Chromatography

Chromatography is an important biophysical technique that enables the separation, identification,

and purification of the components of a mixture for qualitative and quantitative analysis.
There are two essential components (phases) of all chromatography techniques.
Stationary phase
The stationary phase in chromatography is the phase that is either a solid or liquid particle
attached to a glass or a metal surface on which the components of the mixture to be separated is
absorbed selectively.
Mobile phase
The mobile phase in chromatography is the phase that is either liquid or gas that is passed
through a chromatographic system where the components of the mixture are separated at
different rates by adsorbing them to the stationary phase.
Types of Chromatography
• Paper chromatography
• Column chromatography
• Liquid chromatography
• Thin-layer chromatography
• Ion exchange chromatography
• Affinity chromatography
• Anion exchange chromatography
• Cation exchange chromatography
• Flash chromatography
• Gas chromatography
• Gel-filtration/ permeation chromatography
• High-performance liquid chromatography
• Hydrophobic interaction chromatography
• Reverse-phase chromatography
Paper chromatography
Paper chromatography is a separation technique where the separation is performed on a
specialized paper.
 Principle
• Paper chromatography is of two types based on two different principles.
• The first is the paper adsorption chromatography that is based on the varying degree of
interaction between the molecules and the stationary phase.
• The molecules having higher affinity remain adsorbed for a longer time decreasing their speed
of movement through the column.
• However, the molecules with lower affinity move with a faster movement.

Applications
• Paper chromatography is performed to detect the purity of various pharmaceutical products.
• It can also be employed to detect contamination in various samples, like food and drinks.
• This method can also be used for the separation of impurities from various industrial products.
• The analysis of the reaction mixtures in chemical labs is also conducted via paper
chromatography.
Column chromatography
Column chromatography is the separation technique where the components in a mixture are
separated on the basis of their differential adsorption with the stationary phase, resulting in them
moving at different speeds when passed through a column.

It is a solid-liquid chromatography technique in which the stationary phase is a solid & mobile
phase is a liquid or gas.

Principle
• This technique is based on the principle of differential adsorption where different molecules in
a mixture have different affinities with the absorbent present on the stationary phase.
• The molecules having higher affinity remain adsorbed for a longer time decreasing their speed
of movement through the column.
• However, the molecules with lower affinity move with a faster movement, thus allowing the
molecules to be separated in different fractions.
• Here, the stationary phase in the column chromatography also termed the absorbent, is a solid
(mostly silica) and the mobile phase is a liquid that allows the molecules to move through the
column smoothly.

Applications
• Column chromatography is routinely used for the separation of impurities and purification of
various biological mixtures.
• This technique can also be used for the isolation of active molecules and metabolites from
various samples.
• Column chromatography is increasingly used for the detection of drugs in crude extracts.
Liquid chromatography
Liquid chromatography is a separation technique where the mobile phase used is liquid, and the
separation can take place either in a column or a plain surface.
 Principle
• The process of liquid chromatography is based on the principle for the affinity of the molecules
to the mobile phase.
• If the components to be separated have a higher affinity to the mobile phase, the molecules
move along with the mobile phase and come out of the column faster.
• However, if the components have a lower degree of interaction with the mobile phase, the
molecules move slowly and thus come out of the column later.
• Thus, if two molecules in a mixture have different polarities and the mobile phase is of a
distinct polarity, the two molecules will move at different speeds through the stationary phase.

Applications
• Liquid chromatography is an effective method for the separation of a colored solution as they
form two separate bands after separation.
• This method can also be used over other techniques as it is quite simple and less expensive.
• It can be used for the separation of solid molecules that are insoluble in water.
Thin-layer chromatography

Thin-layer chromatography is a separation technique where the stationary phase is applied as a

thin layer on a solid support plate with a liquid mobile phase.

Principle
• This chromatography technique is based on the principle that components of a mixture are
separated when the component having an affinity towards the stationary phase binds to the
stationary phase. In contrast, other components are eluted with the mobile phase.
• The substrate/ ligand is bound to the stationary phase so that the reactive sites for the binding
of components are exposed.
• Now, the mixture is passed through the mobile phase where the components with binding sites
for the substrate bind to the substrate on the stationary phase while the rest of the components
are eluted out with the mobile phase.
• After separation, the molecules are seen as spots at a different location throughout the
stationary phase.
• The detection of molecules is performed by various techniques.

Applications
• Thin-layer chromatography is routinely performed in laboratories to identify different
substances present in a mixture.
• This technique helps in the analysis of fibers in forensics.
• TLC also allows the assay of various pharmaceutical products.
• It aids in the identification of medicinal plants and their composition.

Affinity chromatography
Affinity chromatography is a separation technique where the components of a mixture are
separated based on their affinity towards the stationary phase of the system.

