1、Designation: E388 04 (Reapproved 2015)Standard Test Method forWavelength Accuracy and Spectral Bandwidth ofFluorescence Spectrometers1This standard is issued under the fixed designation E388; the number immediately following the designation indicates the year oforiginal adoption or, in the case of r
2、evision, 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 test method covers the testing of the spectralbandwidth and wavelength accuracy of fluoresce
3、nce spectrom-eters that use a monochromator for emission wavelengthselection and photomultiplier tube detection. This test methodcan be applied to instruments that use multi-element detectors,such as diode arrays, but results must be interpreted carefully.This test method uses atomic lines between 2
4、50 nm and 1000nm.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
5、to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Summary of Test Method2.1 The difference between the apparent wavelength and theknown wavelength for a series of atomic emission lines is usedas a test for wavelength accu
6、racy. The apparent width of someof these lines is used as a test for spectral bandwidth.3. Apparatus3.1 Fluorescence Spectrometer to be tested.3.2 Atomic Discharge Lamps, Low-pressure, sufficientlysmall to be placed in the sample cell holder of the instrument.4. Reagent4.1 Scattering SuspensionDisso
7、lve1gofglycogen perlitre of water, or use a dilute microsphere suspension contain-ing 1 mL of a commercially available, concentrated micro-sphere suspension.5. Procedure5.1 The emission lines given for mercury (Hg), neon (Ne),argon (Ar), krypton (Kr), and xenon (Xe) in Table 1 aretypically observabl
8、e using standard commercial fluorometers,although some of them may be too weak to detect on someinstruments.5.1.1 Most fluorescence instruments will not be able toresolve very closely spaced lines such as those for Hg at 312.57nm, 313.15 nm, and 313.18 nm, due to the relatively lowresolution monochr
9、omators used in fluorescence equipmentcompared to those used in absorbance spectrometers. Evenlower resolution fluorometers may not resolve lines separatedby less than several nanometres such as those for Hg at 404.66and 407.78, or at 576.96 and 579.07 nm.5.1.2 In instruments using blazed grating mo
10、nochromators,additional weaker lines are found due to second order diffrac-tion of atomic lines. For instance, lines appear for Hg at 507.30and 593.46 nm, arising from the 253.65 and 296.73 nm lines,respectively.5.2 Calibration and Adjustment of Emission Monochroma-tor:5.2.1 With an atomic arc sourc
11、e properly aligned (see 5.3)inthe sample cell compartment, adjust the position of thewavelength dial to give maximum signal for each of the atomiclines and record the wavelength reading. The difference be-tween the observed value and the corresponding value in Table1 represents the correction that m
12、ust be subtracted algebra-ically from the wavelength reading of the instrument. Thecorrections may be recorded or the monochromator adjusted togive the proper values. Since there may be some backlash inthe wavelength drive of scanning instruments, always approachthe peak position from the same direc
13、tion, if applicable.5.2.2 When calibrating scanning-type instruments, approachthe peak position in the same direction that the motor scans, ifyour instrument does not correct for backlash. Check theposition against that recorded while scanning and, if necessary,correct as in 5.2.1.5.3 In cases where
14、 the monochromator is designed so that alateral displacement of the calibration source from a positiondirectly in front of the entrance slit appears as a wavelengthshift, proceed as follows:1This test method is under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Scie
15、nce and is the direct responsibility ofSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved May 1, 2015. Published June 2015. Originallyapproved in 1969. Last previous edition approved in 2009 as E388 04 (2009).DOI: 10.1520/E0388-04R15.Copyright ASTM I
16、nternational, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.3.1 Instead of placing the atomic lamp in front of theentrance slit of the monochromator, fill a sample cell with adilute scattering suspension, as described in 4.1.5.3.2 Place the cell in the sample
17、position in the instrument.5.3.3 Illuminate the cell transversely with the atomic lamp,either from the side or from above.5.3.4 Adjust the wavelength to give the maximum signal foreach of the atomic lines given in Table 1; record the wave-length reading and proceed as in 5.2.5.4 Adjustment of Excita
18、tion Monochromator:5.4.1 After the emission monochromator has beencalibrated, adjust the excitation monochromator to match, asfollows:5.4.2 Place a sample cell containing either the dilute scat-tering suspension described in 4.1 in the sample cell compart-ment.5.4.3 With a continuous source in the n
19、ormal source posi-tion of the instrument, illuminate the suspension.5.4.4 Set the wavelength positions of both excitation andemission monochromators at a previously determined settingused for calibration of the emission monochromator.5.4.5 Adjust the wavelength position of the excitationmonochromato
