Fluorimeter 6285

Application note: A10-006A

Determination of chlorophyll a using fluorimetry

Introduction
The photosynthetic pigment chlorophyll is present in
most plants, algae and cyanobacteria. It can be
measured both by spectrophotometry and fluorimetry
as an indicator of the abundance of photosynthetic
organisms in fresh and salt water. In fresh water,
levels of chlorophyll are also an important factor in
determining water quality. Chlorophyll pigments may
be present in several forms in varying ratios, the most
common being chlorophyll a. Here we demonstrate
two methods of determining chlorophyll a using the
Jenway 6285 fluorimeter based on EPO method
445.0 (1). We also show how the presence of
chlorophyll b can affect the results. In addition we
demonstrate that the model 6285 can detect as little
as 0.01g/l chlorophyll a.

Methods
Chlorophyll a and b from spinach were purchased
from Sigma, part codes C5753 and C5878. The
contents of the vials, containing 1mg of pure
chlorophyll, were each dissolved in 50ml of 90%
acetone (spectrophotometric grade, Sigma 154598) to
give 20mg/l stock solutions. These were stored in
aliquots in the dark at -20C until required.
Stock solutions of chlorophylls a and b were diluted in
90% acetone to give solutions of 1mg/l which were
determined spectrophotometrically using the formulae
of Lichtenthaler and Wellburn (2). Briefly, the
absorbance of each dilution was measured in a
Jenway model 6505 spectrophotometer at the
absorbance maxima for chlorophylls a and b
(662.6nm and 645.6nm respectively). The formulae
given below were used to calculate the chlorophyll
concentrations.
Ca = (11.75 x A662.6) (2.35 x A645.6)
Cb = (18.61 x A645.6) (3.96 A662.6)
where Ca and Cb are the concentrations of chlorophyll
a and b respectively. The solutions were adjusted
until they reached 1mg/l.
Photosynthetic pigments were also extracted from a
sample of slime algae isolated from a domestic
marine fish tank. The algae were ground in a mortar
containing ceramic beads with 90% acetone. The
extract was filtered through Whatman No. 1 filter
paper into a volumetric flask and made up to a
volume of 50ml with 90% acetone. The extract was
stored in aliquots in the dark at -20C until required.

The two methods described in EPO method 445.0 are

as follows:
a.

Corrected
chlorophyll
a
determination,
measuring the fluorescence before and after
acidification of the sample. This uses broad
excitation and emission filters. The filters used in
the model 6285 for this assay were BG28 380500nm band pass filter (part code 627 124) for
excitation and Kodak 29, 610nm cut-off filter (part
code 627 127) for emission.

b.

Uncorrected chlorophyll a determination which

uses narrow band pass filters to eliminate most of
the spectral overlap with pheopyhtin a and
chlorophyll b. The filters used in the model 6285
for this assay were 435nm interference filter (part
code 627 165) for excitation and 680nm
interference filter (part code 627 193) for
emission.

Results
The detection limit of the model 6285 fluorimeter was
first determined by serially diluting the stock
chlorophyll a solution until it could no longer be
detected above the blank (90% acetone). The
fluorimeter was set up with the narrow band pass
filters, 435nm and 680nm and the gain set to 100%
for maximum sensitivity. Each sample was diluted in
triplicate and 2.5ml placed in a glass fluorimeter
cuvette (part code 060 254). The results are
presented in Table 1.
Concentration
(g/l)
10
1.0
0.1
0.05
0.01
0

Average
(RFU)
34.180
3.971
0.949
0.940
0.715
0.655

SD ( RFU)
0.155
0.045
0.011
0.007
0.008
0.006

Table 1: Relative fluorescence unit (RFU) values for

various dilutions of chlorophyll a.
It can be seen from the results that 0.01g/l
chlorophyll a can be clearly distinguished from the
blank by greater than 3 standard deviations from the
mean, demonstrating the very high sensitivity of the
instrument.

