Chromatography

Chromatography

The development of chromatography

Types of chromatographic attraction

Industrial applications of chromatography

Resources

Chromatography refers to a variety of related laboratory techniques for separating mixtures of chemicals into their individual compounds. The basic principle of chromatography is that different compounds will stick to a solid surface, or dissolve in a film of liquid, to different degrees. The differing properties allow each compound to be retained in a distinctive way. As the sample is collected after passage through the chromatography column, the separated compounds can be individually collected.

When a gas or liquid containing a mixture of different compounds is made to flow over such a surface, the molecules of the various compounds can be prone to associate with the surface. The association can be on the basis of charge, with positively charged groups of the sample compound being attracted to negatively-charged surface groups. Or, in the case of a sample that is hydrophobic (water-hating), the association can be to partition away from the watery fluid. Sample compounds may even be attracted to specific proteins positioned on the surface of the chromatography column.

If the attraction between sample and surface is not too strong, a given molecule will become stuck and unstuck hundreds or thousands of times as it is swept along the surface. This repetition exaggerates even tiny differences in the various molecules, and they become spread out along the length of the chromatography column, as some compounds move more slowly than others. After a given time, the different compounds will have reached different places along the surface and will be physically separated from one another.

Using variations of this basic phenomenon, chromatographic methods have become an extremely powerful and versatile tool for separating and analyzing a vast variety of chemical compounds in quantities from picograms (10^-12 gram) to tons.

Chromatographic methods all share certain characteristics, although they differ in size, shape, and configuration. Typically, a stream of liquid or gas (the mobile phase) flows constantly through the column packed with a porous solid material (the stationary phase). A sample of the chemical mixture is injected into the mobile phase at one end of the column, and the compounds separate as they move along. The individual separated compounds can be removed one at a time as they exit (elute) from the column.

Because it usually does not alter the molecular structure of the compounds, chromatography can provide a non-destructive way to obtain pure chemicals from various sources. It works well on very large and very small scales; chromatographic processes are used both by scientists studying micrograms of a substance in the laboratory and by industrial chemists separating tons of material.

The technology of chromatography has advanced rapidly in the past few decades. It is now possible to obtain separation of mixtures in which the components are so similar they only differ in the way their atoms are oriented in space, in other words, they are isomers of the same compounds. It is also possible to obtain separation of a few parts per million of a contaminant from a mixture of much more concentrated materials.

The development of chromatography

The first paper on the subject of chromatography appeared in 1903, written by Mikhail Semyonovich Tsvet (1872-1919), a Russian-Italian biochemist, who also coined the term. Tsvet had managed to separate a mixture of plant pigments, including chlorophyll, on a column packed with finely ground calcium carbonate, using petroleumether as the mobile phase. As the colored mixture passed down the column, it separated into individual colored bands (the term chromatography comes from the Greek words chroma, meaning color, and graphein, meaning writing, or drawing). Although occasionally used by biochemists, chromatography as a science lagged until 1942, when A. J. P. Martin (1910–) and R. L. M. Synge (1914–1994) developed the first theoretical explanations for the chromatographic separation process. Although they eventually received the Nobel Prize in chemistry for this work, chromatography did not come into wide use until 1952, when Martin, this time working with A. T. James, described a way of using a gas instead of a liquid as the mobile phase and a highly viscous liquid coated on solid particles as the stationary phase.

Gas-liquid chromatography (now called gas chromatography) was an enormous advance. Eventually, the stationary phase could be chemically bonded to the solid support, which improved the temperature stability of the column’s packing. Gas chromatographs could then be operated at high temperatures, so even large molecules could be vaporized and would progress through the column without the stationary phase vaporizing and bleeding off. Additionally, since the mobile phase was a gas, the separated compounds were very pure; there was no liquid solvent to remove. Subsequent research on the technique produced many new applications.

The shapes of the columns themselves began to change. Originally vertical tubes an inch or so in diameter, columns began to get longer and thinner when it was found that this increased the efficiency of separation. Eventually, chemists were using coiled glass or fused silica capillary tubes less than a millimeter in diameter and many yards long. Capillaries cannot be packed, but they are so narrow that the stationary phase can simply be a thin coat on the inside of the column.

A somewhat different approach is the set of techniques known as planar or thin layer chromatography (TLC), in which no column is used. The stationary phase is thinly coated on a glass or plastic plate. A spot of sample is placed on the plate, and the mobile phase migrates through the stationary phase by capillary action (the movement of liquid molecules through the support from a region of higher density to a region of lower density).

In the mid-1970s, interest in liquid mobile phases for column chromatography resurfaced when it was discovered that the efficiency of separation could be vastly improved by pumping the liquid through a short, packed column under pressure, rather than allowing it to flow slowly down a vertical column by gravity alone. High-pressure liquid chromatography, also called high performance liquid chromatography (HPLC), is now widely used in industry. A variation on HPLC is Supercritical Fluid Chromatography (SFC). Certain gases (carbon dioxide, for example), when highly pressurized above a certain temperature, become a state of matter intermediate between gas and liquid. These so-called supercritical fluids have unusual solubility properties, some of the advantages of both gases and liquids, and appear very promising for chromatographic use.

