United States
Environmental
Protection Agency
Great Lakes
National Program
Off,ce
EPA 905-R-97-012b
June 1 997
Lake Michigan Mass Balance Study
(LMMB) Methods Compendium
Volume 2: Organic and Mercury
Sample Analysis Techniques
Printed on Recycled Paper
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United States Office of Water EPA-905-R-97-012b
Environmental Protection 4303 October 1997
Agency
FPA Lake Michl9an Mass Balance Study (LMMB)
Methods Compendium
Volume 2: Organic and Mercury Sample
Analysis Techniques
U.S. EPA
MID-CONTINENT ECOLOGY DIVISION
LIBRARY
DULUTH, MN 55804
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4>EPA Lake Michigan Mass Balance Study
(LMMB) Methods Compendium
Volume 2: Organic and Mercury
Sample Analysis Techniques
Printed on Recycled Paper
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Acknowledgments
This compendium was prepared under the direction of Louis Blume of the EPA Great Lakes National
Program Office. The compendium was prepared by DynCorp Environmental and Grace Analytical Lab.
Special thanks are extended to Dr. William Telliard and staff at EPA's Office of Water for technical
assistance and support of this project. The methods contained in this compendium were developed by the
following Principal Investigators (Pis) participating in the Lake Michigan Mass Balance (LMMB) Study:
Eric Crecelius, Ph.D., Battelle Marine Sciences Laboratory, Sequim, WA
David Edgington, Ph.D., Great Lakes Research Facility, Milwaukee, WI
Brian Eadie, Ph.D., NOAA, Ann Arbor, MI
Steven Eisenreich, Ph.D., Rutgers University, New Brunswick, NJ
John Gannon, Ph.D., USGS National Biological Survey, Ann Arbor, MI
Nathan Hawley, Ph.D., NOAA, Ann Arbor, MI
Bob Hesselberg, USGS National Biological Survey, Ann Arbor, MI
Ron Hites, Ph.D., Indiana University, Bloomington, IN
Mark Holey, Fish and Wildlife Service, Green Bay, WI
Alan Hoffman, U.S. EPA AREAL, Research Triangle Park, NC
Tom Holsen, Ph.D., Illinois Institute of Technology, Chicago, IL
Peter Hughes, United States Geological Survey, Madison, WI
Jim Hurley, Ph.D., University of Wisconsin, Madison, WI
Tom Johengen, Ph.D., NOAA, Ann Arbor, MI
Jerry Keeler, Ph.D., University of Michigan, Ann Arbor, MI
Robert Mason, Ph.D., University of Maryland, Solomons, MD
Mike Mullin, U.S. EPA Large Lakes Research Station, Grosse He, MI
Edward Nater, Ph.D., University of Minnesota, Minneapolis, MN
Jerome Nriagu, Ph.D., University of Michigan, Ann Arbor, MI
John Robbins, Ph.D., NOAA, Ann Arbor, Michigan
Ron Rossmann, Ph.D., EPA Large Lakes Research Station, Grosse He, MI
Martin Shafer, Ph.D., University of Wisconsin, Madison, WI
William Sonzogni, Ph.D., Wisconsin State Lab of Hygiene, Madison, WI
Clyde Sweet, Ph.D., Illinois State Water Survey, Champaign, IL
Deborah Swackhamer, Ph.D., University of Minnesota, Minneapolis, MN
Pat Van Hoof, Ph.D., NOAA, Ann Arbor, MI
Glenn Warren, Ph.D., U.S. EPA, GLNPO, Chicago, IL
Marvin Palmer, GLNPO, Chicago, IL
Disclaimer
This document describes sampling and analytical methods used by Pis participating in the LMMB Study.
Due to the nature and low concentrations of pollutants monitored in the study, many of the methods used in
the LMMB Study represent state-of-the art techniques that will be refined further as new technology is
developed and as necessary to resolve matrix interferences. Therefore, the procedures described in this
compendium should be considered to accurately reflect procedures in use by the LMMB Study Pis at the
time of publication. Users of this document should recognize that these procedures are subject to change.
Users of this document also should recognize that these methods do not constitute "approved EPA methods"
for use in compliance monitoring programs. Publication of these methods is intended to assist users of
LMMB Study data and to provide a reference tool for researchers interested in building upon LMMB Study
findings. Mention of company names, trade names, or commercial products does not constitute
endorsement or recommendation tor use.
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Foreword
The Lake Michigan Mass Budget/Mass Balance (LMMB) Study was initiated in late 1993 as part of the
Lakewide Management Plan (LaMP) for Lake Michigan. The Lake Michigan LaMP and the LMMB Study
were developed to meet requirements mandated by Section 118 of the Clean Water Act (CWA); Title III,
Section 112(m) of the Clean Air Act Amendments; and Annex 2 of the Great Lakes Water Quality
Agreement. Organizations participating in the development of these programs included: EPA Region 5,
the EPA Great Lakes National Program Office, the National Oceanic and Atmospheric Administration, the
U.S. Geological Survey, the U.S. Fish and Wildlife Service, the Michigan Department of Natural
Resources, the Wisconsin Department of Natural Resources, the Illinois Department of Natural Resources,
and the Indiana Department of Environmental Management. In general, the primary goal of the LaMP and
the LMMB Study is to develop a sound, scientific base of information with which to guide future toxic load
reduction efforts at the federal, state, and local levels.
This compendium describes the sampling and analytical methods used in the LMMB Study. For ease of
use, the compendium is organized into three volumes. Volume 1 describes sampling procedures used in the
study; Volumes 2 and 3 describe analytical procedures used by each PI. Because sampling apparatus and
techniques are generally geared towards specific matrices, Volume 1 is organized according to sample
matrix (e.g., air, water, sediment, tissue, etc). Volumes 2 and 3 are organized by pollutant type (e.g,
organics, metals, biologicals) because laboratories and instrumentation are typically set up to address
specific pollutants rather than specific matrices.
Each Principal Investigator (PI) was required to follow specific quality control requirements necessary to
meet data quality and measurement quality objectives for the LMMB Study. To assist users of this
document, Appendix A provides the measurement quality objectives (MQOs) established by each PI for
his/her sampling and analysis program.
Finally, EPA has made no attempt to standardize the procedures submitted by Pis for publication in this
compendium. Therefore, the methods provided in this document contain varying levels of detail. Appendix
B provides names, addresses and phone numbers for each PI and for each EPA Project Officer (PO).
Specific questions about the procedures used in the study should be directed to the appropriate PI or PO
listed in Appendix B.
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Table of Contents
Volume 1
Sample Collection Techniques
CHAPTER 1: AIR
LMMB 001 Standard Operating Procedure for Air Sampling for Semivolatile Organic
Contaminants Using the Organics High-Volume Sampler (Sweet, C.) 1-3
LMMB 002 Standard Operating Procedure for Precipitation Sampling Using XAD-2 and
MIC Collectors (Sweet, C.) 1-31
LMMB 003 Standard Operating Procedure for Air Sampling for Metals Using the
Dichotomous Sampler (Sweet, C.) 1-53
LMMB 004 Standard Operating Procedure for Sampling Trace Metals in Precipitation
Using Modified Aerochem Collectors (Vermette, S. and Sweet, C.) 1-71
LMMB 005 Metals Cleaning Procedures for Teflon Bottles and Rigid HOPE (Vermette, S.
and Sweet, C.) 1-87
LMMB 006 Standard Operating Procedure for Sampling of Vapor Phase Mercury (Keeler, G.
and Landis, M.) 1-91
LMMB 007 Standard Operating Procedure for Sampling of Mercury in Precipitation
(Keeler, G and Landis, M.) 1-107
LMMB 008 Standard Operating Procedure for Sampling of Particulate Phase Mercury
(Keeler, G. and Landis, M.) 1-123
LMMB 009 Standard Operating Procedure for Dry Deposition Sampling: Dry Deposition of
Atmospheric Particles (Paode, R. and Holsen, T.) 1-137
CHAPTER 2: WATER
LMMB 010 Standard Operating Procedure for Sample Collection of Atrazine and Atrazine
Metabolites (Eisenreich, S., Schottler, S., and Nines, N.) 1-159
LMMB 011 HOC Sampling Media Preparation and Handling; XAD-2 Resin and GF/F Filters
(Crecelius, E. and Lefkovitz, L.) 1-167
LMMB 012 Standard Operating Procedure for Site Selection and Sampling for Mercury in
Lakewater (Mason, R. and Sullivan, K.) 1-175
LMMB 013 Field Sampling Using the Rosette Sampler (Warren, G.) 1-185
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Table of Contents
LMMB 014 Standard Operating Procedure for the Sampling of Particulate-Phase and
Dissolved-Phase Organic Carbon in Great Lakes Waters (Grace Analytical Lab) . . 1-193
LMMB 015 Standard Operating Procedure for Chlorophyll-a Sampling Method: Field
Procedure (Grace Analytical Lab) 1-199
LMMB 016 Standard Operating Procedure for Primary Productivity Using UC: Field
Procedure (Grace Analytical Lab) 1 -205
LMMB 017 USGS Field Operation Plan: Tributary Monitoring (USGS/Eisenreich, S.) 1-215
LMMB 018 Trace Metal and Mercury Sampling Methods for Lake Michigan Tributaries
(Shafer, M.) 1-221
CHAPTERS: SEDIMENT
LMMB 019 Standard Operating Procedure for Collection of Sediment Samples
(Edgington,^ D. and Bobbins, J.) 1 -239
LMMB 020 Trap Sample Splitting (wet): Use of Sediment Traps for the Measurement of
Particle and Associated Contaminant Fluxes (Eadie, B.) 1-245
CHAPTER 4: PLANKTON
LMMB 021 Standard Operating Procedure for Sampling Lake Michigan Lower Pelagic
Foodchain for PCBs, Nonachlor, and Mercury (Swackhamer, D.,
Trowbridge, A., and Nater, E.) 1 -253
LMMB 022 Sampling Procedure for Collection of Benthic Invertebrates for Contaminant
Analysis (Warren, G.) 1-269
LMMB 023 Standard Operating Procedure for Phytoplankton Sample Collection and
Preservation (Grace Analytical Lab) 1-273
LMMB 024 Standard Operating Procedure for Zooplankton Sample Collection and
Preservation (Grace Analytical Lab) 1-277
CHAPTERS: FlSH
LMMB 025 Fish Processing Method (Hesselberg, R.) 1-285
LMMB 026 Quality Assurance Project Plan for Lake Trout and Forage Fish "Sampling for
Diet Analysis and/or Contaminant Analysis (Brown, E. and Eck, G.) 1-291
LMMB 027 Quality Assurance Project Plan for Coho Sampling for Contaminant and Diet
Analysis (Holey, M. and Elliott, R.) 1-367
IV
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Table of Contents
Volume 2
Organic and Mercury Sample Analysis Techniques
CHAPTER 1: ORGANIC ANALYSIS
LMMB 028 Instrumental Analysis and Quantitation of Polycyclic Aromatic Hydrocarbons
and Atrazine: IADN Project (Cortes, D. and Brubaker, W.) 2-3
LMMB 029 Analysis of PCBs and Pesticides in Air and Precipitation Samples : IADN
Project - Gas Chromatography Procedure (Basu, I.) 2-23
LMMB 030 Analysis of PCBs, Pesticides, and PAHs in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure (Basu, I.) 2-61
LMMB 031 Analysis of PCBs, Pesticides, and PAHs in Air and Precipitation Samples:
Sample Preparation Procedures (Harlin, K. and Surratt, K.) 2-115
LMMB 032 Standard Operating Procedure for the Analysis of PAHs and Atrazine by
GC/lon Trap MS (Peters, C. and Harlin, K.) 2-165
LMMB 033 Standard Operating Procedure for the Analysis of PCBs and Organochlorine
Pesticides by GC-ECD (Harlin, K., Surratt, K., and Peters, C.) 2-189
LMMB 034 Standard Operating Procedure for Isolation, Extraction and Analysis of
Atrazine, DEA and DIA (Eisenreich, S., Schottler, S., and Hines, N.) 2-243
LMMB 035 Standard Operating Procedures for Semivolatile Organic Compounds in Dry
Deposition Samples (Eisenreich, S. and Franz, T.) 2-251
LMMB 036 Extraction and Cleanup of XAD-2 Resin Cartridges for Polychlorinated
Biphenyls and Trans-Nonachlor (Crecelius, E. and Lefkovitz, L.) 2-257
LMMB 037 Extraction and Cleanup of Glass Fiber Filters for Polychlorinated Biphenyls
and Trans-Nonachlor (Crecelius, E. and Lefkovitz, L.) 2-271
LMMB 038 PCB Congener Analysis of XAD-2 Resins and GFF Filters Using GC/ECD
(Crecelius, E. and Lefkovitz, L.) 2-285
LMMB 039 PCBs and Pesticides in Surface Water by XAD-2 Resin Extraction (Wisconsin
State Lab of Hygiene) 2-307
LMMB 040 Extraction and Cleanup of Sediments for Semivolatile Organics Following the
Internal Standard Method (Van Hoof, P. and Hsieh, J.) 2-325
LMMB 041 Analysis of Polychlorinated Biphenyls and Chlorinated Pesticides by Gas
Chromatography with Electron Capture Detection (Van Hoof, P. and Hsieh, J.) .... 2-335
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Table of Contents
LMMB 042 Standard Operating Procedure for the Analysis of PCB Congeners by GC/ECD
and Trans-Nonachlor by GC/MS/ECNI (Swackhamer, D., Trowbridge, A.,
and Nater, E.) 2-347
LMMB 043 Extraction and Lipid Separation of Fish Samples for Contaminant Analysis and
Lipid Determination (Schmidt, L.) 2-381
LMMB 044 Analysis of Total PCBs and PCB Congeners and Trans-nonachlor in Fish by Gas
Chromatography/Negative Chemical lonization Single Ion Mass Spectrometry
(Schmidt, L.) 2-389
CHAPTER 2: MERCURY ANALYSIS
LMMB 045 Standard Operating Procedure for Analysis of Vapor Phase Mercury (Keeler, G.
and Landis, M.) 2-403
LMMB 046 Standard Operating Procedure for Analysis of Mercury in Precipitation (Keeler, G.
and Landis, M.) 2-417
LMMB 047 Standard Operating Procedure for Analysis of Particulate Phase Mercury
(Keeler, G. and Landis, M.) 2-431
LMMB 048 Standard Operating Procedure for Mercury Analysis (Mason, R. and Sullivan, K.) . . 2-445
LMMB 049 Total Mercury Analysis in Aqueous Samples (Hurley, J.) 2-453
LMMB 050 Standard Operating Procedure for Analysis of Sediment for Total Mercury Using
the Cold Vapor Technique with the Leeman Labs, Inc. Automated Mercury
System (Uscinowicz, T. and Rossmann, R.) 2-473
LMMB 051 Mercury in Plankton (Nater, E. and Cook, B.) 2-505
LMMB 052 Versatile Combustion-Amalgamation Technique for the Photometric
Determination of Mercury in Fish and Environmental Samples (Willford, W.,
Hesselberg, R., and Bergman, H.) 2-511
LMMB 053
Analysis of Fish for Total Mercury (Nriagu, J.) 2-527
NOTE: For "Standard Operating Procedure for Lab Analysis of Coho Salmon Stomachs and Data Entry",
see Volume 1, Chapter 5, LMMB 026, Quality Assurance Project Plan for Coho Sampling for Contaminant
and Diet Analysis.
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Table of Contents
Volume 3
Metals, Conventionals, Radiochemistry, and Biomonitoring
Sample Analysis Techniques
CHAPTER 1: METALS
LMMB 054 Laboratory Methods for ICP-MS Analysis of Trace Metals in Precipitation (Talbot, J.
and Weiss, A.) 3-3
LMMB 055 Standard Operating Procedures for Preparation, Handling and Extraction of Dry
Deposition Plates: Dry Deposition of Atmospheric Particles (Paode, R. and
Holsen, T.) 3-25
LMMB 056 Standard Operating Procedure for EPA's LBL Energy Dispersive X-Ray
Fluorescence Spectrometry (Kellogg, R.) 3-43
LMMB 057 Analysis of Surface Waters for Trace Elements by Inductively-Coupled Plasma
Mass Spectrometry (Shafer, M. and Overdier, J.) . 3-83
CHAPTER 2: CONVENTIONALS
LMMB 058 ESS Method 130.1: General Auto Analyzer Procedures (Wisconsin State Lab
of Hygiene) 3-127
LMMB 059 ESS Method 200.5: Determination of Inorganic Anions in Water by Ion
Chromatography (Wisconsin State Lab of Hygiene) 3-135
LMMB 060 ESS Method 140.4: Chloride - Automated Flow Injection Analysis (Wisconsin
State Lab of Hygiene) 3-145
LMMB 061 ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method (Wisconsin State Lab of Hygiene) 3-153
LMMB 062 ESS Method 230.1: Total Phosphorus and Total Kjeldahl Nitrogen,
Semi-Automated Method (Wisconsin State Lab of Hygiene) 3-163
LMMB 063 ESS Method 310.1: Ortho-Phosphorus, Dissolved Automated, Ascorbic Acid
(Wisconsin State Lab of Hygiene) 3-173
LMMB 064 ESS Method 310.2: Phosphorus, Total, Low Level (Persulfate Digestion)
(Wisconsin State Lab of Hygiene) 3-179
LMMB 065 ESS Method 340.2: Total Suspended Solids, Mass Balance (Dried at
103-1053C) Volatile Suspended Solids (Ignited at 550°C) (Wisconsin State Lab
of Hygiene) 3-187
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Table of Contents
LMMB 066 Outline of Standard Protocols for DOC Analyses (Shafer, M.) 3-193
LMMB 067 Outline of Standard Protocols for Particulate Organic Carbon (POC) Analyses
(Baldino, R.) 3-201
LMMB 068 ESS Method 360.2: Silica Dissolved, Automated, Colorimetric (Wisconsin State
Lab of Hygiene) 3-207
LMMB 069 ESS Method 360.3: Silica, Dissolved, Micro Level Automated, Colorimetric
(Wisconsin State Lab of Hygiene) 3-213
LMMB 070 ESS Method 370.2: Sulfates Colorimetric, Automated, Methylthymol Blue
(Wisconsin State Lab of Hygiene) 3-219
LMMB 071 ESS Method 370.3: Sulfates Colorimetric, Automated Flow Injection,
Methylthymol Blue (Wisconsin State Lab of Hygiene) 3-227
LMMB 072 Standard Operating Procedure for Chloride and Silica in Lake Water
(Lachat Method) (Grace Analytical Lab) 3-235
j
LMMB 073 Standard Operating Procedure for Dissolved Reactive Phosphorous
(Lachat Method) (Grace Analytical Lab) 3-247
LMMB 074 Standard Operating Procedure for Ammonia (Lachat Method)
(Grace Analytical Lab) 3-255
LMMB 075 Standard Operating Procedure for Nitrate, Nitrite (Lachat Method)
(Grace Analytical Lab) 3-263
LMMB 076 Standard Operating Procedure for Total Kjeldahl Nitrogen (Lachat Method)
(Grace Analytical Lab) 3-275
LMMB 077 Standard Operating Procedure for Total and Dissolved Phosphorous
(Lachat Method) (Grace Analytical Lab) 3-285
LMMB 078 Analysis of Total Suspended Particles (TSP) and Total Organic Carbon (TOC)
in Air Samples: Integrated Atmospheric Deposition Network (IADN) TSP/TOC
Procedure (Wassouf, M. and Basu, I.) '. 3-297
LMMB 079 Standard Operating Procedures for Determining Total Phosphorus, Available
Phosphorus, and Biogenic Silica Concentrations of Lake Michigan Sediments
and Sediment Trap Material (Johengen, T.) , 3-305
LMMB 080 Standard Operating Procedure for Perkin Elmer CHN Analyzer (Model 2400)
(Eadie, B.) 3-313
LMMB 081
Quality Assurance Plan for the Use of Sediment Traps (Eadie, B.) 3-319
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Table of Contents
CHAPTER 3: RADIOCHEMISTRY
LMMB 082 Standard Operating Procedure for Primary Productivity Using 14C: Laboratory
Procedures (Grace Analytical Lab) 3-327
LMMB 083 Protocol for Standard Analysis for Cesium-137 (Bobbins, J. and Edgington, D.) ... 3-337
LMMB 084 Determination of the Activity of Lead-210 in Sediments and Soils (Edgington, D.
and Bobbins, J.) 3-341
CHAPTER 4: BIOMONITORING
LMMB 085 Standard Operating Procedure for Chlorophyll-a and Pheophytin-a (Turner
Designs Method) (Grace Analytical Lab) 3-349
LMMB 086 ESS Method 150.1: Chlorophyll - Spectrophotometric (Wisconsin State Lab
of Hygiene) 3-357
LMMB 087 Standard Operating Procedure for Phytoplankton Analysis (Grace Analytical Lab) . 3-365
LMMB 088 Standard Operating Procedure for Zooplankton Analysis (Grace Analytical Lab) . . . 3-395
LMMB 089 Quality Assurance Project Plan: Diet Analysis for Forage Fish (Davis, B. and
Savino, J.) 3-417
CHAPTER 5: SHIPBOARD MEASUREMENTS
LMMB 090 Standard Operating Procedure for GLNPO Turbidity: Nephelometric Method
(Palmer, M.) 3-443
LMMB 091 Standard Operating Procedure for GLNPO Total Alkalinity Titration (Palmer, M.) . . 3-451
LMMB 092 Standard Operating Procedure for Electrometric pH (Palmer, M.) 3-457
LMMB 093 Standard Operating Procedure for Meteorological Data Aboard the BV/Lake
Guardian (Palmer, M.) 3-463
LMMB 094 Standard Operating Procedure for GLNPO Specific Conductance: Conductivity
Bridge (Palmer, M.) 3-467
LMMB 095 Total Hardness Titration (Palmer, M.) 3-473
LMMB 096 Standard Operating Procedure for the Analysis of Dissolved-Phase Organic
Carbon in Great Lakes Waters (Grace Analytical Lab) 3-477
LMMB 097 Standard Operating Procedure for the Analysis of Particulate-Phase Organic
Carbon in Great Lakes Waters (Grace Analytical Lab) 3-485
LMMB 098 Standard Operating Procedure for the Sampling and Analysis of Total
Suspended Solids in Great Lakes Waters (Grace Analytical Lab) 3-499
IX
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Volume 2
Chapter 1: Organic Analysis
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Instrumental Analysis and Quantitation
of Polycyclic Aromatic
Hydrocarbons and Atrazine:
IADN Project
Donald Cortes and Wayne Brubaker
School of Public and Environmental Affairs
Indiana University
Bloomington, IN 47405
July 1996
Version 1.3
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Instrumental Analysis and Quantitation of
Polycyclic Aromatic Hydrocarbons
and Atrazine: IADN Project
1.0 Introduction
This document briefly outlines the instrumental analysis and quantitation of polycyclic aromatic
hydrocarbons (PAH) and atrazine collected in air and precipitation samples from three sites on the
Great Lakes. This work is conducted at the School of Public and Environmental Affairs, Indiana
University-Bloomington as a part of the Integrated Atmospheric Deposition Network (IADN).
The following summarizes the gas chromatographic-mass spectrometry (GC-MS) technique used.
its pertinent parameters, and analyte quantitation.
PAH analyses are performed on a Hewlett-Packard (HP) 5890 Series II gas chromatograph and a
HP 5989 mass spectrometer. Chromatographic resolution is achieved with a 30 m x 250 (jm DB-5
capillary column which has a 0.25 urn film thickness (J & W Scientific, Folsom, CA) with helium
carrier gas. PAHs are quantified by GC-MS using selected ion monitoring (SIM) and the method
of internal standards. The PAHs analyzed in this study are listed in Table I along with the primary
and secondary ions from their mass spectra.
Table 1. Target compounds and their monitored ions.
Retention Order
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Compound
dlO-anthracene*
acenaphthylene
acenaphthene
fluorene
d 1 0-phenanthrene**
phenanthrene
anthracene
fluoranthene
pyrene
retene
d!2-benzo (a) anthracene*
benzo (a) anthracene
chrysene
d!2-perylene*
benzo (b) fluoranthene
benzo (k) fluoranthene
benzo (e) pyrene
benzo (a) pyrene
indeno ( 1 .2.3.cd) pyrene
dibenzo (a.h) anthracene
Major Ion
188
152
153
166
188
178
178
202
202
219
240
228
228
264
252
252
252
252
276
27S
Secondary Ion
—
151
154
165
—
176
176
101
101
234
—
114
114
—
126
126
126
126
138
139
2-5
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Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter^
1
Table 1. Target compounds and their monitored ions.
Retention Order
21
22
Compound
benzo (g,h,i) perylene
coronene
Major Ion
276
300
Secondary Ion
138
150
^internal standard
**surrogate standard
2.0 Performance Evaluation
Prior to analyzing a sample set, the GC-MS system performance and calibration are verified for all
analytes. The mass spectrometer is tuned immediately before the running of a sample batch using
the system's operating software programs (AUTOTUNE) with perfluorotributylamine (PFTBA)
calibration gas. Mass spectrometer parameters are adjusted so that masses 69, 219, and 502 and
their respective isotopes meet the target mass-intensity criteria. A sample AUTOTUNE report is
included.
Hexane is injected prior to the running of a sample set to insure the system is free from
contaminants or interfering peaks. The Relative Percent Difference (RPD) between a calibration
standard and a performance standard should be withing 20%.
Sample injections and system maintenance are recorded in the appropriate laboratory logbooks
located near the instrument.
3.0 Instrumental Parameters
The GC oven temperature program is given in Table 2. Other significant gas chromatographic
parameters are:
Carrier gas:
Injector:
Injection volume:
Transfer line:
helium (99.999%; Liquid Carbonic, Chicago)
On Column, Constant Flow
luL
300°C
The mass spectrometer is operated in the electron ionization (El) mode with ion source and
quadrapole temperatures of 250DC and 100°C respectively.
2-6
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Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Table 2. GC oven temperature program for PAH analysis
Initial Temperature: 40DC
Initial Time: 3.00 min
Rate(°C/min)
Final Temperature (°C)
Final Hold Time (min)
30.0
240
0.00
50.0
Total Run Time: 22.70 min
300
II.7
4.0 Sample Analysis
The extracted samples, lab blanks and matrix spikes are stored in 4-mL amber vials at -20°C until
they are ready for analysis. They contain approximately I mL of solvent (hexane) and were
previously spiked with 50 uL of the internal standard solution (see Table 3).
Table 3. Internal standard solution.
Compound
dlO-anthracene
dl 2-benzo(a)anthracene
d!2-perylene
Concentration (ng/uL)
4.00
4.00
4.00
Standards and samples are brought to room temperature before they are injected. After the hexane
blank is injected, a calibration standard (see Table 5) is injected followed by the samples. All
injections are performed manually with 1 uL volumes.
The mass spectrometer is turned on after a four minute solvent delay. Data is acquired in selected
ion mode. Windows and ion ranges are given in Table 6.
5.0 Data Reduction and Analyte Quantitation
Data is collected and stored within the system's HP Apollo 400 computer. Mass chromatograms
are generated, and their peaks are integrated with the accompanying software programs.
Chemstation Enviroquant prepares quantitation reports with individual mass chromatograms and
spectra for all target analytes.
2-7
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Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
For each day of samples run. relative response factors (RRFs) for each analyte are determined from
the calibration standard's peak areas using Equation I.
mass
area
SHl
mass
area ,
ISM
Where massu - the analyte's known mass in the injected amount of calibration standard,
areaa - the analyte's peak area,
masSj^j — the known mass of the appropriate internal standard, and
areaisttl = that internal standard's peak area.
With reference to Table 1, the response factors for compounds 2-10, 12-13, and 15-22 are
calculated relative to the internal standards dlO-anthracene, d!2-benzo(a)anthracene, and
d!2-perylene respectively.
An analyte's mass in a sample (massj is calculated from the RRFsll/ above and the internal
standard response in the sample by the following equation:
(mass ) , = (area ) . x RRF
a siiinpl? ^ i.i/.-iiininlt- slt
mass.
area
sample
Where area,, = the analvte 's peak area in the sample,
mussel = the mass of internal standard spiked into the sample, and
arealitd — the internal standard's peak area in the sample.
The analyte concentrations are tabulated by Enviroquant and transferred to an Excel spreadsheet.
6.0 Quality Assurance
Each daily analytical batch includes at least one calibration standard, one performance standard,
one instrument blank, one procedure blank, and one matrix spike. Acceptance criteria are
summarized on the attached Table 3-1. Refer to the IADN Quality Assurance Project Plan
(QAPjP) for more details.
>
7.0 Atrazine
Atrazine is analyzed by GC/MS in the selected ion monitoring mode. The GC temperature
program is given in Table 4 below. The calibration standard contains both atrazine and the
internal standard d 10-anthracene at concentrations of 0.4 ng/pL.
2-8
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Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
The mass spectrometer is turned on after a solvent delay of 8.5 minutes. The ions monitored are
m/z 186, m/z 188, m/z 200. and m/z 215. The ions m/z I 88 and m/z 200 are the quantitation ions
for dlO-anthracene and atrazine respectively, and the other two are for confirmation purposes.
Table 4. GC oven temperature program for atrazine analysis
Initial Temperature: 40°C
Initial Time: 1 .00 min
Rate(°C/min)
25.0
4.0
20.0
Final Temperature (°C)
1 40
240
290
Final Hold Time (min)
0.00
0.00
5.00
Total Run Time: 29.50 min
8.0 Chromatograms
Mass chromatograms from a typical 1 uL injection of calibration standard are given on the
following pages in Figures la-i. The windows on the left-hand side of each page show the
quantitation peak(s) for one or more PAHs, and those on the right give the corresponding
confirmation peaks. Similar mass chromatograms (Figure 2) are given for a 1 u.L injection of the
atrazine calibration standard.
2-9
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
Table 5. Calibration standard.
Retention Order
!
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Compound
dIO-anthracene*
acenaphthylene
acenaphthene
fluorene
phenanthrene
dlO-phenanthrene**
anthracene
fluoranthene
pyrene
retene
d!2-benzo (a) anthracene*
benzo (a) anthracene
chrysene
d!2-perylene*
benzo (b) fluoranthene
benzo (k) fluoranthene
benzo (e) pyrene
benzo (a) pyrene
indeno (l,2,3,cd) pyrene
dibeno (a,h) anthracene
benzo (g,h,i) perylene
coronene
Concentration (ng/uL)
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0,20
0.20
0.20
0.20
0.20
,0.20
0.20
0.20
0.20
0.19
0.20
0.20
0.20
0.20
0.20
^internal standard
**surrogate standard
Table 6. SIM windows for analyte detection.
SIM Window
1
T
3
4
Start Time (min)
6.50
9.80
11.70
14.80
SIM Mass (m/z)
76.83, 151. 152, 153, 154, 165, 166
101,202, 176, 178, 188,204,219,234
114.126,152,228,240,264
138. 139. 150. ->76. 278. 279. 300
2-10
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Auto Tune
Tune File: alune.u Tue Apr 11 35 05.08:05 PM
Instrument Name: hpl Instrument Type: 5389 looization Mode: El
Repeiler 7.00
Ion Focus Cain 255
Ion Focus 34
Entrance Lena 14S
X Ray 40
MASS: 69 00
AB: 563038 PW: 0 SO
C4 65 66 67 68 69 70 71 72 73 74
ultiplier
MUGain
MU Offset
xis gain
xls Offset
AB:
-1 216
1583
167
106
63
4
MASS
Emission 300 Integrmlion 50
Elec. Energy 79 Samples 16
Polarity POSITIVE Averages 1
Source Temp 250 Stepsize 0.12
Quad. Temp 100
213.30
183129 32.501 PW: 052
I
I
218
L
220 222 22
4
MASS: 502.00
AB: 7218 1-28X PW 0 56
I
j
493 SOO 502 504 506
m/z m,"z
MASS
63.00
21890
501 9S
Abundance
422528
158464
(714
Rel. Abund
100.00
37 .SO
1.59
Iso. Mass
70.00
219.90
502.95
Iso. Abund.
4033
6279
672
Iso. Ratio
0.37
3 36
10.01
Scan. 10.00 -800.00 samples: 16 thresh: 20 Tue Apr 11 35 05:08:16 PM
259 peaks Bue. 63.00 Abundance: 422528
100-
30^
Rfl "
ou -
70 -1
60-;
50^
•w-.
30 -i
20^
a
"I
0 — H— i r-
,
'—i ' I—^T 1 1 ! — n 1 r — 1 1 — 1 1 1 : ; ^ i i r~T I i i I 1 i i i • — : \
100 200 300 40Q SOO 600 700 300
m/2
2-11
-------
QA Criteria
precision
accuracy
blanks
completeness
calibration
PCB identification
detection limits
deteclahility
holding lime
QC
Code
FD1/
FT) 2-
LI) I/
LD2
LSS
LMS
FRB
LRB
CI.M
bCl.S
LPC
LCB
RFS
RFS
Table 3.1. Data
Sample Type
field; co-located samplers
laboratory: replicate analyses
surrogate spikes
matrix spikes
9
field matrix blank
lab matrix blank
field samples
multiple point calibration.
4 point
single calibration sld
performance sld
lab calibration blank
GCMS confirmation
MDL study
routine field samples
routine field sample
Quality Objectives for Trace Organic Compounds
Frequency
20%
1 0%
all samples
1 /batch
10% (I/month)
10%
annual
1/10 samples
daily
2/GC run
5%
1 /project
all samples
all samples
Required Objective
at<5xMDL, rsd < 100%
al<5xMDL, rsd <50%
ut <5xMDL, rsd <5()%
M <5xMDL, rsd <30%
5()%MDL
1 year
Control Action Units
re-analyze if sample available; otherwise Hag %
samples FFD or PTS
re-aiicily/e same or all sample; otherwise Hag %
samples FDL or PTS
flag PCB congener range FSS; investigate sources %
of loss
(lag sample FMS; %
investigale sources of loss
Hag samples FFR; find source of contamination mass
run 2nd LRB; elminate source of imprecision; Hag mass
sample FBS
no action; % reported
reoptimi/.e instrument, repeal calibration
reoplimi/e instrument, repeal calibration
regenerate response factors
check for contamination; reoptimize instrument
reported in yearly QA Report
Hag BDL
Hag EHT
^
3
1
»•»
o'
T;
§•
i
0
(A
to
Q.
Jj
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ts
3'
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5.
o
o
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to s»
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c
I!
O" -i
9. ?
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o^ to
° Q.
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Abundance
Ref 50 -
0 -
Scan 228 (8.557 min) : 15129DRC.
1!
76
64 128
v , | . , , ! , , , , | , i , , | ,-, , , |— r-i—
n/z~> 60 80 100 120 140
Abundance
Raw 50 -
0
Scan 912 (8.680 min): 16449DRC
1,
76
1
n/z — > 60 80 100 120 140
Abundance
Sub so :
n .
Scan 912 (8.680 min): 16449DRC.
i:
76
1 1
D (-.*)
>2
1 165
H--T '|-r i-r—
160
-D <*)
12
) 166
1 i | i r I
160
D <-,*)
2
1 166
U I | , I , | , , , , | , , r , , , , , I | , , i I , , , ,
jl/z — > 60 80 100 120 140 160
02
Acenaphthylene
Concen: 374.36 ng
RT: 8.68 min Scan* 912
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug OS 96 12: 03: 1C
Tgt Ion:152 Reap: 75932
Ion Ratio Lower Upper
152 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
80000-
60000 -
40000-
20000-
0-
Time — >8.
Ion 152.00 (151.70
8.63
I
62 8.77
Sundance Scan 238 (8.754 min): 15129DRC.D (-,*)
1
•
Ref 50 -
0 -
1
76
64 128
n/z — > 60 80 100 120 140
4
166
160
ftbundance Scan 947 (8.819 min): 16449DRC.D (*)
Raw 50 .
1
0 J
1!
76
i I i i i ,,,,,,,,,,,,,,, | ,.
3
166
•(•'••
n/z— > 60 80 100 120 140 160
Abundance Scan 947 (8. 819 min): 16449DRC.D (-,*)
Sub ,rt
50 -
-
tt/Z — >
1
76
S~1"r- T — r r ~p r -r -i — i — | — i — i — i — T — j — i — i — r— i — ^ — t — T—
60 80 100 120 140
3
166
' 1 ' ' ' '
160
#3
Acenaphthene
Concen: 359.10 ng
RT: 8.82 min Scantt 947
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:153 Reap: 64434
Ion Ratio Lower Upper
153 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundancelon 153.00 (152.70
80000 -
:
60000-
•
40000 -
20000 -
0 -
Time — >8.
8 . 82
I
\
I
74 8.95
Figure la.
2-13
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
Abundance
Ref 50 -
0
Scan 268 (9.346 min) : 15129DRC.D {-
1
83
64 | 128 154
,*)
16
n/z--> 60 80 100 120 140 160
Abundance
Raw 50 -
0 -
Scan 1053 (9.239 min): 16449DRC.D
1
83
. I 154
*)
16
n/z--> 60 80 100 120 140 160
Abundance
Sub50.
Q
Scan 1053 (9.239 min): 16449DRC.D (-
1
83
| 154
,*)
16
n/z— > 60 80 100 120 140 160
#4
Fluorene
Concen: 359.90 ng
RT: 9.24 min Scan# 1053
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 166 Resp: 64595
Ion Ratio Lower Upper
166 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
80000-
60000 -
40000 -
20000-
0-
time — >9.
Ion 166.00 (165.70
9.24
[
16 9.35
"Abundance
Ref 50 -
0 -
tl/2 — >
Abundance
Raw 50 ;
0-
n/z — >
Scan 358 (10.598 min) :
1'
1
15129DRC.D (-,*)
8
100 150 200
Scan 1242 (10.009 min)
r
101
: 16449DRC.D (*)
8
188 219 244
100 150 200
Abundancescan 1242 (10.009 min): 16449DRC.D (-,*)
Sub 50 -
0 -
n/z — >
1"
101
•-,--, ,•-••, • , - , - r — T
100 150
'8
188 219 244
V- , 1 r-' 1-1 r ,
200
#6
Phenanthrene
Concen: 349.43 ng
RT: 10.01 min Scan* 1242
Delta R.T. ' 0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 178 Reap: 87788
Ion Ratio Lower Upper
178 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
100000-
50000-
0 -
Time — >9.
Ion 178.00 (177.70
10.01
1 \ \
1\J
97 10.04
Figure Ib.
2-14
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
MsundanceScan 364 (10.678 min): 15129DRC.D (-,*)
Ref 50
0 -
n/z — >
r
83
8
I i | l I l ' 1 i r— "i 1 1 , 1 1 , 1-
100 150 200
Abundance scan LZSZ (10.053 min): 16449DRC.D (*)
Raw 50 .
0 -1
1"
101
8
188 219 244
i i 1 i i i i 1 i i H i 1 1 1 1 1 r-
n/z — > 100 150 200
PibundanceScan 1252 (10.053 min): 16449DRC.D (-,*)
Sub ,
50 -
r
101
8
188 219 244
m/z — > 100 150 200
#7
Anthracene
Concen: 370.90 ng
RT: 10.05 min Scan# 1252
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 178 Resp: 80073
Ion Ratio Lower Upper
178 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Pibundance
100000-
50000-
0 -
Time — >9.
Ion 178.00 (177.70
(1 10.05
A
/UL
99 10.14
Abundance
Ref 50 -
0 -
Sc«
1C
in 515 (12.534 min) : 1512
2(
)1
165
9DRC.D (-.*)
2
234
n/z— > 100 120 140 160 180 200 220 240
Abundance
Raw 50
0 -
Scan 1451 (10.935 min): 16
' 2<
101
165 188
449DRC.D (*)
2
219 244
TI/Z-- > 100 120 140 160 180 200 220 240
Abundance
Sub50^
Scan 1451 (10.935 min): 164
2<
101
165 188
49DRC.D (-,*)
12
219 244
0 'i i i1 i i i i i i i i i-[ i i i i | i i i i | i i i i i i i i |
TI/Z— > 100 120 140 160 180 200 220 240
#9
Fluoranthene
Concen: 351.20 ng
RT: 10.94 min Scant* 1451
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:202 Reap: 88151
Ion Ratio Lower Upper
202 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
100000 •
50000-
0 -
Ion 202.00 (201.70
10.94
1
rime— 510.87 11.02
PM
Figure Ic.
2-15
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
Abundance Scan 549 (12.945 min): 15129DRC.D (-,*)
Ref 50 -
0-
n/z — >
Pibundance
Raw 50 -
0 -
n/z — >
21
101
165
12
100 120 140 160 180 200 220 240
Scan 1490 (11.109 min): 16
2
101
165 188
449DRC.D (*)
12
219 244
100 120 140 160 180 200 220 240
WaundanceScan 1490 (11. 109 min): 16449DRC.D (-,*}
Sub50-
0-
1»/Z >
21
1
31
165
12
219 234
100 120 140 160 180 200 220 240
#10
Pyrene
Concen: 350.53 ng
RT: 11.11 min Scan# 1490
Delta R.T. -0.01 min
Lab File: 16449DRC.D
Acq: Mon Aug 0
Tgt Ion: 202 Re.
Ion Ratio Lot
202 100
0 0.0 (
3 96 12:03:10
»p: 91556
-»er Upper
).0 0.0
0 0.0 0.0 0.0
i 0 0.0 0.0 0.0
fcbundanceion 202.00 (201.70
100000-
'
50000-
0-
Time — 511.
11.
i
1
04
11
11.22
toundanceScan 598 (13.537 min): 15129DRC.D (-,*)
Ref 50 -
0 -
2
101 202
165
9
234
n/z— > 100 120 140 160 180 200 220 240
Abundance
Raw 50 -
0-
Scan 1540 (11.331 min): 16449D
21
1(
O f\ A
ZU4
31 165178
1 ' i
RC.D (*)
.9
234
,,,,,,,,, 1 , , , , | , , , , | , i \ , ,.,,.,,,,
TI/Z— > 100 120 140 160 180 200 220 240
toundancescan 1540 (11. 331 min): 16449DRC.D (-,*)
Sub ...
50 -
0-
2
204
101 165178
i t
.9
234
.... , ,,.... . .. i ... i i i i .., ,,.,,,., | ,..
n/z— > 100 120 140 160 180 200 220 240
ffll
Retene
Concen: 389.34
RT: 11.33 min
ng
Scant* 1540
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 219 Reap: 33832
Ion Ratio Lower Upper
219 100
0 0.0
0 0.0
0 0.0
0.0 0.0.
0.0 0.0
0.0 0.0
Maundancelon 219.00 (218.70
40000 -
20000 -
-
11
j
.33
L '•
Time— >11.25
i
11.45
PM
Figure Id.
2-16
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Abundance
Ref 50 -
0 -
Scan 750 (15.375 min): 151291
2;
114
126
Abundance
Raw 50 _
o
5RC.D (-,*)
.8
264
1 ' ' I ' ' ' ' I ' ' ' ' | ' '
150 200 250
Scan 1743 (12.059 min): 1644
2:
114
n/z — >
Abundance
Sub50-
9DRC.D (*)
18
240
i
150 200 250
Scan 1743 (12.059 min): 1644$
2:
114
T»/Z — >
JDRC.D (-,*)
18
240
150 200 250
#13
Benzo (a) anthracene
Concen: 351.22 ng
RT: 12.06 min Scan* 1743
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:228 Reap: 66629
Ion Ratio Lower Upper
228 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
80000-
40000 :
20000-
0-
Ion 228.00 (227.70
12.06 .
Tirae—^.2.01 12.08
Abundancescan 756 (15.448 min): 15129DRC.D (-,*)
1
Ref 50 -
n/z-->
Abundance
Raw 50 .
0-
2;
114
126
8
252
120 140 160 180 200 220 240
Scan 1754 (12.091 tain) : 16449D
2:
114
RC . D ( * )
3
, , , | , , , , | T r i i j i i . i J i . i . | . - ' i | i i • I I i ' |
n/z— > 120 140 160 180 200 220 240
Abundancescan 1754 (12.091 min): 16449DRC.D (-,*)
Sub ,n
50 -
0-
2:
114
i ii | i i i i i i i i i | i i i i | i i i i i i i i i i
8
1 ' ' 1 ' ' ' ' 1
TI/Z— > 120 140 160 180 200 220 240
#14
Chrysene
Concen: 335.06 ng
RT: 12.09 min Scanfl 1754
Delta R.T.
-0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug
05 96 12:03:10 PM
Tgt Ion:228 Resp: 80895
Ion Ratio Lower Upper
228 100
0 0.0
0 0.0
0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
Abundancelon 228.00 (227.70
•
80000-
60000 -
•
40000 -
20000 •
0 -
12.09
ft
\
J
^-*
Time— ^.2. 02
A
1 ^
i
12.20
Figure le.
2-17
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
Abundance Scan
Ref 50 -
0 -
936 (17. 623 min): 15129DRC.D (-,*)
252
126
114
n/z — >
Abundance Scan
Raw 50 .,
0 -
12
n/z — >
AbundanceScan
Sub50-
228
264
150 200 250
2113 (13.184 min): 16449DRC.D (*)
252
>6
2€4
> ) i i ' i | . i i i | i i
150 200 250
2113 (13.184 min): 16449DRC.D (-,*)
21
126
Tt/Z — >
>2
264
150 200 250
#16
Benzo (b) f luoranthene
Concen: 329.91 ng
RT: 13.18 min Scan# 2113
Delta R.T. -0.00 min
Lab File: 16449DRC.
