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Original Article
In situ measurement of leaf chlorophyll concentration:
analysis of the optical/absolute relationship
Christopher Parry1, J. Mark Blonquist Jr.2& Bruce Bugbee1
1Department of Plants, Soils, and Climate; Utah State University, Logan, UT 84322, USA and 2Apogee Instruments, Inc., Logan,
UT 84322, USA
ABSTRACT
In situ optical meters are widely used to estimate leaf chlo-
rophyll concentration, but non-uniform chlorophyll distribu-
tion causes optical measurements to vary widely among
species for the same chlorophyll concentration. Over 30
studies have sought to quantify the in situ/in vitro (optical/
absolute) relationship, but neither chlorophyll extraction nor
measurement techniques for in vitro analysis have been con-
sistent among studies. Here we: (1) review standard pro-
cedures for measurement of chlorophyll; (2) estimate the
error associated with non-standard procedures; and (3)
implement the most accurate methods to provide equations
for conversion of optical to absolute chlorophyll for 22
species grown in multiple environments.Tests of five Minolta
(model SPAD-502) and 25 Opti-Sciences (model CCM-200)
meters, manufactured from 1992 to 2013, indicate that differ-
ences among replicate models are less than 5%. We thus
developed equations for converting between units from these
meter types. There was no significant effect of environment
on the optical/absolute chlorophyll relationship. We derive
the theoretical relationship between optical transmission
ratios and absolute chlorophyll concentration and show how
non-uniform distribution among species causes a variable,
non-linear response. These results link in situ optical meas-
urements with in vitro chlorophyll concentration and
provide insight to strategies for radiation capture among
diverse species.
Key-words: CCM-200; Chla/Chlb; leaf optical properties;
SPAD-502.
INTRODUCTION
Leaf chlorophyll concentration is most accurately measured
by extraction of chlorophyll in a solvent followed by in vitro
measurements in a spectrophotometer. However, non-
destructive, in situ, optical techniques have become widely
used to provide a relative indication of leaf chlorophyll con-
centration. Two commercially available meters are widely
used (Minolta, model SPAD-502 (Spectrum Technologies,
Plainfield, Ill.); and Opti-Sciences, model CCM-200 (Opti-
Sciences, Inc., Hudson, NH)) and results from these meters
have been reported in over 30 studies (Table 1). Neither
meter has a linear relationship with chlorophyll concentra-
tion, and the reported optical/absolute chlorophyll concen-
tration relationship has varied widely, sometimes even within
the same species.
Measurement of absolute chlorophyll
concentration in vitro
The extraction method, extraction solvent, spectropho-
tometric equation and spectrophotometer resolution must
match to accurately determine chlorophyll in vitro (Wellburn
1994). More than 30 studies have been conducted, but
few have used the appropriate combination of analytical
procedures.
Seven organic solvents have been widely used for chloro-
phyll extraction: acetone, methanol, ethanol, chloroform,
diethyl-ether, dimethyl-formamide (DMF) and dimethyl
sulphoxide (DMSO).Acetone has been the most widely used
solvent because it has sharp chlorophyll peaks, but it is con-
sidered to be less efficient at chlorophyll extraction than
methanol and ethanol (Holmhansen & Riemann 1978;
Ritchie 2006). Acetone, methanol and ethanol require grind-
ing of leaf tissue for complete extraction of chlorophyll. DMF
and DMSO have an advantage over other solvents in that
they allow for immersion of intact leaf tissue for chlorophyll
extraction. However, immersion may not be effective for all
plant tissues. Schaper & Chacko (1991) were not able to
completely extract chlorophyll from Cashew and Mango leaf
discs using DMSO. DMSO is less toxic than DMF, and
extracted solutions are stable up to 7 d in the dark at 4 °C
(Barnes et al. 1992). These advantages have led to increasing
use of DMSO as an extraction solvent, but it is absorbed
through the skin and gloves should be worn when handling it
(Barnes et al. 1992).
Matching extraction solvent with
spectrophotometric equation to convert
absorption values to chlorophyll concentration
Wellburn (1994) emphasized the importance of using
spectrophotometric equations that have been derived from
accurate extinction coefficients determined in a reliable
reference solution. Extinction coefficients from Smith &
Benitez (1955) derived for diethyl-ether are generally
accepted as accurate and are recommended for use in deriv-
ing extinction coefficients for other extraction solvents using
the procedures described in Porra et al. (1989). Based on the
Correspondence: C. Parry. e-mail: chris.k.parry@aggiemail.usu.edu
Plant, Cell and Environment (2014) 37, 2508–2520 doi: 10.1111/pce.12324
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© 2014 John Wiley & Sons Ltd2508
magnesium concentration of a known chlorophyll a and b
solution, Porra et al. (1989) confirmed the extinction coeffi-
cients of Smith & Benitez (1955) for both chlorophyll a and
b in diethylether. They found that the error in the original
Smith & Benitez (1955) equation was less than 1%. Several
equations developed for DMSO and DMF solvents have
failed to follow the appropriate Porra et al. (1989) procedure
(Moran & Porath 1980; Moran 1982; Inskeep & Bloom 1985;
Barnes et al. 1992).
