EPA/600/R-94/150
SUMMARY REPORT
Evaluation of a New Fluorometric Technique that Uses
Highly Selective Interference Filters for Measuring
Chlorophyll a in the Presence of Chlorophyll b and
Pheopigments
by
Elizabeth J. Arar
Inorganic Chemistry Branch
Chemistry Research Division
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268

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DISCLAIMER
This report has been reviewed by the Environmental Monitoring Systems
laboratory - Cincinnati, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.

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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts research
to:
o Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in marine and estuarine waters,
drinking waters, surface waters, groundwaters, wastewaters,
sediments, sludges, and solid wastes.
o Investigate methods for the identification and measurement of
viruses, bacteria and other microbiological organisms in aqueous
samples and to determine the responses of aquatic organisms to water
quality.
o Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants
in marine and estuarine waters, drinking water, surface water,
groundwater, wastewater, sediment and solid wastes.
o Develop methods and models to detect and quantify responses in
aquatic and terrestrial organisms exposed to environmental stressors
and to correlate the exposure with effects on chemical and
biological indicators.
Spectral interferences, caused by the presence of pigments such as
chlorophyll b and pheophytin a, degrade the accuracy of conventional
fluorometry for determining chlorophyll a extracted from algae. This EMSL-
Cincinnati report, "Evaluation of a New Fluorometric Technique that Uses
Highly Selective Interference Filters for Measuring Chlorophyll a in the
Presence of Chlorophyll b and Pheopigments," was prepared to inform
environmental monitoring organizations of a recently developed technique that
improves the fluorometric measurement of chlorophyll a.
i \ i

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ABSTRACT
A new fluorometric technique was compared to conventional fluorometry
with and without pheophytin a (pheo a) correction and to spectrophotometry
using Lorenzen's modified monochromatic equations and Jeffrey and Humphrey's
trichromatic equation to calculate chlorophyll a (chl a). The new technique
uses excitation and emission filters that are highly selective for chl a,
eliminating the interference caused by accessory pigments and pheopigment
degradation products.
Four method parameters were evaluated using the new technique,
conventional fluorometry and spectrophotometry. They were (1) sensitivity,
(2) linear dynamic range, (3) precision, and (4) accuracy. Controlled studies
of the interference caused by chl b were conducted, and real world samples of
varying taxonomic composition were analyzed. In laboratory solutions, the new
technique was comparable to conventional fluorometry with respect to
sensitivity and accuracy. The linear dynamic range for the new technique
exceeded that of conventional fluorometry by a factor of three. Interference
caused by chl b was +6% at the highest chl atchl b likely to occur in nature.
Chl a values obtained using the new technique compared well with conventional
fluorometry when pheo a was the only interfering pigment present in the
sample.
iv

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ACKNOWLEDGMENTS
The valuable assistance and technical advice of Susan Mokelke of Turner
Designs (Sunnyvale, CA) was much appreciated throughout this work. Many
thanks also go out to Don Schultz of the USEPA for organizing the Chlorophyll
Workshop in Athens, GA, in February, 1993 and for seeing this report through
to its completion.
v

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FIGURES
NUMBER	PAGE
1	Excitation/Emission Filters 		10
2	Excitation/Emission Spectra of the Chlorophylls 		11
3	Excitation/Emission Spectra of the Pheopigments 		12
4	Chlorophyll a - Spectrophotometry 		13
5	Analog Fluorometer 				14
6	Digital Fluorometer 		15
7	Effect of Chi b on Chi a: Analog vs. Digital
Fluorometer 	 16
8	Effect of Chi b on Chi a: Analog vs. Digital
Fluorometer 	 17
9	Effect of Chi b on Measured Pheophytin Analog
Fluorometer 	 18
10	1:1 Mixtures of Chi a and Pheo a Spectrophotometry 	 19
11	Chlorophyll a - Comparison of Methods 	 20
San Francisco Bay - 1000 ml Samples
12	Chlorophyll a - Comparison of Methods 	 HI
Lake Pontchartrain - 300 ml Samples
13	Chlorophyll a - Comparison of Methods 	 22
New York Bight - 300 ml Samples
14	Chlorophyll a - Comparison of Methods 	 23
New York Bight - 900 ml Sample
15	Chlorophyll a - Comparison of Methods 	 24
New York Bight - 600 ml Sample
vi

