ASTM E579-04(2015)

Standard Test Method for Limit of Detection of Fluorescence of Quinine Sulfate in Solution

SIGNIFICANCE AND USE
4.1 When determining the limiting detectable concentration of a fluorescent substance, it is usually necessary to increase the readout scale of a photoelectric instrument to a point where noise (that is, random fluctuations of the system) becomes apparent. This noise will be superimposed upon the signal from the sample.  
4.2 In molecular fluorescence spectroscopy, the limit of detection for the sample will be determined by the limiting signal-to-noise ratio, S/N, where the signal, S, is the difference between readings obtained with the sample and blank solutions, and N is the total root-mean-square (rms) noise. The limit of detection for the sample will be given by the instrument readings that give a signal equal to three times the rms value of the noise.
Note 2: Factors other than noise affecting the sample concentration corresponding to the limit of detection include: the spectral bandwidths of the excitation and emission monochromators, the intensity of the exciting light that can be concentrated on the sample, the fraction of the fluorescence collected by the detection system, the response time of the detection system, and the purity of the solvent. The size and arrangement of the sample container with respect to the light beams are also important, as they affect both the desired signal and the extraneous signal that only contributes noise.
Note 3: The value of rms noise (N) can be obtained by calculating the standard deviation of a series of readings of the signal from the sample at the peak emission wavelength at approximately 450 nm as follows:
   where:  
   x  =  mean of the series of readings,   x  =  value of the individual reading, and   n  =  number of readings.   Alternatively, rms noise may be estimated by noting the extreme differences between the members of a series of readings (peak-to-peak noise) and dividing by a factor that is usually taken to be 5.6, 7
SCOPE
1.1 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10 nm at or near room temperature.  
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate dihydrate in solution, which are approximately 350 nm and 450 nm, respectively.  
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources. There is no assurance that extremely intense illumination will not cause photodecomposition2 of the compound suggested in this test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo decomposition or adsorption onto containers.  
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.  
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark in a stoppered glass bottle.  
1.5 The values stated in SI units are to be regarded as standard. No...

General Information

• Status: Historical
• Publication Date: 30-Apr-2015
• ICS: 71.080.30 - Organic nitrogen compounds
• Technical Committee: E13 - Molecular Spectroscopy and Separation Science
• Drafting Committee: E13.01 - Ultra-Violet, Visible, and Luminescence Spectroscopy

Standards Content (Sample)

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: E579 − 04 (Reapproved 2015)
Standard Test Method for
Limit of Detection of Fluorescence of Quinine Sulfate in
1
Solution
This standard is issued under the fixed designation E579; 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 1.4 The suggested shelf life of a 1 mg/mL stock solution of
quinine sulfate dihydrate is three months, when stored in the
1.1 This test method employs the signal-to-noise ratio to
dark in a stoppered glass bottle.
determinethesensitivityofafluorescencemeasuringsystemin
1.5 The values stated in SI units are to be regarded as
testing for the limit of detection (LOD) of quinine sulfate
standard. No other units of measurement are included in this
dihydrate in solution. The results obtained with quinine sulfate
standard.
dihydrate in solution are suitable for specifying instrument
performance on samples having excitation and fluorescence 1.6 This standard does not purport to address all of the
safety problems, if any, associated with its use. It is the
bands wider than 10 nm at or near room temperature.
responsibility of the user of this standard to establish appro-
1.1.1 This test method is not intended to be used as (1)a
priate safety and health practices and determine the applica-
rigorous test of performance of instrumentation, or (2), to
bility of regulatory limitations prior to use.
intercompare the quantitative performance of instruments of
different design. Intercomparison of the LOD between instru-
2. Referenced Documents
ments is commonly expressed as the ratio of the water Raman
3
2.1 ASTM Standards:
peakintensitytotheroot-mean-square(rms)noiseasmeasured
E578 Test Method for Linearity of Fluorescence Measuring
on a fluorometer using an excitation wavelength of 350 nm
Systems
This test method uses the excitation and emission peak
wavelengths for quinine sulfate dihydrate in solution, which
3. Summary of Test Method
are approximately 350 nm and 450 nm, respectively.
3.1 To measure the concentration corresponding to the
1.2 This test method has been applied to fluorescence-
LOD, the fluorescence intensity scale and gain on the detector
measuring systems utilizing non-laser, low-energy excitation
are adjusted such that noise observed with pure solvent in the
sources. There is no assurance that extremely intense illumi-
samplecellislargeenoughtomeasure.Thetestsolutionisthen
2
nation will not cause photodecomposition of the compound
diluted until readings on both the test solution and pure solvent
suggested in this test method. For this reason, it is recom-
canbereadatthesameintensity,scale,andinstrumentsettings.
mended that this test method not be indiscriminately employed
The concentration corresponding to the limit of detection is
with high intensity light sources. This test method is not
thatatwhichthenoiseintensity,multipliedbythree,isequalto
intended to determine minimum detectable amounts of other
the signal intensity.
materials. If this test method is extended to employ other
3.2 This test for limit of detection requires an instrument to
chemical substances, the user should be aware of the possibil-
meet the following conditions: stable, free of extraneous noise,
ity that these other substances may undergo decomposition or
electrical pickup, and internal stray light. The sample space
adsorption onto containers.
must be covered to exclude room light. The instrument should
1.3 A typical LOD for conventional fluorometers using this be operated according to the manufacturer’s recommendations,
test method is 1 ng of quinine sulfate per mL.
or, if they are modified, the modifications must be applied
consistently to the test for limit of detection and to the analysis
for which the test is a requirement, so that levels of perfor-
mance are comparable for both. All modifications must be
1
This test method is under the jurisdiction of ASTM Committee E13 on
included in the report outlined in Section 8.
Molecular Spectroscopy and Separation Science and is the direct responsibility of
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved May 1, 2015. Published June 2015. Originally
3
published in 1976. Last previous edition approved in 2009 as E579 – 04 (2009). For referenced ASTM standards, visit the ASTM website, www.astm.org, or
DOI: 10.1520/E0579-04R15. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
2
Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Chemistry, ANCHA, Vol Standards volume information, refer to the standard’s Document Summary page on
45, 1973, p. 511. the ASTM website.
...

