Extracting EPA Method 625.1 Semi-Volatile Analytes from Wastewater

Since 1984, the EPA has developed its own methods and acceptance criteria to regularly monitor organic pollutants within wastewater matrices. The revised version of EPA Method 625 includes the basic, neutral, and acidic extractions in EPA Method 625.1.

As per EPA Method 625.1, the target analytes are classified into three different tables. The non-pesticide/PCB basic/neutral analytes are listed in Table 1, while Table 2 includes the acidic analytes that may be extracted from a sample matrix for quantitative and qualitative determination of analytes. Additional analytes that can be extracted using 625.1 have been listed in Table 3.

There are both acidic and basic analytes to be recovered during the extraction; therefore, the method necessitates two pH adjustment steps when the semi-volatile analytes are extracted from a sample. At first, the pH of the water sample is adjusted to <2 to extract the neutral and acidic analytes.

After the acidic extraction, the basic analytes are extracted from the sample by adjusting the pH to greater than 11. Due to the huge number of analytes given in Tables 1–3 of this method, testing becomes challenging if all analytes are determined at the same time. Thus, only the “analytes of interest” must be determined and quality control (QC) testing must be performed only for those.

Analytes of interest are those necessary to be identified by a control/regulatory authority or in a permit, or by a client. If there is no list of specified analytes, it is essential to determine at least the analytes in Tables 1 and 2, and QC tests must be performed for these analytes. The analytes in Tables 1 and 2, and certain analytes from Table 3 have been determined as Toxic Pollutants (40 CFR 401.15), widened to a list of Priority Pollutants (40 CFR 423, Appendix A).1

The revised version of the EPA Method 625.1 permits laboratories to extract samples through solid-phase extraction. However, once the samples are extracted, they must be dried and concentrated.

This article discusses the extraction of all the analytes listed in Tables 1–2 of 625.1, as well as a few analytes from Table 3, extracted using the Biotage® Horizon 5000 with Atlantic® 8270.

Experimental

For preparing each 1 L of deionized water sample, 1 mL of hydrochloric acid was added to bring the pH of the sample to less than 2. In total, two method blanks were studied. While the first method blank consisted of 50 μg/L of surrogates, the second method blank included 100 μg/L of surrogates.

A 625.1 spike mix and surrogates were used to spike 12 samples in total. Of the 12 samples, 6 were spiked at a concentration of 50 μg/L, while the other 6 were spiked at a concentration of 100 μg/L. The Biotage® Horizon 5000 (P/N SPE-DEX 5000) was used to extract all the samples by employing the method illustrated in Table 1.

Table 1. Biotage® Horizon 5000 extraction method. Note: The method below was written in such a way as to be able to run those samples that may require a 5-μm Pre-filter, glass wool, and a fine mesh screen. Source: Biotage

Step Operation Message Attachment
1 Pause
with
Message
Part 1 of 3: Neutrals and Acids Elution. Have the Fast Flow Sediment Disk Holder (FFSDH) with One-Pass disk, 1 um filter, 5 um filter, top screen over the filters, 250 mL collection flask, and carbon cartridge installed. The down spout of the water in valve must push down on the top screen in the FFSDH. Click “Continue” to start Part 1. None
Step Operation Solvent Approximate
Solvent
Volume
(mL)
Purge Time
(s)
Pump Rate
(#)
Saturation Time Soak Time
(s)
Drain Time
(s)
2 Condition SPE Acetone 40 60 4 2 60 60
3 Condition SPE *Reagent Water 2 20 60 4 2 60 60
Step Operation Sample Flow Rate (#) Done Loading Sample Delay (s)
4 Load Sample 5 45
Step Operation Solvent Approximate
Solvent
Volume
(mL)
Purge Time (s) Pump Rate (#) N2 Blanket Saturation Time Soak Time (s) Drain Time (s)
5 Wash
Sample
Container
Reagent
Water
20 30 4 Off 2 5 30
6 Air Dry
Disk Timer
    360 6 Off      
7 Elute
Sample
Container
Acetone 20 20 4 Off 2 180 180
8 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 2 180 180
Step Operation Solvent Approximate
Solvent
Volume
(mL)
Purge Time
(s)
Pump Rate
(#)
N2 Blanket Saturation Time Soak Time
(s)
Drain Time
(s)
9 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 2 120 120
10 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 2 120 120
11 Elute
Sample
Container
Methylene
Chloride
17 15 6 Off 2 120 180
Step Operation Message Attachment
12 Pause
with
Message
Part 2 of 3: Ion Exchange Elution. Remove the 250 mL collection flask containing the neutrals and acids elution. Stopper the flask and set aside for part 3. Then install a clean 125 mL flask to collect the ion exchange elution. Click “Continue” to start part 2. None
Step Operation Solvent Approximate
Solvent
Volume
(mL)
Purge Time (s) Pump Rate (#) N2 Blanket Saturation Time Soak Time (s) Drain Time (s)
13 Elute
Sample
Container
Acetone 20 20 4 Off 2 0 180
14 Elute
Sample
Container
1% Ammonium Hydoxide 20 30 4 Off 2 120 120
15 Elute
Sample
Container
Acetone 20 20 4 Off 2 180 120
16 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 2 180 180
17 Elute
Sample
Container
Methylene
Chloride
16 15 4 Off 2 120 180
18 Elute
Sample
Container
Methylene
Chloride
16 15 4 Off 2 120 180
19 Elute
Sample
Container
Methylene
Chloride
16 15 6 Off 2 120 180
Step Operation Message Attachment
20 Pause
with
Message
Part 3 of 3: Carbon Cartridge Elution. Remove the carbon cartridge from the tubing lines. Connect the tubing ends together. Using a 20 cc syringe, plunge the carbon cartridge with air through the cap adapter to reseat the carbon bed on the frit. Replace the cap adapter with the funnel on the cartridge. Replace the disk holder with the cartridge. Replace the 125 mL flask with the 250 mL flask containing the neutrals and acids elution from part 1. Stopper the 125 mL flask. Click “Continue” to start part 3. None
Step Operation     Dry Time (s) Pump Rate (#) N2 Blanket      
21 Air Dry
Disk Timer
    60 6 Off      
Step Operation Solvent Approximate
Solvent
Volume
(mL)
Purge Time
(s)
Pump Rate
(#)
N2 Blanket Saturation Time Soak Time
(s)
Drain Time
(s)
22 Elute
Sample
Container
Acetone 25 20 4 Off 3 60 60
23 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 3 60 20
24 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 3 60 20
25 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 3 60 20
26 Elute
Sample
Container
Methylene
Chloride
17 15 4 Off 3 60 20
27 Elute
Sample
Container
Methylene
Chloride
17 15 6 Off 3 60 60

