1、Designation: E2719 09 (Reapproved 2014)Standard Guide forFluorescenceInstrument Calibration and Qualification1This standard is issued under the fixed designation E2719; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of la
2、st revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide (1)2lists the available materials and methodsfor each type of calibration or correction for fluorescenceinstr
3、uments (spectral emission correction, wavelengthaccuracy, and so forth) with a general description, the level ofquality, precision and accuracy attainable, limitations, anduseful references given for each entry.1.2 The listed materials and methods are intended for thequalification of fluorometers as
4、 part of complying with regu-latory and other quality assurance/quality control (QA/QC)requirements.1.3 Precision and accuracy or uncertainty are given at a 1 confidence level and are approximated in cases where thesevalues have not been well established.31.4 The values stated in SI units are to be
5、regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and det
6、ermine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:4E131 Terminology Relating to Molecular SpectroscopyE388 Test Method for Wavelength Accuracy and SpectralBandwidth of Fluorescence SpectrometersE578 Test Method for Linearity of Fluorescence Me
7、asuringSystemsE579 Test Method for Limit of Detection of Fluorescence ofQuinine Sulfate in Solution3. Terminology3.1 Definitions(2):3.1.1 absorption coeffcient (), na measure of absorptionof radiant energy from an incident beam as it traverses anabsorbing medium according to Bouguers law, I/Io= e-b,
8、where I and Ioare the transmitted and incident intensities,respectively, and b is the path length of the beam through thesample. E1313.1.1.1 DiscussionNote that transmittance T = I/Ioandabsorbance A = log T.3.1.2 absorptivity (a), nthe absorbance divided by theproduct of the concentration of the sub
9、stance and the samplepathlength, a = A/bc. E1313.1.3 Beer-Lambert law, nrelates the dependence of theabsorbance (A) of a sample on its path length (see absorptioncoeffcient, ) and concentration (c), such that A = abc.3.1.3.1 DiscussionAlso called Beers law or Beer-Lambert-Bouquer law. E1313.1.4 cali
10、brated detector (CD), noptical radiation detectorwhose responsivity as a function of wavelength has beendetermined along with corresponding uncertainties (3).3.1.5 calibrated diffuse reflector (CR), nLambertian re-flector whose reflectance as a function of wavelength has beendetermined along with co
11、rresponding uncertainties (4).3.1.6 calibrated optical radiation source (CS), nopticalradiation source whose radiance as a function of wavelengthhas been determined along with corresponding uncertainties (5,6).3.1.7 calibration, nset of procedures that establishes therelationship between quantities
12、measured on an instrument andthe corresponding values realized by standards.3.1.8 certified reference material (CRM), nmaterial withproperties of interest whose values and corresponding uncer-tainties have been certified by a standardizing group ororganization. E1313.1.9 certified value, nvalue for
13、which the certifying bodyhas the highest confidence in its accuracy in that all known or1This guide is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and Separation Science and is the direct responsibility of Subcom-mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spec
14、troscopy.Current edition approved May 1, 2014. Published June 2014. Originallyapproved in 2009. Last previous edition approved in 2009 as E271909. DOI:10.1520/E2719-09R14.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3Certain commercial equipment, in
15、struments, or materials are identified in thisguide to foster understanding. Such identification does not imply recommendationor endorsement by ASTM International nor does it imply that the materials orequipment identified are necessarily the best available for the purpose.4For referenced ASTM stand
16、ards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA
17、 19428-2959. United States1suspected sources of bias have been investigated or accountedfor by the certifying body (7).3.1.10 diffuse scatterer, nmaterial that scatters opticalradiation in multiple directions; this includes diffuse reflectors,which are often Lambertian, and scattering solutions, whi
18、ch arenot Lambertian.3.1.11 fluorescence anisotropy (r), nmeasure of the degreeof polarization of fluorescence, defined as r =(Ill I)/(Ill+2I), where Illand Iare the observed fluorescence intensitieswhen the fluorometers emission polarizer is oriented paralleland perpendicular, respectively, to the
