CHAPTER 4

(AN INTRODUCTION TO
CHROMATOGRAPHY SEPARATION)

4.1 GENERAL DISCRIPTION OF CHROMOTOGRAPHY

Method

Basis of Method

Mechanical phase separation

Precipitation and filtration
Difference in solubility of compounds formed
Distillation
Difference in volatility of compounds
Extraction
Difference in solubility in two immiscible liquids
Ion exchange

Difference in interaction of reactants with ionexchange resin

Chromatography

Difference in rate of movement of a solute

through a stationary phase

Electrophoresis

Difference in migration rate of charged species

in an electric field

Field-flow fractionation

Difference in interaction with a field or gradient

applied perpendicular to transport direction
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4.1.1 Definition of Chromatographic Terms

a. Chromatography
A technique in which the components of a mixture are separated
based on differences in the rates at which they are carried
through a fixed or stationary phase by a gaseous or liquid mobile
phase.
b. Stationary Phase
A solid or an immobilized liquid on which analyte species are
partitioned during passage of a mobile phase.
c. Mobile Phase
A liquid or a gas that carries analytes through a liquid or solid
stationary phase.

4.1.2 Classification of Chromatography

Chromatography methods are of two basic types :

Column Chromatography
The stationary phase is held in
a narrow tube called column,
and the mobile phase is forced
through the tube under pressure
or by gravity.

Planar Chromatography
The stationary phase is supported on a flat plate or in the pores
of a paper. The mobile phase moves through the stationary
phase by capillary action or under the influence of gravity.

 Classification of chromatography column

AV X
X A V
X
V
V X A
X
A V
V
A
X A
A

Injection

V
A A
A

V
A

X
V

X
X

V
V

Interaction

A
A

V
V

A
A

X X
X X
X X

V
V

Elution

4.1.3 Elution of Chromatography

4.1.3 (a) Terms
Elution : A process in which solutes are washed through a stationary
phase by the movement of a mobile phase.
Eluate : The mobile phase that exits the column.
Eluent : A fresh solvent used to carry the components of a mixture
through a stationary phase.

4.1.3 (b) Mechanism

Initially, a solution of a mixture of A and B in the mobile phase is
introduced at the head of the column at time to.
The two components separate themselves between the mobile
phase and stationary phase.

 Elution then occurs by forcing the sample components through the

column by continuously adding fresh mobile phase.
With the first introduction of fresh mobile fresh, the eluent moves
down the column, where further partitioning between the mobile
phase and the stationary phase takes occurs (time t1).
Further additions of solvent carry solute molecules down the
column in a continuous series of transfers between the two phases.
Because solute movement can occur only in the mobile phase, the
average rate at which a solute migrates depends on the fraction of
time it spends in that phase.

 This fraction is small for solutes that are strongly retained by the
stationary phase (component B), and large when retention in the
mobile phase is more likely (component A).
The resulting differences in rates cause the components in a
mixture into bands or zones along the length of the column.
Isolation of the separated species is then accomplished by passing a
sufficient quantity of mobile phase through the column to cause the
individual bands to pass out the end, where they can be collected or
detected (times t3 and t4).

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a) Diagram showing
the separation of a
mixture of
components A and
B by elution
chromatography

b) The detector signal

at the various
stages of elution
shown in a
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4.1.3 (c) Chromatograms

If a detector that responds to solute concentration is placed at the
end of the column during elution and its signal is plotted as a
function of time (or of volume of added mobile phase), a series of
peaks is obtained.
Such a plot is called a chromatograms, which is 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.

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14

 On a chromatogram, the perfect elution would have the same as the

graphical representation of the law of Normal distribution of
random errors a (Gaussian curve).
Gaussian Distribution : a theoretical bell-shaped distribution of
results obtained for replicate measurements that are affected by
random errors.

, would correspond to the

retention time of the eluting peak while to the standard deviation of
the peak ( represents the variance 2). y represents the signal as a
function of time, x, from the detector located at the outlet of the
column.

