Automating EPA method 3630C silica gel cleanup for TPH analysis

Introduction

Total petroleum hydrocarbon (TPH) analysis in environmental matrices may require selective cleanup to remove polar compounds while preserving nonpolar hydrocarbon components for accurate quantitation. Silica gel cleanup for TPH analysis, aligned with EPA Method 3630C, remains one of the most effective approaches for preparing extracts prior to GC analysis under EPA Method 8015D, nonhalogenated organics by gas chromatography-flame ionization detection (GC-FID).

EPA Method 3630C describes the use of activated silica gel to remove polar compounds from sample extracts prior to gas chromatographic analysis. Environmental extracts from soils, groundwater, wastewater, and biosolids frequently contain co-extracted polar organic material. The method is widely applied in petroleum hydrocarbon workflows to eliminate naturally derived polar constituents (e.g., fatty, humic, and fulvic acids) that are not representative of the petroleum sample.

ISOLUTE® SI solid-phase extraction (SPE) cartridges provide strong retention of polar compounds via hydrogen bonding and dipole interactions. Nonpolar hydrocarbons elute readily with nonpolar solvents such as pentane or hexane. This polarity-driven selectivity forms the chemical basis for silica gel cleanup for TPH analysis under EPA 3630C principles. Following cleanup, hydrocarbon quantitation is performed using GC-FID.

Analytes

This method is routinely used to measure the following analyte groups:

• Diesel range organics (DRO): C10–C28

• Motor oil range organics (MRO): C17–C44

• Broad range TPH: C6–C44

Sample preparation procedure

1. 460-0050-B: ISOLUTE® SI 500 mg/3 mL

    

2. C44651: test tubes (12 x 75 mm)

3. 414141: 1000 µL clear tips
   

Sample pre-treatment and extraction

1.  Environmental samples are extracted per laboratory method requirements.

    

2. Aqueous samples (e.g. 250 – 1000 mL) are commonly processed utilizing separatory funnel liquid-liquid (EPA 3510C) or solid-phase extraction (EPA 3535A).

    

3. Solid samples (e.g. 2 – 10 g) are commonly processed utilizing microwave (EPA 3546), manual and automated Soxhlet (EPA 3540C/3541), or pressurized fluid extraction (EPA 3545A).

    

4. Concentrate sample extracts to 2 mL. Fortify with the surrogate (n-octacosane) and reverse surrogate (decanoic “capric” acid).

   a. The purpose of the reverse surrogate is to demonstrate the absence of polar analyte breakthrough.

Extract cleanup procedure

TPH sample extracts undergo the silica gel cleanup SPE process on the Biotage® Extrahera™ following EPA Method 3630C.

1. Add 1 mL of sample extract to a 12 x 75 mm test tube.

2.  

    

   Add samples to the Biotage® Extrahera™ 12 x 75 mm sample/collection rack.  Secure into “position 4” of the system.

3. Fill the 24 position 3 mL media rack with the 500 mg/3 mL silica gel cartridges.  Secure into “position 3” of the system.

4. Add the proper number of 12 x 75 mm test tubes to another 12 x 75 mm sample/collection rack.  Place the sample/collection rack onto the carousel in position A, ensuring A1 of the rack is in the bottom right corner.

    

5. Load the silica gel cleanup method outlined in Table 1 onto the Biotage® Extrahera™.

Silica gel cleanup on the Biotage® Extrahera™ using the TPH_ISOLUTE 3 mL/500 mg Silica_Cleanup method.

Table 1: Silica gel cleanup method for the Biotage® Extrahera™.

Post-extraction

1. Once the cleanup method is complete, remove the collected extracts containing methylene chloride:pentane (3:1, v/v, 1.75 mL).  Concentrate to 1 mL in the TurboVap® EH or 96 Dual:

     a.    TurboVap® EH (PN: 415540)

        i.    Bath temperature: 40°C

        ii.    Nitrogen gas flow rate: 1.2 L/min

        iii.    Time: < 5 minutes

     b.    TurboVap® 96 Dual (PN: 418000)

        i.    Time: 2.5 minutes, 2.5 minutes

        ii.    Gas flow: 50 L/min, 50 L/min

        iii.    Gas temperature: 40°C, 40°C

        iv.    Plate temperature: 40°C, 40°C

        v.    Plate height: 55 mm, 55 mm

Analytical conditions

Once extracts are concentrated to 1 mL, analyze extracts via GC-FID.
GC conditions
•    Column: Zebron ZB-1; dimensions: 30 meters x 0.25 mm x 0.25 μm; p/n: 7HG-G001-11
•    Carrier gas: helium 30 cm/sec @ 35 °C (constant flow)
•    Injection: split 20:1 @ 250 °C, 1 μL
•    Oven settings: 35 °C (2 min) to 110 °C @ 6 °C/min
FID conditions
•    Temperature: 300 °C

