LAAN-A-LC-E245

Application News
No.

High Performance Liquid Chromatography

L468

Simultaneous Determination of Polycyclic Aromatic Hydrocarbons Using the Prominence-i Integrated High Performance Liquid Chromatograph
Table 2 Analytical Conditions
Detector : UV at 230 nm RF-20Axs 0.0 - 10.0 min 10.0 - 12.8 min 12.8 - 16.0 min 16.0 - 21.0 min 21.0 - 27.6 min 27.6 - 30.0 min 30.0 - 40.0 min : 28 °C

Many polycyclic aromatic hydrocarbons exhibit fluorescence, and can therefore be detected with high selectivity and high sensitivity using a fluorescence detector. Previously, in Application News No. 393 and No. 441A, we introduced examples of the simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) using a fluorescence detector. However, of the sixteen polycyclic aromatic hydrocarbons designated as "priority pollutants" by the U.S. Environmental Protection Agency (EPA), acenaphthylene alone does not exhibit fluorescence. Therefore, a single fluorescence detector cannot be used for simultaneous analysis of all sixteen of these PAHs. However, the Prominence-i, which incorporates a UV detector, can be connected to the RF-20Axs fluorescence detector as an optional detector, permitting simultaneous analysis of all sixteen polycyclic aromatic hydrocarbons. Here, using two analytical methods, one with the wavelength switching mode and the other using simultaneous measurement at multiple wavelengths, we introduce an example of simultaneous analysis of the 16 PAHs.

Ex. at 270 nm, Em. at 330 nm, Gain : X1 Ex. at 250 nm, Em. at 370 nm, Gain : X1 Ex. at 330 nm, Em. at 430 nm, Gain : X4 Ex. at 270 nm, Em. at 390 nm, Gain : X1 Ex. at 290 nm, Em. at 430 nm, Gain : X1 Ex. at 370 nm, Em. at 460 nm, Gain : X16 Ex. at 270 nm, Em. at 330 nm, Gain : X1

Cell Temp.

n Analysis of Polycyclic Aromatic Hydrocarbons
Using Wavelength Switching Mode Fig. 1 (a) shows the chromatogram obtained using a UV detector for analysis (detection wavelength: 254 nm), and Table 1 shows the analytical conditions that were used. Although good separation was obtained using these conditions, the insufficient sensitivity obtained with UV absorption alone is due to the low absorption of ultraviolet light by the PAHs. F i g . 1 ( b ) s h o w s c h ro m a t o g r a m s o b t a i n e d v i a simultaneous analysis of sixteen polycyclic aromatic hydrocarbons using the wavelength switching mode according to component group, accomplished by connecting the Prominence-i to an RF-20Axs detector. Table 2 shows the analytical conditions used for conducting analysis via combination of the fluorescence detector and UV detector. The non-fluorescent acenaphthylene is detected by the UV absorption detector at 230 nm, in the vicinity of the wavelength at which it exhibits the maximum absorption. Using these analytical conditions, good sensitivity and separation of the sixteen components is achieved, permitting analysis of each component at the optimum wavelength.
Table 1 Analytical Conditions
Column : RESTEK Pinacle Ⅱ PAH (250 mm L. × 4.6 mm I.D., 4 μm) Mobile Phase : A: Water B: Acetonitrile Column Temp. : 40 °C Time Program : B Conc. 60 % (0 - 5 min) → 100 % (30 - 35 min) → 60 % (35 - 40 min) Flowrate : 1.5 mL/min Injection : 5 μL Detector : UV at 254 nm

(a) mV 3.00 2.75 2.50 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 -0.25 0.0 (b) 1.25 1.00 0.75

LC-2030 UV at 254 nm

6 11 2 1 4 3 5 7 8 10 9 12

13

16 1415

5.0

10.0

15.0

20.0

25.0

30.0

min

mV (×1000) LC-2030 UV and RF-20Axs

3

1

0.50
2

4 9 5 6 8 7 10

12 11 13

15 14 230 nm (UV) 16 RF-20Axs

0.25 0.00 0.0 5.0 10.0

15.0

20.0

25.0

30.0

min

1. Naphthalene (1.0 mg/L) 3. Acenaphthene (1.0 mg/L) 5. Phenanthrene (0.1 mg/L) 7. Fluoranthene (0.2 mg/L) 9. Benzo[a]anthracene (0.1 mg/L) 11. Benzo[b]fluoranthene (0.2 mg/L) 13. Benzo[a]pyrene (0.1 mg/L) 15. Benzo[g, h, i]perylene (0.2 mg/L)

2. Acenaphthylene (2.0 mg/L) 4. Fluorene (0.2 mg/L) 6. Anthracene (0.1 mg/L) 8. Pyrene (0.1 mg/L) 10. Chrysene (0.1 mg/L) 12. Benzo[k]fluoranthene (0.1 mg/L) 14. Dibenzo[a, h]anthracene (0.2 mg/L) 16. Indeno[1, 2, 3-cd]pyrene (0.1 mg/L)

Fig. 1 (a) UV Chromatogram of Standard Mixture of 16 PAHs (b) Chromatogram of Standard Mixture of 16 PAHs Analyzed by the RF-20Axs and UV Detector Note: The peak intensity of the chromatogram of Fig. 1 (b) is displayed as 10 times that of the actual chromatogram.

