1、Designation: E 275 01Standard Practice forDescribing and Measuring Performance of Ultraviolet,Visible, and Near-Infrared Spectrophotometers1This standard is issued under the fixed designation E 275; 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 (e) indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the Department of Defense.INTRODUCTIONIn devel
3、oping a spectrophotometric method it is the responsibility of the originator to describe theinstrumentation and the performance required to duplicate the precision and accuracy of the method.It is necessary to specify this performance in terms that may be used by others in applications of themethod.
4、The tests and measurements described in this practice are for the purpose of determining theexperimental conditions required for a particular analytical method. In using this practice an analysthas either a particular analysis for which he describes requirements for instrument performance, or heexpe
5、cts to test the capability of an instrument to perform a particular analysis. To accomplish eitherof these objectives it is necessary that instrument performance be obtained in terms of the factors thatcontrol the analysis. Unfortunately, it is true that not all the factors that can affect the resul
6、ts of ananalysis are readily measured and easily specified for the various types of spectrophotometricequipment.Of the many factors that control analytical results, this practice covers selection of the setting ofanalytical wavelength, selection of slit width, photometric measurements, and character
7、istics ofabsorption cells as the parameters of spectrophotometry that are likely to be affected by the analyst inobtaining data. Other important factors, particularly those primarily dependent on instrument design,are not covered in this practice.1. Scope1.1 This practice covers the description of r
8、equirements ofspectrophotometric performance especially for ASTM meth-ods, and the testing of the adequacy of available equipment fora specific method. The tests give a measurement of some of theimportant parameters controlling results obtained in spectro-photometric methods, but it is specifically
9、not to be concludedthat all the factors in instrument performance are measured.1.1.1 This practice is not to be used ( 1) as a rigorous test ofperformance of instrumentation, or (2) to intercompare thequantitative performance of instruments of different design.1.1.2 This practice is primarily direct
10、ed to dispersive spec-trophotometers used for transmittance measurements ratherthan instruments designed for diffuse transmission and diffusereflection.2. Referenced Documents2.1 ASTM Standards:E 131 Terminology Relating to Molecular Spectroscopy2E 168 Practices for General Techniques of Infrared Qu
11、anti-tative Analysis2E 169 Practices for General Techniques of Ultraviolet-Visible Quantitative Analysis2E 387 Test Method for Estimating Stray Radiant PowerRatio of Spectrophotometers by the Opaque Filter Method2E 958 Practice for Measuring Practical Spectral Bandwidthof UltravioletVisable Spectrop
12、hotometers23. Terminology3.1 Definitions:3.1.1 For definitions of terms used in this practice, refer toTerminology E 131.4. Significance and Use4.1 This practice permits an analyst to compare the generalperformance of his instrument, as he is using it in a specificspectrophotometric method, with the
13、 performance of instru-ments used in developing the method.1This practice is under the jurisdiction of ASTM Committee E13 on MolecularSpectroscopy and is the direct responsibility of Subcommittee E13.01 on Ultravioletand Visible Spectroscopy.Current edition approved Feb. 10, 2001. Published April 20
14、01. Originallypublished as E 275 65 T. Last previous edition E 275 93.2Annual Book of ASTM Standards, Vol 03.06.1Copyright ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.5. Reference to This Practice in Standards5.1 Reference to this practice in any ASTM spectrophoto-me
15、tric method (preferably in the section on apparatus wherethe spectrophotometer is described) shall constitute due noti-fication that the adequacy of the spectrophotometer perfor-mance is to be evaluated by means of this practice. Perfor-mance is considered to be adequate when the instrument can beop
16、erated in a manner to give test results equivalent to thoseobtained on instruments used in establishing the method or incooperative testing of the method.5.2 It is recommended that the apparatus be described interms of the results obtained on application of this practice toinstruments used in establ
17、ishing the method. This descriptionshould give a numerical value showing the wavelength accu-racy, wavelength repeatability, and photometric repeatabilityfound to give acceptable results. A recommended spectral slitwidth maximum should be given along with typical spectra ofthe components to be deter
