Paper chromatography was widely used before the advent of thin-layer chromatography.

The basic principle underlying all methods of chromatography is simple. A substance in either liquid or gaseous solution (known as the mobile phase) passes through a solid stationary phase, consisting of a packed column or the surface of a solid material. Progress of the mobile phase slows down when interaction with the stationary phase occurs.

In effect, the stationary phase acts as a molecular obstacle course. The molecules of one chemical species move faster or slower than those of another, depending on their chemical nature and molecular size. What actually happens with the materials in solution is that the mobile and stationary phases compete for them. The greater the attraction to the stationary phase, the slower the compound in solution moves. Conversely, the greater the attraction to the mobile phase, the faster the compound moves. These differences in rate of movement allow chemicals to be separated from one another. They can then be identified by the rate at which they move under specified conditions. Chromatographic methods are quite valuable, often making it possible to achieve the three analytical objectives of separation, identification, and quantification with one procedure.

In column chromatography, a mixed solution is poured into the column.
A solvent is then trickled through (moving solvent phase) to separate the mixture into bands (solid absorbed phase). The various fractions can then be washed through one at a time using a series of other solvents.

Types of chromatography

The main forms of chromatography are adsorption chromatography and partition chromatography. Adsorption is the process of taking up and holding a gas, liquid, or dissolved substance by spreading it out on a surface in a thin layer of molecules. An ac/sorbed material is spread out on the surface of a substance. By contrast, an absorbed material goes into the substance itself. In adsorption chromatography, the stationary phase is simply a solid material of a uniform fine particle size. Components in solution are progressively adsorbed and desorbed (released from being adsorbed) as they pass over the surface.

In partition chromatography, the stationary phase is a thick liquid of high boiling point that is deposited on the surface of chemically inactive particles, which serve simply as a support material. Separation of substances in the mobile solution depends on the way in which they split up between the mobile and stationary phases.

Early chromatographic separations were applied mainly to colored compounds whose separated zones could be readily observed. However, many separations are now carried out on colorless substances. These are detected after chromatographic separation by passing the solution through special detector cells that measure changes in properties of a solution passing through them.

In descending paper chromatography, solvent (colored yellow) passes down a sheet of filter paper. The paper has been previously “spotted” at the top with the mixture to be separated. Each component in the mixture (A, B, and A + B) moved a different distance, described by its RF value. This is the ratio of the distance traveled by the component to the distance traveled by the solvent front.

Column chromatography

The earliest form of chromatography is still in regular use—adsorption chromatography in columns. The apparatus is easy to set up and can give good results on both a small and large scale. Typically, the chemist uses a glass tube about 1 inch (25 mm) in diameter and 20 inches (50 cm) long, packed with an adsorbent, such as powdered alumina. The mixture to be separated is added as a small concentrated volume at the top of the column, followed by the continuous addition of an appropriate solvent to carry the mixture through the adsorbent.

The simple ascending paper chromatogram shows the results of an analysis of black ink. An elongated blot of black ink was made near the lower edge of a sheet of filter paper. The lower edge of the paper was placed in a trough of solvent On its way upward, the solvent has separated the ink into its various components.

The separated components of the mixture can be dealt with in two ways. They can be run out from the bottom of the column if sufficient solvent is added, or they can be obtained by removing the packing material from the tube. The separated compounds are then extracted from their zones. This form of chromatography is useful for large-scale operations in which sizable quantities of material are to be separated.

In two-phase chromatography, a mixture has been “spotted” near the bottom right-hand corner of a sheet of paper. Solvent A (far left) is then absorbed up the paper. The solvent separates the mixture into regions along a straight line (in the x direction). The paper is then turned through 90° in a second solvent B (near left). This causes each component to travel a different distance in the / direction.

Plate (thin-layer) chromatography (TLC)

Adsorption chromatography can also be carried out on a small scale, using an adsorbent in the form of a thin layer spread on a sheet of glass or aluminum foil. A small volume of the sample to be separated is placed near one edge of the sheet That edge is then placed in a shallow pool of solvent at the bottom of a glass tank. The solvent moves up the plate by capillary and molecular attraction—the same way liquid “travels up” a paper towel when the corner is dipped in a liquid. The solvent also “travels up” the sheet, carrying the sample with it. Components within the sample separate. Measuring their distances from the front of the solvent establishes their identities.

Paper chromatography

Paper chromatography, widely employed before the advent of TLC, is now less commonly used. One advantage of paper chromatography over TLC is that it can be run in either a descending or ascending manner. Although the latter is easier to carry out, the former is preferable. It is faster, due to the effect of gravity on the solvent flow. Paper chromatography works in a fashion similar to TLC, except that a sheet of paper is used in place of the glass or aluminum foil of TLC.

