United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/R-97/072
September 1997
xvEPA Methods for the
Determination of Chemicai
Substances in Marine and
Estuarine Environmental
Matrices - 2nd Edition
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EPA/600/R-97/072
Methods for the Determination of Chemical Substances in
Marine and Estuarine Environmental Matrices - 2nd Edition
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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DISCLAIMER
This manual has been reviewed by the National Exposure Research 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 National Exposure Research
Laboratory - Cincinnati (NERL-Cincinnati) conducts research to:
• Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in marine and estuarine waters,
drinking waters, surface waters, ground waters, wastewaters, sediments,
sludges, and solid wastes.
• 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.
• 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 waters, surface waters, ground
waters, wastewaters, sediments, and solid wastes.
• 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.
This NERL-Cincinnati publication, "Methods for the Determination of Chemical
Substances in Marine and Estuarine Environmental Matrices - 2nd Edition" was
prepared as the continuation of an initiative to gather together under a single cover a
compendium of standardized laboratory analytical methods for the determination of
nutrients, metals, chlorophyll and organics in marine matrices. It is the goal of this
initiative that the methods that appear in this manual will be multilaboratory validated.
We are pleased to provide this manual and believe that it will be of considerable value
to many public and private laboratories involved in marine studies for regulatory or
other reasons.
Alfred P. Dufour, Director
Microbiological and Chemical Exposure
Assessment Research Division,
National Exposure Research Laboratory - Cincinnati
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ABSTRACT
This manual contains eleven methods for determination of nutrients, metals, and
chlorophyll. Since Revision 1.0 appeared in 1992, four new methods have been added, one
deleted and four have been multilaboratory validated. Methods 440.0, 445.0, 446.0 and 447.0
have been multilaboratory validated, and Method 353.4 has been replaced with an improved
method.
The metals methods, Methods 200.10, 200.12 and 200.13 have not changed since the
1992 manual. Method 365.5 has remained the same and Method 440.0, that appeared in 1992,
now contains multilaboratory validation data. Two new chlorophyll methods, Methods 446.0 and
447.0, have been added and all three chlorophyll methods have been multilaboratory validated.
Since the chlorophyll methods validation study was also a comparison study of the methods, that
data has been added to the methods. Anyone interested in obtaining a copy of the full
chlorophyll study final report should contact the Chemical Exposure Research Branch office of
NERL-Cincinnati.
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CONTENTS
Page
Disclaimer ii
Foreword iii
Abstract iv
Acknowledgments vii
Introduction 1
Method Multilab
Number Title Revision Validation Status
200.10 Determination of Trace Elements in 1.6 No
Marine Waters by On-line Chelation
Preconcentration and Inductively
Coupled Plasma - Mass Spectrometry
200.12 Determination of Trace Elements in 1.0 No
Marine Waters by Stabilized Temperature
Graphite Furnace Atomic Absorption
200.13 Determination of Trace Elements in 1.0 No
Marine Water by Off-Line Chelation
Preconcentration with Graphite Furnace
Atomic Absorption
349.0 Determination of Ammonia in Estuarine and 1.0 No
Coastal Waters by Gas Segmented Continuous
Flow Colorimetric Analysis
353.4 Determination of Nitrate and Nitrite in 1.0 No
Estuarine and Coastal Waters by Gas
Segmented Continuous Flow Colorimetric
Analysis
365.5 Determination of Orthophosphate in Estuarine 1.4 Yes
and Coastal Waters by Automated Colorimetric
Analysis
366.0 Determination of Dissolved Silicate in 1.0 No
Estuarine and Coastal Waters by Gas
Segmented Continuous Flow Colorimetric Analysis
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440.0 Determination of Carbon and Nitrogen in 1.4 Yes
Sediments and Particulates of Estuarine/Coastal
Waters Using Elemental Analysis
445.0 In Vitro Determination of Chlorophyll a 1.2 Yes
and Pheophytin a in Marine and Freshwater
Phytoplankton by Fluorescence
446.0 In Vitro Determination of Chlorophylls 1.2 Yes
a, b, Cj+c2 and Pheopigments in Marine and
Freshwater Algae by Visible Spectrophotometry
447.0 Determination of Chlorophylls a and b and 1.0 Yes
Identification of Other Pigments of Interest
in Marine and Freshwater Algae Using High
Performance Liquid Chromatography with
Visible Wavelength Detection
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ACKNOWLEDGMENTS
This manual is dedicated to the memory of Dr. Barbara Metzger, late Director of the
Environmental Services Division of USEPA Region 2. She was the impetus and driving force for
this work.
This manual was prepared by the Chemical Exposure Research Branch of the
Microbiological and Chemical Exposure Assessment Research Division, NERL-Cincinnati. The
metals and chlorophyll methods were authored by in-house scientists and the nutrient methods
were authored under contract by Carl Zimmermann and Carolyn Keefe at the Chesapeake
Biological Laboratory, University of Maryland and under an interagency agreement by Dr. Jia-
Zhong Zhang, National Oceanic and Atmospheric Administration, Atlantic Oceanographic and
Meteorological Laboratory, Ocean Chemistry Division. Dr. Zhang deserves recognition for the
outstanding efforts he put into making these methods both informative and practical.
Special thanks go out to Dr. Margo Hunt of USEPA Region 2 for staying so involved in
the chlorophyll methods study. The need to standardize analytical methods for use in the marine
environment was identified and championed by the USEPA regions. The staff at Regions 2 and 3
were instrumental in identifying resources for this project. They provided insight from the
regional perspective and served as technical advisors. Their input has been valuable.
Diane Shirmann and Helen Brock put a tremendous effort into preparing this manuscript
and we are extremely thankful for their hard work.
VII
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INTRODUCTION
Since the first edition of this manual was published in 1992, the Environmental
Monitoring Systems Laboratory (EMSL) has been reorganized and its name changed to the
National Exposure Research Laboratory (NERL). The principal aim of this manual is to bring
together under one cover a suite of analytical methods specifically adapted or developed for the
examination of coastal and estuarine environmental samples. Many of the methods presented here
are adaptations of analytical techniques which, for many years, have been used routinely by the
marine community. Hallmarks of the methods which appear in this manual, however, are the
integrated quality control/quality assurance requirements, the use of standardized terminology and
the Environmental Monitoring Management Council (EMMC) format. The mandatory
demonstration of laboratory capability and the continuing checks on method performance ensure
the quality and comparability of data reported by different laboratories and programs. Another
distinction of this manual is the multilaboratory validation data for many of the methods.
Multilaboratory validation studies test the ruggedness of methods, provide single-analyst
and multilaboratory precision and accuracy statements, and method detection limits that are
"typical" of what most laboratories can achieve. Methods that reach this level of evaluation have
been thoroughly investigated to the fullest extent possible by a single laboratory and have usually
been informally adopted as standard methods by the analytical community. When a method does
not perform as expected in a multilaboratory study, it must be returned to the development phase.
For example, although widely accepted and routinely used in the marine community, Method
353.4 (Determination of Nitrite + Nitrate in Estuarine and coastal Waters by Automated
Colorimetric Analysis) failed the ruggedness test in 1992 when 50% of the participating
laboratories in the multilaboratory study returned unacceptable data. Review of the data
suggested that the cadmium reduction column chemistry and maintenance required further
investigation. The method was subsequently reevaluated by Dr. Jia-Zhong Zhang, under an
Interagency Agreement between the U.S. EPA and NOAA. The new nitrite/nitrate method is
improved in technical detail and QA/QC requirements.
We are pleased to present this 2nd Edition manual to the public and to research and
monitoring labs in the hope that it contributes to better protection and preservation of our
estuarine and coastal ecosystems.
Elizabeth J. Arar, William L. Budde, Thomas D. Behymer
Microbiological and Chemical Exposure Assessment Research Division
September, 1997
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Method 200.10
Determination of Trace Elements in Marine Waters by
On-Line Chelation Preconcentration and Inductively
Coupled Plasma - Mass Spectrometry
Stephen E. Long
Technology Applications, Inc.
and
Theodore D. Martin
Chemical Exposure Research Branch
Human Exposure Research Division
Revision 1.6
September 1997
Edited by
John T. Creed
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
200.10-1
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Method 200.10
Determination of Trace Elements in Marine Waters by On-Line Chelation
Preconcentration and Inductively Coupled Plasma - Mass Spectrometry
1.0 Scope and Application
1.1 This method describes procedures for
preconcentration and determination of total recoverable
trace elements in marine waters, including estuarine
water, seawater, and brines.
1.2 Acid solubilization is required prior to the
determination of total recoverable elements to facilitate
breakdown of complexes or colloids that might influence
trace element recoveries. This method should only be
used for preconcentration and determination of trace
elements in aqueous samples.
1.3 This method is applicable to the following
elements:
Element
Chemical Abstracts Service
Registry Numbers (CASRN)
Cadmium
Cobalt
Copper
Lead
Nickel
Uranium
Vanadium
(Cd)
(Co)
(Cu)
(Pb)
(Ni)
(U)
(V)
7440-43-9
7440-48-4
7440-50-8
7439-92-1
7440-02-0
7440-61-1
7440-62-2
1.4 Method detection limits (MDLs) for these ele-
ments will be dependent on the specific instrumentation
employed and the selected operating conditions. How-
ever, the MDLs should be essentially independent of the
matrix because elimination of the matrix is a feature of
the method. Reagent water MDLs, which were deter-
mined using the procedure described in Section 9.2.4,
are listed in Table 1.
7.5 A minimum of 6-months experience in the use of
commercial instrumentation for inductively coupled
plasma mass spectrometry (ICP-MS) is recommended.
2.0 Summary of Method
2.1 This method is used to preconcentrate trace
elements using an iminodiacetate functionalized chelating
resin.12 Following acid solubilization, the sample is
buffered prior to the chelating column using an on-line
system. Groups I and II metals, as well as most anions,
are selectively separated from the analytes by elution with
ammonium acetate at pH 5.5. The analytes are
subsequently eluted into a simplified matrix consisting of
dilute nitric acid and are determined by ICP-MS using a
directly coupled on-line configuration.
2.2 The determinative step in this method is ICP-
MS.3"5 Sample material in solution is introduced by
pneumatic nebulization into a radio frequency plasma
where energy transfer processes cause desolvation,
atomization and ionization. The ions are extracted from
the plasma through a differentially pumped vacuum
interface and separated on the basis of their mass-to-
charge ratio by a quadrupole mass spectrometer having
a minimum resolution capability of 1 amu peak width at
5% peak height. The ions transmitted through the
quadrupole are registered by a continuous dynode elec-
tron multiplier or Faraday detector and the ion information
is processed by a data handling system. Interferences
relating to the technique (Section 4) must be recognized
and corrected. Such corrections must include
compensation for isobaric elemental interferences and
interferences from polyatomic ions derived from the
plasma gas, reagents or sample matrix. Instrumental drift
must be corrected for by the use of internal standard-
ization.
3.0 Definitions
3.1 Calibration Blank (CB) — A volume of reagent
water fortified with the same matrix as the calibration
standards but without the analytes, internal standards, or
surrogate analytes.
3.2 Calibration Standard (CAL) - A solution pre-
pared from the primary dilution standard solution or stock
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate the
instrument response with respect to analyte concen-
tration.
3.3 Instrument Detection Limit (IDL) - The mini-
mum quantity of analyte or the concentration equivalent
that gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance line,
etc.
3.4 Instrument Performance Check Solution (IPC)
- A solution of one or more method analytes, surrogates,
internal standards, or other test substances used to
evaluate the performance of the instrument system with
respect to a defined set of criteria.
3.5 Internal Standard (IS) - A pure analyte(s) added
to a sample, extract, or standard solution in known
amount(s) and used to measure the relative responses
Revision 1.6 September 1997
200.10-2
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of other method analytes and surrogates that are compo-
nents of the same sample or solution. The internal
standard must be an analyte that is not a sample compo-
nent.
3.6 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
is in control and whether the laboratory is capable of
making accurate and precise measurements.
3.7 Laboratory Fortified Sample Matrix (LFM) — An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
3.8 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, the
reagents, or the apparatus.
3.9 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.70 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling
precautions.
3.77 Method Detection Limit (MDL) — The minimum
concentration of an analyte that can be identified,
measured, and reported with 99% confidence that the
analyte concentration is greater than zero.
3.72 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations that is used to
fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.73 Stock Standard Solution (SSS) - A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial
source.
3.74 Total Recoverable Analyte (TRA) - The
concentration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following
treatment by refluxing with hot dilute mineral acid(s) as
specified in the method.
3.75 Tuning Solution (TS) - A solution that is used to
adjust instrument performance prior to calibration and
sample analyses.
4.0 Interferences
4.7 Several interference sources may cause inaccu-
racies in the determination of trace elements by ICP-MS.
These are:
4.1.1 Isobaric elemental interferences - Are caused
by isotopes of different elements that form singly or
doubly charged ions of the same nominal mass-to-
charge ratio and that cannot be resolved by the mass
spectrometer in use. All elements determined by this
method have, at a minimum, one isotope free of isobaric
elemental interference. The analytical isotopes recom-
mended for use with this method are listed in Table 1.
4.12 Abundance sensitivity - Is a property defining
the degree to which the wings of a mass peak contribute
to adjacent masses. The abundance sensitivity is af-
fected by ion energy and quadrupole operating pressure.
Wing overlap interferences may result when a small ion
peak is being measured adjacent to a large one. The
potential for these interferences should be recognized
and the spectrometer resolution adjusted to minimize
them.
4.13 Isobaric polyatomic ion interferences — Are
caused by ions consisting of more than one atom that
have the same nominal mass-to-charge ratio as the
isotope of interest and that cannot be resolved by the
mass spectrometer in use. These ions are commonly
formed in the plasma or interface system from support
gases or sample components. Such interferences must
be recognized, and when they cannot be avoided by the
selection of alternative analytical isotopes, appropriate
corrections must be made to the data. Equations for the
correction of data should be established at the time of the
analytical run sequence as the polyatomic ion
interferences will be highly dependent on the sample
matrix and chosen instrument conditions.
4.14 Physical interferences - Are associated with the
physical processes that govern the transport of sample
into the plasma, sample conversion processes in the
plasma, and the transmission of ions through the plasma
mass spectrometer interface. These interferences may
result in differences between instrument responses for
200.10-3
Revision 1.6 September 1997
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the sample and the calibration standards. Physical
interferences may occur in the transfer of solution to the
nebulizer (e.g., viscosity effects), at the point of aerosol
formation and transport to the plasma (e.g., surface
tension), or during excitation and ionization processes
within the plasma itself. Internal standardization may be
effectively used to compensate for many physical
interference effects.6 Internal standards ideally should
have similar analytical behavior to the elements being
determined.
4.1.5 Memory interferences — Result when isotopes of
elements in a previous sample contribute to the signals
measured in a new sample. Memory effects can result
from sample deposition on the sampler and skimmer
cones and from the buildup of sample material in the
plasma torch and spray chamber. The site where these
effects occur is dependent on the element and can be
minimized by flushing the system with a rinse blank
between samples. Memory interferences from the che-
lating system may be encountered especially after
analyzing a sample containing high concentrations of the
analytes. A thorough column rinsing sequence following
elution of the analytes is necessary to minimize such
interferences.
4.2 A principal advantage of this method is the
selective elimination of species giving rise to polyatomic
spectral interferences on certain transition metals (e.g.,
removal of the chloride interference on vanadium). As
the majority of the sample matrix is removed, matrix
induced physical interferences are also substantially
reduced.
4.3 Low recoveries may be encountered in the
preconcentration cycle if the trace elements are
complexed by competing chelators in the sample or are
present as colloidal material. Acid solubilization pretreat-
ment is employed to improve analyte recovery and to
minimize adsorption, hydrolysis, and precipitation effects.
5.0 Safety
5.7 Each chemical reagent used in this method
should be regarded as a potential health hazard and
exposure to these reagents should be as low as reason-
ably achievable. Each laboratory is responsible for
maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals
specified in this method.78 A reference file of material
data handling sheets should also be available to all
personnel involved in the chemical analysis.
5.2 Analytical plasma sources emit radio frequency
radiation in addition to intense UV radiation. Suitable
precautions should be taken to protect personnel from
such hazards.
5.3 The acidification of samples containing reactive
materials may result in the release of toxic gases, such
as cyanides or sulfides. Acidification of samples should be
performed in a fume hood.
5.4 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.5 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Equipment and Supplies
6.7 Preconcentration System - System containing
no metal parts in the analyte flow path, configured as
shown in Figure 1.
6.1.1 Column - Macroporous iminodiacetate chelating
resin (Dionex Metpac CC-1 or equivalent).
6.12 Sample loop - 10-mL loop constructed from
narrow bore, high-pressure inert tubing, Tefzel ethylene
tetra-fluoroethylene (ETFE) or equivalent.
6.13 Eluent pumping system (PI) - Programmable
flow, high pressure pumping system, capable of
delivering either one of two eluents at a pressure up to
2000 psi and a flow rate of 1-5 mL/min.
6.14 Auxiliary pumps — On line buffer pump (P2),
piston pump (Dionex QIC pump or equivalent) for
delivering 2M ammonium acetate buffer solution; carrier
pump (P3), peristaltic pump (Gilson Minipuls or equiva-
lent) for delivering 1% nitric acid carrier solution; sample
pump (P4), peristaltic pump for loading sample loop.
6.15 Control valves — Inert double stack, pneumati-
cally operated four-way slider valves with connectors.
6.1.5.1 Argon gas supply regulated at 80-100 psi.
6.16 Solution reservoirs - Inert containers, e.g., high
density polyethylene (HOPE), for holding eluent and
carrier reagents.
6.17 Tubing — High pressure, narrow bore, inert
tubing (e.g., Tefzel ETFE or equivalent) for interconnec-
tion of pumps/valve assemblies and a minimum length
for connection of the preconcentration system to the ICP-
MS instrument.
6.2 Inductively Coupled Plasma - Mass Spec-
trometer
6.2.1 Instrument capable of scanning the mass range
5-250 amu with a minimum resolution capability of 1 amu
peak width at 5% peak height. Instrument may be fitted
with a conventional or extended dynamic range detection
system.
Revision 1.6 September 1997
200.10-4
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6.2.2 Argon gas supply (high-purity grade, 99.99%).
6.2.3 A mass-flow controller on the nebulizer gas
supply is recommended. A water-cooled spray chamber
may be of benefit in reducing some types of interfer-
ences (e.g., polyatomic oxide species).
6.2.4 Operating conditions - Because of the diversity
of instrument hardware, no detailed instrument operating
conditions are provided. The analyst is advised to follow
the recommended operating conditions provided by the
manufacturer.
6.2.5 If an electron multiplier detector is being used,
precautions should be taken, where necessary, to
prevent exposure to high ion flux. Otherwise changes in
instrument response or damage to the multiplier may
result. Samples having high concentrations of elements
beyond the linear range of the instrument and with
isotopes falling within scanning windows should be di-
luted prior to analysis.
6.3 Labware - For the determination of trace
elements, contamination and loss are of critical concern.
Potential contamination sources include improperly
cleaned laboratory apparatus and general contamination
within the laboratory environment. A clean laboratory
work area, designated for trace element sample han-
dling, must be used. Sample containers can introduce
positive and negative errors in the determination of trace
elements by (1) contributing contaminants through
surface desorption or leaching or (2) depleting element
concentrations through adsorption processes. For these
reasons, borosilicate glass is not recommended for use
with this method. All labware in contact with the sample
should be cleaned prior to use. Labware may be soaked
overnight and thoroughly washed with laboratory-grade
detergent and water, rinsed with water, and soaked for 4
hr in a mixture of dilute nitric and hydrochloric acids,
followed by rinsing with ASTM type I water and oven
drying.
6.3.1 Griffin beakers, 250-mL, polytetrafluoroethylene
(PTFE) or quartz.
6.3.2 Storage bottles - Narrow mouth bottles, Teflon
FEP (fluorinated ethylene propylene), or HOPE, 125-mL
and 250-mL capacities.
6.4 Sample Processing Equipment
6.4.1 Air displacement pipetter - Digital pipet system
capable of delivering volumes from 10 to 2500 jA. with an
assortment of metal-free, disposable pipet tips.
6.4.2 Balances - Analytical balance, capable of
accurately weighing to ±0.1 mg; top pan balance, accu-
rate to ± 0.01g.
6.4.3 Hotplate - Corning PC100 or equivalent.
6.4.4 Centrifuge — Steel cabinet with guard bowl,
electric timer and brake.
6.4.5 Drying oven - Gravity convection oven with
thermostatic control capable of maintaining 105°C±5°C.
6.4.6 pH meter - Bench mounted or hand-held
electrode system with a resolution of ± 0.1 pH units.
7.0 Reagents and Standards
7.1 Water - For all sample preparation and dilu-
tions, ASTM type I water (ASTM D1193) is required.
7.2 Reagents may contain elemental impurities that
might affect the integrity of analytical data. Because of
the high sensitivity of this method, ultra high-purity
reagents must be used unless otherwise specified. To
minimize contamination, reagents should be prepared
directly in their designated containers where possible.
7.2.1 Acetic acid, glacial (sp. gr. 1.05).
7.2.2 Ammonium hydroxide (20%).
7.2.3 Ammonium acetate buffer 1M, pH 5.5 - Add 58-
mL (60.5 g) of glacial acetic acid to 600-mL of ASTM
type water. Add 65 mL (60 g) of 20% ammonium hydrox-
ide and mix. Check the pH of the resulting solution by
withdrawing a small aliquot and testing with a calibrated
pH meter, adjusting the solution to pH 5.5±0.1 with small
volumes of acetic acid or ammonium hydroxide as nec-
essary. Cool and dilute to 1 L with ASTM type I water.
7.2.4 Ammonium acetate buffer 2M, pH 5.5 - Prepare
as for Section 7.2.3 using 116 mL (121g) glacial acetic
acid and 130 mL (120 g) 20% ammonium hydroxide,
diluted to 1000 mL with ASTM type I water.
Note: The ammonium acetate buffer solutions may be
further purified by passing them through the
chelating column at a flow rate of 5.0-mL/min.
With reference to Figure 1, pump the buffer
solution through the column using pump P1, with
valves A and B off and valve C on. Collect the
purified solution in a container at the waste
outlet. Following this, elute the collected contam-
inants from the column using 1.25M nitric acid for
5 min at a flow rate of 4.0 mL/min.
7.2.5 Nitric acid, concentrated (sp.gr. 1.41).
7.2.5.1 Nitric acid 1.25M - Dilute 79 mL (112 g) cone.
nitric acid to 1000-mL with ASTM type I water.
7.2.5.2 Nitric acid 1% - Dilute 10 mL cone, nitric acid to
1000 mL with ASTM type I water.
200.10-5
Revision 1.6 September 1997
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7.2.5.3 Nitric acid (1+1) - Dilute 500 ml cone, nitric acid
to 1000-mL with ASTM type I water.
7.2.5.4 Nitric acid (1 +9) - Dilute 100 ml cone, nitric acid
to 1000-mL with ASTM type I water.
7.2.6 Oxalic aciddihydrate (CASRN 6153-56-6), 0.2M
- Dissolve 25.2 g reagent grade C2H2O4-2H2O in 250-mL
ASTM type I water and dilute to 1000 ml with ASTM type
I water. Caution - Oxalic acid is toxic; handle with care.
7.3 Standard Stock Solutions - May be purchased
from a reputable commercial source or prepared from
ultra high-purity grade chemicals or metals (99.99-
99.999% pure). All salts should be dried for 1 h at 105°C,
unless otherwise specified. (Caution- Many metal salts
are extremely toxic if inhaled or swallowed. Wash hands
thoroughly after handling.) Stock solutions should be
stored in plastic bottles. The following procedures may
be used for preparing standard stock solutions:
Note: Some metals, particularly those that form sur-
face oxides require cleaning prior to being
weighed. This may be achieved by pickling the
surface of the metal in acid. An amount in ex-
cess of the desired weight should be pickled
repeatedly, rinsed with water, dried, and
weighed until the desired weight is achieved.
7.3.1 Cadmium solution, stock 1 ml = 1000 //g Cd:
Pickle cadmium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5-mL (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100-mL with
ASTM type I water.
7.3.2 Cobalt solution, stock 1 ml = 1000 //g Co:
Pickle cobalt metal in (1+9) nitric acid to an exact weight
of 0.100 g. Dissolve in 5 ml (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 ml with ASTM type
I water.
7.3.3 Copper solution, stock 1 ml = 1000 //g Cu:
Pickle copper metal in (1 +9) nitric acid to an exact weight
0.100 g. Dissolve in 5 ml (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 ml with ASTM type
I water.
7.3.4 Indium solution, stock 1 ml = 1000 //gin: Pickle
indium metal in (1+1) nitric acid to an exact weight 0.100
g. Dissolve in 10 ml (1+1) nitric acid, heating to effect
solution. Cool and dilute to 100 ml with ASTM type I
water.
7.3.5 Lead solution, stock 1 ml = 1000 //g Pb: Dis-
solve 0.1599g PbNO3in 5 ml (1+1) nitric acid. Dilute to
100 ml with ASTM type I water.
7.3.6 Nickel solution, stock 1 ml = 1000 //g Ni:
Dissolve 0.100 g nickel powder in 5 ml cone, nitric acid,
heating to effect solution. Cool and dilute to 100 ml with
ASTM type I water.
7.3.7 Scandium solution, stock 1 ml = 1000 ,ug Sc:
Dissolve 0.1534 g Sc2O3 in 5 ml (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100 ml with
ASTM type I water.
7.3.8 Terbium solution, stock 1 ml = 1000 //g Tb:
Dissolve 0.1176 g Tb4O7 in 5 ml cone, nitric acid, heating
to effect solution. Cool and dilute to 100 ml with ASTM
type I water.
7.3.9 Uranium solution, stock 1 ml = 1000 //g U:
Dissolve 0.2110 g UO2(NO3)2-6H2O (Do Not Dry) in 20
ml ASTM type I water. Add 2-mL (1+1) nitric acid and
dilute to 100-mL with ASTM type I water.
7.3.70 Vanadium solution, stock 1 ml = 1000 //g V:
Pickle vanadium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5-mL (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100 ml with
ASTM type I water.
7.3.77 Yttrium solution, stock 1 ml = 1000 ,ug Y:
Dissolve 0.1270 g Y2O3 in 5 ml (1+1) nitric acid, heating
to effect solution. Cool and dilute to 100 ml with ASTM
type I water.
7.4 Multielement Stock Standard Solution - Care
must be taken in the preparation of multielement stock
standards that the elements are compatible and stable.
Originating element stocks should be checked for
impurities that might influence the accuracy of the
standard. Freshly prepared standards should be trans-
ferred to acid cleaned, new FEP or HOPE bottles for
storage and monitored periodically for stability. A
multielement stock standard solution containing the
elements, cadmium, cobalt, copper, lead, nickel, ura-
nium, and vanadium (1 ml = 10 //g) may be prepared by
diluting 1 ml of each single element stock in the list to
100 ml with ASTM type I water containing 1 % (v/v) nitric
acid.
7.4.7 Preparation of calibration standards - Fresh
multielement calibration standards should be prepared
weekly. Dilute the stock multielement standard solution
in 1% (v/v) nitric acid to levels appropriate to the required
operating range. The element concentrations in the
standards should be sufficiently high to produce good
measurement precision and to accurately define the
slope of the response curve. A suggested mid-range
concentration is 10 //g /L.
7.5 Blanks — Four types of blanks are required for
this method. A calibration blank is used to establish the
analytical calibration curve, and the laboratory reagent
blank is used to assess possible contamination from the
sample preparation procedure. The laboratory fortified
blank is used to assess the recovery of the method
Revision 1.6 September 1997
200.10-6
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analytes and the rinse blank is used between samples to
minimize memory from the nebulizer/spray chamber
surfaces.
7.5.1 Calibration blank - Consists of 1 % (v/v) nitric acid
in ASTM type I water (Section 7.2.5.2).
7.5.2 Laboratory reagent blank (LRB) - Must contain
all the reagents in the same volumes as used in process-
ing the samples. The LRB must be carried through the
entire sample digestion and preparation scheme.
7.5.3 Laboratory Fortified Blank (LFB) - To an aliquot
of LRB, add aliquots from the multielement stock stan-
dard (Section 7.4) to produce a final concentration of 10
[2Q/L for each analyte. The fortified blank must be carried
through the entire sample pretreatment and analytical
scheme.
7.5.4 Rinse Blank (RB) - Is a 1% (v/v) nitric acid
solution that is delivered to the ICP-MS between samples
(Section 7.2.5.2).
7.6 Tuning Solution — This solution is used for
instrument tuning and mass calibration prior to analysis
(Section 10.2). The solution is prepared by mixing nickel,
yttrium, indium, terbium, and lead stock solutions (Sec-
tion 7.3) in 1% (v/v) nitric acid to produce a concentration
of 100 ,ug/L of each element.
7.7 Quality Control Sample (QCS) - A quality
control sample having certified concentrations of the
analytes of interest should be obtained from a source
outside the laboratory. Dilute the QCS if necessary with
1% nitric acid, such that the analyte concentrations fall
within the proposed instrument calibration range.
7.8 Instrument Performance Check (IPC) Solution
-- The IPC solution is used to periodically verify instru-
ment performance during analysis. It should be prepared
by combining method analytes at appropriate concentra-
tions to approximate the midpoint of the calibration curve.
The IPC solution should be prepared from the same
standard stock solutions used to prepare the calibration
standards and stored in a FEP bottle. Agency programs
may specify or request that additional instrument perfor-
mance check solutions be prepared at specified concen-
trations in order to meet particular program needs.
7.9 Internal Standards Stock Solution, 1 mL =
100 jj,g — Dilute 10-mL of scandium, yttrium, indium,
terbium, and bismuth stock standards (Section 7.3) to
100-mL with ASTM type I water, and store in a Teflon
bottle. Use this solution concentrate for addition to
blanks, calibration standards and samples (Method A,
Section 10.5), or dilute by an appropriate amount using
1% (v/v) nitric acid, if the internal standards are being
added by peristaltic pump (Method B, Section 10.5).
Note: Bismuth should not be used as an internal
8.0
standard using the direct addition method (Me-
thod A, Section 10.5) as it is not efficiently
concentrated on the iminodiacetate column.
Sample Collection, Preservation, and
Storage
8.1 Prior to the collection of an aqueous sample,
consideration should be given to the type of data re-
quired, so that appropriate preservation and pretreatment
steps can be taken. Acid preservation should be per-
formed at the time of sample collection or as soon
thereafter as practically possible. The pH of all aqueous
samples must be tested immediately prior to aliquoting
for analysis to ensure the sample has been properly
preserved. If properly acid preserved, the sample can be
held up to 6 months before analysis.
8.2 For the determination of total recoverable
elements in aqueous samples, acidify with (1+1) nitric
acid (high purity) at the time of collection to pH<2;
normally, 3 mL of (1+1) acid per liter of sample is
sufficient for most samples. The sample should not be
filtered prior to analysis.
Note: Samples that cannot be acid preserved at the
time of collection because of sampling limita-
tions or transport restrictions, or are >pH2
because of high alkalinity should be acidified
with nitric acid to pH<2 upon receipt in the
laboratory. Following acidification, the sample
should be held for 16 h and the pH verified to be
<2 before withdrawing an aliquot for sample
processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container and acid as used in sample collec-
tion.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an
initial demonstration of laboratory capability and the
periodic analysis of laboratory reagent blanks, fortified
blanks and other laboratory solutions as a continuing
check on performance. The laboratory is required to
maintain performance records that define the quality of
the data generated.
9.2 Initial Demonstration of Performance (Manda-
tory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (determination of
200.10-7
Revision 1.6 September 1997
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linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) prior to samples being analyzed
by this method.
9.2.2 Linear calibration ranges — The upper limit of the
linear calibration range should be established for each
analyte. Linear calibration ranges should be determined
every six months or whenever a significant change in
instrument response is expected.
9.2.3 Quality control sample (QCS) - When beginning
the use of this method, on a quarterly basis or as re-
quired to meet data-quality needs, verify the calibration
standards and acceptable instrument performance with
the preparation and analyses of a QCS (Section 7.7). If
the determined concentrations are not within ± 10% of
the stated values, performance of the determinative step
of the method is unacceptable. The source of the prob-
lem must be identified and corrected before either
proceeding with the initial determination of method
detection limits or continuing with ongoing analyses.
9.2.4 Method detection limit (MDL) - MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.9 To determine MDL
values, take seven replicate aliquots of the fortified
reagent water and process through the entire analytical
method. Perform all calculations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
MDL = (t) x (S)
where: t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S = standard deviation of the replicate analyses.
Note: If the relative standard deviation (RSD) from the
analyses of the seven aliquots is <15%, the
concentration used to determine the analyte
MDL may have been inappropriately high for the
determination. If so, this could result in the
calculation of an unrealistically low MDL. If
additional confirmation of the MDL is desired,
reanalyze the seven replicate aliquots on two
more nonconsecutive days and again calculate
the MDL values for each day. An average of the
three MDL values for each analyte may provide
for a more appropriate MDL estimate. Concur-
rently, determination of MDL in reagent water
represents a best case situation and does not
reflect possible matrix effects of real world
samples. However, successful analyses of LFMs
(Section 9.4) can give confidence to the MDL
value determined in reagent water. Typical
single laboratory MDL values using this method
are given in Table 1.
MDLs should be determined every six months, when a
new operator begins work or whenever there is a signifi-
cant change in the background or instrument response.
9.3 Assessing Laboratory Performance (Manda-
tory)
9.3.1 Laboratory reagent blank (LRB) — The laboratory
must analyze at least one LRB (Section 7.5.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and
the samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
9.3.2 Laboratory fortified blank (LFB) - The laboratory
must analyze at least one LFB (Section 7.5.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that
analyte is judged out of control, and the source of the
problem should be identified and resolved before contin-
uing analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and
the standard deviation (S) of the mean recovery. These
data can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most recent 20-30 data
points. Also, the standard deviation (S) data should be
used to established an ongoing precision statement for
the level of concentrations included in the LFB. These
data must be kept on file and be available for review.
9.3.4 Instrument performance check (IPC) solution —
For all determinations the laboratory must analyze the
IPC solution (Section 7.8) and a calibration blank imme-
diately following daily calibration, after every tenth
sample (or more frequently, if required) and at the end of
the sample run. Analysis of the IPC solution and calibra-
tion blank immediately following calibration must verify
that the instrument is within ±10% of calibration. Subse-
Revision 1.6 September 1997
200.10-8
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quent analyses of the IPC solution must verify the
calibration within ±15%. If the calibration cannot be
verified within the specified limits, reanalyze the IPC
solution. If the second analysis of the IPC solution
confirms calibration to be outside the limits, sample
analysis must be discontinued, the cause determined
and/or in the case of drift the instrument recalibrated. All
samples following the last acceptable IPC solution must
be reanalyzed. The analysis data of the calibration blank
and IPC solution must be kept on file with the sample
analyses data.
9.3.5 The overall sensitivity and precision of this
method are strongly influenced by a laboratory's ability to
control the method blank. Therefore, it is recommended
that the calibration blank response be recorded for each
set of samples. This record will aid the laboratory in
assessing both its long- and short-term ability to control
the method blank.
9.4 Assessing Analyte Recovery and Data
Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and the
quality of the data. Taking separate aliquots from the
sample for replicate and fortified analyses can in some
cases assess these effects. Unless otherwise specified
by the data user, laboratory or program, the following
laboratory fortified matrix (LFM) procedure (Section
9.4.2) is required.
9.4.2 The laboratory must add a known amount of
each analyte to a minimum of 10% of the routine sam-
ples. In each case the LFM aliquot must be a duplicate
of the aliquot used for sample analysis and for total
recoverable determinations added prior to sample
preparation. For water samples, the added analyte
concentration must be the same as that used in the
laboratory fortified blank (Section 9.3.2).
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is less than
25% of the unfortified sample concentration. Percent
recovery may be calculated in units appropriate to the
matrix, using the following equation:
-x 100
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
S = concentration equivalent of analyte added
to sample.
9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range and the laboratory per-
formance for that analyte is shown to be in control
(Section 9.3), the recovery problem encountered with the
LFM is judged to be either matrix or solution related, not
system related.
9.4.5 If analysis of LFM sample(s) and the test rou-
tines above indicate an operative interference and the
LFMs are typical of the other samples in the batch, those
samples that are similar must be analyzed in the same
manner as the LFMs. Also, the data user must be
informed when a matrix interference is so severe that it
prevents the successful analysis of the analyte or when
the heterogeneous nature of the sample precludes the
use of duplicate analyses.
9.4.6 Where reference materials are available, they
should be analyzed to provide additional performance
data. The analysis of reference samples is a valuable
tool for demonstrating the ability to perform the method
acceptably.
10.0 Calibration and Standardization
70.7 Initiate proper operating configuration of ICP-MS
instrument and data system. Allow a period of not less
than 30 min for the instrument to warm up. During this
process conduct mass calibration and resolution checks
using the tuning solution. Resolution at low mass is
indicated by nickel isotopes 60, 61, 62. Resolution at
high mass is indicated by lead isotopes 206, 207, 208.
For good performance adjust spectrometer resolution to
produce a peak width of approximately 0.75 amu at 5%
peak height. Adjust mass calibration if it has shifted by
more than 0.1 amu from unit mass.
70.2 Instrument stability must be demonstrated by
analyzing the tuning solution (Section 7.6) a minimum of
five times with resulting relative standard deviations of
absolute signals for all analytes of less than 5%.
70.3 Priorto initial calibration, setup proper instrument
software routines for quantitative analysis and connect
the ICP-MS instrument to the preconcentration appara-
tus. The instrument must be calibrated for the analytes
of interest using the calibration blank (Section 7.5.1) and
calibration standard (Section 7.4.1) prepared at one or
more concentration levels. The calibration solutions
should be processed through the preconcentration
system using the procedures described in Section 11.
70.4 Demonstration and documentation of acceptable
initial calibration is required before any samples are
analyzed. After initial calibration is successful, a calibra-
tion check is required at the beginning and end of each
period during which analyses are performed and at
requisite intervals.
200.10-9
Revision 1.6 September 1997
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10.4.1 After the calibration has been established, it
must be initially verified for all analytes by analyzing the
I PC (Section 7.8). If the initial calibration verification
exceeds ±10% of the established IPC value, the analysis
should be terminated, the source of the problem identi-
fied and corrected, the instrument recalibrated, and the
new calibration verified before continuing analyses.
10.4.2 To verify that the instrument is properly calibrated
on a continuing basis, analyze the calibration blank
(Section 7.5.1) and IPC (Section 7.8) after every 10
analyses. The results of the analyses of the standards
will indicate whether the calibration remains valid. If the
indicated concentration of any analyte deviates from the
true concentration by more than 15%, reanalyze the
standard. If the analyte is again outside the 15% limit, the
instrument must be recalibrated and the previous 10
samples reanalyzed. The instrument responses from the
calibration check may be used for recalibration purposes.
70.5 Internal Standardization - Internal standardiza-
tion must be used in all analyses to correct for instrument
drift and physical interferences. For full mass range
scans, a minimum of three internal standards must be
used. Internal standards must be present in all samples,
standards and blanks at identical levels. This may be
achieved by directly adding an aliquot of the internal
standards to the CAL standard, blank or sample solution
(Method A), or alternatively by mixing with the solution
prior to nebulization using a second channel of the
peristaltic pump and a mixing coil (Method B). The
concentration of the internal standard should be suffi-
ciently high that good precision is obtained in the mea-
surement of the isotope used for data correction and to
minimize the possibility of correction errors if the internal
standard is naturally present in the sample. Internal
standards should be added to blanks, samples and
standards in a like manner, so that dilution effects result-
ing from the addition may be disregarded.
Note: Bismuth should not be used as an internal
standard using the direct addition method (Me-
thod A, Section 10.5) because it is not efficiently
concentrated on the iminodiacetate column.
11.0 Procedure
77.7 Sample Preparation
Elements
Total Recoverable
77.7.7 Add2-ml_(1+1) nitric acid to the beaker contain-
ing 100-mL of sample. Place the beaker on the hot plate
for solution evaporation. The hot plate should be located
in a fume hood and previously adjusted to provide evapo-
ration at a temperature of approximately but no higher
than 85°C. (See the following note.) The beaker should
be covered with an elevated watch glass or other neces-
sary steps should be taken to prevent sample contamina-
tion from the fume hood environment.
Note: For proper heating, adjust the temperature
control of the hot plate such that an uncovered
Griffin beaker containing 50 ml of water placed
in the center of the hot plate can be maintained
at a temperature approximately but no higher
than 85°C. (Once the beaker is covered with a
watch glass the temperature of the water will rise
to approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20-mL by gentle heating at 85°C. Do Not Boil.
This step takes about 2 h for a 100-mL aliquot with the
rate of evaporation rapidly increasing as the sample
volume approaches 20 ml. (A spare beaker containing
20-mL of water can be used as a gauge.)
77.7.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the
sample for 30 min. (Slight boiling may occur, but vigor-
ous boiling must be avoided.)
77.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
77.7.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight, the
sample contains suspended solids, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination
from filtration.) The sample is now ready for analysis.
Because the effects of various matrices on the stability of
diluted samples cannot be characterized, all analyses
should be performed as soon as possible after the
completed preparation.
77.2 Prior to first use, the preconcentration system
should be thoroughly cleaned and decontaminated using
0.2M oxalic acid.
11.2.1 Place approximately 500-mL 0.2M oxalic acid in
all the eluent/solution containers and fill the sample loop
with 0.2M oxalic acid using the sample pump (P4) at a
flow rate of 3-5 mL/min. With the preconcentration
system disconnected from the ICP-MS instrument, use
the pump program sequence listed in Table 2 to flush the
complete system with oxalic acid. Repeat the flush se-
quence three times.
77.2.2 Repeat the sequence described in Section
11.2.1 using 1.25M nitric acid and again using ASTM
type I water in place of the 0.2M oxalic acid.
11.2.3 Rinse the containers thoroughly with ASTM type
I water, fill them with their designated reagents (see
Figure 1) and run through the sequence in Table 2 once
to prime the pump and all eluent lines with the correct
reagents.
Revision 1.6 September 1997
200.10-10
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11.3 Initiate ICP-MS instrument operating configura-
tion. Tune the instrument for the analytes of interest
(Section 10).
11.4 Establish instrument software run procedures for
quantitative analysis. Because the analytes are eluted
from the preconcentration column in a transient manner,
it is recommended that the instrument software is config-
ured in a rapid scan/peak hopping mode. The instrument
is now ready to be calibrated.
11.5 Reconnect the preconcentration system to the
ICP-MS instrument. With valves A and B in the off
position and valve C in the on position, load sample
through the sample loop to waste using pump P4 for 4
min at 4 mL/min. Switch on the carrier pump (P3) and
pump 1% nitric acid to the nebulizer of the ICP-MS
instrument at a flow rate of 0.8-1.0-mL/min.
11.6 Switch on the buffer pump (P2), and pump 2M
ammonium acetate at a flow rate of 1.0 mL/min.
11.7 Preconcentration of the sample may be achieved
by running through an eluent pump program (P1) se-
quence similar to that illustrated in Table 2. The exact
timing of this sequence should be modified according to
the internal volume of the connecting tubing and the
specific hardware configuration used.
11.7.1 Inject sample — With valves A, B, and C on, load
sample from the loop onto the column using 1M ammo-
nium acetate for 4.5 min at 4.0 mL/min. The analytes are
retained on the column, while the majority of the matrix
is passed through to waste.
717.2 Elute analytes - Turn off valves A and B and
begin eluting the analytes by pumping 1.25M nitric acid
through the column at 4.0 mL/min, then turn off valve C
and pump the eluted analytes into the ICP-MS instrument
at 1.0 mL/min. Initiate ICP-MS software data acquisition
and integrate the eluted analyte profiles.
11.7.3 Column Reconditioning — Turn on valve C to
direct column effluent to waste, and pump 1.25M nitric
acid, 1M ammonium acetate, 1.25M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min. During this process, the next sample can be
loaded into the sample loop using the sample pump (P4).
77.8 Repeat the sequence described in Section 11.7
for each sample to be analyzed. At the end of the analyti-
cal run leave the column filled with 1M ammonium
acetate buffer until it is next used.
77.9 Samples having concentrations higher than the
established linear dynamic range should be diluted into
range with 1% HNO3 (v/v) and reanalyzed.
12.0 Data Analysis and Calculations
72.7 Analytical isotopes and elemental equations
recommended for sample data calculations are listed in
Table 3. Sample data should be reported in units of ug/L.
Do not report element concentrations below the deter-
mined MDL.
72.2 For data values less than 10, two significant
figures should be used for reporting element concentra-
tions. For data values greater than or equal to 10, three
significant figures should be used.
72.3 Reported values should be calibration blank sub-
tracted. If additional dilutions were made to any samples,
the appropriate factor should be applied to the calculated
sample concentrations.
72.4 Data values should be corrected for instrument
drift by the application of internal standardization. Correc-
tions for characterized spectral interferences should be
applied to the data.
72.5 The QC data obtained during the analyses
provide an indication of the quality of the sample data
and should be provided with the sample results.
13.0 Method Performance
73.7 Experimental conditions used for single labora-
tory testing of the method are summarized in Table 4.
73.2 Data obtained from single laboratory testing of
the method are summarized in Tables 5 and 6 for two
reference water samples consisting of National Research
Council Canada (NRCC) Estuarine Water (SLEW-1) and
Seawater (NASS-2). The samples were prepared using
the procedure described in Section 11.1.1. For each
matrix, three replicates were analyzed and the average
of the replicates was used to determine the sample
concentration for each analyte. Two further sets of three
replicates were fortified at different concentration levels,
one set at 0.5 //g/L, the other at 10 ug/L. The sample
concentration, mean percent recovery, and the relative
standard deviation of the fortified replicates are listed for
each method analyte. The reference material certificate
values are also listed for comparison.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that place pollution pre-
200.10-11
Revision 1.6 September 1997
-------
vention as the management option of first choice. When-
ever feasible, laboratory personnel should use pollution
prevention techniques to address their waste generation
(e.g., Section 7.8). When wastes cannot be feasibly
reduced at the source, the Agency recommends recy-
cling as the next best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 1155 16th Street N.W.,
Washington, D.C. 20036, (202)872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management, consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. Siraraks, A., H.M. Kingston, and J.M. Riviello,
Anal Chem. 62,1185 (1990).
2. Heithmar, E.M., T.A. Hinners, J.T. Rowan, and
J.M. Riviello, Anal Chem., 62, 857 (1990).
3. Gray A.L. and A.R. Date, Analyst, 108, 1033
(1983).
4. Houk, R.S., et al. Anal. Chem., 52, 2283 (1980).
5. Houk, R.S., Anal.Chem., 58, 97A (1986).
6. J. J. Thompson and R.S. Houk, Appl. Spec., 41,
801 (1987).
7. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, (Re-
vised, January 1976).
8. Safety in Academic Chemistry Laboratories,
American Chemical Society Publication, Commit-
tee on Chemical Safety, 3rd Edition, 1979.
9. Code of Federal Regulations 40, Ch. 1, Pt. 136
Appendix B.
Revision 1.6 September 1997
200.10-12
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Total Recoverable Method Detection Limits for Reagent Water
Element
Cadmium
Cobalt
Copper
Lead
Nickel
Uranium
Vanadium
Recommended
Analytical Mass
111
59
63
206, 207,208
60
238
51
MDL1
//g/L
0.041
0.021
0.023
0.074
0.081
0.031
0.014
Determined using 10-mL sample loop.
Table 2. Eluent Pump Programming Sequence for Preconcentration of Trace Elements
Time
(min)
0.0
4.5
5.1
5.5
7.5
8.0
10.0
11.0
12.5
Flow
(mL/min)
4.0
4.0
1.0
1.0
4.0
4.0
4.0
4.0
0.0
Eluent
1 M ammonium acetate
1 .25M nitric acid
1 .25M nitric acid
1 .25M nitric acid
1 .25M nitric acid
1 M ammonium acetate
1 .25M nitric acid
1 M ammonium acetate
Valve
A,B
ON
ON
OFF
OFF
OFF
OFF
OFF
OFF
OFF
Valve
C
ON
ON
ON
OFF
ON
ON
ON
ON
ON
Table 3. Recommended Analytical Isotopes and Elemental Equations for Data Calculations
Element Isotope Elemental Equation Note
Cd
Co
Cu
Pb
Ni
U
V
106, 108, 111, 114
59
63,65
206, 207, 208
60
238
51
(1.000)(111C)-(1.073)[(108C)-(0.712)(106C)]
(1 .000)(59C)
(1 .000)(63C)
(1 .000)(206C)+(1 .000)(207C)+(1 .000)(208C)
(1 .000)(60C)
(1 .000)(238C)
(1.000)(51C)
(1)
(2)
C - calibration blank subtracted counts at specified mass.
(1) - correction for MoO interference. An additional isobaric elemental correction should be made if palladium is present.
(2) - allowance for isotopic variability of lead isotopes.
NOTE: As a minimum, all isotopes listed should be monitored. Isotopes recommended for analytical determination are italized.
200.10-13 Revision 1.6 September 1997
-------
Table 4. Experimental Conditions for Single Laboratory Validation
Chromatography
Instrument
Preconcentration column
Dionex chelation system
Dionex MetPac CC-1
ICP-MS Instrument Conditions
Instrument
Plasma forward power
Coolant flow rate
Auxiliary flow rate
Nebulizer flow rate
Internal standards
Data Acquisition
Detector mode
Mass range
Dwell time
Number of MCA channels
Number of scan sweeps
VG PlasmaQuad Type I
1.35 kW
13.5 L/min
0.6 L/min
0.78 L/min
Sc, Y, In, Tb
Pulse counting
45-240 amu
160 MS
2048
250
Table 5. Precision and Recovery Data for Estuarine Water (SLEW-1)
Analyte
Cd
Co
Cu
Pb
Ni
U
V
Certificate
(MP./L)
0.018
0.046
1.76
0.028
0.743
Sample
Cone.
(MS/L)
<0.041
0.078
1.6
<0.074
0.83
1.1
1.4
Spike
Addition
(MS/L)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Average
Recovery
(%)
94.8
102.8
106.0
100.2
100.0
96.7
100.0
RSD
(%)
9.8
4.0
2.7
4.0
1.5
7.4
3.2
Spike
Addition
(MS/L)
10
10
10
10
10
10
10
Average
Recovery
(%)
99.6
96.6
96.0
106.9
102.0
98.1
97.0
RSD
(%)
1.1
1.4
4.8
5.8
2.1
3.6
4.5
- No certificate value
Table 6. Precision and Recovery Data for Seawater (NASS-2)
Analyte
Cd
Co
Cu
Pb
Ni
U
V
Certificate
(MP./L)
0.029
0.004
0.109
0.039
0.257
3.00
Sample
Cone.
(MS/L)
<0.041
<0.021
0.12
<0.074
0.23
3.0
1.7
Spike
Addition
(MS/L)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Average
Recovery
(%)
101.8
98.9
95.8
100.6
102.2
94.0
104.0
RSD
(%)
1.0
3.0
2.3
8.5
2.3
0.7
3.4
Spike
Addition
(MS/L)
10
10
10
10
10
10
10
Average
Recovery
(%)
96.4
99.2
93.1
92.1
98.2
98.4
109.2
RSD
(%)
3.7
1.7
0.9
2.6
1.2
1.7
3.7
-No certificate value
Revision 1.6 September 1997
200.10-14
-------
Waste
| Waste |
Waste
1
Buffer
Pump
2M NhjjOAc
P2
1
P/STALTIC
Pump
Sample
P4 Eluent
Pump
P1
1 1
1MN4jOAc 1. 25 M Nitric Acid
I
Carrier
Pump
1%NtricAcid
P3
Mixing Tee
Figure 1. Configuration of Preconcentration System.
Off
On
200.10-15
Revision 1.6 September 1997
-------
Method 200.12
Determination of Trace Elements in Marine Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption
John T. Creed and Theodore D. Martin
Chemical Exposure Research Branch
Human Exposure Research Division
Revision 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
200.12-1
-------
Method 200.12
Determination of Trace Elements in Marine Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption
1.0 Scope and Application
7.7 This method provides procedures for the
determination of total recoverable elements by graphite
furnace atomic absorption (GFAA) in marine waters,
including estuarine, ocean and brines with salinities of up
to 35 ppt. This method is applicable to the following
analytes:
Analyte
Arsenic
Cadmium
Chromium
Copper
Lead
Nickel
Selenium
(As)
(Cd)
(Cr)
(Cu)
(Pb)
(Ni)
(Se)
Chemical Abstracts
Service Registry
Numbers (CASRN)
7440-38-2
7440-43-9
7440-47-3
7440-50-8
7439-92-1
7440-02-0
7782-49-2
7.2 For determination of total recoverable analytes in
marine waters, a digestion/extraction is required prior to
analysis.
7.3 Method detection limit and instrumental operating
conditions for the applicable elements are listed in Tables
1 and 2. These are intended as a guide and are typical of
a commercial instrument optimized for the element.
However, actual method detection limits and linear work-
ing ranges will be dependent on the sample matrix,
instrumentation and selected operating conditions.
7.4 Users of the method data should state the data
quality objectives prior to analysis. The ultra-trace metal
concentrations typically associated with marine water may
preclude the use of this method based on its sensitivity.
Users of the method must document and have on file the
required initial demonstration performance data described
in Section 9.2 prior to using the method for analysis.
2.0 Summary of Method
2.7 Nitric acid is dispensed into a beaker containing
an accurately weighed or measured, well-mixed,
homogeneous aqueous sample. Then, for samples with
undissolved material, the beaker is covered with a watch
glass and heated, made up to volume, centrifuged or
allowed to settle, and the sample is then analyzed.
2.2 The analytes listed in this method are determined
by stabilized temperature platform graphite furnace
atomic absorption (STPGFAA). In STPGFAA, the sample
and the matrix modifier are first pipetted onto the platform
or a device which provides delayed atomization.
The furnace chamber is then purged with a continuous
flow of a premixed gas (95% argon - 5% hydrogen) and
the sample is dried at a relatively low temperature (about
120°C) to avoid spattering. Once dried, the sample is
pretreated in a char or ashing step which is designed to
minimize the interference effects caused by the concomi-
tant sample matrix. After the char step, the furnace is
allowed to cool prior to atomization. The atomization
cycle is characterized by rapid heating of the furnace to a
temperature where the metal (analyte) is atomized from
the pyrolytic graphite surface into a stopped gas flow
atmosphere of argon containing 5% hydrogen. (Only
selenium is determined in an atmosphere of high purity
argon.) The resulting atomic cloud absorbs the element-
specific atomic emission produced by a hollow cathode
lamp (HCL) or an electrodeless discharge lamp (EDL).
Following analysis, the furnace is subjected to a cleanout
period of high temperature and continuous argon flow.
Because the resulting absorbance usually has a nonspe-
cific component associated with the actual analyte ab-
sorbance, Zeeman background correction is required to
subtract from the total signal the component which is
nonspecific to the analyte. In the absence of interfer-
ences, the background-corrected, absorbance is directly
related to the concentration of the analyte. Interferences
relating to STPGFAA (Section 4.0) must be recognized
and corrected. Suppressions or enhancements of instru-
ment response caused by the sample matrix must be
corrected for by the method of standard addition (Section
11.3).
Revision 1.0 September 1997
200.12-2
-------
3.0 Definitions
3.1 Calibration Blank (CB) - A volume of reagent
water fortified with the same matrix as the calibration
standards, but without the analytes, internal standards, or
surrogate analytes.
3.2 Calibration Standard (CAL) - A solution pre-
pared from the primary dilution standard solution or stock
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate the
instrument response with respect to analyte concen-
tration.
3.3 Field Reagent Blank (FRB) - An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling
site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine it method analytes or other
interferences are present in the field environment.
3.4 Instrument Detection Limit (IDL) - The mini-
mum quantity of analyte or the concentration equivalent
which gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance line,
etc.
3.5 Instrument Performance Check Solution (IPC)
- A solution of one or more method analytes, surrogates,
internal standards, or other test substances used to
evaluate the performance of the instrument system with
respect to a defined set of criteria.
3.6 Laboratory Duplicates (LD1 and LD2) - Two
aliquots of the same sample taken in the laboratory and
analyzed separately with identical procedures. Analyses
of LD1 and LD2 indicate precision associated with
laboratory procedures, but not with sample collection,
preservation, or storage procedures.
3.7 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.8 Laboratory Fortified Sample Matrix (LFM) - An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background concentra-
tions.
3.9 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, the
reagents, or the apparatus.
3.70 Linear Dynamic Range (LDR) - The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.11 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
3.72 Matrix Modifier (MM) - A substance added to
the instrument along with the sample in order to minimize
the interference effects by selective volatilization of either
analyte or matrix components.
3.13 Matrix Performance Check (MPC) - A solution
of method analytes used to evaluate the laboratory's
ongoing capabilities in analyzing high salinity samples.
The reference material NASS-3 or its equivalent is forti-
fied with the same analytes at the same concentration as
the LFB. This provides an ongoing check of furnace
operating conditions to assure the analyte false positives
are not being introduced via elevated backgrounds.
3.74 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified, mea-
sured and reported with 99% confidence that the analyte
concentration is greater than zero.
3.75 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
200.12-3
Revision 1.0 September 1997
-------
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.76 Standard Addition - The addition of a known
amount of analyte to the sample in order to determine the
relative response of the detector to an analyte within the
sample matrix. The relative response is then used to
assess either an operative matrix effect or the sample
analyte concentration.
3.77 Stock Standard Solution (SSS) - A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial
source.
3.78 Total Recoverable Analyte (TRA) - The con-
centration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following treat-
ment by refluxing with hot dilute mineral acid(s) as
specified in the method.
4.0 Interferences
4.7 Several interference sources may cause
inaccuracies in the determination of trace elements by
GFAA. These interferences can be classified into three
major subdivisions: spectral, matrix, and memory.
