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High Performance

Liquid
Chromatography

GARVITA ARORA
I M.PHARM
PHARMACEUTICAL CHEMISTRY
INTRODUCTION TO LIQUID
CHROMATOGRAPHY
• It is a versatile technique available to an analyst.
• It is simple method and has a high capability for high
resolution separations.
• Separations are caused by diverse characteristics like
1) Polarity of the solutes
2) Ionic nature
3) Molecular weight
4) Partitioning ability
5) Ability to form affinity complexes
INTRODUCTION TO LIQUID
CHROMATOGRAPHY
• The word liquid chromatography is used today
is the method in which the separation take
place in a packed column.
• The packaging material is the stationary phase
and a solid with an inert support coated with a
liquid phase.
• A liquid mobile phase is used as eluant.
INTRODUCTION TO LIQUID
CHROMATOGRAPHY

• They were carried out in glass columns


– diameters: 1-5 cm
– lengths: 50-500 cm
• Size of the solid particle in stationary phase
– diameters: 150-200 m
• Flow rate is low ,Separation times long
Diagram of column
chromatography
High
Performance
Priced
Pressure
Liquid
Chromatography
High Performance Liquid Chromatography

• The most widely used analytical separations technique.


• Utilizes a liquid mobile phase to separate components
of mixture.
• Uses high pressure to push solvent through the column.
• Popularity:
– Sensitivity.
– Suitable for separating nonvolatile species .
– widespread applicability to substances that are of prime
interest to industry, to many fields of science.
High Performance Liquid Chromatography is

• Ideally suited for separation and identification of amino


acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, pharmaceuticals, pesticides, pigments,
antibiotics, steroids, and a variety of other inorganic
substances.
Advantages to HPLC
• Higher resolution and high speed of analysis.
• HPLC columns can be reused without repacking or
regeneration.
• Greater reproducibility due to close control of the
parameters affecting the efficiency of separation.
• Easy automation of instrument operation and data
analysis.
• Adaptability to large-scale, preparative procedures.
Advantages to HPLC
• Advantages of HPLC are result of 2 major
advances:
– stationary supports with very small particle
sizes and large surface areas.
– appliance of high pressure to solvent flow.
DIFFERENT TYPES OF HPLC
Modes of Chromatography

Principle of Chromatography

Elution techniques

The scale of operation

Type of Analysis
Types of HPLC Techniques

Modes of
Chromatography
Normal Phase
mode
Reverse
Phase mode
Liquid-liquid partition chromatography
• In this relative method stationary phase is liquid
coated onto a solid support.
• The relative distribution of solutes in between
two phases determines the separation of
substance.
• This can be further classified into
1) Normal phase chromatography – Stationary phase is
polar and mobile is non-polar.

2) Reverse phase chromatography – Stationary phase is


non-polar and mobile phase is polar.
DIAGRAM OF NORMAL PHASE
CHROMATOGRAPHY
Polar
Solutes
Non-
Polar
Solutes

Direction
of
Mobile
Phase
DIAGRAM OF REVERSE PHASE
CHROMATOGRAPHY
Polar
Solutes
Non-
Polar
Solutes

Direction
of
Mobile
Phase
Types of HPLC Techniques

Adsorption Ion exchange


Chromatography Chromatography
Principle of
Chromatography
Size exclusion Affinity
Chromatography Chromatography
ADSORPTION CHROMATOGRAPHY
◊ Adsorption is a surface phenomenon where
interaction takes place only on the surface of
adsorbent.
◊ The interaction is competitive.
◊ Solute having high affinity towards stationary
phase will be adsorbed first compared to less
affinity molecules.
◊ Adsorbed solute is replaced from stationary
phase by mobile phase.
SOLUTES
High Affinity

