Thin Layer Chromatography Stationary phase is solid Mobile

Thin Layer Chromatography Stationary phase is solid Mobile phase is liquid TLC is simple and rapid method carried out on thin layer of adsorbents Advantages of TLC above paper and column are as: 1. Requires little equipments 2. Requires very less time 3. Separation effects are superior than other methods 4. Very less amount of sample is essential for analysis 5. Spraying with corrosive agents also permitted Pritam Jain 1

Technique of TLC: Adsorbents: Two important factors considered while choosing adsorbent 1. Characteristic of compounds to be separated 2. Solubility of compounds Binders are used with adsorbents like gypsum (calcium sulphate), starch. Inorganic adsorbents: 1. Silica gel: Most widely used because of its higher adsorptive strength. The silica gel is prepared by hydrolysis of sodium silicate to silicic acid. This product then gone through vaporisation and condensation produce silica gel (amorphous powder). In adsorption the silanol groups represent the active centers where the intereactions take place which is required for the retention of compounds to be separated. These intereactions are mainly hydrogen bonding, dipole-dipole intereactions and electrostatic intereactions. Available with binder or without binder. 2 Plaster of Paris (Ca. SO 4) mostly used advantageous

2. Aluminum oxides: Al 2 O 3 the aluminum oxides used for precoated layers of TLC are preferentially prepared from aluminium hydroxide. This replaces silica gel. Available with or without binders 3. Kieselguhr: natural product of fossil origin that is formed from the skeletons of dead diatoms. It contains silicic acid. Diatomaceous earth available with or without binders but resolution is lesser.

4. Magnesia: Mg. O is too finely divided and can be mixed with filter aid 5. Magnesium silicate, Calcium silicate: Used for separation of sugars and its acetates 6. Others: Aluminium silicate, bauxite, barium sulphate, calcium hydroxide can be used II) Organic Adsorbents: 1. Cellulose and its acetylates: These are fibrous. Modified cellulose powders are used to obtain ion exchange separations in TLC. Can be used with or without binders 2. Charcoal and Activated Carbon: Adsorb strongly aromatic substances 3. Others: Dextran gels, polyamides, ion exchange resins, sucrose are also used. Pritam Jain 4

2. Preparation of Chromatoplates: Using the suspension of slurry of the coating material to give uniform layer. a) Pouring: Simplest method. Adsorbent made into slurry and poured on the Plate and allowed to flow over it. b) Dipping: Used for small plates. Dipping the two plates back to back in a Slurry of adsorbent. Exact thickness of layer is not known. c) Spraying: Slurry is diluted for the operation of sprayer. Not used now a days, Difficult to get uniform layer on the plate. d) Spreading: Modern methods utilizes the spreading divices for preparation of uniform thin layers on glass plates. Commercial spreaders are of two types Moving spreader Moving plate type 5 Pritam Jain

Pre-coated plates of different adsorbents either on glass or polymeric sheets have been introduced by E. Merck. 3. Activation of plates: After spreading, plate is allowed to dry for 5 -10 mins. And further dried and activated by heating at 1000 C For 45 mins. 4. Solvent system: Choice of mobile phase depends on a) Nature of substances to be separated b) Adsorbent material An organic solvent mixture of low polarity should be used. Highly polar solvent are generally avoided to minimize adsorption of any Components of solvent mixture. Petroleum ether, CCl 4, Benzene, Dichloromethane, Chloroform, Diethyl ether, Ethyl acetate, Pyridine, Acetone, n-propanol, Ethanol, Methanol, Formaldehyde Pritam Jain 6

5. Application of sample: Sample is applied as solution in nonpolar solvent. l l l Polar solvent has tendency to spread out the starting spot. The solvent used for dissolving should be volatile. The area of application should be kept small for great Resolution. Capillary tube or micro-pipette, loop or syringe is used. Care should be taken to prevent the hole through the Adsorbent layer. The spots should not be nearer than 1 cm centre to centre and should be 2 -5 mm in diameter. The loading of sample should be 15 microgram per spot. 7 Pritam Jain

6. Development chambers: The plate is placed vertically in specially designed Containers called chromatography chamber or tank. l Chambers prepared from Glass or stainless steel are used. l Degree of saturation is very imp. factor. l Saturation is done by putting a sheet of filter paper along the broad side of tank and dipping in the solvent. 7. Development of chromatograms: l Ascending development: The flow of solvent is from bottom to top. l Descending development: Flow of solvent from reservoir to the plate is by means of filter paper strip. Pritam Jain 8

l Location of spots: l l l Physical methods include UV, fluorescence or radioactive counting. Chemical methods include locating agents are applied by spraying. Conc. Sulphuric acid is used as it produced colored spots. Iodine vapors are also used for organic substances as locating agents. Evaluation of chromatogram: Qualitative: The Rf value of sample is compared with known, and qualitative identification of sample is made. Pritam Jain 9

Quantitative: l Visual comparison: Visual comparison of spot size, intensity of spot with the standard. l l Spot area and weight relationship: From the area of spot, amount of substance is calculated. l Spot Densitometry: After spraying, color developed is measured in densitometer. l Direct spectrometry: The absorption or fluorescence of separated zones directly on TLC plates at wavelength of maximum absorption of substance. Pritam Jain 10

l Applications l l l l of TLC: Purity of sample Examination of reactions Identification of compounds Biochemical analysis In pharmaceutical chemistry: Detection of impurities in pharmaceutical drugs. Various drugs have been tested qualitatively Separation of multicomponent pharmaceutical formulation Separation and Identification of colors, preservatives, sweetening agent etc. in food and cosmetic industry. Pritam Jain 11

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