Spectrophotochemical Analysis

Introduction to Spectrochemical Methods

Spectroscopy: science of the interaction of radiation and
matter.

Spectroscopic analytical method: based on measuring the

amount of radiation produced or absorbed by molecular or
atomic species of interest

We can classify spectroscopic methods according to

the region of the electromagnetic spectrum used or
produced in the measurement.
•Gamma Ray
•X Ray
•Ultra violet Ray(UV)
•Visible Infrared(IR)
•Microwave
•Radio Frequency(RF)
Direction: Identify the ionizing radiation that can penetrate the
given materials.

Alpha particles
alpha particles are relatively
large they collide readily with matter and quickly loses this
energy, thus they have little penetrating power. Sheets of
paper and skin can easily block or stop alpha particles
Beta particles, on the other hand, are fast moving
electrons ejected from the nuclei. They are smaller than the
alpha particle, thus they have greater penetrating capability.
They can penetrate up to 2 cm of water or human flesh and
can only be stopped by a sheet of aluminum.
X-rays and gamma rays are transmitted through waves. X-rays are
generally artificially produced while gamma rays are generally emitted
from the atomic nucleus. Both have high penetrating
power and can easily pass through the human body. Only thick barriers of
concrete, lead or water can be used to stop them.
Neutrons are particles, that are highly penetrating. They usually
originate from the splitting (nuclear fission) of atoms in a nuclear
reaction. Water and concrete are the most common shields against neutron
radiation.
The amount of ionizing radiation or ‘dose’
received by a person is measured in terms of the
energy absorbed in the body tissue and is
expressed in gray. One gray (Gy) is one
joule
deposited per kilogram of mass. Equal exposure
to different types of radiation expressed as gray
does not necessarily mean the same biological
effects.
For example, one gray of alpha radiation
will have greater effect than one gray of beta
radiation. The unit known as sievert (Sv) is
used to express the radiation effect as
effective dose.

Accordingly, 2-10 sievert doses are believed to

cause severe radiation sickness and can be fatal.
Although radiation is a menace at high levels,
radiation in correct doses has many uses. Medical
and
dental x-rays discern hidden problems. Radiation is
used to diagnose ailments. Cancer patients are
treated with radiation.

Aside from its medical applications, radiation can

also be used as a technique by archeologist to
establish thedates of wooden artifacts and skeleton.
This process is known as carbon dating. The dating
of older, but non-living things like the planet earth is
done using this method.

Further, nuclear reactions can produce large

amounts of energy that could sustain life on earth
for a long time. We all benefit from a multitude of
products and services made possible by the careful
and responsible use of radiation
Application:

•Spectroscopy has played a vital role in the development

of modern atomic theory.

•Spectrochemical methods provided perhaps the most

widely used tools for the elucidation of molecular structure
as well as the quantitative qualitative determination of both
inorganic and organic compounds.

Spectroscopic Instruments
Molecular absorption spectroscopy
Molecular fluorescence spectroscopy
Atomic spectroscopy
Mass spectrometry
SPECTROSCOPIC INSTRUMENT:

Spectroscopists use the interaction of radiation with matter to

obtain information about the sample.

Several of the chemical elements were discovered by

spectroscopy.

Sample is usually stimulated in some way by applying:

• heat
•electrical energy
•light ,particle
• chemical reaction.
Prior to applying the stimulus, the analyte is predominately in
its lowest energy or ground state
The stimulus then causes some of the analyte species to
undergo a transition to a higher-energy or excited state.

We acquire information about the analyte :

•by measuring the electromagnetic radiation emitted as it

return s to the ground state or

•by measuring the amount of electromagnetic radiation

absorbed as a result of excitation.
APPLICATION
The newly refurbish Irvine-Michigan –
Brookhaven underground detector in Ohio
consist of a 6800-cubic meter volume of water
surrounded by 2048 high-sensitivity large –area
photomultiplier tubes and housed in a salt mine
under Lake Eerie. When at least 20 of the
photomultiplier detect a pulse of blue
Cherenkov Radiation from the impact of
neutrinos with water molecules in the
detector within a time window of 55 ns. A
neutrino event is judged to have occurred.

The Lake Eerie detector and others like it were

built in an effort to detect the spontaneous
decay of protons in the water molecules. These
experiment are very long term, and data from
the Lake Eerie Detector are recorded
continuously.
As a result, the detector was poised to monitor
the neutrino burst from Supernova in 1987. The
photomultiplier is an an example of a radiation
detector
The Supernova 1987 was the first
supernova visible to the naked eye to
appear in over 400 yrs. Nearly
coincident with the supernova was an
unusual burst of neutrinos, which
was detected by a facility beneath
Lake Eerie and by a similar facility in
Japan
Types of Spectroscopy

The term emission spectroscopy usually refers to methods in

which the stimulus is heat or electrical energy, while

chemiluminescente spectroscopy refers to the excitation of

the analyte by a chemical reaction.

In both case, measurement of the radiant power emitted

as the analyte returns to the ground state can give
information about the identity and concentration.

