Organic Chemistry Lab Report Example

Isolation and Isomerization of Lycopene from Tomato Paste
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ISOLATION AND ISOMERIZATION OF LYCOPENE FROM TOMATO PASTE 1
Abstract
Lycopene refers to a primary pigment that is principally responsible for the deep-red
characteristic color of a ripe tomato and its products. This lycopene is the primary source of a
useful antioxidant, an exciting product for its physicochemical and biological properties. In this
experiment, tomato paste will be prepared from cultivated tomato in Dezfoul that has been
dehydrated using methanol. After that, lycopene will be extracted using a tetrachloride mixture
of methanol-carbon. In this experiment, crude oil was crystallized twice using benzene and
adding boiling methanol. A technique of liquid extraction will also be used to isolate lycopene
and xanthophyll from the tomato paste. Furthermore, a chromatogram column containing
alumina adsorbent was used in the process of purification. The chemical structure identification
was achieved through isolation of lycopene utilizing IR, UV, NMR, and technology of
spectroscopy. Pure lycopene average quantity was computed and found to be 2.313 mg in 100 g
of tomato paste. The resulting color of isolated effluents was found to relate to radiation's
wavelengths absorbed. Ideally, the lycopene band had red-orange color with a yellow band. TLC
analysis was also performed to evaluate the separated lycopene effluent's column
chromatography quality, Rf OF 0.5 visible on TLC plate. The percentage of trans-lycopene was
determined through UV-vis spectrum (ultraviolet-visible spectrum). The maximum cis-lycopene
wavelength was found to be 466 nm and that of trans-lycopene as 500nm. The wavelength of
trans-lycopene, the isomerized form, was 490 nm. Moreover, the trans-lycopene isomerized form
percent with UV-vis spectrum was 47.4%, while that of trans-lycopene was 92.9%.
Keywords: Antioxidants, Tomato, Lycopene, Carotenoids.

Isolation and Isomerization of Lycopene from Tomato Paste
Over a long period of time, different epidemiological studies have been developed.
According to Caseiro et al. (2020(, feeding on tomatoes and foods that are tomato-based can
reduce cancer of esophagus, oral cavity, rectum, stomach, rectum, breast and prostate in human
beings. In its protective effect, tomato and tomato based food products have been attributed to so
many carotenoids, a major phytochemical class of tomato fruit. Carotenoid is a group of
compounds comprising more than 600 different plant pigments, most of which are fat-soluble,
providing the deep-red color seen with tomatoes. Additionally, carotenoid compounds have very
nutritious content in the human body by providing pro-vitamin A and aiding antioxidant
activities (Elvira-Torales et al., 2019). Lycopene is the abundant and basic carotenoid in a
tomato, although there are contents such as beta-carotene, gamma carotene, and neurosporene.
Lycopene is the red-carotenoid pigment found in a ripe tomato and products made from it. It is
principally responsible for the deep-red characteristic in tomato and tomato-made products. It
has gained substantial interest in finding different biological applications, such as in reducing
coronary heart oxidative stress disease and other notable chronic diseases (Imran at al., 2020).
Lycopene has interesting physical properties. It has a molecular formula of C40H56 with a
molecular mass of 536.89. It is 89.45% carbon and only 10.51% hydrogen. The chemical
structure of lycopene is as shown in figure 1 below.
Figure 1: Lycopene Chemical Structure
Figure 1 above shows that lycopene is an unsaturated hydrocarbon with two unconjugated bonds
and 11 different conjugated bonds. There are several methods that can be used to extract and
quantify lycopene. However, lycopene is highly affected by undesirable degradation, both on the
final product and sensory quality and health benefits of tomato made food products. Lycopene is
essentially found in all fresh tomatoes in different transfigurations. Lycopene degradation mainly
occurs during the process of oxidation and isomerization (Saini et al., 2019).
Isomerization involves converting all-transfiguration isomers into cis-isomers through
addition of energy input resulting into an undesirable into an unstable station that is rich in
energy (Imran at al., 2020). Determination of lycopene isomerization degree is very important.
This process will ensure that health benefits of tomato-based products, especially food has been
measured before processing. Thermal processing, a technique that involves bleaching, freezing
and retorting, will cause some reduction of lycopene content in tomato food products. The heat in
thermal processing will induce isomerization of all cis forms. Thereafter, cis-isomers will

