Tlc (Thin layer chromatography)

Thin layer chromatography ppt

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

2. CONTENT Introduction History When TLC i s 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

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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 Spreading Pouring Dipping Spraying 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

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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

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30. 7) Chromatographic development Different development techniques are used for efficient separation Ascending Descending Two dimensional Circular (radial) 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

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

34. ii i ) 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. i v ) 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 I n a n t i - c i r c u l a r d e v e l o p ment , a f l a t c i r c u l ar s u p p o rt i s u s e d w i t h m o b i l e p h a s e a p p l i ed a t i t s e d g e s . S a m p l e s a r e a p p l i ed n e a r t h e e d g e s a n d Carri ed t o w a r ds t h e c e n t e r b y t h e m o b i l e p h a s e L i k e c i r c u l ar d e v e l opment , anti-circular d e v e l o p ment a l s o h a s a d v a n t a ge o f a v o i ding " e d g e s e f f e c t s " 36

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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 O r 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

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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: Measurement directly on the layer 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 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

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