Download - Intro to Chromatography Sp'14
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INTRODUCTIONTOCHROMATOGRAPHY
PHRM 312: PHARMACEUTICALANALYSIS- III
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SIMPLEMETHODSOFSEPARATION
Evaporation
Precipitation
Crystallization
Filtration
Membrane separation
Distillation
Sublimation
Extraction
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SIMPLEMETHODSOFSEPARATION(CONT)
Evaporation:
Evaporation simply entails vaporizing the solvent
by using heat or by utilizing air currents in a
manner that the material concentrates to a solid
state.
Precipitation:
Changing the concentration of a solute in a
solution so that it exceeds solubility in a given
solvent can bring about precipitation of the solute.- Solvent Precipitation
- Precipitation via Chemical Reaction
- Precipitation by Adjustment of pH
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SIMPLEMETHODSOFSEPARATION(CONT)
Crystallization:
This involves concentrating a solution containing the
component of interest by heating it and then
allowing it to stand (i.e., cooling it) until the crystals
are obtained from the solution.
Filtration:
Separation of particles that are visible to the naked
eye with a filter paper is called filtration; however,
filtration of submicron particle size is also possible.- Simple filter papers are made from cellulose and
exhibit particle retention levels down to 2.5 m (e.g.,
Whatman Grade 5 filter paper)
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SIMPLEMETHODSOFSEPARATION(CONT)
- Membrane filters made from various materials
can offer various pore sizes that allow
separations down to 0.02 m level.
Membrane separation
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SIMPLEMETHODSOFSEPARATION(CONT)
Dialysis
A dialyzer is an apparatus in which one or more
solutes are transported from one fluid to another
through a membrane under a concentration driving
force.
Electrodialysis
In electrodialysis, electrically charged membranes
are used to separate components of an ionic
solution under the driving force of an electric
current.
This process has been used for desalination of
water, recovery of salt from seawater, deashing of
sugar solutions, and deacidification of citrus
juices.
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SIMPLEMETHODSOFSEPARATION(CONT)
Distillation
Distillation is a method of separating mixtures based on
differences in their volatilities in a boiling liquid mixture.
- Origin of distillation can be related to evaporation.
- In distillation, the components of interest are volatile,
whereas in evaporation volatile components are
separated from nonvolatile ones.
- Generally liquids.
Extraction
- relatively simple type of separation process
- a solute is distributed between two immiscible
solvents.
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WHATISCHROMATOGRAPHY?
Greek word: Chroma (colour)Graphein(to write).
Chromatography provides a way to identify unknown compounds
and separate mixtures
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HISTORY
The Russian botanist MikhailTsvet coined the term
chromatography in 1906 to
describe his experiments in
separating different colored
constituents of leaves by passing
an extract of the leaves through a
column
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Chromatography is a physical method of
separation in which components to be
separated are distributed between two
phases
one of which does not move [(stationary
phase)S.P]
the other that moves through S.P in a definite
direction (mobile phase).
DEFINITION
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CHROMATOGRAPHY
Separates components in
mixture:
Based on
- polarity- boiling point
- ionic strength
- size
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CHROMATOGRAPHY
Mobile phase: phase in which sample is
dissolved. It may be gas, liquid, or supercritical
fluid
Stationary phase: phase in which mobilephase is forced through it
Mobile and stationary phases are chosen in amanner so that analyte will distribute itself
between the two phases
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TERMINOLOGY
The analyteis the substance to be separated during
chromatography.
The sampleis the matter analyzed in chromatography. It
may consist of a single component or it may be a mixture
of components.
When the sample is treated in the course of an analysis,
the phase or the phases containing the analytes of
interest is/are referred to as the sample whereaseverything out of interest separated from
the sample before or in the course of the analysis is
referred to as waste.
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TERMINOLOGY
The eluateis the mobile phase leaving the column.
The eluentis the solvent that carries the analyte.
An eluotropic seriesis a list of solvents ranked
according to their eluting power.
The retention timeis the characteristic time it takesfor a particular analyte to pass through the system
(from the column inlet to the detector) under set
conditions.
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TERMINOLOGY
A chromatographis equipment that enables a
sophisticated separation, e.g. gas chromatographic or
liquid chromatographic separation.
The detectorrefers to the instrument used for
qualitative and quantitative detection of analytes after
separation.
A chromatogramis the visual output of the
chromatograph.
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CHROMATOGRAM
Detector signal (conc. of Sample) vs.
retention time or volume of mobilephase
time or volume
DetectorSignal
1 2
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ChromatogramsIf a detector that responds solute concentration is placed at the
end of the column and its signal is plotted as function of time
(or of volume of the added mobile phase), a series of peaks isobtained.
Such a plot is called a chromatogram, useful for both qualitative
and quantitative analysis.
- positions of peaks on the time axis may serve to identify thecomponents of the sample
- areas under the peaks provide a quantitative measure of the
amount of each component.
Concentration of sample
Vs
Time (Volume of mobile phase)
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TYPESOFCHROMATOGRAPHY
According to purpose
1. Analytical chromatography: Itis used to
determine the existence and possibly also the concentrationof analyte(s) in a sample.
2. Preparative chromatography:It is used to purify
sufficient quantities of a substance for further use, ratherthan analysis.
