Supercritical fluid chromatography (SFC) is a green, environmentally friendly, and efficient column chromatography technique developed to expand the application range of high-performance liquid chromatography (HPLC). CO₂ is the most commonly used mobile phase, which can be mixed with organic solvents of different polarities. The characteristic of mobile phase compatibility determines the diversity of stationary phase types. Almost all stationary phases used in liquid chromatography can be applied in SFC, effectively broadening the polarity range of analytes and enabling SFC to play an important role in multiple fields such as pharmaceuticals, food, environment, and natural products. ### Supercritical Fluid Chromatography Supercritical fluid chromatography is a chromatographic technique that uses fluids above the critical temperature and critical pressure as the mobile phase for analysis. SFC employs supercritical fluids as the mobile phase, whose relative kinetic properties are between those of gases and liquids. Its diffusion coefficient and viscosity coefficient are similar to those of gases, and its density is similar to that of liquids. Therefore, SFC combines the advantages of high solubility and high diffusivity. As shown in Figure 1, from the perspective of separation mode, SFC is very similar to HPLC. The mobile phase plays an important role, not only in that analytes are directly dissolved in the mobile phase, but also in that the mobile phase competes with analytes on the surface of the stationary phase, thereby affecting the interaction between analytes and the stationary phase. However, compared with the liquid mobile phase used in HPLC, supercritical fluids have lower viscosity, faster diffusion, and smaller surface tension, so the separation speed of samples on SFC is faster. Supercritical fluids are highly compressible, and the density of the mobile phase has a great impact on the retention of analytes. Therefore, in SFC, column temperature and back pressure are important parameters for adjusting analyte retention. Increasing the back pressure will increase the density of the mobile phase, enhance its dissolving capacity, and shorten the retention time. Column temperature has a dual impact: on the one hand, increasing the temperature will reduce the density of the mobile phase, weaken its dissolving capacity, and prolong the retention time; on the other hand, increasing the temperature can increase the energy of analyte molecules, thus shortening the retention time. Although the retention of compounds can be changed by adjusting the solvent density, the polarity of the solvent cannot be sufficiently altered. At present, the most widely used supercritical fluid is CO₂. As can be seen from the phase diagram in Figure 2, its critical conditions are relatively mild (critical temperature Tc = 31℃, critical pressure Pc = 7.4 MPa). More importantly, CO₂ has good miscibility and can be mixed with strongly polar organic solvents (even trace amounts of water). This property truly improves the polarity range of SFC mobile phases, which can be extended to a wider range than normal-phase chromatography (NPLC) and reversed-phase chromatography (RPLC). In addition to the above unique properties, CO₂-based mobile phases are also compatible with various stationary phases. In HPLC, the classification of stationary phases is relatively clear according to different chromatographic modes. For example, silica gel columns are only suitable for NPLC, while C18 columns can only be used in RPLC. However, in SFC, the polarity range of chromatographic columns can gradually transition from C18 columns to silica gel columns. In addition, benefiting from the development of commercial SFC instruments, their stability, repeatability, and precision have been significantly improved. SFC has gradually become one of the mainstream column chromatography techniques and has shown better separation capabilities than HPLC in various separation scenarios. When using SFC to separate samples, it is first necessary to understand the properties of the analytes. It is generally believed that compounds soluble in common organic solvents can be analyzed by SFC. To scientifically and rigorously indicate the applicable range of analytes that can be applied to SFC, studies on the retention of different types of compounds in SFC have found that compounds with weak to moderate polarity are more suitable for SFC analysis. According to the polarity of the analytes and separation requirements, the most potential stationary phase is selected for subsequent experiments. To obtain appropriate retention time and ideal selectivity, it is generally necessary to add co-solvents and additives. Common co-solvents include methanol, ethanol, isopropanol, and acetonitrile. To improve selectivity, a mixture of two or more common co-solvents is also used. The acidity and basicity of analytes will affect the shape of chromatographic peaks. Generally speaking, when the analyte is acidic, formic acid, trifluoroacetic acid, etc., are selected as additives; when the analyte is basic, triethylamine, ammonia water, etc., are added to improve the peak shape. Sometimes, a small amount of water is used as an additive to increase the polarity of the mobile phase and improve the peak shape of the analytes. In experimental operations, parameters such as back pressure and column temperature can also be adjusted to achieve better separation results. ### Four Characteristics of Supercritical Fluids: ① Density and solvating power are close to those of liquids. ② The diffusion coefficient of supercritical fluids is between that of gases and liquids, and their viscosity is close to that of gases. ③ When the fluid state is close to the critical region, the heat of vaporization drops sharply. At the critical point, the gas-liquid interface disappears, the enthalpy of vaporization is zero, and the specific heat capacity becomes infinitely large. ④ A small change in pressure or temperature near the critical point of the fluid can lead to a significant change in the density of the supercritical fluid, thereby causing a significant change in the solubility of the solute in the fluid. This is the design basis of the supercritical extraction process. Source: Compiled from *Chromatography* and other online sources