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User Benefits Application News Supercritical Fluid Chromatography (SFC) is a separation technique that employs supercritical carbon dioxide as the primary mobile phase. Owing to its high diffusion coefficient and low viscosity, supercritical carbon dioxide allows high flow-rate delivery at lower column backpressure compared to Liquid Chromatography (LC), enabling shorter analysis time. It also provides excellent separation performance for structurally related compounds. However, since supercritical carbon dioxide has a polarity similar to that of hexane, it often lacks sufficient elution strength on its own. Therefore, organic solvents (modifiers) such as methanol, acetonitrile, 2-propanol, and ethanol are commonly added to the mobile phase to adjust polarity. By appropriately selecting and combining these modifiers, the interaction between analytes and the stationary phase can be finely tuned, resulting in diverse separation patterns. Because SFC and LC exhibit different retention behaviors, compounds that are difficult to separate by LC may be effectively separated by SFC. Accordingly, combining both techniques in method development can improve the efficiency of searching for optimal separation conditions. On the other hand, both column and mobile phase selection have a strong influence on retention behavior. To obtain optimal separation, it is ideal to examine multiple types of columns and mobile phases. However, this process is highly labor- and time-intensive. This study demonstrates efficient optimization of separation conditions using Nexera UC/s (LC/SFC switching system), which allows LC and SFC analyses to Introduction Because LC and SFC have different separation characteristics, SFC has the potential to separate compounds that are difficult to resolve by LC. Both LC and SFC analyses can be performed on a single system. By utilizing mobile phase and column switching valves, LabSolutions MD enables automated screening of mobile phases and columns. Diversification of Separation Selectivity Using Supercritical Fluid Chromatography -Efficient Search for Optimal Separation Conditions by Combining LC and SFC- Shinichi Fujisaki Supercritical Fluid Chromatography System / Ultra High Performance Liquid Chromatograph be performed on a single instrument, in combination with the LabSolutions MD, a dedicated software for supporting method development. Column and mobile phase screening were conducted using a mixed sample containing six small-molecule pharmaceutical compounds as a model sample. Nexera UC/s enables both LC and SFC analyses on a single system (flow diagram: Fig. 1). The system is configured by adding a supercritical CO₂ delivery pump and backpressure regulator unit to a standard LC system. By switching the ON/OFF status of the delivery pump and backpressure, both LC and SFC analyses can be carried out. The organic solvent delivery pumps, autosampler, column oven, and detector can be shared between LC and SFC, enabling space-saving and cost-effective operation. Existing LC systems can also be upgraded. In combination with a PDA (photodiode array) detector and the single quadrupole mass spectrometer LCMS-2050, the system allows obtained mass data to be used for identification of known compounds and estimation of unknown impurities. When controlled by LabSolutions MD, up to 12 columns and 7 types of organic solvents (modifiers) can be automatically switched, enabling comprehensive screening for both LC and SFC and significantly reducing labor in method development. Features of LC/SFC Switching System Fig. 1 LC/SFC Switching System (Nexera UC/s) (Green: SFC flow path, Blue: LC flow path, Orange: Common flow path for LC and SFC) CO 2 pump (SFC) CO₂ cylinder Organic solvents pump (LC/SFC) Autosampler Column oven A (SFC) PDA detector Back pressure regulator Aqueous solvents pump (LC) Column oven B (LC) Make-up pump 4 solvents switching valve 7 solvents switching valve Single MS Shut-off valve Manifold Column switching valve Column switching valve Manifold
