Uv Spec

UV-VISSIBLE SPECTROMETRY
Yimer Mekonnen (B.pharm,

MSc)
Specialty: Pharmaceutical QA
& regulatory affairs
School of Pharmacy, Jimma

University
Email: yimer.
[email protected]
Office No: PLB 016 or PC 245
LEARNING OBJECTIVES:
At the end of the chapter the students are be able to:
 Define terms like spectroscopy, spectrophotometer and EMR.
 Differentiate between the wave and particle nature of EMR
 Describe the parameters that characterize light wave
 Understand the relationship between light energy, frequency
and wavelength
 Understand the different types of electronic transitions
 Calculate maxfororg anicc omp o
u ndsb y usingW ood ward a
nd
Kuh n-H auss
e rR ul
es
 Understand and apply Beer-Lambert’s Law to quantitative
analysis
INTRODUCTION
Definition
• Spectroscopy: is the study of interaction between
electromagnetic radiation (EMR) and matter .
• Electromagnetic radiation is a form of energy
whose behavior is described byboth waves and
particles Like properties.
• EM waves are composed of oscillating electric and
magnetic fields at right angles to each other and
both are perpendicular to the direction of
propagation of the wave.
3
INTRODUCTION…
• Spectrometry: The measurement of electromagnetic
radiation as a means of obtaining information about
physical systems and their components.
• Spectrometer: is an instrument used to measure and
study the properties of light. It is also known as
spectrograph or spectroscope
• EM spectrum: The entire range of wavelengths or
frequencies of electromagnetic radiation extending
from gamma rays to the longest radio waves and
including visible light.
INTRODUCTION…
• EMR consists of electromagnetic waves, which are
synchronized oscillations of electric and magnetic fields that
propagate at the speed of light through a vacuum.
• The oscillations of the two fields are perpendicular to each
other and perpendicular to the direction of wave propagation.
Fig. 1: Electromagnetic wave

INTRODUCTION…
The Electromagnetic Spectrum
 The frequency and wavelength of
electromagnetic radiation vary over many
orders of magnitude.
 For convenience, electromagnetic radiation is
divided into different regions based onthe type
of atomic or molecular transition that gives rise
to the absorption or emission of photons (Figure
below).
 The boundaries describing the electromagnetic
spectrum are not rigid, and an overlap between
spectral regions is possible.
6
INTRODUCTION…
• The position of an electromagnetic wave
within the electromagnetic spectrum can be
characterized by either its frequency of
oscillation or its wavelength.
• The electromagnetic spectrum includes, in
order of increasing frequency and
decreasing wavelength: radiowaves,
microwaves, infrared radiation, visible light,
ultraviolet radiation, X-rays and gamma rays.
INTRODUCTION…
Fig.1: Elecrtomagnetic spectrum

INTRODUCTION…
• In a vacuum, EM radiation travels at the speed of lightc,
which is 3x10 8m/s (300,000km/s)
• Electromagnetic radiation moves through a medium
other than a vacuum with a velocity,v, less than that of
the speed of light in a vacuum.
• The interaction of electromagnetic radiation with
matter can be explained using either the electric field or
the magnetic field.
9
INTRODUCTION…
• An electromagnetic wave can be characterized by several
fundamental properties (wave and particle like properties).
A. Wavelength (, lambda)
• Is defined as the distance between successive maxima, or
successive minima of the wave
• Different units of length are used to express wavelengths
 E.g. Angstrom, centimeter, micro and nanometer
 1 m = 102 cm = 103 mm = 106 m = 109 nm = 1010 o.
 10 = 10-1 nm = 10-4 m = 10-7 mm = 10-8 cm = 10‑10 m.
 For ultraviolet and visible electromagnetic radiation the
wavelength is usually expressed in nanometers (nm, 10–9 m)
10
INTRODUCTION…
B. Amplitude (A)
Is the vertical distance from midline of a wave to the
peak or trough of the wave.
Measured by units of distance
C. Frequency (v ,nu)
 is the number of waves that pass through a particular
point in 1 second (Hzt = 1 cycle/s)
D. Wave number(
Number waves of per
centimeter
11
INTRODUCTION…
 Electromagnetic radiation consists of a beam of
energetic particles called photons.
 The energy of a photon, in joules, is related to its
frequency, wavelength, or wave number by the following
equations.
 Whereh is Planck’s constant, which has a value

