EXPT.

5 DETERMINATION OF pKa OF AN INDICATOR USING SPECTROPHOTOMETRY

Structure
5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 Introduction
Objectives

Determination of pKa of an Indicator Using Spectrophotometry

Principle Spectrophotometric Determination of pKa Value of Indicator Requirements Solutions Provided Procedure Observations and Calculations Result Precautions

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.

SpectroscopicMethods Lab.

5.2

PRINCIPLE

HO O N N

Methyl red

2-(4-dimethylaminophenylazo)benzoic acid

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 (acidic) and yellow in its ionised (basic) form. Methyl red is a weak organic acid which can be used as an indicator in the pH range of 4.4 to 6.2. This implies that a solution of methyl red will be red if the pH is lower than 4.8 and yellow if it is above 6.2. On the 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 represented as say, HMR. The dissociation of the indicator can be expressed as given below.
HMR Unionised H
+

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

+ H O O N N

OH

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.

pH = pK a + log It can be rearranged as following,

pK a = pH log

[MR ] [ HMR ]

(5.1)

[MR ] [ HMR ]

(5.2)

or

log

[ MR ] = pH pK a [ HMR ]

(5.3)

Thus, if we know the concentrations of the ionised and unionised forms of the indicator at a given pH, we can determine the pKa value of the indicator. As both, the ionised as well as the unionised forms of methyl red are coloured, their concentrations can be determined by measuring the absorbances at the wavelengths of maximum absorption of the two forms with the help of a spectrophotometer or a colorimeter. These can then be used to compute the pKa value of methyl red using Henderson-Hasselbach equation. This forms the basis of the spectrophotometric determination of the pKa value of the indicator.

Determination of pKa of an Indicator Using Spectrophotometry It may be noted that the ionised form has small absorption at the wavelength of maximum absorption of the unionised form. Similarly, the unionised form also absorbs to some extent at the wavelength of maximum absorption of the ionised form.

5.3

SPECTROPHOTOMETRIC DETERMINATION OF pKa 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 max,HMR , and max, MR respectively. For convenience let us simplify the expressions as HMR and MR ; 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 BeerLamberts law. You know that the expression for the Beer-Lamberts law can be written as follows.

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A = bc

(5.4)

In the expression, A is the absorbance, 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 c

(5.5)

The four molar absorption coefficients for the unionised and ionised forms of the indicator at the two wavelengths, HMR and MR can be defined as follows.

MR , MR ,

AMR, c =

MR

MR

(5.6)

AMR, c

HMR

HMR

(5.7)

HMR ,
HMR ,

=
MR

AHMR, c

MR

(5.8)

=
HMR

AHMR, c

HMR

(5.9)

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-Lamberts law for the unionised and ionised forms of the indicator at the wave lengths, HMR and MR
The Beers 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 Beers 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 HMR and MR , 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 step 1-3 to obtain the pKa value
The data obtained in the steps 1-3 can be used to determine the pK a value of the indicator. This can be achieved in a number of ways. Two of these are described as follows.

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., HMR and MR can be written as follows.

Determination of pKa of an Indicator Using Spectrophotometry

A
A

HMR

= MR ,

HMR

[MR ]

+ HMR , HMR [HMR]

(5.10)

MR

= MR , MR [ MR ] + HMR , MR [ HMR ]

(5.11)

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

[ HMR ] =

AMR . MR , HMR AHMR . MR , MR MR , . HMR , MR , . HMR ,

HMR MR MR

(5.12)

HMR

[ MR ] =

AMR . HMR , HMR AHMR . HMR , MR HMR , . MR , HMR , . MR ,

HMR MR MR

(5.13)

HMR

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.

[ MR ] [ HMR ]

AHMR . HMR , MR AMR . HMR , HMR A . MR , A .MR ,

MR HMR HMR MR

(5.14)

This can then be used to obtain the pK a value of the indicator by using the HendersonHasselbach equation, viz., pK a = pH log Thus,
pK a = pH - log [ A
HMR

[ MR ] [ HMR ]

(5.2)

. HMR ,

MR

A . HMR ,
MR

HMR

[ A . MR ,
MR

HMR

HMR

. MR , ]
MR

. (5.15)

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 HMR and MR . These can be obtained by

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extrapolating the linear plots obtained for the verification of Beers law to relative concentration of 1.0. Accordingly, Equations 5.10 and 5.11 get modified as follows.

AHMR = Au
AMR = Au

MR , HMR

[ MR ] +
[MR ] +

u AHMR , HMR [ HMR ]

(5.16)

MR , MR

u AHMR , MR [ HMR ]

(5.17)

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.

pK a = pH log
Graphical Method

u u [ AHMR . AHMR ,MR AMR . AHMR , HMR ] u u [ AMR . AMR , HMR AHMR . AMR ,MR ]

(5.18)

Recall that according to the Henderson- Hasselbach equation,

log [MR ] = pH pK a [HMR ]

(5.3)

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 log [MR ]

[ HMR]

versus pH. Also, the line would cross the pH axis at pH = pK a ( as at this stage the concentrations of the ionised and unionised forms would be equal, [ MR ] = [HMR ] , making the log term equal to zero.

log

Fig. 5.2:

[ MR ] [HMR ] versus pH plot for the indicator

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

Determination of pKa of an Indicator Using Spectrophotometry

5.4

REQUIREMENTS
1 2 1 1 1 8 6 1each 1

Apparatus Spectrophotometer/ Filter photometer

Matched cuvettes pH meter with glass electrode Volumetric flasks (1 litre) Volumetric flasks (250 cm3) Volumetric flasks (100 cm3) Volumetric flasks (50 cm ) Pipettes (10, 20 cm ) Burette stand with clamp
3 3