Application News Table 1 Screening Conditions A case study of screening to find the optimal combination of columns and mobile phases for SFC analysis is presented (screening conditions: Table 1; target compounds: Table 2). Three columns with different stationary phases were evaluated. Three modifier compositions (0%, 50%, and 100% acetonitrile in methanol) were tested. A comprehensive analysis schedule covering all 9 (3×3) combinations of columns and modifiers was generated to identify the optimal combination. LabSolutions MD enables error-free automatic generation of analysis schedules combining columns, mobile phases, and other parameters (steps (1)–(5) in Fig. 2). Column switching valves allow automatic selection of the desired column (step (2) in Fig. 2). Modifier composition changes are automatically prepared using the mobile phase blending function by simply selecting the desired composition (step (1) in Fig. 2). This reduces manual preparation work and prevents errors. LC and SFC switching is performed automatically by executing the analysis schedule generated in LabSolutions MD. The specified switching method (Fig. 3) automatically replaces the flow path, allowing consecutive analyses without manual intervention or influence from the previous analysis. Column and Mobile Phase Screening Table 2 Target Compounds Fig. 2 Easy Creation of Analysis Schedule by LabSolutions MD (2) Select columns. (1) Select organic solvents (modifiers). (3) Enter sample information. (4) Enter gradient conditions, including flowrate and oven temp. (5) Create an analysis schedule. The screening results are shown in Fig. 4. Changing columns or modifier compositions significantly affected retention and peak shapes of all compounds. Results of Screening Fig. 4 Results of Column and Mobile Phase Screening *1 P/N：227-32501-03, *2 P/N：227-32602-09 *3 P/N：227-32608-24 System Column 1 Column 2 Column 3 Column Temp. Injection volume Sample solvent Mobile phases Pump A (CO2) Pump B (Organic) – Line A – Line B – Line C – Line D Flow rate Time program (%B) Detection BPR pressure BPR Temp. : : : : : : : : : : : : : : : : : Nexera UC/s (SFC) Shim-pack UC-Poly BT *1 Shim-pack UC-PBr *2 Shim-pack UC-ODS *3 (150 mm × 3.0 mm I.D., 3.0 µm) 30 °C 5 µL (100 mg/L) Methanol CO2 Methanol Acetonitrile 100 mmol/L ammonium formate in methanol Water 1 mL/min 5% (0 min) →70% (5 - 7 min)→5% (7.01-12 min) 254 nm (SPD-M40 、 high-pressure flow cell) 10 MPa 50 ℃ System : LCMS-2050 Ionization : ESI/APCI (DUIS ) 、 positive and negative mode Mode : SCAN (m/z 100-1000) Nebulizing gas : 2.0 L/min (N2) Drying gas : 5.0 L/min (N2) Heating gas : 7.0 L/min (N2) DL temp. : 200 °C Desolvation temp. : 450 °C Interface Voltage Make-up pump Mobile phase Flow rate : : : +3.0 kV / -2.0 kV Methanol 0.1 mL/min Name Monoisotopic mass * (A) Antipyrine 188 (B) Probenecid 285 (C) Naproxen 230 (D) Hydrocortisone 362 (E) Clozapine 326 (F) Indomethacin 357 * rounded to the nearest unit mass Fig. 3 Method Selection Screen for LC/SFC Switching (8) UC-PBr_ACN 100% (7) UC-PolyBT_ACN 100% (6) UC-ODS_MeOH_ACN 50%_50% (5) UC-PBr_MeOH_ACN 50%_50% (4) UC-PolyBT_MeOH_ACN 50%_50% (3) UC-ODS_MeOH 100% (2) UC-PBr_MeOH 100% (1) UC-PolyBT_MeOH 100% (9) UC-ODS_ACN 100% (1) (2) (3) (6) (5) (4) (8) (7) 0 2.5 5 7.5 10 min AU 0 1.0 2.0 (9) SFC
Application News Because screening generates as many chromatograms as the number of analysis schedule, they must be evaluated to determine which one is the optimal. Checking all chromatograms manually is time-consuming. LabSolutions MD can quickly and easily find optimal condition using equation (Eq. 1) below to quantitatively evaluate the separation. (Evaluation Value) = P × (Rs1 + Rs2 +...