Utilizing 100 ml sample volumes for EPA method 608.3 automated extractions on the Biotage® Horizon 5000

Written by Biotage | Feb 25, 2026 9:36:07 AM

Acknowledgements: A very special thank you to Adirondack Environmental Services for working with us on finding a solution for reducing sample volumes for EPA Method 608.3.

Introduction

Pesticides, including insecticides, fungicides, and herbicides, are used extensively to increase agricultural yield. However, it has since been determined that these pesticides can be detrimental to the health of humans as well as animals. It is important to monitor organochlorine pesticides and polychlorinated biphenyls (PCBs) not just in drinking water (US EPA Method 508), but also in wastewater (US EPA Method 608.3). After the wastewater has gone through the treatment plant, testing needs to be done to ensure that the treated wastewater is safe and will not harm the ecosystems that it flows into.

Some influent wastewater samples can be heavily laden with particulates, sludge, and the like, so it may be difficult to extract an entire liter of this sample when following US EPA Method 608.3. Fortunately, with this method, laboratories have the option of extracting smaller sample volumes, thus reducing the particulates, sludge, etc. that needs to be filtered out of the sample during extraction. This leads to a direct cost savings as smaller solid phase extraction (SPE) disks can be used and less prefilters, filter aids, and solvent are required for each sample extraction. In addition, indirect costs related to shipping can be reduced for all samples types regardless of their particulate content since reduced volumes mean smaller physical sizes and more samples will fit in each shipping container.

The purpose of this application note is to provide a solution for extracting 100 mL of 608.3 wastewater samples utilizing the Biotage® Horizon 5000 to automatically load the sample and rinse the original sample container for solid phase extraction (SPE), DryDisk® Solvent Drying System for drying the residual water from the extracts, and the TurboVap® for concentrating the dried extracts. Samples are extracted in methylene chloride, then solvent exchanged into hexane prior to analysis by Gas Chromatography/Electron Capture Detection (GC/ECD).

Instrumentation

Biotage instruments and consumables

Biotage® Horizon 5000 (P/N SPE-DEX 5000)
DryDisk® Solvent Drying System (P/N SDS-101-19/22)
TurboVap® II (P/N 415001)
Atlantic® ReadyDisk C18 (P/N 47-6005)
DryDisk®-R (P/N 40-1000-HT)
Evaporation Tube TurboVap® II, 200 mL, 1 mL EndPoint (P/N C128506)

Analytical instrument:

Agilent 6890; HP Micro Dual GC-ECD
Column 1: Rtx-CLPesticides (30 m, 0.32 mm ID, 0.32 µm film thickness)
Column 2: Rtx-CLPesticides2 (30 m, 0.32 mm ID, 0.32 µm film thickness)

Method summary

Purge the Biotage® Horizon 5000 using the method found in Table 1.
Obtain the 100 mL samples of DI water that will be extracted. Acidify to a pH <2 with HCl. For Matrix Spike (MS) and Matrix Spike Duplicate (MSD) test samples only, 2 mg of oil and grease (hexadecane and stearic acid) was added to the acidified DI water.
Fortify samples with surrogates decachlorobiphenyl (DCB) and tetrachloro-m-xylene (TCMX).
Fortify necessary samples with the proper amount of spike mix if analyte retention testing will be determined by this sample.
Attach the water inlet valve to the sample bottle (and cap adapter if necessary) and place onto the Biotage® Horizon 5000.
Place an Atlantic® ReadyDisk C18 onto each active station.
Attach the 19/22 tapered collection vessel to each active station. For the purposes of this application note, the VOA vial adapter with a 40 mL VOA vial was used as the collection vessel.
Run the extraction method found in Table 2 with the Biotage® Horizon 5000.
Once the extraction concludes (approximately 30 minutes), remove the collection vessels from the extractor.
Set up a DryDisk® Solvent Drying System (SDS) with a DryDisk®-R for each extract and attach a collection vessel to the bottom.
Pour each extract into a glass reservoir over the DryDisk®-R and open the stopcock. Allow the extract to flow through completely before rinsing the collection vessel three times with methylene chloride into the reservoir (approximately 15 mL in total).
Once all the extract has been transferred, rinse the reservoir and DryDisk®-R once with methylene chloride (approximately 10 mL).
Transfer the dried extract to a concentration tube and concentrate. For the purposes of this application note, the TurboVap® II, 200 mL, 1 mL EndPoint evaporation tube was used in conjunction with the TurboVap® II. Cover the tube with aluminum foil and using a glass pipette, poke three holes into the foil (one on the left, one in the middle, and one on the right). The nitrogen nozzle should sit in the right hole of the foil.
Concentrate to endpoint (approximately 0.7 mL) following the concentration protocol found in Table 3.
Add 10 mL of hexane directly into the tip of the evaporation tube, using a glass pipette, into each concentrated extract. Swirl well for the solvent exchange step.
Replace the foil on the concentration tubes and bring the sample to an end point of 0.7 mL again using the protocol found in Table 4.
Once concentration is complete, bring each extract up to 2 mL with hexane.
Perform any necessary cleanup procedures for PCBs/ Pesticides. For the purposes of this application note, the following cleanups were conducted:
1. For PCB extracts only, perform a sulfuric acid cleanup.
2. For pesticides and PCB MS extracts, perform a copper cleanup.
3. For pesticides and PCB Method Detection Limit (MDL), Laboratory Control Spike (LCS), and MS extracts, perform a florisil cleanup.
Analyze by GC/ECD following the method protocol found in Table 5.

Table 1. Biotage® Horizon 5000 608.3 purge method.

Step	Step Description	Solvent	Volume (mL)	Purge (s)	Pump Speed	Saturate (s)	Soak (s)	Drain/Elute (s)	Done Loading Delay (s)
1	Condition SPE Disk	Reagent Water	15	30	4	0	0	30
2	Condition SPE Disk	Methanol	15	30	4	0	0	30
3	Elute Sample Container	Acetone	15	15	4	0	0	30
4	Elute Sample Container	Methylene Chloride	15	15	4	0	0	30
5	Elute Sample Container	Methylene Chloride	15	15	6	0	0	30

Table 2. Biotage® Horizon 5000 608.3 extraction method.

