ASTM D3731-20

Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters

SIGNIFICANCE AND USE
5.1 Data on the chlorophyll content of the algae have the following applications:  
5.1.1 To provide estimates of algal biomass and productivity.  
5.1.2 To provide general information on the taxonomic composition (major groups) of the algae, based on the relative amounts of chlorophyll a, b, and c, and the physiological condition of algal communities, which is related to the relative abundance of pheopigments.  
5.1.3 To determine long-term trends in water quality.  
5.1.4 To determine the trophic status of surface waters.  
5.1.5 To detect adverse effects of pollutants on plankton and periphyton in receiving waters.  
5.1.6 To determine maximum growth rates and yields in algal growth potential tests.
SCOPE
1.1 These practices include the extraction and the measurement of chlorophyll a, b, and c, and pheophytin a in freshwater and marine plankton and periphyton. Three practices are provided as follows:  
1.1.1 Spectrophotometric, trichromatic practice for measuring chlorophyll a, b, and c.  
1.1.2 Spectrophotometric, monochromatic practice for measuring chlorophyll a corrected for pheophytin a; and for measuring pheophytin a.  
1.1.3 Fluorometric practice for measuring chlorophyll a corrected for pheophytin a; and for measuring pheophytin a.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 8.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-May-2020
• ICS: 13.060.10 - Water of natural resources
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D3731-87(2012) - Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters
• Effective Date: 01-Jun-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-04 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-03a - Standard Terminology Relating to Water
• Effective Date: 10-Aug-2003
• Refers: ASTM D1129-03 - Standard Terminology Relating to Water
• Effective Date: 10-Mar-2003
• Refers: ASTM D1129-02a - Standard Terminology Relating to Water
• Effective Date: 10-Jul-2002
• Refers: ASTM D1129-01 - Standard Terminology Relating to Water
• Effective Date: 10-Jul-2002
• Refers: ASTM D1129-99a - Standard Terminology Relating to Water
• Effective Date: 10-Feb-2002
• Refers: ASTM D1129-02 - Standard Terminology Relating to Water
• Effective Date: 10-Feb-2002

Overview

ASTM D3731-20: Standard Practices for Measurement of Chlorophyll Content of Algae in Surface Waters establishes standardized procedures for extracting and analyzing key algal pigments in both freshwater and marine environments. This essential standard supports accurate assessment of algal biomass and productivity, insight into water quality trends, and evaluation of ecological health in surface waters.

Developed by ASTM International, this standard includes three validated analytical practices for measuring chlorophyll a, b, c, and pheophytin a in plankton and periphyton samples. The procedures use spectrophotometric and fluorometric methods and are internationally recognized for water quality analysis, ecological monitoring, and limnology research.

Key Topics

• Chlorophyll and Pheopigment Analysis: The standard covers protocols for extracting chlorophyll a, b, and c, along with pheophytin a, critical for determining algal community structure and health.
• Analytical Methods:
  • Spectrophotometric, Trichromatic: Simultaneously measures chlorophyll a, b, and c.
  • Spectrophotometric, Monochromatic: Measures chlorophyll a, corrected for pheophytin a, and pheophytin a.
  • Fluorometric: Highly sensitive measurement for chlorophyll a and pheophytin a.
• Sample Preparation:
  • Highlights the need for effective cell disruption (e.g., using tissue grinders) and optimal use of glass-fiber filters to ensure precise pigment recovery.
• Data Application: Utilizes chlorophyll measurements for estimating algal biomass, tracking water trophic status, and identifying impacts of pollutants on aquatic communities.
• Quality Considerations: Provides guidelines to minimize interferences, such as correcting for light sensitivity, addressing instrument resolution, and managing pigment degradation.

Applications

Adopting ASTM D3731-20 provides value for a range of professionals involved in environmental monitoring, water resource management, and ecological research, including:

• Water Quality Assessment: Enables consistent estimation of algal biomass and productivity, which are critical for evaluating ecosystem health and detecting eutrophication.
• Long-term Monitoring: Supports detection of changes in water quality over time, informing policy and management decisions for surface water bodies.
• Ecological Classification: Assists in determining the trophic status of lakes and rivers, and inferring taxonomic composition of algae based on pigment profiles.
• Pollution Detection: Aids in identifying adverse effects of pollutants by monitoring changes in plankton and periphyton communities.
• Algal Growth Potential: Facilitates tests for maximum algal growth rates and yields, useful for studies related to nutrient cycling and productivity.
• Research and Academic Purposes: Fundamental for limnology, oceanography, and aquatic biology studies requiring accurate chlorophyll content determination.

