1

Ultraviolet and Visible (UV-Vis) Absorption Spectroscopy

The word ‘spectroscopy’ is used as a collective term for all the analytical
techniques based on the interaction of light and matter. Spectrophotometry is
one of the branches of spectroscopy where we measure the absorption of light
by
molecules that are in a gas or vapour state or dissolved molecules/ions.
Spectrophotometry investigates the absorption of the different substances
between the wavelength limits 190 nm and 780 nm (visible spectroscopy is
restricted to the wavelength range of electromagnetic radiation detectable by
the
human eye, that is above ~360 nm; ultraviolet spectroscopy is used for shorter
wavelengths). In this wavelength range the absorption of the electromagnetic
radiation is caused by the excitation (i.e. transition to a higher energy level) of
the bonding and non-bonding electrons of the ions or molecules. A graph of
absorbance against wavelength gives the sample’s absorption spectrum. Modern
spectrophotometers draw this automatically. The measured spectrum is
continuous, due to the fact that the different vibration and rotation states of the
molecules make the absorption band wider.
Spectrophotometry is used for both qualitative and quantitative investigations of
samples. The wavelength at the maximum of the absorption band will give
information about the structure of the molecule or ion and the extent of the
absorption is proportional with the amount of the species absorbing the light.
Quantitative measurements are based on Beer’s Law (also known as “Lambert-
Beer Law” or even “Bouguer-Lambert-Beer Law”) which is described as follows:
A = ec l
where A = absorbance [no units, because it is calculated as A = log10(I0/I),
where I0 is the incident light’s intensity and I is the light intensity
after it passes through the sample];
e = molar absorbance or absorption coefficient [in dm3 mol-1 cm-1
units];
c = concentration (molarity) of the compound in the solution [in mol
dm-3 units];
l = path length of light in the sample [in cm units].
Note 1.: Instead of the molar absorbance you can also use the specific
absorbance, but you have to make sure that the proper concentration unit is
applied.
Note 2.: Molar absorbance is a function of wavelength, so Beer’s Law is always
applied at one (or several) specific wavelength values.
The instruments used for spectrophotometry are called photometers and
spectrophotometers (fig. 1). The difference between them is that we can only
make measurements at a particular wavelength with a photometer, but
spectrophotometers can be used for the whole wavelength range.
2
Figure 1: Photograph of a spectrophotometer
Both types of instruments have suitable light sources, monochromator (that
selects the light with the necessary wavelength) and a detector. The solution is
put into a sample tube (called a “cuvette”). The light intensity measured by the
detector is converted into an electric signal and is displayed as a certain
absorbance on the readout (fig. 2.).
Figure 2: Principle of UV/Vis spectrometer, source:
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UVVis/
uvspec.htm#uv1
3
The quantitative determinations made by spectrophotometry could be divided in
two groups according to the number of substances to be measured. These are
the analytical measurements of systems that consist of only one absorbing
component and systems that consist of more than one absorbing component.
In the case of systems containing one absorbing component we measure the
absorption of light at a particular wavelength (which is usually identical with the
absorption maximum of the analyte). We either calculate the concentration from
the results by using the molar absorptivity found in the literature or the
unknown
concentration can be determined by comparing the results with a working curve
of absorbance versus concentration (calibration curve) derived using standards.
However the determination of concentration could be influenced by the following
factors even in the simplest cases:
Selectivity problems: the absorbance of other substance or substances
being present might be added to the absorbance of the compound under
investigation. The effect of this phenomenon could be eliminated (or at
least reduced) either by increasing the selectivity by chemical reaction or
by preliminary separation using either extraction or chromatography.
Deviations from the Beer’s Law: if using a “not properly monochromatic”
light beam, the high absorbencies are decreased compared to the real
values. The association of molecules in the sample (which depends on the
concentration) also could cause deviations.
When samples contain more than one absorbing component, the determination
of concentration can be done only by an indirect method, because the
absorbencies of two or more component are added together. In this case we
have the following options:
Determination of concentrations by using systems of equations
Derivative spectrophotometry
Author: Sore Ferenc
Photograph: Vámos István
Institution: Petrik Lajos Vocational School for Chemistry, Environmental
Sciences and Information Technology, Budapest, Hungary
For further information see:
http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/uvvisab1.htm
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm
Reference textbooks:
D. A. Skoog - D. M. West - F. J. Holler: Fudamentals of Analytical
Chemistry (Saunders College Publishing, Fort Worth, US 1992.)
J. Kenkel: Analytical Chemistry for Technicians (Lewis Publishers, Boca

Raton, US 1994.)

