384 Biochimica et BiophysicaActa, 975 (1989) 384-394

Elsevier

BBABIO43036

Determination of accurate extinction coefficients

and simultaneous equations for assaying chlorophylls a
and b extracted with four different solvents:
verification of the concentration of chlorophyll standards
by atomic absorption spectroscopy
R.J. Porra, W.A. T h o m p s o n * and P.E. K r i e d e m a n n *
Dioision of Plant Industry, CommonwealthScientific and Industrial Research Organization, Canberra (.4ustralia)

(Received15 December1988)
(Revised manuscriptreceived11 April 1989)

Key words: Chlorophylldetermination;Chlorophylla/b ratio; Correctionfactor; Arnon equation;

Magnesiumatomicabsorption spectroscopy

The extinction coefficients for chlorophylls a and b in diethylether (Smith, J.H.C. and Benitez, A. (1955) in Modem
Methods of Plant Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143-196, Springer-Verlag, Berlin), used in
this paper as primary standards, were verified, to within an error of less than 1%, by magnesium determination using
atomic absorbance spectrophotometry. We also report the determination of accurate extinction coefficients for
chlorophylls a and b in N,N'-dimethylformamide, methanol or buffered 80% aqueous acetone. Highly purified
chlorophylls were used and methods were employed which not only minimize errors due to evaporation of the volatile
solvents employed in their estimation but also eliminate variable micro-contamination by chlorophyll degradation
products, a potential source of inconsistency between the extinction coefficients obtained in each of these three
solvents. Using these new coefficients, expressed as both millimolar and specific coefficients, we have derived new
simultaneous equations to obtain chlorophyll concentrations as n m o l / m l and p g / m l , respectively. These equations
were applied to data obtained with leaf discs from spinach and Flindersia brayleyana extracted with the three specified
solvents and to a concentrated solution (in N,N'-dimethylformamide) of a chlorophyll a + b mixture added to the three
solvent systems. The validity of these equations is proven by the consistency of the chlorophyll determinations and of
the chlorophyll a / b ratios. New simultaneous equations, compatible with the equations derived for the three solvents,
are presented for the assay of chlorophylls a and b converted to their cyclic hydroxylactone derivatives by extraction
with alkaline pyridine reagent (2.1 M pyridine in 0.35 M NaOH). Most chlorophyll analyses in higher plants, including
the chlorophyll content and chlorophyll a / b ratios of plant thylakoids and chlorophyll-protein complexes, have been
obtained in 80% aqueous acetone with the much used simultaneous equations of Arnon (Amon, D.I. (1949) Plant
Physiol. 24, 1-15). For this reason we include conversion factors which correct these earlier data and make it
compatible with data calculated with the simultaneous equations presented in this paper. The importance of these
corrections to the formulation of meaningful models of the photosynthetic apparatus is demonstrated. Our results also
indicate that grinding leaf discs with N,N'-dimethylformamide is a more reliable method for extracting all chlorophylls
than shaking with this solvent for 24 h.

Introduction
* Permanent address: Division of Forestry and Forest Products,
Commonwealth Scientific and Industrial Research Organization,
P.O. Box 4008, Canberra ACT 2600, Queen Victoria Terrace, Three different solvents, namely DMF [1], methanol
Australia. [2] or buffered 80% aqueous acetone [3], were used in a
Abbreviations: Chl, chlorophyll;DMF, N,N'-dimethylformamide.
search for a convenient extraction and assay procedure
Correspondence: R.J. Porra, Divisionof Plant Industry,CSIRO,GPO for Chls a and b in leaf discs from Queensland Maple
Box 1600, Canberra ACT 2601, Australia. seedlings (Flindersia brayleyana) grown under a variety
0005-2728/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
 385

of conditions. It was found that the Chl a / b ratios, Preparation of pure chlorophylls. Chlorophylls were
determined in buffered 80% aqueous acetone extracts of extracted from maize leaves with buffered aqueous
leaf discs from F. brayleyana using the simultaneous acetone. Chls a and b were then separated and purified
equations of Arnon [3] to determine the Chl a and b by column chromatography on sucrose [9] after precipi-
concentrations, differed significantly from the ratios tation with dioxane [10] as described by Porra et al. [11].
obtained in DMF or methanol extracts using the simul- Chl a and b zones of the column were transferred to
taneous equations of Inskeep and Bloom [4] and of sintered glass funnels and the chlorophylls completely
BSger [2], respectively. We have also noted that Chl a / b removed from the sucrose with diethylether by reduced
ratios determined in buffered aqueous acetone using the pressure filtration. Diethylether was removed by
equations of Arnon [3] are drastically lower than those evaporation under reduced pressure and the purified
obtained with the equations of other workers [5-7]. Chl a and b samples redissolved in petroleum spirit
In view of these considerations and to obtain reliable and rechromatographed on a fresh sucrose column as
and consistent chlorophyll assays with the three speci- described above. Rechromatographed Chls a and b
fied solvents which are routinely used in these laborato- were again removed from the sucrose with diethylether
ries, we redetermined the extinction coefficients for and these ethereal solutions were kept in the dark under
Chls a and b in these solvents and derived from them oxygen-free nitrogen at - 1 5 ° C to minimize chloro-
three new sets of compatible simultaneous equations to phyll degradation by demetallation and oxidation reac-
calculate their concentrations. The coefficients used by tions: the solutions were used for spectrophotometric
Arnon [3] for Chl a and especially for Chl b were found determination of extinction coefficients within approx.
to be low. 9 h. All preparative procedures were carried out in dim
Porra and Grimme [8] earlier presented simultaneous light to minimize light-associated degradation of chloro-
equations for the assay of Chls a and b, converted to phylls.
their cyclic hydroxylactone derivatives (see Fig. 2, struc- Determination of extinction coefficients of Chl a and b
ture IV in Discussion section) by extraction with al- in various solvents. To eliminate errors due to evapora-
kaline pyridine, which were compatible with the equa- tive losses from all volatile solutions, absorbance mea-
tions of Arnon [3]. The original data [8] have now been surements were made using a cuvette (1 cm light path)
reprocessed to give equations v~hich are compatible with fused to a B14 socket with a total capacity of 7 ml when
those presented for use with the other three extractants. stoppered but marked with a 5 ml graduation line. To
The past formulation of more accurate equations the cuvette was added exactly 5 ml of a diethylether
[5-7] than those of Arnon [3] does not appear to have solution of Chl a or b with absorbance at the major red
diminished the use of the latter. This may be due to the peak between 0.7 and 1.0. The cuvette was immediately
failure to simultaneously provide conversion factors to closed with a B14 stopper secured with two extension
quickly and conveniently correct earlier data coupled springs. The spectrum was recorded from 400 to 750 nm
with a reluctance to forego the ability to compare newly in a Hitachi model U3200 recording spectrophotometer
gathered data with the old. To overcome such resis- with a corrected baseline. The spectrophotometer
tance, correction factors have been derived and pre- searched for all peaks; because spectra were automati-
sented in this paper. Replacement of Arnon's equations caUy zeroed at 750 rim, all extinction coefficients are
[3] by the new equations for use with buffered 80% difference coefficients (i.e., peak maxima minus 750
aqueous acetone now enjoys the added advantage that nm). The cuvette was then opened under dim light,
DMF, methanol and alkaline pyridine can be used partly immersed in a warm water bath (approx. 55 ° C)
when alternative extractants are required and, with the and the diethylether removed by evaporation under a
equations provided in this paper, compatible results will stream of oxygen-free nitrogen. The chlorophyll residue
be produced. was then dissolved in a new replacement solvent such as
DMF, cold methanol or buffered aqueous acetone and
Experimental the volume adjusted to the 5 ml mark. The cuvette was
quickly reclosed and secured with springs, the spectrum
Chemicals and reagents. Acetone, diethylether and recorded between 400 and 750 nm and the peaks auto-
methanol were all Analar grade reagents supplied by matically recorded and measured.
BDH Chemicals (Australia Pty.), Port Fairy, Australia. For Chls a and b dissolved in Analar quality diethyl-
DMF was supplied by May and Baker, West Footscray, ether, containing approx. 0.2% H20, the major peak in
Australia. (DMF is a liver toxin which is readily ab- the red region of the spectrum occurred at 660.8 and
sorbed through the skin: disposable gloves should be 642.6 nm, respectively. Smith and Benitez [12] found
used for protection). Buffered aqueous acetone is 80% that the specific extinction coefficients (a) at these
aqueous acetone containing 2.5 mM sodium phosphate peaks are 100.9 and 62.0 1-g -1 .cm -1, respectively:
buffer pH 7.8 to minimize conversion of chlorophylls to using molecular weights of 893.48 and 907.46 for Chls a
phaeophytins. and b, respectively, the corresponding millimolar ex-
386

