It is the separation technique in which the mixture
to be separated is in mobile phase which is made to move in contact
with a selectively absorbent stationary phase.
It can be an analytical method,
examining the number and nature of the components in a very small
amount of a mixture, but does not actually isolate them. Or it can
be a preparative method,
which investigates a large quantity of the mixture to obtain useable
amounts of each component.
It is the
physical technique for the study of matter and its properties by
examining the light, sound or particles emitted, absorbed or
scattered by the matter by the application of electromagnetic
radiations, which is represented in form of spectrum.
A mixture is separated into its
components by distribution between two phases; one phase is immobile
which may be in the form of a porous bed, bulk liquid, layer or film
is generally named as stationary phase, while the other phase is a
mobile phase (fluid) that percolates through or over the stationary
phase. Then separation occurs due to the repeated
sorption/desorption by the movement of sample over the stationary
phase in the direction of mobile phase. Effective separations
require an adequate difference in the strength of the physical
interactions of the sample components within the two phases, along
with transport properties of the system that control sample movement
within and between phases. (
REF _Ref277195303 \r \h \* MERGEFORMAT 1)
Intermolecular interaction between the
two phases which can be predicted by the equilibrium
Extent of dispersion of solute molecules
over the stationary phase
The radiations which the matter absorbs
or emit are analyzed by a device called spectrometer which
separates it into various frequency components, measures intensity
and plot a graph of intensity versus wavelength or frequency called
is a device which separates the radiation into its component colors.
It is simply a box with a slit at one end for the inlet of radiation
and a light-separating device at the other end.
Based on phases and distribution process.
REF _Ref277173799 \r \h \* MERGEFORMAT 1)
For practical utility
transport processes at least one phase must be reasonably fast
that’s why solid-solid chromatography is impractical which
is a very slow process.
Two distinct phases are
required to set up the distribution component of the separation
mechanism, which explains why gas-gas chromatography does not exist
and liquid-liquid separations are restricted to immiscible solvents.
a non volatile liquid or a solid.
GC has five basic components and they
are as follows:
Supplies gas (like helium and neon) and
also controls its flowrate.
2) Injection system:
A sample of volatile mixture is injected
into the carrier gas and the heated injector port,
vaporises the sample if needed.
Carrier gas carries the sample to the
coiled column where separation occurs. The separated components in
order of their increasing interaction with the stationary phase
emerges from the column
The column is placed in a
thermostatically controlled oven.
It is an integral detector or a link to
a mass spectrometer. There are two types of detectors
flame ionization detector
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
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.
HPLC is separates and detect components
of the mixture and is widely used in drug testing, testing for
vitamins in food and growth promoters in meat.
1) Solvent Reservoir: stores the
2) The Pump System controls the
flow and measures the volume of solvent (the mobile phase).
3) The Injector System: The
sample to be separated is injected into the liquid phase at this
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
5) The Detector: When the
components reach the end of the column they are analyzed by a
detector. The amount passing through the column is small, that’s why
solutes are analyzed as they leave the column. Therefore we link
HPLC to a spectrometer.
The time a compound take to reach the
detector allows the component to be identified. Like in GC, once the
retention time of a solute has been determined 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.
solvent or mixture of different solvents
a finely divided solid, such as silica gel or alumina
A small volume of the sample is placed
on top of the column. The sample should b soluble in the chosen
mobile phase solvent. If the sample is highly soluble in the mobile
phase solute will move quickly with the solvent resulting in non
separation of certain components. The difference in the rate of
movement through the medium is calculated to the retention time of
The chromatography columns vary in size
and polarity. The type of column and suitable solvent for the
separation of mixture into its constituents is chosen by hit and
Type of column for chromatography:
The stationary bed coated with liquid stationary phase fill the
inside of the column
The stationary phase fills the column with a path in the centre of
the column for the mobile phase (iupac
In this technique of separation the
stationary phase is present as a plane or on a plane. When the
mixture of sample is placed on the plane, different compounds travel
different distances according to the interactions between the mobile
and stationary phase. The specific Retention factor (Rf)
of each component is used in the identification of an unknown
Distance the solute moves
Distance the solvent front moves
There are two types of planar
Paper chromatography: (the plane is a
Thin layer chromatography: (the plane is
a glass plate)
In this type of chromatography the
mobile phase is solvent and water held in the
fibre of chromatographic paper is the stationary phase. With
the help of the dropper the mixture is spotted on the strip of
chromatography paper. Strip is suspended in the tank containing the
solvent with the lower end of the strip dipped inside in the
solvent. But the sample spot is kept above the solvent. The solvent
is drawn up the paper by capillary action. The solvent moves up the
paper with the components of mixture at different rates due to
difference in the interaction between the two phases.
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 as it involves spotting the mixture on the
plate and the solvent moves up the plate in the chromatography tank.
