Lab 22
Chemical properties of sheet pigments
The most important components of the photosynthetic apparatus of plants are pigments. Pigments are divided into two classes: tetrapyrrole compounds ( chlorophylls and phycobilins) and polyisoprenoid ( carotenoids).
Phycobilins are algae pigments. Higher plants have chlorophyll "a", chlorophyll "b" and carotenoids. The main functional pigment is chlorophyll "a" , which is found in all photosynthetic organisms (except bacteria). It serves as a direct donor of energy for photosynthetic reactions. The rest of the pigments only transfer the absorbed energy to chlorophyll "a" .
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Rice. 17 Structural formulas of carotenoids and the sequence of their transformations
Moreover, in cyclic carotenes, six-membered rings are represented by two types: β-ionone and α-ionone.
In photosynthetic organisms, this group of yellow pigments is represented by lycopene, α-carotene, β-carotene, and γ-carotene. In higher plants, β-carotene is the main carotene.
Xanthophylls are oxygen-containing carotene derivatives, including lutein (C40H56O2), zeaxanthin (C40H56O4), violaxanthin (C40H56O4), neoxanthin (C40H56O4) (Fig. 17). Among the named xanthophylls, lutein predominates, which is very close in chemical structure to α-carotene, but unlike it is a dihydric alcohol, i.e., in each ionic ring, one hydrogen atom is replaced by a hydroxyl group.
Functions of carotenoids: 1) are additional pigments; 2) protect chlorophyll molecules from photooxidation; 3) play a role in oxygen exchange during photosynthesis.
Method principle: pigments from plant tissue are extracted with polar solvents (ethyl alcohol, acetone), which destroy the bond of chlorophylls and xanthophylls with plastid lipoproteins and ensure their complete extraction. Non-polar solvents (petroleum ether, hexane, gasoline, etc.) do not break the bond of these pigments with proteins.
Purpose of work: get acquainted with the chemical properties of sheet pigments.
Progress: 1. Getting an alcohol solution of pigments. To obtain the extract of pigments, both raw and dry plant material is used. The dried leaves are pretreated with hot water to facilitate subsequent extraction of the pigments.
Finely chop fresh plant leaves (1 g) with scissors, place in a mortar and grind with a small amount of CaCO3. Gradually add 2 ... 3 ml of ethyl alcohol into the mortar and thoroughly grind the sample until a homogeneous mass is obtained. Then add another 5 ... 8 ml of alcohol, mix the contents. Lubricate the nose of the mortar from below with petroleum jelly and transfer the contents of the mortar to a paper filter with a glass stick. Place the resulting filtrate in a test tube. Alcoholic extract contains a sum of green and yellow pigments.
2.Separation of pigments according to Kraus based on the different solubility of pigments in alcohol and gasoline. These solvents do not mix when draining, but form two phases, upper gasoline and lower alcohol, due to which the components of the mixture of pigments are separated.
Pour 2 ... 3 ml of alcohol extract of pigments into a test tube and add 3 ... 4 ml of gasoline. Shake the contents of the tube vigorously, having previously closed it with a cork or thumb, and leave to stand. For better separation, add 1 ... 2 drops of water.
As the emulsion stratifies, the upper gasoline layer will turn green due to the better solubility of chlorophylls in it. In addition, carotene is converted into gasoline, but its color is masked by chlorophyll. Xanthophyll remains in the lower alcohol layer, giving it a golden yellow color.
If the pigments do not separate clearly enough, add 1 ... 2 drops of water and shake again. With an excess of water, the lower layer may become cloudy, then you should add a little ethyl alcohol and shake the contents of the test tube.
Sketch the distribution of pigments in alcohol and gasoline, draw conclusions about their different solubility.
3. Saponification of chlorophyll with alkali. When chlorophyll is treated with alkali, the ester groups are saponified, that is, the residues of methyl alcohol and phytol are split off (Fig. 18). The sodium salt of chlorophyllic acid is formed, which retains the green color and optical properties of chlorophyll, but is more hydrophilic than the native pigment.
Rice. 18 Saponification of chlorophyll with alkali
Pour 1 ml of 20% NaOH solution into a test tube with 2 ... 3 ml of alcohol solution of pigments and shake. After mixing the extract with alkali, place the test tube in a boiling water bath, bring to a boil and cool.
Add an equal volume of gasoline and a few drops of water to the cooled solution for better separation of the mixture. Then shake the contents of the test tube and let it settle.
Carotene and xanthophyll will pass into the gasoline layer, and the sodium salt of chlorophyllic acid will go into the alcohol layer.
Sketch the color of the layers, indicating the distribution of the pigments 3. Obtaining pheophytin and reverse replacement of hydrogen by a metal atom. The magnesium atom is weakly retained in the porphyrin core of chlorophyll and, under careful action of strong acids, is easily replaced by two protons, which leads to the formation of brown pheophytin.
