ACID DISSOCIATION CONSTANT OF AN

INDICATOR DYE
by
Clarissa A. Gomez
CHE121L

ABSTRACT

Methyl red is the indicator dye used in this experiment. The maximum absorbance
obtained in its acidic form is 520 nm while 420 nm in its basic form. In the determination of
molar absorptivities using 10, 25 and 40 ml aliquots; the absorbance increases both in the acidic
and basic solution while the volume of aliquots escalates. Buffer solutions were prepared using
the Beer’s and Henderson-Hasselbalch’s equation. The isosbestic point appears at near 420 nm
which means that there is only one wavelength where two species have the same molar
absorptivity. By plotting the pH vs. absorbance of the prepared buffer solutions at two
maximum absorbance (acidic and basic), it was deduced that the spectrum of each solution is
dependent on the pH range. The obtained pka of methyl red using these data should be
compared to the theoretical pKa of methyl red which is 5.05 ± 0.05. However, this is not
achieved due to data deficiency.
INTRODUCTION
Indicators are weak organic acids (HIn)
that change color when deprotonated (In-). A
few drops of indicator added to the analyte
solution before the beginning an acid-base
titration. When enough base titrant is added to
the analyte solution the equilibrium expressed
in equation 1 will shift toward products.
HIn(aq) In– (aq) + H+(aq) (1)
The result is the formation of more of the
deprotonated indicator (In ) and a
corresponding color change of the analyte
solution (the endpoint). A good indicator for a
specific acid-base titration has an endpoint with
a pH at or near the pH of the equivalence point.
In this experiment, phenolphthalein indicator
will be used for each titration. The pH range of
the color change will be observed and
compared with the pH of the equivalence point
to determine if the indicator is an appropriate
choice for each titration. The acid dissociation
constant, Ka, is a measure of an acid’s strength.
For weak acids these values are less than 1 and
typically so small that they are expressed with
scientific notation. Taking the negative log of
the Ka results in more easily expressed pKa
values ranging from 0 to 14 for weak acids.
A spectrophotometer separates light into its
separate colors. It is able to separate the light
into colors because each color of light has a
different wavelength than the other colors. The
spectrophotometer can shine a narrow band of
wavelengths (essentially one specific color) of
light on a sample of substance and then
measure how much of that light is absorbed by
the sample. Different colored substances
absorb varying amounts of specific wavelengths
of light. Therefore, a spectrophotometer can be
Used to measure how much of a substance is
present. A calibration curve is a graph of
absorbance, how much of a particular
wavelength of light is absorbed, versus
concentration (Beer’s law). A calibration curve
is specific to a particular substance, and must
be created by measuring the absorbance of a
large number of solutions of known
concentration.
An acid is a substance which dissociates
+
to produce hydrogen ions, H , when dissolved in
+
aqueous solution. Once in solution, the H ion,
which is simply a proton, immediately combines
+
with water to form the hydronium ion, H O . So
3
+
when aqueous H appears in a chemical
equation, it is understood that the actual
+
species exists as H O . An acid is classified as
3
strong or weak depending on the extent to
which the molecules of the acid dissociate into
+ −
H and its anion, A . A strong acid dissociates
completely in water (e.g., HCl dissociates
+ −
virtually100% into H and its anion, Cl ), while a
weak acid dissociates only partially and forms
+
very little H (e.g., only about 3% of the
dissolved molecules of acetic acid, CH COOH,
3
+
dissociate into H and the acetate anion,
−
CH COO ). Weak acid dissociation can be
3
represented as a generic reversible reaction:
+ –
HA(aq) ⇔ H + A where HA is the weak acid
-
and A is the anion, or conjugate base, of HA.
For this reversible reaction, equilibrium
constant can be defined:
(2)
The equilibrium constant is called the acid
dissociation constant or acid ionization
constant. If the undissociated acid (HA) is
favored and the acid is weak, K is measurable
a
and will be much less than one. For strong
+ −
acids, the dissociated products, (H and A ) are
so strongly favored, that [HA] approaches zero
and K approaches infinity. Thus strong acids, of
a
which there are very few, do not have values of
K associated with them. Mathematically, it is
a
found that the amount of light absorbed by a
specific sample depends on three things: 1) the
concentration of the solution; 2) the distance
the light travels through the sample; and 3) the
natural ability of the specific substance to
absorb light. Thus an equation can be written
relating these things:
A=bc (3)
where A = absorbance
 = molar absorbtivity -- how well the
material absorbs light
b = path length through which the light
passes
c = concentration of the solution
In general, the value for b will remain the same
and the value for  is constant for a specific
chemical at a given wavelength of light.
Because the general equation for a straight line
is
y = mx + b (4)
If A is graphed against c, the result will be a
straight line with a slope of b and a y-intercept
of zero. A solution that contains two colored
species can also be analyzed using Beer’s Law
(A= bc). Mathematically, the concentrations
of the two species can be determined if two
simultaneous equations can be developed that
relate the two. The two unknown
concentrations are determined by taking
readings on each solution twice, using two
different wavelengths of light. The two species
that are being studied in this experiment are
the two colored forms of specific indicators that
are weak acids. If there are two species, HIn
and In, in a solution with absorbance AHIn and
AIn, respectively, the total absorbance is A = AHIn
+ AIn . If the sample path length is combined
with, then  = b and Beer’s Law for a two
component mixture becomes
A1 = A1HIn + A1In = 1Hin [Hin] + 1In [In]
at 1 (5)
The constant  does not change with
concentration, but will change at different
wavelengths and/or with a different absorbing
species. So, at a second wavelength 2, the
following equation for Beer’s Law would be
true:
A2 = A2HIn + A2In = 2HIn [HIn] + 2In [In]
at 2 (6)
There are now two equations that can be used
to determine the concentrations of the two
different colored unknowns that are present in
the solution. These equations can be
rearranged to allow determination of each
concentration.
[HIn] = ((A12In)  ( A21In)) / ((2In 1HIn) 
(1In 2HIn)) (7)
[In ] = ((A2 1HIn) – ( A1 2HIn)) / ((2In 1HIn) –
(1In 2HIn)) (8)
The best wavelengths for the experiment are
selected by measuring the absorbance vs.
wavelength for each of the pure substances.
The actual wavelengths are then chosen such
that they will maximize the absorbance for each
species. The values for the four  constants
can be determined by doing Beer’s Law plots of
absorbance vs. concentration (of pure samples)
at both wavelengths and determining the slopes
of the lines generated. The final set of
measurements will be collected by measuring
the absorbance of solutions of the weak acid at
different pH’s.
EXPERIMENTAL MEHOD

