CHEM 110 LABORATORY ASSIGNMENT 4

SPECTROPHOTOMETRY OF INDICATORS
1 SHEET OF GRAPH PAPER WILL BE SUPPLIED DURING DATA COLLECTION.

BEFORE COMING TO THE LAB: Read through the assignment carefully and complete the
Pre-Lab 4 Quiz on Canvas.
1. INTRODUCTION
For students in semester 1, you will come in to the lab to collect data, but complete the write-up
in your own time. You will need to sign-up for a data collection session on Canvas (Calendar –
Find Appointment – CHEM110 – Lab 4 Data Collection). These sessions are INSTEAD of your
regular lab session for this cycle.

For students in semester 2, this will run as a regular lab, entirely within your scheduled laboratory
session.

2. WEAK ACIDS AND THEIR SALTS

2.1 Acidic and basic substances
Acids are molecules or ions that have a proton (H+) which is can be removed by a base. The product
arising from loss of a proton (H+) from an acid is known as its conjugate base. The charge on the
conjugate base is one unit more negative than on the acid.
+
-H
Summarising: Acid + Conjugate base .
+H
Following on from this definition, bases are compounds which can take a proton from an acid.
Bases have atoms with nonbonding pairs available to bond with H+.

Substances such as H2PO4- and water can act as both

an acid and a base. Such species are said to be
amphiprotic.

2.2 Water as an acid or base and pH

Water molecules can react with each other. Although
hydronium and hydroxide ions are present in pure water 2H2O ⇌ H3O + OH
+ -
(1)
their concentrations are too low to be detected except by
very sensitive conductivity experiments.
The equilibrium concentration of H3O+ in water or in an aqueous solution can be measured using
an electrochemical cell known as a pH meter. The potential of the cell varies with the hydronium
ion concentration and is displayed in pH units.
By definition: pH = -log([H3O+]/mol L-1) or alternatively [H3O+]/mol L-1 = 10-pH
Thus, if the pH of a solution is 2.58, [H3O+] is 2.63 x 10-3 mol L-1. Try this on your calculator,
noting the definition of pH involves both the log function and a sign change. Note that because of
the negative sign, solutions having lower pH have higher [H3O+].

2.3 Aqueous solutions of acids and bases.

Aqueous solutions of acids have a higher
concentration of H3O+ than pure water as a acid + H2O ⇌ H3O + conjugate base (2)
+

consequence of reaction (2).

This means that the pH of an aqueous solution of an acidic solute is lower than that of pure water.
For strong acids, reaction (2) is product-favoured, and the major species in their aqueous solution
are hydronium ions and the conjugate base of the acid. For weak acids, the reaction is reactant-
Ó School of Chemical Sciences, The University of Auckland
 CHEM 110 Laboratory Assignment 4 [2020] 2
favoured and the major species in their aqueous solution is the unreacted acid, with hydronium
and the conjugate base being the minor species.

A solution of a weak acid in water is an equilibrium system. Adding a strong acid (increasing the
concentration of H3O+) will push the equilibrium to the left. Thus at lower pH (more acidic), you
have more of the acid form. Adding a strong base, which will react with and decrease the
concentration of H3O+ will push the equilibrium to the right. Thus at high pH (more basic) you
have more of the base form. At a certain pH, the concentration of acid and conjugate base will be
exactly equal. This pH is determined by the nature of the acid, and described as the pKa of the acid.
You will learn more about pKa and how it related to (2) in lectures – but you don’t need it for this
lab.

2.4 Indicators
Indicators are simply compounds that are weak acids, and whose acid form (HIn) and conjugate
base form (In-) are different in colour.
HIn + H2O ⇌ H3O+ + In-

Thus indicators can provide a visual information about the pH of a solution. If you decrease the pH
of the system, the equilibrium will move to the left and you will get more of HIn. If you increase
the pH, the equilibrium moves to the right and you get more of the In-. In this experiment you will
use bromothymol blue, which is yellow in the acid form and blue in the base form. At pH values
were both acid and conjugate base form are present in a significant amount the solution will appear
green. Note that the pH at which the colour change occurs is related to the point at which [HIn] =
[In-] – that is, the pKa of the indicator.

