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LU5:
CHROMATOGRAPHIC
METHODS
High Performance Liquid Chromatography: HPLC

Introduction
 form of liquid chromatography that employs a liquid
mobile phase and a very finely divided stationary
phase used to separate compounds that are
dissolved in solution.
 The liquid mobile phase adds an extra dimension
of control to chromatography since the analyte
interacts with the mobile phase as well as with the
stationary phase
 It is also sometimes referred to as high-pressure
liquid chromatography

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Introduction
 most widely used separation technique
 HPLC can handle nonvolatile samples and thermally sensitive samples
which cannot be analyzed with a GC
 HPLC instruments consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector.

Types of HPLC
 Classifications of HPLC are actually based on the types of
interactions between the analyte and the stationary and mobile
phases.

1. Normal phase chromatography

2. Reversed phase chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography

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Types of HPLC

1. Normal phase chromatography

 Normal phase HPLC (NP-HPLC) separates analytes based

on polarity.
 This method uses a polar stationary phase and a non-polar
mobile phase, and is used when the analyte of interest is
fairly polar in nature.
 The polar analyte associates with and is retained by the
polar stationary phase.
 Adsorption strengths increase with increase in analyte
polarity, and the interaction between the polar analyte and
the polar stationary phase (relative to the mobile phase)
increases the elution time.

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1. Normal phase chromatography

 The interaction strength not only depends on the functional groups in
the analyte molecule, but also on steric factors and structural isomers
are often resolved from one another.
 Use of more polar solvents in the mobile phase will decrease the
retention time of the analytes while more hydrophobic solvents tend to
increase retention times.
 Particularly polar solvents in a mixture tend to deactivate the column
by occupying the stationary phase surface.
 Typical stationary phases are silica (SiO2) and alumina (Al2O3 ).

2. Reverse Phase Chromatography

 Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary

phase and an aqueous, moderately polar mobile phase.
 One common stationary phase is a silica which has been treated with
RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or
C8H17.
 The retention time (RT) is therefore longer for molecules which are
more non-polar in nature, allowing polar molecules to elute more
readily.
 R.T is increased by the addition of polar solvent to the mobile phase
and decreased by the addition of more hydrophobic solvent.

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2. Reverse Phase Chromatography

 RPC operates on the principle of hydrophobic interactions, which
result from repulsive forces between a polar eluent, the relatively non-
polar analyte, and the non-polar stationary phase.
 The binding of the analyte to the stationary phase is proportional to the
contact surface area around the non-polar segment of the analyte
molecule upon association with the ligand in the aqueous eluent.

2. Reverse Phase Chromatography

 Reversed phase columns are quite difficult to damage compared with
normal silica columns,
 however, many reversed phase columns consist of alkyl derivatized
silica particles and should never be used with aqueous bases as
these will destroy the underlying silica particle.
 They can be used with aqueous acid, but the column should not be
exposed to the acid for too long, as it can corrode the metal parts of
the HPLC equipment.

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2. Reverse Phase Chromatography

 For reverse phase HPLC, typical solvents are
 water,
 alcohols,
 acetonitrile, and
 hexane,
 all of which are UV-VIS transparent.

 Often the pH is controlled with added acid or buffers.

3. Ion exchange chromatography

 In ion-exchange chromatography, retention is based on the attraction
between solute ions and charged sites bound to the stationary
phase.
 Ions of the same charge are excluded.
 Uses an ionic stationary phase to separate ionic analytes.
 The stationary phase can be a polymer with anionic (e.g. -SO3- or
COO- ) or cationic (e.g. –NR3+ ) sites.

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3. Ion exchange chromatography

Some types of Ion Exchangers include:
a. Polystyrene resins
 allows cross linkage which increases the stability of the chain.
 Higher cross linkage reduces swerving, which increases the
equilibration time and ultimately improves selectivity.
b. Cellulose and dextran ion exchangers (gels)-
 These possess larger pore sizes and low charge densities making
them suitable for protein separation.
c. Controlled-pore glass or porous silica.

3. Ion exchange chromatography

 In general, ion exchangers favor the binding of ions of higher charge
and smaller radius.
 An increase in counter ion (with respect to the functional groups in
resins) concentration reduces the retention time.
 An increase in pH reduces the retention time in cation exchange while
a decrease in pH reduces the retention time in anion exchange

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4. Size exclusion chromatography (SEC)

 SEC, also known as gel permeation chromatography or gel filtration

chromatography, separates particles on the basis of size.
 It is generally a low resolution chromatography and thus it is often
reserved for the final, "polishing" step of a purification.
 Uses a polymeric stationary phase with well-defined pores.
 The analyte molecules that partly penetrate the pores are retained
longer in the stationary phase.
 Molecules that are too large will all elute with the dead volume.

