EXPT.

5 DETERMINATION OF pKa OF AN
INDICATOR USING
SPECTROPHOTOMETRY
Structure
5.1 Introduction
Objectives
5.2 Principle
5.3 Spectrophotometric Determination of pKa Value of Indicator
5.4 Requirements
5.5 Solutions Provided
5.6 Procedure
5.7 Observations and Calculations
5.8 Result

5.1 INTRODUCTION
You have so far learnt about and performed the quantitative determination of inorganic
and organic species using UV-VIS spectrophotometry in this laboratory course. In this
experiment you would learn about an application of spectrophotometry in the
determination of a physical constant for an organic compound. You would learn about
and carry out the determination of the pKa of an acid-base indicator. You know that an
indicator is used for the visual detection of the end point of a titration. The indicator
used in acid-base titrations is either a weak acid or weak base which has distinctly
different colours in the ionised and unionised form. The end point in an acid-base
titration is indicated by a sharp change in the colour of the indicator due to a steep
change in the pH of the solution near the equivalence point of the titration.
Spectrophotometry can be used to determine the concentrations of the ionised (basic)
and unionised (acidic) forms of the indicator which in turn is used for the determination
of the acid dissociation constant using Henderson-Hasselbach equation.

In the next experiment you would learn about the application of IR spectrometry in the
detection of the functional group in an organic compound.

Objectives
After studying and performing this experiment you should be able to:

• explain the principle underlying the spectrophotometric determination of pKa of

an acid-base indicator,

• state and explain Henderson-Hasselbach equation,

• prepare a series of buffer solutions and measure the absorbance of the indicator
solution as a function of pH,

• compute the relative concentrations of the ionised and unionised forms of the
indicator by simultaneous equation method and determine the pKa value of the
indicator using Henderson-Hasselbach equation, and

• determine graphically, the pKa of an acid-base indicator using the pH versus

absorbance data.

31
 Spectroscopic Methods
Lab 5.2 PRINCIPLE
As mentioned in the introduction, an acid-base indicator is either a weak acid or a weak
base that has distinctly different colours in the ionised and unionised forms. One form
of an indicator may be colourless but the other must be distinctly coloured. Let us take
the example of the indicator methyl red. It is a two colour indicator; red in its unionised
HO (acidic) and yellow in its ionised (basic) form. Methyl red is a weak organic acid which
O Methyl red can be used as an indicator in the pH range of 4.4 to 6.2. This implies that a solution of
N methyl red will be red if the pH is lower than 4.8 and yellow if it is above 6.2. On the
N N other hand, if the pH of the solution is in this range (4.4< pH > 6.2), the colour will be
an appropriate mixture of both the colours. Methyl red is a weak acid and can be
2-(4-dimethylaminophenylazo)benzoic acid
represented as say, HMR. The dissociation of the indicator can be expressed as given
below.
+
HMR H + MR
Unionised Ionised

MR‒ represents the ionised or the basic form of the indicator. The acid form (HMR) of
the indicator is zwitterionic in nature and is a resonance hybrid of two closely related
structures; the basic form on the other hand is an anionic species. The structures of the
acidic and basic forms and the equilibrium between them are as given below.

O O
O O

N N
+ +
N N N N
H H
Acid form (RED)

+
H OH

O
O

N
N N

Base form (YELLOW)

You have learnt earlier that Henderson-Hasselbach equation provides the relationship
between pH and pKa value of an indicator. For methyl red we can write the
Henderson-Hasselbach equation as given below.
[MR − ]
pH = pK a + log … (5.1)
[HMR ]
It can be rearranged as following,
[ MR − ]
pK a = pH − log … (5.2)
[HMR ]

[MR − ]
or log = pH − pK a … (5.3)
[HMR ]

