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1. Chromatography
Chromatography is the term used to describe a separation technique in which a mobile
phase carrying a mixture is caused to move in contact with a selectively absorbent stationary
phase. There are number of different kinds of chromatography, which differ in the mobile
and the stationary phase used.
Chromatography is derived from a Greek word χρῶμα means chroma (colour) and γράφειν
means graphein (to write). Chromatography is the laboratory techniques used for the
separation of mixtures.
The mixture is dissolved in a fluid called the mobile phase, which carries it through a
structure holding another material called the stationary phase. The various constituents of the
mixture travel at different speeds, causing them to separate. The separation is based on
differential partitioning between the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on the stationary phase and
thus changing the separation.
Chromatography may be preparative or analytical. The purpose of preparative
chromatography is to separate the components of a mixture for more advanced use (and is
thus a form of purification). Analytical chromatography is done normally with smaller
amounts of material and is for measuring the relative proportions of analytes in a mixture.
The two are not mutually exclusive.

2. History of chromatography.......
Chromatography, literally "color writing", was first employed by Russian scientist Mikhail
Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th
century, primarily for the separation of plant pigments such as chlorophyll, carotenes,
and xanthophylls. Since these components have different colours (green, orange, and yellow,
respectively) they gave the technique its name. New types of chromatography developed
during the 1930s and 1940s made the technique useful for many separation processes.
Chromatography technique developed substantially as a result of the work of Archer John
Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They
established the principles and basic techniques of partition chromatography, and their work
encouraged the rapid development of several chromatographic methods: Paper
Chromatography, Gas Chromatography, and what would become known as high
performance liquid chromatography. Since then, the technology has advanced rapidly.
Researchers found that the main principles of Tsvet's chromatography could be applied in
many different ways, resulting in the different varieties of chromatography described below.
Advances are continually improving the technical performance of chromatography, allowing
the separation of increasingly similar molecules.
Techniques by chromatographic bed shape.......
1. paper chromatography
In Paper Chromatography, the mobile phase is a
solvent and the stationary phase is water held in the
fibres of chromatography paper. A solution of the
mixture to be separated is spotted onto a strip of
chromatography paper (or filter paper) with a dropper.
The chromatogram is developed by placing the bottom
of the paper (but not the sample spot) in a tank
containing suitable solvent. The solvent is drawn up the
paper by capillary action. The components of the mixture move up the paper with the solvent
at different rates due to their differing interactions with the stationary and mobile phases.
FormulaRf = Distance the solute moves
Distance the solvent front move

3. 2. Column chromatography
In column chromatography, the mobile phase
is again a solvent, and the stationary phase is a
finely divided solid, such as silica gel or
alumina. Chromatography columns vary in size
and polarity. There is an element of trial and
error involved in selecting a suitable solvent
and column for the separation of the
constituents of a particular mixture. A small
volume of the sample whose constituents are to
be separated is placed on top of the column.
The choice of the eluting solvent should ensure
that the sample is soluble. However, if the
sample was too soluble the mobile phase
(solvent) would move the solutes too quickly,
resulting in the non-separation of the different
constituents.
3. Thin layer chromatography
In thin layer chromatography, the
mobile phase is also a solvent, and
the stationary phase is a thin layer
of finely divided solid, such as
silica gel or alumina, supported on
glass or aluminium. Thin layer
chromatography is similar to paper
chromatography in that it involves
spotting the mixture on the plate
and the solvent (mobile phase) rises up the plate in the chromatography
tank. It has an advantage over paper chromatography in that its

4. separations are very efficient because of the much smaller size of the
particles in the stationary phase.
Thin layer chromatography is particularly useful in forensic work, for example
in the separation of dyes from fibres. Gas chromatography and high
performance liquid chromatography are more sophisticated chromatographic
techniques.
4. Gas chromatography
A gas is the mobile phase and the stationary phase can be either a solid or a
non- volatile liquid.
There are five basic GC components:
1) Pneumatic system – gas supply (flow control and measurement).
2) Injection system – a heated injector port, where the sample is vaporised
if necessary.
3) Column – where the separation occurs.
4) Oven –The coiled column is wholly contained in a thermostatically
controlled oven.
5) Detector – integral detector or link to a mass spectrometer.
How does gas chromatography work.........?
1) A carrier gas, examples of which are Helium and Neon flows through the
system. A valve controls the flow rate.
2) A sample of the volatile mixture is injected into the carrier gas. The
sample is vaporised in the heated injector port.

