Chem- 131
Chromatography
Chromatography is a non-destructive procedure for resolving a complex mixture into its individual
components. It’s a separation procedure and the separated entities are identified by other analytical
techniques like UV-visible, Infrared, NMR (nuclear magnetic resonance), Mass spectrometry etc.
Chromatography was invented by Russian botanist Mikhail Tswett. The term chromatography came
from Greek word chroma (means color) and graphein (means write).
Definition: Chromatography is a technique is which the components of a mixture are separated based
upon the rates at which they are carried through the stationery phase by a gaseous or liquid mobile phase.
IUPAC recommended definition: Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of which is stationary, the other
(the mobile phase) moves in a definite direction.
Classification of Chromatography Methods
In general the Chromatography methods are of two types;
1. Planar Chromatography
2. Column Chromatography
1. Planar Chromatography: In this chromatography the stationary phase is supported on a flat place
or in the pores of a paper. Here the mobile phase moves through the stationary phase by capillary action
or under intense of gravity.
This chromatographic method includes-
i). Paper chromatography
ii). Thin layer chromatography (TLC)
iii) High performance thin layer chromatography (HPTLC)
Paper chromatography:
Paper Chromatography is one of the types of chromatography procedures which runs on a piece of
specialized paper. It is a planar chromatography system wherein a cellulose filter paper acts as a
stationary phase on which separation of compounds occurs.
Principle:
The principle involved is partition chromatography where in the substances are distributed or partitioned
between to liquid phases. One phase is the water which is held in pores of filter paper used and other
phase is that of mobile phase which moves over the paper. The compounds in the mixture get separated
due to differences in their affinity towards water (in stationary phase) and mobile phase solvents during
the movement of mobile phase under the capillary action of pores in the paper. The principle can also
be adsorption chromatography between solid and liquid phases, where in the stationary phase is the solid
surface of paper and the liquid phase is of mobile phase. But most of the applications of paper
chromatography work on the principle of partition chromatography i.e. partitioned between to liquid
phases.
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Chem- 131
Figure: Paper chromatography
Uses/applications of paper chromatography
1. Paper chromatography is specially used for separation of mixture having polar and non polar
compounds.
2. For separation of amino acids.
3. It is used to determine organic compounds, biochemicals in urine etc.
4. In pharma sector for determination of hormones, drugs.
5. Sometimes used for evaluation of inorganic compounds like salts and complexes.
Thin Layer Chromatography (TLC)
TLC is a type of planar chromatography. TLC is routinely used by researcher in the field of phyto-
chemicals, biochemistry etc. to identify the components in a compound mixture like alkaloids,
phospholipids, amino acids etc.
Principle:
This technique involves the use of an inert piece of glass, plastic or metal. A drop of solution to be
analyzed is added to the TLC glass plate, which has already been coated with a thin layer of silica gel.
This silica coated glass plate acts as the stationary phase. The sheet is then placed in a glass chamber
containing a solvent, which forms the mobile phase. Via capillary action, the solvent moves upwards
and carries the molecules of the solution to be detected at different rates. The components of the solution
will appear in the form of a series of spots at various locations on the plate. Calculations are made based
on ratio of the distance that the substance travels to the distance that the solvent travels up the plate.
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TLC chromatography System components: TLC System consists of
TLC plates preferably ready made with stationary phase: These are stable and chemically inert plates
on to whose surface a thin layer of stationary phase is applied. The stationary phase on the plates is of
uniform thickness and consists of fine particle size.
TLC chamber: This is used for the development of TLC plate. The chamber maintains uniform
environment inside for proper development of spots. It also prevents the evaporation of solvents and
keep the process dust free.
Mobile phase: This comprises of a solvent or solvent mixture recommended for the purpose. The
mobile phase used should be particulate free and of highest purity for proper development of TLC
spots. The solvents recommended are chemically inert with the sample, stationary phase.
A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps uniform rise
in mobile phase over the length stationary phase
Figure: Thin layer chromatography.
Advantages of TLC
1. It is simple process with short development time.
2. It helps in visualization of separated compound spots easily.
3. The method helps to identify the individual compounds.
4. It helps in isolation of most of the compounds.
5. The separation process is faster and the selectivity for compounds is higher.
6. The purity standards of the given sample can be assessed easily.
7. It is a cheaper chromatographic technique.
Applications of Thin layer chromatography
1. To check purity of given samples.
2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics etc.
