Chromatography
 Chromatography- a physical method to separate components
of mixtures
 In 1906, the Russian botanist Mikhail Tsvett separated
pigments in leaves
 Chemist determines if a particular substance is present
 Examples: cocaine in urine, alcohol in blood, lead in water
 The analysis of analyte (the substance to be analyzed) is
referred to as trace analysis
o 1 g or less
 Paper chromatography is simple –
o strip of paper = stationary phase
o solvent = mobile phase (or eluent)
o solvent moves up the paper due to capillary action
o carries with it the various components in the spot
o sample dissolves in the solvent vs. adheres to the
adsorbent
o separation  different solubility in a given solvent
adhere to a solid adsorbent to a different degree
different components in mixture carried along by the
solvent at different rates  separation
o differences in solubility due to different sizes and shapes
of molecules as well as difference in polarity (polar
molecules have positive and/or negative ends).
o two compounds are started at the same place and the
solvent passed over them (elution), one compound will
move along the strip faster than the other
 Paper chromatography tells whether or not the sample is pure
o positions of each spot can be compared with standards
o compound is indicated by an Rf value
o distance the compound moves relative to the distance the
mobile phase moves is a characteristic of that compound
(it doesn’t change) and is known as the Rf value.
 solvent must dissolve components in the mixture and there
must be at least slight differences in solubility of components
 No movement = no separation = no resolution
o usually found that one pure solvent will not give resolution
and a mixture of solvents must be found for each sample
to be analyzed
 resulting pattern is called a chromatogram.
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Definitions
Solubility – amount of solute that will dissolve in a given solvent
Solvent – dissolves another substance (the solute)
Solute – gets dissolved (by the solvent)
Miscible – when 2 things do not mix (oil & water are miscible)
Rf – migration value found by dividing the solvent distance by the
solvent distance
Rf 
solute_dis tance
solvent_di stance
Affinity – tendency for a solute to dissolve in a solvent
o If Rf = 1, the solute is extremely soluble in the solvent.
o You would say the solute has an affinity for the solvent.
o If Rf = 0, the solute is extremely insoluble in the solvent.
o You would say the solute does not have an affinity for the
solvent.
Rf = Distance moved by pigment
Distance from pigment origin to solvent front
or
Rf 
pigment_distance
solvent_distance
Example: If the solvent front is 6 cm then the Rf value
for the pigment at 3 cm would simply be:
Rf 
3cm
 0.5
6cm
Table 1: How to calculate the Rf value.
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Chromatography Lab Procedure
1) Your lab group will be performing chromatography on 2 different markers. One of the
markers should be black. Then select another pen of a different color.
2) Create 2 pieces of chromatography paper by cutting a filtration paper into 5cm x 11 cm
rectangular strips. Make them as rectangular as possible. Then, using a pencil draw a line about
1.5 cm from the end of the paper (make sure to use a pencil!!).
3) Tape the chromatography strips to a pencil by wrapping the top (opposite from the pencil line
you drew) around the pencil and taping it. Make sure the pencil will support the chromatography
paper by placing it in a dry (NOT wet!) 250 mL beaker to see if it hangs. It should just barely
touch the bottom of the beaker. If it does not, adjust the wrap at the top until it does.
4) On each of the pieces of paper make several distinct, round markings (or dots) on the pencil
line for each of the 2 markers chosen in part 1. Space the dots about 1 cm apart. Make sure you
place the "dots" in the same order on each of the pieces of paper. Your 2 pieces of
chromatography paper should be exact replicas of each other.
5) Place 25 mL of each solvent (water and alcohol) in 2 different 250 mL beakers. Suspend each
pieces of chromatography paper in a different solvent- note the solvent each paper was placed
into. DO NOT LET THE SOLVENT COVER THE DOTS OF INK WHEN YOU FIRST
PLACE THE PAPER INTO THE SOLVENT!
6) After 5-10 minutes note the colors that have been separated from each ink dot. When the
solvent front has almost reached the top take it the paper out of the solvent. Write the initials of
you and your lab partner at the top of the paper and tape it to a cabinet to dry overnight.
Tomorrow you will do the Experimental Analysis on the paper.
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Experimental Analysis
Table 2. Description of ink mixtures and chromatography.
#
Description of
Solvent 1:________
ink type
Solvent 2:_______
Example
Red
Permanent
Marker
Did not separate
into colors at all.
No migration from
the start line.
Separated into two
colors: yellow and
red.
1
2
Table 3. Rf. values of pigments within each ink mixture.
#
Description of
ink type
Solvent
1:________
Solvent
2:________
Example
Black
Permanent
Marker
Red Rf = 0.5
Yellow Rf = 0.35
Rf = 0
1
2
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Lab Summary
Answer on a separate piece of paper.
A. Using the 2 completed tables you created, explain in detail the
results of the experiment. Include a comparison of the solvents.
Also include which pigment(s) had the greatest affinity for each
solvent.
B. If you accidentally pressed any of the markers onto your
clothing, which of the solvents would you use to remove the ink?
And why?
C. How does chromatography determine if an ink is a mixture of
not? How does this apply to the definition of a mixture?
D. What makes an ink travel farther in a certain solvent?
E. Just because the ink of the marker did not break up into
separate colors on a chromatogram, does that mean that the ink
is not necessarily a mixture? Explain.
What should you turn in?
1. Experimental Analysis Tables
2. Lab Summary Questions
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