Chapter 33
High-Performance Liquid
Chromatography
1
• 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.
• In liquid chromatography, the mobile phase is a liquid solvent
containing the sample as a mixture of solutes.
2
• The types of high-performance liquid chromatography are often
classified by the separation mechanism or by the type of stationary
phase.
• These include
1) partition, or liquid-liquid, chromatography
2) adsorption, or liquid-solid, chromatography
3) ion-exchange, or ion, chromatography
4) size-exclusion chromatography
5) affinity chromatography
6) chiral chromatography
3
• Early in the development of the theory of LC, it was recognized
that large decreases in plate heights would be realized if the
particle size of packings were reduced.
• The minimum shown in Figure is not reached. It is because that
diffusion in liquids is much slower than in gases and its effect
on plate heights is observed only at extremely low flow rates.
4
• In the late 1960s, particle diameters are developed as small as 3 to
10 mm.
• The instruments was required of much higher pumping pressures
than the simple devices that preceded them.
• Detectors were developed for continuous monitoring of column
effluents.
• High-performance liquid chromatography (HPLC) 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.
5
• Applications of LC.
• For analytes having molecular masses greater than 10,000, one
of the two size-exclusion methods is often used:
 Gel permeation for
nonpolar compound
 Gel filtration for polar or
ionic compounds.
6
Instrumentation
• Pumping pressures of several hundred atmospheres are
required to achieve reasonable flow rates with packings in the 3to 10-mm size range.
• Because of the high pressures required, the equipment for
HPLC tends to be considerably more elaborate and expensive.
7
Mobile-Phase Reservoirs and Solvent
Treatment System
• A modern HPLC instrument is equipped with one or more glass
reservoirs, each of which contains 500 mL or more of a solvent.
• Provisions such as degassers are often included to remove
dissolved gases and dust from the liquids.
• Dissolved gases can lead to irreproducible flow rates and band
spreading.
• Both bubbles and dust interfere with the performance of most
detectors.
• Sparging is a process in which dissolved gases are swept out of
a solvent by bubbles of an inert, insoluble gas.
8
Mobile-Phase Reservoirs and Solvent
Treatment System
• An elution with a single solvent or
solvent mixture of constant composition
is termed an isocratic elution.
• In gradient elution, two (or more)
solvent systems that differ significantly
in polarity are used and varied in
composition during the separation.
• Modern HPLC are equipped with
proportioning valves that introduce
liquids from two or more reservoirs at
ratios that can be varied continuously.
9
Pumping System
• The requirements for liquid chromatographic pumps include:
1) the generation of pressures of up to 6,000 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
5) resistance to corrosion by a variety of solvents
• Two major types of pumps are used in HPLC instruments: 1) the
screw driven syringe type and 2) the reciprocating pump.
• Reciprocating types are used in almost all commercial instruments.
• Syringe-type pumps produce a pulse-free delivery whose flow rate
is easily controlled.
10
Pumping System
• The screw driven syringe pump:
 Advantage: flow rate is easily controlled
 Disadvantage: relatively low capacity, inconvenient when
solvents must be changed.
11
Pumping System
• The reciprocating pump consists of a small cylindrical chamber
that is filled and then emptied by the back-and-forth motion of a
piston.
• Modern HPLC uses dual pump heads or elliptical cams to
minimize pulsations.
•
A tandem-piston
pump provides a
continuous output flow.
12
Pumping System
• The advantages of reciprocating pumps:
 small internal volume (35 ~ 400 mL),
 high output pressure (up to 10,000 psi),
 adaptability to gradient elution,
 constant flow rate.
• Computer-controlled device measures the flow rate by
determining the pressure drop across a restrictor located at the
pump outlet.
13
Sample Injection Systems
• The most widely used method of sample introduction in liquid
chromatography is based on a sampling loop.
• An interchangeable loops are capable of providing a choice of
sample sizes ranging from 1 to 100 mL or more.
• Many HPLC instruments incorporate an auto sampler with an
automatic injector.
• These injectors can introduce continuously variable volumes from
containers on the auto sampler.
14
Columns for HPLC
• Liquid chromatographic columns are usually constructed from
stainless steel tubing, although glass and polymer tubing, such as
polyetheretherketone (PEEK), are sometimes used.
PEEK
Analytical Columns
• Most columns range in length from 5 to 25 cm and have inside
diameters of 3 to 5 mm.
• The most common particle size of packings is 3 ~ 5 mm.
• The common columns are 10 ~ 15cm long, 4.6 mm in inside diameter,
and packed with 5-mm particles. Columns of this type provide 40,000
~ 70,000 plates/m.
15
Columns for HPLC
• In 1980s, microcolumns became available with inside diameters of 1
to 4.6 mm and length of 3 to 7.5 cm.
• Microcolumn, which are packed with 3- or 5-mm particles, contain as
many as 100,000 plates/m and have the advantage of speed and
minimal solvent consumption.
 Example: a separation based on a microbore column. MS/MS
was used to monitor the separation of rosuvastatin from human
plasma.
16
Columns for HPLC
Precolumns
• Two types of precolumns are used.
1) A precolumn between the mobile phase reservoir and the
injector is used for mobile-phase conditioning and is termed
a scavenger column.
2) A second type of precolumn is a guard column, positioned
between the injector and the analytical column.
• A guard column is a short column packed with a similar
stationary phase as the analytical column.
• The purpose of the guard column is to prevent impurities, such
as highly retained compounds and particulate matter, from
reaching and contaminating the analytical column.
• The guard column is replaced regularly and serves to increase
the lifetime of the analytical column.
17
Columns for HPLC
Column Temperature Control
• For some applications, close control of column temperature is not
necessary and columns are operated at room temperature.
• Better and 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 constanttemperature bath to give precise temperature control.
18
Columns for HPLC
Column Packings
• Two types of packings are used in HPLC: 1) Pellicular Particles
and 2) Porous Particles.
•
Pellicular Particles - spherical, nonporous, glass or polymer
beads with typical diameters of 30 to 40 mm.

A thin, porous layer of silica, alumina, a polystyrene-divinyl
benznene synthetic resin, or an ion-exchange resin was
deposited on the surface of these beads.

In recent year, only small pellicular packings (~5 mm) was
developed for separation of proteins and large biomolecules.
19
Columns for HPLC
•
Porous Particles - microparticles having diameters ranging
from 3 to 10 mm.

The particles are composed of silica, alumina, the
synthetic resin polystyrene-divinyl benzene, or an ionexchange resin.

Silica is the most common packing in LC.

Silica particles are often coated with thin organic films ,
which are chemically or physically bonded to the surface.
20
HPLC Detectors
• The ideal detector for HPLC should have all the characteristics of
the ideal GC detector expect that it has no need to have great
temperature range.
• HPLC detector must have low internal volume (dead volume) to
minimize extra-column band broadening.
• The detector should be small and compatible with liquid flow.
21
HPLC Detectors
• The most widely used detectors for liquid chromatography are
based on absorption of ultraviolet or visible radiation.
• Both photometers and spectrophotometers are available for HPLC.
• Photometers often make use of the 254- and
280- nm lines from a mercury source because
many organic functional groups absorb in the
region.
• Deuterium sources or tungsten-filament
sources with interference filters also provide a
simple means of detecting absorbing species.
• Spectrophotometeric detectors are widely
used in high-performance instruments.
• Diode-array detector (DAD) can display an
entire spectrum as an analyte exits the column.
22
HPLC Detectors
• The combination of HPLC with a mass spectrometry detector
produces a very powerful analytical tool.
• LC/MS systems can identify the analytes exiting from the HPLC
column.
• The refractive index detector (RI) is general rather than selective
and responds to the presence of all solutes.
• RI has limited sensitivity.
• Potentiometric, conductometric,
and voltametric measurements can
also be used in HPLC.
23
Five Mechanical LC Techniques
Partition Chromatography
• The most widely used type of HPLC is partition chromatography in
which the 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.
 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.
• The bonded-phase method predominate because of their greater
stability and compatibility with gradient elution.
25
Bonded-Phase Packings
• 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 organic group R is often a straight chain octyl- (C8) or
octyldecyl- (C18) group.
