ASTM E2719-09(2022)
(Guide)Standard Guide for Fluorescence—Instrument Calibration and Qualification
Standard Guide for Fluorescence—Instrument Calibration and Qualification
SIGNIFICANCE AND USE
4.1 By following the general guidelines (Section 5) and instrument calibration methods (Sections 6 – 16) in this guide, users should be able to more easily conform to good laboratory and manufacturing practices (GXP) and comply with regulatory and QA/QC requirements, related to fluorescence measurements.
4.2 Each instrument parameter needing calibration (for example, wavelength, spectral responsivity) is treated in a separate section. A list of different calibration methods is given for each instrument parameter with a brief usage procedure. Precautions, achievable precision and accuracy, and other useful information are also given for each method to allow users to make a more informed decision as to which method is the best choice for their calibration needs. Additional details for each method can be found in the references given.
SCOPE
1.1 This guide (1)2 lists the available materials and methods for each type of calibration or correction for fluorescence instruments (spectral emission correction, wavelength accuracy, and so forth) with a general description, the level of quality, precision and accuracy attainable, limitations, and useful references given for each entry.
1.2 The listed materials and methods are intended for the qualification of fluorometers as part of complying with regulatory and other quality assurance/quality control (QA/QC) requirements.
1.3 Precision and accuracy or uncertainty are given at a 1 σ confidence level and are approximated in cases where these values have not been well established.3
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 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.
1.6 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.
General Information
Relations
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: E2719 − 09 (Reapproved 2022)
Standard Guide for
Fluorescence—Instrument Calibration and Qualification
This standard is issued under the fixed designation E2719; 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 4
1.1 This guide (1) lists the available materials and methods 2.1 ASTM Standards:
for each type of calibration or correction for fluorescence E131 Terminology Relating to Molecular Spectroscopy
instruments (spectral emission correction, wavelength E388 Test Method for Wavelength Accuracy and Spectral
accuracy, and so forth) with a general description, the level of Bandwidth of Fluorescence Spectrometers
quality, precision and accuracy attainable, limitations, and E578 Test Method for Linearity of Fluorescence Measuring
useful references given for each entry. Systems
E579 Test Method for Limit of Detection of Fluorescence of
1.2 The listed materials and methods are intended for the
Quinine Sulfate in Solution
qualification of fluorometers as part of complying with regu-
latory and other quality assurance/quality control (QA/QC)
3. Terminology
requirements.
3.1 Definitions (2):
1.3 Precision and accuracy or uncertainty are given at a 1 σ
3.1.1 absorption coeffıcient (α), n—a measure of absorption
confidence level and are approximated in cases where these
of radiant energy from an incident beam as it traverses an
values have not been well established.
-αb
absorbing medium according to Bouguer’s law, I/I = e ,
o
1.4 The values stated in SI units are to be regarded as
where I and I are the transmitted and incident intensities,
o
standard. No other units of measurement are included in this
respectively, and b is the path length of the beam through the
standard.
sample. E131
3.1.1.1 Discussion—Note that transmittance T = I/I and
1.5 This standard does not purport to address all of the
o
absorbance A = –log T.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
3.1.2 absorptivity (a), n—the absorbance divided by the
priate safety, health, and environmental practices and deter-
product of the concentration of the substance and the sample
mine the applicability of regulatory limitations prior to use.
pathlength, a = A/bc. E131
1.6 This international standard was developed in accor-
3.1.3 Beer-Lambert law, n—relates the dependence of the
dance with internationally recognized principles on standard-
absorbance (A) of a sample on its path length (see absorption
ization established in the Decision on Principles for the
coeffıcient, α) and concentration (c), such that A =abc.
Development of International Standards, Guides and Recom-
3.1.3.1 Discussion—Also called Beer’s law or Beer-
mendations issued by the World Trade Organization Technical
Lambert-Bouquer law. E131
Barriers to Trade (TBT) Committee.
3.1.4 calibrated detector (CD), n—opticalradiationdetector
whose responsivity as a function of wavelength has been
This guide is under the jurisdiction of ASTM Committee E13 on Molecular
determined along with corresponding uncertainties (3).
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
3.1.5 calibrated diffuse reflector (CR), n—Lambertian re-
mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Nov. 1, 2022. Published November 2022. Originally
flector whose reflectance as a function of wavelength has been
approved in 2009. Last previous edition approved in 2014 as E2719–09 (2014).
determined along with corresponding uncertainties (4).
DOI: 10.1520/E2719-09R22.
The boldface numbers in parentheses refer to the list of references at the end of
this standard.
