1、Designation: E3029 15Standard Practice forDetermining Relative Spectral Correction Factors forEmission Signal of Fluorescence Spectrometers1This standard is issued under the fixed designation E3029; the number immediately following the designation indicates the year oforiginal adoption or, in the ca
2、se of revision, the year of last 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 practice (1)2describes three methods for determin-ing the relative spectral correctio
3、n factors for grating-basedfluorescence spectrometers in the ultraviolet-visible spectralrange. These methods are intended for instruments with a0/90 transmitting sample geometry. Each method uses dif-ferent types of transfer standards, including 1) a calibrated lightsource (CS), 2) a calibrated det
4、ector (CD) and a calibrateddiffuse reflector (CR), and 3) certified reference materials(CRMs). The wavelength region covered by the differentmethods ranges from 250 to 830 nm with some methods havinga broader range than others. Extending these methods to thenear infrared (NIR) beyond 830 nm will be
5、discussed briefly,where appropriate. These methods were designed for scanningfluorescence spectrometers with a single channel detector, butcan also be used with a multichannel detector, such as a diodearray or a CCD.1.2 The values stated in SI units are to be regarded asstandard. No other units of m
6、easurement are included in thisstandard.1.3 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 applica-bility of regulatory
7、limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3E131 Terminology Relating to Molecular SpectroscopyE388 Test Method for Wavelength Accuracy and SpectralBandwidth of Fluorescence SpectrometersE578 Test Method for Linearity of Fluorescence MeasuringSystemsE2719 Guide for Fluorescen
8、ceInstrument Calibration andQualification3. Significance and Use (Intro)3.1 Calibration of the responsivity of the detection systemfor emission (EM) as a function of EM wavelength (EM), alsoreferred to as spectral correction of emission, is necessary forsuccessful quantification when intensity ratio
9、s at different EMwavelengths are being compared or when the true shape orpeak maximum position of an EM spectrum needs to beknown. Such calibration methods are given here and summa-rized in Table 1. This type of calibration is necessary becausethe spectral responsivity of a detection system can chan
10、gesignificantly over its useful wavelength range (see Fig. 1). It ishighly recommended that the wavelength accuracy (see TestMethod E388) and the linear range of the detection system (seeGuide E2719 and Test Method E578) be determined beforespectral calibration is performed and that appropriate step
11、s aretaken to insure that all measured intensities during this cali-bration are within the linear range. For example, when usingwide slit widths in the monochromators, attenuators may beneeded to attenuate the excitation beam or emission, thereby,decreasing the fluorescence intensity at the detector
12、. Also notethat when using an EM polarizer, the spectral correction foremission is dependent on the polarizer setting. (2)Itisimportant to use the same instrument settings for all of thecalibration procedures mentioned here, as well as for subse-quent sample measurements.3.2 When using CCD or diode
13、array detectors with aspectrometer for EMselection, the spectral correction factorsare dependent on the grating position of the spectrometer.Therefore, the spectral correction profile versus EMmust bedetermined separately for each grating position used. (3)3.3 Instrument manufacturers often provide
14、an automatedprocedure and calculation for a spectral correction function foremission, or they may supply a correction that was determinedat the factory. This correction can often be applied duringspectral collection or as a post-collection correction. The user1This practice is under the jurisdiction
15、 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 Spectroscopy.Current edition approved Sept. 1, 2015. Published October 2015. DOI: 10.1520/E3029-152The boldface numbers in parent
16、heses refer to a list of references at the end ofthis standard.3For 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 websit
17、e.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1should be advised to verify that the automated vendor proce-dure and calculation or supplied correction are performed anddetermined according to the guidelines given within thisstandar
18、d.4. Calibrated Optical Radiation Source (CS) Method (seeTest Method E578,(4-6, 14)4.1 Materials:4.1.1 A calibrated tungsten lamp is most commonly used asa CS in the visible region due to its high intensity and broad,featureless spectral profile. Its intensity falls off quickly in theultraviolet (UV
19、) region, but it can typically be used down to350 nm or so. It also displays a high intensity in the nearinfrared, peaking at about 1000 nm. Its intensity graduallydecreases beyond 1000 nm, but continues to have significantintensity out to about 2500 nm. A calibrated deuterium lampcan be used to ext
20、end farther into the UV with an effectiverange from about 200 to 380 nm. The effective range of a CSis dependent on the intensity of the CS and the sensitivity of thedetection system. This range can be determined by measuringthe low-signal regions where the signal profile of the light fromthe CS bec
21、omes flat or indistinguishable from the backgroundsignal, implying that the signal afforded by the CS is notmeasurable in these EMregions.4.1.2 A calibrated reflector (CR) is often used to reflect thelight from the CS into the emission detection system. A diffusereflector made of compressed or sinte
22、red polytetrafluoroethyl-ene (PTFE) is most commonly used as a CR, due to its nearlyLambertian reflectance, which prevents both polarization andspatial dependence of the reflectance. In addition, PTFEpossesses a reflectance profile that is nearly flat, changing byless than 10 % from 250 to 2500 nm.
