ASTM E169-16(2022)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E169 − 16 (Reapproved 2022)
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 Visible Spectrophotometers whose Spectral Bandwidth
does not Exceed 2 nm
1.1 These practices are intended to provide general infor-
E958Practice for Estimation of the Spectral Bandwidth of
mation on the techniques most often used in ultraviolet and
Ultraviolet-Visible Spectrophotometers
visible quantitative analysis. The purpose is to render unnec-
essary the repetition of these descriptions of techniques in
3. Summary of Practice
individual methods for quantitative analysis.
3.1 Quantitative ultraviolet and visible analyses are based
1.2 The values stated in SI units are to be regarded as
upontheabsorptionlaw,knownasBeer’slaw.Theunitsofthis
standard. No other units of measurement are included in this
law are defined in Terminology E131. Beer’s law (Note 1)
standard.
holds at a single wavelength and when applied to a single
1.3 This standard does not purport to address all of the
component sample it may be expressed in the following form
safety concerns, if any, associated with its use. It is the
(see Section 11):
responsibility of the user of this standard to establish appro-
A 5 abc (1)
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
Whenappliedtoamixtureof nnon-interactingcomponents,
1.4 This international standard was developed in accor-
it may be expressed as follows:
dance with internationally recognized principles on standard-
A 5 a bc 1a bc 1….1a bc (2)
1 1 2 2 n n
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
mendations issued by the World Trade Organization Technical
NOTE 1—Detailed discussion of the origin and validity of Beer’s law
maybefoundinthebooksandarticleslistedinthebibliographyattheend
Barriers to Trade (TBT) Committee.
of these practices.
2. Referenced Documents
3.2 This practice describes the application of Beer’s law in
typical spectrophotometric analytical applications. It also de-
2.1 ASTM Standards:
scribes operating parameters that must be considered when
E131Terminology Relating to Molecular Spectroscopy
using these techniques.
E168Practices for General Techniques of Infrared Quanti-
tative Analysis
4. Significance and Use
E275PracticeforDescribingandMeasuringPerformanceof
Ultraviolet and Visible Spectrophotometers
4.1 These practices are a source of general information on
E387TestMethodforEstimatingStrayRadiantPowerRatio
the techniques of ultraviolet and visible quantitative analyses.
of Dispersive Spectrophotometers by the Opaque Filter
Theyprovidetheuserwithbackgroundinformationthatshould
Method
help ensure the reliability of spectrophotometric measure-
E925Practice for Monitoring the Calibration of Ultraviolet-
ments.
4.2 These practices are not intended as a substitute for a
thorough understanding of any particular analytical method. It
These practices are under the jurisdiction of ASTM Committee E13 on
Molecular Spectroscopy and Separation Science and are the direct responsibility of is the responsibility of the users to familiarize themselves with
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy.
the critical details of a method and the proper operation of the
Current edition approved Nov. 1, 2022. Published November 2022. Originally
available instrumentation.
approved in 1960. Last previous edition approved in 2016 as E169–16. DOI:
10.1520/E0169-16R22.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or 5. Sample Preparation
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
5.1 Accurately weigh the specified amount of the sample
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. (solid or liquid). Dissolve in the appropriate solvent and dilute
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E169 − 16 (2022)
tothespecifiedvolumeinvolumetricglasswareoftherequired uncertainty in the absorbance measurement is within accept-
accuracy, ensuring that all appropriate temperature range able limits at the extremes of this wavelength uncertainty
tolerancesaremaintained.Ifneeded,adilutionshouldbemade range, then single point measurements on a slope can be used.
with a calibrated pipet and volumetric flask, using adequate For example, the use of isoabsorptive or isosbestic points is
volumes for accuracy. With the availability of modern wide frequently useful.
range electronic balances, (capable of reading kg quantities to
NOTE 3—If the sample matrix includes fluorescent compounds, the
four or five decimal places), gravimetric dilution should be
measured signal usually will contain a contribution from fluorescence.
considered as a more accurate alternative to volumetric, if
7.2 Record the absorbance readings at the specified analyti-
available. Fill the absorption cell with the solution, and fill the
cal wavelengths, operating the instrument in accordance with
comparisonorblankcellwiththepuresolvent,atleast2×to3×
the recommendations of the manufacturer or Practice E275.
(if sufficient sample or solvent is available), before measuring.
7.3 Absorbance values should be used only if they fall
5.2 The solution should be visibly clear, and free from
within the acceptably accurate range of the particular spectro-
particulate matter. However, there may still be present sus-
photometerandmethodemployed.Iftheabsorbanceistoolow,
pended particles not visible to the naked eye, and these will
either use a longer absorption cell or prepare a new solution of
stillscatterlightbytheTyndalleffect,causingadecreaseinthe
higher concentration. If the absorbance is too high, use a
measured intensity that increases as the wavelength decreases.
shorter cell or make a quantitative dilution. If different cells
Unless there is no alternative, absorbance should not be
are used, a new base-line must be obtained.
determinedonturbidorlightscatteringsamples.Anymeasure-
7.4 Theprecisionandbiasofthewavelengthandphotomet-
ments performed on a light scattering solution are highly
ric scales of the instrument must be adequate for the method
instrument specific and can be used only for comparative
being used. Procedures for checking precision and accuracy of
purposes in the same system.
these scales are presented in Practices E275 and E925.
