350

PLASTIDS

[34]

glycerol and MGDG substrates containing specific fatty acids. Thus, the
extent of the observable galactosylation reactions in any tissue preparation depends upon the molecular species of diacylglycerol and MGDG
available in the preparation. The type and amount of substrate available in
the plant preparation may be altered by both biotic and abiotic factors,
Factors which may alter the rate of galactolipid biosynthesis and the
nature (molecular species) of the galactolipids formed include (1) the temperature of growth of plant tissue,~4 (2) herbicide treatment of plant material, 13 (3) addition of drugs to the assay mixture, ~5etc. The light intensity
during growth of the plant appears to have little effect on galactolipid
metabolism. H
14j. p. Williams, M. U. Khan, and K. Mitchell, in "Structure, Function and Metabolism of
Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 123. Elsevier, Amsterdam, 1984.
1~N. W. Lem and P. K. Stumpf, Plant Physiol. 75, 700 (1984).

[34] ChlorolShylls a n d C a r o t e n o i d s : P i g m e n t s of
Photosynthetic Biomembranes
B y HARTMUT K . LICHTENTHALER

Introduction
The photosynthetic pigments chlorophylls and carotenoids, together
with sterols, prenylquinones, and prenols, belong to the group of isoprenoid plant lipids, which in 1976 were named prenyl lipids by Goodwin ~
and Lichtenthaler. 2 Carotenoids as tetraterpenoids are simple or pure
prenyl lipids, the carbon skeleton of which is made up solely of isoprenoid
units. Chlorophyll a and b are mixed prenyllipids: they possess an isoprenoid phytyl chain which is bound to a nonisoprenoid porphyfin ring system. The possession of the phytyl side chain, which is esterified to the
carboxyl group of the ring, gives the chlorophylls their lipid character.
Because of their biogenetic relationship (isopentenoid pathway), chlorophylls and carotenoids are also referred to as prenyl pigments.
1 T. W. Goodwin, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and H. K.
Lichtenthaler, eds.), p. 29. Springer-Verlag, Berlin and New York, 1977.
2 H. K. Lichtenthaler, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and
H. K. Lichtenthaler, eds.), p. 231. Springer-Verlag, Berlin and New York, 1977.

METHODS IN ENZYMOLOGY, VOL. 148

Copyright 1987 by Academic Press, Inc.

All fights of reproduction in any form reserved.

[34]

CHLOROPHYLLS AND CAROTENOIDS

351

Chlorophylls
The chlorophylls of higher plants, ferns, mosses, and green algae, as
well as of the prokaryotic organism Prochloron, consist of chlorophyll a
as the major pigment and of chlorophyll b as an accessory pigment. Both
chlorophylls are genuine components of the photosynthetic membranes
and occur in a ratio (a/b) of approximately 3 to 12-5 Growth conditions
and environmental factors can modify this a/b ratio. High-light and sunexposed plants (high-light chloroplasts) exhibit a/b ratios of 3.2 to 4,
whereas shade plants (low-light chloroplasts) possess lower values for the
a/b ratio (e.g., 2.5 to 2.9). 4-6 In older literature minor amounts of an
additional chlorophyll (a') were found. 7 This is a C-10 epimer of chlorophyll a and is now regarded in the form of the a' dimer as identical
with the reaction center chlorophyll P-700 of the photosynthetic photosystem 1.8

Carotenoids
The group of primary plant carotenoids can be divided (1) into the
oxygen-free carotenes and (2) into the xanthophylls, which contain oxygen in different forms, such as one or several hydroxy or epoxy groups.
There are two types of isomers on each oxidation level, the a-carotene
derivatives (one e-ionone and one/3-ionone ring) and the/3-carotene derivatives (2/3-ionone rings). Introduction of hydroxy and epoxy functions
into a-carotene (ionone rings) gives rise to lutein and lutein epoxide./3Carotene is the precursor of the xanthophylls zeaxanthin, violaxanthin,
and antheraxanthin. The carotenoids of green, photosynthetically active
plant tissue, which are needed for photosynthetic function, are classified
as primary carotenoids, whereas those of red fruits and flowers have been
termed secondary carotenoids.
The carotenoids of functional chloroplasts include/3-carotene, lutein,
violaxanthin, and neoxanthin as major and regular components of the
photochemically active thylakoids of chloroplasts of higher plants and
3 R. Willst~itter and A. Stoll, "Untersuchungen tiber Chlorophyll." Springer-Verlag,
Berlin, 1913.
4 A. Seybold and K. Egle, Planta 26, 491 (1970).
5 H. K. Lichtenthaler, C. Buschmann, M. D611, H.-J. Fietz, T. Bach, U. Koze|, and U.
Rahmsdorf, Photosynth. Res. 2, 115 (1981).
6 H. K. Lichtenthaler, G. Kuhn, U. Prenzel, C. Buschmann, and D. Meier, Z. Naturforsch., C: Biosci. 37C, 464 (1982).
7 H. H. Strain, B. T. Cope, and W. A. Svec, this series, Vol. 23, p. 452.
8 T. Watanabe, M. Kobayashi, A. Hongu, M. Nakazato, T. Hiyama, and N. Murata, FEBS
Lett. 191, 252 (1985).

352

PLASTIDS

[34]

d-Carotene

i-Corotene

H O ~ d ~ ~ ~ ~ V ~

0H Zeoxonthin

OH

Lutein

Violoxanthin

HO

.~:]~
HO

OH

Neoxanthln

OH

FIG. 1. Chemicalstructure of the main chloroplastcarotenoidsof higher plants.

green algae (Fig. 1). Depending on growth conditions and stress factors,
the percentage composition of carotenoids (weight %) can vary within the
ranges: /3-carotene 25-40% lutein, 40-57%, violaxanthin, 9-20%, and
neoxanthin, 5-13%. The xanthophylls antheraxanthin, luteinepoxid, and
zeaxanthin are regular but minor carotenoid components. In green algae
a-carotene shows up as an additional carotene to/3-carotene. Under highlight conditions the xanthophyll violaxanthin (two epoxy, two hydroxy
groups) can by deepoxidized via antheraxanthin to zeaxanthin, the corresponding deepoxy derivative, which exhibits a chromatographic mobility
similar to lutein. Though this xanthophyll cycle is well established, 9-11 its
9 A.
l0 D.
H.
" K.

Hager, in "Pigments in Plants" (F. C, Czygan, ed.), p. 57. Fischer, Stuttgart, 1980.
Siefermann-Harms, in "Lipids and Lipid Polymers in Higher Plants" (M. Tevini and
K. Lichtenthaler, ds.), p. 218. Springer-Verlag, Berlin and New York, 1977.
Grumbach, Z. Naturforsch., C: Biosci. 38C, 393 (1983).

[34]

CHLOROPHYLLS AND CAROTENOIDS

353

complete physiological role is not yet known. Lutein and neoxanthin

apparently only function as accessory pigments in the photosynthetic
light absorption, r-Carotene may partially serve as a light-absorbing pigment; its main function, however, seems to be the protection of chlorophyll a from photooxidation in or near the reaction center.
The main paths of biosynthesis of chlorophylls and carotenoids are
established. Details concerning chlorophylls may be found in the reviews. 12,13An open question seems to be whether chlorophyll b (--CHO
instead o f - - C H 3 in the second pyrrole ring of the porphyrin ring) derives
via oxidation of chlorophyll a or by prenylation of a special precursor
(chlorophyllide b?), which has not yet been detected. Basic information
on carotenoid biosynthesis can be found in Goodwin I and Davies. 14
Though certain aspects of compartmentalization are not yet fully clear,
there is agreement that at least the final steps of carotenoid biosynthesis
(including dimerization, desaturation, and cyclization) proceed within the
chloroplasts.
The aim of this chapter on chlorophylls and carotenoids is to give
practical directions toward their quantitative isolation and determination
in extracts from leaves, chloroplasts, thylakoid particles, and pigment
proteins. Hence the main emphasis is placed on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively
determine them. Many data in this field were based on rather old observations and old instrumentation and needed revision. Therefore the specific
absorption coefficients of the pigments were reevaluated and in part also
first established for the purpose of this chapter. This was achieved by
using a two-beam spectrophotometer of the new generation, which allows
programmed automatic recording and printing out of the proper wavelengths and absorbancy values.

Chlorophyll-Carotenoid Proteins
Much of the present research work on photosynthetic pigments concerns isolation and characterization of pigment proteins. The main features of their isolation and pigment content will therefore be treated here.
12 H. A. W. Schneider, Ber. Dtsch. Bot. Ges. 88, 83 (1975).
13 W. Riidiger and J. Benz, in "Chloroplast Biogenesis" (R. J. Ellis, ed.), p. 225. Cambridge
Univ. Press, London and New York, 1984.
14 B. H. Davies, in "Chemistry and Biochemistry of Plant Pigments" (T. W. Goodwin, ed.),
2nd ed., Vol. 2, p. 38. Academic Press, New York, 1976.

354

PLASTIDS

[34]

A
I1. n

~-~

C
",,I

0..'

