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.
solid-liquid (column, thin-layer, & paper)
liquid-liquid ( liquid chromatography – LC or HPLC)
liquid-gas (gas chromatography - GC)
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 can’t be seen. (Colorless components)
If you can’t 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
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:
O–Al RCOO– salt formation strongest
\ carboxylic acids attraction
Oδ––Alδ+---:N–R coordination interactions
\ | (Lewis bases)
Oδ– H amines
\ (hydroxylic compds.)
R \ dipole-dipole interaction
\ Oδ– (polar molecules)
Oδ––Alδ+ London forces weakest
\ (nonpolar) attraction
Oδ– only very high MM
fastest solutes –stay on absorbant shortest time
alkanes least polar
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.
petroleum ether (pentanes) least polar
t-butyl methyl ether
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
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 don’t pack tight enough for
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.
NEVER ALLOW COLUMN TO DRY OUT
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 – don’t 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.
Don’t do TLC (thin layer chromatography part of Ch 11, Exp 1)