A Review: HPLC an introduction and it’s

application
Author : Vibhor kumar jain*, Neeraj kumar sethiya, Dinesh chandra

Corresponding author:
Vibhor kumar jain
Lecturer
Deptt. Of pharmaceutical chemistry
Faculty of Pharmacy
KNIMT , Faridipur
Sultanpur. Distt. Sultanpur
Mob. +919410411583
HPLC (High Performance/Pressure Liquid Chromatography)

Before to the 1970, few chromatographic methods were commercially

available to the laboratory scientist. During the 1970, most chemical
separations were carried out using a variety of techniques including open
column chromatography, paper chromatography, and thin layer
chromatography. However, these chromatographic techniques were
inadequate for the quantification of the compound and resolution between
very similar compounds. During this, time pressure liquid chromatography
began to be used to decrease flow through time, thus, reducing purification
times of the compounds being isolated by column chromatography (35)

HPLC was developed in the mid 1990’s and the efficiency quickly
improved with the development of column packing material in addition to
the convenience of online detectors. In the later 1970’s, new method
including reverse phase liquid chromatography allowed improved separation
between very similar compounds.
HPLC
A liquid chromatography instrument consist of 4 basic parts: (33)
i. The column ii.The detector iii.The injection system
iv Mobile phase pump system
The multiple valves allow the selection of suitable mobile phase for a
continuous flow or a gradient flow into a small mixing chamber which is
placed between the pump and the reservoir pushes it through the column and
into the detector while maintaining the constant reproducible flow rate. A
coil placed between the pump and the column dampens the pulsating action
of the pump. The injection system is inserted above the column.
Hence high pressure liquid chromatography is thus a method of
separation in which stationary phase is contained in a column, one end of
which is attached to a sources of pressurized liquid eluent (mobile phase).
Modes of Chromatography:
Chromatographic separation of two components depends on the fact
that they have different partition or distribution coefficient between the
stationary phase and the mobile phase.
i. Adsorption chromatography has widely been used in the nonpolar or

moderately polar organic molecule. During the chromatographic run, the

solids are eluted down on an adsorbent bed, usually silica or alumina,
with an organic solvents such as hexane, dichloromethane (mobile phase)
and are retained according to their affinity for the adsorbent surface.
In adsorption chromatography, molecules which are highly polar
give rise to a problem of long retention time and peak tailing. This
problem can be solved by chromatographing the compound, by the
making use of a technique known as reversed phase chromatography in
which a nonpolar stationary phase is used in conjunction with polar
mobile phase. In this technique both diamond and hydrocarbon polymers
coated on pellicular material have been used as the nonpolar supports.

ii. Ion Pair Partition chromatography (IPP) may be regarded as an

alternative procedure to ion exchange chromatography for the separation
of ionic and ionisable molecules.
a. In normal phase ion pair chromatography, silica is used as the support
with an aqueous stationary phase containing an acid (if bases are to be
separated) or a base such as tetraethyl ammonium hydroxide (if acids are
to be separated) with and an organic mobile phase such as butanol/
chloroform.
b. In reversed phase ion chromatography, a chemically bonded silica
support is used along with an aqueous phase containing the required
basic or acidic counter ions.
c. Soap chromatography is the form of IPP in which the counter ions of
the ionisable compound to be separated is a detergent.

iii. In Ion exchange chromatography, the chromatographic support

contains ion which can be exchanged with ionic solute in mobile phase.
In HPLC, the ion exchangers used for the separation of anions are the
bonded the quaternary ammonium groups whereas those for the
separation of cations are the bonded sulphonic acid groups.
iv. In exclusion chromatography, separation occurs because of the
difference in the molecular weights and the shape of the solute being
separated. Formerly the term gel permeation chromatography was used to
describe such type of separation occurring in organic solutes e.g.
Tetrahydrofuran with polystyrene matrix.

v. Affinity chromatography is similar to ion exchange chromatography

and has a great biological potential. In this technique, the ligand which
binds specifically to one particular compound or a small group of
compounds is bonded to an ionisable support such as polystyrene beds or
silica. When a complex biological mixture of a compound is percolated
down a column of such a material only those compounds which have
the affinity to the bonded groups are retained. After the elution of other
components of a mixture, the retained compound, under the analysis are
eluted by changing the mobile phase composition.

