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.
Types of Chromatography

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
 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 “Like attracts like.”
• dipole-dipole interactions
• hydrogen bonding
• complex formation
• Van der Waals or London forces
 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
 Lower Petroleum Ether
Elutropic Series polarity
Hexane
Columns are generally Toluene
eluted with a series of
Dichloromethane
solvents or solvent
mixtures so that the Chloroform
polarity of the eluent Diethyl ether
increases (normal-phase
Ethyl acetate
chromatography)
Acetone
1-Propanol
(List to right: Lowest Polarity
at top – first eluent ) Ethanol
Higher Methanol
polarity
Water
 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
slowly sif t
in alumnia
• Wash any alumnia while
tapping the
that sticks to walls 10 mL Pet Ether add approx column
1 cm sand with hand
down with petroleum
ether
• Cover the alumnia glass wool
plug
with a 1-cm layer 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….
add 1 mL
pet. ether and
drain down
pipette fluorene: until liquid
drain pet.ether fluorenone level is even
down to top extract drain extract with add approx
of stationary onto top down onto stationary 1/2 cm of
phase of column alumina phase sand

do not
allow liquid level
to drop below
top of alumina
 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 Chromatography
1.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 Solvent
surface with your fingers. front
2. Don’t use ink to mark the
origin.
3. Mark the leading edge of each
spot with pencil and measure
the distance traveled. Origin
4. Measure the distance the
solvent traveled.
5. Calculate the rate of flow (Rf )

Rf = distance the spot traveled (cm) Know how to

max distance the eluent traveled (cm) 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.