TOPIC: CHROMATOGRAPHY

SUBMITTED TO: SUBMITTED BY

DR.RENU SHARMA MAYANK ROY
CALSS SEC:264
ROLL NO.:29
REGISTRATION NO.10800597
ACKNOLEDGEMENT
I would like to thank my organic chemistry teacher
Dr. Renu Sharma for helping me to complete my term
Paper on topic chromatography and I am very
thankful to her for her valuable guidance and support
to complete my term paper without her my term
paper would not have been a success
Chromatography
Chromatography is a chemical technique for separating mixtures of coloured chemicals.
This technique is important in biology as well as chemistry; it is also used by forensic
scientists.
Introduction
The Experiment
The Secret
Introduction:
We all know that green plants are green because they contain chlorophyll; we know that
chlorophyll is green. Hang on; why are plants different shades of green if they all contain
the same green pigment? Well perhaps it is not quite as simple as you were told by your
biology teacher. !n fact there are se"eral different kinds of chlorophyll and some other
photosynthetic pigments in green plants. #o exactly what colour they are depends upon
which types of chlorophyll and other pigments their lea"es contain.
What has all this to do with chromatography?
What is chromatography and what can we do with it?
Well for a start we could separate the different pigments in lea"es and find out a bit more
about how photosynthesis works. $ea"es contain a mixture of two or more of the
following pigments% chlorophyll a chlorophyll b chlorophyll c xanthophyll carotene
phaeophytin and other pigments. The lea"es of different plants contain different
pigments hence their different colours. We could also use chromatography to separate the
different pigment in writing ink% a forensic scientist might do this to find out if all the
writing on a cheque was written with the same pen.
We can e"en use chromatography to separate mixtures of colourless chemicals% it is
possible to use the technique to separate the amino&acids produced when a protein is
digested. 'nce we ha"e separated the mixture it is necessary to stain the amino&acids
using a coloured chemical. (inhydrin is used; unfortunately this is not a "ery nice
chemical. #moke cigarettes if you must but don)t use carcinogenic chemicals without
taking the proper precautions.
Well, quite simply, it is a broad range of physical
methods used to separate and or to analyze complex
mixtures. The components to be separated are
distributed beteen to phases: a stationary phase
bed and a mobile phase ! mixture of "arious
components enters a chromatography process, and
the different components are flushed through the
system at different rates. These differential rates of
migration as the mixture mo"es o"er adsorpti"e
materials pro"ide separation. #epeated
sorption$desorption acts that ta%e place during the
mo"ement of the sample o"er the stationary bed
determine the rates. The smaller the affinity a
molecule has for the stationary phase, the shorter
the time spent in a So, Why Is It So Special&
In any chemical or bioprocessing industry, the need to separate and
purify a product from a complex mixture is a necessary and
important step in the production line. Today, there exists a wide
market of methods in which industries can accomplish these
goals. Chromatography is a v!" special separation process for
a multitude of reasons! First of all, it can separate complex
mixtures with great precision. Een ery similar components,
such as proteins that may only ary by a single amino acid, can
be separated with chromatography. In fact, chromatography
can purify basically any soluble or olatile substance if the right
adsorbent material, carrier fluid, and operating conditions are
employed. !econd, chromatography can be used to separate
delicate products since the conditions under which it is
performed are not typically seere. For these reasons,
chromatography is "uite well suited to a ariety of uses in the
field of biotechnology, such as separating mixtures of proteins.
column. which percolates The Different Types of Chromatography
There are four main types of chromatography. These are $iquid Chromatography *as
Chromatography Thin&$ayer Chromatography and +aper Chromatography.
'iquid (hromatography is used in the world to test water samples to look for pollution
in lakes and ri"ers. !t is used to analy,e metal ions and organic compounds in solutions.
$iquid chromatography uses liquids which may incorporate hydrophilic insoluble
molecules.
)as (hromatography is used in airports to detect bombs and is used is forensics in
many different ways. !t is used to analy,e fibers on a persons body and also analy,e
blood found at a crime scene. !n gas chromatography helium is used to mo"e a gaseous
mixture through a column of absorbent material.
Thin*layer (hromatography uses an absorbent material on flat glass or plastic plates.
This is a simple and rapid method to check the purity of an organic compound. !t is used
to detect pesticide or insecticide residues in food. Thin&layer chromatography is also used
in forensics to analy,e the dye composition of fibers.
+aper (hromatography is one of the most common types of chromatography. !t uses a
strip of paper as the stationary phase. Capillary action is used to pull the sol"ents up
through the paper and separate the solutes.
The analyte is the substance that is to be separated during chromatography.
