High Sensitivity Detection of Polycyclic Aromatic Hydrocarbons using Fluorescence Detection in SFC

Download PDF January 21, 2021

Introduction

Polycyclic Aromatic Hydrocarbons (PAHs) are produced by the incomplete combustion of organic compounds from various sources including diesel exhaust, coal dust, and cigarette smoke; many are strongly carcinogenic. PAHs are subject to regulation by various organizations including the EPA (United States Environmental Protection Agency). These regulations strictly control the impact of PAHs on environmental pollution and their effects on human health.

PAHs exhibit strong natural fluorescence due to the aromatic structure with many conjugated double bonds. This makes the high sensitivity of fluorescence detection necessary for the low levels often found in the environment.

In this application note, 16 PAHs were separated by supercritical fluid chromatography (SFC) and detected using a FP-4020 fluorescence detector (FL detector) with a high pressure flow cell (previously introduced in LC application data No.032006U). Data was collected using both UV-Vis and fluorescence detectors for comparative purposes.

Experimental

Equipment
CO2 PumpPU-4380
Modifier PumpPU-4180
Modifier Pump Option SV Unit
MX Unit
AutosamplerAS-4350
Column OvenCO-4060
UV/Vis DetectorUV-4070
UV/Vis Detector Flow CellAnalytical High Pressure
FL DetectorFP-4020
FL Detector Flow CellAnalytical High Pressure
Back Pressure RegulatorBP-4340
Conditions
Column2-Ethylpyridine (Princeton Chromatography)
(4.6 mm I.D. x 250 mm L, 5 µm)
Eluent ACO2
Eluent B Acetonitrile
Gradient(A/B), 0 min (95/5) → 1.5 min (95/5)
→ 12 min (80/20) → 14 min (80/20)
→ 15 min (95/5) → 20 min (95/5)
Flow Rate 3.0 mL/min
Column Temp. 40 ̊C
WavelengthRefer to Figure 1
Back Pressure15 MPa
Injection Volume5 µL
Standard Sample Mixture of 16 PAHs standards in acetonitrile

Keywords

Polycyclic Aromatic Hydrocarbons, PAHs, Ethylpyridine column, SFC, Fluorescence detector, carcinogenicity, UV-Vis, absorbance detector

Results

Figure 1 shows the separation of a 16-component PAHs standard mixture with UV-visible detection (500 pg/µL each) and fluorescence detection (5 pg/µL each). Since Acenaphthylene does not fluoresce, it was not detected in the fluorescence chromatogram and was co-eluted with Fluorene (peak No. 3) overlapping in the UV chromatogram. Table 1 shows the detection limits of each component.

Figure 1. UV and FL chromatogram of PAHs 16-component (UV: 500 pg/µL, FL: 5 pg/µL) 1: Naphthalene, 2: Acenaphthene, 3: Fluorene, 4: Acenaphthylene (non-fluorescent), 5: Anthracene, 6: Phenanthrene, 7: Fluoranthene, 8: Pyrene, 9: Benzo[a]anthracene, 10: Chrysene, 11: Benzo[k]fluoranthene, 12: Benzo[b]fluoranthene, 13: Benzo[a]pyrene, 14: Dibenzo[a,h]anthracene, 15: Indeno[1,2,3-cd]pyrene, 16: Benzo[g,h,i]perylene The measurement wavelength used for the UV/Visible detector is the same as the excitation wavelength of the fluorescence detector. The gain of the fluorescence detector is x100.
Using the fluorescence detector, the PAHs were detected at the low detection limit of  0.17 pg to 4.6 pg, and the sensitivity ratio compared with the UV detector is approx. 20 to 400 times. Figure 2 shows a calibration curve for the standard PAH mixture at concentrations from 1 to 100 pg/µL measured with the fluorescence detector. From these results (Table 1), excellent linearity with a correlation coefficient of 0.9997 or higher was confirmed for all components.

Peak No.CompoundDetection Limit [pg]Sensitivity Ratio*3Correlation coefficient (FL)*4
UV*1FL*2
1Naphthalene1202.04591.0000
2Acenaphthene13.60.293460.9998
3Fluorene*521.30.369580.9999
5Anthracene8.670.166520.9998
6Phenanthrene17.80.676261.0000
7Fluoranthene58.82.34250.9999
8Pyrene75.20.3572110.9998
9Benzo[a]anthrancene32.50.432750.9999
10Chrysene31.51.06301.0000
11Benzo[k]fluoranthene93.00.2363930.9997
12Benzo[b]fluoranthene81.91.46560.9999
13Benzo[a]pyrene53.60.2332300.9998
14Dibenz[a,h]anthracene32.60.737441.0000
15Indeno[1,2,3-cd]pyrene79.24.61170.9998
16Benzo[g,h,i]perylene65.50.697940.9998
*1 Calculated from the measurement of 500 pg/mL standard sample (S/N=3)
*2 Calculated from the measurement of 5 pg/mL standard sample (S/N=3)
*3 Lower limit of detection of UV detector/Lower limit of detection of FL detector
*4 Calibration curve was made using standards at 1, 5, 10, 50, 100 pg/mL
*5 The value of the Fluorene peak using UV detection includes Acenaphthylene which is co-eluted.

Figure 2. FL chromatogram of PAHs 16-components 1: Naphthalene, 2: Acenaphthene, 3: Fluorene, 4: Acenaphthylene (non-fluorescent), 5: Anthracene, 6: Phenanthrene, 7: Fluoranthene, 8: Pyrene, 9: Benzo[a]anthracene, 10: Chrysene, 11: Benzo[k]fluoranthene, 12: Benzo[b]fluoranthene, 13: Benzo[a]pyrene, 14: Dibenzo[a,h]anthracene, 15: Indeno[1,2,3-cd]pyrene, 16: Benzo[g,h,i]perylene Measurement wavelength and gain set for the fluorescence detector is the same as Figure 1.
Part NumberModelDescription
7032-J002APU-4380CO2 Delivery Pump
7002-J004APU-4180RHPLC Pump (Base Unit)
7008-H350ASV-4300Stop valve unit
7007-H063AMX-4300-NCDynamic Mixing Unit (for SFC/E)
7007-H146BMixing chamber, 1.5 mL, for Analytical
7038-J002AAS-4350SFC Autosampler
7021-J002ACO-4060Column Oven
6990-J002ABP-4340Automatic Back Pressure Regulator
7025-J002AUV-4070UV/VIS detector
7025-H201BAnalytical high pressure flow cell
6996-J001AFP-4020Fluorescence detector
7052-H201AAnalytical high pressure flow cell
7139-J002ABS-4500-4Bottle stand
Custom orderPrinceton SFC 2-Ethylpyridine 60 Å (4.6 mm I.D. x 250 mm L, 5 µm)
6688-H564ALC-NetCGCG cable (x6)
6925-H262APreheat coil 1/16", 0.8 µm I.D. x 5 m L
7059-J012AChromNAV V2 ChromNAV 2.0 Chromatography Data System
SFC tubing kit
7001-H405BMaintenance tool kit

 

 

 

About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.