Dr.

Habib Nasir

▪ Chapter 31
▪ Fundamentals of Analytical
Chemistry
9th Edition
Douglas A. Skoog, Donald M. West, F. James,
Holler, Stanley R. Crouch
Dr. Habib Nasir
School of Natural Sciences
National University of Sciences and Technology 1 2

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▪ History ▪ Russian botanist Tswett is

▪ Chromatographic separation generally referred to as the
▪ General description of chromatography father of chromatography.
▪ Classifying chromatographic methods ▪ His work was published in
1906.
▪ Chromatogram
▪ Separated plant pigments
▪ Relative migration rates of solutes
by column containing
▪ Distribution constant calcium carbonate
▪ Retention time

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❑ Tswett coined the term ▪ Chromatography is a widely used

chromatography. method for the separation,
identification, and determination of
❑ Chromatography means the chemical components in
"color writing“ complex mixtures.
▪ No other separation method is as
powerful and generally applicable
as chromatography.

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➢ The basic principle is based on the

concentration equilibrium of the ▪ Components of a mixture are carried
components of interest, between two through the stationary phase by the flow
immiscible phases. of the mobile phase, and
➢ The phases are chosen such that ▪ separations are based on differences in
components of the sample have differing migration rates among the mobile-phase
solubilities in each phase. components.
➢ The differential migration of compounds
leads to their separation.

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▪ The stationary phase in chromatography

is a phase that is fixed either in a column
or on a planar surface.
▪ The mobile phase in chromatography is a Gas Gas/Gas Liquid/Gas Solid/Gas
phase that moves over or through the
stationary phase, carrying with it the
Liquid Gas/Liquid Liquid/Liquid Solid/Liquid
analyte mixture.
▪ The mobile phase may be a gas, a liquid,
Solid Gas/Solid Liquid/Solid Solid/Solid
or a supercritical fluid.

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Chromatographic methods are of two basic types.

▪ In column chromatography, the stationary phase is
held in a narrow tube, and the mobile phase is forced
through the tube under pressure or by gravity.
▪ In planar chromatography, the stationary phase is
supported on a flat plate or in the pores of a paper.
Here the mobile phase moves through the stationary
phase by capillary action or under the influence of
gravity.
▪ Column chromatographic methods can be further
subdivided according to the nature of the mobile
phase, specifically liquid, gas, and supercritical fluid.

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▪ Partition
▪ Adsorption
▪ Absorption
▪ Sorption

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Adsorption versus Absorption

The elution of a solute through a chromatographic system

2009,
Prentice-

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▪ A chromatograph is equipment that

enables a sophisticated separation e.g. ▪ Analytical chromatography
gas chromatographic or liquid
chromatographic separation. ▪ Preparative chromatography
▪ The retention time is the characteristic
time it takes for a particular analyte to
pass through the system (from the
column inlet to the detector) under set
conditions.

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▪ Elution is a process in which solutes are

CALIBRATION washed through a stationary phase by
CURVE the movement of a mobile phase.
▪ The mobile phase that exits the column
is called the eluate.
▪ An eluent is a solvent used to carry the
components of a mixture through a
stationary phase.

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CHROMATOGRAPHIC
PROCESS

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Chromatograph
Separation of dye Nile red by column The 5 Basic Components of a Chromatograph
chromatography.

Different components are visible.

under UV light.

Nile red is pink when traveling through the

silica but orange once it’s released in the
solvent.

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Methods for Improving Column Performance

▪ Plot of signals as a function of time CHROMATOGRAM
▪ Useful for both qualitative and quantitative
analysis.
▪ The positions of the peaks on the time axis
can be used to identify the components of
the sample.
▪ The area under the peak provides a
quantitative measure of the amount of each
species.

A chromatogram is a plot of some function of solute concentration versus

elution time or elution volume.

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Methods for Improving Column Performance

Chromatography Nomenclature

CHROMATOGRAM peak maximum

Band Separation
Band broadening

baseline

injection point

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▪ The baseline is any part of the chromatogram ▪ The retention time (tr) is the time elapsed
where only mobile phase is emerging from between the injection point and the peak
maximum.
the column.
▪ Each solute has a characteristic retention
▪ The peak maximum is the highest point of the time.
peak.
▪ The retention volume (Vr) is the volume of
▪ The injection point is that point in time / mobile phase passed through the column
position time when/where the sample is between the injection point and the peak
placed on the column. maximum.
Vr = Q x tr
where Q is the flow rate in ml/min.

