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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
Principle of Chromatography
Elution techniques
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.
Direction
of
Mobile
Phase
DIAGRAM OF REVERSE PHASE
CHROMATOGRAPHY
Polar
Solutes
Non-
Polar
Solutes
Direction
of
Mobile
Phase
Types of HPLC Techniques
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
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.
ELUTION
TECHNIQUES Gradient separation
Qualitative Analysis
BASED ON TYPE
OF ANALYSIS Quantitative Analysis
INSTRUMENTATION
MOBILE PHASE
Buffers
Acidic Mobile Phases
Ion-Pairing Additives
High pH Mobile Phase
MOBILE PHASE RESERVOIRS
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 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
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
COMPRESSION CALTRIDGE
FITTING FITTING
Length 15-25 cm
Internal diameter 1mm
Particle size 3-8 µm
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
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
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