Step	Step Description	Solvent	Volume (mL)	Purge (s)	Pump Speed	Saturate (s)	Soak (s)	Drain/Elute (s)	Done Loading Delay (s)
1	Condition SPE Disk	Methylene Chloride	20	60	4	1	60	60
2	Condition SPE Disk	Acetone	20	60	2	1	60	30
3	Condition SPE Disk	Methanol	20	60	2	2	60	10
4	Condition SPE Disk	Reagent Water	20	60	2	1	45	10
5	Load Sample				2				45
6	Wash Sample Container	Reagent Water	15	60	6	2	20	30
7	Air Dry				6			360
8	Elute Sample Container	Acetone	3	15	2	1	60	45
9	Elute Sample Container	Methylene Chloride	5	15	2	1	60	45
10	Elute Sample Container	Methylene Chloride	5	15	6	1	60	60

Table 3. TurboVap® II evaporation parameters for methylene chloride (Pre-Solvent Exchange).

Settings
Gradient Type	End-Point Detection	Bath Temperature
Ramp
Method Step	Flow (L/min)	Time (min)
Step 1	3.0	9
Step 2	5.0	1

Table 4. TurboVap® II evaporation parameters for hexane (Post-Solvent Exchange).

Settings
Gradient Type	End-Point Detection	Bath Temperature
Ramp	On	60 °C
Method Step	Flow (L/min)	Time (min)
Step 1	4.2	2
Step 2	5.0	1

Table 5. GC/ECD conditions.

Inlet Settings
Pesticides Injection Volume	PCB Injection Volume	Injection Mode	Gas Type
1 µL	4 µL	Pulsed Splitless	Helium
Temperature	Pulse Pressure	Pulse Time	Total Purge Flow
50°C	28psi	1 min	60.4 mL/min
Purge Time Gas Saver Flow Gas Saver Time
0.75 min	20 mL/min	2 min
Oven Settings
Initial Oven Temperature	Initial Oven Time	Post Oven Temperature	Maximum Oven Temperature
125°C	0.5 min	120°C	325°C
Method Step	Rate (°C/min)	Temperature (°C)	Hold Time (min)
Step 1	45	200	0
Step 2	12.5	230	0
Step 3	30	300	1.5

Results and discussion

Demonstration of low background

In order to confirm that the instrumentation utilized for the entire preparation workflow was free from background
contamination, a 100 mL laboratory reagent blank (LRB) was processed and analyzed for trace levels of PCBs and Pesticides. The result of this test demonstrated that the instrumentation used was free from PCBs and Pesticides. In addition, the workflow demonstrated excellent extraction performance with surrogate recoveries in the 90% range (Table 6).

Table 6. Blank results (n=1).

Analyte	Lowest Calibration Point (ng/L)	Blank Results (ng/L)	Acceptance Criteria
PCB	1016	25	N.D
PCB	1260	25	N.D
PCB	1221	25	N.D
PCB	1254	25	N.D
PCB	1242	25	N.D
PCB	1248	25	N.D
PCB	1268	25	N.D
4,4´-DDD	33	N.D	PASS
4,4´-DDE	33	N.D	PASS
4,4´-DDT	33	N.D	PASS
Aldrin	16	N.D	PASS
alpha-BHC	16	N.D	PASS
alpha-Chlordane	16	N.D	PASS
beta-BHC	16	N.D	PASS
delta-BHC	16	N.D	PASS
Dieldrin	33	N.D	PASS
Endosulfan	I	16	N.D
Endosulfan	II	33	N.D
Endosulfan	sulfate	33	N.D
Endrin	33	N.D	PASS
Endrin	aldehyde	33	N.D
Endrin	Ketone	33	N.D
gamma-BHC	16	N.D	PASS
gamma-Chlordane	16	N.D	PASS
Heptachlor	16	N.D	PASS
Heptachlor	epoxide	16	N.D
Methoxychlor	16	N.D	PASS
Chlordane	16	N.D	PASS
Toxaphene	50	N.D	PASS
DCB	(Surr.)	33	94%
TCMX	(Surr.)	33	90%

Surrogate recovery acceptance criteria is within 60–140%.
N.D. = Not Detected.

Determination of the method detection limits (MDL)

To demonstrate that acceptable method detection limits can be achieved with 100 mL reduced sample volume extractions, laboratory defined practical quantitation limits (PQLs) were utilized as a benchmark for acceptance criteria. Utilizing Adirondack Environmental Services’ PQL values, seven replicate blanks were fortified at low spike levels to demonstrate the reduced sample volume extraction workflow was capable of passing the MDL study for the laboratory’s current limits (Table 7). Results for each individual MDL replicate are provided in the appendix section (Tables 12 and 13). Per EPA Method 608.3 method requirements, acceptance criteria must be met using at least one GC/ECD column. The data here demonstrates that an MDL study was passed on both GC/ECD columns.

Table 7. Calculated method detection limits (n=7).

Analyte	Spike Amount (ng/L)	Column 1 - MDL (ng/L)	Column 2 - MDL (ng/L)	Laboratory Maximum PQL (ng/L)	Acceptance Criteria
PCB 1016	100	32	60	65	PASS
PCB 1260	100	42	39	65	PASS
PCB 1221	200	68	116	130	PASS
PCB 1254	100	16	22	65	PASS
PCB 1242	100	22	29	65	PASS
PCB 1248	100	12	12	65	PASS
PCB 1268	100	19	23	65	PASS
4,4´-DDD	100	14	29	100	PASS
4,4´-DDE	100	13	17	100	PASS
4,4´-DDT	100	26	21	100	PASS
Aldrin	100	14	12	50	PASS
alpha-BHC	100	35	14	50	PASS
alpha-Chlordane	100	23	14	50	PASS
beta-BHC	100	33	29	50	PASS
delta-BHC	100	11	30	50	PASS
Dieldrin	100	13	11	100	PASS
Endosulfan I	100	10	10	50	PASS
Endosulfan II	100	22	20	100	PASS
Endosulfan sulfate	100	28	13	100	PASS
Endrin	100	29	13	100	PASS
Endrin aldehyde	100	37	31	100	PASS
Endrin Ketone	100	64	11	100	PASS
gamma-BHC	100	17	15	50	PASS
gamma-Chlordane	100	14	36	50	PASS
Heptachlor	100	21	15	50	PASS
Heptachlor epoxide	100	19	10	50	PASS
Methoxychlor	100	47	31	500	PASS
Chlordane	200	32	44	50	PASS
Toxaphene	1000	281	273	1000	PASS