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CONTENTS
Disclaimer	ii
Foreword	iii
Abstract		iv
Acknowledgments 		v
Figures	vi
1.	Introduction 		1
2.	Experimental 		1
3.	Results		3
4.	Conclusions		7
References		9
vi i

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INTRODUCTION
The high sensitivity of fluorometry makes it a choice technique for
measuring chlorophyll a (chl a). However, spectral interferences caused by
pheophytin a (pheo a) and chlorophyll b (chl b) can cause substantial over- or
underestimation of chl a. Conventional fluorometry with acidification
correction for pheo a results in underestimation of chl a and overestimation
of pheo a if chl b is present (caused by the conversion of chl b to pheo b
which is determined as pheo a), whereas if no pheo a correction is applied,
overestimation of chl a results.1 A simple fluorometric method for
measuring chl a in the presence of pheo a and chl b recently has been
developed4 and commercially introduced (Turner Designs, Sunnyvale, CA). The
new technique utilizes excitation and emission filters that are highly
selective for chl a, thereby eliminating the need for acidification (Figures
1-3). The result is a direct relationship between chl a and fluorescence with
a maximum interference from chl b or pheo a of +10%.
At a chl a measurement and interpretation workshop held by USEPA Region 4
in Athens, GA, Turner Designs (a leading manufacturer of fluorometers)
presented laboratory results obtained by Dr. Nicholas Welschmeyer of Moss
Landing Marine Laboratories, CA using the newly selected interference
filters.4 EMSL-Cincinnati personnel present at the workshop arranged to
evaluate the new technique using a Turner Designs Model 10-AU digital
fluorometer. Solutions of pure pigments, mixtures of pigments and natural
sample extracts were analyzed using the 10-AU fluorometer, a Turner Designs
Model 10 analog fluorometer and a Beckman DU-6 spectrophotometer. The results
of those analyses are presented here.
EXPERIMENTAL
Instrumentation Specifications
Spectrophotometer - Beckman DU-6, multiwavelength. Wavelength accuracy -
+0.5 nm, Wavelength resolution - 2 nm, Lamp - tungsten. Unless otherwise
indicated, a 1-cm glass cell was used for all analyses.
Analog Fluorometer - Turner Designs Model 10. F4T5 blue lamp, red-
sensitive photomultip!ier and filters for excitation (CS-5-60) and emission
(CS-2-64). 13 mm diameter borosilicate glass culture tubes were used for all
analyses.
Digital Fluorometer - Turner Designs Model 10-AU. Daylight white lamp,
red-sensitive photomultiplier, and filters for excitation (436FS10) and
emission (680FS10). 13 mm diameter borosilicate glass culture tubes were used
for all analyses.
Preparation of Standards and Samples
Chl a and chl b obtained from Sigma Chemical were prepared in 90%
acetone:10% water. Pheo a was prepared by the mild acidification of chl a
solutions with .1 N HC1 and subsequent 1:1 molar neutralization with 0.1 NaOH.
All standard solutions were stored in the dark at -70°C.
1

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The technique that was used for sample collection, handling and
extraction is described in detail in USEPA Method 445.0.5 Natural water
samples were vacuum-filtered through 47-mm Whatman GF/F glass fiber filters.
Filters were folded once with particulate matter inward, blotted with a
laboratory tissue to remove excess moisture, and stored in petri dishes in the
dark at -70°C until extraction.
Samples were extracted with 90% acetone using a motor-driven Teflon
pestle and glass grinding tube. Final dilution volume in all cases was 10 mL.
Extracted filters were allowed to steep from 4 to 18 hours. The supernatant
of centrifuged samples was analyzed by the three techniques.
Cal1 bration
Chi a and chl b were determined using the trichromatic equations of
Jeffrey and Humphrey.6 The spectrophotometer was used to verify the
concentration of chl a. Lorenzen's modified monochromatic equation was used
to determine corrected chl a for fluorometer calibration.
The fluorometers were always calibrated on the day of use and with the
same dilutions of stock chl a solution. The following set of equations were
used for calibration of the analog fluorometer:
= Ca/Rs
where:
Fs = response factor for sensitivity setting, S.
Rs = fluorometer reading for sensitivity setting, S.
Ca - concentration of chl a (from spectrophotometric
analysis).
and, r - Rb/Ra
where:
Rb = fluorescence of pure chl a standard solution before
acidification.
Ra = fluorescence of pure chl a standard solution after
acidification.
The following equations were used for sample data reduction:8
Uncorrected chl a, iig/L = Rsb X Fs
Corrected chl a, jug/L = Fs (r/r-1) (Rsb - Rsa)
Pheo a, M9/L - Fs(r/r-l) (rRsa - Rsb)
2

