SFC Technology for Preparative Chiral Separation

SFC Technology for Preparative Chiral Separation

The famous “Thalidomide Incident” in the 1960’s, which lead to the birth of about 12,000 defected babies, has raised worldwide attention to the importance of identification and separation for chiral drugs. This incident further urged FDA to impose strict requirements on submitting chirality research data for new drug application. Such data include the separation method, efficacy, toxicity and side effects of a chiral drug for both the pure enantiomer and racemic form. Because of the strong influence of chirality on drug efficacy and safety, an efficient and effective separation of the chiral structures is inevitably necessary throughout the drug development process. 

Among various chiral separation techniques, chromatographic approach is by far the predominant one. Chromatographic chiral separation method includes thin layer chromatography (TLC), gas chromatography (GC), high performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Drawbacks of these methods include limited separating ability by TLC, irreversible damage on thermally unstable compounds caused by high separating temperature during GC, limited solvent options offered by HPCE, and large organic solvents consumption when using HPLC. On the other hand, SFC offers advantages such as rapid analysis, good resolution, high sample load and minimal organic solvent consumption, making it the superior chiral separating method. 

With years of success in developing preparative HPLC system, Sepiatec launched the latest Prep SFC 100 system, endeavor to delivering the best solution for preparative separation of chiral compounds.

SFC Technology

Sepiatec Prep SFC 100 is a highly efficient and environmental friendly compact bench top unit which also comes with a very user-friendly software. Sepiatec Prep SFC 100 tackles many of the technical bottlenecks encountered in other preparative SFC systems as shown in the following table:

Conventional SFC System LimitationsSepiatec Prep SFC 100 Features
System pressure fluctuationsRobust design of the backpressure regulator maintains precise backpressure control even under the most intense workload.
Offline modifier preparationProgrammable precise and reproducible online pre-mixing of modifiers is now achieved.
Low recoveryEven under extreme separation conditions, such as low organic solvent ratio of about 5%, the newly designed gas-liquid separator can still maintain its high recovery at above 95%.



The preparative separation of the chiral compound Trans-Stilbene Oxide (TSO) by stack injection method using Sepiatec Prep SFC 100 is illustrated below. Experimental conditions are shown in Table 1.


Table 1: The experimental conditions for the chiral separation of TSO

EluentCO2 / Methanol 70:30
ColumnReprosil Chiral NR, 8µm, 250 x 20mm (Dr. Maisch GmbH)
Column Oven Temperature40℃
Back pressure150bar
Detection wavelengthUV 235nm
Sample size50mg TSO/mL
Injection volume200μL
Stack time1.3 min

Stack injection is a unique feature of SFC which can tremendously shorten the processing time. Typical range for preparative separation of one sample requires 20 to 50 sample injections. The stacking time can be set in the range of 1 to 5 minutes depending on the separating condition, which can save up to 4 hours. In the TSO example as shown in Figure 1, 15 stack injections of TSO were performed at a stacking time of 1.3min, and the cumulative saving time is 19.5 min.

Apart from separation efficiency, repeatability is also one of the key performance indices of a SFC system. Figure 2 shows the overlay chromatogram of 15 stacked injections of the TSO sample, of which all are almost identical, demonstrating the excellent reproducibility and stability of the system. The separation details and results are shown in Table 2. The recovery of TSO was 48.9mg (97.2%) for peak 1 (enantiomer 1) and 48.5mg (96.4%) for peak 2 (enantiomer 2). The HPLC-UV purity test results of these two compounds showed that all the purity figures reached more than 99%.

Fig. 1 Total run of the 15 stack injections of the TSO

Fig. 2 Overlay view of the 15 stack injections of TSO


Table 2. The experimental results of TSO chiral separation.

Starting material100.6mg TSO
Total runtime21 min
Total CO2 consumption1,176mL
Total methanol consumption504mL
 Peak 1Peak 2
HPLC-UV purity99.97%99.93%
HPLC chromatogram
Mass spectrometer Coupling

Sepiatec Prep SFC 100 could be optionally coupled with a high performance compact mass spectrometer (CMS) from Advion. With the fully integrated SFC 100-MS software, controlled mass directed fractionation can be easily achieved by inputting the mass of the targeted compound. This function is extremely useful in the case of separating targeted chiral molecules from a mixture of other unwanted compounds (Figure 3).

Fig. 3 TIC, XIC and UV signals from Sepiatec Prep SFC 100 system coupled with an Advion expression Compact Mass Spectrometer

  • Sepiatec
    Prep SFC 100


Sepiatec Prep SFC 100 system offers rapid and easy preparative chiral separation for a diverse range of compounds such as natural product, synthetic products and by-products, and even heat sensitive compounds. Substantial application results show that the Sepiatec Prep SFC 100 system has high recovery efficiency and reproducibility, meeting all the needs for research, production and quality control.

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