In 1957, while doing a theoretical study of gas chromatographic columns, Marcel J.E. Golay, as a consultant for the Perkin-Elmer Corporation, concluded that a very long column (90 to 180 metres [300 to 600 feet]) of narrow-diameter tubing (internal diameter of 0.25 millimetres [0.0098 inch]) with its wall coated with a thin film of liquid would yield superior separations. Fortunately, at about this same time, detectors with extremely low limits of detection became available, which could sense the small sample sizes required by these new columns. These capillary, or Golay, columns, now called open-tubular columns and characterized by their open design and an internal diameter of less than one millimetre, had an explosive impact on chromatographic methodology. It is now possible to separate hundreds of components of a mixture in a single chromatographic experiment.

Molecular sieves are porous substances that trap a mobile-phase gas. Large molecules cannot enter the pores, and so they flow largely unimpeded through the system. Small molecules are interrupted in their migration as they meander in and out of the pores by diffusion. Molecules of intermediate sizes show different rates of migration, depending on their size. In 1959 Per Flodin and Jerker Porath in Sweden developed cellulose polymeric materials that acted as molecular sieves for substances dispersed in liquids. This extended the molecular weight range of chromatography to polypeptides, proteins, and high-molecular-weight polymers. The generic term for such separations is size-exclusion chromatography.

In 1964 the American chemist J. Calvin Giddings, referring to a theory largely worked out for gas chromatography, summarized the necessary conditions that would give liquid chromatography the resolving power achievable in gas chromatography—that is, very small particles with a thin film of stationary phase in small-diameter columns. The development of the technique now termed high-performance liquid chromatography (HPLC) depended on (1) the development of pumps that would deliver a steady stream of liquid at high pressure to the column to force the liquid through the narrow interstitial channels of the packed columns at reasonable rates, and (2) detectors that would sense the small sample sizes mandated. At first, only adsorptive solids were used as the stationary phase, because liquid coatings were swept away by the mobile phase. Previously gas chromatography had employed chemical bonding of an organic stationary phase to solids to reduce adsorptive activity; István Halász of Germany exploited these reactions to cause a separation based on liquid solution effects in the bonded molecular layers. These and similar reactions were employed to give firmly attached molecules that acted as a thin film of solvent in liquid systems. These bonded phases gave high-performance liquid chromatography such scope and versatility that the technique is now the dominant method for separations.

Ion exchangers are natural substances—for example, certain clays—or deliberately synthesized resins containing positive ions (cation exchangers) or negative ions (anion exchangers) that exchange with those ions in solution having a greater affinity for the exchanger. This selective affinity of the solid is called ion, or ion-exchange, chromatography. The first such chromatographic separations were reported in 1938 by T.I. Taylor and Harold C. Urey, who used a zeolite. The method received much attention in 1942 during the Manhattan Project as a means of separating the rare earths and transuranium elements, fission products of uranium, and other elements produced by thermonuclear explosions. Ion-exchange chromatography can be applied to organic ion separations and has particular importance for the separation of amino and nucleic acids.

As early as 1879, the solubility of solids in gases at high pressure had been observed. In 1958 the British scientist James Lovelock suggested that gases above their critical temperature (i.e., the temperature above which the appearance of a liquid phase cannot be produced by increasing the pressure) might be used at high pressure as mobile phases. A substance in this state is termed a supercritical fluid. At very high pressure, the density of the fluid can be 90 percent or more of the liquid density. The German chemist Ernst Klesper and his colleagues working at Johns Hopkins University were the first to report separation of the porphyrins with dense gases in 1962. Carbon dioxide at 400 atmospheres is a typical supercritical-fluid mobile phase. (One atmosphere equals 760 millimetres, or 29.92 inches, of mercury; standard sea-level pressure is one atmosphere.) In an extreme case, Giddings and his group used gases at pressures of up to 2,000 atmospheres to chromatograph carotenoids, sugars, nucleosides, amino acids, and polymers. Supercritical-fluid chromatography bridges a gap between gas chromatography and liquid chromatography. In gas chromatography, concentration of solutes in the gas phase is achieved with increased temperature. Supercritical-fluid chromatography achieves this result with increased pressure so that thermally unstable compounds may be analyzed. Additional advantages include increased speed and resolution.

A technique exhibiting great selectivity, affinity chromatography, was first described by Pedro Cuatrecasas and his coworkers in 1968. In these separations, a biomolecule such as an enzyme binds to a substrate attached to the solid phase while other components are eluted. The retained molecule can subsequently be eluted by changing the chemical conditions of the separation.

