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Seminar
On
HPLC
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Table of Content
 How it Looks
 Introduction
 Principle of HPLC
 Instrumentation pf HPLC
 Types of HPLC
 Comparison of HPLC with different Chromatography
 Why We use HPLC
 Applications
 Advantages
 Limitation
 References
How it Looks

3
Introduction
 High performance liquid chromatography or commonly known as HPLC is
an analytical technique used to separate, identify or quantify each
component in a mixture.
 The mixture is separated using the basic principle of column 
chromatography and then identified and quantified by spectroscopy. 
 In the 1960s the column chromatography LC with its low-pressure suitable
glass columns was further developed to the HPLC with its high-pressure
adapted metal columns.
 HPLC is thus basically a highly improved form of column liquid
chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up to 400
atmospheres.
 Principle
 The purification takes place in a separation column between a stationary
and a mobile phase.
 The stationary phase is a granular material with very small porous
particles in a separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture
which is forced at high pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary
made of stainless steel, the sample is injected into the mobile phase flow
from the pump to the separation column using a syringe.
Cont...

 Subsequently, the individual components of the sample migrate through the

column at different rates because they are retained to a varying degree by
interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on
the computer is obtained.
 The chromatogram allows the identification and quantification of the
different substances. 
Diagrammatic View of HPLC Principle
Instrumentation of High-Performance
Liquid Chromatography (HPLC)
 The Pump
 Injector
 Column
 Detector
 Recorder
 Degasser
 Column Header
Schematic of an HPLC Instrument
The Pump

 The development of HPLC led to the development of the pump system.

 The pump is positioned in the most upper stream of the liquid chromatography
system and generates a flow of eluent from the solvent reservoir into the system.
 High-pressure generation is a “standard” requirement of pumps besides which, it
should also to be able to provide a consistent pressure at any condition and a
controllable and reproducible flow rate.
 Most pumps used in current LC systems generate the flow by back-and-forth motion
of a motor-driven piston (reciprocating pumps). Because of this piston motion, it
produces “pulses”.
Injector
 An injector is placed next to the pump.
 The simplest method is to use a syringe, and the sample is introduced to
the flow of eluent.
 The most widely used injection method is based on sampling loops.
 The use of the autosampler (auto-injector) system is also widely used
that allows repeated injections in a set scheduled-timing. 
Column

 The separation is performed inside the column.

 The recent columns are often prepared in a stainless steel housing,
instead of glass columns.
 The packing material generally used is silica or polymer gels
compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
 Most column housing is made of stainless steel since stainless is
tolerant towards a large variety of solvents.
Detector

 Separation of analytes is performed inside the column,

whereas a detector is used to observe the obtained separation.
 The composition of the eluent is consistent when no analyte is
present. While the presence of analyte changes the
composition of the eluent. What detector does is to measure
these differences.
 This difference is monitored as a form of an electronic signal.
There are different types of detectors available.
Recorder

 The change in eluent detected by a detector is in the form of an

electronic signal, and thus it is still not visible to our eyes.
 In older days, the pen (paper)-chart recorder was popularly used.
Nowadays, a computer-based data processor (integrator) is more
common.
 There are various types of data processors; from a simple system
consisting of the in-built printer and word processor while those with
software that are specifically designed for an LC system which not only
data acquisition but features like peak-fitting, baseline correction,
automatic concentration calculation, molecular weight determination,
etc.
Degasser

The eluent used for LC analysis may contain gases such as oxygen that
are non-visible to our eyes.
 When gas is present in the eluent, this is detected as noise and causes
an unstable baseline.
 Degasser uses special polymer membrane tubing to remove gases.
 The numerous very small pores on the surface of the polymer tube
allow the air to go through while preventing any liquid to go through
the pore.
Column Heater

The LC separation is often largely influenced by the column

temperature.
 In order to obtain repeatable results, it is important to keep
consistent temperature conditions.
 Also for some analysis, such as sugar and organic acid, better
resolutions can be obtained at elevated temperatures (50 to 80°C).
 Thus columns are generally kept inside the column oven (column
heater).
BLOCK DIAGRAM OF HPLC
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Reservoir
Reservoir Recorder

Pump Gradient
controller Pump
Fraction Detector

collector
Mixing Analytical
chamber column

Solvent Injector
Conditioning Precolumn
column
Types of High-Performance Liquid
Chromatography (HPLC)
 Normal phase
 Reverse phase
 Ion exchange
 Size exclusion
Normal phase:

 This method separates analytes on the basis of polarity. NP-HPLC

uses polar stationary phase and non-polar mobile phase.
 Therefore, the stationary phase is usually silica and typical mobile
phases are hexane, methylene chloride, chloroform, diethyl ether, and
mixtures of these.
 Polar samples are thus retained on the polar surface of the column
packing longer than less polar materials.
Reverse Phase HPLC

