:4 1:1 4:6 3:7 2:8 1:9

Acid : base ratio

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 Medical Chemistry, First Year General Medicine 2005/2006

1.2 Calculation of pH of buffer after addition of strong acid or base

Example:
Let’s have 600 ml of a sodium phosphate buffer of concentration c = 0.25 mol/l and the
ratio acid : base is 2:3. Next, we add 150 ml of HCl, c = 0.2 mol/l. How much the pH
changes after this addition? (pKa of sodium dihydrogen phosphate is 7.21)

Solution:
In order to calculate the pH of this buffer before and after addition of HCl, we need to
know the ratio of acid : base for both conditions. The ratio is given for the original buffer
as 2:3, so we can easily use the Henderson-Hasselbalch equation to calculate the original
pH:
pH = pKa + log 3/2 = 7.21 + log 1.5 = 7.21 + 0.17609 = 7.386.
Next, we need to consider how the ratio acid : base changes following addition of HCl.
The strong acid actually titrates the basic component of the buffer, converting it into the
acidic component:
HCl + Na2HPO4 → NaH2PO4 + NaCl

before addition of HCl:

Number of moles for total phosphate: (0.6 x 0.25) = 0.15 mol
NaH2PO4: 0.15 /5 x 2 = 0.06 mol
Na2HPO4: 0.15 /5 x 3 = 0.09 mol

after addition of HCl 0.15 x 0.2 = 0.03 mol:

Number of moles for total phosphate: (0.6 x 0.25) = 0.15 mol
NaH2PO4: 0.06 mol + 0.03 mol = 0.09 mol
Na2HPO4: 0.09 mol − 0.03 mol = 0.06 mol
The new ratio of acid : base is 0.09:0.06, i.e. 3:2.
And the resulting pH is:
pH = pKa + log 2/3 = 7.21 + log 0.6667 = 7.21 − 0.17609 = 7.034.

The pH change (effect of addition): ∆pH = 7.386 − 7.034 = − 0.352

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 Medical Chemistry, First Year General Medicine 2005/2006

2 EXPERIMENTAL

2.1 Directions for use of the pH meter EIM

The pH meter has been already switched on, checked and calibrated by technicians using
standard buffer solutions pH 7.0 and 4.0. Do not change any settings!

pH measurement:
a. The electrode is resting in buffer of pH 7.00 (the green solution). For measurement of
pH pull the electrode out from the buffer pH 7.0, rinse it with distilled water, wipe
gently with a piece of tissue paper (just touch, avoid rubbing the glass bulb that would
charge it with static electricity), and place it into the sample solution in plastic
container.
b. Wait until stable reading is achieved (the usual response time of glass electrode is
about one minute). Write down the value.
c. Pull the electrode out, rinse again with distilled water, wipe with a piece of tissue
paper, and place it into another sample, or, when the experiment is complete, into the
buffer pH 7.0.

NOTE: In the meantime in case of repetitive measurements and when the work is
finished, always leave the electrode immersed in the standard buffer pH 7.00. The
electrode can be irreversibly damaged when it is allowed to get dry, it must never
happen!

2.2 Evaluation of titration curves for strong and weak acid

Titration curve means a plot of pH against amount of alkali added to sample of acid (see
also figure in the Practical Lesson 1). Comparison and analysis of titration curves for a
strong and weak acid is useful for understanding of concept of buffering.
From technical reasons, you will not obtain your own data here, but rather, the task is to
evaluate previous data that we provide.
Description of experimental procedure:
20 ml of either HCl (c = 0.1 mol/l), or CH3COOH (c = 0.1 mol/l) was measured into
titration flask. The initial pH was measured. Then, NaOH (c = 0.1 mol/l) was added step
by step in amounts as indicated, and pH was measured after each addition.
Tasks for evaluation:
a. For both acids, plot measured pH values against volume (ml) of added base (as in
figure in Pract. Lesson 1).
b. Calculate pH of hydrochloric acid (activity coefficient f = 0.796) and compare with
the initial measured value.
c. Calculate pH of acetic acid 0.1 mol/l (pKa = 4.75) and compare with the initial
measured value.

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 Medical Chemistry, First Year General Medicine 2005/2006

d. Compare the shapes of both curves – which of the mixtures used could work as a
buffer and for what pH range ?
e. From the inflection point of your titration curve, determine pKa of acetic acid.

2.3 Buffer capacity

Procedure:
a. Number the test tubes and prepare the initial mixtures 1-4 as shown in the following
table:
Tube No 1 2 3 4
Na2HPO4 0.1 mol/l ml 5 1 9 -
NaH2PO4 0.1 mol/l ml 5 9 1 -
NaCl 0.1 mol/l ml - - - 10
acid/conjugate base ratio ml
pH measured ml
pH calculated ml
Add to all tubes
acid or base 0.1 mol/l 1.0 1.0 1.0 1.0
pH measured
pH calculated1
b. Using the Henderson-Hasselbalch equation, calculate the pH values of prepared
phosphate buffers (pKa = 7.21) before as well as after addition of 1 ml of HCl 0.1
mol/l.
c. Measure pH of the initial mixtures and compare these values with the calculated ones.
d. Then add to each of the four tubes 1 ml of HCl 0.1 mol/l, mix and measure pH again.
Compare the obtained values with calculated ones.
e. Conclude which of the prepared solutions shows the highest buffer capacity. Do all
the examined solutions behave as buffers?
f. Alternatively, you can test the buffer capacity of the prepared solutions by adding 1
ml of NaOH 0.1 mol/l instead of HCl.

1
Only in cases when the buffering capacity has not been exceeded (over-titrated)

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