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Thin Layer Chroatography

  1. 1. THIN LAYER CHROMATOGRAPHY ADSORBENTS FOR TLC, PREPARATION TECHNIQUES, MOBILE PHASE SELECTION, REVERSED PHASE TLC -: Presented By :- Amruta S. Sambarekar 1st Year M.Pharm Dept. of Pharmaceutics M M C P, BELGAUM 10 February 2013 Dept. of Pharmaceutics 1
  2. 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. 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. 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 Paper cannot be heated in an oven for a long long time time. Separation is more sharp Separation is less sharp. Thin layer have physical strength. Ascending Due to lack of physical strength in paper, techniques are preferred for this type descending techniques are used. In TLC, chromatoplates are prepared by Here paper is generally used. Silica gel or coating thin layers of silica gel or alumina on alumina cannot be used because they cannot glass plates. be made into sheets. in TLC, corrosive reagents may be coated on Here, corrosive reagents destroy the paper. glass plates. Sensitivity of detection of the fraction on Less sensitivity of detection. Spots easily plate is high. The spots are less diffused diffused TLC can be evaluated under UV. Paper chromatography cannot be evaluated under UV.
  5. 5. • COMPARISION OF TLC & HPTLC: Parameter TLC HPTLC Chromatographic plate used Hand made /pre- Pre-coated coated Adsorbent layer thickness 250 μm 100-200μm Particle size range 5-20μ 4-8μ Pre-washing of the plate Not followed Must Application of sample Manual/Semi Semi automatic automatic/Autom mmcp,belgaum atic Shape Spot Spot/Band Spot size 2-4mm 0.5-1mm Sample volume 1-10μL 0.2-5μL Application of larger volume Spotting which Can be applied as leads to over bands 5 loading
  6. 6. No. of samples/plate (20X20) 15-20 40-50 Optimum development 10-15cm 5-7cm distance Development time Depends on 40% Less than mobile phase TLC mmcp,belgaum Reproducibility of results Difficult Reproducible 6
  7. 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. 10 February 2013 Dept. of Pharmaceutics 7
  8. 8. Factors to be considered while choosing the adsorbents 1. Characteristics of compound to be separated. 2. Solubility of compounds. 3. Nature of substance to be separated i.e. acidic, basic, amphoteric. 4. To see whether compound is liable to react chemically with adsorbent (or solvent), or not. 10 February 2013 Dept. of Pharmaceutics 8
  9. 9.  Two general properties that decide its application are : 1. Particle size 2. Homogeneity  Particle size of 1-25 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. 10 February 2013 Dept. of Pharmaceutics 9
  10. 10. TYPES OF ADSORBENTS 1. Inorganic adsorbents 2. Organic adsorbents 10 February 2013 Dept. of Pharmaceutics 10
  11. 11. INORGANIC ADSORBENTS • Silica gel • Alumina • Magnesia • Magnesium silicate • Calcium silicate 10 February 2013 Dept. of Pharmaceutics 11
  12. 12. ORGANIC ADSORBENTS • Cellulose & its acetylates • Charcoal & activated carbon 10 February 2013 Dept. of Pharmaceutics 12
  13. 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 10 February 2013 Dept. of Pharmaceutics 13
  14. 14. Methods for application of adsorbent. Pouring Dipping Spraying Spreading. 10 February 2013 Dept. of Pharmaceutics 14
  15. 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. 10 February 2013 Dept. of Pharmaceutics 15
  16. 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. 10 February 2013 Dept. of Pharmaceutics 16
  17. 17. ACTIVATION OF PLATES • After spreading plates are allowed to dry in air and further dried and activated by heating at 0 about 100 c for 30 mins. • By removing the liquids associated with layer completely, the adsorbent layer is activated. 10 February 2013 Dept. of Pharmaceutics 17
  18. 18. SOLVENT SYSTEM • The choice of the mobile phase is depends upon the following factors:- 1. Nature of the substance to be separated 2. Nature of the stationary phase used 3. Mode of chromatography ( Normal phase or reverse phase) 4. Separation to be achieved- Analytical or preparative. 10 February 2013 Dept. of Pharmaceutics 18
  19. 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. 10 February 2013 Dept. of Pharmaceutics 19
  20. 20. • n-Hexane • Cyclohexene • Toluene • Benzene • Diethyl ether • Chloroform • Dichloromethane • 1,2 dichloroethane Increasing • Acetone polarity • Ethyl acetate • Acetonitrile • Propanol • Methanol • Acetic acid • Water. 10 February 2013 Dept. of Pharmaceutics 20
  21. 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. 10 February 2013 Dept. of Pharmaceutics 21
