CHEM 2921

Food Chemistry

2020

Lab Manual

School of Chemistry

UNSW

© School of Chemistry 2020

 Table of Contents

Safety Regulations........................................................................................................................ iii

General Rules for all Chemistry Laboratories .............................................................................. iv

Waste Disposal ............................................................................................................................. 2

EXPERIMENTS ............................................................................................................................ x

Protein by Dye Binding Method .................................................................................................... 1

Enzymatic Determination of Starch in Flour ................................................................................. 3

Analysis of Antioxidants in Oils by HPLC ..................................................................................... 8

Sucrose in Condensed Milk ........................................................................................................ 16

Fat by Soxhlet Extraction and the Fatty Acid Composition of an Edible Oil ............................... 20

Iodine Value of an Edible Oil ....................................................................................................... 28

Available Carbohydrates in Foods .............................................................................................. 31

Saponification Value of an Edible Oil .......................................................................................... 34

Guide to Professional and Ethical Conduct for Students in Chemistry ........................................ 36

ii
 SAFETY REGULATIONS

Students MUST COMPLY with the School of Chemistry safety regulations. You have been

issued with a sheet giving these in detail but note the following in particular:

(i) Safety glasses or safety goggles must always be worn in the laboratory.

(ii) Note that it is inadvisable to wear contact lenses while working in a chemical laboratory.

(iii) Bare feet, thongs and open sandals are prohibited. Closed shoes are strongly

recommended. A laboratory coat is a requirement; bulky and/or elaborate clothing is not

recommended. Long hair should be tied back.

(iv) Eating, drinking and chewing in the laboratory are prohibited. Foodstuffs and drinks must

not be brought into the laboratory.

(v) Keep your work area neat and uncluttered. Clean up chemical and water spills at once.

(vi) Avoid skin contact with chemicals.

(vii) If in any doubt about the general physical properties, fire hazards, and toxicities of

chemicals that are stored in the laboratory, consult a senior demonstrator.

(viii) Learn the location and proper use of fire extinguishers, fire blankets, and safety showers.

(ix) You MUST REPORT ALL ACCIDENTS IMMEDIATELY to a demonstrator.

(x) Visitors are not permitted, except with the consent of a senior demonstrator.

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 GENERAL RULES FOR ALL CHEMISTRY LABORATORIES

BE ON TIME!

This is very important: you need the allotted time to complete your lab work.

Valuable tips about your experimental work may be given in the first 10 minutes of

the lab, and you will often work in groups. You might be refused admittance to the

laboratory if you are late, and make-up labs are not available.

• Do not walk around with pipettes (empty or full). Be careful. Do not to wave

pipettes or any other sharp objects around!

• Do not play with wash bottles.

• Do not run or fool around in the lab.

• Turn off hot-plate stirrers, gases, vacuum, and cooling water after you finish using

them.

Balances

o All balances must be kept clean.

o Clean up all spills immediately on the balance pan and on the bench and floor

nearby.

Condensers

o Be careful to connect the condenser to the appropriate inlet / outlet of the cooling

water.

o Turn off all water after every use.

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Handwash Units

o Do not pour chemical waste of any kind down the handwash unit.

o Do not leave spatulas, gloves or other contaminated items on or in the handwash

unit.

Floors

o If water is spilled, mop it up immediately.

o In case of a large flood, call for help immediately to prevent serious damage.

o If an organic solvent is spilled, call for help immediately so it can be cleaned up

appropriately.

Lockers

o All lockers are shared. If glassware is found to be dirty at the start of a laboratory

session, report this to the lab staff. All glassware used by you is to be washed by

you and put back into the locker CLEAN.

Sinks

o Clean and rinse the sinks after each use.

o Do not leave ANY glassware in the sink at any time.

o Do not leave gloves, paper or sharps in or around the sink.

v
 Waste Disposal

Type 1 - Ordinary rubbish such as paper towel used to dry hands etc. goes into the general waste

bins near the lab doors.

Type 2 - Used disposable gloves go into the Used disposable gloves containers (clear buckets

with yellow lids) on top of each work bench.

Type 3 - Broken glassware including all used disposable glass Pasteur pipettes goes into the

white Broken glass bins (near the end of each bench row).

Type 4 - Pour waste solvents into the appropriate Waste solvent containers; there are Waste

solvent containers for Aqueous solutions and for Halogenated and Non-Halogenated organic

solvents located at the fumehoods at both ends of the labs.

NEVER POUR WASTE SOLVENTS OR SOLUTIONS DOWN THE SINK!

Type 5 - Solid chemical waste goes into the appropriately-labelled Solid waste containers. This

includes used silica gel and drying agents, etc.

MISSED LAB SESSIONS

• You can miss one lab session/experiment over the term. Failure to attend 7 laboratories may

result in failure of the course

• Evidence must be provided in writing for absence due to illness or another UNSW approved

reason, e.g. letter from general practitioner, funeral notice etc., and you must apply for special

consideration through the UNSW central system

• Your mark for the practical course will be calculated based on the reports you submit for the

practical sessions you attend.

• To pass this course you must achieve a satisfactory performance in the practicals, i.e. 50%.

Failure to accomplish this mark may result in an unsatisfactory fail grade.

INSTRUCTIONS FOR PREPARATION AND SUBMISSION OF LABORATORY REPORTS

Reports should be written up and submitted electronically through Moodle. This means

that:

1. Reports must be submitted via Moodle up to one week after the scheduled

completion date. Students submitting late reports will have 10% of their report mark deducted

for each day late. Reports submitted more than 5 working days late will not be accepted but

reports after this date can still be submitted for feedback (but no marks).

In general, reports should be set out on the following lines:

(a) Experimental readings, observations, etc., as they are made.

(b) Aim of experiment (brief statement).

(c) Experimental details (brief outline of essential operations involved including notes of any

changes from the published procedure).

(d) Results and Calculations. All results obtained must be given, e.g. all weighings, burette

readings, spectrophotometer readings, etc. Calculations should be neatly set out so that the

marker can easily trace errors. All chemical equations on which calculations are based must be

given.

(e) Discussion. This should clearly indicate that the student understands the purpose

and results of the experiment.

All relevant chemical equations should be given for reactions involved in the experiment.

Information derived from references should be indicated. An u nduly long discussion is not

required and it is not necessary to reproduce long extracts from lectures or texts. On the other

hand, poorly presented arguments in the discussion will earn poor marks.
The student should assess their results in this section and if they are unexpected, an attempt

to explain the variations noted should be given. Possible errors should be discussed. The

uncertainty associated with each result must be calculated.

Note: The practical work counts for a significant proportion of the composite mark for the course.

It must be performed to the satisfaction of the School before a pass can be awarded for the course.

ASSESSABLE COMPONENTS

The practical course allows students to complete seven experiments and to submit

seven practical reports. A minimum attendance requirement of six laboratory classes,

and six reports, is required for a pass in the course. All experiments and reports carry the

same weighting towards the final assessment. Note that lab is 30% of overall grade for

CHEM2921.
EXPERIMENTS
 CHEM2921 LABORATORY MANUAL

PROTEIN BY DYE BINDING METHOD

This method depends on the principle that polar groups in proteins (such as the amino groups

of lysine and arginine and the carboxylate groups of aspartic and glutamic acids) can bind

dyes of the opposite charge to yield insoluble protein-dye complexes. The insoluble complex is

then removed by centrifugation or filtration and the concentration of the unbound dye is

assessed from a standard curve relating absorbance and protein content as determined by

the reference Kjeldahl procedure (see additional information on Moodle). Dye binding methods

have greater success in dairy products than in other foods. Milk is an ideal substrate with

proteins in solution, ready for complex formation. The most common dyes employed are amido

black and orange G.

