CHM-507 4(3-1)
FUNDAMENTAL ANALYTICAL CHEMISTRY
CHROMATOGRAPHY Principle,
Classification
Applications
ZAHID ASGHAR BAJWA
DEPARTMENT OF CHEMISTRY
GCUF
Chromatography
Mikhail Tswett: A Russian Botanist & Chemist
Produced a colorful separation of plant
pigments through a column of calcium
carbonate in 1903
He first time used the term chromatography in
1906 for this novel separation
(Chroma-Graphein- Colour writing )
Chromatography
Chromatography is a broad range of physical methods
used to separate complex mixtures by their distribution
b/w two immiscible phases – one that is stationary and
one that moves.
• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.
Terminology:
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)
• Stationary Medium – the part of the apparatus that does
not move with the sample (stationary phase)
Definition of Chromatography
Simplified Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile
phase and the stationary phase.
Explanation:
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases
Chromatography
• Distribution of components of mixture between two phases is
governed by distribution constant
K = C m Cs
• Mechanism - selective retardation caused by interactions with
bonded phase of stationary phase
Chromatography
Chromatogram - Detector signal vs.
retention time or volume
Detector Signal 1 2
time or volume
Classification of Chromatographic Methods
Classification of Chromatographic Methods
Separation
Equipment Mobile Phase Application
Mechanism
Shape based Based based
based
classification Classification classification
classification
Types Chromatography Based on
Shape of Equipment
Planar Chromatography is a
technique in which We make
use of a stationary phase in
the form of a sheet or flat
surface of a paper or any
other material such as metal,
glass or plastic plate coated
with suitable adsorbent with
the help of a binder
Column chromatography is a
technique in which we make use
of a stationary phase in the
form of a packed column using
suitable adsorbent and binder
Gas Chromatography
11
13
Classification Based on Phases
Classification on Mechanism of Separation
•Partition Chromatography
•Adsorption Chromatography
•Size Exclusion Chromatography
•Ion Exchange Chromatography
•Affinity Chromatography
Classification on Mechanism of Separation
• Adsorption - for polar non-ionic compounds
• Ion Exchange - for ionic compounds
• Anion - analyte is anion; bonded phase has positive charge
• Cation – analyte is cation; bonded phase has negative charge
• Partition - based on the relative solubility of analyte in mobile
and stationary phases
• Normal – analyte is polar organic; stationary phase MORE polar than
the mobile phase
• Reverse – analyte is nonpolar organic; stationary phase LESS polar
than the mobile phase
• Size Exclusion - stationary phase is a porous matrix; sieving
• Affinity - Analyte and SP has key & loch pH depending
relation
Partition Chromatography
Solute equilibriates
between the mobile phase
and the liquid stationary
phase.
Adsorption Chromatography
probably one of the oldest
types of chromatography.
The equilibration of the solute
components between the
mobile and solid stationary
phase accounts for the
separation.
Size Exclusion Chromatography
• This type of chromatography
lacks an attractive
interaction between the
stationary phase and solute.
• Porous gel separates the
molecules according to its
size.
Affinity Chromatography
• Most selective type of
chromatography
• It utilizes the specific
interaction between one kind
of analyte molecule and a
second molecule that is
immobilized on a stationary
phase
• The immobilized molecule
may be an antibody to some
specific protein
Ion-exchange Chromatography
• Ion-exchange separations are carried out in columns packed
with an ion-exchanger.
• Analyte ions of the opposite charge in the mobile liquid phase
are attracted to the ion-exchanger by electrostatic forces.
Cation Exchanger
RSO-3 …… Na+ + +
NH3R/ RSO-3 …… +NH3R/ + Na +
exchanger counter ion charged molecule bound molecular exchanged
to be changed ion ion
Anion Exchanger
(R)4N+ …… Cl- + -OOCR/ (R)4N+ …… -OOCR/ + Cl-
exchanger counter ion charged molecule bound molecular exchanged
to be changed ion ion
Applications of Ion Exchange
Chromatography
1- Water softening:
Removal of Ca2+, Mg2+ & other multivalent ions causing
hardness of water by filtration through a layer of strong
cation resin.
2-Water demineralization:
Removal of cations & anions dissolved in water. Usually carried
by the two step technique in which two columns of strongly
acid cation exchanger in [H+] form & strongly basic anion
exchanger in [OH-] form are used in sequence.
3- Neutralization:
Cationic exchanger in [H+] can be used to neutralize alkali
hydroxide & anionic exchanger in [OH+] form to neutralize
the acidity.
Separation and Retention Theory
Detector Response
Time scale
t0 = hold up time tR= retention time W= width at base
t/R = Corrected or adjusted retention time=t R - t0
Theoretical Plate
• Theoretical plate number (N) & Efficiency of a Column
N = 16 (tR/wb)2
• HETP = L/N
Different Types of chromatography
Mode or type Stationary phase Mobile phase Mechanism
Adsorption Solid that attracts Liquid or gas Solutes move at different rates
Chromatography the solutes according to the forces of attraction
to the stationary phase.
Partition Thin film of liquid Liquid or gas Solutes equilibrate between the 2
Chromatography formed on the phases according to their partition
surface of a solid coefficients
inert support
Ion Exchange Solid resin that Liquid Solute ions of charge opposite to the
Chromatography carries fixed ions containing fixed ions are attracted to the resin
& mobile electrolytes by electrostatic forces & replace the
couterions of mobile counterions.
opposite charge
attached by
covalent bonds
Gel filtration or Porous gel with no Liquid Molecules separate according to
Molecular Exclusion attractive action their size:
Chromatography on solute 1.Smaller molecules enter the pores
molecules of the gel, and need a larger volume
of eluent.
2.Larger molecules pass through the
column at a faster rate.
Affinity Solid on which Liquid or gas Special kind of solute molecules
Chromatography specific molecules interact with those immobilized on
are immobilized the stationary phase
Classification based on Applications
Analytical
Chromatography
Long & thin column-
Separation, Identification,
Quantification
Semi-preparative Preparative
Chromatography Chromatography
Short & fat Short & fat
column- for small column--for large
scale Purification scale purification
