Biochromatography :-

Bio chromatography is a technique that separates biomolecules based on

their physical properties, such as their size, shape, polarity, electric charge,
or hydrophobicity. It's used in many fields, including protein analysis, drug
research, diagnostics, and the production of biopharmaceuticals.
Size exclusion chromatography (SEC) is a technique used to separate
molecules by size. It is also known as gel filtration chromatography (GFC)
or gel permeation chromatography (GPC).
SEC works by passing a mixture of molecules through a column with many
pores. Smaller molecules flow slowly and pass through the pores, while
larger molecules flow faster and exit the column without passing through
the pores.
SEC has many applications, including:
 Protein analysis: SEC is a popular method for determining the size of
proteins, as well as for protein purification, analyzing purity, and studying
protein interactions.
 Polymer analysis: SEC is a well-developed technique for analyzing
polymers, including natural polymers like polysaccharides that are found in
food products.
 Nanomaterial characterization: SEC can be used to characterize
nanomaterials.
SEC was invented in 1955 by Grant Henry Lathe and Colin R Ruthven at
Queen Charlotte's Hospital in London. They later received the John Scott
Award for their invention.
Some advantages of SEC include:
 High speed: SEC can process samples in less than five minutes.
 High recovery: Recovery is often greater than 95%, depending on the
biomolecule, column format, and purification protocol.
 High capacity: SEC can process even relatively large sample volumes
rapidly and efficiently.

Ion exchange chromatography

(or ion chromatography) is a process that allows the

separation of ions and polar molecules based on their
affinity to ion exchangers.
 The principle of separation is thus by reversible exchange of ions
between the target ions present in the sample solution to the ions
present on ion exchangers.
1. Cationic exchangers possess negatively charged group, and
these will attract positively charged cations. These exchangers
are also called “Acidic ion exchange” materials, because their
negative charges result from the ionization of acidic group.
2. Anionic exchangers have positively charged groups that will
attract negatively charged anions. These are also called “Basic
ion exchange” materials.
 Ion exchange chromatography is most often performed in the
form of column chromatography. However, there are also thin-
layer chromatographic methods that work basically based on the
principle of ion exchange.
This form of chromatography relies on the attraction between
oppositely charged stationary phase, known as an ion exchanger,
and analyte.
 The ion exchangers basically contain charged groups covalently
linked to the surface of an insoluble matrix.
 The charged groups of the matrix can be positively or negatively
charged.
 When suspended in an aqueous solution, the charged groups of
the matrix will be surrounded by ions of the opposite charge.
 In this “ion cloud”, ions can be reversibly exchanged without
changing the nature and the properties of the matrix.
Typical IC instrumentation includes: pump, injector, column,
suppressor, detector and recorder or data system.
1. Pump
The IC pump is considered to be one of the most important
components in the system which has to provide a continuous
constant flow of the eluent through the IC injector, column, and
detector.
2. Injector
Sample introduction can be accomplished in various ways. The
simplest method is to use an injection valve. Liquid samples may
be injected directly and solid samples need only to be dissolved in
an appropriate solvent. Injectors should provide the possibility of
injecting the liquid sample within the range of 0.1 to 100 ml of
volume with high reproducibility and under high pressure (up to
the 4000 psi).
3. Columns
Depending on its ultimate use and area of application, the column
material may be stainless steel, titanium, glass or an inert plastic
such as PEEK. The column can vary in diameter from about 2mm
 to 5 cm and in length from 3 cm to 50 cm depending on whether
it is to be used for normal analytical purposes, microanalysis, high
speed analyses or preparative work.
Guard column is placed anterior to the separating column. This
serves as a protective factor that prolongs the life and usefulness
of the separation column. They are dependable columns designed
to filter or remove particles that clog the separation column
4. Suppressor
The suppressor reduces the background conductivity of the
chemicals used to elute samples from the ion-exchange column
which improves the conductivity measurement of the ions being
tested. IC suppressors are membrane-based devices which are
designed to convert the ionic eluent to water as a means of
enhancing the sensitivity.
5. Detectors
Electrical conductivity detector is commonly use.
6. Data system
In routine analysis, where no automation is needed, a pre-
programmed computing integrator may be sufficient. For higher
control levels, a more intelligent device is necessary, such as a
data station or minicomputer.
Advantages of ion exchange
chromatography
1. It is one of the most efficient methods for the separation of
charged particles.
2. It can be used for almost any kind of charged molecule including
large proteins, small nucleotides and amino acids.
3. Ion exchange is used for both analytical and preparative
purposes in the laboratory, the analytical uses being the more
common.
4. Inorganic ions also can be separated by ion-exchange
chromatograph.y
Affinity chromatography
is a technique that uses the specific binding of a molecule to a ligand to
purify a target molecule from a complex mixture:
 Principle
A biologically active molecule, called a ligand, is attached to a solid
support, called a matrix, to create a stationary phase. A sample containing
the target molecule is passed through the column, and the target molecule
binds to the ligand. Other molecules in the sample that don't bind to the
ligand pass through the column or are washed out. The target molecule is
 then recovered by washing the column with a solution that disrupts the
binding.
 Ligands
Ligands can be antibodies, metals, lectins, biotin, aptamers, or other
molecules.
 Matrices
Matrices can be made from porous materials like agarose, cellulose,
silica, or polymethacrylate, or non-porous materials. The matrix should be
chemically and physically inert, insoluble in solvents and buffers, and
have a large surface area.
 Applications
Affinity chromatography is often used in the early stages of protein
purification. It's the most expensive chromatographic method because the
ligand is often a highly purified protein that must be manufactured
separately.

