 Chromatography is an important biophysical technique that enables the

separation, identification, and purification of the components of a mixture

for qualitative and quantitative analysis.
 The Russian botanist Mikhail Tswett coined the term chromatography in
1906.
 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.
 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

 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. Separated molecules

The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on the
separation of molecules from each other.
TYPES OF CHROMATOGRAPHY-

1.

 Gas chromatography differs from other forms of chromatography in that

the mobile phase is a gas and the components are separated as vapors.
 It is thus used to separate and detect small molecular weight compounds
in the gas phase.
 The sample is either a gas or a liquid that is vaporized in the injection port.
The mobile phase for gas chromatography is a carrier gas, typically helium
because of its low molecular weight and being chemically inert.
 The pressure is applied and the mobile phase moves the analyte through
the column. The separation is accomplished using a column coated with a
stationary phase.
The equilibrium for gas chromatography is partitioning, and the components of
the sample will partition (i.e. distribute) between the two phases: the stationary
phase and the mobile phase.
Compounds that have a greater affinity for the stationary phase spend more time
in the column and thus elute later and have a longer retention time (Rt) than
samples that have a higher affinity for the mobile phase.
Affinity for the stationary phase is driven mainly by intermolecular interactions
and the polarity of the stationary phase can be chosen to maximize interactions
and thus the separation.
Ideal peaks are Gaussian distributions and symmetrical, because of the random
nature of the analyte interactions with the column.
 The separation is hence accomplished by partitioning the sample between
the gas and a thin layer of a nonvolatile liquid held on a solid support.
 A sample containing the solutes is injected into a heated block where it is
immediately vaporized and swept as a plug of vapor by the carrier gas
stream into the column inlet.
 The solutes are adsorbed by the stationary phase and then desorbed by a
fresh carrier gas.
 The process is repeated in each plate as the sample is moved toward the
outlet.
 Each solute will travel at its own rate through the column.
 Their bands will separate into distinct zones depending on the partition
coefficients, and band spreading.
 The solutes are eluted one after another in the increasing order of their kd,
and enter into a detector attached to the exit end of the column.
 Here they register a series of signals resulting from concentration changes
and rates of elution on the recorder as a plot of time versus the
composition of carrier gas stream.
 The appearance time, height, width, and area of these peaks can be
measured to yield quantitative data.

