1、Designation: E169 04 (Reapproved 2014)Standard Practices forGeneral Techniques of Ultraviolet-Visible QuantitativeAnalysis1This 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 Performance ofUltraviolet and Visible SpectrophotometersE925 Practice for Monitoring the Calibration of Ultrav
6、iolet-Visible Spectrophotometers whose Spectral Bandwidthdoes not Exceed 2 nmE958 Practice for Estimation of the Spectral Bandwidth ofUltraviolet-Visible Spectrophotometers3. Summary of Practice3.1 Quantitative ultraviolet and visible analyses are basedupon the absorption law, known as Beers law. Th
7、e 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 follow
8、s:A 5 a1bc11a2bc21.1anbcn(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 applications. I
9、t 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 shouldhelp
10、 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 operati
11、on 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 rangetole
12、rances 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 should b
13、econsidered as a more accurate alternative to volumetric, ifavailable. Fill the absorption cell with the solution, and fill the1These practices are under the jurisdiction of ASTM Committee E13 onMolecular Spectroscopy and Separation Science and are the direct responsibility ofSubcommittee E13.01 on
14、Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Aug. 1, 2014. Published August 2014. Originallyapproved in 1960. Last previous edition approved in 2009 as E169 04(2009). DOI:10.1520/E0169-04R14.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcont
15、act ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1comparison or blank cel
16、l with the pure solvent, at least 2 to 3(if sufficient sample or solvent is available), before measuring.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-beamspectrophotometer by
17、 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 thecorresponding wavelengths.6.3
18、 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 andautomatically incorporated in
19、to 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 leadingmanufacturers can guarantee
20、 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. To minimize the effect
21、 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 the extremes of this wa
22、velength 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 withthe recommendations o
23、f 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 ofhigher concentration.
24、If the absorbance is too high, use ashorter cell or make a quantitative dilution.3If 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. Procedures for checking pre
25、cision 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 mechanicalbandwidth indicator,
26、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 analytical method does no
27、t 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 nm, the resolution sho
28、uld 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 values of resolution o
29、r 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 components, deposits on the
30、windows 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 the chemical structures
31、involved. 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 solvents leads to a broad-e
32、ning 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 short wavelengths in the
33、beam 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. Availabilityof a particular solv
34、ent may be restricted by internationalagreement, and the users attention is directed to 1.3 of thesepractices. 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 hydrochloric acid, sul
35、furicacid, and sodium hydroxide also are commonly used assolvents. Buffered solutions, involving non-absorbing3The errors associated with cell path lengths are significantly less than thosegenerated by volumetric dilution, and therefore where possible, different path lengthcells should be used in pr
36、eference to volumetric procedures.E169 04 (2014)2materials, are frequently used; both the composition of thebuffer and the measured pH should be specified. Mixtures of0.1 M di-hydrogen sodium phosphate and 0.1 M hydrogendi-sodium phosphate are useful in the 4.5 to 8.9 pH range. Atable of non-absorbi
37、ng buffers has been presented by Abbott(3).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! 3bc (3)where:A = absorbance,a = absorptivity,b = cell l
38、ength, 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 sample as received. This i
39、s 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 must be included. Dilution
40、 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, have the dimensions of gra
41、ms 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, is obtained by examinati
42、on 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 “percentageagainst calibration m
43、aterial” 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 effect ofsample fluorescence
44、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 Component, Simple Background Cor
45、rection:C 5A12 A2! 3fa1bCs(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 awavelength longer than that of A
46、1, preferably where a2is muchless than a1.10.4.3 One Component, with Slope-Type Background Cor-rection:C 5A12 A21S22 1!# fa1bCs(9)where:S = slope between wavelengths 1 and 2 for the background.10.4.3.1 The background absorption is usually not linearbetween the analytical wavelength and the wavelengt
47、h 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 for the background absorption.1
48、0.4.4 One Component, With Linear Background Correc-tion:10.4.4.1 The equation for the general case is as follows:C 5A12FA31A22 A3# 332 132 2GfabCc(10)The absorptivity a is here the effective absorptivity asdetermined on a pure sample, using the corrections, and issomewhat lower than the true or abso
49、lute absorptivity.4The boldface numbers in parentheses refer to a list of references at the end ofthis standard.TABLE 1 SolventsASolvent Cutoff, nmPyridine 305Tetrachloroethylene 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 (1, 2). Solvents of high purity for