Sample

Diluted condensed milk. Please note down your sample number in your lab book. Return

sample to refrigerator at end of class.

Procedure

Weigh 5 ± 0.05 g of milk sample in a 50 mL beaker. Dissolve it in approximately 20 mL

of distilled water and transfer quantitatively to a 100 mL volumetric flask, making up to the

mark, using additional portions of distilled water. From this sample of diluted milk, carry out

the experiment as follows in duplicate. Pipette out 5 mL of this solution and 10 mL of the dye

solution into a 25 mL centrifuge tube and mix.

Similarly, set up a blank containing 5 mL of distilled water plus 10 mL of the dye solution. After

allowing the blank and samples to stand for 10 minutes, centrifuge at 2,500 rpm for 5 minutes.

Pipette out 3 mL of the supernatant liquid into a 100 mL volumetric flask and dilute to the

mark with distilled water. Measure the absorbance at 485 nm in the case of orange G against

water.
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 CHEM2921 LABORATORY MANUAL

Record the difference in absorbance (D) between blank (Do) and sample (Dx) and read the

protein content from a standard curve supplied for the absorbance D. Calculate the uncertainty

in your answer.

Questions

1 Give an equation for the reaction between orange G and the protein.

2 Comment on the protein concentration in the milk.

References

1. R.M. Dolby, J. Dairy Res., 28, 43 (1961).

2. U.S. Ashworth, J. Dairy Sci., 49, 133 (1966).

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 CHEM2921 LABORATORY MANUAL

ENZYMATIC DETERMINATION OF STARCH IN FLOUR

Starch determination methods are broadly grouped into acid hydrolysis or enzymic procedures.1

Acid hydrolysis procedures can only be applied to pure starch samples and thus have limited

application. Enzymic procedures vary in pre-treatment steps,2 starch gelatinisation, liquefaction

and dextrinisation, hydrolysis of dextrins to glucose, and glucose measurement. The American

Association of Cereal Chemists (AACC) Method 76-113 specifies starch gelatinisation under

aqueous conditions in an autoclave, followed by starch conversion to glucose with

amyloglucosidase and glucose measurement.

This AACC Method 76-11 underestimates starch content in a range of samples and materials,

including high amylose maize starches, and many processed cereal products. Most methods

in use today incorporate treatment with thermostable α-amylase either during, or immediately

following the starch gelatinisation step.4,5 For samples which are difficult to gelatinise (such as

high amylose maize starch) solvents such as sodium hydroxide or dimethyl sulfoxide (DMSO)6,7

have been employed. In a procedure to ensure complete solubilisation of starch in dietary

fibre determination, Englyst and Cummings (1988)6 included treatment with the starch

debranching enzyme, pullulanase.

This technology has been incorporated into a total starch analysis kit by Megazyme (McCleary

et al.7) and this method received First Approval status by AACC (AACC Method 76-12) following

extensive inter-laboratory evaluation (McCleary et al.8).

Principles

The Megazyme total starch analysis procedure using α-amylase/ amyloglucosidase (AA/AMG)

allows the measurement of total starch in most cereal products (natural or processed). In

the assay format described, starch hydrolysis proceeds in two phases. In phase I, starch is

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 CHEM2921 LABORATORY MANUAL

partially hydrolysed and totally solubilised. In phase II, the starch dextrins are quantitatively

hydrolysed to glucose by amyloglucosidase.

Samples containing high levels of glucose and maltodextrins are washed with aqueous ethanol

before analysis.

Risk and Safety Information

Glucose: may be harmful by inhalation, in contact with skin and if swallowed

Ethanol: highly flammable; toxic; poison; may be fatal or cause blindness if swallowed
α-Amylase (heat stable): may cause sensitisation by inhalation.
Amyloglucosidase: avoid contact and inhalation.
Glucose oxidase: irritating to eyes, respiratory system and skin.
Peroxidase: avoid contact and inhalation.
4-Aminoantipyrine: harmful if swallowed; irritating to eyes, respiratory system and skin.
MOPS: data on hazards not available.
Acetic acid - (glacial): flammable; corrosive; causes severe burns; harmful by inhalation, in
contact with the skin and when swallowed; lachrymator
Sodium acetate: may be harmful by inhalation, in contact with skin and if swallowed

Procedure

Weigh out the sample of wheat flour accurately (~100 mg each) into two glass test tubes. Record

the weights. One of these test tubes will serve as the sample while the other one as the sample

blank. Tap the tubes to ensure all sample falls to the bottom of the tube.

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 CHEM2921 LABORATORY MANUAL

Wet both samples with 0.2 mL of aqueous ethanol (80% (v/v)) to aid dispersion and stir the

tubes on a vortex mixer.

To the sample tube immediately add 3 mL of thermostable α-amylase in MOPS buffer, and stir

vigorously on a vortex mixer.

To the sample blank tube immediately add 3 mL of distilled water, and stir vigorously on

a vortex mixer.

Incubate both the test tubes on a hot plate for 5 minutes. Remove the tubes from the hot

plate and place them in a water bath at 50 °C and add acetate buffer (4 mL).

To the sample tube add 0.1 mL of amyloglucosidase and stir on a vortex mixer.

To the sample blank tube add 0.1 mL of distilled water and stir on a vortex mixer.

Incubate the tubes at 50 °C for 30 minutes.

Remove the tubes from the water-bath and dry with a tissue. Transfer the entire contents

of the sample and sample blank quantitatively into separate 100 mL volumetric flasks and dilute

to volume with distilled water. Mix thoroughly. Centrifuge an aliquot of each diluted solution.

Transfer duplicate aliquots (0.1 mL) of the diluted and centrifuged sample and sample blank

solutions to the bottom of glass test tubes. Ensure all the 0.1 mL reaches the bottom of each

tube.

In glass test tubes, prepare a glucose standard of 0.1 mL of glucose solution (1 mg/mL)

and a reagent blank solution of 0.1 mL of distilled water. Add 3.0 mL of GOPOD reagent

(glucose oxidase/peroxidase) to each of the six tubes (including the glucose standard and

reagent blanks), and incubate the test tubes at 50 °C for 20 minutes.

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 CHEM2921 LABORATORY MANUAL

Within 60 minutes after the incubation, read the absorbance at 510 nm for each sample,

each sample blank and the glucose standard, against the reagent blank.

Calculation

1 100 162
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑆𝑡𝑎𝑟𝑐ℎ (𝑎𝑠 𝑖𝑠) = ∆𝐸 × 𝐹 × 100 × 10 × × ×
1000 𝑊 180

𝐹
= ∆𝐸 × 90
𝑊

where ΔE = Absorbance (sample – sample blank) read against the reagent blank
100(𝜇𝑔 𝑔𝑙𝑢𝑐𝑜𝑠𝑒)
𝐹 = (𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑓𝑟𝑜𝑚 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑡𝑜 𝜇𝑔)
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑓𝑜𝑟 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑔𝑙𝑢𝑐𝑜𝑠𝑒

100 = volume correction (0.1 mL taken from 10 mL)

10 = dilution factor
E
EFFF
conversion from micrograms to milligrams

EFF
G
= factor to express “starch” as a percentage of flour weight

W = the weight in milligrams (“as is” basis) of the flour analysed

EHI
EJF
= adjustment from free glucose to anhydroglucose (as occurs in starch)

100
𝑆𝑡𝑎𝑟𝑐ℎ 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 (𝑑𝑟𝑦 𝑤𝑖𝑔ℎ𝑡 𝑏𝑎𝑠𝑖𝑠) = 𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑆𝑡𝑎𝑟𝑐ℎ (𝑎𝑠 𝑖𝑠) ×
100 − 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡(%)

Discuss the various errors involved in the determination and make an estimate of the uncertainty

in your answer.

