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An Introduction to Thin Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a technique that utilizes a thin layer of substrate (such as Al2O3 or SiO2) for analytical separation of mixtures. Introduced in 1938, it relies on principles of adsorption and partition to separate components. With advantages like simplicity, quick development time, and easy visualization, TLC allows for effective component separation, aided by factors like retention factor and partition coefficient. Specific procedures involve selecting coating materials, preparing thin layers, activating adsorbents, and employing suitable solvent systems for plate development and component detection. This technique finds applications in various fields, including forensic analysis of petroleum and explosive residues.

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An Introduction to Thin Layer Chromatography (TLC)

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  1. TLC: Introduction • Thin layer chromatography (TLC) employs a thin layer (0.25 mm) as a substrate. • Thin Layer: Al2O3, SiO2, MgO etc. • Introduced by Izmailov and Scraiber in 1938 • Introduced as a procedure for analytical adsorption chromatography by Stahl in 1958 • Other Names: drop, strip, spread layer, surface chromatography and open column chromatography

  2. TLC: Theory • Resolution of mixtures of solutes on filter paper may depend upon: 1. surface adsorption 2. Partition between two solvents (predominant factor of separation) Adsorption Adsorptivity varies for different components of mixture Partition The substances are distributed between two liquids, i.e., one is stationary liquid, held in thin layer (stationary phase) and other is moving liquid or developing solvent (mobile phase).

  3. Retention Factor • Partition coefficient (Kd) describes the way in which a compound distributes itself between two phases. • ‘R’ is a function of partition coefficient and is related to the migration of solute front relative to the solvent. • RFdefines the movement of the substances relative to the solvent front in a given chromatographic system.

  4. TLC: Advantages • Simple requirement • Short development time • Wide choice of stationary phase • Easy recovery of separated components • Superior separation effect • Easy visualization of separated components • Sensitivity • Variable thickness of thin layers • Chemically inert stationary phase

  5. TLC: Disadvantages • Can be used only for small scale preparative work • Sensitivity is low compared chromatographic techniques to other modern and sophisticated

  6. TLC: Experimental Procedure • Selection of appropriate coating material • Preparation of Thin layer in plates • Activation of adsorbent • Purification of Silica Gel ‘G’ layers • Application of sample • Plate development with solvent system • Detection of components

  7. Coating Material Adsorbent Acidic/Basic Activity Separatory Mechanism Components to be Separated Silica gel Acidic Active Adsorption Partition Acidic and Neutral substances Alumina Basic Active Adsorption Partition Basic and Neutral Kiesleguhr Neutral Inactive Partition Strongly Hydrophilic substances Cellulose Powder Neutral None Partition Water soluble compounds

  8. Preparation of Thin Layers Various methods of preparing layers are: a. Pouring b. Dipping c. Spraying d. Spreading using applicator e. Pre-coated Plates a. Operation of thin layer spreader b. Aligning tray with glass-slide partially coated

  9. Activation and Purification of Adsorbent Activation • Removal of any liquid associated with thin layer • Done by drying thin layer plate for 30 minutes in air and then in an oven at 110⁰C for another 30 minutes • To obtain very active layers, plates are heated to 150⁰C for about 4 hours Purification • Iron free Silica layers are obtained by giving air-dried plates a preliminary development with methanol-conc. HCl (9:1, v/v)

  10. Sample Application • Agla microsyringe • Capillary tubes

  11. Solvent System If chemical nature of components is unknown • Best eluent is found by trial and error using small, very rapid running TLC plates If chemical nature is known • Stahl’s triangle: Inter-relate adsorbent activity, nature of solute and nature of solvent

  12. Plate Development • Plate is placed in a development chamber at an angle of 45⁰ • Bottom is covered upto nearly 1 mm by solvent • A drop of sample is placed near edge of plate • After sample solvent has evaporated, plate is placed in a closed container saturated with vapors of developing solvents

  13. TLC Plate Development

  14. Detection of Components Coloured substances • Visual assessment Colourless substances • Treating plates with visualizing reagent • Under UV light

  15. Evaluation of Chromatogram • After detection of separated solutes on plate, position is marked and analyzed qualitatively and quantitatively Qualitative Analysis • RFvalues for spots Quantitative Analysis • Direct Method (directly on the layer) • Indirect Method (substances are removed from adsorbent and then determined after elution)

  16. TLC: Forensic Analysis of Petroleum • About 2 ml volumes, each of Petrol (P), Kerosene (K) and Diesel are spotted either on TLC (Silica Gel-G) plates along with the respective case work samples. • Run chromatogram upto 10 cm distance • Visualization under UV light (254 nm) Solvent System Petrol Diesel Kerosene Hexane: Toulene: Acetic Acid (50:50:2) Pink or orange colour RF- 0.49 and 0.51 Violet Blue colour RF- ~0.4