32
Thus, if we know the concentrations of the ionised and unionised forms of the indicator
It may be noted that the
at a given pH, we can determine the pKa value of the indicator. As both, the ionised as
ionised form has small
well as the unionised forms of methyl red are coloured, their concentrations can be absorption at the
determined by measuring the absorbances at the wavelengths of maximum absorption wavelength of maximum
of the two forms with the help of a spectrophotometer or a colorimeter. These can then absorption of the
be used to compute the pKa value of methyl red using Henderson-Hasselbach equation. unionised form. Similarly,
This forms the basis of the spectrophotometric determination of the pKa value of the the unionised form also
indicator. absorbs to some extent at
the wavelength of
maximum absorption of
5.3 SPECTROPHOTOMETRIC DETERMINATION OF pKa the ionised form.
VALUE OF INDICATOR
A typical spectrophotometric determination of the pKa value of the indicator consists of
the following steps.

Step 1: Obtaining the absorption spectra of the pure unionised and ionised forms
of the indicator to determine the wavelengths of their maximum
absorption and the corresponding molar absorption coefficients
A solution of a known concentration of the indicator is prepared in acidic solution (low
pH) such that the indicator exists almost exclusively in the unionised form and the
spectrum is obtained. Similarly, a spectrum is obtained for a solution of a known
concentration of the indicator in a basic solution (high pH) such that the indicator exists
almost exclusively in the ionised form. The schematic spectra of the unionised and the
ionised forms of the indicator are given in Fig. 5.1.

Fig. 5.1: Schematic spectra of ionised (basic) form (blue curve) and unionised (acidic)
form (black curve) of methyl red indicator
These spectra are then analysed to determine the wavelengths of maximum absorption
respectively for the unionised and ionised forms of the indicator. Let these be
represented as λmax,HMR , and λmax,MR − respectively. For convenience let us simplify the
expressions as λHMR and λMR ; the subscript max and the charge on the ionised form
being dropped.

The molar absorption coefficients of the unionised and ionised forms at the two
wavelengths of maximum absorption obtained above are determined using Beer-

33
Spectroscopic Methods Lambert’s law. You know that the expression for the Beer-Lambert’s law can be written
Lab as follows.
A = εbc … (5.4)
In the expression, A is the absorbance, ε is the molar absorption coefficient, b is the
thickness or the path length (in cm) of the sample and c is the concentration of the
absorbing species in moles per litre. For a unit path length at a given concentration the
molar absorption coefficient can be written as given below.
A
ε= … (5.5)
c
The four molar absorption coefficients for the unionised and ionised forms of the
indicator at the two wavelengths, λ HMR and λ MR can be defined as follows.
AMR, λ
εMR , λ = MR
… (5.6)
MR c
AMR, λ
ε MR , λ = HMR
… (5.7)
HMR c
AHMR, λ
ε HMR , λ = MR
… (5.8)
MR c
AHMR, λ
ε HMR , λ = HMR
… (5.9)
HMR c
These are determined by using the absorption values at the wavelengths of maximum
absorption of the unionised and ionised forms in the spectra obtained above.

Step 2: Verification of the Beer-Lambert’s law for the unionised and ionised
forms of the indicator at the wave lengths, λHMR and λMR
The Beer’s law can be verified by measuring the absorbances of a series of solutions of
varying concentration obtained by diluting the stock solution of the indicator in the
unionised and ionised forms using the cuvettes of path length equal to 1cm. These
absorbance values are then plotted against relative concentrations of the solution. The
linear plot so obtained establishes the validity of the Beer’s law. The slope of the line
obtained gives the molar absorption coefficients.

Step 3: Obtaining the absorption values of the indicator at different pH values

A small but fixed amount of the indicator solution is added to a series of buffer
solutions having pH spread over the indicator range (pKa ± 1) such that the indicator
exists in varying proportions of the ionised and unionised form. As the Ka value for
acetic acid is in the same range as that for methyl red, we will use acetic acid-acetate
buffers to control the pH. The absorption values of these solutions at λHMR and λMR , the
wavelengths of maximum absorption of the unionised and ionised form of the indicator
respectively are measured with the help of a suitable spectrophotometer or colorimeter.

