Assignment Analytical Chemistery BY Muhammad Pervaiz Roll Number 1039 Bs Chemistry 4 (M)

ASSIGNMENT
ANALYTICAL CHEMISTERY
BY
MUHAMMAD PERVAIZ
ROLL NUMBER 1039
BS CHEMISTRY 4th (M)
Chromatography
Introduction:
Chromatography is based on the principle where molecules in mixture applied onto the
surface or into the solid, and fluid stationary phase (stable phase) is separating from each other
while moving with the aid of a mobile phase. The factors effective on this separation process
include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and
affinity or differences among their molecular weight. Because of these differences, some
components of the mixture stay longer in the stationary phase, and they move slowly in the
chromatography system, while others pass rapidly, and leave the system faster.
The type of interaction between stationary phase, mobile phase, and substances contained in
the mixture is the basic component effective on separation of molecules from each other.
Chromatography methods based on partition are very effective on separation, and identification
of small molecules as amino acids, carbohydrates, and fatty acids.
Components:
Based on this approach three components form the basis of the chromatography technique.
 Stationary phase: This phase is always composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface a solid support”.
 Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
 Separated molecules
Types:
 Column chromatography
 Ion-exchange chromatography
 Gel-permeation (molecular sieve) chromatography
 Affinity chromatography
 Paper chromatography
 Thin-layer chromatography
 Gas chromatography
Column chromatography
Since proteins have difference characteristic features as size, shape, net charge, stationary
phase used, and binding capacity, each one of these characteristic components can be purified
using chromatographic methods. Among these methods, most frequently column
chromatography is applied. This technique is used for the purification of biomolecules. On a
column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are
applied. Their flow through inside column material placed on a fiberglass support is ensured. The
samples are accumulated at the bottom of the device in a time-, and volume-dependent manner.
Ion- exchange chromatography

Ion- exchange chromatography is based on electrostatic interactions between charged
protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the
protein to be separated, and the affinity of the protein to the column is achieved with ionic ties.
Proteins are separated from the column either by changing pH, concentration of ion salts or ionic
strength of buffer solution. Positively charged ion- exchange matrices are called anion-exchange
matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged
groups are known as cation-exchange matrices, and adsorb positively charged proteins.
Affinity chromatography
This chromatography technique is used for the purification of enzymes, hormones,
antibodies, nucleic acids, and specific protein. 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.
Paper chromatography
In paper chromatography support material consists of the layer of cellulose highly
saturated with water. In this method thick filter paper comprised the support, and water drops
settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate
fluid placed in a developing tank. Paper chromatography is “liquid-liquid” chromatography.
Thin-layer chromatography
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method
stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all
solid substances used. In column chromatography (alumina, silica gel, and cellulose) can be
utilized. In this method, the mobile phase travels upward through the stationary phase the solvent
travels up the thin plate soaked with the solvent by means of capillary action. During this
procedure, it also drives the mixture priory dropped on the lower parts of the plate with a pipette
upwards with different flow rates. Thus the separation of analytes is achieved. This upward
travelling rate depends on polarity of material, solid phase, and of the solvent.
Gas chromatography
In this method stationary phase is a column which is placed in the device, and contains a
liquid stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography
is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N 2. Mobile phase
which is an inert gas is passed through a column under high pressure. The sample to be analyzed
is vaporized, and enters into a gaseous mobile phase. The components contained in the sample
are dispersed between mobile phase, and stationary phase on the solid support. Gas
chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the
extremely excellent separation of molecules. It is used in the separation of very little amounts of
analytes.

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Ion- exchange chromatography

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