Most chemical compounds are not highly colored, as were the ones Tsvet used. A chromatographic separation of a colorless mixture would be fruitless if there were no way to tell exactly when each pure compound eluted from the column. All chromatographs thus must have a device attached for this purpose and some kind of recorder to capture the output of the detector—usually a chart recorder or its computerized equivalent. In gas chromatography, several kinds of detectors have been developed; the most common are the thermal conductivity detector, the flame ionization detector, and the electron capture detector. For HPLC, an ultraviolet detector is standardized to the concentration of the separated compound. The sensitivity of the detector is of special importance, and research has continually concentrated on increasing this sensitivity, because chemists often need to detect and quantify exceedingly small amounts of a material.

Within the last few decades, chromatographic instruments have been attached to other types of analytical instrumentation so that the mixture’s components can be identified as well as separated (this takes the concept of the “detector” to its logical extreme). Most commonly, this second instrument has been a mass spectrometer, which allows identification of compounds based on the masses of molecular fragments that appear when the molecules of a compound are broken up. Currently, chromatography as both science and practical tool is intensively studied, and several scientific journals are devoted exclusively to chromatographic research.

Types of chromatographic attraction

Absorption chromatography (the original type of chromatography) depends on physical forces such as dipole attraction to hold the molecules onto the surface of the solid packing. In gas chromatography and HPLC, however, the solubility of the mixture’s molecules in the stationary phase coating determines which ones progress through the column more slowly. Polarity can have an influence here as well. In gel filtration (also called size-exclusion or gel permeation) chromatography, the relative sizes of the molecules in the mixture determine which ones exit the column first. Large molecules flow right through; smaller ones are slowed down because they spend time trapped in the pores of the gel. Ion exchange chromatography depends on the relative strength with which ions are held to an ionic resin. Ions that are less strongly attached to the resin are displaced by more strongly attached ions. Hence the name ion exchange: one kind of ion is exchanged for another. This is the same principle upon which home water softeners operate. Affinity chromatography uses a stationary phase composed of materials that have been chemically altered. In this type of chromatography, the stationary phase is attached to a compound with a specific affinity for the desired molecules in the mobile phase. This process is similar to that of ion exchange chromatography and is used mainly for the recovery of biological compounds. Hydrophobic Interaction Chromatography is used for amino acids that do not carry a positive or negative charge. In this type of chromatography, the hydrophobic amino acids are attracted to the solid phase, which is composed of materials containing hydrophobic groups.

Chemists choose the mobile and stationary phases carefully because it is the relative interaction of the mixture’s compounds with those two phases that determines how efficient the separation can be. If the compounds have no attraction for the stationary phase at all, they will flow right through the column without separating. If the compounds are too strongly attracted to the stationary phase, they may stick permanently inside the column.

Industrial applications of chromatography

Chromatography of many kinds is widely used throughout the chemical industry. Environmental testing laboratories look for trace quantities of contaminants such as PCBs in waste oil and pesticides such as DDT in groundwater. The Environmental Protection Agency uses chromatography to test drinking water and to monitor air quality. Pharmaceutical companies use chromatography both to prepare large quantities of extremely pure materials, and also to analyze the purified compounds for trace contaminants.

A growing use of chromatography in the pharmaceutical industry is for the separation of chiral compounds (compounds that are mirror images of each other but which cannot be superimposed on one another). These compounds have molecules that differ slightly in the way their atoms are oriented in space. Although identical in almost every other way, including molecular weight, element composition, and physical properties, the two different forms—called optical isomers, or enantiomers—can have enormous differences in their biological activity. The compound thalidomide, for example, has two optical isomers. One causes birth defects when women take it early in pregnancy; the other isomer does not. Because this compound looks promising for the treatment of certain drug-resistant illnesses, it is important that the benign form be separated completely from the dangerous isomer.

Chromatography is used for quality control in the food industry, by separating and analyzing additives, vitamins, preservatives, proteins, and amino acids. It can also separate and detect contaminants such as aflatoxin, a cancer-causing chemical produced by a mold on peanuts. Chromatography can be used for various purposes, from finding drug compounds in urine or other body fluids to looking for traces of flammable chemicals in burned material from possible arson sites.

Resources

BOOKS

Grob, Robert L. and Eugene F. Barry. Modern Practice of Gas Chromatography. New York: Wiley-Interscience, 2004.

Miller, James M. Chromatography: Concepts and Contrasts. New York: Wiley-Interscience, 2004.

Poole, Colin F. The Essence of Chromatography. St. Louis: Elsevier Science, 2002.

Gail B. C. Marsella