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where:
Fs = response factor for the sensitivity setting used.
Rsb » fluorescence of sample extract before acidification.
Rsa = fluorescence of sample extract after acidification.
The digital fluorometer was calibrated directly with a single standard
solution. Readout was in concentration units of ^tg/L. Pheo a is optically
excluded, for the most part, from the chl a measurement. For users of analog
instrumentation equipped with the new interference filters, calibration is
affected as with conventional fluorometry without acidification and subsequent
corrections (i.e., "uncorrected" chl a equation).
Four parameters were evaluated for the spectrophotometry and
fluorometric techniques as a baseline comparison. They were (1) sensitivity,
(2) linear dynamic range, (3) precision, and (4) accuracy. Accuracy of the
fluorometers determined by comparison to spectrophotometry chl a results.
Interference caused by the presence of chl b or pheo a was also investigated.
Finally, comparisons in the determination of chl a were made using real
samples. Filter samples from Lake Pontchartrain, Louisiana (primarily green
and bluegreen algae), San Francisco Bay (unknown mixed assemblage) and New
York Bight (mixed assemblage, predominantly diatoms) were extracted and
analyzed by all three methods.
Sensitivity (Instrumental Detection Limit) - A stock solution of chl a
(50 mg/L) was serially diluted and analyzed until a response of .005 AU was
obtained using the spectrophotometer. Dilutions were continued and analyzed
fluorometrically until the normalized FU response for the analog fluorometer
was .00013 (.4 FU non-normalized on the most sensitive setting) and the direct
readout response of the digital fluorometer was .026. The results can be
summarized as follows:
Determined using a 1-cm cell.
Linear Dynamic Range (LDR) - The LDR is the concentration at which the
instrument's response to chl a is no longer linear. Sixteen serial dilutions
of chl a (.09 to 50 mg/L) were analyzed spectrophotometrically and 38 serial
dilutions (.15 to 730 fig/l) were analyzed fluorometrically. Linear
regressions of absorbance/fluorescence versus concentration were plotted and
the concentration at which the response deviated more than 10% of the expected
response was judged to be the upper limit of the LDR (Figures 4-6). The
RESULTS
Instrumental Detection Limit
DU-6 Spectrophotometer
Model 10 Analog Fluorometer
Model 10-AU Digital Fluorometer
.08 mg/L
.05 ng/l
.03 M9/L
3

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upper limit of the LDR for the digital fluorometer was the first dilution from
a 1000 fig/l solution that was on scale. (NOTE: The LDR should not be
confused with a calibration range. Calibration relying upon a non-weighted
linear regression should be over 1 to 2 orders of magnitude, at most, to
minimize lack-of-fit inaccuracies. This type of inaccuracy becomes especially
apparent at low concentrations.) The results can be summarized as follows:
linear Dynamic Range
DU-6 Spectrophotometer	25 mg/L
Model 10 Analog Fluorometer	250 (ig/l
Model 10-AU Digital Fluorometer 700 ng/i
The higher LDR for the Model 10-AU fluorometer can probably be attributed
to the interference filters, which block out all but a narrow band of
excitation and emission light. Quenching effects caused by the presence of
accessory pigments and other compounds not of interest are thus minimized.
The upper limit of the LDR for the digital fluorometer also proved to be
an acceptable calibration point (direct readout of concentration agreeing well
with the calculated concentration from serial dilution). (NOTE: Quenching
effects caused by the presence of other compounds can cause underestimation of
the true chl a concentration. Since the presence of these compounds cannot be
predicted, real world samples should not be analyzed at concentrations
approaching the LDR.)
Precision - Precision was calculated as the percent relative standard
deviation (%RSD) of repeated measurements of standard solutions and natural
samples. Precision for real world samples are presented later in this
summary. Precision obtained using pure solutions of chl a obtained from the
USEPA is summarized below:
Corrected Chl a	Uncorrected Chl a
%RSD	iRSfi
DU-6 Spectrophotometer*	5.1%	2.2%
(N=9)	mean chl a = 69 ng/l mean chl a = 74 fig/l
Model 10 Analog Fluorometer	4.7%	4.5%
(N-3)	mean chl a = 67 ng/l mean chl a = 69 ftg/L
Model 10-AU Digital Fluorometer	4.6%
(N-3)	mean chl a = 68 fig/l
Determined using a 10-cm cell.
The fluorometers were calibrated on the day of use using the same
standard solutions (chl a prepared from a pure pigment obtained from Sigma
Chemical). Three fluorometric QC samples obtained from the USEPA were then
analyzed. The spectrophotometry results were obtained from the analyses of
nine fluorometric QC samples using a 10-cm cell.
4

