ASTM E3029-15(2023)
(Practice)Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
Standard Practice for Determining Relative Spectral Correction Factors for Emission Signal of Fluorescence Spectrometers
SIGNIFICANCE AND USE
3.1 Calibration of the responsivity of the detection system for emission (EM) as a function of EM wavelength (λEM), also referred to as spectral correction of emission, is necessary for successful quantification when intensity ratios at different EM wavelengths are being compared or when the true shape or peak maximum position of an EM spectrum needs to be known. Such calibration methods are given here and summarized in Table 1. This type of calibration is necessary because the spectral responsivity of a detection system can change significantly over its useful wavelength range (see Fig. 1). It is highly recommended that the wavelength accuracy (see Test Method E388) and the linear range of the detection system (see Guide E2719 and Test Method E578) be determined before spectral calibration is performed and that appropriate steps are taken to insure that all measured intensities during this calibration are within the linear range. For example, when using wide slit widths in the monochromators, attenuators may be needed to attenuate the excitation beam or emission, thereby, decreasing the fluorescence intensity at the detector. Also note that when using an EM polarizer, the spectral correction for emission is dependent on the polarizer setting. (2) It is important to use the same instrument settings for all of the calibration procedures mentioned here, as well as for subsequent sample measurements.
FIG. 1 Example of Relative Spectral Responsivity of Emission Detection System (Grating Monochromator-PMT Based), (see Test Method E578) for which a Correction Needs to be Applied to a Measured Instrument-Specific Emission Spectrum to Obtain its True Spectral Shape (Relative Intensities).
3.2 When using CCD or diode array detectors with a spectrometer for λEM selection, the spectral correction factors are dependent on the grating position of the spectrometer. Therefore, the spectral correction profile versus λEM must be determined separately for each grating position ...
SCOPE
1.1 This practice (1)2 describes three methods for determining the relative spectral correction factors for grating-based fluorescence spectrometers in the ultraviolet-visible spectral range. These methods are intended for instruments with a 0°/90° transmitting sample geometry. Each method uses different types of transfer standards, including 1) a calibrated light source (CS), 2) a calibrated detector (CD) and a calibrated diffuse reflector (CR), and 3) certified reference materials (CRMs). The wavelength region covered by the different methods ranges from 250 nm to 830 nm with some methods having a broader range than others. Extending these methods to the near infrared (NIR) beyond 830 nm will be discussed briefly, where appropriate. These methods were designed for scanning fluorescence spectrometers with a single channel detector, but can also be used with a multichannel detector, such as a diode array or a CCD.
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
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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:E3029 −15 (Reapproved 2023)
Standard Practice for
Determining Relative Spectral Correction Factors for
Emission Signal of Fluorescence Spectrometers
This standard is issued under the fixed designation E3029; 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 2. Referenced Documents
2 3
1.1 This practice (1) describes three methods for determin- 2.1 ASTM Standards:
ing the relative spectral correction factors for grating-based E131 Terminology Relating to Molecular Spectroscopy
fluorescence spectrometers in the ultraviolet-visible spectral E388 Test Method for Wavelength Accuracy and Spectral
range. These methods are intended for instruments with a Bandwidth of Fluorescence Spectrometers
0°/90° transmitting sample geometry. Each method uses dif- E578 Test Method for Linearity of Fluorescence Measuring
ferenttypesoftransferstandards,including 1)acalibratedlight Systems
source (CS), 2) a calibrated detector (CD) and a calibrated E2719 Guide for Fluorescence—Instrument Calibration and
diffuse reflector (CR), and 3) certified reference materials Qualification
(CRMs). The wavelength region covered by the different
3. Significance and Use (Intro)
methods ranges from 250 nm to 830 nm with some methods
havingabroaderrangethanothers.Extendingthesemethodsto
3.1 Calibration of the responsivity of the detection system
the near infrared (NIR) beyond 830 nm will be discussed
for emission (EM) as a function of EM wavelength (λ ), also
EM
briefly, where appropriate. These methods were designed for
referred to as spectral correction of emission, is necessary for
scanning fluorescence spectrometers with a single channel
successful quantification when intensity ratios at different EM
detector, but can also be used with a multichannel detector,
wavelengths are being compared or when the true shape or
such as a diode array or a CCD.
peak maximum position of an EM spectrum needs to be
known. Such calibration methods are given here and summa-
1.2 The values stated in SI units are to be regarded as
rized in Table 1. This type of calibration is necessary because
standard. No other units of measurement are included in this
the spectral responsivity of a detection system can change
standard.
significantly over its useful wavelength range (see Fig. 1). It is
1.3 This standard does not purport to address all of the
highly recommended that the wavelength accuracy (see Test
safety concerns, if any, associated with its use. It is the
MethodE388)andthelinearrangeofthedetectionsystem(see
responsibility of the user of this standard to establish appro-
Guide E2719 and Test Method E578) be determined before
priate safety, health, and environmental practices and deter-
spectral calibration is performed and that appropriate steps are
mine the applicability of regulatory limitations prior to use.
