PLANNAR

CHROMATOGRAPHY
 It
includes two types:
1- Thin Layer Chromatography
(TLC).
2- Paper Chromatography (PC).
 Thin Layer Chromatography
(TLC)
 In this type a thin layer of a solid
coating material is spread on a
suitable supporting surface.
 Types Supporting Surfaces:

1- Glass Plates.
2- Plastic sheets.
3- Aluminum sheets.
 Coating materials:
1-Adsorption:
a- silica gel (silicic acid).
b- Alumina (Aluminum oxide).

c- Magnesium Silicate (florisil) for Lipids.

2-Partition:
a- Cellulose.

3-Ion Exchange TLC:

a- Cellulose phosphate

4-Reversed – phase partition:

a- C-18 silica gel
b- C-8 silica gel
 5- Polyamides:
a- E-poly caprolactam
b- Poly acrylonitrite

6- Gel chromatography (Size

exclusion):
a- Sephadex G25
b- Sephadex G50
c- Sephadex G75
d- Sephadex G100
e- Sephadex LH20
Binders:
These are materials used to hold the thin
layer of the coating material into the surface
of the supporting plates.

Types of binders:
a- CaSO4 (Plaster of Paris) Gypsum (10-15%)

b- Silicon dioxide
c- Starch (1-3 %)
d- Organic polymers e.g. polyvinyl alcohol.
Indicators:
These are materials mixed with the
coating material and binder to help
locating the spots on the TLC. The most
common used indicator is the fluorescent
materials (silica gel 60 F254).
 Sample Application (Spotting):

 Samples are applied as a solution in any

volatile solvent using glass Capillaries for
Qualitative, Preparative applications.
Graduated syringes are used for Qualitative
analyses.

 Thespots must be about 1-1.5cm away from

the bottom of the plate and 0.5 cm away
from the plate sides and 0.5 cm away from
each other.
 Development:

Chromatographic Jars (Tanks) made of Glass

with air-tight lids of different sizes containing
the mobile phase are used for developments.
The solvent must be left in the Jars enough
time before developing the plates for
saturation.
 Developing system:
(Mobile phase – developing solvent)
Using a single solvent (very rare) or mixture of solvents to allow the
separation. The type of adsorbent used will affect the choice of the
developing system.

Adsorption:
Usually mixture of non polar organic solvents are used.

Partition:
More polar organic solvents such as butanol- acetic acid – water are
used. Buffer solution are also used in partition chromatography.

Ion Exchange:
Acidic or basic solutions are used.(HCl, NaOH, NaCl, LiCl)

Reversed phase:
Methanol- acetonitril- water- acetone-acetic acid are used as
mixtures.

Polyamide:
Mixtures of Water – ethanol- acetone can be used.

Gel:
 Types of developments:
A- Ascending:
1- Single development:
The solvent system is allowed to move through
the stationary phase one time only against
gravity.

2- Repeated developments:
a- Multiple developments:
The plated are developed more than one time
using the same solvent system. The plates
must be completely dried after each
development.
b- Stepwise developments:
The plated are developed more than one time
3- Two-dimensional development:

Is used to verify if a given spot on TLC

using the above methods of
development (one Dimensional) is one
pure compound or mixture of two
closely related compounds. The spots
are applied to one corner and the plate
developed as usual. The plate is then
rotated 90 ˚C and then developed again.
This method allow better separation of
related compounds.
 One compound

.
Two closely related compounds
.
B- Centrifugal (chromatotron):

This method of development require the

use of Chromatotron. Simply it is
composed of motor rotate in high speed
(about 1000 rpm) to accelerate the speed
of the mobile phase. Circular plates are
used and the mixture is applied to the
center of the plate. Mobile phase is also
allowed to flow from the plate center to
the edges. The separated materials will
appear as concentric zones.
Chromatotron is used only for
preparative work.
 Mobile Phase

Circular plate Sample

application

Motor
1000 rpm
 Visualization (Detection of spots):
A- Universal methods:
1- Destructive methods:
The plated are sprayed with corrosive reagents and
then heated in oven where organic compounds will
give charred spots. After this treatment the materials
can not be recovered.
e.g. Anisaldehyde / H2SO4
Vanillin / H2SO4
 2- Non – Destructive methods:
In these methods the materials can be
recovered.
 Day light for colour compounds.
 UV light for fluorescent compounds
(conjugated double bonds).
 I2 vapour for any compounds contain at
least one double bond
 Spray with water where organic
compounds appear as white opaque
spots.
B- Specific Methods:
 These reagents are used for the detection
of certain classes of compounds. They are
usually destructive.

 Dragendorff΄s reagent for Alkaloids.

 Ferric Chloride (FeCl3) for phenolic

compounds.

 Aniline phthalate for sugars.

 Ninhydrine for nitrogenous compounds as

Amines, Amino acids.
Rate of flow (Rf Value):
Distance traveled by the spots
Rf = -----------------------------------------
Distance traveled by the solvent
The Rf of any compound must be less than one.
Solvent front

Distance travelled by
the solvent

Distance travelled by
the spot

Start line
 Tailing in TLC:
In some cases instead of getting round spots a Tailed
or comet like spots are obtained leading to
overlapping of the spots and poor resolution.

Tailed or comet like spot

Reasons and solution for tailing
problem:

1-Ionic characters of acids and bases when they

are chromatographed under neutral
conditions.
Solution: add acids or bases to the developing
system.

2-Application of large amounts of material.

Solution: decrease conc. of material.

3-Unproper choice of solvent system.

Solution: change the solvent system.
 Applicatio
n:
1- Qualitative:
 Identification through comparison of the Rf value
with that of Reference material.
 Determination of Complexity of mixtures. That will
be indicated from number of spots.
 Determination the purity of materials.
 Monitoring the progress of Chemical reactions.
 Monitoring of column chromatography.
 Development of finger print TLC for extracts,
volatile oils or pharmaceutical preparation for
future identification and comparison.
In this application plates 5×5, 5×10 cm with thin film
of coating material are usually used.
 2- Quantitative:
In this case an accurate volume of samples are
applied using syringes. The dimensions of plates
range from 5x10 to 20x20 according to the number
pf spots used. The plates are developed as usual in
the chromatographic tanks. After development the
concentration of material can be determined by:
 Spot area measurement: Which is directly proportional
to the conc. of materials.
 Photodensitometry: Measure transmittance, reflection
or fluorescence of spots.
 Radioactivity: For radioactive material.

These measurements are done using TLC Scanner

connected to computer that perform all
calculations.
3- Preparative TLC:
In preparative application 20×20
plates with thick layer of adsorbent
0,25m are used. The mixture is apply
as bands and a pilot or guide spots
may be used in one side of the plate
to enable the detection of the spots
location.
Paper Chromatography
(PC)
Stationary phase:
Papers (cellulose), mechanism of
separation is through partition.

Mobile phase:
As TLC but more polar mixtures are usually
used. Buffers can also be used.

Sample application:
A line drawn by pencil, spot places are
determined as dots. Apply sample as in TLC.
 Development:
1- Ascending: The mobile phase move against
Gravity.
2-Descending: The mobile phase move with
Gravity.
3-Horizontal.
4- Radial.

Visualization:
As TLC but must be non-destructive or specific
with no use of heat.

Applications: