SPECTROPHOTOMETRIC DETERMINATION OF THE ACID

DISSOCIATION CONSTANT OF METHYL RED

G.M.L. CHUNGUNCO1 AND J.R.N. GENTOLEA2

1
DEPARTMENT OF MINING, METALLURGICAL, AND MATERIALS ENGINEERING, COLLEGE OF ENGINEERING
2
DEPARTMENT OF CHEMICAL ENGINEERING, COLLEGE OF ENGINEERING
UNIVERSITY OF THE PHILIPPINES, DILIMAN, QUEZON CITY, PHILIPPINES
DATE PERFORMED: NOVEMBER 9, 2017
INSTRUCTOR’S NAME: MELLA, L.

1. Discuss the application of Beer’s law on baseline corrected. Thus, Beer’s law can be
the analysis of a multi-component system. modified to give total absorbance A as

If all analytes in the system absorb in A λx =∑ A n , λ =∑ ε n , λ b c n

x x
the range of the spectrophotometer with (3)
minimal interference from other absorbing
species in the solvent, spectrophotometry is Thus, the total absorbance of all
still a valid method of analysis to use since the
species at the λ max of species x is equal to the
wavelength at peak absorbance λ max is
summation of all individual absorbances of
characteristic to a species. Therefore, every
other components n at that same λ max of
component will have its own λ max; however,
species x1. This in turn is equal to the sum of
at λ max of one component, there exists some the product of each component’s molar
degree of interference from other absorbing absorptivity at λ max of species x, the constant
species. Beer’s law, expressed as path length of the cuvette, and the
concentration of the component n. In other
A=abc words, total absorbance at a certain
(1) wavelength is just the sum of individual
absorbances at that same wavelength.
where A is absorbance, a is absorptivity of a
species at a certain wavelength, b is the path Equation 3 must be replicated for
length of the cuvette, and c is concentration. every component in the system; a system of
For our purposes, we will express equation 1 equations would be created with number of
as equations equal to the number of n
components. Thus, for a two-component
A=εbc system,
(2)
A λx =ε x , λ b c x + ε y , λ b c y
where ε is molar absorptivity in M -1cm-1 and c x x

(4)
is concentration expressed in mol/L. Beer’s
law as it is can only be used for one
A λy =ε x , λ b c x + ε y , λ b c y
y y
(5)
component at a time; however, calibration
curves that follow the linearity of Beer’s law If either molar absorptivities or
can be combined based on the concept that concentrations of each component are known
absorbance values A read by a (although the latter is the usual case in multi-
spectrophotometer are inclusive of all component analysis due to the presence of
absorbing species not removed, zeroed, or standard solutions), the other unknown

Page 1 of 7
quantity can be calculated by solving the same would then be done for the three
system of equations. solutions with A-.

2. How do we determine the molar 3. Explain the importance of using

absorptivity of each component in a multi- matched cells in the experiment.
component system?
First, λ max for each component must Even when made by the same
be determined via spectral scanning using manufacturer, different cells have slight
solutions containing only that component. deviations in size, thickness, material, and
optical properties such as transparency or
Molar absorptivity must then be refractive index, all of which are unaccounted
determined from standard solutions of for or impossible to detect when used in an
known analyte concentration. More experiment. These could in turn cause
specifically, each standard solution should absorbance errors due to differences in path
contain only one component among the length and transmittance due to the amount
analytes (ensured by addition of a reagent of light manipulated (scattered, absorbed,
that prevents analyte reaction, such as a reflected, refracted, etc.) by the cuvette itself.
strong acid added to a weak acid analyte), and Using matched cells allows all absorbance
each component should be present in more errors of a particular cuvette to be excluded
than two solutions in order to construct a via baseline correction or auto-zero; however,
calibration curve (two points cannot be used using another cell after correction contributes
to form a realistically linear relationship since errors again.
they always form a perfect line). Thus, there
should be three isolated solutions of species Mismatched cells cause greater y-
x, three of only species y, etc. Each solution intercepts to appear in the absorbance vs.
must be run through a spectrophotometer at concentration curves, thus turning equation 2
the λ max for each component to obtain the into
total absorbance at the λ max of each
A=εbc +k
component.
(6)
Calibration curves should be
where k is some y-intercept1. In essence, a
constructed for solutions containing the same
blank solution can still exhibit absorbance
analyte, but there should be one absorption
due to the optical activity of a different cell
spectrum per wavelength to isolate the
unaccounted for in previous calibrations.
absorbance values. Thus, each component
Note that even when using the same cell, a y-
should give n molar absorptivities at n
intercept can appear from repeated handling,
wavelengths.
though these errors are smaller and more
random in nature.
For example, in a two-component
system, if three standard solutions contain
Using the same cell also makes
varying concentrations of only HA, a
computations easier, since all values of b in
calibration curve following equation 2 should
equations 3 and 4 are equal and can be
be made for the absorbance of HA at λ HA and
cancelled out when the system is solved.
another should be made for absorbance of HA Attempting to cancel out path lengths of
at λ A ¿ The slope of the first equation divided
−¿.
different actual values will cause errors. A
by the path length is the molar absorptivity of longer path length increases individual
HA at λ HA while that of the second equation is absorbance while a shorter one decreases
the molar absorptivity of HA at λ A .¿ The
−¿ absorbance.