of 6.63 10 –34 J · s.
 Example-- What is the energy per photon of the sodium D line (
λ= 589 nm)? (ans: = 1.13 x 10-18J
12
 useful equations
c=× v
E=hxv
 Constants
C = 3.00 × 108 m/s
H = 6.63 x 10-34 J.s
HOME TAKE ASSINGMENT
1. For light with a wavelength ( λ ) of 408 nm
determine:
a) The frequency
b) The wave number
c) The wavelength in Å
d) The total energy (in Joules) associated with 1 mole
of photons
UV-Visible spectrophotometer
 A spectroscopic technique which utilizes the UV/Visible
region of the EMR is known as UV/visible spectroscopy/
spectrophotometer ??
 Near UV region-200 nm-400 nm
 Visible region-400-800 nm
 Absorption of light in these region mainly causes
electronic transition.
 The outer electrons in an organic molecule may occupy
one of three different energy levels (- , - or n-
energy level).
 Accordingly there are three types of electrons (-
electrons , -electrons and n- electrons)
15
INSTRUMENTATION FOR UV-VIS
Spectrophotometer
16
UV-Visible spectrophotometer…
INSTRUMENTATION…
 Today a wide range of instruments are available for
making molecular absorption measurements in the UV-
visible range.
 These vary from simple and inexpensive machines for
routine work to highly sophisticated devices.
 However, the basic components of these instruments
remain the same.
 The five essential components of UV-VIS instruments are
– A stable radiation source
– Wavelength selector
– Sample holder
– Radiation detector or transducer , and
– Signal processing and output device 17
INSTRUMENTATION…
The general layout of the essential components in a
simple absorption instrument is
18
INSTRUMENTATION…
 Radiation Sources
• A deuterium discharge lamp for UV region (160-375 nm)
• A tungsten filament lamp or tungsten-halogen lamp for Visible and
NIR regions (350 - 2500 nm)
• The instruments automatically swap lamps when scanning between
the UV and VIS-NIR regions
19
INSTRUMENTATION…
 Wavelength Selectors
 In spectrophotometric measurements we need to usea
narrow band of wavelengths of light.
 This enhances the selectivity and sensitivity of the
instrument and give a more linear relationship between
the optical signal and concentration of the substance to
be determined
 There are different types of wavelength selectors.
– These includeFilters and moncochromators
20
INSTRUMENTATION…
A. Filters
 Either absorption or interference filters are used for
wavelength selection:
Absorption filters
Usually function via selective absorption of unwanted
wavelengths and transmitting the complementary color.
They are inexpensive and widely used for band selection
in the visible region.
21
INSTRUMENTATION…
A. Filters….
Interference filters
As the name implies, an interference filter relies on
optical interference to provide a relatively narrow band
of radiation.
It consists of a transparent material (calcium or
magnesium fluoride) sandwiched between two
semitransparent metallic films coated on the inside
surface of two glass plates.
When it is subjected to a perpendicular beam of light, a
fraction passes through the first metallic layer and the
other is reflected.
22
INSTRUMENTATION…
A. Filters….
Interference filters…
Fraction that is passed undergoes a similar partitioning
upon passing through the second metallic film, thus
narrower bandwidths are obtained.
23
INSTRUMENTATION…
B. Monochromators
 An alternative approach to wavelength selection, which
provides for a continuous variation of wavelength, is the
monochromator.
 These are of two types;the prism and grating
monochromators.
24
INSTRUMENTATION…
B. Monochromators…
Prisms
 The radiations of different colors having different
wavelengths are refracted to different extent due to
the difference in the refractive index of glass for
different wavelengths.
25
INSTRUMENTATION…
Prisms
 In a prism monochromator, shown below fine beam of the light from
the source is obtained by passing through an entrance slit. This is
then collimated on the prism with the help of a lens.
 The refracted beams are then focused on an exit slit. The prism is
then rotated in a predetermined way to provide the desired
wavelength from the exit slit.
26
INSTRUMENTATION…
Gratings
 A grating is made by cutting or etching a series of closely spaced
parallel grooves on the smooth reflective surface of a solid material
as shown below
 The surface is made reflective by making a thin film of aluminium on
it and the etching is done with the help of a suitably shaped diamond
tool.
27
INSTRUMENTATION…
Gratings
 In grating monochromator (Fig. above), a fine beam of
the light from the source falls on a concave mirror
through an entrance slit.
 This is then reflected on the grating which disperses it.
The dispersed radiation is then directed to an exit slit.
 The range of wavelengths isolated by the
monochromator is determined by the extent of
dispersion by the grating and the width of the exit slit.
 Rotation of the grating in a predetermined way can be
used to obtain the desired wavelength from the exit slit.
28
 Sample cells
 The UV-VIS absorption spectra are usually determined
either in vapor phase or in solution.
 In order to take the UV spectrum of the analyte it is
taken in a cell called a cuvette which is transparent to the
wavelength of light passing through it.
 A variety of quartz cuvettes are available for the
spectral determination .These are of varying path
lengths and are equipped with inlet and outlets.
 For measurements in the visible region the cuvettes made
of glass can also be used.
 However, since glass absorbs the ultraviolet radiations,
these cannot be used in the UV region.
29
 Sample cells…
 Therefore, most of the spectrophotometers employ
quartz cuvettes (Fig below), as these can be used for
both visible and UV region.
 Usually square cuvettes having internal path length 1.0
cm are used, though cuvettes of much smaller path
lengths say of 0.1 mm or 0.05 mm are also available.
The faces of these cells