12.
It.

U
IZ

IIII
IIII

.10

",-'r
-J-~

,<

t.2
"1-.I

t.

rQ

IX.

'..,It

o~

fit.

(.2
"1-

..a

o-"1

it
ffl
.13

<

I~igrolion

(~

FIG. 2. Densitometer scans of the pigment proteins of Raphanus chloroplasts separated

by SDS-PAGE [(A) Scan at 672 nm and (B) scan at 470 nm]. As compared to 672 nm,
absorbance of the light-harvesting proteins (LHCPs) increases at 470 nm due to their high
content in xanthophylls and chlorophyll b. In contrast, the relative absorbance of the chlorophyll a proteins CPI, CPIa, and CPa, which contain only little chlorophyll b, decreases at
470 nm. With respect to the free pigment zone (FP), the scan at 672 nm shows only the peak
of free chlorophylls; this peak is broadened by the free carotenoids, when the scan is
performed at 470 nm.

Except for violaxanthin, which to some extent is bound to the chloroplast

envelope,15 all carotenoids and chlorophylls are bound to the thylakoids,
the photochemically active photosynthetic biomembranes. ~6 Within the
thylakoids the pigments are associated with several chlorophyll-carotenoid proteins which possess differential chlorophyll and carotenoid composition. 16-24 By polyacrylamide gel electrophoresis (PAGE) of digitonin
or sodium dodecyl sulfate-treated chloroplasts or thylakoid preparations
15 H. K. Lichtenthaler, U. Prenzel, R. Douce, and J. Joyard, Biochim. Biophys. Acta 641, 99

(1981).
,6 j . p. M a r k w e l l , J. P. Thornber, and R. T. B o g g s , Proc. Natl. Acad. Sci. U.S.A. 76, 1223

(1979).

[34]

CHLOROPHYLLS AND CAROTENOIDS

355

TABLE I
PRENYL PIGMENT RATIOS AND PERCENTAGE COMPOSITION OF CAROTENOIDS
IN PIGMENT PROTEINS
Protein

Chloroplasts

CPIa

CPI

CPa

LCHPI_3

FP

4-7
5-6
12

9-21
7-9
9

3-8
3-5
4-8

1.1-1.3
3-5
60-180

2.6
3
14

56
24
9
5
6

70
15
4
6
5

75
13
5
4
3

Pigment ratios

Chlorophyll a / b

3.2
4
12
Percentage carotenoid composition c
fl-Carotene
30
Lutein
45
Neoxanthin
6
Violaxanthin
11
Others
8
a + b/x + cb
a/c b

1-2

56-65
21-29
4-5
6

24
37
10
20
9

Isolated by S D S - P A G E from radish chloroplasts. (After Lichtenthaler et al. 23,24)

b a + b / x + c = weight ratio chlorophylls to carotenoids; a / c = weight ratio chlorophyll
a/ fl-carotene.
c The enrichment of particular carotenoids in a pigment protein as compared to whole
chloroplasts is indicated in boldface.

(Fig. 2), one obtains the following: (1) the P700-containing chlorophyll

a/fl-carotene proteins CPI and CPIa of photosystem I, (2) CPa, the presumable reaction center of photosystem II, which is also a chlorophyll

a/13-carotene protein, and (3) several forms of LHCPs (LHCP~, LHCP2,

LHCP3, LHCPy, etc.), the light-harvesting chlorophyll a/b-xanthophyll
proteins. 16-24
The xanthophylls lutein and neoxanthin are preferentially bound together with chlorophyll b and some chlorophyll a to the LHC-proteins,
which contain 13-carotene only in traces. 13-Carotene is, however, the
main carotenoid component of the chlorophyll a-proteins CPI, CPIa, and
CPa. The exact composition of the different pigment proteins has been
established for Raphanus chloroplasts (Table I). Violaxanthin is present
~7j. p. Thornber, A n n u . Reo. Plant Physiol. 26, 127 (1975).
18 j. M. Anderson, J. C. Waldron, and S. W. Thorne, F E B S Lett. 92, 227 (1978).
19 O. Machold, D. J. Simpson, and B. L. MOiler, Carlsberg Res. C o m m u n . 44, 235 (1979).
2o R. Bassi, O. Machold, and D. Simpson, Carlsberg Res. Cornmun. 50, 145 (1985).
21 j. Argyroudi-Akoyunoglou and G. Akoyunoglou, F E B S Lett. 104, 78 (1979).
22 H. K. Lichtenthaler, G. Burkard, G. Kuhn, and U. Prenzel, Z. Naturforsch., C: Biosci.
36C, 421 (1981).
23 H. K. Lichtenthaler, U. Prenzel, and G. Kuhn, Z. Naturforsch., C: Biosci. 37C, 10 (1982).
z4 U. Prenzel and H. K. Lichtenthaler, in "Biochemistry and Metabolism of Plant Lipids"
(J. F. G. M. Wintermans and P. C. Kuiper, eds.), p. 565. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982.

356

PLASTIDS

[34]

in the various chlorophyll-carotenoid proteins only in minor amounts,

which does not account for the total violaxanthin present in the thylakoids. Violaxanthin, however, is found in a rather high proportion in the
free pigment fraction. 24 From this it is assumed that this apparently not
protein-bound pool of violaxanthin is identical with those 70 to 80% of
chloroplast violaxanthin, which can be transformed at high light intensities into zeaxanthin. 9,1
A similar carotenoid distribution among different pigment proteins has
also been found using the isoelectric focusing technique as a method for
pigment protein isolation. 25-27
The individual levels of the pigment proteins can vary depending on
the developmental stage and on the environmental conditions. At lowlight growth conditions and in the shade a higher proportion of LHCPs are
found in comparison to CPI + CPIa. 6,28,29This fully explains the lower
values for the ratios chlorophyll a/b and the higher values for the ratios
chlorophyll a/fl-carotene (a/c) and a + b/x + c in low-light plants.
There are different methods for the isolation of pigment particles and
pigment proteins, such as the PAGE technique 16-22or isoelectric focusing, 26,27including the classical differential centrifugation of partially detergent-digested chloroplast or thylakoid preparations. The latter often precedes the gel electrophoretic methods. Details of the procedures may be
found in the original older literature 16,1s-27and the most recent results in
the current journals of plant physiology and plant biochemistry.
Mild procedures (low detergent concentrations, short incubation
times, and low temperatures) allow the detection of larger pigment protein
aggregates (e.g., CPIa and LHCPs 1 and 2), whereas at higher concentrations primarily the oligomeric forms (e.g., CPI and LHCP3) show up. By
choosing the right conditions it is also possible to detect several transitional stages between the larger aggregates and the oligomeric forms.
Very pure and well-characterized pigment proteins are those of Machold
et al. 19,20A recent overview on thylakoid proteins including pigment proteins and molecular weights is found in Critchley. 3
The general problem with the isolation of pigment proteins is that the
free chlorophyll portion often exceeds 15 or even 20% of the total thylakoid chlorophyll applied and that it should be well beyond 10% to obtain
representative results for the pigment composition of a thylakoid. An25 D. Siefermann-Harms, Biochim. Biophys. Acta 811, 1 (1985).
26 D. Siefermann-Harms and N. Ninnemann, FEBS Lett. 104, 71 (1979).
27 T. Omata, N. Murata, and K. Satoh, Biochim. Biophys. Acta 765, 403 (1984).
H. K. Lichtenthaler, G. Kuhn, U. Prenzel, and D. Meier, Physiol. Plant. 56, 183 (1982).
T.-Y. Leong and J. M. Anderson, Biochim. Biophys. Acta 723, 391 (1983).
3o C. Critchley, Biochim. Biophys. Acta 811, 33 (1985).

[34]

CHLOROPHYLLS AND CAROTENOIDS

357

other problem which is not yet fully resolved is the quantitative extraction
of chlorophylls and carotenoids from the pigment proteins on the polyacrylamide gels. Repetitive extraction of the material seems to be the
method of choice.
Isolation and Chromatography of Pigments

Extraction
All prenyl pigments, chlorophylls and carotenoids, are fat-soluble
compounds that can be extracted from water-containing living plant tissue
by organic solvents (grinding with quartz sand or use of a homogenizer)
such as acetone, methanol, or ethanol, which can take up water. Though
aqueous solutions of these solvents are also suitable (and may sometimes
be preferable) for extraction, their water content should not exceed 5 or
10%. The widely used method to extract isolated chloroplasts by 80%
aqueous acetone3~ does not fully extract the less polar pigment chlorophyll a and the polyene/3-carotene. An addition step of extraction with
100% acetone is needed to guarantee complete extraction. By addition of
petroleum hydrocarbon (or hexane) and water the pigments are transferred to the water-free epiphase, and concentrated in a rotary evaporator
to a certain standard volume. In a water-free organic solvent, pigments
can be stored longer than in aqueous solution.
Freeze-dried plant material can be extracted directly with hexane or
petroleum hydrocarbon; this, however, will mainly remove fl-carotene
and some chlorophyll a. Addition of small amounts of a polar solvent
(acetone) to the extracting hydrocarbon solvent will fully extract the chlorophylls and carotenoids. Since carotenoids and chlorophylls are extremely light sensitive and easily become photobleached, the extraction
and concentration procedures should be performed in dim light. For correct pigment values the quantitative determination of chlorophylls and
total carotenoids in the whole leaf extract should be carried out immediately after preparation of the extract. If storage of the extract is required,
it should be done in concentrated form and under nitrogen.

