“Development of Teaching Kit on Chemical Testing for New Senior

Secondary Curriculum”

Student Laboratory Manual

Department of Chemistry
Hong Kong Baptist University

A Project commissioned by
the Innovation and Technology Commission and
Hong Kong Council for Testing and Certification

1
 Content Page

Experiment 1 – Determination of NO2 in Air: Air Pollutant 4

Analysis
Experiment 2 – Analysis of Copper in Wastewater 13
Experiment 3 – Determination of Sulphur Dioxide (SO2) in 22
Dried Food Using Optimized
Monier-Williams Method
Experiment 4 – Is The Dairy Product Safe? Gold 30
Nanoparticles As A Visual Detection Tool
of Melamine
Experiment 5 – Differentiation of Chinese Herb Danshen 40
(丹參) from Other Similar Herbs Using
Facile Test-Tube Scale Chemical Test
Method
Experiment 6 – Determination of Vitamin C in Commercial 47
Sample and Fresh Fruit Juice by
Iodometric Titration

2
 Important Note

Students must read the safety precaution of each experiment in this

Manual carefully beforehand and take all necessary safety
precautions in conducting the experiments carefully. Advice and
information offered in this Manual are by no means exhaustive and
do not preclude the need for exercising care and good judgement at
all times in safeguarding against accidents. When in doubts, please
seek instructions from your teachers immediately.

3
Experiment 1 – Determination of NO2 in Air: Air Pollution Analysis
Introduction
The air pollutant examined in this experiment is nitrogen dioxide, NO2. NO2 is one of

the precursors in photochemical smog formation. In the presence of sunlight, NO2

dissociates to form highly reactive atomic oxygen. The atomic oxygen released

further initiates a series of chain reactions with hydrocarbons (i.e., volatile organic

compounds, VOCs), nitrogen oxides (usually in the presence of light) to form

compounds such as ozone and other oxidants including aldehydes, peroxyacetylnitrate

(PAN). Aldehydes are toxic and can condense to form aerosols which limit

atmospheric visibility. Ozone and PAN are not only hazardous to human but also

extremely toxic to plants and can cause oxidative damage to many materials such as

fabrics, plastics and rubber. In addition, they are also very powerful lachrymators or

eye irritants.

To analyze airborne substances, the initial step is to sample them from the atmosphere.

This can be achieved by using either the active or passive sampling techniques. A

rather expensive constant flow rate pump is required in the active sampling process to

pull airborne materials into an absorbent device. On the other hand, passive samplers

are the preferred sampling devices for taking airborne molecules for subsequent

instrumental analysis. Commercial passive sampling devices are available in the

market. In this laboratory activity, a passive sampler covered by a Chinese Patent is

used [1]. The passive sampler (Fig. 1) consists of a glass vial, a Teflon membrane and a

screw cap which can hold a fixed amount of liquid absorbent. It allows natural

diffusion of gas into the liquid. It is simple and easy to fabricate. They can be used to

monitor simultaneously a large number of sampling sites. The sampling rate can be

determined experimentally by the direct active sampling method and it is normally

4
provided by the supplier.

NO2 on the other hand can be generated indoors from the flue gas of burning stove

and tobacco smoking. Thus, it has been identified as one of the indoor pollutants.

High concentration of NO2 in the indoor environment may cause eye, nose and throat

irritation and could also impair lung functions and increase the risk of respiratory

infections. For promoting health, the Environmental Protection Department (EPD) has

launched an Indoor Air Quality Certification Scheme (www.iaq.gov.hk). Through the

[2]
Hong Kong Laboratory Accreditation Scheme (HOKLAS) , a number of local

commercial testing laboratories have been accredited for measuring the indoor NO2

level. Most accredited laboratories are testing indoor air samples collected by the

passive sampling method. NO2 trapped in the sampler will be extracted by water and

converted to nitrite ion for quantification with different instrumental analytical

methods such as “flow injection method” or “ion chromatographic method”. However,

the instruments used in the commercial laboratories are not available in secondary

schools. On the basis of a literature procedure, a colorimetric method for the

determination of NO2 is used in both the indoor and outdoor environments.

In this laboratory work, NO2 in air (either indoor or outdoor environment) is collected

by a passive sampler. When NO2 and the colourless absorbent reagent are brought

together, a pink-colour solution is developed. The colour is due to the formation of an

azo dye complex. Fig. 2 shows the absorption spectrum of the azo dye. Since the

complex is the only coloured species in the system, the concentration of NO2 can be

determined by colorimetry. The absorbance is directly proportional to the

concentration of the coloured constituent as governed by the Beer’s Law [3].

5
Fig. 1. Passive samplers. Fig. 2. Visible absorption spectrum of the azo dye.

Intended Learning Outcomes

After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong;

2. isolate NO2 in the air samples using passive sampling techniques;

3. analyze NO2 quantitatively using colorimetry;

4. acquire the basic concepts of accuracy, precision, and detection limit of

analytical methods.

Experimental
Apparatus
 1x 4-LED photometer

 2x passive sampler with stand

 1x membrane

 1x glass cuvette

 4x volumetric flask of 25 mL

 1x pipette of 5 mL

 1x 100 – 1000 μL auto-pipette with pipette tubes or 1 mL graduated pipette

 1x 50 – 200 μL auto-pipette with pipette tubes or 0.2 mL graduated pipette

6
 1x scissors

 1x beaker of 100 mL

 1x beaker of 250 mL

 2x dropper

Reagents and chemicals

 2.5 g sulphanilic acid

 0.025 g N-(1-naphthyl)-ethylenediamine dichlorohydrate

 5 mL 1-propanol

 0.75 g sodium nitrite

 2L Deionized water

Sample pretreatment method

1. Set-up of gas samplers
i. Pipet 5 mL of absorbent reagent into the sampling vial (i.e., sampler).

Absorbent reagent: A - Dissolve 2.5 g of sulphanilic acid, B - 0.025 g of N-

(1-naphthyl)-ethylenediamine dichlorohydrate, 5 mL of 1-propanol, and
make up to 500 mL with deionized water.

ii. Mount a membrane onto the opening of the vial.

iii. Screw the hole-cap to fix the membrane in position.

iv. Wrap the vial with aluminum foil. Invert the sampler and let it stand near
the roadside for 1.5 hours.

