School of Chemistry

CHM 2922
Spectroscopy and Analytical Chemistry
2022

LABORATORY MANUAL
 TABLE OF CONTENTS
SAFETY RULES 1

OCCUPATIONAL HEALTH AND SAFETY RULES FOR UNDERGRADUATE STUDENTS .................................................. 2

MONASH UNIVERSITY PLAGIARISM AND CHEATING POLICY .......................................................................................... 7

INTRODUCTION TO CHM2922 PRACTICAL WORK................................................................................................................. 9

EXPERIMENT 1. MEASURING THE ALCOHOL CONTENT OF WINE USING GAS

CHROMATOGRAPHY ...................................................................................................................... 15

EXPERIMENT 2. MEASURING IRON IN “CONCRETE BOOTS” USING ATOMIC ABSORPTION

SPECTROMETRY .............................................................................................................................. 22

EXPERIMENT 3. MEASURING HERBICIDE RESIDUE IN SOIL USING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC) ......................................................................................................... 27

EXPERIMENT 4. DETECTING GUNSHOT DISCHARGE RESIDUES USING UV-VISIBLE

SPECTROPHOTOMETRY ................................................................................................................. 33

EXPERIMENT 5. MEASURING CHLORIDE IN MARINE WATER USING FLUORESCENCE

QUENCHING AND INVESTIGATING USING FLUORESCENCE TO MEASURE
QUININE SULFATE IN TONIC WATER ......................................................................................... 37

EXPERIMENT 6. IDENTIFYING ADULTERANTS AND CONTAMINANTS IN FOOD AND DRUGS

USING FTIR AND RAMAN SPECTROSCOPY ............................................................................... 45

EXPERIMENT 7. INVESTIGATING PROBLEMS OF FLUORIDE ANALYSIS BY ION SELECTIVE

ELECTRODE ...................................................................................................................................... 58

EXPERIMENT 8. MEASURING TRACE HEAVY METALS IN ENVIRONMENTAL SAMPLES FROM

THE MT. ISA MINING DISTRICT USING SQUARE WAVE STRIPPING
VOLTAMMETRY .............................................................................................................................. 65

EXPERIMENT 9. DETERMINE ANALYTICAL FIGURES OF MERIT OF A ELECTROCHEMICAL

GLUCOMETER .................................................................................................................................. 74

Cover photo – Discharge of a handgun with the bullet visible at left. The plume deposits gunshot residue
(GSR) on the shooter’s hands, leaving behind a tell-tale elemental fingerprint containing traces of barium
and antimony.

Edited by Rodney Hall, Kellie Vanderkruk Version 3.1. This revision Jul 2019
Copyright©, Chemistry Department, Monash University 2019
 1

SAFETY RULES
• Safety glasses must be worn at all times in laboratories where chemicals are
stored and are in use. Please consult the coordinator (Dr Irene Ling) if you
wear contact lenses.
• Closed shoes must be worn (never thongs or open sandals).

• Long hair must be tied back.

• Eating and drinking in the laboratory are strictly prohibited.

• Using mobile phones in the laboratory is strictly prohibited.

• Keep your work area neat and uncluttered. Clean up chemical and water spills
at once, seek advice from your demonstrator as necessary.

• Avoid skin contact with chemicals, wash your hands after every practical
session and NEVER pipette by mouth.

• Learn the location and proper use of safety showers and eyewashes.

• Visitors are NOT permitted to undergraduate laboratories.

• Risk Assessment must be carried out for each experiment before coming to the
practical session, and the completed forms signed by a demonstrator, before
work commences. The completed Risk Assessment Form must be submitted
with the signed Practical Report Cover Sheet for each experimental report.
 2

OCCUPATIONAL HEALTH AND SAFETY RULES FOR UNDERGRADUATE STUDENTS

• Like all other members of Australian society, you have the right to work and study in a healthy
and safe environment. Monash University has an extensive network of Occupational Health and
Safety staff and facilities whose purpose is to ensure that this goal is achieved.
• As a student at Monash University you also have certain roles and responsibilities in relation to
Occupational Health and Safety to help ensure your own health and safety and that of other
students, staff, visitors to the University and the general public. These are summarised below.
• A wide range of OHS resources are available to you. Some are listed below, but you are also
encouraged to seek out further information in this area. If you have questions or concerns about
this or any other OHS issues, please discuss them with any of the contact people listed below.

Your roles and responsibilities

General
• You must read and understand this information sheet before undertaking any practical classes.
• The School’s academic and technical staff will give you specific information relating to the
location of OHS facilities in your lecture theatre or laboratory, and details of practical classes in
which you are involved. You must listen to this information and comply with any health and
safety instructions.
• If you are ever unsure of the correct and safe procedure to be followed, ask one of the technical or
academic staff. Do not rely on information from other students.

Evacuations
• You should ensure you are aware of the location of fire exits, extinguishers and other emergency
facilities in your laboratory or lecture theatre, and also of the point at which you should assemble
after an evacuation. This information is available from the academic or technical staff in the area
and is also clearly signposted.
• You should also ensure that you are aware of the meaning of the different tones produced by the
alarm system. Drills will be held regularly to assist with this.
• You also have a responsibility to ensure that if an alarm sounds you behave sensibly and do not
panic. This includes heeding the alarms tones, listening carefully to all voice announcements,
complying with instructions from Wardens, and if necessary evacuating in a calm and orderly
fashion and proceeding directly to the nominated assembly point.
 3

First aid
• You should not provide first aid or emergency medical assistance to any person unless you have
received formal First Aid training.
• However, you should be aware that most of the technical staff in your laboratory have been
formally trained in First Aid, etc.
• You should also make sure that you are aware of the location of all emergency facilities in your
laboratory (eye-washers, safety showers, etc.). The technical staff will give you this information at
your first laboratory session, and facilities are also clearly signposted.

Incident reporting
• In the past, some people have been reluctant to report incidents because they feel they might get
someone else into trouble. However, the reporting process is not intended for that purpose! We
would prefer you to think of it as a way of letting us know about an issue so we can make sure it
is resolved before anyone gets into trouble.
• So, if you witness or are involved in an incident which you feel may have safety or security
implications, whether or not anyone was hurt, please report it as soon as practical to one of the
contact people listed below.

Key OHS contact people

• Campus Safety Officers
• Any of the School’s technical staff.

Useful OHS resources

• The School of Chemistry’s OHS website is at http://www.chem.monash.edu.au/safety/ and has
information on safety and detailed information on relevant procedures.
• The University’s OHSE website at http://www.monash.edu.au/ohs/ has a range of information and
links to other sites which you may find useful. This includes a number of policy documents,
information sheets, hazard alerts, procedures and guidelines which you are strongly advised to
look through.
 4

Risk Assessment Guidance Notes/Questionnaire

Revised June 2015.

Please read in conjunction with other information on Risk Assessment such as that provided by
OHSE and the Risk Control Program.

Labelling, referring to MSDSs, undertaking Risk Assessments and control, and record keeping are
all important aspects of risk minimisation which you must be able to perform when carrying out
laboratory work.

You need to have good scientific and common sense (defensible) answers to the following types of
questions.

There is little legal defence for ignoring the foreseeability of risk. You should do your best to minimise it.

- Are you doing/going to do (understand exactly what is expected of you)?

- Is the scale, scope and timeframe of the operation; you need to be aware of
WHAT? matters such as exothermic reactions, etc. as appropriate (what observations
are important).
- Is the classification of the risk A (low), B (medium) or C (high)?
- Are you going to conduct the work; are there existing "standard operating
procedures"; have you the right equipment to conduct the work and has it
been checked that it is in good working order; are any specialist
HOW?
facilities/devices required (e.g. pressure relief valves)? Do not take short
cuts, they can lead to unwanted incidents.
- Well prepared are you for an unplanned incident?
- Will you conduct your work; in the fume-cupboard (volatiles/toxics/
WHERE? operations subjecting you to high exposure); on the bench; in a designated
lab/fume-cupboard?
- Will you be conducting the work; if after "normal business hours" ensure
that you have consulted the appropriate OHS Policy Documents and
WHEN?
particularly the School Safety Manual. Carrying out Undergraduate practical
work always requires a demonstrator being present.
- Will give you advice and training in unfamiliar techniques? (i.e., a
demonstrator must be present whenever you are performing practical work)
WHO?
- Will be able to assist in the case of an unplanned incident? (know where
closest first-aiders are and other appropriately trained personnel)
 5

Risk Assessments in Undergraduate Laboratories

Risk assessments MUST be carried out and the form signed by a demonstrator before proceeding
with any practical work.

THIS PAGE IS AN EXAMPLE OF A COMPLETED FORM.

Lab Course details (e.g. CHM 2922) Date

Experiment name and number

Determine the RISK

Identify the HAZARD CONTROL the Risk
(the PROBABILITY that harm may
(the POTENTIAL to do harm) (PREVENTING an incident)
result)

Keep well clear of sources of ignition

e.g. 1 Use of Diethyl Ether Risk of fire from ignition sources
No flames, sparks etc.
(flammable liquid) (Ether vapour has a v. low flash point)
Use in fume-cupboard
(vapour heavier than air) Medium risk
Use earthed equipment
Wear safety glasses, appropriate
Splash to skin, face, eye Personal Protection Equipment
e.g. 2 Use of acid solution.
Possible acid burn Take care when handling
Corrosive liquid
Medium risk Know location of safety shower, eye
wash
Cut, stab wound from sharp edges by Take care with use
e.g. 3 Broken glass pipette
careless handling; Low/medium risk Listen to instructions

Risks are categorised as A (Low), B (Medium) and C (High).

More details are to be found in the departmental safety manual, page 19 and 20. See next page.
Only Category A & B Risk experiments are to be conducted by undergraduates.
Control Measures:
In summary
Consult MSDSs, Prac notes and e.g. Use of Fume Cupboard.
understand what you are working
References. Wear safety glasses, lab coat.
with, what you are doing, and why.
Categorise risk, A, B or C. Consider spill procedures.
List Materials and Processes.
Discuss with demonstrator. Consider substitution of another
Read all relevant notes.
reagent etc.

Checked and approved by Date

Name Signature
 6

Definition of Risk Categories

RISKS CAN BE CATEGORISED AS A (LOW), B (MEDIUM) AND C (HIGH).

MORE DETAILS ARE TO BE FOUND IN THE SCHOOL SAFETY MANUAL.

NORMALLY UNDERGRADUATES ONLY CONDUCT CATEGORY A & B RISK

EXPERIMENTS.

Category A (low risk)

Operations not involving the handling of chemicals or the use of ionising radiation, potential UV or laser
exposure, etc. except for spectroscopic and other measurements on small quantities of non-hazardous
chemicals.

Category B (medium risk)

B1 (Fume hood advisable)
Procedures involving exposure to low-risk chemicals as in solvent transfers, storage, drying and
extraction; chromatography; cleaning; and small scale* reactions.

B2 (Fume hood or Schlenk line required)

Procedures involving the small scale* use of chemicals that are known to be mildly toxic, irritant,
corrosive, allergenic, etc., or involve non-commercial compounds where no data are available
(low risk based on generic assumptions). Restrictions on working hours or requiring attendance
by a second person may apply.
*small scale – reactions of volume < 500 mL of flammable solvent, or distillation of < 2 litres of
flammable solvent.

Category C (significant risk)

Procedures as for Category B2 plus:
Special precautions must be taken according to the nature of the hazard (e.g. special eye protection
against UV or laser radiation, face shield, safety shield, respirator, experienced colleague in attendance,
etc.). Restrictions on working hours or requiring attendance by a second person may apply.

NB. Category C experiments are not to be conducted by undergraduates.

 7

MONASH UNIVERSITY PLAGIARISM AND CHEATING POLICY

Plagiarism – means to take and use another person’s ideas and or manner of expressing them and to
pass them off as one’s own by failing to give appropriate acknowledgement.

Cheating – means seeking to obtain an unfair advantage in an examination or in other written or

practical work required to be submitted or completed by a student for assessment.

Non-examination (e.g. laboratory reports) Plagiarism and Cheating

If there are no substantial factors to indicate that plagiarism was accidental or unintentional, plagiarism –
non-examination – will be treated as cheating.

A member of the teaching staff who has reasonable grounds to believe that non-examination cheating
has occurred, must report the matter to the chief examiner. Where the chief examiner has reasonable
grounds to believe that non-examination cheating has occurred, the chief examiner must –

• disallow the work concerned by prohibiting assessment; or

• report the matter to the relevant faculty manager.

Where a student's work has been disallowed –

• the chief examiner must give written notice of the disallowance to the student and to the associate dean
(teaching) of the faculty concerned, including advice that the student may appeal within 28 days of the
date of the written notice; and
• the student may appeal to the relevant faculty discipline committee.

Work which has been disallowed must be retained by the faculty until the appeal period has expired.

Collaboration/Collusion (Please note this!)

Plagiarism may take the form of similar work submitted by students who may have worked together. It
is essential that Lecturers provide students with clear instructions as to whether they have been permitted
to work on the assignment (prac report) jointly, or individually. The incidence of collaborative work
should be made absolutely clear.

In this subject, students frequently do experiments in pairs (and occasionally in larger groups). You are
encouraged to discuss your results and their interpretation with your prac partner(s), your demonstrator
 8

and, if necessary, the lecturer in charge of your lab class. This discussion is an important part of the
learning process. However, your lab report, including discussion and answers to questions must be
written in your own words, you must prepare your own graphs and tables and you must record all
references you use.

Coversheet for Non-examination Assessment

To minimise the incidence of plagiarism, students are required to submit a Cover Sheet for non-
examination assessment. These sheets are available from the Moodle site for this subject. ALL practical
reports (including field trip reports) submitted must be accompanied by a signed coversheet; Unsigned
reports will be not be accepted for marking and will be returned. Please note: Late submissions will be
penalized. These cover sheets will normally be on the reverse side of the Risk Assessment form.
 INTRODUCTION 9

INTRODUCTION TO CHM2922 PRACTICAL WORK

**Important Notice**

As is clearly stated in the Monash University undergraduate course outline, a passing grade in the
practical component of this unit is a hurdle requirement for this subject. Consequently, if you fail prac,
you fail the course, irrespective of how well you do on the exam. If you fail prac, you will receive a fail
grade for the whole subject and a maximum mark of 45%. Regular attendance at laboratory classes
combined with on-time submission of lab reports is an excellent start to ensuring you pass the practical
component of the unit.

Objectives of the Practical Component of this unit

The practical component of the CHM2922 course has four main aims:

• to teach instrumental physical and analytical techniques that are used in forensic, environmental
and industrial chemistry and research,

• to develop the skills by which students can critically evaluate the results of their experiments,
especially through consideration of accuracy and precision

• to reinforce concepts and ideas developed during lectures

• to develop and broaden the professional skills required of a graduate working in the area of
analytical chemistry.

