Thin Layer Chroatography

Thin Layer Chroatography

1. 9 August 2012 Dept. of Pharmaceutics 1 THIN LAYER CHROMATOGRAPHY ADSORBENTS FOR TLC, PREPARATION TECHNIQUES, MOBILE PHASE SELECTION, REVERSED PHASE TLC -: Presented By :- Amruta S. Sambarekar 1 st Year M.Pharm Dept. of Pharmaceutics M M C P, BELGAUM

2. THIN LAYER CHROMATOGRAPHY INTRODUCTION: TLC is a technique in which the components of the extract are distributed between two phases, stationary phase & mobile phase. PRINCIPLE: The principle of separation is adsorption.

3. REQUIREMENTS OF TLC: Stationary phase and papers used. Glass plates. Preparation and activation of TLC plates. Application of the sample. Development tank. Mobile phase. Development techniques . Detecting agents.

4. COMPARISION OF THIN LAYER CHROMATOGRAPHY AND PAPER CHROMATOGRAPHY : THIN LAYER CHROMATOGRAPHY PAPER CHROMATOGRAPHY The principle of separation is adsorption. The principle of separation is partition More amount of substance is required. Less amount of substance is required. Less time consuming i.e. 15—45 minutes More time consuming i.e. 2—3 hours TLC plates can be heated in an oven for a long time Paper cannot be heated in an oven for a long time. Separation is more sharp Separation is less sharp. Thin layer have physical strength. Ascending techniques are preferred for this type Due to lack of physical strength in paper, descending techniques are used. In TLC, chromatoplates are prepared by coating thin layers of silica gel or alumina on glass plates. Here paper is generally used. Silica gel or alumina cannot be used because they cannot be made into sheets. in TLC, corrosive reagents may be coated on glass plates. Here, corrosive reagents destroy the paper. Sensitivity of detection of the fraction on plate is high. The spots are less diffused Less sensitivity of detection. Spots easily diffused TLC can be evaluated under UV. Paper chromatography cannot be evaluated under UV.

5. COMPARISION OF TLC & HPTLC: Parameter TLC HPTLC Chromatographic plate used Hand made /pre-coated Pre-coated Adsorbent layer thickness 250 μ m 100-200 μ m Particle size range 5-20 μ 4-8 μ Pre-washing of the plate Not followed Must Application of sample Manual/Semi automatic Semi automatic/Automatic Shape Spot Spot/Band Spot size 2-4mm 0.5-1mm Sample volume 1-10 μ L 0.2-5 μ L Application of larger volume Spotting which leads to over loading Can be applied as bands 5 mmcp,belgaum

6. No. of samples/plate (20X20) 15-20 40-50 Optimum development distance 10-15cm 5-7cm Development time Depends on mobile phase 40% Less than TLC Reproducibility of results Difficult Reproducible 6 mmcp,belgaum

7. ADSORBENTS FOR TLC In the beginning of TLC method, only few coating materials were used as adsorbents such as silica gel, alumina etc. However, now a days , there is variety of adsorbents which can be selectively utilized. 9 August 2012 Dept. of Pharmaceutics 7

8. Factors to be considered while choosing the adsorbents Characteristics of compound to be separated. Solubility of compounds. Nature of substance to be separated i.e. acidic, basic, amphoteric. To see whether compound is liable to react chemically with adsorbent (or solvent), or not. 9 August 2012 Dept. of Pharmaceutics 8

9. Two general properties that decide its application are : Particle size Homogeneity Particle size of 1-25 m m is generally preferred. Adsorbents do not generally adhere to glass plates & hence binders like gypsum, starch are added. Gypsum (calcium sulphate) in 10-15% w/w is widely used as binder. 9 August 2012 Dept. of Pharmaceutics 9

10. TYPES OF ADSORBENTS Inorganic adsorbents Organic adsorbents 9 August 2012 Dept. of Pharmaceutics 10

11. INORGANIC ADSORBENTS Silica gel Alumina Magnesia Magnesium silicate Calcium silicate 9 August 2012 Dept. of Pharmaceutics 11

12. ORGANIC ADSORBENTS Cellulose & its acetylates Charcoal & activated carbon 9 August 2012 Dept. of Pharmaceutics 12

13. PREPARATION OF CHROMATOPLATES Glass plates or flexible plates are commonly used for adsorbent. Size used depends on type of separation to be carried out, the type of chromatographic tank and spreading apparatus available. The standard sizes are 20 x 5 cm, 20 x 10 cm or 20 x 20 cm . The surface should be flat without irregularities. The standard film thickness is 250um 9 August 2012 Dept. of Pharmaceutics 13

14. Methods for application of adsorbent . Pouring Dipping Spraying Spreading. 9 August 2012 Dept. of Pharmaceutics 14

15. Pouring : The adsorbent of finely divided and homogeneous particle size is made into slurry and is poured on a plate and allowed to flow over it so that it is evenly covered. Dipping : This technique is used for small plates by dipping the two plates at a time, back to back in a slurry of adsorbent in chloroform or other volatile solvents. Exact thickness of layer is not known and evenness of layer may not be good. 9 August 2012 Dept. of Pharmaceutics 15

