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Paper Chromatography
PRE-LAB ASSIGNMENTS:
To be assigned by your lab instructor.
STUDENT LEARNING OUTCOMES:

Learn how to identify an unknown compound via paper chromatography.
EXPERIMENTAL GOALS:
The goal of this experiment is to perform a paper chromatography experiment on a set of known
compounds, and use this data to identify an unknown compound.
INTRODUCTION:
The name chromatography (Greek “color” + “writing”) comes from a technique used by
the Russian botanist Michael Tswett in 1903 to separate plant pigments from green leaves.
When leaf pigment extracts were slowly washed through a column packed with insoluble
calcium carbonate, the pigments move through the column at different rates and thus become
separated into colored bands along the packed column, hence the name. The components of a
separated mixture can be detected in a variety of ways often unrelated to color but the technique
of separating is the same.
A mixture to be separated is moved over the absorbing substance (the stationary phase)
by a suitable solvent (the mobile phase). Separation depends on the fact that components of the
mixture are adsorbed onto the surface of the stationary phase with different affinities and
therefore the components will move over the stationary phase at different rates.
In paper chromatography, the mixture is applied as a small spot at one end of a paper
strip. The solvent (mobile phase) will wet the paper and move up through the fibers of the paper
(stationary phase) by capillary action. The components of the mixture will be carried at different
rates over the fibers by the moving solvent.
The longer the solvent is allowed to travel, the further it moves, and the further the
component also travels. However, the ratio of these distances, the relative movement of a
component of the mixture compared to the movement of the mobile phase, can be expressed by a
ratio called the Rf value, which will be a different decimal fraction for different components:
Rf 
distance traveled by a component of the mixture
distance traveled by the solvent
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In this experiment, you will use paper chromatography to separate components of a
mixture of dyes called acid-base indicators. By comparing the Rf values of the components of
your unknown with Rf values for known dyes, you will identify which dyes are present in your
mixture. You will also be able to use the colors of the dyes to aid your identification.
PROCEDURE:
1.
If not already present, draw a pencil line across the paper through the center of the unknown
spot (starting line). Make four evenly spaced labels across the pencil line to indicate where
to place the known compounds, and label them in pencil with the initials of the known dye
compounds. Record the unknown number written on the chromatogram on your report
sheet.
#
X
X
X
X
2.
Use capillary pipets to make small spots of the four known components on the pencil line.
3.
Suspend the paper in a beaker by hanging a small fold at the top of the paper from a rubber
band stretched around the beaker. The paper should almost touch the bottom of the beaker.
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4.
Using a long-stemmed funnel, add enough developing solution to touch the bottom of the
paper. The solvent must not cover pencil line and spots.
5.
Immediately cover the beaker with plastic wrap to keep the atmosphere inside the beaker
saturated with solvent vapor so that the solvent will not evaporate from the paper fibers as
the solvent moves up the paper. Do not move the developing chamber once the paper has
been immersed in the developing solution.
6.
Wait for the solvent front to get high enough so that all components of the mixture are well
separated, (about 45 minutes to 1 hour), then remove the chromatogram and immediately
mark the solvent front with a pencil. Pour the developing solvent into the bottle labeled
“Recovery Bottle” and wash out the beaker in the hood.
7.
When the solvent has dried, measure the distance from the starting line to the leading edge
of each spot on the chromatogram, and measure the distance that the solvent moved above
the starting line. Make all measurements in units of mm.
#
solvent front
distance solvent
moved
distance spot
moved
X
X
X
X
8.
Calculate Rf values for the knowns and for each component of the unknown mixture, and
record the color of each dried spot.
9.
Hold the chromatogram over an open container of concentrated hydrochloric acid (HCl)
under the hood. Record the color of each spot.
10. Hold the chromatogram over an open container of ammonium hydroxide (NH4OH) under
the hood. Record the color of each spot.
11. By comparing the Rf values and colors of your unknown spots with those for the known
spots, identify the components present in your unknown mixture.
12. Pour the remaining developing solvent in the Recovery Bottle in the hood. Wash and dry
the beaker, and return the apparatus to the stockroom.
14. Staple your chromatogram to your report sheet.
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LAB REPORT: Paper Chromatography
Name ________________________________
Date _________
Partner ________________________________
Section _________
Distance
Solvent
Moved
(mm)
Distance
Sample
Moved
(mm)
Rf
Congo Red
Phenol Red
Bromcresol Green
Methyl Violet
Unknown No._____
Unknown contains:
______________________________
______________________________
______________________________
______________________________
Show Rf calculations on the back of this sheet
Color
When
Dry
Report Grade ______
Color in
HCl
Vapor
Color in
NH4OH
Vapor
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Rf Calculations:
Congo Red
Phenol Red
Bromcresol Green
Methyl Violet
Unknowns