MOLECULAR ULTRAVIOLET

AND VISIBLE SPECTROSCOPY

 Molecular absorption spectroscopy

• Molecular absorption spectroscopy in the UV-

Vis spectral regions is widely used for the
quantitative determination of a large number
of inorganic, organic and biological species.
• based on electromagnetic radiation in the
wave length region of 190 to 800nm.
• Ultraviolet: 190~400nm
• Violet: 400 - 420 nm
• Indigo: 420 - 440 nm
• Blue: 440 - 490 nm
• Green: 490 - 570 nm
• Yellow: 570 - 585 nm
• Orange: 585 - 620 nm
• Red: 620 - 780 nm
 Ultraviolet/Visible Spectroscopy
(UV/Vis)
• It measures the absorption of ultraviolet and
visible light by  bonds in an organic
compound.
• Bonds of different types and with different
extents of conjugation (C=C, C=O, C=C–C=C,
aromatic) absorb energy of different
wavelengths.
 Instrumentation
Instrument Components
• Sources
• Wavelength selectors
• Sample containers
• Radiation transducers
• Signal processors and readout devices
 Sources
• For the purpose of molecular absorption
measurements ,
– a continuum source is required
– whose radiant power does not change sharply
over a considerable range of wave length.
 Sources- Light sources
▪ Deuterium and Hydrogen Lamps
▪ Tungsten Filament Lamps
▪ Tungsten Halogen lamps
▪ Light emitting Diodes
▪ Xenon Arc Lamp
Deuterium and Hydrogen Lamps

Fig: Deuterium lamp used in spectrophotometers

 Deuterium and Hydrogen Lamps
• A continuum spectrum in the UV region is produced
by electrical excitation of deuterium or hydrogen at
low pressure.
• Initial formation of excited molecule.
• The excited molecule dissociates into atomic species
and UV photons.
• D2 + Ee D2* D’ + D’’ + hn ,
Where, Ee--electrical energy absorbed by the
molecule
D2*is the excited deuterium molecule
 Deuterium and Hydrogen Lamps
• The energetics of the overall process
• Ee = ED2* = ED′ + ED′′ + hν
• Where, ED2* is the fixed quantized energy of D2*
ED′ and ED′′ are kinetic energies of the two deuterium
atoms
• The sum of above K.E. can vary continuously from
zero to ED2*
• Thus the energy and the frequency of the photon can
also vary continuously . That is when the two K.E. are
small, hν will be large , and conversely.
 Deuterium and Hydrogen Lamps
• Produce a useful
continuum spectrum in
the region of 160nm to
beginning of visible
region.

Fig: Spectrum of deuterium lamp

 Deuterium and Hydrogen Lamps

• Both deuterium and hydrogen lamps produce

output in the range of 160-800 nm.
• In UV region (190-400nm)-continuum spectra exists.
(maximum intensity occurs at ～225nm )
• At longer wavelengths (>400nm) , the lamps
produce emission lines, which are superimposed on
the weak continuum.
• Quartz windows
 Tungsten Filament Lamps
• Are commonly used in the visible and near-infrared
region.
• Useful in wavelength range (350 to 2500 nm)
• Energy distribution approximates that of black body-
thus temperature dependent.
• In most absorption instruments, the operating
filament temperature is about 3000 K (2870K) .
• Bulk of energy is thus emitted in IR region.
 Tungsten Filament Lamps
• For stable radiation source, voltage should be
closely controlled by electronic voltage regulators or
constant voltage transformers
 Tungsten Filament Lamps
Tungsten halogen Lamps
• A quartz envelope houses tungsten filament
containing small amount of iodine. (quartz can resist
high temperature ≈3500K)
• higher intensities
• extends to UV
• This has double life time than ordinary tungsten
lamp, because of iodine gas.
• W gas + I2 = WI2 (volatile compound), when WI2 strikes the
filament, tungsten is redeposited by deposition.
 Tungsten Filament Lamps
Tungsten halogen Lamps
• Tungsten halogen Lamps are significantly more
efficient and extend the output wavelength range
well into ultraviolet.
• For these reasons, they are found in many modern
spectroscopic instrumetns.
Light-Emitting Diodes
Xenon arc lamp:
•Radiation is produced by passing current through xenon atmosphere.
•It is applicable for 200-1000nm, has intense peak around 500nm.
 Wavelength selector
• Are the device/s which restricts radiation to a
narrow band, that is absorbed or emitted
from analyte.
• Determine sensitivity and selectivity of the
method
• These are filter, monochromator,
spectrograph etc.
 Wavelength selector
• Filters: Filters are used for wavelength
selection.
• absorb all but a restricted band of radiation
from a continuum source.
• Absorption filter- restricted to visible region
• Interference filters (Fabry-Perot filters):
available for UV, Vis, IR
 Filter

