Experiment 9: Isolation and Isomerization of Lycopene from Tomato Paste

Date Performed: 6th Feb 2014

Date Submitted: 11th Feb 2014
Introduction:

Objective: We wanted to extract lycopene from tomato paste and measure its
absorption at a specific wavelength (360nm) using a Ultra-violet Spectrum to find
out whether isomerization occurred or not.

Hypothesis: The isomerization did not occur and our lycopene is in an all trans
conformation.

Experimental Operational Techniques:

1) Gravity Filtration: Filtration is a technique used to separate a solid from a liquid.

The solid is separated from the liquid phase by passing the mixture over a filtering
media. Filtering media is characterized by being chemically inert to the mixture, and
having small pathways for the liquid to pass through, but these pathways should be
smaller than the solid particle size. Filtering media can be prepared using paper,
fritted-glass, or any porous material. The mixture is forced through the filter by
gravity.

2) Solid-liquid Extraction: In liquid-solid extraction, a solvent (hydrophilic or

hydrophobic, acidic, neutral or basic) is added to a solid. Insoluble material can be
separated by gravity or vacuum filtration, and soluble material is 'extracted' into the
solvent. A sequence of solvents, of varying polarity or pH, can be used to separate
complex mixtures into groups. The filtered solution can be used as a solution (for
example, to be injected into the GC/MS system) or the solvent can be evaporated to
recover the solute(s) in powder or crystalline form.

3) Solid-Liquid Chromatography: Liquid chromatography is a technique used to

separate a sample into its individual parts. This separation occurs based on the
interactions of the sample with the mobile and stationary phases. Because there are
many stationary/mobile phase combinations that can be employed when separating
a mixture, there are several different types of chromatography that are classified
based on the physical states of those phases. Liquid-solid column chromatography,
the most popular chromatography technique and the one discussed here, features a
liquid mobile phase, which slowly filters down through the solid stationary phase,
bringing the separated components with it.

4) Ultraviolet Visible Spectra: Ultraviolet and visible (UV-Vis) absorption

spectroscopy is the measurement of the attenuation of a beam of light after it passes
through a sample or after reflection from a sample surface. Absorption
measurements can be at a single wavelength or over an extended spectral range.
Ultraviolet and visible light are energetic enough to promote outer electrons to
higher energy levels, and UV-Vis spectroscopy is usually applied to molecules or
inorganic complexes in solution. The UV-Vis spectra have broad features that are of
limited use for sample identification but are very useful for quantitative
measurements. The concentration of a compound in solution can be determined by
measuring the absorbance at some wavelength and applying the Beer-Lambert Law

Since the UV-Vis range spans the range of human visual acuity of approximately 400
- 750 nm, UV-Vis spectroscopy is useful to characterize the absorption,
transmission, and reflectivity of a variety of technologically important materials,
such as pigments, coatings, windows, and filters. This more qualitative application
usually requires recording at least a portion of the UV-Vis spectrum for
characterization of the optical or electronic properties of materials.

5) Melting point: Temperature at which the solid and liquid forms of a pure
substance can exist in equilibrium. As heat is applied to a solid, its temperature will
increase until the melting point is reached. More heat then will convert the solid into
a liquid with no temperature change. When the entire solid has melted, additional
heat will raise the temperature of the liquid. The melting temperature of crystalline
solids is a characteristic figure and is used to identify pure compounds and
elements.

Chemical Reaction

Results:

Please refer to end of report for spectra.

{(b/a-0.4)x100%}/0.4 = % of all trans lycopene .

On our spectra , the 13-cis-lycopene (iodine added solution) was higher than our all
trans lycopene .
So using b as our all trans=
{(2.5mm/4.1mm)x100%}/0.4 = 52.439% of all trans lycopene

Discussion:

Our objective was to extract Lycopene from tomato paste and then measure its
absorption at 360nm using an Ultraviolet visible spectrum to find out whether
isomerization of all trans lycopene to 13-cis-lycopene occurred.