Low Affinity
STATIONARY
Direction of
Mobile Phase PHASE
◊ The stationary phase in adsorption
Chromatography is called "Adsorbent"
Types of adsorbents: (Stationary phase)
◊ Usually a solid such as silica gel, alumina
(Al2O3) or porous glass beads are used.
Types of Mobile Phases:
◊ The mobile phases such as heptane, octane,
chloroform etc are used in adsorption
chromatography.
SIZE EXCLUSION CHROMATOGRAPHY

• Size-exclusion chromatography is a separation


mode based solely on the analyte’s molecular
size.
• In this a large molecule is excluded from the
pores and migrates quickly, whereas a small
molecule can penetrate the pores and migrates
more slowly down the column.
SIZE EXCLUSION CHROMATOGRAPHY
It is often called Gel permeation chromatography (GPC) or
Gel-filtration chromatography (GFC)

Gel permeation chromatography (GPC) - when used to


determine the molecular weights of organic polymers.
The column is packed with cross-linked polystyrene beads
of controlled pore sizes.
It is eluted with common mobile phases such as toluene
and tetrahydrofuran.

Gel-filtration chromatography (GFC) when used in the


separation of water-soluble biological materials.
Ion exchange Chromatography
• In ion-exchange chromatography, the separation mode
is based on the exchange of ionic analytes with the
counter-ions of the ionic groups attached to the solid
support.
• These resins are coated on to the porous glass bead.
Separation takes place by exchange of ion of resin with
counter ion of mobile phase.
• These are of two types.
1) Cation-ion exchange resin.
2) Anion-ion exchange resin.
P+

Na+
Ion-exchange
chromatography
(IEC), showing the
P+ exchange of analyte
ion p+ with the
sodium counter ions
Na+ of the
bonded sulfonate
groups

P+
• Typical stationary phases are cationic
exchange (sulfonate) or anionic exchange
(quaternary ammonium) groups bonded to
polymeric or silica materials.
• Mobile phases consist of buffers, often with
increasing ionic strength, to force the
migration of the analytes.
• Common applications are the analysis of ions
and biological components such as amino
acids, proteins/peptides, and polynucleotides
Other Separation Modes
• Affinity chromatography: Based on a receptor/ligand
interaction in which immobilized ligands (enzymes,
antigens, or hormones) on solid supports are used to
isolate selected components from a mixture. The retained
components can later be released in a purified state.
• Hydrophilic interaction chromatography (HILIC):
This is somewhat similar to normal phase
chromatography using a polar stationary phase such as
silica or ion-exchange materials but eluted with polar
mobile phases of organic solvents and aqueous buffers. It
is most commonly used to separate polar analytes and
hydrophilic peptides.
Other Separation Modes
• Chiral chromatography: For the separation of
enantiomers using a chiral-specific stationary phase. Both
NPC and RPC chiral columns are available.

• Hydrophobic interaction chromatography: Analogous


to RPC except that mobile phases of low organic solvent
content and high salt concentrations are used for the
separation of proteins that are easily denatured by mobile
phases with high concentrations of organic solvents used
in RPC.
Other Separation Modes
• Electro chromatography: Uses capillary electrophoresis
(CE) equipment with a packed capillary HPLC column.
The mobile phase is driven by the electromotive force
from a high-voltage source as opposed to a mechanical
pump. It is capable of very high efficiency.

• Supercritical fluid chromatography (SFC): Uses


HPLC packed columns and a mobile phase of pressurized
supercritical fluids (i.e., carbon dioxide modified with a
polar organic solvent). Useful for non-polar analytes and
preparative applications where purified materials can be
recovered easily by evaporating the carbon dioxide.
HPLC pumps and GC-type detectors are often used.
Types of HPLC Techniques:
BASED ON Isocratic separation

ELUTION
TECHNIQUES Gradient separation

BASED ON THE Analytical HPLC


SCALE OF
OPERATION Preparative HPLC

Qualitative Analysis
BASED ON TYPE
OF ANALYSIS Quantitative Analysis
INSTRUMENTATION
MOBILE PHASE

The mobile phase is the solvent that moves


the solute (analyte) through the column. In
HPLC, the mobile phase interacts with both
the solute and the stationary phase and has a
powerful influence on solute retention and
separation.
HPLC mobile phases should have these
characteristics:

• High solubility for the sample components.


