What is the key to the success of chloroplast pigment extraction and isolation?

The principle of pigment extraction in chloroplast is: because the photosynthetic pigment is located on the sac-like structure in the chloroplast, in order to extract it, it is necessary to destroy the leaf epidermis, cell wall, cell membrane, and chloroplast double membrane, so it should be cut and crushed with silica to grind in order to fully extract the pigment.

And because the pigment of chloroplast is insoluble in water, but soluble in organic solvents, acetone is added to dissolve the pigment to extract various pigments. In addition, calcium carbonate is added to protect the pigments in the chloroplast, because calcium carbonate is added to regulate the pH of the liquid and prevent chlorophyll from being destroyed. The pigments in chloroplasts are dissolved in organic solvents such as alcohol or acetone (similar to each other) to form a pigment solution.

The principle of separation is related to the solubility of the four pigments in the chromatography. Chromatography solution is a very fat-soluble organic solvent, due to the different chemical composition and structure of the four pigments, so each pigment molecule under the action of the chromatography molecules, overcome the gravitational force between the pigment molecules, and the diffusion speed to the chromatography solution is different, that is, the solubility in the chromatography solution is different, so the diffusion speed of the four pigments with the chromatography solution on the filter paper strip is different, among which carotene has the highest solubility in the chromatography solution and the fastest diffusion rate, followed by lutein and chlorophyll a, and chlorophyll b has the lowest solubility, Spreads the slowest. According to this principle, the four pigments on the filter strip on the same filtrate line are separated in diffusion.

Extract. 1.Shredded.

2.Add silica grinding key!!

3.Acetone is added to dissolve the pigment.

4.Calcium carbonate to protect the pigments in chloroplasts KEY!!

Separate. 1。Grind well.

2。The lines drawn on the filter paper should be thin or the dots should be smaller, and should not be immersed in the chromatography solution.

There are a lot of things to pay attention to, as long as you do it well, you can bring up all four belts. First of all, after adding silica, calcium carbonate should be added (to protect and prevent the pigment from being destroyed), and it should be slower when loading alumina to make it uniform and the surface is flat, the most important thing is the control of the rate during chromatography, which is the key to whether all your four bands can be precipitated, and if the rate is too slow, the time is too long. If it is too fast, only three bands can be precipitated.

I don't know what the rate is, but the lab textbook or the teacher will definitely tell you, just do that, don't be too anxious. The chromatography solution is replenished at any time throughout the process, so that the chromatography fluid is always not over the upper layer of silica.

This is what I summarized when I did this experiment, and I hope it will be helpful to you.

Grind well. Dissolve in place.

1. Extraction: pigment in green leaves - grinding method. Weigh and cut 5g of leaves and put them in a mortar. Add a little silica.

Calcium carbonate, 5 ml acetone.

Grind quickly and thoroughly.

According to chloroplasts.

pigments in organic solvents (ethanol.

acetone, etc.), the pigment can be extracted from the leaf with acetone (if 95 alcohol is used as a solvent, anhydrous sodium carbonate should be added to the pigment extract to remove the water in the pigment extract.

If acetone is used as a solvent, the dosage should not be too much, 2 5ml is appropriate, too much will affect the pigment concentration, too little is not easy to filter), collect the filtrate after filtration, collect the filtrate into a small test tube, and plug the test tube tightly with a tampon in time for later use.

2. Separation: Prepare filter paper strips, cut the dried qualitative filter paper into strips of 6cm lcm, cut off two corners at one end, and draw a very thin horizontal line with a pencil from the end of lcm. Draw a small amount of filtrate with a capillary pipette, draw a thin and straight filtrate line evenly along the pencil line, and repeat the drawing 2 3 times after drying (the more times you do, the more pigment).

Experimental Principle:

1. Principle of pigment extraction: pigment is soluble in organic solvent but not in water, and 95% organic solvents such as ethanol and acetone can be used to dissolve pigment, so as to extract pigment in green leaves. Pigment extraction can also be extracted by water bath heating, that is, the leaves are cut and placed in 95% ethanol for water-proof heating.

Generally speaking, when the leaves are heated to a yellowish-white color, the pigment in the leaves is basically extracted.

