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What is Chromatography?
Greek word: Chroma (colour)
Graphein (to write).
Chromatography is a technique for separating mixtures into
Introduction to their components in order to analyze, identify, purify, and/or
quantify the mixture or components.

Chromatography
Dr. Hasina Yasmin
Analyze
Separate Identify
Purify
Quantify
Mixture Components

USES FOR CHROMATOGRAPHY

Uses for Chromatography
Real-life examples of 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 a mixture
and/or the components present in the sample
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Definitions of Chromatography Definitions of Chromatography

Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.
Terminology:
Differential showing a difference, distinctive
Affinity natural attraction or force between things
Mobile Medium gas or liquid that carries the components
(mobile phase)
Stationary Medium the part of the apparatus that does
not move with the sample (stationary phase)
Simplified Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile
phase and the stationary phase.
Explanation:
Compound is placed on stationary phase
Mobile phase passes through the stationary phase
Mobile phase solubilizes the components
Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases

History of Chromatography
Illustration of Chromatography
The Russian botanist Stationary Phase
Mikhail Tsvet coined the
term chromatography in
1906 to describe his Separation
experiments in separating
different colored constituents
of leaves by passing an
extract of the leaves through
a column Mobile Phase

Mixture Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase
Blue ---------------- Insoluble in Mobile Phase

Black

Red

Yellow
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Rf = distance moved by substance

distance moved by solvent front

For substances that are very soluble in the liquid Rf

will be close to ....
1

For substances that are rather insoluble in the liquid Rf

will be close to ....
0

Chromatography
TypesChromatography
of Chromatography
Mobile phase: phase in which sample is dissolved. The
Separates components mobile phase may be a liquid or a gas. This phase
moves through the chromatography column which is
in mixture: packed with the stationary phase.
Based on
- polarity Stationary phase: phase in which mobile phase is forced
through it. It may be Solid, liquid coated on solid, etc. The
- ionic strength
stationary phase is the substance which is fixed in a
- size column or on a flat plate or surface. The mobile phase
is passed through the stationary phase where the
sample interacts and is separated. Examples are silica gel,
aluminum oxide or cellulose.

Mobile and stationary phases are chosen in a manner so that

analyte will distribute itself between the two phases

Principle of Chromatography:
The mixture to be separated is dissolved in a
fluid called the mobile phase, which carries it
through a structure (column, TLC plateholding )
The various components of the mixture
travel at different speeds through the
stationary phase causing them to separate.
So that the separation is actually based on
differential partitioning between the mobile and
stationary phases.
TERMINOLOGY
The analyte is the substance to be separated during
chromatography.
The sample is the matter analyzed in chromatography.
It may consist of a single component or it may be a
mixture of components.
Elution: Purification or separation by washing.
The eluent is the mobile phase leaving the column.
The eluate is the solvent that carries the analyte.
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The speed of a migrating sample component
depends on whether the component has an
affinity for the stationary or mobile phase.
This affinity can be of different types such
as: Adsorption, Partition, Ion exchange etc.
Components that have a higher affinity for the
mobile phase compared with the stationary phase
migrate more rapidly, while components that
have a higher affinity for the stationary
phase are eluted later from the column.
TERMINOLOGY
The retention time is the characteristic time it
takes for a particular analyte to pass through the
system (from the column inlet to the detector)
under set conditions.
A chromatograph is equipment that enables a
sophisticated separation, e.g. gas chromatographic
or liquid chromatographic separation.
Chromatogram: It is the visual output of the
chromatograph. In the case of an optimal
separation, different peaks on the chromatogram
correspond to different components of the separated
mixture.
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TERMINOLOGY Chromatogram

Concentration of sample
The detector refers to the instrument used for
Vs
qualitative and quantitative detection of analytes
Time (Volume of mobile phase)
after separation.

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 the components of the sample
- areas under the peaks provide a quantitative measure
of the amount of each component.

CHROMATOGRAM
CHROMATOGRAM
Detector signal (conc. of Sample)
vs.
retention time or volume of
mobile phase

1 2
Detector Signal

time or volume
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Based on the physical means of bringing the

stationary and mobile phases into contact
chromatographic methods can be classified
into 2 broad categories.