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 CHEM2921 LABORATORY MANUAL

Questions

1. Explain the reason for using the reagent blank solution.

2. Explain the reason for using the sample blank solution.

3. Why is it necessary to use an adjustment of 162/180 to convert from "free glucose" to

"anhydrous glucose"?

References

1. O.R. Fennema, Food Chemistry, 3rd ed., pp. 199-201.

2. Anon, (1987). Measurement of the starch content of commercial starches. Starch 39:
414.

3. J. Karkalis, (1985). An improved enzymic method for the determination of native and
modified starch. J. Sci. Fd. Agric., 36: 1019.

4. American Association of Cereal Chemists: "Approved Methods of the AACC". Methods

76- 11, approved October 1976.

5. O. Theander, and P. Aman, (1979), Studies on dietary fibres. I. Analysis and chemical
characterisation of water-soluble and water-insoluble dietary fibres. Swedish J. Agric.
Res. 9: 97.

6. I.L. Batey, (1982), Starch analysis using thermostable 〈-amylase.

7. Starch 34: 125.

8. H.N. Englyst, and J.H. Cummings, (1988), Improved method for measurement of
dietary fibre as non-starch polysaccharides in plant foods. J.Assoc.Off.Anal.Chem. 71:
808.

9. B.V. McCleary, V. Solah, and T.S. Gibson, (1994), Quantitative measurement of total
starch in cereal flours and products. J.Cereal Science, 20: 51.

10. B.V. McCleary, T.S. Gibson, V. Solah, and D.C. Mugford, (1994), Total starch
measurement in cereal products: inter-laboratory evaluation of a rapid enzymic test
procedure. Cereal Chemistry, 71: 501.
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 CHEM2921 LABORATORY MANUAL

ANALYSIS OF ANTIOXIDANTS IN OILS BY HPLC

The term antioxidant mainly refers to non-nutrient compounds in foods that have a proven

antioxidant capacity in vitro. Other than the antioxidant vitamins: A, C and E, no food compounds

have been proven to exhibit antioxidant efficacy in vivo. Accordingly, regulatory agencies like the

Food and Drug Administration (FDA) of the United States and the European Food Safety Authority

(EFSA) have published guidelines disqualifying food product labels that claim antioxidant benefits

when no such physiological evidence exists. Indeed, many common foods are rich sources of

phenols that have antioxidant properties only in test tube studies, i.e. they have little or no

antioxidant value following digestion and are poorly conserved (< 5%), with most destined for

rapid excretion. Thus, it is likely that most of their physiological benefits result from their

preservation of other nutrients in the food stuff.

Two non-naturally occurring antioxidants added to foodstuffs are BHA (butylated hydroxyanisole,

additive 320) and BHT (butylated hydroxytoluene, additive 321), see below.

Pick up a bag of chips, a bottle of vegetable oil, some sausages, or a box of cereal and you’ll

probably find BHA and/or BHT in the ingredients list. Both are widely used by the food industry

as preservatives, mainly to prevent oils from oxidizing (cf. antioxidant) and becoming rancid.

Oxidation affects the flavour, colour and odour of edible oils and can deplete some nutrients. BHT

is even sold in supplements as an antioxidant.

The safety of BHA and BHT is a topic of ongoing controversy. Most research has been in animals

or in vitro. The FDA categorises these food additives as GRAS, i.e. they are Generally

8
 CHEM2921 LABORATORY MANUAL

Recognized As Safe, for their use in specified amounts without pre-market review. A subsequent

review by an independent committee supported their general safety, but concluded that

“uncertainties exist, requiring that additional studies be conducted.” To this end, based on animal

studies, the U.S. National Toxicology Program concluded that BHA “is reasonably anticipated to

be a human carcinogen,” while BHT has been linked to increases and decreases of cancers in

animals. The consumer group the Centre for Science in the Public Interest cites BHA as an

additive to “avoid” and puts BHT in its “caution” column. The possible beneficial effects of these

materials may result from their antioxidant scavenging of damaging free radicals or by stimulating

the production of enzymes that detoxify carcinogens.

Industrially, one of the simplest cost effective ways to analyse for BHA and BHT is high

performance liquid chromatography (HPLC). The experiment herein is based on an Association

of Official Analytical Chemists (AOAC) HPLC method for the analysis of these antioxidant

preservatives.

An Introduction to HPLC

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid

chromatography, is an analytical chemistry technique used to separate, identify, and quantify

components in a mixture. It relies on pumps to pass a pressurised (30-350 bar) liquid solvent

containing the sample mixture through a thin column filled with a solid adsorbent material

(together known as “the column”). Each component in the sample interacts slightly differently with

the adsorbent material, causing different flow rates for the different components and leading to

the separation of the components as they flow through the column (see Figure 1 below). HPLC

is used prodigiously for manufacturing, legal, research, and medical applications.

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 CHEM2921 LABORATORY MANUAL

Figure 1. Example separation of three analytes through a HPLC column (blue, red and yellow)
over time. All images sourced from 2017 WATERS©, all rights reserved: http://www.waters.com

The active component of the column is typically a granular material, e.g. silica or modified silica,

made of solid particles of specific size (ca. 1-50 μm)1 known as the “stationary phase”. Sample

separation occurs due to different degrees of interaction with the adsorbent particles. These

interactions are physical in nature, such as hydrophobic, dispersive, and ionic forces. The

pressurized2 liquid is typically a mixture of solvents (e.g. water, acetonitrile, methanol) referred to

as the "mobile phase".

The image below provides a schematic of a typical HPLC instrument (Figure 2). This includes a

sampler to automate sample introduction to the mobile phase, pumps, and a detector. The pumps

deliver the desired flow and composition of the mobile phase through the column (pure solvent or

solvent combinations or solvent gradients).

1
HPLC is distinguished from benchtop gravity liquid chromatography by its operational pressures, small volumes of sample (mL versus L),
small column dimensions (2-5 mm diameter, 30–250 mm length versus 20-150 mm diameter, >500 mm length), and small adsorbent particle
size (1-50 μm versus 60-200 μm). This gives HPLC superior resolving power relative to benchtop chromatography.
2

10
 CHEM2921 LABORATORY MANUAL

Figure 2. A schematic of a conventional HPLC set-up.

The detector generates a signal (see Figure 3) proportional to the amount of sample emerging

from the column, hence allowing for quantitative analysis of the components of the sample.

Figure 3. The detector flow cell computer output (note colour order relative to Figure 1). Detection
can be accomplished using IR, UV or fluorescence spectroscopy or mass spectrometry cf. GCMS.
All images sourced from 2017 WATERS©, all rights reserved: http://www.waters.com

Overall, the footprint of a standard HPLC set-up is very small and the necessary components are

often housed in a consolidated piece of apparatus with a high capacity for automation (see Figure

4).

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 CHEM2921 LABORATORY MANUAL

Figure 4. A consolidated automated HPLC apparatus with column temperature and mobile phase
variation.

12
 CHEM2921 LABORATORY MANUAL

Risk and Safety Information

n-hexane: highly flammable, toxic by inhalation and if swallowed, irritant.

acetonitrile: flammable, irritant. Harmful if swallowed, inhaled or in contact with skin. Irritant to
eye.

isopropyl alcohol: highly flammable liquid and vapour, irritant. Causes serious eye irritation, may
cause drowsiness or dizziness.