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TYPESOFCHROMATOGRAPHY
In general:
1. Column chromatography
2. Paper chromatography
3. Thin-layer chromatography (TLC)
4. Gas chromatography (GC)5. High pressure / High performance liquid
chromatography (HPLC)
6. Ion exchange chromatography
7. Gel filtration chromatography
8. Affinity chromatography
9. Super critical fluid chromatography
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TYPESOFCHROMATOGRAPHY
Paper
HPLC Gas
Thin layer
Column
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TYPESOFCHROMATOGRAPHY
On the basis of stationary and mobile phase.
- liquid-solid chromatography
- liquid-liquid chromatography
- gas-solid chromatography
- gas-liquid chromatography
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TYPESOFCHROMATOGRAPHY
On the basis of type of equilibrium process:
1. Adsorption chromatography
2. Partition chromatography
3. Ion Exchange chromatography
4. Molecular/size exclusion chromatography
5. Affinity chromatography
6. Gel Electrophoresis
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ADSORPTIONCHROMATOGRAPHY
The stationary phase is solid on which the sample
components are adsorbed.The mobile phase may be a
- liquid (liquid-solid chromatography) or,
- gas (gas-solid chromatography)The components distribute
between the two phases through
the combination of
- adsorption &
- desorption
e.g. Column chromatography, TLC
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PARTITIONCHROMATOGRAPHY
The stationary phase is a liquid supported on an inert solid
The mobile phase is a
- liquid (liquid-liquid partition chromatography) or,
- gas ( gas-liquid partition chromatography)
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Paper chromatography is a type of partition
chromatography in which the stationary phase isa layer of solvent adsorbed on a sheet of paper.
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IONEXCHANGECHROMATOGRAPHY
An ion exchange resin isused as the stationaryphase.
Separation of either
cations or anions Separtion based on
relative strength of ionicbond
Mobile phases used isalways liquid (liquid
chromatography)
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MOLECULAR/SIZEEXCLUSIONCHROMATOGRAPHY
Separation based on size
Stationary phase ispolymeric substancecontaining numerouspores of molecular
dimension. Larger molecules that will
not fit into the poresremain in the mobilephase and are not
retained.
Small molecules gettrapped in pores & takelonger to get out
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AFFINITYCHROMATOGRAPHY
A method of separating
biochemical mixtures
Based on a highly specific biological
interaction such as that between
- antigen and antibody,
- enzyme and substrate, or- receptor and ligand.
Stationary phase is typically a gel
matrix, often of agarose.
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GELELECTROPHORESIS
a separation technique in
which the flow of themobile phase or buffer is
driven through a
chromatographic column
by an electric field, ratherthan by an applied
pressure
Separation based on size
and charge
Smaller molecules will
migrate further, less
tangled
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APPLICATIONSOFCHROMATOGRAPHY
Forensics
Research
Pharmaceutical industry
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APPLICATIONOFCHROMATOGRAPHY
It is a technique for separating mixtures of compounds
identifying unknown compounds
establishing the purity or concentration of
compounds
monitoring product formation in the pharmaceutical
and biotechnology industries.
It is widely used by forensic teams to analyse
blood and urine samples for drugs.
T C ( )
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TYPESOFCHROMATOGRAPHY(CONT.)
Classification on the basis of the sample
introduction and migration through thechromatographic bed:
1. Frontal Analysis
2. Displacement Analysis
3. Elution Analysis
F A
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FRONTALANALYSIS
Sample is introduced into the bed continuously,
rather than in small portions and sample itself
constitutes the mobile phase.
The sample components are selectively retarded;
and fronts, rather than bands, are formed as a
result of the separation.
The less retained component emerges from the
column first, followed by the mixture of the first
component plus the next most strongly retained
component.
F A ( )
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FRONTALANALYSIS(CONT.)
Frontal analysis
cannot accomplish
complete recovery of
the pure sample;however, it is useful
for concentrating
trace impurities and
for purifying large
volumes of liquids
D A
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DISPLACEMENTANALYSIS
Sample mixture,
dissolve in a smallvolume of solvent
C
O
LU
M
N
Mobile phase, containing
a displacing agent
The displacement agentis asubstance that is retained more
strongly by the stationary phase
than any of the components in the
sample mixture and thereforeforces them off the surface of the
stationary phase into the mobile
phase.
D A ( )
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DISPLACEMENTANALYSIS(CONT.)
As each of the displaced solute pass through the column
in the mobile phase, it in turn acts as a displacing agent
for less strongly retained compounds.
The final result is that, the compound that is least firmly
bound is eluted first, followed in order by those more
tightly bound and finally by the displacing agent.
E A
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ELUTIONANALYSIS
Elution analysis is carried out by introducing the sample in
as small volume as possible onto the head of the column. The mobile phase is then allowed to flow through the
system.
The components with larger partition
coefficients will be retarded and willelute later.
Compounds can be isolated in a
relatively pure state.
- Isocratic elution
- Gradient elution
S O S S
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STATIONARYPHASES
Alumina:Aluminum oxide
- Surface is highly polar- Capable of adsorbing practically all polar molecules.
- Judicious selection of the eluent allows to separate any
pairs of solute on alumina column.
-Adsorbent power of alumina is markedly affected by its
water content, with freshly dried alumina having the
highest adsorbent activity.
- Adsorbent power of alumina is specified by its ability toretard the migration of certain dyestuffs through a
column prepared with the alumina.
Grades I-V
STATIONARY PHASES
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STATIONARYPHASES
Silicic acid or silica gel, SiO2
- can be activated by heating or prewashing the bed withan anhydrous solvent to remove adosorbed water.
Some others stationary phase:
1. Fullers earth 5. Potassium carbonate2. Activated charcoal 6. Talc
3. Magnesium oxide 7. Starch
4. Calcium carbonate