Analytical Absorption Spectrophotometry in the Visible and Ultraviolet: The Principles

303 Pages·1989·32.08 MB·English

by  L. SOMMER (Eds.)

STUDIES IN ANALYTICAL CHEMISTRY
Editorial Board
E. PUNGOR (Budapest), W. SIMON (Zurich),
H. MALISSA (Vienna), J. INCZEDY (Veszprem)
Volume 1 Structural Analysis of Organic Compounds by Combined Application of Spectroscopic
Methods (Clerc, Pretsch, Seibel)
Volume 2 The Principles of Ion-Selective Electrodes and of Membrane Transport (Morf)
Volume 3 Nondestructive Activation Analysis (Amiel, Editor)
Volume 4 Guide-Lines to Planning Atomic Spectrometric Analysis (Magyar)
Volume 5 Quantitative Analysis of Steroids (Gorog)
Volume 6 Solvation, Ionic and Complex Formation Reactions in Non-Aqueous Solvents.
Experimental Methods for their Investigation (Burger)
Volume 7 Problem Solving with Microbeam Analysis (Kiss)
Volume 8 Analytical Absorption Spectrophotometry in the Visible and Ultraviolet. The Principles
(Sommer)
STUDIES IN ANALYTICAL CHEMISTRY 8
ANALYTICAL
ABSORPTION
SPECTROPHOTOMETRY
IN THE VISIBLE
AND ULTRAVIOLET
THE PRINCIPLES
L. SOMMER
Professor of Analytical Chemistry
J. E. Purkyne University
Brno, Czechoslovakia
ELSEVIER
Amsterdam-Oxford-New York-Tokyo 1989
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Library of Congress Cataloging in Publication Data
Sommer, L. (Lumir)
Analytical absorption spectrophotometry in the visible and ultraviolet the principles/L. Sommer.
p. cm. — (Studies in analytical chemistry; 8)
Bibliography: p.
Includes index.
ISBN 0-444-98882-3 (U.S.)
1. Absorption spectra. 2. Spectrophotometry.
I. Title. II. Series.
QD96.A2S65 1989 3-37014
543\ 0853—dcl9 CIP
ISBN 0-444-98882-3 (Vol. 8)
ISBN 0-444-41944-6 (Series)
© L. Sommer, 1989
Joint edition published by
Elsevier Science Publishers, Amsterdam, The Netherlands
and Akademiai Kiado, Budapest, Hungary
All rights reserved. No part of this book may be reproduced by any means, or transmitted or translated
into machine language without the written permission of the copyright owner.
Printed in Hungary
by Akademiai Kiado es Nyomda Vallalat, Budapest
PREFACE
Today it is rather difficult to write a book on molecular absorption
spectrophotometry in the ultraviolet (UV) and visible (VIS) regions, because a
comprehensive literature is already available on this frequently used physicochem-
ical analytical method. Quantitative analysis by means of spectrophotometry has
been practised continuously for more than a century. Despite the development of
many competitive analytical techniques, especially atomic absorption spec­
trophotometry, molecular spectrophotometry has remained very popular, e.g. in
biochemical, biomedical, agricultural or environmental analysis. The inherent ease
and relative simplicity of spectrophotometric procedures, as well as the availability
of reliable and highly automated instruments are undoubtedly important reasons of
this popularity.
On the other hand, although comprehensive collections of spectrophotometric
procedures for the determination of elements, inorganic, organic, biochemical or
pharmaceutical substances are readily available, there are few treatments of the
actual trends in spectrophotometry. The spectrophotometry in UV and VIS is far
from losing the analyst's interest. In inorganic elemental analysis this method has
currently been largely replaced by atomic absorption spectrophotometry (AAS) for
several reasons, but there are elements for which atomization remains difficult or the
AAS procedures are insensitive or subject to a considerable matrix effect. It is very
suitable to combine spectrophotometry with extraction procedures. Moreover, the
use of spectrophotometry for the analysis of organic, biochemical or biologically
active compounds, surfactants or macromolecules should be further investigated,
especially in connection with thin layer chromatography (TLC) or high-
performance liquid chromatography (HPLC). The simultaneous use of ion-
exchange or chelating resins and HPLC, improving the selectivity of this method, or
the derivative, difference and high-sensitivity spectrophotometry, as well as the
computer-aided spectrophotometric analysis of multicomponent mixtures open
new horizons for spectrophotometry. Moreover, spectrophotometry in the UV and
VIS regions remains of significance in routine analyses of several kinds such as the
determination of pollutants, drugs and toxic substances, or the analysis of body
tissues, food stuffs, etc. The automation of analytical procedures in solution is also
5
favourable for spectrophotometry. In addition, spectrophotometry in the UV and
VIS regions is closely linked with the development of the theory and practice of
organic analytical reagents and complex formation in solution; however, we are still
unable to predict unambiguously the most suitable reagent for a particular metal
ion.
In this book, I try to show briefly the present status of spectrophotometry in the
UV and VIS regions, and describe the trends in its development, without detailing
procedures for the determination of analytes. For the latter, the reader is referred to
comprehensive books on spectrophotometry.
