pH and Buffers

PRACTICAL 3 pH AND BUFFERS

Structure
3.1 Introduction
3.2 pH and Buffers: Basic Concept
3.2.1 pH
3.2.2 Buffers and Buffer Solutions
3.3 Methods for Determining pH
Experiment 1: Measurement of pH
Experiment 2: Preparation of Phosphate Buffer
Experiment 3: Preparation of Chloride Buffer

3.1 INTRODUCTION
Practical 3 deals with pH and buffer solutions. We already know that the pH of the
system plays a very vital role in success of many chemical and biochemical reactions.
Buffers, on the other hand, are aqueous solutions that tend to resist any pH change
when small amounts of acid or base are added. What is pH? What are buffer solutions?
What is the role of buffers? How to prepare buffer solutions? We shall learn about
these aspects in this practical.

Objectives
After studying this practical and undertaking the experiments given herewith, you will
be able to:

define and measure pH of any solution,

explain the concept of buffers, and

prepare buffer solutions both acidic and basic.

3.2 pH AND BUFFERS: BASIC CONCEPTS

In this section we shall review our knowledge about pH and buffers. We begin with pH.

3.2.1 pH
You have already studied about pH in your undergraduate years and know what it
means. Can you define pH? Write the definition of pH in the space provided.

Yes, mathematically we can define pH as equal the negative log of the hydrogen
ion concentration, or pH = -log [H+]. In simple terms, pH is a measure of the
acidity or alkalinity of a solution.

You have studied that the pH scale is a convenient means of expressing the relative
acidity or alkalinity of solutions. The pH of the system plays a very vital role in
success of many chemical and biochemical reactions. The pH of pure water is 7.0,
therefore, the pH of any water sample may give an indication of the type of impurity
present in water.

So now you know what pH means and also understand its significance. From your
Nutritional chemistry lessons in school and in undergraduate years, you should be
able to distinguish between acids and bases by looking at their pH values. You may 41
Nutritional also determined the pH of solutions using the pH paper. However, pH paper is used
Biochemistry when approximate pH values are required. For a more accurate determination of pH, a
pH meter is used. We shall learn about the pH meter in section 3.3.

Now let us get to know about buffers.

3.2.2 Buffers and Buffer Solutions

Solutions containing both weak acid and their salts or solutions containing weak hydroxides
and their salts are referred to as buffer solutions. They have the capacity of resisting
changes in pH when either acid or alkali is added to them.

Buffers are therfore, aqueous solutions that tend to resist any pH change when small
amounts of acid or base are added. A buffer system consists of a weak acid (proton
donor) and its conjugate base (proton acceptor) or a weak base and its conjugate acid in
comparable concentrations. Because they maintain a constant pH, buffers are good
reference solutions.

A buffer solution can be prepared to have a desired value of pH by controlling the

amount of acid and its salt in case of acidic buffer and the amount of base and its salt in
case of basic buffer. What do we mean by an acidic and basic buffer? An acidic
buffer solution is simply one which has a pH less than 7. Acidic buffer solutions are
commonly made from a weak acid and one of its salts - often a sodium salt. An alkaline
buffer solution, on the other hand, has a pH greater than 7. Alkaline buffer solutions
are commonly made from a weak base and one of its salts. Frequently used example of
acidic and basic buffer include:

_ Acidic buffer: a mixture of CH3COOH (acetic acid) + CH3COONa (sodium

acetate), citric acid and sodium acetate, boric acid and borax.
_ Basic Buffer: a mixture of NH4Cl (ammonium chloride) + NH4OH (ammonium
hydroxide), potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen
phosphate (Na2HPO4), sodium carbonate and sodium bicarbonate.

Let us next understand the mechanism according to which buffers function. You may be
aware of the fact that:
a) A weak acid ionizes as
HA H− + A−
where, HA is acid, H is the positively charged ion and A is the negatively charged
ion.

b) Complete ionization of the salt yields:

NaA Na+ + A−
where, NaA is sodium salt

At equilibrium,
(H+) (A−) where, Ka is constant.
Ka =
(HA)

(HA) (salt)
(H+) = Ka (A−) = Ka (acid)

By taking negative logarithm on both sides we get:

(salt)
−log (H+) = −log Ka + log
(acid)
(salt) (Handerson-Hasselbach equation)
or pH = pka + log
(acid)
42
A buffer has its maximum capacity to resist changes of pH when pH = PK pH and Buffers

Addition of a few drops of an acid produces H+ ions which react with excess of A- ions
present to form unionized HA and there is practically no change in the pH of the buffer.
H+ + A- HA

Addition of a few drops of base produces OH- ions that react with excess of unionized
acid to form A- ions and there is practically no change in the pH of the solution.

