High performance liquid chromatography: A short review

Abstract

High performance liquid chromatography (HPLC) is an important qualitative and quantitative technique, generally used for the estimation of pharmaceutical and biological samples. It is the most versatile, safest, dependable and fastest chromatographic technique for the quality control of drug components. This article was prepared with an aim to review different aspects of HPLC, such as principle, types, instrumentation and application.

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ISSN 0975 – 8542
Journal of Global Pharma Technology
Available Online at www.jgpt.co.in
REVIEW ARTICLE
© 2009, JGPT. All Rights Reserved. 22
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY: A SHORT
REVIEW
Malviya R, Bansal V*, Pal O.P. and Sharma P.K.
Department of pharmaceutical technology, Meerut Institute of Engineering and Technology,
Meerut, India
*For correspondence: Email: vipinbansal1986@gmail.com
Abstract: High performance liquid chromatography (HPLC) is an important qualitative and quantitative
technique, generally used for the estimation of pharmaceutical and biological samples. It is the most
versatile, safest, dependable and fastest chromatographic technique for the quality control of drug
components. This article was prepared with an aim to review different aspects of HPLC, such as principle,
types, instrumentation and application.
Keywords: High performance liquid chromatography, instrumentation, elution, applications, mobile phase.
INTRODUCTION
High-performance liquid chromatography
(or High pressure liquid chromatography,
HPLC) is a specific form of column
chromatography generally used in
biochemistry and analysis to separate,
identify, and quantify the active
compounds. [1] HPLC mainly utilizes a
column that holds packing material
(stationary phase), a pump that moves the
mobile phase(s) through the column, and a
detector that shows the retention times of
the molecules. Retention time varies
depending on the interactions between the
stationary phase, the molecules being
analyzed, and the solvent(s) used. [2] The
sample to be analyzed is introduced in
small volume to the stream of mobile
phase and is retarded by specific chemical
or physical interactions with the stationary
phase. The amount of retardation depends
on the nature of the analyte and
composition of both stationary and mobile
phase. The time at which a specific analyte
elutes (comes out of the end of the
column) is called the retention time.
Common solvents used include any
miscible combinations of water or organic
liquids (the most common are methanol
and acetonitrile). [2, 3] Sepration has been
done to vary the mobile phase composition
during the analysis; this is known as
gradient elution. [3] The gradient separates
the analyte mixtures as a function of the
affinity of the analyte for the current
mobile phase. The choice of solvents,
additives and gradient depend on the
nature of the stationary phase and the
analyte.
TYPES OF HPLC
Types of HPLC generally depend on phase
system used in the process. [3, 4]
Following types of HPLC generally used
in analysis-
Normal phase chromatography: Also
known Normal phase HPLC (NP-HPLC),
this method separates analytes based on
polarity. NP-HPLC uses a polar stationary
phase and a non-polar mobile phase. The
polar analyte interacted with and is
retained by the polar stationary phase.
Adsorption strengths increase with

Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26
increased analyte polarity, and the
interaction between the polar analyte and
the polar stationary phase increases the
elution time.
Reversed phase chromatography:
Reversed phase HPLC (RP-HPLC or RPC)
has a non-polar stationary phase and an
aqueous, moderately polar mobile phase.
RPC operates on the principle of
hydrophobic interactions, which result
from repulsive forces between a polar
eluent, the relatively non-polar analyte,
and the non-polar stationary phase. The
binding of the analyte to the stationary
phase is proportional to the contact surface
area around the non-polar segment of the
analyte molecule upon association with the
ligand in the aqueous eluent.
Size exclusion chromatography: Size
exclusion chromatography (SEC), also
called as gel permeation chromatography
or gel filtration chromatography mainly
separates particles on the basis of size. It is
also useful for determining the tertiary
structure and quaternary structure of
proteins and amino acids. This technique is
widely used for the molecular weight
determination of polysaccharides.
Ion exchange chromatography: In Ion-
exchange chromatography, retention is
based on the attraction between solute ions
and charged sites bound to the stationary
phase. Ions of the same charge are
excluded. This form of chromatography is
widely used in purifying water, Ligand-
exchange chromatography, Ion-exchange
chromatography of proteins, High-pH
anion-exchange chromatography of
carbohydrates and oligosaccharides, etc.
[3, 4]
Bio-affinity chromatography: Separation
based on specific reversible interaction of
proteins with ligands. Ligands are
covalently attached to solid support on a
bio-affinity matrix, retains proteins with
interaction to the column-bound ligands.
Proteins bound to a bioaffinity column can
be eluted in two ways:
• Biospecific elution: inclusion of free
ligand in elution buffer which
competes with column bound ligand.
• Aspecific elution: change in pH, salt,
etc. which weakens interaction protein
with column-bound substrate.
Because of specificity of the interaction,
bioaffinity chromatography can result in
very high purification in a single step (10 -
1000-fold).
PARAMETERS
For the accurate analysis of a compound,
there are some parameters which are used
as a standard for a particular compound. If
there is a change occurs in the parameters
the result may be affected greatly. The
most commonly used parameters are
internal diameter, particle size, pore size,
pump pressure. For different compounds
the parameters can be changed according
to their nature and chemical properties.
Internal diameter: The internal diameter
(ID) of an HPLC column is a critical
aspect that determines quantity of analyte
that can be loaded onto the column and
also influences sensitivity. Larger columns
are usually seen in industrial applications
such as the purification of a drug product
for later use. Low ID columns have
improved sensitivity and lower solvent
consumption at the expense of loading
capacity.
Particle size: Most traditional HPLC is
performed with the stationary phase
attached to the outside of small spherical
silica particles (very small beads). Smaller
particles generally provide more surface
area and better separations, but the
pressure required for optimum linear
velocity increases by the inverse of the
particle diameter squared.
Pore size: Many stationary phases are
porous to provide greater surface area.
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Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26
Small pores provide greater surface area
while larger pore size has better kinetics
especially for larger analytes. Pore size
defines an ability of the analyte molecules
to penetrate inside the particle and interact
with its inner surface. This is especially
important because the ratio of the outer
particle surface to its inner one is about
1:1000. The surface molecular interaction
mainly occurs on the inner particle surface.
Pump pressure: Pumps vary in pressure
capacity, but their performance is
measured on their ability to yield a
consistent and reproducible flow rate.
Modern HPLC systems have been
improved to work at much higher
pressures, and therefore be able to use
much smaller particle sizes in the columns
(< 2 micrometres).
INSTRUMENTATION
Injection of the sample: Septum injectors
are available; using which sample solution
is injected. Sample can be injected when
the mobile phase is flowing or it is
stopped. A new advanced rotary valve and
loop injector can be used to produce
reproducible results.
The detector: There are several ways of
detecting when a substance has passed
through the column. Generally UV
spectroscopy is attached, which detect the
specific compounds. Many organic
compounds absorb UV light of various
wavelengths. The amount of light
absorbed will depend on the amount of a
particular compound that is passing
through the beam at the time.
Interpreting the output from the
detector: The output is recorded as a
series of peaks, each one representing a
compound in the mixture passing through
the detector and absorbing UV light. The
area under the peak is proportional to the
amount of substance, which is passed
through detector, and this area can be
calculated automatically by the computer
linked to the display.
APPLICATION
The information that can be obtained using
HPLC includes identification,
quantification, and resolution of a
compound. Preparative HPLC refers to the
process of isolation and purification of
compounds. This differs from analytical
HPLC, where the focus is to obtain
information about the sample compound.
Chemical Separations It is based on the
fact that certain compounds have different
migration rates given a particular column
and mobile phase, the extent or degree of
separation is mostly determined by the
choice of stationary phase and mobile
phase.
Purification: Purification is defined as the
process of separating or extracting the
target compound from a mixture of
compounds or contaminants. Each
compound showed a characteristic peak
under certain chromatographic conditions.
The migration of the compounds and
contaminants through the column need to
differ enough so that the pure desired
compound can be collected or extracted
without incurring any other undesired
compound.
Identification Generally assay of
compounds are carried using HPLC. The
parameters of this assay should be such
that a clean peak of the known sample is
observed from the chromatograph. The
identifying peak should have a reasonable
retention time and should be well
separated from extraneous peaks at the
detection levels which the assay will be
performed.
Other applications of HPLC: Other
applications of HPLC includes
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Bansal V. et al., Journal of Global Pharma Technology. 2010; 2(5): 22-26
Pharmaceutical applications [5-8]
• Tablet dissolution study of
armaceutical dosages form.
• Shelf-life determinations of
parmaceutical products
• Identification of active ingredients of
dosage forms
• Pharmaceutical quality control
Environmental applications [9-12]
• Detection of phenolic compounds in
Drinking Water
• Identification of diphenhydramine in
sedimented samples
• Bio-monitoring of pollutant
Forensics [13-15]
• Quantification of the drug in biological
samples.
• Identification of anabolic steroids in
serum, urine, sweat, and hair
• Forensic analysis of textile dyes.
• Determination of cocaine and
metabolites in blood
Clinical [16-19]
• Quantification of ions in human urine
Analysis of antibiotics in blood
plasma.
• Estimation of bilirubin and bilivirdin in
blood plasma in case of hepatic
disorders.
• Detection of endogenous
neuropeptides in extracellular fluids of
brain.
FoodandFlavor [20]
• Ensuring the quality of soft drink and
drinking water.
• Analysis of beer.
• Sugar analysis in fruit juices.
• Analysis of polycyclic compounds in
vegetables.
• Trace analysis of military high
explosives in agricultural crops.
CONCLUSION
It can be concluded from the entire review
that HPLC is a versatile, reproducible
chromatographic technique for the
estimation of drug products. It has wide
applications in different fields in term of
quantitative and qualitative estimation of
active molecules.
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