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Once the appropriate gain settings had been

determined, the effects of the presence of chlorophyll
b in the solution were investigated using both the
corrected and uncorrected chlorophyll a methods.
50
45

y = 0.2364x + 0.029
R2 = 1

40
35

RFU

Next, a set of chlorophyll a standards were prepared

ranging from 0.2g/l to 200g/l to determine the linear
dynamic range of the fluorimeter. Essentially this is to
determine a suitable gain setting where all the
standards fit on a linear calibration curve, such that
the top standard is not more or less than 10% from
the calculated concentration based on the linear
regression equation of the line. The RFU values for
each of the standards were read at the gain as set by
the auto gain function and at three lower gain
settings. This test was done using both sets of filters.
The data was plotted for the four lowest standards
and, using the equation of the line, the theoretical
value for the 200g/l standard was calculated. The
results are presented in Tables 2 and 3.

30
25

y = 0.2304x + 0.076
R2 = 1

20
15
10
5
0
0

Std conc.
(g/l)
0
0.2
2.0
5.0
20
200
Calc. conc.
(g/l)
2
R value

RFU
52%
0.038
0.174
1.295
3.245
13.22
112.1
169.87

RFU
50%
0.027
0.134
0.991
2.487
10.16
90.67
178.73

RFU
45%
0.012
0.064
0.472
1.185
4.852
47.31
195.26

RFU
40%
0.005
0.027
0.205
0.516
2.126
21.33
200.87

0.9997

0.9999

Table 2: Test of linearity at different gain settings

using the BG28 and Kodak 29 filters.
Std conc.
(g/l)
0
0.2
2.0
5.0
20
200
Calc. conc.
(g/l)
2
R value

RFU
81%
0.145
0.271
1.413
3.350
13.26
110.6
168.11

RFU
75%
0.085
0.159
0.818
1.947
7.757
71.25
185.02

RFU
70%
0.050
0.094
0.506
1.204
4.803
46.14
193.41

RFU
65%
0.027
0.052
0.299
0.722
2.870
28.30
198.34

0.9997

0.9999

Table 3: Test of linearity at different gain settings

using the 435nm and 680nm interference filters.
For the BG28 and Kodak 29 filters, the 45% gain
setting gave a good linear relationship over the whole
standard range with the calculated top standard
fluorescence within 2.5% of the expected calculated
value. This gain setting was therefore used in
following experiments. Likewise, the 70% gain setting
was used with the narrow band pass filters, with the
top standard within 3.5% of the calculated value. The
standard curves produced with both sets of filters are
shown in Figure 1.
From the gradient of these lines, the response factor
described in EPO method 445.0 can be derived. The
response factor, Fs, is equivalent to 1/gradient of the
standard curve at the chosen gain setting, S.

50

100

150

200

Concentration (ug/l)

Figure 1: Chlorophyll a standards curves measured

using the BG28 and Kodak 29 filters (red) and 435nm
and 680nm interference filters (blue).
The conventional method of chlorophyll a
determination described in EPO method 445.0 relies
on acidification of the sample to correct for the
contribution of any pheophytin a (the magnesium-free
derivative of chlorophyll a) present in the samples.
Spectral overlap of pheophytin a and chlorophyll b
with chlorophyll a can lead to errors in the estimation
of chlorophyll a.
A number of mixtures were prepared containing
different ratios of chlorophyll a and b; the total amount
of chlorophyll remained the same in each case. Table
4 lists the composition of the mixtures. A 1 in 1000
dilution of the slime algae extract was also measured.
Sample

Chl a
conc.
(g/l)

Chl b
conc.
(g/l)

A
B
C
D
E
F

10
9
8
6
5
0

0
1
2
4
5
10

Vol.
20g/l
Chl a
(ml)
2
1.8
1.6
1.2
1.0
0

Vol.
20g/l
Chl b
(ml)
0
0.2
0.4
0.8
1.0
2.0

Vol.
90%
acetone
(ml)
2.0
2.0
2.0
2.0
2.0
2.0

Table 4: Mixtures of chlorophyll a and b tested.