The equations developed by Arnon (1949) have often
been used to quantify chlorophyll a and b concentration in
higher plants and green algae. These equations were devel-
oped for use with 80% acetone in water. Several authors
(Lichtenthatler & Wellburn 1983; Barnes et al. 1992; Porra
2002) have reported that equations from Arnon (1949) are
inaccurate because they used the less accurate extinction
coefficients of Mackinney (1941). Also, the chlorophyll a/b
ratios obtained from the equations of Arnon (1949) under-
estimate the true a/b chlorophyll ratio (Porra et al. 1989;
Wellburn 1994). Porra et al. (1989) developed an equation to
convert a/b chlorophyll ratios determined by the equations of
Arnon (1949) to correct values.
Several authors have used DMSO as an extracting solvent,
but used spectrophotometric chlorophyll equations devel-
oped for 80% acetone (Monje & Bugbee 1992; Richardson
et al. 2002).This has been justified by citing other publications
that suggest that the absorption spectra for chlorophylls a and
b are identical for 90% acetone and DMSO (Shoaf & Lium
1976; Hiscox & Israelstam 1979; Ronen & Galun 1984).
However, equations from Arnon (1949) were developed for
80% (not 90%) acetone. Furthermore, Barnes et al. (1992)
showed that the peak absorption wavelength for chlorophylls
a and b is at a longer wavelength in DMSO than 80% acetone
and found that equations from Arnon (1949) underestimated
chlorophyll concentration using DMSO extracts by approxi-
mately 10%.
Matching spectrophotometric chlorophyll
equations with instrument resolution
Wellburn (1994) discussed differences in chlorophyll meas-
urement among spectrophotometers with differing spectral
bandwidth resolution. Early spectrophotometer models used
to derive equations were capable of only 1–4 nm resolution.
High-quality modern spectrophotometers have a resolution
of 0.1–0.5 nm and have been used to derive recently devel-
oped spectrophotometric chlorophyll equations. Wellburn
(1994) compared three types of spectrophotometers (Uvikon
model 941 Plus (Kontron, [U.K] Ltd.), 0.5 nm resolution;
Table 1. Summary of publications on the
optical/absolute chlorophyll concentration
relationship
Meter type/Author (year) Species
SPAD-501
Yadava (1986) Twenty-two unrelated species
Marquard & Tipton (1987) Twelve unrelated species
Schaper & Chacko (1991) Eight tropical and subtropical fruit-tree species
Dwyer et al. (1991) Maize
Fanizza et al. (1991) Twelve wine-grape cultivars
SPAD-502
Gratani (1992) Six Sclerophyllous species
Monje & Bugbee (1992) Rice, soybean, wheat
Markwell et al. (1995) Soybean and maize
Xu et al. (2000) Sorghum
Bindi et al. (2002) Potato
Richardson et al. (2002) Paper birch
Netto et al. (2002) Papaya
Yamamoto et al. (2002) Sorghum and pigeon pea
Esposti et al. (2003) Four citrus species
Wang et al. (2004) Peace lily
Netto et al. (2005) Coffee
Jifon et al. (2005) Six citrus species
Cartelat et al. (2005) Wheat
Uddling et al. (2007) Birch, wheat and potato
Marenco et al. (2009) Six Amazonian tree species
Naus et al. (2010) Tobacco
Imanishi et al. (2010) Flowering cherry
Coste et al. (2010) Thirteen tree species of tropical rainforest
Ling et al. (2011) Arabidopsis thaliana
Cerovic et al. (2012) Kiwi, grape, wheat, and maize
CCM-200
Richardson et al. (2002) Paper birch
van den Berg & Perkins (2004) Sugar maple
Jifon et al. (2005) Six citrus species
Goncalves et al. (2008) Four tropical wood species
Cerovic et al. (2012) Kiwi, grape, wheat and maize
The optical/absolute chlorophyll relationship 2509
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
Hewlett-Packard model HP8452A (Hewlett Packard Corpo-
ration, Palo Alto, CA), diode array 2 nm fixed resolution; and
Pye Unicam model SP30 (Pye Ltd., Cambridge England),
1–4 nm variable resolution) and determined chlorophyll con-
centrations in six solvents. He omitted data from the diode
array spectrophotometer because it had values that almost
always deviated more than 10% from values of the other two
instruments. Wellburn (1994) concluded that diode array
spectrophotometers are not appropriate for use with equa-
tions derived by non-diode array spectrophotometers, and
emphasized that equations derived with one spectro-
photometer should not be used with a spectrophotometer
with a different spectral resolution.
Although the goal of previous studies has been to develop
standard curves to convert optical measurements to absolute
chlorophyll concentration, measurement techniques vary
widely. Predicted chlorophyll concentration from optical
measurements of wheat leaves, measured with the same
model of meter, has varied up to 80% among studies (Monje
& Bugbee 1992; Uddling et al. 2007). These differences have
not been widely acknowledged in the literature.
Optical meters used to determine
chlorophyll concentration
The two most widely used chlorophyll concentration meters
are the Konica Minolta, model SPAD-502 (Konica Minolta
Sensing, Inc., Sakai, Osaka, Japan) and the Opti-Sciences,
model CCM-200 (Opti-Sciences, Inc., Hudson, NH, USA).