4.2 Spectral interferences are caused by absorbance
of light by a molecule or atom which is not the analyte of
interest or emission from black body radiation.
4.2.1 Spectral interferences caused by an element only
occur if there is a spectral overlap between the wave-
length of the interfering element and the analyte of
interest. Fortunately, this type of interference is relatively
uncommon in STPGFAA because of the narrow atomic
line widths associated with STPGFAA. In addition, the use
of appropriate furnace temperature programs and high
spectral purity lamps as light sources can minimize the
possibility of this type of interference. However, molecular
absorbances can span several hundred nanometers
producing broadband spectral interferences. This type of
interference is far more common in STPGFAA. The use
of matrix modifiers, selective volatilization, and
background correctors are all attempts to eliminate un-
wanted nonspecific absorbance. Table 2 contains typical
background absorbances associated with the analysis of
the MPC solution (NASS-3) which has a salinity of 35 ppt.
These background absorbances were obtained using the
suggested matrix modifiers and the appropriate furnace
charring conditions. Figure 1 is a plot of integrated
background absorbance vs. char temperature for Ni, Cd,
Pb, and Se. Figure 1 indicates that the background
absorbance in a saline matrix is strongly affected by the
char temperature. Therefore, char temperature optimi-
zation is a critical part of the successful analysis of metals
in saline water by GFAA. The elevated backgrounds
associated with ocean water can produce false positives.
For this reason, the char temperature profiles shown in
Figure 1 should be constructed for each analyte prior to
using this method for saline water analysis.
Note: False analyte positives can be generated by large
backgrounds. Figure 2 is an atomization profile for Pb
using a 800°C char temperature. The background shown
in the figure has exceeded the capabilities of the Zeeman
corrector. This profile can be used as a guide in screening
other analyses which may have background absorbances
which exceed the Zeeman capability. The background
profile is characterized by a smooth baseline in the
beginning of the atomization cycle followed by a sharp
increase. During this sharp increase the background peak
profile may remain relatively smooth, but when the
background exceeds the Zeeman correction capability,
the background profile will appear extremely erratic. The
atomic profile is also erratic during this part of the atomi-
zation cycle. These types of background/atomic profiles
obtained during atomization result in false positives.
Since the nonspecific component of the total absorbance
can vary considerably from sample type to sample type,
to provide effective background correction and eliminate
the elemental spectral interference of palladium on cop-
per and iron on selenium, the exclusive use of Zeeman
background correction is specified in this method.
4.2.2 Spectral interferences are also caused by black
body radiation produced during the atomization furnace
cycle. This black body emission reaches the
photomultiplier tube, producing erroneous results. The
magnitude of this interference can be minimized by
proper furnace tube alignment and monochromator
design. In addition, atomization temperatures which
adequately volatilize the analyte of interest without
producing unnecessary black body radiation can help
reduce unwanted background emission produced during
atomization.
4.3 Matrix interferences are caused by sample com-
ponents which inhibit the formation of free atomic analyte
atoms during atomization. In this method the use of a
delayed atomization device which provides a warmer gas
Revision 1.0 September 1997
200.12-4
-------
phase environment during atomization is required. These
devices provide an environment which is more conducive
to the formation of free analyte atoms and thereby
minimize this type of interference. This type of
interference can be detected by analyzing the sample
plus a sample aliquot fortified with a known concentration
of the analyte. If the determined concentration of the
analyte addition is outside a designated range (Section
9.4.3), a possible matrix effect should be suspected. In
addition, the matrix can produce analyte complexes which
are lost via volatilization during the char. These losses will
result in poor recovery of the analyte within the matrix and
should be corrected by adjusting the char temperature.
4.4 Memory interferences result from analyzing a
sample containing a high concentration of an element
(typically a high atomization temperature element) which
cannot be removed quantitatively in one complete set of
furnace steps. The analyte which remains in the furnace
can produce false positive signals on subsequent
sample(s). Therefore, the analyst should establish the
analyte concentration which can be injected into the
furnace and adequately removed in one complete set of
furnace cycles. If this concentration is exceeded, the
sample should be diluted and a blank analyzed to assure
the memory effect has been eliminated before reanalyz-
ing the diluted sample.
4.5 Specific Element Interferences. - The matrix
effects caused by the saline water can be severe. In order
to evaluate the extent of the matrix suppression as a
function of increasing salinity a plot of normalized inte-
grated absorbance vs. microliters NASS-3 (Reference
Material from the National Research Council of Canada)
is constructed. Figure 3 is a plot of relative response of
As, Se, Cd, Ni, Cu, and Pb in waters containing salinity of
3.5 ppt (1 uL NASS-3) to 35 ppt (10 uL NASS-3). Figure
3 indicates that the matrix effects caused by the increas-
ing salinity are minor for Pb, Cu, and Ni. The relative
responses of Pb, Ni, and Cu shown in Figure 3 are within
± 5% of the 1% HNO3 standard or zero uL of matrix.
Figure 3 indicates that the increasing salinity does cause
a substantial matrix interference for Se and Cd. This
suppression must be compensated for by methods of
standard addition or the use of matrix matched standards
where applicable.
4.5.1 Cadmium: The background level associated with the
direct determination of Cd in NASS-3 exceeds the
Zeeman background correction. Therefore, NH4 NO3 is
used as a matrix removing modifier in addition to the Pd/
Mg(NO3)2.1 Figure 4 is a plot of the relative Cd response
vs. the amount of seawater on the platform. A similar
response profile is observed in a solution containing
10,000 ppm NaCI. Therefore, in well-characterized
samples of known salinity it is possible to effectively matrix
match the standards with NaCI and perform the analysis
directly using matrix matched standards, thereby avoiding
the time consuming method of standard additions. If the
matrix matched standards are going to be used, it is
necessary to document that the use of NaCI is indeed
compensating for the suppression. This documentation
should include a response plot of increasing matrix vs.
relative response similar to Figure 4.
4.5.2 Selenium: The background level associated with
the direct determination of Se in NASS-3 exceeds the
Zeeman correction capability. Therefore, HNO3 is used as
a matrix removing modifier in addition to the Pd/ Mg(NO3)2
for the determination of Se in saline waters. Figure 5 is a
plot of relative response vs. the amount of seawater on
the platform. A similar suppression is observed in a
solution containing 10,000 ppm NaCI. Therefore, in well-
characterized samples of known salinity it is possible to
effectively matrix match the standards with NaCI and
perform the analysis directly using matrix matched
standards, thereby avoiding the time consuming method
of standard additions. If the matrix matched standards are
going to be used, it is necessary to document that the use
of NaCI is indeed compensating for the suppression. This
documentation should include a response plot of
increasing matrix vs. relative response similar to Figure 5.
4.5.3 Arsenic: The elevated char temperatures
possible with the determination of As minimize the
interferences produced by the marine water background
levels. Figure 3 is a plot of relative response vs. the
amount of seawater on the platform. Figure 3 indicates a
matrix suppression on As caused by the seawater.
Although this suppression does cause a slight bias as
shown in the recovery data in Table 3, the suppression
does not warrant the method of standard additions (MSA)
given the recovery criteria of 75-125% for LFMS.
5.0 Safety
5.7 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible for
maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in
this method.2"5 A reference file of material data handling
sheets should also be made available to all personnel
200.12-5
Revision 1.0 September 1997
-------
involved in the chemical analysis. Specifically,
concentrated nitric and hydrochloric acids present various
hazards and are moderately toxic and extremely irritating
to skin and mucus membranes. Use these reagents in a
fume hood whenever possible and if eye or skin contact
occurs, flush with large volumes of water. Always wear
safety glasses or a shield for eye protection, protective
clothing, and observe proper mixing when working with
these reagents.
5.2 The acidification of samples containing reactive
materials may result in the release of toxic gases, such as
cyanides or sulfides. Acidification of samples should be
done in a fume hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 The graphite tube during atomization emits in-
tense UV radiation. Suitable precautions should be taken
to protect personnel from such a hazard.
5.5 The use of the argon/hydrogen gas mixture
during the dry and char steps may evolve a considerable
amount of HCI gas. Therefore, adequate ventilation is
required.
5.6 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Equipment and Supplies
6.1 Graphite Furnace Atomic Absorption Spec-
trometer
6.1.1 The GFAA spectrometer must be capable of
programmed heating of the graphite tube and the
associated delayed atomization device. The instrument
must be equipped with Zeeman background correction
and the furnace device must be capable of utilizing an
alternate gas supply during specified cycles of the
analysis. The capability to record relatively fast (< 1 s)
transient signals and evaluate data on a peak area basis
is preferred. In addition, a recirculating refrigeration unit is
recommended for improved reproducibility of furnace
temperatures.
6.12 Single element hollow cathode lamps or single
element electrodeless discharge lamps along with the
associated power supplies.
6.1.3 Argon gas supply (high-purity grade, 99.99%) for
use during the atomization of selenium, for sheathing the
furnace tube when in operation, and during furnace
cleanout.
6.1.4 Alternate gas mixture (hydrogen 5%-argon 95%)
for use as a continuous gas flow environment during the
dry and char furnace cycles.
6.15 Autosampler capable of adding matrix modifier
solutions to the furnace, a single addition of analyte, and
completing methods of standard additions when required.
6.2 Analytical balance, with capability to measure to
0.1 mg, for preparing standards, and for determining
dissolved solids in digests or extracts.
6.3 A temperature adjustable hot plate capable of
maintaining a temperature of 95°C.
6.4 An air displacement pipetter capable of delivering
volumes ranging from 100 to 2500 uL with an assortment
of high quality disposable pipet tips.
6.5 Labware - All reusable labware (glass, quartz,
polyethylene, PTFE, FEP, etc.) should be sufficiently
clean for the task objectives. Several procedures found to
provide clean labware include washing with a detergent
solution, rinsing with tap water, soaking for 4 h or more in
20% (v/v) nitric acid or a mixture of HCI and HNO3, rinsing
with reagent water and storing clean. Chromic acid
cleaning solutions must be avoided because chromium is
an analyte.
Note: Glassware having ground glass stoppers, etc.
should be avoided because the ground glass surface is
difficult to clean properly and can contain active sites
which adsorb metals.
6.5.1 Glassware — Volumetric flasks, graduated cylin-
ders, funnels and centrifuge tubes (glass and/or metal-
free plastic).
6.5.2 Assorted calibrated pipettes.
6.5.3 Griffin beakers, 250-mL with 75-mm watch
glasses and (optional) 75-mm ribbed watch glasses.
Revision 1.0 September 1997
200.12-6
-------
6.5.4 Narrow-mouth storage bottles, FEP (fluorinated
ethylene propylene) with screw closure, 125-mL to 1-L
capacities.
6.5.5 One-piece stem FEP wash bottle with screw clo-
sure, 125-mL capacity.
7.0 Reagents and Standards
7.7 Reagents may contain elemental impurities which
might affect analytical data. Only high-purity reagents that
conform to the American Chemical Society specifications6
should be used whenever possible. If the purity of a
reagent is in question, analyze for contamination. All acids
used for this method must be of ultra high-purity grade or
equivalent. Suitable acids are available from a number of
manufacturers. Redistilled acids prepared by sub-boiling
distillation are acceptable.
7.2
Nitric acid, concentrated (sp.gr. 1.41) HNO3.
7.2.1 Nitric acid (1+1) - Add 500 ml concentrated
HNO3 to 400 ml reagent water and dilute to 1 L.
7.2.2 Nitric acid (1+5) - Add 50 ml concentrated
HNO3 to 250 ml reagent water.
7.2.3 Nitric acid (1+9) - Add 10 ml concentrated
HNO3 to 90 ml reagent water.
7.3 Reagent water. All references to water in this
method refer to ASTM Type I grade water.7
7.4 Ammonium
(sp.gr.0.902).
hydroxide,
concentrated
7.5 Matrix Modifier, dissolve 300 mg palladium (Pd)
powder in concentrated HNO3 (1 ml of HNO3, adding 10
uL of concentrated HCI if necessary). Dissolve 200 mg of
Mg(NO3)2-6H2O in ASTM Type I water. Pour the two
solutions together and dilute to 100 ml with ASTM Type
I water.
Note: It is recommended that the matrix modifier be
analyzed separately in order to assess the contribution of
the modifier to the absorbance of calibration and reagent
blank solutions.
7.6 Standard stock solutions may be purchased or
prepared from ultra-high purity grade chemicals (99.99 to
99.999% pure). All compounds must be dried for 1 h at
105°C, unless otherwise specified. It is recommended
that stock solutions be stored in FEP bottles. Replace
stock standards when succeeding dilutions for prepara-
tion of calibration standards cannot be verified.
Caution: Many of these chemicals are extremely toxic if
inhaled or swallowed (Section 5.1). Wash hands thor-
oughly after handling.
Typical stock solution preparation procedures follow for
1-L quantities, but for the purpose of pollution prevention,
the analyst is encouraged to prepare smaller quantities
when possible. Concentrations are calculated based
upon the weight of the pure element or upon the weight
of the compound multiplied by the fraction of the analyte
in the compound.
From pure element,
Concentration = weight (mq)
volume (L)
From pure compound,
Concentration = weight (mq) x gravimetric factor
volume (L)
where:
gravimetric factor = the weight fraction of the analyte
in the compound.
7.6.7 Arsenic solution, stock, 1 ml = 1000 ug As: Dis-
solve 1.320 g of As2O3 (As fraction = 0.7574), weighed
accurately to at least four significant figures, in 100 ml of
reagent water containing 10.0 ml concentrated NH4OH.
Warm in solution gently to effect dissolution. Acidify the
solution with 20.0 ml concentrated HNO3 and dilute to
volume in a 1-L volumetric flask with reagent water.
7.6.2 Cadmium solution, stock, 1 ml = 1000 ug Cd:
Dissolve 1.000 g Cd metal, acid cleaned with (1+9) HNO3,
weighed accurately to at least four significant figures, in
50 ml (1+1) HNO3with heating to effect dissolution. Let
solution cool and dilute with reagent water in a 1-L
volumetric flask.
7.6.3 Chromium solution, stock, 1 mL = 1000 ug Cr:
Dissolve 1.923 g CrO3 (Cr fraction = 0.5200), weighed
accurately to at least four significant figures, in 120 mL (1
+5) HNO3. When solution is complete, dilute to volume in
a 1-L volumetric flask with reagent water.
7.6.4 Copper solution, stock, 1 mL = 1000 ug Cu: Dis-
solve 1.000 g Cu metal, acid cleaned with (1+9) HNO3,
200.12-7
Revision 1.0 September 1997
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weighed accurately to at least four significant figures, in
50.0 ml (1+1) HNO3 with heating to effect dissolution. Let
solution cool and dilute in a 1-L volumetric flask with
reagent water.
7.6.5 Lead solution, stock, 1 mL = 1000 ug Pb:
Dissolve 1.599 g Pb(NO3)2 (Pb fraction = 0.6256),
weighed accurately to at least four significant figures, in a
minimum amount of (1+1) HNO3. Add 20.0 mL (1+1)
HNO3 and dilute to volume in a 1-L volumetric flask with
reagent water.
7.6.6 Nickel solution, stock, 1 mL = 1000 ug Ni:
Dissolve 1.000 g of nickel metal, weighed accurately to at
least four significant figures, in 20.0 mL hot concentrated
HNO3, cool, and dilute to volume in a 1 -L volumetric flask
with reagent water.
7.6.7 Selenium solution, stock, 1 mL = 1000 ug Se:
Dissolve 1.405 g SeO2 (Se fraction = 0.7116), weighed
accurately to at least four significant figures, in 200 mL
reagent water and dilute to volume in a 1-L volumetric
flask with reagent water.
7.7 Preparation of Calibration Standards - Fresh
calibration standards (CAL Solution) should be prepared
weekly, or as needed. Dilute each of the stock standard
solutions to levels appropriate to the operating range of
the instrument using the appropriate acid diluent. The
element concentrations in each CAL solution should be
sufficiently high to produce good measurement precision
and to accurately define the slope of the response curve.
The instrument calibration should be initially verified using
a IPC sample (Section 7.9).
7.8 Blanks — Four types of blanks are required for
this method. A calibration blank is used to establish the
analytical calibration curve, the laboratory reagent blank
(LRB) is used to assess possible contamination from the
sample preparation procedure and to assess spectral
background, the laboratory fortified blank is used to
assess routine laboratory performance, and a rinse blank
is used to flush the instrument autosampler uptake sys-
tem. All diluent acids should be made from concentrated
acids (Section 7.2) and ASTM Type I water.
7.8.1 The calibration blank consists of the appropriate
acid diluent in ASTM Type I water. The calibration blank
should be stored in a FEP bottle.
7.8.2 The laboratory reagent blanks must contain all
the reagents in the same volumes as used in processing
the samples. The preparation blank must be carried
through the entire sample digestion and preparation
scheme.
7.8.3 The laboratory fortified blank (LFB) is prepared
by fortifying an aliquot of the laboratory reagent blank with
all analytes to provide a final concentration which will
produce an absorbance of approximately 0.1 for each
analyte. The LFB must be carried through the complete
procedure as used for the samples.
7.8.4 The rinse blank is a 0.1% HCI and 0.1% HNO3
solution used to flush the autosampler tip and is stored in
the appropriate plastic containers.
7.9 Instrument Performance Check (IPC) Solution -
The IPC solution is used to periodically verify instrument
performance during analysis. It should be prepared in the
same acid mixture as the calibration standards by com-
bining method analytes at appropriate concentrations to
approximate the midpoint of the calibration curve. The
IPC solution should be prepared from the same standard
stock solutions used to prepare the calibration standards
and stored in a FEP bottle. Agency programs may specify
or request that additional instrument performance check
solutions be prepared at specified concentrations in order
to meet particular program needs.
7.70 Quality Control Sample (QCS) - For initial and
periodic verification of calibration standards and instru-
ment performance, analysis of a QCS is required. The
QCS must be obtained from an outside source different
from the standard stock solutions and prepared in the
same acid mixture as the calibration standards. The
concentration of the analytes in the QCS solution should
be such that the resulting solution will provide an absor-
bance reading of approximately 0.1. The QCS solution
should be stored in a FEP bottle and analyzed as needed
to meet data-quality needs. A fresh solution should be
prepared quarterly or as needed.
7.11 Matrix Performance Check (MPC) - The MPC
solution is used to periodically evaluate the laboratory/
instrument performance in saline samples. It should be
prepared in the same acid mixture as the calibration
standards by combining method analytes at appropriate
concentrations in a seawater matrix (NASS-3, or its
equivalent) to produce an absorbance of 0.1. The MPC
solution should be prepared from the same standard
stock solutions used to prepare the calibration standards
and stored in a FEP bottle. The MPC sample should be
analyzed after every 10 samples to assure saline matrix
is not producing false positives.
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200.12-8
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8.0 Sample Collection, Preservation and
Storage
8.1 Prior to collection of an aqueous sample,
consideration should be given to the type of data required.
Acid preservation should be performed at the time of
sample collection or as soon thereafter as practically
possible. The pH of all aqueous samples must be tested
immediately prior to aliquoting for analysis to ensure the
sample has been properly preserved. If properly acid-
preserved, the sample can be held up to 6 months before
analysis.
8.2 For determination of total recoverable elements
in aqueous samples, acidify with (1+1) nitric acid at the
time of collection to pH<2. Normally, 3 ml of (1+1) nitric
acid (ultra high purity) per liter of sample is sufficient for
most ambient water samples. The sample should not be
filtered prior to analysis.
Note: Samples that cannot be acid-preserved at the
time of collection because of sampling limitations or
transport restrictions, or are > pH 2 because of high
alkalinity should be acidified with nitric acid to pH < 2 upon
receipt in the laboratory. Following acidification, the
sample should be held for 16 h and the pH verified to be
<2 before withdrawing an aliquot for sample processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container and acid as used in sample collec-
tion.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability, and the periodic
analysis of laboratory reagent blanks, fortified blanks and
other laboratory solutions as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
thus generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (determination of
linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) prior to samples being analyzed
by this method.
9.2.2 Linear dynamic range (LDR) - The upper limit of
the LDR must be established for the wavelength utilized
for each analyte by determining the signal responses
from a minimum of six different concentration standards
across the range, two of which are close to the upper limit
of the LDR. Determined LDRs must be documented and
kept on file. The linear calibration range which may be
used for the analysis of samples should be judged by the
analyst from the resulting data. The upper LDR limit
should be an observed signal no more than 10% below
the level extrapolated from the four lower standards. New
LDRs should be determined whenever there is a
significant change in instrument response, a change in
instrument analytical hardware or operating conditions.
Note: Multiple cleanout furnace cycles may be neces-
sary in order to fully define or utilize the LDR for certain
elements such as chromium. For this reason, the upper
limit of the linear calibration range may not correspond to
the upper operational LDR limit.
Measured sample analyte concentrations that exceed the
upper limit of the linear calibration range must either be
diluted and reanalyzed (with concern for memory effects
Section 4.4) or analyzed by another approved method.
9.2.3 Quality control sample (QCS) — When beginning
the use of this method, on a quarterly basis or as required
to meet data-quality needs, verify the calibration stan-
dards and acceptable instrument performance with the
preparation and analyses of a QCS (Section 7.10). If the
determined concentrations are not within ± 10% of the
stated values, performance of the determinative step of
the method is unacceptable. The source of the problem
must be identified and corrected before either proceeding
on with the initial determination of method detection limits
or continuing with ongoing analyses.
9.2.4 Method detection limit (MDL) - MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.8 To determine MDL
values, take seven replicate aliquots of the fortified
reagent water and process through the entire analytical
method. Perform all calculations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
200.12-9
Revision 1.0 September 1997
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MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1 degrees of
freedom [t = 3.14 for seven replicates].
S = standard deviation of the replicate analyses.
Note: If the percent relative standard deviation (% RSD)
from the analyses of the seven aliquots is < 15%, the
concentration used to determine the analyte MDL may
have been inappropriately high for the determination. If
so, this could result in calculation of an unrealistically low
MDL. If additional confirmation of the MDL is desired,
reanalyze the seven replicate aliquots on two more
nonconsecutive days and again calculate the MDL values
for each day. An average of the three MDL values for
each analyte may provide a more appropriate MDL
estimate. Concurrently, determination of MDL in reagent
water represents a best case situation and does not
reflect possible matrix effects of real world samples.
However, successful analyses of LFMs (Section 9.4) and
the analyte addition test described in Section 9.5.1 can
give confidence to the MDL value determined in reagent
water. Typical single laboratory MDL values using this
method are given in Table 2.
MDLs should be determined every six months, when a
new operator begins work or whenever there is a signifi-
cant change in the background or instrument response.
The MDLs reported in Table 2 were determined in forti-
fied NASS-3 samples. It is recommended that a certified
saline matrix such as NASS-3 be used to determine
MDLS.
9.3 Assessing Laboratory Performance (Mand-
atory)
9.3.1 Laboratory reagent blank (LRB) - The laboratory
must analyze at least one LRB (Section 7.8.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and the
samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
9.3.2 Laboratory fortified blank (LFB) - The laboratory
must analyze at least one LFB (Section 7.8.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that
analyte is judged out of control, and the source of the
problem should be identified and resolved before
continuing analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required
control limits of 85-115%. When sufficient internal perfor-
mance data become available (usually a minimum of 20-
30 analyses), optional control limits can be developed
from the percent mean recovery (x) and the standard
deviation (S) of the mean recovery. These data can be
used to establish the upper and lower control limits as
follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most recent 20-30 data
points. Also, the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.3.4 Instrument performance check (IPC) solution -
For all determinations the laboratory must analyze the
IPC solution (Section 7.9) and a calibration blank
immediately following daily calibration, after every tenth
sample (or more frequently, if required) and after the last
sample in the batch is analyzed. Analysis of the I PC
solution and calibration blank immediately following
calibration must verify that the instrument is within ± 5% of
calibration. Subsequent analyses of the IPC solution must
verify the calibration within ± 10%. If the calibration cannot
be verified within the specified limits, reanalyze the IPC
solution. If the second analysis of the IPC solution con-
firms calibration to be outside the limits, sample analysis
must be discontinued, the cause determined and/or, in
the case of drift, the instrument recalibrated. All samples
following the last acceptable IPC solution must be reana-
lyzed. Data for the calibration blank and IPC solution
must be kept on file with associated sample data.
9.3.5 Matrix performance check (MFC) solution — For
all determinations, the laboratory must analyze the MPC
solution (Section 7.11) immediately following daily cali-
bration, after every tenth sample (or more frequently, if
required) and after the last sample in the batch is ana-
lyzed. Analysis of the MPC must verify that the instrument
Revision 1.0 September 1997
200.12-10
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is within ± 15% of calibration and confirm that the matrix
is not causing matrix/background interferences. If the
MPC is not within ± 15%, reanalyze the MPC solution. If
the second analysis of the MPC solution is outside the
limits, sample analysis must be discontinued the cause
determined and/or, in the case of drift, the instrument
recalibrated. All samples following the last acceptable
MPC solution must be reanalyzed. The analysis data for
the calibration blank and MPC solution must be kept on
file with the sample analyses data.
9.4 Assessing Analyte Recovery and Data Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and the
data quality. Taking separate aliquots from the sample for
replicate and fortified analyses can in some cases assess
these effects. Unless otherwise specified by the data user,
laboratory or program, the following laboratory fortified
matrix (LFM) procedure (Section 9.4.2) is required. Also,
the analyte addition test (Section 9.5.1) can aid in
identifying matrix interferences. However, all samples
must have a background absorbance < 1.0 before the
test results obtained can be considered reliable.
9.4.2 The laboratory must add a known amount of
each analyte to a minimum of 10% of the routine
samples. In each case the LFM aliquot must be a
duplicate of the aliquot used for sample analysis and for
total recoverable determinations added prior to sample
preparation. For water samples, the added analyte
concentration must be the same as that used in the
laboratory fortified blank (Section 9.3.2).
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is less than
25% of the unfortified sample concentration. Percent
recovery may be calculated in units appropriate to the
matrix, using the following equation:
R= C,-C x100
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte
added to sample.
9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range (but is still within the
range of calibration and the background absorbance is <
1.0 abs.) and the laboratory performance for that analyte
is shown to be in control (Section 9.3), the recovery
problem encountered with the LFM is judged to be either
matrix or solution related, not system related. A flowchart
of the remainder of this section can be found in Figure 6.
This flowchart may clarify the verbal discussion given
below.
If the background absorbance is > 1 abs., the sample and
the LFM should be diluted 1:3 and reanalyzed until the
background absorbance is < 1, at which point a percent
recovery of the LFM should be calculated. If the fortified
analyte in the diluted LFM is found to be < 25% of the
sample concentration or the diluted LFM produces an
atomic signal of <10 times the MDL, the diluted sample
should be analyzed by methods of standard addition. If
the calculated recovery of the diluted sample is within the
designated range, the sample concentration should be
calculated from the diluted sample. If the calculated
recovery of the diluted sample is outside the designated
range, follow the directions given below. If the back-
ground is reduced and/or the matrix effect is reduced by
dilution, all samples of a similar matrix should be diluted
and analyzed in a similar fashion. The result should be
flagged indicating the methods sensitivity has been re-
duced by the dilution. If dilution is unacceptable because
of data quality objectives the sample should be flagged
indicating the analysis is not possible via this analytical
procedure.
If the analyte recovery on the LFM is <75% and the
background absorbance is <1, complete the analyte
addition test (Section 9.5.1) on the original sample (or its
dilution). The results of the test should be evaluated as
follows:
1. If recovery of the analyte addition test (< 85%)
confirms a low recovery for the LFM, a suppressive
matrix interference is indicated and the unfortified
sample aliquot must be analyzed by method of
standard additions (Section 11.3).
2. If the recovery of the analyte addition test is between
85% to 115%, a low recovery of the analyte in the
LFM (< 75%) may be related to the heterogeneity of
the sample, sample preparation or a poor transfer,
etc. Report the sample concentration based on the
unfortified sample aliquot.
200.12-11
Revision 1.0 September 1997
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3. If the recovery of the analyte addition test is less
than recovery calculated for the LFM, matrix sup-
pression is confirmed. The unfortified sample
should be analyzed by MSA (Section 11.3).
Significantly lower recoveries (relative to the LFM)
associated with the analyte addition test are
unlikely unless the sample is heterogeneous.
4. If the recovery of the analyte addition test is >115%,
the dramatic change in analyte response should be
verified by fortifying the LFM. The recovery in the
sample and the recovery in the LFM should be
compared. If the recoveries verify the dramatic
response difference, the sample results should be
flagged indicating the sample matrix is not homoge-
neous.
If the analyte recovery in the LFM is > 125% and the
background absorbance is < 1, complete the analyte
addition test (Section 9.5.1) on the unfortified sample (or
its dilution) aliquot.
1 . If the percent recovery of the analyte addition test is
> 115% and the LFB does not indicate laboratory
contamination, an enhancing matrix interference
(albeit rare) is indicated, and the unfortified sample
aliquot must be analyzed by method of standard
additions (Section 11.3).
2. If the percent recovery of the analyte addition test is
between 85% to 115%, either random sample con-
tamination of the LFM, an incorrect analyte concen-
tration was added to the LFM prior to sample
preparation, or sample heterogeneity should be
suspected. Report analyte data determined from the
analysis of the unfortified sample aliquot.
3. If the percent recovery of the analyte addition test is
< 85%, a heterogeneous sample with matrix inter-
ference is suspected. This dramatic change in re-
sponse should be verified by performing the analyte
addition test to the LFM. The recovery in the sample
and the recovery in the LFM should be compared. If
the recoveries verify the dramatic response differ-
ence the sample results should be flagged indicating
the sample matrix is not homogeneous.
9.4.5 If the analysis of a LFM sample(s) and the test
routines above indicate an operative interference and the
LFMs are typical of the other samples in the batch, those
samples that are similar must be analyzed in the same
manner as the LFMS. Also, the data user must be
informed when a matrix interference is so severe that it
prevents successful determination of the analyte or when
the heterogeneous nature of the sample precludes the
use of duplicate analyses.
9.4.6 Where reference materials are available, they
should be analyzed to provide additional performance
data. Analysis of reference samples is a valuable tool for
demonstrating the ability to perform the method accept-
ably. It is recommended that NASS-3 or its equivalent be
fortified and used as an MPC.
9.5 Matrix interference effects and the need for MSA
can be assessed by the following test. Directions for using
MSA are given in Section 11.3.
9.5.1 Analyte addition test: An analyte standard added
to a portion of a prepared sample or its dilution should be
recovered to within 85-115% of the known value. The
analyte addition should occur directly to sample in the
furnace and should produce a minimum absorbance of
0.1. The concentration of the analyte addition plus that in
the sample should not exceed the linear calibration range
of the analyte. If the analyte is not recovered within the
specified limits, a matrix effect should be suspected and
the sample must be analyzed by MSA.
10.0 Calibration and Standardization
10.1 Specific wavelengths and instrument operating
conditions are listed in Table 1. However, because of
differences among makes and models of spectropho-
tometers and electrothermal furnace devices, the actual
instrument conditions selected may vary from those listed.
70.2 Prior to the use of this method, the instrument
operating conditions must be optimized. The analyst
should follow the instructions provided by the manufac-
turer while using the conditions listed in Table 1 as a
guide. The appropriate charring condition for each of the
analytes is a critical part of the metal analysis in saline
waters; therefore, the char temperature profiles should be
determined in a saline water matrix. The appropriate
charring temperature should be chosen so as to minimize
background absorbance while providing some furnace
temperature variation without the loss of analyte. For
analytical operation, the charring temperature is usually
set at least 100°C below the point at which analyte begins
to be lost during the char. Because the background
absorbance can be affected by the atomization tempera-
ture, care should be taken in the choice of an appropriate
atomization temperature. The optimum conditions se-
Revision 1.0 September 1997
200.12-12
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lected should provide the lowest reliable MDLs and be
similar to those listed in Table 2. Once the optimum
operating conditions are determined, they should be
recorded and available for daily reference. The effective-
ness of these operating conditions are continually evalu-
ated by analyzing the MPC.
70.3 Prior to an initial calibration the linear dynamic
range of the analyte must be determined (Sect 9.2.2)
using the optimized instrument operating conditions. For
all determinations allow an instrument and hollow cath-
ode lamp warm-up period of not less than 15 min. If an
EDL is to be used, allow 30 min for warm-up.
70.4 Before using the procedure (Section 11.0) to ana-
lyze samples, there must be data available documenting
initial demonstration of performance. The required data
and procedure are described in Section 9.2. This data
must be generated using the same instrument operating
conditions and calibration routine to be used for sample
analysis. These documented data must be kept on file
and be available for review by the data user.
11.0 Procedure
77.7 Aqueous Sample Preparation - Total Re-
coverable Analytes
11.1.1 Add 2 ml (1+1) nitric acid to the beaker
containing 100 ml of sample. Place the beaker on a hot
plate for solution evaporation. The hot plate should be
located in a fume hood and previously adjusted to provide
evaporation at a temperature of approximately but no
higher than 85°C. (See the following note.) The beaker
should be covered with an elevated watch glass or other
necessary steps should be taken to prevent sample
contamination from the fume hood environment.
Note: For proper heating adjust the temperature control
of the hot plate such that an uncovered Griffin beaker
containing 50 ml of water placed in the center of the hot
plate can be maintained at a temperature approximately
but no higher than 85°C. (Once the beaker is covered
with a watch glass the temperature of the water will rise
to approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20 ml by gentle heating at 85°C. DO NOT BOIL.
This step takes about 2 h for a 100-mL aliquot with the
rate of evaporation rapidly increasing as the sample
volume approaches 20 ml. (A spare beaker containing
20 ml of water can be used as a gauge.)
77.7.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the
sample for 30 min.
77.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
77.7.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight the
sample contains suspended solids, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination from
filtration.) The sample is now ready for analysis. Because
the effects of various matrices on the stability of diluted
samples cannot be characterized, all analyses should be
performed as soon as possible after the completed
preparation.
77.2 Sample Analysis
11.2.1 Prior to daily calibration of the instrument, in-
spect the graphite tube and contact rings for salt
buildup, etc. Generally, it will be necessary to clean the
contact rings and replace the graphite tube daily. The
contact rings are a cooler environment in which salts
can deposit after atomization. A cotton swab dipped in a
50/50 mixture of isopropyl alcohol (IPA) and H2O (such
that it is damp but not dripping) can be used to remove
the majority of the salt buildup. A second cotton swab is
dipped in IPA and the contact rings are wiped down to
assure they are clean. The rings are then allowed to
thoroughly dry and then a new tube is placed in the
furnace and conditioned according to instrument
manufacturer's specifications.
77.2.2 Configure the instrument system to the selected
optimized operating conditions as determined in Sections
10.1 and 10.2.
77.2.3 Before beginning daily calibration the instrument
should be reconfigured to the optimized conditions. Ini-
tiate the data system and allow a period of not less than
15 min for instrument and hollow cathode lamp warm up.
If an EDL is to be used, allow 30 min for warm up.
11.2.4 After the warm up period but before calibration,
instrument stability must be demonstrated by analyzing a
standard solution with a concentration 20 times the IDL a
minimum of five times. The resulting relative standard
deviation of absorbance signals must be < 5%. If the
200.12-13
Revision 1.0 September 1997
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relative standard deviation is > 5%, determine and correct
the cause before calibrating the instrument.
712.5 For initial and daily operation, calibrate the in-
strument according to the instrument manufacturer's
recommended procedures using the calibration blank
(Section 7.8.1) and calibration standards (Section 7.7)
prepared at three or more concentrations within the
usable linear dynamic range of the analyte (Sections 4.4
and 9.2.2).
11.2.6 An autosampler must be used to introduce all
solutions into the graphite furnace. Once the sample and
the matrix modifier are injected, the furnace controller
completes a set of furnace cycles and a cleanout period
as programmed. Analyte signals must be reported on an
integrated absorbance basis. Background absorbances,
background heights and the corresponding peak profiles
should be displayed to the CRT for review by the analyst
and be available as hard copy for documentation to be
kept on file. Flush the autosampler solution uptake sys-
tem with the rinse blank (Section 7.8.4) between each
solution injected.
11.2.7 After completion of the initial requirements of
this method (Section 9.2), samples should be analyzed
in the same operational manner used in the calibration
routine.
11.2.8 During sample analyses, the laboratory must
comply with the required quality control described in
Sections 9.3 and 9.4.
11.2.9 For every newer unusual matrix, when practical,
it is highly recommended that an inductively coupled
plasma atomic emission spectrometer be used to screen
for high element concentration. Information gained from
this may be used to prevent potential damage to the
instrument and to better estimate which elements may
require analysis by graphite furnace.
11.2.10 Determined sample analyte concentrations that
are > 90% of the upper limit of calibration must either
be diluted with acidified reagent water and reanalyzed
with concern for memory effects (Section 4.4), or
determined by another approved but less sensitive
procedure. Samples with background absorbances > 1
must be diluted with appropriate acidified reagent water
such that the background absorbance is < 1 (Section
9.4.4). If the method of standard additions is required,
follow the instructions described in Section 11.3.
11.2.11 When it is necessary to assess an operative
matrix interference (e.g., signal reduction due to high
dissolved solids), the test described in Section 9.5 is
recommended.
11.2.12 Report data as directed in Section 12.
11.3 Standard Additions - If the method of standard
addition is required, the following procedure is recom-
mended:
11.3.1 The standard addition technique9 involves pre-
paring new standards in the sample matrix by adding
known amounts of standard to one or more aliquots of the
processed sample solution. This technique compensates
for a sample constituent that enhances or depresses the
analyte signal, thus producing a different slope from that
of the calibration standards. It will not correct for additive
interference, which causes a baseline shift. The simplest
version of this technique is the single-addition method.
The procedure is as follows: Two identical aliquots of the
sample solution, each of volume Vx, are taken. To the first
(labeled A) is added a small volume Vs of a standard
analyte solution of concentration Cs. To the second
(labeled B) is added the same volume Vs of the solvent.
The analytical signals of A and B are measured and
corrected for nonanalyte signals. The unknown sample
concentration Cx is calculated:
c =-
(sA-sB)vx
where, SA and SB are the analytical signals (corrected
for the blank) of solutions A and B, respectively. Vs and
Cs should be chosen so that SA is roughly twice SB on
the average. It is best if Vs is made much less than Vx,
and thus Cs is much greater than Cx, to avoid excess
dilution of the sample matrix. If a separation or
concentration step is used, the additions are best made
first and carried through the entire procedure. For the
results from this technique to be valid, the following
limitations must be taken into consideration:
1. The analytical curve must be linear.
2. The chemical form of the analyte added must re-
spond in the same manner as the analyte in the
sample.
3. The interference effect must be constant over the
working range of concern.
Revision 1.0 September 1997
200.12-14
-------
4. The signal must be corrected for any additive inter-
ference.
12.0 Data Analysis and Calculations
72.7 Sample data should be reported in units of//g/L
for aqueous samples.
72.2 For total recoverable aqueous analytes (Section
11.1), when 100-mL aliquot is used to produce the 100
ml final solution, round the data to the tenths place and
report the data in //g/L up to three significant figures. If a
different aliquot volume other than 100 ml is used for
sample preparation, adjust the dilution factor accordingly.
Also, account for any additional dilution of the prepared
sample solution needed to complete the determination of
analytes exceeding the upper limit of the calibration curve.
Do not report data below the determined analyte MDL
concentration or below an adjusted detection limit
reflecting smaller sample aliquots used in processing or
additional dilutions required to complete the analysis.
72.3 The QC data obtained during the analyses
provide an indication of the quality of the sample data and
should be provided with the sample results.
13.0 Method Performance
73.7 Instrument operating conditions used for single
laboratory testing of the method and MDLs are listed in
Tables 1 & 2.
73.2 Table 3 contains precision and recovery data ob-
tained from a single laboratory analysis of four fortified
sample replicates of NASS-3. Five unfortified replicates
were analyzed, and their average concentration was used
to determine the sample concentration. Samples were
prepared using the procedure described in Section 11.1.
Four samples were fortified at the levels reported in Table
3. Average percent recovery and percent relative
standard deviation are reported in Table 3 for the fortified
samples.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the
next best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202)872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section 14.2.
16.0 References
1. Pruszkowska, E., G. Carnrick, and W. Slavin. Anal.
Chem. 55,182-186,1983.
2. Carcinogens - Working With Carcinogens,
Department of Health, Education, and Welfare,
Public Health Service, Centers for Disease Control,
National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
200.12-15
Revision 1.0 September 1997
-------
4. Safety in Academic Chemistry Laboratories,
American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. Proposed OSHA Safety and Health Standards,
Laboratories, Occupational Safety and Health
Administration, Federal Register, July 24,1986.
6. Rohrbough, W.G. et al. Reagent Chemicals,
American Chemical Society Specifications, 7th
edition. American Chemical Society, Washington,
DC, 1986.
7. American Society for Testing and Materials.
Standard Specification for Reagent Water, D1193-
77. Annual Book of ASTM Standards, Vol. 11.01.
Philadelphia, PA, 1991.
8. Code of Federal Regulations 40, Ch. 1, Pt. 136,
Appendix B.
9. Winefordner, J.D., Trace Analysis: Spectroscopic
Methods for Elements, Chemical Analysis, Vol. 46,
pp. 41-42, 1976.
Revision 1.0 September 1997 200.12-16
-------
17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Furnace Conditions for Determination of Metals in Seawater1
Element
As
Cd
Cr
Cu
Ni
Pb
Se
Wavelength (nml
Slit Width (nm)
193.7
0.7
228.8
0.7
357.9
0.7
324.8
0.7
232.4
0.2
283.3
0.7
196.0
2.0
Method of
Analysis
Direct
Matrix Match
Standard
or
Std. Addition
Direct
Direct
Direct
Direct
Matrix Match
Standard
or
Std. Addition
Modifier 2'3
Pd/Mg
Pd/Mg
+
600 fj.g
NH4N03
Pd/Mg
Pd/Mg
Pd/Mg
Pd/Mg
Pd/Mg
9% HNO3on
Platform
Furnaces5
Cycle
Dry
Char
Atomization
Dry
Char!
Char 2
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Dry
Char
Atomization
Temp
°C
130
14004
2200
130
350
850
1500
130
1500
2600
130
1300
2600
130
14004
2600
130
1200
2200
130
1000
2100
Temp
Ramp
1
10
0
1
45
1
0
1
5
0
1
10
0
1
10
0
1
10
0
1
5
0
Hold Time (sec)
60
60
5
60
30
30
5
60
30
5
60
30
5
60
30
7
60
45
5
60
60
5
10-,uL sample size.
2 5/j.L of (30 mg Pd Powder and 20 mg Mg(NO3)2-6H2O to 10 ml).
3 A gas mixture of 5% H2 in 95% Ar is used during the dry and char.
4 Sodium emission is visibly exiting from the sample inlet port.
5 The furnace program has a cool down step of 20° between char and atomization and a clean out step of 2600° C after atomization.
Table 2. MDLs and Background Absorbances Associated with a Fortified NASS-31"3
Element
Cd
Cr
Cu
Ni
Pb
Se4
As4
MDL5
//g/L
0.1
2.8
1.8
2.4
9.5
2.6
Typical
Integrated
Background
Absorbances6
1.2
0.2
0.2
0.1
0.4
1.4
0.3
1 Matrix Modifier = 0.015 mg Pd + 0.01 mg Mg(NO3)2.
2 A 5% H2 in Ar gas mix is used during the dry and char steps at 300 mL/min for all elements.
3 10-//L sample size.
4 An electrodeless discharge lamp was used for this element.
5 MDL calculated based on fortifying NASS-3 with metal analytes.
6 Background absorbances are affected by the atomization temperature for analysis, therefore, lowering atomization temperatures may be
advantageous if large backgrounds are observed.
- Not Determined.
200.12-17
Revision 1.0 September 1997
-------
Table 3. Precision and Recovery Data for Fortified NASS-3
Element
As
Cd1
Cr
Cu
Pb
Ni
Se1
Certified
Value
1.65 ±0.1 9
0.029 ± 0.004
0.1 75 ±0.010
0.1 09 ±0.011
0.039 ± 0.006
0.257 ± 0.027
0.024 ± 0.004
Observed
Value
-------
3.221 -I
•
•
i • »
dL
Time (sec)
Figure 2. Pb atomization Profile Utilizing a 800° Char.
Current Atomic
Current Backgrd
5.00
13
03
£5
en
I
CO
no
100
90
80
70
60
50
40
30
+ Pb
All Samples Fortified with 5 ul of Standard
0246
Microliters of Fortified NASS-3
Figure 3. Normalized Integrated Absorbance vs. Microliters of Fortified NASS-3.
10
200.12-19
Revision 1.0 September 1997
-------
s
<
T3
I
110
105
100
95
90
85
80
75
70
5 jul of a Cd Standard Added
+NASS-3
Microliters of Matrix
Figure 4. Cd Response in NASS-3 and 10,000 ppm NaCI.
8
_o
<
-a
I
110
100
90
80
70
60
50
5 pi of Se Standard Added
+ Seawater
4 6
Microliters of Matrix
10
Figure 5. Se Response in Seawater vs 10,000 ppm NaCI
Revision 1.0 September 1997
200.12-20
-------
(1) Poor Transfer
(2) Sample Heterogeneity
(3) Digestion/Precipitation
(4) Matrix Suppression/Enhancements
(5) Contamination
IFA = In Furnace Analyte Addition
Report Results on Diluted Sample
No
% Recovery
85-115
Yes
Calculate %
Recovery
Background
Absorbance
< 1.0 abs
Reanalyze
Dilute 1:3
Sample & LFM
No
Background
Absorbance
< 1.0 abs
Start
No
Yes
Report Results on
Unfortified Sample
85% < IFA < 115
IFAs > LFM/ Compare /IFA = LFM
MSA -^ < Recoveries \ >• MSA
IFAs to LFM/IFA > 115
Fortified x
Cone
<10MDL
or
< 25% Sample/
\ Cone
Yes
Suspected Matrix
Interference
Recovery
75% > LFM > 125%
MSA
\/
IFA Analysis
on
Sample
See 9.5
Yes
LFM>125%
LFM < 75%
^
Yes
r
IFA Analysis
on
Sample
See 9.5
IFAs < LFM/ compare \ IFA < 85
MSA *4 / Recoveries > ^ MSA
\IFAstoLFM
(4)
I FAs > 85%
3
Report Results on Unfortified Sample
Figure 6. Matrix Interference Flowchart.
200.12-21
Revision 1.0 September 1997
-------
Method 200.13
Determination of Trace Elements in Marine Waters by Off-Line
Chelation Preconcentration with Graphite Furnace Atomic Absorption
John T. Creed and Theodore D. Martin
Chemical Expsoure Research Branch
Human Exposure Research Division
Revision 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
200.13-1
-------
Method 200.13
Determination of Trace Elements in Marine Waters by Off-Line Chelation
Preconcentration with Graphite Furnace Atomic Absorption
1.0 Scope and Application
1.1 This method describes procedures for pre-
concentration and determination of total recoverable trace
elements in marine waters, including estuarine water,
seawater and brines.
1.2 Acid solubilization is required prior to determina-
tion of total recoverable elements to facilitate breakdown
of complexes or colloids which might influence trace
element recoveries. This method should only be used for
preconcentration and determination of trace elements in
aqueous samples.
1.3 This method is applicable to the following
elements:
Element
Chemical Abstracts Service
Registry Numbers (CASRN)
Cadmium
Cobalt
Copper
Lead
Nickel
(Cd)
(Co)
(Cu)
(Pb)
(Ni)
7440-43-9
7440-48-4
7440-50-8
7439-92-1
7440-02-0
1.4 Method detection limits (MDLs) for these
elements will be dependent on the specific
instrumentation employed and the selected operating
conditions. MDLs in NASS-3 (Reference Material,
National Research Council of Canada) were determined
using the procedure described in Section 9.2.4 and are
listed in Table 1.
1.5 A minimum of 6-months experience in graphite
furnace atomic absorption (GFAA) is recommended.
2.0 Summary of Method
2.1 Nitric acid is dispensed into a beaker containing
an accurately weighed or measured, well-mixed,
homogeneous aqueous sample. The sample volume is
reduced to approximately 20 ml and then covered and
allowed to reflux. The resulting solution is diluted to
volume and is ready for analysis.
2.2 This method is used to preconcentrate trace ele-
ments using an iminodiacetate functionalized chelating
resin.1'2 Following acid solubilization, the sample is buff-
ered using an on-line system prior to entering the chelat-
ing column. Group I and II metals, as well as most
anions, are selectively separated from the analytes by
elution with ammonium acetate at pH 5.5. The analytes
are subsequently eluted into a simplified matrix consisting
of 0.75 M nitric acid and are determined by GFAA.
3.0 Definitions
3.1 Calibration Blank (CB) — A volume of reagent
water fortified with the same matrix as the calibration
standards, but without the analytes, internal standards, or
surrogate analytes.
3.2 Calibration Standard (CAL) - A solution pre-
pared from the primary dilution standard solution or stock
standard solutions and the internal standards and surro-
gate analytes. The CAL solutions are used to calibrate
the instrument response with respect to analyte concen-
tration.
3.3 Field Reagent Blank (FRB) - An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling
site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.4 Instrument Performance Check Solution (IPC)
- A solution of one or more method analytes, surrogates,
internal standards, or other test substances used to
evaluate the performance of the instrument system with
respect to a defined set of criteria.
3.5 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology
Revision 1.0 September 1997
200.13-2
-------
is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.6 Laboratory Fortified Sample Matrix (LFM) - -
An aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
3.7 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
the reagents, or the apparatus.
3.8 Linear Dynamic Range (LDR) - The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.9 Matrix Modifier (MM) - A substance added to
the instrument along with the sample in order to minimize
the interference effects by selective volatilization of either
analyte or matrix components.
3.70 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified, mea-
sured and reported with 99% confidence that the analyte
concentration is greater than zero.
3.77 Quality Control Sample - A solution of method
analytes of known concentrations which is used to fortify
an aliquot of LRB or sample matrix. The QCS is obtained
from a source external to the laboratory and different
from the source of calibration standards. It is used to
check laboratory performance with externally prepared
test materials.
3.72 Standard Addition - The addition of a known
amount of analyte to the sample in order to determine the
relative response of the detector to an analyte within the
sample matrix. The relative response is then used to
assess either an operative matrix effect or the sample
analyte concentration.
3.73 Stock Standard Solution (SSS) - A concen-
trated solution containing one or more method analytes
prepared in the laboratory using assayed reference ma-
terials or purchased from a reputable commercial source.
3.74 Total Recoverable Analyte (TRA) - The con-
centration of analyte determined to be in either a solid
sample or an unfiltered aqueous sample following treat-
ment by refluxing with hot dilute mineral acid(s) as
specified in the method.
4.0 Interferences
4.7 Several interference sources may cause
inaccuracies in the determination of trace elements by
GFAA. These interferences can be classified into three
major subdivisions: spectral, matrix, and memory. Some
of these interferences can be minimized via the pre-
concentration step, which reduces the Ca, Mg, Na and Cl
concentration in the sample prior to GFAA analysis.
4.2 Spectral interferences are caused by absorbance
of light by a molecule or atom which is not the analyte of
interest or emission from black body radiation.
4.2.1 Spectral interferences caused by an element only
occur if there is a spectral overlap between the wave-
length of the interfering element and the analyte of
interest. Fortunately, this type of interference is relatively
uncommon in STPGFAA (Stabilized Temperature Plat-
form Graphite Furnace Atomic Absorption) because of
the narrow atomic line widths associated with STPGFAA.
In addition, the use of appropriate furnace temperature
programs and high spectral purity lamps as light sources
can minimize the possibility of this type of interference.
However, molecular absorbances can span several hun-
dred manometers, producing broadband spectral inter-
ferences. This type of interference is far more common
in STPGFAA. The use of matrix modifiers, selective
volatilization, and background correctors are all attempts
to eliminate unwanted nonspecific absorbance. Because
the nonspecific component of the total absorbance can
vary considerably from sample type to sample type, to
provide effective background correction and eliminate the
elemental spectral interference of palladium on copper
and iron on selenium, the exclusive use of Zeeman
background correction is specified in this method.
4.2.2 Spectral interferences are also caused by emis-
sions from black body radiation produced during the
atomization furnace cycle. This black body emission
200.13-3
Revision 1.0 September 1997
-------
reaches the photomultiplier tube, producing erroneous
results. The magnitude of this interference can be mini-
mized by proper furnace tube alignment and monochro-
mator design. In addition, atomization temperatures
which adequately volatilize the analyte of interest without
producing unnecessary black body radiation can help re-
duce unwanted background emission produced during
atomization.
4.3 Matrix interferences are caused by sample com-
ponents which inhibit formation of free atomic analyte
atoms during the atomization cycle. In this method the
use of a delayed atomization device which provides
warmer gas phase temperatures is required. These
devices provide an environment which is more conducive
to the formation of free analyte atoms and thereby
minimize this type of interference. This type of interfer-
ence can be detected by analyzing the sample plus a
sample aliquot fortified with a known concentration of the
analyte. If the determined concentration of the analyte
addition is outside a designated range, a possible matrix
effect should be suspected (Section 9.4).
4.4 Memory interferences result from analyzing a
sample containing a high concentration of an element
(typically a high atomization temperature element) which
cannot be removed quantitatively in one complete set of
furnace steps. The analyte which remains in the furnace
can produce false positive signals on subsequent
sample(s). Therefore, the analyst should establish the
analyte concentration which can be injected into the
furnace and adequately removed in one complete set of
furnace cycles. If this concentration is exceeded, the
sample should be diluted and a blank analyzed to assure
the memory effect has been eliminated before reanalyz-
ing the diluted sample.
4.5 Low recoveries may be encountered in the
preconcentration cycle if the trace elements are
complexed by competing chelators (humic/fulvic) in the
sample or are present as colloidal material. Acid solubi-
lization pretreatment is employed to improve analyte
recovery and to minimize adsorption, hydrolysis and
precipitation effects.
4.6 Memory interferences from the chelating system
may be encountered, especially after analyzing a sample
containing high analyte concentrations. A thorough col-
umn rinsing sequence following elution of the analytes is
necessary to minimize such interferences.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible for
maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in
this method.3"6 A reference file of material data handling
sheets should also be made available to all personnel
involved in the chemical analysis. Specifically,
concentrated nitric and hydrochloric acids present various
hazards and are moderately toxic and extremely irritating
to skin and mucus membranes. Use these reagents in a
fume hood whenever possible and if eye or skin contact
occurs, flush with large volumes of water. Always wear
safety glasses or a shield for eye protection, protective
clothing and observe proper mixing when working with
these reagents.
5.2 Acidification of samples containing reactive mate-
rials may result in release of toxic gases, such as cya-
nides or sulfides. Samples should be acidified in a fume
hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 The graphite tube during atomization emits in-
tense UV radiation. Suitable precautions should be taken
to protect personnel from such a hazard.
5.5 The use of the argon/hydrogen gas mixture
during the dry and char steps may evolve a considerable
amount of HCI gas. Therefore, adequate ventilation is
required.
5.6 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
6.0 Equipment and Supplies
6.1 Graphite Furnace Atomic Absorption
Spectrometer
6.1.1 The GFAA spectrometer must be capable of
programmed heating of the graphite tube and the
Revision 1.0 September 1997
200.13-4
-------
associated delayed atomization device. The instrument
should be equipped with an adequate background
correction device capable of removing undesirable non-
specific absorbance over the spectral region of interest.
The capability to record relatively fast (< 1 sec) transient
signals and evaluate data on a peak area basis is
preferred. In addition, a recirculating refrigeration unit is
recommended for improved reproducibility of furnace
temperatures. The data shown in the tables were
obtained using the stabilized temperature platform and
Zeeman background correction.
6.12 Single element hollow cathode lamps or single
element electrodeless discharge lamps along with the
associated power supplies.
6.1.3 Argon gas supply (high-purity grade, 99.99%).
6.14 A 5% hydrogen in argon gas mix and the
necessary hardware to use this gas mixture during
specific furnace cycles.
6.1.5 Autosampler- Although not specifically required,
the use of an autosampler is highly recommended.
6.16 Graphite Furnace Operating Conditions — A
guide to experimental conditions for the applicable
elements is provided in Table 1.
6.2 Preconcentration System - System containing
no metal parts in the analyte flow path, configured as
shown with a sample loop in Figure 1 and without a
sample loop in Figure 2.
6.2.1 Column - Macroporous iminodiacetate chelating
resin (Dionex Metpac CC-1 or equivalent).
6.2.2 Control valves — Inert double stack, pneumati-
cally operated four-way slider valves with connectors.
6.2.2.1 Argon gas supply regulated at 80-100 psi.
6.2.3 Solution reservoirs - Inert containers, e.g., high
density polyethylene (HOPE), for holding eluent and
carrier reagents.
6.2.4 Tubing - High pressure, narrow bore, inert tubing
such as Tefzel ETFE (ethylene tetra-fluoro ethylene) or
equivalent for interconnection of pumps/ valve assemblies
and a minimum length for connection of the pre-
concentration system with the sample collection vessel.
6.2.5 Eluent pumping system (Gradient Pump) - Pro-
grammable flow, high-pressure pumping system, capable
of delivering either one of three eluents at a pressure up
to 2000 psi and a flow rate of 1-5 mL/min.
6.2.6 System
Figure 1).
setup, including sample loop (See
6.2.6.1 Sample loop — 10-mL loop constructed from
narrow bore, high-pressure inert tubing, Tefzel ETFE or
equivalent.
6.2.6.2 Auxiliary pumps — On-line buffer pump, piston
pump (Dionex QIC pump or equivalent) for delivering 2M
ammonium acetate buffer solution; carrier pump, peri-
staltic pump (Gilson Minipuls or equivalent) for delivering
1% nitric acid carrier solution; sample pump, peristaltic
pump for loading sample loop.
6.2.7 System
Figure 2).
setup without sample loop (See
6.2.7'A Auxiliary Pumps - Sample pump (Dionex QIC
Pump or equivalent) for loading sample on the column.
