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

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 make fluorescence detection extremely sensitive and 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 an FP-4020 fluorescence detector (FL detector) with high pressure cell. For a sensitivity evaluation, a comparison of detection using fluorescence and UV-visible absorbance (UV/Vis detector) was performed.

LC-4000 SFC system

Experimental

Experimental conditions
Column: 2-Ethylpyridine (Princeton Chromatography) (4.6 mm I.D. x 250 mm L, 5 µm)
Eluent A: CO₂
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
Wavelength: Refer to Figure 1.
Back pressure: 15 MPa
Injection volume: 5 µL
Standard sample: Mixture of 16 PAHs standards in acetonitrile

Structure of PAHs

SFC System Schematic

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).

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.）

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.

Table 1　 Lower limit of detection of PAHs in each detector and linearity in FL detector

Peak No.	Compound	Detection limit [pg]		Sensitivity ratio^*3	Correlation coefficient (FL)^*4
Peak No.	Compound	UV^*1	FL^*2	Sensitivity ratio^*3	Correlation coefficient (FL)^*4
1	Naphthalene	120	2.04	59	1.0000
2	Acenaphthene	13.6	0.293	46	0.9998
3	Fluorene^*5	21.3	0.369	58	0.9999
5	Anthracene	8.67	0.166	52	0.9998
6	Phenanthrene	17.8	0.676	26	1.0000
7	Fluoranthene	58.8	2.34	25	0.9999
8	Pyrene	75.2	0.357	211	0.9998
9	Benzo[a]anthrancene	32.5	0.432	75	0.9999
10	Chrysene	31.5	1.06	30	1.0000
11	Benzo[k]fluoranthene	93.0	0.236	393	0.9997
12	Benzo[b]fluoranthene	81.9	1.46	56	0.9999
13	Benzo[a]pyrene	53.6	0.233	230	0.9998
14	Dibenz[a,h]anthracene	32.6	0.737	44	1.0000
15	Indeno[1,2,3-cd]pyrene	79.2	4.61	17	0.9998
16	Benzo[g,h,i]perylene	65.5	0.697	94	0.9998
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.

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.

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.)