Chromatography

Chromatography is a family of 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. Chromatography is used extensively in forensics, from analyzing body fluids for the presence of illicit drugs, to fiber analysis, blood analysis from a crime scene, and at airports to detect residue from explosives.

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 will tend to stick to the surface. If the stickiness 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' stickiness, and they become spread out along the "track," because the stickier compounds move more slowly than the less sticky ones do. After a given time, the different compounds will have reached different places along the surface and will be physically separated from one another. Or, they can all be allowed to reach the far end of the separation surface and be detected or measured one at a time as they emerge.

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 a tube (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 (or "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.

In gas-liquid chromatography (now called gas chromatography), the material that separates components is chemically bonded to the solid support, which improves the temperature stability of the column's packing. Gas chromatographs can be operated at high temperatures, so even large molecules can be vaporized and progress through the column without the stationary phase vaporizing and bleeding off. Additionally, since the mobile phase is a gas, the separated compounds are very pure; there is no liquid solvent to remove.

The shapes of chromatographic columns, originally vertical tubes an inch or so (2–3 cm) in diameter, became 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 at all. 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.

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 super-critical 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 "supercritical fluids" have unusual solubility properties, some of the advantages of both gases and liquids, and appear very promising for chromatographic use.

All chromatographs must have a detection device attached, 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, the UV 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.

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.