• Mobile Phase: Liquid

• Stationary Phase Separation

Mechanism
High- - Solid Adsorption
Performance - Liquid Layer Partition
Liquid - Ion exchange resin Ion exchange
Chromatography - Microporous beads Size
Exclusion
- Chemically modified resin Affinity
 Not limited by sample volatility or
thermal stability

HPLC Two interacting phases

Advantage
s vs GC Room temperature analysis

Ease of sample recovery

Instrumentation

Solvent Sample
Pump
Reservoirs Injector

Data
Column(s) Detector
System
Mobile Phase Reservoirs
Inert container with inert lines leading to the pump are required.

Reservoir filters (2-10 mm) at reservoir end of solvent delivery lines

- Vacuum filtration
- Sparge with inert gas
Degassed solvent (N2 or He)
- Ultrasonic under vacuum

Elevate above pumps

Isocratic elution: A separation that employs a single solvent or solvent
mixture of constant composition.

Gradient elution: Here two or more solvent systems that differ

significantly in polarity are employed. After elution is begun; the ratio
of the solvents is varied in a programmed way, sometimes
continuously and sometimes in a series of steps. Separation efficiency
is greatly enhanced by gradient elution.
 • Constructed of materials inert toward
solvents to be used
• Deliver high volumes (flow rates) of solvent
(to 10 mL/min)
HPLC • Deliver precise and accurate flow (<0.5%
Pump variation)
Criteria • Deliver high pressure (to 6000 psi)
• Deliver pulse free flow
• Have low pump-head volume
• Be reliable
 Reciprocating pumps

HPLC Pumps: Types Syringe pumps

Constant pressure
pumps
Reciprocating Pumps
• One, two, or three pump heads
- more heads, less pulse
• Small head volumes (50 to 250 mL)
• Short piston stroke
• Inert pistons (generally sapphire)
• Continuous use (no refill time)
• Pulse dampeners
 Constant flow rate pump

Non-pulsating flow
Syringe
Low flow rates (1 to 100 mL/min)
Pumps
Isocratic flow only

Refill required when reservoir (~50mL) expended

 Constant pressure pump, not constant flow

Constant Can deliver high pressures

Pressure Stable flow during delivery stroke
Pump Stop flow on refill stroke

Low cost
Sample Introduction

• Valve-type injectors
- Six port fixed volume Rheodyne
reproducible injection volumes
variable loop size
easy to use, reliable
- Six port variable volume Waters
variable injection volumes without loop change increased
maintenance, operator skill required more expensive
 Continuous injections operator free

Comparable precision and accuracy to

Auto manual

Injectors Much more expensive initially

Much more convenient Up 100 samples

and standards with microprocessor
control
Liquid-Chromatographic Columns
Liquid-chromatographic columns are ordinarily
constructed from smooth-bore stainless steel
tubing, although heavy-walled glass tubing is
occasionally encountered. The latter is
restricted to pressures that are lower than
about 600 psi.
Analytical Columns

Liquid-chromatographic columns range in length from 10 to 30

cm. Normally, the columns are straight, with added length, where
needed, being gained by coupling two or more columns together.
The inside diameter of liquid columns is often 4 to 10 mm; the
most common particle size of packings is 5 or 10 m. The most
common column currently in use is one that is 25 cm in length,
4.6 mm inside diameter, and packed with 5 m particles. Columns
of this type contain 40,000 to 60,000 plates/meter.
Guard Columns