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▪ The corrected retention time (t'r) is the time ▪ Peak width at half height (w0.5) - distance
elapsed between the dead point and the peak between each side of a peak measured at half
maximum.
▪ The corrected retention volume (V'r) is the the peak height.
volume of mobile phase passed through the
column between the dead point and the peak
maximum. It will also be the retention volume ▪ Peak width at the base (wB) - distance
minus the dead volume. between the intersections of the tangents
V'r = Vr - Vo = Q(tr - to) where Q is the flow rate drawn to the sides of the peak and the peak
in ml/min. base geometrically produced.
▪ The peak height (h) is the distance between the
peak maximum and the base line geometrically
produced beneath the peak.

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▪ The dead time (to) tM is the time it takes for an

unretained species to pass through a column.
▪ The retention time tR is the time between injection
of a sample and the appearance of a solute peak
at the detector of a chromatographic column.
▪ The average linear rate of solute migration, , in
centimeters per second is
 = L / tR
▪ where L is the length of the column packing.
Similarly, the average linear velocity, u, of the
molecules of the mobile phase is
u = L / tM

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▪ All chromatographic separations are based on

differences in the extent to which solutes are
▪ The effectiveness of a distributed between the mobile and the stationary
chromatographic column in phase.
▪ For the solute species A, the equilibrium involved is
separating two solutes depends on described by the equation:
the relative rates at which the two
A(mobile) A(stationary)
species are eluted.
▪ These rates are in turn determined ▪ The equilibrium constant Kc for this reaction is
called a distribution constant, which is defined as
by the ratios of the solute
concentrations in each of the two  A S cS
Kc = =
phases.  A M cM
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▪ The amount of band broadening that ▪ Two related terms are widely used as quantitative
measures of chromatographic column efficiency:
occurs as a solute passes through a ➢ plate height H and
chromatographic column strongly affects ➢ plate count or number of theoretical
the column efficiency. plates N.

▪ The two are related by the equation

N=L/H
where L is the length (usually in centimeters) of
the column packing.
▪ The efficiency of chromatographic columns
increases as the plate count N becomes greater
and as the plate height H becomes smaller.

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Determining the Number of Plates in a Column

▪ The number of theoretical plates, N, and the
plate height, H, are widely used in the
literature and by instrument manufactures
as measures of column performance.
▪ N can be determined from a chromatogram.
▪ The retention time of a peak tR and the width
of the peak at its base W (in units of time)
are measured. The number of plates can
then be computed by the simple
relationship.
N = 16 (tR / W)2
▪ To obtain H, the length of the column L is
measured and H = L / N equation is applied.

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The plate heights can be decreased, and thus

column efficiency increased:
▪ by keeping the flow rate in an optimum
range.
▪ by decreasing the particle size of column
packings,
▪ by employing thinner layers of film (for GLC
where the stationary phase is a liquid
adsorbed on a solid), and
▪ by lowering the viscosity of the mobile
phase.
▪ Increase in temperature also reduces band
broadening in most cases.

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EXAMPLE
A gas chromatographic method for the separation of a mixture
of cyclohexane, t-butanol and benzene on a 10 m capillary
column gave the following data:

Parameter Cyclohexane t-Butanol Benzene

tR 3 m 20 s 3 m 30 s 3 m 45 s
Wb 8s 9s 11 s

Calculate (a) the plate numbers, (b) the plate heights, and
(c) the resolution between adjacent pairs of solutes.

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CYCLOHEXANE
▪ The resolution Rs of a column provides a
quantitative measure of its ability to separate
N = 16 (tR / W)2 two analytes. The resolution is defined as
2 Z 2( tR) B − ( tR) A
N =16 (200/8)2 RS = =
WA + WB WA + WB
= 10 000 ▪ A resolution of 1.5 gives an essentially
complete separation of A and B, whereas
H=L/N ▪ a resolution of 0.75 does not.
▪ At a resolution of 1.0, zone A contains about 4%
B and zone B contains about 4% A.
H = 10000 / 10000 ▪ The resolution for a given stationary phase can
be improved by lengthening the column and
H = 1 mm thus increasing the number of plates.

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Column Resolution

Problem

In a chromatographic analysis of lemon

oil a peak for limonene has a retention
time of 8.36 min with a baseline width of
0.96 min. g-Terpinene elutes at 9.54 min,
with a baseline width of 0.64 min.

What is the resolution between the two

peaks?

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▪ Chromatography is a powerful and

versatile tool for separating closely
related chemical species.
▪ In addition, it can be employed for
the qualitative identification and
quantitative determination of
separated species.

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