4. Size exclusion chromatography (SEC)

 It is also useful for determining the tertiary structure and
quaternary structure of purified proteins.
 This technique is widely used for the molecular weight
determination of polysaccharides.

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DIFFERENCES BETWEEN GC AND LC/HPLC

1. Mobile phase is liquid not gas
2. Liquid may require different detectors
3. High pressure requires different injectors

HPLC SYSTEM

HPLC components:
mobile phase
reservoirs, pump,
an injector, a
separation column,
and a detector.

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Mobile Phase
 The mobile phase in HPLC refers to the solvent being
continuously applied to the column, or stationary phase.
 The mobile phase acts as a carrier for the sample solution.
 A sample solution is injected into the mobile phase of an
assay through the injector port.
 As a sample solution flows through a column with the mobile
phase, the components of that solution migrate according to
the non-covalent interactions of the compound with the
column

Mobile Phase
 The chemical interactions of the mobile phase and sample,
with the column, determine the degree of migration and
separation of components contained in the sample.
 For example, those samples which have stronger interactions
with the mobile phase than with the stationary phase will elute
from the column faster, and thus have a shorter retention time,
while the reverse is also true.
 The mobile phase can be altered in order to manipulate the
interactions of the sample and the stationary phase.

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Mobile Phase reservoirs

 Solvents (mobile phase) – are stored in special reservoirs connected to
the pumping system – must be free of particles that can clog
components & free of bubble forming gases that get trapped in column
or detector.
 Three basic ways to degas solvents (to remove dissolved O and N)

1) vacuum or suction filter (0.4 or 0.2 μm)

2) ultrasonicate (with vacuum)
3) He purge (sparging units often built in)

Mobile Phase
There are several types of mobile phases, these include:
 Isocratic elution: An elution with a single solvent of constant composition

 This type of elution is both simple and inexpensive,

 But resolution of some compounds is questionable and elution may
not be obtained in a reasonable amount of time

 Gradient elution: Two (and sometimes more) solvent systems that differ
significantly in polarity are employed. The ratio of the two solvent are
varied in a preprogrammed way, sometimes continuously and
sometimes in a series of steps.

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Mobile Phase
Gradient elution
 In gradient elution different compounds are eluted by
increasing the strength of the organic solvent.
 The sample is injected while a weaker mobile phase is being
applied to the system.
 The strength of the mobile phase is later increased in
increments by raising the organic solvent fraction, which
subsequently results in elution of retained components.
 This is usually done in a stepwise or linear fashion.

Mobile Phase
Gradient elution
 At the onset of sample introduction, the compounds are initially
retained at the inlet of the column.
 As the solute capacity, or k', for the compound decreases, the
compound begins to migrate through the stationary phase.
 Each of the other compounds in the sample subsequently
migrate as their k' values decrease.
 Compared with isocratic elution, resolution and separation are
improved, and bandwidths are nearly equal.

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Mobile Phase

Gradient elution
dramatically improves
the efficiency of
separation

Mobile Phase
Polytyptic Mobile Phase
 Polytyptic Mobile Phase (also referred as mixed-mode
chromatography) is a versatile method in which several types
of chromatographic techniques can be employed using the
same column.
 These columns contain rigid macroporous hydrophobic resins
covalently bonded to a hydrophilic organic layer.
 SEC, IEC, hydrophobic or affinity chromatography are some of
the methods that may be utilized

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Mobile Phase
Polytyptic Mobile Phase
 By changing the mobile phase, the mode of separation is

thereby changed which allows the chromatographer to achieve

the desired selectivity in the separations.

Pumping systems
 The requirements for LC pumps are severe and include:
(1) generation of pressures of up to 6000 psi,
(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

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Pumping systems
 Two types of mechanical pumps are employed:
(1) a screw driven syringe type and

(2) a reciprocating pump.

 A screw driven type: Produces a pulse free delivery whose flow rate is

readily controlled; inconvenient when solvents must be changed

 Reciprocating pumps: more widely used; usually consists of a small

cylindrical chamber that is filled and then emptied by the back-and-

forth motion of a piston.

