SBT 431:Food Biochemistry

Practicals

Qualitative tests for Lipids

Elizabeth Laryea-Akrong
 Objective
• To qualitatively detect the presence of a lipid

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Lipids
• They are naturally-occurring molecules which includes fats,
oils, waxes, sterols monoglycerides, diglycerides, phospholipids.

• Lipids may be broadly defined as hydrophobic or amphiphilic small

molecules.

• They are generally soluble in organic solvents eg: ether, chloroform

• Fried foods, animal fats, and dairy products like cream, butter, and
cheese are examples of foods that contain lipids
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Qualitative tests
• There are some tests that can be performed to identify the presence of
lipids. These tests helps us determine the presence or absence of lipid,
depending upon the colour change and the characteristics of the lipids.
These include
• Solubility test
• Grease spot test
• Fatty acid test for unsaturation
• Salkowski test
• Lieberman-Burchard Test
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Solubility test
• It is the preliminary test that detects the presence of all lipids.

• Solubility test detects lipid solubility in various solvents, to determine

whether it is miscible or immiscible in polar or non-polar solvents

• It is based on the property of lipid solubility in different solvents.

• Lipids are readily miscible in non-polar solvents like chloroform

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Solubility test-Procedure
1.In two clean dry test tubes add 1 ml of oil samples into each tube.

2.Add to the first tube 1ml of chloroform

3.Add to the second tube 1ml of distilled water

4.Shake both tubes vigorously for 2 minutes

5.Allow the tubes to stand and record your observation

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Solubility tests- Observation

Positive test Negative test

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Grease spot test

• Lipids have the tendency to make paper translucent.

• The fat connects the fibers in the paper with a liquid which can transmit by
refraction (rather than scatter) light that falls upon it. As a result, the paper (if
thin enough) seems almost transparent.

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Grease spot test-Procedure

1. Place a drop of oil sample on a piece

of brown paper.

2. Place a drop of chloroform on the

opposite side of the first spot.

3. Allow the spots to air dry

4. Hold the paper up to light and record

your observation.
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Fats and oils

Fats Oils
• These are mostly solids in nature • These are mostly liquids at room
due to how well the structures temperature.
are able to pack.

• They are generally obtained • They are generally obtained

from animal sources from plant sources
• They are saturated fatty acids • The are unsaturated fatty acids

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 Unsaturated lipid test
• This is a test to determine the presence of
saturation or unsaturation in the fatty acid
molecule.

• Reagent used:10%KI solution

• Principle: The iodine will attach itself to one

of the double bonds which causes de-
colorization of the iodine.

• The amount of halogen added is a measure of

the degree of unsaturation.
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 Cholesterol
• Cholesterol is a steroid lipid built
from four linked hydrocarbon
rings.
• Sources: milk, eggs, meat etc.
• It is an amphipathic molecule,
with
• a polar head group ( the hydroxyl
group at C-3 ) and
• nonpolar hydrocarbons body ( the
steroid nucleus and the
hydrocarbon side chain at C-17 )
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• It is synthesized by the liver.
• It is an important component of
animal cell membrane(regulates
membrane fluidity)
• A precursor of very important
biomolecules in the body eg:
hormones
• Sources: milk, eggs, meat etc.
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 Salkowski test
• Salkowski test is used to detect cholesterol in a solution.
• This test depends on the colors observed that yield from the
reaction of cholesterol with concentrated sulfuric acid.
• Procedure
1. Take few drops of oil samples into their respective tubes.
2. Add 2ml volume of chloroform into each tube
3. Add 2ml volume of Conc. Sulphuric acid into each tube gently along
the wall.
NB: Two distinct layers form. The upper layer turns bluish red to violet
and the sulphuric acid layer shows a yellow colour with a green
fluorescence.
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Salkowski test
Principle:
• Sulphuric acid leads to the dehydration of the cholesterol and
formation of new double bonds. Two cholesterol molecules bind
together, bluish red colour as a result of the sulphonic acid of the
bisterol.

