Designation: E169 − 16

Standard Practices for

General Techniques of Ultraviolet-Visible Quantitative
Analysis1
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.

1. Scope 3. Summary of Practice

1.1 These practices are intended to provide general infor- 3.1 Quantitative ultraviolet and visible analyses are based
mation on the techniques most often used in ultraviolet and upon the absorption law, known as Beer’s law. The units of this
visible quantitative analysis. The purpose is to render unnec- law are defined in Terminology E131. Beer’s law (Note 1)
essary the repetition of these descriptions of techniques in holds at a single wavelength and when applied to a single
individual methods for quantitative analysis. component sample it may be expressed in the following form
1.2 The values stated in SI units are to be regarded as (see Section 11):
standard. No other units of measurement are included in this A 5 abc (1)
standard.
When applied to a mixture of n non-interacting components,
1.3 This standard does not purport to address all of the it may be expressed as follows:
safety concerns, if any, associated with its use. It is the
A 5 a 1 bc1 1a 2 bc2 1….1a n bcn (2)
responsibility 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. NOTE 1—Detailed discussion of the origin and validity of Beer’s law
may be found in the books and articles listed in the bibliography at the end
2. Referenced Documents of these practices.

2.1 ASTM Standards:2 3.2 This practice describes the application of Beer’s law in
E131 Terminology Relating to Molecular Spectroscopy typical spectrophotometric analytical applications. It also de-
E168 Practices for General Techniques of Infrared Quanti- scribes operating parameters that must be considered when
tative Analysis (Withdrawn 2015)3 using these techniques.
E275 Practice for Describing and Measuring Performance of
Ultraviolet and Visible Spectrophotometers 4. Significance and Use
E387 Test Method for Estimating Stray Radiant Power Ratio 4.1 These practices are a source of general information on
of Dispersive Spectrophotometers by the Opaque Filter the techniques of ultraviolet and visible quantitative analyses.
Method They provide the user with background information that should
E925 Practice for Monitoring the Calibration of Ultraviolet- help ensure the reliability of spectrophotometric measure-
Visible Spectrophotometers whose Spectral Bandwidth ments.
does not Exceed 2 nm 4.2 These practices are not intended as a substitute for a
E958 Practice for Estimation of the Spectral Bandwidth of thorough understanding of any particular analytical method. It
Ultraviolet-Visible Spectrophotometers is the responsibility of the users to familiarize themselves with
the critical details of a method and the proper operation of the
available instrumentation.
1
These practices are under the jurisdiction of ASTM Committee E13 on
Molecular Spectroscopy and Separation Science and are the direct responsibility of
Subcommittee E13.01 on Ultra-Violet, Visible, and Luminescence Spectroscopy. 5. Sample Preparation
Current edition approved April 1, 2016. Published April 2016. Originally 5.1 Accurately weigh the specified amount of the sample
approved in 1960. Last previous edition approved in 2014 as E169 – 04(2014). DOI:
10.1520/E0169-16. (solid or liquid). Dissolve in the appropriate solvent and dilute
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or to the specified volume in volumetric glassware of the required
contact ASTM Customer Service at [email protected]. For Annual Book of ASTM accuracy, ensuring that all appropriate temperature range
Standards volume information, refer to the standard’s Document Summary page on
tolerances are maintained. If needed, a dilution should be made
the ASTM website.
3
The last approved version of this historical standard is referenced on with a calibrated pipet and volumetric flask, using adequate
www.astm.org. volumes for accuracy. With the availability of moderin wide

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States

1
 E169 − 16
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
comparison or blank cell with the pure solvent, at least 2× to 3× 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- photometer and method employed. If the absorbance is too low,
pended particles not visible to the naked eye, and these will either use a longer absorption cell or prepare a new solution of
still scatter light by the Tyndall effect, causing a decrease in the 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.4 If different cells
Unless there is no alternative, absorbance should not be are used, a new base-line must be obtained.
determined on turbid or light scattering samples. Any measure- 7.4 The precision and bias of the wavelength and photomet-
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 For single beam instruments, either use the same cell for
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
6.5 An accurate determination of cell path length in the manufacturer’s literature to calculate the bandwidth that cor-
1-cm range is not practical in most laboratories, and common responds to the specified resolution.
practice is to purchase cells of known path length. Modern cell 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 1-cm 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
7. Analytical Wavelengths and Photometry spectrum, set the resolution or bandwidth of the instrument to
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- 5 nm.
rections. To minimize the effect of wavelength error, the
analytical wavelengths are frequently chosen at absorption 9.3 If the method neither specifies resolution or bandwidth
maxima, but this is not always necessary. If the wavelength nor provides an illustrative spectrum, use the smallest resolu-
accuracy of the spectrophotometer is such that the calculated tion or bandwidth that yields an acceptable signal-to-noise
uncertainty in the absorbance measurement is within accept- ratio. Record this value for future reference.
able limits at the extremes of this wavelength uncertainty
range, then single point measurements on a slope can be used. 4
The errors associated with cell path lengths are significantly less than those
For example, the use of isoabsorptive or isosbestic points is generated by volumetric dilution, and therefore where possible, different path length
frequently useful. cells should be used in preference to volumetric procedures.