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Introduction
Ultraviolet and visible spectrometers have been in general use for the last 35 years and over this period have become
the most
important analytical instrument in the modern day laboratory. In many applications other techniques could be
employed but none
rival UV-Visible spectrometry for its simplicity, versatility, speed, accuracy and cost-effectiveness.
This description outlines the basic principles for those new to UV-Visible spectrometry. It is intended purely as a brief
introduction to the technique and it is Thermo Spectronic's policy to continually add to this range of documentation for
further
details, as they become available.
Definitions and Units
Radiation is a form of energy and we are constantly reminded of its presence via our sense of sight and ability to feel
radiant
heat. It may be considered in terms of a wave motion where the wavelength, λ, is the distance between two
successive peaks.
The frequency, ν, is the number of peaks passing a given point per second. These terms are related so that:
c =νλ
where c is the velocity of light in a vacuum.
Figure 1 The wavelength λ of electromagnetic radiation
The full electromagnetic radiation spectrum is continuous and each region merges slowly into the next. For
spectroscopy
purposes, we choose to characterize light in the ultraviolet and visible regions in terms of wavelength expressed in
nanometers.
Other units which may be encountered, but whose use is now discouraged, are the Angstrom (Å) and the millimicron
(mμ).
1nm = 1mμ = 10Å = 10-9 meters

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For convenience of reference, definitions of the various spectral regions have been set by the Joint Committee on
Nomenclature
in Applied Spectroscopy:
Region Wavelength (nm)
Far ultraviolet 10-200
Near ultraviolet 200-380
Visible 380-780
Near infrared 780-3000
Middle infrared 3000-30,000
Far infrared 30,000-300,000
Microwave 300,000-1,000,000,000
The human eye is only sensitive to a tiny proportion of the total electromagnetic spectrum between approximately 380
and 780
nm and within this area we perceive the colors of the rainbow from violet through to red. If the full electromagnetic
spectrum
shown in Figure 2 was redrawn on a linear scale and the visible region was represented by the length of one
centimeter, then
the boundary between radio and microwaves would have to be drawn approximately 25 kilometers away!
Figure 2 The electromagnetic spectrum
Radiation Sources
Besides the sun, the most conveniently available source of visible radiation with which we are familiar is the tungsten
lamp. If
the current in the circuit supplying such a lamp is gradually increased from zero, the lamp filament at first can be felt
to be
emitting warmth, then glows dull red and the gradually brightens until it is emitting an intense white light and a
considerable
amount of heat.

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The radiation from normal hot solids is made up of many wavelengths and the energy emitted at any particular
wavelength
depends largely on the temperature of the solid and is predictable from probability theory. The curves in Figure 3
show the
energy distribution for a tungsten filament at three different temperatures. Such radiation is known as 'black body
radiation'. Note
how the emitted energy increases with temperature and how the wavelength of maximum energy shifts to shorter
wavelengths.
More recently it has become common practice to use a variant of this - the tungsten-halogen lamp. The quartz
envelope
transmits radiation well into the UV region. For the UV region itself the most common source is the deuterium lamp
and a UVVisible
spectrometer will usually have both lamp types to cover the entire wavelength range.
Figure 3 Tungsten filament radiation
Quantum Theory
To gain an understanding of the origins of practical absorption spectrometry, a short diversion into quantum theory is
necessary.
For this purpose, it is best to think of radiation as a stream of particles known as photons instead of the waves
considered
earlier. Atoms and molecules exist in a number of defined energy states or levels and a change of level requires the
absorption
or emission of an integral number of a unit of energy called a quantum, or in our context, a photon.
The energy of a photon absorbed or emitted during a transition from one molecular energy level to another is given
by the
equation
e=hν
where h is known as Planck's constant and ν is the frequency of the photon. We have already seen that c= νλ ,
therefore, E= hc/λ

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Thus, the shorter the wavelength, the greater the energy of the photon and vice versa.
A molecule of any substance has an internal energy which can be considered as the sum of the energy of its
electrons, the
energy of vibration between its constituent atoms and the energy associated with rotation of the molecule.
The electronic energy levels of simple molecules are widely separated and usually only the absorption of a high
energy photon,
that is one of very short wavelength, can excite a molecule from one level to another.
Figure 4 Energy levels of a molecule
In complex molecules the energy levels are more closely spaced and photons of near ultraviolet and visible light can
effect the
transition. These substances, therefore, will absorb light in some areas of the near ultraviolet and visible regions.
The vibrational energy states of the various parts of a molecule are much closer together than the electronic energy
levels and
thus protons of lower energy (longer wavelength) are sufficient to bring about vibrational changes. Light absorption
due to only
to vibrational changes occurs in the infrared region. The rotational energy states of molecules are so closely spaced
that light in
the far infrared and microwave regions of the electromagnetic spectrum has enough energy to cause these small
changes.

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Figure 5 Idealized absorption spectrum
For ultraviolet and visible wavelengths, one should expect from this discussion that the absorption spectrum of a
molecule (i.e.,
a plot of its degree of absorption against the wavelength of the incident radiation) should show a few very sharp lines.
Each line
should occur at a wavelength where the energy of an incident photon exactly matches the energy required to excite
an
electronic transition.
In practice it is found that the ultraviolet and visible spectrum of most molecules consists of a few humps rather than
sharp lines.
These humps show than the molecule is absorbing radiation over a band of wavelengths. One reason for this band,
rather than
line absorption is that an electronic level transition is usually accompanied by a simultaneous change between the
more
numerous vibrational levels. Thus, a photon with a little too much or too little energy to be accepted by the molecule
for a 'pure'
electronic transition can be utilized for a transition between one of the vibrational levels associated with the lower
electronic
state to one of the vibrational levels of a higher electronic state.
If the difference in electronic energy is 'E' and the difference in vibrational energy is 'e', then photons with energies of
E, E+e,
E+2e, E-e, E-2e, etc. will be absorbed.
Furthermore, each of the many vibrational levels associated with the electronic states also has a large number of
rotational
levels associated with it. Thus a transition can consist of a large electronic component, a smaller vibrational element
and an
even smaller rotational change. The rotational contribution to the transition has the effect of filling in the gaps in the
vibrational
fine structure.
In addition, when molecules are closely packed together as they normally are in solution, they exert influences on
each other
which slightly disturb the already numerous, and almost infinite energy levels and blur the sharp spectral lines into
bands. These
effects can be seen in the spectra of benzene as a vapor and in solution. In the vapor, the transitions between the
vibration
levels are visible as bands superimposed on the main electronic transition bands.
In solution they merge together and at high temperature or pressure even the electronic bands can blur to produce
single wide
band such as that enclosed by the dotted line in Figure 6.

