HPLC

INTRODUCTION
HPLC is derived from classical column chromatography. HPLC is a high
resolution and high-speed liquid chromatography. It is known as high
performance liquid chromatography as it has improved performance than
classical column chromatography. It is also known as high pressure liquid
chromatography as it uses high pressure as compared to classical method. It is a
method in which stationary phase is contained in a column and one end of
which is attached to a high-pressure mobile phase. In HPLC, the particle size is
very small as compared to classical column chromatography. Smaller particle
size gives more surface area and hence better Performance. HPLC 1S a type of
liquid chromatography which utilizes high pressure
pumps provide mobile phase at high pressure. Basic principle of HPLC and
liquid chromatography is same i.e. it is based on the principle of adsorption and
partition.

TYPES OF HPLC

HPLC technique is classified into following types

1) Depending upon Mode of Separation
According to the polarity of stationary phase and mobile phase, HPLC is of two
types
a) Normal phase (NP)
b) Reverse phase (RP)

Normal phase (NP) Chromatography

In this mode, stationary phase is polar and mobile phase is non-polar. Non-polar
compounds travel faster with non-polar mobile phase and eluted out first. There
exists less affinity between solute and stationary phase. Polar compounds have
more affinity for polar stationary phase so they retained for longer time in the
column and hence eluted out of the
column after a long time. This method is of less importance in pharmacy as
most of the pharmaceutical compounds and drugs are polar in nature and will
take more time for elution and detection
n-polar compounds

Reverse phase (NP) Chromatography

In this mode, stationary phase is non- polar and mobile phase is polar in nature.
It is reverse of normal phase chromatography. Polar compounds eluted first (i.e.
have high affinity for mobile
HPLC
phase) while non-polar compounds retained on the column. It is most
advantageous for the analysis of pharmaceuticals as most of the drugs are polar
in nature and will eluted out of the column quickly.

2) Depending upon elution technique

Based upon the elution technique HPLC is classified into following types
a) Isocratic Separation
In this method, same mobile phase combination is used during the entire
separation technique. The mobile phase of same polarity and composition is
maintained throughout the process.
b)gradient separation
In this mobile phase of low polarity or composition is used and polarity is
gradually increased from time to time i.e. there is step by step increase in
polarity and elution strength of mobile phase.
3) Depending upon type of Operation
a) Analytical HPLC
this method only analysis of the samples are done. Recovery of the sample is
not possible as quantity of the sample used is very small i.e. micrograms
quantity.
b) Preparative HPLC
Recovery of the sample is the main aim of this type of HPLC. In this technique
individual components of pure compounds can be collected and the collected
samples can be reused.
4) Depending upon type of analysis
a) Qualitative Analysis
It is done to identify the compounds and to detect the presence of impurities in
the given sample. This method is helpful to check the quality of the sample.
Qualitative analysis is done by calculating retention time values.
b) Quantitative Analysis
This is done to determine the quantity of each component of the sample. This is
done by comparing the peak area of the standard with that of the sample.

PRINCIPLE OF HPLC
The basic principle of HPLC in normal phase and reverse phase mode is
adsorption. The sample is introduced into HPLC column, diferent components
of the sample move according to their affinities towards the stationary phase.
Components having high affinity gets adsorb on stationary phase while
components having less affinity towards stationary phase travels faster down the
column. As no two components have the same adsorption
HPLC
and affinity towards stationary phase, the components get separated by this
method.

INSTRUMENTATION
The main components of HPLC system are
1) Mobile phase Reservoir
2) A High Pressure Pump
3) Sample Injection System
4) Column
5) Mobile phase
6) Detectors
7) Recorder and Integrator