2
 E169 − 16
NOTE 5—Changes in the day-to-day values of resolution or bandwidth di-sodium phosphate are useful in the 4.5 to 8.9 pH range. A
obtained with a given gain, or changes in signal-to-noise ratio at a given table of non-absorbing buffers has been presented by Abbott
resolution, are indicative of present or potential problems. Increased
resolution or a lowering of the S/N ratio may result from a lower output
(3).5
of the light source, deterioration of optical components, deposits on the 11. Calculations
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- 11.1 Quantitative analysis by ultraviolet spectrophotometry
nent. depends upon Beer’s law. The terms and symbols used are
those defined in Terminology E131. According to Beer’s law:
10. Solvents and Solvent Effects
A 5 abc 5 ~ ε/M ! 3 bc (3)
10.1 The ultraviolet absorption spectrum of a compound
will vary in different solvents depending on the chemical where:
structures involved. Non-polar solvents have the least effect on A = absorbance,
the absorption spectrum. Non-polar molecules in most in- a = absorptivity,
stances are not affected in polar solvents. However, polar b = cell length, cm,
molecules in polar solvents may show marked differences in c = concentration, g/L,
their spectra. Any interaction between solute and solvents leads ε = molar absorptivity, and
to a broadening and change in structural resolution of the M = molecular weight.
absorption bands. Ionic forms may be created in acidic or basic 11.1.1 In practice, a distinction must be made between c, the
solutions. In addition, there are possible chemical reactions concentration of the absorbing material in the cell at the time
between solute and solvent, and also photochemical reactions of observation, and the concentration of the absorbing material
arising from either room illumination or the short wavelengths in the sample as received. This is here designated as a mass
in the beam of the spectrophotometer. It is important that the fraction C (g/g). The solution to be examined has a concentra-
solvent used be specified in recording spectral data. (The tion of sample in solution, Cs, which is in units of grams per
change in spectra between acidic and basic conditions may litre.
sometimes be employed in multicomponent analysis.) c 5 A/ab (4)
10.2 Reference solvent data is shown in Table 1. Availabil- C 5 c/C s 5 A/ ~ abCs ! (5)
ity of a particular solvent may be restricted by international 11.2 If one or more dilutions are then made, the quantity
agreement, and the users’ attention is directed to 1.3 of these called the dilution factor must be included. Dilution factor, f, is
practices. The short wavelength limit is approximate, and the ratio of the final volume to the initial volume. If more than
refers to the wavelength at which a 1-cm light path length gives one dilution is performed, the dilution factor is the product of
an absorbance of unity. the factors from each dilution. If dilutions are made, the
10.3 Water, and 0.1 M solutions of hydrochloric acid, equation becomes the following:
sulfuric acid, and sodium hydroxide also are commonly used as C 5 cf/C s 5 Af/ ~ abCs ! (6)
solvents. Buffered solutions, involving non-absorbing
materials, are frequently used; both the composition of the Note that c and Cs, have the dimensions of grams per litre.
buffer and the measured pH should be specified. Mixtures of If dilution is made, Cs is not the concentration in the cell at the
0.1 M di-hydrogen sodium phosphate and 0.1 M hydrogen time the absorbance is determined; the concentration in the cell
is Cs/ f.
11.3 Chemical Calibration—The absorptivity of the absorb-
TABLE 1 SolventsA ing material, the concentration of which it is desired to
Solvent Cutoff, nm determine, is obtained by examination of a series of quantita-
Pyridine 305 tive dilutions of a neat sample of this material. However, if no
Tetrachloroethylene 290 such neat sample is available, the best available material is
Benzene 280
N,N-Dimethylformamide 270 used, or a value of the absorptivity is taken from the literature.
Carbon tetrachloride 265 Take care to specify this, by reporting values as “percentage
Methyl formate 260 against calibration material” or by noting that the accuracy of
Chloroform 245
Dichloromethane 235 the analysis is dependent upon a published value of the
Ethyl ether 220 absorptivity or molar absorptivity. (A reference must be cited.)
Acetonitrile 215 11.3.1 Some sample materials are highly fluorescent which
Isopropyl alcohol 210
Ethyl alcohol 210 can significantly reduce the measured absorbance. The effect of
Methyl alcohol 210 sample fluorescence may vary depending upon the spectropho-
Cyclohexane <210 tometer and wavelength chosen. Sample fluorescence may be a
Isooctane <210
A
particular problem when using published absorptivity values.
Procedures for special purification of solvents for further improvement in the
wavelength limit are given in Refs (1, 2). Solvents of high purity for use in
5
absorption spectroscopy also are available commercially. The boldface numbers in parentheses refer to a list of references at the end of
this standard.

