ITS A SEPARATION TECHNIQUE

1. High – Performance
Liquid Chromatography (HPLC)

2. • High Performance Liquid Chromatography (HPLC) is the most
widely used type of elution chromatography
• In Liquid Chromatography, the columns were packed with 50 to
500 cm lengths of solid particles coated with an adsorbed liquid
that formed the stationary phase and the mobile phase is a liquid
solvent containing the sample as a mixture of solutes
• The types of high – performance liquid chromatography are often
classified by separation mechanism or by the type of the
stationary phase
(a) Partition, or liquid – liquid chromatography
(b) Adsorption, or liquid – solid chromatography
(c) Ion – exchange, or ion chromatography
(d) Size exclusion chromatography
(e) Affinity chromatography
(f) Chiral chromatography

3. • High–Performance Liquid Chromatography (HPLC) is a type of
chromatography that employs a liquid mobile phase and a finely
divided stationary phase
• To obtain satisfactory flow rates, the liquid must be pressurized
to several hundreds or more pounds per square inch
 Applications of Liquid Chromatography
• Molecular masses greater than 10,000:
Size exclusion: gel permeation for non-
polar compounds & gel filtration for
polar compounds
• Ionic compounds of lower masses:
Ion exchange chromatography
• Smaller but non-ionic: Partition
methods

4. High – Performance Partition Chromatography
• Partition Chromatography
(a) Mobile Phase: Liquid
(b) Stationary Phase: A second liquid that is immiscible with the
liquid mobile phase
• Partition Chromatography is sub-divided into
(a) Liquid–Liquid Chromatography
The stationary phase is a solvent that is held in place by
adsorption on the surface of packing particles
(b) Liquid bonded–phase chromatography
The stationary phase is an organic species that is attached to
the surface of the packing materials by chemical bonds

5. Instrumentation

6. • Pumping pressures of several hundred atmospheres are required
to achieve reasonable rates with packings in the 3- to 10-mm size
range
1. Mobile phase reservoirs and solvent treatment systems
2. Pumping systems
3. Sample injection system
4. Columns
5. Detectors
Important Components of HPLC
Instrumentation Contd…..

7. 1. Mobile phase reservoirs and solvent treatment systems
• A modern HPLC apparatus is equipped with one or more glass
reservoirs which contains 500 mL or more of a solvent
• Gases and dust are present in the solvents and they are to be
removed. Gases produce bubbles in the column and thereby cause
band spreading
• Degassers may consist of vacuum pumping system, a distillation
system, a device for heating and stirring – mainly causes sparging
Sparging: It is a process in which dissolved gases are swept out of
a solvent by fine bubbles of an inert gas that is not soluble in the
mobile phase

8. • A isocratic elution in HPLC is one in which the solvent
composition remains constant
• A gradient elution in HPLC is one in which the composition of the
solvent in changed continuously or in a series of steps

9. Pumping Systems
• Requirements for liquid chromatographic pumps include
(a) ability to generate of up to 6000 psi (lb/in2)
(b) pulse-free output
(c) flow rates ranging from 0.1 to 10 mL/min
(d) flow reproducibilities of 0.5% relative or better
(e) resistance to corrosion by a variety of solvents
There are three major types of pumps:
(a) The screw driven syringe pump
(b) The reciprocating pump
(c) The pneumatic or constant pressure pump
• The most widely used pump is reciprocating pump

10. Pumping Systems
• Consists of small cylindrical
chamber that is filled and
then emptied by the back-
and-forth motion of a piston
• The pumping motion
produces a pulsed flow that
must be subsequently
damped
Advantages
• Small internal volume
• High output pressure
• Gradient elution is possible
• Constant flow rates
A reciprocating pump for HPLC

11. Sample Injection System

12. Columns for HPLC
• Most columns range in length from 10 to 30 cm and have inside
diameters of 2 to 5 mm
• The most common packing for liquid chromatography is prepared
from silica particles, which are synthesized by agglomerating
submicron silica particles under conditions that lead to larger
particles with highly uniform
• The resulting particles are often coated with thin organic films,
which are chemically or physically bonded to the surface
Detectors
• The detector should have low dead volume to minimize extra
column band broadening
• Should be small and compatible with liquid flow

13.  Bonded – Phase Packings
• Most bonded – packings are prepared by the reaction of an
organochlorosilane with the –OH groups on the surface of silica
particles
where R = straight chain octyl or octyldecyl group
• Other organic functional groups include: aliphatic amines, ethers,
and nitriles, as well as aromatic hydrocarbons
• Bonded – phase packings have the advantage of markedly greater
stability than the physically held stationary phases. With the
later, periodic recoating of the solid surfaces is required because
the stationary phase is gradually dissolved away in the mobile
phase.

