Hello Respected
I am trying to measure the total chlorophyll, but I am unable to calculate its amount.
I am following the papers written by (LICHTENTHALER and WELLBURN, 1983) and (Arnon, 1949) but those equations are not clear to me.
Please help me, with how to use the mentioned papers equations to estimate chlorophyll.
Regards
Procedure
1. 0.2 g of fresh leaf sample (FW) was homogenized in 2 mL of 80% acetone
2. After homogenization, centrifuge the solution at 11,500 × g for 12 min and separate the supernatant in another tube.
3. The absorbance of the collected supernatant was measured at 663 nm, 645 nm and 470 nm
4. Use 80% acetone solution as a blank
Calculation
Chl a(mg g−1 FW) = (0.0127 × D663 – 0.00269 × D645) × V (2 mL)/W (0.2 g)
Chl b(mg g−1 FW) = (0.0229 × D645 – 0.00468 × D663) × V (2 mL)/W (0.2 g)
Total Chls (mg g−1 FW) = (20.2 × D645 + 8.02 × D663) × V (2 mL)/ [1000 × W (0.2 g)]
Carotenoids (mg g−1 FW) = ((1000 × A470 – 2.270 × Chl a – 81.4 × Chl b)/227)) × V (2 mL)/ [1000 × W (0.2 g)]
where, D663, D645 and D470 denote absorbance reading, V is the used volume of 80% (v/v) acetone (mL) for homogenization, and W is the FW of leaf samples (g).
References
1. Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology, 24(1), 1.
2. Lichtenthaler, H. K. & Wellburn, A. R. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592 (1983).
This method is applicable to the determination of chlorophyll a in surface waters. 2. PRINCIPLE AND THEORY The sample is filtered and the algae are collected on a filter and extracted in 90% acetone. The concentration of chlorophyll a is determined by measuring the fluorescence emitted at a wavelength > 665 nm, following excitation at a wavelength of 340 - 500 nm. The wavelength may vary slightly depending on the model of fluorometer used.
Dear Uzzal
Have a Good Day!
Will you please share with us the reference in which they talked about the 0.2g weight of leaves because whatever the paper you have mentioned as a reference does not have that information?
Regards
Harsh
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