3. The word Chromatography is derived from two Greek words ,
Chroma means colour and graphein to write.
Chromatography is a laboratory technique for the separation of a
mixture.
Chromatography is an important biophysical technique that enables
the separation, identification, and purification of the components of
a mixture for qualitative and quantitative analysis.
Chromatography, literally "color writing", was used , and named in
the first decade of the 20th century, primarily for the separation of
plant pigments such as chlorophyll.
4. • 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.
5. Chromatography is a technique for separating mixtures into their
components to analyze, identify, purify, and/or quantify the mixture or
components. The components to be separated are distributed between two
phases i.e. stationary phases and mobile phase.
IMPORTANT TERMS
• Analyte – the substance to be separated during chromatography. It is also
normally what is needed from the mixture.
• Analytical chromatography – the use of chromatography to determine the
existence and possibly also the concentration of analyte(s) in a sample.
• Bonded phase – a stationary phase that is covalently bonded to the
support particles or to the inside wall of the column tubing.
• Chromatogram – the visual output of the chromatograph. In the case of an
optimal separation, different peaks or patterns on the chromatogram
correspond to different components of the separated mixture.
6. • Chromatograph – an instrument that enables a sophisticated separation, e.g. gas
chromatographic or liquid chromatographic separation.
• Eluent (sometimes spelled eluant) – the solvent or solvent mixure used in elution
chromatography and is synonymous with mobile phase.[7]
• Eluate – the mixture of solute (see Eluite) and solvent (see Eluent) exiting the column.[7]
• Effluent – the stream flowing out of a chromatographic column. In practise, it is used
synonymously with eluate, but the term more precisely refers to the stream independent
of separation taking place.[7]
• Eluite – a more precise term for solute or analyte. It is a sample component leaving the
chromatographic column.[7]
• Eluotropic series – a list of solvents ranked according to their eluting power.
• Immobilized phase – a stationary phase that is immobilized on the support particles, or
on the inner wall of the column tubing.
• Mobile phase – the phase that moves in a definite direction. It may be a liquid (LC and
Capillary.
• Preparative chromatography – the use of chromatography to purify sufficient quantities
of a substance for further use, rather than analysis.
7. • Retention time – the characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set conditions.
See also: Kovats' retention index
• Sample – the matter analyzed in chromatography. It 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 is/are referred to as the sample whereas everything out of interest
separated from the sample before or in the course of the analysis is referred to as
waste.
• Solute – the sample components in partition chromatography.
• Solvent – any substance capable of solubilizing another substance, and especially
the liquid mobile phase in liquid chromatography.
• Stationary phase – the substance fixed in place for the chromatography procedure.
Examples include the silica layer in thin-layer chromatography
• Detector – the instrument used for qualitative and quantitative detection of
analytes after separation.
8. • The Russian botanist Mikhail S. Tsvet coined the term chromatography in 1906.
He introduced the concept of liquid chromatography to separate plant pigments.
• 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.
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, for which they won the 1952 Nobel Prize in Chemistry.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.
9. PRINCIPLE OF
CHROMATOGRAPHY
• 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.
Three components thus form the basis of the chromatography technique.
1.Stationary phase: This phase is always composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface solid support”.
2.Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3.Supporting medium: A soild surface on which the stationary phase is bound or
coated.
10. TWO WAYS TO CLASSIFY METHODOLOGY OF CHROMATOGRAPHY:
A. Based on interactions between sample component and stationary phase:
I. Partition chromatography
II. Adsorption chromatography
III. Ion-exchange chromatography
IV. Gel-filtration chromatography
V. Affinity chromatography
VI. High performance liquid chromatography (HPLC)
11. BASE ON NATURE OF STATIONARY PHASE OR MOBILE PHASE IT IS OF
TWO TYPES
I. Planar chromatography - It may be Paper or Thin layer.
II. Column chromatography - it may be Gas or Liquid.
12. It is used for the separation of mixture of amino acids and peptides.
The molecules of a mixture get partitioned between the stationary and the mobile
phase depending on the relative affinity of each one of the phases.
IT IS UNDERTAKEN IN TWO WAYS:
1) Paper chromatography
2) Thin layer chromatography
13. Paper chromatography is an analytical technique for separating and identifying
both colored and colorless mixtures.
It is a liquid partition chromatography.
Used for separation of amino acids, sugars, sugar derivatives & peptides.
A. The stationary phase is water held on a solid support of filter paper (cellulose).
B. The mobile phase is a mixture of immiscible solvents which are mixtures of
water, a non polar solvent and an acid or base. e.g. Butanol, acetic acid, water or
phenol-water-ammonia.
14. The samples are subjected to flow by mobile liquid onto or through the stable
stationary phase. The sample components are separated into fractions based on their
relative affinity towards the two phases during their travel.
Material used:
Jar or beaker, Lid , Distilled water, Graduated cylinder, Strips of filter paper, Assorted
colors, Pencil, Ruler, Scissors, Tape.
PROCEDURE:
I. In the first step, chromatography strips are prepared through filter paper by using
scissors.
II. Then, the paper is placed in a jar containing a solvent such as ethanol or water
then sealed.
III. Then, a small concentrated spot of solution that contains the sample of the solute
is applied to a strip of chromatography paper about 2 cm away from the base of the
plate.
IV. As the solvent rises through the paper, it meets the sample mixture which starts to
travel up the paper with the solvent.
V. Paper chromatography takes from several minutes to several hours.
15.
16. After development, the spots corresponding to different compounds may be located
by their color, ultraviolet light, ninhydrin or by treatment with iodine vapors.
The paper remaining after the experiment is known as the Chromatogram. The
components which have been separated differ in their retention factor i.e, Ratio of
distance traveled from the spot or origin by the solute component to that of the
distance traveled from the spot or origin by the solvent.
