Reduce Acetonitrile Dependence by Analyzing Polycyclic Aromatic Hydrocarbons with Methanol Based Mobile Phase

The recent acetonitrile shortage has resulted in limited availability and significantly increased solvent costs. In response, many labs are interested in alternate mobile phases for HPLC methods. Here we demonstrate an effective method for analyzing polycyclic aromatic hydrocarbons (PAHs) using methanol, instead of acetonitrile, resulting in a cost savings of approximately US$40 per liter. Baseline resolution of 16 target PAHs was easily obtained in approximately 23 minutes using a Pinnacle® II PAH column with a methanolic mobile phase.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants found in air, water, soil, and sludge. PAHs are an increasing human health concern, as this group of chemicals includes several known or suspected carcinogens. Analysis of PAHs often is done by high pressure liquid chromatography (HPLC) because it provides a higher degree of selectivity between structural isomers than gas chromatography (GC). Chromatographic separation of these isomers is critical, because they cannot be distinguished by a mass spectrometer. Acetonitrile is commonly used in the mobile phase when analyzing PAHs by HPLC; however, the recent acetonitrile shortage has limited its availability and significantly increased analytical costs. Here we detail conditions using a methanolic mobile phase and a Pinnacle® II PAH column that result in baseline resolution of 16 priority PAHs, offering labs a cost-effective alternative to using acetonitrile.

While switching to alternate solvent systems can be an attractive way to reduce costs, several points must be considered first, including: mode of separation, detector, and analyte compatibility. Here, reverse phase chromatography is used, so a polar mobile phase solvent, such as methanol, must be used with a nonpolar stationary phase. Since an ultraviolet (UV) detector is used, the UV cutoff of the solvent also must be considered. PAHs are analyzed at 254nm, so methanol can be used without producing interference, because its UV cutoff is 205nm (acetonitrile is 190nm). Finally, preliminary injections should be performed to ensure that the solvent does not react with target analytes in a way that causes undesirable effects, such as peak tailing, degradation peaks, or poor response. These points should always be considered when evaluating an alternate mobile phase solvent.

Procedure

A mixed standard containing 16 priority PAHs at 10µg/mL each was prepared in methanol. Note that it is important to prepare samples and standards in mobile phase or a compatible solvent, otherwise poor peak shape may be observed. The mixed standard was analyzed using a mobile phase gradient consisting of HPLC grade methanol and deionized water under constant flow conditions (1mL/min.). Since methanol is a weaker mobile phase than acetonitrile, a slightly higher starting percentage was used in order to achieve the desired resolution (75% methanol vs. 65% acetonitrile in typical methods). A Pinnacle® II PAH column (150mm x 3.2mm, 4µm) was chosen for this analysis, because it has a unique selectivity and is known to perform well for PAHs. All analyses were performed on a Shimadzu Prominence HPLC equipped with a UV detector set at 254nm. Column temperature was maintained at 30°C.

Results

All 16 PAHs were baseline resolved on the Pinnacle® II PAH column using a methanolic mobile phase (Figure 1). One elution order change was noted: when using acetonitrile, dibenzo(ah)anthracene elutes before benzo(ghi)perylene, however the opposite elution order is observed when using methanol. Total analysis time was approximately 23 minutes, which is slightly longer, but comparable, to common acetonitrile-based methods. Since acetonitrile is currently much more expensive than methanol, this methanol-based procedure offers labs an opportunity to considerably reduce solvent costs, while maintaining complete resolution of all target compounds.

Figure 1: Reduce acetonitrile costs by analyzing PAHs on Pinnacle® II PAH columns with a methanolic mobile phase.

naphthalene
acenaphthylene
acenaphthene
fluorene
phenanthrene
anthracene
fluoranthene
pyrene
benzo(a)anthracene
chrysene
benzo(b)fluoranthene
benzo(k)fluoranthene
benzo(a)pyrene
benzo(ghi)perylene
dibenzo(a,h)anthracene
indeno(1,2,3-cd)pyrene

Column:

Pinnacle® II PAH

Cat. #:

9219463

Dimensions:

150mm x 3.2mm

Particle size:

4µm

Pore size:

110Å

Sample:

Inj.:

10µL

Conc.:

10µg/mL each component

Sample diluent:

methanol

Conditions:

Instrument:

Shimadzu Prominence®

Mobile phase:

A: water
B: methanol

Time (min.)	%B
0.0	75
10.0	90
22.0	100

Flow:

1.0mL/min.

Temp.:

30ºC

Det.:

UV @ 254nm

LC_EV0480

Conclusion

The goal of this experiment was to establish conditions for analyzing PAHs using an alternate mobile phase in order to reduce acetonitrile use and its associated costs. Using a methanol-based mobile phase and a Pinnacle® II PAH column, all 16 target PAHs were baseline resolved in 23 minutes. The use of alternate solvent systems—such as the methanol-based PAH analysis shown here—should be considered by labs interested in reducing acetonitrile costs.

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