D663=82.04Ca9.27Cb........................(1)
D645=16.75Ca45.6Cb........................(2)
In the formula, D663 and D664 respectively measure the optical density of chlorophyll solution at wavelengths of 663 nm and 645 nm; Ca and Cb are the concentrations of chlorophyll a and b, respectively (g/L); 82.04 and 9.27 are respectively chlorophyll a and b at a wavelength of 663 nm. The specific absorption coefficients, 16.75 and 46.6, are the specific absorption coefficients of chlorophyll a and b at a wavelength of 645 nm, respectively.
Solving the simultaneous equations (1) and (2) gives:
Ca=0.0127D663-0.00269D645...............(3)
Cb=0.0229D645-0.00468D663............(4)
For example, if the unit of chlorophyll content is changed from g/L to mg/L, (3) and (4) can be rewritten as:
Ca(mg/L)=12.7D663-2.69D645............(5)
Cb(mg/L)=22.9D645-4.68D663............(6)
Total chlorophyll C (mg/L) = CaCb = 20.2D6458.02D663......(7)
The total amount of chlorophyll can also be derived according to the following formula: D652=34.5×C...............(8)
652 nm is the intersection of the absorption spectrum curves of chlorophyll a and b in the red region (equal absorption point), and 34.5 is the absorption coefficient of chlorophyll a and b at a wavelength of 652 nm.
DC=D652/34.5(g/L)Cd
DC=D652×1000/34.5(mg/L)............(9)
Therefore, the chlorophyll a and b contents can be calculated by using the formulas (5) and (6), respectively, and the total amount of chlorophyll can be calculated by the formula (7) or (9).
The chloroplast pigment extract contains lutein and carotene in addition to chlorophyll a and b. Since the absorption spectra of lutein and carotene are in the blue region, the spectrophotometric determination of chlorophyll content in the red region is not disturbed.
Equipment and reagents 722 spectrophotometer, equipment required for chloroplast pigment extraction.
Method and Step 1. Extract chlorophyll.
2. Determine the optical density.
In a 1 cm cuvette, inject the chlorophyll extract (diluted when the concentration is large). A chlorophyll extraction medium was also injected as a reference solution into the same cuvette. According to the experimental requirements, the corresponding wavelength is selected (the total amount is 652 nm, the chlorophyll a, b content and the total amount are 663 nm and 645 nm), and the optical density is measured on a spectrophotometer.
3. Calculation results: The chlorophyll content in the cuvette can be determined by substituting the measured optical density into the above formula. To calculate the chlorophyll content of a sample, the dilution factor needs to be considered.
Chlorophyll content in the sample (mgChl/g fresh weight or mgChl/ml chloroplast)
D = chlorophyll content in the cuvette × dilution factor / sample size (g fresh weight or ml chloroplast)
Precautions When measuring the substance content by spectrophotometry, the wavelength adjustment of the spectrophotometer should be accurate, and the transmittance of the reference cup and the measuring cup should be the same.
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