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Figure 6 Vapor and solution spectra of Benzene
General Chemical Origins
When white light falls upon a sample, the light may be totally reflected, in which case the substance appears white or
the light
may be totally absorbed, in which case the substance will appear black. If, however, only a portion of the light is
absorbed and
the balance is reflected, the color of the sample is determined by the reflected light. Thus, if violet is absorbed, the
sample
appears yellow-green and if yellow is absorbed, the sample appears blue. The colors are described as
complementary.
However, many substances which appear colorless do have absorption spectra. In this instance, the absorption will
take place in
the infra-red or ultraviolet and not in the visible region. Table 1 illustrates the relationship between light absorption
and color.
Basic UV-Vis Theory, Concepts and Applications
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Table 1 Relationship between light absorption and color
Color absorbed Color observed Absorbed radiation(nm)
Violet Yellow-green 400-435
Blue Yellow 435-480
Green-blue Orange 480-490
Blue-green Red 490-500
Green Purple 500-560
Yellow-green Violet 560-580
Yellow Blue 580-595
Orange Green-blue 595-605
Red Blue-green 605-750
A close relationship exists between the color of a substance and its electronic structure. A molecule or ion will exhibit
absorption
in the visible or ultraviolet region when radiation causes an electronic transition within its structure. Thus, the
absorption of light
by a sample in the ultraviolet or visible region is accompanied by a change in the electronic state of the molecules in
the sample.
The energy supplied by the light will promote electrons from their ground state orbitals to higher energy, excited state
orbitals or
antibonding orbitals.
Potentially, three types of ground state orbitals may be involved:
i) σ (bonding) molecular as in
ii) π (bonding) molecular orbital as in

Basic UV-Vis Theory, Concepts and Applications

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iii) n (non-bonding) atomic orbital as in
In addition, two types of antibonding orbitals may be involved in the transition:
i) σ* (sigma star) orbital
ii) π* (pi star) orbital
(There is no such thing as an n* antibonding orbital as the n electrons do not form bonds).
A transition in which a bonding s electron is excited to an antibonding σ orbital is referred to as σ to σ* transition. In
the same
way π to π* represents the transition of one electron of a lone pair (non-bonding electron pair) to an antibonding π
orbital. Thus
the following electronic transitions can occur by the absorption of ultraviolet and visible light:
σ to σ*,
n to σ*
n to π*
π to π*.
Figure 7 illustrates the general pattern of energy levels and the fact that the transitions are brought about by the
absorption of
different amounts of energy.
Figure 7 Energy and molecular transitions
Both s to σ* and n to σ* transitions require a great deal of energy and therefore occur in the far ultraviolet region or
weakly in the
region 180-240nm. Consequently, saturated groups do not exhibit strong absorption in the ordinary ultraviolet region.
Transitions
of the n to π* and π to π* type occur in molecules with unsaturated centers; they require less energy and occur at
longer

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wavelengths than transitions to σ* antibonding orbitals. Table 2 illustrates the type of transition and the resulting
maximum
wavelength.
Table 2 Examples of transitions and resulting λmax
It will be seen presently that the wavelength of maximum absorption and the intensity of absorption are determined by
molecular structure. Transitions to π* antibonding orbitals which occur in the ultraviolet region for a particular
molecule may well
take place in the visible region if the molecular structure is modified. Many inorganic compounds in solution also show
absorption in the visible region. These include salts of elements with incomplete inner electron shells (mainly
transition metals)
whose ions are complexed by hydration e.g. [Cu(H204)]2+. Such absorptions arise from a charge transfer process,
where
electrons are moved from one part of the system to another by the energy provided by the visible light.

