Thin layer chromatography

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THIN LAYER CHROMATOGRAPHY

• PRESENTED BY
• Miss. Surabhi R Singh
• B.Pharm
• SGSPS Institute of Pharmacy, Akola

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Thin layer chromatography

• Introduction
• Thin layer chromatography ( TLC ) is widely used
  technique.
• Essential for analyst and research workers.
• It is universal analytical technique in chemical
  analysis for organic and inorganic matter.
• It is simple and rapid method carried out using
  thin layer of adsorbents on plates.

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History

• In 1938, Izmailov and Shraiber described the
• basic principle underlying the process and used
  it
• for separation of plant extracts.
• Consdon, Gorden and Martin 1944 started using
  filter paper instead of open column.
• Attempts were made using adsorption
• chromatography on impregnated filter paper and
• later on glassfibre, paper coated with silicic
  acid
• or alumina.
• Stahl in 1958 credited with bringing out the
• work on preparing plates and separation of wide
• variety of compounds.

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Definition

• CHROMATOGRAPHY
• It represents a group of methods for
• separating molecular mixtures that depend on
• the differential affinities of the solute
  between
• two immiscible phases.
• Minute differences in a compound's partition
• coefficient result in differential retention on
  the stationary
• phase and thus resulting in the separation
• One of the phase is a fixed bed of large surface
• area called as STATIONARY PHASE.
• It may be
• 1)Porous
• 2)Finely divided solid or liquid, that has been
  coated as thin layer on an inert support material.

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• While the other phase is fluid or gas which
• moves through or over the surface, which is
  called
• as the MOBILE PHASE.
• It may be-
• 1) Pure liquid
• 2) Mixture of solvents
• 3) Gas or mixture of gases
• THIN LAYER CHROMATOGRAPHY is a
• simple and rapid method carried out using
• thin layer of adsorbents on plates for the
• separation of components from molecular
• mixture .
• It is also called as Flat bed chromatography
• or Planer chromatography or open column
• Chromatography.

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• ADVANTAGES
• 1) Requires little equipment, little time for
  separation
• 2) Requires small amount of sample
• 3) More sensitive
• 4) Corrosive agents for identification is also
  permitted
• 5) It can be used for adsorption, partition and
  ion
• exchange chromatography
• 6) Components separated can be recovered easily
  by
• scrapping
• DISADVANTAGES
• It does not give a quantitative information of
  high
• precision and accuracy

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Principle

• In the classification of chromatographic
• methods TLC has been included under both
• adsorption and Partition chromatography.
• As various materials of different adsorptive
• power are used in TLC, the separation not
• always by adsorptive phenomenon.
• Separation may result due to adsorption or
• partition or by both phenomenon depending
• upon the nature of adsorbents used on
• plates and solvent system used for
• development.

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Technique

• In TLC the separation is carried on a glass or
• plastic plate which is coated with a thin
  uniform
• layer of finely divided inert adsorbent such as
• silica gel or alumina.
• The plates are activated, the solution of the
• sample in a volatile solvent is applied by using
  a
• capillary tube or a micropipette to a spot
  keeping
• 1-2 cm from the bottom of TLC plate.
• The position of the sample spot is indicated by
  
• marking a origin line on the plate with the
  lead
• pencil.
• When the spot has dried, the plate is placed
• vertically in a suitable tank with its lower
  edge
• immersed in selected mobile phase.

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• The solvent rises by capillary action, resolving
  the sample mixture into discrete spots.
• At the end, the run solvent is allowed to
  evaporate from the plate and the separated spots
  are located and identified by various physical
  and chemical methods.
• In short-
• Thin layer chromatography (TLC) is a simple,
  inexpensive method which requires a minimum of
  instrumentation and can be used for separation of
  sample mixtures.
• It is possible to separate up to 70 samples and
  standards on a single plate, which makes analysis
  quick and inexpensive

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TLC - DIAGRAM
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VARIOUS STEPS IN TLC

• Selection of adsorbents
• Preparation of chromatoplates
• Activation of plates
• Solvent system
• Application of sample
• Development chambers
• Development of chromatograms
• Location of spots
• Evaluation of chromatogram

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1) Selection of adsorbents

• Depends on-
• Characteristic of compounds to be
  separated
• Solubility of compounds
• Nature of substance to be separated i.e. acidic,
  basic, amphoteric
• Whether compound liable to react chemically with
  adsorbent or solvent

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Requirements for adsorbents

• Should not react with sample- Inertness
• Should not catalyze or decompose the sample.
• Should not deteriorate during run
• Should be immiscible with mobile phase.
• Should have high degree of mechanical stability.
• Should be inexpensive.