Acq: Mon Aug 05 96 12
D
:03:10
Tgt Ion:252 Resp: 55725
Ion Ratio Lower Upper
252 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundancelon 252.00 (251.70
40000-
20000-
0 -
13.18
A
/ \
/ \
/ \
/
y
/"
Time— =13.13 13.21
AbundanceScan 941 (17. 683 min): 15129DRC.D (-,*)
Ref 50 -
0 -
. 2:
1
126
114
n/z— >
Abundance Scan
Raw 50 .
o
12
n/z— >
AbundanceScan
Sub - _
50 -
0 -
1
•n/z — >
2
264
150 200 250
2123 (13. 218 min): 16449DRC.D (*)
2:
>6
>2
264
150 200 250
2123 (13.218 min): 16449DRC.D (-,*)
2.
26
•2
264
i | i , i i | . , , ,
150 200 250
#17
Benzo (k) f luoranthene
Concen: 371.03 ng
RT: 13.22 min Scan# 2123
Delta R.T.
-0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug
05 96 12:03:10
Tgt Ion:252 Resp: 78719
Ion Ratio Lower Upper
252 100
0 0.0
0 0.0
0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
BJaundancelon 252.00 (251.70
40000-
20000 -
0-
13.22
A
A
J
rime— 513.12
\
v_
' ' 1
13.37
PM
Figure If.
2-18
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitatlon of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
^bundanceScan
Ref 50 •
0 -
986 (18.227 min) : 15129DRC.D (-,*)
2i
126
114
u/z — >
Abundance Scan
Raw 50 J
o
226
>2
• • | .,,,,,, , , | , ,
150 200 250
2219 (13.544 rain): 16449DRC.D (*)
2;
126
n/z — >
Abundances can
Sub50-
0-
,2
264
ISO 200 250
2219 (13.544 min) : 16449DRC.D (-.*)
2,
126
n/z — >
12
264
T • | . 1 J , | ... I .1
150 200 250
#18
Benzo (e)pyrene
Concen: 337.11 ng
RT: 13.54 min Scan# 2219
Delta R.T. -0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:252 Resp: 69063
Ion Ratio Lower Upper
252 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundancelon 252.00 (251.70
40000-
20000-
0-
13.54
A
/ \
/ \ A
/ \ /
/ \ /
/ V J
J ^T
1
rime-->13.47 13.60
Abundance Scan
Ref 50 -
996 (18.348 min): 15129DRC.D (-,*)
2!
126
114
Sundance Scan
Raw 5Q _
01
12
Jd/Z >
Abundances can
Sub -„
50 -
1-
- — i — i —
n/z — >
228
2
264
150 200 250
2240 (13. 615 min): 16449DRC.D (*)
2*9
1
!6
*.
264
150 200 250
2240 (13. 615 min): 16449DRC.D (-,*)
2!
26
'2
264
150 200 250
#19
Benzo (a)pyrene
Concen: 398.
13 ng
RT: 13.62 min Scant* 2240
Delta R.T.
Lab File:
-0.01 min
16449DRC.D
Acq: Mon Aug 05 96 12:03:10 PM
Tgt Ion: 252
Ion Ratio
252 100
0 0.0
0 0.0
0 0.0
Abundancelon
40000-
,
20000-
0-
-I
rime — H3.46
Resp: 63486
Lower Upper
0.0 0.0
0.0 0.0
0.0 0.0
252.00 (251.70
.3.62
1
1
\ \
V v_
13.87
Figure Ig.
2-19
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
BibundanceScan
Ref 50 -
0 -
138
1340 (22. 506 min): 15129DRC.D (-,*)
21
'6
300
n/z--> 150 200 250 300
\bundance Scan
Raw 50 .j
0
138
n/z— > 1,
AbundanceScan
Sub50
0-
138
2681 (15. 531 min): 16449DRC.D (*)
2
6
300
k .
50 200 250 300
2681 (15. 531 min): 16449DRC.D (-,*)
21
6
300
. . , , ,,.,,,,,,11
n/z — > 150 200 250 300
#20
Indeno(l,2,3-cd)pyrene
Concen: 615.39 ng
RT: 15.53 min Scan* 2681
Delta R.T. 0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:276 Reap: 66114
Ion Ratio Lower Upper
276 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundancelon 276.00 (275.70
20000-
15000-
10000-
5000-
0-
15.53
ti
ft
\
\
1 \_ I
, . . , ,
Time— -515.25 15.98
PM
&bundanceScan
Ref 50 -
0 -
139
1273 (21. 696 min): 15129DRC.D (-,*)
21
8
n/z--> 150 200 250 300
Abundance Scan
Raw 50 i
0-
139
2687 (15. 579 min): 16449DRC.D (*)
2'
8
300
I
, . . | ,,.,,,,., |
n/z— > 150 200 250 300 ^
l\bundanceScan
Sub50-
0 -
139
2687 (15. 579 min): 16449DRC.D (-,*)
2|8
i
I 30°
II i *
, , , i i , i i , i . , |
n/z— > 150 200 250 300
#21
Dibenzo(a,h) anthracene
Concen: 464.76
RT: 15.58 min
ng
Scan* 2687
Delta R.T. -0.01 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion:278 Reap: 47193
Ion Ratio Lower Upper
278 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0
0.0 0.0
Msundancelon 278.00 (277.70
15000-
10000 -
5000 -
0 -
15
)
.58
i
1
\
rime-->15l29
i
15.97
PM
Figure Ih.
2-20
-------
Volume 2, Chapter 1
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Abundance
Ref 50 -
0 -
oc
13
in 1340 (22.506 min): 15129DRC
2'
18
•D (-,*)
re
" i i | i i i i | r i — i 1 [
n/z — > 150 200 250 300
Abundance
Raw 50 .
0 -
Sc
i:
:an 2752 (16. 093 min): 16449DRI
2'
i8
6
300
n/z — > 150 200 250 300
Abundance
Sub50-
0 -
Sc
i:
an 2752 (16.093 min): 16449DRC
2'
58
•D (-,*)
6
300
n/z— -> 150 200 250 300
»22
Benzo (g,h,i)perylene
Concen: 363.31 ng
RT: 16.09 min Scan* 2752
Delta R.T. 0.00 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 276 Resp: 64079
Ion Ratio Lower Upper
276 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
Abundance
20000-
15000
10000-
5000-
0-
Time — 515.
Ion 276.00 (275.70
16.09
1
86 16.40
Abundance
Ref 50-
Q
Scan
1!
1993 (30. 396 min): 15129DRC.D (-
31
0
278
,*>
0
n/z— > 150 200 250 300
Abundance
Raw 50 .
Q
Scan
If
3339 (20. 740 min): 16449DRC.D («
31
»0
279
ii
)
0
n/z~> 150 200 250 300
Abundance
Sub50-
Scan
1.
3339 (20. 740 min): 16449DRC.D (-
3(
30
,*)
10
n/z— > 150 200 250 300
#23
Coronene
Concen: 328.96 ng
RT: 20.74 min Scan# 3339
Delta R.T. -0.01 min
Lab File: 16449DRC.D
Acq: Mon Aug 05 96 12:03:10
Tgt Ion: 300 Resp: 44543
Ion Ratio Lower Upper
300 100
0 0.0 0.0 0.0
0 0.0 0.0 0.0
0 0.0 0.0 0.0
/Abundance
8000 :
6000-
4000-
2000-
0 -
rime — :20
Ion 300
2C
/
.00 (299.70
.74
V
.43
21.02
PM
Figure li.
2-21
-------
Instrumental Analysis and
Quantitation of Polycyclic
Aromatic Hydrocarbons and Atrazine: IADN Project
Volume 2, Chapter 1
Ion 200.00 amu from 13S88wwta.d
100% -1584
.
120-
100-
80 -i
60-
40-
20:
0-
CD
at
0
nUa,
10 11 12 13 14 15 16 17 18 19
Ion 188.00 amu from 13588wwb.d
100% -4366
-
120:
-
100:
80:
60-
40-j
20:
- 0-
•*
r>
to
v
JlO-wthracaM
10 11 12 13 14 15 16 17 IB 13
Ion 215.00 amu from 13588wwto.d
100% -1033
-
120-
-
100-
80^
60-
40:
20 -j
0-
8
0
f»
(
1
.l^- i
10 11 12 13 14 15 16 17 18 13
Ion 186.00 amu from 13S88wwb.d
100% - 834
-
120:
-
100:
80 -_
60-
40:
20:
0-
r*.
(D
T"
10 11 12 13 14 15 16 17 18 13
Figure 2. Mass chromatograms for atrazine calibration standard.
2-22
-------
Analysis of PCBs and Pesticides in Air and
Precipitation Samples:
IADN Project
Gas Chromatography Procedure
ilora Basu
School of Public and Environmental Affairs
Indiana University
Bloomington, IN 47405
September 1995
Version 1.0
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
1.0 Routine GC Maintenance
l.l Gas Tanks
1.1.1 Check gas tanks. Tanks should not go dry. While changing the tank, lower the
temperature of the oven down to 40°C. Leave it at 40°C for about half an hour after
changing the tank to get rid of air or oxygen that was drawn in.
1.1.2 Check column head pressure. It should be at 24 psi. If pressure falls, tighten the septum
nut. If the pressure is still low check for leaks and tighten other connections.
1.2 GC Baking
After every GC run bake the oven at 280°C for one hour. After every other run also bake the
injector and the detector at 280°C and 380°C.
1.3 Septum
1.3.1 Preparation of Septum
1.3.1.1 Place the septa in a small beaker. Cover with aluminum foil.
1.3.1.2 Place the beaker in GC oven and bake the septa while baking the column at
280°C.
1.3.1.3 Store the clean septa in the beaker keeping the foil lid intact and use as needed.
Always use tweezers to get clean septum out.
1.3.2 Changing of Septum
1.3.2.1 After every 60 samples or so change the septum.
1.3.2.2 Cool the oven down to 40°C.
1.3.2.3 Remove autosampler towers.
1.3.2.4 Remove septum nut and take the old septum out. Discard.
1.3.2.5 Using clean Q-tips soaked in hexane, wipe off the septum holder until no more
dirt is seen on Q-tips.
1.3.2.6 Put in pre-cleaned septum and replace the nut. Nut should he snug but not too
tight. Column head pressure should go up to 24 psi if nut is tight enough. Check
the tightness of the nut after injecting the first sample. Make sure that the head
pressure is still 24 psi.
2-25
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure Volume 2, Chapter 1
1.4 Checking Background
1.4.1 Background signal in GC varies from 28 to 32. Hexane is analyzed at the start of every
GC run to monitor the baseline stability. If the signal goes up or hexane run produces
noisy chromatogram GC should be cleaned.
1.5 Checking Standard
1.5.1 Mullin 94 standard and Mixed Pesticide Standard should be monitored to check peak
detection and peak broadening or tailing. If the peak shapes are not satisfactory, column
should be clipped. More than 90 peaks should be detected in PCB standards and cong. 17
and 18 should be separated. If not, install a new column.
1.6 Checking Leaks and Gas Flow
1.6.1 Check leaks once in two weeks with a leak detector. Check, around the septum, at the
injector end, and at the detector end of the column.
1.6.2 Check the gas flow once in two weeks with a flow meter. Approximate gas flow are as
follows :
Split vent 120mL/min.
Purge vent 2 mL/min.
Total flow through detector 22 mL/min.
2.0 GC Cleaning: Clipping Old Column or Installing a New Column
2.1 Taking Apart
2.1.1 Turn oven, injector and detector off.
2.1.2 Turn hydrogen and nitrogen off. Wait until everything cools down.
2.1.3 Take the autosampler tower off.
2.1.4 Undo the small nut covering the septum and the large nut underneath it to expose the
injection liner. Take the liner out.
2.1.5 Open the oven. Take the columns out (by detaching from injector and detector ends).
2.1.6 Unscrew the nuts from both injector and detector ends of columns and plug the column
ends with septum. Open end of column should not be exposed to air.
2.1.7 Place the columns on the workbench.
2.1.8 Unscrew the holder nut underneath the injection liner. There is one gold seal and a
washer in it. Washer and seal need to be replaced each time it is taken apart. Clean these
parts by ultrasonication with Dichloromethane and Hexane.
2-26
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
Volume 2, Chapter 1 IADN Project - Gas Chromatography Procedure
2.1.9 Put a beaker inside the oven underneath the injection port and pour some hexane through
the injection port. Clean the injection port with Q-tips and rinse again with hexane.
2.2 Assembling Injection Port and Liner
2.2.1 After ultrasonication air dry each part. Assemble the holder nut. Place the gold washer
first and then the seal. The tapered opening of the seal will face downward. (The tapered
end will hold the end of the ferrule from the column.) Screw the nut in before placing the
injection liner.
2.2.2 Insert a new liner.
2.2.3 Put a viton O-ring on the liner. Put the big nut on and tighten it. Put a clean septum.
Cover the septum with septum nut. Tighten with wrench.
2.3 Clipping Column
2.3.1 Take the nut off the injector end of the column. Carefully scrape out all the ferrules from
the column nuts. Clean all different parts with Q-tips soaked in DCM and ultrasonicate
these with DCM and Hexane for 10 minutes with each solvent. Onto the column, insert
the nut first and then the ferrule with conical end pointing towards the open end of the
column.
2.3.2 Clip the column. Make a clean cut with diamond tip score or ceramic square. Examine
the hole with magnifying glass. It should be a clean hole without any jagged end. Always
clip the column after putting the nut and the ferrule on.
2.3.3 Measure 25mm from the tip of the column. Mark this point with Liquid Paper®.
2.3.4 Carefully insert the column with nut and ferrule through the holder nut and screw it in.
As soon as it feels tight, pull the column out gently until the white mark is seen. Hand
tighten the screw more and make it tight with wrench 1A turn after hand tight. Do not
overtighten.
2.3.5 Take the nut off the detector end of the column. Put the nut and the ferrule on the column
in the same way as in the injector end. Clip the column and check for the nice clean cut.
Insert the column all the way up until it does not go any more. Pull down about 1 mm and
tighten the screw. Turn hydrogen on and check the flow of gas through the column by
inserting the cut end in a beaker of hexane. Turn hydrogen off.
2.4 Checking Leaks and Gas Flow
2.4.1 Turn H, and N, on. Check leaks with a leak detector. Check around the septum, at the
injector and at the detector end of the column. Check that the head pressure is 24 psi.
2.4.2 Check the gas flow with a bubble meter Approximate gas flow are as foil
Split vent 120 mL/min.
Purge vent 2 mL/min.
Total flow through detector 22 mL/min.
2-27
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure Volume 2, Chapter 1
2.5 Assembling
2.5.1 Replace autosampler tower.
2.5.2 Turn heated zones on.
2.5.3 Turn oven on and set the temperature to 40°C for an hour. Change oven temperature to
70°C and leave another hour.
2.5.4 If it is an old column, bake the column, injector and detector for an hour.
Baking temperature :
Oven: 280°C
Injector A: 28-0°C
Injector B: 280 °C
Detector A: 380°C
Detector B: 380°C
2.5.5 If it is a new column, bake injector and detector. Column can be baked by ramping it 13
or 2° per minute to 280°C. Hold there for one hour.
2.5.6 If blank run looks satisfactory, put in a standard and check.
3.0 Routine GC Operation
3.1 GC condition and oven temperature program:
PCBs, Hexachlorobenzene, and DDE are eluted in the hexane fraction, whereas the other
chlorinated pesticides are eluted in the 50% dichloromethane in hexane fraction after silica gel
column chromatography. The procedure for nitrogen blowdown, spiking with internal standard,
and making microvials for the autosampler are described in IADN Project Sample Preparation
Procedure, Version 1.0, June 1995.
Gas Chromatograph used for analysis of PCBs and pesticides is Hewlett Packard 5890 with 7673 A
Autosampler. Operation is controlled by the Integrator Hewlett Packard 3396. Data acquisition is
done by Hewlett Packard Peak 96. Finally, data analyses are done in Hewlet Packard 3365
ChemStation.
Carrier gas: Hydrogen
Make up gas: Nitrogen
Split vent: 120mL/min.
Purge vent: 2 mL/min. *•
Total flow through the detector: 22 mL/min.
Column: DB-5. 60m. 0.25mm i.d, 0.1 u film thickness
2-28
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Temperature Program:
Initial time:
Rate:
Final temp.:
Rate A:
Final temp A:
Final time:
Purge on:
Purge off:
Run time:
Initial temp. !00°C
1 min.
l°C/min.
240° C
10°C
280°C
20 min.
3 min.
150 min.
165 min.
The GC condition, column type, and the oven temperature program was specified by Mike
Mullin. The method name is Mullin.met.
3.2 GC Pre-run
3.2.1 Check to see if there is sufficient H2 for operation. If not, change tanks.
3.2.2 If necessary, change septum.
3.2.3 Bake oven at 280°C for one hour. Bake injector and detector at 280°C and 380°C
respectively about every other time the oven is baked.
3.2.4 Cool oven to 100°C, injector to 250°C, and detector to 350°C.
3.2.5 Make the samples ready in microvials and load the autosampler tray.
3.3 Preparing Sequence in ChemStation
3.3.1 Editing Sequence
3.3.1.1 Sequence Parameters
Sequence
Edit Sequence Parameters
Type in operator's name and subdirectory name. Type in information about
calibration standard and spikes in comment.
3.3.1.2 Sequence Table
Sequence
Edit Sequence
Table
Enter "From Vial" and "To Vial" number.
2-29
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
3.3.1.3 Sample Table
Sequence
Edit Sample Table
Type in sample ID's. First vial should be hexane. Second vial should be
calibration standard. Third vial should be a Performance standard. Other vials
are "actual samples. At the end of each sample ID indicate whether the sample is a
hexane fraction or 50% fraction (H,Fl). Repeat hexane blank and a fresh
standard after every set of samples.
3.3.1.4 Print Sequence
Sequence
Print
x Sequence Parameters
x Sample Table
3.4 Starting a GC run
3.4.1 Programming integrator
Set runnum 1
Set Date mo/day/year
Shift edit seq
The integrator will ask questions about method to load and autosampler sequence. Enter
all information.
3.4.2 Programming peak 96
Integrator 2
Follow the keys:
Data Acquisition
Set up integrator
Select old method
Download method
Mullin.met
3.4.3 Set up PC
Follow the kevs:
Generate new
2-30
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Data prefix J3095 (June 30, 95. It will pick up the vial number from the integrator)
Check export path/peak/export 2
Data to ChemStation Y
3.4.4 Transfer information from integrator to PC
Follow the keys:
Integrator to PC
IBCOL.SEQ
3.4.5 Start a GC run
Follow the keys:
Data Acquisition
3.4.6 Post GC run
After the GC run is over copy the .d files on floppy disks.
2-31
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
Sequence
27 Apr 95 05:05 PM
age 1
Sequence: C:\HPCHEM\1\SEQUENCE\DEFAULT.SEQ
Operator: Ilora Basu
Sequence preparation date: 15 Jun 92 09:14 AM
Data File Subdirectory: ON94CH
Part of methods to run: full method
On a barcode mismatch: inject anyway
Comment:
was spiked with 8 ng of 30 and 6 ng of 204
Seq. Cal. Method
Inj. Line Line Name
FRONT
1
REAR
1
Sequence Table
From To Inj/
Vial Vial Vial
1 16 l
Sample Table
Vial Sample Name
Num.
1 hexane blank
2 pcbcalst950427
3 pcbperfst950427
4 LBC 950113,H
5 MSC 950112,H
6 EH02C1 941024,H
7 EH02C2 941024,H
8 EH01C 941105,H
9 SH01C 941024,H
10 SH02C 941025B,H
11 SH01C 941105,H
12 TH01C 941024,H
13 TH02C 941024,H
14 TH01C 941105,H
15 PCBCALST950427
16 Hexane blank
Sample
Amount
Multiplier ISTD
Amount
Chart 1
2-32
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
4.0 HP 3365 Chemstation Initialization and Baseline Correction
4.1
4.2
4.3
Copy the .d files from Peak 96 on to the floppy disks.
Copy the .d files to HP 3365 ChemStation.
Load IBPCBN.MTH or IBPEST2.MTH
Loading Chromatogram
Load Data File
Sub directory
Sample Vial Number
Chromatogram will appear on screen.
4.4 Baseline Correction
4.4.1 By Integration Event
Go to "command line" and type in Clrevents.
Correct starting parameters and then make baseline corrections by using Baseline Now,
and Area Sum.
Starting Parameters
Integration Events
Initial Area Reject
Initial Peak Width
Shoulder Detection
Initial Threshold
Integrator On
Negative peak on
10 Initial
0.04 Initial
OFF Initial
-4 Initial
0.0
0.0
4.4.2 Integrate the Chromatogram
Change the scale ims for close view of each peak.
4.4.3 Baseline Now:
This command is used to get a straight base line
Integration
Possible Events
Baseline Now
2-33
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
Click cursor where you want the baseline.
Integration
If do not like changes go to and
delete.
4.4.4 Area Sum and Negative Peak:
This command is used to split a peak.
Integration Events
Turn negetive peak on and off in the area to split the peak
Move the cursor and click on the area to split.
Notice: Area Sum OrAOff does not work with Neg. Peak On.
5.0 Pesticide Data Reduction in 50% Fraction
5.1 Standard
5.1.1 Integration and Peak Identification
Call standard chromatoaram and correct baseline and integrate
Compounds
a-HCH
Y-HCH
Cong65(ISTD)
GC Retention time
min.(approx.)
35
40
59
concentration
ng/mL
20
20
20
2-34
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Compounds
y-Chlordane
cong 155(ISTD)
a-Chlordane
t-Nonachlor
Dieldrin
ODD
DDT
Dibutylchlorendate
GC Retention time
min.(approx.)
70
72
73
74
77
86
93
HO
concentration
ng/mL
20
20
20
20
20
20
20
10
.1.2 Printing Report and Saving Text File
Type in the sample name and add it on the chromatogram.
Check Calibration Settings under Reports.
Standard.
Mixed Pest.
Ref. Window
Nonref. Window
Units of Amount
Sample ISTD
Fit: Linear
I
ng
20
Origin: Force
Check report on screen before printing
Specify Report
X File (X Auto)
X Screen
X Report
XArea
XISTD(ifcalib. is replaced).
X Percent (if new calib is made).
If the report looks alright, print the report.
Print Report
To print report on paper go back to Specify Report and add Printer and Chromatogram to
the options selected.
2-35
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
Print integration events and calibration settings
File
Method
Return to Top
File
Integration Events
5.1.3 Preparing New Calibration
Set up initial calibration table by identifying the standard peaks in the area percent report.
Prp Calib/Recalib.
Manually type in the amount and name of each analyte with the retention times from the
area percent printout.
Highlight "yes" in Reference ISTD peak boxes for Congener 65
Do you want to delete all the peaks with the amount of zero?
Save to method?
You will get a printout for calibration table.
5.1.4 Replacing Previous Calibration
For subsequent standard runs with analyte peaks all correctly identified in ISTD report the
calibration table is replaced.
Reports
Prep Calib/Recalib.
Recalibrate
Replace
OK
Save to method'1
Will get a printout of calibration table.
2-36
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
If the GC column has been clipped or running conditions have been changed the analyte
peaks shift so much that they are not found in the internal standard report and then a new
calibration table will have to be created.
5.2
5.3
5.1.5 Saving Calibration on Data File
Command Line
Type in: SAVETBL C:\HPCHEM\l\DATA\SUBDIR.\0**RO101.CAL
5.1.6. Saving Event on Data File
Command Line
Type in: SAVE C:\HPCHEM\1\DATA\SUBDIR.\0**R0101 .EVT
Samples, 50% Fraction
5.2.1 Integration - See Section 4.4.
5.2.2 Printing Report and Saving Text File.
See previous Section 5.1.2 with the following exceptions:
Note: Sometimes it is necessary to increase the window more than 0.25% to find internal
standard. If it goes more than 0.5%, rerun the sample in GC.
5.2.3 Saving Events - See Section 5.1.6
Copying .Txt Files
After one set of data is reduced write comments in the text files.
Program manager
Click on C, HPCHEM 1, data, subdirectory and .txt files.
Call .txt file one by one and write down comments about spike, dilution, reinjection etc.
Save .txt files after writing comments.
2-37
-------
SOOO -,
o
•5'
&'
"*
o'
en
Q>
1
IV-JL
4 O
6O
OO
1 00
Sig. 1 i r-i A:\SO94CF^ 1\J S4 952 D
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
09 Aug 95 08:33 AM
Method: C:\HPCHEM\1\METHODS\IBPEST2.MTH
Pk*
i
2
3
4
5
6
7
8
9
10
11
RT
34.931
40.469
59.180
70.521
71.658
73.377
74.405
76.972
86.359
92.832
110.300
Lvl
1
1
1
1
1
1
1
1
1
1
1
ng
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
10.0
ISTD
Calibration Table
Amt/Area Ref Istd
1.27446-003
1.39240-003
7.8613e-004 Ref
1.04228-003
8.3836e-004
9.91216-004
1.07126-003
1.44316-003
2.48226-003
3.87776-003
2.21056-003
If Name
1 A-HCH
1 G-HCH
1 CONG 65
1 G-CHLORDANE
1 COKG 155
1 A-CHLORDANE
1 T-NONA
DIELDRIN
ODD
DDT
DBC
Title:
Reference window:
Non-reference window:
Units of amount:
Multiplier:
RF uncal peaks:
Sample Amount:
It
1
Amount
20.0
Calibration Settings
0.500 *
0.500 %
ng
1.0
0.0
o.o
Sample ISTD Information
Multilevel Information
Fit: Linear
Origin: Force
Chart 2
2-39
-------
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-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Internal Standard Report
Data File Name
Operator
Instrument
Sample Name
Run Time Bar Code
Acquired on
Report Created on
Last Recalib on
Multiplier
C:\HPCHEM\l\DATA\J95CFl\U-8957.D
HP5890A
JUL 19, 1995 02:43:09
09 Aug 95 12:33 PM
09 AUG 95 10:20 AM
1
Sig. 1 in C:\HPCHEM\1\DATA\J95CF1\L18957.D
Ret Time Area Type Width Ref# ng
Page Number
Vial Number
Injection Number
Sequence Line
Instrument Method
Analysis Method
Sample Amount
ISTD Amount
Name
IBPEST2.MTH
0
20
34
39
58
69
70
72
73
76
85
91
109
1
.169
.662
.314
.780
.775
.366
.506
.043
.473
.939
.416
* not
* not
* not
* not
70370
3866
22586
142
19005
545
found
found
found
found
13402
'PV '
PV
w
w -
BV
BVA
*
*
*
*
PV
1 —
0.
0.
0.
0.
0.
0.
0.
— 1
093
096
123
000
132
109
128
(.
1
1
1
1
1-IR
1
1
1
1
1
1
71
4
20
0
20
0
25
1
.797
.038
.759
.142
.000
.549
.822
A-HCH
G-HCH
CONG 65
G-CHLORDANE
CONG 155
A-CHLORDANE
T-NONACHLOR
DIELDRIN
ODD
DDT
DBC
Time Reference Peak
5
Expected RT
70.785
Actual RT
70.775
Difference
-0.0%
Not all calibrated peaks were found
Chart 3
2-41
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure Volume 2, Chapter 1
09 Aug 95 12:33 PM
Method: C:\HPCHEM\1\METHODS\IBPEST2.MTH
Integration Events
Events: Value: Time:
Initial Area Reject 50 INITIAL
Initial Peak Width 0.040 INITIAL
Shoulder Detection OFF INITIAL
Initial Threshold -4 INITIAL
Baseline Now 0.000
Negative Peak ON 0.000
Baseline Now 33.943
Baseline Now 62.680
Negative Peak OFF 72.300
Area Sum ON 72.400
Area Sum OFF 72.722
Negative Peak ON 72.730
Baseline Now 81.033
Baseline Now 82.107
Baseline Now 87.837
Baseline Now 88.403
Baseline Now 89.180
Calibration Settings
Title:
Reference window: 0.500 %
Non-reference window: 0.500 %
Units of amount: ng
Multiplier: 1.0
RF uncal peaks: 0.0
Sample Amount: 0.0
I# Amount
1 20.0
Fit: Linear
Origin: Force
Sample ISTD Information
Multilevel Information
Chart 4
2-42
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
6.0 PCB and Pesticide Data Reduction in Hexane Fraction
6.1 Mullin 94 Standard
6.1.1 Integration and Peak Identification
Load standard chromatogram and correct baseline according to Section 4.2 and 4.3.
Integrate and divide the chromatogram in five to six sections. Identify PCBs from
Mullin's 94 chromatogram and pesticides (HCB and DDE) from Mixed Pesticide
Standard.
6.1.2 Printing Report and Saving Text Files
Click in text box and type in sample name. Click on I Add I , and then on chromatogram
where you want sample name to be placed.
Check Calibration Settings under Reports.
1.0
1 .0
Ref. Window
Nonref. Window
Units of Amount ng
Sample ISTD#1 8
Sample ISTD #2 6
Fit: Linear Origin: Force
Check report on screen before printing
Specify
Reports
X File (X Auto)
X Screen
X Report
X Area
XISTD(ifcalib. is replaced).
X Percent (if new calib is made).
Print Reports
To print report on paper go back to Specify Report and add Printer and Chromatogram to
the options selected.
2-43
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
Print integration events and calibration settings:
File
Return to Top
Method
Print
I
Calibration Setting
Integration Events
6.1.3 Preparing New Calibration
Set up initial calibration table by identifying congener peaks from Mullin's chromatogram
in the area percent report.
Reports
Prep Calib/Recalib
New Table
OK
Manually type in and enter the amount and name of each analyte with the retention times
from the area percent printout.
Highlight "yes" in Reference ISTD peak boxes for Congener 30 and 204.
Ref.ISTD#l will be used up to cong.l 10.
Ref.ISTD#2 will be used from cong.82.
OK |
Do you want to delete all the peaks with the amount of zero?
Yes |
Save to method?
I Yes
6.1.4 Replacing Previous Calibration
For subsequent standard runs with analyte peaks all correctly identified in ISTD report the
calibration table is replaced.
Reports
Prep Calib/Recalib
Recalibration
Replace
OK
2-44
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Save to method?
| Yes |
Will get a printout of calibration table.
If the GC column has been clipped or running conditions have been changed, the retention
times of analyte peaks shift and they are not identified in the ISTD report. A new
calibration table will have to be created. This can be done either by following Section
6.1.3 or by inserting the new retention time in old calibration table and creating a
temporary calibration table. Get ISTD report of the new standard with temporary
calibration table and replace the calibration according to Section 6.1.4.
6.1.5 Saving Calibration on Data File
File
Command Line
Type in SAVETBL C:\HPCHEM\1\DATA\SUBDDRECTORY\0**R0101.CAL
6.1.6 Saving Event
Command Line
6.2
Type in: SAVE C:\HPCHEM\1\DATA\SUBDIR.\0**R0101.EVT Enter with keyboard.
Samples, Hexane Fraction
6.2.1 Integration - See previous Section 4.4
6.2.2 Printing Report and Saving Text File
See previous Section 5.1.2 with the following exceptions:
Calibration Settings Ref. and Nonref. Windows should be changed to 0.25%.
Note: Sometimes it is necessary to increase the window more than 0.25% to find internal
standard. If it goes more than 0.5% rerun the sample in GC.
6.2.3 Saving Events - See previous Section 5.1.6
2-45
-------
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5
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
;. Jul 95 02:52 PM
Method: C:\HPCHEM\1\METHODS\IBPCBN.MTH
Calibration Table
48.0
28 .0
13 . 6
4.8
7.6
56.0
20.0
20. 0
1. 12
8 . 0
0.68
0. 39
14.8
14.8
0.52
0.52
8.0
7 . 6
0.21
2.8
1. 3
18 . 8
18. 8
13.2
2. 6
0.72
11.6
3.56
1.6
18.0
1. 1
9.2
4.0
4 .0
5.0
17.2
4.8
5. 6
9 .2
7. 2
3 .76
0 . 44
0. 84
7 . 6
13.6
20. &
8.0
2.04
14 .0
7 . 2
0. 4
7.2
2.93
o. na
o.a
2.24
Amt/Area Ref Istd
1.0132e-002
2.2807e-002
3.6808e-003
9.3258e-004
1.6816e-003
3 .05966-003
3.63996-004
2.24366-003
9.3145e-004
8.4154e-004 Ref ISTD
1.7346-003
8 . 11066-004
8.68196-004
1.75056-003
2.23856-003
4.58446-004
1.2027e-003
1. 39796-003
1. 0343e-003
1. 12526-003
1.03636-003
1. 05696-003
1. 1076-003
1.0978e-003
7.78526-004
7.32066-004
1. 6826-003
9.13436-004
1.02236-003
9.99446-004
1.0759e-003
7 . 17176-004
8 .06196-004
6.86216-004
7.53446-004
9 . 4466e-004
1.65826-003
7 . 105e-004
1.11256-003
6. 3997e-004
9 . 46526-004
8 .02996-004
1. 127e-003
7. 905e-004
7 . 13196-004
1. 3476e-003
9 .40676-004
9 . 51416-004
9.71136-004
1.47466-003
8 .74536-004
7 .93846-004
6. 6508e-004
2. 60716-004
6.7352e-004
5.4267e-004
If
i 1
1 3
1 4 + 10
1 7+9
1 6
1 5+8
1 HCB
1 14
1 19
1 30
1 12
1 13
1 18
1 17+15
1 24
1 27
1 16
1 32
1 29
1 26
1 25
1 31
1 28
1 33
1 53
1 51
1 22
1 45
1 46
1 52
1 43
1 49
1 47
1 48
1 65
1 44
1 37
1 42
1 41+71
1 64
1 40
1 100
1 63
1 74
1 70+76
1 66
1 95
1 91
1 56+60
1 84+92
1 89
1 101
1 99
1 119
1 83
1 97
Name
Chart 5
2-47
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
11 Jul 95 02:52 PM
Method: C: \HPCHEM\1\METHODS\IB1PCBN.MTH
1.1439e-003
7.3726-004
9.01026-004
17436-003
10616-003
0.00125
6996e-004
0807e-004
57
58
59
60
- 61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
76
77
77
78
78
78
78
80
81
81
82
82
83
83
84
85
86
87
89
91
91
92
92
93
94
94
94
95
96
96
97
97
99
100
100
101
101
102
102
103
103
104
104
105
105
108
110
110
111
111
114
116
117
120
120
126
130
.768
.055
.665
.061
.260
.596
.788
.467
.135
.903
.187
.730
.249
. 524
.783
.727
.796
.813
.856
.096
.398
.301
.663
.503
.033
. 411
.927
.471
.296
.711
.464
.877
.284
.118
.850
.080
. 616
.194
.445
. 240
.432
. 309
.737
. 361
.905
.970
.083
.714
.510
.732
.055
.223
.547
.001
.770
.056
.915
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.64
4.0
2.8
3.0
20.0
0.92
7.6
1.8
6.8
3.56
0.22
0.52
11.2
4.8
0.29
0.56
1.56
17.2
6.8
1.04
0.3
10.8
1.0
0.052
4.4
5.0
0.8
14.4
6.8
0.4
0.196
1.9
12.8
6.8
3.16
0.26
0.152
1.56
6.0
2.24
0.44
24 .4
1. 68
0.48
1.72
6.8
0.48
16.8
8. 6
8.6
0.16
3.2
0.37
7.2
0.44
2.72
0. 048
1
9
1
1
7
7
7
1
5
7
6
6
3
1
9
6
5
4
1
9
9
2
7
1
6
8.
4
1,
(
7
8,
(
5,
1
8
8
1
1
7
8
1
8
1
5
1
9
1
1
6
6
1
7
7
4
45296-004
06356-003
12626-004
61936-004
82466-004
97066-004
30276-004
52686-003
0246e-004
84566-004
09336-004
77466-004
30916-003
64166-004
93196-004
89246-004
1.1636-003
7.49556-004
0108e-003
24316-004
8.14566-004
73256-004
1.99146-003
6.2556-004
7.77246-004
8.8769e-004
6.067e-004
5.35336-004
42426-003
8.4592e-004
8.74296-004 Ref ISTD
15576-003
22686-003
67526-004
8.66036-004
48266-003
8.98336-004
6.3936-004
5.7375e-004
1407e-003
33436-004
5565e-003
57066-003
1544e-004
94446-004
6.306e-004
7.34616-004
7.3361e-004
4.72336-004
1 81
1 87
1 85
1 136
1 DDE
1 77
1 110
2 82
2 151
2 135+144
2 124+147
2 107
2 123+149
2 118
2 134
2 114+131
2 146
2 105+132+153
2 141
2 137+176
2 130
2 163+138
2 158
2 129
2 178
2 166
2 175
2 187+182
2 183
2 128
2 167
2 185
2 174
2 177
2 202+171
2 156
2 173
2 157+200
2 204
2 172
2 197
2 180
2 193
2 191
2 199
2 170+190
2 198
2 201
2 203
2 196
2 189
2 208+195
2 207
2 194
2 205
2 206
2 209
Chart? (Cont'd)
2-48
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
11 Jul 95 02:52 PM
Method: C:\HPCHEM\1\METHODS\1BPCBN.MTH
'Title:
Reference window:
Non-reference window:
Units of amount:
Multiplier:
RF uncal peaks:
Sample Amount:
Calibration Settings
0.500 %
0.500 %
ng
1.0
0.0
0.0
Sample ISTD Information
It
1
2
Amount
8.0
6.0
Multilevel Information
Fit: Linear
Origin: Force
Chart 5 (Cont'd)
2-49
-------
Ul
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0) Cl)
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
I AON Project - Gas Chromatography Procedure
Internal Standard Report
Data File Name C:\HPCHEM\1\DATA\ON94CH\A279514.D
Operator Page Number 1
Instrument HP5890A Vial Number 0
Sample Name Injection Number
Run Time Bar Code Sequence Line
Acquired on APR 28, 1995 17:32:09 Instrument Method
Report Created on 22 Jun 95 01:39 PM Analysis Method IBPCBN.MTH
Last Recalib on 21 JUN 95 11:36 AM Sample Amount 0
Multiplier 1 ISTD Amount 8
Sig. 1 in C:\HPCHEM\1\DATA\ON94CH\A279514.D
Ret Time
1
19.225
24.692
27.971
32. Ill
33.752
34.697
34.958
37.071
37.950
39.522
41. 195
41.464
42. 141
42.378
43.807
43.922
45.191
45.367
47 .763
48.833
49.142
50.160
50. 304
51.964
52.238
53. 020
53.236
54.087
55.485
56.910
57.204
57.566
57.941
58. 058
58.291
60.300
60.588
60.756
62.234
62. 393
63 . 560
65.155
65.997
66.702
67.507
Area Type
_ 1 |
1 I
50 VBA
* not found *
868 PV
4297 PVA
2840 PP
6,752 PV
235283 W
9'754 VP
398 PP
10418 PP
250 VP
1313 PP
9515 W
5080 W
* not found *
662 PV
3020 PV
2080 W
* not found *
1350 W
890 PV
3441 PV +
11483 VP
12077 W
1138 VP
710 PV
3123 W
1466 PV
625 W
9430 W
908 W
4527 W
1458 W
1937 W
7669 W
4996 W
1785 W +
2317 WA
1399 PV
Width Reft
____ _
0.039
0.110
0. 110
0.168
0.113
0.116
0.116
0.095
0.109
0. HO
0.095
0.123
0.122
0.1Q4
0.115
0.1Q8
0.113
0.119
o.odo
0.137
0. 125
0. H3
0. 108
0.131
0.142
0.138
0.137
o.uo
0.122
0.110
0.115
0.125
0.118
0.000
0.133
0.116
2446 WA 0.128
306— PP 0-.-084-
* not found *
1094 PP_^-
1344 W
3229 PVA
-0.178
0.135
0.130
1
1
1
1
1
1
1
1
1
1-IR
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-1
1
-1 -
1
1
ng Name
i i
1 ^|
0.496 1
3
3.027 4+10
3.839 7+9
4.459 6
19.228 5+8
80.586 HCB
20.720 14
0.327 19
8.000 30
0.393 12
0.835 13
7.438 18
8.112 17+15
24
0.272 27
3.319 16
2.622 32
29
1.390 26
0.804 25
3.219 31
11.717 28
11.951 33
0.779 53
0.446 51
4.763 22
1.178 45
0.555 46
8.396 52
0.866 43
2.889 49
1.044 47
1.181 48
5.113 65
4.236 44
2.658 37
1.532 42
1.401 41+71
1.405 64
- — 0-r253 40
100
1-.-692- 63
0.977 74
2.106 70+76
Chart 6
2-51
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
67.
68.
69.
70.
71.
72.
72.
73.
74.
75.
76.
76.
77.
77 .
78.
78.
78.
78.
80.
81.
81.
82.
82.
83 .
83.
84 .
85.
86.
87.
89.
91.
91.
92.
92.
93.
94 .
94 .
94.
95.
96.
96.
97 .
97.
99.
100.
100.
101.
101.
102.
102.
103 .
103.
104 .
104 .
105.
105.
103 .
110.
110.
111.
Ill .
114 .
986
207
243
796
659
058
621
438
559
229
122
759
050
661
056
257
605
786
467
131
906
216
713
246
522
792
586
783
809
850
130
387
311
655
488
054
404
960
468
299
698
488
911
281
114
853
108
644
226
442
270
458
311
764
299
932
986
270
709
539
712
035
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
* not
1823
6262
1042
1228
2814
2434
5809
2358
74
205
1239
111
1895
730
507
10906
found
3549
300
679
252
found
94
2093
1475
152
1290
480
2728
723
W
W
PV
PV
W
WA
PV
BV
BV
PV
PV
BV
PV
BV
W
W
*
W
PB
PV
PV
*
PV
PV
W
PV
BV
W
BB
BV
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
127
132
137
136
134
122
028
138
087
088
126
097
129
140
095
125
131
116
107
080
088
128
133
111
125
150
163
127
found *
109
1195
157
72
111
7584
BB
BV
PB
PV
BV
PV
0.
0.
0.
0.
0.
0.
112
131
103
116
097
127
found *
562
202
170
found
found
305
123
112
found
found
found
8529
found
found
361
found
106
found
225
1318
105
found
found
found
BV
PV
PV
*
*
PB
BV
PV
*
*
*
BV
*
*
PB
*
PB
*
PV
PBA
PB
*
*
*
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
125
128
115
138
094
104
133
132
112
167
132
100
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2-IR
2
2
2
2
2
2
2
2
2
2
2
2
2
5
0
1
3
2
4
1
0.
0
0
0
1
0
0
11
2
0
0
0
0.
1
0
0.
2
0
1
0
0
0
0
.236
.290
.924
.131
.904
.361
.210
.450
0427
.205
.708
.191
.371
.658
.572
.329
.766
.224
.420
.219
0876
.156
.924
0981
.220
.333
.495
.303
.117
.944
.117
0.0122
0
4
0
0
0.
0
0.
0.
6
0
0.
