1、Designation: E169 04 (Reapproved 2014)E169 16Standard 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 revis
2、ion, 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 These practices are intended to provide general information on the techniques most often used in ultr
3、aviolet and visiblequantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methodsfor quantitative analysis.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standa
4、rd.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced
5、Documents2.1 ASTM Standards:2E131 Terminology Relating to Molecular SpectroscopyE168 Practices for General Techniques of Infrared Quantitative Analysis (Withdrawn 2015)E275 Practice for Describing and Measuring Performance of Ultraviolet and Visible SpectrophotometersE387 Test Method for Estimating
6、Stray Radiant Power Ratio of Dispersive Spectrophotometers by the Opaque Filter MethodE925 Practice for Monitoring the Calibration of Ultraviolet-Visible Spectrophotometers whose Spectral Bandwidth does notExceed 2 nmE958 Practice for Estimation of the Spectral Bandwidth of Ultraviolet-Visible Spect
7、rophotometers3. Summary of Practice3.1 Quantitative ultraviolet and visible analyses are based upon the absorption law, known as Beers law. The units of this laware defined inTerminology E131. Beers law (Note 1) holds at a single wavelength and when applied to a single component sampleit may be expr
8、essed in the following form (see Section 1011):A 5abc (1)When applied to a mixture of n non-interacting components, it may be expressed as follows:A 5a1bc11a2bc21.1anbcn (2)NOTE 1Detailed discussion of the origin and validity of Beers law may be found in the books and articles listed in the bibliogr
9、aphy at the end ofthese practices.3.2 This practice describes the application of Beers law in typical spectrophotometric analytical applications. It also describesoperating parameters that must be considered when using these techniques.4. Significance and Use4.1 These practices are a source of gener
10、al information on the techniques of ultraviolet and visible quantitative analyses. Theyprovide the user with background information that should help ensure the reliability of spectrophotometric measurements.1 These practices are under the jurisdiction ofASTM Committee E13 on Molecular Spectroscopy a
11、nd Separation Science and are the direct responsibility of SubcommitteeE13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.Current edition approved Aug. 1, 2014April 1, 2016. Published August 2014May 2016. Originally approved in 1960. Last previous edition approved in 20092014 asE169 04(2
12、009).(2014). DOI: 10.1520/E0169-04R14.10.1520/E0169-16.2 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This d
13、ocument is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions
14、as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14.2 These practices are not intended as a substitute f
15、or a thorough understanding of any particular analytical method. It is theresponsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the availableinstrumentation.5. Sample Preparation5.1 Accurately weigh the specified amount of the sample
16、(solid or liquid). Dissolve in the appropriate solvent and dilute to thespecified volume in volumetric glassware of the required accuracy, ensuring that all appropriate temperature range tolerances aremaintained. If needed, a dilution should be made with a calibrated pipet and volumetric flask, usin
17、g adequate volumes for accuracy.With the availability of moderin wide range electronic balances, (capable of reading kg quantities to four or five decimal places),gravimetric dilution should be considered as a more accurate alternative to volumetric, if available. Fill the absorption cell withthe so
18、lution, and fill the comparison or blank cell with the pure solvent, at least 2 to 3 (if sufficient sample or solvent isavailable), before measuring.5.2 The solution should be visibly clear, and free from particulate matter. However, there may still be present suspendedparticles not visible to the n
19、aked eye, and these will still scatter light by the Tyndall effect, causing a decrease in the measuredintensity that increases as the wavelength decreases. Unless there is no alternative, absorbance should not be determined on turbidor light scattering samples. Any measurements performed on a light
20、scattering solution are highly instrument specific and can beused only for comparative purposes in the same system.NOTE 2To avoid the dilution step, the instrument may contain an automatic system which will allow adjustment of the path length of the measurementcell to optimize the measured absorbanc
