Planar Chromatography Thin Layer Chromatography (TLC)

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Planar Chromatography Thin Layer Chromatography (TLC). 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. - PowerPoint PPT Presentation

Transcript of Planar Chromatography Thin Layer Chromatography (TLC)

  • Planar Chromatography

    Thin Layer Chromatography(TLC)

  • The beginning: Paper chromatographyDeposition of a drop of colourDeposition of a drop of solventDeposition of more solventChromatography = Chroma (colour) + Graphein (Writing)Chromatography = Colour Writing

  • The beginning: Paper chromatography

  • Evolution: Vertical paper chromatographySample is deposited at the bottom line of the paper

    Paper is placed in a tank filled with 1 cm solventSolvent migrates in the paper and elutes the solutes

    The solute migrate depending on their affinity for the solvent

  • 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

  • Thin Layer chromatography (TLC)Sample volume: a few nL to a few LSample is preferably deposited using an automated apparatus in a band-shape with a drying gas spraying the sampleDeposit is thiner and more evenBetter resolutions can be achievedSample deposition

  • Thin Layer chromatography (TLC)Use of concentration zone

  • Effect of pre-equilibrium of a TLC plate

    Solvent front migrates less rapidly

    Better separations can be achievedThin Layer chromatography (TLC)Pre-saturation of the layer is often preferableSolvent is volatile

    Allows pre-saturating the layer with solvent vapors prior to development

  • Thin Layer chromatography (TLC)Vertical DevelopmentSolvent in Liquid-Vapour equilibrium

    Solvent in Vapour adsorbs on the layer

    Solvent migrating in the layer vaporizes Effect of gravity

  • 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 DevelopmentNo effect of gravityMigration speed is constantBetter resolutions can be achieved

  • Thin Layer chromatography (TLC)Horizontal DevelopmentBetter control of the operating conditions(saturation, evaporation)Possibility to develop both sides of the plate= Twice more samples

  • Spotting the plate1st elution2nd elution90 rotation2D separationAnalogy: 2D-gel electrophoresis used in biotechnologyThin Layer chromatography (TLC)

  • Solvent frontStarting linedsolventdsoluteTotally retained soluteTotally unretained soluteThin Layer chromatography (TLC)Reading the TLC

  • Thin Layer chromatography (TLC)Detection of the analytesColoured analytes

    Derivatisation procedures

    Densitometry with UV scannerUV light beamReflected beamDetectorPseudo-chromatogram

  • Thin Layer chromatography (TLC)Detection of the analytesAbsorption of UV radiation is proportional to concentration

    Quantification is possible

  • Thin Layer chromatography (TLC)Detection of the analytesMass spectrometry (Analogous to Matrix Assisted Laser Desorption Ionisation)

  • Advantages:

    Easy to useCheapPossible multiple analysisPossible recovery of the productsNo sample preparation required2-dimensional analysisDrawbacks:

    Slow (typically 30-60 minutes)Limited quality of the separationLimited reproducibilityEvaporation of the mobile phase (composition varies during the analysis)Thin Layer chromatography (TLC)

  • Example: ginsenoside solutesVanhaelen-Fastr et al., J. Chromatogr. A, 868 (2006) 269-276 ginsenosides standards solution

    Triterpene glycosidesUsed in traditional asian medicineand occidental phytotherapy

    extract of Panax ginseng Silica gel1,2-dichloroethane ethanol methanol water56.8:19.2:19.2:4.8 (v/v/v/v), 4CUV-densitometry at 275 nm

  • Example: anthraquinone derivativesSingh et al., J. Chromatogr. A, 1077 (2005) 202-206 RP-18 thin layermethanol water formic acid 80:19:1 (v/v/v)UV-densitometry at 445 nmCompoundR1R2

    PhyscionHOCH3ChrysophanolHHEmodinHOHChrysophanolGlcHGlycoside4 major anthraquinone derivatives found in a species of indian rhubarb

    Varied bioactivities (antioxidant, antifungal, antimicrobial, antiviral, etc)