• Noncorrosive to HPLC system components.
• High purity, low cost, UV transparency.
• Other desirable characteristics include low
viscosity, low toxicity, and non-flammability.
DIFFERENT TYPES OF MOBILE PHASES

 Buffers
 Acidic Mobile Phases
 Ion-Pairing Additives
 High pH Mobile Phase
MOBILE PHASE RESERVOIRS

• The most common type of solvent reservoir is a


glass bottle.
• Most of the manufacturers supply these bottles
with special caps, Teflon tubing and filters to
connect to the pump inlet and to the sparge gas
(helium) used to remove dissolved air.
• Filtration is needed to eliminate suspended
particles and organic impurities.
• The main culprit is oxygen (from the air) that
dissolves in polar solvents, particularly water.
• The dissolved gases in the mobile phase will
come out at column exit or in the detector
resulting in sharp peaks.
• The microscopic bubbles can change the
nature of the flowing stream making it
heterogeneous.
• But peaks can also occur when these
microscopic bubbles gradually collect in the
detector cell.
DEGASSING TECHNIQUES

Degassing may be accomplished by one or a


combination of the following methods:

1) Apply a vacuum to the liquid.


2) By Heating.
3) Sonication.
4) Sparging.
PUMPS
INTRODUCTION
Pumps are used to pass mobile phase at high
pressure and at controlled flow rates.
The pump must be capable of generating
pressure of up to 5000 psi at flow rates up to 3
ml/min for analytical situations.
Pumps used in preparative scale HPLC may be
required to pump at flow rates of up to
20ml/min.
IDEAL CHARACTERSTIC OF PUMP
• Non corrosive & compatible with solvent.
• Provide high pressure to push the mobile phase.
• High pressure generated by pump should not lead to
an explosion.
• Provide constant flow rate to mobile phase.
• Easy to change from one mobile phase to another.
• Should not leak.
• Should have reproducible flow rate and independent
of column back pressure.
• It should be easy to dismantle or repair.
TYPES OF PUMP USED IN HPLC
1.Constant flow pump
A). Syringe pump.
B). Reciprocating pump.
-Single piston
-Dual piston
2). Constant pressure pump.
A). Pneumatic pump
-Amplifier pump
A) SYRINGE PUMP
ADVANTAGES

• Flow is pulse free.


• Provide high pressure up to 200-475 atm.
• Independent of column back pressure and
viscosity of solvent.
• Simple operation.

DISADVANTAGES
• Limited solvent capacity.
• Gradient elution is not easy.
CONSTANT FLOW PUMP
B) Reciprocating pump(single piston)
RECIPROCATING PUMP (Dual piston)
DIAGRAM OF RECIPROCATING PUMP
ADVANTAGES
• Generate high output pressure.
• Ready adaptability to gradient elution.
• Provide constant flow rate.
• Pressure generated is so high that any back pressure
generated in the column due to mobile phase can be
easily overcome.
DISADVANTAGES
Pulsed flow must be damped as they produce a base
line noise on chromatogram.
PNEUMATIC PUMP
PULSE DAMPER
• A TRIPLE HEADED PUMP.

• A TUBE AN AIR SPACE OR A FLEXIBLE BELLOWS


OR TUBE.

• A RESTRICTOR.
TRIPLE HEADED PUMP
Two heads in different
stages of filling as the third
is pumping.
2
3
COLUMN

MOBILE
1 2 3
PHASE
RESERVOIR PUMPS
Flow of 1
mobile phase
A TUBE AN AIR SPACE OR A FLEXIBLE
BELLOWS OR TUBE
Where a flexible metal(air space or a
flexible metal vessel) takes up some of the
solution energy. When pump refills, this
energy is released and a smooth pressure
pulsation results.

