INTRODUCTION TO

CHROMATOGRAPHY
INTENDED LEARNING OUTCOMES (LLO)

At the end of this lecture, student should be

able to:

1. Explain terms used in chromatography analysis.

2. Explain the basic principles of chromatography.
3. Explain different types of chromatography.
CHROMATOGRAPHY
The term Chromatography and its principles were first discovered by
Mikhail Tswett, Russian, 1872-1919.

In 1906 Tswett used to chromatography to separate plant pigments.

He called the new technique chromatography because the result of

the analysis was 'written in color' along the length of the adsorbent
column Chroma means “color” and graphein means to “write”.
 Chromatography Terms
TERMS
Chromatogram Is the visual output of the chromatograph. In the case of an ideal
separation, different peaks or patterns on the chromatogram represent
different components of the separated mixture.
Analyte The analyte is the substance to be separated during chromatography.
Solute The term for the sample components being analysed.
 Commonly used terms in chromatography
TERMS

Eluate Is the mobile phase leaving the column.

Eluent The solvent that carries the analyte.

Elution Elution then is the process of removing analytes

from the adsorbent by running a solvent, called an
"eluent", past the adsorbent/analyte complex.

Retention Is the time required for the mobile phase to sweep

time a component from the stationary phase.
Commonly used terms in chromatography
TERMS
Mobile phase Is the phase that moves over stationary phase. It may be liquid or
a gas. It moves through the stationary phase where the sample
interacts with the stationary phase and its separated.
Stationary phase Is the substance fixed in place for the chromatography procedure.
It will be solid, gel or a liquid,i.e: silica, cellulose

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Principles of chromatography
• Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases

• One of which is stationary (stationary phase) while the other (the mobile phase)
moves through it in a definite direction.

• The chromatographic process occurs due to differences in the distribution constant

of the individual sample components.

•
Works by allowing the molecules present in the mixture to distribute themselves
between a stationary and a mobile medium.

•
Molecules that spend most of their time in the mobile phase are carried along faster.
Principles of chromatography

• In a chromatographic separation of any type, different components

of a sample are transported in a mobile phase (a gas, a liquid, or a
supercritical fluid).
• The mobile phase penetrates or passes through a solid or
immiscible stationary phase.
• Solutes (eluates) in the sample usually have differential partitioning
or interactions with the mobile and stationary phases.
• Since the stationary phase is the fixed one then those solutes which
have stronger interactions with the stationary phase will tend to
move slower (have higher retention times) than others which have
lower or no interactions with the stationary phase will tend to move
faster.
 Principle of Chromatography

• Therefore, chromatographic separations are a consequence of differential

migration of solutes.

• It should be remembered that maximum interactions between a solute and a

stationary phase take place when both have similar characteristics, for example
in terms of polarity.

• However, when their properties are so different, a solute will not tend to stay
and interact with the stationary phase and will thus prefer to stay in the mobile
phase and move faster; a polar solvent and a non polar stationary phase is a
good example.
 Principle of Chromatography

• A small volume of the sample is first introduced at the top of

the chromatographic column.

• Elution involves passing a mobile phase inside the column

whereby solutes are carried down the stream but on a
differential scale due to interactions with the stationary phase.

• As the mobile phase continues to flow, solutes continue to

move downward the column.

• Distances between solute bands become greater with time and as solutes start to leave the
column they are sequentially detected.
 Principle of Chromatography

• The time a solute spends in a column (retention

time) depends on the fraction of time that solute
spends in the mobile phase.

• As solutes move inside the column, their

concentration zone continues to spread and the
extent of spreading (band broadening) depends on
the time a solute spends in the columns.

