Photosynthesis Research 73: 149–156, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

149

Minireview

The chequered history of the development and use of simultaneous

equations for the accurate determination of chlorophylls a and b

Robert J. Porra
Division of Plant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra, P.O. Box
1600, ACT 2601, Australia; Botanisches Institut, Ludwig-Maximilians-Universität München, Menzinger Str. 67,
D-80638 München, Germany (e-mail: r.porra@pi.csiro.au; fax: +61-2-62465000)

Received 4 July 2001; accepted in revised form 24 October 2001

Key words: absorption spectroscopy, accurate chlorophyll a and b determinations, algebraic correction method for
Arnon’s Chl determinations, chlorophyll a/b ratios, Daniel Arnon, LHC II, light-harvesting complex of photosytem
II, G. Mackinney, magnesium atomic absorption spectroscopy, molar and specific extinction coefficients, Richard
Willstätter

Abstract
Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the
Arnon assay [Arnon DI (1949) Plant Physiol 24: 1–15], employed simultaneous equations for determining the
concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials.
These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early
inaccurate spectrophotometric data. Thus, Arnon’s equations give inaccurate chlorophyll a and b determinations
and, therefore, inaccurate chlorophyll a/b ratios, which are always low. This paper describes how the ratios are
increasingly and alarmingly low as the proportion of chlorophyll a increases. Accurate extinction coefficients
for chlorophylls a and b, and the more reliable simultaneous equations derived from them, have been published
subsequently by many research groups; these new post-Arnon equations, however, have been ignored by many
researchers. This Minireview records the history of the development of accurate simultaneous equations and some
difficulties and anomalies arising from the retention of Arnon’s seriously flawed equations.

Abbreviations: Chl – chlorophyll; DMF – N,N
-dimethylformamide; DMSO – dimethylsulfoxide; LHC – light-
harvesting complex; PS I – Photosystem I; PS II – Photosystem II

Introduction Comar and F.P. Zscheile (1942) and Daniel Arnon

(1949) became available in the 1940s because, as dis-
During the last half century, plant biochemists stud- cussed in the next section, the Chl assays of the first
ied the effects of different light regimes, nutrients and half of the century (see R. Willstätter and A. Stoll
other growth conditions on the efficiency of various 1913) were slower, more difficult, and not especially
photosynthetic reactions including O2 evolution, CO2 accurate (cf. E. I. Rabinowitch 1945; J.H.C. Smith and
fixation, or carbohydrate biosynthesis. Because of the A. Benitez 1955; H.H. Strain and W.A. Svec 1966).
fundamental role of chlorophylls (Chls) in photosyn- My interest in the extraction and assay of Chls
thesis, the rates of these reactions were often presented was triggered by my colleagues W.A. Thompson and
per unit of Chl expressed in mass or molar terms; P.E. Kriedemann of the CSIRO-Division of Forestry
thus, reliable assays for Chls were required. It was and Forestry Products, Canberra, who were study-
fortunate, therefore, that the fast and convenient sim- ing the effects of various nutrients on the growth
ultaneous equation assays for Chls a and b of C.L. of Queensland Maple (Flindersia brayleyana); they
150

