1Isolation of Lycopene from Tomato
Lycopene is a carotenoid hydrocarbon that may be found in tomatoes as well as other red
fruits and vegetables such as red carrots, watermelons, and grapefruits. It has antioxidant
properties (from the neo-Latin Lycopersicum). Both the strawberry and cherry flavors are
devoid of this flavoring. Despite the fact that lycopene is chemically a carotene, it does not
possess any vitamin A action. Lycopene, a pigment found in tomato sauce, is responsible for
the orange color ation of white plastic cookware. To the contrary of water, it is not soluble in
organic solvents or oils. The lack of polarity of lycopene means that it will stain any porous
object, including most plastics, when used in culinary dishes. It is possible to eliminate the
stains on the plastics with a solution that contains only a small amount of chlorine bleach.
The bleach oxidizes the material, allowing it to be broken down and dissolved. The
antioxidant qualities of lycopene have previously been the focus of research, owing to its
biological features, which are regarded to be the primary source of its health benefits.
Lycopene may be extracted and isolated from a variety of sources using a variety of
techniques. There have been a number of research conducted on the extraction and
quantification of lycopene from a variety of natural sources.
Fruits and vegetables contain a high concentration of antioxidants. The protection provided
by antioxidants against potentially dangerous free radicals reduces the chance of developing
cancer and cardiovascular disease. Lycopene, an antioxidant carotenoid, has a very strong
antioxidant effect. In addition to lowering the effects of oxidative stress, lycopene, a naturally
occurring pigment, helps to protect the body from the disease. Lycopene, like beta-carotene
and cryptoxant, is a carotenoid that plays an important role in the synthesis of vitamin A and
the growth of plants (Semenova et al. 1997). Lycopene is a fat-soluble pigment produced by
plants and microorganisms and found in red fruits and vegetables. Vitamin E, selenium, and
lycopene have all been shown to lower the risk of prostate cancer when lycopene-rich meals
are consumed on a regular basis, according to research. For these reasons, the antioxidant
lycopene is essential for cancer prevention as well as LDL cholesterol lowering and heart
disease prevention (Moghimipour et al. 2012). It is a carotenoid, which means that it gives
foods a crimson tint. Among the several forms of lycopene found in processed foods, isomers
are the most abundant. It has the chemical formula C40 H56 and a molecular weight of
536.88, making it one of the lightest substances known (Rahmat et al. 2002). A hydrocarbon
that does not include saturating bonds. It has been found that lycopene contains 11
conjugated bonds and 13 double bonds, according to the literature. The antioxidant effects of
lycopene are enhanced by the presence of conjugated bonds in the molecule, making it more
beneficial to human health. A substantial amount of the antioxidant lycopene can be found in
tomato and watermelon fruits and vegetables. Lycopene has been isolated from a wide variety
of fruits and berries. Harsten was the first to isolate it from Tamuscommunis, in 1873
(Rahmat et al. 2002). There are no known side effects associated with ingesting up to 150 mg
of lycopene per day from a variety of sources, according to research. According to current
research, the body is capable of processing lycopene that has been ingested. Metabolites have
been found and are currently being explored.
Aims and objectives
Traditional fruits’ chemical and therapeutic characteristics will be investigated.
To separate and detect lycopene from diverse fruits, TLC, UV, spectroscopy, and
infrared spectroscopy are utilized.
To test properties of lycopene and its important factors
For lab preparation, it was vital to have a thorough understanding of the potential
risks. Acetone and petroleum ethers can cause skin burns, eye pain, and respiratory
irritation when used in high concentrations. It should be noted that hexanes is a highly
flammable substance. Everything that contains a chemical must be disposed of in the
proper waste bins.
Benzene extraction method
The watermelon, tomato, and papaya were pureed in separate containers. 100 grams
of paste from each of the three fruits should be weighed separately in the laboratory.
A 100-gram sample of watermelon was placed in a beaker with a capacity of 250 mL.