nl = d (sin I + sin r)
 Monochromator
• Generally have a diffraction grating
• disperse the radiation into its component
wavelengths
Contains
• Entrance slit
• Collimating lens or mirror
• A dispersion medium (prism or grating)
• Focusing element
• Exit slit.
 Components of Monochromator

(I) an entrance slit that provides a rectangular

optical image,
(2) a collimating lens or mirror that produces a
parallel beam of radiation,
(3) a prism or a grating that disperses the radiation
into its component wavelengths,
(4) a focusing element that reforms the image of the
entrance slit and focuses it on a planar surface called
a focal plane, and
(5) an exit slit in the focal plane that isolates the
desired spectral band.
 Monochromator
• A refraction grating disperse radiation into its
component wavelength.
• Different wavelength are focused to exit slit by
rotating the grating.
• Can provide radiation of effective bandwidth
(spectral band) of less than 1 nm (expensive) to
about 20nm (inexpensive)
(a) grating monochromator

(b) prism monochromator

Cuvettes (sample containers):

1. Virtually all UV spectra are recorded in solution-phase

2. Cells can be made of plastic, glass or quartz
3. Only quartz is transparent in the full 200-700 nm range; plastic and
glass are only suitable for visible spectra
 Photon Transducers
• Photovoltaic cells Which contains a
• Photo tubes In which
photoemmisive
In which
which the
chargesradiant
absorption energy
surface
developedas
of
In
In which radiation
which photons causes
cause the
generates
well
radiation
in a assiliconcurrent
several -scrystal
by at the
additional
as a
• Photomultiplier tubes emission
formation of e from
-
ofthee semi- a
hole pairs
interface
surfaces
semiconductor
result of of that
absorption
provides
emit e-a
of
s
• Photoconductivity photosensitive
and a current solid across a
conductor
cascade
and
photons
holes, of layer-
areethus and
scollected
when a
leading
struck
and
to
transducers surface
reverse biased n
P junction
metal
by
enhanced
measured -
e s conductivity
from the
• Silicon photon diodes photosensitive area
• Charge-transfer
transducers
 Radiation Transducers

• Phototube - electrons produced by irradiation

of cathode travel to anode. response depends
on cathode material (200-1000 nm)
 Radiation Transducers
• Photomultiplier tube
(PMT) - irradiation of
cathode produces
electrons, series of
additional electrodes
(dynodes) increases
gain to 105-107
electrons per photon.
 Types of Instruments
• Single Beam Instruments
• Double Beam Instruments
– UV-Vis Double Beam Spectrophotometer (in
time)
– UV-Vis Double Beam Spectrophotometer (in
space)
• Multichannel instruments
 Single Beam Instruments

•Two sources are used, a tungsten halogen and a deuterium lamp

•The wavelength selector is a grating or prism monochromator
•The detector is usually a vacuum phototube or a photomultiplier
tube in higher cost instruments
 Double Beam Instruments ( in space)