 Parts of Gas chromatography

Gas chromatography is mainly composed of the following parts:
1. Carrier gas in a high-pressure cylinder with attendant pressure
regulators and flow meters
 Helium, N2, H, Argon are used as carrier gases.
 Helium is preferred for thermal conductivity detectors because of its high
thermal conductivity relative to that of most organic vapors.
 N2 is preferable when a large consumption of carrier gas is employed.
 Carrier gas from the tank passes through a toggle valve, a flow meter, (1-
1000 ml/min), capillary restrictors, and a pressure gauge (1-4 atm).
 Flow rate is adjusted by means of a needle valve mounted on the base of
the flow meter and controlled by capillary restrictors.
 The operating efficiency of the gas chromatograph is directly dependant
on the maintenance of constant gas flow.
2. Sample injection system
 Liquid samples are injected by a microsyringe with a needle inserted
through a self-scaling, silicon-rubber septum into a heated metal block by
a resistance heater.
 Gaseous samples are injected by a gas-tight syringe or through a by-pass
loop and valves.
 Typical sample volumes range from 0.1 to 0.2 ml.
3. The separation column
 The heart of the gas chromatography is the column which is made of
metals bent in U shape or coiled into an open spiral or a flat pancake
shape.
  Copper is useful up to 2500
 Swege lock fittings make column insertion easy.
 Several sizes of columns are used depending upon the requirements.
4. Liquid phases
 An infinite variety of liquid phases are available limited only by their
volatility, thermal stability and ability to wet the support.
 No single phase will serve for all separation problems at all temperatures.
Non-Polar – Parafin, squalane, silicone greases, apiezon L, silicone gum rubber.
These materials separate the components in order of their boiling points.
Intermediate Polarity – These materials contain a polar or polarizable group on
a long non-polar skeleton which can dissolve both polar and non-polar solutes.
For example. diethyl hexyl phthalate is used for the separation of high boiling
alcohols.
Polar – Carbowaxes – Liquid phases with a large proportion of polar groups.
Separation of polar and non-polar substances.
Hydrogen bonding – Polar liquid phases with high hydrogen bonding e.g.
Glycol.
Specific purpose phases – Relying on a chemical reaction with solute to achieve
separations. e.g AgNO3 in glycol separates unsaturated hydrocarbons.
5. Supports
 The structure and surface characteristics of the support materials are
important parameters, which determine the efficiency of the support and
the degree of separation respectively.
 The support should be inert but capable of immobilizing a large volume of
liquid phase as a thin film over its surface.
 The surface area should be large to ensure the rapid attainment of
equilibrium between stationary and mobile phases.
 Support should be strong enough to resist breakdown in handling and be
capable of packed into a uniform bed.
 Diatomaceous earth, kieselguhr treated with Na 2CO 3 for 900 0 C causes
the particle fusion into coarser aggregates.
 Glass beads with a low surface area and low porosity can be used to coat
up to 3% stationary phases.
 Porous polymer beads differing in the degree of cross-linking of styrene
with alkyl-vinyl benzene are also used which are stable up to 250 0
6. Detector
  Detectors sense the arrival of the separated components and provide a
signal.
 These are either concentration-dependent or mass dependant.
 The detector should be close to the column exit and the correct
temperature to prevent decomposition.
7. Recorder
 The recorder should be generally 10 mv (full scale) fitted with a fast
response pen (1 sec or less). The recorder should be connected with a
series of good quality resistances connected across the input to attenuate
the large signals.
 An integrator may be a good addition.

The procedure of Gas Chromatography

Step 1: Sample Injection and Vapourization

1. A small amount of liquid sample to be analyzed is drawn up into a syringe. 
2. The syringe needle is positioned in the hot injection port of the gas
chromatograph and the sample is injected quickly. 
3. The injection of the sample is considered to be a “point” in time, that is, it
is assumed that the entire sample enters the gas chromatograph at the
same time, so the sample must be injected quickly.
4. The temperature is set to be higher than the boiling points of the
components of the mixture so that the components will vaporize.
5. The vaporized components then mix with the inert gas mobile phase to be
carried to the gas chromatography column to be separated. 
Step 2: Separation in the Column
 Components in the mixture are separated based on their abilities to adsorb
on or bind to, the stationary phase.
 A component that adsorbs most strongly to the stationary phase will spend
the most time in the column (will be retained in the column for the longest
time) and will, therefore, have the longest retention time (Rt). It will emerge
from the gas chromatograph last.
 A component that adsorbs the least strongly to the stationary phase will
spend the least time in the column (will be retained in the column for the
 shortest time) and will, therefore, have the shortest retention time (Rt).  It
will emerge from the gas chromatograph first.
 If we consider a 2 component mixture in which component A is
more polar than component B then:
1. component A will have a longer retention time in a polar column than
component B
2. component A will have a shorter retention time in a non-polar column
than component B
Step 3: Detecting and Recording Results
1. The components of the mixture reach the detector at different times due to
differences in the time they are retained in the column.
2. The component that is retained the shortest time in the column is detected
first. The component that is retained the longest time in the column is
detected last.
3. The detector sends a signal to the chart recorder which results in a peak on
the chart paper. The component that is detected first is recorded first.  The
component that is detected last is recorded last.