16. Spraying : Slurry is diluted further for the operation of sprayer. But this technique is not used now a days as it is difficult to get uniform layer. Spreading : All the above methods fail to give thin and uniform layers. Modern methods utilize the spreading devices for preparation of uniform thin layers on glass plates. Commercial spreaders are of two types (a) Moving spreader, (b) Moving plate type. It gives layer thickness from 0.2 to 2.0 mm. 9 August 2012 Dept. of Pharmaceutics 16

17. ACTIVATION OF PLATES After spreading plates are allowed to dry in air and further dried and activated by heating at about 100 c for 30 mins. By removing the liquids associated with layer completely, the adsorbent layer is activated. 9 August 2012 Dept. of Pharmaceutics 17

18. SOLVENT SYSTEM The choice of the mobile phase is depends upon the following factors:- Nature of the substance to be separated Nature of the stationary phase used Mode of chromatography ( Normal phase or reverse phase) Separation to be achieved- Analytical or preparative. 9 August 2012 Dept. of Pharmaceutics 18

19. The organic solvent mixture of low polarity is used Highly polar solvents are avoided to minimize adsorption of any components of the solvent mixture. Use of water as a solvent is avoided as it may loosen the adhesion of a layer on a glass plate. Solvents with an increasing degree of polarity are used in liquid-solid or adsorption chromatography. The solvents listed in elutropic series are selected. 9 August 2012 Dept. of Pharmaceutics 19

20. n-Hexane Cyclohexene Toluene Benzene Diethyl ether Chloroform Dichloromethane 1,2 dichloroethane Acetone Ethyl acetate Acetonitrile Propanol Methanol Acetic acid Water. 9 August 2012 Dept. of Pharmaceutics 20 Increasing polarity

21. 9 August 2012 Dept. of Pharmaceutics 21 APPLICATION OF SAMPLE Sample solution in a non polar solvent is applied. The concentration of a sample or standard solution has to be minimum of a 1% solution of either standard or test sample is spotted using a capillary tube or micropipette. The area of application should be kept as small as possible for sharper and greater resolution.

22. TLC plate “finishing line”  1 cm. “starting line”  1 cm. B. Dissolve solid sample in CH 2 Cl 2 C. Use TLC capillary to transfer and spot dissolved sample T- stillbene benzoic acid 9-fluorenone unknown Sample Application (spotting) A. Draw “guide lines” lightly with pencil Process

23. DEVELOPMENT CHAMBERS TLC plates are placed vertically in a rectangular chromatography tank (as in fig) 9 August 2012 Dept. of Pharmaceutics 23

24. They are classified according to the separation technique used. (a) Tanks for ascending development (b) Tanks for descending development (c) Tanks for horizontal development (d) Tanks for thin layer electrophoresis For first three methods glass or S.S is most suitable. Degree of saturation will affect Rf value 9 August 2012 Dept. of Pharmaceutics 24

25. DEVELOPMENT OF CHROMATOGRAMS Generally ascending method is used to greater extent but various other methods are also used. They are Ascending Development : The plates after spotting of the sample are placed in chamber containing solvent at bottom. Flow of solvent is from bottom to top. (as in fig.) 9 August 2012 Dept. of Pharmaceutics 25

26. Descending : 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 (as in fig.) 9 August 2012 Dept. of Pharmaceutics 26

27. Two dimensional development : It is used if the component of the mixture are not completely separated by development in a single direction. In these sample spot is applied at corner of plate. First development is carried out by ascending method in one solvent. The plate is taken out, solvent allowed to evaporate. second development is carried out in another solvent by changing the edge of plate at 90 (as in fig.) 9 August 2012 Dept. of Pharmaceutics 27

28. 9 August 2012 Dept. of Pharmaceutics 28 Two dimensional development .

29. REVERSED PHASE TLC Here stationary phase is non polar in nature and mobile phase is polar in nature. These chromatoplates are prepared by immersing the adsorbent layer very slowly in 5-10% of paraffin, silicone oil, undecane in petroleum ether or diethyl ether. After removing the plate and evaporating the solvent , the plate is ready for chromatography. Paraffin and silicon oil provides the permanent impregnation whereas undecane can be removed after development after heating the plate at 120 0. 9 August 2012 Dept. of Pharmaceutics 29

30. 9 August 2012 Dept. of Pharmaceutics 30 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Multiple development technique &quantitative method for TLC/HPTLC

31. 9 August 2012 Dept. of Pharmaceutics 31 INTRODUCTION HPTLC or High-Performance Thin Layer Chromatography is an analytical technique based on thin layer chromatography, but with enhancements intended to increase the resolution of the compounds to be separated and to allow quantitative analysis of the compounds.

