HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
DEFINITION:
High Performance Liquid Chromatography (HPLC) is a form of
column chromatography that pumps a sample mixture or analyte in a solvent
(known as the mobile phase) at high pressure through a column with chromatographic
packing material (stationary phase).
PRINCIPLE:
All chromatographic separations, including 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.
Instrumentation of HPLC:
HPLC instrumentation includes a
pump, injector, column, detector and integrator or acquisition and display
system. The heart of the system is the column where separation occurs.
1. Solvent
Reservoir
Mobile phase contents are
contained in a glass reservoir. The mobile phase, or solvent, in HPLC is
usually a mixture of polar and non-polar liquid components whose respective
concentrations are varied depending on the composition of the sample.
2. Pump 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. 3. Sample Injector: 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).
4. Columns: 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.
5.
Detector: The HPLC detector, located at the
end of the column detect the analytes as they elute from the chromatographic
column. Commonly used detectors are UVspectroscopy, fluorescence,
mass-spectrometric and electrochemical detectors.
6.
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.
WORKING
OF INSTRUMENTATION:
The reservoir holds the solvent,
which is referred to as the mobile phase because it moves. There are usually a
minimum of two reservoirs in a system, fitted with a gas diffuser through which
helium can be bubbled. A pump is used to generate a specified flow of the
mobile phase. Most HPLCs are now fully automated and controlled by computer.
The injector, or auto sampler, introduces the solvent into a phase stream that
carries the sample into the high pressure (up to400 bar) column, which is
referred to as stationary phase. A detector is needed to see the separated
compound bands as they elute from the high pressure column. The information is
sent from the detector to a computer which generates the chromatogram.
Applications
of HPLC:
The information that can be obtained by HPLC
includes resolution, identification and quantification of a compound. It also
aids in chemical separation and purification. The other applications of HPLC
include
v Pharmaceutical Applications
1. To
control drug stability.
2. Tablet
dissolution study of pharmaceutical dosages form.
3. Pharmaceutical
quality control.
v Environmental Applications
1. Detection
of phenolic compounds in drinking water.
2. Bio-monitoring
of pollutants.
v Applications in Forensics
1. Quantification
of drugs in biological samples.
2. Identification
of steroids in blood, urine etc.
3. Forensic
analysis of textile dyes
4. Determination
of cocaine and other drugs of abuse in blood, urine etc.
v Food and Flavor
1. Measurement
of Quality of soft drinks and water.
2. Sugar
analysis in fruit juices.
3. Analysis
of polycyclic compounds in vegetables.
4. Preservative
analysis.
v Applications in Clinical Tests
1. Urine
analysis, antibiotics analysis in blood.
2. Analysis
of bilirubin, biliverdin in hepatic disorders.
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