As with any scientific discipline, there are some important ground rules which must be followed if
experimental results are to be valid. These include:

• Accurate recording of samples collected, field measurements, analytical methods used, and
results obtained.
• Correct, SAFE, use of prescribed methods and instrumentation. This includes the appropriate
assessment of risk before undertaking the exercise.
• Use of “Quality Assurance” (or “Quality Control”) techniques to maintain checks on the
accuracy of the measured experimental data. This is fundamentally important! Without such
information and consideration of it, no confidence can be attached to the results obtained.
Forensic applications (i.e. matters that go to court) cannot proceed without such confirmation of
accuracy.
• Careful and unambiguous labelling of sample vials in the lab and in the field.
• Reporting of results/experimental data to an appropriate number of significant figures.
 INTRODUCTION 10

• Thoughtful consideration of potential sources of error and limitations of the techniques.

Many of these issues are discussed in detail below. Please read the following information carefully
and refer to it constantly when writing up your reports.

Report Writing and Assessment

The recording of laboratory notes and the writing of chemical reports are widely regarded as important
skills. Laboratory reports are therefore an integral part of each experiment.

Before attempting an experiment

• Find out which experiment you are to perform. Lists will be posted on the CHM2922 MOODLE
site. These lists will show which experiments have been assigned to you and when.
• Experiments are all performed in Chemistry Lab (Level 7, Building 3)
• Read the laboratory notes for your experiment and complete any pre-laboratory exercises. This
should be done before entering the laboratory each week. All these experiments can be completed
well within the allowed time if the necessary preparation has been done.
• Read the safety sheets indicated for each experiment, complete the Risk Assessment analysis sheet
and have it checked by a demonstrator, BEFORE you begin any practical work. The risk
assessment sheet also contains the Cover sheet for your experiment (downloadable from
MOODLE) that must be attached to the front of each report.

Assessment

Performance is assessed in terms of (a) results in laboratory work (accuracy and precision); (b) the ability
to communicate the results in written reports; (c) general laboratory techniques; and (d) attitude to and
understanding of the experimental work.

Reports

The reports for the introduction exercises are to be handed in at the end of each of those practical
sessions. Subsequent practical reports are to be submitted on-line before the commencement of the
next practical session. Reports are to be submitted through CHM2922 Moodle page, using the
correct assignment drop box for that particular practical. There is a penalty of 10% PER DAY,
including weekends and holidays, for reports up to one week late. Please contact the academic
 INTRODUCTION 11

supervisor in charge of your session immediately (BEFORE THE PRAC REPORT IS DUE!) if
you have problems causing the report to be late. Medical certificates must be provided in the case
of illness/incapacity. Extensions can be granted in extenuating circumstances (e.g. very heavy
coursework commitments such as major assignments due, mid-semester tests etc.). Such
extensions must be applied for in writing (email is fine) to the Academic supervisor of your
session, again, before the practical report is due. Without an exemption, reports later than one week
after the due date cannot be accepted for marking.

There are three different types of report which all students will gain experience in completing. These
are: proforma reports, oral reports, and an oral (group) PowerPoint presentation.

Proformas
Most reports in this unit consist of extended proforma reports that contain prompts and guidelines for
each section of the report. The proformas can be downloaded from MOODLE. To see the prompts for
each section, you must set Microsoft Word to show hidden text. To do this, go to “File” tab, and click on
OPTIONS. Click on DISPLAY and tick the box for HIDDEN TEXT.

The proforma MUST be submitted electronically as a “.docx” file only. Signed risk assessment,
coversheet, lab notes, prelabs and excel spreadsheets can be submitted using common file extensions,
such as .pdf, .jpeg or .xls.

An example proforma with nonprinting text displayed in red (will appear grey in greyscale printing) is
shown on the next page:

Proforma reports are important because they provide clearly and accessibly very detailed examples of the
best scientific practice in recording and presenting scientific data and discussion.
 INTRODUCTION 12

CHM2922 LABORATORY REPORT NAME…………………………

Experiment #: Title
AIM:
List the main aim/s of this experiment (1-2 lines).
OUTLINE OF METHOD:
Give a brief description of the basis of the method used for analysis (3-4 lines).
EXPERIMENTAL SECTION:
There is no need to reproduce the instructions from the manual. It is sufficient to state “as in the
laboratory notes”. However, if changes were made, or if the demonstrators suggested additional
experiments or tests, these should be detailed here (4-5 lines maximum)
PRELIMINARY QUESTIONS:
Should be answered in your Carbon Copy book before the experiment is commenced and appended to this
report with your raw data.
CALCULATIONS:
Show a sample calculation only. Do not include repetitive calculations (use a spreadsheet to perform
these or write them in your carbon copy notebook).
SAMPLE DATA:
Tables, graphs, etc.
Calibration graphs
Paste your calibration graph from EXCEL (or other graphing program) here, attach it on the page
following. Include the regression equation and R2 value. Ensure that error bars of ± 1 standard
deviation are plotted where appropriate.
DISCUSSION AND CONCLUSIONS:

REFERENCES:

PLEASE APPEND YOUR CARBON COPY BOOK PAGES AT THE END OF THIS REPORT.

LABORATORY RESULTS: Results pages from your Carbon copy practical note book must be scanned in and
submitted with your proforma. It does not matter the file format, so long as it can be read by both PC and
Mac computers. Do not use loose sheets of paper - All experimental notes must be written directly in your
laboratory notebook. These notes are a record of the progress of the experiment; and should contain
sample weights, burette readings, instrument settings, observations, etc. This section must be signed by a
demonstrator at the completion of each day’s work.
 INTRODUCTION 13

Oral Reports

Some exercises require an oral report to be completed at the end of the lab session (for example Ex. 9).
Oral reports are usually carried out by group, but assessment includes an individual evaluation of how
well you understood the exercise and prelab questions. Oral reports are important because you receive
immediate feedback about the practical exercise, because as a professional chemist you will often be called
upon to discuss your results face to face in formal and informal settings and because once the oral is
completed so is your practical exercise: you have nothing more to hand in.

PowerPoint Presentation Report

Clayton campus: One exercise (Ex 5) is completed over two sessions and requires completing a group
PowerPoint presentation report as assessment. Details about this report can be found in Exercise 5. Such
reports are important because this is a very common way for professional scientists to report and share
data. In preparing and creating this report you will have more opportunity to understand infrared
spectroscopy principles in depth, reinforcing the lecture material if you have the lectures before doing the
exercise, or preparing you to better understand the lecture material if you have the lectures afterwards.
Malaysia campus: Ex 9 report is to be in Power-point Presentation form.

Keeping Laboratory Records

All experimental records and field data must be recorded in a record book (with a strong preference for
the carbon copy type – this will allow you to keep a copy of your records after handing in the originals
with your practical report). Recording results on the practical manual, pieces of paper towel, backs of
hands, mobile phones and even more unlikely media is strictly forbidden.
A typical lab notebook entry should include:
• Name of Analyst (and Partner(s)).
• Title of Experiment (and reference to the method used)
• Date commenced and finished
• All experimental observations and calculations. Preparing tables beforehand will save you time
in the lab.

It is also helpful to make notes of any unusual events or outcomes (e.g. unexpected results) and possible
explanations discussed with your demonstrator. This makes writing your discussion section of your
report much easier.
 INTRODUCTION 14

Laboratory Practice
The quality of your experimental results obviously depends on how well you adhere to the prescribed
experimental procedure. Some general tips are given below:

• Always use clean glassware (i.e. rinsed with deionised water). If in any doubt, assume that it is
dirty!
• Label all glassware reagents, samples and standards with waterproof marker pen.
• When the method calls for accurate measurement of volume or mass, use volumetric glassware
(auto pipettes, pipettes, burettes and volumetric flasks) and the analytical (4-place) balance
respectively. For less accurate measurements, measuring cylinders and top-pan balance can be
used.
• Ensure that all instruments are properly zeroed and calibrated before collecting.
• Any deviations from the prescribed method should be noted and explained.

Moot Court Exercise

The final exercise (carried out in Weeks 11 and 12) is a ‘moot court’ case. In this exercise you will apply
(in groups) everything you have learned during the CHM2922 practical course: recording and keeping
accurate scientific records, evaluating and presenting scientific data, explaining and defending results in
front of others, working as part of a group - and some chemistry! A ‘moot’ case is a mock or made-up
court case which needs to be argued. Here the case will be based on one of the lab exercises you have
completed during semester. You will be part of either a prosecution or defence team in a committal hearing
where a jury will decide if your case should proceed to court. You will prepare and present not legal but
scientific argument about the quality of your own and others’ data and what conclusions can validly be
drawn from it – exactly what scientists do, but hopefully in a fun and entertaining way. Full details will
be available before Week 11.
 EXPERIMENT 1 15

EXPERIMENT 1. MEASURING THE ALCOHOL CONTENT OF WINE USING GAS

CHROMATOGRAPHY

Forensic Context
As an analytical chemist, you have been approached by the legal firm, Argue and Phibbs, on behalf of
their client, to determine whether there are grounds for a civil damages case against a wine producer. The
client has lost his license because he was found guilty of driving with a blood alcohol content (BAC) over
0.05%. His defence, which was not upheld in court, was that although he had only consumed three standard
drinks1 in the space of two hours, his BAC was over the limit because the wine was incorrectly labelled,
and had a higher alcohol content than specified, for reasons unknown. The client has a cask of wine from
which the drinks were dispensed and has asked that the alcohol content be determined preparatory to
pressing his claim for damages for loss of license and resultant loss of income.

Objectives
• To determine the content of wine by gas chromatography using two methods of quantification:
by the calibration and internal standard methods.
• To determine the chromatographic elution parameters: capacity factor, and resolution.

Safety
Use of ethanol and acetone. Check flammability
Use of GC – care should be taken to avoid needle injuries or burns from the heated injection port or oven.

Introduction

Principle
Gas-Liquid Chromatography (GLC) is a technique that enables separation and quantification of a multi-
component mixture. A sample of a mixture of compounds of varying volatility is injected into a stream
of inert carrier gas (generally N2 or He) which flows at a constant rate through a tubular column. The
column may be packed with solid siliceous particles coated with a liquid, or it may consist of a fused silica
capillary that has an internal coating of a liquid stationary phase. The components of the mixture are

1 A “standard drink” is deemed to contain 10 g of ethanol.

Number of standard drinks = Volume of container (glass, can etc.) in L x Alcohol content (%v/v) x 0.789
www.alcohol.gov.au/internet/alcohol/publishing.nsf/Content/standard
 EXPERIMENT 1 16

carried along by the carrier gas, but on reaching the column, partition into the liquid stationary phase to
varying degrees.
The rates of transport (elution) of the different
components of the mixture through the column
will differ according to how strongly they
partition into the liquid phase. This will depend on
the relative polarities of the substance and of the
liquid phase. A non-polar substance will partition
into a non-polar liquid-coated stationary phase
more than a polar substance ("like dissolves like"),
and hence the non-polar component will have a longer retention time, tR, than that of a polar substance.

As the separated components exit the column, a sensitive flame ionisation detector registers changes in
the composition of the carrier gas stream, giving a series of signal peaks, or a chromatogram that is
recorded on a chart recorder. The area under each peak in the chromatogram is proportional to the quantity
of that component in the original mixture. If the peaks were triangles or Gaussian (normal curves) then
the peak height would be proportional to the area. However, the peaks often have an unsymmetrical shape,
and the height cannot be used as an accurate measure of concentration.

Quantifying elution behaviour

The retention time, tR, for each peak depends on:

• the partition constant or capacity factor, k', for the compound between the stationary (liquid)
phase and the mobile gas phase,

• the flow rate of the gas,

• the temperature of the column.

Provided conditions are identical (i.e. same column packing, column temperature and gas flow rate) a
particular compound should always be eluted with the same retention time. Thus, by measurement of
retention times it is possible to identify the unknown components of a mixture by comparison with the
measured retention times of a standard.

Retention time for a given substance is calculated, as shown below.

 EXPERIMENT 1 17

tR Figure 1: tM = retention time for unretained material

tM (min), called variously the "void", "air" or
A B "injection" peak, tR= retention time of solute (min),
The adjusted retention time is given by

0 t'R = tR – tM
Time (min)

The capacity factor, k', can be calculated from the following:

t R − t M t R
k = =
tM tM

For rapid chromatography, k' should ideally be in range 2 - 5; if k' <2 little or no separation from injection
 if k' >5, long retention times (slow chromatography) is observed. In
(void) peak is observed, while
practice, 1<k'<15 is considered acceptable.

When two or more components are present in the mixture, it is desirable that each is properly separated,
or resolved, from the other; overlap of one peak onto another may increase the measured peak area or
height and lead to inaccurate quantitation.

Figure 2: Resolution, R, is a parameter that is used

to measure the degree of peak separation.
2(t R B − t R A ) 2Z
R= =
wA + wB wA + wB
For baseline resolution of perfectly triangular
peaks, R should ≥1, but because the peaks are more

Gaussian in shape, R ≈ 1.5 (corresponds to < 2%
overlap).

Quantification techniques
There are several possible techniques for determining the concentration of the separated component
(analyte). These are:

The calibration technique, where prepared analyte standards are injected, and the peak area or height used
to prepare a calibration graph from which the unknown concentration of analyte in samples can be
 EXPERIMENT 1 18

obtained. This method is quite susceptible to operator error, especially if injection technique is not
reproducible, and fluctuations in flow rate.

The standard addition technique in which a sample is analysed and the peak response analysed. The
sample is then spiked with a small volume of concentrated analyte standard and reanalysed. From the
enhanced signal response caused by the known addition, the unknown analyte concentration can be
calculated.

