ASTM E388-04(2023)

Standard Test Method for Wavelength Accuracy and Spectral Bandwidth of Fluorescence Spectrometers

ABSTRACT
This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. The method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. Atomic lines between 250 nm and 1000 nm are used in the method. The difference between the apparent wavelength and the known wavelength for a series of atomic emission lines is used as a test for wavelength accuracy. The apparent width of some of these lines is used as a test for spectral bandwidth.
SCOPE
1.1 This test method covers the testing of the spectral bandwidth and wavelength accuracy of fluorescence spectrometers that use a monochromator for emission wavelength selection and photomultiplier tube detection. This test method can be applied to instruments that use multi-element detectors, such as diode arrays, but results must be interpreted carefully. This test method uses atomic lines between 250 nm and 1000 nm.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 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.  
1.4 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-Dec-2022
• 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

Overview

ASTM E388-04(2023) - Standard Test Method for Wavelength Accuracy and Spectral Bandwidth of Fluorescence Spectrometers provides a systematic approach for evaluating the wavelength accuracy and spectral bandwidth of fluorescence spectrometers. This standard is essential for laboratories, instrument manufacturers, and organizations seeking reliable, repeatable performance from fluorescence spectrometers used in molecular spectroscopy and luminescence analyses.

The test method is designed specifically for instruments utilizing a monochromator for emission wavelength selection and photomultiplier tube (PMT) detection. It is also applicable to spectrometers with multi-element detectors, such as diode arrays, though the results may require careful interpretation.

Key features of the method:

• Utilizes atomic emission lines between 250 nm and 1000 nm for calibration and testing
• Focuses on both emission and excitation monochromator alignment
• Specifies the use of SI units throughout
• Encourages adherence to safety and regulatory practices before application

Key Topics

• Wavelength Accuracy: Determined by measuring the difference between the observed wavelength and the known wavelength of select atomic emission lines (e.g., mercury, neon, argon, krypton, xenon).
• Spectral Bandwidth: Evaluated by assessing the apparent width of atomic emission lines at different intensity points (50% and 5% of the maximum).
• Calibration Procedures: Provides detailed steps for calibrating both emission and excitation monochromators using atomic discharge lamps and scattering suspensions.
• Instrument Requirements: Covers both traditional single PMT detector spectrometers and newer systems with multi-element detectors.
• Slit Width Considerations: Emphasizes the importance of using the narrowest practical slit widths for accurate results.
• Data Recording and Adjustments: Instructs on systematic recording of measurement corrections and instrument adjustments to ensure reliability across measurements.

Applications

The practical value of ASTM E388-04(2023) lies in its ability to standardize the performance testing of fluorescence spectrometers for various scientific and industrial applications. Key use cases include:

• Quality Control in Laboratories: Ensures the reliability and repeatability of spectroscopic measurements for chemical, pharmaceutical, environmental, and biochemical analysis.
• Instrument Manufacturing and Maintenance: Guides manufacturers and service technicians in validating and recalibrating spectrometer performance to meet established accuracy and bandwidth criteria.
• Research and Development: Supports scientists in generating reproducible fluorescence spectroscopy data critical for molecular identification, quantification, and material characterization.
• Regulatory and Accreditation Compliance: Helps organizations fulfill documentation and audit requirements by providing standard methods for instrument calibration and performance verification.
• Educational Settings: Used as a training tool in teaching precise methods for spectroscopic instrument validation.

Related Standards

For comprehensive fluorescence and molecular spectroscopy practices, consider consulting the following ASTM and international standards:

• ASTM E1309 - Standard Guide for Experiments to Characterize Fluorescence Spectrometers
• ASTM E1620 - Standard Terminology Relating to Molecular Spectroscopy
• ISO 17025 - General requirements for the competence of testing and calibration laboratories
• ASTM E2414 - Standard Test Method for Determining the Performance of Fluorescence Spectrometers

These standards, together with ASTM E388-04(2023), provide a robust framework for ensuring the accuracy, reliability, and comparability of spectrometric data across a variety of scientific and industrial disciplines.

Keywords: fluorescence spectrometers, wavelength accuracy, spectral bandwidth, ASTM E388, spectrofluorometer calibration, molecular spectroscopy, emission monochromator, photomultiplier tube detection, atomic emission lines, instrument validation.