For the corrected method, which is based on
acidification of the solution, the fluorescence of a set
of standard dilutions plus a number of mixtures of
chlorophyll a and b were measured before and 90
seconds after the addition of 0.1N HCl to a final
concentration of 0.003N. Briefly, 2.5ml of each
sample were placed in a glass fluorimeter cuvette and
the fluorescence determined. 75l of 0.1N HCl was
then added to the cuvette, mixed and incubated for
90s before reading the fluorescence again.

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only about 44% that of the equivalent concentration of

chlorophyll a.

Using a standard solution of chlorophyll a, a beforeto-after acidification response ratio, r, can be

determined:
r = Rb/Ra
where:
Rb

Fluorescence of pure chlorophyll a

standard solution before acidification

Ra

Fluorescence of pure chlorophyll a

standard solution after acidification

On studying the corrected values (Cc) it can be seen

that the top standard (200 g/l) appears to contain a
large proportion of pheophytin a; this could indicate
some degradation of the sample. It is also clearer
from these results the presence of the added
chlorophyll b in the mixed samples A to F. Chlorophyll
b is not degraded at such a rapid rate as chlorophyll a
in the presence of acid, therefore the before-to-after
ratio is not as great. In these experiments, the amount
of chlorophyll b present is being measured as an
overestimation of the pheophytin a concentration.

The acidification response ratio for the mid-range

chlorophyll a standards (2, 5 and 20g/l) averaged at
1.59. Table 5 gives the calculated results for
uncorrected chlorophyll a, corrected chlorophyll a
and pheophytin a, based on the following formulae:

Sample
0 g/l
0.2 g/l
2.0 g/l
5.0 g/l
20 g/l
200 g/l
A
B
C
D
E
F
Slime
algae

Uncorrected chlorophyll a:
Cu = Rb x Fs
where:
Cu

Uncorrected chlorophyll a concentration

(g/l)

Rb

Fluorescence of the sample solution

before acidification

Fs

Fluorescence response factor at gain

setting S

Rb
(RFU)
0.012
0.066
0.474
1.192
4.881
47.49
2.441
2.478
2.363
2.195
1.969
1.082
4.257

Ra
(RFU)
0.012
0.046
0.301
0.750
3.051
31.69
1.531
1.596
1.568
1.518
1.438
1.042
2.962

Cu
(g/l)
0.049
0.270
1.945
4.892
20.027
194.86
10.02
10.17
9.698
9.008
8.079
4.440
17.47

Cc
(g/l)
-0.005
0.222
1.907
4.884
20.237
174.72
10.06
9.753
8.794
7.487
5.864
0.443
14.32

P
(g/l)
0.086
0.077
0.060
0.012
-0.333
32.028
-0.076
0.658
1.437
2.417
3.521
6.356
5.011

Table 5: Fluorescence of standards and samples

before and after acidification and the calculated
uncorrected and corrected chlorophyll a and
pheophytin a concentrations

Corrected chlorophyll a:
Cc = Fs(r/r-1) (Rb Ra)
where:
=

Corrected chlorophyll a concentration

(g/l)

Fs

Fluorescence response factor at gain

setting S

The before-to-after acidification

response ratio of a pure chlorophyll a
solution

Rb

Fluorescence of the sample solution

before acidification

Ra

Fluorescence of the sample solution

after acidification

Pheophytin a (P, g/l):

Figure 2 shows the correlation of chlorophyll a

concentration in samples A to F compared to the
corrected chlorophyll a value. Also in the graph,
chlorophyll b is correlated to the calculated amount of
pheophytin a.
12

"Corrected" concentration

Cc

10

Chl a

Chl b

8
6
4
2
0
0

10

Chlorophyll concentration (ug/l)

P = Fs(r/r-1) (rRa Rb)