BHA: harmful if swallowed, causes skin irritation, eye irritation, may cause respiratory irritation,
suspected carcinogen.

BHT: hazardous to the aquatic environment. Avoid release to the environment. Very toxic to
aquatic life with long lasting effects.

Procedure

Accurately weigh 20 ± 0.1g oil into a 50 mL beaker and transfer it to a 100 mL volumetric flask.

Rinse the beaker with several small volumes of n-hexane saturated with acetonitrile and dilute to

the 100 mL mark. Accurately pipette 25 mL into a 125 mL separatory funnel, and extract with two

50 mL portions of acetonitrile saturated with n-hexane [SAFETY: let your demonstrator show you

how to safely use a separatory funnel]. Add 5 mL n-hexane saturated with acetonitrile to the

hexane layer and extract with a third 50 mL portion of acetonitrile saturated with n-hexane.3 To

ease removal of n-hexane droplets, combine the acetonitrile extracts in a 250 mL separatory

funnel and let them drain slowly into a 250 mL round bottom flask. Using a rotary evaporator,

evaporate the extracts to 3-4 mL at ≤ 40 °C. Ask a demonstrator to check the set-up of your

evaporation to ensure it doesn’t take too long.

3
If non-separable emulsions form, break them by removing the stopper from the funnel and

warming the glass of the funnel under a warm water tap. It is advisable to ask your demonstrator

to show you if you have not done this before.

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 CHEM2921 LABORATORY MANUAL

Using a Pasteur pipette, transfer the acetonitrile/oil mixture to a 10 mL volumetric flask. Rinse the

round bottom flask with small portions of acetonitrile and transfer the washings to the same

volumetric flask with a Pasteur pipette until ≈ 5 mL is collected. Rinse the Pasteur pipette, and

continue to rinse the round bottom flask with small portions of isopropyl alcohol until the mark is

reached. Stopper the volumetric flask and mix well. The sample is now ready for HPLC and should

be fully labelled with your surname, locker number and sample number, and submitted for

analysis.

HPLC Data

The results of the HPLC analysis will be posted on Moodle within 48 hours of your practical

session, these data will include a chromatogram of your sample (Figure 5) in which your sample’s

BHA and BHT has been quantified.

Figure 5. Example chromatogram from BHA, BHT HPLC analysis.

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 CHEM2921 LABORATORY MANUAL

Calculation

From the concentration of each component in the 10 mL volumetric flask, calculate the total weight

of each component in the volumetric flask. Then calculate the weight of each component in your

accurately weighed 20±0.1g oil sample allowing for the aliquots taken at different stages of the

procedure. Express the BHA and BHT content of your oil in ppm (mg BHA/BHT per kg of oil).

Questions

1. Are the concentrations of BHA (200 ppm) and BHT (100 ppm) within the limits permitted by

law?

2. What is the difference between normal phase and reversed phase chromatography? (Hint:

Typical chromatography uses a polar stationary phase and a non-polar mobile phase).

3. Explain the order of elution of BHA (1st) and BHT (2nd) by HPLC.

15
 SUCROSE IN CONDENSED MILK

Sucrose is an unusual disaccharide in that it does not have reducing properties. The reason

for this is that the glycosidic bond linking glucose to fructose is between the anomeric

(hemiacetal) carbon atoms of both sugars. This prevents ring opening to the straight chain

aldehyde or ketone forms and results in a lack of reducing properties.

However, the glycosidic bond in sucrose is much weaker than in most other disaccharides

such as maltose and lactose. The relative weakness of this bond allows it to be hydrolysed under

conditions which do not hydrolyse other disaccharides. This may be exploited so as to analyse

sucrose in the presence of other disaccharides such as lactose.

Sucrose has a specific rotation ([α]D) of +66° whereas the products of hydrolysis, glucose and

fructose, together have a specific rotation of -20°. Since the sign of rotation changes from

positive to negative during the hydrolysis, the reaction is known as ‘inversion’ and the equimolar

mixture of glucose and fructose as invert sugar. Under the conditions of this reaction lactose

(also optically active) is not hydrolysed at all and its specific rotation does not change.

Thus, in a mixture of sucrose and lactose, the change in optical rotation during the reaction

is due solely to hydrolysis of sucrose and is related to the concentration of sucrose in the formula

16
below.

Before the sucrose is hydrolysed, it must be separated from the protein and fat because the

protein is optically active and the fat gives turbid solutions.

Therefore the first part of the experiment involves the co-precipitation and removal by filtration of

the protein and fat. This leaves a clear solution of sucrose and lactose which is then hydrolysed

selectively.

Risk and Safety Information

Acetic acid (glacial): flammable; corrosive; causes severe burns; harmful by inhalation, in
contact with the skin and when swallowed; lachrymator.

Ammonia: corrosive; causes burns; harmful by inhalation, in contact with skin and if swallowed;
risk of explosion if heated under confinement.

Hydrochloric acid: corrosive; causes burns; poison; reacts violently with water.

Potassium ferrocyanide: may be harmful by inhalation, in contact with skin and if swallowed;
may cause cyanosis (blue-gray colouring of skin and lips) caused by lack of oxygen.

Zinc acetate: harmful by inhalation, in contact with skin and if swallowed.

Sample

A milk sample is provided. Mix well and record the sample number. Return the sample at the end

of the class for refrigeration.

Procedure

Transfer to a 100 mL beaker and accurately weighed quantity (approximately 40 ± 0.1 g) of the

well-mixed sample. Add 50 mL of hot distilled water (80-90 °C), mix, transfer to a 200 mL

volumetric flask washing in with successive quantities of distilled water at 60 °C until the total

volume is approximately 100 mL.

Mix, cool to room temperature and then add 5 mL of dilute ammonia solution (provided). Again
17
mix and allow to stand for 15 minutes. During this time, determine the quantity of dilute acetic

acid required to neutralise 5 mL of ammonia using the concentrations of acid and base provided

and phenol red as indicator. Add this amount of dilute acetic acid to the milk sample and mix.

Add with gentle stirring 12.5 mL (measuring cylinder) of zinc acetate solution and in the same

manner 12.5 mL of potassium ferrocyanide solution. N.B: Be sure the zinc acetate is thoroughly

mixed in before adding the ferrocyanide, which should then be mixed in well before water is added.

Bring the contents of the flask to 20 °C and add distilled water at 20 °C up to the 200 mL mark.

(Care must be taken to avoid the formation of air bubbles.) Close the flask with a dry stopper and

mix thoroughly by shaking. Allow to stand for a few minutes and then filter through dry filter

paper in a 25 cm diameter funnel discarding the first 25 mL of filtrate.

Determine the rotation of the filtrate at 20 °C. This is the direct polarimeter reading (‘D’ in

the formula below).

Pipette 40 mL of the filtrate into a 50 mL volumetric flask and add 6 mL of 6.34 M HCl.

Immerse for twelve minutes the entire bulb of the flask in a water bath thermostatically

maintained at 60 °C, mixing by rotation during the first three minutes, during which time the

contents of the flask should have attained the temperature of the bath.

Cool, dilute to 50 mL with distilled water at 20°C mix and allow to stand for 10 minutes.

