CONVENTIONAL SEPARATION

TECHNIQUES

Topic 6
Planar Chromatography-Paper &
thin layer chromatography

22nd March 2011

DrSabiha/CHM421/Dec 10-Apr11

CHROMATOGRAPHY
Chromatography technique is widely used for the
separation, identification and determination of the
chemical components in complex mixtures.
Therefore , chromatography is used for QUALITATIVE and
QUANTITATIVE analysis.
Is defined as a technique in which the components of a
mixture are separated based upon the rates at which they
are carried through a stationary phase (S) by a gaseous or
liquid mobile phase (M).

Classification of chromatographic techniques

Adsorption Stationary phase is a solid on which sample
components are adsorbed. Mobile phase may be liquid
(LSC) or a gas (GSC)
Partition Stationary phase is a liquid supported on an inert
solid. Mobile phase may be liquid (i.e. LLC) or a gas
(GLC)
Ion-exchange Ion-exchange resin as stationary phase.
Mechanism of separation is based on ion-exchange
equilibrium.
Pore penetration solvated molecules are separated based on
on their sizes by their ability to penetrate a sievelike
structure (the stationary phase).

LSC or GSC
LLC
The stationary phase is solid 1. Normal-phase LLC
and is supported in the
2. Reversed-phase LLC
pores of a paper ( in Paper
(more widely used)
Chromatography) or on a
flat plate of silica ( in TLC).
Here the mobile phase
moves through the
stationary phase by capillary
action or under the
influence of gravity.

Normal-phase LLC
Polar stationary phase
(e.g. methanol on silica)
and non-polar liquid
phase (hexane)
for retention of polar
component and elution
of non-polar
component

Reversed-phase LLC
(more widely used)
Non-polar stationary
phase (e.g. hexane on
silica) and polar liquid
phase (methanol)
for retention of non-polar
component and elution of
polar component

PLANAR CHROMATOGRAPHY
Paper Chromatography (PC)
-Solid stationary phase, Liquid mobile phase (LSC)
In PC the stationary phase is the paper itself which contain
cellulose fibers made-up of hydroxyl groups which are polar.
The mobile phase liquid will be less polar; it is usually a
mixture of organic solvents (alcohols, ketones, aldehydes etc.).
Advantages: PC is cheap, fast , sensitive and have wide
applications. However the reproducibility of the results is low.
Disadvantage is it cannot withstand corrosive chemicals.

Principles and techniques of PC.

1. Sample (analyte) application.
A sample is dissolved in a volatile solvent. The sample is
spotted using the capillary tube on a line previously
drawn on one end of the PC as shown.

The spot should be dried first before you spot again the
second time. This is to make sure that the diameter of the
spot is less than 5 mm. Solute is "spotted" on paper
Chromatograph is "developed by dipping one end in the
mobile phase. Two modes: Ascending or Descending

2. Development of chromatogram
After spotting all the standards and samples are
completed , all the spots are allowed to dry first before it
is dipped into the development solvent (mobile phase).
The development tank should be close so that the
development chamber is saturated with the development
solvent.
This is to make sure that the spots move upward
smoothly by capillary action without interruption.
The solvent will carry the various spots upward at
different rate until the solvent front is about of the PC.

3.Identification Retention factor (Rf)

Rf =

Height of spot
Height of solvent

By comparing the Rf of samples to that of standards, the

identity of the samples can be identified.
Sample with same Rf value with the standard confirms its
presence.

The solvent moves through

the paper, drawn by
capillary action
Solutes move as spots with
a rate depending upon
how much time they spend
in the stationary phase vs.
the mobile phase which is
determined by their
partition coefficient (Rf
value).
A

Standard

unknown
sample

What is wrong with this student's paper

chromatography chamber setup?

A student has developed a chromatogram as

shown in the picture above. Will this
chromatogram yield good results?

A student removed this chromatogram from the

development chamber and allowed the solvent to
dry.What did he forget to do?