tinction coefficients (aAM) are 90.2 and 56.3 1. mmo1-1 sorbance spectrophotometer (model AA 360). The
• cm -1, respectively. The reliability of these extinction method used is sensitive to 0.05 p.p.m.
coefficients has been confirmed by Falk [13] who found
that the spectrophotometric data of Smith and Benitez Results
[12] were consistent with this data based on magnesium
microanalysis with titanium yellow. In addition, we Confirmation of our primary standards by magnesium
have also confirmed these extinction coefficients on the determinations using atomic absorption spectroscopy
basis of magnesium content but determined by atomic The extinction coefficients of Chls a and b in dieth-
absorption spectroscopy (see Results section). ylether, determined by Smith and Benitez [12], were
Extinction coefficients (emM or a) for Chl a and b in used as primary standards in this work. The validity of
the new replacement solvent were then calculated as these primary standards was confirmed by taking 10 ml
follows: of an approx. 20 /xM solution of each chlorophyll in
diethylether in a stoppered 10 ml graduated cuvette.
Ot ~mM The precise micromolar concentration was then predic-
Cl'd a 100.9 X A--L 90.2 × A1 ted on the basis of the extinction coefficients of Smith
A2 A2 and Benitez [12]. The magnesium was extracted exhaus-
Chl b 62.0 × A---L 56.3 X A~ tively from the diethylether with 1.4% (w/v) perchloric
A3 A3 acid and the pooled aqueous washings made up to a
final volume of 10 ml. Complete conversion to
where A~ is the absorbance of each chlorophyll in the phaeophytins a and b took approx. 30 and 120 rain,
new replacement solvent at the peak maximum of either respectively. The magnesium concentration was then
Chl a or Chl b in that solvent, and A 2 and A 3 are the determined by atomic absorbance spectroscopy using
absorbances of the Chl a and Chl b solutions, respec- magnesium standards which were verified by complexi-
tively, in diethylether at 660.8 nm (Chl a) and at 642.5 metric titration with EDTA using Eriochrome Black T
nm (Chl b), respectively. as indicator. For two samples of Chl a the predicted
Extraction of chlorophylls from leaf discs with DMF, concentrations were 12.97/~M and 16.75 /~M and the
methanol or buffered 80% aqueous acetone. Seedling magnesium concentrations were determined as 13.04 +
trees (up to 1 m high) of well or poorly nourished F. 0.25 ~tM and 16.67 +__0.33 ~tM, respectively. Similarly,
brayleyana were grown in pots in a shade house. Spinach the predicted concentrations for two samples of Chl b
plants (Spinacia oleracea) were grown hydroponically in were 12.97 /~M and 15.31 /~M and the magnesium
a glass house. For chlorophyll analysis, circular discs concentrations were found to be 13.06 + 0.25 /~M and
were cut with a 16 mm diameter punch (approx. 200 15.32 + 0.29/~M. The errors quoted here are maximum
mm 2 surface) from both well nourished Flindersia and errors for individual determinations and include all
spinach leaves: for the pale green leaves of poorly errors associated with the atomic absorption determina-
nourished Flindersia plants, discs of 23 mm diameter tion, verification of the magnesium standard and volu-
(approx. 400 mm2 surface) were used. These discs were metric manipulations. The magnesium determinations
extracted with DMF, cold methanol or buffered aque- have confirmed the extinction coefficients of Smith and
ous acetone by grinding with 2 ml of each solvent in a Benitez [12] for both Chls a and b in diethylether to
mortar with pestle. The homogenate, combined with a within an error of less than 1.0%.
further three washings of the pestle and mortar (each of
1.5 ml) with the same solvent, was centrifuged at 2500 Extinction coefficients of chlorophylls and equations for
r.p.m, in an MSE bench centrifuge for 10 rain. The determination of their concentrations in DMF, methanol
pellet was then extracted with a further 1 ml of solvent or buffered 80 %" aqueous acetone
in a Potter-Elvejhem homogenizer and the pooled su- During our initial attempts to obtain accurate extinc-
pernatants adjusted to a final volume of 8 ml. The tion coefficients, diethylether solutions (10 ml) of Chls
spectrum was recorded between 750 and 600 nm and a and b, with appropriate absorption of its major red
the major red absorption peak automatically de- peak (see Experimental), were prepared in a 10 ml
termined by the Hitachi Model U3200 recording spec- graduated flask fitted with a ground glass stopper.
trophotometer zeroed at 750 nm. The absorbance at the Approx. 3 ml was transferred by Pasteur pipette to a
major red absorption peak of Chl b was also de- Hellma (type ll0QS) cuvette and tightly stoppered prior
termined; Chls a and b and Chls a + b concentrations to spectrophotometric examination. When the contents
were then calculated using the equations described be- of the cuvette were returned to the flask, a loss of about
low in Table III. 0.6 ml of diethylether was noted. It seemed reasonable
Magnesium determination. The magnesium contents to assume that half the loss (0.3 ml) occurred during the
of Chls a and b were determined by atomic absorbance transfer to the cuvette so that it could be anticipated
spectrophotometry using a Perkin Elmer atomic ab- that absorbance of the diethylether solution could be
 387

