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ASTM E578-07(2021)

(Test Method)

Standard Test Method for Linearity of Fluorescence Measuring Systems

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
Current Stage
Ref Project

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ASTM E578-07(2021) - Standard Test Method for Linearity of Fluorescence Measuring SystemsASTM E578-07(2021) - Standard Test Method for Linearity of Fluorescence Measuring Systems
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ASTM E578-07(2021) - Standard Test Method for Linearity of Fluorescence Measuring Systems
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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
...

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4.3 An infrared procedure must include a description of the instrumentation and of the performance needed to duplicate the precision and accuracy of the method.
SCOPE
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SCOPE
1.1 This practice is intended to serve as a guide for the testing of the performance of a photometric detector (PD) used as the detection component of a liquid-chromatographic (LC) system operating at one or more fixed wavelengths in the range 210 nm to 800 nm. Measurements are made at 254 nm, if possible, and are optional at other wavelengths.  
1.2 This practice is intended to describe the performance of the detector both independently of the chromatographic system (static conditions) and with flowing solvent (dynamic conditions).  
1.3 For general liquid chromatographic procedures, consult Refs (1-9).2  
1.4 For general information concerning the principles, construction, operation, and evaluation of liquid-chromatography detectors, see Refs (10 and 11) in addition to the sections devoted to detectors in Refs (1-7).  
1.5 This standard does not purport to address all of the safety problems, 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.  
1.6 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.

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