• Other organic functional groups that have been bonded to silica
surfaces include aliphatic amines, ethers, and nitriles as well as
aromatic hydrocarbons.
• Many different polarities for the bonded stationary phase are
available.
26
Normal- and Reversed-Phase Packings
• Two types of partition chromatography are distinguishable based on
the relative polarities of the mobile and stationary phases.
• Early work in LC was based on highly polar stationary phases
(triethylene glycol or water) and a relatively nonpolar solvent
(hexane or i-propyl ether) served as the mobile phase.
• For historic reasons, this type of chromatography is called normalphase chromatography (NPLC).
 In normal-phase partition chromatography, the stationary phase
is polar and the mobile phase is nonpolar.
 The least polar analyte is eluted first.
 Increasing the polarity of the mobile phase then decreases the
elution time.
27
Normal- and Reversed-Phase Packings
• In reversed-phase partition chromatography (RPLC), the stationary
phase is non-polar, and the mobile phase is a relatively polar
solvent.
 The least polar analyte is eluted last.
 The most polar component elutes first.
 Increasing the mobile phase polarity increases the elution time.
• More than ¾ of all HPLC separations are performed with reversedphase, bonded, octyl- or octyldecyl- siloxane packings.
• The long-chain hydrocarbon groups are aligned parallel to one
another and perpendicular to the surface of the particle giving a
nonpolar hydrocarbon surface.
• The mobile phase used is often an aqueous solution containing
various concentrations of MeOH, Acetonitrile, or Tetrahydrofuran.
28
Normal- and Reversed-Phase Packings
• 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.
• The separation mechanism is:
 The counterion forms an uncharged ion pair with a solute ion
of opposite charge in the mobile phase.
 The ion pair then partitions into the nonpolar stationary phase
giving differential retention of solutes based on the affinity of
the ion pair for the two phases.
 Separation of organic solute ions of the opposite charge then
occurs by formation of reversible ion-pair complexes with the
more strongly retained solutes forming the strongest
complexes with the stationary phase.
29
Normal- and Reversed-Phase Packings
• An example illustrates the
separation of ionic and
nonionic compounds
using alkyl sulfonates of
various chain lengths as
ion-pairing agents.
• A mixture of C5- and C7alkyl sulfonates gives the
best separation results.
30
Choice of Mobile and Stationary Phases
• Successful partition chromatography requires a proper balance of
intermolecular forces among the three participants in the separation
process: 1) the analyte, 2) the mobile phase, and 3) the stationary
phase.
• These intermolecular forces are described qualitatively in terms of
the relative polarity possessed by each of the 3 components.
• The polarities of common organic functional groups in increasing
order are:
hydrocarbons < ethers < ketones < aldehydes < amides < alcohols
• Water is more polar than compounds containing any of these
functional groups.
31
Choice of Mobile and Stationary Phases
• The polarity of the stationary phase is matched roughly with that of
the analytes; a mobile phase of considerably different polarity is
then used for elution.
• If the polarities of the analyte and the mobile phase are matched
but are different from that of the stationary phase, the stationary
phase cannot compete successfully for the sample components.
 Retention times will be too short for practical application.
• The order of polarities of common mobile phase solvents are:
Water > acetonitrile > methanol > ethanol > tetrahydrofuran >
propanol > cyclohexane > hexane
32
Applications
Gradient elution
• Figure shows typical
applications of bonded-phase
partition chromatography for
separating soft drink additives
and organophophate
insecticides.
C8 bondedphase column
Isocratic elution
Nitile bondedphase column
33
Applications
• Table illustrates the variety of samples to which the technique is
applicable.
34
Adsorption Chromatography
• Adsorption, or liquid-solid, chromatography is the classic form of
liquid chromatography first introduced by Tswett at the beginning of
the 20th century.
• In many normal-phase separations, adsorption/displacement
processes govern retention.
• Finely divided silica and alumina are the only stationary phases that
find use for adsorption chromatography.
• Silica is preferred because of its higher sample capacity.
• For both silica and alumina, retention times become longer as the
polarity of the analyte increases.
• Because of the versatility and ready availability of bonded stationary
phase, traditional adsorption chromatography with solid stationary
phases has seen decreasing use in recent years in favor of normalphase chromatography.
