Transcript PLANNAR CHROMATOGRAPHY

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.
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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
c- C-4
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):
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Samples are applied as a solution in any volatile
solvent using glass Capillaries for Qualitative,
Preparative applications. Graduated syringes are
used for Qualitative analyses.
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The spots 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.
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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
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:
Buffer solutions and aqueous acidic or basic solutions can be used.
used.
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 using
different solvent systems.
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.
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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:
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These reagents are used for the detection of
certain classes of compounds. They are usually
destructive.
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Dragendorff΄s reagent for Alkaloids.
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Ferric Chloride (FeCl3) for phenolic compounds.
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Aniline phthalate for sugars.
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Ninhydrine for nitrogenous
Amines, Amino acids.
compounds
as
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
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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.
Application:
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.
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2- Quantitative:
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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:
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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
determined as dots. Apply sample as in TLC.
are
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:
As in TLC.