Experiments for the quantitative determination of chlorophyll
According to the absorption spectrum of chlorophyll on visible light, the use of spectrophotometer at a particular wavelength to determine its optical density, and then calculated by the formula of the chlorophyll content, this method is highly accurate, and can be measured in the absence of separation of chlorophyll a, b content, respectively.
Operation method
Experiments for the quantitative determination of chlorophyll
Principle
According to the absorption spectrum of chlorophyll on visible light, the use of spectrophotometer at a particular wavelength to determine its optical density, and then calculated by the formula of the chlorophyll content, this method is highly accurate, and can be measured in the absence of separation of chlorophyll a, b content, respectively. According to Lambert-Beer law, the optical density D of a colored solution is directly proportional to its concentration C and the thickness of the liquid layer L, i.e.: D = kCL in which k is a constant of proportionality, when the concentration of the solution in units of weight percent concentration, the thickness of the liquid layer of 1 cm, k is known as the specific absorption coefficient of the substance. If there are several light-absorbing substances in a solution, the total optical density of this solution at a certain wavelength is equal to the sum of the optical densities of the components at the corresponding wavelengths, which is the additivity of the optical density. Chlorophyll a, b in the red light region of the maximum absorption peak is located in 663 nm and 645 nm, respectively, at a wavelength of 663 nm, chlorophyll a, b of 80% acetone solution of the specific absorption coefficient of 82.04 and 9.27, respectively, at a wavelength of 645 nm, respectively, 16.75 and 45.6, according to which the following equation can be set out: the formula of the D663 and D645 for the chlorophyll solution at a wavelength of 663 nm and 645 nm. D663 and D645 are the extinction of 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/liter. The total amount of chlorophyll CT is obtained by adding Ca and Cb. In addition, since chlorophyll a and b have the same specific absorption coefficient at 652 nm (both 34.5), the total amount of chlorophyll a and b can also be determined by measuring the extinction once at this wavelength (D652):
Materials and Instruments
Fresh leaves acetone mortar funnel balance scissors beaker filter paper measuring cylinder spectrophotometer Move Weigh 0.5 g of fresh leaves, cut into pieces and place in a mortar, add a small amount of calcium carbonate and quartz sand, and add 2-3 ml of 80% acetone, research into a homogenate, then add 10 ml of 80% acetone, and continue to grind sufficiently, filtered with a layer of acetone moistened filter paper, and then a small amount of acetone will be filtered and rinsed clean of the pigment on the filter paper and the mantle, and then fixed to 25 ml volumetric flasks, and then shaken well. For more product details, please visit Aladdin Scientific website.
Pipette 2 ml of chlorophyll acetone extract, add 80% acetone 2 ml diluted, poured into a 1 cm colorimetric cup, using a spectrophotometer to determine the optical density at wavelengths of 645 nm, 663 nm and 652 nm, respectively, with 80% acetone as a blank control.
The chlorophyll a and b concentrations and the total concentration (mg/L) were calculated according to equations (3), (4) and (6), respectively.
The content of chlorophyll in the leaves was calculated according to the following formula: 