Chromatographic Procedures
Several procedures have been developed for the separation of the
photosynthetic pigments including column (CC), paper (PC), and thinlayer chromatography (TLC) and also high-pressure liquid chromatogra3t D. I. Arnon, Plant Physiol. 24, 1 (1949).

358

PLASTIDS

[34]

-111-rain
_.

.c_
"-.r" 0

II

>,,

--"

",.

!
start

detection:

1.50 n m

FIG. 3. High-performance liquid chromatography of a leaf pigment extract (reversedphase HPLC). Column: 25 cm, Nucleosil 5 C-8; solvent: 9% water in methanol; flow rate:
1.3 ml/min, na, Neoxanthin a.

phy (HPLC). Basic and detailed information on these methods is found in

a recent extensive chromatography review by Lichtenthaler. 32
The separation of a plant pigment extract on CaCO3 and MgO columns
into green (chlorophylls) and yellow pigments (carotenoids) by Tswett
can be regarded as the beginning of chromatography.33Today TLC is the
preferred method for the isolation of the photosynthetic pigments of
higher plants. Chromatography on precoated silica gel plates using 70 ml
petroleum hydrocarbon (bp 40-60), 30 ml dioxane, and 10 ml 2-propanol
as developing solvent results in the separation of the two chlorophylls and
the major carotenoids. 34 The chromatographic sequence (Rf values) is/3carotene (0.85), a trace of pheophytin (0.67), chlorophyll a (0.6), chlorophyll b (0.5), lutein + zeaxanthin (0.4), antheraxanthin (0.34), violaxanthin (0.3), and neoxanthin (0.2). The extract should be applied to the plate
in a water-free solvent. Elution of the pigments is performed with ethanol
or acetone. A very suitable TLC method for carotenoids, which also
separates a- and fl-carotene derivatives, e.g., lutein and zeaxanthin, is
given by Hager and Meyer-Bertenrath.35
32 H. K. Lichtenthaler, in "CRC Handbook of Chromatography: Lipids" (H. K. Mangold,
ed.), Vol. 2, p. 115. CRC Press, Boca Raton, Florida, 1984.
33 M. Tswett, Ber. Dtsch. Bot. Ges. 24, 384 (1906).
34 H. K. Lichtenthaler, D. Meier, G. Retzlaff, and R. Hamm, Z. Naturforsch., C: Biosci.
37C, 889 (1982).
3s A. Hager and T. Meyer-Bertenrath, Planta 76, 149 (1967).

[34]

CHLOROPHYLLS AND CAROTENOIDS

359

(l

mln

start
detection:/,10 nm
FIG. 4. Reversed-phase HPLC of leaf pigments including pheophytins. Column: 12.5 cm,
Nucleosil 7 C-18; solvent: methanol; flow rate: 1.8 ml/min.

An example of the HPLC of prenyl pigments is shown in Fig. 3.

Adsorption and reversed-phase HPLC have been applied. 36-41For further
examples and details see Table 3 in Lichtenthaler. 32HPLC also allows the
separation of pheophytin a and b (Fig. 4).
The Chlorophylls

Spectral Characteristics of Chlorophylls

The absorption maxima of chlorophylls are found in the red and blue
regions of the visible spectrum (Figs. 5 and 6). The position for krnax of
chlorophyll b (e.g., 641.8 and 452.2 nm in dried diethyl ether) lies between
those of chlorophyll a (660 and 428 nm in dried diethyl ether) in all sol36 K. Eskins, C. R. Scholfield, and H. J. Dutton, J. Chromatogr. 135, 217 (1977).
37 H. Stransky, Z. Naturforsch., C: Biosci. 33C, 836 (1978).
3s U. Prenzel and H. K. Lichtenthaler, in "Advances in the Biochemistry and Physiology of
Plant Lipids" (L.-A. Appelqvist and C. Liljenberg, eds.), p. 319. Elsevier/North-Holland
Biomedical Press, Amsterdam, 1979.
39 T. Braumann and L. Grimme, J. Chromatogr. 170, 264 (1979).
4o M. Tevini, W. Iwanzik, and U. Thoma, Planta 153, 388 (1981).
4~ T. Watanabe, M. Nakazato, H. Mazaki, A. Hongu, M. Konno, S. Saitoh, and K. Honda,
Biochim. Biophys. Acta 807, 110 (1985).

360

PLASTIDS

[34]

{'I

I'

350.0

450.0

550.0

650.0

750.0

Wavelength (nm)
FIG. 5. Absorption spectrum of freshly isolated chlorophyll a (solid line) and chlorophyll
b (broken line) in diethyl ether (pure solvent). The positions of the absorption maxima are
given in Table II.

vents assayed (see Table II). The shortest wavelength for hmax in the blue
and red is found for both chlorophylls in water-free diethyl ether. The two
major absorption maxima in the red and blue of both chlorophylls shift to
longer wavelengths with increasing polarity and/or water content of the
solvent. The shift at hmax red is less pronounced than that at hmax in the
blue region and it is larger for chlorophyll b than for chlorophyll a. The
wavelengths of the much smaller, minor absorption maxima also undergo
this shift.
The specific absorption coefficients are highest in dried (water-free)
diethyl ether and decrease via diethyl ether (pure solvent), water-saturated diethyl ether, to acetone (100 ---> 80%), ethanol (95%), and methanol
(100 ~ 90%) (Table II). The absorption in the red and blue maxima is
highest in freshly isolated chlorophylls and also decreases in older solutions, particularly in those which are light exposed. Quantitative chlorophyll determinations should therefore be carried out immediately after
extraction. In the case of chlorophyll a the decrease in absorbance in the
blue is stronger than in the red region, which is documented by a continu-

[34]

CHLOROPHYLLS AND CAROTENOIDS

1

361
1

o
III

0.500 T

0.500

//i

3SQ.O

450.0

550.0

6S0.0

750.0

Wavelength (rim)
FIG. 6. Absorption spectrum of freshly isolated chlorophyll a (solid line) and chlorophyll
b (broken line) in aqueous ethanol (95%), The wavelengths of the absorption maxima are
given in Table II.

ous decrease of the values for the ratio of absorption coefficients of the
blue and the red maximum from 1.28 to 0.96 (Table IV). For chlorophyll b
the decrease is about the same in the blue and in the red region, as seen
from the ratio of the absorption coefficients, which remains about the
same with values around 2.7 (Table IV).
With increasing polarity and water content of the solvent, the absorption maxima in the blue and red not only become smaller, but at the same
time broader. Parallel to the broadening of the red maximum of chlorophyll a there occurs a considerable increase in the absorption coefficients
of the minor maxima at about 614 to 618 nm and 576 to 582 nm. Due to the
shift in the wavelengths, the specific absorption coefficients of chlorophyll a and b at 470 nm, the reference point of simultaneous carotenoid
determination in a whole leaf pigment extract, rise continuously with
increasing polarity and water content of the solvent. In ethanol (95%)
and in methanol (100 and 90%), the two minor absorption maxima (in
the range of 576 to 582 nm and 531 to 537 nm) do not show up (see also
Fig. 6).

362

PLASTIDS

[34]

TABLE II
SPECIFIC ABSORPTION COEFFICIENTS FOR CHLOROPHYLL a AND b REDETERMINED a

Diethyl ether
(water free)
(nm)

(sp. coeff.)

Diethyl ether
(pure solvent)
(nm)

(sp. coeff.)

Diethyl ether
(H20 saturated)
(nm)

Acetone
(100%)

(sp. coeff.)

(nm)

(sp. coeff.)

Chlorophyll a
660

101.9

660.6

101c

661.6

98.46

661.6

92.45

641.8
614.4
576.2
531.0
470
460
450
428.0
408.8

15.20
15.7
8.7
4.67
1.3
1.5
3.6
130.4
83.15

642.2
614.8
576.6
531.6
470
460
450
428.4
409.4

15.0
15.88
8.80
4.48
1.43
1.71
3.87
127.21
82.83

643.2
615.8
578.2
532.6
470
460
450
429.4
410.0

15.31
16.07
8.86
4.19
1.38
1.79
5.47
120.96
81.22

644.8
616.0
579.0
534.2
470
460
450
429.6
410.6

19.25
17.25
9.92
4.34
1.90
2.44
7.40
112.36
83.18

Chlorophyll b
660
641.8

4.7
62.3

660.6
642.2

6.0
62.0~

661.6
643.2

7.20
58.29

661.6
644.8

9.38
51.64

593.2
470
460
450
452.2
428.2

11.57
33.12
122.17
162.3
171.33
60.13

593.6
470
460
450
452.6
428.4

12.35
35.87
121.70
158.51
167.87
62.09

594.2
470
460
450
453.8
430.2

12.02
48.05
128.88
139.70
156.69
58.57

595.8
470
460
450
455.8
--

12.11
63.14
133.86
122.08
145.36
--

By use of a UV-260 Shimadzu UV-visible recording spectrometer, which allows autoabsorption maxima of the pigments, sp. coeff., Specific absorption coefficient.
b Wavelengths (nm) of the two major absorption maxima in the red and blue specThe solvents used for the determination of the absorption coefficients were of purest
stadt, FRG, No. 929; diethyl ether (pure solvent), diethyl ether extra pure DAB7 qualresidue analysis, Merck No. 12; ethanol (95%), ethanol for fluorometry, Merck No.
water to 95%; methanol, methanol z.A. (analytical grade), Merck No. 6009, or methac Accepted from Smith and Benitez 49 (for a light path of I cm).