18、mined to indicate the resolutionfound to be adequate to perform the analysis. If it is considerednecessary in a particular analysis, the use of only the linearportion of an analytical curve (absorbance per centimetreversus concentration) may be specified, or if nonlinearity isencountered, the use of
19、 special calculation methods may bespecified. However, it is not permissible to specify the amountof curvature if a nonlinear working curve is used.6. Parameters in Spectrophotometry6.1 Any spectrophotometer may be described as a source ofradiant energy, a dispersing optical element, and a detectort
20、ogether with a photometer for measuring relative radiantpower. Accurate spectrophotometry involves a large number ofinterrelated factors that determine the quality of the radiantenergy passing through a sample and the sensitivity andlinearity with which this radiant energy may be measured.Assuming p
21、roper instrumentation and its use, the instrumentalfactors responsible for inaccuracies in spectrophotometry in-clude resolution, linearity, stray radiant energy, and cell con-stants. Rigorous measurement of these factors is beyond thescope of this practice. The measurement of stray radiant energyis
22、 described in Method E 387.6.2 Modern spectrophotometers are capable of more accu-racy than most analysts obtain. The problem lies in theselection and proper use of instrumentation. In order to ensureproper instrumentation and its use in a specific spectrophoto-metric method, it is necessary for an
23、analyst to evaluate certainparameters that can control the results obtained. These param-eters are wavelength accuracy and precision, spectral slitwidth, photometry, and absorption-cell constants. Unsatisfac-tory measurement of any of these parameters may be due toimproper instrumentation or to impr
24、oper use of availableinstrumentation. It is therefore first necessary to determine thatinstrument operation is in accordance with the manufacturersrecommendations. Tests shall then be made to determine theperformance of an instrument in terms of each of the param-eters in 6.1 and 6.2.7. Instrument O
25、peration7.1 In obtaining spectrophotometric data, the analyst mustselect the proper instrumental operating conditions in order torealize satisfactory instrument performance. Operating condi-tions for individual instruments are best obtained from themanufacturers literature because of variations with
26、 instrumentdesign. A record should be kept to document the operatingconditions selected so that they may be duplicated.7.2 Because tests for proper instrument operation vary withinstrument design, it is necessary to rely on the manufacturersrecommendations. These tests should include documentationof
27、 the following factors in instrument operation, or theirequivalent:7.2.1 Ambient temperature,7.2.2 Response time,7.2.3 Signal-to-noise ratio,7.2.4 Mechanical repeatability,7.2.5 Scanning parameters for recording instruments, and7.2.6 Instrument stability.7.3 Each of the factors in instrument operati
28、on is importantin the measurement of analytical wavelength and photometricdata. For example, changes in wavelength precision andaccuracy can occur because of variation of ambient tempera-ture of various parts of a monochromator. The correspondenceof the absorbance to wavelength and any internal calc
29、ulations(or corrections) can affect wavelength measurement for digitalinstruments. In scanning spectrophotometers there is alwayssome lag between the recorded reading and the correct reading.It is necessary to select the conditions of operation to make thiseffect negligible or repeatable. Scanning s
30、peeds should beselected to make sure that the detecting system can follow thesignal from narrow emission lines or absorption bands. Toorapid scanning will displace the apparent wavelength towardthe direction scanned and peak absorbance readings will varywith speed of scanning. A change in instrument
31、 response-timemay produce apparent wavelength shifts. Mechanical repeat-ability of the various parts of the monochromator and recordingsystem are important in wavelength measurement. Instructionson obtaining proper mechanical repeatability are usually givenin the manufacturers literature.7.4 Digital
32、 spectrophotometers and diode array spectropho-tometers may require a calibration routine to be completedprior to measurement of wavelength or absorbance accuracy.Consult the manufacturers manual for any such procedures.WAVELENGTH ACCURACY AND PRECISION8. Nature of Test8.1 Most spectrophotometric me
33、thods employ pure com-pounds or known mixtures for the purpose of calibratinginstruments photometrically at specified analytical wave-lengths. The wavelength at which an analysis is made is readfrom the dial of the monochromator, from the digital readout,from an attached computer, or from a chart in
34、 recordinginstruments. To reproduce measurements properly, it is neces-sary for the analyst to state the wavelength limits within whichthe analytical wavelength is known.8.2 The accompanying spectra are given to show the loca-tion of selected reference wavelengths which have been foundE 2752useful.
