ASTM E2719-09
(Guide)Standard Guide for Fluorescence—Instrument Calibration and Qualification
Standard Guide for Fluorescence<span class='unicode'>—</span>Instrument Calibration and Qualification
SIGNIFICANCE AND USE
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.
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) 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.
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 and health practices and determine the applicability of regulatory limitations prior to use.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: E2719 − 09
StandardGuide 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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E578Test Method for Linearity of Fluorescence Measuring
Systems
1.1 Thisguide (1) liststheavailablematerialsandmethods
E579TestMethodforLimitofDetectionofFluorescenceof
for each type of calibration or correction for fluorescence
Quinine Sulfate in Solution
instruments (spectral emission correction, wavelength
accuracy, and so forth) with a general description, the level of
3. Terminology
quality, precision and accuracy attainable, limitations, and
3.1 Definitions(2):
useful references given for each entry.
3.1.1 absorption coeffıcient (α), n—a measure of absorption
1.2 The listed materials and methods are intended for the
of radiant energy from an incident beam as it traverses an
qualification of fluorometers as part of complying with regu-
-αb
absorbing medium according to Bouguer’s law, I/I = e ,
o
latory and other quality assurance/quality control (QA/QC)
where I and I are the transmitted and incident intensities,
o
requirements.
respectively, and b is the path length of the beam through the
1.3 Precision and accuracy or uncertainty are given at a 1 σ
sample. E131
confidence level and are approximated in cases where these
3.1.1.1 Discussion—Note that transmittance T = I/I and
o
values have not been well established.
absorbance A = –log T.
1.4 The values stated in SI units are to be regarded as
3.1.2 absorptivity (a), n—the absorbance divided by the
standard. No other units of measurement are included in this
product of the concentration of the substance and the sample
standard.
pathlength, a = A/bc. E131
1.5 This standard does not purport to address all of the
3.1.3 Beer-Lambert law, n—relates the dependence of the
safety concerns, if any, associated with its use. It is the
absorbance (A) of a sample on its path length (see absorption
responsibility of the user of this standard to establish appro-
coeffıcient, α) and concentration (c), such that A =abc.
priate safety and health practices and determine the applica-
3.1.3.1 Discussion—Also called Beer’s law or Beer-
bility of regulatory limitations prior to use.
Lambert-Bouquer law. E131
3.1.4 calibrated detector (CD), n—opticalradiationdetector
2. Referenced Documents
whose responsivity as a function of wavelength has been
2.1 ASTM Standards: determined along with corresponding uncertainties (3).
E131Terminology Relating to Molecular Spectroscopy
3.1.5 calibrated diffuse reflector (CR), n—Lambertian re-
E388Test Method for Wavelength Accuracy and Spectral
flector whose reflectance as a function of wavelength has been
Bandwidth of Fluorescence Spectrometers
determined along with corresponding uncertainties (4).
3.1.6 calibrated optical radiation source (CS), n—optical
radiation source whose radiance as a function of wavelength
This guide is under the jurisdiction of ASTM Committee E13 on Molecular
hasbeendeterminedalongwithcorrespondinguncertainties (5,
Spectroscopy and Separation Science and is the direct responsibility of Subcom-
6).
mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
Current edition approved Oct. 1, 2009. Published November 2009. DOI:
3.1.7 calibration, n—set of procedures that establishes the
10.1520/E2719-09.
relationshipbetweenquantitiesmeasuredonaninstrumentand
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the corresponding values realized by standards.
this standard.
Certain commercial equipment, instruments, or materials are identified in this
3.1.8 certified reference material (CRM), n—material with
guide to foster understanding. Such identification does not imply recommendation
properties of interest whose values and corresponding uncer-
or endorsement by ASTM International nor does it imply that the materials or
tainties have been certified by a standardizing group or
equipment identified are necessarily the best available for the purpose.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
organization. E131
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3.1.9 certified value, n—valueforwhichthecertifyingbody
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. has the highest confidence in its accuracy in that all known or
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2719 − 09
suspected sources of bias have been investigated or accounted 3.1.22 photobleaching, n—loss of emission or absorption
for by the certifying body (7). intensity by a sample as a result of exposure to optical
radiation.
3.1.10 diffuse scatterer, n—material that scatters optical
3.1.22.1 Discussion—Thislosscanbereversibleorirrevers-
radiationinmultipledirections;thisincludesdiffusereflectors,
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—Whenaquantumcounteriscombined
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—Thisartifactshouldbeknowntoyield
3.1.14 fluorescence quantum effıciency, n—ratioofthenum-
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 (thewavelengthsofthescatteredandincidentradiationarenot
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
molecular vibration. (See Terminology E131, Raman spec-
3.1.15.1 Discussion—This quantity is an innate property of
trum.)
the species and is typically calculated for a sample as the ratio
3.1.26.1 Discussion—Theradiationbeingscattereddoesnot
of the number of molecules that fluoresce to the number of
molecules that absorbed. have to be in resonance with electronic transitions in the
sample, unlike fluorescence (11).
3.1.16 flux (or radiant flux or radiant power), n—rate of
3.1.27 Rayleigh scattering, n—elasticscatteringofradiation
propagation of radiant energy typically expressed in Watts.
byasample,thatis,thescatteredradiationhasthesameenergy
3.1.17 grating equation, n—relationship between the angle
(same wavelength) as the incident radiation.
ofdiffractionandwavelengthofradiationincidentonagrating,
3.1.28 responsivity, n—ratio of the photocurrent output and
that is, mλ = d(sinα + sinβ), where d is the groove spacing on
the radiant power collected by an optical radiation detection
thegrating; αand βaretheanglesoftheincidentanddiffracted
system.
wavefronts, respectively, relative to the grating normal; and m
is the diffraction order, which is an integer (8).
