Jeffrey 1975

Biocbem. Pbysiol. Pflanzen (BPP), Bd. 167, S.
191-194 (1975)
Short Communication
New Spectrophotometric Equations

for Determining Chlorophylls a, b, c1 and c2
in Higher Plants, Algae and Natural Phytoplankton
S. W. JEFFREY and G. F. HuMPHREY
CSIRO Marine Biochemistry Unit, Botany Building (A 12), Sydney University

Sydney, Australia
Key Term Index: chlorophylls, spectrophotometry, determination, equations.
Summary
New equations are presented for spectrophotometric determination of chlorophylls, based on
revised extinction coefficients of chlorophylls a, b, c1 and c2 • These equations may be used for deter-
mining chlorophylls a and b in higher plants and green algae, chlorophylls a and c1 + c2 in brown
algae, diatoms and chrysomonads, chlorophylls a and c2 in dinoflagellates and cryptomonads, and
chlorophylls a, b, and c1 + c2 in natural phytoplankton.
Spectrophotometric equations currently used for determining chlorophylls a and b

in higher plants (ARNON 1949; VERNON 1960), chlorophylls a and C in algae (HUMPHREY
and JEFFREY in JEFFREY and HAxo 1968) and chlorophylls a, b and c in phytoplankton
(PARSONS and STRICKLAND 1963; SCOR-UNESCO 1966) are based on extinction coeffi-
cients of MACKINNEY (1941) and SMITH and BENITEZ (1955) for chlorophylls a and b,
and that of JEFFREY (1963) for chlorophyll c (Table 1). JEFFREY (1968, 1969) subse-
quently found that chlorophyll c was a mixture of two components (c1 and c2), which
polymerize when purified (JEFFREY and SHIBATA 1969) to give lowered intensities of
absorption bands and therefore lowered extinction coefficients. Each component of
chlorophyll c has now been carefully crystallized to prevent polymerization, and extinc-
tion coefficients determined (JEFFREY 1972). The new extinction coefficients for chloro-
phylls c1 and c2 are approximately twice the former value for unresolved chlorophyll c.
Table 1 Extinction coefficients of ch!orophylls used to derive spectrophotometric equations in current use
Chlorophyll Extinction Coefficients in diethyl ether

at red maximum (I· gm-1 cm-1 )
MACKINNEY (1941) SMITH and BENITEZ STRAIN et al. JEFFREY (1963)
(1955) (1963)
chlorophyll a 90.1 102.0 96.0

chlorophyll b 54.9 59.0 59.0
chlorophyll c 15.8
192 S. W. JEFFREY and G. F. HuMPRHEY
Table 2 Extinction coefficients of chlorophylls used to derive the new equations
Chlorophyll Extinction Coefficients at the red maximum

(I· gm-1 cm- 1 )
Diethyl ether 100% acetone 90% acetone
chlorophyll a* 98.07 88.15 87.67

(660.7 nm) (662.7 nm) (664.3 nm)
chlorophyll b* 62.00 51.36
(643.3 nm) (646.8 nm)
chlorophyll c1 + 39.2 44.8
(629.1 nm) (630.6 nm)
chlorophyll c2 + 37.2 40.4
(629.6 nm) (630.9 nm)
* JEFFREY unpublished
+ JEFFREY (1972)
Thus determinations of chlorophyll c using the old equations give results approximately
twice as high as those indicated by the present work. Extinction coefficients for crystal-
line chlorophylls a and b have been also redetermined for a variety of solvents most
frequently used for making pigment assays (JEFFREY in preparation). Extinction coeffi-
cients of these highly purified preparations of chlorophylls a, b, c1 and c2 at their red
maxima, are given in Table 2. These new values for a and b are almost identical to the
most careful recently determined coefficients of STRAIN and co-workers (1963), (Table 1).
The early MAc KINNEY values used in the original ARNON (1949) higher plant equations
are some 8% lower than the new values of both STRAIN and JEFFREY. The VERNON
values, based on the SMITH and BENITEZ coefficients, which were also used in the algal
a + c and phytoplankton a + b + c equations, are about 5% higher than the STRAIN
and JEFFREY values. There are independent reasons for believing that these values are
a few percent too high (JEFFREY, in preparation).
The new sets of equations have therefore been written using the revised extinction
coefficients of JEFFREY for chlorophylls a, b, c1 and c2 (Table 2). Since the extinction
coefficients of chlorophylls c1 and c2 differ only by 5% and 10% in 100% and 90%
acetone respectively, and because of the smallness of the errors involved, equations
utilizing chlorophylls c1 and c2 were derived for 1 : 1 mixtures of these two pigments. In
the many algal species which have been examined which contain both chlorophylls c1
and c2, the most common proportions of chlorophylls c1 and c2 are c2 : c1 = 1: 0. 7 and
c2 : c1 = 1: 1 (JEFFREY, unpublished). The equations may be employed to assay a
variety of mixtures of chlorophylls which are typically found in higher plants, green
algae, brown algae, diatoms, chrysomonads, dinoflagellates and cryptomonads (JEFFREY
1968, 1969, 1972), and natural mixtures of these algae. We present here equations derived
for chlorophyll estimation in 90% and 100% acetone. Results are expressed in /)-g · ml-1
(using 1 em cell path-length). The symbol Ex denotes the extinction at wavelength x.
. Spectrophotometric Equations for Chlorophyll Determinations 193
1. Higher plants and green algae containing chlorophylls a and b

