Plant chloroplast pigment content directly affects plant photosynthesis and crop yield, and is also closely related to the supply of plant water and mineral nutrients, adversity conditions and plant senescence. Chloroplast content is also an important physiological indicator reflecting the growth and development of leaves. This experiment is to learn and master the quantitative determination of chloroplast pigments.
Principle
The basic principle of quantitative determination of chloroplast pigments is based on the absorption of chloroplast pigment extracts on the visible spectrum, the use of spectrophotometer at a particular wavelength to determine its extinction, can be calculated using the formula for the content of each pigment in the extract.
According to the Lambert-Beer law, the absorbance A of a colored solution is proportional to the concentration of solute c in it and the thickness of the liquid layer L, i.e.:
A = kcL (14-1)
Where: A an absorbance;
c - solute concentration;
L-thickness of the liquid layer;
k a proportionality constant.
When the concentration of the solution in percent concentration units, the thickness of the liquid layer is 1 cm, the specific absorption coefficient of the substance. The specific absorption coefficients of various colored substance solutions at different wavelengths can be obtained by determining the extinction of pure substances of known concentration at different wavelengths.
If there are several light-absorbing substances in the solution, the total extinction of the mixture at a certain wavelength is equal to the sum of the extinction of each component at the corresponding wavelength, which is the summation of extinction. Determination of chlorophyll a, chlorophyll b and carotenoids in the chloroplast pigment mixture extract content, just determine the extract at three specific wavelengths of absorbance A, and according to chlorophyll a, chlorophyll b and carotenoids at the wavelength of the specific absorption coefficient can be derived from its concentration. In the determination of chlorophyll a, chlorophyll b, in order to exclude the interference of carotenoids, the wavelength of the monochromatic light used to select the maximum absorption peak of chlorophyll in the red light region.
Known chlorophyll a, chlorophyll b of 80% acetone extract in the red region of the maximum absorption peak were 663 nm and 645 nm, and know that at a wavelength of 663 nm, chlorophyll a, chlorophyll b in the solution than the absorption coefficient were 82.04 and 9.27, respectively, at a wavelength of 645 nm, respectively, 16.75 and 45.60, according to the principle of additivity to exude the following equation A663 = 82.04 ca
A663 = 82.04 ca + 9.27 cb ( 14-2)
A645 = 16.75 ca + 45.60 cb (14-3)
Where: A663, A645 - chlorophyll solution in the wavelength of 663 nm and 645 nm extinction;
ca, cb a chlorophyll a and chlorophyll b concentration, mg・L-1.
Solve the system of equations (14-2) and (14-3) to obtain:
ca = 12.72 A663 - 2.59 A645 (14-4)
cb = 22.88 A645 - 4.67 A663 (14-5)
Total chlorophyll cT is obtained by adding ca to cb
cT = ca + cb = 20.29 A645 + 8.05 A663 (14-6)
In addition, because the absorption peaks of chlorophyll a and chlorophyll b at 652 nm are intersected, and they have the same specific absorption coefficient (both 34.5), it is also possible to determine the total amount of chlorophyll a and chlorophyll b by measuring the extinction degree ( A652 ) at this wavelength:
cT = ( A652 × 1 000)/34.5 (14-7)
In the presence of chlorophyll conditions, the spectrophotometric method can be simultaneously determined in the solution of carotenoids content. li-chten-thaler et al. on the Arnon method for the correction, proposed 80% acetone extract in the content of the three pigments of the calculation formula:
ca = 12.21 A663 - 2.81 A646 (14-8)
cb = 20.13 A646 - 5.03 A663 (14-9)
cx. c = (1 000 A470 - 3.27 ca - 104 cb )/229 (14-10)
Where: ca, cb-Concentration of chlorophyll a, b;
cx. c-total concentration of one class of carotenoids;
A663, A645 and A470 - extinction of chloroplast pigment extracts at 663 nm, 646 nm and 470 nm. Since the absorption spectra of chloroplast pigments differ in different solvents, the formulae are different when using other solvents for pigment extraction. The wavelengths of the maximum absorption peaks of chlorophyll a and chlorophyll b in 95% ethanol are 665 nm and 649 nm, respectively, and that of carotenoids is 470 nm, according to which the following equation can be listed:
ca = 13.95 A665 - 6.88 A649 (14-11)
cb = 24.96 A649 - 7.32 A665 (14-12)
cx. c = (1 000 A470 - 2.05 ca - 114.8 cb )/245 (14-13)
Operation method
extraction by means of a mixture of liquids
Principle
According to the absorption of chloroplast pigment extracts on the visible spectrum, using a spectrophotometer at a particular wavelength to determine its optical density, you can use the formula to calculate the content of each pigment in the extract. According to Lambert-Beer law, the optical density D of a colored solution is directly proportional to the solute concentration C and the thickness L of the liquid layer, i.e., D = kCL where: k is a constant of proportionality. When the concentration of the solution in percent concentration units, the thickness of the liquid layer is 1cm, k is the specific absorption coefficient of the substance. The specific absorption coefficients of various colored substance solutions at different wavelengths can be obtained by determining the optical densities of known concentrations of pure substances at different wavelengths. If there are several light-absorbing substances in the solution, the total optical density of this mixture at a certain wavelength is equal to the sum of the optical densities of the components at the corresponding wavelengths, which is the summation of optical density. Determination of chlorophyll a, b and carotenoids in the chloroplast pigment mixture extract content, only need to determine the extract at three specific wavelengths of optical density D, and according to the chlorophyll a, b and carotenoids at this wavelength than the absorption coefficient can