Download Column Chromatography notes

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
• dipole-dipole interactions
• hydrogen bonding
• complex formation
• Van der Waals or London forces
“Like attracts like.”
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
Elutropic Series
Lower Petroleum Ether
polarity
Hexane
Columns are generally
eluted with a series of
solvents or solvent
mixtures so that the
polarity of the eluent
increases (normal-phase
chromatography)
Toluene
Dichloromethane
Chloroform
Diethyl ether
Ethyl acetate
Acetone
1-Propanol
(List to right: Lowest Polarity
at top – first eluent )
Ethanol
Higher
polarity
Methanol
Water
Reverse Phase Chromatography
•
•
•
•
Stationary phase is more non-polar;
Mobile phase is more polar.
Common in HPLC
Adsorbent glass beads coated with nonpolar, 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
•
Wash any alumnia
that sticks to walls
down with petroleum
ether
•
Cover the alumnia
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….
drain pet.ether
down to top
of stationary
phase
pipette fluorene:
fluorenone
extract
onto top
of column
drain extract
down onto
alumina
do not
allow liquid level
to drop below
top of alumina
add 1 mL
pet. ether and
drain down
until liquid
level is even
with
stationary
phase
add approx
1/2 cm of
sand
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
front
surface with your fingers.
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)
max distance the eluent traveled (cm)
Know how to
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.