Isolation of Carotenoids

By Jonathan Harcourt
Introduction
-Carotene and lycopene are organic compounds known as carotenoids. Carotenoids are
natural phytochemicals of the tetraterpenoid family, which means that they are comprised of 8
isoprene units, hence the 40 carbon atoms in each molecule. Carotenoids naturally occur in the
chloroplasts of plants and other photosynthetic organisms.3 Carotenoids are of interest because
they are great antioxidants, stimulate the immune system, and are required for the production of
provitamin A. -Carotene and lycopene are the most studied carotenoids since they are the most
abundant in human diet.2
-Carotene is the most common form of carotene, since -rings are preferred over a-rings
in its structure.1,3 It was discovered by Heinrich Wilhelm Ferdinand Wackenroder in 1831;
Wackenroder isolated it from the roots of carrots. The structure was determined a century later
by Paul Karrer.1 -Carotene is yellow-orange in color. The most common side effect of
excessive -carotene consumption is carotenodermia; which is a harmless condition where the
subjects skin turns orange. This is a result of -carotene being deposited in the outmost layer of
the skin.3 There is mixed results on weather -carotene prevents or facilitates cancer. High doses
of -carotene have been associated with increased risk of lung cancer in people who smoke or
were exposed to asbestos.2,3 However, dietary levels of -carotene have been known to reduce
the risk of many types of cancer.2,3
Lycopene was first isolated from a European yam, Tamus communis, in 1873.4 Lycopene
is a red-orange color; excess amounts of lycopene can accumulate in the adipose tissue under the
1

skin, producing lycopenemia, a variant of carotenodermia.5 Unlike -carotene, lycopene can not
be converted into vitamin A, in the human body. However, lycopene is still a biologically
important substance.5 Lycopene is two times more effective as an antioxidant than -carotene;
and it is associated with the reduced risk of prostate, lung and stomach cancers.4
The purpose of this experiment was to isolate lycopene and -carotene from a mixture of
tomato paste and carrot baby food. Column chromatography (abbreviated CC) was used to
separate the two carotenoids. Thin layer chromatography (abbreviated TLC) was used to
monitor the progress of the column, the purity of the column fractions, as well as which color
band was -carotene. The technique of ultraviolet and visible spectroscopy (abbreviated
UV/Vis) was used to confirm the identities of the isolated products.
Experimental
A tomato paste and carrot baby food mixture (10g) was placed into a beaker with 95%
ethanol (30mL). The solution was stirred for about 6 minutes. The solid was isolated via
vacuum filtration and placed into a 25mL round-bottom flask with dichloromethane (abbreviated
DCM) (10mL). The solution was refluxed for about 5 minutes. The extracted solid was isolated
once again by vacuum filtration. The solid was extracted similarly two more times. The three
DCM solutions were combined and washed with brine solution. The organic solution was dried
with sodium sulfate. The DCM was evaporated from the organic solution and hexane (1mL) was
added. TLC was used to determine if -carotene was present in the organic solution. The
solution was separated on an alumina gel column using three different mobile phases. The first
mobile phase, hexanes, was used to separate the color bands. The second mobile phase, 2%

DCM in hexanes, was used to elute the first color band, which was yellow (-carotene). The
third mobile phase, DCM, was used to elute the second color band, which was scarlet (lycopene).
TLC Data: Figure 1
Fraction
Number
5
6
10

Rf Value
0.196
0.327
0.18

-Carotene
Rf Value
0.217
0.327
0.35

% Difference from
-Carotene Rf Value
9.7%
0%
48.6%

Identity
-Carotene
-Carotene
Lycopene

UV/Vis Data: Figure 2

Sampl
e
1
2

Color
yello
w
scarlet

Sample Absorbed
wavelengths (nm)
426,450,476

-Carotene Absorbed
wavelengths (nm)
426, 448, 474

Lycopene Absorbed
wavelengths (nm)
444, 473, 502

Identity

444,472,502

426, 448, 474

444, 473, 502

Lycopene

-Carotene

Results and Discussion

The alumina gel column was very effective in separating the two carotenoids. There was
a 3 fraction gap between the last fraction containing -carotene and the first fraction containing
lycopene. In addition, there was only one spot found for any of the fractions, which means that
each carotenoid was completely separated from the other. This is most likely the cause of using
the 3 different mobile phases, which allowed for good band separation, as well as timely elution
of the carotenoids. In addition, problems with CC encountered in previous experiment were
addressed and prevented. For example, the column never came close to running dry, which was
a problem faced in the Synthesis and Separation of Fluorenone from Fluorene experiment.

The TLC data shows that only 3 fractions contained carotenoids. The first column
fraction to show the presence of a carotenoid was fraction 5, which had a spot that almost lined
up with the -carotene spot. The sixth fraction had a spot that had the same Rf value as the carotene spot. As a result the fifth fraction was most likely -carotene with some impurities,
while the sixth fraction was pure -carotene. This makes sense because -carotene should elute
first; and fractions 5 and 6 were both yellow in color. The tenth fraction had a spot with an Rf
value that was half (48.6% difference) that of the -carotene spot. As a result the tenth fraction
was most definitely not -carotene, which means that it was lycopene. This makes sense because
the tenth fraction was scarlet in color, which corresponds to the color of lycopene.
The UV/Vis data confirms the TLC data findings. The absorbed wavelengths
(abbreviated max) for the fifth and sixth column fractions were found to be 426, 450, and 476
nm, which is no more than a 2nm difference from the max for pure -carotene. The max for the
tenth column fraction were found to be 444, 472, and 502, which is no more than a 1nm
difference from the max for pure lycopene.
In conclusion, this experiment was successful for many reasons. Both lycopene and carotene were successfully isolated from the tomato paste and carrot baby food mixture. CC was
successfully used to separate and purify the two carotenoids, which the TLC and UV/Vis data
confirms. In addition, lessons learned from previous experiments were applied to acquire
desired results.

References
1

Beta Carotene. University of Bristol. < http://www.chm.bris.ac.uk/motm/carotene/beta-

carotene_home.html > [accessed December 5 2010]

2

Beta-carotene and other carotenoids as antioxidants. Paiva S. A; Russell R. M. J Am Coll

Nutr. 1999 Oct;18(5):424-5. < http://www.ncbi.nlm.nih.gov/pubmed/10511324> [accessed

December 5 2010]
3

Haugen, Leiv; Bjornson, Terje. Beta Carotene: Dietary Sources, Cancer and Cognition. Nova

Biomedical. 2009.
4

"lycopene." Encyclopdia Britannica. 2010. Encyclopdia Britannica Online. 06 Dec. 2010

<http://www.britannica.com/EBchecked/topic/352572/lycopene>.
5

The carotenoid lycopene. Nutrition Research Newsletter. Jan, 1997.

<http://findarticles.com/p/articles/mi_m0887/is_n1_v16/ai_19094812/?tag=content;col1>
[accessed December 7 2010]