increase as processing and temperature increases with time. Generally, powdered and dehydrated
tomato products are characterized with lycopene's poor stability unless they have been processed
carefully and promptly kept within a sealed hermetically inert environment for storage(Saini et
al., 2019).
There will be a significant increase in cis-isomers if kept in such a manner and a
subsequent reduction in all-transfiguration isomers, which can be seen in samples of dehydrated
tomatoes employing different methods of dehydration. Frozen heat-sterilized food, for example,
will exhibit improved lycopene stability when stored in normal temperature shelf-life. Column
chromatography is primarily used to separate pigments. There are two hexane mobile phases and
10 % acetone which will be eluted from this column using gravity at various rates. Solubility and
polarities of lycopene usually determine these rates. Application of alumina as a stationary phase
is significant as it allows isolation and separation of lycopene segments. The eluents can then be
tested with the lycopene extracts using a TLC analysis that ensures complete separation.
Over a long period of time, lycopene has been prepared from different berries and fruits
(Zardini et al., 2018). Lycopene was first prepared from the Tanus communis in 1873 by Harsten
(Saini et al., 2019). Escher and willstter later improved this model. They had processed 75 kg of
tomato concentrate, obtaining 11 g of recrystallized Lycopene (Saini et al., 2019). All the
lycopene extracted was impure, necessitating stabilizing, and purification. They introduced
different purification methods, including a preparative HPLC, TLC, and extractive solid-phase
technique. Even though it is possible to obtain lycopene through techniques of chromatography,
the use of liquid-liquid extraction techniques is preferred, especially when the always
complicated chromatography instruments are not effective or not available.
Natural products could be excellent antioxidants sources and economic costs evaluation.
They also simple to extract and have a useful economic costing. Moreover, the required methods
of extraction are readily available. Currently, the main method of quantification and economical
extraction of lycopene from tomato is Dezfoul cultivation, which will be used in this experiment
before extracting lycopene using a tetrachloride mixture methanol-carbon. Crude oil will then be
crystallized twice using benzene and adding boiling methanol before using a chromatogram
column containing alumina adsorbent for the process of purification. The chemical structure
identification will be achieved through lycopene's isolation utilizing IR, UV, NMR, and
technology of spectroscopy. The quality of Lycopene extract will be calculated as an all-trans
lycopene percentage.
Material and Methods
Plant Materials
Tomato fruits from Dezfoul farms. These tomatoes were identified as Lycopersicum
esculentum.
Procedure

Isolation. The first technique was done by use of a 50 g tomato paste sample was first
dehydrated in a beaker by addition of methanol, 65 ml. The resulting mixture was then shaken
vigorously, almost immediately to prevent it from forming hard lumps. A period of 2 hours, the
suspension which had resulted was filtered off. The remaining dark-red cake was then vigorously
shaken for 15 minutes with a 75 ml carbon tetrachloride and methanol mixture, then separated
through filtration. The carbon tetrachloride phase was then transferred into a separating funnel
where it was added one volume of water then shaken properly before doing phase separation.
After the phase separation, carbon tetrachloride had to be evaporated and the remaining residue
diluted using 2 ml of benzene. A volume of 1 ml methanol, which had been heated to boil, was
then taken using a dropper and added to the diluted residue. Crude oil residue resulted and
immediately crystallized, maintaining room temperature by keeping it in an ice bath. Crystals
formed were then washed 10 times using a mixture of boiled methanol and benzene. Lycopene's
very long and dark-red prisms were formed as observed using a microscope with some traces of
colorless impure substances.
The second technique involved the use of 10 ml, 50 % hexane/ acetone mixture to do the
separation and determine the percentage of lycopene extract. The 10 ml, 50 % hexane/ acetone
mixture was first added into 3.907 g of specially prepared tomato paste and filtered in a
separating funnel. This step was repeated two more times, without changing the 10 ml, 50 %
hexane/ acetone condition. The resulting mixture was then decanted and adequately washed on a
special filter paper. It was then pressed to increase the quantity of the final pigment product.
Thereafter, 10 % sodium carbonate solution was added (20 ml) then washed using 20 ml sodium
chloride, which had been saturated, followed by 10 ml hexane. The resulting extract was later
dried using magnesium sulfate, gravity filtration in a round flask, and then rotary evaporation
was employed to complete the process drying process. A 0.5 ml hexane was then added to this
extract to ensure complete wetting of 5cm alumina into the column chromatography (TLC).
Purification. For the purification procedure, the column chromatography (TLC)
technique was used with active acidic alumina, using toluene. From this purification step, a deep-
red zone was formed. This zone was subjected to evaporation to eliminate its solvent portion.
The remaining residue was then dissolved into 2 ml of benzene solution. The process of
recrystallization followed using a boiled methanol solvent recrystallization process, and there
was no formation of colorless impure substances indicating that the sample had been purified
completely. However, the crystalline lycopene was not isomerized. Instead, it shows air
oxidation or autoxidation tendencies, especially when placed in the light. Crystalline lycopene is
therefore stored in specially evacuated dark glass tubes before it can be used. Chemical reactions
of color were done for primary identification. The chemical structure of lycopene was identified
by isolating it using UV, NMR, IR, and mass spectroscopy techniques. From the analysis of UV-
vis spectroscopy, the percentage of lycopene all-transfiguration could be determined, employing
absorption maximum technique for the present stereoisomers.
Results and Identification