20、r to give a maximum signal, and record thewavelength reading. The difference between the observedvalue on the dial and the corresponding value in 5.1 representsthe correction that must be subtracted algebraically from thereading of the instrumentation. The corrections may be eitherrecorded, or the m
21、onochromator may be adjusted to give theproper value. It is possible to perform the latter using thecontrol software for some spectomaters. As stipulated in 5.2.1,always approach the desired wavelength position in the samedirection that the scan motor scans, if applicable.5.4.6 The match of the exci
22、tation monochromator with theemission monochromator may be checked at wavelengthsabove or below that used in 5.4.5.NOTE 1Some fluorescence spectrometers are designed to allow theuser to place an atomic lamp before the excitation monochromator. Thelamp is installed into the instrument either by the m
23、anufacturer or by theuser as specified by the manufacturer. In this case, wavelength accuracycan be calibrated for the excitation monochromator using a procedure thatparallels that given in 5.2 for the emission monochromator. A scatteringsolution or other scattering media can then be placed at the s
24、ampleposition to scatter atomic lamp light into the emission monochromator tocalibrate emission wavelength accuracy using a procedure that parallelsthat given in 5.4 for the excitation monochromator.5.5 Slit Width Effects:5.5.1 Use the narrowest practical slit widths in calibratingthe wavelength sca
25、le. In cases when monochromator slits arenot filled, or when intensity of fluorescence varies rapidly withwavelength, there may be an apparent wavelength error withwide slits. Under the most unfavorable conditions this errormay approach one spectral bandwidth, so that narrow slitsshould be used for
26、accurate wavelength measurements. As themagnitude of the error may depend on characteristics of boththe instrument and the sample, a generally applicable correc-tion for slit widths is not practical.5.5.2 For greatest accuracy at important wavelengths ofspecific compounds, measure the peak wavelengt
27、h as a func-tion of slit width and plot a correction curve.5.6 Spectral Bandwidth of Monochromator:5.6.1 Use the well-separated atomic lines such as those forHg at 253.65, 435.84, and 546.07 nm. Do not use second orderlines.5.6.2 Take sets of readings at wavelengths on both sides ofthe reading of ma
28、ximum intensity.TABLE 1 Atomic Emission LinesAfor Wavelength AccuracyHg Ne Ar Kr Xe253.65 336.99 633.44 830.03 696.54 427.40 645.63 450.10296.73 341.79 638.30 836.57 706.72 428.30 722.41 458.28302.15 345.42 640.11 837.76 727.29 431.96 758.74 462.43312.57 346.66 640.22 841.72 738.40 436.26 760.15 467
29、.12313.15 347.26 650.65 841.84 750.39 437.61 768.53 469.70313.18 350.12 653.29 846.34 751.47 440.00 769.45 473.42334.15 352.05 659.90 857.14 763.51 442.52 785.48 480.70365.02 359.35 667.83 859.13 772.38 445.39 805.95 482.97404.66 533.08 671.70 863.46 794.82 446.37 810.44 484.33407.78 534.11 692.95 8
30、64.70 800.62 450.24 811.29 491.65435.84 540.06 703.24 865.44 801.48 557.03 819.00 492.32546.07 576.44 717.39 865.55 810.37 564.96 826.32 711.96576.96 582.01 724.52 867.95 811.53 567.25 828.10 764.20579.07 585.25 743.89 868.19 826.45 583.29 829.81 823.16588.19 783.91 870.41 840.82 587.09 850.89 828.0
31、1594.48 792.71 877.17 842.46 599.39 877.67 834.68597.55 793.70 878.06 912.30 601.21 975.18 840.92603.00 794.32 885.39 922.45 605.61 881.94607.43 808.25 920.18 965.78 895.22609.62 811.85 930.09 979.97614.31 812.89 932.65 992.32616.36 813.64 942.54621.73 825.94 948.67626.65 826.61 953.42630.48 826.71A
32、Wavelength values have been obtained from Harrison, G.R., MIT Wavelength Tables, Wavelengths by Element, Vol 2, MIT Press, Cambridge, MA, 1982; and Zaidel,A.N., Prokofev, V.K., Raiskii, S.M., Slavnyi, V.A., and Shreider, E.Ya., Tables of Spectral Lines, Plenum Press, New York, NY, 1970.E388 04 (2015
33、)25.6.3 Record the wavelength positions on both sides of thepeak maximum that correspond 50 % and 5 % of the maxi-mum. The wavelength interval between the 50 % points is thespectral bandwidth of the monochromator. This test is used asan indication of the approximate resolving power that may beexpect
34、ed from the instrument. The wavelength interval be-tween the 5 % points is an indication of the degree of isolationthat may be achieved between adjacent wavelengths.6. Precision and Bias6.1 The precision of this test method is undetermined, but ismost likely limited by the wavelength repeatability o
35、f thefluorescence spectrometer.6.2 Bias does not have meaning for this test method.7. Keywords7.1 fluorescence spectrometers; fluorometer; molecular lu-minescence; molecular spectroscopy; spectrofluorometerASTM International takes no position respecting the validity of any patent rights asserted in
36、connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the res
37、ponsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive caref
38、ul consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International
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