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(Chuangrui Peristaltic Pump )Paper chromatography for determination of chlorophyll

2022-05-31

Paper chromatography is a kind of chromatographic analysis with filter paper as the carrier, its principle is mainly using the mixture of each component; They are separated by differences in the distribution ratio (solubility) between the mobile phase and the stationary phase. The adsorbed water on the filter paper is the stationary phase (the filter paper fiber can absorb about 20% water), the organic solvent such as ethanol is the mobile phase, and the pigment extract is the chromatographic sample. The sample point on the filtrate fine line position of the filter paper, when the mobile phase solvent in the filter paper under the action of the capillary, continuously along with the filter paper forward through the filtrate fine line, the components of the sample will move forward with the mobile phase solvent, and in the mobile phase and fixed phase solvent continuous distribution. Results The material with a larger distribution ratio moved faster and farther. The material with small distribution moves slowly and moves closer, and the components of the sample gather in different positions on the filter paper, to achieve separation.
Keywords: paper chromatography, chlorophyll, separation, speed
To master the method of extracting and separating pigment from the chloroplast. There are several pigments in chloroplasts. Carotene, lutein, chlorophyll A and chlorophyll B could be separated into four pigment bands with different diffusion rates of chromatographic solution on filter paper.
Materials and Methods:
Appliance: A pair of scissors, dry qualitative filter paper, 3 50ml beakers, and 3 100ml beakers, 3 pieces of white paper, 1 test-tube holder, 1 mortar, 1 glass funnel, nylon cloth or gauze, 1 capillary, 1 medicine spoon, 1 10ml measuring cylinder, 1 balance, 3 test tubes, 1 cardboard, 3 cotton plugs, 3 Petri dishes, scale, 1 syringe, cover glass
Reagents: acetone, anhydrous ethanol, layer absorbent (20 parts petroleum ether, 2 parts acetone, 1 part benzene configuration), silica, calcium carbonate, sodium carbonate materials: fresh spinach leaves, green vegetable leaves
(II) Methods and steps:
1. Extract pigment from green leaves (1) Take several green leaves, remove petioles and main veins, weigh 5g leaves with a balance, cut them into pieces, and put them into a mortar.
(2) Put a little silica and calcium carbonate into a mortar and grind it thoroughly. Use a measuring cylinder to measure 5 ml acetone into a mortar and grind quickly and thoroughly.
(3) Quickly pour the abrasive solution into a small glass funnel for filtering. Collect the filtrate into a small test tube and close the tube with a cotton plug.
2. Preparation of filter paper take a piece of pre-dried qualitative filter paper, cut the filter paper 10 cm long and 1cm wide, and draw a thin horizontal line 1cm away from one end of the filter paper with a pencil.
3. Draw the fine line of filtrate, draw a small amount of filtrate with a capillary pipette, and draw a thin and straight line of filtrate evenly along the pencil line. After filtrate, draw 2~3 times.
4. Separate the pigments from the chloroplast and pour 3 mL chromatographic solution into the beaker. Gently insert the filter paper (with the thin line of filtrate facing down) against the inner wall of the beaker, and then cover the beaker with a petri dish. (Note that the thin lines of the filtrate on the filter paper should not touch the chromatography. An organic substance used in paper chromatography
Solvents such as acetone, are generally volatile and have certain toxicity, pay attention to sealing chromatography when using, and avoid inhaling too much harmful volatiles.) Results: After 10-15 minutes, the distribution of pigment bands after separation was observed. The top orange is carotene, followed by
The yellow is lutein, the lower part is chlorophyll-a, and the lower part is chlorophyll-B.
Discussion and analysis:
Analysis: Paper chromatography is based on the principle of polarity similarity compatibility, which uses the combined water of filter paper fiber as the stationary phase and the organic solvent as the mobile phase. Due to the different distribution coefficients of each material in the sample, the diffusion rate is different, to achieve the purpose of separation. Generally used for the separation of pigments in chloroplasts, pigments in chloroplasts mainly include carotene, lutein,
Chlorophyll A and chlorophyll B have different solubility in the chromatographic solution. The solubility of chlorophyll A and chlorophyll B diffuses quickly with the chromatographic solution on the filter paper, while that of chlorophyll B diffuses slowly with the chromatographic solution on the filter paper. The pigment band is wider when the content is higher. Four pigment bands were left on the filter paper, so the pigment in chloroplast could be separated clearly by paper chromatography.
Discussion: 1. Chlorophyll and other pigment molecules are fat-soluble organic molecules. According to the principle of similar phase dissolution, chlorophyll and other pigment molecules are soluble in organic solvents but not in polar water. Therefore, when grinding and collecting chlorophyllin, organic solvents such as acetone or ethanol should be used instead of water.
2. Chlorophyll is distributed on the lamellar film of the grana, and a little silica is added to ground the cell wall, plasma membrane, chloroplast envelope, and photosynthetic tablet so that the pigment is dissolved in acetone. 3. The broken cells contain organic acids such as oxalic acid, and the Mg element contained in chlorophyll molecules is in an unstable combination state. The combination of magnesium ions and organic acids will lead to the destruction of pigment molecules. Adding a little calcium carbonate makes calcium ions bind to organic acids, reduces the transfer of magnesium ions, and prevents the destruction of chloroplast pigments during grinding. So the same goes for adding calcium carbonate as well as sodium carbonate when grinding.
4, choose absorbent cotton or gauze when filtering, rather than filter paper. The main reasons are as follows :(1) the pigment molecules are relatively large and it is not easy to pass through the filter paper; (2) The filter paper has strong absorbability so that the pigment is adsorbed on the filter paper, thus reducing the concentration of pigment and affecting the experimental effect; (3) chlorophyll is fat-soluble. According to the principle of similarity and compatibility, absorbent cotton can reduce the loss of pigment in the experimental process and enhance the experimental effect.
5. According to the capillary phenomenon in physics, the filter paper must be dried before drawing the filter paper fine line, to prevent water molecules from blocking the capillary in the filter paper and affecting the diffusion of the chromatography liquid. But if it is roasted with fire, it will deform the filter paper fiber and destroy the capillary, and affect the diffusion of the chromatography liquid.
6. Because the surface tension is different at different positions of the liquid surface, when the paper is close to the liquid surface, the surface tension at the edge of the paper is larger, and the chromatography liquid spreads too fast along the edge of the filter paper, resulting in the irregular separation of the pigment band. Therefore, two corners are cut off at one end of the filter paper and inserted into the chromatography liquid.
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