1、Designation: E 388 04Standard Test Method forWavelength Accuracy of Spectral Bandwidth ofFluorescence Spectrometers1This standard is issued under the fixed designation E 388; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year
2、 of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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 fluorescence spectrom-ete
3、rs 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 250 nm and 1000nm
4、.1.2 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. Summary of
5、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 accuracy. The apparent width of someof these lines is used as a test for spectral bandwidth.3. Apparatus3.1 Fluorescence Spectrometer to be test
6、ed.3.2 Atomic Discharge Lamps, Low-pressure, sufficientlysmall to be placed in the sample cell holder of the instrument.4. Reagent4.1 Scattering SuspensionDissolve1gofglycogen perlitre of water, or use a dilute microsphere suspension contain-ing 1 mL of a commercially available, concentrated micro-s
7、phere suspension.5. Procedure5.1 The emission lines given for Hg, Ne, Ar, Kr, and Xein Table 1 are typically observable using standard commercialfluorometers, although some of them may be too weak to detecton some instruments.5.1.1 Most fluorescence instruments will not be able toresolve very closel
8、y spaced lines such as those for Hg at 312.57nm, 313.15 nm, and 313.18 nm, due to the relatively lowresolution monochromators used in fluorescence equipmentcompared to those used in absorbance spectrometers. Evenlower resolution fluorometers may not resolve lines separatedby less than several nanome
9、tres 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 monochromators,additional weaker lines are found due to second order diffrac-tion of atomic lines. For instance, lines appear for mercury at507.30 and 593.46 nm, arising from the 253.6
10、5 and 296.73 nmlines, respectively.5.2 Calibration and Adjustment of Emission Monochroma-tor:5.2.1 With an atomic arc source properly aligned (seesection 5.3) in the sample cell compartment, adjust the positionof the wavelength dial to give maximum signal for each of theatomic lines and record the w
11、avelength reading. The differencebetween the observed value and the corresponding value inTable 1 represents the correction that must be subtractedalgebraically from the wavelength reading of the instrument.The corrections may be recorded or the monochromatoradjusted to give the proper values. Since
12、 there may be somebacklash in the wavelength drive of scanning instruments,always approach the peak position from the same direction, ifapplicable.5.2.2 When calibrating scanning-type instruments, approachthe peak position in the same direction that the motor scans, ifyour instrument does not correc
13、t for backlash. Check theposition against that recorded while scanning and, if necessary,correct as in 5.2.1.5.3 In cases where 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, pr
14、oceed as follows:5.3.1 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 section 4.1.5.3.2 Place the cell in the sample position in the instrument.5.3.3 Illuminate the cell transversely with th
15、e atomic lamp,either from the side or from above.1This test method is under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Chromatography and is the direct responsibility ofSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Nov.
16、1, 2004. Published December 2004. Originallyapproved in 1969. Last previous edition approved in 1998 as E 388 72 (1998).1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.3.4 Adjust the wavelength to give the maximum signal foreach of
17、 the atomic lines given in Table 1; record the wave-length reading and proceed as in 5.2.5.4 Adjustment of Excitation Monochromator:5.4.1 After the emission monochromator has been cali-brated, adjust the excitation monochromator to match, asfollows:5.4.2 Place a sample cell containing either the dil
18、ute scat-tering suspension described in section 4.1 in the sample cellcompartment.5.4.3 With a continuous source in the normal 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 set
19、tingused for calibration of the emission monochromator.5.4.5 Adjust the wavelength position of the excitationmonochromator 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
20、 must be subtracted algebraically from thereading of the instrumentation. The corrections may be eitherrecorded, or the monochromator 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
21、the desired wavelength position in the samedirection that the scan motor scans, if applicable.5.4.6 The match of the excitation 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 theu
22、ser to place an atomic lamp before the excitation monochromator. Thelamp is installed into the instrument either by the manufacturer or by theuser as specified by the manufacturer. In this case, wavelength accuracycan be calibrated for the excitation monochromator using a procedure thatparallels tha
23、t given in section 5.2 for the emission monochromator. Ascattering solution or other scattering media can then be placed at thesample position to scatter atomic lamp light into the emission monochro-mator to calibrate emission wavelength accuracy using a procedure thatparallels that given in section
24、 5.4 for the excitation monochromator.5.5 Slit Width Effects:5.5.1 Use the narrowest practical slit widths in calibratingthe wavelength scale. In cases when monochromator slits arenot filled, or when intensity of fluorescence varies rapidly withwavelength, there may be an apparent wavelength error w
25、ithwide slits. Under the most unfavorable conditions this errormay approach one spectral bandwidth, so that narrow slitsshould be used for accurate wavelength measurements. As themagnitude of the error may depend on characteristics of boththe instrument and the sample, a generally applicable correc-
26、tion for slit widths is not practical.5.5.2 For greatest accuracy at important wavelengths ofspecific compounds, measure the peak wavelength 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 former
27、cury at 253.65, 435.84, and 546.07 nm. Do not use secondorder lines.5.6.2 Take sets of readings at wavelengths on both sides ofthe reading of maximum intensity.5.6.3 Record the wavelength positions on both sides of thepeak maximum that correspond 50 % and 5 % of the maxi-mum. The wavelength interval
28、 between the 50 % points is thespectral bandwidth of the monochromator. This test is used asan indication of the approximate resolving power that may beexpected from the instrument. The wavelength interval be-tween the 5 % points is an indication of the degree of isolationthat may be achieved betwee
29、n adjacent wavelengths.TABLE 1 Atomic Emission Lines2for Wavelength AccuracyHb 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
30、.15 467.12313.15 347.26 650.65 841.84 750.39 437.61 768.53 469.70313.18 350.12 653.29 846.34 751.47 440.00 769.45 473.42334.15 352.05 659.90 857.14 763.51 442.52 785.48 480.70365.02 359.35 667.83 859.13 772.38 445.39 805.95 482.97404.66 533.08 671.70 863.46 794.82 446.37 810.44 484.33407.78 534.11 6
31、92.95 864.70 800.62 450.24 811.29 491.65435.84 540.06 703.24 865.44 801.48 557.03 819.00 492.32546.07 576.44 717.39 865.55 810.37 564.96 826.32 711.96576.96 582.01 724.52 867.95 811.53 567.25 828.10 764.20579.07 585.25 743.89 868.19 826.45 583.29 829.81 823.16588.19 783.91 870.41 840.82 587.09 850.8
32、9 828.01594.48 792.71 877.17 842.46 599.39 877.67 834.68597.55 793.70 878.06 912.30 601.21 975.18 840.92603.00 794.32 885.39 922.45 605.61 881.94607.43 808.25 920.18 965.78 895.22609.62 811.85 930.09 979.97614.31 812.89 932.65 992.32616.36 813.64 942.54621.73 825.94 948.67626.65 826.61 953.42630.48
33、826.71E3880426. Precision and Bias6.1 The precision of this test method is undetermined, but ismost likely limited by the wavelength repeatability of thefluorescence spectrometer.6.2 Bias does not have meaning for this test method.7. Keywords7.1 fluorescence spectrometers; fluorometer; molecular lu-
34、minescence; molecular spectroscopy; spectrofluorometerASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent r
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37、ng you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).E388043