3 4
Certain commercial equipment, instruments, or materials are identified in this For referenced ASTM standards, visit the ASTM website, www.astm.org, or
guide to foster understanding. Such identification does not imply recommendation contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
or endorsement by ASTM International nor does it imply that the materials or Standards volume information, refer to the standard’s Document Summary page on
equipment identified are necessarily the best available for the purpose. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2719 − 09 (2022)
3.1.6 calibrated optical radiation source (CS), n—optical quantum efficiency to be dependent on the absorbance,
radiation source whose radiance as a function of wavelength concentration, and excitation and emission path lengths of the
hasbeendeterminedalongwithcorrespondinguncertainties (5, sample (9, 10).
6).
3.1.19 Lambertian reflector, n—surface that reflects optical
3.1.7 calibration, n—set of procedures that establishes the radiation according to Lambert’s law, that is, the optical
relationship between quantities measured on an instrument and
radiation is unpolarized and has a radiance that is isotropic or
the corresponding values realized by standards. independent of viewing angle.
3.1.8 certified reference material (CRM), n—material with
3.1.20 limit of detection, n—estimate of the lowest concen-
properties of interest whose values and corresponding uncer-
tration of an analyte that can be measured with a given
tainties have been certified by a standardizing group or
technique, often taken to be the analyte concentration with a
organization. E131
measured signal-to-noise ratio of three.
3.1.9 certified value, n—value for which the certifying body
3.1.21 noise level, n—peak-to-peak noise of a blank.
has the highest confidence in its accuracy in that all known or
3.1.22 photobleaching, n—loss of emission or absorption
suspected sources of bias have been investigated or accounted
intensity by a sample as a result of exposure to optical
for by the certifying body (7).
radiation.
3.1.10 diffuse scatterer, n—material that scatters optical
3.1.22.1 Discussion—Thislosscanbereversibleorirrevers-
radiation in multiple directions; this includes diffuse reflectors,
ible with the latter typically referred to as photodegradation or
whichareoftenLambertian,andscatteringsolutions,whichare
photodecomposition.
not Lambertian.
3.1.23 qualification, n—process producing evidence that an
3.1.11 fluorescenceanisotropy(r),n—measureofthedegree
instrument consistently yields measurements meeting required
of polarization of fluorescence, defined as r=(I – I )/(I +
ll ' ll
specifications and quality characteristics.
2I ), where I and I are the observed fluorescence intensities
' ll '
3.1.24 quantum counter, n—photoluminescent emitter with
when the fluorometer’s emission polarizer is oriented parallel
a quantum efficiency that is independent of excitation wave-
and perpendicular, respectively, to the direction of the polar-
length over a defined spectral range.
ized excitation.
3.1.24.1 Discussion—When a quantum counter is combined
3.1.12 fluorescence band, n—region of a fluorescence spec-
with a detector to give a response proportional to the number
truminwhichtheintensitypassesthroughamaximum,usually
of incident photons, the pair is called a quantum counter
corresponding to a discrete electronic transition.
detector.
3.1.13 fluorescence lifetime, n—parameter describing the
3.1.25 quasi-absolute fluorescence intensity scale,
time decay of the fluorescence intensity of a sample compo-
n—fluorescence intensity scale that has been normalized to the
nent; if a sample decays by first-order kinetics, this is the time
intensity of a fluorescent reference sample or artifact under a
required for its fluorescence intensity and corresponding ex-
fixed set of instrumental and experimental conditions.
cited state population to decrease to 1/e of its initial value.
3.1.25.1 Discussion—This artifact should be known to yield
3.1.14 fluorescence quantum effıciency, n—ratio of the num-
a fluorescence intensity that is reproducible with time and
beroffluorescencephotonsleavinganemittertothenumberof
between instruments under the fixed set of conditions.
photons absorbed.
3.1.26 Raman scattering, n—inelasticscatteringofradiation
3.1.15 fluorescence quantum yield (Φ), n—probabilitythata
(the wavelengths of the scattered and incident radiation are not
molecule or species will fluoresce once it has absorbed a
equal) by a sample that occurs because of changes in the
photon.
polarizability of the relevant bonds of a sample during a
3.1.15.1 Discussion—This quantity is an innate property of
molecular vibration. (See Terminology E131, Raman spec-
the species and is typically calculated for a sample as the ratio
trum.)
of the number of molecules that fluoresce to the number of
3.1.26.1 Discussion—Theradiationbeingscattereddoesnot
molecules that absorbed.
have to be in resonance with electronic transitions in the
3.1.16 flux (or radiant flux or radiant power), n—rate of
sample, unlike fluorescence (11).
propagation of radiant energy typically expressed in Watts.