1) Mobile phase Reservoir

It consists of a glass or stainless-steel reservoir which contains upto 1000ml of
mobile phase. Degassing is required in HPLC systems so, reservoirs are
equipped with degassers. Degassers
may consist of-
a) Vacuum pumping system
b) Distillation system
c) Devices for heating and stirring of solvents
d) Sparging Unit ( in this the dissolved gases are removed from the solution
by passing fine bubbles of an inert gas of low solubility)
In HPLC mobile phase can be aqueous organic mixtures, mixtures of organic
solvents or buffers solutions. Mixing of different solvents is done by using a
static mixture or a dynamic mixture which uses magnetic stirrer and operates
under high pressure. Several gases are soluble in organic solvents. So when
such solvents are passed through column under high pressure, gas bubbles
are formed which causes interference with separation. A steady base line is not
formed hence degassing is required and can be done by using one of the
following techniques –
A) by ultra sonification
B) By filtration method
HPLC
A) by ultra sonification

In this dissolved gas is removed by an ultra-sonification. This can be done by

two ways
1) By placing an ultrasonicator into the solvent which produce ultrasonic waves.
These waves burst the gas bubbles and gas gets expelled out.
2) The solvent beaker is placed in an ultrasonicator trough. The water in the
sonicator produces an ultrasound wave which helps in degassing.
B) By filtration method
IT is highly effective and can be done
IT is highly effective and can be done by using-
1) Vaccum filtration - it removes air bubbles and entrapped air during passage
of solvent through filter pores.
2) Helium purging - in this method helium is passed through the solvent which
removes entrapped air and gas bubbles.
3)Mechanical stirring-this method provides high quality degassing as
compared toother method.
In actual there is synergistic action of the above three methods for efficient
solvent degassing
2) A high pressure pump
In HPLC pumps are used to pass mobile phase through the column at high
pressure and at controlled flow rate. The particle size of stationary phase is very
small and resistance to
flow of the solvent is high hence high pressure is required. Generally a pressure
of about 1000 - 3000psi is required. HPLC pumps should have following
qualities -
a) It must generate pressure up to 6000psi.
b) It must have proper flow control and flow reproducibility.
c) It should give a pulse free output.
d) It should be easy to change from one mobile phase to another.
e) It should provide constant flow rate ranging from 0.1 ml - 10 ml/min.
Various types of pumps are available. These are mechanical g which operate
with constant flow rate and uses a piston. These types of pumps are il in
analytical purpose. Pneumatic pumps which operate with constant pressure and
uses highly compressed gases.
Broadly, pumps are categorized into following types -
1) Constant displacement pumps
2) Reciprocating pumps
3) Pneumatic pumps
1) Displacement pumps
HPLC
These are also known as syringe pumps. It consists of large syringe like
chamber which is equipped with a plunger which is operated by a screw driver
mechanism operated by a motor.
So displacement pump is a motor driven syringe pump where fixed volume of
solvent is forced from the pumnp to the column by a piston.
Advantages
1) It provides uniform flow rate.
2) It gives pulse less solvent flow.
3) Flow is independent of viscosity and column pressure.
Disadvantages
1) It has limited solvent capacity.
2) It is not useful for the purpose of gradient elution.

2) Reciprocating pumps (Constant flow pumps)

It consists of a small chamber in which a piston is moved back and forth with
the help of a motor which pushes the solvent at a high pressure into the column.
The entry and exit of the solvent to the column is regulated by check valves. On
compression the solvent is forced from the pumps into the column. During its
return the exit check valve closes and solvent is drawn in via the entry valve to
the pump chamber ready to be pumped into the column in the next step. These
pumps have inbuilt safety cut out mechanism and preset limits to avoid any
accident.
Advantages
1) It has small internal volume and provides high output pressure.
2) These are adaptable to gradient elution.
3) They provide constant flow rate.
4) Flow rate does not depend upon the column pressure and solvent viscosity.
Disadvantages
1) It does not provide pulse less flow rate.
2) It produces base line noise.

3) Pneumatic pumps
It is also known as constant pressure pump. In this mobile phase is placed a
collapsible container which is pressurized by a compressed gas. These pumps
operate by the introduction
of high pressure gas into the pump and this gas pushes the solvent from pump
chamber into the column.
Advantages
1) It provides pulse free flow.
2) It is cheap.
Disadvantages
HPLC
1) In this flow rate depends upon solvent viscosity and column pressure.
2) Gradient elution is not possible with this type of pump.
3) It gives less pressure output i.e. below 2000psi.