HA + OH- H2 O + A -

The pH of the buffer depends not on the buffer but on the thermodynamic quantity
called activity. This parameter is strongly affected by the total concentration of ions in
the solution so that the pH of the buffer will vary both with its own concentration and
with concentration of other salts in solution.

Let us next understand the mechanism according to which buffers function by taking an
example of acidic buffer - sodium acetate + acetic acid.
Sodium acetate dissociates completely in water:

CH3COONa CH3COO + Na

If an acid is added, the H+ ions will be consumed by the conjugate base in the buffer
as shown herewith:

CH 3COOH + HCl CH3COOH + H2O

CH3COONa

If a base is added, say sodium hydroxide (NaOH), the OH- ions will be neutralized by
the acid in the buffer as shown herewith:

CH 3COOH + NaOH CH3COONa + H2O

CH 3COONa

In this way, the buffer solution prevents appreciable increase of OH- ions from NaOH
and H + ions from HCl. Thus, the addition to a buffer solution of small amounts of base
results merely in the production of more salt ions at the expense of an equivalent amount
of the weak acid present. The addition of acid similarly results in the production of
more
weak acid at the expense of the salt. Remember, more concentrated the buffer is, the
smaller will be the change in pH on addition of a given amount of strong acid or base i.e.
the greater will be the buffer power or buffer capacity of the solution.

According to Handerson-Hasselbach equation:

pH = pKa + log (salt) For anionic buffer

(acid)

(salt) For cationic buffer

pH = pKa + log
(base)
A buffer has its maximum capacity to resist changes of pH when pH = pKa. Thus, the
maximum efficiency of a buffer with the given total of salt plus acid is greatest when
the ratio of salt to acid is equal to 1.

Another property of buffer solution is that they may be diluted considerably without
appreciable change in pH, since the pH depends upon a concentration ratio rather than
upon concentration itself. In this context, let us get to know about buffer capacity. 43
Nutritional Buffer capacity
Biochemistry
Buffer capacity can be defined as, ‘the capacity of a solution to resist changes in pH on
the addition of strong acid or strong base which may be expressed numerically as the
number of moles of strong acid or strong base required to change the pH by one unit
when added to one liter of the specified buffer solution’.
Thus,
Buffer Capacity (a) = The number of moles of H+ that must be added to 1 litre of the
buffer in order to decrease the pH by 1 unit = the buffer capacity in the acid direction,
and
Buffer Capacity (b) = The number of moles of OH- that must be added to one litre of
the buffer in order to increase the pH by 1 unit = the buffer capacity in the alkaline
direction.
Buffer capacity as a quantitative unit for measuring buffering effects is therefore
expressed as:
δ (HA)
Buffering capacity =
δ (pH)
where δ is delta and HA is acid.
Buffer capacity is maximum when pH = pKa
With this basic understanding about buffers, we end our discussion about pH and buffers
here. Next, let us get to know about the methods we can use for measuring pH.

3.3 METHODS FOR DETERMINING pH

There are two general methods for determining pH of a solution. One is the colorimetric
method and the other most common method is the use of pH meter for determining the
pH. In this practical we shall focus only on the principle, method involved in the use of
pH meter for determining the pH. So let us get started by first getting to know about the
pH meter.

What is a pH meter?
A pH meter, we have already studied in Practical 1, is a device used for measuring. pH
of any unknown solution. It is composed of :
a) A reference electrode
b) Glass electrode whose potential depends on the pH of the solution surrounding it
c) An electrometer, a device capable of measuring very small potential difference in a
circuit of extremely high resistance.
Figure 3.1 (a) shows you the front view of a typical, direct reading pH meter. Look at
Figure 3.1 (b), which shows the digital pH meter. Measurement of pH of a solution with
such an instrument can be made following the procedure given in a stepwise manner
under the procedure section in Experiment 1 later in this practical. But, if the pH meter,
which you have in your laboratory, is of a different type, go through its instruction manual
and then use it accordingly.

