Chapter Two

Introduction to Chromatographic Separation

Historical Background

Chromatography was introduced first by Russian Botanist, Tswett in 1960. He devised a method
to separate components of plant extract.
Petroleum ether (mobile phase)
Plant extract
░░░░
░░░░
░░░░ CaCO3 (stationary phase)
░░░░ Column (tube)
░░░░

Petroleum ether which is the mobile phase eluted the extract down the column that is filled with
finely pulverized CaCO3. Finally he observed colour zones on the column.

Chlorophyll (green)

Xanthophyll (yellow)

Carotene (red)

It is clear that CaCO3 is polar compound and petroleum ether is slightly polar solvent. It is
expected that the red carotene is less polar for it has moved fast with the slightly polar solvent
and chlorophyll should be most polar because it has stayed for longer time with the polar packing
material, CaCO3. Michael Tswett named the process chromatography.

The term ‘chromatography’ was emanated from two Greek terms ‘chroma’ and ‘graphien’ .
Chroma means colour and graphien means writing. The modern definition of chromatography
has nothing to with colour.

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Definition of Chromatography

Chromatography is a means of separating components of a mixture (a sample) based on their

affinity to the stationary and mobile phase. Chromatography makes use of two phases.

Namely:

a. Stationary Phase
b. Mobile phase
A. Stationary Phase
Stationary phase is fixed in a particular place (on a plane or in a column).
B. Mobile Phase
Mobile phase can be gas, liquid or super critical fluid that moves through/over the stationary
phase. In chromatography, components of a sample are eluted (carried) by the mobile phase
across the stationary phase. Components of the mixture are then separated while traversing the
stationary phase based on the difference in partitioning behaviour between the stationary and
mobile phase. Separation is based on the relative retention of components of a sample (a
mixture) on the two phases.
Separation of components of a mixture can be achieved by their difference in
 Bond polarities
 Molecular size
 Molecular charge etc…
In chromatographic process, the two phases are chosen so that the components of the mixture
have different solubilities in each phase based on the above criteria.
Classification of Chromatography
The classification can be in various ways.
A. Based on the type of material on/in which the stationary phase is fixed/kept
Based on the type of material on/in which the stationary phase is fixed/kept, chromatography is
classified in to planar and column chromatography.
I. Planar Chromatography
In planar chromatography, the stationary phase is supported by a flat plate or pores of a paper.
The mobile phase moves on the stationary phase by capillary action. Planar chromatography is
also known as internal method because after the constituents of the sample are separated, they
are located within the separation bed and then they will be detected over there.
II. Column Chromatography
In column chromatography, the stationary phase is packed in a narrow tube (column). The
mobile phase is forced to move through the column under pressure or gravity. Column
chromatography is also known as external method because components of a sample travel
through separation bed and then they appear at the end of the column at different times where
they can be collected and detected based on physical and chemical properties.

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 B. Based on the type of Mobile Phase
Based on type of mobile phase, chromatography is classified in to
I. Liquid Chromatography (L.C.)
It makes use of liquid mobile phase.
Liquid chromatography is classified in to liquid-liquid chromatography (LLC) and liquid –solid
chromatography (LSC). In liquid-liquid chromatography, both the mobile and the stationary
phase are liquids. In liquid- solid chromatography, the mobile phase is liquid while the stationary
phase is solid. Liquid chromatography can be carried out either in column or planar
chromatographic modes.
II. Gas Chromatography
Gas chromatography makes use of gaseous mobile phase.
Gas chromatography is classified in to gas-liquid chromatography (GLC) and gas-solid
chromatography (GSC). In gas –liquid chromatography, the mobile phase is gas and the
stationary phase is liquid whereas in gas-solid chromatography, the mobile phase is gas and the
stationary phase is solid. Gas chromatography is conducted only in column.
C. Based on the Difference in Polarities between the Two Phases
In chromatographic process, the two phases should have different polarities so as to decrease the
interaction that is found between them and to enable the movement of the mobile phase on the
stationary phase. Based on the difference in polarities between the two phases, chromatography
is classified in to normal phase and reversed phase chromatography.
I. Normal Phase Chromatography
In normal phase chromatography, the stationary phase is more polar than the mobile phase.
Example: ether mobile phase and silica (SiO2) or alumina (Al2O3) stationary phase
II. Reversed Phase Chromatography
In reversed phase chromatography, the stationary phase is less polar than the mobile phase.
Example: stationary phase : Alkane with 18 carbon atoms
N.B. In both techniques, solutes (analytes) are eluted in the order of polarity. i.e.in normal phase
chromatography, the least polar component is eluted first while in in reversed phase
chromatography, the least polar component is eluted last.
D. Based on the Mode of Separation of the Sample Components
I. Adsorption Chromatography
In adsorption chromatography, separation is based on interaction of the analyte with the surface
of the solid. Gas-solid chromatography (GSC) and liquid-solid chromatography (LSC) are
examples of adsorption chromatography.
II. Partition/distribution Chromatography
In partition chromatography, separation of components of a sample is based on difference in
their distribution (dissolution) between the mobile phase and liquid stationary phase. Liquid-
liquid chromatography (LLC) is an example of partition chromatography. The process of
chromatographic separation considers column chromatographic separation of a mixture

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containing components A and B or more than two components. Sample is introduced at the top
of the column that is packed with stationary material.

sample
░░░
Stationary phase
░░░
:::::::
░░░ column
Type equation here.