0
.108
.354
.294
.141
0841
.202
0918
0569
.000
.242
0882
.139
0.924
0.107
66
95
91
56+60
84+92
89
101
99
119
83
97
81
87
85
136
ODE
77
110
82
151
135+144
124+147
107
123+149
118
134
114+131
146
105+132+153
141
137+176
130
163+138
158
129
178
166
175
187+182
183
128
167
185
174
177
202+171
156
173
157+200
204
172
197
180
193
191
199
170+190
198
201
203
196
189
Chart 6 (Cont'd)
2-52
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
116.256 * not found *
117.576 * not found *
120.031 * not found *
120.791 * not found *
125.982 306 BV
130.854 92 BV
T9«
2O8-H95
207
194
205
-O-s-215 206
-2 OvOl-40 209
Time Reference Peak
10
95
Expected RT
39.556
102.477
Actual RT
39.522
102.442
Difference
-O. 1%
-O. 0%
Not all calibrated peaks were found
Chart 6 (Cont'd)
22 Jun 95 Ol:41 PM
Method: C: \HPCHEM\1\METHODS\IBPCBN.MTH
Events:
Initial Area Reject
Initial Peak Width
Shoulder Detection
Initial Threshold
Negative Peak ON
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
Baseline Now
Baseline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Negative Peak OFF
Area Sum ON
Area Sum OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Negative Peak OFF
Area Sum ON
Area Sum OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Title:
Integration Events
Value: Time:
50 INITIAL
0.040 INITIAL
OFF INITIAL
-4 INITIAL
O.OOO
IB.23O
18.483
19.250
2O.61O
31.O27
31.886
32.158
32.279
32.302
33.447
43.747
45.733
50.081
50.O92
SO.224
5O.558
SO.O26
6O.479
6O.687
6O.885
61.O37
61.386
62.O1O
62.492
62.643
62.8O3
67.328
67.663
67.85O
68.652
71.281
71.318
71.523
72.138
72.325
73.705
92.O57
92.O8O
11O.463
111.157
Calibration Settings
Chart 7
2-53
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure Volume 2, Chapter 1
22 Jun 95 01:41 PM
Method: C:\HPCHEM\1\METHODS\IBPCBN.MTH
Reference window: 0.500 %
Non-reference window: 0.500 %
Units of amount: ng
Multiplier: 1.0
RF uncal peaks: 0.0
Sample Amount: 0.0
If Amount
1 8.0
2 6.0
Fit: Linear
Origin: Force
Sample ISTD Information
Multilevel Information
Chart 7 (Cont'd)
7.0 Creating Excel File for PCB Analysis
7.1 Call *.txt files
7.2 Select "fixed width" and click on "next"
7.3 Click on line and "finish"
7.4 Delete al..a5
7.5 Insert six rows
7.6 Delete Column C
7.7 Change column width b to 10
7.8 Edit and replace *not found* to 0
7.9 Copy O's to C
7.10 Type at A I
Subdirectory
Sample name
Vial id
Date
2-54
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
Volume 2, Chapter 1 IADN Project - Gas Chromatography Procedure
7. II Delete rows A124 to II31
7.12 Delete negative peaks amount
7.13 At 124 type "total pcbs"
7.14 At C124 put formula =((sum(c9..cl 21 ))-(cl5+c!6+c43+c69+c90)
7.15 At A126 type % recovery of 14 = c 16/20* 100
7.16 At A127 type % recovery of 65 = c43/5 * 100
7.17 At 128 type % recovery of 166 = c90/5* 100
7.18 At 130 type ratio of 30:204 = B18/B103
7.19 At A132typeHCB = cl5
7.20 At A133 type DDE = c69
Write comments
Save as .xls files
2-55
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project • Gas Chromatography Procedure
Volume 2, Chapter 1
subdirectory
MrnptomrrM
vtalkS
(M*
ret time
19.225
24.692
27.971
32.111
33.752
34.697
34.956
37.071
37.95
39.522
41.195
41 464
42.141
42.378
43.907
43.922
45.191
45.367
47.763
48.833
49.142
50.16
50.304
51.964
52.238
53.02
53.236
54.087
55.485
56.91
57.2O4
57.566
57.941
58.058
58.291
60.3
60.588
60.756
62.234
62.393
53.56
65.155
65.997
Am
[•—•••• — ••(
50
0
888
4297
2840
6752
235283
9754
398
10418
250
1313
9515
5080
0
662
3020
2080
0
1350
890
3441
11483
12077
1138
710
3123
1466
625
9430
908
4527
1458
1937
7669
4996
1785
2317
1399
2446
0
0
0
on94ch
th02c941024,
14
22-Jur>-a5
f* ng
I " T
0.496
0
3.027
3.839
4.459
18.228
60.586
20.72
0.327
-IR 8.000
0.393
0.83S
7.438
8.112
0
0.272
3.319
2.622
0
1.39
0.804
3.219
11.717
11.951
0.779
0.446
4.763
1.178
0.555
8.396
0.866
2.889
1.044
1.181
5.113
4.236
2.658
1.532
1.401
1.405
0
0
0
1
Name
1
1
3
4*10
7+9
6
5*8
HCB
14
19
30
12
13
18
17*15
24
27
16
32
29
26
25
31
28
33
53
51
22
45
46
52
43
49
L_ 47
48
65
44
37
42
41*71
64
40
100
63
i
1
Chart 8
2-56
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
66.702
67.507
67.986
. 68.207
69.243
70.796
71.659
72.058
72.621
73.438
74.559
75.229
76.122
76.759
77.05
77.661
78.056
78.257
78.605
78.786
80.467
81.131
81.906
82.216
82.713
83.246
83.522
84.792
85.586
86.783
87.809
89.85
91.13
91.387
92.311
92.655
93.488
94.054
94.404
94.96
95.468
96.299
96.698
97.488
97.911
99.281
100.114
100.853
101.108
101.644
102.226
1344
3229
1823
6262
1042
1228
2814
2434
5809
2358
74
205
1239
111
1895
730
507
10906
0
3549
300
679
252
0
94
2093
1475
152
1290
480
2728
723
0
109
1195
157
72
111
7584
0
562
202
170
0
0
305
123
112
0
0.977
2.106
2.236
5.29
0.924
1.131
3.904
2.381
4.21
1.45
0.0427
0.205
0.708
0.191
1.371
0.658
0.572
11.329
0
2.766
0.224
0.42
0.219
0
0.0876
1.156
0.924
0.0981
2.22
0.333
1.495
0.303
0
0.117
0.944
0.117
0.0122
0.108
4.854
0
0.294
0.141
0.0841
0
0
0.202
0.0918
0.0569
r
Ol 0
o! o
74
70*76
66
95
91
56*60
84+92
89
101
99
119
83
97
81
87
85
136
ODE
77
110
82
151
135+144
124+147
107
123+149
118
134
114+131
146
105+132+153
141
137+176
130
163+138
158
129
178
166
175
187+182
183
128
167
185
174
177
202+171
156
173
157+200
Chart 8 (Cont'd)
2-57
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Volume 2, Chapter 1
102.442
103.27
103.458
. 104.311
104.784
105.299
105.932
106.966
110.27
110.709
111.539
111.712
114.085
116.256
117.576
120.031
120.791
125.982
130.854
total PCBs
%rec14
%rec65
%rec166
30:204
hcb
dde
comments
8529
0
0
361
0
106
0
225
1316
105
0
0
0
0
0
0
0
0
0
IR 6
0
0
0.242
0
0.0882
0
0.139
0.924
0.107
0
0
0
0
0
0
0
0
0
159.0276
103.6
102.26
97.08
1.22148
80.586
11.329
204
172
197
180
193
191
199
170*190
198
201
203
196
189
208+195
207
194
205
206
209
sample saver worked property
Chart 8 (Cont'd)
8.0 Data Storage and Data Retrieval
8.1 Copy the complete files (*.d, *.txt, *.evt,*.cal, and *.xls) on to the floppy disks. Leave one copy
on hard disks too.
8.2 Loading Old Chromatogram
Same as Loading chromatogram in Section 4.3
8.3 Loading Old Calibration
Program Manager
File Manager
2-58
-------
Volume 2, Chapter 1
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project - Gas Chromatography Procedure
Click on c:Vhpchem\I\subdirectory\0**RO 101 .cal
To C:\hpchem\l\methods\appropriate method.cal
Replace Calib
Close File manager and Return to TOP
Click on Method and Load proper method.
Make sure that you got correct calibration table by clicking on
Edit Calibration Table
8.4 Loading Old Integration Event
Program Manager
File Manager
Click on c:\hpchem\l\data\subdirectory\0**R0101.evt
.5 Data Storage and Data Retrieval
To C:\hpchem\l\methods\appropriate method.evt
Replace Event
Close File manager and Return to TOP
Click on Method and Load proper method.
Make sure that you got correct integration event by clicking on
2-59
-------
Analysis of PCBs and Pesticides in Air and Precipitation Samples:
IADN Project • Gas Chromatography Procedure
Volume 2, Chapter 1
Close File manager and Return to TOP
Click on Method and Load proper method.
Make sure that you got correct calibration table by clicking on
Edit Calibration Table
2-60
-------
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project
Sample Preparation Procedure
llora Basu
School of Public and Environmental Affairs
Indiana University
Bloomington, IN 47405
June 1995
Version 1.0
-------
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
1.0 Summary
This guide is written for chemists and technical assistants so that everybody follows the same
details in preparation of IADN samples. We process Air (both vapor and particle phase) and
Precipitation samples for analysis of PCBs, pesticides and PAHs. A brief description of the
12 sections covered in this volume of SOP follows:
1.1 Section I: Cleaning
This section describes the procedures of soap and water cleaning, muffling, and ultrasonic
cleaning of glassware and other tools.
1.2 Section II: Precleaning
This section covers the long procedure of precleaning XAD-, with different types of solvents. The
procedure was originally standardized by Steve Eisenreich and later on it was modified in our
laboratory. Besides XAD2, we have also described the precleaning procedure of glass wool, silica
gel, boiling chips, and glass fibre filters.
1.3 Sections III and IV: Extraction
Soxhlet extraction of air vapor, air particle and precipitation samples from XAD: cartridges and
OFF are described in these two sections. Detailed procedures for concentration by rotary
evaporation, solvent exchange and back extraction are documented. We also mention QC samples
and spiking samples with recovery standard etc.
1.4 Section V: Silica Column Chromatography
After extraction, the extracts are cleaned up from interfering compounds and fractionated into
three different fractions through silica gel deactivated to 4%. First fraction is collected with
Hexane which contains all PCBs and pesticides like HCB and DDE. The second fraction which is
collected with 50% CH2C1, in Hexane contains all PAHs and pesticides like a and y HCHs,
Dieldrin. ODD, DDT, y Chlordane, a Chlordane, and T-Nonachlor. The third fraction is collected
in Methanol and contains atrazine.
1.5 Sections VI and VII: Transfer and Nitrogen Blow Down
These t\vo sections describe the procedure for final transfer of prepared samples from flasks to
4 mL amber vials. The samples are then concentrated to desired volume by a slow stream of ultra-
pure nitrogen. Final volumes are adjusted depending on types of samples and time of collection to
ensure GC chromatograms are not off scale.
2-63
-------
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
\ .6 Sections VIII and IX: Spiking and Making Microvials
After proper concentration by Nitrogen blow down, each sample is spiked with known amount of
internal standard or quantitation standard. Subsamples are then transferred to autosampler
microvials for GC analysis.
1.7 Section X: Standards
Procedures for preparation of all stock standards, working standards, calibration standards,
recovery standards, and spiking internal standards are compiled in this section.
1.8 Section XI: Safety
Some of the safety rules that we follow for day to day laboratory work are mentioned here.
Procedure for waste disposal is also included in this section.
2-64
-------
Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
2.0 The Flow Chart of Sample Preparation
AIR SAMPLES
FRACTION
Hexane
ROTARY EVAPORATION
TRANSFER TO VIAL
NITROGEN SLOWDOWN
RAIN SAMPLES
ADD SURROGATES
24 HOURS SOXHLET EXTRACTION
375 mL Acetone/Hexane
ROTARY EVAPORATION AND
SOLVENT EXCHANGE TO HEXANE
SILICA COLUMN CHROMATOGRAPHY
FRACTION 2
50<7r CH2CL2
ROTARY EVAPORATION
TRANSFER TO VIAL
NITROGEN BLOWDOWN
ADD ISTD
65. I 55, and
dIO. d!2 PAHs
GC HP5X90. ECD
GC/V1S. HP5989
PESTICIDES
PAHs
FRACTION 3
Methanol
ROTARY EVAPORATION
TRANSFER TO VIAL
NITROGEN BLOWDOWN
ADD ISTD
d 10 anthracene
GC/MS, HP5989
2-65
-------
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
3.0 Cleaning
3.1 Glassware
3.1.1 Supplies
3.1.1.1 Glassware:
Assemble what is to be cleaned
3.1.1.2 Non-glassware:
Micro cleaning solution
DI water
Dish washing brush
3.1.1.3 Equipment:
Drying oven
Muffle furnace
Acid bath: 50/50 H2SO4 and HNO3
3.1.2 Procedures
1) Wash/Dry
Wash glassware thoroughly with soap and water. Use brush if necessary.
Glassware with bad stains should be rinsed with MeOH or CH:C1: before using
the soap and water procedure. If still not clean, soak in acid bath overnight, then
wash thoroughly with soap and water.
Volumetric pipettes used for standards must soak in acid bath overnight.
Rinse glassware thoroughly with tap water.
Rinse glassware thoroughly with DI water.
Dry glassware in air.
Cover all open ends with foil.
2) Muffle glassware at 450°C for four hours. If glassware is not clean after muffling
at 450°C for four hours, muffle at 500°C for four hours.
3) Allow glassware to cool to 100°C before removing from furnace.
4) Store.
3.1.3 Comment
Always use dull side of foil towards glassware.
3.2 Stainless Steel Tools
Supplies
2-66
-------
Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
3.2.1.1 Glassware:
-none-
3.2.1.2 Non-glassware:
Items to be cleaned: forceps, spatula, scissors, etc.
CH:CU in teflon bottle
Cl waste bottle
3.2.1.3 Equipment:
drying oven
3.2.2 Procedures
I)
2)
3)
4)
5)
6)
Wash with soap and water.
Rinse well with tap water.
Rinse thoroughly with DI water.
Dry at room temperature overnight.
Wrap each tool separately in foil.
Store.
3.2.3 Comment
ALWAYS rinse with CH2Cl, before use.
3.3 Amber Glass Vials and Pasteur Pipettes
3.3.1 Supplies
3.3.1.1 Glassware:
400 mL beaker
3.3.1.2 Non-glassware:
Foil
3.3.1.3 Equipment:
Muffle furnace
3.3.2 Procedure
I) Wrap glass in foil or place in beaker and cover beaker with foil
2) Muffle at 450°C for four hours.
3) Cool to lOO'C; remove from oven.
4) Insert teflon liner into vial cap and cap the vial as soon as the \ial comes out of
the oven.
5) Store in a beaker covered with foil.
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3.4 Teflon liners
3.4.1. Supplies
3.4.1.1 Glassware:
400 mL beaker
3.4.1.2 Non-glassware:
Foil
CH,Cl,
Cl" waste bottles
3.4.1.3 Equipment:
Ultrasonicator
3.4.2 Procedures
1) Place teflon liners in glass beaker; cover with CH2C12.
2) Ultra-sonicate for 15 minutes. Drain CH2C12.
3) Repeat.
4) Place in 70°C drying oven for two hours.
5) Store in sealed jar.
3.5 Micropipette Tubes, GC Microvials, and Stainless N-, Blowdown Needles
3.5.1 Supplies
3.5.1.1 Glassware:
400 mL or larger beaker
3.5.1.2 Non-glassware:
CH2CI2
Cl waste bottle
3.5.1.3 Equipment:
Muffle furnace
3.5.2 Procedures
1 i Micropipette tubes
Before using, rinse with CH-CK and air dry.
1} GC microvials
Place microvials, open end up, in a clean beaker. Cover vials with CH,Ck
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Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
making sure NO air bubbles remain in the microvials. Cover loosely with foil.
Sonicate microvials for 10 minutes.
Drain solvent, and repeat twice more. (The microvials should be sonicated a total
of three times.)
Drain all solvent and transfer microvials to clean beaker; cover with foil. Muffle
at 450°C for four hours. After furnace returns to !00°C (or the next morning)
remove vials from furnace.
Store in sealed container.
3) Stainless N2 blowdown needles
Place needles in a clean beaker and cover with CH:Cl:. Cover loosely with foil.
Sonicate needles for 10 minutes.
Drain solvent, and repeat twice more. (The needles should be sonicated a total of
three times.)
Drain all solvent and transfer needles to clean beaker. Cover beaker with foil.
Label beaker "CLEAN"; store near the N, blowdown unit.
3.6 Teflon Stopcocks and Lids for Sample Jars
3.6.1 Supplies
3.6.1.1 Glassware:
-none-
3.6.1.2 Non-glassware:
Alconox
DI water
kimwipes
3.6.1.3 Equipment
-none-
3.6.2 Procedure
I) Wash stopcocks and lids with Alconox and tap water.
2) Rinse stopcocks and lids with DI water.
3) Air dry on kimwipes.
4) Storage
Store the stopcocks in muffled jar or beaker covered with foil.
Place lids on muffled sample jars or wrap them in foil.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure ___ Volume 2, Chapter 1
4.0 Precleaning
4.1 Glass Wool
4.1.1 Supplies
4. 1 . 1 . 1 Glassware:
Sample jar and lid
Glasswool
4.1.1.2 Non-glassware:
Foil
4.1.1.3 Equipment:
Scissors
Muffle furnace
4. 1 .2 Procedures
1 ) Cut glass wool into 2" pieces.
2) Put into muffled glass sample jar; cover jar with foil.
3) Muffle at 450 °C for four hours.
4) Screw lid on jar (do not remove foil).
5) Store.
4.2 Teflon Boiling Chips
4.2.1 Supplies
4.2.1.1 Glassware:
Soxhlet extractor: extra large (71/60 and 29/42 joints)
large (55/50 and 24/40 joints)
Condenser: 7 1 /60 joint for extra large soxhlet
55/50 joint for large soxhlet
Round bottom flask: I liter for extra large soxhlet
500 ml for large soxhlet
Adaptor (for extra large soxhlet. converts 29/42 joint to 24/40 joint)
Sample jar and lid
1 L beaker
4.2. 1 .1 iVo
Boiling chips
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IADN Project - Sample Preparation Procedure
CH:C1: in squirt bottle
Methanol in squirt bottle
Cr solvent waste container
Non-Cl~ solvent waste container
Cellulose thimbles: 60 x 180 mm for extra large soxhlet
43 x 123 mm for large soxhlet
Foil
Cork ring for round bottom flask
4.2.1.3 Equipment:
Variable autotransformer (aka variac)
Heating mantle for either 1 L or 500 mL round bottom flask
Drying oven
4.2.2. Procedures
Day /
1)
2)
3)
4)
5)
6)
7)
Thoroughly rinse inside of condenser and outside of joint with solvent in squirt
bottles: first with methanol, then with CH2C12. Cover joint and exhaust tube with
foil.
Add five or six boiling chips to flask. Add appropriate amount of CH2C1, to flask.
Place new teflon boiling chips in appropriate cellulose thimble. Place thimble in
soxhlet extractor.
large
soxhlet
extra
large
soxhlet
Thimble
Size
43 x 123
60 x 180
Flask
Size
(mL)
500
1000
CFLCK
(mL)
300
600
Assemble flask/soxhlet/condenser/adaptor (if necessary) apparatus.
Turn on heater to give proper boiling (set variac to 40-45).
Turn on chilled water for condenser.
Extract for 18 to 24 hours.
D«y 2
1)
2)
3)
4}
Turn heat off; let cool 15 to 30 minutes.
Turn off condenser water.
Drain as much solvent from soxhlet as possible.
Remove thimble from soxhlet, place upside down in a
with foil.
L beaker, cover loosely
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IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
5) Place boiling chips in a 70°C oven: Every 10 to 15 minutes, check boiling chips,
shaking beaker to determine if all solvent has evaporated. Let boiling chips
remain in oven two to four hours, until dry.
***WARNING: BEWARE OF SOLVENT FUMES.***
6) Wrap thimble in foil and store for future use.
7) Place in boiling chips in clean sample jar; cover with foil and lid.
8) Store on shelf.
Note: Boiling chips can be directly placed in soxhlet plugged with glasswool instead of
using cellulose thimble.
4.3 Sodium Sulfate (Na2SO4)
4.3.1 Supplies
4.3.1.1 Glassware:
500 mL beaker
Sample jar and lid
4.3.1.2 Non-glassware:
Sodium sulfate (Na2SO4)
Foil
4.3.1.3 Equipment:
Muffle oven
Drying oven
Desiccator
4.3.2 Procedures
1) NewNa,SO4
Put Na2S04 in a clean muffled beaker and bake at 450°C for four hours or
overnight.
Cool to 100°C in oven. Remove.
Place in clean sample jar; cover with foil and lid.
Store in desiccator.
2) Reconditioning Na2SO4 (to be done every two weeks):
Place Na2SO4 in 100°C drying oven overnight.
Remove from oven; cover with foil and lid.
Store in desiccator.
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Analysis of PCBs, Pesticides, and PAHs
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Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
4.4 XAD:
4.4.1 Supplies
4.4.1.1 Glassware:
Soxhlet extractor and condenser 71/60 and 29/42 joints
Six 1 L round bottom flasks with 24/40 joint
Six glass stoppers (24/40 joint)
One 1 L beaker
Two 400 mL beakers (one need not be clean)
Adaptor to convert 29/42 to 24/40
4.4.1.2 Non-glassware.:
Boiling chips
CH:C1:
Hexane
Methanol
Acetone
HPLC grade water: EM Science
CH2C1: in squirt bottle
Methanol in squirt bottle
Cl~ solvent waste container
Non-Cl~ solvent waste container
Foil
Glass wool
Six cork rings
4.4.1.3 Equipment:
Heating mantle for 1 L flask
Variable autotransformer (aka variac)
Refrigerator or freezer
4.4.2 Procedures for dry XAD2 for air sample cartridges
Day I
1) Place XAD2 in extractor plugged with glass wool.
2) Rinse XAD, with tap water many times, stirring to remove foam and small
particles. Use kimwipes to remove foam.
3) Rinse with small amount of methanol three times to remove water.
4) Add 500 mL of methanol to 1 L flask.
5) Add about 20 boiling chips to flask.
6) Assemble flask/soxhlet/condenser apparatus.
7) Turn on heater to give proper boiling (set variac to 60-65 for methanol i
8) Turn on chilled water for condenser.
9) Cover soxhlet and flask with foil.
10) Extract with methanol for 24 hours.
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IADN Project - Sample Preparation Procedure Volume 2, Chapter^
Day 2
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much methanol from soxhlet as possible.
3) Add 500 mL acetone to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 45 for acetone).
6) Cover soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 3
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from soxhlet as possible.
3) Add 500 mL hexane to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-45 for hexane).
6) Cover soxhlet and flask with foil.
7) Extract with hexane for 24 hours.
Day 4
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from soxhlet as possible.
3) Add 500 mL CH2C12 to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-50 for CH:C12).
6) Cover soxhlet and flask with foil.
7) Extract with Ch2Cl2 for 24 hours.
Day 5
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much CH2C12 from soxhlet as possible. Wait 15 minutes. Drain as much
solvent as possible.
3) Add 100 mL hexane to the soxhlet. Wait 15 minutes, then hand flush. Repeat at
least three times, until the level of the solvent in the siphon tube is the same as in
the soxhlet.
4) Add 500 mL hexane to 1 L flask.
5) Add about 20 boiling chips to flask.
6) Turn on heater (set variac to 40-45 for hexane).
7) Cover soxhlet and flask with foil.
8) Extract with hexane for 24 hours. Flushing may need to be induced twice before
it flushes on its own.
Day 6
]) Turn off heater; cool 15 to 30 minutes.
2) Flush us much hexane from soxhlet us possible.
3) Add 500 mL 50<7c acetone/507c hexane to 3 L flask.
4) Add boiling chips to flask.
5) Turn on heater (set variac to 40-45 for acetone/hexane).
6) Co\er soxhlet and flask with foil.
7) Extract with acetone/hexane for 24 hours.
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Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
Day 7
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone/hexane from soxhlet as possible.
3) Pour XAD2 in a beaker and dry overnight in 65°C oven.
4) Store in amber bottle in freezer at -20°C for up to three months.
5) Keep subsample in separate jar for checking lab blank and matrix spike.
4.4.3 Procedures for wet XAD, for precipitation sample cartridges
Day 1
1) Place XAD2 in soxhlet plugged with glass wool.
2) Rinse XAD2 with water many times, stirring to remove foam and small particles.
Use kimwipes to remove foam.
3) Rinse with small amount of methanol three times to remove water.
4) Add 500 mL methanol to 1 L flask.
5) Add about 20 boiling chips to flask.
6) Assemble flask/soxhlet/condenser apparatus.
7) Turn on heater to give proper boiling (set variac at 60-65 for methanol).
8) Turn on chilled water for condenser.
9) Cover soxhlet and flask with foil.
10) Extract for 24 hours.
Day 2
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much methanol from soxhlet as possible.
3) Add 500 mL acetone to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-45 for acetone).
6) Cover soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 3
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from soxhlet as possible.
3) Add 500 mL hexane to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-45 for hexane).
6) Cover soxhlet and flask with foil.
7) Extract with hexane for 24 hours.
Day 4
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from soxhlet as possible.
3) Add 500 mL CH,C1, to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40 for CH:C1:).
6) Cover soxhlet and flask with foil.
7) Extract with CH2C12 for 24 hours.
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IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
Day 5
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much CHiCK from soxhlet as possible. Wait 15 minutes. Drain as much
solvent as possible.
3) Add 100 mL hexane mixture to the soxhlet. Wait 15 minutes, then drain solvent.
Repeat at least three more times, until level of solvent in the siphon tube is the
same as in the soxhlet.
4) Add 500 mL hexane to 1 L flask.
5) Add about 20 boiling chips to flask.
6) Turn on heater (set variac at 40-45 for hexane).
7) Cover soxhlet and flask with foil.
8) Extract with hexane for 24 hours.
Day 6
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from soxhlet as possible.
3) Add 500 mL acetone to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac at 40-45 for acetone).
6) Cover soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 7
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from soxhlet as possible.
3) Add 500 mL methanol to 1 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 60-65 for methanol).
6) Cover soxhlet and flask with foil.
7) Extract with methanol for 24 hours.
Day 8 or so
1) Turn off heater; cool 15 to 30 minutes.
2) Turn off condenser water.
3) Flush as much methanol from soxhlet as possible.
4) Rinse XAD: at least three times with EM Science HPLC grade water (until XAD,
does not smell of methanol).
5) Store the clean XAD2 in DI water in amber bottle in the refrigerator at 4 C. (The
resin may be stored in this manner for up to three months.)
4.4.4 Comments
4.4.4.1 Variac settings may vary from autotransformer to autotransformer. Check that the
solvent is boiling properly (nice rolling boil).
4.4.4.2 If XAD: is re-used after sample extraction, it is not necessary to rinse with DI
water before extracting. The cleaning process can begin by extracting with
methanol.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
4.4.4.3 Sometime solvent does not siphon very well. Induce siphoning by hand as many
times as possible. Allow extra time in case of improper flushing.
4.4.5 Flowchart of XAD2 precleaning procedure
rinse XAD, with
DI water to remove
fines
methanol
extract
24
hours
acetone
extract
24
hours
hexane
extract
24
hours
CH,Cl,
extract
24
hours
hexane
extract
24
hours
50% acetone/50% hexane
extract
24 hours
oven dry at 65 °C
store at -20°C in amber
bottle
AIR SAMPLES
acetone
extract
24 hours
methanol
extract
24 hours
exchange to HPLC
water; store at 43C
in amber bottles
PRECIPITATION
SAMPLES
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
4.5 Silica and Quartz Fiber Filters (QF)
4.5.1 Silica
It has been determined silica is adequately cleaned during the activation process therefore
no additional processing is necessary.
4.5.2 Quartz fiber filters (QF)
Each QF is wrapped up by aluminum foil separately and then muffled to 450°C for four
hours. After it reaches ambient temperature, about 25 are wrapped again in aluminum foil
and stored in freezer in a sealed plastic bag.
5.0 Air Samples, Particle and Vapor Phase: QF and XAD2 Cartridges
5.1 Extraction
5.1.1 Supplies
5.1.1.1 Glassware:
Large soxhlet extractor (55/50 and 24/40 joints)
Condenser (55/50 joint)
500 mL round bottom flask (24/40 joint)
Glass stopper (24/40 joint)
400 mL beaker
Micro-dispenser (50 or 100 jaL) and 1 mL pipette
5.1.1.2 Non-glassware:
Boiling chips
Acetone
Hexane
Spiking standards:
Standard
PCB surrogate standard
pesticide recovery standard
PAH recovery standard
Dibutylchlorendate
Terbutvluzme
Atruzine
d|() Phenanthrene
PCB reco\erv standard
Concentration
Congener 14: 200 ng/mL
Congener 65: 50 ng/mL
Congener 166: 50 ng/mL
100 ng of each pesticide/mL
2 |ag of each PAH/mL
500 ng/mL
5600 ng/mL
2000 ng/mL
2 |ag/mL
683 na of PCBs/mL
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Analysis of PCBs, Pesticides, and PAHs
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Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
CH:Cl: in squirt bottle
Methanol in squirt bottle
Cl~ solvent waste bottle
Non-Cl" solvent waste bottle
Cork ring (size #2)
Glasswool
12" rod (glass or metal)
Large tweezers
Small tweezers
Foil
Scissors
5.1.1.3 Equipment:
Heating mantle and variable autotransformer or multi-unit extraction heater
5.1.2 Procedures
One set of sample is extracted in two days. The set includes 10-12 samples (including one
duplicate), one field blank, one lab blank, and one combination matrix spike. In
combination matrix spike, the matrix is spiked with known amount of PCBs,
Pesticides,PAHs, and atrazine to calculate recovery of each compound. A name will be
assigned to each set on the day of extraction (month, year and type of sample), such as
S94C, in which:
S = Month of sample collection, such as September
94 = year of sample collection
C = Type of sample, such as cartridge
Day 1
1) Remove spiking standards from freezer. Standards must be at ambient
temperature before using. (Ambient temperature is achieved in about two hours.)
surrogate PCB standard- PCB #14, 65, 166
surrogate pesticide standard- dibutylchlorendate
surrogate atrazine standard- trbutylazine
surrogate PAH standard- d,0 phenenthrene
pesticide recovery standard
PAH recovery standard
PCB recovery standard
Atrazine recovery standard
2) Thoroughly rinse inside of condenser and outside of joint with solvent in squirt
bottles: first with methanol, then with CH,CL Cover joint and exhaust tube with
foil.
3) Assemble supplies and samples under hood and/or utility cart. Label flasks.
4) Add five to six clean teflon chips into 500 mL round bottom flask.
5) Pour solvent into round bottom flask: 175 mL of acetone and 175 mL of hexane.
6) Transfer sample to soxhlet extractor:
Vapor sample - XAD,
Place glass wool plug at the siphon tube opening of the soxhlet extractor
using glass or metal rod.
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Carefully pour XAD2 in soxhlet extractor. Rinse container with solvent
(50% acetone/50% hexane) to remove all XAD2; pour solvent rinse into
soxhlet.
Assemble flask/soxhlet/condenser. Place on heating mantle.
Particle sample-Composite QF
Unwrap one QF at a time.
Trim off the number at the corner with clean scissors.
Use two pairs of blunt tweezers to fold one QF; place in soxhlet.
Rinse tweezers and scissors with CH:Cl:.
Repeat procedure for all QFs in composite sample.
Assemble flask/soxhlet/condenser. Place on heating mantle.
7) Spike extraction:
XAD2 and QF Samples
Using a micropipette dispenser, spike each sample with 100 |jL of the
PCB surrogate standard. (One standard solution contains all three
congeners.) 50 uL of dibutylchlorendate, 1-00 uL of terbutylazine, and
200 pL of d,0 phenenthrene
Lab Blank
Using a micropipette dispenser, spike the extraction medium with lOOuL
of the PCB surrogate standard, 50 |uL of dibutylchlorendate, 100 pL of
terbuylazine, and 200 (aL of d,0phenenthrene.
Combination matrix spike:
Spike sample medium with 1 mL of PCB recovery standard (683 ng of
PCBs), 200 uL of Mixed Pesticide Recovery standard (20 ng of each),
200 uL of mixed PAH congeners (400 ng of each), 500 |aL of atrazine
(lOOOng), 100 uL of PCB surrogate standard (14=20 ng, 65=5 ng,
166=5 ng), 50 uL of dibutyl chlorendate (25 ng), 100 uL of terbutylazine
(560 ng), and 200 uL of d,0 phenanthrene (400 ng ). PCB recovery
standard contains 683 ng of PCB in 1 mL (diluted from Michael D.
Mullin 94 mix). These data are used for the recovery of individual PCB
congeners, individual pesticides and PAHs.
8) Assemble flask/soxhlet/condenser unit. Place on heating mantle.
9) Turn on heating mantles: set Staco heating mantles to 45 or the multi-unit
extraction heater to 5.
10) Turn on condenser water.
11) Cover soxhlet and flask with foil.
12) Extract for 18 to 24 hours.
Day 2
\) Turn heating mantle off. Let cool 15 to 30 minutes. Siphon off as much solvent
from soxhlet extractor into flask as possible.
2) Detach the flask and insert stopper.
3) Turn off condenser water.
4) Store in cool dark place.
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IADN Project - Sample Preparation Procedure
5.1.3 Comments
5.1.3.1 If XAD: gets into the flask, see Section 5.2.2. Removing XAD2 from flask.
5.1.3.2 If condensation is a problem, wrap condensers with foil wrapped insulation or
with kimwipes.
5.1.4 Flow charts for air sample extraction
5.1.4.1 Setting-up extraction
350 mL of acetone/hexane (50:50) in 500 mL
round bottom flask with boiling chips
put sample in soxhlet with rinse
spike sample with 100 jjL PCB surrogate ,
50 pL dibutylchlorendate, and 100 |aL of
terbutylazine 200 jaL of dlO phenenthrene
turn on heater
turn on condenser water
cover soxhlet and flask with foil
extract for 24 hours
5.1.4.2 Taking down extraction
turn off heater
after '/z hour, turn off water
siphon off as much solvent as possible
stopper flask and store in cool dark place
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5.2 Rotary Evaporation
5.2.1 Supplies
5.2.1.1 After extraction/before column clean-up
5.2.1.1.1 Glassware:
Splash guard with 24/40 joint
100 or 200 mL beaker
Waste container for used boiling chips
5.2.1.1.2 Non-glassware:
Hexane
Clean large forceps
Cl" and non-Cl" waste bottles
CH2C12 in teflon bottle
5.2.1.1.3 Equipment:
Rotary evaporator
Aspirator pump
Chiller circulator
5.2.1.2 After column clean-up
5.2.1.2.1 Glassware:
Splash guards: one with 24/40 joint and one with 14/20 joint
25 mL beaker
5.2.1.2.2 Non-glassware:
Hexane
Cl and non-Cl waste bottles
CH:C1, in teflon bottle
5.2.1.2.3 Equipment:
Rotary evaporator
Aspirator pump
Chiller circulator
5.2.1.3 Removing XAI), from Flask
5.2.1.3.1 Glassware:
500 mL round bottom flask
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IADN Project - Sample Preparation Procedure
5.2.1.3.2
5.2.1.3.3
Non-glassware:
Cork ring
Hexane
Equipment:
-none-
5.2.2 Procedures
5.2.2.1 Set-up
1)
2)
3)
Fill chamber with DI water.
Turn on the chiller circulator.
Set bath temperature:
Solvent
hexane
acetone
acetone/hexane
methanol
CH,CU
Temperature (°C)
30-32
30-32
30-32
40
30
4)
5)
6)
Rinse joint of steam duct with CH2C1:.
Attach appropriate splash guard(s) to steam duct. Clamp each joint.
Turn on vacuum. Check vacuum of system.
5.2.2.2 Evaporation
1) Remove boiling chips with large forceps. If XAD2 is in flask, see
Section 5.2.2.4. Removing XAD2 From Flask.
2) Attach flask to splash guard. Clamp joint.
3) Turn on motor of rotator to predetermined rotation speed (usually to the
bottom of the indicator line, or about 50 rpm). Turn flask to start rotation.
Evaporation should begin in approximately one minute; solvent should
not boil.
4) Evaporate sample down to approximately 2 mL (in a 500 mL round
bottom flask, area of liquid should be about the size of a quarter).
5) Open stopcock of rotary evaporator to release vacuum.
6) Detach the flask:
If exchanges are necessary, add specified amount of hexane from
Section 5.2.2.3. Solvent Exchanges, then return flask to splash
guard and clamp.
If additional exchanges are not necessary, stopper flask. Store
flask under cabinet.
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
7) Empty receiving flask into proper waste bottle as needed.
8) Rinse splash guard with CH,C1: before using with a different sample.
Muffle splash guard at the end of a set of samples. Splash guards should
be washed and muffled after every three or four sets of samples.
5.2.2.3 Solvent exchanges
after
extraction
after column
clean-up
Fraction
—
hexane
50%
Amount of
Hexane to
Add
75 mL
—
25 mL
No. of
Exchanges
2
0
I
Total # of
Rotary
Evaporations
3
1
2
Final
volume
2-5 mL
1 mL
1 mL
5.2.2.4 Removing XAD2 from flask
1) Label another 500 mL flask with sample ID.
2) Decant sample from original flask into clean flask.
3) Rotary evaporate new flask using above procedures.
4) Add hexane for the exchanges to original flask with XAD,; swirl hexane
in flask to remove any remaining items of interest.
5) Decant hexane wash from original flask into new flask as needed to
complete exchanges.
5.2.2.5 Clean-up
1) Turn off heater on rotary evaporator.
2) Turn off motor on rotary evaporator.
3) Turn off chiller.
4) Empty receiving flask into proper waste solvent bottle.
5) Cover steam duct with foil.
6.0 Rain Samples
6.1 Extraction
6.1.1 Supplies
6.1.1.1 Glassware:
Large soxhlet extractor (55/50 and 24/40 joints)
Condenser (55/50 joint)
500 mL round bottom flask
Glass stopper (24/40 joint)
Micro-dispenser (50 or 100 |_iL) and 1 mL pipette
200 mL (or larger) beaker
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
6.1.1.2 Non-glassware:
Boiling chips
Acetone
Hexane
CHiCU in squirt bottle
Methanol in squirt bottle
Cl~ solvent waste bottle
Non-Cl~solvent waste bottle
Cork ring for 500 mL flask
Glasswool
12" rod (glass or metal)
Large tweezers
Small tweezers
Foil
Spiking standards:
Standard
PCB surrogate standard
pesticide recovery
standard
dibutylchlorendate
terbutylazine
atrazine
PAH recovery standard
d|0phenanthrene
PCB recovery standard
Concentration
Congener 14: 200 ng/ml
Congener 65: 50 ng/mL
Congener 166: 50 ng/mL
1 00 ng of each
pesticide/mL
500 ng/mL
5600 ng/mL
2000 ng/mL
2pgofeachPAH/mL
2 |Jg/mL
683 n^ofPCBs/mL
6.1.1.3 Equipment:
Heating mantle for 500 mL round bottom flask
Variable autotransformer or multi-unit extraction heater
6.1.2 Procedures
6.1.2.1 Extraction of Rain Samples from XAD: cartridges
One set of samples (usually one month's sample from all different site) is extracted
on Day 1. A name is assigned to that set. An example of set name is Au94P-
month of collection, year, and type of sample. In this case, P stands for
precipitation sample. One set will include approximately six to eight samples, at
least one duplicate sample, one field blank, one lab blank, and one combination
matrix spike.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
Day 1
1) Remove spiking standards from freezer. Standards must be at ambient
temperature before using. (Ambient temperature is achieved in about two
hours.)
surrogate PCB standard
surrogate pesticide standard - dibutylchlorendate
surrogate atrazine standard - terbutylazine
surrogate PAH standarad - d,0 phenanthrene
pesticide recovery standard
PAH recovery standard
PCB recovery standard
Atrazine recovery standard
2) Thoroughly rinse inside of condenser and outside of joint with solvent in
squirt bottles: first with methanol, then with CH,Ck Cover joint and
exhaust tube with foil.
3) Assemble supplies and samples under hood and/or utility cart. Label
flasks.
4) Add five to six clean teflon chips into 500 mL round bottom flask.
5) Measure 175 mL acetone in a beaker.
6) Place glass wool plug at the siphon tube opening of the soxhlet extractor
using glass or metal rod. Assemble soxhlet extractor and flask.
7) Put XAD: sample in soxhlet extractor. Rinse container with acetone from
beaker; add this and remaining acetone from beaker to soxhlet.
8) Add 175 mL hexane to top of soxhlet.
9) Spike extraction:
Samples
Using micropipette dispenser, spike each sample with 100 |jL of
the PCB surrogate (One standard solution contains all three
congeners), 50 pL of dibuylchlorendate, 100 (jL of terbutylazine,
and 200 uL of d,0phenanthrene.
Lab Blank
Using micropipette dispenser, spike approximately 8 g of clean
XAD, with 100 |aL of the PCB surrogate standard, 50 uL of
dibuylchlorendate, 100 uL of terbutylazine, and 200 uL of
d,0 phenanthrene.
Combination Matrix Spike or MS
Spike sample medium with 1 ml of PCB recovery standard
(683 ng of PCBs), 200 jaL of Mixed Pesticide Recovery standard
(20 ng of each), 200 uL of mixed PAH standard (400 ng of each),
500 pL of atrazine( 1000 ng), 100 uL of PCB surrogate standard
(14=20 ng, 65=5 ng, 166=5 ng), 50 uL of dibutyl chlorendate
(25 ng). 100 |jL of terbutylazine (560 ng). and 200 |jL of
d,,, phenanthrene (400 ng). PCB recovery standard contains
683 ng of PCB in 1 mL (diluted from Michael D. Mullin 94 mix).
These data are used for the recovery of individual PCB
congeners, individual pesticides, each PAHs and atrazine.
10) Assemble flask/soxhlet/condenser apparatus. Place on heating mantle.
11) Turn on heating mantles: set Staco heating mantle to 45 or the multi-unit
extraction heater to 5.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
12) Turn on condenser water.
13) Cover soxhlet and flask with foil.
14) Extract for 24 hours.
Note: The sample has water in it, thus it may not siphon on its own the first two
or three times depending on the amount of water present. Induce siphoning until
the level of solvent in the soxhlet and in the syphon tube are the same.
Day 2
1)
2)
3)
4)
Turn heating mantle off. Let cool 15 to 30 minutes. Siphon off as much
solvent from soxhlet extractor into flask as possible.
Detach the flask and insert stopper.
Turn off condenser water.
Store in cool dark place.
6.1.3 Flow chart for the extraction of rain samples
rain on XAD^ in soxhlet
assemble soxhlet and flask
add 175ml acetone on XAD, in soxhlet
add 175 ml hexane on XAD, in
soxhlet
spike with 100 |aL PCS surrogate,
50 uL of dibutylchlorendate, lOOuL
of terbutylazine,and 200 uL of
dlO phenanthrene
turn on heater and condenser water
hand induce siphoning for the initial
three to four flushes
extract for 24 hours
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
6.2 Rotary Evaporation
6.2.1 Supplies
6.2.1.1 After extraction/before column clean-up
6.2.1.1.1 Glassware:
Splash guard with 24/40 joint
100 mL or 200 mL beaker
Waste container for used boiling chips
6.2.1.1.2 Non-glassware:
Hexane
Clean large forceps
Cl and non-Cl waste bottles
CH2C12 in teflon bottle
6.2.1.1.3 Equipment:
Rotary evaporator
Aspirator pump
Chiller circulator
6.2.1.2 After column clean-up
6.2.1.2.1 Glassware:
Splash guards: one with 24/40 joint and one with 14/20 joint.
25 mL beaker
6.2.1.2.2 Non-glassware:
Hexane
Cl and non-Cl" waste bottles
CH:C1: in tenon bottle
6.2.1.2.3 Equipment:
Rotary evaporator
Chiller circulator
621.3 Back Extraction (in addition to the items listed in Section 6.2.1, Supplies. After
extraction/before column clean-up)
2-g
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
6.2.1.3.I
6.2.1.3.2
6.2.1.3.3
Glassware:
125 mL separately funnel
Pasteur pipettes
10 mL graduated pipette
Non-glassware:
Rubber pipette bulb
Equipment:
Three-prong clamp with support
6.2.2 Procedures
6.2.2.1 XAD2 Cartridges
6.2.2.1.1
6.2.2.1.2
Set-up
1)
2)
3)
Fill chamber with DI water.
Turn on the chiller circulator.
Set bath temperature:
Solvent
hexane
methanol
acetone/hexane
CH,C1,
Temperature (°C)
30-32
40
30-32
30
4)
5)
6)
Rinse joint of steam duct with CH:CK.
Attach appropriate splash guard(s) to steam duct. Clamp
each joint.
Turn on vacuum. Check vacuum of system.
Evaporation
1)
2)
3)
4)
Remove boiling chips with large forceps. If XAD2 is in
flask, see Section 6.2.2.1.3. Removing XAD; from flask.
Attach flask to splash guard. Clamp joint.
Turn on motor of rotator to predetermined rotation speed
(usually to the bottom of the indicator line, or about
50 rpm). Turn flask to start rotation. Evaporation should
begin in approximately one minute; solvent should not
boil.
Evaporate sample until the evaporation slows down.
Note: If rate of evaporation slows down, DO NOT
continue. There is water in the sample.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
6.2.2.1.3 Removing XAD, from the flask
1) Label another 500 mL flask with the samples ID
2) Decant sample from original flask into the clean flask;
wash with 10 mL hexane twice.
3) Rotary evaporate the new flask until evaporation begins
to slow down.
6.2.2.1.4 Back extraction and solvent exchanges
1) Add 75 mL hexane to sample flask. Rotavap again to
lOOmL. Transfer the content to separatory funnel. Add
I gm of sodium sulfate. Shake vigorously. Wait for
20 minutes.
2) First Extract:
Transfer the original hexane layer to the flask.
Add 25 mL hexane to the water in separatory
funnel. Then add approximately 1 gofNa:SO4
Shake vigorously; let stand at least 20 minutes.
While waiting for first extract to separate, rotary
evaporate the original sample to approximately
5mL.
After 20 minutes or so, pipette the hexane out
and add it to the original sample flask.
3) Second Extract:
Add 25 mL hexane to the water layer in the
separatory funnel. Shake vigorously; let stand at
least 20 minutes.
Pipette out the hexane layer from the separatory
funnel; add it to the original flask.
4) Third Extract:
Add 25 mL hexane to the water layer in the
separatory funnel. Shake vigorously; let stand at
least 20 minutes.
Pipette out the hexane layer from the separatory
funnel; add it to the original flask.
5) Rotary evaporate the combined extract to 2 mL.
Note: *More water may separate out after the addition of
the first and second extract. Pipette the water out and
add it to the separatory funnel.
*It is possible trace amounts of water may be in the final
sample - ignore it! The NaSO4 on the top of the silica
column will take care of it.
*If an emultion forms in the separatory funnel, add extra
Na,SO4 to the funnel. This will facilitate the separation
of the water.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
6.2.2.1.5 Clean-up
1) Turn off heater on rotary evaporator.
2) Turn off motor on rotary evaporator.
3) Turn off chiller.
4) Empty receiving flask into proper waste solvent bottle.
5) Cover steam duct with foil.
6.2.2.1.6
Flow Chart of Rotary Evaporation and Back Extraction
rotary evaporate rain extract until it
slows down. Add 75 ml hexane.
Shake and wait 20 minutes
transfer hexane layer to the original flask
add 25 mL hexane to separator/ funnel
shake and wait 20 minutes
1st extract
[rotary evaporate original extract to 5 mL|
pipette out hexane layer from 1 st extract
add to original extract
add 25 mL hexane to separatory funnel.
Shake and wait 20 minutes
pipette out hexane layer from 2nd
extract
add to original extract
add 25 mL of hexane to separatory
funnel. Shake and wait 20 minutes
pipette out hexane layer from 3rd extract
add to original extract
rotary evaporate combined extract to
2mL
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
6.2.2.2 Rotary evaporation after column chromatography
I) Attach flask to splash guard. Clamp joint.
2) Turn on motor of rotator to pre-determined rotation speed (usually to the
bottom of the indicator line, or about 50 rpm). Turn flask to start rotation.
Evaporation should begin in approximately one minute; solvent should
not boil.
3) Evaporate sample down to approximately 2 mL.
4) Open stopcock of rotary evaporator to release vacuum.
5) Detach the flask:
If exchanges are necessary, add specified amount of hexane as
listed in Section 5.2.2.3, Solvent Exchanges, then return flask to
splash guard and clamp.
If additional exchanges are not necessary, stopper flask and store
it under the cabinet.
