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CHEM 333L

Organic Chemistry Laboratory


Revision 1.2

Isolation of Lycopene from Tomato Paste


using Column Chromatography
In this laboratory exercise we will isolate the pigment Lycopene from tomato paste. In a follow
up lab, we will examine the UV-VIS spectrum of Lycopene, isomerize it and then examine the
isomers spectrum for comparison.

Lycopene, the red pigment of the tomato, is a C40-carotenoid made up of eight Isoprene units;
making it a tetraterpene.

Other sources of the compound include:

Vegetable Source
Gac
Tomato
Tomato Juice

g Lycopene per Gram Wet Weight


2000 3000
8.8 42
86 100

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Tomato Sauce
Tomato Ketchup
Watermelon
Pink Grapefruit
Pink Guava
Papaya

63 131
124
23 72
3.6 34
54
20 - 53

-Carotene, the yellow pigment of the carrot is an isomer of Lycopene in which the double
bonds at C1-C2 and C'1-C'2 are replaced by bonds extending from C1 to C6 and from C'1 to C'6 to
form rings, and is also a constituent of the tomato.

Each of these compounds is classified as a Carotenoid.


Carotenoids are organic pigments that are naturally occurring in the chloroplasts and
chromoplasts of plants and some other photosynthetic organisms like algae, some types
of fungus and some bacteria.
There are over 600 known carotenoids; they are split into two classes, xanthophylls
(which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no
oxygen). Carotenoids in general absorb blue light. They serve two key roles in plants and
algae: they absorb light energy for use in photosynthesis, and they protect chlorophyll
from photodamage. In humans, four carotenoids (-carotene, -carotene, -carotene, and
-cryptoxanthin) have vitamin A activity (meaning they can be converted to retinal), and
these and other carotenoids can also act as antioxidants.
Wikipedia

Vitamin A Aldehyde (Retinal) binds to the protein Opsin in rod cells in the eye. Photons striking
this chromophore attached to the Opsin cause it to isomerize from 11-cis-Retinal into 11-transRetinal. This isomerization causes a signal to be sent to the brain that is interpreted as a visual
event.

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This is the first step in the overall Visual Cycle associated with night vision.
We will isolate Lycopene from tomato paste, which as noted above, contains high levels of this
pigment, using Column Chromatography. Like other forms of chromatography, Column
Chromatography is based on a two phase system where the stationary phase is a column of
adsorbant and the mobile phase is a liquid eluent.

http://en.wikipedia.org/wiki/File:Column_chromatography_sequence.png

The theory of column chromatography is analogous to that of thin-layer chromatography. The


most common adsorbents, silica gel and alumina, are the same ones used in TLC. The sample is
applied to the top of the column. The eluent, instead of rising by capillary action up a thin layer,
flows down through the column filled with the adsorbent. Just as in TLC, there is an equilibrium
established between the solute adsorbed on the silica gel or alumina and the eluting solvent
flowing down through the column. Under some conditions, the solute may be partitioning
between an adsorbed solvent and the elution solvent, the partition coefficient, just as in the
extraction process, determines the efficiency of separation chromatography. The partition
coefficient is determined by the solubility of the solute in the two phases.
In general, the amount of alumina or silica gel used should weigh at least 30 times as much as the
sample, and the column, when packed, should have a height at least 10 times the diameter. The
density of silica gel is 0.4 g/mL and the density of alumina is 0.9 g/mL, so the optimal size for
any column can be calculated.

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Uniform packing of the chromatography column is critical to the success of this technique. The
sample is adsorbed onto a small quantity of adsorbent as a pure liquid or, if it is a solid, as a very
concentrated solution in the solvent that will dissolve it best, regardless of polarity. As elution
takes place, this narrow band of sample will separate into several bands corresponding to the
number of components in the mixture and their relative polarities and molecular weights. It is
essential that the components move through the column as a narrow horizontal band in order to
come off the column in the least volume of solvent and not overlap with other components of the
mixtures. Therefore, the column should be vertical, and the packing should be perfectly uniform,
without voids caused by air bubbles.
The preferred method for packing silica gel and alumina columns is the slurry method, whereby
a slurry of the adsorbent and the first eluting solvent is made and poured into the column. When
nothing is known about the mixture being separated, the column is prepared in ligroin or
hexanes, the least polar of the eluting solvents.
We will extract crude Lycopene from tomato paste and apply the extract to our column. Tomato
paste, as a source for our Lycopene, has the advantage that its Lycopene concentration is
significantly higher, gram for gram, than that of a ripe tomato. The main drawback of using
tomato paste is that trans-Lycopene may isomerize during the cooking process during which the
paste is produced. We will then pack a column for purifying the Lycopene. We will use neutral
Alumina (Grade II-III), a fairly active adsorbant, to separate the Lycopene from other tomato
paste pigments.
Lycopene, with its 13 double bonds, is attracted to alumina more strongly than are
-carotene and related carotenes, which have 11 to 12 double bonds. Therefore, the yellow
carotene band will move down the column faster than the orange-red lycopene band. Yellow
xanthophyll pigments will trail behind the lycopene band because they contain polar hydroxyl
groups that are strongly attracted to Alumina.
Operational Organic Chemistry; 4th Ed.
by John W. Lehman

We will then examine the Lycopene spectroscopically and crystallize it to obtain a melting point.