A guard column is introduced before the analytical column to increase the life
of the analytical column by removing not only particulate matter and
contaminants from the solvents but also sample components that bind
irreversibly to the stationary phase. The guard column serves to saturate
the mobile phase with the stationary phase so that losses of this solvent
from the analytical column are minimized. The composition of the guard-
column packing is similar to that of the analytical column; the particle size
is usually larger. When the guard column has become contaminated, it is
repacked or discarded and replaced with a new one.
Detectors: Unlike gas chromatography, liquid chromatography has
no detectors that are as universally applicable and as reliable as
the flame ionization and thermal conductivity detectors. A major
challenge in the development of liquid chromatography has been
in detector improvement.
• Types of Detectors: Liquid chromatographic detectors are of two
basic types. Bulk property detectors respond to a mobile-phase
bulk property, such as refractive index, dielectric constant, or
density.
• In contrast, 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.
• Absorbance Detectors: Is a Z-shaped, flow-through cell for
absorbance measurements on eluents from a chromatographic
column. Many absorbance detectors are double-beam devices in
which one beam passes through the eluent cell and the other
through a filter to reduce its intensity.
• Ultraviolet Absorbance Detectors with Filters: The simplest UV
absorption detectors are filter photometers with a mercury lamp
as the source. Most commonly the intense line at 254 nm is
isolated by filters. Deuterium or tungsten filament sources with
interference filters also provide a simple means of detecting
absorbing species.
• UV Absorbance Detector with Monochromator: There are
detectors that consist of a scanning spectrophotometer with
grating optics. Some are limited to uv radiation; others
encompass both uv and visible radiation. The most powerful uv
spectrophotometric detectors are diode-array instruments.
• Infrared Absorbance Detectors: Two types of infrared detectors
are offered commercially. The range of the first instrument is
from 2.5 to 14.5 m or 4000 to 690 cm-1. The second type of
infrared detector is based upon Fourier transform instruments.
• Fluorescence Detectors: Fluorescence is observed by a
photoelectric detector located at 90 deg to the excitation beam.
The simplest detectors employ a mercury excitation source and
one or more filters to isolate a band of emitted radiation. More
sophisticated instruments are based upon a Xenon source and
employ a grating monochromator to isolate the fluorescent
radiation. An inherent advantage of fluorescence methods is
their high sensitivity, which is typically greater by more than an
order of magnitude than most absorbance procedures.
• Refractive-Index Detectors: Refractive-index detectors have the
significant advantage of responding to nearly all solutes. That is
they are general detectors analogous to flame or thermal
conductivity detectors in gas chromatography. In addition, they are
reliable and unaffected by flow rate. They are, however, highly
temperature sensitive and must be maintained at a constant
temperature to a few thousandths of a degree centigrade.
Furthermore, they are not as sensitive as most other types of
detectors.
• Electrochemical Detectors: These devices are based upon
amperometry, polarography, coulometry, and conductometry.
They appear to offer advantages, in many instances, of high
sensitivity, simplicity, convenience, and widespread applicability.
• Mass Spectrometric Detectors: A problem in coupling liquid chromatography with
mass spectrometry is the enormous mismatch between the relatively large solvent
volumes and the vacuum requirements. Several interfaces have been developed for
solving this problem. In a first type the eluent from the column is split, with only a
tiny fraction being introduced directly into the mass spectrometer. In a second type
of interface the effluent is deposited on a continuous, moving-belt or moving-wire
that transports the solvent and analyte to a heated chamber for removal of the
former by volatilization.
Partition Chromatography
Partition chromatography can be subdivided into
(i) liquid-liquid chromatography and
(ii) bonded-phase chromatography.

• With liquid-liquid, a liquid stationary phase is retained on the

surface of the packing by physical adsorption.
• With bonded-phase, the stationary phase is bonded chemically
to the support surfaces.
 Early partition chromatography was the liquid-liquid type; now the
bonded-phase method has become predominate because of certain
disadvantages of liquid-liquid systems.
• One of these disadvantages is the loss of stationary phase by
dissolution in the mobile phase, which requires periodic recoating of
the support particles.
• Furthermore, stationary-phase solubility problems prohibit the use
of liquid-phase packings for gradient elution.
Columns for Bonded-Phase Chromatography

The supports for the majority of bonded-phase packings for partition

chromatography are prepared from rigid silica, or silica-based,
compositions. These solids are formed as uniform, porous,
mechanically sturdy particles commonly having diameters of 3, 5, or
10m. The surface of fully hydrolyzed silica is made up of chemically
reactive silanol groups. The most useful bonded-phase coatings are
siloxanes formed by reaction of the hydrolyzed surface with an
organochlorosilane.
Reversed-Phase and Normal-Phase Packings
• Two types of partition chromatography are distinguishable based upon
the relative polarities of the mobile and stationary phases.