With HPLC adsorption, partition, ion exchange and exclusion type of

separations are possible with a great success.
Applications of HPLC:

1. Preparative HPLC refers to the process of isolation and purification of

compounds. The degree of solute purity and the throughput, which is

the amount of compound produced per unit time, is important. This
differs from analytical HPLC where the focus is to obtain information
about the sample compound.
2. Chemical separation can be accomplished using HPLC by utilizing

the fact that, certain compounds have different migration rates given
in a particular column and a mobile phase. Thus chromatographer can
separate compounds (more on chiral separation) from each other using
HPLC.
3. Purification refers to the process of separating or extracting the target

compound from the other (possibly structurally related) compounds or

contaminants. Each compound should have a characteristic peak
under certain chromatographic conditions. Depending upon what
needs to be separated and how closely the related samples are,
chromatographer may choose the condition.
4. Identification of the compounds by the HPLC is a crucial part of any

HPLC assay. In order to identify any compound by HPLC, a detector

must be selected first. Identifying a compound by HPLC is
accomplished by surveying the literatures and by trial and error
method. A sample of a known compound must be utilized in order to
assure identification of unknown compound.
5. Quantification of the compound by HPLC is the process of

determining the unknown concentration of the compound in a known

solution. It involves injecting a series of known concentration of the
 standard compound compound solution into the HPLC for the
detection. The chromatogram of this known concentration will give a
series of peaks that correlates to the concentration of compound
injected. Using area of triangle equation (A=1/2b*h) to calculate the
area under each peak, a set of data generated to develop a calibration
curve.

Advantages of HPLC:

1. Separation is fast and efficient.

2. Continuous monitoring of the column effluent.
3. Can be applied to the separation and analysis of very complex mixture

of compounds.
4. Accurate quantitative measurement.
5. Both aqueous and non aqueous samples can be analyzed for the

measurement.
6. It provides a mean of multiple component analysis in a single
determination.
7. Repetitive and reproducible analysis using the same column.
8. Separated components can easily be collected and used for further

analysis.
Instrumentation:

The essential feature of HPLC shown below and comprise of the following
basic components-
1. A solvent delivery system, including pump.
2. A sample injector
3. A chromatographic column
4. A detector and recording system
1. Mobile phase is pumped under the pressure from the solvent reservoir

and flows through the column at a constant rate. It is advisable to

deareated mobile phase solvent mixture using a vacuum pump or other
suitable means of deareation that has no effect on the composition of the
mixtures. The choice of mobile phase is very important in HPLC. For
normal phase separation, eluting power increases with the increasing
polarity of the solvent but for reversed phase separations eluting power
decreases with increasing solvent polarity.
2. Another most important part of HPLC is pump, because its

performance directly affects the retention time, reproducibility and

detector sensitivity. Most of the work in analytical HPLC is done using
pressure between about 400-150 psi, but for analytical applications in
which columns of 25-50 cm in length (4.0-10 mm id), packed with the
particles of 5-10 micron. The pump should be capable of delivering the
mobile phase at a flow rates 1-5 ml/min and pressure reaching 5000 psi.

There are four important types of pumps that have been used.
a. Pneumatic pumps
b. Syringe type pumps
c. Reciprocating pumps
d. Hydraulic amplifier pumps

3. The amount of sample to be injected into the column, depends upon

the fact that, the component of interest has to be detected with

adequate accuracy after the separation on the column, thus the amount
of the sample to be injected depends upon the two factors-
 # The sensitivity of the detector for these components.
# The extent of dilution undergone in the column.

Accordingly, a good injection system should have the smallest possible

contribution to peak broadening. The injection system should be –
a. Convenient to use.
b. Able to operate at high pressure.
c. Chemically inert with the eluent and the sample.
d. Reproducible.

In some cases, the injection should be possible at high temperature

required for the sample solution as for the steric exclusion chromatographic
analysis of polyethylene sample.

There are three important ways of introducing the sample into the
injection port:
a. Fixed volume valve injection
b. Variable volume valve injection.
c. On column injection.

4. The column is usually made up of heavy glass or stainless steel tubing to

withstand high pressure. The column are usually long (10-30 cm), narrow
tube contains the stationary phase at particular diameters of 25 micron or
less. The tubing must have smooth, precision bore internal diameter to
ensure that well packed column will not channel near the wall/packing
interface because of wall irregularities.
 The maximum sample volume and the sample concentration should
not exceed the linear capacity of the column. The linear capacity is the
weight of the sample per gram of packing which causes a 10% reduction in
the specific retention volume relative to constant retention volume observed
for smaller samples.
Column with internal diameter of 5 mm give good results because of
compromise between sample capacity, the amount of packing, the solvent
required and column efficiency.
The most efficient HPLC column packings are based on fully porous
particles with diameter of about 5 micron. It is desirable for the particles to
have fairly wide porousity i.e. >5nm, otherwise solute molecules may get
trapped in small pores and give rise to tailed peaks.
Silica and alumina are the two most widely used adsorbents, although
metal oxides such as ceria, zirconia and thoria have also been used as
packing materials with more alkali resistant than either silica or alumina.
The most common substances employed so far in HPLC are
hydrocarbons especially octadecylsilyl (ODS) group i.e. C18H37Si-, cation
exchanger (-Si(CH2)n-C6H4.SO3H), anion exchangers (-Si(CH2)nNH2 or –
Si(CH2)NR3+X-) and polar group such as cyanopropyl or the bonded ether
phase , gama-glycidoxyl propyl. A useful range of potential bond phases
should comprise of two hydrocarbon phases (a long and a short chain
material), a cation and anion exchanger and one or two polar bonded phases.