Both meters measure the transmission of two wavelengths of
radiation through plant leaves: red at approximately 650 nm,
and near infrared (NIR) at approximately 900 nm. Increased
chlorophyll concentration increases the absorption of red
radiation.All plants transmit a high fraction of NIR radiation
as these wavelengths are not absorbed by photoreceptors
and this transmission is used as a reference wavelength.
Another hand-held, optical chlorophyll meter was recently
introduced, the Dualex 4 Scientific (Dx; FORCE-A, Orsay,
France).This meter measures the transmission of radiation at
710 and 850 nm and converts the measurement into a value
of chlorophyll in μgcm
−2.
The sampling area differs between meters. The CCM-200
samples 71 mm2, the SPAD-502 samples 6 mm2and the Dx4
samples 20 mm2. Larger areas provide a larger spatial
average, but smaller areas can measure narrower leaves.
Description of the optical differences
between meters
The output of the CCM-200 is the ratio of transmission of
radiation from a light emitting diode (LED) centred at
931 nm to transmission of radiation from an LED cantered at
653 nm (CCM-200 user manual). This ratio is defined as the
chlorophyll content index (CCI).
CCI =%
%
transmission nm
transmission nm
931
653 (1)
The SPAD-502 measures radiation centred at 940 and
650 nm (Minolta Manual), but the equation to convert these
measurements to a ‘SPAD’ value has been reported differ-
ently in four publications. The most complete equation is
given by Naus et al. (2010):
SPAD k C=× ⎛
⎝
⎜⎞
⎠
⎟+log %
%
transmission nm
transmission nm
940
650 (2)
where kis a confidential slope coefficient and Cis a confi-
dential offset value. Three other publications have reported
less complete equations to calculate the SPAD value.
Uddling et al. (2007) reported this equation, but without the
Coffset. Cerovic et al. (2012) and Markwell et al. (1995)
reported the equation without either kor C.As the slope and
offset values are confidential, it is not possible to derive
SPAD values from transmission measurements, and it is not
possible to mathematically derive a conversion equation
between meters. However, as both the SPAD values and the
CCI are based on a ratio of the transmission at two closely
related wavelengths:
SPAD k CCI C≈×
()
+log (3)
Studies on the optical/absolute chlorophyll
concentration relationship
Four studies have reported empirical relationships that relate
optical measurements to absolute chlorophyll concentration
for a meter (model SPAD-501) that was a predecessor to the
SPAD-502 (Yadava 1986; Marquard & Tipton 1987; Fanizza
et al. 1991; Schaper & Chacko 1991). The SPAD-501 used
slightly different wavelengths and is thus not directly compa-
rable with the SPAD-502.
Monje & Bugbee (1992) appear to have been the first to
develop an equation that relates the output from the SPAD-
502 to absolute chlorophyll concentration in mg m−2. Since
then, numerous other relationships for a range of species
have been proposed (Schaper & Chacko 1991; Markwell
et al. 1995; Xu et al. 2000; Bindi et al. 2002; Netto et al. 2002,
2005; Richardson et al. 2002; Yamamoto et al. 2002; Esposti
et al. 2003; Wang et al. 2004; Cartelat et al. 2005; Jifon et al.
2005; Uddling et al. 2007; Marenco et al. 2009; Coste et al.
2010; Imanishi et al. 2010; Naus et al. 2010; Ling et al. 2011;
Cerovic et al. 2012). The acronym ‘SPAD’ refers to the divi-
sion of Minolta that developed the meter, special products
analysis division.As the acronym implies,SPAD has no direct
relationship to chlorophyll concentration.
Like SPAD, CCI values returned by the CCM-200 are
only relative indicators of chlorophyll concentration, as CCI
has no direct relationship to chlorophyll concentration.
Several studies have also developed chlorophyll prediction
equations using CCI measurements from the CCM-200
meter (Richardson et al. 2002; van den Berg & Perkins
2004; Jifon et al. 2005; Goncalves et al. 2008; Cerovic et al.
2012).
2510 C. Parry et al.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
Variation in experimental techniques
among studies
Extraction and measurement techniques have not been con-
sistent among studies.Because chlorophyll concentration can
have significant spatial variation it is important to remove the
leaf disk from exactly the same location as the optical meas-
urement. This precaution has not always been described
in experimental procedures. Multiple extraction solvents,
measurement wavelengths, spectrophotometric equations
and instruments with varying resolution have been used to
measure absolute chlorophyll. Sampling and measurement
differences likely have caused significant variation among
studies.
Most studies that have sought to determine the optical/
absolute relationship have used only a single meter with the
assumption that all meters of the same model are uniform. In
an early study, Marquard & Tipton (1987) found 5% differ-
ences between two SPAD-501 meters. Markwell et al. (1995),
mentioned that three SPAD-502 meters at the same univer-
sity differed by ±5% and recommended that separate equa-
tions be developed for individual meters, but they did not
indicate if optics in the meters had been cleaned before use.
A comprehensive evaluation of uniformity among replicate
meters has not been done. Two studies have attempted to
estimate the prediction error associated with an individual
measurement. Richardson et al. (2002) examined the error
associated with individual optical measurements for paper
birch leaves. They compared CCM-200 and SPAD-502
meters and found similar errors for both meters (19% for the
SPAD meter and 20% for the CCM-200 meter). This relative
error was calculated by dividing the root mean square error
(RMSE) by average chlorophyll concentration across all
samples. Cerovic et al. (2012) compared the Dx4 meter to
SPAD-502 and CCM-200 meters and reported similar
RMSEs for all three meters.