Determine the rotation at 20°C. This is the invert polarimeter

reading (‘I’ in the formula below). Calculate the percentage of

sucrose from the formula:

5
𝐷 − (4 × 𝐼) 𝑉−𝑣
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑠𝑢𝑐𝑟𝑜𝑠𝑒 = ×
𝑄×𝐿 𝑊

where D = direct polarimeter reading

I= invert polarimeter reading
18
 L = length of polarimeter tube in dm
V = volume to which the sample is diluted before filtration, (i.e. 200
mL)

Correction for volume of precipitate produced during clarification,

G
𝑣 = EFF (1.08𝐹 + 1.55𝑃)

where W = weight of sample in g

F = percent fat in sample
P = percent protein in sample
= W x 0.22 (fat and protein ~9% each)

Q = inversion divisor factor, which equals 0.8825 because the polarimetric readings are angular
degrees for sodium light. (It is 1.0392 if the readings are angular degrees for mercury green
line and 2.549 if the readings are international sugar scale degrees for (j) light.)

Compare your value with that expected in condensed milk.

References

1. Methods of Analysis - A.O.A.C. 12th Edition, 1975.

2. H. Egan, R.S. Kirk, R. Sawyer, Pearson's Chemical Analysis of Foods, (8th Ed.), Chap.
6, (1981).

19
FAT BY SOXHLET EXTRACTION AND THE FATTY ACID COMPOSTION OF

AN EDIBLE OIL

This experiment has two distinct parts. Part 1 (extraction) is assessed by the submission of

calculations only. Part 2 (fatty acid composition) is to be submitted as a report using the

marking rubric for guidance. The calculation of % by weight fat content for part 1

(extraction) should be included with your report for part 2 (e.g. on a separate sheet of

paper with full working and your sample number). The report must be submitted to your

demonstrator at the next lab session.

Part 1 - EXTRACTION

The fat content of foods by weight can be determined by extracting the dried ground

material with a hydrocarbon (hydrophobic) solvent using a Soxhlet extraction apparatus

(see Figure 6). This device permits a low solubility solid to be exposed to large quantities

of a warm solvent that dissolves the component of interest. In this part, the fat in a food

stuff is extracted into such a solvent (n-hexane), and then the solvent is evaporated to

dryness using a rotary evaporator so that the percentage fat in the food (by weight) can

be measured.

20
 Apparatus Used:

The Soxhlet extractor was invented in 1879 by

Franz von Soxhlet for the extraction of fats/lipids

from food. During operation, the solvent is heated

to boiling and its vapour travels up a distillation

arm into the chamber housing the thimble of food.

The condenser ensures that the solvent

condenses into the chamber. As the chamber

slowly fills with warm solvent, some of the fat is

extracted into the warm solvent. When the

chamber is almost full, it is emptied by a siphon

(like the mechanism on a toilet!), returning the

solvent to the flask. During each cycle, a portion

of the fat dissolves. After many cycles the fat is

concentrated in the distillation flask. The

Figure 6. Soxhlet extractor setup
advantage of this system is that instead of many

portions of warm solvent being passed through the

food, just one batch of solvent is recycled.

Risk and Safety Information

n-hexane: highly flammable, toxic by inhalation and if swallowed, irritant.

Procedure

Accurately weigh 2 g of the ground food sample (Note the sample number and accurate mass in

your lab book) into an extraction thimble and place it in a Soxhlet extraction apparatus. Add

80 mL of n-hexane to the round bottom flask and extract by heating to reflux for 90 minutes

(undertake part 2 during this time). Once extracted, transfer the solvent into a pre-weighed

21
 round bottom flask (>200 mL) and rinse the Soxhlet apparatus into the same flask with a small

amount of n-hexane (no more than 20 mL). Evaporate the contents of the flask to dryness on a

rotary evaporator and accurately reweigh the flask to deduce the weight of the fat extracted from

the sample.

Calculation

The calculation to determine the percentage fat by weight is as follows:

𝑤I − 𝑤E
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝐹𝑎𝑡 = × 100
𝑤Z

where w1 = weight of empty flask (g)

w2 = weight of flask + fat (g)

w3 = weight of ground food sample (g)

Ensure you use the correct balance and methodology to get the most accurate percent fat

content possible. Ask your demonstrator if you are unsure. Submit your calculation and values

for w1, w2 and w3 to your demonstrator as a separate and distinct addition to your report on

part 2 of this practical. Please include your sample number so that the demonstrator can

compare your outcomes to those expected based on the foodstuff used.

References

1. C.S. James, Analytical Chemistry of Foods (1995).

Part 2 – FATTY ACID COMPOSITION

Edible oils and fats are composed of triacylglycerols that contain various fatty acids (see

below). The percent composition of these acids is characteristic of the plant source for a

specific oil. Although there can be some seasonal variation in the relative amounts of

different fatty acids, and a degree of geographic variation, each plant oil typically has a

limited range of values. It is therefore possible to characterize an unknown edible oil by

determining the relative amounts of its individual fatty acids simply by comparing these data

22
 to those acquired on known oils. These kinds of data can also distinguish olive oil from virgin

olive oil and extra virgin olive oil.

In this part of the experiment, a sample of an unknown edible oil will be reacted with sodium

methoxide (cf. similar to the saponification process conducted earlier in term) leading to the

transesterification of its fatty acids as illustrated below.

Risk and Safety Information

n-hexane: highly flammable, toxic by inhalation and if swallowed, irritant.

sodium methoxide: Danger!‼ corrosive, causes burns to any area of contact, extremely
destructive to upper respiratory tract, eyes and skin; harmful if swallowed or inhaled.

methanol: highly flammable, toxic, can be fatal or cause blindness if swallowed.

Procedure

Accurately weigh 0.25 g of the oil sample into a small screw-capped tube (Note the sample

number and accurate mass in your lab book). Add 3 mL of n-hexane, replace the cap and

shake to dissolve the fat. Remove the cap and carefully add 0.15 mL of the provided sodium

methoxide solution using an autopipette. Replace the cap and, making sure the cap is very

secure, shake vigorously for 5 seconds. The mixture should first go clear and then turbid

(cloudy) as the sodium glyceroxide precipitates. Leave to se ttl e for 5 minutes and then take

1.0 mL of the clear supernatant (upper) solution and dilute it to 10.0 mL with n-hexane in

a suitable flask. Transfer 1 mL of the diluted solution into an autosampler vial and submit for

23
 GC-MS analysis to the laboratory servery ensuring it is labelled with your name, locker

allocation AND sample number.

GC-MS Data

The results of the GC-MS analysis will be posted on Moodle within 48 hours of your practical

session, these data will include a chromatogram of your sample in which some (but sometimes

not all) of the identifiable methyl ester components will be named/highlighted (as determined by

their mass spectrometry) with their respective peak areas also given. The proportion of each

methyl ester can be calculated using the following equation:

𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎 𝑓𝑜𝑟 𝐹𝑎𝑡𝑡𝑦 𝐴𝑐𝑖𝑑

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝐹𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑 = × 100
∑(𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎 𝑓𝑜𝑟 𝑎𝑙𝑙 𝐹𝑎𝑡𝑡𝑦 𝐴𝑐𝑖𝑑𝑠)

How to Identify the Source of Your Oil

It is important focus on the major peaks for the purposes of oil identification, especially the relative

amounts of the C18 derived acids and the relative amount of palmitic (C16) acid.

Figure 7. Chromatogram from a GCMS with Peak Areas.

24
For example, in Figure 7, the peaks at retention times of 8.688 (16:0, palmitic acid), 11.154 (18:1,

oleic acid, deduced due to placement between C18 and C18:2) and 11.696 (18:2, linoleic acid)

allow for its identification as explained below (see Table of common fatty acids and fatty acid oil

compositions on later pages):

Calculations

Total peak area for identified methyl esters of fatty acids = 3071910504

% Palmitic Acid Methyl Ester:

457695588
𝟏𝟎𝟎 × = 𝟏𝟒. 𝟗%
3071910504

% Oleic Acid Methyl Ester:

1448886649
𝟏𝟎𝟎 × = 𝟒𝟕. 𝟐%
3071910504

% Linoleic Acid Methyl Ester:

616992370
𝟏𝟎𝟎 × = 𝟐𝟎. 𝟏%
3071910504

With small amounts of C18 (stearic) and C18:3 (linolenic) acid.