5. The function of the detector in HPLC is to monitor the mobile phase as it

emerges from the column. The detector for HPLC consists of a photometric
detector fitted with a low volume flow cell (about 10 µL suitable). The
detector response is usually represented as a record trace displaying the
components as a peak on a time scale. A no. of detectors used in LC is also
suitable in the case of HPLC. These are of two types:

a. Bulk property detector which compares an overall change in

physical property of the mobile phase with and without an eluting solute.
Examples of such detectors are refractive index detector and conductivity
detector.
b. Solute property detector which respond to a physical property of the
solute which is not exhibited by the pure mobile phase. Such types of
detectors are about 1000 times more sensitive, giving a detectable signal for
a few nanograms of the sample. UV visible adsorption, fluorescence and
electrochemical detectors have achieved popularity in this category of
detectors.
Sensitivity linear response and the type of response are the various
characteristics of a detector to be used in HPLC. Sensitivity is expressed as
the noise equivalent concentration or the solute concentration [Cn] that
produces a signal equal to the detector noise level. The lower the value of
Cn, for a solute, the more sensitive is the detector. It may be regarded as the
concentration range over which the response is proportional to the
concentration of the solute. Whether a detector is universal or sensitive
depends upon the type of response given by the detector. (38)

Some detectors used in HPLC:

A. Refractive index detector:

The bulk property detectors are based on the changes in the

repetitive index of the eluent from the column w.r.t the pure mobile
phase. These detectors no doubt have the advantage that many UV non
absorbing compounds may be directly measured without derivatization
and in cases where sensitivity is not very important, they are very useful.
Infect RI detector can be used in gradient elution technique,
because the base line continuously varies with the changing solvent
mixture and causes the RI variation. In order to avoid this, a step gradient
system may be used where the mobile phase is changed abruptly.
The important types of RI detectors are:
i. Deflection refractometer
ii. Fresnel refractometer

1. UV detectors:

UV adsorption detectors have widely been used in HPLC

because they are based on the principle that adsorption of UV-visible
light occurs as the effluent from the column is passed through the
small flow cell held in the radiation beam.
The UV detectors measure the changes in the UV adsorption as
the solute passes through a flow cell (usually 10 µL volume) in the UV
transparent solvent.
In order to increase the sensitivity of detection, variable
wavelength detectors have been used. They incorporate the deuterium
lamp and sometimes tungsten lamp to extend the wavelength range to the
visible domain.
The light detector is a silicon photodiode. Most diodes have
good response down to below 200nm.
 - Both single beam and double beam UV detector are
available commercially.
- Double array (Multichannel) detector is another UV
detector in which polychromatic light is passed through the flow cell.

C. Fluorescence detector:
When molecules absorb high frequency electromagnetic
radiations they are excited to higher electronic states from which they
retain to normal or fundamental state in one or several steps by the
emission of different protons. This process is called fluorescence. Light
emitted in perpendicular direction is collected on a photocell and its
intensity is measured as a function of concentration of elute in the
effluent stream.
In comparison to other detection methods, fluorescence provide
a greater detection sensitivity, lower detection limit and less sensitivity to
fluctuation of flow rate, temperature and pressure because it measures the
intensity of the emitted light.
In order to deal with the optics of a substance, some other
optical detectors have also been used-
a. Induced couple plasma
b. Polarimetry
c. Photoascaustic detectors
d. Laser thermal lens
D. Electrochemical detectors:
The popularity of the EC detector is probably due to their high
sensitivity and selectivity. Without the use of secondary reaction the no.
of compounds which can undergoe electrolysis at a pre-selected potential
is limited. Moreover, they are very suitable for the operation with micro
bore columns.
An EC detector is a transducer which permits a direct
conversion of chemical information into electric current. The EC reaction
takes place at the electrode surface which makes it possible to reduce the
cell volume to a thin layer of minimum thickness. In this way, cell
column of less than a µL can be easily obtained. In recent years, EC
detectors with a cell volume of less than 1µl have been developed and
used in conjunction with capillary columns.
Depending upon the electrolysis, EC detectors can be
subdivided into amperometric and coulometric detectors, which can
measure the current associated with the oxidation or reduction of the
solute
A large no. of other detectors has also been used in recent years for
specific application. Some of them include transparent detectors, spray
impact detectors, radioactivity detectors, electric conductivity detectors,
gas chromatography detectors, electrical conductivity detectors, gas
chromatography detectors, polymer specific detectors etc.