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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
18
Reservoir
Reservoir Recorder

Pump Gradient Pump

controller
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 High Performance Liquid

Chromatography Chromatography

2 Planar Chromatography Column Chromatography

3 Principle based on Adsorption Principle is based on Adsorption and
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 BEH

4.6mm 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
62
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