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Correlation of Molecular Structure and Spectra Conjugation
π to π * transitions, when occurring in isolated groups in a molecule, give rise to absorptions of fairly low intensity.
However,
conjugation of unsaturated groups in a molecule produces a remarkable effect upon the absorption spectrum. The
wavelength of
maximum absorption moves to a longer wavelength and the absorption intensity may often increase.
Figure 8 The effect of increasing conjugation on the absorption spectrum
The same effect occurs when groups containing n electrons are conjugated with a π electron group; e.g.,
Aromatic systems, which contain p electrons, absorb strongly in the ultraviolet:

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In general, the greater the length of a conjugated system in a molecule, the nearer the λmax comes to the visible
region.
Thus, the characteristic energy of a transition and hence the wavelength of absorption is a property of a group of
atoms rather
than the electrons themselves. When such absorption occurs, two types of groups can influence the resulting
absorption
spectrum of the molecule: chromophores and auxochromes.
Chromophores
A chromophore (literally color-bearing) group is a functional group, not conjugated with another group, which exhibits
a
characteristic absorption spectrum in the ultraviolet or visible region. Some of the more important chromophoric
groups are:
If any of the simple chromophores is conjugated with another (of the same type or different type) a multiple
chromophore is
formed having a new absorption band which is more intense and at a longer wavelength that the strong bands of the
simple
chromophores.
This displacement of an absorption maximum towards a longer wavelength (i.e. from blue to red) is termed a
bathochromic shift.
The displacement of an absorption maximum from the red to ultraviolet is termed a hypsochromic shift.
Auxochromes
The color of a molecule may be intensified by groups called auxochromes which generally do not absorb significantly
in the 200-
800nm region, but will affect the spectrum of the chromophore to which it is attached. The most important
auxochromic groups
are OH, NH2, CH3 and NO2 and their properties are acidic (phenolic) or basic.
The actual effect of an auxochrome on a chromophore depends on the polarity of the auxochrome, e.g. groups like
CH3-,
CH3CH2
- and Cl- have very little effect, usually a small red shift of 5-10nm. Other groups such as -NH2 and -NO2 are very
popular and completely alter the spectra of chromophores such as:

Basic UV-Vis Theory, Concepts and Applications

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In general it should be possible to predict the effect of non-polar or weakly polar auxochromes, but the effect of
strongly polar
auxochromes is difficult to predict. In addition, the availability of non-bonding electrons which may enter into
transitions also
contributes greatly to the effect of an auxochrome.
Steric Effects
Steric hindrance will also affect the influence of an auxochrome on a chromophore. Electron systems conjugate best
when the
molecule is planar in configuration. If the presence of an auxochrome prevents the molecule from being planar then
large effects
will be noticed in the spectrum; e.g., m- and p-methyl groups in the diphenyls have predictable but slight effects on
the spectra
compared with that of diphenyl itself. However, methyl groups in the o-position alter the spectrum completely.
Cis and trans isomers of linear polyenes also show differences in their spectra. The all-trans isomer has the longer
conjugated
system. λ max is at a longer wavelength and ε max (molar absorptivity or molar extinction coefficient) is higher than for
the all cis or
mixed isomer.
Visible Spectra
In general a compound will absorb in the visible region if it contains at least five conjugated chromophoric and
auxochromic
groups; e.g.,
The ability to complex many metals, particularly the transition elements, with complex organic and inorganic
molecules which
absorb in the visible region provides the basis for their quantitative spectrometric analysis. The absorptions are due to
movement of electrons between energy levels of the organo-metal complex. These complexing systems are termed
spectrometric reagents. The most common are dithizone, azo reagents (PAN, thoron, zincon), dithiocarbamate, 8-
hydroxyquinoline, formaldoxime and thiocyanate. In addition, many inorganic ions in solution also absorb in the
visible region
e.g. salts of Ni, Co, Cu, V etc. and particularly elements with incomplete inner electron shells whose ions are
complexed by
hydration e.g. (Cu(H2O)4)2+. Such absorptions arise from a charge transfer process where electrons are moved from
one part of
the system to another due to the energy provided by the visible light.

Basic UV-Vis Theory, Concepts and Applications

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Solvents
The effect on the absorption spectrum of a compound when diluted in a solvent will vary depending on the chemical
structures
involved. Generally speaking, non-polar solvents and non-polar molecules show least effect. However, polar
molecules exhibit
quite dramatic differences when interacted with a polar solvent. Interaction between solute and solvent leads to
absorption band
broadening and a consequent reduction in structural resolution and ε max. Ionic forms may also be created in acidic or
basic
conditions. Thus care must be taken to avoid an interaction between the solute and the solvent.
Figure 9 illustrates the effect of iso-octane and ethanol on the spectrum of phenol, a change from hydrocarbon to
hydroxylic
solvent. The loss of fine structure in the latter is due to broad band h-bonded solvent-solute complexes replacing the
fine
structure present in the iso-octane. The fine structure in the latter solvent illustrates the principle that non-solvating or
nonchelating
solvents produce a spectrum much closer to that obtained in the gaseous state.
Figure 9 Spectra of Phenol in Iso-octane and in Ethanol
Commercially available solvents of 'spectroscopic purity' are listed in Table 3 accompanied by their cut-off
wavelengths, based
on a 10mm pathlength. Water and 0.1N solutions of hydrochloric acid and sodium hydroxide are commonly used
solvents for
absorption spectrometry. Again care has to be taken to avoid interaction. Where methodology requires buffering,
solutions have
to be non-absorbing and generally both the composition and pH will be specified. However, if this information is not
available
lists can be found in the literature. For reactions in the 4.2 to 8.8 pH region, mixtures of 0.1N dihydrogen sodium
phosphate and
0.1N hydrogen disodium phosphate are generally used.

