HIGH

PERFORMANCE
LIQUID
CHROMATOGRAPHY
[HPLC]

Prepared By: Helyati Abu Hassan Shaari

 INTRODUCTION

 HPLC stands for “High-performance liquid chromatography”

(sometimes referred to as High-pressure liquid chromatography)

 HPLC is a form of liquid chromatography used to separate compounds that are

dissolved in solution

 High performance liquid chromatography is a powerful tool in analysis:

*it yields high performance and
*high speed compared to traditional columns chromatography
 These are due to forcibly pumped mobile phase

 HPLC is a type of liquid chromatography where the sample is forced through a

column (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.
High Pressure
Stationary Phase:
Mobile Phase: Liquid
Column
 INTRODUCTION

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

Individual components of the sample are moved down the

packed tube (column) with a liquid (mobile phase) forced through
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”

Preparation for HPLC INTRODUCTION

Preparation of solvents
•Correct solvent preparation is very important.
• It can save vast amounts of time spent troubleshooting such spurious peaks,
baseline noise, etc…

Quality
•All reagents and solvents should be of the highest quality.
• HPLC grade reagents may cost slightly more than lower grade reagents
•HPLC grade reagents contain no impurities to produce spurious peaks in a
chromatogram baseline.

Buffers
•All buffers should be prepared freshly on the day required.
-to ensures that the buffer pH is unaffected by prolonged storage and
that there is no microbial growth present.
 INTRODUCTION

Filtration
•All HPLC solvents should be filtered through a 0.45 µm filter before use.
•This removes any particulate matter that may cause blockages.
•After filtration, the solvents should be stored in a covered reservoir to
prevent contamination with dust, etc…
•Pump plungers, seals and check valves will perform better and lifetimes will
be maximized.

Degassing
•Before the freshly prepare mobile phase is pumped around the HPLC
system, it should be thoroughly degassed to remove all dissolved gasses.
•Dissolved gas can be removed from solution by:
• Bubbling with helium
• Sonication
• Vacuum filtration
 INTRODUCTION

•If the mobile phase is not degassed, air bubbles can form in the high-
pressure system resulting in problems with system instability, spurious
baseline peaks, etc.

The most efficient form of degassing is bubbling with helium or another low
solubility gas.
It is recommended that the mobile phase is continually degassed at very low
levels throughout the analysis.
-This will inhibit the re-adsorption of gases over the analysis time.
HPLC INSTRUMENT
 HPLC INSTRUMENT

Reservoir Pump

Injector
Column system

Detector
 HPLC INSTRUMENT

HPLC instruments consist of;

•a reservoir of mobile phase
• a pump,
•an injector,
•a separation column,
•a detector.
•Waste collector
•Recorder (Data collection)

Compounds are separated by injecting a sample 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.
 Reservoir of mobile phase

Degasser - to remove bubbles in the column that will cause band spreading
Isocratic elution system – An elution with a single solvent
Gradient elution system – Two and sometime more solvents system that differ significantly in polarity.

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)
During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic)
or an increasing mobile phase composition (gradient).
The type of pump; the screw driven syringe type, the reciprocating pump, and the penumatic pump

Injector system
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 .
 HPLC INSTRUMENT
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

•Delivers four solvents
HPLC INSTRUMENT
 Column

Construct form stainless steel tubing with range in range from 10 cm to 30 cm

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 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.
Most common packing for column is silica particles which are often coated with thin organic films

Detector

The most widely detector are based on absorption of ultraviolet or visible radiation
 PRINCIPLE

Principle of separati on
•When a mixture of components are introduced into the column ,
various chemical and/or physical interactions take place between
the sample molecules and the particles of the column packing .

•They travel according to their relative affinities towards the

stationary phase. The component which has more affinity
towards the SP, travels slower.

•The component which has less affinity towards the SP travels

faster.

•Since no two components have the same affinity towards the

stationary phase, the components are separated

•Mechanism of separation of HPLC is divided into 2;

*NORMAL PHASE MECHANISM
*REVERSE PHASE MECHANISM
 TYPES OF HPLC PACKINGS

NORMAL PHASE

REVERSE PHASE
 MECHANISM

Normal phase (NPC)

●
● SP – polar
●
● MP – non polar or less polar
●
● The least polar compound elutes first

Reverse phase (RPC)

●
● SP – Non polar or less polar
●
● MP - polar
●
● The most polar elutes first

NPC – most polar elutes

last
RPC – most polar elutes
first

PST351-POLYMER CHARACTERISATION 16
 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 stationary phase must be

more polar than the mobile phase with respect to the sample.

Silica or alumina possess polar sites that interact with polar molecules.

Components elute in increasing order of polarity.

 NORMAL PHASE

SP = Polar
(hydrophilic)
(ex: Silica gel or
alumina backbone)

MP = Non-polar
(hydrophobic)
(ex:hexane mixed with
polar solvent
Most polar
elute last
 REVERSE PHASE

In this column the packing material is relatively non polar and the solvent
is polar with respect to the sample.

Separation is the result of the interaction of the polar components of the

solutes and the non polar stationary phase.

Typical stationary phases are:

-non polar 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.

Components elute in decreasing order of polarity.

 REVERSE PHASE

SP = non polar (hydrophobic)

=Ex :liquid coated on a solid
support (hydrocarbon)

MP = Polar (hydophilic)
(Ex: water, methanol,
acetonitrile etc)

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

groups, they interact strongly with non-polar molecules.
MECHANISM NPC VS RPC

PST351-POLYMER CHARACTERISATION 21
MECHANISM

PST351-POLYMER CHARACTERISATION 22
 POLARITY ORDER

Choice of Mobile and Stationary Phase

Less polar
Most separation are achieved by
matching the polarity of the
Hydrocarbons Olefins
analyte to that ofAromatic
the stationary
hydrocarbons
phase; a mobile phase of
considerably different polarity
is then used.

Sulfides Halides

Most polar

Esters
Nitro
Ethers (aldehyde,
compound
ketone)

Amines alcohols

Carboxylic
Sulfones Amides
acids
How can We Analyze the
Sample?

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

2
3
Zorbax NH2 (4.6 x 250 mm) mAU
4
70/30 Acetonitrile/Water 1
6
1 mL/min Detect=Refractive Index

time

PST351-POLYMER CHARACTERISATION 24
 Separations
Separation in based upon
Injector
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
Column through the column.

Detector

Waste
Solvents

High Performance Liquid Chromatograph

PST351-POLYMER CHARACTERISATION 25
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

High Performance Liquid Chromatograph

PST351-POLYMER CHARACTERISATION 26
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 27
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 28
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 29
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 30
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 31
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 32
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime
Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 33
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 34
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 35
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 36
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 37
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 38
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 39
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 40
 Injector Chromatogram

Mixer

Pumps

Start Injectiontime

Column

Detector

Solvents

PST351-POLYMER CHARACTERISATION 41
CHROMATOGR
AM

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
PST351-POLYMER CHARACTERISATION 42
Modes of High Performance
Liquid Chromatography

Types of Mode Stationary Mobile Phase

Compounds Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases

Ionics, Bases, Acids Ion C-18, C-8 Water/Organic

Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin
High Molecular Weight Size Polystyrene Gel Filtration-
Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic

PST351-POLYMER CHARACTERISATION 43
APPLICATIONS

Bioscience
Chemical proteins
peptides
polystyre nucleotides
nes
dyes
phthalate
s

tetracyclines
corticosteroids
Pharmaceuticals
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine

PST351-POLYMER CHARACTERISATION 44
 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
 APPLICATIONS

HPLC is optimum for 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

•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
The end