High Performance Liquid
Chromatography (HPLC)
HPLC is used in pharmaceuticals for
analysis of dosage forms before send these to market because HPLC is most
accurate method for analysis.
High performance liquid chromatography
(HPLC) is a fast
column liquid chromatography method where a solvent is passed through a column
under high pressure (of up to 400atms). Like every other form of
chromatography, HPLC separates a liquid sample into its constituent parts on
the basis of the differences between molecules of the subject mixture and the
molecules mobile and stationary phases.
For chromatography to occur, there
must be a stationary phase and a mobile phase. The adsorbent normally is a
solid (or a liquid) in nature and the mobile-phase normally a liquid or a gaseous
matter.
The solvent is responsible for
carrying the constituents of the subject mixture through the stationary phase.
More lagging is experienced in components which interact more with the
stationary phase.
In HPLC, a
pressure pump forces the solvent (mobile phase) together with the subject
mixture through a column with the stationary phase material (normally a solid).
In the column, each component of the mixture will interact differently with the
stationary phase.
Due to the interaction with the stationary
phase, these components in the mixture will separate, each exiting the column
on its own. It is important that the temperature of both the phases be kept
constant.
Components of an HPLC Instrument:
Any HPLC instrument generally
comprises of:
Columns: This is where the stationary-phase
material is placed. It is about 5 mm in diameter and can be as long as 300m.
Pumps: These supply high pressure of up to
400 atms that forces the mixture and solvent through the column.
Sampler
Injector: This
delivers the mixture in the subject to the mobile phase.
Detector: This device is located at the and of
the column. It facilitates quantitative analysis of the different components of
the mixture. The device detects the components as they flow out of the column.
UV-spectroscopy is a commonly used detector.
Different
Types Of High Performance Liquid Chromatography
The different types of HPLC exist on
the basis of the stationary phase system. Different materials used in the
stationary phase have different methods of interaction with components. The
following are the different types of HPLC.
Size-exclusion
HPLC: The material
used in the stationary phase in this type operates on the basis of components'
molecular size. The material has pores of specific sizes. The larger molecules
are eluted faster than the smaller ones.
Ion-exchange
HPLC: This type of
HPLC operates on the basis of ionic charges. The adsorbent has ionic charges
that are opposite to the subject constituents' ionic charges. Constituents with
a higher ionic charge will experience more attraction and so they will lag
through the column. Those with a lower ionic charge will experience lesser
attraction and they will be eluted fast.
Normal
phase HPLC: The basis
for the operation of normal phase HPLC is polarity. The solvent is non-polar
(normally an organic compound) and adsorbent polar. Polar materials will
interact more with each other as opposed to polar and non-polar interactions.
The less polar components of a mixture will be eluted faster than the more polar
components.
Reverse
Phase HPLC: In this
type, the solvent polar (hydrophilic) and adsorbent non-polar. It is the
opposite of the normal phase HPLC. The non-polar components will take longer to
exit the column.
Uses
Of HPLC In Pharmaceuticals:
In the pharmaceutical industry, it is mainly used for analytical studies.
Manufactured drugs are always under constant analysis to check for compliance
with the required standards and determine their dosage.
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