Supercritical Fluid Chromatography (SFC)

Supercritical Fluid Chromatography is a powerful technique transforming analytical and preparative separations across multiple industries By harnessing the power of the supercritical fluid state, SFC delivers faster and more sustainable results.

Overview

What is Supercritical Fluid Chromatography?

Supercritical fluid chromatography (SFC) is an advanced separation technique that uses carbon dioxide (CO₂) above or near its critical state (temperature >31.1 °C and pressure >73.8 bar) as the primary mobile phase, often combined with a small percentage of organic modifiers such as methanol. Under these conditions, CO₂ exhibits both gas-like diffusivity and liquid-like solvating power, making it ideal for rapid and efficient separations.

This relatively non-polar mobile phase can be used with stationary phases commonly employed in normal-phase HPLC; however, SFC is a highly versatile technique that can also operate under conditions similar to reversed-phase or HILIC mode, depending on the choice of stationary phase, and mobile phase/modifier composition. Compared to conventional solvents, the low viscosity and high diffusivity of supercritical CO₂ enable much faster separations. SFC columns can be used in two main modes highlighting the unique flexibility of SFC in analytical separations:

Although still an emerging technique, supercritical fluid chromatography (SFC) is gaining traction in pharmaceutical, environmental, and food industries. It offers significant economic advantages through faster run times and lower solvent usage, and is recognized as a more sustainable, “green” alternative to traditional normal-phase liquid chromatography. The economic benefits of SFC are especially evident in preparative LC separations, where lower solvent consumption and shorter purification cycles reduce operational costs and enhance throughput.

By shortening analysis times while maintaining high resolution, SFC provides a sustainable and cost-effective alternative to traditional HPLC methods.

Explore Phenomenex Solutions for SFC

Phenomenex offers a comprehensive range of column technologies optimized for supercritical fluid chromatography.

What Can SFC Separate?

SFC is a highly versatile technique capable of separating a wide variety of analytes. It is particularly effective for compounds that are difficult to separate using traditional normal or reversed-phase HPLC. For example, chiral compounds, lipids, fatty acids, and other structurally similar molecules can be efficiently resolved using SFC columns. The ability to tune selectivity through changes in pressure, mobile phase additives, temperature, and modifier composition makes SFC highly adaptable for complex mixtures.

Pharmaceutical applications often rely on SFC columns to achieve high-resolution separations of chiral drug candidates, while environmental and food testing laboratories use the technique to analyze bioactive compounds, pesticides, and natural products. Compared to conventional methods, SFC offers reduced run times, lower solvent usage, and excellent peak shapes, making it a preferred choice for both preparative and analytical separations. In addition, its minimal use of organic solvents contributes to a lower environmental impact compared with conventional LC techniques. Whether using a single SFC column or advanced systems with multiple columns, the flexibility of this method supports diverse research and industrial needs.

Procedure of Supercritical Fluid Chromatography

SFC follows concepts like liquid and gas chromatography but with distinct advantages due to the use of a supercritical mobile phase.

  • Mobile phase: Supercritical CO₂ serves as the mobile phase, modified with small amounts of organic solvents (e.g., methanol, IPA or ethanol) to enhance solubility and selectivity.

  • Sample introduction: The mixture to be separated is introduced into the flow of the supercritical fluid.

  • Stationary phase: The mobile phase, carrying the analytes, passes over the stationary phase—commonly silica gel packed in SFC columns.

  • Separation mechanism:

    • Constituents partition differently between the mobile and stationary phases based on their physical and chemical properties.
    • Analytes with greater affinity for the mobile phase move faster, while those favoring the stationary phase travel slower.
    • This difference in partitioning leads to effective separation according to partition coefficients.
  • Control parameters: Pressure, temperature, mobile phase additives and modifier percentage are carefully optimized to achieve the desired resolution.

  • Detection methods: Commonly coupled with UV, mass spectrometry (MS), or flame ionization detectors (FID) for precise qualitative and quantitative analysis.

  • Sample recovery: Following SFC preparative separations, the carbon dioxide is vaporized into gas form allowing for easy collection of purified compounds.

FAQs

To safely transfer a Lux column from normal phase to polar organic or reverse phase conditions, flush the column with methanol:ethanol 9:1 (V/V) as transition solvent at a flow rate of 0.5 mL/min. First, flush the column with at least ten column volumes (i.e. 25 mL for a 250 x 4.6 mm i.d. column or 15 mL for a 150 x4.6 mm i.d. column) to completely remove the initial mobile phase. When the column has been flushed, equilibrate the column with at least ten column volumes of the polar organic or reverse phase solvent mixture to condition the column. In addition, when the buffer salt additive of the reverse phase mobile phase is insoluble in methanol/ethanol, flush the column briefly with water before switching to the buffered mobile phase. When the column has been flushed equilibrate the column with at least ten column volumes with the reverse phase solvent mixture. To safely transfer a column from polar organic to normal phase conditions flush the column with at least ten column volumes of methanol:ethanol 9:1 (V/V) as transition solvent at a flow rate of 0.5 mL/min. When the column has been flushed with methanol/ethanol equilibrate the column with at least ten column volume of the normal phase solvent mixture to condition the column. We do not recommend switching from reverse phase mode back to normal phase mode.
While both form helical structures that are maintained in coating, the amylose can be described as more tightly coiled. The looser cellulose helical structure may more readily accommodate enantiomer interaction. Practically, this results in different selectivity even if the same chiral selector is substituted, as the 3-dimensional orientation around the CSP will differ.
To switch a Lux column from SFC back to LC mode you need to run a gradient up to 100% of the co-solvent which was previously used in SFC mode. Once the column is in 100% co-solvent you can introduce the polar organic solvents to it. Particular attention should be paid to the back pressure when introducing solvents again into the Lux column to ensure you exceed the maximum pressure stability of the column
Make up a 1 Liter solution of 5 mM Ammonium Bicarbonate and mix well. Transfer to a larger flask or beaker and follow the procedure. The solution will easily get to pH 9 with a small amount of concentrated Ammonium Hydroxide. In order to get to pH 11 you should expect to add an amount of concentrated Ammonium Hydroxide around 5-10% of the total volume of your solution. For example if you were to make a 1 Liter solution of 5 mM Ammonium Bicarbonate then expect to add as much as 50-100 mL of concentrated Ammonium Hydroxide.