Thin layer chromatography (TLC) is a technique used to separate mixtures by distributing the components between a stationary phase and a mobile phase. The document discusses the principles, requirements, procedures, and applications of TLC. It explains that TLC involves applying a sample as a spot on a thin layer of adsorbent material like silica coated on a plate, then developing the plate in a mobile phase which separates the components by traveling up the plate at different rates based on their interactions with the phases. The document provides details on the materials, equipment, development techniques and evaluation of TLC.

1. THIN LAYER
CHROMATOGRAPHY
1
PREPARED BY :- 02-MANSI GANGWAR
BRANCH :- PHARMACEUTICS (sem1)
SUBJECT:-MODERN PHARAMACEUTICAL ANALYTICAL
TECHNIQUES

2. CONTENT
Introduction
History
When TLC is used?
Principle of TLC
Practical requirements
How to run TLC
Evaluation of Chromatography
Advantages of TLC
Disadvantages of TLC
Application of TLC
Common problems in TLC
Difference between paper chromatography and TLC
2

3. Chromatography
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases, one of which is stationary
(stationary phase) while other (the mobile phase) moves
in definite direction
3

4. INTRODUCTION
Thin Layer Chromatography is similar to Paper Chromatography,
except that a thin(0.25 mm) layer of some inert material like silica is
used as the substrate instead of paper.
A layer of inert material is applied in the form of slurry over a flat
surface (glass) and dried , it may be spread manually or mechanically.
TLC is often named by other names such as drop, strip, spread layer,
surface chromatography and open column chromatography.
4

5. HISTORY
The technique of TLC was first introduced by Izmailov and Shraiber in
1938.These workers used this technique for separating plant extract on
2mm thick and firm adhesive layer of alumina set on glass plates .
Consden, Gordon and Martin(1944) started using filter papers. Their
work in the field of amino acid analysis met with considerable success.
Williams carried out chromatography on adsorbent layer sandwich
between two glass plates.
TLC as a procedure for analytical adsorption chromatography was first
introduced by Stahl(1958) who was mainly responsible for bringing out
a standard equipment for preparing thin layers.
5

6. When TLC is used ?
 TLC is used if
The substance are nonvolatile or of low volatility
The substance are strong polar, of medium polarity, non polar
or ionic
A large number of sample must be analyzed simultaneously,
cost effective, and within a limited period of time
No source of electricity is available
The substance in the material being analyzed cannot be
detected by the methods of liquid chromatography or gas
chromatography or with great difficulty.
6

7. 7

8. Principle
It is based on the principal of adsorption chromatography or
partition chromatography or combination of both, depending
on adsorbent and nature of solvent employed.
If the solid phase is used as stationary phase then the
adsorption will be the principle and if the liquid coated in solid
support is used then partition will be the principle.
The absorbent is coated on a glass slide or plastic sheet
creating a thin layer of the particular stationary phase.
A solution of the sample containing a mixture of compounds is
applied to the layer of absorbent, near the edge, as a small
spot
8

9. When the plate comes in contact with the mobile phase in a container under
closed condition, the solvent, travels up the layer of the adsorbent by capillary
action.
Depending upon the solubility, and rate of migration, compounds in the mixture
move up on the plate at different rates resulting in separation of the
compounds.
The separation relies on the relative affinity of compounds towards both the
phases.
The compounds which have a higher affinity to the stationary phase move
slowly and the compounds with the less affinity towards stationary phase
travels faster.
Therefore the separation of the mixture is attained
9

10.  PRACTICAL REQUIREMENTS
1.Stationary phase
2.Glass plates
3.Preparation and activation of TLC plates
4.Application of sample
5.Development tank
6.Mobile phase
7.Development technique
8.Detecting or visualizing agents
10

11. 1. Stationary phase
• The stationary phase used in tlc is a finely divided powder in the
size range of 10-50 (micrometer) coated on a glass plate.
• The thickness of the thin layer of stationary phase is 100-250
micrometer.
• Slurry of stationary phase powder (adsorbent) is prepared using
water.
• Substance like gypsum, calcium sulphate or plaster of paris, is
added to the stationary phase powder.
• These all substance known as binding agent, which help it to
adhre to backing material.
11

12. • The slurry is spread on the plate in a thin film using glass rod
or applicators .
• The solvent is evaporated by keeping coated plates at room
temperature for 30 minutes and it is activated by keeping it
in an oven at 110°c for 30 minutes.
12

13. 2. Glass plate
• In TLC the glass plate are used.
• They are available in different sizes.
• Based upon on size of the plate they are
divided into 3 types
1. 20×20cm (full plate)
2. 30×10cm (half plate)
3. 20×5cm (quater plate)
13

14. 3. preparation of slurry
• Powder and solvent (water) is mixed and slurry is prepared
• NOTE
• If the slurry is too thick there will be no separation of
compound will occur
• If the compound if too thin during spotting compound will
spread and no exact or accurate spots can be detected
14

