High Performance

Liquid
Chromatography
Presented By : Bilal Siddique
Presented To: Dr. Kiran
Shahzadi
MS-Chemistry IInd
Sem.
University of
 Backgroun
d
● Chromatography and its
Principle
• Invented by M. Tsvet in 1900
• Used it for separation of plants pigments like chlorophyll, carotenes
etc.
• Combination of two Greek word
• Chromatos means color
• Graphy means writing
• Chromatography is a separation technique which is used to separate
a mixture of compounds into its individual components based on
certain physical and chemical properties.
Some important terms:
● Mobile phase: The solvent system which carries the mixture to be
separated.
● Stationary phase: Immobile surface which is particulate in nature. This is the region
over which the compound gets separated.
 Introductio
● HPLC is a
nform of liquid chromatography used
to separate compounds that are dissolved in solution.
● HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
● Compounds are separated by injecting a sample mixture onto
the column.
● The different component in the mixture pass through the
column at differentiates due to differences in their partition
behavior between the mobile phase and the stationary phase.
● The mobile phase must be degassed to eliminate the formation
of air bubbles.
 Terminologies for HPLC
● HPLC : High Performance Liquid Chromatography : High Pressure LC

● Now, before we go in depth of principle, lets have a basic look at few

terms as follows:

● Resolving Power: The extent of separation of the compounds

present in the mixture across the column.

● Theoretical plates : An imaginary division of the column into

equilength plates.
 Principles of HPLC
Principle:

● The table shows relation between various

parameters of HPLC.
● Trendline:
Column length No. of theoretical plates
per unit area
Resolving power Column length
Particle size Surface area

● Stationary phase have small particulate size and

high surface areas.
● Columns: 20 cm or less
● Mobile phase pumped at high pressures of
200Bar, 3000 psi.
● Flow rates: 1-3 cm3 per min
 What is HPLC?
● HPLC is a separation technique that involves:
• 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.
● 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. An output
from this 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.
HPLC
system

Flow chart of HPLC mechanism

Components OF A LIQUID CHROMATOGRAPH SYSTEM

Solvent
Solvent Delivery System (Pump)
Injector
Sample Column
Detectors Waste
Collector
Recorder (Data
Collection)
 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
1.
Pump:
• 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).
Pump Module–types:
● Isocratic pump - Delivers constant mobile phase composition;
• solvent must be pre-mixed;
• lowest cost pump
● Gradient pump - Delivers variable mobile phase composition;

• can be used to mix and deliver an isocratic mobile phase or a

gradient mobile phase
–Binary gradient pump –delivers two solvents

–Quaternary gradient pump –four solvents

2. Injector:

• The injector serves to introduce the liquid sample into the flow stream of the
mobile phase.

• Typical sample volumes are 5-to 20-microliters (μL).

• The injector must also be able to withstand the high pressures of the
liquid system.

• An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
3. Column:
•Considered the “heart of the chromatograph” the column’s stationary phase
separates the sample components of interest using various physical and chemical
parameters.

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

• The pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within the
chromatograph.
 Several Column Types
( can be classified
as)
● Normal phase

● Reverse phase

● Size exclusion

● Ion exchange
 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
 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

28
 Reverse
with respect phase
● In this column the packing material is relatively nonpolar and the solvent is polar
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
 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

30
 Size
●
exclusion
In size exclusion the HPLC column is consisted of
substances which have controlled pore sizes and is
able to be filtered in an ordinarily phase according to
its molecular size.
● Small molecules penetrate into the pores within the
packing while larger molecules only partially
penetrate the pores. The large molecules elute
before the smaller molecules.
 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 lslowly (long path length)

Larger molecules…….Smaller molecules

32
 Ion
●
exchange
In this column type the sample components are
separated based upon attractive ionic forces
between molecules carrying charged groups of
opposite charge to those charges on the
stationary phase.
● Separations are made between a polar mobile
liquid, usually water containing salts or small
amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.
 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
a with acid (H+)

Most +ve…….Least +ve

23
 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
M
e

-ve
- charged species adhere to the support and t
are later eluted
a with acid (H+)

Most -ve…….Least -ve

24
 HPLC s
Column
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.
4.
Detector:
•The detector can see (detect) the individual molecules that come out
(elute) from the column.
•A detector serves to measure the amount of those molecules so
that the chemist can quantitatively analyze the sample
components.
•The detector provides an output to a recorder or computer that
results in the liquid chromatogram(i.e., the graph of the detector
response).
5. 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:

1. determine the time of elution (retention time) of the

sample components (qualitative analysis) and
2. the amount of sample ( quantitative analysis) .
 What is HPLC used
for ?
Separation and analysis of non-volatile
or thermally
HPLC unstable
is optimum for compounds
the separation of chemical and biological
compounds that are non-volatile .

NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in

gasoline, etc.), gas chromatography is a better separation technique .

Typical non-volatile compounds are:

□ Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
□ Salts like sodium chloride and potassium phosphate
□ Proteins like egg white or blood protein
□ Organic chemicals like polymers (e.g. polystyrene, polyethylene)
□ Heavy hydrocarbons like asphalt or motor oil
□ Many natural products such as ginseng, herbal medicines, plant extracts
□ Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
Qualitative Analysis of HPLC
Quantitative Analysis of
HPLC
HPLC Applications

Bioscience
Chemical

Pharmaceuticals
Consumer Products

Environmental
Clinical
• THANK YOU…