FUNDAMENTAL OF ANALYTICAL CHEMISTRY

Course Code: 2011

Dr. Huusain Ullah

Ph.D (Chemistry)
Email: chem.hussain@gmail.com/chem_hussain@uo.edu.pk

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 Summary
Separation Techniques
Advanced Separation Techniques Classical Separation Techniques

Evaporation Extraction Precipitation

G. C H.P.L.C Capillary Distillation

electrophoresis
Crystallization

Applications

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 Modern/Advance Separation Techniques
Chromatographic
The word chromatography is a combination of two Greek words chroma “colour and
Graphein/Graphy "to write“, collectively it means colour writing. It is a collective term
used for the separation, identification and quantification of components of a mixture

Definition: According to Keuleman, chromatography may be defined as a “physical

method of separation of a mixture of solutes brought about by the dynamic partitions or
distribution of dissolved materials between two immiscible phases, one of which (called
the mobile phase) moves past the other (called stationary phase)
According to Micheal Tswett, Chromatography is a process used in separating
substances by filtering their solution through a column of a finely powdered adsorbent
and then developing the column with a solvent. 3
 History

The history of chromatography ranges back to Runge (8150). However, a Rusian

botanist Mikhail Tswett in 1906 is credited for the world chromatography.
Chromatography, literally “colour writing", was first employed by Russian Scientist
Mikhail Tswett in 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 by passing the solution of these
compounds into the glass column (adsorption chromatography) which was packed
with finely divided chalk and then washed with light petroleum.

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 Since these components have different colours (green, orange,
and yellow, respectively) and begin to separate in the form of Sand
bands they gave the technique its name.
Chlorophyl
However, his initial experiment could not attract much attention Β-Carotene
as it was published in Rushian. Silica
The next development was found in 1930s when Redever and his
co-worker (1931) separated Lutien and Zeaxanthine in CS2 using
CaCO3 as column materials and it led to solid-liquid Sand
chromatography. Glass wool
Pinch Clamp
Tubing
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Later, the concept of Liquid-liquid chromatography was introduced by Martin and
Synge and isolated amino acids from proteins in 1941. this was the beginning of
partition chromatography, and both were awarded by a Nobel prize for his work in
1952. Tiselius was also awarded Nobel prize in 1948 for his contribution to
chromatography. Tiselius in 1948 and claessen in 1946 developed the classical
procedures and classified them into three basic groups.
1. Frontal analysis
2. Displacement analysis
3. Elution Technique

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 Classification of Chromatography

Chromatography can be classified in three ways:

(i) Based on the physical state of the mobile phase and stationary phase.
(ii) By the method of contact between the mobile phase and the stationary phase.
(iii) By the chemical or physical mechanism responsible for the separation of the sample’s
constituents.

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Classification based on the physical state
(i) The first method of classification is based on the physical state of the mobile phase and
stationary phase. The stationary phase may be a solid or a liquid film coated on a solid
surface while the mobile phase may be either a liquid or a gas. They fall into four categories
such as; a) Gas-Liquid, b) Gas-Solid, c) Liquid-Solid, d) Liquid-Liquid
i. Types of Chromatography based on physical state of mobile phase and stationary phase
Type Mobile Phase Stationary Phase Name of the method
s
1 Liquid Solid Liquid-solid chromatography (LSC)
2 Liquid Liquid Liquid-Liquid chromatography (LLC)
3 Gas Solid Gas-solid chromatography (GSC)
4 Gas Liquid Gas-Liquid chromatography (GLC)

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Classification based on contact between mobile phase and stationary phase

(ii) The second method of classification is based on contact between the mobile phase and stationary phase.
ii. Types of Chromatography based on contact between mobile phase and stationary phase
Types Stationary Phase Name of the method
1. Liquid-solid (i) Solid stationary phase held in a tubular column (i) Column chromatography
chromatography through which the mobile phase moves under the (ii) Ion-exchange chromatography
(LSC) influence of gravity or pressure.
(ii) Stationary phase being finely divided ion-
exchange resin held in a tubular column.
2 Liquid-Liquid Liquid stationary phase is held in the pores (cellulose Paper Chromatography
chromatography molecules) of a thick paper. Mobile phase moves through
(LLC) the stationary phase by capillary action or under the
influence of gravity.
3 Gas-solid Solid stationary phase held in a tubular column Gas-solid chromatography (GSC)
chromatography
(GSC)
4 Gas-solid Liquid stationary phase adsorbed on a porous solid held in Gas-Liquid chromatography (GLC)
chromatography a tube or adsorbed on the inner surface of a capillary tube.
(GSC) 9
 ii. Types of Chromatography based on contact between mobile phase and stationary phase
Types Mechanism involved Name of the method
1. (LSC) (i) Adsorption (i) Adsorption chromatography
i. Column Chromatography (ii) Ion-Exchange (ii) Ion-exchange
ii. Ion-Exchange chromatography
2 (LLC): Liquid-Liquid chromatography Partition of solute between two immiscible liquids Paper Chromatography
3 (GSC): Gas-solid chromatography Adsorption chromatography Adsorption chromatography