1. Planar Chromatography: Here the

stationary phase is supported on a flat plate or in
the fibres of a paper. Here the mobile phase
moves through the stationary phase by capillary
action or by gravity.
E.g.: Paper chromatography, Thin layer
chromatography (TLC).

2. Column Chromatography: Here the stationary Types of Chromatography

phase is packed in a test tube / boiling tube / glass
column of any convenient size. The substances to be
According to purpose
separated are introduced onto the top of a column and
pass through the column at different rates that
depend on the affinity of each substance for the 1. Analytical chromatography: It is used to
adsorbent and for the solvent or solvent mixture. determine the existence and possibly also the
concentration of analyte(s) in a sample. It is done in
smaller amount of material.

2. Preparative chromatography: It is used

to purify sufficient quantities of a substance for
further use, rather than analysis. It is done in
larger amounts of material.
Figure schematic diagram
of a packed column
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Types of Chromatography Types of Chromatography

On the basis of physical state of stationary and mobile phase. On the basis of method of separation liquid
1. liquid-solid chromatography: In liquid-solid chromatography can be classified into:
chromatography the stationary phase is a solid such as calcium
carbonate or more likely these days silica or alumina (Al2O3). 1. Adsorption chromatography
2. liquid-liquid chromatography: In liquid-liquid
chromatography, the stationary phase is a liquid, e.g. a C18 silane, 2. Partition chromatography
covalently bonded to an inert silica support.
3. gas-solid chromatography: Conventionally columns are 3. Ion Exchange chromatography
packed with porous polymers or materials such as activated
carbon, molecular sieves, silica and alumina powder, etc.
4. Molecular/size exclusion chromatography
2. gas-liquid chromatography: In gas-liquid chromatography
the stationary phase is a low volatility liquid coated on to a solid
support e.g. Carbowax 20M - a polyethylene glycol with a 5. Affinity chromatography
molecular mass of 20,000 g/mole.

TYPES OF CHROMATOGRAPHY ADSORPTION CHROMATOGRAPHY

The stationary phase is solid on which the sample

components are adsorbed.
The mobile phase may be a

- liquid (liquid-solid chromatography) or,

Thin layer - gas (gas-solid chromatography)
Paper The components distribute

between the two phases through

the combination of
- adsorption &
- desorption

HPLC Gas Column

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ADSORPTION CHROMATOGRAPHY
Adsorption chromatography is based on the
adsorption of analyte molecules onto the surface If silica, a silicon-oxygen polymer as
of a solid (stationary phase). This attachment or illustrated in the figure is the stationary
phase, the surface hydroxyl groups interact
interaction depends on the polarity of
with the analytes, the more the interaction
solutes. the more the analytes are retained and Figure-
E.g.: Column chromatography (CC), Thin therefore the longer they take to pass structure of silica
through the column.
Layer chromatography (TLC), High
Performance Liquid chromatography
(HPLC), etc.

Figure binding of analytes to silica.

Some analytes (pink) will bind more
than others (blue) and hence
separation is obtained. 30

The mobile phase (solvent) and APPLICATIONS OF ADSORPTION

analyte molecules compete for the
adsorbent sites of the stationary CHROMATOGRAPHY
phase. The more polar the
molecules the longer they are
retained.
Separation of relatively non-polar, water insoluble compounds

Modification of the adsorbent sites Separation of isomers

can make it possible to separate
isomers. Purification of antibody fragments

Strong solvents result in short

retention times as the solvent
molecules successfully compete for
the stationary phase active sites
which means there are fewer
available sites to retain the analyte
molecules. Figure competition for adsorption
sites

31 32
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PARTITION CHROMATOGRAPHY
Paper chromatography is a type of partition
In partition chromatography a
solid support with a high surface chromatography in which the stationary phase is
area such as crushed firebrick or a layer of solvent adsorbed on a sheet of paper.
keiselguhr is coated with a high
boiling liquid which acts as the
stationary phase. Separation Solid
occurs because of the differences in support
solubility for the analytes in the
stationary and mobile phases.

