1、Designation: E 169 04Standard Practices forGeneral Techniques of Ultraviolet-VisibleQuantitative Analysis1This standard is issued under the fixed designation E 169; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last r
2、evision. 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 These practices are intended to provide general infor-mation on the techniques most often used in ultraviolet andvisible qu
3、antitative analysis. The purpose is to render unnec-essary the repetition of these descriptions of techniques inindividual methods for quantitative analysis.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 o
4、f 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:2E 131 Terminology Relating to Molecular SpectroscopyE 168 Practices for General Techniques of Infrared Quanti-tativ
5、e AnalysisE 275 Practice for Describing and Measuring the Perfor-mance of Ultraviolet, Visible, and Near-Infrared Spectro-photometersE 925 Practice for Monitoring the Calibration of Ultra-Violet Visible Spectrophotometers Whose Spectral SlitWidth Does Not Exceed 2nmE 958 Practice for Measuring Pract
6、ical Spectral Bandwidthof Ultraviolet-Visible Spectrophotometers3. Summary of Practice3.1 Quantitative ultraviolet and visible analyses are basedupon the absorption law, known as Beers law. The units of thislaw are defined in Terminology E 131. Beers law (Note 1)holds at a single wavelength and when
7、 applied to a singlecomponent sample it may be expressed in the following form(see Section 10):A 5 abc (1)When applied to a mixture of n non-interacting components,it may be expressed as follows:A 5 a1bc11 a2bc21 1 anbcn(2)NOTE 1Detailed discussion of the origin and validity of Beers lawmay be found
8、 in the books and articles listed in the bibliography at the endof these practices.3.2 This practice describes the application of Beers law intypical spectrophotometric analytical applications. It also de-scribes operating parameters that must be considered whenusing these techniques.4. Significance
9、 and Use4.1 These practices are a source of general information onthe techniques of ultraviolet and visible quantitative analyses.They provide the user with background information that shouldhelp ensure the reliability of spectrophotometric measure-ments.4.2 These practices are not intended as a sub
10、stitute for athorough understanding of any particular analytical method. Itis the responsibility of the users to familiarize themselves withthe critical details of a method and the proper operation of theavailable instrumentation.5. Sample Preparation5.1 Accurately weigh the specified amount of the
11、sample(solid or liquid). Dissolve in the appropriate solvent and diluteto the specified volume in volumetric glassware of the requiredaccuracy, ensuring that all appropriate temperature rangetolerances are maintained. If needed, a dilution should be madewith a calibrated pipet and volumetric flask,
12、using adequatevolumes for accuracy. With the availability of moderin widerange electronic balances, (capable of reading kg quantities tofour or five decimal places), gravimetric dilution should beconsidered as a more accurate alternative to volumetric, ifavailable. Fill the absorption cell with the
13、solution, and fill thecomparison or blank cell with the pure solvent, at least 2 to 33(if sufficient sample or solvent is available), before measuring.1These practices are under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Science and are the direct responsibility o
14、fSubcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Nov. 1, 2004. Published December 2004. Originallyapproved in 1960. Last previous edition approved in 1999 as E 169 99.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact
15、ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6. Cell and Base-Line Chec
16、ks6.1 Clean and match the cells. Suggested cleaning proce-dures are presented in Practice E 275.6.2 Establish the base line of a recording double-beamspectrophotometer by scanning over the appropriate wave-length region with pure solvent in both cells. Determineapparent absorbance of the sample cell
17、 at each wavelength ofinterest. These absorbances are cell corrections that are sub-tracted from the absorbance of the sample solution at thecorresponding wavelengths.6.3 For single beam instruments, either use the same cell forpure solvent and sample measurements, use matched cells, orapply appropr
18、iate cell corrections.6.4 On most software-controlled instruments, the cell cor-rections or the blank cell absorbance is stored in memory andautomatically incorporated into the sample absorbance mea-surement.6.5 An accurate determination of cell path length in the1-cm range is not practical in most