23、For a CS and a CR,“calibrated” implies that the spectral radiance and the spectralreflectance, respectively, are known (calibrated wavelengthdependence of the spectral radiant factor including measure-ment uncertainty) and traceable to the SI (International Systemof Units). This is commonly done thr
24、ough certification of thesevalues by a national metrology institute (NMI). (15, 16, 7)4.2 Procedure:4.2.1 Direct the optical radiation from a CS into the EMdetection system by placing the CS at the sample position. Ifthe CS is too large to be placed at the sample position, place aCR at the sample po
25、sition to reflect the optical radiation fromthe CS into the EM detection system. Ensure that the CS isaligned such that its light is centered on the entrance slit of theEMselector, and on all optics it encounters before the entranceslit. Ideally, the light should fully and uniformly fill theTABLE 1
26、Summary of Methods for Determining Spectral Correction of Detection System ResponsivityNOTE 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
27、immediately-usable format; “Uncertainty” is the estimated expanded (k=2) totaluncertainty; “Caveats” refer to important information that a user should know about the method before attempting to use it; “Certified Values” refers towhether or not the material/hardware is supplied with appropriate valu
28、es 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 EMDrop-In Off-Shelf Uncertainty Caveats Certified Values Ref.CS UV-NIR N Y 5% difficultsetup Y E578,(3-6)CD+CR UV-NIR N Maybe 1
29、0 % difficult setup Y E578,(4, 5, 7)CRMs UV-NIR Y Y 5 % Y E131,(8-13)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 Spectrumto
30、 Obtain its True Spectral Shape (Relative Intensities).E3029 152entrance slit. Make sure that the detection system is stilloperated within its linear range (see 3.1).NOTE 1Correction factors, supplied by the manufacturer and auto-matically applied by the software to the collected spectrum, must besw
31、itched off for the signal channel during this procedure.4.2.2 Scan the EM-selector over the EM region of interest,using the same instrument settings as employed with thesubsequent measurement of the fluorescence of the sample, andcollect the signal channel output (S).4.2.3 Use the known radiance of
32、the CS incident on thedetection system (L) to calculate the relative correction factor(CCS), such that CCS= L/S. Note that Lmay be replaced by thespectral irradiance or the spectral radiant flux, since thecorrection factors determined herein are relative, not absolute.The corrected EM intensity is e
33、qual to the product of the signaloutput of the sample (S) and CCS. Since CCSvalues are relativecorrection factors, they can be scaled by any constant. Forinstance, it is often useful to scale them with a constant thatgives a CCSvalue of one at a particular EM.4.2.4 Note that L is given in power unit
34、s, not photon units,whereas, the units for S and S are either in power or photonunits depending on whether your detector measures an analogor a digital (photon counting) signal, respectively. In eithercase, the corrected signal will be in power units, so aconversion, that is, dividing the corrected
35、signal by EM,isnecessary if photon units are needed.5. Calibrated Detector (CD) with Calibrated ReflectorMethod (see Test Method E578,(4, 5, 17)5.1 Materials:5.1.1 A calibrated photodiode, by itself, as part of a trapdetector or mounted in an integrating sphere, is most com-monly used as a calibrate
36、d detector (CD). Using a trap detectoror photodiode with integrating sphere is typically more accu-rate than using a photodiode alone. A Si photodiode covers therange from 200 to 1100 nm. An InGaAs or Ge photodiode canbe used in the NIR from 800 to 1700 nm. For a CD,“calibrated” implies that the wav
37、elength dependence of thespectral responsivity is known, its associated uncertaintieshave been determined and the measurements are traceable tothe SI. This is typically done through values certified by anNMI. (18, 19) A photodiode usually outputs a current that isproportional to the power of the lig
