Introduction Principle Methodology Rf values advantages disadvantages and applications Dr Nisha Sharma Associate Professor School of Pharmaceutical Sciences CSJM University Kanpur ID: 1018518
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1. UNIT –IIIThin layer chromatography Introduction, Principle, Methodology, Rf values, advantages, disadvantages and applicationsDr. Nisha Sharma, Associate Professor School of Pharmaceutical SciencesC.S.J.M. University, Kanpur1
2. THIN LAYER CHROMATOGRAPHYM. Tsvet- Father of Liq. Chrom. 1900’sIntroduced by Izmailov and Shraiber in 1938- separation of plant extractsKirchner: 1950- coated plates- ident. terpenesE. Stahl in 1958 Designed equipmentAlso known as surface, strip, open column, spread layer chromatography2
3. Based on separation by adsorptionDepends on the relative affinity of components towards stationary and mobile phaseSuppose, affinity of component A has strong towards adsorbent, then the movement will be less. Hence the different constituents separate.Liquid – solid chromatographyPRINCIPLE3
4. Competition b/w molecules of analyte & solventBoth binds with the adsorbent surfaceDegree of retention depends upon:Binding strength of analyte with supportSurface area of supportAmount of mobile phase displaced by the analyteBinding strength of solvent phase to adsorbentPRINCIPLE4
5. The retention may be affected by Electrostatic interactionsHydrogen bondingDipole dipole interactionsVan der-waals forcesPRINCIPLE5
6. MethodologyAlmost similar to paper chromatographyCoating material: AdsorbentNatureActivityMechanismSubstances Silica gelAcidicActiveAdsorption/ PartitionAcidic / neutral AluminaBasicActiveAdsorption/ partitionBasic & neutralKeisulguhrNeutralInactivePartitionSt. hydrophilic substCellulose powderNeutralNonePartitionWater soluble compounds6
7. MethodologyCoating material: other examples: CaPO4, Mg Trisilicate, silica gel alumina (1:1), acetylated cellulose etc. Adsorbent : must adhere to plateBinders: CaSO4, starch, hydrated silica OGypsum- widely used binderEx: Silica gel G, Alumina G--- G stands for GypsumZinc silicate- inert fluorescent indicator Ex. Silica Gel GF7
8. 2. PREPARATION OF TLC PLATESSuspension of slurry is preparedMethods to prepare:Pouring method: back & forthDipping: Peifer (1962)- 2 plates at a time- CHCl3 or CHCl3— MeOHSpraying: Reitsema– SprayerSpreading- applicator- aligning tray, spreader, developed by DESAGA (west G)0.1-0.5 mm thick for analytical work0.5-2 mm – preparative worke) Precoated plates- ready to use- 0.1-0.2mm8
9. 3. Activation of Adsorbent: To remove liq. from TLC, Dry for 30min. in air, & then in oven at 110°C fro 30 min, For very active plates- 150°C- 4 hrs4. Purification of silica gel G layers: To remove Iron as impurity, run plates in MeOH:HCl:: 9:1 v/v, Fe gets migrated to solvent front, again activate at 110°C, but if CaSO4 dissolves reused by adding Suitable binder 9
10. 5. Sample application: MicrosyringeSample solution: non polar/ volatile solvent6. Developing tank: tank + lid, saturation of tank is must, or lack of reproducibility in Rf 7. Solvent system: Stahl’s triangle: better separation-mix. of solventsEx: Ethyl acetate : methanol:: 99:1Trial error basis – best solvent system10
11. 11SMEEluting solventMixturePolarNon polarHydrophilicLipophilicinactiveactiveAdsorbent activityStahl’s triangle
12. 8. Development methods: Solvent allowed to rise at ht of 15-18cm on 20 cm plate, 20-40 minutesSolvent front marked and dried9. Determination of components: Colorless--- U.V. Visualizing reagentCorrosive reagents – chromic acid, H2SO4 10. Evaluation: Qualitative (R.f)Quantitative : Direct and indirect methodsDirect: Visual, spot areas, densitometry, direct spectrometryIndirect: Elution- Instrumental12
13. 13After 5 minAfter 15 minabTHIN LAYER CHROMATOGRAPHYsampleSolvent front i.e. distance travelled by solventDistance travelled by Sample
14. Saturation of TLC Tank14Cover plateThin layer plateSolvent mixtureSolvent wick for saturation
15. AdvantagesCombines advantages of PC & CCEquipment simpleShort development time compared to PC & CCSeparation is fairly good on inorganic adsorbent materialWide choice of stationary phase- adsorption, partition, ion exchange15
16. AdvantagesFast recovery of separated components – powdery coating of plate, scrapping, spot/ zone, quantitative removal, dissolve, spect./colorimeterEasy visualization of separated components, florescent compds. Detected easily under U.V. because inorganic background don’t produce fluorescence 16
17. AdvantagesSensitivity: Delineated, sharp spots, 10 to 100 folds as compared to PCVariable thickness of layers: Thin layers for qualitative and thicker layers for preparative Chemically inert stationary phase- application of strong heat or corrosive reagent like sulfuric acid Cost effective17
18. DisadvantagesReproducible results are difficultNot automated procedureOnly non volatile substance or subst. with low volatility can be separatedLength of plate is limitedSeparation is in open system- humidity, temp.Quality of separation is limited 18
19. ApplicationsUsed for separation of all types of natural compoundsTo check the purity of samplesAs a purification process of isolated compdTo examine the reaction: intermediate productsTo identify organic compds.As a check on process- progress of procedure19
20. ApplicationsUsed for separation of inorganic ions- cationic, anionic, covalent species. organic deriv. of metalsSep. of vitaminsAmino acidsSep. of alcohols, glycols, alkaloids, amines, proteins, antibioticsUsed in all type of Industries, Medicine, Forensic science etc20
21. ReferencesJeffry G.H., J. Mendham et al. Vogel’s Text book of Quantitative Chemical Analysis, 5th Edition, 1989, Longman Scientific & Technical, Bath Press, Great Britain. G.R. Chatwal, SK Anand. Instrumental methods of chemical analysis (Analytical Chemistry), ed. 1995, Himalaya Publishing House, Bombay.A.H. Beckett & J.B. Stenlake. Practical Pharmaceutcal Chemistry, Part Two, ed. 4th, 1997, (reprint 2003) CBS Publishers & Distributors, New Delhi.21