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ASTM E578-83(1998)

(Test Method)

Standard Test Method for Linearity of Fluorescence Measuring Systems

Standard Test Method for Linearity of Fluorescence Measuring Systems

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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. 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 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.
1.4 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
09-Feb-2001
ICS
17.180.30 - Optical measuring instruments
Technical Committee
E13 - Molecular Spectroscopy and Separation Science
Current Stage
Ref Project

Relations

Replaced By
ASTM E578-01 - Standard Test Method for Linearity of Fluorescence Measuring Systems
Effective Date
10-Feb-2001
Referred By
ASTM E2143-01(2021) - Standard Test Method for Using Field-Portable Fiber Optics Synchronous Fluorescence Spectrometer for Quantification of Field Samples for Aromatic and Polycyclic Aromatic Hydrocarbons
Effective Date
10-Feb-2001
Referred By
ASTM E579-04(2023) - Standard Test Method for Limit of Detection of Fluorescence of Quinine Sulfate in Solution
Effective Date
10-Feb-2001
Referred By
ASTM D5412-93(2017)e1 - Standard Test Method for Quantification of Complex Polycyclic Aromatic Hydrocarbon Mixtures or Petroleum Oils in Water
Effective Date
10-Feb-2001
Referred By
ASTM E2719-09(2022) - Standard Guide for Fluorescence—Instrument Calibration and Qualification
Effective Date
10-Feb-2001
Referred By
ASTM E3029-15(2023) - Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
Effective Date
10-Feb-2001

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ASTM E578-83(1998) - Standard Test Method for Linearity of Fluorescence Measuring SystemsASTM E578-83(1998) - Standard Test Method for Linearity of Fluorescence Measuring Systems
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ASTM E578-83(1998) - 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 discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 578 – 83 (Reapproved 1998)
Standard Test Method for
Linearity of Fluorescence Measuring Systems
This standard is issued under the fixed designation E 578; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope can be employed (Note 1). The standard used to determine
linearity should be stated in the report. The fluorescence of the
1.1 This test method covers a procedure for evaluating the
test solution is measured in the measuring system with the
limits of linearity of fluorescence-measuring systems under
cuvettes, slits, or filters that are to be employed in projected
operating conditions. Particular attention is given to slit widths,
use.
filters, and sample containers. This test method can be used to
test the overall linearity under a wide variety of instrumental
NOTE 1—A substitute standard should have the following properties:
and sampling conditions. The results obtained apply only to the
(1) It should have a large quantum yield at very high dilution; (2) it should
be stable to the exciting radiation during spectral measurements; (3) its
tested combination of slit width and filters, and the size, type
fluorescence and its absorption spectra overlap should be small; (4) its
and illumination of the sample cuvette, all of which must be
quantum yield should not be strongly concentration dependent; and (5)it
stated in the report. The sources of nonlinearity may be the
should have a broad emission spectrum, so that little error is introduced
measuring electronics, excessive absorption of either the ex-
when wide slits are used.
citing or emitted radiation, or both, and the sample handling
2.2 Upper Limit of Linearity—The fluorescence intensity of
technique, particularly at low concentrations.
a series of standard solutions is measured, the resultant
1.2 This test method has been applied to fluorescence-
instrument readings are plotted against concentration on log-
measuring systems utilizing continuous and low-energy exci-
log graph paper, and a smooth curve is drawn through the data
tation sources (for example, an excitation source of 150-W
points. The point (concentration) at which the upper end of the
electrical input or less). There is no assurance that extremely
curve deviates by more than 5 % of the signal from the straight
intense illumination will not cause photodecomposition of the
2 line (defined by the center region of the curve) is taken as the
compounds suggested in this test method. For this reason it is
upper limit of linearity. The limit is expressed in micrograms
recommended that this test method not be indiscriminately
per milliliter of quinine sulfate dihydrate.
employed with high-intensity light sources. It is not a test
method to determine the linearity of response of other materi-
NOTE 2—Absorption of the exciting radiation at high solute concentra-
als. If this test method is extended to employ other chemical tions is dependent on instrument geometry, and can result in fluorescence
signal nonlinearity.
substances, the user should be aware of the possibility that
these other substances may undergo decomposition, or adsorp-
2.3 Lower Limit of Linearity—The lower limit of linearity is
tion onto containers.
taken as the point (concentration) at which the lower end of the
1.3 This standard does not purport to address all of the
curve deviates from the straight line defined by the central
safety problems, if any, associated with its use. It is the
portion of the curve by more than twice the average percent
responsibility of the user of this standard to establish appro-
deviation of the points that determine the straight line.
priate safety and health practices and determine the applica-
3. Significance and Use
bility of regulatory limitations prior to use.
3.1 The range of concentration of a fluorescing substance in
2. Summary of Test Method
solution over which the fluorescence varies linearly with the
2.1 This procedure is used for testing the linearity of
concentration is the range most useful for quantitative analysis.
fluorescence-measuring systems by using solutions of quinine
This range is affected by properties of the solution under
sulfate dihydrate in sulfuric acid as standard test solutions.
analysis and by features of the measuring system. This test
Other stable solutions which may be more suitable to the user
method provides a means of testing the performance of a
fluorescence measuring system and of determining the concen-
tration range over which the system is suitable for making a
This test method is under the jurisdiction of ASTM Committee E-13 on
Molecular Spectroscopy and is the direct responsibility of Subcommittee E13.06 on
given quantitative analysis.
Molecular Luminescence.
3.2 This test method is not meant for comparing the
Current edition approved Aug. 26, 1983. Published October 1983. Originally
published as E 578 – 76. Last previous edition E 578 – 76.
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, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 578
performance of different fluorescence measuring instruments. standard solutions, ending with the stock solution described in
5.1.
4. Apparatus
7. Calculation of Results and Data P
...