Carrier pump (Dionex QIC Pump or equivalent) used to
flush collection line between samples.
6.3 Labware - For determination of trace elements,
contamination and loss are of critical consideration.
Potential contamination sources include improperly
cleaned laboratory apparatus and general contamination
within the laboratory environment. A clean laboratory
work area, designated for trace element sample handling
must be used. Sample containers can introduce positive
and negative errors in determination of trace elements by
(1) contributing contaminants through surface desorption
or leaching and (2) depleting element concentrations
through adsorption processes. For these reasons, boro-
silicate glass is not recommended for use with this
method. All labware in contact with the sample should be
cleaned prior to use. Labware may be soaked overnight
and thoroughly washed with laboratory-grade detergent
and water, rinsed with water, and soaked for 4 h in a
mixture of dilute nitric and hydrochloric acids, followed by
rinsing with ASTM type I water and oven drying.
6.3.1 Griffin beakers, 250 mL, polytetrafluoroethylene
(PTFE) or quartz.
6.3.2 Storage bottles - Narrow mouth bottles, Teflon
FEP (fluorinated ethylene propylene), or HOPE, 125-mL
and 250-mL capacities.
200.13-5
Revision 1.0 September 1997
-------
6.4 Sample Processing Equipment
6.4.1 Air displacement pipetter - Digital pipet system
capable of delivering volumes from 100 to 2500 |jl_ with
an assortment of metal-free, disposable pipet tips.
6.4.2 Balances — Analytical balance, capable of
accurately weighing to ± 0.1 mg; top pan balance,
accurate to ± 0.01 g.
6.4.3 Hotplate - Corning PC100 or equivalent.
6.4.4 Centrifuge - Steel cabinet with guard bowl,
electric timer and brake.
6.4.5 Drying oven - Gravity convection oven with ther-
mostatic control capable of maintaining 105°C ± 5°C.
6.4.6 pH meter - Bench mounted or hand-held elec-
trode system with a resolution of ± 0.1 pH units.
6.4.7 Class 100 hoods are recommended for all
sample handling.
7.0 Reagents and Standards
7.7 Reagents may contain elemental impurities which
might affect analytical data. Only high-purity reagents
that conform to the American Chemical Society specifi-
cations7 should be used whenever possible. If the purity
of a reagent is in question, analyze for contamination. All
acids used for this method must be of ultra high-purity
grade or equivalent. Suitable acids are available from a
number of manufacturers. Redistilled acids prepared by
sub-boiling distillation are acceptable.
7.1.1 Nitric acid, concentrated (sp.gr. 1.41).
7.1.1.1 Nitric acid 0.75M - Dilute 47.7 ml (67.3g) cone.
nitric acid to 1000 ml with ASTM type I water.
7.1.1.2 Nitric acid (1+1)-Dilute 500 ml cone, nitric acid
to 1000 ml with ASTM type I water.
7.1.1.3 Nitric acid (1+9)-Dilute 100 ml cone, nitric acid
to 1000 ml with ASTM type I water.
7.1.2 Matrix Modifier, dissolve 300 mg Palladium (Pd)
powder in a minimum amount of concentrated HN03 (1
ml of HNO3, adding concentrated HCI only if necessary).
Dissolve 200 mg of Mg(NO3)2«6H2O in ASTM type I water.
Pour the two solutions together and dilute to 100 ml with
ASTM type I water.
Note: It is recommended that the matrix modifier be
analyzed separately in order to assess the contribution of
the modifier to the overall laboratory blank.
7.1.3 Acetic acid, glacial (sp.gr. 1.05). High purity acetic
acid is recommended.
7.14 Ammonium hydroxide (20%). High purity ammo-
nium hydroxide is recommended.
7.1.5 Ammonium acetate buffer 1M, pH 5.5 - Add 58
ml (60.5 g) of glacial acetic acid to 600 ml of ASTM type
I water. Add 65 ml (60 g) of 20% ammonium hydroxide
and mix. Check the pH of the resulting solution by
withdrawing a small aliquot and testing with a calibrated
pH meter, adjusting the solution to pH 5.5 ±0.1 with small
volumes of acetic acid or ammonium hydroxide as nec-
essary. Cool and dilute to 1 L with ASTM type I water.
7.1.6 Ammonium acetate buffer 2M, pH 5.5 - Prepare
as for Section 7.1.5 using 116 ml (121 g) glacial acetic
acid and 130 ml (120 g) 20% ammonium hydroxide,
diluted to 1000 ml with ASTM type I water.
Note: If the system is configured as shown in Figure 1,
the ammonium acetate buffer solutions may be further
purified by passing them through the chelating column at
a flow rate of 5.0 mL/min. Collect the purified solution in
a container. Following this, elute the collected contami-
nants from the column using 0.75M nitric acid for 5 min at
a flow rate of 4.0 mL/min. If the system is configured as
shown in Figure 2, the majority of the buffer is being
purified in an on-line configuration via the clean-up col-
umn.
7.17 Oxalic acid dihydrate (CASRN 6153-56-6),
0.2M - Dissolve 25.2 g reagent grade C2H2O4«2H2O in
250 ml ASTM type I water and dilute to 1000 ml with
ASTM type I water. CAUTION - Oxalic acid is toxic;
handle with care.
7.2 Water- For all sample preparation and dilutions,
ASTM type I water (ASTM D1193) is required.
7.3 Standard Stock Solutions - May be purchased
from a reputable commercial source or prepared from
ultra high-purity grade chemicals or metals (99.99 -
99.999% pure). All salts should be dried for one hour at
105°C, unless otherwise specified. (CAUTION - Many
metal salts are extremely toxic if inhaled or swallowed.
Wash hands thoroughly after handling.) Stock solutions
should be stored in plastic bottles. The following proce-
dures may be used for preparing standard stock solu-
tions:
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200.13-6
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Note: Some metals, particularly those which form sur-
face oxides require cleaning prior to being weighed. This
may be achieved by pickling the surface of the metal in
acid. An amount in excess of the desired weight should
be pickled repeatedly, rinsed with water, dried and
weighed until the desired weight is achieved.
7.3.1 Cadmium solution, stock 1 mL = 1000 /jg Cd -
Pickle cadmium metal in (1+9) nitric acid to an exact
weight of 0.100 g. Dissolve in 5 ml (1+1) nitric acid,
heating to effect solution. Cool and dilute to 100 ml with
ASTM type I water.
7.3.2 Cobalt solution, stock 1 mL = 1000 [jg Co -
Pickle cobalt metal in (1+9) nitric acid to an exact weight
of 0.100 g. Dissolve in 5 ml (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 ml with ASTM type
I water.
7.3.3 Copper solution, stock 1 mL = 1000 /jg Cu -
Pickle copper metal in (1 +9) nitric acid to an exact weight
of 0.100 g. Dissolve in 5 ml (1+1) nitric acid, heating to
effect solution. Cool and dilute to 100 ml with ASTM type
I water.
7.3.4 Lead solution, stock 1 mL = 1000 [jg Pb -
Dissolve 0.1599 g PbNO3 in 5 ml (1+1) nitric acid. Dilute
to 100 ml with ASTM type I water.
7.3.5 Nickel solution, stock 1 mL = 1000 ug Ni -
Dissolve 0.100 g nickel powder in 5 ml cone, nitric acid,
heating to effect solution. Cool and dilute to 100 ml with
ASTM type I water.
7.4 Multielement Stock Standard Solution - Care
must be taken in the preparation of multielement stock
standards that the elements are compatible and stable.
Originating element stocks should be checked for the
presence of impurities which might influence the accuracy
of the standard. Freshly prepared standards should be
transferred to acid cleaned, new FEP or HOPE bottles for
storage and monitored periodically for stability. A
multielement stock standard solution containing cad-
mium, cobalt, copper, lead, and nickel may be prepared
by diluting an appropriate aliquot of each single element
stock in the list to 100 ml with ASTM type I water
containing 1% (v/v) nitric acid.
7.4.1 Preparation of calibration standards — Fresh
multielement calibration standards should be prepared
weekly. Dilute the stock multielement standard solution
in 1% (v/v) nitric acid to levels appropriate to the required
operating range. The element concentrations in the stan-
dards should be sufficiently high to produce good mea-
surement precision and to accurately define the slope of
the response curve.
7.5 Blanks - Four types of blanks are required for
this method. A calibration blank is used to establish the
analytical calibration curve, the laboratory reagent blank
(LRB) is used to assess possible contamination from the
sample preparation procedure and to assess spectral
background. The laboratory fortified blank is used to
assess routine laboratory performance, and a rinse blank
is used to flush the instrument autosampler uptake sys-
tem. All diluent acids should be made from concentrated
acids (Section 7.1) and ASTM type I water.
7.5.1 The calibration blank consists of the appropriate
acid diluent in ASTM type I water. The calibration blank
should be stored in a FEP bottle.
7.5.2 The laboratory reagent blanks must contain all
the reagents in the same volumes as used in processing
the samples. The preparation blank must be carried
through the entire sample digestion and preparation
scheme.
7.5.3 The laboratory fortified blank (LFB) is prepared
by fortifying an aliquot of the laboratory reagent blank with
all analytes to provide a final concentration which will
produce an absorbance of approximately 0.1 for each
analyte. The LFB must be carried through the complete
procedure as used for the samples.
7.5.4 The rinse blank is prepared as needed by adding
1.0 ml of cone. HNO3 and 1.0 ml cone. HCI to 1 L of
ASTM Type I water and stored in a convenient manner.
7.6 Instrument Performance Check (IPC) Solution
- The IPC solution is used to periodically verify instrument
performance during analysis. The IPC solution should be
a fortified seawater prepared in the same acid mixture as
the calibration standards and should contain method
analytes such that the resulting absorbances are near the
midpoint of the calibration curve. The IPC solution should
be prepared from the same standard stock solutions used
to prepare the calibration standards and stored in a FEP
bottle. Agency programs may specify or request that
additional instrument performance check solutions be
prepared at specified concentrations in order to meet
particular program needs.
7.7 Quality Control Sample (QCS) - A quality con-
trol sample having certified concentrations of the analytes
of interest should be obtained from a source outside the
200.13-7
Revision 1.0 September 1997
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laboratory. Dilute the QCS if necessary with 1% nitric
acid, such that the analyte concentrations fall within the
proposed instrument calibration range.
8.0 Sample Collection, Preservation and
Storage
8.1 Prior to collection of an aqueous sample,
consideration should be given to the type of data required,
so that appropriate preservation and pretreatment steps
can be taken. Acid preservation, etc., should be
performed at the time of sample collection or as soon
thereafter as practically possible. The pH of all aqueous
samples must be tested immediately prior to aliquot!ng for
analysis to ensure the sample has been properly
preserved. If properly acid-preserved, the sample can be
held up to 6 months before analysis.
8.2 For determination of total recoverable elements
in aqueous samples, acidify with (1+1) nitric acid at the
time of collection to pH < 2. Normally 3 ml of (1+1) acid
per liter of sample is sufficient. The sample should not be
filtered prior to analysis.
Note: Samples that cannot be acid-preserved at the
time of collection because of sampling limitations or
transport restrictions, or have pH > 2 because of high
alkalinity should be acidified with nitric acid to pH < 2 upon
receipt in the laboratory. Following acidification, the
sample should be held for 16 h and the pH verified to be
<2 before withdrawing an aliquot for sample processing.
8.3 For aqueous samples, a field blank should be
prepared and analyzed as required by the data user. Use
the same container type and acid as used in sample
collection.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and periodic
analysis of laboratory reagent blanks, fortified blanks and
other laboratory solutions as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used to
characterize instrument performance (determination of
linear dynamic ranges and analysis of quality control
samples) and laboratory performance (determination of
method detection limits) prior to samples being analyzed
by this method.
9.2.2 Linear dynamic range (LDR) — The upper limit of
the LDR. must be established for the wavelength utilized
for each analyte by determining the signal responses
from a minimum of 6 different concentration standards
across the range, two of which are close to the upper limit
of the LDR. Determined LDRs must be documented and
kept on file. The linear calibration range which may be
used for analysis of samples should be judged by the
analyst from the resulting data. The upper LDR. limit
should be an observed signal no more than 10% below
the level extrapolated from the four lower standards. New
LDRs should be determined whenever there is a
significant change in instrument response, a change in
instrument analytical hardware or operating conditions.
Note: Multiple cleanout furnace cycles may be necessary
in order to fully define or utilize the LDR. for certain
elements such as nickel. For this reason, the upper limit
of the linear calibration range may not correspond to the
upper LDR limit.
Measured sample analyte concentrations that exceed the
upper limit of the linear calibration range must either be
diluted and reanalyzed with concern for memory effects
(Section 4.4) or analyzed by another approved method.
9.2.3 Quality control sample (QCS) - When beginning
the use of this method, on a quarterly basis or as required
to meet data-quality needs, verify the calibration stan-
dards and acceptable instrument performance with the
preparation and analyses of a QCS (Section 7.7). If the
determined concentrations are not within ± 10% of the
stated values, performance of the determinative step of
the method is unacceptable. The source of the problem
must be identified and corrected before either proceeding
on with the initial determination of method detection limits
or continuing with ongoing analyses.
9.2.4 Method detection limit (MDL) - MDLs must be
established for all analytes, using reagent water (blank)
fortified at a concentration of two to three times the
estimated instrument detection limit.8 To determine MDL
values, take seven replicate aliquots of the fortified
Revision 1.0 September 1997
200.13-8
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reagent water and process through the entire analytical
method. Perform all calculations defined in the method
and report the concentration values in the appropriate
units. Calculate the MDL as follows:
MDL = (t) x (S)
where, t = Student's t value for a 99% confidence level
and a standard deviation estimate with n-1
degrees of freedom [t = 3.14 for seven
replicates].
S= standard deviation of the replicate
analyses.
Note: If the relative standard deviation (RSD) from the
analyses of the seven aliquots is < 15%, the concentration
used to determine the analyte MDL may have been in
appropriately high for the determination. If so, this could
result in the calculation of an unrealistically low MDL. If
additional confirmation of the MDL is desired, reanalyze
the seven replicate aliquots on two more nonconsecutive
days and again calculate the MDL values for each day.
An average of the three MDL values for each analyte may
provide for a more appropriate MDL estimate. Determi-
nation of MDL in reagent water represents a best case
situation and does not reflect possible matrix effects of
real world samples. However, successful analyses of
LFMs (Section 9.4) can give confidence to the MDL value
determined in reagent water. Typical single laboratory
MDL values using this method are given in Table 1. MDLs
should be determined every 6 months, when a new
operator begins work, or whenever there is a significant
change in the background or instrument response.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory reagent blank (LRB) - The laboratory
must analyze at least one LRB (Section 7.5.2) with each
batch of 20 or fewer samples. LRB data are used to
assess contamination from the laboratory environment.
LRB values that exceed the MDL indicate laboratory or
reagent contamination should be suspected. Any deter-
mined source of contamination must be corrected and the
samples reanalyzed for the affected analytes after
acceptable LRB values have been obtained.
9.3.2 Laboratory fortified blank (LFB) - The laboratory
must analyze at least one LFB (Section 7.5.3) with each
batch of samples. Calculate accuracy as percent recov-
ery (Section 9.4.3). If the recovery of any analyte falls
outside the required control limits of 85-115%, that
analyte is judged out of control, and the source of the
problem should be identified and resolved before
continuing analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each 5-10
new recovery measurements, new control limits can be
calculated using only the most recent 20-30 data points.
Also, the standard deviation (S) data should be used to
establish an ongoing precision statement for the level of
concentrations included in the LFB. These data must be
kept on file and be available for review.
9.3.4 Instrument Performance Check (IPC) Solution -
For all determinations the laboratory must analyze the
IPC solution (Section 7.6) and a calibration blank imme-
diately following each calibration, after every tenth sample
(or more frequently, if required) and at the end of the
sample run. The IPC solution should be a fortified
seawater matrix. Analysis of the IPC solution and
calibration blank immediately following calibration must
verify that the instrument is within ±10% of calibration.
Subsequent analyses of the IPC solution must be within
±10% of calibration. If the calibration cannot be verified
within the specified limits, reanalyze the IPC solution. If
the second analysis of the IPC solution confirms
calibration to be outside the limits, sample analysis must
be discontinued, the cause determined and/or in the case
of drift the instrument recalibrated. All samples following
the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution
must be kept on file with the sample analyses data.
9.3.5 The overall sensitivity and precision of this
method are strongly influenced by a laboratory's ability to
control the method blank. Therefore, it is recommended
that the calibration blank response be recorded for each
set of samples. This record will aid the laboratory in
assessing both its long- and short-term ability to control
the method blank.
200.13-9
Revision 1.0 September 1997
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9.4 Assessing Analyte Recovery and Data
Quality
9.4.1 Sample homogeneity and the chemical nature of
the sample matrix can affect analyte recovery and data
quality. Taking separate aliquots from the sample for
replicate and fortified analyses can, in some cases,
assess these effects. Unless otherwise specified by the
data user, laboratory or program, the following laboratory
fortified matrix (LFM) procedure (Section 9.4.2) is
required.
9.4.2 The laboratory must add a known amount of
each analyte to a minimum of 10% of routine samples.
In each case, the LFM aliquot must be a duplicate of the
aliquot used for sample analysis and for total recoverable
determinations added prior to sample preparation. For
water samples, the added analyte concentration must be
the same as that used in the laboratory fortified blank
(Section 7.5.3). Over time, samples from all routine
sample sources should be fortified.
9.4.3 Calculate the percent recovery for each analyte,
corrected for concentrations measured in the unfortified
sample, and compare these values to the designated
LFM recovery range of 75-125%. Recovery calculations
are not required if the concentration added is <25% of the
unfortified sample concentration. Percent recovery may
be calculated in units appropriate to the matrix, using the
following equation:
where, R = percent recovery.
Cs = fortified sample concentration.
C = sample background concentration.
s = concentration equivalent of analyte
added to sample.
9.4.4 If the recovery of any analyte falls outside the
designated LFM recovery range (but is still within the
range of calibration and the background absorbance is <
1 abs.) and the laboratory performance for that analyte is
shown to be in control (Section 9.3), the recovery problem
encountered with the LFM is judged to be either matrix or
solution related, not system related. This situation should
be rare given the matrix elimination preconcentration step
priorto analysis. If a low recovery is found, check the pH
of the sample plus the buffer mixture. The resulting pH
should be about 5.5. The pH of the sample strongly
influences the column's ability to preconcentrate the
metals; therefore, a low recovery may be caused by a low
pH. If the pH for the LFM/buffer mixture is about 5.5, the
analyst is advised to make an in furnace analyte addition
to the LFM using the preconcentrated standard solution.
If recovery of the in furnace analyte addition is shown to
be out of control, a matrix interference is confirmed and
the sample must be analyzed by MSA.
9.5 Utilizing Reference Materials
9.5.1 It is recommended that a reference material such
as NASS-3 (from the Research Council of Canada) be
fortified and used as an Instrument Performance Check
Solution.
10.0 Calibration and Standardization
10.1 The preconcentration system can be configured
utilizing a sample loop to define the sample volume
(Figure 1) or the system can be configured such that a
sample pump rate and a pumping time defines the
sample volume (Figure 2). The system illustrated in
Figure 1 is recommended for sample sizes of <10 mL. A
thorough rinsing of the sample loop between samples
with HNO3 is required. This rinsing will minimize the
cross-contamination which may be caused by the sample
loop. The system in Figure 2 should be used for sample
volumes of >10 mL. The sample pump used in Figure 2
must be calibrated to assure that a reproducible/defined
volume is being delivered.
70.2 Specific wavelengths and instrument operating
conditions are listed in Table 1. However, because of
differences among makes and models of spectropho-
tometers and electrothermal furnace devices, the actual
instrument conditions selected may vary from those listed.
10.3 Priorto the use of this method, instrument operat-
ing conditions must be optimized. The analyst should
follow the instructions provided by the manufacturer while
using the conditions listed in Table 1 as a guide. Of
particular importance is the determination of the charring
temperature limit for each analyte. This limit is the fur-
nace temperature setting where a loss in analyte will
occur prior to atomization. This limit should be deter-
mined by conducting char temperature profiles for each
analyte and when necessary, in the matrix of question.
The charring temperature selected should minimize back-
ground absorbance while providing some furnace tem-
perature variation without loss of analyte. For routine
analytical operation the charring temperature is usually
Revision 1.0 September 1997
200.13-10
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set at least 100°C below this limit. The optimum condi-
tions selected should provide the lowest reliable MDLs
and be similar to those listed in Table 1. Once the
optimum operating conditions are determined, they
should be recorded and available for daily reference.
70.4 Prior to an initial calibration, the linear dynamic
range of the analyte must be determined (Section 9.2.2)
using the optimized instrument operating conditions. For
all determinations allow an instrument and hollow cath-
ode lamp warm-up period of not less than 15 min. If an
EDL is to be used, allow 30 min for warm-up.
70.5 Before using the procedure (Section 11.0) to ana-
lyze samples, data must be available to document initial
demonstration of performance. The required data and
procedure are described in Section 9.2. This data must be
generated using the same instrument operating
conditions and calibration routine (Section 11.4) to be
used for sample analysis. These documented data must
be kept on file and be available for review by the data
user.
11.0 Procedure
77.7 Sample Preparation - Total Recoverable
Elements
11.1.1 Add 2 ml (1+1) nitric acid to the beaker
containing 100 ml of sample. Place the beaker on the
hot plate for solution evaporation. The hot plate should
be located in a fume hood and previously adjusted to
provide evaporation at a temperature of approximately but
no higher than 85°C. (See the following note.) The
beaker should be covered with an elevated (ribbed) watch
glass or other necessary steps should be taken to prevent
sample contamination from the fume hood environment.
Note: For proper heating adjust the temperature control
of the hot plate such that an uncovered Griffin beaker
containing 50 ml of water placed in the center of the hot
plate can be maintained at a temperature approximately
but no higher than 85°C. (Once the beaker is covered
with a watch glass the temperature of the water will rise
to approximately 95°C.)
11.1.2 Reduce the volume of the sample aliquot to
about 20 ml by gentle heating at 85°C. DO NOT BOIL.
This step takes about 2 hr for a 100-mL aliquot with the
rate of evaporation rapidly increasing as the sample
volume approaches 20 ml. (A spare beaker containing
20 ml of water can be used as a gauge.)
77.7.3 Cover the lip of the beaker with a watch glass to
reduce additional evaporation and gently reflux the
sample for 30 min. Slight boiling may occur, but vigorous
boiling must be avoided.
77.1.4 Allow the beaker to cool. Quantitatively transfer
the sample solution to a 100-mL volumetric flask, dilute
to volume with reagent water, stopper and mix.
77.7.5 Allow any undissolved material to settle over-
night, or centrifuge a portion of the prepared sample until
clear. (If after centrifuging or standing overnight the
sample contains suspended solids that would clog or
affect the sample introduction system, a portion of the
sample may be filtered prior to analysis. However, care
should be exercised to avoid potential contamination from
filtration.) The sample is now ready for analysis. Because
the effects of various matrices on the stability of diluted
samples cannot be characterized, all analyses should be
performed as soon as possible after the completed
preparation.
77.2 Prior to first use, the preconcentration system
should be thoroughly cleaned and decontaminated using
0.2M - oxalic acid.
77.2.7 Precleaning the Preconcentration System
11.2.1.1 Place approximately 500 ml 0.2M - oxalic acid
in each of the sample/eluent containers. Flush the entire
system by running the program used for sample analysis
3 times.
11.2.1.2 Rinse the containers with ASTM type I water
and repeat the sequence described in Section 11.2.1.1
using 0.75M nitric acid and again using ASTM type I water
in place of the 0.2M - oxalic acid.
11.2.1.3 Rinse the containers thoroughly with ASTM type
I water, fill them with their designated reagents and run
through the program used for sample analysis in order to
prime the pump and all eluent lines with the correct
reagents.
77.2.2 Peak Profile Determination
11.2.2.1 The peak elution time or the collection window
should be determined using an ICP-AES (Inductively
Coupled Plasma Atomic Emission Spectrometer) or
Flame AA. Figure 3 is a plot of time vs. emission intensity
forCd, Pb, Ni, and Cu. The collection window is marked
in Figure 3 and should provide about 30 sec buffer on
200.13-11
Revision 1.0 September 1997
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either side of the peak. If an ICP-AES is not available, it
is recommended that the peak profile be determined by
collecting 200-|jL samples during the elution part of the
preconcentration cycle and then reconstructing the peak
profile from the analysis of the 200-uL samples.
11.3 Sample Preconcentration
11.3.1 Preconcentration utilizing a sample loop.
11.3.1.1 Loading Sample Loop - With valve 1 in the off
position and valve 2 in the on position, load sample
through the sample loop to waste using the sample pump
for 4 min at 4 mL/min. Switch on the carrier pump and
pump 1 % nitric acid to flush the sample collection line.
11.3.1.2 Column Loading - With valve 1 in the on
position, load sample from the loop onto the column
using 1 M ammonium acetate for 4.5 min at 4.0 mL/min.
Switch on the buffer pump, and pump 2M ammonium
acetate at a flow rate of 1 mL/min. The analytes are
retained on the column, while the majority of the matrix is
passed through to waste.
11.3.1.3 Elution Matrix - With valve 1 in the on position
the gradient pump is allowed to elute the matrix using the
1M ammonium acetate. During which time the carrier,
buffer and the sample pumps are all off.
11.3.1.4 Elute Analytes - Turn off valve 1 and begin
eluting the analytes by pumping 0.75M nitric acid through
the column and turn off valve 2 and pump the eluted
analytes into the collection flask. The analytes should be
eluted into a 2-mL sample volume.
11.3.1.5 Column Reconditioning - Turn on valve 2 to
direct column effluent to waste, and pump 0.75M nitric
acid, 1M ammonium acetate, 0.75M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min. Each solvent should be pumped through the
column for 2 min. During this process, the next sample
can be loaded into the sample loop using the sample
pump.
11.3.1.6 Preconcentration of the sample may be a-
chieved by running through an eluent pump program.
The exact timing of this sequence should be modified
according to the internal volume of the connecting tubing
and the specific hardware configuration used.
713.2 Preconcentration utilizing an auxiliary pump to
determine sample volume.
11.3.2.1 Sample Loading - With the valves 1 and 2 on
and the sample pump on, load the sample on the column
buffering the sample utilizing the gradient pump and the
2M buffer. The actual sample volume is determined by
knowing the sample pump rate and the time. While the
sample is being loaded the carrier pump can be used to
flush the collection line.
11.3.2.2 Elution Matrix - With valve 1 in the off position
the gradient pump is allowed to elute the matrix using the
1M ammonium acetate. During which time the carrier,
buffer and the sample pumps are all off.
11.3.2.3 Elution of Analytes - With valves I and 2 in the
off position the gradient pump is switched to 0.75M HNO3
and the analytes are eluted into the collection vessel.
The analytes should be eluted into a 2 mL sample
volume.
11.3.2.4 Column Reconditioning - Turn on valve 2 to
direct column effluent to waste, and pump 0.75M nitric
acid, 1M ammonium acetate, 0.75M nitric acid and 1M
ammonium acetate alternately through the column at 4.0
mL/min.
Note: When switching the gradient pump from nitric
acid back to the ammonium acetate it is necessary to
flush the line connecting the gradient pump to valve 2 with
the ammonium acetate prior to switching the valve. If the
line contains nitric acid it will elute the metals from the
cleanup column.
11.3.2.5 Preconcentration of the sample may be a-
chieved by running through an eluent pump program.
The exact timing of this sequence should be modified
according to the internal volume of the connecting tubing
and the specific hardware configuration used.
77.4 Repeat the sequence described in Section 11.3.1
or 11.3.2 for each sample to be analyzed. At the end of
the analytical run leave the column filled with 1M ammo-
nium acetate buffer until it is next used.
77.5 Samples having concentrations higher than the
established linear dynamic range should be diluted into
range and reanalyzed.
77.6 Sample Analysis
11.6.1 Prior to daily instrument calibration, inspect the
graphite furnace, the sample uptake system and auto-
sampler injector for any change that would affect
instrument performance. Clean the system and replace
Revision 1.0 September 1997
200.13-12
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the graphite tube and/or platform when needed or on a
daily basis. A cotton swab dipped in a 50/50 mixture of
isopropyl alcohol (I PA) and H2O (such that it is damp but
not dripping) can be used to remove the majority of the
salt buildup. A second cotton swab is dipped in IPA and
the contact rings are wiped down to assure they are
clean. The rings are then allowed to thoroughly dry and
then a new tube is placed in the furnace and conditioned
according to instrument manufacturers specifications.
716.2 Configure the instrument system to the selected
optimized operating conditions as determined in Sections
10.1 and 10.2.
11.6.3 Before beginning daily calibration the instrument
should be reconfigured to the optimized conditions. Ini-
tiate data system and allow a period of not less than 15
min for instrument and hollow cathode lamp warm-up. If
an EDL is to be used, allow 30 min for warm-up.
11.6.4 After the warm-up period but before calibration,
instrument stability must be demonstrated by analyzing a
standard solution with a concentration 20 times the IDL a
minimum of five times. The resulting relative standard
deviation of absorbance signals must be <5%. If the
relative standard deviation is >5%, determine and correct
the cause before calibrating the instrument.
11.6.5 For initial and daily operation calibrate the instru-
ment according to the instrument manufacturer's recom-
mended procedures using the calibration blank (Section
7.5.1) and calibration standards (Section 7.4) prepared at
three or more concentrations within the usable linear
dynamic range of the analyte (Sections 4.4 & 9.2.2).
11.6.6 An autosampler must be used to introduce all
solutions into the graphite furnace. Once the standard,
sample or QC solution plus the matrix modifier is injected,
the furnace controller completes furnace cycles and
cleanout period as programmed. Analyte signals must be
integrated and collected as peak area measurements.
Background absorbances, background corrected analyte
signals, and determined analyte concentrations on all
solutions must be able to be displayed on a CRT for
immediate review by the analyst and be available as hard
copy for documentation to be kept on file. Flush the
autosampler solution uptake system with the rinse blank
(Section 7.5.4) between each solution injected.
11.6.7 After completion of the initial requirements of this
method (Section 9.2), samples should be analyzed in the
same operational manner used in the calibration routine.
11.6.8 During sample analyses, the laboratory must
comply with the required quality control described in
Sections 9.3 and 9.4.
11.6.9 Determined sample analyte concentrations that
are >90% of the upper limit of calibration must either be
diluted with acidified reagent water and reanalyzed with
concern for memory effects (Section 4.4), or determined
by another approved test procedure that is less sensitive.
Samples with a background absorbance > 1.0 must be
appropriately diluted with acidified reagent water and
reanalyzed (Section 9.4.6). If the method of standard
additions is required, follow the instructions described in
Section 11.5.
11.6.10 Report data as directed in Section 12.
77.7 Standard Additions- If the method of standard
addition is required, the following procedure is recom-
mended:
11.7.1 The standard addition technique9 involves pre-
paring new standards in the sample matrix by adding
known amounts of standard to one or more aliquots of the
processed sample solution. This technique compensates
for a sample constituent that enhances or depresses the
analyte signal, thus producing a different slope from that
of the calibration standards. It will not correct for additive
interference, which causes a baseline shift. The simplest
version of this technique is the single addition method.
The procedure is as follows: Two identical aliquots of the
sample solution, each of volume Vx, are taken. To the
first (labeled A) is added a small volume Vs of a standard
analyte solution of concentration Cs. To the second
(labeled B) is added the same volume Vs of the solvent.
The analytical signals of A and B are measured and
corrected for nonanalyte signals. The unknown sample
concentration Cx is calculated:
Cx = SoVoCo
(SA-SB)VX
where, SAand SB are the analytical signals (corrected for
the blank) of solutions A and B, respectively. Vs and Cs
should be chosen so that SA is roughly twice §, on the
average. It is best if Vs is made much less than Vx, and
thus Cs is much greater than Cx, to avoid excess dilution
of the sample matrix. If a separation or concentration
step is used, the additions are best made first and carried
through the entire procedure. For the results from this
technique to be valid, the following limitations must be
taken into consideration:
200.13-13
Revision 1.0 September 1997
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1. The analytical curve must be linear.
14.0 Pollution Prevention
2. The chemical form of the analyte added must re-
spond in the same manner as the analyte in the
sample.
3. The interference effect must be constant over the
working range of concern.
4. The signal must be corrected for any additive inter-
ference.
12.0 Data Analysis and Calculations
72.7 Sample data should be reported in units of ug/L
for aqueous samples.
72.2 For total recoverable aqueous analytes (Section
11.1), when 100-mL aliquot is used to produce the 100
ml final solution, round the data to the tenths place and
report the data in ug/L up to three significant figures. If an
aliquot volume other than 100 ml is used for sample
preparation, adjust the dilution factor accordingly. Also,
account for any additional dilution of the prepared sample
solution needed to complete the determination of ana-
lytes exceeding the upper limit of the calibration curve.
Do not report data below the determined analyte MDL
concentration or below an adjusted detection limit
reflecting smaller sample aliquots used in processing or
additional dilutions required to complete the analysis.
72.3 The QC data obtained during the analyses
provide an indication of the quality of the sample data and
should be provided with the sample results.
13.0 Method Performance
73.7 Experimental conditions used for single
laboratory testing of the method are summarized in Table
1.
73.2 Table 2 contains precision and recovery data ob-
tained from a single laboratory analysis of a fortified and
a non-fortified sample of NASS-3. The samples were
prepared using the procedure described in Section 11.1.
Four replicates of the non-fortified samples were
analyzed and the average of the replicates was used for
determining the sample analyte concentration. The forti-
fied samples of NASS-3 were also analyzed and the
average percent recovery and the percent relative stan-
dard deviation is reported. The reference material certi-
fied values are also listed for comparison.
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation (e.g., Section 7.8). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 1155 16th Street N.W., Wash-
ington D.C. 20036, (202)872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in the Section 14.2.
16.0 References
1. A. Siraraks, H.M. Kingston and J.M. Riviello, Anal
Chem. 621185(1990).
2. E.M. Heithmar, T.A. Hinners, J.T. Rowan and J.M.
Riviello, Anal Chem. 62 857 (1990).
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
Revision 1.0 September 1997
200.13-14
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4. Carcinogens - Working With Carcinogens, Depart-
ment of Health, Education, and Welfare, Public
Health Service, Centers for Disease Control, Na-
tional Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
5. Proposed OSHA Safety and Health
Standards,Laboratories, Occupational Safety and
Health Administration, Federal Register, July 24,
1986.
6. Safety in Academic Chemistry Laboratories, Ameri-
can Chemical Society Publication, Committee on
Chemical Safety, 3rd Edition, 1979.
7. Rohrbough, W.G. et al. Reagent Chemicals,
American Chemical Society Specifications, 7th
edition. American Chemical Society, Washington,
DC, 1986.
8. Code of Federal Regulations 40, Ch. 1, Pt. 136
Appendix B.
9. Winefordner, J.D., Trace Analysis: Spectroscopic
Methods for Elements, Chemical Analysis, Vol. 46,
pp. 41-42,1976.
200.13-15 Revision 1.0 September 1997
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Method Detection Limits for Total Recoverable Analytes in Reagent Water1
Element
Cadmium
Cobalt
Copper
Lead
Nickel
Slit,
nm
0.7
0.2
0.7
0.7
0.2
Recommended
analytical
Wavelengths, nm
228.8
242.5
324.8
283.3
232.4
Char
Temp, °C
800
1400
1300
1250
1400
Atomization
Temp, °C
1600
2500
2600
2000
2500
MDL2,
ug/L
0.016
-
0.36
0.28
*
MDLs were calculated using NASS-3 as the matrix.
2 MDLs were calculated based on a 10-mL sample loop.
* MDL was not calculated because the concentration in the matrix exceeds the MDL spike level.
- Not Determined.
Table 2. Precision and Recovery Data for NASS-3 Using System Illustrated in Figure 11
Analyte
Cd
Co
Cu
Pb
Ni
Certified
Value,
ug/L3
0.029 ± 0.004
0.004 ± 0.001
0.1 09 ±0.011
0.039 ± 0.006
0.257 ± 0.027
Sample
Cone.,
ug/L3
0.026 ±0.01 2
<0.36
<0.28
0.260 ± 0.04
Fortified
Cone.,
ug/L
0.25
5.0
5.0
5.0
Avg.
Recovery, %
93
87
90
117
%RSD
3.3
1.4
3.7
8.3
1 Data collected using 10-mL sample loop.
2 Matrix modifier is Pd/Mg(NO3)2/H2.
3 Uncertainties based on 95% confidence limits.
- Not determined.
Revision 1.0 September 1997 200.13-16
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Sample Loop
Loading
Column
Loading
Elution of
Matrix
Elution of
Analytes
Column
Recondition
1
Off
On
On
Off
Off
Valves
2
On
On
On
Off
On
Buffer
Pump
Off
On
Off
Off
Off
Carrier
Pump
On
Off
Off
Off
Off
Sample
Pump
On
Off
Off
Off
Off
Off
On
Waste
Waste
X
\
*
Buffer
Pump
Mixing Tee
Figure 1. Sample Loop Configuration.
200.13-17
Revision 1.0 September 1997
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Event
Sample
Loading
Elution of
Matrix
Elution of
Analytes
Column
Recondition
1
On
Off
Off
Off
Valves
2
On
On
Off
On
Carrier
Pump
On
Off
Off
Off
Sample
Pump
On
Off
Off
On
Off
On
Waste
*
* .
\
X
,,'
•»
Sample
Pump
Mixing Tee
Figure 2. System Diagram without Sample Loop.
Revision 1.0 September 1997 200.13-18
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3.5
•3- 2'5
13
1.5
o>
c
0.5
Start of Collection
40
End of Collection
80
Time (sec)
120
160
Figure 3. Peak Collection Window from ICP-AES.
200.13-19
Revision 1.0 September 1997
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Method 349.0
Determination of Ammonia in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies
Rosenstiel School of Marine and Atmospheric Science/AOML, NOAA
University of Miami, Miami, FL 33149
Peter B. Ortner, Charles J. Fischer, and Lloyd D. Moore, Jr., National Oceanic and
Atmospheric Administration, Atlantic Oceanographic and Meteorological Laboratory,
Ocean Chemistry Division, Miami, FL 33149
Project Officer
Elizabeth J. Arar
Version 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
349.0-1
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Method 349.0
Determination of Ammonia in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for the
determination of ammonia in estuarine and coastal
waters. The method is based upon the indophenol
reaction,1"5 here adapted to automated gas-segmented
continuous flow analysis.
The term ammonia as used in this method denotes total
concentration of ammonia, including both chemical
forms, NH3 and NH4+. Because ionization of NH,+ has a
pK value of about 9.3, NH4+ is the dominant chemical
form in natural waters. At pH of 8.2 and 25°C only 8.1%
is present as NH3, the form that can be toxic to fish and
other aquatic organisms.
The concentration of ammonia in estuarine and coastal
water shows considerable temporal and spatial variability.
It rarely exceeds 0.005 mg N/L in oxygenated, unpolluted
estuarine and coastal water, but in anoxic water, the
amount of ammonia can be as high as 0.28 mg N/L.6
Although other forms of nitrogen contribute to primary
productivity and nutrient cycling in marine and estuarine
waters, ammonia is particularly important. Because
ammonia represents the most reduced form of inorganic
nitrogen available, it is preferentially assimilated by
phytoplankton. Whereas nitrate is the source of nitrogen,
it must first be reduced to ammonia before it can be
assimilated and incorporated into amino acids and other
compounds. Ammonia is released during the
decomposition of organic nitrogen compounds by
proteolytic bacteria, but also excreted directly by
invertebrates along with urea and peptides.7 In regions of
coastal upwelling, ammonia released by zooplankton can
play a significant role in supplying the nitrogen that
supports phytoplankton production.8
Chemical Abstracts Service
Analyte Registry Numbers (CASRN)
Ammonia
7664-41-7
1.2 A statistically determined method detection limit
(MDL)9 of 0.3 ug N/L has been determined by one
laboratory from seawaters of four different salinities. The
method is linear to 4.0 mg N/L using a Flow Solution
System (Alpkem, Wilsonville, Oregon).
1.3 Approximately 60 samples per hour can be
analyzed.
1.4 This method should be used by analysts both
experienced in the use of automated gas segmented
continuous flow colorimetric analyses, and also familiar
with matrix interferences and the procedures used in their
correction. A minimum of 6-months experience under the
close supervision of a qualified analyst is recommended.
2.0 Summary of Method
2.1 The automated gas segmented continuous flow
colorimetric method is used for the analysis of ammonia
concentration. Ammonia in solution reacts with alkaline
phenol and NaDTT (Sect. 7.2.5) at 60°C to form
indophenol blue in the presence of sodium
nitroferricyanide as a catalyst. The absorbance of
indophenol blue at 640 nm is linearly proportional to the
concentration of ammonia in the sample. A small
systematic negative error caused by differences in the
refractive index of seawater and reagent water, and a
positive error caused by the matrix effect on the color
formation, may be corrected for during data processing.
3.0 Definitions
3.1 Calibration Standard (CAL) - A solution
prepared from the primary dilution standard solution or
stock standard solution containing analytes. The CAL
solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.2 Laboratory Fortified Blank (LFB) - An aliquot of
reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed
Version 1.0 September 1997
349.0-2
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exactly like a sample, and its purpose is to determine
whether method performance is within acceptable control
limits, and whether the laboratory is capable of making
accurate and precise measurements. This is a standard
prepared in reagent water that is analyzed as a sample.
3.3 Laboratory Fortified Sample Matrix
(LFM)-/\r\ aliquot of an environmental sample to which
known quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
3.4 Laboratory Reagent Blank (LRB) - An aliquot of
reagent water that is treated exactly as a sample including
exposure to all labware, equipment, and reagents that are
used with other samples. The LRB is used to determine
if method analytes or other interferences are present in
the laboratory environment, the reagents, or apparatus.
3.5 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.6 Method Detection Limit (MDL) — The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.9
3.7 Reagent Water (RW) — Type 1 reagent grade
water equal to or exceeding the standards established by
the American Society for Testing and Materials (ASTM).
Reverse osmosis systems or distilling units followed by
Super-Q Plus Water System that produce water with 18
megohm resistance are examples of acceptable water
sources. To avoid contamination of ammonia from the air,
the reagent water should be stored in a sealed or a
collapsible container and used the day of preparation.
3.8 Refractive Index (Rl) — The ratio of the velocity
of light in a vacuum to that in a given medium. The
relative refractive index is the ratio of the velocity of light
in two different media, such as estuarine or sea water
versus reagent water. The correction for this difference is
referred to as refractive index correction in this method.
3.9 Stock Standard Solution (SSS) - A concentrated
solution of method analyte prepared in the laboratory
using assayed reference compounds or purchased from
a reputable commercial source.
3.70 Primary Dilution Standard Solution (PDS) - A
solution prepared in the laboratory from stock standard
solutions and diluted as needed to prepare calibration
solutions and other needed analyte solutions.
3.77 Quality Control Sample (QCS) - A solution of
method analyte of known concentrations which is used to
fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.72 Synchronization Peak Solution — A
synchronization peak is required by most data acquisition
programs to initialize the peak finding parameters. The
first cup in every run must always be identified as a SYNC
sample. The SYNC sample is usually a high
concentration standard, but can be any sample that
generates a peak at least 25% of full scale.
3.73 Color SYNC Peak Solution - A colored solution
used to produce a synchronization peak in the refractive
index measurement in which no color reagent is pumped
through system.
3.74 Sensitivity Drift — The change in absorbance for
a given concentration of analyte due to instrumental or
chemical drift during the course of measurement.
3.75 Matrix Effect — The change of absorbance in
different matrices due to the effect of ionic strength and
composition on the kinetics of color forming reactions.
4.0 Interferences
4.1 Hydrogen sulfide at concentrations greater than 2
mg S/L can negatively interfere with ammonia analysis.
Hydrogen sulfide in samples should be removed by
acidification with sulfuric acid to a pH of about 3, then
stripping with gaseous nitrogen.
4.2 The addition of sodium citrate and EDTA
complexing reagent eliminates the precipitation of
calcium and magnesium hydroxides when calcium and
349.0-3
Version 1.0 September 1997
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magnesium in seawater samples mix with high pH (about
13) reagent solution.4
4.3 Sample turbidity is eliminated by filtration or
centrifugation after sample collection.
4.4 As noted in Section 2.1 refractive index and salt
error interferences occur when sampler wash solution
and calibration standards are not matched with samples
in salinity, but are correctable. For low concentration
samples (< 20 ug N/L), low nutrient seawater (LNSW)
with salinity matched to samples, sampler wash solutions
and calibration standards is recommended to eliminate
matrix interferences.
5.0 Safety
5.7 Water samples collected from the estuarine and
coastal environment are rarely hazardous. However, the
individual who collects samples should use proper
technique.
5.2 Good laboratory technique should be used when
preparing reagents. Laboratory personnel should obtain
material safety data sheets (MSDS) for al chemicals used
in this method. A lab coat, safety goggles, and gloves
should be worn when handling the concentrated acid.
5.3 Chloroform is used as a preservative in this
method. Use in a properly ventilated area, such as a fume
hood.
6.0 Equipment and Supplies
6.1 Gas Segmented Continuous Flow Autoanalyzer
Consisting of:
6.1.1 Automatic sampler.
6.1.2 Analytical cartridge with reaction coils and heater.
6.13 Proportioning pump.
6.1.4 Spectrophotometer equipped with a tungsten lamp
(380-800 nm) or photometer with a 640 nm interference
filter (maximum 2 nm bandwidth).
6.15 Strip chart recorder or computer based data
acquisition system.
6.16 Nitrogen gas (high-purity grade, 99.99%).
6.2 Glassware and Supplies
6.2.1 Gaseous ammonia concentration in the laboratory
air should be minimal to avoid sample or reagent
contamination. Remove any NH4OH solution stored in the
laboratory. Smoking should be strictly forbidden. An air
filtration unit might also be used to obtain ammonia-free
lab air.
6.2.2 All labware used in the analysis must be free of
residual ammonia to avoid sample or reagent
contamination. Soaking with laboratory grade detergent,
rinsing with tap water, followed by rinsing with 10% HCI
(v/v) and then thoroughly rinsing with reagent water was
found to be sufficient when working at moderate and high
concentration of ammonia. Ammonia is known for its high
surface reactivity.10 When working at low levels of
ammonia (< 20 ug N/L), further cleaning of labware is
mandatory. Plastic bottles and glass volumetric flasks
should be cleaned in an ultrasonic bath with reagent
water for 60 minutes. Bottles and sample tubes made of
glass can be easily cleaned by boiling in reagent water.
Repeat the cleaning process with fresh reagent water
prior to use if necessary.
6.2.3 Automatic pipetters with disposable pipet tips
capable of delivering volumes ranging from 100 uL to
1000 uL and 1 mL to 10 mL.
6.2.4 Analytical balance, with accuracy to 0.1 mg, for
preparing standards.
6.2.5 60-mL glass or high density polyethylene sample
bottles, glass volumetric flasks and glass sample tubes.
6.2.6 Drying oven.
6.2.7 Desiccator.
6.2.8 Membrane filters with 0.45 urn nominal pore size.
Plastic syringes with syringe filters.
6.2.9 Centrifuge.
6.2.10 Ultrasonic water bath cleaner.
7.0 Reagents and Standards
Note: All reagents must be of analytical reagent grade.
Version 1.0 September 1997
349.0-4
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7.1 Stock Reagent Solutions
7.1.1 Complexing Reagent - Dissolve 140 g of sodium
citrate dihydrate(Na3C6H5O7.2H2O, FW 294.11), 5 g of
sodium hydroxide (NaOH, FW40) and 10 g of disodium
EDTA (Na2C10H1408N2.2H20, FW 372.24), in
approximately 800 ml of reagent water, mix and dilute to
1 L with reagent water. The pH of this solution is
approximately 13. This solution is stable for 2 months.
7.1.2 Stock Ammonium Sulfate Solution (100 mg N/L) -
Quantitatively transfer 0.4721 g of pre-dried (105°C for 2
hours) ammonium sulfate ((NH4)2SO4, FW 132.15) to a
1000 ml glass volumetric flask containing approximately
800 ml of reagent water and dissolve the salt. Add a few
drops of chloroform as a preservative. Dilute the solution
to the mark with reagent water. Store in a glass bottle in
the refrigerator at 4°C. It is stable for 2 months.11
7.1.3 Low Nutrient Sea Water (LNSW) - Obtain natural
low nutrient seawater from surface water of the Gulf
Stream or Sargasso Sea (salinity 36 %o, < 7 ug N/L) and
filter it through 0.3 micron pore size glass fiber filters. If
this is not available, commercial low nutrient sea water
( < 7 ug N/L) with salinity of 35 %o (Ocean Scientific
International, Wormley, U.K.) can be substituted. NOTE:
Don't use artificial seawater in this method.
7.2 Working Reagents
7.2.1 Brij-35 Start-up Solution - Add 2 mL of Brij-35
surfactant (ICI Americas, Inc.) to 1000 mL reagent water
and mix gently.
Note: Brij-35 is a trade name for polyoxyethylene(23)
lauryl ether (C^hUOCH.CH^OH, FW=1199.57, CASRN
9002-92-0).
7.2.2 Working Complexing Reagent - Add 1 mL Brij-35
to 200 mL of stock complexing reagent, mix gently.
Prepare this solution daily. This volume of solution is
sufficient for an 8-hour run.
7.2.3 Sodium Nitroferricyanide Solution - Dissolve 0.25
g of sodium nitroferricyanide (Na2Fe(CN)5NO.2H2O, FW
297.97) in 400 mL of reagent water, dilute to 500 mL with
reagent water. Store in an amber bottle at room
temperature.
7.2.4 Phenol Solution - Dissolve 1.8 g of solid phenol
(C6H5OH, FW 94.11) and 1.5 g of sodium hydroxide
(NaOH, FW40) in 100 mL of reagent water. Prepare this
solution fresh daily.
7.2.5 NaDTT Solution - Dissolve 0.5 g of sodium
hydroxide (NaOH, FW40) and 0.2 g dichloroisocyanuric
acid sodium salt (NaDTT, NaC3CI2N3O3, FW 219.95) in
100 mL of reagent water. Prepare this solution fresh daily.
7.2.6 Colored SYNC Peak Solution - Add 50 uL of blue
food coloring solution to 1000 mL reagent water and mix
thoroughly. Further dilute this solution to obtain a peak of
between 25 to 100 percent full scale according to the
AUFS setting used for refractive index measurement.
7.2.7 Primary Dilution Standard Solution - Prepare a
primary dilution standard solution (5 mg N/L) by diluting
5.0 mL of stock standard solution to 100 mL with reagent
water. Prepare this solution daily.
Note: This solution should be prepared to give an
intermediate concentration appropriate for further dilution
in preparing the calibration solutions. Therefore, the
concentration of a primary dilution standard solution must
be adjusted according to the desired concentration range
of calibration solutions.
7.2.8 Calibration Standards - Prepare a series of
calibration standards (CAL) by diluting suitable volumes
of a primary dilution standard solution (Section 7.2.7) to
100 mL with reagent water or low nutrient seawater.
Prepare these standards daily. The concentration range
of calibration standards should bracket the expected
concentrations of samples and not span more than two
orders of magnitude. At least five calibration standards
with equal increments in concentration should be used to
construct the calibration curve.
When working with samples of a narrow range of
salinities (± 2 %o) or samples containing low ammonia
concentration (< 20 ug N/L), it is recommended that the
CAL solutions be prepared in Low Nutrient Seawater
(Section 7.1.4) diluted to the salinity of samples, and the
Sampler Wash Solution also be Low Nutrient Seawater
(Section 7.1.4) diluted to the same salinity. NOTE: If this
procedure is employed, it is not necessary to perform the
matrix effect and refractive index corrections outlined in
Sections 12.2 and 12.3.
349.0-5
Version 1.0 September 1997
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When analyzing samples of moderate and high ammonia
concentration (> 20 |jg N/L) with varying salinities,
calibration standard solutions and sampler wash
solutions can be prepared in reagent water. The
corrections for matrix effect and refractive index should be
subsequently applied (Sections 12.2 and 12.3).
7.2.9 Saline Ammonia Standards - If CAL solutions are
not prepared to match sample salinity, then saline
ammonia standards must be prepared in a series of
salinities in order to quantify the matrix effect (the change
in the colorimetric response of ammonia due to the
change in the composition of the solution). The following
dilution of Primary Dilution Standard Solution (Section
7.2.7) and LNSW with reagent water to 100 ml in
volumetric flasks, are suggested.
Salinity Volume of
(%0) LNSW(mL)
Volume of Cone.
PDS(mL) mg N/L
0
9
18
27
35
0
25
50
75
98
2
2
2
2
2
.10
.10
.10
.10
.10
8.0 Sample Collection, Preservation and
Storage
8.1 Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Go-Flo or equivalent) attached at fixed intervals to
a hydro wire. These bottles are sent through the water
column open and are closed either electronically or via a
mechanical messenger when the bottles have reached
the desired depth.
8.1.2 In a submersible pump system, a weighted hose
is sent to the desired depth in the water column and water
is pumped from that depth to the deck of the ship for
sample processing.
8.1.3 For collecting surface samples, an acid - cleaned
plastic bucket or a large plastic bottle can be used as
convenient samplers. Wash the sampler three times with
sample water before collecting samples.
8.14 Turbid samples must be filtered through a 0.45
urn membrane filter as soon as possible after collection.
Wash the filter with reagent water before use. Pass at
least 100 ml of sample through the filter and discard
before taking the final sample. Care must be taken to
avoid the contamination of ammonia especially handling
low concentrations of ammonia (< 20 ug N/L) samples.10
An alternative technique to remove particulate is
centrifugation.
8.1.5 60-mL glass or high density polyethylene bottles
are used for sample storage. Sample bottles should be
rinsed 3 times with about 20 ml of sample, shaking with
the cap in place after each rinse. Pour the rinse water into
the cap to dissolve and rinse away salt crusts trapped in
the threads of the cap. Finally, fill the sample bottle about
3/4 full, and screw the cap on firmly.
8.2 Sample Preservation - After collection and
filtration or centrifugation, samples should be analyzed as
soon as possible. If samples will be analyzed within 3
hours then keep refrigerated in tightly sealed, glass or
high density polyethylene bottles in the dark at 4°C until
the analysis can be performed.
8.3 Sample Storage - At low concentrations of
ammonia (< 20 ug N/L), no preservation technique is
satisfactory. Samples must be analyzed within 3 hours of
collection. At moderate and high concentrations of
ammonia (> 20 ug N/L) samples can be preserved by the
addition of 2 mL of chloroform per liter of sample and
refrigerated in the dark at 4°C. Samples can be stored in
either glass or high density polyethylene bottles. A
maximum holding time for preserved estuarine and
coastal water samples with moderate to high
concentrations of ammonia is two weeks.12
9.0 Quality Control
9.1 Each laboratory using this method is required to
implement a formal quality control (QC) program. The
minimum requirements of this program consists of an
initial demonstration of performance, continued analysis
of Laboratory Reagent Blanks (LRB), laboratory
duplicates and Laboratory Fortified Blanks (LFB) with
each set of samples as a continuing check on
performance.
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9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance by determining
the MDL and LDR and laboratory performance by
analyzing quality control samples prior to analysis of
samples using this method.
9.2.2 A method detection limit (MDL) should be
established for the method analyte, using a low level
seawater sample containing, or fortified at, approximately
5 times the estimated detection limit. To determine MDL
values, analyze at least seven replicate aliquots of water
which have been processed through the entire analytical
method. Perform all calculations defined in the method
and report concentration in appropriate units. Calculate
the MDL as follows:
MDL = (t)(S)
where, S = the standard deviation of the
replicate analyses
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees
of freedom.
MDLs should be determined every 6 months or whenever
a significant change in background or instrument
response occurs or a new matrix is encountered.
9.2.3 The LDR should be determined by analyzing a
minimum of eight calibration standards ranging from
0.002 to 2.00 mg N/L across all sensitivity settings
(Absorbance Units Full Scale output range setting) of the
detector. Standards and sampler wash solutions should
be prepared in low nutrient seawater with salinities similar
to that of samples to avoid the necessity to correct for salt
error, or refractive index. Normalize responses by
multiplying the response by the Absorbance Units Full
Scale output range setting. Perform the linear regression
of normalized response vs. concentration and obtain the
constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher
concentration until the measured absorbance response,
R, of a standard no longer yields a calculated
concentration Cc, that is within 100 ± 10% of known
concentration, C, where Cc = (R-b)/m. That concentration
defines the upper limit of the LDR for the instrument.
Should samples be encountered that have a
concentration that is > 90% of the upper limit of LDR,
then these samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB with each set of
samples. LRB data are used to assess contamination
from the laboratory environment. Should an analyte value
in the LRB exceed the MDL, then laboratory or reagent
contamination should be suspected. When the LRB value
constitutes 10% or more of the analyte concentration
determined for a sample, duplicates of the sample must
be prepared and analyzed again after the source of
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2 Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB with each set of
samples. The LFB must be at a concentration within the
daily calibration range. The LFB data are used to
calculate accuracy as percent recovery. If the recovery of
the analyte falls outside the required control limits of 90
-110%, the source of the problem should be identified
and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB data to assess
laboratory performance against the required control limits
of 90 -110%. When sufficient internal performance data
become available (usually a minimum of 20 to 30
analyses), optional control limits can be developed from
the percent mean recovery (x) and standard deviation (S)
of the mean recovery. These data can be used to
establish the upper and lower control limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and available for review.
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9.4 Assessing Analyte Recovery -Laboratory
Fortified Sample Matrix (LFM)
9.4.1 A laboratory should add a known amount of
analyte to a minimum of 5% of the total number of
samples or one LFM per sample set, whichever is
greater. The analyte added should be 2-4 times the
ambient concentration and should be at least four times
greater than the MDL.