If pheophytin is acted upon by salts of copper, zinc or mercury, then instead of two protons, the corresponding metal enters the nucleus and the green color is restored again. However, it is somewhat different from the color of chlorophyll. Therefore, the color of chlorophylls depends on the organometallic bond in their molecule.
Pour 2 ... 3 ml of alcohol extract of pigments into a test tube and add 1 ... 2 drops of 10% hydrochloric acid solution. During the reaction, the green color changes to brown, while chlorophyll is converted to pheophytin. Dispense the contents of the test tube into two test tubes.
Leave one tube with pheophytin for control, and in the second place a few crystals of acetic acid copper and heat the solution in a water bath until boiling. After heating, the brown color of the solution changes to green as a result of the formation of a chlorophyll-like copper derivative.
Sketch the color of pheophytin and copper chlorophyll.
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chlorophyll-like copper derivative
Equipment and materials: 1) fresh plant leaves; 2) ethyl alcohol; 3) gasoline; 4) 20% NaOH solution; 5) 10% hydrochloric acid in a dropper; 6) 10% hydrochloric acid; 7) water bath; 8) a rack with test tubes; 9) 1 ml pipettes or volumetric tubes; 10) funnels; 11) filter paper; 12) a mortar with a pestle; 13) glass rods; 14) scissors.
Why are plants green?
Complexity:
Danger:
Do this experiment at home
Reagents
Security
- Wear protective gloves and goggles before starting the experiment.
- Run the experiment on a tray.
- Conduct the experiment in a well-ventilated area, away from sources of ignition.
General safety rules
- Do not allow chemicals to come into contact with eyes or mouth.
- Keep people without safety glasses, and small children and animals away from the testing area.
- Store the experimental kit out of the reach of children under 12 years of age.
- Wash or clean all equipment and accessories after use.
- Make sure all reagent containers are tightly closed and stored properly after use.
- Make sure all disposable containers are properly disposed of.
- Use only equipment and reagents supplied in the kit or recommended by the current instructions.
- If you used a food container or utensil for experiments, discard it immediately. They are no longer suitable for storing food.
First aid information
- If reagents come in contact with your eyes, rinse your eyes thoroughly with water, keeping your eyes open if necessary. See a doctor immediately.
- If swallowed, rinse your mouth with water and drink some clean water. Do not induce vomiting. See a doctor immediately.
- If reagents are inhaled, remove to fresh air.
- In the event of skin contact or burns, rinse the affected area with copious amounts of water for 10 minutes or longer.
- If in doubt, consult a doctor immediately. Take the chemical and its container with you.
- Always see a doctor in case of injury.
- Improper use of chemicals can cause injury and damage to health. Perform only the experiments specified in the instructions.
- This set of experiences is only for children 12 years of age or older.
- Children's abilities vary significantly even within the age group. Therefore, it is up to parents who experiment with their children to decide which experiments are appropriate and safe for their children.
- Parents should discuss safety rules with the child or children before starting experiments. Particular attention should be paid to the safe handling of acids, alkalis and flammable liquids.
- Before starting experiments, clear the testing area of objects that may interfere with you. Storing food near the test site should be avoided. The test site should be well ventilated and close to a tap or other source of water. A stable table is required to conduct experiments.
- Substances in single-use packaging should be used completely or disposed of after one experiment, i.e. after opening the package.
FAQ
Where to get 96% alcohol (ethanol) solution?
Alcohol can be bought at a pharmacy or obtained by laboratory methods. To do this, you need three candles and strong alcohol or 40-60% ethanol solution. The rest can be found in the Plant Chemistry box and starter kit.
- Insert the metal adapter into the one-hole plug.
- Slide the silicone tube over the adapter.
- Insert a funnel into a flask and pour 40 ml of strong alcohol or 40-60% ethanol solution.
- Stopper the flask.
- Pour cold water into a glass (halfway down). Place the test tube in the beaker.
- Place three candles on the burner and light them. Cover the burner with a flame deflector.
- Place the flask on the flame diffuser. Dip the free end of the tube into the test tube. Wait until the tube is two-thirds full of liquid.
- Put out the candles.
- Pour the liquid from the test tube into a glass with crushed green leaves and continue the experiment as instructed.
Other experiments
Step-by-step instruction
Chlorophyll is the substance that gives the leaves their green color. It is practically insoluble in water, but it dissolves in many organic solvents such as ethyl alcohol.
When enough chlorophyll is dissolved in alcohol, take two samples of the solution.
The chlorophyll molecule contains a magnesium ion Mg 2+ (green). In the presence of acid, it easily “leaves” the molecule. Formed pheophytin - a compound with a less bright and saturated color.