(a) pH meter (b) Digital pH meter

44 Figure 3.1: pH meter
Let us next understand the principle behind the functioning of a pH meter. pH and Buffers

Principle
Hydrogen ions in solution, like other ionic species, conduct an electric current. When a
glass electrode is dipped in a solution containing hydrogen ions, a potential difference
develops across a very thin glass membrane separating two solutions of different
hydrogen ion concentrations - one within and the other outside the glass electrode. A
pH meter is an electronic voltmeter that measures this difference of potential and through
its internal calibration, converts it to a pH reading which is displayed on a scale. The
scale is normally taken extending from 0 to 14 pH units with a least count of 0.05 pH
units or better. A pointing needle moves across the graduated scale and the pH of the
solution can be read directly on the scale. These days digital pH meters, as illustrated in
Figure 3.1 (b), are becoming more popular as compared to scale - needle instruments.

With a review of the principle and functions of the pH meter, we are now ready to get
started with the experiments related to pH and buffer solutions. There are 3 experiments
in this practical. So let us carry out these experiments and learn as we go about conducting
the experiments.

45
Nutritional
EXPERIMENT
Biochemistry

1 MEASUREMENT OF pH
Date: ..........................

Aim: To measure the pH of an unknown solution with the help of a pH meter.

Principle
(Write the principle behind the working of a pH meter in the space provided herewith.
You may like to look up section 3.3 for reference)

Requirements
Apparatus
pH meter - 1
Glass electrode - reference electrode assembly - 1
Beakers 100 ml - 3

Reagents
Buffer solutions of pH 4, pH 7 and pH 9.18 for calibration and an unknown solution (i.e.
solution of unknown pH).
You can prepare these buffer solutions (of pH 4, 7 and 9.18) by following the steps
enumerated herewith:

1. Buffer solution of pH 4: It is prepared by dissolving buffer tablet of pH 4 in a 100 ml

volumetric flask and diluting it up to the mark with distilled water. Alternatively, it may
be prepared by dissolving 1.021 g of potassium hydrogen phthalate in distilled water in a
100 ml volumetric flask and making the solution up to the mark.

2. Buffer solution of pH 7: Dissolve a buffer tablet of pH 7 in distilled water in a 100 ml

volumetric flask and make up the solution up to the mark with distilled water.
Alternatively, dissolve 0.340 g of potassium dihydrogen phosphate (KH2PO4) and 0.3550 g
of disodium hydrogen phosphate (Na2HPO4) in distilled water in a 100 ml volumetric flask
and make up the solution to the mark.

3. Buffer solution of pH 9.18: Dissolve a buffer tablet of pH 9.18 in distilled water in

a 100 ml volumetric flask and make up the solution up to the mark. Alternatively, dissolve
1.906 g of borax in distilled water in a 100 ml volumetric flask and make up the solution
up to the mark.
46
Procedure pH and Buffers

To ensure reliability of the pH readings, you should first calibrate the pH meter. You
should adjust the pH meter so that the readings agree with a known standard buffer.
The pH value of a solution changes with temperature, hence the instrument must be set
to the temperature of the solution. For accurate measurement of the pH, a buffer of
appropriate pH must be used for calibration.

