Individual Assignment

Course name;Analysis II
Instructor;Mona
Submission date 20/10/2014EC
BY
Ahmanur Sule Abrahim ID 171/12
Department;C/Pharmacy
Program;degree Regular 3rd year 2nd semister
 TITLES OF ASSIGNMENT
Individual assignment (10%)
1. Infrared spectroscopy
2. Chromatography
3. Gas Chromatography (GC)
4. High performance liquid chromatography
 Infrared Spectroscopy
• Infrared spectroscopy is the measurement of the wavelength
and intensity of the absorption of mid-infrared light by a
sample. Mid-infrared is energetic enough to excite molecular
vibrations to higher energy levels.
• The wavelength of infrared absorption bands is characteristic
of specific types of chemical bonds, and infrared spectroscopy
finds its greatest utility for identification of organic and
organometallic molecules. The high selectivity of the method
makes the estimation of an analyte in a complex matrix
possible.
Example of IR
 Theory of Infrared Absorption
Spectroscopy
• For a molecule to absorb IR, the vibrations or rotations within
a molecule must cause a net change in the dipole moment of
the molecule. The alternating electrical field of the radiation
(remember that electromagnetic radiation consists of an
oscillating electrical field and an oscillating magnetic field,
perpendicular to each other) interacts with fluctuations in the
dipole moment of the molecule.
• If the frequency of the radiation matches the vibrational
frequency of the molecule then radiation will be absorbed,
causing a change in the amplitude of molecular vibration.
 Molecular Rotations
• Rotational transitions are of little use to the
spectroscopist. Rotational levels are
quantized, and absorption of IR by gases
yields line spectra.
• However, in liquids or solids, these lines
broaden into a continuum due to molecular
collisions and other interactions.
 Molecular Rotations (cont)

,
 Vibrational-Rotational Transitions
• In general, a molecule which is an excited vibrational state
will have rotational energy and can lose energy in a transition
which alters both the vibrational and rotational energy
content of the molecule.
• The total energy content of the molecule is given by the sum
of the vibrational and rotational energies. For a molecule in a
specific vibrational and rotational state, denoted by the pair
of quantum numbers (v, J), we can write its energy as:
E(v, J)=Evib(v) + Erot(J)
 Transitions (cont)
• The energies of these three transitions form a very
distinctive pattern. If we consider the lower
vibrational state to be the initial state, then we can
label the absorption lines as follows.
• Transitions for which the J quantum number
decreases by 1 are called P-branch transitions, those
which increase by 1 are called R-branch transitions
and those which are unchanged are called Q-branch
transitions.
 Molecular Vibrations
• In order to predict equilibrium stable-isotope fractionations, it is
necessary to know the characteristic frequencies of molecular vibrations. 
It is also necessary to know how much each vibrational frequency in a
molecule changes when a heavy isotope is substituted for a light one. 
Vibrational frequencies for isotopically substituted molecules are not
always known, so it is often necessary to use some type of force-field
model to predict them. 
• Molecular vibrations are also important in understanding infrared
absorption and the mechanisms and kinetics of chemical reactions. 
Frequencies are most commonly measured with infrared or Raman
spectroscopy.  Rotational-vibrational spectroscopy, isotope substitution,
and many forms of force-field modeling are used to determine
characteristic atomic motions. 
 Vibrational Motion
There are two types of vibrational motion.
1.Stretching .
2.bending.
Stretching and Bending
Stretching Vibrations
Bending Vibrations
 Quantum Treatment of Vibrations
• Transitions in vibrational energy levels can be
brought about by absorption of radiation, provided
the energy of the radiation exactly matches the
difference in energy levels between the vibrational
quantum states and provided also that the vibration
causes a fluctuation in dipole.
• Infrared measurements permit the evaluation of the
force constants for various types of chemical bonds.
 Infrared Instruments
• An infrared spectrophotometer is an instrument that passes
infrared light through an organic molecule and produces a
spectrum that contains a plot of the amount of light.
Transmitted on the vertical axis against the wavelength of
infrared radiation on the horizontal axis. In infrared spectra
the absorption peaks point downward because the vertical
axis is the percentage transmittance of the radiation through
the sample.
• Absorption of radiation lowers the percentage transmittance
value. Since all bonds in an organic molecule interact with
infrared radiation, IR spectra provide a considerable amount
of structural data.
 2.Chromatography

