Chromatography is a widely used analytical technique in chemistry and biochemistry. It is a method for separating and identifying the individual components of a mixture based on their physical and chemical properties. The word "chromatography" itself means "color writing," and it was initially developed for separating and analyzing colored compounds, but it is now applied to a broad range of substances. Chromatography is a powerful tool in research, quality control, and various industries because it allows for the separation and identification of complex mixtures, helping scientists and analysts understand the composition of substances and solve various analytical problems.

2.  Chromatography is a crucial biophysical process that permits
separate, recognition and purification of a mix for an analysis
that is both qualitative as well as quantitative. Chromatography
was invented by the Russian botanist Mikhail Tswett coined the
term Chromatography in the year 1906.
 The first use for analytical purposes of chromatography was first
described in 1952 by James as well as Martin in the year 1952, to
describe gas chromatography as a method to study fat acid
mixtures. The vast array of chromatographic methods make use
of variations in binding affinities, size charges, size, and other
characteristics to distinguish different substances. It is a highly
effective separation instrument used in every field of science. It
is usually the sole method of segregating elements of complex
mix-ups.

3.  Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture
for qualitative and quantitative analysis.
 The Russian botanist Mikhail Tswett coined the term chromatography in
1906.
 The first analytical use of chromatography was described by James and
Martin in 1952, for the use of gas chromatography for the analysis of fatty
acid mixtures.
 A wide range of chromatographic procedures makes use of differences in
size, binding affinities, charge, and other properties to separate materials.
 It is a powerful separation tool that is used in all branches of science and is
often the only means of separating components from complex mixtures.

4.  Chromatography is based on the principle where molecules
in mixture applied onto the surface or into the solid, and
fluid stationary phase (stable phase) is separating from each
other while moving with the aid of a mobile phase.
 The factors effective on this separation process include
molecular characteristics related to adsorption (liquid-
solid), partition (liquid-solid), and affinity or differences
among their molecular weights.
 Because of these 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 the mobile phase, and leave the system faster.

5.  Three components thus form the basis of the
chromatography technique.
 Stationary phase: This phase is always composed of a
“solid” phase or “a layer of a liquid adsorbed on the surface
solid support”.
 Mobile phase: This phase is always composed of “liquid”
or a “gaseous component.”
 Separated molecules
 The type of interaction between the stationary phase,
mobile phase, and substances contained in the mixture is
the basic component effective on the separation of
molecules from each other.

6.  Pharmaceutical sector
 To identify and analyze samples for the presence of
trace elements or chemicals.
 Separation of compounds based on their molecular
weight and element composition.
 Detects the unknown compounds and purity of
mixture.
 In drug development.
 Chemical industry
 In testing water samples and also checks air quality.
 HPLC and GC are very much used for detecting
various contaminants such as polychlorinated
biphenyl (PCBs) in pesticides and oils.
 In various life sciences applications

7.  Food Industry
 In food spoilage and additive detection
 Determining the nutritional quality of food
 Forensic Science
 In forensic pathology and crime scene testing like analyzing blood and
hair samples of crime place.
 Molecular Biology Studies
 Various hyphenated techniques in chromatography such as EC-LC-MS
are applied in the study of metabolomics and proteomics along with
nucleic acid research.
 HPLC is used in Protein Separation like Insulin Purification, Plasma
Fractionation, and Enzyme Purification and also in various departments
like Fuel Industry, biotechnology, and biochemical processes.


9.  To perform a separation, the gel filtration medium is packed
into a column to form a packed bed. The medium is a
porous matrix in the form of spherical particles that have
been chosen for their chemical and physical stability, and
inertness (lack of reactivity and adsorptive properties).
 The packed bed is equilibrated with a buffer which fills the
pores of the matrix and the space in between the particles.
The liquid inside the pores is sometimes referred to as the
stationary phase and this liquid is in equilibrium with the
liquid outside the particles, referred to as the mobile phase.

11.  The stationary phase used is a porous polymer matrix
whose pores are completely filled with the solvent to be
used as the mobile phase.
 The molecules in the sample are pumped through
specialized columns containing such microporous packing
material (gel).
 The basis of the separation is that molecules above a certain
size are totally excluded from the pores, while smaller
molecules access the interior of the pores partly or wholly.
 The flow of the mobile phase hence will cause larger
molecules to pass through the column unhindered, without
penetrating the gel matrix, whereas smaller molecules will
be retarded according to their penetration of the gel.

12.  High-performance liquid chromatography is a modified form of
column chromatography where the components of a mixture are
separated on the basis of their affinity with the stationary phase.
 Principle of HPLC
 This technique is based on the principle of differential adsorption
where different molecules in a mixture have a varying degree of
interactions with the absorbent present on the stationary phase.
 The molecules having higher affinity remain adsorbed for a longer time
decreasing their speed of movement through the column.
 However, the molecules with lower affinity move with a faster
movement, thus allowing the molecules to be separated in different
fractions.
 This process is slightly different from the column chromatography as in
this case; the solvent is forced under high pressures of up to 400
atmospheres instead of allowing it to drip down under gravity.

