Chromatography 1686504920

Chromatography
Presented By:
Muhammad Younus
Medical Laboratory Technology
Chromatography:
▪ Chromatography (Greek Chroma = Color and Graphein = To write)
▪ A collective term for a set of Lab techniques for the Separation of mixture
▪ Chromatography is a technique for separating mixtures into their

components in order to analyze, identify, purify, and/or quantify the
mixture or components.
Principle
• Chromatography usually consist of
1. Mobile Phase = A liquid or a gas which flows

through stationary phase & carries the
components of mixture with it.
2. Stationary Phase = The solid or liquid phase

of a chromatography system on which the
materials to be separated are selectively
adsorbed.
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
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 patient’s 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 of Chromatography
There are several types of Chromatography based on type of
equipment and based on type of principle. However, we will discuss
1. Paper Chromatography
2. Thin Layer Chromatography
3. Column Chromatography
4. Gas Chromatography
5. High Performance Liquid Chromatography (HPLC)
Paper Chromatography
• Paper chromatography is a technique that involve placing a
small dot on to a strip of chromatography paper
• Then the paper is placed in a jar containing a shallow layer of

solvent
• As the solvent rises through the paper, it meets the sample

mixture, which starts to travel up the paper with solvent
Principles of Paper Chromatography:
▪ Capillary Action – the movement of liquid within the spaces of a porous
material due to the forces of adhesion, cohesion, and surface tension. The
liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.
▪ Solubility – the degree to which a material (solute) dissolves into a solvent.

Solutes dissolve into solvents that have similar properties. (Like dissolves like)
This allows different solutes to be separated by different combinations of
solvents.
▪ Separation of components depends on both their solubility in the mobile phase

and their differential affinity to the mobile phase and the stationary phase.
Procedure:
▪ Cut strip of filter paper
▪ Draw a line 0.5cm above the bottom edge of the strip
with the pencil
▪ Draw another line of 0.5cm above the first line
▪ Place a spot from each pen on your second line
▪ Place the strips in the beakers
▪ Make sure the solution does not come above your
start line
▪ Keep the beakers covered
▪ Let strips develop until the ascending solution front is
about 2 cm from the top of the strip
▪ Remove the strips and let them dry
Rf Values=Distance traveled by solute/Distance traveled by solvent
❑ Rf Value helps for the identification of unknown

Thin Layer Chromatography (TLC)
TLC is a widely employed laboratory technique similar to paper

Chromatography
However, instead of using a stationary phase of paper, it involves

a stationary phase of thin layer of absorbent like silica gel,
alumina and cellulose
Compared to paper it has advantage of faster runs, better

separations, and the choice between different absorbents.
Thin Layer Chromatography (TLC)
• TLC is a widely employed laboratory technique similar to paper

Chromatography
• However, instead of using a stationary phase of paper, it

involves a stationary phase of thin layer of absorbent like silica
gel, alumina and cellulose
• Compared to paper it has advantage of faster runs, better

separations, and the choice between different absorbents
Column Chromatography
• It is the most widely used chromatography which find daily

use in research labs and industries. It is easy to operate and
less expensive technique
• As the name indicates, there is a lengthy column which is

suspended in the air with the help of a stand
• Into the column, the stationary phase is packed and mobile

phase is allowed to flow taking the sample through the solid
column.
Gas Chromatography
• Gas chromatography also sometimes known as Gas Liquid Chromatography (GLC), is

a separation technique in which the mobile phase is a Gas
• In Gas Chromatography a sample is rapidly heated and vaporized
• The sample is transported through the column by a mobile phase consisting of an
inert gas
• Sample components are separated based on their boiling points and relative affinity
for the stationary phase. The higher a component’s affinity for the stationary phase,
the slower in comes off
• The components are then detected and represented as peaks
High Performance Liquid Chromatography.
(HPLC)
• Liquid chromatography is a separation technique in which the mobile phase
is a liquid
• Liquid chromatography that generally utilizes very small packing particles
and a relatively high pressure is referred to as High Performance Liquid
Chromatography
• Since the chromatographic techniques are slow and time consuming, hence
the separation can be improved by using high pressure in the range of 5000-
10,000 psi (pounds per square inch
• In HPLC the sample is forced by a liquid (mobile phase) at high pressure
through a column that is packed with a stationary phase
• The interaction between the mobile phase and the stationary
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Chromatography

▪ Chromatography is a technique for separating mixtures into their

1. Mobile Phase = A liquid or a gas which flows

2. Stationary Phase = The solid or liquid phase

▪ Chromatography is used by scientists to:

• Analyze – examine a mixture, its components and their

• Pharmaceutical Company – determine amount of each

• Then the paper is placed in a jar containing a shallow layer of

• As the solvent rises through the paper, it meets the sample

▪ Solubility – the degree to which a material (solute) dissolves into a solvent.

▪ Separation of components depends on both their solubility in the mobile phase

❑ Rf Value helps for the identification of unknown

TLC is a widely employed laboratory technique similar to paper

However, instead of using a stationary phase of paper, it involves

Compared to paper it has advantage of faster runs, better

• TLC is a widely employed laboratory technique similar to paper

• However, instead of using a stationary phase of paper, it

• Compared to paper it has advantage of faster runs, better

• It is the most widely used chromatography which find daily

• As the name indicates, there is a lengthy column which is

• Into the column, the stationary phase is packed and mobile

• Gas chromatography also sometimes known as Gas Liquid Chromatography (GLC), is

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