Chemicals Ethanol
Hydrochloric acid Sodium acetate Acetic acid Methyl red indicator

5.5
i)

SOLUTIONS PROVIDED
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. 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. 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. 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. 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. 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. 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.

ii) iii)

iv)

v) vi)

vii)

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, 7

SpectroscopicMethods Lab.

b) c) d)

Verification of Beers law for unionised (HMR) and ionised (MR-) forms at the wavelengths of their maximum absorption, Obtaining the absorption values of the indicator at different pH values, 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. 2. Record the absorption spectrum of solution A in the range 350 610 nm against 0.01 M HCl. 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. 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. 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. Select the wavelength which gives maximum absorbance for solution A and solution B and record the same as HMR and MR respectively.

3.

4.

5.

b)

Verification of Beers law for HMR and MR at HMR and 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. Similarly, pipette out 40.0 cm3, 20.0 cm3and 10.0 cm3 of 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. Measure the absorbances of the solutions A1, A2 and A3 at HMR and MR using 0.01 M HCl as the reference and record your observations in Table 5.2. Similarly, Measure the absorbance values for the solutions B1, B2 and B3 at HMR and MR using 0.01 M sodium acetate as the reference and record your observations in Observation Table 5.2. Plot the absorbance values obtained in step 3 and 4 against the corresponding relative concentrations in the graph provided in Fig.5.4. The linearity of the plot so obtained establishes the validity of the Beers law. Extrapolate the linear plots obtained above to compute the absorbance values of the unionised and ionised forms of the indicator at HMR and MR and record the same.

2.

3.

4.

5. 6. 7.

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. 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. Measure the absorbance values of these solutions at HMR and MR against water as a blank. Record the same under column 7 and 8 of Observation Table 5.3.

Determination of pKa of an Indicator Using Spectrophotometry

2. 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. 3. 4. Use these to calculate pKa value with the help of Henderson-Hasselbach equation and record in Observation Table 5.4. Find average value and report the result.
Plot a graph between pH (x-axis) and log [MR ] (y-axis) in Fig.5.5.

[HMR ]

Determine the pK a 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
A.

OBSERVATIONS AND CALCULATIONS

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 1 Wavelength (nm) 2 3 Absorbance Solution Solution B A Column 4 5 6 Wavelength Absorbance (nm) Solution Solution B A
440 450 460 470 480 490 500 510 520

7 Wavelength (nm)

8 9 Absorbance Solu- Solution tion B A

350 360 370 380 390 400 410 420 430

530 540 550 560 570 580 590 600 610

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B.

Spectra for unionised form of the indicator (solution A) and ionised form of 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, HMR = nm For unionised form, MR =.nm C.

Verification of Beers law for HMR and MR- at the HMR and MR Observation Table 5.2: Absorbance values of the solution A and solution B at different wavelengths For Unionised form, HMR

Solution
A1 A2 A3

Volume of Solution A
40 20 10

Volume of 0.01 M HCl

10 30 40

Relative concentration

Absorbance

HMR

MR

0.8 0.4 0.2 For ionised form, MR-

Solution

Volume of Solution B
40 20 10

B1 B2 B3

Volume of 0.01 M CH3COONa 10 30 40

Relative concentration
0.8 0.4 0.2

Absorbance

HMR

MR

10

Determination of pKa of an Indicator Using Spectrophotometry

Absorbance
0

0.2

0.4

0.6

0.8

1.0

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

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

Au HMR, MR = Au HMR, HMR = Au MR, HMR Au MR, MR

D.

= =

Obtaining the absorption values at HMR and 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 S.No. 0.04 M CH3COONa (cm3)
1 2 3 4 25.0 25.0 25.0 25.0

3 Volume of 0.02 M CH3COOH (cm3)

50.0 25.0 10.0 5.0

6 pH

7 HMR

8 MR

Absorbance Indicator Water Stock (cm )

10.0 10.0 10.0 10.0 To make up to the mark To make up to the mark To make up to the mark To make up to the mark
3

(cm )

11

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E.

Calculation of pKa value for the indicator from the data obtained
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.

[HMR] =

u A . AMR,
MR

HMR

HMR

u . AMR,

MR

u AMR,

HMR

u u u . AMR, MR AMR, MR . AHMR, HMR

u u A . AHMR,, A . AHMR, MR MR HMR HMR [MR] = u u u u AHMRHMR . AMR, MR AHMR, MR . AMR, HMR

u u [MR - ] AHMR . AHMR,, MR AMR . AHMR, HMR = u [HMR] A . AMR, HMR A . Au MR HMR, MR, MR
u u [ AHMR . AHMR, MR AMR . AHMR, HMR ] u u [ AMR . AMR, HMR AHMR . AMR, MR ]

pK a = pH log

Observation Table 5.4: Computation of the pKa values of methyl red indicator using Handerson-Hesselbalch equation. S.No. pH [MR] [HMR]
[MR ] [ HMR] [ MR ] [ HMR] [ MR ] [ HMR]

log

pK a = pH log

1 2 3 4 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]

12

Determination of pKa of an Indicator Using Spectrophotometry

4.5

5.0 pH

5.5

6.0

6.5

Fig. 5.5: Plot of

log

[MR ] [HMR]

versus pH to determine the pKa of methyl red.

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

5.8

RESULT
..

The pK a of the indicator (methyl red) using simultaneous equation method is found to be = The pK a of the indicator (methyl red) using graphical method is found to be =

..

13