Protocol

Authors

Hendrik Poorter, Yvonne de Jong-Van Berkel

Author Affiliations

Hendrik Poorter IBG-2, Forschungszentrum Jülich

Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University

Overview

Chlorophyll plays an important role in the absorption of light energy of the plant. Chlorophyll can be extracted with a range of solvents. After extraction of the chlorophyll pigments with 80% acetone or 100% methanol, they can be measured in the spectrophotometer. When measured in the red light part of the spectrum there is no interference through absorbance by carotenoids.

Extractions

Materials/Equipment

Reagents

• 80% Acetone (cooled) (the remaining 20% is water)
• Methanol (cooled)
• Quartz sand

Equipment

• Centrifuge for large tubes, labtop
• Large centrifuge tubes (25 ml)
• Volumetric flasks (25 ml for MACRO, 10 ml for MICRO extraction)
• Box impenetrable to light
• Leaf area meter and / or balance
• Box with ice (facultative for MICRO extraction)
• Glass pipet (10 ml)
• Mortar and pestle (MACRO)
• Potter tissue grinder (MICRO)
• Razor blade/scissors (MACRO)
• Cork borer (diam. ± 1.0 cm, MICRO)

Procedure

Depending on the amount of plant tissue that has to be extracted, you can choose for the “MACRO” (> 150 mg fresh weight) or “MICRO” (leaf disk diam. ± 1.0 cm) extraction.

Macro extraction

1. Cool 80% acetone or methanol. The easiest way is to permanently store a bottle with acetone or methanol in a fridge.
2. Place a mortar and pestle on ice or in the freezer.
3. Cut leaf material in pieces to get a representative sample and determine weight (± 150 mg) and / or area (± 8 cm²) as fast as possible.
4. Put the material in the mortar that was placed on ice.
5. Keep the tubes and 80% acetone or methanol cooled on ice during the extractions.
6. The flasks also have to be kept on ice and in the dark (in the light impenetrable box).
7. Add to mortar a bit of quartz sand and a few ml of 80% acetone or methanol (use less than 10 ml).
8. Grind until you have a homogeneous suspension.
9. Put the suspension quantitatively in a large centrifugation tube.
10. Centrifuge for about 5 minutes at maximum speed (4500 rpm – 2649 g) and decant the supernatant in a volumetric flask of 25 ml.
11. Extract the pellet with 5 ml of 80% acetone or methanol and centrifuge again at 3000 rpm (1250g) for 5 minutes.
12. Repeat this procedure until the green colour of the pellet has gone (do not exceed 25 ml 80% acetone or methanol in total, otherwise your volumetric flask is too small).
13. When you have prepared all of your samples (it is prudent to have an extract measured within 1 hour after preparation) let the samples warm up by taking them out of the ice(but keep them in the dark).
14. At room temperature (± 20ºC) the supernatant in the volumetric flaks is made up to 25 ml with 80 % acetone or methanol.
15. Now the chlorophyll extract is ready to be measured in the spectrophotometer.

Micro extraction

1. Calculate the exact area of the cork borer (area circle = πr²) you are planning to use.
2. Pierce a leaf disk with help of the cork borer (place the leaf on a piece of rubber before piercing: otherwise the cork borer gets blunt).
3. The area is known; if you want, you can also weigh the disk (quickly).
4. Put the disk in the potter tissue grinder.
5. Add less than 5 ml 80% acetone or methanol.
6. Grind until you have a homogeneous suspension.
7. Put the suspension quantitatively in a centrifugation tube, use less than 10 ml in total of 80% acetone or methanol.
8. Centrifuge for about 5 minutes at maximum speed (4500 rpm – 2649 g) and decant the supernatant in a volumetric flask of 10 ml.
9. Make the supernatant in the volumetric flasks up to 10 ml with 80% acetone or methanol.
10. Put the volumetric flasks in the light impenetrable box.
11. Now the chlorophyll is ready to be measured.

Note: If you’re planning to do more samples than you can prepare within half an hour, put the volumetric flasks on ice in this box (it is prudent to have an extract measured within 1 hour after preparation). Note: The samples kept on ice should first warm up to room temperature (± 20 ºC) before measurement in the spectrophotometer otherwise condensation on your cuvette disturbs the measurements (take them out of the ice, but leave them in the box).

Notes and troubleshooting tips

• A few years ago, an extraction method with 80% acetone was commonly used. However Porra et al (1989) showed that the extraction of chlorophyll b with 80% acetone is not complete. In the calculations, chlorophyll b is underestimated, resulting in a higher chlorophyll a-b ratio (>4.00). With methanol, chlorophyll b is extracted completely, resulting in a better determination of the chlorophyll a:b ratio.
• All preparations should be done as cold, as quickly and as dark as possible. Chlorophyll disintegrates due to warmth and light.
• 150 mg of leaf material and a volume of 25 ml is used for plants grown under optimum circumstances.
• If you have a sample that has already been dissolved in acetone (because of prior measurements), you can use an 80% acetone chlorophyll measurement. You will have to look up the coefficients necessary for the calculation in Lichtenthaler, 1987 (not in Porra et al. 1989, because they used buffered 80% acetone).
• When you prepare 80% acetone, make sure that you do that in the right way: measure the deionized water and the 100% acetone first separately, than mix them together and cover it well (acetone evaporates fairly quickly).
• When using acetone never take plastic cuvettes.
• Acid in the extract can be neutralized by adding a little bit of chalk.
• Oxidation can be avoided by adding a bit of ascorbic acid.