Page 2 of 7
4. Why is it right for the HMR solution to [HMR]. log ¿ ¿ increases, which raises the pH
have a pH of approximately 2, and for the above pKa 5.05. pH should be approximately
MR- solution to have a pH of 8 to exhibit the yellow color with a noticeable
approximately 8? λ max difference from that of the pink HMR.
Only a weak basic solution was prepared
The HMR solution contains HMR and because methyl red is still yellow in highly
HCl, which is a strong acid. Since the HCl basic solution; increasing [MR-] would
dissociates readily, the solution has a high increase the absorbance of the solution at
concentration of H+. This prevents the HMR roughly the same wavelength. Also,
from dissociating into H+ and MR- by the spectrophotometry is most effective for dilute
following acid dissociation reaction solutions since changes in light phenomena
−¿¿
caused by refractive index and in electrostatic
HMR+ H 2 O ⇌ H 3 O +¿+MR ¿
attractions are minimized1. With a higher
(7) absorbance curve, there is greater
interference of MR- at the HMR curve. Below
by shifting the equilibrium backwards. Since are the absorbance spectra taken for various
both a weak and a strong acid are present in pH of methyl red solution.
solution, the pH depends mostly on the H +
from the strong acid HCl. The solution
contains 0.01 M HCl after dilution, which
gives a pH of approximately 2. Additionally,
by the Henderson-Hasselbalch equation,

pH= pKa+log ¿ ¿
(8)

as log ¿ ¿ decreases, pH decreases. Since

[MR-] is very low while [HMR] is Figure 1. Absorbance spectra of methyl red
predominant in solution, the pH would be at various pH2
much lower than pKa 5.05. The pH of HMR
solution must be 2 to exhibit the pink color Notice how much more interference
necessary for getting an accurate there is between the pH 2.0 curve and the pH
experimental pKa. An HMR solution with 13.1 curve than there is between the pH 2.0
higher acidic pH is red, which would absorb curve and the pH 8.5 curve despite the two
light at a smaller wavelength (dark green basic curves having the same peak
instead of light green) and give a lower λ max wavelength. As discussed, greater
value for HMR. At this wavelength, there is interference causes greater chance of random
greater interference from MR-, which is more error. Calculating the estimated pH from
susceptible to errors due to different known values gives a pH of approximately
instantaneous equilibrium from the dynamic 7.659, considering only the MR- concentration
equilibrium of equation 7. and assuming zero HMR concentration. Since
some acetate would not be hydrogenated, the
The MR- solution contains pH would further increase to a value near 8
NaCH3COO, a salt of a strong base and weak since the estimated pH from just the
acid that ionizes totally into Na + and CH3COO-,
a weak base that forms a weak basic solution
by decreasing H+ concentration in the
solution, forcing the equilibrium in equation 7
forward to increase [MR-] and decrease

Page 3 of 7
concentration of acetate is around 8.375. 6. Why do we measure the pH of the
Thus, a value in between is to be expected. solutions 7 to 10?