The place freed from magnesium can easily be taken by the copper ion Cu 2+ (brown) from the copper salt CuSO 4. The resulting copper complex of pheophytin is similar in color to chlorophyll.
The copper complex of pheophytin is more stable than chlorophyll. If both samples are left in the light, the chlorophyll will tarnish and the difference between the substances will be clearly visible.
Disposal
Dispose of solid waste from the experiment together with household waste. Drain the solutions into a sink and then rinse thoroughly with water.
What happened
What do we use a solvent for?
The alcohol helps extract the chlorophyll from the crushed leaves. The chlorophyll molecule has a long hydrophobic ("water-fearing") tail that prevents the substance from dissolving in water. But in alcohol (or, for example, in acetone), the solubility of chlorophyll is already quite high.
To learn more
Chlorophyll also dissolves in fats. Because of this, some vegetable oils, such as canola and olive, often have a pronounced green tint. To discolor such oils, an alkali treatment is carried out. As a result, the chlorophyll molecule loses its hydrophobic tail, and with it the ability to dissolve in fats.
Better than acetone and alcohol, chlorophyll dissolves only in liquids such as gasoline. But gasoline cannot extract pigment from leaves as efficiently. The fact is that in a plant, chlorophyll molecules are closely associated with protein molecules. To break the bond with a protein, the solvent must contain water that does not mix with hydrocarbons (gasoline, kerosene, petroleum ether).
Why did the green solution turn pale after adding citric acid?
The color of the solution became less saturated, because in an acidic environment, hydrogen ions H + displaced magnesium ions Mg 2+ and chlorophyll turned into pheophytin. Compared to the original substance, pheophytin has a darker, but at the same time less bright color.
To learn more
Feophytinization is a very common phenomenon. This terrible word is called the process of chlorophyll discoloration due to the loss of magnesium ions Mg 2+ in the presence of acids. You may have noticed that fresh green vegetables get darker when cooked. The effect of pheophytinization is especially evident when pickling cucumbers: after adding the marinade, the bright green skin of the fruit becomes brownish.
What happens when CuSO 4 is added?
When we add a solution of copper sulfate CuSO 4, copper ions Cu 2+ appear in the test tube. They occupy a place in the chlorophyll molecule, from which magnesium Mg 2+ was previously displaced. The chlorophyll-copper complex has a bright green color, so the solution again acquires a pronounced green color. Even after a few days, when the magnesium-containing chlorophyll has already been destroyed, the color of the chlorophyll copper complex solution remains saturated.
To learn more
The product of the interaction of the pheophytin solution with copper ions Cu 2+ has a stern name - "copper chlorophyll complex". This substance is registered under the code E141 as a permitted food coloring. Such a substance can be used only in strictly limited doses, because the copper contained in it is a heavy metal that is dangerous to health in quantities of more than 5 mg per day. The US Food and Drug Administration (FDA) allows the use of E141 in food solely for coloring dry mixes in citrus-based drinks. In this case, the proportion of the dye should be no more than 0.2% by weight of the dry product. In Europe, Russia and most countries in Asia, Africa and South America, it is allowed to use the copper chlorophyll complex in the production of confectionery, canned vegetables, cosmetic products and medicines.
What other metals can replace magnesium in chlorophyll?
Not only copper Cu 2+ can return the color to the acidified chlorophyll solution. Zinc salts Zn 2+ and mercury Hg 2+ also form green-colored compounds with chlorophyll. However, reactions with these ions are much slower and require special conditions, and the color of complexes with chlorophyll is not as saturated as with copper. It is also worth remembering that mercury salts are extremely toxic and are not at all intended for home experiments.
Why did the chlorophyll solution turn pale?
Over time, photochemical oxidation occurs in a solution of the magnesium complex of chlorophyll. Because of this, the solution loses its rich color. The chlorophyll copper complex is much more stable than its natural predecessor. It does not undergo oxidation as quickly, and therefore its solution retains its color longer.
Which plant leaves are best for the experiment?
Many fresh green leaves will do. Make sure the plant is not poisonous before testing. Also, do not use plant leaves with milky juice (euphorbia, dandelion, mother's favorite ficus and others). To check if the plant contains milky sap, look at the leaf cut: protruding white (sometimes yellow, beige or reddish) opaque droplets indicate that it is better not to take such material for the experiment. With juicy fleshy leaves (sedum, Kalanchoe, Tradescantia, and others), the solution will turn out pale, because there is too little chlorophyll in the leaf pulp of such plants.
lives by control. The position of the dark stripes in the experimental spectrum is used to determine which rays are absorbed by the investigated pigment.
