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