• Chromatography is the most frequently used analytical

technique in
pharmaceutical analysis
• Refers to any separation method in which the components
are distributed
between a stationary phase (SP) and mobile phase(MP)
Mobile phase: a solvent that flows through the column
Stationary phase: a coating on the supporting medium that interacts with
the analytes (immobilized in the column
 SEPARATON

Principles of separation
• The samples are subjected to flow by mobile liquid phase through the
stationary phase
• Separation is based on their relative affinity towards the two phases
• Separations occur because sample components have different affinities
for stationary phase and mobile phase.,

those components of the sample having great affinity for the

stationary phase will move very slowly and with less affinity travels
faster
• The mobile phase is usually a liquid or a gas, and the stationary phase
is a
 Classification Chromatography

Classification of chromatography
Chromatographic methods can be classified in various ways:
1. Based on geometry of the system
2. Based on separation mechanism
3. Based on Mobile phase
 Planner chromatography
– A form of chromatography in which the stationary phase is
immobilized on a flat
surface .

– the stationary phase coats a flat glass, metal, or plastic plate and is
placed in a
developing chamber
 Chromatographic Methods
chromatographic methods are classified as:
1. Adsorption chromatography
2. Partition chromatography
3. Ion-exchange chromatography
4. Size-exclusion chromatography
 3.Gas-chromatography
Gas chromatography (GC), also called gas-liquid
chromatography
• Partition of molecules between gas (mobile phase)
and liquid (stationary phase.
 Gass chromatography

Gas chromatography (GC) is a common type of chromatography

extensively
used in the field of pharmacy and chemistry for separating and analyzing
compounds that can be vaporized without decomposition
• Gas chromatography (GC), also called gas-liquid chromatography
• Partition of molecules between gas (mobile phase) and liquid
(stationary phase).
• GLC is the most widely used technique for separation of volatile
species.
Criteria for compounds to be analyzed
 Principle of GC analaysis

A sample is vaporized and injected into the chromatographic

column
• As the carrier gas sweeps the analyte molecules through the
column
• GLC is based on the partition of the analyte between a
gaseous mobile
phase and a liquid stationary phase
• Polar compounds interact strongly with a polar stationary
phase, hence
have a longer retention time than non-polar columns
• The mobile phase doesn't interact with the analyte ; its only
function is to
transport the analyte through column
INSTRUMENTAION

A. Carrier gas
B. Injector
C. Column
D. Detector
E. Display system
printer/monitor
 Carrier gas supply

.Carrier gas supply

• It is compressed gas cylinder which supplies carrier gas to the column
of GC
Carrier gas
– It is the mobile phase in Gc
– It transports analytes through the column
– Most common carrier gas are Helium(He), Nitrogen gas(N2),
Hydrogen
gas(H2), Carbon dioxide gas (CO2) and Argon
– GC requires a supply of carrier gas of sufficient quality and pressure to
achieve the desired separations
 Carrier gas… cont’d

• The carrier gas should be

– chemically inert and should not interact with sample and stationary
phase
– readily available, cheap, and of high purity
2. Sample injector system:
• mainly used to introduce the sample into the carrier gas flow
• The sample usually in the form of the solution (0.5 μl) is introduced
into the
chromatography with
– a micro syringe
– auto samplers in which the syringe and injection procedure are totally
automated
 Temperature programing in Gc
• A technique in which the column temperature is increased either
continuously
or in steps as the separation proceeds
• Column temperature is an important variable that must be controlled
for
precise work
• The optimum column temperature depends upon the boiling point of
the
sample and the degree of separation required
• Roughly a temperature equal to or slightly above the average boiling
point of a
sample results in a reasonable elution time 2 -30 min
• For samples with a broad boiling range, it is often desirable to employe
temperature programming
 Gradient elution

• Increase the column temperature during the analysis

• Rate of temperature increase
• It allows adequate separation for the analyte that
elute early in the
analysis, and
• shortening the time it takes for late-eluting analyte to
pass through the
column
 DETECTOR

• The detector is placed at the exit of the column

• Gc detectors senses the solute vapor in the mobile phase as they
emerge from the column
• The solutes emerging from the column interact with the detector and
the detector converts this interaction into an electronic signal
• Detectors give response that is dependent on the conc. Of the analyte
in the carrier gas
• The magnitude of the signal is plotted versus time and a
chromatogram
is generated
 Detectors…cont’d