Basic UV-Vis Theory, Concepts and Applications

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Table 3 Commonly used solvents and their 'cut-off' wavelengths
Solvent Cut-off (nm)
Iso-octane 202
Ethyl alcohol 205
Cyclohexane 200
Acetone 325
Tetrachloroethylene 290
Benzene 280
Carbon tetrachloride 265
Chloroform 245
Ethyl ether 220
Isopropyl alcohol 210
Methyl alcohol 210
General Interactions of Light and Matter
When a beam of radiation strikes any object it can be absorbed, transmitted, scattered, reflected or it can excite
fluorescence.
These processes are illustrated in Figure 10. With scattering it can be considered that the radiation is first absorbed
then almost
instantaneously completely re-emitted uniformly in all directions, but otherwise unchanged. With fluorescence a
photon is first
absorbed and excites the molecule to a higher energy state, but the molecule then drops back to an intermediate
energy level
by re-emitting a photon. Since some of the energy of the incident photon is retained in the molecule (or is lost by a
non-radiative
process such as collision with another molecule) the emitted photon has less energy and hence a longer wavelength
than the
absorbed photon. Like scatter, fluorescent radiation is also emitted uniformly in all directions.

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Figure 10 Interaction of light and matter
The processes concerned in absorption spectrometry are absorption and transmission. Usually the conditions under
which the
sample is examined are chosen to keep reflection, scatter and fluorescence to a minimum. In the ultraviolet and
visible regions
of the electromagnetic spectrum, the bands observed are usually not specific enough to allow a positive identification
of an
unknown sample, although this data may be used to confirm its nature deduced from its infrared spectrum or by other
techniques. Ultraviolet and visible spectrometry is almost entirely used for quantitative analysis; that is, the estimation
of the
amount of a compound known to be present in the sample. The sample is usually examined in solution.
Lambert's (or Bouguer's) Law
Lambert's Law states that each layer of equal thickness of an absorbing medium absorbs an equal fraction of the
radiant energy
that traverses it.
The fraction of radiant energy transmitted by a given thickness of the absorbing medium is independent of the
intensity of the
incident radiation, provided that the radiation does not alter the physical or chemical state of the medium.
If the intensity of the incident radiation is Io and that of the transmitted light is l, then the fraction transmitted is:
l/lo = T
The percentage transmission is
%T = l/lo x 100

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If a series of colored glass plates of equal thickness are placed in parallel, each sheet of which absorbs one quarter
of the light
incident upon it, then the amount of the original radiation passed by the first sheet is:
(1 - 1/4)/1 x 100 = 75%
and by the second sheet is 56.25%, i.e. 75% of 75%, and by the third sheet is 42.19%, i.e. 75% of 56.25%, and by
the nth sheet
is (0.75)^n x 100%.
Now imagine a container with parallel glass walls 10mm apart filled with an absorbing solution. If monochromatic light
is incident
on one face and 75% of the light is transmitted, Lambert's Law states that if a similar cell is put next to the first the
light
transmitted will be reduced to 56.25%. If the contents of the two containers are evaporated to half their volume,
thereby doubling
their concentration, and then measured in a single container, it will be found that the transmission will again be
reduced to
56.25%.
It can be immediately seen that to determine the concentration of an unknown sample the percentage transmittance
of a series
of solutions of known concentration or 'standards' can be plotted and the concentration or the unknown read from the
graph. It
will be found that the graph is an exponential function which is obviously inconvenient for easy interpolation.
The Beer-Lambert Law
The Beer-Lambert Law states that the concentration of a substance in solution is directly proportional to the
'absorbance ', A, of
the solution.
Absorbance A = constant x concentration x cell length
The law is only true for monochromatic light, that is light of a single wavelength or narrow band of wavelengths, and
provided
that the physical or chemical state of the substance does not change with concentration.
When monochromatic radiation passes through a homogeneous solution in a cell, the intensity of the emitted
radiation depends
upon the thickness (l) and the concentration (C) of the solution.
Io is the intensity of the incident radiation and I is the intensity of the transmitted radiation. The ratio I/Io is called
transmittance.
This is sometimes expressed as a percentage and referred to as %transmittance.

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Mathematically, absorbance is related to percentage transmittance T by the expression:
A = log10(Io/I) = log10(100/T) = kcL
where L is the length of the radiation path through the sample, c is the concentration of absorbing molecules in that
path, and k
is the extinction coefficient - a constant dependent only on the nature of the molecule and the wavelength of the
radiation.
Now, in the example above, the transmittance of our sample fell from 75 to 56.25% when the concentration doubled.
What
happens to the absorbance in the same circumstance?
A = log10(100/T) = log10(100) - log10 (T) = 2 - log10 (T)
When T = 75%, A = 2 - 1.875 = 0.125
When T = 56.25% A = 2 - 1.750 = 0.250
Quite clearly as the absorbance doubles for twice the concentration, it is far more convenient to work in absorbance
than
transmittance for the purposes of quantitative analysis.
It is useful to remember that
0%T = ∞A
0.1% = 3.0A
1.0%T = 2.0A
10%T = 1.0A
100% = 0A
Absorbance in older literature is sometimes referred to as 'extinction' or 'optical density' (OD).
Figure 11 (a) %T vs concentration (b) Absorbance vs concentration