Using the C18:1 and C18:2 major components identifies avocado, canola, macadamia and olive

oil as possible identities (all contain C18:1>C18:2). The small relative amount of palmitic acid

(C16) in canola oil rules that oil out. The presence of C18 and C18.3 in this oil and the relative

quantities of oleic and linoleic (C18:1 and C18:2) acid versus palmitic suggest this is most likely

avocado oil.

25
Some common fatty acids

Systematic Name Common Formula Shortened

Name Name
n-butanoic butyric CH 3(CH 2)2COOH C4
n-hexanoic caproic CH 3•(CH 2)4 COOH C6
n-octanoic caprylic CH 3•(CH 2)6 COOH C8
n-decanoic capric CH 3(CH 2 )8COOH C10
n-dodecanoic lauric CH 3(CH 2)10COOH C12
n-tetradecanoic myristic CH3(CH2)12COOH C14
n-hexadecanoic palmitic CH3(CH2)14COOH C16
n-octadecanoic stearic CH3(CH2)16COOH C18
9-octadecenoic oleic CH3(CH2)7CH=CH(CH2)7COOH C18:1
9,12-octadecadienoic linoleic CH3(CH2)4(CH=CHCH2)2(CH2)6COOH C18:2
9,12,15- linolenic CH3CH2(CH=CHCH2)3(CH2)6COOH C18:3
n-eicosanoic
octadecatrienoic arachidic CH 3(CH 2)18COOH C20
5,8,11,14- arachidonic CH 3(CH 2)4(CH=CHCH 2)4(CH 2)2COOH C20:4
The acids in bold are the ones most pertinent to this practical.
eicosatetraenoic

Component fatty acids of some vegetable oils (wt. %)

Oil/Fatty Acid C14 C16 C16.1 C18 C18.1 C18.2 C18.3

avocado <1 18 1 56 24 1
canola <1 5 2 60 22 11
corn 13 4 29 54
cottonseed <1 29 2 4 24 40
grapeseed 14 12 27 45 2
macadamia 4 21 12 53 10
olive <1 14 2 1 72 11
palm 1 48 4 38 9
peanut 9 2 42 44 1
safflower <1 8 3 13 75 1
sesame 10 5 40 45
soybean <1 11 4 25 51 9
sunflower 11 6 30 53

Those oils listed in bold are the oils provided as the samples in this practical.

Questions (to be addressed in your report)

1 Using your chromatogram, identify the fatty acid methyl esters in your sample and their

relative amounts.

26
 i.e. relative peak areas. Focus on peaks for C14, C16 (also look for C16:1 if present) and

the C18 acids, including C18:1, C18:2 and C18:3 if they are present.

2 Using the data from Q1, identify your sample oil using the data provided for the candidate

oils (see bold oils in the table above). Tips: (a) look at relative quantities of C18, C18:1 and

C18:2 acids to flag likely oils, then

(b) discriminate between the candidate oils using C16 and C18:3 content and/or the

presence of C14 (macadamia) or C16:1 (olive) esters. If your sample identity is

ambiguous, describe the ambiguity and highlight the most likely candidate oils.

3 Give a general mechanism for the transesterification reaction used to generate the

methyl esters in this practical experiment.

4 Account for the order of elution of the C18, C18:1, C18:2 and C18:3 esters during GCMS

(see above for an example chromatogram).

References

1. J.M. de Mann, Principles of Food Chemistry, p. 47 (1979).

2. H. Egan, R.S. Kirk, R. Sawyer, Pearson’s Chemical Analysis of Foods, (8th Ed.).

27
 IODINE VALUE OF AN EDIBLE OIL

The iodine value of a fixed oil is the weight of iodine absorbed by 100 parts by weight of the

substance when determined by a standard method. It provides a means of comparing the

keeping properties of fixed oils.

Thus the iodine value is related to the content of unsaturated fatty acids since addition takes

place at double bonds. The addition does not go to completion during the determination and it is

necessary to standardise carefully: (i) the amount of oil used, (ii) the concentration of the reagent

and (iii) the time allowed for absorption.

Since iodine adds very slowly to unsaturated acids, iodine monochloride, which reacts faster, is

more commonly used for the addition. This reagent consists of a solution of iodine

monochloride in glacial acetic acid (Wijs' solution).

Risk and Safety Information

dichloromethane: harmful, irritant; possible risk of irreversible effects; possible

carcinogen/mutagen. neurological hazard.

acetic acid (glacial): flammable; corrosive; causes severe burns; harmful by inhalation, in
contact with the skin and when swallowed; lachrymator.

iodine monochloride: corrosive; causes severe burns to every area of contact; may be fatal
if swallowed or inhaled; severe irritant to skin, eyes and respiratory tract.

magnesium acetate: not a dangerous substance or mixture according to the Globally Harmonised
System (GHS)..

potassium iodide: harmful by inhalation, in contact with the skin and when swallowed; harmful
to eyes; possible risk of irreversible effects; possible teratogen.

sodium thiosulfate: may be harmful by inhalation, in contact with skin and if swallowed.

28
Procedure

N.B. Do in duplicate, plus a reagent blank.

Weigh accurately a clean, dry iodine flask. Transfer 3-4 drops (about 0.15 to

0.2 g) of the oil to the flask and reweigh.

Add roughly 10 mL of dichloromethane to the oil in the iodine flask (see image

adjacent) and mix by swirling gently. Accurately add 20 mL of the iodine

monochloride in glacial acetic acid solution (sometimes referred to as Wijs

solution) using a burette with swirling (make a note of the exact concentration of

the solution), followed by approximately *5 mL of 3% magnesium acetate

catalyst solution. Insert the stopper after moistening the ground glass in a solution of potassium

iodide (dip the end in the provided 10% KI solution). Store the reaction in your cupboard (a dark

place) for 5 minutes during which the double bonds in your oil will be halogenated.

At the end of the exposure period, add approximately * 15 mL of 10% potassium iodide solution

to the reservoir of the iodine flask (with the stopper still in place) and slowly raise the stopper

to allow the iodide solution to enter the flask and displace air to vent/bubble up (this represents

quenching of the absorption reaction). Then add roughly 100 mL of water and titrate the liberated

iodine with 0.1 M sodium thiosulfate (note the accurate concentration of the solution) until the

solution becomes a pale yellow colour (if you assume your oil has an iodine value of 100, this

will be ca. 20 mL of solution). Then add starch indicator and complete the titration swirling

vigorously between each addition of thiosulfate.

Complete the duplicate and blank experiments in the same manner. Calculate the iodine value

*
these quantities are for reagents that are added in excess and are either catalytic or non-yield limiting. Thus, they
do not affect the analysis outcome.

29
from the formula below:

(𝑏 − 𝑎) × 1.269
𝑖𝑜𝑑𝑖𝑛𝑒 𝑣𝑎𝑙𝑢𝑒 =
𝑤𝑡. 𝑜𝑓 𝑜𝑖𝑙 𝑢𝑠𝑒𝑑

where b = mL of thiosulfate used in blank experiment

a = mL of thiosulfate used in determination

Questions

1. Outline the reactions involved in determining the iodine value.

2. Derive the expression for the iodine value from the relevant chemical equations and atomic

mass of iodine (126.90 g/mol).