through which the radiation
passes are highly polished
to keep reflection and
scatter losses to a
minimum.
30
 Sample cells…
 Now a days ‘spectral grade ’ solvents are available which
have high purity and have negligible absorption in the
region of absorption by the chromophore.
 In a typical measurement of a UV spectrum, the solution
of the sample is taken in a suitable cuvette and the
spectrum is run in the desired range of the wavelengths.
The absorption by the solvent, if any , is compensated by
running the spectrum for the solvent alone in the sameor
identical cuvette and subtracting it from the spectrum of
the solution.
 This gives the spectrum only due to the absorbing species
under investigation.
 In double beam spectrometers, the sample and the solvent
are scanned simultaneously
31
 Detectors
 The detectors are used to convert a light signal to an
electrical signal which can be suitably measured and
transformed into an output.
 The detectors used in most of the instruments generate a
signal, which is linear in transmittance i.e. they respond
linearly to radiant power falling on them.
 The transmittance values can be changed logarithmically
into absorbance units by an electrical or mechanical
arrangement in the signal read out device.
There are three types of detectors which are used in
modern spectrophotometers.
32
 Detectors…
A. phototube
• A phototube consists of a photoemissive cathode and an
anode in an evacuated tube with a quartz window.
• These two electrodes are subjected to high voltage (about
100 V) difference.
• When a photon enters the tube and strikes the cathode, an
electron is ejected and is attracted to the anode resulting
in flow
a of current which can be amplified and measured.
33
Detectors…
B. Photomultiplier (PM) Tube
 A photomultiplier tube consists of a series of electrodes,
called dynodes.
 The voltage of successive electrodes is maintained 50
to 90 volt more positive than the previous one.
 When a radiation falls on the cathode an electron is
emitted from it. This is accelerated towards the next
photoemissive electrode by the potential difference
between the two. Here, it releases many more
secondary electrons.
34
Detectors…
B. Photomultiplier (PM) Tube…
 These, in turn are accelerated to the next electrode
where each secondary electron releases more electrons.
 The process continuous up to about 10 stages of
amplification. The final output of the photomultiplier
tube gives a much larger signal than the photocell.
35
Detectors…
C. Diode Array Detector
• These detectors employ a large number of silicon diodes arranged
side by side on a single chip.
• When a UV-VIS radiation falls on the diode, its conductivity increases
significantly. This increase in conductivity Is proportional to the
intensity of the radiation and can be readily measured.
• Since a large number of diodes can be arranged together, the
intensity at a number of wavelengths can be measure simultaneously.
36
 Signal Processing and Output Devices
 The electrical signal from the transducer is suitably
amplified or processed before it is sent to the recorder
to give an output.
 The subtraction of the solvent spectrum from that of
the solution is done electronically.
 The output plot between the wavelength and the
intensity of absorption is the resultant of the
subtraction process and is characteristic of the
absorbing species.
37
TYPES OF UV-VISIBLE SPECTROMETERS
Broadly speaking thereare two types of spectrometers.
1. Single Beam Spectrometers
As the name suggests, these instruments contain a single
beam of light.
The same beam is used for reading the absorption of the
sample as well as the reference.
The radiation from the source is passed through a filter or
a suitable monochromator to get a band on monochromatic
radiation.
It is then passed through the sample (or the reference)
and the transmitted radiation is detected by the
photodetector.
The signal so obtained is sent as a read out or is recorded.
38
Typically, two operations have to be performed – first,
the cuvette is filled with the reference solution and the
absorbance reading at a given wavelength or the
spectrum over the desired range is recorded.
Second, the cuvette is taken out and rinsed and filled
with sample solution and the process is repeated.
The spectrum of the sample is obtained by subtracting
the spectrum of the reference from that of the sample
solution.
39
Advantages of Single Beam Systems
• Single beam instruments are less expensive
• High energy throughput due to non-splitting of
source beam results in high sensitivity of
detection
Disadvantages
• Instability due to lack of compensation for
disturbances like electronic circuit fluctuations,
voltage fluctuations, mechanical component’s
instability or drift in energy of light sources.
2. Double Beam Spectrometers
 In a double beam spectrometer, the radiation coming
from the monochromator is split into two beams with the
help of the mechanical chopper (beam splitter).
 These are passed simultaneously through the reference
and the sample cell.
 The reference beam monitors the lamp energy whereas
the sample beam reflects sample absorption.
 The transmitted radiations are detected by the
detectors and the difference in the signal at all the
wavelengths is suitably amplified and sent for the
output.
41
• The observed absorbance measurement is
the ratio of the sample and reference
beams which are recombined before moving
to the monochromator.
Advantages of Double Beam Systems
• Modern improvements in optics permit high level
of automation and offer better level of detection
as compared to earlier single beam systems.
• Instability factors due to lamp drift, stray light,
voltage fluctuations do not affect the
measurement in real-time.
• Little or no lamp warm up time is required. This not
only improves throughput of results but also
conserves lamp life
Disadvantages of Double Beam Systems
• Double Beam spectrophotometers are very
expensive
The cost factor is more than offset by the
advantages offered by modern double beam systems
and therefore these have become the preferred
choice
FACTORS GOVERNING ABSORPTION
OF RADIATION IN THE UV/
VISIBLE REGION
The concept of Chromophore and
Auxochrome
• Chromophore: A covalently unsaturated group
responsible for electronic absorption.
 or Any group of atoms that absorbs light whether or not a
color is thereby produced.
 A compound containing chromophore is called
chromogen.
 There are two types of chromophore:
I. Independent chromophore:
 single chromophore is sufficient to import color to the
compound e.g. Azo group
II. Dependent chromophore:
 When more than one chromophore is required to produce color.
e.g. acetone having one ketone group is colorless where as
diacetyl having two ketone group is yellow.
• Some of the 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.
• Auxochromes: The absorption of a molecule may be
intensified by groups called auxochromes which
generally do not absorb significantly in the 200-800
nm region, but will affect the spectrum of the
chromophore when attached to it.
• Auxochromes are saturated group with non-bonding
electron when attached to chromophore alters both
wavelengths as well as intensity of absorption.
e.g. OH, NH2, NHR etc.
Absorption intensity and frequency
shifts
• The absorption intensity and frequency of any
chemical substance can be shifted depending on the
addition or removal of functional groups
• Changes in the environment also lead to changes in
the absorption spectrum of molecules and materials
Frequency shift (λm a)
x
⁻ Bathochromic (Red) shift: shift of absorption to

