High Sensitivity Fluorescence Detector for Supercritical Fluid Chromatography

Download PDF May 24, 2018

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

Fig. 1 The JASCO SFC system

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 PumpPU-4380
Modifier Solvent Delivery PumpPU-4180 (with SV*1, LV*2 and MX*3 unit)
AutosamplerAS-4350
Column OvenCO-4065 (with 2 switching valve unit*4)
Detector 1UV-4070 (with Analytical High Pressure cell)
Detector 2FP-4020 (with Analytical High Pressure cell)
Back Pressure RegulatorBP-4340
Chromatography Data SystemChromNAV Ver. 2
*1 Stop valve unit *2 Solvent switching unit *3 Mixing unit *4 10 position-11port valve unit
Fig. 2 Schematic diagram of the SFC-UV-FL

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.

Fig. 3 Structures and differences between the conventional and novel fluorescence flow cell

 

Results

SFC/UV vs. SFC/FL

Fig. 4 Sensitivity of anthracene by SFC/UV and SFC/FL
Analysis Conditions
ColumnSFCpak DIOL-5 (JASCO)
(4.6 mmI.D. x 250 mmL, 5 µm)
Mobile PhaseCO2 / methanol (90 / 10)
Flow Rate3.0 mL/min
Column Temp.40º C
UV Detection250 nm
FL DetectionEx. 250 nm, Em. 402 nm
Back Pressure15 MPa
Injection Volume5 µL

SFC/FL Applications

Polycyclic Aromatic Hydrocarbons (PAH)

Fig. 5 Measurement results of 16 mixture of PAH by SFC/UV and SFC/FL

Analysis Conditions
Column2-Ethylpyridine (Princeton)
(4.6 mmI.D. x 250 mmL, 5 µm)
Mobile PhaseCO2 / acetonitrile (gradient elution)
Flow Rate3.0 mL/min
Column Temp.40º C
Back Pressure15 MPa
Injection Volume5 µL
Sample16 mixture of PAH in acetonitrile

Vitamin E (α, β, γ, δ-Tocopherol)

Fig. 6 Measurement results of isomer mixture of tocopherol by SFC/UV and SFC/FL

Analysis Conditions
ColumnSFCpak NH2-5 (JASCO)
(4.6 mmI.D. x 250 mmL, 5 µm)
Mobile PhaseCO2 / ethanol (93 / 7)
Flow Rate3.0 mL/min
Column Temp.40º C
UV Detection295 nm
FL DetectionEx. 295 nm, Em. 330 nm
Back Pressure15 MPa
Injection Volume5 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

Fig. 7 Measurement results of 3 mixture of quinolone antibiotics by SFC/UV and SFC/FL
Analysis Conditions
ColumnSFCpak C18T-5 (JASCO)
(4.6 mmI.D. x 250 mmL, 5 µm)

Mobile PhaseCO2 / methanol with 100mM ammonium acetate and 0.1% diethylamine (gradient elution)
Flow Rate3.0 mL/min
Column Temp.40º C
UV Detection290 nm
FL DetectionEx. 290 nm, Em. 450 nm
Back Pressure15 MPa
Injection Volume5 µL
Sample3 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: satoe.iijima@jasco.co.jp

About the Author

Ms Iijima is a member of the chromatography separations team at JASCO Corporation main applications laboratory in Tokyo Japan.