Liquid-Solid Chromatography

Liquid-solid chromatography originally was developed for the separation of colored substances, hence the name chromatography, which stems from the Greek word chroma meaning color. In a typical examination, a colored substance suspected of containing colored impurities is dissolved in a suitable solvent and the solution allowed to percolate down through a column packed with a solid adsorbent, such as alumina or silica, as shown in Figure 9-3. The "chromatogram" then is "developed" by passing through a suitable solvent that washes the adsorbate down through the column. What one hopes for, but may not always find, is that the components of the mixture will be adsorbed unequally by the solid phase so distinct bands or zones of color appear. The bands at the top of the column contain the most strongly adsorbed components and the bands at the bottom the least strongly held components. The zones may be separated mechanically, or sufficient solvent can be added to wash, or elute, the zones of adsorbed materials sequentially from the column for further analysis.

Liquid-solid chromatography in the form just described was developed first by the Russian biochemist M. S. Tswett, about 1906. In recent years, many variations have been developed that provide greater convenience, better separating power, and wider applicability. In thin-layer chromatography, which is especially useful for rapid analyses, a solid adsorbent containing a suitable binder is spread evenly on a glass plate, a drop of solution to be analyzed is placed near one edge and the plate is placed in a container with the edge of the plate below the spot, dipping into an eluting solvent. The solvent ascends the plate and the materials in the spot move upward at different rates, as on a Tswett column. Various detecting means are used - simple visual observation for colored compounds, differential fluorescence under ultraviolet light, and spraying of the plate with substances that will give colored materials with the compounds present. In favorable cases, this form of liquid-solid chromatography can be carried out with submicrogram quantities of materials.

Figure 9-3: A simple chromatographic column for liquid-solid chromatography

An extremely important improvement on the Tswett procedure is high-pressure solid-liquid chromatography (HPLC). Increasing the input pressure on the system to 20-70atm improves the speed of separations by permitting the use of much smaller solid particles (with more surface area) than would be practical for gravity-flow Tswett columns. Automatic monitoring of the column effluent by ultraviolet spectroscopy ) or by changes in the refractive index usually provides an effective means of determining how the separation is proceeding. With such techniques chromatograms similar to Figure 9-2 are obtained. High-pressure liquid chromatography (hplc) has great advantages for analysis and separation of high-molecular-weight heat-sensitive compounds that are unsuitable for glc.

An ingenious variation of solid-liquid chromatography is to use a solid support to which material is attached that has a specific affinity for a particular substance to be separated. The technique is especially useful for separating enzymes, and the immobile phase can be constructed from compounds known to react with, or be complexed by, the enzyme.

Observation of a single peak in a given chromatographic procedure is evidence, albeit not definitive evidence, for purity. Contaminants with nearly the same properties may be very difficult to separate and, if knowing the degree of purity is highly important, one can run chromatograms with a variety of different adsorbents to see if each gives the same result. If they do, the presumption of purity improves, although it is desirable to determine whether the spectroscopic techniques to be described in the following section permit the same conclusion.