To obtain the spectra of acid and base forms of the dye, first these two forms were prepared.
For the acidic solution of the dye, 0.05M HCl was used. While, 0.05M borax was utilized solution for the
basic solution. Then the maximum absorbances of the solutions were determined at 20-nm intervals
within the 340 – 625 nm wavelength range. In order to identify the molar absorptivities dilute solutions
were prepared. Through obtaining 10, 25, and 40 mL aliquots of the solution these were diluted in a 50
mL volumetric flasks using 0.01M HCl for the acidic solution while 0.01M borax solution for dilution of
basic solution. The absorbances of the six solutions were measured at the wavelengths of maximum
absorption, λHIn and the λIn . After this in order to recognize the pKa of the dye using Henderson-
Hasselbalch equation, five solutions at various pH values but with constant total dye concentration were
prepared. It was followed by obtaining 10 mL of the original dye solution and placing it into a 100 mL
volumetric flask. Then it was diluted to the mark with the buffer solution. Afterwards, their
absorbanceswere obtained at λHIn and the λIn-.
RESULTS

Table 1. Wavelength and Maximum Absorption of Acidic Dye and Basic Dye Solution

(With 20 nm intervals)

λ (nm) Acidic Dye Solution Basic Dye Solution

380 - 0.034
400 0.003 0.045
420 0.008 0.050
440 0.019 0.048
460 0.042 0.042
480 0.077 0.027
500 0.110 0.011
520 0.129 0.001
540 0.118 -0.003
560 0.075 -0.004
580 0.021 -0.004
600 0.004 -0.003
620 0.001 -0.003
625 0.001 -0.003

