Column and Thin Layer Chromatography

Two separation techniques --

Chromatography is a separation technique used to obtain pure chemical compounds from a mixture of compounds on a scale from micrograms up to kilograms.

The prefix chroma derives from the original separations of colored plant pigments by this method.

All forms of chromatography are based on the same underlying principal -- i.e. partition coefficient.

Characterized by the nature of the mobile phase (eluant) and stationary phase (adsorbent) –

• liquid – solid = column chromatography, HPLC (high performance liquid chromatograhy), TLC (thin layer chromatography)

• gas – liquid = GLC (gas-liquid chromatograhy)

• liquid-liquid = paper chromatography

Types of Chromatography

Chromatography Basics (1)

The components of a mixture will distribute themselves unequally between two immiscible phases.

Mobile phase (eluent) – a liquid or gas which flows continuously over the stationary phase

Stationary phase (adsorbent) – a solid or liquid held in place and for which the components of the mixture have different affinities

Chromatography Basics (2)The mixture is applied to the column to create a narrow band (migratory band) of material in dynamic equilibrium between the mobile phase and the stationary phase.

As the mobile phase moves, the components of the mixture which are the most soluble in the mobile phase move with it – those most attracted to the stationary phase “bind” and are held up. New equilbria are established and the process repeats.

Chromatography Basics (3)

With time, the component which is the most soluble in the mobile phase moves farther away from the other = separation.

Collecting fractions of the solution exiting the column allows one to separate the components of the mixture from the mobile phase.

Forces of Attraction

The components of the mixture are attracted to the stationary phase by forces such as –

• electrostatic forces

• dipole-dipole interactions

• hydrogen bonding

• complex formation

• Van der Waals or London forces

“Like attracts like.”

Selection of Adsorbent

Components of mixture must have different affinities for the adsorbent.

Common organic adsorbents are:

• Alumina (Al2O3) – more polar – highly reactive – three forms are neutral, basic, acidic

• Silica gel (silicic acid , SiO2●xH2O) – slightly acidic

• Others include – chitin, derivatives of cellulose, ion exchange resins, etc

Packing Rules

• Adsorbent must be dry and have uniform particles size

• Start with at least 10 g adsorbent per 1 g of mixture

• Packing about 10 x higher than wide

• No air pockets or irregularities

Elutropic SeriesColumns are generally eluted with a series of solvents or solvent mixtures so that the polarity of the eluent increases (normal-phase chromatography)

(List to right: Lowest Polarity at top – first eluent )

Petroleum Ether

Hexane

Toluene

Dichloromethane

Chloroform

Diethyl ether

Ethyl acetate

Acetone

1-Propanol

Ethanol

Methanol

Water

Lower polarity

Higher polarity

Reverse Phase Chromatography

• Stationary phase is more non-polar; Mobile phase is more polar.

• Common in HPLC

• Adsorbent glass beads coated with non-polar, hydrocarbon film or non-polar polymeric material

• Elute with decreasing polarity solvents

(For more on chromatography go to: http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html)

Detection of Compounds

Unless the components of the mixture were colored, how to do you know when they are coming off the column?

Detection Methods include not only visual but also

• Refractive Index

• Ultraviolet (UV) spectroscopy

• Thin layer chromatography tracking

• Evaporation and collection of a residue

Overview of Procedural Steps• Clamp upright a dry 50-mL chromatography column with either a

stopcock or hose and clamp on the tip. Ensure the stopcock is closed.

• Loosely insert a small plug of either cotton or glass wool at the

• Add enough sand to give a column 1-cm high

• Add 10 mL petroleum ether via a funnel

• Slowly sift in 8 g of dry neutral alumina while constantly tapping the walls of the column

glass woolplug

10 mL Pet Ether add approx1 cm sand

slowly sif t in alumnia while tapping the column with hand

• Wash any alumnia that sticks to walls down with petroleum ether

• Cover the alumnia with a 1-cm layer of sand

drain pet.etherdown to top of stationaryphase

pipette fluorene:fluorenone extractonto top of column

drain extract down onto alumina

do not allow liquid level to drop below top of alumina

add 1 mLpet. ether and drain down until liquid level is evenwith stationaryphase

add approx1/2 cm of sand

Procedural Steps cont’d• Put ~0.1 g of fluorene:fluorenone mixture in a test tube.• Add 0.5 mL of pet ether to mixture & then 1 drop of dichloromethane with shaking to get the solid mixture to dissolve.• Then….

Procedural Steps cont’d

• Add 20 mL clean petroleum ether without disturbing the sand

• Open the stopcock and collect the eluent in a graduated cylinder

• Monitor the eluent every 5 mL.

Monitoring the Eluent

1. Every 5 mL that elutes, catch a drop or two on a watch glass and determine if a solid residue remains after evaporation.

2. When no residue appears in the last drop evaporated, run 5 mL more petroleum ether.

3. Shut the stopcock and switch to dichloromethane.

4. When the eluant is no longer yellow, stop the column. The separation is complete.

Thin Layer Chromatography1.Using a small capillary, place a tiny drop of the eluent from each collected sample on a thin layer strip. Do the same for the original mixture.

2. Develop the strips in a mixture of 1.5 mL dichloromethane to 8.5 mL petroleum ether until the solvent is within a cm or so of the top of the strip.

3. Dry the strip and visualize the spots – iodine or UV light

Thin Layer Chromatography

1. Don’t touch the separating surface with your fingers.

2. Don’t use ink to mark the origin.

3. Mark the leading edge of each spot with pencil and measure the distance traveled.

4. Measure the distance the solvent traveled.

5. Calculate the rate of flow (Rf )

Origin

Solvent front

Rf = distance the spot traveled (cm) max distance the eluent traveled (cm)

Know how to calculate the Rf

of a spot!

Procedural Steps• Evaporate the solvent from both eluted

substances.• Remember to make sure that you know the

mass of the container before evaporation! (Weigh-by-difference technique)

• Put the dry solids properly labeled bottles and submit to your instructor .

• Results:• %-recoveries of each compound

• Rf values of each compound

What do you expect?

Adsorbent is alumnia – neutral, polar

Which compound has the higher affinity for the adsorbent and why?

O

Fluorene Fluorenone

Preparing your NB…• Chemicals in Table:

– Fluorene, 9-fluorenone, petroleum ether, dichloromethane, alumina, water

• Figures:– Fig. 6.3 (pg. 181)

– Fig. 6.7 (pg. 189) combined with Fig. 6.11 (pg. 193)

• Follow the procedure given in the Handout! You may print and securely tape the procedure in your NB.

• Make sure that you’ve read over the procedure and have a good idea of what you’ll be doing!

• No chemical reactions.