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Thinlayerchromatography(TLC)
Biological Techniques
Introduction of TLC
 It is a technique to separate components of non-
volatile mixtures using a thin stationary phase on
some plates (glass)
 It may be used for analytical or preparatory scale
 It is performed on a sheet of glass, plastic or
aluminium foil which is coated with a thin layer
of adsorbent material like silica gel, aluminium
oxide (alumina) or cellulose
 This layer of adsorbent acts as the stationary phase
Introduction of TLC
 A mixture is separated on the basis of affinities for
the mobile and stationary phases
 A solvent (mobile phase) draws up the plate
via capillary action and separates components of a
mixture
 Different components rise TLC plate at different rates
which results in the separation
 Rate of upward movement of the sample on the plate
depends on how tightly the sample binds with the
stationary phase
Introduction of TLC
 Spots of the sample on the plate are visualized by
various methods
 Distance travelled by a component is measured as
compared with the distance covered by the solvent in
a given time
 This is named as retardation factor (Rf) which ranges
from zero to one
 This technique is being widely used due to its
simplicity, low cost, high sensitivity and fast speed
Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Principle
Principle of TLC
 Principle of TLC is similar to paper chromatography
 Different compounds in the sample travel at
different rates due to different affinities for the
stationary phase and the solvent
 Separation is based on the competition between the
solute and the mobile phase for binding places on
the stationary phase
 Rf values are used to identify the compounds
because of their uniqueness
Principle of TLC
 Rf is the distance travelled by
a component divided by the
distance by the solvent
 Rf values depend on polarity of
the components, stationary as
well as mobile phase
 More polar compound have
stronger interaction with the
silica (polar stationary phase)
as compared with the less
polar compounds
 As a consequence the less polar
compound moves higher up
the plate and have higher Rf
values
Principle of TLC
 For more polar mobile phase all compounds on TLC
plate move high up
 Generally a substance whose structure resembles to
that of the stationary phase will have low Rf value
 Rf values depend on layer thickness, moisture on the
TLC plate, temperature, nature of mobile phase,
nature of TLC plate, sample size, etc.
 Mobile phase must not be allowed to reach the end
of the stationary phase
Principle of TLC
 Rf values of the known compounds or standards are
calculated
 Unknown compounds are identified by comparing
with Rf values of the known ones
 Compounds which are more soluble in the mobile
phase (solvent) will travel high up on the plate
 Similarly compounds more similar to the stationary
phase will travel less on the plate than the different
ones during separation
Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Components
Components of TLC
Stationary phase
 TLC plates are prepared by mixing the adsorbent such
as silica or alumina with a small amount of inert
calcium sulfate and water
 This mixture is spread as a thick slurry on an
unreactive sheet
 A compound which fluoresces under UV may also be
used
 Plate is dried and activated by heating in an oven
 Choice of stationary phase depends on the sample
Mobile phase
 Selection of a good solvent system is very important
 TLC is started with a nonpolar solvent such as ligroin
followed by a polar solvent such as ether or ethyl
acetate
 Rf values should vary between zero to one
 Separation depends on solubility of the compound in
a solvent
 Acids, bases or polar compounds require special
solvent composition
Components of TLC
Spotting the plate
 Once the sample is prepared a spotting capillary is
used to add the sample to the plate
 Spotting capillaries must be extremely small
 simply touch the end of the capillary tube at the start
line of the plate
 Smearing, smudging and spots that overlap will result
in inefficient separation
 Solvent is allowed to evaporate
 Spots should be separate
Components of TLC
Rf values
 Rf value is retardation factor and it is the distance
traveled by the individual component as compared
with the solvent
 Components of a mixture may have different Rf
values
 Rf values of pure compounds is used as a reference
value
 Unknown compounds are identified by comparing
their Rf values
 Rf values depend on how tightly the sample binds to
the stationary phase
Components of TLC
Components of TLC
Spot visualization
 It is easy to visualize spots of colored compounds
 However, since most organic compounds are
colorless they are observed by UV light or by some
other methods
 TLC plates glow green under UV light due to presence
of fluorescent indicator
 Plates are also placed in jars containing iodine stain
which turns organic spots to brown
 Permanganate and other stains are also used
Components of TLC
Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Method
Sample application
 Different samples are applied in a row of spots at the
same distance from the bottom edge
 Each sample will move in its own lane from the starting
point
 Samples are applied just above the solvent layer so
that they are not dissolved
 A standard is also applied for the comparison
 A co-spot is also applied by applying the sample on the
standard
Method of TLC
Solvent and capillary action
 A small amount of solvent is poured in a container
 The container is closed with a cover glass to achieve
the equilibrium and to avoid the evaporation
 Solvent is used according to the sample being
analyzed
 Solvent is allowed to rise up the plate by capillary
action about one cm below the upper end of the
plate
 Solvent is never allowed to run the end of the plate
Method of TLC
Plate visualization
 TLC plates are pre-coated with a fluorescent material
such as zinc sulfide which allows some of the
compounds to be visualized when examined under UV
light
 Plates can also be visualized by applying other
chemicals after elution
 Various visualizing agents react with the spots to
produce different results
 Use of different agents depends on the types of
samples
Method of TLC
Thin layer
chromatography (TLC)-
Common problems
Advanced Analytical Techniques
Common problems in TLC
 Since length of the stationary phase is limited in TLC,
there may be limited separation as compared with
other methods
 It may not withstand high temperature required for
the separation
 Similarly strong acidic and oxidizing stains may also
damage the stationary phase
 There may be problems of contaminations during
processing and handling of the TLC plates
Stationary phase
Common problems in TLC
 Mobile phase may advance with uneven flow
 This may lead to inaccurate Rf values
 It may be avoided by placing TLC plates vertically
without any contact
 There should be enough solvent so that it can travel
upto most of length of plate
 TLC plate should be cut evenly for the separation
 Choice of a suitable solvent system is critical
Mobile phase
Common problems in TLC
 Concentrated samples may lead to streaks instead of
isolated spots
 This is due to limited separation by the mobile phase
 It may be avoided by diluting the sample for efficient
elution
 Samples should be applied above the solvent level
 Too big spots of samples on the TLC plate may also
lead to inadequate separation
 Samples should be spotted with care
Streaking
Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Applications
Measure of sample purity
 Purity of sample is analyzed by direct comparison of
the sample and the standard
 In case of impurities extra spots are detected on
visualization of the plates
Identification of compounds
 TLC can be used for purification, isolation and
identification of the natural products like alkaloids,
steroids, etc.
