Rationale

Chlorophylls possess a phytyl chain which is esterified to the carboxyl group of a porphyfin ring system, giving the chlorophylls their lipid character. Therefore, chlorophylls can be separated and extracted from water-containing living plant tissue by organic solvents such as acetone, methanol, or ethanol.

Different pigment species have their own unique absorbance spectrum. By measuring absorbance values at different wavelength, we can quantify the amount of pigments using equations established by previous studies.

<aside> ❄️ In all times, the original sample must be preserved at –80°C or colder. When you are handling the samples, put them in dry ice or liquid nitrogen. Whey they are not in use, store them in the –80°C right away.

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Preparation

<aside> 💡 Always prepare enough reagents for extra 10 samples.

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• [ ] For each lyophilised sample, prepare the extraction buffer as follows and store at 4°C:
  • [ ] 4.8 ml acetone
  • [ ] 1.2 ml Tris-Cl solution (0.25 M, pH 7.8), or
    • [ ] 0.3 ml Tris-Cl solution (1M)
    • [ ] 0.9 ml water
• [ ] Grind the frozen samples in a 2 ml Eppendorf tube with a steel bead with a TissueLyzer at 20/s for 1 minute.

Extraction

<aside> ❄️ Set the benchtop centrifuge to 4°C. You can usually proceed with 24 samples as one batch.

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• [ ] On ice, away from light: add ~1 ml cold acetone/Tris extraction buffer to each sample and vortex vigorously. Store at –80°C for maximum 3 days.
• [ ] Quickly spin down the buffer on the lid. Transfer all content to a 15 ml Falcon tube. You may use more cold acetone/Tris buffer to wash the Eppendorf tube and collect in the Falcon tube.
• [ ] On ice, away from light: top up the sample to 6 ml in the Falcon tube.

<aside> ⚠️ For light-coloured sample, use 3 ml instead of 6 ml.

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• [ ] Centrifuge the Falcon tubes at 3,200 g at 4°C ****for 5 min.

Quantification

• [ ] Use the acetone/Tris extraction buffer as a blank.