What about the separation of chloroplast pigments by thin layer chromatography? 20

Do you want to know the steps?

1. Extract the pigment in the green leaves.

1. Weigh 5 grams of green leaves, cut them into pieces, and put them in a mortar.

2. Add silica and calcium carbonate to the mortar, add 10ml of absolute ethanol, and grind quickly.

3. Pour the slurry into a glass funnel (put a single-layer nylon cloth at the bottom of the funnel) for filtration. Collect the filtrate into the test tube, and plug the test tube tightly with a cotton plug in time.

2. Prepare filter paper strips.

Cut off two corners of one end of the filter strip and draw a thin horizontal line with a pencil 1 cm away from this end.

3. Draw a fine line of filtrate.

Aspirate a small amount of filtrate with a capillary pipette and draw a thin line evenly along the pencil line. Once the filtrate is dry, draw it once or twice.

4. Isolate the pigments in green leaves.

Pour 3 ml of chromatography into the tube, gently insert the filter paper strip (with the filtrate line with the end facing down) into the chromatography, and then plug the mouth of the test tube tightly with a tampon. Note: Do not allow filtrate filtrate lines to touch the chromatography.

5. Observation and Recording.

Observe the presence of several pigment bands on the filter paper strip inside the tube, as well as the color of each pigment band.

There are four color bands of different widths and colors on the filter strip, from top to bottom: orange-yellow carotene, yellow lutein, blue-green chlorophyll a, and yellow-green chlorophyll b

There can be a number of reasons for the failure of the experiment, such as not letting the chromatography reach the filtrate line during the pigment separation. Otherwise, the chromatography will dissolve the pigment in the filtrate fine line, causing it to enter the chromatography in the beaker, and as a result, the pigment separation will fail.

1. Separation principle: paper chromatography method is based on the principle of polar similarity and dissolution, with the bound water of the filter paper fiber as the stationary phase and the organic solvent as the mobile phase. Due to the different partition coefficients of each substance in the sample, the diffusion velocity is different, so as to achieve the purpose of separation.

2. Extraction principle: extraction with organic solvent, and then filtration, vacuum concentration and vacuum drying and refining to obtain the final product. The solvents used to extract pigments have different choices according to the properties of pigments and the raw materials used, and commonly used are water, acid and alkali solutions, organic solutions, such as ethanol, acetone, alkanes, benzene, etc., oils and carbon dioxide.

The role of pigments:The pigments in chloroplasts are divided into two categories. One is pigments that can absorb and transmit light energy, including most chlorophyll a, all chlorophyll b, all carotene and lutein.

The other type is a small number of chlorophyll a in a special state, which can not only absorb light energy, but also convert light energy, that is, convert light energy into electrical energy. Therefore, the role of synthetic chloroplast pigments is to absorb, transfer, and convert light energy.

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.

In chlorophyll extraction and bending separation experiments, the reasons for the overlap of the color bands may be as follows:

1.During the extraction process, the grinding of the green leaves is not sufficient, resulting in the chlorophyll not being fully released.

2.When grinding green leaves, too much calcium carbonate is added, so that the pigment extract contains no or very little chlorophyllin, resulting in the chlorophyll not being completely separated.

3.During the experiment, the chromatography was not properly spotted, so that the different pigments were not separated.

4.During the experiment, the separation was not done correctly with filter paper, allowing the different buried pigments to mix together.

5.During the experiment, the filtrate lines on the filter strip are dissolved by the chromatography, resulting in overlapping pigment bands.

To prevent the ribbon from overlapping, the following measures can be taken:

1.Proper grinding and chlorophyll release operations.

2.The correct number of rolls controls the amount of calcium carbonate added to the grinding process.

3.Correct dispensing and separation.

4.Properly control the filtrate line on the filter strip from being dissolved by the chromatography.

The overlapping phenomenon of pigment on the filter paper strip is due to the fact that the filtrate line is not dried when drawing, so that the filtrate drawn out can not meet the requirements of fineness and uniformity, so that the pigment diffusion is inconsistent and the pigment overlap occurs.

It should also be noted that the chromatography solution of Songduyan is not dispersed with the fine lines of the filtrate, and the pigment will be dissolved in the chromatography solution, and no pigment band will appear.

The filtrate fine line is not straight! Bu Liang!!

Absolute! Kai cavity!

Why should the filtrate line be straight? Why redraw a few times?

It is to prevent the overlapping of the type and width of the pigment band; Increase the amount of pigment to make the pigment band darker.

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.

When separating pigments in chloroplasts by paper chromatography, the resulting filter strip should have four pigment bands, from top to bottom, carotene.

orange-yellow), lutein.

Yellow), chlorophyll a (blue-green), chlorophyll b (yellow-green) So it's chlorophyll b, so choose: b

Extraction and isolation of photosynthetic pigments from green leaves. The main purpose of this course is to let students master the experimental principles, methods and steps of "extraction and separation of pigments in experimental green leaves" and the analysis of experimental results, so as to improve students' self-learning ability.

Design ideasIn order to allow students to master the experiment in a short period of time, I directly cut into the topic, guided the students step by step to understand the principle and method of the experiment, and designed several key questions to improve the thinking ability of the students who regretted it, and then analyzed the results of the experiment.

The extraction and separation of pigments in green leaves is chlorophyll b.

Solubility affects the diffusion rate, high solubility and fast diffusion rate, carotene solubility is the highest, followed by lutein, then chlorophyll a, chlorophyll b, so the pigment bands are arranged like this from top to bottom. The width of the pigment band is the number of pigments represented, and solubility is not concerned.

Among the four pigments, chlorophyll a is the most abundant and therefore the widest, followed by chlorophyll b, and then lutein and carotene, and the width and narrowness of the pigment band are different.

Chemical Properties:

There are two main types of chlorophyll in chloroplasts of higher plants: chlorophyll a and chlorophyll b. They are insoluble in water while soluble in organic solvents such as ethanol, acetone, ether, chloroform, etc. Chlorophyll a molecular formula:

c55h72o5n4mg；Chlorophyll b molecular formula: C55H70O6N4MG

In color, chlorophyll a is blue-green, while chlorophyll b is yellowish-green. Chemically speaking, chlorophyll is an ester of chlorophyllin that undergoes saponification. Chlorophyllin is a dicarboxylic acid in which one carboxyl group is esterified by methanol and the other by leaf alcohol.