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Accuracy of Fluorometric Techniques (versus spectrophotometry) -
Measurement of pure chl a by fluorometry is only as accurate as the
spectrophotometer used to standardize the calibration solutions. As a
baseline comparison, however, the data gathered from the sensitivity study is
used to compare the accuracy of the two fluorometers. The spectrophotometry
result for corrected chl a was regarded as the nominally "true" concentration,
69 /xg/L (±5%). Comparing the mean fluorometric values we obtained: -2.9%
error using the analog fluorometer with pheo a correction, and -1.4% error
with the digital fluorometer (no pheo a correction is necessary).
Effect of Chl b on the Measurement of Chl a
Spectrophotometry - The trichromatic equation of Jeffrey and Humphrey is
fairly accurate in determining chl a in the presence of chl b.9 However,
spectrophotometry often is not used because of poor sensitivity.
Fluorometry - Six concentrations of chl a ranging from 1.8 to 180 nq/L
were evaluated. Chl b was present at varying ratios up to a chl a:chl b ratio
of 1:1, the maximum likely to occur in nature. Figures 7 and 8 illustrate the
error observed over a 1:0 to a 1:1 ratio, at 180 /ig/L and 1.8 /jg/L (the
highest and lowest concentrations used in the study). "Corrected" and
"uncorrected" chl a results refer to pheo a-corrected and non-corrected chl a,
respectively, as determined by the analog fluorometer. No error would be
indicated by a line with zero slope. In Figure 7 note that the conventional
fluorometric method, with or without pheo a correction, results in significant
error (-19% and +30%, respectively). The underestimation of chl a is caused
by the overestimation of pheo a which is actually pheo 6. The overestimation
of pheo a caused when chl b is converted to pheo b is illustrated in Figure 9.
The slightly nonlinear trend observed in Figure 8 cannot be explained,
although it could be a dilution error since it is observed for all three
techniques. Chl a as determined by the new fluorometric method was
overestimated by ca. +6%. In summary, the effect of chl b on the
determination of chl a, by the new fluorometric method, across six
concentrations ranging from 1.8 to 180 (ig/L, was approximately +6%.
Effect of Pheophytin a on the Measurement of Chl a
Spectrophotometry - When the trichromatic equation was used to calculate
chl a in the presence of pheo a, a positive bias was observed (Figure 10).
The monochromatic equation, on the other hand, was fairly accurate even in the
presence of chl b.
Fluorometry - The results obtained when chl a was calculated in the
presence of pheo a at various ratios up to a 1:1 ratio were erratic. These
erratic results are thought to be due to the manner in which the pheo a was
prepared. Solutions of pure chl a were acidified to convert the chl a to pheo
a. The solutions were then neutralized 1:1 molar with NaOH. The results
indicated that residual acid was present which converted the chl a to pheo a
when the two solutions were mixed. Regrettably, the borrowed digital
fluorometer was returned prior to evaluating the data gathered in that study.
Dr. Welschmeyer, however, reports an error no greater than +10% at a chl a:
5