*“Reagent Water 2” is added to the list of configured solvents with the Waste Destination of “Solvent Waste” rather than using the factory programmed “Reagent Water”, which is sent to “Water Waste”.

 

Atlantic® 8270 One-Pass Disks were used in combination with the 8270 Carbon Cartridge Max-Detect as the solid-phase extraction consumables. Each Atlantic® 8270 One-Pass Disk includes a mixed-mode chemistry, which avoids the need for secondary adjustment of the sample’s pH.

One Pass solid-phase extraction disks (P/N 47-2346-11), 8270 Carbon Cartridges Max-Detect cartridges (P/N 49-2620-01), and 1.0 Micron Atlantic® Fast Flow Pre-Filters (P/N FFAP-100-HS1) for analyte extraction. The DryDisk® Solvent Drying System and the TurboVap® II are used for solvent drying and concentration and analysis is by GC-MS.

Figure 1. One Pass solid-phase extraction disks (P/N 47-2346-11), 8270 Carbon Cartridges Max-Detect cartridges (P/N 49-2620-01), and 1.0 Micron Atlantic® Fast Flow Pre-Filters (P/N FFAP-100-HS1) for analyte extraction. The DryDisk® Solvent Drying System and the TurboVap® II are used for solvent drying and concentration and analysis is by GC-MS. Image Credit: Biotage

By contrast, the pH of the disk can be adjusted by a rinse with 1% ammonium hydroxide. Once the analytes retained on the disk are collected, more volatile analytes in a sample are recovered by eluting the 8270 Carbon Cartridge Max-Detect (P/N 49-2620-01).

After completing the extraction, the DryDisk® Solvent Drying System (P/N SDS-101-19/22) was used in combination with DryDisks® (P/N 40-705-HT) to dry the samples using the parameters illustrated in Table 2.

Table 2. Drying parameters via the DryDisk® Solvent Drying System. Source: Biotage

Parameter Setting
Vacuum: -8 "Hg

 

The dried extracts were added to 200 mL evaporation tubes with an endpoint of 0.9 mL (P/N C128506) to be used in the TurboVap® II (P/N 415001). The final volume of the dried extracts went beyond 200 mL, so the samples were filled in the evaporation tubes in two portions.

TurboVap® II solvent evaporator.

Figure 2. TurboVap® II solvent evaporator. Image Credit: Biotage

Biotage® Horizon 5000.

Figure 3. Biotage® Horizon 5000. Image Credit: Biotage

Before the final extract volume and glassware rinses could be added, the first portion was concentrated to about 15 mL. The parameters mentioned in Table 3 were used to evaporate the samples.

Table 3. Evaporation parameters for drying via the Biotage TurboVap® II. Source: Biotage

Parameter Setting
Inlet Nitrogen Pressure: 87 psi
Gas Flow: 2.8 mL/min
Water Bath Temperature: 40 °C

 

Once the samples were evaporated on the TurboVap® II, methylene chloride was used to bring them to 1 mL, following which they were transferred to GC-MS vials. To each 1 mL of the completed extracts in an aliquot, internal standard was added and analysis was performed using the GC/MS using instrument parameters described in Table 4.

