CHAPTER 6

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
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Lesson Outcomes:
• Explain the principles of liquid chromatography
• Draw and label the schematic diagram of the
components in HPLC
• Able to explain the functions of each component in
HPLC
• Discuss the difference between each component in
terms of parts and functions
• Interpret analytical information from chromatographic
data for HPLC
• Recognize problems arising from resultant
chromatograms and suggest probable solutions
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Syllabus
• 6.1 Scope of liquid chromatography
• 6.2 Apparatus
• 6.2.1 Mobile phase reservoirs and solvent treatment systems
• 6.2.2 Pumping systems
• 6.2.3 Sample injection systems
• 6.2.4 Liquid chromatography columns
• Analytical columns
• Guard columns
• 6.2.5 Column thermostats
• 6.2.6 Detectors
• 6.3 High performance partition chromatography
• 6.3.1 Normal-and reversed-phase packings
• 6.3.2 Choice of mobile and stationary phases
• 6.3.3 Applications
• 6.4 High performance ion-exchange chromatography
• 6.4.1 Ion chromatography (anion and cation)
• 6.4.2 Applications
• 6.5 High-performance size-exclusion chromatography
• 6.5.1 Packings
• 6.5.2 Applications
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Introduction
• HPLC utilizes a liquid mobile phase to separate
the components of a mixture
• Analytes are first dissolved in a solvent, & then
forced through the column under high pressure
• HPLC is very versatile because of its ability to
easily separate a wide variety of chemical
mixtures which include non volatile & thermally
unstable compounds (eg: organic, inorganic,
biological samples, synthetic or natural
polymers & thermal labile compound)
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Advantages of HPLC:
• Greater sensitivity – various detectors
• Reusable column – many analyses
• Ideal for ionic species and large molecules
• Sample recovery – fraction collectors

HPLC applications - any compound with solubility

in a liquid that can be used as a mobile phase
• Analytical or preparative (highly purified compound
in small or large quantities)
• Applications include analysis of sugars, pesticide
residues, organic acids, lipids, amino acids, toxins &
vitamin)
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Principles
• Begins by injecting the solute (analytes) onto the
top of the column
• Separations occurs as the analytes and mobile
phase are pumped through the column
• Each component elutes from the column as peak
on the recorder
• The response of detector to each component is
displayed and known as a chromatogram
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Guard
column

Analytical
column
Solvent reservoir

Data system and

display

Schematic Diagram of HPLC

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Instrumentation

• Mobile phase
resevoir, filtering
• Pump
• Injector
• Columns:
analytical & guard
column
• Detector
• Data system
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6.2.1 Mobile phase

Mobile phase reservoir, filtering:
• The common type of reservoir is a glass bottle with
special caps, Teflon tubing and filters to connect
the pump inlet and to the purge gas (He) used to
remove gas (degasser eg: sparger)
Mobile Phase:
• Readily available in pure form
• Low viscosity
• Compatibility with the detection method
• Able to dissolve the sample components
• No chemical reaction with the components
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Mobile phase Elution:

1. Isocratic elution 2. Gradient elution
A separation that employs A separation in which the
solvent/s of a constant composition of solvent is
composition changed continously/steps
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Degasser
• To removed dissolved gases and dust from the
liquid
• Dissolved gases can lead to irreproducible flow
rates and band spreading
• Both bubbles and dust also will interfere with
the performance of the detector
• The presents of particles will damage the
pumping and injection systems and/or clog the
column
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How to degas the solvent?

[Link] (sonicator)
2. Sparging (rapid bubbling of helium through the solvent)

Sparging:
• Definition: A process where the dissolved gases are swept out of
the solution by fine bubbles of an inert gas of low solubility.
• The system also contain means of filtering dust and particulate
matter
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Effect of dissolved gases to the HPLC system

if they are not removed from mobile phase
solvent:
• Air bubbles interfere with the performance of
detector by causing band spreading.
• Will generate peaks when pass through the
detector.
• Will interfere with pump function
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Why must the solvent be dust and

particulate free?
• the presents of this particles will damage the
pumping and injection systems.
• the dust and particulate will clog the column.