a and b were freshly extracted from maize leaves and

used immediately after chromatographic purification
(Porra et al. 1989); some previous studies employed
stored dried solid samples of Chls without further puri-
fication. The unexpected difficulty of extracting Chls
from some algae, an irritating problem for some of my
colleagues, was another challenge that furthered my
interest in Chl extraction and assay procedures.
The derivation of simultaneous equations from
molar rather than specific extinction coefficients was a
rather new innovation for Chl assays at the time (Porra
et al. 1989). It was inspired by the elegant work of
electron crystallographers who needed to accurately
determine the numbers of Chl a and Chl b molecules
located on each Chl a/b-polypeptide of LHC II (rather
than the mass of each Chl) to formulate realistic mo-
lecular models (see later section ‘Molecular modeling
of the major Chl a/b protein of LHC II’).
The determination of Chls a and b and of Chl
a/b ratios has also played an important role in in-
vestigations of how higher plants and algae adapt
their photosynthetic apparatus during acclimation to
new light regimes to make optimal use of ambient
Figure 1. Daniel Arnon (1910–1994) at his desk in the University of light intensities and qualities (see later section ‘Light
California at Berkeley in 1988. His simultaneous equation assay for acclimation studies’).
chlorophylls was the most frequently used after 1950. Photograph
reproduced with the kind permission of Dr R. Buchanan, University
of California at Berkeley.
The derivation of the simultaneous equation
method for the assay of Chls a and b
used Chl concentrations in leaves as one indicator
of plant health. They found that Chl a and b deter- The longest and most used assay to determine the con-
minations in the tough leathery leaves when extracted centrations of Chls a and b in plant and algal materials
and determined in N,N
-dimethylformamide (DMF) was that of Arnon (1949). In this method, pigments
(Inskeep and Bloom 1985) or in methanol (Böger, were extracted in aqueous 80% acetone and determ-
1964) differed from those obtained with aqueous 80% ined in the same solvent. The concentration of each
acetone (Arnon 1949). Further, the Chl a/b ratios Chl was determined by measuring the extinction of the
obtained by Arnon’s method were much lower than extract at the major red absorption (QY ) maxima of
those obtained in aqueous acetone using the more Chl a (∼664 nm) and b (∼647 nm) and inserting these
accurate equations of Ziegler and Egle (1965) and values into the simultaneous equations [6] and [7] (see
H. Lichtenthaler (1987). Consequently, I decided below). Acetone was diluted with 20% (v/v) water so
to determine accurate extinction coefficients, both that further dilution by extracted cell sap would be
specific (α = l·g−1 ·cm−1 ) and millimolar ( mM = insignificant and thus leave the wavelength and intens-
l·mmol−1·cm−1 ), in all three solvents to derive reli- ity of the QY maxima of Chl a and b unaffected (cf.
able simultaneous equations giving compatible Chl a Porra et al. 1989). Later, buffering the aqueous acet-
and b concentrations and Chl a/b ratios (Porra et al. one, at pH 7.8, was introduced to minimize pheophytin
1989); it should be noted here that the above definition formation by loss of the Mg atom in the presence of
of the specific extinction coefficient (α) used in tet- extracted metabolic acids.
rapyrrole chemistry (Smith and Benitez 1955; Porra et Daniel Arnon (see Figure 1) used the spectro-
al. 1989) differs from that in more general use (i.e. 100 photometric data of G. Mackinney (1941) shown in
ml·g−1 ·cm−1 ). To achieve the required compatibility Table 1 to develop his assay. Since no other pigment
of Chl a and b concentrations and Chl a/b ratios, Chls extracted by these solvents, including carotenoids, in-
 151
Table 1. Errors in the specific extinction coefficients of Mackinney (1941). The specific extinction coefficients of Chls a and b in aqueous
80% acetone obtained by Mackinney (1941) and Porra et al. (1989) are compared. The percentage errors are calculated assuming that the
coefficients of Porra et al. (1989) are correct

Workers Wavelength Chl a Chl b

(nm)a Spec. ext. Error Spec. ext. Error
(α) (α)

Porra et al. (1989) 663.6 85.95 0 10.78 0

646.6 20.79 0 51.84 0
Mackinney (1941) 663 82.04 –4.55% 9.27 –14.01%
645 16.75 –19.43% 45.60 –12.03%

a The wavelengths of the Q peaks of Chls a and b are variously reported in the literature but are near 664 and 647 nm, respectively.
Y

