CHEM103 – Chemistry for Engineers – 2023 1

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CHEM103 – Chemistry for Engineers - 2023

Week 4 Practical 2: Spectrophotometry – determination of iron in water

Face-to-face students: Before the laboratory: complete the pre-lab quiz on Moodle
(worth 2.5% of your total mark). During the laboratory: WORK in groups of four and
complete the report template (you will need this to complete the post-lab quiz). After the
laboratory complete the post-lab quiz on Moodle (worth 5.0% of your total mark).
Online students: complete the pre-lab quiz (worth 2.5% of your total mark) and post-lab
quiz (worth 5.0 % of your total mark) – all via Moodle.

This section applies to face-to-face students

To ensure that we can operate this subject for the full session there are some important requirements
of you. These are listed in categories below.
Foremost we ask that if you are feeling unwell and showing any symptoms of COVID-19 that
you stay home and get tested. This is important in maintaining operations through the session.
Inform your subject coordinator (Prof Marc in het Panhuis) by email (panhuis@uow.edu.au).
Entry to the laboratory
• After entering the building proceed upstairs to the level 3 First year Chemistry Laboratories
• Optional: you can include a FACE MASK as part of your PPE
•
• Lockers: We are using the lockers outside the lab, alternatively your belongings should be placed
under the lab bench well out of the way – there is plenty of room. You need only your device
(labtop/tablet), paper, pen(s) and calculator out of your bag.
• Please follow the requests of any staff during your laboratory sessions.

Conduct in the laboratory

• We expect that you take the same lab location, with the same demonstrator group for the 2 lab
sessions.
• You are required to abide by and floor and bench signage as well as laboratory staff instructions.
• Optional: You can put on a face mask at any time.
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• Adhere to equipment/glassware cleaning protocols as directed.
• Ensure you are aware of the changes to emergency evacuation procedures; this will be presented
in class.

Exit of the laboratory

• At completion of lab work and equipment/glassware cleaning you are requested to wash your
hands before exiting.
• Exit via the main stairs of the building

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The following applies to all students, i.e. face-to-face and online students

1 Introducing Spectrophotometry
1.1 Outline
In this experiment you will use absorption of visible light to determine the concentration of iron in
water samples by instrumental analysis. Instrumental analysis relies on a direct or indirect
interaction of a species with some measurable phenomenon, in this case light. Within the system it
is necessary to achieve a selective response (nothing else present gives this same signal) and that the
signal can be quantified (e.g. here the photons are “counted” at the detector of the
spectrophotometer).
Outcomes:
1. Preparation and use of calibration standards to determine instrument response.
2. Determination of concentration by comparison of sample measurement to that of known
standards.
3. To familiarise yourself with the essentials of an instrumental determination to ensure sound
data are obtained.

1.2 Principle of analysis

When matter is exposed to electromagnetic radiation, absorption of certain wavelengths of the
radiation is possible, whereas other wavelengths do not interact at all. The nature of the interaction
depends on the wavelength (energy of the photons) involved. The atoms or molecules of the matter
absorbing the radiation are excited to higher energy levels in a variety of ways. The energy
imparted to the matter may cause changes in nuclear spin, molecular vibrations or electronic states.
A molecule initially in a ground state or a lower state is said to be excited to a higher energy level
or an excited state. Generally, where photons of visible or near UV light are absorbed by molecular
species, electronic transitions occur, where electrons move from the ground state (or lower states) to
excited states.
Where species absorb visible light, they appear coloured to the human eye, and we see those
photons which were not absorbed. Phenolphthalein in base appears magenta, not because it absorbs
magenta (red) wavelengths of light, rather it absorbs green light (wavelength of max absorption is
553 nm) and the photons of magenta wavelength are absorbed by your retina. Copper ions, solvated
by water molecules in aqueous solution, appear blue because they absorb yellow light
(complementary colours which you may have seen as opposite colours on a colour wheel).
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The record of absorption of radiation (y axis) vs. wavelength (x axis) is called an absorption
spectrum, and this spectrum provides information about the electronic nature of the molecular
species.
The amount of light (number of photons) absorbed depends on the number of absorbing species in
the path of the light, therefore the greater the population of the absorbing species in the light path,
the greater the absorption. This gives rise to methods of quantitative analysis based on the linear
relationship between concentration and absorption given by the Beer Lambert Law (Beer’s law):
I
Absorbance = log o = a b c
I
Where Io = light intensity entering solution
I = light intensity leaving solution
a = absorptivity (a proportionality constant)
b = path length (light path through solution) in cm
c = molar concentration
Thus a spectrophotometer supplies electromagnetic radiation to a sample of the solution which is
held in the path of the radiation. The spectrophotometer effectively measures the number of photons
entering the solution and the number emerging from the solution, to provide the absorbance. A plot
of absorbance (y axis, dependent variable) vs concentration (x axis, independent variable) called a
Beer’s Law plot, is generated from absorbances of solutions of known concentration (standard
solutions) of the species of interest called the analyte. One then measures the absorbance of the
solution of unknown concentration and, from the plotted relationship, determines the concentration
of the analyte.
The basic components of a spectrophotometer are shown below in fig 1 with a typical Beer’s law
plot in fig 2 for a species X, which absorbs light at a wavelength of 510 nm.