Table 4. GC/MS Method. Source: Biotage

Parameter Setting
Injection Volume 1 µL
Inlet Temperature 280 °C
Injection Mode Split
Split Ratio 10:1
Split Flow 12.5 mL/min
Gas Type Helium
GC Column Zebron™ ZB-Semi Volatiles (Phenomenex),
30 m, 0.25 mm, 0.25 µm
GC Mode Constant Flow: 1.3 mL/min
Oven Program 45 °C hold for 1.0 minutes
Ramp 15 °C/min to 270 °C
Ramp 6 °C/min to 318 °C
MS Ions Monitored 35–550 AMU

 

Results and discussions

The total time taken to concentrate each sample using the TurboVap® II is illustrated in Table 5.

Table 5. Concentration times on the TurboVap® II. Source: Biotage

Sample Concentration (µg/L) Time (Hour:Min.:Sec.)
1 50 1:51:56
2 50 1:42:00
3 50 1:56:36
4 50 1:37:25
5 50 1:47:41
6 50 1:55:53
7 100 1:48:43
8 100 1:39:12
9 100 1:48:30
10 100 1:53:53
11 100 1:46:59
12 100 1:55:49

 

During sample concentration, each sample vial was covered with aluminum foil to restrict the reaction of the evaporating acetone with the water vapor molecules from the warm water bath.

Both sides of the foil caps were pierced with two holes to insert the nozzle (N2 flow) into the vials, as well as for escaping of evaporated solvent. There was only a slight variation in the concentration times between the samples. On average, the samples that consisted of about 303 mL of solvent at first were concentrated on the TurboVap® II to about 0.9 mL in 1 hour and 49 minutes.

Table 6 outlines the data for the spiked samples and method blanks, which includes %RSD for each level (50 μg/L and 100 μg/L) and average percent recovery. In Table 6, the analytes have been color-coded based on the legend outlined in the header. Table 6 lists all the analytes from Tables 1 and 2 and a few from Table 3 of the EPA Method 625.1.

Table 6. Average percent recovery for 625.1 analytes spiked at 50 μg/L and 100 μg/L using the Biotage® Horizon 5000 and the TurboVap® II. Source: Biotage

 

Tables 4 and 5 in EPA Method 625.1 outline the EPA acceptance criteria for the analytes in Tables 1 and 2 analytes, respectively. For the compounds listed in Tables 3 and 8, the EPA has chosen not to set particular acceptance criteria themselves. Rather, the laboratories are allowed to develop the suitable acceptance criteria in one of multiple ways: using the range 60%–140%, based on laboratory control charts, or using the guidelines described in section 8.4.5 of the 625.1 method itself.

All the analytes listed in Tables 1 and 2 of the EPA method met the acceptance criteria set by the EPA method for both concentration levels. For each sample set, the percent relative standard deviations showed minimal deviation in percent recovery.

Section 6.8.1 of the EPA Method 625.1 specifies that at least three surrogates are needed for analysis given that they do not interfere with target analytes. The six surrogates used in this article met the acceptance criteria of the EPA method for three of the samples at 100 μg/L and all the samples at 50 μg/L.

The recovery of two surrogates, phenol-d6 and 2-fluorophenol, from the other three samples at 100 μg/L was short of the lower passing limit of 60%, which led to a lower average. But only a minimum of three surrogates is required for each sample in the EPA method.

Although two surrogates were short of the lower limit, four surrogates reached the limits. The estimated percent relative standard deviation for each surrogate in the list specified in Table 7 was 7.23%, 5.48%, 1.99%, 2.93%, 3.66%, and 2.36%, which denotes minimal deviation between the samples.

Extraction of the two method blanks was performed with surrogates at different levels. For the first blank, the concentration of the surrogates was 50 μg/L, and that for the second blank was 100 μg/L. Neither of the blanks exhibited any false positives for target analytes beyond the detection limit of 10 μg/L. This is specified as N.D, or “not detected.”

References

  1. United States Environmental Protection Agency, Method 625.1: Base/Neutrals and Acids by GC/MS, available at www.epa.gov, December 2016.

About Biotage

Biotage offers solutions, knowledge, and experience in the areas of analytical chemistry, medicinal chemistry, peptide synthesis, separation and purification. Customers include pharmaceutical, clinical and biotech companies, companies within the food industry and leading academic and government institutes. The company is headquartered in Uppsala and has offices in the US, UK, China, S. Korea, India, and Japan. Biotage has approx. 460 employees and had sales of 1,101 MSEK in 2019. Biotage is listed on the NASDAQ Stockholm.

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Biotage is a global Life Science company that develops innovative and effective solutions for separation within organic and analytical chemistry, as well as for industrial applications. We help shape the sustainable science of tomorrow and our future society for the benefit of humankind. Our mission is to help our customers to make the world more sustainable, healthier, and cleaner.

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The company has a strong customer base of industry and academic partners, which include the world’s top 20 pharmaceutical companies and prestigious academic and government institutes such as the US National Institutes of Health, the US Centers for Disease Control and Prevention and the Karolinska Institute in Sweden.

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Headquartered in Uppsala, Sweden, Biotage AB also has facilities in Lund, Sweden; Charlotte, NC, USA; San Jose, CA, USA; Salem, NH, USA; Cardiff, UK; Bundang, S. Korea; New Dehli, India; Tokyo and Osaka, Japan; and Shanghai, China.


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Last updated: Dec 4, 2020 at 5:49 AM

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