References

1. C.S. James, Analytical Chemistry of Foods, (1995).

30
 AVAILABLE CARBOHYDRATES IN FOODS

Available carbohydrates in foods may be determined by hydrolysis of carbohydrates to reducing

sugars. In alkaline solution, reducing sugars convert 3,5-dinitrosalicylic acid (DNS) into the red-

brown 3-amino-5-nitrosalicylic acid which absorbs at 540 nm.

Risk and Safety Information

3,5-Dinitrosalicylic acid: harmful if swallowed; irritating to eyes, respiratory system and skin
Sulfuric acid: toxic; contact with combustible material may cause fire; may cause cancer by
inhalation; causes burns; reacts violently with water.

Sodium hydroxide: corrosive; causes burns; harmful by inhalation, in contact with the skin and
if swallowed.

Procedure

Weigh accurately about 0.1-0.2 g of the powdered food sample into a large test tube. Add 10 mL

of 1.5 M sulfuric acid and heat on a hotplate for 20 minutes, stirring occasionally, to hydrolyse

polysaccharides and other non-reducing sugars.

Cool and add carefully 12 mL of 10% NaOH. Mix and filter into a 100 mL volumetric flask,

washing the residue in the tube into the flask with distilled water. Make up to volume with

distilled water and mix well by inversion. This is the hydrolysate.

Standard glucose solutions: Prepare solutions of 0.3, 0.6, 0.9, 1.2 and 1.5 mg/mL in 25 mL

volumetric flasks by diluting 0.5, 1.0, 1.5, 2.0 and 2.5 mL respectively of the stock glucose
31
solution (15 mg/mL) with distilled water.

ONLY DO ONE STANDARD PER GROUP

Absorbance Measurements

Standard glucose solutions. Pipette 1.0 mL of distilled water into a test tube (blank) and into 5

other labelled test tubes pipette 1.0 mL of each standard glucose solution (0.3-1.5 mg). Add 1.0

mL of DNS reagent and 2.0 mL water to each tube.

Hydrolysate. Pipette 1.0 mL of the hydrolysate into a test-tube and add 2.0 mL of water and 1.0

mL of DNS reagent.

Standards and hydrolysate. Heat all tubes on a hotplate for 1 5 minutes. Cool, transfer the

contents to a 25 mL volumetric flask, make up to the mark with distilled water and mix well.

Read the absorbance of each solution at 540 nm.

Calculation

Prepare a standard curve of absorbance at 540 nm versus mg glucose per 25 mL

N.B. The standard curve should be a straight line.

10
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝐴𝑣𝑎𝑖𝑙𝑎𝑏𝑙𝑒 𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠 (𝑎𝑠 𝑔𝑙𝑢𝑐𝑜𝑠𝑒) = 𝐶 ×
𝑊

where C = concentration, in mg glucose per 25 mL

W = weight of sample (g)

Calculate the uncertainty in your answer.

Questions

1. Draw up a table of percentage composition of protein, fat and available carbohydrates and

try to identify your biscuit from the table below.

32
 2. What are ‘available’ carbohydrates?

Percentage composition (g/100 g) of some biscuits

Protein Fat Carbo (total)

Arrowroot 6.3 11.1 78.8
Glengarry 5.5 27.6 63.6
Granita 6.5 21.7 67.4
Wheatmeal 9.1 12.1 66.4
Lattice 9.6 28.6 57.7
Nice 5.5 15.1 75.5

Reference

1. C.S. James, Analytical Chemistry of Foods (1995)

33
 SAPONIFICATION VALUE OF AN EDIBLE OIL

The saponification value of an oil or fat is the number of milligrams of potassium hydroxide

required to neutralise the fatty acids resulting from the complete hydrolysis of 1 gram of oil or

fat.

The saponification value varies according to the molecular weight of the component fatty acids.

Butter fat with a considerable proportion of glycerides of butyric (C3H7COOH), caproic

(C5H11COOH) and caprylic (C7H15COOH) acids (low m.w.) - has a higher saponification value

than other oils where oleates and stearates (C18 acids) (higher m.w.) - form the main bulk of

the fatty acids.

Risk and Safety Information

potassium hydroxide: corrosive; causes burns; harmful by inhalation, in contact with the skin
and if swallowed.

hydrochloric acid: corrosive; causes burns; poison; reacts violently with water.

Procedure

Weigh accurately about 2 g (analytical balance) of oil into a clean dry, 100 mL round bottom

flask and add exactly 25 mL of 0.5 M alcoholic potassium hydroxide. Put a few boiling chips

into the flask.

Pipette another 25 mL of alcoholic potassium hydroxide into a second 100 mL flask to be used

34
 as a blank.

Attach a reflux condenser to the sample flask and boil on a hotplate fitted with a heat-on

block for 30 minutes, shaking occasionally to ensure mixing. Cool the sample flask, add a

few drops of phenolphthalein indicator and titrate the excess alkali with 0.5 M hydrochloric

acid. Titrate the blank similarly.

(𝑏 − 𝑎) × 28.05
𝑆𝑎𝑝𝑜𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑣𝑎𝑙𝑢𝑒 =
𝑤𝑡. 𝑜𝑓 𝑜𝑖𝑙 (𝑔)

where a = mL of 0.5 M HCl required for excess alkali in the sample

b = mL of 0.5 M HCL required in the blank

Calculate the uncertainty in your answer.

Questions

1 Why is phenolphthalein the most suitable indicator for this titration?

2 Derive the expression for the saponification value from the relevant chemical equations.

References

1. C.S. James, Analytical Chemistry of Foods, (1995)

35
 Guide to Professional and Ethical Conduct for Students in Chemistry

These guidelines are intended to inform students of their responsibility to act in a professional

and ethical manner, and to abide by the principles of academic and scientific honesty. They

include extracts from official UNSW policy in this area along with more specific examples and

guidelines relevant to different types of work which students in Chemistry will carry out during

the course of their studies.

These principles apply not only in a university environment but will also be expected of you

in your future career. The various professional bodies to which scientists belong all subscribe to

codes of ethical behaviour.

Excerpts from the Official UNSW Policy on Academic Misconduct

Students are expected to familiarize themselves with all aspects of the University policies on

misconduct and to adhere to these policies during their university career. Some important

aspects relevant to handing in work for assessment are reproduced below.

The UNSW Learning Centre also has a guide to plagiarism and how to avoid it – see

https://student.unsw.edu.au/plagiarism

Academic Misconduct

“Students and staff are governed by the normal laws which regulate our daily lives. But in

addition the University has its own code of rules and conduct. This is because good conduct and

academic honesty are fundamental to the mission of the University as an institution devoted to

the pursuit of excellence in scholarship and research, and to the service of society. These

principles apply not only to students but to the whole University community, including staff

engaged in research. They have been developed over many years and are widely supported

by staff and students. Staff and students are committed to good conduct and academic honesty

and are keen to see that these values and principles are upheld.”

36
“Some aspects of academic misconduct are obvious (e.g. cheating in exams), while others are

perhaps less obvious.”

“It is important that students realise just how broad the definition of academic misconduct may

be. It certainly covers practices such as cheating or copying. Furthermore, practices which may

be acceptable in other situations are considered to be misconduct according to current

academic usage within a University.”

Misconduct Concerning Academic Works

“Failing to acknowledge the source of material in an assignment; quoting without the use of

quotation marks even if the source is acknowledged; plagiarism submitting work for assessment

knowing it to be the work of another person.”