7
 v. When time is up, replace the membrane cover with a septum cap and let it
stand for 10 min.

vi. Repeat the above set-up steps on the other two sample vials and use the vial
with a solid cap as control.

2. Preparation of a series of NO2 standard solution in 25 mL volumetric flask as

follows:
Standard Solution 1 2 3 4
Concentration of NO2 (ppm) 0.02 0.04 0.08 0.12
Volume of 10 ppm NO2 added (mL) 0.05 0.1 0.2 0.3
Then all the solutions are filled up to the mark with the absorbent reagent.

10 ppm NO2: Dilute 5 mL of 1000 mg/L (ppm) NO2 solution to 500 mL in a

volumetric flask with deionized water.
1000 ppm NO2: Dissolve 0.7499 g NaNO2 in a 500 mL volumetric flask and
dilute to the mark with deionized water.

Analytical method
Let the colour develop for 10 min and then measure the

absorbance of the blank (absorbent reagent), sample and

standard solution using the photometer with a green

LED light source (or other types of colorimeter).

Safety precaution
 Observe the standard safety procedures for laboratory activity;

 Put on the safety goggles, laboratory coats, and gloves;

 Be careful when cutting the membrane with a pair of scissor;

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com.

8
Data Sheet

I. Analysis of NO2 content near road side

E 
A = Absorbance, Eo = Detected potential of the blank ∴ A = log o 
 E 

Eo
Absorbance = log using green LED measurement, Eo = ______________V
E
Sampling time: ___________________min
Sampling location: __________________
Sampling rate (provided): 2.3 x 10-5 m3/min
Descriptions E (V)
Blank Eo =
0.02 ppm NO2 standard
0.04 ppm NO2 standard
0.08 ppm NO2 standard
0.12 ppm NO2 standard
Sample 1st trial
Sample 2nd trial
Sample 3rd trial
Control

Data Treatment
1. Calculate the absorbance (A) for the standards, sample solutions and control.

Descriptions E (V) Absorbance

Blank Eo = 0
0.02 ppm NO2 standard
0.04 ppm NO2 standard
0.08 ppm NO2 standard
0.12 ppm NO2 standard
Sample 1st trial
Sample 2nd trial
Sample 3rd trial

9
 Sample Mean -----------------
Control

2. Plot a calibration curve {absorbance vs. concentration of NO2 (ppm)} and find
out the concentration of NO2 (mg/L) in the samplers and the control.

The concentration of NO2 (mg/L) in the sampler: _____________________

The concentration of NO2 (mg/L) in the control: ______________________

10
3. Calculate the weight of NO2 collected in the samplers and the control:

Weight of NO2 in sampler (μg):

Conc. of NO2 (mg/L) in sampler × 103 μg/mg × 5 mL × 10-3 L/mL

Weight of NO2 in control (μg):

Conc. of NO2 (mg/L) in control × 103 μg/mg × 5 mL × 10-3 L/mL

Net weight of NO2 collected (μg):

Weight of NO2 in sampler (μg) - Weight of NO2 in control (μg)

4. Calculate the volume of air sample (m3):

Volume of air sample (m3): Diffusion rate (m3/min) x sampling time (min)
= 2.3 x 10-5 m3/min x sampling time (min)

5. Calculate the concentration of NO2 in atmosphere with unit μg/m3.

Weight of NO 2 (µ g)
NO 2 (µ g/ m3 ) =
Volume of Air Sampled in m3

11
Questions and Answer
1. What is the colour change of absorbent reagent after standing near roadside for

1.5 hours?

2. Green light source is used for taking measurements in the experiment. Suggest

the reasons.

3. What are the major differences between passive and active samplers for NO2

analysis?

4. Why must the sampler be wrapped with aluminum foil?

5. Find out about the “flow injection method” or “ion chromatographic method”

that the testing and certification industry uses to measure NO2 in IAQ. What are

the advantages of using these methods?

References
[1] 陳永康,肖丹,王柯敏,<快速簡便氣體採集和測定裝置> 中國專利<ZL 00 2
25023.3>, 22/04/2001. The Intellectual Property holder (i.e., Wing Hong Chan)
of this Patent agrees to permit the use of the Patent for education purpose in
Hong Kong.
[2] http://www.hkas.gov.hk/
[3] D. Xiao, L. Lin, H. Yuan, M. F. Choi, W. H. Chan, J. Chem. Educ. 2005, 82,
1231-1233.

12
Experiment 2 – Analysis of Copper in Wastewater
Introduction
Industrial effluents and wastewater may pose potential risks and hazards to human

beings and the environment. In general, effluents from electroplating plants may

contain toxic metal ions such as copper (II) ions. Repeated or prolonged exposure to
[1]
Cu (II) species such as copper sulfate can cause kidney and liver damage . To

comply with the local legislation, companies can engage testing laboratories in Hong

Kong which are capable of analysing the copper content to ensure that the effluents do
[2]
not exceed the upper limit of 0.2 mg/L before discharging into the environment.

The concentration of Cu (II) in industrial effluents can be determined by

“colorimetric” method.

Cu (II) reacts with oxalic acid bis(cyclohexylidene hydrazide) (cuprizone) to form a

[3]
complex with a broad band absorption in the visible light range (Fig. 1) . The

absorbance of this complex is insensitive to pH change and is therefore commonly

used for the determination of copper. Classically, the concentration of Cu (II) can be

determined by comparing visibly the intensity of the orange colouration with Cu (II)

standards. For a more accurate quantitative result, colorimetric method can be used.

O O

N NH C C NH N

Oxalic acid bis(cyclohexylidenehydrazide) – (Cuprizone)

The deepness of the colour, usually measured as the absorbance (A) of the solution

containing the absorbing analyte, is proportional to the extent of the absorption of

13
characteristic light by the coloured compound. The absorbance of a coloured species

can be correlated with the concentration of the species according to the Beer’s Law,

which states that: A = εbc where A is the absorbance, ε is the molar extinction

coefficient, c is the concentration of the species and b is the path length of the optical

cuvette. The absorbance of a solution is defined by A = log(Io/I) where Io and I are the

initial and final light intensity detected after passing through the analyte solution,

respectively. The deepness of the colour of the copper-cuprizone complex is

proportional to the copper contents. The absorbance of the analyte solution will be

measured by a colorimeter or a spectrophotometer and compared with those obtained

from standard copper solutions.