6) Empty receiving flask into proper waste bottle as needed.
7) Rinse splash guard with CH2Cl2 before using with a different sample.
Muffle splashguard a the end of a set of samples. Splash guards should
be washed and muffled after every three or four sets of samples.
6.2.2.3 Solvent Exchanges
after column
chromatography
Fraction
hexane
507c
meth.
Amount of
Hexane to
Add
—
25 mL
No. of
Exchanges
0
1
0
Total # of
Rotary
Evaporations
1
•>
0
Final
Volume
1 mL
1 mL
1 mL
7.0 Silica Column Chromatography
7.1 Supplies
7.1.1 Activation/Deactivation
7.1.1.1 Glassware:
100 mL or 250 mL beaker
Powder funnel
250 mL or 500 mL round bottom flask
Stopper to fit round bottom flask
1 mL graduated pipette
25 mL beaker
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Analysis of PCBs, Pesticides, and PAHs
In Air and Precipitation Samples:
Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
7.1.1.2 Non-glassware:
*Silica
Pipette bulb
Cork ring to fit round bottom flask
7.1.1.3 Equipment:
Muffle furnace
Desiccator
Calculator
Balance
Particle mask
7.1.2 Column clean-up (for a three-fraction column clean-up of one sample)
7.1.2.1 Glassware:
Column
Three 100 mL pear shaped flasks with 14/20 joints
Three glass stoppers with 14/20 joints
Pasteur pipettes (9!/2 inch and/or 514 inch): minimum of one for each sample and
six additional pipettes for each set of samples fractionated
Graduated cylinders: 50 mL and 10 mL
Funnel
100 mL beaker
250 mL beaker OR waste jar (need not be clean)
Three 250 mL beakers
7.1.2.2 Non-glassware:
Rubber pipette bulbs
Hexane
50% hexane/50% CH2C12
CH2C1:
Methanol
Two cork rings for 100 mL pear shaped flasks (size #1)
Rubber hammer
Stainless steel spatula
20" rod
Teflon stopcock
Glass wool
4c/c deactivated silica
NaSO4
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
7.1.2.3 Equipment
Ultrasonicator
7.1.3 Supply chart for each sample
Item
amount of silica to activate/deactivate
column size
amount of NaSO4
elution volume (1st and 2nd fraction)
switching volume
elution volume (3 rd fraction)
Air Particle (QF)
4-6 g
3.5"
0.5
25 mL
4mL
30 mL
Air Vapor (XAD:)
4-6 g
3.5"
0.5"
25 mL
4mL
30 mL
Rain (XAD,)
4-6 g
3.5"
1.5"
30 mL
5mL
35 mL
7.2
Procedures
7.2.1 General procedures
7.2.1.1 Activation/Deactivation of silica
Day 1
1)
2)
Day 2
1)
3)
Place approximate amount of silica needed in a beaker. Cover beaker
with foil.
Place beaker in 100°C oven, turn thermostat to 300°C; keep in oven
overnight.
DO NOT PUT SILICA INTO 300°C OVEN!
Turn oven temperature down to 100°C;
DO NOT REMOVE SILICA FROM OVEN.
When oven has cooled to 100°C, remove beaker from oven; let cool on
counter top until warm (approximately 5 to 10 minutes): place in
desiccator.
When silica has reached ambient temperature (approximately two hours),
deactivate it:
Working quickly, weigh out desired amount of silica in the round
bottom flask. Stopper flask immediately after pouring silica.Add
4% weight/volume of DI water to silica, using the following
equation:
ileactivcition
mL DI \\atcr
100
r'r ileactivation weight of fili
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
Volume 2, Chapter 1 IADN Project - Sample Preparation Procedure
For precipitation samples use 3% deactivation.
SHAKE WELL. Shake flask until all clumps are broken-up.
Store in desiccator overnight for equilibration.
Use deactivated silica in desiccator within three days. Any
unused silica may be reused after re-activating and
re-deactivating).
7.2.1.2 Preparation and packing of column(s)
I) Assemble stopcock(s) on column(s).
2) Stuff glass wool plug (approximately I cm) into lower end of the each
column with 20" rod.
3) Measure and mark appropriate distance from top of glass wool plug.
4) Clamp column(s) securely onto frame in ventilation hood. Place empty
glass container under each column (100 mL minimum size; it need not be
clean).
5) Close stopcock(s); fill column(s) half full with hexane.
6) Make a slurry of hexane and deactivated silica. Pour slurry into each
column. DO NOT ALLOW SILICA TO DRY OUT: rinse column
and beaker with hexane via Pasteur pipette. (Use of a funnel may
facilitate process.) Open stopcock(s). Tap column(s) with rubber
hammer to pack silica. Add silica/hexane as needed until desired length
is loaded.
7) Cap column(s) with W Na:SO4 for XAD-, and QF samples, 0.5" Na:SO4
for rain samples.
8) Wash column(s) with 25 mL hexane for conditioning.
9) When hexane level reaches I cm above top of Na2SOj, close stopcock(s)
to prevent further dripping. NEVER LET COLUMN RUN DRY.
10) If column(s) is/are not going to be used immediately, stopper column(s)
and cover tip(s) of column(s) with foil.
7.2.1.3 Set-up
I) Label one 100 mL pear-shaped flask for each fraction which is to be
collected.
2) On a cart, assemble pear shaped flasks and remaining supplies listed in
Section 7.1, Supplies.
3) Place sample flask in front of column.
4) Place a 50 mL or 100 mL beaker in front of sample flask.
5) Add hexane to 50 mL or 100 mL beaker; cover with foil. (For volume of
hexane, see chart in Section 7.1.3, Supply Chart.)
7.2.1.4 Column chromatography
7.2.1.4.1 First fraction
1) Ultrasonicate sample flask before loading the sample
onto the column to detach the particles \\hich arc sticking
to the walls of the flask.
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in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
2) Remove stopper from sample flask. Assemble pipette
and rubber bulb; place pipette in sample flask.
3) Place Fraction #l (hexane) pear shaped flask under the
column.
4) Open stopcock and let column drip until hexane level is
at the top of the Na2SO4.
5) Load sample into column with Pasteur pipette.
6) Set drip rate to approximately one drip per second. Add
approximately 5 mL hexane to sample flask from the
beaker. Swirl solvent in flask..
7) When sample has drained down to the top of the Na;SO4,
add the hexane from the sample flask to the column.
Add an additional 5 mL hexane to the sample flask from
the beaker. Swirl solvent in flask.
8) When solvent has drained down to the top of the Na2SO4,
add the second 5 mL hexane to the column. Add the
remaining hexane from the beaker to the sample flask.
Swirl solvent in sample flask.
9) When solvent has drained down to the top of the Na,SO4,
add the remaining hexane from the sample flask. (If
reservoir on top of the column cannot hold entire amount,
add as much as possible, then refill as space becomes
available.)
Note: Stagger the timing of the column loadings such
that the changing of the flasks are not concurrent.
7.2.1.4.2 Second fraction
1) While the hexane is dripping (from the first fraction),
measure the hexane/CH:Cl, and put it into the
appropriate containers.
2) When the hexane drips down to the top of the Na,SO4,
add the switching volume hexane/CH^CU from the
sample flask to the column.
3) Transfer the hexane/CH:Cl2 from the beaker to the
sample flask. Swirl the solvent in the flask.
4) Place the appropriate pear shaped flask (labeled '50%'
fraction) next to the flask under the column.
5) When the hexane/CH2Cl2 level in the column is to the top
of the NaSO4, quickly switch flasks and pour as much of
the remaining hexane/CH2Cl2 into the column as
possible. Add hexane/CH:Cl: to the column as space
permits.
6) Continue to monitor the rate of drip (approximately
one drip per minute).
7) Place the pear shaped flask from the first fraction on the
supply cart. Stopper the flask.
8) Once the column has stopped dripping, remove flask
from second fraction, stopper it, and put it on the supply
cart.
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
9) collect another fraction with 30 ml of methanol in case of
air samples and 35 mL of methanol in case of rain
samples.
7.2.1.4.3 Clean-up
I) Remove stopcock from column.
2) Turn column upside down and secure it with clamps.
Place container under column to catch NaSO., and silica.
3) After column has dried out, use vacuum (air or water) to
remove glass wool plug.
4) Pour silica and NaSO4 into used glove or foil before
discarding into trash can.
7.2.2 Specific procedures by sample type
7.2.2.1 XAD: (vapor) and OFF
Follow General Procedures.
7.2.2.2 Rain samples
Procedure is essentially the same as XAD, (vapor and particles) and QF with the
following exceptions:
I) NaSO4 should be activated no more than 2 to 3 days before using; cap
should be 1.5"
2) Elution volumes:
Solvent
hexane
50% CH^CU in hexane
switching volume
methanol
Solvent needed (mL)
for rain samples
30
30
5
35
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
I AON Project - Sample Preparation Procedure
Volume 2, Chapter 1
7.2.3 Summary flow-chart
4% deactivated silica
silica slurry in hexane
3.5" column
top column with Na3SQ4
equilibrate column with hexane
load sample
elute with hexane: 25 mL for air,
30 mL for rain collect eluent in
pear shaped flask
add switching volume: 4 mL for air
and 5 ml for rain,
collect in same flask
change flask
elute with 50^ CH,Cl, in hexane
.Mute with methano!
1 st fraction contains
PCBs, DDE, and HCB.
2nd fraction contains a and y HCH.
dieldnn, DDD, DDT, a-chlordane.
y-chlordane, t-nonachlor, and all
PAHs.
Atrazine
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project • Sample Preparation Procedure
8.0 Transfer of Solution
8.1 Supplies for Each Sample
8.1.1 Glassware:
Pasteur pipettes (9'/2 inch and/or 51A inch): minimum of one for each fraction and one
additional pipette for hexane
4 mL amber glass vial for each fraction
50 mL beaker
8.1.2 Non-glassware:
Vial file for 4 mL vials
Rubber pipette bulbs
Hexane (minimum 2 mL for each fraction)
8.1.3 Equipment:
-none-
8.2 Procedures
8.2.1 Preparation
1) Label each amber vial with sample ID and fraction ID.
2) Put hexane in 50 mL beaker (approximately 2 mL per sample).
3) Concentrate all fractions by rotary evaporation to 1 mL. 50% CH:CL: fraction
needs to be solvent exchanged to hexane (see Section 5.2.3).
8.2.2 Transferring sample
1) Using a Pasteur pipette, transfer entire sample from flask to amber vial.
2) Add approximately 1 mL hexane to flask and swish solvent around to clean out
flask; transfer to amber vial.
3) Repeat.
Note: Do not add so much solvent as to fill the vial. If it is too full, there is a
chance of splashing at the time of N: blowdown.
4) Close amber vial tightly, place in vial file, and store in freezer. Label the vial file
with sample set name, type of samples, and site of collection.
9.0 N2 Blowdown
9.1 Supplies
9.1.1 Glassware:
-none-
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
Volume 2, Chapter
9.2
9.1.2 Non-glassware:
CH2C12
Sample in amber vial
9.1.3 Equipment:
N2 blowdown unit
Procedures
9.2.1
9.2.2
Set-up
1) Remove all nozzle plugs from unit.
2) Turn on N: at tank and trap (do NOT touch primary or secondary controls on
regulator). Let N, flush out for approximately five minutes.
3) Turn heater on LOW.
4) Attach clean needle to each nozzle to be used.
Blowdown
1) Place amber vials in slot; adjust N, flow such that there are gentle (barely
detectable) ripples in the vials.
2) Evaporate down to the approximate predetermined volume. (See following chart.)
Type of sample
rain
QF
XAD,
winter
summer
winter
summer
winter
summer
Approximate volume after N:
blowdown (mL)
hexane
fraction
0.4
0.4
0.4
0.4
0.4-0.8
0.8-1.0
50%
fraction
0.4
0.4
0.4
0.8
1.0-2.0
1.5-2.0
methanol
fraction
0.4
0.4
0.4
0.4
0.4
0.4
9.2.3 Closing-up unit
1) Turn off N: at trap and near the regulator.
2) Replace the nozzle caps.
3) Rinse nozzle extension tubes with CH:CI:. After three to four uses or after a
highly contaminated batch of samples, ultrasonicate nozzle extension tubes.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
10.0 Spiking Samples with ISTD
10.1 Supplies
10.1.1 Glassware:
-none-
10.1.2 Non-glassware:
Samples in 4 mL amber glass vials
Internal standards (see next page)
Hexane
CHjCl,
Cl~ and non-CP waste containers
10.1.3 Equipment:
25, 50, and 100 uL microdispensers
10.2 Procedures
1) Remove ISTDs from freezer; equilibrate to ambient temperature (approximately
two hours).
Fraction
hexane
50%
50%
methanol
Compound
PCBs and
pesticides
PAHs
Pesticide
atrazine
Type of
sample
vapor,
particle,
and rain
vapor,
particle,
and ram
vapor
particle
rain
vapor
particle
ram
Internal Standard
PCB30
PCB 204
D|0 anthracene,
Dn benzo(a)anthracene
triphenylmethane
d,2 perylene
PCB 65
PCB 155
d|(J anthracene
Spike
Volume
(uL)
100
100
50
Final
Mass in
Sample
(ng)
8
6
200
200
134.6
180
20
20
200
Color of Dot
on Label
red
blue
green
Clean micropipette
Remove glass tube used to cover plunger.
Rinse plunger with CH,CL Wave pipette to evaporate solvent.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
Without touching glass tubes, insert plunger into new glass capillary; tighten tube in
place.
Rinse the capillary with dichloromethane twice and air dry. Draw spiking standard. Make
sure that there is no air bubble.
3) Spike sample vial (see above chart for surrogate and amount).
4) Mark each amber vial label with a appropriate color of dot (use a water-proof marker).
5) Replace glass tube used to cover plunger of micropipette. Store micropipette.
11.0 Making Microvials for GC Analysis
II. I Supplies
11.1.1 Glassware:
Conical microvials
Pasteur pipettes
11.1.2 Non-glassware:
Vial racks
Septa (vial caps)
11.1.3 Equipment:
Crimper
11.2 Procedures
1)
2)
Label conical microvials with sample Ids. In addition, label extra microvial for hexane
and the appropriate calibration standard for every set of samples.
Using a Pasteur pipette, remove approximately 200 uL of each sample and put in labeled
conical microvial. (The level of liquid will be at the shoulder of the microvial.) Also
place 200 uL of hexane and 200 jaL of the appropriate standard into the labeled
microvials. (See following chart for the appropriate standard.)
Fraction
hexane
50%
50%
methanol
Target
Compounds
PCBs
pesticides
PAH
atrazine
Calibration Standard
Mullin 94: 683 ng/mL
mixed pesticide standard: 20 ng/mL ea
mixed PAH standard: 200 ng/mL ea (approx )
1000 ng/mL
Note: Make one vial with performance standard for each set of analyte.
3) Crimp septa (into microvial.
4) Load microMuls into autosampler or store in t'ree/cr.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
12.0 Standards
12.1 PCB Standards
12.1.1 Mullin's94mix: 170.8 jag/mL: Mixture of 1232, 1248, and 1262 in 25:18:1 8.
12.1.2. Surrogate standards
12.1.2.1 Congener 14: Primary stock: 100 |Jg/mL in isooctane: Accustandard.
12.1.2.2
12.1.2.3
12.1.2.4
Secondary stock
Primary Stock
1 mL(100|ag)
Dilution
to 100 mL hexane
Final
Concentration
lOOOng/mL
Congener 65: Primary stock: 100 |Jg/mL in isooctane: Accustandard
Secondary stock
Primary Stock
1 mL(100|ag)
Dilution
to 1 00 mL hexane
Final
Concentration
lOOOng/mL
Congener 166: Primary stock: 100 |Jg/mL in isooctane: Accustandard
Secondary stock
Primary Stock
1 mL(lOO)ag)
Dilution
to 100 mL hexane
Final
Concentration
lOOOng/mL
PCB mix surrogate recovery standard: To be used for spiking each
sample.
Congener
14
65
166
Stock
Concentration
lOOOng/mL
lOOOng/mL
1 000 ng/mL
Mix
10 mL
2.5 mL
2.5 mL
Final
Concentration
200 ng/mL
50 ng/mL
50 ne/mL
Volume was made up to 50 mL with hexane.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
12.1.3 Internal Standards (ISTD)
12.1.3.1 Congener 30: Primary stock: 100 (Jg/mL in isooctane: Accustandard
Secondary stock
Primary Stock
0.5 mL (50 |ag)
Dilution
50 mL
Final Concentration
lOOOng/mL
12.1.3.2
Congener 204: Primary stock: 100 |ag/mL in isooctane: Accustandard
Secondary stock
Primary Stock
0.5mL(50|ag )
Dilution
50 mL
Final Concentration
1000 ng/mL
12.1.3.3
PCB spiking standard:
Congener
30
204
Stock
Concentration
1000 ng/mL
1000 ng/mL
Mix
8 mL
6 mL
Final
Concentration
80 ng/mL
60 ng/mL
Volume was made up to 100 mL with hexane.
12.1.4 PCB calibration standard for PCBs
Congener
Mullin 94
14
65
166
30
204
DDE
HCB
Stock Concentration
170.8jag/mL
1 000 ng/mL
1 000 ng/mL
1000 ng/mL
1 000 ng/mL
1000 ng/mL
2000 ng/mL
2000ng/mL
Mix
400 |uL
2mL
0.5
0.5
0.8
0.6
1 mL
1 mL
Final Concentration
683.2 ng/mL
20 ng/mL
5 ng/mL
5 ng/mL
8 ng/mL
6 ng/mL
20 ng/mL
20 ng/mL
Volume made up to 100 mL with hexane.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
12.1.5 PCB recovery standard for PCBs: used for matrix spike.
Congener
Mullin 94
Stock Concentration
170.8(jg/mL
Mix
400 uL
Final Concentration
683.2 ng/mL
12.1.6 PCB performance standard: used for instrument calibration check.
Congener
Mullin 94
14
65
166
30
204
DDE
HCB
Stock Concentration
170.8 ug/mL
1000 ng/mL
1000 ng/mL
1000 ng/mL
1000 ng/mL
1000 ng/mL
2000 ng/mL
2000ng/mL
Mix
300 uL
1 mL
1 mL
1 mL
0.8
0.6
0.5 mL
0.5 mL
Final Concentration
5 12.4 ng/mL
10 ng/mL
1 0 ng/mL
10 ng/mL
8 ng/mL
6 ng/mL
10 ng/mL
10 ng/mL
Volume made up to 100 mL with hexane
12.2 Pesticide Standards
12.2.1 Stock solutions
12.2.1.1 Primary stock
12.2.1.2 Stock
Pesticide
dieldrin
a-HCH
Y-HCH
HCB
Ultra Sc. ampule
concentration
100 ug/mL in MeOH
100 ug/mL in MeOH
100 ug/mL in MeOH
100 ug/mL in
methylene chloride
Dilution
1 mL - 50 mL
hexane
1 mL - 50 mL
hexane
1 mL - 50 mL
hexane
1 mL - 50 mL
hexane
Stock
Concentration
2 ug/mL
2 ug/mL
2 ug/mL
2 ug/mL
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
Pesticide
4-4'DDT
4-4'DDD
4-4'DDE
a-chlordane
y-chlordane
t-nonachlor
atrazine
Ultra Sc. ampule
concentration
lOOug/mLinMeOH
lOOug/mLinMeOH
lOOug/mLinMeOH
100|jg/mLin MeOH
lOOug/mLinMeOH
100(Jg/mLinMeOH
lOOfag/mLMeOH
Dilution
1 mL - 50 mL
hexane
1 mL - 50 mL
hexane
1 mL - 50 mL
nexane
1 mL - 100 mL
lexane
1 mL— lOOmL
n hexane
1 mL— - lOOmL
n hexane
1 rnL - 50 mL
icxane
Stock
Concentration
2 Mg/mL
2 |Jg/mL
2 ug/mL
1 Mg/mL
1 (Jg/mL
1 Mg/mL
2 |Jg/mL
12.2.2 Pesticide spiking standard: Cong 65, 155
Compound
Congener 65
Congener 155
Stock Concentration
lOOOng/mL
1 000 ng/ mL
Mix
10 mL
lOmL
Final Concentration
200 ng/mL
200 ng/mL
Volume made up to 50 mL with hexane
12.2.3 Pesticide surrogate standard
Dibutylchlorendate: 100 ug/ mL in methanol
Stock:
Spiking standard:
1 mL of above diluted to 100 mL in hexane = 1000 ng/ mL
25 mL of stock solution diluted to 50 mL with hexane =
500 ng/ mL
Terbutylazine: 2.8 mg was weighed and diluted to 100 mL with MeOH
Stock: 28000 ng/ mL
Spiking standard: 10 mL of stock was diluted to 50 mL with CH.CL, = 5600
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
12.2.4 Mixed pesticide calibration standard: MPS 65, 155
This is used for analysis of pesticides in 50% CH:Cl: fraction.
Compound
a-HCH
y-HCH
dieldrin
DDT
DDD
cc-chlordane
y-chlordane
t-nonachlor
Cong. 1 55
Cong. 65
Stock Concentration
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
1 000 ng/mL
1 000 ng/mL
1 000 ng/mL
1 000 ng/mL
1 000 ng/mL
Mix
I mL
I mL
I mL
I mL
I mL
2mL
2mL
I mL
2mL
2mL
Final Concentration
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
20 ng/mL
Volume made up to 1 00 mL with hexane
12.2.5 Mixed pesticide performance standard
Compound
Pest Recovery
standard B5
from ISWS
Dibutyl
chlorendate
cong 65
cong 155
Stock Concentration
1 00 ng of each / mL
1 000 ng/ mL
1 000 ng/ mL
1 000 ng/ mL
Mix
5 mL
0.5 mL
0.5 mL
0.5 mL
Final Concentration
10 ng of each/ mL
1 0 ng/ mL
10 ng/mL
1 0 ng/ mL
Volume was made up to 50 mL with hexane.
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter 1
12.2.6 Pesticide recovery standard
Compound
HCB
a-HCH
Y-HCH
dieldrin
4-4' DDE
4-4' DDD
4-4' DDT
a-chlordane
y-chlordane
t-nonachlor
Stock Concentration
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
2000 ng/mL
1 000 ng/mL
1000 ng/mL
1000 ng/mL
Mix
2.5 mL
2.5 mL
2.5 mL
2.5 mL
2.5 mL
2.5 mL
2.5 mL
5 mL
5 mL
25 mL
Final Concentration
1 00 ng/mL
100 ng/mL
100 ng/mL
, 100 ng/mL
100 ng/mL
100 ng/mL
100 ng/mL
100 ng/mL
100 ng/mL
100 ng/mL
Volume made up to 50 mL with hexane
12.3 PAH Standard
12.3.1 PAH mixed GC/MS calibration standard
solvent = hexane
PAH
acenapthene
acenapthylene
anthracene
benzo(a)anthracene
benzo(a)pyrene
benzo(b)fluoranthene
benzo(e)pyrene
benzo(g,h,i (perylene
benzo(k)fluoranthrene
chrysene
coronene
d|,,anthracene-ISTD
Stock Cone.
(Hg/mL)
1.97
1.97
1.97
1.97
1.97
1.97
1.91
1.97
1.97
1.97
1 .93
4.00
mL stock
10
10
10
10
10
10
10
10
10
10
10
4.2
Final Volume
(mL)
100
100
100
100
100
100
100
100
100
100
100
100
Final Cone.
(|jg/mL)
0.20
0.20
0.20
0.20
0.20
0.20
0.19
0.20
0.20
0.20
0.19
0.17
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
PAH
d,0 perylene
d,,benzo(a)anthracene-
ISTD
dibenzo(a,h)anthracene
fluoranthene
fluorene
indeno( 1 ,2,3,cd)pyrene
napthalene
phenanthrene
pyrene
retene
triphenylmethane-ISTD
Stock Cone.
(ug/mL)
3.6
4.00
1.97
1.97
1.97
1.97
1.97
1.97
1.97
1.98
2.69
mL stock
4.2
4.2
10
10
10
10
10
10
10
10
4.2
Final Volume
(mL)
100
100
100
100
100
100
100
100
100
100
100
Final Cone.
(Mg/mL)
0.15
0.17
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.11
12.3.2 PAH matrix spike recovery standard, Batch 2A
Analyte
Acenapthene
Acenapthylene
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluroanthene
Benzo(a)pyrene
Benzo(e)pyrene
Benzo(g,h,i)perylene
Chrysene
Coronene
Dibenz(a.h (anthracene
Fluoranthene
Fluorene
Stock Cone.
(Hg/mL)
100
100
100
100
.100
100
100
96.5
100
100
98.2
100
100
100
Stock Amt. (mL)
1.97
1.97
1.97
1.97
1.97
1.97
1.97
1.98
1.97
1.97
1.97
1.97
1.97
1.97
Final Cone.
(jag/mL)
1.97
1.97
1.97
1.97
1.97
1.97
1.97
1.91
1.97
1.97
1.93
1.97
1.97
1.97
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Analysis of PCBs, Pesticides, and PAHs
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IADN Project - Sample Preparation Procedure
Volume 2, Chapter
Analyte
Indeno( 1 ,2,3,cd)pyrene
Naphthalene
Phenanthrene
Pyrene
Retene
Stock Cone.
(|ag/mL)
50
95.24
100
100
158.95
Stock Amt. (mL)
1.97
1.97
1.97
1.97
1.98
Final Cone.
(|jg/mL)
1.97
1.97
1.97
2.0
1.98
Volume was made up to 100 mL with hexane.
12.3.3 PAH internal standard
12.3.4 PAH surrogate standard
d 10 Phenanthrene: 2.13 ug/ mL of hexane
13.0 Safety
13.1 Emergency Numbers
Name
IU Fire Department
Ronald A. Hites
Jer'ferv White
Telephone numbers
911
812-855-0193(0)
812-334-1323 (H)
812-855-1466(0)
812-336-1462 (H)
Compound
d|0anthracene
d,2benzo(a)anthracene
d|2perylene
triphenylmethane
Stock (ug/mL)
1000
1000
2000
136
Mix (mL)
0.2
0.2
0.09
0.99
Final Concentration
(Ug/mL)
4
4
3.60
2.69
Volume was made up to 50 mL with hexane
2-110
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure
13.2 Chemists Numbers
Name
Ilora Basu
Barbara Hillery
James M. O'Dell
Tom Stanko
Mary Tankard
Mike Wassouf
Charles Alan Long
13.3 Working in the Laboratory
Telephone Numbers
812-855-5040(0)
812-855-2926(0)
812-334-2184 (H)
812-855-1005(0)
812-334-4151 (H)
812-855-5040(0)
812-824-7962 (H)
812-855-2926(0)
812-336-8546 (H)
812-855-5035(0)
812-824-1863 (H)
812-855-2926(0)
812-330-1517 (H)
812-855-2926(0)
812-333-9535 (H)
Chemists working in the laboratory should follow certain safety rules :
Individual is required to wear a lab coat whenever working in the lab.
Eye protection with splash resistant safety glasses or safety goggles is required. Contact
lens is forbidden.
Protective gloves should be used while handling samples or standards. Special solvent
resistant gloves should be used while handling large amount of solvents.
All solvent work should be done inside fume hood.
Open shoes are not allowed in the laboratory.
Particle mask is required when using dry silica.
Generally nobody should work alone in the laboratory. If work must be performed after
hours or in the weekend inform supervisor or other lab mates so that your presence is
known and will be accounted for in case of an emergency.
Chemicals and solvents are stored in separate storage area. One week's supply is kept in
the lab. Solvents are stored in special solvent cabinet. Acids must be separated from
bases. A rubber bucket needs to be used to carry any chemical.
Gas cylinders should be well secured at all times. Flammable gases are stored in separate
cage.
Wash your hands well after work. Protective hand cream "Soft guard" is supplied.
No food or drink is allowed in the laboratory.
3)
4)
5)
6)
7)
8)
9)
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure Volume 2, Chapter^
12) In case of minor spillage, get spillage kit to clean the area. A major spill requires the
University Health and Safety Division to be contacted and the working area evacuated.
13) MSDS are filed in a three ring binder.
14) All chemicals and standard should be labeled properly with scientific name, date, and
initials of person to contact.
15) Empty chemical bottles should be flushed out with water, or, in case of liquid, allowed to
evaporate under a hood before discarding.
13.4 Safety Equipment
13.4.1 Fume hood
IADN sample preparation requires frequent use of solvent. Therefore, all extraction,
column chromatography, standard preparation, sample transfer, Nitrogen blow down and
preparation of microvials should be done in the hood. It is real important to check hood
from time to time to ensure that it is working properly. A flow of 80-120 linear feet per
second must cross the hood.
13.4.2 Safety showers
Emergency showers are located in strategic areas of the laboratory to provide to provide
immediate emergency protection against fire or chemical injury. It is operated by pulling
the hanging ring down. It delivers 30 gallons of water per minute.
13.4.3 Eye Wash
Emergency eye wash is located in the laboratory. It is operated by pushing the lever
backward.
13.5 Waste Disposal
13.5.1 Solvents
1) Label 2 containers, 'CHLORINATED WASTE' and 'NON-CHLORINATED
WASTE'.
2). Containers may be empty glass bottles from solvents or poly jericans (10 L or
less).
3) When in use they are to be placed inside a fume hood with the sash pulled down.
4) University Health and Safety Department will pick up the waste solvent on
Friday. Label the bottle properly and sign it.
13.5.2 Silica
After solvent has evaporated, pour silica into a separate bottle. When the bottle is full
label it. University Health and Safety will pick it up together with the waste solvent.
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
Volume 2, Chapter 1 lADN Project - Sample Preparation Procedure
13.5.3 Teflon boiling chips
Place in waste container (i.e., beaker) under hood until solvent evaporates, then empty into
trash can.
13.5.4 Glass
Place in 'Broken Glass Disposal Containers'. When containers are full, close according to
directions on box; leave for janitors to pick-up or take out to the trash dumpster.
13.5.5 Foil
Place in trash can.
13.5.6 Fiberglass
Place in waste container (i.e., beaker) under hood until solvent evaporates, then empty into
trash can.
13.5.7. XAD,andQF
Leave in soxhlet under hood until solvent has evaporated. Pour XAD-, into container
labeled 'USED XAD2. Discard QF into trash can.
14.0 References
Following publications were consulted for the development of methods of PCB, pesticides and
PAHs analysis in Air and Precipitation samples. Experimental procedure was modified according
to our need.
Baker, J.E.; Eisenreich, S.J. PCBs and PAHs as Tracers of Particulate Dynamics in Large Lakes.
J. Great Lakes Res.. 1989, 15(1),84-103.
Bidleman, T.F.; Mathews, J.R.; Olney, C.E.; and Rice, C.P Separation of Polychlorinated
Biphenyl, Chlordane and p-p DDT from Toxaphene by silicic acid column chromatography.
J. Ass, off, analvt. chem.. 1978, 61, 820-828.
Hermanson, M.H. and Hites, R.A. Long-Term Measurement of Atmospheric Polychlorinated
Biphenyls in the Vicinity of Superfund dumps. Environ. Sci. Technol.. 1989, 23. No. 10,
1253-1258.
Marti. E.A. Armstrong D.E. Polychlorinated Biphenyls In Lake Michigan Tributaries.
J. Great Lakes Res.. 1990, 16(3): 396-405
Me Veety, B.D. and Hites. R.A. Atmospheric Deposition of Polycychc Aromatic Hydrocarbons to
Water Surfaces: A Mass Balance Approach. Atmos. Environ.. 198S. 22. 51 1-536.
2-113
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Analysis of PCBs, Pesticides, and PAHs
in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure Volume 2, Chapter 1
Mullin. M.D. PCB Workshop, U.S. EPA Large Lakes Research Station. Grosse He. MI,
June 1985.
Murphy. T. J. and Rzeszutko, C.P. Precipitation inputs of PCBs to Lake Michigan. J. Great Lakes
Res.. December 1977. Internal. Assoc. Great Lakes Res.. 3(3-4): 305-312.
Swackhamer, D.L.; Me Veety, B.D.; and Hites R.A. Deposition and Evaporation of
Polychlorinated Biphenyl congeners to and from Siskiwit Lake, Isle Royale. Lake Superior.
Environ. Sci. Technol.. 1988, 22, 664-672.
Sweet. C.W.; Vermette, S.J.; and Gatz, D.F. Atmospheric Deposition of Toxic Materials:
A Compound of the Green Bay Mass Balance Study. 1992, Contract Report 530, Illinois State
Water Survey, Champaign, IL 61820.
Personal communication with:
Hites, R.A. and his group from Indiana University, 1990-1993
Eisenreich S.J. and his group, 1990-1993
Swackhamer, D.L., 1990-1993
2-114
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Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
Karen Harlin and Kaye Surratt
Illinois State Water Survey
Office of Atmospheric Chemistry
2204 Griffith Drive
Champaign, IL 61820
SOP#CH-PR-001.3
March 1995
Revision 3.0
-------
Analysis of PCBs, Pesticides, and PAHs in Air and
Precipitation Samples:
Sample Preparation Procedures
SOP #CH-PR-001.3, Revision 3.0
1.0 Scope and Applications
1.1 This procedure details the sample preparation methods utilized at the ISWS, Office of
Atmospheric Chemistry, Trace Organic Toxicants Lab as applied to the Lake Michigan Mass
Balance (LMMB) and Lake Michigan Loading Study (LMLS) projects. The procedures apply to
XAD-2 cartridge, filter, and XAD-2 precipitation samples. The following analytes are measured
by this SOP:
Polychlorinated Biphenyls (PCBs) - Total and 105 congener peaks
congener (BZ)
1
3
4+10
6
7+9
8+5
12
13
15+17
16
18
19
21
">->
27
25
24
CAS#
205 1 -60-7
2051-62-9
13029-08-8,
33146-45-1
25569-80-6
33284-50-3,
34883-39-1
34883-43-7,
16605-91-7
2974-92-7
2974-90-5
2050-68-2, 37680-66-3
28444-78-9
37680-65-2
39444-73-4
55702-46-0
38444-85-8
38444-76-7
55712-37-3
55702-45-9
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures
Volume 2, Chapter 1
Polychlorinated Biphenyls (PCBs) Continued
congener (BZ)
26
29
31+28
32
33
37
40
41+71
42
43
44
45
46
47
48
49
51
52
53
56+60
63
64
66
70+76
74
77
81
82
CAS# •
38444-81-4
15862-07-4
16606-02-3,
7012-37-5
38444-77-8
38444-86-9
38444-90-5
8444-93-8
52663-59-9,
41464-46-4
36559-22-5
70362-46-8
41464-39-5
70362-45-7
41464-47-5
2437-79-8
70362-47-9
41464-40-8
68194-04-7
35693-99-3
41464-41-9
41464-43-1,
33025-41-1
74472-34-7
52663-58-8
32598-10-0
32598-11-1,
70362-48-0
32690-93-0
32598-13-3
70362-50-4
52663-62-4
2-118
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
Polychlorinated Biphenyls (PCBs) Continued
congener (BZ)
83
87
89
91
92+84
95
97
99
100
101
107
110
114+131
118
119
123+149
128
129
130
132+153+105
134
135+144
136
137+176
141
146
151
156
157+200
158
163+138
CAS#
60145-20-2
38380-02-8
73575-57-2
68194-05-8
52663-61-3,52663-60-2
38379-99-6
41464-51-1
38380-01-7
39485-83-1
37680-73-2
70424-68-9
38380-03-9
74472-37-0,61798-70-7
31508-00-6
56558-17-9
65510-44-3,38380-04-0
38380-07-3
55215-18-4
52663-66-8
38380-05-1,35065-27-1,
32598-14-4
52704-70-8
52744-13-5,68194-14-9
38411-22-2
35694-06-5, 52663-65-7
52712-04-6
51908-16-8
52663-63-5
38380-08-4
69782-90-7, 52663-73-7
74472-42-7
74472-44-9, 35065-28-2
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures
Volume 2, Chapter 1
Polychlorinated Biphenyls (PCBs) Continued
congener (BZ)
167
170+190
174
175
177
178
180
183
185
201
202+171
196
203
205
206
207
208+195
209
CAS#
52663-72-6
35065-30-6,
41411-64-7
38411-25-5
40186-70-7
52663-70-4
52663-67-9
35065-29-3
52663-69-1
52712-05-7
40186-71-8
2136-99-4,52663-71-5
42740-50-1
52663-76-0
4472-53-0
40186-72-9
52663-79-3
52663-77-1,
52663-78-2
2051-24-3
Pesticide
atrazine
desethylatrazine (DEA)
desisopropylatrazine (DIA)
dieldrin
a-chlordane
g-chlordane
t-nonachlor
a-hexachlorocyclohexane
(a-HCH)
g-hexachlorocyclohexane
(g-HCH)
hexachlorobenzene (HCB)
p'p'-DDD
p.p'-DDH
p.p'-DDT
CAS#
1912-24-9
6190-65-4
1007-28-9
60-57-1
5103-71-9
5103-74-2
39765-80-5
319-84-6
58-89-9
118-74-1
72-54-8
72-55-9
50-29-3
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
1.2
1.3
Polycyclic Aromatic
Hydrocarbons (PAHs)
acenaphthene
acenaphthylene
anthracene
benzo(a)anthracene
benzo(a)pyrene
benzo(b)fluoranthene
benzo(e)pyrene
benzo(ghi)perylene
benzo(k)fluoranthene
chrysene
coronene
dibenzo(a,h)anthracene
fluoranthene
fluorene
indeno( 1 23cd)pyrene
phenanthrene
pyrene
retene
CAS#
83-32-9
208-96-8
120-12-7
56-55-3
50-32-8
205-99-2
192-97-2
191-24-2
207-08-9
218-01-9
191-07-1
53-70-3
206-44-0
86-73-7
193-39-5
85-01-8
129-00-0
483-65-8
Method detection limits (MDL) are defined in CFR, Vol 49, No. 209, October 26, 1984,
Appendix B to Part 136. Matrix specific MDLs are determined by spiking 7-10 clean matrix
samples with the analytes of interest and processing them through the entire extraction, cleanup,
and analysis procedure.
The instrument detection limit (IDL) refers to the smallest signal above background noise that an
instrument can reliably detect. The IDL is determined from a data set comprised of three separate
chromatographic runs of a low level calibration standard; each run contains 7-10 analyses of the
standard. The IDL equals the Student's t value (n-1) multiplied by the standard deviation of this
data set.
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Sample Preparation Procedures
Volume 2, Chapter^
1.4 Method Flow Diagram
hi-volume sampler
precipitation sampler
_L
particle: QF filter
vapor: XAD-2 resin
precipitation: XAD-2 resin
spike sample with surrogate
samples
(PCBs 65, 166)
2,4,7-trichloro-9-fluorenone
benzo(a)pyrene-d-|2, atrazine-d5
extract and concentrate
cleanup and fractionation
Fraction 1
(hexane)
Fraction 2
(CH2CI2: hexane 40:60)
concentrate
I
concentrate
add PCB internal
standards
(PCBs 30 + 204)
add pesticide internal standards
(PCB65, PCB155,p,p'DDE)
GC-ECD
(PCBs, HCB, p,p' DDE)
GC-ECD
(a-HCH, g-HCH, dieldrin,
p,p' ODD, p,p' DDT, a-chlordane,
g-chlordane, trans-nonachlor)
add internal standards
(dig-anthracene,
triphenylmethane,
di2-benzo(a)anthracene,
acenaphthylene
acenaphthene
fluorene
phenanthrene
anthracene
fluoranthene
pyrene
retene
benzo(a)anthracene
_L
Fraction 3
(Methanol)
add internal standards
(d-|Q-anthracene,
triphenylmethane,
d-| 2-benzo(a)anthracene,
d-|2-perylene)
_L
GC-MS for atrazine
desethylatrazine,
desisopropylatrazine
GC-MS for PAHS
chrysene
benzo(b)fluoranthene
benzo(k)fluoranthene
benzo(e)pyrene
benzo(a)pyrene
ideno(123cd)pyrene
dibenzo(ah)anthracene
benzo(ghi)perylene
coronene
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
2.0 Summary of Method
The analytes are extracted from XAD-2 resin or filter samples by Soxhlet extraction with hexane:
acetone (50:50) followed by concentration by rotary evaporation. After extraction, interfering
compounds are removed and analytes separated into different fractions with silica gel
(3% deactivated). The first fraction (hexane) contains all PCBs and the pesticides HCB and DDE.
The second fraction (40% DCM, 60% hexane) contains all PAHs and pesticides a and y HCHs,
dieldrin, DDD, DDT, y chlordane, a chlordane, and t-nonachlor. Fraction three (methanol)
contains atrazine and two metabolites (DEA, DIA). The samples are then concentrated to the
desired volume with a slow stream of ultra-pure nitrogen. Final volumes depend on sample
matrix, site, and date. Each sample is spiked with a known amount of internal standard.
Subsamples are then transferred to autosampler microvials for capillary GC-ECD or GC-Ion Trap
MS analysis.
3.0 Definitions
3.1 Internal Standard (IS) - A pure analyte(s) added to a sample extract, or standard solution in
known amount(s) and used to measure the relative responses of other method analytes and
surrogates that are components of the same solution.
3.2 Surrogate Analvte (SA) — A pure analyte(s), which is extremely unlikely to be found in any
sample, and which is added to a sample aliquot in known amount(s) before extraction or other
processing, and is measured with the same procedures used to measure other sample components.
The purpose of the SA is to monitor method performance with each sample.
3.3 All other terms are defined in the QAPjP, Revision 5, July 1995.
4.0 Interferences
Method interferences may be caused by contaminants in solvents, the sampling matrix, reagents,
glassware, and other sample processing apparatus that lead to anomalous peaks or elevated
baselines in gas chromatograms. Laboratory equipment and reagents will be monitored by the
inclusion of quality control samples with each batch of samples prepared. Individual samples may
contain interferences which will require additional sample preparations. All sample preparation
details will be documented.
5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been
precisely defined. However, each one must be treated as a potential health hazard, and exposure to
these chemicals should be minimized. Some method analytes have been tentatively classified as
known or suspected human or mammalian carcinogens. Pure standard materials and stock
standard solutions of these compounds should be handled with suitable protection to skin,
eyes. etc.
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Sample Preparation Procedures Volume 2, Chapter^
5.2 Chemists working in the laboratory should follow ISWS safety rules :
5.2.1 A lab coat is required when working in the lab.
5.2.2 Eye protection with splash resistant safety glasses or safety goggles are required.
5.2.3 Protective gloves should be used while handling samples or standards. Special solvent
resistant gloves should be used while handling large amount of solvents.
5.2.4 All solvent work should be done in fume hoods.
5.2.5 Open shoes are not allowed in the laboratory.
5.2.6 Particle mask is required when using dry silica.
5.2.7 Avoid working alone in the laboratory. If work must be performed after hours or in the
weekend inform the supervisor or other staff so that your presence is known and will be
accounted for in case of an emergency.
5.2.8 Chemicals and solvents are stored under the hoods. Acids must be separated from bases.
A rubber bucket is required to transport any chemical.
5.2.9 Gas cylinders should be well secured at all times. Flammable gases are stored in separate
storage areas.
5.2.10 Wash hands well after work.
5.2.11 No food or drink is allowed in the laboratory.
5.2.12 In case of minor spillage, get spillage kit to clean the area. A major spill requires the
University of Illinois Fire Department to be contacted and the working area evacuated.
5.2.13 MSDS sheets are stored in the laboratory and a copy placed on file with the office
administrator.
5.2.14 All chemicals and standards must be labeled with chemical name, date, and initials of
person to contact.
5.2.15 Empty chemical bottles should be flushed out with water, or, in case of liquid, allowed to
evaporate under a hood before discarding.
5.3 Waste disposal
5.3.1 Solvents
Label waste containers. Chlorinated Waste and Non-Chlorinated Waste. Glass bottles
used for waste are placed under hoods for convenience. When full, transfer waste to 10 L
currun containers in solvent cabinet. Contact the ISWS Waste Coordinator for removal.
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Analysis of PCBs, Pesticides, and PAHs in
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Volume 2, Chapter 1 Sample Preparation Procedures
5.3.2 Silica
After solvent has evaporated, pour silica into a disposable glove and discard.
5.3.3 Teflon Boiling Chips
Allow solvent to evaporate then discard.
5.3.4 Glass
Place in 'Broken Glass Disposal Containers'. When containers are full, close according to
directions on box and discard per university instructions.
5.3.5 Aluminum Foil
Recycle
5.3.6 Glass Wool
Allow solvent to evaporate then discard.
5.3.7 XAD-2 and Filters
Leave XAD from air sample in Soxhlet under hood until solvent has evaporated then pour
into container labeled Used XAD-2. Allow filter to dry in hood before discarding. XAD-
2 (precip) is discarded.
6.0 Equipment and Supplies
6.1 Glassware -- General requirements
All glassware must be meticulously cleaned. Large glassware is thoroughly washed with
laboratory detergent and hot water. Glassware with bad stains should be rinsed with MeOH or
CH2CI: before using the soap and water procedure. If still not clean, soak in H:SO4:HNO, (50:50)
acid bath overnight, then wash thoroughly with soap and water. Volumetric pipettes used for
standards must soak in acid bath overnight. Glassware is thoroughly rinsed with tap water, then
with DI water and allowed to air dry. The glassware is foil wrapped and heated 450 "C for four
hours. If glassware is not clean after muffling at 450°C for four hours, muffle at 500 JC for four
hours. The glassware is cooled to ambient temperature and stored in a clean location.
Small glassware such as stoppers, vials, and disposables are wrapped in foil or placed into a
beaker and covered with foil and heated to 450"C for four hours, cooled to ambient temperature,
and stored in a clean location. Vials are capped as soon as they are removed from the oven. Note:
Always use dull side of foil towards glassware. Set initial temperature of furnace to 200"C if
possible.