Chemical Determination

Materials/Equipment

• Spectrophotometer (double beam)

Procedure

Determination of absorption 80% acetone extracts

1. The absorption of the extracts is measured with a spectrophotometer at the wavelengths 663, 646 and 710 nm.
2. Determine the zero absorption with 80% acetone at every wavelength.
3. The wavelength of 710 nm is used to determine the absorption of possible compounds that may interfere with the measurements of the chlorophyll.
4. For total chlorophyll content, the absorption at 652 nm is sufficient

Determination of absorption methanol extracts

1. The absorption of the extracts is measured with a spectrophotometer by taking the spectrum between the wavelenghts 750 nm and 640 nm.
2. Measure in 1 cm light path cuvettes (only when those cuvettes are used, you can calculate the chlorophyll concentration with the underneath formulas).
3. After you have taken the spectrum of a sample, search for the peak of chlorophyll a (see the options menu of your spectrophotometer) which should be at c. 665.2 nm.
4. Then subtract from this wavelength 13.2 nm to obtain the wavelength necessary to calculate chlorophyll b (somewhere around 652 nm).
5. Again use the options menu of your spectrophotometer to go to that peak wavelength so you can measure the absorption.

Calculations 80 % Acetone extracts

• Concentration in extract (mg l^-1)
  Chl_a = 12.21 * (A₆₆₃ – A₇₁₀) – 2.81 * (A₆₄₆ – A₇₁₀)
  Chl_b = 20.13 * (A₆₄₆ – A₇₁₀) – 5.03 * (A₆₆₃ – A₇₁₀)
  Chl_a+b = 7.18 * (A₆₆₃ – A₇₁₀) + 17.32 * (A₆₄₆ – A₇₁₀) or
  Chl_a+b = 27.80 * (A₆₅₂ – A₇₁₀)

• Concentration in the leaf:
  leaf = ((C_extract * Vol_extract) / DW_leaf)

Methanol extracts

• Concentration in extract (μmol l^-1)
  Chl_a = 18.22 * A_{peak Chla} – 9.55 * A_{peak Chla – 13.2nm}
  Chl_b = 33.78 * A_{peak Chla – 13.2 nm} – 14.96 * A_{peak Chla}
  Chl_a+b = 24.23 * A_{peak Chla + 13.2nm} + 3.26 * A_{peak Chla}
• Concentration in extract (mg l^-1)
  Chl_a = 16.29 * A_{peak Chla} – 8.54 * A_{peak Chla – 13.2nm}
  Chl_b = 30.66 * A_{peak Chla – 13.2 nm} – 13.58 * A_{peak Chla}
  Chl_a+b = 22.12 * A_{peak Chla – 13.2 nm} – 2.71 * A_{peak Chla}

Notes and troubleshooting tips

• All preparations should be done as cold, as quickly and as dark as possible. Chlorophyll disintegrates rapidly due to warmth and light.
• Never use ice-cold solutions for the measurements in the spectrophotometer, because condensate on the cuvette influences the measurement.
• Never use plastic cuvettes with acetone.
• All absorbance measurements at indicated wavelengths must have the absorbance at 750 nm subtracted.
• These coefficients are valid until 6 mg l^-1
• For extraction solutions other than 80% acetone or 100% methanol there are different calculations (Lichtenthaler and Wellburn, 1983)
• Chl_b disintegrates more rapidly than Chl_a. A high Chl_a:b ratio may indicate problems (but different methods may also yield different a:b ratios).
• Shade leaves usually have a higher chlorophyll content per unit total N than sun leaves

Reference values

• Chl_a+b: 200 – 800 μmol m²
• The Chl_a:b ratio is usually 2 – 3.5 in 80% acetone extracts and 3 – 4 in 100% methanol extracts

Literature references

Lichtenthaler, HK, 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. In: Methods in Enzymology, Pacher, L & Douce, A.(eds), Academic Press New York, 148: 350-382.

Lichtenthaler, H.K. & Wellburn, A.A., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans Ac. 603: 591-592.

MacKinney, G., 1941. Absorption of light by chlorophyll solutions. J. BioI. Chem. 140: 315-322.

Porra, R.J., Thompson, WA & Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BBA 975: 384-394.

Sesták, Z., 1966. Construction of a simple nomogram for evaluating two-length spectrophotometric determination of chlorophylls. Biol. Plant 8: 97-109.

Sesták, Z., Catsky, J. & Jarvis, P.G., 1971. Plant Photosynthetic Production, Manual of Methods, Den Haag.

Steubing, L, 1965. Pflanzenökologisches Praktikum, Methoden und Geräte zur Bestimmung wichtiger Standortsfaktoren, Verlag Parey, Berlin.