•Two sources are existent namely a tungsten halogen lamp and a

deuterium lamp.
•A lamp selection mechanism is present.
•The beam from the source is split into two beams by a beam splitter
which is a semi-permeable mirror.
•The detector is usually a pair of photomultiplier tubes connected to a
difference amplifier and the wavelength selector is a grating or prism
monochromator.
 log(I0/I) = A
UV-VIS sources I0 I

sample
200 700
l, nm

detector
monochromator/
reference

beam splitter optics I0 I0

UV-Vis Double Beam Spectrophotometer
(in time)
UV-Vis Double Beam Spectrophotometer
(in time)
• Two sources are existent & a lamp selection
mechanism is present.
• Chopper split the beam into two parts .
• Transmitted beam is passed to the detector
through a semi-permeable splitter.
• The detector is PMT and the wavelength
selector is a grating or prism monochromator.
 Advantages of a double-beam over a
single-beam instrument
• Compensate for variations in the source
intensity.
• Compensate for drift in the detector and
amplifier.
• Compensate for variation in intensity as a
function of wavelength.
Multichannel Instruments
 Multichannel Instruments
•The most widely used multichannel instrument is that
based on
photodiode array detector.
•The monochromator slit width is made identical to the
width of a single silicon diode.
•The signal from any of the silicone diodes represents a
single wavelength of the monochromator setting.
•Therefore, resolution of such an instrument can be
increased by increasing the number of the silicon diodes of
the photodiode array (i.e. decreasing the slit width of the
monochromators).
The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum of
the UV radiation
2. If a particular electronic transition matches the energy of a certain band
of UV, it will be absorbed
3. The remaining UV light passes through the sample and is observed
4. From this residual radiation a spectrum is obtained with “gaps” at these
discrete energies – this is called an absorption spectrum

*

*



 ABSORBING SPECIES
• The absorption of ultraviolet or visible radiation by a
molecular species M can be considered to be a two-step
process,
(i) Excitation
M + hn M*
• The lifetime of the excited species is brief (10-8 to 10-9 s).
(ii) Relaxation involves conversion of the excitation energy
to heat/photochemical process
M* M + heat
• The absorption of ultraviolet or visible radiation generally
results from excitation of bonding electrons.
 ABSORBING SPECIES

• The absorption of UV or Vis radiation results

from excitation of bonding electrons.
• Wave length of absorption band– correlated
with the types of bonds under study
• Valuable for identifying functional group in a
molecule
• Application-quantitative determination of
compounds containing absorbing groups.
 ABSORBING SPECIES
• Absorption of UV and visible radiation by molecules
generally occurs in one or more electronic
absorption bands, each of which is made up of
closely packed but discrete lines.
Fig: Ultra violet absorption
spectra for 1,2,4,5-tetrazine

(A)Gas phase,
many line due to
electronic ,vibrational, rotational
transitions can be seen.

(B)In non polar solvent:

the electronic transitions can
be observed but the vibrational
and rotational has been lost.

(C) In a polar solvent:

the strong intermolecular
forces cause the electronic
peak to blend, giving only
a single smooth absorption band.
ABSORBING SPECIES (CONTD..)

❖Absorption by Organic Compounds

❖Absorption by Inorganic Species
❖Charge Transfer Absorption
 ABSORBING SPECIES (CONTD..)
Electronic Transitions
There are three types of electronic transitions. The
three include transitions involving:
(1) , , and n electrons
(2) d and f electrons
(3) charge transfer electrons.
Electronic Transitions and UV-visible
Spectra in Molecules
The lowest energy transition (and most often observed by UV)
is typically that of an electron in the Highest Occupied
Molecular Orbital (HOMO) to the Lowest Unoccupied
Molecular Orbital (LUMO)

*
Unoccupied levels
*

Atomic orbital Atomic orbital

Energy n

Occupied levels



Molecular orbitals
From the molecular orbital diagram, there are several possible
electronic transitions that can occur, each of a different relative
energy:

*
 * alkanes
*
 * carbonyls

 * unsaturated cmpds.
Energy
n
n * O, N, S, halogens

n * carbonyls



➢The energies for the various types of molecular orbitals
differ significantly.