Principle

• This chromatography technique is based on the principle that components of a mixture are
separated when the element having an affinity towards the stationary phase binds to the
stationary phase. In contrast, other components are eluted with the mobile phase.
• The substrate/ ligand is bound to the stationary phase so that the reactive sites for the binding
of components are exposed.
• Now, the mixture is passed through the mobile phase where the components with binding sites
for the substrate bind to the substrate on the stationary phase while the rest of the components
are eluted out with the mobile phase.
• The components attached to the stationary phase are then eluted by changing the pH, ionic
strength, or other conditions.

Applications
• It is used as a staple separation technique from enzymes and other proteins.
• This principle is also applied in the in vitro antigen-antibody reactions.
• This technique is used for the separation of components as well as the removal of impurities
from a mixture.
• can be used in the detection of mutation and nucleotide polymorphisms in nucleic acids.
Ion exchange chromatography
Ion exchange chromatography is the separation technique for charged molecules by their
interaction with the oppositely charged stationary phase in the form of ion-exchange resin.

Principle
• This technique is based on the principle of attraction of charged resin and the oppositely
charged analyte. Here the exchange of negatively/ positively charged ions takes place to
remove the charged molecules.
• The stationary phase is first coated with particular charges where the components of the
mixture with opposite charges will bind.
• A cation or anion exchange resin with a higher affinity to the charged components then binds
the components, displacing the oppositely charged resin.
• The cation or anion exchange resin-component complex then is removed by using different
buffers.

Applications
• Ion exchange chromatography is used in the purification of water where the positively charged
ions are replaced by hydrogen ions, and the negatively charged ions are replaced by hydroxyl
ions.
• This method also works as an effective method for the analysis of the products formed after
hydrolysis of nucleic acids.
• The separation of metals and other inorganic compounds is also facilitated by the ion-exchange
chromatography.
Anion exchange chromatography
Anion exchange chromatography is the separation technique for negatively charged molecules
by their interaction with the positively charged stationary phase in the form of ion-exchange
resin.

Principle
• This technique is based on the principle of attraction of positively charged resin and the
negatively charged analyte. Here the exchange of positively charged ions takes place to
remove the negatively charged molecules.
• The stationary phase is first coated with positive charges where the components of the mixture
with negative charges will bind.
• An anion exchange resin with a higher affinity to the negatively charged components then
binds the components, displacing the positively charged resin.
• The anion exchange resin-component complex then is removed by using different buffers.
Application
• Itis used to separate proteins and amino acids from their mixtures.
• Negatively charged nucleic acids can be separated, which helps in further analysis of the
nucleic acids.
• This method can also be used for water purification where the anions are exchanged for
hydroxyl ions.
• It can be used for the separation of metals as they usually have negatively charged complexes
that are bound to the anion exchangers.
Cation exchange chromatography
Cation exchange chromatography is the separation technique for positively charged molecules by
their interaction with negatively charged stationary phase in the form of ion-exchange resin.
Principle
• This technique is based on the principle of attraction of negatively charged resin and the
positively charged analyte. Here the exchange of negatively charged ions takes place to
remove the positively charged molecules.
• The stationary phase is first coated with negative charges where the components of the mixture
with positive charges will bind.
• A cation exchange resin with a higher affinity to the positively charged components then binds
the components, displacing the negatively charged resin.
• The cation exchange resin-component complex then is removed by using different buffers.
Applications
• It is used for the analysis of the products obtained after the hydrolysis of nucleic acids.
• This can also be used for the separation of metals where the metal ions themselves bind to the
negatively charged resins to remove the negatively charged complexes.
• It helps in purification of water by exchanging the positively charged ion by the hydrogen ions.
• It is also used to analyze the rocks and other inorganic molecules.
Flash chromatography
Flash chromatography is a separation technique where smaller sizes of gel particles are used as
stationary phase, and pressurized gas is used to drive the solvent through the column.

Principle
• The principle of flash chromatography is similar to that of column chromatography, where the
components are separated on the basis of their differential adsorption to the stationary phase.
• The sample applied is passed by using a pressurized gas that makes the process faster and more
efficient.
• Molecules bind to the stationary phase on the basis of their affinity while the rest of the solvent
is eluted out by applying the pressured gas which quickens the process.
• Here, the stationary phase is solid, the mobile phase and the elution solution are liquid, and an
additional pressurized gas is used.
Applications
• Flash chromatography is used as a rapid and more efficient method of separation of
components of different mixtures.
• It is used for the removal of impurities from crude extracts of natural and synthetic mixtures.
Gas chromatography
Gas chromatography is a separation technique in which the molecules are separated on the basis
of their retention time depending on the affinity of the molecules to the stationary phase.The
sample is either liquid or gas that is vaporized in the injection point.

Principle
• Gas chromatography is based on the principle that components having a higher affinity to the
stationary phase have a higher retention time as they take a longer time to come out of the
column.
• However, the components having a higher affinity to the stationary phase have less retention
time as they move along with the mobile phase.
• The mobile phase is a gas, mostly helium, that carries the sample through the column.
• The sample once injected in converted into the vapor stage is then passed through a detector to
determine the retention time.
• The components are collected separately as they come out of the stationary phase at different
times.