 Standards are used for the comparison
Applications of TLC
Analysis of reactions
 Reaction mixture is examined to assess whether
certain reaction is complete or not
 Formation of specific spots on the plates indicate
the presence of certain products
Biochemical analysis
 TLC is useful in separation of biochemical
metabolites from body fluids like blood plasma,
urine, etc.
 Other samples are also used to identify the
metabolites
Applications of TLC
Pharmaceutical industry
 TLC technique is used for detection of impurities in
the pharmaceutical products
 Various medicines like sedatives, antihistaminics,
analgesics, local anaesthetics, may be tested
qualitatively by TLC method
 It is also possible to analyze various intermediate
products formed in the development of
pharmaceutical products
 TLC can be applied in studying kinetics of the
products
Applications of TLC
Chemical analysis
 TLC is used for separation and identification of
various compounds closely related to each other
 It is also used in the identification of cations and
anions in inorganic chemistry
Food and cosmetics
 TLC method is used for separation and identification
of colors, preservatives, etc.
 It is also used in analysis of various cosmetic
products
Applications of TLC

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3. Thin Layer CHROMATOGRAPHY Bio Techniques.pptx

  • 2. Introduction of TLC  It is a technique to separate components of non- volatile mixtures using a thin stationary phase on some plates (glass)  It may be used for analytical or preparatory scale  It is performed on a sheet of glass, plastic or aluminium foil which is coated with a thin layer of adsorbent material like silica gel, aluminium oxide (alumina) or cellulose  This layer of adsorbent acts as the stationary phase
  • 3. Introduction of TLC  A mixture is separated on the basis of affinities for the mobile and stationary phases  A solvent (mobile phase) draws up the plate via capillary action and separates components of a mixture  Different components rise TLC plate at different rates which results in the separation  Rate of upward movement of the sample on the plate depends on how tightly the sample binds with the stationary phase
  • 4. Introduction of TLC  Spots of the sample on the plate are visualized by various methods  Distance travelled by a component is measured as compared with the distance covered by the solvent in a given time  This is named as retardation factor (Rf) which ranges from zero to one  This technique is being widely used due to its simplicity, low cost, high sensitivity and fast speed
  • 5. Advanced Analytical Techniques Thin layer chromatography (TLC)- Principle
  • 6. Principle of TLC  Principle of TLC is similar to paper chromatography  Different compounds in the sample travel at different rates due to different affinities for the stationary phase and the solvent  Separation is based on the competition between the solute and the mobile phase for binding places on the stationary phase  Rf values are used to identify the compounds because of their uniqueness
  • 7. Principle of TLC  Rf is the distance travelled by a component divided by the distance by the solvent  Rf values depend on polarity of the components, stationary as well as mobile phase  More polar compound have stronger interaction with the silica (polar stationary phase) as compared with the less polar compounds  As a consequence the less polar compound moves higher up the plate and have higher Rf values
  • 8. Principle of TLC  For more polar mobile phase all compounds on TLC plate move high up  Generally a substance whose structure resembles to that of the stationary phase will have low Rf value  Rf values depend on layer thickness, moisture on the TLC plate, temperature, nature of mobile phase, nature of TLC plate, sample size, etc.  Mobile phase must not be allowed to reach the end of the stationary phase
  • 9. Principle of TLC  Rf values of the known compounds or standards are calculated  Unknown compounds are identified by comparing with Rf values of the known ones  Compounds which are more soluble in the mobile phase (solvent) will travel high up on the plate  Similarly compounds more similar to the stationary phase will travel less on the plate than the different ones during separation
  • 10. Advanced Analytical Techniques Thin layer chromatography (TLC)- Components
  • 11. Components of TLC Stationary phase  TLC plates are prepared by mixing the adsorbent such as silica or alumina with a small amount of inert calcium sulfate and water  This mixture is spread as a thick slurry on an unreactive sheet  A compound which fluoresces under UV may also be used  Plate is dried and activated by heating in an oven  Choice of stationary phase depends on the sample
  • 12. Mobile phase  Selection of a good solvent system is very important  TLC is started with a nonpolar solvent such as ligroin followed by a polar solvent such as ether or ethyl acetate  Rf values should vary between zero to one  Separation depends on solubility of the compound in a solvent  Acids, bases or polar compounds require special solvent composition Components of TLC