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Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures Volume 2, Chapter j
6.2 Sample vials and bottles -- amber glass vials with Teflon-lined screw caps. Amber colored glass is
used for extract and standard storage since some of the method analytes are sensitive to light.
6.3 Volumetric flasks -- Various sizes.
6.4 Volumetric and graduated pipets -- Various sizes.
6.5 Forceps, spatulas, scissors and other stainless steel laboratory supplies. Metal supplies are washed
with soap and water, rinsed with tap water, then rinsed with DI water and allowed to air dry. They
are then rinsed with CH2C1:, wrapped in foil, and stored in a clean location.
Note: Always rinse with CH2C12 immediately before use.
6.6 Micropipettor (Drummond or equiv.), glass capillaries -- Various sizes. Preclean capillaries with
CH2C12 prior to use.
6.7 Teflon stopcocks and Teflon lined caps. Stopcocks and caps are washed with laboratory detergent
and hot water, rinsed with tap then DI water, and air dried on kimwipes. The stopcocks are stored
in a clean jar or beaker and covered with foil. Caps are rinsed with hexane, air dried, then placed
onto their associated bottle or vial.
6.8 Cork Rings
6.9 Muffle Oven
6.10 Drying Oven
6.11 Soxhlet extraction apparatus, glassware and heating assembly.
6.12 Desiccator
6.13 Analytical and top loading balances with check weights.
6.14 Rotary evaporator with aspirator pump and chiller circulator
6.15 Separator}' funnel, various sizes
6.16 Round bottom flask and stoppers, various sizes
6.17 Chromatography column for silica cleanup - large columns for cartridge samples are 1 I x 300 mm
(Kimax # 178001 1300) with removable PTFE stopcocks, replaceable large bore elass tips, and
100 mL reservoirs. Small columns for rain and filters samples are 11 x 300 mm
(Kimax # 4205300214) with size 2 PTFE stopcock plug. A 22 x 155 mm joint (# 668500-1922) is
added to the top of the column.
6.18 Misc. lab supplies including: Pasteur pipets. beakers, funnels, pipet bulbs, glass rods, rubber
hammer
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Analysis of PCBs, Pesticides, and PAHs in
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Volume 2, Chapter 1 Sample Preparation Procedures
6.19 Ultrasonic bath
6.20 Pierce Reactitherm, Model 18800, with stainless steel needles
6.21 Autosampler vials, caps and inserts
7.0 Reagents and Standards
7.1 Solvents - Pesticide quality or equivalent
Methylene Chloride (CH2C12, DCM)
Methanol (MeOH)
Acetone
Hexane
7.2 Reagents - Residue grade or equivalent
7.2.1 Organic free DI water
7.2.2 Glass Wool -- Cut glass wool into 2" pieces, place into a muffled beaker and cover with
foil. Muffle at 450°C for four hours. Store in a clean location.
7.2.3 Teflon Boiling Chips — Boiling chip cleanup requires a Soxhlet extraction step as follows:
Thoroughly rinse inside of condenser and outside of joint with methanol then CH:C1: from
wash bottles. Cover joint and exhaust tube with foil.
Add five or six boiling chips to flask. Add appropriate amount of CFLC1, to flask.
Place glasswool plug at the siphon tube opening of the Soxhlet extractor using large
tweezers.
Place new teflon boiling chips in Soxhlet extractor.
Assemble flask/Soxhlet apparatus.
Turn on heater to give proper boiling (set variac to 40-45).
Turn on chilled water for condenser.
Extract for 18 to 24 hours.
Turn heat off; let cool 15 to 30 minutes.
Turn off condenser water.
Drain as much solvent from Soxhlet as possible.
Remove boiling chips from Soxhlet and place in a 1 L beaker, cover loosely with foil.
Place boiling chips in a 70°C oven:
Every 10 to 15 minutes, shake beaker to accelerate solvent evaporation.
Let boiling chips remain in oven two to four hours, until dry.
***Wcirnint>: Beware of Solvent Fumes. ***
Place boiling chips in clean sample jar; cover with foil and lid.
Store on shelf.
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures Volume 2, Chapter^
7.2.4 Sodium Sulfate (Na,SO^) - Place Na.SO., into a clean muffled beaker and heat to 450°C
for four hours or overnight, cool to 100°C in the oven. Place into a clean sample jar,
cover with foil and cap. Store in a desiccator. If not used within two weeks, recondition in
a 100°C drying oven overnight. Remove from oven; cover with foil and cap then store in
desiccator.
7.2.5 Silica Gel (Davisil, grade 634, 100-200 mesh or equivalent)
7.3 Standards
7.3.1 PCB Stock Standards
7.3.1.1 LMMB PCB Intermediate PCB Stock Standard (provided by Mullin June 1994).
Stock solution provided in sealed ampules with a total congener concentration of
183 ug/mL. (Aroclor 1232 = 75 ug/mL, Aroclor 1248 = 54 ug/mL, Aroclor
1262 = 54 ug/mL). Per M. Mullin the PCB concentration of the intermediate
PCB Stock Std is 170.8 ug/mL due to the presence of biphenyl and other
components in the mix.
7.3.1.2 Custom PCB Standard (Ultra Scientific #CUS-937) for Method Detection Limit
studies (provided by US EPA July 1994). Stock solution provided in sealed
ampules with a certificate of analysis as follows: Lot # 0858
PCB 1 = 12.00 ug/mL
PCB 6= 14.20 ug/mL
PCB 29 = 6.30 ug/mL
PCB 49 = 5.86 ug/mL
PCB 101 =4.93 ug/mL
PCB 141 =2.19 ug/mL
PCB 180 = 2.21 ug/mL
PCB 194= 1.69 ug/mL
PCB 206 = 2.05 ug/mL
PCB 209 = 1.36 ug/mL
7.3.1.3 2-chlorobiphenyl (PCB 1), Ultra Scientific (RPC-0069) Stock Standard for
enhancement of PCB 1 in the Ultra CUS-937 mix described above. Stock
Solution provided by EPA in a sealed ampule with a concentration for PCB 1 =
100 ug/mL (Lot No. H 0039)
7.3.2 Pesticide and PAH Stock Standards
Stock standard solutions are purchased from commercial sources (Ultra Scientific.
Accustandard, Chem Service, Cresent Chemical) or are obtained from the USEPA
repository. When stock solutions are not available, pesticides are purchased as the neat
material and gravimetrically prepared in house.
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Volume 2, Chapter 1
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
7.3.3 Surrogate Standard Solutions
7.3.3.1 Surrogate standards are purchased from commercial sources (Aldrich Chemical,
Cambridge Isotope) or are obtained from the USEPA repository. The following
surrogate standards are utilized.
PCB 65 (PCB, HCB, DDE surrogate)
PCB 166 (PCB surrogate)
2,4,7-trichloro-9-fluorenone (pesticide surrogate)
atrazine-d5 (atrazine surrogate)
benzo-(a)pyrene-d!2 (PAH surrogate)
7.3.3.2 If stock solutions are not commercially available, they are gravimetrically
prepared from the neat material. Individual stock solutions are serially diluted in
volumetric flasks to obtain the surrogate spike standard/s. A combined surrogate
spiking standard may be prepared to save sample preparation time during the
extraction procedure.
7.3.3.3 All samples are spiked with surrogate standards prior to extraction using
volumetric pipets or a Drummond pipet and the spike volumes recorded on the
sample preparation log.
7.3.4 Internal Standard Solutions (ISTDs)
7.3.4.1 Internal Standards are purchased commercially (Ultra Scientific) as a stock
standard or as the neat material. The following ISTDs are utilized.
PCB 30 (PCB ISTD)
PCB 204 (PCB ISTD)
PCB 65 (Pesticide ISTD)
PCB 155 (Pesticide ISTD)
DDE (Pesticide ISTD)
anthracene-dlO (PAH and atrazine ISTD)
benzo(a)anthracene-d!2 (PAH ISTD)
perylene-d!2(PAHISTD)
triphenylmethane (PAH ISTD)
7.3.4.2 If stock solutions are not commercially available, they are gravimetrically
prepared from the neat material. Individual stock solutions are serially diluted in
volumeteric flasks to obtain the ISTD spiking standard/s.
7.3.4.3 ISTDs are added to the appropriate sample fraction (PCBs in hexane, pesticides
and PAHs in 40% DCM, and atrazine in MeOH) prior to GC-ECD or GC-MS
analysis. A Drummond micropipet is used for ISTD addition.
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Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures Volume 2, Chapter_r
7.3.5 Chromatographic Calibration Standards
Combined instrument calibration standards are prepared from the individual stock
standards by volumetric dilution to obtain five concentration levels. The calibration
standard concentrations bracket the expected analyte amounts in samples assayed and are
within the working linear range of the detectors. Calibration mixes are prepared
specifically for the appropriate instrument and fraction analyzed. The following
calibration mixes are prepared.
PCBs, DDE, HCB in the hexane fraction, with surrogate and ISTDs
chlorinated pesticides in the 40% DCM fraction, with surrogate and ISTDs
PAHs in the 40% DCM fraction, with surrogate and ISTDs
atrazine, DEA, and DIA in the MeOH fraction, with surrogate and ISTDs.
7.3.6 Matrix Spiking Solutions
7.3.6.1 Combined matrix spiking solutions are prepared from the individual stock
standards by volumetric dilution. Combined matrix spike solutions are prepared
for each analyte group. The following matrix spike mixes are prepared.
PCBs
chlorinated pesticides
PAHs
atrazine, DEA, DIA
7.3.6.2 The matrix spike solutions will be added to clean sample matrix material prior to
extraction to calculate the recovery of individual analytes. One matrix spike will
be extracted with each batch of samples. The matrix spike will be added to the
sample using a Drummond micropipet or a volumetric pipet and the spiking
amounts reported in the sample preparation log.
7.3.7 Standard Evaluation
New working standards will be assayed prior to use by comparison with existing
standards. Standards must agree within 10% prior to use.
8.0 Sample Collection, Preservation and Storage
8.1 Sample collection, storage, and storage limits are defined in the QAPjP, Revision 5.0, Jul\ 1995.
8.2 Extracts are stored in amber vials at -10 to -20 3C before and after GC analyses.
9.0 Quality Control/Quality Assurance (QC/QA)
9.1 QA/QC requirements are described in the QAPjP. Revision 5.0. Julv 1995.
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Analysis of PCBs, Pesticides, and PAHs in
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Volume 2, Chapter 1 Sample Preparation Procedures
9.2 Quality control samples include: field and laboratory blanks, laboratory matrix spikes, laboratory
surrogate spikes, and field and laboratory duplicate samples. The laboratory maintains all sample
preparation and data records to document the quality of the data generated.
10.0 Calibration
All instrument calibration and analysis are detailed in the following ISWS SOPs:
Standard Operating Procedure for the Analysis of PAHs and Atrazine by GC/Ion Trap MS,
July 1995.
Standard Operating Procedure for the Analysis of PCBs and Organochlorine Pesticides b\
GC-ECD, Revision 3.0, November 1995.
11.0 Filter and XAD-2 Resin Precleaning Procedure
11.1 XAD-2 Resin
11.1.1 The following supplies are required:
Soxhlet extractor and condenser custom made by Crown Glass Company
six 3 L round bottom flasks with 29/42 joint
six glass stoppers (29/42 joint)
one 1L beaker
two 400 mL beakers
heating mantle for 3 L flask
variable autotransformer
11.1.2 Procedure for vapor sample cartridges
Day 1
1) Place approximately 1 kg XAD-2 in extractor plugged with glass wool.
2) Rinse XAD-2 with tap water many times, stirring to remove foam and small
particles. Use kimwipes to remove foam.
3) Rinse with a small amount of methanol three times to remove water.
4) Add 2000 mL of methanol to 3 L flask.
5) Add about 20 boiling chips to flask.
6) Assemble flask/Soxhlet/condenser apparatus.
7) Turn on heater to give proper boiling (set variac to 60-65 for methanol).
8) Turn on chilled water for condenser.
9) Co\er Soxhlet and flask with foil.
10) Extract with methanol for 24 hours.
Day 2
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much methanol from Soxhlet as possible.
3) Add 2000 mL acetone to 3 L flask.
4) Add about 20 boiling chips to flask.
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures Volume 2, Chapter
5) Turn on heater (set variac to 45 for acetone).
6) Cover Soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 3
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from Soxhlet as possible.
3) Add 2000 mL hexane to 3 L flask.
4) Add five or six boiling chips to flask.
5) Turn on heater (set variac to 40-45 for hexane).
6) Cover Soxhlet and flask with foil.
7) Extract with hexane for 24 hours.
Day 4
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from Soxhlet as possible.
3) Add 2000 mL CH2C12 to 3 L flask.
4) Add five or six boiling chips to flask.
5) Turn on heater (set variac to 40-50 for CH2CU).
6) Cover Soxhlet and flask with foil.
7) Extract with CH2CI2 for 24 hours.
Day 5
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much CH2C12 from Soxhlet as possible. Wait 15 minutes. Drain as
much solvent as possible through stopcock (remove stopcock if necessary).
3) Add 300 mL hexane to the Soxhlet. Wait 15 minutes, then drain through
stopcock. Repeat at least three times, until the level of the solvent in the siphon
tube is the same as in the Soxhlet.
4) Add 2000 mL hexane to 3 L flask.
5) Add five or six boiling chips to flask.
6) Turn on heater (set variac to 40-45 for hexane).
7) Cover Soxhlet and flask with foil.
8) Extract with hexane for 24 hours. Flushing may need to be induced twice before
it flushes on its own.
Day 6
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from Soxhlet as possible.
3) Add 2000 mL 50% acetone/50% hexane to 3 L flask.
4) Add five or six boiling chips to flask.
5) Turn on heater (set variac to 40-45 for ucetone/hexane).
6) Cover Soxhlet and flask with foil.
7) Extract with acetone/hexane for 24 hours.
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Du\ 7
I) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone/hexane from Soxhlet as possible.
3) Pour XAD-2 in a beaker and dry overnight in 65°C oven.
4) Store in amber bottle in freezer at -20°C for up to three months.
5) Keep subsample in separate jar for use in preparation of lab blank and matrix
spike.
11.1.3 Procedure for precipitation sample cartridges
Day 1
1) Place XAD-2 in Soxhlet plugged with glass wool.
2) Rinse XAD-2 with water many times, stirring to remove foam and small particles.
Use kimwipes to remove foam.
3) Rinse with small amount of methanol 3 times to remove water.
4) Add 2000 mL methanol to 3 L flask.
5) Add about 20 boiling chips to flask.
6) Assemble flask/Soxhlet/condenser apparatus.
7) Turn on heater to give proper boiling (set variac at 60-65 for methanol).
8) Turn on chilled water for condenser.
9) Cover Soxhlet and flask with foil.
10) Extract for 24 hours.
Day 2
\) Turn off heater; cool 15 to 30 minutes.
2) Flush as much methanol from Soxhlet as possible.
3) Add 2000 mL acetone to 3 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-45 for acetone).
6) Cover Soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 3
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from Soxhlet as possible.
3) Add 2000 mL hexane to 3 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 40-45 for hexane).
6) Cover Soxhlet and flask with foil.
7) Extract with hexane for 24 hours.
Day 4
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from Soxhlet as possible.
3) Add 2000 mL CH:CI: to 3 L flask.
4) Add about 20 boiling chips to flask.
5} Turn on heater (set variac to 40 tor CTI.C1-,).
6) Cover Soxhlet and flask with foil.
7) Extract with CFKCK for 24 hours.
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Day 5
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much CH2C12 from Soxhlet as possible. Wait 15 minutes. Drain as
much solvent as possible through stopcock (remove stopcock if necessary).
3) Add 300 mL hexane mixture to the Soxhlet. Wait 15 minutes, then drain solvent
through stopcock. Repeat at least three more times, until level of solvent in the
siphon tube is the same as in the Soxhlet.
4) Add 2000 mL hexane to 3 L flask.
5) Add about 20 boiling chips to flask.
6) Turn on heater (set variac at 40-45 for hexane).
7) Cover Soxhlet and flask with foil.
8) Extract with hexane for 24 hours.
Day 6
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much hexane from Soxhlet as possible.
3) Add 2000 mL acetone to 3 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac at 40-45 for acetone).
6) Cover Soxhlet and flask with foil.
7) Extract with acetone for 24 hours.
Day 7
1) Turn off heater; cool 15 to 30 minutes.
2) Flush as much acetone from Soxhlet as possible.
3) Add 2000 mL methanol to 3 L flask.
4) Add about 20 boiling chips to flask.
5) Turn on heater (set variac to 60-65 for methanol).
6) Cover Soxhlet and flask with foil.
7) Extract with methanol for 24 hours.
Day 8
1) Turn off heater; cool 15 to 30 minutes.
2) Turn off condenser water.
3) Flush as much methanol from Soxhlet as possible.
4) Rinse XAD-2 at least three times with organic-free DI water.
5) Store the clean XAD-2 in DI water in amber bottle in the refrigerator at 4 C.
(The resin may be stored in this manner for up to three months.)
6) Vacuum filter about 50 gm of XAD-2 for use in the preparation of lab blank and
matrix spike. Store it in a separate jar at 4;C.
11.1.4 Notes: Settings may vary from autotransformer to autotransformer.
Check that the solvent is boiling properly (nice rolling boil).
Solvent ma> not siphon well. Induce siphoning as many times as possible. Allow extra
extraction time when improper syphoning occurs.
If XAD-2 is re-used after sample extraction, it is not necessary to rinse with DI water
before extracting.
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Sample Preparation Procedures
Extract used XAD with CH:Cl: and acetone:hexane (I: I) for three days each. XAD for
precipitation collection requires an additional extraction in methanol for two days.
11.1.5 Flowchart of XAD-2 Precleaning Procedure
50% acetone/50% hexane
oven dry
extract
24 hours
at65°C
store at -20 JC in amber
bottle
use for vapor sampling
rinse XAD-2 with
DI water to remove
fines
methanol
extract
24
hours
acetone
extract
24
hours
hexane
extract
24
hours
CH,CU
extract
24
hours
hexane
extract
24
hours
exc
wate
use
acetone
extract
24 hours
methanol
extract
24 hours
nange to milh-Q
r; store at 4 C in
imber bottles
for precipitation
sampling
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Volume 2, Chapter 1
11.1.6 Flow Chart of Used XAD, Precleaning Procedure
CH.Cl.-72h
50% acetone/50%
hexane-72h
oven dry at
65°C
methanol-
48h
store at (-) 20°C in
amber bottle
exchange to milli-Q
water; store at 4°C in
amber botte
Air Samples
11.2 Glass fiber (OFF) or Quartz fiber (QFF) Filters
Precipitation Samples
Each filter is wrapped with aluminum foil separately, and muffled to 450°C for four hours. After
returning to ambient temperature, about 25 are wrapped in aluminum foil and stored.
12.0 Sample Preparation - Extraction
12.1 Air Samples, Paniculate and Vapor Phase (Filter and XAD-2 Cartridges)
12.1.1
Samples are extracted as a set. A sample set includes up to 12 samples (including one
duplicate, one field blank, one lab blank, and one matrix spike). The matrix spike is
spiked with known amount of PCBs, Pesticides, PAHs, and atrazine and is used to
calculate the recovery of each analyte for that set. A batch code is assigned to each sample
set based on the date of extraction (YY, MM, DD) and sample matrix, such as 951 107C
(where C = cartridge). All information about the sample set will be recorded on sample
preparation log sheets (see Appendix A & B).
12.1.2 Sample extraction requires the following:
large Soxhlet extractor (55/50 and 24/40 joints)
condenser (55/50 joint)
500 mL round bottom flask (24/40 joint)
glass stopper (24/40 joint)
400 mL beaker
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Remove spiking standards from freezer. Standards must he at ambient temperature
before using. (Ambient temperature is achieved in about two hours.)
Thoroughly rinse inside of condenser and outside of joint with methanol then CFLCK from
wash bottles. Cover joint and exhaust tube with foil.
Assemble supplies and samples under hood and/or utility cart. Label flasks.
Add five to six clean teflon chips into 500 mL round bottom flask.
Pour solvent into round bottom flask: 175 mL of acetone and 175 mL of hexane.
12.1.3 Procedure for compositing XAD-2 samples.
Composite information is included on the Sample Log worksheet. Group the samples to
be composited for each site on the lab cart.
Label a 400mL beaker with the same site, year and month as the individual samples with
"00" in place of the day. eg. VH01C9503QO.
Check and record the balance calibration using a 50g and lOg weight. If off by greater
than 0.1 g, recalibrate.
Tare the labelled beaker.
Weigh out approximately the same amount of each individual sample so the composite
final weight is close to 40g.
Record the initial and final weights of the individual samples as well as the total weight of
the composite sample on the sample prep, sheet.
Immediately cover beaker with aluminum foil.
XAD-2 Procedure:
Place glass wool plug at the siphon tube opening of the Soxhlet extractor using glass or
metal rod.
Carefully pour XAD-2 into Soxhlet extractor. Rinse container with solvent (acetone/
hexane 1:1) to remove all XAD-2; pour solvent rinse into Soxhlet.
Assemble flask/Soxhlet/condenser apparatus. Place on heating mantle.
12.1.4 Procedure for Compositing Filters
Unwrap one filter at a time.
Trim off the black number at the corner with clean scissors.
Use two pairs of blunt tweezers to fold one filter: place in Soxhlet.
Repeat procedure for all filters in composite sample.
Assemble flask/Soxhlet/condenser apparatus. Place on heating mantle.
Rinse tweezers and scissors with CH:C1:.
12.1.5 Surrogate Standard Addition
Using a micropipette dispenser, spike each sample with the PCB, Pesticide, PAH. and
atrazine surrogate standards. Record the amount spiked.
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12.1.6 Matrix Spike (CMS)
Spike sample medium with the PCB, PAH, Pesticide and atrazine matrix standards. Add
surrogate standards used in 12.1.5. Record the amounts added.
12.1.7 Lab Blank (LB)
Spike sample medium with surrogate standards (12.1.5). Record amount added.
12.1.8 Assemble flask/Soxhlet/condenser unit. Place on heating mantle.
Turn on heating mantles: set Staco heating mantles to 45 or the multi-unit extraction
heater to 5. Turn on condenser water. Cover Soxhlet and flask with foil. Extract for
24 hours.
12.1.9 Turn heating mantle off. Let cool 15 to 30 minutes. Siphon off as much solvent from
Soxhlet extractor into flask as possible. Detach the flask and insert stopper. Turn off
condenser water. Store in cool dark place.
Notes: If XAD-2 gets into the flask, see Removing XAD-2 from flask (12.1.10.3). If
condensation is a problem, wrap condensers with foil wrapped insulation or with
kimwipes.
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Sample Preparation Procedures
12.1.9.1 Flow Charts for Air Sample Extraction
Setting-up extraction:
350 mL of acetone/hexane (50:50) in 500 mL
round bottom flask with boiling chips
put sample into Soxhlet. include rinse
spike sample with PCB surrogate standards
turn on heater
turn on condenser water
cover Soxhlet and flask with foil
extract for 24 hours
Taking down extraction:
turn off heater
after '/2 hour, turn off water
siphon off as much solvent as possible
stopper flask and store in cool dark place
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Volume 2, Chapter 1
12.1.10 Rotary Evaporation
12.1.10.1 Fill chamber with DI water. Turn on the chiller circulator.
Set bath temperature:
SOLVENT
hexane
acetone
acetone/hexane
CH.CK
Methanol
TEMPERATURE (°C)
30-32
30-32
30-32
30
40
Rinse joint of steam duct with CH2CU. Attach appropriate splash
guard(s) to steam duct. Clamp each joint. Turn on and check vacuum
system.
12.1.10.2 Evaporation
Remove boiling chips with large forceps. If XAD-2 is in flask, remove it
as described in 12.1.10.3.
Attach flask to splash guard. Clamp joint.
Turn on motor to predetermined rotation speed (usually to the bottom of
the indicator line, or about 50 rpm). Evaporation should begin in
approximately one minute; solvent should not boil.
Evaporate sample down to approximately 2 mL (in a 500 mL round
bottom flask, area of liquid should be about the size of a quarter).
Open stopcock of rotary evaporator to release vacuum.
Detach the flask
If exchanges are necessary, add specified amount of hexane from table
below, then return flask to splash guard and clamp.
If additional exchanges are not necessary, stopper flask. Store flask under
cabinet.
If \ucuum unit get hot, turn on cold tap \\ater and allow it to cycle
through the bath.
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Sample Preparation Procedures
Empty receiving flask into proper waste bottle as needed.
Rinse splash guard with CH-,Cl, before using with a different sample.
Splash guards should be washed and muffled after every set of samples.
After
extraction
After
column
cleanup
Fraction
40%
methanol
Amount
of hexane
to add
75 mL
25 mL
25 mL
#of
exchanges
2
0
I
1
Total # of
rotary
evaporations
3
1
2
3
Final
volume
2-5 mL
1 ml
1 mL
1 mL
12.1.10.3
12.1.10.4
Removing XAD-2 From Flask
Label another 500 mL flask with sample ID. Decant sample from original
flask into clean flask. Hexane rinse the XAD-2 remaining in flask and
add rinse to new flask. Rotary evaporate new flask using above
procedures.
Clean-up
Turn off heater and motor on rotary evaporator. Turn off chiller. Empty
receiving flask into proper waste solvent bottle. Cover steam duct with
foil. Turn off water supply to the vacuum unit, if used.
Precipitation Samples
12.2.1 Sample extraction requires the following:
large Soxhlet extractor (55/50 and 24/40 joints)
condenser (55/50 joint)
500 mL round bottom flask
glass stopper (24/40 joint)
200 mL (or larger) beaker
"> 7
Samples are extracted as a set. A sample set will include approximately 12 samples
(including at least one duplicate sample, one field blank, one lab blank, and one matrix
spike). All information about the sample set will be recorded on the sample preparation
log sheets (see Appendix A & B). An example of a set name is 941 107P (year, month.
day of sample extraction: P = precipitation sample).
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12.2.3 Remove spiking standards from freezer. Standards must be at ambient temperature
before using. (Ambient temperature is achieved in about two hours.)
Thoroughly rinse inside of condenser and outside of joint with methanol then CH2C12 from
wash bottles. Cover joint and exhaust tube with foil.
Assemble supplies and samples under hood and/or utility cart. Label flasks.
Add five to six clean teflon chips into 500 mL round bottom flask.
Measure 175 mL acetone in a beaker.
Place glass wool plug at the siphon tube opening of the Soxhlet extractor using glass or
metal rod. Assemble Soxhlet extractor and flask.
Put XAD-2 sample into Soxhlet extractor. Rinse container with acetone from beaker, add
this and remaining acetone from beaker to Soxhlet.
Add 175 mL hexane to top of Soxhlet.
12.2.4 Surrogate Standard Addition
Using micropipette dispenser, spike each sample with the PCB, Pesticide, PAH and
Atrazine surrogate standards.
12.2.5 Matrix Spike (CMS)
Spike clean XAD-2 with the PCB, Pesticide, PAH, and Atrazine matrix standards. Add
surrogate standards as in 12.2.4 above. Record the amount added.
12.2.6 Lab Blank (LB)
Spike clean matrix with the surrogate standards (12.2.4). Record the amount added.
12.2.7 Assemble flask/Soxhlet/condenser apparatus. Place on heating mantle. Turn on heating
mantles: set Staco heating mantle to 45 or the multi-unit extraction heater to 5. Turn on
condenser water. Cover Soxhlet and flask with foil. Extract for 30 hours.
Note: The sample has water in it, thus it may not siphon on its own the first two or three
times depending on the amount of water present. Induce siphoning until the level of
solvent in the Soxhlet and in the syphon tube are the same.
12.2.8 Turn heating mantle off. Let cool 15 to 30 minutes. Siphon off as much solvent from
Soxhlet extractor into flask as possible. Detach the flask and insert stopper. Turn off
condenser water. Store in cool dark place.
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Sample Preparation Procedures
12.2.9 Flow Chart for the Extraction_of Precipitation Samples
precipitation on XAD-2 in Soxhlet
assemble Soxhlet and flask
add 175 mL acetone on XAD-2 in
Soxhlet
add 175 mL hexane on XAD-2 in
Soxhlet
spike with surrogate standards
turn on heater and condenser water
induce siphoning for the initial three to
four flushes
extract for 30 hours
12.2.10 Rotary Evaporation
12.2.10.1 Fill chamber with DI water.
Turn on the chiller circulator.
Set bath temperature:
SOLVENT
hexane
acetone
acetone/hexane
CH,CI,
methanol
TEMPERATURE (C)
30-32
30-32
30-32
30
40
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Rinse joint of steam duct with CH2C1:. Attach appropriate splash
guard(s) to steam duct. Clamp each joint. Turn on and check vacuum
system.
12.2.10.2 Evaporation
Remove boiling chips with large forceps. If XAD-2 is in flask, remove it
as described in 12.2.10.3. Attach flask to splash guard. Clamp joint.
Turn on motor of rotator to predetermined rotation speed (usually to the
bottom of the indicator line, or about 50 rpm). Turn flask to start rotation.
Evaporation should begin in approximately one minute; solvent should
not boil. Evaporate sample until sample is at half the original volume. If
vacuum unit gets hot, turn on cold tap water and allow it to cycle through
the bath.
Note: If rate of evaporation slows down, do no! continue. There is water
in the sample.
12.2.10.3 Removing XAD-2 from the Flask.
Label another 500 mL flask with the sample ID. Decant sample from
original flask into the clean flask; wash with 10 mL hexane twice. Rotary
evaporate the new flask with rinses until evaporation begins to slow
down.
12.2.10.4 Back Extraction and Solvent Exchanges
Add 75 mL hexane to sample flask. Shake vigorously, let stand at least
20 minutes. Rotary evaporate to a volume of approximately 100 mL or
more if evaporation slows. Transfer entire sample plus 2-15 mL hexane
rinses to a separatory funnel using a pipet. Rinse sample flask with 2-
15 mL acetone and discard. Allow flask to dry. Add 25 mL hexane to
separatory funnel. Add approximately 1 gm Na,SO4. Shake vigorously.
Let stand at least 20 minutes then transfer the upper hexane layer back to
original flask. Back extract the water layer twice more with hexane.
Combine all hexane extracts.
12.2.10.5 Rotary evaporate the combined extract to 2 mL.
12.2.10.6 Clean-up
Turn off heater on rotary evaporator, motor on rotary evaporator, and
chiller. Empty receiving flask into proper waste solvent bottle. Cover
steam duct with foil.
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Sample Preparation Procedures
12.2.10.7
Flow Chart of Rotary Evaporation and Back Extraction
rotary evaporate precipitation extract half way
add 75 mL hexane
shake and wait 20 minutes
rotary evaporate to -100 mL or
more if evaporation slows
Transfer entire sample plus 2-15 mL
hexane rinses to separatory funnel
add 25 mL hexane to separatory funnel
shake and wait 20 minutes
rinse sample flask with 2-15 mL
acetone, discard acetone
remove hexane layer from 1 st extract
add to original sample flask
add 25 mL hexane to separatory funnel
shake and wait 20 minutes
remove hexane layer from 2nd extract
add to original extract
add 25 mL of hexane to separatory funnel
shake and wait 20 minutes
remove hexane layer from 3rd extract
add to original extract
rotary evaporate combined extract to 2 mL
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Volume 2, Chapter 1
13.0 Sample Preparation - Silica Cleanup
13.1 Glassware required:
columns
three 100 mL pear shaped flasks with 14/20 joints
three glass stoppers with 14/20 joints
Pasteur pipettes (9'/z inch)
graduated cylinders: 50, 25, and 10 mL
funnel
100 mL beaker
250 mL beaker OR sample jar (need not be clean)
three 250 mL beakers
50 mL beakers
13.2 Column packing and elution amounts
Item
amount of silica to
activate/deactivate
column size
amount of Na;SO4
elution volume
(hexane)
switching volume
eiution volume
(407c DCM)
DCM
switching volume
elution volume
(methanol)
Air Paniculate
(filter)
4-6 gms
3'/2"
'/2"
23 mL
4mL
23 mL
15
4
23
Air
Vapor
(XAD-2)
8-10 gms
7"
'/a"
50 mL
8mL
50 mL .
30
8
50
Precipitation
(XAD-2)
4-6 gms
31/2"
1 l/2"
23 mL
4mL
23 mL
15
4
23
13.3 Silica gel activation
Place approximate amount of silica needed in a beaker. Cover beaker with foil.
Place beaker in 100°C oven, turn thermostat to 300°C; keep in oven overnight.
Do \<>t Put Silica Into 300 C Oven.'
Turn t>ven temperature down to 100 C:
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Do Not Remove Silica From Oven.
When oven has cooled to 100°C, remove beaker from oven; let cool on counter top until warm
(approximately five to 10 minutes); place in desiccator.
When silica has reached ambient temperature (approximately two hours), deactivate it:
13.4 Silica gel deactivation
Working quickly, weigh out desired amount of silica into a round bottom flask. Stopper flask
immediately after pouring silica.
Add .Wr weight/volume of DI water to silica, using the following equation:
% deactivation mL DI water
100 - % deactivation weight of silica (grri)
Shake Well. Shake flask until all clumps are broken-up.
Store in desiccator overnight for equilibration.
Use deactivated silica in desiccator within three days. Any unused silica may be reused after
re-activating and re-deactivating.
13.5 Preparation and packing of column(s)
Assemble stopcock(s) on column(s).
Stuff glass wool plug (approximately 1 cm) into lower end of the each column with 20" rod.
Measure and mark appropriate distance from top of glass wool plug for silica and Na,SO4 with
information from 13.2.
Clamp column(s) securely onto frame in ventilation hood. Place empty glass container under each
column (100 mL minimum size; it need not be clean).
Close stopcock(s); fill column(s) half full with hexane.
Make a slurry of hexane and deactivated silica. Pour slurry into each column. Do Not Allow
Silica to Dry Out: rinse column and beaker with hexane via Pasteur pipette. (Use of a funnel may
facilitate process.) Open stopcock(s). Tap column(s) with rubber hammer to pack silica. Add
silica/hexane as needed until desired length is loaded.
Cap column(s) with '/z" Na2SO4 for XAD-2 and filter samples, IW Na:SO4 for precipitation
samples.
Wash column(s) with 25 or 50 mL hexane for equilibrium.
When hexane level reaches top of Na^SOj, close stopcock(s) to prevent further dripping. Never
Let Column Run Dry.
If column(s) is/are not going to be used immediately, stopper column(s) and cover tip(s) of
column(s) with foil.
13.6 Cleanup procedure
13.6.1 Label one 100 mL pear-shaped flask for each fraction which is to be collected.
On a cart, assemble pear shaped flasks and remaining supplies.
Place sample flask in front of column.
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Place a 50 or 100 mL beaker in front of sample flask.
Add hexane to 50 or 100 mL beaker; cover with foil (see 13.2 for hexane volume
required).
13.6.2 Column chromatography
13.6.2.1 First Fraction (hexane)
Sonicate sample flask before loading the sample onto the column to
detach the particles adhering to the walls of the flask.
Remove stopper from sample flask. Assemble pipette and rubber bulb;
place pipette in sample flask.
Place fraction #1 (hexane) pear shaped flask under the column.
Load sample into column with Pasteur pipette.
Set drip rate to approximately one drip per second. Add approximately
5 mL hexane to sample flask from the beaker. Swirl solvent in flask.
When sample has drained down to the top of the Na,SO4add the hexane
from the sample flask to the column. Add an additional 5 mL hexane to
the sample flask from the beaker. Swirl solvent in flask.
When solvent has drained down to the top of the Na2SO4add the second
5 mL hexane to the column. Add the remaining hexane from the beaker
to the sample flask. Swirl solvent in sample flask.
When solvent has drained down to the top of the Na:SO4 add the
remaining hexane from the sample flask (If reservoir on top of the column
cannot hold entire amount, add as much as possible, then refill as space
becomes available).
Note: Stagger the timing of the column loadings such that the changing
of the flasks are not concurrent.
13.6.2.2 Second Fraction (40% CH2C12/60% hexane)
While the hexane is eluting (from the first fraction), measure the
hexane/CH:Cl: and put it into the appropriate containers. (See the
following table.) Swirl the hexane/CH2Cl: in the sample flask.
column size (in)
3'/2
7
hexane/CH2Cl: in
beaker (mL)
23
50
switching volume in
flask (mL)
4
L_ §
When the hexane level reaches the top of the Na,SO4 add the switching
volume he\ane/CH:CI: from the sample flask to the column.
Transfer the he\ane/CH2Cl: from the beaker to the sample flask. Swirl
the solvent in the flask.
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13.6.2.3
13.7 Clean-Up
Analysis of PCBs, Pesticides, and PAHs in
Air and Precipitation Samples:
Sample Preparation Procedures
Place the appropriate pear shaped flask (labeled '40%' fraction) next to
the flask under the column.
When the hexane/CH2C!2 level in the column is to the top of the Na2SO4
quickly switch flasks and pour as much of the remaining hexane/CH2Cl2
into the column as possible. Add hexane/CH2C!2 to the column as space
permits.
Continue to monitor the rate of drip (approximately one drip per minute).
Place the pear shaped flask from the first fraction on the supply cart.
Stopper the flask.
DCM column conditioning: While the hexane/CH2CK is dripping,
measure out 15 mL DCM for 3'/2" columns or 30 mL for 7" columns and
put into beaker.
Place a waste jar next to the flask under the column.
When the hexane/CH2Cl, reaches the top of the Na: SO4, add the DCM to
the column and quickly switch the flask and waste jar.
Stopper the second fraction flask and place on the supply cart.
Third Fraction (MeOH)
While the DCM is eluting, measure the methanol and put into the
appropriate containers (See the following table).
column size (in)
3'/2
7
methanol in
large beaker
23
50
switching volume in
small beaker
4
8
When DCM reaches the top of Na,SO4, add the switching volume to the
column.
Place the appropriate flask (labeled "MeOH fraction") next to the waste
jar under the column.
When the methanol level in the column is to the top of the Na:SO4,
quickly switch the flask and waste jar and pour the remaining methanol
onto column.
Open the stopcocks fully so the methanol elutes as fast as possible.
Discard DCM from the waste jars.
Once the column has stopped dripping, remove flask and stopper it.
Remove stopcock from column.
Turn column upside down and secure it with clamps. Place container under column to catch
Na:SO.,and silica.
After column has dried out, use vacuum (air or water) to remove glass wool plug.
Pour silica and Na,S04 into used gloxe or foil before discarding into trash can.
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Volume 2, Chapter 1
13.8 Flow-Chart
3% deactivated silica
silica slurry in hexane
3.5" column (filter, and precipitation)
7" column for cartridge
i" NaSO4 (filter and cartridge)
11/2" NaSO4 for precipitation
equilibrate column with hexane
load sample
elute with hexane:
23 mL for filter
23 mL for precipitation
50 mL for cartridge
1st fraction contains PCBs,
DDE, and HCB
add 40% CH,C1: in hexane switch
volume: 4 mL for filter and precipitaiton
and 8 mL for cartridge
change to a new flask
elute with 40% CH,CI, in hexane:
23 mL for filter
23 for precipitation
50 mL for cartridge
2nd fraction contains a and g HCH,
dieldrin, ODD. DDT. a-chlordane,
g-chlordane. t-nonachlor. and all PAHs.
change flask to waste jar
add CH:C12:
15" mL for filter
15 mL for precipitation
30 mL for cartridge
add methanol switch volume:
4 mL for filter
4 mL for precipitation
8 mL for cartridge
change to new flask
clute with methanol: 23 mL for filter
23 mL for precipitation
50 mL loi Lailndge
3rd fraction contains atrazine,
descth\latra/ine. and
JcMMipropylatrazine
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14.0 Rotary Evaporation after Column Chromatography
14.1 Attach flask to splash guard. Clamp joint.
Turn on motor of rotator to pre-determined rotation speed (usually to the bottom of the indicator
line, or about 50 rpm). Evaporation should begin in approximately one minute; solvent should
not boil.
Evaporate sample down to approximately 2 mL.
Open stopcock of rotary evaporator to release vacuum.
Detach the flask:
If vacuum unit gets hot, turn on cold tap water and allow it to cycle through the bath.
If exchanges are necessary, add specified amount of hexane as listed in 14.2, then return flask to
splash guard and clamp.
If additional exchanges are not necessary, stopper flask and store it under the cabinet.
Empty receiving flask into proper waste bottle as needed.
Rinse splash guard with CH2C12 before using with a different sample. Splash guards should be
washed and muffled after every set of samples.
14.2 Solvent Exchange
after column
chromatography
fraction
hexane
40%
methanol
amount of
hexane
to add
—
25 mL
25 mL
#of
exchanges
0
1
2
total # of rotary
evaporations
1
2
3
final
volume
1 mL
1 mL
1 mL
15.0 Extract Transfer
15.1 Label each 4 mL amber vial with sample ID and fraction ID.
15.2 Using a Pasteur pipette, quantitatively transfer entire sample from flask to amber vial rinsing with
hexane.
Note: Do not add so much solvent as to fill the vial. If it is too full, there is a chance of splashing
at the time of N: blowdown.
Close amber vial tightly, place in vial file, and store in freezer. Label the vial file with sample set
name, type of samples, and site of collection.
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Sample Preparation Procedures Volume 2, Chapter
16.0 N2 Blow Down
16.1 Set-up
Let N, flush the system for approximately five minutes. Ensure the N2 cylinder pressure is >500
psi.
Turn heater on Low, 5.5 setting
Attach clean needle to each nozzle to be used.
16.2 Blow down
Place amber vials in heater block; adjust N2 flow such that there are gentle (barely detectable)
ripples in the vials.
Evaporate samples down to a final volume of 0.5 to 4.0mL according to sample site, matrix, and
season. Document the actual amount on the sample preparation log sheet.
Note: Volumes may be changed as analyte amounts and/or interferences fluctuate.
16.3 Cleanup
Turn off N2.
Replace the nozzle caps.
Soak used needles in MeOH. Sonicate needles with CH2C12 three times prior to reuse.
Store in a foil covered beaker.
17.0 Spiking Samples with ISTD
17.1 Remove ISTDs from freezer; equilibrate to ambient temperature (approximately two hours).
17.2 Clean micropipette
Remove glass tube used to cover plunger.
Rinse plunger with CH2C12. Allow to dry
Without touching glass tubes, insert plunger into new glass tube;
Rinse pipette tube:
1) Draw-up CH2C1: into pipette and discard in solvent waste container.
2) Draw-up hexane into pipette and discard in solvent waste container. Repeat five times.
3) Vortex standard bottle to mix solution.
4) Draw standard into pipette and discard in solvent waste container. Repeat two times.
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Sample Preparation Procedures
17.3 Spike sample vial (as described).
fraction
hexane
40%
40%
MeOH
analyte group
PCBs
OC pesticides
PAHs
atrazine
ISTDs
PCB 30
PCB 204
DDE
PCB 65
PCB 155
anthracene-dlO
benzo(a)anthracene-d 1 2
perylene-d!2
triphenylmethane
anthracene-d 1 0
color code
red
blue
black
black
17.4 Mark each amber vial label with a appropriate color of dot (use a water-proof marker).
17.5 Rinse tip of micropipet with DCM and hexane, replace glass tube used to cover plunger. Store
micropipette.
17.6 Label vial file with the following information (use same color of pen as dots on microvials):
date microvials spiked fraction spiked initials of chemist spiking
17.7 Mix samples vigorously after spiking.
18.0 Preparing Samples for GC Analysis
18.1 Glassware required
disposable GC vials
200 uL disposable inserts
18.2 Label GC vials with sample IDs. In addition, label an extra GC vial for hexane and the
appropriate calibration and performance standards for every set of samples.
Place inserts into the GC vials.
Using a Pasteur pipette, remove approximately 200 uL of each sample and put into the appropriate
vials. (The level of liquid should be at least half the volume of the insert.) Also place 200 uL of
hexane and 200 uL of the appropriate standards into individual vials (See following chart for the
appropriate standard).
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Volume 2, Chapter 1
fraction
hexane
40%
MeOH
40%
target
compounds
PCBs
PAHs
atrazine
OC
pesticides
calibration standard
PCB calibration
standard with DDE and
HCB
PAH standards
atrazine standards
mixed pesticide
standard
Cap microvial.
Load microvials into autosampler.
19.0 References
19.1 Baker, J.E., Eisenreich, S.J. PCBs and PAHs as Tracers of Paniculate Dynamics in Large Lakes.
J. Great Lakes Res., 1989, 15(1), #84-103.
19.2 Bidleman. T.F.; Mathews, J.R.; Olney, C.E.; Rice, C.P Separation of Polychlorinated Biphenyl,
Chlordane and p-p DDT from Toxaphene by silicic acid column chromatography. JAOAC, 1978,
61,820-828.
19.3 Harlin, K.S.; Surratt, K; and Peters, C. Standard Operating Procedure for the Analysis of PCBs
and Organochlonne Pesticides by GC-ECD. Illinois State Water Survey, Champaign, IL 61820,
November 1995.
19.4 Hermanson. M.H.; Hites, R.A. Long-Term Measurement of Atmospheric Polychlorinated
Biphenyls in the Vicinity of Superfund dumps. Environ. Sci. Technoi 1989 23 No 10
1253-1258.
19.5 Lake Michigan/Superior Load Monitoring Quality Assurance Program Plan (QAPP), Feb. 1993.
19.6 Marri, E.A.; Armstrong D.E. Polychlonnated Biphenyls In Lake Michiaan Tributaries J Great
Lakes Res., 1990, 16(3): 396-405
19.7 McVeety, B.D., Hites, R.A. Atmospheric Deposition of Polycyclic Aromatic -Hydrocarbons to
Water Surfaces: A Mass Balance Approach. Atmos. Environ., 1988, 22, 51 1-536.
19.8 Miillin. M.D. Personal Communication. June 1995
19.9 Mullin.M.D. PCB Workshop, U.S. EPA Large Lakes Research Station. Grossc He. MI,
June 19S5.
