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 sample

Deposit 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

PhyscionHOCH3ChrysophanolHHEmodinHOHChrysophanolGlcH

Glycoside4 major anthraquinone derivatives found in a species of indian rhubarb

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