(solvent 90% acetone)
Chlorophyll a = 11.93 E 664 - 1.93 E 6 47
Chlorophyll b .· . 20.36 E 647 - 5.50 E 664
2. Diatoms, chrysomonads and brown algae containing chlorophylls a, and
c1 and c2 in equal proportions (solvent 90% acetone)
Chlorophyll a = 11.47 E 664 - 0.40 E 630
Chlorophylls c1 + c2 = 24.36 E 630 - 3. 73 E 664
3. Dinoflagellates and cryptomonads containing chlorop4ylls a and c2
(solvent 100% acetone)
Chlorophyll a = 11.43 E 663 - 0.64 E 630
Chlorophyll c2 = 27.09 E 630 - 3.63 E 663
4. Mixed phytoplankton populations containing chlorophylls a and band
equal amounts of chlorophylls c1 and c2 (solvent 90% acetone)
Chlorophyll a 11.85 E 664 - 1.54 E 647 - 0.08 E 630
Chlorophyll b = - 5.43 E 664 + 21.03 E 647 - 2.66 E 630
Chlorophylls c1 + c2 = - 1.67 E 664 - 7.60 E 647 + 24.52 E 630
The recovery of chlorophyll a from known mixtures using all four equations was
usually in the range of 98-102 %· The recovery of chlorophyll b from mixtures of
a+ b, using the a+ b equations, was from 98-106%. The recovery of chlorophyll c
from mixtures of a+ c, using both sets of a+ c equations, averaged 95-104% when
chlorophyll c was present in amounts greater than one-fifth the chlorophyll a concen-
tration. Decreasing proportions of chlorophyll c with respect to chlorophyll a gave
increasing errors in recovery, due to the swamping of the small absorption band of
chlorophyll c by the relatively large absorptio"!l. band of chlorophyll a at the wavelength
of estimation (630 nm). Likewise in mixtures of a + b + c1 + c2 the recovery of band c
using the fourth set of equations, were in the range 95-110%, where band c were each
greater than one quarter the a concentration. However, in mixtures increasingly
overloaded with chlorophyll a recoveries of b and c were significantly over estimated.
Very low concentrations of all pigments in mixtures (less than 0.2,ug · mi-1 ) also gave
large errors in recovery (up to 60%).
The preparative methods used for obtaining the revised extinction coefficients of
chlorophylls a and b, the sensitive tests for purity of the chlorophylls, and data on the
accuracy and precision of the equations will be presented in detail elsewhere. Alsothe
effects on accuracy of common degradation products of chlorophyll a (chlorophyllide a,
pheophytin a and pheophorbide a) which occur in natural phytoplankton populations
(JEFF'REY 1974) will be discussed.
References
ARNON, D. 1., Copper enzymes in isolated chloroplasts. Polypheil.oloxidase in ·Beta vulgaris. Plant
Physiol. 24, 1-15 (1949).
194 S. W. JEFFREY and G. F. HuMPHREY, Spectrophotometric Equations
JEFFREY, S. W., Purification and properties of chlorophyll c from Sargassum flavicans. Biochem. J.
86, 313-318 (1963).
Quantitative thin-layer chromatography of chlorophylls and carotenoids from marine algae.
Biochim. biophys. Acta 162, 271-285 (1968).
Properties of two spectrally different components in chlorophyll c preparations. Biochim. biophys.
Acta. 177, 456-467 (1969).
Preparation and some properties of crystalline chlorophyll c1 and c2 from marine algae. Biochim.
biophys. Acta 279, 15-33 (1972).
