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CHROMATOGRAPHY
CHROMATOGRAPHY

DEFINITION:

the

compounds

with

in

separation

of

individual

a

complex

mixture by

partitioning between a stationary phase , and a

mobile phase.

CHROMATOGRAPHY TECHNIQUES
CHROMATOGRAPHY
TECHNIQUES
GAS CHROMATOGRAPHY  GC is used used as an analytical tool .  It is used
GAS CHROMATOGRAPHY
GC is used used as an analytical tool .
It
is
used to separate volatile components of
mixture .
In this the stationary phase is a high boiling liquid
and the mobile phase is an inert gas.
TYPES OF GAS CHROMATOGRAPHY 1. Gas Solid Chromatography 2. Gas Liquid chromatography
TYPES OF GAS
CHROMATOGRAPHY
1.
Gas Solid Chromatography
2. Gas Liquid chromatography
INSTRUMENTAL COMPONENTS
INSTRUMENTAL COMPONENTS
CARRIER GAS The most important requirements of carrier gas: It should be inert. It should be
CARRIER GAS
The most important requirements of carrier
gas:
It should be inert.
It should be available at low cost.
It should allow the detector to respond in an
adequate manner.
SAMPLE INJECTION PORT
SAMPLE INJECTION PORT
: COLUMNS
:
COLUMNS
DETECTORS Types Of Detectors FID TCD NPD ECD
DETECTORS
Types Of Detectors
FID
TCD
NPD
ECD
FACTORS EFFECTS OF GC  Volatility of compound  Polarity of compound  Flow rate of
FACTORS EFFECTS OF GC
Volatility of compound
Polarity of compound
Flow rate of the gas
APPLICATIONS  Analysis of purity of organic compound  Analysis of pesticide  Biochemical and clinical
APPLICATIONS
Analysis of purity of organic compound
Analysis of pesticide
Biochemical and clinical field
In cosmetics and perfumes field
In plastic industry
GAS CHROMATOGRAPHY  It is an analytical tool  It is used to separate volatile components
GAS CHROMATOGRAPHY
It is an analytical tool
It is used to separate volatile components of a
mixture.
In this stationary phase is a high-boiling liquid
&the mobile phase is an inert gas
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
INTRODUCTION:HPLC  HPLC is a form of liquid chromatography used to separate compounds that are dissolved
INTRODUCTION:HPLC
 HPLC is a form of liquid chromatography used
to separate compounds that are dissolved in
solution. HPLC instrument consists of a
reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.
 Compounds
are
separated
by
injecting
a
sample mixture on 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.
FLOW CHAT
FLOW CHAT
INSTRUMENTATION
INSTRUMENTATION
MOBILE PHASE  The mobile phase in HLPC refers to the solvent being continuously applied to
MOBILE PHASE
The mobile phase in HLPC refers to the solvent
being continuously applied to the column or
stationary phase.
STATIONARY PHASE
The stationary phase in HPLC refers to the solid
support contained within the column over which the
mobile phase continuously flow
INJECTORS FOR HPLC  Samples are injected into HPLC via injection port  Injection port consist
INJECTORS FOR HPLC
Samples are injected into HPLC via injection port
Injection port consist of a injection valve and
sample loop
The sample dissolved in mobile phase before
injection
The sample drawn into syringe and injected into
loop via injector valve
Injection of sample range 10ul to over 500ul
In modern HPLC sample injection is typically
automatic
HPLC COLUMN
HPLC COLUMN
TYPES OF DETECTORS  Absorbance (UV with filters, UV with monochromators)  IR Absorbance  Fluorescence
TYPES OF DETECTORS
Absorbance
(UV
with
filters,
UV
with
monochromators)
IR Absorbance
Fluorescence
Refractive-index
Evaporative Light Scattering Detector (ELSD)
Electrochemical
Mass Spectrometric
Photo-diode Array
THE COLUMN AND THE SOLVENT  They are two varients in use in HPLC depending on
THE COLUMN AND THE
SOLVENT
They are two varients in use in HPLC depending
on the relative polarity of the solvent and the
stationary phase
1. NORMAI PHASE HPLC
2.REVERSED PHASE HPLC
NORMAL PHASE  In this column type, the retention is governed by the interaction of the
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 sample. Retention
Reverse phase
 In this column the packing material is relatively
nonpolar and the sample. Retention is the result
of the interaction of the nonpolar stationary
phase. Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc) and the
solvents are polar aqueous-organic mixtures such
as methanol-water or acetonitrile-water.
ISOCRATIC AND GRADIENT ELUTIONS  A Separation in which the mobile phase composition remain constant throughout
ISOCRATIC AND GRADIENT
ELUTIONS
A Separation in which the mobile phase
composition remain constant throughout is
termed isocratic
The mobile phase composition does not have to
remain constant is termed Gradient elution
USES OF HPLC  This technique is used for chemistry and biochemistry research analyzing complex mixtures,
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 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
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