The internal standard method, in which a known amount of another compound is mixed with the sample
(spiking) and can be used as a reference provided that a calibration is carried out beforehand. The internal
standard chosen should have a similar nature to the analyte substance and elute in the same proximity but
be fully resolved from the analyte. Addition of internal standard to both the sample and standard enables
compensation for any variations in T, carrier flowrate, and injection technique. An example of the
technique is shown in the figure below:

Figure 3: If A is the measured peak area or height

of each peak, and the same concentration of
internal standard, Y is used in both cases, then the
response ratios of X:Y can be used to calculate

the unknown concentration, CX

135
128 Cx
 
 = 20 , and hence the unknown concentration, CX = 16.9 mg/L
150
120
 

In this experiment you will:

• measure the concentration of ethanol in a wine sample using both the calibration and internal
standard methods of quantification
• determine the chromatographic parameters: tR, t'R, k' and R
 EXPERIMENT 1 19

Experimental
Reagents
Absolute ethanol Acetone
Quality Control Wine samples
Equipment
8 x 10 or 25 mL standard flasks
200-1000 µL and 1-5 mL auto pipettes
1 µL syringe
Perkin Elmer GC with BP20 capillary column (25 m x 0.32 mm id)

Setting up and operating the GC

A Set of Standard Operating Procedures will be available next to the instrument. Your demonstrator will
help you program the instrument to run your samples. Some of your analyses will be done using the GC
auto sampler. Practising manual injections can be done on a separate manually operated GC. Operating
conditions used in your session may vary from the typical operating conditions listed below for an analysis
of wine. Analysis of calibration standards containing only ethanol does not require column heating to 200
˚C
Carrier Gas: He; FID gases: H2 and Air
Detector: FID
Temperatures (ºC):
Column: Initial: 80 ˚C (hold for 4 minute). Rate: 40.0 ˚C/min, Final: 200˚C
Injector: 240 ˚C
Detector: 280 ˚C

Flow: Flow1: Pressure: 36, Rate: ~116, Velocity: 40

Equilibrium time: 3min (default)

Procedure
Dead volume determination and peak identification
Include among your samples:

• an injection of 1 µL of pentane to determine tM. If this is run manually, ensure that the syringe is
quickly inserted into the septum, and injected very rapidly. Record the retention time in minutes.
 EXPERIMENT 1 20

• 1 µL injections of pure ethanol and acetone, again measuring and recording the retention time, tR,
for each peak. Unknown peaks in the sample chromatograms can be identified using these tR
values. (Why do you need to inject the two solutions separately)?

Calibration method
Accurately prepare standard solutions of ethanol in water with concentrations of 5, 10, 15 and 20 % (v/v)
ethanol. Make triplicate injections of at least one of the ethanol standards. Include injections of the wine
and QC samples provided, and make sure you measure and record the peak areas for the ethanol peaks.
See below instructions for preparation details. Automated GC injection is usually both accurate and precise
and so, depending on the time available, you may not be required to do triplicates of every injection.

Internal standard method

Prepare a 15 % (v/v) ethanol standard that also contains 15 % (v/v) acetone. This is called the acetone
standard. In addition, for each wine sample, add the required amount of acetone to a dry standard flask to
make 15% (v/v), and dilute to the mark with the sample. These are called acetone-spiked samples because
they have been spiked with the internal standard acetone.

Make 1 µL injections of the 15 % (v/v) ethanol-15 % (v/v) acetone standard and of the acetone-spiked
wine samples. A QC is also provided, with 15% (v/v) ethanol and 15% (v/v) acetone, to determine
accuracy. Run the QC 15% (v/v) ethanol and 15% (v/v) acetone and compare the resultant data with that
generated when you ran the 15 % (v/v) ethanol-15 % (v/v) acetone standard you prepared. This
comparison is the basis of the %RE measurement.

Analysis of results
Elution parameters

Record the following information in table form: tM, tR and t'R for ethanol and acetone respectively.
Calculate k' for both ethanol and acetone. Are these within acceptable limits? Using the t R values and
estimated peak widths for the acetone and ethanol peaks to determine the resolution, R.

Ethanol quantification-calibration method

In a table, list the mean and standard deviation for the peak areas for each standard. Plot a calibration
graph of Peak Area vs Concentration, and use this, or the regression equation for the plot, to determine
the concentration of ethanol in each sample. Report the average ethanol concentration (%v/v), and the %
RSD for each sample. You do not need to use the acetone peaks in the section. You will need the acetone
peaks only for the “internal standard method”.
 EXPERIMENT 1 21

Ethanol quantification-internal standard method (acetone-spiked)

Average the ethanol and acetone peak areas for your acetone-standard. Using these average values,
calculate the concentration (% (v/v)) of ethanol in both wine and QC (see Figure 3 above). Adjust your
final ethanol concentrations based on any dilution factors. That is, any dilution to the original sample
which occurred when you were preparing your sample for analysis.

Report
Compare the ethanol concentrations obtained by the two methods with the concentrations reported on the
wine labels and suggest which is the most effective method of analysis. Are measured alcohol
concentrations consistent with the alcohol content specified on the wine label?

References
Background Reading: Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). 'Principles of Instrumental
Analysis' (Thomson, CA)
Method: Williams S. (Ed.) (1984). ‘Official Methods of Analysis of the Association of Official Analytical
Chemists (AOAC), Inc.
 EXPERIMENT 2 22

EXPERIMENT 2. MEASURING IRON IN “CONCRETE BOOTS” USING ATOMIC

ABSORPTION SPECTROMETRY
Objectives
• To learn the principles of Atomic Absorption Spectroscopy.
• To determine the iron content of three cement samples and hence solve a murder case.
• To use EXCEL to plot calibration data and carry out a linear regression analysis.

Background
A body with the feet encased in cement has been found in the Yarra River. The Forensic pathologists have
determined that the body has been in the river for 5-6 weeks and is that of a lawyer known for shady
dealings in the building industry. On the day of his disappearance, his secretary had arranged visits for
him to two major building sites in and around Melbourne. Both building companies deny that he visited
them on that day. The detective in charge decides to sample the concrete at the 2 building sites and carry
out chemical tests to determine whether the concrete is the same as that in the “concrete boots” of the
victim. You have been given ground cement samples and asked to determine the iron content of the three
samples involved (one from each building site and one from the victim’s new footwear) and report your
results. There are a number of methods of determining the Fe content and you decide that AAS should
give you the most accurate result.

Portland cement is made up mainly of lime (CaO), silica (SiO2) with small amounts of alumina (Al2O3),
iron(III) oxide (Fe2O3) and minor amounts of other oxides. The iron oxide content is typically 3.5 – 4.5%
by mass although it can vary from 1 – 8%.

Safety
Great care should be taken with concentrated HCl and when using the glass rod to break up any lumps in
the cement (remembering the tube contains almost conc. HCl).
The AAS uses flammable gases and produces a flame ~2500°C.
 EXPERIMENT 2 23

Atomic absorption Analysis

The Atomic Absorption Spectrometer, after calibration, can accurately determine concentration of Fe
solutions as low as 1 mg/L. The method for preparing Fe calibration standards, described below is suitable
for concentrations of Fe in solution between 25 – 100 mg/L.

Experimental

Reagents
• 5.5M HCl
• 2 M HCl
• 100 mg Al/L stock solution
• 100 mg Ca/L stock solution
• 1000 mg Fe/L stock solution
• Quality assurance standard: 85 mg Fe/L
• Standard Reference Material (SRM) 18 mg Fe/g

Equipment
• 12 large test tubes (2  15 cm)
• 3 large wooden pegs (as test tube holders)
• 10mL autopipette with tips
• 100 mL beaker
• 6  100 mL volumetric flasks
• 9  250 mL volumetric flasks
• 3  500 mL volumetric flasks
• Vacuum filtration system and GF/C filter paper
• Sonicator (in fume cupboard)
• 3  Glass stirring rods
• Side-arm filter flask

Procedure

Preparation of Calibration Solutions

1. Prepare a series of five calibration standards of 0 (blank), 25, 50, 75 and 100 mg/L of Fe in 100
mL volumetric flasks by appropriately diluting the 1000 mg Fe/L standard.
 EXPERIMENT 2 24

Extraction of Iron from cement by acid digestion

This needs to be done in triplicate for each concrete sample and the Standard Reference Material (SRM).
This means there will be 12 samples in total, 3 each of the 2 known and 1 unknown concrete samples, and
3 of the SRM. You should treat the SRM in exactly same way as the cement sample (except where
indicated otherwise in the instructions below).

1. Weigh out approximately 0.4 g of the cement sample onto weighing paper or a piece of filter paper.
Before weighing, gently crush any lumps of the cement into a finer powder. This will aid in dissolving
the cement sample in acid later. The SRM is provided in a plastic vial, partially crushed. Weigh out
the SRM and treat as for the cement sample.

2. Weigh the paper and cement contents on an analytical balance and then carefully pour the cement into
a large test tube (2  15cm). Reweigh the paper to determine exactly how much cement was put into
the test tube.

3. Add 10 mL 5.5M hydrochloric acid (not 2M HCl) to the test tube and agitate carefully until the cement
is dissolved. (Add another 2 ml (approximately) of 5.5M HCl if some of the cement is not yet
dissolved, although most should have dissolved.)

4. Sonicate for 10 minutes in a beaker of hot water placed in the sonicator. This will ensure most of the
cement is dissolved. A precipitate a fine suspension of silica may also form. Add a further 3 ml
(approximately) of 5.5M HCl and sonicate for another 5 minutes. Use a vacuum filtration assembly
and GF/C filter to filter the sample or alternatively, centrifuge the solution for 10-15 min at 4000 g.
Decant the liquid from the test tube into the funnel or a volumetric flask. (For the SRM use a 250 ml
filter flask as filtering could cause substantial frothing.) Use a glass rod to prevent any liquid from
running down the outside of the test tube. Rinse the test tube with 2  20mL of 2M HCl and transfer
each to the filter flask (or a volumetric flask) and then rinse with ultrapure water.

5. For the cement samples: If you are using the centrifuged method, make up to the mark with ultrapure
water in a 100 mL volumetric flask. If you are using the vacuum filtration method, quantitatively
transfer the contents of the side-arm filter flask into the 100 mL volumetric flask. Rinse the filter flask
with about 10 mL of ultrapure water and transfer this rinse quantitatively to the volumetric flask.
Repeat this water rinse step and finally make up to the mark with ultrapure water. {Note: You may
need to dilute the sample solution 5-10 times in order to have absorbance reading in the range 0.1-
1.0}. For the SRM: quantitatively transfer the side-arm flask contents into a 100 ml volumetric flask.
If frothing occurs (very unlikely for the cement sample, but almost certain for the SRM), add a small
drop of n-butanol as an anti-frothing agent.
 EXPERIMENT 2 25

Using the AAS Spectrometer

The AAS software is capable of calculating the sample Fe concentration in samples after calibration.
However, in this experiment, the GBC software is used only to take three replicate absorption readings
and to calculate the mean and relative standard deviation of both the calibration standards and unknowns.
All of these are then recorded on an adjacent printer and the results analysed by you on the laboratory PCs
using EXCEL

The analysis of Fe is performed at a wavelength of 372 nm. Initialise the instrument, with the help of a
demonstrator. Safety instructions for use of the AAS are beside the instrument. Briefly, with a
demonstrator present, light the flame, optimize the chosen wavelength and lamp orientation to achieve
maximum signal and adjust the GAIN. Zero the detector by aspirating with ultrapure water.

• Aspirate calibration standards to establish the instrument calibration, and then aspirate blanks and
samples, including the 85 mg-Fe/L QC.

• If the absorbance readings for the unknowns exceeds the absorbance of the highest calibration
standard, then you will need to adjust your dilution factors to produce absorbances within the
calibration region.

Lastly, aspirate the 100 mg/L standards of calcium, aluminium, and iron, and record the flame colour,
along with that for the samples.

Treatment of Data (to be carried out during laboratory time)

Using Excel on a PC, plot a calibration graph of the mean absorbances of the calibration standards versus
their concentration. Plot error bars of ±1 n-1. Is the calibration plot linear? If so, then fit a straight line
and carry out a linear regression. From the linear regression equation calculate the Fe concentration and
standard deviation of the solutions. Then calculate the Fe concentration of the QC, 3 concrete samples
and SRM before dilution, as well as their concentration on a sample dry weight basis. If the calibration
plot is clearly not linear, then you may need to do a polynomial fit (one of the fitting options with
“Trendline”) instead.

Compare the results obtained for the QA sample and SRM with the accepted concentration, and comment
on any discrepancies between the results.
 EXPERIMENT 2 26

Report
Submit the report proforma listing the analytical results (including uncertainties), an estimate of accuracy
together with the AAS printout, calibration graph, calculations and answers to preliminary questions. You
also need to report to the detective whether the Fe concentrations you have determined allow him to decide
from which building site the concrete boots may have come.

Reference
Background Reading: Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). 'Principles of Instrumental
Analysis' (Thomson, CA)
 EXPERIMENT 2 27

EXPERIMENT 3. MEASURING HERBICIDE RESIDUE IN SOIL USING HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Objectives

• To become familiar with reverse phase HPLC as an analytical technique.

• To become familiar with the technique of solvent extraction as part of the process of chemical
analysis.
• To analyse a sample of contaminated sand for the herbicide Atrazine.

Introduction
In this experiment a solution of the sample in methanol is injected into a stream of solvent which is a
mixture of methanol and water. The solvent is pumped through the injection chamber and into the column,
which is a tube packed with a solid that can absorb the relatively non-polar herbicide (Atrazine) from the
solution. After leaving the column the solvent passes through the detector which is a small, but simple
ultra-violet spectrometer. As a large number of substances absorb UV radiation the detector signals
whenever the composition of the solution passing through it differs from that of the solvent. The
instrument determines the area of the signal. This area is proportional to the amount of the substance
being measured.

Overview of HPLC
High performance liquid chromatography (HPLC) is one of the most widely used separation and analysis
techniques in environmental monitoring, food and beverage, and clinical and biochemical analysis. HPLC
chromatography can involve one of four separation processes, namely; partition chromatography,
adsorption, size exclusion and ion exchange.

Partition chromatography is one of the most commonly used separation modes of HPLC. This is
conceptually similar to that of solvent extraction, where a solute, A, is preferentially partitioned into a
phase of similar polarity or miscibility.
 EXPERIMENT 2 28

In the case of partition, or liquid-liquid chromatography, partitioning of the solute occurs between the
mobile phase and the stationary phase, consisting of a solid core material to which is chemically bonded
a coating which acts as a de facto liquid phase.
The affinity of the solute for the mobile phase can be quantified by the equilibrium constant for the process
(called the partition coefficient), K, which is given by:

CS
K = C
M

where CS and CM are the equilibrium concentrations of solute A in the stationary and mobile phases
respectively. Separation of solutes A, B, C, etc., therefore requires that KA, KB, KC, etc., have differing
values. This can be achieved by selection of the mobile and stationary phases of appropriate polarities.

The polarity of the bonded phase depends on the type of functional groups attached to it, as shown below,
and this in turn influences the nature of the separation.

Designation of Separation Normal Phase Reverse Phase

Typical bonded phase attached groups amino, cyano octadecyl (C18), octyl (C8), phenyl

Polarity of Stationary Phase High Low

Typical mobile phase composition Heptane/CHCl3 CH3OH/H2O

Polarity of mobile phase Low-medium Medium-high

Order of elution of solutes Least polar first Most polar first

To increase retention of solutes Decrease mobile phase polarity Increase mobile phase polarity

Separations can be performed using either a constant mobile phase polarity throughout (called isocratic
elution, i.e. mobile phase composition held constant), or by changing the mobile phase polarity by varying
its composition during the course of elution (called gradient elution).

