Why Does Chromatography Work?

Chromatography identifies different chemicals based on the properties and mobility of the molecules in the compound being analyzed. Chromatography lets scientists separate liquids and gases ranging from petroleum and DNA to chlorophyll and pen inks. Students can also use chromatography for experiments and fun projects.

"Chromat-" comes from the Greek word "chroma," which means color. "-Graphy" comes from the Latin "-graphia" or Greek "graphein" and means (per Merriam-Webster) "writing or representation in a (specified) manner or by a (specified) means or of a (specified) object." Chromatography therefore literally means to write or represent with color. A more formal definition from Merriam-Webster states that chromatography is "a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the solutes as they flow around or over a stationary liquid or solid phase."

Chromatography works because of differences in the properties of molecules in materials. Some molecules, like water, have polarity, so they act like little magnets. Some molecules are ionic, meaning that the atoms are held together by their charge differences, again like little magnets. Some molecules differ in shape and size. These differences in molecular properties allow scientists to separate compounds into individual molecules using chromatography.

Chromatography also depends on the mobility of the molecules. In other words, the ability of the molecules to be moved determines whether chromatography works. Putting molecules into a mobile phase requires either dissolving the substance in a solvent or having the substance in a liquid or gaseous stage. If a solvent used, the solvent depends on the material to be separated. Liquid and gas mixtures can be pushed or pulled through a material that absorbs the molecules as they pass through. No matter what material is being analyzed, for chromatography to work the material must have a mobile phase.

Although chromatography techniques differ, they all depend on a combination of molecular differences and material mobility. Chromatography works by passing the dissolved material, liquid or gas through a filter material. The molecules separate into layers as the molecules pass through the filter. The mechanism of separation depends on the filtering method, which is determined by the kinds of molecules to be separated. But no matter which method is used, the molecules travel at different rates through the filter, separating the molecules into layers that often appear as colored lines in the filter material.

In general, the larger or heavier molecules travel through the filter material more slowly than the smaller or lighter molecules. The molecules separate as they move because they travel at different speeds, falling out like sediments dropping out of water as the volume or energy of the water drops.

While many chromatography tests require special equipment and techniques, chromatography can be used in some home and school experiments using simple materials.

Pen Ink Analysis

A simple demonstration of chromatography uses coffee filters and a variety of marker pens. If the pens use water-soluble inks, the solvent used is water. If the markers use permanent ink, isopropyl alcohol often works as a solvent. Begin by flattening out a coffee filter. Place the coffee filter on a disposable plate or other material to prevent staining underlying surfaces. Use a variety of pens to make dots around the center portion of the filter. Add water or alcohol to the center of the coffee filter. A teaspoon works well for this. Do not add enough liquid to create a puddle; the water or alcohol should expand out from the center. As the liquid moves out from the center, the inks will dissolve and move outward from center. Different pigments in the inks will separate, carried out from initial ink spot and deposited in rows based on the pigment molecules.

Chlorophyll Chromatography

A little more complicated but equally interesting chromatography project separates the chlorophyll found in leaves. Chlorophyll occurs in the leaves of plants. Although chlorophyll is green, most leaves also contain additional pigments such as carotenoids, which create the red and orange colors you see in autumn. These carotenoids and other pigments are revealed as the green chlorophyll degrades, which is why deciduous plant leaves show different colors in the fall. Begin by selecting several green leaves. Crush the leaves and soak the pieces in isopropyl alcohol or acetone (also called propanone). The chlorophyll will leach out of the leaves and turn the liquid green.

To separate the pigments, cut about an inch-wide strip from the center of a flattened coffee filter or use chromatography paper. Tape one end of the paper to a pencil. Pour about 1 inch of the liquid into a container slightly shorter than the paper strip. Lay the pencil across the top of the container so that the bottom of the paper is in the liquid. The liquid will rise up in the paper due to capillary action, carrying the chlorophyll and other pigment molecules along. As the liquid evaporates the molecules are left behind on the paper, creating lines of pigment. Remove the paper when the lines become distinct because if the paper is left too long the liquid will eventually carry all the pigment molecules to the top of the paper.