Eluent (mobile phase) is applied to the sample. The eluent forces the sample to move down the
column. The movement of the sample across the column is known as elution. As the sample
passes through the column, its ingredients (A and B) distribute themselves between the mobile
and the stationary phase. Elution is the process in which the solute (analyte) is eluted (washed)
through the stationary phase by the movement of mobile phase.
flow
Distribution ___________________________________________________

The sample is applied initially.

A (M) A(S) Where M and S are mobile phase and stationary phase respectively. As the
components of a sample travel longer through the stationary phase, concentration of components
in the mobile phase increases. When the distribution is repeated several times, A and B will be
separated. The component that is retained strongly by the stationary phase lags behind and the
one that is retained weakly by the stationary phase will be eluted first.

A
B

The eluted components are detected (identified) at the end of the column. Detection can be
carried out in two ways.

A. Chemical Detection
B. Instrumental Detection
A. Chemical Detection
In chemical detection, each separated component is treated with a reagent solution. This is
classical qualitative analysis.

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 B. Instrumental Detection
In instrumental detection, each component of the sample that is eluted from the column is
detected by instrumental detectors. Detectors produce information that denotes each substance
using absorption of ultraviolet radiation, oxidation or reduction potential etc… This information
is plotted in the form of a series of symmetrical peaks called chromatograms.
Detector response, DR

B
A

t0 tB tA elution time

The smallest peak (the first one) represents compounds that are not retained by the stationary
phase at all. It is solvent front. They have moved together with the solvent (mobile phase).
Peaks A and B represent components A and B respectively. Since component A is retained
more strongly than component B, its peak appeared next to component B.

Distribution Equilibria of Solutes

Distribution (partition) of a solute is the distribution (partition) of a particular analyte

between the mobile and the stationary phase.

A (M) A(S) The constant of this equilibrium is given by K = where CS and CM are
molar concentrations of the solute in the stationary and mobile phase respectively and K is
partition coefficient ratio. If >> , then the solute is strongly retained by the stationary
phase. This indicates large value of K and the retention time of the solute is large.

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 Consider the following chromatogram

tR2
Detector response, DR

tR1

tM

Retention time (minute)

Solvent front
The reason why the baseline doesn’t coincide with the x-axis is that the detector responds to
the solvent.

A. Mobile Time, tM
 Mobile time or hold up time is the elution time of solvent that is not retained.
 Mobile time is the time that is required by molecules in the mobile phase to pass through the
column and reach the detector.
B. Retention time, tR
Retention time is the time that is required by the analyte to reach the detector. It is the time that
is found between sample introduction and the arrival of solute to the detector.
C. Adjusted Retention Time,
Adjusted retention time is the time that is required by the solute to travel the length of the
column beyond the time that is required by unretained solvent.
= −
D. Hold up Volume or Mobile Volume (VM)
 Hold up volume is the volume of the mobile phase that reaches the detector.
 Hold up volume is the volume of the mobile phase that passes simply through the column and
reaches the detector without being retained.
E. Retention Volume(VR)

Retention volume is the volume of the mobile phase (solvent) that is required to elute (carry) the
solute (component of the sample) through the column.

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 F. Adjusted Retention Volume,
Adjusted retention volume is the difference in volume between retention volume and mobile
volume. = −

and are related as follows:

= where is flow rate of the mobile phase.

Flow rate is the rate at which the mobile phase travels across the column. Peaks in
chromatography are identified by their retention time, and retention volume, but these
parameters vary with the length of the column, flow rate of the mobile phase and temperature.

Relationship between K and Retention Time,

Linear velocity of migration of mobile phase is given by the following formula:

U= Where U is linear velocity of the mobile phase; L is length of the column and is
mobile time of the solvent.

Average linear velocity of migration of solute is given by the following formula:

̅= where ̅ is average linear velocity of analyte, L is length of column and is retention time

Fraction of time a solute spends in the mobile phase is given by:

̅
f = ------------------------(1)

f can also be expressed as :

f= where is amount of solute in the mobile phase and is total amount of solute in the
column. = + where is amount of solute in the mobile phase is amount of
retained solute in the stationary phase.

f= but n = CV where C is concentration of analyte and V is volume of mobile phase.

f= where is concentration of analyte in the mobile phase, is volume of

mobile phase is concentration of analyte on the stationary phase and is volume of
stationary phase

f= multiplying both the denominator and the numerator by

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f= but =K

Partition coefficient (distribution ratio) is defined as the amount of analyte that is partitioned (
distributed ) between two immiscible phases (as two liquids or a stationary and a mobile phase)

f= let = -------------------------------(2)

is capacity factor

Capacity factor (retention factor) is a means of measuring the retention of analyte on the
chromatographic column.

f= ------------------------------------------(3)

Substituting equation 3 in to equation 1.