Light Monochronmator Detector Output

source (wavelength
selection)

Sample cell

Fig 1 Components of a spectrophotometer

Absorbance
at 510 nm

[X] / mol L-1

Fig 2 Beer’s Law Plot Fig 3 o-phenthroline structure

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In this practical you will react Fe2+(aq) ions in the standards and samples with orthophenanthroline
(also o-phenanthroline and often informally called o-phen), to produce a highly coloured complex
of iron. You will use the absorption of green light by the highly coloured iron complex to determine
the concentration of iron in water samples. The molecular structure of the o-phenanthroline is
shown fig 3 above.
Generally the level of iron in natural waters is low, so the units most commonly used to express
concentration of iron are mg/L or ppm. The concentration of iron found in a particular lake or
stream depends on the geology of the catchment, microbial activity and the presence of oxygen.
Typical values of iron in well oxygenated surface waters would be < 0.10 mg/L. Because Fe2+ is
more soluble in the absence of oxygen, ground waters can accumulate dissolved iron from minerals.
Such waters can easily be ten or even up to one hundred times higher in iron concentration.

1.3 Summary of Procedures:

All glassware will require a dilute hydrochloric acid rinse before use, follow the procedure.
You will serially dilute a stock standard solution of iron to generate a series of iron solutions of
known concentration, 0.00, 1.00, 2.00, and 4.00 mg Fe2+/ L. You will also be supplied with water
samples. All solutions and samples used in the measurement of iron concentration will be treated
with:
• hydroxylamine hydrochloride solution (5% w/v) to ensure all iron is present in the form of
Fe2+(aq).
• sodium acetate solution (1 mol/L) to maintain the pH within the range 4-9.
• o-phenanthroline monohydrate solution (0.15 %w/v) to form the highly coloured iron
complex.
The absorbance of the solutions is measured using the spectrophotometers.
Used a pipette before? One of your group will need to, maybe one of you has. Filled a
volumetric flask just to the mark before – practice! Check techniques with
demonstrator.

BEFORE LABORATORY (face-to-face and online students) – worth 2.5% of your total mark
• Complete the pre-lab quiz via Moodle (you will need to watch video and record
information from the pre-lab in the Laboratory Report Template)

DURING Online LABORATORY class (online students) – worth 5% of your total mark
• Complete the Laboratory Report Template (see page 8) below during the online
laboratory activity using the laboratory videos on Moodle.
• Please upload your Laboratory Report Template to Moodle

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DURING LABORATORY class (face-to-face students) – worth 5% of your total mark
• Complete the Laboratory Report Template (see page 8) below during the face-to-face
laboratory class using the information below.
• Please upload your Laboratory Report Template to Moodle

2 Procedures
Person 1 and Person 2 Preparation of calibration standards
Supplied:
• Stock working solution: 10.0 mg Fe2+(aq) / L
• Dilute 1 mol/L HCl(aq) for glassware preparation
• hydroxylamine hydrochloride solution (5% w/v)
• sodium acetate solution (1.2 mol/L)
• o-phenanthroline monohydrate solution (0.15 %w/v)
Procedure for preparing glassware: Wash all glassware well with tap water, rinse with tap water,
rinse with dilute HCl(aq) and recycle the acid, rinse well with tap water again then finally with
distilled water.

2.1 Calibration curve preparation:

1. In a clean acid washed 100 mL beaker (rinsed
twice with about 5 – 10 mL of the stock iron
solution) collect about 100 mL of the stock iron
solution which is 10.0 mg Fe2+ / L.
2. Prepare 4 x 100 mL volumetric flasks
appropriately. Label the flasks with the
concentrations of each standard.
3. Rinse a 50 mL burette twice with dil. HCl(aq) and
then with small volumes of your stock 10.0 mg / L
iron solution and then fill the burette to the mark.
Dispense the four tabulated volumes of the
working standard from the burette into the four
flasks. Do not dilute to the mark yet, reagents need to be added.
4. Do not dilute to the mark yet. Are your flasks labelled? Complete the template table.

Person 3 and Person 4: Preparation of sample solutions

2.2 Preparation of sample solutions with unknown Fe concentration:
1. Three water samples, A, B, and C, have been collected for your analysis. You will analyse
each and you will analyse sample B in triplicate. You also need to carry a sample “blank”.
2. Prepare six 100 mL volumetric flasks to receive the samples. Procedure for preparing
glassware: Wash all glassware well with tap water, rinse with tap water, rinse with dilute
HCl(aq) and recycle the acid, rinse well with tap water again then finally with distilled
water. Label flasks for samples: A, B1, B2, B3, C, Blank.
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3. Transfer 25.0 mL of each sample to each of the prepared flasks using a 25.0 mL volumetric
pipette. Place 0.0 mL sample in your Blank. Do not dilute to the mark yet as reagents need
to be added. Don’t forget to replicate sample B three times, ie, measure sample B in
triplicate as B1, B2 and B3.

