CHM260

CHAPTER 6:
HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
NOR AKMALAZURA JANI
 LEARNING OUTCOMES
After completing this chapter,
students should be able to:
- explain the concepts of mobile phase
and its connection with samples
polarity
- State the component in high
performances liquid chromatography
(HPLC)
- State the functions of each
component in high performances
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 Subtopics
Scope of liquid chromatography
LC instrumentation:
- Mobile phase reservoirs and solvent treatment
system
- Pumping systems
- Sample injection systems
- LC columns
- Detectors
Type of HPLC:
- partition chromatography
- ion-exchange chromatography
- size-exclusion chromatography
 SCOPEOF
SCOPE OFLIQUID
LIQUIDCHROMATOGRAPHY
CHROMATOGRAPHY

Most widely used in analytical separation

techniques because of its:
i) sensitive
ii) ready adaptability to accurate
quantitative determinations
iii) ease of automation
iv) widespread applicability to substances
that are important to industry, science such
as amino acids, proteins, nucleic acids,
hydrocarbons, carbohydrates, drugs,
terpenoids, pesticides, antibiotics, steroids,
metal-organic species and variety of
inorganic substances.
 Selection of LC modes:
i) Solutes having molecular masses greater
than 10,000
- Size-exclusion chromatography
ii) Lower-molecular-mass ionic species
- Ion-exchange chromatography
iii) Small polar but nonionic species
(members of a homologous series:
- Reversed phase methods
iv) Nonpolar species, structural isomers,
compound classes such as aliphatic
hydrocarbons from aliphatic alcohols.
- Adsorption chromatography
v) Isolation and preparation of biomolecules
- Affinity chromatography
 LCINSTRUMENTATION
LC INSTRUMENTATION

BASIC COMPONENTS OF HPLC:

Mobile phase reservoirs and solvent

treatment systems.
Pumping systems.
Sample injection systems.
Liquid chromatography columns.
- Analytical columns
- Guard columns
- Column temperature control.
Detectors.
 Block diagram of HPLC

Guard
column

Analytical
column
Solvent reservoir

Data system and

display
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 Mobile phase reservoirs and solvent
treatment systems
Mobile phase reservoirs:
- contain mobile phase (solvent) with different
polarities such as water, methanol and
acetonitrile.
- capacity: 200 1000 mL
- constructed of glass or stainless steel and
contain an exit port open to air.
- Each reservoir is usually fitted with a gas
diffuser which helium can be bubbled.
 Characteristics of mobile phase:

Readily available in pure form

An acceptable level of toxicity
Low viscosity
Compatibility with the detection
method
Able to dissolve the sample
components
No chemical reaction with the
components
 Degassing system:
Consist of a vacuum pumping system, a
distillation system, a device for heating and
stirring or a system for sparging.
The purpose of degassing the solvent is to
removed dissolved gases (O2 and N2 from air) and
dust from the liquid.
Effect of dissolved gases to the HPLC system if
they are not removed from mobile phase solvent:
i) Air bubbles interfere with the performance of
detector by causing band spreading.
ii) Will generate peaks when pass through the
detector.
iii) Will interfere with pump function.
 Why must the solvent be dust and particulate
free?
i) the presents of this particles will damage the
pumping and injection systems.
ii) the dust and particulate will clog the
column.

Method to remove dust and particulate from

mobile phase solvents:
i) Filtration through a membrane or
milipore filter (0.45 m) under
vacuum.
 Method to remove dissolved gas from
mobile phase solvents:
i) Sonication with ultrasonic or sonicator.

ii) Sparging system

- A process where the dissolved gases
are swept out of the solution by fine
bubbles of an inert gas such as He that
is not soluble in the mobile phase.
- The system contain a means of
filtering dust and particulate matter.
 Elution of the mobile phase:

Isocratic elution
A single solvent or solvent mixture of
constant composition. Example, 50:50
(v/v) methanol-water

