Chlorophyll determination

Protocol

Author

Adrienne Nicotra

Overview

Procedure for the extraction of pigments from leaf material for the calculation of chlorophylls a and b and carotenoid concentrations.

Materials/Equipment

  • 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
  • Ascorbate
  • Mortar and pestle
  • Test tube
  • Vortex
  • Eppendorf tubes
  • Centrifuge
  • Cuvettes
  • Spectrophotometer
  • Ice
  • Pipette

Units, terms, definitions

Blank
Spectrophotometer
Eppendorf tube
Cuvette
Chlorophyll

Procedure

  1. 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).
  2. Grind disks with a mortar and pestle and a couple of drops of the acetone solution.
  3. 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.
  4. Vortex briefly to homogenise, before transferring to 2x replicate eppendorf tubes (to later obtain 2 cuvette readings).
  5. Keep cold, on ice, throughout procedure.
  6. Spin for 1 min in eppendorf centrifuge (or 2 min to get a hard pellet).
  7. Pipette liquid to 2 cuvettes to measure absorbance.
  8. Prepare spectrophotometer for your recordings using a blank (cuvette with buffered acetone solution only).
  9. 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)

Chla = 13.7A(663.6-750) – 2.85A(646.6-750)Chlb = 22.39(646.6-750) – 5.42(663.6-750)

Chlsa + b = 19.54(646.6-750) + 8.29(663.6-750)

Equations for chlorophyll concentrations in µg/ml

Chla = 12.25A(663.6-750) – 2.55A(646.6-750)Chlb = 20.31(646.6-750) – 4.91(663.6-750)

Chlsa + b = 17.76(646.6-750) + 7.34(663.6-750)

Equation for the determination of carotenoids (Lichtenthaler 1987)

[1000A470-750 – 1.82Ca – 85.02Cb] / 198

Notes and troubleshooting tips

  • When freezing eppendorf tubes, pierce the lids so that they don’t open during the freezing process.

Literature references

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.