In this experiment a yellow Light Emitting Diode (LED) is used as the radiation

source (Fig. 2). The radiation after passing through the absorbing analyte is allowed to

fall on a photo-transistor which converts light energy into an electric signal. The

signal is proportional to the irradiation intensity and can be amplified and measured.

Fig. 1. Visible spectrum of Cu (II)-Cuprizone.

14
Fig. 2. Emission spectrum of four coloured LED.

Intended Learning Outcomes

After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong;

2. analyze Cu (II) quantitatively using colorimetry;

3. acquire the basic concepts of accuracy, precision, and detection limit of

analytical methods.

Experimental
Apparatus
 2x Spatula

 4x beaker of 100 mL

 1x Pyrex bottle of 100 mL

 1x volumetric flask of 250 mL

 7x volumetric flask of 25 mL

 2x dropper

 2x rubber teats

 1x 100 – 1000 μL auto-pipette with pipette tubes or 1 mL graduated pipette

 1x plastic cuvette

 1x hot plate
15
 1x 4-LED photometer

Reagent and chemicals

 Oxalic acid bis(cyclohexylidene hydrazide)

 Copper (II) sulfate

 Ethanol

 Citric Acid

 25 % Ammonia Solution

Laboratory preparation
1. Preparation of cuprizone reagent

Dissolve 0.5 g oxalic acid bis(cyclohexylidene hydrazide) in 100 mL 50%

ethanol with heating. The reagent solution is stable for about three months if

stored in well-closed containers in a cool place.

2. Preparation of citrate buffer

Dissolve 37 g citric acid to 100 mL deionized (D.I.) water in a 250 mL beaker.

Treat the solution with 95 mL 25% ammonia solution with stirring, let the

solution cool to room temperature, and transfer the resulting solution to a 250

mL volumetric flask and make up to the mark with D.I. water.

3. Preparation of 100 ppm of copper standard solution

Dissolve 0.251 g copper (II) sulfate in 25 mL D.I. water. Transfer the resulting

solution to a 100 mL volumetric flask and make up to the mark with D.I. water.

This stock solution will have a copper concentration of 1000 ppm. Transfer 2.5

mL 1000 ppm of copper standard solution in a 25 mL volumetric flask and add

D.I. water to the mark.

16
Analytical method
1. Measurement of Cu concentration by Cu (II)-Cuprizone complex

i. Preparation of standard Cu (II) solutions and unknown solution

Prepare a series of Cu (II)-Cuprizone standard solutions by mixing

different amounts of 100 ppm Cu (II) stock solution and citrate buffer,

then followed by adding 1.00 mL cuprizone according to the table below:

Volume of 100 ppm Volume of Volume of Final Final

Cu (II) stock Cuprizone citrate buffer concentration volume
solution (mL) (mL) (mL) (ppm) (mL)
Standard #1 0.10 1.00 2.5 0.40 25.0
Standard #2 0.20 1.00 2.5 0.80 25.0
Standard #3 0.30 1.00 2.5 1.20 25.0
Standard #4 0.40 1.00 2.5 1.60 25.0
Standard #5 0.50 1.00 2.5 2.00 25.0
Blank 0.00 1.00 2.5 0.00 25.0
Unknown * 1.00 2.5 -- 25.0
* Pipette 1.00 mL unknown solution to a 25 mL volumetric flask.

2. Spectrophotometric measurements

i. Turn on the power of the colorimeter, turn on the Yellow LED and allow 15

minutes to stabilize.

ii. Click to the “Calib” position

iii. Adjust the “Light Level” knob until the LCD display reads about 4.50 V.

iv. Fill the sample tube with the reagent blank.

v. Remove the cover of cell holder.

vi. Click to the “Detect” and measure the blank sample by inserting the blank
17
 into the cell compartment.

vii. Record the reading (Eo) shown on the LCD display.

viii. Rinse the cuvette and then fill it with the standard solution and record the

reading (E) again.

ix. Repeat the procedure with series of standard solutions and sample solution.

(Caution: Do not adjust the “Light Level” knob while taking the standard

and sample measurements)

Detect/Calib switch

Light Level knob

LCD display

Wavelength Selector knob

VR2
VR3

3. Data treatment
Eo
Plot a calibration graph of the standards using A = log ( ) . Determine the
E
concentration of the sample solution from the calibration curve using
Eo
Asample = log ( )
Esample

18
 Eo − E∞
A = log ( )
E − E∞
where E is the potential measured with the sample placed in the sample holder,
A is Absorbance
Eo is the potential measured with the blank solution, and
E∞ is the potential in the absence of light (dark current) (assuming = 0 V)
Eo
∴ A = log ( )
E

Data Sheet
Photometer ( Yellow LED )
Calibration Voltage =
E [Cu] /ppm
(E) log 
 E 
Blank Eo = 0.000 0.000
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5

19
 Sample
Linear coefficient (R2) =

Safety precaution
 Observe the standard safety procedures for laboratory activity

 Put on the safety goggles, laboratory coats, and gloves

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com

20
Questions and Answer
1. In the electroplating industry, metals other than copper may be released into the

surrounding water. State the names of the metals.

2. Cuprizone is used to form complex with Cu2+ before measurement. Do you think

that Cu2+ can be determined directly without addition of cuprizone? Why?

3. Will the calibration curve change if a Green LED (wavelength of roughly

520–570 nm) is used instead of the yellow one?

References
[1] Canadian Centre for Occupational Health and Safety, Material Safety Data

Sheets: Copper Sulfate Pentahydrate, Hamilton, 2013.

[2] Environmental Protection Department, HKSAR, Water Pollution Control

Ordinance, Chapter 358 Proposed Amendments to the Technical Memorandum

on Effluent Standards, HKSAR, 2001.

[3] E. B. Sandekk, Photometric Determination of Traces Metals, 4th edition; Wiley:

New York, 1989.

21
Experiment 3 – Determination of Sulphur Dioxide (SO2) in Dried
Food Using Optimized Monier-Williams Method

Introduction
Fresh and raw food are limited in supply. Moreover, food is not always consumed

immediately after harvest or slaughter. Food has to be transported from where it is

produced to the consumer. Food spoilage and corresponding prevention is, therefore, a

global concern of human health and economy. Food scientists and technologists are

working hard to find ways to preserve food, so that the food can be stored and

transported without deterioration in quality over a period of time. There are many

preservation techniques, such as heat treatment, irradiation, drying, chilling or

freezing, sugaring, and salting, the use of chemical preservatives is so far the most

common method adopted in the food industry. The basic principle of using chemical

preservatives is to kill microorganisms and to inhibit microbial growth on food items.