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EXAMPLES

• Inorganic Adsorbents
• Silica gel
• Kieselghur
• Alumina
• Magnesia
• Magnesium silicate, calcium silicate etc.
• Organic Adsorbents
• Cellulose and its acetylates
• Charcoal and activated carbon
• Others- dextran gels, ion exchange resins,
  polyamides etc.

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2)Preparation of chromatoplates

• Glass plates or flexible plates are used commonly
  for spreading adsorbent.
• Standard sizes used are 205 cm, 2010 cm or
  2020 cm.
• The surface of plates should be flat and without
  irregularities. The standard thickness is 250 µm.
• Suspension or slurry of the coating material
  is used to give uniform thickness over the
  plates.
• Following are the 3 methods of applying
  thin layer of adsorbent-
• 1) Pouring,
• 2) Dipping,
• 3) Spreading

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• 1) POURING-
• Slurry of adsorbent made and poured on a plate
• and allowed to flow over it so that it is evenly
  covered.
• 2) DIPPING-
• It is used for small plates by dipping 2
• plates at a time, in a slurry. But in this exact
  
• thickness of layer is not known.
• 3)SPREADING-
• Modern methods utilise the spreading
• devices for preparation of uniform thin layers
• on glass plates .
• The spreading was done by means of
• Spatula or by glass rod.
• Nowadays commercial spreaders such as-
• Moving spreader,
• Moving plate type are used mostly.

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• Precoated Plates
• Glass Support- It is resistant to heat and
• chemicals, easy to handle and offers superior,
  flat
• and smooth surface for chromatographic work.
• Disadvantage- Fragility, relatively high
  weight,
• additional packing material and higher
  production
• cost.
• Polyester Sheets- These are unbreakable,
• require less packing material, less shelf space
  for
• storage and can be cut to required format.
• Disadvantage- Charring reactions are possible if
  
• temperature exceeds 120C.
• Aluminum Sheets- They have increased
• temperature resistance.
• Disadvantage- Eluents containing high
  concentrations of
• mineral acids and concentrated ammonia should
  not be
• used, as they chemically attack aluminum.

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• Commonly available Percolated plates-
• 1.Silica gel(unmodified)More than 80analysis
  are done on this layer.
• 2.Aluminum oxide These are used for basic
  substances, alkaloids and steroids.
• 3.High purity Silica gel 60 These are used for
  Aflotoxins.
• 4.Cellulose (microcrystalline) Used for Amino
  acid, dipeptides, sugars, antibiotics and other
  labile compounds which cannot be chromatographed
  on active layers of silica gel.
• 5.Silica gel- chemically modified
• a)Amino Used for carboxylic acids, phenols,
  nucleotides, vitamins(B1,B6,B12), Uric acid and
  xanthine derivatives.
• b)Cyano It is used for pharmaceutical
  preservatives.
• c)Chiral It is used for resolution of
  enantiomeric substances for optical purity such
  as amino acids, dipeptides, lactones.

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• 4) ACTIVATION OF PLATES
• After spreading plates are allowed to dry
• in air (5-10 mins) and it is further dried and
• activated by heating at about 100ºC for 30 mins.
• 5) SOLVENT SYSTEM
• The choice of mobile phase depends on-
• 1)Nature of substance to be separated.
• 2)Adsorbent material to be used.
• Organic solvent mixture of as much low
• polarity as possible . Highly polar solvent are
• avoided to minimize adsorption of any component
• Use of water as a solvent is avoided as it
• may loosen the adhesion of a layer on a glass
• plate. Following solvents are generally used-
• Petroleum ether , carbon tetrachloride ,benzene,
• Chloroform , acetone ,ethanol , methanol etc.

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5)APPLICATION OF SAMPLE-

• The area of sample application should be kept
• as small as possible for sharper and greater
  
• resolution.
• For preparative work , the sample is applied in
• narrow band.
• The pipette, loop or syringe can be used for
• applying sample.
• The spot diameter should be between 2-5 mm.
• Before the sample application, the starting
• point and finish line is usually marked.