Maximum Wavelength of Absorption for Acidic Dye Solution: 520 nm

Maximum Wavelength of Absorption for Basic Dye Solution: 420 nm

Table 2A. Wavelengths and Absorbance 2A. Wavelengths and Absorbance

of Acidic DyeTable of Basic Dye

Acid Dye Solution Basic Dye Solution

λ (nm) Absorbance λ (nm) Absorbance
505 0.119 380 0.034
510 0.126 385 0.037
515 0.128 390 0.040
520 0.129 395 0.044
525 0.128 400 0.045
530 0.126 405 0.046
535 0.123 410 0.048
540 0.118 415 0.050
420 0.050
Table 3A. Determination of Molar Table 3B. Determination of Molar

Absorptivities Absorptivities

Acidic Dye Solution Basic Dye Solution

λ (nm) 10 mL 25 mL 45 mL λ (nm) 10 mL 25 mL 40 mL
520 0.027 0.067 0.107 420 0.011 0.026 0.043

Figure 1. Wavelength vs. Absorbance graph (Acidic Dye Solution)

Absorbance of the acidic solution

0.14
0.12

0.1
Absorbance

0.08

0.06
0.04

0.02
0
0 100 200 300 400 500 600 700
Wavelength

Figure 2. Wavelength vs. Absorbance graph (Basic Dye Solution)

Absorbance of the basic solution

0.06

0.05

0.04
Absorbance

0.03

0.02

0.01

0
0 100 200 300 400 500 600 700
-0.01
Wavelength
Figure 3. Wavelength vs. Absorbance graph (Acidic and Basic Dye Solution)