21、e.6. Cell and Base-Line Checks6.1 Clean and match the cells. Suggested cleaning procedures are presented in Practice E275.6.2 Establish the base line of a recording double-beam spectrophotometer by scanning over the appropriate wavelength regionwith pure solvent in both cells. Determine apparent abs
22、orbance of the sample cell at each wavelength of interest.These absorbancesare cell corrections that are subtracted from the absorbance of the sample solution at the corresponding wavelengths.6.3 For single beam instruments, either use the same cell for pure solvent and sample measurements, use matc
23、hed cells, or applyappropriate cell corrections.6.4 On most software-controlled instruments, the cell corrections or the blank cell absorbance is stored in memory andautomatically incorporated into the sample absorbance measurement.6.5 An accurate determination of cell path length in the 1-cm range
24、is not practical in most laboratories, and common practiceis to purchase cells of known path length. Modern cell manufacturing techniques employed by a number of leading manufacturerscan guarantee the path length of a 1-cm cell to 60.01 mm or better.7. Analytical Wavelengths and Photometry7.1 Analyt
25、ical wavelengths are those wavelengths at which absorbance readings are taken for use in calculations. These mayinclude readings taken for purposes of background corrections. To minimize the effect of wavelength error, the analyticalwavelengths are frequently chosen at absorption maxima, but this is
26、 not always necessary. If the wavelength accuracy of thespectrophotometer is such that the calculated uncertainty in the absorbance measurement is within acceptable limits at the extremesof this wavelength uncertainyuncertainty range, then single point measurements on a slope can be used. For exampl
27、e, the use ofisoabsorptive or isosbestic points is frequently useful.NOTE 3If the sample matrix includes fluorescent compounds, the measured signal usually will contain a contribution from fluorescence.7.2 Record the absorbance readings at the specified analytical wavelengths, operating the instrume
28、nt in accordance with therecommendations of the manufacturer or Practice E275.7.3 Absorbance values should be used only if they fall within the acceptably accurate range of the particular spectrophotometerand method employed. If the absorbance is too low, either use a longer absorption cell or prepa
29、re a new solution of higherconcentration. If the absorbance is too high, use a shorter cell or make a quantitative dilution.3 If different cells are used, a newbase-line must be obtained.7.4 The precision and bias of the wavelength and photometric scales of the instrument must be adequate for the me
30、thod beingused. Procedures for checking precision and accuracy of these scales are presented in Practices E275 and E925.3 The last approved version of this historical standard is referenced on www.astm.org.3 The errors associated with cell path lengths are significantly less than those generated by
31、volumetric dilution, and therefore where possible, different path length cellsshould be used in preference to volumetric procedures.E169 1628. Stray Radiant Energy (Stray Light)8.1 The acceptable absorbance range for any given instrument will be governed not only by the specification of thespectroph
32、otometer, but also by the condition at time of measurement.8.2 Given that the measurement is fundamentally a difference in energy in an optical system, the factors affecting themeasurement may include, but not be limited to: the output from the source(s), efficiency of the grating, cleanliness of th
33、e mirrors,etc.8.3 Stray radiant energy in any instrument system may begin to cause a negative deviation error, long before the transmittance(absorbance) limit is reached. An effective estimation may be performed using Practice E387.9. Resolution and Bandwidth9.1 If the analytical method specifies a
34、resolution or a spectral slit width, set the resolution of the instrument to the specifiedvalue. If the instrument has only a mechanical bandwidth indicator, use the information provided in the manufacturers literatureto calculate the bandwidth that corresponds to the specified resolution.NOTE 4The
35、accuracy of resolution and mechanical bandwidth indicators can be determined using the procedure given in Practice E958.9.2 If the analytical method does not state a required resolution or a bandwidth value but includes an illustrative spectrum, setthe resolution or bandwidth of the instrument to ob
