Chromatography and Polarity
1. Chromatography Introduction
2. Pigment Mixtures
3. Chromatography in Action
4. Interpret the Results
5. Chemical Structure and Polarity
6. Calculating Rf Values
7. Rf Values with Broad Bands
8. Real Rf Values
1. Chromatography Introduction
1. solvent
2. chromatography paper
3. pigment mixture
4. individual pigments
5. solvent front
6. starting line
Chromatography is a method of separating mixtures. Different components within mixtures each have their own unique properties, such as molecular weight and polarity. Chromatography utilizes these differences to sort out the individual components within a mixture.

In this lesson we will study paper chromatography, which was the first method of chromatography invented. For this method, a drop of a mixture is put onto one end of a piece of chromatography paper. The place where the mixture starts is called the staring line. The paper is then set in a jar with a small amount of solvent. The solvent will wick vertically up the paper, much like a paper towel soaks up a spilled drink. The farthest it moves up the paper during the experiment is called the solvent front.

In paper chromatography, polarity is the key factor separating the mixture's components. In the image to the left, the solvent in the base of the jar is non-polar. Polar components of the mixture will not dissolve in the solvent and thus will not travel very far. Non-polar components will dissolve and will move with the solvent as it travels up the paper.

In the image to the left, which pigment is the most polar?

Which pigment is the least polar?

2. Pigment Mixtures
In the following examples, 4 different marker pigments were separated on strips of chromatography paper. The markers were black, blue, brown, and yellow. A starting line was drawn with each marker on a separate strip of chromatography paper, and each strip was placed in a glass jar with a small amount of solvent. The solvent wicks up the paper and moves the pigments along with it. The solvent in each of these examples is water, which is polar.

The results of the 4 different marker chromatography experiments are shown in the image above. From left to right, the markers are black, blue, brown, and yellow.
Because water is a polar solvent, what can we tell about the different marker pigments?

Which markers contained only one pigment?

Which markers contained a mixture of pigments?

Rank the pigments from most polar to least polar.

3. Chromatography in Action
In the following chromatography experiment, kale leaves were rubbed on the base of a strip of chromatography paper. The strip was then placed in a glass jar with a non-polar solvent, petroleum ether. Watch the video below to see the experiment in action.
4. Interpret the Results
The image below shows results of a chromatography experiment using kale leaves, just like the video you just saw. The four plant pigments shown in the chromatogram are beta-carotene (yellow orange), xanthophyll (lighter yellow), chlorophyll a (bright green), and chlorophyll b (olive green). Use the chromatogram to answer the following questions.

Which band represents beta-carotene?

Which band represents chlorophyll a?

What is represented by E?

Which pigment is the most polar? (Remember, the solvent is non-polar.)

5. Chemical Structure and Polarity
In the last section, we saw that chlorophyll b is more polar than chlorophyll a. We also saw that beta-carotene is the most non-polar of the pigments. When looking at the chemical structures of the different pigments, we can see that they are very similar to one another. Chlorophyll a and chlorophyll b differ by only a couple oxygen groups. The same is true for beta-carotene and xanthophyll.
Based on the chemical structures of these pigments, which of the following is true?

6. Calculating Rf Values
In paper chromatography, you can calculate the different retention factors (abbreviated Rf) for each pigment. The Rf value for each substance is the distance the substance traveled divided by the distance the solvent traveled.

Rf = distance substance traveled / distance solvent traveled

What is the distance the solvent traveled?
7. Rf Values with Broad Bands
Not all chromatograms have clear, compact bands like in the previous example. Quite often bands will have broad, smeared sections. How are Rf values calculated from these bands?

Although there are several way to calculate Rf values for smeared bands, one of the best ways is to determine the center of each band. To do this, you need to know the highest and lowest points of the band. The center of the band is then calculated by the following equation:

Center = [ (highest point - lowest point) / 2 ] + lowest point

The band in the image above extends from 5 to 7.
What is the value of "highest point" for this equation?
8. Real Rf Values
Now let's use a real chromatogram to calculate Rf values.
What is the center of the chlorophyll b band? Use the red lines to measure the band.
If the solvent front traveled 8, what is the Rf value for chlorophyll b? Round to the nearest hundredth.
9. Lesson Done