Chromatography

Chromatography is an important biophysical technique that enables the

separation, identification, and purification of the components of a
mixture for qualitative and quantitative analysis. All Chromatography is
base on two phases, stationary phase and mobile phase.

There are primarily four different types of chromatography:

1) Paper chromatography.
2) Thin-layer chromatography (TLC)
3) Gas chromatography,
4) High-performance liquid chromatography(HPLC)
 Stationary phase in chromatography is the one which does not
move with the sample whereas mobile phase in chromatography is
one which moves with the sample.

Example: In Paper Chromatography:

The paper strip acts as "stationary phase" while the solvent act as
"mobile phase" in paper chromatography.

In paper chromatography and in thin-layer chromatography, the

retention factor (Rf) is used to compare and help identify
compounds. The Rf value of a compound is equal to the distance
traveled by the compound divided by the distance traveled by the
solvent.
Paper chromatography: is one type of chromatography
that enables the separation, identification, and purification of
the components of a mixture for qualitative and quantitative
analysis. Paper chromatography is also an example of
Partition chromatography.

In paper chromatography Stationary Phase is a paper (filter

paper) which does not move and is fixed firmly while
mobile phase is a solvent (water or organic solvents) that
rises up the filter paper (stationary phase) and reacts with the
sample.

Components of the mixture react with both phases. The

higher a components polarity the faster it reacts with and
move with the water or organic solvent (mobile phase).Less
polar components will react better with the stationary phase
(the paper) and travel slower.
 Chromatographic experiment is basically a three-step process:
1) Application of the sample,
2) Developing the chromatogram by allowing the mobile phase to
move up the paper,
3) Calculating Rf (Retardation factor) values and making conclusions

Rf = distance travel by solute (analyte)

distance travel by solvent

Rf value is always less than one because solutes must have some attractive
qualities with stationary phases, while the solvent always travels more
than the solute.
Rf values can be used to identify unknown chemicals if they can be compared to a
range of reference substances.

The Rf value for a particular substance is always the same if the same solvent,
same thickness of stationary phase, same amount of spotted samples are used at
same temperature.

The stronger a compound is bound to

the adsorbent , the slower it moves up
the TLC plate. Non-polar compounds
move up the plate most rapidly (higher
Rf value), whereas polar substances
travel up the TLC plate slowly or not at
all (lower Rf value).
Thin-layer chromatography is a chromatography technique
that separates components in non-volatile mixtures. It is
performed on a TLC plate made up of a non-reactive
solid(glass or alumina) coated with a thin layer of adsorbent
material(silica gel). This is called the stationary phase

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.
Application of TLC
TLC is extremely useful in Biochemical analysis
such as separation or isolation of biochemical
metabolites from its blood plasma, urine, body
fluids, serum, etc. Thin layer chromatography
can be used to identify natural products like
essential oils or volatile oil, fixed oil,
glycosides, waxes, alkaloids, etc.
The Advantages of Chromatography
• Precise separation, analyses, and purification is
possible using chromatography.
• It requires very low sample volumes.
• It works on a wide range of samples including drugs,
food particles, plastics, pesticides, air and water
samples, and tissue extracts.
• Chromatography is used for the separation of a mixture.
• It is used to test drug levels and water purity.
• It is also used to determine the nutritional value of the
food sample.
• It is used to determine the type of chlorophyll in various
photosynthetic organisms.

(Application of Chromatography - Pharmaceuticals,

clinical trials, environmental and chemical safety, food
and beverage, drug testing, forensics, petroleum
creation, and molecular biology)
 Column chromatography consists of a stationary solid phase that
adsorbs and separates the compounds passing through it with the help
of a liquid mobile phase. On the basis of their chemical nature,
compounds get adsorbed and elution is based on differential
adsorption of a substance by the adsorbent.

Column chromatography in chemistry is a chromatography method used

to isolate a single chemical compound from a mixture.

Eluent is slowly passed through the column to advance the organic

material. The individual components are retained by the stationary phase
differently and separate from each other while they are running at
different speeds through the column with the eluent. At the end of the
column they elute one at a time.
Gas chromatography (GC) is a common analytical technique used to separate
and analyze volatile and semi-volatile compounds in a mixture.

