Photosynthetic Pigments Lab 2010
Absorption Spectra of Photosynthetic Pigments In today’s experiment you will use paper chromatography and spectrophotometry to separate and analyze the photosynthetic pigments found in plant leaves. Leaf pigments you will separate in this lab are broadly divided into two groups, chlorophylls and carotenoids. Chlorophylls are a class of photosynthetic porphyrin pigments that intercept and utilize light energy for the photosynthetic process. The chlorophylls are responsible for the green colour of most plants and are so basic to photosynthesis that photosynthetic rate is sometimes expressed on a ‘per unit chlorophyll’ basis rather than the more usual denominations (i.e., leaf area, tissue weight, or total protein). Carotenoids are a widespread group of highly unsaturated yellow, orange, red and brownish pigments. Their universal association with photosynthetic tissue suggests some function in this process (i.e., accessory light absorption, protective agents against photo-oxidation, etc.). Two kinds of carotenoids occur in cells: the yellow xanthophylls, which are carotenoids containing oxygen as hydroxyl or keto groups; and the orange-yellow carotenes, which contain no oxygen. Chromatography The word chromatograph comes from the Greek word “khromatokos”, meaning “colour of pigment” and the Greek word “grapho”, meaning “to write”. It used to mean “to write with colour”. In its current usage, chromatography refers to a variety of methods used for separating mixtures. In chromatography the separation is based mainly upon differential solubilities in different media or phases. There is often a stationary (non-mobile) phase, which is often a solid or a liquid attached to a solid phase. Another liquid with different solvent characteristics usually makes up the mobile phase. The sample then distributes itself between the two solvents. A sample with many solutes will have its solutes spread out or separated along the solid support based upon the solutes’ relative solubilities in the mobile and in the stationary phases. Paper Chromatography In paper chromatography, the paper is hydrophilic and water adheres to it. Water will rise up in the paper as the hydrophilic surface of the paper attracts the water molecules. This makes up the stationary phase through which the other solvents pass. Hydrophobic solutes in the mixture (in the case of this lab, a crude pigment extract) will be able to pass faster since they are not interacting with the immobilized hydrophilic phase. Hydrophilic solutes in the mixture will be slowed because they are more easily dissolved by the immobilized hydrophilic phase. The mixture is always applied at the origin, which is usually about 2 cm above the bottom of the paper. It is essential that the mixture not be immersed in the developing solvent at the start of the chromatography. The developing solution must climb up to the origin and sweep solutes from the mixture up from there.
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The solvent front is the highest migration of the solvent(s) and is represented by drawing a line in pencil on the paper. The solvent front is drawn immediately after the chromatogram is taken out of its tank and before the solvent(s) evaporate. In a proper chromatogram you will find that that the higher the solvent front, the greater the separation of solutes. The relative solubilities of pigments can be compared by calculating the Rf (Relative to the front) value. The Rf value is calculated as the distance from the origin (o) to the sample (s) divided by the distance from the origin (o) to the solvent front (f). Rf = (s – o) / (f – o) Spectrophotometry Spectrophotometry is a means of measuring the amount of light that a sample absorbs. In the study of photosynthetic pigments, this is important information since only light (energy) that is absorbed may be used to do work. If you examine the inside of a Spectronic-20, an example of a spectrophotometer, you will be able to identify the four main components of any spectrophotometer: the light source, the prism, the sample tube and the photo-multiplier tube. The light source produces white light. The white light passes through the prism which allows only a specific wavelength of light (i.e., a specific colour of light) to pass through. The light that passes through the prism is either absorbed or transmitted by the sample. The light that is transmitted through the sample reaches the photo-multiplier tube which accurately counts the rate of photons (discrete packets of light) that hit it.
