Chromatography

Definition:
A method for the separation of components of a sample, in which the
components are distributed, between two phases, one of which is
stationary while the other moves in definite direction, is known as
chromatography.
Types:
 Adsorption chromatography
i) Column chromatography
ii) Thin layer chromatography(TLC)
iii) Gas-solid chromatography(GSC)
 Ion exchange chromatography
i) Cation exchange chromatography
ii) Anion exchange chromatography
 Partition chromatography
i) Column chromatography
ii) Paper chromatography
(1) Single
(a)Ascending
(b) Descending
(c)Circular
(2) 2-Dimensional
iii) Thin layer chromatography(TLC)
iv) Gel filtration
v) Gas- liquid chromatography
 Size exclusion chromatography
 i) Gel permeation
ii)Gel filtration
 Affinity chromatography
i) Metal chelate chromatography
ii) Dye ligand chromatography
iii) Immuno affinity chromatography
iv) Lectin affinity chromatography
v) Covalent chromatography

A. Adsorption chromatography:
Adsorption chromatography is a technique for the separation of a
component in a mixture by adsorption from a mobile phase into the
stationary solid surface. 

Basic Principle:
In Adsorption Chromatography the analytical separation of a chemical
mixture is involved based on how the adsorbate and the adsorbent
interact with each other. A mixture of gas or liquid gets separated when
it is passed over the adsorbent bed that has different adsorbing capacity
and rate for different compounds.
Simply put, an Adsorbent is a substance which is usually porous in
nature with a high surface area to adsorb substances on its surface by
intermolecular forces like non-ionic attractive forces and hydrogen
bonding is called adsorbent. Some commonly used adsorbents are
Silica, alumina and carbon.
Applications:
Adsorption chromatography has many applications. Generally, it is used
for determining the concentration of a compound (or its purity),
separating out a mixture into individual components, and identifying
what is in a mixture.

 Separation of amino acids:

Amino acids are the building blocks of proteins. Each amino acid
has a slightly different chemical structure. These are separated out
using TLC (Thin Layer Chromatography) - a type of adsorption
chromatography. Standard solution of amino acids are prepared
and applied on TLC plates in the form of spots and plates are air
dried. After development of the mobile phase, the plates are dried
and sprayed with the locating agent. Colors are always observed
visually. Detection limits of the amino acids gives their
 characterization and thus amino acids are separated out from the
sample solution.
 Isolation of antibiotics:
Silica gel adsorption chromatography is used to separate major
pigment components from the biologically active fractions of
antibiotics, using an eluting solvent of benzene: methanol:
chloroform. Further purification using silica gel TLC plates
developed with benzene: methanol: chloroform (30:20:9, v/v/v)
separated the active fractions into different components. The
molecular recognition capability for antibiotics is evaluated by
static and dynamic adsorption curves.
 Identification of carbohydrates:
 Carbohydrates are one of the most important components in many
foods. Carbohydrates may be present as isolated molecules or they
may be physically associated or chemically bound to other
molecules. Individual molecules can be classified according to the
number of monomers that they contain as monosaccharides,
oligosaccharides or polysaccharides. Carbohydrates are separated
on the basis of their differential adsorption characteristics by
passing the solution to be analyzed through a column.
 Identification of fats and fatty acids:
HPLC on adsorption columns of silica gel can be used for the
analysis or isolation of fatty acids with polar functional groups,
especially oxygenated moieties such as hydroperoxides. With
care, isomers differing in the position of hydroperoxy or hydroxy
groups on an aliphatic chain can be separated.
 Isolation and determination of peptides and proteins:
Proteins can be characterized by their amino acid content,
nutritional value, and functional properties. Adsorption column
 chromatography is one of the most common methods of protein
purification.
 In the examination of explosives:
 Forensic identification of explosives is a major problem in the
criminalistics investigation of a bombing which involves
connecting the type of explosive used with the suspect. The
detection and identification of explosive residues in debris
material constitutes a highly difficult task. The thermal instability
of most explosives, along with the requirements of high sensitivity
of the analysis, limit the adsorption chromatography for such
determinations.
 Detection of drugs in clinical samples:
Adsorption chromatography is also applicable in the detection of
drugs in different clinical samples like food, urine, blood etc. in
forensic labs.
 In pharmaceutical industries:
Adsorption chromatography also found its applications in quality
control and pharmaceutical formulations.
 In food and cosmetic industries:
Adsorption TLC is used in the identification and quantification of
colors, ingredients, preservatives, and sweetening agents in food
and cosmetic products.
 Chemical kinetics:
Adsorption chromatography is used for the examination of
chemical reactions for completion.

