uv/visible spectroscopy

1. Submitted by: Savita
M Sc. Forensic (Final)
Roll No. - 1301
Submitted to: Diksha Mam

3. It is the branch of science that deals
with the study of interaction of matter
with light.
or
It is the branch of science that deals
with the study of interaction of
electromagnetic radiation with the
matter.

4.  Electromagnetic Radiation

5.  Electromagnetic radiation are different forms
of energy which have a dual character; they
have the properties of both wave and particle.
 They are called electromagnetic radiations
because they consist of oscillating electric and
magnetic field which are perpendicular to
each other and perpendicular to the direction
of motion of the radiations.
 Electromagnetic radiation consist of discrete
packages of energy which are called photons

7. The energy of these radiation is
inversely proportional to their wavelength
and directly proportional to their
frequency.
These radiation do not require any
physical medium and can be transmitted
through empty space.
All types of electromagnetic radiations
travel through space with the same
velocity which is nearly same as the
speed of light.

8. Wavelength It is the distance between
two adjacent crest or trough in a particular
wave. It is denoted by the symbol λ
Usually expressed in meters, micrometers
nanometers and angstrom.
Frequency It is the number of waves
which passes across a point in one
second.
Generally expressed as hertz

10. The arrangement of entire range of
electromagnetic radiations in
increasing order of their wavelengths
or decreasing order of their
frequencies is known as
electromagnetic radiation.

12.  The principle is based on the measurement
of spectrum of a sample containing atoms/
molecules.
 Spectrum is a graph of intensity of absorbed
or emitted radiation by sample versus
frequency or wavelength.
 Spectrometer is a device design to measure
the spectrum of a compound.

13.  Absorption spectroscopy- An analytical
technique which concerns with the
measurement of absorption of
electromagnetic radiation.
 E.g. UV(185-400nmm)/visible(400-800)
spectroscopy, IR spectroscopy.

14.  Emission spectroscopy is a analytical
technique in which emission (of a radiation
or a particle ) is dispersed according to some
property of the emission and the amount of
dispersion is measured.
 E.g. mass spectroscopy
.

15.  When a beam of electromagnetic radiation is
passed through a substance, the radiation
may be either absorbed or transmitted
depending upon the wavelenth of the
radiation and molecular structure of the
substance.
 Absorption of radiation would generally
increase in the energy of the molecule; the
increase in the energy may lead to electronic
excitation whereby electrons are raised to
higher energy level. Alternatively it may
bring about increase in the vibrational and
rotational energy of the level.

17.  Ultraviolet spectroscopy involves the
measurement of absorption light in the
visible and ultraviolet region by a substance.
 Absorption of light involves transition from
one electronic energy level to another within
a molecule , so also known as electronic
spectroscopy

19.  A uv spectrometer consist of the following
parts:
 A source of radiation which emits all
wavelengths of UV. The common used source of
light in the uv region are hydrogen or deuterium
discharge lamp while a tungsten filament is used
for producing visible light.
 Monochromator – which separates the radiation
into two individual wavelength. The single
wavelength of radiation is split into two beams
one of which passes through the sample and
other passes through the reference sample.

20.  Detector- continuously measures the
intensity ratio of the beams transmitted
through the sample and the solvent
respectively.
 Recorder- Automatically records the
absorption of light at each wavelength as a
graph

23.  Absorption of visible and uv light produces
change in electronic states in the molecule
associated with excitation of a electron
from lower to higher energy level.
 Each electronic level in a molecule is
associated with no of vibrational sub level
 Each vibrational level in turn are associated
with no. of rotational sub-levels.

25. Optical system- spectrometers are
instrument that provide information about
the intensity of light absorbed or
transmitted as a function of wavelength.
Both single and double beam optical
system are used in molecular absorption
spectroscopy.

27.  Double beam system is used extensively for
spectroscopic studies. The individual
component of system have same function as
in single beam system, important difference
is radiation from source is split into two
beams of approximately equal intensity using
a beam splitter.
 The intensity of the two beams after passing
through the cells are then compared.

29.  Radiation source for molecular absorptions
must produce light over a continuum of
wavelengths. Traditionally the two most
common radiation source used for UV
spectroscopy were:
 Tungsten lamp
 Deuterium lamp

30.  Tungsten lamp is similar in functioning to the
ordinary electric light bulb. It contains a
tungsten filament heated electrically to
white light, and generates a continuum
spectrum.
 It has two shortcomings:
 The intensity of radiation at short
wavelengths is too low; furthermore to
maintain constant intensity, electrical bulb
current to the bulb must be carefully
controlled

32.  The deuterium lamp consist of deuterium gas
in a quartz bulb through which there is
electrical discharge. The molecules are
excited electrically and the excited molecule
dissociates emitting UV radiation.

34.  A monochromator consist of an dispersion
element, an entrance slit, and an exit slit plus
lenses and mirror for collimating and focusing
the beam of radiation.
 The two most common type of dispersion
element are prism and gratings.
 Entrance slit allows light from the source to fall
on the dispersion element. The dispersed light
falls on the exit slit of the monochromator. The
function of the slit is to permit only a very
narrow band of light to pass through to the
sample and detector

35.  • The purpose of the detector is convert the transmitted
radiant energy into an equivalent amount of electrical
energy .
 • Two types of detectors are frequently used :-
 1- Photomultiplier tube ( PMT ) :
 • Incident light strikes the coated cathode , emitting
electrons .
 • The electrons are attracted to a series of anodes , known
as dynodes , each having a
Successively higher positive voltage .
 • Theses dynodes are of a material that gives off many
secondary electrons when hit by single electrons .
 • Initial electron emission at the cathodes triggers a multiple
cascade of electrons within the PMT itself .
 • Because of this amplification , PMT are used in instruments
designed to be extremely sensitive to very low light levels .
 • the accumulation of electrons striking the anode produces
a current signal that can be fed into a meter or recorder .