Related Standards

Professionals implementing ASTM D3731-20 may also reference these related ASTM standards for broader water analysis and terminology consistency:

• ASTM D1129 – Terminology Relating to Water
• ASTM D1193 – Specification for Reagent Water

For international harmonization, ASTM D3731-20 aligns with principles recognized by the World Trade Organization Technical Barriers to Trade (TBT) Committee, ensuring it meets high-level global standardization requirements.

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Keywords: chlorophyll measurement, algae, surface water, water quality, algal biomass, spectrophotometric analysis, fluorometric analysis, trophic status, ASTM D3731-20, water monitoring, pigment extraction, periphyton, plankton, pheophytin

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D3731 − 20
Standard Practices for Measurement of
Chlorophyll Content of Algae in Surface Waters
This standard is issued under the fixed designation D3731; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.1 For definitions of terms used in this standard, refer to
Terminology D1129.
1.1 These practices include the extraction and the measure-
mentofchlorophyll a, b,and c,andpheophytin ainfreshwater 3.2 Definitions of Terms Specific to This Standard:
and marine plankton and periphyton. Three practices are 3.2.1 chlorophyll, n—a class of pigments involved in pho-
provided as follows: tosynthesis.
1.1.1 Spectrophotometric, trichromatic practice for measur-
3.2.2 periphyton, n—microorganisms growing on sub-
ing chlorophyll a, b, and c.
merged objects, commonly consisting of bacteria, algae,
1.1.2 Spectrophotometric,monochromaticpracticeformea-
protozoa, and rotifers.
suring chlorophyll a corrected for pheophytin a; and for
3.2.3 pheopigments (phaeopigments), n—a non-
measuring pheophytin a.
photosynthetic pigment which is the degradation product of
1.1.3 Fluorometric practice for measuring chlorophyll a
algal chlorophyll pigments.
corrected for pheophytin a; and for measuring pheophytin a.
3.2.4 plankton, n—nonmotile or weakly swimming
1.2 The values stated in SI units are to be regarded as
organisms, usually microscopic, that drift or are carried along
standard. No other units of measurement are included in this
bycurrentsinsurfacewaters,commonlyconsistingofbacteria,
standard.
algae, protozoa, rotifers, and microcrustacea.
1.3 This standard does not purport to address all of the
4. Summary of Practices
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4.1 The chlorophyll and related compounds are extracted
priate safety, health, and environmental practices and deter-
from the algae with 90% aqueous acetone. The concentration
mine the applicability of regulatory limitations prior to use.
of the pigments is determined by measuring the light absorp-
Specific precautionary statements are given in Section 8.
tion or fluorescence of the extract.
1.4 This international standard was developed in accor-
dance with internationally recognized principles on standard- 5. Significance and Use
ization established in the Decision on Principles for the
5.1 Data on the chlorophyll content of the algae have the
Development of International Standards, Guides and Recom-
following applications:
mendations issued by the World Trade Organization Technical
5.1.1 To provide estimates of algal biomass and productiv-
Barriers to Trade (TBT) Committee.
ity.
5.1.2 To provide general information on the taxonomic
2. Referenced Documents
composition (major groups) of the algae, based on the relative
2.1 ASTM Standards:
amounts of chlorophyll a, b, and c, and the physiological
D1129Terminology Relating to Water
condition of algal communities, which is related to the relative
D1193Specification for Reagent Water
abundance of pheopigments.
5.1.3 To determine long-term trends in water quality.
3. Terminology
5.1.4 To determine the trophic status of surface waters.
3.1 Definitions:
5.1.5 Todetectadverseeffectsofpollutantsonplanktonand
periphyton in receiving waters.
These practices are under the jurisdiction ofASTM Committee D19 on Water
5.1.6 To determine maximum growth rates and yields in
and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
algal growth potential tests.
Current edition approved June 1, 2020. Published June 2020. Originally