0.14

0.12

0.1

0.08
Absorbance

0.06 Basic

0.04
Acidic
0.02

0
380 400 420 440 460 480 500 520 540 560 580 600 620 625
-0.02 Wavelength

Table 4A. pH vs. Absorbance At maximum Table 4B. pH vs. Absorbance At maximum

Absorbance of Acidic Form Absorbance of Basic Form

0.06
0.12

0.05
0.1

0.04
0.08
Absorbance
Absorbance

0.03
0.06

0.02
0.04

0.01
0.02

0
0
4.4 4.8 5.6 6 6.4
4.4 4.8 5.6 6 6.4
pH
pH
Figure 4. Absorbance of acid and basic form of methyl red at two wavelengths
DISCUSSION
After the preparation of the acidic and
basic solutions, their wavelength at maximum
absorbance. Based on the data in Table 1, the
maximum wavelength of absorption for acidic
dye solution is 520 nm while 420 nm for the
basic solution. The wavelength at which the
absorbance is greatest needs to be determined
for the reason that the spectrophotometer is
more sensitive to absorbance changes at this
wavelength. Although a substance will have an
extinction coefficient at every wavelength,
concentrations are typically measured at
maxima in the absorbance spectra because this
is where the absorbance changes least with
changes in wavelength. From Figure 2 and 3,
the Wavelength vs. Absorbance graph of acidic
dye solution tends to lean on the left due to the
large presence of acidic forms while in the
Wavelength vs. Absorbance graph of basic dye
solution tends to lean on the left ro to the large
presence of its basic forms.
In the case of the dilute solutions, their
molar absorptivies were obtained by using the
maximum absorbance of the acidic and basic
dye solution, which is 520 and 420 nm
respectively. From the data gathered in Table
3A and 3B, one can see that the absorptivities
of the dilute solutions by using the maximum
absorbance in acidic solutions are higher than
the basic solution. This can be explained
through Beer’s law from equation 3. Based in
this, the molar absorptivity is directly
proportional to the absorbance. The capacity of
the material to absorb light or its absorptivity
increases as its absorbance increases as well.
The Henderson-Hasselbalch equation
[ ]
which is [ ]
was used in
preparing the buffer solutions. Using the
of acetic which is 4.7, buffer solutions with pH
4.4, 4.8, 5.6, and 6.4 were prepared. The ratio
[In–]/[HIn] at different pH values and at
constant total dye concentration of 0.1 M were
determined first, then from this the moles of
the acid which is acetic acid and the base which
is sodium acetate can be determined as well. In
order to be prepared, their volumes must be
known by using the equation for molarity,
which is moles/Liter. However, after calculating
the appropriate volumes for the different pH, it
was found out that the amount of volumes
were very small making it very difficult for the
acid and the base to be pipette. So instead
preparing the buffer solutions by volume, it was
prepared by mass with the help of analytical
balance and diluting both the acid and the base
to 250 ml with distilled water.
Methyl red (4-dimethylaminobenzene-
2’-carboxylic acid) is a commonly used indicator
for acid-base titrations. By following the change
in absorbance as a function of pH of the
prepared buffer solutions we will determine the
acid dissociation constant, or pKa. In this
experiment we will determine this equilibrium
constant, pKa', by varying the pH and measuring
the ratio [In–]/[HIn . We will use acetic acid-
acetate buffers to control the pH, since the Ka
value for acetic acid is in the same range as the
Ka' value for methyl red. The pH of these
buffers force methyl red to distribute itself
somewhat evenly between the two colored
forms. The absorption of light is governed by
the Beer-Lambert Law. The absorbance of
mixtures is the sum of the separate
absorbencies.
The best wavelengths to choose for the analysis
are where one form absorbs strongly which is
the maximum absorbance. We need to set up
two equations in two unknowns, one equation
for each wavelength. Call the two wavelengths
and . The two measurements then
provide two simultaneous equations with two
unknowns:
[ ] [ ]
[ ] [ ]
The molar absorbance coefficients are
determined from standard solutions that
contain one component alone. This equations
provide two equations in two unknowns. For an
unknown solution, the absorbances at the two
wavelengths, and , are determined
and then the two equations are solved for the
unknown concentrations [ ] and [ ] at
each given pH. Methyl red has a pKa of 5.1
An isosbestic point is defined as the wavelength
where two species have the same molar
absorptivity. At the isosbestic point the total
absorbance of a solution of the two ions is
independent of their relative concentrations
but is dependent only upon the total dye
concentration. The appearance of an isosbestic
point is evidence that only two species are
involved. In this experiment from Figure 3, the
isosbestic point is near 420 nm.
By looking at Table 4A, one can see that the
absorbance decreases as the pH increases using
the maximum absorbance at acidic conditions.
While in Table 4B, the absorbance increases as
the pH increases using the maximum
absorbance at basic conditions. Equilibrium
constants involving ionic species are especially
sensitive to ionic strength. The
ionic strength is a measure of the total ion
concentration in solution. The activity of all the
species in solution are a function of the ionic
strength. The spectral changes are explained in
terms of shifts in equilibria between different
molecular and ionic species in the solutions.
Spectrophotometry involves the use of a
spectrophotometer. A spectrophotometer is a
photometer (a device for measuring light
intensity) that can measure intensity as a
function of the color, or more specifically, the
wavelength of light. UV/Vis spectroscopy is
routinely used in the quantitative
determination of solutions of transition metals
and highly conjugated organic compounds.
Spectrophotometers are most commonly used
for the measurement of transmittance or
reflectance of a solution or transparent
material, like polished glass. However they can
also be designed to measure the diffusivity on
any of the listed light ranges that usually cover
around 250nm - 2500nm using different
controls and calibrations. Within these ranges
of light, calibrations are needed on the machine
using standards that vary in type depending on
the wavelength of the photometric
determination.
Historically, spectrophotometers use a
monochromator containing a diffraction grating