In a separate bowl, combine 30 mL of heated (400°C) benzene and stir well. Decant
the benzene layer after it has been gently swirled.
It is important to add 30 mL of warm benzene and decant the benzene a second time
after the first decanting. This has occurred around five times. Benzene was removed
from the mixture and just a trace amount of Lycopene was left over.
The residue that had been ether-recrystallized was weighed. The results of the
experiment were based on additional tomato and papaya samples. Chemical testing
was used to identify the separated Lycopene, and microscopic examinations and a
visual spectrophotometer were used to determine the chemical structure of the
Methanol extraction method
Methanol was used to dehydrate 50 grams of tomato paste. To avoid solid lumps, the
mixture was forcefully shook as soon as it was incorporated. After two hours, the dark
crimson cake was filtered out of the thick solution after being agitated for another 15
minutes in a 75 cc combination of methanol and carbon tetrachloride.
One volume of water was added to the carbon chloride phase and aggressively
agitated before being transferred to a separatory funnel. The carbon tetrachloride
phase was diluted with benzene after it had evaporated during phase separation. It was
done using a dropper, and the crude lycopene crystals appeared immediately after 1
minute. The crystallization process was finished by chilling the liquid in a room
temperature ice bath. The crystals were washed ten times with benzene and boiling
A microscope revealed long, crimson lycopene prisms in several colorless impurity
compounds. To further purify the sample, column chromatography with toluene as an
eluent on active acidic alumina was used. The terrifying deep red zone has been
gathered for research.
The remainder was dissolved in 2 cc of benzene after the solvent had completely
evaporated. Only white powder was obtained after recrystallizing boiling methanol.
Because crystalline lycopene is susceptible to autoxidation (or air oxidation), it was
stored in dark evacuated glass.
Hexane-Ethanol- Acetone extraction method
The extraction efficiencies of acetone, ethanol, and hexane were compared using three
different solvents. Each solvent was extracted in a shaker for 30 minutes (1:10 v/v).
Covering the jars with aluminum foil shielded them from the sun’s rays while they
were extracted at room temperature. Using UV–Vis spectroscopy at 472 nm, the
purity and content of lycopene were analyzed (maximum of absorption for lycopene)
Acetone-petroleum ether extraction
The substance was extracted with 10mL of a 50/50 mixture of acetone and petroleum
ether (1.0-1.5 g powder). The lycopene-containing organic layer was pipette-separated
and collected in a test tube. Multiple extractions were carried out. The mixed extracts
were washed with aqueous sodium chloride (NaCl) and the aqueous wash was
removed using a micropipette.
The water used to wash the extract with potassium carbonate (K2CO3) was discarded.
Using a drying agent, the organic layer containing lycopene was eliminated (calcium
chloride). A few minutes were required for the additional solvent to evaporate at room
temperature. After being wrapped in aluminum foil and stored in the freezer, the tubes
containing the lycopene extracts were ready for examination.
Hexane extraction method
In a beaker containing powdered material, 5 mL of a BHT-acetone solution (0.05
percent, weight-to-volume), 5 mL of ethanol, and 10 mL of hexane were weighed
(0.3-0.6 grams). After 15 minutes of spinning on a magnetic stirring plate, 3 mL of
distilled water was added to the beaker.
The two phases were separated after 5 minutes of shaking and room-temperature
incubation. Using a pipette, the lycopene-containing layer was extracted. Lycopene
extract tubes were wrapped in aluminum foil and stored in the freezer for subsequent
Fig 1: Lycopene structure
Fig 2: IR- spectra of lycopene
Rf values are more likely to be higher in substances with less polarity. In general, the polarity
of a substance increases its capacity for adsorption (i.e. the more polar the compound then the
stronger it binds to the adsorbent).
Analysing IR spectra of lycopene
The maximum wavelength of the UV spectrum is s472.2nm, which is closer to the
maximum wavelength of pure slycopene documented in the literatures.
