ASTM E169-04(2009)
(Practice)Standard Practices for General Techniques of Ultraviolet-Visible Quantitative Analysis
Standard Practices for General Techniques of Ultraviolet-Visible Quantitative Analysis
SIGNIFICANCE AND USE
These practices are a source of general information on the techniques of ultraviolet and visible quantitative analyses. They provide the user with background information that should help ensure the reliability of spectrophotometric measurements.
These practices are not intended as a substitute for a thorough understanding of any particular analytical method. It is the responsibility of the users to familiarize themselves with the critical details of a method and the proper operation of the available instrumentation.
SCOPE
1.1 These practices are intended to provide general information on the techniques most often used in ultraviolet and visible quantitative analysis. The purpose is to render unnecessary the repetition of these descriptions of techniques in individual methods for 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 standard.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E169 − 04 (Reapproved2009)
Standard Practices for
General Techniques of Ultraviolet-Visible
Quantitative Analysis
This standard is issued under the fixed designation E169; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope holds at a single wavelength and when applied to a single
component sample it may be expressed in the following form
1.1 These practices are intended to provide general infor-
(see Section 10):
mation on the techniques most often used in ultraviolet and
visible quantitative analysis. The purpose is to render unnec-
A 5 abc (1)
essary the repetition of these descriptions of techniques in
Whenappliedtoamixtureof nnon-interactingcomponents,
individual methods for quantitative analysis.
it may be expressed as follows:
1.2 The values stated in SI units are to be regarded as
A 5 a bc 1a bc 1….1a bc (2)
standard. No other units of measurement are included in this 1 1 2 2 n n
NOTE 1—Detailed discussion of the origin and validity of Beer’s law
standard.
maybefoundinthebooksandarticleslistedinthebibliographyattheend
1.3 This standard does not purport to address all of the
of these practices.
safety concerns, if any, associated with its use. It is the
3.2 This practice describes the application of Beer’s law in
responsibility of the user of this standard to establish appro-
typical spectrophotometric analytical applications. It also de-
priate safety and health practices and determine the applica-
scribes operating parameters that must be considered when
bility of regulatory limitations prior to use.
using these techniques.
2. Referenced Documents
4. Significance and Use
2.1 ASTM Standards:
E131Terminology Relating to Molecular Spectroscopy
4.1 These practices are a source of general information on
E168Practices for General Techniques of Infrared Quanti-
the techniques of ultraviolet and visible quantitative analyses.
tative Analysis
Theyprovidetheuserwithbackgroundinformationthatshould
E275PracticeforDescribingandMeasuringPerformanceof
help ensure the reliability of spectrophotometric measure-
Ultraviolet and Visible Spectrophotometers
ments.
E925Practice for Monitoring the Calibration of Ultraviolet-
Visible Spectrophotometers whose Spectral Bandwidth 4.2 These practices are not intended as a substitute for a
does not Exceed 2 nm thorough understanding of any particular analytical method. It
E958Practice for Measuring Practical Spectral Bandwidth is the responsibility of the users to familiarize themselves with
of Ultraviolet-Visible Spectrophotometers the critical details of a method and the proper operation of the
available instrumentation.
3. Summary of Practice
3.1 Quantitative ultraviolet and visible analyses are based 5. Sample Preparation
upontheabsorptionlaw,knownasBeer’slaw.Theunitsofthis
5.1 Accurately weigh the specified amount of the sample
law are defined in Terminology E131. Beer’s law (Note 1)
(solid or liquid). Dissolve in the appropriate solvent and dilute
tothespecifiedvolumeinvolumetricglasswareoftherequired
accuracy, ensuring that all appropriate temperature range
These practices are under the jurisdiction of ASTM Committee E13 on
Molecular Spectroscopy and Separation Science and are the direct responsibility of
tolerancesaremaintained.Ifneeded,adilutionshouldbemade
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
with a calibrated pipet and volumetric flask, using adequate
Current edition approved Oct. 1, 2009. Published December 2009. Originally
volumes for accuracy. With the availability of moderin wide
approved in 1960. Last previous edition approved in 2004 as E169–04. DOI:
10.1520/E0169-04R09.
range electronic balances, (capable of reading kg quantities to
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
four or five decimal places), gravimetric dilution should be
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
considered as a more accurate alternative to volumetric, if
Standardsvolumeinformation,refertothestandard’sDocumentSummarypageon
the ASTM website. available. Fill the absorption cell with the solution, and fill the
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E169 − 04 (2009)
comparisonorblankcellwiththepuresolvent,atleast2×to3× being used. Procedures for checking precision and accuracy of
(if sufficient sample or solvent is available), before measuring. these scales are presented in Practices E275 and E925.