be derived from its concentration. In the determination of chlorophyll a, b in order to exclude the interference of carotenoids, the wavelength of the monochromatic light used to select the maximum absorption peak of chlorophyll in the red light region. Known chlorophyll a, b of 80% acetone extract in the red region of the maximum absorption peak for 663nm and 645nm, respectively, and know that at a wavelength of 663nm, chlorophyll a, b in the solution than the absorption coefficient of 82.04 and 9.27, respectively, at a wavelength of 645nm, respectively, 16.75 and 45.60, according to the principle of additivity, listed in the following equation: D663 = 82.04Ca + 9.27Cb (1) D645 = 16.75Ca + 45.60Cb (2) D663 and D645 in equations (1) and (2) are the optical densities of the chlorophyll solution at wavelengths of 663 nm and 645 nm, and Ca and Cb are the concentrations of chlorophylls a and b respectively, in mg/L. Solve the system of equations (1), (2), get: Ca = 12.72D663 - 2.59D645 (3) Cb = 22.88D645 - 4.67D663 (4) Ca and Cb will be added to obtain the total amount of chlorophyll (CT): CT = Ca + Cb = 20.29D645 + 8.05 D663 (5) In addition, due to the intersection of chlorophyll a and b absorption peaks at 652 nm, the two have the same specific absorption coefficient (both 34.5), can also be measured at this wavelength once the optical density (D652) and find out the total amount of chlorophyll a and b: CT = (D652 × 1000) / 34.5 (6) in the presence of chlorophyll under the conditions of spectrophotometric determination of carotenoids in the solution can be determined simultaneously Lichtenthaler et al. modified Arnon's method and proposed a formula for calculating the contents of the three pigments in an 80% acetone extract: Ca = 12.21D663 - 2.81D646 (7) Cb = 20.13D646 - 5.03 D663 (8) Cx-c = (1000D470 - 3.27Ca - 104Cb)/ 229 (9) Where: Ca and Cb are the concentrations of chlorophylls a and b, respectively; Cx-c is the total concentration of carotenoids; D663, D646 and D470 are the concentrations of chlorophyll pigment extracts at wavelengths of 663 nm, 646 nm and 470 nm, respectively. Since the absorption spectra of chloroplast pigments differ in different solvents, the formulae are different when using other solvents for pigment extraction. The wavelengths of the maximum absorption peaks of chlorophylls a and b in 96% ethanol are 665 nm and 649 nm, respectively, and 470 nm for the carotenoids, from which the following relation can be made: Ca = 13.95D665 - 6.88D649 (10) Cb = 24.96D649 - 7.32D665 ( 11) Cx-c = (1000D470 - 2.05Ca - 114.8Cb) / 245 (12)
Materials and Instruments
Plant leaves Move 1. Take fresh plant leaves (or other green tissues) or dry materials, wipe off the surface dirt, cut (remove the midrib), and mix well. 2. Weigh 0.2g of the freshly cut sample, a total of 3 portions, respectively, into the mortar, add a small amount of quartz sand and calcium carbonate powder and 2 - 3ml 96% ethanol (or 80% acetone) into a homogeneous pulp, then add 10ml of ethanol, continue to grind until the tissue becomes white, and leave it to stand for 3 - 5min. -5min. 3. 3. Take 1 piece of filter paper, placed in a funnel, moistened with ethanol, along the glass rod to pour the extract into the funnel, filtered into a 25 ml brown volumetric flask, with a small amount of ethanol to rinse the mantle, rod and residue for several times, and finally together with the residue poured into the funnel. 4. Pipette the ethanol and wash all the chloroplast pigments on the filter paper into the volumetric flask. Until there is no green color in the filter paper and residue. Finally, the volume was fixed with ethanol to 25 ml and shaken well. 5. Pour the chloroplast pigment extract into a colorimetric cup. The optical density was measured at wavelengths 665 nm, 649 nm and 470 nm using 96% ethanol as blank. 6. Calculate the concentration of chlorophyll a, b and carotenoids (mg/L) according to equations (10), (11), (12) (if 80% acetone is used, then according to equations 7, 8, 9), respectively, (10), (11) formula is summed up to obtain the total chlorophyll concentration. 7. Calculate the content of each pigment in the tissue after finding the concentration of the pigment. Caveat 1. In order to avoid photodecomposition of chlorophyll, the operation should be carried out in low light and the grinding time should be as short as possible. 2. The chloroplast pigment extract should not be turbid. Optical density can be measured at 710nm or 750nm wavelength, the value should be less than when the wavelength of chlorophyll a absorption peak time density value of 5%, otherwise it should be re-filtered. 3. Determination of chlorophyll content by spectrophotometer method, the wavelength accuracy of the spectrophotometer requires a high degree of accuracy. If the wavelength and the original absorption peak wavelength difference of 1nm, the determination of chlorophyll a error of 2%, chlorophyll b for 19%, the use of the spectrophotometer must be corrected before the wavelength. Calibration method in addition to the instrument instructions, but also should be pure chlorophyll a and b to correct. 4. In the use of low-grade models of spectrophotometer (such as: 72, 125, 721, etc.) to determine the chlorophyll a, b content, due to the instrument's slit is wider, the spectral performance is poor, the purity of monochromatic light is low (± 5 - 7nm), with high-grade instruments such as Shimadzu UV-120, UV-240, etc. Compared with the results of the determination, the measured value of chlorophyll a bias. Chlorophyll a value is low, chlorophyll b value is high, a/b ratio is seriously small. Therefore, it is necessary to use a high-grade spectrophotometer to correct the low-grade spectrophotometer. Common Problems Grinding should be done in low light and the grinding time should be as short as possible. The extract should not be cloudy, otherwise it should be re-filtered. For more product details, please visit Aladdin Scientific website.
96% ethanol Quartz sand Calcium carbonate powder
Spectrophotometer Mantle Scissors Brown volumetric flask Small funnel Dropper Electronic top-loading balance