The results of such a measurement are often expressed

graphically by a spectrum, which is a plot of the emitted
radiation as a function of frequency or wavelength
MOLECULAR ABSORPTION
SPECTROSCOPY:

Every molecular species is capable of

absorbing its own characteristic
frequencies of electromagnetic
radiation.

This process transfers energy to the

molecule and results in a decrease in the
intensity of the incident
electromagnetic radiation.

Absorption of the radiation thus

attenuates (transmitted radiant power).the
beam in accordance with absorption law.
The absorption law , also known as the Beer-Lambert Law or
just Beer’s Law, tells us quantitatively how the amount of
attenuation depends on the concentration of the absorbing
molecule and the path length over which absorption occurs. .
Application: Glass making is among the
oldest technologies, dating from the
Neolithic period nearly 10,000 years ago.
Ordinary glass is transparent because
valence electrons in the silicate structure do
not receive sufficient energy from visible
light to be excited from their ground states
in the valence band of the silicate structure
to the conduction band.

Beginning with the Egyptians in the second

millennium BCE glassmakers learned to add
various compounds to glasses to produce
colored glass.

These additives often contain transition

metals to provide accessible energy levels
so that absorption of light occurs and the
resulting glass is colored. Colored glass is
used widely in art and architecture such as
in the instances of stained glass windows.
Molecular fluorescence spectroscopy:
Fluorescence and phosphorescence are analytically
important emission process in which atoms or
molecules are excited by the absorption of a beam
of a electromagnetic radiation.

Fluorescence is a photoluminescence in which

atoms or molecules are excited by absorption of
electromagnetic radiation.

The excited species then relax back to the ground

state, giving up their excess energy.
Application: In the instance of immune
fluorescent light micrograph of Hela
cancer cells, the chromosomes have
condensed before dividing to form two
nuclei.

As such, the cells are stained to reveal

actin micro f ilaments and
microtubules of the cytoskeleton,
which appear as the filamentary
structures surrounding the cell nuclei.

The nuclei of the cells are visualized by

Immunofluorescence im
exposing the cells to structure-specific
age of HeLa cells grown
fluorescent antibodies prepared by
in tissue culture and
covalently attaching ordinary antibodies
stained with antibody
to fluorescent molecules.
to actin .
HeLa (/ˈheɪlɑː/;
also Hela or hela) is an
immortal cell line used in
scientific research.
It is the oldest and most
commonly used
human cell line.
The line was derived from
cervical cancer cells taken on
February 8, 1951 from
Henrietta Lacks, a patient who
died of cancer on October 4,
1951.
HeLa cells also led to
breakthroughs in the study
of herpes, leukemia,
Influenza, hemophilia,
Parkinson's disease, certain
types of genetic diagnoses,
cancer, AIDS, cloning,
the effects of radiation and
toxic substances, and
in vitro fertilization.
Atomic spectroscopy: Atomic spectroscopic
methods are used for the qualitative and
quantitative determination of more than 70
elements.

Typically, this methods can detect parts-per billion

to parts-billion amounts, and in some cases even
smaller concentration. Atomic Spectroscopic are
also rapid, convenient, and usually of high
selectivity.
Atomic spectroscopy:
This method can only be performed on a
gaseous medium in which the
individual atoms or elementary ions,
such as Fe + , Mg- or Al+ are well
separated from one another.

Atomization, is a process in which a

sample is volatilized and decompose in
such a way as to produce gas-phase
atoms and ions. This is a critical step in
atomic spectroscopy.
• coupled plasma
•Flames
•Electrothermal atomizer

Application: Water pollution remains a

serious problem any industrial country.
Single element technique such as atomic
absorption spectrometry is used.
Mass spectrometry: is capable of
providing information about the
structure of inorganic, organic, and
biological molecules and about the
qualitative and quantitative
composition of complex mixtures.

Analyte molecules are converted to ions

by applying energy to them.

The ions formed are separated on the

basis of their mass-charge ratio(m/z)
and directed to a transducer that
converts the number of
ions(abundance) into an electrical
signal.
 Application: Mass spectrometers
are being used to study the impact of
air pollution on remote areas of the
planet such as Arctic Region. The
amount and types of airborne
particulates are measured by the mass
spectrometer to study the influence of
pollution to climate change.

Used in chemistry and biology to

determine the structure o complex
molecules and to identify the molecules
present in many different samples. It
has also become very important in
geology ,in paleontology in forensic
science, and in clinical chemistry.
THANK YOU!!
Your (20) points Post Quiz starts now!!!

