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.