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pheo a ratio of 1:1, the maximum likely to occur in nature. The results
obtained in this study using real world samples containing chl a and pheo a,
do not contradict those findings.
Comparison of Techniques Using Real World Samples
Filtered samples from various locales and of various taxonomic
composition were analyzed by the three techniques. In most cases the samples
that contained a sufficient concentration of chl a were analyzed first by
spectrophotometry, then diluted and analyzed fluorometrically. In all cases
the samples that were analyzed by the two fluorometers were the exact same
dilution.
The results are presented in Figures 11 - 15. The following annotation
is used:
Digital Fl.
Fl.Uncorra.
Fl.Corra
Methods
Sp.Tri.
Sp.Hono.
Macerated
Unmacerated
-	the new fluorometric method under evaluation.
-	conventional fluorometry without pheo a correction.
-	conventional fluorometry with pheo a correction.
-will indicate the mean or %RSD of the results from all
the methods that are presented on that particular
figure.
-	spectrophotometry, trichromatic equations.
-	spectrophotometry, monochromatic equations.
-	indicates that filters were macerated using a tissue
grinder prior to steeping.
-	indicates that samples were not macerated prior to
steeping.
Unless otherwise indicated, all samples were macerated prior to steeping
in 90% acetone.
Figure 11 - San Francisco Bay, 1000 ml samples. The taxonomic
composition of these samples was unknown. Previous laboratory experiments
have indicated that the spectrophotometry, monochromatic equation (Sp.Mono.)
performs well even in the presence of chl b. Previous studies also have
indicated that the spectrophotometry, trichromatic equation (Sp.Tri.)
overestimates chl a in the presence of pheo a. There was pheo a present in
the sample and the extremely low results for chl a using the Fl.Corra method
indicated the presence of chl b. This information leads to the conclusion
that the most confidence may be placed in the Sp.Mono results. The Digital
Fl. results agree closely with the Sp.Mono. results. The relative percent
difference (RPD) between the Fl.Corra and Digital Fl results was 34%.
6

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Figure 12 - Lake Pontchartrain, 300 mL samples. These samples were
primarily green and blue-green algae. Blue-green algae do not contain chl b
or chl c and the green algae do not contain chl c. Four filtered samples were
and three filters were not macerated prior to steeping in 90% acetone.
Concentrations of chl a were normalized to the spectrophotometry results
(actual concentrations for fluorometric analyses were 10X less).
Spectrophotometry results indicated the presence of chl a and pheo a but very
little chl b. In this case close agreement was observed between the Digital
F1. and FL.Corra methods (RPD = 7%, for macerated samples). Maceration of the
filters increased the quantity of pigment that was extracted. The fact that
calculated pheo a, relative to calculated chl a, was considerably different
for the FL.Corra and the Sp.Mono techniques gives reason to believe that
either chl b is present in the sample in a higher concentration than indicated
by the spectrophotometry method or another pigment that interferes with
conventional fluorometry is present in the sample. Previous work performed in
this laboratory has demonstrated that chl b is underestimated when the chl a
concentration is greater than 4X the chl b concentration.
Figure 13 - New York Bight, 300 mL samples. This was a mixed assemblage
with diatoms being the predominant species. Diatoms contain the pigments chl
a and c, however, chl c is not a major spectral interferant for the
spectrophotometry method or the fluorometric methods. Three filter samples
were extracted. For this sample all the techniques performed comparably. The
RPD between the Digital F1. technique and the Fl.Corra technique was 5.6%.
Figure 14 - New York Bight, 900 mL sample. This was a one month old
frozen extract that was analyzed once spectrophotometrically, diluted three
times and analyzed fluorometrically. Results were normalized to the
fluorometric concentrations. The Sp.Tri. results reflect the fact that the
trichromatic equation overestimates chl a in the presence of pheo a. The RPD
between the Digital F1. and Fl.Corra methods was 7.7%.
Figure 15 - New York Bight, 600 mL. This was a one month old frozen
extract that was analyzed once spectrophotometrically, diluted three times and
analyzed fluorometrically. The chl arpheo a ratio was approximately 3:1.
Concentrations were normalized to the fluorometric results. The 56RPD between
the Digital F1. results and the Fl.Corra results was 10%.
CONCLUSIONS
A new fluorometric technique that uses highly selective interference
filters to optically exclude the fluorescence of pheo a, chl b and pheo b was
compared to the spectrophotometry and conventional fluorometric techniques
for determining chl a. A baseline comparison was made using the following
parameters: (1) sensitivity, (2) linear dynamic range, (3) precision, and (4)
accuracy. The new technique was comparable in sensitivity to conventional
fluorometry. The upper limit of the linear dynamic range was 700 pg/L,
compared to 250 fig/l for conventional fluorometry. Precision compared with
spectrophotometry and conventional fluorometry. Accuracy, using standard
solutions of chl a that were verified spectrophotometrically, compared well
with conventional fluorometry.
7