Purpose of work: to get acquainted with the optical properties of pigments
Determination of the absorption spectrum of chlorophyll ... Set the spectroscope in relation to the light so that all spectral regions have the same brightness. Pour chlorophyll alcohol extract into the spectrophotometric cuvette, place it in front of the spectroscope slit and determine the position of the dark bands that correspond to the rays absorbed by chlorophyll.
The width of the stripes depends on the concentration of the pigment or the thickness of the layer of its solution. To observe the absorption spectra of solutions with different concentrations of chlorophyll, dilute the extract with alcohol in the ratios 1: 1, 1: 3, 1: 5, etc. and investigate the optical properties of the resulting solutions. From a comparison of the absorption spectra of solutions of various concentrations, we find out that the strongest absorption occurs in the red rays (the most concentrated extract). At the end of the experiment, draw a conclusion about the dependence of the absorption spectrum of chlorophyll on its concentration and explain the established fact.
Absorption spectrum of carotene and xanthophyll. To obtain the absorption spectrum of carotenoids with a pipette, carefully take a gasoline solution into which carotene and xanthophyll passed after chlorophyll saponification, transfer it to a cuvette and place it in front of the spectroscope slit. Examine the absorption spectrum and compare it with the absorption spectrum of chlorophyll. Sketch both spectra.
Chlorophyll fluorescence. Fluorescence is the emission of light by an excited chlorophyll molecule. Its essence is as follows. At room temperature and in the dark, the chlorophyll molecule is in the ground state, i.e. its energy corresponds to the lower singlet level (So) .: The absorption of a quantum of light is accompanied by the transition of one of the π-electrons to a higher energy level. As a result, a singlet electronically excited state of the molecule arises. A singlet state is such an excited state in which the transition of an electron to a higher energy level is not accompanied by a change in the spin sign. One line corresponds to it in the absorption spectra. If, in this case, a quantum of red light is absorbed, then the electron goes over to the first singlet level (S1) with an energy of 1.7 eV and a lifetime of 10–8 –10–9 s. In the case of the capture of a quantum of blue light, the electron is at the second singlet level (S2) with an energy of 2.9 eV, and the lifetime of this state decreases to 10–12 –10–13 s. However, no matter what kind of electric
the throne-excited state of the molecule was transferred by the absorbed quantum; it ultimately goes over to the lowest vibrational sublevel of the first singlet excited state (S1). The energy of this state can be used to carry out photochemical processes, migrate from one chlorophyll molecule to another, and be wasted in the form of heat or fluorescent radiation.
Thus, regardless of the length of the exciting light, chlorophyll fluoresces only in the red part of the spectrum. The decrease in the energy of a quantum emitted by an excited molecule in comparison with the energy of an absorbed quantum is called the Stokes shift. Only chlorophyll "a" and chlorophyll "b" fluoresce; carotenoids do not have this ability. In a living leaf, the main fluorescent pigment is chlorophyll a. At the same time, fluorescence in leaves is much less pronounced than in solution, since part of the absorbed energy is used to sensitize photochemical reactions. Therefore, an increase in the intensity of photosynthesis, as a rule, entails a weakening of fluorescence. Fluorescence not only provides valuable information on the use of energy in photochemical processes, but is also an important characteristic of the interaction of molecules of various pigments in chloroplast thylakoid lamellae, energy migration in photosystems, etc.
Progress . To determine fluorescence, an alcoholic extract of pigments or a solution of chlorophyll in gasoline, obtained by separating pigments according to Kraus, should be placed on dark paper near
Fig. 10. Consideration of chlorophyll alcohol extract:
A - in reflected rays; B - in transmitted rays; a - light source; b - a test tube with a hood; into the eye; d - incident rays; d, e
- reflected rays; g - rays passed through chlorophyll
light source and view in reflected light (Fig. 10). The chlorophyll extract will be dark red in color.
Fluorescence can also be observed in a living leaf. To do this, take Canadian Elodea (Elodea canadensis Michx.), Place the object on the microscope stage and illuminate it with blue-violet rays, under the influence of which green plastids begin to glow with red light.
Materials and equipment: 1) alcohol extract of leaf pigments; 2) a solution of carotene and xanthophyll (gasoline layer obtained after saponification of chlorophyll); 3) pipettes for 1 ml; 4) cuvettes; 5) spectroscopes.
3.3. Separation of pigments by paper chromatography
The proposed method allows partially separating plastid pigments on paper. Complete separation of pigments can be obtained with special chromatographic paper using several solvents.
In this work, the separation of pigments is based on their different advancement with a solvent, which is due to the different adsorbing capacity of pigments on paper and partly their different solubility in gasoline.
Purpose of the work: to carry out a complete separation of a mixture of pigments into individual components using a two-dimensional chromatogram.
