HPLC: Principle, Parts, Types,

Uses, Diagram
May 24, 2024 by Sagar Aryal
Edited By: Sagar Aryal

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.

Image Source: Sartorius AG.

Table of Contents
 HPLC Principle
 Instrumentation of HPLC
 Types of HPLC
 Applications of HPLC
 Advantages of HPLC
 Limitations of HPLC
 References

HPLC Principle
 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 HPLC
 Instrumentation of 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).

Types of HPLC
1. Normal phase:

Column packing is polar (e.g silica) and the mobile phase is

non-polar. It is used for water-sensitive compounds,
geometric isomers, cis-trans isomers, and chiral compounds.
1. Reverse phase:

The column packing is non-polar (e.g C18), the mobile phase

is water+ miscible solvent (e.g methanol). It can be used for
polar, non-polar, ionizable, and ionic samples.
1. Ion exchange:

Column packing contains ionic groups and the mobile phase

is buffer. It is used to separate anions and cations.
1. Size exclusion:
 Molecules diffuse into pores of a porous medium and are
separated according to their relative size to the pore size.
Large molecules elute first and smaller molecules elute later.

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

Limitations of HPLC
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.