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If, in the expression A = kcl, c is expressed in molar-1 and l in m, then k is replaced by the symbol τ and is called the
molar
absorption coefficient. The units of τ are mol-1m2. τ was formerly called the molar extinction coefficient and
concentrations were
often expressed as mol l-1, mol dm-3 or M and the cell length in cm to give units mol-1lcm-1, mol-1dm3cm-1 and M-1cm-1
respectively.
Alternatively, if the relative molecular mass (molecular weight) of the substance is unknown, then a 1% w/v solution is
prepared
and the absorbance is measured in a 1 cm cell. In this case, k is replaced by E1% Sometimes the wavelength is
included:
E1%(325 nm).
C Sometimes is expressed in g dm-3(gl-1) and l in cm. In this case, k is replaced by A (sometimes E). A is known as
the specific
absorption coefficient.
General Applications
Having looked, albeit very briefly, at the theory and origins of UV-Visible spectra, let us now investigate how we can
apply the
technique to chemical analysis starting with consideration of sample state.
With solid sample it is usually found that the material is in a condition unsuitable for direct spectrometry. The
refractive index of
the material is high and a large proportion of the radiation may be lost by random reflection or refraction at the
surface or in the
mass. Unless the sample can be easily made as an homogenous polished block or film, it is usual to eliminate these
interfaces
by dissolving it in a transparent solvent.
Liquids may be contained in a vessel made of transparent material such as silica, glass or plastic, known as a cell or
cuvette.
The faces of these cells through which the radiation passes are highly polished to keep reflection and scatter losses
to a
minimum. Gases may be contained in similar cells which are sealed or stoppered to make them gas tight. With the
sample now
ready for measurement, the Io (incident intensity) can be set by moving the sample out of the beam and letting the
light fall
directly on the detector. On today's modern instrumentation, Io setting is generally accomplished by an 'autozero'
command. In
practice, such a method does not account for the proportion of radiation which is reflected or scattered at the cell
faces. It also
does not account for the radiation which is absorbed by any solvent and thus does not effectively pass through the
sample.
Therefore it is usual to employ a reference or blank cell, identical to that containing the sample, but filled only with
solvent and to
measure the light transmitted by this reference as a true or practical Io.
Having established the Io or reference position, the procedure adopted for the analysis will depend on the analytical
information
required. In general terms there are two major measurement techniques; how much analyte is in the sample
(quantitative
analysis) and which analyte is in the sample (qualitative analysis).
Quantitative Analysis
For quantitative analysis, it is normally chosen to use radiation of a wavelength at which k, the extinction coefficient,
is a
maximum, i.e. at the peak of the absorption band, for the following reasons:
The change in absorbance for a given concentration change is greater, leading to greater sensitivity and accuracy in
measurement

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The relative effect of other substances or impurities is smaller.
The rate of change of absorbance with wavelength is smaller, and the measurement is not so severely effected by
small errors in the wavelength setting.
The extinction coefficient must have units relating to the units used for concentration and pathlength of the sample.
It is usually written as:
the value of k for a 1% sample concentration of 1 cm thickness.
or
ε the molar absorptivity previously described as the molar
extinction coefficient; the value of k for a sample concentration of
1 gram molecule per liter and a 1 cm thickness.
To carry out an analysis of a known material it should be possible in theory to measure the absorption of a sample at
its
absorption maximum, measure the thickness, obtain values of e from tables and calculate the concentration. In
practice, values
of E depend on instrumental characteristics, so published values from tables are not accurate enough, and a reliable
analyst will
usually plot the absorbance values of a series of standards of known concentration; the concentrations of actual
samples can
then be read directly from the calibration graph. Using today's modern software driven instruments, the analyst will
usually only
require one 'top' standard which can be used to calibrate the instrument so that it displays the concentration of
subsequent
samples directly in the required units. If the calibration curve is non-linear due to either instrumental or chemistry
considerations,
then some instruments will automatically construct a 'best fit' calibration when presented with a number of standards.
Rate Measurements
Rate studies involve the measurement of the change in concentration of a participant in the reaction as a function of
time.
Whereas we have previously considered the measurement of a sample whose absorption characteristics are
constant,
spectrometric rate measurement involves the measurement of a fall or rise in absorption at a fixed wavelength. This
measurement provides information on the change in concentration of either the reactants or products. The most
widely
encountered application of spectrometric rate measurements is for study of enzymes (proteins that are present in all
living
tissue). Enzymes cannot be measured directly but their catalytic properties allow their estimation from the speed of
the reactions
which they catalyse. Enzymes have many uses as reagents or as labels that can be attached to other molecules to
permit their
indirect detection and measurement. However, their widest use is in the field of clinical diagnostics as an indicator of
tissue
damage. When cells are damaged by disease, enzymes leak into the bloodstream and the amount present indicates
the severity
of the tissue damage. The relative proportions of different enzymes can be used to diagnose disease, say of the liver,
pancreas
or other organs which otherwise exhibit similar symptoms. The reaction most used in these clinical assays is the
reduction of
nicotinamide adenine dinucleotide (NAD) to NADH2. The spectra of NAD and NADH2 are shown in Figure 12 and it
can be seen
that if the absorbance is monitored at the NADH2 peak at 340nm the reading will increase as the reaction progresses
towards
NADH2.

