Supercritical fluid chromatography (SFC)

Supercritical fluid chromatography abbreviated as SFC is considered an environment-friendly chromatographic technique. SFC is particularly valuable for the separation and analysis of temperature-sensitive analytical components. The distinguishing feature of supercritical fluid chromatography is as its name implies, the application of supercritical fluid in it. What is a supercritical fluid, what is the working principle of SFC, and how is it a green chromatographic protocol, all this and other relevant information will be discussed in this article.

What is a supercritical fluid

A supercritical fluid is a substance that exists at an intermediate stage between a gas and a liquid.  The supercritical fluid possesses both liquid as well as gaseous characteristics. It has the density and viscosity of a liquid, but it flows like a gas. The critical temperature is the temperature at which a gas cannot be converted to a liquid without applying some extra pressure. If a liquid or gas is used above its critical temperature then it is known as a supercritical fluid.

When a substance is heated to its critical temperature, keeping the pressure constant in a closed container, a liquid expands and converts into a gas. The vapor pressure increases until a saturation point is achieved.

At a particular point, the liquid and gas molecules achieve a dynamic equilibrium. At this equilibrium stage, the liquid and gas molecules cease to exist.

This stage is called the supercritical stage and the substance is known as a supercritical fluid.

What is supercritical fluid chromatography

Supercritical fluid chromatography is a type of normal-phase column chromatography. It is a large-scale chromatographic separation technique. It uses the supercritical fluid as a mobile phase against a solid stationary phase packed into a column. The supercritical mobile phase allows a faster chromatographic separation. The working principle of SFC is a combination of HPLC with gas chromatography (GC).

Historical perspective of supercritical fluid chromatography

Supercritical fluid chromatography was proposed back in 1958 by James Ephraim Lovelock, a well-known British scientist, and environmentalist. The early development of SFC  continued to build up pace in the 1960s and the 1970s. The first successful supercritical fluid chromatography however was performed in 1982.

Working principle of supercritical fluid chromatography

Supercritical fluid chromatography employs a high-pressure stream of supercritical fluid. The complex analyte mixture is introduced into this fast-moving mobile phase stream. The stream carries the mixture into the analytical column. Individual analyte components interact specifically with the stationary phase packed in the column. Those with high affinity towards the supercritical fluid readily pass out of the column while the others get retained onto the stationary phase. In this way, the required analyte separation takes place.

The mobile phase in supercritical fluid chromatography

A supercritical fluid is used as the main mobile phase in SFC. Carbon dioxide (CO₂) has a low critical temperature (31.3° C) and pressure (72.9 atm) thus it is the most practically usable supercritical fluid. Other than CO₂, research is also in progress on the use of nitrous oxide (N₂O), ammonia (NH₃), water (H₂O), etc., as supercritical fluids.

H₂O for instance attains a supercritical state at a much higher critical temperature (374°C) and pressure (217.7 atm), both quite difficult to achieve which makes the overall process uneconomical. Thus, it is not frequently utilized as a supercritical fluid in chromatography and CO₂ is undoubtedly the best supercritical fluid choice in SFC, so far.

The stationary phase in supercritical fluid chromatography

Supercritical fluid chromatography is often performed in the normal-phase mode of liquid chromatography. Polar stationary phase materials such as silica or alumina are usually used to develop a normal phase in the chromatographic column. Polysiloxane, cyanopropyl polysiloxane, and/or polymethyl siloxane can also be used as polar stationary phases for selective analyte retention in SFC, depending upon the functional groups present.

How to perform supercritical fluid chromatography

SFC instrument setup and operation are similar to high-performance liquid chromatography (HPLC).

Below is a step-by-step guide that introduces you to all the stages and components of supercritical fluid chromatography.

Step I: Column packing

Both packed as well as capillary columns can be used in supercritical fluid chromatography. Packed columns have an internal diameter of about 10 µm while the SFC capillary columns have an internal diameter of 50 µm. Column length can range from 10 to 20 m. The SFC column is situated in a thermostatic oven to maintain the precise temperature necessary for the supercritical mobile phase.

Step II: Mobile phase flow

99.99 %  pure CO₂ gas is directly introduced into the SFC system and converted into supercritical CO₂ using special temperature and pressure conditions.

Step III: Pressure pumping

Pumps are important to maintain constant high pressure while controlling the mobile phase flow rate inside the column. The mobile phase flow rate in SFC for small-scale analysis is maintained at 20 mL/min while that for large-scale purification, the flow rate is maintained as high as 200 mL/min. Syringe pumps are used in SFC. Additionally, a restrictor is used for back pressure regulation inside the column.

