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