In keeping the classical notation,

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4.1.4 Methods of Improving Column Performance

By changing experimental conditions, non-separated bands can be
separated :
(a) Adjust migration rates for A and B (increase band separation)
(b) Adjust zone broadening (decrease band spread)

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4.2 MIGRATION RATES OF SOLUTE

The effectiveness of a chromatographic column in separating two
solutes depends on the relative rates at which the two species are
eluted.
These rates in turned are determined by the ratios of the solute
concentrations in each of the two phases.
Other factors:

Distribution constant

Retention times

Retention factor

Selectivity factor

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4.2.1 Distribution Constant

Distribution constant for a solute in chromatography is equal to the
ratio of its molar concentration in the stationary phase (cs) to its
molar concentration in the mobile phase (cm).
Analyte A in equilibrium with two phases :

A mobile A stationary
K=

Cstationary
C mobile

where K is distribution constant

K is constant over a wide range of solute concentration; that is Cs is

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directly proportional to Cm.

4.2.2 Retention Time

In chromatograms, the small peak on the left is for a species that is
not retained by the stationary phase.
Time tM between sample injection and the appearance of this peak is
called dead time or void time.
Dead time/ void time : time it takes for an unretained species to pass
through a chromatographic column.

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 The larger peak on the right is that of an analyte species.

The time required for this zone to reach the detector after sample
injection is called the retention time, tR.
The analyte has been retained because it spends a time tS in the
stationary phase. The retention time is then :

t R = t M + tS
Retention time : is the time between injection of a sample and the
appearance of a solute peak at the detector of a chromatographic
column In chromatograms.

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 Average linear rate of solute migration :

L
u =
tM
where L = length of column packing
Average linear rate of mobile-phase migration :

L
=
tR
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4.2.3 Retention Factor

The retention factor is an important experimental parameter that is
widely used to compare the migration rates of solutes on columns.
Retention factor for a solute A:

tR tM tS
kA =
=
tM
tM
Retention factor : Retention factor kA for a solute A is related to the
rate at which A migrates through a column. It is the amount of time a
solute spends in the stationary phase relative to the time it spends
in the mobile phase.
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 Value of kA :
when kA is 1.0 separation is poor

when kA is > 30 separation is slow

when kA is = 2 10 separation is optimum

4.2.4 Selectivity Factor

Selectivity factor : the ratio of the distribution constant of the more
strongly retained solute (B) to the distribution constant for the
less strongly held solute (A).
Selectivity factor of a column for the two solutes is defined as :

K
=
K

B
A

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where KB is the distribution constant for the more strongly retained

species B.

KA is the constant for the less strongly held or more rapidly

eluted species A.

is always greater than unity and larger better separation

Selectivity factor can also be described with retention factor. :

k
=
k

B
A

where kB and kA are the retention factors for B and A.

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4.3 THE EFFICIENCY OF CHROMATOGRAPHIC

COLUMN
The efficiency of a chromatographic column is affected by the
amount of band broadening that occurs as a compound passes
through the column.

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4.3.1 Quantitative Description of Column Efficiency

Two related terms are widely used as quantitative measures of
chromatographic column efficiency :

Plate height H

Plate count or number of theoretical plates N

The two are related by the equation :

L
N =
H
where L is length (in cm) of column packing.
The efficiency of chromatographic columns increases as the plate
count N becomes greater and as the plate H becomes smaller.
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 Thus, H is needed to be as small as possible more plates can be

fitted into a column.

Plate

L
N =
H

L, column
length

5 Plates
(Larger H)
Thicker

10 Plates
(Smaller H)
Thinner

H No of Plate Efficiency
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 Because chromatographic bands are usually Gaussian and because

the efficiency of a column is reflected in the breadth of
chromatographic peaks, the variance per unit length of column is
used by chromatographers as a measure of column efficiency.
That is the column efficiency is defined by :

H =

Efficient column has small plate height less zone broadening.

Thus, unit H is in cm.