Initial demonstration of accuracy and precision

To determine the accuracy and precision of the sample preparation process, four samples were prepared at mid to high level concentrations. The precision and accuracy can be found in Tables 8 and 9, as well as illustrated in Figures 1 and 2. Due to the varying values of acceptance criteria for each analyte, the interim acceptance criteria of 60–140% (Section 8.4.5) was used as the limits on Figure 1 for ease of observation. The lowest %SD limit within 608.3 is 22%. This percent was used as the limit on Figure 2 for ease of observation. The remaining analytes have limits higher than 22%, but it is worth noting that all analytes pass utilizing 22% for the %SD, illustrating the precision with the instrumentation used for conducting this study. Since all analytes recovered within the recovery range of the method, this study also demonstrates that the reduced sample volume (100 mL) extraction provides exceptional precision and accuracy. Results for individual precision and accuracy replicates are provided in the appendix section (Tables 14 and 15). It should be noted here that while the method requires only one GC/ECD column meet the given acceptance criteria, this study yielded data showing that both columns are able to pass the precision and accuracy requirements.

Table 8. Initial demonstration of accuracy (n=4).

Analyte	Column 1 - X̅	Column 2 - X̅	X̅ Recovery Range	Acceptance Criteria
PCB 1016	96.9%	100.9%	61–103%	PASS
PCB 1260	117.8%	117.3%	37–130%	PASS
PCB 1221	106.5%	119.8%	44–150%	PASS
PCB 1254	83.2%	107.0%	44–130%	PASS
PCB 1242	72.0%	104.5%	50–139%	PASS
PCB 1248	62.8%	95.2%	58–140%	PASS
PCB 1268	82.7%	126.2%	60–140%	PASS
4,4´-DDD	90.0%	84.5%	48–130%	PASS
4,4´-DDE	98.9%	93.3%	58–130%	PASS
4,4´-DDT	112.1%	115.4%	46–137%	PASS
Aldrin	90.1%	89.1%	54–130%	PASS
alpha-BHC	117.0%	108.9%	49–130%	PASS
alpha-Chlordane	95.4%	94.5%	55–130%	PASS
beta-BHC	99.2%	106.3%	39–130%	PASS
delta-BHC	95.9%	101.3%	51–130%	PASS
Dieldrin	106.2%	107.4%	58–130%	PASS
Endosulfan I	85.6%	90.8%	57–141%	PASS
Endosulfan II	103.8%	94.7%	22–171%	PASS
Endosulfan sulfate	101.0%	96.4%	38–132%	PASS
Endrin	102.5%	102.5%	51–130%	PASS
Endrin aldehyde	109.0%	117.1%	60–140%	PASS
Endrin Ketone	103.0%	99.4%	60–140%	PASS
gamma-BHC	104.3%	103.2%	43–130%	PASS
gamma-Chlordane	98.7%	98.4%	55–130%	PASS
Heptachlor	98.4%	97.1%	43–130%	PASS
Heptachlor epoxide	95.5%	94.4%	57–132%	PASS
Methoxychlor	94.1%	102.6%	60–140%	PASS
Chlordane	97.1%	96.6%	60–140%	PASS
Toxaphene	95.2%	99.4%	53–130%	PASS
DCB (Surr.)	79.9%	95.9%	60–140%	PASS
TCMX (Surr.)	77.5%	77.3%	60–140%	PASS

Table 9. Initial demonstration of precision (n=4).

Analyte	Column 1 - %SD	Column 2 - %SD	Limit for %SD	Acceptance Criteria
PCB 1016	4.8%	2.1%	24%	PASS
PCB 1260	2.0%	1.2%	28%	PASS
PCB 1221	5.0%	5.8%	50%	PASS
PCB 1254	2.5%	4.5%	34%	PASS
PCB 1242	4.5%	3.4%	26%	PASS
PCB 1248	5.2%	7.7%	32%	PASS
PCB 1268	7.2%	5.4%	30%	PASS
4,4´-DDD	7.4%	9.8%	32%	PASS
4,4´-DDE	7.8%	7.4%	30%	PASS
4,4´-DDT	6.2%	12.2%	39%	PASS
Aldrin	6.2%	7.5%	25%	PASS
alpha-BHC	8.1%	7.6%	28%	PASS
alpha-Chlordane	7.2%	7.1%	24%	PASS
beta-BHC	6.9%	8.7%	38%	PASS
delta-BHC	6.7%	7.6%	43%	PASS
Dieldrin	8.1%	8.9%	42%	PASS
Endosulfan I	7.7%	9.5%	25%	PASS
Endosulfan II	7.2%	13.2%	63%	PASS
Endosulfan sulfate	7.5%	8.0%	32%	PASS
Endrin	9.0%	9.4%	42%	PASS
Endrin aldehyde	3.0%	8.0%	30%	PASS
Endrin Ketone	11.3%	9.2%	30%	PASS
gamma-BHC	7.2%	7.3%	29%	PASS
gamma-Chlordane	6.3%	8.1%	24%	PASS
Heptachlor	6.8%	6.3%	28%	PASS
Heptachlor epoxide	6.4%	6.4%	22%	PASS
Methoxychlor	10.9%	19.1%	30%	PASS
Chlordane	6.7%	7.8%	30%	PASS
Toxaphene	2.8%	3.6%	30%	PASS
DCB (Surr.)	8.8%	11.3%	30%	PASS
TCMX (Surr.)	7.6%	7.4%	30%	PASS

Figure 1. Initial demonstration of accuracy (n=4).
Figure 2. Initial demonstration of precison (n=4)

Matrix spike and matrix spike duplicate accuracy and precision

To demonstrate the effectiveness of an extraction with matrix interferences (EPA 608.3 - Section 8.1.2.1.2.e) , two duplicate samples were created in-house with 2 mg of oil and grease standard (hexadecane and stearic acid). The results in Table 10 prove that the reduced sample volume (100 mL) provides robust extraction performance of the target analytes. The precision of matrix sample extractions is demonstrated in Table 11 with Relative Percent Differences (RPDs) well within the method requirements for duplicate matrix spikes. Figure 3 provides a visual of the MS/MSD accuracy using acceptance criteria of 60–140% (section 8.4.5). Figure 4 provides a visual of the MS/MSD precision using RPD of 30% (EPA 608.3 section 8.1.2.1.2.e). Results for individual MS/MSD precision and accuracy replicates are provided in the appendix section (Tables 16 and 17). It should be noted here that while the method requires only one GC/ECD column meet the given acceptance criteria, this study yielded data showing that both columns are able to pass the MS/MSD requirements.