➢The energy level of a nonbonding electron lies between the

energy levels of the bonding and the antibonding  and 
orbitals.

➢Electronic transitions among certain of the energy levels can

be brought about by the absorption of radiation.

➢ Four types of transitions are possible:

 *, n *, n *, and  *.

Absorption by Organic Compounds

• All organic compounds are capable of absorbing

electromagnetic radiation because all contain
valence electrons that can be excited to higher
energy levels.
• The excitation energies associated with electrons
forming most single bonds are sufficiently high –
vacuum UV region (λ < 185nm)
Absorption by Organic Compounds
• n * transition ,molar absorptivity low to
intermediate (100 to 3000 L/mol/cm)
• Special equipment to study vacuum or far UV is
required.
• Routine organic UV spectra are typically collected
from 200-700 nm.
• Most application to organic compounds involved
transition from n or electrons to * .
• Require presence of unsaturated functional group
to provide pi orbitals.
Absorption by Organic Compounds
Chromophores
▪ Molecules containing unsaturated functional groups
and capable of absorbing UV-Visible radiation and
having characteristic electronic transitions are called
chromophores (color loving) .
▪ Structural or electronic changes in the chromophore
can be quantified and used to predict shifts in the
observed electronic transitions.
  * Transition
• An electron in a bonding  orbital of a molecule
is excited to the corresponding antibonding
orbital by the absorption of radiation.
• The energy required to induce a  *
transition is large.
• Methane can undergo only  * transitions,
exhibits an absorption maximum at 125 nm.
• Absorption maxima due to  * transitions
are never observed in the ordinarily accessible
ultraviolet region.
Alkanes – only posses -bonds and no lone pairs of electrons, so only the
high energy   * transition is observed in the far UV

This transition is destructive to the molecule, causing cleavage of the -

bond

C C
*

 C C
 n * Transitions
• Saturated compounds containing atoms with
unshared electrons are capable of n*
transitions.
• These transitions require less energy than the
 * type and can be brought about by
radiation in the region of between 150 and 250
nm, with most absorption peaks appearing
below 200 nm.
• The molar absorptivities are low to intermediate
in magnitude and range between 100 and 3000
L cm-1 mol -1.
Alcohols, ethers, amines and sulfur compounds – in the cases of
simple, aliphatic examples of these compounds the n  * is the
most often observed transition; like the alkane   * it is most
often at shorter wavelength than 200 nm

Note how this transition occurs from the HOMO to the LUMO

C N
*CN

C N

nN sp3 C N anitbonding
orbital

CN C N
 n * and  * Transitions
• Most applications of absorption spectroscopy are based
upon transitions for n or  electrons to the * excited
state
• Because the energies required for these processes bring
the absorption peaks into an experimentally convenient
spectral region (200 to 700 nm).
• Both transitions require the presence of an unsaturated
functional group to provide the  orbitals.
• The molar absorptivities for peaks associated with
excitation to the n  * state are generally low and
ordinarily range from 10 and 100 L cm-1 mol -1;
• values for  * transitions, on the other hand,
normally fall in the range between 1000 and 10,000 L
cm-1 mol -1.
Alkenes and Alkynes – in the case of isolated
examples of these compounds the   * is observed
at 175 and 170 nm, respectively

Even though this transition is of lower energy than 

 *, it is still in the far UV – however, the transition
energy is sensitive to substitution

*


Carbonyls – unsaturated systems incorporating N or O
can undergo n  * transitions (~285 nm) in
addition to   *

Despite the fact this transition is forbidden by the

selection rules (e = 16), it is the most often observed
and studied transition for carbonyls

This transition is also sensitive to substituents on the

carbonyl

Similar to alkenes and alkynes, non-substituted

carbonyls undergo the   * transition in the
vacuum UV (188 nm, e = 900); sensitive to
substitution effects
Carbonyls – n  * transitions (~285 nm);   * (188 nm)