Applications

 GC analysis is used to calculate the content of a chemical product, for

example in assuring the quality of products in the chemical industry; or
measuring toxic substances in soil, air or water.
 Gas chromatography is used in the analysis of:
(a) air-borne pollutants 
(b) performance-enhancing drugs in athlete’s urine samples 
(c) oil spills 
(d) essential oils in perfume preparation
 GC is very accurate if used properly and can measure picomoles of a
substance in a 1 ml liquid sample, or parts-per-billion concentrations in
gaseous samples.
 Gas Chromatography is used extensively in forensic science. Disciplines as
diverse as solid drug dose (pre-consumption form) identification and
quantification, arson investigation, paint chip analysis, and toxicology
 cases, employ GC to identify and quantify various biological specimens and
crime-scene evidence.

Advantages

 The use of longer columns and higher velocity of carrier gas permits the
fast separation in a matter of a few minutes.
 Higher working temperatures up to 5000C and the possibility of converting
any material into a volatile component make gas chromatography one of
the most versatile techniques.
 GC is popular for environmental monitoring and industrial applications
because it is very reliable and can be run nearly continuously.
 GC is typically used in applications where small, volatile molecules are
detected and with non-aqueous solutions. 
 GC is favored for non-polar molecules.

Limitations

 Compound to be analyzed should be stable under GC operation

conditions.
 They should have a vapor pressure significantly greater than zero.
 Typically, the compounds analyzed are less than 1,000 Da, because it is
difficult to vaporize larger compounds.
 The samples are also required to be salt-free; they should not contain ions.
 Very minute amounts of a substance can be measured, but it is often
required that the sample must be measured in comparison to a sample
containing the pure, suspected substance known as a reference standard
 High-Performance Liquid
2.

Chromatography (HPLC)
 High performance liquid chromatography or commonly known as HPLC is
an analytical technique used to separate, identify or quantify each
component in a mixture.
 The mixture is separated using the basic principle of
column chromatography and then identified and quantified by
spectroscopy. 
 In the 1960s the column chromatography LC with its low-pressure suitable
glass columns was further developed to the HPLC with its high-pressure
adapted metal columns.
 HPLC is thus basically a highly improved form of column liquid
chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up to
400 atmospheres.
  The purification takes place in a separation column between a stationary
and a mobile phase.
 The stationary phase is a granular material with very small porous particles
in a separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which
is forced at high pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary
made of stainless steel, the sample is injected into the mobile phase flow
from the pump to the separation column using a syringe.
 Subsequently, the individual components of the sample migrate through
the column at different rates because they are retained to a varying degree
by interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a
suitable detector and passed on as a signal to the HPLC software on the
computer.
 At the end of this operation/run, a chromatogram in the HPLC software on
the computer is obtained.
 The chromatogram allows the identification and quantification of the
different substances. 