Pumping systems
 Reciprocating pumps: more widely used
 usually consists of a small cylindrical chamber that is filled and then emptied
by the back-and-forth motion of a piston.
 On the back stroke, the separation column valve is closed, and the piston pulls
in solvent from the mobile phase reservoir.
 On the forward stroke, the pump pushes solvent out to the column from the
reservoir.

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Pumping systems
Reciprocating pumps
 A wide range of flow rates can be attained by altering the

piston stroke volume during each cycle, or by altering the stroke

frequency.
 Dual and triple head pumps consist of identical piston-chamber

units which operate at 180 or 120o out of phase.

 This type of pump system is significantly smoother because one
pump is filling while the other is in the delivery cycle

Pumping systems
 Advantages of reciprocating pumps: include small internal volume, high
output pressures (up to 10 000 psi), ready adaptability to gradient
elution, constant flow rates

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Sample injection system (injector)

 Widely used method of
sample introduction in LC
is based upon sampling
loops.
 Integral part of modern
LC system
 Have interchangeable
loops with choice of
sample sizes ranging
from 5 to 500 µL.

Sample injection system (injector)

 Samples are injected into the HPLC via an injection port.
 The injection port of an HPLC commonly consists of an injection
valve and the sample loop.
 The sample is typically dissolved in the mobile phase before
injection into the sample loop.
 The sample is then drawn into a syringe and injected into the
loop via the injection valve.
 The syringe containing the sample is then injected into the
valve in the usual manner, and the pump is turned on.

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Stationary Phase
 The stationary phase in HPLC refers to the solid support
contained within the column over which the mobile phase
continuously flows.
 The sample solution is injected into the mobile phase of the
assay through the injector port.
 As the sample solution flows with the mobile phase through the
stationary phase, the components of that solution will migrate
according to the non-covalent interactions of the compounds
with the stationary phase.

Stationary Phase
 The chemical interactions of the stationary phase and the sample with
the mobile phase, determines the degree of migration and separation
of the components contained in the sample.
 For example, those samples which have stronger interactions with the
stationary phase than with the mobile phase will elute from the column
less quickly, and thus have a longer retention time, while the reverse is
also true.

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Stationary Phase
 Columns containing various types of stationary phases are
commercially available.
 Some of the more common stationary phases include: Liquid-Liquid,
Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse
Phase, Ion Exchange, and Affinity

Columns

 Usually constructed from stainless steel

 Range of length: 10-30 cm and have inside diameters of 4 to 10 mm.
 Column packing: particle sizes of 5 or 10 µm
 High performance micro columns with inside diameters of 1 to 4.6 mm
and lengths of 3 to 7.5 cm have become available. (Advantage:
speed and minimal solvent consumption)

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Columns
 There are various columns:
 Guard,
 Derivatizing,
 Capillary,
 Fast, and
 Preparatory Columns

Columns
Guard columns: normally used before the analytical column to
protect & increase lifetime of column – operator usually slurry or dry
packs short guard column regularly with same or similar packing used in
analytical column – can purchase guard systems, cartridges, etc

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Columns

Capillary columns
 Advances in HPLC led to smaller analytical columns.
 Also known as microcolumns, capillary columns have a diameter much less
than a millimeter and there are three types: open-tubular, partially
packed, and tightly packed.
 They allow the user to work with nanoliter sample volumes, decreased
flow rate, and decreased solvent volume usage which may lead to cost
effectiveness.
 However, most conditions and instrumentation must be miniaturized, flow
rate can be difficult to reproduce, gradient elution is not as efficient, and

Columns
Microbore and small-bore columns
 Microbore and small-bore columns are also used for analytical
and small volumes assays.
 A typical diameter for a small-bore column is 1-2 mm.
 Like capillary columns, instruments must usually be modified to
accommodate these smaller capacity columns (i.e., decreased
flow rate).
 However, besides the advantage of smaller sample and mobile
phase volume, there is a noted increase in mass sensitivity
without significant loss in resolution

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Columns
Fast columns
 One of the primary reasons for using these columns is to obtain
improved sample throughput (amount of compound per unit
time).
 For many columns, increasing the flow or migration rate through
the stationary phase will adversely affect the resolution and
separation.
 Therefore, fast columns are designed to decrease time of the
chromatographic analysis without forsaking significant
deviations in results.