approved in 1979. Last previous edition approved in 2012 as D3731–87 (2012).
6. Interferences and Special Considerations
DOI: 10.1520/D3731-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
6.1 Pigment Extraction—The chlorophylls are only poorly
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
extracted, if at all, from some forms of algae, such as the
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. coccoid green algae, unless the cells are disrupted, whereas
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D3731 − 20
other algae, such as the diatoms, give up their pigments very 6.6 Spectrophotometer Resolution—The absorption peak of
readily when merelysteepedinacetone.Sincenaturalcommu- acetone solutions of chlorophyll extracts is relatively narrow,
nities of algae usually consist of a wide variety of taxa that and a spectrophotometer with a resolution of 2 nm or better is
differ in their resistance to extraction, it is necessary to disrupt required to obtain accurate results. If instruments of lower
the cells routinely to ensure maximum recovery of the chloro- resolution are employed, the concentration of chlorophyll a
phylls. Failure to do so may result in a systematic underesti- may be significantly underestimated depending on the band
mation of 10% or more in the chlorophyll content of the width. At a spectral band width of 20 nm, the error in the
samples. (1-3) estimate of the chlorophyll a concentration may be as large as
40%.
6.2 Grinders—The cells of many common coccoid green
algae resist disruption by most methods, but usually yield their 6.7 Fluorometer Filters—In the fluorometric practice, inter-
ferences from light emitted by chlorophyll b and chlorophyll c
pigments after maceration with a tissue grinder. The routine
use of grinders, therefore, is recommended. Glass-to-glass are greatly reduced by the use of a sharp cut-off red filter that
blocks all light with a wavelength of less than 650 nm (6).
grinders are more rigorous in disrupting cells in plankton
concentrated by centrifugation, and in periphyton scrapings,
6.8 Light Sensitivity of Extracts—Chlorophyll solutions de-
than are TFE-fluorocarbon-to-glass grinders, and their use for
grade rapidly in strong light. Work with these solutions,
this purpose is preferred. However, TFE fluorocarbon-to-glass
therefore, should be carried out in subdued light, and all
grinders perform well with glass-fiber filters. Other cell dis-
vessels, tubes, and so forth, containing the pigment extracts
ruption methods, such as sonication, may be used if, for each
should be covered with aluminum foil or other opaque sub-
typeofsample,itisdemonstratedthatthechlorophyllrecovery
stance.
is comparable to that obtained with tissue grinders (4).
7. Apparatus
6.3 Filters—Glass-fiber filters usually provide a higher re-
covery of chlorophyll than is obtained with membrane filters 7.1 Filters—Glass-fiber filters, providing quantitative reten-
when extraction-resistant algae are present in the samples and tion of particles equal to or greater than 0.45 µm in diameter.
should be employed routinely (4).
7.2 Filtering Apparatus suitable for use with glass-fiber
filters.
6.4 Chlorophyll-Related Pigments—Naturally occurring,
structurally related chlorophyll precursors and degradation
7.3 Tissue Homogenizer—Tissue grinder consisting of a
products, such as the chlorophyllides, pheophytins, and
motor-driven pestle and enclosing glass tube (glass to glass or
pheophorbides, commonly occur in pigment extracts and may
TFE-fluorocarbon-to-glass grinder).
absorb light in the same region of the spectrum as the
7.4 Spectrophotometer suitable for use over the range from
chlorophylls.Thesecompoundsmayinterferewiththeanalysis
600 to 750 nm, with a resolution of 2 nm or better, and
by indicating falsely high chlorophyll concentrations.
equipped with sample cells having a light path of 1, 5, and 10
6.4.1 This practice includes a correction for pheophytin a
cm, with a capacity of 10 mL or less.
only. Pheophytin a is similar in structure to chlorophyll a, but
7.5 Fluorometer (Optional):
lacks the magnesium atom (Mg) in the porphyrin ring. The
7.5.1 Spectrophotofluorometer that provides an excitation
magnesium can be removed from chlorophyll in the laboratory
wavelengthof430nmanddetectionofemissionovertherange