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Interference from chl b was evaluated over six concentrations and varying
chl a:chl b ratios, up to 1:1. The superiority of the new technique was most
pronounced in these comparisons. Whereas conventional fluorometry severely
underestimated chl a in the presence of chl b, by overestimating pheo a, the
new fluorometric technique exhibited an average +6% bias. Interference
studies of pheo a were unsuccessful due to incorrect preparation of pheo a,
however, real world samples containing pheo a and negligible quantities of chl
b allowed an estimate of error of the new method in the presence of pheo a.
The bias of the technique was less than +10% for real world samples.
Real world samples from three locales and of varying taxonomic
composition were analyzed by all three techniques. The degree of difference
in the results was correlated with taxonomic differences in the samples. If
chl b was present, the new fluorometric technique compared well with the
spectrophotometric technique using the monochromatic equation. In those
cases, however, chl a was severely underestimated by conventional fluorometry.
For a sample that was known to contain chl a and pheo a, the new fluorometric
method compared well to pheopigment-corrected conventional fluorometry.
8

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REFERENCES
1.	Neveux.J., D. Delmas, J.C. Romano, P. Algarra, L.
Ignatiades, A. Herbland, P. Morand, A. Neori, 0. Bonin, J.
Barbe, A. Sukenik and T. Berman, "Comparison of
chlorophyll and pheopigment determinations by
spectrophotometric, fluorometric, spectrofluorometric and
HPLC methods," Marine Microbial Food Webs, 4(2), (1990)
pp. 217-238.
2.	Trees, C.C., M.C. Kennicutt, and J.M. Brooks, "Errors associated
with the standard fluorometric determination of chlorophylls and
pheopigments", Mar. Chem., 17 (1985) pp. 1-12.
3.	Weber, C.I., L.A. Fay, G.B. Collins, D.E. Rathke, and J. Tobin, "A
Review of Methods for the Analysis of Chlorophyll in Periphyton and
Plankton of Marine and Freshwater Systems", work funded by the
Office of Sea Grant, NOAA, Department of Commerce and the Ohio Sea
Grant Program, Grant No.NA84AA-D-00G79, 1986, 54 pp.
4.	Weischmeyer, N., "Fluorometric Analysis of Chlorophyll a in the
Presence of Chlorophyll b and Pheopigments." In Press, Limnology
and Oceanography.
5.	USEPA Method 445.0, "In vitro determination of chlorophyll a and
pheophytin a in marine and freshwater phytoplankton by
fluorescence," Methods for the Determination of Chemical Substances
in Marine and Estuarine Environmental Samples, EPA/600/R-92/121.
6.	Jeffrey, S.W. and G.F. Humphrey, "New Spectrophotometric
Equations for Determining Chlorophylls a, b, c, + c2 in
Higher Plants, Algae and Natural Phytoplankton," Biochem.
Physiol. Pflanzen. Bd, 167, (1975), S. pp. 191-4.
7.	Lorenzen, C.J., "Determination of Chlorophyll and Pheo-
pigments: Spectrophotometric Equations," Limnol.
Oceanogr., 12 (1967), pp. 343-6.
8.	Strickland, J.D.H. and T.R. Parsons, A Practical Handbook of
Seawater Analysis, Bull. Fish. Res. Board Can., 1972, No.167, p.
201.10.
9.	USEPA Method 446, "In vitro Determination of Chlorophylls a, b, c, +
cz and Pheopigments in Marine and Freshwater Phytoplankton by
Visible Spectrophotometry," to appear in Methods for the
Determination of Chemical Substances in Marine and Estuarine
Environmental Samples in 1995.
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0.9
0.8-
0.7-
0.6
a>
a
« 0.5
ti
£ 0.4
(0
k_
I-
0.3-
0.2-
0.1
0
Excitation/Emission Filters
i i	i i	i	r	i"

436FS10
M
5-60

2-64
X
/
J 680FS10
I
1
350 400
TTT-T-T-rTT-rTT~T_T-T-T-T-T-r-rnrT^rT_Ti*r-r
450 500 550 600 650 700
i i i i i i
750 800
Wavelength (nm)
Figure 1. Transmittance Characteristics of Excitation/Emission Filters
used in Conventional Fluorometric Acidification Technique
(Corning 5-60/Corning 2-64) and Newly Described Fluorometric Method
(436FS10/680FS10 Interference Filters, Andover Corp.).
Reprinted courtesy of Nicholas Welschmeyer, Moss Landing Marine Laboratories,
CA.
10

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Fluorescence Excitation/Emission
(Chlorophylls)
Chi c2
Chi b
Chi a
Chi c2
Chi b
— Chi a
0.9-
G.6-
0.3-'
0.2-
0.1-
400
450
500
600
650
350
550
700
750
Wavelength (nm)
Figure 2. Excitation/Emission Spectra of the Chlorophylls
Reprinted courtesy of Nicholas Weischmeyer, Moss Landing Marine Laboratories,
CA.
11