B. Ion exchange Chromatography:

 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.
Basic Principle:
The principle of separation is reversible exchange of ions between the
target ions present in the sample solution to the ions present on ion
exchangers.

Applications:
 Analysis of amino acid mixtures:
Ion exchange chromatography is used in routine analysis of amino acid
mixtures to determine the amino acid content of amino acid-, peptide-
and protein-containing samples. The 20 principal amino acids from
blood serum or from the hydrolysis of proteins are separated and used
in clinical diagnosis using ion- exchange chromatography. Also,
separation of proteins from foods, for example, to investigate the
effects of individual food components on health – this type of analysis
is used in nutrition research, is also done by ion-exchange
chromatography.
 Purification of water:

Ion-exchange chromatography is the most effective method for water

purification. Complete deionization of water (or) a non-electrolyte
solution is performed by exchanging solute cations for hydrogen ions
and solute anions for hydroxyl ions. This is usually achieved by
method is used for softening of drinking water.

 In the analysis of nucleic acids:

Ion exchange chromatography also find its application in the
analysis of products of hydrolysis of nucleic acids. In this way,
information is gained about the structure of these molecules and
how it relates to their biological function as carriers of hereditary
information. 
 Separation of trace metals:
Chelation resins in ion exchange chromatography are used to
extract the trace metals from seawater.
 In Petrology:
Ion exchange chromatography is used to analyze lunar rocks and
rare trace elements on Earth.
 In aquatic ecosystems:
Ion exchange chromatography is highly applicable in the
determination of water chemistries in aquatic ecosystems. It is
 also helpful in the analysis of pollution and other toxic
constituents in water samples.
 In food industry:
Most of the food related industries use ion exchange
chromatography for the determination of sugar and salt content in
food samples. In an even older use of ion exchange, salts are
removed from sugar juices to raise the yield of crystallized sugar.
Deionization also can improve the flavor and storage time of
pineapple juice and wine. In these and other beverage
applications, ion exchange removes traces of heavy metals, which
not only taste bad but also catalyze oxidation.
 Separation of organic molecules:
Ion exchange chromatography is helpful in segregating and
purifying organic molecules from natural sources. The non-solvent
extractable natural products can be followed up.
 In Hydrometallurgy:
In hydrometallurgy, the treatment of ores with water solutions, ion
exchange helps to recover valuable metals like copper, silver, and
gold from waste waters. Uranium can be recovered from low-
grade ores by leaching with dilute sulfuric acid—oxidizing if
necessary to convert uranium(IV) to uranium(VI)—and then
absorbing the negatively charged uranium sulfate complex ions
on a quaternary-base anion-exchange resin. This highly selective
absorption process thereby separates the uranium from iron and
other metals. The uranium is later removed from the resin with
dilute nitric acid.
 Ion exchangers as catalysts:
Ion exchangers can function as catalysts. Strong-acid cation-
exchange resins loaded with hydrogen ions catalyze certain
 chemical reactions carried out in the liquid phase, such as
hydrolysis and esterification (ester formation). The advantage of
the resin over hydrochloric acid as a catalyst in these reactions is
that it is present as a separate phase that does not contaminate the
product. In addition, the ion-exchange process lends itself to
continuous-flow techniques. Gas-phase reactions catalyzed
by metal ions, like the cracking of petroleum fractions to produce
gasoline, also can be catalyzed by metal-loaded inorganic
exchangers, the molecular sieves being particularly suitable for
this purpose since their open crystalline structure makes every
metal ion accessible.
 In medicine:
Ion-exchange resins have a limited use in medicine. Carboxylic
resins containing hydrogen or ammonium ions, taken by mouth,
remove sodium ions from the gastrointestinal tract and
control edema; other resins are consumed to lower acidity in the
stomach and hence to soothe stomach ulcers. Interest in these
treatments has declined, however, because of the resins’
undesirable side effects. Resins also are incorporated into artificial
kidneys outside the body to remove ammonium
and potassium ions from the blood. The most important medical
applications of ion exchange, however, have been made in clinical
analysis procedures that depend on ion-exchange
chromatography.
 Miscellaneous uses:
 Miscellaneous analytical uses of ion exchange include the
dissolving of sparingly soluble salts like calcium sulfate, the
determination of total dissolved salts in natural waters (by passing
them through hydrogen-loaded, cation-exchange resins and
titrating the acid formed), and the identification of minute traces
 of ions (by absorbing them onto a single resin bead along with a
colour-producing reagent).
C. Size exclusion chromatography:
Size-exclusion chromatography (SEC), also known as molecular
sieve chromatography, is a chromatographic method in which
molecules in solution are separated by their size, and in some
cases molecular weight.

