Exercise # 08
Determination of Acrylamide content
Introduction
• Acrylamide is a chemical with the chemical formula C 3H5NO. that can
form in some foods during high-temperature cooking processes, such
as frying, roasting, and baking.
• Acrylamide in food forms from sugars and an amino acid that are
naturally present in food; it does not come from food packaging or the
environment.
Background
• Acrylamide was first synthesized in 1949 as an industrial chemical
used for water treatment, paper industry, glues, flocculants, dyes and
gels.
• In 2002 Swedish researches found acrylamide in food.
• In 2009 Health Canada assessed if acrylamide is hazardous to human
health or not.
• In 2010, EChA added acrylamide to potentially harmful agents in
food.
Effects
• Acrylamide can be absorbed into body by inhalation, skin
contact and ingestion.
• It’s short term exposure can cause irritation in eyes, skin and the
respiratory tract and may also affect central nervous system.
• Long term or repeated exposure can lead to peripheral nerve
damage.
• Acrylamide is carcinogenic to humans and can cause heritable
genetic damage in humans.
Introduction
• Acrylamide is formed mainly in foods made from plants,
such as fried potato products, grain products, or coffee.
• Generally, acrylamide is more likely to accumulate when
cooking is done for longer periods or at higher temperatures.
• High amount of acrylamide content in food can increase risk
of cancer.
• Acrylamide content can be determined through high
performance liquid chromatography technique (HPLC).
Procedure - Equipment
Equipment
Vortex Mixer
Centrifuge machine
Nitrogen concentrator
HPLC w/ UV detector
Hamilton Syringe
Micropipette
Centrifuge tubes
Filter paper
Procedure - Equipment
Vortex Mixer
A vortex mixer, or vortexer, is a simple device
used commonly in laboratories to mix small
vials of liquid. It consists of an electric motor
with the drive shaft oriented vertically and
attached to a cupped rubber piece mounted
slightly off-center.
Procedure - Equipment
Centrifuge machine
A centrifuge machine can be described as a
machine with a rapidly rotating container
that applies centrifugal force to its contents.
This is achieved by spinning the fluid at high
speed within a container, thereby separating
fluids of different densities or liquids from
solids.
Procedure - Equipment
Nitrogen Concentrator
The nitrogen sample concentrator is used
for concentrating or preparing samples in
batches. It is used in processes such as drug
screening, hormone analysis, liquid phase
and mass spectrometry in the analysis of
sample preparation.
Procedure - Equipment
HPLC
High Performance Liquid Chromatography
(HPLC) is a form of column
chromatography that pumps a sample
mixture or analyte in a solvent (known as the
mobile phase) at high pressure through a
column with chromatographic packing
material (stationary phase).
Procedure - Equipment
HPLC Principle
The separation principle of HPLC is based on the distribution of the
analyte (sample) between a mobile phase (eluent) and a stationary phase
(packing material of the column). Depending on the chemical structure
of the analyte, the molecules are retarded while passing the stationary
phase.
Procedure - Equipment
UV Detector
A UV detector is an in-line device that measures the UV absorbance of
the HPLC eluent and provides a continuous signal that can be used to
quantify the amount of chromophoric compounds emerging from the
HPLC column.
Hamilton Syringe
Hamilton syringes are manufactured to be accurate within ±1% of
nominal volume, and with precision within 1%, measured at 80% of
total scale volume.
Procedure - Equipment
Micropipette
Micropipettes are utilized in the laboratory to transfer
small quantities of liquid, usually down to 0.1 uL.
Centrifuge tubes
Centrifuge tubes are used to contain liquids
during centrifugation, which separates the sample into its
components by rapidly rotating it around a fixed axis.
Filter Paper
Filter paper is a semi-permeable paper barrier placed
perpendicular to a liquid or air flow. It is used to separate
fine solid particles from liquids or gases.
Procedure - Reagents
Reagents
Acrylamide stock standard (Acrylamide 13C3 -AA)
Acetonitrile
n-Hexane
Procedure
Sample preparation
1. Accurately weigh 1.0g of fried oil sample and transfer into 50-
mL centrifuge tube.
2. Add 20 µL of internal standard (13C3 -AA, concentration 1
µg/mL) and 10 mL acetonitrile, and vortex the mixture for 5
min.
3. Now defat the sample by adding 5 mL of n-hexane in
centrifuge tube and centrifuging at 7000 RPM for 5 min. After
this, remove the n-hexane layer from the mixture.
Procedure
4. Evaporate the residual acetonitrile from the mixture using
nitrogen via concentrator, followed by reconstitution with 1
mL acetonitrile.
5.Finally, filter the sample solution through 0.2-µm filter paper
before HPLC analysis.
Procedure
HPLC Analysis
1. Setup high pressure liquid chromatography equipped with
temperature reading 30ºC, flow rate 0.6 ml/minute and UV-visible
detector at 200 nm.
2. Run the standard stock solution of known concentration in HPLC to
get the standard reading.
3. Collect 1 µl of sample with the help of Hamilton syringe from lower
layer of vial having sample in aqueous solution and inject into HPLC
and run the system for analysis of acrylamide content.
Procedure
HPLC Analysis
4. Compare peak values of standards and sample graphs to obtain value
of acrylamide content in fried oil sample.
Example graph
Sample
Standard
