COLUMN CHROMATOGRAPHY

 

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

solute

 

Principle

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

 

O O

|| ||

OAlOAlO alumina

 

+

OH

/

OAl RCOO salt formation strongest

\ carboxylic acids attraction

O

 

 

 

 

Oδ H

/ |

OδAlδ+---:NR coordination interactions

\ | (Lewis bases)

Oδ H amines

 

Oδ---HOR

/

OδAlδ+ H-bonding

\ (hydroxylic compds.)

Oδ alcohols

 

Oδ

/

OδAlδ+

R \ dipole-dipole interaction

\ Oδ (polar molecules)

Cδ+=Oδ ketones

/

R

 

Oδ

/

OδAlδ+ London forces weakest

\ (nonpolar) attraction

Oδ only very high MM

Hδ+CδH2

\

CH2

/

CH3

 

fastest solutes stay on absorbant shortest time

alkanes least polar

alkyl halides

alkenes

dienes

aromatic HCs

aromatic halides

ethers

esters

ketones

aldehydes

amines

alcohols

phenols

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

solutes.

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

dichloromethane

ethyl acetate

acetone

2-propanol

ethanol

methanol

water

acetic acid most polar

 

 

 

 

 

 

 

 

 

 

 

 

Practical The experiment

We have chosen the absorbant

solvent

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

bubbles

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.

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 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)