1、Designation: E169 04 (Reapproved 2009)Standard Practices forGeneral Techniques of Ultraviolet-VisibleQuantitative Analysis1This standard is issued under the fixed designation E169; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, th
2、e 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 These practices are intended to provide general infor-mation on the techniques most often used in ultraviole
3、t andvisible quantitative analysis. The purpose is to render unnec-essary the repetition of these descriptions of techniques inindividual methods for quantitative analysis.1.2 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.3
4、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. Referenced Docum
5、ents2.1 ASTM Standards:2E131 Terminology Relating to Molecular SpectroscopyE168 Practices for General Techniques of Infrared Quanti-tative AnalysisE275 Practice for Describing and Measuring Performanceof Ultraviolet and Visible SpectrophotometersE925 Practice for Monitoring the Calibration of Ultrav
6、iolet-Visible Spectrophotometers whose Spectral Bandwidthdoes not Exceed 2 nmE958 Practice for Measuring Practical Spectral Bandwidthof Ultraviolet-Visible Spectrophotometers3. Summary of Practice3.1 Quantitative ultraviolet and visible analyses are basedupon the absorption law, known as Beers law.
7、The units of thislaw are defined in Terminology E131. Beers law (Note 1)holds at a single wavelength and when 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 foll
8、ows:A 5 a1bc11 a2bc21 1 anbcn(2)NOTE 1Detailed discussion of the origin and validity of Beers lawmay be found 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 application
9、s. It also de-scribes operating parameters that must be considered whenusing these techniques.4. Significance 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 should
10、help ensure the reliability of spectrophotometric measure-ments.4.2 These practices are not intended as a substitute 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 ope
11、ration of theavailable instrumentation.5. Sample Preparation5.1 Accurately weigh the specified amount of the 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 range
12、tolerances are maintained. If needed, a dilution should be madewith a calibrated pipet and volumetric flask, using adequatevolumes for accuracy. With the availability of moderin widerange electronic balances, (capable of reading kg quantities tofour or five decimal places), gravimetric dilution shou
13、ld beconsidered as a more accurate alternative to volumetric, if1Precision and Bias These practices are under the jurisdiction of ASTMCommittee E13 on Molecular Spectroscopy and Separation Science and are thedirect responsibility of Subcommittee E13.01 on Ultra-Violet, Visible, and Lumi-nescence Spe
14、ctroscopy.Current edition approved Oct. 1, 2009. Published December 2009. Originallyapproved in 1960. Last previous edition approved in 2004 as E169 04. DOI:10.1520/E0169-04R09.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. F
15、or 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.available. Fill the absorption cell with the solution, and fill theco
16、mparison or blank cell with the pure solvent, at least 23 to33 (if sufficient sample or solvent is available), beforemeasuring.6. Cell and Base-Line Checks6.1 Clean and match the cells. Suggested cleaning proce-dures are presented in Practice E275.6.2 Establish the base line of a recording double-be
17、amspectrophotometer by scanning over the appropriate wave-length region with pure solvent in both cells. Determineapparent absorbance of the sample cell at each wavelength ofinterest. These absorbances are cell corrections that are sub-tracted from the absorbance of the sample solution at thecorresp
18、onding wavelengths.6.3 For single beam instruments, either use the same cell forpure solvent and sample measurements, use matched cells, orapply appropriate cell corrections.6.4 On most software-controlled instruments, the cell cor-rections or the blank cell absorbance is stored in memory andautomat
19、ically 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 laboratories, and commonpractice is to purchase cells of known path length. Modern cellmanufacturing techniques employed by a number of leadingmanuf
20、acturers 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 wavelengths at whichabsorbance readings are taken for use in calculations. Thesemay include readings taken for purposes of background cor-rections.
