- Uses and Application in Microscopy -
Essentially, chromatography is a versatile
method through which different kinds of chemical mixtures of substance can be separated. Here, the
word versatile is included in the definition because there are a number of
techniques that can be used to separate a chemical substance into its
individual components. Various chemical substances are made up of a number of
For instance, such substances as food colorings, plants dyes and inks contain several components. Using chromatograph techniques, it becomes possible to separate these components depending on the needs of the technician. Here, I will focus on different types of chromatography, their uses and application in microscopy.
Basically, separation of compounds is achieved by dissolving the mixture in a mobile phase and
passing it over a stationary phase. Here, the molecules that interact more
strongly with the stationary phase, with which they have greater affinity move
slowly through the resin while those that have a weak interaction move through
it much faster.
Ultimately, this results in the components within the substance
being separated. Chromatography may be used to either analyze or purify
molecules of a given substance. Therefore, there are two major categories of chromatography: Analytical
chromatography and Preparative chromatography.
Preparative Chromatography and Analytical Chromatography
Preparative chromatography is largely concerned
with the isolation and purification of given molecules within a substance.
It's therefore largely used for various purification
purposes as is the case with laboratory-scale protein purification in
biochemical characterization in the biopharmaceutical industry.
Analytical chromatography is different from
preparative chromatography in that the separation of molecules in a substance
is for the purposes of identifying and quantifying the components of the
substance. It therefore serves as the best technique for
observing what happens to a substrate in a chemical reaction or testing the
presence of a given substance or component of interest in a given mixture among
Before looking at the different types/techniques, it is important to know some of the terms used.
Mobile phase - Also referred to as the
carrier, the mobile phase refers to the solvent that moves
through the column
The stationary phase - The stationary phase is
also often referred to as the adsorbent and is the substance that remains fixed
in the column
Eluent and Eluate - The eluent
refers to the fluid that enters the column, the eluate is the fluid that
collects in flasks after exiting the column
Analyte - This is the mixture that
has been separated into individual components for analysis.
In chromatography (normal-phase) the stationary
phase is always hydrophilic in nature, meaning that it is polar while the mobile phase is non-polar which
means that it is hydrophilic. In some cases, technicians will use reverse-phase where the stationary phase is non-polar while the mobile phase
Although there are different types of chromatography that vary
depending on the type of stationary and mobile phase used, the basic principle
is the same. That is, differential affinities of different components in the
substance towards the stationary and mobile phases causes differential
separation of the components.
The following are some of the most common techniques:
This is one of the most common types. Paper chromatography is an analytical method used for the purposes of separating colored constituents in a substance. With
paper chromatography, the stationary phase is typically solid cellulose while
the mobile phase is liquid.
With paper chromatography, the paper (cellulose
paper) is typically suspended in a container that contains a shallow layer of
the solvent (or in some cases a mixture of solvents). A line with spots is made
near the bottom of the paper (spots with the substance) and the solvent has to
be just below this line. As the solvent slowly travels up the paper, different
constituents of the substance (in the spots) also travel up the paper at
different rates as they separate. After the separation, the different
components of the compound can be seen directly on the cellulose
The distance travelled relative to the solvent
is referred to as the Rf value. For different compounds, this may be worked out
using the following formula
Rf = distance travelled by the compound/distance travelled by the solvent
This is also used to identify the type of components.
Some of the main uses of paper chromatography
- Qualitative method to
identify components of a mixture
- Crime scene investigation
and DNA/RNA sequencing
- In analytical chemistry to
identify and separate coloured mixtures.
- In scientific studies to
identify unknown organic and inorganic compounds from a mixture.
Thin Layer Chromatography
This technique is a type of planar
chromatography where the stationary phase is on a flat plate while the mobile phase
travels through the stationary phase by capillary action. Thin layer
chromatography is also a qualitative analytical chromatography method that is
commonly used for the purposes of separating nonvolatile molecules.
This technique uses solid silica or
alumina for the solid phase and a mobile liquid phase (such as cyan). The
substance of interest is separated on the basis of the polarity of the
molecules. Unlike paper chromatography, glass is coated with a thin layer of
silica in thin layer chromatography on which the compound is spotted for
separation. As is the case with paper chromatography, the solvent travels up
the plate through capillary action along with the analyte as its components are
Uses of TLC
Thin layer chromatography can be used for:
- Determine the number of components
in a given mixture
- To monitor reaction
- To compare compounds
- To determine the
effectiveness of a separation achieved on a column
- To determine the
appropriate solvent for column chromatography
Liquid Column Chromatography
Liquid column chromatography also uses solid
silica or alumina for the stationary phase and a liquid phase. Unlike TLC,
liquid column chromatography uses microporous beads of silica. Liquid
chromatography may be used for either analytical or preparative applications.
