Chromatography:
the process at which a chemical mixture carried by a liquid or gas is separated
into components because of differential distribution of the solutes as they
flow around or over a stationary liquid or solid phase. To many this may sound
like complete non-sense but other in the science world many knows how important
this procedure is in this field of study. Chromatography has developed into a
crucial laboratory tool for the separation and identification of compounds. Separation
of compounds in chemicals is essential in any chemical analysis. Samples must
first be simplified as much as it could possibly break down to its most simple
components. This now allows the researcher to be able to characterize ad
identify each of its parts.

The
word Chromatography translates to Chroma and
Graphein, which literally means to
write with colors. Chromatography was discovered by a Botanist names Mikhail
Tswett. In the year of 1901 this Russian scientist produced a separation of colorful
pigments of a plant through a column of calcium carbonate. Tswett recognized
the physiochemical basis of the separation and applied to the separation of
pigments, the carotenoids and the chlorophylls. What Tswett did was pack a vertical
glass column with an adsorptive material, like powdered sugar, then he added a
solution of the plant pigments to the top of the column. He then washed the
pigments through the column with an organic solvent. The different pigments separated
into a series of discrete colored band on the column, which later became
chromatography method that we know and still use today. It wasn’t until 1931,
when other scientist got a hold unto Tswett’s method. This was because during
this century he published his findings in strictly German or Russian Journal
articles. In 1931 a German chemist known as Richard Kuhn and a French Chemist
known as Edgar Lederer, reported the use of the chromatography method in multiple
important biological publications. It wasn’t until Almost 4 decades later that
chromatography finally got the recognition that it deserved. Two British
Chemist named, Archer J.P. martin and Richard L.M. Synge, first came up with failed
method of Chromatography called liquid-liquid countercurrent distribution. This
method failed to give them the sufficient enough separation of compounds. They
then came up with a similar idea that Tswett discovered. They both tried breaking
down compounds by their electrical charges, by being able to see this physical
movement with in some type of medium that allows the charges to flow apart. The
main difference between Tswett’s method and the Brit’s method was the type of
medium they used in the cylinder. This Brits used a clear water-like gel medium,
silica gel. Surprisingly their method was very successful and innovative for
using a water and a silica gel. With the result the solute molecules
partitioned between stationary liquid and a separate mobile liquid phase.
Martian and Synge were the chemist who were awarded the Noble Prize for their research.
They didn’t receive the award for
discovering chromatography method, but for their technique with the silica liquid.

So how
does Chromatography work exactly? A great way to visualize what is does, just
think about what happens to ink on wet paper. The ink particles separate into
smaller ones throughout the water and you can physically see this separation on
the white paper. Chromatography is a surface effect. Surface Effect is also
known as hydrophobic effect, which means something into a surface effect
substance is “water fearing”, which will literally repel anything with water or
with anything containing the H20 molecule. As the liquid starts
to move past the solid, some of its molecules are sucked toward the surface of the solid
and stick there temporarily before being pulled back again into the
liquid they came from. This exchange of molecules between the surface of the
solid and the liquid is a kind of adhesive or gluing effect called adsorption. Now remember that our
liquid is a mixture of quite a few different liquids. Each one undergoes
adsorption in a slightly different way and spends time in either the solid or
the liquid phase. One of the liquids might spend much longer in the solid phase
than in the liquid, so it would travel more slowly over the solid; another one
might spend less time in the solid and more in the liquid, so it would go a bit
faster. Another way of looking at it is to think of the liquid as a mixture of
glue-like liquids, some of which stick more to the solid than others. This is
what causes the different liquids within our original liquid mixture to spread
out on the solid. or chromatography to work
effectively, we obviously need the components of the mobile phase to separate
out as much as possible as they move past the stationary phase. That’s why the
stationary phase is often something with a large surface area, such as a
sheet of filter paper, a solid made of finely divided particles, a liquid
deposited on the surface of a solid, or some other highly adsorbent
material.         

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            Since all researchers are different, just as every
molecular compound is different, there are many alternate was to use liquid
chromatography to separate compounds. Usually the type of Chromatography
depends on the physiochemical characteristics of the molecule being broken down.
Here are a couple ways one would decipher which technique to implement.