In order to achieve high separation efficiencies, columns containing stationary phase particles with
diameters typically of 4 - 10 µm are used. Consequently, the pressures required to force mobile phase
through the columns are high (e.g. 300-400 atm), and a high pressure piston pump is required. Some
pumps have the facility to vary mobile phase composition with time and are suitable for gradient elution.
Highly reproducible sample injection is achieved using a high pressure rotary injection valve. Detection
of eluting solutes can be performed using any of a variety of flow-through liquid chromatographic
detection devices (UV absorbance, fluorescence, refractive index, electrochemical), and of these, the UV
absorbance detector is the most commonly used.
 EXPERIMENT 2 29

Experimental
There will be 3 samples of materials contaminated with atrazine. Analyse two of them for atrazine.

The experimental is divided into four sections:

• preparation of the solutions for analysis
• learning to use the instrument
• analysing the solution
• calculation and reporting of the results

The questions associated with each section are to be answered during the practical
Reagents
80:20% Methanol:Water 100 mg/L Atrazine stock solution
Atrazine Quality Control Solution Unknown samples
Equipment
Centrifuge & centrifuge tubes Sonicator 10 ml standard flasks
Glass vials auto-pipettes syringe and syringe filters
Perkin Elmer HPLC with C18 column and autosampler Autosample vials
Shimadzu HPLC (manual injection) Manual HPLC injection syringe

Preparation of Standard Solutions

You are supplied with a standard solution containing 100 mg/L (0.1g /L) of Atrazine in 80%
methanol/water solution. Transfer the appropriate calculated volumes from your prelab exercise into
column 2 in Table 1, have your demonstrator check them, and use the automatic pipettes and 10 mL
standard flasks to prepare the standard solutions listed:

Table 1
Volume of 100 mg/L Made up to volume with
Concentration solution of Atrazine required 80% methanol/water (±
required (± Typical max error for Typical max error for
autopipette2) autopipette)

1 mg/L ± 0.003 mL to 10.00±0.02 mL

5 mg/L ± 0.010 mL to 10.00±0.02 mL

10 mg/L ± 0.010 mL to 10.00±0.02 mL
15 mg/L ± 0.015 mL to 10.00±0.02 mL

2 DIN 12650 error limits for single channel air displacement pipettes under normal laboratory conditions.
 EXPERIMENT 2 30

Question 1 You have made up the solutions to the nominal concentrations given. Estimate the
uncertainty in the concentration of each of the solutions in Table 1. Estimating uncertainty
is discussed in the “Prac Tips” section in the CHM2922 Clayton Laboratory Information
section on Moodle.

Extraction of the atrazine from sand (rutile)

Question 2 Draw up a table numbering the steps 1 – 7 in the following procedure. Consider each of the
steps and identify those that it is important to carry out carefully (briefly explain why) and
those which don't require precise quantities.

Step Must be carried out Reason

quantitatively (Y/N)
1.
2.

1. Weigh accurately 0.5 g of the soil into a 15 mL centrifuge tube (4 place balance).
2. Add 5 mL of 80% methanol/H2O and place in the ultrasonic bath for 5 minutes. (Why?)
3. Centrifuge for 5 min at 2500 rpm. (Check that the centrifuge is set up properly)
4. Quantitatively transfer the clear solution into a 50 mL standard flask.
5. Repeat steps (ii) to (iv) two more times.
6. Fill the flask to the mark with 80% methanol/H2O.
7. Use a small syringe to filter the sample through a 0.45 m filter into a clean, dry glass vial.

Investigating Instrument Variables and Practising manual injection technique

Sample Injection
This can be carried out on the manual injection HPLC while the rest of your analysis is being carried out
one of the auto sampling HPLCs. Your demonstrator will help you use the manual injection HPLC. With
your demonstrator’s help, carry out a run using the 20 mg/L standard. Repeat this at least four more times
until the results are consistent. Report the results in a table headed run number peak area
Calculate the mean and standard deviation of the final four measurements.
Question 3.

(a) What is the retention time of atrazine under the conditions of the analysis?

(b) What would the retention time be if the flow rate were 0.5 mL/min?
 EXPERIMENT 2 31

Analysis of solutions using auto sampling

Analyse each standard solution and unknowns using the auto sampling HPLC. A standard operating
procedure will be available beside the instrument. Your TA will help you program the auto sample and
show you how to analyse your results using the HPLC software to measure retention time and peak areas.
Auto sampling injections are usually both precise and accurate and you may not need to carry out triplicate
measurements on every standard or sample, although you should carry out a triplicate analysis on at least
one solution. Your demonstrator will guide you about this. For each solution you run, calculate the
concentration the measured area and then determine mean and standard deviation of the concentrations.
Report your results in a table.

Solution Peak area Concentration Mean Concentration Relative Standard

(mg/L) (mg/L) Deviation (%RSD)

Calculation of results
• Using EXCEL, generate a plot of peak area versus concentration of atrazine in the standard.
• Calculate the concentration of atrazine in aqueous extract.
• Report the atrazine concentration in the original contaminated soil sample expressed as mg/kg.

Report
Your report should consist of the tables of results and the answers to the questions – including the general
ones below.

Question 4.

(a) What is the structural formula of atrazine?

(b) The herbicide simazine is more polar than atrazine. In reverse phase chromatography of a solution
containing a mixture of the two substances which would elute first?

(c) The instrument used in this practical has a UV spectrophotometer as a detector. Give two other
types of chromatographic detector that might be used.

References

Lindsay, S. (1987). 'High Performance Liquid Chromatography' (John Wiley and Sons, Chichester) 299
pp.
Background Reading: Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). 'Principles of Instrumental
Analysis' (Thomson, CA).
 EXPERIMENT 5 33

EXPERIMENT 4. DETECTING GUNSHOT DISCHARGE RESIDUES USING UV-

VISIBLE SPECTROPHOTOMETRY

Objectives
• To apply a suitable sampling method for collecting gunshot discharge residue from the hands of a
suspect.
• To use spectrophotometry for the determination of nitrite in gunshot discharge or explosive
residues.
• To determine who fired the weapon!

Introduction
The cartridge primers used in small arms ammunition commonly contain substances such as barium,
antimony and lead nitrates. The smokeless powders used as propellants consist of either nitrocellulose or
a mixture of nitrocellulose and nitroglycerine. When a rifle or handgun is fired, residue from the rapid
detonation of the primer or propellant material is usually deposited on the firing hand and the forearm.
Thus, the presence on the skin of the firing hand of elevated concentrations of antimony, lead, barium and
nitrate from the primer, or nitrate or nitrite residue from the propellant may be detected may be used as
evidence in crimes involving firearms.

To collect this evidence, the suspect's hands are either swabbed with cotton swabs dipped in 1 M nitric
acid, or alternately, the skin is sampled by applying adhesive tape and the adhering material extracted in
suitable solvent prior to detection. Minute amounts of barium, antimony and lead are usually detected
using graphite furnace atomic absorption spectrometry, but nitrates and nitrites can be determined at low
levels by either by direct UV spectrophotometry or a colorimetric reaction that is specific to nitrite
(Steinberg et al. 1984).

This colorimetric reaction involves the reaction of nitrite with sulfanilamide under acidic conditions to
form a diazonium salt that is then coupled with N-(1-naphthyl) ethylenediamine dihydrochloride to form
a strongly coloured pink dye. In this experiment, you will measure nitrite concentrations on the hands of
two persons suspected of a firearms offence using the colorimetric nitrite method.
 EXPERIMENT 5 34

Experimental
Equipment
UV-vis Spectrophotometer, with 1 cm glass cuvettes
Transparent Adhesive tape (approx. 1 cm wide)
15 screw top 15 mL centrifuge tubes.
1-5 mL and 200-1000 µL adjustable autopipette
17 × 50 mL standard flasks
Marking pen
Disposable gloves
Forceps
Stopclock or stopwatch

Reagents (Those marked * will be provided ready for use)

* 10 mM NaNO2 stock solution (0.6899 g/L)

* 100 µM NaNO2 QC solution to be diluted 1:10 to give a 10 µM NaNO2 Quality Control (QC).

Deionized water.

* Diazotizing Reagent: Dissolve 0.5 g of sulphanilamide in 100 mL of 20% (v/v) hydrochloric acid. This

solution is stable for several months if stored in an amber borosilicate glass bottle at <4˚C.

* Coupling Reagent: Dissolve 0.3 g of N-(1-naphthyl) ethylenediamine dihydrochloride in 100 mL of

20% (v/v) hydrochloric acid. This solution is stable for a one month if stored in an amber borosilicate
glass bottle at <4˚C. Discard this reagent if discoloration has occurred.

Safety
• Refer to MSDS for Sodium nitrate, sodium nitrite, N(1-naphthyl) ethylenediaminedihydrochloride,
sulfanilamide.

• **SKIN CONTACT WITH THIS REAGENT MUST BE AVOIDED**

 EXPERIMENT 5 35

Ensure that all centrifuge tubes and glassware used

is first rinsed with ultrapure deionized water
Collect samples
with adhesive tape
Place three pieces of the adhesive tape supplied on
from this area.
the left and right hand of each suspect, in the
approximate positions A, B and C shown in Figure
1. Ensure that the person carrying out the sampling
uses forceps to avoid contaminating the samples.
Gently remove the tape from each of the suspect's
hands, and place each portion of tape into a
prewashed, labelled centrifuge tube along with 10
mL of deionized water (use the autopipette). Shake
well for 1 ±0.1 minute and quantitatively transfer the
extract into a 50 mL standard flask and make up to
approximately 45 ml with ultrapure water.

Also treat blank extractions for 3 strips of unused

adhesive tape in an identical manner.

Procedure for detecting nitrite

1. Prepare a working standard of 100 µM nitrite in a 100 mL volumetric flask. Use serial dilutions
starting from the 10 mM NaNO2 stock solution provided.
2. Prepare a set of five nitrite standards (0, 2.5, 5, 10 and 15 µM) in 50 mL standard flasks and make
up to about 45 ml.
3. Using an autopipette, add 5 ml of the QC to a separate 50 ml standard flask and make up to about
45 ml.
4. Add 1.0 mL of Diazotizing Reagent to each standard solution and the Quality Control solution,
wait 5 minutes, and then add 1.0 mL of Coupling Reagent, make up to 50 ml with deionized water
and wait another 10 minutes for the colour to develop.
5. Allow 10 minutes for the complete formation of the diazo dye product, and measure the absorbance
of each sample
6. Using the 15 µM standard, obtain a visible spectrum from 400 to 700 nm (use deionized water to
zero the instrument). From this spectrum, select the optimum wavelength for all subsequent
absorbance measurements. Take care when filling cuvettes with liquid samples. Make sure to fill
cuvettes NO MORE THAN 2/3 full and always wipe the outside faces and bottom of the cuvette
with a tissue before inserting it so that no solution inadvertently enters the instrument.
 EXPERIMENT 5 36

7. Set the spectrophotometer to the optimum wavelength in the PHOTOMETRIC mode. Take
absorbance measurements IN TRIPLICATE for all standards and samples.
8. Plot a graph of Absorbance vs Concentration (use Excel, and if linear, obtain gradient, intercept
and R2 all to the appropriate number of significant figures), and use the regression equation from
this calibration plot to determine the concentration of nitrite in each sample.

Report

Calculate the mean concentration and standard deviation for each set of samples from the two suspects,
and the blanks. Remember to include any dilution factors in your calculations.

Show the results for both in tabular form:

X, n−1 X n−1 X n−1

Suspect 1 Suspect 1 Suspect 2 Suspect 2 Blank Blank

Left Hand

Right Hand

Use Excel to perform a t-test (Tools, Data analysis, Two samples assuming unequal variances) (Microsoft
1998; Miller and Miller 1993) to determine whether there is any statistical difference between left and
right hand samples, and between suspects at the 95% confidence interval (a = 0.05). Note: there are useful
files on using Excel and performing t-tests on Moodle.

On this basis, which, if any, of your suspects should be charged?

References
Microsoft (1998). Excel (Tools, Data Analysis).
Miller, J.C., and Miller, J.N. (1993). 'Statistics for Analytical Chemistry' (Ellis Horwood Limited,
Chichester.) 233 pp.
Steinberg, M., Leist, Y., Goldschmidt, P., and Tassa, M. (1984). Spectrophotometric determination of
nitrite in gunpowder residue on shooters' hands. J. Forensic Sci. 29, 464-70.
 EXPERIMENT 5 37

EXPERIMENT 5. MEASURING CHLORIDE IN MARINE WATER USING

FLUORESCENCE QUENCHING AND INVESTIGATING USING FLUORESCENCE TO
MEASURE QUININE SULFATE IN TONIC WATER

Inquiry Exercise: This exercise has two parts. In Part 1 (1 - 1 ½ hours), you will use fluorescence
quenching to measure the concentration of chloride in sea water using a Stern-Volmer plot. In Part 2
(inquiry exercise) you will evaluate a method describing how quinine in tonic water was analysed for a
court case by investigating how pH might affect fluorescence measurements. NB: in Part 2 marks will be
awarded for your inquiry, reasoning and explanation of your investigation and any measurements you
have taken.
The prelab exercise: is designed to help you understand fluorescence. Completing it will help you
complete Parts 1 & 2.
The report for Part 1: is a proforma. The report for Part 2: is a short briefing given to your TA by the
end of your lab session. (This applies to Clayton students only)

Introduction
Fluorescence spectroscopy and spectroscopic techniques based on light scattering (e.g. Raman
spectroscopy, static and dynamic light scattering) are being increasingly used in chemical and biochemical
research and in industrial applications. Some examples: fluorescent proteins are now routinely used in
biological imaging, fluorescent sensors are used for low level detection of analytes, and ultrafast
fluorescence spectroscopy is used to measure phenomena on nanosecond and picosecond timescales.
Fluorescence-based assays are also playing a rapidly increasing role in areas traditionally served by
radioactive assays. In another chemistry unit, CHM2951, you may have used probes which use
fluorescence quenching to measure dissolved oxygen in water samples. Major reasons for the wide uptake
of fluorescence based spectroscopic and analytical techniques include the high sensitivity of fluorescence
detection, non-invasiveness and the method’s environmental friendliness.

Both fluorescence and light scattering involve the irradiation of a sample with light and the detection of
the light emitted by the sample. Because the experimental set-up for the observation of both phenomena
is the same, fluorescence and light scattering are often both observed in a single experiment. Therefore,
to avoid misinterpretation of the data obtained it is important to recognize the origin of any particular
signal.
 EXPERIMENT 5 38

Objectives

• Learn how to optimise instrument parameters for a given measurement.

• Determine the concentration of chloride in a marine water sample using fluorescence quenching.
• Learn about writing experimental methods by evaluating a method description for determining
the concentration of quinine sulphate in Indian Tonic Water by direct fluorescence measurement.
• Investigate if pH changes affect the quantitative analysis of quinine sulfate content, and if so how
you can improve the accuracy of measurements.