32. 9 August 2012 Dept. of Pharmaceutics 32 Pre-conditioning (chamber saturation) Pre-conditioning has pronounced influence on the separation profile What happen plate are introduced in non saturated chamber- Lager quantity of solvent are required for given distance Increase in Rf values

33. 9 August 2012 Dept. of Pharmaceutics 33 Reason for chamber saturation If chamber is saturated prior to development solvent vapour get uniformly distributed in chamber As plate are introduced in such chamber it soon get pre-loaded with solvent Less solvent required to travel particular distance Lower the Rf values

34. 9 August 2012 Dept. of Pharmaceutics 34 Development &drying Techniques used for devolvement in HPTLC/TLC Ascending Descending Two dimensional Horizontal (continuous) Gradient Radial Multidimensional

35. 9 August 2012 Dept. of Pharmaceutics 35 Drying- After development the plate are removed form the chamber & mobile phase removed completely as quickly as possible] Drying preferably be performed in fume cup board to avoid contamination of lab atmosphere During drying plate should always laid horizontally

36. 9 August 2012 Dept. of Pharmaceutics 36 Usually hand dryer are used ( cold or hot). The following considerations are taken care of during the drying process:- Essential oil component may evaporate. Compound sensitive to oxygen may get destroyed. Particle of dust from lab. may deposit on chromatogram. It is precisely for this reason the drying of chromatogram should preferably be done in dessicator with protection from heat and light.

37. 9 August 2012 Dept. of Pharmaceutics 37 Detection and visualization One of the most characteristic feature of the HPTLC is possibility to utilize post chromatographic off-line derivatization. The zones can be located by various physical, chemical, biological, physiological methods. There is no difficulty in detecting the colored substance or colorless substance absorbing the short wave in UV region ( 254nm ). Contd…

38. 9 August 2012 Dept. of Pharmaceutics 38 Or with intrinsic fluorescence such as riboflavin or quinine sulphate. The substance which do not have above properties have to be transferred into detectable substance by means of chromogenic and flurogenic reagent. Iodine is the universal detecting agent. Derivatization is essentially required for detection when individual compounds does not respond to UV or does not intrinsic fluorescence. Contd… Contd…

39. 9 August 2012 Dept. of Pharmaceutics 39 Other detection methods based on wetting and solubility phenomena. As aluminium oxide, kieselguhr or silica gel are hydrophilic adsorbent, on dipping or spraying the chromatogram with water lipophilic such as steroid hydrocarbon appears as a white spot against semi transparent background. Instead of water one can employ hydrophilic or lipophilic dye solution for spraying or dipping. Contd… Contd…

40. 9 August 2012 Dept. of Pharmaceutics 40 In case of hydrophilic dyes such as methylene blue the background is stained blue, whereas non-wetted zone appear pale. While in case of lipophilic dyes non-wetted zone appears as a deeply colored against as a pale background. Fluorescent chemicals are used for the detection of lipophilic substances by wetting or non-wetting technique. E.g. Rhodomin B. Contd… Contd…

41. 9 August 2012 Dept. of Pharmaceutics 41 Several corrosive reagents are also used for detection of organic compounds. These compounds produce color or fluroscent zone on heating. E.g. 10% v/v alcoholic sulphuric acid, 5% potassium dichromate. Contd…

42. Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. C. Mark spots with a pencil while viewing under UV. UV B. View results under UV light. look for grayish spots on the fluorescent green background  

43. 9 August 2012 Dept. of Pharmaceutics 43 Quantitative method in TLC and HPTLC Techniques for quantitative analysis Analysis of fraction on plate:- Visual comparison with standard. Precise measurement of spot area or spot size. Photo densitometry. Direct spectrometry. Contd…

44. 9 August 2012 Dept. of Pharmaceutics 44 Determination of fraction after elution from coating material Scooped area containing adsorbed compound with vacuum cleaner and then analyzed by various analytical methods like gravimetry, polagraphy, chlorometry. Radioactive substance can be assayed by Photo densitometry of x-ray film. Direct radio-scanning of chromatoplates. Contd…

45. QUALITATIVE ANALYSIS: Rf value is calculated for identifying the spots i.e in qualitative analysis. Rf value ranges from 0 – 1. but the ideal values are from 0.3 to 0.8 . when Rf value of a sample and reference standard. APPLICATIONS OF TLC: Separation of mixtures of drugs of chemical or biological origin,Plant extracts etc. Separation of carbohydrates, vitamins, antibiotics, glycosides, etc. Identification of related compounds in the drugs. To detect the presence of foreign substances in drugs.

46. IMPORTANT QUESTIONS What do you mean by reversed phase TLC? (10) Explain various techniques used for the visualisation of spot in TLC? (5) Difference between TLC and HPTLC?(5) 9 August 2012 Dept. of Pharmaceutics 46

47. REFERENCES ‘Instrumental method of chemical analysis’ by B.K. Sharma. ‘Instrumental method of chemical analysis’ by Chatwal. Text book of pharmaceutical analysis by Dr. Ravi shankar. Pharmaceutical Analysis Volume – ii Instrumental Methods by Dr. A. V Kasture, Dr. S. G Wadodkar, Dr. K. R. Mahadik, Dr. H. N. More. Internet source. 9 August 2012 Dept. of Pharmaceutics 47

48. 9 August 2012 Dept. of Pharmaceutics 48 Thank you