!nalytical chromatography is used to determine the existence and possibly also
the concentration of analyte-s. in a sample.
/ bonded phase is a stationary phase that is co"alently bonded to the support
particles or to the inside wall of the column tubing.
/ chromatogram is the "isual output of the chromatograph. !n the case of an
optimal separation different peaks or patterns on the chromatogram correspond to
different components of the separated mixture.
+lotted on the x&axis is the retention time and plotted on the y&axis a signal -for
example obtained by a spectrophotometer mass spectrometer or a "ariety of other
detectors. corresponding to the response created by the analytes exiting the
system. !n the case of an optimal system the signal is proportional to the
concentration of the specific analyte separated.
/ chromatograph is equipment that enables a sophisticated separation e.g. gas
chromatographic or liquid chromatographic separation.
(hromatography is a physical method of separation in which the components to
be separated are distributed between two phases one of which is stationary
-stationary phase. while the other -the mobile phase. mo"es in a definite
direction.
The effluent is the mobile phase lea"ing the column.
/n immobilized phase is a stationary phase which is immobili,ed on the support
particles or on the inner wall of the column tubing.
The mobile phase is the phase which mo"es in a definite direction. !t may be a
liquid -$C and C0C. a gas -*C. or a supercritical fluid -supercritical&fluid
chromatography #1C.. / better definition% The mobile phase consists of the
sample being separated2analy,ed and the sol"ent that mo"es the sample through
the column. !n one case of H+$C the sol"ent consists of a carbonate2bicarbonate
solution and the sample is the anions being separated. The mobile phase mo"es
through the chromatography column -the stationary phase. where the sample
interacts with the stationary phase and is separated.
+reparati"e chromatography is used to purify sufficient quantities of a
substance for further use rather than analysis.
The retention time is the characteristic time it takes for a particular analyte to
pass through the system -from the column inlet to the detector. under set
conditions. #ee also% 3o"at)s retention index
The sample is the matter analysed in chromatography. !t may consist of a single
component or it may be a mixture of components. When the sample is treated in
the course of an analysis the phase or the phases containing the analytes of
interest is2are referred to as the sample whereas e"erything out of interest
separated from the sample before or in the course of the analysis is referred to as
waste.
The solute refers to the sample components in partition chromatography.
The sol"ent refers to any substance capable of solubili,ing other substance and
especially the liquid mobile phase in $C.
The stationary phase is the substance which is fixed in place for the
chromatography procedure. 0xamples include the silica layer in
Chromatography4Thin layer chromatography
thThe analyte is the substance that is to be separated during chromatography.
!nalytical chromatography is used to determine the existence and possibly also
the concentration of analyte-s. in a sample.
/ bonded phase is a stationary phase that is co"alently bonded to the support
particles or to the inside wall of the column tubing.
/ chromatogram is the "isual output of the chromatograph. !n the case of an
optimal separation different peaks or patterns on the chromatogram correspond to
different components of the separated mixture.
+lotted on the x&axis is the retention time and plotted on the y&axis a signal -for
example obtained by a spectrophotometer mass spectrometer or a "ariety of other
detectors. corresponding to the response created by the analytes exiting the
system. !n the case of an optimal system the signal is proportional to the
concentration of the specific analyte separated.
/ chromatograph is equipment that enables a sophisticated separation e.g. gas
chromatographic or liquid chromatographic separation.
(hromatography is a physical method of separation in which the components to
be separated are distributed between two phases one of which is stationary
-stationary phase. while the other -the mobile phase. mo"es in a definite
direction.
The effluent is the mobile phase lea"ing the column.
/n immobilized phase is a stationary phase which is immobili,ed on the support
particles or on the inner wall of the column tubing.
The mobile phase is the phase which mo"es in a definite direction. !t may be a
liquid -$C and C0C. a gas -*C. or a supercritical fluid -supercritical&fluid
chromatography #1C.. / better definition% The mobile phase consists of the
sample being separated2analy,ed and the sol"ent that mo"es the sample through
the column. !n one case of H+$C the sol"ent consists of a carbonate2bicarbonate
solution and the sample is the anions being separated. The mobile phase mo"es
through the chromatography column -the stationary phase. where the sample
interacts with the stationary phase and is separated.
+reparati"e chromatography is used to purify sufficient quantities of a
substance for further use rather than analysis.
The retention time is the characteristic time it takes for a particular analyte to
pass through the system -from the column inlet to the detector. under set
conditions. #ee also% 3o"at)s retention index
The sample is the matter analysed in chromatography. !t may consist of a single
component or it may be a mixture of components. When the sample is treated in
the course of an analysis the phase or the phases containing the analytes of
interest is2are referred to as the sample whereas e"erything out of interest
separated from the sample before or in the course of the analysis is referred to as
waste.