[34]

CHLOROPHYLLS AND CAROTENOIDS

363

FOR ORGANIC SOLVENTS OF DIFFERENT POLARITY AND WATER CONTENTb

Acetone,
80% (v/v)
(aqueous sol.)
(nm)

663.2
646.8
618.2
582.4
537.0

470
460
450
431.2
412.6

663.2
646.8
597.6

470
460
450
459.0

(sp. coeff.)

Ethanol,
95% (v/v)
(aqueous sol.)

Methanol,
90% (v/v)
(aqueous sol.)

Methanol
(100%)

(nm)

(sp. coeff.)

(nm)

(sp. coeff.)

(nm)

(sp. coeff.)

86.3
20.49
18.15
10.35
4.10
1.82
2.87
11.62
95.82
78.76

664.2
648.6

84.60
25.06
19.14

665.2
652.4

79.24
35.52
18.68

665,2
652.4
618.2

78.95
35.36
18.84

11.20
49.18
12.80
85.02
134.14
94.89
134.50

664.2
648.6

617.8

.
.
470
460
450
431.6
414.4

600.6

470
460
450
463.6

.
.

617.6

.
.
2.13
4.32
18.42
83.89
76.60

16.0
41.2
11.81
97.64
103.32
69.95
107.45

.
.
470
460
450
431.8
--

.
.
1.63
6.11
28.80
77.05
--

470
460
450
431.8

1.91
5.94
26.37
75.97

417.6

--

665.2
652.4

21.28
38.87
12.74
104.96
87.48
63.07
105.36

665.2
652.4

19.84
35.97
11.78
95.15
82.03
58.83
99.51

602.6

470
460
450
469.2

.
.

602.8

470
460
450
469.2

matic recording and printing of wavelength and absorbance in the major and minor
tral region are in boldface, those of the minor absorption maxima (italics).
obtainable quality: Diethyl ether (water free), diethyl ether dried GR, Merck, Darmity, Merck No. 926; acetone (100%), acetone extra pure, Merck No. 13, and acetone for
973, or ethanol absolute, DABs quality, Roth Karlsruhe, FRG, No. 5054 diluted with
nol dried, Merck No. 6012.

364

PLASTIDS

[34]

Quantitative Determination o f Chlorophylls

T h e r e exist various equations from different authors for different solvents for the determination o f chlorophylls in leaf pigment extracts (see
reviews o f Szestak 42 and Holden43). The disadvantage of these equations
is that the pigment values obtained in one solvent are not comparable with
those o f another. The discrepancies are particularly large in the values for
the ratio o f chlorophyll a/b. The cause o f this is that the equations are
based on rather early determinations of the specific absorption coefficients at times where the resolution o f spectrophotometers was not as
good as today. The widely used equations given by Arnon 31 for 80%
acetone are still based on the specific absorption coefficients of Mackinn e y / 4 Though the equations o f A r n o n may still be used for estimating the
total chlorophyll a + b content, they can never be applied to determine
the ratio o f chlorophyll a/b o f a pigment extract. Several attempts have
been made to correct and modify the equations for acetone (I00 and 80%),
e.g., by Vernon, 45 Ziegler and Egle, 46 and Wintermans and de Mots. 47 Yet
there remained differences in the wavelength position of the red peak
maxima and in the relative absorption o f chlorophyll a at hrn~ of chlorophyll b and vice versa. The same holds true for the determination of
chlorophylls in diethyl ether solutions. The original absorption coefficients o f C o m a r and Zscheile, 48 were redetermined by Smith and Benitez, 49 and their data were confirmed by Falk, 5 who applied a different
technique o f magnesium determination. The equations given by Smith
and Benitez 49 for diethyl ether,
Ca = 10.1A662- 1.01A644
Cb = 1 6 . 4 A ~ - 2.57.4662

were later slightly modified by Ziegler and Egle46:

Ca = 10.05A662 - 0.89A644
Cb = 16.37A644 - 2.68A662

42z. Szestak, in "Plant Photosynthetic Production: Manual of Methods" (Z. ~est,4k, J.

Catsky, and P. G. Jarvis, eds.), p. 672. Dr. W. Junk Publ., The Hague, 1971.
43M. Holden, in "Chemistry and Biochemistry of Plant Pigments" (T. W. Goodwin, ed.),
2nd ed., Vol. 2, p. 1. Academic Press, New York, 1976.
44G. Mackinney, J. Biol. Chem. 140, 315 (1941).
45L. P. Vernon, Anal. Chem. 32, 1144 (1960).
46R. Ziegler and K. Egle, Beitr. Biol. Pflanz. 41, 11 (1965).
47j. F. G. M. Wintermans and A. de Mots, Biochim. Biophys. Acta 109, 448 (1965).
4s C. L. Comar and F. P. Zscheile, Plant Physiol. 17, 198 (1942).
49j. H. C. Smith and A. Benitez, in "Modern Methods of Plant Analysis" (K. Paech and
M. V. Tracey, eds.), Vol. 4, p. 142. Springer-Verlag, Berlin and New York, 1955.
5oH. Falk, Planta 51, 49 (1958).

[34]

CHLOROPHYLLS AND CAROTENOIDS

365

More recently, in establishing equations for the simultaneous determination of total carotenoids, together with chlorophyll a and b, of leaf extracts in different solvents 51 we reinvestigated and confirmed the absorption coefficients of Smith and Benitez and adjusted the equations for a
Unicam SP 8000 spectrophotometer:
Ca = 10.05A662 - 0.766A644
Cb = 16.37A644 - 3.14A662
The differences found by authors for the same solvent are mainly due
to (1) the resolution properties of the spectrophotometers applied in the
red spectral region (which will determine the position of hmax of the red
absorption maxima of chlorophyll a/b and the relative absorption at hmax
of the other chlorophyll), (2) the accuracy with which a particular wavelength can be chosen for the absorbancy readings, and (3) the purity of the
solvents: slight differences in the content of water or organic material of
the solvent will largely influence the refractive index as well as height and
broadness of the maxima.
By applying a spectrophotometer of the new generation, which exhibits automatic baseline correction and can be programmed for peak
picking, automatic readings, and printing out of absorbancy readings at
certain wavelengths, the absorption coefficients were again redetermined
using pure solvents of different sources (cf. footnote of Table II). These
new spectrophotometers guarantee a better selection of wavelengths (or
parts of wavelengths) and absorbancy readings. The position of hmaxfor
chlorophyll a and b in diethyl ether, as determined by repetitive recording
of spectra (in a UV-260 Shimadzu spectrometer) of freshly isolated chlorophyll from several plants (with five aliquots per isolation) are at shorter
wavelengths (660.6 and 642.2 nm) than so far reported using other instruments (662 and 644 nm). The new equations for diethyl ether (pure solvent) for the determination of chlorophylls a and b in a UV-260 spectrophotometer are as follows:

Ca = 10.05A660.6 - 0.97A642.2
Cb = 16.36A642.2 -' 2.43A660.6
Based on the redetermined specific absorption coefficients in various
other solvents (Table II), equations are given for the simultaneous determination of chlorophyll a and b in a pigment extract solution (Table III).
The validity of the equations was cross-checked by dissolving the same
amount of plant extract in each of the solvents. The advantage and progress of the new equations is that the pigment values obtained in one
5, H. K. Lichtenthaler and A. R. Wellburn, Biochem. Soc. Trans. 603, 591 (1983).

