10 Thin Layer Chromatography.ppt

Planar Chromatograph Chromatography y 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-00 !m" of stationary phase (silica gel# alumina" deposited on a rectangular glass plate (10-0 cm large" $urrent evolution% &igh-Performance T'$ (&PT'$" $ontrolled sie of stationary phase particles )odified stationary phases (bonded silica% *DS# chiral+"  ,utomated procedures for better reproducibility

Thin Layer chromatography (TLC) ample !epo"ition

Sample volume% a few n' to a few !' 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 etter resolutions can be achieved

Thin Layer chromatography (TLC) #"e o$ concentration %one

Thin Layer chromatography (TLC) Pre-saturation of the layer is often preferable

Solvent is volatile

.ffect of pre-e/uilibrium of a T'$ plate

 ,llows pre-saturating the layer with solvent vapors prior to development

Solvent front migrates less rapidly etter separations can be achieved

Thin Layer chromatography (TLC) Vertical &evelopment

1 Solvent in 'i/uid-apour e/uilibrium  Solvent in apour adsorbs on the layer  2 Solvent migrating in the layer vapories

E$$ect o$ gravity

)igration distance

3n presaturated chamber  3n non saturated chamber 

 ,nalysis time

Thin Layer chromatography (TLC) 'ori%ontal &evelopment

1 &PT'$ plate (layer facing down"  glass plate for sandwich configuration 2 reservoir for developing solvent 4 glass strip 5 cover plate 6 conditioning tray

o e$$ect o$ gravity igration "pee! i" con"tant *etter re"olution" can be achieve!

Thin Layer chromatography (TLC) 'ori%ontal &evelopment

*etter control o$ the operating con!ition" ("aturation evaporation) Po""ibility to !evelop both "i!e" o$ the plate = T,ice more "ample"

Thin Layer chromatography (TLC) -& "eparation

Spotting the plate

1st elution

708 rotation

nd elution

Different mobile phases 9 different principles of separation

 ,nalogy% D-gel electrophoresis used in biotechnology

Thin Layer chromatography (TLC) .ea!ing the TLC

Totally unretained solute Solvent front

 R f  

=

d  solute

dsolvent

d  solvent 

dsolute Starting line Totally retained solute

Thin Layer chromatography (TLC) &etection o$ the analyte"

$oloured analytes Derivatisation procedures Densitometry with : scanner 

#V light beam

&etector 

.e$lecte! beam

P"eu!o/chromatogram

Thin Layer chromatography (TLC) &etection o$ the analyte"

0b"orption o$ #V ra!iation i" proportional to concentration 1uanti$ication i" po""ible

Thin Layer chromatography (TLC) &etection o$ the analyte"

a"" "pectrometry (0nalogou" to atri2 0""i"te! La"er &e"orption 3oni"ation)

Laser 

Desorption

Desolvation

+

+

H+

Proton transfer 

Thin Layer chromatography (TLC)

0!vantage":

&ra,bac4":

.asy to use $heap Possible multiple analysis Possible recovery of the products ;o sample preparation re/uired -dimensional analysis

Slow (typically 20-60 minutes" 'imited /uality of the separation 'imited reproducibility .vaporation of the mobile phase (composition varies during the analysis"

E2ample: gin"eno"i!e "olute"

ginsenosides standards solution Triterpene glycosides :sed in traditional asian medicine and occidental phytotherapy

e%17%17%4> (v?v?v?v"# 48$ :-densitometry at @5 nm

E2ample: anthra5uinone !erivative"

4 maCor anthra/uinone derivatives found in a species of indian rhubarb aried bioactivities (antio