chapter two

Introduction to chromatographic
separation

1
Historical background
 Chromatography, literally "color writing", was first employed by
Russian scientist Mikhail Tswett in 1903/1906.

 He continued to work with chromatography in the first decade of

the 20th century, primarily for the separation of plant pigments
such as chlorophyll, carotenes, and xanthophylls.

 Since these components have different colors (green, orange, and

yellow, respectively) they gave the technique its name.

2
Chromatography -- what does it mean?

• "Chromatography is a physical method of separation in which

the components to be separated are distributed between two
phases;

• one of which is stationary (stationary phase) while the other

moves in a definite direction (mobile phase)".

• Chromatography derives its name from two Greek words as;

(chroma) meaning "color" and (graphy) meaning "writing".

• Chromatography is the collective term for a family of

laboratory techniques for the separation of mixtures.
• Chromatography is not restricted to analytical separations and
identifications.
3
• Why use chromatography? The key here is separation. But
what is the importance of separation in the lab?

• Separation of chemical components is vital in any type of

chemical analysis.

• trying to identify an unknown substance, the sample must first

be simplified as much as possible into its constituent
compounds.

• The unknown can then be characterized by individual

identification of its parts.

• This does not imply that the separated chemical components

are recovered after the separation and analyzed.

4
 General description of chromatography
• Chromatography is based on a flow system containing two
phases,

• mobile and stationary, and the sample components are separated

according to differences in their distribution between the two
phases.

• In all chromatographic separations, the sample is dissolved in a

mobile phase.

• This mobile phase is then forced through an immiscible stationary

phase.
5
 The two phases are chosen so that the components of the
sample distribute themselves between the mobile and
stationary phase to varying degree.

 As continues moving of the mobile phase, the components in

the mixture move through the system at different rates due to
the difference in their interaction between the mobile and
stationary phases.

Mobile phase is the phase which moves in a definite direction.

 It may be a liquid (LC), a gas (GC), or a supercritical fluid
(supercritical-fluid chromatography, SFC).

6
 Cont…
 The mobile phase consists of the sample being separated/
analyzed and the solvent that moves the sample through the
column.

 In the case of high performance liquid chromatography

(HPLC) the mobile phase consists of a non-polar solvent(s)

 such as hexane in normal phase or polar solvents in reverse

phase chromatography and the sample being separated.

 The mobile phase moves through the chromatography column

(the stationary phase) where the sample interacts with the
stationary phase and is separated
7
 Con…
• Q? Which kind of phases can be used as mobile phase?
 Answer: gases and liquids, because gases and liquids are fluids
that can flow easily but solid is not fluid which can‟t be used to
transport sample.

• Stationary phase is the substance which is fixed in place for the

chromatography procedure. Examples include the silica layer in
thin layer chromatography.
• Q? Which kinds of phases can be used for stationary phase?

 Answer: liquid and solid. Liquid can be used as stationary phase

by immobilizing the liquid on a solid surface.
 Gases can‟t be used for stationary phase preparation because it
is difficult to fix the gas on fixed space.
8
 Con…
 In a chromatographic separation the mobile and stationary
phase must have low interaction.
 Always when we select a mobile phase we should consider the
following:
 The mobile phase must be the least eluted species.
 The mobile phase could not wash the stationary phase.

 Supporting medium: a solid surface on which the stationary

phase is bound or coated
 A chromatogram is the visual output of the chromatograph.

 In the case of an optimal separation, different peaks or

patterns on the chromatogram correspond to different
components of the separated mixture.
9
 2.2. Types (Classification) of Chromatography

 Chromatographic methods can be categorized in several ways:

 1. Based on the physical state of the mobile and stationary

phases.

 Mobile phase could be gas, liquid or a supercritical fluid.

Stationary phase could be liquid or solid.

 In this classification, chromatography is classified as:

A. Column Chromatography
• In column chromatography, the stationary phase is held in a
narrow tube through which the mobile phase is forced under
pressure.
10
 Cont…

 The particles of the solid stationary phase or the support

coated with a liquid stationary phase may fill the whole
inside volume of the tube (packed column).

 or be concentrated on or along the inside tube wall leaving an

open, unrestricted path for the mobile phase in the middle
part of the tube (open tubular column).

 Differences in rates of movement through the medium are

calculated to different retention times of the sample and the
mobile phase moves in the column either due to gravity or by
the force of pump.
11
B. Planar Chromatography

 Planar chromatography is a separation technique in which the

stationary phase is present as or on a plane.

 The plane can be a paper, serving as such or impregnated by a

substance as the stationary bed (paper chromatography)

 or a layer of solid particles spread on a support such as a glass

plate (thin layer chromatography) .

 Different compounds in the sample mixture travel different

distances according to how strongly they interact with the
stationary phase as compared to the mobile phase.
12
 Con…
 The specific Retention factor (Rf) of each chemical can be
used to aid in the identification of an unknown substance.