• Liquid chromatography based upon highly polar stationary phases such

as water or triethyleneglycol supported on silica or alumina particles; a
relatively nonpolar solvent such as hexane or I-propylether then
served as the mobile phase. This type of chromatography is referred to
as normal-phase chromatography.
In reversed-phase chromatography, the stationary phase is nonpolar,
often a hydrocarbon, and the mobile phase is relatively polar (such as
water, methanol, or acetonitrile).
In normal-phase chromatography, the least polar component is
eluted first because it is the most soluble in the mobile phase;
increasing the polarity of the mobile phase has the effect of
decreasing the elution time.
In contrast, in the reversed-phase method, the most polar
component appears first, and increasing the mobile phase polarity
increases the elution time.
HPLC Derivatization Methods
• Why derivatize?
• Enhance detector response
• Improve analyte resolution
• Improve analyte peak shape
• Improve analyte sensitivity
• Establish analyte identity
• Improve analyte stability during analysis
• Change analyte physical properties
Preparative Chromatography
• Separation and isolation of relatively large quantities (> 0.1g) of solute
• Similar systems to analytical chromatography
- higher flow rates (10 to 200 mL/min)
- Large sample loops
- preparative columns
- larger dimensions and packing size
- detection not critical
- not trace analysis
- but non-destructive
Chromatographic Separations
• Open column chromatography
- Very slow analysis
- Poor chromatographic efficiency
- Inconvenient sample recovery
• HPLC
- More rapid analysis
- High efficiency packing materials
- continuous flow-through detection
Gel Permeation LC
• Also known as size exclusion or gel filtration
• Separation by effective size in solution
- dependent on molecular size and shape
- molecules large enough to be excluded from pores all co-
elute at tm
- smaller molecules permeate the pores to differing degrees
based on relative size and are proportionally retarded.
GPLC Stationary Phases
• Stationary phase controls retention or selection
• Two types: rigid cross-linked polymer gels and other
polymer gels
• Classification by pore size, or range of pore sizes
• Small molecules - pores 60 to 100Å
• Mixed bed columns for wide size ranges
 GPLC Mobile Phases
• Mobile phase not generally used to control
retention or selection
• Selection primarily based on solubility
- low viscosity solvent
- detector compatible
- column compatible
polstryrene - less polar solvents
silica gel - water, wide range of
solvents, limited pH
 Ion Exchange Chromatography

• Reversible exchange of ionic species between the

stationary phase and mobile phase
• Ionic species chemically bound to insoluble matrix
serves as exchange site (adsorption)
IEC Separations
• Insoluble matrix (M+) and counter ion (E-) as
stationary phase
• Analyte ion (A-) in mobile phase

M+E- + A- M+A- + E-
 IEC Stationary Phases
• Silica based materials
- Pellicular particles
• Organic materials
- porous beads
- styrene/divinylbenzene crosslinked co-
polymers
- methacrylic acid/divinylbenzene
crosslinked co-polymers
• Inorganic materials
IEC Stationary Phases

• Functional group addition through surface

reactions with appropriate reagents to produce
the desired cation or anion exchange resin
• Cation exchangers (strong & weak)
- Acid functional groups
• Anion exchangers (strong & weak)
- Basic functional groups
 Resin Cross-linkage

• Pore size a degree of crosslinkage

• High degrees of Crosslinkage:
- increase mechanical strength of the resin
- decrease the degree of swelling
- decrease the permeability of the resin
IEC Mobile Phases

• Aqueous solutions of salt or salts with a small % of

organic modifier and perhaps a buffer
Retention & Selectivity Factors

• Size and shape of the solvated molecule

• Mobile phase pH
• Concentration & type of competing ion
• Addition of organic solvent to mobile phase
• Column temperature
Cation M-A+ Bond Strengths

• Ce3+ > Al3+ >> Ba2+ > Pb2+ > Ca2+ >
Ni2+ > Cu2+ > Mg2+ > UO22+ >> Tl+ >
Ag+ > K+ > NH4+ > H+
Anion M+A- Bond Strengths

• Citrate > SCN- > CrO42- > SO42- > NO3- >
Br- > CN- > Cl- > HCO3- > HCOO- > OH-

Anion selectivities are not as rigidly

defined as those of cations
Prediction of MxAy Bond Strengths
• Factors:
- charge on the solute ion
- size of the solvated ion
- degree of resin crosslinkage
- polarizability of the solute molecule
- ion-exchange capacity of the resin
- functional group on the resin
- degree of interaction between the
solute and the resin
Ion Chromatography

• Separation of inorganic cations and anions and low

molecular weight water-soluble organic acids and
bases
• Non-suppressed IC methods
• Suppressed IC methods
Ion Exclusion Chromatography

• Separation of ionic solutes from weakly ionized of

neutral solutes using strong cation and anion
exchange resins
 Factors Affecting Retention in IC
• Degree of ionization of the solute
- solute pKa
- eluent pH
- organic modifier content
• hydrophobic interactions between solute and sp
• molecular size of the solute
• degree of X-linkage of the sp
• system temperature
Affinity Chromatography
• Separation of proteins and biomolecules through
the use of a biologically specific immobilized ligand
(sp).
• Biologically specific ligand is chemically attached to
a gel matrix
• pH of the system is controlled to control the
strength of the interaction between the ligand and
the solute molecule
Complexation Chromatography

• Separation based on the rapid reversible formation

of a complex between metal ions and ligands
• Suitable ligands are immobilized on sp
 Separation based on the use of chiral
stationary phases which interact to varing
degrees with optical isomers of the solute

Chiral Systems differ little from conventional

Chromatography HPLC systems

Limited applications for a given sp