Differences among plant groups and species
Related species may share leaf optical properties. Monocots
have a larger fraction of vascular tissue per unit surface area
and dicots have a thicker adaxial cuticle with more palisade
and spongy tissue. Cerovic et al. (2012) measured two
monocot and two dicot species, and suggested that optical/
absolute chlorophyll relationships could be grouped into
separate monocot and dicot categories.
Chlorophyll a/b ratio
Considering that chlorophyll a and b can be easily distin-
guished in vitro, there has been a surprising lack of litera-
ture reporting differences among species. Few of the 30
studies on the optical/absolute relationship have reported
the a/b chlorophyll ratio. Chang and Troughton (1972)
pointed out that the chlorophyll a/b ratio can be affected by
the species, environment, phase of leaf and plant growth
and nutrient status on the chlorophyll a/b ratio. Their data
indicate that chlorophyll a/b ratios are higher in C4than C3
plants.
Chlorophyll a/b ratios are known to decrease during leaf
senescence (Watts & Eley 1981; Castro & Sanchez-Azofeifa
2008), but several studies have found that drought stress has
no effect on the chlorophyll a/b ratio (Martin & Warner 1984;
Mafakheri et al. 2010). Several authors have suggested that
chlorophyll a/b ratio should increase as leaf nitrogen content
decreases, and the data of Kitajima & Hogan (2003) support
this conclusion.
Cultivar differences within a species
Markwell et al. (1995) developed a single optical/absolute
chlorophyll relationship for multiple strains of soybeans and
maize, Uddling et al. (2007) found that a single curve could be
used for multiple wheat cultivars grown over multiple
seasons, and Dwyer et al. (1991) found that six maize (corn)
hybrids had similar relationship curves. However, signifi-
cantly different relationships were observed among citrus
cultivars (Jifon et al. 2005). Cate & Perkins (2003),
Richardson et al. (2002), and van den Berg & Perkins (2004)
have all cautioned against treating a single optical/absolute
chlorophyll relationship as universal.
The objectives of this study were: (1) estimate the magni-
tude of differences associated with the use of non-standard
combinations of solvents and equations; (2) to implement
the most correct methods for chlorophyll measurement to
provide improved equations for conversion of optical
measurements to absolute chlorophyll concentration; (3) to
examine uniformity among two meter models (Opti-Sciences,
model CCM-200; and Minolta, SPAD-502) manufactured
from 1992 to 2013; (4) to develop equations for inter-
converting between units (CCI and SPAD units) from the two
most common chlorophyll meters (Opti-Sciences, model
CCM-200; and Minolta, SPAD-502); (5) estimate environ-
mental effects on the optical/absolute chlorophyll concentra-
tion relationship; and (6) use optical and mathematical
principles to better understand the underlying causes of non-
linearity in the optical/absolute chlorophyll concentration
relationship.
MATERIALS AND METHODS
Collection and extraction of samples
Leaves of multiple ages and intensity of green colour were
measured and sampled from 22 plant species (five monocots
and 17 dicots,11 deciduous species, and 11 annual crop plants)
grown in greenhouse and field environments. Leaves were
visually selected for a wide range of the intensity of greenness,
which varied due to leaf age, position on the plant, and nutri-
ent deficiencies. A common nutrient deficiency was lack of
either nitrogen or iron, which was caused by high root-zone
pH. Measurements were made near midday to minimize
potential effects of light intensity on chloroplast movement.
CCI, using a CCM-200 meter, was measured at least three
times in the same location on each leaf and averaged. A leaf
disk was extracted from the exact same location as the meas-
urement. Leaf disks were immediately extracted using a
The optical/absolute chlorophyll relationship 2511
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
number 4 cork borer with an area of 90 mm2to replicate the
area measured by the chlorophyll meter and placed in a vial
containing 10 mL of DMSO.Vials were incubated in an oven
at 65 °C until all of the chlorophyll was in solution and the
disk became transparent. This extraction occurred in less
than 30 min for some species, but required 3 h for other
species. After incubation, a 3 mL aliquot was transferred to
an optical-grade analysis cell to measure light absorbance at
646.6 and 663.6 nm (Porra, 1989 acetone equation), and at
649.1 and 665.1 nm (Wellburn 1994;DMSO equation) using a
Shimadzu UV-2401PC (Shimadzu Corporation, Kyoto,
Japan) spectrophotometer with a resolution of 0.1 nm. Chlo-
rophyll a and b concentrations were determined from spec-
tral measurements using the equations developed by
Wellburn (1994) for DMSO and for 0.1–0.5 nm spectral
resolution:
Chlorophyll a mL nm
nm
µg A
A
−
()
=×
()
−×
()
112 47 665 1
3 62 649 1
..
.. (4)
Chlorophyll b mL nm
nm
µg A
A
−
()
=×
()
−×
()
125 06 649 1
6 5 665 1
..
.. (5)
where Ais the absorption at the referenced wavelength and
chlorophylls a and b are summed to obtain the total chloro-
phyll concentration.
