Chromatography Techniques
Techniques
of chromatography
Column chromatography
Column chromatography is a separation technique in which the stationary bed
is within a tube. The particles of the solid stationary phase or the support
coated with a liquid stationary phase may fill the whole inside volume of the
tube or be concentrated on or along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the middle part of the tube.
Differences in rates of movement through the medium are calculated to different
retention times of the sample
Planar chromatography
Planar chromatography- It is a separation technique in which the
stationary phase is present as or on a plane. The plane can be a paper, serving
as such or impregnated by a substance as the stationary bed or a layer of solid
particles spread on a support such as a glass plate. Different compounds in the
sample mixture travel different distances according to how strongly they
interact with the stationary phase as compared to the mobile phase.
Thin layer
chromatography
Thin layer chromatography (TLC) is a widely employed laboratory technique and
is similar to paper chromatography. However, instead of using a stationary phase
of paper, it involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper,
it has the advantage of faster runs, better separations, and the choice between
different adsorbents. For even better resolution and to allow for
quantification, high-performance TLC can be used.
Paper chromatography
Paper chromatography is a technique that involves placing a small dot or line
of sample solution onto 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 starts to travel up
the paper with the solvent. This paper is made of cellulose, a polar substance,
and the compounds within the mixture travel farther if they are non-polar. More
polar substances bond with the cellulose paper more quickly, and therefore do
not travel as far. Techniques
by physical state of mobile phase
Gas chromatography
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC),
is a separation technique in which the mobile phase is a gas. Gas chromatography
is always carried out in a column, which is typically "packed" or
"capillary" Gas chromatography (GC) is based on a partition equilibrium of
analyte between a solid stationary phase (often a liquid silicone-based
material) and a mobile gas (most often Helium). The stationary phase is adhered
to the inside of a small-diameter glass tube (a capillary column) or a solid
matrix inside a larger metal tube (a packed column).
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile
phase is a liquid. Liquid chromatography can be carried out either in a column
or a plane. Present day liquid chromatography that generally utilizes very small
packing particles and a relatively high pressure is referred to as high
performance liquid chromatography (HPLC).
In the HPLC technique, the sample is forced through a column that is packed
with irregularly or spherically shaped particles or a porous monolithic layer
(stationary phase) by a liquid (mobile phase) at high pressure. HPLC is
historically divided into two different sub-classes based on the polarity of the
mobile and stationary phases.
Techniques
by separation mechanism
Ion exchange
chromatography
Ion exchange chromatography uses ion exchange mechanism to separate analytes.
It is usually performed in columns but can also be useful in planar mode. Ion
exchange chromatography uses a charged stationary phase to separate charged
compounds including amino acids, peptides, and proteins. In conventional methods
the stationary phase is an ion exchange resin that carries charged functional
groups which interact with oppositely charged groups of the compound to be
retained. Ion exchange chromatography is commonly used to purify proteins using
FPLC.
Size exclusion
chromatography
Size exclusion chromatography (SEC) is also known as gel permeation
chromatography (GPC) or gel filtration chromatography and separates
molecules according to their size (or more accurately according to their
hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to
enter the pores of the media and, therefore, take longer to elute, whereas
larger molecules are excluded from the pores and elute faster. It is generally a
low-resolution chromatography technique and thus it is often reserved for the
final, "polishing" step of a purification. It is also useful for
determining the tertiary structure and quaternary structure of purified
proteins, especially since it can be carried out under native solution
conditions.
Reference from wikipedia
Posted by Student of Pharmacy BMEF college of Surat.
BHAVESH(Silent Killer)
of chromatography
Column chromatography
Column chromatography is a separation technique in which the stationary bed
is within a tube. The particles of the solid stationary phase or the support
coated with a liquid stationary phase may fill the whole inside volume of the
tube or be concentrated on or along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the middle part of the tube.
Differences in rates of movement through the medium are calculated to different
retention times of the sample
Planar chromatography
Planar chromatography- It is a separation technique in which the
stationary phase is present as or on a plane. The plane can be a paper, serving
as such or impregnated by a substance as the stationary bed or a layer of solid
particles spread on a support such as a glass plate. Different compounds in the
sample mixture travel different distances according to how strongly they
interact with the stationary phase as compared to the mobile phase.
Thin layer
chromatography
Thin layer chromatography (TLC) is a widely employed laboratory technique and
is similar to paper chromatography. However, instead of using a stationary phase
of paper, it involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper,
it has the advantage of faster runs, better separations, and the choice between
different adsorbents. For even better resolution and to allow for
quantification, high-performance TLC can be used.
Paper chromatography
Paper chromatography is a technique that involves placing a small dot or line
of sample solution onto 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 starts to travel up
the paper with the solvent. This paper is made of cellulose, a polar substance,
and the compounds within the mixture travel farther if they are non-polar. More
polar substances bond with the cellulose paper more quickly, and therefore do
not travel as far. Techniques
by physical state of mobile phase
Gas chromatography
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC),
is a separation technique in which the mobile phase is a gas. Gas chromatography
is always carried out in a column, which is typically "packed" or
"capillary" Gas chromatography (GC) is based on a partition equilibrium of
analyte between a solid stationary phase (often a liquid silicone-based
material) and a mobile gas (most often Helium). The stationary phase is adhered
to the inside of a small-diameter glass tube (a capillary column) or a solid
matrix inside a larger metal tube (a packed column).
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile
phase is a liquid. Liquid chromatography can be carried out either in a column
or a plane. Present day liquid chromatography that generally utilizes very small
packing particles and a relatively high pressure is referred to as high
performance liquid chromatography (HPLC).
In the HPLC technique, the sample is forced through a column that is packed
with irregularly or spherically shaped particles or a porous monolithic layer
(stationary phase) by a liquid (mobile phase) at high pressure. HPLC is
historically divided into two different sub-classes based on the polarity of the
mobile and stationary phases.
Techniques
by separation mechanism
Ion exchange
chromatography
Ion exchange chromatography uses ion exchange mechanism to separate analytes.
It is usually performed in columns but can also be useful in planar mode. Ion
exchange chromatography uses a charged stationary phase to separate charged
compounds including amino acids, peptides, and proteins. In conventional methods
the stationary phase is an ion exchange resin that carries charged functional
groups which interact with oppositely charged groups of the compound to be
retained. Ion exchange chromatography is commonly used to purify proteins using
FPLC.
Size exclusion
chromatography
Size exclusion chromatography (SEC) is also known as gel permeation
chromatography (GPC) or gel filtration chromatography and separates
molecules according to their size (or more accurately according to their
hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to
enter the pores of the media and, therefore, take longer to elute, whereas
larger molecules are excluded from the pores and elute faster. It is generally a
low-resolution chromatography technique and thus it is often reserved for the
final, "polishing" step of a purification. It is also useful for
determining the tertiary structure and quaternary structure of purified
proteins, especially since it can be carried out under native solution
conditions.
Reference from wikipedia
Posted by Student of Pharmacy BMEF college of Surat.
BHAVESH(Silent Killer)
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