+ RsP-1) (Eq. 1) Evaluation Value is calculated as the number of peaks detected (P) multiplied by the sum of resolution factor (Rs) for all peaks. Fig. 5 shows Evaluation Value obtained through column and mobile phase screening and listed in the order from the highest to the lowest. Fig. 6 shows the chromatogram of the highest Evaluation Value. Quickly Find Optimal Condition Fig. 5 Ranking of Each Condition by Evaluation Value Fig. 6 Chromatogram with Highest Evaluation Value (A) Antipyrine、(B) Probenecid、(C) Naproxen (D) Hydrocortisone、(E) Clozapine、(F) Indomethacin SFC UC-PBr_MeOH 100% 0 2.5 5 7.5 10 min AU 0 0.3 (E) m/z 327 ([M+H]⁺) (D) m/z 363 ([M+H]⁺) (A) m/z 189 ([M+H]⁺) (B) m/z 284 ([M+H] - ) (C) m/z 229 ([M+H] - ) (F) m/z 358 ([M+H]⁺) SFC The optimal condition was a UC-PBr column with 100% methanol as the modifier. The clozapine peak (Fig. 6E) exhibited slight tailing. Subsequently, additives were added to the modifier in an attempt to improve separation and peak shape. Based on the condition with the highest Evaluation Value (UC- PBr, 100% methanol), ammonium formate (20 mmol/L) was added to methanol, and water was further added at 0%, 2%, and 4% to evaluate the effect on separation of each compound. The resulting chromatograms are shown in Fig. 7. Adding ammonium formate sharpened the clozapine peak (Fig. 7(2)). Increasing water content improved separation between clozapine (E) and indomethacin (F), with the best separation observed at 4% water (Fig. 7(4)). Improvement of Separation and Peak Shape Using Additives (4) UC-PBr_20 mM AF in MeOH 96%_Water 4% (3) UC-PBr_20 mM AF in MeOH 98%_Water 2% (2) UC-PBr_20 mM AF in MeOH 100%_Water 0% (1) UC-PBr_MeOH 100% SFC Fig. 7 Chromatograms with Varying Modifier Compositions (A) Antipyrine、(B) Probenecid、(C) Naproxen (D) Hydrocortisone、(E) Clozapine、(F) Indomethacin Fig. 8 Comparison of Chromatograms After Modifier Composition Optimization SFC (Upper), LC (Lower) (A) Antipyrine、(B) Probenecid、(C) Naproxen (D) Hydrocortisone、(E) Clozapine、(F) Indomethacin SFC LC (B) m/z 284 ([M+H] - ) (C) m/z 231 ([M+H]⁺) (F) m/z 358 ([M+H]⁺) (E) m/z 327 ([M+H]⁺) (D) m/z 363 ([M+H]⁺) (A) m/z 189 ([M+H]⁺) AU 0 0.5 AU 0 2.0 3 5 7 min UC-PBr_20 mM AF in MeOH 96%_Water 4% Scepter C18_0.1% formic acid in water / MeOH 100% (E) m/z 327 ([M+H]⁺) (D) m/z 363 ([M+H]⁺) (A) m/z 189 ([M+H]⁺) (B) m/z 284 ([M+H] - ) (C) m/z 229 ([M+H] - ) (F) m/z 358 ([M+H]⁺) 2 4 6 min AU 0 1.0 2.0 (1) (2) (3) (4) (A) (B) (C) (D) (E) (F) (E) (F) (E) (F) (E) (F) Fig. 8 compares SFC chromatograms after modifier optimization (Upper) and LC chromatograms using water-based (0.1% formic acid) and methanol-based mobile phases (Lower). Mass data obtained by LCMS-2050 indicated that elution orders of compounds differ significantly between LC and SFC. Mass detection enables identification even when elution orders differ. SFC with ammonium formate and water in the modifier achieved good separation and peak shapes for all compounds. The results suggest that compounds difficult to separate by LC have the potential to be separated by SFC.
Application News www.shimadzu.com/an/ Shimadzu Corporation © Shimadzu Corporation, 2025 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. First Edition: Nov. 2025 01-01058-EN Nexera, LabSolutions, DUIS and Shim-pack are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries. Because SFC and LC have different separation characteristics, combining both techniques in method development can improve the efficiency of finding optimal separation conditions. Nexera UC/s allows automated switching between LC and SFC analyses on a single system. Using the LCMS-2050, mass-based identification of known compounds and estimation of unknown impurities is possible. Combining this with LabSolutions MD enables automated switching of up to 12 columns and 7 modifiers, allowing comprehensive condition screening for both LC and SFC and significant labor savings. This article demonstrated the use of column and mobile phase screening for efficient method development using both LC and SFC. In SFC, gradient conditions, column oven temperature, and backpressure settings also contribute to separation selectivity, and optimization of these parameters is important. For details on a complete workflow for efficient SFC method development, please refer to Application News “01-01057.” Conclusion
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