35
Ion Chromatography
• Ion chromatography was first developed in the mid-1970s when it was
shown that anion or cation mixtures can be resolved on HPLC
columns packed with anion-exchange or cation-exchange resins.
• The detection was generally performed with conductivity
measurements, but it is not ideal because of high electrolyte
concentrations in the mobile phase.
• The development of low-exchange-capacity columns allowed the use
of low-ionic-strength mobile phase that could be deionized to allow
high sensitivity conductivity detection.
• Currently, spectrophometric and eletrochemical detectors are
available for ion chromatography.
36
Ion Chromatography
• 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.
37
Ion Chromatography Based on Suppressors
• Conductivity detectors are highly sensitive, universal for charged
species, and respond in a predictable way to concentration
changes.
• Conductivity detectors are simple to operate, inexpensive to
construct and maintain, easy to miniaturize and usually give
prolonged, trouble-free service.
• The only limitation to the use of conductivity detectors was due to
the high electrolyte concentrations required to elute most analyte
ions in a reasonable time.
• The conductivity from the mobile-phase components tends to
swamp that from the analyte ions greatly reducing the detector
sensitivity.
38
Ion Chromatography Based on Suppressors
• The problem created by the high conductance of eluents was solved
by the introduction of an eluent suppressor column immediately
following the ion-exchange column.
• The suppressor column is packed with a second ion-exchange resin.
• The resin effectively converts the ions of the eluting solvent to a
molecular species of limited ionization without affecting the
conductivity due to analyte ions.
• For example: when cations are being separated and determined,
hydrochloric acid is chosen as the eluting reagent, and the suppressor
column is an anion-exchange resin in the hydroxide form. The product
of the reaction in the suppressor is water,
H+(aq) + Cl-(aq) + resin+OH-(s)  resin+Cl-(s) + H2O
the analyte cations are not retained in the 2nd anion-exchange column.
39
Ion Chromatography Based on Suppressors
• For anion separations, the suppressor packing is the acid form of a
cation-exchange resin and sodium bicarbonate or carbonate is the
eluting agent,
Na+(aq) + HCO3-(aq) + resin-H+(s)  resin-Na+(s) + H2CO3(aq)
• The disadvantage of the suppressor columns is the need to
regenerate them periodically (typically every 8 to 10 hr) to convert
the packing back to the original acid or base form.
• Micromembrane suppressors operates continuously and they are
capable of removing all Na+ from a 0.1M NaOH solution with an
eluent flow rate of 2 mL/min.
40
Ion Chromatography Based on Suppressors
• Figure shows two applications of IC based on a suppressor column
and conductometric detection.
• Ions are present in ppm range,
the sample size is 50 mL in one
case and 100 mL in the 2nd one.
41
Single-Column Ion Chromatography
• Single-Column IC depends on the small differences in
conductivity between sample ions and the prevailing eluent ions.
• In single-column ion-exchange chromatography, analyte ions
are separated on a low-capacity ion exchanger by means of a
low-ionic strength eluent that does not interfere with the
conductometric detection of analyte ions.
• The advantage offered is that no special equipment is required
for suppression.
• However, it is a somewhat less sensitive method for determining
anions than suppressor-column methods.
42
Size-Exclusion Chromatography
• Size-exclusion, or gel chromatography, is a powerful technique that
is particularly applicable to high-molecular-mass species.
• Packings for size-exclusion chromatography consist of small
(~10mm) silica or polymer particles containing a network of uniform
pores into which solute and solvent molecules can diffuse.
• While in the pores, molecules are trapped and removed from the
flow of the mobile phase.
• The average residence time of analyte molecules depends on their
effective size.
43
Size-Exclusion Chromatography
• Molecules that are larger than the average pore size of the packing
are excluded and thus suffer no retention, that is, they travel
through the column at the rate of the mobile phase.
• Molecules that are appreciably smaller than the pores can
penetrate throughout the pore maze and are entrapped for the
greatest time; so they are last to elute.
• Intermediate-size molecules penetrate into the pores of the
packing depending on their diameters.
• Fractionation is based on molecular size and to some extent
molecular shape.