by acidifying the extract. When a solution of pure chlorophyll
from 600 to 700 nm, or:
a is converted to pheophytin a by acidification, the absorption
7.5.2 Filter Fluorometer equipped with a blue light source
peakisreducedtoapproximately60%ofitsoriginalvalueand
6 4
and blue excitation filter and a sharp cut off filter.
shifts from 664 to 665 nm, resulting in a before:after acidifi-
cation absorption peak ratio (OD664/OD665) of 1.70. This
7.6 Centrifuge that can provide a centrifugal force of 1000
phenomenon is utilized in correcting the apparent concentra-
g; head with swing-out buckets preferred.
tion of chlorophyll a for the presence of pheophytin a.
7.7 Centrifuge Tubes, screw-cap or stoppered, conical,
Unwanted degradation of chlorophyll to pheophytin in the
graduated, 15-mL. Avoid cap liners soluble in acetone and
phytoplankton on filters, or in periphyton samples, or in the
neoprene rubber stoppers.
acetone extract, by the occurrence of acidic conditions can be
prevented by the addition of a magnesium carbonate suspen-
8. Reagents and Materials
siontotheplanktonsamplebeforefilteringortotheperiphyton
8.1 Aqueous Acetone, 90 %—Add 1 volume of distilled or
samples before grinding, and by adding a small amount of a
deionized water (see Specification D1193) to 9 volumes of
sodium bicarbonate solution to the aqueous acetone when it is
reagent grade acetone.Add 5 drops of 1 N sodium bicarbonate
prepared. Addition of magnesium carbonate may also aid in
clarifying the samples following steeping (5).
Corning CS-2-64 filter or its equivalent, has been found suitable for this
6.5 Turbidity—The optical density of the extract is mea-
purpose.AvailablefromCorningGlassWorks,388BeartownRd.,PaintedPost,NY
sured at 750 nm to correct for turbidity.
14870.
Kontes type C, glass-to-glass grinder or its equivalent, has been found suitable
for this purpose. Available from Kontes Manufacturing Co., Spruce St., Vineland,
NJ 08360.
3 6
The boldface numbers in parentheses refer to a list of references at the end of Corning CS-5-60 filter has been found satisfactory. Equivalent filters may be
this standard. used.
D3731 − 20
solutionperlitre.(Warning—Thevolume:volumerelationship extract by centrifuging 20 min at 1000 g, or by filtering. If the
between the acetone and water must be strictly followed to clarified extract is not analyzed immediately, store in the dark
prevent shifts in the absorption peaks.) at−20°C in a tightly stoppered tube.
8.2 Hydrochloric Acid (1 N)—Add one volume of concen- 10.4 After clarification, decant the extract directly into a
tratedhydrochloricacid(HCl,spgr1.19)toelevenvolumesof cuvetteorascrew-caporstopperedtube.Iftheanalysiscannot
distilled or deionized water (see Specificat
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D3731 − 87 (Reapproved 2012) D3731 − 20
Standard Practices for Measurement of
Chlorophyll Content of Algae in Surface Waters
This standard is issued under the fixed designation D3731; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 These practices include the extraction and the measurement of chlorophyll a, b, and c, and pheophytin a in freshwater and
marine plankton and periphyton. Three practices are provided as follows:
1.1.1 Spectrophotometric, trichromatic practice for measuring chlorophyll a, b, and c.
1.1.2 Spectrophotometric, monochromatic practice for measuring chlorophyll a corrected for pheophytin a; and for measuring
pheophytin a.
1.1.3 Fluorometric practice for measuring chlorophyll a corrected for pheophytin a; and for measuring pheophytin a.
1.2 The values stated in SI units are to be regarded as the standard. The values given in parentheses are provided for information
purposes only.standard. No other units of measurement are included in this standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use. Specific precautionary statements are given in Section 78.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
3.2 Definitions:Definitions of Terms Specific to This Standard:
3.2.1 chlorophyll, n—a class of pigments involved in photosynthesis.
3.2.2 periphyton, n—microorganisms growing on submerged objects, commonly consisting of bacteria, algae, protozoa, and
rotifers.
3.2.3 pheopigments (phaeopigments), n—a non-photosynthetic pigment which is the degradation product of algal chlorophyll