I am very much obliged to my co-worker, Dr. M. Langova, C. Sc., assistant
professor of this Department, who greatly helped me in the preparation of the text
and figures and compiled with me the chapter on the evaluation of spec­
trophotometric data. Drs. V. Otruba and V. Kuban, C. Sc., senior research fellows
of this Department, contributed to the preparation of the chapters on in­
strumentation (V. O.) and automation in spectrophotometry (V. K.) The chapter
on the application of spectrophotometric procedures in the clinical laboratory was
discussed with Dr. P. Popov, C. Sc., Head of the Laboratory of Clinical
Biochemistry, Municipal Institute of National Health, Brno.
I am indebted to my distinguished friend, Professor J. Inczedy, head of the
Institute of Analytical Chemistry of the University of Chemical Engineering,
Veszprem, for the careful reading of the manuscript and a number of comments, and
to Dr. Cristina Kallay, the responsible editor of the Akademiai Kiado, Budapest,
who carefully followed the text and found various errors. Special thanks are due to
Professor E. Pungor, Member of the Hungarian Academy of Sciences and Head of
the Institute of General and Analytical Chemistry of the Technical University,
Budapest, and to the Staff of Akademiai Kiado for their work and kind help in
publishing this book.
L. Sommer
6
CHAPTER 1
BASIS OF SPECTROPHOTOMETRY
IN THE UV AND VIS REGIONS
1.1. Interaction of species in solution with radiation
of wavelengths between 200 and 800 nm
If radiation of wavelength between 200 and 800 nm is absorbed by molecules or
solvated ions, the main energy changes occur at electronic levels but may be
accompanied by changes in the vibrational quantum levels of atomic groups
comprising the molecule. The whole energy change during the transition of a
molecule into its excited state is formally described by
J£ = hc// = /v = (£<f-£<l>) + (£tf>-£;t{») + ( £ < 2^ 0, O-D
1 r r
where h = 6.6256 χ 1034 Jsandc = 3x 1010 cm s The result of all the interactions
of the molecule with the absorbed radiation is the actual absorption spectrum in the
VIS and UV regions, which can be represented as a plot of a measure of the
absorbed radiant energy vs. λ, ν, or ν, where λ (nm=10~9 m), v = c/x (s1) and
v = l/A =107//l .
cm nm
Some characteristic energy transitions in a molecule are shown in Fig. 1.1. The
area where electronic transitions are localized is called the chromophore. This can
be a hydrated cation, a complex or chelate with inorganic or organic ligands, a
π-complex, an electron donor and acceptor complex, an organic molecule, etc.
The incident coherent radiation power /, can interact with a particular molecule
0
in an homogeneous or heterogeneous medium limited by two planeparallel
boundaries according to
/ο = Λ* + /ρ + Ά + / (1-2)
Γ
or
l=/R//0 + /p// + /A//0 + /r//0, (13)
0
where /, /, / and J are the radiation powers of the reflected, transmitted,
R P A r
absorbed or scattered intensity. In the context of particular methods of
measurement, / is related to the direct reflectance, / and / with molecular
R P A
absorption spectrophotometry and turbidimetry, / with photoacoustic spectrome­
A
try, fluorimetry and phosphorimetry and I with light scattering, nephelometry and
T
diffuse reflectance. The absorbed radiant energy is transformed mainly into heat,
but is sometimes emitted as secondary radiation.
The important spectrophotometric variables are collected in Table 1.1.
13
> Rotational levels of E2 53000
J Vibrational levels of E
2 45000
50000
Electronic level E
2
4000
Vibr.+Rot. levels Fi
2000
Electronic level E^ Vibrational Electronic
transitions transitions
Fig. 1.1. Possible energy levels of a molecule (adapted from Manning, R. L., Introduction to Spectroscopy,
Pye Unicam, Ltd., Cambridge 1969, with permission); numbers in the figure mean wavenumbers
Table 1.1. Spectrophotometric variables for monochromatic radiation
Variable Formula Limiting values
Transmittance r=/// O^T^l
0
T%=100 I// O^TSIOO
0
Absorptance a=l-T l^a^O
a%=100-T lOO^a^O
Absorbance ,4 = log (lo/l)
A = 2- logT%
1.2 Bouguer-Lambert-Beer (BLB) law
The absorption of incident monochromatic radiation by a sample is controlled
by
-dl = kl dl (1.4)
which when integrated over the path length, /, gives
fd/// = fcf
dl (1.5)
/ο 0
14
or
ln^=fc/, (1.6)
where k is a constant depending on concentration. For a solution of a molar
concentration c of the absorbing analyte we obtain
Iny =ec. (1.7)
x
Finally, combination of Eqs (1.6) and (1.7) yields
Iny = φ (1.8)
or
I = Ie~£Alc, (1.9)
0
where In (///) = >l = - In T.