Safety
Usual laboratory care should always be observed. Fluorescence cuvettes should not be overfilled because
spillage into the fluorimeter can cause serious damage. Fill the cuvettes NO MORE THAN 2/3 full and
always wipe the outside faces and bottom of the cuvette with a paper towel before inserting the cuvette so
that no solution inadvertently enters the instrument.

Part 1 Measuring chloride in seawater by fluorescence quenching

Equipment
Fluorimeter (Perkin-Elmer)
10 mm plastic fluorescence cells
100 mL volumetric flasks
Autopipettes

Reagents
0.05M H2SO4
5.0 mg/L quinine sulfate QA solution
QA containing known amounts of quinine sulfate and Cl-.
Sea water samples.
Chloride stock solution, 10,000 mg/L Cl-*
Standard solution of 100mg/L quinine sulphate**

*If extra 10,000 mg/L Cl- chloride stock solution is required, dissolve 16.485 g NaCl in deionized water
and make to 1 L in a volumetric flask.
**The STANDARD SOLUTION of 100 mg/L quinine sulphate needs to be prepared fresh each time.
Weigh out accurately 0.1000 g of quinine sulphate and dissolve it in water in a 1 L volumetric flask.
Sonicate the mixture for a few minutes to assist dissolution of the solid.
 EXPERIMENT 5 39

Cuvette cleanliness and alignment

To analyse a sample by fluorescence, the excitation light must enter one face of the cuvette and the
emission must pass out through the face perpendicular to the detector. Dirt, scratches, fingerprints on the
walls of the cuvette, and random changes in its orientation with respect to the incident beam all affect the
quality of the readings that can be obtained. Wipe the faces clean before inserting the cuvette into the
fluorimeter and make sure the same side is facing the light beam each time. There might be some
fluorescence from the plastic cuvette and this will appear as a small fluorescence reading from the blank
and can be subtracted from future measurements. It is best to use the one cuvette for all your
measurements, working from lowest to highest concentration and rinsing the cell several times before each
new sample is measured.

Importance of pH
In Part 2 you will investigate whether pH may affect fluorescence. Here (Part 1) the emphasis is rather on
fluorescence quenching. To observe the effects of quenching clearly without introducing another possible
variable, you should check that the solutions you are using are all at the same pH (here pH 3) and adjust
with 0.05M H2SO4 as needed.

Quenching
Fluorescence quenching is an important consideration which must be investigated in the development of
any analytical fluorimetric method. A chloride ion can quench quinine fluorescence, and in some cases,
this can be used as the basis for analysis of the quenching substance. In Part 1, the chloride concentration
in a sample of estuarine water is determined from the chloride quenching of quinine sulfate fluorescence.

The relationship between fluorescence and quenching is given by the Stern-Volmer equation:

F0
F = KSV[Q] + 1

where: F0 = fluorescence intensity without quenching,

F = fluorescence intensity quenched by [Q],
[Q] is the concentration of the analyte, and
KSV = Stern-Volmer constant.

The Stern Volmer constant, KSV = kqt0,

where kq = rate of quenching, and t0 = fluorescence lifetime (sec)

 EXPERIMENT 5 40

F0
Thus a plot of F vs [Q] should yield a linear plot (Figure 4).

Fo/F A plot of this type can be used to

determine:
(a) the concentration of quenching
Gradient
agent in the presence of a known
Unknown
= K SV
amount of a fluorescent species, and
(b) the rate of quenching, kq from

KSV = kqt0 if t0 is known.

[Q]
[Q] unknown

Figure 4: Example Stern-Volmer plot

Procedure
Optimisation of instrument conditions using a commercial fluorimeter
High end commercial fluorimeters are sophisticated and flexible (but costly) instruments that require user
knowledge and operational expertise. Fluorescence emission and excitation spectra are recorded by
scanning the appropriate wavelength range to yield fluorescence intensity as a function of either the
emission or excitation wavelength. Control of the excitation and emission bandpasses (the spectral “width”
of the excitation or emission light) allows the extent of fluorescence intensity reaching the detector to be
controlled. This allows for collection of spectra with optimal signal to background ratios and spectral
resolution for the application at hand. Further control over fluorescence collection conditions is available
via the detector voltage and integration time.

• Prepare a 10 mg/L quinine sulfate standard solution using the correct amount of 0.05M H2SO4 to
give a solution of pH 3 and fill a fluorescence cell. (This solution is the highest concentration
standard you will use for this exercise – see below)
• Using your knowledge (see pre-lab #4!) of the absorption and emission maximum wavelengths of
quinine, choose suitable excitation wavelength (ex) and a suitable range for emission collection
for your samples.
• Collect the spectrum. Take care to avoid Raman and Rayleigh (including 2nd order) scattering and
ensure that you are using the full dynamic range of the instrument. This can be achieved by
 EXPERIMENT 5 41

variation of the excitation and emission bandpasses in the first instance or adjustment of the
detector voltage if necessary.

Measuring Seawater samples

• Prepare six solutions containing 0, 50, 100, 300, 1000 and 2000 mg/L chloride, 0.10 mg/L quinine
sulfate and the correct amount of 0.05M H2SO4 ( 1.00 mL into a 100 mL flask) to give solutions of pH
3.
• In triplicate, pipette 50 mL of the unknown chloride solution into a 100 mL volumetric flask, ensuring
that the solutions also contain 5 mg/L quinine sulfate and the correct amount of 0.05M H2SO4 to give
a solution of pH 3 when it is diluted to the mark with deionized water.
• For one of the triplicates for each concentration of chloride, record a fluorescence emission spectrum.
You should aim to use the full dynamic range of the instrument, i.e. start with the “least quenched”
solution (check with your demonstrator if you unsure!) since this will have the highest intensity of
quinine fluorescence and adjust the measurement conditions so that the intensity of this solution is
>900 counts at the emission maximum.
• Once you have measured spectra at each of the [Cl-] conditions, you can use the point measurement
function (which gives intensity at a user designated wavelength) to measure the other two solutions at
each [Cl-]. You should zero the instrument before each reading or run a control measurement of the
fluorescence of an H2SO4 solution at pH 3 under the same conditions and subtract any fluorescence
intensity of this solution from your quinine standards.
• Measure the fluorescence intensity of each of these solutions thrice.
• Plot F0/F against the molar concentration of chloride and calculate the Stern-Volmer constant, Ksv.

• Calculate the quenching rate constant if the fluorescence lifetime of quinine sulfate in water in the
absence of quencher is 18.9 nanoseconds.
• Calculate the Chloride concentration of your seawater sample.

Part 2. Inquiry exercise: Investigating the effect of pH on measuring quinine in tonic water using
fluorimetry.

Equipment and reagents available.

All reagents and equipment for Part 1, plus:
Tonic water.
Quinine sulfate.
Dilute acids and bases.
pH buffers 4, 6.8 and 9.
 EXPERIMENT 5 42

The Problem.
You and your team are expert analytical chemists. You have been approached by Argue, Phibbs and Lise,
a legal team defending Schweppes against accusations of supplying Schweppes Indian Tonic Water with
0.2g/L less quinine added to each bottle than required. They have supplied you with a copy of the method
(see below) used by prosecution chemists to provide the quantitative data on which the court case rests.
Your challenge is to use your chemical expertise to discover, demonstrate and explain any significant
flaws in the provided method so that the defence can present this information in court in defence of their
client. You may also suggest an improved method for the determination which would give more accurate
and precise results.

How you proceed and the approach you take to solve this challenge is up to you. Recall what you have
learnt in the prelab and from Part 1. You can also ask your TA questions about why measured fluorescence
might change with pH.
And remember, the most important thing here is what you learn, demonstrate and can explain about
fluorescence, fluorescence measurement, its quantitation, its errors, and the factors which influence them.

Method used by prosecution chemists to quantitatively determine quinine (as quinine sulfate) in
Schweppes Indian tonic water.

Reagents: 0.05M sulfuric acid, 5.0 mg /L Quinine sulfate Quality Control standard,
100 mg/L standard solution of Quinine Sulfate (100 mg/L), Schweppes Indian Tonic Water.
Equipment: Agilent Cary Eclipse fluorimeter, 10 mm plastic fluorescence cells, 100 mL volumetric
flasks, autopipettes.
Procedure:
A set of quinine sulfate (QS) standard solutions (0, 0.02, 0.04, 0.06, 0.08, and 0.10 mg/L were made to
pH 4 in 100 mL volumetric flasks. (Pipette 0.10 mL 0.05 M sulphuric acid into 100 mL flask)

For each standard, use the point measurement function (gives intensity at a user designated wavelength)
to measure the emission intensity. This is repeated four more times by refilling the cuvette each time. The
mean and standard deviation was calculated. The instrument was zeroed with H2SO4 solution at pH 4
before each reading. The data was plotted as an intensity vs QS concentration calibration curve, with error
bars determined by the calculated standard deviations, and a line of best fit. The QC (you need to dilute
it) was measured.
 EXPERIMENT 5 43

0.10 mL of the Indian tonic water was diluted to 100 mL and fluorescence was measured at pH 4 and
recorded five times as for the standards above. Taking the dilution factor into account and using the
calibration curve, the quinine concentration in the tonic water sample, mean and standard deviations were
calculated.

To help you begin your investigation:

Initial Experiment

To help you start off your investigation, you should know a something (from theory) about the effect of
pH on fluorescence.
Fill a cuvette 2/3rds full with the 0.06 mg/L quinine sulphate solution. Measure the fluorescence using the
appropriate emission parameters and note the fluorescence intensity.

Carefully add 1small drop by pasteur pipette of 0.05 M H2SO4 to the cuvette taking care not to spill any
solution into the instrument. Make sure the solution is mixed. Measure the intensity under the same
conditions as before. What can you say about the effect of pH on emission intensity? Add another drop of
0.05M H2SO4 and repeat the measurement. How in principle might this effect be used to measure pH?
Carry on your investigations from here…

NB: Before finishing Part 2 make sure that you have the data needed to discuss your investigation
and results.

Guide to reporting
Part 1
The report for Part 1 of this exercise is a proforma. Complete the proforma and hand in as usual.
Part 2
The report for Part 2 is a briefing given to your TA, who for the purposes of the assessment is a member
of Argue, Phibbs & Lise, the legal team representing Schweppes. You will need to explain to the TA if
there is a shortcoming in the prosecution’s method and if so, the reasons for it and why the shortcoming
is important. You will also need to present any data you have which support, in as much detail as possible,
your criticisms of the method. You will need to be able to answer questions and demonstrate an
understanding of underlying scientific principles when asked about them.
For Malaysia students, write the report according to proforma format.
 EXPERIMENT 5 44

References
Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). Principles of Instrumental Analysis (Thomson, CA)
J. R. Lakowicz, Principles of Fluorescence Spectroscopy (2nd ed.), Kluwer/Plenum, New York, 1999
 EXPERIMENT 6 45

EXPERIMENT 6. IDENTIFYING ADULTERANTS AND CONTAMINANTS IN FOOD

AND DRUGS USING FTIR AND RAMAN SPECTROSCOPY

Inquiry Exercise. In this two week exercise you will investigate FTIR and Raman spectroscopy and use
what you learn to identify contaminants in supermarket products (week 1). In week 2 lab you (as a group)
will create and present a PowerPoint report explaining how you applied FTIR and Raman theory and
principles to identify the contaminants.

Assessment. A ten minute PowerPoint presentation about IR and Raman theory, your procedure and
findings.

1. Introduction
Chemicals added to foods (food additives) are heavily regulated because many chemicals are toxic and
when added to food, accidentally or deliberately, can lead to illness or death. Sometimes economic
imperatives can make food adulteration seem tempting. For example, cheap cooking oils may be added to
much more expensive culinary oils to increase profits. This practice, while harmless to health, cheats
consumers. Other examples, however, are not so benign. Infant milk formula has been adulterated with
toxic melamine (a cheap way to increase milk’s apparent protein content), while expensive dessert wines
have been sweetened with cheap but toxic ethylene glycol. Recently, olive oil shipped to the US was
detained because of pesticide contamination fears (see http://www.oliveoiltimes.com/olive-oil-
business/north-america/olive-oil-detained/33430 ).

All this makes the problems of detecting and identifying contaminants and adulterants in food and drugs
important to solve. Among the useful techniques employed for detecting contaminants, because of their
low cost and ease of use, are Infrared (FTIR) and Raman spectroscopy. In this exercise you will use these
techniques to analyse supermarket products (analgesics and olive oils) suspected of being contaminated.

Exercise timeline
Timeline for Week 1

• A minimum of 2 hours before the start of the practical exercise: complete the prelab exercise on
Moodle. You will be penalised marks and not be allowed to commence the exercise until you have
done so.
 EXPERIMENT 6 46

• Complete Investigation 1: investigating how FTIR and Raman theory can be used to identify
chemical compounds. IR spectra are provided to give you some practice in identifying some known
compounds.
• Complete Investigation 2: using FTIR and Raman techniques to detect and identify
contaminants/adulterants in supermarket analgesics or olive oils.
• Begin preparing your PowerPoint presentation if time allows.
• By the end of the first practical session you should have (as a minimum):
o Saved hard copy or digital versions of all spectra collected
o Identified the contaminant/adulterant in your sample/s
o Labelled the Raman or IR spectral features important in your identification
o Began planning your PowerPoint presentation and know what material you will need
to have on hand to create and deliver it in Week 2*
o Exchanged email addresses with your partners and/or arranged to meet to finalize any
preparation and extra materials/additional research required to make your PowerPoint
presentation.
o Discussed and confirmed each of the above points with your demonstrator before you
leave the practical session.

*What is required in your PowerPoint presentation:

• A short introduction (2 to 3 minutes) on the basics of IR and Raman theory as it applies to
identifying functional groups and other molecular structures using IR and Raman spectra.

• Which contaminants you found in the supermarket products.

• Your reasoning supporting your contaminant identification, including NB: any IR and Raman
spectral peak assignments supporting your claims, referring as necessary to the IR and Raman
spectra you have collected.

o A spectral assignment usually includes which bond in a compound is vibrating and often
what sort of vibrational motion is involved. You will need to discuss this with your
demonstrator some time during Week 1.

If you identified pharmaceuticals contaminants you must include:

• The functional groups and spectral regions which allowed you to uniquely distinguish your
identified contaminant/s from other possible contaminants and the pharmaceutical’s active
constituent.
 EXPERIMENT 6 47

If you identified food oil contaminants you must include:

• How you can be certain that the claimed adulterant is not naturally part of the product.

• How you could determine the concentration of the adulterating oil in the contaminated olive oil.

For Week 2

In this session you will complete and give your PowerPoint presentation.

Timeline for Week 2

• See your demonstrator at the session’s start to discuss your progress to date.
• Continue making your PowerPoint demonstration.
o By the end of Week 1 you should have already have finalized the materials/files you need
to complete the presentation and decided approximately who will be presenting what parts
of it. Every group member needs to have contributed and presented some part of the
presentation.

