EXPERIMENT 5: IDENTIFICATION OF AN UNKNOWN INDICATOR

BACKGROUND

An acid-base indicator exists in equilibrium between its acidic (HIn) and basic (In-) forms:

HIn + H 2O " H 3O + + In#

Each form has its own unique color. For example, the traditional indicator used in acid-base titration,
phenolphthalein, is colorless in its acidic form and pink in its basic form.

The equilibrium constant for this reaction, called the acid dissociation constant, Ka, can be written as:

[H ][In ]
=
+

Ka

"

[ HIn]

where the square brackets indicate concentrations. We can rearrange this equation, in cases where
have both the acid form (HIn) and conjugate base form (In-) of the indicator present, to yield
Henderson-Hasselbalch equation.
! This equation can be used to describe the relationship between
relative concentrations of the acidic and basic forms of the indicator, the pKa of the indicator, and
measured pH of the solution:

[In ]
+ log

we
the
the
the

"

pH = pK a

[ HIn]

(1)

This expression can be rewritten to give:

[In ] + pK
pH = log
"

[ HIn]

(2)

This linear equation (y = mx + b) can be used to determine the pKa of an unknown, if the pH and

[In ]
"

concentrations of the acidic and

! basic forms are known. A plot of pH (on the y-axis) vs. log

[ HIn]

(on

the x-axis) will yield a straight line with a slope (m) of +1 and a y-intercept (b) equal to the pKa of the
unknown acid.

! requires the use of

The determination of the relative concentrations of the acidic and basic forms
techniques studied in previous experiments. Because both of the species are colored, spectrophotometric
techniques can be used to determine the concentrations of both the acidic and basic forms of the indicator
in each solution with a different pH.
Lets look at the acidic form first. In highly acidic solution, where there is an abundance of H3O+ ions,
the equilibrium lies to the left and the indicator is primarily in the acidic (HIn) form:

HIn( aq ) + H 2O( ) H 3O(+aq ) + In(aq )

(3)

Similarly, in a highly basic solution, the equilibrium lies to the right and the indicator is predominantly in
its basic form (In-). A plot of absorbance vs. wavelength will show a peak that corresponds to the acidic

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form of the indicator in solutions with low pH; this peak will decrease in intensity as the pH increases
and, at very high pH values, will disappear completely. A peak that corresponds to the basic form of the
indicator, will gain intensity as the pH is raised. Because the acidic and basic forms of the indicator have
different colors, the peaks will be at different wavelengths. At pH values intermediate between these
extremes, both forms (HIn and In-) will contribute peaks to the spectrum.
By monitoring the absorbance of the solution at two wavelengths (one that corresponds to HIn, 1, the
other to In-, 2) as a function of pH, the relative concentrations of the two species can be determined.
If the absorbance spectra of both the acidic and basic forms overlap, then the absorbance measurement at
a single wavelength will be the sum of the absorbances of each species that exists at that wavelength.
Consider the absorbance spectra of the acidic and basic forms of unknown indicator A, as shown below:
The absorbance at 443 nm, where the basic (In-)
form of the indicator maximally absorbs, will
include a contribution from the acidic (HIn) form of
the indicator (blue line), which has non-zero
absorbance at this wavelength.
Likewise, the
absorbance at 523, where the acidic (HIn) form of
the indicator absorbs, will include a contribution
from the basic (In-) form of the indicator (red line).
In order to correctly figure out the [In-]/[HIn] ratio,
we need to determine the absorbance contribution
from only the acidic and basic forms at the
appropriate wavelength, and not the contribution from the overlapping spectrum. As this is complicated,
the simpler case will be considered first in which the peaks for the acidic and basic forms do not overlap
at all.
The Case of Zero Overlap Between Indicator Spectra
At 1, the absorbance at can be written as

A"1 = # HIn b[ HIn]

(4)

where A1 is the absorbance at wavelength 1, HIn is the molar absorptivity constant for HIn, b is the cell
path length (assumed to be constant), and [HIn] is the concentration of the acidic form of the indicator.
The total concentration, at any time, C!
total is given by:

Ctotal = [ HIn ] + [ In " ]

(5)

However, at low pH values, the indicator is primarily in its acidic form, HIn, so the total concentration is
given by:
!
(6)
Ctotal = [ HIn ]
If we substitute Ctotal into the Beers Law expression (eqn 4), we get

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A"1,acid = # HIn bCtotal

(7)

where A1,acid is the absorbance of the most acidic sample at 1. The ratio of these two equation (4) and
(7) yields

A1
A1,acid

HIn b[ HIn] [ HIn]

=
HIn bCTotal
CTotal

(8)

Similarly, for the basic form of the indicator, In-:

A 2

A 2,basic

[In ]
=

CTotal

(9)

Here 2 is the wavelength where the In- absorbs maximally, A2 is the absorbance of a sample at
intermediate pH values, A2,basic is the absorbance of the most basic solution, and [In-] is the concentration
of the conjugate base. Each of these equations assumes that the total concentration of indicator in the

solutions studied is constant.