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: E579 − 04 (Reapproved 2009) E579 − 04 (Reapproved 2015)
Standard Test Method for
Limit of Detection of Fluorescence of Quinine Sulfate in
1
Solution
This standard is issued under the fixed designation E579; 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 This test method employs the signal-to-noise ratio to determine the sensitivity of a fluorescence measuring system in testing
for the limit of detection (LOD) of quinine sulfate dihydrate in solution. The results obtained with quinine sulfate dihydrate in
solution are suitable for specifying instrument performance on samples having excitation and fluorescence bands wider than 10
nm at or near room temperature.
1.1.1 This test method is not intended to be used as (1) a rigorous test of performance of instrumentation, or (2), to intercompare
the quantitative performance of instruments of different design. Intercomparison of the LOD between instruments is commonly
expressed as the ratio of the water Raman peak intensity to the root-mean-square (rms) noise as measured on a fluorometer using
an excitation wavelength of 350 nm This test method uses the excitation and emission peak wavelengths for quinine sulfate
dihydrate in solution, which are approximately 350 nm and 450 nm, respectively.respectively.
1.2 This test method has been applied to fluorescence-measuring systems utilizing non-laser, low-energy excitation sources.
2
There is no assurance that extremely intense illumination will not cause photodecomposition of the compound suggested in this
test method. For this reason, it is recommended that this test method not be indiscriminately employed with high intensity light
sources. This test method is not intended to determine minimum detectable amounts of other materials. If this test method is
extended to employ other chemical substances, the user should be aware of the possibility that these other substances may undergo
decomposition or adsorption onto containers.
1.3 A typical LOD for conventional fluorometers using this test method is 1 ng of quinine sulfate per mL.
1.4 The suggested shelf life of a 1 mg/mL stock solution of quinine sulfate dihydrate is three months, when stored in the dark
in a stoppered glass bottle.
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 problems, 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.
2. Referenced Documents
3
2.1 ASTM Standards:
E578 Test Method for Linearity of Fluorescence Measuring Systems
3. Summary of Test Method
3.1 To measure the concentration corresponding to the LOD, the fluorescence intensity scale and gain on the detector are
adjusted such that noise observed with pure solvent in the sample cell is large enough to measure. The test solution is then diluted
until readings on both the test solution and pure solvent can be read at the same intensity, scale, and instrument settings. The
concentration corresponding to the limit of detection is that at which the noise intensity, multiplied by three, is equal to the signal
intensity.
1
This test method is under the jurisdiction of ASTM Committee E13 on Molecular Spectroscopy and Separation Science and is the direct responsibility of Subcommittee
E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Oct. 1, 2009May 1, 2015. Published December 2009June 2015. Originally published in 1976. Last previous edition approved in 20042009 as
E579 – 04.E579 – 04 (2009). DOI: 10.1520/E0579-04R09.10.1520/E0579-04R15.
2
Lukasiewicz, R. J., and Fitzgerald, J. M., Analytical Chemistry, ANCHA, Vol 45, 1973, p. 511.
3
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
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