Because several publications have extracted with DMSO,
but incorrectly used the equation of Porra et al. (1989) that
was developed for 80% acetone, chlorophyll was calculated
using both procedures to determine the magnitude of error
between equations.
Uniformity among meters
Five replicate Minolta SPAD-502 meters, manufactured from
1992 to 2008, and 25 replicate Opti-Sciences CCM-200
meters, manufactured from 2007 to 2013, were examined for
uniformity of output by making replicate measurements on
six coloured filters. These filters provided a consistent,
uniform standard over a range of readings from 2 to 72 CCI
units and from 6 to 62 SPAD units. The filters were Roscolux
filters: #88, ‘Light Green’; #3204, ‘Half Blue’; #86, Pea Green;
#92, ‘Turquoise’; #89, ‘Moss Green’; and #4490, ‘CalColor 90
Green’.
Conversion between meters
Optical measurements were made in multiple identical loca-
tions on leaves of 10 plant species using a SPAD-502 and a
CCM-200 meter. These measurements were supplemented
with measurements made on 16 Roscolux filters to provide a
wide range of SPAD and CCI values. Measured SPAD values
were plotted against corresponding CCI measurements to
obtain a relationship curve for the output of the two meters.
Multiple wheat cultivars
Four diverse wheat cultivars (Golden Spire, Lewjain, Green-
ville and Wanser) were grown in a greenhouse under three
nutrient treatments: optimal nutrient availability, nitrogen
deficient and iron deficient to determine relationships among
cultivars and environmental conditions.
RESULTS
Summary of previous studies
Relationships between SPAD-502 and CCM-200 meters and
absolute chlorophyll concentration from 17 previous studies
indicate a wide range of relationships among species
(Fig. 1a,b).
Relationships among similar species in
different studies
Wheat is the most widely studied species with four SPAD-502
curves reported in four studies (Monje & Bugbee 1992;
Cartelat et al. 2005; Uddling et al. 2007; Cerovic et al. 2012).
The difference in the optical/absolute relationship among
studies was as high as 80% between Uddling et al. (2007) and
Monje & Bugbee (1992) (Fig. 2).
Figure 1. Relationship between meter output and chlorophyll
concentration (μmol m−2). (a) Twelve representative studies using
special products analysis division (SPAD) units, and (b) four
studies and this study using chlorophyll content index (CCI).
Species and analytical methods differed among studies.
2512 C. Parry et al.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
Some of our measurements on wheat were made with the
SPAD-502 meter; others were made with the CCM-200
meter. All data were converted to SPAD units to develop a
comprehensive curve for wheat (Fig. 2). No significant differ-
ence among cultivars or nutrient stress treatments was found
in the optical/absolute chlorophyll relationship. Our meas-
urements were close to the average of the other studies
across all chlorophyll concentrations.
Mean percentage difference between relationship
curves of this study and others was also calculated for
soybean (29%) (Markwell et al. 1995) and sorghum (40%)
(Yamamoto et al. 2002; data not shown).
Paper birch was the only species that was common among
studies using the CCM-200 meter. Richardson et al. (2002)
used DMSO as the extractant, and the equation of Porra et al.
(1989) that was developed for acetone extractants.We deter-
mined the magnitude of the error associated with this incor-
rect match of extraction solvent and spectrophotometric
equation. Based on calculations for each of the 22 species in
this study, we found that the mean difference between abso-
lute chlorophyll concentrations calculated for a DMSO
extractant using the DMSO equation of Wellburn (1994) and
the acetone equation of Porra et al. (1989) is 7.84% (SD
0.28%; data not shown).We thus corrected the equation from
Richardson et al. (2002) for paper birch by multiplying it by
7.84%. This correction resulted in a nearly identical fit to our
derived equation for paper birch (Fig. 3).
Differences among species
The 22 species in this study had a wide range of optical/
absolute chlorophyll relationships (Fig. 4a). A single univer-
sal relationship for all species was derived (Fig. 4b), along
with individual equations for each species (Table 2; Fig. 5).
Although it appears that some cultivars within a species
can be expressed by a single relationship, we found signifi-
cantly different optical/absolute chlorophyll concentration
relationships between two lettuce cultivars (cv. Waldman’s
Green and cv. Buttercrunch; Lactuca sativa; Fig. 5). However,
our data indicate that the monocots barley, wheat, and rice
have a similar optical/absolute chlorophyll concentration
relationship (Fig. 5).
Uniformity of replicate meters
Output from each individual meter was plotted against the
mean of all meters of the same type to determine variation
among studies because of variation among replicate
meters (Fig. 6). Mean coefficient of variation was 2.60%
for the CCM-200 meter and 1.10% for the SPAD-502
meter.
Inter-conversion between units
Our results indicate that that universal relationships can be
used to inter-convert between CCI and SPAD units
(Figs 7A & 3B; r2= 0.98, 0.99). A similar relationship was
developed by Richardson et al. (2002) for converting SPAD
units to CCI units (r2= 0.97). However, the meter conver-
sion relationship created by Richardson et al. (2002) was
based on measurements on paper birch leaves with a
narrow range of chlorophyll (SPAD units of 0–40). It was
also developed for a prototype CCM-200 meter, which had
a different wavelength for the red absorption wavelength.