Step 4: Manipulating the data obtained in steps 1-3 to obtain the pKa value
The data obtained in the steps 1-3 can be used to determine the pKa value of the
indicator. This can be achieved in a number of ways. Two of these are described as
follows.

34
A. Simultaneous Equations Method
You would recall from Experiment 3 of this course that when the analyte contains a
mixture of two species whose spectra overlap to certain extent then the concentrations
of these can be obtained by solving a set of simultaneous equations. As in the present
case also the two species present in the solutions of the indicator at a given pH have
overlapping regions in their spectra, we can compute their concentrations in the same
way. The relevant equations can be worked out as follows.

In the mixtures of the acidic and basic forms of methyl red, the total absorbance at the
wavelengths of maximum absorptions of the two forms viz., λHMR and λMR can be
written as follows.
−
Aλ = εMR , λ [MR ] + εHMR , λHMR [HMR] … (5.10)
HMR HMR

Aλ = εMR , λ [ MR − ] + εHMR , λMR [ HMR ] … (5.11)

MR MR

Solving these simultaneous equations we get the expressions for the concentrations of
the two species as follows.

AλMR . ε MR , λHMR − AλHMR . ε MR , λMR

[ HMR ] = … (5.12)
ε MR , λHMR . ε HMR , λMR − ε MR , λMR . ε HMR , λHMR

AλMR . εHMR , λHMR − AλHMR . εHMR , λMR

[ MR ] = … (5.13)
εHMR , λHMR . εMR , λMR − εHMR , λMR . εMR , λHMR
These equations can be used to obtain an expression for the ratio of the ionised and the
unionised form of the indicator in a given mixture. The expression comes out to be as
follows.
− AλHMR . ε HMR , λMR − AλMR . εHMR , λHMR
[ MR ]
= … (5.14)
[ HMR ] AλMR . εMR , λHMR − AλHMR .εMR , λMR
This can then be used to obtain the pKa value of the indicator by using the Henderson-
Hasselbach equation, viz.,
−
[ MR ]
pK a = pH − log … (5.2)
[ HMR ]

Thus,
[ AλHMR . ε HMR, λMR − AλMR . ε HMR, λHMR ]
pK a = pH − log …. (5.15)
[ AλMR . ε MR , λHMR − AλHMR .ε MR , λMR ]

In this experiment the concentration of the indicator is to be kept constant in all the
absorbance measurements. As we do not need the absolute values of the concentrations
of the unionised and ionised forms of the indicator we just need their ratio. Therefore,
we can do away with the determination of the molar absorption coefficients mentioned
above, instead use the absorbance values for the total concentration of the indicator in
the unionised and ionised forms at the λHMR and λMR . These can be obtained by

35
Spectroscopic Methods extrapolating the linear plots obtained for the verification of Beer’s law to relative
Lab concentration of 1.0.

Accordingly, Eq. (5.10) and (5.11) get modified as follows.

−
AλHMR = Au [ MR ] +
u
AHMR , λHMR
[ HMR ] … (5.16)
λ
MR , HMR

−
Aλ = Au [ MR ] +
u
AHMR ,λ
[ HMR ] … (5.17)
MR λ
MR , MR MR

Where, the terms containing the superscript ‘u’ pertain to the absorbance values at
relative concentration of 1.0 or unity. The final expression for the pKa value then can be
written as follows.
u
[ AλHMR . AHMR , λMR
− AλMR . AHMR
u
, λHMR
]
pK a = pH − log … (5.18)
u
[ AλMR . AMR , λHMR
− AλHMR . AMR
u
, λMR
]

B. Graphical Method
Recall that according to the Henderson-Hasselbach equation,
[MR − ] … (5.3)
log = pH − pK a
[HMR ]

This represents an equation of a straight line of the type, Y= mX + C. where,

− −
Y equals log [MR ] ; X = pH and C = − pK a . Thus, in a plot of log [MR ] versus pH the
[HMR ] [ HMR ]
slope would be equal to 1 and the intercept would be equal to − pK a as shown in
Fig.5.2. Thus, the pKa can be found by determining the intercept of the plot of [MR − ]
log
[HMR ]
versus pH. Also, the line would cross the pH axis at pH = pKa ( as at this stage the
concentrations of the ionised and unionised forms would be equal, [MR − ] = [HMR ] ,
making the log term equal to zero.