In this technique the mobile phase is transported across the

stationary phase by a capillary action.

This type of chromatography can be divided into:

I. Paper Chromatography

Paper chromatography is a technique that involves placing a

small dot or line of sample solution onto a strip of
chromatography paper 13
 Cont…

 The paper is placed in a jar containing a shallow layer of

solvent and sealed.

 As the solvent rises through the paper, it meets the sample

mixture which starts to travel up the paper with the solvent.

 This paper is made of cellulose, a polar substance, and the

compounds within the mixture travel farther if they are non-
polar.

 More polar substances bond with the cellulose paper more

quickly, and therefore do not travel as far.
14
II. Thin Layer Chromatography (TLC)

 Thin layer chromatography (TLC) is a widely employed

laboratory technique and is similar to paper chromatography

 However, instead of using a stationary phase of paper, it

involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate.

 Compared to paper, it has the advantage of faster runs, better

separations, and the choice between different adsorbents.

 For even better resolution and to allow for quantification,

high-performance TLC can be used.
15
 Q? How you can identify if invisible spots could occur
when you do TLC?

 Answer: by applying Ultraviolet light or Iodine vapors to stain

spots.

 Q? Which types of mobile phases are used for column and

planar chromatography?

 Answer: for column chromatography a liquid and gas mobile

phase is used.

 For planar chromatography only liquid mobile phase is used.

Because in a planar stationary phase, a gas molecule could not
transport linearly and the transportation is not good.
16
2. Classification of chromatography based on the type of mobile
phase and stationary phases

 In this case the classification is on the physical state of the mobile

and stationary phase.

 During naming using this classification the first latter represents

the type of the mobile phase while the second letter represents the
stationary phase used.

 With these bases, chromatography can be divided in to three as

major part. These are liquid, gas and supercritical fluid
chromatography

 Gas chromatography (GC) includes all chromatographic methods

in which gas is used as mobile phase. 17
 Cont…

 It uses only stationary phases either coated or packed on a

column.

 Liquid chromatography (LC) includes all chromatographic

techniques in which liquid is used as mobile phase.

 For LC a stationary phase is either packed on a column or coated

on a column or a planar plate can be used.

 Supercritical fluid chromatography (SFC) includes

chromatographic techniques which uses supercritical fluid as
mobile phase.

 For this technique the stationary phase could be organic species

bonded on a solid surface.
18
Table 2.1. Classification of chromatography based on the type of
mobile phase and stationary phases

19
 3. Classification of chromatography based on the type of
interaction occur between the sample and stationary phase

(a) Partition chromatography: Separation based on solubility.

Stationary phase is liquid.

(b) Adsorption chromatography: Separation based on polarity.

Stationary phase is solid.

(c) Ion exchange chromatography: Separation based on charge.

(d) Size exclusion chromatography: Separation based on

molecular size.

20
 Note: Separation in chromatography is based on the elution of
the sample across the stationary phase by the mobile phase.

 Across the movement the components of the sample interacts

with the stationary phase differently.

 The solute which interacts strongly with the stationary phase

will be strongly retained.

 So the speed of this solute in the column or plane is very slow

and takes more time to be eluted.

 The solute, which interacts with the stationary phase weakly,

will be weakly retained. So the solute will be eluted easily.

21
22
23
• Figure 2.1. (a) Diagram showing the separation of a mixture of components
A and B by column elution chromatography. A -is a solute which has a
weak interaction with the stationary phase and weakly retained. B –is a
solute which has a strong interaction with the stationary phase and strongly
retained. (b) The detector signal at the various stages of elution shown in
(a).

24
25
 Chromatogram is the plot (representation) of the variation
with time of the amount of the analyte in the mobile phase
exiting the chromatographic column.

 It is a curve that has a baseline which corresponds to the trace

obtained in the absence of a compound being eluted.

 The separation is complete when the chromatogram shows as

many chromatographic peaks as there are components in the
mixture to be analyzed.

26
 Figure 2.2. A typical Chromatogram
 Where : tM = retention time of mobile phase (dead time)
 tR = retention time of analyte (solute)
 tS = time the solute spent in stationary phase (adjusted
retention time)

27
 A chromatogram is a plot of the detector signal as a function of
time taken for the resolution.

It is useful for qualitative and quantitative analysis.

 Every peak represents one component.

 The position of the peaks on the time axis (retention time) may
serve to identify the components of the sample.

 The area under each peak provides the quantitative measure of the
amount of each component
Note:
 During elution weakly retained species elute first and reach the
detector first and detected first while strongly retained species
elute late to reach the detector. 28
 Efficiency of Separation (Column Resolution, Rs)

 Resolution (Rs ) is a term used to describe the degree of

separation between neighboring solute bands or peaks
(distance between the peaks).

 It is the ratio of the distance between two peaks to the average

width of these peaks, and this descriptor encompasses both the
efficiency and selectivity.