THIN LAYER CHROMATOGRAPHY (TLC)

Thin-layer chromatography (TLC) is a chromatographic
technique that is useful for separating organic
compounds.
Because of the simplicity and rapidity of TLC, it is often
used to monitor the progress of organic reactions and to
check the purity of products.
Thin layer chromatography ( also known as open-column
chromatography or thin-film chromatography) is a
technique whereby separation occurs on a thin layer of
adsorbent which is attached to an inert support usually
glass.

Advantages of TLC over paper

1. The separation occurs much faster,
2. The spots are more discrete, and
3. It can withstand corrosive detection
reagents, without harming the substrate
or adsorbent.

ADSORBENTS FOR TLC

1.

Silica gel
Silica gel can be obtained with small admixture
(additive) of calcium sulphate ( increases the binding to
the plates ), and also with flourescent indicator. It can
also be obtained without binder.

2.

Kieselguhr
Kieselguhr is less polar than silica gel and thus is less
strongly adsorbent.
Thick layers cannot be made easily, and the amount of
substance that can be applied is less than with silica gel.

3. Alumina
Alumina has the adsorbent properties of silica
gel but the degree of adsorption depends on its
pH and the amount of activation.
4. Cellulose
Although the chromatographic principles
involved in using cellulose layers are mainly
those of paper chromatography, the spots are
sharper on TLC than paper and give better
separation. It is available with or without
binder.

PREPARATION OF ADSORBENTS
1. Silica gel and Kieselguhr plates may be prepared by making
a slurry of 50 g of the substrate with 110 mL of water and
stirring continuously for 1 minute. The mixture sets in 2 4
minutes, although this may be prolonged by adding more
water. This amount will cover five 20 x 20 cm plates with
deposition thickness of 0.25mm.
2. Alumina plates with or without binder and with or without
flourescent indicator, 30 g is mixed with 40 mL of water.
This amount cover five 20 x 20 cm plates.
3. For cellulose plates , 25 g is mixed with 90 mL of water. It is
mixed thoroughly and rapidly for 2 minutes. This cover five
20 x 20 cm plates.

How does thin layer chromatography

work?
The stationary phase - silica gel
Silica gel is a form of silicon dioxide
(silica). The silicon atoms are joined
via oxygen atoms in a giant covalent
structure. However, at the surface of
the silica gel, the silicon atoms are
attached to -OH groups. So, at the
surface of the silica gel you have SiO-H bonds instead of Si-O-Si bonds.
The diagram shows a small part of the
silica surface.

Polar OH will form

H-bond or have van
der waal / dipoledipole interaction
with other polar
compound

surface

What separates the compounds as a chromatogram

develops?
1. As the solvent begins to soak up the plate, it first dissolves
then carried up the compounds in the spot that you have put
on the base line.
2. The separation of compounds depends on ;
- how much attraction there is between the molecules of the
compound and those of the solvent
- how much attraction there is between the molecules of the
compound and the silica gel
the more strongly a compound is adsorbed, the less distance it
can travel up the plate

PREPARATION OF THE THIN-LAYER PLATE

All glass plates should be cleaned by acid washing
and, just prior to use, rubbing the surface with
acetone to remove any grease.
Spreading apparatus is available commercially.
The spreader have rollers which rest on an
inflatable bag.
The plates are placed on the rollers and when the
bag is inflated, the plates are firmly pressed
against a metal ridge

 The apparatus can therefore be used to coat plates of

differing thickness.
All spreading apparatus consist of a flat base into which
the plates may be inserted and firmly held.
Most spreader can cope with different sizes plates usually
available, that is 5 x 20 cm, 10 x 20 cm and 20 x 20 cm.
The prepared adsorbent or substrate is poured into a
spreading tray which is then pulled from one end of the
row of plates to the other.

Manual TLC Plate coater

ACTIVATION OF PLATES
Before the plates can be used , they must be heated to
drive off water which acts as an impurity and prevents
good separation.
Often the water is firmly bound to the adsorbent and so
the plate must be heated strongly.
The amount of heating depends on the type of separation
needed.
For hydrophilic or polar compounds air dry or drying
with hair dryer is often adequate, but for hydrophobic or
non-polar compounds stronger heating is necessary.

