23-Nov-18

High-performance liquid chromatography (HPLC)

Dr. Eddy Owaga

IFBT, DeKUT

CHROMATOGRAPHY

 Laboratory technique for the Separation of mixtures

 Chroma -"color" and graphein - "to write”.

 Colour bands - separation of individual compounds

 Measured or analyzed

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TSWETT EXPERIMENT

Liquid chromatography

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Recall- Paper chromatography

•Chromatography : physical method in which separation of

components takes place between two phases-a stationary phase and
a mobile phase

•Stationary phase : The substance on which adsorption of the

analyte (the substance to be separated during chromatography)
takes place . It can be a solid, a gel, or a solid liquid combination

•Mobile phase : solvent which carries the analyte (a liquid or a gas)

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Types of chromatography

What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange
• Elution technique- isocratic, gradient
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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What is HPLC?

 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

 ready adaptability to accurate quantitative determination

 suitability for separating nonvolatile species or thermally

fragile ones

 Popularity:
 widespread applicability to substances that are of prime interest to
industry, to many fields of science, and to the public

 Ideally suited for separation and identification of,

 proteins,
 amino acids
 nucleic acids,
 hydrocarbons,
 carbohydrates,
 pharmaceuticals,
 pesticides,
 pigments,
 antibiotics,
 steroids, and a variety of other inorganic substances

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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Principle of HPLC
• HPLC is a type of liquid chromatography where the sample is
forced through a column that is packed with a stationary phase
composed of irregularly or spherically shaped particles, a porous
monolithic layer , or a porous membrane by a liquid (mobile
phase) at high pressure.
• the injection of a small volume of liquid sample into a tube
packed with tiny particles (3 to 5 micron (μm) in diameter called
the stationary phase).
• where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced
through the column by high pressure delivered by a pump.
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Principle of HPLC contd…

• These components are separated from one another by the
column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.
• These separated components are detected at the exit of this
tube (column) by a flow-through device (detector) that
measures their amount.
• The output from the detector is called a liquid chromatogram. In
principle, LC and HPLC work the same way except the speed ,
efficiency, sensitivity and ease of operation of HPLC is vastly
superior .

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How can We Analyze the Sample?

For example:
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose 5
6. isomaltose

2
3
mAU
4
1
6

time

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Separations
Injector
Separation is based upon differential
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Pumps Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse phase
chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange
• Elution technique- isocroaitic, gradient elution

HPLC instrumentation- mobile phase reservoir, pump,

injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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(i) Normal phase chromatography

Stationary Phase – Polar nature.Eg: SiO2,Al2O3; Mobile Phase – Non-Polar
nature.
Eg:heptane,hexane,cyclohexane,CHCl3,CH3OH.
Mechanism: Polar compounds travels slower & eluted slowly due to higher
affinity to stationery phase.
Non-polar compounds travels faster & eluted 1st due to lower affinity to
stationery phase.

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Normal phase
 In this column type, the retention is governed by
the interaction of the polar parts of the stationary
phase and solute.
 For retention to occur in normal phase, the
packing must be more polar than the mobile
phase with respect to the sample

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STATIONARY PHASES
(NORMAL POLARITY)

Silica or alumina possess polar sites that

interact with polar molecules.
silica
O
Polar Group HO Si
O

Components elute in increasing

order of polarity.

Most polar…….Least polar

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(ii) Reverse phase chromatography

Stationary Phase – Non-Polar nature.
Eg: n-octadecyl, n-octyl, ethyl, phenyl diol, hydrophobic polymers.
Mobile Phase – Polar nature.
Eg: methanol or acetonitrile/water or buffer sometimes with additives
of dioxane.
Mechanism: Polar compounds travels faster & eluted 1st due to lesser
affinity to stationery phase .
Non-Polar compounds travels slower & eluted slowly due to higher
affinity to stationery phase

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Reverse phase
 In this column the packing material is relatively nonpolar and the solvent is
polar with respect to the sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the nonpolar stationary phase.
 Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-
organic mixtures such as methanol-water or acetonitrile-water.

Common Reverse Phase Solvents –

Methanol CH3OH

Acetonitrile CH3CN

Tetrahydrofuran

Water H2O
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STATIONARY PHASES
(REVERSE POLARITY)

If the polar sites on silica or alumina are capped with non-polar

groups, they interact strongly with non-polar molecules.
silica
C18 phase Me O
Si O Si
Me O

Components elute in decreasing

order of polarity.