9.4.2 Calculate percent recovery of analyte, corrected
for background concentration measured in a separate
unfortified sample. These values should be compared
with the values obtained from the LFBs. Percent
recoveries may be calculated using the following
equation:
(CS-C)
x 100
where, R = percent recovery
Cs = measured fortified sample
addition in mg N/L
C = sample background
concentration (mg N/L)
S = concentration in mg N/L added
to the environmental sample.
9.4.3 If the recovery of the analyte falls outside the
required control limits of 90-110%, but the laboratory
performance for that analyte is within the control limits,
the fortified sample should be prepared again and
analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is
judged to be matrix related and the sample data should
be flagged accordingly.
10.0 Calibration and Standardization
10.1 At least five calibration standards should
prepared fresh daily for system calibration.
be
70.2 A calibration curve should be constructed for
each sample set by analyzing a series of calibration
standard solutions. A sample set should contain no more
than 60 samples. For a large number of samples make
several sample sets with individual calibration curves.
10.3 Analyze the calibration standards, in duplicate,
before the actual samples.
10.4 The calibration curve containing five data points
or more that bracket the conentrations of samples should
have a correlation coefficient, r, of 0.995 or better and the
range should not be greater than two orders of
magnitude.
70.5 Use a high CAL solution followed by two blank
cups to quantify system carryover. The difference in peak
heights between two blank cups is due to the carryover
from the high CAL solution. The carryover coefficient, k,
is calculated as follows:
k =
P - P
rb1 rb2
high
where, Phigh = the peak height of the high
ammonia standard
Pb1 = the peak height of the
first blank sample
Pb2 = the peak height of the
second blank sample
The carryover coefficient, k, should be measured in seven
replicates to obtain a statistically significant number. The
carryover coefficient should be remeasured with any
change in manifold plumbing or upon replacement of
pump tubes.
The carryover correction (CO) of a given peak, i, is
proportional to the peak height of the preceding sample,
PM-
CO = (k)x(PM)
To correct a given peak height reading, P,, subtract the
carryover correction.1314
Pi, = P:-CO
where Pic is corrected peak height. The correction for
carryover should be applied to all the peak heights
throughout a run. The carryover coefficient should be less
than 5% in this method.
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70.6 Place a high standard solution at the end of each
sample run to check for sensitivity drift. Apply sensitivity
drift correction to all the samples. The sensitivity drift
during a run should be less than 5%.
Note: Sensitivity drift correction is available in most data
acquisition software supplied with autoanalyzers. It is
assumed that the sensitivity drift is linear with time. An
interpolated drift correction factor is calculated for each
sample according to the sample position during a run.
Multiply the sample peak height by the corresponding
sensitivity drift correction factor to obtain the corrected
peak height for each sample.
11.0 Procedure
77.7 If samples are stored in a refrigerator, remove
samples and equilibrate to room temperature prior to
analysis.
77.2 Turn on the continuous flow analyzer and data
acquisition components and warm up at least 30 minutes.
in
77.3 Set up cartridge and pump tubes as shown i
Figure 1.
77.4 Set spectrophotometer wavelength to 640 nm,
and turn on lamp.
77.5 Set the Absorbance Unit Full Scale (AUFS) range
on the spectrophotometer at an appropriate setting
according to the highest concentration of ammonia in the
samples. The highest setting appropriate for this method
is 0.2 AUFS for 6 mg N/L.
77.6 Prepare all reagents and standards.
77.7 Choose an appropriate wash solution for sampler
wash. For analysis of samples with a narrow range of
salinities (± 2 %o) or for samples containing low ammonia
concentrations (< 20 ug N/L), it is recommended that the
CAL solutions be prepared in Low Nutrient Seawater
(Section 7.1.4) diluted to the salinity of samples, and that
the Sampler Wash Solution also be Low Nutrient
Seawater diluted to the same salinity. For samples with
varying salinities and higher ammonia concentrations (>
20 ug N/L), it is suggested that the reagent water used for
the sampler wash solution and for preparing calibration
standards and procedures in Section 12.2 and 12.3 be
employed.
77.8 Begin pumping the Brij-35 start-up solution
(Section 7.2.1) through the system and obtain a steady
baseline. Place the reagents on-line. The reagent
baseline will be higher than the start-up solution baseline.
After the reagent baseline has stabilized, reset the
baseline.
Note: To minimize the noise in the reagent baseline,
clean the flow system by sequentially pumping the
sample line with reagent water, 1 N HCI solution, reagent
water, 1 N NaOH solution for few minutes each at tahe
end of the daily analysis. Make sure to rinse the system
well with reagent water after pumping NaOH solution to
prevent precipitation of Mg(OH)2 when seawater is
introduced into the system.
samples free of particulate.
samples if necessary.
Keep the reagents and
Filter the reagents and
If the baseline drifts upward, pinch the waste line for a few
seconds to increase back pressure. If absorbance drops
down rapidly when backpressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach
a syringe at the waste outlet of the flowcell. Air bubbles
in the flowcell can often be eliminated by simply attaching
a syringe for a few minutes or, if not, dislodged by
pumping the syringe piston. Alternatively, flushing the
flowcell with alcohhol was found to be effective in
removing air bubbles from the flowcell.
77.9 The sampling rate is approximately 60 samples
per hour with 30 seconds of sample time and 30 seconds
of wash time.
77.70 Use cleaned sample cups or tubes (follow the
procedures outlined in Section 6.2.2). Place CAL
solutions and saline standards (optional) in sampler.
Complete filling the sampler tray with samples, laboratory
reagent blanks, laboratory fortified blanks, laboratory
fortified sample matrices, and QC samples. Place a blank
after every ten samples.
77.77 Commence analysis.
12.0 Data Analysis and Calculations
72.7 Concentrations of ammonia in samples are
calculated from the linear regression, obtained from the
standard curve in which the concentrations of the
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Version 1.0 September 1997
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calibration standards are entered as the independent
variable, and their corresponding peak heights are the
dependent variable.
72.2 Refractive Index Correction for Estuarine and
Coastal Samples
12.2.1 If reagent water is used as the wash solution, the
operator has to quantify the refractive index correction
due to the difference in salinity between sample and wash
solution. The following procedures are used to measure
the relationship between the sample salinity and refractive
index on a particular detector.
72.2.2 First, analyze a set of ammonia standards in
reagent water with color reagent using reagent water as
the wash and obtain a linear regression of peak height
versus concentration.
12.2.3 Second, replace reagent water wash solution with
Low Nutrient Seawater wash solution.
Note: In ammonia analysis absorbance of the reagent
water is higher than that of the LNSW. When using
reagent water as a wash solution, the change in refractive
index causes the absorbance of seawater to become
negative. To measure the absorbance due to refractive
index change in different salinity samples, Low Nutrient
Seawater must be used as the wash solution to bring the
baseline down.
72.2.4 Third, replace the phenol solution (Section 7.2.4)
and NaDTT solution (Section 7.2.5) with reagent water.
All other reagents remain the same. Replace the
synchronization sample with the colored SYNC peak
solution (Section 7.2.6).
12.2.5 Prepare a series of different salinity samples by
diluting the LNSW. Commence analysis and obtain peak
heights for different salinity samples. The peak heights for
the refractive index correction must be obtained at the
same AUFS range setting and on the same
spectrophotometer as the corresponding standards
(Section 12.2.2).
72.2.6 Using LNSW as the wash water, a maximum
absorbance will be observed for reagent water. No
change in refractive index will be observed in the
seawater sample. Assuming the absolute absorbance for
reagent water (relative to the seawater baseline) is equal
to the absorbance for seawater (relative to reagent water
baseline), subtract the absorbances of samples of various
salinities from that of reagent water. The results are the
apparent absorbance due to the change in refractive
index between samples of various salinities relative to the
reagent water baseline.
72.2.7 For each sample of varying salinity, calculate the
apparent ammonia concentration due to refractive index
from its peak height corrected to reagent water baseline
(Section 12.2.5) and the regression equation of ammonia
standards obtained with color reagent being pumped
through the system (Section 12.2.2). Salinity is entered as
the independent variable and the apparent ammonia
concentration due to refractive index is entered as the
dependent variable. The resulting regression allows the
operator to calculate apparent ammonia concentration
due to refractive index when the sample salinity is known.
Thus, the operator would not be required to obtain
refractive index peak heights for all samples.
72.2.8 The magnitude of refractive index correction can
be minimized by using a low refractive index flowcell. An
example of a typical result using a low refractive index
flowcell follows:
Salinity Apparent ammonia cone, due
(%0) to refractive index (ug N/L)
0.0
4.5
9.1
13.9
17.9
27.6
36.2
0.00
0.18
0.45
0.66
0.86
1.30
1.63
Note: You must calculate the refractive index correction
for your particular detector. The refractive index must be
redetermined whenever a significant change in the design
of the flowcell or a new matrix is encountered.
12.2.9 An example of a typical equation is:
Apparent ammonia (ug N/L) = 0.0134 + 0.0457S
where S is sample salinity in parts per thousand. The
apparent ammonia concentration due to refractive index
so obtained should then be added to samples of
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349.0-10
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corresponding salinity when reagent water was used as
the wash solution for samples analysis.
If a low refractive index flowcell is used and ammonia
concentration is greater than 200 ug N/L, the correction
for refractive index becomes negligible.
72.3 Correction for Matrix Effect in Estuarine and
Coastal Samples
12.3.1 When calculating concentrations of samples of
varying salinities from standards and wash solution
prepared in reagent water, it is necessary to first correct
for refractive index errors, then correct for the change in
color development due to the differences in composition
between samples and standards (matrix effect). Even
where the refractive index correction may be small, the
correction for matrix effect can be appreciable.
12.3.2 Plot the salinity of the saline standards (Section
7.2.9) as the independent variable, and the apparent
concentration of ammonia (mg N/L) from the peak height
(corrected for refractive index) calculated from the
regression of standards in reagent water, as the
dependent variable for all saline standards. The resulting
regression equation allows the operator to correct the
concentrations of samples of known salinity for the color
enhancement due to matrix effect. An example of a
typical result follows:
Salinity
(%o)
0
4.5
9.1
13.9
17.9
27.6
36.2
Peak height of
0.140mgN/L
2420
2856
2852
2823
2887
2861
2801
UncorrectedNH3
cone, calculated
from standards
in reagent water
(mg N/L)
0.1400
0.1649
0.1649
0.1635
0.1673
0.1663
0.1633
Corrected concentration (mg N/L)
= Uncorrected concentration /1.17(mg N/L)
12.3.4 Results of sample analyses should be reported
in mg N/L or in ug N/L.
mg N/L = ppm (parts per million)
ug N/L = ppb (part per billion)
13.0 Method Performance
73.7 Single Laboratory Validation
13.1.1 Method Detection Limit- A method detection limit
(MDL) of 0.3 ug N/L has been determined by one
laboratory from spiked LNSW of three different salinities
as follows:
Salinity
(%o)
36.2
36.2
36.2
36.2
17.9
17.9
17.9
17.9
4.5
4.5
4.5
0.0
0.0
0.0
[NH3]
(ug N/L)
0.7
0.7
1.4
1.4
0.7
0.7
1.4
1.4
0.7
1.4
1.4
0.7
0.7
1.4
SD
(ug N/L)
0.0252
0.0784
0.0826
0.0966
0.0322
0.0182
0.0938
0.0882
0.0672
0.1008
0.126
0.077
0.0784
0.0854
Recovery
(%)
95.4
100.8
104.7
105.6
106.5
92.2
109.1
100
95.1
94.1
106.7
98.2
100.8
101.9
MDL
(ug N/L)
0.0792
0.2463
0.2595
0.3035
0.1012
0.0572
0.2947
0.2771
0.2111
0.3167
0.3959
0.2419
0.2463
0.2683
13.1.2 Single Analyst Precision - A single laboratory
analyzed three samples collected from the Miami River
and Biscayne Bay, Florida. Seven replicates of each
sample were processed and analyzed with salinity
ranging from 4.8 to 35.0. The results were as follows:
72.3.3 Using the reagent described in Section 7.0, as
shown above, matrix effect becomes a single factor
independent of sample salinity. An example of a typical
equation to correct for matrix effect is:
349.0-11
Version 1.0 September 1997
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Sample
1
2
3
Salinity
(%o)
35.5
20.0
4.8
Concentration
(M9 N/L)
6.3
72.1
517.6
RSD
(%)
7.19
1.57
0.64
13.1.3 Laboratory Fortified Sample Matrix - Laboratory
fortified sample matrices were processed in three
different salinities ranging from 4.8 to 35.0 and ambient
ammonia concentrations from 0.0 to 72.1 |jg N/L. Seven
replicates of each sample were analyzed and the results
were as follows:
Salinity Concentration RSD Recovery
ambient fortified
(%0) (ug N/L)
35.5
20.0
4.8
6.3
72.1
0.0
70
140
280
5.01
1.71
1.81
98.3
98.3
98.1
13.1.4 Linear Dynamic Range - A linear dynamic range
(LDR) of 4.0 mg N/L has been determined by one
laboratory from spiked LNSW using a Flow Solution
System (Alpkem, Wilsonville, Oregon).
13.1.5 Sample Preservation Study - Natural samples
have been preserved by freezing, acidification and
addition of chloroform and phenol as preservatives to the
samples stored in glass and high density polyethylene
bottles. Table 1 summarized the results of preservation
study.
There is no significant difference in recovery of ammonia
from samples stored in glass and high density
polyethylene bottles, suggesting either glass or high
density polyethylene bottles can be used for storage of
ammonia samples.
For low concentration of ammonia samples (< 20 ug N/L,
sample 1 in table 1), no preservation technique is
satisfactory. Samples must be analyzed within 3 hours of
collection.
Freezing cannot preserve ammonia in samples for more
than one week. Acidified samples must be neutralized
with NaOH solution prior to analysis. Addition of NaOH to
acidified samples induces the precipitation of Mg(OH)2
and Ca(OH)2. Centrifuging the samples cannot
completely eliminate the interference, therefore,
acidification is not suitable preservation technique.
Addition of phenol increases the absorbance of samples.
Phenol is not recommended as a suitable preservative
although samples preserved with phenol were stable as
those preserved by chloroform.12
For moderate and high concentrations of ammonia (> 20
ug N/L) samples, it is suggested samples be preserved
by the addition of 2 mL of chloroform per liter of sample
and refrigerated in the dark at 4°C. A maximum holding
time for preserved estuarine and coastal water samples
with moderate to high concentrations of ammonia is two
weeks.10
13.2 Multi-Laboratory Validation
Multi-laboratory data is unavailable at this time.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the agency recommends recycling as the next
best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society, Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
75.7 The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
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349.0-12
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For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
16.0 References
1. Solorzano, L. 1969. Determination of ammonia in
natural waters by the phenylhypochlorite method.
Limnol. Oceanogr., 14:799-801.
2. Head, P.C., 1971. An automated
phenolhypochlorite method for the determination of
ammonia in sea water. Deep-Sea Research,
18:531-532.
3. Slawyk, G., and Maclsaac, J.J., 1972. Comparison
of two automated ammonia methods in a region of
coastal upwelling. Deep-Sea Research, 19:521-
524.
11. Aminot A. and R. Kerouel, 1996. Stability and
preservation of primary calibration solutions of
nutrients. Mar. Chem. 52:173-181.
12. Degobbis, D. 1973. On the storage of seawater
samples for ammonia determination. Limnol.
Oceanogr., 18:146-150.
13. Angelova, S, and H.W.Holy. 1983. Optimal speed
as a function of system performance for continuous
flow analyzers. Analytica Chimica Acta, 145:51-58.
14. Zhang, J.-Z. 1997. Distinction and quantification of
carry-over and sample interaction in gas
segmented continuous flow analysis. Journal of
Automatic Chemistry, 19(6):205-212.
6.
Hansen, H.P. and Grasshoff, K. 1983, Automated
Chemical Analysis, In Methods of Seawater
Analysis (Grasshoff, K., M. Ehrhardt and K.
Kremling, Eds) Weinheim, Verlag Chemie,
Germany. pp363-365.
Mautoura, R.F.C. and E.M.S. Woodward, 1983.
Optimization of the indophenol blue method for the
automated determination of ammonia in estuarine
waters. Estuarine, Coastal and Shelf Science,
17:219-224.
Zhang J-Z, and F. J. Millero 1993. The chemistry of
anoxic waters in the Cariaco Trench. Deep-Sea
Res.,40:1023-1041.
Raymont, J.E.G. 1980. Plankton and productivity in
the oceans. Pergamon Press, Oxford, England.
Smith, S.L. and T.E. Whitledge. 1977. The role of
zooplankton in the regeneration of nitrogen in a
coastal upwelling off northwest Africa. Deep-Sea
Res. 24: 49-56.
9. Code of Federal Regulations 40, Ch. 1, Pt. 136
Appendix B. Definition and Procedure for the
Determination of Method Detection Limit. Revision
1.11.
10. Eaton, A.D. and V. Grant, 1979. Sorption of
ammonium by glass frits and filters: implications for
analyses of blakish and freshwater. Limnol.
Oceanogr. 24:397-399.
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Debubbler
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1 1 S
J Detector
^ 640nm
Coil ^
C
aste
O 1
10 O-
o
_7~ ~
6
f^^^^^^~
--0--5--
4
o~ "
0 3
r\ ^_ ^
1
Manifold
Wash To Sarr
o Waste \
/
\
Heater
B ^
J
\
iplei \
0.41
0.41
0.10
0.10
0.10
1.01
0.25
0.32
1.57
)
r
Nitroferricyanide
NaDTT
Phenol
—| Sample
1 i
Nitrogen
Complexing Reagent
Reagent Water
or Low Nutrient Seawater
Pump
mL/min
Sample:Wash = 30":30"
Figure 1. Manifold Configuration for Ammonia Analysis.
Version 1.0 September 1997
349.0-14
-------
Table 1 . Percentage RecoveryA of Ammonia From Natural Water Samples Preserved by Freezing, Acidification,
Addition of Chloroform and Phenol.
sample8
1
1 +
2
2+
3
method0
none
freezing
H2S04E
CHCI3
phenol F
freezing
H2S04E
CHCI3
phenol F
none
freezing
H2S04E
CHCI3
phenol F
freezing
H2S04E
CHCI3
phenol F
none
freezing
H2S04E
CHCI3
phenol F
bottle0
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
glass
HOPE
0
100
100
100
200
200
193
193
153
153
100
100
95
95
96
96
130
130
100
100
100
252
252
99
99
108
108
99
99
100
100
99
99
117
117
100
100
100
101
101
100
100
112
112
7
349
100
102
564
113
135
193
36
36
101
97
105
91
105
102
133
128
32
109
107
162
193
114
98
107
101
108
106
107
102
106
107
121
124
104
-
108
106
108
96
93
106
112
time (day)
14
345
0
0
285
64
29
18
44
0
82
76
69
91
85
85
110
102
0
93
82
66
45
83
80
88
83
109
95
51
39
116
98
106
107
14
116
105
44
111
98
97
107
108
21
18
0
0
73
45
47
44
0
0
77
61
54
88
78
78
148
103
0
77
67
62
41
75
70
74
74
111
78
86
98
94
95
105
106
1
64
65
74
106
96
95
112
110
28
91
0
0
55
36
36
36
0
0
102
81
37
116
89
92
123
118
0
88
91
50
27
96
83
93
86
106
91
88
107
105
103
116
117
0
106
75
61
109
94
95
125
112
Recovery is calculated based on the ammonia concentration in non-preserved sample at day 0. Samples with
recoveries higher than 100% are subject to interference either from precipitation or phenol.
For salinity and concentration of ammonia in samples 1,2,3 see Section 13.1.2.
349.0-15
Version 1.0 September 1997
-------
Sample 1+ and 2+ are the fortified samples 1 and 2 at ammonia concentrations 76.3 and 202.1 |jg N/L,
respectively.
Methods of preservation:
A/one: stored the samples in high density polyethylene carboys at room temperature without any
preservative added.
Freezing: Frozen and stored at -20°C.
H2SO4: Acidified to pH 1.8 with H2SO4, and stored at 4°C. Neutralized the acid with
NaOH solution before analysis.
CHC/3/Added 2 ml chloroform per 1000 ml sample, and stored at 4°C.
Phenol: Added 8 g phenol per 1000 ml sample, and stored at 4°C.
Glass and high density polyethylene bottles were compared to determine the effect of sample
bottle type on the preservation.
Adding NaOH to neutralize acidified samples induced the precipitation of Mg(OH)2 and Ca(OH)2.
Centrifuging the samples can not completely eliminate the interference, therefore, acidification is
not suitable preservation technique.
Although samples preserved with phenol were stable as those preserved by chloroform,
an absorbance increase was observed, therefore, this is not recommended as a suitable
preservation technique.
Version 1.0 September 1997 349.0-16
-------
Method 353.4
Determination of Nitrate and Nitrite in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel
School of Marine and Atmospheric Science/AOML, NOAA, University of Miami, Miami,
FL33149
Peter B. Ortner and Charles J. Fischer, Ocean Chemistry Division, Atlantic
Oceanographic and Meteorological Laboratory, National Oceanic and Atmospheric
Administration, Miami, FL 33149
Project Officer
Elizabeth J. Arar
Revision 2.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
353.4-1
-------
Method 353.4
Determination of Nitrate and Nitrite in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for determining
nitrate and nitrite concentrations in estuarine and coastal
waters. Nitrate is reduced to nitrite by cadmium,1"3 and the
resulting nitrite determined by formation of an azo dye.4"6
In most estuarine and coastal waters nitrogen is thought
to be a limiting nutrient. Nitrate is the final oxidation
product of the nitrogen cycle in natural waters and is
considered to be the only thermodynamically stable
nitrogen compound in aerobic waters.7 Nitrate in
estuarine and coastal water is derived from rock
weathering, sewage effluent and fertilizer run-off. The
concentration of nitrate usually is high in estuarine waters
and lower in surface coastal waters.
Nitrite is an intermediate product in the microbial
reduction of nitrate or in the oxidation of ammonia. It may
also be excreted by phytoplankton as a result of excess
assimilatory reduction. Unlike nitrate, nitrite is usually
present at a concentration lower than 0.01 mg N/L except
in high productivity waters and polluted waters in the
vicinity of sewer outfalls.
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Nitrate
Nitrite
14797-55-8
14797-65-0
1.2 A statistically determined method detection limit
(MDL)8 of 0.075 ug N/L has been determined by one
laboratory in seawaters of five different salinities. The
method is linear to 5.0 mg N/L using a Flow Solution
System (Alpkem, Wilsonville, Oregon).
1.3 Approximately 40 samples per hour can be
analyzed.
1.4 This method requires experience in the use of
automated gas segmented continuous flow colorimetric
analyses, and familiarity with the techniques of
preparation, activation and maintenance of the cadmium
reduction column. A minimum of six-months training is
recommended.
2.0 Summary of Method
2.1 An automated gas segmented continuous flow
colorimetric method for the analysis of nitrate
concentration is described. In the method, samples are
passed through a copper-coated cadmium reduction
column. Nitrate in the sample is reduced to nitrite in a
buffer solution. The nitrite is then determined by
diazotizing with sulfanilamide and coupling with N-1-
naphthylethylenediamine dihydrochloride to form a color
azo dye. The absorbance measured at 540 nm is linearly
proportional to the concentration of nitrite + nitrate in the
sample. Nitrate concentrations are obtained by
subtracting nitrite values, which have been separately
determined without the cadmium reduction procedure,
from the nitrite + nitrate values. There is no significant salt
error in this method. The small negative error caused by
differences in the refractive index of seawater and
reagent water is readily corrected for during data
processing.
3.0 Definitions
3.1 Calibration Standard (CAL) - A solution
prepared from the primary dilution standard solution or
stock standard solution containing analytes. The CAL
solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.2 Laboratory Fortified Blank (LFB) - An aliquot of
reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine
whether method performance is within acceptable control
limits, and whether the laboratory is capable of making
accurate and precise measurements. This is a standard
prepared in reagent water that is analyzed as a sample.
Revision 2.0 September 1997
353.4-2
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3.3 Laboratory Fortified Sample Matrix (LFM) - An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
3.4 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water that is treated exactly as a sample
including exposure to all labware, equipment, and
reagents that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
the reagents, or apparatus.
3.5 Linear Dynamic Range (LDR) - The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.6 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.8
3.7 Reagent Water (RW) - Type 1 reagent grade
water equal to or exceeding standards established by
American Society for Testing and Materials (ASTM).
Reverse osmosis systems or distilling units followed by
Super-Q Plus Water System that produce water with 18
megohm resistance are examples of acceptable water
sources. To avoid contamination, the reagent water
should be used the day of preparation.
3.8 Refractive Index (Rl) - The ratio of velocity of
light in a vacuum to that in a given medium. The relative
refractive index is the ratio of the velocity of light in two
different media, such as estuarine or sea water versus
reagent water. The correction for this difference is
referred to as the refractive index correction in this
method.
3.9 Stock Standard Solution (SSS) -A concentrated
solution of method analyte prepared in the laboratory
using assayed reference compounds or purchased from
a reputable commercial source.
3.70 Primary Dilution Standard Solution (PDS) - A
solution prepared in the laboratory from stock standard
solutions and diluted as needed to prepare calibration
solutions and other needed analyte solutions.
3.11 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.72 SYNC Peak Solution - A colored solution used
to produce a synchronization peak in the refractive index
measurement. A synchronization peak is required by the
data acquisition programs to initialize the peak finding
parameters. The first cup in every run must always be
identified as a SYNC sample. The SYNC sample is
usually a high standard, but can be any sample that
generates a peak at least 25% of full scale.
4.0 Interferences
4.1 Hydrogen sulfide at concentrations greater than
0.1 mg S/L can interfere with nitrite analysis by
precipitating on the cadmium column .9 Hydrogen sulfide
in samples must be removed by precipitation with
cadmium or copper salt.
4.2 Iron, copper and other heavy metals at
concentrations larger than 1 mg/L alter the reduction
efficiency of the cadmium column. The addition of EDTA
will complex these metal ions.10
4.3 Phosphate at a concentration larger than 0.1
mg/L decreases the reduction efficiency of cadmium11.
Dilute samples if possible or remove phosphate with ferric
hydroxide12 prior to analysis.
4.4 Particulates inducing turbidity should be removed
by filtration after sample collection.
4.5 This method corrects for small refractive index
interference which occurs if the calibration standard
solution is not matched with samples in salinity.
5.0 Safety
5.1 Water samples collected from the estuarine and
coastal environment are generally not hazardous.
353.4-3
Revision 2.0 September 1997
-------
However, the individual who collects samples should use
proper technique.
5.2 Good laboratory technique should be used when
preparing reagents. Laboratory personnel should obtain
material safety data sheets (MSDS) for all chemicals
used in this method. A lab coat, safety goggles, and
gloves should be worn when handling the concentrated
acid.
6.0 Equipment and Supplies
6.7 Gas Segmented Continuous Flow Autoanalyzer
Consisting of:
6.1.1 Autosampler.
6.12 Analytical cartridge with reaction coils for nitrate
analysis.
6.1.3 Open Tubular Cadmium Reactor (OTCR,
Alpkem, OR) or laboratory prepared packed copper-
coated cadmium reduction column (prepared according
to procedures in Section 7.4 - 7.5).
6.14 Proportioning pump.
6.1.5 Spectrophotometer equipped with a tungsten
lamp (380-800 nm) or photometer with a 540
interference filter (2 nm bandwidth).
nm
6.16 Strip chart recorder
acquisition system.
or computer based data
6.17 Nitrogen gas (high-purity grade, 99.99%).
6.2 Glassware and Supplies
6.2.1 All labware used in the analysis must be low in
residual nitrate to avoid sample or reagent contamination.
Soaking with lab grade detergent, rinsing with tap water,
followed by rinsing with 10% HCI (v/v) and thoroughly
rinsing with reagent water is sufficient.
6.2.2 Automatic pipetters capable of delivering volumes
ranging from 100 uL to 1000 uL and 1 ml to 10 ml with
an assortment of high quality disposable pipet tips.
6.2.3 Analytical balance, with capability to measure to
0.1 mg, for preparing standards.
6.2.4 60 ml high density polyethylene sample bottles,
glass volumetric flasks and plastic sample tubes.
6.2.5 Drying oven.
6.2.6 Desiccator.
6.2.7 Membrane filters with 0.45 urn nominal pore size.
Plastic syringes with syringe filters.
6.2.8 A pH meter with a glass electrode and a
reference electrode. A set of standard buffer solutions for
calibration of the pH meter.
7.0 Reagents and Standards
7.7 Stock Reagent Solutions
7.1.1 Stock Sulfanilamide Solution - Dissolved 10 g of
sulfanilamide (C6H8N2O2S, FW 172.21) in 1 L of 10%
HCI.
7.12 Stock Nitrate Solution (100 mg-N/L) -
Quantitatively transfer 0.7217 g of pre-dried (105°C for 1
hour) potassium nitrate (KNO3, FW 101.099) to a 1000-
ml_ glass volumetric flask containing approximate 800 ml
of reagent water and dissolve the salt. Dilute the solution
to the mark with reagent water. Store the stock solution in
a polyethylene bottle in refrigerator at 4°C. This solution
is stable for six months.
7.13 Stock Nitrite Solution (100 mg-N/L)
Quantitatively transfer 0.4928 g of pre-dried (105°C for 1
hour) sodium nitrite (NaNO2, FW 68.99) to a 1000 ml
glass volumetric flask containing approximate 800 ml of
reagent water and dissolve the salt. Dilute the solution to
the mark with reagent water. Store the stocksolution in a
polyethylene bottle in a refrigerator at 4°C- Thls solutlon
is stable for three months.
Note: High purity nitrite salts are not available. Assays
given by reagent manufacturers are usually in the range
of 95-97%. The impurity must be taken into account for
calculation of the weight taken.
7.1.4 Low Nutrient Sea Water (LNSW) - Obtain natural
low nutrient seawater from surface water of the Gulf
Stream or Sargasso Sea (salinity 36 %o, < 7 ug N/L) and
filter it through 0.3 micron pore size glass fiber filters. If
this is not available, commercial low nutrient sea water
(< 7 ug N/L) with salinity of 35 %o (Ocean Scientific
International, Wormley, U.K.) can be substituted.
Revision 2.0 September 1997
353.4-4
-------
7.2 Working Reagents
7.2.1 Brij-35 Start-up Solution - Add 2 ml of Brij-35
surfactant (ICI Americas, Inc.) to 1000 ml reagent water
and mix gently.
Note: Brij-35 is a trade name for polyoxyethylene(23)
lauryl ether (C^H^OCH^H^OH, FW=1199.57, CASRN
9002-92-0).
7.2.2 Working Sulfanilamide Solution - Add 1 ml of
Brij- 35 solution to 200 ml of stock sulfanilamide solution,
mix gently.
Note: Adding surfactant Brij-35 to sulfanilamide solution
instead of to the buffer solution is to prevent the Brij from
being adsorbed on the cadmium surface, which may
result in decreasing surface reactivity of the cadmium and
reduce the lifetime of the cadmium column.
7.2.3 NED Solution - Dissolve 1 g of NED (N-1-
naphthylethylenediamine Dihydrochloride, C12H14N2.2HCI,
FW 259.18) in 1 L of reagent water.
7.2.4 Imidazole Buffer Solution - Dissolve 13.6 g of
imidazole (C3H4N2, FW 68.08) in 4 L of reagent water.
Add 2 ml of concentrated HCI. Adjust the pH to 7.8 with
diluted HCI while monitoring the pH with a pH meter.
Store in a refrigerator.
7.2.5 Copper Sulfate Solution (2%) - Dissolve 20 g of
copper sulfate (CuSO4.5H p, FW 249.61) in 1 L of
reagent water.
7.2.6 Colored SYNC Peak Solution - Add 50 uL of red
food coloring solution to 1000 ml reagent water and mix
thoroughly. Further dilute this solution to obtain a peak
between 25 to 100 percent full scale according to the
AUFS setting used for the refractive index measurement.
7.2.7 Primary Dilution Standard Solution - Prepare a
primary dilution standard solution (5 mg N/L) by dilution of
5.0 ml of stock standard solutions to 100 ml with reagent
water. Prepare this solution daily.
Note: This solution should be prepared to give an
appropriate intermediate concentration for further dilution
to prepare the calibration solutions. Therefore the
concentration of a primary dilution standard solution
should be adjusted according to the concentration range
of calibration solutions.
7.2.8 Calibration Standards - Prepare a series of
calibration standards (CAL) by diluting suitable volumes
of a primary dilution standard solution (Section 7.2.7) to
100 ml with reagent water or low nutrient seawater.
Prepare these standards daily. The concentration range
of calibration standards should bracket the expected
concentrations of samples and not exceed two orders of
magnitude. At least five calibration standards with equal
increments in concentration should be used to construct
the calibration curve.
If nitrate + nitrite and nitrite are analyzed simultaneously
by splitting a sample into two analytical systems, a nitrate
and nitrite mixed standard should be prepared. The total
concentration (nitrate+nitrite) must be assigned to the
concentrations of calibration standards in the
nitrate+nitrite system.
When analyzing samples of varying salinities, it is
recommended that the calibration standard solutions and
sampler wash solution be prepared in reagent water and
corrections for refractive index be made to the sample
concentrations determined (Section 12.2).
7.2.9 Saline Nitrate and Nitrite Standards - If CAL
solutions will not be prepared to match sample salinity,
then saline nitrate and nitrite standards must be prepared
in a series of salinities in order to quantify the salt error,
the change in the colorimetric response of nitrate due to
the change in the composition of the solution. The
following dilutions of Primary Dilution Standard Solution
(Section 7.2.7) to 100 ml in volumetric flasks with
reagent water, are suggested:
Salinity
(%c)
0
9
18
27
35
Volume of
LNSW(mL)
0
25
50
75
98
Volume of
PDS(mL)
2
2
2
2
2
Cone.
mgN/L
.10
.10
.10
.10
.10
7.3 Open Tubular Cadmium Reactor
7.3.1 Nitrate in the samples is reduced to nitrite by
either a commercial Open Tubular Cadmium Reactor
(OTCR, Alpkem, OR) or a laboratory-prepared packed
copper-coated cadmium reduction column.
353.4-5
Revision 2.0 September 1997
-------
7.3.2 If an OTCR is employed, the following procedures
should be used to activate it.10
Prepare reagent water, 0.5N HCI solution and 2% CuSO4
solution in three 50 ml beakers. Fit three 10-mL plastic
syringes with unions. First flush the OTCR with 10 ml
reagent water. Then flush it with 10 ml 0.5N HCI
solution in 3 seconds, immediately followed by flushing
with a couple of syringe volumes of reagent water. Slowly
flush with CuSO4 solution until a large amount of black
precipitated copper come out of OTCR, then stop the
flushing. Finally flush the OTCR with reagent water. Fill
the OTCR with imidazole buffer for short term storage.
7.4 Packed Cadmium Reduction Column
The following procedures are used for preparation of a
packed cadmium reduction column.13
7.4.1 File a cadmium stick to obtain freshly prepared
cadmium filings.
7.4.2 Sieve the filings and retain the fraction between 25
and 60 mesh size (0.25-0.71 mm).
7.4.3 Wash filings two times with 10% HCI followed with
reagent water.
7.4.4 Decant the reagent water and add 50 ml of 2%
CuSO4 solution. While swirling, brown flakes of colloidal
copper will appear and the blue color of the solution will
fade. Decant the faded solution and add fresh CuSO4
solution and swirl. Repeat this procedure until the blue
color does not fade.
7.4.5 Wash the filings with reagent water until all the
blue color is gone and the supernatant is free of fine
particles. Keep the filings submersed under reagent water
and avoid exposure of the cadmium filings to air.
7.4.6 The column can be prepared in a plastic or aglass
tube of 2 mm ID. Plug one end of column with glass wool.
Fill the column with water and transfer Cd filings in
suspension using a 10 ml pipette tip connected to one
end of column. While gently tapping the tube and pipette
tip let Cd filings pack tightly and uniformly in the column
without trapping air bubbles.
7.4.7 Insert another glass wool plug at the top of the
column. If a U- shape tube is used, the pipette tip is
connected to the other end and the procedure repeated.
Connect both ends of the column using a plastic tube
filled with buffer solution to form a closed loop.
7.4.8 If an OTCR or a packed cadmium column has
not been used for several days, it should be reactivated
prior to sample analysis.
7.5 Stabilization of OTCR and Packed Cadmium
Reduction Columns
7.5.1 Pump the buffer and other reagent solutions
through the manifold and obtain a stable baseline.
7.5.2 Pump 0.7 mg-N/L nitrite standard solution
continuously through the sample line and record the
steady state signal.
7.5.3 Stop the pump and install an OTCR or a packed
column on the manifold. Ensure no air bubbles have
been introduced into the manifold during the installation.
Resume the pumping and confirm a stable baseline.
7.5.4 Pump 0.7 mg-N/L nitrate solution continuously
through the sample line and record the signal. The signal
will increase slowly and reach steady state in about 10-15
minutes. This steady state signal should be close to the
signal obtained from the same concentration of a nitrite
solution without the OTCR or packed cadmium column
on line.
7.5.5 The reduction efficiency of an OTCR or a packed
cadmium column can be determined by measuring the
absorbance of a nitrate standard solution followed by a
nitrite standard solution of the same concentration.
Reduction efficiency is calculated as follows:
Reduction Efficiency =
Absorbance of Nitrate
Absorbance of Nitrite
8.0 Sample Collection, Preservation and
Storage
8.1 Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Go-Flo or equivalent) that are attached at fixed
intervals to a hydro wire. These bottles are sent through
the water column open and are closed either
Revision 2.0 September 1997
353.4-6
-------
electronically or via a mechanical messenger when the
bottles have reached the desired depth.
8.1.2 In a submersible pump system, a weighted hose is
sent to the desired depth in the water column and water
is pumped from that depth to the deck of the ship for
sample processing.
8.13 For collecting surface samples, an acid - cleaned
plastic bucket or a large plastic bottle can be used as a
convenient sampler. Wash the sampler three times with
sample water before collecting samples.
8.14 Turbid samples should be filtered as soon as
possible after collection.
8.15 60-mL high density polyethylene bottles are used
for sample storage. Sample bottles should be rinsed 3
times with about 20 ml of sample, shaking with the cap
in place after each rinse. Pour the rinse water into the cap
to dissolve and rinse away salt crusts trapped in the
threads of the cap. Finally, fill the sample bottle about 3/4
full, and screw the cap on firmly.
8.2 Sample Preservation - After collection and
filtration, samples should be analyzed as soon as
possible. If samples will be analyzed within 3 hours then
keep refrigerated in tightly sealed, high density
polyethylene bottles in the dark at 4°C until analysis can
be performed.
8.3 Sample Storage - Natural samples usually
contain low concentrations of nitrite (< 14 g N/L) and no
preservation techniques are satisfactory.14 Samples must
be analyzed within 3 hours of collection to obtain reliable
nitrite concentrations.15
Samples containing high concentrations of ammonia or
nitrite may change in nitrate concentration during storage
due to microbial oxidation of ammonia and nitrite to
nitrate. These samples should be analyzed as soon as
possible.
Natural samples containing low concentrations of nitrite
and ammonia ( < 10% of the nitrate concentration ) can
be preserved for nitrate analysis by freezing. A maximum
holding time for preserved estuarine and coastal water
samples for nitrate analysis is one month.16
The results of preservation of natural samples are shown
in Tables 1 and 2 for nitrate and nitrite, respectively.
9.0 Quality Control
9.1 Each laboratory using this method is required to
implement a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of performance, continued analysis of
Laboratory Reagent Blanks (LRB), laboratory duplicates
and Laboratory Fortified Blanks (LFB) with each set of
samples as a continuing check on performance.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance by determining
the MDL and LDR and laboratory performance by
analyzing quality control samples prior to analysis of
samples using this method.
9.2.2 A method detection limit (MDL) should be
established for the method analytes using a low level
seawater sample containing, or fortified at, approximately
5 times the estimated detection limit. To determine MDL
values, analyze at least seven replicate aliquots of water
which have been processed through the entire analytical
method. Perform all calculations defined in the method
and report concentration in appropriate units. Calculate
the MDL as follows:
MDL = (t)(S)
where, S = the standard deviation of the
replicate analyses
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees
of freedom.
MDLs should be determined every six months or
whenever a significant change in background or
instrument response occurs or a new matrix is
encountered.
9.2.3 The LDR should be determined by analyzing a
minimum of eight calibration standards ranging from
0.002 to 2.00 mg N/L across all sensitivity settings
(Absorbance Units Full Scale output range setting) of the
detector. Standards and sampler wash solutions should
be prepared in low nutrient seawater with salinities similar
to that of samples, therefore a correction factor for salt
error, or refractive index, will not be necessary. Normalize
353.4-7
Revision 2.0 September 1997
-------
responses by multiplying the response by the Absorbance
Units Full Scale output range setting. Perform the linear
regression of normalized response vs. concentration and
obtain the constants m and b, where m is the slope and
b is the y-intercept. Incrementally analyze standards of
higher concentration until the measured absorbance
response, R, of a standard no longer yields a calculated
concentration Cc, that is within 100 ± 10% of known
concentration, C, where Cc = (R-b)/m. That concentration
defines the upper limit of the LDR for the instrument.
Should samples be encountered that have a
concentration that is > 90% of the upper limit of LDR,
then these samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB with each set of
samples. LRB data are used to assess contamination
from the laboratory environment. Should an analyte value
in the LRB exceed the MDL, then laboratory or reagent
contamination should be suspected. When the LRB value
constitutes 10% or more of the analyte concentration
determined for a sample, duplicates of the sample must
be prepared and analyzed again after the source of
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2 Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB with each set of
samples. The LFB must be at a concentration that is
within the daily calibration range. The LFB data are used
to calculate accuracy as percent recovery. If the recovery
of the analyte falls outside the required control limits of
90 -110%, the source of the problem should be identified
and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required
control limits of 90-110%. When sufficient internal
performance data become available (usually a minimum
of 20 to 30 analyses), optional control limits can be
developed from the percent mean recovery (x) and
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing
Laboratory
(LFM)
Analyte Recovery
Fortified Sample Matrix
9.4.1 A laboratory should add a known amount of
analyte to a minimum of 5% of the total number of
samples or one sample per sample set, whichever is
greater. The analyte added should be 2-4 times the
ambient concentration and should be at least four times
greater than the MDL.
9.4.2 Calculate percent recovery of analyte, corrected
for background concentration measured in a separate
unfortified sample. These values should be compared
with the values obtained from the LFBs. Percent
recoveries may be calculated using the following
equation:
(CS-C)
R = x100
where,
R = percent recovery
Cs = measured fortified sample concentration
(background + addition in mg N/L)
C = sample background concentration (mg N/L)
S = concentration in mg N/L added to the
environmental sample.
9.4.3 If the recovery of the analyte falls outside the
required control limits of 90-110%, but the laboratory
performance for that analyte is within the control limits,
the fortified sample should be prepared again and
analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample
is judged to be the matrix related and the sample data
should be flagged.
Revision 2.0 September 1997
353.4-8
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10.0 Calibration and Standardization
CO = (k)(PM)
10.1 At least five calibration standards should be
prepared fresh daily for system calibration. The
calibration concentrations should bracket the
concentrations of samples and the range should not be
over two orders of magnitude.
70.2 A calibration curve should be constructed for
each sample set by analyzing a series of calibration
standard solutions. A sample set should contain no more
than 60 samples. For a large number of samples make
several sample sets with individual calibration curves.
10.3 Analyze the calibration standards, in duplicate,
before actual samples.
10.4 The calibration curve containing five or more data
points should have a correlation coefficient, r, of 0.995 or
better.
70.5 Place a high CAL solution followed by two blank
cups to quantify the carry-over of the system. The
difference in peak heights between two blank cups is due
to the carry over from the high CAL solution. The carry-
over coefficient, k, is calculated as follows:
PM -
k =
high
where,
high = the peak height of the high
nitrate standard
Pb1 = the peak height of the
first blank sample
Pb2 = the peak height of the
second blank sample.
The carry over coefficient, k, for a system should be
measured in seven replicates to obtain a statistically
significant number, k should be remeasured with any
change in manifold plumbing or upon replacement of
pump tubung.
The carry over correction (CO) on a given peak i is
proportional to the peak height of the preceding sample,
To correct a given peak height reading, Ph subtract the
carry over correction,1718
PI.C = PI - CO
where Pic is corrected peak height. The correction for
carry over should be applied to all the peak heights
throughout a run. The carry over coefficient should be
less than 5% in this method.
10.6 Place a high standard nitrate solution followed by
a nitrite standard solution of same concentration at the
beginning and end of each sample run to check for
change in reduction efficiency of OTCR or a packed
cadmium column. The decline of reduction efficiency
during a run should be less than 5%.
70.7 Place a high standard solution at the end of each
sample run (60 samples) to check for sensitivity drift.
Apply sensitivity drift correction to all the samples. The
sensitivity drift during a run should be less than 5%.
Note: Sensitivity drift correction is available in most data
acquisition software supplied with autoanalyzers. It is
assumed that the sensitivity drift is linear with time. An
interpolated drift correction factor is calculated for each
sample according to the sample position during a run.
Multiply the sample peak height by the corresponding
sensitivity drift correction factor to obtain the corrected
peak height for each sample.
11.0 Procedure
77.7 If samples are frozen, thaw the samples at room
temperature. If samples are stored in a refrigerator,
remove samples and equilibrate to room temperature.
Mix samples thoroughly prior to analysis.
77.2 Turn on the continuous flow analyzer and data
acquisition components and warm up at least 30 minutes.
77.3 Set up the cartridge according to the type of
cadmium reductor used for nitrate + nitrite analysis
(configuration for OTCR shown in Figure 1 and packed
cadmium column in Figure 2). Configuration for analysis
of nitrite alone is shown in Figure 3.
Note: When a gas segmented flow stream passes
through the OTCR, particles derived from the OTCR
were found to increase baseline noise and to cause
353.4-9
Revision 2.0 September 1997
-------
interference at low level analysis. Packed cadmium
columns are, therefore, preferred for nitrate analysis at
low concentrations.
77.4 Set spectrophotometer wavelength at 540 nm.
77.5 Set the Absorbance Unit Full Scale (AUFS) range
on the spectrophotometer at an appropriate setting
according to the highest concentration of nitrate in the
samples. The appropriate setting for this method is 0.2
AUFS for 0.7 mg N/L.
77.6 Prepare all reagents and standards.
77.7 Begin pumping the Brij-35 start-up solution (Section
7.2.1) through the system and obtain a steady baseline.
Place the reagents on-line. The reagent baseline will be
higher than the start-up solution baseline. After the
reagent baseline has been stabilized, reset the baseline.
NOTE: To minimize the noise in the reagent baseline,
clean the flow system by sequentially pumping the
sample line with reagent water, 1 N HCI solution, reagent
water, 1 N NaOH solution for a few minutes each at the
end of the daily analysis. Make sure to rinse the system
well with reagent water after pumping NaOH solution to
prevent precipitation of Mg(OH)2 when seawater is
introduced into the system. Keep the reagents and
samples free of particulate. Filter the reagents and
samples if necessary.
If the baseline drifts upward, pinch the waste line for a few
seconds to increase back pressure. If absorbance drops
down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach
a syringe at the waste outlet of the flowcell. Air bubbles
in the flowcell can often be eliminated by simply attaching
syringe for a few minutes or, if not, dislodged by pumping
the syringe piston. Alternatively, flushing the flowcell with
alcohol was found to be effective in removing air bubbles
from the flow cell.
For samples of varying salinities, it is suggested that the
reagent water used for the sampler wash solution and for
preparing calibration standards and procedures in
Sections 12.2 and 12.3 be employed.
77.8 Check the reduction efficiency of the OTCR or
packed cadmium column following the procedure in
Section 7.5.5. If the reduction efficiency is less than 90%
follow the procedure in Section 7.5 for activation and
stabilization. Ensure reduction efficiencies reach at least
90% before analysis of samples.19
77.9 A good sampling rate is approximately 40 samples
per hour for 60 second sample times and 30 second
wash times.
77.70 Use cleaned sample cups or tubes (follow the
procedures outlined in Section 6.2.2). Place CAL
solutions and saline standards (optional) in sampler.
Complete filling the sampler tray with samples, laboratory
reagent blanks, laboratory fortified blanks, laboratory
fortified sample matrices, and QC samples. Place a blank
after every ten samples.
77.77 Commence analysis.
12.0 Data Analysis and Calculations
72.7 Concentrations of nitrate in samples are
calculated from the linear regression, obtained from the
standard curve in which the concentrations of the
calibration standards are entered as the independent
variable, and their corresponding peak heights are the
dependent variable.
72.2 Refractive Index Correction for Estuarine and
Coastal Samples
12.2.1 If reagent water is used as the wash solution and
to prepare the calibration standard solutions, the operator
has to quantify the refractive index correction due to the
difference in salinity between sample and standard
solutions. The following procedures are used to measure
the relationship between sample salinity and refractive
index for a particular detector.
12.2.2 First, analyze a set of nitrate or nitrite standards
in reagent water with color reagent using reagent water
as the wash and obtain a linear regression of peak height
versus concentration.
Note: The change in absorbance due to refractive index
is small, therefore low concentration standards should be
used to bracket the expected absorbances due to
refractive index.
12.2.3 Second, replace reagent water wash solution with
Low Nutrient Seawater wash solution.
Revision 2.0 September 1997
353.4-10
-------
Note: In nitrate and nitrite analysis absorbance of the
reagent water is higher than that of the LNSW. When
using reagent water as a wash solution, the change in
refractive index causes the absorbance of seawater to
become negative. To measure the absorbance due to
refractive index change in different salinity samples, Low
Nutrient Seawater must be used as a wash solution to
bring the baseline down.
72.2.4 Replace NED solution (Section 7.2.4) with
reagent water. All other reagents remain the same.
Replace the synchronization sample with the colored
SYNC peak solution (Section 7.2.6).
12.2.5 Prepare a set of different salinity samples with
LNSW. Commence analysis and obtain peak heights for
different salinity samples. The peak heights for the
refractive index correction must be obtained at the same
AUFS range setting and on the same spectrophotometer
as the corresponding standards (Section 12.2.2).
12.2.6 Using Low Nutrient Seawater as the wash water,
a maximum absorbance will be observed for reagent
water. No change in refractive index will be observed in
the seawater sample. Assuming the absolute absorbance
for reagent water (relative to the seawater baseline) is
equal to the absorbance for seawater (relative to reagent
water baseline), subtract the absorbances of samples of
various salinities from that of reagent water. The results
are the apparent absorbance due to the change in
refractive index between samples of various salinities
relative to the reagent water baseline.
12.2.7 For each sample of varying salinity, calculate the
apparent nitrate or nitrite concentrations due to refractive
index from its peak height corrected to reagent water
baseline (Section 12.2.5) and the regression equation of
nitrate or nitrite standards obtained with color reagent
being pumped through the system (12.2.2). Salinity is
entered as the independent variable and the apparent
nitrate or nitrite concentration due to refractive index is
entered as the dependent variable. The resulting
regression allows the operator to calculate apparent
nitrate or nitrite concentration due to refractive index when
sample salinity is known. Thus, the operator would not be
required to obtain refractive index peak heights for all
samples.
12.2.8 An example of typical results follows:
Salinity Apparent concentration (ug N/L)
(%„) Nitrate Nitrite
0.0
3.8
9.2
13.8
18.1
26.8
36.3
0.000
0.026
0.096
0.142
0.190
0.297
0.370
0.000
0.015
0.040
0.055
0.086
0.153
0.187
Note: You must calculate the refractive index correction
for your particular detector. Moreover, the refractive index
must be redetermined whenever a significant change in
the design of flowcell or a new matrix is encountered.
12.2.9 An example of typical linear equations is:
Apparent nitrate (ug N/L) = 0.01047S
Apparent nitrite (ug N/L) = 0.00513S
where S is sample salinity. The apparent nitrate and nitrite
concentration due to refractive index so obtained should
be added to samples of corresponding salinity when
reagent water is used as wash solution and standard
matrix.
If nitrate and nitrite concentrations are greater than 100
and 50 ug N/L respectively, the correction for refractive
index is negligible and this procedure can be optional.
72.3 Correction for Salt Error in Estuarine and
Coastal Samples
12.3.1 When calculating concentrations of samples of
varying salinities from standards and the wash solution
prepared in reagent water, it is common to first correct for
refractive index errors, and then correct for any change in
color development due to the differences in composition
between samples and standards (so called salt error).
12.3.2 Plot the salinity of the saline standards (Section
7.2.9) as the independent variable, and the apparent
concentration of analyte (mg N/L) from the peak height
(corrected for refractive index) calculated from the
regression of standards in reagent water, as the
dependent variable for all saline standards. The resulting
regression equation allows the operator to correct the
353.4-11
Revision 2.0 September 1997
-------
concentrations
enhancement
Following are
systems:
Salinity
(%o)
0.0
3.8
9.2
13.8
18.1
26.8
36.3
of samples of
known salinity for the color
27.5
0.0335
100.1
0.1052
due to matrix effect, e.g., salt error.
typical results
for the nitrate and nitrite
18.6
18.6
18.6
18.6
0.0167
0.0170
0.0229
0.0229
105.8
101.6
106.4
104.5
0.0523
0.0534
0.0720
0.0719
Apparent concentration (ug N/L)
Nitrate
569.64
570.50
572.74
568.96
566.44
558.74
559.86
Nitrite
558.15
565.50
563.00
564.94
563.00
559.06
554.67
9.4
9.4
9.4
0.0
0.0
0.0
0.0
0.0222
0.0229
0.0197
0.0260
0.0306
0.0160
0.0248
105.3
106.4
91.5
103.9
106.9
111.0
109.5
0.0698
0.0720
0.0620
0.0817
0.0961
0.0501
0.0780
12.3.3 As shown in above results, salinity has no
systematic effect on the nitrate and nitrite signal and
therefore salt error correction is not recommended.
72.4 Results of sample analyses should be reported
in mg N/L or in ug N/L.
mg N/L = ppm (parts per million)
ug N/L = ppb (part per billion)
13.0 Method Performance
13.1 Single Laboratory Validation
13.1.1 Method Detection Limit- A method detection limit
(MDL) of 0.075 ug N/L has been determined by one
laboratory from LNSW of five different salinities fortified at
a nitrate concentration of 0.28 ug N/L.
Salinity SD Recovery MDL
(%0) (ugN/L) (%) (ugN/L)
36.5
36.5
36.5
36.5
27.5
27.5
27.5
0.0234
0.0298
0.0148
0.0261
0.0203
0.0321
0.0314
103.5
98.9
110.3
103.6
105.4
102.3
103.8
0.0734
0.0935
0.0464
0.0819
0.0638
0.1009
0.0986
73.12 Single analyst precision - A single laboratory
analyzed three samples collected from the Miami River
and Biscayne Bay, Florida. Seven replicates of each
sample were processed and analyzed with salinity
ranging from 0.019 to 32.623%o. The results were as
follows:
Sample
1
2
3
1
2
3
Salinity
32.623
13.263
0.019
32.623
13.263
0.019
Concentration
(ug N/L)
Nitrate
48.22
206.41
276.38
Nitrite
5.21
31.03
54.07
RSD
2.59
1.07
1.99
1.62
0.58
0.49
13.1.3 Laboratory fortified sample matrix - Laboratory
fortified sample matrices were processed in three
different salinities ranging from 0.019 to 32.623 and
ambient nitrate concentrations from 48.22 to 276.38 ug
N/L. Seven replicates of each sample were analyzed and
the results were as follows:
Revision 2.0 September 1997
353.4-12
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Salinity Concentration RSD Recovery
ambient fortified
(%0) (ug N/L)
32.623
13.263
0.019
48.22
206.41
276.38
139.94
139.94
139.94
1.50
1.25
1.19
106.4
102.6
102.3
73.2 Multi-Laboratory Validation
Multi-laboratory data is unavailable at this time.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the agency recommends recycling as the next
best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society, Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
75.7 The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
16.0 References
1. Morris, A. W. and Riley, J.P., 1963. Determination
of nitrate in sea water. Anal. Chim. Acta. 29:272-
279.
2. Brewer P. G. and J. P. Riley 1965. The automatic
determination of nitrate in seawater. Deep-Sea
Res., 12:765-772.
3. Wood, E.O., Armstrong, F.A.J., and Richards, F.A.,
1967. Determination of nitrate in seawater by
cadmium-copper reduction to nitrite. J. Mar. Biol.
Assn. U.K., 47:23-31.
4. Bendschneider, K. and R. J. Robinson, 1952. A
new spectrophotometric method for the
determination of nitrite in sea water. J. Marine Res.,
11:87-96.
5. Fox, J.B. 1979. Kinetics and mechanisms of the
Griess reaction. Analytical Chem. 51:1493-1502.
6. Norwitz, G., P.M. Keliher,, 1984.
Spectrophotometric determination of nitrite with
composite reagents containing sulphanilamide,
sulphanilic acid or 4- nitroaniline as the diozotisable
aromatic amine and N-(1-
naphthyl)ethylenediamine as the coupling agent.
Analyst, 109:1281-1286.
7. Spencer, C.P. 1975, The micronutrient elements.
In Chemical Oceanography (Riley, J. P. and G.
Skirrow, Eds.), Academic Press, London and New
York, 2nd Ed. Vol 2, Chapter 11.
8. 40 CFR, 136 Appendix B. Definition and Procedure
for the Determination of Method Detection Limit.
Revision 1.11.
9. Timmer-ten Hoor, A., 1974. Sulfide interaction on
colorimetric nitrite determination. Marine Chemistry,
2:149-151.
353.4-13
Revision 2.0 September 1997
-------
10. Alpkem Corporation. 1990. RFA Methodology:
Nitrate+Nitrite Nitrogen. Method A303-S170.
Alpkem Corporation, Clackamas, Oregon.
11. Olson, R.J. 1980. Phosphate interference in the
cadmium reduction analysis of nitrate. Limnol.
