Chromatography can be described as a broad class of biochemical techniques in which a mixture of substances can be separated by various properties of which include charge, size, hydrophobicity, non-covalent binding affinities or some other properties by allowing the mixture to partition between a moving phase and a stationary phase. (Alberts et al., 2008).
Paper Chromatography is a technique involving two phases; the stationary phase and the mobile phase. As the names suggest the stationary phase does not move. In this experiment the stationary phase was the filter paper while the mobile phase was the solvent.
In ascending chromatography the solvent moves upwards while in descending chromatography the solvent moves downwards. In this experiment ascending chromatography is used. The solvent (the mobile phase) rises up the paper by capillary action. The other components in the mixture rise up the paper at a rate proportional to the partitioning into the solvent. The process when the mobile phase moves along the stationary phase is called development. Since different components travel at different rates the end resultant chromatograph will be dotted with the components at different distances.
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Using this information the Rf values of the components could then be calculated. The formula used is:
Since no two components have the same Rf values the components can then be identified. (Clark, 2007).
Size Exclusion Chromatography is a chromatographic technique in which the molecules are separated according to their size, or more accurately their hydrodynamic volume. In this experiment the sugar was the stationary phase while the petroleum ether with the pigments was the mobile phase. As the mobile phase moves down the column components of different sizes start to separate as they move at different speeds. The advantage of Size Exclusion Chromatography is that the components are separated while retaining their biological properties. (McNaught & Wilkinson, 2006).
Experiment 1A: The Identification of an Unknown Amino Acid by Paper Chromatography
Butanol/Glacial acetic/water (12/3/5) solvent
Various Amino Acids
A pencil line was first drawn across a piece of filter paper. This line was approximately 2cm away from the edge. A toothpick was used to spot 5 drops of amino acid solution. These drops were then dried and the next drop with a different amino acid was then applied.
The above procedure was repeated for every solution of amino acid as well as for the unknown.
The chromatography tank was then filled with butanol/glacial acetic acid/ water (12/3/5) solvent mixture to a depth of approximately 0.5cm. The spotted paper was placed into the tank with the spots at the base.
The solvent was allowed to rise up the paper to within 2cm of the top of the paper. The paper was removed and the solvent front was marked with a pencil.
The paper was allowed to dry. Then it was sprayed by ninhydrin solution. The paper was allowed to dry again.
The distance was measured from the point of application to the solvent front and to the centre of any spots revealed by the ninhydrin solution. These values were recorded and the general colour of the spots observed was also observed.
Using the results obtained, the Rf values of the amino acid standards and of the unknown were calculated. Hence the composition of the unknown could be identified.
The line on the filter paper was drawn using a pencil. This was done because if ink was used it would have been dissolved in the solvent.
The spots were not allowed to touch the solvent in the chromatography tank.
The filter paper was handled from the edges so as to prevent contaminating the results.
The filter paper was kept vertical in the chromatography tank.
Ninhydrin was sprayed in a fume hood since it is a known carcinogen.
The container containing the solvent was sealed.
Solvent front does not move up in a perfectly straight line.
Leaks of the solvent from the container.
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The equation used to calculate the Rf values is:
Distance moved (cm)
Total distance travelled by solvent = 9.00cm
Unknown A with an Rf of 0.611 is Phenylalanine.
Unknown B with an Rf of 0.267 is Threonine.
Amino acids are compounds which are the monomers of proteins. There are about 20 different types of amino acids. Each amino acid is built around the same core structure through which linking in a standard way to other amino acids can easily be done.
The basic structure of an amino acid can be seen below:
Figure 1. Basic Structure of an Amino Acid
All amino acids possess a carboxylic group and an amino group which are both linked to a carbon atom called the ÎÂ±-carbon. Each protein or polypeptide is essentially a chain of amino acids strung together. The protein however is then folded into a three-dimensional structure which is unique to each type of protein. The covalent linkage between two adjacent amino acids in the protein chain forms an amide. (Alberts et al., 2008).