Basic Principle:
The underlying principle of SEC is that particles of different sizes
elute (filter) through a stationary phase at different rates. This results
in the separation of a solution of particles based on size.
Applications:
 Biochemical applications:
In general, SEC is considered a low-resolution chromatography as
it does not discern similar species very well, and is therefore often
reserved for the final step of a purification. The technique can
determine the quaternary structure of purified proteins that have
slow exchange times, since it can be carried out under native
solution conditions, preserving macromolecular interactions. SEC
can also assay protein tertiary structure, as it measures the
hydrodynamic volume (not molecular weight), allowing folded
and unfolded versions of the same protein to be distinguished. For
example, the apparent hydrodynamic radius of a typical protein
domain might be 14 Å and 36 Å for the folded and unfolded
forms, respectively. SEC allows the separation of these two forms,
as the folded form elutes much later due to its smaller size.
 Polymer Synthesis:
SEC can be used as a measure of both the size and
the polydispersity of a synthesized polymer, that is, the ability to
find the distribution of the sizes of polymer molecules. If
standards of a known size are run previously, then a calibration
curve can be created to determine the sizes of polymer molecules
of interest in the solvent chosen for analysis (often THF). In
alternative fashion, techniques such as light scattering
and/or viscometry can be used online with SEC to yield absolute
molecular weights that do not rely on calibration with standards of
known molecular weight. Due to the difference in size of two
polymers with identical molecular weights, the absolute
determination methods are, in general, more desirable. A typical
SEC system can quickly (in about half an hour) give polymer
 chemists information on the size and polydispersity of the sample.
The preparative SEC can be used for polymer fractionation on an
analytical scale.
 Purification:
The main application of size exclusion chromatography is in the
purification of biological macromolecules. Viruses, proteins,
enzymes, hormones, antibodies, nucleic acids, and
polysaccharides have all been separated and purified by the use of
appropriate gels or glass granules.
  Molecular weight determination:
The effluent volumes of globular proteins are largely determined
by their molecular weight. It has been shown, that over a
considerable molecular weight range, the effluent volume is
approximately a linear function of the logarithm of the molecular
weight. Thus, SEC is also used for this purpose.
 Solution concentration:
SEC is used in solution concentration. Solution of high molecular
weight substances can be concentrated by the addition of dry
sephadex G-25 (coarse). Water and low molecular weight
substance remain in solution. After ten minutes the gel is removed
by centrifugation, leaving the high molecular material in a
solution whose concentration has increased but whose pH and
ionic strength are unaltered.
 Desalting:
Applications of SEC are also found in desalting. By use of a
column of sephadex G-25, solutions of high molecular weight
compounds may be desalted. The high molecular weight
substances move with the void volume while the low molecular
 weight components are distributed between the mobile phase and
hence move slowly.

 Protein building studies: Size exclusion chromatography is

one of a number of methods commonly used to study the
reversible binding of a ligand to a macromolecular such as
proteins including receptor proteins. A sample of the
protein/ligand mixture is applied to a column of a suitable gel (e.g.
G-25) which has previously been equilibrated with a solution of
the ligand of the same concentration as that in the mixture.
 Analysis of water samples:
Another use of size exclusion chromatography is to examine the
stability and characteristics of natural organic matter in water. In
this method, Margit B. Muller, Daniel Schmitt, and Fritz H.
Frimmel tested water sources from different places in the world to
determine how stable the natural organic matter is over a period of
time.
 Fractionation of proteins:
The main application of gel-filtration chromatography is
the fractionation of proteins and other water-soluble polymers,
while gel permeation chromatography is used to analyze the
molecular weight distribution of organic-soluble polymers. Either
technique should not be confused with gel electrophoresis, where
an electric field is used to "pull" or "push" molecules through the
gel depending on their electrical charges. The amount of time a
solute remains within a pore is dependent on the size of the pore.
Larger solutes will have access to a smaller volume and vice
versa. Therefore, a smaller solute will remain within the pore for a
longer period of time compared to a larger solute.
 In pharmaceutical development:
 Size exclusion chromatography provides a method to continuously
monitor all the development stages of Nano-particulate drug
delivery systems, thereby, ensuring the quality of the starting
materials used and the final product.
D. Partition chromatography:
Partition Chromatography technique is defined as the separation of
components between two liquid phases via original solvent and the
film of solvent used in the column.
Basic Principle:
In partition chromatography, the separation of the components from
the sample takes place through the process of partition the
components between two phases, where both the phases are present in
liquid form.

Applications:
 Isolation and Purification:

Partition chromatography is used for the purification of natural

extracts, biological mixtures and synthetic matrices. This technique
is useful in the purification and isolation of components of mixtures.
Here, the separated components on the paper are cut, dissolved in
suitable solvents and using spectroscopic methods, their absorption is
characterized at specific wavelengths.