15.  Techniques of coating TLC plate
15
I. Spreading
II. Pouring
III. Dipping
IV. Spraying
V. Pre-Coated

16. i) Spreading
• Slurry of coating material is poured on a plate. It is
spreaded with the help of glass rod.
• The uniform and smooth layer of material is air dried
for at least 15-20 min.
• TLC plate can be prepared by spreading slurry with
applicator.
• The slurry is filled in a applicator.
• It is moved over Stationary phase and the coating of
glass plate is performed.
16

17. 17

18. ii) Pouring
• The slurry of coating material is poured on
plate.
• The slurry is evenly distributed on glass plate
by tilting the glass plate without touching the
edges.
• The plate is air dried for 15-20 min.
18

19. iii) Dipping
• Two plates are held together and dipped into a
beaker containing coating powder slurry.
• The plates are withdrawn and allowed to drain.
• The plates are air dried.
19

20. iv) Spraying
• The prepared slurry is sprayed using spray
gun on a plate .
20

21. v) Pre-coated plates
• Pre coated plates are available in market which is
prepared by automatic spreader or applicator.
• Advantage:- of using pre-coated plate is uniform and
reproducible coating thickness of the plate.
• The stationary phase is activated by heating the pre-
coated plates at 110°c for 30 min ,which removes the
adsorbed water molecule.
21

22. Disadvantage of techniques
• Pouring:- uniformity in thickness can not be
ensured.
• Dipping:-larger quantity of slurry is required
even for preparing fewer plates.
• Spraying:- layer thickness cannot be
maintained uniformity all over the plate.
22

23. Activation
• Activation of tlc plates is nothing but removing
water/moisture and other absorbed substance from the
surface of any adsorbent by heating at hight temperature
so that adsorbent activity is retained.
• The activated plates can be stored in thermostatically
controlled oven or in desiccator and can be used
whenever required (activation occurs at 105 degree
celcius for 30 minutes)
23

24. 4) Sample application
• Usually a sample solution is applied as a spot 1-2 cm
from the efge of the plate.
• Sample may be applied manually using capillary,
microsyringe or hypodermic syringe.
• Mechanical applicators are available which can apply
samples precisely and accurately.
24

25. 5) Mobile phase
• The choice of solvent depends on nature of solute molecules to
be separated and the nature of the stationary phase used.
• In adsorption chromatography eluting power of solvents
increases as their polarity increases.
• A single solvent or a mixture of solvent can be used as mobile
phase.
• The solvent used as mobile phase must be pure .
• A small amount of water or other Impurities may interfere in the
analysis.
25

26. • Two types of mobile phase
• Hydrophilic Mobile phase
• (a mixture of organic solvent and water with the addition
of acid and base or complexing agent to optimize the
solubility of the component of a mixture can be used)
• Eg :-
1. n-butanol : glacial acetic acid : water (4:1:5)
Isopropanal : ammonia : water (9:1:2)
• Hydrophobic mobile phase
• Eg:- cyclohexane, di-ethyl ether, benzene, chloroform
26

27. 6) Developing chamber
• It is used for the purpose of TLC plate run in mobile
phase.
• After the mobile phase is poured into the chamber it
is kept closed with lid.
• This is done to equilibrate the atmosphere of empty
space in chamber with the mobile solvent.
• This is known as saturation of TLC chamber.
• Edge effect occurs when the solvent front in the
middle of TLC plate moves fasters than that the edges
of plate.
27

28. 28

29. 29

30. 7) Chromatographic development
• Different development techniques are used for
efficient separation
1. Ascending
2. Descending
3. Two dimensional
4. Circular (radial)
5. Anti circular
30

31. i) Ascending
• Sample is applied near one edge of the plate and its position is marked
with a pencil.
• After the sample solvent has evaporated, the plate is placed in MP
chamber.
• One end of the plate is immersed in MP.
• The mobile phase travels up to the plate by capillary action.
• Sample distributes itself between MP and SP.
• After the mobile phase has travelled 2/3rd of the plate, the plate is
removed from the chamber and dried.
• The position of the developed components is determined.
31

32. 32

33. ii)Descending:-
• Descending chromatography is a technique in which the
mobile phase moves to the downside.
33

34. iii) Two dimensional
• A sample spot is applied at the bottom of the plate.
• The plate is allowed to develop.
• After the development with a given mobile phase, the plate is turned to
90° and further development is carried out.
34

35. iv) Circular
• The plate is fixed in a horizontal position and Mobile Phase is
applied to the center of the plate via syringe.
• Sample spots are applied on the center of the plate around the
solvent inlet.
• The development of tlc plate occurs radially in all directions.
35

36. v) Anti circular
• In anti-circular development, a flat circular
support is used with mobile phase applied at
its edges.
• Samples are applied near the edges and
Carried towards the center by the mobile
phase
• Like circular development, anti-circular
development also has advantage of avoiding
"edges effects"
36

37. 37

38. 8) Detection of analyte spot
• After developing TLC plate it is removed from the mobile phase
chamber.
• Developed plate is dried in air and heated in oven to remove Mobile
Phase.
1. Exposing developed plate to UV light:-
• If a solute molecule fluorescence it can be detected by exposing
developed plate under UV light.
• The compound will appear as a shinning spot.
2. Spraying with reagent :-
• Chemical reagent are sprayed on the developed chromatogram
which chemically reacts with the solute on the plate and produces a
spot of different color which helps in the location of the spot.
38