6. Cell and Base-Line Checks 8. Resolution and Bandwidth
8.1 If the analytical method specifies a resolution or a
6.1 Clean and match the cells. Suggested cleaning proce-
dures are presented in Practice E275. spectral slit width, set the resolution of the instrument to the
specified value. If the instrument has only a mechanical
6.2 Establish the base line of a recording double-beam
bandwidth indicator, use the information provided in the
spectrophotometer by scanning over the appropriate wave-
manufacturer’s literature to calculate the bandwidth that cor-
length region with pure solvent in both cells. Determine
responds to the specified resolution.
apparent absorbance of the sample cell at each wavelength of
interest. These absorbances are cell corrections that are sub-
NOTE 2—The accuracy of resolution and mechanical bandwidth indi-
cators can be determined using the procedure given in Practice E958.
tracted from the absorbance of the sample solution at the
corresponding wavelengths.
8.2 If the analytical method does not state a required
resolution or a bandwidth value but includes an illustrative
6.3 Forsinglebeaminstruments,eitherusethesamecellfor
spectrum, set the resolution or bandwidth of the instrument to
pure solvent and sample measurements, use matched cells, or
obtain comparable data. As a rule of thumb, the resolution
apply appropriate cell corrections.
should be less than one-eighth of the bandwidth; thus for a
6.4 On most software-controlled instruments, the cell cor-
peak of bandwidth 40 nm, the resolution should not exceed 5
rections or the blank cell absorbance is stored in memory and
nm.
automatically incorporated into the sample absorbance mea-
8.3 If the method neither specifies resolution or bandwidth
surement.
nor provides an illustrative spectrum, use the smallest resolu-
6.5 An accurate determination of cell path length in the
tion or bandwidth that yields an acceptable signal-to-noise
1-cm range is not practical in most laboratories, and common
ratio. Record this value for future reference.
practiceistopurchasecellsofknownpathlength.Moderncell
NOTE 3—Changes in the day-to-day values of resolution or bandwidth
manufacturing techniques employed by a number of leading
obtained with a given gain, or changes in signal-to-noise ratio at a given
manufacturers can guarantee the path length of a 1-cm cell to
resolution, are indicative of present or potential problems. Increased
60.01 mm or better.
resolution or a lowering of the S/N ratio may result from a lower output
of the light source, deterioration of optical components, deposits on the
7. Analytical Wavelengths and Photometry
windows of the cell compartment or on the inside wall of the reference
cell, an absorbing impurity in the solvent, or a faulty electronic compo-
7.1 Analytical wavelengths are those wavelengths at which
nent.
absorbance readings are taken for use in calculations. These
may include readings taken for purposes of background cor-
9. Solvents and Solvent Effects
rections. To minimize the effect of wavelength error, the
9.1 The ultraviolet absorption spectrum of a compound will
analytical wavelengths are frequently chosen at absorption
varyindifferentsolventsdependingonthechemicalstructures
maxima, but this is not always necessary. If the wavelength
involved. Non-polar solvents have the least effect on the
accuracy of the spectrophotometer is such that the calculated
absorption spectrum. Non-polar molecules in most instances
uncertainty in the absorbance measurement is within accept-
are not affected in polar solvents. However, polar molecules in
ablelimitsattheextremesofthiswavelengthuncertainyrange,
polar solvents may show marked differences in their spectra.
then single point measurements on a slope can be used. For
Any interaction between solute and solvents leads to a broad-
example, the use of isoabsorptive or isosbestic points is
ening and change in structural resolution of the absorption
frequently useful.
bands. Ionic forms may be created in acidic or basic solutions.
7.2 Record the absorbance readings at the specified analyti-
In addition, there are possible chemical reactions between
cal wavelengths, operating the instrument in accordance with
solute and solvent, and also photochemical reactions arising
the recommendations of the manufacturer or Practice E275.
from either room illumination or the short wavelengths in the
beam of the spectrophotometer. It is important that the solvent
7.3 Absorbance values should be used only if they fall
used be specified in recording spectral data. (The change in
within the acceptably accurate range of the particular spectro-
spectrabetweenacidicandbasicconditionsmaysometimesbe
photometerandmethodemployed.Iftheabsorbanceistoolow,
employed in multicomponent analysis.)
eitherusealongerabsorptioncellorprepareanewsolutionof
higher concentration. If the absorbance is too high, use a
9.2 Reference solvent data is shown in Table 1.Availability
shorter cell or make a quantitative dilution . If different cells
of a particular solvent may be restricted by international
are used, a new base-line must be obtained.
agreement, and the users’ attention is directed to 1.3 of this
Practices. The short wavelength limit is approximate, and
7.4 Theprecisionandbiasofthewavelengthandphotomet-
referstothewavelengthatwhicha1-cmlightpathlengthgives
ric scales of the instrument must be adequate for the method
an absorbance of unity.