 Quality analysis of water:

Partition chromatography is used to examine the quality of water.
Polluted water contains different contaminants like pesticides,
phenols and insecticides etc. These contaminants are separated out
with the help pf partition chromatography.
 In analytical chemistry:
In research laboratories, partition chromatography is used to
concentrate out trace metals from aqueous solutions. This is
mainly done by paper partition chromatography.
 Biochemical applications:
Paper chromatography is used to examine the reduction mixtures
in biochemical laboratories. Cholesterol is a steroid found in
mammals that is needed for the formation of cell membranes, bile
acids, and several hormones. Bile salts are secreted into the small
intestine to aid in the digestion of fats. Many other steroids, bile
acids and mycotoxins are separated out from clinical samples for
examination using partition chromatography.
 In food industry:
Partition chromatography is used in the separation of aromatic
molecules from wines and other beverages. Paper chromatography
is also used to detect the contaminants in drinks and food
 products. Analysis of food colors in synthetic drinks and beverages,
ice creams, sweets, etc. Only edible colors are permitted for use, this
is why identification and quantification are of utmost importance.
 In Petroleum industry:
For fractionation of complex crude extracts, partition
chromatography is highly applicable. For example; petroleum
fractions are fractionized from crude oil using partition
chromatography.
 In Cosmetics industry:
Paper chromatography is majorly used to inspect the constituents
of cosmetic products in cosmetic industry.
 Detection of drugs:
Paper partition chromatography is used to determine dopes and
drugs in animals and humans.
 In Pharmaceutical industry:
Paper chromatography is used to check the purity of medicines in
pharmaceutical industry. It provides information related to the
development of new drugs molecules, reaction completion and
progress of manufacturing processes. This process is cost-effective
and hence used as an alternative method in monitoring the active
ingredients present in the drug forms. Paper chromatography is also
applicable in colour identifications of pharmaceutical formulations.
 In Forensics:
Partition chromatography provides a basis for identification and
comparison against reference standards for drugs and their
metabolites. Paper chromatography offers a vital role in the viable
analysis of samples that are available in milligrams or microliter
quantities. 
E. Affinity Chromatography:
Affinity chromatography is a separation method based on a specific
binding interaction between an immobilized ligand and its binding
partner.
Basic Principle:
The principle of affinity chromatography is that the stationary phase
consists of a support medium (e.g. cellulose beads) on which the
substrate (or sometimes a coenzyme) has been bound covalently, in
such a way that the reactive groups that are essential for enzyme
binding are exposed.

Applications:
 Separation of biological molecules:
The major application of affinity chromatography is that it is used
for separation and purification of all biological macromolecules
 i.e. carbohydrates, lipids, proteins, enzymes and nucleic acids
from different biological samples.
 In Pharmaceutical industry:
Affinity chromatography is an adaptable and valuable separation
technique for pharmaceutical and biomedical analysis. This
method is based on the use of a biologically-related agent as a
stationary phase to selectively retain analytes or to study
biological interactions.
 Clinical applications:
Affinity chromatography is used in a number of applications,
including nucleic acid purification, protein purification from cell
free extracts, and purification from blood.
 Enzyme purification:
Affinity chromatography is used to purify and concentrate on an
enzyme solution. In affinity chromatography, the enzyme to be
purified is passed through a column containing a cross-linked
polymer or gel to which a specific competitive inhibitor of the
enzyme has been covalently attached. All proteins without
substantial affinity for the bound inhibitor will pass directly
through the column, whereas one that recognizes the inhibitor will
be retarded in proportion to its affinity constant. Elution of the
bound enzyme is readily achieved by changing such parameters as
salt concentration or pH, or by addition of a competitive inhibitor
in solution.
 In analytical chemistry:
Affinity chromatography is used in analytical chemistry to
studying the kinetics of biological interactions.
 Reduction of substances:
 Affinity chromatography is used to reduce the number of
unnecessary substances in the sample mixture.
 Purification of biological molecules:
Affinity chromatography is often selected method for purification
of biomolecules due to its ease of operation, specificity, yield, and
throughput.
 Concentration of substances :
Affinity chromatography is used for purification and concentrates
the substance from a sample mixture into a buffering solution.
 Isolation of antibodies:
Metal chelate chromatography is very suitable for the isolation of
antibodies and the separation of antibody conjugate complexes.
Interestingly the use of Ni columns is recommended for this
application area .The binding of antibodies is significantly better
when immobilized nickel ions are used.
 Protein purification:
Dye-ligand affinity chromatography is a widely used technique
in protein purification. The utility of the reactive dyes as affinity
ligands results from their unique chemistry, which confers wide
specificity towards a large number of proteins.