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PIGMENT SEPARATION USING PAPER CHROMATOGRAPHY

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Desmond Goh

on 16 August 2014

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Transcript of PIGMENT SEPARATION USING PAPER CHROMATOGRAPHY

1.0 INTRODUCTION
1.1 PROBLEM STATEMENTS
1.2 OBJECTIVES
To prepare an extract of plant tissue
To apply the technique of paper chromatography as a method of separating individual plant pigments contained in plant tissue extracts containing pigment blends.
To identify the presence of different pigments in chlorophyll's.
To identify the R
f
values which are affected by the solubility and the molecular size of the pigments.
1.3 HYPOTHESIS
The pigments can be identified using paper chromatography method.
The more soluble the pigment,the further the movement of the pigment,thus the higher the R
f
values.
The smaller the molecular size of the pigment, the higher R
f
value.
2.0 Literature Review
A "pigment" is simply a molecule that absorbs and reflects light.
ChlorophyII is a pigment that reflect green light,but absorbs red and blue wavelength and is critical for the light reaction for photosynthesis.
Paper chromatography is a useful technique in the separation and identification of different plant pigment. the history of chromatography spans from the mid-19 century to the 21st.
The first true chromatography is usually attributed to the Russian-Italian botanist Mikhail Tsvet.Tsvet applied his observations with filter paper extraction to the new methods of column fractionation that had been developed in the 1980s for separating the components of petroleum.
A photosynthetic pigment (accessory pigment;chloroplast pigment;antenna pigment) is a pigment that is present in chloroplasts or photosynthetic bacteria and capture the light energy necessary for photosynthesis. Green plant have six closely related photosynthesis pigments(in order of increasing polarity): Carotene (orange pigment),XanthophyII (yellow pigment), Phaeophytin
a
(grey-brown pigment),Phaeophytin
b
(yellow-brown pigment), ChlorophyII
a
(blue-green pigment) and ChorophyII
b
(yellow-green pigment)
The retention factor (R
f)
may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent
3.0 METHODOLOGY
3.1 MATERIAL AND APPARATUS
3.2 ARRANGEMENT OF APPARATUS
CHEMISTRY
PBS

How to identify the presence of difference pigments in chlorophyll's
What are the factors affecting these R
f
values.
PIGMENT SEPARATION
USING PAPER CHROMATOGRAPHY

80% of Acetone
"Cekur Manis" or Red Spinach Leaves
Mortar and Pestle
Muslin (cheese cloth)
Boiling Tube/Large Test Tube With Stopper (24x150mm)
Solvent ( 1 Part 80% Acetone and 9 Part Petroleum Ether)
Ruler
Whatman no.1 Chromatography Paper Strip (20 X 130mm)
Siccors
Pin
Dark Paper/Black Sugar Paper
Retort Sand/ Test Tube Rack
Diagram 1: Assembling of chromatography apparatus
3.3 PROCEDURE
1. Switch off the fans while conducting the experiment.
2. Prepare the pigment extract by grinding up fresh leaves with 5 ml acetone using a mortar and pestle.
3. Squeeze out a thick pigment extract using a cheese cloth.
4. Cut out one end of the chromatography strip to form a pointed tip. Rule a pencil line across the strip of paper, 20 mm from the pointed tip.
5. Next, transfer the pigment extract by spotting onto the centre of the pencil line of a chromatography strip using the head of a small pin as a dropper.
6. Let the spot dry. Repeat the process 15 to 20 times over on the same spot(smaller spot gives better results) and then put enough solvent into a large boiling tube.
7. After that, hang the chromatography strip to the stopper using a pin/clip(pointed end down into the solvent). Place the stopper to seal the boiling tube(Diagram 1).
8. Make sure that the strip is in a perfectly vertical position. Solvent level must be 1 cm from the pigment spot. Later, place the boiling tube on a rack or stand it using retort stand.
9. The next step would be letting solvent moves until it reaches near the pin/clip.
10. Take out the chromatography strip when the solvent front comes close to the top. Mark the solvent front with a pencil.
11. Quickly dry it and measure the distance between the pigment spot and the solvent front.
12. Observe and mark the positions of the separated pigments.

