Cliffs AP Chemistry, 3rd Edition

Experiment 18: Separation by Chromatography

Background:Methods to separate mixtures can be based on many principles such as differ-
ences in solubility, boiling and freezing points, polarity, density, and so on. In chromatography,
differences in polarity can be used to separate mixtures and identify their components.
Molecular sizes and shapes are also used in sieving (gel) and recognition (host-guest) chro-
matography. Common methods include: 1) paper; 2) thin layer; 3) column; 4) high perfor-
mance liquid (HPLC); and 5) gas (GC) or vapor phase chromatography (VPC). No matter what
method of chromatography is employed, it is a powerful analytical tool that involves a station-
ary phase (either a liquid or solid) and a mobile phase (either a liquid or gas). The stationary
phase attracts the components of the mixture. The medium of the stationary phase may be po-
lar, attracting polar components of the mixture, or it may be nonpolar, attracting nonpolar and
excluding polar components. In paper chromatography, the mobile phase or solvent travels
through the stationary phase (the paper) depositing components of the mixture along the way
on the stationary phase based upon differences in intermolecular interactions. The leading edge
of the solvent is known as the solvent front. The movement of the mobile phase, due to capil-
lary action, through the stationary phase and selectively depositing components is characterized
by determining a retention or retardation factor (Rf):

()

()

R traveled by solvent front cm

traveled by component cm

Distance

Distance

f=

Rfvalues can range from 0.0 which indicates that the component did not travel at all, to a maxi-
mum of 1.0, which indicates that the component traveled to the solvent front, resulting in no or
little retention. It is best to determine the solvent characteristics for a good separation prior to
determining the Rf.

Scenario:A student was given a sample containing a mixture of food dyes. The sample
appeared green. The student was also given four commercial grade samples of FDC food dyes
including Blue 1, Blue 2, Red 3 and Yellow 5. The student spotted the chromatography paper
with the known dyes and the unknown mixture, making sure the paper dried before each appli-
cation, and then placed the paper into a developing chamber that contained a small amount of
eluting solvent (see Figure 1). After the solvent front had reached a point near the top of the
paper, the paper was removed, a line was marked to show the solvent front and the paper dried.
The chromatogram is shown in Figure 2.

Part III: AP Chemistry Laboratory Experiments