5. 3) The carrier gas carries the vaporised sample into the column. The columns
are stainless steel or glass tubes. They can be up to 25 m in length and are
of narrow bore (2-10 mm). Therefore the column is often wound into a
coil. The packed columns contain porous support material. The sample
mixture undergoes a series of interactions between the stationary and
mobile phases as it is carried through the system by the carrier gas. Due
to the wide choice of materials available for the stationary and mobile
phases, it is possible to separate molecules that differ only slightly in their
physical and chemical properties.
4) The coiled column is contained in the thermostatically controlled oven.
5) Separated components emerge in the order of increasing interaction with
the stationary phase. The least retarded component comes through first.
Separation is obtained when one compound is sufficiently retarded to
prevent overlap with another component of the sample, as it emerges
from the column.
6) Two types of detector can be used:
(1) Thermal Conductivity detectors which respond to changes in the thermal
conductivity of the gas leaving the column and
(2) Flame Ionisation detection (FID), which is more commonly used. In
thermal conductivity, as the carrier gas leaves the column, it cools the
detector. When a solute emerges with the carrier gas, it does not cool the
detector to the same extent. Alternatively, samples can be passed from the
oven directly into a mass spectrometer, where they are analysed.
Retention time is defined as the time taken for a component to go from injection
to detection. This varies depending on
a) The nature of and the interactions between the solute and the stationary
and mobile phases.
b) The flow rate of the carrier gas,
c) The temperature of the column (shorter retention times are obtained at
higher temperatures),
d) The length and diameter of the column,

6. Once GC has separated a mixture, the components can be identified using
known retention times. For unknown compounds the solutes are collected
individually and analysed using another method, e.g. mass spectrometry.
For each compound in a mixture one peak is observed on the chromatogram. In
the particular set of operating conditions relating to the column, the retention
time will increase with the size and polarity of the compound. To find the
concentration of a particular compound, the peak height should be measured.
GC is used to analyse blood samples for the presence of alcohol. It is also used
to analyse samples taken from athletes to check for the presence of drugs. In
each case, it separates the components of the mixture and indicates the
concentrations of the components. Water companies test samples of water for
pollutants using GC to separate the pollutants, and mass spectrometry to
identify them.
GC is used to analyse blood samples for the presence of alcohol. It is also used
to analyse samples taken from athletes to check for the presence of drugs. In
each case, it separates the components of the mixture and indicates the
concentrations of the components. Water companies test samples of water for
pollutants using GC to separate the pollutants, and mass spectrometry to
identify them.

7. 5. High performance liquid chromatography
Basic Components:
1) Solvent Reservoir.
2) The Pump System controls the flow and measures the volume of solvent
(the mobile phase). The flow rates of HPLC columns are slow – often in
3
-1
the range of 0.5 - 5 cm min .
3) The Injector System: The sample to be separated is injected into the
liquid phase at this point.
4) The Column is made of steel and packed usually with porous silica
particles (the stationary phase). Different materials can be used depending
on the nature of the liquid. A long column is not needed because
separation in HPLC is very efficient. Columns are usually 10 –30 cm
long, with an internal diameter of 4 mm.
Different components of the sample are carried forward at different
rates by the moving liquid phase, due to their differing interactions
with the stationary and mobile phases.
5) The Detector: When the components reach the end of the column they
are analysed by a detector. The amounts passing through the column are
small, so solutes are analysed as they leave the column. Therefore HPLC
is usually linked to a spectrometer (e.g. ultra violet or mass
spectrometry).
The length of time it takes for a compound to reach the detector allows
the component to be identified. Like the GC, once the retention time
of a solute has been established for a column using a particular set of
operating conditions, the solute can be identified in a mixture. A
chromatogram is obtained for the sample.

8. Uses
HPLC has many uses such as drug testing, testing for vitamins in food and
growth promoters in meat. In each case components of the mixture are
separated and detected.
Comparison of HPLC over GC
Less volatile and larger samples can be used with HPLC.

9. Chromatogram and Mass Spectrometry Data
6. Affinity chromatography
Affinity chromatography is based on selective non-covalent
interaction between an analyte and specific molecules. It is very
specific, but not very robust. It is often used in biochemistry in the
purification of proteins bound to tags. These fusion proteins are
labelled with compounds such as his-tags, biotin or antigens, which
bind to the stationary phase specifically. After purification, some of
these tags are usually removed and the pure protein is obtained.
Affinity chromatography often utilizes a bio-molecule's affinity for a
metal (Zn, Cu, Fe, etc.). Columns are often manually prepared.
Traditional affinity columns are used as a preparative step to flush out
unwanted bio-molecules. However, HPLC techniques exist that do
utilize affinity chromatography properties. Immobilized Metal
Affinity Chromatography (IMAC) is useful to separate
aforementioned molecules based on the relative affinity for the metal
(i.e. Dionex IMAC). Often these columns can be loaded with different
metals to create a column with a targeted affinity.

