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ASTM E578-07(2013)

(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 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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

Status
Historical
Publication Date
30-Apr-2013
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(2013) - Standard Test Method for Linearity of Fluorescence Measuring SystemsASTM E578-07(2013) - Standard Test Method for Linearity of Fluorescence Measuring Systems
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ASTM E578-07(2013) - Standard Test Method for Linearity of Fluorescence Measuring Systems
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E578 − 07 (Reapproved 2013)
Standard Test Method for
1
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 10-mm pathlength
cuvette formats and instruments covering a wavelength range
1.1 This test method covers a procedure for evaluating the
within 190 to 900 nm.The use of other sample formats has not
limitsofthelinearityofresponsewithfluorescenceintensityof
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 and health practices and determine the applica-
electronics, excessive absorption of either the exciting or
bility of regulatory limitations prior to use.
emitted radiation, or both, and the sample handling technique,
2. Summary of Test Method
particularly at low concentrations.
2.1 This procedure is used for testing the linearity of
1.2 This test method has been applied to fluorescence-
fluorescence-measuring systems by using solutions of quinine
measuring systems utilizing continuous and low-energy exci-
sulfate dihydrate in sulfuric acid as standard test solutions.
tation sources (for example, an excitation source of 450-W
Other stable solutions which may be more suitable to the user
electrical input or less). There is no assurance that extremely
can be employed (Note 1). The standard used to determine
intense illumination will not cause photodecomposition of the
2
linearity should be stated in the report.The fluorescence of the
compounds suggested in this test method. For this reason it is
test solution is measured in the measuring system with the
recommended that this test method not be indiscriminately
cuvettes, slits, or filters that are to be employed in projected
employed with high-intensity light sources. It is not a test
use.
method to determine the linearity of response of other materi-
als. If this test method is extended to employ other chemical
NOTE1—Asubstitutestandardshouldhavethefollowingproperties:(1)
substances, the principles within can be applied, but new Itshouldhavealargequantumyieldatveryhighdilution;(2)itshouldbe
stable to the exciting radiation during spectral measurements; (3) its
material parameters, such as the concentration range of
fluorescence and its absorption spectra overlap should be small; (4) its
linearity, must be established. The user should be aware of the
quantum yield should not be strongly concentration dependent; and (5)it
possibility that these other substances may undergo
should have a broad emission spectrum, so that little error is introduced
3
decomposition, or adsorption onto containers.
when wide slits are used.
2.2 Upper Limit of Linearity—The fluorescence intensity of
1.3 This test method has been applied to fluorescence-
a series of standard solutions is measured, the resultant
measuring systems utilizing a single detector, that is, a photo-
instrument readings are plotted against concentration on a
multiplier tube or a single photodiode. It has not been demon-
log-log graph, and a smooth curve is drawn through the data
strated if this method is effective for photo-array instruments
such as those using a CCD or a diode array detector. points.The point (concentration) at which the upper end of the
curvedeviatesbymorethan5%ofthesignalfromthestraight
line (defined by the center region of the curve) is taken as the
upper limit of linearity. The limit is expressed in micrograms
1
This test method is under the jurisdiction of ASTM Committee E13 on
per millilitre of quinine sulfate dihydrate.
Molecular Spectroscopy and Separation Science and is the direct responsibility of
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
NOTE 2—Absorption of the exciting radiation at high solute concentra-
Current
...

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4.1 These practices should be used by a person who develops an analytical method to ensure that the spectral bandwidths cited in the practice are actually the ones used.
Note 2: The method developer should establish the spectral bandwidths that can be used to obtain satisfactory results.  
4.2 These practices should be used to determine whether a spectral bandwidth specified in a method can be realized with a given spectrophotometer and thus whether the instrument is suitable for use in this application. If accurate absorbance measurements are to be made on compounds with sharp absorption bands (natural half band widths of less than 15 nm) the spectral bandwidth of the spectrometer used should be better than 1/8th of the natural half band width of the compound’s absorption.  
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