Chromatography, a technique for separating (purification and identification) components, or

solutes, of a mixture for qualitative and quantitative analysis on the basis of the relative amounts of
each solute distributed between a moving fluid stream, called the mobile phase, and a contiguous
stationary phase. The mobile phase may be either a liquid or a gas, while the stationary phase is
either a solid or a liquid. Protein purification employs multiple chromatography techniques that
separate products according to differences of their properties such as molecular size and shape,
solubility, electric charge, binding affinity, adsorption properties and Hydrophobicity.The factors that
involve in separation process include adsorption (liquid-solid), partition (liquid-solid), and affinity and
due to this differences, some components of the mixture stay longer in the stationary phase, and they
move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave
the system faster. And in the end, various chromatography methods have been developed.

One of the most common methods of protein purification is Column Chromatography.

Column Chromatography is a preparative technique used to purify compounds depending on their
polarity or hydrophobicity. In column chromatography, a mixture of molecules is separated based on
their differentials partitioning between a mobile phase and a stationary phase. It is a solid-liquid
method where the solid is the stationary phase and mobile phase is a liquid that is used in the process
of separation. The next type of chromatography on the list is the ion exchange chromatography
which is a matrix consists of positively(exchange matrices are called anion-exchange matrices, and
adsorb negatively charged proteins) and while negatively(matrices bound with negatively charged
groups are known as cation-exchange matrices, and adsorb positively charged proteins) charged ions.
This is a process that allows the separation of ions and polar molecules based on their affinity to ion
exchangers. Essentially, molecules undergo electrostatic interactions with opposite charges on the
stationary phase matrix. Then, gel permeation chromatography (GPC) is a type of molecular sieving
chromatography, where a sample is separated into its constituent parts based on their molecular
sizes. This is accomplished by dissolving the sample in a mobile phase (solvent) and passing it through
a porous column packing. Affinity chromatography technique is used for the purification of enzymes,
hormones, antibodies, nucleic acids, and specific proteins. It composed of a stationary phase (solid
phase) and a mobile phase which is the mobile phase is your cell lysate or any mixture that contains
biomolecules and A ligand that binds the target molecule is attached covalently to the solid phase.
The interaction between the solid and the mobile phase are exploited by affinity chromatography to
get your desired substance in a pure form. The target molecule binds to the ligand and the target
biomolecule is eluted by changing conditions (pH or salt concentrations) or by competition with a free
ligand. The simplest chromatography is carried out on paper, it is the paper chromatography which is
a “liquid-liquid” chromatography. It is a technique for separating dissolved chemical substances by
taking advantage of their different rates of migration across sheets of paper.Thin-layer
chromatography, in analytical chemistry, technique for separating dissolved chemical substances by
virtue of their differential migration over glass plates or plastic sheets coated with a thin layer of a
finely ground adsorbent, such as silica gel or alumina, that is mixed with a binder such as starch or
plaster of paris.

Gas chromatography is a “gas-liquid” chromatography. It is an analytical technique used in

the separation of a mixture of volatile compounds. It uses a gaseous mobile phase and a liquid
stationary phase. The simpler and more inert compounds come out of the column quickly while
heavier and polar compounds take some time for the elution.Dye affinity chromatography is a
purification technique offering unique selectivities and high purification factors. Dye ligands may act
as substrate analogs, offering affinity interactions with their corresponding enzymes. It is the
interaction of Cibacron Blue F3GA with proteins and enzymes. The dye Cibacron Blue F3GA has a high
affinity for many proteins and enzymes. Hydrophobic interaction chromatography (HIC) separates
proteins according to differences in their surface hydrophobicity. HIC utilizes a reversible interaction
between the proteins and the hydrophobic ligand of a HIC resin. The proteins are bound to the
hydrophobic ligand on the HIC resin in a binding buffer with a high salt concentration. When the ionic
strength of the buffer is reduced, the interaction is reversed. In similar fashion, pseudo-affinity
chromatography utilizes dyes as ligands in order to target proteins. The dyes used mimic the ligands,
but they do not display high specificity. Therefore, these dyes are able to capture a variety of
proteins.Lastly, High Performance Liquid Chromatography (HPLC) is a form of column chromatography
that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure
through a column with chromatographic packing material (stationary phase). The sample is carried by
a moving carrier gas stream of helium or nitrogen. HPLC has the ability to separate, and identify
compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as
low as parts per trillion. Because of this versatility, HPLC is used in a variety of industrial and scientific
applications, such as pharmaceutical, environmental, forensics, and chemicals.It affects many aspects
of our lives, from the food that we eat and the medication that we use, to the better understanding of
life outside of our planet. Without chromatography the world would be a less wealthy, less clean, and
a less healthy place.