38、ht incident on it.5.1.2 Alternatively, a quantum counter solution can be usedinstead of a CD. (6, 20, 8) This is a dye solution at a sufficientlyhigh concentration such that all photons incident on it areabsorbed. In addition, it must have an emission (EM) spectrumwhose shape and intensity do not ch
39、ange with excitation (EX)wavelength, that is, its fluorescence quantum yield is indepen-dent of excitation wavelength. Note that there are severaldrawbacks to using a quantum counter (QC) instead of a CD.Firstly, QCs tend to have a more limited range than CDs anduncertainties that are not certified
40、or even well known. Inaddition, a QC is prone to polarization and geometry effectsthat are concentration and solvent dependent, thus requiringthat the ideal concentration for proper functioning be deter-mined for the measurement geometry to be used. It should alsobe noted that the output measured fr
41、om the QC will beproportional to the quantum flux (number of photons persecond) at the sample, not the flux in power units. This canresult in enhanced measurement uncertainties compared to theuse of a calibrated detector.5.2 Procedure:5.2.1 Unlike the CS method, this is a two-step method. Thefirst s
42、tep uses a CD (or a QC) placed at the sample position,which measures the excitation intensity incident on the sampleas a function of EX wavelength by scanning the EX wave-length selector over the desired range. The second step uses aCR with reflectance RCRto reflect a known fraction of the fluxof th
43、e EX beam into the detection system. Follow the proce-dures in either 5.2.1.1 or 5.2.1.2 depending upon whether youare using a CD or a QC, respectively.NOTE 2Correction factors, supplied by the manufacturer and auto-matically applied by the software to the collected spectrum, must beswitched off for
44、 signal and reference channels during this procedure.5.2.1.1 Step 1 with Calibrated DetectorPlace the CD atthe sample position and scan the EX-selector over the EXregion of interest while collecting the signal from the CD (SCD)as a function of EX. Be sure to use the same instrumentsettings as those
45、employed with the sample. Calculate the fluxof the EX beam (x), using x= SCD/RCD, where RCDis theknown responsivity of the CD. Note that if the instrument hasits own reference detector with output (Rf) for monitoring theexcitation intensity, then the correction factor for the respon-sivity of the re
46、ference detector CR= x/Rf can be calculated.Multiplying an Rf value by CRat a particular EXwill give acorrected Rf value in the same units as x, typically Watts.5.2.1.2 Step 1 with Quantum CounterPlace the QC solu-tion at the sample position in a cuvette (typically fused silica)that transmits the ex
47、citation and emission wavelengths ofinterest. If front face detection is possible, then use a standardcuvette with the EX beam at normal incidence. If 90 detectionis chosen, then use a right-triangular cuvette with the excitationbeam at 45 incidence to the hypotenuse side and one of theother sides f
48、acing the detector. Scan the EX-selector over theEX region of interest with the EMfixed at a positioncorresponding to the long-wavelength tail of the emission bandand collect the signal intensity (SQC) as a function of EX.Besure to use the same instrument settings for the excitation beamas those emp
49、loyed with the sample. SQCis the relative quantumflux of the excitation beam at the sample. Note that if theinstrument has its own reference detector with output (Rf) formonitoring the excitation intensity, then the correction factorfor the responsivity of the reference detector CR= SQC/Rf canbe calculated. Multiplying an Rf value by CRat a particular EXwill give a corrected Rf value in units of relative quantum flux.5.2.1.3 Step 2 Using a Calibrated ReflectorPlace the CRat the sample position at a 45 angle relative to the excitationbeam, a
