Having previously discussed the similarities and differences between HPLC and SFC, I thought I would dig deeper into what is possible with modern SFC. Many developments have been made over the years since SFC’s inception that many chemists are unaware of – especially those, like myself, who come from an HPLC background. Modern instruments and methods are pushing the boundaries of what was previously thought possible with SFC, a technique synonymous with chiral compounds. Today, almost all the applications associated with HPLC can be performed with SFC, though not in the same way, so if you want to get hands-on and find out more about what is possible with SFC and how then read on!

My family recently went to visit some friends who had twins, and I found out that they were ‘mirror twins’. I had not come across this term before and was instantly fascinated. Mirror twins are a subset of twins that develop when the fertilized egg splits later in the embryonic stage. These twins exhibit mirrored physical characteristics. In the case of my friend’s children, they have opposite dominant hands (one is right-handed, the other left), their hair whorls swirl in opposite directions, and they have birthmarks on opposite sides of their bodies. This mirroring, although not the same phenomenon, reminded me of chirality in chemistry, a subject I also find fascinating. The word chiral comes from the Greek word for hand and refers to objects that cannot be superimposed on their mirror images – just like our hands. The thing that I find fascinating is that chiral pairs can act very differently. Chirality is often described as ‘handedness’, with objects displaying either left or right-handedness, a phenomenon known as homochirality. Most amino acids (except for glycine) are chiral and are of the L-configuration (left-handed), whereas sugars in DNA and RNA are predominantly of the D-configuration (right-handed).

In biochemistry, the spatial arrangement of atoms can significantly impact their function, as was proven by the cautionary tale of Thalidomide, a drug administered to alleviate morning sickness. While the R-enantiomer had the desired therapeutic effect, the S-enantiomer caused severe birth defects. The original drug was a racemic mixture containing an equal amount of left- and right-handed enantiomers. This highlights the importance of chromatography to be able to separate chiral compounds efficiently so that the pharmaceutical industry can develop enantiomerically pure drugs.

When it comes to chiral compounds, SFC has long been the method of choice due to its efficiency and versatility. SFC offers a higher resolution for chiral separations than HPLC, which is essential for obtaining pure enantiomers. Chiral compounds are notoriously difficult to separate, just as it can be hard to distinguish between twins. Enantiomers have similar physical and chemical properties, such as melting points, boiling points, solubilities, and densities. It takes a method with a high degree of flexibility to distinguish and separate chiral compounds, and here SFC shines. For a mobile phase to be effective, it must be able to solubilize the sample and transport the target compound through the stationary phase at just the right speed. The high solvation and diffusivity, and low viscosity of supercritical CO₂, used in SFC can be finely tuned by adjusting pressure and temperature or adding a modifier. This high level of control is key to separating tricky chiral compounds.

So, what about achiral and polar compounds? Due to the nonpolar nature of CO₂, SFC was traditionally not deemed an effective technique for separating polar compounds; however, extensive research has been performed using different modifiers that increase the polarity of the mobile phase. Even highly polar compounds can now be separated at high resolution with the use of modifiers; however, this sometimes results in a sub-critical rather than supercritical fluid. Polar modifiers are generally organic solvents that are completely miscible in carbon dioxide, such as alcohols or cyclic ethers, but can be any liquid, even water. As a rule, if something is soluble in methanol or a less polar solvent, it is a good candidate for SFC.

When approaching SFC from a background in flash or HPLC, transitioning from normal-phase chromatography is a simple switch as the CO₂ mobile phase is also non-polar. When transitioning from reverse-phase chromatography, column screening is usually required, and the column with the best selectivity will have to be determined. The optimal stationary phase should provide a balance between retention and selectivity while minimizing peak broadening and tailing. Method development involves sample screening and evaluation before optimizing the run conditions, including the type and amount of modifier; then, the pressure and temperature need to be optimized, as well as separation parameters, such as the number of stacked injections or whether a 1 or 2 shot separation is required.

With the correct screening and method development, almost all compounds can be separated with SFC, though there are a few exceptions that may become easier and more viable in time. Currently, proteins are still purified by prep HPLC due to the risk of denaturation and their poor solubility in CO₂. Water-soluble vitamins and lipids are also mainly purified with prep HPLC based on their mobile phase. Apart from these few exceptions, all other compound classes are viable with SFC, including peptides, fat-soluble vitamins, natural products/ extracts, carbohydrates, small molecule drugs, and the list goes on…In fact, most things can be achieved by getting hands-on with SFC, and the benefits offered make all the effort worthwhile as it is likely you can achieve a faster, higher resolution separation that is more environmentally friendly than traditional liquid chromatography methods.

Till next time,