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Figure 12 Absorption spectra of NAD and NADH
Other reactions can be monitored by coupling them together so that the product of one reaction feeds the next which
can then
be optically monitored.
The general equation for an enzyme reaction is:
In this reaction enzyme X catalyses the conversion of compound A to B. A coupled reaction utilises NAD in a
secondary
reaction, the completion of which depends on the concentration of a product of the primary reaction. e.g.
This equation, however, will only hold true when the reaction between B and NADH2 is virtually instantaneous.
Consider the
following coupled reaction:
Reaction 1
The initial reaction is the conversion of L-alanine and a-ketoglutarate to pyruvate in the presence of alanine
transaminase. The
indicator reaction is the conversion of pyruvate (and NADH2) to lactate which is catalysed by the enzyme lactate
dehydrogenase.

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The effect of this reaction on absorbance is illustrated in Figure 13. The actual decrease in absorbance caused by the
oxidation
of NADH2 can be seen in Figure 12. The rate of the reaction is determined by the average drop in absorbance over a
short
period of time following the addition of a-ketoglutarate. Other reactions involve the reduction of NAD to NADH2 with a
resultant
increase in absorbance.
Figure 13 Enzyme reaction based on oxidation of NADH to NAD
The conversion of glucose-6-phosphate to 6-phosphogluconalactone (a reaction catalysed by glucose-6-phosphate
dehydrogenase in the presence of NADH2) is an example of this type of reaction.
The equation for this reaction would be as follows:
Reaction 2
Figure 14 Enzyme reaction based on reduction of NAD to NADH

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The change on absorbance is shown as in Figure 14. In case of reaction 1, i.e. alanine transaminase, the rate of
reaction is
calculated by measuring the average decrease in absorbance over a period of time and expressing it as an average
drop in
absorbance per minute. With reaction 2, the increase in absorbance is measured.
A variety of parameters will influence the rate of reaction. The majority of these i.e. substrate concentration, enzyme
concentration and pH have to be accounted for in the chemistry. However, one of the most important is temperature
and it is
important to precisely control the temperature of the sample and reagents immediately prior to and during
measurement. For
this requirement, a whole range of thermostatted cell holders and temperature control systems is available.
Analysis of Mixtures
It is relatively rare to find a practical problem in which one has a mixture to be analysed with only one component
which absorbs
radiation. When there are several such components which absorb at the same wavelength their absorbances add
together, and
it is no longer true that the absorbance of the sample is proportional to the concentration of one component (see
Figure 15).
In these cases, several approaches can be adopted with the most important being chemical reaction and multi-
wavelength
measurements.
Figure 15 Error caused by superimposed absorption in a mixture
Chemical Reaction
A common method of analysis is to change the required component by adding a chemical reagent which reacts with it
specifically to form a highly absorbing compound. An example of this is shown in Figure 16. A quantity of the reagent
added to
the mixture reacts only with one component and both increases its absorption and changes the wavelength of the
absorption
maximum so that there is no longer interference between the components. The analysis is then reduced to a simple
case and its

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sensitivity is improved. Many hundreds of such specific reagents are now available for all sorts of analyses and
sample matrices
and are thoroughly detailed in the literature.
Figure 16 Improving sensitivity by adding a chemical reagent
Multiwavelength Measurements
In a mixture of components, the observed absorption at any wavelength is the sum of the individual absorption
spectra of the
components thus:
Measure absorbance at wavelength 1
A1 = EAaL + EBbL + ECcL ...
Similarly at a wavelength 2
A' = E'AaL + E'BbL + E'CcL ...
where EA and E'A are the absorptivity of components A at wavelength 1 and wavelength 2 and A is its concentration,
etc.
The cell pathlength L is generally constant and therefore cancels. If we then take measurements at a number of
different
wavelengths equal to the number of components, and if the value of the absorptivities are known by measurements
of the pure
components at each of the wavelengths concerned, we can solve the simultaneous equations to find the required
concentrations.
The multi-wavelength or multicomponents analysis technique has seen a resurgence of interest over the last few
years. This
has been due to data processing techniques. A variety of algorithms is available and the analyst is generally required
to input
the number of components, measurement wavelengths and concentration values of the standards. Having measured
the

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standards, samples can be processed and results presented appropriately. Programs of this type, including
measurement
parameters, can then be stored on disk and recalled and run with a minimum of operator intervention, providing
results on
complex mixtures containing up to ten components.
Instrumentation
In earlier sections we have seen that the purpose of a spectrophotometer is to provide a beam of monochromatic
radiation to
illuminate a sample and so measure the ratio I / Io. Any spectrophotometer will consist of the component parts
illustrated in
Figure 17.
Figure 17 Schematic diagram of a spectrophotometer
There are many combinations of sources, monochromators, measuring systems etc. which can be assembled to form
integrated spectrophotometers with varying degrees of accuracy and suitability for particular applications. The various
optical
components which comprise a spectrometer will not be discussed here.