This meter was replaced with the current version in late
2002. The meter conversion curves for this study were
developed from multiple species over a wide range of chlo-
rophyll concentrations.
Monocot and dicot species differences
The absolute/optical relationships between CCI and
chlorophyll concentration for the mean of five monocot
Figure 2. Relationship between special products analysis division
(SPAD) units and chlorophyll concentration (μmol m−2) for wheat
from four prior studies and this study.The chlorophyll content
index relationship from this study was converted for use with
SPAD units using the equation in Fig. 7a.
Figure 3. Relationship between chlorophyll content index (CCI)
and chlorophyll concentration (μmol m−2) for paper birch (Betula
papytifera) leaves from two studies.The original relationship from
Richardson et al. (2002) was corrected for the underestimation of
chlorophyll concentrations derived from the equation of Porra
et al. (1989) for dimethyl sulphoxide extractants.
The optical/absolute chlorophyll relationship 2513
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
species and the mean of 17 dicot species were not signifi-
cantly different as indicated by the 95% prediction intervals
(Fig. 8).
Chlorophyll a/b ratio
The mean chlorophyll a/b ratio for C3and C4plants was 3.2
and 6.3, respectively (Table 2). These results are similar to the
values of Chang and Troughton (1972) when corrected for
the underestimation of the Arnon (1949) equation (C3: 3.9
and C4: 5).
There was a small positive relationship between
chlorophyll concentration and the a/b ratio. The coefficient
of determination between absolute chlorophyll concentra-
tion and a/b ratio was 0.68 for Lilac, 0.48 for Japanese
Maple and less than 0.20 for all other species (data not
shown).
DISCUSSION
Relationship between transmission and absolute
chlorophyll and cell wall content of leaves
Output of both Minolta SPAD-502 and Opti-Sciences
CCM-200 meters is based on the ratio of transmission of
NIR to red wavelengths. Transmission of radiation is non-
linearly related to the amount of absorbing compound in
leaf tissue and linearly related to the absorbance of com-
pound (Atkins 1990). Absorbance is the negative log of
transmittance.
Non-chlorophyll compounds (primarily cell walls) absorb
radiation similarly at both red and NIR wavelengths,
so transmission of red light is similarly affected by both
compounds. Transmission of NIR radiation is not affected
by chlorophyll and is thus primarily determined by the
amount of non-chlorophyll compounds.Assuming a uniform
Figure 4. Relationship between chlorophyll content index (CCI) and chlorophyll concentration (μmol m−2) for (a) 22 individual species and
(b) all 22 species combined. The molar mass of the chlorophyll molecule is about 900 grams per mole. These measurements can easily be
converted to mass per unit area.
2514 C. Parry et al.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
distribution of chlorophyll in leaves, the absolute amount of
cell wall and chlorophyll in leaves can be determined from
the ratio of percentage transmission by the following rela-
tionship based on the Beer–Lambert law:
CCI e
e
=≈
−
()
−
%
%
Transmission NIR
Transmission RED
cell wall
chlorrophyll cell wall
chlorophyll cell wall
cell wall
+
()
+
()
()
=e
e
(6)
CCI e e=−
+
()()
chlorophyll cell wall cell wall (7)
ln ln lnCCI e e
()
=−
[][]
+
()()
chlorophyll cell wall cell wall (8)
ln CCI
()
=+
()
−
()
chlorophyll cell wall cell wall (9)
SPAD CCI≈
()
=
()
ln chlorophyll (10)
As shown by the final equation, if chlorophyll is uniformly
distributed, SPAD values would be linearly related to chlo-
rophyll concentration of leaves and CCI values would be
related to chlorophyll concentration as a logarithmic func-
tion. Chlorophyll, however, is not uniformly distributed in
leaves and this causes concentration estimates based on
transmission measurements to deviate from the equations
shown earlier. The optical changes caused by non-uniform
distribution are caused by the sieve and detour effects.
The sieve effect and the detour effect
The transmission of light through a leaf is affected by
pigment concentration and pigment spatial distribution in
leaves. Non-uniform chlorophyll distribution (clumping of
chlorophyll molecules) decreases transmission of light at
lower chlorophyll concentrations and increases transmission
of light at higher chlorophyll concentrations. Distribution of
chlorophyll within a leaf is influenced by structural organiza-
tion of grana within chloroplasts, chloroplasts within cells,
and cells within tissue layers (Fukshansky et al. 1993).
When light passes through leaf tissue without encountering
an absorber it is known as the sieve effect, which increases
with increasing non-uniformity of chloroplasts. As chloro-
plast uniformity increases, efficiency of red light absorption
increases.
The detour effect (light scattering) increases the optical
path-length through the leaf, which reduces light transmis-
sion. The leaf reflectance at the reference NIR wavelength is
much higher than the leaf reflectance at the red chlorophyll
absorption wavelength. This causes the detour effect to be
more pronounced for the reference NIR wavelength. The
detour effect reduces transmission per unit chlorophyll
(Monje & Bugbee 1992; Uddling et al. 2007; Naus et al. 2010).
Differing optical/absolute chlorophyll relationships among
species are likely due to different chlorophyll distribution
patterns, and thus differing sieve and detour effects.