Most non-polar…….Least non-polar

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse phase
chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange, size exclusion
• Elution technique- isocroaitic, gradient elution

HPLC instrumentation- mobile phase reservoir, pump,

injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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(i) Adsorption chromatography

• In the Adsorption Chromatography solute molecules bond directly to

the surface of the stationary phase
• component which has more affinity towards mobile phase elutes first &
the component which has less affinity towards stationary phase elutes
later.
• No two components have same affinity towards mobile phase &
stationary phase.

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(ii) Ion exchange chromatography

• Ion exchange chromatography is a process that allows the separation of ions
and polar molecules based on their charge. It can be used for almost any kind
of charged molecule including large proteins, small nucleotides and amino
acids.

• Retention is based on the attraction between solute ions and charged sites
bound to the stationary phase. Ions of the same charge are excluded.

• The use of a resin (the stationary solid phase) is used to covalently attach
anions or cations onto it. Solute ions of the opposite charge in the mobile
liquid phase are attracted to the resin by electrostatic forces.

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STATIONARY PHASES
(CATION EXCHANGE)

Silica is substituted with anionic residues that interact

strongly with cationic species (+ve charged)
Cations exchange Na+ silica
O
Na O S
O

+ve charged species adhere to the support

and are later eluted with acid (H+)

Most +ve…….Least +ve

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STATIONARY PHASES
(ANION EXCHANGE)

Silica is substituted with cationic residues that interact

strongly with anionic species (-ve charged)
Anions exchange Cl- silica
Me
Cl Me N CH2
Me

-ve charged species adhere to the support

and are later eluted with acid (H+)

Most -ve…….Least -ve

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(iii) Size exclusion

• A mixture of components with different molecular sizes are
separated by using gels. The gels used acts as molecular
sieve & hence a mixture of substances with different
molecular sizes are separated.
• Soft gels like dextran, agarose or poly acrylamide are used.
Semi-rigid gels like polystyrene, alkyl dextran in non
aqueous medium are also used.

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• Specific pore sizes. average residence time in the pores

depends on the effective size of the analyte molecules
– larger molecules
– smaller molecules
– intermediate size molecules

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STATIONARY PHASES
(SIZE EXCLUSION)

Size exclusion gels separate on the basis of molecular size.

Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.

Large molecules elute fast (restricted path),

while small molecules elute slowly (long path length)

Larger molecules…….Smaller molecules

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SEC column packing-

• Small (~10 µm) silica or polymer particles containing a
network of uniform pores
• Two types (diameters of 5 ~ 10 µm)
Polymer beads
silica-based particles

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(v) Affinity Chromatography

• This is the most selective type of chromatography employed. It utilizes the
specific interaction between one kind of solute molecule and a second
molecule that is immobilized on a stationary phase.
• For example, the immobilized molecule may be an antibody to some
specific protein. When solute containing a mixture of proteins are passed
by this molecule, only the specific protein is reacted to this antibody,
binding it to the stationary phase.
• This protein is later extracted by changing the ionic strength or pH.

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse phase
chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange
• Elution technique- isocratic, gradient elution

HPLC instrumentation- mobile phase reservoir, pump,

injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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Elution technique-
(i) Isocratic elution
• A separation in which the mobile phase composition remains
constant throughout the procedure is termed isocratic elution
• In isocratic elution, peak width increases with retention time
linearly with the number of theoretical plates. This leads to the
disadvantage that late-eluting peaks get very flat and broad.
• Best for simple separations ;
• Often used in quality control applications that support and are in
close proximity to a manufacturing process

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Isocratic System

Column
Detector
Injector
Pump Oven

Mobile Phase
Data
processor

Simple system with one pump and one solvent reservoir.

If more than one solvent is used, solvents should be premixed.

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(ii) gradient elution

•A separation in which the mobile phase composition is changed during the
separation process is described as a gradient elution
•Gradient elution decreases the retention of the later-eluting components
so that they elute faster, giving narrower peaks . This also improves the peak
shape and the peak height
• Best for the analysis of complex samples; Often used in method
development for unknown mixtures . Linear gradients are most popular

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High-pressure Gradient System

pump

B
Mixer
Column Detector
pump Injector
Oven
Data
processor
C

pump • Excellent gradient accuracy.

• 2-3 pumps required - one pump per solvent used.
• On-line degassing may not be critical.

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse phase
chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange
• Elution technique- isocratic, gradient elution
HPLC instrumentation- mobile phase reservoir, pump, injector,
column, detector, data system
Advantages of HPLC
Application of HPLC
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Instrumentation of HPLC
( Describing the 5 major components and their functions….)