But separation is more efficient as very small
paricles are in stationary phase. The plate is able to
separate a number of samples concurrently within a relatively short
It is particularly useful in forensic
work, for example in the separation of dyes from
Classification According To The Mechanism Of Separation:
( REF _Ref277174898 \r \h 4)
Mobile phase: liquid or gas
Stationary phase: solid
It is one of the oldest types of
chromatography. The mobile phase is adsorbed onto the surface of a
stationary phase. The equilibrium between the mobile and stationary
phase accounts for the separation of different solutes. Separation
occurs due to the differences between the adsorption affinities of
the components of sample for the surface of an active solid.
Forces responsible for chromatographic
Forces of attraction:
Weak covalent bond
Vander Waal forces
Forces causing solute movement:
In this type of chromatography a thin
film is formed on the surface of a solid support by a liquid
stationary phase. Solute forms equilibrium between the mobile phase
and the stationary liquid. Separation occurs to the relative
solubilities of component in both
In this type of chromatography, resin
is used as a stationary phase. Anions and
cations covalently attach onto the resin. Electrostatic
forces are responsible for the attraction of solute ions of the
opposite charge in the mobile liquid phase to the resin. Separation
occurs due to difference in the ion exchange affinities of the
components of the sample.
Molecular Exclusion Chromatography:
Also known as gel permeation or gel
filtration. In this type of chromatography there is no an attractive
interaction between the stationary phase and solute. Liquid or
gaseous phase passes through a porous gel which are separated based
on molecular size. The pores are normally small and exclude the
larger solute molecules, but allow 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.
This is a selective chromatography
technique based on highly specific interaction between one specific
kind of solute molecule and the second molecule that is immobilized
on a stationary phase. Usually used for purification of proteins.
For example, an antibody, to some specific protein, can be taken as
the immobilized molecule. When a mixture of protein is passed by
this molecule, only the specific protein is reacted to this
antibody, binding it to the stationary phase. Then by changing the
ionic strength or pH protein is extracted.
types of spectroscopy based on different energy sources.
spectroscopy the power of a beam of light is measured before and
after interaction with a sample and then compared. Specific
absorption techniques tend to be referred to by the wavelength of
radiation measured such as ultraviolet, infrared or microwave
gives information about bond vibration in molecules.
reveal electronic energy levels in molecules.
reveals motion and rotation of molecules.
The result of this kind of measurement
is an absorption spectrum, a plot of energy absorbed as a
function of wavelength.
spectroscopy higher energy photons are used to excite a sample,
which then emit lower energy photons. This technique has found its
applications in the biochemical and medical field and can also be
used for confocal microscopy, fluorescence resonance energy transfer
and fluorescence lifetime imaging.
x ray spectroscopy, X-rays are used for excitation of the atoms,
inner shell electrons in the atom are excited to outer empty
orbitals or they may be completely removed, ionizing the atom. The
inner shell "hole" will then be filled by electrons from surrounding
outer orbitals. Energy available in this de-excitation process will
be emitted as radiation (fluorescence) or will remove other
less-bound electrons from the atom (Auger effect). The X-rays
frequencies and auger energy are measured and used
for determining the
electronic structure of materials.
In flame spectroscopy,
liquid solution samples are subjected into a burner or
nebulizer/burner combination to desolvate,
atomize, and sometimes to excite to a higher energy electronic
state. Flame used during analysis requires fuel and oxidant usually
in the form of gases. This method can be used for analyzing metallic
element analytes in the part per
million, billion, or possibly lower concentration ranges. Light
detectors are used to detect light with the analysis information
coming from the flame.
The usage of flame for solvation, atomization and excitation of
atoms is used in many spectroscopic techniques.
Atomic Emission Spectroscopy
- Atoms are excited from the heat of the flame to emit light. A
total consumption burner with a round burning outlet is used. A high
resolution polychromator /
monochromator can be used to produce
emission intensity vs. wavelength spectrum over a range of
wavelengths for detecting the elements of sample.
Atomic absorption spectroscopy
(often called AA) - A pre-burner nebulizer is used to create mist
and a slot-shaped burner that gives a longer path length flame. The
nebulizer and flame are used to desolvate
and atomize the sample and the excitation of the atoms is done by
the use of lamps shining through the flame at various wavelengths.
The amount of light absorbed after going through the flame
determines the amount of analyte in the
Atomic Fluorescence Spectroscopy:
A burner with a round burning outlet is used. The flame is used to
solvate and atomizes the sample, but a lamp shines light at a
specific wavelength into the flame to excite the
analyte atoms in the flame. The atoms of
certain elements can then emit light in a different direction. The
intensity of this fluorescing light is used for quantifying the
amount of analyte element in the sample.