3. To evaluate reaction process by assessment of intermediates, reaction course etc.
4. To purify samples i.e for purification process.
5. To keep a check on the performance of other separation processes.
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Some important terms:
Stationery phase: The stationery phase in Chromatography is a phase that is fixed in place either in a
column or on a planar surface.
Mobile phase: The mobile phase in Chromatography is a phase that moves over or through the
stationary phase, carrying the analyses with it. The mobile phase may be gas, liquid or supercritical
liquid.
Elution: Elution is a process in which shuts are washed through a stationary phase by the movement of
a mobile phase.
Eluent: An eluent is a solvent used to carry the components of a mixture through the stationary phase.
Chromatogram: Chromatogram is the visual output of a chromatographic separation. It can be defined
as a graph showing the quantity of a substance leaving a chromatography column as a function of time.
Figure: A Chromatogram
Principles of Chromatographic Separations:
The samples are subjected to flow by mobile liquid phase onto or through the stable stationary phase.
The separation of fractions of mixture based on their relative affinity towards the two phases during
their travel. The fraction with greater affinity to stationary phase travels slower and shorter while that
with less affinity travels faster and longer.
On moving with mobile phase the components of the sample equilibrate between the mobile phase and
the stationary phase.
The more the interaction of the component interest with the stationary phase to different degrees.
Xm Xs
The distribution equilibrium is described by the distribution constant.
Kc Distribution co-efficient
[X]𝑠
Kc = [X] [X]𝑠 Concentration of X is the stationary
𝑚
[X]𝑚 Concentration of X is the mobile phase
It depends on temperature, the type of compound and the stationary and mobile phases.
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Chem- 131
2. Columnar Chromatography:
Columnar chromatography is the technique used to separate the components of a mixture using a column
of suitable adsorbent (stationary phase) packed in a glass tube. The mixture is placed on the top of the
column, and an appropriate eluent (mobile phase) is made to flow down the column slowly.
Column chromatography is basically a type of adsorption chromatography techniques. Here the
separation of components depends upon the extent of adsorption to stationary phase. The component
with the highest absorptivity has the slowest movement, while the other flow down to different speeds
accordingly.
In this method, the stationary phase is held in a narrow glass or metallic tube and the mobile phase is
forced through the tube under pressure or by gravity. Column Chromatography methods fall in to
following categories based upon the nature of mobile phase.
A. Liquid Chromatography (LC) (where mobile phase is liquid)
These subdivisions are based on the nature of the stationary phase and by the types of equilibria between
phases. e.g.
i. Liquid –Liquid or Partition chromatography
ii. Liquid – Solid or adsorption chromatography
iii. High performance liquid chromatography (HPLC)
iv. Ion– exchange chromatography
B. Gas Chromatography (GC) (where mobile phase is a gas)
i. Gas –Liquid chromatography (GLC)
iii. Gas- Solid chromatography (GSC)
Principle of Column Chromatography:
This technique consists of a powdered adsorbent as the stationary phase, placed in a vertical glass
column. The mixture to be analyzed is placed on top of this vertical glass column. The solvent forming
the mobile phase is poured from the top of the column and flow down the column. In most cases, either
silica (SiO2) or alumina (Al2O3) is added to the solvent which act as the stationary phase. As the solvent
flows, the solvent elutes the sample through the column and the components of the mixture begin to
flow at their respective rates down the column. The solute molecules adsorb to the column in a reversible
manner.
Procedure:
The stationary phase material is suitably moistened with mobile phase and packed sufficiently in the
column with a cotton or asbestos pad at the bottom. The extract material or sample to be separated is
placed on the top of packed stationary phase with a second cotton or asbestos pad in between. The
mobile phase is poured into the column over the sample. A collecting beaker is placed at the bottom of
column to collect the elute.
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Figure: Column chromatograph
Applications
Column chromatography is best suited to separate active principle from plant materials.
In separation of compounds after organic synthesis to obtain desired molecule.
To separate or purify natural compound mixtures like alkaloids, glycosides.