terfered with the red absorption of these two Chls, The Chls were displaced into diethylether by dilution
Arnon reasoned that the extinction (E) of these mixed with water and the ethereal phase was then washed
Chl extracts at 663 and 645nm could be described as free of acetone with more water before drying over
follows: anhydrous sodium sulphate for spectrophotometric
analysis.
E 663 = 82.04 · [Chl a] + 9.27 · [Chl b] (1)
The simpler one-step extraction method of Arnon
(1949) quickly replaced the earlier multistep technique
E 645 = 45.60 · [Chl b] + 16.75 · [Chl a] (2)
(Comar and Zscheile 1942). Both these methods, how-
[Chl a] and [Chl b] represent Chls a and b concentra- ever, rapidly supplanted the earlier and more difficult
tions expressed in g·l−1 . From Equation (2), assay of Willstätter and Stoll (1913) in which Chls a
and b were acidified to form phaeophytins a and b,
E645 − 45.60 [Chl b] which were treated with KOH-methanol to open the
[Chl a] = (3)
16.75 isocyclic ring and form rhodochlorins a and b (i.e.
chlorin e6 and rhodin g7 , respectively, using earlier H.
By inserting Equation (3) for [Chl a] in Equation (1)
Fischer nomenclature). The two rhodochlorins were
and solving for [Chl b], Equation (4) is obtained:
transferred to diethylether, and the green rhodochlorin
[Chl b] = 0.0229·E645 – 0.00468·E663 (4) a was extracted exhaustively with 3% HCl and the
red rhodochlorin b with 12% HCl and both were de-
By inserting Equation (4) for [Chl b] into Equation (2)
termined colorimetrically against standard solutions of
and solving for [Chl a], Equation (5) is obtained:
known concentration. Although this assay gave low
[Chl a] = 0.0127·E663 – 0.00269·E645 (5) Chl a/b ratios (cf. Rabinowitch 1945), it produced
much useful information (see section ‘Light acclima-
Equations (4) and (5) are usually multiplied by 103 as
tion studies’). Richard Willstätter (see Figure 2) was
shown in Equations (6) and (7), respectively, and the
awarded the Nobel Prize for Chemistry in 1915 for
addition of Equations (6) and (7) gives Equation (8)
his investigations of plant pigments, especially the
for total Chl, designated [Chls a + b]. Equations (6),
chlorophylls.
(7) and (8), which express [Chl a], [Chl b] and [Chls a
Later, Chls a and b, as Chls or as their pheophytins,
+ b] in µg·ml−1 , are those published by Arnon (1949).
were assayed photometrically after chromatographic
[Chl b] = 22.90·E645 – 4.68·E663 (6) separation on sucrose columns (Seybold and Egle
1938). Prior to the 1940s, Chls a+b were often assayed
[Chl a] = 12.70·E663 – 2.69·E645 (7) colorimetrically as Chls, pheophytins, or rhodochlor-
ins against relevant standards, but with little or no
[Chls a + b] = 20.21·E645 + 8.02·E663 (8) allowance for differences in Chl a/b ratios between
samples and standards. The development of simple,
Arnon (1949) was not the first, however, to assay fast, and accurate simultaneous equation assays was,
Chls a and b using simultaneous equations. Previously, therefore, a great and much-needed advance.
Comar and Zscheile (1942) assayed Chls a and b in
diethylether after extraction from leaves with acetone.
152

