Chromatography was first described in 1850 by a German chemist, Friedlieb Ferdinand Runge. It was not until the early twentieth century, however, that Mikhail Semenovich Tsvet became the first to explain the phenomenon and methods of this analytical tool.
Chromatography and Photosynthesis
Tsvet’s chromatography of plant leaf pigments prompted scientific investigations of photosynthesis—the all-important biochemical reaction that transforms inorganic to organic energy and therefore is at the base of most life. Chromatography has revealed that many different pigments, not only green ones, are simultaneously present in leaves.
Each pigment absorbs only certain colors of light from sunlight, rather than absorbing all the incident light energy that falls upon it. Each pigment behaves as though it has a tiny “window” that allows the energy of certain wavelengths of light to be harvested.
These little bundles of energy are quantized, or set, amounts of energy, and they are unique for each different type of pigment. (White sunlight is actually composed of a broad range of wavelengths, with the visible wave lengths appearing as a rainbow of colors when passed through a prism.)
Paper chromatography has allowed for the discovery of many specialized pigments, including at least five forms of chlorophyll. Chlorophyll pigments are now known to include chlorophylls a through e.
Also, many different forms of carotenes and xanthophylls exist. Paper chromatography reveals that red and yellow pigments are always present in the leaves of deciduous trees and shrubs and not just during the fall color change.
Because of the high abundance of the green chlorophyll pigments, as compared with the bright reds of carotenes or yellows of xanthophyll, only the dominant green hues are generally seen. In the fall, deciduous trees show a loss of chlorophyll pigments, thereby revealing the brilliant foliage associated with an autumn forest.
Once pigments are separated from one another, they can be chemically characterized and further studied. Carotenes and xanthophylls have been discovered to be of similar chemical composition, with each being made of forty carbon atoms covalently bonded to one another. Different arrangements of these covalent bonds produce the different colors of red and orange.
Chromatography has allowed scientists the opportunity to trace the path that carbon atoms follow through every tiny increment of the photosynthetic process.
Paper chromatography, coupled with radioisotopic studies of carbon-labeled (with radioactive carbon 14) compounds, eventually led to the ability to describe the carbon-containing products of each step in the series of reactions of photosynthesis.Today this pathway is called the Calvin cycle.
A classical demonstration of chromatographic principles utilizes techniques that allow plant pigments to be isolated. Spinach leaves are an excellent tool for the identification of four pigments: chlorophyll a, chlorophyll b, carotene, and xanthophyll.
The stationary phase is a piece of chromatography paper with a dried spot of the plant extract near one end. The mobile phase is an acetone-ligroin mixture, a nonpolar (hydrophobic) solvent mixture.
The paper is placed with a small portion of the end with the pigment spot in the solvent, the mobile phase. As the acetone-ligroin mobile phase comes into contact with the paper, capillary action allows the liquid to travel upward, against gravity.
The mobile phase has a migrating moisture line, or leading line of wetness, which is called the solvent front. As the solvent travels over the spot, each of the pigments will travel with the mobile phase at different rates from the original spot. Some pigments will adhere to the paper more strongly than others, and thus travel shorter distances along the paper.
Yellow-green chlorophyll b travels the least distance with the mobile phase. Chlorophyll b is a more polar (water-loving) pigment than the other pigments found in spinach extracts and is therefore more strongly attracted to the polar surface of the paper than to the nonpolar solvent.
The remaining pigments travel increasing distances with respect to chlorophyll b, beginning with blue-green chlorophyll a, followed by yellow-orange xanthophyll and, finally, the orange pigment of carotene.
Carotene moves the farthest because it is the most nonpolar of the pigments and it is attracted more strongly to the acetone-ligroin mixture (mobile phase) than to the paper. This stronger, nonbonded interaction with the mobile phase indicates that carotene is the most nonpolar pigment found in spinach chloroplasts.
Once the solvent front is about half an inch from the top of the paper strip, the strip is removed from the chamber. A pencil line must be drawn immediately across the top of the strip to indicate how far up the paper the mobile phase traveled. The paper strip is then referred to as a chromatogram.
The Rf value is a numerical constant that is unique for each of the four pigments identified in spinach. The ratio of the distance each pigment travels, as compared with the distance traveled by the mobile phase (from the start to finish lines),will be unique to that pigment alone.
Thus, chlorophyll b will not switch places with carotene on the chromatogram because of the unique interactions it has with the stationary and mobile phases. For this reason, the Rf values determined by the method described above can be generated repeatedly by anyone using this method.
Types of Chromatography
As performed by Runge and Tsvet, chromatography has evolved from the days of paper, chalk, and dyes into a computerized and versatile instrumentation requiring expert training and a significantly larger budget.
Thin-layer chromatography (TLC) is useful in protein chemistry. The stationary phase of this method consists of thin gel applied to a plastic or glass plate (strip). Various gels can be used to coat the plate. Some coatings may be polar, while others may be nonpolar.
Column chromatography can look for the amounts and types of vitamins in food or diet supplement tablets. Pigments, steroids, alkaloids, and carbohydrates all can be identified and measured using an appropriate column-chromatographic system. Many recent advances in column chromatography now allow for isolation and purification of proteins, DNA, RNA, and many other biological molecules.
High-pressure liquid chromatography (HPLC) can purify biologically important enzymes from living systems without destroying the biological activity of the enzyme. In gas chromatography (GC), an inert gas such as helium or nitrogen flows through several feet of a packed and coiled column. The gas acts as the mobile phase by sweeping the sample through the column.
The packing is often a solid material, but liquid-coated solid particles are also used. Paper chromatography continues be a popular method for analysis of plant pigments, dyes, inks, and food colorings. It is largely used, however, in academic settings to demonstrate the principles of chromatography.