21、To minimize the effect of wavelength error, theanalytical wavelengths are frequently chosen at absorptionmaxima, but this is not always necessary. If the wavelengthaccuracy of the spectrophotometer is such that the calculateduncertainty in the absorbance measurement is within accept-able limits at t
22、he extremes of this wavelength uncertainy range,then single point measurements on a slope can be used. Forexample, the use of isoabsorptive or isosbestic points isfrequently useful.7.2 Record the absorbance readings at the specified analyti-cal wavelengths, operating the instrument in accordance wit
23、hthe recommendations of the manufacturer or Practice E275.7.3 Absorbance values should be used only if they fallwithin the acceptably accurate range of the particular spectro-photometer and method employed. If the absorbance is too low,either use a longer absorption cell or prepare a new solution of
24、higher concentration. If the absorbance is too high, use ashorter cell or make a quantitative dilution3. If different cellsare used, a new base-line must 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. Proc
25、edures for checking precision and accuracy ofthese scales are presented in Practices E275 and E925.8. Resolution and Bandwidth8.1 If the analytical method specifies a resolution or aspectral slit width, set the resolution of the instrument to thespecified value. If the instrument has only a mechanic
26、albandwidth indicator, use the information provided in themanufacturers literature to calculate the bandwidth that cor-responds to the specified resolution.NOTE 2The accuracy of resolution and mechanical bandwidth indi-cators can be determined using the procedure given in Practice E958.8.2 If the an
27、alytical method does not state a requiredresolution or a bandwidth value but includes an illustrativespectrum, set the resolution or bandwidth 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
28、 nm, the resolution should not exceed 5nm.8.3 If the method neither specifies resolution or bandwidthnor provides an illustrative spectrum, use the smallest resolu-tion or bandwidth that yields an acceptable signal-to-noiseratio. Record this value for future reference.NOTE 3Changes in the day-to-day
29、 values of resolution or bandwidthobtained with a given gain, or changes in signal-to-noise ratio at a givenresolution, are indicative of present or potential problems. Increasedresolution or a lowering of the S/N ratio may result from a lower outputof the light source, deterioration of optical comp
30、onents, deposits on thewindows of the cell compartment or on the inside wall of the referencecell, an absorbing impurity in the solvent, or a faulty electronic compo-nent.9. Solvents and Solvent Effects9.1 The ultraviolet absorption spectrum of a compound willvary in different solvents depending on
31、the chemical structuresinvolved. Non-polar solvents have the least effect on theabsorption spectrum. Non-polar molecules in most instancesare not affected in polar solvents. However, polar molecules inpolar solvents may show marked differences in their spectra.Any interaction between solute and solv
32、ents leads to a broad-ening and change in structural resolution of the absorptionbands. Ionic forms may be created in acidic or basic solutions.In addition, there are possible chemical reactions betweensolute and solvent, and also photochemical reactions arisingfrom either room illumination or the s
33、hort wavelengths in thebeam of the spectrophotometer. It is important that the solventused be specified in recording spectral data. (The change inspectra between acidic and basic conditions may sometimes beemployed in multicomponent analysis.)9.2 Reference solvent data is shown in Table 1. Availabil
34、ityof a particular solvent may be restricted by internationalagreement, and the users attention is directed to 1.3 of thisPractices. The short 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
35、hydrochloric acid, sulfuricacid, and sodium hydroxide also are commonly used assolvents. Buffered solutions, involving non-absorbing materi-als, are frequently used; both the composition of the buffer and3The errors associated with cell path lengths are significantly less than thosegenerated by volu
36、metric dilution, and therefore where possible, different path lengthcells should be used in preference to volumetric procedures.E169 04 (2009)2the measured pH should be specified. Mixtures of 0.1 Mdi-hydrogen sodium phosphate and 0.1 M hydrogen di-sodiumphosphate are useful in the 4.5 to 8.9 pH rang
37、e. A table ofnon-absorbing buffers has been presented by Abbott (1).410. Calculations10.1 Quantitative analysis by ultraviolet spectrophotometrydepends upon Beers law. The terms and symbols used arethose defined in Terminology E131. According to Beers law:A 5 abc 5 /M! 3 bc (3)where:A = absorbance,a
38、 = absorptivity,b = cell length, cm,c = concentration, g/L, = molar absorptivity, andM = molecular weight.10.1.1 In practice, a distinction must be made between c, theconcentration of the absorbing material in the cell at the timeof observation, and the concentration of the absorbing materialin the
39、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, 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
40、must be included. Dilution factor, f,isthe ratio of the final volume to the initial volume. If more thanone dilution is performed, the dilution 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,
41、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 concentration in the cellis Cs/ f.10.3 Chemical CalibrationThe absorptivity of the absorb-ing material, the concentration of which it is desired todetermine
42、, is obtained by examination of a series of quantita-tive dilutions of a neat sample of this material. However, if nosuch neat sample is available, 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 “perce
43、ntageagainst calibration material” or by noting that the accuracy ofthe analysis is dependent upon a published value of theabsorptivity or molar absorptivity. (A reference must be cited.)10.3.1 Some sample materials are highly fluorescent whichcan significantly reduce the measured absorbance. The ef
44、fect ofsample fluorescence may vary depending upon the spectropho-tometer and wavelength chosen. Sample fluorescence may be aparticular problem 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 Compo
45、nent, Simple Background Correction:C 5A1 A2! 3 fa1bCs(8)where the subscripts refer to analytical wavelengths. Theterm A2is the absorbance at the wavelength used for making asimple subtractive correction. It is usually selected fromexamination of the spectral curve of the reference material at awavel
46、ength 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 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
47、 wavelength and the wavelength atwhich a simple subtractive background correction may beobtained. When it is possible to determine the slope betweenwavelengths 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 fo
48、r 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:4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.TABLE 1 SolventsASolvent Cutoff, nmPyridine 305Tetrachloroethyle
49、ne 290Benzene 280N,N-Dimethylformamide 270Carbon tetrachloride 265Methyl formate 260Chloroform 245Dichloromethane 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 (2, 3). Solvents of high purity for use inabsorption spectroscopy also are available commercially.E169 04 (2009)3C 5A1A31 A2 A3# 3l3 l1l3l2fabCc(10)The absorptivity a is here the effective absorptivity asdetermi
