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CHAPTER 6:
HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
NOR AKMALAZURA JANI
LEARNING OUTCOMES
After completing this chapter,
students should be able to:
- explain the concepts of mobile phase
and its connection with samples
polarity
- State the component in high
performances liquid chromatography
(HPLC)
- State the functions of each
component in high performances
3/2/17 copyright 2006 www.brainybetty.com 2
Subtopics
Scope of liquid chromatography
LC instrumentation:
- Mobile phase reservoirs and solvent treatment
system
- Pumping systems
- Sample injection systems
- LC columns
- Detectors
Type of HPLC:
- partition chromatography
- ion-exchange chromatography
- size-exclusion chromatography
SCOPEOF
SCOPE OFLIQUID
LIQUIDCHROMATOGRAPHY
CHROMATOGRAPHY
Guard
column
Analytical
column
Solvent reservoir
Isocratic elution
A single solvent or solvent mixture of
constant composition. Example, 50:50
(v/v) methanol-water
Gradient elution
Two (and sometimes more) solvent
systems that differ significantly in
polarity and varied in composition.
Example: 40:60 methanol-water, the
methanol concentration was the
increased at the rate of 8%/min.
Gradient elution shortened the time of
separation without sacrificing the
resolution of the early peaks
Properties of Common Chromatographic Mobile Phases
Pumping systems
Used to force the mobile phase to flow
through the packing.
Requirements for LC pumps:
i) The generation of pressures of up to 6000
psi (lb/in2) or 414 bar.
ii) Pulse-free output.
iii)Flow rates: 0.1-10 mL/min.
iv) Flow reproducibility of 0.5% relative.
v) Resistance to corrosion by a variety of
solvents.
) Types of pumps:
i) Reciprocating pumps (most widely used)
ii) Displacement pumps
Reciprocating Pumps HPLC pumps
for HPLC
Sample-Injection Systems
Sample are introduced via a manual injector or
autosampler.
Syringe injection through a self-sealing elastomeric
septum:
- Earliest and simple mean of sample introduction.
- Microsyringes designed to withstand pressure up to 1500
psi.
Sampling loop
Most widely used method of sample introduction.
Sample size 5 to 500 l.
Permit introduction of sample at pressure up to
7000 psi.
Microsample injection valves also available
(sampling volumes of 0.5 to 5 l).
o Manual injector:
- the knob is manually operated to deliver
the sample to the column.
- Step 1: The knob is set at the "LOAD"
position for sample injection, as shown in
the left image. Using a microsyringe, the
sample can be injected into the sample
loop, which is separated from the flow
path.
- Step 2: The knob is turned to the
"INJECT" position. The eluent travels
through the loop from the pump then
delivers the sample to the column.
HPLC sample injection valves
(sampling loop)
Sampling loop
Autosampler
Liquid Chromatography Columns
LC columns
Analytical Pellicular
columns particle
Guard Porous
columns particle
Types of LC Columns
Analytical column
Column body
Guard column
Types of Column Packing
Pellicular particles
Type of
HPLC
Normal Gel
Anionic
phase filtration
Gel
Reversed Cationic permeati
phase on
Partition Chromatography
Stationary phase is second liquid that is immiscible with
the liquid mobile phase.
Most widely used type of liquid chromatography.
Applications to non-ionic, non-polar and polar compounds
of low to moderate molecular weight (usually < 3000).
Two types:
1. Liquid-liquid chromatography:
liquid stationary phase is retained on the surface of
packing by physical adsorption.
2. Bonded-phase chromatography:
the stationary phase is bonded chemically to the
support group, resulting in highly stable packings which
insoluble in the mobile phase.
Earlier partition chromatography was
exclusively liquid-liquid type.
Stationary phase
Solute polarities: A
>B>C
*** Increasing the polarity of
the mobile phase, elution time
will decreased
Reversed-Phase Chromatography
Stationary phase:
- Non Polar (hydrocarbon)
- most common bonded-phase are n-octyldecyl
(C18), or n-decyl (C8) chains, or phenyl groups.
Mobile phase:
- Polar solvent (e.g. methanol, acetonitrile, water,
or tetrahydrofuran or mixture of water with one
of the organic solvents).
- The organic solvent is called the modifier.
- Water content is varied for adjusting the polarity.
- Methanol is used for acidic compounds.
- Acetonitrile is used for basic compounds.
- Tetrahydrofuran for compounds with large dipoles.
Solute polarities: A
>B>C
*** Increasing mobile
phase polarity will
increased the elution
time
Normal-phase
chromatography
Reversed-phase
chromatography
Packings:
- Consists of small (~10 m) silica or polymer
particles containing a network of uniform
pores into which solutes and solvent can
diffuse.
Exclusion limit:
1. The MW of a species beyond which
no retention occurs.
2. All species having greater molecular
weight than the exclusion limit are so
large that they are not retained and
elute together to give a peak.
Size exclusion separations are different from
other chromatographic procedures in the
respect that no chemical or physical
interactions between analytes and stationary
phase are involved, such interaction will lead
to impaired column efficiency.
Column packings
Two types of packing for size-exclusion
chromatography:
i) Polymer beads
ii) Silica-based particles
Advantages:
- greater rigidity, which leads to easier
packing and permits the use of higher pressures.
- greater stability, which permits the use of a
wider range of solvents, including water.
- more rapid equilibration with new solvents.
- stability at higher temperatures.
Disadvantages:
- tendency to retain solutes by adsorption.
- have potential for catalyzing the degradation
of solute molecules.
Types of Size Exclusion Chromatography