• The dark colors at the center of the solute zones in

the above figure represent higher concentrations
than are concentrations at the sides. This can be
represented schematically as:
Classification of chromatography

• Analytical - determine chemical composition of a

sample

• Preparative - purify and collect one or more

components of a sample
Types of Chromatography
There are several types of chromatography include:

• Paper chromatography
• Thin Layer Chromatography (TLC)
• Column Chromatography
• Ion Exchange Chromatography
• Gas chromatography
• HPLC Chromatography
• Gel permeation/Filtration/Size Exclusion Chromatography
• Affinity Chromatography
 Types of chromatography
Paper chromatography
• Paper chromatography is a techniques that involves placing a small dot or line
of sample on to a strip of chromatography paper.
• The paper is placed in a jar containing a
shallow layer of solvent and sealed.

• As the solvent rises through the paper, it

meets the sample mixture, which start to
travel up the paper with the solvent.
 Types of chromatography
Thin Layer Chromatography (TLC)

• It is similar to paper chromatography but, the stationary phase is a thin layer of a solid such as
alumina or silica supports on an inert base such as glass, aluminium foil or insoluble plastic.

The mixture is spotted at the bottom

of the TLC plate and allowed to dry.
The plate is placed in a closed vessel
containing solvent ( the mobile
phase) so that the liquid level is
below the spot.
 Types of chromatography
Column Chromatography
A solvent acts as the mobile phase and finely divided
solid surface act as the stationary phase.
The stationary phase will absorb the components of
the mixture to varying degress.

As the solution containing the mixture passes over the

adsorbent, the component are distributed between
the solvent and absorbent surface.
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A weakly adsorbed compound will spend more in the
solvent and be eluted first.
 Types of chromatography
Ion exchange chromatography
• Ion exchange is used to remove ions of one
type from a mixture and replace them by ions
of another type.
• The column is packed with porous beads of a
resin that will exchange either cation or anions.
Types of chromatography
GAS CHROMATOGRAPHY

• Gas chromatography or sometimes known as Gas- Liquid Chromatography.

• It is a type of chromatography in which the mobile phase is a carrier gas, usually an inert
gas such as helium or an unreactive gas such as nitrogen, and the stationary phase is a
microscopic layer of liquid or polymer on an inert solid support, inside glass or metal
tubing, called a column. 
• Sample is heated rapidly and vaporized at injection port. The sample is transported
through the column by a mobile phase consisting of an inert gas. The sample components
are separated based on their boiling point and relative affinity for the stationary phase .
• The more affinity the component towards stationary phase, the slower it comes off the
column. The component are detected and presented as peaks on chromatogram
Types of chromatography
 Types of chromatography

High performance Liquid Chromatography

• HPLC involves a liquid sample being passes over a solid
adsorbent material packed into column using a flow of
liquid solvent under pressure.
• The interaction between the mobile and the stationary
phase leads to the separation of the mixture.
• The molecules having higher affinity remain adsorbed
for a longer time decreasing their speed of movement
through the column.
• However, the molecules with lower affinity move with a
faster movement, thus allowing the molecules to be
separated in different fractions.
 Types of chromatography
Gel Permeation/Gel Filtration/ Size Exclusion Chromatography

• The stationary column (the gel) typically consist of particle of a cross –linked
polyamide which contain pores.

• The solution containing molecules of different dimensions are passed

continuously with a constant flow rate through the column.

• Molecules larger than pores can not permeate into gel particles, and they are
retained between particles within a restricted area.

• Larger molecules pass through spaces between porous particles, and move
rapidly through inside the column.

• Molecules smaller than the pores are diffused into pores, and as molecules
get smaller, they leave the column with proportionally longer retention times
 Types of chromatography
• AFFINITY CHROMATOGRAPHY

• This chromatography technique is used for the purification of

enzymes, hormones, antibodies, nucleic acids, and specific
proteins.
• A ligand which can make a complex with specific protein
(dextran, polyacrylamide, cellulose etc) binds the filling material
of the column.
• The specific protein which makes a complex with the ligand is
attached to the solid support (matrix), and retained in the
column, while free proteins leave the column. Then the bound
protein leaves the column by means of changing its ionic
strength through alteration of pH or addition of a salt solution.
Thank You