This protocol was contributed by Robin Martin in conjunction with the Carnegie Institution for Science. Please visit the Spectranomics website at http://spectranomics.stanford.edu/ for more information and spectranomics tools.
- PerkinElmer Lambda 25 UV-Vis Spectrophotometer
- Talboys High Throughput Homogenizer
- Sartorius Balance (0.0001 g)
- 2 ml centrifuge tubes with threaded, rubber sealed O-ring caps
- 15 ml centrifuge tubes
- Chilled acetone
- Magnesium carbonate (MgCO3)
- 5/8" threaded lugs ¼" width, stainless steel and small cone bearings
- Chlorophyll A Standard stock in 100% Acetone
Units, terms, definitions
- Weigh two leaf disks (total area = 0.7693 cm2) while keeping other samples cold on dry ice or in liquid nitrogen, with minimum light exposure.
- Place the leaf disks in 2 ml centrifuge tubes prepared with 0.05 mg MgCO3.
- Add 0.75 ml acetone and ball bearings and lugs to each, cap and homogenize for 2 minutes at high intensity.
- Add 0.75 ml acetone, shake each sample to homogenize then centrifuge 1-2 minutes.
- Aliquot exactly 0.75 ml extract into 15 ml centrifuge tube containing 3.0 ml of acetone; centrifuge 3 minutes.
- Run at least one blank and one chlorophyll (a or b) standard measurement to check the instrument.
- Using and autosampler or quartz cuvette, measure absorbance of the solution at 470, 662, 645, and 710 nm as part of a full scan of the sample (400-800 nm; 960 nm min-1 scan speed) using a Perkin Elmer Lambda 25 spectrophotometer.
- If samples have too much sediment (i.e. absorbance reading at 710 nm > 0.05), centrifuge and re-run those samples.
Data preparation and finalization
- From the measured absorptance, calculate chlorophyll A, B and bulk carotenoid concentrations on a weight (mg g-1) and area (μg cm-2) basis following the equations of Litchenthaler, H.K. and Buschmann, C. (2001) Current protocols in Food and Analytical Chemistry F4.3.8-F4.2.4 and Lichtenthaler (1987) Chlorophylls and carotenoids – pigments of photosynthetic biomembranes. In Methods in Enzymology Eds. SP Colowick and NO Kaplan) pp350-382. (Academic Press: Sydney):
Chl A concentration (μg ml-1) = [11.24*(A662 – A710) – 2.04*(A645-A710)]*dilution factor
Chl B concentration (μg ml-1) = [20.13*(A645-A710) – 4.19*(A662-A710)]*dilution factor
Bulk Carotenoids (μg ml-1) = [(1000*(A470-A710) – 1.90*Chl Aconc – 63.14*Chl Bconc)/214]*dilution factor
Area basis (μg cm-2) – Concentration (μg ml-1) * Extract Volume (ml) / Disk Area (cm2)
Dry weight basis (mg g-1 dry leaf) – Concentration (μg ml-1) * Extract Volume (ml) / (Disk dry weight (g)*1000)
Disk dry weight = [Disk wet weight – (disk wet weight *%H2O from total leaf)]; H2O is from bulk leaf sample measured during the field collection.
Notes and troubleshooting tips
Links to resources and suppliers
Litchenthaler, H.K. and Buschmann, C. (2001) Current protocols in Food and Analytical Chemistry F4.3.8-F4.2.4
Lichtenthaler (1987) Chlorophylls and carotenoids – pigments of photosynthetic biomembranes. In Methods in Enzymology Eds. SP Colowick and NO Kaplan) pp350-382. (Academic Press: Sydney)
Health, safety & hazardous waste disposal considerations