Spectrophotometric Determination of pKa

Eugenio, F.D., Dequia, R.L., Mendoza, J.

Department of Chemical Engineering, Faculty of Engineering, University of Santo Tomas, Espaa, Manila
ABSTRACT
Optical methods of analysis rely on the principle of colorimetry. It works on the principle that a certain colored
chemical species allows a certain wavelength of electromagnetic radiation to pass through it, allowing the chemist to
assume that the amount of light absorbed is directly proportional to the concentration of the chemical species being
tested. The principle of equilibrium and the interaction of light will be demonstrated in this experiment. A calibrated
spectrophotometer was used to measure the amount of light absorbed by the sample.

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1

Introduction
Spectroscopy is a broader form of colorimetry which
deals with the measurement of the concentration of a
chemical species by monitoring the relative
absorbance of an unknown sample. Unlike colorimetry
whose emitted light is limited to the visible spectrum,
the light generated in spectroscopy can encompass
the entire spectrum of electromagnetic radiation.[1]
The idea began to develop in the 18th century after
Isaac Newton published his observations on the
spectrum and the nature of light in 1704. The idea
further developed after Frederick William Herschel
discovered the existence of electromagnetic radiation
beyond the visible light spectrum/region.[2] All
spectrophotometric methods that measure absorption,
including various enzyme assays, detection of
proteins, nucleic acids and different metabolites, reside
upon two basic rules, which combined are known as
the Beer-Lambert law. This law states that the fraction
of light absorbed by a transparent medium is
independent of the incident light intensity, and each
successive layer of the medium absorbs an equal
fraction of the light passing through it.[3] The law
quantitatively describes how the amount of light
attenuated is dependent on the concentration of the
absorbing molecules and the path length over which
absorption occurs.
When a monobasic indicator (e.g. Methyl Red) is
prepared in an aqueous solution, the indicator
dissociates according to the equation
HIn + H2O

H3O+ + In-

The absorbance indices of HIn and In may be

calculated from the absorbencies A1 and A2 at
wavelengths, 1 and 2, using the equation for the
equilibrium constant of dissociation:

Experimental Detail

Table 1. Composition of the Aqueous Solutions

Vol. of
Vol of
Solution No.
Indicator
NaH2PO4
2
1
5
3
1
5
4
1
5
5
1
10
6
1
5
7
1
1
8
1
1
9
1
0

Vol. of

NaHPO4
0
1
5
5
10
5
10
5

Results and Discussion

The aim of this experiment is to obtain a value for the
equilibrium constant of an indicator in water. For the
experiment, two data sets were used. The
absorbencies A1, for the acidic solution, and A2 for the
basic solution were determined experimentally.
Using Microsoft Excel software, the following
absorption spectrum was prepared:
Figure 1. Absorption Spectrum of the Solutions
40

The acid and base forms of the indicator, HIn and In-,
absorb strongly in the visible range, and thus may be
determined spectrophotometrically. The absorption
spectra of the indicator in acidic and basic solutions
are determined at two wavelengths.

A1 = a1,HIn [HIn] + a1, In [In]

A2 = a2,HIn [HIn] + a2, In [In]

For the first run of the experiment, the following

solutions were prepared in Erlenmeyer flasks for the
first part: 50 mL of 0.01 M NaH 2PO4 (fresh), 50 mL of
0.1 M NaHPO4 (fresh), 25 mL of concentrated HCl, 50
mL of 0.05% methyl red indicator, and 50 mL of 0.1M
NaOH. Table 1 shows the amounts of reagents
weighed to prepare the solutions.

eq. [4]
eq. [5]

30
20
10
0
365 385 405 425 445 465 485 505 525
-10

Wavelength in nm
Upon investigation, it was determined that the
minimum and maximum wavelengths are 385 and 515
nanometres,
respectively.
The
U-5100

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Spectrophotometer was again used to measure the

absorbance at the maxima and minima of the plot in
Figure 1.
Table 2. Tabulated Data
Solution
No.
1
2
3
4
5
6
7
8
9
10

pH
measured
1.00
4.86
6.33
7.20
7.04
7.65
8.03
8.16
10.54
13

Absorbance
at 1, A1
4.624
6.66
21.792
17.045
14.286
15.176
26.673
20.188
30.209
5.968

Absorbance
at 2, A2
5.341
27.003
6.344
2.967
2.602
2.421
3.608
2.728
4.428
0.938

Using the two equations for absorbencies discussed

earlier, the absorbancies value can be computed (as
shown in the Appendix). The data set produced a pKa
value of 6.015, which has a relative error of 17.94%
compared to the true value from literature which is 5.05
at around 298 Kelvin.
Conclusion
This experiment successfully facilitated an
understanding of spectrophotometric analysis
techniques. The use of the two equations for the
calculation of the absorbencies was useful and
effective in determining the concentration of the acid
and base forms of the indicator with good accuracy.
The aim of the experiment is the quantitative
determination of the acid-dissociation constant of the

indicator, methyl red using spectrophotometric

analysis. A source of error could be the resolution of
the spectrophotometer which is limited by the
sensitivity of the instrument at a certain wavelength.
Another source of error is the lack of experience of the
researchers in handling the spectrophotometer and the
computer software bundled with it.
If statistical methods are available to the researchers,
the standard deviations can be computed in testing the
reliability of the values. If the calculated value for the
standard deviations is small, it can be said that the
values produced are close to one another. Hence, the
precision of the method of analysis is concluded. Other
methods for the determination of the acid-dissociation
constant of methyl red could also be used. One such
method that could be employed is potentiometric
titration. In colored solutions, this type of analysis
provides more reliable data than titrations using
indicators.
References
[1]

Schmidt, W. (2005). Optical spectroscopy in

chemistry and life sciences: An introduction.
Wiley.
[2] Baptista, M. (n.d.). History of infrared
spectroscopy.
Retrieved
from
http://www.quimica3d.com/EN/IR/history.phpP
ayumo, E. M. and Castillo, E. S. Phil. J. Nutr.
37(2), (1979), p82
[3] Vanasse, G.A. Spectrometric Techniques,
(Academic Press, 1984), Vol. 3, p. 104.

Appendix

Calculation of mass needed to produce the solutions:

Na2HPO4
mass = (142 g mol-1)(0.05L) = 7.1g Na2HPO4
volume = (1)(0.05L) = 0.5L Na2HPO4

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NaH2PO4
mass = (12)(0.04L) = 0.48g NaH2PO4
NaOH
mass = (4)(0.024L) = 0.096g Na2HPO4

Calculation of Absorbancies and pKa:

Using equation [4] and [5]:
A1 = a1,HIn [HIn] + a1, In [In]
A2 = a2,HIn [HIn] + a2, In [In]

eq. [4]
eq. [5]

4.264 = aHIn (0.1) + aIn (0) Equation 1

5.968 = aHIn (0) + aIn (0.1) Equation 2
solving: aHin = 42.64, aIn = 59.68 (value 1) for value 2: aHin = 53.41, aIn = 9.38
pKa = pH log (In- / HIn)
pKa = 4.86 log (0.006897/0.0987)
pKa=6.015

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