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Lecture 11. Chromatography Introduction Ch 7: Thin-Layer Chromatography Lecture Problem. This Week In Lab: Ch 6: Procedure 2 - Caffeine from Tea Due: Ch 5 Final Report Next Week in Lab: Ch 7 PreLab Quiz 4 Ch 10 Spectral Unknown Progress Check. Chromatography.

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Lecture 11

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Lecture 11

  • Chromatography Introduction
  • Ch 7: Thin-Layer Chromatography
  • Lecture Problem
  • This Week In Lab:
  • Ch 6: Procedure 2 - Caffeine from Tea
  • Due: Ch 5 Final Report
  • Next Week in Lab:
  • Ch 7 PreLab
  • Quiz 4
  • Ch 10 Spectral Unknown Progress Check


A separation/purification technique.

  • Two main types:
  • Thin-Layer Chromatography (TLC)
  • 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

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


Mobile Phase:

Organic solvent(s) of varying


Polar Stationary Phase:

alumina or silica gel with

your sample loaded onto it -

can accommodate a larger

amount of sample vs. TLC

Small Erlenmeyers to collect


A Packed Column


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 compounds
  • have different polarities. Non-polar compounds will elute first and
  • polar compounds will elute last.

Silica gel, [SiO2]n


Polarity & Intermolecular Attractive Forces

Example: Separate a mixture of butyl amine and cyclohexane using TLC

Things to consider:

Polarity of each compound in the mixture

Butyl amine is polar; cyclohexane is non-polar

Polarity of stationary phase

Silica gel (or alumina) is polar - predict that butyl amine will

interact with it more strongly

Polarity of the mobile phase - the solvent: you determine

what solvent to use


Cyclohexane will elute

first/faster through the stationary


Butyl amine will elute last/slower.


TLC Separation

Example: Separate a mixture of butyl amine and cyclohexane using TLC

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.

Note the separation

of 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 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

(b) Use capillary tubes to load on



TLC plate (labeled)

with samples loaded

Predict the order

of elution for these



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 with
  • varying mobile phases.

Identifying Unknowns via TLC

Compare the Rfs of the known analgesics (standards) with

the Rfs of

the analgesics in your “unknown” tablet.

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.