Organic Chemistry Laboratory

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CHEM 333L
Organic Chemistry Laboratory
Revision 1.1

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 isomer’s 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

µg 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

β-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 (β-carotene, α-carotene, γ-carotene, and β-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.

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.

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

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concentrated solution in the solvent that will dissolve it...
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