TLC
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TLC

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TLC Presentation Transcript

  • 1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar.
  • 2.  Izmailov & Shraiber separated plant extracts using 2mm thick and firm layer of alumina set on glass plate.  In 1944, Consden, Gorden & Martin used filter papers for separating amino acids.  In 1950, Kirchner identified terpenes on filter paper  In 1958, Stahl developed standard equipment for analysing byTLC.
  • 3.  Adsorption  Separation based on the affinity of the compounds towards the stationary phase.  Stationary phase: thin layer of adsorbent coated on chromatographic plate.  Mobile phase solvent flows through b’cos of capillary action (against gravity)  The components with more affinity towards the stationary phase travels slower.  Lesser affinity towards the stationary phase travels faster.
  • 4.  1. Simple method & less expensive  2. Rapid technique  3. Separation upto µgms  4.Any type of compound can be analysed  5. Efficiency of separation: depends upon the particle size ( small) flow rate ( less viscous)  6. Detection is easy and not tedious  7. Capacity of the thin layer can be altered.  8. Needs less solvent, stationary phase & time  9. Corrosive spray reagents can be used without damaging the plates.
  • 5.  Stationary phases  Glass plates  Preparation and activation ofTLC plates  Application of sample  Development tank  Mobile phase  Development technique  Detecting or visualising agents
  • 6. NAME COMPOSITION ADSORBENT: WATER RATIO Silicagel H Silicagel without binder 1:1.5 Silicagel G Silicagel +CaSO4 1:2 Silicagel GF Silicagel+Binder+f luorescent indicator 1:2 Alumina Neutral, Basic & Acidic Al2O3 without binder 1:1.1
  • 7. Al2O3 G Al2O3 + binder 1:2 Cellulose powder Cellulose without binder 1:2 Cellulose powder Cellulose with binder 1:6 Kieselguhr G Diatomaceous earth + binder 1:2 Polyamide powder Polyamide 1:9 (CHCl3:CH3OH=2:3)
  • 8.  Glass plates 20cmX20cm(full plate), half plate (20x10), quarter plate (20x5)  These dimensions are used since the width of the TLC spreader is 20cm  Microscopic slides can be used for some applications like monitoring the progress of a chemical reaction  The development time is 5 min  Good quality & should withstand temperatures used for drying the plates
  • 9.  1. Pouring: slurry is prepared & poured on glass plate & spread uniformly  Demerit: uniformity in thickness cannot be ensured  2. Dipping: two plates are dipped into the slurry and are separated after removing from slurry and later dried.  Demerit: larger quantity of slurry is needed  3. Spraying: using a sprayer  Demerit: layer thickness cannot be maintained uniformly  4. Spreading : the best technique  Normal thickness 0.25mm for analytical purposes  2mm thickness for preparative purposes  Oven temperature for drying should be 100-120 for 1 hr.
  • 10.  To get good spots, the conc of the sample has to be minimum  2-5µl of a 1% solution is spotted using a capillary tube or micropipette.  Spots should be kept atleast 2cm above the base of the plate  Spotting area should not be immersed in the mobile phase
  • 11.  Glass beakers, specimen jars  The tank should be lined inside with filter paper moistened with mobile phase so as to saturate the atmosphere  If saturation is not done, edge effect occurs where the solvent front in the middle ofTLC plate moves faster than that of the edge.
  • 12.  The solvent or mobile phase used depends upon  1. nature of the substances to be separated  2. nature of the stationary phase used  3. mode of chromatography ( normal / reverse phase)  4. separation to be achieved ( analytical / preparative)  Eg: petroleum ether, CCl4, cyclohexane, CS2, Ether, acetone, Benzene,Toluene, Ethylacetate, CHCl3, alcohols, water, pyridine, organic acids,
  • 13.  1. One dimensional development (vertical)  2.Two dimensional development  3. Horizontal development  4. Multiple development  One dimensional development:The plates are kept vertical & the solvent flows against gravity b’cos of capillary action.  Two dimensional development:  This tech is used for complex mixtures  First, the plates are developed in one axis & the plates after drying are developed in the other axis.
  • 14.  After the development ofTLC plates, the spots should be visualised.  Detecting coloured spots can be done visually.  For colourless spots   1. Non-specific methods: no. of spots can be detected, but not the exact nature or type of compound  2. Specific methods: specific spray reagents or detecting agents or visualising agents are used to find out the nature of compounds or for identification purposes.
  • 15.  Where the no. of spots can be detected but not the exact nature / type of compound  1. Iodine chamber method: where brown or amber spots are observed  2. H2SO4 spray reagent: 70-80% of H2SO4 with few mg of either K2Cr2O7 or KMnO4 or few ml of HNO3 as oxidising agent is used.  After spraying onTLC plates is heated in an oven  Black spots are seen due to charring of compounds
  • 16.  3. UV chamber for fluorescent compounds: when compounds are viewed under UV chamber, at 254nm (short wavelength) or at 365nm (longer) fluorescent compounds can be detected. Bright spots are seen under a dark background.  4. Using fluorescent stationary phase: when the compounds are non fluorescent, a fluorescent stationary phase is used.When the plates are viewed under UV chamber, dark spots are seen on a fluorescent background. Eg: such stationary phase is Silicagel GF
  • 17.  Specific spray reagents or detecting agents or visualising agents are used to find out the nature of compounds or for identification purposes.  Ex: 1. Ninhydrin  for amino acids  2. FeCl3  phenolic compounds & tannins  3. Dragendroff’s reagent for alkaloids  4. 3,5- Dinitrobenzoic acid cardiac glycosides  5. 2,4-Dinitorphenylhydrazine  aldehydes & ketones
  • 18.  2 types  1. Destructive technique: samples are destroyed  Specific spray reagents, con H2SO4  2. Non-Destructive technique: Sample is not destroyed even after detection.This is used in preparativeTLC  Eg: UV-chamber method, Iodine chamber method, densitometric method
  • 19.  Measures the density of the spots  When the optical density of the spots for the standard & the test solution are measured , the quantity of the substance can be calculated.  The plates are neither destroyed nor eluted with solvents to get the compounds.  This method is also called in-situ method
  • 20.  1. Separation of mixtures of drugs of chemical or biological origin, plant extracts  2. Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids, glycosides  3. identification of drugs  4. identification of related compounds in drugs  5. to detect the presence of foreign substances in drugs  6. to detect decomposition products in drugs
  • 21.  HPTLC is a sophisticated & automated form ofTLC.  Features:  1. the use of precoated plates with stationary phase particle size of <10µ diameter.  2. wide choice of stationary phases like Silicagel, for normal phase Reverse phase:C18,C8 3. Auto sampler instead of manual spotting and streaking for preparative purposes. 4. New type of development chambers which requires less amt of solvents for developing. 5. More efficiency b’cos smaller & uniform size of adsorbents 6.The use of UV/Vis/Fluorescence scanner which scans the entire chromatogram qualitatively & quantitatively. The scanner is an advanced type of densitometer. 7. Increased data processing capabilities by the use of computers.
  • 22.  Rf value is the ratio of distance travelled by the solute to the distance travelled by the solvent front.  The Rf value ranges from 0 to 1.0 (0.3-0.8)  Rx value: is nothing but the ratio of distance travelled by the sample and the distance travelled by the standard.  Rx value is always closer to 1.0  Rm value is used in qualitative analysis to find out whether the compounds belong to a homologous series.