3) Sample injection system

Introduction of the sample into the column is most important part of HPLC.
Overloading of the sample causes band broadening so minimum amount of
sample must be introduced. Generally sample is introduced without
depressurizing the system. Sample is injected at the head of the column with
minimum disturbance of the column packing material.
Various types of devices are available for injection of the sample. Injectors are
of two types -
a) Manual injection
b) Auto injection

Different types of devices used for injection are -

1) Syringe injection
It is simplest and earliest technique. In this sample is injected through a self-
sealing elastomeric septum. Syringes are made to withstand pressures up to
2000psi. These injectors are generally
fixed volume injections. The main disadvantage of this system is that
reproducibility is poor.
2) Stop flow injection
It is also known as online injection system. This system also utilizes a syringe
injection but here the flow of mobile phase is stopped for a while and the
sample is injected directly on the head of the column packing at atmospheric
pressure and system is again pressurized. This is Very simple technique and
sometimes valve devices are used to inject the sample into the column,
3) Solvent flowing injectors
In this sampling valves or loops are used. It is also known as Rheodyme
injector. it is generally used when sample volumes are more than 10l. It is most
popular injector and has two mode-
a) Load position
in this sample in the loop is filled at atmospheric pressure i.e. sample is loaded
in the loop.
b Inject mode
In this the sample is injected into the column.
The sample is first loaded into an external loop in the valve and then introduced
into mobile phase by rotating the valve. Automatic sample injectors are also
available.
HPLC
Valve injectors are better than syringe injectors but valve injectors produces
more band Broadening as compared to syringe injectors. In spite of this fact,
due to convenience of the use of valves and their capability these are most
widely used.

4) Columns
The column is made up of heavy glass or stainless steel which can withstand
high pressures. The length of the column is generally 10-30cm with diameter of
25um. Columns are of two types
1) Analytical Columns
2) Preparative Columns
For analytical columns the ratio of size : length is 25:100 with internal diameter
of 2-6mm. For preparative columns ratio of size : length is 25:100 and internal
diameter is 6mm or more.
Columns decide the efficiency of separation. There are various types of
stationary phases for columns depending upon the technique and mode of
separation.
Column packing material
Column packing material is of three types
1) Microporous Supports
In this branched microporous particles of diameter 5-10um are used. This type
of packing material is not widely used for adsorption chromatography.
Adsorbents like silica or alumina are available as microporous forms.
2)Pellicular Supports (Superficially porous)
It consists of non-porous, spherical glass or polymer beads of diameter 30-
40um. A thin porous layer of silica or alumina is coated over it. Sometimes
polymer beads may be treated chemically
to produce an organic surface. It is further of three types
a) Porous polymeric beads
It is based on styrene and divinyl benzene copolymer. It is mainly used for ion
exchange and size exclusion chromatography.
b) Porous layer beads
It consists of thin shell of silica or modified silica on a spherical inert core. c)
Totally porous silica particles
It is widely used for analytical purposes. The mechanical strength of the
packing and particle
size of the stationary phase decides the separation efficiency.
Bonded phase column
In this stationary phase is chemically bonded to an inert support. The major
disadvantage of the support coated with liquid phase is that the mobile phase is
gradually washed off the liquid phase with repeated use. To overcome this
HPLC
problem bonded phase have been developed in which liquid phase has been
covalently bonded to the supporting material which may be silica or silicon
polymer. Silicon polymer bonded phases are chemically, hydrolytically and
thermally stable. So they do not wash off with the developing solvent.
Bonded phases are prepared by the reaction of silica with an alcohol. In this
process the reactive silanol groups (Si-OH) on silica gel reacts with alcohol and
gets esterified i.e.
Si-OH-----→Si-OR
Such stationary phases are known chemically bonded stationary phases. The Si-
O-R bonds formed are hydrolytically unstable. So conversion of Si-OH into
the other types of bonded groups is carried out. Other bonded phase can be
prepared by first converting Si-OH groups into Si-Cl groups (by using thionyl
chloride) or conversion of Si-OH into Si-N (by using silica chloride and
amines). These bonded phases on reaction with Grignard or Organolethium
reagents produce Si-R bonds which have very high hydrolytic stability.
Commercially available bonded materials are prepared by reaction of
Organochloro silianes or organoalkoxy silanes with surface silanol groups of
silica.
In these two types of reactions are involved-