Table 10. Matrix duplicate spike (MS/MSD) accuracy (n=2).

Analyte	Column 1 - MS/MSD % R	Column 2 - MS/MSD % R	Range for Recovery (P)	Acceptance Criteria
PCB 1016	106.0%	103.0%	50–140%	PASS
PCB 1260	117.0%	122.5%	8–140%	PASS
PCB 1221	96.5%	107.5%	15–178%	PASS
PCB 1254	93.5%	97.5%	29–140%	PASS
PCB 1242	66.5%	94.0%	39–150%	PASS
PCB 1248	91.5%	93.0%	38–158%	PASS
PCB 1268	81.0%	123.5%	60–140%	PASS
4,4´-DDD	77.0%	77.4%	31–141%	PASS
4,4´-DDE	81.4%	82.0%	30–145%	PASS
4,4´-DDT	104.0%	108.4%	25–160%	PASS
Aldrin	87.0%	87.2%	42–140%	PASS
alpha-BHC	107.0%	101.6%	37–140%	PASS
alpha-Chlordane	95.2%	76.8%	45–140%	PASS
beta-BHC	94.2%	97.8%	17–147%	PASS
delta-BHC	90.2%	96.0%	19–140%	PASS
Dieldrin	88.2%	90.4%	36–140%	PASS
Endosulfan I	64.0%	76.4%	45–153%	PASS
Endosulfan II	89.6%	80.0%	D–202%	PASS
Endosulfan sulfate	86.4%	87.0%	26–144%	PASS
Endrin	90.2%	89.6%	30–147%	PASS
Endrin aldehyde	108.2%	92.2%	60–140%	PASS
Endrin Ketone	97.4%	89.2%	60-140%	PASS
gamma-BHC	98.4%	96.6%	32–140%	PASS
gamma-Chlordane	79.6%	75.4%	45–140%	PASS
Heptachlor	96.4%	98.4%	34–140%	PASS
Heptachlor epoxide	84.4%	59.8%	37–140%	PASS
Methoxychlor	92.2%	96.4%	60–140%	PASS
Chlordane	87.4%	76.1%	60–140%	PASS
Toxaphene	90.5%	116.9%	41–140%	PASS
DCB (Surr.)	79.3%	90.8%	60–140%	PASS
TCMX (Surr.)	82.9%	81.6%	60–140%	PASS

Table 11. Matrix duplicate spike (MS/MSD) relative percent difference (n=2).

Analyte	Column 1 - RPD (%)	Column 2 - RPD (%)	Maximum MS/MSD RPD (%)	Acceptance Criteria
PCB 1016	1.4%	4.1%	36%	PASS
PCB 1260	8.6%	9.1%	38%	PASS
PCB 1221	1.1%	17.2%	48%	PASS
PCB 1254	7.7%	5.2%	45%	PASS
PCB 1242	2.1%	6.3%	29%	PASS
PCB 1248	5.5%	6.5%	35%	PASS
PCB 1268	8.4%	10.7%	30%	PASS
4,4´-DDD	3.6%	3.6%	39%	PASS
4,4´-DDE	16.2%	7.8%	35%	PASS
4,4´-DDT	6.9%	9.6%	42%	PASS
Aldrin	2.3%	0.9%	35%	PASS
alpha-BHC	1.1%	2.4%	36%	PASS
alpha-Chlordane	7.6%	2.1%	35%	PASS
beta-BHC	2.1%	9.4%	44%	PASS
delta-BHC	4.0%	1.7%	52%	PASS
Dieldrin	3.2%	3.5%	49%	PASS
Endosulfan I	12.5%	2.1%	28%	PASS
Endosulfan II	4.5%	3.0%	53%	PASS
Endosulfan sulfate	3.7%	1.4%	38%	PASS
Endrin	3.1%	3.6%	48%	PASS
Endrin aldehyde	9.2%	4.8%	30%	PASS
Endrin Ketone	6.2%	5.4%	30%	PASS
gamma-BHC	1.6%	5.4%	39%	PASS
gamma-Chlordane	2.0%	2.7%	35%	PASS
Heptachlor	3.3%	0.8%	43%	PASS
Heptachlor epoxide	0.9%	12.7%	26%	PASS
Methoxychlor	27.3%	5.8%	30%	PASS
Chlordane	5.0%	0.3%	30%	PASS
Toxaphene	2.6%	3.0%	41%	PASS
DCB (Surr.)	14.4%	8.4%	30%	PASS
TCMX (Surr.)	14.7%	15.7%	30%	PASS

Figure 3. Matrix duplicate spike (MS/MSD) accuracy (n=2).

Figure 4. Matrix duplicate spike (MS/MSD) relative percent difference. (n=2).

This application note demonstrates the ability to extract and analyze 100 mL samples of PCBs and pesticides for EPA Method 608.3 with the automated SPE format of the Biotage® Horizon 5000, the DryDisk® Solvent Drying System, and the TurboVap® II. When reducing sample volumes from 1 L to 100 mL, the extraction and concentration time is reduced and most importantly the amount of methylene chloride used for extraction is significantly reduced. See Figures 5 and 6 illustrating the differences in time and volume between extracting 1 L and 100 mL samples.

Additional information

Due to the lingering nature of the pesticides and PCBs within this method, it is very important that good cleaning practices are performed not only for quality control samples, but also is most important for when a laboratory adopts the method for real sample extractions. Conducting the purge method found in Table 1 at the start of the day, between samples, and at the end of the day is very important to ensure the cleanliness of the Biotage® Horizon 5000 and its supporting components.

When utilizing a reusable disk holder or collection vessel, it is very important that they are cleaned well. The best practice for cleaning these components is to use warm, soapy water to remove any remaining residue, followed by three rinses with acetone, and three rinses with methylene chloride. Since the Atlantic® ReadyDisk C18 and VOA vials were used for this application note, time and solvent were saved, as this manual cleaning procedure was not required.