3.1.29 sensitivity, n—measure of an instrument’s ability to
detect an analyte under a particular set of conditions.
3.1.18 inner filter effects, n—decrease in the measured
3.1.30 spectral bandwidth (or spectral bandpass or resolu-
quantum efficiency of a sample as a result of significant
absorptionoftheexcitationbeam,reabsorptionoftheemission tion) , n—measure of the capability of a spectrometer to
separate radiation or resolve spectral peaks of similar wave-
of the sample by itself, or both, and this causes the measured
quantum efficiency to be dependent on the absorbance, lengths. (See Terminology E131, resolution.)
concentration, and excitation and emission path lengths of the
3.1.31 spectral flux (or spectral radiant flux or spectral
sample (9, 10).
radiant power), n—flux per unit spectral bandwidth typically
expressed in W/nm.
3.1.19 Lambertian reflector, n—surface that reflects optical
radiation according to Lambert’s law, that is, the optical
3.1.32 spectral responsivity, n—responsivity per unit spec-
radiation is unpolarized and has a radiance that is isotropic or
tral bandwidth.
independent of viewing angle.
3.1.33 spectral slit width, n—mechanical width of the exit
3.1.20 limit of detection, n—estimate of the lowest concen-
slitofaspectrometerdividedbythelineardispersionintheexit
tration of an analyte that can be measured with a given
slit plane. E131
technique, often taken to be the analyte concentration with a
3.1.34 traceability, n—linking of the value and uncertainty
measured signal-to-noise ratio of three.
of a measurement to the highest reference standard or value
3.1.21 noise level, n—peak-to-peak noise of a blank. through an unbroken chain of comparisons, where highest
E2719 − 09
TABLE 1 Spectral Transmission Characteristics of
refers to the reference standard whose value and uncertainty
Cuvette Materials
are not dependent on those of any other reference standards,
Wavelength Range (nm)
and unbroken chain of comparisons refers to the requirement
Glass 350–2500
that any intermediate reference standards used to trace the
Near Infrared Quartz 220–3800
measurement to the highest reference standard must have their
Far UV Quartz 170–2700
values and uncertainties linked to the measurement as well
Polystyrene 400–1000
Acrylic 280–1000
(12).
3.1.35 transfer standard, n—reference standard used to
transfer the value of one reference standard to a measurement
powder-free, disposable gloves are recommended. Cuvettes
or to another reference standard.
shouldberinsedseveraltimeswithsolventafteruseandstored
3.1.36 transition dipole moment, n—oscillating dipole mo-
wet in the normal solvent system being used. For prolonged
ment induced in a molecular species by an electromagnetic
storage, cuvettes should be stored dry, wrapped in lens tissue
wave that is resonant with an energy transition of the species,
and sealed in a container. To clean a cuvette more thoroughly,
for example, an electronic transition.
it should be filled with an acid, such as 50% concentrated
3.1.36.1 Discussion—Its direction defines that of the transi-
nitric acid, and allowed to sit for several hours. It should then
tion polarization and its square determines the intensity of the
be rinsed with deionized water several times to remove all
transition.
traces of acid.
5.3 Selection of Solvent—Solvents can change the spectral
4. Significance and Use
shape, cause peak broadening, and alter the wavelength posi-
4.1 By following the general guidelines (Section 5) and
tionofafluorophore (13).Checktoinsurethatthesolventdoes
instrument calibration methods (Sections 6-16) in this guide,
not itself absorb or contain impurities at the analytical wave-
usersshouldbeabletomoreeasilyconformtogoodlaboratory
length(s). As standard practice, when optimizing a procedure,
and manufacturing practices (GXP) and comply with regula-
oneshouldfirstscanthesolventusingtheanalyticalparameters
tory and QA/QC requirements, related to fluorescence mea-
to see if the solvent absorbs or fluoresces in the analytical
surements.
wavelength range. This will also identify the position of the
Raman band of the solvent and any second order bands from
4.2 Each instrument parameter needing calibration (for
example, wavelength, spectral responsivity) is treated in a the grating. It is essential to examine the quality of solvents
periodicallysincesmalltracesofcontaminantsmaybeenough
separatesection.Alistofdifferentcalibrationmethodsisgiven
for each instrument parameter with a brief usage procedure. to produce high blank values.
5.3.1 Water is the most common solvent and deionized-
Precautions, achievable precision and accuracy, and other
useful information are also given for each method to allow distilled water should always be employed.All other reagents
used in the assay should be carefully controlled and high
users to make a more informed decision as to which method is
the best choice for their calibration needs. Additional details quality or spectrophotometric grades are recommended.
5.3.2 Solvents should not be stored in plastic containers
for each method can be found in the references given.
sinceleachingoforganicadditivesandplasticizerscanproduce
5. General Guidelines high blank values.
5.3.3 Reagent blanks should be measured during the ana-
5.1 General areas of concern and precautions to minimize
lytical procedure and the actual value of the blank determined
errors for fluorescence measurements are given by topic. All
before the instrument is zeroed.
topics applicable to a user’s samples, measurements and
analysis should be considered. 5.4 Other Contaminants:
5.4.1 Soaking glassware in detergent solutions is a general
5.2 Cuvettes—Various types of cuvettes or optical “cells”
method of cleaning. Some commercial preparations are
are available. They vary in material composition and in size.
strongly fluorescent. Before use, the fluorescence characteris-
The former will determine the effective spectral range of the
ticsofadilutesolutionofthedetergentshouldbemeasured,so
cuvette. To check the spectr
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