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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
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
A separation/purification technique.
Chromatography
Thin-Layer Chromatography
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TLC Bottle/Chamber
Filter Paper
TLC Plate: containsA polar stationary phase(alumina or silica gel) &a very small amountof your sample
Mobile Phase: organic solvent(s) of varying polarity
Column Chromatography
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A Packed Column
Polar Stationary Phase:alumina or silica gel withyour sample loaded onto it -can accommodate a largeramount of sample vs. TLC
Mobile Phase:Organic solvent(s) of varyingpolarity
Funnel
Small Erlenmeyers to collectfractions
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.
• 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 compoundshave different polarities. Non-polar compounds will elute first andpolar compounds will elute last.
Si
OH
O
O
O
Siδ+
δ-
δ-
δ-
δ+
Silica gel, [SiO2]n
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-polar2. Polarity of stationary phase
Silica gel (or alumina) is polar - predict that butyl amine willinteract with it more strongly
3. Polarity of the mobile phase - the solvent: you determine what solvent to use
Si
OOδ+
δ-
N
H
Hδ-δ+
H2C
H2CCH2
CH2
CH2
H2C
Prediction:Cyclohexane will elutefirst/faster through the stationaryphase.Butyl amine will elute last/slower.
TLC Separation
Example: Separate a mixture of butyl amine and cyclohexane using TLC
Si
OOδ+
δ-
N
H
Hδ-δ+
H2C
H2CCH2
CH2
CH2
H2C
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Mobile Phase: Typically use a mixed solventsystem. If the mobile phase is non-polar,cyclohexane will travel along with it, butbutyl amine will not as readily.If the mobile phase is polar, both cyclohexaneand butyl amine will travel with it, butbutyl amine will be slower because it’ll beinteracting with silica gel as it’s traveling.
Note the separationof spots
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 bestseparation 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.
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Chapter 7: TLC Experiment/Separation of AnalgesicsThe Experimental Steps
1. Load sample onto stationary phase/TLC plate (labeled)
(a) Dissolve sample in asmall amount of organic solvent
(b) Use capillary tubes to load onSample
The smaller the spot, the better. Why?
TLC plate (labeled)with samples loaded
depth of mobile phase
Ac As C I
Aceaminophen spot
Aspirin spot
Caffeine spot
Ibuprofen spot
pencil mark 1 cmfrom bottomPredict the order
of elution for thesecompounds.
CO2H
O CH3
O
Aspirin
OH
HN
CH3
O
AcetaminophenCO2H
Ibuprofen
N
N N
N
O
O
CH3
CH3
H3C
Caffeine
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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.
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 withvarying mobile phases.
Origin Line
Solvent Front Line
Distance traveledby solvent
Distance traveled by spot
Rf = distance spot traveled from origin line/distance of solvent front
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.