ASTM E578-07(2021)

Standard Test Method for Linearity of Fluorescence Measuring Systems

SIGNIFICANCE AND USE
3.1 The range of concentration of a fluorescing substance in solution over which the fluorescence varies linearly with the concentration is the range most useful for quantitative analysis. This range is affected by properties of the solution under analysis and by features of the measuring system. This test method provides a means of testing the performance of a fluorescence measuring system and of determining the concentration range over which the system is suitable for making a given quantitative analysis.  
3.2 This test method is not meant for comparing the performance of different fluorescence measuring instruments.
SCOPE
1.1 This test method covers a procedure for evaluating the limits of the linearity of response with fluorescence intensity of fluorescence-measuring systems under operating conditions. Particular attention is given to slit widths, filters, and sample containers. This test method can be used to test the overall linearity under a wide variety of instrumental and sampling conditions. The results obtained apply only to the tested combination of slit width and filters, and the size, type and illumination of the sample cuvette, all of which must be stated in the report. The sources of nonlinearity may be the measuring electronics, excessive absorption of either the exciting or emitted radiation, or both, and the sample handling technique, particularly at low concentrations.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing continuous and low-energy excitation sources (for example, an excitation source of 450 W electrical input or less). There is no assurance that extremely intense illumination will not cause photodecomposition of the compounds suggested in this test method.2 For this reason it is recommended that this test method not be indiscriminately employed with high-intensity light sources. It is not a test method to determine the linearity of response of other materials. If this test method is extended to employ other chemical substances, the principles within can be applied, but new material parameters, such as the concentration range of linearity, must be established. The user should be aware of the possibility that these other substances may undergo decomposition, or adsorption onto containers.  
1.3 This test method has been applied to fluorescence-measuring systems utilizing a single detector, that is, a photomultiplier tube or a single photodiode. It has not been demonstrated if this method is effective for photo-array instruments such as those using a CCD or a diode array detector.  
1.4 This test method is applicable to 10 mm pathlength cuvette formats and instruments covering a wavelength range within 190 nm to 900 nm. The use of other sample formats has not been established with this test method.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 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.  
1.7 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-Mar-2021
• ICS: 17.180.30 - Optical measuring instruments
• Technical Committee: E13 - Molecular Spectroscopy and Separation Science
• Drafting Committee: E13.01 - Ultra-Violet, Visible, and Luminescence Spectroscopy

Overview

ASTM E578-07(2021), Standard Test Method for Linearity of Fluorescence Measuring Systems, establishes a standardized procedure for assessing the linearity of fluorescence measuring instruments. The linearity range-the span of analyte concentration over which fluorescence response is directly proportional to concentration-is a critical performance characteristic for quantitative analysis in molecular spectroscopy. This standard focuses on evaluating the limits of linear response within defined instrumental and operating conditions, addressing key system parameters such as slit widths, filters, sample containers, and excitation source intensity.

This test method is primarily applicable to fluorescence measuring systems using single-detector setups (such as photomultiplier tubes or photodiodes) and 10 mm pathlength cuvette formats that operate in the 190 nm to 900 nm wavelength range. The outcomes of this test are essential for ensuring confidence in quantitative fluorescence measurements and verifying instrument suitability for specific applications.

Key Topics

• Linearity Assessment: Provides a method to determine the upper and lower concentration limits where fluorescence response remains linear, crucial for accurate quantitative measurements.
• Instrument Configuration Impact: Results are specific to the instrument configuration used in the test, including slit width, filter selection, sample cuvette size, and illumination conditions.
• Sources of Nonlinearity: Identifies potential causes such as electronic limitations, excessive absorption of excitation or emission light, and sample handling practices, particularly at low analyte concentrations.
• Scope and Applicability:
  • Designed for single-channel detectors (not validated for array detectors like CCDs).
  • Applicable only to solution samples in 10 mm cuvettes and those operating under continuous, low-energy excitation sources.
  • Not intended for direct comparison between different instruments or different chemical analytes without recalibrating linearity parameters.
• Safety and Compliance: Highlights user responsibility for adhering to safety, health, and regulatory requirements during method application.

Applications

The ASTM E578-07(2021) standard method is widely used in laboratory settings where fluorescence spectrometers are key analytical instruments. Typical applications include:

• Quantitative Chemical Analysis: Ensuring accurate determination of analyte concentration in solution by validating the instrument’s linear response range.
• Instrument Calibration and Verification: Assessing the performance and suitability of fluorescence spectrometers for specific analytical tasks.
• Method Validation in Molecular Spectroscopy: Supporting the development and validation of new analytical methods and protocols where fluorescence detection is used.
• Quality Control: Implementing standard checks for system performance to maintain data integrity in research, quality assurance, and regulatory compliance environments.

This standard is particularly valuable in chemical, pharmaceutical, environmental, and life science laboratories where reliable measurement of fluorescence intensity is fundamental.

Related Standards

Professionals working with ASTM E578-07(2021) may also consider the following related standards to broaden their compliance and understanding of molecular spectroscopy procedures:

• ASTM E169 – General Chemistry Test Methods
• ASTM E275 – Practice for Describing and Measuring Performance of Ultraviolet, Visible, and Near-Infrared Spectrophotometers
• ASTM E2877 – Guide for Molecular Luminescence Laboratory Procedures
• ISO 17025 – General requirements for the competence of testing and calibration laboratories

Understanding and implementing these related standards alongside ASTM E578-07(2021) ensures comprehensive coverage of fluorescence spectroscopy measurement practices and instrumental performance assessment.

