Introduction

Supercritical fluid chromatography (SFC) is an instrumental technique in which a small volume of a sample is entrained in a solvating mobile phase, held near its critical point, and is swept through a stationary bed of particles, or a tube coated with a liquid film. The components of the mixture are separated from each other through differential solvation by the mobile phase, and retention by the stationary phase. SFC can be viewed as a transition technique between GC and HPLC.

A fluid is 'supercritical' when it is raised above its characteristic 'critical point' (a critical temperature, Tc, and a critical pressure, Pc). Most fluids used as SFC mobile phases are gases at room temperature and pressure but all are easily liquefied by raising the pressure. In a supercritical fluid, the molecules are forced close together by an externally applied pressure. Some but not all supercritical fluids act as a solvent. Changing the density of such fluids changes their solvent strength.

Packed vs. capillary columns There are two SFC

techniques, using either open tubular (capillary), or packed columns, each of which exploits a different aspect of the technology. Although in 1958 Lovelock originally proposed SFC as a capillary technique, for its first 20+ years SFC was exclusively performed with packed columns. The first commercial instrumentation for packed-column SFC was introduced in 1982 as a modification kit for a HPLC. Capillary SFC was not viable until the late 1970s when stationary phases were first bonded to the surface of the column. Earlier stationary phases were washed out of the column by the SFC mobile phase. In 1981, the groups of Lee at Brigham Young University and Novotny at Indiana University, combined bonded stationary phases with SFC and published the first realistic capillary (open tubular column) SFC chromatograms.

Subsequently, a rather unfortunate controversy raged for some years as to whether capillary or packed columns were 'better'. However, the two techniques are sufficiently different that almost any attempt to compare them will show one or the other unfavourably.

Open tubular or capillary SFC (CSFC) is almost always performed using pressure programming of pure carbon dioxide as the mobile phase. CSFC can be thought of as an extension of gas chromatography (GC) to higher molecular masses, since the solvation energy of the fluids can replace thermal energy. Elu-tion temperatures can be 200°C or more lower than in GC (i.e. carbon dioxide at 150°C vs. GC with hydrogen at 400°C). The column temperature is almost always maintained at >50°C above the critical temperature.

The fluid solvent strength can be changed in a predictable manner by programming the fluid density (usually pressure, sometimes temperature, rarely both). Carbon dioxide is compatible with the flame ionization detector (FID). The most common uses of CSFC are for the separation of light polymers, surfactants and other homologous series.

Carbon dioxide has a solvent strength similar to hexane or a fluorocarbon. Relatively nonpolar solutes such as hydrocarbons are miscible with carbon dioxide. Polar solutes such as sugars or amino acids are virtually insoluble in carbon dioxide.

Packed columns are, inherently, 10 to 100 times more retentive than capillary columns, due to their higher surface area to void volume ratio. This makes it more difficult to elute molecules from packed columns using pure carbon dioxide. On the other hand, the low polarity of carbon dioxide is incompatible with the solvation of polar molecules.

The most widely used approach to extending SFC to more polar solutes has involved the use of packed columns with binary or ternary mobile phases. Binary fluids consist of a normal liquid such as acetonitrile or methanol mixed with carbon dioxide. Ternary mobile phases consist of a fraction of a percent to several percent of a third, even more polar component, such as trifluoroacetic acid or isobutylamine, added to the liquid modifier. The use of modifiers was pioneered by Jentoff and Gouw in 1969. The use of additives was pioneered by Berger et al. starting in 1988.

Once a modifier is added to the mobile phase, the composition of the fluid tends to dominate over its density in determining solvent strength. Pressure programming becomes a secondary control variable. The enhanced solvent strength of modified fluids tends to be less compatible with the inherent low retentivity of capillary columns.

Packed-column SFC can be thought of as an extension of HPLC, using modifier concentration gradients of binary or ternary mixtures near (within 50°C above or below!) their critical temperature. Packed-column SFC is a form of normal-phase chromatogra-phy, complementing reversed-phase HPLC, but with superior speed, and efficiency. The low temperatures used make packed-column SFC ideal for separation of moderately polar, thermally labile molecules. The primary detection scheme with packed columns is UV absorption.

Packed-column and capillary SFC are two different techniques sharing part of a name. As with GC and HPLC there is clearly an overlap between the two. Packed columns are sometimes used with pure carbon dioxide, pressure programming and FID detection, to perform some of the applications more commonly performed by capillary SFC. Some relatively polar solutes are soluble enough in carbon dioxide to allow elution with capillaries and pure carbon dioxide.

Unified chromatography A further problem in defining these techniques is the fact that there are no real borders between GC, SFC and HPLC. One can start an experiment, above the mobile phase critical temperature (Tc) (i.e. as GC), then raise the pressure above the critical pressure (Pc) (to SFC), then drop the temperature below Tc (to LC). In the process the name of the technique changes from GC, to SFC, to HPLC, but there is never more than one phase present, and there is never a phase transition. Changes in solute retention, viscosity, diffusion coefficients, etc., are smooth and continuous, even when the definition of the fluid changes. In packed-column SFC, modifier concentration can be programmed from 0 to 100%. At some intermediate (but not obvious) concentration, the definition of the technique changes from SFC to HPLC. The characteristics of interest in SFC are summarized in Table 1. SFC has additional special advantages when scaled up for preparative use.

There has been some effort to promote SFC as a form of unified chromatography. SFC instrumentation can typically also perform HPLC, and at least

Table 1 Characteristics of interest in SFC

A compressible fluid that acts as a solvent

Solvent strength changes with a physical parameter (pressure)

Very wide range of solvent strength using one binary pair of solvents

Low viscosity long, high efficiency columns (i.e. >2 m with 5 ^m particles) low pressure drop at high flow with smaller particles Higher speed than HPLC - higher diffusion coefficients 3-10 x higher throughput much faster re-equilibration rapid method development Compatibility with GC detectors, like the FID, ECD, NPD Transparent in the UV

Complementary selectivity to reversed-phase HPLC Low cost, environmentally friendly solvents, easily removed

Figure 1 A summary of solute families which have been separated by SFC.

some GC, whereas instrumentation for the other techniques cannot perform much SFC.

Solutes Typical samples separated by packed column SFC consist of low to moderate polarity organic solutes with relative molecular masses ) 10 000 Da. As a 'rule of thumb', solutes that can be dissolved in methanol or a less polar solvent are good candidates for SFC. On the other hand, solutes requiring water or buffered or pH adjusted water environments are a poor choice. Solute families which have been separated by SFC are summarized in Figure 1. Low polarity solutes are to the left, higher polarity solutes to the right. At the bottom of the figure, there is an attempt to relate the nature of the mobile phase needed to elute the various solute families.

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