ASTM E388-04(2015)

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 and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 30-Apr-2015
• 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

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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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 (Reapproved 2015)
Standard Test Method for
Wavelength Accuracy and 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. Procedure
1.1 This test method covers the testing of the spectral
5.1 The emission lines given for mercury (Hg), neon (Ne),
bandwidth and wavelength accuracy of fluorescence spectrom-
argon (Ar), krypton (Kr), and xenon (Xe) in Table 1 are
eters that use a monochromator for emission wavelength
typically observable using standard commercial fluorometers,
selection and photomultiplier tube detection. This test method
although some of them may be too weak to detect on some
can be applied to instruments that use multi-element detectors,
instruments.
such as diode arrays, but results must be interpreted carefully.
5.1.1 Most fluorescence instruments will not be able to
This test method uses atomic lines between 250 nm and 1000
resolveverycloselyspacedlinessuchasthoseforHgat312.57
nm.
nm, 313.15 nm, and 313.18 nm, due to the relatively low
resolution monochromators used in fluorescence equipment
1.2 The values stated in SI units are to be regarded as
compared to those used in absorbance spectrometers. Even
standard. No other units of measurement are included in this
lower resolution fluorometers may not resolve lines separated
standard.
by less than several nanometres such as those for Hg at 404.66
1.3 This standard does not purport to address all of the
and 407.78, or at 576.96 and 579.07 nm.
safety concerns, if any, associated with its use. It is the
5.1.2 In instruments using blazed grating monochromators,
responsibility of the user of this standard to establish appro-
additional weaker lines are found due to second order diffrac-
priate safety and health practices and determine the applica-
tion of atomic lines. For instance, lines appear for Hg at 507.30
bility of regulatory limitations prior to use.
and 593.46 nm, arising from the 253.65 and 296.73 nm lines,
respectively.
2. Summary of Test Method
2.1 The difference between the apparent wavelength and the 5.2 Calibration and Adjustment of Emission Monochroma-
known wavelength for a series of atomic emission lines is used tor:
as a test for wavelength accuracy. The apparent width of some
5.2.1 Withanatomicarcsourceproperlyaligned(see5.3)in
of these lines is used as a test for spectral bandwidth.
the sample cell compartment, adjust the position of the
wavelength dial to give maximum signal for each of the atomic
3. Apparatus
lines and record the wavelength reading. The difference be-
tween the observed value and the corresponding value in Table
3.1 Fluorescence Spectrometer to be tested.
1 represents the correction that must be subtracted algebra-
3.2 Atomic Discharge Lamps, Low-pressure, sufficiently
ically from the wavelength reading of the instrument. The
small to be placed in the sample cell holder of the instrument.
corrections may be recorded or the monochromator adjusted to
give the proper values. Since there may be some backlash in
4. Reagent
thewavelengthdriveofscanninginstruments,alwaysapproach
4.1 Scattering Suspension—Dissolve1gof glycogen per
the peak position from the same direction, if applicable.
litre of water, or use a dilute microsphere suspension contain-
5.2.2 Whencalibratingscanning-typeinstruments,approach
ing 1 mL of a commercially available, concentrated micro-
the peak position in the same direction that the motor scans, if
sphere suspension.
your instrument does not correct for backlash. Check the
position against that recorded while scanning and, if necessary,
correct as in 5.2.1.
1
This test method is under the jurisdiction of ASTM Committee E13 on
Molecular Spectroscopy and Separation Science and is the direct responsibility of
5.3 In cases where the monochromator is designed so that a
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
lateral displacement of the calibration source from a position
Current edition approved May 1, 2015. Published June 2015. Originally
directly in front of the entrance slit appears as a wavelength
approved in 1969. Last previous edition approved in 2009 as E388 – 04 (2009).
DOI: 10.1520/E0388-04R15. shift, proceed as follows:
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
E388 − 04 (2015)
A
TABLE 1 Atomic Emission Lines for Wavelength Accuracy
Hg Ne Ar Kr Xe
253.65 336.99 633.44 830.03 696
...

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: E388 − 04 (Reapproved 2009) E388 − 04 (Reapproved 2015)
Standard Test Method for
Wavelength Accuracy and 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
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 and health practices and determine the applicability of regulatory
limitations prior to use.
2. Summary of Test Method
2.1 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.
3. Apparatus
3.1 Fluorescence Spectrometer to be tested.
3.2 Atomic Discharge Lamps, Low-pressure, sufficiently small to be placed in the sample cell holder of the instrument.
4. Reagent
4.1 Scattering Suspension—Dissolve 1 g of glycogen per litre of water, or use a dilute microsphere suspension containing 1 mL
of a commercially available, concentrated microsphere suspension.
5. Procedure
5.1 The emission lines given for mercury (Hg), neon (Ne), argon (Ar), krypton (Kr), and xenon (Xe) in Table 1 are typically
observable using standard commercial fluorometers, although some of them may be too weak to detect on some instruments.
5.1.1 Most fluorescence instruments will not be able to resolve very closely spaced lines such as those for Hg at 312.57 nm,
313.15 nm, and 313.18 nm, due to the relatively low resolution monochromators used in fluorescence equipment compared to those
used in absorbance spectrometers. Even lower resolution fluorometers may not resolve lines separated by less than several
nanometres such as those for Hg at 404.66 and 407.78, or at 576.96 and 579.07 nm.
5.1.2 In instruments using blazed grating monochromators, additional weaker lines are found due to second order diffraction of
atomic lines. For instance, lines appear for Hg at 507.30 and 593.46 nm, arising from the 253.65 and 296.73 nm lines, respectively.
5.2 Calibration and Adjustment of Emission Monochromator:
5.2.1 With an atomic arc source properly aligned (see 5.3) in the sample cell compartment, adjust the position of the wavelength
dial to give maximum signal for each of the atomic lines and record the wavelength reading. The difference between the observed
value and the corresponding value in Table 1 represents the correction that must be subtracted algebraically from the wavelength
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 October 2009June 2015. Originally approved in 1969. Last previous edition approved in 20042009 as
E388 – 04.E388 – 04 (2009). DOI: 10.1520/E0388-04R09.10.1520/E0388-04R15.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
E388 − 04 (2015)
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 35
...