TABLE I each investigation with each of the solvents: this was

Absorption maxima and QRs ratios for chlorophylls a and b in diethyl- done to minimize inconsistency due to variable micro-
ether contamination by chlorophyll degradation products
The absorption spectra of Chls a and b dissolved in diethylether after arising from demetallation and oxidation reactions
rechromatography on sucrose columns were recorded on a spectro- which can occur during storage if the pigments are not
photometer automatically zeroed at 750 nm (see Experimental). The kept in the dark under oxygen-free nitrogen at - 1 5 ° C.
QRS ratios, which represent the absorbance at the indicated peaks
divided by the absorbance at the Soret peak, are compared with those
In these early studies, Chls a and b were separated by
of purified chlorophylls from other sources. only one passage down a sucrose column; a small but
variable contamination of Chl a with pheophytin a and
Maxima QRS ratio of Chl b with pheophytin b and Chl a was subse-
(rim) quently found to be responsible for this lack of con-
Ref. 12 Ref. 7 this work
Chl a sistency between results obtained in each solvent. To
660.8 0.767 0.794 0.794 overcome this problem a second purification by sucrose
615.4 0.124 0.125 0.123 chromatography was introduced (see Experimental) and
577.4 0.070 0.069 0.065 by examining the ratios (QRs) of all absorption peaks to
532.4 0.032 0.035 0.030
the Soret peak (Table I), the purity of the Chl a and
429.0 (Soret) 1.0 1.0 1.0
409.8 0.648 0.651 0.650 Chl b samples produced by rechromatography was
found to be better than that used by Smith and Benitez
Chl b
[12] and at least equivalent to that used by Lich-
643.0 0.355 0.369 0.366
594.2 0.073 0.074 0.075 tenthaler [7]: the lower ratios we have obtained for our
454.0 (Soret) 1.0 1.0 1.0 Chl a at 532A and 409.8 mn indicate smaller con-
430.0 0.359 0.370 0.363 tamination by phaeophytin a (cf. Ref. 12) and hydro-
xylactone derivatives (oxidation products) of Chl a (cf.
Refs. 11 and 8), respectively. To eliminate inconsisten-
overestimated by as much as 10% which would then cies due to variable microcontamination by the chloro-
lead to an underestimate of 10% in the extinction coeffi- phyll degradation products mentioned above, sufficient
cients in the replacement solvents. The error would be freshly purified Chls a and b were prepared to enable
somewhat mitigated if the replacement solvent was also the extinction coefficients in all three solvents to be
very volatile. Such errors were overcome by using a 5 ml obtained from a single ethereal solution of each chloro-
graduated cuvette fused to a, B14 ground-glass socket phyll; the coefficients were obtained as quickly as possi-
and stopper (see Experimental). ble (within approx. 9 h) during which time the ethereal
Early studies with the graduated cuvette still failed to solutions were kept in the dark and under oxygen-free
provide consistent Chls a and b concentrations and Chl nitrogen at - 1 5 ° C to minimize all degradation reac-
a/b ratios for leaf discs extracted with the three solvents. tions including those mentioned above (see Discussion
Ethereal solutions of Chls a and b for these studies section). Each coefficient, given as both millimolar (emM)
were freshly prepared from maize leaves just prior to and specific (a) extinction coefficients (Table II), is the

TABLE II
Corrected millimolar and specific difference coefficients for chlorophylls a and b in N,N'-dimethylformamide, methanol and buffered 80 ~ aqueous
acetone

As the spectrophotometer was zeroed at 750 nm, the extinction coefficients listed below are difference coefficients as shown in column 2. Each
coefficient is the mean of three separate determinations. The percentage variation about the mean ( m ) is presented as l O 0 . o / m , where o
represents the standard deviation. The percentage variation is presented (in brackets) beside the millimolar (emM) coefficients but would be
identical for the specific (a) coefficients which are derived from the same data.

Solvent Wavelengths appro- Difference extinction coefficients

priate to the differ- Chl a Chl b
ence coefficients
(nm) (emM) Oc (EmM) a
(l.mmol-l.cm -] ) ( I . g - l . c m -1) (1. m m o l - I.cm -1 ) ( l . g - l . c m -1 )

DMF 663.8 minus 750 79.29 (+0.34) 88.74 12.03 (5:4.4) 13.26
646.8 minus 750 18.62 ( + 1.47) 20.84 46.49 ( ::k 0.92) 51.23

Methanol 665.2 minus 750 71.43 ( ___0.91) 79.95 20.20 ( + 0.99) 22.26
652.0 minus 750 31.65 ( + 0.66) 35.42 38.55 (+0.60) 42.48

Aqueous 80% 663.6 minus 750 76.79 ( 5: 0.52) 85.95 9.79 ( + 0.51) 10.78
acetone 646.6 minus 750 18.58 (5:0.59) 20.79 47.04 ( 5: 0.34) 51.84
388

TABLE III
Corrected equations for the determination of chlorophylls in N,N' -dimethylformamide, methanol and buffered 80 % aqueous acetone
These equations (1-18) are derived from the difference extinction coefficients presented in Table II; therefore all absorbance measurements at the
indicated wavelengths must have the absorbance at 750 nm subtracted.