longer wavelength due to substitution and solvent
effects.
⁻ Hypsochromic (Blue) shift: it is shift of absorption
to shorter wavelength.
 Absorption intensity shift
⁻ Hyperchromic effect: it is an increase in
absorption intensity ( molar absorptivity)
⁻ hypochromic effects: it is decrease in absorption
intensity ( molar absorptivity)
Effect of pH on absorption spectra
 The spectra of compounds containing acidic or basic
groups are dependent on the pH of the medium (e.g.)
phenols and amines.
 UV-spectrum of phenol in acid medium (where the
molecular form predominates) is completely different
from its spectrum in alkaline medium (where the
phenolate anion predominates).
 Spectrum in alkaline medium exhibits bathochromic shift
with hyperchromic effect.
 The red shift is due to the participation of the pair of
electrons in resonance with the  electrons of the
benzene ring, thus increasing the delocalization of the 
electrons.
51
Effect of pH on absorption spectra…
 On the other hand, UV spectrum of aniline in acid
medium shows hypsochromic (blue) shift with
hypochromic effect (decrease in absorption intensity).
 This blue shift is due to the protonation of the amino
group, hence the pair of electrons is no longer available
and the spectrum in this case is similar to that of
benzene (thus called benzenoid spectrum).
52
Effect of Solvent on absorption spectra
• The solvent in which the absorbing species is
dissolved also has an effect on the spectrum
of the species.
• Different compounds may have very
different absorption maxima and
absorbances.
• Intensely absorbing compounds must be
examined in dilute solution, so that
significant light energy is received by the
detector, and this requires the use of
completely transparent (non-absorbing)
solvents.
53
• Typical solvents are water, ethanol, hexane
and cyclohexane.
• Solvents having double or triple bonds, or
heavy atoms (e.g. S, Br & I) are generally
avoided.
Effect of Solvent on absorption spectra…
 For example, the figure below shows that the
absorption maximum of acetone in hexane appears at
279 nm which in water is shifted to 264 nm, with a blue
shift of 15 nm.
55
QUIZE1
1. Define the following terms