*
It has been determined
from spectral studies,
n that carbonyl oxygen
C O

more approximates sp
rather than sp2 !
 O

CO transitions omitted for clarity

 Substituent Effects
 The attachment of substituent groups (other
than H) can shift the energy of the transition.
 Substituents that increase the intensity and
often wavelength of an absorption are called
auxochromes
 Common auxochromes include alkyl, hydroxyl,
alkoxy and amino groups and the halogens
Substituents may have any of four effects on a chromophore

i. Bathochromic shift (red shift) – a shift to longer l; lower energy

ii. Hypsochromic shift (blue shift) – shift to shorter l; higher energy

iii. Hyperchromic effect – an increase in intensity

iv. Hypochromic effect – a decrease in intensity

Hyperchromic

e
Hypsochromic Bathochromic
Hypochromic

200 nm 700 nm
Effect of Conjugation on Chromophores

•  electrons are considered to be further delocalized by

conjugation; the orbitals involve four (or more) atomic
centers.
• The effect of this delocalization is to lower the energy
level of the * orbital and give it less antibonding
character.
• Absorption maxima are shifted to longer wavelengths as
a consequence.
• Conjugation of chromophores, has a profound effect on
spectral properties.
Effect of Conjugation on Chromophores

• 1,3-butadiene, CH2=CHCH=CH2, has a strong absorption

band that is displaced to a longer wavelength by 20 nm
compared with the corresponding peak for an
unconjugated diene.
Fig: Absorption Spectra for typical organic compounds
ABSORPTION BY INORGANIC
SPECIES
 Absorption by inorganic species

• A number of inorganic anions exhibit UV

absorption bands that are result of exciting
nonbonding electrons.
• Example:
– Nitrate 313nm
– Carbonate 217nm
– nitrite 360 and 280 nm
– azido 230 nm
– trithiocarbonate 500nm
Absorption Involving d and f Electrons
• Most transition-metal ions absorb in the
ultraviolet or visible region of the spectrum.
• For the lanthanide and actinide series, the
absorption process results from electronic
transition of 4f and 5f electrons.
• For elements of the first and second
transition-metal series, the 3d and 4d
electrons are responsible.
Absorption by Elements of the First and
Second Transition-Metal Series
• The ions and complexes of the first two
transition series tend to absorb visible radiation
in one if not all of their oxidation states.
• The absorption bands are often broad and are
strongly influenced by chemical environmental
factors.
• The spectral characteristics of transition metals
involve electronic transitions among the various
energy levels of d orbitals.
 z z z z

x x x x x
y y y y y

dxz dyz dx2 - y2 dz2

dxy

dx2- y2
dz2 dx2- y2
dxy dxz dyz
dxy

 dz2
Energy

dxz dyz

dxy dxz dyz dz2 dx2- y2

dxy dxz dyz
dz 2 dx 2 - y 2
Tetrahedral Square planar
No ligand Octahedral ligand field
field ligand field ligand field
 Properties of d-d transition

➢ At least, at one oxidation state, transition element

absorb visible radiation.
➢ The absorption involves transitions between filled
and unfilled d-orbitals with energies that depend on
the ligands bonded to the metal ions.
➢ The absorption band is broad & is highly influenced
by chemical environmental factors; for eg, aq. Cu2+
[Cu(H2O)4]2+ is pale blue and ammonical Cu2+
[Cu(NH3)] 2+ is deep blue
 Properties of d-d transition