Oceanogr., 25(4)758-760.
12. Alvarez-Salgado, X.A., F.Fraga and F.F.Perez.
1992, Determination of nutrient salt by automatic
methods both in seawater and brackish water: the
phosphate blank. Marine Chemistry, 39:311-319.
13. Grasshoff, K. 1983, Determination of Nitrate, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp143-150.
14. Takenaka, N., A.Ueda and Y. Maeda 1992,
Acceleration of the rate of nitrite oxidation by
freezing in aqueous solution. Nature, Vol. 358,
p736-738.
15. Grasshoff , K. 1983, Determination of Nitrite, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp139-142.
16. MacDonald, R.W. and F.A. Mclaughlin. 1982. The
effect of Storage by freezing on dissolved inorganic
phosphate, nitrate, and reactive silicate for samples
from coastal and estuarine waters. Water
Research, 16:95-104.
17. Angelova, S, and H.W.Holy. 1983. Optimal speed
as a function of system performance for continuous
flow analyzers. Analytica Chimica Acta, 145:51-58.
18. Zhang, J.-Z. 1997. Distinction and quantification of
carry-over and sample interaction in gas
segmented continuous flow analysis . Journal of
Automataic Chemistry, 19(6):205-212.
19. Garside, C. 1993. Nitrate reductor efficiency as an
error source in seawater analysis. Marine
Chemistry 44: 25-30.
Revision 2.0 September 1997
353.4-14
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Debubbler
P/
Detector
540nrrf J
I
To Waste
/
OTCR
_j \
\
\
i
N
(
3
r\
U
10
9
8
— f~\ —
0
6
4
~0—
W
7
0
5
TT~
\J
3
2 p.
1
^v
To)
6
iA/aste (
) ;
)
^
X
Manifold
Wash To Sam
/
\
/
\
i /
plei \
0.41
0.41
0.10
0.10
0.32
0.25
1.12
1.57
)
r
NED
Sulfanilamide
— i Sample
1 — i
Nitrogen
Buffer
Reagent Water
or Low Nutrient Seawater
Pump
mL/min
Sample:Wash = 60":30"
Figure 1. Manifold configuration for nitrate + nitrite analysis using an Open Tubular Cadmium Reactor.
353.4-15
Revision 2.0 September 1997
-------
Detector
540nrrf J
T f
To Waste
Cd
Column
1
Debubbler
3/ /
\
-(
1 (
J
10 0~
-C\ -^ -
\J
8
-o=
6
0
0 5
4
- — — /<-s
o~
0
2 p>
1
Manifold
Wash To S
i o wastes
\
)
} ^
J ^
amplei \
0.41
0.41
0.10
0.10
0.25
0.32
1.12
1.57
)
r
NED
Sulfanilamide
Nitrogen
~~| Sample
i — i
Buffer
Reagent Water
or Low Nutrient Seawat
Pump
mL/min
Sample:Wash = 60":30"
Figure 2. Manifold configuration for nitrate + nitrite analysis using a homemade packed copper-coated cadmium
reduction column.
Revision 2.0 September 1997
353.4-16
-------
Detector
540nm(
Debubbler
S/ _..../
>
? ^
'
aste /^
V
t
-•L
n_ 9
U
8
-Ct—
\j
0 7
6
0
0 5
4
_/~\
~U—
0 3
2 O=
1
Manifold
Wash To S
i o vvdsie \
/
) ;
\
/
)
\
amplei \
0.41
0.10
0.10
0.25
1.01
1.57
_)
NED
Sulfanilamide
Air
Sample
Reagent Water
or Low Nutrient Seawater
Pump
mL/min
Sample:Wash = 60":30"
Figure 3. Manifold configuration for nitrite analysis.
353.4-17
Revision 2.0 September 1997
-------
Table 1 . Percentage recovery of nitrate from natural water samples preserved by freezing and refrigeration.
MethodA
Sample13
Salinity
Time (Day)
14
21
28
35
46
62 92
25C, P river
estuary
coast
25C, G river
estuary
coast
4C,P river
estuary
coast
4C,G river
estuary
coast
4C,P, river+
estuary+
coast+
4C,G, nver+
estuary+
coast+
Fr,P river
estuary
coast
Fr,P, river+
estuary+
coast+
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
192.5
108.5
102
257
108.8
98
105
104.5
127.6
158.2
103.1
100.9
105.5
110.2
112.7
100.5
114.1
130.5
101.9
102
103.2
279
106.2
128.8
294.9
108.5
135.2
90
90.4
65.7
88.1
84.5
54.4
99.2
116.4
112.7
100.4
115.5
100.9
103.2
106.7
111.1
287,
124
153,
316,
122,
150,
111
107,
149,
108,
107,
123
106,
104,
103,
105,
100,
104,
103,
105,
128,
103,
102,
101
o
.J
.8
.4
.5
.9
.6
.1
.1
.4
.4
.1
.8
.8
.7
.1
.4
.9
.6
2
.1
.4
o
.J
267.5
103.9
93.3
298.2
90.6
98.5
100.7
102.6
82.3
99.4
95.9
68.9
96.2
102.9
93.3
98.3
98
93.6
95.8
97.9
92.7
95.4
97.4
91.5
262.4
139.3
89
225.4
79.2
84.3
82.7
95.9
93.3
91
93
90.6
101
93.3
90.2
88.6
104.6
98.5
91.2
95
92.1
300.7
258.9
44.2
135.4
81.5
36.9
112.2
109
43.3
114.8
110.9
102.4
114.5
109.1
99.5
98.8
42.2
82.5
78.5
104.7
228.1
188.5
72.4
77.6
56.2
56.1
97.3
82.4
73.5
98.4
85
75.4
85.7
72.8
50.9
87.4
78
69.6
260.8
229.1
84.9
66.9
128.2
66.6
104.7
101.4
89.2
96.9
99.7
98.6
95.9
87.6
87.5
90.2
94.7
92.3
Revision 2.0 September 1997
353.4-18
-------
Table 2 . Percentage recovery of nitrite from natural water samples preserved by freezing and refrigeration
MethodA
Sample5
Salinity
14
Time(day)
21 28
35 46 62
92
25C, P river
estuary
coast
25C, G river
estuary
coast
4C,P river
estuary
coast
4C,G river
estuary
coast
4C,P river+
estuary+
coast+
4C,G river+
estuary+
coast+
Fr,P river
estuary
coast
Fr,P river+
estuary+
coast+
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.263
0.019
13.263
32.623
0.019
13.263
32.623
0.019
13.263
32.623
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
220
110.6
104.1
182.8
108.5
100
104.2
102.8
68.4
104.9
104.4
94.3
47.6
95.4
0
70.6
1.3
78.6
97
103.5
99.7
0.3
456.8
92.2
0.3
519.1
87.8
88.2
101.8
65.7
97.8
98.8
87
98.9
21.1
0
86.2
0.7
4.9
87.2
98.6
95.9
0
920.2
74.1
0
1026.3
73.8
31.8
38.9
33.2
99.8
100.6
71.1
98.5
0
0
97.9
100.6
69.5
98
0
0
95.4
95.9
56.5
0
957.8
89,
0
.5
1079.1
89,
93,
0
70,
96,
91
97,
97,
0
0
95,
91
97,
77,
0
0
75,
52
92,
.5
.9
.5
.7
.6
.2
.8
.6
.6
.3
.9
.2
0
661.
74.1
0
867.
73.5
0
91
50.5
67.8
0
0
84.6
94.1
65.9
68.1
0
0
75.9
90.5
67
Cont
0
5 58.7
94.6
0
5 843.1
95.9
65
17.8
0
0
2.7
0
85.9
100
87.6
96
8.6
63.1
74.2
100.5
'don
0
0
72.2
0
705.7
85.7
84.1
8.5
0
2.2
0
0
74.9
13.3
80
75.2
0
80
0
0
0
0
209.2
66.5
0
0
0
75.0
0
0
77.3
57.3
27.8
69.2
77.6
65.9
next page
353.4-19
Revision 2.0 September 1997
-------
Cont'd
A Methods of preservation:
25C,P and G: Store the samples in high density polyethylene carboys (P) or glass bottles (G)
at room temperature (~25°C).
4C, P and G: Store samples in high density polyethylene bottles (P) or glass bottles (G) in a
refrigerator (4°C) in the dark.
Fr,P and Fr,P: Freeze the samples in high density polyethylene bottles (P) and store at -20°C
in a freezer in the dark.
Glass and high density polyethylene bottles were used to study the effect of type of sample
bottles on the recovery of nitrite and nitrate from refrigeration.
B For salinity and concentration of nitrate in river, estuary and coast samples see section 13.1.2.
Sample river+, estuary+ and coast+ are the fortified river, estuary and coast samples,
respectively, at nitrate concentrations 139.94 jig N/L.
Revision 2.0 September 1997 353.4-20
-------
Method 365.5
Determination of Orthophosphate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis
Carl F. Zimmeimann
Carolyn W. Keefe
University of Maryland System
Center for Environmental and Estuarine Studies
Chesapeake Biological Laboratory
Solomons, MD 20688-0036
Revision 1.4
September 1997
Edited by
Elizabeth J. Arar
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
365.5 -1
-------
Method 365.5
Determination of Orthophosphate in Estuarine and Coastal
Waters by Automated Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for the deter-
mination of low-level orthophosphate concentrations
normally found in estuarine and/or coastal waters. It is
based upon the method of Murphy and Riley1 adapted for
automated segmented flow analysis2 in which the two
reagent solutions are added separately for greater
reagent stability and facility of sample separation.
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Phosphate
14265-44-2
1.2 A statistically determined method detection limit
(MDL) of 0.0007 mg P/L has been determined by one
laboratory in 3 parts per thousand (ppt) saline water.3 The
method is linear to 0.39 mg P/L using a Technicon
AutoAnalyzer II system (Bran & Luebbe, Buffalo Grove,
IL).
1.3
lyzed.
Approximately 40 samples per hour can be ana-
1.4 This method should be used by analysts experi-
enced in the use of automated colori metric analyses, and
familiar with matrix interferences and procedures for their
correction. A minimum of 6-months experience under
experienced supervision is recommended.
2.0 Summary of Method
2.1 An automated colorimetric method for the
analysis of low-level orthophosphate concentrations is
described. Ammonium molybdate and antimony potas-
sium tartrate react in an acidic medium with dilute solu-
tions of phosphate to form an antimony-phospho-molyb-
date complex. This complex is reduced to an intensely
blue-colored complex by ascorbic acid. The color
produced is proportional to the phosphate concentration
present in the sample. Positive bias caused by differ-
ences in the refractive index of seawater and reagent
water is corrected for prior to data reporting.
3.0 Definitions
3.1 Calibration Standard (CAL) - A solution
prepared from the stock standard solution that is used to
calibrate the instrument response with respect to analyte
concentration. One of the standards in the standard
curve.
3.2 Dissolved Analyte (DA) — The concentration of
analyte in an aqueous sample that will pass through a
0.45-//m membrane filter assembly prior to sample
acidification or other processing.
3.3 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is ana-
lyzed exactly like a sample, and its purpose is to deter-
mine whether method performance is within acceptable
control limits. This is basically a standard prepared in
reagent water that is analyzed as a sample.
3.4 Laboratory Fortified Sample Matrix (LFM) — An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background concentra-
tions.
3.5 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water that is treated exactly as a sample
including exposure to all glassware, equipment, and
reagents that are used with other samples. The LRB is
used to determine if method analytes or other interfer-
ences are present in the laboratory environment, the
reagents, or apparatus.
3.6 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.7 Method Detection Limit (MDL) — The minimum
concentration of an analyte that can be identified, mea-
sured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.8 Reagent Water (RW) — Type 1 reagent grade
water equal to or exceeding standards established by
American Society of Testing Materials (ASTM). Reverse
osmosis systems or distilling units that produce 18
megohm water are two examples of acceptable water
sources.
Revision 1.4 September 1997
365.5-2
-------
3.9 Refractive Index (Rl) - The ratio of the velocity
of light in a vacuum to that in a given medium. The
relative refractive index is the ratio of the velocity of light
in two different media, such as sea or estuarine water
versus reagent water. The correction for this difference
is referred to as the refractive index correction in this
method.
3.70 Stock Standard Solution (SSS) - A concen-
trated solution of method analyte prepared in the labora-
tory using assayed reference compounds or purchased
from a reputable commercial source.
4.0 Interferences
4.7 Interferences caused by copper, arsenate and
silicate are minimal relative to the orthophosphate deter-
mination because of the extremely low concentrations
normally found in estuarine or coastal waters. High iron
concentrations can cause precipitation of and subsequent
loss of phosphate from the dissolved phase. Hydrogen
sulfide effects, such as occur in samples collected from
deep anoxic basins, can be treated by simple dilution of
the sample since high sulfide concentrations are most
often associated with high phosphate values.4
4.2 Sample turbidity is removed by filtration prior to
analysis.
4.3 Refractive Index interferences are corrected for
estuarine/coastal samples (Section 12.2).
5.0 Safety
5.7 Water samples collected from the estuarine
and/or ocean environment are generally not hazardous.
However, the individual who collects samples should use
proper technique.
5.2 Good laboratory technique should be used when
preparing reagents. A lab coat, safety goggles, and
gloves should be worn when preparing the sulfuric acid
reagent.
6.0 Equipment and Supplies
6.7 Continuous Flow Automated Analytical Sys-
tem Consisting of:
6.1.1 Sampler.
6.12 Manifold or Analytical Cartridge equipped with
37°C heating bath.
6.13 Proportioning pump.
6.14 Colorimeter equipped with 1.5 X 50 mm tubular
flow cell and a 880 nm filter.
6.15 Phototube that can be used for 600-900 nm
range.
6.16 Strip chart recorder or computer based data
system.
6.2 Phosphate-Free Glassware and Polyethylene
Bottles
6.2.1 All labware used in the determination must be
low in residual phosphate to avoid sample or reagent
contamination. Washing with 10% HCI (v/v) and thor-
oughly rinsing with distilled, deionized water was found to
be effective.
6.2.2 Membrane or glass fiber filters, 0.45 //m nominal
pore size.
7.0 Reagents and Standards
7.7 Stock Reagent Solutions
7.1.1 Ammonium Molybdate Solution (40 g/L) —
Dissolve 20.0 g of ammonium molybdate tetrahydrate
((NH4)6Mo7024'4H20, CASRN 12027-67-7) in approxi-
mately 400 ml of reagent water and dilute to 500 ml.
Store in a plastic bottle out of direct sunlight. This
reagent is stable for approximately three months.
7.12 Antimony Potassium Tartrate Solution (3.0 g/L) -
Dissolve 0.3 g of antimony potassium tartrate
[(K(SbO)C4H406«1/2H20, CASRN 11071-15-1] in approxi-
mately 90 ml of reagent water and dilute to 100 ml. This
reagent is stable for approximately three months.
7.13 Ascorbic Acid Solution (18.0 g/L) - Dissolve 18.0
g of ascorbic acid (C6H6O6, CASRN 50-81-7) in approxi-
mately 800 ml of reagent water and dilute to 1 L. Dis-
pense approximately 75 ml into clean polyethylene
bottles and freeze. The stability of the frozen ascorbic
acid is approximately three months. Thaw overnight in
the refrigerator before use. The stability of the thawed,
refrigerated reagent is less than 10 days.
7.14 Sodium Lauryl Sulfate Solution (30.0 g/L) -
Sodium dodecyl sulfate (CH3(CH2)11OSO3Na, CASRN
151-21-3). Dissolve 3.0 g of sodium lauryl sulfate (SLS)
in approximately 80 ml of reagent water and dilute to
100 ml. This solution is the wetting agent and its
stability is approximately three weeks.
7.15 Sulfuric Acid Solution (4.9 N) - Slowly add 136
ml of concentrated sulfuric acid (H2SO4, CASRN 7664-
93-9) to approximately 800 ml of reagent water. After
the solution is cooled, dilute to 1 L with reagent water.
7.16 Stock Phosphorus Solution — Dissolve 0.439 g
of pre-dried (105°Cfor 1 hr) monobasic potassium phos-
phate (KH2PO4, CASRN 7778-77-0) in reagent water and
365.5-3
Revision 1.4 September 1997
-------
dilute to 1000 ml. (1.0 ml_= 0.100 mg P.) The stability of
this stock standard is approximately three months when
kept refrigerated.
7.17 Low Nutrient Seawater - Obtain natural low
nutrient seawater (36 ppt salinity; <0.0003 mg P/L) or
dissolve 31 g analytical reagent grade sodium chloride,
(NaCI, CASRN 7647-14-5); 10 g analytical grade magne-
sium sulfate, (MgSO4) CASRN 10034-99-8); and 0.05 g
analytical reagent grade sodium bicarbonate, (NaHCO3,
CASRN 144-55-8), in 1 L of reagent water.
7.2 Working Reagents
7.2.1 Reagent A - Mix the following reagents in the
following proportions for 142 ml of Reagent A: 100 ml of
4.9 N H2SO4 (Section 7.1.5), 30 ml of ammonium
molybdate solution (Section 7.1.1), 10 ml of antimony
potassium tartrate solution (Section 7.1.2), and 2.0 ml of
SLS solution (Section 7.1.4). Prepare fresh daily.
7.2.2 Reagent B — Add approximately 0.5 ml of the
SLS solution (Section 7.1.4) to the 75 ml of ascorbic acid
solution (Section 7.1.3). Stability is approximately 10 days
when kept refrigerated.
7.2.3 Refractive Reagent A - Add 50 ml of 4.9 N
H2SO4 (Section 7.1.5) to 20 ml of reagent water. Add 1
ml of SLS (Section 7.1.4) to this solution. Prepare fresh
every few days.
7.2.4 Secondary Phosphorus Solution — Take 1.0 ml
of Stock Phosphorus Solution (Section 7.1.6) and dilute
to 100 ml with reagent water. (1.0 ml = 0.0010 mg P.)
Refrigerate and prepare fresh every 10 days.
7.2.5 Prepare a series of standards by diluting suitable
volumes of standard solutions (Section 7.2.4) to 100 ml
with reagent water. Prepare these standards daily. When
working with samples of known salinity, it is recom-
mended that the standard curve concentrations be pre-
pared in low-level natural seawater (Section 7.1.7) diluted
to match the salinity of the samples. Doing so obviates
the need to perform the refractive index correction
outlined in Section 12.2. When analyzing samples of
varying salinities, it is recommended that the standard
curve be prepared in reagent water and refractive index
corrections be made to the sample concentrations (Sec-
tion 12.2). The following dilutions are suggested.
ml of Secondary
Phosphorus Solution (7.2.4)
0.1
0.2
0.5
1.0
2.0
4.0
5.0
Cone.
mg P/L
0.0010
0.0020
0.0050
0.0100
0.0200
0.0400
0.0500
8.0 Sample Collection, Preservation and
Storage
8.1 Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems. Filtration of the sample
through a 0.45-//m membrane or glass fiber filter imme-
diately after collection is required.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Nansen, Go-Flo or equivalent) that are attached
at fixed intervals to a hydro wire. These bottles are sent
through the water column open and are closed either
electronically or via a mechanical "messenger" when the
bottles have reached the desired depth.
8.12 When a submersible pump system is used, a
weighted hose is sent to the desired depth in the water
column and water is pumped from that depth to the deck
of the ship for processing.
8.1.3 Another method used to collect surface samples
involves the use of a plastic bucket or large plastic bottle.
While not the most ideal method, it is commonly used in
citizen monitoring programs.
8.2 Sample Preservation - After collection and
filtration, samples should be analyzed as quickly as
possible. If the samples are to be analyzed within 24 hr
of collection, then refrigeration at4°C is acceptable.
8.3 Sample Storage - Long-term storage of frozen
samples should be in clearly labeled polyethylene bottles
or polystyrene cups compatible with the analytical sys-
tem's automatic sampler (Section 6.1.1). If samples
cannot be analyzed within 24 hr, then freezing at -20°C
for a maximum period of two months is acceptable.5"8
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability, the continued
analysis of LRBs, laboratory duplicates, and LFBs as a
continuing check on performance.
9.2 Initial Demonstration
(Mandatory)
of Performance
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (MDLs and linear
dynamic range) and laboratory performance (analysis of
QC samples) prior to analyses of samples using this
method.
9.2.2 MDLs should be established using a low-level
estuarine water sample fortified to approximately five
Revision 1.4 September 1997
365.5-4
-------
times the estimated detection limit.3 To determine MDL
values, analyze seven replicate aliquots of water and
process through the entire analytical method. Perform all
calculations defined in the method and report the con-
centration values in the appropriate units. Calculate the
MDL as follows:
MDL = (t)(S)
where, S= the standard deviation of the
replicate analyses.
t = the Student's t value for n-1
degrees of freedom at the 99%
confidence limit, t = 3.143 for six
degrees of freedom.
MDLs should be determined every six months or when-
ever a significant change in background or instrument
response occurs or when a new matrix is encountered.
9.2.3 Linear Dynamic Range (LDR) - The LDR should
be determined by analyzing a minimum of five calibration
standards ranging in concentration from 0.001 mg P/L to
0.20, mg P/L across all sensitivity settings of the auto-
analyzer. Normalize responses by dividing the response
by the sensitivity setting multiplier. Perform the linear
regression of normalized response vs. concentration and
obtain the constants m and b, where m is the slope and b
is the y-intercept. Incrementally analyze standards of
higher concentration until the measured absorbance
response, R, of a standard no longer yields a calculated
concentration Cc, that is ± 10% of the known concentra-
tion, C, where Cc = (R - b)/m. That concentration defines
the upper limit of the LDR for your instrument. Should
samples be encountered that have a concentration that
is >90% of the upper limit of the LDR, then these samples
must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB (Section 3.5) with each
set of samples. LRB data are used to assess contamina-
tion from the laboratory environment. Should an analyte
value in the LRB exceed the MDL, then laboratory or
reagent contamination should be suspected. When LRB
values constitute 10% or more of the analyte level deter-
mined for a sample, fresh samples or field duplicates of
the samples must be prepared and analyzed again after
the source of contamination has been corrected and
acceptable LRB values have been obtained.
9.3.2 Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB (Section 3.3) with each
batch of samples. Calculate accuracy as percent recov-
ery. If the recovery of the analyte falls outside the re-
quired control limits of 90-110%, the analyte is judged out
of control and the source of the problem should be
identified and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB data to assess
laboratory performance against the required control limits
of 90-110% (Section 9.3.2). When sufficient internal per-
formance data become available (usually a minimum of
20 to 30 analyses), optional control limits can be devel-
oped from the percent mean recovery (x) and the stan-
dard deviation (S) of the mean recovery. These data can
be used to establish the upper and lower control limits as
follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also, the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery - Laboratory
Fortified Sample Matrix
9.4.1 A laboratory should add a known amount of
analyte to a minimum of 5% of the routine samples or one
sample per sample set, whichever is greater. The analyte
concentration should be two to four times the ambient
concentration and should be at least four times the MDL.
9.4.2 Calculate the percent recovery of the analyte,
corrected for background concentrations measured in the
unfortified sample, and compare these values with the
values obtained from the LFBs.
Percent recoveries may be calculated using the following
equation:
R=
-x 100
where,
R =
C =
percent recovery
measured fortified sample
concentration (background +
concentrated addition in mg P/L)
sample background concentration
(mg P/L)
concentration in mg P/L added to
the environmental sample.
9.4.3 If the recovery of the analyte falls outside the
designated range of 90-110% recovery, but the laboratory
performance for that analyte is in control, the fortified
365.5 - 5
Revision 1.4 September 1997
-------
sample should be prepared again and analyzed. If the
result is the same after reanalysis, the recovery problem
encountered with the fortified sample is judged to be
matrix related, not system related.
10.0 Calibration and Standardization
10.1 Calibration (Refer to Sections 11.5 and 12.0).
70.2 Standardization (Refer to Section 12.2).
11.0 Procedure
11.1 If samples are frozen, thaw the samples to room
temperature.
11.2 Set up manifold as shown in Figure 1. The tubing,
flow rates, sample:wash ratio, sample rate, etc., are
based on a Technicon AutoAnalyzer II system. Specifi-
cations for similar segmented flow analyzers vary, so
slight adjustments may be necessary.
11.3 Allow both colorimeter and recorder to warm up
for 30 min. Obtain a steady baseline with reagent water
pumping through the system, add reagents to the sample
stream and after the reagent water baseline has equili-
brated, note that rise (reagent water baseline), and adjust
baseline.
For analysis of samples with a narrow salinity range, it is
advisable to use low nutrient seawater matched to
sample salinity as wash water in the sampler in place of
reagent water. For samples with a large salinity range, it
is suggested that reagent wash water and procedure
(Section 12.2) be employed.
11.4 A good sampling rate is approximately 40 sam-
ples/hrwith a 9:1, sample:wash ratio.
11.5 Place standards (Section 7.2.5) in sampler in
order of decreasing concentration. Complete filling the
sampler tray with samples, LRBs, LFBs, and LFMs.
11.6 Commence analysis.
11.7 Obtain a second set of peak heights for all
samples and standards with Refractive Reagent A
(Section 7.2.3) being pumped through the system in place
of Reagent A (Section 7.2.1). This "apparent" concentra-
tion due to coloration of the water should be subtracted
from concentrations obtained with Reagent A pumping
through the system.
12.0 Data Analysis and Calculations
72.7 Concentrations of orthophosphate are calculated
from the linear regression obtained from the standard
curve in which the concentrations of the calibration
standards are entered as the independent variable and
the corresponding peak height is the dependent variable.
72.2 Refractive Index Correction for Estuarine/
Coastal Systems
12.2.1 Obtain a second set of peak heights for all
samples and standards with Refractive Reagent A (Sec-
tion 7.2.3) being pumped through the system in place of
Reagent A (Section 7.2.1). Reagent B (Section 7.2.2)
remains the same and is also pumped through the
system. Peak heights for the refractive index correction
must be obtained at the same Standard Calibration
Setting and on the same colorimeter as the correspond-
ing samples and standards.9
72.2.2 Subtract the refractive index peak heights from
the heights obtained for the orthophosphate determina-
tion. Calculate the regression equation using the cor-
rected standard peak heights. Calculate the concentra-
tion of samples from the regression equation using the
corrected sample peak heights.
12.2.3 When a large data set has been amassed in
which each sample's salinity is known, a regression for
the refractive index correction on a particular colorimeter
can be calculated. For each sample, the apparent or-
thophosphate concentration due to refractive index is
calculated from its peak height obtained with Refractive
Reagent A (Section 7.2.3) and Reagent B (Section 7.2.2)
and the regression of orthophosphate standards obtained
with orthophosphate Reagent A (Section 7.2.1) and
Reagent B (Section 7.2.2) for each sample. Its salinity is
entered as the independent variable and its apparent
orthophosphate concentration due to its refractive index
in that colorimeter is entered as the dependent variable.
The resulting regression equation allows the operator to
subtract an apparent orthophosphate concentration when
the salinity is known, as long as other matrix effects are
not present. Thus, the operator would not be required to
obtain the refractive index peak heights for all samples
after a large data set has been found to yield consistent
apparent orthophosphate concentrations due to salinity.
An example follows:
Salinity (ppt)
Apparent orthophosphate
cone, due to refractive
index (mg P/L)
1
5
10
20
0.0002
0.0006
0.0009
0.0017
12.2.4 An example of a typical equation is:
mg P/L apparent PO43" = 0.000087 X Salinity
(ppt) where, 0.000087 is the slope of the line.
where, 0.000087 is the slope of the line.
Revision 1.4 September 1997
365.5-6
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72.3 Results should be reported in mg PO43"- P/L or ug
PCX3'- P/L.
mg PO43' - P/L = ppm (parts per million)
ug PO43- - P/L = ppb (parts per billion)
13.0 Method Performance
73.7 Single Analyst Precision - A single laboratory
analyzed three samples collected from Chesapeake Bay,
Maryland, and East Bay, Florida. Seven replicates of
each sample were processed and analyzed randomly
throughout a group of 75 samples with salinities ranging
from 3 to 36 ppt. The results were as follows:
Sample
1
2
3
Salinity
(ppt)
36
18
3
Concentration
(mg P/L)
0.0040
0.0024
0.0007
Percent Relative
Standard Deviation
6.5
10
24
73.2 Multilaboratory Testing
13.2.1 This method was tested by nine laboratories
using reagent water, high salinity seawater from the
Sargasso Sea (36 ppt) and three different salinity waters
from Chesapeake Bay, Maryland (8.3 ppt, 12.6 ppt, and
19.5 ppt). The reagent water and the Sargasso Seawater
were fortified at four Youden pair concentrations ranging
from 0.0012 mg P/L to 0.1000 mg P/L10 The Chesa-
peake Bay waters were fortified at three Youden pair
concentrations ranging from 0.0050 mg P/L to 0.0959 mg
P/L with the highest salinity waters containing the lowest
Youden pair and the lowest salinity waters containing the
highest Youden pair. Analysis of variance (ANOVA) at the
95% confidence level found no statistical differences
between water types indicating that the refractive index
correction for different salinity waters is an acceptable
procedure. Table 1 contains the linear equations that
describe the single-analyst standard deviation, overall
standard deviation, and mean recovery of orthophosphate
from each water type.
13.2.2 Pooled Method Detection Limit (p-MDL) - The p-
MDL is derived from the pooled precision obtained by
single laboratories for the lowest analyte concentration
level used in the multilaboratory study. The p-MDLs using
reagent water and Sargasso Sea water were 0.00128 and
0.00093 mg P/L, respectively.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the next
best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better. Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 1155 16th Street N.W., Wash-
ington, D.C. 20036, (202)872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management, consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. Murphy, J. and J.P. Riley. 1962. A Modified Single
Solution Method for the Determination of Phos-
phate in Natural Waters. Analytica Chim. Acta 27,
31-36.0.
2. Technicon Industrial Systems. 1973. Orthophos-
phate in Water and Seawater. Industrial Method
155-71 W. Technicon Industrial Systems, Tarry-
town, NY 10591.
3. 40 CFR, 136 Appendix B. Definition and Procedure
for the Determination of the Method Detection
Limit. Revision 1.11.
4. Grasshoff, K., M. Ehrhardt, and K. Kremling. 1983.
Methods of Seawater Analysis. Verlag Chemie,
Federal Republic of Germany, 419 pages.
5. Klingamann, E.D. and D.W. Nelson. 1976. Evalua-
tion of Methods for Preserving the Levels of Solu-
ble Inorganic Phosphorus and Nitrogen in Unfil-
tered Water Samples. J. Environ. Qua/., 5:1, 42-
46.
365.5-7
Revision 1.4 September 1997
-------
6. MacDonald, R.W. and F.A. Mclaughlin. 1982. The
Effect of Storage by Freezing on Dissolved Inorganic
Phosphate, Nitrate, and Reactive Silicate for Samples
from Coastal and Estuarine Waters. Water Re-
search, 16:95-104.
7. Thayer, G.W. 1979. Comparison of Two Storage
Methods for the Analysis of Nitrogen and Phosphorus
Fractions in Estuarine Water. Ches. Sci., 11:3, 155-
158.
8. Salley, B.A., J.G. Bradshaw, and B.J. Neilson. 1986.
Results of Comparative Studies of Preservation
Techniques for Nutrient Analysis on Water Samples.
VIMS, Gloucester Point, VA 23062. 32 pp.
9. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic
Absorbance Errors with Technicon AutoAnalyzer II
Colorimeters. Water Research 12: 599-603.
10. Edgell, K.W., E.J. Erb, and J.E. Longbottom, "De-
termination of Orthophosphate in Estuarine and
Coastal Waters by Automated Colorimetric Analysis:
Collaborative Study," submitted in November 1992
for publication in Marine Chemistry.
Revision 1.4 September 1997 365.5-8
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Single-Analyst Precision, Overall Precision and Recovery from Multilaboratory Study
Reagent Water
(0.0012-0.100 mg P/L)
Mean Recovery
Overall Standard Deviation
Single-Analyst Standard Deviation
Sargasso Sea Water
(0.0012-0.100 mg P/L)
Mean Recovery
Overall Standard Deviation
Single-Analyst Standard Deviation
Chesapeake Bay Water
( 0.005-0.100 mg P/L)
Mean Recovery
X = 1.0190-0.000871
Overall Standard Deviation
Single-Analyst Standard Deviation
X = 0.9720-0.000018
SR = 0.033X + 0.000505
Sr = 0.002X + 0.000448
X = 0.971 C - 0.000002
SR = 0.021 X +0.000550
Sr = 0.01 OX+ 0.000249
SR = 0.066X + 0.000068
Sr = 0.030X +0.000165
C True value of spike concentration, mg P/L
X Mean concentration found, mg P/L, exclusive of outliers.
SR Overall standard deviation, mg P/L, exclusive of outliers.
Sr Single-analyst standard deviation, mg P/L, exclusive of outliers.
mL/min
) Sample Wash Receptacle
37°C 5 Turns
Heating
Bath
arm
Debubbler
Colorimeter
880 nm filters
5 Turns
arm
2.0
0.32
1.2
0.23
0.10
0.42
Pump
Water (GRN/GRN)
Air (Blk/Blk)
Sample (YEL/YEL)
Sampler
40/hr.
9:1
Reagent A (ORN/WHT)
Reagent B (ORN/GRN)
Waste from F/C (ORN/ORN)
F/C to waste
50x1.5 mm ID F/C
199-B021-04 Phototube
Figure 1. Manifold Configuration for Orthophosphate.
365.5-9
Revision 1.4 September 1997
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Method 366.0
Determination of Dissolved Silicate in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
Jia-Zhong Zhang, Cooperative Institute for Marine and Atmospheric Studies,
Rosenstiel School of Marine and Atmospheric Science, Atlantic Oceanographic and
Meteorological Laboratory, National Oceanic and Atmospheric Administration,
University of Miami, Miami, FL 33149
George A. Berberian, National Oceanic and Atmospheric Administration, Atlantic
Oceanographic and Meteorological Laboratory, Ocean Chemistry Division, Miami, FL
33149
Project Officer
Elizabeth J. Arar
Version 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
366.0-1
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Method 366.0
Determination of Dissolved Silicate in Estuarine and Coastal Waters
by Gas Segmented Continuous Flow Colorimetric Analysis
1.0 Scope and Application
1.1 This method provides a procedure for the
determination of dissolved silicate concentration in
estuarine and coastal waters. The dissolved silicate is
mainly in the form of silicic acid, H4SiO4, in estuarine and
coastal waters. All soluble silicate, including colloidal
silicic acid, can be determined by this method. Long chain
polymers containing three or more silicic acid units do not
react at any appreciable rate1, but no significant amount
of these large polymers exists in estuarine and coastal
waters.23 This method is based upon the method of
Koroleff,4 adapted to automated gas segmented
continuous flow analysis.5"7
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Silicate
12627-13-3
1.2 A statistically determined method detection limit
(MDL) of 0.0012 mg Si/L has been determined by one
laboratory in seawaters of three different salinities.8 The
method is linear to 6.0 mg Si/L using a Flow Solution
System (Perstorp Analytical Inc., Silver Spring, MD).
1.3 Approximately 60 samples per hour can be
analyzed.
1.4 This method should be used by analysts
experienced in the use of automated gas segmented
continuous flow colorimetric analyses, and familiar with
matrix interferences and procedures for their correction.
A minimum of 6-months experience under supervision is
recommended.
2.0 Summary of Method
2.1 An automated gas segmented continuous flow
colorimetric method for the analysis of dissolved silicate
concentration is described. In the method, P-
molybdosilicic acid is formed by reaction of the silicate
contained in the sample with molybdate in acidic solution.
The p-molybdosilicic acid is then reduced by ascorbic
acid to form molybdenum blue. The absorbance of the
molybdenum blue, measured at 660 nm, is linearly
proportional to the concentration of silicate in the sample.
A small positive error caused by differences in the
refractive index of seawater and reagent water, and
negative error caused by the effect of salt on the color
formation, are corrected prior to data reporting.
3.0 Definitions
3.1 Calibration Standard (CAL) - A solution
prepared from the primary dilution standard solution or
stock standard solution containing analytes. The CAL
solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.2 Dissolved Analyte (DA) - The concentration of
analyte in an aqueous sample that will pass through a
0.45 fj,m membrane filter assembly prior to sample
acidification or other processing.
3.3 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine
whether method performance is within acceptable control
limits, and whether the laboratory is capable of making
accurate and precise measurements.
This is basically a standard prepared in reagent water that
is analyzed as a sample.
3.4 Laboratory Fortified Sample Matrix (LFM) - An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background
concentrations.
Version 1.0 September 1997
366.0-2
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3.5 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water that is treated exactly as a sample
including exposure to all labware, equipment, and
reagents that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
the reagents, or apparatus.
3.6 Linear Dynamic Range (LDR) - The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.7 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.8
3.8 Reagent Water (RW) - Type 1 reagent grade
water equal to or exceeding standards established by
American Society for Testing and Materials (ASTM).
Reverse osmosis systems or distilling units followed by
Super-Q Plus Water System that produce water with 18
megohm resistance are examples of acceptable water
sources.
3.9 Refractive Index (Rl) - The ratio of velocity of
light in a vacuum to that in a given medium. The relative
refractive index is the ratio of the velocity of light in two
different media, such as estuarine or sea water versus
reagent water. The correction for this difference is
referred to as refractive index correction in this method.
3.70 Stock Standard Solution (SSS) - A
concentrated solution of method analyte prepared in the
laboratory using assayed reference compounds or
purchased from a reputable commercial source.
3.77 Quality Control Sample (QCS) - A solution of
method analyte of known concentrations which is used to
fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
3.72 SYNC Peak Solution - A colored solution used
to produce a synchronization peak in the refractive index
measurement. A synchronization peak is required by
most data acquisition programs to initialize the peak
finding parameters. The first cup in every run must always
be identified as a SYNC sample. The SYNC sample is
usually a high standard, but can be any sample that
generates a peak at least 25% of full scale.
4.0 Interferences
4.1 Interferences caused by hydrogen sulfide, such
as occur in samples taken from deep anoxic basins can
be eliminated by oxidation with bromine or stripping with
nitrogen gas after acidification. Interferences of
phosphate at concentrations larger than 0.15 mg P/L is
eliminated by the use of oxalic acid in the color
development step of this method. Interferences of fluoride
at concentrations greater than 50 mg F/L can be reduced
by complexing the fluoride with boric acid.4
4.2 Glassware made of borosilicate glass should be
avoided for use in silicate analysis. Plastic labware such
as polyethylene volumetric flasks and plastic sample
vials, should be used.
4.3 Sample turbidity and particles are removed by
filtration through a 0.45 urn non-glass membrane filters
after sample collection.
4.4 This method corrects for refractive index and salt
error interferences which occur if sampler wash solution
and calibration standards are not matched with samples
in salinity.
4.5 Frozen samples should be filled about 3/4 full in
the sample bottles. The expansion of water on freezing
will squeeze some of the brine out of the bottle if the
bottle was overfilled. The overfill of the sample bottle
during freezing will drastically alter the nutrient
concentrations in the sample that remains.
5.0 Safety
5.7 Water samples collected from the estuarine and
coastal environment are generally not hazardous.
However, the individual who collects samples should use
proper technique.
5.2 Good laboratory technique should be used when
preparing reagents. A lab coat, safety goggles, and
gloves should be worn when preparing the sulfuric acid
reagent.
6.0 Equipment and Supplies
6.7 Gas Segmented Continuous Flow
Autoanalyzer Consisting of:
6.1.1 Autosampler.
366.0-3
Version 1.0 September 1997
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6.1.2 Analytical cartridge with reaction coils for silicate
analysis.
6.13 Proportioning pump.
6.1.4 Monochromator or spectrophotometer equipped
with a tungsten lamp (380-800 nm) and a low refractive
index flowcell.
6.15 Strip chart recorder or computer based data
acquisition system.
6.2 Glassware and Supplies
6.2.1 All labware used in the analysis must be low in
residual silicate to avoid sample or reagent
contamination. Soaking with lab grade detergent, rinsing
with tap water, followed by rinsing with 10% HCI (v/v) and
thoroughly rinsing with reagent water was found to be
effective.
6.2.2 Glassware made of borosilicate glass should be
avoided for storage of solutions for silicate analysis.
Plastic containers are preferable for silicate analysis.
6.2.3 Non-glass membrane filters with 0.45 urn
nominal pore size. Plastic syringes with syringe filters,
pipets, 60 ml polyethylene bottles, and polyethylene
volumetric flasks, plastic sample vials.
6.2.4 Drying oven, desiccator and analytical balance.
7.0 Reagents and Standards
7.1 Stock Reagent Solutions
7.1.1 Sulfuric Acid Solution (0.05 M) - Cautiously add
2.8 ml of concentrated Analytical Reagent Grade sulfuric
acid (H2SO4) to approximately 800 ml of reagent water,
mix then bring up to 1 L with reagent water.
7.12 Ammonium Molybdate Solution (10 g/L) -
Dissolve 10 g of ammonium molybdate (VI) tetrahydrate
((NH4)6Mo7O24.4H2O) in approximately 800 ml of 0.05 M
sulfuric acid solution and dilute to 1000 ml with 0.05 M
sulfuric acid solution. Store in an amber plastic bottle.
This solution is stable for one month. Inspect the solution
before use. If a white precipitation forms on the wall of
container, discard the solution and make a fresh one.
7.13 Stock Silicate Solution (100 mg Si/L) -
Quantitatively transfer 0.6696 g of pre-dried (105°C for 2
hours) sodium hexafluorosilicate (Na2SiF6) to a 1000 ml
polypropylene flask containing approximate 800 ml of
reagent water, cover with plastic film and dissolve on a stir
plate using a Teflon-coated stirring bar. Complete
dissolution usually takes 2-24 hours. Dilute the solution to
1000 ml in polyethylene volumetric flask with reagent
water. Store the stock solution in a plastic bottle. This
solution is stable for one year if care is taken to prevent
contamination and evaporation.
7.1.4 Low Nutrient Sea Water (LNSW) - Obtain natural
low nutrient seawater from surface seawater in the Gulf
Stream or Sargasso Sea (salinity 36 %o, < 0.03 mg Si/L)
and filter through 0.45 urn pore size non-glass membrane
filters. In addition, commercially available low nutrient sea
water ( < 0.03 mg Si/L) with salinity of 35 %o (Ocean
Scientific International, Wormley, U.K.) can be used.
7.2 Working Reagents
7.2.1 Dowfax Start-up Solution - Add 2 mL of Dowfax
2A1 surfactant (Dow Chemical Company) to 1000 mL
reagent water and mix gently.
Note: Dowfax 2A1 contains (w/w) 47% benzene, 1,1-
oxybis, tetrapropylene derivatives, sulfonate, sodium salt,
1% sodium sulfate, 3% sodium chloride and 49% water.
7.2.2 Working Molybdate Reagent - Add 0.5 mL
Dowfax 2A1 to 250 mL of ammonium molybdate solution,
mix gently. Prepare this solution daily. This volume of
solution is sufficient for an 8-hour run.
7.2.3 Ascorbic Acid Solution - Dissolve 4.4 g of
ascorbic acid (C6H8O6) in 200 mL of reagent water and
12.5 mL of acetone(C3H6O), dilute to 250 mL with reagent
water. Store in a plastic container. This solution is stable
for one week if stored at 4°C. Discard the solution if it
turns brown.
7.2.4 Oxalic Acid Solution - Dissolve 50 g of oxalic acid
(C2H2O4) in approximately 800 mL of reagent water and
dilute to 1000 mL with reagent water. Store in a plastic
container. This solution is stable for approximately 3-
months.
7.2.5 Refractive Index Matrix Solution - Add 0.5 mL
Dowfax 2A1 to 250 mL of 0.05 M sulfuric acid solution
and mix gently.
7.2.6 Colored SYNC Peak Solution - Add 50 uL of blue
food coloring solution to 1000 mL reagent water and mix
thoroughly. The solution should give a peak of between
25 to 100 percent full scale, otherwise the volume of food
coloring added needs to be adjusted.
7.2.7 Calibration Standards - Prepare a series of
calibration standards (CAL) by diluting suitable volumes
of Stock Silicate Solution (Section 7.1.3) to 100 mL with
Version 1.0 September 1997
366.0-4
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reagent water or low nutrient seawater. Prepare these
standards daily. The concentration range of calibration
standards should bracket the expected concentrations of
samples and not exceed two orders of magnitude. At
least five calibration standards with equal increments in
concentration should be used to construct the calibration
curve.
When working with samples of a narrow range of
salinities (± 2 %o), it is recommended that the CAL
solutions be prepared in Low Nutrient Seawater (Section
7.1.4) diluted to the salinity of samples, and the Sampler
Wash Solution also be Low Nutrient Seawater (Section
7.1.4) diluted to that salinity. If this procedure is
performed, it is not necessary to perform the salt error
and refractive index corrections outlined in Sections 12.2
and 12.3.
When analyzing samples of varying salinities, it is
recommended that the calibration standard solutions and
sampler wash solution be prepared in reagent water and
corrections for salt error and refractive index be made to
the sample concentrations (Section 12.2 and 12.3).
7.2.8 Saline Silicate Standards - If CAL solutions will
not be prepared to match sample salinity, then saline
silicate standards must be prepared in a series of
salinities in order to quantify the salt error, the change in
the colorimetric response of silicate due to the change in
the ionic strength of the solution. The following dilutions
prepared in 100 mL volumetric flasks, diluted to volume
with reagent water, are suggested.
Salinity Volume of
(%0) LNSW(mL)
0
9
18
27
35
0
25
50
75
98
Volume(mL) Cone.
Si stock std mg Si/L
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
8.0 Sample Collection, Preservation
and Storage
8.1 Sample Collection - Samples collected for
nutrient analyses from estuarine and coastal waters are
normally collected using one of two methods: hydrocast
or submersible pump systems.
8.1.1 A hydrocast uses a series of sampling bottles
(Niskin, Go-Flo or equivalent) that are attached at fixed
intervals to a hydro wire. These bottles are sent through
the water column open and are closed either
electronically or via a mechanical messenger when the
bottles have reached the desired depth.
8.1.2 In a submersible pump system, a weighted hose
is sent to the desired depth in the water column and water
is pumped from that depth to the deck of the ship for
sample processing.
8.1.3 For collecting surface samples, an acid - cleaned
plastic bucket or a large plastic bottle can be used as
convenient samplers. Wash the sampler three times with
sample water before collecting samples.
8.1.4 Samples must be filtered through a 0.45 urn non-
glass membrane filters as soon as possible after
collection.
8.1.5 60-mL high density polyethylene bottles are used
for sample storage. Sample bottles should be rinsed 3
times with about 20 mL of sample, shaking with the cap
in place after each rinse. Pour the rinse water into the cap
to dissolve and rinse away salt crusts trapped in the
threads of the cap. Finally, fill the sample bottle about 3/4
full, and screw the cap on firmly. The expansion of water
on freezing will squeeze some of the brine out of the
bottle if the bottle was overfilled.
8.2 Sample Preservation - After collection and
filtration, samples should be analyzed as soon as
possible. If samples will be analyzed within 24 hours then
keep refrigerated in tightly sealed, high density
polyethylene bottles in the dark at 4°C until analysis can
be performed.
8.3 Sample Storage - If samples are to be frozen
for long-term storage ensure that none of the sample
bottles are filled more than 3/4 full and the cap is firmly
screwed on. Place the bottles upright on a rack and store
in the freezer (-20°C).
Before analysis, frozen samples must be taken out of the
freezer and allowed to thaw in a refrigerator at 4°C in the
dark. Thawing times depend upon sample salinities. It
was found that the frozen low salinity estuarine water took
4 days to thaw. After completely thawing, take samples
out of the refrigerator and mix thoroughly. Keep samples
in the dark at room temperature overnight before
analysis.
Effects of thawing conditions on the recoveries of frozen
samples are more pronounced in low salinity estuarine
366.0-5
Version 1.0 September 1997
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waters than high salinity coastal waters as shown in
following results:
each set of
performance.
samples as a continuing check on
Day
0
7
14
21
27
35
42
49
56
84
91
Recovery
3=35.85
100.00
102.44
98.59
99.51
98.86
98.70
100.87
103.47
104.12
99.35
100.65
100.22
3=18.07
100.00
102.65
101.06
99.30
98.86
98.66
102.44
103.92
99.92
100.80
99.90
%}
3=2.86
100.00
89.37
86.49
83.49
91.43
92.98
79.12
79.10
89.68
91.71
93.81
Remark
a
a
a
a
b
b
b
b
c
d
e
c
d
e
f
g
f
g
3 = Salinity
a, overnight thawing at room temperature
b, 20 hours thawing at room temperature
c, 24 hours thawing at room temperature
d, 8 hours thawing at room temperature then
heating at 80°C for 16 hours
e, 24 hours thawing at room temperature in the dark
f, 4 days thawing at room temperature in the dark
g, 4 days thawing at 4°C in a refrigerator in the dark
To ensure a slow process of depolymerization of
polysilicate to occur, thawing the frozen samples in the
dark at 4°C for 4 days is critical condition for obtaining
high recoveries of silicate in frozen samples. A maximum
holding time for frozen estuarine and coastal waters is
two months.9"11
9.0 Quality Control
9.1 Each laboratory using this method is required to
implement a formal quality control(QC) program. The
minimum requirements of this program consists of an
initial demonstration of performance, continued analysis
of Laboratory Reagent Blanks (LRB), laboratory
duplicates and Laboratory Fortified Blanks (LFB) with
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The Initial demonstration of performance is used
to characterize instrument performance by determining
the MDL and LDR and laboratory performance by
analyzing quality control samples prior to analysis of
samples using this method.
9.2.2 Method Detection Limits (MDLs) should be
established using a low level seawater sample containing,
or fortified at, approximately 5 times the estimated
detection limit. To determine MDL values, analyze at least
seven replicate aliquots of water which have been
processed through the entire analytical method. Perform
all calculations defined in the method and report
concentration in appropriate units. Calculate the MDL as
follows:
MDL = (t)(S)
where, 3 = the standard deviation of the
replicate analyses
t = Student's t value for n-1 degrees of
freedom at the 99% confidence
limit; t = 3.143 for six degrees of
freedom.
MDLs should be determined every 6-months or whenever
a significant change in background or instrument
response occurs or a new matrix is encountered.
9.2.3 The LDR should be determined by analyzing a
minimum of eight calibration standards ranging from 0.03
to 5.00 mg Si/L across all sensitivity settings (Absorbance
Units Full Scale) of the detector. Standards and sampler
wash solutions should be prepared in low nutrient
seawater with salinities similar to that of samples,
therefore a correction factor for salt error, or refractive
index, will not be necessary. Normalize responses by
multiplying the response by the Absorbance Units Full
Scale output range setting. Perform the linear regression
of normalized response vs. concentration and obtain the
constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher
concentration until the measured absorbance response,
R, of a standard no longer yields a calculated
concentration Cc, that is within 100 ± 10% of the known
concentration, C, where Cc = (R-b)/m. That concentration
Version 1.0 September 1997
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defines the upper limit of the LDR for the instrument.
Should samples be encountered that have a
concentration that is > 90% of the upper limit of LDR, then
these samples must be diluted and reanalyzed.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) - A laboratory
should analyze at least one LRB with each set of
samples. LRB data are used to assess contamination
from the laboratory environment. Should an analyte value
in the LRB exceed the MDL, then laboratory or reagent
contamination should be suspected. When the LRB value
constitutes 10% or more of the analyte concentration
determined for a sample, duplicates of the sample must
be prepared and analyzed again after the source of
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2 Laboratory Fortified Blank (LFB) - A laboratory
should analyze at least one LFB with each set of
samples. The LFB must be at a concentration that is
within the daily calibration range. The LFB data are used
to calculate accuracy as percent recovery. If the recovery
of the analyte falls outside the required control limits of
90 -110%, the source of the problem should be identified
and resolved before continuing the analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required
control limits of 90 -110%. When sufficient internal
performance data become available (usually a minimum
of 20 to 30 analyses), optional control limits can be
developed from the percent mean recovery (x) and
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 90-110%. After each 5 to 10
new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery -
Laboratory Fortified Sample Matrix
(LFM)
9.4.1 A laboratory should add a known amount of
analyte to a minimum of 5% of the total number of
samples or one sample per sample set, whichever is
greater. The analyte added should be 2-4 times the
ambient concentration and should be at least four times
greater than the MDL.
9.4.2 Calculate percent recovery of analyte, corrected
for background concentration measured in a separate
unfortified sample. These values should be compared
with the values obtained from the LFBs. Percent
recoveries may be calculated using the following
equation:
(CS-C)
where, R = percent recovery
Cs = measured fortified sample
concentration (background +
addition in mg Si/L)
C = sample background concentration
(mg Si/L)
S = concentration in mg Si/L added to the
environmental sample.
9.4.3 If the recovery of the analyte falls outside the
required control limits of 90-110%, but the laboratory
performance for that analyte is within the control limits,
the fortified sample should be prepared again and
analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is
judged to be matrix related and the sample data should
be flagged.
10.0 Calibration and Standardization
10.1 At least five calibration standards should
prepared daily for system calibration.
be
70.2 A calibration curve should be constructed for
each run by analyzing a set of calibration standard
solutions. A run should contain no more than 60 samples.
366.0-7
Version 1.0 September 1997
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It is suggested that a large set of samples be analyzed in
several sets with individual calibration curves.
70.3 Place the calibration standards before samples
for each run. All the calibration solutions should be
analyzed in duplicate.
70.4 The calibration curve containing five data points
or more should have a correlation coefficient > 0.995.
70.5 Place a high standard solution cup and follow by
two blank cups to quantify the carry-over of the system.
The difference in peak heights between two blank cups is
due to the carry over from the high standard cup. The
carry-over coefficient, k, is calculated as follows:
p _ p
rb1 rb
X100
high
where, Phigh = the peak height of the high
silicate standard
Pb1 = the peak height of the first
blank sample
Pb2 = the peak height of the second
blank sample.
The carry over coefficient, k, for a system should be
measured in seven replicates in order to obtain a
statistically significant number. The k should be
remeasured when a change in the plumbing of the
manifold or replacement of pump tube occur.
The carry over correction (CO) on a given peak, i, is
proportional to the peak height of the preceding sample,
PM-
CO = kxPM
To correct a given peak height reading, Ph one subtracts
the carry over correction.12 13
PI.C = PI - CO
where Pic is corrected peak height. The correction for
carry over should be applied to all the peak heights
throughout a run. The carry over should be less than 2%.
70.6 Place a high standard solution at the end of a run
to check sensitivity drift. The sensitivity drift should be ±
5% during the run.
11.0 Procedure
77.7 If samples are frozen, thaw the sample at 4°C in
the dark as outlined in Section 8.3. Mix samples
thoroughly prior to analyses.
77.2 Turn on the continuous flow analyzer and PC
components and warm up at least 30 minutes.
77.3 Set up the cartridge and pump tubes as shown in
Figure 1.
Note: Fluctuation of ambient temperature can cause
erratic results due to the effect of temperature on kinetics
of color development. The laboratory temperature should
be maintained as close to a constant temperature as
possible. The cartridge should be away from the direct
path of air flow from a heater or air conditioner. In cases
such as on a ship where the fluctuation of temperature
can be extreme, the temperature effect can be minimized
by increasing the length of mixing coil 1 (Figure 1) to bring
the formation of silicomolybdic acid reaction to
completion.
77.4 Set the wavelength at 660 nm on the
spectrometer/monochrometer.
Note: The absorption spectra of silicomolybdeum blue
complex has two maxima at 820 nm and 660 nm with 820
nm higher than 660 nm. This method measures
absorbance at 660 nm because the detector works in the
range of 380 to 800 nm. The sensitivity of the method is
satisfactory at 660 nm. The sensitivity, however, can be
improved by using 820 nm if this wavelength is available
on the detector.
77.5 On the monochromator, set the Absorbance Unit
Full Scale at an appropriate setting according to the
highest concentration of silicate in the samples. The
highest setting used in this method was 0.2 for 6 mg Si/L.
77.6 Prepare all reagents and standards.
77.7 Begin pumping the Dowfax start-up solution
(Section 7.2.1) through the system and obtain a steady
baseline. Place the reagents on-line. The reagent
baseline will be higher than the start-up solution baseline.
After the reagent baseline has stabilized, reset the
baseline.
NOTE: To minimize the noise in the reagent baseline,
clean the flow system by sequentially pumping the
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366.0-8
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sample line with reagent water, 1 N HCI solution, reagent
water, 1N NaOH solution for a few minutes each at the
end of the daily analysis. Make sure to rinse the system
well with reagent water after pumping NaOH solution to
prevent precipitation of Mg(OH)2 when seawater is
introduced into the system. Keep the reagents and
samples free of particulate. Filter the reagents and
samples if necessary.
If the baseline drifts upward, pinch the waste line for a few
seconds to increase back pressure. If absorbance drops
down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach
a syringe at the waste outlet of the flowcell. Air bubbles
in the flowcell can often be eliminated by simply attaching
a syringe for a few minutes or, if not, dislodged by
pumping the syringe pistion. Alternatively, flushing the
flowcell with alcohol was found to be effective in removing
air bubbles from the flowcell.
For analysis of samples with a narrow range of salinities
(± 2 %o), it is recommended that the wash water in the
sampler be prepared in Low Nutrient Seawater diluted to
the salinity of samples in place of reagent water. For
samples with varying salinities, it is suggested that reagent
waters and procedures in Sections 12.2 and 12.3 be
employed.
77.8 A good sampling rate is approximately 60
samples per hour with 40 seconds of sample time and 20
seconds of wash time.
77.9 Use 10% HCI followed by reagent water to rinse
sample cups. Place CAL solutions and saline standards
(optional) in sampler. Complete filling the sampler tray
with samples, laboratory reagent blanks, laboratory
fortified blanks, laboratory fortified sample matrices, and
QC samples. Place a blank every ten samples and
between samples of high and low concentrations.