Indicated by the existence of a peak at 2922.97 on the graph, the sample has the
The sample contains the structure –C=C-, as evidenced by the peak at 1564.79 on the
The occurrence of a peak at 1020.99 denotes the presence of the structure –C-C- in
Fig 3: TLC plate for lycopene
The surface of silica gel is composed of Silicon dioxide (silica) which is transformed into an
irregular three-dimensional structure called silica gel by nanometer-scale voids and holes.
The surface is non-polar and that bears the main reason why it absorbs the lycopene. Polarity
of a molecule allows us to differentiate -carotene from the other carotenoids in our mixture.
Because Hexane is non-polar, it can be used to extract -Carotene from other compounds. Due
to its absence of functional groups and high degree of conjugation, this hydrocarbon exhibits
a rich color and a high level of lipophilicity.
Calculation of retardation factor
Retardation factor: Mobile phase – methanol
chloroform – 9.5: 0.5
Formula: retardation factor= distance travelled by solute / distance travelled by solvent = 2.5
/6.1 = 0.4098
Lycopene contains antioxidants naturally. Using the cup-plate method, the bactericidal
activity of tomato methanol extract was evaluated. The antimicrobial activity of Bacillus
Substilis is significantly more potent (Moghimipour et al. 2012). The therapy slowed the
development of Bacillus substilis by around 25 mm, or the inhibition zone. Several methods
have been used to extract lycopene, and the yield of lycopene from natural sources has been
found to be high. Initially, the benzene test assists in identifying lycopene in waste. To extract
lycopene with the least amount of organic solvent possible, a simple liquid-liquid extraction
method was adopted (Rahmat et al. 2002). To improve the crystals, they were recrystallized
from ether. Using a projection microscope, the scientists examined the recently discovered
crystals. It was discovered that lycopene in acetone has three distinct absorption peaks at 445,
471 and 500 nm. Similar peak characteristics were seen at 445, 471 and 502 nm when hexane
was used. The extraction of lycopene from tomato paste using acetone as a solvent was a
success. The hexane extraction generated less crude lycopene than the acetone-petroleum
ether extraction (Moghimipour et al. 2012). Due to its high degree of unsaturation, lycopene
was eluted after yellow-orange carotene pigments following purification by column
chromatography. The maximum absorbance of lycopene, for instance, is 476 nm, which is
typical of molecules of carotenoid pigments. L-lycopene exhibited maximal absorbance at
476nm and 503nm, according to spectrophotometry.
Properties of lycopene
Lycopene’s health advantages cannot be overstated. The chemical aids in digestion and
regulates the exchange of cholesterol. Antioxidant-rich goods protect against cardiovascular
disease by regulating appetite, maintaining acid-base balance, and lowering pathogenic gut
flora. The antifungal and antibacterial characteristics of lycopene enable it to revitalize and
nourish the skin by strengthening capillaries and blood vessels (Moghimipour et al. 2012).
Various liver illnesses can be prevented with the aid of a plant pigment.
Lycopene is an anti-oxidant capable of neutralizing free radicals, which are chemicals
linked to a variety of ailments. A diet rich in lycopene is beneficial to the circulatory
system and internal organs, hence reducing the risk of numerous chronic diseases.
Lycopene lowers the amount of oxidized DNA components, hence preventing
stomach and prostate cancer (Semenova et al. 1997). Lycopene is the only carotenoid
with anti-cancer properties.
Recent scientific research indicates that frequent lycopene consumption inhibits
cancer cell development by 70 percent. If you consume tomatoes many times per
week, you can minimize your risk of developing cancer by 35 percent.
Summary and Conclusion
According to the study’s findings, the fruits examined had a significant amount of lycopene.
Tomatoes contain a lot of lycopene. Lycopene can be extracted and purified from tomato
paste using simple, novel, and eco-friendly sorbents like nano and biomaterials.
