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
The beginning: Paper chromatography
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
Thin Layer chromatography (TLC)
The modern version of paper chromatography
Paper is replaced by a layer (100-200 μm) 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 μL
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
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 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
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 chamberIn non saturated chamber
Analysis time
Migration distance
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 gravityMigration speed is constant
Better resolutions can be achieved
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
Spotting the plate 1st elution 2nd elution90° rotation
Different mobile phases= different principles of separation
2D separation
Analogy: 2D-gel electrophoresis used in biotechnology
Thin Layer chromatography (TLC)
Solvent front
Starting line
dsolvent
dsolutesolvent
solutef d
dR
Totally retained solute
Totally unretained solute
Thin Layer chromatography (TLC)
Reading the TLC
Thin Layer chromatography (TLC)
Detection of the analytes
Coloured analytes
Derivatisation procedures
Densitometry with UV scanner
UV light beam
Reflected beam
Detector
Pseudo-chromatogram
Thin Layer chromatography (TLC)
Detection of the analytes
Absorption of UV radiation is proportional to concentration
Quantification is possible
Thin Layer chromatography (TLC)
Detection of the analytes
+
+
Laser
Desorption Desolvation
H+
Proton transfer
Mass spectrometry (Analogous to Matrix Assisted Laser Desorption Ionisation)
Advantages:
Easy to useCheapPossible multiple analysisPossible recovery of the productsNo sample preparation required2-dimensional analysis
Drawbacks:
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 solutes
Vanhaelen-Fastré 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), 4°CUV-densitometry at 275 nm
Example: anthraquinone derivatives
Singh 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 nm
Compound R1 R2
1. Physcion H OCH3
2. Chrysophanol H H3. Emodin H OH4. Chrysophanol Glc H
Glycoside
4 major anthraquinone derivatives found in a species of indian rhubarb
Varied bioactivities (antioxidant, antifungal, antimicrobial, antiviral, etc)