Table 2. Equations to determine chlorophyll concentration (μmol m−2) from chlorophyll content index (CCI), r2values for each equation
and mean chlorophyll a/b ratio for 22 species
Conversion Equation
(μmol m−2 from CCI) r2
Mean Chlorophyll
a/b ratio
Standard Deviation
of a/b ratio
Deciduous Species
European Birch −76 + 85*(CCI)0.64 0.89 3.3 0.5
Lilac −98 + 93*(CCI)0.51 0.95 2.6 0.5
Norway Maple −95 + 96*(CCI)0.57 0.94 3.9 0.7
Quaking Aspen −128 + 106*(CCI)0.50 0.92 3.3 0.3
Purple Leaf Sand Cherry −144 + 113*(CCI)0.55 0.96 2.5 0.7
Crab Apple −124 + 117*(CCI)0.47 0.93 4.4 1.4
Paper Birch −120 + 135*(CCI)0.48 0.94 2.5 0.4
Crimson King Maple −160 + 144*(CCI)0.50 0.90 2.6 0.3
Japanese Maple −150 + 150*(CCI)0.43 0.97 1.9 0.1
Boxelder −191 + 182*(CCI)0.38 0.92 2.7 0.3
Forsythia −486 + 477*(CCI)0.18 0.93 2.6 0.5
Annual Crop Plants
Sorghum (C4) −8 + 29*(CCI)0.80 0.90 6.9 2.0
Pepper −19 + 39*(CCI)0.69 0.92 3.7 0.7
Rice −64 + 57*(CCI)0.68 0.82 5.0 1.5
Wheat −84 + 79*(CCI)0.60 0.87 4.3 0.4
Soybean −103 + 123*(CCI)0.47 0.95 4.2 0.6
Maize (C4) −121 + 129*(CCI)0.42 0.84 5.7 1.4
Barley −132 + 146*(CCI)0.43 0.95 3.1 0.7
Kohlrabi −150 + 162*(CCI)0.34 0.83 3.1 0.8
Tomato −328 + 304*(CCI)0.26 0.87 2.9 0.7
Pea −334 + 316*(CCI)0.24 0.84 3.8 0.9
Lettuce
Waldman’s Green −2204 + 2204*(CCI)0.04 0.98 2.7 0.2
Buttercrunch −29 + 32*(CCI)0.74 0.98 2.5 0.2
The optical/absolute chlorophyll relationship 2515
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
Figure 5. Relationship between chlorophyll content index (CCI) and chlorophyll concentration (μmol m−2) for 22 species. Equations for
each relationship are provided in Table 2.
2516 C. Parry et al.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
The sieve effect causes transmission to increase and thus
the optical chlorophyll measurement is lower than a sample
with uniform chlorophyll distribution (Monje & Bugbee
1992; Richardson et al. 2002; Jifon et al. 2005; Uddling et al.
2007; Marenco et al. 2009). The detour effect decreases trans-
mission of light compared with a sample with uniform
chlorophyll distribution and thus increases the optical chlo-
rophyll measurement (Uddling et al. 2007). Uddling et al.
(2007) observed a noticeable deviation caused by the sieve
affect above a SPAD value of 20 and a relatively larger
deviation caused by the detour effect below a SPAD value of
20.The combined effects of these relationships on the optical/
absolute chlorophyll relationship cause a predictable devia-
tion from the theoretical relationship (Fig. 9).
Environmental effects on optical measurements
Changes in leaf environment have the potential to alter leaf
morphology, leaf thickness and chloroplast distribution.
Changes in specific leaf area, often caused by water or tem-
perature stress, have the potential to alter the optical/
absolute chlorophyll relationship. Light scatter is higher in
thicker leaves (Naus et al. 2010); however, unlike other
studies (Campbell et al. 1990; Jifon et al. 2005), we did not
find a different optical/absolute chlorophyll relationship
between leaves of the same species (tomatoes, peppers,
maize, peas) grown in greenhouse versus outdoor environ-
ments. Our data for paper birch leaves match the corrected
data of Richardson et al. (2002), in spite of measurements
made on seedlings in a greenhouse (Richardson et al. 2002),
and our measurements on mature trees in an arid environ-
ment in Utah. Collectively, these findings do not suggest a
significant environmental effect on the optical/absolute chlo-
rophyll concentration relationship.
Figure 6. Uniformity of the two most common chlorophyll
meters. The output of individual meters was compared with the
mean of all meters of the same type. Measurements were made on
coloured filters to provide a uniform reference. (a) Five Minolta
model SPAD-502 meters with special products analysis division
(SPAD) output manufactured from 1992 to 2008. (b) 25
Opti-Sciences model CCM-200 meters with chlorophyll content
index (CCI) output manufactured from 2007 to 2013. The
coefficient of variation (SD/mean) was 1.1% among meters with
SPAD unit output, and 2.6% among meters with CCI output. Both
types of meters were highly uniform and differences among meters
are much smaller than differences in genetic, environmental and
extraction/analytical techniques.
Figure 7. Equations to convert (a) special products analysis
division (SPAD) units to chlorophyll content index (CCI) and
(b) CCI to SPAD units. Data are from replicate measurements of
multiple species. Each comparison measurement was made on the
same spot on each leaf.