Gas chromatography (GC) is one of the popular chromatography techniques to

separate volatile compounds or substances. The mobile phase is a gas such as
helium, and the stationary phase is a high-boiling liquid that is adsorbed on a
solid

The food industry relies on the technique of gas chromatography for several
applications, including the quantitative and qualitative analysis of food, the
analysis of food additives, components of flavor and aroma, and the detection
and analysis of contaminants such as environmental pollutants, pesticides,
fumigants, ..
It was obvious that gas phase separation and analysis of very polar high
molecular weight biopolymers was impossible.

GC was ineffective for many life science and health applications for
biomolecules, because they are mostly non-volatile and thermally unstable
at the high temperatures of GC. As a result, alternative methods were
hypothesized which result in the development of HPLC.
High-performance liquid chromatography (HPLC), formerly
referred to as high-pressure liquid chromatography, is a
technique in analytical chemistry used to separate, identify, and
quantify specific components in mixtures. The mixtures can originate
from food, chemicals, pharmaceuticals, biological, environmental
and agricultuer etc, which have been dissolved into liquid solutions.

It relies on high pressure pumps, which deliver mixtures of various

solvents, called the mobile phase, which flows through the
system, collecting the sample mixture on the way, delivering it into
a cylinder, called the column, filled with solid particles, made
of adsorbent material, called the stationary phase.
Each component in the sample interacts differently with the adsorbent material,
causing different migration rates for each component. These different rates lead to
separation as the species flow out of the column into a specific detector such as UV
detectors. The output of the detector is a graph, called a chromatogram.

Chromatograms are graphical representations of the signal intensity versus time or

volume, showing peaks, which represent components of the sample. Each sample
appears in its respective time, called its retention time, having area proportional to its
amount.
A modern self-contained HPLC
 Chromatography can be described as a mass transfer process
involving adsorption and/or partition . As mentioned, HPLC relies on
pumps to pass a pressurized liquid and a sample mixture through a
column filled with adsorbent, leading to the separation of the sample
components. The active component of the column, the adsorbent, is
typically a granular material made of solid particles (e.g., silica gel,
polymers, etc.), 1.5–50 μm in size
Applications
Research
Gas chromatography is fundamental to many research areas, in particular,
for the analysis of meteorites and natural products.
use gas chromatography to analyze the composition of meteorites that fall
to the earth.
This provides vitally important information regarding the nature of life
outside of earth, as well as revealing details about primitive life on earth.
Specifically, many studies have been conducted with gas chromatography
to determine the presence of ribose in meteorites, the building block of
RNA.
Forensics
For many years gas chromatography has been used in forensic
science. Mostly, it is used to determine the circumstances of a
person’s death, such as whether they ingested poison, or consumed
drugs or alcohol in the hours prior. Scientists take samples of blood
and fibers from the crime scene and analyze them using gas
chromatography to help investigators piece together the facts.
Quality control
Manufacturing relies on gas chromatography for quality control, companies that
produce cars, chemicals, and pharmaceuticals, in particular, are big adopters of the
technology.

The pharmaceutical industry uses gas chromatography to help produce pure products
in large quantities. The method is used to ensure the purity of the produced material,
eliminated inconsistencies in pharmaceutical products.

The industry also uses gas chromatography to analyze compounds to check for trace
contaminants. Currently, there is also a growing use of the method within the
pharmaceutical industry to separate chiral compounds.

Producers of chemicals also heavily rely on gas chromatography, particularly when

making emulsifiers, solvents, and co-solvents, to ensure that they maintain the same
quality when production is scaled up.
Studies have shown that the interiors of new automobiles
release a significant amount of volatile organic compounds
(VOCs). For this reason, gas chromatography has been adopted
by the automotive industry to identify and measure the
chemicals that are released into the air inside the car from its
carpets, door linings, pedals, seat covers, and other interior
materials. Scientists aim to reduce the levels of harmful toxins
released into the interiors of new vehicles.
Partition chromatography was one of the first kinds of
chromatography that chemists developed, and is barely used
these days. The partition coefficient principle has been applied in
paper chromatography, thin layer chromatography, gas
chromatography and liquid - liquid applications.

Partition chromatography uses a retained solvent, on the

surface or within the grains or fibers of an "inert" solid
supporting matrix as with paper chromatography interaction with
the stationary phase. Analyte molecules partition between a
liquid stationary phase and the eluent.