❏ Chromatography is the technique by which components or solutes from a
mixture are separated depending on the comparative amount of each solute
which has been dispersed between a moving fluid stream, called the mobile
phase, and a stationary phase.
❏ High performance liquid chromatography or commonly known as HPLC is
an analytical technique used to separate, identify or quantify each
component in a mixture.
❏ Mixture is separated using the basic principle of column chromatography
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
➢ High performance liquid chromatography is basically an improvised variety
of column chromatography.
➢ Instead of mobile phase being permitted to drip down through a column
under the force of gravity, it is forced through a column under high pressures
of up to 400 atmospheres, making it a much faster technique. This promotes
enhanced separation of the constituents of the mixture.
➢ Another major development over column chromatography is the use of
highly sensitive detectors
TYPES OF HPLC
❏ Normal Phase HPLC :
This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary
phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and
typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and
mixtures of these.Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials.
❏ Reverse Phase HPLC :
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar
liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of
hydrophobic interactions hence the more nonpolar the material is, the longer it will be
❏ Size-exclusion HPLC :
The column is filled with material having precisely controlled pore sizes, and the
particles are separated according to its their molecular size. Larger molecules are
rapidly washed through the column; smaller molecules penetrate inside the porous
of the packing particles and elute later.
❏ Ion-Exchange HPLC :
The stationary phase has an ionically charged surface of opposite charge to the
sample ions. This technique is used almost exclusively with ionic or ionizable
samples.The stronger the charge on the sample, the stronger it will be attracted to
the ionic surface and thus, the longer it will take to elute. The mobile phase is an
aqueous buffer, where both pH and ionic strength are used to control elution time.
HPLC operate under the same basic principle; separation of a sample into its
constituent parts because of the difference in the relative affinities of different
molecules for the mobile phase and the stationary phase used in the separation.
The separation principle of HPLC is based on the distribution of the analyte (sample)
between a mobile phase (eluent) and a stationary phase (packing material of the
column). Depending on the chemical structure of the analyte, the molecules are
retarded while passing the stationary phase. The specific intermolecular interactions
between the molecules of a sample and the packing material define their time
“on-column”. Hence, different constituents of a sample are eluted at different times.
Thereby, the separation of the sample ingredients is achieved
HPLC INSTRUMENTATION AND WORKING
It consists of a solvent reservoir, degasser,injector, pump, column,
detector and an integrator and display system
● The sample is injected through a port in the high pressure liquid carrier
stream between the pump and the column.
● The separation takes place in the column which varies from 3- 30 cm in
length and 3 mm in diameter.
● Typical flow rates fall between 1-2 ml/min with pressure up to several
● The column effluent passes through a non-destructive detector where a
property such as ultraviolet absorbance, refractive index and molecular
fluorescence is monitored.
● The signal is then amplified and recorded as a detector response v/s
● The graph thus obtained is called chromatogram.
● The effluent may be discarded, recycled or saved for any further research or
studies in a fraction collector which is synchronized with the detector.
Mobile phase contents are stored in a glass reservoir In HPLC, usually a mixture of polar and
non-polar liquid constituents is used as the mobile phase, or solvent, where the concentration
of constituents varies based on the composition of sample
Degassing is an important action performed prior to mobile phase delivery into HPLC system.
Solvents remain in contact with the atmosphere and therefore essentially consist of an
equilibrium level of dissolved air. When solvents are mixed, bubbles are formed due to
dissolved air which can interfere with the pump operation and sensitivity of detectors. Also
the solubility of analytes in air saturated solvents is lower as compared to pure solvents.
Degassing of the solvent before its entry into pump can resolve all these issues.
The pump is considered as the heart of this chromatography system.A pump
aspirates the mobile phase from the solvent reservoir and forces it through the system’s
column and detector. Depending on a number of factors including column dimensions,
particle size of the stationary phase, the flow rate and composition of the mobile phase,
operating pressures of up to 42000 kPa (about 6000 psi) can be generated.
The injector can be a single injection or an automated injection system. An injector for an
HPLC system should provide injection of the liquid sample within the range of 0.1-100
mL of volume with high reproducibility and under high pressure (up to 4000 psi).
Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have
an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase
with a particle size of 3–10 µm.
Columns with internal diameters of less than 2 mm are often referred to as microbore columns.
Ideally the temperature of the mobile phase and the column should be kept constant during an
The HPLC detector, located at the end of the column detect the analytes as they elute from the
chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence,
mass-spectrometric and electrochemical detectors.
Data Collection Devices
Signals from the detector may be collected on chart recorders or electronic integrators that
vary in complexity and in their ability to process, store and reprocess chromatographic
data. The computer integrates the response of the detector to each component and places it
into a chromatograph that is easy to read and interpret.
TYPES OF COLUMN
There are different types of chromatographic columns on basis of composition and method
1 Normal phase columns
2 Reverse phase columns
3 Ion exchange columns
4 Size exclusion columns