Gas Chromatography (GC)
Principle: This technique involves the packing of a stationary phase in a long, narrow glass column.
The stationary phase is a high-boiling liquid into which the mixture to be analyzed is loaded with the
help of a syringe. An inert carrier gas (Helium or Nitrogen) acts as the mobile phase, which flows
through the column with the stationary phase. Once the gas is passed, the components of the mixture
will begin to distribute between the stationary high-boiling liquid and the inert gas and begin to reveal
themselves in the form of different peaks on a recorder.
Instrumentation: In gas chromatography the injection port, column and detector are to be heated to a
pre-specified temperature. Since the mobile phase here is a gas (carrier gas) the components present in
the analyte mixture should be vaporized, so that it can be effectively carried through the column. The
basic instrumentation for GC includes a carrier gas cylinder with regulator, a flow controller for the gas,
an injection port for introducing the sample, the column, the detector and the recorder.
The injection port, column oven and detector are hot zones. The success of this technique requires the
appropriate selection of the column and the temperature conditions at which the column to be maintained
throughout the analysis. Basically the columns for GC are classified as analytical columns and
preparative columns. The analytical columns are of two types: packed column and open-tubular or
capillary column. Both differ in the way the stationary phases are stacked inside. An outlay is as follow:
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Figure: Schematic diagram of gas chromatograph.
In the instrumentation of GC detectors play unique role. There are a number of detectors, which vary in
design, sensitivity and selectivity. Detectors in GC are designed to generate an electronic signal when a
gas other than the carrier gas elutes from the column. Few examples and applications of the detectors
are:
Thermal Conductivity Detector (TCD) - this operates on the principle that gases eluting from the
column have thermal conductivity different from that of the carrier gas. It is the universal detector
(detects most of the analytes) and is non-destructive and hence used with preparative GC, but less
sensitive than other detectors.
Flame Ionization Detector (FID) - effluent is passed into a hydrogen flame and the flammable
components are burned. In this process a fraction of the molecules gets fragmented into charged species
as positive and negative. While positively charged ions are drawn to a collector, negatively charged ions
are attracted to positively charged burner head, this creates an electric circuit and the signal is amplified.
It is restricted for preparative GC and for inorganic substances which do not burn.
Electron Capture Detector (ECD) – It utilizes the beta emissions of a radioactive source, often nickel-
63, to cause the ionization of the carrier gas molecules, thus generating electrons which constitute an
electrical current. It is especially useful for the analysis of halogenated pesticide residues found in
environmental and bio-medical samples. It is extremely sensitive. It does not destroy the sample and
thus may be used for the preparative work.
Nitrogen/Phosphorus Detector (NPD) - the design of the detector is same to that of the FID detector
except that a bead of alkali metal salt is positioned just above the flame. It is also known as ‘Thermionic
Detector’. It is useful for the phosphorus and nitrogen containing pesticides, the organophosphates and
carbamates. The sensitivity for these compounds are very high since the fragmentation of the other
organic compounds is minimized.
Flame Photometric Detector (FPD) - here a flame photometer is incorporated into the instrument. The
principle is that the sulfur or phosphorus compounds burn in the hydrogen flame and produce light
emitting species. It is specific for organic compounds containing sulphur or phosphorus. It is very
selective and very sensitive.
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Electrolytic Conductivity Detector (ECD Hall)- It is also known as ‘Hall detector’, converts the
eluting gaseous components into ions in liquid solution and then measures the electrolytic conductivity
of the solution in a conductivity cell. The conversion to ions is done by chemically oxidizing or reducing
the components with a “reaction gas” in a small reaction chamber. This detector is used in the analysis
of organic halides and has excellent sensitivity & selectivity, but is a destructive detector.
The recent developments allow the GC to be coupled with other analytical techniques like Infra-Red
Spectrometry (Gas Chromatography-Infrared Spectrometry, GC-IR) and Mass Spectrometry (Gas
Chromatography- Mass Spectrometry, GC-MS). These are termed as ‘hyphenated techniques’, and are
very efficient for qualitative analysis as very accurate and precise information like mass or IR spectrum
of the individual sample components are readily obtained as they elute from the GC column. It saves
time and reduces the steps involved for a component to be separated and analyzed.
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