This experiment focused on extracting and separating pigments of Chloroplast. For the procedure green leaves were grinded in a mortar with some chemicals and the fluid was filtrated to use for further analysis. Stripes of this solution were put on a filter paper and later, after dried placed into a beacon of solvent. After this the chloroplast pigments were separated by the solvent into groups of more or less soluble pigments.
How many pigment types are present in a green leaf?
It is hoped to be able to identify the four different pigments types of a leaf. As the filter paper with the solvent will separate the pigments in terms of solubility, a clear segmentation of each is expected to show off.
As various chemicals were used in the whole process of this lab, certain variables might have influenced the results in terms of the purity of each chemical or purity of the used filtrate.
The chloroplast, basically, is the organelle responsible for photosynthesis. Structurally it is very similar to the mitochondrion. It contains a permeable outer membrane, a less permeable inner membrane, a intermembrane space, and an inner section called the stroma. However, the chloroplast is larger than the mitochondria. It needs to have the larger size because its membrane is not folded into cristae. Also the inner membrane is not used for the electron transport chain. Instead it contains the light-absorbing system, the electron transport chain, and ATP synthetase in a third membrane that forms a series of flattened discs, called the thylakoids.
Purpose: The purpose of this lab was to separate plant pigments using chromatography, calculate Rf values using the collected data, and study photosynthesis with isolated chloroplasts. Light energy Light energy Background Information (Activity A): In photosynthesis, plant cells convert light energy into chemical energy that is stored in sugars and other organic compounds. It is an endergonic and ...
Test tubes, Water, small beacon, Pipette, Stand, Mortar, green leaves, Filter paper, capillary tube, funnel, silicon dioxide, calcium carbonate, acetone, solvent.
1) To extract pigments from green Leaves
– Weigh 5g green Leaves, cut into pieces, and put in mortar.
– Add silicon dioxide + calcium carbonate + 5ml acetone and grind (Try not to inhale fumes)
– Pour the ground liquid to a small glass funnel and filter, and collect into a small test tube (if there is a plug use it).
2) Prepare filter paper strips
– cut filter paper into pieces 6cm long, 1cm wide. Mark a line with pencil at 1cm to the tip.
3) Draw filtrate fine Line
– use a capillary tube to draw a fine line along the pencil line
– repeat 3 times after the pigments have dried.
4) Separation of pigments
– pour 3ml of solvent in beaker. Place your strip in it.
– pigment fine line should not be in the solid.
– when the solvent front is close to the top, remove and let it dry. Mark it before it dries
– Observe the number of pigment bonds and colors.
1) 2)+3) 4)
Data Collection and Data Analysis
Sketch Data Table 1
Band # Distance (mm) Band colour
1 8 Bluish green
2 32 Yellowish green
3 n/a n/a
4 n/a n/a
Data Table 2
n/a RF for carotene
n/a RF for Xanthophyll
0.11111 RF for Chlorophyll A
0.44444 RF for Chlorophyll B
After having placed the filter paper into the beacon with the solvent, it took a whole while till any real results could be viewed. In fact, the first filtrate we used wasn’t even containing enough pigments which reacted to the solvent to create any useful results. After redoing the methods for the filtrate and paper strip the solvent started to do its job. Within about 10 minutes the solvent had slowly sucked up into the paper till the grey pencil line. Then, after having dried the strip one could very scarcely identify two distinct colors, each only a couple of millimeters away of each other: a bluish green and a yellowish green line.
The majority of chemical processes in chemical synthesis are carried out in solution and organic solvents usually are the first choice. To satisfy this demand, 15 billion kilograms of organic are produced worldwide each year.  These solvents often bear considerable risk due to their toxicity, flammability or environmental hazards. Furthermore, they have to be separated from the products and ...
Teacher Glen then gave us a scale upon which we should identify each pigment strip with its individual color, measure its distance in relation to the origin (the first line drawn with the filtrate) and then calculate the RF. First one had to identify the precise color of each, this was a bit problematic, due to the gamut of different tones present. As presented in figure 1 or Data Table 2, there is a set order of the distribution of each color band.
The RF was calculated upon the following formula: RF = Distance Pigment migrated (mm)
Distance solvent front migrated (mm)
– Carotene (orange)
– Carotenoid – Xanthophylls (yellow)
Pigments in Chloroplast
– Chlorophyll – Chlorophyll A (bluish green)
– Chlorophyll B (yellowish green)
As expected the solvent has caused the pigments to migrate into their groups of solubility. The yellowish green Chlorophyll B was the first band we identified, only migrated 8mm and the bluish green Chlorophyll A migrated 32mm. This told us that there was only Chlorophyll present in the green leave, although other students who had performed this experiment as well achieved different results.
The only issue which could be mentioned about the overall lab design is the rooms’ ventilation system. After having opened the bottle with the solvent, within only a couple of minutes the whole room had a very intense smell which made a few students feel dizzy or get a headache.
There are no real suggestions for this problem, except talking to the schools maintenance crew and tell them to get the fans fixed.
The reasons for the strange results could only be derived due to the fact, that maybe the chemicals used for this experiment weren’t pure enough, as well as the self made filtrate.