ASTM_E_3029_-_15.pdf

Designation:E302915Standard 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 case 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 correction 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 detector(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 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 measurement 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 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 FluorescenceInstrument 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 ratios 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 changesignificantly 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 steps 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.Also notethat when using an EM polarizer,the spectral correction foremission is dependent on the polarizer setting.(2)It isimportant 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 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 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 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 parentheses 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 website.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 should be advised to verify that the automated vendor proce-dure and calculation or supplied correction are performed anddetermined according to the guidelines give