Plasma Emission Spectroscopy:
It is the method of
analysis of various elements in a sample, by passing the sample
solution through a plasma (high temperature ionized gas) source,
thereby exciting the outer orbital
electrons of the analyte with
simultaneous emission of electromagnetic radiations (in the form of
lights), which are analyzed by means of a spectrograph, which
separates various wavelengths of analytes
in the sample. It is
somewhat similar to optical emission spectroscopy, but is preferred
Plasma are divided into three classes,
depending on the technique of production
Direct current plasma (DCP)
Inductively coupled plasma (ICP)
Spark or arc (emission) spectroscopy:
This method is used for
the analysis of metallic elements in solid samples. For analyzing
non-conductive materials, the sample is ground with graphite powder
to make it conductive. In traditional arc spectroscopy method, the
sample solid is ground. An electric arc or spark is passed through
the sample, heating the sample to a high temperature to excite the
atoms in it. The excited analyte atoms
glow, emitting light at various wavelengths that are detected by
spectroscopic methods. The conditions producing the arc emission are
not controlled quantitatively; the analysis for the elements is
qualitative.Nowadays, the spark sources
with controlled discharges under an argon atmosphere making this
method quantitative, and is use in control laboratories of foundries
and steel mills.
uses the inelastic scattering of light to analyze vibrational and
rotational modes of molecules and is a
type of spectroscopy that is complementary to infrared spectroscopy.
The resulting 'fingerprints' are an aid to analysis.
is a recent technique that has high
sensitivity and powerful applications for ''in vivo'' spectroscopy
resonance spectroscopy analyzes the magnetic properties of atomic
nuclei to determine different electronic local environments of
hydrogen, carbon or other atoms in an organic compound or other
compound. It is used to help determine the structure of the
A mass spectrometer source produces
ions. Information about a sample may be obtained by analyzing the
dispersion of ions when they interact with the sample, generally
using the mass-to-charge ratio.
In this type of spectroscopy, each
optical wavelength that is recorded is encoded with an audio
frequency containing the original wavelength information. A
wavelength analyzer can then reconstruct the original spectrum.
( REF _Ref277206326 \r \h
In this technique is
based on Mössbauer effect which uses
gamma rays for analyzing the resonant absorption frequency. It is
used for analyzing the properties of specific isotope nuclei in
different atomic environments.
Uses of chromatography:
In chemical plant,
chromatography is used to separate particles and contaminates like
pesticides and insecticides.
Government agencies use
chromatography to separate toxic material from drinking water and
for monitoring of air quality.
companies chromatography is used for preparing pure raw materials
and checking contaminants from manufactured compounds.
In organic chemistry many compounds
are very close to each other in terms of atomic or molecular
weight, composition and physical property. Therefore
chromatography is used for identification of compounds.
In food industry, chromatography
technique is used for proper food maintenance.
Examining of the molecular
Estimation of the energy
levels of the ions and complexes in a chemical system along with
Study of the structure making and
structure breaking processes in solutions.
Examining the intrinsic
configuration and relative association and chemical shifts
The identification of
substances through the spectrum emitted from them or absorbed in
Widely used in astronomy
and remote sensing.
For the identification of sample and
detection the combination of chromatography with spectroscopy is
We use combination of gas chromatography
with mass spectroscopy. Individually these devices are sensitive and
bulky, difficult to handle, using their combination simplifies its
structure and improved its operating time.
GC-MS is used for
drug detection, fire investigation,
environmental analysis, explosives
investigation, and identification of unknown samples.
Modern liquid chromatography can
separate very complex mixture but has problem in elucidating
structure of the eluted component. The solute eluted from the
column, in the past, were collected as fractions, concentrated and
then examined by suitable spectroscopic techniques. Now elute is
directly send to the spectrometer and data is collected concurrent
with the separation process. There are two methods for it depending
on the speed at which data can b acquired and on the type of the
spectroscopic data obtained. The most common spectroscopic
techniques employed for the elucidation of molecular structure are:
Nuclear magnetic resonance
If the spectroscopic data can be
obtained rapidly then mass spectrometer (LC-MS) or Fourier Transform
infrared spectrometer (LC-FTIR) is used. If the scanning rate of
spectrometer is slow then nuclear magnetic resonance (NMR) or IR
spectrometer are used.
NMR is the only spectroscopic technique
that in any circumstances, without the aid of the any supplementary
spectroscopic technique can identify the unknown mixture. When it is
employed with liquid chromatography; it forms a powerful analytical
system that is used for separation and identification of the unknown
Although, it is a very useful technique;
but it has some serious difficulties in association of these two
Intensity of NMR signal is dependent on
the flow rate of solvent. As the flow rate increase the signal
For high resolution of NMR the magnetic
field must be homogeneous that requires the sample tube to spin at
high speed; which make it impossible for flow in the NMR cell.
It would sufficiently reduce the solvent
consumption but that demands very small cell volume.
The LC/MS is another important technique
in structure elucidation of eluted solutes but the system is not as
comprehensive as LC/NMR as it requires other spectroscopic
information. For example the IR spectrum of substance for
identification of certain functional groups is used. But it also has
certain advantages over other spectroscopic methods that make it
ideal to combine with LC.
Mass spectra is obtained rapidly
Small amount of material is required to
form the spectra.
Data collected is highly informative
with respect to molecular structure.
C.;”Encyclopedia of Chromatography”;3rd Ed; Volume
chromatography detector” ;2nd Ed; volume 33(181-191)