For extract drying, a new DryDisk®-R was used for each sample and all the parts of the SDS were rinsed three times with acetone and three times with methylene chloride. Lastly, the TurboVap® II, 200 mL, 1 mL EndPoint evaporation tubes were rinsed three times with acetone and three times with methylene chloride as well to prevent cross contamination. Following these cleaning procedures will help to reduce the chance of contamination from sample to sample for PCBs and Pesticides.

Another tip when working in implement this extraction procedure in the laboratory is to eliminate solvent squeeze bottles entirely. It is recommended that any rinsing or transferring action within the process be replaced with glass. For example, 10 mL glass pipettes were used in the production of this application note.
Figure 5. Total extraction and concentration time.

Figure 6. Total methylene chloride extraction volume.

Appendix

This section provides all the additional raw data for each sample extracted within this application note.

Table 12. Method Detection Limit (MDL) Results - Column 1.

Analyte	Spike Level (µg/L)	MDL 1 (µg/L)	MDL 2 (µg/L)	MDL 3 (µg/L)	MDL 4 (µg/L)	MDL 5 (µg/L)	MDL 6 (µg/L)	MDL 7 (µg/L)	Standard Deviation (µg/L)	Calculated MDL (µg/L)	Calculated MDL (ng/L)
PCB-1016	0.10	0.130	0.128	0.106	0.134	0.116	0.128	0.114	0.01001	0.0315	31.5
PCB-1260	0.10	0.103	0.107	0.098	0.119	0.108	0.128	0.088	0.01332	0.0419	41.9
PCB-1221	0.20	0.230	0.215	0.262	0.256	0.255	0.281	0.244	0.02158	0.0678	67.8
PCB-1254	0.10	0.089	0.086	0.091	0.099	0.095	0.099	0.095	0.00519	0.0163	16.3
PCB-1242	0.10	0.126	0.114	0.125	0.125	0.124	0.137	0.120	0.00683	0.0215	21.5
PCB-1248	0.10	0.047	0.049	0.054	0.046	0.045	0.055	0.048	0.00377	0.0118	11.8
PCB-1268	0.10	0.059	0.066	0.071	0.064	0.059	0.075	0.071	0.00597	0.0188	18.8
4,4´-DDD	0.10	0.072	0.076	0.084	0.082	0.080	0.080	0.084	0.00439	0.0138	13.8
4,4´-DDE	0.10	0.100	0.092	0.092	0.098	0.098	0.102	0.092	0.00423	0.0133	13.3
4,4´-DDT	0.10	0.104	0.096	0.094	0.096	0.082	0.108	0.094	0.00828	0.0260	26.0
Aldrin	0.10	0.072	0.072	0.074	0.076	0.078	0.084	0.080	0.00443	0.0139	13.9
alpha-BHC	0.10	0.098	0.096	0.096	0.074	0.070	0.088	0.090	0.01810	0.0569	56.9
alpha-Chlordane	0.10	0.094	0.102	0.112	0.106	0.112	0.108	0.096	0.00725	0.0228	22.8
beta-BHC	0.10	0.136	0.126	0.114	0.140	0.112	0.120	0.120	0.01065	0.0335	33.5
delta-BHC	0.10	0.080	0.082	0.078	0.088	0.080	0.086	0.080	0.00365	0.0115	11.5
Dieldrin	0.10	0.106	0.104	0.104	0.112	0.108	0.114	0.104	0.00412	0.0129	12.9
Endosulfan I	0.10	0.084	0.084	0.086	0.090	0.088	0.090	0.082	0.00315	0.0099	9.9
Endosulfan II	0.10	0.092	0.092	0.102	0.110	0.108	0.100	0.102	0.00701	0.0220	22.0
Endosulfan sulfate	0.10	0.102	0.094	0.098	0.106	0.088	0.116	0.098	0.00898	0.0282	28.2
Endrin	0.10	0.086	0.110	0.104	0.110	0.106	0.114	0.104	0.00908	0.0285	28.5
Endrin aldehyde	0.10	0.138	0.114	0.120	0.136	0.116	0.144	0.132	0.01182	0.0372	37.2
Endrin Ketone	0.10	0.054	0.072	0.070	0.102	0.100	0.102	0.104	0.02044	0.0642	64.2
gamma-BHC	0.10	0.106	0.104	0.104	0.118	0.106	0.114	0.106	0.00547	0.0172	17.2
gamma-Chlordane	0.10	0.122	0.120	0.122	0.126	0.122	0.130	0.116	0.00443	0.0139	13.9
Heptachlor	0.10	0.104	0.104	0.104	0.112	0.114	0.122	0.112	0.00678	0.0213	21.3
Heptachlor epoxide	0.10	0.112	0.112	0.116	0.120	0.114	0.122	0.104	0.00594	0.0187	18.7
Methoxychlor	0.10	0.070	0.064	0.084	0.104	0.080	0.102	0.082	0.01494	0.0470	47.0
Chlordane	0.20	0.216	0.222	0.234	0.232	0.234	0.238	0.212	0.01012	0.0318	31.8
Toxaphene	1.00	1.174	1.238	1.136	1.022	1.294	1.221	1.238	0.08923	0.2805	280.5

Table 13. Method Detection Limit (MDL) Results - Column 2.

