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) be created by measuring the absorbance of a
that change color when deprotonated (In-). A large number of solutions of known
few drops of indicator added to the analyte concentration.
solution before the beginning an acid-base
titration. When enough base titrant is added to An acid is a substance which dissociates
+
the analyte solution the equilibrium expressed to produce hydrogen ions, H , when dissolved in
in equation 1 will shift toward products. +
aqueous solution. Once in solution, the H ion,
which is simply a proton, immediately combines
HIn(aq) In– (aq) + H+(aq) (1) +
with water to form the hydronium ion, H O . So
3
The result is the formation of more of the +

deprotonated indicator (In ) and a when aqueous H appears in a chemical

corresponding color change of the analyte equation, it is understood that the actual
+
solution (the endpoint). A good indicator for a species exists as H O . An acid is classified as
3
specific acid-base titration has an endpoint with strong or weak depending on the extent to
a pH at or near the pH of the equivalence point. which the molecules of the acid dissociate into
In this experiment, phenolphthalein indicator + −
will be used for each titration. The pH range of H and its anion, A . A strong acid dissociates
the color change will be observed and completely in water (e.g., HCl dissociates
+ −
compared with the pH of the equivalence point virtually100% into H and its anion, Cl ), while a
to determine if the indicator is an appropriate weak acid dissociates only partially and forms
+
choice for each titration. The acid dissociation
very little H (e.g., only about 3% of the
constant, Ka, is a measure of an acid’s strength.
dissolved molecules of acetic acid, CH COOH,
For weak acids these values are less than 1 and 3
+
typically so small that they are expressed with dissociate into H and the acetate anion,
scientific notation. Taking the negative log of −
CH COO ). Weak acid dissociation can be
the Ka results in more easily expressed pKa 3
values ranging from 0 to 14 for weak acids. represented as a generic reversible reaction:
+ –
HA(aq) ⇔ H + A where HA is the weak acid
A spectrophotometer separates light into its -
separate colors. It is able to separate the light and A is the anion, or conjugate base, of HA.
into colors because each color of light has a For this reversible reaction, equilibrium
different wavelength than the other colors. The constant can be defined:
spectrophotometer can shine a narrow band of
wavelengths (essentially one specific color) of
light on a sample of substance and then
(2)
measure how much of that light is absorbed by
the sample. Different colored substances The equilibrium constant is called the acid
absorb varying amounts of specific wavelengths dissociation constant or acid ionization
of light. Therefore, a spectrophotometer can be constant. If the undissociated acid (HA) is
Used to measure how much of a substance is favored and the acid is weak, K is measurable
present. A calibration curve is a graph of a

absorbance, how much of a particular and will be much less than one. For strong
+ −
wavelength of light is absorbed, versus acids, the dissociated products, (H and A ) are
concentration (Beer’s law). A calibration curve so strongly favored, that [HA] approaches zero
is specific to a particular substance, and must and K approaches infinity. Thus strong acids, of
a
which there are very few, do not have values of A1 = A1HIn + A1In = 1Hin [Hin] + 1In [In]
K associated with them. Mathematically, it is
a
at 1 (5)
found that the amount of light absorbed by a
specific sample depends on three things: 1) the The constant  does not change with
concentration of the solution; 2) the distance concentration, but will change at different
the light travels through the sample; and 3) the wavelengths and/or with a different absorbing
natural ability of the specific substance to species. So, at a second wavelength 2, the
absorb light. Thus an equation can be written following equation for Beer’s Law would be
relating these things: true:

A=bc (3) A2 = A2HIn + A2In = 2HIn [HIn] + 2In [In]

where A = absorbance at 2 (6)

 = molar absorbtivity -- how well the There are now two equations that can be used
material absorbs light to determine the concentrations of the two
different colored unknowns that are present in
b = path length through which the light
the solution. These equations can be
passes
rearranged to allow determination of each
c = concentration of the solution concentration.

