ASTM E388-04

Standard Test Method for Wavelength Accuracy of Spectral Bandwidth 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 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: 31-Oct-2004
• 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

Relations

• Replaces: ASTM E388-72(1998) - Standard Test Method for Spectral Bandwidth and Wavelength Accuracy of Fluorescence Spectrometers
• Effective Date: 01-Nov-2004
• Replaced By: ASTM E388-04(2009) - Standard Test Method for Wavelength Accuracy and Spectral Bandwidth of Fluorescence Spectrometers ^,
• Effective Date: 01-Oct-2009
• Referred By: ASTM E2056-04(2016) - Standard Practice for Qualifying Spectrometers and Spectrophotometers for Use in Multivariate Analyses, Calibrated Using Surrogate Mixtures
• Effective Date: 01-Nov-2004
• Referred By: ASTM E3029-15(2023) - Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
• Effective Date: 01-Nov-2004
• 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: 01-Nov-2004
• 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: 01-Nov-2004
• Referred By: ASTM E2719-09(2022) - Standard Guide for Fluorescence—Instrument Calibration and Qualification
• Effective Date: 01-Nov-2004

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:E388–04
Standard Test Method for
Wavelength Accuracy of Spectral Bandwidth of
1
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 5.1.1 Most fluorescence instruments will not be able to
resolveverycloselyspacedlinessuchasthoseforHgat312.57
1.1 This test method covers the testing of the spectral
nm, 313.15 nm, and 313.18 nm, due to the relatively low
bandwidth and wavelength accuracy of fluorescence spectrom-
resolution monochromators used in fluorescence equipment
eters that use a monochromator for emission wavelength
compared to those used in absorbance spectrometers. Even
selection and photomultiplier tube detection. This test method
lower resolution fluorometers may not resolve lines separated
can be applied to instruments that use multi-element detectors,
by less than several nanometres such as those for Hg at 404.66
such as diode arrays, but results must be interpreted carefully.
and 407.78, or at 576.96 and 579.07 nm.
This test method uses atomic lines between 250 nm and 1000
5.1.2 In instruments using blazed grating monochromators,
nm.
additional weaker lines are found due to second order diffrac-
1.2 This standard does not purport to address all of the
tion of atomic lines. For instance, lines appear for mercury at
safety concerns, if any, associated with its use. It is the
507.30 and 593.46 nm, arising from the 253.65 and 296.73 nm
responsibility of the user of this standard to establish appro-
lines, respectively.
priate safety and health practices and determine the applica-
5.2 Calibration and Adjustment of Emission Monochroma-
bility of regulatory limitations prior to use.
tor:
2. Summary of Test Method 5.2.1 With an atomic arc source properly aligned (see
section 5.3) in the sample cell compartment, adjust the position
2.1 The difference between the apparent wavelength and the
of the wavelength dial to give maximum signal for each of the
known wavelength for a series of atomic emission lines is used
atomic lines and record the wavelength reading.The difference
as a test for wavelength accuracy. The apparent width of some
between the observed value and the corresponding value in
of these lines is used as a test for spectral bandwidth.
Table 1 represents the correction that must be subtracted
3. Apparatus
algebraically from the wavelength reading of the instrument.
The corrections may be recorded or the monochromator
3.1 Fluorescence Spectrometer to be tested.
adjusted to give the proper values. Since there may be some
3.2 Atomic Discharge Lamps, Low-pressure, sufficiently
backlash in the wavelength drive of scanning instruments,
small to be placed in the sample cell holder of the instrument.
always approach the peak position from the same direction, if
4. Reagent
applicable.
5.2.2 Whencalibratingscanning-typeinstruments,approach
4.1 Scattering Suspension—Dissolve1gof glycogen per
the peak position in the same direction that the motor scans, if
litre of water, or use a dilute microsphere suspension contain-
your instrument does not correct for backlash. Check the
ing 1 mL of a commercially available, concentrated micro-
position against that recorded while scanning and, if necessary,
sphere suspension.
correct as in 5.2.1.
5. Procedure
5.3 In cases where the monochromator is designed so that a
lateral displacement of the calibration source from a position
5.1 The emission lines given for Hg, Ne, Ar, Kr, and Xe
directly in front of the entrance slit appears as a wavelength
in Table 1 are typically observable using standard commercial
shift, proceed as follows:
fluorometers,althoughsomeofthemmaybetooweaktodetect
5.3.1 Instead of placing the atomic lamp in front of the
on some instruments.
entrance slit of the monochromator, fill a sample cell with a
dilute scattering suspension, as described in section 4.1.
1
This test method is under the jurisdiction of ASTM Committee E13 on
5.3.2 Place the cell in the sample position in the instrument.
Molecular Spectroscopy and Chromatography and is the direct responsibility of
5.3.3 Illuminate the cell transversely with the atomic lamp,
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Nov. 1, 2004. Published December 2004. Originally
either from the side or from above.
approved in 1969. Last previous edition approved in 1998 as E388 – 72 (1998).
DOI: 10.1520/E0388-04.
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