CHE106: C h e m i c a l
S c i e n c e
Chromatography
C o n c e p t s
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Name
Partners
Date
Objectives
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To separate a water-soluble ink into it component colors using
paper chromatography
To separate and identify the components of a mixture using thin
layer chromatography (TLC)
To calculate the Rf value (ratio to front) of the spots on a
developed plate and paper
Interpret a TLC plate to determine if a “white powder”is an
illegal substance or an over the counter analgesic
Chromatography and Forensic Science
Thin layer chromatography (TLC) is a separation technique frequently
used in a forensic laboratory to screen for controlled substances,
identify explosives and to analyze inks from forgeries and ransom
notes. TLC by itself is not a confirmation technique, however, by
comparing unknown samples with standard reference samples, compounds
can be identified. TLC data must be supported by other analytical
techniques. Paper chromatography is generally not used in a crime lab.
Introduction
Chromatography is a method used to separate, purify and identify the
compounds in a mixture. This is accomplished by distributing
(partitioning) the mixture between two different phases called the
stationary phase (a fixed phase) and the mobile phase (moving phase).
The stationary phase is usually a solid; the mobile phase is usually a
gas or liquid.
Most chromatography methods work because of a difference in polarity.
The stationary phase holds back the components of a mixture of similar
polarity causing them to move along more slowly than the mobile phase.
Because of differences in solubility of compounds in the mobile phase,
and because of the strength of attraction to the stationary phase, some
components move faster than others. This results in separation of the
compounds. In this experiment you will use paper and thin layer
chromatography methods to separate mixtures.
Chromatography methods are classified according to the physical states
of the stationary and mobile phases. The term “paper chromatography” is
used for the practice of separating mixtures by partitioning the
compounds between a mobile liquid phase (the solvent) and a stationary
solid phase (the paper).
Thin Layer Chromatography (TLC) is used for identifying compounds and
determining their purity. The process is carried out on a plate that is
CHE106: C h e m i c a l
S c i e n c e
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covered with a thin layer of adsorbent (stationary phase). In both
processes, the liquid solvent (mobile phase) creeps up the plate or
paper by capillary action. Small amounts of samples are spotted in a
row near one end of the plate or paper, which is placed, sample end
down, in a developing chamber containing the solvent. As the solvent
rises, the compounds in the samples move to varying heights on the
plate. The individual compounds appear as spots although often either a
UV light or an iodine chamber is needed to visualize the spots.
The relative intensity of the spots is not an accurate indication of
the amount of compound present. The distance the spot travels on the
plate is expressed as “ratio to front” or Rf value given in the
equation below.
distance traveled by spot
Rf = 
distance traveled by solvent
The Rf value can be affected by many experimental conditions. The only
way to be reasonably sure that a TLC of an unknown is identical to a
known compound is to spot a solution of the known compound on the same
TLC plate. Figure 1 illustrates how to determine the Rf values of the
spots.
solvent front
distance traveled by spot
Rf = 
distance traveled by solvent
4 cm
2 cm
starting line
2 cm
Rf =  0.5
4 cm
Figure 1. Measuring The Rf Value Of A Spot On A Developed Chromatogram
The objective of part A is to develop a strip of chromatography paper
that has been spotted with a water-soluble ink, and to calculate the
Rfvalue of each spot.
CHE106: C h e m i c a l
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Analgesic drugs are compounds that relieve pain. Some nonprescription
analgesics are aspirin, ibuprovin and acetaminophen. Sometimes caffeine
is added to these compounds to alleviate drowsiness.
The objective of part B of this experiment is to determine if a white
powder found at a crime scene is an analgesic drug by comparing the TLC
of your unknown to solutions of aspirin, acetaminophen and caffeine.
Crime Scene
Several packages of mysterious white powders were found on a table in the student
center, in the bookstore, library and in some classrooms. A crime scene investigation
team documented and packaged the evidence. Preliminary tests were performed on the
samples and the team narrowed down the identification to either heroin or some type of
over the counter analgesic compound. Your job is to analyze the powders and determine
the identity of the compounds. Write the name of each compound under the structure
given below. The compound names are acetaminophen, caffeine, ibuprofen, heroin, and
aspirin.
