Spectrophotometric Determination of pKa

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

Department of Chemical Engineering, Faculty of Engineering, University of Santo Tomas, España, 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.

Introduction
The acid and base forms of the indicator, HIn and In-,
Spectroscopy is a broader form of colorimetry which absorb strongly in the visible range, and thus may be
deals with the measurement of the concentration of a determined spectrophotometrically. The absorption
chemical species by monitoring the relative spectra of the indicator in acidic and basic solutions
absorbance of an unknown sample. Unlike colorimetry are determined at two wavelengths.
whose emitted light is limited to the visible spectrum,
the light generated in spectroscopy can encompass The absorbance indices of HIn and In− may be
the entire spectrum of electromagnetic radiation.[1] calculated from the absorbencies A1 and A2 at
The idea began to develop in the 18th century after wavelengths, λ1 and λ2, using the equation for the
Isaac Newton published his observations on the equilibrium constant of dissociation:
spectrum and the nature of light in 1704. The idea
further developed after Frederick William Herschel A1 = a1,HIn [HIn] + a1, In− [In−] eq. [4]
discovered the existence of electromagnetic radiation A2 = a2,HIn [HIn] + a2, In− [In−] eq. [5]
beyond the visible light spectrum/region.[2] All
spectrophotometric methods that measure absorption, Experimental Detail
including various enzyme assays, detection of
proteins, nucleic acids and different metabolites, reside For the first run of the experiment, the following
upon two basic rules, which combined are known as solutions were prepared in Erlenmeyer flasks for the
the Beer-Lambert law. This law states that the fraction first part: 50 mL of 0.01 M NaH2PO4 (fresh), 50 mL of
of light absorbed by a transparent medium is 0.1 M NaHPO4 (fresh), 25 mL of concentrated HCl, 50
independent of the incident light intensity, and each mL of 0.05% methyl red indicator, and 50 mL of 0.1M
successive layer of the medium absorbs an equal NaOH. Table 1 shows the amounts of reagents
fraction of the light passing through it.[3] The law weighed to prepare the solutions.
quantitatively describes how the amount of light
attenuated is dependent on the concentration of the Table 1. Composition of the Aqueous Solutions
absorbing molecules and the path length over which Vol. of Vol of Vol. of
Solution No.
absorption occurs. Indicator NaH2PO4 NaHPO4
2 1 5 0
When a monobasic indicator (e.g. Methyl Red) is 3 1 5 1
prepared in an aqueous solution, the indicator 4 1 5 5
dissociates according to the equation 5 1 10 5
6 1 5 10
HIn + H2O = H3O+ + In- 7 1 1 5
8 1 1 10
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1
 9 1 0 5 shown in the Appendix). The data set produced a pKa
value of 6.015, which has a relative error of 17.94%
Results and Discussion compared to the true value from literature which is 5.05
at around 298 Kelvin.
The aim of this experiment is to obtain a value for the
equilibrium constant of an indicator in water. For the Conclusion
experiment, two data sets were used. The
absorbencies A1, for the acidic solution, and A2 for the This experiment successfully facilitated an
basic solution were determined experimentally. understanding of spectrophotometric analysis
techniques. The use of the two equations for the
Using Microsoft Excel software, the following calculation of the absorbencies was useful and
absorption spectrum was prepared: effective in determining the concentration of the acid
and base forms of the indicator with good accuracy.
Figure 1. Absorption Spectrum of the Solutions The aim of the experiment is the quantitative
determination of the acid-dissociation constant of the
40 indicator, methyl red using spectrophotometric
515
510520 analysis. A source of error could be the resolution of
30 470
490
485
480
475 495
505 525
500 the spectrophotometer which is limited by the
465
460 sensitivity of the instrument at a certain wavelength.
20
455
Another source of error is the lack of experience of the
450
445
researchers in handling the spectrophotometer and the
10 440 computer software bundled with it.
435
430
425
0 365
370 410
420
415 If statistical methods are available to the researchers,
375
380 405
400
365
385 395
390
415 465 515
the standard deviations can be computed in testing the
reliability of the values. If the calculated value for the
-10
standard deviations is small, it can be said that the
Wavelength in nm values produced are close to one another. Hence, the
precision of the method of analysis is concluded. Other
Upon investigation, it was determined that the methods for the determination of the acid-dissociation
minimum and maximum wavelengths are 385 and 515 constant of methyl red could also be used. One such
nanometres, respectively. The U-5100 method that could be employed is potentiometric
Spectrophotometer was again used to measure the titration. In colored solutions, this type of analysis
absorbance at the maxima and minima of the plot in provides more reliable data than titrations using
Figure 1. indicators.
Table 2. Tabulated Data References
Solution pH Absorbance Absorbance
No. measured at λ1, A1 at λ2, A2
[1] Schmidt, W. (2005). Optical spectroscopy in
1 1.00 4.624 5.341
2 4.86 6.66 27.003
chemistry and life sciences: An introduction.
3 6.33 21.792 6.344
Wiley.
4 7.20 17.045 2.967 [2] Baptista, M. (n.d.). History of infrared
5 7.04 14.286 2.602 spectroscopy. Retrieved from
6 7.65 15.176 2.421 http://www.quimica3d.com/EN/IR/history.phpP
7 8.03 26.673 3.608 ayumo, E. M. and Castillo, E. S. Phil. J. Nutr.
8 8.16 20.188 2.728 37(2), (1979), p82
9 10.54 30.209 4.428 [3] Vanasse, G.A. Spectrometric Techniques,
10 13 5.968 0.938 (Academic Press, 1984), Vol. 3, p. 104.

Using the two equations for absorbencies discussed

earlier, the absorbancies value can be computed (as

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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

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−] eq. [4]

A2 = a2,HIn [HIn] + a2, In− [In−] 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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