CHROMATOGRAPHY

INTRODUCTION
THEORY & TECHNIQUES
APPLICATIONS
Introduction
What is chromatography?
Powerful separation method that has wide
applications in all branches of science

Method of separating a mixture of compounds

into its component
…………“History”
1906:Michael Tswett (Russian Botanist)
Crushed chalk placed in a column and leaf
extract passed through the column with a
solvent
Observed solution separated into
different coloured pigments (chlorophyll &
carotene)
Named it Chromatography (Greek word)
“Chroma” colour
“Graphos” writing
https://i.ytimg.com/vi/VgIiAwQZAZc/maxresdefault.jpg
HISTORY
1944: Martin & Synge published paper
about theory of chromatography (liquid
chromatography)

1952: Martin & Synge reinforce the theory

of chromatography with
demonstration on the separation of
amino acids
Then & Now?
Then… separation method followed by
identification & measurement
Now… Coupled with other major
analytical field
Infrared – GC-IR
Mass Spectrometry GC MS
LC MS

Absorption HPLC - AAS

Some definitions..
Definition

Chromatography is a separation technique based on the different

interactions of compounds with two phases, a mobile phase and a
stationary phase, as the compounds travel through a supporting
medium.

Components:
mobile phase: a solvent that flows through the supporting
medium

stationary phase: a layer or coating on the supporting medium that

interacts with the analytes

supporting medium: a solid surface on which the stationary phase

is bound or coated
Chromatography involves a sample (or sample extract) being dissolved in a
mobile phase (which may be a gas, a liquid or a supercritical fluid).

The mobile phase is then forced through an immobile, immiscible stationary

phase.
The phases are chosen such that components of the sample have
differing solubilities in each phase.
A component which is quite soluble in the stationary phase
will take longer to travel through it than a component which is not
very soluble in the stationary phase but very soluble in the mobile
phase.
As a result of these differences in mobilities, sample
components will become separated from each other as they travel
through the stationary phase.
The analytes interacting most strongly with the
stationary phase will take longer to pass through the
system than those with weaker interactions.

These interactions are usually chemical in nature, but

in some cases physical interactions can also be used.
The distribution of analytes between phases can often
be described quite simply. An analyte is in equilibrium
between the two phases.
The equilibrium constant, K, is termed the partition
coefficient; defined as the molar concentration of
analyte in the stationary phase divided by the molar
concentration of the analyte in the mobile phase
Chromatography:
Distribution Constant (recommended by IUPAC)
(old term: partition coefficient)

A mobile ↔ A stationary
cS
Kc  stationary
cM mobile
K ~ constant  linear chromatography

>>>K >>> Retention

CS = nS/VinS,theCstationary phase  Retention times
M = nM/VM

How to manipulate K?
Classical Model of Chromatographic
Column
Mobile Phase ( CO2 )
Mixture
Detector
Inject

Stationary Phase (Polymer)

Separation No Separation
Polymeric No
Stationary Stationary
Phase Phase

Time Time
Principles of Separation Techniques
AB 3.1
Molecular Physical property Separation Technique
Characteristic

Polarity Volatility Gas-liquid chromatography

Solubility Liquid-liquid chromatography
Adsorptivity Liquid-solid chromatography

Ionic Charge Ion-exchange chromatography

Electrophoresis

Size (mass) Diffusion Gel permeation

chromatography
Dialysis

Shape Sedimentation Ultracentrifugation

Liquid binding Affinity chromatography 1
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 POLARITY (affinity of like molecules
for each other)
PARTITION BETWEEN TWO PHASES

Solid/Liquid Liquid/Liquid Liquid/Vapour

A MAJOR FACTOR IN SEPARATION IS

Adsorption Solubility-Partition

AND THE METHODS INVOLVE

Solid adsorbents Two immiscible A solution and

liquids Its vapour

THE METHODS ARE GENERALLY KNOWN AS

Adsorption Liquid Gas-liquid 6

chromatography chromatography chromatography

 Different Kinds of Chromatography
(characterized by the mobile phase)
 Liquid chromatography (includes column
chromatography, thin-layer, and HPLC)
– Stationary phase: silica, alumina, etc.
– Mobile phase (moving phase): organic solvents
– Important properties: polarity
 Gas chromatography
– Stationary phase: a film of a polymer or a wax. The
film must have a high boiling point
– Mobile phase:gas (Helium is the usual carrier gas)
– Important properties: boiling point
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 Modes of Chromatography
(characterized by shape of stationary
phase
 Column chromatography
• Stationary phase is packed into a column

 Thin-Layer chromatography
• Stationary phase is coated onto glass, metallic or plastic
plate.