Work progress: 1. Prepare an acetone extract from fresh plant leaves. The weighed amount of plant material should be 2-3 g, the volume of the acetone extract of pigments - 25 ml (100% acetone).
2. Cut a strip 1.5-2.0 cm wide and 20 cm long from chromatographic paper.Holding the paper strip vertically, the tip
her lower for a few seconds in a pigment drawer poured into a bottle or porcelain cup. With a short immersion, the hood rises on the paper by 1.0-1.5 cm (starting line). The paper is then dried in a stream of air and immersed again in the pigment solution. This operation is carried out 5-7 times.
3. After that, the lower end of the paper strip is immersed in pure acetone for a few seconds so that all pigments rise by 1.0-1.5 cm.Thus, a colored zone (in the form of a green strip) is obtained on the chromatographic paper, where the mixture of pigments is concentrated, which should be split.
4. Having well dried a strip of paper in a stream of air (until the smell of acetone disappears), place it in a strictly vertical position in a cylinder, on the bottom of which gasoline with a boiling point of 80-1200 C is poured, so that the solvent does not touch the pigment zone. The cylinder is hermetically sealed with a well-fitted stopper. After 15 minutes, the solvent rises by 10-12 cm.At the same time, the mixture of pigments is separated into
individual components in the form of
los, which are located in |
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next | order: first |
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below chlorophyll "b", above it |
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chlorophyll "a", then xantho- |
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moves |
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with the front | solvent |
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faster than other components, and |
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its zone on paper is |
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Rice. 11. Distribution of pigments | other pigments |
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(fig. 11). Make a drawing. |
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on the paper |
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Materials and equipment: 1) plant leaves; 2) acetone; 3) gasoline; 4) petroleum jelly; 5) cups or porcelain cups; 6) porcelain mortars with pestles; 7) funnels; 8) glass rods; 9) paper filters; 10) strips of chromatographic paper; 11) tall glasses or cylinders; 12) scissors.
3.4. Determination of carotene content in carrot roots
To carry out this work, a photometric method is used. It is based on converting the analyte in solution into a light-absorbing compound and measuring the light absorption of the resulting compound.
If a light flux is directed to a cuvette with a colored solution, then part of it will be absorbed, while the other will pass through the solution. By-
absorption will depend on the number of molecules encountered in the path of the light flux.
When working, you need to choose the light filter that would transmit the rays absorbed by the solution: the maximum transmission of the light filter should coincide with the maximum absorption of the solution. Light filters on the FEK are installed with different wavelengths in the region of the maximum transmission. For measurement, they are selected according to the principle of additional color: when working with a yellow-colored compound - blue, with a blue compound - red, etc.
The cuvettes are characterized by a working length (the distance between the edges, which is indicated on the wall facing the transmitted light): 5, 10, 20, 30, 50 mm. When analyzing weakly colored solutions, take cuvettes with a longer working length, strongly colored - with a shorter one. They strive for readings to be obtained on an optical density scale of no more than 0.8.
Purpose of work: to determine the amount of carotene in carrot roots.
Work progress: 1. Finely chop a weighed portion of carrots (1 g) and grind in a mortar with sand and 0.3 g of CaO (to remove water) until smooth. Add solvent in small portions to the mortar
- acetone and continue rubbing. Pour the resulting extract into a 25 ml volumetric flask. At the end of the extraction, top up the flask with solvent up to the mark. If the carotene solution is cloudy, it is filtered.
2. A solution of azobenzene is used as a standard (it corresponds to 0.00235 g of carotene per 1 ml of solution).
3. After receiving the experimental and standard solutions, proceed to their colorimetry. To do this, an experimental solution is poured into one cuvette, and a standard solution is poured into another cuvette and colorimetric on FEC with a blue light filter. The calculation is made according to the formula:
(K D1 | V 100) | |||
where X is the amount of carotene in mg per 100 g of carrots;
K is the amount of carotene for the standard (0.00235 g); V is the volume of the solution in ml (25 ml);
D1 is the optical density for the carotene solution; D2 is the optical density for the standard.
4. Determine the daily human need for carrots, based on the rate of 5 mg of carotene per day.
Materials and equipment: 1) carrot root vegetable; 2) acetone; 3) azobenzene solution; 4) 25 ml flasks; 5) porcelain mortars with pestles; 6)
filters; 7) funnels; 8) photoelectric colorimeter with cuvettes; 9) glass rods.
3.5. Determination of the intensity of photosynthesis by the assimilation flask method (according to L.A. Ivanov and N.L. Kossovich)
The method is based on determining the amount of carbon dioxide absorbed by leaves during photosynthesis. A shoot or a separate leaf is placed in a glass flask turned upside down (Fig. 12) and exposed to light for 15-20 minutes. Some of the carbon dioxide in the flask is consumed during photosynthesis. Then they bind CO2 not absorbed by the leaves, pouring some excess of alkali solution into the flask. Then the remaining alkali is titrated with hydrochloric or oxalic acid. The same is done with the control flask (without plant) and the titration results are compared.