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4.1 This practice permits an analyst to compare the performance of an instrument to the manufacturer's supplied performance specifications and to verify its suitability for continued routine use. It also provides generation of calibration monitoring data on a periodic basis, forming a base from which any changes in the performance of the instrument will be evident.
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4.1 This practice permits an analyst to compare the general performance of an instrument on any given day with the prior performance of an instrument. This practice is not necessarily meant for comparison of different instruments with each other even if the instruments are of the same type and model. This practice is not meant for comparison of the performance of one instrument operated under differing conditions.
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Standard Practice for Estimation of the Spectral Bandwidth of Ultraviolet-Visible Spectrophotometers

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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.  
4.3 These practices allow the user of a spectrophotometer to estimate the actual spectral bandwidth of the instrument under a given set of conditions and to compare the result to the spectral bandwidth calculated from data given in the manufacturer's literature or indicated by the instrument.
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1.1 This practice describes procedures for estimating the spectral bandwidth of a spectrophotometer in the wavelength region of 185 nm to 820 nm.  
1.2 These practices are applicable to all modern spectrophotometer designs utilizing computer control and data handling. This includes conventional optical designs, where the sample is irradiated by monochromatic light, and ‘reverse’ optic designs coupled to photodiode arrays, where the light is separated by a polychromator after passing through the sample. For spectrophotometers that utilize servo-operated slits and maintain a constant period and a constant signal-to-noise ratio as the wavelength is automatically scanned, and/or utilize fixed slits and maintain a constant servo loop gain by automatically varying gain or dynode voltage, refer to the procedure described in Annex A1. This procedure is identical to that described in earlier versions of this practice.  
1.3 This practice does not cover the measurement of limiting spectral bandwidth, defined as the minimum spectral bandwidth achievable under optimum experimental conditions.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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ASTM E578-07(2021)(Test Method)
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.
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1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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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.

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SIGNIFICANCE AND USE
4.1 Although it is possible to observe and measure each of the several characteristics of a detector under different and unique conditions, it is the intent of this practice that a complete set of detector specifications should be obtained under the same operating conditions. It should also be noted that to completely specify a detector’s capability, its performance should be measured at several sets of conditions within the useful range of the detector. The terms and tests described in this practice are sufficiently general that they may be used regardless of the ultimate operating parameters.  
4.2 Linearity and response time of the recorder or other readout device used should be such that they do not distort or otherwise interfere with the performance of the detector. This requires adjusting the gain, damping, and calibration in accordance with the manufacturer's directions. If additional electronic filters or amplifiers are used between the detector and the final readout device, their characteristics should also first be established.
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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