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)