77.70 Commence analysis.
77.77 If the reagent water is used as wash solution
instead of Low Nutrient Seawater and an operator wants
to quantify the refractive index correction due to the
difference in salinities between sample and wash solution,
the following procedures are used. Replace ammonium
molybdate solution (Section 7.1.2) with refractive index
matrix solution (Section 7.2.5). All other reagents remain
the same. Replace the synchronization cup with the
colored SYNC peak solution (Section 7.2.6). Commence
analysis and obtain a second set of peak heights for all
standards and samples. The peak heights obtained from
these measurements must be subtracted from the peak
heights of samples analyzed with color developing
reagent pumping through the system. If a low refractive
index flowcell is used, the correction for refractive index is
negligible. This procedure is optional.
12.0 Data Analysis and Calculations
72.7 Concentrations of silicate are calculated from the
linear regression, obtained from the standard curve in
which the concentrations of the calibration standards are
entered as the independent variable, and their
corresponding peak heights are the dependent variable.
72.2 Refractive Index Correction for Estuarine and
Coastal Samples (optional)
12.2.1 Obtain a second set of peak heights for all
standards and samples with refractive index matrix
solution being pumped through the system in place of
color reagent (ammonium molybdate solution). All other
reagents remain the same. The peak heights for the
refractive index correction must be obtained at the same
Absorbance Unit Full Scale range setting and on the
same monochromator as the corresponding samples and
standards.
72.2.2 Subtract the refractive index peak heights from
the peak heights obtained from the silicate determination.
12.2.3 An alternative approach is to measure the
relationship between the sample salinity and refractive
index on a particular detector.
First analyze a set of silicate standards in reagent water
with color reagent and obtain a linear regression from the
standard curve.
Prepare a set of different salinity samples with LNSW.
Analyze these samples with refractive index matrix
solution being pumped through the system in place of
color reagent (ammonium molybdate solution). All other
reagents remain the same. The peak heights for the
refractive index correction must be obtained at the same
Absorbance Unit Full Scale setting and on the same
monochromator as the corresponding standards.
For each sample, the apparent silicate concentration due
to refractive index is then calculated from its peak height
obtained with refractive index reagent and the regression
of silicate standards obtained with color reagent pumping
366.0-9
Version 1.0 September 1997
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through the system. Salinity is entered as the independent
variable and the apparent silicate concentration due to
refractive index in that detector is entered as the
dependent variable. The resulting regression allows the
operator to calculate apparent silicate concentration due
to refractive index when the salinity is known. Thus, the
operator would not be required to obtain refractive index
peak heights for all samples.
72.2.4 Refractive index correction can be minimized by
using a low refractive index flowcell. An example of typical
results using a low refractive index flowcell follows:
Salinity
(%c)
4.5
9.0
18.0
27.0
36.0
Apparent silicate cone.
to refractive index (mg
0.0003
0.0005
0.0016
0.0027
0.0042
. due
Si/L)
12.2.5 An example of a typical equation is:
Apparent silicate (mg Si/L) = 0.00001953*S1 5
where S is sample salinity. The form of fitted equation
might vary as the design of flowcell, so the operators are
advised to obtain the appropriate equation which has the
best fit of their own data with the least fitting coefficients.
72.3 Correction for Salt Error in Estuarine and
Coastal Samples
12.3.1 When calculating concentrations of samples of
varying salinities from standards and wash solution
prepared in reagent water, it is usual to first correct for
refractive index errors, then correct for the change in color
development due to the differences in ionic strength
between samples and standards (salt error). The
refractive index correction is negligible, so is optional, but
correction for salt error is necessary.
12.3.2 Plot the salinity of the saline standards (Section
7.2.8) as the independent variable, and the apparent
concentration of silicate (mg Si/L) from the peak height
(corrected for refractive index) calculated from the
regression of standards in reagent water, as the
dependent variable for all 1.50 mg Si/L standards. The
resulting regression equation allows the operator to
correct the concentrations of samples of known salinity
for the color suppression due to salinity effect, e.g., salt
error. An example of typical results follows:
Salinty
(%o)
0
9
18
27
36
Peak height of
1 .50 mg Si/L
2503
2376
2282
2250
2202
Un corrected Si
cone. calculated
from standards
in reagent water
1.50
1.32
1.27
1.25
1.23
12.3.3 An example of a typical equation to correct for
salt error is:
Corrected mg Si/L =
Uncorrected mg Si/L
1 - 0.02186x/S
where S is salinity.
12.3.4 Results of sample analyses should be reported
in mg Si/L or in ug Si/L.
mg Si/L = ppm (parts per million)
ug Si/L = ppb (part per billion)
13.0 Method Performance
73.7 Single Laboratory Validation
13.1.1 Method Detection Limit - A method detection limit
(MDL) of 0.0012 mg Si/L has been determined by one
laboratory in seawaters of three different salinities.
Salinity
36
36
27
27
27
18
18
18
3
3
3
3
SD
(ug/L)
0.3924
0.4980
0.2649
0.3362
0.4671
0.3441
0.2809
0.2432
0.3441
0.2331
0.1963
0.2809
Recovery
105
107
104
104
100
101
105
104
101
102
98
99
MDL
(ug/L)
1.233
1.565
0.832
1.056
1.468
1.081
0.883
0.764
1.081
0.733
0.617
0.883
13.1.2 Single Analyst Precision - A single laboratory
analyzed three samples collected from the Miami River
and Biscayne Bay areas of Florida. Seven replicates of
Version 1.0 September 1997
366.0-10
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each sample were processed and analyzed with salinities
ranging from 2.86 to 35.85. The results were as follows:
Sample Salinity
(%o)
1 35.85
2 18.07
3 2.86
Concentration
(mg Si/L)
0.097
1.725
3.322
RSD
(%)
1.2
1.4
0.9
13.1.3 Laboratory Fortified Sample Matrix - Laboratory
fortified sample matrixes were processed in three different
salinities ranging from 2.86 to 35.85 and ambient
concentrations from 0.095 to 3.322 mg Si/L with three
fortified levels at each salinity. Seven replicates of each
sample were analyzed and the results were as follows:
Salinity
(%c)
Concentration
(ma S\/L)
Ambient I Fortified
RSD Recovery
(%) (%)
35.85
35.85
35.85
18.07
18.07
18.07
2.86
2.86
2.86
0.095
0.095
0.095
1.725
1.725
1.725
3.322
3.322
3.322
0.1647
0.2196
0.2747
0.5517
1.1008
1 .6508
0.5421
1.0801
1.6188
0.82
1.34
1.74
1.11
0.77
0.98
0.99
1.26
0.98
99.37
100.61
99.62
107.18
104.69
103.62
101.03
103.22
100.59
13.2 Multi-Laboratory Validation
Multi-laboratory validation has not been conducted for this
method and, therefore, multi-laboratory data is currently
unavailable.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the next
best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society, Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
75.7 The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
16.0 References
1 Chow, D. T-W., and Robinson, R.J. 1953, Forms of
silicate available for colorimetric determination.
Analytical Chemistry. 25, 646-648.
2. Burton, J. D., T.M. Leatherland and P.S. Liss, 1970.
The reactivity of dissolved silicon in some natural
waters. Limnology and Oceanography, 15, 473-
476.
3 Isshiki, K., Sohrin, Y, and Nakayama, E., 1991.
Form of dissolved silicon in seawater. Marine
Chemistry, 32, 1-8.
4. Koroleff, F. 1983, Determination of silicon, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp174 -187.
366.0-11
Version 1.0 September 1997
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5. Grasshoff, K. 1965. On the automatic
determination of silicate, phosphate and fluoride in
seawater. I.C.E.S. Hydrographic Committee
Report, No. 129, Rome. (Mimeographed).
6. Brewer P. G. and J. P. Riley. 1966. The automatic
determination of silicate-silicon in natural water with
special reference to sea water. Anal. Chim. Acta,
35,514-519.
7. Hansen, H.P., K.Grasshoff, Statham and P.J.LeB.
Williams. 1983, Automated chemical analysis, In
Methods of Seawater Analysis (Grasshoff, K., M.
Ehrhardt and K. Kremling, Eds) Weinheim, Verlag
Chemie, Germany. pp374 -395.
8. 40 CFR, 136 Appendix B. Definition and Procedure
for the Determination of Method Detection Limit.
Revision 1.11.
9. MacDonald, R.W. and F.A. Mclaughlin. 1982. The
effect of storage by freezing on dissolved inorganic
phosphate, nitrate, and reactive silicate for samples
from coastal and estuarine waters. Water
Research, 16:95-104.
10. MacDonald, R.W. , F.A. Mclaughlin and C. S.
Wong. 1986. The storage of reactive silicate
samples by freezing. Limnol. Oceanogr.,
31 (5): 1139-1142.
11. Salley, B.A., J.G. Bradshaw, and B.J. Neilson.
1987. Results of comparative studies of
preservation techniques for nutrient analysis on
water samples. Virginia Institute of Marine Science,
Gloucester Point, VA 23062. USEPA, CBP/TRS
6/87, 32pp.
12. Angelova, S, and H.W.Holy. 1983. Optimal speed
as a function of system performance for continuous
flow analyzers. Analytica Chimica Acta, 145:51-58.
13. Zhang, J.-Z. 1997. Distinction and quantification of
carry-over and sample interaction in gas
segmented continuous flow analysis. J. Automatic
Chemistry, 19(6):205-212.
Version 1.0 September 1997 366.0-12
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Debubt
/
s
( jDetector
\-^ 660nm
T
To Waste
Coil2 §
C
)ler
T
o
10 O__
0 A
8 O
- ~ 1 -
c\
\J
6
( \
\J
"0—5- -
4
CT -
0 3
2 Q^
1
Manifold
Wash To Sar
o Wable S
^ CoilS
— /
N
/
g coin
\
^N /
)
/
\
0.41
0.41
0.32
0.41
0.25
0.41
1.57
Pump
ml/min
)
Ascorbic Acid
Oxalic Acid
Sample
Air
Molybdate
Reagent Water
or Low Nutrient Seawater
Sample:Wash = 20":40"
Figure 1. Manifold Configuration for Silicate Analysis.
366.0-13
Version 1.0 September 1997
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Method 440.0
Determination of Carbon and Nitrogen in Sediments and Particulates
of Estuarine/Coastal Waters Using Elemental Analysis
Carl F. Zimmeimann
Carolyn W. Keefe
University of Maryland System
Center for Environmental Estuarine Studies
Chesapeake Biological Laboratory
Solomns, MD 20688-0038
and
Jerry Bashe
Technology Applications, Inc.
26 W. Martin Luther King Drive
Cincinnati, OH 45219
Revision 1.4
September 1997
Work Assignment Manager
Elizabeth J. Arar
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
440.0-1
-------
Method 440.0
Determination of Carbon and Nitrogen in Sediments and Particulates
of Estuarine/Coastal Waters Using Elemental Analysis
1.0 Scope and Application
7.7 Elemental analysis is used to determine particu-
late carbon (PC) and participate nitrogen (PN) in estua-
rine and coastal waters and sediment. The method
measures the total carbon and nitrogen irrespective of
source (inorganic or organic).
Analyte
Chemical Abstracts Service
Registry Numbers (CASRN)
Carbon
Nitrogen
7440-44-0
1333-74-0
7.2 The need to qualitatively or quantitatively deter-
mine the particulate organic fraction from the total
particulate carbon and nitrogen depends on the data-
quality objectives of the study. Section 11.4 outlines
procedures to ascertain the organic/inorganic particulate
ratio. The method performance presented in the method
was obtained on particulate samples with greater than
80% organic content. Performance on samples with a
greater proportion of particulate inorganic versus organic
carbon and nitrogen has not been investigated.
7.3 Method detection limits (MDLs)1 of 10.5 ug/L and
62.3 ug/L for PN and PC, respectively, were obtained for
a 200-mL sample volume. Sediment MDLs of PN and
PC are 84 mg/kg and 1300 mg/kg, respectively, for a
sediment sample weight of 10.00 mg. The method has
been determined to be linear to 4800 ug of C and 700 ug
of N in a sample. Multilaboratory study validation data are
in Section 13.
7.4 This method should be used by analysts experi-
enced in the theory and application of elemental analysis.
A minimum of 6 months experience with an elemental
analyzer is recommended.
7.5 Users of the method data should set the data-
quality objectives prior to analysis. Users of the method
must document and have on file the required initial
demonstration of performance data described in Section
9.2 prior to using the method for analysis.
2.0 Summary of Method
2.7 An accurately measured amount of particulate
matter from an estuarine water sample or an accurately
weighed dried sediment sample is combusted at 975°C
using an elemental analyzer. The combustion products
are passed over a copper reduction tube to convert the
oxides of N into molecular N. Carbon dioxide, water vapor
and N are homogeneously mixed at a known volume,
temperature and pressure. The mixture is released to a
series of thermal conductivity detectors/traps, measuring
in turn by difference, hydrogen (as water vapor), C (as
carbon dioxide) and N (as N2). Inorganic and organic C
may be determined by two methods which are also
presented.
3.0 Definitions
3.7 Sediment Sample - A fluvial, sand, or humic
sample matrix exposed to a marine, brackish or fresh
water environment. It is limited to that portion which may
be passed through a number 10 sieve or a 2-mm mesh
sieve.
3.2 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling pre-
cautions.
3.3 Instrument Detection Limit (IDL) - The mini-
mum quantity of analyte or the concentration equivalent
which gives an analyte signal equal to three times the
standard deviation of the background signal at the se-
lected wavelength, mass, retention time, absorbance line,
etc.
3.4 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified, mea-
sured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.5 Linear Dynamic Range (LDR) - The absolute
quantity over which the instrument response to an analyte
is linear.
3.6 Calibration Standard (CAL) - An accurately
weighed amount of a certified chemical used to calibrate
the instrument response with respect to analyte mass.
3.7 Conditioner- A standard chemical which is not
necessarily accurately weighed that is used to coat the
surfaces of the instrument with the analytes (water vapor,
carbon dioxide, and nitrogen).
3.8 External Standards (ES) - A pure analyte(s)
that is measured in an experiment separate from the
experiment used to measure the analyte(s) in the sample.
The signal observed for a known quantity of the pure
external standard(s) is used to calibrate the instrument
Revision 1.4 September 1997
440.0-2
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response for the corresponding analyte(s). The instru-
ment response is used to calculate the concentrations of
the analyte(s) in the sample.
3.9 Response Factor (RF) — The ratio of the re-
sponse of the instrument to a known amount of analyte.
3.70 Laboratory Reagent Blank (LRB) - A blank
matrix (i.e., a precombusted filter or sediment capsule)
that is treated exactly as a sample including exposure to
all glassware, equipment, solvents, and reagents that are
used with other samples. The LRB is used to determine
if method analytes or other interferences are present in
the laboratory environment, the reagents, or the appa-
ratus.
3.77 Field Reagent Blank (FRB) - An aliquot of
reagent water or other blank matrix that is placed in a
sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling
site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.72 Laboratory Duplicates (LD1 and LD2) - Two
aliquots of the same sample taken in the laboratory and
analyzed separately with identical procedures. Analyses
of LD1 and LD2 indicate precision associated with labo-
ratory procedures, but not with sample collection, preser-
vation, or storage procedures.
3.73 Field Duplicates (FD1 and FD2) - Two sepa-
rate samples collected at the same time and place under
identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of
FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well
as with laboratory procedures.
3.74 Laboratory Fortified Blank (LFB) - An aliquot
of reagent water or other blank matrices to which known
quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the method is in
control, and whether the laboratory is capable of making
accurate and precise measurements.
3.7 5 Laboratory Fortified Sample Matrix (LFM) -An
aliquot of an environmental sample to which known
quantities of the method analytes are added in the
laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must
be determined in a separate aliquot and the measured
values in the LFM corrected for background concentra-
tions.
3.76 Standard Reference Material (SRM) - Material
which has been certified for specific analytes by a variety
of analytical techniques and/or by numerous laboratories
using similar analytical techniques. These may consist of
pure chemicals, buffers or compositional standards.
These materials are used as an indication of the accuracy
of a specific analytical technique.
3.77 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. The QCS is
obtained from a source external to the laboratory and
different from the source of calibration standards. It is
used to check laboratory performance with externally
prepared test materials.
4.0 Interferences
4.7 There are no known interferences for estua-
rine/coastal water or sediment samples. The presence of
C and N compounds on laboratory surfaces, on fingers,
in detergents and in dust necessitates the utilization of
careful techniques (i.e., the use of forceps and gloves) to
avoid contamination in every portion of this procedure.
5.0 Safety
5.7 The toxicity or carcinogenicity of each reagent
used in this method has not been fully established. Each
chemical should be regarded as a potential health hazard
and exposure to these compounds should be as low as
reasonably achievable. Each laboratory is responsible for
maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in
this method.2"5 A reference file of material safety data
sheets (MSDS) should also be made available to all
personnel involved in the chemical analysis.
5.2 The acidification of samples containing reactive
materials may result in the release of toxic gases, such as
cyanides or sulfides. Acidification of samples should be
done in a fume hood.
5.3 All personnel handling environmental samples
known to contain or to have been in contact with human
waste should be immunized against known disease
causative agents.
5.4 Although most instruments are adequately
shielded, it should be remembered that the oven tem-
peratures are extremely high and that care should be
taken when working near the instrument to prevent
possible burns.
5.5 It is the responsibility of the user of this method to
comply with relevant disposal and waste regulations. For
guidance see Sections 14.0 and 15.0.
440.0-3
Revision 1.4 September 1997
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6.0 Apparatus and Equipment
6.7 Elemental Analyzer
6.1.1 An elemental analyzer capable of maintaining a
combustion temperature of 975°C and analyzing particu-
late samples and sediment samples for elemental C and
N. The Leeman Labs Model 240 XA Elemental Analyzer
was used to produce the data presented in this method.
6.2 A gravity convection drying oven. Capable of
maintaining 103-105°C for extended periods of time.
6.3 Muffle furnace. Capable of maintaining 875°C ±
15°C.
6.4 Ultra-micro balance. Capable of accurately
weighing to 0.1 ug. Desiccant should be kept in the
weighing chamber to prevent hygroscopic effects.
6.5 Vacuum pump or source capable of maintaining
up to 10 in. Hg of vacuum.
6.6 Mortar and pestle.
6.7 Desiccator, glass.
6.8 Freezer, capable of maintaining -20°C ± 5°C.
6.9 47-mm or 25-mm vacuum filter apparatus made
up of a glass filter tower, fritted glass disk base and 2-L
vacuum flask.
6.70 13-mmSwinlok filter holder.
6.77 Teflon-tipped, flat blade forceps.
6.72 Labware - All reusable labware (glass, quartz,
polyethylene, PTFE, FEP, etc.) should be sufficiently
clean for the task objectives. Several procedures found
to provide clean labware include washing with a detergent
solution, rinsing with tap water, soaking for 4 hr or more
in 20% (v/v) HCI, rinsing with reagent water and storing
clean. All traces of organic material must be removed to
prevent C-N contamination.
6.12.1 Glassware - Volumetric flasks, graduated
cylinders, vials and beakers.
6.12.2 Vacuum filter flasks - 250 ml and 2 L, glass.
6.12.3 Funnel, 6.4 mm i.d., polyethylene.
6.12.4 Syringes, 60-mL, glass.
7.0 Reagents and Standards
7.7 Reagents may contain elemental impurities which
affect analytical data. High-purity reagents that conform
to the American Chemical Society specifications6 should
be used whenever possible. If the purity of a reagent is in
question, analyze for contamination. The acid used for
this method must be of reagent grade purity or equivalent.
A suitable acid is available from a number of manu-
facturers.
7.2
HCI.
Hydrochloric acid, concentrated (sp. gr. 1.19)-
7.3 Acetanilide, 99.9% + purity, C8H19NO (CASRN
103-84-4).
7.4 Blanks - Three blanks are used for the analysis.
Two blanks are instrument related. The instrument zero
response (ZN) is the background response of the instru-
ment without sample holding devices such as capsules
and sleeves. The instrument blank response (BN) is the
response of the instrument when the sample capsule,
sleeve and ladle are inserted for analysis without standard
or sample. The BN is also the laboratory reagent blank
(LRB) for sediment samples. The LRB for water samples
includes the capsule, sleeve, ladle and a precombusted
filter without standard or sample. These blanks are
subtracted from the uncorrected instrument response
used to calculate concentration in Sections 12.3 and 12.4.
7.4.1 Laboratory fortified blank (LFB) - The third blank
is the laboratory fortified blank. For sediment analysis,
add a weighed amount of acetanilide in an aluminum
capsule and analyze for PC and PN (Section 9.3.2). For
aqueous samples, place a weighed amount of acetanilide
on a glass fiber filter the same size as used for the
sample filtration. Analyze the fortified filter for PC and PN
(Section 9.3.2)
7.5 Quality Control Sample (QCS) - For this meth-
od, the QCS can be any assayed and certified sediment
or particulate sample which is obtained from an external
source. The Canadian Reference Material, BCSS-1, is
just such a material and was used in this capacity for the
data presented in this method. The percent PC has been
certified in this material but percent PN has not.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection - Samples collected
for PC and PN analyses from estuarine/coastal waters
are normally collected from a ship using one of two
methods; hydrocast or submersible pump systems. Fol-
low the recommended sampling protocols associated
with the method used. Whenever possible, immediately
filter the samples as described in Section 11.1.1. Store
the filtered sample pads by freezing at -20°C or storing in
a desiccator after drying at 103-105° C for 24 hr. No
significant difference has been noted in comparing the
two storage procedures for a time period of up to 100
days. If storage of the water sample is necessary, place
Revision 1.4 September 1997
440.0-4
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the sample into a clean amber bottle and store at 4°C
until filtration is done.
8.1.1 The volume of water sample collected will vary
with the type of sample being analyzed. Table 1 provides
a guide for a number of matrices of interest. If the matrix
cannot be classified by this guide, collect 2 x 1L of water
from each site. A minimum filtration volume of 200 ml is
recommended.
8.2 Sediment Sample Collection - Estua-
rine/coastal sediment samples are collected with benthic
samplers. The type of sampler used will depend on the
type of sample needed by the data-quality objectives.7
Store the wet sediment in a clean jar and freeze at -20°C
until ready for analysis.
8.2.1 The amount of sediment collected will depend on
the sample matrix and the elemental analyzer used. A
minimum of 10 g is recommended.
9.0 Quality Control
9.1 Each laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and the continued
analysis of laboratory reagent blanks, laboratory dupli-
cates, field duplicates and calibration standards analyzed
as samples as a continuing check on performance. The
laboratory is required to maintain performance records
that define the quality of data thus generated.
9.2 Initial Demonstration
(Mandatory)
of Performance
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (MDLs, linear dy-
namic range) and laboratory performance (analysis of QC
samples) prior to the analyses conducted by this method.
9.2.2 Linear dynamic range (LDR) - The upper limit of
the LDR must be established by determining the signal
responses from a minimum of three different concentra-
tion standards across the range, one of which is close to
the upper limit of the LDR. Determined LDRs must be
documented and kept on file. The LDR which may be
used for the analysis of samples should be judged by the
analyst from the resulting data. The upper LDR limit
should be an observed signal no more than 10% below
the level extrapolated from the lower standards. Deter-
mined sample analyte concentrations that are 90% and
above the upper LDR must be reduced in mass and
reanalyzed. New LDRs should be determined whenever
there is a significant change in instrument response and
for those analytes that periodically approach the upper
LDR limit, every 6 months or whenever there is a change
in instrument analytical hardware or operating conditions.
9.2.3 Quality control sample (QCS) (Section 7.5) -
When beginning the use of this method, on a quarterly
basis or as required to meet data quality needs, verify the
calibration standards and acceptable instrument perfor-
mance with the analyses of a QCS. If the determined
concentrations are not within ± 5% of the stated values,
performance of the determinative step of the method is
unacceptable. The source of the problem must be iden-
tified and corrected before either proceeding with the
initial determination of MDLs or continuing with analyses.
9.2.4 Method detection limits (MDLs) - MDLs should
be established for PC and PN using a low level estuarine
water sample, typically three to five times higher than the
estimated MDL. The same procedure should be followed
for sediments. To determine MDL values, analyze seven
replicate aliquots of water or sediment and process
through the entire analytical procedure (Section 11).
These replicates should be randomly distributed through-
out a group of typical analyses. Perform all calculations
defined in the method (Section 12) and report the con-
centration values in the appropriate units. Calculate the
MDL as follows:1
where, S =
t =
MDL = (t) X (S)
Standard deviation of the repli-
cate analyses.
Student's t value for n-1
degrees of freedom at the
99% confidence limit; t = 3.143
for six degrees of freedom.
MDLs should be determined whenever a significant
change in instrumental response, change of operator, or
a new matrix is encountered.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory reagent blank (LRB) - The laboratory
must analyze at least one LRB (Section 3.10) with each
batch of 20 or fewer samples of the same matrix. LRB
data are used to assess contamination from the labora-
tory environment. LRB values that exceed the MDL
indicate laboratory or reagent contamination. When LRB
values constitute 10% or more of the analyte level deter-
mined for a sample, fresh samples or field duplicates of
the samples must be prepared and analyzed again after
the source of contamination has been corrected and
acceptable LRB values have been obtained. For aque-
ous samples the LRB is a precombusted filter of the
same type and size used for samples.
440.0-5
Revision 1.4 September 1997
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9.3.2 Laboratory fortified blank (LFB) - The laboratory
must analyze at least one LFB (Section 7.4.1) with each
batch of samples. Calculate accuracy as percent recov-
ery. If the recovery of any analyte falls outside the
required control limits of 85-115%, that analyte is judged
out of control, and the source of the problem should be
identified and resolved before continuing analyses.
9.3.3 The laboratory must use LFB analyses data to
assess laboratory performance against the required con-
trol limits of 85-115% (Section 9.3.2). When sufficient
internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can
be developed from the percent mean recovery (x) and the
standard deviation (S) of the mean recovery. These data
can be used to establish the upper and lower control
limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than
the required control limits of 85-115%. After each five to
ten new recovery measurements, new control limits can
be calculated using only the most recent 20-30 data
points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the
level of concentrations included in the LFB. These data
must be kept on file and be available for review.
9.4 Assessing Analyte Recovery and Data
Quality
9.4.1 Percent recoveries cannot be readily obtained
from particulate samples. Consequently, accuracy can
only be assessed by analyzing check standards as
samples and quality control samples (QCS). The use of
laboratory fortified matrix samples has not been as-
sessed.
10.0 Calibration and Standardization
70.7 Calibration - After following manufacturer's
installation and temperature stabilization procedures,
daily calibration procedures must be performed and
evaluated before sample analysis may begin. Single
point or standard curve calibrations are possible, de-
pending on instrumentation.
10.1.1 Establish single response factors (RF) for each
element (C,H, and N) by analyzing three weighed portions
of calibration standard (acetanilide). The mass of
calibration standard should provide a response within
20% of the response expected for the samples being
analyzed. Calculate the (RF) for each element using the
following formula:
Response factor (uv/ug) =
RN-ZN-BN
WTN
where, RN =
ZN =
BN =
Average instrument response to
standard (uv)
Instrument zero response (uv)
Instrument blank response (uv)
and, WTN = (M)(Na)(AW/MW)
where, M = The mass of standard material in
ug
Na = Number of atoms of C, N or H, in
a molecule of standard material
AW = Atomic weight of C (12.01), N
(14.01) or H (1.01)
MW = Molecular weight of standard
material (135.2 for acetanilide)
If instrument response is in units other than uv, then
change the formula accordingly.
10.1.2 For standard curve preparation, the range of
calibration standard masses used should be such that the
low concentration approaches but is above the MDL and
the high concentration is above the level of the highest
sample, but no more than 90% of the linear dynamic
range. A minimum of three concentrations should be
used in constructing the curve. Measure response versus
mass of element in the standard and perform a
regression on the data to obtain the calibration curve.
11.0 Procedure
77.7 Aqueous Sample Preparation
11.1.1 Water Sample Filtration - Precombust GF/F
glass fiber filters at 500°C for 1.5 hr. The diameter of filter
used will depend on the sample composition and instru-
ment capabilities (Section 8.1.1). Store filters covered if
not immediately used. Place a precombusted filter on
fritted filter base of the filtration apparatus and attach the
filtration tower. Thoroughly shake the sample container
to suspend the particulate matter. Measure and record
the required sample volume using a graduated cylinder.
Pour the measured sample into the filtration tower, no
more than 50 mL at a time. Filter the sample using a
vacuum no greater than 10 in. of Hg. Vacuum levels
greater than 10 in. of Hg can cause filter rupture. If less
than the measured volume of sample can be practically
filtered due to clogging, measure and record the actual
volume filtered. Do not rinse the filter following filtration.
It has been demonstrated that sample loss occurs when
the filter is rinsed with an isotonic solution or the filtrate.8
Air dry the filter after the sample has passed through by
continuing the vacuum for 30 sec. Using Teflon-coated
flat-tipped forceps, fold the filters in half while still on the
fritted glass base of the filter apparatus. Store filters as
described in Section 8.
Revision 1.4 September 1997
440.0-6
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11.1.2 If the sample has been stored frozen, place the
sample in a drying oven at 103-105° C for 24 hr before
analysis and dry to a constant weight. Precombust one
nickel sleeve at 875° C for 1 hr for each sample.
11.1.3 Remove the filter pads containing the particulate
material from the drying oven and insert into a pre-
combusted nickel sleeve using Teflon-coated flat-tipped
forceps. Tap the filter pad using a stainless steel rod.
The sample is ready for analysis.
11.2 Sediment Samples Preparation
11.2.1 Thaw the frozen sediment sample in a 102-
105°C drying oven for at least 24 hr before analysis and
dry to a constant weight. After drying, homogenize the
dry sediment with a mortar and pestle. Store in a desic-
cator until analysis. Precombust aluminum capsules at
550°C in a muffle furnace for 1.5 hr for each sediment
sample being analyzed. Precombust one nickel sleeve
at 875°C for 1 hr for each sediment sample.
712.2 Weigh 10 mg of the homogenized sediment to
the nearest 0.001 mg with an ultra-micro balance into a
precombusted aluminum capsule. Crimp the top of the
aluminum capsule with the Teflon-coated flat-tipped for-
ceps and place into a precombusted nickel sleeve. The
sample is ready for analysis.
11.3 Sample Analysis
11.3.1 Measure instrument zero response (Section 7.4)
and instrument blank response (Section 7.4) and record
values. Condition the instrument by analyzing a condi-
tioner. Calibrate the instrument according to Section 10
and analyze all preliminary QC samples as required by
Section 9. When satisfactory control has been estab-
lished, analyze samples according to the instrument
manufacturer's recommendations. Record all response
data.
11.3.2 Report data as directed in Section 12.
11.4 Determination of Particulate Organic and
Inorganic Carbon
11.4.1 Method 1: Thermal Partitioning - The difference
found between replicate samples, one of which has been
analyzed for total PC and PN and the other which was
muffled at 550°C and analyzed is the particulate organic
component of that sample. This method of thermally
partitioning organic and inorganic PC may underestimate
slightly the carbonate minerals' contribution in the
inorganic fraction since some carbonate minerals
decompose below 500°C, although CaCO3 does not.9
11.4.2 Method 2: Fuming HCI - Allow samples to dry
overnight at 103-105°C and then place in a desiccator
containing concentrated HCI, cover and fume for 24 hr in
a hood. The fuming HCI converts inorganic carbonate in
the samples to water vapor, CO2 and calcium chloride.
Analyze the samples for particulate C. The resultant data
are particulate organic carbon.10
12.0 Data Analysis and Calculations
72.7 Sample data should be reported in units of ug/L
for aqueous samples and mg/kg dry weight for sediment
samples.
72.2 Report analyte concentrations up to three signifi-
cant figures for both aqueous and sediment samples.
72.3 For aqueous samples, calculate the sample con-
centration using the following formula:
Corrected
Concentration (ug/L) = sample response (uv)
Sample volume (L) x RF (uv/ug)
where, RF = Response Factor (Section 10.1.1)
Corrected Sample Response (Section
7.4)
72.4 For sediment samples, calculate the sample con-
centration using the following formula:
Corrected
Concentration (mg/kg) = sample response (uv)
Sample weight (g) x RF (uv/ug)
where, RF = Response Factor (Section 10.1.1)
Corrected Sample Response (Section
7.4)
Note: Units of ug/g = mg/kg
72.5 The QC data obtained during the analyses
provide an indication of the quality of the sample data and
should be provided with the sample results.
13.0 Method Performance
73.7 Single Laboratory Performance
13.1.1 Single laboratory performance data for aqueous
samples from the Chesapeake Bay are provided in Table
2.
13.1.2 Single-laboratory precision and accuracy data for
the marine sediment reference material, BCSS-1, are
listed in Table 3.
73.2 Multilaboratory Performance
13.2.1 In a multilab study, 13 participants analyzed
sediment and filtered estuarine water samples for
particulate carbon and nitrogen. The data were analyzed
440.0-7
Revision 1.4 September 1997
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using the statistical procedures recommended in ASTM
D2777-86 for replicate designs. See Table 4 for
summary statistics.
13.2.2 Accuracy as mean recovery was estimated from
the analyses of the NRC of Canada Marine Sediment
Reference Material, BCSS-1. Mean recovery was 98.2%
of the certified reference carbon value and 100% of the
noncertified nitrogen value.
73.2.3 Overall precision for analyses of carbon and
nitrogen in sediments was 1-11% RSD, while the
analyses of both particulate carbon and nitrogen in
estuarine water samples was 9-14% RSD.
13.2.4 Single analyst precision for carbon and nitrogen
in sediment samples was 1-8% RSD and 4-9% for water
samples.
73.2.5 Pooled method detection limits (p-MDLs) were
calculated using the pooled single analyst standard
deviations. The p-MDLs for particulate nitrogen and
carbon in estuarine waters were 0.014 mg N/L and 0.064
mg C/L , respectively. The p-MDLs for percent carbon
and nitrogen in estuarine sediments were not estimated
because the lowest concentration sediment used in the
study was still 20 times higher than the estimated MDLs.
Estimates of p-MDLs from these data would be
unrealistically high.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of environ-
mental management techniques that places pollution
prevention as the management option of first choice.
Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the next
best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and
regulations, and by complying with all solid and hazard-
ous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For
further information on waste management consult The
Waste Management Manual for Laboratory Personnel,
available from the American Chemical Society at the
address listed in Section 14.2.
16.0 References
1. 40 CFR, Part 136, Appendix B. Definition and
Procedure for the Determination of the Method
Detection Limit. Revision 1.11.
2. Carcinogens - Working With Carcinogens,
Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control,
National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. OSHA Safety and Health Standards, General
Industry, (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised,
January 1976).
4. Safety in Academic Chemistry Laboratories,
American Chemical Society Publication, Committee
on Chemical Safety, 3rd Edition, 1979.
5. Proposed OSHA Safety and Health Standards,
Laboratories, Occupational Safety and Health
Administration, Federal Register, July 24, 1986.
6. Rohrdough, W.G. et al. Reagent Chemicals,
American Chemical Society Specifications, 7th
Edition. American Chemical Society, Washington,
DC, 1986.
7. Holme, N.A. and A.D. Mclntyre (eds). 1971.
Methods for the Study of Marine Benthos.
International Biome Program. IBP Handbook #16.
F.A. Davis Co., Philadelphia, PA.
8. Hurd, D.C. and D.W. Spencer (eds). 1991. Marine
Particles: Analysis and Characterization.
Geophysical Monograph: 63, American Geophysical
Union, Washington, DC 472p.
9. Hirota, J. and J.P. Szyper. 1975. Separation of total
particulate carbon into inorganic and organic
components. Limnol. and Oceanogr. 20:896-900.
10. Grasshoff, K., M. Ehrhardt and K. Kremling (eds).
1983. Methods of Seawater Analysis. Verlag
Chemie.
Revision 1.4 September 1997
440.0-8
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17.0 Tables, Diagrams, Flowcharts, and
Validation Data
Table 1. Filter Diameter Selection Guide
Sample matrix
47mm
Filter diameter
25mm
13mm
Sample matrix volume
Open ocean
Coastal
Estuarine
(low particulate)
Estuarine
(high particulate)
2000 ml
1000mL
500-700 ml
100-400 ml
500ml
400-500 ml
250-400 ml
75-200 ml
100ml
100ml
50ml
25ml
Table 2. Performance Data-Chesapeake Bay Aqueous
Samples
Sample
1
2
3
4
Measured
nitrogen
concentration
(ug/L)
147
148
379
122
S.DA
(ug/L)
± 4
± 11
±51
±9
Measured
carbon
concentration
(ug/L)
1210
1240
3950
1010
S.DA
(ug/L)
±49
±179
±269
±63
k Standard deviation based on 7 replicates.
Table 3. Precision and Accuracy Data - Canadian
Sediment Reference Material BCSS-1
Element
Carbon
Nitrogen
T.VA
2.19%
0.195%
Mean
measured
value (%)
2.18
0.194
%RSDB
±3.3
±3.9
% Recovery0
99.5
99.5
True value. Carbon value is certified; nitrogen value is listed but
not certified
B Percent relative standard deviation based on 10 replicates.
c As calculated from T.V.
440.0-9
Revision 1.4 September 1997
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Table 4. Overall and Single Analyst Precision Estimates from Collaborative Study
Analyte
Participate
Nitrogen
(as N) in
Estuarine
Waters
Sample
A
B
C
D
E
N(1)
11
12
12
12
11
Mean(2)
Cone.
0.0655
0.0730
0.0849
0.126
0.182
Overall
Std. Dev.
0.0081
0.0076
0.0110
0.0138
0.0245
Overall
%RSD
12.4%
10.3%
12.9%
11.0%
13.5%
Analyst
Std. Dev.
0.0050
0.0057
0.0060
0.0071
0.0157
Analyst
%RSD
7.6%
7.7%
7.1%
5.6%
8.6%
Nitrogen
(as %N) in
Estuarine
Water
1
2
3
4
5
10
10
10
10
10
0.178
0.295
0.436
0.497
0.580
0.0190
0.0114
0.0178
0.0183
0.0207
10.7%
3.9%
4.1%
3.7%
3.6%
0.0131
0.0046
0.0104
0.0082
0.0150
7.3%
1 .6%
2.4%
1 .6%
2.6%
Particulate
Carbon
(as C) in
Estuarine
Waters
B
A
D
C
E
12
12
12
12
12
0.369
0.417
0.619
0.710
0.896
0.0505
0.0490
0..0707
0.0633
0.1192
13.7%
11.8%
1 1 .4%
8.9%
13.3%
0.0222
0.0230
0.0226
0.0367
0.0569
6.0%
5.5%
3.6%
5.2%
6.4%
Carbon
(as %C) in
Estuarine
Sediments
1
2
3
4
5
13
13
13
13
13
1.78
2.55
3.18
4.92
5.92
0.1517
0.0372
0.0435
0.1201
0.0621
8.5%
1 .5%
1 .4%
2.4%
1.1%
0.1346
0.0204
0.0348
0.0779
0.0547
7.6%
0.8%
1.1%
1 .6%
0.9%
(1) N = Number of participants whose data was used.
(2) Concentration in mg/L or percent, as indicated.
Revision 1.4 September 1997
440.0-10
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Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytin a
in Marine and Freshwater Algae by Fluorescence
Elizabeth J. Arar
and
Gary B. Collins
Revision 1.2
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
445.0-1
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Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytin a
in Marine and Freshwater Algae by Fluorescence
1.0 Scope and Application
1.1 This method provides a procedure for low level
determination of chlorophyll a (chl a) and its magnesium-
free derivative, pheophytin a (pheo a), in marine and
freshwater phytoplankton using fluorescence detection.'12)
Phaeophorbides present in the sample are determined
collectively as pheophytin a. For users primarily
interested in chl a there is currently available a set of very
narrow bandpass excitation and emission filters (Turner
Designs, Sunnyvale, CA) that nearly eliminates the
spectral interference caused by the presence of pheo a
and chlorophyll b. The difference between the modified
method and the conventional fluorometric method is that
the equations used for the determination of chlor a
without pheo a correction (uncorrected chlor a), are used
instead of the equations for "corrected chlor a". This EPA
laboratory has evaluated the modified filters and found
the technique to be an acceptable alternative to the
conventional fluorometric method using pheo a
correction.(3)
Chemical Abstracts Service
Analyte Registry Number (CASRN)
Chlorophyll a
479-61-8
1.2 Instrumental detection limits (IDL) of 0.05 ug chl
a/L and 0.06 ug pheo a/L in a solution of 90% acetone
were determined by this laboratory. Method detection
limits (MDL) using mixed assemblages of algae provide
little information because the fluorescence of other
pigments interferes in the fluorescence of chlorophyll a
and pheophytin a.(4) A single lab estimated detection limit
for chlorophyll a was determined to be 0.11 ug/L in 10 ml
of final extraction solution. The upper limit of the linear
dynamic range for the instrumentation used in this
method evaluation was 250 ug chl a/L.
1.3 This method was multilaboratory validated in
1996.(5) Results from that study may be found in Section
13. Additional QC procedures also have been added as
a result of that study.
1.4 This method uses 90% acetone as the extraction
solvent because of its efficiency for most types of algae.
There is evidence that certain chlorophylls and
carotenoids are more thoroughly extracted with
methanol'5"8' or dimethyl sulfoxide.(9) Bowles, et al.(8)
found that for chlorophyll a, however, 90% acetone was
an effective extractant when the extraction period was
optimized for the dominant species present in the sample.
1.5 Depending on the type of algae under
investigation, this method can have uncorrectable
interferences (Sect. 4.0). In cases where taxomonic
classification is unavailable, a spectrophotometric or high
performance liquid chromatographic (HPLC) method may
provide more accurate data for chlorophyll a and
pheophytin a.
1.6 This method is for use by analysts experienced in
the handling of photosynthetic pigments and in the
operation of fluorescence detectors or by analysts under
the close supervision of such qualified persons.
2.0 Summary of Method
2.7 Chlorophyll-containing phytoplankton in a
measured volume of sample water are concentrated by
filtering at low vacuum through a glass fiber filter. The
pigments are extracted from the phytoplankton in 90%
acetone with the aid of a mechanical tissue grinder and
allowed to steep for a minimum of 2 h, but not to exceed
24 h, to ensure thorough extraction of the chlorophyll a.
The filter slurry is centrifuged at 675 g for 15 min (or at
1000 g for 5 min) to clarify the solution. An aliquot of the
supernatant is transferred to a glass cuvette and
fluorescence is measured before and after acidification to
0.003 N HCIwith 0.1 N HCI. Sensitivity calibration factors,
which have been previously determined on solutions of
Revision 1.2 September 1997
445.0-2
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pure chlorophyll a of known concentration, are used to
calculate the concentration of chlorophyll a and
pheophytin a in the sample extract. The concentration in
the natural water sample is reported in ug/L.
3.0 Definitions
3.1 Estimated Detection Limit (EDL) - The
minimum concentration of an analyte that yields a
fluorescence 3X the fluorescence of blank filters which
have been extracted according to this method.
3.2 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.3 Instrument Detection Limit (IDL) - The
minimum quantity of analyte or the concentration
equivalent which gives an analyte signal equal to three
times the standard deviation of the background signal at
the selected wavelength, mass, retention time,
absorbance line, etc. For this method the background is
a solution of 90% acetone.
3.4 Stock Standard Solution (SSS) - A
concentrated solution containing one or more method
analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable
commercial source.
3.5 Primary Dilution Standard Solution (PDS) - A
solution of the analytes prepared in the laboratory from
stock standard solutions and diluted as needed to
prepare calibration solutions and other needed analyte
solutions.
3.6 Calibration Standard (CAL) - A solution
prepared from the primary dilution standard solution or
stock standard solutions containing the internal standards
and surrogate analytes. The CAL solutions are used to
calibrate the instrument response with respect to analyte
concentration.
3.7 Response Factor (RF) - The ratio of the
response of the instrument to a known amount of analyte.
3.8 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
reagents, or apparatus.
3.9 Field Duplicates (FD1 and FD2) - Two separate
samples collected at the same time and place under
identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of
FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well
as with laboratory procedures.
3.70 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. Ideally, the
QCS is obtained from a source external to the laboratory
and different from the source of calibration standards. It
is used to check laboratory performance with externally
prepared test materials.
3.11 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling
precautions.
4.0 Interferences
4.1 Any substance extracted from the filter or
acquired from laboratory contamination that fluoresces in
the red region of the spectrum may interfere in the
accurate measurement of both chlorophyll a and
pheophytin a.
4.2 The relative amounts of chlorophyll a, b and c
vary with the taxonomic composition of the phytoplankton.
Chlorophylls b and c may significantly interfere with
chlorophyll a measurements depending on the amount
present. Due to the spectral overlap of chlorophyll b with
pheophytin a and chlorophyll a, underestimation of
chlorophyll a occurs accompanied by overestimation of
pheophytin a when chlorophyll b is present in the sample.
The degree of interference depends upon the ratio of a:Jb.
This laboratory found that at a ratio of 5:1, using the
acidification procedure to correct for pheophytin a,
chlorophyll a was underestimated by approximately 5%.
Loftis and Carpenter'10' reported an underestimation of
16% when the a:Jb ratio was 2.5:1. A ratio of 1:1 is the
highest ratio likely to occur in nature. They also reported
overestimation of chlorophyll a in the presence of
chlorophyll c of as much as 10% when the a:c ratio was
1:1 (the theoretical maximum likely to occur in nature).
The presence of chlorophyll c also causes the under-
445.0-3
Revision 1.2 September 1997
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estimation of pheophytin a. The effect of chlorophyll c is
not as severe as the effect of chlorophyll b on the
measurement of chlorophyll a and pheophytin a.
Knowledge of the taxonomy of the algae under
consideration will aid in determining if the
spectrophotometric method using trichromatic equations
to determine chlorophyll a, b, and c or an HPLC method
would be more appropriate.(11"16) In the presence of
chlorophyll b or pheopigments, the modified fluorometric
method described here is also appropriate.'5'
4.3 Quenching effects are observed in highly
concentrated solutions or in the presence of high
concentrations of other chlorophylls or carotenoids.
Minimum sensitivity settings on the fluorometer should be
avoided; samples should be diluted instead.
4.4 Fluorescence is temperature dependent with
higher sensitivity occurring at lower temperatures.
Samples, standards, LRBs and QCSs must be at the
same temperature to prevent errors and/or low precision.
Analyses of samples at ambient temperature is
recommended in this method. Ambient temperature
should not fluctuate more than ± 3°C between
calibrations or recalibration of the fluorometer will be
necessary.
4.5 Samples must be clarified by centrifugation prior
to analysis.
4.6 All photosynthetic pigments are light and
temperature sensitive. Work must be performed in
subdued light and all standards, QC materials and filter
samples must be stored in the dark at -20°C or -70°C to
prevent degradation.
5.0 Safety
5.7 The toxicity or carcinogenicity of the chemicals
used in this method have not been fully established.
Each chemical should be regarded as a potential health
hazard and handled with caution and respect. Each
laboratory is responsible for maintaining a current
awareness file of Occupational Safety and Health
Administration (OSHA) regulations regarding the safe
handling of the chemicals specified in this method.(17"20) A
file of MSDS should also be made available to all
personnel involved in the chemical analysis.
5.2 The grinding of filters during the extraction step of
this method should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.
6.0 Apparatus and Equipment
6.7 Fluorometer — Equipped with a high intensity
F4T.5 blue lamp, red-sensitive photomultiplier, and filters
for excitation (CS-5-60) and emission (CS-2-64). A
Turner Designs Model 10 Series fluorometer was used in
the evaluation of this method. The modified method
requires excitation filter (436FS10) and emission filter
(680FS10).
6.2 Centrifuge, capable of 675 g.
6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)
with grooves in the tip with 1/4" stainless steel rod long
enough to chuck onto a suitable drive motor and 30-mL
capacity glass grinding tube.
6.4 Filters, glass fiber, 47-mm or 25-mm, nominal
pore size of 0.7 urn unless otherwise justified by data
quality objectives. Whatman GF/F filters were used in this
work.
6.5 Petri dishes, plastic, 50 X 9-mm, or some other
solid container for transporting and storing sampled
filters.
6.6 Aluminum foil.
6.7 Laboratory tissues.
6.8 Tweezers or flat-tipped forceps.
6.9 Vacuum pump or source capable of maintaining
a vacuum up to 6 in. Hg.
6.70 Room thermometer.
6.11 Labware — All reusable labware (glass,
polyethylene, Teflon, etc.) that comes in contact with
chlorophyll solutions should be clean and acid free. An
acceptable cleaning procedure is soaking for 4 h in
laboratory grade detergent and water, rinsing with tap
water, distilled deionized water and acetone.
6.11.1 Assorted Class A calibrated pipets.
6.712 Graduated cylinders, 500-mL and 1-L.
6.11.3 Volumetric flasks, Class A calibrated, 25-mL, 50-
ml_, 100-mL and 1-L capacity.
6.11.4 Glass rods.
Revision 1.2 September 1997
445.0-4
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6.715 Pasteur type pipets or medicine droppers.
6.11.6 Disposable glass cuvettes for the fluorometer.
6.11.7 Filtration apparatus consisting of 1 or 2-L filtration
flask, 47-mm fritted glass disk base and a glass filter
tower.
6.11.8 Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.11.9 Polyethylene squirt bottles.
7.0 Reagents and Standards
7.1 Acetone, HPLC grade, (CASRN 67-64-1).
7.2 Hydrochloric acid (HCI), concentrated (sp. gr.
1.19), (CASRN 7647-01-0).
7.3 Chlorophyll a free of chlorophyll b. May be
obtained from a commercial supplier such as Sigma
Chemical (St. Louis, MO). Turner Designs (Sunnyvale,
CA) supplies ready-made standards.
7.4 Water - ASTM Type I water (ASTM D1193) is
required. Suitable water may be obtained by passing
distilled water through a mixed bed of anion and cation
exchange resins.
7.5 0.1 N HCI Solution - Add 8.5 ml of
concentrated HCI to approximately 500 ml water and
dilute to 1 L.
7.6 Aqueous Acetone Solution - 90% acetone
/10% water. Carefully measure 100 ml of water into the
1-L graduated cylinder. Transfer to a 1-L flask or storage
bottle. Measure 900 ml of acetone into the graduated
cylinder and transfer to the flask or bottle containing the
water. Mix, label and store.
7.7 Chlorophyll Stock Standard Solution (SSS) -
Chlorophyll a from a commercial supplier will be shipped
in an amber glass ampoule which has been flame sealed.
This dry standard should be stored at -20 or -70°C in the
dark and the SSS prepared just prior to use. Tap the
ampoule until all the dried chlorophyll is in the bottom of
the ampoule. In subdued light, carefully break the tip off
the ampoule. Transfer the entire contents of the
ampoule into a 50-mL volumetric flask. Dilute to volume
with 90% acetone, label the flask and wrap with
aluminum foil to protect from light. The concentration of
the solution must be determined spectrophotometrically
using a multiwavelength spectrophotometer.(10) When
stored in a light and airtight container at freezer
temperatures, the SSS is stable for at least six months.
The concentration of all dilutions of the SSS must be
determined spectrophotometrically each time they are
made.
7.8 Laboratory Reagent Blank (LRB) - A blank
filter which is extracted and analyzed just as a sample
filter. The LRB should be the last filter extracted of a
sample set. It is used to assess possible contamination
of the reagents or apparatus.
7.9 Chlorophyll a Primary Dilution Standard
Solution (PDS) - Add 1 ml of the SSS (Sect. 7.8) to a
clean 100-mL flask and dilute to volume with the aqueous
acetone solution (Sect. 7.7). If exactly 1 mg of pure
chlorophyll a was used to prepare the SSS, the
concentration of the PDS is 200 ug/L. Prepare fresh just
prior to use.
7.70 Quality Control Sample (QCS) - Since there
are no commercially available QCSs, dilutions of a stock
standard of a different lot number from that used to
prepare calibration solutions may be used.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection — Water may be
obtained by a pump or grab sampler. Data quality
objectives will determine the depth at which samples are
taken. Healthy phytoplankton, however, are generally
obtained from the photic zone (depth at which the
illumination level is 1% of surface illumination). Enough
water should be collected to concentrate phytoplankton
on at least three filters so that precision can be assessed.
Filtration volume size will depend on the particulate load
of the water. Four liters may be required for open ocean
water where phytoplankton density is usually low,
whereas 1 L or less is generally sufficient for lake, bay or
estuary water. All apparatus should be clean and acid-
free. Filtering should be performed in subdued light as
soon as possible after sampling since algal poulations,
thus chlorophyll a concentration, can change in relatively
short periods of time. Aboard ship filtration is highly
recommended.
Assemble the filtration apparatus and attach the vacuum
source with vacuum gauge and regulator. Vacuum
filtration should not exceed 6 in. Hg (20 kPa). Higher
445.0-5
Revision 1.2 September 1997
-------
filtration pressures and excessively long filtration times (>
10 min) may damage cells and result in loss of
chlorophyll.
Prior to drawing a subsample from the water sample
container, thoroughly but gently agitate the container to
suspend the particulates (stir or invert several times).
Pour the subsample into a graduated cylinder and
accurately measure the volume. Pour the subsample into
the filter tower of the filtration apparatus and apply a
vacuum (not to exceed 20 kPa). A sufficient volume has
been filtered when a visible green or brown color is
apparent on the filter. Do not suck the filter dry with the
vacuum; instead slowly release the vacuum as the final
volume approaches the level of the filter and completely
release the vacuum as the last bit of water is pulled
through the filter. Remove the filter from the fritted base
with tweezers, fold once with the particulate matter inside,
lightly blot the filter with a tissue to remove excess
moisture and place it in the petri dish or other suitable
container. If the filter will not be immediately extracted,
then wrap the container with aluminum foil to protect the
phytoplankton from light and store the filter at -20 or
-70°C. Short term storage (2 to 4 h) on ice is acceptable,
but samples should be stored at -20 or -70°C as soon as
possible.
8.2 Preservation - Sampled filters should be stored
frozen (-20°C or -70°C) in the dark until extraction.
8.3 Holding Time - Filters can be stored frozen at
-20 or -70°C for as long as 31/2 weeks without significant
loss of chlorophyll a.(21)
9.0 Quality Control
9.1 Each Laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and the continued
analysis of laboratory reagent blanks, field duplicates and
quality control samples as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
thus generated.
9.2 Initial Demonstration
(Mandatory)
of Performance
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (instrumental
detection limits, linear dynamic range and MDLs) and
laboratory performance (analyses of QCSs) prior to
sample analyses.
9.2.2 Linear Dynamic Range (LDR) - The LDR should
be determined by analyzing a minimum of 5 calibration
standards ranging in concentration from 0.2 ug/L to 200
ug chl a/L across all sensitivity settings of the fluorometer.
If using an analog fluorometer or a digital fluorometer
requiring manual changes in sensitivity settings, normalize
responses by dividing the response by the sensitivity
setting multiplier. Perform the linear regression of
normalized response vs. concentration and obtain the
constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher
concentration until the measured fluorescence response,
R, of a standard no longer yields a calculated
concentration, Cc, that is ± 10% of the known
concentration, C, where Cc = (R - b)/m. That
concentration defines the upper limit of the LDR for your
instrument. Should samples be encountered that have a
concentration which is 90% of the upper limit of the LDR,
these samples must be diluted and reanalyzed.
9.2.3 Instrumental Detection Limit (IDL) - Zero the
fluorometer with a solution of 90% acetone on the
maximum sensitivity setting. Pure chlorophyll a in 90%
acetone should be serially diluted until it is no longer
detected by the fluorometer on a maximum sensitivity
setting.
9.2.4 Estimated Detection Limit (EDL) - Several blank
filters should be extracted according to the procedure in
Sect. 11, using clean glassware and apparatus, and the
fluorescence measured. A solution of pure chlorophyll a
in 90% acetone should be serially diluted until it yields a
response which is 3X the average response of the blank
filters.
9.2.5 Quality Control Sample (QCS) - When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify the calibration standards
and acceptable instrument performance with the analysis
of a QCS (Sect. 7.10). If the determined value is not
within the confidence limits established by project data
quality objectives, then the determinative step of this
method is unacceptable. The source of the problem
must be identified and corrected before continuing
analyses.
9.2.6 Extraction Proficiency - Personnel performing
this method for the first time should demonstrate
proficiency in the extraction of sampled filters (Sect. 11.1).
Revision 1.2 September 1997
445.0-6
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Twenty to thirty natural samples should be obtained using
the procedure outlined in Sect. 8.1 of this method. Sets
of 10 or more samples should be extracted and analyzed
according to Sect. 11.2. The percent relative standard
deviation (%RSD) of uncorrected values of chlorophyll a
should not exceed 15% for samples that are
approximately 10X the IDL. RSD for pheophytin a might
typically range from 10 to 50%.
9.2.7 Corrected Chi a - Multilaboratory testing of this
method revealed that many analysts do not adequately
mix the acidified sample when determining corrected chl
a. The problem manifests itself by highly erratic pheo-a
results, high %RSDs for corrected chl a and poor
agreement between corrected and uncorrected chl a. To
determine if a new analyst is performing the acidification
step properly, perform the following QC procedure:
Prepare 100 ml of a 50 ppb chl a solution in 90%
acetone. The new analyst should analyze 5-10 separate
aliquots, using separate cuvettes, according to
instructions in Section 11.2. Process the results
according to Section 12 and calculate separate means
and %RSDs for corrected and uncorrected chl a. If the
means differ by more than 10%, then the stock chl a has
probably degraded and fresh stock should be prepared.
The %RSD for corrected chl a should not exceed 5%. If
the %RSD exceeds 5%, repeat the procedure until the
%RSD 5%.
9.3
Assessing
(Mandatory)
Laboratory Performance
9.3.1 Laboratory Reagent Blank (LRB) - The
laboratory must analyze at least one blank filter with each
sample batch. The LRB should be the last filter
extracted. LRB data are used to assess contamination
from the laboratory environment. LRB values that exceed
the IDL indicate contamination from the laboratory
environment. When LRB values constitute 10% or more
of the analyte level determined for a sample, fresh
samples or field duplicates must be analyzed after the
contamination has been corrected and acceptable LRB
values have been obtained.