Equations for chlorophyll concentrations in n m o l / m l Equations for chlorophyll concentrations in t t g / m l

In D M F
Chl a = 13.43 A 665.8 _ 3.47 A ~6.s (1) Chl a =12.00 A 663"8 - 3 . 1 1 /I 646.8 (10)
Chl b = 22.90 A 6'~'s - 5.38 A 663"8 (2) Chl b = 20.78 A 646"8 - 4 . 8 8 A 663"8 (11)
Chls a + b = 19.43 A 646s + 8.05 A 663'8 (3) Chls a + b = 17.67/1646.8 + 7.12 X 663"8 (12)

In methanol
Chl a = 18.22,4665.2 - 9.55 A 652"0 (4) Chl a = 16.29 A 665"2 - 8.54 A 652"0 (13)
Chl b = 33.78 A 652"0 -14.96/1665.2 (5) Chl b = 30.66 A 652"0 - 13.58 A 665"2 (14)
Chls a + b = 24.23 A 652"0 + 3.26 A 665"2 (6) Chls a + b = 22.12 A 652"0 + 2.71 A 665"2 (15)

In buffered 80% aqueous acetone

Chl a = 13.71 A 663"6 - 2.85/1646.6 (7) Chl a = 12.25 A 663"6 - 2.55 A 646"6 (16)
Chl b = 22.39 A 646"6 - 5.42 A 663"6 (8) Chl b = 20.31 /1646.6 - 4 . 9 1 A 663"6 (17)
Chls a + b = 19.54 A 646"6 + 8.29 A 663"6 (9) Chls a + b = 17.76 A 646"6 + 7.34/1663.6 (18)

mean of three separate determinations. The percentage function of the various component complexes of the
variation about the mean (see Table II) seldom reached photosynthetic process, has been expressed in molar
+ 1.0% and exceeded it only when absorbance measure- terms: for this reason, equations are presented in Table
ments were made on steeply sloping segments of Chl a III expressing Chls a and b and Chls a + b concentra-
and Chl b spectra; data have been obtained (see below) tions in both n m o l / m l (Eqns. 1-9) and # g / m l (Eqns.
which verify the reliability of all the coefficients. 10-18).
Although it has been customary since Amon's early
work [3] to express chlorophyll concentrations in/~g/ml, The effect of aqueous dilution of D M F and methanol on
chlorophyll composition in modem photosynthesis re- the calculation of chlorophyll concentrations and ratios
search, especially that dealing with the composition and When undiluted solvents such as DMF or methanol

TABLE IV
Chlorophyll content and chlorophyll a / b ratios using three solvent systems for the extraction of three different types of leaves
Discs were taken from specific positions where L and R refer to left and right of the midrib and a, b and c to positions equally spaced from leaf tip
to stem. The discs were 16 m m diameter (equals 200 mrn2 surface) for experiments with spinach and well nourished Flindersia but 23 mm diameter
(equals 400 mm 2 surface) for the remaining experiment. The chlorophylls were extracted as described in Experimental and concentrations were
calculated using equations 1 - 9 in Table III; consequently, Chl a / b are in molar terms (see Eqn. 19).

Leaf type Solvent Disc Chlorophyll c o n t e n t / u n i t leaf area 0zmol .m - 2 ) Chl a / b

position Chl a Chl b Chls a + b ratios ( R ' )

Spinach leaf DMF aL 294.5 79.0 373.5 3.72

DMF bR 285.9 76.8 362.7 3.73
80% Acetone bL 250.0 67.0 317.0 3.73
80% Acetone cR 278.8 75.2 354.0 3.71
Methanol cL 310.8 83.4 394.2 3.73
Methanol aR 288.4 73.1 361.5 3.94

Flindersia DMF aL 399.0 128.4 527.4 3.11

(well DMF bR 403.6 132.3 535.9 3.05
nourished) 80% Acetone bL 382.1 123.3 505.4 3.10
80% Acetone cR 325.3 97.9 423.2 3.32
Methanol cL 408.3 135.2 543.5 3.02
Methanol aR 400.5 131.4 531.9 3.04

Flindersia DMF aL 85.8 25.2 111.0 3.41

(poorly DMF bR 107.9 32.0 139.9 3.38
nourished) 80% Acetone bL 90.8 27.6 118.4 3.29
80% Acetone cR 88.3 25.3 111.5 3.42
Methanol eL 106.7 33.6 140.3 3.18
Methanol aR 103.2 31.0 134.2 3.33
 389

are employed as extractants of chlorophylls from leaf TABLE V

discs using the assay conditions described in this paper Determination of chlorophyll concentrations and chlorophyll a / b ratios
(see Experimental), dilution by tissue water up to 10% of a solution of a chlorophyll a and b concentrate diluted with three
can be expected. Stepwise increase of water up to 10% solvents
was found to decrease the extinction coefficients of A concentrate of Chls a and b was prepared by extracting a spinach
both Chls a and b in both D M F and methanol by less chloroplast pellet with DMF. An aliquot (40 t~l) of this concentrate
was diluted with 3.5 ml of DMF, methanol or buffered 80,% aqueous
than 0.5% thus having little influence on the calculation
acetone. The chlorophyll concentrations were calculated using Eqns.
of chlorophyll concentrations or ratios. 1-9 of Table Iii.
The stepwise dilution of D M F solutions with water
up to 10% has a stepwise effect on the position of the Solvent Chlorophyll concentration Chl a / b ratio
major red peaks of Chls a and b: the major red peak of (nmol/ml) ( R' )
Chl a moved from 663.8 to 664.6 nm and of Chl b from Chl a Chl b Chl a + b
646.8 to 647.6 nm. Thus, when the observable Chl a DMF 7.03 2.04 9.07 3.45
peak of a chlorophyll extract in D M F is located at 664.2 6.98 2.04 9.02 3.42
rather than 663.8 nm it is suggested that the second Methanol 7.06 2.07 9.13 3.41
reading for the simultaneous equations for D M F in 6.94 2.07 9.01 3.35
Table III also be read a further 0.4 nm towards longer
Buffered 80,% 7.15 2.14 9.29 3.34
wavelengths at 647.2 rather than 646.8 nm. aqueous acetone 7.22 2.12 9.34 3.41
With methanol solutions, the major red peak maxima
of Chls a and b were moved stepwise in the opposite
direction toward shorter wavelengths but only by 0.2 to
0.4 nm; thus, if the observable Chl a peak in a chloro- each of the three solvent systems (Table V). The con-
phyll extract in methanol has moved 0.2 nm towards sistency of these results indicates that our new chloro-
shorter wavelengths it is suggested that the second phyll-concentration equations in all three solvent sys-
wavelength in the simultaneous equations for methanol tems (Table III), like the new extinction coefficients
solutions (Table III) should also be read a further 0.2 from which they are derived (Table II), are reliable
nm towards shorter wavelengths. while those of Arnon [3] are incorrect: our equations for
use with aqueous acetone extracts, like those of Ziegler
Determination o f chlorophyll concentrations and Chl a / b and Egle [5], Jeffrey and H u m p h r e y [6] and Lich-
ratios in leaves extracted with O M F , methanol or buffered tenthaler [7], produce higher Chl a / b ratios than those
80 % aqueous acetone of Arnon [3]. Our coefficients (Table II) must, of course,
T o prove that our extinction coefficients (Table II) be consistent with those in diethylether [12], since the
and the equations derived from them (Table III) will latter were used in their derivation (see Experimental).
gi~,e reliable and consistent results, discs of spinach When using a full scale absorbance range of 0-1.0,
leaves and of well and poorly nourished Flindersia other data (not shown) indicated that consistent results,
leaves were extracted with the three solvents by grinding including Chl a / b ratios, are more reliably obtained if
in a mortar (see Experimental): using this technique, it the area of leaf tissue sampled or the volume of extrac-
was found that the re-extracted pellet (see Experimen- tant used is adjusted to give Chls a + b concentrations
tal) was no longer green. Concentrations of Chls a and not lower than about 6.5 n m o l / m l (approx. 6.0 ~tg/ml).
b and of Chls a + b are expressed in / z m o l / m z of leaf
area (Table IV). Chl a / b ratios ( R ' ) are also presented Derivation o f new simultaneous equations for use with
in Table IV in molar terms: the relationship of this alkaline pyridine as extractant
molar ratio ( R ' ) to that expressed in terms of mass ( r ) Porra and G r i m m e [8] derived simultaneous equa-
is expressed in Eqn. 19 below: tions for assaying Chls a and b converted to their cyclic
hydroxylactones by extraction with 2.1 M pyridine in
[ Mr(Chl b) ) 0.35 M N a O H at 6 0 ° C for 15-30 min. They found that
R ' = r ~ a ) =1.0156.r (19)
this procedure, which gave extremely reliable results [8],
was exceedingly useful when chlorophylls were difficult
The chlorophyll content of the leaves varies some- to extract as in regreening nitrogen-deficient Chlorella
what depending on the position from which the disc fusca [14]; however, the extinction coefficients in this
was taken (see also Table VII) but the Chl a/b. ratios earlier work [8] were not calculated by comparison with
remained remarkably constant over the entire leaf and diethylether solutions but with 80% aqueous acetone
in all three solvents. More compelling evidence that the solutions using Arnon's earlier equations [3]. Using the
equations also give consistent chlorophyll concentra- new equations (Table III, Eqns. 7-9) for use with
tions and ratios was obtained by adding 40 /~1 of a buffered aqueous acetone, the original data (see Ref. 8;
concentrated D M F extract of spinach chloroplasts to pp. 261-262) were reprocessed. The new extinction
390