4 Gas-solid Partition of solute between the gaseous mobile Partition chromatography

chromatography phase and a liquid stationary phase adsorbed on a
(GSC) porous solid held in a tube or adsorbed on the inner
surface of a capillary tube.
Note:
Thin layer chromatography can also be called as Liquid-Liquid chromatography (partition chromatography). When a
liquid is adsorbed on a finely divided solid coated on a glass plate then the liquid act as a stationary phase and the
solid act as a support. The moving phase is a liquid called a mobile phase that may be any solvent of interest.

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 Classification of Chromatography-1
Based on physical state of mobile and stationary phase Based on contact between Mechanism of
mobile phase and separation of the
Column Chromatography
Super Critical Liquid
stationary phase sample’s constituents.
Chromatography
Gas Chromatography
Planar Chromatography
Liquid
Paper/partition Thin Layer
G.S.C G.L.C Chromatography
Chromatography
Liquid under
critical pressure (L.L.C) Solid
Gas Solid Liquid Liquid
Liquid Liquid
Gas

Mobile Phase
(L.S.C)
Mobile Phase stationary phase as
Liquid Solid-stationary Occluded in cellulose layer Mobile Phase thin layer
Stationary phase Mobile Phase Mobile Phase
hold in tube (L.L.C)
column Liquid stationary Mobile Phase Stationary
HPLC phase adsorbed on a Solid-beads (Liquid) (Liquid)
porous solid held in a Solid-Silica Solid-ion exchange resin
tube or adsorbed on Size Exclusion
Liquid Solid the inner surface of a Adsorption Ion Exchange Affinity Chromatography
Chromatography
capillary Chromatography Chromatograph
(L.S.C)
Liquid phase used under Stationary phase (L.S.C) (L.S.C) Gel Permeation Sieve separation
pressurized conditions hold in tube Chromatography Chromatography 11
 Classification of Chromatography-2
Classification based on developmental procedure

1. Elution Technique
2. Displacement Technique 3. Frontal Technique
In partition and adsorption
In adsorption chromatography, It is used for the purification of
chromatography, it is a
one component has a strong the least retained solute. If a
stepwise removal of
affinity to the stationary phase mixture contains two components
different components of a
and displaces the loosely bonded A + B and A has less affinity than
mixture due to differences
component to the stationary B, the A will be eluted first then
in their rate of migration
phase. The displacer is eluted last B and then A + B (some mixture).
along the stationary phase.
then all the other components.

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1. Elution Technique
This technique was first used by Tswett in 10903 for the separation of leaf pigments.
This is the most important chromatographic technique and is most widely used in all
methods of chromatography e.g., GC, GLC, LLC, and LSC.
In fact, it is the only technique used in partition chromatography. It is a stepwise removal
of different components of the mixture from each other due to their different rate of
migration along the stationary phase.
In this technique, the stationary phase is used in the form of a column and the sample is
applied to it and then eluted with the mobile phase.

Elution: The stepwise addition of solvent until all the components of a mixture are
Remember removed or stepped out from the column is called elution. The eluate is a combination of
the mobile phase and the components emerging from the chromatographic column

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The mobile phase in this case has a greater affinity towards the component
of as compared to the stationary phase. So, these are washed away with
the mobile phase but as they have different rates of migration over the
stationary phase, therefore separation is achieved, and the components of
the mixture move slowly as compared to the solvent (mobile
phase/eluent). The concentration of solute in the mobile and stationary
phases are represented as Gausian curve in shape.
Suppose, we have a mixture of A, B and C, these have different migration
rates as A > B > C. If this mixture is eluted, then component A will be
Note: Eluent: the solvent
eluted/collected first at the end of the column as eluate because it has a
carrying the analyte is
faster migration rate. called eluent.