Profiles of photosynthetic pigments in the ocean, using thin-layer chromatography. Marine Biol.
26, 101-110 (1974).
HAxo, F. T., Photosynthetic pigments of symbiotic dinoflagellates (zooxanthellae) from corals
and clams. Biol. Bull. 135, 149-165 (1968).
JEFFREY, S. W., SHIBATA, K., Some spectral characteristics of chlorophyll c from Tridacna crocea
zooxanthellae. Bioi. Bull. 136, 54-62 (1969).
MACKINNEY, G., Absorption of light by chlorophyll solutions. J. biol. Chern. 140, 315-322 (1941).
PARsoNs, T. R., STRICKLAND, J.D. H., Discussion of spectrophotometric determination of marine
plant pigments, with revised equations for ascertaining chlorophylls and carotenoids. J. mar.
Res. 21, 155-163 (1963). ·
SCOR-UNESCO, Determination of photosynthetic pigments in seawater. Monographs on Oceano-
graphic Methodology I, 11-18. UNESCO, Paris, 1966.
SMITH, J. H. C., BENITEZ, A., Chlorophylls: Analysis in plant materials. In: Modern Methods of
Plant Analysis, 4, 142-196, PAECH, K., and TRACEY, M., (eds.) Springer, Heidelberg 1955.
STRAIN, H. H., THOMAs, M. R., KATZ, J. J., Spectral absorption properties of ordinary and fully
deuterated chlorophylls a and b. Biochim. biophys. Acta 75, 306-311 (1963).
VERNON, L. P., Spectrophotometric determination of chlorophylls and pheophytins in plant extracts.
Anal. Chern. 32, 1144-1150 (1960).
Received October 10, 1974.
Authors' address: Dr. S. W. JEFFREY and G. F. HuMPHREY, CSIRO Marine Biochemistry Unit,
Botany Building (A 12), Sydney University, Sydney, N.S. W., 2006 (Australia).
Verantwortlich fiir die Redaktion: Dr. B. Parthier, 401 Halle (Saale). Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2,
Telelon 24141, 24142. Alleinige Anzeigenannahme: DEWAG·Werbung Leipzig, 701 Leipzig, Briihl 34-40, Telefon 297 40. Flir
Auslandsanzeigen: Interwerbung GmbH, DDR -104 Berlin, TucholskystraJ.le 40, Postfach 230. Satz und Druck: Druckerei
,Magnus Poser", 69 Jena.- Veriiffentlicht unter der Lizenznu=r 1067a des Presseamtes beim Vorsitzenden des Ministerrates
der Deutschen Demokratischen Republik. Alle Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Ver·
lagos und des Verlassers sowie mit Angabe der Quelle gestattet. Printed in the German Democratic Republic.

Jeffrey 1975

Uploaded by

Copyright:

Available Formats

Jeffrey 1975

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Jeffrey 1975

Uploaded by

Copyright:

Available Formats

Biocbem. Pbysiol. Pflanzen (BPP), Bd. 167, S.

New Spectrophotometric Equations

CSIRO Marine Biochemistry Unit, Botany Building (A 12), Sydney University

Key Term Index: chlorophylls, spectrophotometry, determination, equations.

Spectrophotometric equations currently used for determining chlorophylls a and b

Chlorophyll Extinction Coefficients in diethyl ether

chlorophyll a 90.1 102.0 96.0

Table 2 Extinction coefficients of chlorophylls used to derive the new equations

Chlorophyll Extinction Coefficients at the red maximum

chlorophyll a* 98.07 88.15 87.67

1. Higher plants and green algae containing chlorophylls a and b

Received October 10, 1974.

You might also like