CHE106: C h e m i c a l
S c i e n c e
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EXPERIMENTAL
A. Paper Chromatography: Separation of the Dyes in Fiber Tipped Pens.
Equipment:
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Developing solvent: 5 mL distilled water
Developing chamber: gas collecting bottle plus a 5 cm x 5 cm
piece of cardboard
Chromatography paper: 3 cm x 12 cm
Pencil
Ruler
Fiber tipped pen
Procedure:
Spotting
Draw a “starting line” in pencil, about 2 centimeters above the bottom
edge of a 3 cm x 12 cm piece of precut chromatography paper. Apply two
different colored ink spots (about 2 mm in diameter) from the fiber
tipped pen on the starting line about one centimeter from the edges of
the paper. (See Figure 2.) While the spots air dry, prepare your
developing chamber.
Chromatography Paper
Starting Line
2 cm
Figure 2. Preparation of Chromatography Paper
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Developing Chamber
Add enough water to a gas-collecting bottle to give a liquid depth of
about 1 cm. Slosh the solvent around for about one minute.
Development
Support the spotted chromatography paper in the slit in the square
piece of cardboard. Make sure the paper just barely touches the bottom
of the bottle, and does not touch the sides of the bottle. Also make
sure the solvent line is below the starting line.
When the solvent front moves to approximately 3 cm from the top of the
paper, remove the strip from the developing chamber. Mark the solvent
front with a pencil before the paper has time to dry. Let the strip air
dry and calculate the Rf of the dye spots. (See Figure 1.)
Attach your chromatogram here. Calculate the Rf value of each spot on
your chromatogram. Show your work for credit.
CHE106: C h e m i c a l
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B. TLC of an Unknown White Powder
Equipment:
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Developing Chamber - 250 mL beaker
Developing solvent: petroleum ether, methylene chloride ethyl
acetate (1:1:2)
watch glass
Filter paper
Capillary micropipets
Pencil
Ruler
Procedure:
Sample Preparation
Dissolve half of your unknown in 1 mL of methanol. Three reference
solutions (acetaminophen. aspirin and caffeine) are provided. Draw a
starting line, in pencil, about one centimeter above the bottom edge of
a 5 cm x 8 cm TLC plate. Draw four evenly spaced dots, in pencil, on
the starting line about a half of a centimeter from the edge of the
plate. Spot your unknown on the third dot. Spot the reference solutions
on the remaining dots using a clean micropipet for each spot.
Developing Chamber
Place a piece of folded filter paper (Figure 3) and about one
centimeter (depth) of developing solvent in a 250 mL beaker. Cover the
beaker with a watch glass, and slosh the solvent so that the filter
paper is wet.
Figure 2. Preparation of Chromatography Paper
Development
When the spots are dry, place the spotted plate in the developing
chamber. Make sure the solvent level is below the starting line. When
the solvent front is about one centimeter from the top of the plate,
remove it from the developing chamber. Immediately mark the solvent
front height with a pencil. Let the plate air dry. Hold the
chromatogram under a UV lamp and circle the spots. Identify the spots
in the unknown lane by comparing the unknown spots with the known
spots. Identify your unknown from the following list of brand name
drugs.
CHE106: C h e m i c a l
Brand Name
Anacin®
Bayer® Aspirin
Excedrin®
Tylenol®
NoDoz
S c i e n c e
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Ingredients
aspirin, caffeine
aspirin
acetaminophen, aspirin, caffeine
acetaminophen
caffeine
Unknown number: ________
Draw your TLC plate and identify the spots. Your TLC plate should be
discarded when your analysis is complete.
List the ingredients in your unknown.
Identify the name brand of your unknown: ___________________
Explain how you determined the identity of the white powder.
CHE106: C h e m i c a l
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Questions
1. What does the technique of chromatography allow you to do?
2. Explain the importance of the following statements.
a. The level of the solvent must be below the starting line.
b.The spots should be concentrated yet small in diameter.
3. Why is the starting line drawn for positioning the spots drawn in
pencil, never in ink?
4. The identity of a suspect material is not considered definitive.
Explain this statement.
5. Often a forensic scientist has to explain the TLC technique to a
jury. Discuss how you would present the following chromatography
concepts so that everyone would understand.
a. Capillary action
b. Visualization using a UV light