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Fluorescence Excitation/Emission
(Pheopigments)
Pheo a
Pheo a (B)
Pheo c2
Pheo b
Pheo c2
Pheo b
0.9-
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
500
550
600
650
700
750
350
400
450
Wavelength (nm)
Figure 3. Excitation/Emission Spectra of the Pheopigments
Reprinted courtesy of Nicholas Welschmeyer, Moss Landing Marine Laboratories,
CA.
12

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Chlorophyll a - Spectrophotometry
Linear Dynamic Range (.08 - 25 ppm)
Absorbance at 664 nm
2.5
1.5
0.5
0
-0.5
/
/'


v
10	20	30
Concentration (ppm)
40
50
Raw Data
Quadratic Model
Linear Model
Figure 4
13

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Analog Fluorometer
Linear Dynamic Range (250 ug/L)
Response (Fluorescence Units)
r ~ 		~ 		"	
0
X
r

,4/
X/'
J?
/



T

i

-1
0
200	400	600
Concentration (ug/L)
800
Raw Data
Linear Model
Quadratic Model
Figure 5
14

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Digital Fluorometer
Linear Dynamic Range (730 ug/L)
Response (Fluorescence Units)
800
600
400
200
-200
200
400
600
800
Concentration (ug/L)
Raw Data	-)- Linear Model
Quadratic Model
Figure 6
15

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Effect of ChI b on ChI a
Ana Iog vs. D T g i taI FIuorometer
Concentrat1 on ChI a CPPtO
240
220
200
160
140
100
15D
200
0
50
Concentration Chi b CPP^O
Uncorrected Chi a	+ Corrected Chi a
* Droits I Readout
Average %Error (Slope X 100)
Uncorrected chl a	+29.8%
Corrected chl a	-18.9%
Digital chl a	+6.3%
Chlorophyll a True Value = 180 ppb
Figure 7
16

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Effect of ChI b on Cht a
Analog vs. DlgTtal FIuorometer
Concentration Chi a CPP^O
~
1.5
2
0.5
1
Concentration Chi b CPPtO
* Uncorrected Chi a	+ Corrected Chi a * Digital Roadout
Slope X 100 (Average %Error)
Uncorrected chl a	+28.9%
Corrected chl a	-11.5%
Digital chl a	+2.3%
Chlorophyll a True Value = 1.8 ppb
Ratios chl a:chl b were 1:0, 3:1, 2.5:1, 2:1, 1.7:1 and 1:1
Average %Error for digital method for six concentrations
(1.8 ppb to 180 ppb) was +6%
Figure 8
17

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Effect of Chi b on Measured Pheophytin
Analog Fluorometer
Measured Pheophytin a (ppb)
80
60 h
0
/•
/
/
0	20	40	60	80
Concentration chl b (ppb)
¦ Pheophytin a
Figure 9
18

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1:1 Mixtures of Chi a and Pheo a
Spectrophotometry
Pigment Concentration (ppm)
1.5
~
0.5
c
0
X-
ik
~

0
0.2 0.4 0.6 0.8	1
Concentration pheo a and chl a (ppm)
1.2
— True Chl a Value
* Corrected chl a
+ Trichromatic chl a
~ Pheo a
Figure 10
19

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Chlorophyll a - Comparison of Methods
San Francisco Bay - 100G mL samples
Concentration CPpbD
209
207
Sp.Trl.	Sp.Mono Digital Fl. FI.Uncorr.a Fl.Corr.a
Method
* Average = 173 ppb
N»3, Samp (es were macftrated

MEAN (ppb)
RSD
Sp.Tri.
209
6.1%
Sp.Mono
169
9.1%
Digital Fl.
161
4.5%
Fl.Uncorr.a
207
5.7%
Fl.Corr.a
120
4.8%
METHODS
173
21.3%

Figure 11


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ChlorophylI a - Comparison of Methods
Lake PontchatraIri - 300 mL samples
Concentration CPPtO
600 "
500 "
400 -
300 ~
200 "
100 -
5p.Trl .	Sp.Mono.
Digital FL, FL.Uncorr.a FL.Corr.a
Method
U™netc«rot»d CN-3D
Unmacarated Phooa
Wm& Mac«rat«d
"'X	 Macerated Pheo. a
ALL METHODS
MEAN (ppb)	RSD
Macerated	503	11.1%
Urimacerated 3 07	14.3%
Figure 12
21