−
Fig. 5.2: log [MR ] versus pH plot for the indicator
[HMR]

36
The pKa can be obtained either as the point of intersection of the line with the X-axis or
from the intercept on the Y-axis.

5.4 REQUIREMENTS
Apparatus Chemicals
Spectrophotometer/ Filter photometer 1 Ethanol
Matched cuvettes 2 Hydrochloric acid
pH meter with glass electrode 1 Sodium acetate
Volumetric flasks (1 litre) 1 Acetic acid
Volumetric flasks (250 cm3) 1 Methyl red indicator
Volumetric flasks (100 cm3) 8
Volumetric flasks (50 cm3) 6
Pipettes (10, 20 cm3) 1each
Burette stand with clamp 1

5.5 SOLUTIONS PROVIDED

i) Sodium acetate (0.04M): It is prepared by accurately weighing 3.28 g of
anhydrous sodium acetate and transferring to a 1 dm3 volumetric flask containing
about 100 cm3 of distilled water. After dissolving the salt the volume is made up
to the mark with distilled water.
ii) Sodium acetate solution (0.01M): It is prepared by diluting 250 cm3 of the
0.04 M sodium acetate solution prepared above to 1 dm3 by distilled water.
iii) Acetic acid solution (0.02M): It is prepared by mixing 1.2 cm3 of glacial acetic
acid with 100 cm3 of distilled water in a 1dm3 volumetric flask and making up the
volume with distilled water.
iv) Hydrochloric acid solution (0.1M): It is prepared by transferring 9 cm3 of
concentrated hydrochloric acid to a 1dm3 volumetric flask containing 500 cm3 of
distilled water. After mixing the volume is made up by distilled water.
v) Hydrochloric acid solution (0.01M): It is prepared by diluting 100 cm3 of the
0.1 M hydrochloric acid solution prepared above to 1 dm3 by distilled water.
vi) Methyl red indicator (stock) solution: It is prepared by dissolving 0.1 g of pure
crystalline methyl red in 30 cm3 of 95% ethanol and making up to 100 cm3 with
distilled water.
vii) Methyl red in acidic form (Solution A): It is prepared by mixing 10 cm3 of the
indicator solution prepared in (vi) above with 10 cm3 of 0.1 M HCl solution and
diluting to 100 cm3 with distilled water in a volumetric flask.
viii) Methyl red in basic form (Solution B): It is prepared by diluting 10 cm3 of the
indicator solution prepared in vi) above with 0.01 M sodium acetate solution to
100 cm3 in a volumetric flask.

5.6 PROCEDURE
You would recall from section 5.3 that a typical spectrophotometric determination of
the pKa value consists of four steps. These are as follows.
a) Determine the wavelengths of maximum absorption for the unionised and ionised
forms of the indicator,

37
Spectroscopic Methods b) Verification of Beer’s law for unionised (HMR) and ionised (MR-) forms at the
Lab wavelengths of their maximum absorption,
c) Obtaining the absorption values of the indicator at different pH values,
d) Manipulating the data obtained in step 1-3 to obtain the pKa value of the
indicator.
Follow the instructions given below in the sequential order to accomplish these tasks.

a) Determination of the wavelengths of maximum absorption for the unionised

and ionised forms of the indicator
1. Record the absorption spectrum of ‘solution A’ in the range 350 – 610 nm
against 0.01 M HCl.
2. In case the instrument is of manual type, measure the absorption value after
every 10 nm over the spectral range and record the readings in columns 2,
5 and 8 of the Observation Table 5.1.
3. Similarly, measure the absorption value for solution B against 0.01 M
sodium acetate after every 10 nm over the spectral range and record the
readings in columns 3, 6 and 9 of Observation Table 5.1.
4. Draw the spectrum of solution A and solution B by plotting the absorbance
as a function of the wavelength in the graph provided in Fig.5.3. You may
use two different colours to draw the spectra for solution A and solution B
respectively.
5. Select the wavelength which gives maximum absorbance for solution A
and solution B and record the same as λHMR and λMR respectively.

