Unit: Lab Instructional Demonstrations Module: Lab Demos [page 1 of 2]

CIA Demonstration: Chromatography

• Micropipettes can be created from disposable pipettes.

• Chromatography is a group of procedures for separating components of a

mixture.

• The separation in thin layer chromatography (TLC) is based on differences in

polarities.

Thin layer chromatography (TLC) requires a

micropipette to apply the spot of mixture to be
separated. Micropipettes can be created from
disposable pipettes.
First, the disposable pipette is heated in the flame of
a Bunsen burner. The disposable pipette must be
twisted back and forth to ensure uniform heating.
When the glass is softened, the disposable pipette
is removed from the flame and stretched apart. This
creates a very thin tube of glass.
After the tube cools, it is cut into several
micropipettes.

Chromatography is a group of procedures for

separating components of a mixture. Types of
chromatography include gas chromatography (GC),
high performance (or high pressure) liquid
chromatography (HPLC), and thin layer
chromatography (TLC).
All types of chromatography involve three basic
components. The stationary phase is the support
on which the chromatography is performed. For
example, filter paper is a stationary phase used in
TLC. The mobile phase is the moving solvent front
which carries the mixture. For example, water is a
mobile phase used in TLC. The substrate is the
mixture being separated. For example, grape drink
can be separated using TLC.

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Copyright  2001, Thinkwell Corp. All Rights Reserved. 7657.doc –rev 07/12/2001
 Unit: Lab Instructional Demonstrations Module: Lab Demos [page 2 of 2]

CIA Demonstration: Chromatography

The distance moved by a pigment of grape drink is
the distance from the baseline (where the pigment
was applied) to the center of the pigment smear at
the end of the chromatography. In this
demonstration, the red pigment (lower) moved
38 mm, while the blue pigment (upper) moved
55 mm.
Rf values are calculated by dividing the distance the
sample moved by the distance the solvent front
moved. Rf values of components of a mixture can
be compared to known Rf values to identify
components. For example, if a particular blue
pigment has a known Rf of 55 mm / 67 mm = 0.82,
the blue pigment of grape drink could be identified
as that pigment.
The separation in TLC is based on differences in
polarities. Water is a polar molecule, while the
molecules making up the filter paper are nonpolar.
The blue pigment is more polar than the red
pigment, and thus is more soluble in water than on
the surface of the filter paper. Therefore, the blue
pigment travels further.

www.thinkwell.com info@thinkwell.com
Copyright  2001, Thinkwell Corp. All Rights Reserved. 7657.doc –rev 07/12/2001