The partition coefficient is defined Stationary phase

Analyte
as:
n
Conc . in stationary phase Figure coated support
K = particle
Concn. in mobile phase

The mobile phase is a

- liquid (liquid-liquid partition chromatography) or,
- gas ( gas-liquid partition chromatography) 33

APPLICATIONS OF PARTITION ION EXCHANGE CHROMATOGRAPHY

CHROMATOGRAPHY
There are an immense number of possible applications of partition
chromatography. The list below gives just a few examples of where this
technique is routinely applied:
Ionic mixtures can be separated by this technique.
Here the stationary phase is composed of ion-
exchange resins and the mobile phase is a buffered
aqueous solution.

Determination of water quality;

Separation of aroma molecules of wine;

Determination of pesticide residues;

Quality control of pharmaceutical preparations;

Identification and measurement on petroleum fractions.

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APPLICATIONS OF ION EXCHANGE

Solute ions of the opposite charge in the mobile
phase attach to the resin by electrostatic forces. CHROMATOGRAPHY
Ion exchange columns are of 2 types.

1. Cation exchange columns, contains ()ve Separation of vitamins

ly charged groups.
Separation of inorganic cations and anions
Used for anion separation
Separation low molecular weight organic acids

2. Anion exchange columns, contains (+)ve ly Analysis of serum

charged groups.
Used for cation separation Analysis of drugs

E.g.: CC, HPLC.

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MOLECULAR/SIZE EXCLUSION CHROMATOGRAPHY APPLICATION OF SIZE EXCLUSION

Also known as Gel CHROMATOGRAPHY
permeation or Gel Determination of the molecular mass distribution of synthetic polymers
filtration chromatography.
Separation based on size. Here no
interactions occur between the Analysis of sugars
stationary phase and the solute
molecules.
Analysis and isolation of lipid polymers
Stationary phase is polymeric
substance containing numerous
pores of molecular dimension. Purification, identification, and quantification of protein mixtures

Larger molecules that will not fit Study of polymer reaction kinetics
into the pores remain in the
mobile phase and are not
retained. Separation and purification of large biomolecules (molecular mass >
Small molecules get trapped in 10,000
pores & take longer to get out g/mole)
40
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AFFINITY CHROMATOGRAPHY
Affinity Chromatography: A method of separating biochemical mixtures
Based on a highly specific biological
This technique also requires the interaction between interaction such as that between
the solute molecules and active sites on the - antigen and antibody,
stationary phase. - enzyme and substrate, or
- receptor and ligand.
According to the theory of this technique, those Stationary phase is typically a gel matrix,
compounds whose shape matches perfectly to the shape often of agarose.
of the stationary molecules, bind well to the stationary
phase. This interaction is of covalent type and thus
taking longer period to elute where as other compounds
elute earlier.

APPLICATIONS OF AFFINITY
This technique is mainly used in antibody
testing assays. Test sample contains a CHROMATOGRAPHY
mixture of proteins and immobilized
(stationary) molecule is an antibody to some Purification of proteins
specific protein.
Study of drug and hormone interactions with proteins

Immunoassays

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STATIONARY PHASES STATIONARY PHASES

Alumina: Aluminum oxide Silicic acid or silica gel, SiO2
- Surface is highly polar - can be activated by heating or prewashing the bed
with an anhydrous solvent to remove adsorbed water.
- Capable of adsorbing practically all polar molecules.
- Judicious selection of the eluent allows to separate
Some others stationary phase:
any pairs of solute on alumina column.
1. Fullers earth (hydrous aluminum silicate)
- Adsorbent power of alumina is markedly affected by 2. Activated charcoal
its water content, with freshly dried alumina having 3. Magnesium oxide
the highest adsorbent activity. 4. Calcium carbonate
- Adsorbent power of alumina is specified by its ability 5. Potassium carbonate
to retard the migration of certain dyestuffs through a 6. Talc
column prepared with the alumina. 7. Starch
Grades I-V