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Introduction
Carotenoids are organic, fat-soluble pigments that are naturally occurring in plants
and some other photosynthetic organisms, called terpenes. These compounds are
responsible for the red, yellow, and orange color of fruits and vegetables. There are over
600 different carotenoids known, they are split into two classes, xanthophylls and
carotenes. Animals are incapable of synthesizing carotenoids, and must obtain them
through their diet.
Structurally carotenoids are tetraterpenes (eight isoprene units) of a 40- carbon atoms
( C4O H56 ) which could be considered the backbone of the molecule. The chain may be
terminated by cyclic end-groups (rings) and may be complemented with oxygencontaining functional groups. The hydrocarbon carotenoids with molecules containing
oxygen are known as xanthophylls . The unoxygenated (oxygen free) carotenoids are
known as carotenes. Carotenes typically contain only carbon and hydrogen.
Some familiar examples of carotenoids coloration are the orange of carrots and citrus
fruits, the reds of peppers and tomatoes. The two commonest carotenoids are lycopene, a
red pigment found in tomato and beta-carotene, a yellow pigment found in carrots.
Other pigments also occur in the leaves of plants but they are not obvious because their
colors masked by the chlorophylls in live, healthy leaves. These pigments become visible
in the fall when the leaf dies and the chlorophyll rapidly decompose.
Carotenes act as a precursor of vitamin A. Only alpha-carotene and beta-carotene are
converted to significant amounts of vitamin A in the body, and beta-carotene is by far the
most plentiful carotenoid found in fruits and vegetables. Beta-carotene cleaves to form
two molecules of vitamin A when it is ingested. Also called retinol, vitamin A plays an
important role in vision. Beta-carotene is also a powerful antioxidant, and has been
shown to help guard against cancer and heart disease.
Lycopene, is an antioxidant stop free radical production, and it may lower the risk of
prostate cancer. The best food source of lycopene are processed tomato products such as
ketchup, tomato paste, and tomato juice.
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Principle
This experiment performed to try and tentatively identify and
isolate lycopene and beta-carotene from two foods rich in them,
tomato paste and carrots using refluxing, column
chromatography and thin layer chromatography. Analyze the
fractions by thin-layer chromatography to determine if the
fraction contains more than one component. Since both lycopene
and -carotene are colored pigments that absorb light in the UV
and visible range, they can be identified from their maximum wavelength.
PART I: Dehydration and Extraction
In this part you will extract pigments from tomato paste and carrots, using ethanol and
chloroform as an organic solvents.
Condenser
Hot plate
Beaker
Filter paper
Separation funnel
Glass funnel
Round-bottom flask
2 Conical flask
Procedure
Organized by: Badahdah, M. S. & Alghazzawi, W. M.
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Principle
Column chromatography (LSC) is one of the most useful methods
in biochemical work in separation and purification of both solids
and liquids. The theory of column chromatography is analogous
to that of thin-layer chromatography.
In TLC, the stationary phase (adsorbent) is a thin layer of the most
common stationary phase, silica gel (SiO2) or alumina (Al2O3) on
a glass, metal or plastic plate. The cellulose powder has often
been used in the past.
In column chromatography the same materials (adsorbent) is
packed into a vertical glass column. A fine powder of the
adsorbent in a tube (called a column) serves as the stationary
phase, and mixtures of substances in appropriate solvents are
applied to the top of the column and passed through the column, more or less like
filtration. The less-polar components of the mixture emerge from the bottom of the
column first, followed by the more-polar components.
The solvent (eluent), instead of rising by capillary action up a TLC, flows down through
the column filled with the adsorbent by gravity action or by the application of air
pressure.
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Just
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The stationary phase (column packing) in the column is polar.The polar compound
interacts more strongly with the stationary phase and moves more slowly. The non-polar
compound moves more rapidly.
The stationary phase is non-polar. Non-polar compounds will move more slowly
because they are attracted to the column packing. The more polar compound moves more
quickly down the column.
Components of the sample separate from each other by partitioning between the
stationary phase (silica or alumina) and the mobile phase (eluent). Molecules with
different polarity partition to different extents, and therefore move through the column at
different rates. The eluent is collected in fractions.
Column chromatography
2 Beakers
Glass rod
Conical flask
Glass funnel
Pasteur pipette
Small vial
Procedure
Organized by: Badahdah, M. S. & Alghazzawi, W. M.
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Packing of column
1. Quickly and carefully pour the slurry (10-12 cm) into the
top of a clean, dry column using a glass funnel. Stir and
pour immediately to maximize the amount of alumina that
goes into the column instead of remaining behind in the
beaker. You may find a clean spatula or glass rod helpful in
transferring the alumina. The column is tapped constantly
and gently by tubing or rubber stopper on the side during
the pouring of slurry to help the alumina settle uniformly,
compact, and to remove air bubbles inside the column.
2. Place a beaker under the column outlet, open the screw clamp, and allow the solvent to
drain into it. Add more solvent as necessary. The solvent collected prior to the sample
can be re-used.