COLUMN
MOBILE
PHASE
RESERVOIR
A RESTRICTOR
In this method, 25 cm length of 4mm bore, stainless
steel tubing packed with 20 micro meter glass beads, is
placed between the pump and the column.

25 cm

COLUMN 4 mm PUMP

MOBILE PHASE
PRE COLUMN
• Pre-columns are packed with inexpensive, coarse silica for
mobile phase conditioning function.
• Placed in the HPLC system prior to the sample injector.
• Could have larger particle size.
• Used to saturate the mobile phase with silica so that the silica
or bonded silica of analytical column packing is not dissolved
during use.
• Reduces the problems due to change in the pH of the mobile
phase and extends the life of the column.
• Used to remove impurities.
SAMPLE INJECTION SYSTEM
• SEPTUM INJECTION.
• MICRO VOLUME SAMPLING VALVES/
SAMPLING LOOP.
• RHEODYNE INJECTOR/STOP FLOW.
SEPTUM INJECTION

• Syringe is used to inject the


sample through an self
sealing inert septum
directly in to the mobile
phase
• DRAWBACK - Leaching
effect of the mobile phase
with the septum resulting
in the formation of ghost
peaks.
SYRINGE INJECTOR
RHEODYNE INJECTOR/STOP FLOW
• Uses syringe, the pumping is stopped and
sample is introduced from the head of the
column and pumping of the solvent is
continued.
RHEODYNE
INJECTOR/STOP
FLOW
MICRO VOLUME SAMPLING
VALVES/ SAMPLING LOOP
• Highly sophisticated modern HPLC method for
sampling makes use of micro volume sampling
devices.
• No interruption of the mobile phase flow.
• Volume of sample ranges between 2 µl to over 100µl.
Operation of sample loop
1)Sampling mode
2)injection mode
Sampling mode and injection mode
COLUMNS

• The column is the main part in HPLC.


• Column composed of porous graphite or particles
of polymeric materials like styrene divinyl
benzene copolymer material are stable over wide
range.
• The column containing small particles of
stationary phase because surface area of
stationary phase is exposed to sample
component in HPLC column.
• Porous plugs of stainless steel or teflon are
used in the end of the column to retain the
packaging material.
• It is important in some separation like liquid
partition and ion exchange that the column
temperature is thermostatically controlled
during analysis.
PROPERTIES OF PUMP
• It should withstand the operating pressure.
• Materials of column should not be corroded.
• It should be non- reactive with mobile phase and the
solute. 
• It should show satisfactory peak shapes (minimal tailing).
• It should show satisfactory peak width (narrower or
better).
• It should give a stable detector base line.
• It should have good resolving power.
CLASSIFICATION
OF COLUMN

A) GUARD COLUMN

B) MAIN COLUMN
GUARD COLUMN
• Guard column are used in HPLC to protect the
analytical column from contaminating substances
that may be present in sample or in solvent system.
• The guard column are packed with the same
material used in analytical column but have larger
particle size than analytical column packaging
material.
• Guard column connected between injectors and
analytical column.
Main column
1)Analytical column
2) Preparative column
3)Micro bore
4)Semi-preparative column
5) New column technology
• Radial compression column
• Axially compressed column
ANALYTICAL COLUMN
The separation takes place in a stainless steel
tube usually 3-25cm in length with an internal
diameter 3-4.6 mm . It is packed with
stationary phase.
1) Superficially porous or pellicular
Materials used to pack column 2) Totally porous

Particle size 3-10 µm

spherical, uniformed sized porous


Particle nature material
The price of analytical column depend on the
particle size that is a 5nm column cost is more
than 10 nm column length, diameter and
nature of the packaging material(reverse
phase packing cost more ).
TYPE OF ANALYTICAL COLUMN
1)COMPRESSION
2)CALTRIDGE