§  Specific
binding interactions (affinity chromatography)

§  Charge
(ion exchange chromatography)

§  Size
(size exclusion chromatography/gel filtration chromatography)

§  Hydrophobic
surface area (hydrophobic interaction chromatography and reverse phase
chromatography)

§  Multiple
properties (multimodal or mixed-mode chromatography)

 

 

Affinity
Chromatography

Affinity
chromatography is one of the most diverse and powerful chromatographic methods
for purification of a specific molecule or a group of molecules from complex
mixtures. It is based on highly specific biological interactions between two
molecules, such as interactions between enzyme and substrate, receptor and
ligand, or antibody and antigen. These interactions, which are typically
reversible, are used for purification by placing one of the interacting
molecules, referred to as affinity ligand, onto a solid matrix to create a
stationary phase while the target molecule is in the mobile phase. Successful
affinity purification requires a certain degree of knowledge and understanding
of the nature of interactions between the target molecule and the ligand to
help determine the selection of an appropriate affinity ligand and purification
procedure. 

 

Ion-Exchange Chromatography

Ion Exchange Chromatography relies on charge-charge interactions
between the proteins in your sample and the charges immobilized on the resin of
your choice. Ion exchange chromatography can be subdivided into cation exchange
chromatography, in which positively charged ions bind to a negatively charged
resin; and anion exchange chromatography, in which the binding ions are
negative, and the immobilized functional group is positive. Once the solutes
are bound, the column is washed to equilibrate it in your starting buffer,
which should be of low ionic strength, then the bound molecules are eluted off
using a gradient of a second buffer which steadily increases the ionic strength
of the eluent solution. Alternatively, the pH of the eluent buffer can be
modified as to give your protein or the matrix a charge at which they will not
interact and your molecule of interest elutes from the resin. If you know the
pH you want to run at and need to decide what type of ion exchange to use paste
your protein sequence into the titration curve generator. If it is negatively charged at the pH you wish, use an anion exchanger;
if it is positive, use a cation exchanger. Of course, this means that your
protein will be binding under the conditions you choose. In many cases, it may
be more advantageous to select conditions at which your protein will flow
through while the contaminants will bind. This mode of binding is often
referred to as “flow through mode”. 

 

Size Exclusion Chromatography/Gel Filtration Chromatography

SEC is a preparative, non-destructive analytical technique that permits
the separation of molecules by their size.  This is especially useful in
protein purification because while there may be many proteins in a sample,
their molecular weights can vary widely.  This allows one to separate a
mixture of proteins based on this wide size distribution.  It also
provides a simple procedure for removing salt (desalting) from a protein
sample. 

Molecules
smaller than the exclusion limit of the gel material will become trapped in the
gel beads.  Those of larger molecular weight will not be trapped but will
flow through the gel.  The larger molecules are retarded only by their
shape and their ease of passing by the beads.  Thus, larger molecules
elute first, while smaller molecules are held longer inside the beads and elute
last. In addition to the separation of a protein mixture based on size, gel filtration
chromatography also allows one to estimate the molecular weight of an unknown
globular protein.  

 

Hydrophobic Interaction Chromatography

Hydrophobic
interaction chromatography (HIC) is a milder form of reversed-phase liquid
chromatography (LC). Separation of analytes is based on hydrophobic
interactions with the stationary phase; therefore, the elution order in HIC
enables proteins to be ranked on the basis of their relative hydrophobicity.
HIC employs no denaturing conditions, does not require the use of organic
solvents or high temperatures, and separations are carried out at physiological
pH, which allows the preservation of protein structure. Historically HIC has
been used for the determination of the relative hydrophobicity of proteins and
was applied on a preparative scale for protein purification; it is now applied
at all stages of the purification process, including high-yield capture,
polishing monoclonal antibodies (mAbs), characterization of mAbs and
antibody–drug conjugates (ADCs), removal of truncated species from full-length
forms, separation of active and inactive forms, and clearing viruses.

Multimodal or mixed-mode Chromatography

Mixed-mode chromatography materials contain ligands of
multimodal functionality that allow protein adsorption by a combination of
ionic interactions, hydrogen bonds, and/or hydrophobic interactions. Complex
mixtures like fermentation supernatants or cell lysates can be applied directly
at relatively high conductivity, and elution is usually achieved by
electrostatic charge repulsion. We used mixed-mode materials for capturing and
intermediate purification of several recombinant therapeutic proteins from various
expression systems like yeast, Escherichia coli, and mammalian cells.
Product-related impurities as well as process-related impurities from
fermentation media were efficiently removed while the desired product was bound
with high selectivity. Because these purification protocols can be scaled up
easily to production scale, mixed-mode materials are being considered as
potential elements of a general purification platform for recombinant
therapeutic proteins produced in various expression systems.