TABLE VI
Corrected millimolar and specific extinction coefficientsfor chlorophylls a and b in alkaline pyridine reagent
The data from Ref. 8 were reprocessed using Eqns. 7 and 8 of Table III to obtain corrected coefficients for the cyclic hydroxylactone derivatives of
Chls a and b produced by addition of 2.1M pyridine in 0.35 M NaOH.

Section Source Wavelength Extinction coefficient

(nm) Chl a Chl b
((~mM) a (EmM)
(l.mmol-l.cm -a ) (l.g-Lcm -1 ) (1.mmol- Lcm -1 ) (l.g-l.cm -1 )
A ref. 8 419 131.9 - 65.5 -
454 15.8 - 159.0 -
B this work 419 136.4 152.7 93.5 103.0
454 16.4 18.4 227.0 250.1

coefficients ( T a b l e V I B ) for the Chl a a n d b h y d r o x y - p h y l l c o n c e n t r a t i o n s i n / ~ g / m l [3] h a v e b e e n c o n v e r t e d ,

l a c t o n e derivatives at 419 a n d 454 n m in a l k a l i n e p y r i - u s i n g m i l l i m o l a r e x t i n c t i o n coefficients, to e x p r e s s all
d i n e were h i g h e r t h a n the o r i g i n a l values ( T a b l e V I A ) c o n c e n t r a t i o n s in n m o l / m l a n d a r e p r e s e n t e d b e l o w
f r o m the o r i g i n a l w o r k [8]. T h e s e n e w e x t i n c t i o n coeffi- (see Eqns. 2 6 - 2 8 ) :
cients were u s e d to d e r i v e n e w s i m u l t a n e o u s e q u a t i o n s
( T a b l e V I I ) to c a l c u l a t e c o n c e n t r a t i o n s of the Chls a Chl a = 14.21 A 663 - 3.01 / 1 6 4 5 (26)
a n d b derivatives e x p r e s s e d b o t h in n m o l / m l (Eqns.
Chl b = 25.23 A 645 - 5.16 A 663 (27)
2 0 - 2 2 ) a n d / ~ g / m l (Eqns. 2 3 - 2 5 ) . T h e s e n e w e q u a t i o n s
a r e n o w c o m p a t i b l e w i t h the o t h e r e q u a t i o n s in T a b l e Chl a + b = 22.22 A64s - 9.05 A663 (28)
I I I for use with the o t h e r three solvents.
U s i n g M a c k i n n e y ' s coefficients, the a b s o r b a n c e s o f
Comparison of results obtained with the new and with s o l u t i o n s o f C h l s a a n d b m i x t u r e s o f 5 : 1, 4 : 1, 3 : 1,
Arnon's simultaneous equations 2 : 1 a n d 1 : 1 (all c o n t a i n i n g 10 n m o l / m l o f Chl a ) w e r e
A r n o n ' s s i m u l t a n e o u s e q u a t i o n s for d e t e r m i n i n g C h l s c a l c u l a t e d at 663 a n d 645 nm. T h e s e a b s o r b a n c e s were
a a n d b were d e r i v e d using the specific e x t i n c t i o n t h e n i n s e r t e d i n t o Eqns. 7 - 9 o f T a b l e I I I to o b t a i n the
coefficients ( a ) o f M a c k i n n e y [15] in the s a m e solvent. true t o t a l c h l o r o p h y l l c o n c e n t r a t i o n s , (Chl a + b ) w. T h e
T h e s e coefficients w i t h their m i l l i m o l a r e q u i v a l e n t s r e l a t i o n s h i p o f ( C h l a + b ) T to the t o t a l c h l o r o p h y l l
s h o w n in b r a c k e t s a r e as follows: d e r i v e d f r o m the A r n o n e q u a t i o n s , (Chl a + b ) A, is
e x p r e s s e d b e l o w in Eqn. 29:
Chl a: a 663 = 82.04 (73.30); a ~5 =16.75 (14.97)
Chl b: a 663= 9.27 (8.41); a 64s=45.60 (41.38) (Chl a + b) T = 0.895(Chl a + b) A (29)