COMPRESSION CALTRIDGE
FITTING FITTING

Length 3-25cm 7.5 to 10cm

Inner diameter 3-4.6mm 3-4.6 mm

Particle size 3-10 µm 3-10 µm


PREPARATIVE COLUMN
• These column are larger as compared to
analytical column because large amount of
sample to be loaded. They have length 10-
25cm and particle size 5-20 micrometer.
• The packaging are attached to the internal
column by support includes oxides , carbon ,
polymer & silica is the most common type.
• TYPES:-
 Magnum 9
 Magnum 20
 Magnum 40
MICRO BORE COLUMN
The micro bore HPLC technique first introduced in 1979.
Column with internal diameter less than 2 mm are often referred
to as micro bore column.

Length 15-25 cm
Internal diameter 1mm
Particle size 3-8 µm

Micro bore column is to get high sensitivity.


We compare a typical HPLC separation (10 min at 2ml /min)with a
microbore separation consumes 20 times less solvent than
conventional separation.
ADVANTAGES
1. Highest possible efficiency.
2. Decrease in solvent consumption.
3. Small sample requirement.
4. Can interface with mass spectrometer.
5. Lower detection limits.
SEMIPREPARATIVE COLUMN
These are designed to isolate milligrams to
multigram quantities . These are packed with
number of packaging materials for
adsorption, reverse phase, size exclusion, ion
exchange.

Length 10-25 cm
Internal diameter 0.8-1 cm
Particle size 5-20 µm
NEW COLUMN TECHNOLOGY
These column have been incorporated for
separating mixture 1-50gm range.
These are:-
1) Radial compression column
2) Axially compressed column
Have advantages that both column length &
packing type can be readily changed by
operator.
CRITERIA TO
CHOOSE Particle Shape

PACKING
Surface Area

MATERIALS
Particle Size

Bonding Type

Pore size

Carbon load


Particle Shape
Spherical particles offer reduced back
pressures and longer column life.
Particle Size
Smaller particles offer higher efficiency, but also
cause higher backpressure. Choose 3µm
particles for resolving complex, multi-
component samples. Otherwise, choose 5 or
10µm particle size.
Surface Area
High surface area generally provides
greater retention, capacity and resolution for
separating complex, multi-component
samples.
Pore Size
Larger pores allow larger solute molecules
to be retained longer through maximum
exposure to the surface area of the particles.
Choose a pore size of 150Å or less for sample
MW  2000. Choose a pore size of 300Å or
greater for sample MW > 2000.
Bonding Type
Monomeric bonding offers increased mass transfer rates,
higher column efficiency, and faster column equilibration.

CH3 R
monomeric
OH + X Si (CH2 )17 CH3 Si (CH2)17 CH 3
bonding
CH3 R

OH CH3 O CH3
polymeric
+ X Si (CH2)17CH3 Si
bonding
OH X O (CH2)17CH3

Polymeric bonding offers increased column stability, particularly when


highly aqueous mobile phases are used. Polymeric bonding also enables
the column to accept higher sample loading.
Carbon Load
Higher carbon loads generally offer greater
resolution and longer run times. Low carbon
loads shorten run times and many show a
different selectivity.
PACKING MATERIALS
Silica–based packing material
• Silica can be altered by reaction with
organochlorosilanes or organoalkoxysilanes to give Si–
O–Si–R linkages with the surface.
• The attachment of hydrocarbon chains to silica produces
a non–polar surface suitable for RPC in which mixtures
of water and organic solvents are used as eluents.
• The most popular material is octadecylsilica (ODS),
which contains C18 chains, but materials with C1, C2, C4,
C6, C8 and C22 chains are also available.
• The latest silica–based bonded phase to be introduced is
a long C30 phase, which make it one of the most
retentive phases available.
Different types of packing materials
Special Silica Miscellaneous Chemical Moieties are Bound To Silica,
They are Designed To Purify Specific Compounds.