3.1.27 Rayleigh scattering, n—elasticscatteringofradiation
3.1.17 grating equation, n—relationship between the angle
byasample,thatis,thescatteredradiationhasthesameenergy
ofdiffractionandwavelengthofradiationincidentonagrating,
(same wavelength) as the incident radiation.
that is, mλ = d(sinα + sinβ), where d is the groove spacing on
3.1.28 responsivity, n—ratio of the photocurrent output and
thegrating; αand βaretheanglesoftheincidentanddiffracted
the radiant power collected by an optical radiation detection
wavefronts, respectively, relative to the grating normal; and m
system.
is the diffraction order, which is an integer (8).
3.1.29 sensitivity, n—measure of an instrument’s ability to
3.1.18 inner filter effects, n—decrease in the measured
detect an analyte under a particular set of conditions.
quantum efficiency of a sample as a result of significant
absorptionoftheexcitationbeam,reabsorptionoftheemission 3.1.30 spectral bandwidth (or spectral bandpass or
of the sample by itself, or both, and this causes the measured resolution), n—measure of the capability of a spectrometer to
E2719 − 09 (2022)
separate radiation or resolve spectral peaks of similar wave- cuvette. To check the spectral transmission characteristics,
lengths. (See Terminology E131, resolution.) measure a cuvette’s transmittance in a UV/Vis
spectrophotometer, after filling it with a solvent of interest.
3.1.31 spectral flux (or spectral radiant flux or spectral
Check to insure that the cuvettes being used transmit energy
radiant power), n—flux per unit spectral bandwidth typically
through the entire analytical wavelength range. Many organic
expressed in W/nm.
solvents dissolve plastic, so plastic cuvettes should not be used
3.1.32 spectral responsivity, n—responsivity per unit spec-
in these cases. Standard cuvettes have inner dimensions of
tral bandwidth.
10 mm by 10 mm by 45 mm. If only a small amount of sample
3.1.33 spectral slit width, n—mechanical width of the exit
is available, then microcuvettes can be used. Black self-
slitofaspectrometerdividedbythelineardispersionintheexit
masking quartz microcuvettes are recommended since they
slit plane. E131
require no masking of the optical beam. Cuvette caps or
3.1.34 traceability, n—linking of the value and uncertainty stoppers should be used with volatile or corrosive solvents.
5.2.1 Handling and Cleaning—For highest quality work,
of a measurement to the highest reference standard or value
through an unbroken chain of comparisons, where highest windows should never be touched with bare hands. Clean,
refers to the reference standard whose value and uncertainty powder-free, disposable gloves are recommended. Cuvettes
shouldberinsedseveraltimeswithsolventafteruseandstored
are not dependent on those of any other reference standards,
and unbroken chain of comparisons refers to the requirement wet in the normal solvent system being used. For prolonged
storage, cuvettes should be stored dry, wrapped in lens tissue
that any intermediate reference standards used to trace the
measurement to the highest reference standard must have their and sealed in a container. To clean a cuvette more thoroughly,
it should be filled with an acid, such as 50 % concentrated
values and uncertainties linked to the measurement as well
(12). nitric acid, and allowed to sit for several hours. It should then
be rinsed with deionized water several times to remove all
3.1.35 transfer standard, n—reference standard used to
traces of acid.
transfer the value of one reference standard to a measurement
or to another reference standard. 5.3 Selection of Solvent—Solvents can change the spectral
shape, cause peak broadening, and alter the wavelength posi-
3.1.36 transition dipole moment, n—oscillating dipole mo-
tionofafluorophore (13).Checktoinsurethatthesolventdoes
ment induced in a molecular species by an electromagnetic
not itself absorb or contain impurities at the analytical wave-
wave that is resonant with an energy transition of the species,
length(s). As standard practice, when optimizing a procedure,
for example, an electronic transition.
oneshouldfirstscanthesolventusingtheanalyticalparameters
3.1.36.1 Discussion—Its direction defines that of the transi-
to see if the solvent absorbs or fluoresces in the analytical
tion polarization and its square determines the intensity of the
wavelength range. This will also identify the position of the
transition.
Raman band of the solvent and any second order bands from
the grating. It is essential to examine the quality of solvents
4. Significance and Use
periodically since small traces of contaminants may be enough
4.1 By following the general guidelines (Section 5) and
to produce high blank values.
instrument calibration methods (Sections6–16) in this guide,
5.3.1 Water is the most common solvent and deionized-
usersshouldbeabletomoreeasilyconformtogoodlaboratory
distilled water should always be employed. All other reagents
and manufacturing practices (GXP) and comply with regula-
used in the assay should be carefully controlled and high
tory and QA/QC requirements, related to fluorescence mea-
quality or spectrophotometric grades are recommended.
surements.
5.3.2 Solvents should not be stored in plastic containers
4.2 Each instrument parameter needing calibration (for
sinceleachingoforganicadditivesandplasticizerscanproduce
exam
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