In chromatography, the stationary phase and mobile phase are two phases
that separate a mixture:
 Stationary phase
A solid or liquid phase that remains in place while the mobile phase
passes over it. The stationary phase can be a porous solid, like glass,
silica, or alumina, packed into a tube or coating the walls of a capillary.
 Mobile phase
A liquid or gas that flows over the stationary phase. The type of mobile
phase determines whether the chromatography technique is called liquid
chromatography (LC) or gas chromatography (GC).
Here are some examples of stationary and mobile phases in different types
of chromatography:
 Thin-layer chromatography (TLC)
The stationary phase is a thin layer of solid material, usually silica-based,
and the mobile phase is a liquid.
 Gas–liquid chromatography
The mobile phase is a gas and the stationary phase is a liquid film coated
on a solid substrate.
 Ion exchange chromatography
Separates components based on their charge. The stationary phase is a
positively charged resin, and the mobile phase is a buffer.
 Chromatographic techniques are often named by listing the type of mobile
phase followed by the type of stationary phase. For example, gas
chromatography is named for its mobile phase, which is a gas.

Supercritical fluid chromatography

(SFC) is a technique that separates and purifies substances using a
supercritical fluid as the mobile phase:
 Supercritical fluid
A fluid that is above its critical temperature and pressure, where the
kinetic energy of its molecules is greater than the intermolecular
forces. This means there is no distinct liquid phase.
 Separation
The mixture to be separated is introduced into the supercritical fluid, which
then passes over a stationary phase. The components of the mixture
separate based on how they partition between the two phases.
 Properties
Supercritical fluids have properties that make them useful for
chromatography, including:
 Viscosity: Supercritical fluids are less viscous than liquids.
 Diffusion coefficient: The diffusion coefficient of substances in supercritical
fluids is much greater than in liquids.
 Density: The density and properties of supercritical fluids change with
pressure and temperature.

Instrumentation: - The instrumentation for supercritical fluid chromatography

(SFC) includes:
 Mobile phase container: Holds the mobile phase, which is usually
supercritical carbon dioxide mixed with a small amount of organic solvent
 Injector: Uses high pressure to inject the mobile phase
 Column: Similar to those used in HPLC, and can be open-tubular or
packed
 Oven: Controls the temperature of the column
 Pressure regulator: Controls the pressure of the system, which affects the
density of the mobile phase
 Back pressure regulator: Maintains a single dense phase throughout the
system
 Detector: SFC can use a variety of detectors, including ultraviolet-visible
absorbance, flame ionization, and ELS
  Microprocessor: Collects data on pressure, temperature, and detector
performance to control the instrument
SFC is similar to high performance liquid chromatography (HPLC), but SFC
is faster and uses less toxic solvents. SFC is used to analyze and purify
compounds in many fields, including pharmaceuticals, food, and
environmental science.

 Applications :-
 Drug discovery
SFC is used to separate and analyze complex mixtures during the drug
discovery process.
 Quality control
SFC helps ensure that only high-quality medicines reach consumers by
detecting impurities, degradants, and chiral purity.
 Lipophilicity determination
SFC determines the lipophilicity of drug molecules, which is an important
property that influences drug absorption and distribution in the human
body.
 Lipid analysis
SFC is used to analyze lipids, especially separating difficult-to-separate
isomers.
 Metabolite analysis
SFC is used to analyze metabolites with increasing polarity.
SFC is a versatile analytical technique that uses a supercritical fluid,
typically carbon dioxide, as the mobile phase. It's often used for
separations involving non-volatile or thermally labile species that cannot be
separated by gas chromatography (GC) or liquid chromatography (LC).
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