  22. 22. Sample Application (spotting) TLC plate 1 cm. Process “finishing line” A. Draw “guide lines” lightly with pencil B. Dissolve solid sample in CH2Cl2 C. Use TLC capillary to transfer and spot dissolved sample “starting line” 1 cm. T-stillbene benzoic acid 9-fluorenone unknown
  23. 23. DEVELOPMENT CHAMBERS • TLC plates are placed vertically in a rectangular chromatography tank (as in fig) 10 February 2013 Dept. of Pharmaceutics 23
  24. 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 10 February 2013 Dept. of Pharmaceutics 24
  25. 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.) 10 February 2013 Dept. of Pharmaceutics 25
  26. 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.) 10 February 2013 Dept. of Pharmaceutics 26
  27. 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 0 another solvent in by changing the edge of plate at 90 (as in fig.) 10 February 2013 Dept. of Pharmaceutics 27
  28. 28. Two dimensional development. 10 February 2013 Dept. of Pharmaceutics 28
  29. 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 0. heating the plate at 120 10 February 2013 Dept. of Pharmaceutics 29
  30. 30. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Multiple development technique &quantitative method for TLC/HPTLC 10 February 2013 Dept. of Pharmaceutics 30
  31. 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. 10 February 2013 Dept. of Pharmaceutics 31
  32. 32. Pre-conditioning (chamber saturation) • Pre-conditioning has pronounced influence on the separation profile • What happen plate are introduced in non saturated chamber- 1. Lager quantity of solvent are required for given distance 2. Increase in Rf values 10 February 2013 Dept. of Pharmaceutics 32
  33. 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 10 February 2013 Dept. of Pharmaceutics 33
  34. 34. Development &drying • Techniques used for devolvement in HPTLC/TLC 1. Ascending 2. Descending 3. Two dimensional 4. Horizontal (continuous) 5. Gradient 6. Radial 7. Multidimensional 10 February 2013 Dept. of Pharmaceutics 34
  35. 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 10 February 2013 Dept. of Pharmaceutics 35
  36. 36. • Usually hand dryer are used ( cold or hot). • The following considerations are taken care of during the drying process:- i. Essential oil component may evaporate. ii. Compound sensitive to oxygen may get destroyed. iii. 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. 10 February 2013 Dept. of Pharmaceutics 36
  37. 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… 10 February 2013 Dept. of Pharmaceutics 37
  38. 38. Contd… • 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. 10 February 2013 Dept. of Pharmaceutics Contd… 38
  39. 39. Contd… • 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. 10 February 2013 Dept. of Pharmaceutics Contd… 39
  40. 40. Contd… • 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. 10 February 2013 Dept. of Pharmaceutics Contd… 40
  41. 41. Contd… • 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. 10 February 2013 Dept. of Pharmaceutics 41
  42. 42. Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. B. View results under UV light. look for grayish spots on the UV fluorescent green background C. Mark spots with a pencil while viewing under UV.
  43. 43. Quantitative method in TLC and HPTLC • Techniques for quantitative analysis A. Analysis of fraction on plate:- i. Visual comparison with standard. ii. Precise measurement of spot area or spot size. iii. Photo densitometry. iv. Direct spectrometry. 10 February 2013 Dept. of Pharmaceutics Contd… 43
  44. 44. Contd… B. Determination of fraction after elution from coating material i. Scooped area containing adsorbed compound with vacuum cleaner and then analyzed by various analytical methods like gravimetry, polagraphy, chlorometry. C. Radioactive substance can be assayed by i. Photo densitometry of x-ray film. ii. Direct radio-scanning of chromatoplates. 10 February 2013 Dept. of Pharmaceutics 44
  45. 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. 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) 10 February 2013 Dept. of Pharmaceutics 46
  47. 47. REFERENCES 1. ‘Instrumental method of chemical analysis’ by B.K. Sharma. 2. ‘Instrumental method of chemical analysis’ by Chatwal. 3. Text book of pharmaceutical analysis by Dr. Ravi shankar. 4. Pharmaceutical Analysis Volume – ii Instrumental Methods by Dr. A. V Kasture, Dr. S. G Wadodkar, Dr. K. R. Mahadik, Dr. H. N. More. 5. Internet source. 10 February 2013 Dept. of Pharmaceutics 47
  48. 48. 10 February 2013 Dept. of Pharmaceutics 48