If you have prepared your material as advised and followed the above advice you should easily be ready
to give your PowerPoint presentation by 5.30 pm, if not earlier. Arrange the time with your demonstrator
as soon as you are ready. You should expect to be finished by 6.00 pm.

WEEK 1
INTRODUCTION: BASIC THEORY AND PRINCIPLES OF FTIR AND RAMAN
SPECTROSCOPY

Approach
In order to identify compounds by FTIR and Raman spectroscopy you will need to understand some basic
spectroscopic theory. Read the theory in this section and discuss with your demonstrator both the prelab
exercise and the main points in this section. You may want to refer to the Animol exercise you completed
in Week 1 or Week 2 of semester to help you apply IR and Raman theory. After reading the basic theory
below, practise analysing and interpreting IR spectra of known compounds using the spectra provided
(Investigation 1). Finally, apply the theory and principles you have learned to identify contaminants in the
supermarket drug or food products (Investigation 2).

Basic IR Theory
 EXPERIMENT 6 48

The theory of vibrational spectroscopy can be approached on many levels and further discussions are
available from the references below and many others:
• The quantum mechanical theory and symmetry necessary to fully understand the background can
be found in simplified form in books such as “Modern Spectroscopy” – J. M. Hollas, John Wiley
and sons.
• A short description can also be found in “Principles of Instrumental Analysis”, Skoog Holler and
Nieman 7th edition or the 4th edition by Skoog and Leary, Saunders press.
• Toby Bell’s CHM2922 lectures on FTIR and Raman spectroscopy (in the Lecture Notes section
of CHM2922 on MOODLE).

Very simply, every molecular compound has a unique arrangement of atoms and bonds – its molecular
structure. The molecular bonds in this structure are constantly vibrating in different directions. Each bond
vibration requires energy. The exact amount of energy required depends upon which atoms make up the
molecule and the unique arrangement of their bonds. FTIR and Raman spectroscopy measure the
vibrational energies of these bonds and produce them as IR and Raman spectra.

Greater detail about the basic terms and concepts involved in infrared spectroscopy, including its physical
basis in terms of the vibrations of molecular bonds, is provided below.

The vibrations within a molecule.

A non-linear molecule with N atoms in a molecule can move along any of the x, y or z Cartesian axes and
so the molecule has 3N degrees of freedom of movement. For a non-linear molecule, 3 of these total
degrees of freedom are the result of translation of the whole molecule along three axes and three are the
result of rotation of all atoms around the 3 axes. The remaining 3N-6 degrees of freedom are due to inter-
atomic motions and so represent the number of possible vibrations within the molecule.

These possible motions are termed normal modes (in a normal mode of vibration all the nuclei vibrate at
the same frequency but with differing amplitudes).

Group vibrations.
In some cases, these normal modes consist of motions which are essentially localized to parts of the
molecule and are then called group vibrations. For example, in the ethanol molecule, the O-H group
consists of a light H atom attached to a heavy O atom and the motion consisting of vibration of the O-H
group is essentially independent of the rest of the molecule.
 EXPERIMENT 6 49

Functional groups and the functional group region.

Substituting the CH3CH2- part of the molecule (in this case ethanol) with a different hydrocarbon group
has a minimal effect on the vibrational frequency and so the O-H stretching frequency in all alcohols is a
similar value. Above 1500 cm-1, where the group vibrations occur, is often termed the functional group
region.

Skeletal vibrations and the fingerprint region.

Many normal modes consist of coupled motions of straight chains, branched chains or rings. These are
called skeletal vibrations and the IR frequencies tend to be specific to a particular type of molecule. The
region where the skeletal vibrations occur is called the fingerprint region and occurs from about 1300 –
200 cm-1.

Correlation tables.
Group vibration and skeletal vibration wavenumber values are tabulated in correlation tables. Extensive
correlation tables can be found in (for example): Spectroscopic Methods in Organic Chemistry, D.H.
Williams and I. Fleming, McGraw Hill. A shortened table of group wavenumber values is given in Table
1 below.

Compound identification by IR spectroscopy.

The vibrating movement of a particular bond, such as the carbon-oxygen double bond C=O, will always
result in the absorbance of energy of nearly the same frequency, with slight differences being caused by
the type of functional group it is (carboxylic acid, ether, ester, aldehyde). That vibrational frequency is
characteristic of the bond length, bond strength and the masses of the atoms which form the bond. It is
therefore possible to infer the presence of particular bonds and so elucidate the molecular structure of a
compound by analysing the IR absorbance (or transmittance) spectrum of a compound provided that the
spectrum is collected at sufficient resolution. The greater the resolution the more structural detail of a
compound may be inferred.

Modern Infrared spectrometers are termed Fourier Transform Infrared (FTIR) Spectrometers and are
based on the principle of recording an interference pattern produced from a Michelson interferometer and
mathematically transforming the output to produce a plot of absorption intensity versus wavenumber. A
description of the basis of this instrument can be found in “Modern Spectroscopy” – J. M. Hollas. The
 EXPERIMENT 6 50

sample is irradiated with an infrared beam and radiation is absorbed by the sample at wavenumber values
that coincide with the allowed vibrational energy changes.

In a modern Raman spectrometer, a laser beam is used to irradiate the sample and the Raman scattered
radiation is collected and dispersed on to a 2 dimensional CCD (charge coupled device) array detector.
The small amount of Raman scattered light is shifted in frequency from the original radiation and the shifts
depend on the changes in vibrational energy. The Raman spectrum is thus a plot of intensity versus Raman
shift.

Raman and FTIR spectra are complementary in that they both contain spectral information on
vibrational energies. The group frequency correlation tables can thus be used to understand both Raman
spectra and IR spectra. In general, bands that are weak in the IR are strong in the Raman and vice versa.

INVESTIGATION 1: INTERPRETING THE IR SPECTRA OF KNOWN COMPOUNDS

On the following pages the IR spectra and molecular structures of three compounds are provided: Caffeine,
Xanthin and Phenacatin. Using any or all of the resources provided below you should:

1. Identify and clearly label the important peaks on each spectrum.

2. Write down the important structural features and label the peaks in the spectra which you could
use to identify these drugs using infrared spectroscopy.

Question. Which spectral features could you use to definitively differentiate among these compounds?
Explain. Make use of the provided structural diagrams in your explanation if necessary.

Table 1 below provides contains the typical vibrational frequencies of some organic functional groups.
 EXPERIMENT 6 51

TABLE 1.
Bond Type of Compound Frequency Intensity
Range,cm-1

C–H Alkanes 2850-2970 Strong

1340-1470 Strong

H
C–H Alkenes C C 3010-3095 Medium
675-995 Strong

C–H Alkynes (–CC–H) 3300 Strong

C–H Aromatic rings 3010-3100 Strong

690-900 Variable

O–H Monomeric alcohols, phenols 3590-3650 Variable

Hydrogen-bonded alcohols, phenols 3200-3600 Variable, sometimes broad

Monomeric carboxylic acids 3500-3650 Medium

Hydrogen-bonded carboxylic acids 2500-2700 Broad

N–H Amines, amides 3300-3500 Medium

C=C Alkenes 1610-1680 Variable

C=C Aromatic rings 1500-1600 Variable

CC Alkynes 2100-2260 Variable

C–N Amines, amides 1000-1360 Strong

CN Nitriles 2210-2280 Strong

C–O Alcohols, ethers, carboxylic acids, esters 1050-1300 Strong

C=O Aldehydes, ketones, carboxylic acids, esters 1690-1760 Strong

NO2 Nitro compounds 1500-1570 Strong

1300-1370 Strong

Many books on IR Spectroscopy or Organic Chemistry have such tables. A more comprehensive table can
be found at: http://www2.ups.edu/faculty/hanson/Spectroscopy/IR/IRfrequencies.html

Many websites provide useful tools to help with IR spectra identification. One such website is provided
by the University of Potsdam at http://www.science-and-fun.de/tools/. Enter a number in the “IR-Wizard”
for possible suggestions to help with peak identification
 EXPERIMENT 6 52

IR and Raman Spectra databases can be very helpful in interpreting spectra and identifying compounds.
A website with a very powerful searchable database, from which the spectra below (and many others) are
available, can be found at: http://sdbs.riodb.aist.go.jp/sdbs/cgi-bin/cre_index.cgi?lang=eng

To use this website, agree to the disclaimer, then enter a compound name or formula and select for an IR
or Raman spectrum.

The following papers detailing FTIR and Raman spectra and vibrational assignments of (i) sodium
salicylate and (ii) xanthine, caffeine and related molecules can be found in CHM2922 on Moodle, in the
“Clayton Lab Information” section under “Additional info for practicals”:

• (i) Philip D. et al. (2001) Spectrochimica Acta Part A vol. 57 pages 1561-1566.
• (ii) Ucun F. et al. (2007) Spectrochimica Acta Part A vol. 67 pages 342-349.

Discuss your labelled spectra with a demonstrator before continuing to the next section.

Caffeine
 EXPERIMENT 6 53

Xanthine

Phenacetin
 EXPERIMENT 6 54

INVESTIGATION 2: COLLECTING AND INTERPRETING IR SPECTRA OF MIXTURES OF

KNOWN AND UNKNOWN COMPOUNDS: ANALGESICS AND OLIVE OILS

Once you have become familiar with IR and Raman theory and are comfortable identifying, interpreting
and labelling peaks observed in IR and Raman spectra, you should begin considering how to identify
compounds in supermarket analgesics and olive oils which are complicated mixtures of known and
unknown compounds. To help you with this a range of information and materials is provided, including
molecular structures, some published papers on olive oil adulterant identifications, and materials and
samples relevant to supermarket analgesics and olive oils. Your demonstrator will assign you a
supermarket product for investigation. NB: it is worth remembering that often, bonds which show up
poorly in IR usually show up clearly in Raman and vice versa, so you may need to use both IR and Raman
spectra to identify an unknown contaminant.

Some questions to consider:

Analgesics
• Analgesics do not contain just an active ingredient but may contain other materials as well. How
will you argue that a particular peak on an IR or Raman spectrum belongs to a suspected
contaminant rather than to a legitimate ingredient?
• Analgesics could contain more than one contaminant. Having identified one contaminant, might
there be more in the product you are investigating? How could you argue one way or the other?
• How will you argue that a particular IR or Raman peak belongs to a contaminant if the contaminant
is structurally very similar to the active ingredient?
• It is not enough in this exercise simply to compare the IR and Raman spectra of the uncontaminated
and contaminated materials to identify contaminants. Uncontaminated (reference) materials or
identical uncontaminated products will not always be available to you. Product batches may
change and products alter as they age. Does a suspected contaminant have unique structural
features allowing you to identify it with certainty? Where would these show up on an IR or Raman
spectrum? How can you use such features to make your arguments and draw conclusions more
convincingly?
• How can you use IR and Raman theory to make your arguments more convincing?

Some useful molecular structures relevant to analgesics are provided below:

 EXPERIMENT 6 55

You may wish to refer to simulated spectra from the Animol program to help with your contaminant
identification. However, Animol is only a simulation program, so its assigned vibrational frequencies
cannot be taken as completely accurate. In particular, Hydrogen bonding between molecules often
changes the absorbance wavenumber of a particular bond, usually to lower wavenumber - especially
important when comparing the theoretical peaks from Animol with the real spectra. This also explains
why some peaks are translated slightly in the unknowns.

Olive oils
Some questions you consider when identifying olive oil and its adulterants:
• Olive oils are natural materials, and such materials are usually complex mixtures. So, what is the
expected makeup of olive oils? Is it always the same?
• What are the most common adulterants in olive oils?
• Are there any compounds in the adulterating oils which are different to those found in pure olive
oils? If not, what other differences are there between olive oils and adulterating oils? How could
you use IR and Raman spectroscopy to detect such differences?
• What bonds and molecular structures in olive oils and adulterants can be identified using Raman
or IR spectroscopy?
• The greater the amount of cheap adulterating oil added to an expensive olive oil, the greater the
profit realised when selling it, so olive oil adulterants are often present in quite high concentrations.
How does this help you in identifying an adulterant?
 EXPERIMENT 6 56

Some useful websites:

• Information about olive oils and their makeup can be found at:
http://en.wikipedia.org/wiki/Olive_oil.
• Because they are very cheap, coconut oil, sunflower oil or canola oil are some common adulterating
oils. A useful table listing and comparing constituents and their relative amounts in all these oils
can be found at http://en.wikipedia.org/wiki/Coconut_oil.

Some useful papers on identifying adulterants using Raman and FTIR:

• Poiana et al. (2012) on FTIR identification of olive oil adulterants

• Zou et al. (2009) on Raman identification of adulterants.

You can access them via CHM2922 on Moodle, in the “Clayton Lab Information” section under
“Additional info for practicals”.

Talk over your ideas on how to identify contaminants with your demonstrator if necessary and then
proceed.

Staff will be on hand to help you use the IR and Raman instruments, master ATR techniques and to discuss
choosing appropriate values for instrument parameters including resolution, number of scans,
wavenumber range etc. Collect the necessary IR and Raman spectra to identify the contaminant(s) in your
assigned supermarket product.

After acquiring satisfactory spectra, you may wish to improve them to make identifying functional groups
within molecules easier. This may involve baseline correction, peak picking and labelling (ask a
demonstrator for help if necessary).

You will need to refer to the IR and Raman spectra you have collected, molecular structures, functional
groups and/or other vibrational modes etc., so make sure you have saved this information in a suitable
format for your PowerPoint report.

REPORT.
The report for this exercise is a 20 minutes (maximum) PowerPoint presentation. You will produce your
presentation as a group, but your final mark will be assessed individually.
 EXPERIMENT 7 58

EXPERIMENT 7. INVESTIGATING PROBLEMS OF FLUORIDE ANALYSIS BY ION

SELECTIVE ELECTRODE

This is an inquiry experiment. In it you will investigate the theory and problems associated with using
Ion Selective Electrodes (ISEs).

In Part 1 relevant background theory is provided to allow you to investigate the problems of making
accurate measurements with a Fluoride ISE.

In Part 2 you will investigate how to analyse water samples quantitatively.

In Part 3 you will consider how to analyse more complex solid samples.

Apply to Clayton students only: There is no formal write-up required for this practical. Assessment will
involve a short oral question and answer session during the practical, completion of a spreadsheet and a
short discussion of your results and conclusions. For details see the REPORT section below.

**Each student should ensure they have their own record of all the measurements taken**

Introduction
Fluoride is added worldwide to drinking water to help prevent dental cavities. In Melbourne its addition
is required by the Fluoride Act 1973, an acceptable concentration being about 1 mg/L or 1 ppm. At much
higher levels, fluoride can cause chronic health problems or become acutely toxic, so the concentration of
fluoride in water supplies and other consumable products (wine or toothpaste for example) must be
accurately known. Automated on-line systems using fluoride ion selective electrodes are widely used to
monitor drinking water.