b) Verification of Beer’s law for HMR and MR‒ at λ HMR and λ MR

1. Pipette out 40.0 cm3, 20.0 cm3 and 10.0 cm3 of solution A into three
separate 50 cm3 volumetric flasks and make up the volume in each case
with 0.01 M HCl solution. Label these solutions as A1, A2 and A3
respectively. These solutions would have concentrations equal to 0.8, 0.4
and 0.2 times the concentration of the stock solution A.
2. Similarly, pipette out 40.0 cm3, 20.0 cm3and 10.0 cm3of solution B into
three separate 50 cm3 volumetric flasks and make up the volume in each
case with 0.01 M sodium acetate solution. Label these solutions as B1, B2
and B3 respectively. These solutions would have concentrations equal to
0.8, 0.4 and 0.2 times the concentration of the stock solution B.
3. Measure the absorbances of the solutions A1, A2 and A3 at λHMR and λMR
using 0.01 M HCl as the reference and record your observations in Table
5.2.
4. Similarly, Measure the absorbance values for the solutions B1, B2 and B3
at λHMR and λMR using 0.01 M sodium acetate as the reference and record
your observations in Observation Table 5.2.
5. Plot the absorbance values obtained in step 3 and 4 against the
corresponding relative concentrations in the graph provided in Fig.5.4.
6. The linearity of the plot so obtained establishes the validity of the Beer’s
law.
7. Extrapolate the linear plots obtained above to compute the absorbance
values of the unionised and ionised forms of the indicator at λHMR
and λMR and record the same.

38
c) Obtaining the absorption values of the indicator at different pH values
1. Prepare four solutions of the indicator in buffer solution of different pH
values by mixing sodium acetate, acetic acid, methyl red and water as
detailed in column 2 to 5 of the Observation Table 5.3. Use 100 cm3
volumetric flasks labelled as 1, 2, 3 and 4 for this purpose.
2. Measure the pH values of these solutions with the help of a suitably
calibrated pH meter. Record these values in the column 6 of Table 5.3.
3. Measure the absorbance values of these solutions at λ HMR and λ MR against
water as a blank. Record the same under columns 7 and 8 of Observation
Table 5.3.

d) Calculation of pKa value for the indicator from the data obtained
−
1. Calculate the values of [MR], [HMR], and [MR ] respectively from the
[HMR ]
observed absorbance values at different pH values using equations given
under step D of Section 5.6. Record the same in Observation Table 5.4.
2. Use these to calculate pKa value with the help of Henderson-Hasselbach
equation and record in Observation Table 5.4.
3. Find average value and report the result.
−
4. Plot a graph between pH (x-axis) and log [MR ] (y-axis) in Fig.5.5.
[HMR ]
Determine the pKa value from the point of intersection of the line and the
pH axis and also in terms of the intercept on the y-axis and report the
result.

5.7 OBSERVATIONS AND CALCULATIONS

A. Determination of the wavelengths of maximum absorption for the unionised
and ionised forms of the indicator

Observation Table 5.1: Absorbance values of the solution A and solution B

at different wavelengths
Column
1 2 3 4 5 6 7 8 9
Wavelength Absorbance Wavelength Absorbance Wavelength Absorbance
(nm) Solution Solution (nm) Solution Solution (nm) Solu- Solu-
A B A B tion tion
A B
350 440 530
360 450 540
370 460 550
380 470 560
390 480 570
400 490 580
410 500 590
420 510 600
430 520 610

39
Spectroscopic Methods B. Spectra for unionised form of the indicator (solution A) and ionised form of
Lab the indicator (solution B) using the data recorded in Table 5.1

Absorbance

350 400 450 500 550 600 650

Wavelength (nm)
Fig. 5.3: Visible spectra for methyl red in the unionised and ionised forms
From the spectra obtained above, the wavelengths of maximum absorption for the
unionised and ionised forms of the indicator methyl red are as follows.