Along with being a crucial role in the
science world for research and lab experiments, Chromatography is involved with
plenty of different aspects of our everyday life. Chromatography plays an important role in many pharmaceutical industries and also in the chemical and food industry. Environmental testing laboratories generally
want to identify for very small quantities of contaminants such as PCBs in
waste oil, and pesticides. The Environmental Protection Agency makes the method
of chromatography to test drinking water and to monitor air quality.
Pharmaceutical industries use this method both to prepare huge quantities of
extremely pure materials, and to analyze the purified compounds for trace
contaminants. The other applications of chromatography especially HPLC is used
in Protein Separation like Insulin Purification, Plasma Fractionation and Enzyme
Purification. These separation techniques like chromatography gain importance in
different kinds of companies, different departments like Fuel Industry,
biotechnology, biochemical processes, and forensic science. Chromatography is
used for quality analyses and checker in the food industry, by identifying and
separating, analyzing additives, vitamins, preservatives, proteins, and amino
acids. Chromatography like HPLC is used in DNA fingerprinting and bioinformatics.

 

Uses of Chromatography in Chemistry

Chromatography
has gained immense importance in the field of chemistry from detecting the
optical isomer to determining the amount of mixture present in a sample.
Following are some of the uses of chromatography in chemistry.

Chromatography is used to figure out the relation of
different mixtures with one another.
It is very effective technique to test the purity of
the sample.
The amount of mixture present in a sample can be
calculated by using chromatography.
Chiral compounds which are very similar in molecular
weight, elemental composition, and physical properties and differ only in
optical isomers can be separated using chromatography.
It is used for the separation of mixture of compounds.
Paper chromatography is particularly very effective in detection and
separation of mixture of compounds.

 

Uses of Chromatography in Medicine

Chromatography
in field of medicines has an extended use. Some of the uses
of chromatography in medicine are:

In pharmaceutical companies, large numbers of pure
chemicals for making further medicines is prepared by using
chromatography.
Paper chromatography is used to separate the various
inks or dyes from the mixture.
Presence of alcohol or some other drugs in blood or
urine are detected by using gas chromatography.
Chiral compounds resemble to each other greatly in
terms of molecular weight, physical composition and elemental weight. But
they have different optical isomers due to which they have different
biological activities. Chromatography is very effective technique to
separate the isomers. For example, thalidomide is compound with two
isomers one of them causes birth defects, chromatography is used to
separate the isomer from its harmful counterpart.
In pharmacy chromatography is very important to analyze
whether correct medicine is manufactured or not.
In forensic science, it helps in solving many cases by
detecting residual burnt particles and flammable chemicals present in the
body parts in case of fire or explosions.
Paper chromatography and Gas chromatography are
employed in finger print, DNA RNA analysis.

 

Uses of Chromatography in Everyday Life

Chromatography
is used in almost everywhere around us. Some examples are given below:

·           It is used in the laboratories for
making pure sample of any compound.

·           It is also used to derive the number
of reactants or products.

·           Percentage purity is also found by
chromatography.

·           Analytical chemistry use
chromatography for various experiments

·           The detection and separation of
pure compound is carried by chromatography.

·           It helps in checking the level of
pesticides, fungicides and contaminants in the food and drinking water.

·           It is also used to check the level
of adulterants in the manufactured food

·           It is applied in forensic science
for investigation.

 

Uses of Chromatography in Industry

There are many industrial uses
of chromatography. Different chromatography techniques are used in various
industries like food industries, drinking water treatment plants and other
industries. Some of the uses of chromatography in industry are:

 

It is used in the food industries for analysis of
different additives in the food. For example, milk is consumed all over
the world. The common adulterant that can be added in milk is pyruvic
acid. Pyruvic acid is derived from lactic acid bacteria. Chromatography is
employed acid bacteria. Chromatography is employed to check the quality of
milk.

Paper chromatography is particularly used to check the
quality of food by examining different vitamins, preservatives, amino
acids and proteins.

Chromatography is also used to separate the
contaminants, traces of harmful chemicals and other micro-organisms in
food.

Gas chromatography is used in the manufacture and
separation of essential oils.

It is used in industries for separating different
components whose amounts can range from milligrams to tons.

The manufactured food is checked for quality. For example,
malic acid is added in apple juice to prevent it from getting spoiled and
to maintain its taste. As malic acid is found in apple juice, therefore it
is very difficult to check its content in apple juice. To check the level
of maleic acid in apple juice, chromatography is employed. Fumaric acid is
a contaminant of malic acid, so to check the quantity of synthetic malic
acid, the level of fumaric acid is determined.

Thin layer chromatography is used to check and remove
Polychlorinated biphenyls, pesticides and insecticides in ground water and
fish contaminated by these.

Environmental and governmental agencies also use
chromatography to test drinking water.

Gas chromatography is also used in the environment
field. Fixed monitors are employed to check the emission levels of
pollutants such as nitrogen dioxide, carbon dioxide and carbon monoxide.
It is also used to detect the quality of air.

Gas chromatography is used to monitor variation present
if any in the industrial processes.

 

 

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