By taking the reciprocal of equation (9) and multiplying both sides by both sides of equation (8), the ratio
of the concentrations of [In-] to [HIn] in any sample can be determined:

[In ] = A

A1,acid
A 2,basic A1
2

[HIn]

(10)

This equation can be used to determine the relative concentrations of the conjugate base form of the
indicator to the acidic form of the indicator, which will ultimately assist us in determining the pKa of an

unknown indicator.
The Real Case Where Indicator Spectra Overlap (with thanks to Dr. Kahn)
Where spectra overlap, we need to subtract the contribution from the species that we are not interested in
to find the contribution of the species that we are interested in to the absorbance value at that point. This
is complicated, but relies on the fact that, for a single species, the ratio of two absorbances at two
wavelengths is equal to the ratio of the extinction coefficients at these wavelengths.
The algebra is somewhat complicated, but it can be shown that:

(
" "A
%% +
* A 2 $$ A1 $ 2,acidic ''' [In ] A1,acidic *
# # A1,acidic && =
.*
"
"A
%% [HIn] A 2,basic *
1,basic
A $A $
'' * 1 $# 2 # A 2,basic &'& )
,

(11)

It is better to use equation 11 when analyzing your data as opposed to the derived equation 10 above.

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POSSIBLE QUIZ QUESTIONS

1. What is the objective of this experiment? What is being measured? How will the data collected be
used to determine the final result?
2. What two variables will be plotted in order to determine the pKa of the unknown indicator?
3. How will the relative concentrations of the acidic and basic forms of the indicator be determined?

[In ] . Each solution contains the same amount of the indicator

"

4. Given the following data, determine

[HIn]

bromothymol blue.
Sample
1 !
2
3

Measured
pH
1.0
6.85
13

A,
430 nm
1.097
0.7333
---

A,
555 nm
--0.569
1.428

5. How would you prepare 50 mL of a 0.20 M solution starting with solid NaH2PO4.H2O?
6. How would you prepare 50 mL of a 0.20 M solution starting with solid Na2HPO4.7H2O?

MATERIALS & METHODS

SAFETY CONCERNS

There are no known safety concerns with this laboratory exercise.
Preparation of a Buffer Series
Work in groups of 4
1. Obtain
a. Eight 50-mL volumetric flasks (depending
on the number of flasks in the room, you Table 1: Solutions
Volume of
might need to use four then make solutions Solution Volume of
NaH
PO
,
Na2HPO4,
2
4
for the other four samples)
mL
mL
b. Two 5.0-mL graduated measuring pipettes
1
5.0
0
(one for each phosphate solution)
5.0
1.0
c. Two 10.0-mL graduated measuring pipettes 2
3
10.0
5.0
(one for each phosphate solution)
4
5.0
10.0
d. Two micropipettes, previously calibrated
5
1.0
5.0
(be sure to note the micropipette number in
6
1.0
10.0
your notebook)
7
10.0
1.0
e. Two small beakers (each should hold ~45
8
0
5.0
mL of solution)
*Solution
8
should
also
include
4 drops of
f. Number the flasks 1 8.
3
M
NaOH
g. Label the beakers and pipets (NaH2PO4 or
Na2HPO4). Any tape must be removed from the glassware before returning to the cabinet.
2. Record the formula weight of the salts to be used in your notebook. Prepare the solutions according to
your calculations (50 mL of ~0.20 M; what glassware should you use?) and pour them into the
appropriately labeled beaker and mix thoroughly.

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3. Prepare the solutions listed in Table 1.

a. Add the appropriate amounts of NaH2PO4 and Na2HPO4 (see Table 1) to the 50-mL
volumetric flask.
b. For solution 8 only, add 4 drops of 3 M NaOH.
c. Use the micropipette to add 2.0 mL of the unknown indicator to the flask. Be sure to note
the unknown number in your notebook.
d. Dilute each buffer with distilled water to the etched mark on the flask. You should use a
dropper pipette to add the final few drops.
e. Stopper and gently invert the flask 10-15 times to mix thoroughly.
Measuring pH
Teams of 4
Each group will analyze all 8 samples.
1. Use the pH meter directions and standardize the meters as directed in the LabQuest User Guide. Be
sure that the voltage stabilizes during calibration.
2. Measure and record the pH of each solution. Do three trials for each solution. Carefully rinse the pH
probe between each sample with DI water using a squirt bottle into a waste beaker.
3. Record the color of the most acidic and most basic samples.
Determination of [HIn] and [In-]
Absorbance measurements will be performed on the spectrophotometers.
1. Follow the instructions provided for using the SpectroVis Plus and LabQuest2 (posted to Canvas and
will also be available in lab).
2. Use a 50/50 mixture of phosphate buffer as the blank.
3. Using samples 1 and 8, determine the wavelength of maximum absorbance ( max) for the acidic and
basic forms of the unknown indicator. Once you have determined the max, you will switch to
Events with Entry mode to measure absorbance at those two wavelengths.
4. Determine [HIn] and [In-] for samples 1 8.
a. Set the spectrophotometer at the wavelength determined for the acidic sample (1). Record
the absorbance. Change the wavelength to 2 and record the absorbance. Record three trials
for each sample. Use a new sample each time. For each sample, rinse the cuvet with a small
amount of sample, discard, refill, and then record the absorbance. Use the same cuvet for
each measurement.
b. Measure and record the absorbance of each sample 1 8 at both wavelengths.
Before leaving lab:

(
" "A
%% +
2,acidic
*
$
A

A
$
''' 2
$ 1 A
In ]
[
[In ] A1,acidic *
# # 1,acidic && =
.*
Determine log
for each solution using
.
"
"A
%% [HIn] A 2,basic *
[ HIn]
A $ A $ 1,basic '' * 1 $# 2 # A 2,basic &'& )
,
Turn in copies of your data before leaving lab. You should leave with 48 absorbance values and 24

pH measurements.

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