pigments.
3.2.4 plankton—plankton, n—nonmotile or weakly swimming organisms, usually microscopic, that drift or are carried along by
currents in surface waters, commonly consisting of bacteria, algae, protozoa, rotifers, and microcrustacea.
2.1.2 periphyton—microorganisms growing on submerged objects, commonly consisting of bacteria, algae, protozoa, and
rotifers.
4. Summary of Practices
4.1 The chlorophyll and related compounds are extracted from the algae with 90 % aqueous acetone. The concentration of the
pigments is determined by measuring the light absorption or fluorescence of the extract.
These practices are under the jurisdiction of ASTM Committee D19 on Water and are the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Sept. 1, 2012June 1, 2020. Published October 2012June 2020. Originally approved in 1979. Last previous edition approved in 20042012 as
D3731 – 87 (2004).(2012). DOI: 10.1520/D3731-87R12.10.1520/D3731-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D3731 − 20
5. Significance and Use
5.1 Data on the chlorophyll content of the algae have the following applications:
5.1.1 To provide estimates of algal biomass and productivity.
5.1.2 To provide general information on the taxonomic composition (major groups) of the algae, based on the relative amounts
of chlorophyll a, b, and c, and the physiological condition of algal communities, which is related to the relative abundance of
pheopigments.
5.1.3 To determine long-term trends in water quality.
5.1.4 To determine the trophic status of surface waters.
5.1.5 To detect adverse effects of pollutants on plankton and periphyton in receiving waters.
5.1.6 To determine maximum growth rates and yields in algal growth potential tests.
6. Interferences and Special Considerations
6.1 Pigment Extraction—The chlorophylls are only poorly extracted, if at all, from some forms of algae, such as the coccoid
green algae, unless the cells are disrupted, whereas other algae, such as the diatoms, give up their pigments very readily when
merely steeped in acetone. Since natural communities of algae usually consist of a wide variety of taxa that differ in their resistance
to extraction, it is necessary to disrupt the cells routinely to ensure maximum recovery of the chlorophylls. Failure to do so may
result in a systematic underestimation of 10 % or more in the chlorophyll content of the samples. (11-3, 2, 3)
6.2 Grinders—The cells of many common coccoid green algae resist disruption by most methods, but usually yield their
pigments after maceration with a tissue grinder. The routine use of grinders, therefore, is recommended. Glass-to-glass grinders
are more rigorous in disrupting cells in plankton concentrated by centrifugation, and in periphyton scrapings, than are
TFE-fluorocarbon-to-glass grinders, and their use for this purpose is preferred. However, TFE fluorocarbon-to-glass grinders
perform well with glass-fiber filters. Other cell disruption methods, such as sonication, may be used if, for each type of sample,
it is demonstrated that the chlorophyll recovery is comparable to that obtained with tissue grinders (4).
6.3 Filters—Glass-fiber filters usually provide a higher recovery of chlorophyll than is obtained with membrane filters when
extraction-resistant algae are present in the samples,samples and should be employed routinely (4).
6.4 Chlorophyll-Related Pigments—Naturally occurring, structurally related chlorophyll precursors and degradation products,
such as the chlorophyllides, pheophytins, and pheophorbides, commonly occur in pigment extracts and may absorb light in the
same region of the spectrum as the chlorophylls. These compounds may interfere with the analysis by indicating falsely high
chlorophyll concentrations.
6.4.1 This practice includes a correction for pheophytin a only. Pheophytin a is similar in structure to chlorophyll a, but lacks
the magnesium atom (Mg) in the porphyrin ring. The magnesium can be removed from chlorophyll in the laboratory by acidifying
the extract. When a solution of pure chlorophyll a is converted to pheophytin a by acidification, the absorption peak is reduced
to approximately 60 % of its original value and shifts from 664 to 665 nm, resulting in a before:after acidification absorption peak