taken to insure that all measured intensities during this cali-
1.4 This international standard was developed in accor-
bration are within the linear range. For example, when using
dance with internationally recognized principles on standard-
wide slit widths in the monochromators, attenuators may be
ization established in the Decision on Principles for the
needed to attenuate the excitation beam or emission, thereby,
Development of International Standards, Guides and Recom-
decreasing the fluorescence intensity at the detector.Also note
mendations issued by the World Trade Organization Technical
that when using an EM polarizer, the spectral correction for
Barriers to Trade (TBT) Committee.
emission is dependent on the polarizer setting. (2)Itis
important to use the same instrument settings for all of the
calibration procedures mentioned here, as well as for subse-
This practice is under the jurisdiction of ASTM Committee E13 on Molecular quent sample measurements.
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Jan. 1, 2023. Published January 2023. Originally
approved in 2015. Last previous edition approved in 2015 as E3029-15. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E3029-15R23. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3029−15 (2023)
TABLE 1 Summary of Methods for Determining Spectral Correction of Detection System Responsivity
NOTE 1—“Drop-In” refers to whether or not the material/hardware can be put in the sample holder and used like a conventional sample; “Off-Shelf”
refers to whether or not the material/hardware can be purchased in an immediately-usable format; “Uncertainty” is the estimated expanded (k=2) total
uncertainty; “Caveats” refer to important information that a user should know about the method before attempting to use it; “Certified Values” refers to
whether or not the material/hardware is supplied with appropriate values as a function of emission wavelength and their corresponding total uncertainties;
the references (Ref.) give examples and more in-depth information for each method.
Method λ Drop-In Off-Shelf Uncertainty Caveats Certified Values Ref.
EM
CS UV-NIR N Y <±5% difficultsetup Y E578,(3-6)
CD+CR UV-NIR N Maybe ± 10 % difficult setup Y E578,(4, 5, 7)
CRMs UV-NIR Y Y ± 5 % Y E131,(8-13)
FIG. 1Example of Relative Spectral Responsivity of Emission Detection System (Grating Monochromator-PMT Based),
(see Test Method E578) for which a Correction Needs to be Applied to a Measured Instrument-Specific Emission Spectrum
to Obtain its True Spectral Shape (Relative Intensities).
3.2 When using CCD or diode array detectors with a decreases beyond 1000 nm, but continues to have significant
spectrometer for λ selection, the spectral correction factors intensity out to about 2500 nm. A calibrated deuterium lamp
EM
are dependent on the grating position of the spectrometer.
can be used to extend farther into the UV with an effective
Therefore, the spectral correction profile versus λ must be range from about 200 nm to 380 nm. The effective range of a
EM
determined separately for each grating position used. (3)
CS is dependent on the intensity of the CS and the sensitivity
of the detection system. This range can be determined by
3.3 Instrument manufacturers often provide an automated
measuring the low-signal regions where the signal profile of
procedure and calculation for a spectral correction function for
the light from the CS becomes flat or indistinguishable from
emission, or they may supply a correction that was determined
the background signal, implying that the signal afforded by the
at the factory. This correction can often be applied during
CS is not measurable in these λ regions.
EM
spectral collection or as a post-collection correction. The user
4.1.2 A calibrated reflector (CR) is often used to reflect the
should be advised to verify that the automated vendor proce-
light from the CS into the emission detection system.Adiffuse
dure and calculation or supplied correction are performed and
reflector made of compressed or sintered polytetrafluoroethyl-
determined according to the guidelines given within this
ene (PTFE) is most commonly used as a CR, due to its nearly
standard.
Lambertian reflectance, which prevents both polarization and
4. Calibrated Optical Radiation Source (CS) Method (see
spatial dependence of the reflectance. In addition, PTFE
Test Method E578,(4-6, 14))
possesses a reflectance profile that is nearly flat, changing by
less than 10 % from 250 nm to 2500 nm. For a CS and a CR,
4.1 Materials:
4.1.1 Acalibrated tungsten lamp is most commonly used as “calibrated” implies that the spectral radiance and the spectral
reflectance, respectively, are known (calibrated wavelength
a CS in the visible region due to its high intensity and broad,
featureless spectral profile. Its intensity falls off quickly in the dependence of the spectral radiant factor including measure-
ultraviolet (UV) region, but it can typically be used down to ment uncertainty) and traceable to the SI (International System
350 nm or so. It also displays a high intensity in the near of Units).This is commonly done through certification of these
infrared, peaking at about 1000 nm. Its intensity gradually values by a national metrology institute (NMI). (15, 16, 7)
E3029−15 (2023)
4.2 Procedure: dent of excitation wavelength. Note that there are several
4.2.1 Direct the optical radiation from a CS into the EM drawbacks to using a quantum counter (QC) instead of a CD.
detection system by placing the CS at the sample position. If Firstly, QCs tend to have a more limited range than CDs and
the CS is too large to be placed at the sample position, place a uncertainties that are not certified or even well known. In
CR at the sample position to reflect the optical radiation from
addition, a QC is prone to polarization and geometry effects
the CS into the EM detection system. Ensure that the CS is that are concentration and solvent dependent, thus requiring
aligned such that its light is centered on the entrance slit of the
that the ideal concentration for proper functioning be deter-
λ selector,andonallopticsitencountersbeforetheentrance mined for the measurement geometry to be used. It should also
EM
slit. Ideally, the light should fully and uniformly fill the
be noted that the output measured from the QC will be
entrance slit. Make sure that the detection system is still proportional to the quantum flux (number of photons per
operated within its linear range (see 3.1).
