ULTRAVIOLET

AND VISIBLE
SPECTROSCOPY
Chap 6&7-T1
Chap 1-T2
Chap 13&14-R1

when a molecule is subjected to UV (or Vis)

radiation transitions of valence electrons occurs in
the molecule.
i.e excitation of an electron from a filled
molecular orbital to the next higher energy orbital.
since UV energy is quantized, the absorption
spectrum arising from a single electronic transition
should consist of single discrete line.
but due to superimposition of rotational and
vibrational sublevel a discrete line never obtains

Lambert-Beer law: For monochromatic

radiation absorbance is directly proportional to the
pathlength and the concentration of the
absorbing species.
Absorbance A of a sample is defined
as: A = -log10 T = log P0/P

=cl

where,

Po = intensity of incident radiation energy

P = intensity of transmitted radiation energy
c = molar concentration of solute
l = internal length of the cell

Molar absorptivity/Molar extinction

coefficient:
if the concentration of species is expressed in
molar concentration and pathlength is in
centimeter, the constant is c/a molar extinction
coefficient
Its unit = liter mole-1cm-1 and it is constant for a
given absorbing molecule
For Bovine Serum Albumin(BSA)
2801% =6.67

Deviation from BL law: fall into 3 categories:

1. Real deviation:At high concentration,
- the average distance between the species molecules
decreases affecting the charge distribution of
neighboring molecule
- the refractive index of solution (>10-3M) varies
considerably.
2. Instrumental deviation: arise from finite bandpass of
filters or monochromators. Means truly
monochromatic radiation( single wavelength) is
seldom practical

3. Chemical deviation: arises when a species

dissociates,associates or react with a solvent to
form a product having different absorption.
ex. dichromate ion absorbs in the visible region at
450 nm.
2CrO42- + 2H+ 2HCrO4- Cr2O72- + H2O
Upon diluting the equilibrium shifts to the left.The
equilibrium can be controlled by converting all the
chromium to CrO42- by making the solution of
0.05M in KOH

Instrumentation

1. Radiation sources: Hydrogen or Deuterium Lamps

for UV regions, Tungsten Filament lamps for vis
and near-IR
2. Wavelength selector:filters and monochromators
3. Cells: for UV quartz, for visible plastic etc
4. Photodetector: photomultiplier tubes
5. Readout device

Type of Instrument
a) Single beam instrument: simplest and least
expensive one consists of a battery-operated
tungsten bulb as the source, a set of glass filters for
wavelength selection, test tubes for sample holders,
a photovoltic cell as the detector and a small
microammeter a the readout device

b) double beam instrument:

In double-beam-in-space instrument two beams
are formed in space by a V-shaped mirror c/a a
beam splitter
one beam passes through the reference cell and
other through the sample cell, simultaneously, to
respective detector

b) double beam instrument:

In double-beam-in-time instrument two beams are
separated in time by a rotating sector mirror
that direct entire beam first through the reference
cell and then sample cell

Application: 1. Qualitative analysis

have some what limited application for qualitative
analysis because the number of absorption maxima
and minima are relatively few
nevertheless it is useful for detecting the presence
of certain functional groups that act as chromophore
a weak absorption band in the region of 280 to
290nm, which is shifts towards shorter wavelengths
with increased solvent polarity, strongly indicates
the presence of the carbonyl group.

Application: 2. Quantitative analysis

one of the most useful and widely used tools
available for quantitative analysis. (95% in the
health field)
any organic compound containing one or more
chromophore can easily be quantitated
a number of inorganic species also absorb and can
be directly determined
nonabsorbing species can also be quantitated by
reacting selectively with certain reagents

Procedural Details:involve establishment of working

conditions and the preparation of a calibration curve
1. Selection of wavelength: max from spectrum
2. Variables that influences absorbance: solvent, pH
temperature, interfering substances.
3. Cell handling: lens paper soaked in spectrograde
methanol,careful reposition a cuvette
4. Preparation of calibration curve: from a series of
standard solutions that bracket the conc. range
expected for the unknown sample

Analysis of Mixtures of absorbing substances:

total absorbance of a
solution is equal to the sum
of absorbances of the
individual component present

Atotal = AM + AN
= McMl + NcNl
By measuring the absorbance at 2 wavelength (1
and 2) A1

= 1M c1M l + 1N c1N l (at 1)

A2 = 2M c2M l + 2N c2N l

(at 2)

 4 molar absorptivities 1M 2M 1N 2N can be obtained

from individual standard solutions of M and N
l is the cell thickness, constant
Absorbance of mixture at 2 wavelengths ( 1 and 2)
are experimentally determined
Thus from the above two equations conc. of
individual species, cM and cN, can be calculated

Photometric Titration:

photometric titration curve is a plot of absorbance

vs. volume of titrant and can be employed to
measure the equivalence point of a titration
change in absorbance of a solution is used to follow
the change in concentration of a absorbing species
during a titration
absorbance is directly proportional to the conc. of
the absorbing species
in ideal condition the plot consist of two straight
lines that intersect at the end point
Some of the typical titration curves are:

a)Where titrant alone absorbs ex: titration

of arsenic(III) with bromate-bromide,
where absorbance is measured at the
wavelength where the bromine absorbs

b)Where the product of the reaction

absorbs ex: titration of Copper(II) with
EDTA
c

c) Where species is converted into nonabsorbing product ex: titration of ptoluidine in butanol with perchloric acid

d) Where a colored species is converted to

a colorless product by a colored titrant ex:
bromination of a red dyestuff

Advantages over direct photometric determination:

presence of other absorbing impurities at the
analytical wavelength does not cause interference
accuracy is more because data from several
measurements are used to calculate the end point
relatively dilute solution can be used
nonabsorbing species can also be quantitated

Application:
Determination of the dissociation constants of weak
acids or bases
Determination of the composition of complex ions
Determination of chlorine in water

Determination of the total, calcium and magnesium

hardness of water samples by photometric titration
Determination of silicon, calcium, magnesium, iron
and aluminum in cements by photometric titration of
the solubilized product
Analysis of jams, fruit and vegetable juices and
their concentrates