2.3 Reaction to generate the coloured iron complex.

Add the colour development
reagents using 10 mL measuring
cylinders. Clean these first.
To all flasks add 10 mL
hydroxylamine hydrochloride.
Next, to all flasks add 10 mL
sodium acetate buffer.
Lastly, to all flasks add 10 mL o-
phenanthroline solution.
Make the flasks up to the mark with
distilled water, stopper and shake well
to mix completely. Leave to stand for 10 minutes to maximise colour development. You are now
ready to use the spectrophotometer to measure the absorbance of each of your samples and
standards. Complete the samples dilution table in the template.

Flask label Blank Sample A Sample B1 Sample B2 Sample B3 Sample C

Dilution -

2.4 Spectrophotometry
From discussion with your group and demonstrator, make notes about the use of the
spectrophotometer. You will find this instrument frequently used in laboratories. Note especially
your demonstrator’s comments on problems of making absorbance measurements.
With direction from your demonstrator, zero the spectrophotometer on distilled water. Measure the
absorbance of each standard solution, including 0 ppm Fe. Measure the absorbance of the samples
including the sample blank.
You will then construct the calibration graph and determine the concentration of samples from that
graph. If you wish to calculate the slope and intercept using your calculator you may. You must
complete the Beer’s law plot and show how concentration is derived from the plot even if you use a
program to calculate the least squares data.

Question time: may include calculations from graphs or data; consideration of the sample
concentrations found, precision, principles or procedures or techniques in use of the
spectrophotometer, what can give falsely high or low concentration results.

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3 OH&S notes for these experiments

Disposal of solutions:

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Week 4: Laboratory report template
Your name: Your lab partners:

Part 1: Prelab: online

1. Flow diagram summary for standards and samples from Prelab standards and samples
process: NOTE you are supplied with working solution 10.0 mg/L Fe in this lab.
Standards samples

Standard Working solution 10.0 mg/L Using 25.0 mL samples delivered by

supplied pipette

2. Write explanation and annotate

graph to explain how to use the
calibration graph to determine the
concentration of a sample
solution.

Beer’s Law: Abs = a b c

Note how this relationship aligns with y = m x + c

3. Calculation of sample results, complete the following from the prelab.

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Remember you have diluted your samples from 25.0 mL to 100.0 mL.
• For Dilutions Use: C1V1 = C2V2

Using Coriginal Voriginal = CmeasuredVmeasured

Coriginal =

Part 2: Record preparation of calibration curve:

Flask label

Volume of stock working 0.00 mL 10.00 mL 20.00 mL 40.00 mL

standard dispensed
Final volume 100.0 mL 100.0 mL 100.0 mL 100.0 mL
Flask concentration

Part 3: Record preparation of samples

Flask label Blank Sample A Sample B1 Sample B2 Sample B3 Sample C
Sample
volume
Final volume 100 mL 100 mL 100 mL 100 mL 100 mL 100 mL
Dilution -

Notes about the spectrophotometer: Sketch or notes of instrument controls

(i) Record the absorbance measurement process:

Record things to watch out for:

(i) Cleanliness of cells – why

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(ii) Placing cell in spectrophotometer

(iii) Depth of solution in cell

(iv) Any time lag required for chemical reactions

(v) Other…

Part 4: Record calibration data:

Flask label 0 mg/L 1 mg/L 2 mg/L 4 mg/L
Absorbance at 510 nm

Part 5: Record Water Sample data

Flask label Blank Sample A Sample B1 Sample B2 Sample B3 Sample C
Dilution
Absorbance at
510 nm
sample abs
– blank abs

Part 6: Results:
Plot absorbance versus concentration for the standards, with appropriate labelling on the graph on
the next page – alternatively do this in Excel and insert a screenshot of the graph.

Plot a best fit straight line by eye or use Excel linear fit.

The straight line fit can be used with Beer’s Law relationship: Abs = a b c
where a b is the slope of your straight line fit (assume b = 1 cm) and c the concentration of Fe2+
(aq) in mg /L.

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Using the absorbance of each sample – any blank absorbance, read the concentrations of the sample
solutions from the Beer’s law plot. Remember that your samples were diluted four fold during this
analysis. You need to take that into account to calculate your sample concentrations.

Check significant figures!

Use the graph below for plotting by hand (alternatively use Excel)

Sample Sample Abs – Blank Abs mg Fe /L (diluted sample) read mg Fe / L original

from graph calculated, give one
dilution calculation below
A
B
B
B
C

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Calculate and report mean for sample B

RESULTS: Sample A: ___________ B: ___________ C: ___________ mg Fe /L

Have a think about the following questions:

Review the steps taken to produce the standard calibration curve.
• what could be contributing most to any scatter of calibration points on the calibration
graph?

• How could you improve this?

End of Lab:
• Upload a copy of this template to Moodle (online and face-to-face students)
• Answer the questions in the post-lab quiz on Moodle

Checklist

My bench / work area is clean and tidy. All glassware cleaned and stored,
concrete mini-slab completed

All common areas are wiped down: sinks, balance area, fumehood.

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Week 4 – Spectrophotometry: determination of iron in water