H Igh

BY SANELISO FUTURE MOYO

Performance
L iquid
C hromatography
1
 H igh
Pressure
L iquid
C hromatography
HPLC uses high pressure to force solvent through closed columns
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containing very fine particles that give high-resolution separations.
H igh
Priced
L iquid
C hromatography
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 HPLC
• High-performance liquid chromatography (HPLC) is the
most versatile and widely used type of elution
chromatography.
• The technique is used by scientists for separating and
determining species in a variety of organic, inorganic, and
biological materials
• HPLC, is a type of chromatography that combines a liquid
mobile phase and a very finely divided stationary phase. In
order to obtain satisfactory flow rates, the liquid must be
pressurized to several hundred or more pounds per square
inch.

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 Why is high pressure needed in HPLC?

Small particles (in the stationary phase) give increased resistance to

flow. High pressure is required to obtain a usable flow rate

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6
 The HPLC system consists of a solvent delivery system, a
sample injection valve, a high-pressure column, a detector,
and a computer to control the system and display results.

Detector

Column
Pump Column oven
(thermostatic
Eluent Sample injection column Drain
(mobile unit chamber)
(injector) Data processor
phase) Degasser
7
Block diagram showing components of a typical apparatus for HPLC.

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 Instrumentation

• Pumping pressures of several hundred atmospheres are required to

achieve reasonable flow rates with packings in the 3- to 10-mm
size range, which are common in modern liquid chromatography.
• Because of these high pressures, the equipment for high-
performance liquid chromatography tends to be considerably more
elaborate and expensive than that encountered in other types of
chromatography.

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Mobile-Phase Reservoirs and Solvent Treatment Systems
A modern HPLC instrument is equipped with one or more glass reservoirs, each of
which contains 500 mL or more of a solvent.
Provisions are often included to remove dissolved gases and dust from the liquids.
Dissolved gases can lead to irreproducible flow rates and band spreading.
In addition, both bubbles and dust interfere with the performance of most detectors.
Degassers may consist of a vacuum pumping system, a distillation system, a device for
heating and stirring, a system for sparging in which the dissolved gases are swept out
of solution by fine bubbles of an inert gas that is not soluble in the mobile phase

Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of
an inert, insoluble gas.

An elution with a single solvent or solvent mixture of constant composition is termed

an isocratic elution.
In gradient elution, two (and sometimes more) solvent systems that differ significantly
in polarity are used and varied in composition during the separation. The ratio of the
two solvents is varied in a preprogrammed way, sometimes continuously and
sometimes in a series of steps

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Comparison of gradient elution and isocratic elution

. As shown in the Figure,

gradient elution frequently
improves separation efficiency,
just as temperature
programming helps in gas
chromatography.

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 Pumping Systems

The requirements for liquid chromatographic pumps include :

(1) the generation of pressures of up to 6000 psi (lb/in2),
(2) pulse-free output,
(3) flow rates ranging from 0.1 to 10 mL/min,
(4) flow reproducibilities of 0.5% relative or better, and
(5) Resistance to corrosion by a variety of solvents.

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Two major types of pumps are used in HPLC instruments: the screw-driven Syringe type and the
reciprocating pump.
Syringe-type pumps
• Produce a pulse-free delivery whose flow rate is easily controlled.
• Limitation is they have relatively low capacity (250 mL)
• They are inconvenient when solvents must be changed.

• Figure illustrates the operating principles of the reciprocating pump. This device consists of a small cylindrical chamber that is filled and then
emptied by the back and-forth motion of a piston. The pumping motion produces a pulsed flow that must be subsequently damped because the
pulses appear as baseline noise on the chromatogram.

Advantages of reciprocating pumps include:

small internal volume (35 to 400 mL), high output pressure (up to 10,000 psi), ready adaptability to gradient elution, and constant flow rates,
which are largely independent of column back-pressure and solvent viscosity.

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 Sample Injection Unit (Injector)

 Performance Requirements
 No sample remaining in unit
 Minimal broadening of sample band
 Free adjustment of injection volume
 Minimal loss
 Superior durability and pressure resistance

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 Manual Injector

From pump

To column
LOAD position
From pump

To column
INJECT position
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 Manual Injector:
Operating Principle of Sample Injection

From pump From pump

Loop

Loop
To column
To column

LOAD INJECT

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 Manual Injector:
Injection Method
 Syringe measurement method
 It is desirable that no more than half the loop
volume is injected.
 Loop measurement method
 It is desirable that at least 3 times the loop
volume is injected.