In general, the value for b will remain the same [HIn] = ((A12In)  ( A21In)) / ((2In 1HIn) 
and the value for  is constant for a specific (1In 2HIn)) (7)
chemical at a given wavelength of light.
Because the general equation for a straight line [In ] = ((A2 1HIn) – ( A1 2HIn)) / ((2In 1HIn) –
is (1In 2HIn)) (8)
y = mx + b (4)

If A is graphed against c, the result will be a The best wavelengths for the experiment are
straight line with a slope of b and a y-intercept selected by measuring the absorbance vs.
of zero. A solution that contains two colored wavelength for each of the pure substances.
species can also be analyzed using Beer’s Law The actual wavelengths are then chosen such
(A= bc). Mathematically, the concentrations that they will maximize the absorbance for each
of the two species can be determined if two species. The values for the four  constants
simultaneous equations can be developed that can be determined by doing Beer’s Law plots of
relate the two. The two unknown absorbance vs. concentration (of pure samples)
concentrations are determined by taking at both wavelengths and determining the slopes
readings on each solution twice, using two of the lines generated. The final set of
different wavelengths of light. The two species measurements will be collected by measuring
that are being studied in this experiment are the absorbance of solutions of the weak acid at
the two colored forms of specific indicators that different pH’s.
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
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 constant total dye concentration of 0.1 M were
basic solutions, their wavelength at maximum determined first, then from this the moles of
absorbance. Based on the data in Table 1, the the acid which is acetic acid and the base which
maximum wavelength of absorption for acidic is sodium acetate can be determined as well. In
dye solution is 520 nm while 420 nm for the order to be prepared, their volumes must be
basic solution. The wavelength at which the known by using the equation for molarity,
absorbance is greatest needs to be determined which is moles/Liter. However, after calculating
for the reason that the spectrophotometer is the appropriate volumes for the different pH, it
more sensitive to absorbance changes at this was found out that the amount of volumes
wavelength. Although a substance will have an were very small making it very difficult for the
extinction coefficient at every wavelength, acid and the base to be pipette. So instead
concentrations are typically measured at preparing the buffer solutions by volume, it was
maxima in the absorbance spectra because this prepared by mass with the help of analytical
is where the absorbance changes least with balance and diluting both the acid and the base
changes in wavelength. From Figure 2 and 3, to 250 ml with distilled water.
the Wavelength vs. Absorbance graph of acidic
dye solution tends to lean on the left due to the Methyl red (4-dimethylaminobenzene-
large presence of acidic forms while in the 2’-carboxylic acid) is a commonly used indicator
Wavelength vs. Absorbance graph of basic dye for acid-base titrations. By following the change
solution tends to lean on the left ro to the large in absorbance as a function of pH of the
presence of its basic forms. prepared buffer solutions we will determine the
acid dissociation constant, or pKa. In this
In the case of the dilute solutions, their experiment we will determine this equilibrium
molar absorptivies were obtained by using the constant, pKa', by varying the pH and measuring
maximum absorbance of the acidic and basic the ratio [In–]/[HIn . We will use acetic acid-
dye solution, which is 520 and 420 nm acetate buffers to control the pH, since the Ka
respectively. From the data gathered in Table value for acetic acid is in the same range as the
3A and 3B, one can see that the absorptivities Ka' value for methyl red. The pH of these
of the dilute solutions by using the maximum buffers force methyl red to distribute itself
absorbance in acidic solutions are higher than somewhat evenly between the two colored
the basic solution. This can be explained forms. The absorption of light is governed by
through Beer’s law from equation 3. Based in the Beer-Lambert Law. The absorbance of
this, the molar absorptivity is directly mixtures is the sum of the separate
proportional to the absorbance. The capacity of absorbencies.
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 The best wavelengths to choose for the analysis
of acetic which is 4.7, buffer solutions with pH are where one form absorbs strongly which is
4.4, 4.8, 5.6, and 6.4 were prepared. The ratio the maximum absorbance. We need to set up
[In–]/[HIn] at different pH values and at two equations in two unknowns, one equation
for each wavelength. Call the two wavelengths Spectrophotometry involves the use of a
and . The two measurements then spectrophotometer. A spectrophotometer is a
provide two simultaneous equations with two photometer (a device for measuring light
unknowns: 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
The molar absorbance coefficients are reflectance of a solution or transparent
determined from standard solutions that material, like polished glass. However they can
contain one component alone. This equations also be designed to measure the diffusivity on
provide two equations in two unknowns. For an any of the listed light ranges that usually cover
unknown solution, the absorbances at the two around 250nm - 2500nm using different
wavelengths, and , are determined controls and calibrations. Within these ranges
and then the two equations are solved for the of light, calibrations are needed on the machine
unknown concentrations [ ] and [ ] at using standards that vary in type depending on
each given pH. Methyl red has a pKa of 5.1 the wavelength of the photometric