a) Chloro or alkoxy silane is hydrolyzed and partialy polymerized and then

chemically bonded to the surface of silica via hydrolytically stable siloxane
bonds (Si-O-Si) for
e.g permaphase support.
b) Direct reaction of chloro or alkoxy silane with the anhydrous support to
produce monomolecular siloxane bonds. For e.g. Bonda pack C-18, Li Chrosorb
RP-8 etc. are prepared by this method. The most common substituents in HPLC
are hydrocarbons for e.g. octadecyl silyl (ODS) groups ie. C,H, silica.
Reaction for chemically modified HPLC column packing are

Guard column
These are made up of same material as that of analytical column. It is generally
placed before the column to improve the life of the column. It has very small
quantity of adsorbent. It acts as a pre filter to remove impurities or any dust
HPLC
particle if present in the solution or Sample. It acts as a safety guard for the
column.
Column Packing 'procedure
various methods are available for packing columns which depends upon the
nature of packing material and dimensions of the particle. Main purpose is to
obtain a uniform bed of packing material with no cracks. The most widely used
technique is high pressure slurry packing. In this method a suspension of
packing material is made in a solvent of equal density. The slurry is then poured
at high pressure into the column. This packing is generally used in case of rigid
solids and hard gels.
Soft gels cannot be packed under pressure but they are allowed to pack in the
column under gravitational sedimentation only. For hard gels they are first
allowed to swell in the solvent before packing under pressure.
5) Mobile Phase
Mobile phase is pumped under high pressure through the column at constant
rate. Deaerated mobile phase solvent mixture is generally used. The choice of
mobile phase is very important in HPLC as it act as a carrier for the sample
solution. The chemical interaction of mobile phase and sample with the column
determines the degree of migration and separation of components present in the
sample. There are various types of elution techniques like-
Isocratic- in this separation is made with a single solvent or two or more
solvents mixed in a fixed ratio.
Gradient- in this the composition of mobile phase is continuously changed
using a gradient programmer.
Mobile phase should have following properties
1) Non-hazardous
2) Low cost
3) Sample components should be miscible with mobile phase
4) Detectors should not respond to any changes in mobile phase composition

Mobile phase generally consists of water-organic organic solvents, aqueous

buffers or mixtures of solvents with or without modifier. The choice of mobile
phase depends on the mode of HPLC. Mobile phase in normal phase
chromatography consists of non-polar solvents like hexane, heptane, isooctane
used in combination with slightly more polar solvents like ethyl acetoacetate or
chloroform. Retention time increases on increasing the amount of non-polar
solvents.
In reverse phase chromatography the mobile phase is water. Other polar
solvents like methanol, acetonitrile or tetrahydrofuran are used. pH is adjusted
by using buffer to modify the separation of ionisable substances. lon pairing
reagents also increases separation and retention on hydrophobic bonded phases.
HPLC
Most of the common solvents used in HPLC are flammable. So special grades
of solvents are available for HPLC and these must be purified in order to be
remove UV absorbing impurities and any particulate matter.

6) Detectors
Detectors are required to detect the presence and amount of sample components
present in the column effluent. The output of detector is an electrical signal
which is proportional to some property of mobile phase and solute.
Depending upon the property of component to be separated detectors are of
following types -
a) Bulk property detector
A detector which measures the property which is possessed by both mobile
phase and solute is known as bulk property detector. For e.g. refractive index
detector (RID).
Refractive index detector (RID) - Refractive index is the ratio of sped of life
in a vacuum to the speed in the medium. These detectors measure the change in
refractive index eluent as the solute passes through the sample cell. This is non-
specific or universal detector. In this a differential refractometer measures the
difference in refractive index b/w the pure mobile phase and column effluent.