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Volume 2, Chapter 1 Sample Preparation Procedures
19.10 Murphy, T.J.; Rzeszutko, C.P Precipitation inputs of PCBs to Lake Michigan. J. Great Lakes
Res., December 1977. Internal. Assoc. Great Lakes Res., 3(3-4): 305-312.
19.11 Peters, C. and Harlin, K.S. Standard Operating Procedure for the Analysis of PAHs and Atrazine
by GC/Ion Trap MS. Illinois State Water Survey, Champaign, IL 61820, July 1995.
19.12 Quality Assurance Project Plan (QAP,P) Atmospheric Monitoring for the Lake Michigan Mass
Balance and the Lakes Michigan and Superior Loading Studies, July 1995, Revision 5.
19.13 Swackhamer, D.L.; Me Veety, B.D.; Hites R.A. Deposition and Evaporation of Polychlorinated
Biphenyl congeners to and from Siskiwit Lake, Isle Royale, Lake Superior. Environ, Sci. TechnoL,
1988,22,664-672.
19.14 Sweet, C. W., Vermette, S.J.; Gatz, D.F Atmospheric Deposition of Toxic Materials: A
Compound of the Green Bay Mass Balance Study. 1992, Contract Report 530, Illinois State Water
Survey, Champaign, IL 61820.
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Volume 2, Chapter 1 Sample Preparation Procedures
Appendix A.
Sample Preparation Log Form
Filter and Precipitation Samples
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures
LAKE MICHIGAN LOADING STUDY (LMLS)
SAMPLE PREPARATION LOG
SAMPLE SET NAME:
FIELD ID
LAB ID
COMMENTS
NOTES:
SAMPLE SET NAME:
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DATE BY
LAB PROCEDURES PERFORMED (INITIALS) COMMENTS
Extraction
Extract Concentration
(hexane/acetone)
Back Extraction (if Appl.)
Silica Column Cleanup
Concentration of Fraction
hexane
40%
MeOH
Nitrogen Slowdown
hexane
40%
MeOH
ADDITIONAL COMMENTS:
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Volume 2, Chapter 1 Sample Preparation Procedures
Appendix B.
Preparation Log Form
Composited Cartridge Samples
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Analysis of PCBs, Pesticides, and PAHs in
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Sample Preparation Procedures
LAKE MICHIGAN LOADING STUDY (LMLS)
SAMPLE PREPARATION LOG
SAMPLE SET NAME:
FIELD ID/WT (g)
SUB SAMPLE
WT(g)
LAB ID/TOTAL WT
EXTRACTED (g)
COMMENTS
NOTES:
SAMPLE SET NAME:
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DATE BY
LAB PROCEDURES PERFORMED (INITIALS) COMMENTS
Extraction
Extract Concentration
(hexane/acetone)
Back Extraction (if Appl.)
Silica Column Cleanup
Concentration of Fraction
hexane
40%
MeOH
Nitrogen Slowdown
hexane
40%
MeOH
ADDITIONAL COMMENTS:
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Standard Operating Procedure for the
Analysis of PAHs and Atrazine by
GC/lon Trap MS
Cathy Peters and Karen Harlin
Illinois State Water Survey
Office of Atmospheric Chemistry
2204 Griffith Drive
Champaign, IL 61820
SOP # CH-IN-003.1
July 1995
-------
Standard Operating Procedure for the Analysis of
PAHs and Atrazine by GC/lon Trap MS
SOP # CH-IN-003.1
1.0 Scope And Application
This method is used to determine the concentrations of polycyclic aromatic hydrocarbons (PAHs),
atrazine, and degradation products in extracts from vapor, particulate, and precipitation samples.
The method is specific for the IADN and LMMB/LMLS projects. The following analytes are
measured by this Standard Operation Procedure (SOP).
Analyte CAS#
atrazine 1912-24-9
desethylatrazine (DEA) 6190-65-4
desisopropylatrazine (DIA) 1007-28-9
acenaphthene 83-32-9
acenaphthylene 208-96-8
anthracene 120-12-7
benzo(a)anthracene 56-55-3
benzo(a)pyrene 50-32-8
benzo(b)fluoranthene 205-99-2
benzo(e)pyrene 192-97-2
benzo(ghi)perylene 191-24-2
benzo(k)fluoranthene 207-08-9
chrysene 218-01-9
coronene 191-07-1
dibenzo(a,h)anthracene 52-70-3
fluoranthene 206-44-0
fluorene 86-73-7
indeno(123cd)pyrene 193-39-5
phenanthrene 85-01-8
pyrene 129-00-0
retene 483-65-8
2.0 Summary of Method
This method describes equipment and procedures for operating the GC-Ion trap MS. and
instrument optimization specific for PAHs. atrazine and atrazine metabolites. The method is
specific for the IADN and LMLS/LMMB atmospheric deposition research projects.
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2.1 Personnel Restrictions
This method is restricted to use by or under the supervision of an analyst trained and experienced
in the operation of gas chromatographs, mass spectrometers, mass spectral and capillary
chromatogram interpretation, and data reduction. Each analyst must demonstrate the ability to
generate acceptable results with this method.
2.2 Working Linear Range
A multipoint calibration curve will be constructed for each analyte to document the working linear
range.
2.3 Limit of Detection
2.3.1 IDL
The instrument detection limit (IDL) refers to the smallest signal above background noise
that an instrument can reliably detect. The IDL is determined from a data set comprised of
three separate chromatographic runs of a low level calibration standard; each run contains
7-10 analyses of the standard. The IDL equals the Student's t value (n-1) multiplied by
the standard deviation of this data set.
2.3.2 MDL
Method detection limits (MDL) are defined in CFR, Vol 49, No. 209, October 26, 1984,
Appendix B to Part 136. Matrix specific MDLs are determined by spiking 7-10 clean
matrix samples with the analytes of interest and processing them through the entire
extraction, cleanup, and analysis procedure.
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2.4 Flow Diagram
hi-volume sampler
precipitation sampler
_L
particle: QF filter
vapor: XAD-2 resin
Fraction 1
(hexane)
concentrate
add PCB internal
standards
(PCBs 30 + 204)
GC-ECD
(PCBs, HCB, p.p1 DDE)
I
precipitation: XAD-2 resin
Spike sample with surrogate
samples
(PCBs 65, 166)
2,4,7-trichloro-9-fluorenone
benzo(a)pyrene-di2, atrazine-d5
extract and concentrate
cleanup and fractionation
Fraction 2
(CH2CI2: hexane 40:60)
Fraction 3
(Methanol)
concentrate
add pesticide internal standards
(PCB 65, PCB155, p,p'DDE)
add internal standards
(d-(Q-anthracene,
triphenylmethane,
d-| 2-benzo(a)anthracene,
GC-ECD
(a-HCH, g-HCH, dieldrin,
p,p' ODD, p,p' DDT, a-chlordane,
g-chlordane, trans-nonachlor)
GC-MS for atrazine
desethylatrazine,
desisopropylatrazine
add internal standards
(diQ-anthracene,
triphenylmethane,
d-| 2-benzo(a)anthracene,
acenaphthylene
acenaphthene
fluorene
phenanthrene
anthracene
fluoranthene
pyrene
retene
benzo(a)anthracene
GC-MS for PAHS
chrysene
benzo(b)fluoranthene
benzo(k)fluoranthene
benzo(e)pyrene
benzo(a)pyrene
ideno(123cd)pyrene
dibenzo(ah)anthracene
benzo(ghi)perylene
coronene
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3.0 Safety Precautions
3.1 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been
precisely defined. However, each one must be treated as a potential health hazard, and exposure to
these chemicals should be minimized. Some method analytes have been tentatively classified as
known or suspected human or mammalian carcinogens. Pure standard materials and stock
standard solutions of these compounds should be handled with suitable protection to skin, eyes,
etc.
3.2 Chemists working in the laboratory should follow ISWS safety rules:
3.2.1 A lab coat is required when working in the lab.
3.2.2 Eye protection with splash resistant safety glasses or safety goggles are required.
3.2.3 Protective gloves should be used while handling samples or standards. Special solvent
resistant gloves should be used while handling large amount of solvents.
3.2.4 All solvent work should be done in fume hoods.
3.2.5 Open shoes are not allowed in the laboratory.
3.2.6 Particle mask is required when using dry silica.
3.2.7 Avoid working alone in the laboratory. If work must be performed after hours or in the
weekend inform the supervisor or other staff so that your presence is known and will be
accounted for in case of an emergency.
3.2.8 Chemicals and solvents are stored under the hoods. Acids must be separated from bases.
A rubber bucket is required to transport any chemical.
3.2.9 Gas cylinders should be well secured at all times. Flammable gases are stored in separate
storage areas.
3.2.10 Wash hands well after work.
3.2.11 No food or drink is allowed in the laboratory.
3.2.12 In case of minor spillage, get spillage kit to clean the area. A major spill requires the
University of Illinois Fire Department to be contacted and the working area evacuated.
MSDS sheets are stored in the laboratory and a copy placed on file with the office
administrator.
3.2.! 3 All chemicals and standards must be labeled with chemical name, date, and initials of
person to contact.
3.2.14 Empty chemical bottles should be Hushed out with water, or, in case of liquid, allowed to
evaporate under a hood before discarding.
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3.3 Waste Disposal
Solvents: Label waste containers, Chlorinated Waste and Non-Chlorinated Waste. Glass bottles
used for waste are placed under hoods for convenience. When full, transfer waste to 10 L carboy
containers in solvent cabinet. Contact the ISWS Waste Coordinator for removal.
4.0 Apparatus and Materials
4.1 Glassware: General Requirements
All glassware, must be meticulously cleaned. Large glassware is thoroughly washed with
laboratory detergent and hot water. Glassware with bad stains should be rinsed with MeOH or
CH2CU before using the soap and water procedure. If still not clean, soak in H2SO4:HNO3 (50:50)
acid bath overnight, then wash thoroughly with soap and water. Volumetric pipettes used for
standards must soak in acid bath overnight. Glassware is thoroughly rinsed with tap water, then
with DI water and allowed to air dry. The glassware is foil wrapped and heated 450°C for four
hours. If glassware is not clean after muffling at 450°C for 4 hours, muffle at 500°C for four
hours. The glassware is cooled to ambient temperature and stored in a clean location. Small
glassware such as stoppers, vials, and disposables are wrapped in foil or placed into a beaker and
covered with foil and heated to 450°C for four hours, cooled to ambient temperature, and stored in
a clean location. Vials are capped as soon as they are removed from the oven. Note: Always use
dull side of foil towards glassware. Set initial temperature of furnace to 200°C if possible.
4.2 Vortex Mixer
4.3 Volumetric flasks and pipets - Class A, various sizes
4.4 Auto sample vials, amber, 2 mL with 200 uL inserts, caps and teflon-lined septa
4.5 Positive displacement micro pipet, glass capillaries (Drummond or equivalent)
4.6 GC-Ion Trap MS (see Section 9.0 for detailed information)
4.6.1 Model: Varian Saturn 3, capillary, GC/lon trap-MS system
4.6.2 Injector: SPI, 8200 autosampler, LB2 Thermogreen septa (Supelco)
4.6.3 Column: 30 m x 0.25 urn x 0.25 mm, DB-5 MS (J & W Scientific)
4.7 Chemicals/Standards
Pesticide residue quality or equivalent. All reagents are evaluated for interferences using
laboratory blanks.
4.7.1 Solvents. EM Omnisolve or equivalent
4.7.1.1 Hexane
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4.7.1.2 Methanol
4.7.1.3 Acetone
4.7.2 Standards
4.7.2.1 Atrazine and PAH Stock Standards
Stock standard solutions are purchased from commercial sources (Ultra Scientific,
Accustandard, Chem Service, Cresent Chemical) or are obtained from the USEPA
repository. When stock solutions are not available, pesticides are purchased as the
neat material and gravimetrically prepared in house.
4.7.2.2 Surrogate Standard Solutions
4.7.2.2.1 Surrogate standards are purchased from commercial sources
(Aldrich Chemical, Cambridge Isotope) or are obtained from the
USEPA repository. The following surrogate standards are
utilized:
atrazine-d5 (atrazine surrogate)
benzo-(a)pyrene-d!2 (PAH surrogate)
4.7.2.2.2 If stock solutions are not commercially available, they are
gravimetrically prepared from the neat material. Individual stock
solutions are serially diluted in volumetric flasks to obtain the
surrogate spike standard/s. A combined surrogate spiking
standard may be prepared to save sample preparation time during
the extraction procedure.
4.7.2.2.3 All samples are spiked with surrogate standards prior to
extraction using volumetric pipets or a Drummond pipet and the
spike volumes recorded on the sample preparation log.
4.7.2.3 Internal Standard Solutions (ISTDs)
4.7.2.3.1 Internal Standards are purchased commercially (Ultra Scientific)
as a stock standard or as the neat material. The following ISTDs
are utilized:
anthracene-d 10 (PAH and atrazine ISTD)
benzo(a)anthracene-d!2 (PAH ISTD
perylene-d!2(PAH ISTD)
triphenylmethane (PAH ISTD)
4.7.2.3.2 If stock solutions are not commercially, available they are
gravimetnculK prepared from the neat material. Individual stock
solutions are serially diluted in volumetric flasks to obtain the
ISTD spiking standard/s.
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4.7.2.3.3 ISTDs are added to the appropriate sample fraction (PAHs in
40% DCM, and atrazine in MeOH) prior to GC-MS analysis. A
Drummond micropipet is used for ISTD addition.
4.7.2.4 Chromatographic Calibration Standards
Combined instrument calibration standards are prepared from the individual stock
standards by volumetric dilution to obtain five concentration levels. The
calibration standard concentrations bracket the expected analyte amounts in
samples assayed and are within the working linear range of the detectors.
Calibration mixes are prepared specifically for the appropriate instrument and
fraction analyzed. The following calibration mixes are prepared.
PAHs with surrogate and ISTDs
atrazine, DEA, and DIA with surrogate and STDs.
4.7.2.5 Matrix Spiking Solutions
4.7.2.5.1 Combined matrix spiking solutions are prepared from the
individual stock standards by volumetric dilution. Combined
matrix spike solutions are prepared for each analyte group. The
following matrix spike mixes are prepared:
PAHs
atrazine, DEA, DIA.
4.7.2.5.2 The matrix spike solutions will be added to clean sample matrix
material prior to extraction to calculate the recovery of individual
analytes. One matrix spike will be extracted with each batch of
samples. The matrix spike will be added to the sample using a
Drummond micropipet or a volumetric pipet and the spiking
amounts reported in the sample preparation log. Detailed sample
preparation procedures can be found in SOP #CH-PR-001.3,
March 1995.
4.7.2.6 Standard Evaluation
New working standards will be assayed prior to use by comparison with existing
standards. Standards must agree within 10% prior to use.
4.7.3 Gases
4.7.3.1 Helium, carrier gas, 99.9999% chromatographic grade
4.7.3.2 Carbon Dioxide, SPI coolant, general grade
4.7.3.3 Air. autosampler pressurizing gas. general grade
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5.0 GC/MS System Evaluation
Prior to each run the GC/IT-MS system performance and calibration are verified for all analytes.
A mass spectrometer tune is performed prior to each run using system software with
perfluorotributylamine (PFTBA) calibration gas. Adjustments are made or maintenance is
performed such that selected calibration masses and their respective isotopes meet the target mass-
intensity criteria.
Hexane is injected immediately prior to each run to ensure the system is free of contaminants or
interfering peaks.
Records of daily system performance and maintenance are maintained.
Note: The system is evaluated and tuned with the column and injector set at the highest
temperature attained during a normal acquisition (i.e., column at 300 °C and injector at 289 °C).
This is to assure optimal conditions for the high-hoiling compounds (such as coronene}.
5.1 Daily Evaluation
5.1.1 Check air/water for leaks.
Set mass range to 10-45 m/z; insure AGC is off; set ion time to 300/^sec; set filament
emission current to 10 uA; turn on the filament, multiplier, and RF; normalize the peaks.
The following conditions indicate there is no evidence of a significant air leak or water
background present:
a) There are discrete peaks at 18, 28, and 32.
b) The peak at 28 is higher than that at 18 and the 28:32 ratio is about 4:1.
c) The ratio of peak 18 to peak 19 is 10:1 or greater.
d) The 100% counts value is less than 500.
e) The TIC value is less than 2000.
5.1.2 Check PFTBA calibration gas level.
The ionization time should read between 500-1000 usec.
5.1.3 Check the valley to isotope % of ions 131 and 132.
The value should be around 25%. This means the 132 isotope peak is four times the
height of the valley between ions 131 and 132. Adjust tune parameters, if necessary, to
obtain the proper valley/isotope %:
a) Begin by setting the mass range to 127-137 m/z. Turn on the multiplier, filament,
RF, calibration gas, and AGC. Set the AGC target value to 20000.
b) Adjust the A/M (axial modulation) voltage until the valley/isotope 9r appears to
be close to 25%. The axial modulation value is typically between 2.5 and 5 volts.
c) Run the Set AGC Target program. The AGC target should be optimally set to
20000 - 25000. If not, the A/M voltage and/or the electron multiplier voltage may
need to he (re)adjusted.
5.1.4 Run a mass calibration with PFTBA calibration gas. Verify valid calibration prior to
proceeding.
2-174
-------
SOP for the Analysis of
Volume 2, Chapter 1 PAHS and Atrazine by GC/lon Trap MS
5.2 Weekly Evaluation
5.2.1 Check integrator zero.
When both the RF and the multiplier are on, the measured signal should be between 0.2
and 0.8 ADC counts. If the integrator zero is not within these limits, it must be adjusted.
5.2.2 Set AGC target.
The Set AGC Target program should be run to verify the valley/isotope % is actually 25%.
This program is also a check on the electron multiplier value. The optimal target value is
20000.
5.3 Monthly Evaluation
5.3.1 Check multiplier voltage.
5.3.2 Check filament emission current.
The filament emission current is set to 10 |jA. The Set Filament Emission Current
program should be ran to verify instrument performance. If the program wants to set the
filament emission current higher, this is usually indicative of a high level of background.
5.3.3 Check RF voltage ramp
The RF voltage ramp should rise gradually in a generally straight line from low mass to
high mass throughout the entire mass range, without any sudden rises in the ramp. The
average response value should be 300 - 500 ADC counts and the highest response value
should be 500-900 ADC counts.
5.3.4 Check carrier gas flow rate.
The optimum carrier gas volumetric flow rate into the ion trap is 1 mL/min. at the
maximum temperature reached during an analysis. This is determined by measuring the
time required for an unretained compound, such as air, to elute from the column, and then
calculating the volumetric flow rate using the following formula:
2 .
Volumetric flow rate (mL/min)
Where:
n is 3.14
r is the radius of the column (cm)
I is the length of the column (cm)
t is the retention time of the unretained compound (min)
6.0 Periodic GC/MS Maintenance
6.1 Change injector septa after approximately 100 injections.
6.2 Vent rough pump a minimum of two times per month.
6.3 Change hexane in injector rinse reservoir every month.
2-175
-------
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap MS Volume 2, Chapter^
6.4 Change waste arm septa every month.
6.5 Change rough pump oil every three to six months.
7.0 Preparation of Autosampler Vials for GC/MS Analysis
7.1 Remove standards and samples from freezer; equilibrate to ambient temperature (approximately
two hours).
7.2 Initiate sample log sheet for GC/MS run. Each analytical run consists of a hexane blank, standards
to establish a calibration curve, a performance check standard if available, samples, a calibration
check standard every five to seven samples, and a final calibration check and hexane blank.
7.3 Label autosampler vials for samples, standards, and hexane blanks.
7.4 Insert 200 uL glass insert into each vial (except in cases of sample dilutions).
7.5 Spike samples with ISTD if necessary. (See Section 8.0 for spiking procedure.)
7.6 Before transferring samples and standards to the corresponding labeled autosampler vials, mix
contents of each vial and bottle well by holding on a vortex mixer for 5-10 seconds. Use muffled
Pasteur pipettes for transfer. The glass insert should be filled approximately half full.
7.7 Place cap onto autosampler vial after it is filled.
7.8 Load autosampler vials onto autosampler.
8.0 Spiking Samples with ISTD
8.1 Remove ISTDs from freezer; equilibrate to ambient temperature (approximately two hours).
Vortex ISTD solution to mix.
8.2 Clean micropipette:
Remove glass tube used to cover plunger.Rinse plunger with CH,C12. Allow to dry. Without
manually touching glass tubes, insert plunger into a new glass tube. Tighten tube in position.
Rinse pipette tube:
1) Draw up CH,CU into pipette and discard into solvent waste container.
2) Draw up hexane into pipette and discard into solvent waste container. Repeat five times.
Allow to dr>.
3) Draw up internal standard into pipette and discard into solvent waste container. Repeat
two times.
4) Fill micropipette.
2-176
-------
Volume 2, Chapter 1
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap MS
8.3 Spike sample vial. See chart below for internal standard and amount.
Fraction
Hexane
40%
40%
MeOH
Compound
PCBs and
Pesticides
Pesticides
PAHs
Atrazine
Type of
Sample
vapor, particle,
and rain
vapor, particle,
and rain
vapor, particle,
and rain
vapor, particle,
and rain
Internal Standard
PCB30
PCS 204
PCB65
PCB 1 55
DDE
d 10- Anthracene
Triphenylmethane
d!2-Benzo(a)anthracene
d!2-Perylene
dlO-Anthracene
Spike
Volume
(ML)
100
100
100**
100
Final Mass
in Sample
(ng)*
8
6
23.7
17.5
20
200
100
200
200
200
Color of
Dot on
Label
Red
Blue
Black
Black
8.4
8.5
8.6
*Values in this column are approximate and may change slightly depending on the exact concentrations of
the compounds in the stock solutions.
**If the sample is expected to be high in concentration of PAHs or if the final sample volume is greater than
2 mL, more ISTD solution may be added in increments of lOOpL.
Mark each sample vial label with the appropriate color of dot to verify sample has been spiked.
(Use water-proof marker. )
Rinse with solvent and replace glass tube used to cover plunger of micropipette. Store
micropipette.
Mark sample box with the following information using the same color of pens as dots on vial
labels:
Date sample vials spiked.
Fraction spiked.
Initials of chemist spiking.
9.0 Normal Operating Parameters for Saturn 3 GC/MS
9.1 Operating software:
9.2 Operating mode:
9.3 AGC (automatic gain control):
Version 5.0 or later
El (electron impact)
ON
2-177
-------
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap MS
Volume 2, Chapter 1
9.4 AGC prescan factors:
Background mass:
Data steps:
Weight factor:
9.5 Scan rate variables:
Scan time (msec):
Micro-scans:
9.6 EI/AGC parameters:
El background mass (m/z):
El maximum ionization time (usec):
AGC prescan ionization time (psec):
AGC prescan storage level (m/z):
Data steps in AGC prescan:
RF dump value (m/z):
AGC weight factor:
9.7 Scan segment breaks:
Seg masses
Segment 1: 10-98
Segment 2: 99-310
Segments: 311-399
Segment 4: 400-650
9.8 PAH Operating Conditions
9.8.1 SPI Injector
Injection volume:
Hot needle time:
Injection time:
Injection rate:
Lower air gap
Upper air gap
Needle depth
Initial temperature:
Ramp 1:
98
50
1
1000
15
25000
100
20.0
50
650.0
1
Seg time
100
100
100
100
IpL
0.03 minutes
0.01 minutes
0.5 uL/second
yes
yes
90%
50°C, hold 0.10 minutes
200°C/min. to 290°C, hold 48.70 minutes
9.8.2 The GC temperature program is optimized to achieve >50<7c resolution for all analyte
peaks and any known interferant. Typical conditions are as follows:
Oven Program:
Initial temperature:
Ramp 1:
Ramp 1:
Rump 3:
Ramp 4:
50 minutes
75°C, hold 2.0 minutes
25°C/min. to 150°C
4°C/min. to235:C
3:C/min. to 265 :C
50"C/min. to 300'C. hold 11.94 minutes
2-178
-------
SOP for the Analysis of
Volume 2, Chapter 1 PAHs and Atrazine by GC/lon Trap MS
9.8.3 Miscellaneous Parameters
Column head pressure: 15 psi
Transfer line: 300°C
Column linear velocity: 1.0 mL/min. at 300°C
Septum purge flow: 4.25 mL/min.
Manifold set temperature: 240°C
Mass range: 98-310 m/z
Mass defect: 50 mu/100|j
9.8.4 Calibration Information
A multipoint calibration curve is prepared for each analyte prior to each run. A calibration
check is performed every 5-10 samples and at the end of each run. Recalibration is
required if a shift of >20% is observed. The method of internal standard calibration is
utilized with four internal standards for PAHs and one for atrazine. The chromatogram is
divided into four time segments with each segment calibrated relative to one internal
standard response for the PAH runs.
Data is collected in the full-scan mode, however, selected ions are used for quantitation.
2-179
-------
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap
MS
Volume 2, Chapter 1
Retention
Time
8.03
8.46
9.95
13.54
13.68
13.76
18.02
19.36
20.5
22.58
27.38
27.5
27.7
34.25
34.44
35.89
36.09
36.21
36.59
40.68
40.87
41.56
48.51
ISTD#
1
1
1
1
1
1
2
2
2
3
3
3
3
4
4
4
4
4
4
4
4
4
4
Name of Compound
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
dlO-Anthracene(ISTD#l)
Anthracene
Triphenylmethane (ISTD #2)
Fluoranthene
Pyrene
Retene
d!2-Benzo(a)anthracene (ISTD #3)
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(e)pyrene
d!2-Benzo(a)pyrene (surrogate)
Benzo(a)pyrene
d!2-Perylene(ISTD#4)
Indeno( 1 23cd)pyrene
Dibenzo(ah)anthracene
Benzo(ghi)perylene
Coronene
Quantitating
Masses
152
153
165+166
178
188
178
1 65+244
202
202
219+234
240
228
228
252
252
252
264
252
130+132+264
138+276+277
139+278+279
138+276+277
150+300+301
2-180
-------
itoyram IMot C :SDATrtM>OPS950B090'l Dote.'
Cuwmont: STD 500 CrtL
S'con: 1900 Sey : 1 Grout*: 0 Retention: 22.1G NIC: 1815
Motlcil: 400 lo 1900 Range: 1 to 1ZB5
§
Masses: 100-2U!
JBRx - 55972
oo
TOT
'—V-«i'
•s_ X'.L,
j , r_
(:>(!)()
7.00
98W
1280
13.99
1500
17.49
1860
21.80
VO
°
X
o
3-
3
3
o
7Q
3
3
O
O.
ft)
I
(D
-N
§
T]
^
a:
M
0)
01
fci. en
O
TJ
(ft
o
-------
TJ en
oo
ro
C: \Dr»Tf»SS01»X950B090<1
Date: BB/B9/95 22:09:
tuyrari I* lot
C tinmen I : STI) 500 CrtL
Scnn: -1000 Seg : 1 Group: B Retention: 46.66 RIG: 19581 Masses: 99-3B'
Plotted: 2200 to '1B0B Range: 1 to 4205 IBBx - 55972
IHBx
TOT-
J
I U
/
3
1
21HH
r
2DUO
32.G6
320B
:iv. 33
3680
'11.99
u
i
Q)
^
O" (/>'
< r»
"
I
f?
-------
Volume 2, Chapter 1
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap MS
9.9 Atrazine Operating Conditions
9.9.1 SPI Injector
Injection volume:
Hot needle time:
Injection time:
Injection rate:
Lower air gap
Upper air gap
Needle depth
Initial temperature:
Ramp I:
9.9.2 Oven Program: 32 minutes
Initial temperature:
Ramp 1:
Ramp 2:
Ramp 3:
9.9.3 Miscellaneous Parameters
Column head pressure:
Transfer line:
Column linear velocity:
Septum purge flow:
Manifold set temperature:
Mass range:
Mass defect:
9.9.4 Calibration
2uL
0.03 minutes
0.10 minutes
0.2 uL/second
yes
yes
90%
50°C, hold 0.85 minutes
200°C/min. to 290°C, hold 29.95 minutes
75°C, hold 2.0 minutes
25°C/min. to 150°C
4°C/min. to 190°C
50°C/min. to 300°C, hold 14.80 minutes
15psi
300°C
1.0 mL/min. at 300°C
4.25 mL/min.
240 °C
98-310m/z
50mu/100u
See Section 9.8.4 for a general discussion of the calibration procedure.
Retention Time
10.73
10.93
12.61
12.69
13.64
Name of Compound
Desisopropylatrazine
Desethylatrazine
d5-Atrazine (surrogate)
Atrazine
dlO-Anthracene(ISTD)
Quantitating Masses
158+173
172
205+220
200+2 1 5
188
2-183
-------
vo
O
•fl
i!
CD
S. I.
Chromatograw Plot
Connent: ATR 1830 CAL
Scan: 1258 Seg: 1
Plotted: 980 to 1250
lOBx
C:SSATURNSDATAS951B12B5
U/588 DIA.DSA AND 499 DEA
Group: 0 Retention: 14.58 RIG: 1254
Range: 1 to 2742
Date: 18/12/95 15:IB:37
Masses: 181-295
188X = 36858
ry
00
IOT-
988
18.49
958
11.88
' ' i ' '
1888
11.66
1858
12.24
' ' | ' ' ' l
1188
12.83
i i | i i
1158
13.41
' i i i i i i
1288
13.99
3
n
3-
3
O
CTQ
3
in
r>
63
3
O
CL
n
O- '
O
§
I
n
3
-------
SOP for the Analysis of
Volume 2, Chapter 1 pAHs and Atrazine by GC/lon Trap MS
10.0 Data Evaluation
10.1 View the hexane blank total ion chromatogram to determine if the system is clean.
10.2 Integrate each standard using the previous calibration curve. Print a hard copy of each standard
data report.
10.3 Build a new calibration curve daily or for each sample set using a minimum of three standards.
10.4 Evaluate the performance check standard (US 106) for PAHs. Print a hard copy of the data report.
10.5 Integrate each sample, peak observing each individual compound. Print a hard copy of
compounds that are manually integrated and a hard copy of the final sample report.
10.6 Continue evaluating each sample, each calibration check (every five to seven samples), and the
final hexane blank chromatogram.
10.7 Print a hard copy of the tune conditions for the run.
10.8 After the entire run has been integrated and evaluated, run the macro Print.prc (see Appendix A)
to convert Saturn data files to a format for import into Quattro® Pro.
10.9 Import the Saturn data to a Quattro® Pro worksheet.
10.10 Check the data in the worksheet against the Saturn hard copies. Correct the worksheet and add
extraction batch codes and any necessary comments.
10.11 Print out a hard copy of the Quattro® Pro worksheet and compare the data again to the Saturn hard
copies. Initialize the folder containing the Saturn hard copies as being checked.
10.12 Copy the worksheet data into the appropriate spreadsheet.
10.13 Send the Saturn hard copies and a disk copy of the updated Quattro® Pro spreadsheet to the lab
supervisor for final review.
10.14 Saturn data files must be backed up before they are deleted from the Saturn system hard drive.
Make two backups using optical disks or magnetic tapes.
10.15 The data will be considered valid if the calibration check standards and the performance check
standard are within 20%.
10.16 Formula for manual-calculations of GC/MS Data
Some over-range peaks may require manual calculations to determine the analyte concentration
., , I Mass] I Area\ I Mass]
Mass Area x { > x \ > x < >
'"""' """'' \Area\ . l/V/
-------
SOP for the Analysis of
Volumne 2, Chapter 1 PAHs and Atrazine by GC/lon Trap MS
Appendix A. Macros
PRINT.PRC MACRO
screen I:
\PRINT
# Spathname = "D:\DATAV
#$areafile = "B:\AR"
#$amntfile = "B:\AM"
screen 2:
\PRINT
CLS
ROW 10
COLUMN 10
PRINT "Enter subdirectory: "
INPUT-STRING
CHOP-TRAILING-BLANKS-FROM SSTRING
JOIN-STRINGS Spathname $STRESfG
JOIN-STRINGS Spathname "\"
JOIN-STRINGS $areafile $STRING
JOIN-STRINGS Sareafile ".PRN"
JOIN-STRINGS $amntfile SSTRING
JOIN-STRINGS Samntfile ".PRN"
screen 3:
\PRINT
CLS
ROW-COL 3 15
FILE-LIST-OF "*.QD"
$FILE-LIST-PATH = $pathname
FILE-LIST-TITLE "Select Quant, files"
FILE-LIST-SIZE = 10
SHOW-FILE-LIST
screen 4:
\PRINT
CREATE-FILE Sareafile
WRITE-TO-FILE
FOR J = I TO #FILES-IN-LIST
USE-DATA-FILE $FILE-LIST-NAME# J
PRINT I "
PRINT $DATA-SAMPLE-ID
PRINT I "
PRINT I .
PRINT I '
PRINT SDATA-FILE-NAME
2-187
-------
SOP for the Analysis of
PAHs and Atrazine by GC/lon Trap MS Volume 2, Chapter^
Appendix A. Macros (Cont'd)
PRINT 1 "
PRINT 1 ,
PRINT 1 "
PRINT-DATE( 1 )-OF DATA-FILE-DATE
PRINT 1"
PRINT ", ,"
USE-QUAN-FILE $FILE-LIST-NAME# J
screen 5:
\PRINT
FOR I = 1 TO #QUAN-COMPOUNDS
READ-QUAN-COMPOUND# I
FIELD L7.0 PRINT QUAN-PEAK-AREA
PRINT 1 ,
NEXT
CR
NEXT
CLOSE-FILE
2-188
-------
Standard Operating Procedure for the
Analysis of PCBs and Organochlorine
Pesticides by GC-ECD
Karen Harlin, Kaye Surratt, and Cathy Peters
Illinois State Water Survey
Office of Atmospheric Chemistry
2204 Griffith Drive
Champaign, IL 61820
Standard Operating Procedure CH-IN-002.3
November 1995
Revision 3.0
-------
Acknowledgments
The authors wish to acknowledge the contributions of Monte Wilcoxon in the generation of
chromatograms and data tables for this SOP.
-------
Standard Operating Procedure for the Analysis of PCBs and
Organochlorine Pesticides by GC-ECD
1.0 Scope and Application
This procedure details the analysis and data reduction methods utilized to determine
polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) in vapor, paniculate, and
precipitation samples. The method is specific to the Lake Michigan Mass Balance (LMMB) and
Lake Michigan Loading Study (LMLS) projects. The following analytes are measured by this
SOP using gas chromatograph-electron capture detection methods:
Polychlorinated Biphenyls (PCBs)-Total and 105 congener peaks
congener (BZ)
1
3
4+10
6
7+9
8+5
12
13
15+17
16
18
19
21
22
24
27
25
26
29
31+28
32
33
37
CAS #
2051-60-7
2051-62-9
13029-08-8
,33146-45-1
25569-80-6
33284-50-3
34883-43-7
, 34883-39-1
, 16605-91-7
2974-92-7
2974-90-5
2050-68-2,
37680-66-3
38444-78-9
37680-65-2
38444-73-4
55702-46-0
38444-85-8
55702-45-9
38444-76-7
55712-37-3
38444-81-4
15862-07-4
16606-02-3
,7012-37-5
38444-77-8
38444-86-9
38444-90-5
2-193
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter 1
congener (BZ)
40
41+71
42
43
44
45
46
47
48
49
51
52
53
56+60
63
64
66
70+76
74
77
81
82
83
85
87
89
91
92+84
95
97
99
100
101
107
110
1 14+131
CAS #
8444-93-8
52663-59-9,
41464-46-4
36559-22-5
70362-46-8
41464-39-5
70362-45-7
41464-47-5
2437-79-8
70362-47-9
41464-40-8
68194-04-7
35693-99-3
41464-41-9
41464-43-1,
33025-41-1
74472-34-7
52663-58-8
32598-10-0
32598-11-1,
70362-48-0
32690-93-0
32598-13-3
70362-50-4
52663-62-4
60145-20-2
65510-45-4
38380-02-8
73575-57-2
68194-05-8
52663-61-3,
52663-60-2
38379-99-6
41464-51-1
38380-01-7
39485-83-1
37680-73-2
70424-68-9
38380-03-9
74472-37-0,
61798-70-7
2-194
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
congener (BZ)
118
119
123+149
128
129
130
132+153+105
134
135+144
136
137+176
141
146
151
156
157+200
158
163+138
167
170+190
172
173
174
175
177
178
180
183
185
187+182
189
191
193
194
197
CAS #
31508-00-6
56558-17-9
65510-44-3,
38380-04-0
38380-07-3
55215-18-4
52663-66-8
38380-05-1.
32598-14-4
35065-27-1,
52704-70-8
52744-13-5,
68194-14-9
38411-22-2
35694-06-5,
52663-65-7
52712-04-6
51908-16-8
52663-63-5
38380-08-4
69782-90-7,
52663-73-7
74472-42-7
74472-44-9,
35065-28-2
52663-72-6
35065-30-6,
41411-64-7
52663-74-8
68194-16-1
38411-25-5
40186-70-7
52663-70-4
52663-67-9
35065-29-3
52663-69-1
52712-05-7
52668-68-0,
60145-23-5
39635-31-9
74472-50-7
69782-91-8
35694-08-7
33091-17-7
2-195
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter 1
congener (BZ)
198
199
201
202+171
196
203
205
206
207
208+195
209
CAS #
68194-17-2
52663-75-9
40186-71-8
2136-99-4, 52663-71-5
42740-50-1
52663-76-0
4472-53-0
40186-72-9
52663-79-3
52663-77-1,52663-78-2
2051-24-3
Pesticides
dieldrin
a-chlordane
g-chlordane
t-nonachlor
a-hexachlorocyclohexane
(a-HCH)
g-hexachlorocyclohexane
(g-HCH)
hexachlorobenzene (HCB)
p'p'-DDD
p,p'-DDE
p,p'-DDT
CAS #
60-57-1
5103-71-9
5103-74-2
39765-80-5
319-84-6
58-89-9
118-74-1
72-54-8
72-55-9
50-29-3
2.0 Summary of Method
This method describes equipment and procedures for performing gas chromatographic analysis
with an electron capture detector (GC-ECD). It includes instrument optimization specific for
PCBs and OCs and data reduction.
2.1 Personnel Restrictions
This method is restricted to use by or under the supervision of an analyst trained and experienced
in the operation of gas chromatographs, electron capture detectors, and capillary chromatogram
interpretation, and data reduction. Each analyst must demonstrate the ability to generate
acceptable results with this method.
2.2 Working Linear Range
A multi point calibration curve will be constructed for each analyte to document the working
linear ran tie.
2-196
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
2.3 Method Flow Diagram
hi-volume sampler
precipitation sampler
particle: QF filter
vapor: XAD-2 resin
1
precipitation: XAD-2 resin
spike sample with surrogate
samples
(PCBs 65, 166)
2,4,7-trichloro-9-fluorenone
benzo(a)pyrene-d-)2, atrazine-
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD . Volume 2, Chapter^
3.0 Definitions
3.1 Limits of Detection
3.1.1 Instrument detection limit
The instrument detection limit (IDL) refers to the smallest signal above background noise
that an instrument can reliably detect. The IDL is determined from a data set comprised of
three separate chromatographic runs of a low level calibration standard; each run contains
7-10 analyses of the standard. The IDL equals the Student's t value (n-1) multiplied by
the standard deviation of this data set.
3.1.2 Method detection limit
Method detection limits (MDL) are defined in CFR, Vol 49, No. 209, October 26, 1984,
Appendix B to Part 136. Matrix specific MDLs are determined by spiking 7-10 clean
matrix samples with the analytes of interest and processing them through the entire
extraction, cleanup, and analysis procedure.
3.2 Internal standard (ISTD)
A pure analyte(s) added to a sample extract, or standard solution in known amount(s) and used to
measure the relative responses of other method analytes and surrogates that are components of the
same solution.
3.3 Laboratory surrogate spike (LSS)
A pure analyte(s), which is extremely unlikely to be found in any sample, and which is added to a
sample aliquot in known amount(s) before extraction or other processing, and is measured with the
same procedures used to measure other sample components. The purpose of the LSS is to monitor
method performance with each sample.
3.4 All other terms are defined in the QAPjP, Revision 5, July 1995.
4.0 Interferences
Method interferences may be caused by contaminants in solvents, the sampling matrix, reagents,
glassware, and other sample processing apparatus that lead to anomalous peaks or elevated
baselines in gas chromatograms. Laboratory equipment and reagents will be monitored by the
inclusion of quality control samples with each batch of samples prepared. Individual samples may
contain interferences which will require additional sample preparations. All sample preparation
details will be documented.
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Volume 2, Chapter 1 by GC-ECD
5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been
precisely defined. However, each one must be treated as a potential health hazard, and exposure to
these chemicals should be minimized. Some method analytes have been tentatively classified as
known or suspected human or mammalian carcinogens. Pure standard materials and stock
standard solutions of these compounds should be handled with suitable protection to skin, eyes,
etc.
5.2 Chemists working in the laboratory should follow ISWS safety rules :
5.2.1 A lab coat is required when working in the lab.
5.2.2 Eye protection with splash resistant safety glasses or safety goggles are required.
5.2.3 Protective gloves should be used while handling samples or standards. Special solvent
resistant gloves should be used while handling large amount of solvents.
5.2.4 All solvent work should be done in fume hoods.
5.2.5 Open shoes are not allowed in the laboratory.
5.2.6 Particle mask is required when using dry silica.
5.2.7 Avoid working alone in the laboratory. If work must be performed after hours or in the
weekend inform the supervisor or other staff so that your presence is known and will be
accounted for in case of an emergency.
5.2.8 Chemicals and solvents are stored under the hoods. Acids must be separated from bases.
A rubber bucket is required to transport any chemical.
5.2.9 Gas cylinders should be well secured at all times. Flammable gases are stored in separate
storage areas.
5.2.10 Wash hands well after work.
5.2.11 No food or drink is allowed in the laboratory.
5.2.12 In case of minor spillage, get spillage kit to clean the area. A major spill requires the
University of Illinois Fire Department to be contacted and the working area evacuated.
5.2.13 MSDS sheets are stored in the laboratory and a copy placed on file with the office
administrator.
5.2.14 All chemicals and standards must be labeled with chemical name, date, and initials of
person to contact.
5.2.15 Empty chemical bottles should be flushed out with water, or, in case of liquid, allowed to
evaporate under a hood before discarding.
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by GC-ECD Volume 2, Chapten
5.3 Waste Disposal
Solvents: Label waste containers, 'chlorinated waste' and 'non-chlorinated waste'. Glass bottles
used for waste are placed under hoods for convenience. When full, transfer waste to 10 L carboy
containers in solvent cabinet. Contact the ISWS Waste Coordinator for removal.
6.0 Equipment and Supplies
6.1 Glassware, General Requirements
All glassware, must be meticulously cleaned. Large glassware is thoroughly washed with
laboratory detergent and hot water. Glassware with bad stains should be rinsed with MeOH or
CH2C12 before using the soap and water procedure. If still not clean, soak in H2SO4:HNO, (50:50)
acid bath overnight, then wash thoroughly with soap and water. Volumetric pipettes used for
standards must soak in acid bath overnight. Glassware is thoroughly rinsed with tap water, then
with DI water and allowed to air dry. The glassware is foil wrapped and heated 450°C for 4
hours. If glassware is not clean after muffling at 450°C for 4 hours, muffle at 500°C for 4 hours.
The glassware is cooled to ambient temperature and stored in a clean location. Small glassware
such as stoppers, vials, and disposables are wrapped in foil or placed into a beaker and covered
with foil and heated to 450°C for 4 hours, cooled to ambient temperature, and stored in a clean
location. Vials are capped as soon as they are removed from the oven. Note: Always use dull
side of foil towards glassware. Set initial temperature of furnace to 200°C if possible.
6.2 Vortex Mixer
6.3 Volumetric flasks and pipets - Class A, various sizes
6.4 Autosampler vials, 2 mL, caps, teflon-lined septa, and 200 /uL glass inserts.
6.5 Positive displacement micro pipet, glass capillaries (Drummond or equivalent)
6.6 GC-ECD (see section 11.0 for detailed information)
6.6.1 Hewlett-Packard 5890A capillary gas chromatograph with split/splitless capillary inlet,
and electron capture detector (15 m Curie h'Ni source)
6.6.2 Hewlett-Packard 3365 ChemStation, Version B.01.02
6.6.3 Autosampler: Hewlett-Packard 7673 or 7673A autosampler
6.6.4 Column:
For OC pesticides-30 m x 0.25 /urn x 0.25 mm, DB-5 (J & W Scientific)
For PCBs-60 m x 0.1 ^m x 0.25 mm. DB-5 (J & W Scientific)
6.7 J & W AccuRATErM 1000 flowmeter (PN2201 I 70. Folson. CA)
6.8 GL Sciences Inc..LD223 flowmeter (Tokyo, Japan)
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7.0 Reagents and Standards
7.1 All reagents will be pesticide residue quality or equivalent. All reagents are evaluated for
interferences using laboratory blanks.
7.2 Solvents, EM Omnisolve or equivalent
7.2.1 Hexane
7.2.2 Methylene chloride
7.3 Standards
7.3.1 PCB and pesticide stock standards
Stock standard solutions are purchased from commercial sources (Ultra Scientific,
Accustandard, Chem Service, Cresent Chemical), are obtained from the USEPA
repository, or from the USEPA (PCB Mullin stock standard, 1994). When stock solutions
are not available, analytes are purchased as the neat material and gravimetrically prepared
in house. Stock solutions and mixed working standards are prepared volumetrically by
serial dilution in volumetric flasks. A standards preparations are detailed in laboratory log
books.
7.3.2 Chromatographic Calibration Standards
7.3.2.1 Mixed instrument calibration standards are prepared from the individual
stock standards by volumetric dilution to obtain five concentration levels.
