Extraction of Plant Pigment

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INTRODUCTION
Plant pigments consist of four main chemicals. These are Chlorophylls, The carotenoids, Anthocyanin and Betacaine. The aims of this experiment is to separate the plant pigment into different colours using solvents and column chromatography. The hypothesis of this experiment is that this plant pigment from spinach will contain the chlorophyll pigment or may contain the others too.

Chlorophyll:
Chlorophyll a is the main component of the plant pigments. This is used for the most important process, Photosynthesis. Chlorophyll a makes up about 75% of the plant pigment. The other 25% is the chlorophyll b.

Figure 1: Chlorophyll a (http://brsmblog.com/wp-content/uploads/2011/12/0-chlorophyll-a-1024x735.jpg)

Figure 2: Chlorophyll b (http://patentimages.storage.googleapis.com/EP0071991B1/imgb0003.png) Chlorophyll a and b provide the green colour of the plants. They absorb most of the light required for photosynthesis. The Carotenoids

These pigments can be found in many fruits and vegetables. They are commonly the yellow and orange pigments.

Figure 3: The carotenoids (http://chemwiki.ucdavis.edu/@api/deki/files/7280/=Zeaxantina.jpg) Anthocynins
This pigment associates with the colours pink, red, mauve, violet and blue which are found in flowers, fruit and vegetables. These have a function in reproduction.

Figure 4: Malvidin present in grapes (http://www.friedli.com/herbs/phytochem/anthocyanins.gif)

The Visible Spectrum
The mixture of chlorophyll found in spinach absorbs many wavelengths of the visible light. The absorbance peaks ranges 400 – 500nm is the blue range and 600 -700 nm the yellow- red range. The two principles of this light absorption stated (Practical Skills in Chemistry, 2011) are: 1) the absorption of light is exponentially related to the number of the molecules of the absorbing solute that are encountered, i.e. the solute concentration. 2) The absorption of light is exponentially related to the length of the light path through the absorbing solution, l.

Figure 5: Absorption spectrum of plant pigment
When a white light is passed through or is reflected by a colored substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelength absorbed. (Reusch, 2013)

MATERIALS AND METHOD
Materials
* Pasteur Pipette
* Modified pipette
* Rubber tubing
* A teat
* 40/60 Petroleum
* Clip
* Small funnel
* Small piece of cotton
* 4 Sample tubes
* Piece of wire
* Sand (silica)
* Small spatula
* Some alumina ( 2 – 5g)
* Acetone
* Labels
* Clamp holder
* Retort stand

Figure 6: Column chromatography (http://www.chemguide.co.uk/analysis/chromatography/column1.gif)

Method:
1. Firstly, the pipette was clamped in a vertical position and rubber tube is fitted at the bottom of the pipette below the mark. 2. Some 40/60 petroleum was added using a Pasteur pipette through a funnel into the modified pipette. 3. A small piece of cotton was out in to a sample tube with a very little amount of 40/60 Petroleum to help get rid of any air bubbles. 4. The cotton wool was transferred into the modified pipette and was pushed into the narrow end of the pipette. 5. The pipette was filled with fresh petroleum to make sure there are no air bubbles. 6. Little sand was added to the pipette through a funnel so that a layer of 0.3 cm thick above the cotton was formed. 7. A small spatula was used to add alumina slowly so that column was about 5 to 6cm height was formed. 8. The pipette was then tapped with a pencil to ensure a flat surface for the alumina column. 9. Another layer of sand was added to the pipette on top of the alumina. 10. A fresh sample tube was kept under the pipette ready for collection. 11. The tubing clip was opened and adjusted its position so that the flow of rate of petroleum passing through the column is1...
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