 The stationary phase is nonpolar (hydrophobic) in nature, while the

mobile phase is a polar liquid, such as mixtures of water and methanol
or acetonitrile.
 It works on the principle of hydrophobic interactions hence the more
nonpolar the material is, the longer it will be retained.
Ion-Exchange HPLC

 The stationary phase has an ionically charged surface of opposite

charge to the sample ions. This technique is used almost exclusively
with ionic or ionizable samples.
 The stronger the charge on the sample, the stronger it will be attracted
to the ionic surface and thus, the longer it will take to elute. The mobile
phase is an aqueous buffer, where both pH and ionic strength are used
to control elution time.
Size-exclusion HPLC

 The column is filled with material having precisely controlled pore

sizes, and the particles are separated according to its their
molecular size.
 Larger molecules are rapidly washed through the column; smaller
molecules penetrate inside the porous of the packing particles and
elute later.
COMPARISION BETWEEN HPTLC AND HPLC
Sr.no. HPTLC HPLC

1 High Performance Thin Layer Performance Liquid

High Chromatography Chromatography

2 Planar Chromatography Column Chromatography

3 Principle based Adsorption Principle is based on Adsorption and
on Chromatography Partition Chromatography

4 Simultaneous method for test as well as Not simultaneous method for test as
reference material well as reference material

5 It is simple, rapid, reproducible method It is Tedious method

6 Sample preparation is simple Sample preparation is complex

7 Limited Flexibility Extreme Flexibility
8 Semiautomatic Technique Automatic (Instrumental) Technique
59
9 Determination of Surface Area Determination of Retention Time
COMPARISION BETWEEN GC AND HPLC

SR.NO. GC HPLC

1 Gas Chromatography High Performance Liquid

Chromatography
2 Less resolution High resolution
3 Limited Flexibility Extreme Flexibility
4 Determination of Volatile Determination of Volatile and Non
compounds Volatile Compounds

60
COMPARISION BETWEEN HPLC AND UPLC
Parameters HPLC Assay UPLC Assay

Column XTerra,C18,50 × AQUITY UPLC

4.6mm BEH C18,50
×2.1mm
Particle size 4µm particles 1.7µm particles

Flow rate 3.0 ml per min 0.6 ml per min

Injection volume 20 µl 3 µl partial loop fill or

5 µl full loop fill
Total run time 10 min 1.5 min

Theoretical Plate 2000 7500

count 61
 Parameters HPLC Assay UPLC
Assay
Lower limit of 0.2 µg/ml 0.054µl/ml
quantization
Total solvent Acetonitrile:10.5ml, Acetonitrile:0.53ml,
consumption water:21ml water:0.66ml
Delay volume 720 µl 110 µl
Column temperature 30 °C 65 °C
Maximum back 35-40 Mpa 103.5 Mpa
pressure less more
Resolution Less High
Method development High Low
cost
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WHY USE HPLC

 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Moderate analysis condition
 Easy to fractionate and purify
 Not destructive
Applications of HPLC

The information that can be obtained by HPLC includes resolution, identification and
quantification of a compound. It also aids in chemical separation and purification.
The other applications of HPLC include :
Pharmaceutical Applications
 To control drug stability.
 Tablet dissolution study of pharmaceutical dosages form.
  Pharmaceutical quality control.
Cont...

Environmental Applications
 Detection of phenolic compounds in drinking water.
 Bio-monitoring of pollutants.
Applications in Forensics
  Quantification of drugs in biological samples.
 Identification of steroids in blood, urine etc.
  Forensic analysis of textile dyes.
 Determination of cocaine and other drugs of abuse in blood, urine etc.
Cont.…

Food and Flavour

 Measurement of Quality of soft drinks and water.
 Sugar analysis in fruit juices.
 Analysis of polycyclic compounds in vegetables.
 Preservative analysis.
Applications in Clinical Tests
 Urine analysis, antibiotics analysis in blood.
 Analysis of bilirubin, biliverdin in hepatic disorders.
 Detection of endogenous Neuropeptides in extracellular fluid of brain
etc.
Advantages of HPLC

 Separations fast and efficient (high resolution power)

 Continuous monitoring of the column effluent
 It can be applied to the separation and analysis of very complex mixtures
 Accurate quantitative measurements.
 Repetitive and reproducible analysis using the same column.
 Adsorption, partition, ion exchange and exclusion column separations are
excellently made.
Limitations

 Cost: Despite its advantages, HPLC can be costly, requiring large

quantities of expensive organics. 
 Complexity
 HPLC does have low sensitivity for certain compounds, and some
cannot be detected as they are irreversibly adsorbed.
 Volatile substances are better separated by gas chromatography.
References

 Wikipedia
 Google
 Studymafia.org
Thanks