Zirconia Packing Materials A Wider pH range and Are Especially Useful For Basic
Separations At Ph 10 Or Higher But At Higher Ph The
Silica Gel Starts To Dissolve

Polymer–based Packing Packing Materials Based On Organic Polymers Are


Materials Available. For Ex. Unmodified Styrene–divinylbenzene
Co–polymers Have A Hydrophobic Character And Can
Be Used For Rpc.
Different types of packing materials
Special Silica Miscellaneous Chemical Moieties Are Bound To Silica,
They Are Designed To Purify Specific Compounds

Zirconia Packing Materials A Wider Ph Range And Are Especially Useful For Basic
Separations At Ph 10 Or Higher But At Higher Ph The
Silica Gel Starts To Dissolve

Polymer–based Packing Packing Materials Based On Organic Polymers Are


Materials Available. For Ex. Unmodified Styrene–divinylbenzene
Co–polymers Have A Hydrophobic Character And Can
Be Used For Rpc.
SPECIAL SILICA
A vast range of materials have intermediate
surface polarities that arise from the bonding
to silica of organic compounds that contain
groups such as phenyl, cyano, nitro, amino,
fluoro, sulfono and diols. There are also
miscellaneous chemical moieties bound to
silica, as well as polymeric packings, designed
to purify specific compounds.
Zirconia packing materials
• Zirconia is a metal oxide that is more chemically and thermally
stable than silica.
• It is unaffected by changes in ionic strength or organic content of
the mobile phase.
• Zirconia packings have a wider pH range and are especially useful
for basic separations at pH 10 or higher, where silica gel starts to
dissolve.
• Zirconia can be used for RPC and is extremely stable and efficient
through surface modification with polymer or carbon coatings.
• Other chemical modifications of zirconia produce packing
materials suitable for normal–phase or ion–exchange
chromatography.
Polymer–based packing materials
• Packing materials based on organic polymers are available. For
ex. unmodified styrene–divinylbenzene co–polymers have a
hydrophobic character and can be used for RPC.
• Polymeric materials are best when a mobile phase that can go
beyond the upper pH limits of silica gel (usually pH 6.5 to 7), as
they are stable over a wide pH range.
• They also provide different selectivity and retention
characteristics to silica–based reversed phase packings. They
avoid problems associated with residual silanol groups (e.g. peak
tailing).
• Ion–exchange materials of the styrene–divinylbenzene type are
available in which sulfonic acids, carboxylic acids or quaternary
ammonium groups are incorporated in the polymeric matrix.
NORMAL PHASE PACKAGING MATERIAL

System consist of:-


Stationary phase- Polar
Mobile phase - non polar

STATIONARY PHASE - it is made up of either


silica, alumina of siloxane with polar
functional group (cyno, diol, amino,
dimethylamino)
MOBILE PHASE:- n-hexane ,methylene chloride,
chloroform.
The unmodified silica most widely used in HPLC.
It has high efficiency and high permeability
which allow normal operating pressure less
than 2000psi to be used .It has also relative
high surface area.
HPLC grade silica consist of totally porous micro
particles with spherical irregular shape and
diameter 3,5,7or 10 micrometer.
REVERSE PHASE CHROMATOGRAPHY

• The opposite arrangement of normal phase


chromatography that is stationary phase non
polar and mobile phase polar is found in reverse
phase.
• Stationary phase:-it is made up of octadecyl(c-
18)or octyl(C-8) chain or butyl(C-4) or phenyl
groups.
• Mobile phase:- Methanol, acetonitrile,
tetrahydrofuran, water.
DETECTORS
Comparison of classical Gas
chromatography and HPLC
Parameter Gas HPLC
Stationary phase large small
Particle size 60-200micron 3-20micron
Column size large small
Column material glass mostly metal
Column packing low pressure often gravity high pressure
Analysis time 5-60min 1-5min
Operating pressure low < 20psi 500-6000psi
Flow rates Low medium-high
Cost Economical Not Economical
Summary
Thank you

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