High Performance
Liquid Chromatography
Chem. 331
Introduction
HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile phase,
a pump, an injector, a separation column, and a detector.
Compounds are separated by injecting a sample mixture
onto the column. The different component in the mixture
pass through the column at differentiates due to
differences in their partition behavior between the mobile
phase and the stationary phase. The mobile phase must
be degassed to eliminate the formation of air bubbles.
HPLC system
FOUR TYPES OF LIQUID
CHROMATOGRAPHY
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel, chromatography
COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)
Picture of HPLC instrument
Uses of HPLC
This technique is used for chemistry and biochemistry
research analyzing complex mixtures, purifying chemical
compounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the purity
of raw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate product
stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational safety
or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use HPLC
to survey food and drug products, for identifying confiscated
narcotics or to check for adherence to label claims.
HPLC Chromatograph injectors
The function of the injector is to place the sample into
the high-pressure flow in as narrow volume as possible
so that the sample enters the column as a
homogeneous, low-volume plug. To minimize spreading
of the injected volume during transport to the column, the
shortest possible length of tubing should be used from
the injector to the column.
When an injection is started, an air actuator rotates the
valve: solvent goes directly to the column; and the
injector needle is connected to the syringe. The air
pressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is lowered
to the vial.
HPLC columns
The column is one of the
most important components
of the HPLC chromatograph
because the separation of
the sample components is
achieved when those
components pass through
the column. The High
performance liquid
chromatography apparatus
is made out of stainless
steel tubes with a diameter
of 3 to 5mm and a length
ranging from 10 to 30cm.
Normally, columns are filled
with silica gel because its
particle shape, surface
properties, and pore structure
help to get a good separation.
Silica is wetted by nearly every
potential mobile phase, is inert
to most compounds and has a
high surface activity which can
be modified easily with water
and other agents. Silica can be
used to separate a wide variety
of chemical compounds, and
its chromatographic behavior is
generally predictable and
reproducible.
Picture of an HPLC column
WHAT AFFECTS SYSTEM
Column Parameters
Instrument Parameters
Column Material
Deactivation
Stationary Phase
Coating Material
Temperature
Flow
Signal
Sample Sensitivity
Detector
WHAT AFFECTS SYSTEM
Sample Parameters
Concentration
Matrix
Solvent Effect
Sample Effect
Several column types
(can be classified as )
Normal
phase
Reverse
Size
Ion
phase
exclusion
exchange
Normal phase
In
this column type, the retention is
governed by the interaction of the polar
parts of the stationary phase and solute.
For retention to occur in normal phase, the
packing must be more polar than the
mobile phase with respect to the sample
Reverse phase
In
this column the packing material is relatively
nonpolar and the solvent is polar with respect to
the sample. Retention is the result of the
interaction of the nonpolar components of the
solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.
Size exclusion
In
size exclusion the HPLC column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according to
its molecular size. Small molecules
penetrate into the pores within the packing
while larger molecules only partially
penetrate the pores. The large molecules
elute before the smaller molecules.
Ion exchange
In
this column type the sample components
are separated based upon attractive ionic
forces between molecules carrying charged
groups of opposite charge to those charges
on the stationary phase. Separations are
made between a polar mobile liquid, usually
water containing salts or small amounts of
alcohols, and a stationary phase containing
either acidic or basic fixed sites.
Selectivity Factor
K values
tell us where bands elute relative
to the void volume. These values are
unaffected by such variables as flow rate
and column dimensions. The value tell us
where two peaks elute relative to each
other. This is referred to as the selectivity
factor or separation factor (now and then
as the chemistry factor).
Types of Liquid Column
Chromatography
(LCC)
LLC (Liquid Liquid)
LSC (Liquid Solid adsorption)
SEC (Size Exclusion)
GLC
GSC
SFC (Supercritical
Fluid)
Types of Detectors
Absorbance (UV
with Filters, UV with
Monochromators)
Evaporative Light
Scattering Detector
(ELSD)
Electrochemical
IR Absorbance
Fluorescence
MassSpectrometric
Refractive-Index
Photo-Diode Array
EVALUATION PARAMETERS
EFFICIENCY
RESOLUTION
INERTNESS
RETENTION INDEX
COLUMN BLEED
CAPACITY FACTOR
References
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Skoog, Holler, and Neiman. Principles of Instrumental Analysis.
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