Gradient elution
Two (and sometimes more) solvent
systems that differ significantly in
polarity and varied in composition.
Example: 40:60 methanol-water, the
methanol concentration was the
increased at the rate of 8%/min.
Gradient elution shortened the time of
separation without sacrificing the
resolution of the early peaks
Properties of Common Chromatographic Mobile Phases
 Pumping systems
Used to force the mobile phase to flow
through the packing.
Requirements for LC pumps:
i) The generation of pressures of up to 6000
psi (lb/in2) or 414 bar.
ii) Pulse-free output.
iii)Flow rates: 0.1-10 mL/min.
iv) Flow reproducibility of 0.5% relative.
v) Resistance to corrosion by a variety of
solvents.
) Types of pumps:
i) Reciprocating pumps (most widely used)
ii) Displacement pumps
Reciprocating Pumps HPLC pumps
for HPLC
 Sample-Injection Systems
Sample are introduced via a manual injector or
autosampler.
Syringe injection through a self-sealing elastomeric
septum:
- Earliest and simple mean of sample introduction.
- Microsyringes designed to withstand pressure up to 1500
psi.

Sampling loop
Most widely used method of sample introduction.
Sample size 5 to 500 l.
Permit introduction of sample at pressure up to
7000 psi.
Microsample injection valves also available
(sampling volumes of 0.5 to 5 l).
o Manual injector:
- the knob is manually operated to deliver
the sample to the column.
- Step 1: The knob is set at the "LOAD"
position for sample injection, as shown in
the left image. Using a microsyringe, the
sample can be injected into the sample
loop, which is separated from the flow
path.
- Step 2: The knob is turned to the
"INJECT" position. The eluent travels
through the loop from the pump then
delivers the sample to the column.
 HPLC sample injection valves
(sampling loop)

Sampling loop
Autosampler
 Liquid Chromatography Columns

LC columns

Types of LC Types of column

columns packings

Analytical Pellicular
columns particle

Guard Porous
columns particle
 Types of LC Columns
Analytical column

Straight, stainless steel calibrated tube

Length : 5 to 25 cm. Sometimes length is
added by coupling two or more columns.
Inside diameter (i.d.): 3 to 5 mm
Column packing particle size : 3 or 5 m
Common columns: 10-15 cm long, 4.6 mm
in inside diameter, packed with 5 m
particles (generate 40,000 to 70,000
plates/meter i.e. typically about 10,000
plates/column).
 Waters HPLC Analytical Column 4.6 x 150 mm

Column Inlet frit

Column body

Female end fitting

Male end fitting
 Guard column / pre column
Short column placed before the analytical
column in HPLC.
Function:
- To increase the life time of the analytical
column (prevent
clogging) by removing particulate matter
and contaminants
from the solvent and sample components
that bind
irreversibly to the stationary phase.
The composition of guard column packing
should be closely similar to that of the
analytical column and particle size is larger
Guard column

Guard column
 Types of Column Packing
Pellicular particles

Consists of spherical, non-porous, glass or polymer

beads with typical diameters of 30-40 m.
A thin, porous layer of silica, alumina, a
polystyrene-divinyl-benzene synthetic resin, or an
ion-exchange resin is deposited on the surface of
these beads.
The beads may be treated chemically to give an
organic surface layer.
Currently, pellicular packings are used largely for
guard column and not for analytical column.
Used for separation of proteins and large
biomolecules (~ 5 m).
 Porous particle:

Consists of porous microparticles having

diameter ranging from 3 10 m.
The particles are composed of silica, alumina,
the synthetic resin polystyrene-divinyl
benzene, or an ion exchange resin.
Silica is most common packing in LC.
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.
 Column Temperature Control
Close control of column temperature is not
necessary.
Columns are operated at room temperature.
More reproducible chromatograms are obtained
by maintaining constant column temperature.
Modern commercial instruments:
- equipped with heaters that control column
temperatures to a few tenths of degree from
near ambient to 150C.
Columns is fitted with water jackets fed from a
constant-temperature bath to give precise
temperature control.
 Detectors
Characteristic of ideal detector for LC:
Adequate sensitivity.
Good stability and reproducibility.
A linear response to solutes that extends over
several orders of magnitude.
Short response time.
High reliability and ease of use.
Similarity response towards all solutes or
alternatively a high predictable and selective
response towards one or more classes of solute.
Detector should be nondestructive.
Have minimal internal volume to reduce zone
broadening.
Compatible with liquid flow.
 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.
 Most widely used are based on
absorption of UV or visible radiation.