For the purpose of the discussion the amino acid to be used in the reaction with Ninhydrin is is alanine. Alanine is a simple amino acid with the formula C3H7NO2.
Figure 2. Alanine Figure 3. Ninhydrin
Ninhydrin was the dye used in this experiment. Since the amino acids are not coloured a dye was required so that the amino acids could be seen and the Rf values could be calculated.
Figure 4. Ninhydrin Reaction Mechanism with an Amino Acid
The Rf values of a chemical or component are highly irreproducible and as can be observed from the results, even though the same chemical was used on the same sheet, the results obtained were not exactly the same. In this case the most similar Rf value was taken to be the unknown. From the results obtained one can conclude that Unknown A was most probably Phenylalanine while Unknown B was Threonine.
Experiment 1B: Size Exclusion Chromatography
Methanol/Petroleum Ether (2/1)
Finely powdered sugar
The green leaves were first cut into very small pieces with a sharp knife.
To 5 grams of the material, 150mL of a solvent mixture made up of two parts absolute methanol and one part petroleum ether were added. This was left for 5 to 15 minutes to ensure all or most of the pigments were extracted.
The extract was then filtered through a cloth into a one litre separating funnel.
500-600mL of filtered 10% Sodium Chloride solution were then added.
All of the pigments were transferred to the top layer, the petroleum ether which had separated from methanol.
The contents were mixed by gently rotating the funnel.
The dark green petroleum ether layer was then drawn off and chromatographed on a column of an adsorbent powder on an adsorbent paper.
A small wad of cotton was put in the glass tube provided and pressed flat using a plunger.
About 2-3cm of powdered sugar were placed in the tube and shaken down by tapping the side of the tube while using the plunger to pack down firmly down the tube.
The tube was filled with sugar until the column was 2cms from the top.
A glass rod was inserted into the tube until the end of the rod touched the sugar.
A small sample of pigment was poured down the glass rod. This was done until the top 0.5cm to 1.5cm of the sugar column dark green. Most of the solvent was allowed to move into the sugar.
A small amount of 0.5 percent solution of n-propyl alcohol in petroleum ether was added.
The developing solution caused the pigments to separate from each other.
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The rate of movement of the solvent through the sugar column depended on the pigments, the solvent, the packing of the tube and the force of suction applied.
The various pigments were adsorbed to the sugar particles with varying tightness and were this carried along the column at different rates.
The sugar column was compressed as much as possible in the column.
The sugar was kept from becoming dry by constantly adding the solution containing the pigments.
There were some holes in the packing due to having a wide glass column.
The layers did not move evenly causing the slanting of bands. This could have been due to the packing not being close enough.
Photosynthesis is the process by which Carbon Dioxide is converted into organic compounds for the plants using the energy from sunlight. To absorb this energy from sunlight photosynthetic pigments are required. Since a pigment can only absorb a limited range the organisms which photosynthesise usually have a number of different compounds so that they make the best use out of all the incident sunlight.
The three main types of pigments found in photosynthetic plants are Carotenoids, Chlorphylls and Phycobilins, the latter being found in some unicellular photosynthetic organisms.
Below is a diagram showing the different absorption frequencies of these different pigments:
Figure 5. Absorption Frequencies of Different Photosynthetic Pigments
As can be observed from the graph the major pigments found in plants do not absorb much of the green-yellow frequencies and this is reflected hence giving the typical green colour of plants.
From the results obtained it can be seen that the Carotenoids are the smallest since they moved the fastest down the glass column. Chlorophyll b is the largest and Chlorophyll A is medium sized.
The experiments were successfully carried out. Both methods of chromatography used are important as they are used in different applications.
Experiment A: The unknown compounds were found to be Threonine and Phenylalanine.
Experiment B: Three bands were produced in the glass column with the Carotenoids being found at the lower band and the Chlorophyll B being found at the higher band. The middle layer was found to be Chlorophyll A.