Procedure for the extraction of pigments from leaf material for the calculation of chlorophylls a and b and carotenoid concentrations.
- Leaf disks, 6 per plant – collected and stored in liquid N2 until extraction takes place (or frozen in liquid N2 and stored in deep freeze)
- 80% acetone
- Mortar and pestle
- Test tube
- Eppendorf tubes
Units, terms, definitions
- Extract pigments from leaf disks in 5 ml 80% acetone (400 ml acetone + 100 ml distilled water) + a tiny bit of ascorbate (a pinch per litre) (buffered acetone).
- Grind disks with a mortar and pestle and a couple of drops of the acetone solution.
- Pour the ground material into a test tube, rinsing with buffered acetone to transfer all leaf material and to make up the mixture to 5 ml.
- Vortex briefly to homogenise, before transferring to 2x replicate eppendorf tubes (to later obtain 2 cuvette readings).
- Keep cold, on ice, throughout procedure.
- Spin for 1 min in eppendorf centrifuge (or 2 min to get a hard pellet).
- Pipette liquid to 2 cuvettes to measure absorbance.
- Prepare spectrophotometer for your recordings using a blank (cuvette with buffered acetone solution only).
- Read absorbance at 750, 663.6 and 646.6 nanometers (replicates should be similar and not vary by >10%).
Note: absorbance at 750 nm should be close to zero, and the value at 663.6 nm should be ~2x greater than the value at 646.6 nm.
Equations for chlorophyll concentrations in nmol/ml (Porra et al. 1989)
Chlsa + b = 19.54(646.6-750) + 8.29(663.6-750)
Equations for chlorophyll concentrations in µg/ml
Chlsa + b = 17.76(646.6-750) + 7.34(663.6-750)
Equation for the determination of carotenoids (Lichtenthaler 1987)
Notes and troubleshooting tips
- When freezing eppendorf tubes, pierce the lids so that they don’t open during the freezing process.
Lichtenthaler, H.K. (1987) Chlorophylls and carotenoids: pigments of the photosynthetic biomembranes. Methods in Enzymology 148: 350-382.
Porra, R.J., W.A. Thomson and P.E. Kriedemann (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted wth four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. ”Biochimica et Biophysica Acta 975: 384-394.
Health, safety & hazardous waste disposal considerations
- Take care when handling liquid nitrogen and DO NOT inhale or gt it in contact with your skin.