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Pre-Lab Questions
1.

Squalene is a triterpene found in shark liver oil that is a precursor to many steroids.
a) How is Squalenes structure related to that of Lycopene?
b) What is the purpose of Squalene in shark oil?
c) What is the biosynthetic precursor of Squalene?

2.

We have indicated the first step in the visual cycle involves isomerization of11-cis-Retinal
into the all trans form. How is trans-Retinal converted back into 11-cis-Retinal?

3.

Using Silica or Alumina in packing the column results in Normal chromatography. What
is reversed-phase chromatography and why is it useful?

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Procedure
Packing the Column

Assemble the column as depicted above. Use Alumina (Grade II-III) as your adsorbant. To
measure the adsorbent, fill the column one-half to two-thirds full, and then pour the powder into
a 10-mL Erlenmeyer flask. Clamp the column in a vertical position, and close the valve.
Always grasp the valve with one hand while turning it with the other. Flll the column with

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ligroin or hexanes to the top of the glass column. Add about 8 mL hexanes to the adsorbent in
the flask, stir the mixture to eliminate air bubbles, and then (this is the hard part) swirl the
mixture to get the adsorbent suspended in the solvent and immediately pour the entire slurry into
the funnel. Open the valve, drain some solvent into the flask that had the adsorbent in it, and
finish transferring the slurry to the column. Place an empty flask under the column, and allow
the solvent to drain to about 5 mm above the top surface of the adsorbent. Never allow the
column to dry out. This creates channels that will result in uneven bands and poor separation.

Extracting the Lycopene


Weigh 1.0-1.5 g of tomato paste into a 25-mL Erlenmeyer flask. Extract the solid material by
swirling the Erlenmeyer flask with a 5-mL portion of a 50% (by volume) mixture of acetone and
low-boiling petroleum ether. Break up any large clumps with a spatula. Filter the solution in a
Pasteur pipette and collect the filtrate in a large test tube. Extract with a second 5-mL portion of
acetone-petroleum ether and pass through the Pasteur pipette. Wash the combined extracts with
5 mL of saturated aqueous NaCl. Remove the aqueous wash with a Pasteur pipette. Then wash
with 5 mL of 10% aqueous K2CO3. Again, remove the aqueous wash. Finally, wash with
another 5 mL of saturated aqueous NaCl and remove the aqueous wash. (The above described
washing and removing technique is a microscale extraction. Your instructor will demonstrate
this technique.)
Dry the lycopene-containing organic layer with a drying agent. Remove the solution with a
Pasteur pipette away from the drying agent into a 50-mL reaction flask. Rinse with 5 mL of
acetone-petroleum ether and repeat removal into the 50-mL reaction flask. Evaporate the
lycopene of solvents on the rotavap.

Running the Column


Dissolve in the very minimum volume of eluting solvent (1 mL of ligroin or hexanes).
Then:
(1) open the valve, allow the solvent to drain close to the top of the adsorbent, close the valve
(2) carefully add the lycopene sample to the prepared column,
in such a manner that the top surface of the column is not disturbed
(3) repeat (1)
(4) carefully add ~1 mL of fresh ligroin or hexanes to the top of the column
(5) repeat (1)
(6) carefully add a second ~1 mL of fresh ligroin or hexanes to the top of the column
(7) repeat (1)
(8) fill the column with the solvent and elute the sample from the column
Collect the yellow carotene band eluted by ligroin or hexanes. Remember to not let the solvent
front move below the top of the adsorbent. Add more ligroin or hexanes as needed.

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Switch to 10% acetone-hexanes to elute the lycopene from the column. Analyze the Lycopene
spectroscopically as soon as possible. If you cannot analyze the sample immediately, stopper
your sample stightly, wrap them in aluminum foil and place them in the freezer for
spectrophotometric analysis next week.

Crystallizing the Lycopene


Concentrate some Lycopene containing eluate to a small volume by evaporating it with a stream
of Air at Room Temperature. Cool the mixture on ice to obtain crystals of Lycopene. Allow the
crystals to dry and obtain a melting point. The reported melting point of Lycopene is ~175oC.

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Post Lab Questions


1.

What would be the effect if the chromatographic column was not clamped vertically?

2.

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?

3.

Why would you not use a larger quantity of ligroin (instead of the recommended 1 mL) to
apply the sample to the column? What would be the effect if too much ligroin was used?

4.

What would be the effect of adding more eluting solvent before the level of the sample has
dropped to the level of the alumina?

5.

Why is it better to collect smaller rather than larger fractions?