The solute refers to the sample components in partition chromatography.
The sol"ent refers to any substance capable of solubili,ing other substance and
especially the liquid mobile phase in $C.
The stationary phase is the substance which is fixed in place for the
chromatography procedure. 0xamples include the silica layer in
Chromatography4Thin layer chromatography
Techniques by chromatographic bed shape
Column chromatography
Column chromatography is a separation technique in which the stationary bed is within a
tube. The particles of the solid stationary phase or the support coated with a liquid
stationary phase may fill the whole inside "olume of the tube -packed column. or be
concentrated on or along the inside tube wall lea"ing an open unrestricted path for the
mobile phase in the middle part of the tube -open tubular column.. 5ifferences in rates of
mo"ement through the medium are calculated to different retention times of the sample.
678
!n 79:; W. C. #till introduced a modified "ersion of column chromatography called
flash column chromatography -flash..
6<8
The technique is "ery similar to the traditional
column chromatography except for that the sol"ent is dri"en through the column by
applying positi"e pressure. This allowed most separations to be performed in less than <=
minutes with impro"ed separations compared to the old method. >odern flash
chromatography systems are sold as pre&packed plastic cartridges and the sol"ent is
pumped through the cartridge. #ystems may also be linked with detectors and fraction
collectors pro"iding automation. The introduction of gradient pumps resulted in quicker
separations and less sol"ent usage.
/ spreadsheet that assists in the successful de"elopment of flash columns has been
de"eloped. The spreadsheet estimates the retention "olume and band "olume of analytes
the fraction numbers expected to contain each analyte and the resolution between
ad?acent peaks. This information allows users to select optimal parameters for
preparati"e&scale separations before the flash column itself is attempted.
6@8
!n expanded bed adsorption a fluidi,ed bed is used rather than a solid phase made by a
packed bed. This allows omission of initial clearing steps such as centrifugation and
filtration for culture broths or slurries of broken cells.
#lanar Chromatography
Thin layer chromatography is used to separate components of chlorophyll
+lanar chromatography is a separation technique in which the stationary phase is
present as or on a plane. The plane can be a paper ser"ing as such or impregnated by a
substance as the stationary bed -paper chromatography. or a layer of solid particles
spread on a support such as a glass plate -thin layer chromatography.. 5ifferent
compounds in the sample mixture tra"el different distances according to how strongly
they interact with the stationary phase as compared to the mobile phase . The specific
Aetardation factor -Af. of each chemical can be used to aid in the identification of an
unknown substance.
+aper (hromatography
For more details on this topic, see Paper chromatography.
+aper chromatography is a technique that in"ol"es placing a small dot of sample solution
onto a strip of chromatography paper. The paper is placed in a ?ar containing a shallow
layer of sol"ent and sealed. /s the sol"ent rises through the paper it meets the sample
mixture which starts to tra"el up the paper with the sol"ent. This paper is made of
cellulose a polar substance and the compounds within the mixture tra"el farther if they
are non&polar. >ore polar substances bond with the cellulose paper more quickly and
therefore do not tra"el as far.
Thin layer chromatography
For more details on this topic, see Thin layer chromatography.
Thin layer chromatography -T$C. is a widely&employed laboratory technique and is
similar to paper chromatography. Howe"er instead of using a stationary phase of paper
it in"ol"es a stationary phase of a thin layer of adsorbent like silica gel alumina or
cellulose on a flat inert substrate. Compared to paper it has the ad"antage of faster runs
better separations and the choice between different adsorbents. 1or e"en better resolution
and to allow for quantitation high&performance T$C can be used.
Techniques by physical state of mobile phase
$as chromatography
For more details on this topic, see Gas chromatography.
*as chromatography -*C. also sometimes known as *as&$iquid chromatography
-*$C. is a separation technique in which the mobile phase is a gas. *as chromatography
is always carried out in a column which is typically BpackedB or BcapillaryB -see below. .
*as chromatography -*C. is based on a partition equilibrium of analyte between a solid
stationary phase -often a liquid silicone&based material. and a mobile gas -most often
Helium.. The stationary phase is adhered to the inside of a small&diameter glass tube -a
capillary column. or a solid matrix inside a larger metal tube -a packed column.. !t is
widely used in analytical chemistry; though the high temperatures used in *C make it
unsuitable for high molecular weight biopolymers or proteins -heat will denature them.
frequently encountered in biochemistry it is well suited for use in the petrochemical
en"ironmental monitoring and industrial chemical fields. !t is also used extensi"ely in
chemistry research.
