Plant Physiology

Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu

Measuring Chlorophyll (& Anthocyanin) Concentration

Background Information:
Chlorophyll can easily be quantified with a spectrophotometer based on the Beer-Lambert Law and the extinction coefficient for chlorophyll. In a classic paper, Arnon (1949) reported the following equations for quantification of the total chlorophyll, chlorophyll a and chlorophyll b content in an 80% acetone extract:

Total chlorophyll (�g/ml) = 20.2 (A₆₄₅) + 8.02 (A₆₆₃)
Chlorophyll a (�g/ml) = 12.7 (A₆₆₃) - 2.69 (A₆₄₅)
Chlorophyll b (�g/ml) = 22.9 (A₆₄₅) - 4.68 (A₆₆₃)

where A₆₆₃is the solution absorbance at 663 nm andA₆₄₅is the absorption at 645.

Unfortunately, the Arnon equations are not particularly accurate (Porra, 2002). Other equations have been derived that minimize the problems with the Arnon equations. For example, Lichtenthaler & Welburn (1983) report the following equations to determine chlorophyll a, chlorophyll b and carotenoid content in 80% acetone extracts:

Chlorophyll a (�g/ml) = 12.21 (A₆₆₃) - 2.81 (A₆₄₆)
Chlorophyll b (�g/ml) = 20.13 (A₆₄₆) - 5.03 (A₆₆₃)
Carotenoids (�g/ml) = (1000A₄₇₀ - 3.27[chl a] - 104[chl b])/227

And, more recently Porra (2002) reports the following equations in buffered aqueous 80% acetone:

Chlorophyll a (�g/ml) = 12.25 (A_663.6) - 2.55 (A_646.6)
Chlorophyll b (�g/ml) = 20.31 (A_646.6) - 4.91 (A_663.6)
Total chl (�g/ml) = 17.76 (A_646.6) + 7.34 (A_663.6)

If the absorbance is greater than 0.8 then the solutions should be diluted with fresh 80% acetone and remeasured.

Sims and Gamon (2002) used a solution acetone/Tris buffer (80:20 volume; pH = 7.8) to extract tissue and reported equations corrected for the presence of anthocyanins. They report:

anthocyanin (�mol ml^-1) = 0.08173 A₅₃₇ - 0.00697 A₆₄₇ - 0.002228 A₆₆₃
Chl a (�mol ml^-1) = 0.01373 A₆₆₃ - 0.000897 A₅₃₇ - 0.003046 A₆₄₇
Chl b (�mol ml^-1) = 0.02405 A₆₄₇ - 0.004305 A₅₃₇ - 0.005507 A₆₆₃
Carotenoids (�mol ml^-1) = (A₄₇₀ - (17.1 x (Chl a + Chl b) - 9.479 x anthocyanin))/119.26

Since the anthocyanin concentration estimated in the extraction medium (80% acetone) is not reliable, they report equations for determining anthocyanin in methanol/HCL/water (90:1:1, vol:vol:vol):

Anthocyanin absorbance (corrected) = A₅₂₉ - (0.228 A₆₅₀) and then insert the corrected anthocyanin absorbance in the Beer-Lambert expression, A = ecl, assuming a molar absorbance coefficient at 529 nm or 30,000 l mol^-1 cm^-1.

References:

Arnon, D (1949) Plant Physiology 24: 1- 15.
Lichtenthaler, HK and AR Wellburn (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochemical Society Transactions 11: 591 - 592.
Porra, RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynthesis Research 73: 149 - 156.
Sims, DA and JA Gamon (2002) Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages. Remote Sensing of Environment 81: 337 - 354.