The calibration standard concentrations bracket the expected analyte
amounts in samples assayed and are within the working linear range of
the detectors. Calibration mixes are prepared specifically for the
appropriate instrument and fraction analyzed. Calibration mixes are
described in "Standard Operating Procedure for the Analysis of PCBs,
Pesticides, and PAHs in Air and Precipitation Samples," SOP #CH-PR-
001.3, Revision 3.0, March 1995, Harlin and Surratt, ISWS, Champaign,
IL61820).
7.3.2.2 Standard Evaluation : New working standards will be assayed prior to
use by comparison with existing standards. Standards must agree within
10% prior to use.
7.4 Gases
7.4.1 Hydrogen, carrier gas, 99.9995%- chromatographic grade
7.4.2 Make-up gas. argon/methane (P5)
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8.0 GC-ECD System Evaluation and Maintenance
8.1 System Evaluation
Before each run GC-ECD system performance and calibration are verified for all analytes. Hexane
is injected at the beginning of each run to ensure the system is free from contaminants or
interfering peaks. Records of daily system performance and maintenance are maintained.
8.1.1 Conditioning
A conditioning program is run prior to every analytical GC run with a hexane injection.
The GC oven Is set to 280°C, the injector at 280°C, and the ECD at 380°C for about half
an hour (See Conditioning method, Appendix A; Method 1).
8.1.2 Injector
8.1.2.1 Septum: Prior to every analytical GC run the septum is changed. Prior to
removing the septum the heated zones are cooled and the column oven cooled to
40°C.
Note: Many septa are rated to last for about 100 injections, however many are
soft and small fragments frequently break off and enter the injection port or the
split/splitless weldment. More frequent changes, such as before each run, help
minimize this problem.
8.1.3 ECD Baseline Signal
The ECD baseline signal is usually below 30. After conditioning the system, hexane is
analyzed at the start of every GC run to monitor the baseline stability. If the baseline
signal is elevated or the hexane run produces a noisy chromatogram, the system should be
evaluated for leaks or contamination.
8.1.4 Calibration standard performance
Inject a calibration standard to evaluate peak resolution, peak shape, and identification
using the reference chromatograms in this SOP for evaluation. If the peak shapes are not
satisfactory inspect the system for proper flow rates and leaks. If these are acceptable.
remove 0.5-1 meter from the injector end of the column or install a new column.
8.1.4.1 PCBs: Over 90 peaks should resolved with congeners 77 and 1 10 separated from
adjacent peaks. Monitor the ratio of the areas of the 30/204 ISTD peaks. This
ratio will increase as the sensitivity for peaks in the later portion of the
chromatogram are reduced (observe the peak area of congener 209). When this
ratio exceeds 1.2 (a value of about I or less is desirable), the injection insert
should be changed. In a run with a new injection insert, the peak area of 209 in a
^95 ng/niL standard is easily seen. With repeated injections this peak area will
decrease and can be used in addition to the 30/204 area ratio for determining the
need for an injection liner change.
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8. 1 .5 Leak checking and gas flows (See Appendix B for detailed instructions)
When the chromatography performance deteriorates check for gas leaks. Check around
the septum, the injector connection, and at the detector connection of the column. Use of
a leak detection fluid is discouraged because of the possibility of drawing the fluid into the
system. A pure solvent such as isopropyl alcohol may be used as a leak detection fluid,
however, an electronic leak detector is recommended such as the J & W AccuRATE™,
1000 flowmeter (PN2201 170, Folson, CA)
Check the gas flow after any routine maintenance with a flowmeter. An electronic
flowmeter is recommended such as the GL Sciences Inc., LD223 flowmeter (Tokyo,
Japan).
8.2 Gases
Replace all tanks at 500 psig. Prior to changing a gas tank, lower GC oven temperature to 40° C
for approximately 30 min. Maintain a 40°C oven temperature for approximately 30 min. after
changing a tank to eliminate air from the system prior to heating the column and detector.
8.2.1 Carrier gas (hydrogen) flow rate.
The optimum carrier gas volumetric flow rate is specified by the column manufacturer.
This is determined by measuring the time required for an unretained compound to elute
from the column (such as methylene chloride vapor for an BCD), and then calculating the
volumetric flow rate using the following formula:
Volumetric flow rate (mL/min) =
t
where:
n is 3. 14
r is the radius of the column (cm)
I is the length of the column (cm)
t is the retention time of the unretained compound (min)
8.2.2 Column head pressure should be at the preset value, usually 20/25 psi for 30/60 m
columns. If the pressure is low, tighten the septum nut. If the pressure is still low check
for leaks and tighten other connections.
9.0 Preparation of Autosampler Vials for GC-ECD Analysis
9. 1 Initiate sample sequence table for the GC run. Each analytical run consists of a hexane blank,
standard/s to establish a calibration table, a performance check standard (LPC), samples, a
calibration check standard every 7-10 samples (if necessary), and a final calibration check (CLC).
9.2 Label autosampler vials for samples, standards, and hexane blanks; add inserts to vials.
9.3 Spike samples \\ith ISTD if necessary (See Section 10.0 for spiking procedure).
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Volume 2, Chapter 1
9.4 Before transferring samples and standards to the corresponding labeled autosampler vials, mix
contents of each vial and bottle well by holding on a vortex mixer for 5-10 seconds. Use muffled
Pasteur pipettes for transfer.
10.0 ISTD Addition
10.1 Remove ISTDs from freezer; equilibrate to ambient temperature (approximately two hours).
Vortex ISTD solution to mix.
10.2 Clean micropipette as follows: Remove glass capillary used to cover plunger. Rinse plunger with
CH2C12 and allow to dry. Without manually touching glass capillaries, insert plunger into a new
capillary, position and tighten. Rinse capillary in the following order: CH2C12, hexane (five times),
air dry, ISTD solution (two times). Fill micropipette with the ISTD solution and dispense into
sample vial.
10.3 Internal standard amount:
Fraction
Hexane
40%
40%
MeOH
Compound
PCBs and
Pesticides
Pesticides
PAHs
Atrazine
ISTD
PCB30
PCB 204
PCB65
PCB 155
DDE
dlO-Anthracene
Triphenylmethane
d!2-
Benzo(a)anthracene
d!2-Perylene
d 10- Anthracene
Spike
Volume
(AiL)
100
100
100**
100
Amount in
Sample
(ng)*
8
6
23.7
17.5
20
200
100
200
200
200
Color
code
Red
Blue
Black
Black
*Values in this column are approximate and may change slightly depending on the exact
concentrations of the compounds in the stock solutions.
**If the sample is expected to be high in concentration of PAHs or if the final sample volume
greater than 2 mL, more ISTD solution may be added in increments of 100/^L.
10.4 Mark each sample vial label with the appropriate color code verify ISTD addition with a water-
proof marker.
Rinse the micropipette with solvent and replace glass capillary used to cover plunger.
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Volume 2, Chapter 1 by GC-ECD
10.6 Label the sample set with the following information (using the same color code as dots on vial
labels):
Date sample vials spiked.
Fraction spiked.
Initials of chemist spiking.
11.0 GC-ECD Analytical Run Conditions
11.1 Initial Check
Ensure sufficient carrier (hydrogen) and make-up gas (argon/methane, P5) to complete the run.
Change the septum as described in 8.0. Change the solvent in the autosampler wash vials.
11.2 ChemStation Control
The ChemStation software controls the GC. Analyzer I controls GCl and Analyzer 2 controls
GC2. Load the analysis method. "MULLIN.MTH" for PCBs and "ISWS'PES.MTH" for OC
pesticides. Verify method parameters are correct. Edit the method if changes are necessary and
resave the method. Copies of these methods are included in Appendix A- Method 2 or Method 3.
11.3 Load the method sequence. "ISWS'PCB.SEQ" for PCBs and "ISWS'PEST.SEQ" for OC
pesticides. Edit the sequence parameters to include:
• operator's name
• subdirectory name
• calibration standard information
• spike target levels
• final sample volume
• sample batch code
• comments
In the sequence table ensure the correct injector is selected (rear vs front) and enter the "from vial"
and "to vial" numbers for selected methods. The first hexane injection will utilize the
"BAKE.MTH" method to condition the system.
Edit the sequence sample table to enter sample ID numbers (the ID#=sample name with a ", hex"
or ", 40" suffix to indicate the hexane or 40%DCM fraction respectively). Hexane is always the
first vial followed by two vials of the calibration standard, a calibration performance check (LPC).
Samples usually begin with the 5th vial. Each sample batch will include a hexane blank, a
calibration standard, a LPC, and an end of run calibration check.
Verify the correct method name is entered in the sample log table and the multiplier is entered for
each sample (usually I). Verify the start and end vial numbers. Save the method and sequence on
disk using the path for the directory created for this sample batch. Print copies of the sequence for
the sequence log and the raw data file for the sample batch. An example of a PCB sequence is
shown in Appendix C.
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by GC-ECD . Volume 2, Chapten
11.4 GC Conditions, typical run conditions are listed below.
PCBs:
Oven Program: 158 minutes
Initial temperature: 100°C
Rampl: rc/min. to 235°C
Ramp 2: 15°C/min. to 280°C, hold 20 min.
Injector temp. 250°C
BCD: 350°C
OC pesticides:
Oven Program: 147 minutes
Initial temperature: 70°C, hold 1 min.
Rampl: 5°C/min. to 140°C
Ramp 2: 0.2°C/min. to 160°C
Ramp 3: 10°C/min. to 280°C, hold 20 min.
Injector temperature 250°C
ECD: 350°C
11.5 Calibration
A multipoint calibration curve is prepared yearly for each analyte to document the linear range. A
single point calibration standard is injected in duplicate immediately prior to a sample run.
Compare the duplicate injections of the calibration standard to ensure the system has stabilized and
is reproducible. Inject a calibration performance standard (LPC) immediately after the calibration
standard. The acceptable ranges for the LPC are defined in the QAPjP for the project. Inject a
calibration standard every 5-10 samples (for multi-batch runs) and at the end of each run.
Recalibration is required if a shift of >20% is observed for OC pesticides and >25% for congeners
1, 6, 29, 49, 101, 141. 180. 194. 206, and 209 for PCBs. The methods of internal standard
calibration is utilized. The chromatogram is divided into t\vo time segments with each segment
calibrated relative to one internal standard response for the PCB runs.
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Volume 2, Chapter 1 by GC-ECD
12.0 Data Reduction
12.1 Data Files
The electronic data are converted to compacted (zipped) format for storage and transfer to other
computers for data reduction.
12.2 Chromatogram and ISTD report
The data files are expanded (unzipped) and the appropriate method (*.MTH) and data files loaded
to view the chromatogram, generate calibration tables, and ISTD reports. Each analyte peak is
evaluated for proper peak integration, baseline correction, and peak identification. The final
integrated data and all associated files are saved on removable disks. The chromatograms and
ISTD reports are also printed as hard copy reports and compiled as a sample batch data file (filed
by the GC sequence name). The hard copy files are initialed by the data analyst. PCB and OC
pesticide macro programs convert the ChemStation data into a spreadsheet format for further data
storage and retrieval. All data are reviewed by the laboratory supervisor prior to release.
A properly integrated and identified chromatograms for a 595 ng/mL PCB standard and a 20
ng/mL OC pesticide standard are presented in Appendix C. Calibration tables and sample data are
included.
12.3 Datastorage
All chromatography data are archived including: ChemStation raw data files, processed data files,
and associated calibration and integration files along with associated spreadsheet files. These files
are stored on 5.25" and 3.5" data disks and are filed by the GC sequence name. Duplicate copies
of the raw data disks are stored in two separate buildings.
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by GC-ECD
Injection Source:
Injection Location:
Front:
Rear:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
Appendix A. GC Methods
METHOD 1- System Conditioning
Injector Information
Auto
Dual
3
5
1 stop
0 sec.
6
6
No
3
5
1 stop
0 sec.
6
6
No
Purge A/B:
A (Valve 3)
A (Valve 4)
Init Value
On
On
On Time (Min.)
0.00
0.00
Off Time (Min.)
40.00
40.00
Zone Temperatures:
Inl. A
ml. B
Det. A
Det. B
Oven Parameters:
Oven Equib. Time:
Oven Max:
Oven
Crvo
Temperature Information
Set Point
280 C.
280 C.
380 C.
380 C.
1.00 Min.
300 C.
On
Off
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Appendix A. GC Methods (Cont'd)
METHOD 1 (Cont'd)
Oven Program:
Set Point
Initial Temp: 280 C.
Initial Time: 20.00 Min.
Final Final
Level Rate(C./Min.) Temp. (C.) Time. (Min.)
1 0.00
2 (A)
3(B)
Next Run Time: 20.00 Min.
Signal Information
Save Data: Both
Signal 1
Source: Del. A
Peak Width: 0.053 Min.
Data Rate: 5.000 Hz.
Data Storage: All
Signal 2
Source: Del. B
Peak Width: 0.053 Min.
Data Rate: 5.000 Hz
Data Storage: All
Detector Information
Detector Type State
A ECD On
B ECD On
Signal Plot Information
Signal Attn. (2A) Offset (%) Time (Min.)
1 2 20 20
2 2 20 20
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by GC-ECD
Appendix A. GC Methods (Cont'd)
METHOD 2- PCBs
Method Information
This method is for IL H2O Survey.
Pre-Run Program:
Name:
Parameter:
Data Acquistion:
Data Analysis:
Sig. 2Mth:
Post-Run Program:
Name:
Parameter:
Injection Source:
Injection Location:
Front:
Rear:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
Run Time Checklist
None
On
On
None
None
Injector Information
Auto
Dual
3
5
2 stops
0 sec.
6
6
No
3
5
2 stops
0 sec.
6
6
No
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Volume 2, Chapter 1
Appendix A. GC Methods (Cont'd)
METHOD 2 (Cont'd)
Purge A/B:
A (Valve 3)
A (Valve 4)
Init Value
On
On
Zone Temperatures:
Inl. A
Inl. B
Det. A
Del. B
Oven Parameters:
Oven Equib. Time:
Oven Max:
Oven
Cryo
Oven Program:
Initial Temp:
Initial Time:
On Time (Min.)
0.00
0.00
Temperature Information
Set Point
250 C.
250 C.
350 C.
350 C.
3.00 Min.
280 C.
On
Off
Set Point
100C.
0.00 Min.
Off Time (Min.)
40.00
40.00
Level
I
2 (A)
3(B)
Next Run Time:
Rate (C./Min.
1.00
15.0
0.00
Save Data:
Signal
Source:
Peak Width:
Data Rate:
Data Storage:
Final
Temp. (C.)
235
280
158.00 Min.
Signal Information
Both
Det. A
0.053 Min.
5.000 Hz.
All
Final
Time. (Min.
0.00
20.0
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Appendix A. GC Methods (Cont'd)
Signal 2
Source:
Peak Width:
Data Rate:
Data Storage:
METHOD 2 (Cont'd)
Det. B
0.053 Min.
5.000 Hz
All
Detector Information
Detector
A
B
Cal.
Line
Cal.
Level
Type State
ECD On
ECD On
Sequence Recalibration Table
Update
Response
Factor
Update
Retention
Times
Recalib
Interval
Signal
Attn. (2A)
2
7
Signal Plot Information
Offset (%)
20
20
Time (Min.)
20
20
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Volume 2, Chapter 1
Appendix A. GC Methods (Cont'd)
METHOD 3- OC Pesticides
Method Information
This method is for IL H2O Survey.
Run Time Checklist
Pre-Run Program:
Name:
Parameter:
Data Acquistion:
Data Analysis:
Sig. 2 Mth:
Post-Run Program:
Name:
Parameter:
Injection Source:
Injection Location:
Front:
Rear:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
Sample Washes:
Sample Pumps:
Sample Volume:
Viscosity Delay:
Solvent A Washes:
Solvent B Washes:
On-Column:
None
On
On
None
None
Injector Information
Auto
Dual
3
5
1 stop
0 sec.
6
6
No
3
5
1 stop
Osec.
6
6
No
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Appendix A. GC Methods (Cont'd)
METHOD 3 (Cont'd)
Purge A/B:
A (Valve 3)
A (Valve 4)
Init Value
Off
Off
On Time (Min.)
0.50
0.50
Off Time (Min.
140.00
140.00
Zone Temperatures:
Inl. A
Inl. B
Del. A
Det. B
Oven Parameters:
Oven Equib. Time:
Oven Max:
Oven
Cryo
Oven Program:
Initial Temp:
Initial Time:
Level
1
2 (A)
3(B)
Next Run Time:
Rate(C./Min.)
5.00
0.20
10.0
Temperature Information
Set Point
250 C.
250 C.
350 C.
350 C.
1.00 Min.
300 C.
On
Off
Set Point
70 C.
1.00 Min
Final
Temp. (C.)
140
160
280
Final
Time. (Min.)
0.00
0.00
20.0
Save Data:
Signal
Source:
Peak Width:
Data Rate:
Data Storage:
147.00 Min.
Signal Information
Both
Det. A
0.053 Min.
5.000 Hz.
All
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Appendix A. GC Methods (Cont'd)
METHOD 3 (Cont'd)
Signal 2
Source: Del. B
Peak Width: 0.053 Min.
Data Rate: 5.000 Hz
Data Storage: All
Detector Information
Detector Type State
A BCD On
B ECD On
Sequence Recalibration Table
Update Update
Cal. Cal. Response Retention Recalib
Line Level Factor Times Interval
Signal Plot Information
Signal Attn. (2A) Offset (%) Time (Min.)
1 2 20 20
2 2 20 20
2-216
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
Volume 2, Chapter 1 by GC-ECD
Appendix B. GC-ECD Maintenance
B.1 Injection Port Cleaning and Liner Installation
B.I.I Turn oven, injector and detector off
B. 1.2 After everything cools, turn hydrogen off
B. 1.3 Remove autosampler towers
B. 1.4 Remove the septum retainer nut and the split/splitless weldment (the large nut underneath the
septum) to expose the injection liner. Remove the liner
B. 1.5 Open the oven and disconnect the column from injector end of the GC. The open end of the
column should not be exposed to air so plug the open end with a septum
B.I.6 Unscrew the reducing nut (inside the oven, below the injection liner). There is one gold seal and a
washer in it. Washer and seal need to be replaced each time it is taken apart
B. 1.7 Put a beaker inside the oven underneath the injection port and rinse the injector with hexane.
Clean the injection port with Q-tips and rinse again with hexane
B.I.8 Check inside the split/splitless weldment, below where the septum sits. Some septa particles may
be inside. Complete removal may be impossible, however some particles may be removed using a
small tool similar to a dental tool. Also washing hexane through the opening with a Pasteur pipet
helps, being careful not to break pipet tips off inside this cavity
B. 1.9 Place a new washer followed by a new gold seal in the reducing nut. The tapered opening of the
seal will face downward (the tapered end will be fitted to the ferrule from the column). Tighten
this reducing nut before placing the injection liner in the injection port
B. 1.10 Insert a new liner. Some liners are not symmetrical but require a specific orientation. Take care to
insert properly
B.1.11 Place a viton O- ring (rated for the injector temperature range) on the liner. Put the split splitless
weldment (big nut) on and tighten it. Install in a new septum. Replace and tighten the septum nut
B.2 Column Installation
B.2.1 Remove the column from the injector end. Remove the ferrule and all particles from the column
nut. Before removing a section of the column, insert a clean column nut onto the column,
followed by a new ferrule, with conical end pointing towards the open end of the column (small
pieces of ferrule material can get inside the column, therefore, a new section of column must be
exposed after the ferrule is on).
2-217
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD Volume 2, Chapter^
Appendix B. GC-ECD Maintenance (Cont'd)
B.2.2 Cut off the injector end of the column. Remove 0.5-1 meter from an existing column. For a new
column, remove a few inches. Make a clean cut with diamond tip score or ceramic wafer.
Examine the cut under a magnifier to ensure it is square
B.2.3 Measure 23 mm from the tip of the column. Mark this point with Liquid Paper® Before marking,
the ferrule should be between the end of the column and the 23 mm point
B.2.4 Carefully insert the column, fitted with nut and ferrule, through the reducing nut into the injection
port. Insert column far enough that the white mark is at least even with the end of the column nut,
or a little further in so that white mark is up inside column nut, and tighten slightly. As soon as the
ferrule starts gripping the column, pull the column out gently just until the white mark is seen.
Hand tighten column nut then turn at least 1A additional turn
B.2.5 Detector end of the column (if needed): Remove the column from the detector end. If a portion of
the ferrule is stuck inside the makeup gas adaptor remove by turning a threaded tool (such as a
small file) into the ferrule and pulling it out. The makeup gas adaptor may need to be removed to
remove the ferrule. When replacing the makeup gas adaptor, a 1/4 inch vespel ferrule is used.
Place a column nut and ferrule on the column in the same way as described for the injector end.
Remove a portion of the column and check for cut as described above. Measure 70 mm from the
end of the column and put a white mark on the column, place the ferrule between the end of the
column and the 70 mm point before marking. Turn hydrogen on and check the flow of gas
through the column by inserting the cut end in a beaker of hexane. Turn hydrogen off
B.2.6 Carefully fit the column into the detector, slightly tighten the column nut and pull the column out
until you see the white mark. Tighten with wrench 1A turn after hand tight.
B.3 Leak Checking and Gas Flow Measurement
B.3.1 Turn hydrogen and argon/methane on. Check leaks with a leak detector An electronic leak
detector is recommended such as the J & W AccuRATE™, 1000 flowmeter (PN2201170, Folson,
CA). Check around the septum, and at the injector and at the detector end of the column. Check
the column head pressure for the appropriate reading
B.3.2 Check the gas flow with a flowmeter. An electronic flowmeter is recommended such as the GL
Sciences Inc.,LD223 flowmeter (Tokyo. Japan).Approximate gas flow in both GCs are as follows:
Split vent 130 mL/min
Purge \ent 2 mL/min.
Total (low through detector 22 mL/min
2-218
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
Volume 2, Chapter 1 by GC-ECD
Appendix B. GC-ECD Maintenance (Cont'd)
B.4 Reassembly
B.4.1 Replace autosampler towers
B.4.2 Turn heated zones on
B.4.3 If injector was allowed to cool, retighten the septum retainer nut to avoid a leak at that point
B.4.4 Turn oven on and set to 40°C for about an hour; then increase oven temperature to 70°C for an
hour.
B.4.5 If a used column, bake the column, injector and detector until baseline stabilizes. If the baseline
has not stabilized within an hour, other problems probably require identification and correction.
B.5 System conditioning
B.5.1 Set the temperature setting to the following (see Appendix A for GC method):
Oven: 280°C
Injector A: 280°C
Injector B: 280°C
Detector A: 380°C
Detector B: 380°C
B.5.2 Run approx. 6 hexane blanks by the instrumental method being evaluated
B.5.3 If a new column, bake injector and detector by ramping oven temp 1 -2°/min. to 280°C and hold
there for 1 hour. After conditioning, run 6 hexane blanks by the method being evaluated
B.5.4 If the hexane blank runs look satisfactory, evaluate a calibration standard before resuming analysis
of samples.
2-219
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Appendix C-1. PCB Sequence
_;..ien_e : C • \ HPCHEMX 1 \ SEQUENCEX ISW5 ' PCB. SEQ
•'-aerator: MONTE
Se.:n.i=:r,ce prepa ra t ion date: 03 Mar 95 03:56 PM
Data Tile Subdirectory: 950303H
Part of methods to run: full method
On j barcode mismatch: inject anyway
Comment:
Preparation dates:
595 t- PES STD = 05 AUG 94
3O,204 I.S. - 21 NOV 94
=05 AUG 94
342 QC CHK
Slowdown volumne: 1 ml
Injection: 2 ul, column: DB5, 6O m.
Sample set: 941222F
•^q. Vial Sample
as Num. Name
FRONT
1
2
*
2
2
2
3-
o
2
2
2
2
2
2
2
2
2
2
2
2
o
2
2
2
2
21
21
22
24
25
2 6
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
4 5
HEXANE
HEXAME
595 STD 950303
595 STD 950303
342QCCHK 9503O3
XPFB 950227 HX
XP01 950227 HX
LB 950206C HX
L3XT4 950206C HX
LBX14 950206C HX
HEXAME
595 STD 950303
CMS 941222F HX
LB 941222F HX
BH01F 941100 HX
CH01F 941100 HX
IHOIF 94110O HX
IH01F 941117 HX
IH02F 941117 HX
JH01F 941100 HX
MH01F 9411OO HX
UH01F 941100 HX
VH01F 941100 HX
WHOIF 941100 HX
HEXANE
595 STD 950303
Sample Log Table
Sample Multiplier
Amount
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ISTD Cal. Method
Amount Line Name
BAKE
MULLIN
MULL IN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
MULLIN
Inj/
Vial
I
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
I
2-221
-------
u
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01
rn a
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C3
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter 1
Appendix C-3. PCB Calibration Table
Method: C:\HPCHEM\1\METHODS\PCB1.MTH
Calibration Table
Amt/Area Ref Istd
1.3056e-003
3.0529e-003
3.9365e-004
1.6441e-004
83S9e-OO4
1031e-OO4
0083e-005
4475e-OO4
8.7173e-OO5
1.3793e-004 Ref ISTD
1.89O6e-004
1-0793e-004
1.34O3e-OO4
2.9468e-004
9.177e-O05
l.O768e-004
1.6519e-004
2.2082e-004
1.1757e-OO4
1.8308e-004
1.713Se-004
.8577e-004
8.6374e-005
1.7372e-004
9.398e-005
7.6311e-005
2.8382e-004
1.1769e-004
1.1351e-004
1.4022e-004
1.3413e-OO4
1.0043e-004
1.1243e-004
8.9794e-005
1.1166e-004
1.3076e-004
1.967e-004
1.1316e-004
1.4252e-004
1908e-005
.204e-004
0325e-004
4191e-004
1965e-004
0377e-004
0515e-004
1703e-004
1-0315e-004
1.4375e-004
1.713e-OO4
6.4548e-005
9.747 5e-005
^i995e-005
>
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
"!3
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
16
47
48
49
50
51
52
53
RT
18.270
23. 510
26. 667
30. 633
32.320
33.226
33. 481
35.533
36. 402
37 . 938
39.578
39.772
4O. 5O1
40.738
42 . 112
42.259
43. 516
43. 678
45. 988
47 .085
47 . 393
48 . 5O4
49. 827
50.173
5O. 457
51.210
51. 444
52 .288
53. 676
55.064
55.403
55.714
56.089
56. 199
56. 441
58.432
58.756
58 . 905
60. 351
60. 510
61 .758
63. 198
64 . 142
64 .781
6 5 . 57 6
66. 055
66. 278
67 . 309
68 . 855
69 . 720
70.133
70 - 667
7i : 4 65 "
Lvl
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
ng
42.0
24 . 5
11 . 9
4 .2
6.65
49.0
40.0
22.19
O. 98
9. 12
0.595
0. 3395
12. 95
12. 95
0. 175
0.735
7.0
6. 65
0. 1855
2 . 52
1. 12
32 . 9
O.112
11.55
2.24
0. 63
10. 15
3. 115
1. 4
15.75
0. 945
8.05
3. 5
3. 5
4.74
15.05
4 .2
4 . 9
8.05
6. 3
3.29
0.385
0.735
6. 65
11. 9
18.2
7 .0
1 .785
12 .25
6. 3
0 . 35
6 . 3
~2 ."59
A
1.
3.
3.
1 .
2 .
6 .
7 .
4 .
8.
1.
1 .
1 .
1.
2.
9
1.
1.
2 .
1 .
1 .
1.
1 .
8.
1.
9
7 .
2 .
1.
1.
1.
1.
1 .
1.
8 .
1.
1 .
1
1.
1 .
9.
:
i .
i .
i .
i.
2 .
1
1
1
6
9
" i
I# Name
1 1 (14)
1 3
1 4 + 10
17 + 9
1 6
18 + 5
1 HCB
1 14 surrogate
1 19
1 30 internal std
1 12
1 13
1 18
1 15 + 17
1 24
1 27
1 16
1 32
1 29
1 26
1 25
1 31 + 28
1 21
1 33
1 53
1 51
1 22 (65)
1 45
146
1 52
1 43
149
1 47
1 48
1 65 surrogate
1 44
1 37
1 42
1 41 + 71
1 64
1 40
1 10O
1 63
1 74
1 70 + 76
1 66
1 95
1 91
1 56 + 60
1 92 + 84
189
1 101
1 "99
2-228
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Appendix C-3. (Cont'd)
Method: C:\HPCHEM\1\METHODS\PCB1.MTH
54
55
36
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
72.
73.
74 .
74 .
75.
75.
76.
76.
76.
76.
78.
79.
79.
8O.
81.
81.
82.
83.
84 .
85.
87.
89.
89.
90.
90.
91.
91.
92 .
92 .
93.
94.
94 .
95.
95.
97 .
98 .
98.
99.
99.
100.
100.
101.
101.
102.
102.
547
267
158
786
081
690
087
281
557
807
489
137
903
72O
247
512
777
171
778
791
838
077
377
273
627
476
996
377
886
430
251
673
409
834
241
073
802
Oil
560
141
394
182
376
248
667
103.291
103.
106.
846
907
02 108.001
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104
105
106
107
108
109
108.
642
109.427
109.
111.
114.
115.
117
532
955
.145
. 463
906
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
2
0
3
0
0
1
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1 . 9S
0. 56
3 . 5
2.45
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9. 8
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2775e-OO5
76746-005
4062e-005
. 424e-004
1511e-005
1276e-004
1117e-004
0671e-004
. 189e-004
0175e-004
6984e-005
3983e-005
. 094e-OO4
I219e-004
5132e-005
O255e-004
6548e-005
6698e-004
2233e-005
9865e-005
3325e-005
. 633e-005
. 995e-005
0607©-004
0594e-O04
6136e-005
1609e-004
2456e-OO5
O278e-OO4
7425e-005
6864e-005
5002e-OOS
1549e-004
5655e-005
9148e-005
9665e-005
7744e-005
1043e-005
6.771e-005
7.299e-005
7
1
8
8
9
1
7
7
.
.
.
.
.
5.
1
1
1
9
7
6
8
8638e-005 Ref ISTD
1516e-O04
7529e-OO5
7835e-005
8563e-005
1757e-004
8345e-005
8308e-005
5328e-005
2464e-004
0391e-OO4
6017e-OO4
87O5e-005
1185e-005
4941e-005
7518e-005
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
119
33
97
81
87
85
136
DDE
77
110
82
151
135
107
123
118
(166)
+ 144
+ 149
134R
114
146
132
141
137
130
163
158
129
178
166
175
187
183
128
167
185
174
177
202
156
173
157
2O4
172
197
18O
193
191
199
170
198
201
203
196
189
208
207
+ 131
+ 153 + 105
+ 176
+ 138
surrogate
+ 182
+ 171
+ 200
internal std
+ 190
+ 195
2 194
2-229
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD Volume 2, Chapter 1
Appendix C-3. (Cont'd)
Method: C:\HPCHEM\1\METHODS\PCB1.MTH
110 118.652 1 0.385 9.8299e-OO5 2 205
ill 123.947 1 2.38 9.5285e-OO5 2 2O6
12 128.795 1 0.042 5.172e-OO5 2 2O9
Calibration Settings
Title:
Reference window: O.2 SO %
Non—reference window: O.25O %
Units of amount:
ng
Multiplier: l.O
RF uncal peaks: O.O
Sample Amount: O.O
I# Amount
1 9. 12
2 6. 03
Fit: Linear
Origin: Force
Sample ISTD Information
Multilevel Information
2-230
-------
a.0e4-,
r\3
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CO
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BHO1C 94O6OOA HX
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50
100
1 in C:\HPCHEM\CHECKING\JN94CH\02'?'F'0201 .D
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter!
Appendix C-5. PCB Sample Report
Internal Standard Report
r^ta File Name
orator
Instrument
Sample Name
Run Time Bar Code
Acquired on
Report Created on
Last Recalib on
Multiplier
C:\HPCHEM\1\DATA\JN94CHN027F0201.D
MONTE
ANALYZER1
BHO1C 940600A HX
12 Nov 94 09:52 AM
26 Jan 95 11:OO AM
18 JAN 95 02:25 PM
Page Number
Vial Number
Injection Number :
Sequence Line :
Instrument Method:
Analysis Method
Sample Amount
ISTD Amount
1
27
1
2
MULLIN.MTH
ISWS 'PCB.MTH
0
8 . 09
Sig. 1 in C:
Ret Time
18.
23.
26.
30.
32.
33.
33.
35.
36.
37.
39.
39.
40.
40.
42.
42 .
43.
43.
46.
47 .
47 .
48 .
49.
50.
50.
51.
51.
52 .
53.
55.
55.
55.
56.
56.
56.
58.
58 .
\HPCHEM\1\DATA\JN94CH\027FO201.D
Area Type Width Ref# ng
270 * not found *
51O * not found *
667 * not found *
633 * not found *
345
256
516
564
429
976
651
812
533
775
146
291
558
718
040
130
436
597
908
234
509
268
499
337
723
119
454
770
143
256
499
485
.807
58. 958
60.
6O.
61.
. 398
, 559
, 805
12677
31808
412806
22467
16004
26312
1799
1324
96535
52131
3918
17953
33925
96117
3301
21909
10143
328313
128O9
46002
30698
1O702
77745
33277
15803
129655
7688
84873
32633
41157
12988
120051
26628
47016
64729
66442
22329
VP
w
VP
w
pp
pp
PVA
W +
W
W
PV
VP
PV
vv
VP
w
VP
WA
W
W
VBA
PV
VP
VP
VP
PV
w
VP
PV
w
VP
w
w
w
PV
w
BV
o.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
o.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
o.
0.
142
126
114
114
125
110
123
000
122
124
089
116
105
117
131
121
116
141
137
128
126
109
107
117
126
118
118
116
100
114
113
112
111
116
116
113
114
1
1
1
1
1
1
1
1
1
1-IR
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8.
43.
64.
22.
3.
8.
0.
0.
28.
34 .
0.
4 .
12.
47.
0.
8.
3.
135.
2.
17.
6.
1.
49.
8.
3.
40.
2.
18.
8.
8 .
3.
34.
11.
11 .
20.
13.
5.
013
271
487
272
110
090
758
318
841
242
801
309
492
309
865
940
874
949
48O
813
431
82O
184
730
998
523
299
999
178
238
233
990
675
859
564
612
993
Name
1 (14)
3
4 + 10
7 + 9
6
8 + 5
HCB
14 surrogate
19
30 internal std
12
13
18
15 + 17
24
27
16
32
29
26
25
31 + 28
21
33
53
51
22 (65)
45
46
52
43
49
47
48
65 surrogate
44
37
42
41 + 71
64
40
2-232
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Appendix C-5. (Cont'd)
63.240
64 . 196
64 . 831
65. 628
66. 107
36.322
67 . 353
68 . 895
69.759
70. 168
70.710
71. 508
72 .586
73. 304
74 .194
74.825
75. 119
75.725
76.121
76.315
76. 585
76.846
78.515
79.173
79. 935
80.750
81 .280
81. 545
32. 806
83.195
84 .812
85. 828
87 .851
89.103
89. 401
90. 310
90. 659
91. 500
92 .025
92. 404
92 . 911
93. 462
94 .280
94 - 696
95. 429
95.852
97.268
98. 100
98.817
99.033
99.584
00. 16O
.LOO. 420
101 . 204
101. 391
102.272
102 . 694
YbVYYb
4783 WA
2246 PV
472O6 VP
93861 PV
8O996 W
100355 W
23132 VBA
75O78 W
60811 W
6948 W
126376 W
56295 W
3491 PV
9134 PV
53186 W
4815 W
85185 W
32140 W
17911 W
5312 W
1O8O3 W
205206 W
27812 W
3O6O3 W
34593 PV
19518 WA
158360 W
263380 WA
16415 W
8442 W
59396 PP
640338 PVA
122682 PV
8579 W
43527 W
643704 PV
82819 WA
43235 WA
14459 W
27226 W
4843 W
74608 W
43770 BVA
15O638 WA
25738 W
9196 W
76068 W
42381 PV
29417 PV
57397 WA
3408 W
3235 W
39158 W
1O858 W
1154 W
1OO8O9 W
5435 W
"3
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter 1
Appendix C-5. (Cont'd)
103.
106.
107.
108 .
109.
J9.
Ill .
114 .
115.
117 .
118.
123.
128 .
866
925
987
661
446
558
969
161
458
920
720
962
801
2834
51283
2283
12476
7106
6104
1011
4010
561
4071
392
760
153
W
W
PV
W
BV
W
W
PV
W
PV
PV
PV
W
0 .
0.
0.
O.
O.
0.
0.
0.
0.
0.
0.
0.
0.
118
135
141
130
118
112
130
134
141
135
167
117
100
2
2
2
2
2
2
2
2
2
2
2
2
2
0.433
7.838
0.247
3.035
1 . 441
1. 90S
0. 195
O. 557
0.0710
0. 695
0.0752
0. 141
0.0155
19O
199
170
198
201
203
196
189
208 + 195
207
194
2O5
206
209
Time Reference Peak
1O
94
Expected RT
37.938
100.394
Actual RT
37.976
1OO.420
Difference
0.1%
0.0%
Not all calibrated peaks were found
2-234
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Appendix C-6. PCB Integrator Event Report
hod: C: \HPCHEM\1\METHODS\PCB1.MTH
Events:
initial Area Reject
Initial Peak Width
Shoulder Detection
Initial Threshold
Integrator OFF
Area Reject
Integrator ON
Negative Peak ON
Baseline Now
Baseline Now
Baseline Now
Baseline Hold ON
Baseline Hold OFF
Baseline Now
Baseline Now
Baseline Now
Negative Peak OFF
Baseline Now
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
seline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
Baseline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
Baseline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Baseline Now
Baseline Now
Negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
seline Now
negative Peak OFF
Area Sum ON
Area Sum OFF
Negative Peak ON
Negative Peak OFF
Area Sum ON
Integration Events
Value: Time:
200 INITIAL
0.040 INITIAL
OFF INITIAL
-6 INITIAL
0.000
100 1.150
15.107
15.107
19.190
24.907
25.960
25.960
26.549
26.550
28.887
30.360
30.422
30.513
30.517
30.693
30.956
31.797
32.950
32.967
32.968
33.129
33.855
36.273
37.730
39.559
39.726
39.894
40.085
43.413
48.157
48.160
48.837
49.329
49.432
50.727
61.550
63.083
63.329
63.554
63.862
67.777
74.509
74.550
74.756
77.176
79.801
80.950
2-235
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume2, Chapter!
Appendix C-6. (Cont'd)
Method: C:\HPCHEM\1\METHODS\PCB1.MTH
Area Sum
A r-ea Sum
seline
Area Sum
Area Sum
Negative
Negative
Area Sum
Area Sum
Negative
Negative
Area Sum
Area Sum
Area Sum
Area Sum
Negative
Baseline
Negative
Area Sum
Area Sum
Area Sum
Area Sum
Negative
Negative
Area Sum
ea Sum
Area Sum
Area Sum
Negative
Baseline
Baseline
Baseline
Title:
ON
OFT
Now
ON
OFF
Peak ON
Peak OFF
ON
OFF
Peak ON
Peak OFF
ON
OFF
ON
OFF
Peak ON
Now
Peak OFF
ON
OFF
ON
OFF
Peak ON
Peak OFF
ON
OFF
ON
OFF
Peak ON
Now
Now
Now
OFF
81
82
82
82
82
83
85
86
86
88
90.
90.
91.
91 .
91.
92.
93.
93.
94 .
94 .
94 .
95.
96.
98 .
99.
99.
100.
101.
1O1.
1O3.
109 .
120.
13O.
.893
.499
. 500
.522
.706
. 975
.388
.O6O
.408
.425
.025
.860
.300
. 6O2
.857
.204
. 977
. 981
. 384
. 496
.876
.234
.107
.456
. 149
. 440
.828
.074
.700
.053
.260
78O
OOO
Calibration Settings
Reference window:
Non—reference window:
Units of amount:
Multiplier:
RF uncal peaks:
Sample Amount:
It
1
2
Fit :
Amount
9. 12
6.03
Li nea r
0.250 %
0.250 %
ng
1.0
0.0
O.O
Sample ISTD Information
Multilevel Information
2-236
-------
.4e4-
IV!
OJ
1 . 1 e4-
.Oe4-
9OOO -
80OO-
VOOO-
6OOO-
5OOO -
4OOO -
a —HCH
g-HCH
Mixed.
cong. B5
Standard
; — chlor~dane
cong. 155
dieldrin
I DDE
DDD
DDT
40
60
80
1 OO
Sig. 1 in C:\HPCHEM\CHECKING\95O3O34F\O43FO2O1.D
^
•o
T3
-------
SOP lor the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter!
Appendix C-8. Pesticide Calibration Table
Method: C:\HPCHEM\1\METHODS\PEST1.MTH
PkO
1
2
3
4
5
6
7
8
9
10
11
RT
2O.477
23.517
39.765
56.179
58.529
61.385
63.406
68.317
74.299
92.31O
111.059
Lul
1
1
1
1
1
1
1
1
1
1
1
Calibration Table
ng Ant/Area Ref Istd
28.0 3.4963e-OO4
20.0 3.9724e-OO4
4.74 4.2976e-005
20.0 2.2182e-004
14.99 1.5123e-004
20.0 2.0852e-O04
20.0 2.0738e-004
20.8 2.816e-004
20.0 2.6304e-004 Ref ISTD
20.0 4.7227e-004
2B.O 8-94*1e~004
Iff Name
1 A-HCH
1 G-HCH
1 CONG 65(1.S)
1 G-CHLORDAHE
1 CONG 155(1.S)
1 fl-CHLORDANE
1 T-NONACHLOR
1 DIELDRIN
1 DDECI.S)
1 ODD
1 DDT
2-238
-------
2.Oe4-
.5e4
rv>
CO
to
.Oe4-
5OOO -
A— a—HCH
B- g—HCH
C— cong. 65
D— g —.
E— eong. 155
F1— a — chlordane
G— t —:
H— dieldrin.
I— DDE
J- DDD
K— DDT
>— Vapor-
IHO1C 94O8OO
Jlliiiiiiiin mi IAI iiini i
I I
A B
I I I I I
DE FG H
I
K
4O
eo
so
100
Sifi. 1 in C:\HF>CHEM\CHECKING\94 1 O2&C4XO4QFO2O 1 .D
T3
TJ
(D
D
a
O
to
(D
(0
(D
CO
•g.
a>
O
3-
o
ED
O
CO
Ni
?
O
B
•n
O
oo
(n
Q)
a
18
O T>
o -j.
II
O o> ^-
^?l.
O (n
-------
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Volume 2, Chapter!
Appendix C-10. Pesticide Sample Report
Internal Standard Report
Data File Name
Operator
Instrument
Sample Name
Run Tine Bar Code
Acquired on
Report Created on
Last Recalib on
Multiplier
D :\HPCHEN\2\DATA\941 025C4\049F0201 .D
MONTE Page Nunber
ANALVZER2 Dial Hunber
IH01C 940800 40 Injection Number
Sequence Line
06 Nou 94 02:21 PM Instrument Method
14 Feb 95 03:44 PM Analysis Method
14 FEB 95 03:19 PM Sanple Anount
1 ISTD Anount
1
49
1
2
ISWS'PES.MTH
PEST2.HTH
0
20
Sig. 1 in D:
Ret Tine
I
20.676
23.773
40.294
56.960
59.336
62.249
64.293
69.273
75.279
93.453
112.349
:\HPCHEM\2\DATA\941025C4\049F0201 .D
Area Type Width Reffl ng Name
77562 UP
27066 PU
25633 PU
57957 UU
35618 PU
50086 UU
32679 PP
127351 PU
29322 PU
936 PP
2646 PP
I I 1
8.088 1
0.094 1
0.181 1
0.328 1
0.311 1
0.347 1
0.340 1
0.371 1
0.373 1-IR
0.278 1
0.374 1
1 - _ _
51.647 A-HCH
19.116 G-HCH
13.009 CONG 65
28.780 G-CHLORDANE
15.320 CONG 155
23.779 A-CHLORDAKE
15.738 T-NONACHLOR
92.475 DIELDRIN
20.000 DDE
1.-33 ODD
5.127 DDT
Tine Reference Peak
9
Expected RT
75.220
Actual RT
75.279
Difference
O.tt
2-240
-------
Volume 2, Chapter 1
SOP for the Analysis of
PCBs and Organochlorine Pesticides
by GC-ECD
Appendix C-11. Pesticide Integrator Event Report
Method: C:\HPCHEM\1\METHOOS\PEST2.MTH
Events:
Initial Area Reject
Initial Peak Width
Shoulder Detection
Initial Threshold
Integrator OFF
Integrator ON
Negative Peak ON
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Baseline Now
Integrator OFF
Title:
Reference window:
t-reference window:
o.iits OF amount:
Multiplier:
RF uncal peaks:
Sample Anount:
Itt
1
Amount
4.74
Integration Euents
Ualue: Tine:
100 INITIAL
0.040 INITIAL
OFF INITIAL
-6 INITIAL
0.000
19.845
20.000
23.647
25.503
28.757
35.383
43.350
52.060
68.057
115.000
Calibration Settings
0.250 %
0.250 *
ng
1.0
0.0
0.0
Sample ISTD Information
Hultileuel Information
Fit: Linear
Origin: Force
2-241
-------
Standard Operating Procedure for
Isolation, Extraction and Analysis of
Atrazine, DEA and DIA
Steven Eisenreich, Shawn Schottler, and Neal Mines
Department of Environmental Sciences
Rutgers University
P.O. Box 231
New Brunswick, NJ 08903
1994
-------
Standard Operating Procedure for
Isolation, Extraction and Analysis of Atrazine, DEA and DIA
1.0 General Principal
Atrazine, DEA and DIA will be isolated from Lake Michigan water samples using Carbopak SPE
cartridges. Analytes will be extracted from SPE cartridges with 7 mL of 859r dichloromethane
(DCM), I5<7c methanol (MeOH) solution (vol:vol), followed by 2 mL of MeOH. Concentrated
extracts will be spiked with internal standard and analyzed by gas chromatography-mass
spectrometry.