0 nat
The "natural" absorbance may easily be transformed into the decadic
absorbance
,4 = 2.303 4 = 2.303 logy =2.303 e/c, (1.10)
nat A
where /, / are the intensities of the incident and transmitted radiation, / is the cell
0
path length, c is the analyte concentration in mol 1 ~1 and ε the molar absorption
λ
coefficient in 1 mol"1 cm'1. The decadic absorbance introduced already by Bunsen
is much more convenient for analytical practice in accordance with the following
widely used relationships
/ = / ιο-^ = / ΗΓ", (1.11)
0 0
logy =A= -log T=sjc. (1.12)
Various mathematical treatments have been published in the literature [1, 3-8].
The BLB law is usually valid for dilute analyte solutions, for strongly monochroma­
tic, parallel and coherent radiation and in optically homogeneous (isotropic) media.
Luminescence and scattering of the solution should be absent and the optical path
strictly defined. Its validity is demonstrated by strictly linear plots of A vs. cell path
length or concentration of the absorbing analyte species in solution [7]. The
absorbance is proportional to the number of absorbing particles and depends on
their nature [2], so that the BLB law might be valid also for heterogeneous media or
turbid solutions having particles of a certain number and size, but scattering of
15
radiation causes deviations. The BLB law is also valid for mixtures of absorbing
species providing that there are no mutual interactions which would contravene the
principle of additivity of absorbances
^χ = ει€ι +«2C2 + FI3C3+ · · · + Vn (/= 10 mm). (1.13)
The BLB law is not obeyed if radiation over a broad range of wavelengths is used
or by concentrated analyte solutions, solutions where physical and chemical
interactions take place among the components or where some other scattering is
involved. However, polychromatic radiation may not influence the validity of the
BLB law when a double beam arrangement is used, comprising two cells containing
solutions of different concentrations of absorbing species and whose path length
may be changed in order to match the radiation intensity. This is the case in the
Duboscq colorimeter, where a simple relationship pertains for equal radiation
intensities
= 0-14)
where the indices χ and s denote two analytes [6].
No difficulties are generally observed when highly precise spectrophotometers
are used for solutions in which a single absorbing analyte species is formed
quantitatively and no other interactions take place. The same instrument should be
used for performing the A vs. c (calibration) plot as well as for determination of
anaIyte
unknown analyte concentrations, at the selected wavelength and under optimum
conditions.
13 Molar absorption coefficient
This is the effective cross-section of 1 mol of absorbing species for radiation of a
particular wavelength and corresponds to the absorbance of a solution containing
1 mol Ρ1 of an absorbing species in a path length of 1 cm. It is easily calculated from
s = A/lc (in 1 mol'1 cm'1). (1.15)
x
This relationship is valid for highly monochromatic radiation, but ε is dependent on
the band pass width for certain spectrophotometers, especially in sharp absorption
bands of the analyte. Conditional values, ε', are usually available depending on the
λ
spectrophotometer used. Various special absorption coefficients can be defined if
the concentration of analyte is expressed in another way, e.g.
eJAt. mass (g"11cm"1) (1.15a)
or
eJAt. massx 1000 (mg'Mcm"1) (1.15b)
16
which are independent of the atomic mass of the elements and correspond to the
absorbance of the element at a concentration of 1 mg ml-1 or 1 μgml"1 [12].
Furthermore, the expression A\xj£M may be useful for the absorption coefficient of
not well defined substances or those with unknown stoichiometry.
The molecular absorption coefficient may be calculated as follows: 6.02 χ 1023
absorbing molecules are present in a 1000 cm2 cross-section for an 1-cm path
length, according to the definition of molar absorption coefficient. This means 6.02
χ 1023/103 = 6.02 χ 1020 molecules in a 1cm2 cross-section. Assuming ε
λ
the molecular absorption coefficient is
105 cm2 mol1 ,
=A r 1.6 4χgx 1160"16 cm2.
6.02 xlO20 mol -1
Formally, the absorbing surface of the molecule in cm2 corresponds with the
molecular absorption coefficient. This is ca. 1% of the total molecular surface, if the
average molecular size is 10"7 cm or a surface area of nr2 = 10"14 cm2 is assumed
[11].
13.1 Calculation of molar absorption coefficient
There are various ways to evaluate the molar absorption coefficient of an
absorbing species, i.e.: (a) from the BLB equation (1.15) if a strict linear relationship
between the absorbance and analyte concentration is maintained; (b) from the slope
of the linear calibration plot using regression analysis; (c) from absorbance plateaus
of various absorbance plots, under conditions where the absorbing species is formed
quantitatively in solutions with sufficient reagent excess (A vs. c or A vs. pH);
reagent
(d) from the intercept of various slope-intercept transformations of equilibrium
constants describing the formation of a particular absorbing analyte complex
[13, 14]; (e) from spectrophotometric data for mixtures of absorbing complexes in
solution using various minimizing procedures with the aid of computers (cf. [15,
16]); (/) from isosbestic points on absorption spectra related to solutions where two
absorbing species are mutually transformed. The concentration of the master
variable (ligand or metal ion concentration in sufficient excess or pH of the solution)
is here changed
^iso = ^iso Kci+c). (1.16)
2
c, c being the concentrations of absorbing complexes and c +e = c or c, the
x 2 l 2 M L
total concentration of the minor component.
2 Analytical 17