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: E388 − 04 (Reapproved 2023)
Standard Test Method for
Wavelength Accuracy and Spectral Bandwidth of
Fluorescence Spectrometers
This standard is issued under the fixed designation E388; 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 3.2 Atomic Discharge Lamps, Low-pressure, sufficiently
small to be placed in the sample cell holder of the instrument.
1.1 This test method covers the testing of the spectral
bandwidth and wavelength accuracy of fluorescence spectrom-
4. Reagent
eters that use a monochromator for emission wavelength
selection and photomultiplier tube detection. This test method 4.1 Scattering Suspension—Dissolve1gof glycogen per
can be applied to instruments that use multi-element detectors,
litre of water, or use a dilute microsphere suspension contain-
such as diode arrays, but results must be interpreted carefully. ing 1 mL of a commercially available, concentrated micro-
This test method uses atomic lines between 250 nm and
sphere suspension.
1000 nm.
5. Procedure
1.2 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
5.1 The emission lines given for mercury (Hg), neon (Ne),
standard.
argon (Ar), krypton (Kr), and xenon (Xe) in Table 1 are
typically observable using standard commercial fluorometers,
1.3 This standard does not purport to address all of the
although some of them may be too weak to detect on some
safety concerns, if any, associated with its use. It is the
instruments.
responsibility of the user of this standard to establish appro-
5.1.1 Most fluorescence instruments will not be able to
priate safety, health, and environmental practices and deter-
resolve very closely spaced lines such as those for Hg at
mine the applicability of regulatory limitations prior to use.
312.57 nm, 313.15 nm, and 313.18 nm, due to the relatively
1.4 This international standard was developed in accor-
low resolution monochromators used in fluorescence equip-
dance with internationally recognized principles on standard-
ment compared to those used in absorbance spectrometers.
ization established in the Decision on Principles for the
Even lower resolution fluorometers may not resolve lines
Development of International Standards, Guides and Recom-
separated by less than several nanometres such as those for Hg
mendations issued by the World Trade Organization Technical
at 404.66 and 407.78, or at 576.96 and 579.07 nm.
Barriers to Trade (TBT) Committee.
5.1.2 In instruments using blazed grating monochromators,
2. Summary of Test Method additional weaker lines are found due to second order diffrac-
tion of atomic lines. For instance, lines appear for Hg at 507.30
2.1 The difference between the apparent wavelength and the
and 593.46 nm, arising from the 253.65 and 296.73 nm lines,
known wavelength for a series of atomic emission lines is used
respectively.
as a test for wavelength accuracy. The apparent width of some
of these lines is used as a test for spectral bandwidth. 5.2 Calibration and Adjustment of Emission Monochroma-
tor:
3. Apparatus
5.2.1 Withanatomicarcsourceproperlyaligned(see5.3)in
the sample cell compartment, adjust the position of the
3.1 Fluorescence Spectrometer to be tested.
wavelength dial to give maximum signal for each of the atomic
lines and record the wavelength reading. The difference be-
tween the observed value and the corresponding value in Table
This test method is under the jurisdiction of ASTM Committee E13 on
Molecular Spectroscopy and Separation Science and is the direct responsibility of
1 represents the correction that must be subtracted algebra-
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
ically from the wavelength reading of the instrument. The
Current edition approved Jan. 1, 2023. Published January 2023. Originally
corrections may be recorded or the monochromator adjusted to
approved in 1969. Last previous edition approved in 2015 as E388 – 04 (2015).
DOI: 10.1520/E0388-04R23. give the proper values. Since there may be some backlash in
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E388 − 04 (2023)
A
TABLE 1 Atomic Emission Lines for Wavelength Accuracy
Hg Ne Ar Kr Xe
253.65 336.99 633.44 830.03 696.54 427.40 645.63 450.10
296.73 341.79 638.30 836.57 706.72 428.30 722.41 458.28
302.15 345.42 640.11 837.76 727.29 431.96 758.74 462.43
312.57 346.66 640.22 841.72 738.40 436.26 760.15 467.12
313.15 347.26 650.65 841.84 750.39 437.61 768.53 469.70
313.18 350.12 653.29 846.34 751.47 440.00 769.45 473.42
334.15 352.05 659.90 857.14 763.51 442.52 785.48 480.70
365.02 359.35 667.83 859.13 772.38 445.39 805.95 482.97
404.66 533.08 671.70 863.46 794.82 446.37 810.44 484.33
407.78 534.11 692.95 864.70 800.62 450.24 811.29 491.65
435.84 540.06 703.24 865.44 801.48 557.03 819.00 492.32
546.07 576.44 717.39 865.55 810.37 564.96 826.32 711.96
576.96 582.01 724.52 867.95 811.53 567.25 828.10 764.20
579.07 585.25 743.89 868.19 826.45 583.29 829.81 823.16
588.19 783.91 870.41 840.82 587.09 850.89 828.01
594.48 792.71 877.17 842.46 599.39 877.67 834.68
597.55 793.70 878.06 912.30 601.21 975.18 840.92
603.00 794.32 885.39 922.45 605.61 881.94
607.43 808.25 920.18 965.78 895.22
609.62 811.85 930.09 979.97
614.31 812.89 932.65 992.32
616.36 813.64 942.54
621.73 825.94 948.67
626.65 826.61 953.42
630.48 826.71
A
Wavelength values have been obtained from Harrison, G. R., MIT Wavelength Tables, Wavelengths by Element, Vol 2, MIT Press, Cambridge, MA, 1982; and Zaidel,
A. N., Prokofev, V. K., Raiskii, S. M., Slavnyi, V. A., and Shreider, E. Ya., Tables of Spectral Lines, Plenum Press, New York, NY, 1970.
thewavelengthdriveofscanninginstruments,alwaysapproach 5.4.5 Adjust the wavelength position of the excitation
the peak position from t
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