Rice. 12. Device L.A. Ivanova and N.L. Kossovich to determine the intensity of photosynthesis: a - flask; b - a rod with a sheet; c - cork
If the experimental and control flasks have the same volume and if the same amount of Ba (OH) 2 solution is poured into both flasks, then the amount of carbon dioxide absorbed by the plant will be directly proportional to the difference in the results of titration of the contents of these flasks. In order to establish what amount of CO2 corresponds to 1 ml of acid used for titration, let us compare the reactions in which the alkali poured into the flask enters:
Ва (ОН) 2 + СО2 = ВаСО3 ↓ + Н2 О,
Ba (OH) 2 + 2HCI = BaCI2 + 2H2 O.
1M HCl corresponds to 0.5M CO2, i.e. 44: 2 = 22 g CO2. At a concentration of 0.025N HCI, 1 ml of this solution contains
0.000025M HCI, which is equivalent to 22 × 0.000025 = 0.00055 g or 0.55 mg CO2. This method gives sufficiently accurate results only in
if all operations for opening and closing the flasks are carried out without touching the glass with your hands (otherwise the air, expanding when heated, will partially escape from the flasks).
Purpose of work: to determine the intensity of photosynthesis of plants. Workflow: 1. Take two identical flasks and keep them in
under identical conditions open for 10-20 minutes to fill with air. Then simultaneously insert plugs with holes closed with glass plugs (No. 1) into them, not allowing the flasks to heat up by touching the hands.
2. Cut off a leaf or a plant shoot, update the cut with a razor under water and place in a test tube filled with water (take boiled water so that there are no air bubbles) attached to a stick inserted into a cork (No. 2).
3. With a quick but calm movement, remove the stopper No. 1 from the flask and insert the stopper No. 2 (with the plant).
4. Expose the flask to the light and mark the start time of the experiment. During the experiment, monitor the temperature inside the flask and, in case of overheating, cool the flask with water. It is especially important that at the end of the experiment the temperature is the same as at the beginning, otherwise air can enter
v flask or exit. The duration of the experiment should be such that the leaves had time to absorb no more than 25% of the containing
Xia in a CO2 flask. In good lighting for a 1 L flask, the exposure should not exceed 5 minutes, for larger flasks
- 15-20 minutes.
5. At the end of the experiment, remove the plant from the flask and quickly close it with stopper No. 1, marking the time. Also open the control flask for a few seconds. Pour 25 ml into flasks through the hole in the stopper
0.025N solution of Ba (OH) 2 and 2-3 drops of phenolphthalein and immediately close the hole with a stopper.
Table 8 |
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Intensity of photosynthesis | |||||||||
HCl consumption, ml | Intensive |
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infused | |||||||||
photosynthesis |
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dm2 | Wa (OH) 2, | for, mgСО2 / |
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6. To increase the surface of contact of Ba (OH) 2 with air, carefully moisten the walls of the flasks with this solution.
shake it periodically for 3 minutes, after which titration with 0.025N hydrochloric acid solution is carried out through the hole in the cork until the pink color disappears.
7. Determine the area of the sheet using the squares method. Results for
write to table 8. | ||||
The intensity of photosynthesis J f (ml CO2 / g | hour) is calculated by |
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(A B) K | ||||
where A is the amount of HCI used to titrate barite in a test flask, ml;
B - the amount of HCI used for titration of barite in the control flask, ml;
K - correction to HCI titer;
0.55 is the number of mg CO2 corresponding to 1 ml of 0.025H HC1; S — leaf area, dm2;
t - exposure, min;
60 - conversion factor from minutes to hours.
Materials and equipment: 1) leaves or shoots of plants; 2) 0.025N solution of Ba (OH) 2; 3) 0.025N HCI solution; 4) phenolphthalein; 5) conical flasks with a capacity of 1 l (2 pcs.); 6) paper; 7) rubber plugs (3 pcs.); 8) two stoppers with a hole closed with a glass stopper, a glass or metal rod with a small test tube and a thermometer attached to it is inserted into the third stopper; 9) a stand for installing the flask in an inverted position; 10) electric lamp 200-300 W; 11) scissors; 12) paper; 13) scales with weights.
