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Lycopene is a pigment principally responsible for the
characteristic deep-red color of ripe tomato fruits and
products.
It, as a natural source of antioxidants, has attracted
attentions due to its biological and physicochemical
properties.Recent epidemiological studies have suggested that the
consumption of tomatoes and tomato-based food products
reduce the risk of cancer
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Because of presence of 11 conjugated carbon-carbon double
bonds, lycopene is susceptible to chemical change when exposed
to light, heat and oxygen.
During food processing, lycopene may undergo degradation
and isomerization simultaneously, and the formation of cis
isomer of lycopene reduce it colour intensity and biological
activity.
In tomatoes product, all trans-, 13-cis, 15-cis lycopene are
reported to occur.
Resonance effect of cis-lycopene lower than all trans, the
antioxidative ability of the former should be inferior to the latter.
Thus, to develop a method for all trans-lycopene and its cis
isomers in food products is extremely important.
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Ripe tomato fruitwere immersed inboiling water for
1-2 min
Rapidlycooled, then
hand-peeled
The peels werepartially dried in
air for a few
hours and storedat 4C
The skins were
hand-separatedfrom the seeds and
other impurities
Each peel sample
was characterizedfor moisture andtotal lycopene
content
Moisture wasdetermined by ovendrying at 105C to
constant weight
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Approximately 0.05 g of tomato powder (weight measured exactly to
1/1000 g) was added to 10 ml of ACS grade acetone in a 250 ml glass
beaker.
The solution was agitated gently for 1 minute, followed by vacuum-
filtration through a 5.5 cm paper filter.
The filter paper was transferred to the original 250 ml beaker along
with 10 ml acetone. The solution was agitated and filtered as before.
This step was repeated for a total of 4 filtrations of the powder.
A total of 40 ml acetone was used, but evaporation caused the final
solution to be less than 40 ml. Volume of solution was noted before
injection into the HPLC.
All steps were performed in subdued light, as suggested by Thurnham
et al. (1988).
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Based on
2 phasessystem
Analogous
to the theoryof TLC
Most commonadsorbents aresilica gel and
alumina
The sample isapplied to the
top of thecolumn
Equilibriumestablished betweensolute adsorbed on
alumina and elutingsolvent flowingdown through the
column
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(nonpolar liquid phase, polar modified solid phase) Retention of an analyte under normal phase
conditions is primarily due to interactions between
polar functional groups of the analyte and polar
groups on the sorbent surface
Hydrophilic interactions
polar-polar interactions
hydrogen bonding
pi-pi interactionsdipole-dipole interactions
dipole-induced dipole interactions
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Mobile phase :
Hexane
Tetrahydrofuran stabilised
with 0.025% BHT
N-Ethyl-diisopropylamine
Lycopene standard (purity
95% or higher; available from
CaroteNature GmbH)
Apparatus:
Spectrophotometer with a 1-cmcuvette
HPLC system with a suitable
pump, A phenyl-hexyl silicon
column (250 4.6mm i.d.5 m
particle size) , injector,
thermostated column
compartment, and integrator
Column: Two serially-connected
two stainless steel
columns (250x4.0 mm)
Stationary phase: Nucleosil 300-5, 5 m (Macherey-Nagel or
equivalent)
Detector: UV/VIS or VIS
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HPLC standard solution:
Accurately weigh between 5.5 and
6.5 mg of the lycopene standard
into a 100-ml volumetric flask.Dissolve in 5 ml of
tetrahydrofuran stabilised with
BHT and make up to volume with
hexane. This is a standard solution
for the HPLC assay.
Spectrophotometric determination
of lycopene:
Measure the absorbance of the
spectrophotometric standard solutionin a 1-cm cuvette at the wavelength
of maximum absorption
(approximately 470 nm). Use hexane
as the blank
HPLC conditions:
Flow rate: 0.8 ml/min
Injection volume: 20l
Pressure: approx. 80 bar
Column temperature: 20
Detection: 470 nm
Mobile phase: 0.15% solutionof N-ethyl-diisopropylamine in
hexane (v/v)
Run time: 30 min
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Amount of Nucleosil 300-5used should weigh at least30 times as much as the
sample
The column should have aheight at least 10 times of
the diameter when packed
The sample is
adsorbed onto a smallquantity of adsorbent
as a pure liquid
When elution, narrow bandof sample separate into
several bandscorresponding to no. of
components in the mixture
It is essential that thecomponents move through thecolumn as a narrow horizontalband to come off the column inthe least volume of solvent and
not overlap with othercomponents of the mixtures
The column should bevertical and the
packing should beperfectly uniform
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Number of plate, N
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Retention factor, k
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Resolution, R
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Separation factor,
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