  17. TLC: Forensic Analysis of Explosive Residues Cations • Thin Layer Chromatography of cations such as Aluminium, Zinc, Arsenic, Barium, Mercury and Lead compounds • Solvent system: n-butanol and glacial acetic acid (8:2) • Visualization: Dimethylaminobenzaldehyde Reagent, Pyridine Reagent Anions • Chlorate, Nitrate, Suphide, Thiocynate, Nitrite, Chloride, Sulphate • Solvent System: n-Butanol: Acetone: Ammonia: : 3:2:1 • Visualization: 5% Diphenylamine in acetone

  18. TLC: Cations Spraying Reagent and Colour of Spot RF Metal Ion Dimethylaminob enzaldehyde Reagent Pyridine Reagent n-Butanol: Glacial acetic acid (8:2) Aluminium Brown Pink 0.28 Zinc Orange Brown 0.58 Arsenic Yellow Light Brown 0.30 Barium Violet Brownish Pink 0.38 Cobalt Green Blue 0.63 Copper Light Brown Parrot Green 0.66 Mercury Yellow Faint Pink 0.84 Lead Deep Violet Dark Brown 0.34

  19. TLC: Anions Anions Colour RF Chlorate Light Blue 0.62 Nitrate Yellowish Brown 0.36 Sulphide Blue 0.28 Thiocyanate Indigo 0.80 Nitrite Dark Blue 0.40 Chloride Royal Blue 0.26 Sulphate Pinkish Blue 0.10

  20. TLC: Opium and Heroin • Stationary Phase: Activated Silica gel ‘G’ (0.25/0.20 thickness) • Mobile phase/ Solvent systems: a. Toluene: Acetone: Ethanol: Conc. Ammonia (45:45:7:3) b. Ethyl acetate: Methanol: Conc. Ammonia (85:10:5) c. Ethanol: Chloroform: Dioxane: Petroleum ether: benzene: ammonium hydroxide: ethyl acetate (5:10:50:15:10:15:5) d. Ethanol: benzene: ammonium (5:50:5:40) e. Chloroform: Methanol (90:10) f. Diethyl ether: Acetone: Diethylamine (85:8:7) • Visualization: Dragendorff’s reagent spray, UV light at 254 nm, acidified potassium iodoplatinate reagent spray hydroxide: Dioxane

  21. TLC: Marijuana • Stationary Phase: substance) TLC plates of 0.25/0.20 mm thickness. • Mobile Phase/Solvent systems: [1] – System A : Petroleum ether & Diethyl ether (80:20) – System B : Cyclohexane, Di-isopropyl ether & Di-ethylamine (52:40:8) – System C : n-Hexane, Dioxane & Methanol (70:20:10) • Visualization : Fast blue B salt Activated silica gel (with or without fluorescing

  22. TLC: Cocaine Developing solvent systems: - • System A: Chloroform, Dioxane, Ethyl acetate, Ammonia (29%) (25:60:10:5) • System B: Methanol, Ammonia (29%) (100:1.5) • System C: Cyclohexane, Toluene, Diethylamine (75:15:10) • System D: Ethyl acetate, Benzene, Ammonium hydroxide (60:35:5) • System E: Chloroform, Ethyl acetate, Ammonium hydroxide (40:10:10 drops) Visualization UV light 254 Acidified potassium iodo-platinate reagent. Dragendorff's reagent

  23. TLC: Adulteration in Edible Oils Oil Mobile Phase Visualization RF 0.70-0.75 Rice Bran oil Benzene and Acetic Acid (99:1) Iodine Fuming Petroleum ether (40-600C), diethyl ether and acetic acid (60:40:2, v/v) Castor oil Iodine Vapours 0.25 Argemone oil 1. Butanol, acetic acid, and water (7:2:1). 2. Hexane and acetone (6:4) 3. Heptane UV Light 0.8 Bright yellow spot Petroleum ether (40-600C) , diethyl ether and acetic acid( 6:4:1) Karanza oil UV Light 0.34, 0.22, 0.17 Yellow spot Petroleum ether (40-600C or 60- 800C) Mineral oil fluorescin solution Yellow

  24. References • Chatwal, G.R., Anand, S.K. (2019). Instrumental methods of chemical analysis. Himalaya Publishing House. • Lundanes, E., Reubsaet, L., Greibrokk, T. (2012). Chromatography: Basic principles, sample preparations and related methods. Wiley-VCH. • Wolstenholme, R., Jickells, S., Forbes, S. (2021) Analytical methods in Forensic Sciences. Wiley. • DFS Manual NOTE: These slides are only for academic purposes

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