Table 5 illustrates as expected that the uncorrected
values (Cu) for the standard solutions are very close
to the concentrations of the standard dilutions. For
samples A to F where the total chlorophyll
concentration remained at 10g/l, the values did drop
slightly with increasing amount of chlorophyll b, since
the fluorescence of chlorophyll b can be seen to be

Figure 2: Correlation of diluted chlorophyll a and b

solutions with the corrected values based on the
fluorescence readings before and after acidification.
Using these formulae, the sample extracted from the
marine slime algae was calculated to have a ratio of
approximately 1:0.35 chlorophyll a to pheophytin

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a/chlorophyll b and an
concentration of 14.32mg/l.

undiluted

chlorophyll

An alternative to the acidification assay is to measure

chlorophyll a using narrow band pass interference
filters. If this method is used, then only the
uncorrected chlorophyll a is calculated. As for the
acidification assay, a series of standards and mixtures
of chlorophylls a and b were measured using these
filters at the 70% gain setting as determined previously.
The chlorophyll a concentrations were calculated using
the formula for uncorrected chlorophyll a and the
results are shown in Table 6.
Sample
0 g/l chl a
0.2 g/l chl a
2.0 g/l chl a
5.0 g/l chl a
20 g/l chl a
200 g/l chl a
10 g/l chl a
10 g/l chl a + 2.5g/l chl b
10 g/l chl a + 5.0g/l chl b
10 g/l chl a + 10g/l chl b
Slime algae

RFU
0.050
0.094
0.506
1.204
4.803
46.14
2.379
2.411
2.418
2.870
4.742

Cu (g/l)
0.21
0.39
2.12
5.05
20.14
193.5
9.98
10.11
10.14
12.03
19.89

Table 6: RFU values and derived chlorophyll a

concentrations calculated from the formula for
uncorrected chlorophyll a.
Using this method, the presence of added chlorophyll
b in the sample leads only to very little chlorophyll a
overestimation. At a 2:1 ratio of chlorophyll a to b, the
amount is overestimated by only 1.6%, however if the
ratio is increased to 1:1 (the highest likely to be found
naturally) then the overestimation is around 20%.

Conclusions
The Jenway model 6285 fluorimeter has a redenhanced PMT detector which allows the detection of
molecules which fluoresce at wavelengths up to
850nm. Using this instrument, we have demonstrated
it is possible to detect chlorophyll a down to a
concentration as low as 0.01g/l with a large linear
dynamic range, making it several orders of magnitude
more sensitive than a spectrophotometer.
Based on methods described in EPO method 445.0,
chlorophyll a can be determined using either narrow
band pass interference filters allowing an
uncorrected chlorophyll a calculation, or by using
wider band pass filters and including an acidification
step. In the former method, we have shown that
chlorophyll b can be present up to as much as 30% of
the total chlorophyll and have a minimal effect on
chlorophyll a estimation.
The acidification method has the advantage in that
the amount of pheophytin a and/or chlorophyll b can
also be estimated. With the narrow band pass filters,
although a large amount of chlorophyll b was added,
this could not be measured. With the acidification
method the results calculated for pheophytin a
correlated well with the amount of chlorophyll b spiked
into the sample. Since there is a large degree of
spectral overlap between chlorophyll b and
pheophytin a, it would not possible to distinguish
between them in natural samples.
In summary, the model 6285 fluorimeter is an
extremely sensitive instrument ideal for the estimation
of chlorophyll a in the study of marine and freshwater
algae as described in EPO method 445.0.

References
(1) EPA Method 445.0: In vitro determination of
chlorophyll a and pheophytin a in marine and
freshwater algae by fluorescence. Arar, E. J. and
Collins, G. B. (1997).
(2) Lichtenthaler, H.K., and Wellburn, A.R.,
Determination
of
total
carotenoids
and
chlorophylls a and b of leaf in different solvents.
Biol. Soc. Trans. 11: 591-592 (1983).

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