For unionised form, λHMR = ………nm

For unionised form, λMR =……….nm

C. Verification of Beer’s law for HMR and MR- at the λ HMR and λ MR

Observation Table 5.2: Absorbance values of the solution A and solution B

at different wavelengths
For Unionised form, HMR

Solution Volume of Volume of Relative Absorbance

Solution A 0.01 M HCl concentration λ HMR λ MR
A1 40 10 0.8
A2 20 30 0.4
A3 10 40 0.2
For ionised form, MR-

Solution Volume of Volume of Relative Absorbance

Solution B 0.01 M concentration λ HMR λ MR
CH3COONa
B1 40 10 0.8
B2 20 30 0.4
B3 10 40 0.2

40
 Absorbance

0 0.2 0.4 0.6 0.8 1.0

Relative concentration
Fig. 5.4: Absorbance values (at λ HMR and λ MR ) versus relative concentration plot for
methyl red in the unionised and ionised forms

The absorbance values at λ HMR and λ MR for unionised and ionised forms of methyl red
at relative concentration of 1.0 are found to be as given below.

Au HMR, λMR =
Au HMR, λHMR =
Au MR, λHMR =
Au MR, λMR =
D. Obtaining the absorption values at λ HMR and λ MR for the indicator at
different pH values.

Observation Table 5.3: Absorbance values of the indicator solution in buffer

solutions of different pH values

Column
1 2 3 4 5 6 7 8
S.No. Volume of Absorbance
0.04 M 0.02 M Indicator Water pH λ HMR λ MR
CH3COONa CH3COOH Stock (cm )3
(cm3) (cm3) (cm3)
1 25.0 50.0 10.0 To make up to the mark
2 25.0 25.0 10.0 To make up to the mark
3 25.0 10.0 10.0 To make up to the mark
4 25.0 5.0 10.0 To make up to the mark

41
Spectroscopic Methods E. Calculation of pKa value for the indicator from the data obtained
Lab
a) Simultaneous Equation Method
−
The values of [MR], [HMR], [MR ] , can be calculated from the observed
[HMR ]
absorbance values at different pH values using the following equations.

u u
Aλ . AMR, λ − Aλ . AMR, λMR
[HMR] = u
MR
u
HMR
u
HMR
u
AMR, λ HMR
. AMR, λMR − AMR, λMR . AHMR, λHMR

u u
Aλ . AHMR, , λHMR − AλHMR . AHMR, λMR
[MR] = u MR
u u u
AHMRλHMR . AMR, λMR − AHMR, λMR . AMR, λHMR

[MR - ] AλHMR . AHMR,, λMR − AλMR . AHMR,λHMR

u u

= u u
λHMR − Aλ HMR, . AMR, λMR
[HMR] Aλ . AMR,
MR

u u
[ AλHMR . AHMR, λMR − AλMR . AHMR, λHMR ]
pK a = pH − log u u
[ AλMR . AMR, λHMR − AλHMR . AMR, λMR ]

Observation Table 5.4: Computation of the pKa values of methyl red

indicator using Handerson-Hesselbalch equation.

S.No. pH [MR] [HMR] [MR − ] [MR − ] [MR − ]

log pK a = pH − log
[HMR] [HMR] [HMR]

Average value of pKa

The average value of pKa from the simultaneous equation method is found
to be =

b) Graphical method
−
Graph between pH and log [ MR ]
[ HMR]

42
 4.5 5.0 5.5 6.0 6.5

pH

Fig. 5.5: Plot of [MR − ] versus pH to determine the pKa of methyl red.
log
[HMR]

The value of pKa from the graphical equation method is found to be =

5.8 RESULT
The pKa of the indicator (methyl red) using
simultaneous equation method is found to be = …………………..

The pKa of the indicator (methyl red) using

graphical method is found to be = …………………..

43