Analyte	Spike Level (µg/L)	MDL 1 (µg/L)	MDL 2 (µg/L)	MDL 3 (µg/L)	MDL 4 (µg/L)	MDL 5 (µg/L)	MDL 6 (µg/L)	MDL 7 (µg/L)	Standard Deviation (µg/L)	Calculated MDL (µg/L)	Calculated MDL (ng/L)
PCB-1016	0.10	0.0953	0.1057	0.0904	0.1251	0.1183	0.1303	0.0792	0.01907	0.05994	59.9
PCB-1260	0.10	0.1031	0.1139	0.1004	0.1125	0.1144	0.1312	0.0927	0.01244	0.03911	39.1
PCB-1221	0.20	0.2796	0.1968	0.1928	0.2549	0.2086	0.2715	0.2597	0.03700	0.11629	116.3
PCB-1254	0.10	0.075	0.0756	0.0831	0.08	0.0816	0.0959	0.0816	0.00693	0.02179	21.8
PCB-1242	0.10	0.0951	0.0849	0.106	0.1139	0.0933	0.101	0.0985	0.00933	0.02931	29.3
PCB-1248	0.10	0.0639	0.0691	0.0706	0.0646	0.0633	0.0739	0.0669	0.00393	0.01234	12.3
PCB-1268	0.10	0.0864	0.0946	0.102	0.094	0.0857	0.1035	0.1006	0.00723	0.02273	22.7
4,4´-DDD	0.10	0.074	0.062	0.086	0.086	0.084	0.088	0.08	0.00924	0.02904	29.0
4,4´-DDE	0.10	0.094	0.086	0.084	0.09	0.092	0.1	0.088	0.00538	0.01691	16.9
4,4´-DDT	0.10	0.096	0.09	0.086	0.096	0.09	0.102	0.082	0.00678	0.02131	21.3
Aldrin	0.10	0.086	0.076	0.078	0.082	0.08	0.086	0.084	0.00390	0.01226	12.3
alpha-BHC	0.10	0.078	0.068	0.07	0.076	0.078	0.08	0.076	0.00445	0.01399	14.0
alpha-Chlordane	0.10	0.096	0.09	0.094	0.096	0.098	0.104	0.092	0.00454	0.01427	14.3
beta-BHC	0.10	0.082	0.076	0.1	0.08	0.08	0.078	0.07	0.00930	0.02923	29.2
delta-BHC	0.10	0.082	0.076	0.064	0.088	0.09	0.09	0.078	0.00944	0.02967	29.7
Dieldrin	0.10	0.106	0.104	0.106	0.112	0.108	0.112	0.104	0.00341	0.01072	10.7
Endosulfan I	0.10	0.096	0.088	0.094	0.094	0.092	0.088	0.092	0.00306	0.00962	9.6
Endosulfan II	0.10	0.088	0.08	0.086	0.098	0.094	0.096	0.09	0.00626	0.01968	19.7
Endosulfan sulfate	0.10	0.096	0.092	0.09	0.098	0.094	0.086	0.09	0.00407	0.01279	12.8
Endrin	0.10	0.102	0.102	0.102	0.108	0.106	0.11	0.098	0.00416	0.01307	13.1
Endrin aldehyde	0.10	0.08	0.086	0.106	0.098	0.098	0.098	0.082	0.00985	0.03096	31.0
Endrin Ketone	0.10	0.096	0.096	0.098	0.102	0.1	0.102	0.092	0.00365	0.01147	11.5
gamma-BHC	0.10	0.092	0.092	0.092	0.1	0.096	0.104	0.092	0.00486	0.01527	15.3
gamma-Chlordane	0.10	0.08	0.082	0.078	0.106	0.09	0.102	0.098	0.01131	0.03555	35.5
Heptachlor	0.10	0.072	0.076	0.068	0.072	0.074	0.082	0.068	0.00488	0.01534	15.3
Heptachlor epoxide	0.10	0.098	0.094	0.096	0.102	0.096	0.102	0.096	0.00315	0.00990	9.9
Methoxychlor	0.10	0.088	0.09	0.102	0.112	0.102	0.108	0.088	0.00991	0.03115	31.1
Chlordane	0.20	0.176	0.172	0.172	0.202	0.188	0.206	0.19	0.01394	0.04381	43.8
Toxaphene	1.00	1.206	1.25	1.186	1.106	1.168	1.37	1.29	0.08676	0.27270	272.7

Table 14. Initial Demonstration of Capability (DOC) Results - Column 1.

Analyte	Spike Level (µg/L)	LCS 1 (µg/L)	Recovery	LCS 2	Recover	LCS 3	Recovery	LCS 4	Recovery	Average Recovery	Standard Deviation
Analyte	Spike Level (µg/L)	LCS 1 (µg/L)	(%)	(µg/L)	(%)	(µg/L)	(%)	(µg/L)	(%)	(%)	(%)
PCB-1016	1.0	0.998	99.8	1.02	102.0	0.918	91.8	0.938	93.8	96.9	4.8
PCB-1260	1.0	1.154	115.4	1.198	119.8	1.168	116.8	1.19	119.0	117.8	2.0
PCB-1221	1.0	1.0542	105.4	1.0642	106.4	1.1312	113.1	1.0115	101.2	106.5	5.0
PCB-1254	0.5	0.412	82.4	0.421	84.2	0.4308	86.2	0.401	80.2	83.2	2.5
PCB-1242	0.5	0.3579	71.6	0.3628	72.6	0.3319	66.4	0.3864	77.3	72.0	4.5
PCB-1248	0.5	0.3023	60.5	0.2882	57.6	0.3157	63.1	0.3489	69.8	62.8	5.2
PCB-1268	0.5	0.3868	77.4	0.3874	77.5	0.4168	83.4	0.4634	92.7	82.7	7.2
4,4´-DDD	0.5	0.42	84.0	0.438	87.6	0.438	87.6	0.504	100.8	90.0	7.4
4,4´-DDE	0.5	0.466	93.2	0.48	96.0	0.48	96.0	0.552	110.4	98.9	7.8
4,4´-DDT	0.5	0.53	106.0	0.554	110.8	0.554	110.8	0.604	120.8	112.1	6.2
Aldrin	0.5	0.426	85.2	0.44	88.0	0.44	88.0	0.496	99.2	90.1	6.2
alpha-BHC	0.5	0.552	110.4	0.572	114.4	0.572	114.4	0.644	128.8	117.0	8.1
alpha-Chlordane	0.5	0.45	90.0	0.464	92.8	0.464	92.8	0.53	106.0	95.4	7.2
beta-BHC	0.5	0.466	93.2	0.486	97.2	0.486	97.2	0.546	109.2	99.2	6.9
delta-BHC	0.5	0.45	90.0	0.47	94.0	0.47	94.0	0.528	105.6	95.9	6.7
Dieldrin	0.5	0.498	99.6	0.518	103.6	0.518	103.6	0.59	118.0	106.2	8.1
Endosulfan I	0.5	0.396	79.2	0.416	83.2	0.416	83.2	0.484	96.8	85.6	7.7
Endosulfan II	0.5	0.486	97.2	0.51	102.0	0.51	102.0	0.57	114.0	103.8	7.2
Endosulfan sulfate	0.5	0.458	91.6	0.506	101.2	0.506	101.2	0.55	110.0	101.0	7.5
Endrin	0.5	0.476	95.2	0.498	99.6	0.498	99.6	0.578	115.6	102.5	9.0
Endrin aldehyde	0.5	0.546	109.2	0.534	106.8	0.534	106.8	0.566	113.2	109.0	3.0
Endrin Ketone	0.5	0.47	94.0	0.496	99.2	0.496	99.2	0.598	119.6	103.0	11.3
gamma-BHC	0.5	0.492	98.4	0.51	102.0	0.51	102.0	0.574	114.8	104.3	7.2
gamma-Chlordane	0.5	0.47	94.0	0.482	96.4	0.482	96.4	0.54	108.0	98.7	6.3
Heptachlor	0.5	0.466	93.2	0.48	96.0	0.48	96.0	0.542	108.4	98.4	6.8
Heptachlor epoxide	0.5	0.45	90.0	0.468	93.6	0.468	93.6	0.524	104.8	95.5	6.4
Methoxychlor	0.5	0.394	78.8	0.482	96.4	0.482	96.4	0.524	104.8	94.1	10.9
Chlordane	1.0	0.92	92.0	0.946	94.6	0.946	94.6	1.07	107.0	97.1	6.7
Toxaphene	10.0	9.468	94.7	9.178	91.8	9.844	98.4	9.598	96.0	95.2	2.8
DCB (Surr.)	1.0	0.82	82.0	0.73	73.0	0.73	73.0	0.914	91.4	79.9	8.8
TCMX (Surr.)	1.0	0.776	77.6	0.72	72.0	0.72	72.0	0.882	88.2	77.5	7.6