37. Photodiode arrays ( PDA ) :
 Are new detectors being used in modern spectrometers.
 photodiodes are composed of silicon crystals that are
sensitive to light in the wavelength range 170-1100 nm.
 Upon photon absorption by the diode , a current is
generated in the photodiode that is proportional to the
number of photons.
 Although photodiodes are not as sensitive as PMT
because of the lack of internal amplification , their
excellent linearity , speed , and small size make them
useful in applications where light levels are adequate .
 PDA detectors are available in integrated circuits
containing 256 to 2,048 photodiodes in a linear
arrangement .
 Each photodiode responds to a specific wavelength, and
as a result, a complete UV/visible spectrum can be
obtained in less than 1 second .

38.  Are most often used to obtain quantitative
information , such as the concentration of a solute in
solution or the absorption coefficient of a
chromophore .
 • For fixed-wavelength measurements with a single-
beam instrument, a cuvette containing solvent only is
placed in the sample beam and the instrument is
adjusted to read “Zero” absorbance .
 • A matched cuvette containing sample plus solvent is
then placed in the sample chamber and the
absorbance is read directly from the display .
 • The adjustment to zero absorbance with only
solvent in the sample chamber allows the operator to
obtain a direct reading of absorbance for the sample

39.  Fixed-wavelength measurements using a double-
beam spectrophotometer are made by first
zeroing the instrument with no cuvette in either
the sample or reference holder .
 Alternatively , the spectrophotometer can be
balanced by placing matched cuvettes containing
water or solvent in both sample chambers .
 Then, a cuvette containing pure solvent is placed
in the reference position and a matched cuvette
containing solvent plus sample is set in the
sample position .
 The absorbance reading given by the instrument
is that of the samples; that is, the absorbance
due to solvent is subtracted by the instrument

40.  For recording the UV spectrum, the given
compound is generally dissolved in some
suitable solvents which does not absorb light in
the region under examination.
 The commonly employed solvents are 95%
ethanol, hexane, and water.
 The solution of the compound is placed in a
suitable transparent container which does not
absorb light in the region being studied; a
quartz cell of 1cm path length is generally used
for this purpose.( glass cannot be used since it
absorbs strongly in the ultra violet region)

41.  CHROMOPHORE: The term chromophore was
previously used to denote a functional group of
some other structural feature of which gives a color
to compound. For example- Nitro group is a
chromophore because its presence in a compound
gives yellow color to the compound. But these days
the term chromophore is used in a much broader
sense which may be defined as “any group which
exhibit absorption of electromagnetic radiation in a
visible or ultra-visible region “It may or may not
impart any color to the compound. Some of the
important chromophores are: ethylene, acetylene,
carbonyls, acids, esters and nitrile groups etc.

42.  AUXOCHROMES: It is a group which itself does not
act as a chromophore but when attached to a
chromophore, it shifts the adsorption towards
longer wavelength along with an increase in the
intensity of absorption. Some commonly known
auxochromic groups are: -OH, -NH2, -OR, -NHR,
and –NR2. For example: When the auxochrome –
NH2 group is attached to benzene ring. Its
absorption change from λ max 225 (ɛmax 203) to
λmax 280 (εmax1430)

44.  When absorption maxima (λmax) of a
compound shifts to longer wavelength, it is
known as bathochromic shift or red shift.
 The effect is due to presence of an
auxochrome
or by the change of solvent.
 e.g. An auxochrome group like –OH, -
OCH3
 causes absorption of compound at longer
wavelength.

45.  When absorption maxima (λmax) of a
compound shifts to shorter wavelength, it is
known as hypsochromic shift or blue shift.
 The effect is due to presence of an group
causes removal of conjugation or by the
change of solvent.

46.  • When absorption intensity (ε) of a compound is
increased, it is known as hyperchromic shift.
 • If auxochrome introduces to the compound,
the intensity of absorption increases.
 Pyridine 2-methyl pyridine
 λmax = 257 nm λmax = 260 nm

47.  When absorption intensity (ε) of a compound
is
decreased, it is known as hypochromic shift.
 Naphthalene 2-methyl naphthalene
 ε = 19000 ε = 10250

49.  Ultraviolet (UV) light technologies are used for
multiple purposes in forensic investigations,
including authenticating paintings and other fine
art, authenticating signatures,
analyzing questioned documents , illuminating
latent fingerprints at crime scenes and trace
evidence on clothing, analyzing ink stains, and
revealing residual stains of body fluids.
 Forensic technicians also use UV light technologies,
such as ultraviolet monochromators or
optical spectroscopy , to detect the presence of
illegal or controlled substances or their residues in
unidentified samples, or to determine how many
types of ink or pens were used in a forged
document.

50. o https://www.slideshare.net/422459/uv-visible-
spectroscopy-
o https://en.wikipedia.org/wiki/Ultraviolet
o https://www.spectroscopyeurope.com/article/s
hedding-light-evidence-forensic-applications-
uvvisible-spectroscopy
o Undergraduate instrumental analysis-James W.
Robinson