3.1 INTRODUCTION
HPLC was derived from classical column chromatography and has found an
important place in analytical techniques71. The major advancement in HPLC
was found by the use of efficient separators. These separators used small
particles and high pumping pressures.
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HPLC
K d × Vs Cs × Vm tms − tm t s
k'= = = =
Vm Cm × Vs tm tm
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HPLC
Small values of k’ show that the compound is poorly retained and elutes near
the void volume. Large k’ values imply a good separation but downfalls of this
are longer analysis times with peak broadening and decreases in sensitivity.
Ideal separations occur with a capacity factor of between 1 and 5.
3.2.2 Resolution
tms1 and tms 2 : Gross retention times for peaks 1 and 2 respectively
w1 and w2: Peak widths along the baseline of peak 1 and 2 respectively
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HPLC
L
N=
H
2
⎛t ⎞
N = 16⎜ ms ⎟
⎝W ⎠
tms 2 − tm
α=
tms1 − tm
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HPLC
Cs and Cm: Molar concentrations of the solute in the stationary and mobile
phases respectively
3.2.6 Factors Affecting Band Broadening74;75
Various factors affect the peak variance, σ2, and thus the bandwidth. It is
important to consider these processes in the correct design of any
chromatographic system so that the variance and thus peak width can be
minimised and the efficiency can be maximised. The extent of band
broadening can be expressed in terms of plate height as follows:
B
H = A+ + Cstationary u + Cmobile u
u
a. Eddy Diffusion
The A term in the above equation is called eddy diffusion. This term describes
the multitude of pathways that the solute molecules can follow in a packed
column. Each of the paths is a different length and molecules will pass
through at various rates thus leading to band broadening. Eddy diffusion can
be minimised with a column that is uniformly packed with particles of constant
size. Band broadening is independent of the flow rate of the eluent.
b. Longitudinal Diffusion
Molecules in a sample band tend to diffuse out of this band during it passage
through the chromatographic column. This process is known as longitudinal
diffusion and is the B term in the above equation. Longitudinal diffusion occurs
in both the direction of flow of the mobile phase as well as in the opposite
direction. Longitudinal diffusion increases at low eluent flow rates, as diffusion
is a time dependant process.
c. Mass Transfer
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HPLC
Chromatography can be divided into three subsections namely gas, gel and
liquid chromatography (Figure 3.2). Gas chromatography is used for the
analysis of volatile samples, gel chromatography for non-volatile samples with
a molecular weight larger than 2000 and liquid chromatography for non-
volatile samples with a molecular weight smaller than 2000.
CHROMATOGRAPHY
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HPLC
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HPLC
In this technique the sample migrates through the column under the influence
of gravity, individual fractions are then collected and analysed.
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HPLC
In this technique the analyte ions are separated on a low capacity ion
exchange column, followed by a micromembrane suppressor, which removes
the ionic background to facilitate the conductometric detection of the analyte
ions. The stationary phases used in these columns are discussed later in
section 3.5.1.
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HPLC
The requirements for HPLC pumps are as follows: they must be able to
generate high pressures, have a pulse-free output, deliver flow rates ranging
from 0.1 to 10 ml/min, have flow reproducibility’s of 0.5 % relative or better
and they must be resistant to corrosion by a variety of solvents. Various types
of pumping systems exist. These include:
b. Syringe-type Pumps
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HPLC
reliable although they are expensive, solvent changing is tedious and they
have a finite capacity (~250 ml).
Pneumatic intensifier pumps are operated via gas pressure. A large area
piston drives a small area piston when acted on by pressure from a gas line.
The gas pressure is thus amplified in the ratio of the areas of the forces of the
pistons and a high-pressure liquid at constant pressure is introduced into the
system (Figure3.5). If a partial blockage occurs in this system a drop in flow
rate occurs but the pressure remains constant. The flow sensitivity of the
detector cell will determine how much pulse damping is required in the system
to suppress the detector signal caused when the flow stops during the return
stroke.
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HPLC
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HPLC
The ideal method for sample introduction should enable the sample to be
injected as a narrow plug onto the column so that peak broadening is
negligible. The injection system should contain no void volume, as this would
cause a loss of resolution.
The most widely used methods are those based on sampling valves and
loops. Here the sample loop is filled with sample by means of a syringe. A
rotation of the valve rotor causes the eluent stream to pass through the
sample loop thus injecting the sample onto the column without a noticeable
change in flow (Figure 3.7). These valves have interchangeable loops and
reproducibility is a few tenths of a percent relative.
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HPLC
Figure 3.7: Flow paths of the load and inject positions of an injection valve76
In stopped-flow injection, the eluent flow is stopped and the sample is injected
directly onto the head of the column by means of a syringe. The pump is then
switched on again.
Heavy-wall glass, stainless steel and plastic are among materials that can
withstand high pressures and are thus used to construct HPLC columns. They
must also be able to resist the chemical action of the mobile phase. Wall
irregularities will cause a well-packed column to channel near the wall or
packing interface thus the tubing must have a smooth, precision bore internal
diameter. Channels would cause peak broadening and a decrease in
efficiency.
Column connections are made with low dead-volume fittings, which prevent
stagnant pockets of eluent.
Usually a short guard column is placed in front of the analytical column. This
serves to increase the life of the analytical column by removing particulate
matter and contaminants from the solvents.
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HPLC
Various stationary phases are available for HPLC and are discussed below.
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HPLC
Latex Particle
The length of the diffusion path as well as the rate of diffusion is determined
by the particle size of the latex material.
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HPLC
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HPLC
Chelating resins, which are able to separate metal ions, are made up of a
suitable ligand immobilized onto a stationary phase. Many chelating resins
have been synthesised using styrene-divinylbenzene polymers or silica as the
support material. The ligands are chemically bound to the stationary phase by
an appropriate reaction. Solute retention is altered by manipulating the eluent
pH or by adding a competing ligand to the eluent. The success of using
chelating resins is dependant on the rate at which the metal-ligand complex is
formed and dissociated. Broad peaks are characteristic of slow formation and
dissociation rates.
There is no one highly sensitive, universal detector system used for HPLC.
The system used is thus based on the requirements which need to be met
such as detection limits, expense etc. A summary of detection methods, which
are used with HPLC separation, can be seen in Figure 3.10 and some
methods are discussed below.
Detection Methods
ELECTROCHEMICAL SPECTROSCOPIC
Fluorescence
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HPLC
a. Conductivity Detection
ΔG =
(λ s−
− λε − )Cs
10 − 3 K
anions respectively
Cs: Concentration of the analyte anion
K: Cell constant
The above equation shows that when conductivity detection is used to monitor
the effluent from an anion-exchange column the observed signal for an eluted
solute is proportional to the solute concentration as well as the difference in
the limiting equivalent ionic conductances between the eluent and solute ions.
A similar equation can be derived for the conductimetric detection of a cation-
exchange separation. It can be seen from the above equation that sensitive
detection is possible as long as there is a considerable difference in the
limiting equivalent ionic conductances of the solute and eluent ions. The
resulting difference can be positive or negative depending on whether the
eluent ion is weakly or strongly conducting resulting in direct or indirect
detection respectively.
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HPLC
b. Amperometric Detection
c. Potentiometric Detection
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HPLC
i. UV Detectors
The refractive index of a medium is the ratio of the speed of light in a vacuum
to the speed in the medium. These detectors measure the change in refractive
index in the eluent as the solute passes through the sample cell. This method
of detection is less sensitive than UV detection although non-chromatographic
compounds can be measured directly without derivitisation.
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HPLC
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