With a spectrophotometer we can easily measure two things: the transmittance and absorbance of a sample. The scale on the Spectronic-20 going across in one direction is a linear scale for transmittance (%). Going in the other direction is the logarithmic scale of absorbance (O.D.). Transmittance (or a transmission spectrum) is a measure of the relative ability of photons of different colours to pass through (be transmitted through) a specific medium. It is easy to measure transmittance but it is rarely very useful to a physiologist. Percent (%) transmittance is defined as I (the light intensity received at the photomultiplier tube after passing through the sample) divided by Io (the original light intensity coming from the light source through the prism) then multiplied by 100%. % Transmittance = I/Io x 100%
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Absorbance is defined as the inverse log of transmittance, Log (Io/I) and, although absorbance is dimensionless, scientists often use arbitrary units called optical density (O.D.) units. The absorbance of a solution measured at a particular wavelength (λ) measured in nanometers (nm), using a 1 cm wide cuvette (sample tube), is written as Aλ. For example, if you found that absorbance of a solution is 0.52 O.D. using a wavelength of 663 nm, you would write A663 = 0.52 O.D. Absorbance is very useful to physiologists because it can be used to measure the concentration of chemicals that absorb light. It can be shown that Log (Io/I) varies with the concentration of chemical in the light beam of the spectrophotometer using the following equation: Absorbance = Log (Io/I) = Σ · concentration · cuvette width where sigma (Σ) is the extinction coefficient for a particular pigment (L · mol-1 · cm-1) exposed to the wavelength of light that is maximally absorbed by that pigment. Rearranging the equation we get: concentration = Log (Io/I) / (Σ · cuvette width) or concentration = Absorbance/(Σ · cuvette width)
Part 1: Separation of Pigments from Swiss Chard 1. The pigment extract (mixture) has been prepared for you by dissolving dried, powdered Swiss chard in acetone and filtering the extract. 2. Each group of four should run 4 chromatograms (1 per student) so that samples of the separated pigments can be pooled to provide enough pigment for subsequent spectrophotometery. Handle the chromatography paper by the edges. Fingerprints will interfere with the separation. 3. Using a pencil, draw a thin, faint line 2cm from one of the narrow ends of the chromatography paper. This will be the bottom of the chromatogram. 4. Elevate the lined end of the chromatography paper using a pencil or glass pipette. If the filter paper is lying flat on the desk the extract applied in the following step will smear. 5. Apply the extract in a narrow line, using the pencil line as a guide. Note that the sample should be applied as a very concentrated solution using a thin capillary tube to apply the mixture so that the sample does not flow away from the origin. The larger the spot made by the sample, the more difficult it will be to separate the pigments. Repeated applications of the sample with thorough drying between applications will produce the best chromatogram. Allow the extract on the filter paper to dry before proceeding. 6. With the lid tightly closed, swirl the solvent (9 parts petroleum ether to 1 part acetone) in the chromatography jar to allow the solvents to evaporate and saturate the air in the jar. 7. Working quickly as not to let the solvents dissipate into the surrounding air, open the jar, place the chromatogram in the jar and tightly seal the chromatogram in the jar. The paper must not touch the sides of the jar because the glass surface will interfere with the separation. Also, the origin must not immediately touch the solvent as this will cause the sample to dissolve in the Photosynthetic Pigments
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solvent and result in a continuous smear of colour on the chromatogram rather than discrete separations of the photosynthetic pigments. 8. Watch carefully as the solvents and pigments rise up the paper. In this solvent mixture the orange-yellow carotenes move the fastest followed by one or more bands of yellow xanthophylls. The green chlorophylls (a and b) move the slowest. 9. When the orange-yellow carotenes have nearly reached the top of the chromatogram (~1 cm from the edge) remove it from the jar. Using a pencil, immediately mark the line of the solvent front before the paper dries. 10. After the chromatograph has dried and before proceeding to the spectrophotometry step (where you will need to destroy your chromatograph) make sure that you take the necessary measurement to determine the Rf values for each of the separated pigments. You will need to measure: ▪ the distance from the origin (o) to the solvent front (f) ▪ the distance from the origin (o) to each of the four pigment samples (s) 11. Cut out the four major bands of pigment from your chromatograph. You can combine xanthophyll bands if more than one is visible. The dark grey band (pheophytin) that may appear between the top 2 bands should be discarded. Combine the bands from all four chromatograms created by your group members. 12. Clearly label a 50 mL Erlenmeyer flask for each of the four pigments. Place pieces of each band in the appropriate flask containing a few ml of 80% acetone. You will want to use minimal volumes of acetone to obtain as concentrated a pigment solution as possible (it can always be diluted later if too concentrated). Swirl the flasks to help extract the pigments from the paper. 13. Label the test tubes provided for each of the four pigments and pour the pigment solutions into the appropriate test tube.