One of the common chemical preservatives is sulphur dioxide. Sulphur dioxide is

commonly used to dehydrate and preserve food items because it can also prevent

browning of food and help to preserve the natural colour and flavor during the drying

process. Trace amount of sulphur dioxide is, therefore, found in dried fruits and

vegetables; and the fruit juices and alcoholic beverages made from dried fruits.

Sulphur dioxide (SO2) is a gas produced by the combustion of elemental sulphur. The

gas gives an unpleasant smell similar to rotten eggs. The gas is water soluble resulting

in sulfurous acid (H2SO3). Exposure to high level of sulphur dioxide through

inhalation and ingestion can cause breathing problems, emphysema, and chronic

bronchitis over time. Although the amount of sulphur dioxide in food is not high

enough to give rise to any of these respiratory diseases, individuals who are

hypersensitive to sulphur dioxide may have allergic problems after ingestion.

22
Symptoms include shortness of breath, headache, and nausea. Serious allergic

reactions may even result in death. It is, therefore, necessary to determine the amount

of sulphur dioxide in food.

The determination is one of the important aspects in food analysis of the testing and

certification industry. Standard methods of AOAC Official Method 990.28 and 990.29

are widely used in local commercial testing laboratories. According to the Food and

Drugs (Composition and Labelling) Regulations of the Laws of Hong Kong (Cap

132W), the functional class of sulphur dioxide and the corresponding name shall be

specified in the list of ingredients if a food consists of or contains sulphur dioxide in a

concentration of 10 mg/kg or more. The public, who are concerned with potential

health risks associated with sulphur dioxide in food, should read the food label

carefully prior to consumption of the food.

In this experiment, the optimized Monier-Williams method will be used for

quantitative determination of the sulphur dioxide content in dried food. The

determination follows the standard method of AOAC Official Method 990.28. Dried

food is purchased from the local market and is used as real-life sample. The sample is

heated with reflux in a water-ethanol mixture. A stream of air is used to sweep sulphur

dioxide through a condenser via bubbler to the receiver with hydrogen peroxide

solution (H2O2), where sulphur dioxide is oxidized to sulfuric acid. The amount of

sulfuric acid produced is directly proportional to the sulphur dioxide content in the

sample and is determined by titration with pre-standardized sodium hydroxide

solution (NaOH). The principle and the content of this experiment could confer

students with the importance of analytical technique of acid-base titration and

knowledge of sampling and quantifying the sulfur dioxide in real-life sample of dried
23
food using appropriate tests.

Intended Learning Outcomes

After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong;

2. isolate sulphur dioxide from dried food using reflux distillation;

3. analyze sulphur dioxide quantitatively using acid-base titration method;

4. acquire the basic concepts of accuracy and precision of analytical methods.

Experimental
Apparatus
 1x two necked round bottom flask of 500 mL

 1x water condenser

 1x still head

 1x screw adapter

 3x dropper
Fig. 1. Glass apparatus and air pump.
 3x 1 meter long rubber tubing

 1x air pump

 1x heater

 4x conical flask of 250 mL

 2x beaker of 250 mL

 1x measuring cylinder of 100 mL

 1x burette of 50 mL

 1x scissors Fig. 2. Gas collecting tube.

Reagents and chemicals

 20 g dried food sample
24
 250 mL water-ethanol solution (95:5 v/v)

 50 mL 3% hydrogen peroxide

 250 mL 0.01 M sodium hydroxide solution

 50 mL 6 M hydrochloric acid

 2g potassium hydrogen phthalate

 10 mL methyl red Fig. 3. Reagents required.

 10 mL phenolphthalein

 1L deionized water

Sample pretreatment method

1. Scissor the dried food sample into small pieces and

weigh approximately 12.5 g sample into a two necked

round bottom flask of 500 mL. (Fig. 4).

2. Add 200 mL water-ethanol mixture (95:5 v/v) to the

round bottomed flask.

3. Add 30 mL 3% hydrogen peroxide solution to a conical

flask of 250 mL. Fig. 4. Sample in the two necked

round bottom flask.
4. Add a few drops of methyl red to the 3% hydrogen

peroxide solution.

5. Then, add a few drops of 0.01 M sodium hydroxide

solution until the colour of the solution turns yellow.

6. Assemble the setup as shown in Fig. 5.

7. Disconnect the air purge stopcock and add 30 mL 6 M

hydrochloric acid to the round bottom flask and then

connect the air purge immediately.

Fig. 5. Experimental setup
25
8. Heat the solution for 100 minutes.

Analytical method
Standardization of sodium hydroxide solution

1. Weigh approximately 0.05 g potassium hydrogen phthalate into a conical flask of

250 mL.

2. Dissolve the potassium hydrogen phthalate using 50 mL deionized water and add

several drops of phenolphthalein.

3. Titrate the solution with 0.01 M sodium hydroxide solution until the solution

changes to permanent faint pink colour.

4. Record the volume of sodium hydroxide solution used and calculate the real

concentration of the sodium hydroxide solution (standardization).

5. Repeat the steps of 1 – 4 two times more in order to obtain the mean and standard

deviation of the real concentration of sodium hydroxide.

Determination of sulphur dioxide content by titration

1. After heating of 45-60 minutes, titrate the 3% hydrogen peroxide solution with the

standardized sodium hydroxide solution until the solution changes to yellow

colour.

2. Record the volume of sodium hydroxide solution used and calculate the sulphur

dioxide content (mg/kg) in the dried food sample.