Consequently, lycopene can be harvested and separated in numerous methods. As an
antioxidant, lycopene protects the body from free radicals, which can damage DNA and other
cell components. The powerful antioxidant properties of lycopene may slow or prevent the
development of certain types of cancer. Lycopene supplementation may also reduce
Moghimipour, E.; Aghel, N.; Zarei Mahmoudabadi, A.; Ramezani, Z.; Handali, S.
Preparation and Characterization of Liposomes Containing Essential Oil of
Eucalyptus Camaldulensis Leaf. Jundishapur Journal of Natural Pharmaceutical
Products 2012, 7 (3), 117–122. https://doi.org/10.17795/jjnpp-5261.
Rahmat, A.; Rosli, R.; Mohd. Zain, W. N. I. W.; Endrini, S.; Sani, H. A.
Antiproliferative Activity of Pure Lycopene Compared to Both Extracted Lycopene
and Juices from Watermelon (Citrullus Vulgaris) and Papaya (Caricapapaya) on
Human Breast and Liver Cancer Cell Lines. Journal of Medical Sciences 2002, 2 (2),
Semenova, S. I.; Ohya, H.; Soontarapa, K. Hydrophilic Membranes for Pervaporation:
An Analytical Review. Desalination 1997, 110 (3), 251–286.
Isolation of beta-Carotene From Carrot Paste
Dr. James Cho
Purpose: To learn the technique of column chromatography, and to isolate-carotene from carrot
Safety Information/Physical Data:
Dichloromethane:This chemical is a colorless liquid, and it has a sweet, ether-like odor. This
chemical is not flammable and is moderately soluble in water. The melting range for this
substance starts at about -96.7°C.
Acetone: This chemical is used for cleaning glassware (among other things) and has a string,
fruit-like, sweet odor. This chemical is also highly flammable and should be kept away from
heat. This substance is miscible with things like water, methanol, benzene, ethanol, and
chloroform. The melting range for acetone is from -94.6°C to -95.6°C.
Hexane: This chemical is a colorless liquid that has a gasoline-like odor. This chemical is highly
volatile and the melting range is from about -96 °to -94°C.
Sodium Sulfate: This chemal is a white, crystalline solid, and it does not have an odor. This
substance is insoluble in ethanol but is soluble in water.
The procedure begins by weighing 5 grams of carrots. After this, the participants in the
experiment use a mortar and pestle to mush the carrots and acetone until a paste was formed (A
stainless steel spatula was used to help with this process). After this, vacuum filtration was done
so that all of the yellow liquid would be separated from the carrot paste that was made. After the
solid paste was separated from the initial yellow liquid, the paste was then mixed well with 5 mL
of dichloromethane.Vacuum filtration was again performed until the second mixture was rid of
liquid. In the yellow substance that was leftover, 10 drops of dichloromethane, 10 drops of
hexane, and a lot of sodium sulfate until it turned clear yellow. A microscale chromatography
column was assembled next, and a dry packing technique was used to add powdered alumnia
through the funnel and into the column. This process continued until the alumina was packed
two-thirds of the way full in the column. Hexane was poured into the alumina packed column
until it filtered out the other end. Then, the clear yellow substance that was obtained in previous
steps was poured into the alumina column, and then hexane was added again after that. To help
the alumni flow to the bottom, air was used to push it down in the direction of the 10 mL
Erlenmeyer flask that was placed under the column. Following this, the residue that was obtained
from this step was redissolved with five drops of ethyl acetate. On a thin-layer of
chromatography plate, a 1 cm line was measured away from the end of the plate and traced
gently with a pencil. Two drops of solution were dropped on this line. In a 100 mL beaker, 1.8
mL of hexane and 0.2 mL of ethyl acetate was added, and the plate was placed into the beaker
with a watch class covering the top. When the line was about 0.2 cm away from the top of the
thin-layer chromatography plate, the thin-layer chromatography plate was removed from the
beaker and placed into the fume hood for two minutes to dry. After the plate was dry, a pencil
was used to mark the original dots and the dots that traveled. After this, the spots were measured,
and the measurements were written down for further calculations.
