Paper chromatography is one method for testing the purity
of compounds and identifying substances. Paper chromatography
is a useful technique because it is relatively quick and requires
small quantities of material.
A paper chromatography experiment.
Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile
phase. The stationary phase is usually a piece of high quality
filter paper. The mobile phase is a developing solution that
travels up the stationary phase, carrying the samples with it.
Components of the sample will separate on the stationary phase
according to how strongly they adsorb to the stationary phase versus
how much they dissolve in the mobile phase.
Video: Chromatography process (same as TLC Process video) ( 5.83 M )
Preparing the Chamber
Choose a developing chamber that can be sealed well. The chamber
should be large enough to hold the paper that is to be
The chamber should be clean and dry before use.
Add the mobile phase to the chamber so that it is about 2
cm deep. Seal the chamber tightly and let the chamber stand overnight
if possible. Why? The larger the chamber,
the longer it should stand.
What is wrong with this student's paper chromatography chamber
Preparing the Stationary Phase
Cut a square piece of high-quality filter paper to fit into your development chamber.
With a pencil, draw a straight line about 3 cm from the bottom edge of the paper.
Video: Prepare the paper ( 4.61 M )
Spotting the Samples
First, each sample should be dissolved in an appropriate solvent
to make about a one percent solution (0.01 g sample/1 g solvent). Less than one milliliter
of solution will be needed for the experiment. Then the dissolved
samples may be spotted to the paper.
Video: Spotting the samples ( 3.13 M ) Text description
If a larger quantity of sample is needed for the experiment than is
provided by one application, the solution may be re-spotted.
Video: Re-spotting ( 1.12 M ) Text description
All spots on the chromatogram should be 2 to 2.5 cm away from
the edges of the paper and from each other.
Developing the Chromatograms
After preparing the chamber and spotting the samples, the
paper is ready for development. Be careful to handle the paper
only by its edges, and try to leave the development chamber uncovered
for as little time as possible.
Initially, the chromatogram should be suspended in the chamber without touching the solvent. To suspend the chromatogram, to the top of the paper and thread a piece of string throught the paper clip. Then tape the string to the outside of the chamber to hold the chromatogram in place. The paper should hang in the development chamber overnight, if possible.
Video: Hanging the chromatogram ( 5.41 M )
After the chromatogram has hung in the chamber, immerse the paper's
bottom edge into the developing solvent.
Video: Immerse the chromatogram ( 5.83 M ) Text description
Allow the chromatogram to dry in a well-ventilated area.
A student has developed a chromatogram as shown in the picture above.
Will this chromatogram yield good results?
A student removed this chromatogram from the development chamber and allowed the solvent to dry. What did he forget to do?
Identifying the Spots
If the spots can be seen, outline them with a pencil.
If the spots are not obvious, the most common visualization
technique is to hold the paper under an ultraviolet lamp. (Caution:
Do not look directly into the lamp!) Many organic compounds can
be seen using this technique. Outline the spots with a pencil.
Interpreting the Data
The Rf value for each spot should be calculated. Rf stands
for "ratio of fronts" and is characteristic for any
given compound. Hence, known Rf values can be compared to those
of unknown substances to aid in their identifications.
(Note: Rf values often depend on the temperature, solvent, and type
of paper used in the experiment; the most effective way to identify a
compound is to spot known substances next to unknown substances on the
In addition, the purity of a sample may be estimated from
the chromatogram. An impure sample will often develop as two or
more spots, while a pure sample will show
only one spot.
Copyright © 1995-1996 NT Curriculum Project, UW-Madison