̅ = Uf = U but ̅ = and U =

Multiplying both sides by

= = = (1+ ) = + − =

= = -------------------------------------(4)

As increases, elution takes long time. A convenient value of capacity factors falls between one
and five. Equate equation 2 and equation 4.

=K = K=( --------------------------(5)

Separation or Selectivity Factor, α

Selectivity is a measure of preference of stationary phase that shows for one solute over another
and is expressed as follows:

α= where and are partition coefficients of solutes B and A respectively. For

convenience, the numerator is greater than the denominator; . Hence, analyte B moves
slower than analyte A. Therefore, the retention time of analyte A is less than that of analyte B.

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 α= , > α=

α is always greater than one. The greater the value of α, the greater the separation between the
two components. α is fairly independent of flow rate and can be used to identify peaks when
flow rate is changed.

Column Efficiency

In chromatography, the separation between solutes and the performance of the chromatographic
process can be evaluated from recorded chromatogram based on:

I. Space between adjacent peaks that is measured as resolution,

Components of a sample are said to be well resolved (separated), if there is no overlapping
between their peaks. i.e peaks are well resolved.
II. The width of the peak should be very small. i.e. sharp peak
As the width of the chromatographic peak increases, then the peak becomes broad. This results
in broadening of band. Broadening of band elongates elution time of the solute. This may trigger
overlapping of peaks. i.e. broad peak may be the combination of two peaks.
Optimal chromatographic separations are signaled by sharp and symmetrical peaks.
Detector response, DR

sharp peaks

Broad peaks

Two approaches (theories) are known which explain the way by which column efficiency can be
described. They are:

I. Plate theory
II. Rate/kinetic Theory
I. Plate Theory
This theory was proposed by Martin and Synge in 1941. It assumes that chromatographic
columns contain large number of segments called theoretical plates.

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 Column length

theoretical plate

Theoretical plates are ideal. Separate equilibrations of the sample between the stationary and
mobile phase occur in each plate. The analyte moves down a column by the transfer of
equilibrated mobile phase from one plate to the next. The efficiency of chromatographic column
is described by the height of theoretical plate, H and number of theoretical plates, N. The height
of theoretical plate or height equivalent of theoretical plate is how thick each plate is. The two
quantities are related by N = where ‘N’ is number of theoretical plates, ‘L’ is length of the
column and ‘H’ is height of the theoretical plate.

------------------------------
Detector Response

h
baseline

Retention time (minute)

2
N=( )

A column is said to have high efficiency if its N is large and its H is very small. N and H of a
particular column may differ as the mobile phase and even the solute are changed. Resolution
measures the space that is found between adjacent peaks or it describes how well they are
separated.

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 a
Detector response,DR

Retention time (min)

a) The two peaks are overlapped together. The resolution is poor.
b) The two peaks are separated partially.
c) The two peaks are separated completely. The components are resolved completely. The
resolution is good. No overlapping of adjacent peaks
Quantitative Measurement of Resolution
Resolution (Rs) = for analytes (components) A and B
( (
Rs = The value of resolution should be positive.
(

Assume = W= ½ (W +W) = ½ (2W) =W

( ( (
Rs = but N =16( )2
(
For peak B W=
√
( ( √
Rs = Generally = +
(

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 ( ( ( ( √
Rs = ×
(

Multiplying both the numerator and the denominator by

( (
√
Rs = (
× but =

√
× Multiplying both the numerator and the denominator by

√
× but = α where >

√ ( √
Rs = × Rs = × where is capacity factor of fast moving

component.
(
N=
(
Generally, when Rs = 0.5 the bands are quite overlapped. For example, as it has been
shown in chromatogram ‘a’. If Rs =1, then the bands are still overlapped and the triangles
that approximate the two peaks meet at baseline. For example the one that is shown in
chromatogram ‘b’. If resolution is 1.5, then overlapping between the two peaks is 1%.
For example, in chromatogram ‘c’.
Example: Substances ‘A’ and ‘B’ have retention times of 16.40 and 17.63 minutes
respectively on a 30.0 cm column. Unretained species passes through the column in 1.30
minutes. The widths of the peaks at the base for ‘A’ and ‘B’ are 1.11and 1.21 minutes
respectively. Calculate;
a. column resolution.
b. average number of theoretical plates.
c. plate height.
d. length of column that is required to achieve a resolution of 1.5(assuming the same
height).
e. the time that is required to elute component ‘B’ on the long column.

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