CHM 130

Paper Chromatography
Introduction
Chromatography is one of many techniques to separate the compounds in a mixture and to identify
unknown substances. It is widely used in chemistry and biology. All types of chromatography involve two
different immiscible phases in contact with each other. The mobile phase is moving and the other phase, stationary
phase, is not moving. In paper chromatography, a solvent moves from one end of a paper to the other end, as the
paper absorbs it. Since it is moving, the solvent is the mobile phase. The paper is the stationary phase. (More
specifically, the cellulose molecules in the paper contain many polar –OH (hydroxyl) groups that serve as the
stationary phase). Chromatography works by capillary action.

To separate a mixture of chemicals, a small amount of the mixture is first placed near the edge of an
absorbent paper. That same edge is wetted with solvent. The solvent travels up the paper by capillary action,
carrying the mixture with it. The various chemicals in the mixture travel different distances. The distance a
chemical travels is determined by its interaction with the solvent (mobile phase) and with the hydroxyl groups
attached to the paper. Substances that dissolve more readily in the solvent will move farther than the substances that
have a higher attraction for the paper. When the solvent has moved the entire length of the paper, the paper is
removed from the solvent and dried. The different chemicals in the mixture will be located at different positions on
the chromatogram. At this point, we can calculate the Rf (ratio of fronts) factor for each substance. For a
particular substance the Rf factor is defined as

Rf =

Retention factors are useful in comparing the results of one chromatogram to the results of another. If the
conditions in which the chromatogram are run remain unchanged (same mobile and stationary phases),
the retention factor for a given material should remain constant. This allows unknowns to be compared to
known materials. If the retention factor of an unknown does not match that of a known material, they are
not the same compound.

The various chemicals visible on the chromatogram can often be identified by their positions or their colors
or both. If the mixture contains colored compounds, each different compound will appear on the chromatogram as
a colored spot or a streak in a particular place. The color and location of each compound can be used as a basis for
identification. The color and location of unknown compounds separated under specific chromatographic conditions
can be matched with the color and location of known compounds subjected to the same conditions.
Chromatograms with uncolored compounds can sometimes be treated with chemicals to color them for
identification.

Materials / Equipment
chromatography paper pencil
solvent (0.1% NaCl) ruler
600 mL beaker one set of colored marking pens (crayola or cra-Z-Art)
plastic wrap or watch glass stapler
hair dryer

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Procedure
1. Mark the chromatography paper as shown below. Use only black pencil to write on the paper.
19.0cm

9.0cm

pencil marks
pencil line numbered and
1.0cm from 2.0cm apart
the bottom

1 2 3 4 5 6 7 8 9 10

first and last numbers 0.5cm from edges

1. Obtain a set of marking pens and draw a small dot of ink from a red pen on the first pencil mark on the piece
of chromatography paper. Continue placing colored dots on the pencil marks in the same order as the visible
spectrum: red, orange, yellow, green, blue, and violet. Place other colors such as black, brown, and pink at
the end. (If you cannot find every color, that is OK, try to get a range of colors.) Record the spot number, ink
color, and brand of the pen in the data table.

2. Staple the paper into a cylinder, dot side out, being careful not to allow the edges to touch one another (Figure
B).

3. Pour around 20 mL of the solvent into a large 600mL beaker. Place the paper cylinder into the beaker with the
dots at the bottom. Initially, the colored dots should be above the solvent as shown in Figure C. Be very
careful that the solvent does not touch the colored dots. Cover the beaker with plastic wrap or a watch glass.

4. Wait for the solvent to move up the paper. It will take several minutes.

5. Remove the paper cylinder when the solvent reaches ~2.0 cm from the top of the chromatography paper.
Carefully remove the staples and set the chromatography paper on a paper towel. Draw a line along the
highest point the solvent reached with a pencil and quickly blow dry the paper with a hair dryer.

6. Measure the distance traveled from the pencil line at the bottom of the paper to the solvent line at the top of the
paper. Record this as “Solvent Distance” on the data table.

7. Measure the distance traveled from the pencil line at the bottom to the approximate center point of each
colored splotch. Record each distance as “Spot Distance” on the data table. If the original ink spot has
separated into multiple colors, then each new color must be measured and recorded separately.
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Name_____________________ Section____________________ Lab Partner____________________________

Paper Chromatography Lab Questions

Answer each of the following questions. Base your answers (conclusions) on reasonable interpretations
of your data and provide justification.

1. Why is it important to keep the spots applied to the chromatography paper small?

The spots spread out, so if they are too close, the ink will run together

2. Why did some ink dyes travel to the top of the paper and other ink dyes remained near the bottom of
the paper? Explain in terms of the attractions of the particles for either the mobile liquid phase or the
solid paper stationary phase.

Ink that is attracted to the solvent will move towards the top of the paper. Ink that is attracted to the
paper will stay near the bottom. Attractions are most commonly due to polarity of the molecules:
Polar inks are attracted to polar solvent, nonpolar inks are attracted to nonpolar paper.

3. If a spot travels 2.3 cm during paper chromatography while the mobile phase travels 6.6 cm, what is
the Rf value of the substance in the spot?

4. What is the stationary and mobile phase used in this lab?

Stationary phase is the chromatography paper, because it does not move during the experiment
Mobile phase is the salt solution, because it moves up the paper

5. In preparing the chromatography paper, a baseline is drawn in pencil for the positioning the spots.
Why is ink never used for drawing the baseline?

The ink in the pen may also move with the solvent. This will create a mixed spot with the ink that is
being tested. It also eliminates the reference line that is needed to measure the distance the ink and
solvent travelled for the Rf equation.

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DATA TABLE

You may need to divide some of the boxes into multiple rows if the ink separated into more than one color.

Spot
Ink Color Solvent Distance (cm) Spot Distance (cm) Rf value
Number

10

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