Columns
Fast columns
 These columns have the same internal diameter but much shorter
length than most other columns, and
 they are packed with smaller particles that are typically 3 µm
in diameter.
 Advantages include increased sensitivity, decreased analysis
time, decreased mobile phase usage, and increased
reproducibility

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Columns
Preparatory Columns
 These columns are utilized when the objective is to prepare bulk
(milligrams) of sample for laboratory preparatory applications.
 A preparatory column usually has a large column diameter which is
designed to facilitate large volume injections into the HPLC system.
 Accessories important to mention are the back-pressure regulator
and the fraction collector.
 The back-pressure regulator is placed immediately posterior to the
HPLC detector.

Columns
Preparatory Columns
 It is designed to apply constant pressure to the detector outlet which
prevents the formation of air bubbles within the system.
 This, in turn, improves chromatographic baseline stability.

 It is usually devised to operate regardless of flow rate, mobile phase, or

viscosity.
 The fraction collector is an automated device that collects uniform
increments of the HPLC output.
 Vials are placed in the carousel and the user programs the time interval
in which the machine is to collect each fraction.
 Each vial contains mobile phase and sample fractions at the
corresponding time of elution

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Detectors
 The detector for an HPLC is the component that emits a response
due to the eluting sample compound and subsequently signals a
peak on the chromatogram.
 It is positioned immediately posterior to the stationary phase in
order to detect the compounds as they elute from the column.

Detectors
 Some of the more common detectors include:
 Refractive Index (RI),
 Ultra-Violet (UV),
 Fluorescent,
 Radiochemical,
 Electrochemical,
 Near-Infra Red (Near-IR),
 Mass Spectroscopy (MS),
 Nuclear Magnetic Resonance (NMR),

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Refractive Index (RI)

 RI detectors measure the ability of sample molecules to bend or
refract light.
 This property for each molecule or compound is called its
refractive index.
 For most RI detectors, light proceeds through a bi-modular flow-
cell to a photodetector.
 One channel of the flow-cell directs the mobile phase passing
through the column while the other directs only the mobile phase.

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Refractive Index (RI)

 Detection occurs when the light is bent due to samples eluting
from the column, and this is read as a disparity between the two
channels.

Ultra-violet (UV) detector

 UV detectors measure the ability of a sample to absorb light.
 This can be accomplished at one or several wavelengths:
 Fixed Wavelength measures at one wavelength, usually 254 nm
 Variable Wavelength measures at one wavelength at a time, but can
detect over a wide range of wavelengths
 Diode Array measures a spectrum of wavelengths simultaneously
 UV detectors have a sensitivity to approximately 10-8 or 10-9 gm/ml.

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Detectors

Detectors

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Mass Spectrometry (MS) Detector

 In MS detectors, the sample compound or molecule is ionized, it
is passed through a mass analyzer, and the ion current is
detected.

Mass Spectrometry (MS) Detector

 There are various methods for ionization:
 Electron Impact (EI)- An electron current or beam created
under high electric potential is used to ionize the sample
migrating off the column.
 Chemical Ionization- A less aggressive method which utilizes
ionized gas to remove electrons from the compounds eluting
from the column.
 Fast Atom Bombardment (FAB)- Xenon atoms are propelled at
high speed in order to ionize the eluents from the column.

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Applications of HPLC
 Preparative HPLC refers to the process of isolation and purification
of compounds.
 Important is the degree of solute purity and the throughput, which is
the amount of compound produced per unit time.
 This differs from analytical HPLC, where the focus is to obtain
information about the sample compound.
 The information that can be obtained includes identification,
quantification, and resolution of a compound.

Applications of HPLC
 Chemical Separations can be accomplished using HPLC by
utilizing the fact that certain compounds have different
migration rates given a particular column and mobile phase.
 Thus, the chromatographer can separate compounds (more on
chiral separations) from each other using HPLC;
 the extent or degree of separation is mostly determined by the
choice of stationary phase and mobile phase.

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Applications of HPLC
 Purification refers to the process of separating or extracting
the target compound from other (possibly structurally related)
compounds or contaminants.
 Each compound should have a characteristic peak under certain
chromatographic conditions.
 Depending on what needs to be separated and how closely
related the samples are, the chromatographer may choose the
conditions, such as the proper mobile phase, to allow adequate
separation in order to collect or extract the desired compound
as it elutes from the stationary phase.

Applications of HPLC
 The migration of the compounds and contaminants through the
column need to differ enough so that the pure desired
compound can be collected or extracted without incurring any
other undesired compound

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