44
Size-Exclusion Chromatography
• The size-exclusion separation differ from the other
chromatographic methods in the respect that there are no chemical
or physical interactions between analyte and the stationary phase.
• Unlike other chromatographic methods, size-exclusion separation
has an upper limit to retention time because no analyte species is
retained longer than those small molecules that totally permeate
the stationary phase.
45
Column Packings for Size-Exclusion
• Two types of packing for size-exclusion chromatography are
encountered: polymer beads and silica-based particles, both of
which have diameters of 5 to 10 mm.
• Silica particles are more rigid, which leads to easier packing and
permits higher pressures to be used.
• Silica particles are more stable, allowing a great range of solvents
to be used and exhibiting more rapid equilibration with new solvent.
46
Column Packings for Size-Exclusion
• Packings for size-exclusion: some are hydrophilic for use with
aqueous mobile phase; hydrophobic are used with nonpolar
organic solvents.
• Gel Filtration is a type of size-exclusion chromatography in which
the packing is hydrophilic. It is used to separate polar species.
• Gel Permeation is a type of size- exclusion chromatography in
which the packing is hydrophobic. It is used to separate nonpolar
species.
• Generally, size-exclusion packings accommodate a 2- to 2.5decade range of molecular mass.
47
Applications of Size-Exclusion
• Figure illustrates typical applications of size-exclusion
chromatography.
Fatty acids separation
Epoxy resin separation
48
Applications of Size-Exclusion
• Another important application of size-exclusion chromatography is
the rapid determination of the molecular mass or the molecular mass
distribution of large polymers or natural products.
• Calibrations can be accomplished by means of standards of known
molecular mass.
• Molecules separated in size-exclusion are according to
hydrodynamic volume.
• A universal calibration curve can
be obtained by plotting logM vs.
the retention volume VR.
• Absolute calibration can be
achieved by using a molar
mass-sensitive detector such as
a low-angle light-scattering
detector (LSD).
49
Affinity Chromatography
• In affinity chromatography, a reagent called an affinity ligand is
covalently bonded to a solid support.
• Typical affinity ligands are antibodies, enzyme inhibitors, or other
molecules that reversibly and selectively bind to analyte
molecules in the sample.
• When the sample passes through the column, only the
molecules that selectively bind to the affinity ligand are retained.
• Molecules that do not bind pass through the column with the
mobile phase.
• After the undesired molecules are removed, the retained
analytes can be eluted by changing the mobile phase conditions.
50
Affinity Chromatography
• The stationary phase is a solid, such as agarose, or a porous glass
bead to which the affinity ligand is immobilized.
• The mobile phase in affinity
chromatography has two
distinct roles.
 It must support the strong
binding of the analyte
molecules to the ligand.
 Once the undesired
species are removed, the
mobile phase must
weaken or eliminate the
analyte-ligand interaction
so that the analyte can be
eluted.
51
Affinity Chromatography
• Changes in pH or ionic strength are used to change the elution
conditions during the two stages of the process.
• Affinity chromatography has the major advantage of extraordinary
specificity.
• The primary use is in the rapid isolation of biomolecules during
preparative work.
52
Chiral Chromatography
• Chiral compounds are compounds with non-superimposable
mirror images of each other.
• Such mirror images are called enantiomers.
• Chiral mobile-phase additives or chiral stationary phases are
required for these separations.
• A chiral resolving agent is a chiral mobile-phase additive or a
chiral stationary phase that preferentially complexes one of the
enantiomers.
• Chiral stationary phases have the chiral agent immobilized on
the surface of a solid support and it is the more popular way for
chiral compounds separation.
53
Chiral Chromatography
• Several different modes of interaction occur between the chiral
resolving agent and the solute.
 The interaction are due to attractive forces such as those
between p bonds, hydrogen bonds or dipoles.
 The solute can fit into chiral cavities in the stationary phase
to form inclusion complex.
• Figure shows the separation of a racemic mixture of an ester on a
chiral stationary phase.
54
Comparison of HPLC and GC
• GC offers the advantage of speed and simplicity of equipment.
• HPLC is applicable to nonvolatile substances (including inorganic
ions) and thermally unstable materials.
55