36、tain comparable data. As a rule of thumb, the resolution should be less thanone-eighth of the bandwidth; thus for a peak of bandwidth 40 nm, the resolution should not exceed 5 nm.5 nm.9.3 If the method neither specifies resolution or bandwidth nor provides an illustrative spectrum, use the smallest
37、resolution orbandwidth that yields an acceptable signal-to-noise ratio. Record this value for future reference.NOTE 5Changes in the day-to-day values of resolution or bandwidth obtained with a given gain, or changes in signal-to-noise ratio at a givenresolution, are indicative of present or potentia
38、l problems. Increased resolution or a lowering of the S/N ratio may result from a lower output of the lightsource, deterioration of optical components, deposits on the windows of the cell compartment or on the inside wall of the reference cell, an absorbingimpurity in the solvent, or a faulty electr
39、onic component.10. Solvents and Solvent Effects10.1 The ultraviolet absorption spectrum of a compound will vary in different solvents depending on the chemical structuresinvolved. Non-polar solvents have the least effect on the absorption spectrum. Non-polar molecules in most instances are notaffect
40、ed in polar solvents. However, polar molecules in polar solvents may show marked differences in their spectra. Anyinteraction between solute and solvents leads to a broadening and change in structural resolution of the absorption bands. Ionicforms may be created in acidic or basic solutions. In addi
41、tion, there are possible chemical reactions between solute and solvent,and also photochemical reactions arising from either room illumination or the short wavelengths in the beam of thespectrophotometer. It is important that the solvent used be specified in recording spectral data. (The change in sp
42、ectra betweenacidic and basic conditions may sometimes be employed in multicomponent analysis.)10.2 Reference solvent data is shown in Table 1. Availability of a particular solvent may be restricted by internationalagreement, and the users attention is directed to 1.3 of these practices. The short w
43、avelength limit is approximate, and refers tothe wavelength at which a 1-cm light path length gives an absorbance of unity.TABLE 1 SolventsASolvent Cutoff, nmPyridine 305Tetrachloroethylene 290Benzene 280N,N-Dimethylformamide 270Carbon tetrachloride 265Methyl formate 260Chloroform 245Dichloromethane
44、 235Ethyl ether 220Acetonitrile 215Isopropyl alcohol 210Ethyl alcohol 210Methyl alcohol 210Cyclohexane 210Isooctane 210A Procedures for special purification of solvents for further improvement in thewavelength limit are given in Refs (1, 2). Solvents of high purity for use inabsorption spectroscopy
45、also are available commercially.E169 16310.3 Water, and 0.1 M solutions of hydrochloric acid, sulfuric acid, and sodium hydroxide also are commonly used as solvents.Buffered solutions, involving non-absorbing materials, are frequently used; both the composition of the buffer and the measuredpH shoul
46、d be specified. Mixtures of 0.1 M di-hydrogen sodium phosphate and 0.1 M hydrogen di-sodium phosphate are useful inthe 4.5 to 8.9 pH range. A table of non-absorbing buffers has been presented by Abbott (3).411. Calculations11.1 Quantitative analysis by ultraviolet spectrophotometry depends upon Beer
47、s law. The terms and symbols used are thosedefined in Terminology E131. According to Beers law:A 5abc5/M! 3bc (3)where:A = absorbance,a = absorptivity,b = cell length, cm,c = concentration, g/L, = molar absorptivity, andM = molecular weight.11.1.1 In practice, a distinction must be made between c, t
48、he concentration of the absorbing material in the cell at the time ofobservation, and the concentration of the absorbing material in the sample as received. This is here designated as a mass fractionC (g/g). The solution to be examined has a concentration of sample in solution, Cs, which is in units
49、 of grams per litre.c 5A/ab (4)C 5c/Cs 5A/abCs! (5)11.2 If one or more dilutions are then made, the quantity called the dilution factor must be included. Dilution factor, f, is theratio of the final volume to the initial volume. If more than one dilution is performed, the dilution factor is the product of the factorsfrom each dilution. If dilutions are made, the equation becomes the following:C 5cf/Cs 5Af/abCs! (6)Note that c and Cs, have the dimensions of grams per litre. If dilution is made, Cs is not the concentration in the cell at the timethe absorbance
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