Applications
• This technique is used to calculate the concentration of different chemicals in various samples.
• This is used in the analysis of air pollutants, oil spills, and other samples.
• It can also be used in forensic science to identify and quantify various biological samples
found in the crime scene.
Gel-filtration/ permeation chromatography
Gel-filtration chromatography is a form of partition chromatography used to separate molecules
of different molecular sizes.

This technique has also frequently been referred to by various other names, including gel-
permeation, gel-exclusion, size- exclusion, and molecular- sieve chromatography.

Principle
• Molecules are partitioned between a mobile phase and a stationary phase as a function of their
relative sizes.
• The stationary phase is a matrix of porous polymer which have pores of specific sizes.
• When the sample is injected with the mobile phase, the mobile phase occupies the pores of the
stationary phase.
• If the size of the molecules is appropriate enough to enter the pores, they remain in the pores
partly or wholly.
• However, molecules with a larger size are retained from entering the pores, causing them to be
moved with the mobile phase, out of the column.
• If the mobile phase used in an aqueous solution, the process is termed gel filtration
chromatography.
• If the mobile phase used is an organic solvent, it is termed as gel permeation chromatography.

Applications

• One of the principal advantages of gel-filtration chromatography is that separation can be

performed under conditions specifically designed to maintain the stability and activity of the
molecule of interest without compromising resolution.
• The absence of a molecule-matrix binding step also prevents unnecessary damage to fragile
molecules, ensuring that gel-filtration separations generally give high recoveries of activity.
• Because of its unique mode of separation, gel-filtration chromatography has been used
successfully in the purification of proteins and peptides from various sources.
• Gel-filtration chromatography has been used to separate various nucleic acid species such as
DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine, cytosine,
and uracil.
High-performance liquid chromatography
High-performance liquid chromatography is a modified form of column chromatography where the
components of a mixture are separated on the basis of their affinity with the stationary phase.
High-performance liquid chromatography is a modified form of column chromatography where
the components of a mixture are separated on the basis of their affinity with the stationary phase.

Principle
• This technique is based on the principle of differential adsorption where different molecules in
a mixture have a varying degree of interactions with the absorbent present on the stationary
phase.
• The molecules having higher affinity remain adsorbed for a longer time decreasing their speed
of movement through the column.
• However, the molecules with lower affinity move with a faster movement, thus allowing the
molecules to be separated in different fractions.
• This process is slightly different from the column chromatography as in this case; the solvent is
forced under high pressures of up to 400 atmospheres instead of allowing it to drip down under
gravity.

Applications
• High-performance liquid chromatography is used in the analysis of pollutants present in
environmental samples.
• It is performed to maintain product purity and quality control of various industrial productions.
• This technique can also be used to separate different biological molecules like proteins and
nucleic acids.
• The increased speed of this technique makes the process faster and more effective.
Hydrophobic interaction chromatography

Hydrophobic interaction chromatography is the separation technique that separates molecules on

the basis of their degree of hydrophobicity.

Principle
• The principle of hydrophobic interaction chromatography is based on the interaction between
two molecules with hydrophobic groups.
• Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic
groups.
• The solvent molecules containing hydrophobic regions interact with the hydrophobic groups,
thus separating them from the molecules with hydrophilic groups.
• The interaction is then reversed by applying an elution solution with decreasing salt gradient,
which causes the molecules with hydrophobic groups to be separated from the stationary
phase.

Applications
• It is extremely important for the separation of proteins with hydrophobic groups.
• This technique is more appropriate than other methods, as this technique results in minimum
denaturation activities.
• Similarly, this method can also be applied to the separation of other organic compounds with
hydrophobic groups.
• This allows the separation of hydrophilic and hydrophobic biological molecules from each
other.
Reverse-phase chromatography
Reverse-phase chromatography is a liquid chromatography technique where the separation of
molecules is achieved through hydrophobic interaction between the liquid mobile phase and the
stationary phase.

Principle
• The principle of reverse phase chromatography is based on the interaction between two
molecules with hydrophobic groups.
• Here, the stationary phase is solid support applied with both hydrophobic and hydrophilic
groups.
• The solvent molecules containing hydrophobic regions interact with the hydrophobic groups,
thus separating them from the molecules with hydrophilic groups.
• The interaction is then reversed by applying an elution solution with decreasing salt gradient,
which causes the molecules with hydrophobic groups to be separated from the stationary
phase.

Applications
• Reverse chromatography, in combination with high-performance liquid chromatography, is
increasingly used for the separation of biomolecules.
• This is also used in the study of the analysis of drugs, metabolites, and active molecules.
• It can also be used to remove impurities from various environmental samples.