13. Then, measure the distance from the pigment spot to the leading edge of each pigment.
14. Cover the chromatography strip with a dark paper to protect the pigments.
15. Calculate the Rf value of each pigment using the formula:
Rf = (Distance moved by pigment )/ (Distance moved by solvent)
16. Last but not least to record all the data in a table.
4.0 DATA INTERPRETATION AND DICUSSION
Band Colour
Plant Pigment
Distance moved by the pigment (mm)
Distance moved by the solvent (mm)
R
f
value
Green
ChlorophyII a
31
109
31/109=0.28
Blue Green
ChlorophyII b
80
109
80/109=0.73
Yellow
XanthophyII
86
109
86/109=0.79
Grey
Phaeophytin
92
109
92/109=0.84
Yellow
Orange
Carotene
102
109
102/109=0.94
4.1 RESULT/TABULATION OF DATA
4.0 DISCUSSION
From the experiment,the pigments that exist in the plant based on their colors were chlorophyll a which show green color, chlorophyll b which was blue green,xanthophyII which was grey and carotene which was orange in color.
the factors that affecting the movement of pigment during chromatography were porosity of the chromatography paper,solubility of the solvent as well as the molecular size of the solute.
the pigment which moved the furthest was carotene by 102 nm in distance, this indicates that the soluble pigment carotene dissolved into the acetone easiest and thus moved furthest.
it is very soluble in the solvent being used and because it forms no hydrogen bond with cellulose.
beside the large value of R
f
value also indicates that carotene was smallest in size.
The molecular size of the pigment is inversely proportional to the R
f
value.
Conclusion
Precautions
To obtain a clear and accurate chromatogram the students were advised to use gloves. This was because the student fingerprints excreted amino acids which would make the experiment and the results inaccurate.
Strict safety measures were taken in the adding of solvent. This was to ensure that the solvent didnt go above the origin line. The reason for this is that if the solvent and the samples mixed this would cause unreliable and faulty results.
Prepared By:
Andyriene Christy Slyvester
Desmond Goh Kai Hong
Joanna Wang Joo Ying
Jason Kiew Kian Ming
Reference
From Internet:
- Paper Chromatography
http://lhschem351%20%20Paper%20Chromatography%20Lab.htm
-Carriying out the paper chromatography,
http://Phtotosynthetic%20pigment%20%wikipedia,520the%20free%20encyclopedia.htm
-Separation of plant Pigment
http://Seperation%20of%20Plant%20Pigment%29(Chromatography)-procedures.htm#.U7qrtfmsyws
From book:
-Lee Ching. (2006). “Longman Pre-U STPM Biology Volume 1: Analysis Technique on Paper Chromatography.” Selangor DarulEhsan: Pearson Malaysia Sdn. Bhd.
-Britta Knefelkamp. (30th January 2007). “Comparison of different filter types on chlorophyll-a retention and nutrient measurements.” Helgoland, Germany: BiologischeAnstalt Helgoland, Foundation Alfred-Wegener-Institute for Polar and Marine Research, Marine Station.
-Mary Jones, Richard Fosbery. (2008). “AS Level and A Level Biology Second Edition”.Dubai: Kamae Design, Oxford.
-Y K Richard (2013).“Pre-U STPM Biology: Paper Chromatography”. Bangi, Selangor DarulEhsan: PenerbitanPelangiSdn. Bhd.

The chlorophyll in the leaf of the plant contains different pigments which are chlorophyll a, chlorophyll b, xanthophyll, phaeophytin and carotene.
First of all pieces of Chromatography paper was taken and aline was drawn 2cm above the bottom of the page. this was the origin line.On this line 10 crosses were drawn which would be the starting point of the samples this paper was measured so that it would fit into the Chromatography tank.
After this the Chromatography paper was laid down and 6 drops of the appropriate was added using a capillary tube. This was done 6 times so that the spot could be as concentrated as it could without it being too big.
After the Chromatogram was dried and sprayed with Ninhydren the spots were examined.The students didn't spray the Chromatogram because it was a real toxic solvent therefore the spraying was one by the lab technicians.
Graf of the distance moved by the pigment
Graph of R
f
values against plant pigments
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