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Paper Chromatography

To be assigned by your lab instructor.
Learn how to identify an unknown compound via paper chromatography.
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.
The name chromatography (Greek color ! writing" comes from a techni#ue used by
the $ussian botanist %ichael Tswett in &'() to separate plant pigments from green leaves. *hen
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 techni#ue of
separating is the same.
+ mixture to be separated is moved over the absorbing substance (the stationary phase"
by a suitable solvent (the mobile phase". ,eparation 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.
-n 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. .owever, 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 $f value, which will be a different decimal fraction for different components/
solvent by the traveled distance
mixture the of component a by traveled distance
-n this experiment, you will use paper chromatography to separate components of a
mixture of dyes called acid0base indicators. 1y comparing the $f values of the components of
your unknown with $f values for known dyes, you will identify which dyes are present in your
mixture. 2ou will also be able to use the colors of the dyes to aid your identification.
&. -f not already present, draw a pen!" line across the paper through the center of the unknown
spot (starting line". %ake four evenly spaced labels across the pencil line to indicate where
to place the known compounds, and label them !n pen!" with the initials of the known dye
compounds. $ecord the unknown number written on the chromatogram on your report
3 3 3
5. 6se capillary pipets to make #ma"" spots of the four known components on the pencil line.
). ,uspend 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.
7. 6sing a long0stemmed funnel, add enough developing solution to touch the bottom of the
paper. The solvent must not cover pencil line and spots.
8. -mmediately 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.
9. *ait for the solvent front to get high enough so that all components of the mixture are well
separated, (about 78 minutes to & hour", then remove the chromatogram and immediately
mark the solvent front with a pencil. :our the developing solvent into the bottle labeled
$ecovery 1ottle and wash out the beaker in the hood.
;. *hen 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. %ake all measurements in units of mm.
distance solvent
distance spot
3 3 3
solvent front
<. =alculate $f values for the knowns and for each component of the unknown mixture, and
record the color of each dried spot.
'. .old the chromatogram over an open container of concentrated hydrochloric acid (.=l"
under the hood. $ecord the color of each spot.
&(. .old the chromatogram over an open container of ammonium hydroxide (>.7?." under
the hood. $ecord the color of each spot.
&&. 1y comparing the $f values and colors of your unknown spots with those for the known
spots, identify the components present in your unknown mixture.
&5. :our the remaining developing solvent in the $ecovery 1ottle in the hood. *ash and dry
the beaker, and return the apparatus to the stockroom.
&7. ,taple your chromatogram to your report sheet.
LAB REPORT: Paper Chromatography
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,how $f calculations on the back of this sheet
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