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(a)
(b)

Gaussian distribution of sample molecules

Column length as the distance from the sample entrance point to the
detector
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 In simple relationship, N can be approximated from the width, W

(unit in time) of the base of the chromatographic peak :

tR
N = 16

W
B

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4.3.2 Other Variables That Affect Columns Efficiency

Band broadening reflects a loss of column efficiency.
The slower the rate of mass transfer processes occurring while a
solute migrates through a column, the broader the band at the
column exit.
Some of the variables that affect mass-transfer rates are
controllable can improve the separation.

4.3.2 (a) The Effect of Mobile-Phase Flow Rate

Higher mobile phase velocity, less time in column less zone
broadening.
However, plate height, H, can changes with flow rate. The plot of H
versus u Van Deemter plot.
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H = A+

B
+ Cu
u

B
H = A + + Cu
u

where H is the plate height, u is the average linear velocity of the

mobile phase, and A, B, and C are (positive) constants determined
by various physical properties of the mobile and stationary phases.
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33

34

i. Multipath Term/ Eddy Diffusion (A)

Eddy diffusion, which is caused by non-uniform packing of
chromatographic columns.

35

 Molecules moves through different paths.

Larger difference in pathlengths for a larger particles.
At low flow rates, diffusion allows particles to switch between paths
quickly and reduces variation in transit time.

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ii. Longitudinal Diffusion Term (B)

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 Longitudinal diffusion, which is caused by the tendency of a solute

species to diffuse from regions of high concentration (such as the
center of a chromatographic band) to regions of low concentration
(such as the leading or trailing edge of a chromatographic band).
Proportional to mobile phase diffusion coefficient.
Inversely proportional to flow rate high flow rate, less time for
diffusion.

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iii. Mass Transfer Coefficients (C)

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H = A+

B
+ (CS + CM )u
u

Mass transfer in the form of partitioning of the solute into the

stationary phase, which does not occur instantaneously and
depends on the solutes partition and diffusion coefficients.
All species need a certain amount of time to equilibrate between the
stationary and mobile phases.
High flow rates species with strong affinity for stationary phase
will be slowed and the molecules with no affinity to the stationary
phase move ahead which causes band broadening.

B
H = A+
+ (C S + C M )u
u
Cs is rate for adsorption onto stationary phase
CM is rate for analyte to desorb from stationary phase.
Effect proportional to flow rate at high flowrate less time to
approach equilibrium.

4.4 COLUMN RESOLUTION

The resolution Rs of a column how far apart two bands are relative
to their widths.
The resolution provides a quantitative measure of the ability of the
column to separate two analytes.
The chromatogram consists species A and B on three columns with
different resolving powers.
Resolution :

0.75 = no separation occur

1.0 = zone A contain ~ 4% B and zone B contain ~ 4% A

1.5 = complete separation of A and B (overlap 0.3%)

The resolution for a given stationary can be improved by

lengthening column increasing the no of plates.
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2Z
RS =
WA + WB
=

2 (t R ( B ) t R ( A ) )
WA + WB

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4.4.1 Effect of Retention and Selectivity Factors on

Resolution
Resolution equation of a column that relates to the
no of plates with the retention time and selectivity
factors of a pair of solutes on the column :

N 1 k B

Rs =

4 1 + k B
where kB = retention factor of the slower moving
species and = the selectivity factor

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4.4.2 Effect of Resolution on Retention Time

The goal of chromatography is the highest possible resolution in the
shortest possible elapsed time.
Unfortunately, these goals tend to be incompatible, and a
compromise between the two usually necessary.
The time (tR)B required to elute the two species with a resolution Rs
is given by:

(t R )B

16 R H (1 + k B )
=

2
u
1 (k B )
2
S

where u is the linear velocity of the mobile phase

The resolution will increase if the u is low.
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4.5 APPLICATION OF CHROMATGOGRAPHY

Gas Chromatography (GC)
High Liquid Performance Chromatography (HPLC)

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