PLANNAR CHROMATOGRAPHY

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PLANNAR CHROMATOGRAPHY
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• It includes two types
• 1- Thin Layer Chromatography (TLC).
• 2- Paper Chromatography (PC).

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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

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• 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

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• 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.

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• 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).

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• 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.
• 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.

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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
• Buffer solutions and aqueous acidic or basic
  solutions can be used.

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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.

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• 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.

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• 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.

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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

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• 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.

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• 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.

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• 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.

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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.

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• 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.

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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 55, 510 cm with thin
  film of coating material are usually used.

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• 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.

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• 3- Preparative TLC
• In preparative application 2020 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.

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Paper Chromatography (PC)
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• 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.

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• 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.