19、laboratories, and commonpractice is to purchase cells of known path length. Modern cellmanufacturing techniques employed by a number of leadingmanufacturers can guarantee the path length of a 1-cm cell to60.01 mm or better.7. Analytical Wavelengths and Photometry7.1 Analytical wavelengths are those
20、wavelengths at whichabsorbance readings are taken for use in calculations. Thesemay include readings taken for purposes of background cor-rections. To minimize the effect of wavelength error, theanalytical wavelengths are frequently chosen at absorptionmaxima, but this is not always necessary. If th
21、e wavelengthaccuracy of the spectrophotometer is such that the calculateduncertainty in the absorbance measurement is within accept-able limits at the extremes of this wavelength uncertainy range,then single point measurements on a slope can be used. Forexample, the use of isoabsorptive or isosbesti
22、c points isfrequently useful.7.2 Record the absorbance readings at the specified analyti-cal wavelengths, operating the instrument in accordance withthe recommendations of the manufacturer or Practice E 275.7.3 Absorbance values should be used only if they fallwithin the acceptably accurate range of
23、 the particular spectro-photometer and method employed. If the absorbance is too low,either use a longer absorption cell or prepare a new solution ofhigher concentration. If the absorbance is too high, use ashorter cell or make a quantitative dilution3. If different cellsare used, a new base-line mu
24、st be obtained.7.4 The precision and bias of the wavelength and photomet-ric scales of the instrument must be adequate for the methodbeing used. Procedures for checking precision and accuracy ofthese scales are presented in Practices E 275 and E 925.8. Resolution and Slit Width8.1 If the analytical
25、method specifies a resolution or aspectral slit width, set the resolution of the instrument to thespecified value. If the instrument has only a mechanical slitwidth indicator, use the information provided in the manufac-turers literature to calculate the slit width that corresponds tothe specified r
26、esolution.NOTE 2The accuracy of resolution and mechanical slit width indica-tors can be determined using the procedure given in Practice E 958.8.2 If the analytical method does not state a requiredresolution or a slit width value but includes an illustrativespectrum, set the resolution or slit width
27、 of the instrument toobtain comparable data. As a rule of thumb, the resolutionshould be less than one-eighth of the bandwidth; thus for apeak of bandwidth 40 nm, the resolution should not exceed 5nm.8.3 If the method neither specifies resolution or slit widthnor provides an illustrative spectrum, u
28、se the smallest resolu-tion or slit width that yields an acceptable signal-to-noise ratio.Record this value for future reference.NOTE 3Changes in the day-to-day values of resolution or slit widthobtained with a given gain, or changes in signal-to-noise ratio at a givenresolution, are indicative of p
29、resent or potential problems. Increasedresolution or a lowering of the S/N ratio may result from a lower outputof the light source, deterioration of optical components, deposits on thewindows of the cell compartment or on the inside wall of the referencecell, an absorbing impurity in the solvent, or
30、 a faulty electronic compo-nent.9. Solvents and Solvent Effects9.1 The ultraviolet absorption spectrum of a compound willvary in different solvents depending on the chemical structuresinvolved. Non-polar solvents have the least effect on theabsorption spectrum. Non-polar molecules in most instancesa
31、re not affected in polar solvents. However, polar molecules inpolar solvents may show marked differences in their spectra.Any interaction between solute and solvents leads to a broad-ening and change in structural resolution of the absorptionbands. Ionic forms may be created in acidic or basic solut
32、ions.In addition, there are possible chemical reactions betweensolute and solvent, and also photochemical reactions arisingfrom either room illumination or the short wavelengths in thebeam of the spectrophotometer. It is important that the solventused be specified in recording spectral data. (The ch
33、ange inspectra between acidic and basic conditions may sometimes beemployed in multicomponent analysis.)9.2 Reference solvent data is shown in Table 1. Availabilityof a particular solvent may be restricted by internationalagreement, and the users attention is directed to 1.2 of thisPractices. The sh
34、ort wavelength limit is approximate, andrefers to the wavelength at which a 1-cm light path length givesan absorbance of unity.9.3 Water, and 0.1 M solutions of hydrochloric acid, sulfuricacid, and sodium hydroxide also are commonly used assolvents. Buffered solutions, involving non-absorbing materi
