What is Chromatography?
Chromatography is a method for separating the parts of a mixture containing different chemicals. The
different properties of each molecule type are exploited to accomplish this task.
This technique uses three components:
1. Mixture of chemicals
2. Mobile phase: a gas or liquid that transports the solution or mixture being tested across the
stationary phase (water, rubbing alcohol are examples).
3. Stationary phase: the solid through which the tested substance is carried by the mobile phase
(coffee filter, paper, paper towels and cotton are examples).
To separate the chemicals in a mixture through chromatography, the chemicals need to have different
properties such as molecule size or variable solubility in different solvents. The stationary phase will
absorb or slow down different components of the tested mixture to different degrees creating
separation as the components of the solution move across the stationary phase.
To separate the chemicals in a mixture using chromatography, the chemicals need to have different
affinities for the mobile and stationary phases. These differing affinities might be due to
intermolecular force differences, molecular size or shape differences, different solubility, or different
electrical charge interactions. The substances with higher affinity for the stationary phase will move
more slowly across or through the stationary phase and those substances with greater affinity for the
mobile phase will move more rapidly, creating separation as the components of the solution move
across the stationary phase.
Chemists use this process to separate unknown substances into fractions so they can be used or
further studied in their pure form. This method also can be used in forensic science to help analyze
and compare samples found at crime scenes. By comparing known samples to unknown samples, a
case can be made as to their similarities. The process of gel electrophoresis uses the properties of
molecules such as size and charge to separate mixtures of DNA or proteins as they move through a
gel by electricity. This technique has many applications in molecular biology and forensic science
including paternity testing, biological fluid identification and DNA typing.
The Procedure:
Paper Chromatography: A drop of pigment is placed onto the chromatography paper approximately 3
cm from the bottom. The solvent of choice is added to the container so that it will not touch the
pigment. The chromatography paper strip is placed into the mobile phase not allowing the pigment
spot to go into the mobile phase. Different mobile phases can be used to explore the best medium to
separate the pigments in purple mixture (See Figure 1).
Column Chromatography works like paper chromatography, but the column is the container (such as a
syringe) that holds the stationary phase (such as cotton) that was added to the column. It is often
“packed” into the column or pushed down until it is at the bottom of the syringe. The stationary phase
is generally saturated with the solvent before the mixture is added. Once the mixture is added, then
more solvent is added to move the mixture through the column by gravity. Various solvents can be
passed over the column either individually or in sequence (See Figure 2). Collect the different
mixtures as they separate using different containers such as test tubes.
Mobile Phase
Stationary
Phase
Mobile Phase
Figure 1
Figure 2
1. What is the purpose for chromatography?
2. Compare and contrast the paper and column chromatography.
3. What sort of properties or affinities in the substances allows the separation to take place?
Figure 1: Paper Chromatography Image http://www.nsta.org/publications/news/story.aspx?id=48631
Figure 2: Column chromatography Image from Wiki Commons
Source: NGSS Rollout meeting, Yucaipa, May 27, 28, 2014 H1
https://www.youtube.com/watch?v=a2wwgLV80U8
https://www.youtube.com/watch?v=qH-AJDqsSII