P e r u s a l o f the c o r r e s p o n d i n g coefficients in T a b l e I I T r u e i n d i v i d u a l c o n c e n t r a t i o n s of C h l s a a n d b were

shows t h a t all M a c k i n n e y ' s coefficients are l o w e r a n d then d e r i v e d f r o m (Chl a + b ) T b y u s i n g the true Chl
e s p e c i a l l y t h a t o f Chl b so t h a t the c o n c e n t r a t i o n o f a / b r a t i o , ( C h l a / b ) T, o b t a i n e d b y u s i n g Eqns. 7 a n d 8
b o t h c h l o r o p h y l l s c a l c u l a t e d b y the A r n o n e q u a t i o n s , ( T a b l e III). A q u i c k e r a n d m o r e c o n v e n i e n t m e t h o d to
e s p e c i a l l y t h a t o f C h l b, will b e o v e r e s t i m a t e d , a n d o b t a i n ( C h l a,/b) T is b y r e f e r r a l to Fig. 1 w h e r e the (Chl
c o n s e q u e n t l y the A r n o n C h l a / b r a t i o will b e low. F o r a / b ) T r a t i o is p l o t t e d d i r e c t l y a g a i n s t the A r n o n ratio,
c o n v e n i e n c e , the A r n o n e q u a t i o n s e x p r e s s i n g c h l o r o - (Chl a / b ) A. Fig. 1 also s h o w s the c o r r e c t i o n f a c t o r ( O )

TABLE VII
Corrected equations for the determination of chlorophylls in alkaline pyridine reagent
Eqns. 20-25 below have been derived for the determination of Chls a and b and of Chls a + b converted to their cyclic hydroxylactone derivatives
by solution in 2.1 M pyridine in 0.35 M NaOH.

Equations for chlorophyll concentrations in nmol/ml Equations for chlorophyll concentrations in Fg/ml
Chl a = 7.71 A419-3.I7A 4s4 (20) Chl a = 6.89 A 419 - - 2.84/1454 (23)
Chl b = 4.63 A 4s4 -0.56/1419 (21) Chl b = 4.21 A 454 -0.51 A 419 (24)
Chl a + b=1.46 A 4~ +7.15 A 419 (22) Chl a + b = l . 3 7 A 454 +6.38 A 419 (25)
 391

9.0 c a l c u l a t e d u s i n g Eqns. 31 a n d 32 b e l o w :

so
(Chlb) T - (Chla+b) T (31)
(Chl a/b)T +l

7.0 (Chl a) T = (Chl a + b)T(Chl a/b) x (32)

O (Chl a/b)T + l
n-" 6.o T h e v a l i d i t y of the a b o v e e q u a t i o n s for c a l c u l a t i n g true
5.0 1.8 c o n c e n t r a t i o n s of total c h l o r o p h y l l s , o f Chls a a n d b,
a n d C h l a / b r a t i o s was p r o v e n b y c o m p a r i s o n of the
d a t a o b t a i n e d for leaf discs e x t r a c t e d with a q u e o u s
a c e t o n e ( T a b l e IV) w i t h the s a m e d a t a p r o c e s s e d with
3.0 , ~ the A r n o n e q u a t i o n s . Eqns. 29, 31 a n d 32 as well as Fig.
1 m a y b e used to c o r r e c t c h l o r o p h y l l c o n c e n t r a t i o n s a n d
2.0 o r a t i o s e x p r e s s e d in either m a s s o r m o l a r terms.

1.0
1.0
l
2.0
i
3.0
l
4.0
J
5.0
1'0 "~ An investigation of chlorophyll extraction procedures using
D M F as extractant
A r n o n ' s Chl a / b R a t i o M u c h has b e e n m a d e of the s i m p l e p r o c e d u r e de-
Fig. 1. True Chl a/b ratios (II) and correction factors (II) plotted s c r i b e d b y I n s k e e p a n d B l o o m [4] w h e r e i n t a c t leaf discs
against Arnon's Chl a/b ratios. The values of A 663 a n d A 645 for Chl
are s h a k e n for 24 h w i t h D M F . W e c o m p a r e d the
a + b solutions having Chl a/b ratios of 1.0, 2.0, 3.0, 4,0 and 5.0
were calculated using the coefficients of Mackinney [15] expressed in efficiency o f this p r o c e d u r e with s i m p l e g r i n d i n g a n d
millimolar terms given in the text. These absorbance values were then c e n t r i f u g a t i o n (see E x p e r i m e n t a l ) using discs f r o m t o u g h
inserted into Eqns. 7 and 8 (Table III) to obtain true Chl a/b ratios. a n d l e a t h e r y leaves o f well n o u r i s h e d Flindersia p l a n t s .
The correction factor (tg) is defined in Eqn. 30 (see text). After the I n e a r l i e r e x p e r i m e n t s ( n o t s h o w n ) using the s h a k i n g
true total chlorophyll has been obtained with Eqn. 29, the true Chl
t e c h n i q u e , we f o u n d t h a t the discs were still a very p a l e
a/b ratios can be used with Eqns. 31 and 32 to obtain the correct
concentrations of Chl a and b. green c o l o u r a n d t h a t s u b s e q u e n t g r i n d i n g with D M F
failed to e x t r a c t m o r e c h l o r o p h y l l . Even w h e n the discs
were finely c u t w i t h scissors p r i o r to s h a k i n g for 24 h,
the leaf f r a g m e n t s o f t e n r e t a i n e d a p a l e green c o l o u r
a n d n o m o r e c h l o r o p h y l l c o u l d be e x t r a c t e d b y s u b s e -
w h i c h is d e s c r i b e d in Eqn. 30 b e l o w : q u e n t g r i n d i n g with D M F ( T a b l e V I I I ) . T h e s e results
w i t h this m o d i f i c a t i o n o f the t e c h n i q u e of I n s k e e p a n d
B l o o m ( T a b l e V I I I ) s h o w e d that the r e c o v e r y of total
O ~- (Chl a/b) T
(30) c h l o r o p h y l l is a l m o s t 100% n e a r the tip o f the leaf b u t
(Chl a/b) A falls to a b o u t 84% closer to the stem; however, the Chl
a / b ratios remain constant indicating that both chloro-
Thus, true Chl a a n d b c o n c e n t r a t i o n s m a y n o w b e p h y l l s are e q u a l l y e x t r a c t a b l e b y this solvent. A g a i n , in

TABLE VIII
Comparison of chlorophyll extraction with N,N'-dimethylformamidefrom sficed leaf discs by shaking or by grinding with the soloent
Leaf discs were taken from specific positions of a well nourished Flindersia leaf where L and R refer to left and right of the midrib and a, b, c and
d refer to positions equally spaced from leaf tip to stem. The discs were extracted with DMF by shaking the sliced leaf discs for 24 h with DMF or
by grinding and centrifugation (see Experimental). Chlorophyll concentrations were calculated using Eqns. 1-3 of Table III. Av, average.