Instrumentation of High-Performance Liquid

Chromatography (HPLC

The Pump
  The development of HPLC led to the development of the pump system.
 The pump is positioned in the most upper stream of the liquid
chromatography system and generates a flow of eluent from the solvent
reservoir into the system.
 High-pressure generation is a “standard” requirement of pumps besides
which, it should also to be able to provide a consistent pressure at any
condition and a controllable and reproducible flow rate.
 Most pumps used in current LC systems generate the flow by back-and-
forth motion of a motor-driven piston (reciprocating pumps). Because of
this piston motion, it produces “pulses”.
 Injector
 An injector is placed next to the pump.
 The simplest method is to use a syringe, and the sample is introduced to
the flow of eluent.
 The most widely used injection method is based on sampling loops.
 The use of the autosampler (auto-injector) system is also widely used that
allows repeated injections in a set scheduled-timing. 
Column
 The separation is performed inside the column.
 The recent columns are often prepared in a stainless steel housing, instead
of glass columns.
 The packing material generally used is silica or polymer gels compared to
calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
 Most column housing is made of stainless steel since stainless is tolerant
towards a large variety of solvents.
Detector
 Separation of analytes is performed inside the column, whereas a detector
is used to observe the obtained separation.
 The composition of the eluent is consistent when no analyte is present.
While the presence of analyte changes the composition of the eluent. What
detector does is to measure these differences.
 This difference is monitored as a form of an electronic signal. There are
different types of detectors available.
Recorder
 The change in eluent detected by a detector is in the form of an electronic
signal, and thus it is still not visible to our eyes.
  In older days, the pen (paper)-chart recorder was popularly used.
Nowadays, a computer-based data processor (integrator) is more common.
 There are various types of data processors; from a simple system consisting
of the in-built printer and word processor while those with software that
are specifically designed for an LC system which not only data acquisition
but features like peak-fitting, baseline correction, automatic concentration
calculation, molecular weight determination, etc.
Degasser
The eluent used for LC analysis may contain gases such as oxygen that are non-
visible to our eyes.
 When gas is present in the eluent, this is detected as noise and causes an
unstable baseline.
 Degasser uses special polymer membrane tubing to remove gases.
 The numerous very small pores on the surface of the polymer tube allow
the air to go through while preventing any liquid to go through the pore.
 Column Heater
The LC separation is often largely influenced by the column temperature.
 In order to obtain repeatable results, it is important to keep consistent
temperature conditions.
 Also for some analysis, such as sugar and organic acid, better resolutions
can be obtained at elevated temperatures (50 to 80°C).

Thus columns are generally kept inside the column oven (column heater).

Applications of High-Performance Liquid

Chromatography (HPLC)

The HPLC has developed into a universally applicable method so that it finds its
use in almost all areas of chemistry, biochemistry, and pharmacy.
 Analysis of drugs
 Analysis of synthetic polymers
 Analysis of pollutants in environmental analytics
 Determination of drugs in biological matrices
 Isolation of valuable products
 Product purity and quality control of industrial products and fine chemicals
  Separation and purification of biopolymers such as enzymes or nucleic
acids
 Water purification
 Pre-concentration of trace components
 Ligand-exchange chromatography
 Ion-exchange chromatography of proteins
 High-pH anion-exchange chromatography of carbohydrates and
oligosaccharides

Advantages of High-Performance Liquid

Chromatography (HPLC)

1. Speed
2. Efficiency
3. Accuracy
4. Versatile and extremely precise when it comes to identifying and
quantifying chemical components.

Limitations

1. Cost: Despite its advantages, HPLC can be costly, requiring large quantities

of expensive organics. 
2. Complexity
3. HPLC does have low sensitivity for certain compounds, and some cannot
be detected as they are irreversibly adsorbed.
4. Volatile substances are better separated by gas chromatography.
3. Thin Layer Chromatography
 It is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for
qualitative and quantitative analysis.
 In this physical method of separation, the components to be separated are
distributed between two phases, one of which is stationary (stationary
phase) while the other (the mobile phase) moves in a definite direction.
Depending upon the stationary phase and mobile phase chosen, they can
be of different types.
 Thin Layer Chromatography can be defined as a method of separation or
identification of a mixture of components into individual components by
using finely divided adsorbent solid / (liquid) spread over a plate and liquid
as a mobile phase.
Principle of Thin Layer Chromatography (TLC)
 Thin-layer chromatography is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin layer of adsorbent material,
usually silica gel, aluminium oxide (alumina), or cellulose. This layer of
adsorbent is known as the stationary phase.
 After the sample has been applied on the plate, a solvent or solvent
mixture (known as the mobile phase) is drawn up the plate via capillary
action. Because different analytes ascend the TLC plate at different rates,
separation is achieved.
 It is thus based on the principle of adsorption chromatography or partition
chromatography or combination of both, depending on adsorbent, its
treatment and nature of solvents employed. The components with more
affinity towards stationary phase travels slower. Components with less
affinity towards stationary phase travels faster.
 Once separation occurs, the individual components are visualized as spots
at a respective level of travel on the plate. Their nature or character is
identified by means of suitable detection techniques.