to produce the analytical spectrum. There are
also spectrophotometers that use arrays of
photosensors. Especially for infrared
spectrophotometers, there are
spectrophotometers that use a Fourier
transform technique to acquire the spectral
information quicker in a technique called
Fourier Transform InfraRed.
The spectrophotometer quantitatively
compares the fraction of light that passes
through a reference solution and a test
solution. Light from the source lamp is passed
through a monochromator, which diffracts the
light into a "rainbow" of wavelengths and
outputs narrow bandwidths of this diffracted
spectrum. Discrete frequencies are transmitted
through the test sample. Then the photon flux
density (watts per metre squared usually) of the
transmitted light is measured with a
photodiode or other light sensor, and the
transmittance value for this wavelength is then
compared with the transmission through a
reference sample.t
In short, the sequence of events in a
spectrophotometer is as follows:
1. The light source shines through a
monochromator.
2. An output wavelength is selected and
beamed at the sample.
3. A fraction of the monochromatic light is
transmitted through the sample and to
the photodetector.
Many spectrophotometers must be calibrated
by a procedure known as "zeroing." The
absorbancy of a reference substance is set as a
baseline value, so the absorbancies of all other
substances are recorded relative to the initial
"zeroed" substance.
The spectrophotometer then displays %
absorbancy (the amount of light absorbed
relative to the initial substance).
Beer's law relates the absorbance, the
concentration of the absorbing species and the
path length, such that:
A = εbc
where A is the absorbance, ε, is the extinction
coefficient, b is the path length (normally 1 cm)
and c is the concentration of the absorbing
species. Beer's law applies to solutions
containing one or more absorbing species, if
there is no interaction between the various
species in the solution. In the case of a solution
containing n species which absorb, the above
equation becomes:
Atot = A1+A2+...+An = ε1bc1 + ε2bc2 + ...+
εnbcn
Beer's law in the case of a fixed path length, b,
and extinction coefficient, ε, is a linear
relationship between absorbance and the
concentration This is not generally the case.
Beer's law is successful in describing the
absorption behavior of dilute solutions. One of
the fundamental assumptions in the derivation
of the law is that the average distance between
atoms is large enough such that the charge
distributions of neighboring atoms or molecules
are not affected by those of its neighbors. This
can alter a species' ability to absorb a given
wavelength of radiation. This causes a
deviation from the linear relationship because
the extent of interaction depends on
concentration. A similar situation can occur
when the concentration of the absorbing
species is low compared with the
concentrations of other species. The effects of
these molecular interactions become negligible
at concentrations below 0.01M. Dilute solutions
must therefore be used.
The linearity of the Beer-Lambert law is limited
by chemical and instrumental factors. Causes of
nonlinearity include:
 deviations in absorptivity coefficients at
high concentrations (>0.01M) due to
electrostatic interactions between
molecules in close proximity
 scattering of light due to particulates in
the sample
 fluoresecence or phosphorescence of
the sample
 changes in refractive index at high
analyte concentration
 shifts in chemical equilibria as a
function of concentration
 non-monochromatic radiation,
deviations can be minimized by using a
relatively flat part of the absorption
spectrum such as the maximum of an
absorption band
 stray light
When an isosbestic plot is constructed by the
superposition of the absorption spectra of two
species (whether by using molar absorptivity for
the representation, or by using absorbance and
keeping the same molar concentration for both
species), the isosbestic point corresponds to a
wavelength at which these spectra cross each
other.
A pair of substances can have several isosbestic
points in their spectra.
When a 1-to-1 (one mole of reactant gives one
mole of product) chemical reaction (including
equilibria) involves a pair of substances with an
isosbestic point, the absorbance of the reaction
mixture at this wavelength remains invariant,
regardless of the extent of reaction (or the
position of the chemical equilibrium). This
occurs because the two substances absorb light
of that specific wavelength to the same extent,
and the analytical concentration remains
constant.
An isosbestic point is observed in overlaid
spectra when a chromophoric precursor is
converted to a product with a different
spectrum, so that it is often assumed that an
isosbestic point occurs only when the precursor
is quantitatively converted to a single product.
We show experimentally and by computer
simulations that more complex reactions also
exhibit isosbestic points and that the
wavelength of the isosbestic point may change.
Such wavelength changes will occur if either (i)
the molar absorbtivity of the precursor changes
or (ii) the fraction of the precursor that is
converted to multiple products changes. In the
latter case, the isosbestic wavelength and molar
absorbtivities of the precursor and product can
be used to calculate the fraction of the
precursor that is converted to products from
the relationship, f =
epsilon(Precursor)(M)/epsilon(Product)(M),
where f is the fractional conversion,
epsilon(Precursor)(M) is the molar absorbtvity
of the precursor, and epsilon(Product)(M) is the
molar absorbtivity of the product.
The requirement for an isosbestic point to occur
is that the two species involved are related
linearly by stoichiometry, such that the
absorbance is invariant for one particular
wavelength. Thus, other ratios than one to one
are possible. The presence of an isosbestic point
typically does indicate that only two species
that vary in concentration contribute to the
absorption around the isosbestic point. If a third
one is partaking in the process the spectra
typically intersect at varying wavelengths as
concentrations change, creating the impression
that the isosbestic point is 'out of focus', or that
it will shift as conditions change.[2] The reason
for this is that it would be very unlikely for three
compounds to have extinction coefficients
linked in a linear relationship by chance for one
particular wavelength.