NOTE 2—To avoid the dilution step, the instrument may contain an
automatic system which will allow adjustment of the path length of the
8. Stray Radiant Energy (Stray Light)
measurement cell to optimize the measured absorbance.
8.1 The acceptable absorbance range for any given instru-
6. Cell and Base-Line Checks
ment will be governed not only by the specification of the
spectrophotometer, but also by the condition at time of mea-
6.1 Clean and match the cells. Suggested cleaning proce-
surement.
dures are presented in Practice E275.
8.2 Given that the measurement is fundamentally a differ-
6.2 Establish the base line of a recording double-beam
ence in energy in an optical system, the factors affecting the
spectrophotometer by scanning over the appropriate wave-
measurement may include, but not be limited to: the output
length region with pure solvent in both cells. Determine
from the source(s), efficiency of the grating, cleanliness of the
apparent absorbance of the sample cell at each wavelength of
mirrors, etc.
interest. These absorbances are cell corrections that are sub-
tracted from the absorbance of the sample solution at the 8.3 Stray radiant energy in any instrument system may
corresponding wavelengths.
begin to cause a negative deviation error, long before the
transmittance (absorbance) limit is reached. An effective esti-
6.3 Forsinglebeaminstruments,eitherusethesamecellfor
mation may be performed using Practice E387.
pure solvent and sample measurements, use matched cells, or
apply appropriate cell corrections.
9. Resolution and Bandwidth
6.4 On most software-controlled instruments, the cell cor-
9.1 If the analytical method specifies a resolution or a
rections or the blank cell absorbance is stored in memory and
spectral slit width, set the resolution of the instrument to the
automatically incorporated into the sample absorbance mea-
specified value. If the instrument has only a mechanical
surement.
bandwidth indicator, use the information provided in the
manufacturer’s literature to calculate the bandwidth that cor-
6.5 An accurate determination of cell path length in the
responds to the specified resolution.
1cm range is not practical in most laboratories, and common
practiceistopurchasecellsofknownpathlength.Moderncell
NOTE 4—The accuracy of resolution and mechanical bandwidth indi-
manufacturing techniques employed by a number of leading
cators can be determined using the procedure given in Practice E958.
manufacturers can guarantee the path length of a 1cm cell to
9.2 If the analytical method does not state a required
60.01 mm or better.
resolution or a bandwidth value but includes an illustrative
spectrum, set the resolution or bandwidth of the instrument to
7. Analytical Wavelengths and Photometry
obtain comparable data. As a rule of thumb, the resolution
7.1 Analytical wavelengths are those wavelengths at which
should be less than one-eighth of the bandwidth; thus for a
absorbance readings are taken for use in calculations. These
peak of bandwidth 40 nm, the resolution should not exceed
may include readings taken for purposes of background cor-
5nm.
rections. To minimize the effect of wavelength error, the
analytical wavelengths are frequently chosen at absorption
The errors associated with cell path lengths are significantly less than those
maxima, but this is not always necessary. If the wavelength
generatedbyvolumetricdilution,andthereforewherepossible,differentpathlength
accuracy of the spectrophotometer is such that the calculated cells should be used in preference to volumetric procedures.
E169 − 16 (2022)
9.3 If the method neither specifies resolution or bandwidth 0.1 M di-hydrogen sodium phosphate and 0.1 M hydrogen
nor provides an illustrative spectrum, use the smallest resolu- di-sodium phosphate are useful in the 4.5pH to 8.9 pH range.
tion or bandwidth that yields an acceptable signal-to-noise Atable of non-absorbing buffers has been presented byAbbott
ratio. Record this value for future reference. (3).
NOTE 5—Changes in the day-to-day values of resolution or bandwidth
11. Calculations
obtained with a given gain, or changes in signal-to-noise ratio at a given
resolution, are indicative of present or potential problems. Increased
11.1 Quantitative analysis by ultraviolet spectrophotometry
resolution or a lowering of the S/N ratio may result from a lower output
depends upon Beer’s law. The terms and symbols used are
of the light source, deterioration of optical components, deposits on the
those defined in Terminology E131. According to Beer’s law:
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-
A 5 abc 5 ~ε/M! 3bc (3)
nent.
where:
10. Solvents and Solvent Effects
A = absorbance,
a = absorptivity,
10.1 The ultraviolet absorption spectrum of a compound
b = cell length, cm,
will vary in different solvents depending on the chemical
c = concentration, g/L,
structuresinvolved.Non-polarsolventshavetheleasteffecton
ε = molar absorptivity, and
the absorption spectrum. Non-polar molecules in most in-
M = molecular weight.
stances are not affected in polar solvents. However, polar
molecules in polar solvents may show marked differences in 11.1.1 Inpractice,adistinctionmustbemadebetweenc,the
theirspectra.Anyinteractionbetweensoluteandsolventsleads concentration of t
...