Apparatus - light from the source is passed on to the cell which contains
sample and reference chambers separated by a glass sheet. After passing
through cell, light is diverted to beam splitter. A change in refractive index of
the sample causes a change in amount of light falling on two photo cells which
produces a difference in their relative output. The difference is
amplified and produces a signal

b) Solute property detector

In this, detector measures the property possessed by the solute only for e.g. UV-
VIS detector.
HPLC
1) UV detector

This is based upon the light absorption property of the sample. It measures the
change in UV absorption as the solute passes through the cell. These are of
further two types i.e. fixed wavelength detector which operates at 254nm
(where most of drug compounds absorb UV). The other is the variable
wavelength detector which operates in region 190 - 600nm. These
detectors are selective and detect only that solute which absorbs UV radiation
for eg. alkanes, aromatic compounds and compounds having multiple bonds.
This detector is 1000 times more sensitive than refractive index detector.

2) Fluorometric detector
It is based upon the fact that fluorescent radiations are emitted by some
compounds. For each compound excitation and emission wavelength can be
selected. This detector has more sensitivity and specificity. Those compounds
which are not fluorescent can by not be detected this detector.

3)Amperometric detector
It is based upon the principle of oxidation and reduction of compounds when
potential is applied. The diffusion current recorded is proportional to
concentration of compound eluted. It is highly sensitive. This detector is
applicable to those compounds which have functional groups and can be
oxidized and reduced.
4)Conductivity Detector
It is based upon electrical conductivity. This detector is used for those samples
which have anions or cations to produce conductance.
5)Photo Diode Detector (PDA Detector)
It is similar to UV detector which operates from 190 - 260nm. The resulting
spectra are three-dimensional plot of response Vs time Vs wavelength. The
HPLC
advantage is that the wavelength needs not to be selected, but detector deflects
the response of all the compounds.

7) Recorder and integrator

Recorders are used to record the responses obtained from detectors after
amplification. They record baseline of all the peaks obtained with respect to
time. Retention time can be calculated but area of each peak cannot be
measured. Integrators can record the individual peak with retention time, height
and width of peaks, peak area and % age of area. Thee are improved versions of
recorders.
Applications
HPLC is a versatile and sensitive technique which is useful in various ways.
Some of these are
1) Quantitative analysis
It involves identification of compounds in a mixture. It is done by comparing
the retention time of the sample with that of the standard. If there is any
deviation in their retention time then they are not same compounds.
2) To check purity of compound
In this the chromatogram of the standard and that of the sample is compared. If
additional peaks are obtained it means impurities are present and the compound
is not pure. By calculating the area of the peaks obtained, the %age purity of a
given sample can be determined.
3) Quantitative analysis
The quantity of a particular component in the given mixture can be determine
easily by using one of the following methods –

A) Calibration curve method

this method a series of different concentration of standard is prepared and peak
area of each solution is determined. A calibration curve of peak area vs
concentration of the drug is plotted. From peak area of unknown sample, by
interpolation the concentration can be determined.
B) Internal standard method
According to this method, a known concentration of the internal standard is
added to the sample solution whose concentration is to be measured. The
chromatogram is recorded and Peak areas are determined, The concentration of
unknown sample is determined by same formula as in case of gas
chromatography.

C)Stability studies
HPLC
HPLC is useful for studying the stability of various pharmaceutical compounds
ie. Analysis of various degradation products is carried out. For example,
stability studies of atropine_.
d) Multicomponents analysis
It can be done easily and is similar to quantification of a single drug. The
quantity of each component is determined and simultaneous estimation of two
or more drugs in marketed formulation can be carried out easily.
e) As a compliment to Bioassays
Many drugs like antibiotics and hormones are analysed by bioassays but it is a
costly technique and gives poor precision. So HPLC can be used as a
compliment to bioassays and gives accurate results. HPLC is useful for analysis
of various antibiotics and peptide hormones. It also gives idea about the bio
pharmaceutics of the dosage form and pharmacokinetics of the drugs.
f) Separation of natural products
HPLC is useful for separation of various components present in the plant extract
which resemble in structure and hence require very sensitive method for
analysis for e.g, analysis of ergot extract, digitalis, cinchona etc.
g) Cosmetic industry
HPLC is useful in checking the quality of various cosmetics like lipstick,
shampoos, lotions etc. It is helpful in determining the quality control of various
cosmetic products