➢ The energy difference between these d-orbitals

(position of corresponding absorption maximum) i.e. the
magnitude of Crystal field splitting depends on the:
I. Nature of ligand bonded to it i.e. Spectrochemical
series =>[I-< Br-<Cl-<F-<OH-<C2O42-≈H2O<SCN-<NH
<et diamine<o phenonthroline<NO2-<CN-]
II. Charge possessed by the metal i.e. Oxidation state
III.Position of metal in the periodic table.
Fig: Absorption Spectra of Aqueous Solutions of Transition Metal Ions
Absorption by Lanthanide and Actinide
Ions
• The ions of most lanthanide and actinide elements
absorb in the ultraviolet and visible regions.
• Their spectra consist of narrow, well-defined, and
characteristic absorption peaks.
• The transitions responsible for absorption by
elements of the lanthanide series appear to involve
the various energy levels of 4f electrons, while it is
the 5f electrons of the actinide series that interact
with radiation.
Absorption by Lanthanide and Actinide
Ions
• 4f and 5f electrons are shielded from external
influence by electrons that occupy orbitals with
larger principal quantum numbers.
• As a result, the bands are narrower and relatively
unaffected by the species bonded by the outer
electrons.
Fig: Absorption Spectra of Aqueous Solutions of Rare Earth Ions
CHARGE-TRANSFER ABSORPTION
 Charge-Transfer Absorption

• Species that exhibit charge-transfer absorption are

of particular importance because their molar
absorptivities are very large (emax > 10,000).
• quantitative
• Thus, these complexes provide a highly sensitive
means for detecting and determining absorbing
species.
• Many inorganic and organic complexes exhibit
charge transfer absorption and are called charge-
transfer complexes.
 Charge-Transfer Absorption

• In order for a complex to exhibit a charge-transfer

spectrum, it is necessary for one of its components
to have electron-donor characteristics and for the
other component to have electron-acceptor
properties.
• Absorption of radiation then involves transfer of an
electron from the donor to an orbital that is largely
associated with the acceptor.
• eg. FeSCN2+, Fe(phen)32+, Starch-I5- are shown
in figure in next slide.
Fig: Absorption Spectra of Aqueous Charge Transfer Complex
 Charge-Transfer Absorption
• Other example includes: Phenolic complex of Fe(III),
1,10- phenanthroline complex of Fe(II), iodide
complex of molecular iodine,
• [Fe(CN)6]3-- [Fe(CN)6]4- are purssian blue,
• Fe(III)-SCN complex (red color)
– Absorption of photons results in the transfer of an
electron from thiocyanate ion to orbital of Iron (III)
– SCN- to Fe(III) → SCN + Fe(II) [Excited species] → may
relax to ordinary condition or dissociates to give redox
product.
 Charge-Transfer Absorption
• In most charge transfer complex involving a metal
ion, metal serves as electron acceptor
• Exception : 1,10- phenanthroline complex of Fe(II)
and copper (I)
• Interesting charge transfer complex of organic
compounds:
– Quinhydrone (1:1 complex of quinine and
hydroquinone) , which exhibits strong absorption in the
visible region.
– Iodine complex with amines, aromatics and sulfides.
 APPLICATION OF UV/VISIBLE
MOLECULAR ABSORPTION
SPECTROMETRY

❖Application of absorption measurement to

qualitative analysis
❖Application of absorption measurement to
quantitative analysis
 APPLICATION OF ULTRAVIOLET/VISIBLE
MOLECULAR ABSORPTION SPECTROMETRY

• Absorption measurements based upon ultraviolet

and visible radiation find widespread application for
the identification and determination of infinite
inorganic and organic species.
• Molecular ultraviolet/visible absorption methods are
perhaps the most widely used of all quantitative
analysis techniques in chemical and clinical
laboratories throughout the world.
 APPLICATION OF ABSORPTION
MEASREMENT TO QUALITATIVE ANALYSIS
• Useful for detecting chromophoric groups.
• Large parts of even the most complex organic molecules
are transparent to radiation longer than 180nm
• The appearance of one or more peaks in the region from
200nm to 400nm indicates presence of unsaturated
groups or atoms as S,X.
• Often , the identity of absorbing groups can be
determined by comparing the spectrum of an analyte
with those of simple molecules containing various
chromophoric groups.
 APPLICATION OF ABSORPTION
MEASREMENT TO QUALITATIVE ANALYSIS

• However Ultraviolet spectra do not have enough fine

structure to permit an analyte to be identified
unambiguously.
• Thus UV qualitative data must be supplemented with
other physical or chemical evidences, as IR, NMR, Mass
spectra, solubility, melting point , boiling point
information.
 APPLICATION OF ABSORPTION
MEASREMENT TO QUALITATIVE ANALYSIS

