# The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b

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Govindjee, J. T. Beatty, H. Gest and J.F. Allen (eds): Discoveries in Photosynthesis, pp. 633–640

© Springer 2005

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: [email protected]; 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

634

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-

635

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

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

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.

636

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).

637

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.

638

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

© Springer 2005

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: [email protected]; 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

634

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-

635

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

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

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.

636

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).

637

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.

638

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