QY peaks, but DMF and DMSO are more toxic than

aqueous acetone. The QY absorption bands of Chls
in methanol are broad and less sharp than in aqueous
acetone; although an efficient extractant of Chls, meth-
anol enhances degradation of Chls by opening the
isocyclic ring, especially in alkaline conditions (Porra
1990a, 1991).
Because Chl determinations in DMF (Inskeep and
Bloom 1985), methanol (Böger, 1964), and aqueous
80% acetone (Arnon 1949) were incompatible (see
‘Introduction’), Porra et al. (1989) obtained accurate
molar () and specific (α) extinction coefficients for
freshly prepared samples of chromatographically pure
Chls a and b in these three solvents (see Table 2). The
concentrations of the standard Chl a and b solutions
used to determine these coefficients were verified to
within an error of 1% or less, by magnesium determ-
ination using atomic absorption spectrometry (Porra
et al. 1989). The simultaneous equations derived from
the accurate coefficients given in Table 2 are shown in
Figure 2. Richard Willstätter (1872–1942), Nobel Laureate in Table 3. Using the equations in Table 3, good agree-
Chemistry, 1915. In 1913, at the Kaiser Wilhelm Institut für ment was now obtained for Chl a and b concentrations
Chemie, Berlin, Willstätter developed an assay for chlorophylls with and Chl a/b ratios when the pigments were extracted
Arthur Stoll which was in general use until 1950. © Nobel Found-
ation, Stockholm. Photograph reproduced with the kind permission
and assayed in these three solvents (see Porra et al.
of the Foundation. 1989).
New solvents, other than aqueous acetone, were
usually sought to extract Chls from difficult tissues,
Inaccuracy of the chlorophyll extinction such as tough leathery leaves, by simple immersion for
coefficients used by Daniel Arnon extended periods; however, finely cutting with scissors
followed by grinding with extractant produced more
Over several decades, many researchers (Vernon 1960; exhaustive extraction and more satisfactory results
Ziegler and Egle 1965; Delaporte and Laval-Martin than prolonged immersion which can cause oxidative
1971 a, b; Lichtenthaler 1987; Porra et al. 1989; Well- degradation of the photolabile Chls (cf. Porra et al.
burn, 1994) discovered that Mackinney’s (1941) spe- 1989). Wellburn (1994) has presented accurate extinc-
cific extinction coefficients for Chls a and b in aqueous tion coefficients and relevant simultaneous equations
80% acetone were grossly inaccurate. Mackinney’s for use with various solvents including DMSO, which
coefficients were obtained using dried solid samples is sometimes used as an alternative extractant.
of Chls a and b without further purification to re- The following three special extractants were de-
move oxidation products formed during storage. These signed to remove Chls a and b from some green al-
are compared with accurate coefficients, confirmed gae and marine micro-algae which were unexpectedly
by Mg-atomic absorption spectrometry, obtained by difficult to extract. A review of these three special ex-
Porra et al. (1989) with chromatographically pure Chls tractants (Porra 1991) describes their application, the
(see Table 1): some of the errors are very large. formation of derivatives and the relevant simultaneous
equations for their use.
1. Aqueous 2.1 M pyridine containing 0.35 M
The use of alternative extractants NaOH extracts Chls a and b from regreening
nitrogen-starved Chlorella fusca cells as their
Alternatives to aqueous acetone for Chl extractants are Mg-hydroxylactones formed by opening the five-
DMF (N,N
-dimethylformamide), DMSO (dimethyl- membered isocyclic ring to reclose around an
sulfoxide) and methanol. In DMF and DMSO, as in O atom in a six-membered ring (see Porra and
aqueous 80% acetone, Chls a and b exhibit sharp Grimme 1974; Porra 1991).
 153
Table 2. Corrected specific (α) and millimolar ( mM ) extinction coefficients for Chls a and b in buffered aqueous 80% acetone, DMF
(N,N
-dimethylformamide) and methanol. The spectrophotometer was zeroed at 750 nm so that all coefficients shown are difference coef-
ficients between the QY maximum wavelength specified and 750 nm. Each coefficient is the mean of three determinations: the standard
deviations are presented in Porra et al. (1989)

Solvent Wavelength Difference extinction coefficients

(nm) Chl a Chl b
Millimolar Specific Millimolar Specific
( mM ) (α) ( mM ) (α)

Buffered aqueous 663.6 minus 750 76.79 85.95 9.79 10.78

80% acetone (pH 7.8) 646.6 minus 750 18.58 20.79 47.04 51.84

DMF 663.8 minus 750 79.29 88.74 12.03 13.26

646.8 minus 750 18.62 20.84 46.49 51.23

Methanol 665.2 minus 750 71.43 79.95 20.20 22.26

652.0 minus 750 31.65 35.42 38.55 42.48

Table 3. Simultaneous equations for the determination of Chls a and b concentrations in buffered aqueous 80% acetone, DMF and methanol
using the extinction coefficients presented in Table 2

Solvent Equations for Chl concentrations Equations for Chl concentrations

(nmol/ml) (µg/ml)

In buffered [Chl a] = 13.71 E663.6 – 2.85 E646.6 [Chl a] = 12.25 E663.6 – 2.55 E646.6
aqueous [Chl b] = 22.39 E646.6 – 5.42 E663.6 [Chl b] = 20.31 E646.6 – 4.91 E663.6
80% acetone [Chl a +b] = 19.54 E646.6 + 8.29 E663.6 [Chl a + b] = 17.76 E646.6 + 7.34 E663.6

