ASTM E388-04 (Reapproved 2015)

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 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 test method covers the testing of the spectralbandwidth and wavelength accuracy of fluorescence 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 250 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 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 accuracy. 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 Suspension—Dissolve1gofglycogen 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 observable 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 monochromators 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 monochromators,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 source 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 must 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 direction, 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 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 Science 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 International, 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 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 Excitation 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 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 settingused 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 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 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 1—Some 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 manufacturer 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 sampleposition 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 scale. 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 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 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 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 maximum intensity.TABLE 1 Atomic Emission LinesAfor Wavelength AccuracyHg Ne Ar Kr Xe253.65 336.99 633.44 830.03 696.54 427.40 645.63 450.10296.73 341.79 638.30 836.57 706.72 428.30 722.41 458.28302.15 345.42 640.11 837.76 727.29 431.96 758.74 462.43312.57 346.66 640.22 841.72 738.40 436.26 760.15 467.12313.15 347.26 650.65 841.84 750.39 437.61 768.53 469.70313.18 350.12 653.29 846.34 751.47 440.00 769.45 473.42334.15 352.05 659.90 857.14 763.51 442.52 785.48 480.70365.02 359.35 667.83 859.13 772.38 445.39 805.95 482.97404.66 533.08 671.70 863.46 794.82 446.37 810.44 484.33407.78 534.11 692.95 864.70 800.62 450.24 811.29 491.65435.84 540.06 703.24 865.44 801.48 557.03 819.00 492.32546.07 576.44 717.39 865.55 810.37 564.96 826.32 711.96576.96 582.01 724.52 867.95 811.53 567.25 828.10 764.20579.07 585.25 743.89 868.19 826.45 583.29 829.81 823.16588.19 783.91 870.41 840.82 587.09 850.89 828.01594.48 792.71 877.17 842.46 599.39 877.67 834.68597.55 793.70 878.06 912.30 601.21 975.18 840.92603.00 794.32 885.39 922.45 605.61 881.94607.43 808.25 920.18 965.78 895.22609.62 811.85 930.09 979.97614.31 812.89 932.65 992.32616.36 813.64 942.54621.73 825.94 948.67626.65 826.61 953.42630.48 826.71AWavelength 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)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 beexpected 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 of 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 connection with any item mentionedin this standard. 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