35、-als, are frequently used; both the composition of the buffer andthe measured pH should be specified. Mixtures of 0.1 Mdi-hydrogen sodium phosphate and 0.1 M hydrogen di-sodium3The errors associated with cell path lengths are significantly less than thosegenerated by volumetric dilution, and therefo
36、re where possible, different path lengthcells should be used in preference to volumetric procedures.E169042phosphate are useful in the 4.5 to 8.9 pH range. A table ofnon-absorbing buffers has been presented by Abbott (10).410. Calculations10.1 Quantitative analysis by ultraviolet spectrophotometryde
37、pends upon Beers law. The terms and symbols used arethose defined in Terminology E 131. According to Beers law:A 5 abc 5 e/M! 3 bc (3)where:A = absorbance,a = absorptivity,b = cell length, cm,c = concentration, g/L,e = molar absorptivity, andM = molecular weight.10.1.1 In practice, a distinction mus
38、t be made between c, theconcentration of the absorbing material in the cell at the timeof observation, and the concentration of the absorbing materialin the sample as received. This is here designated as a massfraction C (g/g). The solution to be examined has a concentra-tion of sample in solution,
39、Cs, which is in units of grams perlitre.c 5 A/ab (4)C 5 c/Cs5 A/abCs! (5)10.2 If one or more dilutions are then made, the quantitycalled the dilution factor must be included. Dilution factor, f,isthe ratio of the final volume to the initial volume. If more thanone dilution is performed, the dilution
40、 factor is the product ofthe factors from each dilution. If dilutions are made, theequation becomes the following:C 5 cf/Cs5 Af/abCs! (6)Note that c and Cs, have the dimensions of grams per litre.If dilution is made, Csis not the concentration in the cell at thetime the absorbance is determined; the
41、 concentration in the cellis Cs/ f.10.3 Chemical CalibrationThe absorptivity of the absorb-ing material, the concentration of which it is desired todetermine, is obtained by examination of a series of quantita-tive dilutions of a neat sample of this material. However, if nosuch neat sample is availa
42、ble, the best available material isused, or a value of the absorptivity is taken from the literature.Take care to specify this, by reporting values as “percentageagainst calibration material” or by noting that the accuracy ofthe analysis is dependent upon a published value of theabsorptivity or mola
43、r absorptivity. (A reference must be cited.)10.3.1 Some sample materials are highly fluorescent whichcan significantly reduce the measured absorbance. The effect ofsample fluorescence may vary depending upon the spectropho-tometer and wavelength chosen. Sample fluorescence may be aparticular problem
44、 when using published absorptivity values.10.4 Types of Analyses (see Fig. 1):10.4.1 One Component, No Background Correction:C 5 Af/abCs! (7)10.4.2 One Component, Simple Background Correction:C 5A1 A2! 3 fa1bCs(8)where the subscripts refer to analytical wavelengths. Theterm A2is the absorbance at th
45、e wavelength used for making asimple subtractive correction. It is usually selected fromexamination of the spectral curve of the reference material at awavelength longer than that of A1, preferably where a2is muchless than a1.10.4.3 One Component, with Slope-Type Background Cor-rection:C 5A1 A21 Sl2
46、 l1!# fa1bCs(9)where:S = slope between wavelengths 1 and 2 for the back-ground.10.4.3.1 The background absorption is usually not linearbetween the analytical wavelength and the wavelength atwhich a simple subtractive background correction may beobtained. When it is possible to determine the slope be
47、tweenwavelengths 1 and 2 by observation of the samples that do notcontain the absorbing material that is to be determined, thismay be used as a correction for the background absorption.10.4.4 One Component, With Linear Background Correc-tion:10.4.4.1 The equation for the general case is as follows:C
48、 5A1A31 A2 A3# 3l3 l1l3l2fabCc(10)4The boldface numbers in parentheses refer to the list of references at the endof this standard.TABLE 1 SolventsASolvent Cutoff, nmPyridine 305Tetrachloroethylene 290Benzene 280N,N-Dimethylformamide 270Carbon tetrachloride 265Methyl formate 260Chloroform 245Dichloro
49、methane 235Ethyl ether 220Acetonitrile 215Isopropyl alcohol 210Ethyl alcohol 210Methyl alcohol 210Cyclohexane 210Isooctane 210AProcedures for special purification of solvents for further improvement in thewavelength limit are given in Refs (11, 12). Solvents of high purity for use inabsorption spectroscopy also are available commercially.E169043The absorptivity a is here the effective absorptivity asdetermined on a pure sample, using the corrections, and issomewhat lower than the true or absolute absorptivity.10.4.4.2 This method is especially effective wi
