Basics of Chromatography
CHROMATOGRAPHY : Analyze Identify Purify Quantify Separate
Components
Chromatography is a combination of two words; * Chromo – Meaning color *
Graphy – representation of something on paper
History of Chromatography
Chromatography, literally "color writing", was first employed by Russian
scientist Mikhail Tswett in 1903/1906. He continued to work with
chromatography in the first decade of the 20th century, primarily for the
separation of plant pigments such as chlorophyll, carotenes, and xanthophylls.
Since these components have different colors (green, orange, and
yellow,respectively) they gave the technique its name.
DEFINITIONIt is a physical separation method of separation in which the
components of a mixture are separated by differences in their distribution
between two phases, one of which is stationary (stationary phase) while the
other (mobile phase) moves through it in a definite direction. The substances
must interact with the stationary phase to be retained and separated by it.
Definition of chromatography
IUPAC definition (International Union of pure and applied Chemistry)
(1993):Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of which is
stationary while the other moves in a definite direction.The stationary phase
may be a solid, or a liquid supported on a solid or gel, the mobile phase may be
either a gas or a liquid.
Uses for Chromatography
Chromatography is used by scientists to:Analyze – examine a mixture, its
components, and their relations to one anotherIdentify – determine the identity
of a mixture or components based on known componentsPurify – separate
components in order to isolate one of interest for further studyQuantify –
determine the amount of the a mixture and/or the components present in the
sample
1. 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 of Column chromatography
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.
Figure: Column chromatography.
Steps of Column chromatography
The column is prepared by taking a glass tube that is dried and
coated with a thin, uniform layer of stationary phase (cellulose,
silica).
Then the sample is prepared by adding the mixture to the
mobile phase. The sample is introduced into the column from
the top and is allowed to pass the sample under the influence of
gravity.
The molecules bound to the column are separated by elution
technique where either solution of the same polarity is used
(isocratic technique), or different samples with different
polarities are used (gradient technique).
The separated molecules can further be analyzed for various
purposes.
Uses of Column chromatography
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.
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 of Gas chromatography
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.
Figure: Gas chromatography. Image Source: Bitesize Bio.
Steps of Gas chromatography
The sample is injected into the column where it is vaporized into
a gaseous state. The vapourised component than mixes with the
mobile phase to be carried through the rest of the column.
The column is set with the stationary phase where the molecules
are separated on the basis of their affinity to the stationary
phase.
The components of the mixture reach the detector at different
times due to differences in the time they are retained in the
column.
Uses of Gas chromatography
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.
Gas chromatography can also be used in forensic science to
identify and quantify various biological samples found in the
crime scene.
10. 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 of Ion exchange chromatography
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.
Figure: Ion exchange chromatography.
Steps of Ion exchange chromatography
A column packed with charged resin that can either be positively
charged or negatively charged is taken as the stationary phase.
The mixture with the charged particles is then passed down the
column where the charged molecules bind to the oppositely
charged resins.
If a cation exchange resin is used, the positively charged
molecules now bind to the cation exchange resin displacing the
negatively charged resin.
Similarly, if an anion exchange resin is used, the negatively
charged molecules bind to the anion exchange resin displacing
the positively charged resin.
Now an appropriate buffer is applied to the column to separate
the complex of charged exchange resins and the charged
molecules.
Uses of Ion exchange chromatography
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.
Paper chromatography
Paper chromatography is a separation technique where the
separation is performed on a specialized paper.
Principle of Paper chromatography
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, thus allowing the molecules to be separated in
different fractions.
The second type of paper chromatography is the paper partition
chromatography. It is based on the principle that the moisture
on the cellulose paper acts as a stationary phase for the
molecules moving with the mobile phase.
The separation of the molecules is thus based on how strongly
they adsorb onto the stationary phase.
An additional concept of ‘retention factor’ is applied during the
separation of molecules in the paper chromatography.
The retention value for a molecule is determined as a ratio of
distance traveled by the molecule to the distance traveled by
the mobile phase.
The retention value of different molecules can be used to
differentiate those molecules.
Figure: Paper chromatography. Image Source: Enyoh Christian
Ebere (Researchgate).
Steps of Paper chromatography
The stationary phase is selected as a fine quality cellulosic
paper.
Different combinations of organic and inorganic solvents are
taken as the mobile phase.
About 2-200 µl of the sample solution is injected at the baseline
of the paper, and it is allowed to air dry.
The sample loaded paper is then carefully dipped into the
mobile phase not more than the height of 1 cm.
After the mobile phase reaches near the edge of the paper, the
paper is taken out.
The retention factor is calculated, and the separated
components are detected by different techniques.
Uses of Paper chromatography
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 beverages.
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.
Thin-layer chromatography (TLC)
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 of Thin-layer chromatography
(TLC)
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.
Figure: Thin-layer chromatography (TLC).
Steps of Thin-layer chromatography (TLC)
The stationary phase is uniformly applied on the solid support
(glass, thin plate or aluminum foil) and dried.
The sample is injected as spots on the stationary phase about 1
cm above the edge of the plate.
The sample loaded plate is then carefully dipped into the mobile
phase not more than the height of 1 cm.
After the mobile phase reaches near the edge of the plate, the
plate is taken out.
The retention factor is calculated as in paper chromatography,
and the separated components are detected by different
techniques.
Uses of Thin-layer chromatography (TLC)
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.
