Thin Layer
Chromatography (TLC)
Separatory Techniques: Chromatography
What Is Chromatography?
▪ Chromatography is separating molecules
TLC System: 3 Components
▪ Stationary Phase (Chromatography Plate)
▪ Mobile Phase: Solvent
▪ Analyte: Sample blotted on TLC plate
Stationary and Mobile Phases
▪ Stationary Phase: A
glass/metal/polymer plate most
commonly coated with a polar silica
dioxide or aluminum dioxide matrix.
▪ Mobile Phase: A low polarity solvent
or mix of two or more low polarity
solvents. Ex. Hexanol or 1:1 Mix of
Hexanol and THF
Seperation: Interaction of the analytes, solvent and stationary matrix
1.Prepare
TLC Place
Draw, Blot
and Place
in Vessel
2.Seperate
Add solvent and
allow separation
to occur
3. Results
Add solvent and
allow separation
to occur
Separation: A polar Interaction
Before Separation (0 min)
After Separation (10 min)
Rf: Retention Factor
Rf
Rf A = 8 cm / 10 cm = 0.8
=
Distance solute traveled
----------------------------------Distance solvent front traveled
Rf B = 3 cm / 10 cm = 0.3
Rf C = 2 cm / 10 cm = 0.2
Apply Your
Knowledge
A TLC plate is blotted with 3 samples
and allowed to separate for 10
minutes. In the end the calculated
retention factors are:
0.65
0.30
0.95
Which molecules has the Rf of 0.30?
• Chromatographic method using a polar stationary phase on
a plate and a less polar mobile phase
• Analyte is carried up the stationary matrix by the mobile
phase
Rf = (Distance analyte traveled / distance solvent front)
• The more polar a substance is the less time it will be in the
mobile phase = Travels less, has an Rf closer to 0
• The less polar a sample is the more time it will be in the
mobile phase = Travels more, has an Rf closer to 1
Thin Layer
Chromatography
An Overview