Part 2: Running the Absorption Spectra The following procedure uses the Genesys 20 Spectrophotometer. 1. Plug in and turn on the spectrophotometer using the toggle switch at the back. The spectrophotometer must be allowed to warm-up before you start. 2. Select the wavelength for the spectrophotometric measurement. This is done with the wavelength-control buttons (labeled ‘nm’ with up and down arrows). The first wavelength measured will be 380 nm. For each subsequent reading you will increase the wavelength in 10 nm increments until you reach 700 nm. Each time you change the wavelength you must repeat ALL of the following steps. 3. Fill a sample tube with the reference ‘blank’ solution. For these experiments, the reference ‘blank’ solution is 80% acetone with no extract in it. You will need to fill the tube enough so that the light actually passes through the solution. 4. After wiping the tube with kleenex, insert it into the sample compartment. Light of the selected wavelength will pass through the solution in the tube and strike the photo-multiplier tube.
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5. Close the sample compartment (with the reference ‘blank’ inside) and zero the spectrophotometer using the 0% Abs/100% T button. 6. Remove the reference ‘blank’ and replace it with the crude Swiss chard extract. The crude extract has been prepared and diluted for you so all you need to do is place it in the spectrophotometer and read the absorbance. 7. Close the compartment, allow the reading to stabilize and record the absorbance of the sample in Table 1. 8. Remove the crude extract and place a tube containing one of the pigments in the spectrophotometer. Record the absorbance in the appropriate column in Table 1. Do the same for each of the remaining pigment tubes. You do not need to zero the spectrophotometer until you change the wavelength for the next series of readings. 9. Repeat steps 2-8 for each wavelength used.
References Raven, P.H., Evert, R.E., and Eichhorn, S.E. (1999) Biology of Plants 6th ed. W.H. Freeman and Co. New York. pg. 131. Shepanski, J.F., Williams, D.J., Kalisky, Y. (1984) The triplet exciton of chlorophyll a and carotenoids in solution and in photosynthetic antenna proteins. Biochimica et Biophysica Acta. 766: 116-12
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Table 1. Absorption spectra of the photosynthetic pigments fro Swiss Chard. Wavelength Absorbance (in Optical Density units) Colour Carotenes Xanthophylls Chlorophyll a Chlorophyll b (nm) 380 390 400 Violet 410 420 430 440 Blue 450 460 470 480 Blue-Green 490 500 510 520 530 Green 540 550 560 570 580 Yellow 590 600 610 620 Orange 630 640 650 660 670 Red 680 690 700
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The solvent front is the highest migration of the solvent(s) and is represented by drawing a line in pencil on the paper. The solvent front is drawn immediately after the chromatogram is taken out of its tank and before the solvent(s) evaporate. In a proper chromatogram you will find that that the higher the solvent front, the greater the separation of solutes. The relative solubilities of pigments can be compared by calculating the Rf (Relative to the front) value. The Rf value is calculated as the distance from the origin (o) to the sample (s) divided by the distance from the origin (o) to the solvent front (f). Rf = (s – o) / (f – o) Spectrophotometry Spectrophotometry is a means of measuring the amount of light that a sample absorbs. In the study of photosynthetic pigments, this is important information since only light (energy) that is absorbed may be used to do work. If you examine the inside of a Spectronic-20, an example of a spectrophotometer, you will be able to identify the four main components of any spectrophotometer: the light source, the prism, the sample tube and the photo-multiplier tube. The light source produces white light. The white light passes through the prism which allows only a specific wavelength of light (i.e., a specific colour of light) to pass through. The light that passes through the prism is either absorbed or transmitted by the sample. The light that is transmitted through the sample reaches the photo-multiplier tube which accurately counts the rate of photons (discrete packets of light) that hit it.
