Paper Chromatography Lab/Experiment of the coloured Substances in Leaves
Aim / Objective:
To separate the colored substances in leaves using the process of chromatography
The method of separation used was paper chromatography and the pigments were separated based on rates moved by each pigment. The extract was also separated based on the solubility of the various components and was found to contain green chlorophyll and yellow carotenoids
Chromatography is a technique for separating and identifying mixtures of compounds based upon their different rates of adsorption. All types of chromatography employ two different immiscible phases in contact with each other namely the mobile phase and the stationary phase.
Normally, the stationary phase is a solid such as paper, starch, alumina, or silica and the mobile phase is a liquid such as water, common organic solvents such as ethanol, or solvent mixtures.
The basis for paper chromatography is the fact that porous paper, cellulose, has an enormous surface area to which molecules or ions of substances are attracted (adsorbed) and then released (desorbed) into the solvent as an aqueous solution passes over the paper.
Separation of components occur, that is, they will travel at different speeds in a moving solvent because the varying attractions between these components and the paper. This method of separation is known as partitioning.
The identity of the components can be deduced by comparing a chromatogram of the unknown mixture with chromatograms of mixtures with known composition (standards). An additional aid in the identification of substances is its Rf value. The Rf value of a compound is a characteristic of the compound and solvent used and serves to identify the constituents of a mixture
This can be done by calculation Rf = Ds / Df
Where, Ds = distance traveled by a spot, and
Df = distance traveled by the solvent.
3 Green leaves, mortar and pestle, ethanol, chromatography paper / filter paper, scissors, 10cm3 beaker and cover, glass cover, dropper, pencil
Method / Procedure:
- Use scissors to cut leaves and place in the mortar.
- Add purified sand and 3cm3 ethanol to the leaves and ground with pestle until green extract is obtained.
- Place mixture to one side and prepare chromatography paper.
- Fill the 10cm3 beaker with ethanol to about a height of 2cm.
- Use pencil to draw a light line 1cm from one end of the strip and label as “A”
- Draw a second line, 1cm from line A and label it as “B”. Line A will represent the level to which the strip should be submerged into the solvent, ethanol. Line B will represent the line to which the ink is applied, that is the Line of Origin.
- Using the leaf extract apply thin spots across Line B and allow it to dry.
- Place the chromatography paper in the beaker using the glass cover to hold it in place.
- The solvent was allowed to move up the paper. When the solvent front has almost reached the top of the paper, remove it and mark this line with a pencil.
- Allow paper chromatogram to air dry.
- Measure the distance from line B to each separated dye in the leaf extract.
- Record and tabulate the distances measured.
Discussion / Answers:
>>> Adsorption is the process by which one substance is being attracted and held to the surface of another substance.
2.Distinguish between adsorption and absorption
>>> Adsorption is the process by which atoms, molecules or ions from a substances either a gas, dissolved solid or liquid adhere to the surface of an adsorbent.
Absorption is the process by which a fluid is dissolved by a solid (absorbent) or a liquid.
3.Why was ethanol used as a solvent?
>>> Ethanol is used to decolorize leaves. The separation into constituent parts is dependent on differences in solubility in ethanol.
4.Explain why chlorophyll B moved the shortest distance
>>>The different pigments identified have different polarity and due to this separation in polarity, it is possible to separate. Xanthophyll is non-polar so will travel the highest distance, followed by chlorophyll A. Chlorophyll b is the most polar of the pigments and therefore will travel the shortest distance.
5.How could you ascertain if there are more pigments present in the leaf than the ones identified?
>>>The filter paper could be turned sideways and carry out another chromatography to determine if there are more pigments present in the leaf.
Source of Error/ Limitations/ Assumptions:
- Handling the paper with wet hands allows for additional uncontrolled moisture to the paper which will interfere with separation of components.
- Incorrect drawing of lines and measurements
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