determination.
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 Historically, spectrophotometers use a
the maximum absorbance at acidic conditions. monochromator containing a diffraction grating
While in Table 4B, the absorbance increases as to produce the analytical spectrum. There are
the pH increases using the maximum also spectrophotometers that use arrays of
absorbance at basic conditions. Equilibrium photosensors. Especially for infrared
constants involving ionic species are especially spectrophotometers, there are
sensitive to ionic strength. The spectrophotometers that use a Fourier
ionic strength is a measure of the total ion transform technique to acquire the spectral
concentration in solution. The activity of all the information quicker in a technique called
species in solution are a function of the ionic Fourier Transform InfraRed.
strength. The spectral changes are explained in
terms of shifts in equilibria between different The spectrophotometer quantitatively
molecular and ionic species in the solutions. 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 Beer's law relates the absorbance, the
reference sample.t concentration of the absorbing species and the
path length, such that:
In short, the sequence of events in a A = εbc
spectrophotometer is as follows: 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
1. The light source shines through a
species. Beer's law applies to solutions
monochromator.
containing one or more absorbing species, if
2. An output wavelength is selected and
there is no interaction between the various
beamed at the sample.
species in the solution. In the case of a solution
3. A fraction of the monochromatic light is
containing n species which absorb, the above
transmitted through the sample and to
equation becomes:
the photodetector.
Atot = A1+A2+...+An = ε1bc1 + ε2bc2 + ...+
εnbcn
Many spectrophotometers must be calibrated
Beer's law in the case of a fixed path length, b,
by a procedure known as "zeroing." The
and extinction coefficient, ε, is a linear
absorbancy of a reference substance is set as a
relationship between absorbance and the
baseline value, so the absorbancies of all other
concentration This is not generally the case.
substances are recorded relative to the initial
Beer's law is successful in describing the
"zeroed" substance.
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
The spectrophotometer then displays % these molecular interactions become negligible
absorbancy (the amount of light absorbed at concentrations below 0.01M. Dilute solutions
relative to the initial substance). must therefore be used.
The linearity of the Beer-Lambert law is limited An isosbestic point is observed in overlaid
by chemical and instrumental factors. Causes of spectra when a chromophoric precursor is
nonlinearity include: converted to a product with a different
spectrum, so that it is often assumed that an
 deviations in absorptivity coefficients at
isosbestic point occurs only when the precursor
high concentrations (>0.01M) due to
electrostatic interactions between is quantitatively converted to a single product.
molecules in close proximity We show experimentally and by computer
 scattering of light due to particulates in simulations that more complex reactions also
the sample exhibit isosbestic points and that the
 fluoresecence or phosphorescence of wavelength of the isosbestic point may change.
the sample
Such wavelength changes will occur if either (i)
 changes in refractive index at high
analyte concentration the molar absorbtivity of the precursor changes
 shifts in chemical equilibria as a or (ii) the fraction of the precursor that is
function of concentration converted to multiple products changes. In the
 non-monochromatic radiation, latter case, the isosbestic wavelength and molar
deviations can be minimized by using a absorbtivities of the precursor and product can
relatively flat part of the absorption be used to calculate the fraction of the
spectrum such as the maximum of an
precursor that is converted to products from
absorption band
 stray light the relationship, f =
epsilon(Precursor)(M)/epsilon(Product)(M),
When an isosbestic plot is constructed by the where f is the fractional conversion,
superposition of the absorption spectra of two epsilon(Precursor)(M) is the molar absorbtvity
species (whether by using molar absorptivity for of the precursor, and epsilon(Product)(M) is the
the representation, or by using absorbance and molar absorbtivity of the product.
keeping the same molar concentration for both
species), the isosbestic point corresponds to a
wavelength at which these spectra cross each
other.
The requirement for an isosbestic point to occur
A pair of substances can have several isosbestic is that the two species involved are related
points in their spectra. linearly by stoichiometry, such that the
absorbance is invariant for one particular
When a 1-to-1 (one mole of reactant gives one wavelength. Thus, other ratios than one to one
mole of product) chemical reaction (including are possible. The presence of an isosbestic point
equilibria) involves a pair of substances with an
typically does indicate that only two species
isosbestic point, the absorbance of the reaction
that vary in concentration contribute to the
mixture at this wavelength remains invariant,
regardless of the extent of reaction (or the absorption around the isosbestic point. If a third
position of the chemical equilibrium). This one is partaking in the process the spectra
occurs because the two substances absorb light typically intersect at varying wavelengths as
of that specific wavelength to the same extent, concentrations change, creating the impression
and the analytical concentration remains that the isosbestic point is 'out of focus', or that
constant.
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