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 E169 − 16
11.4 Types of Analyses (see Fig. 1): where the subscripts refer to analytical wavelengths. The
11.4.1 One Component, No Background Correction: term A2 is the absorbance at the wavelength used for making a
C 5 Af/ ~ abCs ! (7) simple subtractive correction. It is usually selected from
examination of the spectral curve of the reference material at a
11.4.2 One Component, Simple Background Correction: wavelength longer than that of A1, preferably where a2 is much
~ A 1 2 A 2! 3 f less than a1.
C5 (8)
a 1 bCs

(5) (6)

(1) One Component, No Background Correction. (2) One Component, Simple Background Correction.
(3) One Component, with Slope-Type Background Correction. (4) One Component, with Linear Background Correction.
(5) Two Components, with Overlapping Absorption for Only One Component. (6) Two Components, with Mutually Overlapping Absorption.

FIG. 1 Plot of Components

4
 E169 − 16
11.4.3 One Component, with Slope-Type Background Cor- one component at a given wavelength is more than 100 times
rection: the product for the other component, allowing the latter to be
@ A 1 2 A 2 1S ~ λ 2 2 λ 1 ! # f neglected.
C5 (9) 11.4.7 Two Components, With Overlapping Absorption for
a 1 bCs
Only One Component—Determine the component with no
where: interference (component x) at an analytical wavelength, λ1,
S = slope between wavelengths 1 and 2 for the background. selected to allow no contribution from component y as follows:
11.4.3.1 The background absorption is usually not linear C x 5 fA1 ~ a 1x bCs ! (13)
between the analytical wavelength and the wavelength at 11.4.7.1 Calculate the contribution of this component to the
which a simple subtractive background correction may be observed absorbance at the other analytical wavelength, λ2,
obtained. When it is possible to determine the slope between where both components are absorbing, as follows:
wavelengths 1 and 2 by observation of the samples that do not
A 2x 5 a 2x bcx 5 a 2x bCx C s /f (14)
contain the absorbing material that is to be determined, this
may be used as a correction for the background absorption. 11.4.7.2 Calculate the concentration of component yas fol-
11.4.4 One Component, With Linear Background Correc- lows:
tion: C y 5 @ ~ A 2 2 A 2x ! 3 f # /a 2y bCs (15)
11.4.4.1 The equation for the general case is as follows:
11.4.8 Two Components, with Mutually Overlapping

C5
F
A 1 2 A 31 @ A 2 2 A 3# 3
λ3 2 λ1
λ3 2 λ 2
f G (10)
Absorption—Use the absorbance-ratio method (graphical) de-
scribed in Ref (4) or by simultaneous equations as follows:
abCc
@ a 2x A 1 2 a 1y A 2 # f
Cx 5 (16)
The absorptivity a is here the effective absorptivity as bCs 3 ~ a 2y a 1x 2 a 1y a 2x !
determined on a pure sample, using the corrections, and is
somewhat lower than the true or absolute absorptivity.
11.4.4.2 This method is especially effective with materials @ a 2x A 1 2 a 1a A 2 # f
Cy 5 (17)
that have sharp bands. Wavelengths 2 and 3 are selected to the bCs 3 ~ a 1y a 2x 2 a 2y a 1x !
long and short wavelength sides of the analytical wavelength 1, 11.4.9 Inverted Matrix Method, for Two or More
usually at absorbance minima. Components, With Mutually Overlapping Absorption—For in-
11.4.4.3 For the special case where the wavelength for A1 is formation on the inverted matrix method, see Sections 10 and
exactly midway between the wavelengths for A1 and A3, the 17 of Practices E168.
equation reduces to the following: 11.5 Computerized Calculations—Newer instruments may