Now for measurement of pH of a solution, follow the procedure given below in a

stepwise manner. But, if the pH meter, which you have in your laboratory, is of a
different type, go through its instruction manual and then use it accordingly.
1. Set the selector switch to ‘zero’ position and adjust the zero position by a screw
driver if the pointer does not indicate zero.
2. Mount the electrode assembly - a glass electrode and a saturated calomel electrode
or a combination electrode in the clip on the stand. Wash the electrodes well with
distilled water.
3. Connect the power cable to a 220 V AC supply. Switch on the instrument and wait
for a few minutes till the instrument warms up.
4. Take about 20 ml of the standard buffer solution of pH 7.0 in a beaker and lower
the electrode assembly into it. You should make sure that the glass electrode
membrane is completely immersed in the solution. The electrodes should not
touch each other or the sides or the bottom of the beaker. Swirl solution in the
region of the glass electrode surface gently so as to bring it into pH equilibrium.
5. Measure the temperature of the solution and adjust the ‘temperature compensation
knob’ to this temperature.
6. Put the selector switch to a suitable pH range (0-7 for acidic or 7-14 for basic
solutions) and adjust ‘set buffer knob’ in a manner that the pointer reads the pH of
the standard buffer solution placed in the beaker, i.e., pH 7.0.
7. Put the selector switch back to the ‘zero’ position. Remove the electrodes from
the buffer solution. Wash the electrodes with distilled water and wipe them
dry with a tissue paper. Transfer the standard buffer to the storage bottle.
8. You will need to make adjustments when precise values are required in the acid
or basic range. To do this, select the pH 4.0 buffer for an acid reading or the pH
9.18 buffer for a basic reading.
9. Take about 20 ml of second buffer solution (of pH 4.0 or 9.18) in another
beaker and place the electrode assembly into it. Set the selector switch in the
suitable pH range position (0-7 or 7-14) and read the pH of the solution on the
scale. If the meter reading does not agree exactly with the known pH of the buffer,
set the ‘slope’ control until the required reading is obtained. The instrument
is now ready for measuring the pH of the unknown solution.
10. Put the selector switch back to zero position and remove the electrodes from
the buffer solution. Wash the electrodes with distilled water and wipe them gently
with tissue paper. Transfer the buffer solution to the storage bottle.
11. Take about 20 ml of unknown solution in another beaker. Introduce the electrodes
into the solution and swirl the solution gently. Put the selector switch back in the
suitable range position and read the pH of the solution on the scale.
12. Put the selector switch back to zero position. Remove the electrodes from the
solution and keep the electrodes in distilled water when not in use.

Precautions
1. Never touch the membrane of the glass electrode with anything else except soft
tissue paper since it is fragile and is easily ruined if scratched or bumped.
2. The electrodes must not be removed from the solution unless the selector switch is
at zero.
3. Never dip the glass electrode in a solution with a dehydrating action.
4. For basic solutions with pH more than 11, glass electrodes of special composition
are required to avoid interference due to sodium ion. 47
Nutritional 5. The glass electrode may be covered with a sleeve to save it from jerks.
Biochemistry
Observations and Result

Record your pH reading of the unknown solution as indicated herewith;

pH of the unknown solution =

Results: The pH of the given solution is…………..……………………………......…

Conclusion
(Write about the usefulness of the pH meter and comment about the acidity or alkalinity
of solutions).

...............................................................................................................................
...............................................................................................................................
...............................................................................................................................
...............................................................................................................................
...............................................................................................................................

Now submit the experiment for evaluation.

....................................
Counsellor signature

48
 pH and Buffers
EXPERIMENT

PREPARATION OF PHOSPHATE BUFFERS 2

Date: ..........................
Aim: To prepare phosphate buffers with pH values of 5.91, 6.47 and 7.17.

Principle
(Write the principle related to buffers as you have studied in section 3.2 above, here in
the space provided).

Requirements
Apparatus
pH meter
Beakers
Volumetric flasks
Pipettes

Reagents
Buffer solutions of pH4, pH7 and pH 9.18
phosphate buffer solution: 0.1 M disodium hydrogen phosphate (Na2HPO4) and 0.1 M
potassium dihydrogen phosphate (KH2 PO4 )

Procedure
i. Preparation of Standard Buffer Solution: Prepare buffer solutions of pH 4, 7 and
9.18 using standard tablets as described in experiment 1 earlier. Write the procedure
for preparing these standard buffer solutions here in the space provided:

a) Preparation of Buffer solution of pH 4:

49
Nutritional b) Preparation of Buffer solution of pH 7:
Biochemistry

c) Preparation of Buffer solution of pH 9.18:

ii) Caliberate the pH meter with the standard buffer before proceeding for the
experiment. (Note as done in the previous experiment).
iii) Preparation of phosphate buffer solution: Prepare 0.1M sodium hydrogen
phosphate and 0.1 M potassium dihydrogen phosphate as explained herewith:
1) 0.1 M Na2HPO 4 – Weigh 14.2 g Na2HPO4 and dissolve in distilled water
in a 1000 ml volumetric flask. Make the volume to 1000 ml mark with
distilled water.
2) 0.1 M KH 2PO 4 - Dissolve 13.6 g of KH 2PO4 in distilled water in a 1000 ml
volumetric flask. Make the volume to 1 L with distilled water.
pH range 5.29 – 8.04
3) For making the phosphate buffer of pH 5.91, 6.47 and 7.17, take calculated
amounts of the solutions of sodium dihydrogen phosphate and potassium
dihydrogen phosphate - for the particular pH solution to be prepared from the
Table 1 given herewith in a beaker.

