Basics of Supercritical Fluid Chromatography

Be careful to discharge exhaust gases into a fume hood to prevent CO₂ concentrations from becoming too high within the laboratory. Installing a CO₂ meter is also recommended for the purpose of detecting CO₂ gas leaks within the room. 

As long as the instruments are used within the manufacturer’s recommended conditions, controlling the room temperature is probably not a significant concern because temperature is controlled in the column oven, but gas cylinders are affected by the ambient temperature where they are installed so care should be given to that temperature.

Yes, if the current Prominence unit is operated in combination with a modifier pump. For more details, contact Shimadzu.

Cylinders of 99.9 % CO₂ gas that are typically sold are more than acceptable for use. However, quality can vary so be sure to contact the gas vendor to confirm detailed specifications. A higher quality is required for MS applications.

Assuming ten minutes per analysis and pumping 100 % CO₂ at a flow rate of 3 mL/min, about 30 g is used per analysis. Use that value to recalculate usage based on appropriate changes to the analysis time, modifier ratio (which reduces the proportion of CO₂), and flow rate

In general, samples must be dissolved in a solution before injection, just like for HPLC. However, by connecting it to a supercritical fluid extraction (SFE) system, even components in solid samples can be analysed.

Due to the polarity of supercritical carbon dioxide, the ideal mobile phase should be n-hexane or a mixture of n-heptane and 2-propanol, but typically samples are dissolved for use in 100 % methanol or another modifier.

Yes, they can. However, for larger injection volumes, the sample solvent could affect peak shape or retention times. 

Supercritical carbon dioxide is known to cause slight acidity in mobile phases, but there is minimal risk of decomposing or otherwise altering compounds.  

We are not aware of any examples of analysing blood by direct injection. However, there are examples of analysing blood soaked in filter paper by SFE-SFC and examples of directly injecting urine samples for SFC-MS analysis.^2-4

SFC analysis of amino acids, soluble vitamins, polyphenols, and other highly polar compounds is possible by increasing the modifier concentration and adding an acid. However, the resulting peak shapes are not as good as when using HPLC. For preparative applications, SFC offers the advantage of not requiring drying or purification steps after fractionation. 

A non-protic solvent is often used in cases where SFC is useful for analysing samples that decompose or change properties when exposed to moisture. Candidate non-protic solvents include acetonitrile and THF.

Yes, waste liquids include the solvent containing dissolved measurement target substances, modifiers, and make-up solutions. However, liquid waste bottles will also contain carbon dioxide gas and some solvent consumption occurs due to evaporation.

There is a report of analysis by using TFA as a method for improving peak shape in peptide analysis by SFC.⁵.

SFC columns are generally compatible with fittings used in Shimadzu HPLC systems so it is possible to connect them. However, confirm the pressure capacity and other requirements for actual use with respective column manufacturers.

Yes, they can. However, it requires particular care regarding purging column liquids after analyses are finished. After using the column for SFC analysis, reduce the pressure inside the column to vaporise any CO₂ in the column or purge it with methanol or ethanol before disconnecting the column. Disconnecting the column with only supercritical carbon dioxide dries out the packing material due to evaporation of liquid in the column. Then if that column is used for HPLC, the high viscosity of the solvent used for the LC mobile phase, such as methanol or water, will cause surface tension around pores in the stationary phase that prevents penetration into the pores and interaction with functional groups. Because the supercritical fluid used for SFC analysis has lower viscosity than the mobile phase solvents used for HPLC, they can penetrate inside pores and interact with functional groups without being affected by surface tension, even if not filled with solvent. If a column used for HPLC analysis is used for SFC, any water in the column must be thoroughly purged with methanol or ethanol, because the water will not mix with the supercritical carbon dioxide.

Due to the lower viscosity of supercritical fluid, it applies less force on the packing material. In other words, it applies less back pressure on the column, which tends to result in longer column life than for HPLC.

For SFC, the same mobile phase parameters can be used for everything from normal phase columns to reversed phase columns. Therefore, we recommend first fixing the mobile phase parameters and then checking separation using multiple columns. Given the multiple columns, solvent delivery parameters, and separation parameters such as temperature and pressure, there is a huge number of possible parameter combinations. To reduce the amount of work involved in considering separation parameters, you are recommended to use a method scouting system because it automatically sets and executes respective parameter combinations.

Repeatability comparisons for HPLC and SFC using identical conditions have resulted in comparable repeatability and quantitative accuracy levels.

Ionisation can be inhibited by the matrix even for SFC-MS. However, since LC and SFC can result in significantly different separation behaviour, sometimes SFC-MS can resolve such LC-MS matrix effect problems.  

Yes, as long as the non-volatile salts are not dissolved by the extraction solvent. However, beware of contamination and precipitation if using a mass spectrometer for detection