19、direction of the polar-ized excitation.3.1.12 fluorescence band, nregion of a fluorescence spec-trum in which the intensity passes through a maximum, usuallycorresponding to a discrete electronic transition.3.1.13 fluorescence lifetime, nparameter describing thetime decay of the fluorescence intensi
20、ty of a sample compo-nent; if a sample decays by first-order kinetics, this is the timerequired for its fluorescence intensity and corresponding ex-cited state population to decrease to 1/e of its initial value.3.1.14 fluorescence quantum effciency, nratio of the num-ber of fluorescence photons leav
21、ing an emitter to the number ofphotons absorbed.3.1.15 fluorescence quantum yield (), nprobability that amolecule or species will fluoresce once it has absorbed aphoton.3.1.15.1 DiscussionThis quantity is an innate property ofthe species and is typically calculated for a sample as the ratioof the nu
22、mber of molecules that fluoresce to the number ofmolecules that absorbed.3.1.16 flux (or radiant flux or radiant power), nrate ofpropagation of radiant energy typically expressed in Watts.3.1.17 grating equation, nrelationship between the angleof diffraction and wavelength of radiation incident on a
23、 grating,that is, m = d(sin + sin), where d is the groove spacing onthe grating; and are the angles of the incident and diffractedwavefronts, respectively, relative to the grating normal; and mis the diffraction order, which is an integer (8).3.1.18 inner filter effects, ndecrease in the measuredqua
24、ntum efficiency of a sample as a result of significantabsorption of the excitation beam, reabsorption of the emissionof the sample by itself, or both, and this causes the measuredquantum efficiency to be dependent on the absorbance,concentration, and excitation and emission path lengths of thesample
25、 (9, 10).3.1.19 Lambertian reflector, nsurface that reflects opticalradiation according to Lamberts law, that is, the opticalradiation is unpolarized and has a radiance that is isotropic orindependent of viewing angle.3.1.20 limit of detection, nestimate of the lowest concen-tration of an analyte th
26、at can be measured with a giventechnique, often taken to be the analyte concentration with ameasured signal-to-noise ratio of three.3.1.21 noise level, npeak-to-peak noise of a blank.3.1.22 photobleaching, nloss of emission or absorptionintensity by a sample as a result of exposure to opticalradiati
27、on.3.1.22.1 DiscussionThis loss can be reversible or irrevers-ible with the latter typically referred to as photodegradation orphotodecomposition.3.1.23 qualification, nprocess producing evidence that aninstrument consistently yields measurements meeting requiredspecifications and quality characteri
28、stics.3.1.24 quantum counter, nphotoluminescent emitter witha quantum efficiency that is independent of excitation wave-length over a defined spectral range.3.1.24.1 DiscussionWhen a quantum counter is combinedwith a detector to give a response proportional to the numberof incident photons, the pair
29、 is called a quantum counterdetector.3.1.25 quasi-absolute fluorescence intensity scale,nfluorescence intensity scale that has been normalized to theintensity of a fluorescent reference sample or artifact under afixed set of instrumental and experimental conditions.3.1.25.1 DiscussionThis artifact s
30、hould be known to yielda fluorescence intensity that is reproducible with time andbetween instruments under the fixed set of conditions.3.1.26 Raman scattering, ninelastic scattering of radiation(the wavelengths of the scattered and incident radiation are notequal) by a sample that occurs because of
31、 changes in thepolarizability of the relevant bonds of a sample during amolecular vibration. (See Terminology E131, Raman spec-trum.)3.1.26.1 DiscussionThe radiation being scattered does nothave to be in resonance with electronic transitions in thesample, unlike fluorescence (11).3.1.27 Rayleigh sca
32、ttering, nelastic scattering of radiationby a sample, that is, the scattered radiation has the same energy(same wavelength) as the incident radiation.3.1.28 responsivity, nratio of the photocurrent output andthe radiant power collected by an optical radiation detectionsystem.3.1.29 sensitivity, nmea
33、sure of an instruments ability todetect an analyte under a particular set of conditions.3.1.30 spectral bandwidth (or spectral bandpass orresolution), nmeasure of the capability of a spectrometer toseparate radiation or resolve spectral peaks of similar wave-lengths. (See Terminology E131, resolutio
34、n.)3.1.31 spectral flux (or spectral radiant flux or spectralradiant power), nflux per unit spectral bandwidth typicallyexpressed in W/nm.3.1.32 spectral responsivity, nresponsivity per unit spec-tral bandwidth.3.1.33 spectral slit width, nmechanical width of the exitslit of a spectrometer divided b
