PLANNAR PLANNAR CHROMATOGRAPHYCHROMATOGRAPHY

It includes two types:It includes two types:

11- - Thin Layer Chromatography Thin Layer Chromatography (TLC).(TLC).

2- Paper Chromatography (PC).2- Paper Chromatography (PC).

Thin Layer Chromatography Thin Layer Chromatography (TLC)(TLC)

In this type a thin layer of a solid In this type a thin layer of a solid coating material is spread on a coating material is spread on a suitable supporting surface.suitable supporting surface.

Types Supporting Surfaces:Types Supporting Surfaces:

1- Glass Plates. 1- Glass Plates.

2- Plastic sheets.2- Plastic sheets.

3- Aluminum sheets.3- Aluminum sheets.

Coating materials:Coating materials: 1-Adsorption:1-Adsorption:

a- silica gel (silicic acid).a- silica gel (silicic acid). b- Alumina (Aluminum oxide). b- Alumina (Aluminum oxide).

c- Magnesium Silicate (florisil) for Lipids.c- Magnesium Silicate (florisil) for Lipids.

2-Partition: 2-Partition:

a- Cellulose.a- Cellulose. 3-Ion Exchange TLC:3-Ion Exchange TLC:

a- Cellulose phosphatea- Cellulose phosphate 4-Reversed – phase partition:4-Reversed – phase partition:

a- C-18 silica gela- C-18 silica gelb- C-8 silica gelb- C-8 silica gelc- C-4 silica gelc- C-4 silica gel

5- Polyamides:5- Polyamides:

a- E-poly caprolactama- E-poly caprolactam

b- Poly acrylonitriteb- Poly acrylonitrite

6- Gel chromatography (Size 6- Gel chromatography (Size exclusion): exclusion):

a- Sephadex G25a- Sephadex G25

b- Sephadex G50b- Sephadex G50

c- Sephadex G75 c- Sephadex G75

d- Sephadex G100d- Sephadex G100

e- Sephadex LH20e- Sephadex LH20

Binders:Binders:

These areThese are materials used to hold the thin materials used to hold the thin layer of the coating material into the surface layer of the coating material into the surface of the supporting platesof the supporting plates..

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

b- Silicon dioxideb- Silicon dioxide

c- Starch (1-3 %) c- Starch (1-3 %)

d- Organic polymers e.g. polyvinyl alcohol.d- Organic polymers e.g. polyvinyl alcohol.

Indicators:Indicators:

These are materials mixed with the These are materials mixed with the coating material and binder to help coating material and binder to help locating the spots on the TLC. The most locating the spots on the TLC. The most common used indicator is the fluorescent common used indicator is the fluorescent materials (silica gel 60 F254).materials (silica gel 60 F254).

Sample Application (Sample Application (SpottingSpotting):):

Samples are applied as a solution in any Samples are applied as a solution in any volatile solvent using volatile solvent using glass Capillariesglass Capillaries for for Qualitative, Preparative applications. Qualitative, Preparative applications. Graduated Graduated syringessyringes are used for Qualitative are used for Qualitative analyses.analyses.

The spots must be about 1-1.5cm away from The spots must be about 1-1.5cm away from the bottom of the plate and 0.5 cm away the bottom of the plate and 0.5 cm away from the plate sides and 0.5 cm away from from the plate sides and 0.5 cm away from each other.each other.

Development:Development:

Chromatographic Jars (Tanks) made of Glass Chromatographic Jars (Tanks) made of Glass with air-tight lids of different sizes containing with air-tight lids of different sizes containing the mobile phase are used for developments. the mobile phase are used for developments. The solvent must be left in the Jars enough time The solvent must be left in the Jars enough time before developing the plates for saturation.before developing the plates for saturation.

Developing system:Developing system:(Mobile phase – developing solvent)(Mobile phase – developing solvent)

Using a single solvent (very rare) or mixture of solvents to allow the 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 separation. The type of adsorbent used will affect the choice of the developing system. developing system.

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

Partition:Partition:MoreMore polar organic solvents such as butanol- acetic acid – water polar organic solvents such as butanol- acetic acid – water

are are used. Buffer solution are also used in partition chromatography.used. Buffer solution are also used in partition chromatography.

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

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

mixtures. mixtures.

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

Gel:Gel: Buffer solutions and aqueous acidic or basic solutions can be Buffer solutions and aqueous acidic or basic solutions can be

used.used.

Types of developments:Types of developments:A- AscendingA- Ascending::

1- Single development: 1- Single development: The solvent system is allowed to move through The solvent system is allowed to move through

the stationary phase one time only against the stationary phase one time only against gravity.gravity.

2- Repeated developments:2- Repeated developments:

a- Multiple developmentsa- Multiple developments: : The plated are developed more than one time The plated are developed more than one time using the same solvent system. The plates must using the same solvent system. The plates must be completely dried after each development. be completely dried after each development.

b- Stepwise developmentsb- Stepwise developments: : The plated are developed more than one time The plated are developed more than one time using different solvent systems.using different solvent systems.

3- Two-dimensional development: 3- Two-dimensional development:

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

..

..

One compound

Two closely related compounds

B- Centrifugal (chromatotron):B- Centrifugal (chromatotron):

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

Sample application

Mobile Phase

Motor1000 rpm

Circular plate

Visualization (Detection of spots):Visualization (Detection of spots):A- Universal methods:A- Universal methods:

1- Destructive methods:1- Destructive methods:

The plated are sprayed with corrosive reagents and The plated are sprayed with corrosive reagents and then heated in oven where organic compounds will then heated in oven where organic compounds will give charred spots. After this treatment the materials give charred spots. After this treatment the materials can not be recovered.can not be recovered.

e.g.e.g. Anisaldehyde / H Anisaldehyde / H22SOSO44

Vanillin / HVanillin / H22SOSO44

2-2- Non – Destructive methods: Non – Destructive methods:

In these methods the materials can be In these methods the materials can be recovered. recovered.