Method to remove dust and particulate

from mobile phase solvents:
• Filtration through a membrane
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6.2.2 Pump
• Function: is to force the mobile phase to flow
through the packing (deliver mobile phase
through the system)
• The pump is installed in order to maintain stable
flow and to avoid pulsation
• Types of pumps
[Link] pumps
[Link] pumps
[Link] pumps
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6.2.3 Injectors
• Function: to put sample in the mobile phase
1. Syringe injection through a self-sealing
elastomeric septum:
➢Earliest & simple mean of sample introduction
➢Micro syringes designed to withstand pressure up to
1500 psi are used
2. Sampling loop:
➢Most widely used method
➢Normal sample size is 5 to 500μL
➢Microsample injection valves also available
(sampling volumes: 0.5 to 5 μL)
➢Permit introduction of sample at pressure up to
7000 psi.
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Sampling Loop
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3. Auto samplers:
➢ can inject a large number of samples
➢ Small vials with a septum placed in a tray
➢ Needle penetrates septum and inject the
sample
➢ Valve introduces sample into the column
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Autosampler
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6.2.4 Column
1. Analytical column:
• Straight, stainless steel calibrated tube
• Column length: 10 to 30cm
• Column i.d: 4 to10mm
• Column packing particle size: 5 to 10μm
2. Guard Column
• Function: to increase the lifetime of a analytical
column
• Prevents deactivation of the analytical column by
adsorption and minimize band broadening
• Prevents clogging of the analytical column while
extending its lifetime
• Guard column should have same internal diameter
(i.d) as analytical column
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• The composition of guard

column packing closely similar
to analytical column
• The particle size are larger in
order to minimize pressure
drop experienced by the
analytical column.
• Length of the column: 2 to
10cm.
• Serves to saturate the mobile
phase with stationary phase so
that losses of this solvent from
the analytical columns are
minimized
• Can be change periodically
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Column Packing
1. Pellicular Packing
Pellicular packings are used largely for
guard column and not for analytical
column
2. Porous Particle Packing
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1. Pellicular packing:
• Consist of spherical, nonporous, glass or
polymer beads with typical diameters of 30-
40 μm
• A thin, porous layer of silica, alumina,
polystyrene-divinyl benzene synthetic resin
or ion-exchange resin is deposited on the
surface of these beads
• The beads may be treated chemically to give
an organic surface layer
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2. Porous particle packing:

• Consists of porous micro particles having
diameter ranging from 3-10μm
• The particles are composed of silica,
alumina, synthetic resin polystyrene-divinyl
benzene or ion exchange resin
• Silica particle is prepared by agglomerating
submicron silica particles under conditions
that lead to larger particles having highly
uniform diameters
• The resulting particles are often coated with
thin organic film, which are chemically or
physically bonded to the surface.
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Porous Particle Packing

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6.2.5 Detectors
• Two basic types of detector
1. Bulk property detectors
Respond to a mobile phase bulk property,
such as refractive index, dielectric constant,
or density, which is modulated by the
presence of solutes.
2. Solute property detectors
Respond to some property of solutes, such
as UV absorbance, fluorescence, or diffusion
current, that is not possessed by the mobile
phase
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• Detectors being chosen depends on the nature

of the sample
• Most widely used detector in HPLC are based
on absorption of UV or visible radiation
• Types of detectors….
a. UV (Absorbance)
b. Refractive index
c. Conductivity
d. Fluorescence
e. Mass-spectrometric (LC/MS)
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Principle class of
Principle of
Detector compound
operation
detected
Absorption of UV absorbing
UV electromagnetic species (organic
radiation chromophore)
Measures change in
Refractometer universal
refractive index
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Types of HPLC
(based on nature of stationary
phase & separation process)

Ion Size
Partition Exclusion
Exchange
Normal Phase
Gel Filtration

Reversed Phase Anionic

Gel Permeation
Cationic
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Partition Chromatography
• Most widely used in liquid chromatography
• Applications are to non-ionic, non-polar and
polar compounds of low to moderate molecular
weight (usually <3000)
• There are 2 types
1. Liquid-liquid – liquid stationary phase is
retained on the surface of packing by
physical adsorption.
[Link] phase – the stationary phase is
bonded chemically to the support group
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• Earlier technique was liquid-liquid type

