intro to chromatography sp'14

8/13/2019 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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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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- 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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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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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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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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Paper

HPLC Gas

Thin layer

Column


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On the basis of stationary and mobile phase.

- liquid-solid chromatography

- liquid-liquid chromatography

- gas-solid chromatography

- gas-liquid chromatography


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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

intro to chromatography sp'14

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