In early 1900`S Mikhail .Tswett a botanist has invented the
He termed this system of colored bands as the chromatogram and
method as chromatography (i.e; chroma -color and graphos -writing )
In 1941 martin and synge introduced partition and paper
chromatography and also laid the foundation for gas liquid
chromatography and high performance liquid chromatography in the
Classic chromatography was slow rate for analysis which lead to delay
and loss of resolution by diffusion this problem was largely overcome
In the year 1969 a very marketed revival of interest in the technique of
liquid chromatography because of development of HPLC by Kirkland
and Huber (waters corporation and named ALC 100 hplc)
It is defined as a method of separating a mixture of components
into individual components through equilibrium distribution between two
HIGH PRESSURE LIQUID CHROMATOGRAPHY:
high performance liquid chromatography is chromatographic
instrumental technique of analytical chemistry.(to separate ,identify and
quantify the each components)
TYPES OF HPLC TECHNIQUES
Based on modes of chromatography
Normal phase mode
Reverse phase mode
Based on principle of separation
Ion exchange chromatography
Ion pair chromatography
Size exclusion or gel permeation chromatography
Chiral phase chromatography
Based on scale of operation
The principle of separation in normal phase mode and reverse
phase mode is absorption. When a mixture of components are
introduced in the hplc column they travel according to their relative
affinities towards the stationary phase. the components which has
more affinity towards the adsorption travel s slower. The components
which has less affinity towards the stationary phase travels faster.
Since no two components have the same affinity towards the
stationary phase ,the components are separated.
A solvent reservoir and mixing system
A high pressure pump
A sample inlet pump
A detector and recording unit
A SOLVENT RESERVIOR
The most common type of solvent
reservoir is a glass bottle. Most of the
manufacturers supply these bottles with
the special caps
The choice of mobile phase to be used
in any separation will depend on the
type of separation to be achieved
Isocratic separation may be made with
a single solvent or two or more solvents,
where as gradient elution system may
be used where developing solvent is
continuously changed .
All solvents for use in hplc system must
be specially purified by the sparing
process for degassing of aqueous
HIGH PRESSURE PUMPS
Pumps performance directly affects the retention time reproducibility
and detector sensitivity
The main feature of a good pumping system is that it is capable of
output of at least 3.4*107 pa(5000 p.s.i)
Flow delivery must be at least 10 Cm3 / min for normal analysis
Four types of pumps
Syringe type pumps
Hydraulic amplifier pumps
SYRINGE TYPE PUMPS
It works on the principle of
positive solvent displacements by
a piston mechanically driven at a
constant rate of about 250-500ml
capacity . They flush solvents
simply by a push button action of
a purge valve.
Double syringe pumps also can
be used in which one piston
delivering solvent to column while
the other is refilling from the
Advantage–potentially quit stable
floe delivery because no check
valve has to be actuated during
RECIPROCAL TYPE PUMP
It employs small volume chamber with reciprocating pistons to work directly on
the solvent or diaphragm,pressurises the liquid from solvent reservoir.
SAMPLE INJECTING SYSTEM
A good injecting system should have the smallest possible contribution
to peak broadening .The injection system should be
Convenient to use
Able to operate at high pressures
Chemically inert with the eluent and the sample
There are 3 important ways of introducing the sample into the
FIXED VOLUME INJECTION :In this a fixed volume is introduced by
making use of a fixed volume loop injector. The sample loaded by means
of load syringe
VARIABLE INJECTION VALVE INJECTION :A variable volume is
introduced by making use of an injection valve .Valve injectors contain a
needle port which can be closed or sealed at high pressure to insert the
syringe then the sample moves to column
COLUMN INJECTION : In this ,a syringe is used to inject the sample
through an inert septum directly into the mobile phase. Commercially high
pressure syringe can inject upto10-20mpa
The columns are generally made of stainless steel.
Withstand pressure of 5.5*107pa 8000p.s.i
Straight columns of 20to50cm in length and 1-4 mm in diameter
Porous plug of stainless steel or Teflon are used in the ends of the
columns to retain the packing material.
COLUMN PACKING MATERIALS
There are three forms
Microporous- they ramify through particles which are 5 to
10micromt eg :silica ,alumina, lichrosorb-si ,micropak-NH2.
Pellicular –particle are coated onto an inert solid core (glass
bead )of 40 mm. eg: inactive silica , zipax,active silica
corasil, polymer coated , permaphase ODS
Bonded phases -stationary phase is chemically bonded onto an
As the quantity of material applied is very small its is imperative that
sensitivity is high and stable.
It response is usually presented as a record trace displaying the
components as peak on time scale.
Mainly two type
Bulk property detectors:
Refractive index and conductivity detector
Solute property detectors:
UV , visible absorption ,fluorescence and electrochemical
RECORDERS AND INTEGRATORS
Recorders are used to record the responses from detectors after
amplification .They record the base line and all the peaks obtained with
respect to time
Integrators are improved version of recorders with some data
Now a days computers and printers are used for recording process.
Separation fast and efficient
Can be applied to the separation and analysis of very complex
Accurate quantitative measurements
Repetitive And reproducible analysis using the same column
Both aqueous and non aqueous samples can be analysed with little
or no sample pretreatments
A variety of solvents and column are available providing a high
degree of selectivity for specific analysis
separated components can be easily collected and isolated from the
mobile phase for further analysis or characterization.
Purification of biological molecules
Separation of compound such as drugs and their metabolites
,peptides , vitamins
Separation of high polar compounds such as amino acids
,organic ,acids and catecholamine's.
Dissolution of pharmaceutical dosages.
Shelf life determinations of pharmaceutical products.
Identification of counterfeit drug products.
Pharmaceutical quality control.
Instrumentation methods of chemical analysis by Gurdeep r. Chatwal and
Sham K. Anand.
-Himalayapublishinghouse(revised fifth edition)
AInstrumental methods of chemical Analysis by B.K.Sharma.
-GOEL Publishing house 27th edition
Text book of pharmaceutical analysisi by Dr.S.Ravi Sankar
-Rx publications third edition