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High Sensitivity Fluorescence Detector for Supercritical Fluid Chromatography

By Satoe Iijima

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

Supercritical fluid chromatography system — **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 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

**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
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)

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

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)

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

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 Kuwajima^{1

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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About the Author

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

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High Sensitivity Fluorescence Detector for Supercritical Fluid Chromatography

Introduction