Table 15. Initial Demonstration of Capability (DOC) Results - Column 1.

Analyte	Spike Level (µg/L)	LCS 1 (µg/L)	Recovery	LCS 2	Recovery	LCS 3	Recovery	LCS 4	Recovery	Average Recovery	Standard Deviation
Analyte	Spike Level (µg/L)	LCS 1 (µg/L)	(%)	(µg/L)	(%)	(µg/L)	(%)	(µg/L)	(%)	(%)	(%)
PCB-1016	1.00	0.978	97.8	1.01	101.0	1.026	102.6	1.02	102.0	100.9	2.1
PCB-1260	1.00	1.178	117.8	1.156	115.6	1.172	117.2	1.184	118.4	117.3	1.2
PCB-1221	1.00	1.1341	113.4	1.1682	116.8	1.2265	122.7	1.2644	126.4	119.8	5.8
PCB-1254	0.50	0.5138	102.8	0.5477	109.5	0.5602	112.0	0.5177	103.5	107.0	4.5
PCB-1242	0.50	0.5288	105.8	0.5255	105.1	0.4978	99.6	0.5369	107.4	104.5	3.4
PCB-1248	0.50	0.458	91.6	0.4382	87.6	0.4794	95.9	0.5282	105.6	95.2	7.7
PCB-1268	0.50	0.6085	121.7	0.6072	121.4	0.6479	129.6	0.6612	132.2	126.2	5.5
4,4´-DDD	0.50	0.398	79.6	0.396	79.2	0.4	80.0	0.496	99.2	84.5	9.8
4,4´-DDE	0.50	0.45	90.0	0.436	87.2	0.46	92.0	0.52	104.0	93.3	7.4
4,4´-DDT	0.50	0.53	106.0	0.562	112.4	0.55	110.0	0.666	133.2	115.4	12.2
Aldrin	0.50	0.418	83.6	0.424	84.8	0.44	88.0	0.5	100.0	89.1	7.5
alpha-BHC	0.50	0.526	105.2	0.516	103.2	0.536	107.2	0.6	120.0	108.9	7.6
alpha-Chlordane	0.50	0.446	89.2	0.454	90.8	0.466	93.2	0.524	104.8	94.5	7.1
beta-BHC	0.50	0.496	99.2	0.504	100.8	0.534	106.8	0.592	118.4	106.3	8.7
delta-BHC	0.50	0.484	96.8	0.48	96.0	0.5	100.0	0.562	112.4	101.3	7.6
Dieldrin	0.50	0.51	102.0	0.506	101.2	0.53	106.0	0.602	120.4	107.4	8.9
Endosulfan I	0.50	0.442	88.4	0.424	84.8	0.426	85.2	0.524	104.8	90.8	9.5
Endosulfan II	0.50	0.436	87.2	0.434	86.8	0.452	90.4	0.572	114.4	94.7	13.2
Endosulfan sulfate	0.50	0.456	91.2	0.454	90.8	0.478	95.6	0.54	108.0	96.4	8.0
Endrin	0.50	0.482	96.4	0.484	96.8	0.502	100.4	0.582	116.4	102.5	9.4
Endrin aldehyde	0.50	0.56	112.0	0.544	108.8	0.61	122.0	0.628	125.6	117.1	8.0
Endrin Ketone	0.50	0.464	92.8	0.47	94.0	0.49	98.0	0.564	112.8	99.4	9.2
gamma-BHC	0.50	0.488	97.6	0.494	98.8	0.514	102.8	0.568	113.6	103.2	7.3
gamma-Chlordane	0.50	0.468	93.6	0.466	93.2	0.482	96.4	0.552	110.4	98.4	8.1
Heptachlor	0.50	0.448	89.6	0.48	96.0	0.49	98.0	0.524	104.8	97.1	6.3
Heptachlor epoxide	0.50	0.452	90.4	0.448	89.6	0.47	94.0	0.518	103.6	94.4	6.4
Methoxychlor	0.50	0.496	99.2	0.516	103.2	0.404	80.8	0.636	127.2	102.6	19.1
Chlordane	1.00	0.914	91.4	0.92	92.0	0.948	94.8	1.08	108.0	96.6	7.8
Toxaphene	10.00	9.692	96.9	9.642	96.4	10.434	104.3	9.994	99.9	99.4	3.6
DCB (Surr.)	1.00	0.868	86.8	0.95	95.0	0.896	89.6	1.12	112.0	95.9	11.3
TCMX (Surr.)	1.00	0.72	72.0	0.77	77.0	0.724	72.4	0.878	87.8	77.3	7.4

Table 16. Matrix Spike and Matrix Spike Duplicate (MS/MSD) Results - Column 1.

