Basics of Supercritical Fluid Chromatography

Welcome Back!

Welcome back for this third session on a brief introduction into supercritical fluid chromatography. Hopefully you are now aware of what a supercritical fluid is and how an SFC instrument needs to be adapted in comparison to an LC to work efficiently. The operating parameters can highly influence the success of the chromatographic separation, therefore, it is important to know which variables need to be investigated. In this section, we will look at the effect of stationary phase type.  

The approach for the SFC analytical condition development process is basically the same as for determining analytical conditions for HPLC. HPLC can generally target any component in compounds that can be dissolved in the mobile phase. In contrast, SFC can be used to analyse any compound that is compatible with supercritical carbon dioxide and can be dissolved in an organic solvent. If chromatography is used for quantitative analysis, then optimal analytical conditions should achieve resolution of 1.5 or more. The basic equation for calculating resolution R_s is indicated below

 

The α, k, and N values that affect R_s are all independent factors that vary depending on the column and mobile phase used, where N represents separation efficiency and α selectivity. In particular, separation can be improved by increasing N and α values. The supercritical carbon dioxide used for SFC offers different properties than mobile phase solvents used for HPLC, therefore when a column used for HPLC is used for SFC, it can sometimes result in different elution behaviour (Fig. 1).

Fig. 1 Separation of three types of pharmaceutical ingredients (upper: HPLC, Lower: SFC)

 

SFC Stationary Phases

The mobile phase is one of the most influential parameters in resolving compounds of interest. While pure supercritical CO₂ is effective at separating non-polar analytes such as fats, triglycerides, esters / ethers and silicones / siloxanes, it is inadequate at separating other more polar compounds, particularly in achiral separations. Historically, the solvating power was adjusted by increasing the supercritical fluid density through higher, thereby reducing retention, however, this approach has a limited impact. A pressure gradient with pure supercritical CO₂ can effectively adjust retention times by exploiting changes in the compressibility of the mobile phase.¹ The most typical way to adjust solvation power in modern laboratories is to include a polar organic modifier such as methanol (typically up to 40% v/v) to adjust selectivity.

When determining the mobile phase for HPLC, factors such as the acid/base level, the addition of ion-pair reagents or other modifiers, and other factors that help separation must be considered. For SFC, separation is affected by the type and amount of modifier, the acid or other additives present in the modifier, as well as the pressure and temperature required to keep the CO₂ in a supercritical state.

Due to the non-polarity of supercritical carbon dioxide, a modifier such as methanol, ethanol, or acetonitrile can be added to provide polarity to the mobile phase (Fig. 1). Increasing the modifier quantity could break down the supercritical fluid state of the CO₂ by changing the conditions required to make CO₂ supercritical. This could potentially change it to a subcritical or liquid state. It is advisable to maintain good chromatography to keep the mobile phase in a single state.

Fig. 1 Polarity range of mobile phases on a range of compound classifications

In HPLC, reversed phase and normal phase chromatography exhibit opposing solvent–analyte interactions, which influence the elution power of the mobile phase. In SFC, however, supercritical CO₂ consistently has low elution strength, while the modifier provides high elution capability in both reversed phase and normal phase modes. Table 1 lists common solvents used as modifiers, and Fig. 2 compares chromatograms obtained with different modifiers.

Fig. 2 Comparison of modifiers

Table 1 Examples of solvents used as modifiers  

• Organic Solvent	LogPOW
  Hexane	3.9
  Toluene	2.69
  Tetrahydrofuran	0.47
  1-propanol	0.33
  2-propanol	0.05

• Organic Solvent	LogPOW
  Acetone	-0.24
  Dioxane	-0.27
  Ethanol	-0.32
  Acetonitrile	-0.34
  Methanol	-0.82

Preparative LC is a technique used to purify samples for specific target components. It is used in a wide range of fields, including chemicals, pharmaceuticals, and foods. Preparative LC serves as a powerful tool for achieving higher purity and recovery rate levels of target components, but it requires drying and powderising steps.

SFC can improve preparative workflow efficiency by significantly reducing the amount of work involved in the powderisation process after preparative purification. The Nexera UC product line includes three systems—a stacked fraction system intended for large-volume fractionation, a multi-fraction system for separating multiple peaks, and an analytical fraction system intended for analysis-scale fractionation.

These products also feature Shimadzu’s unique gas-liquid separator (patented) that inhibits sample dispersion and carryover to obtain high recovery rates. Therefore, excellent recovery rates can be achieved even for highly volatile compounds, such as the fragrance linalool, regardless of the flow rate or modifier concentration. The following sections describe the three product lines and the gas-liquid separator.