Fluorine is an essential element and occurs in animals including human beings in ultra-trace amounts. In
human beings, 99% of body fluoride occurs in bone and teeth. The enamel surface of teeth is composed
of hydroxyapatite [3Ca3(PO4)2.Ca(OH)2]. Tooth decay is caused by reaction between OH groups in the
enamel and acid produced from sugars in foods by bacteria in the mouth. This produces dental caries
(cavities). Scientists have taken advantage of the fact that Fluoride ions are able to substitute some of the
OH anions in hydroxyapatite because their ionic radii are so similar (rF = 129 pm; rOH = 133 pm). This
substitution produces a substance that is resistant to acid attack. Fluoride compounds in toothpastes and
in domestic water supplies thus improve dental health.
 EXPERIMENT 7 59

Nevertheless, while trace amounts of fluorine may be beneficial, chronic overexposure may lead to
harmful medical conditions such as skeletal fluorosis. This occurs when excess fluorine in the body forms
HF, which then reacts with calcium in bone to form CaF2. The CaF2 is then removed from the body,
causing bone weakening. Consequently, many consumer goods are monitored for excess levels of fluorine.
One common example is wine, where the use of the insecticide cryolite (Na3AlF6) on wine grapes can lead
to elevated fluorine levels in wine.

PART 1 INVESTIGATING THE FLUORIDE ISE

Background Theory and Principles of the Fluoride ISE

Measurement by Ion Selective Electrode is a potentiometric method for determining the concentrations
of specific ions in solution. The pH electrode is an early example of an ISE.

The most successful of all ISEs is the fluoride electrode. It is a very stable and reproducible electrode
which can be used to determine fluoride in the 0.02 to 2000 mg/L concentration range. All ISEs operate
on the same fundamental principles.

The Fluoride ISE

The Fluoride ISE comprises an electrode sitting within a sealed membrane filled with an electrolyte
reference solution which contains F-. The membrane used in Fluoride ISEs is crystalline Lanthanum
Fluoride (LaF3) usually doped with a small amount of Europium fluoride (EuF2) to enhance membrane
conductivity. The Europium Fluoride achieves this by creating gaps in the LaF3 lattice just the right size
for Fluoride ions (rF = 129 pm) to move between.

Fluoride ions migrate into or out of the lattice surface depending upon the concentration of fluoride in
solution. When a Fluoride ISE is placed in a solution, the inner lattice surface of the membrane remains
in contact with the reference solution, while the outer lattice surface contacts the solution to be measured.
When the fluoride concentrations of the solutions on either side of the membrane differ, fluoride moves
into or out of the outer membrane surface, causing it to become differently charged compared to the inner
membrane surface. A measurable potential difference is thus created across the membrane surface, its
magnitude related to the difference in Fluoride concentrations between the two solutions.

Measurable Potential and the Nernst Equation

At 25° C, the measured potential is described by the Nernst equation:

 EXPERIMENT 7 60

2.303RT
E=k+ log a −
nF F

or, under the conditions where activity can be replaced by concentration,

E = k '+
2.303RT
nF
log F 
−

Note: F is the Faraday constant; F- represents the fluoride ion; k and k’ are constants, and n is the ion
charge (-1 for F-).
Replacing R and F by their appropriate numeric values, T by 25oC, and n by the F ion charge of -1, gives:

E(mV ) = k'−59.2log F 
−

an equation of the form y = m(x) + c.


Investigation 1. How to measure Fluoride reliably using an ISE
You should spend no more than an hour investigating the problems of how to measure accurately and
reliably fluoride in complex solutions (solutions which may contain a variety of dissolved ions).

For Fluoride measurements to be useful they must be reliable across a variety of measurement conditions.
Drinking water often has aluminium sulphate added to remove suspended solids. It may also contain
significant concentrations of other dissolved salts and vary in pH. These are very frequently encountered
environmental variables and so an obvious question is: do they affect Fluoride measurements? So you
should begin by investigating the reliability of measurements made under these conditions.

Experiments
Transfer (measuring cylinder) about 10 ml or so of the 1 mg/L Fluoride QC Standard (about 10-4 M) into
a 50 ml sample vial, set a stirrer bead stirring, suspend the Fluoride ISE in the solution and record the
potential.

(1) Begin making measurements by adding 0.1 ml (autopipette) of Al2(SO4)3, observing the change in
voltage. Now add 0.1mL of 0.1M EDTA. Is there a noticeable change? (2) Using a fresh QC sample,
add 0.1mL of 4M NaCl. Note any changes with potential. (3) Using another fresh QC sample, add
0.1mL 2M NaOH. Be sure to make notes as you go for you to refer to later.
 EXPERIMENT 7 61

(4) Next, start varying the amounts of the additives and the order of addition to see the effects on the
measured potentials and try to come up with some possible reasons for your observations.

Using the basic knowledge of the fluoride ISE gained above, you should begin trying to explain your
observations and think of simple ways to test your ideas. Discuss your ideas with a demonstrator and if
necessary ask for extra reagents to test them. Remember that the fluoride electrode is selective for fluoride
ions, not other forms of fluoride. What may interfere? Why? What do the forms of the Nernst equation
above predict about electrode behaviour? How can you check?

Additional useful information about how ISEs and the Fluoride ISE work can be found in the CHM2922
Extra Materials folder on a lab laptop. These references go into considerably more detail both about ISEs
and the Fluoride ISE and may help in your investigation.

Once you are satisfied about the requirements for making reliable fluoride measurements under a variety
of conditions, you should discuss it with a demonstrator. Your aim ultimately is to design as simple a
method as possible to allow accurate fluoride measurements in solutions of varying dissolved ion content
and pH range.

PART 2. SAMPLE MEASUREMENT: FLUORIDE IN TAP AND SEAWATER SAMPLES

Once you have completed and discussed Part I with your demonstrator, you should start Part II, the
quantitative analysis of tap and seawater samples.

Investigation 2. How to measure Fluoride quantitatively using an ISE

In this section you are provided with appropriate reagents and glassware, some water samples, and asked
to determine their Fluoride levels accurately.

Question.
How can you use the Nernst equation, or a variation of it, to construct a calibration curve from which to
read off unknown fluoride concentrations in samples?
 EXPERIMENT 7 62

Material available
0.100 M fluoride stock solution
50 ml volumetric flasks
50 ml sample vials (for making measurements)
magnetic stirrers, retort stands etc.
samples: tap and seawater, unknown and QC
TISAB (Total Ionic Strength Buffer)

Overcoming the drift problem when taking ISE measurements

As you may have observed, readings from Ion Selective Electrodes tend to drift before eventually reaching
a stable value. It is possible to work out how to overcome this drift problem when taking measurements
by trial and error but doing so can initially be very time consuming. So to save you some time, a reliable
method for doing this is provided below. You can of course improve upon it if you like.

Starting with the most dilute calibration solution first, add a small magnetic stirring bead and whatever
necessary reagents as determined in Part I (discuss this with a demonstrator). Keep the sample well mixed
using the magnetic stirrer when taking measurements.

To make a measurement of the potential of a standard solution, carefully blot the electrodes dry with a
tissue paper and lower into the standard solution with the stirrer gently stirring. The mV reading will drift
and may take several minutes to stabilize. A reasonable criterion of stability is that the potential should
not change by more than 1 mV over a period of 1 minute. However if this level of stability takes too long
to reach or cannot be reached, take a measurement after a specific period of time has elapsed, then take
each subsequent measurement after the same elapsed time period. Obtain three readings of potential for
each solution so that you can make an error estimate.

Procedure
You will make up a series of 5 standards, ranging from 1 × 10-2 to 1 × 10-7 M using serial dilution. Add
10 mL of the standard and 5 mL of TISAB (Total Ionic Strength Buffer) to 50 mL sample vial and measure
potential. Repeat using same ratios for all standards and samples.
Calibration curve
Record and plot your potential and concentration data in an Excel spreadsheet as you go. Determine the
line of best fit, the regression equation and the R2 value. Note that one point may deviate from the linear
 EXPERIMENT 7 63

trend, and it is sensible to omit this from your regression analysis, if it lies outside the range of your
samples.

Samples
Once you are satisfied with your calibration curve, measure the fluoride concentrations of the QC, tap
water, seawater and unknown samples.

Part 3. Investigating how to analyse solid samples

Measuring fluoride in solid samples adds the complication of ensuring that fluorine in the sample is
released into solution for measurement.

Investigation 3. Measuring Fluoride in a solid sample

For the sample of toothpaste provided, consider how best to extract its available fluorine. How will you
ensure that any reagents which you use to extract the fluoride into solution does not go on to damage the
Fluoride ISE when you are taking measurements? Is the all the fluorine extracted using your extraction
regime? Is the amount of Fluorine extracted the same as would be extracted by someone using the
toothpaste when brushing their teeth? Does that matter?
One method of extracting Fluoride is as follows. Accurately weigh 1.0 g of the toothpaste on an analytical
balance in a small beaker. Half fill the beaker with ultrapure water. Place the beaker on the hot plate in
the fume cupboard and heat the toothpaste gently until it is all dissolved. DO NOT BOIL! Remove the
beaker from the heat, cool and sonicate it for 5 minutes. Pour the contents of the beaker into a 100 mL
volumetric flask and make up with ultrapure water. Note the volume and masses for this section because
you will need to use them for converting the concentration back to mass fluoride per mass of toothpaste.
[ Remember to add TISAB before measuring the potential]
Calculate the Fluoride concentration in the original toothpaste and compare it to the advertised content.
 EXPERIMENT 7 64

Assessment

• There will be a short questions that needs to be answered based on your investigations in Parts 1
and 2.
• For Parts 2 and 3, there is an Excel spreadsheet available on Moodle to assist in data analysis.
This sheet should be filled in and referred to during the Q & A. You will be asked to report on your
calibration curve, measured fluoride concentrations, their accuracy and precision. Make sure you
save your results for the chance you will need them for the Moot Court.

References
Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). 'Principles of Instrumental Analysis' (Thomson, CA)
 EXPERIMENT 8 65

EXPERIMENT 8. MEASURING TRACE HEAVY METALS IN ENVIRONMENTAL

SAMPLES FROM THE MT. ISA MINING DISTRICT USING SQUARE WAVE
STRIPPING VOLTAMMETRY
Objectives

• To become familiar with the operation and use of a potentiostat to carry out electrochemical
measurements.

• To apply an important electro-analytical technique, square wave voltammetry (SWV), to

perform a trace analysis for a suite of heavy metals in an environmental sample using the
method of Standard Additions.

Professional Context
The National Pollutant Inventory (NPI) which can be found at www.npi.gov.au is an initiative of the
Federal Department of the Environment and Water Resources. It is an Internet database that contains
information relating to the emission of a number of substances and gives the source and location of
these emissions. In February this year Mount Isa Mines was reported as being a top emitter of some
of the following substances: sulphur dioxide, lead, copper, zinc, cadmium, arsenic and antinomy
during last year. High levels of lead have been detected in the blood of Mt. Isa children. You have
been engaged as a consultant by the local community to conduct independent testing of samples to
monitor heavy metal contamination in this regional Queensland city. The aqueous samples that you
have been given have come from several locations around the city and these have undergone some
preliminary work-up; these will be investigated using stripping voltammetry.
 EXPERIMENT 8 66

Introduction
Stripping Voltammetry
In the broadest sense, electrochemistry deals with electron transfer processes and their relationship to
chemical changes. In this experiment you will become familiar with stripping voltammetry, an
extremely sensitive trace analysis technique. A stripping experiment consists of two steps, performed
sequentially. The first step involves pre-concentration, in which metal cations are reduced from an
analyte solution and co-deposited onto a metal film (for this experiment, we use bismuth). An
oxidation step follows, in which the metals re-enter solution in a well-defined sequence which is
predictable by using the electrochemical series. Importantly, stripping voltammetry can measure a
number of different trace metals in a single scan. Although methods such as inductively coupled
plasma mass spectrometry (ICP-MS) are now available, electroanalytical instrumentation is easier to
use and far less expensive to implement, particularly if one is interested in a small number of elements.

Electroanalytical experiments are performed in a three electrode electrochemical cell, as illustrated

below in Figure 1. The electrodes within the cell are known as the working electrode (WE), the
counter/auxiliary electrode (CE) and the reference electrode (RE). The solution of interest is placed
into the cell and the cell is connected to a potentiostat. The potentiostat is an instrument that precisely
controls the potential difference between the WE and the RE. It does this by injecting current at the
counter electrode. While this potential control is being maintained the instrument measures the current
at the working electrode. Different “waveforms” are then applied. i.e. (VWE-VRE) varies vs. time
depending on the user’s application.

Figure 1. Diagram of a three electrode electrochemical cell

 EXPERIMENT 8 67

(a) Anodic stripping voltammetry (ASV)

In ASV there is a “deposition period”, during which the working electrode is held at a negative
potential compared to the reference electrode, and metal ions deposit at the Working Electrode (WE).
Thereafter, the potential difference between the WE and the RE is ramped during a computer generated
potential "sweep" as shown in Figure 2. During application of the potential sweep the current is
measured at the WE, so the experimental data appears in the form of a current vs. voltage plot.

Figure 2. Typical sweep waveform for an ASV experiment

(b) Square wave voltammetry (SWV)

SWV is a further improvement of staircase voltammetry (itself a derivative of linear sweep
voltammetry e.g. ASV). In staircase voltammetry the potential sweep is actual a series of staircase
steps. In square wave voltammetry, a square wave (Figure 3) is superimposed on the potential staircase
sweep. The current is measured at the end of each half-wave, just prior to the potential change. The
differential current is then plotted as a function of potential, and the reduction or oxidation of species
is measured as a peak or trough.

Figure 3. The applied potential vs. time waveform that is used in square wave voltammetry (SWV).
 EXPERIMENT 8 68

Risk Assessment
As always, a risk assessment form must be filled out before starting any experimental work.

In this practical experiment you will be working with the nitrates of cadmium, lead, zinc and bismuth.
These are provided as solutions in dilute acid (0.1M HNO3) at concentrations of 10 ppm. Ensure that
you read the MSDS sheets for each of these compounds and comment in your risk assessment on the
recommended acute and chronic toxicity levels (if any) in relation to their use in this experiment.

The computer-controlled potentiostat is a delicate electronic instrument and great care should be taken
to keep solutions away from electrical equipment. The same applies to the laptop that controls the
instrument – be careful not to spill solutions on it!

The experiment also requires you to work with glassware, and fragile electrodes. Please be particularly
careful with the electrochemical cell and its associated electrodes as these are expensive and not easily
replaced.

**AS ALWAYS, SAFETY GLASSES MUST BE WORN AT ALL TIMES**

Experimental

**PLAY “EXPT 8 PRELAB VIDEO” ON MOODLE (IN Prac 8, Clayton Lab Information,
CHM2922)** This video shows you the basics of how set up and operate the Voltammeter.