Results

The laboratory provided their method acceptance criteria for both water and soil as the criteria differed for both matrices. See Table 2 below which outlines this information.

Table 2: Silica gel cleanup method acceptance criteria for water and soil samples.

The acceptance criteria shown in Table 2 were achieved during work conducted on-site at the laboratory. All method blank (MB) background results are summarized in Table 3 and were below the necessary limit of 5 ppb. The solid matrix MB samples were non-detect for both the DRO and MRO ranges. The water matrix MB samples resulted in 1.2 mg/L for the DRO range and varied from 2.8 to 4.5 mg/L for the TPH range. The TPH range covers the entire C6-C44 hydrocarbon range and is used to measure overall method background (< 5 mg/L). These results demonstrate that the workflow had acceptable background levels across the entire range beyond just DRO and MRO (C10–C28 and C17–C44 respectively).

Table 3: Measured DRO, MRO, and TPH background from seven replicate method blanks.

Method blank samples were spiked with 50 mg of n-octacosane surrogate to demonstrate efficient recovery of non-polar hydrocarbons. To demonstrate effective cleanup, solid and aqueous matrix surrogate recoveries must range from 60-138% and 53-151% respectively. Utilizing the strictest of the two ranges (60-138%), both solid and aqueous MB samples passed the surrogate recovery criteria. Samples were also spiked with 500 mg of decanoic (capric) acid reverse surrogate to confirm retention of polar hydrocarbon interferences to the silica gel. Recovery of decanoic acid must be < 1% of the amount spiked into the sample. Figure 1 displays results from the MB sample indicating surrogate recoveries met the required acceptance criteria. In addition, there were no detectable levels of decanoic acid in any sample, confirming effective removal of polar hydrocarbons.

Figure 1: Surrogate recoveries from replicate method blanks (n=7) spiked with 50 mg of n-octacosane.

Laboratory control sample (LCS) tests were conducted with motor oil, soil, and water samples. Each matrix was spiked with 50 mg of n-octacosane surrogate and 500 mg of decanoic acid reverse surrogate. Soil and water samples were spiked with 400 mg of DRO mix (C10-C28). Motor oil and soil samples were spiked with 400 mg of MRO mix (C17-C44). Water samples were also spiked with 400 mg of TPH mix (C6-C44).  All matrices achieved acceptable LCS recoveries for MRO (77-125%), DRO (70-130%), and TPH (70-130%). Results for motor oil, soil, and water samples are shown in Figures 2, 3, and 4 below.

Figure 2. DRO and MRO recoveries from replicate soil LCS samples (n=8) spiked with DRO (400 mg) and MRO (400 mg).

Figure 3. DRO and TPH recoveries from replicate water LCS samples (n=6) spiked with DRO (400 mg) and TPH (400 mg).

Figure 4. MRO recoveries from replicate motor oil LCS samples (n=2) spiked with MRO (400 mg).

The n-octacosane surrogate recovery data associated with the LCS tests were well within passing and are shown in Figures 5, 6, and 7. Motor oil and solid matrix surrogate recoveries must range from 60-138%, while aqueous matrix surrogate recoveries must range from 53-151%. The surrogate data remained consistent with each test resulting in 80-105% range for all matrices.  Reverse surrogate (decanoic acid) recovery was <1% of the amount spiked indicating effective removal of polar hydrocarbons for all three matrices.

Figure 5. Surrogate recoveries from replicate soil LCS samples (n=8) spiked with 50 mg of n-octacosane.

Figure 6. Surrogate recoveries from replicate water LCS samples (n=6) spiked with 50 mg of n-octacosane.

Figure 7. Surrogate recoveries from replicate motor oil LCS samples (n=2) spiked with 50 mg of n-octacosane.