366

PLASTIDS

[34]

TABLE III
EQUATIONS FOR THE DETERMINATIONS OF THE CONCENTRATIONS OF CHLOROPHYLL a
(Ca), CHLOROPHYLL b (Cb), OF TOTAL CHLOROPHYLLS (Ca+b)AND OF TOTAL
CAROTENOIDS (Cx+c) IN LEAF PIGMENT EXTRACTS FOR SOLVENTS OF DIFFERENT
POLARITY AND WATER CONTENT a

Diethyl ether (pure solvent):

Ca = 10.05A660.6- 0.97A642.2
Cb = 16.36A642.2- 2.43Ag,o.6
C~+b = 7.62A660.6 + 15.39-4642,2
Cx+c = 1000A470 - 1.43Ca - 35.87Cb

Acetone, 100% (pure solvent):

Ca = 11.24A661.6- 2.04As44.s
Co = 2 0 . 1 3 A ~ . s - 4.19A661.6
C~+b = 7.05A~1.6 + 18.09A~.s
Cx+c = 1000A470 - 1.90C~ - 63.14Cb
214

205
Diethyl
Ca
Cb
Cab

ether (water free):

= 9 . 9 3 A ~ - 0.75A64Ls
= 16.23A641.s- 2.42A66o
= 7 . 5 1 A ~ + 15.48A64Ls
Cx+c = 1000A470- 1.30C~- 33.12Cb

Acetone, 80% (v/v):

Ca = 12.25A663.2- 2.79A~.s
Cb = 21.50A646.a- 5.10A663.2
Ca+b = 7.15A663.2 + 18.71As46.s
Cx+c = 1000A47o - 1.82Ca - 85.02Cb
198

213
Diethyl ether (water saturated):
Ca
= 10.36A66L6- 1.28A643.2
Cb = 17.49A643.2- 2.72A66L6
Ca+b = 7.64A66L6 + 16.21A643.z
C~+~ = 1000A470- 1 . 3 8 C a - 48.05Cb

Methanol, 100% (pure solvent):

Ca = 16.72A665.2- 9.16A652:
Cb = 34.09A652.4 - 15.28A665.2
Co+b = 1.44A665.2- 24.93A652.4
Cxc = 1000A47o- 1.63Ca- 104.96Cb
221

211
Ethanol, 95% (v/v):
C,
= 1 3 . 3 6 A ~ , 2 - 5.19A64s.6
Cb = 27.43As4s.6 - 8.12A~.2
Co+b = 5.24A~.2 + 22.24A~.6
C~+c = 1000A470 - 2.13C~ - 97.64Cb

Methanol,
Ca =
Cb =
Ca+b =
Cx+c =

90% (v/v):
16.82A665.2- 9.28A652.4
36.92A652.4- 16.54A665.2
0.28A665.2 + 27.64A652.4
1000A47o- 1.91Ca- 95.15Cb
225

209

a The equations are based on the redetermined specific absorption coefficients listed in
Table II. The pigment concentrations obtained by inserting the measured absorbance
values are micrograms per milliliter plant extract solution.

solvent can be compared directly with those of another solvent, including

t h e r a t i o o f c h l o r o p h y l l a/b a n d t h e t o t a l a m o u n t o f l e a f c a r o t e n o i d s .

Spectral Characteristics

of Carotenoids

General Remarks
All chloroplast carotenoids exhibit a typical absorption spectrum
w h i c h is c h a r a c t e r i z e d b y t h r e e a b s o r p t i o n m a x i m a ( v i o l a x a n t h i n , n e o x -

[34]

.Q
o
.Q
<

CHLOROPHYLLS AND CAROTENOIDS

367

0.500

0.500

'\

0
300.0

-~-~
350.0

450.0

0
550.0

Wavelength (nm)
FIG. 7. Absorption spectrum of the major leaf carotenoids in diethyl ether. The wavelengths at the absorption maxima are given in Table V. /3-Carotene (--), lutein ( - - - ) ,
violaxanthin (.-.), and neoxanthin (-'..).

anthin) or two maxima with one shoulder (lutein and/3-carotene) in the

blue spectral region (Fig. 7). The wavelength position of the maxima of
oxygen-free carotenoids such as/3-carotene is oriented to longer wavelengths than those of the oxygen-bearing xanthophylls. With increasing
amounts of hydrophilic groups in the tetraterpenoid carbon skeleton of
carotenoids the maxima (and shoulder) are shifted to shorter wavelengths. The absorption maxima of the pure polyene/3-carotene are 475.4
and 449.2 nm in methanol, whereas those of neoxanthin (with three hydroxy groups and one epoxy group) are 464 and 435.6 nm (Table V).
The identification of pigments can also be performed with the new
method of photoacoustic spectroscopy (PAS method). 52,53 One obtains
52 C. Buschmann, H. Prehn, and H. K. Lichtenthaler, Photosynth. Res. 5, 29 (1984).
53 E. Nagel, C. Buschmann, and H. K. Lichtenthaler, Physiol. Plant. 70, in press (1987).

368

PLASTIDS

[34]

.--.

j:

fll
t

-s

.:
"

t
?
.-

",~
I
t
t

"
:'
~:

to
,<
IX.

6606 0

460
wovelength

[nm]

FIG. 8. Photoacoustic spectra ~3 of leaf pigments as measured on a silica gel plate after
HPTLC of leaf pigments. 34 Chlorophyll a ( ~ ) , chlorophyll b (....), fl-carotene (- - - -).
(Spectra taken by E. Nagel, Karlsmhe.)

photoacoustic spectra which resemble absorption spectra but which are

distinctly different from pure absorption spectra. An example for photoacoustic spectra of fl-carotene as well as chlorophyll a and b is shown in
Fig. 8.
The wavelength position of the absorption maxima of carotenoids also
depends on the type of solvent and its water content. The shortest wavelengths are found for pure methanol. There is an increasing shift of the
maxima to longer wavelengths from methanol via diethyl ether, ethanol
(95%), acetone (100%), to acetone (80%) (Table V). In solvents such as
methanol (90%) or water-saturated diethyl ether, the maxima are shifted
by 0.4 to 0.6 nm to longer wavelengths as compared to pure water-free
solvent. At a higher water content [e.g., acetone (80%) versus acetone
(100%)] the shift can be I nm or more (Table V). In the series methanol,
diethyl ether, ethanol, and acetone the shift of the maxima to longer
wavelengths does not follow the polarity of the solvents. The series of
shift for carotenoids is also different from that of chlorophylls, where the
absorption maxima shift to longer wavelengths in the series diethyl ether,
acetone, ethanol, methanol (cf. Table II).

SpecO~cAbsorption Coefficients
The molecular weights of carotenoids differ due to the differential
oxygen content (B-carotene, 536.85, lutein, 568.85, violaxanthin, 600.85,
neoxanthin, 600.85). The specific absorption coefficients for the individual carotenoids found in literature vary considerably. Most of them lie in

[34]

CHLOROPHYLLS AND CAROTENOIDS

369

the range of 2400 to almost 2600 for a 1% solution and a light path of 1 cm
(A I~
1 cm values). To avoid the problem of the proper coefficient, it was
recommended to apply for the sum of leaf carotenoids 54 and for all individual carotenoids 55 an ,11
AI~cm of 2500 at their main absorption maximum.
This is still the best approach today. The specific absorption coefficients
also vary depending on the type of solvent and its water content. Per
.41%
definition the .,1
~c m of 2500 for the individual carotenoids is determined
here with diethyl ether (pure solvent) as a reference basis.

Determination of Total Carotenoids (x + c)

The sum of carotenoids (xanthophylls +/3-carotene; x + c) can easily
be determined in a total leaf or chloroplast pigment extract together with
chlorophylls by measuring the absorbance not only in the absorption maxima of chlorophyll'a and b, but additionally at a wavelength where carotenoids show good absorption, e.g., at 470 nm (Fig. 7). The absorbance of
the pigment extract measured at 470 nm is mainly due to light absorption
by carotenoids; a smaller amount comes from chlorophyll b, whereas
chlorophyll a contributes very little to the absorption at this wavelength.
The concentration of total carotenoids (Cx+c) can therefore be determined
by deduction of the relative absorption of chlorophyll a and b from the
absorbancy read at 470 nm followed by division by the absorption coefficient of total carotenoids at 470 rim. Equations for determination of Cx+c
in different solvents are shown in Table III. Though the method is quite
elegant and reliable, certain precautions have to be taken.
First of all, this method requires a correct determination of the chlorophyll b concentration beforehand. If the chlorophyll b level were overestimated, the resulting values for total carotenoids would be too low and
vice versa. The equation for Cx+cgiven in Table III is based on measuring
the relative absorbance of the chlorophylls at 470 nm with respect to the
absorption at hma x in the red in a UV-260 Shimadzu spectrometer (cf.
Table IV). The equations presented here are certainly also valid for other
modern two-beam spectrophotometers. Depending on the technical setup of other, often older, two-beam spectrophotometers, the wavelengths
o f hmax of chlorophyll a and b may deviate from the values in Table II by
0.8 to 1.5 nm. This may then cause considerable differences in the absorbancy readings, particularly at hmax of chlorophyll b at 470 rim, which will
affect the determination of total carotenoids. The spectrophotometer applied in a laboratory should therefore be checked with freshly isolated
54 T. W. Goodwin, in " M o d e m Methods of Plant Analysis" (K. Paech and M. V. Tracey,
eds.), Vol. 3, p. 272. Springer-Verlag, Berlin and New York, 1955.
55 H. K. Lichtenthaler, Z. Pflanzenphysiol. 53, 388 (1965).