Figure 2. Individual pH calculations for MR- The goal of the experiment is to

and CH3COO- determine the pKa of methyl red. Measuring
5. Correlate the wavelengths of maximum the pH allows a linear plot to be made of pH
absorption of HMR and MR- to their colors. against the logarithm of the ratio of the
concentrations of the base form to the acid
The coloration of a species depends form, following the Henderson-Hasselbalch
on the presence of unpaired electrons. When equation (equation 7), which relates pH to
electrons absorb energy, they move to a concentration.
higher level farther away from the nucleus.
To stabilize to a preferable state, the The y-intercept of the resulting graph
electrons move to a lower level and release a corresponds to the experimental pKa value of
packet of quantized energy in the process. In methyl red. Only the unknown solutions, 7-
organic compounds like methyl red, electron 10, need pH readings, since none of the
movement from HOMO (highest occupied standard solutions contained both species at
molecular orbital) to LUMO (lowest any given time. Only the unknown solutions
unoccupied molecular orbital) is responsible had a mixture of both HMR and MR-; thus,
for the color. equation 6 could only be used for the pKa
determination of methyl red in the unknown
Because humans observe the trials.
complementary color to the absorbed
wavelength, the maximum wavelength must 7. Account for the deviation or closeness of
be conjugate to the wavelength of the the experimental acid dissociation
observed color. True enough, HMR is purple- constant of methyl red to its literature
pink in solution; it absorbs at around 521 nm, value. What factors might cause the
which corresponds to green light in the discrepancy between the two values?
visible spectrum. Green is the complement of
purple. On the other hand, MR- is yellow in The experimental pKa was found to
solution and absorbs at around 410-428 nm, be 4.63 (Ka=2.36x10-5); the literature pKa
which corresponds to violet light. Violet is the value is 5.05. A negative error of 8.38% was
complement of yellow. Note that maximum obtained. Negative errors could be traced to
absorption wavelengths are considered here various factors.
because they exhibit the highest
independence to concentration and are also For the pKa value to be low, the pH vs.
the only part of an absorbance spectrum that log ¿ ¿ graph must have a smaller y-intercept.
is characteristic to a specific species. For our purposes, we will assume that the pH
meter is correctly calibrated and that all
Figure 3. Minimization of Beer’s Law error by solutions were zeroed or baseline corrected
taking absorbance values at λ max (A) instead (since they were, experimentally). A smaller
of other wavelengths (B)1 y-intercept occurs when the slope of the
graph,log ¿ ¿, is steeper, indicating computed
values for [MR-] are too high, or computed
[HMR] values are too low, for all measured
pH. As ¿ ¿ increases, log ¿ ¿ increases, which
causes a steeper slope that will intersect the
y-axis at a lower point, giving a lower pKa
value.

Page 4 of 7
 For the computed values to end up the same degree in all trials, since the slope
like this, there are several possible factors. depends on the change of values. Other
First, the computed molar absorptivity of MR - random errors, such as the introduction of
has positive error. By equation 2, at constant light-blocking or light-scattering particles in
A, an increase in calculated molar solution or the spectrophotometer itself,
absorptivity indicates a decrease in could cause deviations in the pKa value as
concentration. However, since the well.
concentrations of all standard solutions are
set, calculated molar absorptivity increases Similarly, if the absorbances were not
when absorbance increases. This could arise read at the true λ max values (due to
from the introduction of absorbing or light- interferences of other absorbing or scattering
manipulating species when the cuvette was species), a greater chance of error is possible
taken out for washing or filling, such as (see figure 3). This is true especially for a
bubbles, which can scatter light away from multi-component system. The theoretical λ max
the detector. Dirt and dust in solution could values for HMR and MR- are, respectively, 520
absorb or block light, which increases nm and 435 nm3. Experimental values were
absorbance by increasing the difference 521 nm and 428 nm, respectively.
between transmitted and incident power.
Reading absorbance values at 428 nm
Second, the concentration of HMR has decreases the absorbance of HMR at that
negative error. Light-scattering species wavelength (recall that HMR absorbs at a
introduced after baseline correction can higher wavelength than MR-). This would
scatter light towards the detector, lessening lower the absorbance values for HMR, which
the difference between transmitted and would in turn increase both cHMR and cMR- in
incident power. equation 4, albeit disproportionately due to
the much greater molar absorptivity of HMR
More noticeably, in solutions with at 521 nm. If equation 5 remains the same,
only one species of methyl red, a small MR- would have a higher concentration than
amount of the other is computed. This HMR, causing negative error to the pKa value.
actually happened in the experiment.
Dynamic equilibrium causes methyl red to 8. Discuss possible sources of errors and
shift between HMR and MR- even in solutions their effect on the calculated parameters.
of controlled pH.
Aside from those discussed in the
In the HMR solutions, higher previous numbers, other sources of errors
absorbance of MR- caused higher computed (primarily random) are possible.
molar absorptivity of HMR at MR- wavelength
since the concentrations were taken Dynamic equilibrium constantly
considering only HMR. Likewise, the presence changes the constituent concentrations in a
of HMR in MR- solutions caused higher molar system; thus, taking absorbance
absorptivity of MR- at HMR wavelength; measurements multiple times gives different
however, as seen in the experiment, the instantaneous absorbances of a certain
presence of HMR in the MR- solutions was species. HMR and MR- undergo constant
much lower than the MR- in HMR solutions. changes via acid-base equilibria. In
This situation lowered the computed c HMR standardization, higher instantaneous
(from equation 5) to a greater degree than it concentration of a species increases
increased computed cMR- (from equation 4). absorbance at that moment, which in turn
All of these errors, however, may not decreases molar absorptivity. In unknown
induce a negative overall error if they are analysis, if absorbance differences result in
inconsistent between trials or if they occur in

