Chromatography is an instrumental procedure, used for separating and analyzing the chemical composition of chemical mixtures based upon physical adsorption principles, which is used widely for organic, chemical, biological and medical studies. Chromatography depends upon the selective retardation and separation of substances by a stationary bed of porous sorptive media (the substrate) as they are transported through a column containing the substrate by a moving transport fluid (which may be, for example, gas, liquid or supercritical fluid). The rate of migration of each substance being tested is determined by its relative partitioning affinity between the substrate and the particular transport medium at the particular flow rate of transport medium being used. A detector positioned at the end of the column generates an analog signal from which quantitative and qualitative information can be derived. A time record (or spectrum) of the detector signal is called a chromatogram, and is a signature of the composition being analyzed. This record contains lines, usually in the form of Gaussian peaks. The amplitudes and time spacing (distance) between peaks are characteristic of the substances being analyzed. The resolution of a chromatogram is determined by the distance separating the means of these peaks on the chromatogram as well as the spread of the peak about the peak mean. Since the chromatogram is a function of time, resolution, which is basically based upon the distance between peaks on the chromatogram, increases as the time needed to transport the sample through the column is increased. Consequently, there is a trade off between resolution and time. To obtain ultra high resolution, the chromatographic analyses must be slowed to a relatively slow speed. Conversely, when relatively lower resolution are sufficient, relatively faster analysis times may be employed. However, in many instances, ultra high resolution is not needed, in which case it is desirable to speed up the transport process, so that the analyte solute species will emerge from the column more quickly. This may be done by changing or altering certain properties of the transport fluid, such as, for example, density, in the case of supercritical fluid chromatography.
In the past, to obtain a certain resolution in the fastest time for a given combination of sample substrate and transporting fluid, etc., the alteration needed to be applied to the particular transport fluid could only be arrived at by trial and error methods. Supercritical fluids have the ability to change density markedly when the temperature or pressure is changed. Consequently, by manipulating the temperature and/or pressure in a supercritical fluid chromatography system, the relative affinity for the solutes partitioning between the solid phase and a supercritical fluid can be controlled. This inherently affects the resolution obtained and the transport time of analytes through the column. However, to arrive at the optimum pressure, temperature and density combination needed for a given supercritical fluid and material under analysis, a great deal of trial and error experimentation was still necessary, even for supercritical fluid chromatography. For example, see Retention and Resolution in Density-Programmed Supercritical Fluid Chromatography, by Wilsch and Schneider, J. Chromatography, Vol. 357, pp. 239-252 (1986).