High Sensitivity Fluorescence Detector for Supercritical Fluid Chromatography
January 5, 2024
Download This ApplicationIntroduction
Fluorescence detection has been widely used as a practical detection method in HPLC analysis. It enables the selective detection of fluorescent substances, with sensitivity that can be up to 1000 times greater than with UV detection.
Fluorescence detection can also be applied to non-fluorescent substances with the use of derivatization, with many derivatization agents becoming commercially available in recent years. Therefore, this technique provides the advantages of highly sensitivity and selectivity to many different classes of compound, and broadens the range of applications in many industries. However, fluorescence detection has not been used in SFC analysis due to the difficulty of developing a suitable high pressure flow cell.
We developed a fluorescence detector with a flow cell specifically constructed for operations at the high pressures required for SFC. This fluorescence detector is included in our newer SFC system (SFC-4000 series). In this presentation, we will introduce an application for the analysis of several compounds using SFC separation with fluorescence detection.
Experimental
Apparatus
Figure 1 shows the JASCO SFC system equipped with UV/Visible (UV) and fluorescence (FL) detectors used in this experiment. Figure 2 also shows the schematic diagram of this system. This system enables method scouting analysis to be performed on several mobile phases and columns.
| CO2 Delivery Pump | PU-4380 |
| Modifier Solvent Delivery Pump | PU-4180 (with SV*1, LV*2 and MX*3 unit) |
| Autosampler | AS-4350 |
| Column Oven | CO-4065 (with 2 switching valve unit*4) |
| Detector 1 | UV-4070 (with Analytical High Pressure cell) |
| Detector 2 | FP-4020 (with Analytical High Pressure cell) |
| Back Pressure Regulator | BP-4340 |
| Chromatography Data System | ChromNAV Ver. 2 |
| *1 Stop valve unit *2 Solvent switching unit *3 Mixing unit *4 10 position-11port valve unit |
|
Figure 3 shows the structures and differences between a conventional and novel fluorescence detector flow cell. We developed a unique quartz flow cell design and structure for use with high pressure (up to 20 MPa). The outer surfaces of this flow cell are coated with aluminum, and provides high collection efficiency of fluorescence by functioning as a reflector.
Keywords
032006U
Results
SFC/UV vs. SFC/FL
| Analysis Conditions | |
| Column | SFCpak DIOL-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / methanol (90 / 10) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 250 nm |
| FL Detection | Ex. 250 nm, Em. 402 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
SFC/FL Applications
Polycyclic Aromatic Hydrocarbons (PAH)
| Analysis Conditions | |
| Column | 2-Ethylpyridine (Princeton) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / acetonitrile (gradient elution) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | 16 mixture of PAH in acetonitrile |
Vitamin E (α, β, γ, δ-Tocopherol)
| Analysis Conditions | |
| Column | SFCpak NH2-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / ethanol (93 / 7) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 295 nm |
| FL Detection | Ex. 295 nm, Em. 330 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | ・Isomer mixture of tocopherol in hexane/ethanol (90/10) ・Corn oil 10 mg/mL in hexane/ethanol (90/10) (filtration with 0.45-µm membrane) |
Quinolone Antibiotic
| Analysis Conditions | |
| Column | SFCpak C18T-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / methanol with 100mM ammonium acetate and 0.1% diethylamine (gradient elution) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 290 nm |
| FL Detection | Ex. 290 nm, Em. 450 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | 3 mixture of quinolone antibiotics (Orbifloxacin, enrofloxacin, and ciprofloxacin each 5 pg/µL in methanol) |
Conclusion
- We developed a fluorescence detector with a flow cell specifically constructed for operation at the high pressures required for SFC
- SFC/FL provides highly sensitive detection (from fg to pg) for fluorescent substances, compared to UV/Visible detector.
- SFC using fluorescence detection will open the door to new applications fields.
References
1. V. R. Meyer, “Practical High-Performance Liquid Chromatography”, 4th ed., John Wiley & Sons , 2004, 92 and 279-282.
2. M. Bounoshita, T. Kanomata, CHROMATOGRAPHY, 2011, 32, 23-32.
3. O. A. Adegoke, Afr.J. Pure Appl.Chem., 2012, 6 (14), 129-140.
4. T. Yarita, A. Nomura, K. Abe, Y. Takeshita, J. Chromatogr. A, 1994, 679, 329-334.
Presented at SFC 2017 Rockville, MD
Satoe Iijima1, Akitaka Tearada1, DJ Tognarelli2, John Burchell2, Masao Bounoshita1, Yasuyo Sato1, Miki Kuwajima1
1JASCO Corporation, 2967-5 Ishikawa-machi, Hachioji, Tokyo 192-8537
2JASCO Incorporated, 28600 Mary’s Court, Easton, MD 21601
E-mail: [email protected]
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SFC-4000 Semi-Preparative SFC for semi-preparative scale purifications.