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 Autosampler
(Pressure Injection Method)

From To column From To column

pump pump

Sample Loop

LOAD INJECT 18
 Autosampler
(Total-Volume Injection Method)
From To column From To column
pump pump

Needle

Sample vial
LOAD INJECT
Measuring pump
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 Columns
 Usually constructed from stainless steel tubing, although glass and
polymer tubing, such as polyetheretherketone (PEEK).
 Analytical column
 length from 5 to 25 cm, inside diameters of 3 to 5 mm.. The most
common particle size of packings is 3 or 5 mm.
 Commonly used columns are 10 or 15 cm long, 4.6 mm
 in inside diameter, and packed with 5-mm particles. Columns of
this type provide 40,000 to 70,000 plates/m.
 Microcolumn
 Have
Have inside
inside diameters
diameters ofof 11 to
to 4.6
4.6 mm
mm andand lengths
lengths of
of 33 to
to 7.5
7.5 cm.
cm.
 These
These columns
columns are
are packed
packed with
with 3-
3- or
or 5-mm
5-mm particles,
particles,
 contain
contain as
as many
many asas 100,000
100,000 plates/m
plates/m andand have
have the
the advantage
advantage of of speed
speed and
and minimal
minimal solvent
solvent
consumption.
consumption.
 Minimal
Minimal solvent
solvent consumption
consumption is is of
of considerable
considerable importance
importance because
because the
the high-purity
high-purity solvents
solvents
required
required for
for liquid
liquid chromatography
chromatography are are expensive
expensive toto purchase
purchase andand to
to dispose
dispose of
of after
after use.
use.
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 Precolumns
 There are two types of precolumns.

 A scavenger column between the mobile-phase container and the

injector is used to condition the mobile phase..
 The solvent partially dissolves the silica packing and ensures that the mobile
phase is saturated with silicic acid prior to entering the analytical column.
 This saturation minimizes losses of the stationary phase from the analytical
column.
 A guard column between the injector and the column removes
particulates and other solvent impurities.
 Prolongs the life of the analytical column

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Column Temperature Control

• For some applications, close control of column temperature is not

necessary, and columns are operated at room temperature.
• Often, however, better, more reproducible chromatograms are
obtained by maintaining constant column temperature.
• Most modern commercial instruments are equipped with heaters that
control column temperatures to a few tenths of a degree from near
room temperature to 150°C.
• Columns can also be fitted with water jackets fed from a constant-
temperature bath to give precise temperature control.

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Although temperature does not have nearly the effect on HPLC separations that it has on GC
separations, it nonetheless can play an important role. Discuss how and why temperature might
or might not influence the following separations:
a) a reversed-phase chromatographic separation of a steroid mixture.
b) an adsorption chromatographic separation of a mixture of closely related isomers.

A number of factors that influence separation are clearly temperature dependent including
distribution constants and diffusion rates. In addition, temperature changes can influence
selectivity if components A and B are influenced differently by changes in temperature. Because
resolution depends on all these factors, resolution will also be temperature dependent.

(a) For a reversed phase chromatographic separation of a steroid mixture, selectivity and,
as a consequence, separation could be influenced by temperature dependent changes in
distribution coefficients.

(b) For an adsorption chromatographic separation of a mixture of isomers, selectivity

and, as a consequence, separation could be influenced by temperature dependent changes
in distribution coefficients.

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Column Packings

Two types of packings are used in HPLC, pellicular and porous particle.

• The original pellicular particles were spherical, nonporous, glass or polymer beads with
typical diameters of 30 to 40 mm.
• A thin, porous layer of silica, alumina, a polystyrene-divinyl benzene synthetic resin, or an
ion-exchange resin was deposited on the surface of these beads.
• Small porous microparticles have completely replaced these large pellicular particles. In
recent years, small (<5 mm) pellicular packings have been reintroduced for separation of
proteins and large biomolecules.
 The typical porous particle packing for liquid chromatography consists of porous
microparticles having diameters ranging from 3 to 10 mm; for a given size particle, a very
narrow particle size distribution is desirable.
 The particles are composed of silica, alumina, the synthetic resin polystyrene-divinyl
benzene, or an ion-exchange resin.
 Silica is by far the most common packing in liquid chromatography.
 Silica particles are often coated with thin organic films, which are chemically or physically
bonded to the surface.