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Mainly there are three types of elution procedures.
i. Isochrtaic Elution. ii. Stepwise Elution. iii. Gradient Elution

i. Isochrtaic Elution (single solvent or a constant solvent mixture)

Isocratic elution is a term used in chromatography when the single or mixture of mobile
phases has a constant concentration/composition. Here, the concentration of the mobile
phase is constant (hexane/ethyl acetate) throughout the chromatographic process.

(t = 0 mint) Same composition throughout (t = 20 mint

(10/90) (10/90)

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In this process, we can observe the peak width increasing with retention time linearly in the
chromatogram. However, this leads to a disadvantage – the late-eluting peaks for late elution get
very flat and broad. Therefore, these broad peaks become difficult to be recognized as peaks.

Moreover, in isocratic elution, the selectivity does not change according to the column
dimensions. This means the selectivity does not depend on the changes in column dimensions.
Here, the length and diameter are considered as column dimensions. Therefore, the peaks elute in
the same order.

Definition
Isocratic elution is defined as a static ratio of the mobile phase components for the
entire analysis (10/90).

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ii. Stepwise Elution

In this, method, a set of eluants (solvent) with increasing eluting powers are used to
separate the components which are having a greater affinity towards the stationary
phase. The mobile phase is changed after a certain period of time. These are changes in
such order that the proceeding solvent is a stronger eluting agent or polarity than the
first one and is able to elute a given component from the column more easily.
Example. Suppose, Initially we use benzene, then chloroform, then ethyl acetate and
then methanol (i.e., from nonpolar to more polar).

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Gradient Elution
Gradient elution is a term used in chromatography when the mobile phase has a
varying composition/concentration. In other words, the concentration of the mobile
phase does not have to remain constant.
It is mostly used in adsorption chromatography. For example, in HPLC, a common
separation method uses methanol at 10% initially and ends at 90%, by increasing
the concentration gradually and a concentration gradient (concentration difference)
is produced down the column.

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The mobile phase has two components: a weak solvent and a strong solvent. A weak
solvent allows the solute to elute slowly while a strong solvent causes the rapid elution
of the solute. In reverse-phase chromatography, we use water as the weak solvent and
organic solvent as the strong solvent.
Gradient elution is able to produce high peak heights in a short operation cycle compared
with isocratic elution. For these reasons, gradient elution has been widely used in high-
performance liquid chromatography for analytical purposes.

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 Isocratic Vs Gradient Elution
Isocratic Elution
Definition: Isocratic elution is a term used in
chromatography when the mobile phase has a
constant concentration.
Mobile phase: Has a constant composition
Peak: The peaks is flat and broad.
Selectivity: does not depends on column
dimension.
Gradient Elution
Gradient elution is a term used in
chromatography when the mobile phase
has a varying concentration.
Has a varying concentration.
The peak is narrow.
Depends on column dimension.
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2. Frontal Technique

This is mainly a purification technique and can only be used to develop efficient
distribution in column technique. On this, the diluted solution (most often) of the sample
mixture is added continuously to the adsorption column until it is saturated. This is
contrary to the to displacement and elution procedure, where the discrete samples are
placed in the separating system and further processed. Frontal analysis operates only by
seapating portion of the first compound in a relatively pure state; each subsequent
component component is mixed with those components which were previously eluted.
As the components have different attractions towards the stationary phase so, the component having
the least attraction will move faster and is adsorbed down the column while the component having
strong attraction/adsorption towards the stationary phase will adhere at the start (upper part) of the
column.

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The solvent comes out first followed by the faster moving components that pass out
through the column then the second strongly adsorbed component also starts
migration and so on depending upon their degree of absorptivity.
The first eluted component is in the pure form then a mixture with other components
is obtained which means that this technique is used for the separation of one
component.
Suppose, we have a mixture of three components A, B and C, in dilute solution of
mobile phase that is continuously added into the column. Component A has the least
affinity towards the stationary phase while component C has a strong affinity towards
the stationary phase.