ratio (OD664/OD665) of 1.70. This phenomenon is utilized in correcting the apparent concentration of chlorophyll a for the
presence of pheophytin a. Unwanted degradation of chlorophyll to pheophytin in the phytoplankton on filters, or in periphyton
samples, or in the acetone extract, by the occurrence of acidic conditions can be prevented by the addition of a magnesium
carbonate suspension to the plankton sample before filtering or to the periphyton samples before grinding, and by adding a small
amount of a sodium bicarbonate solution to the aqueous acetone when it is prepared. Addition of magnesium carbonate may also
aid in clarifying the samples following steeping (5).
6.5 Turbidity—The optical density of the extract is measured at 750 nm to correct for turbidity.
6.6 Spectrophotometer Resolution—The absorption peak of acetone solutions of chlorophyll extracts is relatively narrow, and
a spectrophotometer with a resolution of 2 nm or better is required to obtain accurate results. If instruments of lower resolution
are employed, the concentration of chlorophyll a may be significantly underestimated depending on the band width. At a spectral
band width of 20 nm, the error in the estimate of the chlorophyll a concentration may be as large as 40 %.
6.7 Fluorometer Filters—In the fluorometric practice, interferences from light emitted by chlorophyll b and chlorophyll c are
greatly reduced by the use of a sharp cut-off red filter that blocks all light with a wavelength of less than 650 nm (6).
6.8 Light Sensitivity of Extracts—Chlorophyll solutions degrade rapidly in strong light. Work with these solutions, therefore,
should be carried out in subdued light, and all vessels, tubes, and so forth, containing the pigment extracts should be covered with
aluminum foil or other opaque substance.
7. Apparatus
7.1 Filters, Filters—Glass-fiber filters, providing quantitative retention of particles equal to or greater than 0.45 μm in diameter.
7.2 Filtering Apparatus suitable for use with glass-fiber filters.
The boldface numbers in parentheses refer to thea list of references at the end of this standard.
Corning CS-2-64 filter or its equivalent, has been found suitable for this purpose. Available from Corning Glass Works, 388 Beartown Rd., Painted Post, NY 14870.
D3731 − 20
7.3 Tissue Homogenizer—Tissue grinder consisting of a motor-driven pestle and enclosing glass tube (glass to glass or
TFE-fluorocarbon-to-glass grinder).
7.4 Spectrophotometer suitable for use over the range from 600 to 750 nm, with a resolution of 2 nm or better, and equipped
with sample cells having a light path of 1, 5, and 10 cm, with a capacity of 10 mL or less.
7.5 Fluorometer (Optional):
7.5.1 Spectrophotofluorometer that provides an excitation wavelength of 430 nm and detection of emission over the range from
600 to 700 nm, or:
6 4
7.5.2 Filter Fluorometer equipped with a blue light source and blue excitation filter and a sharp cut off filterfilter.
7.6 Centrifuge that can provide a centrifugal force of 1000 g; head with swing-out buckets preferred.
7.7 Centrifuge Tubes, screw-cap or stoppered, conical, graduated, 15-mL. Avoid cap liners soluble in acetone and neoprene
rubber stoppers.
8. Reagents and Materials
8.1 Aqueous Acetone, 90 %—Add 1 volume of distilled water or deionized water (see Specification D1193) to 9 volumes of
reagent grade acetone. Add 5 drops of 1 N sodium bicarbonate solution per litre. (Warning—The volume:volume relationship
between the acetone and water must be strictly followed to prevent shifts in the absorption peaks.Caution—)the volume:volume
relationship between the acetone and water must be strictly followed to prevent shifts in the absorption peaks.)
8.2 Hydrochloric Acid (1 N)—Add one volume of concentrated hydrochloric acid (HCl, sp gr 1.19) to eleven volumes of
distilled water.or deionized water (see Specification D1193).
8.3 Magnesium Carbonate Suspension—Add 1 g of finely powdered magnesium carbonate to 100 mL of distilled water or
deionized water (see Specification D1193) in a stoppered Erlenmeyer flask. Shake immediately before use.
8.4 Sodium Bicarbonate Solution (1 N)—Prepare by dissolving 8.4 g of sodium bicarbonate in 100 mL of distilled water. or
deionized water (see Specification D1193).
Kontes type C, glass-to-glass gri
...