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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 380-500nm 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)
Average (RFU)
SD ( RFU) 10 34.180 0.155 1.0 3.971 0.045 0.1 0.949 0.011 0.05
0.940 0.007 0.01 0.715 0.008
0 0.655 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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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.
Std conc. (g/l)
RFU 52%
RFU 50%
RFU 45%
RFU 40%
0 0.038 0.027 0.012 0.005 0.2 0.174 0.134 0.064 0.027 2.0 1.295
0.991 0.472 0.205 5.0 3.245 2.487 1.185 0.516 20 13.22 10.16 4.852
2.126 200 112.1 90.67 47.31 21.33
Calc. conc. (g/l)
169.87 178.73 195.26 200.87
R2 value 0.9997 0.9999 1 1
Table 2: Test of linearity at different gain settings using the
BG28 and Kodak 29 filters.
Std conc. (g/l)
RFU 81%
RFU 75%
RFU 70%
RFU 65%
0 0.145 0.085 0.050 0.027 0.2 0.271 0.159 0.094 0.052 2.0 1.413
0.818 0.506 0.299 5.0 3.350 1.947 1.204 0.722 20 13.26 7.757 4.803
2.870 200 110.6 71.25 46.14 28.30
Calc. conc. (g/l)
168.11 185.02 193.41 198.34
R2 value 0.9997 0.9999 1 1
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.
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.
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)
Vol. 20g/l Chl a (ml)
Vol. 20g/l Chl b (ml)
Vol. 90%
acetone (ml)
A 10 0 2 0 2.0 B 9 1 1.8 0.2 2.0 C 8 2 1.6 0.4 2.0 D 6 4 1.2 0.8
2.0 E 5 5 1.0 1.0 2.0 F 0 10 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.
y = 0.2304x + 0.076R2 = 1
y = 0.2364x + 0.029R2 = 1
05
101520253035404550
0 50 100 150 200
Concentration (ug/l)
RFU
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Using a standard solution of chlorophyll a, a before-to-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
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:
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
Corrected chlorophyll a:
Cc = Fs(r/r-1) (Rb Ra) where:
Cc = Corrected chlorophyll a concentration (g/l)
Fs = Fluorescence response factor at gain setting S
r = 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):
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
only about 44% that of the equivalent concentration of
chlorophyll a.
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.
Sample Rb (RFU)
Ra (RFU)
Cu (g/l)
Cc (g/l)
P (g/l)
0 g/l 0.012 0.012 0.049 -0.005 0.086 0.2 g/l 0.066 0.046 0.270
0.222 0.077 2.0 g/l 0.474 0.301 1.945 1.907 0.060 5.0 g/l 1.192
0.750 4.892 4.884 0.012 20 g/l 4.881 3.051 20.027 20.237 -0.333
200 g/l 47.49 31.69 194.86 174.72 32.028 A 2.441 1.531 10.02
10.06 -0.076 B 2.478 1.596 10.17 9.753 0.658 C 2.363 1.568 9.698
8.794 1.437 D 2.195 1.518 9.008 7.487 2.417 E 1.969 1.438 8.079
5.864 3.521 F 1.082 1.042 4.440 0.443 6.356
Slime algae
4.257 2.962 17.47 14.32 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
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.
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
0
2
4
6
8
10
12
0 2 4 6 8 10
Chlorophyll concentration (ug/l)
"Co
rrec
ted"
co
nce
ntr
atio
n
Chl a Chl b
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a/chlorophyll b and an undiluted chlorophyll a concentration of
14.32mg/l.
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 RFU Cu (g/l) 0 g/l chl a 0.050 0.21
0.2 g/l chl a 0.094 0.39 2.0 g/l chl a 0.506 2.12 5.0 g/l chl a
1.204 5.05 20 g/l chl a 4.803 20.14
200 g/l chl a 46.14 193.5 10 g/l chl a 2.379 9.98
10 g/l chl a + 2.5g/l chl b 2.411 10.11 10 g/l chl a + 5.0g/l
chl b 2.418 10.14 10 g/l chl a + 10g/l chl b 2.870 12.03
Slime algae 4.742 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 red-enhanced
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).