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When a mixture of the solution is added to the column then A will move faster and so the first
fraction obtained will contain pure A.
Similarly, component B has a greater affinity towards the stationary phase than component A but
least than component C. Component B will also try to move away but behind the component A.
so the second fraction will mainly contain component B but contaminated with some A. because
there is a continuous supply of A from the mixture solution. The third and last one solute will be
C in the purest form like A. this shows that frontal analysis is thus quite inappropriate for most of
the analytical applications of chromatography hence, cannot be applied for separation of a
mixture of components. However, frontal analysis has been completely overtaken by eltion
technique

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3. Displacement Technique
This is the separation procedure by a solvent which is effective only if the stationary phase is solid
and the solute is adsorbed on the stationary phase surface. The sample mixture is first applied to
the column, and each solute component thus competes for the adsorption sites on the solid
stationary phase.
At the start of the procedure, all the nearby adsorption sites will be completely occupied by the
components that are strongly retained in the stationary phase and have low affinity with the mobile
phase.
Moreover, as a band moves through the column/system, the components are distributed in the
column in order of their descending adsorption strength. Hence, the components that are strongly
adsorbed/retained by the stationary phase would not elute through the mobile phase or will elute
very slowly usually at the end of the chromatographic procedure.

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The components which are least strongly held in the mobile phase will have a
greater affinity for the mobile phase and will move rapidly through the flowing
system. The fast-moving component will elute first. However, the strongly held
components on the stationary phase which are difficult to elute by the mobile
phase can be displaced/ removed by any substance which will be more strongly
attracted by the stationary phase than any of the solute components (i., either
strongly adsorbed or less strongly adsorbed). This substance which removes the
strongly adsorbed solute is called a displacer.

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The displacer is allowed to pass through the system and moves the more strongly retained
component first (displaced the strongly attached solute by the displacer which is having
more adsorption property for the adsorbent). This component in turn moves to the next and
so on. Therefore, the displacer removes the adsorbent components (component having less
adsorption than the displacer) progressively through the distribution system, each component
moves forward until all the components are subsequently displaced.
The salute thus eluted are characterized by the order in which they are eluted and the amount
of each eluted component depends upon the length of each band, not on the height.
The main advantage of this method is that large sample quantity can be used. The
disadvantages of this technique is that it can be used for purification rather than quantitative
separation. It can be useful as a preparative technique but is not suitable for analysis.

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 Summary of Different mobile and stationary phases

The main differences in these techniques are either the mobile phase or stationary phase.

Paper Chromatography TLC G. C H.P.L. C

Mobile phase
Stationary phase Stationary phase Mobile phase

H2O etc
H2O occluded Silica gel etc Pet ether, acetone etc Stationary phase (Silica)
in Cellulose Molecules Mobile phase

Stationary phase Mobile phase

Solvent under
pressurized
Carrier gas conditions
Solid-Silica gel etc Liquid adsorbed on
Solid surface etc
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Principal
Chromatography usually consists of mobile phase and stationary
phase. The mobile phase refers any medium that use to separate
mixture of substances to dissolved in it. It may be a gas, a liquid or
supercritical fluid. The stationary phase is a porous solid matrix
through which the sample contained in the mobile phase
percolates/adsorb. The interaction between the mobile phase and
the stationary phase results in the separation of the compound
from the mixture.

For example, increasing the temperature above 31°C and pressure above 73 bar for Carbon dioxide creates a
supercritical phase, neither liquid nor gas but a combination of both properties. High diffusion like a gas but with
the solvation (ability to dissolve substances) of a liquid. Further manipulation of these conditions can be used to
extract a number of natural products. 28
Partition/ Distribution coefficient

The distribution of solute between the mobile and stationary phase. The distribution
coefficient is a constant, which is used to describe the distribution of sample components
between two immiscible liquids. It is equal to the ratio of the molar concentration of a
particular component in the stationary phase to its molar concentration in the mobile
phase.
It can also be estimated by the ratio of the solubility of the desired components in
stationary phase to solubility in mobile phase.

Each component is distributed between two phases with equilibrium and the separation
depends upon the distribution ratio or distribution coefficient.

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Suppose we have a component A of a mixture that is in equilibrium between the stationary
phase and the mobile phase.
(Stationary phase) As Am (Mobile phase)
Where,
[As] is the concentration of A in the stationary phase
[Am] is the concentration of A in the mobile phase
It can be represented by an equation
𝐴𝑠 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝐾= =𝐾=
𝐴𝑚 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
Each component of a mixture to be separated has a specific K value, which depends upon the
relative affinity of component for the stationary phase. The more the value of K for any
component (i.e., A here) the greater the affinity of component for stationary phase

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