Proportions for Na2HPO4 and KH2PO4 to be added.

Table 1:
S. No 0.1MNa2 HPO4 (ml) 0.1MKH2 PO4 (ml) Expected pH
1. 2.5 97.5 5.29
2. 5.0 95.0 5.59
3. 10.0 90.0 5.91
4. 20.0 80.0 6.24
5. 30.0 70.0 6.47
6. 40.0 60.0 6.64
7. 50.0 50.0 6.81
8. 60.0 40.0 6.98
9. 70.0 30.0 7.17
10. 80.0 20.0 7.38
11. 90.0 10.0 7.73
12. 95.0 5.00 8.04
pH range 5.29 – 8.04
iv. Measure the pH of the buffer solution with the help of a pH meter by dipping the
glass electrode in the buffer solution.
v. Note your pH and compare with the expected pH.
50
Precautions pH and Buffers

1. Glass electrodes should always be dried before and after measuring pH

with a tissue paper.
2. The pipettes should be rinsed first.
3. Try to take the amount of reagent as accurately as possible to get nearly
exact pH values.
Observations and Result
Record your observations in the format given herewith:

S.No. Buffer Volume pH (pH Meter)

0.1M Na2HPO4 0.1M KH2PO4 Expected Observed

Results: The pH of the given solution was found to be ………, ……… and ……....
Conclusion
(Comment about the acidity or alkalinity of solutions).

....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................

Now submit the experiment for evaluation.

.................................
Counsellor signature

51
 Nutritional
EXPERIMENT
Biochemistry

3 PREPARATION OF CHLORIDE BUFFERS

Date: ..........................
Aim: To prepare chloride buffers with pH values 1.4, 1.8 and 2.2

Principle
(Write the principle related to buffers as you have studied in section 3.2 above, here in
the space provided).

Requirements
Apparatus:
pH meter
Beakers
Pipettes
Conical flasks
Burette 50 ml

Reagents
Buffer solutions of pH 4, pH 7 and pH 9.18
Chloride buffer solutions - 0.2 M KCl and 0.2 N HCl

Procedure
i. Preparation of Standard Buffer Solution: Prepare buffer solutions of pH 4, 7 and
9.18 using standard tablets as described in experiment 1 earlier. Write the procedure
for preparing these standard buffer solutions here in the space provided:
a) Preparation of Buffer solution of pH 4:

b) Preparation of Buffer solution of pH 7:

52
 c) Preparation of Buffer solution of pH 9.18: pH and Buffers

ii) Calibrate the pH meter with the standard buffer before proceeding for the
experiment. Note as done in the previous experiment.
iii) Preparation of Chloride buffer solution
a) 0.2 M KCl - Dissolve 14.91 g of KCl in distilled water and make upto 1 litre.
b) 0.2 N HCl - Dilute 4.17 ml con. HCl with distilled and make to 250 ml
standardise the solution by titrating against 0.2 N NaOH solution. If the
normality is not exactly 0.2 N make the requisite adjustments in the buffer
solution while adding in the buffer solution while adding HCl.
The pH range is 1.2 - 2.2.
For making the chloride buffer of pH 1.4, 1.8 amd 2.2, calculated amounts of 0.2 M
KCl and 0.2 N HCl solutions are mixed (see the amounts from the Table 2 given
herewith) in a beaker.
Table 2: Proportion for HCl and KCl

S.No. 0.2N HCl (ml) 0.2M KCl (ml) Expected pH

1. 32.25 25 1.2
2. 20.75 25 1.4
3. 13.15 25 1.6

4. 8.3 25 1.8

5. 5.3 25 2.0

6. 3.35 25 2.2

iv) Measure the pH of the solution with the help of a pH meter by dipping the
glass electrode in the buffer solution.

v) Note your pH and compare with the given pH.

Precautions
1. Glass electrodes should always be before and after measuring pH with a tissue
paper.
2. The pipettes should be rinsed first.
3. Try to take the amount of reagent as accurately as possible to get nearly exact
pH values.

Observations and Result

Record your observations in the format given herewith:

53
Nutritional
S.No. Buffer Volume pH (pH Meter)
Biochemistry

Expected Observed

Results

The pH of the given solution is….......……, …................ and ............................

Conclusion

(Comment about the acidity or alkalinity of solutions).

....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................
....................................................................................................................................

Now submit the experiment for evaluation.

..................................
Counsellor signature

54