Gas chromatography

The introduction of partition gas chromatography in 1953 revolutionized chemical analysis.

In this form of chromatography, the sample mixture (as a liquid or vaporl’is injected by a syringe into a continuously flowing gas stream (nitrogen, argon, or helium). This stream passes along a narrow column. The column is packed with an inert solid powder, which acts as a support for a stationary liquid. The substances in the sample distribute themselves between the mobile and stationary phases according to their physical and chemical properties. They are successively carried out by the gas stream at the end of the column. Special electronic detectors at the end of the column measure the individual substances. The whole sequence is also recorded as a series of peaks on a chart. Individual chemicals can be identified by the amount of time they take to pass through the column. The areas of the peaks can also be used to measure the amount of material detected. By this means, very complex mixtures of oils, solvents, drugs, and pollutants can be readily separated.

Gas chromatography and mass spectrometry

One very useful application of gas chromato-gaphy has been in linking it with a mass spectrometer in a process known as interfacing. After a substance has been separated from a mixture by gas chromatography, part of it can be diverted to the mass spectrometer, which then produces its mass spectrum. The substance can be identified by comparing its spectrum with those previously determined.

In modern instruments, this can be done almost instantaneously by computer. The combined technique has become especially useful for qualitative analysis of complex organic mixtures.

A scientist uses gas chromatography to analyze a synthetic fuel (above).

High performance liquid chromatography

The most rapidly expanding area in chromatography, high performance liquid chromatography (HPLC), incorporates all standard forms of chromatographic analysis and can be applied to most chemical substances. The process is carried out on microcolumns, with the solvents pumped through under high pressure (up to 600 atmospheres). The great advantage of HPLC is that only very small samples are required. The detectors used are extremely sensitive, and a very high degree of separation can be achieved between closely related substances.

Preparative chromatography

Traditional methods of chromatography normally deal with very small volumes of samples. Thus, only minute amounts of pure materials are obtained. However, larger quantities of very pure compounds are sometimes needed for use as standards and in the determination of correct chemical constants. This has necessitated the development of preparative chromatographic methods. These fall into two categories: large-scale chromatographic separations and multiple small-scale operations.

Typical of the large-scale separations are the thick-layer chromatograms in which the traditional adsorbent layer is up to 0.4 inch (1 cm) thick. Similarly, gravity-flow adsorption and partition chromatographic columns can be scaled up to 4 inches (10 cm) in diameter and 6.5 feet (2 m) long. There are limitations in all these methods, however. The quality of the separation between the various zones of the column suffers, and the purity required of the extracted chemical limits the maximum sample loading and column length.

The second type of preparative chromatography-multiple small-scale operations—uses gas chromatography. In this process, many small sample volumes are injected. Smaller quantities of pure, separated chemicals are then produced. The system is best when it is automated. A regular injection of sample is followed by a sequence of traps that capture the individual compounds at the end of the column. Although it is relatively time-consuming, this multiple small-scale operation is a useful method for obtaining small quantities of very high-purity chemicals.

In a gas chromatograph, the flow of a dry, inert carrier gas (such as helium or argon) is carefully monitored. Mixed with a sample to be analyzed (sample inlet), the gas flows through a coiled column heated in an oven. The same gas carries the separated components to a detector. This detector usually responds to certain changes in the gas. These changes are noted by the recorder and plotted on a graph. An example of a gas chromatogram is shown below. The four isomers of butanol are plotted.

Other methods of separation

Although not strictly chromatographic, similar separation methods used in analytical chemistry include ion exchange and electrophoresis. Ion exchange uses a column packed with a special synthetic resin or a natural substance such as zeolite. This resin or substance “exchanges” its ions with those of a solution passed through the column. The most familiar application of this technique is in water softeners, which take up calcium ions from hard water and exchange them for sodium ions.

Electrophoresis is more like chromatography. It involves the movement of chemicals in gel columns or on paper strips. But in electrophoresis, the movement of substances relative to the stationary phase is brought about by applying an electric field along the length of the strip of column, blow far the different substances will move depends upon three factors: the power of the electrical charge, the molecular sizes of the substances, and the molecular shapes of the substances. This method of separation is of particular value in the study of biochemicals, such as proteins, peptides, and amino acids.

Research-scale low pressure chromatography is a technique used to separate (fractionate) monoclonal antibodies from a liquid cell culture. The purified fractions are subsequently used to produce new recombinant DNA antibodies.