370

PLASTIDS

[34]

TABLE IV
RATIOS OF THE SPECIFIC ABSORPTION COEFFICIENTS AT ~.maxIN THE BLUE TO hmax IN
THE RED FOR CHLOROPHYLL a AND b AND AVERAGE ABSORPTION COEFFICIENTS Alc~m
AT 470 nm FOR TOTAL LEAF CAROTENOIDS (XANTHOPHYLLS PLUS CAROTENES, X + C) IN
SOLVENTS OF DIFFERENT POLARITY AND WATER CONTENT

Ratio of the
specific coefficients
at 7~m~xblue to hmaxred
Solvent

Chlorophyll a

Chlorophyll b

Total
carotenoids (x + c):
A lcm at 470 nma

Diethyl ether
(water free)
Diethyl ether
(pure solvent)
Diethyl ether
(water saturated)
Acetone
(pure solven0
Acetone, 80% (v/v)
(aqueous solution)
Ethanol, 95% (v/v)
(aqueous solution)
Methanol
(pure solvent)
Methanol, 90% (v/v)
(aqueous solution)

1.28

2.75

2130

1.26

2.71

2050b

1.23

2.69

2110

1.22

2.81

2140

1.11

2.73

1980

0.99

2.61

2100

0.97

2.71

2210

0.96

2.66

2250

a The mean values listed are based on the relative carotenoid composition of green leaf
tissue from several plants (e.g., radish, spinach, tobacco, maize, oat, and wheat).
b This value consists of the relative absorption of the individual leaf carotenoids (/3carotene, lutein, violaxanthin, and neoxanthin) at 470 nm as compared to the absorption maximum. An absorption coefficient Alcm
1~ of 2500 in diethyl ether at the main
absorption maximum (between 436 and 450 nm) was assumed for all individual leaf
carotenoids.

c h l o r o p h y l l a a n d b s o l u t i o n s , a n d t h e w a v e l e n g t h d e v i a t i o n at ~kmax a n d
t h e shift at 470 n m b e a c c o u n t e d f o r ( s e e a d j u s t m e n t b e l o w ) . F u r t h e r
requirements are clear solutions and a correct measurement of the baseline.
T h e r e c o r d i n g o f t h e full a b s o r p t i o n s p e c t r u m o f t h e p i g m e n t e x t r a c t
s o l u t i o n b e t w e e n 750 a n d 450 n m is n e c e s s a r y to b e a b l e to j u d g e f r o m t h e
shape of the spectrum whether one measures a clear pigment solution. In
fully clear pigment extract solutions of normal green leaves the absorb e n c y r e a d i n g s at 750 n m s h o u l d b e z e r o , a n d in t h e r a n g e o f 550 to 510 n m

[34]

CHLOROPHYLLSAND CAROTENOIDS

371

they should be very low and amount to far less than 10% of the absorbancy readings at the red absorption maximum of the extract (in the range
of 660-665 nm). If this is not the case, a centrifugation step (caution: use
closed flasks to avoid evaporation of the solvent) has to be applied.
Strictly sticking to another rule is essential for correct pigment values:
after the recording of the full spectrum the absorbancy readings need to be
performed by setting the proper wavelengths (absorption maxima of chlorophyll a and b as well as 470 nm) on the spectrometer and by marking the
height of the absorbancy by a line with the help of the spectrometer pen.
The measurement of the absorbancy on the recording paper, after the
spectrum had been recorded, by trying to visually determine the proper
wavelengths, is to be avoided. It results in errors up to 20 or 25% of the
chlorophyll b and total carotenoid amounts as well as of the ratio of
chlorophyll a/b.
The determination of the concentration of x + c in the total pigment
extract is the fastest and therefore best method and should always be
applied as a reference before other methods, HPLC or TLC, which allow
determination of individual carotenoids, are applied. TLC with subsequent elution and spectrophotometric determination of separated carotenoids always yields lower values (for B-carotene about - 5 % and for
xanthophylls up to - 13%). The sum of carotenoids determined by evaluation of the individual carotenoid peaks obtained by HPLC often does not
add up to the x + c content obtained by the direct measurement of the leaf
extracts or exceeds it to a large extent, depending on where the baseline is
drawn on the H P L C scans. It is therefore recommended to proof the
values for individual carotenoids obtained by HPLC with the TLC technique 34 mentioned above (see Chapter 2).
The total sum of carotenoids can also be determined in a pigment
extract together with pheophytin a and b, provided that the absorbancy
readings are performed within 10 min after pheophytinization (e.g., with
HC1). A longer standing in acid solution will progressively destroy carotenoids. The x + concentrations obtained using this method (see equations in Table VII) represent a close but only a first approximation. Since
the spectra of pheophytins change with the acid content, one should stick
to the standard conditions (see The Pheophytins a and b, p. 373).

Al%
1 cm o f

the Sum o f Carotenoids

The A [~m values at 470 nm given in Table IV and applied in the equations for Cx+c in Table III are calculated from the relative absorbance of
the individual carotenoids as compared to their absorbance at the main
absorption maximum. For the latter a common absorption coefficient of

372

PLASTIDS

[34]

EL
0
Z

>
.J
0

z
,..1
O
0

e~

>

1.-,
a~
0

E
..g

0
,.J

0
@

N
m
,..1
>

[34]

CHLOROPHYLLS AND CAROTENOIDS

373

2500 in diethyl ether was assumed for the individual carotenoids) 5 In

view of the different wavelength position of the two (or three) carotenoid
maxima and the nearby depressions (cf. Fig. 7), which cancel each other
out in a total carotenoid solution, it is clear that the A [~m value of the sum
of chloroplast carotenoids is lower than 2500. The A i~1
cm value in diethyl
ether of total carotenoids as isolated from green leaf extracts of seven
different plants lies in the range of 2400 to 2450 depending on the carotenoid composition of the plant that is the relative proportions of xanthophylls to/t-carotene. The same arguments (partial compensation of the
second carotenoid maximum near 470 nm of one carotenoid with a nearby
depression of another carotenoid) hold true for the A l%mX + value at 470
nm. The values given in Table IV represent mean values based on the
carotenoid composition of green leaves of seven plants.
The Pheophytins a and b
General Remarks
Chlorophylls a and b are easily transformed by weak acids to their
magnesium-free derivatives, the pheophytins a and b. Their molecular
weight (pheophytin a, 871.18; pheophytin b, 885.16) is 22.3 weight units
smaller than that of the corresponding chlorophylls. Small amounts of
pheophytin a have always been found in plant pigment extracts and seem
already to be present in the intact leaf. In fact, pheophytin a is a functional component of the reaction center of photosystem II (RC II), where
it plays a role in the charge separation and electron transfer from P680.56, 57

Chlorophyll a is more sensitive to pheophytinization than chlorophyll

b, as can be seen from thin-layer chromatography (TLC) of pigment extracts on silica gel plates. Silicic acid, as a weak acid, will transform part
of the chlorophyll a into pheophytin a, whereas chlorophyll b is little
affected. Both pheophytins are formed under the influence of the endogenous organic acids during pigment extract preparation, while grinding the
leaf tissue in the presence of an extracting solvent with quartz sand or by
maceration in a homogenizer. Addition of some magnesium oxide or calcium carbonate will neutralize the endogenous organic acids. This is particularly needed in the case of plants which are especially known to store
organic acids in their vacuoles. Pheophytinization is much enhanced with
increasing temperature. Homogenization of the leaf tissue in a homoge56 V. V. Klimov, A. V. Klevanik, V. A. Shuvalov, and A. A. Krasnovsky, FEBS Lett. 82,
183 (1977).
57 T. Omata, N. Murata, and K. Satoh, Biochim. Biophys. Acta 765, 403 (1984).

374

PLASTIDS

[34]

nizer should therefore be kept very short (20 to 30 sec) and lie far below 1
min. It is also recommended to perform the extraction with solvents
which have been stored in the cold room (e.g., at 3 to 5).
Once a pigment extract has been prepared, the chlorophyll content
should be determined immediately thereafter. The level of pheophytin a
and b will increase with increasing storage time of the extract. This is
particularly the case in organic solvents which are miscible with water
such as acetone, methanol, or ethanol. In cases where the storage of a
pigment extract is required, it should only be done in a water-free organic
solvent, i.e., petroleum hydrocarbon (e.g., bp 40 to 70) to minimize
pheophytinization. This can be accomplished in a separatory funnel by
transferring the pigments of the aqueous extraction solvent, adding petroleum hydrocarbon and water, into the organic hydrocarbon epiphase. The
aqueous acetone (methanol or ethanol) hypophase should contain at least
50% water to guarantee a quantitative transfer of the more polar pigments
(chlorophyll b and xanthophylls) into the water-free epiphase also.