Solvent
reservoirs
and degassing
1

Not shown 2
here 5
3

1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
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Schematic of liquid chromatograph

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Schematic of liquid chromatograph

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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Mobile phase reservoir

•The mobile phase in HPLC refers to the solvent being continuously
applied to the column or stationary phase
•The mobile phase acts as a carrier to the sample solution
•A sample solution is injected into the mobile phase of an assay
through the injector port
•As a sample solution flows through a column with the mobile
phase, the components of that solution migrate according to the
non-covalent interaction of the compound with the column

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Mobile phase reservoir contd…

•The chemical interaction of the mobile phase and sample , with the
column , determine the degree of migration and separation of
components contained in the sample
•The solvents or mobile phases used must be passed through the
column at high pressure at about 1000 to 3000 psi. this is because as
the particle size of stationary phase is around 5-10µ, so the
resistance to the flow of solvent is high.

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Properties of the Mobile Phase

• Must
– dissolve the sample
– have a strong solvent strength leads to reasonable
retention times
– interact with solutes in such a way as to lead to selectivity

• Removal of dissolved gases by degassers

– vacuum pumping system
– heating/stirring of solvents

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
71

Pumps
•The role of the pump is to force a liquid (called the mobile
phase) through the liquid chromatograph at a specific flow rate,
expressed in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000
psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).

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Types of HPLC pumps

There are several types of pumps used for HPLC analysis, most
commonly used are reciprocating piston pump, syringe pump
and constant pressure pump
1.Reciprocating piston pumps:
2.Displacement type pump
3.Pneumatic pump

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Plunger Reciprocating Pump

out

pump head check valve

motor and cam

5 - 50µL

plunger
check valve
plunger seal

in
Mobile phase
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Plunger Reciprocating Pump

out

pump head check valve

motor and cam

5 - 50µL

plunger
check valve
plunger seal

in
Mobile phase
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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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Injector:
•The injector serves to introduce the liquid sample into the
flow stream of the mobile phase for analysis.

•It is equipped with six port valves so that a sample can be

injected into the flow path at continuous pressure

•For a manual injector, the knob is manually operated to

deliver the sample to the column

•The knob is set to LOAD position for sample injection using a

syringe , the sample is injected into the sample loop , which is
separated from the flow path

•The knob is turned to INJECT position and the eluent travels

through the loop from the pump and delivers the sample to
the column
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Injector contd…

• Typical sample volumes for manual injector are 5-to 20-

microliters (μL).

•The injector must also be able to withstand the high

pressures of the liquid system.

•An autos ampler is the automatic version for when the user
has many samples to analyze or when manual injection is not
practical. It can continuously Inject variable volume a of 1 μL –
1 mL

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Sample Injection Systems

• For injecting the solvent through the column
• Minimize possible flow disturbances
• Volumes must be small
• .1-500 L
• Sampling loops
– interchangeable loops (5-500 L at pressures up to
7000 psi)

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Manual Injectors Sample Loop

Load - Inject

Front View Rear View

Inject

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Automatic Injectors

Step 1 Step 2

Step 3
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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
83

Column
•Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest using
various physical and chemical parameters.

•It is usually made of stainless steel to withstand high pressure caused

by the pump to move the mobile phase through the column packing
other material include PEEK and glass

•The small particles inside the column are called the “packing” what
cause the high back pressure at normal flow rates.

•Column packing is usually silica gel because of its particle shape ,

surface properties , and pore structure give us a good separation

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Column contd…
•Other material used include alumina, a polystyrene-divinyl benzene
synthetic or an ion-exchange resin

– Pellicular particle: original, Spherical, nonporous beads,

proteins and large biomolecules separation (dp: 5 μm)

– Porous particle: common used, dp: 3 ~ 10 μm. Narrow size

distribution, porous microparticle coated with thin organic film

•The dimensions of the analytical column are usually

-straight, Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm),
diameter of particle(35 μm). Number (40 k ~ 70 k plates/m)

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HPLC Columns
Within the Column is where separation occurs.
 Key Point –Proper choice of column is critical for success in HPLC

Materials of construction for the tubing

 Stainless steel (the most popular; gives high pressure capabilities)
 Glass (mostly for biomolecules)
 PEEK polymer (biocompatible and chemically inert to most solvents

Packing material:
The packing material is prepared from SILICA particle, ALUMINA particle
and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columns
to retain the packing material.
According to the mode of HPLC , they are available in different size ,
diameters, pore size or they can have special materials attached ( such as
antigen or antibody ) for immuno affinity chromatography.
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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
87

Detector:

•The detector can detect the individual molecules that elute

from the column and convert the data into an electrical signal

•A detector serves to measure the amount of those molecules

•The detector provides an output to a recorder or computer

that results in the liquid chromatogram

•Detector is selected based on the analyte or the sample under

detection

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Commonly used detectors in HPLC

Ultraviolet (UV) Detection

•This type of detector responds to substances that absorb light.