In DMF [Chl a] = 13.43 E663.8 – 3.47 E646.8 [Chl a] = 12.00E663.8 – 3.11E646.8

[Chl b] = 22.90 E646.8 – 5.38 E663.8 [Chl b] = 20.78E646.8 – 4.88E663.8
[Chl a+b] = 19.43 E646.8 + 8.05 E663.8 [Chl a+b] = 17.67E646.8 + 7.12E663.8

In methanol [Chl a] = 18.22 E665.2 – 9.55 E652.0 [Chl a] = 16.29 E665.2 – 8.54 E652.0
[Chl b] = 33.78 E652.0 – 14.96 E665.2 [Chl b] = 30.66 E652.0 – 13.58 E665.2
[Chl a + b] = 24.23 E652.0 + 3.26 E665.2 [Chl a + b] = 22.12 E652.0 + 2.71 E665.2

2. Aqueous 85% methanol containing 2% KOH Correction of data obtained by use of the Arnon
and 1.5 mM sodium dithionite extracts, Chls a simultaneous equations
and b from Nannochloris atomus cells as Mg-
rhodochlorins a and b (i.e., Mg-chlorin e6 and Despite the frequent publication over many decades
Mg-rhodin g7 ) formed by opening the isocyc- of more accurate extinction coefficients and simultan-
lic ring (Porra 1990a). Dithionite prevents Mg- eous equations for Chls a and b (Vernon 1960; Ziegler
hydroxylactone formation. and Egle 1965; Delaporte and Laval-Martin 1971 a, b;
Lichtenthaler 1987; Porra et al. 1989; Wellburn 1994),
3. Aqueous 85% methanol containing 1.5 mM so- these more reliable post-Arnon equations were largely
dium dithionite extracts Chls unchanged from ignored and the Arnon equations retained. Perhaps the
Nannochloris atomus cells (Porra 1990b); presum- magnitude of the errors involved in the Arnon method
ably, the reductant also cleaves disulfide bridges to was not fully appreciated. Perhaps some researchers
relax cell-wall proteins (cf. Thompson and Preston were more interested in trends than in absolute val-
1967, 1968) and render the cells permeable to ues for either Chl concentrations or Chl a/b ratios
methanol.
154

Thus, having obtained Chl a/bT from Figure 3 or

Equation (9) and by calculating [Chl a + b]T from
Equation (10), the true Chl a and b concentrations,
designated [Chl a]T and [Chl b]T , can be calculated
using Equations (11) and (12):

[Chl a + b]T · Chl a/bT

[Chl a]T = (11)
(Chl a/b + 1)

[Chl b]T = [Chls a + b]T/(Chl a/bT + 1) (12)

Figure 3 shows that the quotient of Chl a/bT ÷
Chl a/bA increases from 1.17 to 1.52 to 2.17 for Chl
a/bA values of 1.0, 4.0 and 7.0, respectively; thus, the
higher the Arnon ratio the greater the error. This has
important consequences (see later section on ‘Light
acclimation studies’).