Another separation technique is based on the fact that the velocity of a fluid through a tube is not uniform. In the region immediately adjacent to the wall the fluid is nearly stationary. At distances farther from the wall, the velocity increases, reaching a maximum value at the centre of the channel. In 1966 Giddings conceived the idea that a field, electrical or gravitational, might be used to selectively attract particles to the wall, where they will move slowly through the system. Diffusion away from the high concentrations at the wall into faster inner streams would enhance migration. The net effect would yield differential migration. A thermal gradient between two walls has also been used. This technique is called field-flow fractionation. It has been termed one-phase chromatography because there is no stationary phase. Its main applications are to polymers and particulate matter. The method has been used to separate biological cells, subcellular particles, viruses, liposomes, protein aggregates, fly ash, colloids, and pigments.

The battery of chromatographic techniques, along with field-flow fractionation, provides separations from the level of hydrogen molecules to particulates, encompassing a 1015-fold mass range. An analogous mass range is one of grains of sand to boulders.

Methods

Chromatographic methods are classified according to the following criteria: (1) geometry of the system, (2) mode of operation, (3) retention mechanism, and (4) phases involved.

Geometry

Column chromatography

The mobile and stationary phases of chromatographic systems are arranged in such a way that migration is along a coordinate much longer than its width. There are two basic geometries: columnar and planar. In column chromatography the stationary phase is contained in a tube called the column. A packed column contains particles that either constitute or support the stationary phase, and the mobile phase flows through the channels of the interstitial spaces. Theory has shown that performance is enhanced if very small particles are used, which simultaneously ensures the additional desired feature that these channels be very narrow. The effect of mobile-phase mass transfer on band (peak) broadening will then be reduced (see below discussions of mass transfer and peak broadening in Efficiency and resolution and Theoretical considerations). Constructing the stationary phase as a thin layer or film will reduce band broadening due to stationary-phase mass transfer. Porous particles, either as adsorbents or as supports for liquids, may have deep pores, with some extending through the entire particle. This contributes to band broadening. Use of microparticles alleviates this because the channels are shortened. An alternate packing method is to coat impermeable macroparticles, such as glass beads, with a thin layer of microparticles. These are the porous-layer, superficially porous, or pellicular packings. As the particle size is reduced, however, the diameter of the column must also be decreased. As a result, the amount of stationary phase is less and the sample size must be reduced. Detection methods must therefore respond to very small amounts of solutes, and large pressures are required to force the mobile phase through the column. The extreme cases are known as microbore columns; an example is a column 35 centimetres (14 inches) long of 320-micrometre (1 micrometre = 10−4 centimetre) inside diameter packed with particles of 2-micrometre diameter.

A second column geometry involves coating the stationary phase onto the inside wall of a small-diameter stainless steel or fused silica tube. These are open tubular columns. The coating may be a liquid or a solid. For gaseous mobile phases, the superior performance is due to the length and the thin film of the stationary phase. The columns are highly permeable to gases and do not require excessive driving pressures. Columns in which a liquid mobile phase is used are much shorter and require large driving pressures.

Planar chromatography

In this geometry the stationary phase is configured as a thin two-dimensional sheet. In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase. In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic sheet. One edge of the sheet is dipped in a reservoir of the mobile phase, which, driven by capillary action, moves through the bed perpendicular to the surface of the mobile phase. This capillary motion is rapid compared to solute diffusion in the mobile phase at right angles to the migration path, and so the solute is confined to a narrow path.

Mode of operation

Development chromatography

In terms of operation, in development chromatography the mobile phase flow is stopped before solutes reach the end of the bed of stationary phase. The mobile phase is called the developer, and the movement of the liquid along the bed is referred to as development. With glass columns of diameter in the centimetre range and large samples (cubic-centimetre range), the bed is extruded from the column, the solute zones carved out, and solutes recovered by solvent extraction. Although this is easily done with coloured solutes, colourless solutes require some manner of detection, such as ultraviolet light absorption or fluorescence or the streaking of the column with a reagent that reacts with the solute to form a coloured product.

Planar systems involve placing the samples (in the 10−3 cubic-centimetre range) as spots at an edge of the stationary bed parallel to the developer. Solute zones are located by light irradiation or by spraying the bed with a colour-producing reagent. Migration is reported in terms of the Rf value, the distance moved by the centre of the zone relative to the distance moved by the mobile phase front, where both are measured from the origin. Use of the solvent front as a reference point is frequently inconvenient. A standard solute is often included, and the migration of the solutes relative to the standard reported as the relative R value. If larger samples are required for subsequent manipulation, either simultaneous separations are performed or the sample is applied as a streak across the stationary phase. The final spot or band is carved or cut from the chromatogram. In one type of planar chromatography, the mixture is placed at one corner of a square bed, plate, or sheet and developed, the mobile phase is evaporated, and the plate is rotated 90° so that the spots become the origins for a second development with a different developer. This is termed two-dimensional planar chromatography.