Spectrophotochemical
Analysis
#1 This is a process in which a sample is volatilized
and decomposed in such a way as to produce gas-
phase atoms and ions.
#2 Absorption Law that tells us quantitatively how the amount
of attenuation depends on the concentration of the absorbing
molecule and the path length over which absorption occurs.
 #3. The term ______ spectroscopy usually refers to
methods in which the stimulus is heat or electrical energy
# 4. It is the science of the interaction of
radiation and matter.
#5 Prior to applying the stimulus, the analyte is
predominately in its lowest energy or________
 #6 The stimulus then causes some of the
analyte species to undergo a transition to a
higher-energy or _____________
#7 What was observed in at least 20 of the photomultiplier
in Lake Eerie facility is a pulse of blue _________from the
impact of neutrinos with water molecules .
#8 _______________refers to the excitation of the
analyte by a chemical reaction.
 #9 The results of such a measurement in spectroscopy are
often expressed graphically by a ______, which is a plot of the
emitted radiation as a function of frequency or wavelength.
#10 The _______ was the first supernova visible to
the naked eye to appear in over 400 yrs.
 # 11 _______is among the oldest technologies,
dating from the Neolithic period nearly 10,000 years
ago.
 #12Beginning with the _______in the second
millennium BCE glassmakers learned to add various
compounds to glasses to produce colored glass.
#13 Match the technology: resulting glass is colored

Molecular absorption spectroscopy

Molecular fluorescence spectroscopy
Atomic spectroscopy
Mass spectrometry
# 14 The cells are stained to reveal actin micro f ilaments and
microtubules of the cytoskeleton

Molecular absorption spectroscopy

Molecular fluorescence spectroscopy
Atomic spectroscopy
Mass spectrometry
 #15Amount and types of airborne particulates are
measured by the spectrometer to study the influence
of pollution to climate change

Molecular absorption spectroscopy

Molecular fluorescence spectroscopy
Atomic spectroscopy
Mass spectrometry
 #16 This method can only be performed on a
gaseous medium , This is a technique in solving
water pollution in industrial countries
Molecular absorption spectroscopy
Molecular fluorescence spectroscopy
Atomic spectroscopy
Mass spectrometry
#17 -20 Which of these has been detected impacting the planet
earth in in the supernova phenomenon? Give brief explanation
 Sow a thought, Reap an action,
Sow an action, Reap a habit,
Sow a habit, And reap a character".
 Aristotle says
"We are what we repeatedly do.
Excellence, then, is not an act,
but a habit".
KINETIC METHOD OF SEPARATION
CHROMATOGRAPHIC SEPARATIONS
Chromatography works on the principle that
different compounds will have different
solubilities and adsorption to the two phases
between which they are to be partitioned.
Chromatography is a technique in which the components of
a mixture are separated based on differences in the rates
at which they are carried through a fixed or stationary
phase by a gaseous or liquid mobile phase.

Component of mixture are carried through the stationary

phase by the flow of a mobile phase, and separations are
based on the differences in migration rates among the
mobile-phase component.
Stationary Phase in chromatography
is a phase that is fixed in place either in
a column or on a planar surface.

Mobile phase in chromatography is a

phase that moves over or through the
stationary phase carrying with it the
analyte mixture.

The mobile phase may be :

•Gas
•Liquid
•Supercritical fluid
Classification of Chromatographic Method

Column Chromatography
Planar Chromatography
•Column Chromatography,
the stationary phase is held in a
narrow tube, and the mobile
phase is forced through the
tube under pressure or by
gravity.

•Planar Chromatography, the

stationary phase is supported
on a flat plate or in the pores of
a paper, and the mobile phase
moves through the stationary
phase by capillary action or
under the influence of gravity.
 Column Chromatography
Classification of Column Chromatography

General Classification Specific Method Stationary Phase Type of Equilibrium

1. Gas Gas –Liquid(GLC) Liquid adsorbed or Partition between gas
Chromatography bonded to a solid and liquid
(GC) surface
Gas-Solid Solid Adsorption
2. Liquid Liquid-liquid,or partition Liquid is adsorbed or Partition between
Chromatography bonded to a solid immiscible liquids
(LC) surface
Liquid-solid,or solid adsorption
adsorption
Ion Resin Ion exchange resin Ion exchange
Size exclusion Liquid in interstices of a Partition /sieving
polymeric solid
Affinity Group specific liquid Partition between
bonded to a solid surface liquid and
surface mobile liquid
3.Supercritical fluid Organic species Partition between
Chromatography bonded to a solid supercritical fluid and
P 844

Partition chromatography in which the stationary

phase is the second liquid that is immiscible with the
liquid/gas mobile phase.p900
Adsorption or liquid solid, chromatography is the
classic form of liquid chromatography
P903

Ion Resin in Liquid Chromatography

•Use to separate charged species
•-anion or cation mixture can be resolved on HPLC
columns packed with anion-exchange or cation
exchange resins. P903
•Detection was generally performed with conductivity
measurements.
P903
Size exclusion in Liquid Chromatography
•Or gel chromatography,is a powerful technique
that is particularly applicable to high molecular
mass species.
•Molecules are effectively trapped and removed
from the flow of the mobile phase
p.905

Affinity in Liquid Chromatography

•A reagent called an affinity ligand is covalently
bonded to a solid support
•Typically affinity ligands are antibodies, enzyme
inhibitors, or other molecules that reversibly and
selectively bind to analyte molecule in the sample.
P909
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