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6) Development of chambers

• The TLC plate is placed vertically in a
  rectangular
• Chromatography tank or chamber.
• They are classified according to separation
• technique used as follows-
• Tanks for ascending development.
• Tanks for descending development.
• Tanks for horizontal development.
• Tanks for thin layer electrophoresis
• Glass or stainless steel is most suitable for
  first 3
• methods , plastic are not used widely as they
  are
• not resistant to all organic solvents and
  solutions.
• Saturation of tank is important, development
• should be carried out at room temperature.

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Diagram of chromatographic chambers
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7)Development of chromatograms

• It means running of the mobile phase over the
• stationary phase.
  
• Ascending development
• The plates after spotting the sample are
• placed in chromatography chamber containing
• solvent at the bottom. The flow of solvent is
  from
• bottom to top.
• Diagrams for ascending development

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• DIAGRAM FOR ASCENDING DEVELOPMENT
• B) DESCENDING DEVELOPMENT
• In this method, flow of the solvent from
• reservoir to the plate is by means of a filter
  paper
• strip. Solvent moves from top to bottom of the
• plate.

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8)LOCATION OF SPOTS

• Location of substances are done by using
  PHYSICAL
• METHODS CHEMICAL METHODS.
• a)PHYSICAL METHOD -
• It include ultraviolet, fluorescence or
• radioactive counting.
• b)CHEMICAL METHOD -
• In this method locating agents are applied
• by spraying.
• Concentrated sulfuric acid is used mostly as
  it
• produces colored spots which are visible in
• daylight as well as UV light.
• One more locating agent for organic substances
  
• is iodine vapors. plate is exposed to iodine
• vapors, by placing in closed vessel
  containing
• a few iodine crystals.

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9) Evaluation of chromatogram

• After locating the spots on plates and marking
• their position and size, they are evaluated
• quantitatively or qualitatively.
• A) QUALITATIVE- In this , the RF value of
• standard or authentic sample for the same mobile
• phase is known and is recorded.
• The RF value of the sample is calculated and on
• comparison of RF values for known and unknown
• the qualitative identification of sample is
  made.
• B) QUANTITATIVE- Following 2 methods are
• used-
• Direct method
• Indirect method

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a) Direct methods

• A) visual comparison
• For a quick semiquantitative analysis of
• amount of components, visual comparison of spot
• size, intensity of spot or the combination of
  both is
• made with known standard spots.
• B) spot areas and weight relationship
• From the area of spot, amount of substance
• present is calculated. Usually linear
  relationship is
• found between the spot area and weight of
• compound present in it.
• c) Direct spectrometry
• Quantitative measurements are obtained
• by reading the absorption or fluorescence of
• separated zones directly on TLC plates.

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UV spectra

• UV light
• Detector
• Beam
• Reflected beam

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B) Indirect method

• In this method, the area containing
• substance on TLC plate is marked and scrapped
• or scooped out with the help of vacuum cleaner
• without any loss.
• Then solute from adsorbent is extracted or
• eluted with suitable solvent.
• The eluted is then analysed by suitable
• technique like colorimetry , fluorimetry ,
• spectrophotometry, radiometry or by suitable
• color development method.

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READING THE TLC

• TLC can be read with the help of Rf value
  (Retention Factor)- This measures difference in
  rate of movement of components in chromatography.
• Solvent front
• Starting line

Totally unretained solute
Totally retained solute
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APPLICATION OF TLC

• Purity of sample For this direct comparison of
  the sample and authentic sample is done, if
  impurity is present it shows extra spot.
• Examination of Reactions Reaction mixture is
  examined by TLC to asses whether reaction
  complete or not.
• Identification of Compounds It is employed in
  isolation, purification of various class of
  organic compounds.
• Biochemical Analysis It is used in separation
  of biochemical metabolite.
• Pharmaceutical Industries It is used in
  detection of impurity in pharmacopoeial drug or
  chemical.
• Testing of Drugs Various Drugs like hypnotic,
  sedative, anticonvulsant, analgesic and
  anaesthetics are tested
• Separation of Multi-Component Pharmaceutical
  Formulation.

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reffarences

• 1) Textbook of pharmaceutical analysis and
• HPTLC by P. D. Sethi
• 2) Textbook of Pharmacognosy by Dr. C. K. Kokate
• A. P. Purohit and S. B. Gokhale
• 3) Thin-layer chromatography - Wikipedia, the
  free encyclopedia_files
• 4) htmfacrc03Bthin layer chromatography
  diagramimgdii_images
• 5) Microsoft Powerpoint 97-2003 Presentation at
  listeprix.com

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Thank you