Affinity chromatography

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Affinity chromatography
Affinity chromatography
Separation is a process to separate the substances from mixture. The separation techniques
are based on the chemical and physical properties of the substance such as chemical
reactivity, size, mass, shape etc.
Chromatography is one of the best techniques for the separation of the substances.
Chromatography is a method of separating and identifying various components in a mixture,
which are present in smal trace quantities.
There are several types of chromatography, one of which is paper chromatography. It is
extensively used in research and industry, to analyze and identify different dyes. In its
simplest form, it can be used to separate and identify the different dyes in black ink.
What is chromatography?
It is a technique in which the separation of molecules is based on molecular structure and
molecular composition. Two phases are involved in chromatography method. The one is
stationary phase and the other is mobile phase. The sample molecules show different
interactions with the stationary phase.
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The liquid is in the mobile phase which pumps through bed of particles while the particles are
in the stationary phase.
A mixture of the sample molecules is introduced into the liquid. The mixture of molecules is
migrated by the mobile phase through the stationary phase. Thus the separation is based on
the distribution of solute between mobile phase and the stationary phase.
Gas chromatography
This chromatography is especial y used for analytical purpose. In the gas chromatography, the
carrier gas is worked as mobile phase which have an inert gas. The stationary phase is a
packed column. General y gas-liquid
chromatography is used. It involves a vaporized sample is injected into the chromatographic
The sample is migrated through the column with the flow of carrier gas. The liquid stationary
phase is adsorbed on the surface of the inert solid. The separation method can be affected by
the polarity of stationary phase, temperature, carrier gas flow, length of column, material
amount etc.
Gas Chromatography Equipment :-
Carrier gas :- The chemically inert gas is used as the carrier gas. General y nitrogen, helium,
argon, and carbon dioxide are used as carrier gas. It depends on the type of detector. The
carrier gas system also has a molecular sieve for removing water and impurities.
Sample injection port :- The sample should not too large or to small to introduced onto the
column. It may be reason for band broadening and resolution loss. General y micro syringe is
used for a proper injection of sample. The sample is injected through a rubber septum into a
flash vaporizer port at the head of the column.
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Column temperature :- The column temperature depends on the boiling point of the sample.
The temperature should be controlled in the range of tenths of a degree.
Detectors- Various types of detectors can be used in gas chromatography. Some are non-
selective detector, specific detector, concentration dependent detector, mass flow dependent
detector etc. the commonly used detector is flame ionization detector.
Procedure :- The detector is attached from the column. So the effluent enters in detector from
the column and mix with gas of detector (hydrogen and air). The effluent gets ignited under
the influence of flame ignition coil.
The compound burns in the flame and produces ions and electrons. These ions and electrons
can conduct electricity with the flame. A col ector electrode is located above the flame. The
current due to ionization of organic compounds is measured in recorder.
Paper chromatography :- A piece of specialized paper is used paper chromatography. It is a
planar system in which cel ulose filter paper is act as stationary phase. The separation of
compounds occurs on stationary phase. This is used for separation of amino acids, for
detecting organic compounds, biomolecules, Harmons and drugs etc.
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