Chromatography

Background
In paper chromatography, when you place a colored chemical sample on a filter paper, you can get the colors to separate from the sample by placing one end of the paper in a solvent. As the solvent diffuses up the paper, it dissolves the various molecules in the sample according to the polarities of the molecules and the solvent. If the sample contains more than one color, that means it must have more than one kind of molecule. Because of the different chemical structures of each kind of molecule, the chances are very high that each molecule will have at least a slightly different polarity, and thus, a different solubility in the solvent.

The unequal solubilities cause the various color molecules to leave solution at different places as the solvent continues to move up the paper. The more soluble a molecule is, the higher it will migrate up the paper. If a chemical is very polar it will not dissolve at all in a very nonpolar solvent. The same is true for a very nonpolar chemical and a very polar solvent.

In this lab you will use paper chromatography to separate colorant molecules in pens and plants. In the process, you will be able to identify differences in polarity of pigment (color) molecules of different kinds of markers. You will use vis a vis and permanent markers as well as grass and leaves. The solvents you will use are (in order from most to least polar) water, ethyl alcohol, and hexane.

Chroma means color; so it makes sense that chromatography got its name from the technique you are doing today. There are many kinds of chromatography, however, that do not involve color separation. In each, a mixture of organic molecules is drawn through a medium that separates the mixture by differences in polarity, molecular weight, electrical charge, or a combination of these factors.

As you might recall, you used gel-filtration chromatography to separate three proteins in much the same way an immunologist would separate antibodies. In that technique, even though you separated colors to acquire a calibration curve, the molecules separated because of their different molecular weights--not because of their varying solubilities.

Optional Reading
To describe the molecular structure of a gene, an immunologist might use another form of chromatography called polyacrylamide electrophoresis. As in gel-filtration, this technique separates molecules according to molecular weight. Scientists begin the test by cloning many copies of the same gene. Then, they isolate samples of the gene and add four different compounds, one to each sample. Each compound replaces only one nucleotide: cytosine, guanine, thymine, or adenine. For each gene, the compound reacts at a random spot to replace a nucleotide anywhere along the gene. When it reacts to replace a nucleotide, the compound also terminates the sequence. So, after the terminating compounds react with the samples, they create an array of molecules beginning at the molecular weight of just one nucleotide and increasing all the way up to the molecular weight of the original gene with all the nucleotides in place.

To do the actual electrophoresis, the scientists place four samples at the top of each gel column. Each sample contains the gene pieces terminated by one of the four nucleotide replacement compounds. The scientists turn on an electric field, which forces all the molecules down the gel. The smallest molecules go through first. When all the molecules are spread through the gel from top to bottom, the scientists take a picture of the gel and the bands each protein leaves in it. Finally, they can read the nucleotide sequence starting with the smallest molecule and jumping from column to column as the size increases. In this way they can tell which nucleotide is present in the gene in a given order. In the end, they will have collected the entire sequence of nucleotides in the gene.

Procedure

Cut four 15 cm-high pieces of filter paper wide enough to fit without bending inside a 400 ml beaker.
With a pencil and a ruler, make a dotted line two centimeters from the bottom. Across the top of the line, make about a centimeter square mark with each of the following pens: Vis-a-vis green, blue, and black; and permanent black. Use the pencil to label each pen sample. Write below the line. Include also the name of the solvent used for this sample.
Pour a small amount of the first solvent, water, to cover the bottom of the beaker. Place the marked paper in the water. Observe and record your observations.
While you are waiting for the colors to move up the paper with your first solvent, repeat the pen color samples on two more papers. As above, place these samples in the second and third solvents, ethyl alcohol and hexane.
As the colors near the end of their migration up the columns, answer Processing the Data questions one through four.
Prepare your final paper by rubbing three plant samples above the dotted line. First, smash grass onto the paper to get a good green stain. Likewise, smash a green leaf, then a yellow leaf into the paper. (You may need to scrape the leaves first to get their juices to flow.) Label the paper.
Do the chromatography in the solvent you chose.

Data
Divide the chromatography samples among your group partners. Present your results either by sketching what is on the papers or by taping the actual papers to your report. If they are available, use colored pens, pencils, or crayons to reproduce your results. If not, label what color the marks represent. Label also which solvent was used for each set of colors obtained.

Processing the Data

Which of the pens contain polar pigments? How do you know?
What can you say about the polarity of an ethyl alcohol molecule?
Of all the pens, what color pigment is the most polar?
What solvent is the most logical choice for testing the plant colors?
In plants, the main pigment is chlorophyll. The other colors, present in lesser concentrations, are called carotenoids. They capture some frequencies of light that the chlorophyll cannot. This allows the plant to utilize more sun energy than it could if it had only one pigment. How many different carotenoids can you count in your plant samples?

Extra Credit

Draw the molecular structure of each solvent.
Look up and draw the molecular structure of either chlorophyll or a carotenoid. Indicate the name of the book or magazine where you got the structure. Knowing the molecular structure of the pigment, which solvent would not dissolve it, water or hexane?

Teacher Notes
Chemistry Concepts: Solubility, Polarity, Organic Chemistry

The students will already have done a gel-filtration and so, should be familiar with it when it is mentioned in the background section.
It would be good to have a sample PAGE gel or an actual picture of the electrophoresis gel after separation.
You should find that the hexane is far too powerful a solvent for the nonpolar compounds. The permanent pens and plant juices get carried clear to the top with no separation.
You may want to have the students try the plants in water. They will find the colors do not budge.
Make sure the students know in advance what makes a molecule polar and that like dissolves like.