 Silica gel and alumina plates with binder need to

be air dried for about 30 minutes and then
activated by heating in an oven for about 30
minutes at 100 oC, ( not over 105 oC ).
Silica gel and alumina plates without binder need
to be air dried for 1 - 2 hours and activated at
120 oC for about 60 minutes.
Cellulose plates should be air dried for 30 minutes
and activated for 10 minutes at 105 oC.

STORAGE OF PLATES
Storage racks are available commercially which
take 5 x 20 cm, 10 x 20 cm and 20 x 20 cm plates,
and these can be stored in bulk in a sealed desiccator
cabinet which contains a tray of silica gel to prevent
deactivation.

 Storage in a perfectly dry atmosphere is

essential once the plates have been activated.
It is preferably to air dry the plates, store in a
desiccators cabinet and activate them
immediately before using.
If the plates have been kept for prolonged
periods after activation ( weeks or longer ), they
may have to be reactivated by reheating in an
oven for the necessary time.

SPOTTING THE CHROMATOGRAM

The technique of spotting the chromatogram
is basically the same as in paper
chromatography.
Thin layers generally cannot take as much of
a sample as paper can.

 When spotting the TLC plates, therefore care must be

taken not to overload the spot otherwise streaking will
result.
Another important factor is that
care must also be taken not to
disturb the thin layer during
spotting.
With patience and practise it is
possible to avoid touching the layer
with the end of the micropipette
during spotting or streaking a TLC
plate.

DEVELOPMENT OF THE CHROMATOGRAM

Development tanks

 Development of the chromatogram is carried out

commonly by the ascending technique, although
apparatus is available for descending technique.
The spotted TLC plates are placed in the storage
rack and then dipped into the developing solvent
in a closed tank. The plates are left until the
solvent rises 2/3 of the height of the plates.

DETECTION OF COMPONENTS IN TLC

1. It is generally true to say that all detection
reagents commonly used in paper
chromatography may be used with thin-layer
chromatograms.
2. In addition, however, because of the chemical
inertness of the thin-layer, far more harsh or
corrosive detection reagents can be used than
with paper chromatograms.
3. If the spots are florescence than they can be
viewed under UV light.

DETECTION OF COMPONENTS IN TLC

BESIDES VISUAL OBSERVATION
1. TLC plates normally contain a fluorescent indicator which
makes the TLC plate glow green under UV light of
wavelength 254 nm.
2. Compounds that absorb UV light will quench the green
fluorescence yielding dark purple or bluish spots on the
plate.
3. Simply put the plate under a UV lamp, and the compounds
become visible to the naked eye.
4. Lightly circle the spots, so that you will have a permanent
record of their location for later calculations.
5. Use Retention factor (Rf ) formula.

Spraying cabinet

UV light

Florescent spot

Stationary phase: is solid or an immobilized liquid that is

fixed in place either in column or on a planar surface.
Mobile phase: is liquid or gas that carries analytes
through the stationary phase.
Elution: Process in which solutes are washed through a
stationary phase by the movement of a mobile phase
Retention factor: is used to express the migration rate of
solute on column. It is the amount of time a solute spends
in the stationary phase relative to the time it spends in the
mobile phase.

Concepts used in liquid-liquid extraction and liquidsolid extraction.

Liquid-liquid extraction:
-is a method to separate compounds based on their relative solubilities in
two different immiscible liquids.

Liquid-solid extraction:
- is separation technique between liquid and solid extracting phase. Solid
phase consist of hydrophobic organic compound that is chemically bonded
or coated to silica. Solid phase will attract hydrophobic compound in the
sample by van der Waals interaction and extract them from aqueous
solution.

Techniques to visualize the spots:

1. The spots can be visualized by exposing the plate to

iodine vapour in the tank. The colorless spots are then
marked.
2. Spraying with the concentrated sulphuric acid and
heating to char. The organic compounds will form black
spots.
3. For florescence spots, it can be viewed under UV light.