What is Chromatography?

Chromatography is a technique for separating mixtures into their components in

order to analyze, identify, purify, and/or quantify the mixture or components.

Uses for Chromatography

Chromatography is used by scientists to:

Analyze – examine a mixture, its components, and their relations to one another

Identify – determine the identity of a mixture or components based on known

components

Purify – separate components in order to isolate one of interest for further study

Quantify – determine the amount of the a mixture and/or the components present in
the sample

Detailed Definition

Chromatography is a laboratory technique that separates components within a

mixture by using the differential affinities of the components for a mobile medium and
for a stationary adsorbing medium through which they pass. Chromatography
separates the components of a mixture by their distinctive attraction to the mobile
phase and the stationary phase.

Terminology:

Mobile Medium – gas or liquid that carries the components ( mobile phase )

Stationary Medium – the part of the apparatus that does not move with the sample (
stationary phase )

Explanation:

 Compound is placed on stationary phase

 Mobile phase passes through the stationary phase
 Mobile phase solubilizes the components
 Mobile phase carries the individual components a certain distance through the
stationary phase, depending on their attraction to both of the phases

Types of Chromatography

1. Column chromatography: It is the most widely used chromatography

which find daily use in research labs and industries. It is easy to operate
and less expensive technique. As the name indicates, there is a lengthy
column which is suspended in the air with the help of a stand.
2. High performance liquid chromatography (HPLC): This is similar to
column chromatography but high pressure is employed for effective
separation of compounds. The mobile phase is pumped into the column at
a defined pressure and further the column particles are very small so the
surface area is high and better separation takes place.
3. Gas chromatography (GC): Here gas is used as mobile phase and a
solid or liquid layer as the stationary phase.

4. Ion-exchange chromatography: Here the mobile phase is charged and

sample molecules with similar charge present on the charged stationary
phase get eluted out as the mobile phase molecules with charge displace
them.
5. Size exclusion chromatography. Here the column is loaded with
charged some gel having pores. Sample particles when poured along with
mobile phase have to pass through the sieve like net work of the stationary
phase. In doing so, the larger particles elute out first and smaller ones last.
The reason is the smaller ones take longer path in the column stationary
phase while larger particles take short path to elute out.
6. Thin layer chromatography (TLC): Here the stationary phase is a thin
layer and plate like. The plate is immersed in a solvent up to a small height
so that the mobile phase travels up leading to separation of compounds.

7. High performance thin layer chromatography (HPTLC): Similar to TLC

but more efficient. Read more about HPTLC.
8. Paper chromatography: As the name indicates, here a paper is used as
a stationary phase. The paper can be rectangular or circular one. The
sample is marked on the whatmanns paper and then placed in the solvent
vessel to allow it to percolate through the paper.
9. Super critical fluid chromatography: It is a normal phase
chromatography with instrumentation similar to HPLC. Here mobile phase
is mostly a super critical fluid like carbon dioxide.
10. LC-MS: Liquid chromatography combines with Mass spectroscopy
(detector)
The Application of Electric Field in Chromatography

Gel electrophoresis chromatography

Gel electrophoresis is a form of chromatography. Specifically, it is a kind of

size-exclusion chromatography, a method in which chemical components are
separated by their physical size. In gel electrophoresis, this is accomplished by
applying an electrical current to a gel matrix, which functions as a molecular sieve.

Agarose and polyacrylamide gels are the most commonly used types of
gels, and each has its merits. For instance, polyacrylamide gels have a uniform pore
size and very high resolving power, which allows for greater clarity of results.
Because these gels contain no charge, they are non-reactive. They are more
effective for analyzing single-stranded or very small segments of DNA (those
that are made up of only a few base pairs) than agarose gels. However, their
components are neurotoxic before undergoing the chemical reaction that causes
the gel to set, and the gel must be handled with care throughout the entire
experiment because of potential remaining free acrylamide..
Agarose, a polysaccharide that is naturally derived from seaweed, is more
frequently used in laboratories that perform DNA analysis. It is optimal for fragments
containing 50 to 20,000 base pairs, although it is possible to resolve larger
molecules through the use of a technique called pulse field gel electrophoresis
(PFGE). Though the gel may sometimes break or not solidify evenly (which is rarely
the case for polyacrylamide gels), it is easy to create, sets quickly, and is non-toxic.
In order for agarose gel to set, it is heated to a near-boiling point and then allowed to
cool.

After a type of gel has been selected, its components are mixed with a
buffer solution, poured into a cartridge or cassette, and then allowed to set
around a comb which, once removed, will leave behind wells into which the samples
to be run are loaded. (Usually, these samples are no more than a few micrograms,
and so they require the use of a micropipette or syringe for transfer.) Agarose gel
matrices are oriented horizontally and remain lying flat for the entirety of the
procedure. Conversely, polyacrylamide gels sit vertically in their electrophoretic
chambers.

The addition of buffers is necessary to allow electricity to flow through

the matrix. Therefore, once the gel has been cast, it is perfused with a second
buffer solution before being placed into an electrophoretic chamber. Molecules of
DNA and RNA are negatively charged and require no further preparation, but
some substances, like proteins, are not intrinsically charged and require the use of a
detergent like sodium dodecyl sulfate (SDS, also called sodium lauryl sulfate) to
become so.

The gel must be allowed to set completely before the scientist attaches
electrodes to the apparatus and applies an electrical current, which runs from
the negative to the positive node. In an electrolytic cell, such as the kind created
during gel electrophoresis, this means that the current flows from cathode to anode
(the opposite is true of galvanic cells), and so the cathode must be attached to the
end of the matrix containing the sample wells.

Drawn forth by their electrical charge, the molecules soon begin to move
along in parallel rows. Smaller segments have an easier time getting through the
pores of the gel matrix, and so they travel faster and therefore end up farther along
than those molecules with a greater molecular weight or diameter. In some cases,
substances of a known molecular weight are added to the wells to serve as a
reference for the materials being evaluated.

Often, a dye or stain is added to allow the gel to be more easily read at the
conclusion of the experiment. Ethidium bromide fluoresces under ultraviolet light
and is used for visualization of DNA fragments. If a sample contains any
radioactivity, as is occasionally the case in DNA sequencing, it may be read by a
device called an autoradiogram. For proteins, a silver stain or a dye called
Coomassie Brilliant Blue is conventionally used. A scientist may take photograph of
the results, or, in the case of agarose gel, he or she may simply keep the gel in the
refrigerator for future reference.
 The applications of gel electrophoresis are many and far-reaching. It is used
in forensics labs to analyze DNA and by virologists to study different strains of
viruses. Biochemists use the process to study cellular components. In the field
of genetics, it is a useful tool for isolating a desired sample, which may then be
amplified by a technique called Polymerase Chain Reaction (PCR) and
subjected to further experimentation.