second) at the sample, not the flux in power units. This can
result in enhanced measurement uncertainties compared to the
NOTE 1—Correction factors, supplied by the manufacturer and auto-
use of a calibrated detector.
matically applied by the software to the collected spectrum, must be
switched off for the signal channel during this procedure.
5.2 Procedure:
4.2.2 Scan the λ -selector over the EM region of interest,
EM
5.2.1 Unlike the CS method, this is a two-step method. The
using the same instrument settings as employed with the
first step uses a CD (or a QC) placed at the sample position,
subsequentmeasurementofthefluorescenceofthesample,and
which measures the excitation intensity incident on the sample
collect the signal channel output (Sʹʹ).
as a function of EX wavelength by scanning the EX wave-
4.2.3 Use the known radiance of the CS incident on the
length selector over the desired range. The second step uses a
detection system (L) to calculate the relative correction factor
CR with reflectance R to reflect a known fraction of the flux
CR
(C ),suchthat C = L/Sʹʹ.NotethatLmaybereplacedbythe
CS CS
of the EX beam into the detection system. Follow the proce-
spectral irradiance or the spectral radiant flux, since the
dures in either 5.2.1.1 or 5.2.1.2 depending upon whether you
correction factors determined herein are relative, not absolute.
are using a CD or a QC, respectively.
ThecorrectedEMintensityisequaltotheproductofthesignal
output of the sample (S) and C . Since C values are relative
NOTE 2—Correction factors, supplied by the manufacturer and auto-
CS CS
matically applied by the software to the collected spectrum, must be
correction factors, they can be scaled by any constant. For
switched off for signal and reference channels during this procedure.
instance, it is often useful to scale them with a constant that
gives a C value of one at a particular λ .
CS EM 5.2.1.1 Step 1 with Calibrated Detector—Place the CD at
4.2.4 Note that L is given in power units, not photon units,
the sample position and scan the λ -selector over the EX
EX
whereas, the units for S and Sʹʹ are either in power or photon
regionofinterestwhilecollectingthesignalfromtheCD(S )
CD
units depending on whether your detector measures an analog
as a function of λ . Be sure to use the same instrument
EX
or a digital (photon counting) signal, respectively. In either
settings as those employed with the sample. Calculate the flux
case, the corrected signal will be in power units, so a
of the EX beam (ϕ ), using ϕ = S /R , where R is the
x x CD CD CD
conversion, that is, dividing the corrected signal by λ ,is
EM
known responsivity of the CD. Note that if the instrument has
necessary if photon units are needed.
its own reference detector with output (Rf) for monitoring the
excitation intensity, then the correction factor for the respon-
5. Calibrated Detector (CD) with Calibrated Reflector
sivity of the reference detector C = ϕ /Rf can be calculated.
R x
Method (see Test Method E578,(4, 5, 17))
Multiplying an Rf value by C at a particular λ will give a
R EX
corrected Rf value in the same units as ϕ , typically Watts.
5.1 Materials: x
5.1.1 A calibrated photodiode, by itself, as part of a trap
5.2.1.2 Step 1 with Quantum Counter—Place the QC solu-
detector or mounted in an integrating sphere, is most com- tion at the sample position in a cuvette (typically fused silica)
monly used as a calibrated detector (CD). Using a trap detector
that transmits the excitation and emission wavelengths of
or photodiode with integrating sphere is typically more accu- interest. If front face detection is possible, then use a standard
rate than using a photodiode alone.ASi photodiode covers the cuvette with the EX beam at normal incidence. If 90° detection
range from 200 nm to 1100 nm.An InGaAs or Ge photodiode ischosen,thenusearight-triangularcuvettewiththeexcitation
can be used in the NIR from 800 nm to 1700 nm. For a CD, beam at 45° incidence to the hypotenuse side and one of the
“calibrated” implies that the wavelength dependence of the other sides facing the detector. Scan the λ -selector over the
EX
spectral responsivity is known, its associated uncertainties EX region of interest with the λ fixed at a position
EM
have been determined and the measurements are traceable to correspondingtothelong-wavelengthtailoftheemissionband
the SI. This is typically done through values certified by an and collect the signal intensity (S ) as a function of λ .Be
QC EX
NMI. (18, 19) A photodiode
...
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