Fast and Efficient Separation of 18 PAHs [EPA 610 and EPA 8310 + 2] using an Ascentis^® Express PAH HPLC Column

Petra Lewits, Global Product Manager for HPLC Columns

Article from Analytix Reporter - Issue 15

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds commonly found in the environment because of incomplete combustion of fuels, such as coal, tar, and crude oil, among others. These compounds’ carcinogenic, mutagenic, and teratogenic nature makes them compounds of concern to environmental organizations around the world. Consequently, there are many regulatory methods in place for PAH testing in environmental samples like air, soil, water, as well as food samples to protect human health.

PAH analysis in water at trace levels demands a highly sensitive method. In this application, we demonstrate an analysis for the separation of 16 + 2 standard PAH compounds (Figure 1) mentioned in EPA method 610 and 8310 on an AscentisR Express PAH column.

The Ascentis^® Express PAH is a non-endcapped, trifunctional C18 phase with a proprietary manufacturing process, designed on superficially porous particle (SPP) technology, to provide a fast and efficient separation of PAH compounds. The analysis was completed with a resolution value of at least 1.5 in under 5 minutes for EPA method 610. The column gave better detection sensitivity with fluorescence detection in comparison to UV and a fully porous particle (FPP) sub-2 μm column.

Figure 1. 16 EPA 610 & 8310 + 2 PAH compound structures

Experimental

Here a separation under 5 min for the 16 PAHs in EPA 610 plus 1-methylnapthalene and 2-methylnaphthalene is shown using a 5 cm x 4.6 mm column (Table 1) & is shown (Figure 2).

Separation of 18 PAHs: EPA 610 with UV detection

Table 1. Chromatographic conditions for determination of PAHs by HPLC-UV

Column:	Ascentis® Express 90 Å PAH, 2.7 µm, 5 cm x 4.6 mm (53539-U)
Mobile phase:	[A] Water; [B] acetonitrile
Gradient:	Time	%B
	0.0	50
	4.0	100
	6.0	100
	6.1	50
Flow rate:	1.8 mL/min
Pressure:	256 bar
Column temp:	30 °C
Detector:	UV @ 280 nm; Data rate:  100 Hz; Response time: 0.025 s; Flow cell: 1 µL
Injection:	2 µL
Diluent:	Methanol

Figure 2. HPLC Separation of 18 PAHs with UV detection (peak ID see Table 2).

Table 2. Peak identification for the 18 PAH compounds measured

Peak Number	Compound
1	Naphthalene
2	Acenaphthylene
3	1-methylnaphthalene
4	2-methylnaphthalene
5	Acenaphthene
6	Fluorene
7	Phenanthrene
8	Anthracene
9	Fluoranthene
10	Pyrene
11	Benzo[a]anthracene
12	Chrysene
13	Benzo[b]fluoranthene
14	Benzo[k]fluoranthene
15	Benzo[a]pyrene
16	Dibenzo[a,h]anthracene
17	Benzo[g,h,i]perylene
18	Indeno[1,2,3-c,d]pyrene

Separation of 18 PAHs: UV and Fluorescence detection (FLD)

For this comparison of an FLD to a UV method, the chromatographic conditions were the same as for the above shown UV detection (Table 1), except for the changed injection volume of 0.3 µL. The chosen FLD settings Ex: 260/ Em: 350/440/500.

Figure 3. Separation of 18 PAHs with fluorescence and UV detection (peak ID see Table 2).

FPP vs. SPP: Comparison for PAH Analysis using EPA 8310 + 2 method

Comparison of the superficially porous particle (SPP) Ascentis^® Express PAH column to a fully porous particle column (FFP) in the market, both 5 cm x 4.6 mm I.D. under the conditions outlined in Table 3 is shown in Figure 4.

Table 3. Chromatographic conditions for SPP vs. FFP column comparison

Column:	Ascentis® Express 90 Å PAH, 2.7 µm, 5 cm x 4.6 mm I.D. (53539-U)
Competitor column:	FPP 95 Å PAH, 1.8 µm, 5 cm x 4.6 mm
Mobile phase:	[A] Water; [B] acetonitrile
Gradient:	Time	%B
	0.0	50
	4.0	100
	5.0	100
	5.1	50
Flow Rate:	1.8 mL/min
Pressure Drop:	Ascentis® Express PAH: 256 bar Competitor Column FPP: 344 bar
Column temp.:	30 °C
Detector:	UV @ 280 nm; Data rate: 100 Hz; Response time: 0.025 S; Flow cell: 1 µL
Injection:	2 µL
Diluent:	Methanol

The Ascentis^® Express PAH outperforms a fully porous particle (FPP) sub-2 μm column for a fast, 5 min separation of EPA method 8310 + 2 compounds demonstrating improved speed and resolution (Figure 4).

Figure 4. Comparison of Ascentis® Express PAH (green) and fully porous particle (FPP) sub-2 μm column (yellow) for a fast, 5 min separation of method EPA 8310 + 2 PAHs (Peak IDs see Table 2).

Highly Efficient Separation of 18 PAHs on a 3 mm I.D. Column

A column with 3 mm ID was used for the separation shown in Figure 5 with the conditions described in Table 4. This dimension provided more LC-MS suitability and higher sensitivity of the method.

Table 4. Chromatographic conditions for separation of 18 PAHs on a 10 cm x 3 mm I.D.column

Column:	Ascentis® Express 90 Å PAH, 2.7 µm, 10 cm x 3 mm (53535-U)
Mobile phase:	[A] Water; [B] acetonitrile
Gradient:	Time	%B
	0.0	50
	8.0	100
	10.0	100
	10.1	50
Flow rate:	0.77 mL/min
Pressure drop:	Initial Back Pressure: 263 bar
Column temp.	30 °C
Detector:	UV @ 280 nm; Data rate:  100 Hz; Response time: 0.025 s; Flow cell: 1 µL
Injection:	2 µL
Diluent:	Methanol

Figure 5. Fast and sensitive separation of 18 PAHs on Ascentis^® Express 90 Å PAH, 2.7 μm, 10 cm x 3.0 mm I.D (peak IDs see Table 2).

Conclusion

The Ascentis^® Express PAH column delivers a method-specific, robust, and high-efficiency separation of 16 + 2 standard PAH compounds with a resolution value of at least 1.5 in under 5 minutes for EPA 8310. Using a fluorescence detector, the method’s sensitivity increases substantially in comparison to UV detection.

The Ascentis^® Express PAH outperforms a fully porous particle (FPP) sub-2 μm column for a fast, 5 min separation of method EPA 8310+2 demonstrating improved speed and resolution. A 3 mm I.D. HPLC column enables more results, suitability for LC-MS use, and solvent savings.

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