• Methods of Plotting Spectral Data: Several

different types of spectral plots are
encountered in qualitative molecular
spectroscopy.
– The ordinate is most commonly percent
transmittance, absorbance, log absorbance, or
molar absorptivity.
– The abscissa is usually wavelength or wavenumber,
although frequency is occasionally employed.
 Solvents
• In choosing a solvent, consideration must be given
not only to its transparency, but also to its possible
effects upon the absorbing system.
• Polar solvents such as water, alcohols, esters, and
ketones tend to obliterate spectral fine structure
arising from vibrational effects
• Spectra that approach those of the gas phase are
more likely to be observed in nonpolar solvents such
as hydrocarbons.
 Solvents

• In choosing a solvent, consideration must be given

to
– Transparency
– Possible effects on absorbing system
• In addition, the positions of absorption maxima are
influenced by the nature of the solvent.
• Clearly, the same solvent must be used when
comparing absorption spectra for identification
purposes.
Fig: Effect of solvent on the absorption spectrum of acetaldehyde
 Detection of functional group

• Absorption spectrum in the UV/Visible

regions is useful for detecting the presence of
certain functional groups that act as
chromophores.
 QUANTITATIVE ANALYSIS BY
ABSORPTION MEASUREMENTS
• Absorption spectroscopy is one of the most useful
and widely used tools available to the chemist for
quantitative analysis.
• Important characteristics of spectrophotometric
method include:
(1) wide applicability to both organic and inorganic systems,
(2) typical detection limits of 10-4 to 10-5 M,
(3) moderate to high selectivity,
(4) good accuracy,
(5) ease and convenience of data acquisition.
 Scope

• The applications of quantitative, ultraviolet/visible

absorption methods not only are numerous, but also
touch upon every field in which quantitative
chemical information is required.
• It has been estimated that in the field of health
alone, 95% of all quantitative determinations are
performed by ultraviolet/visible spectrophotometry
and this number represents well over 3,000,000 daily tests carried out in
the United States.
 Procedural Details
• The first steps in a photometric or
spectrophotometric analysis involve the
– establishment of working conditions and
– the preparation of a calibration curve relating
concentration to absorbance.
 Selection of Wavelength
• For high sensitivity , spectrophotometric absorbance
measurements are ordinarily made at a wavelength
corresponding to an absorption maximum
– because the change in absorbance per unit of
concentration is greatest at this point.
– The absorbance is nearly constant at this point (λmax)
which leads to close adherence to Beer’s law
– Uncertainty with wavelength setting also minimized
• the maximum sensitivity is thus realized.
Variables That Influence absorbance

• Common variables that influence the

absorption spectrum of a substance include
– the nature of the solvent,
– the pH of the solution,
– the temperature,
– electrolyte concentrations, and
– the presence of interfering substances.
 Cleaning and Handling of Cells

• Accurate spectrophotometric analysis

requires the use of good-quality, matched
cells.
• These should be regularly calibrated against
one another to detect differences that can
arise from scratches, etching, and wear.
 Determination of the Relationship
Between Absorbance and Concentration
• It is necessary to prepare a calibration curve from a
series of standard solutions that include the
concentration range expected for the samples.
• Ideally, calibration standards should approximate
the composition of the samples to be analyzed not
only with respect to the analyte concentration but
also with regard to the concentrations of the other
species in the sample matrix in order to minimize
the effects of various components of the sample on
the measured absorbance.
 The Standard Addition Method

• The standard addition method involves

adding one or more increments of a standard
solution to sample aliquots of the same size.
• Each solution is then diluted to a fixed
volume before measuring its absorbance.
• It is possible to perform a standard addition
analysis using only two increments of sample.
• Here, a single addition of Vs mL of standard
would be added to one of the two samples.
 The Standard Addition Method

• This approach is based upon the equation:

A1csVs
cx 
 A2  A1Vx