Introduction

Preparative separation by supercritical fluid chromatography (SFC) offers several advantages. It allows for higher throughput compared to HPLC, and the primary solvent of mobile phase, CO₂, becomes a gas upon recovery, making post-processing, such as evaporation and drying, easier. Furthermore, the CO₂ is cost-effective compared to organic solvents, and it can be obtained in high purity (>99.99 %). On the other hand, supercritical CO₂ undergoes an adiabatic expansion approximately 500 times when it is released to atmospheric pressure at the outlet of the back pressure regulator. Consequently, during preparative separation, it is necessary to take measures to prevent contamination such as condensed water caused by scattering of the sample and sudden temperature drops. On this note, by installing MCS-es, a gas-liquid separation nozzle, on the Z-axis arm of the Gilson 223 Sample Changer, we were able to slowly collect only the liquid while preventing scattering and condensation due to CO₂ expansion. Furthermore, this allowed for efficient preparative separation with high recovery rates over a wide flow range. It’s also possible to pour make up solvents directly into MCS-es (patent pending).

In this study, we report the achiral preparation of 3 components including caffeine using the LC-4000 sereis UFC (Unified Fluid Chromatograph) Prep-SFC-L system in combination with the MCS-es.

LC-4000 Semi-prep SFC system

Experimental

Instruments
CO₂ pump: PU-4388
Modifier pump: PU-4088*
Pre-heater: HE-01
Heater controller: HC-4068-01
Autosampler: AS-4358
Column oven: CO-4060*
PDA detector: MD-4010*
BP regulator: BP-4340
Post-heater: HE-02
Heater controller: HC-4068-01
Make up pump: PU-4086*
Fraction valve: FV-4000-06
Fraction collector: Gilson 223
Sample Changer
Fume hood: FH-4388
* with option units

Gilson 223 Sample Changer + MCS-es

Image of collection by MCS-es

SFC Conditions
Column: YMC-Pack Pro C18 (30 mmI.D. x 250 mmL, 10 µm)
Eluent: Carbon dioxide/methanol (95/5)
Flow rate: 120 mL/min
Column temp.: 40 ºC
Pre-heat temp.: 40 ºC
Wavelength: 200-650 nm, 215, 270, 258, 244 nm (CH1, 9, 10, 11)
Back pressure: 10 MPa
Post-heat temp.: 60 ºC
Make up solvent: Methanol
Make up solvent flow rate: 10 mL/min
Inj. volume: 1000 µL
Standard: Caffeine 1000 µg/mL, Anthracene 500 µg/mL, Fluorene 500 µg/mL in methanol

Schematic Diagram

Keywords

Prep SFC, supercritical carbon dioxide, gas-liquid separator, MCS-es, fraction collector, Gilson 223 Sample Changer, achiral preparation

Results

Figure 1 shows a chromatogram of a standard solution of 3 components including caffeine. Based on this chromatogram, a collection method was made and used for preparative separation.

Fig.1 Chromatogram of standard solution of 3 components including caffeine
1: Caffeine, 2: Anthracene, 3: Fluorene

Figure 2 shows the collection results of 3 components including caffeine based on time fractionation, and Figure 3 shows the chromatograms obtained by re-measuring each fraction. The yellow-green section in Figure 2 represents the collection phase, while the pink section indicates the make up phase, and while the purple section indicates the drop waiting time*1.
*1 Time taken to collect the modifier and makeup solvent remaining in the MCS-es after valve switching.

Fig.2 Collection results of 3 components including caffeine
1: Caffeine, 2: Anthracene, 3: Fluorene

Fig.3 Chromatograms obtained by re-measuring each fraction
1: Caffeine (equivalent to 50 µg/mL), 2: Anthracene (equivalent to 25 µg/mL), 3: Fluorene (equivalent to 25 µg/mL)
*2 Solvent peak

Each fraction was diluted to 20 mL by co-washing with methanol so that caffeine, anthracene and fluorene were each 50, 25, 25 µg/mL (equivalent). These solutions were used for recovery rate measurements. Table 1 shows the recovery rates for each component, calculated assuming the area values at the time of measurement of the standard solution of 3 components including caffeine as 100% (Caffeine: 50 µg/mL, Anthracene: 25 µg/mL, Fluorene: 25 µg/mL). Excellent recovery rates were obtained for all components.

Table 1 The recovery rates of 3 components including caffeine

Conclusion

The use of the Prep-SFC-L system in combination with MCS-es successfully achieved achiral preparation of 3 components including caffeine with excellent recovery rates.

MCS-es can be used with a maximum total flow rate of up to 200 mL/min (under specified conditions). It also supports preparation at relatively low flow rates on an analytical scale, so it can be applied to preparation over a wide range of flow rates.