39.  HOW TO RUN TLC
Step 1:- prepare the developing container
Step 2:- prepare the tlc plate
Step 3:- spot the tlc plate
Step 4:- develop the plate
Step 5:- visualize the spot
39

40. Step1:- prepare the developing container
Take a beaker with a watch glass on the
top.
Required quantity of solvent are taken
into the beaker.
Cover the beaker with watch glass and
mix the solvent.
Keep them aside until the plate is
prepared.
40

41. Step2:-Prepare the TLC plate
 Take a TLC plate and cut it to
required length and width Or take a
glass slide and apply Silica gel slurry
on it.
For silica gel slurry, Take silica
powder and add water into it to
make a Spreadable slurry.
41

42. Step 3:- spot the TLC plate
Take the capillary tube and by the
help of heat make it into two so that
the end of the capillary tube will be
thin.
It helps to place the small amount of
sample.
Take the required solution and spot
them at the marked points .
42

43. Step 4:- Develop the plate
Put the tlc plate carefully into the
beaker.
 The solution should not touch the
marked line.
Close the beaker with watch glass.
Do not allow the solvent to run off
the top of the plate.
43

44. Step5:-Visualization of the spot
Take off the TLC plate from beaker
carefully.
Mark the solvent front level.
Let it dry.
Spray the solution.
Observe the spot round it with
pencil carefully.
44

45. 45

46.  Evaluation of chromatography
46
METHOD
QUALITATIVE
ANALYSIS
QUANTITATIVE
ANALYSIS

47. 1) Qualitative analysis
• Qualitative analysis of developed chromatogram is
performed by using calculating retardation factor (Rf).
• Rf = distance traveled by solute
distance traveled by mobile phase (solvent)
• The data from single chromatogram do not provide sufficient
information to permit identification of various species
present in a mixture because of the variability of Rf value
with sample size and thin layer plate.
47

48. • Thickness of stationary
phase, moisture
Content of mobile and
stationary phase,
temperature, degree of
chamber saturation and
sample size are the
different variables that
affect Rf value.
48

49. • Standard substance can be spotted on tlc plate
along with the sample solution.
• A comparison of Rf value of unknown and standard
provides strong evidence as to the identity of
unknown sample.
• Relative retention factor can be used for the
qualitative analysis
• Rf = distance traveled by sample / distance
traveled by standard
49

50. 2) Quantitative analysis
• The quantitative analysis of the developed chromatogram is
performed by two ways:
i. Measurement directly on the layer
ii. Removal of analyte from the plate and analysis
Measurement directly on the layer( direct method)
• Quantitative measurement of compound can be performed by
following ways.
1.Visual comparison:- By visual comparison of the chromatogram
quantitation is performed. But this method is having limited
accuracy. It is used to determine whether impurities are within
certain limits or not
50

51. 2.Area measurement :- It involves measurement of spot
area. Planimetry, tracing spot on writing paper,
photographing the spot for cutting and weighing are the
different techniques used for the measurement of spot area.
3.Densitometery:- It involves in situ measurement of
absorbance characteristics of solute. This technique involves
measurement at wavelength maximum of absorbing
substance to achieve greater sensitivity
51

52. Removal of analyte from the plate and analysis (indirect
method)
• After identifying spots, the area containing analyte is
scraped from the plate.
• It is dissolved in suitable solvent and identification is
performed by suitable techniques like UV spectroscopy,
polography, flame photometry, mass spectroscopy, infrared
spectroscopy etc.
52

53.  Advantage of TLC
• Instrument required is simple compared to liquid
chromatography or gas chromatography.
• This method is speedy and cheaper.
• This method is flexible because TLC plate can be treated with
variety of reagents including corrosive reagent for the
detection of analyte.
• Separation of mg of the substance can be achieved.
• Slow eluting substance may clog the column in hlpc while in
tlc new stationary phase is used for each Analysis.
53

54.  Disadvantage of TLC
• Accurate quantitative analysis may not be
performed by TLC.
• Reproducible results may not ne obtained.
• Number of theoretical plates is less.
• It is having low sensitivity compared to hplc.
• It is not suitable for volatile compound.
54

55.  Application
• Check Purity of sample
• Identification of compound
• Biochemical analysis
• In pharamceutical industry
• In food and cosmetic industry
• 90% of the herbal extracts and preparation are
standardized using tlc
• Separation of multicomponent pharamceutical
formulation
55

56.  Common problems in TLC
1. Over large spot :- spotting size of the sample should not be larger
than 1-2 mm in diameter
• The components spots will never be larger than or smaller than
your sample origin spot
56

57. 2 uneven advance of solvent phase
• No flat bottom
• No enough solvent
• Plate is not cut evenly
3 spotting
• The sample should be above the solvent level
• If the solvent level covers the sample, the sample
spot should be washed off into the solvent before it
travels upto the tlc plate
57

58. 58

59. 59
THANKYOU