|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; email@example.com|
Measuring Chlorophyll (& Anthocyanin) Concentration
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 A663 is the solution absorbance at 663 nm andA645 is 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)
And, more recently Porra (2002) reports the following equations in buffered aqueous 80% acetone:
Chlorophyll a (�g/ml) = 12.25 (A663.6) - 2.55 (A646.6)
Chlorophyll b (�g/ml) = 20.31 (A646.6) - 4.91 (A663.6)
= 17.76(A646.6) + 7.34 (A663.6)
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.
01/07/2009 � Copyright by SG