Lecture 12
• Chromatography Introduction
• Ch 7: Thin-Layer Chromatography
• Lecture Problem 4 Due
This Week In Lab:
• Ch 6: Procedures 2 & 3
• Due: Ch 5 Final Report
Next Week in Lab:
• Ch 7 PreLab
• Quiz 4
Chromatography
A separation/purification technique.
Two main types:
1. Thin-Layer Chromatography (TLC)
2. Column Chromatography (CC)
Uses:
• To separate the components of a mixture - TLC & CC
• To determine the purity of a compound - TLC
• To see if two compounds are identical - TLC
• To monitor the progress of a reaction - TLC
• To follow a column chromatography separation - TLC
Thin-Layer Chromatography
Filter Paper
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
TLC Plate: contains
A polar stationary phase
(alumina or silica gel) &
a very small amount
of your sample
Mobile Phase: organic
solvent(s) of varying polarity
TLC Bottle/Chamber
Column Chromatography
Funnel
Mobile Phase:
Organic solvent(s) of varying
polarity
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Polar Stationary Phase:
alumina or silica gel with
your sample loaded onto it can accommodate a larger
amount of sample vs. TLC
A Packed Column
Small Erlenmeyers to collect
fractions
Chromatography Basics
How it works:
• Your sample is loaded onto the polar stationary phase
• Polar compounds will adsorb onto the stationary phase to a greater
extent than non-polar compounds
• The mobile phase (eluting phase) helps “push” or elute the
compounds either down a column (for CC) or up a plate (for TLC)
The main concept to consider in chromatography is polarity.
Polarity & Intermolecular Attractive Forces
• More polar compounds will be more attracted to silica gel than
non-polar compounds due to intermolecular attractive forces - a
dipole-dipole interaction.
+
Si
Silica gel, [SiO2]n
O
O
-
+
Si
OH
O
• The more non-polar compounds will travel more easily and more
quickly through the stationary phase.
• The mobile phase helps carry the compounds through the stationary
phase.
Separation of compounds in a mixture is possible because compounds
have different polarities. Non-polar compounds will elute first and
polar compounds will elute last.
Polarity & Intermolecular Attractive Forces
Example: Separate a mixture of butyl amine and cyclohexane using TLC
Things to consider:
1. Polarity of each compound in the mixture
Butyl amine is polar; cyclohexane is non-polar
2. Polarity of stationary phase
Silica gel (or alumina) is polar - predict that butyl amine will
interact with it more strongly
3. Polarity of the mobile phase - the solvent: you determine
what solvent to use
H2C
Prediction:
Cyclohexane will elute
first/faster through the stationary
phase.
Butyl amine will elute last/slower.
H2C
O
+
N H
H
+
Si
H2
C
C
H2
O
CH2
CH2
TLC Separation
Example: Separate a mixture of butyl amine and cyclohexane using TLC
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Mobile Phase: Typically use a mixed solvent
system. If the mobile phase is non-polar,
cyclohexane will travel along with it, but
butyl amine will not as readily.
If the mobile phase is polar, both cyclohexane
and butyl amine will travel with it, but
butyl amine will be slower because it’ll be
interacting with silica gel as it’s traveling.
H2C
Note the separation
of spots
H2C
O
+
- H
N
H
+
Si
H2
C
C
H2
O
CH2
CH2
Chapter 7: TLC Experiment/Separation of Analgesics
A one-day experiment:
• Testing and choosing a TLC mobile phase - work in groups. Each
person in a group will test two (2) different solvent systems. Pick
the solvent system that gives you the best
separation of spots.
• TLC analysis on different analgesics (standards). Get Rf
values of these standards.
• Using TLC data of the standards, identify analgesics in an
“unknown” tablet by comparing Rf values.
Chapter 7: TLC Experiment/Separation of Analgesics
The Experimental Steps
1. Load sample onto stationary phase/TLC plate (labeled)
(a) Dissolve sample in a
small amount of organic solvent
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
(b) Use capillary tubes to load on
Sample
The smaller the spot, the better. Why?
TLC plate (labeled)
with samples loaded
Ac As C I
Aspirin spot
Caffeine spot
Aceaminophen spot
Ibuprofen spot
pencil mark 1 cm
from bottom
depth of mobile phase
Predict the order
of elution for these
compounds.
OH
O
CO2H
O
H3C
CH3
O
Aspirin
HN
N
N
O
O
CH3
Acetaminophen
CO2H
Ibuprofen
CH3
N
CH3
Caffeine
N
2. Insert TLC plate into TLC chamber (filled with a layer of
mobile phase & allow mobile phase to “run up” the TLC
plate. Take out when the solvent reaches 1 cm from
top of plate (solvent front). Mark the solvent front line with a pencil.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
3. Detection:
If the spots are not colored and can’t be seen by the eye, use:
• UV lamp for UV-active compounds; most aromatics are
UV-active
• If compounds are not UV-active, use an iodine (I2)
chamber
Once you visualize the spots, circle them with a pencil.
4. Calculate Rf values for each spot/analgesic.
Rf = distance spot traveled from origin line/distance of solvent front
You will obtain Rf values for each analgesic you test. These Rf
values will help you identify analgesics present in an “unknown” tablet.
Make sure to use the same mobile phase as Rf’s will vary with
varying mobile phases.
Rf = distance spot traveled from origin line/distance of solvent front
Solvent Front Line
Distance traveled
by solvent
Distance traveled by spot
Origin Line
Identifying Unknowns via TLC
1. Compare the Rfs of the known analgesics (standards) with
the Rfs of the analgesics in your “unknown” tablet.
2. Use the Rfs to identify the analgesics in your tablet.
Note: More than one analgesic may be in one tablet. Thus,
you may see more than one spot per tablet sample.