HIGH PRESSURE

LIQUID
CHROMATOGRAPHY
(HPLC)
OBJECTIVES
1. Definition
2. Components
3. Materials used to achieve separation
4. How HPLC works
5. Factors affecting performance
6. Applications
7. Advantages and disadvantages
DEFINITION
 Is a form of liquid chromatography used to
separate compounds dissolved in solution.
 The analyte is forced through a column by a
liquid at high pressure.
 Small column size, small beads inside column
and high pressures are used.
COMPONENTS
1. Reservoir of the mobile phase
2. Degasser
3. A pumping system
4. An injector
5. A chromatographic(separation) column
6. Stationary phase
7. Connecting tubings and fittings
8. Detector
9. Data collection device
 Degasser
Solvents are degassed by sparging with Helium
or sonification before pumping to avoid
formation of bubbles in the detector cell.
 The pump

Delivers metered amounts of the mobile phase

at a constant flow rate.
Provides a steady high pressure that moves the
mobile phase and analyte through the
column.
 Injector
It introduces the sample solution into the mobile phase at or near the
head of the column at high pressure.
Can be a fixed loop or variable volume device.
 Separation column
Columns are usually made of
polished stainless steel, 10-30cm long, internal diameter of 2-5mm.
They are filled with a stationary phase with a particle size of 5 - 10μm.
Columns with internal diameters of less than 2 mm are often referred to
as microbore columns. Ideally, the temperature of the mobile phase and
the column should be kept constant during an analysis. Most separations
are performed at ambient temperature but columns may be heated in
order to achieve better efficiency. Normally, columns should not be
heated above 60 °C because of the potential for stationary phase
degradation or changes occurring to the composition of the mobile
phase.
 Connecting tubings and fittings
They should be capable of withstanding the
high pressures developed within the system.
 Detector

Provides a characteristic retention time for the

analyte. Retention time is the time at which
a specific sample elutes.
MATERIALS USED

 MOBILE PHASE
Choice depends on desired retention behaviour
and physicochemical properties of analyte.
Solvents that can be used: acetonitrile, methanol,
acetic acid, isopropanol, THF and water.
The solvents can be mixed together eg 10% water
and 90% ethanol.
Water may contain buffers as salts. They act as
ion pairing agents and assist in separation of
sample components.
Non polar solvents: hexane, silica, toluene.
 STATIONARY PHASE
Can be of different types.
Silica treated with RMe2SiCl is most commonly used.
-Unmodified silica, alumina, or porous
graphite, used in normal-phase chromatography,
where separation is based on differences in
adsorption;
- a variety of chemically modified supports prepared
from polymers, silica, or porous graphite, used in
reverse-phase HPLC, where separation is based
principally on partition of the molecules between
the mobile phase and the stationary phase;
HOW IT WORKS
 The liquid is introduced into a sample loop of
injector with a syringe. The syringe is cleaned
with sample soln before injecting into hplc.
 When loop is filled, the injector can introduce
the sample in small volumes to the stream of
mp then the mixture is introduced to the
densely packed column by liquid at high
pressure provided by the pump.
 The solutions movement through the column is
slowed by chemical or physical interactions with
the sp. velocity of movement is determined by
nature of sample and composition of sp.
 The different components in the mixture
pass thru the column at different rates due
to the difference in their partitioning
behavior between the mp and sp.
FLOW DIAGRAM OF HPLC
TYPES OF HPLC

Depends on polarity of mp and sp.

 NORMAL PHASE HPLC:

The sp is more polar than mp.

MP solvents-hexane, toluene, silica.
Is used for separating analytes readily soluble in
non polar solvents. the analyte associates with
and is retained by the SP. Using more polar
solvents in MP reduces retention time and
hydrophobic solvents do the vv.
Applied in separation of structural isomers since
interaction strength depends on the functional
groups and steric factors.
 REVERSE PHASE HPLC
Is the more common type. Non polar SP and aqueous
moderately polar MP.
SP-silica treated with Rme2SiCl…R is a straight chain alkyl
group.
MP-water/methanol mixture.
Hydrophobic analytes tend to be retained in column and
polar ones elute >readily.
Adding >water to MP incs retention time by making affinity
of the hydrophobic analyte for the hydrophobic SP
stronger, relative to a now more hydrophilic MP; while
adding organic solvents eg methanol, acetonitrile to the
eluent decs RT.
Is used to qualify drugs before their release.
FACTORS AFFECTING
PERFORMANCE
 Internal diameter of hplc column:
It influences:- the detection sensitivity and
separation selectivity in gradient elution.
-the quantity of analyte that can be
loaded on the column.
 Particle size:

Most hplc are performed with sp attached to the

outside of very small silica beads.5micrometers is
the most common bead size.
The smaller the particles the better the separation.
Large particles are used in preparative hplc.
 Pore size
Sp are porous to provide larger surface area.
Smaller pores have a greater surface area
while larger pores have better kinetics esp
for larger analytes.eg proteins
 Pump pressure

Pumps vary in pressure capacity and their

performance is measured in their ability to
yield a consistent and reproducible flow
rate. Pressures in modern hplc could go as
high as 1000atms.
APPLICATIONS
 Molecular biology analysis of DNA- can separate DNA that
differs by as little as one base pair.
 Assay and purifications of biological molecules- used in
separation of oligopeptides and proteins.
 Biochemistry and analytical chemistry to identify, quantify
and purify individual components of a mixture.
 In clinical diagnosis of diseases and disorders eg disorders of
endocrine/exocrine glands are diagnosed by hplc analysis of
the concerned fluids.
 In scientific research and analysis to analyze and quantify
molecules.
 In pharmaceutical and food industry for quality control. The
products are analyzed to determine if they are in
compliance with the pharmacopoeia and other
specifications.
ADVANTAGES
 Speed-it is a faster method of analysis since a
high pressure pump is used to propel the mp
instead of gravity.
 Wide applicability- used in virtually all biological
molecules esp oligopeptides and proteins.
 Higher selectivity-high pressure of mp reduces
time components remain on sp thus they spread
out within column leading to narrower and more
selective peaks.
 High reproducibility.
 High resolution-columns are tightly packed and
of shorter length.
 High sensitivity- separation of constituents is
more complete. tall narrow peaks obtained
are easier to discriminate.
 Permits both instrumentation and
quantification to be automated.
 The stationary columns can be reused
without requiring regeneration.
DISADVANTAGES
 Equipment is very costly.
 Low sensitivity for some compounds due to
high velocity of mp.
 Operation is complex and requires trained
personnel.
 Co-elution(2 compounds escaping from
tubing at once) is difficult to detect hence
inaccuracy.
 Irreversibly adsorbed compounds are not
detected.