Position Grinding in mortar Position Shaking for 24 h Recovery

100. c
on Chl a + b Chl a/b on Chl a + b Chl a/b
leaf (nmol/disc) ratio leaf (nmol/disc) ratio C
(c) (R') (c) (R') (%)
aR 90.28 3.22 aL 88.43 3.11 98.0
bL 97.24 3.19 bR 90.57 3.23 93.1
cR 106.95 3.18 cL 90.08 3.22 84.2
dL 107.01 3.19 dR 90.79 3.16 84.8
Av 3.19 Av 3.18 Av 90.02
392

Table VIII the total chlorophyll content of discs is are firmly embedded in the thylakoid membranes, thus
lower towards the leaf tip while the Chl a / b ratios releasing the chromophores into solution. Alkaline con-
remain reasonably constant (cf. Table IV). In general, ditions, however, have the disadvantage that they render
when D M F is employed, grindingn and centrifugation the chlorophylls susceptible to allomerization; that is
is more reliable (and faster) than the simple procedure irreversible oxidation by molecular oxygen.
which Inskeep and Bloom [4] found successful with Hynninen and Assandri [21] have proposed that al-
soybean leaves. lomerization involves the two types of oxidative reac-
tion sequences described below [21] but other mecha-
Discussion nisms have also been proposed [16]. The first type
results in the formation of C-132-hydroxy (or methoxy)
The purity of the chlorophylls used chlorophylls with absorption spectra virtually identical
Absorption spectroscopy was used to ensure that the to the parent chlorophyll. The proposed sequence in-
chlorophylls employed were free of phaeophytin and volves the removal of the proton at C-132 by base
hydroxylactone contamination (see Results section). ( O H - or O C H 3 ) to give a carbanion which is then
Phaeophytin a has characteristic absorption bands at oxidized by oxygen to a carbonium ion prior to a rapid
532 and 505 nm and pheophytin b at 523 nm not reaction with hydroxyl (or methoxyl) ions to produce
possessed by the parent chlorophylls; similarly the hy- C-132-hydroxy (or methoxy) chlorophylls [21]: structure
droxylactone derivatives (oxidation products) of Chls a I (Fig. 2) shows one stereoisomeric form of C-132-hy -
and b have absorption bands both in the ultra-violet droxychlorophyll. The second type of allomerization
and visible spectra not possessed by the parent chloro- sequence involves the formation, in alkaline solutions,
phylls [8]. If detected, the pheophytins [9] and hydroxy- of the C-131 enolate anion (see Fig. 2, structure II)
lactones [11] can be removed by rechromatography on which possesses a labile double bond between C-131 and
sucrose columns. C-132. This labile bond is susceptible to irreversible
Other oxidation products of Chls a and b, the C- cleavage by molecular oxygen [21,22] which is incorpo-
13:-hydroxy (or methoxy) derivatives, are inseparable rated to form an unstable rhodochlorin-type inter-
from the parent chlorphylls by Sucrose chromatography mediate (Fig. 2, structure III) possessing formic, glyox-
and cannot be detected by absorption spectroscopy alic and propionic acid sidechains at C-13, -15 and -17,
because their spectra [16-18], like those of the chloro- respectively [21]. This structure can subsequently break
phyll epimers [19], are virtually identical to the parent
chlorophylls especially in the red region of the spec- •, / • /

trum; thus, the error (if any) in the determination of

extinction coefficients, even if caused by significant
contamination by these hydroxy (or methoxy) deriva- • ~\\ ,~'
cH3 ~ C, c\'X~___
H ,,, 3

tives, would be minuscule. Nonetheless, the presence of

HO.--C'~-~--~C= O ~:,~' - ,:~_OeH e
these compounds was monitored by reverse phase
HPTLC on Merck RP-8 plates with a m e t h a n o l / H 2 0 COOCHz COOCH 3
(98:2) solvent system: no contamination of Chl b by
I II
the C-132-hydroxy derivative could be detected after 9 h
under oxygen-free nitrogen at - 1 5 °C and with Chl a
it was estimated to be less than 57o. Consistent with Mg. / ", /
this, no more than 57o of Chls a or b resisted conver-
sion to 10 mg rhodochlorms in alkaline methanol as
,2, --/,,
revealed spectrophotometrically.
, ' ooo
~ CH3

The role of alkali, dephytylation and allomerization in the

~73/COO ® cooO CO O C H 3
extraction and assay of chlorophylls
During these studies it was observed that methanol- III IV
extracted chlorophylls from leaf discs marginally more
Fig. 2. The structural differences occurring in rings C, D or E of
readily than DMF, but both these solvents were clearly various chlorophyll derivatives involved in allomerization. Rings A
more effective than buffered 80% aqueous acetone. This and B are unaffected by allomerization. I, one stereoisomeric form of
may account for the use of many methanol procedures, the allomerization product, C-132-hydroxychlorophyU; 11, the enolate
especially hot [2] or alkaline [20] methanol, in situations ion formed in alkaline conditions; III, the tricarboxylic magnesium-
where the chlorophylls are difficult to extract (cf. Refs. rhodochlorin product formed from II by reaction with oxygen (al-
Iomerization); and IV, the cyclic hydroxylactone derivative formed
2 and 14). The use of alkali in such conditions is from III in alkaline pyridine. The tetrapyrrole numbering system and
attractive, since it will hydrolytically release chloro- the use of the nomenclature term 'rhodochlorin' is consistent with the
phylls from their hydrophobic phytyl moieties, which practice of the International Union of Biochemistry [27].
 393