These two wavelengths, λIn– and λ HIn, are
analysis rank among the most widespread and
chosen so that at one, the acidic form has a very
powerful tools for quantitative analysis. The use
large absorbancy index compared with the basic
of a spectrophotometer to determine the
form, and at the other, the situation is reversed.
extent of absorption of various wavelengths of -
visible light by a given solution is commonly The absorbancy indices of [In–] and [HIn] are
known as colorimetry. This method is used to determined at both of these wavelengths, using
determine concentrations of various chemicals several concentrations to determine whether
which can give colors either directly or after Beer’s law is obeyed. From the data gathered in
addition of some other chemicals. Like in his Table 3A and 3B, we can validate that molar
experiment, the acid dissociation constant of absorptivity is directly proportional to the
the indicator dye based on the maximum absorbance. Maximum absorbance was used
absorbance and molar absorptivities of the acid because the wavelength at which the
and base forms of the dye. absorbance is greatest needs to be determined
for the reason that the spectrophotometer is
Spectrophotometer was used to more sensitive to absorbance changes at this
determine the pKa of theindicator solutions, wavelength. This method is useful for
methyl red. The method is a simultaneous studying dyes for use as indicators in acid- base
photometric determination, using Beer's Law titrations, or by an analogous procedure for
and the Henderson - Hasselbalch equation to indicators for oxidation-reduction titrations.
determine the pKa of the indicator. The isosbestic point only appears at near 420
nm which means that there is only one
Methyl red is a weak organic acid which can be wavelength where two species have the same
used as an indicator in the pH range of 4.8 to molar absorptivity. Figure 4 is an example of
6.0. From the prepared solutions done in this absorbance of acid and basic form of methyl red
experiment, we can verify that a solution of at two wavelengths; however, it was not able to
methyl red will be red if the pH is lower than do in his experiment due to the lack of data
4.8, yellow if it is above 6.0 and a mixture of gathered. The pka of methyl red was not able to
both if 4.8< pH > 6.0. The color of these methyl obtain because to determine the ionization
red solutions should vary from the acidic color constant of methyl red, the relative amounts of
to the basic color. HIn and In- present in solution must be
obtained as a function of pH at different
The ionization constant may be calculated from wavelengths.
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.
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http://www.chemistry.adelaide.edu.au/external/soc-rel/content/beerslaw.htm

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

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TITRATION CURVES, INDICATORSAND ACID DISSOCIATION CONSTANTS. "Chemistry with

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