Chromatography

Chromatography Definition

Chromatography is a method of separating the constituents of a solution, based on one or more of its chemical properties. This could be charge, polarity, or a combination of these traits and pH balance. In essence, the solution is passed through a medium which will hinder the movement of some particles more than others. This draws the different molecules apart as they travel through the medium. Oftentimes, different dyes are used to represent the different parts, or fractions of the media.

There are many different types of chromatography, used in a variety of circumstances. While the following is not a definitive list, it is a good overview of the different types and uses of chromatography. As each is discussed, try to picture how it conforms to the broad definition of chromatography. The exact procedures may vary, depending on the circumstance, but all chromatography is centered on moving a solution through a media which slows certain molecules more than others.

Electrophoresis is similar to chromatography, in that a solution is moved through a media. However, in electrophoresis the DNA sample of multiple DNA fragments is dyed the same color. As it travels along the gel, propelled by the electric current, visible bands are seen which represent different sized segments of DNA. In chromatography, many more substances can be tested. The media can be changed, to better retain or expel certain substances, or the solution holding the substance can be altered to improve the separation.

What is Chromatography Used For?

Chromatography is used in many industries, for many purposes. In general, it is used to separate a desired substance from a solution. This could be a specific amino acid from a sample containing many, or a desired chemical from an unknown sample. Chromatography was originally named as it was used to sort plant pigments, which come in many different colors. The pigments sorted when places on a chromatography paper and a solvent allowed to travel with the pigments across the paper. The paper, made of cellulose and having a slightly negative charge, attracts polar substances. This allows the non-polar pigments to travel further, separating from the more polar pigments.

These techniques have expanded to many different industries, the basic principles remaining the same. Upon knowing the characteristics of the desired substance, a medium and solvent can be obtain which will interact with the substance and remove it from the solution. Using a solution with a different pH, or a different composition, the desired substance can then be washed off the media, and collected.

These principles are used to isolate and analyze enzymes, pigments, amino acids, constituents of DNA, and almost any other molecule you can imagine. All molecules have specific interaction with other molecules, and a chromatography experiment can be designed to retrieve almost any molecule from a seemingly homogenous solution.

How Does Chromatography Work?

The core of chromatography lies in the fact that the molecules being separated can move through the media at different speeds. This may be because they have a certain affinity towards the media, or simple because the media is sized to only allow certain molecules through. In some forms of chromatography, the media has such a strong affinity for the desired substance that it binds it as the original solution is poured over, and another solution containing a substance to displace the bound molecules must be introduced.

The affinity for the media could be from a number of properties. Some are designed to attract polar substances, and repel nonpolar substances as in the original chromatography experiment. However, the media could also be designed to hold onto specific ions, or could even be created with antibodies which identify and retain specific proteins. The possibilities of chromatography are nearly endless, but the basic principle of separating molecules by passing them through a selective filter remains the same.

Types of Chromatography

Column Chromatography

Column chromatography is a popular form of the technique, which relies on a solution filled column, filled with a media which will in some way inhibit the movement of particular molecules. As the solution is drained from the bottom, different fractions are obtained as the solution comes out. These fractions will contain different compositions of molecules, determined by how fast the molecules could travel through the column. In basic column chromatography, silicon beads or other negatively charged media are used as a simple way to sort molecules by charge and polarity.

There are several variations of column chromatography, which differ in their media and solvent. They are used to separate different substances from solution. For instance, if the media within the column is meant for ion-exchange chromatography, it will be somehow charged. As a solution passes over it, ions will attract to the media and be drawn from solution. A different solution can then be washed over the media, which will have a higher affinity for the media and displace the ions. They can be washed away and collected, purifying them from the original solution.

Planar Chromatography

Planar chromatography, which includes both the paper and thin-layer methods, depends on a solution moving through a media due to the forces of adhesion and cohesion. If you’ve ever seen a napkin soak up a mess, you’ve seen these forces. As the dry gaps in the material are filled, the solution crawls its way up the napkin. In the same way, planar chromatography uses these forces to pull molecules through a media.

In the same way that gravity forces the solution over the media in column chromatography, adhesion and cohesion pull the solution through the media. In this case, the media is very thin. In paper chromatography, the paper is usually cellulose-based, which is slightly negative. This makes the sorting of polar and nonpolar substances very easy. In thin-layer chromatography, the layer can be made of any substance which has an affinity for the target substance. Solution is allowed to pass through or over it, and the substance is acquired from the solution.

Paper and thin-layer chromatography have been used to sort and identify pigments, amino acids, and many different kinds of organic molecules. Because it is so simple to set up a paper chromatography experiment, this is one of the first laboratory techniques presented in science courses.

Other Forms of Chromatography

Besides these common methods of the technique, there are many more. One, which is used in the more advanced sciences and in criminal forensics is gas chromatography. In gas chromatography, the substance to be sorted is vaporized, and passed through another gas containing various elements. Much like the other forms of chromatography, it is the affinity for particular molecules in the media which causes the separation of the solution.

Still other methods couple chromatography directly with mass spectrometry, a method of identifying the chemical and structural properties of molecules. This is a powerful method which can both separate and identify individual components of a complex solution. The machinery to do this is expensive and sensitive, but necessary for many scientific and forensic applications.

Quiz

1. You are trying to identify an unknown substance dissolved in a sample of pond water. What is the first step?
A. Use chromatography to remove the substance
B. Identify the substance using mass spectrometry
C. Hypothesize the nature of the substance

Answer to Question #1

C is correct. If you don’t know the nature of the substance, you will not be able to extract it efficiently using chromatography. Even by knowing one of its properties, charge or polarity for instance, will help you purify it from solution using chromatography. Remember that chromatography relies on separating molecules by property. You may have to experiment further to learn its properties before extracting it.

2. A scientist places some mushed up plants at the base of a strip of cellulose-based media. He places this strip in a solvent, and the solvent travels up the strip. Which form of chromatography does this represent?
A. Gas
B. Column
C. Planar

Answer to Question #2

C is correct. This is paper chromatography, a form of planar chromatography. This technique is using paper as the media through which the solution travels. The individual pigments in the plant will sort based on how much they are hindered by the cellulose.

3. Which of the following represents a chromatography?
A. An artist allows multiple pigments to separate as they diffuse through the paper
B. A watermelon, being naturally red in the middle and green on the exterior
C. Two dyes are mixed together, allowing new colors to be created

Answer to Question #3

A is correct. Although chromatography is named after the Greek word for color, it is because the original experiment involved separating colors. The true key to chromatography is separation. While the colors in a watermelon are separated, it is not because of chromatography, but rather because genetics directed the creation of those pigments in certain tissues. The pigments could be separated further using a specific chromatography experiment.

References

• Bruice, P. Y. (2011). Organic Chemistry (6th ed.). Boston: Prentice Hall.


• Moore, J. T. (2010). Chemistry Essentials for Dummies. Indianapolis: Wiley Publishing, Inc.


• Nelson, D. L., & Cox, M. M. (2008). Principles of Biochemistry. New York: W.H. Freeman and Company.

Chromatography