With a spectrophotometer we can easily measure two things: the transmittance and absorbance of a sample. The scale on the Spectronic-20 going across in one direction is a linear scale for transmittance (%). Going in the other direction is the logarithmic scale of absorbance (O.D.). Transmittance (or a transmission spectrum) is a measure of the relative ability of photons of different colours to pass through (be transmitted through) a specific medium. It is easy to measure transmittance but it is rarely very useful to a physiologist. Percent (%) transmittance is defined as I (the light intensity received at the photomultiplier tube after passing through the sample) divided by Io (the original light intensity coming from the light source through the prism) then multiplied by 100%. % Transmittance = I/Io x 100%
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Absorbance is defined as the inverse log of transmittance, Log (Io/I) and, although absorbance is dimensionless, scientists often use arbitrary units called optical density (O.D.) units. The absorbance of a solution measured at a particular wavelength (λ) measured in nanometers (nm), using a 1 cm wide cuvette (sample tube), is written as Aλ. For example, if you found that absorbance of a solution is 0.52 O.D. using a wavelength of 663 nm, you would write A663 = 0.52 O.D. Absorbance is very useful to physiologists because it can be used to measure the concentration of chemicals that absorb light. It can be shown that Log (Io/I) varies with the concentration of chemical in the light beam of the spectrophotometer using the following equation: Absorbance = Log (Io/I) = Σ · concentration · cuvette width where sigma (Σ) is the extinction coefficient for a particular pigment (L · mol-1 · cm-1) exposed to the wavelength of light that is maximally absorbed by that pigment. Rearranging the equation we get: concentration = Log (Io/I) / (Σ · cuvette width) or concentration = Absorbance/(Σ · cuvette width)
Part 1: Separation of Pigments from Swiss Chard 1. The pigment extract (mixture) has been prepared for you by dissolving dried, powdered Swiss chard in acetone and filtering the extract. 2. Each group of four should run 4 chromatograms (1 per student) so that samples of the separated pigments can be pooled to provide enough pigment for subsequent spectrophotometery. Handle the chromatography paper by the edges. Fingerprints will interfere with the separation. 3. Using a pencil, draw a thin, faint line 2cm from one of the narrow ends of the chromatography paper. This will be the bottom of the chromatogram. 4. Elevate the lined end of the chromatography paper using a pencil or glass pipette. If the filter paper is lying flat on the desk the extract applied in the following step will smear. 5. Apply the extract in a narrow line, using the pencil line as a guide. Note that the sample should be applied as a very concentrated solution using a thin capillary tube to apply the mixture so that the sample does not flow away from the origin. The larger the spot made by the sample, the more difficult it will be to separate the pigments. Repeated applications of the sample with thorough drying between applications will produce the best chromatogram. Allow the extract on the filter paper to dry before proceeding. 6. With the lid tightly closed, swirl the solvent (9 parts petroleum ether to 1 part acetone) in the chromatography jar to allow the solvents to evaporate and saturate the air in the jar. 7. Working quickly as not to let the solvents dissipate into the surrounding air, open the jar, place the chromatogram in the jar and tightly seal the chromatogram in the jar. The paper must not touch the sides of the jar because the glass surface will interfere with the separation. Also, the origin must not immediately touch the solvent as this will cause the sample to dissolve in the Photosynthetic Pigments
3
