Basics of Supercritical Fluid Chromatography

Analytical Advantages of SFC

The supercritical carbon dioxide used as a mobile phase for SFC offers unique advantages. Five of these advantages are described below.

1. (1) Low polarity
2. The polarity of supercritical carbon dioxide is similar to n-hexane. Normal phase chromatography is a separation method that uses a solvent with low polarity, such as n-hexane, as the mobile phase, and a substance with high polarity, such as silica, as the stationary phase. One advantage of normal phase chromatography is it offers better structure recognition ability than reversed phase chromatography. SFC uses supercritical carbon dioxide, which has similar properties as n-hexane, and also offers excellent structural recognition similar to normal phase chromatography (Fig. 4). By using a column designed for chiral chromatography, which is packed with a stationary phase with optically active functional groups, SFC is suitable for separation of chiral compounds.
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   Fig. 4 Separating vitamins D2 and D3 (UC-Pye column)

    

2. (2) Miscibility with Other Organic Solvents
3. Supercritical carbon dioxide can be mixed with certain organic solvents, such as methanol, ethanol, isopropanol (IPA), acetonitrile, and tetrahydrofuran (THF). In contrast, n-hexane, which offers low polarity similar to supercritical carbon dioxide, cannot be mixed with methanol and similar solvents, due to its relatively high polarity. By mixing the supercritical carbon dioxide with such modifiers, SFC can achieve a wide variety of separation patterns. This allows comprehensive analysis for a wide range of low-to-high polarity compounds not previously possible with HPLC. For example, earlier GC was required for analysing fatty acids and HPLC for glycerides. With SFC, both fatty acids and glycerides can now be analysed simultaneously due to the hexane-like properties of the supercritical carbon dioxide in combination with a column designed for reversed phase chromatography (Fig. 5).
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   Fig. 5 Example of simultaneously analysing fatty acids and triglycerides by SFC

3. (3) Low Viscosity
4. Supercritical carbon dioxide has lower viscosity than solvents such as water, and a high diffusion coefficient. That results in lower column back pressure, assuming the column has the same internal diameter and length, is packed with the same size particles, and supercritical carbon dioxide is pumped through the column at the same rate. That enables analysis with a high linear velocity, which can speed up analysis times. Fig. 6 shows a comparison of linear velocity and column pressure plots for HPLC and SFC.
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   Fig. 6 Linear velocity vs column back pressure

   Though higher linear velocity can shorten HPLC analysis time, it results in increased column pressure. The upper range of column pressure is limited by instrument design and column capability. CO₂ in the supercritical state has lower viscosity than water and other organic solvents typically used for HPLC. That means shorter analysis times are possible for SFC than HPLC without loss of resolution and change in selectivity.

   Supercritical carbon dioxide also has a higher coefficient of diffusion than solvents used for LC. The diffusion coefficient contributes to determining how quickly compounds reach equilibrium between stationary and mobile phases during chromatography and affects resolution. The van Deemter equation below indicates the relationship between physical parameters that determine the height equivalent to a theoretical plate (HETP) value, which is used to indicate column resolution.

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8. Plots of the van Deemter equation for HPLC and SFC are shown in Fig. 7.² In the formula above, Term A is based on the multi-flow diffusion generated by the packing material in the column and is proportional to the packing material particle size.

   Term B is determined by the molecular diffusion in the axial direction of the column. This diffusion phenomenon occurs while compounds stagnate in the stationary or mobile phases and is inversely proportional to linear velocity, regardless of particle size.

   Term C is derived from how easily compounds can penetrate pores in the packing material and the mass transfer diffusion that contributes to linear velocity. The mass transfer diffusion value is proportional to linear velocity and the square of the particle diameter, which results in higher HETP values for small particles with low linear velocities. In the case of HPLC, mobile phases with a high linear velocity will result in larger mass transfer diffusion values, but supercritical carbon dioxide has a higher diffusion coefficient than HPLC solvents and can penetrate packing material pores more easily. That results in lower mass transfer diffusion values even for high linear velocities.

9. That makes SFC an analytical technique that can achieve ultra-fast analysis without sacrificing resolution even at high linear velocities.

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    Fig. 7 van Deemter plot comparison for LC and SFC

    These plots indicate the regions where the optimal linear velocity for SFC is higher than for HPLC. An example comparing the analysis of tocopherols by HPLC and SFC is shown in Fig. 8.³

Fig. 8 Comparison example of analysing tocopherols by HPLC and SFC

1. (4) Vaporisation
2. Supercritical carbon dioxide evaporates at a temperature and pressure lower than the critical point. Under certain temperature and pressure conditions, CO₂ will become a solvent, which is used as a mobile phase for SFC. If an MS is used as a detector, then a back pressure regulator is installed just before it so that only the modifier or make-up solvent reaches the MS. Consequently, unlike conventional LC-MS analysis, this reduces the diluting effects of the mobile phase, potentially increasing the sensitivity while using SFC-MS. This can be seen in the example below where 400 components were analysed with 90% of these showing higher sensitivity with SFC-MS (Fig. 9). Fig. 10 shows a comparison of results from using LC-MS and SFC-MS to measure samples prepared to 10 ppb.

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   Fig. 9 Area comparison for LC-MS and SFC-MS

   

   Fig. 10 Comparison of mass chromatograms of standard pesticide mixture solution measured by LC-MS and SFC-MS

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5. (5) Solvent Cost
6. The similar polarity of supercritical CO₂ and n-hexane makes the former appropriate to be used for normal phase separations. Normal-phase chromatography consumes large amounts of solvent. However, because SFC consumes less solvent and involves treating less liquid waste, the overall solvent cost is lower than for HPLC (Fig. 11)
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   Fig. 11 Comparison of analytical costs and solvent consumption quantities for HPLC and SFC

8. (6) Wide Polarity Range for Samples
9. During the infancy of SFC, applications were typically aimed at apolar, fatty compounds. However, the addition of organic modifiers such as methanol to the mobile phase has expanded the polarity range available for SFC, thereby covering the normal phase and reversed phase range of compounds. Gradients are typically run from 5-40% modifier. This has been further expanded in recent years to include more polar compounds in the typical HILIC range. This expansion is possible with the addition of water (1-10%) in the organic modifier, and the gradient range extended to 100% organic modifier. This would eliminate the need for several methods to analyse a broad range of analytes in a sample.
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    Fig. 12 Polarity range covered by different LC techniques compared to UHPSFC and water rich UHPSFC/UC. Image adapted from Reference 4

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12. (7) Lower Backpressure
13. The lower viscosity of the mobile phase produces a lower backpressure. This allows for  small particle sizes not conventionally employed in HPLC but can be used for SFC methods. For UHPLC applications, 1.8 μm particle morphologies can be used but high backpressures can be expected (>850 bar). These same size particles would have a substantially lower backpressure under SFC conditions. It is also feasible that smaller particle sizes such as 1.3 μm could be used to maximise efficiency in SFC.