Leaf Chlorophyll and Carotenoid Determinations

Please note

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.

Overview

Background

Materials/Equipment

Equipment

PerkinElmer Lambda 25 UV-Vis Spectrophotometer
Talboys High Throughput Homogenizer
Centrifuge
Sartorius Balance (0.0001 g)

Consumable materials

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

Procedure

Sample preparation

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.

Measurement procedure

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):

Equations:

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 A_conc – 63.14*Chl B_conc)/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.

Other resources

Notes and troubleshooting tips

Links to resources and suppliers

Literature references

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)