chromatography - the separation of a mixture of two or more

different compds. (or ions) by a differential

distribution between two phases

(stationary & mobile)

-        used for > 10 grams of material


Works on the same principle as solvent extraction


Separation depends on different solubilities (or absorbtivities)

of substances to be separated relative to the two phases.


Various types

solid-liquid (column, thin-layer, & paper)

liquid-liquid ( liquid chromatography LC or HPLC)

liquid-gas (gas chromatography - GC)


Column Chromatography

stationary phase solid absorbant packed in a column

mixture to be separated solutes (elutants or eluates)

mobile phase solvent (eluent) passing through the

adsorbent in the column, that elutes the




The separation is based on the many equilibrations the solutes

experience between the mobile phase and the stationary phases.




Initially, components of the solute mixture are

absorbed onto the adsorbant (alumina particles)

at the top of the column.


The continuous flow of solvent through the column elutes

(or washes) the solutes off the adsorbant (alumina) and down the column.


Different compds. move down the column at different rates depending on their relative affinity for the adsorbants and the solvent. (The less polar solutes come off first from alumina, which is polar.)


As the components of the mixture are separated,

they begin to form bands,

each band containing a single component.


If the column is long enough & correct parameters (absorbant, polarity of solvent, length & diameter of column, rate of flow of solvent) are chosen, the bands separate from one another leaving gaps of pure solvent between them.


As each band passes out of the column, it can be collected completely before the next band arrives.


If parameters are poorly chosen, bands overlap or coincide, resulting in poor or no separation.


Sometimes the bands cant be seen. (Colorless components)

If you cant see the bands because you have colorless components, collect the solvent in a series of tared flasks & then evaporate the solvent.

Stationary Phase (absorbents)

- can be anything that does not dissolve in the solvent

- usually

alumina, Al2O3 ∙ xH2O (more polar)

- preferred for less polar solutes

silica gel, SiO2 ∙ xH2O (less polar)

-        preferred for more polar solutes

- adsorbents are mixed with a solvent to form a slurry &

- poured into the column


Solutes To Be Separated

The more polar the functional group of the solute, the stronger the bond to the alumina (or silica gel).


Bond strength between solute & adsorbent decreases with change in solute as follows:

salt formation>coordination>H-bonding>dipole-dipole>London



|| ||

OAlOAlO alumina





OAl RCOO salt formation strongest

\ carboxylic acids attraction






Oδ H

/ |

OδAlδ+---:NR coordination interactions

\ | (Lewis bases)

Oδ H amines




OδAlδ+ H-bonding

\ (hydroxylic compds.)

Oδ alcohols





R \ dipole-dipole interaction

\ Oδ (polar molecules)

Cδ+=Oδ ketones






OδAlδ+ London forces weakest

\ (nonpolar) attraction

Oδ only very high MM







fastest solutes stay on absorbant shortest time

alkanes least polar

alkyl halides



aromatic HCs

aromatic halides








carboxylic acids most polar

slowest solutes stay on adsorbant longest



Mobile Phase (solvents)


Sometimes a single solvent can be found that will elute all solutes.

Other times a mixture must be used.

Start with a nonpolar solvent to remove relatively nonpolar


Then gradually increase polarity of the solvent to remove

the more polar solutes.






Chromatography solvents

petroleum ether (pentanes) least polar

ligroin (hexanes)

t-butyl methyl ether

diethyl ether


ethyl acetate






acetic acid most polar













Practical The experiment

We have chosen the absorbant


size of column

You need to be concerned with

packing the column

rate of flow

Experiment 1

Separating solutes ferrocene (yellow)

acetylferrocene (orange) - toxic


packing the column

critical to success of separation

column should be vertical so packing is horizontal

so bands are horizontal for good separation

packing should be uniform without voids caused by air


if slurry is too dilute particles dont pack tight enough for

good separation

if slurry is too thick air bubbles get trapped in the column

resulting in bands that are not horizontal

procedure for packing the column

1. Fill column with 1/2 - 2/3 full with alumina

2. Pour the alumina into 10 mL Erlenmeyer flask

3. Fill the column with (~4 mL) of ligroin or hexane

4. Add ~ 8 mL of hexane to alumina in the flask

5. Stir to eliminate air bubbles

6. Swirl mixture to suspend adsorbent & immediately pour entire

slurry into funnel

7. Open value & allow solvent to drain to about 5 mm above the

top surface of adsorbent.


Why? When more solvent is added to top, air bubbles can form & channels are created that result in uneven bands & poor separation.




using the column to separate the mixture

WORK IN THE HOOD to prepare the ferrocene/acetylferrocene

mixture. [Acetylferrocene is a liver toxin & a mild carcinogen.]

1. Dissolve 90 mg of the 50:50 ferrocene/acetylferrocene mixture

in a minimum volume of dichloromethane (just a few drops)

2. Add 300 mg alumina, stir, evaporate solvent completely in

the hood. (Dichloromethane boils at 55oC)

3. Pour dry powder into funnel of chromatography column

Carefully dont inhale powder.

4. Wash down with a few drops of hexane

5. Tap to remove air bubbles

6. Open value

7. Carefully add new solvent so top of surface of column

is not disturbed

8. Run down and repeat until sample is a narrow band at the top

of the column.


eluting the solute

1. Fill column with solvent and elute samples from column

rate too fast poor separation

too slow sample diffuses up as well as

travels down poor separation


2. Collect yellow ferrocene in 10 mL flask


Continue to follow procedure in lab text page 175


Note: Recrystallization of ferrocene and acetylferrocene takes another period.


Dont do TLC (thin layer chromatography part of Ch 11, Exp 1)