INTRODUCTION

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 (HPLC) is a technique used to separate and analyze
components in a mixture. It involves the use of a column packed with a small particle size
stationary phase, a high pressure mobile phase and a highly sensitive detector. It is an improved
version of column chromatography, which utilizes high pressures, smaller particle size
stationary phase and sensitive detectors to achieve faster and more efficient separations,
enabling the detection of trace amounts of compounds in complex mixtures.

TYPES OF HPLC

There are several different types of HPLC, each with its own set of advantages and applications.
Some common types of HPLC include:

1 Normal-phase HPLC (NP-HPLC): In this type of HPLC, the stationary phase is a polar
material and the mobile phase is a non-polar solvent. This type of HPLC is useful for
separating non-polar compounds, such as fatty acids and pesticides.
2 Reverse-phase HPLC (RP-HPLC): This is the most widely used type of HPLC, in which
the stationary phase is a non-polar or slightly polar material, and the mobile phase is a
polar solvent. This type of HPLC is particularly useful for separating polar compounds,
such as drugs and amino acids.
3 Ion-exchange HPLC: In this type of HPLC, the stationary phase is a solid material that binds
with ions in the mixture, which separates the compound based on their ionic charge.
This type of HPLC is useful for separating charged compounds, such as proteins and
nucleic acids.
4 Size exclusion HPLC (SEC): In this type of HPLC, the stationary phase is a porous material
that separates compounds based on their size. This type of HPLC is useful for separating
large molecules, such as proteins and polymers.
5 Affinity HPLC: In this type of HPLC, the stationary phase is a biomaterial that binds with a
specific target molecule, and separates it from the mixture. This type of HPLC is useful
for purifying specific biomolecules, such as enzymes, antibodies, and nucleic acids.

PRINCIPLE OF HPLC

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.
ADSORPTION CHROMATOGRAPHY

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

INTRUMENTATION
It consists of a solvent reservoir, degasser,injector, pump, column, detector and data system;
1 . Solvent Reservoir: 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
2 . The pump: The pump is responsible for delivering the mobile phase through the column at
a consistent and controlled flow rate. The pump is typically a high-pressure, positive
displacement pump that can generate pressures up to 400 atmospheres.
3 . The injector: The injector is used to introduce the sample into the mobile phase stream. It
can be a manual or automated injector and samples can be in a liquid, gas or solid phase.
4 . The column: The column is where the actual separation of the components of the mixture
takes place. It is typically a long tube packed with a small particle size stationary phase.
The column is typically made of stainless steel or glass, and is maintained at a constant
temperature to control the separation process.
5 . The detector: The detector is used to detect the separated components as they elute from
the column. Various types of detectors can be used, including ultraviolet-visible (UV-
vis) spectrophotometers, fluorescence detectors, and mass spectrometers. The detector
provides a signal proportional to the amount of the separated component present in the
mobile phase stream.
6 . The data system: The data system includes a computer and software that are used to control
the instrument, collect and analyse the data from the detector, and display the results in
graphical form.
7 . Degasser: It is used to remove the bubbles in mobile phase, as bubbles can affect the
pressure in the system and thus the efficiency of separation.
WORKING MECHANISHM
o The sample is injected through a port in the high pressure liquid carrier stream
between the pump and the column.
o The separation takes place in the column which varies from 3- 30 cm in length and 3
mm in diameter.
o Typical flow rates fall between 1-2 ml/min with pressure up to several thousand psi.
o The column effluent passes through a non-destructive detector where a property such
as ultraviolet absorbance, refractive index and molecular fluorescence is monitored.
o The signal is then amplified and recorded as a detector response v/s retention time.
o The graph thus obtained is called chromatogram.
o The effluent may be discarded, recycled or saved for any further research or studies in
a fraction collector which is synchronized with the detector.

TYPES OF COLUMNS

1. Reverse-phase columns: These columns are the most widely used in HPLC. The
stationary phase is a hydrophobic, or non-polar, material, such as C18 or C8. These
columns are useful for separating polar compounds, such as drugs and amino acids.
2. Normal-phase columns: These columns have a polar stationary phase, such as silica or
alumina, and are used with non-polar mobile phases. These columns are useful for
separating non-polar compounds, such as fatty acids and pesticides.
3. Ion-exchange columns: These columns contain a stationary phase that binds with ions
in the mixture, based on their charge. These columns are useful for separating charged
compounds, such as proteins and nucleic acids.
 4. Size-exclusion columns: These columns have a porous stationary phase that separates
compounds based on their size. These columns are useful for separating large
molecules, such as proteins and polymers.
5. Affinity columns: These columns have a biomaterial stationary phase that binds with a
specific target molecule, and separates it from the mixture. These columns are useful
for purifying specific biomolecules, such as enzymes, antibodies, and nucleic acids.

Applications and future Advancement of HPLC

• HPLC is a powerful and versatile analytical technique that is widely used in the
pharmaceutical industry
• Applications in pharmaceutical industry include:
o Analysis and purification of drugs and other compounds
o Testing of the purity and potency of drugs during the manufacturing process
o Analysis of drug compounds during the research and development phase
o Measurement of the concentration of drugs and other compounds in
biological fluids, such as blood, urine, and cerebrospinal fluid to monitor
the pharmacokinetics and pharmacodynamics of drugs
o Bioanalysis, formulation development, impurities and degradation product
analysis, and analyzing complex mixtures
• Versatility and sensitivity of HPLC make it an essential tool for the
pharmaceutical industry from quality control to new drug development
• HPLC is expected to continue to play an important role with new technological
advancements that will lead to more efficient, faster and sensitive separations.