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
