CHROMATOGRAPHIC TECHNIQUES OF SEPARATION
Purpose
In this experiment, you will become familiar with chromatography, a technique used to
separate small quantities of mixtures into their components. In this experiment, paper
chromatography will be used to analyze food dyes, and thin layer chromatography (TLC)
will be used to analyze amino acids.
Theory
In both paper and thin layer chromatography, the mobility of a substance is stated in
terms of its Rf value (retardation factor). The Rf value is the ratio of the distance traveled
by the substance to the distance traveled by the solvent. Each distance is measured from
the point at which the mixture is “spotted” onto the absorbent.
Rf =
Distance traveled by the substance
Distance traveled by the solvent
This ratio remains constant for a particular substance in a particular solvent, regardless of
the distance traveled by the substance and the solvent. It does not matter whether the
component spot travels 10 mm or 100 mm, the same compound under the same
conditions will have the same Rf value each time it is analyzed. Because Rf is a constant,
its value can be used to identify compounds. Vitamin C, for example, has the same Rf
value whether it is part of a mixture of vitamins or if it is the only compound present.
When developed under the same conditions, if a spot produced from the mixture has the
same Rf value as pure vitamin C, then vitamin C is in the mixture.
Ideally, each component in a mixture will have a different Rf value. That means that each
component travels a different distance, as a function of Rf, and the components are
separated from each other by the solvent. The resulting chromatogram is then used to
identify each component in the mixture.
Background Reading
Beran, J. Lab Manual for Principles of General Chemistry (9th Ed.).
pp 69-74 (Experiment 4, Paper Chromatography).
Ebbing D. and Gammon S. General Chemistry (9th Ed).
pp 14-15 (Separation of Mixtures by Chromatography).
McMurry, J. Organic Chemistry (8th Ed.). pg 1058 (Ninhydrin).
Zanger, M. and McKee, J.R. Small Scale Syntheses. pp 52-54 (Planar Chromatography).
Zubrick, J. W. The Organic Chem Lab Survival Manual.
Ch 27 (Chromatography: Some Generalities)
and Ch 28 (Thin-Layer Chromatography).
Key Words
stationary phase, mobile phase, capillary action, Rf value, adsorbent, polarity
Experimental Data






Include all FD&C dyes, amino acids, and solvents used in the Substances table.
The FD&C standards are pure substances (one component each).
Make a table in your Data section of their Rf values.
Most commercial colors have more than one component each.
Make another table in your Data section of each component’s identity
and Rf value for all of the commercial colors.
The amino acid solutions, including the unknown, should have one component each.
Make a table in your Data section of the substances and their Rf values.
Identify the amino acid unknown.
Include ninhydrin in substances, along with its reaction with amino acids
in the Reactions section.
Include the chromatograms or their photos in your report.
Procedure
Part I: Paper Chromatography with Food Colors
In this part of the experiment, you will use paper chromatography
to analyze FD&C colors, and how they are blended together to
obtain blue, green, red, and yellow commercial food colors.
1. In a graduated cylinder, mix 5 ml of DI water, 5 ml of 1-propanol, and 5 ml of
1-butanol. Pour the mixture into the chromatography jar, and swirl the liquid to mix
the developing solvent components. After swirling, tighten the lid onto the jar.
The jar will serve as your developing chamber.
2. Obtain two 8 cm by 14 cm pieces of chromatography paper. Carefully handle the
paper by its edges only at all times. Use a pencil and a ruler to draw a line 2 cm
from the bottom across the long dimension of the paper. Do not use a pen.
Starting in 1 cm from the left edge, mark off five points evenly spaced
from each other on both papers.
3. Four commercial food colors and six FD&C standard colors will be analyzed. The
analysis begins by “spotting” at each of the five points. A total of two trials will be
needed to analyze all ten colors.
Spot the five samples per paper as follows. Draw a small volume of each liquid into a
clean micro-capillary tube, and then lightly touch the tube to the paper to make the
spot. The spot must be < 3 mm, or poor separation will result. Once the samples
have been spotted, set the paper aside for a few minutes to allow the spots to dry.
4. Once the spots are dry, roll the paper into a cylinder and carefully staple the ends
together about 2 cm from the top and bottom of the cylinder.
Also, the edges of the paper should not touch.
5. Place the paper in the developing chamber, and then tighten the lid. The spots must
be above the solvent, and the paper should not touch the wall of the jar.
Do not move the jar, it should be kept as stationary as possible.
6. After the solvent has moved > 3/4 of the distance to the top of the paper, remove the
cylinder and undo the staples. Mark the position of the solvent front with a pencil,
and allow the paper to dry.
7. After the paper has dried, outline each component’s colored spot with a pencil, and
make a dot in each component’s center as well. Food colors often form elongated
spots with a definite head and an extended tail. The dot should be in the center of the
head.
8. Measure and record the distance (in mm) from the starting line to the center of each
dot. Also measure the distance from the starting line to the solvent front. Calculate
the Rf values for each substance. If a sample produces two or more spots, then it is a
mixture. If it produces only one spot, the sample contains only a single compound.
Part II: Thin-Layer Chromatography (TLC) with Amino Acids
In this part of the experiment, you will use TLC to analyze alanine, glycine, and
phenylalanine, along with an unknown sample. This unknown could contain any
one of the three amino acids. Because these substances are colorless in solution,
your instructor will spray the plate with a solution of ninhydrin so that a resulting
product with a purple color will become visible.
9. In a graduated cylinder, mix 2.0 ml of dimethoxyethane (DME) and 18.0 ml of
ethanol. Pour the mixture into the chromatography jar, and swirl the liquid to mix
the developing solvent components. After swirling, tighten the lid onto the jar.
The jar will serve as your developing chamber.
10. Obtain one silica gel plate, handling it only by the edges. Using a pencil and a ruler
to draw a line 2cm from the bottom across the short dimension of the plate.
Do not use a pen. Starting in 1 cm from the left edge, mark off four points evenly
spaced from each other.
11. Spot the four samples as follows. Draw a small volume of each liquid into a clean
micro-capillary tube, and then lightly touch the tube to the plate to make the spot.
The spot must be <3 mm, or poor component separation will result (blurred).
Once the samples have been spotted, set the plate aside for a few minutes to allow the
spots to dry.
12. Place the plate in the developing chamber, and then tighten the lid. The spots must
be above the solvent, and the plate should only touch the wall of the jar at the
top corners. Do not move the jar; it should be kept as stationary as possible.
13. After the solvent has moved >3/4 of the distance up the plate, remove it from the
developing chamber, and allow it to dry for 5 minutes. Mark the position of the
solvent front with a pencil.
14. Bring the plate to the hood so that your instructor can spray it with the ninhydrin
solution. Allow the plate to air dry completely, or in oven at 80oC for 10 minutes if
necessary, so that the spots become visible (purple).
15. Once the plate is dry, outline the spots with a pencil and make a dot at the center of
each spot.
16. Measure and record the distance (in mm) from the starting line to the center of each
dot. Also measure the distance from the starting line to the solvent front. Calculate
the Rf values for each substance.
Post-Lab Questions
1. What would happen to your Rf values if the solvent front was allowed
to reach the top of the paper? Describe the mathematics of the result.
2. What would happen to your chromatogram if you marked it with
a pen instead of a pencil? Describe what you would expect to see.
3. What would happen to your chromatogram if your spots were
too large (> 3 mm)? Describe what you would expect to see.
4. What are your mobile and stationary phases in this experiment?
Describe how they function.
5. What type of mixtures are best suited for separation by paper chromatography?
(See pg 54 in lab manual or ch 27 in Zubrick.)
Describe how the separation happens.