Transcript 10 Thin Layer Chromatography

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
Solvent migrates in the paper and
elutes the solutes
Paper is placed in a tank filled with
1 cm solvent
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 deposition
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
Thin Layer chromatography (TLC)
Use of concentration zone
Thin Layer chromatography (TLC)
Pre-saturation of the layer is often preferable
Solvent is volatile
Effect of pre-equilibrium of a TLC plate
Allows pre-saturating the
layer with solvent vapors
prior to development
Solvent front migrates less rapidly
Better separations can be achieved
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
Migration
distance
In presaturated
chamber
In non
saturated
chamber
Analysis time
Thin Layer chromatography (TLC)
Horizontal Development
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
No effect of gravity
Migration 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
Thin Layer chromatography (TLC)
2D separation
Spotting the plate
1st elution
90° rotation
2nd elution
Different mobile phases
= different principles of separation
Analogy: 2D-gel electrophoresis used in biotechnology
Thin Layer chromatography (TLC)
Reading the TLC
Totally unretained solute
Solvent front
d solute
Rf 
d solvent
dsolvent
dsolute
Starting line
Totally retained solute
Thin Layer chromatography (TLC)
Detection of the analytes
Coloured analytes
Derivatisation procedures
Densitometry with UV scanner
UV light
beam
Detector
Reflected
beam
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
Mass spectrometry
(Analogous to Matrix Assisted Laser Desorption Ionisation)
Laser
Desorption
Desolvation

+
+
H+
Proton transfer


Thin Layer chromatography (TLC)
Advantages:
Drawbacks:
Easy to use
Cheap
Possible multiple analysis
Possible recovery of the products
No sample preparation required
2-dimensional analysis
Slow (typically 30-60 minutes)
Limited quality of the separation
Limited reproducibility
Evaporation of the mobile phase
(composition varies during the
analysis)
Example: ginsenoside solutes
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), 4°C
UV-densitometry at 275 nm
Vanhaelen-Fastré et al., J. Chromatogr. A, 868 (2006) 269-276
Example: anthraquinone derivatives
4 major anthraquinone derivatives found
in a species of indian rhubarb
Varied bioactivities (antioxidant,
antifungal, antimicrobial, antiviral, etc)
Compound
R1
R2
1.
2.
3.
4.
H
H
H
Glc
OCH3
H
OH
H
Physcion
Chrysophanol
Emodin
Chrysophanol
Glycoside
RP-18 thin layer
methanol – water – formic acid 80:19:1 (v/v/v)
UV-densitometry at 445 nm
Singh et al., J. Chromatogr. A, 1077 (2005) 202-206