Control questions
1. The cosmic role of green plants. The significance of the works of K.A. Timiryazev.
2. Pigments of photosynthetic plants. Methods for separating pigments.
3. Chemical and optical properties of pigments.
4. Physicochemical properties of the chlorophyll molecule. Chlorophyll fluorescence.
5. Light stage of photosynthesis. Photosynthetic phosphorylation.
6. The dark stage of photosynthesis. Calvin cycle, Hatch-Slack cycle, photosynthesis like tolstyanka.
7. Intensity of photosynthesis, photorespiration.
8. The influence of environmental factors on the intensity of photosynthesis
4. PLANT BREATHING
The history of the development of the doctrine of breathing. Oxidation and Reduction Theory: A.N. Bach, V.I. Palladin, G. Wieland, O. Warburg, S.P. Kostycheva et al. Classification of enzyme systems of respiration. Enzyme structure. The action of activators and inhibitors. Characterization of dehydrogenases, oxidoreductases, oxidases. Mechanisms of action of catalase, peroxidase, cytochrome oxidase and polyphenol oxidase.
The physiological role of respiration. The specificity of respiration in plants. Mitochondria. Their structure and function.
Ways of oxidation of organic matter in the cell. Unification of sub-
breath striations. The mechanism of activation of respiratory substrates, the ways of their inclusion in the processes of biological oxidation. The main ways of dissimilation of carbohydrates. Pentozomonophosphate pathway of glucose oxidation. Glycolytic oxidation pathway (glycolysis), main stages. G. Krebs cycle, the sequence of the reaction. Glyoxylate cycle.
The electron transport chain of mitochondria: structural organization, main components, their redox potentials. Electron carrier complexes. Alternative catalytic mechanisms of biological oxidation (cyanide-resistant respiration). Extramitochondrial oxidative systems.
Oxidative phosphorylation. Respiratory energy: phosphates and thioesters. The unity of elementary energy processes in living nature. Phosphorylation at the substrate level (substrate) and phosphorylation in the respiratory chain (coenzyme). Oxidative phosphorylation theories: chemical, mechanochemical (Boyer's theory), chemiosmotic (Mitchell's theory). The main provisions of Mitchell's chemiosmotic theory of conjugation. Membrane as a structural basis for bioenergetic processes. Transformation of energy on the conjugating membranes. Electrochemical potential is the driving force behind phosphorylation. Regulation of electron transport and phosphorylation. Dissociation of respiration and phosphorylation. The influence of environmental factors on this process.
Breathing as a central link in metabolism. The importance of respiration in the constructive metabolism of the cell and its connection with other functions of the cell.
Quantitative indicators of gas exchange (oxygen uptake, release of carbon dioxide, respiratory rate, etc.). L. Pasteur effect.
Respiration regulation. The ecology of respiration. The dependence of breathing on external and internal factors.
4.1. Gasometric determination of catalase
Many redox processes in plant tissues involve enzymes.
The method for determining the enzyme activity is based on the ability of catalase to decompose hydrogen peroxide with the release of oxygen gas. Since the amount of hydrogen peroxide decomposed depends on the activity of the enzyme, it is possible to judge the activity of catalase by the amount of oxygen and the rate of its release.
2H2 O2 → 2H2 O + O2.
Purpose of work: determination of the activity of the enzyme catalase in plant material.
Work progress: 1. Take a sample of leaves or plant parts weighing 4 g, add 0.2 g of chalk (to give an alkaline reaction), a pinch of sand and grind thoroughly in a mortar with a small amount of distilled water. Transfer the pounded mass through a funnel into a 100 ml volumetric flask and bring di-
with distilled water up to the mark. 2. A flask with vegetable ex-
leave to stand for 15 minutes. At this time, prepare all parts of the catalasimeter device (Fig. 13) to determine the activity of catalase and check its tightness.
3. After 15 minutes, take 10 ml of the extract together with the suspension from the flask using a measuring pipette and transfer it to one compartment of the reaction vessel (catalase). To another department with
Rice. 13. Catalasimeter the vessels place 5 ml of hydrogen peroxide. Reaction vessel
connect to the rest of the catalasimeter instrument.
Metal compounds with covalent bonds in aprotic solvents change their properties and dissociate, and then form complex compounds, for example: An interesting process of dissolution of TiCl4 in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). Solvent molecules interact with titanium ...Chlorophyll
(Processes of complexation of natural and technogenic origin)Chlorophyll biosynthesis
(Processes of complexation of natural and technogenic origin)(Processes of complexation of natural and technogenic origin)
Chlorophyll
The concept of chlorophyll comes from the Greek words (x ^ sorbs; - green and (poAXov- leaf). This is a green pigment of plants, with the help of which the absorption of sunlight and the process of photosynthesis occurs. Timiryazev was one of the first researchers to pay attention to chlorophyll. group of complex ...(Processes of complexation of natural and technogenic origin)
Chlorophyll biosynthesis
In nature, biosynthesis centers - polyenzyme complexes - are responsible for the biosynthesis of chlorophyll. The biosynthesis process is clear. At the last stage of biosynthesis in higher plants, light-colored protochlorophyllide is converted into chlorophyll under the influence of light. The process takes several ...(Processes of complexation of natural and technogenic origin)
Physicochemical properties of chlorophyll
Chlorophyll a has a high molecular weight of 893.52. At a temperature of 117-120 ° C, blue-black microcrystals of chlorophyll melt. Chlorophyll a dissolves in diethyl ether, ethanol, acetone, benzene, chloroform b pyridine. Its solutions are blue-green in color and highly fluorescent ....(Processes of complexation of natural and technogenic origin)
Target: familiarize with the procedure for performing work; make a conclusion about the chemical properties of sheet pigments.