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Definitions
Absorbance Accuracy
Absorbance accuracy is the agreement between the absorbance displayed on the spectrometer and the true
absorbance value
as given by a standard solution or calibration gauze.
Absorbance Reproducibility
This is the agreement (concordance) of a series of absorbance values obtained when the same solution is scanned,
i.e. it is how
closely the absorbance values agree.
Accuracy
The accuracy of a determination may be defined as the agreement (concordance) between it and the true or most
probable
value.
Black body Radiation
A black body is a hypothetical body or surface which will absorb all radiation incident upon it. Conversely, this black
body
radiates electromagnetic energy proportional to the fourth power of its absolute temperature. The hotter the body the
shorter the
wavelengths of its radiation; thus the colour of a glowing body depends on its temperature.
Bond
A bond is an interaction between two or more atoms or groups of atoms which holds the atoms together. This
interaction results
from the sharing of electrons in (normally) incomplete shells of adjacent atoms.
The predicted paths (orbitals) of the bonding electrons about the nucleus give each atom a particular shape which
affects the
way it bonds with the adjacent atoms. These different bond orientations (orbitals) are termed:
σ (sigma)
σ* (sigma star)
π (pi)
π* (pi star)
n
A molecule can have a combination of different types of bond orientations.
Bonding occurs where there is a single electron in an orbital intended for two electrons. Antibonding occurs where
both
electrons are present in an orbital and this inhibits bonding.
Chelate
A substance which incorporates molecules or ions bonded to a metal ion.

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Complex
A type of compound in which molecules or ions form bonds with a metal atom or ion.
Conjugation
Conjugated compounds are compounds with alternating double and single bonds.
Enzyme
An enzyme is a protein which catalyses a specific biochemical reaction. It mediates the conversion of one or more
substances
(the substrate(s)) to another by combining with the substrate(s) to form an intermediate complex.
Extinction Coefficient
The expression of the absorptivity of a compound of a standard concentration measured in a 10 mm pathlength
cuvette. It can
be expressed as either:
or This is the Absorbance of a 1% w/v solution of a compound
measured in 10 mm pathlength. It is related to molar absorptivity
_ by: = 100 x e /M
Or
Ε the molar absorptivity coefficient. This is the Absorbance of a
molar solution in a 10 mm pathlength cuvette. See Molar
absorptivity.
Hydration
To cause to take up or combine with water or the elements of water.
Ion
An atom or group of atoms which carries an electrical charge as a result of having gained or lost an electron.
Isomer
Any one of the possible compounds arising from the different ways of grouping the atoms in a given molecular
formula.
Compounds can have the same formula but different spatial arrangements of atoms.

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Cis isomer
Groups attached on the same side of a bond; e.g.,
Trans isomer
Groups attached on opposite sides; e.g.,
Molar Absorptivity (e)
The absorbance at a specified wavelength of a solution of a compound of unit molar concentration measured in a 10
mm
pathlength.
It has dimensions of M-1cm-1.
Monochromator
This is a device which isolates a very narrow band of wavelengths of the light coming from a source.
Noise
Noise is the general term used to describe the irregular or random trace obtained when the signal from a
spectrophotometer is
recorded.
Orbital
An orbital is a region round a nucleus where there is a high probability of finding an electron.
Polar
This is a condition where a molecule has no net electrical charge and yet it exhibits polarity - it is more negative at
one end than
the other. This comes about because some elements bind their outer electrons more tightly than others: with a large
atom, the
inner shells of electrons shield the core charge to some extent and the outer electrons tend to be further away from
the core with
a large atom than with a small one.
Precision
Precision may be defined as the agreement (concordance) of a series of measurements of the same quantity; i.e.,
precision
expresses the reproducibility or repeatability of a measurement.

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Probability Theory
It is not possible to specify the location and trajectory of an electron in the region of an atom since any attempt to
observe the
electron will change its location or movement. It is, however, possible to state a probability that the electron will be in
a particular
region. This probability theory allows the orbitals of the various electrons in a shell to be specified.
Reactant
A compound taking part in a chemical reaction.
Reagent
A solvent or solution which reacts with another. The term is usually applied to common laboratory chemicals used for
experiment and analysis, e.g. sodium hydroxide and hydrochloric acid.
Refractive Index
A measure of the amount by which a substance refracts a wave. It is equal to the ratio of the speed of transmission of
the wave
in a reference medium or vacuum to its velocity in the substance.
Rotational Energy States
Just as a fixed quantum of energy is required to change the energy level of an electron, so also is a fixed quantum of
energy
required to change the rotation of a molecule. The allowed energy quanta for a given molecule depend on bond
lengths and
angles within the molecule.
Steric
This relates to the effect which the shape of a molecule has on its reactions. A particular example occurs in
molecules
containing large groups which hinder the approach of a reactant (steric hindrance).
Stray Light
This is the measurable radiation, at the detector, of any wavelength outside the narrow band expected to be
transmitted by the
monochromator.
Vibrational Energy States
Just as a fixed quantum of energy is required to change the energy level of an electron, so also is a fixed quantum of
energy
required to change the vibration of an atom in a molecule. The allowed energy quanta for a given molecule depend
on bond
forces and atomic masses within the molecule.
Wavelength Accuracy
This is the agreement between the value displayed on the spectrophotometer and the true value.
Wavelength Reproducibility
This is the agreement (concordance) of a series of wavelength values obtained when a spectrum is repeatedly
scanned.