down to form a complex mixture of degradation prod- The experience gained here with DMF suggests that
ucts [21] including the cyclic hydroxylactones (Fig. 2, it may also be useful in some conditions where chloro-
structure IV); thus, allomerization can make the assay phyll extraction is difficult: DMF has the advantage
of chlorophylls impossible. Many of the products of this that it is not volatile or pungent and that chlorophylls
second type of allomerization sequence have spectra are relatively stable in DMF in the dark at low tempera-
quite different to the parent chlorophyll. While al- tures [1].
lomerization is well known to occur in methanolic solu-
tion [21,22], the consistency observed between he results Some biological implications of the corrections to Arnon's
of our methanol-extraction assays with those obtained equations
using DMF and buffered aqueous acetone indicates The correction factors (O) which are required to
that allomerization of the second type does not affect correct the Chl a/b ratios obtained in 80% aqueous
the results when the assays are performed rapidly with acetone extracts calculated by Arnon's equations are
cold methanol in the absence of alkali as in this work. not trivial (see Fig. 1) ranging from 1.17 (for a ratio of
Although the use of alkali in conjunction with 1.0) to 1.70 (for a ratio of 5.0). This means that the well
methanol is normally avoided because of the opportun- established differences in Chl a and b content observed
ity for allomerization, Milner et al. [20] devised a simple when higher plants and green algae acclimate to light
colorimetric assay for total chlorophyll by extraction [25] need correction. For example, sun plants or plants
with 5% KOH in methanol at 63°C for 3 min: they acclimated to high irradiance are reported to have Chl
anticipated only the release of 'saponified chlorophylls' a/b ratios ranging from 2.8 to 3.4 but are, in fact,
from the membrane-embedded phytol moiety. While 3.8-4.8: shade plants and plants acclimated to low
dephytylation occurred under their assay conditions, we irradiance are quoted to have Chl a/b ratios of 2.1-2.3,
have found (unpublished results) that allomerization of which are actually 2.7-3.0. Moreover, the chlorophyll
the second type occurred during the extraction of bio- content and Chl a/b ratios of the isolated Chl a- and
logical tissues. This complicated the spectra and may be b-containing protein complexes have also been de-
the reason that this assay was used only to determine termined mainly in 80% aqueous acetone using Arnon's
total chlorophyll and not the individual Chl a and b equations and, therefore, need correction. For example,
concentrations. Surprisingly, working with purified the main light-harvesting Chl a and b protein of Pho-
chlorophylls, we showed that the isocyclic E rings of tosystem II (LHC II) is generally accepted to have a Chl
both Chls a and b were opened by hydrolysis but a/b ratio of 1.15 but, in fact, has a ratio of 1.38 which
without the incorporation of oxygen (i.e., without al- means that the percentage of Chl b is decreased from
lomerization) (cf. Ref. 21) to form two magnesium- the previously accepted value of 46.6 to 42.0%. The
rhodochlorins, known in the past, respectively, as Mg- corresponding pigment protein complex of light-
chlorin e6 and Mg-rhodin g7 [23]: these compounds harvesting complex I (LHC I) has a Chl a/b ratio of
resemble structure III (Fig 2) but the glyoxalate about 3.6 [26] which will actually be 5.25 so that the
sidechain at C-15 is replaced by an acetate. The forma- percentage of Chl b will be 16% rather than 21.7%.
tion of these two tricarboxyl compounds was proved by Thus, it becomes clear that the correction of earlier data
the occurrence of major red peaks at about 642 and 624 is required to assess the true, but lesser, amount of total
nm, respectively, in both KOH-methanol and diethyl- chlorophyll present and its correct distribution between
ether [21,22]; also, the spectra in diethylether of the two Chls a and b before meaningful models of the photo-
Mg-free rhodochlorins, prepared by acidification with synthetic apparatus can be proposed.
HC1, were identical to those of chlorine 6 and rhodin g7
[24]. Thus, it would appear that crude biological ex- Concluding remarks
tracts contain a factor which enhances allomerization of In the assay of a mixture of Chls a and b a major
chlorophylls. problem for the accuracy of the method is the measure-
Alkali has also been used in association with pyridine ment of the absorbance at the wavelength of Chl b
to extract and assay chlorophylls [8] in conditions where which occurs on a steeply sloping segment of the spec-
chlorophyll is difficult to extract [14]. This assay is trum of the mixture. Consequently, it is important to
again based on the removal of the chromophore from measure the position of the observable Chl a peak very
the thylakoid membrane by hydrolysing the phytyl-es- carefully and maintain the appropriate wavelength in-
ter linkage. It is unusual, however, because the second terval (which separates the peaks) because this interval
type allomerization reactions occurring in this solvent appears to be maintained at all relevant water con-
stoichiometrically convert Chls a and b to their respec- centrations. The appropriate interval is 17.0, 13.2 and
tive cyclic hydroxylactones (see Fig. 2, structure IV) by 17.0 nm for the DMF, methanol and buffered 80%
a mechanism described elsewhere (cf. Ref. 21). These aqueous acetone procedures. Maintaining this interval is
photolabile cyclic hydroxylactones are stable for about made considerably easier by the newest microprocessor
60 min at 4 ° C in foil-wrapped, stoppered tubes [8]. controlled spectrophotometers which can locate a peak
394

to the nearest 0.1 nm and enable the operator to read 11 Porra, R.J., Klein, O. and Wright, P.E. (1983) Eur. J. Biochem.
absorbance at any wavelength accurate to the nearest 130, 509-516.
12 Smith, .I.H.C. and Benitez, A. (1955) in Modern Methods of Plant
0.1 nm.
Analysis (Paech, K. and Tracey, M.V., eds.), Vol. 4, pp. 143-196,
Springer-Verlag, Berlin.
Acknowledgements 13 Falk, H. (1956) Planta 51, 49-62.
14 Porra, R.J. and Grinune, L.H. (1974) Arch. Biochem. Biophys.
The authors thank Dr. Jan M. Anderson for en- 164, 312-321.
15 Mackinney, G. (1941) J. Biol. Chem. 140, 315-322.
couragement and advice, Drs. Fred Chow and S.W.
16 Schaber, P.M., Hunt, J.E., Fries, R. and Katz, J.J. (1984) J.
Jeffrey for helpful discussions and Ms. Janice C. Mur- Chromatog. 316, 25-41.
rell for help in identifying inconsistencies in past chlo- 17 Strain, H.H., Thomas, M.R. and Katz, J.J. (1963) Biochim. Bio-
rophyll assays. The authors also thank Mr. G.C. Irving phys. Acta 75, 306-311.
for determining the magnesium content of Chls a and b 18 Pennington, F.C., Strain, H.H., Svec, W.A. and Katz, J.J. (1967) J.
Am. Chem. Soc. 89, 3875-3880.
by atomic absorption spectroscopy.
19 Hynninen, P.H. (1973) Acta, Chem. Scand. 27, 1487-1495.
20 Milner, H.W., French, C.S., Koenig, M.L.G. and Lawrence, N.S.
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21 Hynninen, P.H. and Assandri, S. (1973) Acta Chem. Scand. 27,
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2 B~Sger, P. (1964) Flora 154, 174-211. 22 Holt, A.S. (1958) Can. J. Biochem. Physiol. 36, 439-456.
3 Arnon, D.I. (1949) Plant Physiol. 24, 1-15. 23 Fischer, H. and Stern, A. (1940) in Die Chemie des Pyrrols, Vol. 2,
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