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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 = distance the substance moves
distance the solvent moves
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
Procedure
1. Mark the chromatography paper as shown below. Use only black pencil to write on the paper.
1 2 3 4 5 6 7 8 9 10
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
19.0cm
9.0cm
pencil line
1.0cm from
the bottom
pencil marks
numbered and
2.0cm apart
first and last numbers 0.5cm from edges
