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The below mentioned article includes a list of three experiments on paper chromatography.
1. Experiment to separate amino acids in a mixture by paper strip chromatography:
Principle:
It is based on the fact that paper chromatography separates compounds on the basis of their different rates of migration of filter paper (cellulose). The rates of migration depend upon the solvent which is flowing up or down the paper and on the relative adsorption which holds the molecules more or less tightly to the paper.
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If the solvent flows towards upper side on the paper it is called ascending chromatogram, and if it flows towards lower side then it is known as descending chromatogram.
Requirements:
Chromatography paper, test tubes (2), capillary tubes (3), coupling jar (1), distilled water, amino acids (glycine and aspartic acid), phenol and ninhydrin.
Method:
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1. Take three stips of chromatography paper of equal size (12 cm in length and 1.5 cm in width).
2. Draw a fine line with a lead pencil, parallel to and 1.5 cm from one edge of the paper. This line will indicate the bottom of your chromatogram.
3. On this line draw a circle on each strip, about 1.5 cm from one edge. These circles will indicate the position of your samples.
4. Put a drop of glycine in the circle of one strip, aspartic acid on the second and that of a mixture of both amino acids on the 3rd strip with the help of separate capillary tubes. (Note : Avoid excess handling of the chromatography paper, since your hands may contaminate it with amino acids. Touch it only at the edges).
5. Take 5 ml of 80% phenol in three large test tubes.
6. Fix each strip in the cork as shown in Fig. 40.
7. Insert one strip in each test tube and see carefully that lower edge of the strip touches the phenol. (Note carefully that the amino acid spot should not he touched by the phenol and the paper should not touch the wall of the test tubes).
8. Keep these test tubes in the stand and wait for 20 to 30 minutes or more until the solvent has risen within 0.5 cm from the top of the paper.
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9. Remove the strips from the tube and let it dry in an oven at about 100°C for 3-5 minutes.
10. Dry strips should be sprayed by 0.1 % ninhydrin- acetone reagent and set it aside to dry.
11. Keep it again in the oven for 2 to 3 minutes. Do not overheat the paper.
12. Remove the paper and immediately outline with pencil the spots that you see. Amino acids will appear as purple spots on the filter paper.
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Rf value can be measured by the following formula:
Distance travelled by a given spot is measured from the center of the spot.
2. Experiment to extract free amino acids from germinating seeds and to separate them by paper chromatography method. Also determine the Rf value of each amino acid:
Requirements:
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Germinating seeds, mortar, ethanol, funnel, filter paper, drier, distilled water, Whatman No. 1 filter paper, capillary tube, amino acids, solvent (acetic acid : butanol : distilled water in 1:3:1 ratio), chromatography chamber, ninhydrin solution (0.1%) automizer, sprayer, oven, pencil.
Method:
(a) Extraction of amino acids:
1. Take about 20 gm. of germinating seeds and ground them in mortar with 50 ml of 80% ethanol to make a paste.
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2. Filter the paste and heat the filtrate to evaporate it upto near dryness.
3. Now dissolve the dry precipitate in 10 ml of distilled water and use it as amino acid extract.
(b) Preparation of chromatogram:
1. With the help of a capillary tube load a concentrated spot of amino acids on the strip of Whatman No. 1 filter paper at about 2 cm. away from the base by repeated drying.
2. In the same manner also load known amino acids at 1.5 cm apart from each other on the same filter paper sheet. It is to be used for identification of unknown amino acids.
(c) Preparation of solvent:
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Acetic acid, butanol and distilled water are mixed in the ratio of 1:3:1 and this mixture is used as a solvent.
(d) Isolation of amino acids:
1. Fill about 2 cm. of the chromatography chamber with the solvent and wait for about 10 minutes. (During this period the chromatography chamber would be saturated with the fumes of solvent).
2. Suspend the chromatogram strip in the chromatography chamber in such a way so that the amino acid spot remains in the lower side of the chamber and the strip just touches the solvent. Wait for about 1-2 hours.
3. Remove the chromatogram from the chamber and let it be dried in the air.
4. With the help of automizer spray the dried chromatogram uniformly with the ninhydrin solution (0.1%).
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5. Allow the chromatogram again to get air dried, keep it in an oven at 60°C for about 10 min. and observe.
Observations:
Spots will be clearly visible on the chromatogram.
Mark the spots with pencil and calculate the Rf value of each spot by the following formula:
Results:
Different amino acids (e.g., glycine, alanine, valine, leucine, etc.) present in the seeds are separated on the chromatogram. They can be compared and identified with the known Rf values of various amino acids (e.g., it is 0.26 of glycine. 0.38 of alanine, 0.60 of valine and 0.73 of leucine).
Precautions:
1. Always hold the chromatogram sheet from its edges.
2. Care should be taken that jar is saturated fully with the vapours of solvent.
3. Chromatogram should not touch the walls of the chamber.
4. Never overheat the chromatogram. Amino acids will evaporate if overheated.
3. Experiment to separate the leaf pigments by paper strip chromatography:
Requirements:
Gas jar, chlorophyll extract, Whatman No. 1 filter paper, sliding glass rod, hook, rubber cork, petroleum ether, acetone, lead pencil.
Method:
1. Take a perfectly dry, long strip of Whatman No. 1 filter paper and draw a fine line with a lead pencil, parallel to about 1.5 cm from one edge. This will indicate the bottom of your chromatogram.
2. Draw a circle on this line just in the middle and put a drop of chlorophyll extract just in the circle. Let it dry. (Note—Avoid excess handling of the chromatography paper, since your hands may contaminate it with amino acids. Touch it only at the edges).
3. Repeat the process of putting and drying the drop of chlorophyll extract for 2-3 times.
4. Take the rubber cork of the jar fitted with glass rod having a hook at its lower end, and attach the upper portion of the chromatography paper with the hook (Fig. 41).
5. About 2 cm of the jar is filled with the solvent (petroleum ether: acetone = 100:12).
6. Insert the strip attached with the hook in the jar and see carefully that lower edge of the strip touches the solvent. Note carefully that chlorophyll spot should not be touched by the solvent and paper should not touch the wall of the jar.
7. Keep the whole apparatus as such for 20 to 30 minutes and see that solvent has risen nearly up to the top of the paper.
Observations:
Different pigments separate at different levels on filter paper strip.
In this experiment different pigments from below upward separate in a sequence of chlorophyll b, chlorophyll a, xanthophyll’s and carotene.
Results:
Separation of different pigments on strip is based on the fact that paper chromatography separates compounds on the basis of their different rates of migration on filter paper (cellulose). The rate of migration depends upon the solvent which is flowing up and also on the relative adsorption which holds the molecules more or less tightly to the paper.