• The following are the ideal requirements of the GC

detectors:
– It should have high sensitivity
– It should have high stability
– It should have high reproducibility
– It should be feasible with wide range of temperature
– It should be easy to handle
 Several types of detectors

Several types of detectors

• detectors include
– Thermal conductivity detector (TCD)
– Flame ionization detector (FID)
– Nitrogen phosphorus detectors (NPD)
– Electron capture detector (ECD)
 Applications of Gc

Applications of Gc
• Separation of mixed components in a sample
• Qualitative identification of components
• Quantitative determinations of components in a sample
 Qualitative analysis

• chromatographic data is presented as a graph of detector

response
against retention time, which is called a chromatogram
• Retention time can be used to identify analytes if the method
conditions are constant.
• Gc is widely used as criteria of purity for organic
compounds
o Contaminants, if present, are revealed by the appearance of
additional peaks. The areas under these peaks provide rough
estimates of contamination
 Quantitative Analysis

• The area under a GC peak is proportional to the amount of

analyte
present in the chromatogram
• Concentration can be calculated using a calibration curve
created by
finding the response for a series of concentrations of analyte, or
by
determining the relative response factor of an analyte
 4.High performance Liquid
Chromatoggraphy
HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments
consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.

Compounds are separated by injecting a sample mixture onto the

column. The different component in the mixture pass through the
column at differentiates due to differences in their partition
behavior between the mobile phase and the stationary phase. The
mobile phase must be degassed to eliminate the formation of air
bubbles
 Types of liquid chromatography

Partition chromatography

Adsorption, or liquid-solid

chromatography

Ion exchange chromatography

Size exclusion, or gel, chromatography

Compositon of liquid chromatographic
system

• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors (Diode Array)
• Waste Collector
• Recorder (Data Collection
 Uses of HPLC

This technique is used for chemistry and biochemistry research

analyzing complex mixtures, purifying chemical compounds,
developing processes for synthesizing chemical compounds,
isolating natural products, or predicting physical properties. It is
also used in quality control to ensure the purity of raw materials,
to control and improve process yields, to quantify assays of final
products, or to evaluate product stability and monitor
degradation.

In addition, it is used for analyzing air and water pollutants, for

monitoring materials that may jeopardize occupational safety or
health, and for monitoring pesticide levels in the environment.
Federal and state regulatory agencies use HPLC to survey food
and drug products, for identifying confiscated narcotics or to
check for adherence to label claims
 What affects HPLC system

strument Parameters
Column Parameters
• Temperature
• Column Material
• Flow
• Deactivation
• Signal
• Stationary Phase
• Sample Sensitivity
• Coating Material
• Detector
Types of column phase

• Normal phase

• Reverse phase

• Size exclusion

• Ion exchange
 Normal phase

In this column type, the retention is governed by the interaction of the

polar parts of the stationary phase and solute. For retention to occur in
normal phase, the packing must be more polar than the mobile phase
with respect to the sample
Reverse Phase
In this column the packing material is relatively nonpolar and the solvent
is polar with respect to the sample. Retention is the result of the
interaction of the nonpolar components of the solutes and the nonpolar
stationary phase. Typical stationary phases are nonpolar
 Size exclusion

In size exclusion the HPLC column is consisted of substances which

have controlled pore sizes and is able to be filtered in an ordinarily phase
according to its molecular size. Small molecules penetrate into the pores
within the packing while larger molecules only partially penetrate the
pores. The large molecules elute before the smaller molecules

Ion exchange
In this column type the sample components are separated based upon
attractive ionic forces between molecules carrying charged groups of
opposite charge to those charges on the stationary phase
 Types of Liquid Column Chromatography
(LCC)

LLC (Liquid Liquid)

• GLC GSC

LSC (Liquid Solid - • SFC (Supercritical Fluid

adsorption)

• SEC (Size Exclusion)

 Types of Detectors

• • Absorbance (UV with Filters,

Absorbance (UV with Filters,
UV with Monochromators) UV with Monochromators)
• •
• IR Absorbance • IR Absorbance

• Fluorescence • Fluorescence

• Refractive-Index • Refractive-Index
 EVALUATION PARAMETERS

• EFFICIENCY
• RESOLUTION
• INERTNESS
• RETENTION INDEX
• COLUMN BLEED
• CAPACITY FACTOR
 References

http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html
Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th
ed. Orlando: Harcourt Brace & Co., 1998.
http://weather.nmsu.edu