Transformation of Chlorophylls into Pheophytins by HCI and

Quantitative Determination
There are many cases in present scientific research (e.g., senescent
tissue, leaves from plants fumigated with acid gases such as SO2 or NO2,
plants under heat or light stress, extracts from herbicide-treated chloroplast particles, or from chlorophyll proteins after gel electrophoresis),
where pigment extracts may contain pheophytins together with chlorophylls, and one would like to determine the total amount of pigment in
terms of chlorophyll equivalents. This is possible by quantitatively transforming the remaining chlorophylls into pheophytins under defined conditions (see below). The total amount of pigments in terms of chlorophyll a
and b equivalents is then determined by two-wavelength spectrometry of
the resulting pheophytins at the red absorption maxima of pheophytin a
and b. This method goes back to Willst~itter and Stoll. 3
Equations for the quantitative determination of pheophytin a and b are
given by Bauer 58 for diethyl ether (transformation with HCI) on the basis
of the absorption coefficients for chlorophyll a and b of Smith and
Benitez. 49
Diethyl ether59:
Cph a = 16.7A667 - 3.20A665
Cph b = 25.3A655 - 8.10A667
Cpha+b "~ 8.60A667 + 22.10A655
Bauer, Planta51, 84 (1958).
59Cpha, b,a+b, Concentration of pheophytins a, b, a + b in p.g/rnl extract solution.

5s A.

[34]

CHLOROPHYLLS AND CAROTENOIDS

375

Equations in 80% acetone (transformation with oxalic acid) were determined by Vernon45:
20.15A663 - 5.87A655
Cph b = 31.90A655 - 13.40A666
Cpha+b = 6.75A663 + 26.03A655
Cpha ~-

Though these equations in both solvent systems provide good values

for the total amounts of pheophytin a + b (or chlorophyll a + b), the
relative levels of pheophytin a and b are not accurate, which results in
values for the ratio of pheophytin a to b which are too low.
By use of a modem spectrophotometer (UV-260 Shimadzu), which
allows automatic recording and printing out of the proper wavelengths
and absorbance values at the major and minor absorption maxima, we
redetermined specific absorption coefficients of pheophytin a and b in
diethyl ether and 80% acetone and also established the coefficients for
several other solvents (Table VI). This was performed by transforming
chlorophyll a and b, which were freshly isolated by TLC, 34 into their
pheophytins. The mean values shown in Table VI are based on at least
five chlorophyll isolations per solvent and on the new specific absorption
coefficients for chlorophylls shown in Table II. The repetitive recording
was carried out at different chlorophyll (pheophytin) concentrations (2 to
15/zg/ml). The differences from the older values of Bauer 58 and Vernon 45
are mainly due to a more accurate determination of the proper wavelengths of the main absorption maxima with the new generation of spectrophotometers and to the fact that the absorbance readings, e.g., of
pheophytin a at hmaxof pheophytin b, can be performed automatically.

Determination of Pheophytin a and b

On the basis of the new specific absorption coefficients for pheophytins (Table VI), equations were established which allow the determination
of pheophytin a and b of plant pigment extracts in different solvents
(Table VII). The equations for the different solvents were cross-checked
using pigment extracts from seven different plants. For routine analysis
we highly recommend the solvents diethyl ether (equations given in Table
VII) and 80% acetone (equations in Table VII). In these solvents the
spectral characteristics of pheophytins are almost independent of the acid
content of the solution.

Pheophytinization under Defined Conditions

The transformation of chlorophylls in 5 ml of a leaf pigment extract
solution (range of 5 to 20/.~g a + b/ml) to their pheophytins proceeds

376

PLASTIDS

[34]

Z
ga
t~

I~ ~

t~
0

u~
>
,.d
0
r/)

~l ~ ~i~ ,~ ~l ~ l ~ l
zz

~z~
;0 <

~ r.,.)

e~

~1~ " ~

~l ~

~qeq

r--

m
0

r~

e~

<

6.
m

~g

e q o o

,0

[34]

CHLOROPHYLLS AND CAROTENOIDS

377

TABLE VII
EQUATIONS FOR THE DETERMINATION OF THE CONCENTRATIONS OF PHEOPHYTIN a (Cph a),
PHEOPHYTIN b (Cphb), TOTAL PHEOPHYTINS (Cph a + b), AND OF TOTAL CAROTENO1DS (Cx + c) IN
LEAF PIGMENT EXTRACTS, IN WHICH THE CHLOROPHYLLS a AND b HAD BEEN COMPLETELY
TRANSFORMED TO PHEOPHYTINS a
Diethyl e t h e r (pure solvent) b
Cph,
= 17.87A666.6- 3.58A654.2
Cphb
= 24.62A6s4.2 - 8.46A666.6
Cpha + b = 9.41A666.6 + 21.03Aas4.2
Cx + c = 1000A470 - 4.39A666.6 - 6.44A654.2

A c e t o n e , 80% ( a q u e o u s , v / v )
Cph a
~--- 22.42A66L4 -- 6.81A653.4
Cphb
= 40.17A653.4 - 18.58A665.4
Cph,,+b = 3.84A665.4 + 33.36A653.4
C, + ,. --- 1000A470 - 4.28A~5.4 - 4.78.4653.4
164

174
Diethyl e t h e r ( w a t e r saturated) c
Cpha
= 18.22A666.6- 3.15A654.0
Cphb
= 32.33A654 - 10.14At~6.6
Cph~+b = 7.08A666.6 + 29.18A654
Cx+,, = 1000A47o - 7.09A666.6 - 4.51A654

M e t h a n o l (pure solvent)
Cph a
= 43.77A654.2 - 33.40Ae476
Cphb
= 50.71A647.6 - 36.32A654.2
Cpha+b = 7.45A654.2 + 17.31A6476
C~+,
= I000A470 - 1.53A654.2 - 1.57A647.6
160

135
E t h a n o l , 95% (v/v)
Cpna
= 42.41A666.2
Cphb
= 55.67A654
Cph~+b = 32.39A654
C~ + c = 1000A470 -

- 23.28A654
- 45.53A6622
- 3.12A662.2
4.32A662.2 - 7.30A654

Methanol,
Cpho
Cph b
Cph, + b
Cr + ~

90% ( a q u e o u s , v / v )
= 306.8A655.2 - 294-2A6s3.4
~ 442.2A653.4 - 429.4A655.~
=
148A653.4 -- 122.6A655.2
= 1000/147o - 2.66,4655.2 - 6.38,46.53.4

160

126
Cph ~ + b = 23. IA654

A c e t o n e (pure solvent)
Cpha
= 516.7A653.4
Cob b
= 732.5A652.6
Cpha + b = 321.3A652.6
C~ + c = 1000A470 -

- 501.2A652.6
-- 725. IA~3.4
- 208.4A653.4
2.02A653.4 - 5.97A652.6
148

Cpha + b = 22.3A653

" T h e t r a n s f o r m a t i o n o f c h l o r o p h y l l s into their p h e o p h y t i n s w a s p e r f o r m e d in all s o l v e n t s y s t e m s

by adding o n e d r o p o f a 25% a q u e o u s h y d r o c h l o r i c acid solution (HCI) to 5 ml o f the total leaf
p i g m e n t extract.
b T h e e q u a t i o n s given for solvents o f different polarity a n d w a t e r c o n t e n t are b a s e d o n the newly
d e t e r m i n e d specific a b s o r p t i o n coefficients listed in Table VI. T h e c o n c e n t r a t i o n s o b t a i n e d by
inserting the a p p r o p r i a t e m e a s u r e d extinction ( a b s o r b a n c e ) v a l u e s (at ~,~x o f p h e o p h y t i n a,
p h e o p h y t i n b, and at 470 nm) are m i c r o g r a m s p i g m e n t p e r milliliter extract solution. T h e a m o u n t s
of p h e o p h y t i n a and b are e x p r e s s e d as m i c r o g r a m s c h l o r o p h y l l a a n d b equivalents. By multiplication with the factors 0.975 a n d 0.9754 o n e o b t a i n s the values, in m i c r o g r a m s , of p h e o p h y t i n a
and b, respectively.
c P i g m e n t e x t r a c t s in w a t e r - s a t u r a t e d diethyl e t h e r b e c o m e turbid u p o n addition o f one d r o p o f HC1
solution ( + shaking) and n e e d to be centrifuged (5 min) b e f o r e the a b s o r b a n c y readings.

378

PLASTIDS

[34]

instantaneously after addition of one drop of a 25% HCI solution (mild

shaking) and is terminated after 30 to 60 sec for most solvents. Only
extracts in diethyl ether solutions do not fully absorb the droplet of HCI
solution and become slightly turbid, but become clear after a few minutes.
Only in the case of water-saturated diethyl ether is a short centrifugation
(laboratory centrifuge) needed to clear the solution. In order to avoid
evaporation of diethyl ether with subsequent absorbancy readings, which
are too high, the tubes should be closed. One further precaution: since the
specific absorption coefficients and the wavelength position of the absorption maxima of pheophytins in the solvents acetone (100%), methanol
(I 00 and 90%), and ethanol (95%) are dependent on the acid content of the
pigment solution (see spectral characteristics below) one should stick to
the defined conditions for pheophytinization described here, when one
would like to use the equations listed in Table VII.

