Planar Chromatography Thin Layer Chromatography (TLC)

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Transcript of Planar Chromatography Thin Layer Chromatography (TLC)

  • Slide 1
  • Planar Chromatography Thin Layer Chromatography (TLC)
  • Slide 2
  • The beginning: Paper chromatography Deposition of a drop of colour Deposition of a drop of solvent Deposition of more solvent Chromatography = Chroma (colour) + Graphein (Writing) Chromatography = Colour Writing
  • Slide 3
  • The beginning: Paper chromatography
  • Slide 4
  • Evolution: Vertical paper chromatography Sample is deposited at the bottom line of the paper Paper is placed in a tank filled with 1 cm solvent Solvent migrates in the paper and elutes the solutes The solute migrate depending on their affinity for the solvent
  • Slide 5
  • Thin Layer chromatography (TLC) The modern version of paper chromatography Paper is replaced by a layer (100-200 m) of stationary phase (silica gel, alumina) deposited on a rectangular glass plate (10-20 cm large) Current evolution: High-Performance TLC (HPTLC) Controlled size of stationary phase particles Modified stationary phases (bonded silica: ODS, chiral) Automated procedures for better reproducibility
  • Slide 6
  • Thin Layer chromatography (TLC) Sample volume: a few nL to a few L Sample is preferably deposited using an automated apparatus - in a band-shape - with a drying gas spraying the sample Deposit is thiner and more even Better resolutions can be achieved Sample deposition
  • Slide 7
  • Thin Layer chromatography (TLC) Use of concentration zone
  • Slide 8
  • Effect of pre-equilibrium of a TLC plate Solvent front migrates less rapidly Better separations can be achieved Thin Layer chromatography (TLC) Pre-saturation of the layer is often preferable Solvent is volatile Allows pre-saturating the layer with solvent vapors prior to development
  • Slide 9
  • Thin Layer chromatography (TLC) Vertical Development 1.Solvent in Liquid-Vapour equilibrium 2.Solvent in Vapour adsorbs on the layer 3.Solvent migrating in the layer vaporizes Effect of gravity In pre- saturated chamber In non saturated chamber Analysis time Migration distance
  • Slide 10
  • 1. HPTLC plate (layer facing down) 2. glass plate for sandwich configuration 3. reservoir for developing solvent 4. glass strip 5. cover plate 6. conditioning tray Thin Layer chromatography (TLC) Horizontal Development No effect of gravity Migration speed is constant Better resolutions can be achieved
  • Slide 11
  • Thin Layer chromatography (TLC) Horizontal Development Better control of the operating conditions (saturation, evaporation) Possibility to develop both sides of the plate = Twice more samples
  • Slide 12
  • Spotting the plate1 st elution2 nd elution90 rotation Different mobile phases = different principles of separation 2D separation Analogy: 2D-gel electrophoresis used in biotechnology Thin Layer chromatography (TLC)
  • Slide 13
  • Solvent front Starting line d solvent d solute Totally retained solute Totally unretained solute Thin Layer chromatography (TLC) Reading the TLC
  • Slide 14
  • Thin Layer chromatography (TLC) Detection of the analytes Coloured analytes Derivatisation procedures Densitometry with UV scanner UV light beam Reflected beam Detector Pseudo-chromatogram
  • Slide 15
  • Thin Layer chromatography (TLC) Detection of the analytes Absorption of UV radiation is proportional to concentration Quantification is possible
  • Slide 16
  • Thin Layer chromatography (TLC) Detection of the analytes Mass spectrometry (Analogous to Matrix Assisted Laser Desorption Ionisation)
  • Slide 17
  • Advantages: Easy to use Cheap Possible multiple analysis Possible recovery of the products No sample preparation required 2-dimensional analysis Drawbacks: Slow (typically 30-60 minutes) Limited quality of the separation Limited reproducibility Evaporation of the mobile phase (composition varies during the analysis) Thin Layer chromatography (TLC)
  • Slide 18
  • Example: ginsenoside solutes Vanhaelen-Fastr et al., J. Chromatogr. A, 868 (2006) 269-276 ginsenosides standards solution Triterpene glycosides Used in traditional asian medicine and occidental phytotherapy extract of Panax ginseng Silica gel 1,2-dichloroethane ethanol methanol water 56.8:19.2:19.2:4.8 (v/v/v/v), 4C UV-densitometry at 275 nm
  • Slide 19
  • Example: anthraquinone derivatives Singh et al., J. Chromatogr. A, 1077 (2005) 202-206 RP-18 thin layer methanol water formic acid 80:19:1 (v/v/v) UV-densitometry at 445 nm CompoundR 1 R 2 1.PhyscionHOCH 3 2.ChrysophanolHH 3.EmodinHOH 4.ChrysophanolGlcH Glycoside 4 major anthraquinone derivatives found in a species of indian rhubarb Varied bioactivities (antioxidant, antifungal, antimicrobial, antiviral, etc)
  • Slide 20