10. 7. Super critical fluid chromatography
Supercritical fluid chromatography is a separation technique in which the
mobile phase is a fluid above and relatively close to its critical temperature and
pressure.
Techniques by separation mechanism..........
8. Ion exchange chromatography
Ion exchange chromatography (usually referred to as ion chromatography) uses
an ion exchange mechanism to separate analytes based on their respective
charges. It is usually performed in columns but can also be useful in planar
mode. Ion exchange chromatography uses a charged stationary phase to
separate charged compounds including anions, cations, amino acids, peptides,
and proteins. In conventional methods the stationary phase is an ion exchange
resin that carries charged functional groups that interact with oppositely charged
groups of the compound to retain. Ion exchange chromatography is commonly
used to purify proteins using FPLC.
9. Size exclusion chromatography
Size-exclusion chromatography (SEC) is also known as Gel Permeation
Chromatography (GPC) or Gel Filtration Fhromatography and separates
molecules according to their size (or more accurately according to their
hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able
to enter the pores of the media and, therefore, molecules are trapped and
removed from the flow of the mobile phase. The average residence time in the
pores depends upon the effective size of the analyte molecules. However,
molecules that are larger than the average pore size of the packing are excluded
and thus suffer essentially no retention; such species are the first to be eluted. It
is generally a low-resolution chromatography technique and thus it is often
reserved for the final, "polishing" step of a purification. It is also useful for
determining the tertiary structure and quaternary stucture of purified proteins,
especially since it can be carried out under native solution conditions.

11. Special techniques..........
10. reversed phase chromatography
Reversed-phase chromatography is an elution procedure used in liquid
chromatography in which the mobile phase is significantly more polar than the
stationary phase.
11. two-dimensional chromatography
In some cases, the chemistry within a given column can be insufficient to
separate some analytes. It is possible to direct a series of unresolved peaks onto
a second column with different physico-chemical (Chemical classification)
properties. Since the mechanism of retention on this new solid support is
different from the first dimensional separation, it can be possible to separate
compounds that are indistinguishable by one-dimensional chromatography. The
sample is spotted at one corner of a square plate, developed, air-dried, then
rotated by 90° and usually redeveloped in a second solvent system.
12. pyrolysis gas chromatography
Pyrolysis gas chromatography mass spectrometry is a method of chemical
analysis in which the sample is heated to decomposition to produce smaller
molecules that are separated by gas chromatography and detected using mass
spectrometry.
Pyrolysis is the thermal decomposition of materials in an inert atmosphere or a
vacuum. The sample is put into direct contact with a platinum wire, or placed in
a quartz sample tube, and rapidly heated to 600–1000 °C. Depending on the
application even higher temperatures are used. Three different heating
techniques are used in actual pyrolyzers: Isothermal furnace, inductive heating
(Curie Point filament), and resistive heating using platinum filaments. Large
molecules cleave at their weakest points and produce smaller, more volatile
fragments. These fragments can be separated by gas chromatography. Pyrolysis
GC chromatograms are typically complex because a wide range of different
decomposition products is formed. The data can either be used as fingerprint to

12. prove material identity or the GC/MS data is used to identify individual
fragments to obtain structural information. To increase the volatility of polar
fragments, various methylating reagents can be added to a sample before
pyrolysis.
Besides the usage of dedicated pyrolyzers, pyrolysis GC of solid and liquid
samples can be performed directly inside Programmable Temperature Vaporizer
(PTV) injectors that provide quick heating (up to 30 °C/s) and high maximum
temperatures of 600–650 °C. This is sufficient for some pyrolysis applications.
The main advantage is that no dedicated instrument has to be purchased and
pyrolysis can be performed as part of routine GC analysis. In this case quartz
GC inlet liners have to be used. Quantitative data can be acquired, and good
results of derivatization inside the PTV injector are published as well.
13. fast protein liquid chromatography
Fast protein liquid chromatography (FPLC) is a term applied to several
chromatography techniques which are used to purify proteins. Many of these
techniques are identical to those carried out under high performance liquid
chromatography, however use of FPLC techniques are typically for preparing
large scale batches of a purified product.
14. counter-current chromatography
Counter-current chromatography
(CCC) is a type of liquid-liquid
chromatography, where both the
stationary and mobile phases are
liquids. The operating principle of
CCC equipment requires a column
consisting of an open tube coiled
around a bobbin. The bobbin is
rotated in a double-axis gyratory
motion (a cardioid), which causes a
variable gravity (G) field to act on
(An example of HPCCC system)
the column during each rotation. This motion causes the column to see one
partitioning step per revolution and components of the sample separate in the

13. column due to their partitioning coefficient between the two immiscible liquid
phases used. There are many types of CCC available today. These include
HSCCC (High Speed CCC) and HPCCC (High Performance CCC). HPCCC is
the latest and best performing version of the instrumentation available currently.
15. chiral chromatography
Chiral chromatography involves the separation of stereo-isomers. In the case of
en-antiomers, these have no chemical or physical differences apart from being
three-dimensional mirror images. Conventional chromatography or other
separation processes are incapable of separating them. To enable chiral
separations to take place, either the mobile phase or the stationary phase must
themselves be made chiral, giving differing affinities between the
analytes. Chiral Chromatography HPLC Columns (with a chiral stationary
phase) in both normal and reversed phase are commercially available.

14. Source page
1. www.wikipedia.org
2. www.google.com
3. Physics and Chemistry Support Science (PCSS)