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Lieberman-Burchard Test

• This test is also used to identify cholesterol

• Reagent used: chloroform, acetic anhydride, Conc. Sulphuric acid

• The formation of a green or green-blue colour after a few minutes is

positive The colour is due to the hydroxyl group (-OH) of cholesterol
reacting with the reagents and increasing the conjugation of the un-
saturation in the adjacent fused ring
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Lieberman-Burchard Test
• Procedure
1. Take few drops of oil samples into their respective tubes.
2. Add 2ml volume of chloroform into each tube
3. Add 10 drops acetic anhydride.
4. Add 3 drops Conc. Sulphuric acid

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Qualitative tests for minerals

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Minerals
• Minerals are naturally occurring inorganic elements, with a definite
chemical composition and an ordered atomic structure.
• They are required for growth, repair and regulation of vital body
functions
• The major minerals include calcium, phosphorus, sodium, potassium
and magnesium.
• The trace elements are required in quantities of less than a few
milligrams per day e.g. iron, iodine, fluoride, zinc, copper, cobalt,
chromium, manganese, molybdenum, selenium.

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EDTA titration
• The calcium content of foods is often determined by this method. An
ashed food sample is diluted in water.
• An indicator that can form a colored complex with EDTA is then added to
the solution, and the solution is titrated with EDTA.
• The EDTA-indicator complex is chosen to be much weaker than the EDTA-
mineral complex. Consequently, as long as multivalent ions remain in the
solution the EDTA forms a strong complex with them and does not react
with the indicator.
• However, once all the mineral ions have been complexed, any additional
EDTA reacts with the indicator and forms a colored complex that is used
to determine the end-point of the reaction.
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Procedure
1. Dilute the ashed food sample in demineralized water.
2. Add potassium permanganate as indicator to the solution.
3. Titrate the solution with EDTA (Disodium salt of EDTA).

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 Mohr method for chloride analysis-
Precipitation titration
• When at least one product of a titration reaction is an insoluble (precipitate), it is
referred to as a precipitation titration.
• A titrimetric method commonly used in the food industry is the .
• Silver nitrate is titrated into an aqueous solution containing the sample to be
analyzed and a chromate indicator.
• The interaction between silver and chloride is much stronger than that between
silver and chromate. The silver ion therefore reacts with the chloride ion to form
AgCl, until all of the chloride ion is exhausted. Any further addition of silver
nitrate leads to the formation of silver chromate, which is an insoluble orange
colored solid.

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Procedure

1. Dilute the ashed food sample in demineralized water.

2. Add potassium chromate as indicator to the solution.

3. Titrate the solution with silver nitrate.

4. The end point of the reaction is the first hint of an orange color.

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Sodium hydroxide test
• This test uses sodium hydroxide or aqueous ammonia to test and
identify metal ions by the precipitation formed
• The color of precipitation formed allows for identification of the
compound.
• Several drops of sodium hydroxide (NaOH) solution are added to the
solution being tested.
• If a colored precipitate is formed then stop and find out what the
cation is.
• If white precipitate forms then excess NaOH is added to it and
observed whether the precipitate dissolves.
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Sodium hydroxide test

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 Objective
• To qualitatively detect the presence of antioxidants in food samples
Antioxidants
• Any substance that when present at relatively low concentrations,
compared with those of the oxidizable substrate, significantly delays
or inhibits oxidation of that substrate.
• These are referred to as “free-radical scavengers”
• They are found in vegetables and fruits.
• Examples of antioxidants include vitamins C and E, selenium, and
carotenoids, such as beta-carotene, lycopene, lutein, and zeaxanthin

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 Reactive oxygen species(ROS)
• Free radicals- Reactive oxygen species (ROS)
such as superoxide anion (O2•–), hydroxyl
(•OH), peroxyl (ROO•), alkoxyl radicals (RO•),
and hydrogen peroxide (H2O2) (ROS) are waste
substances produced by cells as the body
processes food and reacts to the environment.
• They can also be produced by cigarette smoke,
air pollution, drinking alcohol
• They may attack biological macromolecules in
vivo, which will rise to protein, lipid, and DNA
damage, cell aging and cause oxidative stress in
the body if they are not kept in a balance.
• This can harm cells and body function and also
cause oxidative stress-originated diseases

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DPPH Assay
• The DPPH free radical is a long-lived organic nitrogen radical with a
deep purple color.
• When a DPPH solution is mixed with an antioxidant, its color turns
from purple to yellow of the corresponding hydrazine.
• The reducing ability of antioxidants toward DPPH can be evaluated by
monitoring the decrease of its absorbance at 515– 528 nm.

