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INSTRUMENTAL METHODS

OF ANALYSIS
Presentation by:
R.Pushpalatha
Dept. of Chemical Engineering
SSN College of Engineering
What is Chromatography?
Elution : always (100%) dilution
What is Chromatography?
sample
in
eluent
in
CaCO3
(adsorption)
column
eluant
out
detector
chromatogram
(mass spect. IR
spect. etc)
Chromatography
The most powerful tool for separating &
measuring the components of a complex
mixture.
The separation of a mixture by distribution of
its components between a mobile and
stationary phase over time
mobile phase = solvent
stationary phase = column packing material
A solute equilibrates between a mobile and a
stationary phase.
The more it interacts with the stationary phase, the
slower it is moved along a column.
Uses for Chromatography
Chromatography is used by scientists to:
Analyze examine a mixture, its components,
and their relations to one another
Identify determine the identity of a mixture or
components based on known components
Purify separate components in order to isolate
one of interest for further study
Quantify determine the amount of the mixture
and/or the components present in the sample
Uses for Chromatography
Real-life examples of uses for
chromatography:
Pharmaceutical Company determine amount of
each chemical found in new product
Hospital detect blood or alcohol levels in a
patients blood stream
Law Enforcement to compare a sample found at
a crime scene to samples from suspects
Environmental Agency determine the level of
pollutants in the water supply
Manufacturing Plant to purify a chemical
needed to make a product
Types
Gas Chromatography
Liquid Column Chromatography
High Performance Liquid Chromatography
(HPLC)
Thin Layer Chromatography (TLC)
Paper Chromatography
Electrophoresis
Affinity Chromatography
Immuno chromatography
Gas Chromatography
Used to determine the chemical composition of
unknown substances, such as the different
compounds in gasoline shown by each separate
peak in the graph below.
Paper Chromatography
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-up,
and many other substances
Liquid Chromatography
Used to identify unknown plant
pigments & other compounds.
Thin-Layer Chromatography
Uses thin plastic or glass trays to
identify the composition of pigments,
chemicals, and other unknown
substances.
Examples of Chromatography
Liquid Chromatography separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)
Gas Chromatography separates vaporized samples
with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary
phase)
Paper Chromatography separates dried liquid
samples with a liquid solvent (mobile phase) and a
paper strip (stationary phase)
Thin-Layer Chromatography separates dried liquid
samples with a liquid solvent (mobile phase) and a glass
plate covered with a thin layer of alumina or silica gel
(stationary phase)
Types of Chromatography
Chromatography
Chromatogram - Detector signal
vs. retention time or volume
time or volume
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Milestones in
Chromatography
1903 Tswett - plant pigments separated on
chalk columns
1931 Lederer & Kuhn - LC of carotenoids
1938 TLC and ion exchange
1950 reverse phase LC
1954 Martin & Synge (Nobel Prize)
1959 Gel permeation
1965 instrumental LC (Waters)
Separation Mechanism
Instrumentation
What is Chromatography?
Factors Governing Retention Time
Retention time of a solute : time taken by the
solute to reach the detector from the moment of
its injection into the column.
Depends upon
Nature of the stationary phase
Composition of the mobile phase
Column length (dia too)
Mobile phase flow rate
Partition Ratio, K
Partition ratio/ coefficient or Distribution ratio /
coeficient : K
How the solute distributes between the stationary
phase & mobile phase
tR = tm ( 1+K (Vs/Vm))
K = Cs / Cm
If K= 1, the solute is equally distributed
If KA = KB, no separation occurs
If KB > KA, then A elutes first
Column Capacity Factor, k
Column Capacity Factor /Column Retention Factor
Also called mass distribution ratio
A normalised quantity used to describe the
migration rates of the solute through the column
Ratio of the total amount of solute present in the
stationary phase to that in the mobile phase
K =(CsVs) / (CmVm) = K /(Vm/Vs) = K /
is Phase ratio
tR = tm ( 1+k) or k = (tR tm) / tm
Value of k should be between 1 & 5
Retention Volume
Retention volume (VR) : Volume of the mobile
phase (in ml) required to elute a solute from the
point of its introduction at the head of the column
to its exit from the column.
VR = tR x F = V0 ( 1 + K(Vs/Vm)) = V0 + KVs
V0 = Vm : void volume or dead volume:tm x F
Separation of Peaks
For pairs of bands
Efficiency : two factors contribute to
how well components are separated :
the widths of the peaks :
the wider the peak, the poorer
separation.
b) Selectivity:
the spacing in time :
the further apart, the better
separation.
Resolution of Column
Tells us how far apart 2 bands are relative to
their widths.
Provides a quantitative measure of the ability of
the column to separate 2 analytes.
Resolution
Factors Governing Resolution
Low flow rates favour increased resolution
Long column and smaller dia particles
packing increase resolution
Temperature
Mobile phase composition
Solution
Change column conditions during
elution
-change in liquid mobile phase
composition - gradient elution
or solvent programming
-change in temperature for gas
chromatography- temperature
programming
Effect of Mobile Phase Composition
Effect of Temperature on Resolution
Elution Method
Gradient Elution
Advantage of Gradient Elution