1、Designation: E2719 09Standard 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 last revision. A num
2、ber 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 fluorescenceinstruments (spectral e
3、mission correction, wavelength accu-racy, 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 part of complyi
4、ng with regu-latory and other quality assurance/quality control (QA/QC)requirements.1.3 Precision and accuracy or uncertainty are given at a 1 sconfidence level and are approximated in cases where thesevalues have not been well established.31.4 The values stated in SI units are to be regarded asstan
5、dard. 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 determine the appl
6、ica-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 MeasuringSystemsE
7、579 Test Method for Limit of Detection of Fluorescence ofQuinine Sulfate in Solution3. Terminology3.1 Definitions (2):3.1.1 absorption coeffcient (a), na measure of absorptionof radiant energy from an incident beam as it traverses anabsorbing medium according to Bouguers law, I/Io= e-ab,where I and
8、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 substance and t
9、he 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, a) and concentration (c), such that A = abc.3.1.3.1 DiscussionAlso called Beers law or Beer-Lambert-Bouquer law. E1313.1.4 calibrated dete
10、ctor (CD), noptical radiation detec-tor whose 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 correspondi
11、ng 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 measured
12、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. E1311This guide is under the jurisdiction of AS
13、TM Committee E13 on MolecularSpectroscopy and Separation Science and is the direct responsibility of Subcom-mittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Oct. 1, 2009. Published November 2009. DOI:10.1520/E2719-09.2The boldface numbers in parentheses
14、refer to the list of references at the end ofthis standard.3Certain commercial equipment, instruments, 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 orequipm
15、ent identified are necessarily the best available for the purpose.4For referenced ASTM standards, 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 web
16、site.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.9 certified value, nvalue for which the certifying bodyhas the highest confidence in its accuracy in that all known orsuspected sources of bias have been investigated or accoun
17、tedfor 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, which arenot Lambertian.3.1.11 fluorescence anisotropy (r), nmeasure of the de-gree of p
18、olarization of fluorescence, defined as r =(Ill I)/(Ill+2I), where Illand Iare the observed fluorescenceintensities when the fluorometers emission polarizer is ori-ented parallel and perpendicular, respectively, to the directionof the polarized excitation.3.1.12 fluorescence band, nregion of a fluor
19、escence 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 intensity of a sample compo-nent; if a sample decays by first-order kinetics, this is the
20、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 leaving an emitter to the number ofphotons absorbed.3.1.15 fluorescence quantum yield (
21、F), 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 number of molecules that fluoresce to the number ofmolecules that absorbed.3.1.16 fl
22、ux (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 grating,that is, ml = d (sina + sinb), where d is the groove spacing onthe gratin
23、g; a and b 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 measuredquantum efficiency of a sample as a result of significantabsorption of the ex
24、citation beam, reabsorption of the emissionof the sample by itself, or both, and this causes the measuredquantum efficiency to be dependent on the absorbance, con-centration, and excitation and emission path lengths of thesample (9, 10).3.1.19 Lambertian reflector, nsurface that reflects opticalradi
25、ation 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 that can be measured with a giventechnique, often taken to be the analyte
26、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 opticalradiation.3.1.22.1 DiscussionThis loss can be reversible or irre-versible with
27、the latter typically referred to as photodegrada-tion or photodecomposition.3.1.23 qualification, nprocess producing evidence that aninstrument consistently yields measurements meeting requiredspecifications and quality characteristics.3.1.24 quantum counter, nphotoluminescent emitter witha quantum
28、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 is called a quantum counterdetector.3.1.25 quasi-absolute fluorescenc
29、e 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 should be known to yielda fluorescence intensity that is reproducible w
30、ith time andbetween instruments under the fixed set of conditions.3.1.26 Raman scattering, ninelastic scattering of radia-tion (the wavelengths of the scattered and incident radiation arenot equal) by a sample that occurs because of changes in thepolarizability of the relevant bonds of a sample duri
31、ng 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 scattering, nelastic scattering of radiationby a sample, that is, the s
32、cattered 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, nmeasure of an instruments ability todetect an analyte under a particula
33、r set of conditions.3.1.30 spectral bandwidth (or spectral bandpass or resolu-tion), nmeasure of the capability of a spectrometer toseparate radiation or resolve spectral peaks of similar wave-lengths. (See Terminology E131, resolution.)3.1.31 spectral flux (or spectral radiant flux or spectralradia
34、nt 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 by the linear dispersion in the exitslit plane. E1313.1.34 traceabi
35、lity, nlinking of the value and uncertaintyof a measurement to the highest reference standard or valuethrough an unbroken chain of comparisons, where highestrefers to the reference standard whose value and uncertaintyare not dependent on those of any other reference standards,and unbroken chain of c
36、omparisons refers to the requirementE2719 092that 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).3.1.35 transfer standard, nreference standard used totransfer the value
37、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 transition of the species,for example, an electronic transition.3.1.36.1 Di
38、scussionIts direction defines that of the tran-sition polarization and its square determines the intensity of thetransition.4. Significance and Use4.1 By following the general guidelines (Section 5) andinstrument calibration methods (Sections 6-16) in this guide,users should be able to more easily c
39、onform 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, spectral responsivity) is treated in aseparate section.Alist of different ca
40、libration 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 informed decision as to which method isthe best choice for their calibration ne
41、eds. 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 applicable to a users samples, measurements andanalysis should be considered.5.2
42、 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 characteristics,measure a cuvettes transmittance in a UV/Vis spectrophotom-eter,
43、after filling it with a solvent of interest. Check to insurethat the cuvettes being used transmit energy through the entireanalytical wavelength range. Many organic solvents dissolveplastic, so plastic cuvettes should not be used in these cases.Standard cuvettes have inner dimensions of 10 mm 3 10 m
44、m3 45 mm. If only a small amount of sample is available, thenmicrocuvettes can be used. Black self-masking quartz microcu-vettes are recommended since they require no masking of theoptical beam. Cuvette caps or stoppers should be used withvolatile or corrosive solvents.5.2.1 Handling and CleaningFor
45、 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 used. For prolongedstorage, cuvettes should be stored dry, wrapp
46、ed 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 several times to remove alltraces of acid.5.3 Selection of Solvent
47、Solvents 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 standard practice, when optimizing a procedure,one should first sc
48、an 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. It is essential to examine the quality of solventsperiodically
49、 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 Reagent blanks should be measured during the ana-lytical procedure and the actual