In chemical kinetics, isosbestic points are used

as reference points in the study of reaction
rates, as the absorbance at those wavelengths
remains constant throughout the whole
reaction. Isosbestic points are used in medicine
in a laboratory technique called oximetry to
determine hemoglobin concentration,
regardless of its saturation. Oxyhaemoglobin
and deoxyhaemoglobin have isosbestic points
at 590 nm and near 800 nm. Isosbestic points
are also used in clinical chemistry, as a quality
assurance method, to verify the accuracy in the
wavelength of a spectrophotometer. This is
done by measuring the spectra of a standard
substance at two different pH conditions (above
and below the pKa of the substance). The
standards used include potassium dichromate
(isosbestic points at 339 and 445 nm),
bromothymol blue (325 and 498 nm) and congo
red (541 nm). The wavelength of the isosbestic
point determined does not depend on the
concentration of the substance used, and so, it
becomes a very reliable reference.
CONCLUSION
Absorption Spectroscopic methods of
analysis rank among the most widespread and
powerful tools for quantitative analysis. The use
of a spectrophotometer to determine the
extent of absorption of various wavelengths of
visible light by a given solution is commonly
known as colorimetry. This method is used to
determine concentrations of various chemicals
which can give colors either directly or after
addition of some other chemicals. Like in his
experiment, the acid dissociation constant of
the indicator dye based on the maximum
absorbance and molar absorptivities of the acid
and base forms of the dye.
Spectrophotometer was used to
determine the pKa of theindicator solutions,
methyl red. The method is a simultaneous
photometric determination, using Beer's Law
and the Henderson - Hasselbalch equation to
determine the pKa of the indicator.
Methyl red is a weak organic acid which can be
used as an indicator in the pH range of 4.8 to
6.0. From the prepared solutions done in this
experiment, we can verify that a solution of
methyl red will be red if the pH is lower than
4.8, yellow if it is above 6.0 and a mixture of
both if 4.8< pH > 6.0. The color of these methyl
red solutions should vary from the acidic color
to the basic color.
The ionization constant may be calculated from
measurements of the ratio [In–]/[HIn] at known
pH values. Since the two forms of methyl red
absorb strongly in the visible range, the ratio
[In–]/[HIn] may be determined
spectrophotometrically. The absorption spectra
of methyl red in acidic and basic solutions are
determined, and two wavelengths are selected
for analyzing mixtures of the two forms.
These two wavelengths, λIn– and λ HIn, are
chosen so that at one, the acidic form has a very
large absorbancy index compared with the basic
form, and at the other, the situation is reversed.
-
The absorbancy indices of [In–] and [HIn] are
determined at both of these wavelengths, using
several concentrations to determine whether
Beer’s law is obeyed. From the data gathered in
Table 3A and 3B, we can validate that molar
absorptivity is directly proportional to the
absorbance. Maximum absorbance was used
because the wavelength at which the
absorbance is greatest needs to be determined
for the reason that the spectrophotometer is
more sensitive to absorbance changes at this
wavelength. This method is useful for
studying dyes for use as indicators in acid- base
titrations, or by an analogous procedure for
indicators for oxidation-reduction titrations.
The isosbestic point only appears at near 420
nm which means that there is only one
wavelength where two species have the same
molar absorptivity. Figure 4 is an example of
absorbance of acid and basic form of methyl red
at two wavelengths; however, it was not able to
do in his experiment due to the lack of data
gathered. The pka of methyl red was not able to
obtain because to determine the ionization
constant of methyl red, the relative amounts of
HIn and In- present in solution must be
obtained as a function of pH at different
wavelengths.
REFERENCES

http://www.ruf.rice.edu/~bioslabs/methods/potein/protein.html#top

http://spinner.cofc.edu/genchemlab/beers.htm?referrer=webcluster&

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/beerslaw.htm

http://sbio.uct.ac.za/Sbio/postgrad/modules/GRD/spectrophotometry/beer1.php

http://www.ncbi.nlm.nih.gov/pubmed/11112281

TITRATION CURVES, INDICATORSAND ACID DISSOCIATION CONSTANTS. "Chemistry with

Computers.Vernier Software, Portland OR,1997