  • 13. Spotting the plate  Once the sample is prepared a spotting capillary is used to add the sample to the plate  Spotting capillaries must be extremely small  simply touch the end of the capillary tube at the start line of the plate  Smearing, smudging and spots that overlap will result in inefficient separation  Solvent is allowed to evaporate  Spots should be separate Components of TLC
  • 14. Rf values  Rf value is retardation factor and it is the distance traveled by the individual component as compared with the solvent  Components of a mixture may have different Rf values  Rf values of pure compounds is used as a reference value  Unknown compounds are identified by comparing their Rf values  Rf values depend on how tightly the sample binds to the stationary phase Components of TLC
  • 16. Spot visualization  It is easy to visualize spots of colored compounds  However, since most organic compounds are colorless they are observed by UV light or by some other methods  TLC plates glow green under UV light due to presence of fluorescent indicator  Plates are also placed in jars containing iodine stain which turns organic spots to brown  Permanganate and other stains are also used Components of TLC
  • 17. Advanced Analytical Techniques Thin layer chromatography (TLC)- Method
  • 18. Sample application  Different samples are applied in a row of spots at the same distance from the bottom edge  Each sample will move in its own lane from the starting point  Samples are applied just above the solvent layer so that they are not dissolved  A standard is also applied for the comparison  A co-spot is also applied by applying the sample on the standard Method of TLC
  • 19. Solvent and capillary action  A small amount of solvent is poured in a container  The container is closed with a cover glass to achieve the equilibrium and to avoid the evaporation  Solvent is used according to the sample being analyzed  Solvent is allowed to rise up the plate by capillary action about one cm below the upper end of the plate  Solvent is never allowed to run the end of the plate Method of TLC
  • 20. Plate visualization  TLC plates are pre-coated with a fluorescent material such as zinc sulfide which allows some of the compounds to be visualized when examined under UV light  Plates can also be visualized by applying other chemicals after elution  Various visualizing agents react with the spots to produce different results  Use of different agents depends on the types of samples Method of TLC
  • 21. Thin layer chromatography (TLC)- Common problems Advanced Analytical Techniques
  • 22. Common problems in TLC  Since length of the stationary phase is limited in TLC, there may be limited separation as compared with other methods  It may not withstand high temperature required for the separation  Similarly strong acidic and oxidizing stains may also damage the stationary phase  There may be problems of contaminations during processing and handling of the TLC plates Stationary phase
  • 23. Common problems in TLC  Mobile phase may advance with uneven flow  This may lead to inaccurate Rf values  It may be avoided by placing TLC plates vertically without any contact  There should be enough solvent so that it can travel upto most of length of plate  TLC plate should be cut evenly for the separation  Choice of a suitable solvent system is critical Mobile phase
  • 24. Common problems in TLC  Concentrated samples may lead to streaks instead of isolated spots  This is due to limited separation by the mobile phase  It may be avoided by diluting the sample for efficient elution  Samples should be applied above the solvent level  Too big spots of samples on the TLC plate may also lead to inadequate separation  Samples should be spotted with care Streaking
  • 25. Advanced Analytical Techniques Thin layer chromatography (TLC)- Applications
  • 26. Measure of sample purity  Purity of sample is analyzed by direct comparison of the sample and the standard  In case of impurities extra spots are detected on visualization of the plates Identification of compounds  TLC can be used for purification, isolation and identification of the natural products like alkaloids, steroids, etc.  Standards are used for the comparison Applications of TLC
  • 27. Analysis of reactions  Reaction mixture is examined to assess whether certain reaction is complete or not  Formation of specific spots on the plates indicate the presence of certain products Biochemical analysis  TLC is useful in separation of biochemical metabolites from body fluids like blood plasma, urine, etc.  Other samples are also used to identify the metabolites Applications of TLC
  • 28. Pharmaceutical industry  TLC technique is used for detection of impurities in the pharmaceutical products  Various medicines like sedatives, antihistaminics, analgesics, local anaesthetics, may be tested qualitatively by TLC method  It is also possible to analyze various intermediate products formed in the development of pharmaceutical products  TLC can be applied in studying kinetics of the products Applications of TLC
  • 29. Chemical analysis  TLC is used for separation and identification of various compounds closely related to each other  It is also used in the identification of cations and anions in inorganic chemistry Food and cosmetics  TLC method is used for separation and identification of colors, preservatives, etc.  It is also used in analysis of various cosmetic products Applications of TLC