CHROMATOGRAPHY &
CENTRIFUGATION
INTRODUCTION
Analytical
chemists
have
few
tools
as
powerful
as
chromatography to measure distinct analytes in complex
samples. The power of chromatography comes from its
ability to separate a mixture of compounds, or “analytes”,
and determine their respective identity (chemical structure)
and concentration. And Centrifugation to collect cells, to
precipitate DNA, to purify virus particles, and to distinguish
subtle differences in the conformation of molecules.
LEARNING OBJECTIVES:
At the end of lesson the student will be able to:
 Define chromatography;
 Understand the process of chromatography ;
 Discuss
The
Different
Types
of
Chromatography;
 Explain centrifugation
 Determine
the
different
types
of
centrifugation
What is Chromatography?
Derived from the Greek word Chroma
meaning colour, chromatography provides
a way to identify unknown compounds
and separate mixtures
How it works
In all chromatography there is a mobile phase and a stationary phase.
The stationary phase is the phase that doesn't move and the mobile
phase is the phase that does move. The mobile phase moves through the
stationary phase picking up the compounds to be tested. As the mobile
phase continues to travel through the stationary phase it takes the
compounds with it. At different points in the stationary phase the
different components of the compound are going to be absorbed and are
going to stop moving with the mobile phase. This is how the results of
any chromatography are gotten, from the point at which the different
components of the compound stop moving and separate from the other
components.
High Performance Liquid
Chromatography
Used in the world to test water samples to look for
pollution in lakes and rivers. It is used to analyze
metal ions and organic compounds in solutions.
Liquid chromatography uses liquids which may
incorporate hydrophilic, insoluble molecules.
Gas Chromatography
Used in airports to detect bombs and is used is
forensics in many different ways. It is used to
analyze fibers on a persons body and also analyze
blood
found
at
a
crime
scene.
In
gas
chromatography helium is used to move a gaseous
mixture through a column of absorbent material.
Thin-layer Chromatography
Uses an absorbent material on flat glass or plastic
plates. This is a simple and rapid method to check
the purity of an organic compound. It is used to
detect pesticide or insecticide residues in food.
Thin-layer chromatography is also used in forensics
to analyze the dye composition of fibers.
Paper Chromatography
One
of
the
most
common
types
of
chromatography. It uses a strip of paper as
the stationary phase. Capillary action is used
to pull the solvents up through the paper and
separate the solutes.
C E N T R I F U G AT I O N
Centrifugation
01
is
a
technique
of
separating
substances which involves the application of
centrifugal force. The particles are separated from
a solution according to their size, shape, density,
the viscosity of the medium and rotor speed.
TYPES OF CENTRIFUGUTION
 Density gradient centrifugation
I. Rate zonal centrifugation
II. Isopynic or sedimentation equilibrium centrifugation
 Differential centrifugation
 Ultra centrifugation
 DENSITY GRADIENT CENTRIFUGATION
It allow separation of many or all components in a mixture and
allows for measurement to be made
There are two forms of Density gradient centrifugation :
Rate zonal centrifugation
Isopynic or sedimentation equilibrium centrifugation
 DENSITY GRADIENT CENTRIFUGATION
RATE ZONAL CENTRIFUGATION
In Rate zonal centrifugation the solution have a density gradient. The
sample has a density i.e. greater than all the layers in the solution.
The sample is applied in a thin zone at the top of the centrifuge tube
on a density gradient. Under centrifugal force, the particles will begin
sedimenting through the gradient.
RATE ZONAL CENTRIFUGATION
The particles will begin sedimenting in
separate zones according to their size
shape and density.
ISOPYNIC OR SEDIMENTATION EQUILIBRIUM CENTRIFUGATION
• In this type of centrifugation , the solution contains a greater range
of densities.
• The density gradient contains the whole range of densities of the
particles in the sample.
• Each particle will sediment only to the position in the centrifuge
tube at which the gradient density is equal to its own density.
ISOPYNIC OR SEDIMENTATION EQUILIBRIUM CENTRIFUGATION
In Isopycnic centrifugation separation of
particles occurs into zones on the basis
of their density differences, independent
of time.
DIFFERENTIAL CENTRIFUGATION
• Differential centrifugation (also known as differential velocity
centrifugation) is a very common procedure in biochemistry and cell
biology, which is used to separate organelles and other sub-
cellular particles based on their sedimentation rate.
ULTRACENTRIFUGATION
• Svedberg coined the term “ultracentrifuge". He was colloid
chemist.
• He used the ultracentrifuge to determine the MW and subunit
structure of hemoglobin , studies which changed the ideas
concerning the structure of proteins.
• The first commercial ultracentrifuge was produced in 1940 by
SPINCO.