Keywords: fluorescence spectrometers, linearity, molecular spectroscopy, ASTM E578-07, quantitative analysis, instrument calibration, laboratory standards, emission intensity, UV-Vis spectroscopy, cuvette testing

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: E578 − 07 (Reapproved 2021)
Standard Test Method for
Linearity of Fluorescence Measuring Systems
This standard is issued under the fixed designation E578; 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 1.4 This test method is applicable to 10mm pathlength
cuvette formats and instruments covering a wavelength range
1.1 This test method covers a procedure for evaluating the
within 190nm to 900 nm.The use of other sample formats has
limitsofthelinearityofresponsewithfluorescenceintensityof
not been established with this test method.
fluorescence-measuring systems under operating conditions.
1.5 The values stated in SI units are to be regarded as
Particular attention is given to slit widths, filters, and sample
standard. No other units of measurement are included in this
containers. This test method can be used to test the overall
standard.
linearity under a wide variety of instrumental and sampling
conditions. The results obtained apply only to the tested
1.6 This standard does not purport to address all of the
combination of slit width and filters, and the size, type and
safety concerns, if any, associated with its use. It is the
illumination of the sample cuvette, all of which must be stated
responsibility of the user of this standard to establish appro-
inthereport.Thesourcesofnonlinearitymaybethemeasuring
priate safety, health, and environmental practices and deter-
electronics, excessive absorption of either the exciting or
mine the applicability of regulatory limitations prior to use.
emitted radiation, or both, and the sample handling technique,
1.7 This international standard was developed in accor-
particularly at low concentrations.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
1.2 This test method has been applied to fluorescence-
Development of International Standards, Guides and Recom-
measuring systems utilizing continuous and low-energy exci-
mendations issued by the World Trade Organization Technical
tation sources (for example, an excitation source of 450W
Barriers to Trade (TBT) Committee.
electrical input or less). There is no assurance that extremely
intense illumination will not cause photodecomposition of the
2. Summary of Test Method
compounds suggested in this test method. For this reason it is
2.1 This procedure is used for testing the linearity of
recommended that this test method not be indiscriminately
fluorescence-measuring systems by using solutions of quinine
employed with high-intensity light sources. It is not a test
sulfate dihydrate in sulfuric acid as standard test solutions.
method to determine the linearity of response of other materi-
Other stable solutions which may be more suitable to the user
als. If this test method is extended to employ other chemical
can be employed (Note 1). The standard used to determine
substances, the principles within can be applied, but new
linearity should be stated in the report.The fluorescence of the
material parameters, such as the concentration range of
test solution is measured in the measuring system with the
linearity, must be established. The user should be aware of the
cuvettes, slits, or filters that are to be employed in projected
possibility that these other substances may undergo
use.
decomposition, or adsorption onto containers.
NOTE1—Asubstitutestandardshouldhavethefollowingproperties:(1)
1.3 This test method has been applied to fluorescence-
Itshouldhavealargequantumyieldatveryhighdilution;(2)itshouldbe
measuring systems utilizing a single detector, that is, a photo-
stable to the exciting radiation during spectral measurements; (3) its
multiplier tube or a single photodiode. It has not been demon-
fluorescence and its absorption spectra overlap should be small; (4) its
strated if this method is effective for photo-array instruments
quantum yield should not be strongly concentration dependent; and (5)it
such as those using a CCD or a diode array detector. should have a broad emission spectrum, so that little error is introduced
when wide slits are used.
2.2 Upper Limit of Linearity—The fluorescence intensity of
a series of standard solutions is measured, the resultant
This test method is under the jurisdiction of ASTM Committee E13 on
instrument readings are plotted against concentration on a
Molecular Spectroscopy and Separation Science and is the direct responsibility of
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
log-log graph, and a smooth curve is drawn through the data
Current edition approved April 1, 2021. Published April 2021. Originally
points.The point (concentration) at which the upper end of the
approved in 1976. Last previous edition approved in 2013 as E578–07 (2013).
DOI: 10.1520/E0578-07R21.
Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Chemistry, ANCHA, Vol
45, 1973, p. 511. Gill, J. E., Photochemistry and Photobiology, PHCBA, Vol 9, 1969, p. 313.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E578 − 07 (2021)
curvedeviatesbymorethan5%ofthesignalfromthestraight 6.4 Rinse the cuvette at least three times and fill with the
line (defined by the center region of the curve) is taken as the reagentblank(0.1Nsulfuricacid)andrecordthereadingusing
upper limit of linearity. The limit is expressed in micrograms the appropriate range setting of the instrument.
per millilitre of quinine sulfate dihydrate.
NOTE 4—When it is necessary to change the measurement settings of
theinstrument,thereadingofthereagentblankshouldalsobedetermined
NOTE 2—Absorption of the exciting radiation at high solute concentra-
using the new setting.
tions is dependent on instrument geometry and pathlength, and can result
in fluorescence signal nonlinearity.
6.5 Discard the blank solution used in 6.4, rinse the cuvette
2.3 LowerLimitofLinearity—Thelowerlimitoflinearityis at least three times with the most dilute of the solutions
takenasthepoint(concentration)atwhichthelowerendofthe
described in Section 4, fill the cuvette with this solution, and
curve deviates from the straight line defined by the central record the fluorescence intensity reading.
portion of the curve by more than twice the average percent
6.6 Discard the more dilute solution, rinse the cuvette at
deviation of the points that determine the straight line.
least three times with the next most concentrated standard
solution, fill the cuvette with this solution, and record the
3. Significance and Use
fluorescenceintensityreading.Proceedsimilarlywiththeother
3.1 The range of concentration of a fluorescing substance in 2
standard solutions, ending with the 10 µg⁄mL solution.
solution over which the fluorescence varies linearly with the
NOTE 5—The 10 µg⁄mL stock solution is not a recommended test
concentr
...