2. Principle of pigment separation: solubility of various pigments in chromatography solution.

In contrast, the diffusion rate of the chromatography solution with high solubility on the filter paper is faster, and vice versa, so that the various pigments are separated from each other.

The extraction and separation of pigments from green leaves are as follows:

The slurry is quickly poured and tampons are used because acetone is added to the grinding, which is easily volatile, and acetone is used to dissolve the pigment in the chloroplast, because chlorophyll can be dissolved in acetone. Because there is residue in the grinding of the leaves.

Chromatography is a highly fat-soluble organic solvent. The solubility of the pigments in the body of the chlorophyllum is different: the high solubility of the chromatography spreads quickly with the chromatography solution;

The low solubility diffuses slowly with the chromatography on the filter paper. Lutein is a diol derived from carotene, which cannot be saponified with alkali and can separate chlorophyll and carotenoids.

1. Extract chlorophyll.

Chlorophyll is an ester of chlorophyllin, under the action of alkali, it can make its ester bond saponified to produce chlorophyllic acid salt, which can be soluble in water, but because it retains the structure of the mg nucleus, it still retains its original green color.

2. Isolate chlorophyll.

The water-soluble anthocyanins can be filtered out by the principle of similar miscibility, and then carotene, lutein and chlorophyll can be separated by thin layer chromatography, column chromatography and high performance liquid chromatography.

As follows:

1) Experimental principle: organic solvent anhydrous ethanol can be used to extract the pigment in green leaves, and the principle of separating pigment is to use the solubility of pigment in the chromatography solution is different, and the diffusion rate of high solubility on the filter paper is fast, and vice versa is slow.

2) Experimental results: After chromatography, four pigment bands will appear on the filter paper strip, and the pigment distribution from top to bottom is carotene, lutein, chlorophyll a, chlorophyll b, the widest color of the pigment band is blue-green, and the narrowest color is orange-yellow.

3) The pigments in green leaves are distributed in the thylakoid membranes of chloroplasts.

Basic introduction

Chlorophyll, the main pigment for photosynthesis in plants, is a family of lipid-containing pigments located in the thylakoid membrane. Chlorophyll absorbs most of the red and violet light but reflects green light, so chlorophyll is green, and it plays a central role in the light absorption of photosynthesis. Chlorophyll is a magnesium porphyrin compound, including chlorophyll a, b, c, d, f, prochlorophyll and bacterial chlorophyll.

Chlorophyll is not very stable, and light, acid, alkali, oxygen, oxidants, etc., will decompose it. Under acidic conditions, the chlorophyll molecule easily loses the magnesium in the porphyrin ring and becomes demagnesium chlorophyll. Chlorophyll has anti-cancer, antibacterial, antioxidant and other effects, and can be used in medical, food, cosmetics and other industries.

In the process of extracting photosynthetic pigments, the key is to be fast. Because photosynthetic pigments are fat-soluble, acetone is volatile and has a certain toxicity, so the extraction process should be fast, and at the same time, it should be covered with a piece of paper to prevent its volatilization; Adding silica during grinding can increase the friction during grinding, speed up the grinding speed, and at the same time make the grinding full and extract more pigments; Since chlorophyll is easy to destroy, calcium carbonate needs to be added to prevent chlorophyll from being destroyed; The purpose of chopping the green leaves is also to speed up the grinding.

Drawing a fine line of filtrate is the key to a clear and clear result in this experiment. The filtrate line must be completely dried for the first time before the second line can be drawn, and the number of repetitions can be more, and the filtrate line should be uniform, thin and straight.

First of all, it is necessary to know the principle: chlorophyll is able to dissolve in pyruvate. There is also the fact that the pigments in chloroplasts have different solubility in the chromatography:

High solubility spreads quickly, low spreads slowly. After understanding the principle, it is time to select materials. Fresh green leaves, such as spinach leaves, are then added and 5mol of acetone is added for a quick and thorough grinding.

Finally, filter, draw the filter line, remember to draw the filter line a few more times, and follow the principle of straight and straight.

Scribing, fold the scribing in half and not flattening, and use the extraction solution to draw in half. In addition, the filtration step can be omitted, and the material can be selected as safflower stepwood, and you will have new discoveries.

Do not avoid light, choose a solvent with low volatility to reduce the degradation of pigments.