Application No. L468 News

n Analysis of Polycyclic Aromatic Hydrocarbons Using
Simultaneous Multichannel Wavelength Mode Fig. 2 shows the results of high-speed analysis of a standard mixture PAHs using the wavelength switching mode of the RF-20Axs fluorescence detector, and Table 3 shows the analytical conditions. Using these conditions, poor separation between "15. Benzo [g, h, i] perylene" and "16. Indeno [1, 2, 3-cd] pyrene" is evident in the vicinity of 13.5 minutes retention time. Next, Fig. 3 shows the results of analysis using the simultaneous multichannel wavelength mode of the RF20Axs, and Table 4 shows the analytical conditions used. The results demonstrate that it is possible to achieve separation with high sensitivity and good selectivity even for components that are difficult to separate using the wavelength switching mode.
Table 3 Analytical Conditions
Column : SUPELCOSILTM LC-PAH (100 mm L. × 3.0 mm I.D., 3 μm) Mobile Phase : A : Water B : Acetonitrile Time Program : B Conc. 50 % (0 - 2 min) → 100 % (13.5 - 14 min) → 50 % (14 - 15 min) Flowrate : 0.7 mL/min Injection : 5 μL Column Temp. : 40 °C Detector : UV at 230 nm RF-20Axs 0.0 - 10.0 min Ex. at 270 nm, Em. at 330 nm, Gain : X1 10.0 - 12.8 min Ex. at 250 nm, Em. at 370 nm, Gain : X1 12.8 - 16.0 min Ex. at 330 nm, Em. at 430 nm, Gain : X4 16.0 - 21.0 min Ex. at 270 nm, Em. at 390 nm, Gain : X1 21.0 - 27.6 min Ex. at 290 nm, Em. at 430 nm, Gain : X1 27.6 - 30.0 min Ex. at 370 nm, Em. at 460 nm, Gain : X16 30.0 - 40.0 min Ex. at 270 nm, Em. at 330 nm, Gain : X1 Cell Temp. : 28 °C

mV (×1000) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 min
1 2 4 56 7 8 9 10 11 12 13 14 16 3
1. Naphthalene 3. Acenaphthene 5. Phenanthrene 7. Fluoranthene 9. Benzo[a]anthracene 11. Benzo[b]fluoranthene 13. Benzo[a]pyrene 15. Benzo[g, h, i]perylene 2. Acenaphthylene 4. Fluorene 6. Anthracene 8. Pyrene 10. Chrysene 12. Benzo[k]fluoranthene 14. Dibenzo[a, h]anthracene 16. Indeno[1, 2, 3-cd]pyrene

15

Fig. 2 Rapid Analysis of 16 PAHs Standard Mixture by UV and Fluorescent Detector Using Wavelength Switching Mode Note: The peak intensity of the chromatogram of Fig. 1 (b) is displayed as 10 times that of the actual chromatogram.

Table 4 Analytical Conditions
Column Temp. : 40 °C Detector : UV at 230 nm RF-20Axs Ex. at 260 nm, Em. at 350 nm Ex. at 260 nm, Em. at 420 nm Ex. at 285 nm, Em. at 440 nm Ex. at 305 nm, Em. at 495 nm Cell Temp. : 28 °C

mV (×1000) 1.50
1. Naphthalene (1.0 mg/L) 2. Acenaphthylene (2.0 mg/L) 3. Acenaphthene (1.0 mg/L) 4. Fluorene (0.2 mg/L) 5. Phenanthrene (0.1 mg/L) 6. Anthracene (0.1 mg/L) 7. Fluoranthene (0.2 mg/L) 8. Pyrene (0.1 mg/L) 9. Benzo [a] anthracene (0.1 mg/L) 10. Chrysene (0.1 mg/L) 11. Benzo [b] fluoranthene (0.2 mg/L) 12. Benzo [k] fluoranthene (0.1 mg/L) 13. Benzo [a] pyrene (0.1 mg/L) 14. Dibenzo [a, h] anthracene (0.2 mg/L) 15. Benzo [g, h, i] perylene (0.2 mg/L) 16. Indeno [1, 2, 3-cd] pyrene (0.1 mg/L)

1.25

3

12 13

1.00

1 11 2 9 5 6 8 7
Ex=260 nm, Em=420 nm

0.75

4

15 230 nm (UV) 10 14
Ex=260 nm, Em=350 nm

0.50

0.25
16

Ex=285 nm, Em=440 nm

0.00 0.0 2.5 5.0 7.5 10.0 12.5

Ex=305 nm, Em=495 nm

15.0

min

Fig. 3 Rapid Analysis of Standard Mixture of 16 PAHs Using Simultaneous Multichannel Wavelength Mode

First Edition: Aug. 2014

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