a) Absorption spectrum
b) Bathochroic shift
c) Chromophore
2. Discuss the principle of uv-vissible
spectroscopic technique in quantitative and
qualitative analysis.
UV-Visible spectrophotometer
TYPES OF ELECTRON IN ORGANIC MOLECULES
 The outer electrons in an organic molecule may
occupy one of three different energy levels (- ,
- or n- energy level).
 Accordingly there are three types of electrons.
 These are:
The σ-electrons
The π-electrons and
n-electrons
Uv-Visible spectrophotometer 57
a) σ-electrons; They are bonding electrons, represent
valence bonds and possess the lowest energy level ( the
most stable)
b) π-electrons; They are bonding electrons, forming the pi-

bonds (double bounds), and possess higher energy than
sigma-electrons.
c) n-electrons; They are nonbonding electrons, present in
atomic orbitals of hetero atoms (N, O, S or halogens).
They usually occupy the highest energy level of the
ground state.
 In excited state the -electrons occupy an anti-bonding

energy level (*) and the transition is termed -*
transition. -electrons occupy an anti-bonding energy
level (*) and the transition is termed -* transition,
While the n-electrons may occupy * or * levels to give
n-* or n-* transition.
58
Organic compounds containing -Electrons:
 Compounds contain -electrons only are the saturated
hydrocarbons, which absorb below 170 nm.
 They are transparent in the near UV region (200 - 400
nm) and this make them ideal solvents for other
compounds studied in this range.
 They are characterized by * transition only.
Organic compounds containing n-Electrons :
 Saturated organic compounds containing hetero atoms,
possess n-electrons in addition to sigma-electrons.
 They are characterized by the  * and n – *
transitions.
 n-electrons can also be transited to * when they exist
in unsaturated compounds
 n – * transitions– 150-250 nm
 n- * transitions---275-300 nm
59
Organic compounds containing -Electrons :
Unsaturated compounds containing no hetero atoms are
characterized by the -* and -* transitions, such as
ethylene (CH2=CH 2)
.
When these compounds containing hetero atoms, they can
undergo -*, -*, n-* and n-* transitions,
example acetone (CH3 - COCH )
3.
-* transitions– 170-190 nm
Many inorganic compounds in solution also show absorption