We initiated the experiment by measuring 4.0 grams of tomato paste and adding to
it three successive 10 ml portions of 50% mixture of acetone and petroleum ether.
The reason we did that is because was wanted to extract the pigments (Lycopene,
Carotenes and Xanthophyll) from the paste. The acetone was used because
Lycopene has a very high affinity for acetone and Carotenes have a very high affinity
for petroleum ether since they are all hydrophobic and “like dissolves like” can be
applied here. We then filtered the extract using gravity filtration and made sure to
cover our extract and not exposed it to any heat , light or chemicals that might
catalyze the isomerization of all trans lycopene to 13-cis lycopene.

We then proceeded to wash our extract with 20 ml of 10% aqueous potassium

carbonate to neutralize any acid that naturally occur in tomatoes and could still be
in our extract. We then washed it with 20 ml of saturated sodium chloride solution
so that we can remove any water remaining in our extract. The saturated sodium
chloride would become unsaturated by removing water from our extract and
forming an aqueous layer that we can easily identify and separate from our organic
layer.

After we separated, we dried our lycopene containing organic layer with anhydrous
sodium sulfate and then we concentrated our pigment solution by evaporating all
the petroleum ether using just room temperature since its boiling point is (35-60℃).

We then packed our chromatography column with neutral Brockmann grade ||-|||
alumina using hexane as our column packing solvent. We made sure our column was
uniform and horizontal and then added our extract and added the first eluent ,
Hexane . Our mobile phase was hexane whereas our stationary phase was the
alumina silica. As soon as we saw the yellow carotene band begin to drain out of the
column we added the second eluent, which was 10% acetone in hexane. The reason
the yellow carotene moved down faster than the lycopene was because the lycopene
was more strongly attracted to the silica (stationary phase) because it was more
polar than carotene due to its 13 double bonds in comparison to the 11-12 double
bonds in carotene. When we added a more polar eluent such as 10% acetone in
hexane, the lycopene moved down the column since it became more attracted to the
mobile phase and we were able to collect it in a vial.

We went to measure its absorption at wavelength of 360nm using the ultraviolet

visible spectra because the 13-cis lycopene peaks at that wavelength whereas the all
trans lycopene shows little absorption; however, our results came out to be
different due to an excessive amount of iodine added to our sample in order to
compare the 13- cis and all trans lycopene. The reason we added iodine was because
iodine can catalyze the isomerization of 13-cis lycopene to all trans lycopene.
Adding iodine will saturate some of the double bonds, breaking up the conjugation
and reducing absorption in the visible region (loss of color). The problem we faced
was we added too much iodine and that gave us an incorrect reading since our
supposedly all cis lycopene came at a higher absorption on the spectra than our all
trans lycopene. So for our results, we should’ve gotten a higher peak for all trans
lycopene rather than for cis lycopene and that’s why our calculations weren’t spot
on.

Conclusion:

We were able to isolate the lycopene from the tomato paste using acetone and
petroleum ether and but unfortunately we couldn’t compare the spectra of the all
trans lycopene and the iodine based 13-cis lycopene.

References:

1)ChemPages. http://chem.wisc.edu/deptfiles/genchem/lab/labdocs/modules/gravfilt/gravfiltdesc.html
(Accessed 7th Feb, 2014)

2)ScientificConsulting. http://www.dsbscience.com/freepubs/forensic_intern/node22.html
(Accessed 7th Feb,2014)

3)ChemWiki
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Liquid_Chrom
atography (Accessed 7th Feb,2014)

4) CHP http://www.files.chem.vt.edu/chem-ed/spec/uv-vis/uv-vis.html (Accessed 7th Feb, 2014)

5) Britannica Academic Edition .

http://www.britannica.com/EBchecked/topic/374185/melting-point (Accessed 7th
Feb, 2014)