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 2.4. Detection
 Requirements of a detector:

1. Sensitive to low concentrations of every analyte.

2. Small volume to avoid peak broadening.
3. Linear response.
4. Insensitive to changes in temperature or solvent composition.

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 2.4.1. UV and Fluorescence Detectors
1. UV detector: using a 2. Fluorescence detector:
flow cell, most common
Fluorescence is measured
type (why?).
after excitation of the eluate
Employ deuterium,
with a laser.
tungsten or xenon lamps
with monochromators to Adv.: very sensitive
choose the optimum λ. Disadv.: response to few
analytes. Derivatization of
A special type of UV
originally non-fluorescing
detectors: the
analytes by covalent
photodiode array
attachment of fluorophors to
detectors (PDA) which
analytes either prior to
records the spectrum of
chromatographic separation,
each solute as it is
or by adding derivatization
eluted. The linearity
The flow reagents to the eluate
range for this type of
cell between column and detector.
detectors is in a 5 orders
of magnitude. 26
 2.4.2. Refractive Index Detector
3. Refractive index detector:
•Responds to most solutes
•Useless in gradient elution,
sensitive to temp. and pressure changes.
•Small linear range only
•Detection limit 1000x
poorer than that of UV detector
It is a deflection-type detector:
Two triangular 5- to 10-L compartments
where pure solvent or eluate passes;
when solute of different refractive index enters
the cell, the beam is deflected and the
photocell output changes.
IR radiation must be removed (avoid heating
of sample soln.!) 27
 2.4.3. Electrochemical Detector
4. Electrochemical detector:
It responds to analytes that can be oxidized or
reduced
aromatic compounds: amines, phenols, halogen
and nitro compounds, peroxides, mercaptans,
ketones, aldehydes, conjugated nitriles.
The eluate is oxidized or reduced at the working
electrode. The potential is maintained at a
selected value with respect to Ag|AgCl (reference)
electrode; and the current is measured between
the working electrode and auxiliary electrode.
Adv: The linear range (current  conc.): 6 orders of
magnitude!
Disadv.: very sensitive to flow rate and temperature
changes, eluate must be free of oxygen, metal ions
from tubing have to be masked. 28
 Partition Chromatography

• The most widely used type of HPLC.

• Partition chromatography: stationary phase is a second liquid that is immiscible with the
liquid mobile phase.
• Partition chromatography can be subdivided into liquid-liquid and liquid bonded- phase
chromatography.
• The difference between the two lies in the way that the stationary phase is held on the
support particles of the packing.
• The liquid is held in place by physical adsorption in liquid-liquid chromatography, while it
is attached by chemical bonding in bonded-phase chromatography.
• Bonded-phase methods offers the following advantages: greater stability and compatibility
with gradient elution.
In liquid-liquid partition chromatography, the stationary phase is a solvent held in place by
adsorption of the surface of the packing particles.
In liquid-bonded-phase chromatography, the stationary phase is an organic species that is
attached to the surface of the packing particles by chemical bonds.
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 Bonded-phase packing
In a bonded-phase packing, the stationary phase liquid is held in place by chemically bonding it to the solid
support.
In a normal-phase packing, the stationary phase is quite polar and the mobile phase is relatively
nonpolar.

• Most bonded-phase packings are prepared by reaction of an

organochlorosilane with the -OH groups formed on the surface of
silica particles by hydrolysis in hot dilute hydrochloric acid.
• The product is an organosiloxane.
• The reaction for one such SiOH site on the surface of a particle can
be written as

where R is often a straight chain octyl- or octyldecyl-

group.

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 Types of partition chromatography

a. Normal phase chromatography

b. Reverse phase chromatography
In normal-phase partition chromatography, the stationary phase is polar and the mobile phase
nonpolar.
In reversed-phase partition chromatography, the polarity of these phases is reversed.

In normal-phase chromatography, the least polar component is eluted first; increasing

the polarity of the mobile phase then decreases the elution time.