Total Carotenoid Content in Pheophytinized Leaf Extracts

The total carotenoid content of the pheophytinized leaf extracts (Cxc)
can be determined in a first approximation by inserting the measured
absorbance at 470 nm--in addition to that at hmaxof pheophytin a and b - into the equations given in Table VII for different solvents. This requires
prompt readings of the absorbancy after pheophytinization before a slow
decomposition of carotenoids takes place. The acid and pH dependency
of the pheophytin spectrum also affects the absorbancy readings of a leaf
extract at 470 nm. When the conditions of pheophytinization mentioned
above are adhered to, one obtains good and fully reproducible carotenoid
amounts which correspond to those found in the pigment extracts containing chlorophyll.

Spectral Characteristics of Pheophytins

As compared to chlorophylls, the red absorption maxima of isolated
pheophytins in acid-free solution are shifted to longer wavelengths with a
distinct decrease of the specific absorption coefficients (Table VI; Fig. 9).
The maxima in the blue spectral region, however, are shifted to shorter
wavelengths, whereby the specific coefficient values of the blue maxima
are either little affected or become larger by comparison to those of chlorophylls. Due to these facts the ratio of the specific absorption coefficients
at hmaxin the blue to hmx in the red are much higher (Table VIII) than
those of chlorophylls (Table IV). These general statements hold true for
the acid-free pheophytins in all tested solvent systems. They are also
valid for diethyl ether and 80% acetone in the presence of some HCI (one
drop 25% HCI/5 ml extract solution).

[34]

CHLOROPHYLLS AND CAROTENOIDS

1

379
1

0
U
C
q

0.500

0.500
iI

<

/ :,

qL~~!ili!l

so.o

4so.o

sso.o

6so.o

7so.o

Wavelength (nm)
FIG. 9. Absorption spectra of freshly isolated pheophytin a (solid line) and pheophytin b
(broken line) in diethyl ether. The positions of the absorption maxima are given in Table VI.

In acetone (100%), methanol (100 and 90%), and ethanol (95%) the red
absorption maximum of chlorophyll a, however, shifts to shorter wavelengths upon pheophytinization with HCI (see Tables II and VI). The blue
maximum only shifts to ca. 418 nm with a considerable increase in the
absorption in the place of 411 nm in acid-free solution. By increasing
addition of water there is a change in the spectral characteristics of
pheophytin a with the appearance of the proper spectra of the acid-free
pheophytin. Correspondingly the coefficient ratio (hrnax blue to ~r,x red) is
much higher for pheophytin a in acid solution than under neutral conditions (see Table VIII). The differential spectral behavior of pheophytin a
in acid and neutral solution is a particular property and has nothing to do
with incomplete pheophytinization. By addition of one drop of HCI to a
neutral solution of isolated pheophytin a, one changes the absorption
spectrum to that of pheophytin a in the presence of acids (see Fig. 10).
For pheophytin b a shift to shorter wavelength only occurs in 100%
methanol. In the other solvents one obtains even in the presence of some
HC1 spectra which are fairly similar in wavelengths and absorption values
to those of the acid-free solvent solution. This indicates that the spectrum
of pheophytin b - - w i t h the exception of pure methanol solutions--is less
sensitive to changes in an acid milieu than that of pheophytin a.

380

PLASTIDS

[34]

TABLE VIII
RATIOS OF THE SPECIFIC ABSORPTION COEFFICIENTS a AT )~rnax
IN THE BLUE TO )Lrn~ IN THE RED SPECTRAL REGION FOR
PHEOPHYTIN a AND b

Ratio of the specific coefficients at

ikmax blue to hmaxred b
Solvent
Diethyl ether
(pure solvent)
Diethyl ether
(water saturated)
Acetone, 80% (v/v)
(aqueous solution)
Acetone
(pure solvent)
Ethanol, 95% (v/v)
(aqueous solution)
Methanol
(pure solvent)
Methanol, 90%
(aqueous solution)

Pheophytin a

Pheophytin b

2.10

5.02

2.08

5.03

2.36

5.07

4.25 (2.15)

5.10 (4.91)

3.33 (2.09)

4.31 (4.99)

4.09 (2.08)

4.91 (4.72)

3.85 (2.12)

4.03 (4.57)

a The coefficients were determined in the presence of some

HCI, which was applied to transform the chlorophylls into
their pheophytins (sec Table 6).
b Values in parantheses represent the ratios of coefficients (at
)tm~xbluc to red) in the acid-free solvent.

Adjustment of Equations to Other Spectrophotometers

In order to check whether the equations for pigment determination
(Table III)can be used with the absorbancy readings of a particular spectrophotorncter present in a laboratory, one should isolate fresh chlorophyll a and b (e.g.,by TLC, see Lichtcnthalcr et al.34) and determine (I)
the wavelength position of their m a x i m u m absorption in the red spectral
region and (2) their relative absorption at hm~x of the othcr chlorophyll as
well as that at 470 nm. This is best pcfforrned at concentrations with
absorbancy values between 0.4 to 0.8/I crn light path.
W h e n the specific absorption coefficient at hr~x red of chlorophyll a
and b in a particular solvent is accepted from Table IIDwhich can be
done in most cases even though the position of hm~,,may bc differentby I
or 1.5 nrn from the wavelengths given in Table II--one can recalculate the
equations (Ca, Cb, Cx+c) for one's o w n instrument by use of the equations

[34]

CHLOROPHYLLSAND CAROTENOIDS

381

1.0'

417!!

!t li

ethanol 95%
--

,. li
ii
,~ Ii

HCt

5%

........

HCI

25%

os
/i

/.."

'

666.6

/-

as0

662-

~g0

/J':

~ "sg0
" "696 ....
7s0
wovelength (nm )
FIG. 10. Absorption spectrum of pheophytin a in aqueous (95%) ethanol (solid line) and
after addition of one droplet (-0.005 ml) of a 5 % ( - - - ) and of one droplet of a 25% HCI
solution (' "").
"

given below. The absorbance of a pigment extract solution (light path 1

cm) at hm~xof chlorophyll a (Amax~), at h~x of chlorophyll b (Amax b), and
at 470 nm is defined by the following three equations:
Amax

aCa + b'Cb
bCb + a' Ca
A470n m = a"Ca + b"Cb + kCx+c
a

Amax b

=
=

(1)
(2)
(3)

a, Specific absorption coefficient of chlorophyll (ch!) a at hmx (mg/liter);

a', specific absorption coefficient of chl a at hm,~ of chl. b (mg/liter); a",
specific absorption coefficient of chl a at 470 nm (mg/liter); b, specific
absorption coefficient of chl b at hmax (mg/liter); b', specific absorption
coefficient of chl b at hmax of chl. a (mg/liter); b", specific absorption
coefficient of chl b at 470 nm (rag/liter); k, specific absorption coefficient
of total carotenoids x + c at 470 nm (mg/liter)(=-~Ja~cm for x + c 0.1);

382

PLASTIDS

[34]

Ca, Cb, concentration o f chl a and b; Cx+c, concentration of total carotenoids.

T r a n s f o r m a t i o n o f Eqs. (1) and (2) results in the equations

(4)
(5)

Ca = [(b/Z)Amaxa] - [(b'/z)Amaxb]
Cb = [(a/Z)Amax b] - [(a'/Z)Amaxa]

where
z = ab - a ' b '

W h e n the specific absorption coefficients a and b (= 100%) are taken

f r o m Table I I the specific coefficients a', a", b', and b" can be calculated
as follows:
a'
a"
b'
b"

=
=
=
=

aEa/lO0
aE470/lO0
bEb/lO0
bE470~ 100

Ea = % a b s o r b a n c e of chl a
E470 = % a b s o r b a n c e o f chl
Eb = % a b s o r b a n c e of chl b
E470 = % a b s o r b a n c e of chl

at hmax chl b
a at 470 nm
at hmx chl a
b at 470 nm

B y inserting the recalculated values for a ' and b' in Eqs. (4) and (5)
one obtains the n e w equations for Ca and Cb, which are adjusted to the
particular s p e c t r o p h o t o m e t e r .
T h e equation to determine the concentration of total carotenoids Cx+c
(/zg/ml) is then obtained using the redetermined a" and b" values:
Cx+c = (1000A470 - a"Ca - b"Cb/O.1 AlUm o f x + c

A recalculation o f the A 1~
cm for x + c at 470 requires isolation o f the
total leaf carotenoids (e.g., b y T L C 33) in diethyl ether t,-,tal%l
c m for x + c at
hm~, = 2450) and m e a s u r e m e n t o f the relative proportion o f their absorption at 470 n m as c o m p a r e d to hmax o f x + c. The "1
A 1~
~cm value in diethyl
ether (2450) is used as reference.
Acknowledgments
The establishment of the absorption coefficients was made possible by the kindness of
Shimadzu Germany, Diisseldorf (Messrs. Steinfeld and Baumann), who for several weeks
provided a new UV-260 UV-visible two-beam spectrometer; this is gratefully acknowledged. My particular thanks are due to Dr. Claus Buschmann for writing the programme for
the HP 85 and to my son, Stefan Lichtenthaler, for programming the Apple IIe computer and
doing most of the calculating and cross-checking. Finally I would like to thank Mr. E. Nagel
for measuring the photoacoustic spectra of pigments as well as Mrs. U. Prenzel and U.
Sieber for excellent assistance.