3. Use a Pasteur pipette to rinse any alumina that is sticking to the
sides of the column. Allow the alumina to settle while eluent
continues to drip into the beaker. Once the alumina has settled,
Organized by: Badahdah, M. S. & Alghazzawi, W. M.
12
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carefully add about 1cm of sand to the top of the column to prevent it from being
disturbed when fresh solvent is added.
4. When finish packing, drain the excess solvent until it just reaches the top level of
alumina (about 0.5 ml). Close the screw clamp. Your column is now packed.
5. Never let the solvent drop below the top of the adsorbent to prevent the column from
going dry out as the column progresses. Cracks will form within the alumina column if
it dries, and compounds can fall down the cracks instead of partitioning between
mobile and stationary phases.
6. The success of your separation will be dependent on how well you pack and load the
column.
5. Fill the column to the top with solvent. The column is now ready to run.
6. The components of the sample (pigment) begin to move down the column and separate
according to their polarity. The yellow beta-carotene moves rapidly through the
column, while the red lycopene moves slowly. Increasingly more polar solvents will be
used to elute the various components from the column.
Sample collection
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Clamp
Petrolum ether
Tomato sample
Alumina
Glass wool
Stopcock
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One gram of silica gel to 3 ml of chloroform is placed in a wide-mouth screw cap jar. The
jar is shaken vigorously to obtain smooth, homogeneous slurry. The slurry may be stored
in the tightly capped screw cap jar until it is to be used.
Preparing (Coating) the Slides
Microscope slides or Silica gel G plates are used as TLC plates.
1. Wash microscope slides with soap and water, then rinse with 50% aqueous methanol
and dry thoroughly on paper towels. Handle by the edge.
2. Stirring the slurry with a glass rod, then dip the slides two by two (as sandwich) in it.
3. Dry the plates at 110C in an oven for about 1 hour.
Applying the sample
1. Using a capillary tubes, spot the solutions to be analyzed on the silica gel side of the
TLC plate (or microscope coated slide) along the pencil line. One spot should be from
the yellow band (i.e., -carotene) from the column. One spot should be from the red
band (i.e., lycopene) from the column. One spot should be from the colored crude
extract that was not ever put on the column. The spots should be very small in
diameter, but clearly visible.
2. If the spots are not visible, check that you have placed enough of each sample on the
plate.
3. Follow the steps as in experiment 1 page 12.
Note: Immidiatly circle the visible spots in pencil since the colors may fade over time.
PART IV: UV/Vis spectroscopy
In this part you will explain the colors of pigment fractions by measuring the absorption
of electromagnetic spectrum of each, using UV-visible spectrophotometer. Also you will
correlate the results of the two types of chromatography with your spectral data.
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solution of carotene into the glass cuvettes. Do not use an ordinary test tube for your
measurement. Care should be taken not to drop the cuvettes.
The following spectrum reveals that the sample absorbs UV radiation strongly at
wavelengths below 240 nm, and absorbs visible light near 410 nm. A
sample transmits light of the wavelengths that it does not absorb. It should not surprise
you that this sample appears orange in color. The sample absorbs light at the blue end of
the visible spectrum, allowing light of higher wavelengths (yellows, oranges, reds) to
pass through and reach the eye of the observer.
Lycopene and -carotene pigments separated from tomato paste and carrots may be
identified from their absorption spectra. For example, lycopene and -carotene absorb
light in the 400-500 nm region of the visible spectrum. Record the spectra of your column
chromatography fractions. Make sure a blank is run first with the appropriate solvent,
100% petroleum ether or 10% petroleum ether in chloroform. Record the spectrum of
your sample from 300-800 nm, ( this is actually just visible light spectroscopy).
Determine the max of any peaks. There are actually several absorption peaks in the
electronic spectrum of lycopene, but the peak of maximum intensity corresponds to a
wavelength near 473 nm. -carotene behaves similarly on irradiation, but the peak of
maximum absorption is found at shorter wavelengths, around 448 nm. The exact position
of maximum absorption absorption depending somewhat on the solvent.
-carotene: max at 426,448,474 nm
Lycopene: max at 444,473,502 nm
Note
Each student will clean their benches and dispose of the waste chemicals.
Each student will submit a lab report the following week to report their findings.
Organized by: Badahdah, M. S. & Alghazzawi, W. M.
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References
1. All pictures are captured by KAU/Biochemistry department.
2. http://www.wfu.edu/academics/chemistry/courses/CC/index.htm
To learn more information click on these links:
Lycopene
Carotenoids
Column chromatography
Column Chromatography Procedures
Separatory Funnel Extraction Procrdure
Result sheet
Title: ..
Test sample:
Group partner:
..
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How many fractions (bands) are separated from the column? Are all they colored? What
the color of each fraction?
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List Rf values for all components off the TLC. (Calculation should be shown).
Rf =
Number of
spots
Color of
spot
Rf
Name of plant
pigment
Extract\
Fraction
Fraction
What information did you get from TLC about the purity of the compounds?
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