lycopene

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

 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.

Moisture was determined by oven drying at 105C to constant weight

Each peel sample was characterized for moisture and total lycopene content

The skins were hand-separated from the seeds and other impurities

Lycopene extraction
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 vacuumfiltration 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).

Based on 2 phases system

Equilibrium established between solute adsorbed on alumina and eluting solvent flowing down through the column

Analogous to the theory of TLC

The sample is applied to the top of the column

Most common adsorbents are silica gel and alumina

Normal Phase liquid-solid chromatography (adsorbtion)

(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 interactions dipole-dipole interactions dipole-induced dipole interactions

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-cm cuvette 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 3005, 5 m (Macherey-Nagel or equivalent) Detector: UV/VIS or VIS

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% solution of N-ethyl-diisopropylamine in hexane (v/v) Run time: 30 min

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 solution in a 1-cm cuvette at the wavelength of maximum absorption (approximately 470 nm). Use hexane as the blank

Amount of Nucleosil 300-5 used should weigh at least 30 times as much as the sample

The column should have a height at least 10 times of the diameter when packed

The sample is adsorbed onto a small quantity of adsorbent as a pure liquid

It is essential that the components move through the column as a narrow horizontal band to come off the column in the least volume of solvent and not overlap with other components of the mixtures

When elution, narrow band of sample separate into several bands corresponding to no. of components in the mixture

The column should be vertical and the packing should be perfectly uniform

Chromatogram

From the Chromatogram

Number of plate, N

Retention factor, k

Resolution, R

Separation factor,