35、y the linear dispersion in the exitslit plane. E1313.1.34 traceability, nlinking of the value and uncertaintyof a measurement to the highest reference standard or valuethrough an unbroken chain of comparisons, where highestE2719 09 (2014)2refers to the reference standard whose value and uncertaintya
36、re not dependent on those of any other reference standards,and unbroken chain of comparisons refers to the requirementthat any intermediate reference standards used to trace themeasurement to the highest reference standard must have theirvalues and uncertainties linked to the measurement as well(12)
37、.3.1.35 transfer standard, nreference standard used totransfer the value of one reference standard to a measurementor to another reference standard.3.1.36 transition dipole moment, noscillating dipole mo-ment induced in a molecular species by an electromagneticwave that is resonant with an energy tr
38、ansition of the species,for example, an electronic transition.3.1.36.1 DiscussionIts direction defines that of the transi-tion polarization and its square determines the intensity of thetransition.4. Significance and Use4.1 By following the general guidelines (Section 5) andinstrument calibration me
39、thods (Sections 616) in this guide,users should be able to more easily conform to good laboratoryand manufacturing practices (GXP) and comply with regula-tory and QA/QC requirements, related to fluorescence mea-surements.4.2 Each instrument parameter needing calibration (forexample, wavelength, spec
40、tral responsivity) is treated in aseparate section.Alist of different calibration methods is givenfor each instrument parameter with a brief usage procedure.Precautions, achievable precision and accuracy, and otheruseful information are also given for each method to allowusers to make a more informe
41、d decision as to which method isthe best choice for their calibration needs. Additional detailsfor each method can be found in the references given.5. General Guidelines5.1 General areas of concern and precautions to minimizeerrors for fluorescence measurements are given by topic. Alltopics applicab
42、le to a users samples, measurements andanalysis should be considered.5.2 CuvettesVarious types of cuvettes or optical “cells”are available. They vary in material composition and in size.The former will determine the effective spectral range of thecuvette. To check the spectral transmission character
43、istics,measure a cuvettes transmittance in a UV/Visspectrophotometer, after filling it with a solvent of interest.Check to insure that the cuvettes being used transmit energythrough the entire analytical wavelength range. Many organicsolvents dissolve plastic, so plastic cuvettes should not be usedi
44、n these cases. Standard cuvettes have inner dimensions of 10mm 10 mm 45 mm. If only a small amount of sample isavailable, then microcuvettes can be used. Black self-maskingquartz microcuvettes are recommended since they require nomasking of the optical beam. Cuvette caps or stoppers shouldbe used wi
45、th volatile or corrosive solvents.5.2.1 Handling and CleaningFor highest quality work,windows should never be touched with bare hands. Clean,powder-free, disposable gloves are recommended. Cuvettesshould be rinsed several times with solvent after use and storedwet in the normal solvent system being
46、used. For prolongedstorage, cuvettes should be stored dry, wrapped in lens tissueand sealed in a container. To clean a cuvette more thoroughly,it should be filled with an acid, such as 50 % concentratednitric acid, and allowed to sit for several hours. It should thenbe rinsed with deionized water se
47、veral times to remove alltraces of acid.5.3 Selection of SolventSolvents can change the spectralshape, cause peak broadening, and alter the wavelength posi-tion of a fluorophore (13). Check to insure that the solvent doesnot itself absorb or contain impurities at the analytical wave-length(s). As st
48、andard practice, when optimizing a procedure,one should first scan the solvent using the analytical parametersto see if the solvent absorbs or fluoresces in the analyticalwavelength range. This will also identify the position of theRaman band of the solvent and any second order bands fromthe grating
49、. It is essential to examine the quality of solventsperiodically since small traces of contaminants may be enoughto produce high blank values.5.3.1 Water is the most common solvent and deionized-distilled water should always be employed. All other reagentsused in the assay should be carefully controlled and highquality or spectrophotometric grades are recommended.5.3.2 Solvents should not be stored in plastic containerssince leaching of organic additives and plasticizers can producehigh blank values.5.3.3