in the visible region.
Increasing order in absorption wavelength
-* <-*< n-* < n-*
60
Calculation of λm
o
f
a
xa
no
r
g
a
ni
c
co
mp
o
u
n
d
I. Woodward's rules:
 Named after Robert Burns Woodward, are several
sets of empirically derived rules
Which attempt to predict the wavelength of the
absorption maximum ( λm ax) in
a
nultravi
olet-
visi
b l
esp ectr um of a given c o
m
p
oun d.
A. Rules for conjugated dienes
 These rules specify a base value (214 nm) for the
parent diene which is 1,3-butadiene.
 The value is red shifted upon alkyl substitution or
attachment of ring carbons
R2
C
=
CR
-C
R
=
C
R
2
62
A. Rules for conjugated dienes…
 It is also affected by the presence of double bonds out
side a ring (exocyclic), extra double bonds in conjugation,
and auxochromes.
63
A. Rules for conjugated dienes…
Examples
64
B. Rules for enones
65
B. Rules for enones…
• Examples
66
B. Rules for enones…
• Examples
67
 α, β -unsaturated aldehydes, acids and esters follow
the same general trends as enones, but have different
base values.
68
C. Rules for Benzoyl Derivatives
69
C. Rules for Benzoyl Derivatives…
• Example
 The Woodward’s rules work well only for conjugated

polyenes having four double bonds or less.
 For conjugated polyenes with more than four double
bonds the Kuhn rules are used.
70
II. Simplified Kuhn and Hausser rule
 According to this rule
λm
=
13
41
/
(
n
)2
+
3
1
a
x
• Where n is the number of conjugated double bonds

Example
• λm
=
4
a
x7
6n
m
• λm
=
4
a
x7
6n
m
•
71
II. Simplified Kuhn and Hausser rules…
 This rule is also useful for calculating number of
double bonds from the observed λm a.
x
Example, a compound with λm
o
f
a
x4
33
n
m
wi
l
l
have9c onju gat
ed double bon
d
s
.
72
Principles of Absorption Spectroscopy
(Beer’s and Lambert’s Law)
• The greater the number of molecules that
absorb light of a given wavelength, the
greater the
extent of light absorption and higher the
peak intensity in absorption spectrum.
• If there are only a few molecules that absorb
radiation, the total absorption of energy is
less and consequently lower intensity peak is
observed.
Principles of Absorption Spectroscopy…
• This makes the basis of Beer-Lambert Law
which states that the fraction of incident
radiation absorbed is proportional to the
number of absorbing molecules in its path.
• When the radiation passes through a solution,
the amount of light absorbed or transmitted is
an exponential function of the molecular
concentration of the solute and also a function
of length of the path of radiation through the
sample.
•
• Therefore, Log Io
/
I=
εc
l
 Where
 Io =Inten s
ity of the inci
dent li
ght (or the l
ight
int ensi
ty passing thro ugh arefere nce cel
l)
I = Intensity of light transmitted through the sample
solution
c = Concentration of the solute in mol/L
l = Path length of the sample in cm
ε = Molar absorptivity or the molar extinction
coefficient of the substance whose light absorption
is under investigation.
• ε is a constant and is a characteristic of a
given absorbing species (molecule or ion) in a
particular solvent at a particular
wavelength.
• ε is numerically equal to the absorbance of
a solution of unit molar concentration (c=1 M)
in a cell of unit length ( l = 1) and its units is
liters.moles-1 . cm-1.
• The ratio I / Io
i
sk
no
w
n
as
t
r
an
sm
it
t
a
n
ce
(
T)
and the log ar i
t
h
mof
t
h
ei
n
v
er
se
ra
t
i
o
I/
oI
i
s know n as th e
ab
s
or
b
a
n
ce
A.
 Therefore
Log I / Io
=
-l
o
gT
=ε
c
l
or
Log Io
/
I=
A=
ε
c
l
•
Practical examples on Beer’s and
Lambert’s Law
1. A solution containing KMnO4, had a % transmittance of 30.9% in
a 1.00 cm cell at 520 nm. If the solution has a conc. of 2.84x10-5
M, calculate the molar absorptivity of KMnO4, at 520 nm.
Solution
• % T for KMnO4 = 30.9% , this means T = 0.309
Therefore A = - log T = - log 0.309 = 0.510
• From the equation, A = ε c l, we have
0.510 = ε * 1.00 cm * 2.84x10 mol/L