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Liquid-Solid Chromatography (Adsorption)
AB 3.2.1

Adsorption (吸附):
Some substances physically bind to the
surface of a solid polar substances

Polar compound
Large surface for adsorption
Often by OH (hydroxy group) to form H-bonding

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 Polarity of Selected Solutes and Solvents
Silica
Adsorption Solvent
Solute Solvent
Energy Strength

Hydrocarbon 0.07 Hexane 0.01

Halogen Derivativ 1.74 Benzene 0.32
Aldehyde 4.97 Chloroform 0.4
Ester Alcohols 5.27 Acetone 0.55
6.5 Pyridine 0.71
Acids/Bases 7.6 Methanol 0.95
Increasing Polarity
Adsorption Energy
the affinity of a solute with an adsorbent ( vary with adsorbant)

Solvent Strength
the affinity of a solvent with an adsorbent ( vary with adsorbant) 10
 Stationary Phase: Alumina

O OH OH OH OH

Al Al Al Al Al
O O O O O O

Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
2
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Examples of Adsorbents and Applications

Adsorbent Strength Application

Silicic acid(silica gel) Strong Steroids,amino acids,lipids

Charcoal Strong Peptides,carbohydrates

Aluminium oxide Strong Steroids,esters,alkaloids

Magnesium carbonate Medium Porphyrins

Calcium phosphate Medium Proteins,polynucleotides

Cellulose Weak Proteins

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2
Thin-layer chromatography and column
chromatography are different types of
liquid chromatography. The principle of
operation is the same!

 The mobile (moving) phase is a liquid.

 The stationary phase is usually silica
or alumina.--- a very polar layer of
adsorbent on an inert, flat support.

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3
Thin Layer Chromatography (薄層層析法)
1. The surface of the plate consists of a very thin layer of
silica on a plastic or aluminum backing. The silica is very
polar the stationary phase.

2. Spot the material at the origin (bottom) of the TLC plate.

3. Place the plate into a glass jar with a small amount of a

solvent in the glass jar. the moving phase.

4. Remove the plate from the bottle when the solvent is close
to the top of the plate.

5. Visualize the spots (Ultraviolet light, color reagent…etc)

Non-polar compounds will be less strongly attracted to the

plate and will spend more time in the moving phase. This
compound will move faster and will appear closer to the
top of the plate.

Polar compounds will be more strongly attracted to the plate

and will spend less time in the moving phase and appear
lower on the plate.
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Thin-Layer Chromatography: A
Two-Component Mixture

solvent front

component B Less polar!

solvent front

component B

component A More polar!

component A

origin origin origin

solvent front
mixture

Increasing Development Time 2

5
 Determination of Rf Values (Rate of Flow)
A measure of the movement of a compound compared with
the movement of solvent
solvent front

Rf of component A = component B
dA
dS dS
dB

Rf of component B =
component A
dB
dS dA
origin
1. Only reported to two decimal places
2
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 Thin-Layer Chromatography:
Qualitative Analysis
Ideally, the Rf value should be the
same of a given compound using
the same solvent
(Practically, the movement depends
on the structure and thickness of
the layer, the amount of water
remaining and effect of the
binding agents.
Advantages
 Simple
 Rapid
A B unknown
 Cheap 17
 Example: Thin-Layer Chromatography

O OH

Fluorene Fluorenone Fluorenol

a) Which one of these compounds is the least polar?

b) Which one of these compounds is the most polar?

c) What would be the relative order of separation on

the TLC plate remembering that CH2Cl2 is not very
polar?
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KLT PREPARATIF

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KROMATOGRAFI KOLOM