As the mobile (liquid) phase travels through the column, components in the
mobile phase interact with the solid phase at varying degrees as the molecules
of interest get separated on the basis of their varying physiochemical
interactions with the mobile and stationary phases.
During the separation
process, the small molecules get trapped in the pores of the stationary phase
while the larger ones flow through the gaps between the beads and have very
small retention rates.
With this technique, there is no chemical or physical
interaction between the analyte and the stationary phase. Some of the main uses
of this technique include purifying individual chemical compounds from a
mixture of compounds and in preparative applications .
This technique is one of the most popular techniques used for the purification of proteins and other
charged molecules. It uses cationic/anionic resign for the solid phase and a
liquid. The separation here is based on the ionic charges of the molecules.
the cation exchange chromatography, the positively charged molecules are
usually attracted to the negatively charged solid support while in anion
exchange chromatography, the negatively charged molecules are usually attracted
to the positively charged solid support.
In this system, the mobile phase is generally a
low medium conductivity solution. Adsorption of molecules to the solid support
is driven by anionic interaction between the oppositely charged ionic groups in
the sample molecule and in the ligand on the support. Here, the strength of the
interaction is largely determined by the number of location of the charges on the
molecule and the functional group.
Overall, the molecules that process opposite
charge as the resin bind tightly to the resin while those that have the same
charge as the resin flow through the column and elute out first.
Because of its ability to separate molecules on
the basis of their total charge, Ion Exchange Chromatography allows for the
separation of similar types of molecules that would otherwise be difficult to
separate using the other techniques. For this reason, it is commonly used to
separate such biological molecules as:
- Amino acids
This technique is often used for:
- Separation of vitamins and
other biological compounds
- Water purification
- To determine the base
competition of nucleic acids
- For analysis of amino acids
Affinity chromatography is a type of
chromatograph that is used to separate given compounds on the basis of specific
binding interaction between immobilized ligand and the component in question.
For this technique, agarose or porous glass beads are used as the solid phase
where separation is based on the binding affinity of the analyte molecule to
the molecule immobilized on the stationary phase.
In case the molecule is a substrate for the
enzyme, then it binds tightly onto the enzyme while the unbound analyte passes
through in the mobile phase, eluting out of the column and leaving the
substrate bound to the enzyme. Using the right solvent, it is also possible to
detach the substrate from the stationary phase and elute it out of the column.
Some of the primary uses of Affinity
- The purification and
concentration of substances from a mixture in to a buffering solution
- To reduce the amount of
substance in a given mixture
- To purify and concentrate
- For discerning the type of
biological compounds that bind a given substrate
Gas chromatography is the technique that entails
the vaporization and injection of the sample in to the head of a
chromatographic column, which is then transported through the column by the
flow of the gaseous mobile phase. Here, the column contains a liquid stationary
phase that is adsorbed on to the surface of an inert solid. Inert gases like
argon or helium are preferred for this technique given that they are not
reactive and would therefore not react with the sample.
This technique works on the basis of the boiling
point of the molecules. Here, the sample has to be volatilized which results in
the molecule with the lowest boiling point coming out of the column first. On
the other hand, the molecule with the highest boiling point comes out last.
Gas chromatography is typically used in:
- Analysis of various body
fluids and secretions containing large amounts of organic volatiles
- Analysis of air samples
- To determine the components
of certain mixtures using retention time and abundance samples in
Chromatography and Microscopy
Recently, new techniques are being developed to
combine chromatic techniques with microscopy. While some of these processes may
be complicated, they are being designed to help scientists and technicians have
a better understanding of various material. One of the best examples of this is
gas chromatic-mass spectrometry and dispersion-relation fluorescence
Fluorescence microscopy has for a long time been
used for studying labeled structures like cells. With new advancements and
developments, this technique is now being used to supply greater spatial and
Whereas gas chromatography-mass spectrometry (GC-MS) is
typically used for the purposes of identifying the different substances in a
sample, fluorescence spectrometry is used to tag given molecules with a
flourophore whose movement helps
create spontaneous fluorescence intensity fluctuations, which can be examined
in order to measure governing mass transport dynamics. This
technique has been increasingly used to examine the molecular transport and
diffusion coefficients at fixed spatial scales.
With current advancements in fluorescent protein
and synthetic flourophore technology, fluorescent live-cell imaging is also
expected to play an important role to examine the localization, assembly and
the role of various components in such systems as the secretion system.
Therefore, microscopy, and particularly fluorescence microscopy can play a very
important role in chromatography where it can be used to not only observe
various components of given compounds, but also compare these components during
In general chromatography allows scientists and other technicians to
separate and analyze the various components of given compounds. By employing
microscopy techniques here, the analysis process is further enhanced given that
scientists and technician will be well able to compare the different samples of
these components for better analysis.
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E. M.; Ferrer, Imma (2003). Liquid chromatography/mass spectrometry, MS/MS and
time of flight MS: analysis of emerging contaminants. Columbus, OH: American Chemical