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Chlorophyll a - Comparison of Methods
N'ew York 3ight - 300 mL samples
Concentration CPPtO
79.5
73.6
72,. 1
Sp.Tr i .
Sp.Mono
Digital Fl. Fl.Uncorr.a Fl.Corr.a
Method
' Average = 74.2
N=3, samples were macerated

Mean (ppb)
RSD
Sp.Tri.
75.9
6.4%
Sp.Mono.
72 .1
9.8%
Digital Fl.
73.6
8.1%
Fl.Uncorr.a
79.5
6.6%
Fl.corr.a
69.7
7.2%
METHODS
74.2
5.1%

Figure 13


22


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ChlorophylI a - Comparison of Methods
New York Bight - 900 mL sample
Concentration chI a CPP^D
196
174
169
143
Sp.Tr I .
Sp.Mono.
Digital Fl, Fl.Uncorr.a
FI.Corr.a
Method
^ Average = 167 ppb
Mean (ppb)	RSD
Digital Fl.	154	2.5%
Fl.Uncorra.	169	2.2%
Fl.Corr.a	143	2.2%
METHODS	167	12.1%
Figure 14
23

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Chlorophyll a - Comparison of Methods
New York Bfght - BOO mL sample
Concentration chl a CPP^O
154
154
130
Sp .Tr I .
Sp.Mono.
Digital Fl
Method
Ft .Uncorr .a
FI.Corr.a
Averag© = 144 ppb
N=3, samples were macerated
Digital Fl.
Fl.Uncorr.a
Fl.Corr.a
METHODS
Mean (ppb)	RSD
143	1.9%
154	1.2%
130 0%
144	7.1%
Figure 15
24

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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing,
1. REPORT NO. 2.
EPA/600/R-94/150
3. RE
4. title and subtitle Summary Report: Evaluation of a New
FLuorometric Technique that uses Highly Selective
Interference Filters for Measuring Chlorophyll a in the
Presence of Chlorophyll b and Pheopigments
5. REPORT DATE
July 1994
6. PERFORMING ORGANIZATION CODE
7. AUTHORiSI
Elizabeth J. Arar
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Inorganic Chemistry Branch, Chemistry Research Division
EMSL-Cineinnati, U.S. Environmental"Protection Agency
26 W. Martin Luther King Dr.
Cincinnati, Ohio 45268
10. PROGRAM ELEMENT NO.
1 1 . CON TRACT/GRANT \ij
12. SPONSORING AGENCY NAME ANO ADDRESS
Same as 9
13.	TYPE OF REPORT AND PERIOD COVEREO
Summary Report
14.	SPONSORING AGENCY CODE
EPA/600/06
15. SUPPLEMENTARY NOTtS
16. ABSTRACT
A new fluorometric technique was compared to conventional fluorometry with and
without pheophytin a (pheo a) correction and to spectrophotometry using Lorenzen's
modified monochromatic equations and Jeffrey and Humphrey's trichromatic equation to
calculate chlorophyll a (chl a). The new technique uses excitation and emission
filters that are highly selective for chl a, eliminating the interference caused by
accessory pigments and pheopigment degradation products.
Four method parameters were evaluated using the new technique, conventional
fluorometry and spectrophotometry. They were (1) sensitivity, (2) linear dynamic
range, (3) precision, and (4) accuracy. Controlled studies of the interference caused
by chl b were conducted, and real world samples of varying taxonomic composition were
analyzed. For laboratory solutions, the new technique was comparable to conventional
fluorometry with respect to sensitivity and accuracy. The linear dynamic range for
the new technique exceeded that of conventional fluorometry by a factor of three.
Interference caused by chl b was +6% at the highest chl a:chl b likely to occur in
nature. Chl a values obtained using the new technique compared well with conventional
fluorometry when pheo a was the only interfering pigment present in the sample.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIF lERS/OPfcN ENDED TERMS
c. Cosati Field/Croup



18. DISTRIBUTION STATEMENT
Release to Public
	-i
19. SECURITY CLASS (This Report)
Unclassi fied
21. NO. OF PAGES
20. SECURITY CLASS ,Thts pagei
Unclassified
22. PRICE
EPA Form 2220-1 (Re*. 4-77) previous eoit'On is obsolete

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