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DPPH Assay
• DPPH- 2,2-diphenyl-1-picrylhydrazyl

• The speed of the reaction depends on the nature of the antioxidant,

and the amount of DPPH-H formed depends on the concentration of
the antioxidant.

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Procedure
• DPPH solution was prepared by taking 7.89 mg of DPPH and dissolving
with 100 ml 99.5% ethanol, and finally kept in dark for 2 hr.
• DPPH solution of 1,000 µl was added with 800 µl of Tris-HCl buffer (pH
7.4) in a test tube, 200 µl of testing sample solution was added and
mixed quickly.
• The solution was kept at room temperature for 30 min. The
absorbance of the solution at 517 nm was recorded. A mixed solution
with 1,200 µl of ethanol and 800 µl of TrisHCl buffer (pH 7.4) was used
as the blank.

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 Objective
• To qualitatively detect the presence of pigments in food samples
Pigments
• Foods, particularly fruits and vegetables, are naturally
colored mainly by four groups of pigments
• The green chlorophylls,
• The yellow-orange-red carotenoids, eg: beta-carotene, alpha-
carotene, beta-cryptoxanthin, lycopene, lutein and zeaxanthin
• The red-blue-purple anthocyanins and
• The red betalains, eg: red-violet betacyanins, betanins and yellow-
orange betaxanthins

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Pigments

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Pigments
• Factors that affect the appearance of the pigment?
• pH
• Temperature
• Age /maturation
• High pressure treatment
• Most tests involve the use of the spectrophotometer to determine
the absorbance relative to a known compound after extraction using
inorganic solvents.

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Thin-layer chromatography (TLC)
• Thin-layer chromatography (TLC) is an extremely valuable analytical technique
in the organic lab.
• It provides a rapid separation of compounds, and thereby gives an indication of
the number and nature of the components of a mixture.
• TLC can also be used to identify compounds by comparison with known
samples, to check the purity of a compound.
• To monitor the progress of a reaction, an extraction, or a purification
procedure.
• There are two phases:
• A stationary phase, which contains a solid and usually polar, the most common are silica
(SiO2) or alumina (Al2O3).
• A mobile phase, which can be either a gas or liquid which can be a pure solvent or a
mixture of solvents.
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Thin Layer Chromatography (TLC)
• This technique is carried out in 3 steps
1. Spotting- The sample mixture is applied near the bottom of the plate as a small
spot.
2. Development- The plate is then placed in a jar containing a few ml of solvent.
The solvent climbs up the plate by capillary action, carrying the sample mixture
along with it. Each compound in the mixture moves at a different rate, depending
on its solubility in the mobile phase and the strength of its absorption to the
stationary phase. When the solvent gets near the top of the plate, it is allowed to
evaporate, leaving behind the components of the mixture at various distances
from the point of origin. The ratio of the distance a compound moves to the
distance the solvent moves is the Rf value (retention factor). This value is
characteristic of the compound, the solvent, and the stationary phase

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Thin-layer chromatography (TLC)
3. Visualization
• Non destructive-Compound unchanged after viewing eg:
Ultravoilet light
• Destructive- Compound is converted into something new after the
process

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Procedure
i. Draw a thin line 2cm on the TLC plate/paper bottom with a pencil. It helps in the application of
sample spots. These spots should be kept at equal distances.
ii. Spot the sample mixture on the line drawn.
iii. Develop the TLC chamber by filling the chamber with the mobile phase up to 0.5cm from its
bottom. Cover the chamber with lid and leave it undisturbed for the mobile phase to evaporate
and saturate the chamber for about 5mins. After pouring the mobile phase, the moistened filter
paper is placed along the inside of the chamber wall. This helps to avoid the edge effect by
maintaining equal humidity.
iv. Place the prepared stationary phase plate (spotted plate) inside the chamber, cover it with the lid
and leave it undisturbed.
v. Once enough time has elapsed or clear separation is observed for the process, remove the plate
from the chamber and allowed to dry. Mark the mobile phase and circle the spots before they
disappear.
vi. The sample spots get analyzed through a suitable method, such as UV light, KMnO4 stain, and
iodine staining.
vii. Calculate the Rf values for each spot. This value changes for each compound, even under the same
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