Some consequences of the continued use of the

Arnon equations
Figure 3. Using the Mackinney’s extinction coefficients (see
Table 1), the extinction values at the red QY absorption peaks of Molecular modeling of the major Chl a/b protein of
Chls a and b were calculated for hypothetical solutions of Chl a and LHC II
b in buffered aqueous 80% acetone with Arnon ratios, Chl a/bA ,
from 1.0 to 7.0. These values were inserted into the appropriate Accurate Chl a and b determinations were required for
equations of Table 3 to obtain true Chl a and b concentrations and
the electron crystallography studies of light-harvesting
hence true ratios, Chl a/bT . The quadratic equation which best fits
the curve is shown: it was determined using the Microcal Origin complex (LHC) II by Werner Kühlbrandt and his
Program (Version 4.0). group. Their goal to build a realistic molecular model
of this Chl a/b-protein complex required that they
and, therefore, retained the Arnon method to permit know the precise number of Chl a and b molecules
comparison between current and previous results. To present in each pigment–protein molecule. Using
remove this relativity obstacle, a quick and precise al- Arnon’s assay, Butler and Kühlbrandt (1988) and
gebraic method was developed to correct Chl a and Kühlbrandt and Wang (1991) found 15 Chl molecules
Chl b determinations obtained by Arnon’s equations per LHC II protein molecule with a Chl a/bA ratio of
without reference to the original spectrophotometric 1.15, which suggested 8 Chl a and 7 Chl b molecules
data (Porra et al. 1989; Porra 1991). per pigment protein. The atomic model derived from
The correction method was developed by plotting their electron-crystallography results (Kühlbrandt and
Arnon Chl a/b ratios, Chl a/bA , against true ratios, Wang 1991) indicated only 12 Chl molecules present
Chl a/bT (see Figure 3). The quadratic equation which and, with a Chl a/bA ratio of 1.15, they deemed them
best fits the curve is shown in Figure 3 and again as to be 7 Chl a and 5 Chl b.
Equation (9): Later, using more accurate equations (Porra et al.
1989), Kühlbrandt et al. (1994) found 14 Chls per
Chla/bT = 0.593 + 0.459· (Chla/bA)+ (9) pigment–protein molecule and a Chl a/bT of about 1.3,
0.229·(Chla/bA)2 suggesting 8 Chl a and 6 Chl b per protein. Disap-
pointingly, the more accurate equations of Porra et al.
Calculations for conversion of Chl a/bA to Chl a/bT
(1989) only reduced the Chl to protein ratio from 15 to
(see legend to Figure 3) showed that the true total Chl,
14. Considering the enormity of the errors in Mackin-
designated [Chl a + b]T, was always 89.5% of the total
ney’s coefficients (see Table 1), it is unlikely that
Chl, Chl[a + b]A , calculated by Arnon’s method [see
sufficient error remains in those of Porra et al. (1989)
Equation (10)]:
to permit elimination of a further 2 Chl molecules to
[Chls a + b]T = 0.895[Chls a + b]A (10) lower the Chl/protein ratio to 12. As suggested by
 155

Kühlbrandt et al. (1994), the discrepancy between the a/b protein of PS I has a Chl a/bA of 3.6 (Thornber
experimentally determined Chl content and the num- 1986). which corresponds to a Chl a/bT of 5.25.
ber of Chl molecules observed in the atomic model is All these corrected Chl a/b values, especially for
probably caused by 2 Chl molecules remaining non- the PS I-enriched fraction, clearly indicate the need to
specifically bound to the LHC II protein throughout specify whether assays were performed by the Arnon
purification and crystallization: this discrepancy of 2 method or a more accurate post-Arnon assay (cf. Ver-
Chl molecules still remains in their more recent work non 1960; Ziegler and Egle 1965; Delaporte and
(Rogl and Kühlbrandt 1999). Laval-Martin 1971a, b; Lichtenthaler 1987; Porra et
al. 1989; Wellburn 1994).