10.0 Calibration and Standardization
10.1 Calibration — Calibration should be performed
bimonthly or when there has been an adjustment made
to the instrument, such as replacement of lamp, filters or
photomultiplier. Prepare 0.2, 2, 5, 20 and 200 ug chl a/L
calibration standards from the PDS (Sect. 7.11). Allow
the instrument to warm up for at least 15 min. Measure
the fluorescence of each standard at sensitivity settings
that provide midscale readings. Obtain response factors
for chlorophyll a for each sensitivity setting as follows:
Fe = CJR,
where:
Fs = response factor for sensitivity setting, S.
Rs = fluorometer reading for sensitivity
setting, S.
Ca = concentration of chlorophyll a.
NOTE: If you are using special narrow bandpass filters
for chl a determination, DO NOT acidify. Use the
"uncorrected" chl a calculation described in Section 12.1.
If pheophytin a determinations will be made, it will be
necessary to obtain before-to-after acidification response
ratios of the chlorophyll a calibration standards as follows:
(1) measure the fluorescence of the standard, (2) remove
the cuvette from the fluorometer, (3) acidify the solution
to .003 N HCI(6) with the 0.1 N HCI solution, (4) use a
pasteur type pipet to thoroughly mix the sample by
aspirating and dispensing the sample into the cuvette,
keeping the pipet tip below the surface of the liquid to
avoid aerating the sample, (5) wait 90 sec and measure
the fluorescence of the standard solution again. Addition
of the acid may be made using a medicine dropper. It will
be necessary to know how many drops are equal to 1 mL
of acid. For a cuvette that holds 5 mL of extraction
solution, it will be necessary to add 0.15 mL of 0.1 N HCI
to reach a final acid concentration of 0.003N in the 5 mL.
Calculate the ratio, r, as follows:
r = Rb/Ra
where:
Rb = fluorescence of pure chlorophyll
standard solution before acidification.
Ra = fluorescence of pure chlorophyll a
standard solution after acidification.
445.0-7
Revision 1.2 September 1997
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11.0 Procedure
11.2 SAMPLE ANALYSIS
11.1 Extraction of Filter Samples
11.1.1 If sampled filters have been frozen, remove them
from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand tissues and squirt bottles
containing water and acetone. Workspace lighting should
be the minimum that is necessary to read instructions and
operate instrumentation. Remove a filter from its
container and place it in the glass grinding tube. The filter
may be torn into smaller pieces to facilitate extraction.
Push it to the bottom of the tube with a glass rod. With a
volumetric pipet, add 4 mL of the aqueous acetone
solution (Sect. 7.6) to the grinding tube. Grind the filter
until it has been converted to a slurry. (NOTE: Although
grinding is required, care must be taken not to overheat
the sample. Good judgement and common sense will
help you in deciding when the sample has been
sufficiently macerated.) Pour the slurry into a 15-mL
screw-cap centrifuge tube and, using a 6-mL volumetric
pipet, rinse the pestle and the grinding tube with 90%
acetone. Add the rinse to the centrifuge tube containing
the filter slurry. Cap the tube and shake it vigorously.
Place it in the dark before proceeding to the next filter
extraction. Before placing another filter in the grinding
tube, use the acetone and water squirt bottles to
thoroughly rinse the pestle, grinding tube and glass rod.
The last rinse should be with acetone. Use a clean tissue
to remove any filter residue that adheres to the pestle or
to the steel rod of the pestle. Proceed to the next filter
and repeat the steps above. The entire extraction with
transferring and rinsing steps takes 5 min. Approximately
500 mL of acetone and water waste are generated per 20
samples from the rinsing of glassware and apparatus.
11.1.2 Shake each tube vigorously before placing them
to steep in the dark at 4°C. Samples should be allowed
to steep for a minimum of 2 h but not to exceed 24 h.
The tubes should be shaken at least once during the
steeping period.
11.1.3 After steeping is complete, shake the tubes
vigorously and centrifuge samples for 15 min at 675 g or
for 5 min at 1000 g. Samples should be allowed to come
to ambient temperature before analysis. This can be
done by placing the tubes in a constant temperature
water bath or by letting them stand at room temperature
for 30 min. Recalibrate the fluorometer if the room
temperature fluctuated ± 3°C from the last calibration
date.
1 1.2. 1 After the fluorometer has warmed up for at least
15 min, use the 90% acetone solution to zero the
instrument on the sensitivity setting that will be used for
sample analysis.
11.2.2 Pour or pipet the supernatant of the extracted
sample into a sample cuvette. The volume of sample
required in your instrument's cuvette should be known so
that the correct amount of acid can be added in the
pheophytin a determinative step. For a cuvette that holds
5 mL of extraction solution, 0.15 mL of the 0.1 N HCI
solution should be used. Choose a sensitivity setting that
yields a midscale reading when possible and avoid the
minimum sensitivity setting. If the concentration of
chlorophyll a in the sample is > 90% of the upper limit of
the LDR, then dilute the sample with the 90% acetone
solution and reanalyze. Record the fluorescence
measurement and sensitivity setting used for the sample.
Remove the cuvette from the fluorometer and acidify the
extract to a final concentration of 0.003 N HCI using the
0.1 N HCI solution. Use a pasteur type pipet to
thoroughly mix the sample by aspirating and dispensing
the sample into the cuvette, keeping the pipet tip below
the surface of the liquid to avoid aerating the sample.
Wait 90 sec before measuring fluorescence again.
NOTE: Proper mixing is critical for precise and accurate
results. Twenty-five to thirty-five samples can be
extracted and analyzed in one 8 hr day.
NOTE: If you are using special narrow bandpass filters
for chl a determination, DO NOT acidify samples. Use
the "uncorrected" chl a calculations described in Section
12.1.
12.0 Data Analysis and Calculations
72.7 For "uncorrected chlorophyll a," calculate the
chlorophyll a concentration in the extract as:
where CEu = uncorrected chlorophyll a concentration
(ug/L) in the extract solution analyzed,
Rt, = fluoresence response of sample extract
before acidification, and
Fs = fluoresence respnse factor for sensitivity
setting S.
Revision 1.2 September 1997
445.0-8
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Calculate the "uncorrected" concentration of chlorophyll
a in the whole water sample as follows:
S,u
CEu x extract volume (L) X DF
sample volume (L)
where CS:U = uncorrected chlorophyll a concentration
(ug/L) in the whole water sample,
extract volume = volume (L) of extraction
prepared before any dilutions,
DF = dilution factor,
sample volume = volume (L) of whole water
sample.
72.2 For "corrected chlorophyll a", calculate the
chlorophyll a concentration in the extract as :
CE,C= Fs(r/r-1)(Rb-Ra)
where:
CEc= corrected chlorophyll a concentration (ug/L) in the
extract solution analyzed,
Fs = response factor for the sensitivity setting S,
r = the before-to-after acidification ratio of a
pure chlorophyll a solution (Sect. 10.1),
Rb = fluorescence of sample extract before
acidification, and
Ra = fluorescence of sample extract after
acidification.
Calculate the "corrected" concentration of chlorophyll a
in the whole water sample as follows:
Cs,c =
CEu x extract volume (L) X DF
sample volume (L)
extract volume = volume (L) of extract prepared
before dilution,
72.3 Calculate the pheophytin a concentration as
follows:
PE = Fs
(rRa-R6)
where CSc = corrected chlorophyll a concetration (ug/L)
in the whole water sample,
PE X extract volume (L) X DF
sample volume (L)
where PE = pheophytin a concentration (ug/L) in the
sample extract; and
Ps = pheophytin a concentration (ug/L) in the
whole water sample.
72.4 LRB and QCS data should be reported with each
sample data set.
13.0 Method Performance
73.7 The single lab EDL forthe instrument used in the
evaluation of this method was 0.05 ug/L for chlorophyll a
and 0.06 ug/L pheophytin a.
73.2 The precision (%RSD) for chlorophyll a in mostly
blue-green and green phytoplankton natural samples
which were steeped for 2 h vs 24 h is reported in Table 1.
Although the means were the same, precision was better
for samples which were allowed to steep for 24 h prior to
analysis. Since pheophytin a was found in the samples,
the chlorophyll a values are "corrected" (Sect. 12.2).
Table 2 contains precision data for pheophytin a. A
statistical analysis of the pheophytin a data indicated a
significant difference in the mean values at the 0.05
significance level. The cause of the lower pheophytin a
values in samples extracted for 24 h is not known.
73.3 Three QCS ampoules obtained from the USEPA
were analyzed and compared to the reported confidence
limits in Table 3. NOTE: The USEPA no longer provides
these QCSs.
73.4 Multilaboratory Testing - A multilaboratory
validation and comparison study of EPA Methods 445.0,
446.0 and 447.0 for chlorophyll a was conducted in 1996
by Research Triangle Institute, Research Triangle Park,
N.C. (EPA Contract No. 68-C5-0011). There were 21
volunteer participants in the fluorometric methods
445.0-9
Revision 1.2 September 1997
-------
component that returned data; 10 that used the modified
fluorometric method and 11 that used the conventional
method. The primary goals of the study were to
determine estimated detection limits and to assess
precision (%RSD) and bias (as percent recovery) for
select unialgal species, and natural seawater.
13.4.1 The term, pooled estimated detection limit (p-
EDL), is used in this method to distinguish it from the EPA
defined method detection limit (MDL). An EPA MDL
determination is not possible nor practical for a natural
water or pure species sample due to known spectral
interferences and to the fact that it is impossible to
prepare solutions of known concentrations that
incorporate all sources of error (sample collection,
filtration, processing). The statistical approach used to
determine the p-EDL was an adaptation of the Clayton,
et.al.(22) method that does not assume constant error
variances across concentration and controls for Type II
error. The statistical approach used involved calculating
an estimated DL for each lab that had the desired Type I
and Type II error rates (0.01 and 0.05, respectively). The
median DLs over labs was then determined and is
reported in Table 4. It is referred to as pooled-EDL (p-
EDL).
Solutions of pure chlorophyll a in 90% acetone were
prepared at three concentrations (0.11, 0.2 and 1.6 ppm)
and shipped with blank glass fiber filters to participating
laboratories. Analysts were instructed to spike the filters
in duplicate with a given volume of solution and to
process the spiked filters according to the method. The
results from these data were used to determine a p-EDL
for each method. Results (in ppm) are given in Table 4.
The standard fluorometric and HPLC methods gave the
lowest p-EDLs while the spectrophotometric
(monochromatic equations) gave the highest p-EDLs.
Due to the large dilutions required to analyze these
solutions, the fluorometric p-EDLs are unrealistically high
compared to what is achievable by a single lab. Typical
single lab EDLs can easily be 1000 fold lower than the p-
EDL reported in Table 4.
13.4.2 To address precision and bias in chlorophyll a
determination for different algal species, three pure
unialgal cultures (Amphidinium, Dunaliella and
Phaeodactylum) were cultured and grown in the
laboratory. Four different "concentrations" of each
species were prepared by filtering varying volumes of the
algae. The filters were frozen and shipped to participant
labs. Analysts were instructed to extract and analyze the
filters according to the respective methods. The "true"
concentration was assigned by taking the average of the
HPLC results for the highest concentration algae sample
since chlorophyll a is separated from other interfering
pigments prior to determination. Pooled precision (as
determined by %RSD) data are presented in Tables 5-7
and accuracy data (as percent recovery) are presented in
Table 8. No significant differences in precision were
observed across concentrations for any of the species. It
should be noted that there was considerable lab-to-lab
variation (as exhibited by the min and max recoveries in
Table 8) and in this case the median is a better measure
of central tendency than the mean.
In summary, the mean and median concentrations
determined for Amphidinium carterae (class
dinophyceae) are similar for all methods. No method
consistently exhibited high or low values relative to the
other methods. The only concentration trend observed
was that the spectrophotometric method-trichromatic
equations (SP-T) showed a slight percent increase in
recovery with increasing algae filtration volume.
For Dunaliella tertiolecti (class chlorophyceae) and
Phaeodactylum tricornutum (class bacillariophyceae)
there was generally good agreement between the
fluorometric and the spectrophotometric methods,
however, the HPLC method yielded lower recoveries with
increasing algae filtration volume for both species. No
definitive explanation can be offered at this time for this
phenomenon. A possible explanation for the
Phaeodactylum is that it contained significant amounts of
chlorophyllide a which is determined as chlorophyll a in
the fluorometric and spectrophotometric methods. The
conventional fluorometric method (FL-STD) showed a
slight decrease in chlorophyll a recovery with increasing
Dunaliella filtration volume. The spectrophotometric-
trichromatic equations (SP-T) showed a slight increase in
chlorophyll a recovery with increasing Dunaliella filtration
volume. The fluorometric and the spectrophotometric
methods both showed a slight decrease in chlorophyll a
recovery with increasing Phaeodactylum filtration volume.
Results for the natural seawater sample are presented in
Table 9. Only one filtration volume (100 mL) was
provided in duplicate to participant labs.
14.0 Pollution Prevention
74.7 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of
Revision 1.2 September 1997
445.0-10
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environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation (e.g., Sect. 11.1.1). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
74.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202)872-4477.
15.0 Waste Management
75.7 The Environmental Protection Agency requires
that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in the Sect. 14.2.
16.0 References
1. Yentsch, C.S. and D.W. Menzel, "A method for
the determination of phytoplankton chlorophyll
and pheophytin by fluorescence", Deep Sea
Res.. 10(1963), pp. 221-231.
2. Strickland, J.D.H. and T.R. Parsons, A Practical
Handbook of Seawater Analysis, Bull. Fish. Res.
Board Can., 1972, No.167, p. 201.
3. Arar, E., "Evaluation of a new technique that uses
highly selective interference filters for measuring
chlorophyll a in the presence of chlorophyll b and
pheopigments," USEPA Summary Report, 1994,
NTISNo. PB94-210622.
4. Trees, C.C., M.C. Kennicutt, and J.M. Brooks,
"Errors associated with the standard fluorometric
determination of chlorophylls and
phaeopigments", Mar. Chem.. 17 (1985) pp. 1-
12.
5. Method 445, "Multi-Laboratory Comparison and
Validation of Chlorophyll Methods," Final Report,
USEPA Contract 68-C5-0011, WA1-03, August
1997.
6. Holm-Hansen, O., "Chlorophyll a determination:
improvements in methodology", OKI OS. 30
(1978), pp. 438-447.
7. Wright, S.W. and J.D. Shearer, "Rapid extraction
and HPLC of chlorophylls and carotenoids from
marine phytoplankton", J. Chrom.. 294 (1984),
pp. 281-295.
8. Bowles, N.D., H.W. Paerl, and J. Tucker,
"Effective solvents and extraction periods
employed in phytoplankton carotenoid and
chlorophyll determination", Can. J. Fish. Aquat.
ScL, 42 (1985) pp. 1127-1131.
9. Shoaf, W.T. and B.W. Lium, "Improved extraction
of chlorophyll a and b from algae using dimethyl
sulfoxide", Limnol. and Oceanoqr.. 21(6) (1976)
pp. 926-928.
10. Loftis, M.E. and J.H. Carpenter, "A fluorometric
method for determining chlorophylls a, b, and c,1"
J. Mar. Res.. 29 (1971) pp.319-338.
11. Standard Methods for the Analysis of Water and
Wastes. 17th Ed., 1989, 10200H, Chlorophyll.
12. Wright, S.W., S.W. Jeffrey, R.F.C. Manntoura,
C.A. Llewellyn, T. Bjornland, D. Repeta, and N.
Welschmeyer, "Improved HPLC method for the
analysis of chlorophylls and carotenoids from
marine phytoplankton", paper submitted for
publication in 1991.
13. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid
determination of algal chlorophyll and carotenoid
pigments and their breakdown products in
natural waters by reverse-phase high
performance liquid chromatography", Anal.
Chim. Acta.. 151 (1983) pp. 297-314.
445.0-11
Revision 1.2 September 1997
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14. Brown, L.M., B.T. Margrave, and M.D.
MacKinnon, "Analysis of chlorophyll a in
sediments by high-pressure liquid
chromatography", Can. J. Fish. Aquat. ScL 38
(1981) pp. 205-214.
15. Bidigare, R.R., M.C. Kennicutt, II, and J.M.
Brooks, "Rapid determination of chlorophylls and
their degradation products by HPLC", Limnol.
Oceanoar.. 30(2) (1985) pp. 432-435.
16. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.
Montano-Asquerino, and J. Garrido-Fernandez,
"Determination of chlorophylls and carotenoids
by HPLC during olive lactic fermentation", ,L
Chrom.. 585 (1991) pp. 259-266.
17. Carcinogens - Working With Carcinogens,
Department of Health, Education and Welfare,
Public Health Service, Center for Disease
Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, 1977.
18. "OSHA Safety and Health Standards, General
Industry", (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, revised
January 1976.
19. Safety in Academic Chemistry Laboratories,
American Chemical Society publication,
Committee on Chemical Safety, 3rd Edition,
1979.
20. "Proposed OSHA Safety and Health Standards,
Laboratories", Occupational Safety and Health
Administration, Federal Register. July 24, 1986.
21. 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 Ohio Sea Grant Program,
Ohio State University. Grant No.NA84AA-D-
00079, 1986,54pp.
22. Clayton, C.A., J.W. Hines and P.O. Elkins,
"Detection limits within specified assurance
probabilities," Analytical Chemistry. 59 (1987),
pp. 2506-2514.
Revision 1.2 September 1997
445.0-12
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77.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. COMPARISON OF PRECISION OF TWO EXTRACTION PERIODS
CORRECTED CHLOROPHYLL a
Mean Concentration
Standard Deviation
(ug/L)
(ug/L)
Relative Standard Deviation (%)
Sample A(1)
2 h(3) 24 h(3)
49.6
4.89
9.9
52.9
2.64
5.0
Sample B(2)
2 h(3) 24 h(3)
78.6
6.21
7.9
78.8
2.77
3.5
Values reported are the mean measured concentrations (n=6) of chlorophyll a in the natural water based
on a 100-mL filtration volume.
Values reported are the mean measured concentrations (n=9) of the extraction solution. Sample filtration
volume was 300 ml.
The length of time that the filters steeped after they were macerated.
445.0-13
Revision 1.2 September 1997
-------
TABLE 2. COMPARISON OF PRECISION OF TWO EXTRACTIONS PERIODS FOR Pheophytin a
Pheophytin a
Mean Concentration (|jg/L)
Standard Deviation (|jg/L)
Relative Standard Deviation (%)
Sample A(1)
2 h(3) 24 h(3)
9.22
2.36
25.6
8.19
3.55
43.2
Samole B(2)
2h(3)
13.1
3.86
29.5
24 h(3)
10.61
2.29
21.6
Values reported are the mean measured concentrations (n=6) of pheophytin a in the natural water based
on a 100-mL filtration volume.
Values reported are the mean measured concentrations (n=9) of pheophytin a the extraction solution.
Sample filtration volume was 300 ml.
The length of time that the filters steeped after they were macerated.
Revision 1.2 September 1997
445.0-14
-------
TABLE 3. ANALYSES OF USEPA QC SAMPLES
ANALYTE
Chlorophyll a
Pheophytin a
ANALYTE
Chlorophyll a
Pheophytin a
REFERENCE VALUE
2.1 |jg/L
0.3 |jg/L
MEAN
MEASURED VALUE
2.8 |jg/L
0.3 |jg/L
CONFIDENCE LIMITS
0.5 to 3.7 |jg/L
-0.2 to 0.8 |jg/L
% Relative Standard1
Deviation
1.5
33
1N = 3
445.0-15
Revision 1.2 September 1997
-------
TABLE 4. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLLS METHODS*
Method'2'
FL -Mod(5)
FL - Std(5)
HPLC
SP-M
SP-T
M(3)
8
9
4
15
15
p-EDL'4' (mg/L)
0.096
0.082
0.081
0.229
0.104
(1) See Section 13.4.1 for a description of the statistical approach used to determine p-EDLs.
(2) FL-Mod = fluorometric method using special interference filters.
FL-Std = conventional fluorometric method with pheophytin a correction.
HPLC = EPA method 447.0
SP-M = EPA method 446.0, monochromatic equation.
SP-T = EPA method 446.0, trichromatic equations.
(3) N = number of labs whose data was used.
(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.
(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are
unrealistically high.
Revision 1.2 September 1997 445.0-16
-------
TABLE 5. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES
ml_s of
culture
Method'1' filtered N(2) Mean (ma chla/D Std. Dev. %RSD
FI-Mod 5 7 0.163 0.037 22.8
10 7 0.298 0.080 26.7
50 7 1.684 0.385 22.9
100 7 3.311 0.656 19.8
Fl-Std
5
10
50
100
8
8
8
8
0.185
0.341
1.560
3.171
0.056
0.083
0.311
0.662
30.4
24.4
19.9
20.9
(1) FI-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2) N = number of volunteer labs whose data was used.
445.0-17 Revision 1.2 September 1997
-------
TABLE 6. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES
ml_s of
culture
Method'1' filtered N(2) Mean (ma chla/D Std. Dev. %RSD
FI-Mod 5 7 0.066 0.010 14.6
10 7 0.142 0.045 31.5
50 7 0.757 0.208 27.5
100 7 1.381 0.347 25.1
Fl-Std
5
10
50
100
8
8
8
8
0.076
0.165
0.796
1.508
0.018
0.040
0.140
0.324
23.2
24.3
17.5
21.5
(1) FI-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 445.0-18
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TABLE 7. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES
Method'1
FI-Mod
ml_s of
culture
filtered
5
10
50
100
N<2
7
7
7
7
Mean (mg chla/D
0.221
0.462
2.108
3.568
Std. Dev.
0.040
0.094
0.491
1.186
%RSD
18.0
20.3
23.3
33.2
Fl-Std
5
10
50
100
8
8
8
8
0.214
0.493
2.251
4.173
0.053
0.091
0.635
0.929
24.8
18.4
28.2
22.3
(1) FI-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2) N = number of volunteer labs whose data was used.
NOTE: The phaeodactylum extract contained significant amounts of chlorophyll c and chlorophyllide a which
interferes in chlorophyll a measurement in the fluorometric method, therefore, the concentration of chlorophyll a is
overestimated compared to the HPLC method which separates the three pigments. The FL-Mod interference filters
minimize this interference more so than the conventional filters.
445.0-19
Revision 1.2 September 1997
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TABLE 8. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND
CONCENTRATION LEVEL
Species
Amphidinium
Dunaliella
Statistic
Minimum
Median
Maximum
Minimum
Median
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
Percent Recovery
Cone.
Level 1
70
66
82
36
21
105
109
102
99
95
121
156
284
141
115
162
179
165
120
167
206
250
252
Cone.
Level 2
73
91
85
48
63
112
107
106
101
96
126
154
210
133
116
159
171
109
188
169
246
228
177
Cone.
Level 3
75
91
87
68
71
105
111
112
101
106
143
148
131
126
119
157
165
64
167
166
227
224
89
Cone.
Level 4
76
90
88
64
70
104
109
105
101
107
146
148
116
125
117
156
164
41
164
165
223
210
80
Revision 1.2 September 1997
445.0-20
-------
Table 8 cont'd
Species
Dunaliella
Phaeodactylum
Statistic
Maximum
Minimum
Median
Maximum
Method
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
Percent Recovery
Cone.
Level 1
240
225
295
439
392
342
291
216
189
150
161
203
292
296
225
287
286
357
371
394
446
357
Cone.
Level 2
247
244
277
385
273
316
283
183
220
119
138
195
285
263
203
274
281
337
415
289
344
316
Cone.
Level 3
247
256
287
276
172
296
283
157
223
84
156
216
250
254
114
254
277
320
415
182
330
318
Cone.
Level 4
243
256
288
261
154
293
283
154
219
75
160
244
245
254
90
253
274
318
334
139
328
299
445.0-21
Revision 1.2 September 1997
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TABLE 9. CHLOROPHYLL A CONCENTRATIONS IN MG/L DETERMINED IN FILTERED SEAWATER
SAMPLES
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
All Methods
Con.(1)
100
100
100
100
100
100
No. Obs.
14
15
10
38
36
113
No. Labs
7
8
5
19
18
57
Mean
1.418
1.576
1.384
1.499
1.636
1.533
Std. Dev.
0.425
0.237
0.213
0.219
0.160
0.251
RSD(%)
30.0
15.0
15.4
14.6
9.8
16.4
Minimum
0.675
1.151
1.080
0.945
1.250
0.657
Median
1.455
1.541
1.410
1.533
1.650
1.579
Maxium
2.060
1.977
1.680
1.922
1.948
2.060
(1) Con = mis of seawater filtered.
Revision 1.2 September 1997
445.0-22
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Method 446.0
In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in
Marine And Freshwater Algae by Visible Spectrophotometry
Adapted by
Elizabeth J. Arar
Revision 1.2
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
446.0-1
-------
Method 446.0
In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in
Marine and Freshwater Algae by Visible Spectrophotometry
1.0 Scope and Application
1.1 This method provides a procedure for
determination of chlorophylls a (chl a), b (chl b), a, + c2
(chl a, + c2) and pheopigments of chlorophyll a (pheo a)
found in marine and freshwater phytoplankton.
Chlorophyllide a is determined as chl a. Visible
wavelength spectrophotometry is used to measure the
pigments in sub-parts per million (ppm) concentrations.
The trichromatic equations of Jeffrey and Humphrey'1' are
used to calculate the concentrations of chl a, chl b, and
chl c.,+c2. Modified monochromatic equations of
Lorenzen(2) are used to calculate pheopigment-corrected
chl a and pheo a.
1.2 This method differs from previous descriptions of
the spectrophotometric technique in several important
aspects. Quality assurance/quality control measures are
described in Sect. 9.0. Detailed sample collection and
extraction procedures are described in Sect. 8.0, and
most importantly, interference data, heretofore only
presented in research journals, is included so the analyst
may know the potential limitations of the method.
Multilaboratory data is included in Section 13.
Chemical Abstracts Service
Analyte Registry Number (CASRN)
Chlorophyll a
Chlorophyll b
Chlorophyll a,
Chlorophyll c2
479-61-8
519-62-0
18901-56-9
27736-03-4
1.3 Instrumental detection limits (IDLs) of 0.08 mg
chl a/L, 0.093 mg chl b/L and 0.085 mg pheo a/L in pure
solutions of 90% acetone were determined by this
laboratory using a 1-cm glass cell. Lower detection limits
can be obtained using 2, 5 or 10-cm cells. An IDL for
chlorophylls c.,+c2 was not determined due to
commercial unavailability of the pure pigments.
Estimated detection limit (EDL) determinations were
made by analyzing seven replicate filtered phytoplankton
samples containing the pigments of interest. Single-
laboratory EDLs (S-EDL) were as follows: chl a - 0.037
mg/L, chl b - 0.07 mg/L, chl a, + £ - 0.087 mg/L,
pheopigment-corrected chl a - 0.053 mg/L, and pheo a -
0.076 mg/L. The trichromatic equations lead to
inaccuracy in the measurement of chlorophylls b and
c.,+c2 at chl a concentrations greater than ~5X the
concentration of the accessory pigment or in the
presence of pheo a. The upper limit of the linear dynamic
range (LDR) for the instrumentation used in this method
evaluation was approximately 2.0 absorbance units (AU)
which corresponded to pigment concentrations of 27 mg
chl a/L, 30 mg chl b/L and approximately 45 mg pheo a/L.
No LDR for chl a, + q was determined. It is highly
unlikely that samples containing chl c.,+c2 at
concentrations approaching the upper limit of the LDR will
be encountered in nature.
1.4 Chl c.,+c2 is not commercially available,
therefore, the minimum indicator of laboratory
performance for this pigment is precision of chl a, + q
determinations in natural samples known to contain the
pigments.
1.5 This method uses 90% acetone as the extraction
solvent because of its efficiency for extracting chl a from
most types of algae. (NOTE: There is evidence that
certain chlorophylls and carotenoids are more thoroughly
extracted with methanol(3~5) or dimethyl sulfoxide.(6) Using
high performance liquid chromatography (HPLC),
Mantoura and Llewellyn'7' found that methanol led to the
formation of chl a derivative products, whereas 90%
acetone did not. Bowles, et al.(5) found that for chl a 90%
acetone was an effective solvent when the steeping
period was optimized for the predominant species
present.)
1.6 One of the limitations of absorbance
spectrophotometry is low sensitivity. It may be preferable
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to use a fluorometric(8-10) or HPLC(11-15) method if high
volumes of water (>4 L) must be filtered to obtain
detectable quantities of chl a. The user should be aware
of the inaccuracies of flu oro metric methods when chl b is
also present in the sample.
1.7 This method is for use by analysts experienced in
handling photosynthetic pigments and in the operation of
visible wavelength spectrophotometers or by analysts
under the close supervision of such qualified persons.
2.0 Summary of Method
2.1 Chlorophyll-containing phytoplankton in a
measured volume of sample water are concentrated by
filtration at low vacuum through a glass fiber filter. The
pigments are extracted from the phytoplankton in 90%
acetone with the aid of a mechanical tissue grinder and
allowed to steep for a minimum of 2 h, but not exceeding
24 h, to ensure thorough extraction of the pigments. The
filter slurry is centrifuged at 675 g for 15 min (or at 1000
g for 5 min) to clarify the solution. An aliquot of the
supernatant is transferred to a glass cell and absorbance
is measured at four wavelengths (750, 664, 647 and 630
nm) to determine turbidity, chlorophylls a, b, and a, + c2,
respectively. If pheopigment-corrected chl a is desired,
the sample's absorbance is measured at 750 and 664 nm
before acidification and at 750 and 665 nm after
acidification with 0.1 N HCI. Absorbance values are
entered into a set of equations that utilize the extinction
coefficients of the pure pigments in 90% acetone to
simultaneously calculate the concentrations of the
pigments in a mixed pigment solution. No calibration of
the instrument with standard solutions is required.
Concentrations are reported in mg/L (ppm).
3.0 Definitions
3.1 Field Replicates — Separate samples collected
at the same time and place under identical circumstances
and treated exactly the same throughout field and
laboratory procedures. Analyses of field replicates give
a measure of the precision associated with sample
collection, preservation and storage, as well as with
laboratory procedures.
3.2 Instrument Detection Limit (IDL) - The
minimum quantity of analyte or the concentration
equivalent that gives an analyte signal equal to three
times the standard deviation of a background signal at the
selected wavelength, mass, retention time, absorbance
line, etc. In this method the instrument is zeroed on a
background of 90% acetone resulting in no signal at the
measured wavelengths. The IDL is determined instead
by serially diluting a solution of known pigment
concentration until the signal at the selected wavelength
is between .005 and .008 AU.
3.3 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
reagents, or apparatus. For this method the LRB is a
blank filter that has been extracted as a sample.
3.4 Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.5 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling
precautions.
3.6 Estimated Detection Limit (EDL) - The EDL
is determined in a manner similar to an EPA MDL. It is
not called an MDL in this method because there are
known spectral interferences inherent to this method that
make 99% confidence that the chlorophyll concentration
is greater than zero impossible.
3.7 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations that is used to
fortify an aliquot of LRB or sample matrix. Ideally, the
QCS is obtained from a source external to the laboratory
and different from the source of calibration standards. It
is used to check laboratory performance with externally
prepared test materials. The USEPA no longer provides
QCSs for this method.
4.0 Interferences
4.1 Any compound extracted from the filter or
acquired from laboratory contamination that absorbs light
between 630 and 665 nm may interfere in the accurate
measurement of the method analytes. An absorbance
measurement is made at 750 nm to assess turbidity in the
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sample. This value is subtracted from the sample's
absorbance at 665, 664, 647 and 630 nm. A 750 nm
absorbance value that is > .005 AU indicates a poorly
clarified solution. This is usually remedied by further
centrifugation or filtration of the sample prior to analysis.
4.2 The relative amounts of chlorophyll a, b and a, +
c2 vary with the taxonomic composition of the
phytoplankton. Due to the spectral overlap of the
chlorophylls and pheo a, over- or underestimation of the
pigments is inevitable in solutions containing all of these
pigments.
Chi a is overestimated by the trichromatic equation of
Jeffrey and Humphrey when pheo a is present (Figure 1).
Lorenzen's modified monochromatic equation only
slightly overestimates chl a in the presence of chl b
(Figure 2). The degree of error in the measurement of
any pigment is directly related to the concentration of the
interfering pigment. Knowledge of the taxonomic
composition of the sample, proper storage and good
sample handling technique (to prevent degradation of chl
a) can aid in determining whether to report trichromatic or
pheopigment-corrected chl a. If no such knowledge
exists, it is advisable to obtain values for all of the
pigments and to compare the chl a results in light of the
apparent concentrations of the other pigments.
Obviously, if the chl a values vary widely, sound
judgement must be used in deciding which pigments, chl
b and chl a, + q, or pheo a, are in greatest abundance
relative to each other and to chl a. The method of
standard additions, explained in most analytical chemistry
textbooks, is recommended when greater accuracy is
required.
Accuracy of chl b measurements is highly dependent
upon the concentration of chl a and pheo a.(16) In pure
solutions of chl a and b, underestimation of chl b is
observed with increasing concentrations of chl a (Figure
3). Using the method of standard additions, the same
phenomenon was confirmed to occur in natural samples.
The underestimation of chl b is due in part to the spectral
component of chl a that is subtracted from chl b as chl a,
+ c2 in the trichromatic equation. Chl a concentrations
that range from 4 to 10 times the concentration of chl b
lead to 13% to 38% underestimation of chl b. The highest
chl Jb:chl a ratio likely to occur in nature is 1:1.
Pheo a:chl a ratios rarely exceed 1:1. Pheo a is
overestimated in the presence of certain carotenoids(16)
and when chl b is converted to pheo b in the acidification
step required to determine pheopigment-corrected chl a
and pheo a. The rate of conversion of chl b to pheo b,
however, is slower than that of chl a to pheo a. It is
important, therefore, to allow the minimum time required
for conversion of chl a to pheo a before measuring
absorbance at 665 nm. Ninety seconds is recommended
by this method.
When a phytoplankton sample's composition is known
(i.e., green algae, diatoms, dinoflagellates) Jeffrey and
Humphrey's dichromatic equations for chl a, b, and a, +
c2 are more accurate than the trichromatic equations
used here.(1)
4.3 Precision and recovery for any of the pigments is
related to efficient maceration of the filtered sample and
to the steeping period of the macerated filter in the
extraction solvent (Table 1). Precision improves with
increasing steeping periods. A drawback to prolonged
steeping periods, however, is the extraction of interfering
pigments. For example, if the primary pigment of interest
is chl a, extended steeping periods may extract more of
the other pigments but not necessarily more chl a.
Statistical analysis revealed steeping period to be a
significant factor in the recovery of chl b and pheo a from
a mixed assemblage containing these pigments in
detectable quantities, but not a significant factor in the
recovery of chl a. Chl b and pheo a are mutual
interferents so that an actual increase in the recovery of
chl b leads to a slight apparent increase in pheo a.
4.4 Sample extracts must be clarified by
centrifugation prior to analysis.
4.5 All photosynthetic pigments are light and
temperature sensitive. Work must be performed in
subdued light and all standards, QC materials, and
filtered samples must be stored in the dark at -20 or
-70°C to prevent rapid degradation.
5.0 Safety
5.1 Each chemical used in this method should be
regarded as a potential health hazard and handled with
caution and respect. Each laboratory is responsible for
maintaining a current awareness file of Occupational
Safety and Health Administration (OSHA) regulations
regarding the safe handling of the chemicals specified in
this method.(17"20) A file of MSDS also should be made
available to all personnel involved in the chemical
analysis.
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5.2 The grinding of filters during the extraction step of
this method should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.
6.0 Apparatus and Equipment
6.1 Spectrophotometer - Visible, multiwavelength,
with a bandpass (resolution) not to exceed 2 nm.
6.2 Centrifuge, capable of 675 g.
6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)
with grooves in the tip with 1/4" stainless steel rod long
enough to chuck onto a suitable drive motor and 30-mL
capacity round-bottomed, glass grinding tube.
6.4 Filters, glass fiber, 47-mm, or 25-mm, nominal
pore size of 0.7 urn unless otherwise justified by data
quality objectives. Whatman GF/F filters were used in
this work.
6.5 Petri dishes, plastic, 50 X 9-mm, or some other
solid container for transporting and storing sampled
filters.
6.6 Aluminum foil.
6.7 Laboratory tissues.
6.8 Tweezers or flat-tipped forceps.
6.9 Vacuum pump or source capable of maintaining
a vacuum up to 6 in. Hg (20 KPa).
6.10 Labware - All reusable labware (glass,
polyethylene, Teflon, etc.) that comes in contact with
chlorophyll solutions should be clean and acid free. An
acceptable cleaning procedure is soaking for 4 h in
laboratory grade detergent and water, rinsing with tap
water, distilled deionized water and acetone.
6.10.1 Assorted Class A calibrated pipets.
6.10.2 Graduated cylinders, 500-mL and 1-L.
6.10.3 Volumetric flasks, Class A calibrated, 25-mL, 50-
ml_, 100-mL and 1-L capacity.
6.10.4 Glass rods.
6.10.5 Disposable Pasteur type pipets or medicine
droppers.
6.10.6 Glass cells for the spectrophotometer, 1, 2, 5 or
10 cms in length. If using multiple cells, they must be
matched.
6.10.7 Filtration apparatus consisting of 1 or 2-L filtration
flask, 47-mm fritted glass disk base and a glass filter
tower.
6.10.8 Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.10.9 Polyethylene squirt bottles.
7.0 Reagents and Standards
7.1 Acetone, HPLC grade, (CASRN 67-64-1).
7.2 Hydrochloric acid (HCI), concentrated (sp. gr.
1.19), (CASRN 7647-01-0).
7.3 Chi a free of chl b and chl b substantially free of
chl a may be obtained from a commercial supplier such
as Sigma Chemical (St. Louis, MO).
7.4 Water - ASTM Type I water (ASTM D1193) is
required. Suitable water may be obtained by passing
distilled water through a mixed bed of anion and cation
exchange resins.
7.5 0.1 N HCI Solution - Add 8.5 mL of
concentrated HCI to approximately 500 mL water and
dilute to 1 L.
7.6 Aqueous Acetone Solution - 90% acetone/10%
ASTM Type I water. Carefully measure 100 mL of the
water into the 1-L graduated cylinder. Transfer to a 1-L
flask or storage bottle. Measure 900 mL of acetone into
the graduated cylinder and transfer to the flask or bottle
containing the water. Mix, label and store.
7.7 Chlorophyll Stock Standard Solution (SSS) -
Chl a (MW = 893.5) and chl b (MW = 907.5) from a
commercial supplier is shipped in amber glass ampules
that have been flame sealed. The dry standards must be
stored at -20°C in the dark. Tap the ampule until all the
dried pigment is in the bottom of the ampule. In subdued
light, carefully break the tip off the ampule. Transfer the
entire contents of the ampule into a 25-mL volumetric
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flask. Dilute to volume with 90% acetone, label the flask
and wrap with aluminum foil to protect from light. Pheo a
may be prepared by the mild acidification of chl a (to .003
N HCI) followed by a 1:1 molar neutralization with a base
such as dilute sodium hydroxide solution. When stored in
a light- and air-tight container at -20°C, the SSS is stable
for at least six months. All dilutions of the SSS must be
determined spectrophotometrically using the equations in
Sect. 12.
7.8 Laboratory Reagent Blank (LRB) - A blank
filter that is extracted and analyzed just as a sample filter.
The LRB should be the last filter extracted of a sample
set. It is used to assess possible contamination of the
reagents or apparatus.
7.9 Quality Control Sample (QCS) - Since there
are no commercially available QCSs, dilutions of a stock
standard may be used.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection — Water may be
obtained by a pump or grab sampler. Data quality
objectives will determine the depth and frequency'21' at
which samples are taken. Healthy phytoplankton,
however, are generally obtained from the photic zone
(depth at which the illumination level is 1% of surface
illumination). Enough water should be collected to
concentrate phytoplankton on at least three filters.
Filtration volume size will depend on the particulate load
of the water. Four liters may be required for open ocean
water where phytoplankton density is usually low,
whereas 1 L or less is generally sufficient for lake, bay or
estuary water. All apparatus should be clean and acid-
free. Filtering should be performed in subdued light as
soon as possible after sampling since algal populations,
thus chlorophyll a concentration, can change in a
relatively short period of time. Aboard ship filtration is
highly recommended.
Assemble the filtration apparatus and attach the vacuum
source with vacuum gauge and regulator. Vacuum
filtration should not exceed 6 in. Hg (20 kPa). Higher
filtration pressures or excessively long filtration times (>10
min) may damage cells and result in loss of chlorophyll.
Care must be taken not to overload the filters. Do not
increase the vacuum during filtration.
Prior to drawing a subsample from the water sample
container, thoroughly but gently agitate the container to
suspend the particulates (stir or invert several times).
Pour the subsample into a graduated cylinder and
accurately measure the volume. Pour the subsample into
the filter tower of the filtration apparatus and apply a
vacuum (not to exceed 20 kPa). Typically, a sufficient
volume has been filtered when a visible green or brown
color is apparent on the filter. Do not suck the filter dry
with the vacuum; instead slowly release the vacuum as
the final volume approaches the level of the filter and
completely release the vacuum as the last bit of water is
pulled through the filter. Remove the filter from the fritted
base with tweezers, fold once with the particulate matter
inside, lightly blot the filter with a tissue to remove excess
moisture and place it in the petri dish or other suitable
container. If the filter will not be immediately extracted,
wrap the container with aluminum foil to protect the
phytoplankton from light and store the filter at -20°C or
-70°C. Short term storage (2 to 4 h) on ice is acceptable,
but samples should be stored at -20°C as soon as
possible.
8.2 Preservation — Sampled filters should be stored
frozen (-20°C or -70°C) in the dark until extraction.
8.3 Holding Time — Filters can be stored frozen at
-20°C for as long as 31/2 weeks without significant loss of
chl a.(22)
9.0 Quality Control
9.1 Each Laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and the continued
analysis of laboratory reagent blanks, field replicates and
QC samples as a continuing check on performance. The
laboratory is required to maintain performance records
that define the quality of the data generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (IDLs and LDRs)
and laboratory performance (MDLs and analyses of
QCSs) prior to sample analyses.
9.2.2 Standard Reference Material (SRM) 930e
(National Institute of Standards and Technology,
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446.0-6
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Gaithersburg, MD) or other suitable spectrophotometric
filter standards that test wavelength accuracy must be
analyzed yearly and the results compared to the
instrument manufacturer's specifications. If wavelength
accuracy is not within manufacturer's specifications,
identify and repair the problem.
9.2.3 Linear Dynamic Range (LDR) - The LDR should
be determined by analyzing a minimum of 5 standard
solutions ranging in concentration from 1 to 15 mg/L.
Perform the linear regression of absorbance response (at
pigment's wavelength maximum) vs. concentration and
obtain the constants m and b, where m is the slope and b
is the y-intercept. Incrementally analyze standards of
higher concentration until the measured absorbance
response, R, of a standard no longer yields a calculated
concentration, Cc, that is ± 10% of the known
concentration, C, where Cc = (R - b)/m. That
concentration and absorbance response defines the
upper limit of the LDR for your instrument. Absorbance
responses for samples should be well below the upper
limit of the LDR, ideally between .1 and 1.0 AU.
9.2.4 Instrumental Detection Limit (IDL) — Zero the
spectrophotometer with a solution of 90% acetone. Pure
pigment in 90% acetone should be serially diluted until it
yields a response at the selected wavelength between
.005 and .008 AU.
9.2.5 Estimated Detection Limit (EDL) — At least seven
natural phytoplankton samples known to contain the
pigments of interest should be collected, extracted and
analyzed according to the procedures in Sects. 8 and 11,
using clean glassware and apparatus. The concentration
of the pigment of interest should be between 2 and 5
times the IDL. Dilution or spiking of the sample extract
solution to the appropriate concentration may be
necessary. Inaccuracies occur in the measurement of
chlorophylls b and a, + c2 when the chl a concentration is
greater than ~5X the concentration of the accessory
pigment. Perform all calculations to obtain concentration
values in mg/L in the extract solution. Calculate the EDL
as fo I lows'23':
EDL = (3) X (S)
S = Standard deviation of the replicate analyses.
9.2.6 Quality Control Sample (QCS) - When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify instrument performance
with the analysis of a QCS (Sect. 7.9). If the determined
value is not within the confidence limits established by
project data quality objectives, then the determinative step
of this method is unacceptable. The source of the
problem must be identified and corrected before
continuing analyses.
9.2.7 Extraction Proficiency — Personnel performing
this method for the first time should demonstrate
proficiency in the extraction of sampled filters (Sect. 11.1).
Twenty to thirty natural samples should be obtained using
the procedure outlined in Sect. 8.1 of this method. Sets
of 10 or more samples should be extracted and analyzed
according to Sect. 11.2. The percent relative standard
deviation (%RSD) of trichromatic chl a should not exceed
15% for samples that are at least 10X the IDL.
9.2.8 Corrected Chl a - Multilaboratory testing of this
method revealed that many analysts do not adequately
mix the acidified sample when determining the corrected
chl a. The problem manifests itself by highly erratic
pheo a results, high %RSDs for correctetd chl a and poor
agreement between corrected and uncorrected chl a. To
determine if a new analyst is performing the acidification
step properly, perform the following QC procedure:
Prepare 100 mL of a 2.0 ppm chl a solution in 90%
acetone. The new analyst should analyze 5-10 separate
aliquots, using carefully rinsed cuvettes, according to
instructions in Section 11.2. Process the results
according to Section 12 and calculate separate means
and %RSDs for corrected and uncorrected chl a. If the
means differ by more than 10%, then the stock chl a has
probably degraded and fresh stock should be prepared.
The %RSD for corrected chl a should not exceed 5%. If
the %RSD exceeds 5%, repeat the procedure until
acceptable results are obtained.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) - The
laboratory must analyze at least one blank filter with each
sample batch. The LRB should be the last filter
extracted. LRB data are used to assess contamination
from the laboratory environment. LRB values that exceed
the IDL indicate contamination from the laboratory
environment. When LRB values constitute 10% or more
of the analyte level determined in a sample, fresh
samples or field replicates must be analyzed after the
contamination has been corrected and acceptable LRB
values have been obtained.
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10.0 Calibration and Standardization
10.1 Daily calibration of the spectrophotometer is not
required when using the equations discussed in this
method. It is extremely important, therefore, to perform
regular checks on instrument performance. By analyzing
a standard reference material such as SRM 930e
(National Institute of Standards and Technology,
Gaithersburg, MD) at least quarterly, wavelength
accuracy can be compared to instrument manufacturer's
specifications. Filter kits that allow stray light, bandpass
and linearity to be evaluated are also commercially
available. Although highly recommended, such kits are
not required for this method if the LDR is determined for
the pigment of interest and QCSs are routinely analyzed.
10.2 Allow the instrument to warm up for at least 30
min. Use a 90% acetone solution to zero the instrument
at all of the selected wavelengths. 750 nm, 664 nm, 647
nm and 630 nm are used for the determination of chl a,
chl b and chl a, + c2. 750 nm, 665 nm and 664 nm are
used for the determination of pheopigment-corrected chl
a and pheo a. The instrument is now ready to analyze
samples.
11.0 Procedure
11.1 Extraction of Filter Samples
11.1.1 For convenience, a 10-mL final extraction volume
is described in the following procedure. A larger
extraction volume may be necessary if using a low-
volume 10-cm cell. On the other hand, a smaller
extraction volume can be used to obtain a concentration
factor. The filter residue retains 2-3 ml of solution after
centrifugation and a 1-cm cell requires approximately 3
ml of solution so that a recommended minimum
extraction volume is 6 ml.
11.1.2 If sampled filters have been frozen, remove them
from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand laboratory tissues and
squirt bottles containing water and acetone. Workspace
lighting should be the minimum that is necessary to read
instructions and operate instrumentation. Remove a filter
from its container and place it in the glass grinding tube.
The filter may be torn into smaller pieces to facilitate
extraction. Push it to the bottom of the tube with a glass
rod. With a volumetric pipet, add 4 ml of the aqueous
acetone solution (Sect. 7.6) to the grinding tube. After the
filter has been converted to a slurry, grind the filter for
approximately 1 min at 500 rpm. (NOTE: Although
grinding is required, care must be taken not to overheat
the sample. Good judgement and common sense will
help you in deciding when the sample has been
sufficiently macerated.) Pour the slurry into a 15-mL
screw-cap centrifuge tube and, using a 6-mL volumetric
pipet, rinse the pestle and the grinding tube with the
aqueous acetone. Add the rinse to the centrifuge tube
containing the filter slurry. Cap the tube and shake it
vigorously. Place it in the dark before proceeding to the
next filter extraction. Before placing another filter in the
grinding tube, use the acetone and water squirt bottles to
thoroughly rinse the pestle, grinding tube and glass rod.
To reduce the volume of reagent grade solvents used for
rinsing between extractions, thoroughly rinse the grinding
tube and glass rod with tap water prior to a final rinse with
ASTM Type I water and acetone. The last rinse should
be with acetone. Use a clean tissue to remove any filter
residue that adheres to the pestle or to the steel rod of the
pestle. Proceed to the next filter and repeat the steps
above. The last filter extracted should be a blank. The
entire extraction with transferring and rinsing takes
approximately 5 min. Approximately 500 ml of acetone
and water waste are generated per 20 samples from the
rinsing of glassware and apparatus.
11.1.3 Shake each tube vigorously again before placing
them to steep in the dark at 4°C. Samples should be
allowed to steep fora minimum of 2 h but not to exceed
24 h. Tubes should be shaken at least once, preferably
two to three times, during the steeping period to allow the
extraction solution to have maximum contact with the filter
slurry.
11.1.4 After steeping is complete, centrifuge samples for
15 min at 675 g or for 5 min at 1000 g.
11.2 Sample Analysis
11.2.1 The instrument must be zeroed on a 90%
acetone solution as described in Sect. 10.2. In subdued
lighting, pour or pipet the supernatant of the extracted
sample into the glass spectrophotometer cell. If the
absorbance at 750 nm exceeds .005 AU, the sample
must be recentrifuged or filtered through a glass fiber
filter (syringe filter is recommended). The volume of
sample required in the instrument's cell must be known if
the pheopigment-corrected chl a and pheo a will be
determined so that acidification to the correct acid
concentration can be performed. For example, a cell that
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holds 3 ml of extraction solution requires .09 ml of the .1
N HCI solution to obtain an acid concentration of .003 N.
Measure the sample's absorbance at the selected
wavelengths for chl a, chl b and chl a, + q. Dilute and
reanalyze the sample if the signal at the selected
wavelength is >90% of the signal previously determined
as the upper limit of the LDR. If pheopigment-corrected
chl a and pheo a will be determined, acidify the sample in
the cell to .003 N HCI using the .1 N HCI solution. Use a
disposable Pasteur type pipet to thoroughly mix the
sample by aspirating and dispensing the sample into the
cuvette, keeping the pipet tip below the surface of the
liquid to avoid aerating the sample, wait 90 sec and
measure the sample's absorbance at 750 and 665 nm.
NOTE: Proper mixing of the acidified sample is critical for
accurate and precise results.
12.0 Data Analysis and Calculations
12.1 Jeffrey and Humphrey's Trichromatic
Equations — Subtract the absorbance value at 750 nm
from the absorbance values at 664, 647 and 630 nm.
Calculate the concentrations (mg/L) of chl a, b, and a, +
c2 in the extract solution by inserting the 750 nm-correcfecf
absorbance values into the following equations:
CEa = 11.85 (Abs 664) -1.54 (Abs 647) - .08 (Abs 630)
CEb = 21.03 (Abs 647) - 5.43 (Abs 664) - 2.66 (Abs 630)
CEC = 24.52 (Abs 630) - 7.60 (Abs 647) -1.67 (Abs 664)
where:
CEa = concentration (mg/L) of chlorophyll a in the
extraction solution analyzed,
CEb = concentration (mg/L) of chlorophyll b in the extract
solution.
CEc = concentration (mg/L) of chlorophyll c., + c2 in the
extract solution analyzed.
12.2 Lorenzen's Pheopigment-corrected Chl a and
Pheo a - Subtract the absorbance values at 750 nm from
the absorbance values at 664 and 665 nm. Calculate the
concentrations (mg/L) in the extract solution, CE, by
inserting the 750 nm corrected absorbance values into
the following equations:
CEa = 26.7(Abs 664b - Abs 665a)
PEa = 26.7 [1.7 X (Abs 665a) - (Abs 664b)]
where,
CEa = concentration (mg/L) of chlorophyll a in the extract
solution measurted,
PEa = concentration (mg/L) of pheophytin a in the
extraction measured.
Abs 664b = sample absorbance at 664 nm (minus
absorbance at 750 nm) measured before acidification,
and
Abs 665a = sample absorbance at 665 nm (minus
absorbance at 750 nm) measured after acidification.
12.3 Calculate the conentration of pigment in the
whole water sample using the following generalized
equation:
Cs = CE (a.b. or c) X extract volume (L) X DF
sample volume (L) X cell length (cm)
where:
Cs = concentration (mg/L) of pigment in the whole water
sample.
CE(aborc) = concentration (mg/l) of pigment in extract
measured in the cuvette.
extract volume = volume (L) of extract (before any
dilutions), typically 0.0104).
DF = any dilution factors.
sample volume = volume (L) of whole water sample that
was filtered, and
cell length = optical path length (cm) of cuvette used
(typically 1 cm).
For example, calculate the conentration of chlorophyll a
in the whole water sample as:
446.0-9
Revision 1.2 September 1997
-------
CEa X extract volume (L) DF
sample volume (L) X cell length (cm)
12.4 LRB and QCS data should be reported with each
sample data set.
13.0 Method Performance
13.1 Single Laboratory Performance
13.1.1 Replicate analyses were performed on low level
dilutions of the pure pigments in 90% acetone. The
results, contained in Table 2, give an indication of the
variability not attributable to sampling and extraction or
pigment interferences.
13.1.2 The IDLs and S-EDLs for the method analytes
are reported in Table 3.
13.1.3 Precision (%RSD) for replicate analyses of two
distinct mixed assemblages are contained in Table 4.
13.1.4 Three QCS ampules were obtained from the
USEPA, analyzed and compared to the reference values
in Table 5. (NOTE: The USEPA no longer provides
pigment QCSs.)
13.2 Multilaboratory Testing - A Multilaboratory
validation and comparison study of EPA Methods 445.0,
446.0 and 447.0 for chlorophyll a was conducted in 1996
by Research Triangle Institute, Research Triangle park,
N.C. (EPA Contract No. 68-C5-0011). There were 24
volunteer participants in the spectrophotometric methods
component that returned data. The primary goals of the
study were to determine detection limits and to assess
precision and bias (as percent recovery) for select
unialgal species, and natural seawater.
13.2.1 The term, pooled-estimated detection limit (p-
EDL), is used in this method to distinguish it from the EPA
defined method detection limit (MDL). An EPA MDL
determination is not possible nor practical for a natural
water or pure species sample due to known spectral
interferences and to the fact that it is impossible to
prepare solutions of known concentrations that
incorporate all sources of error (sample collection,
filtration, processing). The statistical approach used to
determine the p-EDL was an adaptation of the Clayton,
et. al.24 method that does not assume error variances
across concentration and controls for Type II error. The
statistical approach used involved calculating an
estimated DL for each lab that had the desired Type I and
Type II error rates (0.01 and 0.05, respectively). The
median DLs over labs was then determined and is
reported in Table 6. It is referred to as the pooled-EDL
(p-EDL).
Solutions of pure chlorophyll a in 90% acetone were
prepared at three concentrations (0.11, 0.2, and 1.6 ppm)
and shipped with blank glass fiber filters to participating
laboratories. Analysts were instructed to spike the filters
in duplicate with a given volume of solution and to
process the spiked filters according to the method. The
results from these data were used to determine a pooled
EDL (p-EDL) for each method. Results (in ppm) are
given in Table 6. The standard fluorometric and HPLC
methods gave the lowest p-EDLs while the
spectrophotometric (monochromatic equations) gave the
highest p-EDLs.
13.2.2 To address precision and bias in chlorophyll a
determination for different algal species three pure
uniagal cultures (amphidinium, dunnnaliella and
phaeodactylum) were cultured and grown in the
laboratory. Four different "concentrations" of each
species were prepared by filtering varying volumes of the
algae. The filters were frozen and shipped to participant
labs. Analysts were instructed to extract and analyze the
filters according to the respectiave methods. The "true"
concentration was assigned by taking the average of the
HPLC results for the highest concentration algae sample
since chlorophyll a is separatead from other interfereing
pigments prior to determination. Pooled precision data
(%RSD) are presented in Tables 7-9 and accuracy data
(as percent recovery) are presented in Table 10. No
significant differences in precision were observed across
conentrations for any of the species. It should be noted
that there was considerable lab-to-lab variation (as
exhibited by the min and max recoveries in Table 10) and
in this case the median is a better measurement of
central tendency than the mean.
In summary, the mean and median concentrations
determined for Amphidinium carterae (class
dinophyceae) are similar for all methods. No method
consistently exhibited high or low values relative to the
other methods. The only concentration trend observed
was that the spectrophotometric method-trichromatic
Revision 1.2 September 1997
446.0-10
-------
equations (SP-T) showed a slight percent increase in
recovery with increasing algae filtration volume.
For Dunaliella tertiolecti (class chlorophyceae) and
Phaeodactylum tricornutum (class bacillariophyceae)
there was generally good agreement between the
fluorometric and the spectrophotometric methods,
however, the HPLC method yielded lower recoveries with
increasing algae filtration volume for both species. No
definitive explanation can be offered at this time for this
phenomenon. A possible explanation for the
Phaeodactylum is that it contained significant amounts of
chlorophylide a which is determined as chlorophyll a in
the fluorometric and spectrophotometric methods. The
conventional fluorometric method (FL-STD) showed a
slight decrease in chlorophyll a recovery with increasing
Dunaliella filtration volume. The spectrophotometric-
trichromatic equations (SP-T) showed a slight increase in
chlorophyll a recovery with increasing Dunaliella filtration
volume. The fluorometric and tahe spectrophotometric
methods both showed a slight decrease in chlorophyll a
recovery with increasing Phaeodactylum filtration volume.