•The UV detector is mainly to separate and identify the principal

active components of a mixture.

•UV detectors are the most versatile, having the best sensitivity and
linearity.

•UV detectors cannot be used for testing substances that are low in
chromophores (colorless or virtually colorless) as they cannot absorb
light at low range.

•They are cost-effective and popular and are widely used in industry

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UV/Visible
• Mercury lamp
• Photocell measures absorbance

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Fluorescence Detection
•This is a specific detector that senses only those substances
that emit light. This detector is popular for trace analysis in
environmental science.
•As it is very sensitive, its response is only linear over a
relatively limited concentration range. As there are not many
elements that fluoresce , samples must be synthesized to make
them detectable.

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Fluorescence
• For compounds having natural
fluorescing capability
• Fluorescence observed by
photoelectric detector
• Mercury or Xenon source with grating
monochromator to isolate fluorescent
radiation

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Mass Spectrometry Detection

•The mass spectrometry detector coupled with HPLC is called
HPLC-MS. HPLC-MS is the most powerful detector, widely used
in pharmaceutical laboratories and research and
development.
•The principal benefit of HPLC-MS is that it is capable of
analyzing and providing molecular identity of a wide range of
components.

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Refractive Index (RI) Detection

The refractive index (RI) detector uses a monochromator and
is one of the least sensitive LC detectors.

•This detector is extremely useful for detecting those

compounds that are non-ionic, do not absorb ultraviolet light
and do not fluoresce.

•e.g. sugar, alcohol, fatty acid and polymers.

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• Measure displacement of beam with respect to

photosensitive surface of dectector

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What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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Data processing unit (Computer)

•Frequently called the data system, the computer not only
controls all the modules of the HPLC instrument but it takes the
signal from the detector and uses it to determine the time of
elution (retention time) of the sample components (qualitative
analysis) and the amount of sample (quantitative analysis).

•The concentration of each detected component is calculated

from the area or height of the corresponding peak and
reported.

97

What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
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ADVANTAGES OF HPLC
i. Separations fast and efficient (high resolution power)
ii. Continuous monitoring of the column effluent
iii. It can be applied to the separation and analysis of very complex mixtures
iv. Accurate quantitative measurements.
v. Repetitive and reproducible analysis using the same column.
vi. Adsorption, partition, ion exchange and exclusion column separations are excellently
made.
vii. HPLC is more versatile than GLC in some respects, because it has the advantage of
not being restricted to volatile and thermally stable solute and the choice of mobile
and stationary phases is much wider in HPLC
viii. Both aqueous and non-aqueous samples can be analyzed with little or no sample pre
treatment
ix. A variety of solvents and column packing are available, providing a high degree of
selectivity for specific analyses.
x. It provides a means for determination of multiple components in a single analysis. 99

What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel
permeation, affinity, ion-pair exchange
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
100

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Application of HPLC
Clinical
• Analysis of antibiotics.
• Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis.
• Detection of endogenous neuropeptides in brain extracellular fluids.
Food and Flavor
• Analysis of amino acids
• Ensuring soft drink consistency and quality.
• Analysis of vicinal diketones in beer.
• Sugar analysis in fruit juices.
• Polycyclic aromatic hydrocarbons in vegetables and fruits.
• Trace analysis of high explosives in agricultural crops.
Stability of aspartame in the presence of glucose and vanillin

101

Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• Pharmaceutical quality control.
• Phenols in Drinking Water.
• Identification of diphenhydramine in sediment samples.
• Biomonitoring of PAH pollution in high-altitude mountain lakes through the
analysis of fish bile.
• Estrogens in coastal waters - The sewage source.
• Toxicity of tetracyclines and tetracycline degradation products to
• environmentally relevant bacteria.
• Assessment of TNT toxicity in sediment.

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In summary…..

103

What is HPLC?
Principle of HPLC
Types of HPLC
• Mode of separation- normal phase chromatography; Reverse
phase chromatography
• Principle of separation- adsorption, ion exchange, gel permeation,
affinity, ion-pair exchange
• Elution technique- isocratic, gradient
HPLC instrumentation- mobile phase reservoir, pump,
injector, column, detector, data system
Advantages of HPLC
Application of HPLC
104

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