%i"uid chromatography
$iquid chromatography -$C. is a separation technique in which the mobile phase is a
liquid. $iquid chromatography can be carried out either in a column or a plane. +resent
day liquid chromatography that generally utili,es "ery small packing particles and a
relati"ely high pressure is referred to as high performance liquid chromatography
-H+$C..
!n the H+$C technique the sample is forced through a column that is packed with
irregularly or spherically shaped particles or a porous monolithic layer -stationary phase.
by a liquid -mobile phase. at high pressure. H+$C is historically di"ided into two
different sub&classes based on the polarity of the mobile and stationary phases. Technique
in which the stationary phase is more polar than the mobile phase -e.g. toluene as the
mobile phase silica as the stationary phase. is called normal phase liquid
chromatography -(+$C. and the opposite -e.g. water&methanol mixture as the mobile
phase and C7; C octadecylsilyl as the stationary phase. is called re"ersed phase liquid
chromatography -A+$C.. !ronically the Bnormal phaseB has fewer applications and A+$C
is therefore used considerably more.
#pecific techniques which come under this broad heading are listed below. !t should also
be noted that the following techniques can also be considered fast protein liquid
chromatography if no pressure is used to dri"e the mobile phase through the stationary
phase. #ee also /queous (ormal +hase Chromatography.
!ffinity chromatography
For more details on this topic, see Affinity chromatography.
/ffinity chromatography
6D8
is based on selecti"e non&co"alent interaction between an
analyte and specific molecules. !t is "ery specific but not "ery robust. !t is often used in
biochemistry in the purification of proteins bound to tags. These fusion proteins are
labelled with compounds such as His&tags biotin or antigens which bind to the stationary
phase specifically. /fter purification some of these tags are usually remo"ed and the pure
protein is obtained.
!upercritical fluid chromatography
#upercritical fluid chromatography is a separation technique in which the mobile phase is
a fluid abo"e and relati"ely close to its critical temperature and pressure.
Techniques by separation mechanism
Ion exchange chromatography
For more details on this topic, see Ion exchange chromatography.
!on exchange chromatography utili,es ion exchange mechanism to separate analytes. !t is
usually performed in columns but the mechanism can be benefited also in planar mode.
!on exchange chromatography uses a charged stationary phase to separate charged
compounds including amino acids peptides and proteins. !n con"entional methods the
stationary phase is an ion exchange resin that carries charged functional groups which
interact with oppositely charged groups of the compound to be retained. !on exchange
chromatography is commonly used to purify proteins using 1+$C.
!i&e exclusion chromatography
#i,e exclusion chromatography -#0C. is also known as gel permeation
chromatography -*+C. or gel filtration chromatography and separates molecules
according to their si,e -or more accurately according to their hydrodynamic diameter or
hydrodynamic "olume.. #maller molecules are able to enter the pores of the media and
therefore take longer to elute whereas larger molecules are excluded from the pores and
elute faster. !t is generally a low resolution chromatography technique and thus it is often
reser"ed for the final BpolishingB step of a purification. !t is also useful for determining
the tertiary structure and quaternary structure of purified proteins especially since it can
be carried out under nati"e solution conditions.
Special techniques
'eersed(phase chromatography
Ae"ersed&phase chromatography is an elution procedure used in liquid chromatography
in which the mobile phase is significantly more polar than the stationary phase.
+lease help impro"e this section by expanding it. 1urther information might be
found on the talk page. (September 2!"
Two(dimensional chromatography
!n some cases the chemistry within a gi"en column can be insufficient to separate some
analytes. !t is possible to direct a series of unresol"ed peaks onto a second column with
different physico&chemical -Chemical classification. properties. #ince 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.
Fast protein li"uid chromatography
1ast protein liquid chromatography -1+$C. is a term applied to se"eral chromatography
techniques which are used to purify proteins. >any of these techniques are identical to
those carried out under high performance liquid chromatography howe"er use of 1+$C
techniques are typically for preparing large scale batches of a purified product.
) Countercurrent chromatography
.
Countercurrent chromatography -CCC. is a type of liquid&liquid chromatography where
both the stationary and mobile phases are liquids. !t in"ol"es mixing a solution of liquids
allowing them to settle into layers and then separating the layers.
Chiral chromatography
Chiral chromatography in"ol"es the separation of stereoisomers. !n the case of
enantiomers these ha"e no chemical or physical differences apart from being three
dimensional mirror images. Con"entional 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 themsel"es be made chiral gi"ing differing
affinities between the analytes. Chiral chromatography H+$C columns -with a chiral
stationary phase. in both normal and re"ersed phase are commercially a"ailable.
rough the stationary bed.
BIBLIOGRAPHY
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