C5f F A1 2
A 2 1A 3

abCs
2 G (11)
perform automatically many of the calculations described in
11.4. The user should be aware of the algorithms used by the
manufacturer. It is recommended that the user verify the
reliability of computed results by periodically performing the
11.4.5 One Component, With Background Correction from
calculations using the raw analytical data.
Outside Data:
~A 2 X! 3 f 12. Presentation of Data
C5 (12) 12.1 If absorption curves are to be presented with an
abCs
analytical method, it is recommended that one of the following
where X is a background absorbance calculated from outside systems be used, with the wavelength (in nanometers) increas-
data. ing linearly to the right:
11.4.5.1 This is a general case in which some empirical log ε or log a plotted against λ
correction may be derived from data other than A plotted against λ
spectrophotometric, and is applied as an effective absorbance ε × 10-n or a plotted against λ
which is subtracted from the observed. As an example, the where the symbols are as defined in Terminology E131.
concentration of a known interfering material may be deter- Marking the analytical wavelengths and absorptivity values on
mined by titration, and the absorbance due to this calculated, the curve is suggested for clarity, or a separate table of
and then subtracted. analytical wavelengths and absorptivities may be used. (These
11.4.6 Two Components, With No Overlapping data are helpful for others who may wish to use the method in
Absorption—Apply the method in 11.4.1 twice, at the two a somewhat modified form.)
analytical wavelengths. This is an almost impossible case,
except when the relative concentrations of the two components 13. Keywords
are such that the product of absorptivity and concentration of 13.1 molecular spectroscopy; quantitative analysis

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 E169 − 16
REFERENCES

(1) Burke, R. W. and Mavrodineanu, R., Accuracy in Analytical (3) Abbott, R., “Ultraviolet Spectrophotometric Techniques,” Applied
Spectrophotometry, NBS Special Publication 260 – 81, National Bu- Spectroscopy, Vol 4, No. 1, October 1948.
reau of Standards, Washington, DC, 1983. (4) Hirt, R. C., King, F. T., and Schmitt, R. G.,“ Graphical Absorbance
(2) Klevens, H. B., and Platt, J. R., “Ultraviolet Transmission Limits of Ratio Method for Rapid Two-Component Spectrophotometric
Some Liquids and Solids,” Journal of the American Chemical Society, Analysis,” Analytical Chemistry, Vol 26, 1954, p. 1270.
Vol 69, 1947, p. 3055.

BIBLIOGRAPHY

(1) Burgess, C. and Frost, T., eds., Standards and Best Practice in (7) Hershenson, H. M., Ultraviolet and Visible Absorption Spectra:
Absorption Spectrometry, Blackwell Science, London, 1999. Index for 1930–1954, Academic Press, NY, 1956.
(2) Ewing, P. J., Kolthoff, I. M., and Meehan, E. J., editors, Treatise on (8) Lothian, G. F., Absorption Spectrophotometry, Macmillan, NY, 2nd
Analytical Chemistry, Part I, 7th edition, vol. 7, (Chapters 1 through Ed., 1958.
3), Interscience, NY, NY, 1981. (9) Mellon, M. G., Editor, Analytical Absorption Spectroscopy, John
(3) Friedel, R. A., and Orchin, M., Ultraviolet Spectra of Aromatic Wiley & Sons, N. Y.
Compounds, John Wiley & Sons, NY, 1951. (10) Olsen, E. D., Modern Optical Methods of Analysis, (Chapters 1
(4) Graff, M. M., O’Connor, R. T., and Skau, E. L., “Purification of through 3) McGraw-Hill, NY, NY, 1975.
Solvents for Absorption Spectroscopy,” Industrial and Engineering (11) Tunnicliff, D. D., “Solvents for Ultraviolet Spectrophotometry,”
Chemistry, Analytical Edition, IENAA, Vol 16, 1944, p. 556.
Talanta, Vol 2, No. 4, 1959, p. 341.
(5) Gillam, A. E., and Stern, E. S., Electronic Absorption Spectroscopy,
(12) West, W., Editor, Chemical Applications of Spectroscopy, Vol IX or
E. Arnold, London, 2nd Ed., 1957.
Weissberger series, Technique of Organic Chemistry, Interscience,
(6) Hager, L. G. and Howell, J. A., Annual Reviews in Analytical
Chemistry, 54, 171R (1982) and 56 225R (1984). NY, 1956.

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