Experiment #3

Thin Layer Chromatography

(TLC)
Theory

- Purification technique solids and liquids

- Separation of two or more compounds/ions:

- Molecular interactions with two phases:

1. Stationary phase:
Solid: Polar e.g Silica (SiO2), Alumina (Al2O3)
2. Moving phase:
Liquid: Single solvent or Combination of solvents
e.g. Hexanes, dichloromethane, ethyl acetate etc.
Process at the molecular level:

Dynamic equilibrium between solid/liquid phases

Free: dissolved in liquid phase
Adsorbed: surface of solid phase
Mobile Phase

Less Polar Sample

More Polar Sample Stationary Phase
The strength of attraction depends on:

o Size, polarity, hydrogen bonding ability of SAMPLE components.

o Polarity and hydrogen bonding ability of the STATIONARY PHASE.
o Polarity and hydrogen bonding ability of the MOBILE PHASE solvent(s).
Overall Result:

o Non polar molecules---- move faster.

o Polar molecules-----move slower(stronger interaction
with stationary phase).

Butane
Phenyl acetate Butyric acid
Choosing the mobile phase:

o Depends on the polarity of the components.

Spotting the TLC plate:

o TLC is very sensitive. A spot using micro-capillary.

o If too concentrated it may cause smearing.

Visualization:
o Colored components: visible to naked eye.
o Colorless components:
- UV lamp.
- Iodine chamber.

Colored components Colorless components

 Calculating Retardation Factor (Rf )

Rf = Distance spot travel

Distance Solvent travels
What to perform in the lab:
Experiment:
You will use the TLC technique to carry out:

 Identification of an “unknown” analgesic by comparison with known

common analgesics.

I. Identification of an “unkown” analgesic:

Sample preparation:
- Crush part of the “unknown” tablet, transfer to a test tube, mix with a little ethanol
- Try to prepare a 1% solution (the binder will not dissolve)
(1g/100mL = 10 mg/mL)
Preparation of TLC plate:

- Need two plates : Make three spots (spot unknown with two standards in one plat)
Spot the plates : spot <1mm wide
Plate 1: Unknown, Acetaminophen and aspirin
Plate 2: Unknown, Caffeine and Ibuprofen

- Lightly mark a line about 1 cm from the edge of the plate

( see Williamson pp. 182) with graphite pencil (not ink!)

- Lightly mark where you will spot the sample

- Spot the sample: small spot: <1mm wide (Easy to see the spot)

Sample spots under UV-Lamp

Plate Development:
-Prepare development chamber: Solvent
- Carefully place spotted TLC plate:
Spot should be above solvent level!
- Watch carefully as solvent front moves up the plate
- Do not disturb/move the development chamber
Stop when solvent front ~ 1cm from top of the plate
- Remove and immediately mark the position of the
solvent front
- Outline the spots with pencil under UV lamp
- Calculate Rf values of the main spots
- Sketch a picture of the TLC plate in your lab notebook
 Stages of TLC Experiment

Ac Uk C

Dissolved sample
Spotted TLC
Under UV
Running TLC

From TLC 1 and 2 the unknown is?????

II. Separation of paprika pigments (Demo by TA) :

Sample preparation:

- Extraction: TA will extract paprika pigments with diethyl ether (keep

extract in a dark)
- Spot TLC plate
- Carefully place spotted TLC plate:
Spot should be above solvent level!
- Watch carefully as solvent front moves up the plate
- Do not disturb or move the development chamber
during this time!
Pre-Lab Questions:
1. What problem will ensue if the level of the developing liquid is
higher than the applied spot in a TLC analysis?
2. In what order (from top to bottom) would you expect to find
naphthalene, butyric acid, and phenyl acetate on a silica gel TLC
plate developed with dichloromethane?
3. What will be the result of applying too much compound to a TLC
plate?
4. Why is it necessary to run TLC in a closed container and to have the
interior vapor saturated with the solvent?
(Lab Book question # : 3, 4, 7, 8)
Post- lab Questions:

1. Why are the paprika pigments colored, while the common analgesics
you tested are colorless?
2. If you were to test racemic ibuprofen by TLC, how many spots would
you get after developing the plate? Why?