After column chromatography purification, Lycopene crystals' yield was 2.313 mg in 100
g of tomato paste. A few crystals of lycopene extracted had to be placed into sulfuric acid
(concentrated) for lycopene identification, where they imparted a characteristic indigo-blue color
to the sulfuric acid solution. Another identification test had to be done with an antimony tri-
chloride solution. The antimony tri-chloride solution had to be added to the solution containing
lycopene chloroform resulting in an unstable intense blue color product. The two tests proved
that there was lycopene in the primary extract.
A further test was done to confirm the purity of lycopene. This test involved structural
analysis of lycopene extract. This test resulted in a UV spectrum shown in figure 2 below.
Figure 2: Visible UV spectrum of Lycopene Extracts
The spectrum shown in figure 2 above has three maximum wavelengths, 473.2 nm, 447.2 nm,
and 504.2 nm. The standard maximum wavelength of pure lycopene is 504.2 nm. IR spectrum
vibrational wavelengths for purified lycopene extracts were found as shown in figure 3 below.

Figure 3: IR Spectrum for the purified lycopene extracts
The wavelengths of IR Spectrum for the purified lycopene extracts were as shown in
table 1 below.
Table 1: Vibrational Wavelengths for the IR spectrum
Extract Wavelength, (cm-1)
CHstr(SP)2 3100
CHstr(SP)3 2918.92, 2851.05
C=CHstr(Trans) 1670, 1640
CH2(Bending) 1446.92, 1400
CH(Trans OOP) 1101.07,957.33, 1000
R2C=CR 612.84

Using HNMR spectrophotometer, a HNMR spectrum could be recorded as shown in the table 2
below with frequency of Bruker 400 MHZ with ultrasound shield. The HNMR spectra for the
processed lycopene is illustrated in figure 3.
Table 2: Vibrational Wavelengths for the IR spectrum
δ(pmm)
5.12 2H →C2
2.13 4H →C3
2.23 4H →C4
5.96 2H →C6
6.63 2H →C7
6.26 2H →C8
6.11 2H →C10
6.86 2H →C11
6.33 2H →C12
6.11 2H →C14
6.21 2H →C15
1.63 12H →C17
1.83 6H →C18
1.98 12H →C20

Figure 4: HNMR
Lycopene Spectra as
Purified with
Column
Chromatography
The Rf
values for the spots
(for TLC analysis)
were such that
Table 3: Analysis of
TLC plate
Table 3 above shows that yellow eluent Rf values not applicable as it did not appear on the TLC
plate. The value of Rf-1 is for the red-orange spot on the TLC plate while that of yellow spot
corresponds to Rf-2. The values of wavelengths for lycopene isomers in pre-isomerization were
found to be 466 nm and 500 nm for cis-lycopene and all-trans lycopene. The diagram of TLC
plate analysis was as shown in figure 5 below.
All spots Rf Extract Yellow eluent Red-orange eluent
Rf-1 0.55 N/A 0.50
Rf-2 0.78