The principle of extracting chloroplast pigment is that chlorophyll is an ester of chlorophyllin, and under the action of alkali, the ester bond can be saponified to generate a salt of chlorophyllin, which can be dissolved in water.

The principle of separating chloroplast pigments is that among the carotenoids, carotene is an unsaturated hydrocarbon, - carotene hydrolysis can produce 2 molecules of vitamin A, lutein is a diol derived from carotene, which cannot undergo saponification reaction with alkali, according to this, chlorophyll and carotenoids can be separated.

Extended information: Paper chromatography is generally used for the separation of pigments in chloroplasts, which mainly include carotene, lutein, chlorophyll a, chlorophyll b, crush the leaves, leach out the green liquid, mix the liquid with chromatography solution (petroleum ether), and enter the filter paper into the mixed liquid.

The solubility of the four pigments in the chromatography solution is different, and the solubility of the more solubility spreads faster with the chromatography on the filter paper, and vice versa is slower. Finally, four pigment bands were left on the filter paper, from which the relative content and types of various pigments were observed. Therefore, paper chromatography can be used to separate the pigments in chloroplasts.

Chloroplast pigments are soluble in organic solvents, so they can be extracted with absolute ethanol.

Separation: Chloroplast pigments have different solubility in the chromatography, and the large solubility is fast with the diffusion rate of the chromatography, and vice versa, which can be separated.

It is to separate chlorophyll a, chlorophyll b, lutein and carotene in chloroplasts.

Extract chlorophyte.

The principle of body pigment is that chlorophyll is an ester of chlorophyllin, and under the action of alkali, the ester bond can undergo soap specialization to produce a salt belonging to chlorophyllin, which can be dissolved in water.

The principle of separating chloroplast pigments is that among the carotenoids, carotene is an unsaturated hydrocarbon, - carotene hydrolysis can produce 2 molecules of vitamin A, lutein is a diol derived from carotene, which cannot undergo saponification reaction with alkali, according to this, chlorophyll and carotenoids can be separated.

Extracted chlorophyll is easily soluble in organic matter.

Separate different pigments have different solubility in organic matter and therefore move at different rates.

Alcohol can blend with it, so we can see the colors.

The principle of extraction is that the pigment is dissolved in an organic solvent.

The principle of separation is that different pigment molecules have different solubilities in the chromatography.

The solubility of individual chlorophyll varies.

To understand this problem, we must first understand the principle of chromatography, the principle is: use the difference in the partition coefficient of each component of the mixture in the mobile phase and the stationary phase two different solvents, and separate them, the mobile phase in paper chromatography refers to the chromatography liquid, under the action of capillary tension, the chromatography liquid can continue to flow from the bottom to the top. The stationary phase refers to the moisture that is adsorbed between the filter paper fibers.

Because the hydrophilic hydroxyl group on the cellulose binds this part of the water around the cellulose and is not easy to diffuse, it becomes a stationary phase. In the chromatography process, when the chromatography solution flows through the fine line of the pigment filtrate under the action of capillary tension, the pigment on the fine line of the filtrate is successively integrated into the chromatography solution, and as the chromatography solution rises, and the distribution occurs, that is, a part of the pigment is distributed and dissolved into the stationary phase from the chromatography solution until equilibrium. Due to the different partition coefficients, which are related to the size of the molecules, those pigment molecules with large partition coefficients move up quickly with the chromatography solution, and the pigment bands formed are concentrated in the upper part of the filter paper; The pigment molecules with small partition coefficients move slowly upward with chromatography, and the pigment bands formed are concentrated under the filter paper.

From the above principle, we know that in the paper chromatography process, the pigment in the filtrate line does not run downward, but continues to run upward under the action of capillary tension, and because the distribution coefficient of different pigments is different, the distance is different. Paper is made of plant fibers, and there are capillaries in plant fibers, which is a prerequisite for the formation of capillary tension, why plants can transport water to the top of trees up to tens or even hundreds of meters, is because of the tensile effect of capillaries, after making paper, this tensile force still exists.

The pigments are chromatographed at the same time, so no four bands are formed. The experimental phenomenon should be due to the different solubility of the four pigments in the chromatography.

This is because the four pigments have different solubilities in the chromatography.