- 5
ε = 80x10L mol cm
4 - 1 - 1
2. A solution containing the complex formed
between two substances has a molar
absorptivity of 9.32 X 103 L mol-1 cm-1 at 470
nm.
a) What is the absorbance of a 6.24x10-5 M
solution of the complex at 470 nm in a 1.00 cm
cell?
b) What is the percent transmittance of the
solution described in (a)?
c) What is the molar concentration of the complex
in a solution that has the absorbance described
in (a) when measured at 470 nm in a 5.00 cm
cell
3. A 5.00x10–4 M solution of an analyte is placed in a sample
cell that has a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the absorbance of the solution is
found to be 0.338.What is the analyte’s molar absorptivity
at this wavelength? Ans(Molar A. = 676 cm-1 M-1
4. A sample has a percent transmittance of 50.0%. What is
its absorbance? Ans (A= 0.301)
5. The molar absorptivity of a substance is 2.0 × 104 cm-1
mol-1 L. Calculate the transmittance through a cuvette of
path length 5.0 cm containing 2.0 × 10-6 mol L-1 solution of
the substance. Ans (T= 0.63)
80
Limitations to Beer’s Law
 Deviations from the direct proportionality between the
measured absorbance and concentration when path length
is constant may be encountered.
 Assumptions of the absorption law:
The incident beam is monochromatic
The absorbers absorb independently of each other.
Incident radiation consists of parallel rays perpendicular to
the surface of the absorbing medium.
Path length traversed is uniform over the cross section of the
beam.
Absorbing medium is homogenous and does not scatter the
radiation.
81
Limitations to Beer’s Law…
 Deviations from linearity are divided into three
categories: fundamental, chemical, and instrumental.
I. Fundamental Limitations:
• Beer’s law is valid only for low concentrations/
diluted solutions/ of analyte.
• At higher concentrations the individual particles
of analyte no longer behave independently of one
another.
• There will be reflection,
Refraction and scattering
• Positive deviations
82
Limitations to Beer’s Law…
II. Chemical Limitations
 Deviations from Beer’s law also arise when an analyte
associates, dissociates or reacts with a solvent to
produce a product having a different absorption
spectrum from the analyte.
 Depending on the resulting products, it may result in
positive or negative deviations.
III. Instrumental Limitations
• Using polychromatic radiation always gives a negative
deviation from Beer’s
• Stray light causes negative deviations
83
APPLICATIONS OF UV-VISIBLE
SPECTROPHOTOMETER
84
Applications…
 It is one of the most useful tools available to the
chemist for analysis.
 Important advantages of spectrophotometric methods
include:
1-Wide applicability; large number of organic and
inorganic species absorb light in the UV-Visible ranges.
2- High sensitivity; analysis for concentrations in the
range from 10-4 to 10-6 M are ordinary in the
Spectrophotometric determinations.
3- Moderate to high selectivity; Due to selective reactions,
selective measurements and different mathematical
treatments.
4- Good accuracy; Relative errors in concentration
measurement lie in the range of 0.1 to 2 %.
5- Ease and convenience; Easily and rapidly performed
with modern instruments.
85
Qualitative Applications
 In terms of qualitative analysis of the analyte, the UV-
VIS spectrometry is of a secondary importance for the
identification and the determination of structural
details.
 The information obtained from it needs to be
supplemented by that from IR, NMR and mass
spectrometry.
 Nonetheless,it can still provide information about the
presence or absence and the nature of the chromophore
in the molecule.
 Example 1: an absorption band at 254 nm with
characteristic vibrational fine structures may be an
evidence for existence of aromatic structure.
86
Qualitative Applications…
 Example 2: An absorption band at about 280-290 nm, which is
displaced toward shorter wavelength with increasing solvent
polarity strongly, indicates the presence of aromatic carbonyl
group.
 Example 3: Confirmation of presence of aromatic amine or
phenolic structure may be obtained by testing the pH effect on
their spectra.
87
Quantitative Applications
 As discussed above, UV-VIS spectrometry has limited
applications in qualitative analysis of the analytes ;
however, it is probably the most useful tool available for
quantitative determinations in diverse areas.
 Some of the common ones are related to the following
quantitative aspects of solution chemistry.
Analytical determination of metals and non-metals
Analytical determination of organic compounds
 Determination of dissociation constants of organic acids and

dyes
 Determination of metal-ligand formation constants
 Determination of kinetic stability of complexes