Results for the natural seawater sample are presented in
Table 11. Only one filtration volume (100 ml) was
provided in duplicate to partaicpant labs.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation (e.g., Sect. 11.1.1). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
15.1 The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in the Sect. 14.2.
16.0 References
1. Jeffrey, S.W. and G.F. Humphrey, "New
Spectrophotometric Equations for Determining
Chlorophylls a, b, a, + c2 in Higher Plants, Algae
and Natural Phytoplankton," Biochem. Physiol.
Pflanzen. Bd, 167, (1975), S. pp. 191-4.
2. Lorenzen, C.J., "Determination of Chlorophyll
and Pheo-Pigments: Spectrophotometric
Equations," Limnol. Oceanogr., 12 (1967), pp.
343-6.
3. Holm-Hansen, O., "Chlorophyll a determination:
improvements in methodology," OIKOS, 30
(1978), pp. 438-447.
4. Wright, S.W. and J.D. Shearer, "Rapid extraction
and HPLC of chlorophylls and carotenoids from
marine phytoplankton," J. Chrom., 294 (1984),
pp. 281-295.
5. Bowles, N.D., H.W. Paerl, and J. Tucker,
"Effective solvents and extraction periods
employed in phytoplankton carotenoid and
chlorophyll determination," Can. J. Fish. Aquat.
ScL, 42 (1985) pp. 1127-1131.
6. Shoaf, W.T. and B.W. Lium, "Improved extraction
of chlorophyll a and b from algae using dimethyl
sulfoxide," Limnol. and Oceanogr., 21(6) (1976)
pp. 926-928.
446.0-11
Revision 1.2 September 1997
-------
7. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid
determination of algal chlorophyll and carotenoid
pigments and their breakdown products in
natural waters by reverse-phase high
performance liquid chromatography," Anal.
Chim. Ada., 151 (1983) pp. 297-314.
8. Yentsch, C.S. and D.W. Menzel, "A method for
the determination of phytoplankton chlorophyll
and phaeophytin by fluorescence," Deep Sea
Res., 10(1963), pp. 221-231.
9. 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. 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.
11. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,
C.A. Llewellyn, T. Bjornland, D. Repeta, and N.
Welschmeyer, "Improved HPLC method for the
analysis of chlorophylls and carotenoids from
marine phytoplankton," Mar. Ecol. Prog. Ser.,
77:183.
12. Brown, L.M., B.T. Margrave, and M.D.
MacKinnon, "Analysis of chlorophyll a in
sediments by high-pressure liquid
chromatography," Can. J. Fish. Aquat. Sci., 38
(1981) pp. 205-214.
13. Bidigare, R.R., M.C. Kennicutt, II, and J.M.
Brooks, "Rapid determination of chlorophylls and
their degradation products by HPLC," Limnol.
Oceanogr., 30(2) (1985) pp. 432-435.
14. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.
Montano-Asquerino, and J. Garrido-Fernandez,
"Determination of chlorophylls and carotenoids
by HPLC during olive lactic fermentation," J.
Chrom., 585 (1991) pp. 259-266.
15. Neveux.J., D. Delmas, J.C. Romano, P. Algarra,
L. Ignatiades, A. Herbland, P. Morand, A. Neori,
D. 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.
16. Sartory, D.P., "The determination of algal
chlorophyllous pigments by high performance
liquid chromatography and spectrophotometry,"
Water Research, 19(5), (1985), pp. 605-10.
17. Carcinogens - Working With Carcinogens,
Department of Health, Education and Welfare,
Public Health Service, Center for Disease
Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, 1977.
18. "OSHA Safety and Health Standards, General
Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, revised
January 1976.
19. Safety in Academic Chemistry Laboratories,
American Chemical Society publication,
Committee on Chemical Safety, 3rd Edition,
1979.
20. "Proposed OSHA Safety and Health Standards,
Laboratories," Occupational Safety and Health
Administration, Federal Register. July 24, 1986.
21. Marshall, C.T., A. Morin and R.H. Peters,
"Estimates of Mean Chlorophyll-a concentration:
Precision, Accuracy and Sampling design," Wat.
Res. Bull., 24(5), (1988), pp. 1027-1034.
22. 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 Ohio Sea Grant Program,
Ohio State University. Grant No.NA84AA-D-
00079, 1986,54pp.
23. Code of Federal Regulations 40. Ch.1.
Pt.136, Appendix B.
24. Clayton, C.A., J.W. Hines and P.O. Elkins,
"Detection limits within specified assurance
probabilities." Analytical Chemistry. 59(1987),
pp. 2506-2514.
Revision 1.2 September 1997
446.0-12
-------
17.0 Tables, Diagrams, Flowcharts, and Validation Data
Dilutions of a 1:1 Mixture of Chla
and Pheo.a
Concentration pigment Ippiti)
1,5
0,5
0,2
True Chla Value
Corrected chla
0,4 0,6 0,8 1
Concentration pheo.a and chla |ppm|
-i- Trichromatic chla
D Pheophytin a
1,2
FIGURE 1 - The effect of pheo a on calculated pigment
concentrations.
446.0-13
Revision 1.2 September 1997
-------
Corrected Chi a vs, Chi b
Closeness of Fit
3,5
3
2,5
2
1.5
1
0.5
Corrected chla Concentration Ipprn]
0,2
0,4 0,6
chl b Concentration Ippm]
True chl a Value
n a;b-3;l
0,8
a;b-l;l
FIGURE 2 - The effect of Chl b on pheopigment - corrected Chl a.
Revision 1.2 September 1997
446.0-14
-------
Increasing Ratios of chl a:chl b
The Underestimation of chl b
0,5
0,4
0,3
0,2
0,1
Calculated chl b |ppm|
0,1
0,2 0,3
Concentration chl b (ppm|
+ a:b-4:l
X aib-10il
0,4
a:b-6:l
True chl b Value
0,5
FIGURE 3 - The underestimation of Chl b with increasing concentrations of Chl a.
446.0-15
Revision 1.2 September 1997
-------
TABLE 1. COMPARISON OF PRECISION AND RECOVERY OF PIGMENTS FOR 4 h AND 24 h STEEPING PERIODS
N
SD
Mean
%RSD
chl a
4h
6
1.22
26.14
24.67
24h
6
0.88
25.73
3.40
chl b
4h
6
0.42
0.49
6.35
24h
6
0.21
1.72
12.00
chic.,
4h
6
0.44
5.87
7.43
+ C2
24h
6
0.37
5.26
7.04
pheo a
4h 24h
6 6
1.08 1.23
1.38 2.88
78.35 42.62
corr a
4h 24h
6 6
1.46 1.04
24.47 23.29
5.97 4.47
N - Number of samples
SD - Standard deviation
Mean - Concentration in natural water, mg/L
%RSD - Percent relative standard deviation
Revision 1.2 September 1997 446.0-16
-------
TABLE 2. REPLICATE ANALYSES OF PURE PIGMENTS AT LOW CONCENTRATIONS
Modified
Trichromatic Equations
chl a chl b
N 7 7
SD .000612 .009792
Mean .102mg/L .109mg/L
%RSD .60 8.9
N
SD
Mean
%RSD
Monochromatic Equations
chl a chl b
7 6
.010091 .011990
.103mg/L .171 mg/L
9.8 7.0
TABLE 3. INSTRUMENTAL AND METHOD DETECTION LIMITS
INSTRUMENTAL DETECTION LIMITS1
(Concentrations in mg/L)
Trichromatic Equations
chl a
chl b
.080
.093
Modified
Monochromatic Equation
pheo a .085
S-ESTIMATED DETECTION LIMITS1
(Concentrations in mg/L)
Modified
Trichromatic Equations Monochromatic Equation
chl a .0372
chl b .0702
chl q + c2 .0873
chl a .0532
pheo a .0762
1 Determinations made using a 1-cm path length cell.
2 Mixed assemblage samples from San Francisco Bay.
3 Predominantly diatoms from Raritan Bay.
446.0-17
Revision 1.2 September 1997
-------
TABLE 4. ANALYSES OF NATURAL SAMPLES
Trichromatic Equations
chl a chl b
SAN FRANCISCO BAY
chl c.,+c2
N
SD
Mean
%RSD
7
0.0118
0.2097
5.62
7
0.0062
0.04271
14.50
7
0.0096
0.03561
26.82
Modified
Monochromatic Equations
pheo a
7
0.0244
0.0806
30.21
corr a
7
0.0168
0.1582
0.64
RARITAN BAY
Modified
Trichromatic Equations
N
SD
Mean
%RSD
chl a
7
0.0732
1.4484
5.06
chl b
7
0.0223
0.0914
24.43
chl c,+c2
7
0.0277
0.2867
9.65
Monochromatic Equations
pheo a
7
0.0697
0.1720
40.53
corr a
7
0.0521
1.3045
3.99
Mean concentrations (mg/L) reported in final extraction volume of 10 ml. Samples were macerated and allowed to steep for
approximately 24 h.
N - Number of samples
SD - Standard deviation
Mean - Concentration in natural water
%RSD - Percent relative standard deviation
Revision 1.2 September 1997
446.0-18
-------
TABLE 5. ANALYSES OF USEPA QC SAMPLES
Ampule 1 (3 separate ampules, chl a only)
Trichromatic Equations
Mean Reference %RSD
chl a 2.54 mg/L 2.59 .61
Modified
Monochromatic Equations
Mean
chl a 2.56 mg/L
pheo a ND
Reference %RSD
2.70 .8
ND - None detected
Ampule 2 (3 separate ampules, all method pigments)
Trichromatic Equations
Modified
Monochromatic Equations
chl a
chlb
chl q + c2
Mean
4.87 mg/L
1.12 mg/L
.29 mg/L
Reference
4.86
1.02
.37
%RSD
.1
1.3
4.9
Mean
chl a 3.70 mg/L
pheo a 1.79 mg/L
Reference
3.76
1.70
%RSD
2.3
4.4
446.0-19
Revision 1.2 September 1997
-------
TABLE 6. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS*1'
Method*2'
FL -Mod(5)
FL - Std
HPLC
SP-M
SP-T
N(3,
8
9
4
15
15
p-EDL(4) Cma/D
0.096
0.082
0.081
0.229
0.104
(1) See Section 13.2.1 for a description of the statistical approach used to determine p-EDLs.
(2) FL-Mod = fluorometric method using special interference filters.
FL-Std = conventional fluorometric method with pheophytin a correction.
HPLC = EPA method 447.0
SP-M = EPA method 446.0, monochromatic equation.
SP-T = EPA method 446.0, trichromatic equations.
(3) N = number of labs whose data was used.
(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.
(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are
unrealistically high.
Revision 1.2 September 1997 446.0-20
-------
TABLE 7. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES
Method'1'
SP-M
ml_s of
culture
filtered
5
10
50
100
N(2)
17
19
19
19
Mean (mg chla/D
0.068
0.139
0.679
1.366
Std. Dev.
0.026
0.037
0.150
0.205
%RSD
37.8
26.6
22.1
15
SP-T
5
10
50
100
16
18
18
18
0.059
0.130
0.720
1.408
0.021
0.027
0.102
0.175
35.1
20.8
14.2
12.4
(1) SP-M = Pheophytin a - corrected chlorophyll a method using monochromatic equations.
SP-T = Trichromatic equations method.
(2) N = Number of volunteer labs whose data was used.
446.0-21
Revision 1.2 September 1997
-------
TABLE 8. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES
rnLs of
culture
Method'1' filtered N(2) Mean (ma chla/L) Std. Dev. %RSD
SP-M 5 19 0.166 0.043 26.0
10 19 0.344 0.083 24.0
50 19 1.709 0.213 12.5
100 19 3.268 0.631 19.3
SP-T
5
10
50
100
18
18
18
18
0.161
0.339
1.809
3.500
0.030
0.058
0.190
0.524
18.4
17.1
10.5
15.0
(1) SP-M = Pheophytin a corrected chlorophyll a method using monochromatic equations.
SP-T = Trichromatic equationss method.
(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 446.0-22
-------
TABLE 9. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES
rnLs of
culture
Method'1' filtered N(2) Mean (ma chla/L) Std. Dev. %RSD
SP-M 5 19 0.223 0.054 24.1
10 19 0.456 0.091 19.9
50 19 2.042 0.454 22.2
100 19 4.083 0.694 17.0
SP-T
5
10
50
100
18
18
18
18
0.224
0.465
2.223
4.422
0.031
0.077
0.217
0.317
14.0
16.5
9.7
7.2
(1) SP-M = Pheophytin a corrected chorophyll a method using monochromatic equations.
(2) N = number of volunteer labs whose data was used.
446.0-23 Revision 1.2 September 1997
-------
TABLE 10. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND
CONCENTRATION LEVEL
Species
Amphidinium
Dunaliella
Statistic
Minimum
Median
Maximum
Minimum
Median
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
Percent Recovery
Cone.
Level 1
70
66
82
36
21
105
109
102
99
95
121
156
284
141
115
162
179
165
120
167
206
250
252
240
Cone.
Level 2
73
91
85
48
63
112
107
106
101
96
126
154
210
133
116
159
171
109
188
169
246
228
177
247
Cone.
Level 3
75
91
87
68
71
105
111
112
101
106
143
148
131
126
119
157
165
64
167
166
227
224
89
247
Cone.
Level 4
76
90
88
64
70
104
109
105
101
107
146
148
116
125
117
156
164
41
164
165
223
210
80
243
Revision 1.2 September 1997
446.0-24
-------
Table 10 cont'd
Species
Dunaliella
Phaeodactylum
Statistic
Maximum
Minimum
Median
Maximum
Method
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
Percent Recovery
Cone.
Level 1
225
295
439
392
342
291
216
189
150
161
203
292
296
225
287
286
357
371
394
446
357
Cone.
Level 2
244
277
385
273
316
283
183
220
119
138
195
285
263
203
274
281
337
415
289
344
316
Cone.
Level 3
256
287
276
172
296
283
157
223
84
156
216
250
254
114
254
277
320
415
182
330
318
Cone.
Level 4
256
288
261
154
293
283
154
219
75
160
244
245
254
90
253
274
318
334
139
328
299
446.0-25
Revision 1.2 September 1997
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TABLE 11. CHLOROPHYLL A CONCENTRATIONS IN mg/L DETERMINED IN FILTERED SEA WATER
SAMPLES
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
All Methods
Con.™
100
100
100
100
100
100
No. Obs.
14
15
10
38
36
113
No. Labs
7
8
5
19
18
57
Mean
1.418
1.576
1.384
1.499
1.636
1.533
Std. Dev.
0.425
0.237
0.213
0.219
0.160
0.251
RSD(%)
30.0
15.0
15.4
14.6
9.8
16.4
Minimum
0.675
1.151
1.080
0.945
1.250
0.657
Median
1.455
1.541
1.410
1.533
1.650
1.579
Maxium
2.060
1.977
1.680
1.922
1.948
2.060
(1) Con = mis of seawater filtered.
Revision 1.2 September 1997
446.0-26
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Method 447.0
Determination of Chlorophylls a and b and Identification
of Other Pigments of Interest in Marine and Freshwater
Algae Using High Performance Liquid Chromatography
with Visible Wavelength Detection
Elizabeth J. Arar
Version 1.0
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
447.0-1
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METHOD 447.0
DETERMINATION OF CHLOROPHYLLS a AND b AND IDENTIFICATION
OF OTHER PIGMENTS OF INTEREST IN MARINE AND FRESHWATER
ALGAE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
WITH VISIBLE WAVELENGTH DETECTION
1.0 Scope and Application
1.1 This method provides a procedure for
determination of chlorophylls a (chl a) and b (chl Jb) found
in marine and freshwater phytoplankton. Reversed-
phase high performance liquid chromatography (HPLC)
with detection at 440 nm is used to separate the pigments
from a complex pigment mixture and measure them in
the sub-microgram range. For additional reference, other
taxonomically important yet commercially unavailable
pigments of interest are identified by retention time.
1.2 This method differs from previous descriptions of
HPLC methods in several respects. Quality
assurance/quality control measures are described in
Sect. 9.0, sample collection and extraction procedures
are described in Sect. 8.0 and reference chromatograms
of pure pigments and reference algae are provided.
This method has also been evaluated in a multilaboratory
study along with EPA Methods 445.0 and 446.0.
Estimated detection limits, precision and bias are reported
in Section 13.
Analyte
Chemical Abstracts Service
Registry Number (CASRN)
Chlorophyll a
Chlorophyll b
479-61-8
519-62-0
1.3 Instrumental detection limits (IDLs) of 0.7 ng chl
a, and 0.4 ng chl b in pure solutions of 90% acetone were
determined by this laboratory. Method detection limit
(MDL) determinations were made by analyzing seven
replicate unialgal samples containing the chl a and b.
Single-laboratory MDLs were chl a - 7 ng and chl b - 4 ng.
A multilaboratory estimated detection limit (EDL) (in mg/L
of extract is reported in Section 13.
1.4 Most taxonomically important pigments are not
commercially available, therefore, a laboratory must be
willing to extract and purify pigments from pure algal
cultures to quantify and qualitatively identify these very
important pigments. This method contains
chromatographic information of select pure pigments
found either in marine or freshwater algae. The
information is included to aid the analyst in qualitatively
identifying individual pigments and possibly algal species
in natural samples.
1.5 This method uses 90% acetone as the extraction
solvent because of its efficiency for extracting chl a from
most types of algae. (NOTE: There is evidence that
certain chlorophylls and carotenoids are more thoroughly
extracted with methanol'1"3' or dimethyl sulfoxide.)(4) Using
high performance liquid chromatography (HPLC),
Mantoura and Llewellyn'5' found that methanol led to the
formation of chl a derivative products, whereas 90%
acetone did not. Bowles, et al.(3) found that for chl a 90%
acetone was an effective solvent when the steeping
period was optimized for the predominant species
present.)
1.6 One of the limitations of visible wavelength
detection is low sensitivity. It may be preferable to use
fluorometry'6"8' or HPLC(913) with fluorometric detection if
high volumes of water (>4 L) must be filtered to obtain
detectable quantities of chl a or b.
1.7 This method is for use by analysts experienced in
handling photosynthetic pigments and in the operation of
HPLC or by analysts under the close supervision of such
qualified persons.
2.0 Summary of Method
2.1 The HPLC is calibrated with a chl a and b
solution that has been spectrophotometrically quantified
Version 1.0 September 1997
447.0-2
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according to EPA Method 446. Chlorophyll-containing
phytoplankton in a measured volume of sample water are
concentrated by filtration at low vacuum through a glass
fiber filter. The pigments are extracted from the
phytoplankton into 90% acetone with the aid of a
mechanical tissue grinder and allowed to steep for a
minimum of 2 h, but not exceeding 24 h, to ensure
thorough extraction of the pigments. The filter slurry is
centrifuged at 675 g for 15 min (or at 1000 g for 5 min) to
clarify the solution. An aliquot of the supernatant is
filtered through a 0.45 |im syringe filter and 50 to 200 pi
is injected onto a reversed-phase column. Following
separation using a ternary gradient, concentrations are
reported in |jg/L (ppb) or mg/L (ppm) in the whole water
sample. This method is based on the HPLC work of
Wright, et. al.(9)
3.0 Definitions
3.1 Calibration Standard (CAL) - A solution
prepared from dilution of a stock standard solution. The
CAL solution is used to calibrate the instrument response
with respect to analyte concentration or mass.
3.2 Calibration Check Standard (CALCHK) - A
mid-point calibration solution that is analyzed periodically
in a sample set to verify that the instrument response to
the analyte has not changed during the course of
analysis.
3.3 Field Replicates - Separate samples collected
at the same time and placed under identical
circumstances and treated exactly the same throughout
field and laboratory procedures. Analyses of field
replicates give a measure of the precision associated with
sample collection, preservation and storage, as well as
with laboratory procedures.
3.4 Instrument Detection Limit (IDL) - The
minimum quantity of analyte or the concentration
equivalent that gives an analyte signal equal to three
times the standard deviation of a background signal at the
selected wavelength, mass, retention time, absorbance
line, etc.
3.5 Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
reagents, or apparatus. For this method the LRB is a
blank filter that has been extracted as a sample.
3.6 Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling
precautions.
3.7 Method Detection Limit (MDL) - The minimum
concentration of an analyte that can be identified,
measured and reported with 99% confidence that the
analyte concentration is greater than zero.
3.8 Quality Control Sample (QCS) - A solution of
method analytes of known concentrations that is used to
fortify an aliquot of LRB or sample matrix. Ideally, the
QCS is obtained from a source external to the laboratory
and different from the source of calibration standards. It
is used to check laboratory performance with externally
prepared test materials.
4.0 Interferences
4.1 Any compound extracted from the filter or
acquired from laboratory contamination that absorbs light
at 440 nm may interfere in the accurate measurement of
the method analytes.
4.2 Proper storage and good sample handling
technique are critical in preventing degradation of the
pigments.
4.3 Precision and recovery for any of the pigments is
related to efficient extraction, i.e. efficient maceration of
the filtered sample and to the steeping period of the
macerated filter in the extraction solvent. Precision
improves with increasing steeping periods, however, a
drawback to prolonged steeping periods is the possibility
of pigment degradation. The extracted sample must be
kept cold and in the dark to minimize degradation.
4.4 Sample extracts must be clarified by filtration
through a 0.45 |im filter prior to analysis by HPLC to
prevent column fouling.
4.5 All photosynthetic pigments are light and
temperature sensitive. Work must be performed in
subdued light and all standards, QC materials, and
filtered samples must be stored in the dark at -20°C or
-70°C to prevent rapid degradation.
447.0-3
Version 1.0 September 1997
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5.0 Safety
6.9.2 Graduated cylinders, 500-mL and 1-L.
5.1 Each chemical used in this method should be
regarded as a potential health hazard and handled with
caution and respect. Each laboratory is responsible for
maintaining a current awareness file of Occupational
Safety and Health Administration (OSHA) regulations
regarding the safe handling of the chemicals specified in
this method.(15"18) A file of MSDS also should be made
available to all personnel involved in the chemical
analysis.
5.2 The grinding of filters during the extraction step of
this method should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.
6.0 Apparatus and Equipment
6.1 Centrifuge, capable of 675 g.
6.2 Tissue grinder, Teflon pestle (50 mm X 20 mm)
with grooves in the tip with 1/4" stainless steel rod long
enough to chuck onto a suitable drive motor and 30-mL
capacity round-bottomed, glass grinding tube.
6.3 Filters, glass fiber, 47-mm or 25-mm nominal
pore size of 0.7 urn unless otherwise justified by data
quality objectives. Whatman GF/F filters were used in this
work.
6.4 Petri dishes, plastic, 50 X 9-mm, or some other
solid container for transporting and storing sampled
filters.
6.5
Aluminum foil.
6.6 Laboratory tissues.
6.7 Tweezers or flat-tipped forceps.
6.8 Vacuum pump or source capable of maintaining
a vacuum up to 6 in. Hg (20 KPa).
6.9 Labware - All reusable labware (glass,
polyethylene, Teflon, etc.) that comes in contact with
chlorophyll solutions should be clean and acid free. An
acceptable cleaning procedure is soaking for 4 h in
laboratory grade detergent and water, rinsing with tap
water, distilled deionized water and acetone.
6.9.1 Assorted Class A calibrated pipets.
6.9.3 Volumetric flasks, Class A calibrated, 10-mL, 25-
ml_, 50-mL, 100-mL and 1-L capacity.
6.9.4 Glass rods or spatulas.
6.9.5 Pasteur Type pipets or medicine droppers.
6.9.6 Filtration apparatus consisting of 1 or 2-L filtration
flask, 47-mm fritted glass disk base and a glass filter
tower.
6.9.7 Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.9.8 Polyethylene squirt bottles.
6.9.9 Amber 2-mL HPLC autosampler vials with screw
or clamp caps.
6.9.10 Glass syringe, 1 or 2-mL capacity.
6.9.11 HPLC compatible, low-volume, acetone resistant
glass fiber or PTFE syringe filters.
6.10 Liquid Chromatograph
6.10.1 This method uses a ternary gradient thus
requiring a programmable gradient pump with at least
three pump inlets for the three different mobile phases
required. A Dionex Model 4500 chromatograph equipped
with a gradient pump, UV/VIS detector (cell path length,
6 mm, volume 9 |iL) and PC data analysis (Dionex AI450
software, Version 3.32) system was used to generate
data for this method. Tubing was made of polyether ether
ketone (PEEK). A Dionex degas module was used to
sparge all eluents with helium.
6.10.2 Helium or other inert gas for degassing the
mobile phases OR other means of degassing such as
sonication under vacuum.
6.10.3 Sample loops of various sizes (50-200 |iL).
6.10.4 Guard Column - A short column containing the
same packing material as the analytical column placed
before the analytical column to protect it from fouling by
small particles. The guard column can be replaced
periodically if it is noticed that system back pressure has
increased overtime.
Version 1.0 September 1997
447.0-4
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6.10.5 Analytical Column - A C18 reversed-phase
column with end capping. A J.T. Baker 4.6 mm X 250
mm, 5 |im pore size column was used to generate the
data in this method.
6.10.6 A visible wavelength detector with a low volume
flow-through cell. Detection is at 440 nm.
6.10.7 A recorder, integrator or computer for recording
detector response as a function of time.
6.10.8 Although not required, an autosampler
(preferably refrigerated) is highly recommended.
7.0 Reagents and Standards
7.1 Acetone, HPLC grade, (CASRN 67-64-1).
7.2 Methanol, HPLC grade, (CASRN 67-56-1).
Prepare ELUENT A, 80% (v/v) methanol/20% 0.5 M
ammonium acetate, by adding 800 ml of methanol and
200 ml of the 0.5 M ammonium acetate (Sect. 7.5) to an
eluent container.
7.3 Acetonitrile, HPLC grade, (CASRN 75-05-8).
Prepare ELUENT B, 90% (v/v) acetonitrile/10% water, by
adding 900 mL of acetonitrile and 100 mL of water (Sect.
7.7) to an eluent container.
7.4 Ethyl acetate, HPLC grade, (CASRN 141 -78-6).
ELUENT C, 100% ethyl acetate.
7.5 Ammonium acetate, ACS grade (CASRN 631-
61-8). Prepare a 0.5 M solution by dissolving 38.54 g in
approximately 600 mL of water in a 1-L volumetric flask.
After the ammonium acetate has dissolved, dilute to
volume with water.
7.6 Chi a free of chl b and chl b substantially free of
chl a may be obtained from a commercial supplier such
as Sigma Chemical (St. Louis, MO).
7.7 Water - ASTM Type I water (ASTM D1193) is
required. Suitable water may be obtained by passing
distilled water through a mixed bed of anion and cation
exchange resins.
7.8 Aqueous Acetone Solution - 90% acetone/10%
ASTM Type I water. Carefully measure 100 mL of the
water into the 1-L graduated cylinder. Transfer to a 1-L
flask or storage bottle. Measure 900 mL of acetone into
the graduated cylinder and transfer to the flask or bottle
containing the water. Mix, label and store.
7.9 Chlorophyll Stock Standard Solution (SSS) -
Chl a (MW = 893.5) and chl b (MW = 907.5) from a
commercial supplier is shipped in amber glass ampules
that have been flame sealed. The dry standards must be
stored at -20 or -70°C in the dark. Tap the ampule until
all the dried pigment is in the bottom of the ampule. In
subdued light, carefully break the tip off the ampule.
Transfer the entire contents of the ampule into a 25-mL
volumetric flask. Dilute to volume with 90% acetone: (1
mg in 25 mL = 40 mg chl a/L) and (1 mg in 25 ml = 40
mg chl Jb/L), label the flasks and wrap with aluminum foil
to protect from light. When stored in a light- and air-tight
container at -20 or -70°C, the SSS is stable for at least six
months. Dilutions of the SSS should always be confirmed
spectrophotometrically using EPA Method 446.
7.10 Laboratory Reagent Blank (LRB) - A blank
filter that is extracted and analyzed just as a sample filter.
The LRB should be the last filter extracted of a sample
set. It is used to assess possible contamination of the
reagents or apparatus.
7.11 Quality Control Sample (QCS) - Since there
are no commercially available QCSs, dilutions of a stock
standard of a different lot number from that used to
prepare calibration solutions may be used.
8.0 Sample Collection, Preservation and
Storage
8.1 Water Sample Collection — Water may be
obtained by a pump or grab sampler. Data quality
objectives will determine the depth and frequency'21' at
which samples are taken. Healthy phytoplankton,
however, are generally obtained from the photic zone
(region in which the illumination level is 1% of surface
illumination). Enough water should be collected to
concentrate phytoplankton on at least three filters so that
precision can be assessed. Filtration volume size will
depend on the particulate load of the water. Four liters
may be required for open ocean water where
phytoplankton density is usually low, whereas 1 L or less
is generally sufficient for lake, bay or estuary water. All
apparatus should be clean and acid-free. Filtering should
be performed in subdued light as soon as possible after
sampling since algal populations, thus pigment
concentrations, can change in relatively short periods of
time. Aboard ship filtration is highly recommended.
447.0-5
Version 1.0 September 1997
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Assemble the filtration apparatus and attach the vacuum
source with vacuum gauge and regulator. Vacuum
filtration should not exceed 6 in. Hg (20 kPa). Higher
filtration pressures may damage cells and result in loss of
chlorophyll. Care must be taken not to overload the
filters. Do not increase the vacuum during filtration.
Prior to drawing a subsample from the water sample
container, gently stir or invert the container several times
to suspend the particles. Pour the subsample into a
graduated cylinder and accurately measure the volume.
Pour the subsample into the filter tower of the filtration
apparatus and apply a vacuum (not to exceed 20 kPa).
Typically, a sufficient volume has been filtered when a
visible green or brown color is apparent on the filter. Do
not suck the filter dry with the vacuum; instead slowly
release the vacuum as the final volume approaches the
level of the filter and completely release the vacuum as
the last bit of water is pulled through the filter. Remove
the filter from the fritted base with tweezers, fold once
with the particulate matter inside, lightly blot the filter with
a tissue to remove excess moisture and place it in the
petri dish or other suitable container. If the filter will not
be immediately extracted, wrap the container with
aluminum foil to protect the phytoplankton from light and
store the filter at -20°C or -70°C. Short term storage (2 to
4 h) on ice is acceptable, but samples should be stored
at -20°C or -70°C as soon as possible.
8.2 Preservation - Sampled filters should be stored
frozen (-20°C or -70°C) in the dark until extraction.
8.3 Holding Time - Filters can be stored frozen at
-20°C for as long as 31/2 weeks without significant loss of
chl a.(20)
9.0 Quality Control
9.1 Each Laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and the continued
analysis of laboratory reagent blanks, field replicates,
QCSs, and CALCHKs as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
generated.
9.2 Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (IDLs) and
laboratory performance (MDLs, extraction proficiency,
and analyses of QCSs) prior to sample analyses.
9.2.2 Instrumental Detection Limit (IDL) - After a low
level calibration (Sect. 10), prepare a standard solution
that upon injection into the chromatograph yields an
absorbance of 0.002-0.010. If using an autosampler,
variable volumes may be injected and the micrograms
(|jg) injected calculated by multiplying the known
concentration (|ig/|iL) of the standard by the volume
injected (|iL). A practical starting point may be to inject
0.05 |jg (that would be a 50 |iL injection of a 1.0 mg/L
standard solution) and reduce or increase the mass
injected according to the resulting signal. Avoid injecting
really small volumes (< 10 |iL). After the quantity of
pigment has been selected, make three injections and
calculate the IDL by multiplying the standard deviation of
the calculated mass by 3.
9.2.3 Method Detection Limit (MDL) - At least seven
natural phytoplankton samples known to contain the
pigments of interest should be collected, extracted and
analyzed according to the procedures in Sects. 8 and 11,
using clean glassware and apparatus. Mass of the
pigment injected into the chromatograph should be 2 to
5 times the IDL. Dilution of the sample extract solution to
the appropriate concentration or reducing the volume of
sample injected may be necessary. Calculate the MDL
(in micrograms) as follows.(19)
MDL = (t) X (S)
where, t = Student's t-value for n-1 degrees of freedom at
the 99% confidence level, t = 3.143 for six degrees of
freedom.
S = Standard deviation of the replicate analyses.
9.2.4 Quality Control Sample (QCS) - When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify instrument performance
with the analysis of a QCS (Sect. 7.11). If the determined
value is not within +10% of the spectrophotometrically
determined value, then the instrument should be
recalibrated with fresh stock standard and the QCS
reanalyzed. If the redetermined value is still
unacceptable then the source of the problem must be
identified and corrected before continuing analyses.
Version 1.0 September 1997
447.0-6
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9.2.5 Extraction Proficiency - Personnel performing
this method for the first time should demonstrate
proficiency in the extraction of sampled filters (Sect. 11.1).
Fifteen to twenty natural samples should be obtained
using the procedure outlined in Sect. 8.1 of this method.
Sets of 10 samples or more should be extracted and
analyzed according to Sect. 11. The percent relative
standard deviation (%RSD) should not exceed 15% for
samples that are at least 10X the IDL.
10.3 Program the pump with the following gradient:
9.3
Assessing
(Mandatory)
Laboratory Performance
9.3.1 Laboratory Reagent Blank (LRB) - The
laboratory must analyze at least one blank filter with each
sample batch. The LRB should be the last filter
extracted. LRB data are used to assess contamination
from the laboratory environment. LRB values that exceed
the IDL indicate contamination from the laboratory
environment. If the LRB value constitutes 10% or more
of the analyte level determined in a sample, fresh
samples or field replicates must be analyzed after the
contamination has been corrected and acceptable LRB
values have been obtained.
9.3.2 Calibration Check Standard (CALCHK) - The
laboratory must analyze one CALCHK for every ten
samples to verify calibration. If the CALCHK is not+10%
of the spectrophotometrically determined concentration,
then the instrument must be recalibrated.
10.0 Calibration and Standardization
10.1 Allow the visible wavelength detector (440 nm) to
warm up for at least 15 min before calibration. Prepare
ELUENTS A - C and degas by sparging with an inert gas
for 10 minutes or sonicating under vacuum for 5 minutes.
Prime the pump for each eluent taking care to remove all
airthat may be in the liquid lines. Equilibrate the column
for ten minutes with 100% of ELUENT A.
10.2 Remove the SSS from the freezer and allow it to
come to room temperature. Add 1 mL of the SSS to a
10-mL volumetric flask and dilute to 10 mL with 90%
acetone. Prepare the chl a and b separately and
determine the concentrations according to EPA Method
446 using the monochromatic equations for chl a
determination. After the concentration of the SSS is
determined, add 1 mL of the chl a SSS plus 1 mL of the
chl b SSS to a separate 10-mL flask and dilute to volume.
Store the calibration standard in a light tight glass bottle.
Time
0.0
2.0
2.6
13.6
20.0
22.0
25.0
30.0
Flow %1
1
1
1
1
1
1
1
1
.0
.0
.0
.0
.0
.0
.0
.0
100
0
0
0
0
0
100
100
%2
0
100
90
65
31
100
0
0
%3
0
0
10
35
69
0
0
0
Condition
Injection
Linear Gradient
Linear Gradient
Linear Gradient
Linear Gradient
Linear Gradient
Linear Gradient
Equilibration
Flow is in mL/min.
10.4 The first analysis is a blank 90% acetone solution
followed by calibration. Calibrate with at least three
concentrations, covering no more than one order of
magnitude, and bracketing the concentrations of
samples. If an autosampler is used, variable volumes
ranging from 10 - 100% of the sample injection loop
volume are injected to give a calibration of detector
response versus mass of pigment. If doing manual
injections, variable solution concentrations are made and
a fixed sample loop volume is injected for standards and
samples. Calibration can be either detector response
versus mass or detector response versus concentration
(mg/L or |ig/L). Linearity across sensitivity settings of the
detector must be confirmed if samples are analyzed at a
different sensitivity settings from that of the calibration.
10.5 Construct a calibration curve of analyte response
(area) versus concentration (mg/L in solution) or mass
(|jg) of pigment and perform a linear regression to
determine the slope and y-intercept. A typical coefficient
of determination is > 0.99.
10.6 Calibration must be performed at least weekly
although it is not necessary to calibrate daily. Daily mid-
point CALCHKs must yield calculated concentrations
±10% of the spectrophotometrically determined
concentration.
11.0 Procedure
11.1 Extraction of Filter Samples
11.1.1 For convenience, a 10-mL final extraction volume
is described in the following procedure. A smaller
extraction volume may be used to improve detection
limits.
447.0-7
Version 1.0 September 1997
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11.1.2 If sampled filters have been frozen, remove them
from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand laboratory tissues and
wash bottles containing water and acetone. Workspace
lighting should be the minimum that is necessary to read
instructions and operate instrumentation. Remove a filter
from its container and place it in the glass grinding tube.
You may also tear the filter into smaller pieces and push
them to the bottom of the tube with a glass rod. With a
volumetric pipet, add 3 ml of the aqueous acetone
solution (Sect. 7.6) to the grinding tube. Grind the filter
until it has become a slurry. (NOTE: Although grinding
is required, care must be taken not to overheat the
sample. Good judgement and common sense will help
you in deciding when the sample has been sufficiently
macerated.) Pour the slurry into a 15-mL screw-cap
centrifuge tube and, using a 7-mL volumetric pipet, rinse
the pestle and the grinding tube with the aqueous
acetone. Add the rinse to the centrifuge tube containing
the filter slurry. Cap the tube and shake it vigorously.
Place it in the dark before proceeding to the next filter
extraction. Before placing another filter in the grinding
tube, use the acetone and water squirt bottles to
thoroughly rinse the pestle, grinding tube and glass rod.
To reduce the volume of reagent grade solvents used for
rinsing between extractions, thoroughly rinse the grinding
tube and glass rod with tap water prior to a final rinse with
ASTM Type I water and acetone. The last rinse should
be with acetone. Use a clean tissue to remove any filter
residue that adheres to the pestle or to the steel rod of the
pestle. Proceed to the next filter and repeat the steps
above. The last filter extracted should be a blank. The
entire extraction with transferring and rinsing takes
approximately 5 min. Approximately 500 ml of acetone
and water waste are generated per 20 samples from the
rinsing of glassware and apparatus.
11.1.3 Again, shake each tube vigorously before placing
them to steep in the dark at 4°C. Samples should be
allowed to steep fora minimum of 2 h but not to exceed
24 h. Tubes should be shaken at least once, preferably
two to three times, during the steeping period to allow the
extraction solution to have maximum contact with the filter
slurry.
11.1.4 After steeping is complete, centrifuge samples for
15 min at 675 g or for 5 min at 1000 g. Draw
approximately 1 ml into a glass syringe, attach a 0.45 |im
syringe filter, filter the extract into an amber autosampler
vial, cap and label the vial. Protect the filtered samples
from light and heat. If using a refrigerated autosampler,
chill to 10°C.
11.2 Sample Analysis
11.2.1 Draw into a clean syringe 2-3 times the injection
loop volume and inject into the chromatograph. If using
an autosampler, load the sample tray, prepare a
schedule and begin analysis. A typical analyses order
might be: (1) blank 90% acetone, (2) CALCHK, (3) 10
samples, (4) CALCHK, (5) QCS.
11.2.2 If the calculated CALCHK is not ±10 of the
spectrophotometrically determined concentration then
recalibrate with fresh calibration solutions.
12.0 Data Analysis and Calculations
12.1 From the chl a or b area response of the sample,
calculate the mass injected or concentration (CE) of the
solution that was analyzed using the calibration data.
Mass injected must be converted to concentration in
extract by dividing mass by volume injected (|iL) and
multiplying by 1000 to give concentration in mg/L
(mg/L = ug/mL). Concentration of the natural water
sample may be reported in mg/L by the following
formula:
C. X extract volume CD X DF
sample volume (L)
where:
CE = concentration (mg/L) of pigment in extract.
DF = any dilution factors.
L = liters.
12.2 LRB and QCS data should be reported with each
data set.
13.0 Method Performance
13.1 Single Laboratory Performance
13.1.1 An IDL was determined by preparing a mixed chl
a (0.703 ppm) and chl b (0.437 ppm) standard. The
injected mass yielded 0.004 AU for chl a (0.035 |jg) and
0.003 AU for chl b (0.022 |_ig). Seven replicate 50 |_iL
injections were made and the standard deviation of the
calculated concentration was multiplied by three to
determine an IDL. The IDL determined for chl a was 0.76
ng and 0.44 ng for chl b. The %RSDs for chl a and chl b
was 0.45 and 0.67, respectively.
13.1.2 MDLs for chl a and chl b were determined by
spiking seven replicate filtered samples of Pycnacoccus,
extracting and processing according to this method. An
Version 1.0 September 1997
447.0-8
-------
injection volume of 100 pL yielded an MDL for chl a of 7.0
ng and 4.0 ng for chl b. The RSDs were 5.1% for chl b
and 4.7 % for chl a.
13.1.3 Recoveries of chl a and chl b from filtered
samples of phaeodactylum were determined by spiking
three filters with known amounts of the pigments,
extracting, processing and analyzing the extraction
solution according to the method, along with three
unspiked filtered samples (to determine the native levels
in the algae). The spiked levels were 1.1 ppm chl a and
0.53 ppm chl b in the 10 ml extraction volume. Chl a
was 87% recovered and chl b was 94% recovered.
13.1.4 Figures 1-7 are chromatograms of seven
reference unialga cultures processed according to this
method.
13.1.5 Table 1 is a list of pure pigments with retention
times obtained using this method. Purified pigments were
prepared under contract to EPA by Moss Landing Marine
Laboratory, Moss Landing, CA.
13.1.6 Table 2 contains single lab precision data for
seven reference algal suspensions.
13.2 Multilaboratory Testing - A Multilaboratory
validation and comparison study of EPA Methods 445.0,
446.0 and 447.0 for chlorophyll a was conducted in 1996
by Research Triangle Institute, Research Triangle Park,
N.C. (EPA Contract No. 68-C5-0011). There were 8
volunteer participants in the HPLC methods component
that returned data. The primary goals of the study were
to determine estimated detection limits and to assess
precision and bias (as percent recovery) for select
unialgal species, and natural seawater.
13.2.1 The term, pooled estimated detection limit (p-
EDL), is used in this method to distinguish it from the EPA
defined method detection limit (MDL). The statistical
approach used to determine the p-EDL was an adaptation
of the Clayton, et. al.(21) method that does not assume
constant error variances across concentration and
controls for Type II error. The approach used involved
calculating an estimated DL for each lab that had the
desired Type I and Type II error rates (0.01 and 0.05,
respectively). The median DLs over labs was then
determined and is reported in Table 3. It is referred to as
Pooled-EDL (p-EDL).
The p-EDL was determined in the following manner.
Solutions of pure chlorophyll a in 90% acetone were
prepared at three concentrations (0.11, 0.2 and 1.6 ppm)
and shipped with blank glass fiber filters to participating
laboratories. Analysts were instructed to spike the filters
in duplicate with a given volume of solution and to
process the spiked filters according to the method. The
results from these data were used to determine a p-EDL
for each method. Results (in ppm) are given in Table 3.
The standard fluorometric and HPLC methods gave the
lowest p-EDLs while the spectrophotometric
(monochromatic equations) gave the highest p-EDLs.
13.2.2 To address precision and bias in chlorophyll a
determination for different algal species, three pure
unialgal cultures (Amphidinium, Dunaliella and
Phaeodactylum) were cultured and grown in the
laboratory. Four different "concentrations" of each
species were prepared by filtering varying volumes of the
algae. The filters were frozen and shipped to participant
labs. Analysts were instructed to extract and analyze the
filters according to the respective methods. The "true"
concentration was assigned by taking the average of the
HPLC results for the highest concentration algae sample
since chlorophyll a is separated from other interfering
pigments prior to determination . Pooled precision data
are presented in Tables 4-6 and accuracy data (as
percent recovery) are presented in Table 7. No
significant differences in precision (%RSD) were observed
across concentrations for any of the methods or species.
It should be noted that there was considerable lab-to-lab
variation (as exhibited by the min and max recoveries in
Table 7) and in this case the median is a better measure
of central tendency than the mean.
In summary, the mean and median concentrations
determined for Amphidinium carterae (class
dinophyceae) are similar for all methods. No method
consistently exhibited high or low values relative to the
other methods. The only concentration trend observed
was that the spectrophotometric method-trichromatic
equations (SP-T) showed a slight percent increase in
recovery with increasing algae filtration volume.
For Dunaliella tertiolecti (class chlorophyceae) and
Phaeodactylum tricornutum (class bacillariophyceae)
there was generally good agreement between the
fluorometric and the spectrophotometric methods,
however, the HPLC method yielded lower recoveries with
increasing algae filtration volume for both species. No
definitive explanation can be offered at this time for this
phenomenon. A possible explanation for the
Phaeodactylum is that it contained significant amounts of
chlorophyllide a which is determined as chlorophyll a in
the fluorometric and spectrophotometric methods. The
conventional fluorometric method (FL-STD) showed a
slight decrease in chlorophyll a recovery with increasing
Dunaliella filtration volume. The spectrophotometric-
447.0-9
Version 1.0 September 1997
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trichromatic equations (SP-T) showed a slight increase in
chlorophyll a recovery with incresing dunaliella filtration
volume. The fluorometric and athe spectrophotometric
methods both showed a slight decrease in chlorophyll a
recovery with increasing Phaeodactylum filtration volume.
Results for the natural seawater sample are presented in
Table 8. Only one filtration volume (100 ml) was
provided in duplicate to Participant labs.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
USEPA has established a preferred hierarchy of
environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation (e.g., Sect. 11.1.2). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202) 872-4477.
15.0 Waste Management
15.1 The U.S. Environmental Protection Agency
requires that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in the Sect. 14.2.
16.0 References
1. Holm-Hansen, O., "Chlorophyll a determination:
improvements in methodology," OIKOS, 30
(1978), pp. 438-447.
2. Wright, S.W. and J.D. Shearer, "Rapid extraction
and HPLC of chlorophylls and carotenoids from
marine phytoplankton," J. Chrom., 294 (1984),
pp. 281-295.
3. Bowles, N.D., H.W. Paerl, and J. Tucker,
"Effective solvents and extraction periods
employed in phytoplankton carotenoid and
chlorophyll determination," Can. J. Fish. Aquat.
ScL, 42 (1985) pp. 1127-1131.
4. Shoaf, W.T. and B.W. Lium, "Improved extraction
of chlorophyll a and b from algae using dimethyl
sulfoxide," Limnol. and Oceanogr., 21(6) (1976)
pp. 926-928.
5. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid
determination of algal chlorophyll and carotenoid
pigments and their breakdown products in
natural waters by reverse-phase high
performance liquid chromatography," Anal.
Chim. Acta., 151 (1983) pp. 297-314.
6. Yentsch, C.S. and D.W. Menzel, "A method for
the determination of phytoplankton chlorophyll
and phaeophytin by fluorescence," Deep Sea
Res., 10(1963), pp. 221-231.
7. Strickland, J.D.H. and T.R. Parsons, A Practical
Handbook of Seawater Analysis. Bull. Fish. Res.
Board Can., 1972, No.167, p. 201.
8. 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.
9. Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,
C.A. Llewellyn, T. Bjornland, D. Repeta, and N.
Welschmeyer, "Improved HPLC method for the
analysis of chlorophylls and carotenoids from
marine phytoplankton," Mar. Ecol. Prog. Ser,
77:183.
Version 1.0 September 1997
447.0-10
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10. Brown, L.M., B.T. Margrave, and M.D.
MacKinnon, "Analysis of chlorophyll a in
sediments by high-pressure liquid
chromatography," Can. J. Fish. Aquat. Sci., 38
(1981) pp. 205-214.
11. Bidigare, R.R., M.C. Kennicutt, II, and J.M.
Brooks, "Rapid determination of chlorophylls and
their degradation products by HPLC," Limnol.
Oceanogr., 30(2) (1985) pp. 432-435.
12. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.
Montano-Asquerino, and J. Garrido-Fernandez,
"Determination of chlorophylls and carotenoids
by HPLC during olive lactic fermentation," J.
Chrom., 585 (1991) pp. 259-266.
13. Neveux.J., D. Delmas, J.C. Romano, P. Algarra,
L. Ignatiades, A. Herbland, P. Morand, A. Neori,
D. 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.
14. Sartory, D.P., "The determination of algal
chlorophyllous pigments by high performance
liquid chromatography and spectrophotometry,"
Water Research, 19(5), (1985), pp. 605-10.
15. Carcinogens - Working With Carcinogens,
Department of Health, Education and Welfare,
Public Health Service, Center for Disease
Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, 1977.
16. "OSHA Safety and Health Standards, General
Industry," (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, revised
January 1976.
17. Safety in Academic Chemistry Laboratories,
American Chemical Society publication,
Committee on Chemical Safety, 3rd Edition,
1979.
18. "Proposed OSHA Safety and Health Standards,
Laboratories," Occupational Safety and Health
Administration, Federal Register. July 24, 1986.
20. 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,"
Oho State University, Grant No. NA84AA-D-
00079, 1986,54pp.
21. Clayton, C.A., J.W. Hine, and P.O. Elkins,
"Detection Limits within Specified Assurance
Probabilities." Analytical Chemistry. 59(1987), pp.
2506-2514.
19. Code of Federal Regulations 40. Ch.1.
Pt.136, Appendix B.
447.0-11
Version 1.0 September 1997
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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Table 1. Pure Pigments and Retention Times
PIGMENT
19' butanoyloxyfucoxanthin
2,4-divinylpheoporphrin a5
Peridinin
Fucoxanthin
1 9' hexanoyloxyfucoxanthin
Neoxanthin
Chlorophyll C3
Chlorophyll C2
Prasinoxanthin
Violaxanthin
Diadinoxanthin
Chlorophyll b
Myxoxanthophyll
Aphanaxanthin
Chlorophyll a
Monadoxanthin
Lutein
Alloxanthin
Nostaxanthin
Diatoxanthin
Zeaxanthin
RETENTION TIME
8.13
8.60
8.69
8.75
8.90
10.07
10.27
10.40
11.20
12.00
15.20
15.60
17.00
17.20
17.80
17.93
18.00
18.07
18.70
19.07
19.40
Version 1.0 September 1997
447.0-12
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Table 2. Single Lab Precision for Seven Pure Unialgal Cultures
Algae
Pycnacoccus provasolii
Rhodomonas salina
Selenastrum
capricornitum
Amphidinium carterae
Dunaliella tertiolecti
Emiliania huxleyi
Phaeodactylum
tricornutum
N(1)
Mean (mg/L)(2)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
N(1)
Mean (mg/L)
STD DEV
% RSD
Chlorophyll a
3
2.15
0.114
5.31
3
4.0
0.014
0.28
3
4.25
0.199
4.68
3
2.38
0.176
7.40
3
6.68
0.635
9.51
3
1.03
0.008
0.79
3
1.09
0.072
7.07
Chlorophyll b
3
1.47
0.065
4.45
3
ND(3)
ND
ND
3
0.483
0.058
12.01
3
ND
ND
ND
3
1.42
0.0412
2.90
ND
ND
ND
ND
ND
ND
ND
ND
(1) N = Number of filtered samples.
(2) Mean concentration in extract solution.
(3) ND = none detected.
447.0-13
Version 1.0 September 1997
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TABLE 3. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL 4 METHODS*
Method'2'
FL -Mod(5)
FL - Std
HPLC
SP-M
SP-T
M(3)
8
9
4
15
15
p-EDL'4' (mg/L)
0.096
0.082
0.081
0.229
0.104
(1) See Section 13.2.1 for a description of the statistical approach used to determine p-EDLs.
(2) FL-Mod = fluorometric method using special interference filters.
FL-Std = conventional fluorometric method with pheophytin a correction.
HPLC = EPA method 447.0
SP-M = EPA method 446.0, monochromatic equation.
SP-T = EPA method 446.0, trichromatic equations.
(3) N = number of labs whose data was used.
(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.
(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are
unrealistically high.
Version 1.0 September 1997 447.0-14
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TABLE 4. Measured Chlorophyll a (mg/L) in Dunaliella Samples
Method'1'
HPLC
ml_s of
culture
filtered
5
10
50
100
N(2)
5
5
5
5
Mean (mg chla/D
0.172
0.276
0.757
1.420
Std. Dev.
0.064
0.074
0.344
0.672
%RSD
36.8
26.8
45.4
47.3
(1) Not all participants labs followed the EPA method exactly.
(2) N = Number of volunteer labs whose data was used.
447.0-15 Version 1.0 September 1997
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TABLE 5. Measured Chlorophyll a (mg/L) in Amphidinium Samples
ml_s of
culture
Method'1' filtered N(2) Mean (ma chla/D Std. Dev. %RSD
HPLC 5 5 0.104 0.043 56.8
10 5 0.172 0.083 37.5
50 5 0.743 0.213 17.4
100 5 1.394 0.631 14.5
(1) Not all participants labs followed the EPA method exactly.
(2) N = number of volunteer labs whose data was used.
Version 1.0 September 1997 447.0-16
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TABLE 6. Measured Chlorophyll a in Phaeodactylum Samples
ml_s of
culture
Method'1' filtered N(2) Mean (ma chla/D Std. Dev. %RSD
HPLC 5 5 0.193 0.074 38.4
10 5 0.317 0.114 36.1
50 5 1.024 0.340 33.2
100 5 1.525 0.487 29.9
(1) Not all participants labs followed the EPA method exactly.
(2) N = number of volunteer labs whose data was used.
447.0-17 Version 1.0 September 1997
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TABLE 7. Minimum, Median, and Maximum Percent Recoveries by Genera, Method, and Concentration Level
Species
Amphidinium
Dunaliella
Statistic
Minimum
Median
Maximum
Minimum
Median
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
Percent Recovery
Cone.
Level 1
70
66
82
36
21
105
109
102
99
95
121
156
284
141
115
162
179
165
120
167
206
250
252
240
Cone.
Level 2
73
91
85
48
63
112
107
106
101
96
126
154
210
133
116
159
171
109
188
169
246
228
177
247
Cone.
Level 3
75
91
87
68
71
105
111
112
101
106
143
148
131
126
119
157
165
64
167
166
227
224
89
247
Cone.
Level 4
76
90
88
64
70
104
109
105
101
107
146
148
116
125
117
156
164
41
164
165
223
210
80
243
Version 1.0 September 1997
447.0-18
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Table 7. Cont'd.
Species
Dunaliella
Phaeodactylum
Statistic
Maximum
Minimum
Median
Maximum
Method
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
FL-STD
HPLC
SP-M
SP-T
FL-MOD
Percent Recovery
Cone.
Level 1
225
295
439
392
342
291
216
189
150
161
203
292
296
225
287
286
357
Cone.
Level 2
244
277
385
273
316
283
183
220
119
138
195
285
263
203
274
281
337
Cone.
Level 3
256
287
276
172
296
283
157
223
84
156
216
250
254
114
254
277
320
Cone.
Level 4
256
288
261
154
293
283
154
219
75
160
244
245
254
90
253
274
318
447.0-19
Version 1.0 September 1997
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Table 8. Chlorophyll a Concentrations in mg/L Determined in Filtered Seawater Samples
Method
FL-MOD
FL-STD
HPLC
SP-M
SP-T
All Methods
Con.(1)
100
100
100
100
100
100
No. Obs
14
15
10
38
36
113
No. Labs
7
8
5
19
18
57
Mean
1.418
1.576
1.384
1.499
1.636
1.533
Std. Dev
0.425
0.237
0.213
0.219
0.160
0.251
RSD (%)
30.0
15.0
15.4
14.6
9.8
16.4
Minimum
0.675
1.151
1.080
0.945
1.250
0.657
Median
1.455
1.541
1.410
1.533
1.650
1.579
Maximum
2.060
1.977
1.680
1.922
1.948
2.060
(1) Con = ml_s of seawater filtered.
Version 1.0 September 1997
447.0-20
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Figure 1 - Amphidinium carterae
O.O4
0.03
AU
0.02
0.01
0.00
10.00
8.67
18.33
I ' ' '
10
1
15
I '
20
« T.-J
25
Minutes
Figure 2 - Dunaliella tertiolecti
0.08
0.07
0.06
0.06
AU
0.04
0.03
0.02
0.01
0.00
18.27
6.63
16.07
22.93
19.27
I l~l I J I F
5
I II
10
1 ' I
15
I
20
. • |
25
Minute*
3O
3O
447.0 - 21
-------
Figure 3 - Pycnococcus provasolii
0.02
11.60
0.01
AU
0.00
16.53
18.67
9.13
I 1 I I I I i
0 6
I i i i i I i i i i I i
10 16 20
Minute*
i i iiii iiin
26 30
Figure 4 - Selenastrum capricornitum
O.O4
0.03
0.02
AU
0.01
0.00
18.13
23.00
T—n—i -j"i t t i—f~i—i i t .
0 5 10
i—i—i—i—i—i—i—i—i—i—i—i—r~i—i—i
15
1
20
25
30
Minutes
447.0 - 22
-------
Figure 5 - Emiiania huxleyl (slightly senescent)
O.O3
9.07
0.02
AU
0.01
0.00
17.93
21.00
T T I I
I *
10
1 I
15
20
i i * i T r
25
Minutes
Figure 6 - Phaeodactylum tricornufum
AU
0.14
0.12
O.1O
0.08
0.06
0.04
O.O2
.O.OO
I
9.
6.63
I
_^~ A^J
[i ii i j i - f in
) 5
08
14.87
?-73 ft 17.93-
vLJLJL
10 15
22.67
2O
25
Minutes
3O
447.0 - 23 MU-S. GOVERNMENT PRIMING OEBl'CE: 1998 - 650-001/80225
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