Column Chromatography

Chromatography is used to separate compounds in a mixture.

Used to determine what compounds are present

Used to purify and isolate compounds

In all types chromatography, compounds equilibrate between 2 phases, a mobile

phase and a stationary phase. The longer a compounds spends in the mobile phase,
the faster it elutes.

As long as the components elute at different rates, they will be separated.

In TLC, the mobile phase was a liquid and the stationary phase was a solid.

The components spent time on the stationary phase (adsorbent) by adsorbing to

the surface. Adsorption occurred because there was some sort of intermolecular
attraction.

The compounds in the mixture (elutants) spent time in the mobile phase (eluent)
because they were soluble in it. The solubility was also dependent on the strength
of intermolecular forces.

Column chromatography and TLC are just two different techniques for solid-liquid
chromatography. TLC is usually used to identify substances and monitor reactions
(exception preparative TLC). Column chromatography is usually used to purify or
isolate compounds.

The same adsorbents are used, silica gel and alumina (basic, neutral, and acid
washed).
 TLC Column
Chromatography
Flow of Eluent Flow of Eluent

Silica or desorbing Silica or desorbing

Alumina Alumina
adsorbing adsorbing

In TLC, the eluent was drawn up the TLC by capillary action.

In column chromatography, the adsorbent stationary phase is packed in a glass

column (like a buret). The eluent flows down past the adsorbent. The elutants
equilibrate between the phase on their way down. What draws the eluent down in
column chromatography?

As the compounds separate in column chromatography, they form horizontal bands

or regions in the column.
 Eluent

Separated bands
of compounds
Flow

Alumina

Sand

Cotton or glass wool

Stopcock

Individual bands are collected as they come off the column.

As in other types of chromatography, various factors affect separation. In column
chromatography they are:

Adsorbent
Column height and diameter
Eluent
Flow rate

Adsorbents

Although the ability of the stationary phase to adsorb compounds will vary with
the nature of the material, the amount of water present also strongly affects the
adsorption. Water binds tightly to adsorbents through dipole-dipole interactions (H-
bonding). Water molecules occupy sites otherwise available for elutant molecules.
When water is added to an adsorbent, it is deactivated.

When water is removed by heating, it is activated. Highly activated adsorbents can

cause unwanted reactions to occur however.

To insure good separation, the right amount of adsorbent must be used. If too little is
used, good separation will not be achieved. It is often suggested to that the wt. of
the adsorbent be 25 - 30 times greater than the wt. of the sample mixture.

Column Height and Diameter

For good separation is often suggested that the height to diameter ratio of the column
be 8-10 : 1.

Eluent

"Like dissolves like." Nonpolar compounds will spend more time in nonpolar solvents
and pass off the column more quickly. Polar solvents will displace polar compounds
from the adsorbent and dissolve them. They will then pass off the column quickly.

Table 12.2 in the manual lists some common used solvents.

Table 12.3 lists the order of elution for various types of organic compounds based on
their polarity.

Usually one solvent alone will not efficiently elute every component of a mixture off
the column. Often, a nonpolar solvent is used first, and then a polar solvent is used
to elute remaining compounds.

The second solvent is often carefully added as a gradient.

If the switch is from pure hexane to pure diethyl ether, then successively larger
percentages of diethyl ether in hexane are used until it is 100% diethyl ether.

H3C C O C CH3
H2 H2

e.g. text 1, 2, 5, 10, 15, 25, 50, 100%

or 10, 20, 40, 60, 80, 100%

In a change from hexane to diethyl ether, diethyl ether molecules are displacing the
hexane molecules adsorbed to alumina. Since the intermolecular attraction to the
alumina will be stronger with diethyl ether, the process will be significantly
exothermic. This may cause the solvent to actually vaporize and form bubbles that
create cracks and other discontinuities in the adsorbent. As will be seen, this affects
separation.

Actually, in this experiment, an abrupt change from hexane to t-butyl methyl ether
(MTBE) will be used without a problem. This is an exception.

CH3

H3C O C CH3

CH3

For the reasons outlined in the manual, solvents more polar than diethyl ether and
methylene chloride are undesirable.

Flow Rate

As in GC, the flow rate can be too fast or too slow.

If it is too fast, then the separation will be poor.

If it is too slow, then the bands will diffuse (broaden) and potentially lead to
poor separation also.

Column Packing

The adsorbent must be packed into the column so that there are no irregularities
such as bubbles or cracks.

For good separation, the bands in the column must be as horizontal as possible.
(See Fig. 12.5).

Reasons for Non-Horizontal Bands

Surface edge of adsorbent is not horizontal

Column not perpendicular to bench top
Streaming and channeling due to bubbles, cracks, and irregular
adsorbent surface. (See Fig. 12.6)

Packing Your Column

 You may use any of the techniques in the lab separate or lab manual for
packing the column. This method is a version of the dry pack method described in
the lab manual.

It is suggested that you attach the plastic tubing and screw clamp so that you
can control the flow of solvent.

Clamp the column to a ring stand so that it is perpendicular to the bench top.
Add a small wad of cotton as shown in the Figure 1a in the lab separate.
Add a thin layer of sand.
Add solvent and let it drain slowly out.
Slowly add the alumina. It must be added at a rate that prevents it from
clumping or trapping bubbles. Add more solvent to maintain its level above the
adsorbent surface. Do not let the adsorbent go dry!

Now add your mixture as described in the lab separate and follow the remainder of
the procedure.

A general procedure for introducing samples to columns

Dissolve the sample in a minimum amount of polar solvent.

Lower the solvent level to the adsorbent surface.
Add the sample carefully. Avoid disturbing the adsorbent surface. (Often
slowly introducing the sample from a pipet down the side of the column is the
preferred method.)
Drain until the level reaches the adsorbent surface.
Add eluent carefully.
Drain to the adsorbent surface.
Repeat these two procedures until the sample is adsorbed onto the alumina.
Add a layer of sand to protect the adsorbent surface.

When large quantities of solvent are necessary, reservoirs above the column are
used. See Figure 12.10 in the lab manual.

Monitoring the Column

If the compounds have a visible color (yeah!), just place a flask beneath the column
when the band reaches the bottom of the column.

If the compounds fluoresce, then the bands can be followed with an UV lamp.

If the adsorbent if impregnated with a fluorescent dye, then the bands can be
followed with an UV lamp.
Usually, these are not options. Fractions are collected in test tubes. Each tube
tested to determine whether something besides eluent is present. Carousel auto
samplers are available.
One method for testing. Draw a grid in pencil on a TLC plate and number the
intersections. Each number represents a test tube. Spot a sample from each test
tube. Check for which fractions have a spot under UV. Those fractions contain a
component of the mixture.

1 5 6 7

15

In the experiment you will separate ferrocene from acetylferrocene. They are both
examples of organometallic compounds.

C
CH3
Fe Fe

ferrocene acetylferrocene
yellow orange