88
Analysis of binary mixture
The conditions that need to be fulfilled in order for
Beer-Lambert law to be valid are also to be valid
here
• The conditions are:
The absorption spectra of the two components must
be independent of each other i.e. non-interfering,
 the solutions must be homogeneous,
the incident radiation must be monochromatic, and
the radiation must not do any photochemistry or
photophysics with the substances.
• For all practical purposes to make the
situation simple let a cuvette of 1 cm path
length be chosen so that equation becomes
Absorbance = ε C l
• Let a mixture contain two compounds say A
and B and Let the compounds A and B have
their λm axatλ 1a nd λ2
• Let Abs1 and Abs2 be the absorbencies of
compound A at λ1 and λ2 respectively.
Therefore according to the above equation,
the absorbencies at λ1 and λ2
Abs1 = 𝛆𝟏A
C
a
At
λ
1
Abs2 = 𝛆𝟐A
C
a
At
λ
2
• Let Abs3 and Abs4 be the absorbencies of
compound B at λ1 and λ2 respectively.
Therefore again according to the above
equation, the absorbencies at λ1 and λ2 are
Abs3 = 𝛆𝟏B
C
a
Bt
λ
1
Abs4= 𝛆𝟐B
C
a
Bt
λ
2
• Let Abs5 be the total absorbance of the mixture
of the compounds A and B at λ1 and Abs6 is the
total absorbance of the mixture of the compounds
A and B at λ2 respectively.
• Therefore again according to the above equation,
the total absorbencies of the two components in
the mixture at λ1 and λ2 are:
Abs5= 𝛆𝟏A
C
+
A
𝟏
𝛆
C
Ba
t
λ
B1
Abs6= 𝛆2A
C
+
A𝛆
2
C
Ba
t
λ
B2
• 𝛆𝟏A 𝛆
,A
2 𝛆
,B
𝟏
a
nd𝛆2
B, the molar absorp ti
v
i
tie
s a r
e
ind epe nden tly experimen tall
y de te
r
mina bl
e
q uan titi
es of co mpo unds A and B u
s
i
ng B eer-
L am be rtlaw from theco ncentra ti
o
n
d epe nde ncies of absorbvity.
• The two simultaneous equations above with
two unknowns in the mixture i.e. CA and CB
• Using simple algebra one can eliminate one
unknown to calculate the other or substitution
is also possible for each variable.
How to calculate molar absorptivity
coeffient
• The molar absorbance
coefficient (ε) is
calculated from
absorbance and
concentration data
• A = εlC = (εl)C; in a
graph of A vs C, the
slope is εl.
• Slope = ε l= change in A
/change in conc.
• ε is usually written with the units of M-1.
cm-1
•
Quiz-2
1. The absorbance is a dimensionless quantity, that is, a
quantity without units. What will be the units of ∈, the
molar absorptivity.
2. The absorbance of an iron thiocyanate solution containing
0.00500 mg/mL was reported as 0.4900 at 540 nm.
Calculate the specific absorptivity, including units, of iron
thyocyanate on the assumption that a 1.00 cm cuvette was
used.
3. For Q2 above what will be the absorbance if
(a) The solution is diluted to twice its original volume and the
solution is placed in a 5.00 cm cuvette?
THANK YOU

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UV-VISSIBLE SPECTROMETRY

Yimer Mekonnen (B.pharm,

School of Pharmacy, Jimma

Fig. 1: Electromagnetic wave

Fig.1: Elecrtomagnetic spectrum

 Whereh is Planck’s constant, which has a value

The faces of these cells

• If any of the simple chromophores is conjugated

⁻ Bathochromic (Red) shift: shift of absorption to

1. Deﬁne the following terms

b) π-electrons; They are bonding electrons, forming the pi-

 In excited state the -electrons occupy an anti-bonding

Many inorganic compounds in solution also show absorption

 The Woodward’s rules work well only for conjugated

• Where n is the number of conjugated double bonds

0.510 = ε * 1.00 cm * 2.84x10 mol/L

Analytical determination of organic compounds

 Determination of dissociation constants of organic acids and

 Determination of metal-ligand formation constants

 Determination of kinetic stability of complexes

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