Theoretical information. The chloroplast pigment system is represented by two types of pigments: green - chlorophylls a and b and yellow - carotenoids. The main functional pigment is chlorophyll a, serves as a direct donor of energy for photosynthetic reactions, the rest of the pigments only transfer the absorbed energy to it .
Progress:
Getting an alcohol solution (extract) of pigments. Pigments from plant tissue are extracted with polar solvents (ethyl alcohol, acetone), which destroy the bond of chlorophylls and xanthophylls with plastid lipoproteins and ensure their extraction. Dry leaves are placed in a 200 ml conical flask and scalded with boiling water, then the water is drained. 100 ml of ethanol is poured into the flask, closed with a cork stopper with a reflux condenser and placed in a boiling water bath to extract pigments. After boiling for five minutes, the contents of the flask are cooled and carefully poured into another flask. The extract is used in subsequent experiments.
Separation of pigments according to Kraus. The method is based on the different solubility of pigments in alcohol and gasoline. These solvents do not mix in one vessel, but form two phases - upper gasoline, lower alcohol, due to which the components of the mixture of pigments are separated.
2-3 ml of alcohol extract of pigments and 3-4 ml of gasoline are poured into a test tube. The contents of the test tube are shaken, closing it with a stopper or a large polish, and defended. As the emulsion stratifies, the gasoline layer turns green due to the better solubility of chlorophyll in it. Carotene also goes into gasoline, but its color is maximized by chlorophyll. Xanthophyll remains in the alcohol layer of golden yellow color.
If the pigments do not separate, add three to four drops of water and shake again. With an excess of water, the lower layer may become cloudy. In this case, add a little ethyl alcohol and shake the tube.
They draw a picture of the distribution of pigments and draw conclusions.
Saponification of chlorophyll with alkali. By treating chlorophyll with alkali, it is possible to cause saponification of ether groups, i.e. splitting off residues of methyl alcohol and phytol:
The resulting chlorophyllic acid salt retains the green color and optical properties of chlorophyll, but differs from it in greater hydrophilicity.
1 ml of a 20% NaOH solution is poured into a test tube with 2-3 ml of an alcohol solution of pigments and shaken. The test tube is placed in a boiling water bath. As soon as the solution boils, the tube is removed and cooled, then an equal volume of gasoline and a few drops of water are added. The contents of the test tube are shaken sharply and set aside. Carotene and xanthophyll pass into the gasoline layer, and the sodium salt of chlorophyllic acid passes into the alcohol layer. Sketch the color of the layers, indicating the distribution of the pigments.
Obtaining pheophytin and reverse replacement of hydrogen by a metal atom. The magnesium atom is relatively weakly retained in the porphyrin core of chlorophyll and, under careful action of strong acids, is easily replaced by two protons with the formation of brown pheophytin:
If pheophytin is acted upon by salts of copper, zinc or mercury, then instead of two protons, the corresponding metal enters the nucleus and the reaction products turn green. However, the resulting color is somewhat different from that of chlorophyll:
Consequently, the color of chlorophylls is due to the organometallic bond in their molecules. Reverse introduction of magnesium into pheophytin is very difficult. In two test tubes take 2-3 ml of alcohol extract of pigments and add one at a time - two drops of 10% hydrochloric acid solution. When shaken, the green color of chlorophyll turns into a brown one, characteristic of pheophytin. One tube with pheositin is left for control, and a few crystals of copper acetate are introduced into the second and the solution is heated in a water bath to boiling. As it warms, the brown color of the solution changes to green as a result of the formation of a chlorophyll-like copper derivative.
Sketch the color of pheophytin and a copper chlorophyll derivative.
Equipment: Dry or raw leaves, ethyl alcohol, gasoline , 20% NaOH solution, 10% hydrochloric acid solution in a dropper, copper acetate. Reflux conical flasks, water baths, test tube racks, 1 ml pipettes, conical cones, colored pencils.
Literature: 1, p. 63-66
Control questions:
1 What is the role of chlorophyll in the process of photosynthesis?
2 What is the role of carotenoids in the process of photosynthesis?
3 What is the mechanism for converting light energy into chemical energy?