solvent and result in a continuous smear of colour on the chromatogram rather than discrete separations of the photosynthetic pigments. 8. Watch carefully as the solvents and pigments rise up the paper. In this solvent mixture the orange-yellow carotenes move the fastest followed by one or more bands of yellow xanthophylls. The green chlorophylls (a and b) move the slowest. 9. When the orange-yellow carotenes have nearly reached the top of the chromatogram (~1 cm from the edge) remove it from the jar. Using a pencil, immediately mark the line of the solvent front before the paper dries. 10. After the chromatograph has dried and before proceeding to the spectrophotometry step (where you will need to destroy your chromatograph) make sure that you take the necessary measurement to determine the Rf values for each of the separated pigments. You will need to measure: ▪ the distance from the origin (o) to the solvent front (f) ▪ the distance from the origin (o) to each of the four pigment samples (s) 11. Cut out the four major bands of pigment from your chromatograph. You can combine xanthophyll bands if more than one is visible. The dark grey band (pheophytin) that may appear between the top 2 bands should be discarded. Combine the bands from all four chromatograms created by your group members. 12. Clearly label a 50 mL Erlenmeyer flask for each of the four pigments. Place pieces of each band in the appropriate flask containing a few ml of 80% acetone. You will want to use minimal volumes of acetone to obtain as concentrated a pigment solution as possible (it can always be diluted later if too concentrated). Swirl the flasks to help extract the pigments from the paper. 13. Label the test tubes provided for each of the four pigments and pour the pigment solutions into the appropriate test tube.
Part 2: Running the Absorption Spectra The following procedure uses the Genesys 20 Spectrophotometer. 1. Plug in and turn on the spectrophotometer using the toggle switch at the back. The spectrophotometer must be allowed to warm-up before you start. 2. Select the wavelength for the spectrophotometric measurement. This is done with the wavelength-control buttons (labeled ‘nm’ with up and down arrows). The first wavelength measured will be 380 nm. For each subsequent reading you will increase the wavelength in 10 nm increments until you reach 700 nm. Each time you change the wavelength you must repeat ALL of the following steps. 3. Fill a sample tube with the reference ‘blank’ solution. For these experiments, the reference ‘blank’ solution is 80% acetone with no extract in it. You will need to fill the tube enough so that the light actually passes through the solution. 4. After wiping the tube with kleenex, insert it into the sample compartment. Light of the selected wavelength will pass through the solution in the tube and strike the photo-multiplier tube.
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5. Close the sample compartment (with the reference ‘blank’ inside) and zero the spectrophotometer using the 0% Abs/100% T button. 6. Remove the reference ‘blank’ and replace it with the crude Swiss chard extract. The crude extract has been prepared and diluted for you so all you need to do is place it in the spectrophotometer and read the absorbance. 7. Close the compartment, allow the reading to stabilize and record the absorbance of the sample in Table 1. 8. Remove the crude extract and place a tube containing one of the pigments in the spectrophotometer. Record the absorbance in the appropriate column in Table 1. Do the same for each of the remaining pigment tubes. You do not need to zero the spectrophotometer until you change the wavelength for the next series of readings. 9. Repeat steps 2-8 for each wavelength used.
References Raven, P.H., Evert, R.E., and Eichhorn, S.E. (1999) Biology of Plants 6th ed. W.H. Freeman and Co. New York. pg. 131. Shepanski, J.F., Williams, D.J., Kalisky, Y. (1984) The triplet exciton of chlorophyll a and carotenoids in solution and in photosynthetic antenna proteins. Biochimica et Biophysica Acta. 766: 116-12
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Table 1. Absorption spectra of the photosynthetic pigments fro Swiss Chard. Wavelength Absorbance (in Optical Density units) Colour Carotenes Xanthophylls Chlorophyll a Chlorophyll b (nm) 380 390 400 Violet 410 420 430 440 Blue 450 460 470 480 Blue-Green 490 500 510 520 530 Green 540 550 560 570 580 Yellow 590 600 610 620 Orange 630 640 650 660 670 Red 680 690 700
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