Safety precaution
 Observe the standard safety procedures for laboratory activity
26
 Put on the safety goggles, laboratory coats, and gloves

 Be careful when handling corrosive chemicals, such as concentrated acids

 The experiment can be carried out in a fumehood to avoid inhalation of vapours

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com

Data Sheet
Sample pretreatment method
Observations:

Analytical method
Standardization of sodium hydroxide solution

Observations:

1st trial 2nd trial 3rd trial

Mass of KHP (g)

Final volume (mL)

Initial volume (mL)

Volume of NaOH used (mL)

Chemical equation involved:

NaOH (aq) + KHP (aq)  Na+ (aq) + KP- (aq) + H2O (l)

27
 1st trial 2nd trial 3rd trial

Number of mole of KHP (mol)

Number of mole of NaOH (mol)

Concentration of NaOH (M)

∴ The real concentration of the sodium hydroxide solution = ± M

Determination of sulphur dioxide content by titration

Observations:

Mass of dried food sample (g)

Final volume (mL)

Initial volume (mL)

Volume of NaOH used (mL)

Chemical equations involved:

H2O2 (aq) + SO2 (g)  H2SO4 (aq)

2NaOH (aq) + H2SO4 (aq)  Na2SO4 (aq) + 2H2O (l)

Number of mole of NaOH (mol)

Number of mole of SO2 (mol)

Mass of SO2 (g)

SO2 content (mg/kg)

28
∴ The sulphur dioxide content of = mg/kg

Questions and Answer

1. Sulphur dioxide can act as a preservative in food, but Sulphur dioxide gas itself is

an air pollutant. Suggest an instrumentation method to monitor and measure the

sulphur dioxide content in air.

2. In addition to sulphur dioxide, suggest another chemical that can act as a

preservative in food and state the corresponding harmful effect to human.

3. Propose a method to reduce the amount of sulphur dioxide in food prior to

consume.

4. Name some other chemical preservatives that the testing laboratories can test.

References
[1] AOAC International: Official Methods of Analysis of AOAC (2005), 18th Edition.,

ed. Horwitz W., Gaithersburg: MD, Method 990.28.

[2] AOAC International: Official Methods of Analysis of AOAC (2005), 18th Edition.,

ed. Horwitz W., Gaithersburg: MD, Method 990.29.

[3] The European Standard EN 1988-1: 1998, Foodstuffs – Determination of Sulfite –

Part 1: Optimized Monier-Williams method, British Standards Institution, London.

29
Experiment 4 – Is This Dairy Product Safe? Gold Nanoparticles As A
Visual Detection Tool of Melamine
Introduction
Nanotechnology is the utility of nano-sized materials. We can

nowadays easily find the daily applications of nanotechnology

in areas such as textiles, food packaging, and sewage treatment

for their unique advantages. Many researches are being carried

out for their potential uses in the biomedical and clinical field.
TEM (Transmission Electron
Microscopy) image of 13 nm
gold nanoparticles.
The chemical and physical properties of nanoparticles are very

different from their respective elements in bulk. The physical properties of

nanoparticles, in particular their optical (light absorption and emission) properties, are

highly dependent of their chemical compositions, size, and shape. By controlling the

reaction conditions, nanoparticles can be made into different sizes and morphology

for their unique applications.

Food safety has been gaining substantial attention from the public for years including

those products imported from the surrounding regions. One of the incidents was that

melamine, a non-protein chemical rich in nitrogen, was illegally added into infant

formula to increase its apparent protein content as the dairy industry normally checks

the protein level through tests measuring nitrogen content e.g. the Kjeldahl method.

Excessive intake of melamine causes adverse effects in babies including the formation

of kidney stones. The identification and quantification of melamine then drew

considerable attention in food industry. Dairy products, including pasteurized milk,

formula milk, and chocolates, are monitored for melamine contamination in testing
30
laboratories by chromatographic methods. The quantification of melamine can be

performed by liquid chromatography-mass spectrometry/mass spectrometry

(LC-MS/MS) with the detection limit of several parts-per-million (ppm).

To suit the school environment, here we will adopt a simple and sensitive approach to

detect melamine in milk samples using gold nanoparticles. In this experiment, you

will learn about nanomaterial chemistry and food analysis. Gold nanoparticles of the

size of 13 nm will be prepared by citrate reduction of Au (III) to Au (0). The

as-prepared gold nanoparticle is well-dispersed in water and it gives a clear crimson

colour solution. It will be used as a probe to detect the content of melamine in milk

samples. Prior to the test, the milk samples will be pre-treated to remove the protein

and fat contents. Students will thus learn about precipitation chemistry and will gain

hands-on experience of solid-phase extraction, an important technique widely used in

the testing sector of the testing and certification industry.

In the presence of melamine, the individual gold nanoparticles will be “cross-linked”

by the melamine molecules via hydrogen bonding (each melamine molecule offers

three sites of hydrogen bonding) to form a cluster. This results in an observable colour

change from crimson to purple blue because the solution colour is nanoparticle-size

dependent. The higher the content of melamine the higher the extent of the

aggregation cascade and thus a more significant change in visible colour is expected.

31
Intended Learning Outcomes
After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong

and its contribution to food safety;

2. isolate melamine in milk and milk powder using suitable sample pretreatment

methods;

3. acquire the basic concepts of preparation and applications of nanomaterials;

4. understand the development of fast, low-cost, simple yet sensitive technique in

analytical testing.

Experimental
Apparatus
 1x 100-1000 μL auto pipette with pipette tubes or 1 mL graduated pipette

 1x 10-100 μL auto pipette with pipette tubes or 0.2 mL graduated pipette

 1x top pan balance

 1x bench top centrifuge of at least 4000 rpm

 4x PP centrifuge tube of 10 mL

 5x PS cuvette

 1x conical flask of 100 mL

 1x stirrer hot plate

 1x watch glass of 5 cm

 1x volumetric flask of 25 mL

 2x volumetric flask of 1 L

 1x beaker of 100 mL

 1x measuring cylinder of 100 mL

 1x reagent bottle of 1 L

 4x C18 SPE tube

32
Reagents and chemicals
Description Amount
0.1 M HAuCl4 1 mL
38.8 mM sodium citrate 10 mL
10% TCA 10 mL
SPE elution solvent 20 mL
Chloroform 5 mL

Procedures
Part 1. Preparation of 13-nm Spherical Gold Nanoparticles (AuNPs)

1. All glassware used are washed with aqua regia (3 parts HCl, 1 part

HNO3) and rinsed with filter Millipore water. The presence of dirt

and grease affects the size of shape of the resulted nanoparticles.

2. The glassware to be used is oven-dried prior to use.

3. Prepare 50.0 mL of 1 mM HAuCl4 by diluting of 500 μL of 0.10 M

HAuCl4 solution in water and boil in a 100 mL conical flask

which is covered with a watch glass (see Fig. 1). Fig. 1. Set up for
synthesizing 13 nm
4. When the solution boils, rapidly add 5.0 mL of 38.8 mM sodium gold nanoparticles.
citrate to the stirring solution. Put some ice on the top of the watch

glass to condense hot vapour.