The optical/absolute chlorophyll relationship 2517
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
Light-dependent chloroplast movement
Light intensity can alter chloroplast orientation (Hoel &
Solhaug 1998; Naus et al. 2010), which can affect the optical/
absolute chlorophyll relationship. Davis et al. (2011) found
that the effects of chloroplast movement were greatest in
shade species and found that mean maximum percentage
change in red light transmission between low and high light
acclimation was 6.3% (SD 4.7%) for shade-grown leaves and
2.1% (SD 1.6%) for sun-grown leaves. This change in
chloroplast orientation in response to light is small, but
potentially significant in the optical/absolute chlorophyll
relationship.
Davis et al. (2011) hypothesized that the amount of chlo-
rophyll movement was correlated with cell diameter. Nar-
rower, more columnar cells of sun leaves may have a greater
restriction on chloroplast movement than shade leaf cells.
Leaf cell size and shape differ greatly with species (Lee et al.
2000), which may explain varying degrees of chloroplast
movement among species. All measurements in this study
were made in high light to minimize effects from light-
dependent chloroplast movement.
Differences among replicate meters
Previous studies on differences among meters have not pro-
vided a comprehensive test of meter variability (Marquard &
Tipton 1987; Markwell et al. 1995). Our results indicate that
differences among replicate meters were minimal, suggesting
differences among studies in the optical/absolute chlorophyll
concentration relationship are not caused by different
meters.
Most of the variability among optical/absolute chloro-
phyll concentration relationships of similar species is likely
due to the variability of extraction methods, extraction sol-
vents, chlorophyll concentration equations and the resolu-
tion of spectrophotometers. Some studies have determined
chlorophyll concentration using diode array spectropho-
tometers with methanol extinction coefficients from Porra
et al. (1989) (e.g. Cerovic et al. 2012). This is contrary to the
recommendations of Wellburn (1994) and would likely lead
to errors in determination of absolute chlorophyll concen-
tration. Porra et al. (1989) used a Hitachi 3200 spectro-
photometer (Hitachi High-Technologies Corporation,
Tokyo, Japan) with a spectral resolution of 0.1–0.5 nm over
the visible spectrum for extract extinction coefficient deter-
mination. Spectrophotometers with similar resolution
should be used for best accuracy.
Differences among cultivars of the same species
Many studies have shown that cultivars within species have
similar optical/absolute chlorophyll concentration relation-
ships, but this is not always the case. There were significant
Figure 8. Relationship between chlorophyll content index (CCI)
and chlorophyll concentration (μmol m−2) for the mean of five
monocot species and 17 dicot species. In spite of leaf anatomical
differences among species there was no significant difference
between these diverse plant groups.
Figure 9. Impact of the detour (light scattering) and sieve effects
(non-uniform chlorophyll distribution) on the optical/absolute
chlorophyll concentration relationship for (a) special products
analysis division (SPAD) units and (b) chlorophyll content index
(CCI). The black line indicates the theoretical relationship if
chlorophyll was uniformly distributed in the leaf. The range of
units and shapes of the blue dashed lines are from the measured
data in Fig. 1.
2518 C. Parry et al.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2508–2520
differences in the optical/absolute chlorophyll relationship
for the two lettuce cultivars in this study. This difference can
most likely be attributed to the difference in leaf morphology
and anatomy in these two cultivars.
Relationship between monocots and dicots
On the basis of measurements in two monocot and two dicot
species, Cerovic et al. (2012) suggested that there may be a
difference between monocots and dicots.However, no signifi-
cant difference was found between monocot and dicot curves
for the five monocot and 17 dicot species in this study (Fig. 8).
In spite of anatomical differences, it does not appear that
monocot and dicot species have different optical/absolute
chlorophyll concentration relationships.
Chlorophyll a/b ratio
Chlorophyll a/b ratios are often reported to bea3to1ratio,
but this ratio has not been widely studied. Chang and
Troughton (1972) reported typical ratios of C3plants as 3 to 1;
and ratios in C4plants as 5 to 1.They suggest that the a/b ratio
is affected by both genetics and by biotic and abiotic factors.
We found a similar difference in the ratios for C3and C4plants
(Table 2). We did not find a difference in the optical/absolute
chlorophyll relationship between C4and C3plants in spite of
the anatomical difference between these plant groups, and a
significant difference in the a/b chlorophyll ratio.
The slope of the optical/absolute relationship
indicates differences in chlorophyll distribution
and radiation capture
Species with a steep slope in the optical/absolute relationship
poorly intercept light per unit chlorophyll; species with a low
slope efficiently intercept light per unit chlorophyll. It is
likely that increasing non-uniformity of chlorophyll leads to a
steeper slope of this relationship. This study highlights the
enormous differences in chlorophyll distribution among
species and even within species. The lettuce cultivar
(Buttercrunch) had one of the lowest slopes and the other
(Waldman’s Green) had one of the highest slopes.
ACKNOWLEDGMENTS
We thank Mike Hancock for his dedicated technical work.
This work was supported by the Utah Agricultural Experi-
ment Station, Utah State University. Approved as journal
paper no. 8583.
DISCLOSURE OF CONFLICT OF INTEREST
Mark Blonquist and Bruce Bugbee are employees of Apogee
Instruments, which is a reseller of the Opti-Sciences chloro-
phyll meter.
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