13.  Steps of HPLC
 The column is prepared by taking a glass tube that is dried and coated with a thin, uniform layer of
stationary phase (cellulose, silica).
 Then the sample is prepared by adding the mixture to the mobile phase. The sample is introduced
into the column from the top, and a high-pressure pump is used to pass the sample at a constant rate.
 The mobile phase then moves down to a detector that detects molecules at a certain absorbance
wavelength.
 The separated molecules can further be analyzed for various purposes.
 Uses of HPLC
 High-performance liquid chromatography is used in the analysis of pollutants present in
environmental samples.
 It is performed to maintain product purity and quality control of various industrial productions.
 This technique can also be used to separate different biological molecules like proteins and nucleic
acids.
 The increased speed of this technique makes the process faster and more effective.
 Example of HPLC
 High-performance liquid chromatography has been performed to test the efficiency of different
antibodies against diseases like Ebola.

15.  Gas chromatography is a separation technique in which the molecules are separated on
the basis of their retention time depending on the affinity of the molecules to the
stationary phase.
 The sample is either liquid or gas that is vaporized in the injection point.
 Principle of Gas chromatography
 Gas chromatography is based on the principle that components having a higher affinity
to the stationary phase have a higher retention time as they take a longer time to come
out of the column.
 However, the components having a higher affinity to the stationary phase have less
retention time as they move along with the mobile phase.
 The mobile phase is a gas, mostly helium, that carries the sample through the column.
 The sample once injected in converted into the vapor stage is then passed through a
detector to determine the retention time.
 The components are collected separately as they come out of the stationary phase at
different times.


16.  Steps of Gas chromatography
 The sample is injected into the column where it is vaporized into a gaseous state. The
vapourised component than mixes with the mobile phase to be carried through the rest
of the column.
 The column is set with the stationary phase where the molecules are separated on the
basis of their affinity to the stationary phase.
 The components of the mixture reach the detector at different times due to differences in
the time they are retained in the column.
 Uses of Gas chromatography
 This technique is used to calculate the concentration of different chemicals in various
samples.
 This is used in the analysis of air pollutants, oil spills, and other samples.
 Gas chromatography can also be used in forensic science to identify and quantify
various biological samples found in the crime scene.
 Examples of Gas chromatography
 The identification of performance-inducing drug in the athlete’s urine.
 The separation and quantification of a solid drug in soil and water samples.

17.  Liquid chromatography
 Liquid chromatography is a separation technique where the
mobile phase used is liquid, and the separation can take place
either in a column or a plain surface.
 Principle of Liquid chromatography
 The process of liquid chromatography is based on the principle
for the affinity of the molecules to the mobile phase.
 If the components to be separated have a higher affinity to the
mobile phase, the molecules move along with the mobile phase
and come out of the column faster.
 However, if the components have a lower degree of interaction
with the mobile phase, the molecules move slowly and thus
come out of the column later.
 Thus, if two molecules in a mixture have different polarities and
the mobile phase is of a distinct polarity, the two molecules will
move at different speeds through the stationary phase.

18.  Steps of Liquid chromatography
 The column or paper is prepared where the stationary phase (cellulose or silica)
is applied on the solid support.
 The sample is added to the liquid mobile phase, which is then injected into the
chromatographic system.
 The mobile phase moves through the stationary phase before coming out of the
column or the edge of the paper.
 A solution is applied to the system to separate the molecules from the
stationary phase.
 Uses of Liquid chromatography
 Liquid chromatography is an effective method for the separation of a colored
solution as they form two separate bands after separation.
 This method can also be used over other techniques as it is quite simple and
less expensive.
 It can be used for the separation of solid molecules that are insoluble in water.

19.  Thin layer chromatography is a kind of chromatography
used to separate and isolate mixtures that are non-volatile in
nature. Just like other chromatography processes, this one
consists of a mobile phase and a stationary phase.
 The latter one here is a thin layer of absorbent material,
such as aluminium oxide, silica gel, or cellulose. This layer
is applied to plastic, glass, or aluminium foil sheets called
an inert substrate. The mobile phase in the TLC
procedure is a solvent or a mixture of it.
 If you want to learn more about the thin layer
chromatography procedure, you have landed at the right
place. Here we will be discussing its principle, process, and
applications in different industries.

20.  PROCEDURE-
 TLC Plates: These are used for applying the thin layer of stationary phase.
They are inert or stable in nature. The layer of stationary phase is kept even
throughout these plates for better analysis. Usually, ready-to-use plates are
preferred by the people conducting experiments.
 Mobile Phase: This comprises a solvent (or solvent mixture). The taken
solvent needs to be chemically inert, of the highest possible purity, and
particulate-free. Only then can the TLC spots be able to develop.
 TLC Chamber: This is where the thin layer chromatography procedure takes
place. It keeps the dust particles away from the process and does not let the
solvent evaporate. In order to develop the spots appropriately, a uniform
environment is maintained inside this chamber.
 Filter Paper: This gets placed inside the chamber after being moistened with
the mobile phase solution. It ensures that the mobile phase rises uniformly
throughout the TLC plate’s length.