Day light for colour compounds.Day light for colour compounds. UV light for fluorescent compounds UV light for fluorescent compounds

(conjugated double bonds).(conjugated double bonds). II22 vapour for any compounds contain at vapour for any compounds contain at

least one double bondleast one double bond Spray with water where organic Spray with water where organic

compounds appear as white opaque compounds appear as white opaque spots.spots.

B- Specific Methods:B- Specific Methods:

These reagents are used for the detection These reagents are used for the detection of certain classes of compounds. They are of certain classes of compounds. They are usually destructive.usually destructive.

Dragendorff΄s reagent for Alkaloids.Dragendorff΄s reagent for Alkaloids.

Ferric Chloride (FeClFerric Chloride (FeCl33) for phenolic ) for phenolic compounds.compounds.

Aniline phthalate for sugars.Aniline phthalate for sugars.

Ninhydrine for nitrogenous compounds as Ninhydrine for nitrogenous compounds as Amines, Amino acids.Amines, Amino acids.

Rate of flow (Rate of flow (RRff Value):Value): Distance traveled by the spotsDistance traveled by the spots

RRff = -----------------------------------------= ----------------------------------------- Distance traveled by the solventDistance traveled by the solvent

The The RfRf of any compound must be less than one. of any compound must be less than one.

Start line

Solvent front

Distance travelled by the spot

Distance travelled by the solvent

Tailing in TLC:Tailing in TLC:

In some cases instead of getting round spots a In some cases instead of getting round spots a TailedTailed or or comet likecomet like spots are obtained leading to spots are obtained leading to overlapping of the spots and poor resolution.overlapping of the spots and poor resolution.

Tailed or comet like spot

Reasons and solution for tailing Reasons and solution for tailing problem:problem:

1-Ionic characters of acids and bases when 1-Ionic characters of acids and bases when they are they are chromatographed under neutral chromatographed under neutral conditions. conditions.

Solution:Solution: add acids or bases to the developing add acids or bases to the developing system.system.

2-Application of large amounts of material.2-Application of large amounts of material.

Solution:Solution: decrease conc. of material.decrease conc. of material.

3-Unproper choice of solvent system.3-Unproper choice of solvent system.

Solution:Solution: change the solvent system.change the solvent system.

ApplicatioApplication:n:

1- Qualitative:1- Qualitative: Identification through comparison of the Identification through comparison of the RRff value value

with that of Reference material.with that of Reference material. Determination of Complexity of mixtures. That will Determination of Complexity of mixtures. That will

be indicated from number of spots.be indicated from number of spots. Determination the purity of materials.Determination the purity of materials. Monitoring the progress of Chemical reactions.Monitoring the progress of Chemical reactions. Monitoring of column chromatography.Monitoring of column chromatography. Development of finger print TLC for extracts, Development of finger print TLC for extracts,

volatile oils or pharmaceutical preparation for volatile oils or pharmaceutical preparation for future identification and comparison.future identification and comparison.

In this application plates 5×5, 5×10 cm with thin film In this application plates 5×5, 5×10 cm with thin film of coating material are usually used.of coating material are usually used.

2- Quantitative:2- Quantitative:In this case an accurate volume of samples are In this case an accurate volume of samples are applied using syringes. The dimensions of plates applied using syringes. The dimensions of plates range from 5x10 to 20x20 according to the number range from 5x10 to 20x20 according to the number pf spots used. The plates are developed as usual in pf spots used. The plates are developed as usual in the chromatographic tanks. After development the the chromatographic tanks. After development the

concentration of material can be determined by:concentration of material can be determined by: Spot area measurement: Which is directly proportional Spot area measurement: Which is directly proportional

to the conc. of materials.to the conc. of materials. PhotodensitometryPhotodensitometry: : Measure transmittance, reflection Measure transmittance, reflection

or fluorescence of spots.or fluorescence of spots. Radioactivity: For radioactive material.Radioactivity: For radioactive material.

These measurements are done using TLC Scanner These measurements are done using TLC Scanner connected to computer that perform all connected to computer that perform all calculations.calculations.

3- Preparative TLC:3- Preparative TLC:

In preparative application 20×20 In preparative application 20×20 plates with thick layer of adsorbent plates with thick layer of adsorbent 0,25m are used. The mixture is apply 0,25m are used. The mixture is apply as bands and a pilot or guide spots as bands and a pilot or guide spots may be used in one side of the plate may be used in one side of the plate to enable the detection of the spots to enable the detection of the spots location.location.

Paper Chromatography Paper Chromatography (PC)(PC)

Stationary phase:Stationary phase:

Papers (cellulose), mechanism of Papers (cellulose), mechanism of separation is through partition.separation is through partition.

Mobile phase:Mobile phase:

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

Sample application:Sample application:

A line drawn by pencil, spot places are A line drawn by pencil, spot places are determined as dots. Apply sample as in TLC.determined as dots. Apply sample as in TLC.

Development:Development:1- Ascending: The mobile phase move against 1- Ascending: The mobile phase move against

Gravity.Gravity.

2-Descending: The mobile phase move with Gravity.2-Descending: The mobile phase move with Gravity.

3-Horizontal. 3-Horizontal.

4- Radial.4- Radial.

Visualization:Visualization:As TLC but must be non-destructive or specific with As TLC but must be non-destructive or specific with

no use of heat.no use of heat.

Applications:Applications:

As in TLC.As in TLC.