# Chlorophyll experiment - Help!

DOERING.OLAF FB4A043 at phylu.physnet2.uni-hamburg.de
Wed Oct 11 09:12:42 EST 1995

```This is a short description how to measure chlorophylls and
carotenoids taken from a textbook (P. Schopfer, Experimental plant
physiology, Springer, 1989).
Take 500mg of plant material, grind it in a cooled mortar with 1g seasand and
5ml of acetone solution (800 ml acetone + 195ml dist. water + 5 ml
conc. (25% w/w) ammonia sol.). Dont have high light intensity during
the whole procedure because chlorophylls are easily photooxidized
under high-light conditions. Fill the homogenized material in a 20 ml
centrifugation tube and wash the mortar twice with 5ml acetone
solution to have exactly 20ml in your centrifugation tube. Wait for
about 30 min., occasionally shake the tube. After 30 min
ceatrifugation at 10000g for about 15min (the supernatant has to be
clear). Measure the absorbance of the supernatant in a
spectrophotometer at 480, 645, 647, 652, 663, 664, and 750 nm against
air. Subtract the value taken at 750nm from all the other values. The
thus obtained corrected extinctions use for the following
calculations to estimate chlorophyll and carotenoid values:

Chlorophyll a= 11.78*E664 - 2.29*E647 (mg/l)

Chlorophyll b= 20.05*E647 - 4.77*E664 (mg/l)

Chlorophyll a+b = 20.2*E645 + 8.0*E663 (mg/l)
or
Chlorophyll a+b = 27.8*E652 (mg/l)

Carotenoid(approx) = E480 + 0.114*E663 - 0.638*E645

Ennn = Corrected extinction at wavelength nnn

carotenoid determination in:

Goodwin TW ed. (1976) Chemistry and biochemistry of plant pigments.
2nd Ed., Vol. 2, Academic Pr. London

Sestak Z, Catsky J, Jarvis PG eds.: Plant photosynthetic production.
Manual of methods. Junk N.V., Den Haag, pp672-701

Greetings

O. Doering
Univ. Hamburg
Inst. Botany
Ohnhorststr. 18
D-22609 Hamburg, FRG
Phone: +49-40-82282-348
FAX: +49-40-82282-254

```