5. Turn off the heater and continue stirring for 15 min., then cool down to room

temperature.

6. This solution of gold nanoparticles will be used in the following experiments as

the probe to melamine in samples. The following colour changes will happen

within one minute.

33
Part 2. Fast screening the presence of melamine in dairy sample

A. Preparation of testing solution AuNPs

1. Pipette 5.00 mL of the freshly prepared AuNPs into a 25-mL volumetric

flask.

2. Make up to the mark with D.I. water.

B. Sample pre-treatment and clean-up

a. For milk sample (liquid sample)

1. Pipette 500 μL sample into a 10-mL centrifuge tube which contains 7.50 mL

D.I. water, 1.00 mL ~10% trichloroacetic acid (TCA), and 1.00 mL

chloroform.

34
2. The function of TCA and chloroform is to precipitate the proteins and

dissolve the fat existing in the milk sample, respectively.

3. Screw the cap and shake the tube vigorously to ensure the completeness of

the extraction.

4. Repeat step 1.1 & 1.2 for a control sample (free of melamine).

5. Centrifuge the samples and set 4000 rpm for 10 mins. (see Fig. 2).

6. Label the centrifuge tubes according to the data sheet.

Fig. 2. Centrifuge tubes are placed diagonally so that mass is well-balanced.

b. For Milk powder (formula)

1. Mix 0.1 gram milk powder with 1.00 mL D.I. water. Pipette 500 μL sample

into a 10-mL centrifuge tube which contains 7.50 mL D.I. water , 1.00 mL

~10% trichloroacetic acid (TCA), and 1.00 mL chloroform.

2. Screw the cap and shake the vial vigorously.

3. Repeat step 1.1 & 1.2 for a control sample (free of melamine).

4. Centrifuge the samples and set 4000 rpm for 10 mins.

5. Label the centrifuge tubes according to the data sheet.

35
Fig. 3. After the centrifuge treatment, protein is precipitated out from the aqueous

solution.

c. To clean up the supernatant using C18 SPE tube

1. Put a four C18 Solid Phase Extraction (SPE) tubes on the vacuum manifold

and label properly.

2. C18 SPE tube is wetted with 2 mL x 3 acetonitrile and then conditioned with

2 mL x 2 (1:1 acetonitrile/water).
36
 3. Supernatant (aqueous portion) is transferred from the centrifuge tube to the

C18 SPE tube.

4. A clean vial is put under the SPE as shown in the above photo.

5. Start the vacuum and open the tap.

6. Collect the filtrate at the clean vial.

7. Step #1 to #4 are repeated for sample # 1 to sample # 3.

In the absence of vacuum manifold, a disposal plastic syringe can be used to

rinse the SPE tube (on the left) and push the supernatant solution through the

SPE tube to be used in subsequent step shown below.

37
C. Screening test

1. Mix 100 μL filtrate with 2.00 mL AuNPs

testing solution to observe the colour changes.

2. Compare the colour changes between samples.

Record the colour changes and intensity in the

data sheet.

3. Fill out the results in the data sheet.

Fig. 4. AuNPs testing solution (left); milk sample mixed

with AuNPs without melamine (middle); milk sample mixed
with AuNPs in the presence of 10 ppm melamine (right).
Safety precaution
 Observe the standard safety procedures for laboratory activity

 Put on the safety goggles, laboratory coats, and gloves

 Be careful when handling corrosive chemicals, such as concentrated acids

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com

Questions
1. State the colour changes from Gold (III) solution to AuNPs solution.
__ _______________

__ _______________

2. What is the function of sodium citrate in the formation of AuNPs?

__ _______________

__ _______________

38
3. What is the function of C18 SPE tube?

__ _______________

__ _______________

__ _______________

4. Do you think the use of AuNPs as a probe for melamine in milk samples would

be a good method for melamine analysis?

__ _______________

__ _______________

__ _______________

__ _______________

__ _______________

Name Vol. of Vol. of Result

Nature Vol. of Vol. of Colour
Sample of sample 10% positive
of D.I. added CHCl3 Changes/
# sample added TCA or
sample (mL) (mL) Positive
(if any) (mL) (mL) negative
1 Liquid 0.5 7.50 1.00 1.00
2 Liquid 0.5 7.50 1.00 1.00
3 Solid 0.5* 7.50 1.00 1.00
4 Solid 0.5* 7.50 1.00 1.00

* weigh 0.1 g milk powder with 1.00 mL D.I. water

References
[1] http://www.sgs.com/en/Agriculture-Food/Food/Primary-Production/Testing-

and- Analytical-Services/Melamine-Contamination-Testing.aspx

39
Experiment 5 – Differentiation of Chinese Herb Danshen (丹參) from
Other Similar Herbs Using Facile Test-Tube Scale Chemical Test
Method

Introduction
Every day there are tons of Traditional Chinese Medicine (TCM) materials imported

to Hong Kong. These herbal materials are distributed to thousands of Chinese

medicine stores and sold in different ways. In order to identify these TCMs, a quick

and facile scientific based chemical test has been developed. The approach is based on

the identification of selected characteristic chemical constituents in TCM by test tube

scale chemical reaction. Since a single herb may contain over tens or even hundreds

of chemical components, the chemical functional groups of these compounds may

react with certain specified reagents and produce various colours, precipitates, or

crystals. By making use of the result of reactions, preliminary identification can be

achieved. Facile chemical test involves mainly the observation of test-tube reaction. It

refers to the observation of the expected chemical reactions between the extracted

chemical components in TCM with the appropriate reagents in test tubes.

In our developed identification approach, the first step is to convert solid form TCM

into powder form by blending. Powder form has a larger total surface area which will

reduce the extraction time and increase the extraction efficiency. Then, a suitable

solvent is used to extract the target chemicals like organic chemical components from

the sample, e.g., diethyl ether. In general, the extraction efficiency can also be

increased by increasing the temperature using hot water bath. If the chemicals are

thermally unstable, ultrasonication can be used to improve the extraction yield. After

extraction, centrifuge is used to separate the suspended solid from the solution. By
40
spinning down the solid, the aqueous portion can be transferred to another clean and

empty new tube for further analysis. The changed colour and/or the presence of

particles can be more easily observed.