• Currently, bonded phase method has been
widely used due to disadvantages of liquid-
liquid system
1. Loss of stationary phase by dissolution in the
mobile phase, requires recoating of the
support particles for the column
2. Stationary phase solubility problems prohibit
the use of packing for gradient elution
• Partition chromatography divided into two
modes which based on the relative polarities of
the mobile and stationary phase: normal-
phase and reversed-phase
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Normal phase:
• Stationary phase : Polar (eg: silica gel)
• Mobile phase : Less polar (eg. ethyl ether,
chloroform, n-hexane, THF)
• The least polar compound elutes first because
in a relative sense, it is the most soluble in the
mobile phase
• Polar compounds are retained on the polar
surface of the column packing longer
In normal-phase chromatography, the least polar
components is eluted first; increasing the
polarity of the mobile phase will decrease the
elution time
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Reversed phase:
• Covers 90% of all LC application
• Stationary phase : Non Polar (hydrophobic in
nature), R is C8 or C18 hydrocarbon
• Mobile phase : Polar (eg. Methanol or
acetonitrile with water)
• The most polar component appear first
• The more non polar the material is, the longer
it will be retained
In reversed-phase chromatography, the most
polar component elutes first; increasing the
mobile phase polarity, increases the elution
time
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6.4 Ion Exchange Chromatography

• A process by which ions held on a porous
insoluble solid are exchange for ion in solution
that is brought in contact with the solid.
• The retention of the compound in the
stationary phase depends on its charge density.
• Applications depend on the ion exchange resin
use for the analysis. Either
a. Cationic resin
b. Anionic resin
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• Ion exchange resins are useful as stationary

phase for LC and used to separate charged
particles
• Synthetic ion exchange resin are high
molecular weight polymers that contain large
numbers of an ionic functional group per
molecule
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a) Cation Exchange Resin

• Cation-exchange resin contain acidic groups. It
retains positively charged cations because the
stationary phase displays a negatively charged
functional group
xRSO3-H+ + M x+ → (RSO3-)xM x+ + xH+
solid solution solid solution
• M x+ represents a cation
• R represents that part of a resin molecule that
contains a sulfunic acid group
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a) Anion Exchange Resin

• Anion-exchange resin contain basic groups. It
retains anions using positively charged
functional
xRN(CH3)3+OH- + A- → xRN(CH3)3+Ax- + xOH-
solid solution solid solution
• Ax- represents anion
• R represents that part of a resin molecule that
contains a basic group
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6.5 Size Exclusion Chromatography

• Applicable to high molecular weight species
• Divided into gel filtration and gel permeation.
• Size exclusion separations are different from
other chromatographic procedures since there
are no chemical or physical interactions
involved between analytes and stationary
phase. Such interaction will lead to impaired
column efficiency
• The packings consist of small (~10um) silica or
polymer particles containing a network of
uniform pores into which solutes and solvent
can diffuse
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Schematic of a size exclusion column. The larger particles will elute

first because they are too big to fit inside the pores. The smallest
particles will elute last because they fit very well inside the pores.
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• Advantages:
1. Short and well defined separation time
2. Narrow bands, lead to good sensitivity
3. Freedom from sample loss because solutes do not
interact with stationary phase
4. Absence of column deactivation brought about by
interaction of solute with the packing
• Limitations:
1. There is no retention occurs for species with MW
beyond exclusion limit
2. All species having greater molecular weight than
the exclusion limit are so large that they are not
retained and elute together to give a peak
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Types of Size Exclusion

A. Gel Filtration B. Gel Permeation

• Size exclusion • Size exclusion

chromatography with chromatography with
hydrophilic packing are hydrophobic packing and
used with aqueous mobile are used with non-polar
phase organic mobile phase
• Separation of polar species • Separation of non-polar
species
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Applications:
• Separation of high molecular weight, natural
products molecules from low molecular weight
species
• Separation of homologs and oligomers
• Rapid determination of molecular weight
distribution of larger polymers or natural
products