Objectives

This tutorial aims to familiarize you with the following principles of column chromatography:

• What is Chromatography?
• Advantages of this analytical method
• Disadvantages of this analytical method
• The physical basis of column chromatography
• Proper experimental technique

What is Chromatography?

Chromatography is a powerful technique for separating mixtures. There are different types of chromatography, such as paper, thin layer, or column chromatography (amongst others), each with its own strengths and weaknesses. Chromatography systems have a stationary phase (which can be solid or liquid) and a mobile phase (usually liquid or gas). In column chromatography both phases are placed in a column container.

Advantages of Column Chromatography

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only can column chromatography be used to determine the number of components of a mixture, but it can also be used to separate and purify substantial quantities of those components for subsequent analysis. This is in contrast to paper chromatography, which is solely an analytical method.

For example, while paper chromatography is easily applied to see whether a purple coloured beverage contains a mixture of dyes, it is not practical to further analyze the separated dyes given the necessarily very small size of the initial sample. A preparative method like column chromatography allows you to do just that. Separating the purple food dye on an appropriately set up column with good technique will leave you with cleanly separated blue and red dyes in large enough amounts for further investigation. Thus, column chromatography should be used any time you want to separate a mixture of liquids or solutes into its components, and work with these components individually. In fact, it is the most frequently used method of purifying mixtures of products in research laboratories.

Disadvantages of Column Chromatography

With all its advantages and preparative power, column chromatography does have its complications. Properly setting up the column (something that will be done for you prior to experiment) requires some technical skill and manual dexterity, and takes some time. Column chromatography is less foolproof than paper chromatography and requires constant attention while the experiment is being performed: collection vessels must be frequently switched and solvent levels need to be topped up.

Unlike paper chromatography, column chromatography is a much more involved and tedious procedure. It is not as simple as sitting back and watching the mixture separate.

In short, running a column is time-consuming and tedious, especially for large samples. If it is unnecessary to preparatively separate large quantities of sample, analytical methods such as paper chromatography may be more suitable and easier to perform.

Question 1

Which of the following tasks are best suited for column chromatography? Why or why not?

1. Determine if the purple colour in the new GiganticGrapeGuzzler^TM energy drink is the result of one, two, or three food dye additives.
2. Isolate indigo, a blue food dye, from a 50:50 mixture with amaranth, a red food dye.
3. Determine the potassium permanganate content of a faintly reddish-purple coloured solution.
4. Positively identify a red coloured solution as containing FD&C Red #2, and determine its concentration.

How Does Column Chromatography Work?

Like all chromatographic techniques, column chromatography uses a mobile phase to move a mixture of substances through a stationary phase. The different components of the sample have different affinities for the mobile and stationary phases, and emerge from the stationary phase at different times. The stationary phase and mobile phase are chosen based on the nature of the sample mixture in order to achieve the best possible separation of its components.

In most applications in the chemistry laboratory, the stationary phase is either silica (SiO₂) or alumina (Al₂O₃), which is mixed with the solvent being used as the mobile phase to yield a thick white slurry. The mobile phase is a liquid that is chosen to maximize the separation of the sample. This can be water or any organic solvent.

In your food dye experiment, the stationary phase will be silica and the mobile phase will be water. Such a set-up separates substances mostly on the basis of their polarity. The silica and associated solvent forms an extremely polar matrix, more polar than any liquid chosen for the mobile phase. Thus, more polar substances in the sample will adsorb more strongly to the stationary phase and elute slowly from the column. By contrast, less polar substances in the sample adsorb weakly to the stationary phase and are quickly pushed through the column by the mobile phase.

The stationary phase is pre-loaded into the column above a plug of glass wool (to prevent solid material from contaminating products) and a thin layer of sand (to provide a uniform bed for the stationary phase).

Careless addition of sample can disturb the stationary phase and lead to poor separation. For this reason, a second bed of sand is added above the column as a “shock absorber.” Nevertheless, you must be very careful when adding sample or mobile phase to the top of the column. Most importantly, no part of the stationary phase must ever be dry! Air bubbles trapped in the stationary phase can severely impair your separation. To avert disaster, always keep the stationary phase covered with the mobile phase!

Because the silica or alumina gel that makes up the stationary phase is quite dense, column chromatography tends proceed very slowly if gravity is the only force pulling the mobile phase through the gel. The process can be sped up if high gas pressure at the top of the column or a vacuum at the bottom of the column is used to push or pull the mobile phase more quickly. This method is called flash column chromatography. In your food dye experiment, you will have the option of performing flash column chromatography by using a syringe attached to the bottom of the column to provide vacuum suction and thereby quicken elution.

A syringe (bottom left) can be used to expedite elution of the column by providing suction on the outflow end of the column.

A final note is necessary about the versatility of column chromatography. While most organic chemistry laboratories restrict themselves to the usual silica or alumina stationary phase, this is not the case in biochemical applications. Biochemists have been incredibly creative in adapting the column technique for separating macromolecules. For example, by coating the stationary phase with anionic groups, it is possible to selectively adsorb positively charged sample molecules to the column. Or, in an even more advanced application, a stationary phase of cellulose coated with antibodies against a particular molecule can be used to isolate that molecule from a cell extract. There are few separations that column chromatography can't perform!

Question 2

Is it feasible to use column chromatography for quantitative analysis? Why or why not?

Proper Experimental Technique

The Macromedia Flash walk-through below will demonstrate the proper technique for column chromatography.

Summary

This tutorial on column chromatography has presented the following topics:

1. The advantages and disadvantages of column chromatography.
2. The chemical and physical principles underlying column chromatography.
3. Appropriate experimental technique for performing column chromatography.