17. Retention factor (Rf): In paper chromatography, the results are calculated by Rf value
which is basically a distance travelled by a given component divided by the distance
travelled by the solvent front.
Rf= distance travelled by a given component÷ distance travelled by the solvent front
1) If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it
is immobile.
2) If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with
the solvent front.
18. It is a chromatographic technique used to separate mixtures.
It involves a stationary phase consisting of a thin layer of adsorbent material,
usually silica gel, aluminium oxide, or cellulose immobilized onto a flat, inert
carrier sheet.
A liquid phase consisting of the solution to be separated which is dissolved in an
appropriate solvent and is drawn up the plate via capillary action, separating the
solution based on the polarity of the components of the compound in question.
19. In this technique the separation is based on differences in adsorption at the
surface of the solid stationary medium.
The adsorbents such as silica gel, charcoal powder and calcium hydroxyapatite are
packed in to a column in a glass tube. This serves as the stationary phase.
The sample mixture in a solvent is loaded on this column.
The individual components get differentially adsorbed on to the adsorbent.
20. Ion-exchange chromatography (or ion chromatography) is a process that allows the
separation of ions and polar molecules based on the charge properties of the molecules.
It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.
The solution to be injected is usually called a sample, and the individually separated
components are called analytes.
It is often used in protein purification, water analysis, and quality control.
Ion exchange chromatography retains analyte molecules based on coulombic (ionic)
interactions. The stationary phase surface displays ionic functional groups that interact
with analyte ions of opposite charge.
This type of chromatography is further subdivided into cation exchange
chromatography and anion exchange chromatography.
Cation exchange chromatography retains positively charged cations because the
stationary phase displays a negatively charged functional group.
Anion exchange chromatography retains anions using positively charged functional
group.
21. Various types of ion exchanges resins
are commercially available-
Cation Exchange resin- •
Polysterene sulfonate resins,
CMSephadex gel, CM – cellulose,
These bear acidic groups and
immobilize cations from adjacent
solutions.
Anion exchangers:- DEAE cellulose,
Trimethyl amino Polysterene ,
DEAE- Sephadex . All these bear
basic groups ionizing into fixed
positions and immobilize anions from
neighboring solutions.
22.
23. Affinity Chromatography is essentially
a sample purification technique, used
primarily for biological molecules such
as proteins.
It is a method of separating a mixture of
proteins or nucleic acids (molecules) by
specific interactions of those molecules
with a component known as a ligand,
which is immobilized on a support.
For example: If a solution of a mixture
of proteins is passed through the
column, one of the proteins binds to the
ligand based on specificity and high
affinity whereas other proteins in the
solution wash through the column
because they are not able to bind to the
ligand.
24. It is based on highly specific biological
interactions between two molecules
such as interactions between enzyme
and substrate, receptor and ligand, or
antibody and antigen.
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 labeled 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.
25. Also known as gel permeation or gel
filtration, this type of chromatography
lacks an attractive interaction between
the stationary phase and solute.
The liquid or gaseous phase passes
through a porous gel which separates
the molecules according to its size.
The pores are normally small and
exclude the larger solute molecules, but
allows smaller molecules to enter the
gel, causing them to flow through a
larger volume. This causes the larger
molecules to pass through the column
at a faster rate than the smaller ones.
26. Chromatographic techniques are slow and time consuming.
The separation can be greatly improved by applying high pressure in the range of
5000- 10,000 pounds per square inch, hence this technique is also referred to as
high pressure liquid chromatography.
HPLC requires the use of non compressible resin materials and strong metal
columns' It can be applied in the form of partition, adsorption, ion exchange or
molecular sieve chromatography.
The stationary phase consists of an immobilized thin layer of a liquid on the
micro glass or plastic beads, tightly packed in to a narrow column.
The mobile phase - consists of a buffered solvent system which is passed under
high pressure through the column for eluting the solutes of the sample.
27.
28. The method of choice for the separation of volatile substances or volatile
derivatives of certain in volatile substances.
In GLC, the stationary phase is an inert solid material(diatomaceous earth or
powdered firebrick), impregnated with a non volatile liquid(silicon or polyethylene
glycol).
This is packed in a narrow column and maintained at high temperature (around
200 degree C).
A mixture of volatile material is injected in to the column along with the mobile
phase, which is an inert gas (argon, helium or nitrogen). The separation of the
volatile material is based on the partition of the components between the mobile
phase(gas) and stationary phase( liquid), hence the name gas liquid
chromatography.
The separated compounds can be identified and quantitated by a detector. Gas
liquid chromatography is sensitive, rapid and reliable. It is frequently used for the
quantitative estimations of biological materials such as lipids, drugs and vitamins.
29.
30. • Column chromatography is a
separation technique in which the
stationary bed is within a tube. The
particle of solid stationary phase or
support coated with the liquid
stationary phase may fill the whole
inside volume of the tube(packed
column) or to be concentrated on or
along the inside wall leaving an
open, unrestricted path for the
mobile phase in the middle part of
the tube(open tubular column).
Differences in rates of movement
through the medium are calculated
to different retention time of the
sample.
31. Planar chromatography is a separation
technique in which the stationary phase
is present as or on a plane. The plane
can be a paper, serving 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).
Different compounds in the sample
mixture travel different distances
according to how strongly they interact
with the stationary phase as compared
to the mobile phase. The
specific Retention factor (Rf) of each
chemical can be used to aid in the
identification of an unknown substance.
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Chromatography – Wikipedia
Partition Chromatography - Principle, Diagram, Types and Applications
(vedantu.com)
Chromatography: Basics, Principles and Theories (biologydiscussion.com)
Chromatography- definition, principle, types, applications (microbenotes.com)