35、Numerical values are given in wavelength units (na-nometres or micrometres, measured in air). Reference (1)3tabulates additional reference wavelengths of interest.9. Definitions9.1 wavelength accuracythe deviation of the averagewavelength reading at an absorption band or emission bandfrom the known
36、wavelength of the band.9.2 wavelength precisiona measure of the ability of aspectrophotometer to return to the same spectral position asmeasured by an absorption band or emission band of knownwavelength when the instrument is reset or read at a givenwavelength. The index of precision used in this pr
37、actice is thestandard deviation.10. Reference Wavelengths in the Ultraviolet Region10.1 The most convenient spectra for wavelength calibra-tion in the ultraviolet region are the emission spectrum of thelow-pressure mercury arc (Fig. 1), the absorption spectra ofholmium oxide glass (Fig. 2), holmium
38、oxide solution (Fig. 3),and benzene vapor (Fig. 4).10.2 The mercury emission spectrum is obtained by illumi-nating the entrance slit of the monochromator with a quartzmercury arc or by a mercury arc that has a transmittingenvelope (Note 1). It is not necessary, when using an arcsource, that the arc
39、be in focus on the entrance slit of themonochromator. However, it is advantageous to mount thelamp reasonably far from the entrance slit in order to minimizethe scatter from the edges of the slit. Displacement of thesource will not shift the apparent wavelength as long as the slitwidths used are sma
40、ll, that is, less than 0.1 mm. Referencewavelengths for diode array spectrophotometers can be ob-tained by placing a low-pressure mercury discharge lamp in thesample compartment. It is not necessary to put the referencesource in the lamp compartment for systems with the dispers-ing element (polychom
41、ator) located after the sample compart-ment.NOTE 1Several commercially available mercury arcs are satisfactory.They may differ, however, in the number of lines observed and in therelative intensities of the lines because of differences in operatingconditions. Low-pressure arcs have a high-intensity
42、line at 253.65 nm, andother useful lines as seen in Fig. 1 are satisfactory.10.3 The absorption spectrum of holmium oxide glass (Fig.2) is obtained by measuring the transmittance or absorbance ofa piece of holmium oxide glass about 2 to 4 mm thick.4Theabsorption spectrum of holmium oxide solution (F
43、ig. 3) isobtained similarly by measuring an approximately 4 % solu-tion of holmium oxide5in 1.4 M perchloric acid (40 g/L) withair as reference.10.4 The absorption spectrum of benzene is obtained bymeasuring the absorbance of a 1-cm cell filled with vapor ( Fig.6). The sample is prepared by placing
44、1 or 2 drops of liquidbenzene in the cell, pouring out the excess liquid, andstoppering the cell. Some care must be exercised to ensure that3The boldface numbers in parentheses refer to the list of references appended tothis practice.4Holmium oxide glass is available commercially as a polished filte
45、r.5Sealed cuvettes of holmium oxide solution are available from commercialsources and from the National Institute of Standards and Technology (as SRM 2034(2).Line Number Wavelength, nm Line Number Wavelength, nm Line Number Wavelength, nm Line Number Wavelength, nm1 253.65 4 313.16 7 404.66 10 546.0
46、72 296.73 5 334.15 8 407.78 11 576.963 302.15 6 365.01 9 435.84 12 579.07Instrument: Cary Model 14 Slit Width: 0.03 mmScanning Speed: 2.5 A/s Spectral Slit Width: 0.10 to 0.15 nmFIG. 1 Mercury Arc Emission Spectrum in the Ultraviolet and Visible Regions Showing Reference Wavelength (4)E 2753the conc
47、entration of benzene vapor is low enough to permitresolution of the strongest absorption bands.NOTE 2When using complex spectra for wavelength calibration, suchas is exhibited by benzene vapor in the ultraviolet, the approximateconditions of resolution used in obtaining the reference spectra must be
48、achieved in order to depend upon the wavelength values.NOTE 3This test is not recommended for routine use because of thepossible health hazards associated with the use of benzene. If the test mustbe used, it is recommended that the cell be permanently sealed after theconcentration of the benzene vap
49、or has been adjusted.Band Number Wavelength, nm Band Number Wavelength, nm Band Number Wavelength, nm Band Number Wavelength, nm345279.4287.5333.7789360.9385.9418.7101213453.2460.0484.51415536.2637.5Instrument: Cary Model 14 Spectral Slit Width: 0.10 to 0.40 nmScanning Speed: 10A/s Sample Thickness: 2.6 mmSlit Width: 0.025 to 0.105 mmFIG. 2 Spectrum of Holmium Oxide Glass Showing Reference Wavelength (5)Band Number Wavelength, nm Band Number Wavelength, nm Band Number Wavelength, nm Band Number Wavelength, nm1 241.1 5 333.4 9 416.3 13 485.82 249.7 6 345.5 10 450.8 14 536.43
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