HPLC is a technique that uses pumps to pass a pressurized liquid solvent containing a sample mixture through a column filled with adsorbent material. This causes the different components in the sample to interact slightly differently with the adsorbent, leading to separation as they flow out of the column. HPLC provides high-speed and high-performance separation compared to traditional chromatography through the use of high pressure pumps and smaller particle size adsorbents. Key components of an HPLC system include the pump, injector, column, detector, recorder, and optional devices like a degasser and column heater.

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

2.  HPLC is a technique used to separate, identify and separate the components in a
mixture of compounds.
 High performance and high speed compared with traditional column
chromatography because of the forcibly pumped mobile phase.
 It relies on pumps to pass a pressurized liquid solvent containing the sample
mixture through a column filled with a solid adsorbent material.
 Each component in the sample interacts slightly differently with the adsorbent
material, causing different flow rates for the different components and leading to
the separation of the components as they flow out the column.
 The stationary phase of the column, the sorbent, is typically a granular material
made of solid particles like silica, polymers with 2–50 micrometers in size.
 The components of the sample mixture are separated from each other due to their
different degrees of interaction with the sorbent particles.
 The mobile phase is the mixture of solvents like water/acetonitrile/methanol.

3.  The composition and the temperature of the mobile phase plays a major role in
the separation process
 Its composition and temperature play a major role in the separation process by
influencing the interactions taking place between sample components and
stationary phase.
 The interactions includes hydrophobic (dispersive), dipole–dipole and ionic or a
combination of all the types.
 Traditional liquid chromatography relies on the force of gravity to pass the
mobile phase through the column
 In HPLC 50-350 bar pressure is employed, with the column dimensions of 2.1-
4.6 mm diameter and 30-250 mm length. The stationary phase particles are 2-50
µm, which offers an efficient separation.

4. Components of HPLC
 Pump
 Injector
 Column
 Detector
 Recorder
 Degasser
 Column Heater

6. Pump
 Most important part of the system positioned in the upper stream of the
chromatograph.
 Generates a flow of eluent from the solvent reservoir to the system.
 Most LC pumps generates the flow by back-and-forth motion of a motor-
driven piston. This piston motion produces pulses.
 Recent pumps generate much less pulse compared to the older ones.
 These pumps are very important, since a minor change in the flow rate can
influence the analysis.
Injector
 Placed next to the pump, place for injection of sample.
 Hamilton syringe is used for sample injection.
 The use of auto sampler (auto-injector) system is also widely used that allows
repeated injections in a set scheduled-timing.

7. Column
 Depending on the type of application, the column varies in size and make.
 Generally made of stainless steel and packed with high pressure to ensure good
resolution and separation.
 For biomolecules, polyether ether ketone is employed as column material
instead of stainless steel.
 Pre-columns and guard columns – for protecting the main column
 Pre-column – To filter the solvents
 Guard column – TO filter the sample
Detectors
 Used to analyze the column eluents
 Five different types
 UV-visible adsorption detector
 Refractive index detector
 Electrochemical detector
 Fluorescent detector
 Mass spectrometer

8. Recorder
 The change in eluent detected by a detector is in the form of electronic
signal, to read those signals computer based data processor is attached with
the system. Different types are available. Simple system consisting of in-
built printer and word processor
 PC type consisting of display monitor, keyboard and printer.
 Software are also available for data acquisition, peak-fitting, baseline
correction, automatic concentration calculation, molecular weight
determination etc.
Column heater
 The LC separation is often largely influenced by the column temperature.
 In order to obtain repeatable results, it is important to keep the consistent
temperature conditions.
 For sugar and organic acids, better resolutions can be obtained at elevated
temperature (50~80 oC). It is also important to keep stable temperature to
obtain repeatable results even it is analyzed at around room temperature.
 There are possibilities that small different of temperature causes different
separation results.
 Thus columns are generally kept inside the column oven (column heater).

9. Applications
 Medical field - detecting vitamin D levels in blood serum
 Legal issues - detecting performance enhancement drugs in urine
 Research - separating the components of a complex biological sample or
similar synthetic chemicals from each other
 Manufacturing units - production process of pharmaceutical and
biological products