Figure 5: TCL analysis
The plate in figure 5 above represents diagrammatically, the Rf values of. It shows a spot for
yellow eluent and orange-red eluent spreading away from the baseline. The two spots spread
differently with migration of extract on the plate.
For post-isomerization, wavelengths values reduced to 450 nm and 490 nm for cis-lycopene and
all-trans lycopene respectively as shown in table 4 below.
Table 4: Maximum Absorption
The percentage of resulting isomerization for the trans-lycopene were found to be 92.9% for pre-
isomerization and 47.7% for the post-isomerization. These percentages were determined through
the Ultraviolet spectroscopy technique.
Lycopene Isomers Maximum wavelength of cis-
Lycopene
Maximum wavelength of all-
trans lycopene
Post-isomerization 466 490
Pre-isomerization 466 500

Discussion
The purpose of this lab exercise was to evaluate the isolation and isomerization of
lycopene from tomato paste. Lycopene pigments were separated from the tomato through liquid-
liquid extraction using different solutions. The first technique involved use of Dezfoul tomato
paste using a minimum solvent as possible. The resulting mixture had to be shaken vigorously,
almost immediately to prevent it from forming hard lumps. It was then filtered, diluted and
recrystallized forming very long and dark-red prisms of lycopene were formed as observed using
a microscope with some traces of colorless impure substances. The second technique was much
improved as it used 50 % hexane/ acetone mixture to not only do the separation but also
determined the percentage of lycopene extract. The lycopene pigments were placed in an organic
layer hexane-acetone solution mixture and extracted using a potassium carbonate. They had to be
washed using the potassium carbonate to neutralize a citric acid that could be present. The
sodium chloride solution (saturated) was added to ensure that the pigments dried completely to
become unsaturated and form an aqueous immiscible layer that could be separated from which
could be separate easily from the layer (organic layer). The separated organic layer was later
dried using a magnesium sulfate solution. The pigments could then be isolated through rotary
evaporation after gravity filtration.
The main challenge was found in the purification stage with column chromatography,
which incorporated the alumina stationary phase. However, crystals could be observed with a
microscope. Pure lycopene average quantity was computed and found to be 2.313 mg in a 100 g
of tomato paste. The resulting color of isolated effluents was found to relate to radiation's
wavelengths absorbed. Ideally, the lycopene band had red-orange color with a yellow band. The
appearance of a colorless substance had shown the extent of impurity in the lycopene crystals.
When column chromatography was combined with recrystallization, pure lycopene crystals
could be seen without colorless impure substances. The analysis of IR and NMR spectroscopy
revealed some degree of impurity. The lycopene crystals formed through triple recrystallization
had very few lycopene peaks. In fact, the spectrum of UV three maximum wavelengths, 473.2
nm, 447.2 nm, and 504.2 nm. The standard maximum wavelength of pure lycopene is 504.2 nm,
while the IR spectrum had a range of vibrational wavelengths, as shown in table 1, with
CHstr(SP)2 being the highest at 3100 cm-1. HNMR spectrophotometer recorded the frequency of
Bruker 400 MHZ with ultrasound shield. Mass spectra did confirm the presence of lycopene in
the tomato paste. However, column chromatography, when combined with recrystallization using
alumina stationary phase and the toluene, improved the purification process. The crystals
obtained were pure, with recommended transfiguration precautions for storage. This technique
can be enhanced by excluding air then storing the crystals obtained under atmospheric nitrogen
since they are sensitive to light and could get oxidized easily by atmospheric oxygen. After this