In order to confirm the results, duplicate analysis for each sample is required. In

addition, a positive control is also required in the experiment. It is used for comparing

any colour or observational changes.

Radix Salviae Maltiorrhizae (Danshen), is an example to illustrate this technique for

differentiation from closely-related herbs. Danshen is the dried root of Salvia

miltiorrhiza, which is listed in the Pharmacopoeia of the People's Republic of China .

It is commonly used for treatingmenstrual disorder and blood circulation problems,

such as cardiovascular diseases. The chemical constituents of Danshen include both

lipophilic and hydrophilic components. The major hydrophilic components are

phenolic acids including danshensu (DSS), protocatechuic aldehyde (PA), rosmarinic

acid (RA), and salvianolic acids, which are also the major pharmacologically active

constituents [1].

Fig. 1. Chemical Structure of Danshensu.

In general, the qualities and quantities of these compounds are analyzed with High

Performance Liquid Chromatography (HPLC) with ultraviolet detector. However, it

will take several hours to even days for the analysis to complete.
41
For the structure of Danshensu as shown in Fig. 1, it contains typical phenolic

functional group. This characteristic group will react with iron (III) to form a green

complex. Since these substances are water soluble, a hot water bath is used to extract

it first and then tested by iron (III) chloride solution [2]. The formation of the dark

green complex indicates the presence of phenolic functionality. This approach has

been widely used in the TCM industry to test products claimed to be Danshen.

Intended Learning Outcomes

After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong

and how it contributes to TCM development;

2. understand that modern techniques can be applied to TCM analysis;

3. isolate the chemical component in TCM using solvent extraction;

4. analyze the chosen analytes qualitatively using colour test.

Experimental
Apparatus
 1x beaker of 50 mL

 1x beaker of 1000 mL

 1x beaker of 250 mL

 1x volumetric flask of 100 mL

 8x test tube

 8x centrifuge tube of 15 mL

 1x 100 – 1000 μL auto-pipette with pipette tubes or 1 mL graduated pipette

 1x hot plate

 1x centrifuge
42
 1x spatula

 1x balance

Reagents and chemicals

 1,2-dihydroxybenzene

 Iron (III) chloride

 Deionized water

Lab preparation
1. Dissolve 0.33 g of iron (III) chloride hexahydrate (FeCl3 .6H2O) in 10 mL of D.I.

water in a 50 mL beaker.

2. Dissolve 0.05 g of 1,2-dihydroxybenzene in 50 mL D.I. water and then dilute to

the mark of a 100 mL volumetric flask as 500 ppm standard solution.

3. Purchase a Danshen sample (around 30 g) from a local store. Use a blender to

breakdown the solid samples into powder form.

Sample pretreatment and Analytical methods

1. Compare the appearance of the samples.

2. Use a blender to breakdown the solid samples into powder form (Fig. 2).

Fig. 2. A blender is used to breakdown the solid sample into powder.

43
3. 0.1 g of powdered sample is mixed with 4 mL of D.I. water in centrifuge

tubes.

4. Heat the mixture in a water bath for 15 min and then cool down.

5. Use centrifuge to spin down the solid sample.

6. Transfer 1 mL of the supernatant to a new test tube.

7. Add 0.1 mL of FeCl3 indicator solution to each test tube.

8. Record the colour change of each test tube.

Safety precaution
 Observe the standard safety procedures for laboratory activity

 Put on the safety goggles, laboratory coats, and gloves

 Be careful when handling hot water and hot plate

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com

44
Data Sheet
Morphological appearance
Sample A Sample B Sample C

Shape

Colour
Colour of
powder form

Color of the extracted solution

Test tube Content Colour
1 D.I. water
2 Sample A (trial 1)
3 Sample A (trial 2)
4 Sample B (trial 1)
5 Sample B (trial 2)
6 Sample C (trial 1)
7 Sample C (trial 2)
8 Chemical standard

Colour of the extracted solution mixed with 0.1 mL FeCl3 solution

Test tube Content Colour
1 D.I. water
2 Sample A (trial 1)
3 Sample A (trial 2)
4 Sample B (trial 1)
5 Sample B (trial 2)
6 Sample C (trial 1)
7 Sample C (trial 2)
8 Chemical standard

45
Questions and Answer
1. Why is a blender used to pretreat the sample?

2. Suggest another method to extract the water soluble chemicals from Danshen.

3. Why do we need a control test (D.I. water only)?

4. Why do we need to duplicate the experiment?

5. After mixing with FeCl3 solution, explain the difference in observed colour

change between samples and chemical standard.

6. Does Hong Kong has official standards of TCM (中藥材)? Can the standards

serve the purpose of identifying whether a TCM is Danshen? (Hint : Find out

about "Chinese Materia Medica" on the website of the Department of Health)

References
[1] Jin-zhong XU, Jie SHEN, Yi-yu CHENG, Hai-bin QU, Journal of Zhejiang

University SCIENCE B, ISSN 1673-1581 (Print); ISSN 1862-1783 (Online).

[2] www.cmd.gov.hk/hkcmms/vol1/Docs/radix_salviae_miltiorrhizae.pdf

46
Experiment 6 – Determination of Vitamin C in Commercial Sample
of Fresh Fruit Juice by Iodometric Titration
Introduction
Vitamin C (L-ascorbic acid) is essential to our health. A high level of vitamin C is

naturally found in citrus fruits and berries; while vegetables and edible animal internal

organs such as liver and kidney contain lower levels of vitamin C. The human body is

unable to synthesize vitamin C on its own and must depend on diet intake for an

adequate supply. Nowadays, caplets, tablets, capsules, drink mix packets containing

vitamin C as dietary supplement are available in the market. It is recommended from

The National Academy of Sciences for a daily consumption of 60 mg vitamin C in

order to meet the nutritional requirements of a healthy individual.

Vitamin C plays an important role in immune function. Insufficient vitamin C causes

muscles weakness, swollen and bleeding of gums, loss of teeth and bleeding under the

skin as well as tiredness and depression.