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High Sensitivity Fluorescence Detector for Supercritical Fluid Chromatography
Introduction
Fluorescence detection has been widely used as a practical detection method in HPLC analysis. It enables the selective detection of fluorescent substances, with sensitivity that can be up to 1000 times greater than with UV detection.
Fluorescence detection can also be applied to non-fluorescent substances with the use of derivatization, with many derivatization agents becoming commercially available in recent years. Therefore, this technique provides the advantages of highly sensitivity and selectivity to many different classes of compound, and broadens the range of applications in many industries. However, fluorescence detection has not been used in SFC analysis due to the difficulty of developing a suitable high pressure flow cell.
We developed a fluorescence detector with a flow cell specifically constructed for operations at the high pressures required for SFC. This fluorescence detector is included in our newer SFC system (SFC-4000 series). In this presentation, we will introduce an application for the analysis of several compounds using SFC separation with fluorescence detection.
Experimental
Apparatus
Figure 1 shows the JASCO SFC system equipped with UV/Visible (UV) and fluorescence (FL) detectors used in this experiment. Figure 2 also shows the schematic diagram of this system. This system enables method scouting analysis to be performed on several mobile phases and columns.
| CO2 Delivery Pump | PU-4380 |
| Modifier Solvent Delivery Pump | PU-4180 (with SV*1, LV*2 and MX*3 unit) |
| Autosampler | AS-4350 |
| Column Oven | CO-4065 (with 2 switching valve unit*4) |
| Detector 1 | UV-4070 (with Analytical High Pressure cell) |
| Detector 2 | FP-4020 (with Analytical High Pressure cell) |
| Back Pressure Regulator | BP-4340 |
| Chromatography Data System | ChromNAV Ver. 2 |
| *1 Stop valve unit *2 Solvent switching unit *3 Mixing unit *4 10 position-11port valve unit |
|
Figure 3 shows the structures and differences between a conventional and novel fluorescence detector flow cell. We developed a unique quartz flow cell design and structure for use with high pressure (up to 20 MPa). The outer surfaces of this flow cell are coated with aluminum, and provides high collection efficiency of fluorescence by functioning as a reflector.
Results
SFC/UV vs. SFC/FL
| Analysis Conditions | |
| Column | SFCpak DIOL-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / methanol (90 / 10) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 250 nm |
| FL Detection | Ex. 250 nm, Em. 402 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
SFC/FL Applications
Polycyclic Aromatic Hydrocarbons (PAH)
| Analysis Conditions | |
| Column | 2-Ethylpyridine (Princeton) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / acetonitrile (gradient elution) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | 16 mixture of PAH in acetonitrile |
Vitamin E (α, β, γ, δ-Tocopherol)
| Analysis Conditions | |
| Column | SFCpak NH2-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / ethanol (93 / 7) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 295 nm |
| FL Detection | Ex. 295 nm, Em. 330 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | ・Isomer mixture of tocopherol in hexane/ethanol (90/10) ・Corn oil 10 mg/mL in hexane/ethanol (90/10) (filtration with 0.45-µm membrane) |
Quinolone Antibiotic
| Analysis Conditions | |
| Column | SFCpak C18T-5 (JASCO) (4.6 mmI.D. x 250 mmL, 5 µm) |
| Mobile Phase | CO2 / methanol with 100mM ammonium acetate and 0.1% diethylamine (gradient elution) |
| Flow Rate | 3.0 mL/min |
| Column Temp. | 40º C |
| UV Detection | 290 nm |
| FL Detection | Ex. 290 nm, Em. 450 nm |
| Back Pressure | 15 MPa |
| Injection Volume | 5 µL |
| Sample | 3 mixture of quinolone antibiotics (Orbifloxacin, enrofloxacin, and ciprofloxacin each 5 pg/µL in methanol) |
Conclusion
- We developed a fluorescence detector with a flow cell specifically constructed for operation at the high pressures required for SFC
- SFC/FL provides highly sensitive detection (from fg to pg) for fluorescent substances, compared to UV/Visible detector.
- SFC using fluorescence detection will open the door to new applications fields.
Keywords
032006U
References
1. V. R. Meyer, “Practical High-Performance Liquid Chromatography”, 4th ed., John Wiley & Sons , 2004, 92 and 279-282.
2. M. Bounoshita, T. Kanomata, CHROMATOGRAPHY, 2011, 32, 23-32.
3. O. A. Adegoke, Afr.J. Pure Appl.Chem., 2012, 6 (14), 129-140.
4. T. Yarita, A. Nomura, K. Abe, Y. Takeshita, J. Chromatogr. A, 1994, 679, 329-334.
Presented at SFC 2017 Rockville, MD
Satoe Iijima1, Akitaka Tearada1, DJ Tognarelli2, John Burchell2, Masao Bounoshita1, Yasuyo Sato1, Miki Kuwajima1
1JASCO Corporation, 2967-5 Ishikawa-machi, Hachioji, Tokyo 192-8537
2JASCO Incorporated, 28600 Mary’s Court, Easton, MD 21601
E-mail: [email protected]