Analyte	Spike Level (µg/L)	MS (µg/L)	Recovery (%)	MSD (µg/L)	Recovery (%)	Average Recovery (%)	Relative Percent Difference (%)
PCB-1016	0.5	0.534	106.8	0.5267	105.3	106.1	1.4
PCB-1260	0.5	0.559	111.8	0.6096	121.9	116.9	8.6
PCB-1221	1	0.964	96.4	0.9748	97.5	96.9	1.1
PCB-1254	0.5	0.450	90.0	0.486	97.2	93.6	7.7
PCB-1242	0.5	0.328	65.6	0.335	67.0	66.3	2.1
PCB-1248	0.5	0.444	88.8	0.4692	93.8	91.3	5.5
PCB-1268	0.5	0.388	77.6	0.4221	84.4	81.0	8.4
4,4´-DDD	0.5	0.378	75.6	0.392	78.4	77.0	3.6
4,4´-DDE	0.5	0.374	74.8	0.44	88.0	81.4	16.2
4,4´-DDT	0.5	0.502	100.4	0.538	107.6	104.0	6.9
Aldrin	0.5	0.43	86.0	0.44	88.0	87.0	2.3
alpha-BHC	0.5	0.532	106.4	0.538	107.6	107.0	1.1
alpha-Chlordane	0.5	0.458	91.6	0.494	98.8	95.2	7.6
beta-BHC	0.5	0.466	93.2	0.476	95.2	94.2	2.1
delta-BHC	0.5	0.442	88.4	0.46	92.0	90.2	4.0
Dieldrin	0.5	0.434	86.8	0.448	89.6	88.2	3.2
Endosulfan I	0.5	0.3	60.0	0.34	68.0	64.0	12.5
Endosulfan II	0.5	0.438	87.6	0.458	91.6	89.6	4.5
Endosulfan sulfate	0.5	0.44	88.0	0.424	84.8	86.4	3.7
Endrin	0.5	0.444	88.8	0.458	91.6	90.2	3.1
Endrin aldehyde	0.5	0.566	113.2	0.516	103.2	108.2	9.2
Endrin Ketone	0.5	0.472	94.4	0.502	100.4	97.4	6.2
gamma-BHC	0.5	0.488	97.6	0.496	99.2	98.4	1.6
gamma-Chlordane	0.5	0.394	78.8	0.402	80.4	79.6	2.0
Heptachlor	0.5	0.474	94.8	0.49	98.0	96.4	3.3
Heptachlor epoxide	0.5	0.424	84.8	0.42	84.0	84.4	0.9
Methoxychlor	0.5	0.398	79.6	0.524	104.8	92.2	27.3
Chlordane	1	0.852	85.2	0.896	89.6	87.4	5.0
Toxaphene	10	9.162	91.6	8.93	89.3	90.5	2.6
DCB (Surr.)	1	0.85	85.0	0.736	73.6	79.3	14.4
TCMX (Surr.)	1	0.89	89.0	0.768	76.8	82.9	14.7

Table 17. Matrix Spike and Matrix Spike Duplicate (MS/MSD) Results - Column 1.

Analyte	Spike Level (µg/L)	MS (µg/L)	Recovery (%)	MSD (µg/L)	Recovery (%)	Average Recovery (%)	Relative Percent Difference (%)
PCB-1016	0.5	0.503	100.6	0.5243	104.9	102.7	4.1
PCB-1260	0.5	0.585	117.0	0.6408	128.2	122.6	9.1
PCB-1221	1	1.169	116.9	0.9839	98.4	107.6	17.2
PCB-1254	0.5	0.501	100.2	0.4752	95.0	97.6	5.2
PCB-1242	0.5	0.456	91.1	0.4849	97.0	94.0	6.3
PCB-1248	0.5	0.448	89.6	0.4782	95.6	92.6	6.5
PCB-1268	0.5	0.583	116.7	0.649	129.8	123.2	10.7
4,4´-DDD	0.5	0.38	76.0	0.394	78.8	77.4	3.6
4,4´-DDE	0.5	0.394	78.8	0.426	85.2	82.0	7.8
4,4´-DDT	0.5	0.516	103.2	0.568	113.6	108.4	9.6
Aldrin	0.5	0.434	86.8	0.438	87.6	87.2	0.9
alpha-BHC	0.5	0.502	100.4	0.514	102.8	101.6	2.4
alpha-Chlordane	0.5	0.388	77.6	0.38	76.0	76.8	2.1
beta-BHC	0.5	0.466	93.2	0.512	102.4	97.8	9.4
delta-BHC	0.5	0.476	95.2	0.484	96.8	96.0	1.7
Dieldrin	0.5	0.444	88.8	0.46	92.0	90.4	3.5
Endosulfan I	0.5	0.378	75.6	0.386	77.2	76.4	2.1
Endosulfan II	0.5	0.394	78.8	0.406	81.2	80.0	3.0
Endosulfan sulfate	0.5	0.432	86.4	0.438	87.6	87.0	1.4
Endrin	0.5	0.44	88.0	0.456	91.2	89.6	3.6
Endrin aldehyde	0.5	0.472	94.4	0.45	90.0	92.2	4.8
Endrin Ketone	0.5	0.434	86.8	0.458	91.6	89.2	5.4
gamma-BHC	0.5	0.47	94.0	0.496	99.2	96.6	5.4
gamma-Chlordane	0.5	0.372	74.4	0.382	76.4	75.4	2.7
Heptachlor	0.5	0.49	98.0	0.494	98.8	98.4	0.8
Heptachlor epoxide	0.5	0.28	56.0	0.318	63.6	59.8	12.7
Methoxychlor	0.5	0.496	99.2	0.468	93.6	96.4	5.8
Chlordane	1	0.76	76.0	0.762	76.2	76.1	0.3
Toxaphene	10	11.868	118.7	11.52	115.2	116.9	3.0
DCB (Surr.)	1	0.946	94.6	0.87	87.0	90.8	8.4
TCMX (Surr.)	1	0.88	88.0	0.752	75.2	81.6	15.7

Literature number: AN974

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