14. Partition Chromatography:
Normal – and Reversed – Phase Packings
• Two types of partition chromatography are distinguishable based
on the relative polarities of the mobile and stationary phases
(a) Normal Phase Chromatography
The stationary phase is highly polar such as triethylene glycol
or water and relatively nonpolar solvent such as hexane or i-propyl
ether the served as mobile phase.
(b) Reversed Phase Chromatography
The stationary phase is non-polar, often a hydrocarbon, and
the mobile phase is a relatively polar solvent (such as water,
methanol, acetonitrile, or tetrahydrofuran)

15. In normal – phase chromatography, the least polar component is eluted
first; increasing the polarity of the mobile phase decreases the elution
time.
In reverse – phase chromatography, the most polar component elutes
first, and increasing the mobile phase polarity increases the elution time
• It has been estimated that more than three quarters of all HPLC
separations are currently performed with reversed-phase, bonded,
octyl- or octyldecyl-siloxane packings.
• With such preparations, the long chain hydrocarbon groups are aligned
parallel to one another and perpendicular to the surface of the
particle, giving a brush like non-polar, hydro carbon surface
• Mobile phase is water with various conc. of methanol, acetonitrile,
THF

16. Choice of Mobile and Stationary Phases
• Successful partition chromatography requires a proper balance
of intermolecular forces among the three participants in the
separation process – the analyte, the mobile phase, and the
stationary phase
• These intermolecular forces are described qualitatively in terms
of the relative polarity possessed by each of the three
components
• Most chromatographic separations are achieved by matching the
polarity of the analyte to that of the stationary phase; a mobile
phase of considerably different polarity is then used
Increasing polarity

17. Applications of Partition Chromatography
Soft-drink additives
Column is packed with polar (nitrile)
bonded-phase packing
Organophosphate Insecticides
Column is packed with C8 bonded-
phase packing

18. Applications of Partition Chromatography

19. Ion-Exchange Chromatography
• Stationary phase is a resin and mobile phase is a polar solvent
• In most cases, conductivity measurements are used to measure
the eluents
• Two types of Ion-Chromatography are currently in use:
(a) Suppressor-based and
(b) Single-Column
They differ in the method to prevent the conductivity of the eluting
electrolyte from interfering with the measurement of analyte
concentrations

20. Conductivity Detectors – Detectors for Ion–Exchange Columns
• Conductivity detectors are highly sensitive, universal for charged
species
• They respond to concentration changes
• Disadvantage
High electrolyte concentrations are required to elute most
analyte ions in a reasonable time. As a consequence, the
conductivity of the mobile phase components tend to swamp
that from the analyte ions, thus greatly reducing the detector
sensitivity
• So Eluent Suppressor Column is used

21. • The suppressor column is packed with an second ion-exchange
resin that effectively converts the ions of the eluting solvent to a
molecular species of limited ionization without effecting the
conductivity due to analyte ions
Eg: (a) When cations are being separated and determined,
hydrochloric acid is chosen as the eluting reagent, and the
suppressor column is an anion-exchange resin in the hydroxide
form. The product of the reaction in the suppressor is water
The analyte cations are not retained by this second column
Suppressor based Ion-Exchange Column

22. Eg: (b) For anion separations, the suppressor packing is the acid
form of a cation-exchange resin, and sodium bicarbonate or
carbonate is the eluting agent. The reaction in the suppressor is
The largely undissociated carbonic acid does not contribute
significantly to the conductivity

23. Single-Column Ion Chromatography
• No suppressor column is needed
• Analyte ions are separated on a low capacity ion exchanger by
means of a low-ionic strength eluent that does not interfere with
the conductometric detection of analyte ions

24. • Fractionation is based on molecular size
• Powerful technique that is particularly applicable to high-
molecular weight species
 Column Packings
• Packings consist of small (~10 mm) silica or polymer particles
containing a network of uniform pores into which solute and
solvent molecules can diffuse
• In the pores, the molecules are effectively trapped and removed
from the flow of the mobile phase
• The average residence time of the analyte molecules depends on
their effective size
Size-Exclusion Chromatography

25. • Molecules that are significantly larger than the average pore size
of the packing are excluded and thus suffer no retention i.e.,
they travel through the column at the rate of the mobile phase
• Molecules that are appreciably smaller than the pores can
penetrate through the pore maze and thus entrapped for the
greatest time; they are last to elute
• Intermediate size molecules whose average penetration into the
pores of the packing depends on their diameters – undergo
fractionation dependent on molecular size and molecular shape
• No physical and chemical interactions with the stationary phase
Column packings in Size-Exclusion Chromatography

26. • Gel filtration:
Type of size-exclusion chromatography in which the packing
is hydrophilic. It is used to separate polar species
• Gel Permeation:
Type of size-exclusion chromatography in which the packing
is hydrophobic. It is used to separate nonpolar species

27. Affinity Chromatography
• Affinity Chromatography involves covalently bonding a reagent,
called an affinity ligand, to a solid support.
• Typical affinity ligands are
Antibodies, Enzyme inhibitors, or other molecules that
reversibly and selectively bind to analyte molecules in the sample
• Procedure
(1) When sample passes through the column, only the molecules
that selectively bind to the affinity ligand are retained
(2) Molecules that do not bind pass through the column with the
mobile phase
(3) After the desired molecules are removed, the retained analytes
can be eluted by changing the mobile phase conditions

28.  Stationary Phase
• Solid such as a agarose or a porous glass bead to which affinity
ligand is immobilized
 Mobile phase has two important roles
(a) First it should support the binding of the analyte molecules to
the ligand
(b) Second, once the undesired species are removed, the mobile
phase must weaken or eliminate the analyte-ligand interaction
so that the analyte can be eluted
 Applications
(1) Extraordinary specificity
(2) Rapid isolation of biomolecules during preparative work