There are many methods that can be applied to determine vitamin C in fruits or

vitamin supplement. Herein iodometric titration is applied to determine the amount of

vitamin C in either fresh fruits or dietary supplement in tablet form. Since iodine is

not a primary standard, it can be generated by mixing acidified iodate solution with

iodide ions (1). The solubility of iodine is increased by complexation with iodide to

form triiodide (2). Triiodide then oxidizes vitamin C to dehydroascorbic acid (3). The

fast reaction reconverts iodine to iodide immediately when it is generated. When the

limiting agent vitamin C is exhausted, the remaining iodine forms a dark blue

complex with starch.

47
Intended Learning Outcomes
After the activity, the student is expected to be able to

1. understand the operation of the testing and certification industry in Hong Kong;

2. isolate vitamin C from fresh fruit samples using suitable sampling techniques;

3. analyze vitamin C quantitatively using iodometric titration method;

4. acquire the basic concepts of accuracy and precision of analytical methods.

Experimental
Apparatus
 1x burette of 50 mL

 1x burette clamp

 1x stand

 2x pipettes of 25 mL

 1x measuring cylinder of 100 mL

 2x beakers of 600 mL

 1x beaker of 1 L

 2x conical flasks of 250 mL

 2x volumetric flasks of 250 mL

48
Reagents and chemicals
 Potassium iodide

 Potassium iodate

 Sulfuric acid

 L-ascorbic acid (Vitamin C)

 Starch Fig. 1. Reagents required.

Lab preparation
 5.00 g potassium iodide

 0.300 g potassium iodate

 30 mL 3 M sulfuric acid

 0.250 g L-ascorbic acid

 10 mL 1% starch solution

 2L deionized water

Sample pretreatment method

With a fresh orange

1. Slice the orange in half.

2. Grip the one of the orange halves tightly and squeeze it by hand, using a plain

juicer to coax all the juice out (approximately 100 mL).

With a Vitamin C tablet

1. Dissolve one tablet of Redoxon into 100 mL of deionized water.

2. Dilute the solution to 1000 mL with deionized water.

49
Analytical method
Preparation of 0.01 M iodine solution

1. Weight approximately 5.00 g potassium iodide and 290 mg potassium iodate into

a 600 mL beaker.

2. Add 200 mL deionized water to dissolve the mixture.

3. Add 30 mL of 3 M sulfuric acid.

4. Add 270 mL deionized water to the mixture.

Preparation of 1000 ppm vitamin C standard solution

1. Dissolve 0.250 g vitamin C in 100 mL deionized water.

2. Dilute to volume in a 250 mL volumetric flask.

Standardization of the iodine solution with the vitamin C standard solution

1. Pipette 25.00 mL of vitamin C solution into a conical flask of 250 mL and add

several drops of 1 % starch solution.

2. Titrate the solution with iodine solution until the solution mixture changes to

permanent blue colour.

3. Record the volume of iodine solution used and calculate the real concentration of

the iodine solution (standardization).

4. Repeat the steps of 1 – 3 two times more in order to obtain the mean and

standard deviation of the real concentration of iodine solution.

50
Determination of vitamin C content in tablet by titration

1. Dissolve one vitamin C tablet into 1000 mL deionized water in a 1 L beaker.

2. Pipette 25 mL of the resulted vitamin C solution into a conical flask of 250 mL

and several drops of 1 % starch solution.

3. Titrate the solution with the standardized iodine solution until the solution

mixture changes to permanent blue colour.

4. Repeat the steps of 1 – 3 two times more.

Determination of vitamin C content in fresh fruits by titration

1. Measure 25 mL freshly squeezed juice by a measuring cylinder of 100 mL and

transfer it to a conical flask of 250 mL.

2. Add several drops of 1 % starch solution and titrate the solution with iodine

solution until the endpoint is reached.

3. Repeat the steps of 1 – 2 two times more.

Safety precaution
 Observe the standard safety procedures for laboratory activity

 Put on the safety goggles, laboratory coats, and gloves

 Be careful when handling corrosive chemicals, such as concentrated acids

 Be careful when cutting the fruit with a knife

 Material Safety Data Sheet (MSDS) of chemicals are available online on the

website of MSDSonline.com

51
Data Sheet

Weight of potassium iodide = g

Weight of potassium iodate = g

Weight of pure vitamin C = g

Total volume of the freshly squeezed juice = mL

Standardization of the iodine solution with the vitamin C standard solution.

Trial 1 Trial 2 Trial 3

Initial burette reading (mL)

Final burette reading (mL)

Volume of iodine solution used (mL)

The number of moles of ascorbic acid used in each trial = mol

The average volume of iodine solution used = mL

∴The real concentration of the iodine solution = M

Titration of vitamin C tablet solution.

Trial 1 Trial 2 Trial 3

Initial burette reading (mL)

Final burette reading (mL)

Volume of iodine solution used (mL)

The average volume of iodine solution used = mL

The average number of moles of iodine used = mol

52
The average number of moles of vitamin C = mol

The mass of vitamin C content determined (mg) = mg

∴ The vitamin C content of tablet (ppm) = ppm per tablet

Titration of fresh fruit juice.

Trial 1 Trial 2 Trial 3

Initial burette reading (mL)

Final burette reading (mL)

Volume of iodine solution used (mL)

The average volume of iodine solution used = mL

The average number of moles of iodine used = mol

The average number of moles of vitamin C = mol

The mass of vitamin C content (mg) = mg

∴ The vitamin C content of fresh fruit juice (mg) = mg

Questions and Answer

1. How much vitamin C is there in a vitamin C tablet (mg)? Does the value agree

with the label?

2. Is the selected fruit a good source of vitamin C? Explain.

3. Suggest another method that can be used to determine vitamin C.

4. Is vitamin C required to be identified on the nutrition labels for foods sold in

53
 Hong Kong? Do you think Hong Kong's commercial testing laboratories can test

for all the items on the nutrition label?

References
[1] AOAC international: Official Methods of Analysis of AOAC (2005), 18th Edition.,

ed. Horwitz W., Gaithersburg: MD, Method 967.21.

[2] AOAC international: Official Methods of Analysis of AOAC (2005), 18th Edition.,

ed. Horwitz W., Gaithersburg: MD, Method 967.22.

[3] The European Standard EN 14130: 2003, Foodstuffs - Determination of vitamin

C by HPLC, British Standards Institution, London.

[4] GL-CO-33 method accessed from

http://www.itc.gov.hk/en/quality/hkas/doc/scopes/001.pdf

54