Plagiarism

“Plagiarism involves using the work of another person and presenting it as one’s own. Acts

of plagiarism include copying parts of a document without acknowledging and providing the

source for each quotation or piece of borrowed material. These rules against plagiarism apply

whatever the source of the work relied upon may be, whether printed, stored on a compact disc

or other medium, found on the World Wide Web or Internet.”

“Similarly, using or extracting another person’s concepts, experimental results or conclusions,

summarising another person’s work or, where, there is collaborative preparatory work,

submitting substantially the same final version of any material as another student constitutes

plagiarism. It is your responsibility to make sure you acknowledge within your writing where

you have “sourced” the information, ideas and facts, etc. The basic principles are that you

should not attempt to pass off the work of another person as your own, and it should be possible

for a reader to check the information and ideas that you have used by going to the original

source material. Acknowledgment should be sufficiently accurate to enable the source to be

37
located speedily. If you are unsure whether, or how, to make acknowledgment consult your

lecturer.”

The following are some examples of breaches of these principles:

• Quotation without the use of quotation marks.

o It is a serious breach of these rules to quote another’s work without using

quotation marks, even if one then refers to the quoted source. The fact that it is

quoted must be acknowledged in your work.

• Significant paraphrasing, e.g. several, or one very important sentence, which in wording

are very similar to the source. This applies even if the source is mentioned, unless

there is also due acknowledgment of the fact that the source has been paraphrased.

o Unacknowledged use of information or ideas, unless such information or ideas

are commonplace.

Citing sources (e.g. texts) which you have not read, without acknowledging the ‘secondary’

source from which knowledge of them has been obtained. These principles apply to both text

and footnotes of sources. They also apply to sources such as teaching materials, and to any

work by any student (including the student submitting the work) which has been or will be

otherwise submitted for assessment. You must obtain the prior approval of your lecturer if you

wish to submit to that lecturer an essay or report substantially similar to one which has already

been, or will be, submitted to another lecturer.

Using the principles mentioned above proper acknowledgment, you should also proceed on the

general assumption that any work to be submitted for assessment should in fact be your

own work. It ought not be the result of collaboration with others unless your lecturer gives clear

indication that, for that assignment, joint work or collaborative work is acceptable. In this latter

situation, you should specify the nature and extent of the collaboration and the identity of your

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co-workers. Students should note that essays and written assignments may be tested for a match

i.e. source documents on the Internet.”

Scenarios Relevant to Laboratory Courses in Chemical Sciences

Laboratory Data and Reports

The Scenario

Student X makes a mistake preparing standard solutions and as a result does not have time to

complete the experiment during the scheduled lab time.

Three Different Student Responses

1. Student X sees another student’s results lying on the bench, copies their calibration data

and writes up the lab. report using the copied data.

2. The student consults another student who agrees to share the data. Student X writes

up the report using the shared data.

3. The student consults the demonstrator, who agrees that it will not be possible to

repeat the calibration. They approach another student who agrees to share the data.

Student X writes up the report using the shared data.

Which of these (if any) is the best approach and what are the implications?

Copying another student’s data ( 1 ) without permission is unacceptable – this is academic

misconduct.

Sharing data with the agreement of the other person ( 2 ) may be acceptable with some

important provisos:

i. sharing data is not against the specific policies of the laboratory course

ii. the (agreed) use of the second student’s data is clearly acknowledged in Student

X’s report

iii. the reasons for sharing the data AND the possible impact on the experimental

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 results are also acknowledged – otherwise another ethical problem may arise,

that of presenting misleading scientific results.

Consulting the laboratory demonstrator for advice (3) is the best action. If it is agreed that

shared data can be used, the same requirements for acknowledging the use of the data are

required as before. Alternatively, the advice might be to repeat the experiment at another time

or to write up a report on the experiment using incomplete data.

Group Laboratory Work

The Scenario

Laboratory work in a course is carried out in pairs or small groups. All students in the group are

responsible for collecting the data. However, individual lab reports are expected, and students

work is individually assessed.

Students in one group arrange that one person writes the report each week and emails it

to the others, who each modify it slightly and submit their own version for assessment.

Is anything wrong with this? After all, the students each contributed to the same experimental

work.

The Implications

By the act of using copies of a report written by someone else and then ‘individualising’ them,

the students are knowingly concealing the fact that they are each handing in the same work for

assessment. This is definitely academic misconduct. Note that the person who supplies the work

for copying by others is equally guilty of misconduct as those who do the copying.

Unless otherwise notified by the course coordinator, it is normally acceptable (and even

encouraged) for students to consult each other on such things as how to process their joint

experimental results, on whether their method of calculation is correct and on what the

interpretation of the results might be. But if individual laboratory reports are required, they must

each then take the results away and write up individual reports containing their own discussion
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and conclusions.

Laboratory work in pairs or groups can present some uncertainties in terms of what is expected.

Clear guidelines should be provided by the course coordinator. If in doubt, you should always

ask for clarification.

Manipulation of Experimental Data

The Scenario

A student measures a series of data points for a calibration curve. A linear calibration is expected.

One data point in the middle of the calibration is much too high and distorts the entire calibration.

However this is not noticed by the student until after they leave the lab.

Three Different Student Responses

a) The student estimates what the ‘expected’ value should be and substitutes that into the

calibration set. They then write the report using the apparently linear calibration data.

b) The student leaves the suspect data point out of the report and uses the remaining points

to do the calibration.

c) The student performs statistical tests on the data and shows that the suspect point is an

'outlier' at some high level of probability. All the data are recorded and the tests are written

up with a full justification for using the revised data set for the calibration.

Is there anything wrong with any these actions? What is the best approach?

The Implications

Both (a) and (b) are scientifically unacceptable. Both are dishonest in that they seek to conceal

a problem with the data rather than address it.

Deleting data which doesn’t ‘look right’ is not only dishonest, it undermines the entire scientific
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process. It is possible that the odd-looking data point is the only correct one – the problem

might be with the seemingly well-behaved data. The only way to find out is to go back and do

further measurements. Ideally of course the experiment would be repeated in order to determine

what the correct calibration is. If this is not feasible, then the data must be evaluated as best as

possible, within its limitations (see action (c) above).

Statistical procedures are available to test outliers in repeated data sets and in calibrations

(see, for example Miller & Miller Statistics for Analytical Chemistry). However the existence of

a problem point must never be concealed. It should be investigated and rationalised as far as

possible.

Blatant cheating, such as inventing data or observations for an experiment is clearly academic

misconduct. However, it may not always be obvious to students that other forms of data

manipulation are also considered scientifically unsound even if they do not qualify as academic

misconduct. You should not hesitate to get advice on how to handle unusual or unexpected

experimental outcomes.

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Brief Checklist for Submission of Reports or Assignments Individual report / assignment

¨ The submission is all your own work, except where clearly acknowledged.

¨ No sections are copied from another student.

¨ No sections have been copied from work you have previously submitted elsewhere.

¨ All material directly quoted from another source (textbook, lab manual, web or other

source) is in quotation marks or otherwise distinguished from your own writing AND is fully

referenced.

¨ All pictures, graphics or data obtained from a literature or web source are fully referenced.

¨ All material, information or ideas summarized from other sources are fully referenced.

¨ You have not given your report to another student to enable them to copy from it.

¨ All experimental data and observations are honestly presented and not invented or altered

to fit a preconceived outcome.

¨ You have included a signed coversheet with your assignment.

¨ You have kept a copy of the assignment.

Group report / assignment – additional requirements

¨ All contributors to the group work are acknowledged.

¨ Any additional guidelines specific to the particular group work are followed.

See CHEM2921 Course Outline for information on the staff that will attend each lab session.

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