Step IV: Sample injection

Depending upon the internal diameter of the column, a precise amount of sample mixture is injected into the column. Three types of sample injectors are commonly employed:

• Loop injector for injection and transportation of sample components to the analytical column.
• Inline injector for introducing the sample mixture in the supercritical stream.
• In-column injector for injecting the sample mixture directly onto the SFC column.

Step V: Analytical separation

Strongly polar analyte molecules from the sample mixture get retained onto the stationary phase packed in the SFC column through different forces of attraction. On the other hand, non-polar molecules pass out of the column with the non-polar supercritical CO₂.

Step VI: Elution

Polar solvents (ethanol, methanol, isopropanol, acetonitrile, chloroform, etc.) mixed with non-polar supercritical fluids in differing ratios are used while performing elution in supercritical fluid chromatography. A small proportion of this co-solvent is used with a large amount of supercritical fluid in gradient mode.

Supercritical CO₂ offers the same polarity as a non-polar solvent such as n-hexane, but its density can be increased by adding polar solvents. Extremely non-polar analytes pass out of the column with the supercritical mobile phase followed by polar components in increasing order of polarity.

Step VII: Detection

Different types of detectors can be coupled to an SFC column. Particularly useful are the ultraviolet (UV) detector, flame ionization detector (FID), electron-capture detector, and mass spectrometric (MS) detector. The detector sends a signal to the recorder which then plots the chromatogram.

Strengths of supercritical fluid chromatography

• High diffusivity (5 to 50 times higher than liquids) of supercritical fluids allows faster separation and thus a lower analysis time. A chromatographic separation via SFC requires 1/10^th of the separation time required in HPLC.
• Rapid diffusion produces fine, narrow peaks thus high chromatographic resolution.
• SFC offers superior speed and safety for a wide range of applications in the chromatographic world.
• The use of supercritical fluid reduces the need for toxic organic solvents thus supercritical fluid chromatography is largely a green/environment-friendly chromatographic protocol.
• Less solvent consumption also reduces the analysis cost. Conversely, CO₂ is readily available and a cheap gas which makes SFC a relatively inexpensive technique as opposed to other costly chromatographic techniques like UHPLC.

Limitations of supercritical fluid chromatography

• Difficulty in regulating back pressure in the column. Supercritical fluids are highly compressible so even slight pressure changes may lead to a change in the physicochemical properties of the mobile phase.
• Product separation is difficult in SFC.
• Rapid CO₂ depressurization for removing it from the eluate may lead to product dissolution.

Applications of supercritical fluid chromatography

• SFC is particularly useful for the analysis of non-volatile, thermo-sensitive analytical components which are otherwise difficult to separate through GC. These include a wide range of polymer additives.
• SFC can be interfaced with other techniques such as supercritical fluid extraction to detect trace quantities of additives.
• The flexibility of supercritical fluid chromatography allows its coupling with spectrophotometric analytical techniques such as FTIR or NMR. This aids in elucidating the chemical structure of analytical components, especially polymers, in addition to their separation and detection.
• Supercritical fluid chromatography also finds useful applications in the bio-clinical field. It can be employed at the preparative stage during analyses of nucleic acids (DNA and RNA), polar amino acids, and short peptides.
• It can be used for chiral separation and purification of drugs in the pharmaceutical sector.

Conclusion

In conclusion, we would like to emphasize that supercritical fluid chromatography is a versatile analytical technique. Although it is not that new, but scientists have begun to recognize and highlight its benefits only recently. Superior speed, higher safety, and reduced toxic solvent consumption are some of the salient features diverting the attention of the scientific world towards supercritical fluid chromatography (SFC) since environmental protection became a priority.

A specific SFC analysis protocol follows the same rules as that used for HPLC method development and validation.

You may also like to read about another useful pressure-driven chromatographic technique that we discussed in another article, fast protein liquid chromatography (FPLC).

References

1. Berger, T. A. (2007). CHROMATOGRAPHY: SUPERCRITICAL FLUID | Theory of Supercritical Fluid Chromatography. Encyclopedia of Separation Science. I. D. Wilson. Oxford, Academic Press: 1-9.

2. Hofstetter, R. K., M. Hasan, C. Eckert and A. Link (2019). “Supercritical fluid chromatography.” ChemTexts 5(3): 13.

3. Jusforgues, P. and M. Shaimi (2000). CHROMATOGRAPHY: SUPERCRITICAL FLUID | Large-Scale Supercritical Fluid Chromatography. Encyclopedia of Separation Science. I. D. Wilson. Oxford, Academic Press: 809-819.

4. Savale, S. (2018). Supercritical Fluid Chromatography [SFC].