Other HPLC detectors:

Fluorescence
Refractive index
Electrochemical

The JASCO HPLC UV-2077 A UV-visible absorption

dual wavelengthUV- cell for HPLC
 Common HPLC detectors

Detector Principle of Principle class

operation of compound
detected
Absorption of UV absorbing
UV absorption electromagnet species
ic radiation
Refractometer Measure Any
change in
refractive
index
 TYPEOF
TYPE OFHPLC
HPLC

Type of
HPLC

Partition Ion Size-exclusion

chromatogra chromatogr chromatograph
phy aphy y

Normal Gel
Anionic
phase filtration
Gel
Reversed Cationic permeati
phase on
 Partition Chromatography
Stationary phase is second liquid that is immiscible with
the liquid mobile phase.
Most widely used type of liquid chromatography.
Applications to non-ionic, non-polar and polar compounds
of low to moderate molecular weight (usually < 3000).

Two types:
1. Liquid-liquid chromatography:
liquid stationary phase is retained on the surface of
packing by physical adsorption.

2. Bonded-phase chromatography:
the stationary phase is bonded chemically to the
support group, resulting in highly stable packings which
insoluble in the mobile phase.
 Earlier partition chromatography was
exclusively liquid-liquid type.

Currently, bonded phase method has become

predominant due to disadvantages of liquid-
liquid system such as:

o Loss of stationary phase by dissolution in

the mobile phase, requires re-coating of the
support particles.

o Stationary phase solubility problems

prohibit the use of packing for gradient
elution.
 TWO TYPES OF PARTITION CHROMATOGRAPHY
Based on the relative polarities of the mobile and
stationary phase.
Normal Phase Chromatography
Stationary phase:
- Polar
- Stationary phase is a bonded siloxane with polar
functional group.
Mobile phase: non polar (e.g. ethyl ether, chloroform,
n-hexane, methylene chloride).
The polar stationary phase retain polar compounds
compared to non polar compounds (use the principle of
like dissolve like).
The least polar compound elutes first.
Increasing the polarity of the mobile phase, elution time
will decreased.
 Relationship between polarity
and elution time for normal-
phase chromatography

Stationary phase

Solute polarities: A
>B>C
*** Increasing the polarity of
the mobile phase, elution time
will decreased
 Reversed-Phase Chromatography
Stationary phase:
- Non Polar (hydrocarbon)
- most common bonded-phase are n-octyldecyl
(C18), or n-decyl (C8) chains, or phenyl groups.
 Mobile phase:
- Polar solvent (e.g. methanol, acetonitrile, water,
or tetrahydrofuran or mixture of water with one
of the organic solvents).
- The organic solvent is called the modifier.
- Water content is varied for adjusting the polarity.
- Methanol is used for acidic compounds.
- Acetonitrile is used for basic compounds.
- Tetrahydrofuran for compounds with large dipoles.

The most polar component elutes first.

Increasing mobile phase polarity will increased the

elution time.
Relationship between polarity and elution
time for reversed-phase chromatography

Solute polarities: A
>B>C
*** Increasing mobile
phase polarity will
increased the elution
time
 Normal-phase
chromatography

Reversed-phase
chromatography

Solutes polarities: Yellow > red > blue

Typical applications of partition
chromatography
 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.