To further demonstrate the robustness of the silica gel cleanup process, high concentrations of reverse surrogate (decanoic acid) were added to soil LCS samples ranging from 5,000 to 20,000 mg.  In addition, samples were spiked with DRO (400 mg), MRO (400 mg) hydrocarbons, and n-octacosane surrogate (50 mg). The limit for reverse surrogate breakthrough is < 1% of the spiked concentration. No decanoic acid was detected in any LCS test confirming no  reverse surrogate breakthrough even with the addition of up to 20,000 mg. These results demonstrate the robustness of the method for retaining high concentrations of polar hydrocarbons. Figure 8 displays acceptable recoveries of DRO and MRO for samples spiked with high concentrations of decanoic acid.

Figure 8. DRO and MRO recoveries from replicate soil LCS samples (n=4) spiked with DRO (400 mg), MRO (400 mg) and decanoic acid (5,000 – 20,000 mg).

The n-octacosane surrogate recovery data associated with the high concentration reverse surrogate (5,000 – 20,000 mg) LCS tests were also well within passing and are outlined in Figure 9. The surrogate results for the high-level reverse surrogate experiments remained well within the passing range (89-102%), thus further proving that the retention of polars does not affect the recovery of targets.

Figure 9. Surrogate recoveries from replicate soil LCS samples (n=8) spiked with n-octacosane (50 mg) and decanoic acid (5,000 – 20,000 mg).

Four soil samples with unknown quantities of DRO and MRO hydrocarbons were spiked with 50 mg of n-octacosane surrogate, 500 mg of decanoic acid reverse surrogate, and tested with the silica gel cleanup process. These results are shown in Figure 10, while the corresponding surrogate recoveries (Figure 11) demonstrate the reliability of the measured target hydrocarbon concentration. Reverse surrogate was not detected demonstrating the effectiveness of the silica gel cleanup process for sample matrices with unknown concentrations of DRO and MRO hydrocarbons. Figure 11. Surrogate recoveries from soil samples with unknown quantities of DRO and MRO hydrocarbons (n=4) spiked with 50 mg of n-octacosane.

Figure 10. Unknown quantities of DRO and MRO hydrocarbons from soil samples (n=4).

Figure 11. Surrogate recoveries from soil samples with unknown quantities of DRO and MRO hydrocarbons (n=4) spiked with 50 mg of n-octacosane.

Summary and conclusion

This optimized silica gel cleanup protocol for EPA Method 3630C removes polar interferences from soil and water extracts for analysis by EPA Method 8015D. The advanced pipetting capabilities of the Biotage® Extrahera™ allow highly precise and accurate liquid handling of pentane and methylene chloride. The dual-flow positive pressure functionality of the Biotage® Extrahera™ minimizes the need for high amounts of silica gel sorbent and solvent to achieve removal of polar hydrocarbons from TPH extracts. Compared to manual techniques, the Biotage® Extrahera™ met method performance criteria while demonstrating:

• 40x reduction in solvent consumption

• 20x reduction in silica gel sorbent waste

• 8x increase in productivity

Robust removal of polar interferences was demonstrated by the absence of reverse surrogate breakthrough when spiked at high levels ranging from 5,000 to 20,000 ppm while maintaining high recoveries of non-polar target analytes. Method blank background criteria (<5 µg/mL) were met across the total petroleum hydrocarbon range (C6-C44).  In addition, laboratory control sample DRO (C10-C28), MRO (C17-C44), and surrogate (n-octacosane) spike recoveries were within the acceptable matrix-based ranges.

This automated silica gel cleanup technique reduces solvent consumption, shortens processing times, and provides reliable sample preparation. The work described here demonstrates how the Biotage® Extrahera™ can improve operational efficiency while maintaining data precision and accuracy for environmental laboratories performing cleanup on TPH soil and water samples.

Chemicals and reagents 

1.    Fisher Scientific: Methylene Chloride, Optima® grade, (PN: D151-4)

2.    Fisher Scientific: Pentane, pesticide grade, (PN: P400-4)

Additional information

Tips to reduce contamination sources that could contribute to TPH (C6-C44) background:

1.    Verify SPE cleanliness

2.    Soak new Biotage® Extrahera™ solvent reservoirs in a sealed container filled with pentane overnight

3.    Bake all glassware at 400°C for 4 hours

4.    Automate pipette tip cleaning with the Biotage® Extrahera™

Literature number: AN1035

Ordering information