3. SPECTROPHOTOMETRY
Recall from Lab 2 that spectrophotometry is a form of quantitative analysis, which depends on the
fact that the amount of light absorbed at a particular wavelength by a solution of a coloured
compound can be directly related to its concentration. Hence a plot of absorbance vs concentration
of standard solutions gives a straight line (calibration plot). Therefore the concentration of an
unknown solution can be read off from the calibration plot if the absorbance of the solution has
been measured. The relationship between absorbance and concentration is given by Beer’s Law.
A = ebc
If the extinction coefficient (e) and path-length (b) are known, the concentration (c) can be
calculated for a given absorbance value (A). Equally, the slope of such a calibration plot is equal
to e x b. b is related to the size of the cuvette. For the spectrometers used in this experiment, b =
1 cm. See Block 2 for more details.

3.1 Determining the concentration of an indicator by spectrophotometry

As we saw in the previous section, indicators are simply weak acids, where the acid form and base
form have different colours. Thus, the colour of a solution of indicator will be dictated by the pH
of the solution. From a spectrophotometric perspective, the acid form and base form, being
different colours, will have their strongest absorbance (lmax) at different wavelengths, and can thus
be identified and quantified.

While the ratio of acid form (HIn) and base form (In-) changes with pH, the total concentration of
indicator (HIn + In-) does not change. If ONLY the base form absorbs at a particular wavelength,
we can calculate the percentage of the indicator in the base form using the relationship:
%In- = A / Amax *100 (1)
Where A is the absorbance at the pH of interest, and Amax is the absorbance of the solution at a pH
where only the base form is present (%In- = 100). The same calculation can be done for the acid
form, although the wavelength used for measurement would be the lmax of the acid form.
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 CHEM 110 Laboratory Assignment 4 [2020] 3

In practice, while the acid form should have a distinct lmax it is likely to show some absorbance at
lmax for the base form (the absorption peaks are overlapping slightly). Thus, in calculations, we
need to correct for this ‘background’ or ‘baseline’ absorbance. Taking Amin as the absorbance of
the solution at a pH where [In-] = 0 (i.e. only the acid form is present), we can get a %In corrected
for any baseline absorbance:
%In- = (A-Amin) / (Amax-Amin) *100 (2)

All of the indicator in solution is either in the acid form [HIn] or the base form [In-]. This means
the total concentration of the indicator = [HIn]+[In-]. That is: %In- + %HIn = 100%. Using this
relationship, the ratio [HIn]:[In-] can be calculated.

The Henderson-Hasselbach equation gives the relationship between pH, pKa and the
concentrations of the acid and its conjugate base.
pH = pKa + log10([I-]/[HI]) (3)
-
The form of this equation indicates that a graph of pH vs log10([I ]/[HI]) will be linear. The pKa
will be the y-intercept, as pH = pKa when the log10([I-]/[HI]) term is 0.

4. SAFETY INFORMATION
We are required under the Health and Safety in Employment Act, 1992 to make available information
concerning the chemicals used in today’s experiment.
Disposal: Aqueous solutions are disposed of by rinsing them down the sink with plenty of water.
Precautions: Safety glasses, a laboratory coat and appropriate footwear must be worn at all times. If an
accident occurs, and you get any chemicals in your eyes, call for help and use the eye wash
sprays immediately. You should avoid skin contact in all cases, but if you do spill some
on your skin or clothes wash the affected area thoroughly with water.
If chemicals are spilled on the bench, wipe them up immediately.
2 M NaOH solution 2 M HCl solution
Description: Colourless solution Description: Colourless solution
Risk: Corrosive. Risk: Corrosive.
Bromothymol Blue
(in aq. KH2PO4/K2HPO4)
Description: Blue solution
Risk: ----

WHEN YOU HAVE READ THIS SECTION ON SAFETY, AND ARE SURE YOU UNDERSTAND THE
INFORMATION, SIGN IN THE BOX AT THE TOP OF THE REPORT SHEETS (P5).

