Application of Peristaltic Pump in Paper Chromatography Determination of Chlorophyll

Paper chromatography is a kind of chromatographic analysis with filter paper as the carrier, its principle is mainly using the mixture of each component in; 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 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 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 of the filter paper, so as to achieve separation.

Key words: paper chromatography, chlorophyll, separation, speed

To master the method of extracting and separating pigment from 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 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, draw a thin horizontal line 1cm away from one end of the filter paper with a pencil.

3. Draw fine line of filtrate, draw a small amount of filtrate with a capillary pipette, 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, generally volatile, and have a certain toxicity, pay attention to sealing chromatography when using, 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 coefficient of each material in the sample, the diffusion rate is different, so as to achieve the purpose of separation. Generally used for the separation of pigments in chloroplasts, pigments in chloroplasts mainly include carotene, lutein.

If you need a Peristaltic Pump,please contact us:sales01@crpump.com 15633705132