 The goal of chromatography is to separate a sample into a

series of chromatographic peaks, each representing a single
component of the sample.
29
 Cont…

• The resolution provides a quantitative measure of the ability

of the column to separate two analytes, like A and B. The
resolution of each column is defined as:

30
Figure 2.3. A chromatogram of separation of two solutes, A and B

Based on the above equation to increase the resolution:

 We have to increase the difference in the retention time of the

strongly retained species B and the weakly retained species A

 We have to decrease base line width of species A and B. 31

1. increasing z is done by adjusting the migration rate of
the two species.

 That is by increasing retention time of the strongly

retained species, solute B and by decreasing the retention
time of weakly retained species, solute A.

2. decreasing the sum of A W and B W is done by

decreasing the line broadening.

32
Figure 2.4. Chromatogram with: a) poor resolution b) More
separation and c) Less band spread (High resolution)

33
Example:
 Substances A and B have retention times of 16.40 and
17.63 min, respectively & the peak widths (at base) for A
and B are 1.11 and 1.21 min, respectively. Calculate
column resolution of the two peak.

Rs = 2(17.63 - 16.40) / (1.11 + 1.21) = 1.06

34
 Migration Rates of Solutes
 The effectiveness of a chromatographic column in separating
two solutes depends in part on the relative rates at which the
two species are eluted.

 These rates in turn are determined by the ratios of the solute

concentrations in each of the two phases.

 The separation is enhanced by altering the relative flow rate of

solutes.

 i.e. by increasing the flow rate of weakly retained species and

decreasing the flow rate of strongly retained species.
35
 The rate of elution of a solute is determined by the
magnitude of the equilibrium constant for the reaction by
which the solutes distribute themselves between the
mobile phase and the stationary phase.

The following are those factors which affect the migration

rates of a solute
 Distribution Constant, K or D

 All chromatographic separations are based on differences

in the extent to which solutes are distributed between the
mobile and stationary phases.
36
 Cont…
 The distribution constant for a solute in chromatography is
equal to the ratio of its molar concentration in the stationary
phase to its molar concentration in the mobile phase.

 For the solute species A, the equilibrium involved is described

by the equation
A in mobile phase= A in stationary phase
 The equilibrium constant Kc for the distribution of species
A between the two phases is called the distribution constant,
which is defined as

37
 Retention time

 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 dead time (void time) (tM) is the time it takes for an
unretained species to pass through a chromatographic
column.

 All components spend this amount of time in the mobile

phase.

 Separations are based on the different times tS that

components spend in the stationary phase.
38
Figure 2.5. Measurement of the column void time tM, and
the retention time tR

39
• The analyte has been retained because it spends a time tS in
the stationary phase. The retention time is then
tR = tS + tM
The average linear rate of solute migration through the column
v (usually cm/s) is

where L is the length of the column packing. Similarly, the

average linear velocity u of the mobile-phase molecules is

40
 Cont…

• Large retention time indicates the solute takes more time to

reach the detector.

•When we compare the solute B which has large retention time

& solute A which has small retention time ,

•solute B has strong interaction with the stationary phase &

strongly retained than solute A.

41
 The Relationship between Migration Rate and
Distribution Constant
• To relate the rate of migration of a solute to its distribution
constant, we express the rate as a fraction of the velocity of
the mobile phase:

42
 Where KA retention factor

Solving for tR
tR = tM × (1+ k Vs/Vm)

Therefore this equation show the relation between

Retention Time and Distribution Constant

43
 The Retention Factor (K)
• The retention factor k is an important experimental quantity
widely used to compare the migration rates of solutes in
columns.

• It is the amount of time on a solute spends in the stationary

phase stationary phase relative to the time it spends to mobile
phase.
For solute A, the retention factor kA is defined as

where KA is the distribution constant for solute

44
To show how kA can be derived from a chromatogram, we
substitute Equations

This equation rearranges to

= ts/tm

This equation express the relation between retention time &

retention factor 45
46
 selectivity factor ()
•The selectivity factor is the ability of the chromatographic system
to discriminate different analytes.

•The selectivity factor  of a column for the two species A and B

is defined as:

•The distance between the peak maxima reflects the selectivity of

the system.

•The greater the distance, the higher the selectivity.  is always

greater than unity.

47
 Applications of chromatography
• Chromatography has grown to be the premiere method for
separating closely related chemical species.

• In addition, it can be employed for qualitative

identification and quantitative determination of separated
species.
• Modern Chromatographic methods have many applications
including:

48
Qualitative analysis

A chromatogram provides only a single piece of qualitative

information about each species in a sample, its retention time or
its position on the stationary phase after a certain elution period.

it is a widely used tool for recognizing the presence or absence

of components of mixtures containing a limited number of
possible species whose identities are known

Quantitative analysis

Chromatography can provide useful quantitative information

about the separated species.

49
 Quantitative column chromatography is based upon a
comparison of either the height or the area of the analyte
peak with that of one or more standards.

 Quantitative analysis is the determination the

concentration of each constituent in the separated mixture.

50