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 CHEM 110 Laboratory Assignment 4 [2020] 4
5. EXPERIMENT

Spectrophotometry of Bromothymol Blue (in pairs)

1. In a 100 mL beaker, collect 25 mL of bromothymol blue solution.

2. Place the pH probe in the bromothymol blue solution and wait until the pH stabilises.
Record the pH and colour of the solution (solution 4) on page 6.
3. Using a clean pipette, rinse a cuvette with a small amount of this solution, and discard this
into a waste beaker. Then fill the cuvette with the solution.
4. Use the spectrophotometer to run a scan of the sample (the spectrophotometer will measure
absorbance across a range of wavelengths). Record the absorbance at 433 nm and 617 nm in
the table on page 6.
5. Adjust the pH of the remaining solution to approximately pH 8 by adding 2 mol L-1 NaOH
dropwise. Wait for the pH probe to equilibrate and record the pH and colour of the solution
(solution 5) in the table on page 6.
6. Using a clean plastic pipette, repeat steps 3 and 4.
7. In a clean 100 mL beaker, collect a fresh 25 mL portion of bromothymol blue.
8. Adjust the pH of the remaining solution to approximately pH 6.5 by adding 2 mol L-1 HCl
dropwise. Wait for the pH probe to equilibrate an record the pH and colour of the solution
(solution 3) in the table on page 6.
9. Using a clean pipette, repeat steps 3-4.
10. Clean up all equipment and return the probe to the buffer solution provided.
11. Measure the absorbance for the three additional solutions provided (pH 2, 5.5 and 10) and
record on page 6.

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 CHEM 110 Laboratory Assignment 4 [2020] 5
REPORT SHEETS
SEPARATION AND ESTIMATION OF ORGANIC COMPOUNDS
Name:________________________________ Stream#:______ Gp:____ Bench#:_______

Group Supervisor:_______________________

I have read and understood the safety

information for this assignment. Signed: ________________________
Signed:___________________________________
SUPERVISOR USE ONLY

0 (not achieved) 0.5 (progress) 1 (mastery)

Overall Report Quality
Use a
spectrophotometer to
accurately measure
absorbance
Graph experimental
data
Obtain information from
a graph
Perform appropriate
mathematical
manipulations on data
More than 2 questions
unanswered/incomplete
Data incomplete.
Major errors with graph
preparation.
Errors in both elements
of the exercise
Calculations incomplete
AND/OR multiple
significant errors in
calculations.
Major errors or 1-2 All questions answered.
missed questions. Minor errors only.
Data outside the Data within the bounds
bounds of acceptable of acceptable
experimental error. experimental error.
Graph well prepared Graph well prepared
with some minor errors. and labelled.
pKa correctly
pKa correctly determined
determined AND
estimations sensible
All calculations All calculations done
attempted. 1-2 correctly, with units
significant errors. where appropriate.

Issues

Safety non-compliance Incomplete tidy-up Late submission

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 CHEM 110 Laboratory Assignment 4 [2020] 6
7. EXPERIMENTAL RESULTS/CALCULATIONS/QUESTIONS
Follow the instructions in 5 to generate the data for the table.

Solution pH Colour Abs @ Abs @ %[In-] [HIn]:[In-] log([In-]/[HI])

433 nm 617 nm
(Amin)
1 2 yellow ~0% - -

2 5.5 yellow

5
(Amax)
6 10 blue ~100% - -

Using Eq. 2 from section 3.1, calculate %[In-] for each entry. Calculate the ratio [HIn]:[In-] in the
table above (to the nearest integer value), and compute log10([In-/HIn]) for solutions 2-5. (Refer to
3.1 for more details).

Eq. 2 involves a baseline correction. Do you think this correction would be more important for
calculating %[In-] from absorbance at 617 nm or for calculating %[HIn] from absorbance at 433
nm? Explain your reasoning.

On the graph paper provided during the lab, prepare a graph of pH vs log10([In-/HIn]).
On your graph, label the point at which the ratio [HIn] : [In-] = 1:1.

Estimate the pKa of bromothymol blue from your graph. _______________

Estimate the pH above which the concentration of [HI] is less that 10%. _______________
Show your workings.

Estimate the pH above which the concentration of [HI] is less that 1%. _______________

In what (approximate) pH range is the solution green? __________________

This corresponds to a [HIn] to [In-] ratio of between: ______________ and _______________

How does this relate to the pKa of the indicator? ________________________________

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