9.3 Water,and0.1Msolutionsofhydrochloricacid,sulfuric
The errors associated with cell path lengths are significantly less than those
acid, and sodium hydroxide also are commonly used as
generatedbyvolumetricdilution,andthereforewherepossible,differentpathlength
cells should be used in preference to volumetric procedures. solvents. Buffered solutions, involving non-absorbing
E169 − 04 (2009)
A
TABLE 1 Solvents
the factors from each dilution. If dilutions are made, the
Solvent Cutoff, nm
equation becomes the following:
Pyridine 305
C 5 cf/C 5 Af/ abC (6)
~ !
s s
Tetrachloroethylene 290
Benzene 280
N,N-Dimethylformamide 270
Note that c and C , have the dimensions of grams per litre.
Carbon tetrachloride 265
s
Methyl formate 260
Ifdilutionismade, C isnottheconcentrationinthecellatthe
s
Chloroform 245
timetheabsorbanceisdetermined;theconcentrationinthecell
Dichloromethane 235
is C /f.
Ethyl ether 220
s
Acetonitrile 215
10.3 ChemicalCalibration—Theabsorptivityoftheabsorb-
Isopropyl alcohol 210
Ethyl alcohol 210
ing material, the concentration of which it is desired to
Methyl alcohol 210
determine, is obtained by examination of a series of quantita-
Cyclohexane <210
tive dilutions of a neat sample of this material. However, if no
Isooctane <210
A such neat sample is available, the best available material is
Procedures for special purification of solvents for further improvement in the
wavelength limit are given in Refs (2, 3). Solvents of high purity for use in used, or a value of the absorptivity is taken from the literature.
absorption spectroscopy also are available commercially.
Take care to specify this, by reporting values as “percentage
against calibration material” or by noting that the accuracy of
the analysis is dependent upon a published value of the
absorptivity or molar absorptivity. (Areference must be cited.)
materials, are frequently used; both the composition of the
10.3.1 Some sample materials are highly fluorescent which
buffer and the measured pH should be specified. Mixtures of
cansignificantlyreducethemeasuredabsorbance.Theeffectof
0.1 M di-hydrogen sodium phosphate and 0.1 M hydrogen
samplefluorescencemayvarydependinguponthespectropho-
di-sodium phosphate are useful in the 4.5 to 8.9 pH range. A
tometerandwavelengthchosen.Samplefluorescencemaybea
table of non-absorbing buffers has been presented by Abbott
particular problem when using published absorptivity values.
(1).
10.4 Types of Analyses (see Fig. 1):
10. Calculations
10.4.1 One Component, No Background Correction:
10.1 Quantitative analysis by ultraviolet spectrophotometry
C 5 Af/~abC ! (7)
s
depends upon Beer’s law. The terms and symbols used are
those defined in Terminology E131. According to Beer’s law:
10.4.2 One Component, Simple Background Correction:
A 5 abc 5 ~ϵ/M! 3bc (3)
~A 2 A ! 3f
1 2
C 5 (8)
a bC
1 s
where:
A = absorbance,
where the subscripts refer to analytical wavelengths. The
a = absorptivity,
term A is the absorbance at the wavelength used for making a
b = cell length, cm, 2
simple subtractive correction. It is usually selected from
c = concentration, g/L,
´ = molar absorptivity, and examinationofthespectralcurveofthereferencematerialata
M = molecular weight. wavelengthlongerthanthatofA ,preferablywherea ismuch
1 2
less than a .
10.1.1 Inpractice,adistinctionmustbemadebetweenc,the
10.4.3 One Component, with Slope-Type Background Cor-
concentration of the absorbing material in the cell at the time
rection:
ofobservation,andtheconcentrationoftheabsorbingmaterial
in the sample as received. This is here designated as a mass
@A 2 A 1S~λ 2 λ !# f
1 2 2 1
C 5 (9)
fraction C (g/g). The solution to be examined has a concentra-
a bC
1 s
tion of sample in solution, C , which is in units of grams per
s
litre.
where:
c 5 A/ab (4)
S = slopebetweenwavelengths1and2forthebackground.
C 5 c/C 5 A/ abC (5)
~ !
s s
10.4.3.1 The background absorption is usually not linear
between the analytical wavelength and the wavelength at
10.2 If one or more dilutions are then made, the quantity
which a simple subtractive background correction may be
calledthedilutionfactormustbeincluded.Dilutionfactor,f,is
obtained. When it is possible to determine the slope between
the ratio of the final volume to the initial volume. If more than
wavelengths 1 and 2 by observation of the samples that do not
one dilution is performed, the dilution factor is the product of
contain the absorbing material that is to be determined, this
may be used as a correction for the background absorption.
10.4.4 One Co
...
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