In this experiment, you will use Square Wave Voltammetry (SWV) to quantify the levels of trace Pb,
Cd and Zn in samples from the Mt. Isa mining district. The potentiostat that you will use in this
practical experiment is controlled through a software package. The potentiostat connects to the rear
of a BAS cell stand, whose layout is also shown in Figure 4.
 EXPERIMENT 8 69

Figure 4. the BAS cell stand.

There will be a Standard Operating Procedure next to the voltammeter. A demonstrator will show
you how to set up the voltammeter and use the potentiometer software to run the experiment. The
software program should already have the parameters which you need for the experiment loaded.
However these may vary from time to time. Your demonstrator will confirm your parameter set is
correct before you start.

Procedure
Setting up the Voltammeter.

Before proceeding, ensure that you are familiar with the stirring controls on the BAS cell stand. When
required, stirring is activated by moving switch [3] on the cell stand to the “up” position. The stirring
rate should be set to about 400 rpm using dial [4].

1. Remove the glass cell from the BAS cell stand

While supporting the cell, pivot stirrer motor [15] (Refer to Figure 4) to the right, then lower the cell
and place it on the bench.

2. Obtain and insert electrochemical cell electrodes

A glassy carbon working electrode, a Ag/AgCl reference electrode, and a platinum counter electrode
are used in this experiment.

The reference electrode is stored by immersing it in a saturated KCl solution held in a small vial. This
electrode is particularly fragile and expensive – please look after it. At the end of your experiments,
 EXPERIMENT 8 70

this electrode should be carefully rinsed in ultrapure water and returned to storage in the saturated KCl
solution!

The working electrode should be carefully wiped on a tissue before starting the experiment. Before
proceeding carefully check the surface of the glassy carbon electrode – it should be clean, shiny and
lint-free. If this is not the case the electrode may require polishing – if in doubt consult a demonstrator.

Next, insert the three electrodes into the holes in the Teflon cell top. During the experiment these
electrodes all need to dip into the solution.

3. Collect solutions for the experiment

In this experiment you will require the environmental sample, a 1 M acetate buffer solution (pH 4.5)
and a 10 ppm Bi plating solution. During the experiment you will also need a standard solution
containing 10 ppm each of Pb, Cd and Zn. Do not take solutions directly from the original containers
– when required transfer a small quantity of each into a separate, clean container prior to use.

You must ensure that any glassware you use is scrupulously clean (rinse with ultrapure water only)
and dry. Be mindful that you are performing a trace analysis and sloppy work will lead to poor results
that cannot be defended in court!!

4. Fill the cell and place it onto the BAS cell stand

An environmental sample has been provided by a Mt. Isa community action group for analysis. Before
commencing work, record details of the sample you have been given to analyse.

Clean and dry the glass electrochemical cell (the shot glass!), then weigh it on a balance before adding
17 mL of your environmental sample, 2.00 mL of the acetate buffer and 1.00 mL of the 10 ppm Bi
solution (the last two solutions should be delivered by autopipette). Perform separate weighings after
each of these additions so that you can later work out the exact concentration of each of your trace
metals during your standard addition experiments.

Again pivot stirrer motor [15] to the right. Bring the cell up from underneath, around the electrodes
and seat the cell on top. Pivot the stirrer motor back underneath the cell.

5. Make electrical connections to the potentiostat

Ensure that the USB cable from the potentiostat to the PC is disconnected at the PC end when first
making electrical connections to the electrodes. The electrode leads extend through the front panel of
the BAS cell stand. The connectors are a spring-loaded, press-on type. Simply push the connector
over the corresponding pin to make the connection. Each wire is colour coded to the electrode to
which it attaches: white to reference (R); red to counter (C); and black to working (W).
 EXPERIMENT 8 71

6. Start the potentiostat instrument control program and set your parameters

Double-click on the Potentiostat icon that is located on the desktop. This will bring up the control
screen. The required parameter set will usually be set automatically when you access the program, but
occasionally may change. For full instructions, refer to the SOP by the instrument.

7. Overview of the experiment

During each experiment a bismuth film (BiFE) is deposited onto the surface of the glassy carbon
electrode making a BiFE, and metal analyte ions also form an alloy with the BiFE. After the deposition
period the square wave stripping waveform is applied and analyte metals are stripped from the BiFE.
This process also removes the BiFE. The potentiostat is used to control this process.

8. At the end of the run, make sure you have the file.
9. Review your stripping trace
You should review your stripping trace onscreen. During the potential scan any heavy metals that
have accumulated into the BiFE during the deposition period are stripped off – each such metal gives
rise to a distinct peak. Refer to details below on data analysis.

10. Repeat steps 7-9 two more times to generate three replicate scans

11. Perform a standard addition

Use an autopipette to obtain 60 μL of the standard solution containing 10 ppm each of Pb, Cd and Zn
and add this volume of standard to the electrochemical cell. Then follow steps 7 through 10 above.

Do not remove the cell from the BAS stand – simply add the solution via autopipette through the
spare inlet port in the Teflon cell top.

Note: each standard addition increases the amount of Pb, Cd and Zn in the cell by approximately 30
ppb.

12. Perform four further standard additions as described in step 11

A total of 18 stripping scans will have been recorded after you have completed this step (6 runs x 3
replicate scans each). During these experiments you should notice a steady increase in the peak heights
for the Zn, Cd and Pb peaks as the concentration of each metal will have been increased by ~150 ppb
as a consequence of your standard additions.

13. Shutdown procedure

Your stripping voltammetry experiments are now finished but there are some important clean up
procedures that must be completed to prepare the instrument for use by the next group. Your
 EXPERIMENT 8 72

demonstrator will guide you to do this. The shutdown process will include disconnecting the leads to
the three electrodes. Next, detach the cell and empty its contents into an inorganic residues container
via the small funnel. Be careful not to lose the small magnetic stirrer when you do this!
Next rinse the cell and its associated electrodes with ultrapure water from a wash bottle. The working
electrode should then be removed and gently wiped with a tissue. The cell must be left filled with
ultrapure water and attached to the cell stand (containing the magnetic stirrer) and with both the
working and counter electrodes sitting in the water.

Finally the reference electrode should be transferred into the small storage vial containing saturated
KCl.

Data Analysis
The data files from your experiments should have been stored on the PC’s desktop in a Data Files
folder. Your files should be easily identified by the unique filenames you selected (!) and the time of
file creation.

Your demonstrator will guide you on how to use the program to analyse your data (this will involve
some peak integration).

Once you have some integrated peaks go to the CHM2922 Moodle page and open the Excel file
“Standard Additions for tute 1 and prac 8.”

There is a spreadsheet page for each of your metals. Fill in the details of your mean and standard
deviations for the peak areas, mass of the sample and concentration of the standard (highlighted in
yellow cells).

The spreadsheet does the calculations for concentration of your unknown and its uncertainty. Record
these values in your proforma. Submit your completed spreadsheet with your proforma.

For a detailed description of the Standard Addition method it is strongly recommended that you consult
your lecture notes and also refer to section 1D-3 in “Principles of Instrumental Analysis”, 7th ed, by
Skoog, Holler and Crouch.

Report
Your report should include a brief abstract and background discussing the SWV technique. Follow
this with an analysis of your experimental results and present your calculations clearly. Also compare
your observed stripping potentials for each element with the expected values based on comparison
 EXPERIMENT 8 73

with values given in a table of standard reduction potentials, as considered earlier in the prelab
questions.

A proforma is available to assist you in setting out your report.

How do the levels of Zn, Cd and Pb that you have measured compare with what would be considered
safe for drinking? The proforma requires that you write a brief summary of your findings and any
recommendations that you would provide to the community action group on the basis of your results.

References
Skoog, D.A., Holler, F.J., and Crouch, S.R. (2018). 'Principles of Instrumental Analysis'
(Thomson, Ca.)

Good specific literature references on square wave stripping voltammetry using bismuth film
electrodes are: J. Wang, J. Lu, S. B. Hocevar and P. A. M Faria, Anal. Chem. 2000, 72, 3218-3222
and J. Wang, J. Lu, U. A. Kirgoz, A. B. Hocevar and B. Ogorevc. Analytica Chimica Acta 434 (2001)
29-34.
 EXPERIMENT 9 74
EXPERIMENT 9. DETERMINE ANALYTICAL FIGURES OF MERIT OF A
ELECTROCHEMICAL GLUCOMETER
Objectives

• To measure sensitivity, selectivity, interferences, accuracy and precision of a commercial

glucometer.

• To understand how an electrochemical glucometer works.

Introduction:
Diabetes is one of the most common diseases in the world. The body is unable to produce insulin to
convert glucose into energy. The inability to control the glucose concentration in the bloodstream
results in both serious acute and chronic complications. A diabetic needs to measure the glucose levels
in their blood regularly during the day for treatment and self-care. Typically, mobile glucometers are
used to measure glucose concentration in a blood sample.

Glucometers
The first generation of blood glucometers used redox chemistry to produce a colour which was
compared to a colour chart. The second generation blood glucometers use the current associated with
the glucose oxidase/dehydrogenase catalysed redox reaction to determine the glucose concentration.
The typical redox reaction is where the glucose molecule reacts with an enzyme to reduce the enzyme.
This reduced enzyme is then re-oxidised with a mediator, a chemical which transfers those electrons
to an electrode. Figure 1 is a simplified schematic of the electrochemical reaction within a glucometer
test strip.

Reduced
Glucose Oxidised
mediator
enzyme

Oxidised -
Gluco- Reduced e
mediator
lactone enzyme

Figure 1. A schematic of the electrochemistry of a glucometer

The glucometers provided for this practical are either an Accu-Chek Nano or Accu-Chek Performa
Glucometer. The instructions to use this meter will be provided. Take care to wear gloves when
handling the test strips and only remove one from the container at a time to avoid contamination.
 EXPERIMENT 9 75

Risk assessment
Due to the potential biohazard risk which human blood poses,
**DO NOT TEST YOUR OWN BLOOD GLUCOSE LEVELS!

Method:
Chemicals and solutions provided:
0.1 M K2HPO4 solution 0.1 M KH2PO4 solution
100 mM glucose standard solution 10 mM glucose QC
Unknown glucose sample D-(+)-glucose solid
NaCl solid L-Ascorbic acid solid

Fresh solutions to be prepared by students:

▪ 1.5 M NaCl in 100 mL volumetric (refer to pre lab question 2)
▪ pH 7.3 buffer solution (refer to pre lab question 1)
▪ 1000 mg/L ascorbic acid solution in 50 mL volumetric

Glassware required:
10 x 10 mL volumetric flasks 1 x 50 mL volumetric flask
3 x 100 mL volumetric flasks 100 mL measuring cylinder
Autopipettes Pasteur pipettes
4 x small beakers 250 mL beaker
20 x 5 mL glass vials pH meter with calibration solutions

Part 1: Determining the Analytical Figures of Merit

What is the sensitivity?

Prepare a series of glucose standards at 1, 5, 10, 15 and 20 mM concentration of glucose in 10 mL
volumetric flasks as follows. Add 5 mL of the pH buffer and 1.0 mL of the NaCl solution into each
volumetric flask and then add the required amount of glucose solution. Make up to mark with DI water.
Mix well.
Test the glucose concentration in triplicate using the glucometer. Record the data and plot the
calibration curve (your standard values on x-axis and the meter values on y-axis).
 EXPERIMENT 9 76
What is the accuracy?
Record the value of the QC using the glucometer. Calculate %RE from the meter reading and the
reading from your calibration curve.

What is the precision?

Determine the mean and %RSD of an unknown sample by analysing the sample in triplicate.

What is the selectivity? Are there any interferences?

You will be testing the effect of pH, ionic strength and ascorbic acid on the glucose measurements.
Also, you will observe the effect of isomerisation of glucose.
Effect of pH: prepare triplicate 10 mL volumetric flasks with 10 mM glucose and 1 mL NaCl solution.
Make up to the mark with DI water. Test the glucose concentration on the glucometer. Determine the
pH using a calibrated pH meter.
Effect of ionic strength: prepare triplicate 10 mL volumetric flasks with 10 mM glucose and 5 mL
buffer. Make up to the mark with DI water. Test the glucose concentration on the glucometer.
Effect of ascorbic acid: Prepare triplicate 10 mM glucose volumetric flasks with 5 mL buffer, 1.0 mL
NaCl and 1.0 mL of ascorbic acid solution. Test the glucose concentration.
Effect of isomerisation: Prepare a fresh solution of 100 mM glucose by using D-(+)-glucose solid.
Quickly prepare a 10 mM glucose standard using this fresh stock in 10 mL volumetric with the pH
buffer and NaCl solution. Measure the glucose concentration immediately and after 15 min.

Calculations
Calculate the sensitivity, %RE, %RSD and detection limit. Determine the errors associated with the
interferences, isomerisation and selectivity. For assistance, refer to Mike Grace’s lecture notes in week
1.
Present your data appropriately for the oral assessment.

Questions:
1. Accuracy was determined using the calibration curve and the instrumental reading. Which one do
you trust more? Why?
2. Dynamic range could not be determined experimentally due to default settings on the glucometer
stating “HI” or “LO” when glucose levels are too high or too low. Your demonstrator will provide the
dynamic range of the test strips. Define dynamic range and what could be the reasons why the
electrochemical glucometer cannot measure outside this range.
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4. The interferences (including isomerisation) and selectivity of glucometer is important. Do your
results show any change in measured concentrations? Why? Relate these interferences to the
electrochemical reactions in the test strips.

Part 2: Investigating the electrochemistry of a glucometer (1 hour)

Research on-line and utilise papers on the CHM2922 Moodle page to investigate how a typical
electrochemical glucometer detects glucose in blood. Key details to focus on:
• The different types of enzymes and mediators used.
• Is it galvanic, electrolytic or both? Show where the electrons move in the reactions.
• What ways do the meters prevent or reduce the interferences (false readings)?
• What are some of the advantages/disadvantages of these meters over other methods to detect
glucose in blood? For example, hospitals generally don’t use these glucometers for their
patients.

Assessment:
The assessment is an oral Q&A where you will present your results and are marked on the quality of
the data, answers to questions and your findings in part 2.

References:
Cunningham, D.D. Blood Glucose Monitoring, in Medical Instruments and Devices: Principles and
Practices, eds Steven Schreiner Joseph D. Bronzino & Donald R. Peterson, CRC Press, 2016

Diabetes Australia website (accessed 2016), https://www.diabetesaustralia.com.au/

Tang, Z., Du X., Louie R.F. & Kost G.J. (2000), Effects of Drugs on Glucose Measurements With
Handheld Glucose Meters and a Portable Glucose Analyzer. American Journal of Clinical
Pathology 113, 75-86