purification process, 2.313 mg of lycopene was obtained per 100 g of tomato, which is high
compared to other amounts obtained with previous studies.
The process of purification was improved through TCL chromatography. TLC analysis
was also performed to evaluate to identify column chromatography quality of the separated
lycopene effluent. UV spectrum analysis had to be done with this TLC to determine the
percentage of transfigurations of lycopene in the sample. The absorbance of cis-lycopene and
trans-lycopene in post-isomerization and pre-isomerization gave relative values of wavelengths
for lycopene isomers such that the pre-isomerization wavelengths were found to be 466 nm and
500 nm for cis-lycopene and all-trans lycopene. Turning to the post-isomerization, wavelengths
values reduced to 450 nm and 490 nm for cis-lycopene and all-trans lycopene, respectively.
The TLC plate could be represented diagrammatically. Its Rf values showed that yellow
eluent Rf values are not applicable as they did not appear on the TLC plate. The value of Rf-1 is
for the red-orange spot on the TLC plate, while that of the yellow spot corresponds to Rf-2. It
shows a spot for yellow eluent and orange-red eluent spreading away from the baseline. The two
spots spread differently with the migration of extract on the plate. The percentage of resulting
isomerization for the trans-lycopene were found to be 92.9% for pre-isomerization and 47.7% for
the post-isomerization. These percentages were determined through the Ultraviolet spectroscopy
technique. These percentages indicate that when tomato paste is exposed to varying wavelengths
of light, it will have a quality effect on lycopene quality.
Conclusion
This experiment evaluated the isolation and isomerization of lycopene from tomato paste.
Tomato paste was prepared from cultivated, separated, and quantified from Dezfoul tomato that
has been dehydrated using methanol. Lycopene pigments were extracted from Dezfoul tomato
using a tetrachloride mixture of methanol-carbon. Crude oil was later crystallized twice using
benzene and added boiling methanol. A liquid-liquid extraction technique was incorporated to
isolate lycopene and xanthophyll from the tomato paste. A chromatogram column containing
alumina adsorbent was used in the process of purification. The chemical structure identification
was achieved through isolation of lycopene technology of IR, UV, and NMR spectroscopy. Pure
lycopene average quantity was found to be 2.313 mg in 100 g of tomato paste. The resulting
color of isolated effluents was tentatively found to relate radiation's wavelengths absorbed,
lycopene band, having a red-orange color with a yellow band, after performing TLC analysis to
identify column chromatography quality of the separated lycopene effluent, 0.5 Rf visible on
TLC plate. The percentage of trans-lycopene was determined through UV-vis spectrum
(ultraviolet-visible spectrum). The maximum cis-lycopene wavelength of 466 nm and that of
trans-lycopene is 500nm. The wavelength of trans-lycopene, an isomerized form, was 490 nm.
Finally, the trans-lycopene isomerized form percent with UV-vis spectrum had a 47.4% while
that of trans-lycopene was 92.9%. Tomato paste has been presented in this report as a very good
and natural lycopene source. However, this experiment can be improved by incorporating more
new and straightforward extraction techniques environmentally friendly with different sorbents,

including bio and nanomaterials for isolation and subsequent purification stage. The primary
source of error in this experiment was in the isomerization stage. Different and unwanted
appeared, causing a substantial percentage decrease in the final product. Additionally, lycopene
could have been isomerized before the commencement of the experiment, and no amount of
intervention would prevent this isomerization. Delayed collection of final lycopene could have
led to an inaccurate final mass of the final product.

References
Caseiro, M., Ascenso, A., Costa, A., Creagh-Flynn, J., Johnson, M., & Simões, S. (2020).
Lycopene in human health. LWT, 109323.
Elvira-Torales, L. I., García-Alonso, J., & Periago-Castón, M. J. (2019). Nutritional importance
of carotenoids and their effect on liver health: A review. Antioxidants, 8(7), 229.
Imran, M., Ghorat, F., Ul-Haq, I., Ur-Rehman, H., Aslam, F., Heydari, M., & Hashempur, M. H.
(2020). Lycopene as a natural antioxidant used to prevent human health disorders.
Antioxidants, 9(8), 706.
Saini, R. K., A. Bekhit, A. E. D., Roohinejad, S., Rengasamy, K. R., & Keum, Y. S. (2019).
Chemical Stability of Lycopene in Processed Products: A Review of the Effects of
Processing Methods and Modern Preservation Strategies. Journal of Agricultural and
Food Chemistry, 68(3), 712-726.
Zardini, A. A., Mohebbi, M., Farhoosh, R., & Bolurian, S. (2018). Production and
characterization of nanostructured lipid carriers and solid lipid nanoparticles containing
lycopene for food fortification. Journal of food science and technology, 55(1), 287-298.

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