Light acclimation studies

Defining the limitations of the simultaneous equation
A more dramatic anomaly arising from retention of assay for Chls a and b
Arnon’s equations was encountered in investigations
The contribution of Chl b at 647 nm is difficult to
of chloroplast acclimation to ambient light intensities.
measure on a steeply sloping Chl a spectrum when the
Low-light (shade) plants contain more chlorophyll per
Chl a/b ratio is high; thus the Chl content of PS I is
chloroplast and have lower Chl a/b ratios than high-
probably better assayed using the Chl b-oxime method
light (sun) plants (Anderson 1986); incidentally, this
of Ogawa and Shibata (1965). In this assay, the extinc-
had also been established in 1913 by Willstätter and
tion at 666 nm is read before and after the addition of
Stoll (cf. Rabinowitch 1945). In both shade and sun
40% aqueous hydroxylamine (pH 4.0) to the Chls ex-
plants, Arnon’s Chl a/bA ranges and those of Willstät-
tracted in methanol; this converts the 7-formyl group
ter and Stoll (1913) were low. The typical Chl a/bA
of Chl b to form Chl b-oxime which, like Chl a, has
range for shade plants is about 1.6–2.2 which, using
a QY peak at 666 nm. The derivation of the relevant
Figure 3, becomes a true Chl a/bT range of 2.0–2.8: for
simultaneous equations to determine both Chl a and
sun plants the typical Chl a/bA range is approximately
b are described by Ogawa and Shibata (1965). When
2.6–3.4 which becomes a Chl a/bT range of 3.5–4.9
the Chl a/bA ratio exceeds 7.0, which is equivalent to
using Figure 3.
a Chl a/bT value of about 15 (see Figure 3), the Chl
These changes in Chl a/b ratios in shade and sun
b-oxime assay or, alternatively, a spectrofluorimetric
plants reflect the adaptation of the chloroplast to pre-
method (see Talbot and Sauer 1997) should be used.
vailing light intensity through regulation of the amount
of Photosystem I (PS I) relative to Photosystem II
(PS II) and the size and composition of the light-
harvesting complexes (LHCs) of each photosystem.
Shade plants, relative to sun plants, contain more Chl Concluding remarks
in larger grana (Anderson 1986). Relative to PS I,
shade plants have fewer PS II but with very large light- It appears contrary to reason that so many researchers
harvesting antennae compared to those of sun plants. continue to use Arnon’s equations (1949) after more
The most abundant Chl a/b protein, LHC II, in shade- accurate post-Arnon equations have become avail-
plant chloroplasts has a very low Chl a/bA of 1.15 (i.e. able; continuing with Arnon’s assay to retain relativity
Chl a/bT of 1.3); thus, shade plants have lower Chl between their earlier and current results was no longer
a/b ratios than sun plants which contain less Chl and sufficient reason after the simple algebraic method to
possess only small antennae located in more numerous correct the earlier results of Arnon’s assays became
but smaller grana (Anderson 1986). available (Porra et al. 1989).
Sane et al. (1970) and Andersson et al. (1976) As noted above, the error in Arnon’s ratios (Chl
separated PS I- and PS II-enriched fractions from frag- a/bA ) increases as the ratios become higher. The true
mented chloroplasts. With Arnon’s method, the Chl Chl a/b ratio range of 11.4–13.4 for PS I-enriched
a/bA range for the PS I-enriched fraction was approx- fractions is about double the 6.0–6.5 range calculated
imately 6.0–6.5 which, using Figure 3, became a true by the Arnon method (see earlier section on ‘Light
Chl a/bT range of 11.4–13.4. The PS II-enriched frac- acclimation studies’). This starkly emphasizes the ab-
tions have a Chl a/bA < 3.0 (Andersson et al. 1976) surdity of retaining an old inaccurate assay and clearly
which, using Figure 3, becomes Chl a/bT < 4.2. Inter- indicates the potential for even more confusion that its
estingly, the much less abundant light-harvesting Chl further retention could generate.
156

Acknowledgments Porra RJ (1990a) The assay of chlorophylls a and b converted to

their respective magnesium-rhodochlorin derivatives by extrac-
tion from recalcitrant algal cells with aqueous alkaline methanol:
I thank Govindjee for the invitation to write this prevention of allomerization with reductants. Biochim Biophys
minireview, and I am most grateful to Dr W.S. Chow Acta 1015: 493–502
for calculating the quadratic equation best describing Porra RJ (1990b) A simple method for extracting chlorophylls from
the relationship between Chl a/bT and Chl a/bA shown the recalcitrant alga, Nannochloris atomus, without formation of
spectroscopically-different magnesium-rhodochlorin derivatives.
in Figure 3 and Equation (9). Biochim Biophys Acta, 1019: 137–141
I thank the CSIRO-Division of Plant Industry, Porra RJ (1991) Recent advances and re-assessments in chlorophyll
Canberra, for an Honorary Fellowship and the Son- extraction and assay procedures for terrestrial, aquatic, and mar-
derforschungsbereich (Projekt 533) for financial as- ine organisms, including recalcitrant algae. In: Scheer H (ed)
Chlorophylls, pp 31–57. CRC Press, Boca Raton, Florida
sistance during my visit to the Botanisches Institut, Porra RJ and Grimme LH (1974) A new procedure for the determ-
Ludwig-Maximilians Universität, München. I thank ination of chlorophylls a and b and its application to normal and
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of accurate extinction coefficients and simultaneous equations
preparation of this article. I also thank Prof. Hugo for assaying chlorophylls a and b extracted with four different
Scheer for helpful discussions. solvents: verification of the concentration of chlorophyll stand-
ards by atomic absorption spectrometry. Biochim Biophys Acta
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