Components of Thin Layer Chromatography (TLC)

TLC system components consists of:

1. TLC plates, preferably ready made with a stationary phase: These are

stable and chemically inert plates, where a thin layer of stationary phase is
applied on its whole surface layer. The stationary phase on the plates is of
uniform thickness and is in a fine particle size.
2. TLC chamber- This is used for the development of TLC plate. The chamber
maintains a uniform environment inside for proper development of spots.
It also prevents the evaporation of solvents, and keeps the process dust
free.
3. Mobile phase- This comprises of a solvent or solvent mixture The mobile
phase used should be particulate-free and of the highest purity for proper
development of TLC spots. The solvents recommended are chemically inert
with the sample, a stationary phase.
4. A filter paper- This is moistened in the mobile phase, to be placed inside
the chamber. This helps develop a uniform rise in a mobile phase over the
length of the stationary phase.
Procedure of Thin Layer Chromatography (TLC)

The stationary phase is applied onto the plate uniformly and then allowed to dry
and stabilize. These days, however, ready-made plates are more commonly used.
1. With a pencil, a thin mark is made at the bottom of the plate to apply the
sample spots.
2. Then, samples solutions are applied on the spots marked on the line in
equal distances.
3. The mobile phase is poured into the TLC chamber to a leveled few
centimeters above the chamber bottom.
4. A moistened filter paper in mobile phase is placed on the inner wall of the
chamber to maintain equal humidity (and also thereby avoids edge effect).
5. Now, the plate prepared with sample spotting is placed in TLC chamber so
that the side of the plate with the sample line is facing the mobile phase.
Then the chamber is closed with a lid.
6. The plate is then immersed, such that the sample spots are well above the
level of mobile phase (but not immersed in the solvent) for development.
7. Sufficient time is given for the development of spots.
 8. The plates are then removed and allowed to dry.
9. The sample spots are then seen in a suitable UV light chamber, or any
other methods as recommended for the given sample.
Some common techniques for visualizing the results of a TLC plate include
1. UV light
2. Iodine Staining: is very useful in detecting carbohydrates since it turns
black on contact with Iodine
3. KMnO4 stain (organic molecules)
4. Ninhydrin Reagent: often used to detect amino acids and proteins
Retention Factor (Rf ) Value
 The behaviour of a compound on a TLC is usually described in terms of its
relative mobility or Rf value.
 Rf or Retention factor is a unique value for each compound under the
same conditions.
 The Rf for a compound is a constant from one experiment to the next only
if the chromatography conditions below are also constant:
 solvent system
 adsorbent
 thickness of the adsorbent
 amount of material spotted
 temperature
 Since these factors are difficult to keep constant from experiment to
experiment, relative Rf values are generally considered.
 Relative Rf” means that the values are reported relative to a standard.
 The Rf value is calculated using the following equation:

Applications of Thin Layer Chromatography (TLC)

1. In monitoring the progress of reactions
2. Identify compounds present in a given mixture
3. Determine the purity of a substance.
 Analyzing ceramides and fatty acids
 Detection of pesticides or insecticides in food and water
 Analyzing the dye composition of fibers in forensics
 Assaying the radiochemical purity of radiopharmaceuticals
 Identification of medicinal plants and their constituents 

Advantages of Thin Layer Chromatography (TLC)

  It is a simple process with a short development time.
 It helps with the visualization of separated compound spots easily.
 It helps in isolating of most of the compounds.
 The separation process is faster and the selectivity for compounds is higher
(even small differences in chemistry is enough for clear separation).
 The purity standards of the given sample can be assessed easily.
 It is a cheaper chromatographic technique.

Limitations of Thin Layer Chromatography (TLC)

 It cannot tell the difference between enantiomers and some isomers.

 In order to identify specific compounds, the Rf values for the compounds

of interest must be known beforehand.
 TLC plates do not have long stationary phases. Therefore, the length of
separation is limited compared to other chromatographic techniques.