22.  Being a separation process, TLC proves to be highly effective for separating
pharmaceutical formulations that consist of multiple components.
 The process can be used to examine a given product’s purity.
 Medicines like local anaesthetics, analgesics and steroids go through the TLC
procedure for their qualitative testing.
 The cosmetic industry also uses TLC for checking the presence of
preservatives in the products.
 A given compound can be purified using TLC and then compared with a
standard sample.
 TLC also finds its use in Biochemical analysis. Here, it can be used for
biochemical metabolites’ separation from urine, blood plasma, serum, and body
fluids.
 Just like the cosmetic industry, the food industry also utilizes TLC for the
detection of preservatives, artificial colours, and sweetening agents.
 A reaction’s progress can also get tracked with TLC to see whether it is
complete or not.

23.  Mass spectrometry is an analytical technique
that is used to measure the mass-to-charge
ratio of ions. The results are presented as a
mass spectrum, a plot of intensity as a
function of the mass-to-charge ratio.
 . These measurements can often be used to
calculate the exact molecular weight of the
sample components as well.

25.  Importance- Mass spectrometry is a way to calculate the
mass of ions — electrically charged particles, atoms or
molecules formed from them. This is used to describe the
basic atomic and molecular processes, and also those of
immediate interest to cell events.
Application-
 Mass spectrometry is a powerful technique with many
different applications in biology, chemistry, and physics, but
also in clinical medicine and even in space exploration.
 It is used by separating molecular ions on the basis of their
mass and charge to determine the molecular weight of
compounds.

26.  Principle- Fast protein liquid
chromatography (FPLC), is a form of liquid
chromatography that is often used to analyze
or purify mixtures of proteins.
 As in other forms of chromatography,
separation is possible because the different
components of a mixture have different
affinities for two materials, a moving fluid
and a porous solid.

27.  Working Procedure- Fast protein liquid chromatography
(FPLC) is a form of medium-pressure chromatography
that uses a pump to control the speed at which the
mobile phase passes through the stationary phase. FPLC
was introduced in 1982 by Pharmacia as fast performance
liquid chromatography.
 Applications- FPLC is used to purify large biomolecules
such as proteins, nucleotides and peptides. The software
differs between the two techniques as well.
 FPLC include the profiling of proteins used in the
purification of animal venoms and can detect small changes
in single proteins, which has clinical importance

28.  Principle-The molecules separated on the basis of
their charge are eluted using a solution of varying
ionic strength. By passing such a solution through the
column, highly selective separation of molecules
according to their different charges takes place.
 Affinity chromatography is a separation method
based on a specific binding interaction between an
immobilized ligand and its binding partner.
Examples include antibody/antigen, enzyme/substrate,
and enzyme/inhibitor interactions.

29.  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.
 In this process, two types of exchangers i.e., cationic and anionic
exchangers can be used.
 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.
 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.

32.  The main difference between affinity and ion exchange
chromatography is that we can use affinity
chromatography to separate charged or uncharged
components in a mixture whereas we can use ion
exchange chromatography to separate charged
components in a mixture.
 Application- For the separation of high value
proteins from substances. drinking water analysis for
pollution and other constituents. determination of water
chemistries in aquatic ecosystems and determination of
sugar and salt content in foods.

33.  Dialysis is a commonly used laboratory-scale
process to remove salt, or reduce the salt
concentration, from a solution. A semi-permeable
membrane is used to contain the target protein.
 The particles in the areas of high concentration
move towards the area of low concentration. Picture
how a tea bag works: the leaves stay in the bag and the
tea enters the hot water. In dialysis, waste in your blood
moves towards dialysate, which is a drug solution that
has none (or very little waste).

34.  Dialysis is the process of separating molecules in
solution by the difference in their rates of diffusion
through a semipermeable membrane, such as
dialysis tubing.
 Dialysis is a common laboratory technique that
operates on the same principle as medical dialysis

35.  Using LC MS-MS further increases the specificity.
Typically a particular peak from the mass spectrum is
selected and isolated and collisions are induced
within the mass spectrometer to force a characteristic
fragmentation of the selected ion. Because the mass
spectrometer can perform these actions on
timescales of seconds this can all happen while the
LC is performing the initial chemical separation. As
an example, structural isomers that would otherwise
have a very similar chemical behaviour in the column
and give the same ions in a single stage mass
spectrum can give different fragment ions when an
initial ion of a particular m/z is selected and
fragmented.

36.  Partition chromatography is a chromatography method that depends on
the dividing of parts of a combination among static and mobile stages. It
has different sorts, for example, paper chromatography, gas–fluid
chromatography, liquid-liquid chromatography, and so forth. A real-
life example of partition chromatography is water clasped by cellulose,
paper, or silica.
 Fundamentals of Chromatography
 Chromatography is a partitioning strategy where the analyte is held
inside a fluid or gaseous mobile stage, which is siphoned through a
stationary stage.
 Typically, one stage is hydrophilic and the other lipophilic. The parts of
the analyte cooperate diversely with these two stages.
 Contingent upon the extremity they invest pretty much energy
cooperating with the stationary stage and are accordingly impeded to a
more noteworthy or lesser degree.
 Eventually, this contributes to the segregation of various elements
present in the sample.