Synthetic ion exchange solids:

- High molecular weight polymers that contain
large numbers of an ionic functional group per
molecule.
 Cation-exchange resins:

Common active sites for cation-exchange resins:

- sulfonic acid group, -SO3-H+ (strong acid) and the
carboxylic acid group, -COO-H+ (weak acid).
Cation-exchange chromatography retains
positively charged cations because the stationary
phase displays a negatively charged functional
group:
x RSO3-H+ + Mx+ (RSO3-)x Mx+ + xH+
solid solution solid solution

Mx+ represents a cation.

R represents that part of a resin molecule that
contains a sulfonic acid group
 Anion-exchange resins:

Anionic exchangers contain strongly basic

tertiary amine groups, -N(CH 3)3+OH- or weakly
basic primary amine groups, -NH 3+OH-
Retains negatively charged anion because the
stationary phase displays a positively charged
functional group.
x RN(CH3)3+OH- + Ax- [RN(CH3)3+]xAx- + xOH-
solid solution solid solution

Where Ax- represents an anion.

R represents that part of a resin molecule that
contains a basic group.
 Ion-exchange packings:

i) Polymeric bead packing:

- bead surface is coated with a synthetic
ion-exchange resin.

ii) Silica-based packings:

- Coating porous microparticles of silica
with a thin film of the exchanger. Particle
diameters are 3-10 m.
 Mobile phase in ion-exchange
chromatography:

Must dissolve the sample, have a solvent

strength that leads to reasonable retention
times and interact with solutes.

Aqueous solutions that contain moderate

amount of methanol or other water-miscible
organic solvents, in the form of a buffer.
 Applications of ion-exchange
chromatography:

o Applied to organic and biochemical

systems, including drugs and their
metabolites, serums, food
preservatives, vitamin mixtures,
sugars and pharmaceutical
preparations.
 Size-exclusion Chromatography
Applicable to high molecular weight species.

Packings:
- Consists of small (~10 m) silica or polymer
particles containing a network of uniform
pores into which solutes and solvent can
diffuse.

While in the pores, molecules are effectively trapped

and removed from the flow of mobile phase.

The average residence time in the pores depends on

the effective size of the analyte molecules.
 Molecules that are larger than the average
pore size of the packing are excluded and
not retained; they travel through the
column at the rate of the mobile phase.

Molecules that are considerably smaller

than the pores can penetrate throughout
the pores maze and entrapped for the
longest time; they are last to be eluted.

For intermediate size molecules, the

average penetration into the pores
depends on their diameter.
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
 The fractionation occurs between a
group is directly related to:
1. Molecular weight (MW)
2. Molecular shape

Exclusion limit:
1. The MW of a species beyond which
no retention occurs.
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.
 Size exclusion separations are different from
other chromatographic procedures in the
respect that no chemical or physical
interactions between analytes and stationary
phase are involved, such interaction will lead
to impaired column efficiency.
 Column packings
Two types of packing for size-exclusion
chromatography:
i) Polymer beads
ii) Silica-based particles
Advantages:
- greater rigidity, which leads to easier
packing and permits the use of higher pressures.
- greater stability, which permits the use of a
wider range of solvents, including water.
- more rapid equilibration with new solvents.
- stability at higher temperatures.
Disadvantages:
- tendency to retain solutes by adsorption.
- have potential for catalyzing the degradation
of solute molecules.
 Types of Size Exclusion Chromatography

Gel Filtration Gel Permeation

Size exclusion Size exclusion
chromatography chromatography
with hydrophilic with hydrophobic
packings are used packings and are
with aqueous used with non-
mobile phase. polar organic
mobile phase
Separation of Separation of
polar species. non-polar species.
 Application of Size Exclusion
Chromatography
Separation of high-molecular weight,
natural product molecules from low
molecular weight species.

Separation of homologs and

oligomers.

Rapid determination of molecular

weight distribution of larger
polymers or natural products.
Advantages of Size Exclusion
Chromatography
Short and well defined separation time.

Narrow bands, lead to good sensitivity.

Freedom from sample loss because solutes

do not interact with stationary phase.

Absence of column deactivation brought

about by interaction of solute with the
packing.