Isolation and Column Chromatography of Lycopene
Andrew Pany
Chemistry 213H, The Pennsylvania State University, University Park, Pennsylvania
aqp5308@psu.edu
3/25/15
ABSTRACT:
Lycopene was successfully extracted from tomato paste through the utilization of
solid/liquid extraction. The crude lycopene was then purified through column
chromatography. Thin-layer chromatography was used throughout the experiment
to determine the success of the purification process. Moreover, the purified
lycopene underwent isomerization, from trans to cis, in the presence of an iodine
catalyst and strong ultraviolet light.
INTRODUCTION:
Solid/liquid extraction is the most common technique used for isolating compounds
from natural products and is used prevalently in the food industry. For this type of
extraction, the solid is the natural product and the liquid is the solvent into which
the compounds are extracted1. In this experiment, lycopene was isolated from
tomato paste via solid/liquid extraction.
Lycopene 1, the red pigment in tomatoes, is the primary carotenoid contained in
tomatoes and a major dietary antioxidant, with many beneficial effects for human
health2. Solid lycopene was obtained from tomato paste by repeatedly treating the
paste with polar compounds and then drying it through filtration in between each
treatment. After isolation, the crude product was purified through column
chromatography.
Figure 1. The structure of lycopene.
1Masters,
2
K.M.; Williamson, K.L. Macroscale and Microscale Organic Experiments: Extraction and Recrystallization, 6th Edition.
Periago, M. J.; Rinón, F.; Jacob, K.; García-Alonso, J.; Ros, G. J. Agric. Food Chem., 2007, 55 (22), pp 8825–8829
Column chromatography is one of the most useful methods for the separation and
purification of solids in microscale experiments (experiments dealing with 10 grams
or less). This purification process takes advantage of the differences in the elution
rates of the desired product and contaminates through the utilization of adsorbents,
such as silica gel [(SiO2)x] or aluminum [(Al2O3)x], and solvents (usually hexanes)3.
Less polar molecules are not strongly adsorbed and travel through the column
quickly, while more polar molecules are strongly adsorbed and travel through the
column slowly.
Additionally, both the purified and crude lycopene products were analyzed via thinlayer chromatography (TLC). Thin-layer chromatography is a sensitive, fast, simple,
and inexpensive analytical technique used to determine the number of components
in a mixture, the effectiveness of a purification process, and monitor reaction
progress, among other functions. TLC, similarly to column chromatography, takes
advantage of the differences in elution rates of various substances4. Substances that
are like the stationary phase, mostly in polarity, will travel slowly and short
distances. Substances that are unlike the stationary phase will travel fast and
extended distances. These differences allow chemists to determine the identities of
substances.
Furthermore, some of purified lycopene product underwent light-catalyzed
isomerization, using iodine as an added catalyst. Both the isomerized and
unisomerized portions of lycopene were characterized via UV-visual
spectrophotometry.
The objectives of this experiment were to isolate lycopene from tomato paste via
solid/liquid extraction, purify the lycopene mixture via column chromatography,
and to isomerize the lycopene using iodine as a catalyst.
RESULTS AND DISCUSSIONS:
Solid/Liquid Extraction of Lycopene
Tomato paste was initially rinsed with acetone and pressed dry before undergoing
the solid/liquid extraction to help remove water-soluble components from the
paste. The filtrate from the pressing was saved. The solid residue was then shaken
with dichloromethane and filtered, both done three times. Following completion, all
of the filtrates were combined.
Water and sodium chloride were added to the filtrate solution to aid in the breaking
of emulsions. Once the two additional components were added, the solution began
to separate into two layers. A yellow, top layer formed over an orange, bottom
layer. The extraction removed the acetone and any water-soluble components from
the mixture, which were contained in the yellow layer. What remained was the
3Masters,
4
K.M.; Williamson, K.L. Macroscale and Microscale Organic Experiments: Extraction and Recrystallization, 6th Edition.
Masters, K.M.; Williamson, K.L. Macroscale and Microscale Organic Experiments: Extraction and Recrystallization, 6th Edition.
orange organic layer, which contained the crude lycopene still suspended in
dichloromethane.
The orange solution was subsequently dried over anhydrous calcium chloride and
filtered. A portion of this filtrate was analyzed by thin-layer chromatography while
most of the product was evaporated and stored under nitrogen since the
carotenoids are very susceptible to air oxidation.
The TLC analysis gave Rf values of .241 and .769, indicating a mixture of two
products. The first value represents the desired lycopene while the second value
indicates a strong presence of carotene, which is also contained in the tomato paste
and is a byproduct of the extraction in this experiment.
Column Chromatography of Lycopene
The crude lycopene product obtained from extraction was purified through column
chromatography. The chromatography column was prepared via a dry-packing
method, running an 80:20 hexane to water mixture through the column packed with
silica. Sample from the extraction was then added in a dichloromethane solution to
the chromatography column followed by a layer of sand packed on top of the
sample. Hexanes were run through the column continuously, causing the formation
of a yellow and orange band as the sample traveled through the column. The two
bands were separately collected and the submitted for thin-layer chromatography.
The TLC analysis showed an Rf value of .648 for the yellow band. This value closely
matches that of pure carotene. The TLC analysis for the orange band showed a
value of .16, which is very similar to that of pure lycopene. These values indicate
that the purification was a success.
Isomerization of Lycopene
A hexane solution of a portion of the lycopene was prepared for isomerization by
adding a drop of iodine catalyst. The resulting mixture was exposed to strong
ultraviolet light for ten minutes in order to change from a trans-isomer to a cisisomer. Both a solution of nonisomerized and isomerized lycopene were submitted
ultraviolet-visible spectrum analysis.
The unisomerized lycopene had strong absorptions at 448.10nm, 472.30, nm, and
504.40nm. The isomerized lycopene had strong absorptions at 443.00, 466.20, and
498.90. The spectral data verifies that the isomerization was successful since it
shows the presence of a hypochromic effect, a decrease in the visible-band
absorption that typically occurs when a trans-isomer changes to a cis-isomer. The
spectral data also confirms the presence of a hypsochromic shift, a shift to the left,
usually by five to seven nanometers, of the peaks in the visible-band absorption5.
5
Tan, B; Soderstrom, D. N. J. Chem. Educ., 1989, 66 (3), p 258
CONCLUSION:
Solid/liquid extraction was successfully used to isolate a lycopene mixture from
tomato paste. Additionally, column chromatography was successfully used to purify
the mixture, yielding pure carotene and lycopene. The success of the purification
process in addition to the presences of both substances was confirmed by thin-layer
chromatography. Through the use of ultraviolet light and an iodine catalyst, the
lycopene was effectively isomerized from a trans-isomer to a cis-isomer, which was
confirmed by ultraviolet-visible spectrum analysis.
ACKNOWLEDGEMENTS:
The author would like to graciously acknowledge the support from the class
professor, Dr. Masters, and teaching assistant Anthony Nocket.
REFRENCES:
1. Masters, K.M. Chem 213H Food Science Module, Spring 2015.
2. Masters, K.M.; Williamson, K.L. Macroscale and Microscale Organic
Experiments: Extraction and Recrystallization, 6th Edition.
3. Periago, M. J.; Rinón, F.; Jacob, K.; García-Alonso, J.; Ros, G. J. Agric. Food
Chem., 2007, 55 (22), pp 8825–8829
4. Tan, B; Soderstrom, D. N. J. Chem. Educ., 1989, 66 (3), p 258
SUPPORTING DATA:
Please see attached sheets.