ASTM E579-04(2023)
(Test Method)Standard Test Method for Limit of Detection of Fluorescence of Quinine Sulfate in Solution
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:
= 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 o...
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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: E579 − 04 (Reapproved 2023)
Standard Test Method for
Limit of Detection of Fluorescence of Quinine Sulfate in
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, health, and environmental practices and deter-
rigorous test of performance of instrumentation, or (2), to
mine the applicability of regulatory limitations prior to use.
intercompare the quantitative performance of instruments of
1.7 This international standard was developed in accor-
different design. Intercomparison of the LOD between instru-
dance with internationally recognized principles on standard-
ments is commonly expressed as the ratio of the water Raman
ization established in the Decision on Principles for the
peakintensitytotheroot-mean-square(rms)noiseasmeasured
Development of International Standards, Guides and Recom-
on a fluorometer using an excitation wavelength of 350 nm
mendations issued by the World Trade Organization Technical
This test method uses the excitation and emission peak
Barriers to Trade (TBT) Committee.
wavelengths for quinine sulfate dihydrate in solution, which
are approximately 350 nm and 450 nm, respectively.
2. Referenced Documents
1.2 This test method has been applied to fluorescence-
2.1 ASTM Standard:
measuring systems utilizing non-laser, low-energy excitation
E578 Test Method for Linearity of Fluorescence Measuring
sources. There is no assurance that extremely intense illumi-
Systems
nation will not cause photodecomposition of the compound
suggested in this test method. For this reason, it is recom- 3. Summary of Test Method
mended that this test method not be indiscriminately employed
3.1 To measure the concentration corresponding to the
with high intensity light sources. This test method is not
LOD, the fluorescence intensity scale and gain on the detector
intended to determine minimum detectable amounts of other
are adjusted such that noise observed with pure solvent in the
materials. If this test method is extended to employ other
samplecellislargeenoughtomeasure.Thetestsolutionisthen
chemical substances, the user should be aware of the possibil-
diluted until readings on both the test solution and pure solvent
ity that these other substances may undergo decomposition or
canbereadatthesameintensity,scale,andinstrumentsettings.
adsorption onto containers.
The concentration corresponding to the limit of detection is
thatatwhichthenoiseintensity,multipliedbythree,isequalto
1.3 A typical LOD for conventional fluorometers using this
the signal intensity.
test method is 1 ng of quinine sulfate per mL.
3.2 This test for limit of detection requires an instrument to
meet the following conditions: stable, free of extraneous noise,
electrical pickup, and internal stray light. The sample space
This test method is under the jurisdiction of ASTM Committee E13 on
must be covered to exclude room light. The instrument should
Molecular Spectroscopy and Separation Science and is the direct responsibility of
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Jan. 15, 2023. Published January 2023. Originally
published in 1976. Last previous edition approved in 2015 as E579 – 04 (2015). For referenced ASTM standards, visit the ASTM website, www.astm.org, or
DOI: 10.1520/E0579-04R23. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E579 − 04 (2023)
6,7
noise) and dividing by a factor that is usually taken to be 5.
be operated according to the manufacturer’s recommendations,
or, if they are modified, the modifications must be applied
5. Reagents
consistently to the test for limit of detection and to the analysis
for which the test is a requirement, so that levels of perfor-
5.1 Prepare a stock solution of quinine sulfate dihydrate
mance are comparable for both. All modifications must be
(C H O N ) ·H SO ·2H O by transferring 0.100 g of high
20 22 2 2 2 2 4 2
included in the report outlined in Section 8.
purity crystalline dihydrate of quinine sulfate into a 100 mL
volumetric flask and fill the flask to volume using either 0.1
NOTE 1—To obtain the lowest reading (the best instrumental response)
mol/Lsulfuric acid or 0.1 mol/Lperchloric acid as the solvent.
for the limit of detection of fluorescent material, a number of precautions
must be taken. The quality, condition, and position of the sample cell are
This solution contains 1 mg/mL of quinine sulfate dihydrate.
most important. The cell must be made of fused silica that does not
fluoresce at the excitation wavelength and be free of scratches and marks NOTE4—Either0.1mol/Lsulfuricacidor0.1mol/Lperchloricacidcan
be used as a solvent with quinine sulfate dihydrate, but the solvent that is
that scatter light into the fluorescence detection system. Only spectral
grade chemicals and solvents (including water) that do not fluoresce chosen must also be used as the blank. Take note that the quantum yield
of quinine sulfate dihydrate in solution has been shown to be about 13 %
should be used. Dilute solutions of quinine sulfate dihydrate should be
smaller in 0.1 mol/Lsulfuric acid than in 0.1 mol/Lperchloric acid, which
made,justbeforeuse,fromconcentratedstocksolutions.Allsamplesused
will result in a corresponding increase in the concentration of quinine
must be maintained at the same temperature to obviate effects due to
sulfate dihydrate in 0.1 mol/L sulfuric acid versus that in 0.1 mol/L
temperature fluctuations. The average temperature coefficient for fluores-
perchloric acid at the LOD for a particular instrument.
cence intensity in the temperature range from 16 °C to 35 °C is
–0.62 %⁄°C at 450 nm for 1 µg/mLquinine sulfate dihydrate in 0.1 mol/L
5.2 Make serial dilutions by diluting aliquots of the stock
HClO .
solution and successive solutions to ten times their volume
with the solvent. Repeat this process until the desired concen-
4. Significance and Use
tration is obtained. The sixth successive dilution will result in
4.1 When determining the limiting detecta
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