Page 5 of 7
higher MR- and lower HMR, the pKa has Aqueous Solutions: TD-DFT Calculation
negative error. and Experiment Study. Acta Physico-
Chimica Sinica. [Online] 2016, 32 (1),
Not zeroing the spectrophotometer 290-300.
with a blank, performing baseline correction http://www.whxb.pku.edu.cn/full/WHX
before every measurement, or rinsing the B20160124_EN.htm (accessed Nov 12,
cuvette will most likely cause positive error 2017).
from additional absorbing species in the
solvent that were not eliminated. Not baseline [3] Zhang, J.H.; Liu, Q.; Chen, Y.M.; Xu, C.W.;
correcting also produces a different λ max, Determination of Acid Dissociation
which would give the absorbance of the other Constant of Methyl Red by Multi-Peaks
species at the wrong wavelength4. Gaussian Fitting Method Based on UV-
Visible Absorption Spectrum (abstract).
Not calibrating the pH meter could Acta Physico-Chimica Sinica. [Online]
cause either positive or negative errors to the 2012, 68.
pH readings. This systematic error shifts the https://www.researchgate.net/publicati
Henderson-Hasselbalch graph up or down. on/263232302_Determination_of_Acid_D
Shifting it up (i.e. all pH readings increase by issociation_Constant_of_Methyl_Red_by_
the same increment) increases pKa; shifting it Multi-
down (i.e. all pH readings decrease by the Peaks_Gaussian_Fitting_Method_Based_o
same increment) decreases pKa. n_UV-Visible_Absorption_Spectrum
(accessed Nov 12, 2017).
Finally, not ensuring that the HMR
solution has a pH of 2 would increase MR- [4] Shimadzu. UV-1800 UV-Vis
concentration in the solution; spectrum Spectrophotometer.
scanning would give a lower λ max value in the http://www.shimadzu.com/an/molecular
dark green region (instead of light green) that _spectro/uv/uv1800/uv2.html (accessed
is not exclusive to either HMR or MR-. HMR Nov 12, 2017).
absorbance at this wavelength is lower while
MR- absorbance is higher. The same happens
for an MR- solution not at pH 7, except with a
higher λ max value in the blue region (instead
of violet). HMR absorbance at this
wavelength is higher while MR- is lower.

REFERENCES

[1] Skoog, D.A.; West, D.M.; Holler, F.J.;

Crouch, S.R. Fundamentals of Analytical
Chemistry, 9th ed.; Brooks/Cole: USA,
2014; pp 663-673

[2] Zheng, D.; Yuan, X.A.; Ma, J.;

Rationalization of pH-Dependent
Absorption Spectrum of o-Methyl Red in

Page 6 of 7
APPENDIX

0.25

0.2 f(x) = 16653.63 x − 0.02

R² = 0.98
Absorbance

0.15
λ HMR
0.1 Linear (λ HMR)
λ MR-
0.05 Linear (λ MR-)
f(x) = 4532.32 x − 0.02
0 R² = 0.78
0 0 0 0 0 0 0
Molarity of Solution (M)

Figure 4. Calibration curves of HMR

0.08
0.07
0.06 f(x) = 4637.72 x + 0
Absorbance

0.05 R² = 1
0.04 λ HMR
0.03 Linear (λ HMR)
0.02 λ MR-
0.01 Linear (λ MR-)
0 f(x) = 421.61 x − 0
0 R²0= 1 0 0 0 0 0
Molarity of Solution (M)

Figure 5. Calibration curves of MR-

7
6 f(x) = 1.42 x + 4.63
R² = 0.86
5
4
pH

3
2
1
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
log([MR-]/[HMR])

Figure 6. pH vs. log([MR-]/[HMR])

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