COMPETENCY NO. – BC 14.

2
AIM- Describe estimation of pH by pH
meter
OBJECTIVE –
Preparation of various buffer commonly used in
biochemical lab
and determination of pH using pH meter
Preparation of various buffer solution and measurement pH of
a solution requires–
1) Indicator dye
2) Indicator paper
3) pH meter

pH determination is very important and basic requirement

in laboratory. Use of pH paper and pH indicators are not very
accurate, but use of pH meter is the most common and
accurate method for pH determination.
INDICATOR DYE (pH indicator)
Indicator are substances which change their colour
with variation in pH of the solution, in which they are
present. They are usually weak acids or bases having
different colours in ionized form and unionized form.
An indicator which is a weak acid is practically
undissociated in acid solution and gives the acid
colour. In presence of dilute alkali, It will form salt
which will dissociate freely and display the alkaline
colour. At intermediate pH, the colour will be mixture
of an acid and alkali colour.
The pH of solution to which an indicator has been
added, is given by the Handerson – Hasselbalch
equation –
pH = pK + log [salt]
[acid]
 According to this equation besides the pKa value of
indicator, the pH is dependent on the concentration of salt
and acids form of the indicator, represented by its dissociated
and undissociated form respectively.
Each indicator has an effective range of pH over which it
gives detectable change in colour. Usually this range is 1 pH
unit around its pK value , i.e. , pK ± 1.
BUFFER
It is a mixture of weak acid and its salt with strong base or a
mix of weak base and its salt with strong acid.

The pH of a buffer depends upon the pKa value of its acid and
ratio of salt to the acid concentration.
pH = pKa + log [salt]
[acid]

For a given buffer, pKa of acid is constant. Hence, its pH is

independent on the ratio of salt to acid. Routinely buffer of
different pH value are prepared varying this salt to acid ratio.
BUFFER CAPACITY
Buffer resist in pH when small amount of an acid or base is
added to them. It can be defined as the gram equivalent of acid
or alkali required by 1 liter of a buffer, to undergo a change of
pH by 1.

Thus, a buffer requiring more acid or alkali is said to have

more buffer capacity or value.
 pH METER PRINCIPLE
pH meter is based on the principle of measurement of
electromotive force (EMF) [Based on the principle of
potentiometry] generated between the two electrodes due to
the difference in hydrogen ion concentration.

It is known that if a metal plate is placed in a solution of its

own salt, it loses ions into the solution and itself becomes
negatively charged as compared to solution. This generates an
electrical potential on the metal plate or the electrode.
METAL PLATE AS ELECTRODE

Zn Plate

+ +
+ +
+ + that
+ +
+ + ZnSO4 Solution
+ +
+ +
+ +
+ +
So, if two different metal electrodes are connected in
this way, the difference in their electrode potential can
be measured as EMF or it can be calculated as the net
difference of the two electrode potential.
GENERATION
OF EMF
BETWEEN
TWO ELECTRODES
Hence, if one of the electrode is a standard electrode
(with non potential), potential of other electrodes can be
easily measured using following formula-

EMF of the complete cell (E)

E = E (Ref) – E (glass)

In the same way, if there is a standard hydrogen electrode

of unknown potential, electrode potential of unknown
electrode sensitive to H+ ion concentration can be
measured.
 This is the basis of pH meter, the electrode potential
generated by H+ concentration in an unknown solution
is measured against a standard hydrogen electrode
potential.

In pH meter a combined electrode having both

reference and measuring electrode, is used. The pH of
the unknown solution at 25°C is given by the equation –
pH = [E – K]
0.0591

where K is constant and E is measured potential.

Combined electrode used in pH meter
Procedure-I
REAGENTS
(With manual preparation of buffers having known pH)
1.1/15 M Na2HPO4 (salt)
2. 1/15 M KH2PO4 (acid)
3. 0.1 M Acetic Acid
4. 0.1 % Sodium Carbonate
5. pH paper
6. Universal Indicator
7. pH meter
PPREPARATION OF BUFFER SOLUTIONS
Procedure- (phosphate buffer solutions of pH 5.8 and 7.2)
●Take two clean and dry test tubes A & B and add the reagent as
shown in the table-
REAGENT TUBE - A TUBE - B

pH pH – 5.8 pH – 7.2

M/15 Na2HPO4 2.0 ml 14.4 ml

M/15 KH2PO4 23.0 ml 5.6 ml

● Mix the content thoroughly and buffer solution A and B are

ready in Tube A (pH = 5.8) and in Tube B (pH = 7.2).
TYPES OF BUFFER
1) Acetate Buffer (acidic range)
mix 9.2 ml of 0.1N acetic acid + 0.8 ml of 0.1N sodium acetate

2) Carbonate Buffer (basic range)

mix 1.4 ml of 0.2M Na2CO3 + 1.1 ml of 0.2M NaHCO3 and 7.5
ml H2O

3) Phosphate Buffer (neutral range)

mix 6 ml of 0.1M Na2HPO4 + 4 ml of 0.1M KH2PO4

Now we can measure the pH of these buffer solution using pH

paper/ indicator or pH meter.
Procedure-II
REAGENTS
(Using to use pH tablets of known pH)
1) Standard Buffers
pH = 4
pH = 7
pH = 9
Buffer solutions are prepared by dissolving respective pH
tablets in 100ml distilled water.

2) Unknown buffers and solutions.

PROCEDURE
Most instruments have their manuals giving operative
guidelines which should be followed. However, generally,
the method is very simple as given under :

1) Put on the instrument. Most modern instruments do

not require any time for warming up. If required the
instrument should be kept for the specified time period.

2) Note the room temperature and adjust for

temperature correction is required.
3) Take standard buffers of pH 4, 7 and 9 in 100ml
beaker. It should be sufficient so that electrode bulb
can dip into it. Similarly, take the unknown solution in
another beaker.

4) Calibrate the instruments with standard buffer

solution. First, calibrate with pH – 7 buffer. Then
calibrate with buffer pH – 4 (if the pH of unknown
solution is expected to be in the acidic range) or with
buffer of pH – 9 (if the pH of unknown solution is
expected to be in alkaline range).
5) For calibrating, carefully lift the electrode of the
instrument and wipe its tip gently with a soft tissue
paper to remove excess water. Carefully lower the
electrode into the buffer solution (pH = 7) so that the
bulb dips into the solution. Adjust the instrument
coarse and fine adjustment to show pH value of
several instrument. Then remove the electrode gently
and wash with distilled water, gently wipe with soft
tissue paper. Now calibrate with other buffer (pH – 4 or
9).
Recheck the calibration , once again.
6) Now after washing the electrode again with water,
dip the electrode in unknown solution. Allow the
instrument to read the pH value and note it , when the
reading is stable.

7) Remove the unknown solution and after washing the

electrode as before, dip it into distilled water as at the
start of the experiment.

8) Then switch off the instrument as advised in

manual.
PRECAUTIONS

1. Glass electrode is fragile and must be handled with

care. It should not be left to dry.

2. Temperature correction must be entered before

calibration with buffer.

3. The pH meter should be calibrated first with buffer of

pH 7 and then with buffer of pH 4 or 9 as required.
4. After each change, the electrode should be washed
with distilled water and wipe gently.

5. Any specific guidelines outlined in the instrument

manual should be followed.
Thank
You