In contrast, with reversed-phase chromatography, the most polar component elutes first,
and increasing the mobile phase polarity increases the elution time.
Ion-pair chromatography is a subset of reversed-phase chromatography in which easily
ionizable species are separated on reversed-phase columns. In this type of chromatography, an
organic salt containing a large organic counterion, such as a quarternary ammonium ion or alkyl
sulfonate, is added to the mobile phase as an ion-pairing reagent . 31
Why does eluent strength increase as solvent becomes less polar in reversed-phase
chromatography, whereas eluent strength increases as solvent becomes more polar in normal-
phase chromatography?
In reverse-phase chromatography, the solutes are nonpolar and more
soluble in a nonpolar mobile phase. In normal-phase chromatography,
the solutes are polar and more soluble in a polar mobile phase

Indicate the order of elution of the following compounds from a normal-phase packed HPLC
column:
Ethyl acetate, acetic acid, dimethylamine.

ethyl acetate, dimethylamine, acetic acid

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 Comparison of Normal Phase
and Reversed Phase
Normal Phase Reversed Phase
 Effective for separation of  Wide range of applications
structural isomers  Effective for separation of
 Offers separation homologs
selectivity not available  Stationary phase has long
with reversed phase service life
 Stabilizes slowly and is  Stabilizes quickly
prone to fluctuations in  Eluents are inexpensive and
retention time easy to use
 Eluents are expensive

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 Adsorption Chromatography

 A solid such as silica gel is used as the

stationary phase, and differences, mainly in
the degree of adsorption to its surface, are
used to separate the solutes.
 Liquid-solid chromatography
 The retention strength increases with the
hydrophilicity of the solute.

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 Ion Chromatography/ Ion exchange chromatography
Ion-exchange chromatography A form of liquid chromatography in which the stationary phase
is an ion exchange resin.
Two types of ion chromatography are currently in use: suppressor-based and single-column.
They differ in the method used to prevent the conductivity of the eluting electrolyte from
interfering with the measurement of analyte conductivities
The conductivity detector is well suited for ion chromatography.
These detectors are simple to operate, inexpensive to construct and maintain, easy to miniaturize,
and usually give prolonged, trouble-free service. The limitation is high electrolyte concentrations
required to elute most analyte ions in a reasonable time.

Describe the difference between Single-column and suppressor-column ion chromatography .

In suppressor-column ion chromatography the chromatographic column is followed by a
column whose purpose is to convert the ions used for elution to molecular species that are
largely nonionic and thus do not interfere with conductometric detection of the analyte species.
In single-column ion chromatography, low capacity ion exchangers are used so that the
concentrations of ions in the eluting solution can be kept low. Detection then is based on the
small differences in conductivity caused by the presence of eluted sample components.

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Ion Exchange Chromatography
•In ion exchange, the column packing contains ionic groups (e.g. sulfonic,
tetraalkylammonium) and the mobile phase is an aqueous buffer (e.g. phosphate,
formate, etc.).
•Ion exchange is used by about 20% of the liquid chromatographers
•The technique is well suited for:
•the separation of inorganic and organic anions and cations in aqueous solution.
•Ionic dyes, amino acids, and proteins can be separated by ion exchange because
such compounds are salt in brine water,

Application for Ion Exchange

Chromatography: Basic proteins on
strong cation exchanger (-SO3-)
1. RNA polymerase
2. Chymotrypsinogen
3. Lysozyme

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 Size Exclusion Chromatography
A form of liquid chromatography in which the stationary phase is a porous material and
in which separations are based on the size of the solutes.

 Separation is based on the size (bulkiness) of molecules.

 The name varies with the application field!

Size Exclusion Chromatography (SEC)
Gel Permeation Chromatography (GPC) aa type
type of
of size-exclusion
size-exclusion chromatography
chromatography
in
in which
which the
the packing
packing is
is hydrophobic.
hydrophobic. ItIt is
is used
used to
to separate
separate nonpolar
nonpolar species
species
 Application in Chemical industry fields, synthetic polymers,

nonaqueous systems
 Gel Filtration Chromatography (GFC) aa type
type of
of size
size exclusion
exclusion chromatography
chromatography
in
in which
which the
the packing
packing is
is hydrophilic.
hydrophilic. ItIt is
is used
used to
to separate
separate polar
polar species.
species.
 Applications in Biochemical fields, biological
Applications in Biochemical fields, biological macromolecules,
aqueous systems

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 Affinity chromatography

• This most selective kind of chromatography employs specific

. interactions between one kind of solute molecule and a second
molecule that is covalently attached (immobilized) to the stationary
phase.
• For example, the immobilized molecule might be an antibody to a
particular protein. When a mixture containing a thousand proteins is
passed through the column, only the one protein that reacts with the
antibody binds to the column. After all other solutes have been
washed from the column, the desired protein is dislodged by changing
the pH or ionic strength.

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