Carefully blow the residual solvent out of the chromatography column into a small Erlenmeyer
flask with gentle compressed air for 3 min. Pour the liquid into the liquid organic waste
container. Tap to loosen the alumina and dump it into the solid waste container. Wash all of the
glassware that was used with soap, water, and acetone.
Observational Data Presentation: yellow clear looks like oil
(3.15cm distance between the lines)
● 1st traveled 2.75cm, 2nd traveled 2.85cm
● 1st: 2.75/3.15=0.873
● 2nd: 2.85/3.15=0.905
(left is the first, the right is the second)
TLC is carried out against gravity and used for less analytical purposes which also takes less
time for it to separate. TLC also requires more polar solvents but less amounts of it. TLC is also
a solid- liquid based absorption. They are similar because both techniques are solvent and both
are used for separation. They both are organic and do not require a very sophisticated
environment. Both techniques can be handled very easily. Acetone provides a great middle
ground for this process because it is amphipathic. An amphipathic substance has both a polar end
and a nonpolar end. Dichloromethane is more polar, and hexane is more nonpolar. So it can pull
it down the TLC plate. Sodium sulfate is often easier to remove water prior. Powdered alumina is
polar. Hexane was used again to push the yellow substance down the alumina column. The book
explains the reasoning behind how the mixtures in the carrot can be separated, and that involves
the use of appropriate solvents that a.) do not destroy the silica ge, and b.) dissolve like
substances. The solvents chosen for this experiment were chosen via the reasoning of
“like-dissolves-like”. With this, polarity is also a characteristic to take into consideration because
some compounds were absorbed and released over time, and left behind (with the chemicals) in
this experiment. Functional groups and how they react with polar groups is also another example
of how this experiment was able to take place.
Thin-layer Chromatography is in the mobile phase. The stationary phase remains fixed in place
while the mobile phase carries the components of the mixture through the medium being used.
Silica gel with a surface populated with nonpolar chlorophyll. Beta carotene is orange and is
nonpolar because it is a hydrocarbon. Polarity dictates how fast it moves up the TLC plate. There
are barely any differences between the two Rf values.
The Rf values are the same because it was used from the same mixtures. Depowering in this
experiment because the b-carotene is taken away. The ratio between the ethyl acetate and hexane
is 1:9. There might be some errors to this experiment because if an extra drop of hexane or ethyl
acetate was added it might change the distance the solvent traveled.
Side reaction: N/A
Theoretical yield: N/A
Analysis of TLC/IR/NMR: Rf calculation work: 3.15 cm distance between the lines
1st traveled 2.75 cm, 2nd traveled 2.85 cm
1st: 2.75/3.15 = 0.873
2nd: 2.85/3.1 5 = 0.905
Post Lab Questions:
4.) Once the chromatographic column has been prepared, why is it important to allow the level of
the liquid in the column to drop to the level of the alumina before applying the solution of the
compound to be separated?
-If a compound is added while there is still solvent in the column above the alumina, the
compound might dissolve and disperse. or make it very diluted.
5.) A chemist started to carry out column chromatography on a Friday afternoon, reached the
point at which the two compounds being separated were about three-fourths of the way down the
column, and then returned on Monday to find that the compounds came off the column as a
mixture. Speculate the reason for this.
-The column had not dried over the weekend, the column was overloaded, the column was not
packed properly, and the fractions were not calculated properly.
BibGuru. (n.d.). Bibguru.Com. Retrieved April 4, 2022, from
ChemDraw JS Sample Page. (n.d.). Perkinelmer.Cloud. Retrieved April 4, 2022, from
Macroscale, M. (n.d.). Macroscale and Microscale Organic Experiments 7th Edition by
Sketchpad – draw, create, share! (n.d.). Sketchpad.App. Retrieved April 4, 2022, from
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