2.0 Isolation
Samples will be collected on board the EPA research vessel Lake Guardian according to the
Standard Operating Procedure for sample collection. Samples will be transported to the University
of Minnesota and stored at 4°C until processing. Samples will be stored for less than 30 days until
extraction.
Carbopak (Supelco) solid phase extraction cartridges (SPE) will be used for herbicide isolation.
SPE cartridges (250 mg) will be cleaned prior to use with 3 mL of 85% DCM, 15% MeOH
solution (vol:vol). Just prior to isolation cartridges will be conditioned - 2 mL of MeOH, followed
by ~2 mL of Milli Q water. Conditioning the cartridge is critical to achieving complete herbicide
isolation, and facilitating the passing of sample water. Conditioning of SPE cartridges should be
done less than 15 minutes before passing sample water through the cartridge.
Solvents and water are pulled through the SPE cartridge under vacuum. Samples are interfaced to
the SPE cartridge using Teflon tubing and a rubber stopper. One end of the Teflon tubing is
inserted through the stopper about one inch. The stopper and protruding tubing are then pressed
into the reservoir end of the SPE cartridge. The other end of the Teflon tubing is placed in the
sample bottle. The leur end of the extraction cartridge is connected to vacuum, and sample water
is pulled through the cartridge at ~ 20 mL/min. Flow rate is controlled by adjusting the vacuum.
Teflon tubing is cleaned before each extraction by placing one end of the tubing into a reservoir of
MeOH, and inserting the stopper end into a •'dummy" extraction cartridge as though it were a
sample. MeOH is then pulled through the tubing under the vacuum. This procedure is repeated
with Milli-Q water as a rinse.
Prior to isolation the sample will be spiked with 50 uL of a surrogate solution containing
terbutylazine. The sample should be spiked on the same day it is to be passed through the SPE
cartridge. A 50 uL micro-pipetter fitted with a glass capillary tube is used for surrogate spiking.
The same micro-pipetter should be used for spiking all samples.
Sample volumes are determined using an E2000 Mettler top loading balance. The full sample
bottle is placed on the balance and the balance is tared to zero. After the sample has been passed
through the SPE cartridge the empt\ --ample bottle is re-weighed and the sample volume calculated
b\ difference.
2-245
-------
SOP for isolation, Extraction and
Analysis of Atrazine, PEA and DIA Volume 2, CftapteM
Once the sample has been passed through the SPE cartridge, the cartridge is labeled, disconnected
from the vacuum line, wrapped in aluminum foil, and stored at 4°C. Cartridges are labeled with
all information on the sample bottle including: Lake, station, date, depth and code number. The
volume of the sample is also put on the label. All extraction information is also entered into the
lab log book. The lab log book should contain all information transferred from the field sampling
log book, along with the data of sample isolation procedure, volume of sample, and amount of
surrogate added.
3.0 Extraction
SPE cartridges are removed from cold storage, the aluminum foil removed, and allowed to reach
room temperature.
Herbicides are extracted from the SPE cartridge by passing 7 mL of 85% DCM, 15% MeOH
(vol:vol) solution, followed by 2 mL of MeOH through the cartridge. Extraction solvent pass
through the cartridge by gravity. The small amount of solvent remaining in the cartridge after
extraction is forced through using a syringe fitted with a rubber stopper. The bored out stopper is
inserted into the reservoir end of the cartridge and remaining solvent is forced through using the
syringe.
As the extraction solvent moves through the SPE cartridge, a small spatula full of anhydrous
sodium (0.75 g) sulfate is added to the extractant in the centrifuge tube. The sodium sulfate
removes any water remaining from sample isolation. The solvent is then collected by the
centrifuge tube. Care must be taken to insure that the solvent flows evenly through each step of
the extraction, and does not build up and overflow the pipette. Care must also be taken to insure
that the leur end of the cartridge is centered in the pipette, if it is not solvent may drip along the
outsides of the pipette.
Once all the extraction solvent has been collected in the centrifuge tube, the tube is labeled with all
information on the SPE cartridge, sealed with aluminum foil, lined, capped, and stored at 4°C. If
any solvent is lost, or spilled during extraction this should be noted on the label, and in the lab log
book.
Extracted samples should be stored no longer than two weeks in centrifuge tubes. In less than two
weeks the extracted sample should be solvent reduced and concentrated. Sample volume is
reduced by placing the centrifuge tube in a Supelco Visidry Evaporation Manifold, attached to
zero graded nitrogen gas source. Solvent is evaporated under a slow steady stream of nitrogen.
About 25 pounds of pressure is desirable, but all samples should be visually inspected to insure
that solvent is not mixing violently or splashing.
Samples should be blown down to 0.3 mL in the centrifuge tube. When the sample has been
reduced to 0.3 mL it should be removed from the evaporation manifold, capped and placed in a
test tube rack. The sample should then be transferred immediately to a 2 mL amber vial. The
sample should not be stored in the reduced volume condition as it may evaporated to dryness. The
sample should be transferred to a 2 mL via! using a 9 inch disposable pipette and pipette bulb.
The centrifuge tube should be rinsed with S5cf DCM: 159r MeOH solution three times, and the
rinse aKn transferred to the amber vial
2-246
-------
SOP for Isolation, Extraction and
Volume 2, Chapter 1 Analysis of Atrazine, PEA and DIA
The amber vial should be labeled with all information on the centrifuge tube, and stored at 4°C. If
any solvent is spilled or lost during transfer this should be noted on the label and in the lab log
book.
4.0 Analysis
Approximately 15 samples at a time should be prepared for analysis. Amber vials should be
removed from cold storage and allowed to reach room temperature before uncapping. It is
important that samples be allowed to reach room temperature before uncapping, otherwise
condensation forming on the vials may enter the sample, and contaminated the sample with water.
Once the samples have reached room temperature they can be uncapped and placed in the Supelco
Visidry Evaporation Manifold. The 2 mL vials should be concentrated to -150 uL under a slow
steady stream of nitrogen.
After the samples have been concentrated they should be spiked with 2 uL of the internal standard
solution containing deuterated, d,ethyl atrazine and 4,4'-dibromobiphenyl. Samples should be
spiked using a 2 |aL Hamilton syringe fitted with a cheny syringe guide. The cheny syringe guide
assures that every injection spike is identical. The cheny adapter has a stopper fitted on the guide
so that each injection is automatically set to deliver 2 uL. The stopper guide is calibrated at the
beginning of the project to properly set the syringe for exactly a 2 |aL injection. This is
accomplished by making repeated injections of 20°C water onto an analytical balance, and
adjusting the guide stop until the syringe consistently deliver 2 |aL. It is very important the internal
standard solution spikes are accurate and consistent since quantitation is based on these injection
volumes (or mass per injection volume). Once a sample has been spiked with internal standard,
the date and amount should be recorded in the lab log book.
Samples that have been spiked with internal standard should be run within one week after spiking.
As long as samples are stored at 4°C, and in amber vials the internal standards are stable, but
prompt analysis of the samples is recommended to lessen the chance of errors, sample evaporation,
breakage, loss, and to enhance analytical consistency.
When a set of samples has been spiked and is ready for analysis the following steps should be
followed:
1. Remove spiked samples and calibration standards from cold storage, and allow to reach
room temperature.
2. Initiate AutoTune calibration function of GC-ms chem station.
3. Transfer ~ 50 jaL of each sample and each calibration standard to a 2 mL auto sampler vial
with 250 uL micro vial insert.
4. Four calibration standards are run daily. Calibration standards are preparedfrom a stock
solution, and are the same calibration standards used by Schottler and Eisenreich.
5. Load samples and calibration standards into auto sampler tra\. and log samples into
GC-MS chem station sequence function.
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SOP for Isolation, Extraction and
Analysis of Atrazine, PEA and DIA Volume 2, Chapter 1
6. Enter sample order, sample identification, and Adaption values in GC-MS sample run log
book.
7. Fill auto sampler solvent wash vials with DCM and MeOH.
8. Run calibration standard (1) twice, and inspect to make sure all peaks are present and peak
shape is good.
9. Initiate auto sampler and run chem station sequence function.
10. Acquire data and use internal standard method to quantitate, see Section 10.0 of QAPjP.
5.0 Blanks
5.1 Procedural Blanks
One procedural blank should be run with every 20-25 samples.
A procedural blank is a SPE cartridge that is processed identical to a sample with the exception of
water being passed through the cartridge. A procedural blank is cleaned, eluted, concentrated and
analyzed identical to a sample.
5.2 Solvent Blanks
Once every six months, or any time a procedural or field blank shows contamination a solvent
blank should be run.
A solvent blank is simply the analytical reagents analyzed by GC-MSD for possible contaminants.
If a solvent blank shows contamination the auto sampler injection syringe should be changed and
second solvent blank run. Blank contamination often results from dirty syringe needles, and
should be tested as described to eliminate or confirm solvent as contaminated.
6.0 Sample Locations
Remember:
Rinse three times
Fill two bottles per one sample
Label bottle and cap
6.1 Open Water Stations:
6.1.1 If Stratified
"Mid F.pi
"Mid Hypo (If possible, sample h\po at depth that corresponds to mean particle mass
as measured h\ transmissometerv)
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Volume 2, Chapter 1
SOP for Isolation, Extraction and
Analysis of Atrazine, PEA and PI A
6.1.2 If Not Stratified
*2 ft. below surface
*Mid-water column
Collect Duplicates of two open water stations. One station in Northern LM and one in
Southern LM. Put "DUP" on label.
6.2 Master Stations:
6.2.1 If Stratified
Stations 18 and 41
*2 ft below surface plus duplicate
*5 ft below surface
*Mid Epi
*Thermo
*Mid Hypo
*5 ft off bottom plus duplicate
*plus duplicates of all depths at St. 23 and put "BE DUP" on label
6.2.2 If Not Stratified
All Master Stations: (18,23,27,41,47)
*2 ft below surface
*Mid water column
*5 ft off bottom
*Duplicates of all depths at Stations 18,23,41
(Put BE on St. 23 label)
Mark St. 18 and St. 41 Duplicates with "DUP," St. 23 with "BE DUP" in addition to
regular sample label.
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Standard Operating Procedures for
Semivolatile Organic Compounds
in Dry Deposition Samples
Steven Eisenreich and Thomas Franz
Department of Environmental Sciences
Rutgers University
P.O. Box 231
New Brunswick, NJ 08903
1994
-------
Standard Operating Procedures for
Semivolatile Organic Compounds
in Dry Deposition Samples
1.0 Overview
1.1 Tare weights and final weights of dry deposition plates are determined at the Illinois Institute of
Technology (IIT).
1.2 Immediately after the final weight is determined at IIT, two to four deposition plates from each
site during each sampling period will be placed into precleaned 120 mL wide-mouth amber
bottles, capped, labeled and stored frozen in the dark until shipped overnight unfrozen to Rutgers
University/University of Minnesota (RU/UMN) via Federal Express.
1.3 Upon receipt at RU/UMN, samples are again stored frozen in the dark until analyzed.
1.4 Storage of samples will be limited to about one to two years prior to extraction.
2.0 Sample Extraction at RU/UMN
2.1 General Notes
All laboratory glassware and utensils are washed with Alconox and rinsed with tap water, Milli-Q*
water (Millipore) and acetone and baked overnight at 450°C. All equipment is wrapped in
aluminum foil during storage in the laboratory. Only pesticide grade solvents (Baxter) and reagent
grade solids are employed as required during analytical procedures. Whenever clean glassware is
used, the aluminum foil is first removed and the glassware rinsed with dichloromethane (DCM)
prior to use. If at all possible, glassware openings are re-covered with aluminum foil during usage
to minimize exposure to laboratory air. At all transfer steps, sample codes and descriptions are
similarly labeled on the next vial containing that sample. Thus, the same sample code is passed
from the original sample bottle to the final vial containing the final extract for instrument injection
and final archiving. All procedures and sample codes are similarly recorded in laboratory
notebooks and instrument logbooks. Detailed records of daily laboratory progress with all
pertinent observations must be noted in the laboratory notebook by the individual performing the
analytical tasks.
2.2 Specific Procedure
2.2.1 Using forceps, place all deposition strips from an individual amber bottle, representing
one site exposed for one sample cycle (= one month) or two to three month composites.
into a clean 35 mL screw-top amber glass vial.
2.2.2 Rinse the original sample bottle with two 1 mL aliquots of DCM and add to the extraction
vial.
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SOP for Semivolatile Organic Compounds
in Dry Deposition Samples Volume 2, Chapter^
2.2.3 With syringe(s). add an appropriate amount of analytical surrogate standards (3,5-
dichlorobiphenyl [IUPAC #14], 2,3,5.6-tetrachlorobiphenyl [IUPAC #65], and
2,3,4,4',5,6-hexachlorobiphenyl [IUPAC #166] for PCBs and t-nonachlor; d8-naphthalene,
dlo-fluorene, dicrfluoranthene and d,2-perylene for PAHs) to the extraction vial. (The
subset of samples for atrazine analysis will be additionally spiked with the surrogate,
terbutylazine).
2.2.4 Add 30 mL of DCM to the extraction vial to cover the greased deposition strips.
2.2.5 Place the extraction vials in an ultrasonic bath, sonicate for 30 min and store vials
overnight in a freezer.
2.2.6 Transfer the DCM to another clean amber 35 mL vial with two 1 mL hexane (HEX) rinses
of the extraction vial.
2.2.7 Concentrate the DCM extract to = 1 mL under a gentle N2 gas stream.
2.2.8 Meanwhile, add approximately 30 mL of HEX to the original extraction vial with the dry
deposition strips, sonicate for 15 min., and store vials overnight in a freezer.
2.2.9 After the DCM fraction is blown down to = 1 mL, add the HEX fraction to the DCM vial
with two 1 mL HEX rinses of the original extraction vial.
2.2.10 Discard the original extraction vial or reclean.
2.2.11 Concentrate the HEX to = 1 mL under a gentle N, gas stream.
2.2.12 The HEX sample is now ready for cleanup (unless selected for atrazine analysis).
2.3 If samples are selected for atrazine analysis:
2.3.1 General Notes: Since atrazine is not expected to be observed in most dry deposition
samples, only a select set of samples will be analyzed for atrazine. These samples will
primarily include the spring samples (April, May, June) when atrazine is most likely to be
detected on atmospheric particles. Because atrazine may not be recovered from silica
gel/alumina column during sample cleanup for PCBs and PAHs, samples selected for
atrazine analysis will be analyzed prior to clean-up. For this subset of samples, the
following steps apply immediately following Step 12 above (otherwise skip to Sample
Cleanup below).
2.3.2 Specific Procedure
2.3.2.1 Using a syringe, add an appropriate amount of ds-atrazine to the uncleaned
sample (-- 1 mL HEX in 35 mL amber vial) as internal quantification standard
LLS).
2.3.2.2 Concentrate the HEX to 200-500 uL under gentle N\ 2us stream.
2.3.2.3 Transfer the extract to an autosampler \ ial with a 250 uL glass insert and cap.
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SOP for Semivolatile Organic Compounds
Volume 2, Chapter 1 /n Dry Deposition Samples
2.3.2.4 Place the autosampler vials within an autosampler tray and analyze on an
HP-5890GC with an HP-5971A MSD using a 30 m DB-5, 0.25 mm i.d.. 25 |um
film thick glass capillary column (J&W Scientific). The MSD is operated in
selective ion monitoring mode with at least one confirmation ion.
2.3.2.5 Record sample codes in the instrument logbook in the order in which they are
injected.
2.3.2.6 After samples are injected, transfer the samples from the autosampler vial back to
the original HEX vial with four to five complete HEX rinses (= 1-1.5 mL total) of
the glass insert within the autosampler vial.
3.0 Sample Cleanup
3.1 General Notes
Concentrated samples will be cleaned up to remove the grease from the dry deposition plates and
other potential chemical interferences prior to instrumental analysis, using a 0.5 cm i.d. x 20 cm
glass column with a 50 mL glass reservoir on top. Usually, enough columns are assembled to
clean an entire sample set or at least half of a sample set in the course of one day.
3.2 Specific Procedure
3.2.1 Secure glass column to ring stand using clamps, being careful not to break column.
3.2.2 Install teflon stop-cocks.
3.2.3 Place a small clean glasswool plug into bottom of column.
3.2.4 Add pinch (= 0.5 cm) of clean anhydrous Na:SO., to column.
3.2.5 With stopcock open, rinse column with =5-10 mL of DCM, letting the column drain
completely.
3.2.6 Rinse column with =5 mL HEX and let drain to top of Na2SO4.
3.2.7 Close stopcock and add - 5 mL HEX.
3.2.8 Add 5 cm 3% deactivated silica gel as slurry in HEX while slowly letting HEX drum
from column and while tapping the column with rubber hose to help silica gel settle
evenly within column. Do NOT allow HEX to drain below level of silica gel. (Silica gel:
reagent grade. 60-200 mesh, Baker, activated overnight at 200°C, deactivated with MilliQ
water by weight and stored overnight prior to use within dessicator.)
3.2.9 Add another pinch of clean anhydrous Na:SO, to top of silica gel in column.
3.2.10 Add 5 mL HhX to column.
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SOP for Semivolatile Organic Compounds
in Dry Deposition Samples Volume 2, Chapter?
3.2.11 Add -= 5 cm 57c deactivated alumina (as dry solid) while slowly letting HEX drain from
column and while tapping the column with rubber hose to help alumina settle evenly
within column. Do not allow HEX to drain below level of alumina. (Alumina: neutral
Brockman activity 1, 60-325 mesh, Fisher Scientific, activated overnight at 450DC,
deactivated with MilliQ water by weight and stored overnight prior to use within
dessicator).
3.2.12 Add =0.5 cm clean anhydrous Na:SO4 to top of column.
3.2.13 Add 10 mL HEX to clean and condition column and let drain to top of Na:SO4.
3.2.14 Place 25 mL amber vial beneath column. This vial must be labeled with same sample
code as on sample vial to be cleaned.
3.2.15 Add sample extract to top of column and drain solvent to top of column while collecting
eluant in vial. At no time allow solvent to drain beyond level of Na2SO4 at top of column.
3.2.16 When HEX reaches top of column, add two 1 mL HEX rinses of sample vial.
3.2.17 Again, when HEX reaches top of column, add 5 mL HEX to top of column and let drain
to top of column.
3.2.18 When HEX reaches top of column, add 9 mL 1:4 DCM:HEX (v/v) and collect eluant in
the same 25 mL amber vial as above.
3.2.19 When DCM:HEX drains completely from column, cleanup is complete.
3.2.20 Concentrate eluant to = 1 mL under gentle N2 gas stream.
3.2.21 Transfer sample to a 4 mL amber vial with = 2-1 mL HEX rinses of 25 mL vial.
3.2.22 With syringe(s), add appropriate amounts of internal quantification standards (2,4,6-
trichlorobiphenyl [IUPAC #30] and 2,2',3,4,4',5,6,6'-octachlorobiphenyl [IUPAC #204]
forPCBs and t-nonachlor; and d,0-acenaphthalene, d,0-phenanthrene, dlo-pyrene,
dl2-benzo(e)pyrene and d,2-benzo(g,h,i)perylene) for PAHs.
3.2.23 Concentrate the final extract =25-100 uL under gentle N: gas stream prior to injection on
GC-ECD (PCBs and t-nonachlor) or GC-MSD (PAHs).
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Extraction and Cleanup of XAD-2 Resin
Cartridges for Polychlorinated Biphenyls and
Trans-Nonachlor
Eric Crecelius and Lisa Lefkovitz
Pacific Northwest National Laboratory
Battelle Marine Sciences Laboratory
1529 West Sequim Bay Road
Sequim, WA 98382
Standard Operating Procedure MSL-M-091-00
June 1994
-------
Extraction and Cleanup of XAD-2 Resin Cartridges for
Polychlorinated Biphenyls and Trans-Nonachlor
1.0 Scope and Application
This SOP is applicable to the extraction and analysis of polychlorinated biphenyl compounds
extracted from fresh water using XAD-2 polymeric resin cartridges. The target compounds which
can be determined by this method are, generally speaking, the polychlorinated biphenyls (PCBs)
found in Aroclors 1232, 1248, and 1262 and trans-Nonachlor. It may be applied to other
compounds once acceptable method recovery has been demonstrated.
Up to 200 liters of filtered freshwater is passed through a glass column containing approximately
400 grams of pre-cleaned XAD-2 resin at a flow rate of approximately 1 liter per minute. This
resin has been shown to efficiently extract non-polar organic compounds, including PCBs, from
freshwater. The columns are sealed, labeled, and stored under refrigeration until analysis.
At analysis the resin is transferred from the column to an extraction apparatus and rinsed with
acetone to remove external water. The surrogate compounds are then added and the resin is then
extracted with acetone to remove organics and interstitial water. Next the resin is extracted with a
mixed solvent of 50% hexane 50% acetone to remove the remaining organic compounds. The
acetone rinse and acetone extract are combined in a separatory funnel, 300 mL of reagent water is
added, and the mixture is then extracted once with 200 mL of hexane then twice with 100 mL of
hexane to remove PCBs into the hexane extracts. These hexane extracts are then combined with
the hexane-acetone extracts, reduced in volume and exchanged into hexane to a final volume of
approximately 1 mL. The extract is then applied to a cleanup column which is eluted with 60 mL
of hexane. The hexane is reduced in volume to 0.9 mL at which point 0.1 mL of internal standard
is added to complete the sample preparation.
Extracts are then subjected to gas chromatographic analysis with electron capture detection (see
SOP MSL-093-00). The chromatographic separation is performed using a 60 meter capillary
column. Identification and quantitation of the PCB compounds is accomplished by comparison to
calibration standards containing a large number of PCB congeners in known concentration.
2.0 Definitions
The following terms and acronyms are associated with this procedure:
DCM Dichloromethane
GC-ECD Gas chromatography with electron capture detection
K-D Kuderna-Danish
NA;SO4 Sodium Sulfate
rpm Revolutions per minute
SRM Standard reference material
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Extraction and Cleanup of XAD-2
Resin Cartridges for Polychlorinated
Biphenyls and Trans-Nonachlor Volume 2, Chapter 1
3.0 Responsible Staff
Project Manager: A Scientist responsible for 1) administration of the project; 2) providing project
specific quality control requirements to the laboratory, 3) defending the data in a Quality
Assurance Audit; and 4) reporting results to client.
Laboratory Supervisor: A Technical Specialist or Scientist having expertise in the principles
involved with this procedure and in the use of laboratory operations in general. Responsible for 1)
ensuring that analysts are trained in the handling of solvents; 2) that appropriate quality control
samples are included with the sample analysis to monitor precision and accuracy of the analysis;
3) checking the analysts' work to ensure that samples are handled appropriately and that data are
collected and interpreted correctly; 4) making decisions regarding problems with the analysis or
deviations from the SOP; 5) defending the data in a Quality Assurance Audit; and 6) reporting
results to project manager or client.
Analyst: A Technician, Technical Specialist, or Scientist assigned to conduct analyses using
this procedure. Responsible for 1) understanding the proper handing of samples and solvents;
2) recording information regarding extractions and any deviations from the SOP in the appropriate
log books; 3) analyzing the appropriate number of quality assurance samples for each batch of
samples analyzed; 4) reporting results to the Project Manager; and 5) participating in QA audits.
Quality Assurance Representative: A qualified staff member assigned to the Quality Assurance
Unit. Responsible for monitoring the project activities and conducting Quality Assurance Audits
to ensure that 1) analysts have conducted the analysis according to the SOP and that deviations
from the SOP have been noted in project files; 2) instrument use and maintenance records are kept
correctly; and 3) data have been reported and presented accurately.
4.0 Procedure
4.1 Apparatus and Reagents
4.1.1 Chromatography column 50 mm x 300 mm (Ace Glass Co. #5820-50 or equivalent)
4.1.2 Nylon column end caps with FETFE O-Ring (Ace Glass Co. #5845-50 or equivalent)
4.1.3 Teflon column end cap with 1/4" NPT threaded hole (Ace Glass Co. #5844-78)
4.1.4 Roller apparatus capable of rolling 250 mL Qorpak jars
4.1.5 Separatory funnels (sized to fit sample)
4.1.6 Erlenmeyer flasks, various sixes
4.1.7 Chromatography column. I 5 K 250-mm with 250 mL reservoir and No. 2 Teflon stopcock
(Kontes #42080-0222)
4 I ^ Kudema-Danish (K-Du-\,ipi>utor apparatus: 250 mL aiul-'or ^(K) mL reservoir; 3-hall
macro Snyder column; 2 or •> hall micro Snyder column; 10 mL or 25 mL concentrator
lube
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Extraction and Cleanup of XAD-2
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Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
4.1.9 Hot water bath capable of reaching IOO°C, located in a fume hood
4.1.10 Water aspirator vacuum source (Bucchi #B169 or equivalent)
4.1.11 1 Liter Soxhlet extraction apparatus complete with flask and condenser (Ace #6810-10
modified to a 1 L size or equivalent)
4.1.12 1 Liter round-bottom boiling flasks (Ace #6887-53 or equivalent)
4.1.13 Reducing Glass Joint Bushings 34/45 to 24/40 (Ace #5023-21 or equivalent)
4.1.14 Pre-Cleaned XAD-2 Resin, 20-60 mesh, 330 nr surface area, 90A pore diameter
4.1.15 Boiling chips, carborundum, soxhlet extracted or baked at >400° C
4.1.16 Glass wool, soxhlet extracted >4 hrs in 50:50 hexane-acetone
4.1.17 Nitrogen evaporation apparatus, N-Evap or equivalent, heated with a water bath
maintained at 25-35°C
4.1.18 RapidVap Evaporation System (Labconco)
4.1.19 Glass graduated cylinders
4.1.20 Stainless steel and teflon forceps
4.1.21 Steel rod, 3 mm x 50 cm
4.1.22 Microliter syringes or micropipets
4.1.23 Concentrated sulfuric acid
4.1.24 Solvents - pesticide grade or equivalent
Dichloromethane (DCM)
Hexane
Acetone
Reagent Water (Barnstead Organic-Free or equivalent)
4.1.25 Sodium sulfate - anhydrous, reagent grade, heated to 400°C for 24 hr. then cooled to
room temperature and stored in a desiccator
4.1.26 Alumina, Sigma F-20 or equivalent. 80-200 mesh
4.1.27 Silica. Amicon Matrix Silica pore diameter 60 A, particle size 105 .-m
4.1 2X Internal standard solution a hcvanc Dilution containing 100 nL''niL. nl'PCB 30. PCB 204.
andPCB 103
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Extraction and Cleanup of XAD-2
Resin Cartridges for Polychlorinated
Biphenyls and Trans-Nonachlor Volume 2, Chapter^
4.1.29 Surrogate solution - a hexane solution containing 200 ng/mL PCB 14, 50 ng/mL PCB 65,
50 ng/mL PCB 166, and 100 ng/mL dibutyl chlorendate (DEC). PCB numbers refer to
their IUPAC designations.
4.1.30 Matrix spiking solution - a hexane solution having a nominal concentration of
1830 ng/mL as total PCBs prepared from the 1994 Aroclor mixture provided by
M. Mullins, and 100 ng/mL trans-Nonachlor.
4.1.31 Acid silica gel - 30<7r (w/w) sulfuric acid
4.2 Sample Handling
Samples shall be kept cold (2-6°C) until analysis. Samples shall be extracted within 12 months of
receipt at the lab unless specified otherwise by the Project Manager or project-specific plans.
Refer to the project-specific sampling plan for sample collection, preservation, and handling
methods.
4.3 Labware Preparation
Prior to use, all glassware, Teflon, and other labware should be washed with hot, soapy water and
rinsed with tap water, followed by deionized distilled water. Teflon should be solvent rinsed,
teflon stopcocks are sonicated for 30 minutes in dichloromethane. Additionally, glassware must
be baked at 450°C for at least 4 hours.
4.4 XAD-2 Resin Preparation
Prior to use, the XAD-2 resin must be solvent extracted sequentially with a number of solvents to
remove manufacturing impurities. This process is fully described in Battelle SOP MSL-M-090-00
but is also briefly described here. The resin should be placed in a large soxhlet apparatus (1 liter
pot size or bigger) and sequentially extracted for 24 hours with each solvent, first with methanol,
then with acetone, hexane, and finally with dichloromethane. Then the resin is sequentially
extracted for four hours with each solvent, first with hexane, acetone, and finally with methanol.
The methanol is then displaced from the resin by numerous rinses with reagent water and then
stored under reagent water in the dark at cool temperatures. This procedure must be followed
closely with no shortcuts. At this point the resin is ready to be packed into columns.
4.5 XAD-2 Sampling Column Preparation
A detailed description of how the resin-containing columns used for field sampling are prepared is
contained in Battelle SOP MSL-M-090. but a condensed description is also given here. Briefly,
the column is fitted with a teflon end cap adapter which has a 1/4" FPT threaded hole through it, to
which PVC vacuum tubing is connected. The tubing is then connected to a water aspirator
vacuum source. First, several inches of pre-cleaned glass wool is added and then the column is
filled with reagent water. The resin is then added to the column with additional water rinses, and
the \acuum source is activated. The resin is not allowed to go dry and more resin is added with
water washing until approximate!}, 400 cc of resin has been added to the column. Additional pre-
clcancd glass wool is added to the top ol the resin, forcing out the water, so as to a\oid the
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Extraction and Cleanup of XAD-2
Resin Cartridges for Polychlorinated
Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
formation of air pockets. More water is added if needed and a nylon end plug is screwed onto the
column which displaces water. This is done to avoid creating any air pockets. The column is then
inverted and the opposite end is capped in a similar fashion. The columns are then appropriately
labeled and stored horizontally in a cool dark place.
4.6 Resin Sample Extraction
4.6.1 A small wad of pre-cleaned glass wool is first added to the soxhlet extractor body to
prevent any resin from escaping up the siphon tube exit. Next, a column is opened and the
glass wool removed and placed into a soxhlet extractor body. The resin is then
transferred, with acetone rinsing, into the same soxhlet body. The last plug of glass wool
is then added to the soxhlet and the soxhlet is drained of its contents into a 2 liter
separatory funnel. This acetone is saved and put aside until later. The total volume of
acetone used for this process is 400 to 800 mL. The smallest volume that effectively
transfers the resin is the volume that should be used. The soxhlet body containing the
resin is then connected to a I liter boiling flask containing several boiling stones, 800 mL
of acetone is slowly poured into the soxhlet body onto the resin bed and allowed to siphon
into the boiling flask. A 100 /J.L aliquot of surrogate standard (see Sec. 4.1) is then added
to the resin in the soxhlet body and the soxhlet condenser is attached. If the sample is to
be spiked, 100 ,uL of matrix spiking solution is added at this time. Coolant water and the
heating mantle are activated and the resin is extracted for approximately 4 hours, the heat
is then shut off and the system is allowed to cool to room temperature. During extraction
the soxhlet should cycle 4 times per hour. The acetone extraction step is important in
removing water from the resin, which is critical in making the resin hydrophobic
extractable by the non-polar extraction solvent used in the next step.
4.6.2 The soxhlet apparatus is removed from the condenser and tilted to force the acetone
extract to siphon into the 1 liter boiling flask. This extract is then combined with the rinse
acetone in the 2 liter separatory funnel. Note: To avoid contamination of the sample from
material on the exterior of the neck, before transferring extracts from containers with
ground glass joints alwavs rinse the exterior of the necks with the same solvent as that
being transferred. At this point fresh boiling stones and 800-850 mL of 50:50 hexane-
acetone are added to the boiling flask, the flask is connected to the soxhlet body, the
condenser is connected and the cooling water and heating mantle is activated. The resin is
extracted with this solvent system for > 16 hrs after which the heat is then shut off and the
system is allowed to cool to room temperature. During the extraction the soxhlet should
cycle 4 times per hour. After extraction the soxhlet body is tilted to cause the solvent to be
siphoned over into the boiling flask, where it will be evaporated.
4.7 Acetone Extract Back Extraction
To accomplish this step 300 mL of reagent water is added to the combined acetone rinse and
extract contained in the separatory funnel and this mixture is then extracted once with 200 mL
hexane and twice more with 100 mL hexane. This step is performed in order to reduce the amount
of polar or water soluble interferences being carried through the procedure and to remove water
from the acetone prior to evaporation. The hexane extracts are collected in a 1 liter boiling flask
lor evaporation. If emuKions are encountered or the acetone.ualcr and hexane phases do noi
cleanly seperate, additional reaeent water and/or a small amount ot baked sodium sultate may be
added to facilitate separation of the phases.
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Biphenyls and Trans-Nonachlor Volume 2, Chapter^
4.8 Extract Evaporation
Several fresh boiling stones are added to the hexane-acetone extract contained in the boiling flask,
a 24/40 3-ball macro Snyder column is connected to the flask by means of a reducing bushing, the
Snyder column is wetted with hexane, and the extract is evaporated to approximately 300 mL.
This extract is then quantitatively transferred with several hexane rinses (see note in Sec. 4.6.2) to
the boiling flask containing the hexane back-extract of the acetone rinse-extract. Several fresh
boiling stones are added, a 3-ball macro Snyder column is attached and wetted, and the solvent is
evaporated to a volume of approximately 200 mL. The contents are then transferred to a 250 mL
K-D apparatus, fresh boiling stones are added, and the extract is reduced to an apparent volume of
1-5 mL. The K-D is allowed to cool and the 250 mL flask and macro Snyder column are removed
from the concentrator tube. Fresh boiling stones are added, a micro Snyder column is attached and
wetted with hexane, and the volume is reduced to approximately I mL. To effect a complete
exchange into hexane fresh boiling stones and 10 mL of hexane are added and the volume is again
reduced to 1 mL. This final step is then repeated once more and the volume brought to
approximately 1 mL. Any evaporation device (such as the Labconco Rapidvap) that can be
demonstrated to yield acceptable spike recoveries, acceptable precision, and acceptable levels of
contamination can be used in place of the K-D apparatus.
4.9 Column Chromatography Clean-up of Extract
A silica/alumina cleanup column is used to remove polar interfering compounds remaining in the
extract prior to GC analysis.
4.9.1 Prepare 10% deactivated Alumina and 6% deactivated silica by activating a portion of
each by heating to 400°C for at least 4 hours and allowing to cool to room temperature in
a desiccator. Weigh a portion of the alumina or the silica into a glass jar with TFE-lined
lid. Add a weight of water equal to the percent deactivation desired (either 10% or 6%)
based upon the weight of he portion used. Place jar on roller for 30 minutes. Store in a
sealed glass container. The deactivated material must be used within 24 hours of
preparation or this procedure must be repeated. After initial heating to 400°C both the
alumina silica gel must be stored either at approximately 130;C or in a desiccator prior to
deactivation.
4.9.2 Prepare acid silica gel (40% w/w) by thoroughly mixing the appropriate portions of
concentrated sulfuric acid and activated silica gel together in a clean container. The
amount of concentrated sulfuric acid to be used for any weight of activated silica gel can
be calculated by using the following equation:
(0.36) X (gins Silica) = mis cone. H2SO4.
Break up aggregates with a stirring rod or place the container on a roller table until a
uniform mixture is obtained. Store in a glass jar with a TFE-lined lid.
4.9.3 Prepare a column by placing a small portion of glass wool at the bottom of a
chromatography column. Pour 70 mL hexane into the column filling it to a level that is
approximately 1/3 the \nlumo of the ivscnoir. Place a powder funnel in the column and
pour 10 g of the 10% deactivated alumina into a column, swirling the hexane and the
alumina allowing the alumina time to completely settle. Add 3 g of 6% deactivated silica
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Resin Cartridges for Polychlorinated
Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
to the column in the same manner. Before adding the sodium sulfate open the stopcock
and allow the hexane to slowly drain, then add enough sodium sulfate to result in a plug
approximately I cm high. The acid silica gel must be slurry packed in order to prevent
trapping bubbles in the column. Into a small beaker containing 8 g of acid silica gel, pour
a sufficient amount of hexane to immerse the adsorbent. Swirl the beaker to release most
of the bubbles, then with the aid of a squeeze bottle of hexane. pour the slurry into the
column, swirling it to aid settling. Again using the same technique as described above,
add enough sodium sulfate to result in a plug approximately I cm high. Drain the solvent
level down to just above the top of the sodium sulfate. Place a 250 mL K-D apparatus
under the column to collect the eluent.
4.9.4 The I mL sample extract from 4.8.1 is carefully transferred to the column with several
small hexane rinses. A small portion of hexane should be used to rinse down the sides of
the column. Allow each rinse to drain to just above the sodium sulfate layer. Carefully
add a total of 60 mL of hexane to the column (including rinses) and allow the column to
drain completely, collecting all the eluent.
4.9.5 Add boiling stones to the K-D containing the eluent and reduce in volume to 0.9 mL (as
measured in the 10 mL concentrator tube) using macro and micro Snyder columns
followed by nitrogen evaporation using a stream of ultra high punty nitrogen. At this
point add 100 /uL of internal standard solution to bring the volume to 1.0 mL and transfer
to an autosampler vial for analysis.
4.10 Quality Control Sample Frequency
Samples prepared using this procedure should be processed in batches sized in accordance with
the project analytical QAPjP. The QA/QC samples described below and their frequency are
guidelines; the project specific QAPjP should be consulted prior to beginning any analysis of
sample preparation. Additional QA/QC samples may be required as specified in the project
specific QAPjP
4.1 O.I Lab Procedural Blank - prepare one per batch. Prepared by working through the sample
preparation procedure using only solvents and reagents.
4.10.2 Lab Matrix Blank - prepare one per batch. This is a non-field exposed resin sample
prepared in a manner identical to that used for field samples.
4.10.3 Spiked Matrix Blank - prepare one per batch. This is a lab matrix blank fortified with
target analytes and prepared in a manner identical to that used for field samples.
4.10.4 Spiked Procedural Blank - prepare one per batch. This is a lab procedural blank which is
fortified with target analytes and prepared in a manner identical to that used for field
samples.
4.10.5 Sample Replicates - anal\ it one sample per batch in duplicate.
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Biphenyls and Trans-Nonachlor Volume 2, Chapter^
4.1 1 Data Recording and Storage
All standard preparation data will be recorded in accordance with SOP MSL-M-056.
All extraction data and sample extraction information will be recorded on the XAD-2 Resin and
Filter Extraction Data Sheet (Attachment 1).
All transfers of data to forms and data reductions (e.g., concentration calculations, means, standard
deviations) will be checked by the analyst and approved by the project manager. Hard copies of
GC printouts of calibrations and sample data and spreadsheet reports will be kept in the Chemistry
Group Central Files. Analytical electronic data will be archived on magnetic tape.
5.0 Quality Control
Results of quality control samples (e.g., blanks, spikes, intercomparison samples, and replicate
samples) prepared using this procedure will meet the criteria given in the project specific QAPjP.
Recovery of the surrogates will be used to monitor for extraction efficiency, unusual matrix
effects, or sample processing errors. Surrogate recovery criteria will be given in the project
specific QAPjP
Solvents, reagents, glassware, and other sample processing hardware may cause artifacts or
interferences to sample analysis. The analyst must demonstrate that these materials are free from
interferences under the conditions of the procedure by analyzing method blanks.
6.0 Safety
All analysts following this procedure should be aware of routine laboratory safety concerns,
including the following:
6.1 Protective clothing and eyeglasses must be worn at all times when handling samples and
chemicals.
6.2 Proper care must be exercised when handling solvents and acids, and when using syringes.
6.3 Extractions of resin samples are only to be performed in the walk-in fume hood located in Room
MSL 114 or in the fume hood in Room MSL 231. Both of these hoods are equipped with heating
mantles that are equipped with safety earthed ground screens, and with recirculating water chillers
that have been equipped with flow sensing devices.
6.4 The purpose of the safety earthed ground screens is to shut off power to the heating mantle unit
should the screen (which is located just above the heating element) become electrically connected
to the mantle housing, as in the case of a boiling vessel rupturing.
6.5 The (low sensing device on the recirculating chiller will shut off the chiller and the heating mantle
unit should coolant water flow be mierrupted, as in the case of a hose breaking. This will prevent
the hiMhiiL' (Task from boiling dr\
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Resin Cartridges for Polychlorinated
Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
6.6 In addition to these features each hood is equipped with a liquid sensor which, if it detects liquid
present in the hood or in the containment trays (used in the walk-in fume hood), will shut down
power to both the chiller and the heating mantles.
6.7 If overnight unattended extractions are to be performed the project manager must make
arrangements with the Building Director to ensure that at least once during the off-shifts a security
guard checks for any problems in the extraction areas.
7.0 Training Requirements
All staff performing extractions of XAD-2 resin samples for analysis of PCBs and trans-nonachlor
compounds must first read this SOP and then demonstrate proficiency in the process prior to
performing the work. Proficiency will include demonstrating that I) a blank having an acceptably
low level of contaminants can be produced and 2) that blank spike recoveries are within acceptable
recovery range. Documentation of training will be recorded on training assignment and on-the-job
training forms from SOP MSL-A-006. Records of this training will be kept by the laboratory
Quality Assurance Representative.
8.0 References
J. I. Gomez-Bellinchon, Grimalt, J. O., and Albaiges, J., "Intercomparison Study of Liquid-Liquid
Extraction and Adsorption on Polyurethane and Amberlite XAD-2 for the Analysis of
Hydrocarbons, Polychlorobiphenyls, and Fatty Acides Dissolved in Seawater," Environ. Sci.
Technol. 1988,22,677-685.
Quality Assurance Plan Green Bay Mass Balance Study "Cleaning Methods for XAD-2 Resin and
Filters" U.S. Environmental Protection Agency (EPA). 1986.
Analytical Quality Assurance Project Plan (QAPjP) for the EPA Lake Michigan PCB Mass
Balance Study, DRAFT, dated October 25, 1994.
ASTM Method D4059-91, "Standard Test Method for Analysis of Polychlorinated Biphenyls in
Insulating Liquids by Gas Chromatography."
EPA 660/4-81-045, "The determination of Polychlorinated Biphenyls in TQuality Assurance Plan
Green Bay Mass Balance Study' "Cleaning Methods for XAD-2 Resin and Filters" U.S.
Environmental Protection Agency (EPA). 1986.
"Analytical Quality Assurance Plan for the Lake Michigan PCB Mass Balance Study."
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O)
<£>
DA It ANALYST
PRi.JECT/CF*
CRIUSE
BATCH*)
RESIN/FILTER
SOP*
ALUMINA LOT #
GLASS WOOL LOT* and MFG
HEXANE LOT* and MFG
ACETONE LOT* and MFG
SILICA LOT*
Na2S04 LOT*
SURROGATE STD. no/vol
INTERNAL STD. no/vol
SPIKE SOLN A, no/vol
SAMPLE EXTRACTION
SAMPLE
ID
EXTRACTION
DATE
COLUMN
BATCH ID
NO OF
FILTERS
WATER
VOLUME (L)
SPIKE SOLN B. no/vol
COMMENTS
X
>
D
1
i IV)
Tl
Attachment 1.
tesin and Filter Extraction Data Sheet,
EPA PCB Mass Balance Study
Extraction and Cleanup of XAD-2
Resin Cartridges for Polychlorinated
Volume 2, Chapter 1 Biphenvls and Trans-Nonachlor
-------
Extraction and Cleanup of
Glass Fiber Filters for Polychlorinated
Biphenyls and Trans-Nonachlor
Eric Crecelius and Lisa Lefkovitz
Pacific Northwest National Laboratory
Battelle Marine Sciences Laboratory
1529 West Sequim Bay Road
Sequim, WA 98382
Standard Operating Procedure MSL-M-092-00
June 1994
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Extraction and Cleanup of Glass Fiber Filters for
Polychlorinated Biphenyls and Trans-Nonachlor
1.0 Scope and Application
This SOP is applicable to the extraction and analysis of polychlorinated biphenyl compounds from
glass fiber filters used to collect particulates from fresh water. The target compounds which can
be determined by this method are, generally speaking, the polychlorinated biphenyls (PCBs) found
in Aroclors 1232, 1248, and 1262 and trans-Nonachlor. It may be applied to other compounds
once acceptable method recovery has been demonstrated.
Up to 400 liters of freshwater is passed through a series of pre-cleaned 293 mm glass fiber filters
with 0.7^m pore size that are contained in a "Pentaplate" filtration apparatus. The flow rate used
is such that the back pressure on the filters will not exceed 5 psi. The filtrate thus collected is
operationally defined as the suspended paniculate phase. The filters are removed from the
Pentaplate device, replaced in their original pre-cleaned foil pouches, which are sealed, labeled,
and stored frozen until extraction.
At analysis, up to five filters pertaining to a single sampling site are allowed to thaw, removed
from their pouches, placed in an extraction apparatus. Surrogate compounds are then added and
the filters are extracted with acetone to remove organics and interstitial water. The filters are then
extracted with a mixed solvent of 50% hexane, 50% acetone to remove the remaining organic
compounds from the filter. The acetone extract is combined in a separator/ funnel with 300 mL of
reagent water and the mixture is then extracted once with 200 mL of hexane, then twice with
100 mL of hexane, to remove PCBs into the hexane extracts. These hexane extracts are then
combined with the hexane-acetone extracts, reduced in volume, and exchanged into hexane to a
final volume of approximately 1 mL. The extract is then applied to a cleanup column which is
eluted with 60 mL hexane. The hexane is reduced in volume to 0.9 mL, at which point 0.1 mL of
internal standard is added to complete the sample preparation.
Extracts are then subjected to gas chromatographic analysis with electron capture detection (see
SOP MSL-M-093). The chromatographic separation is performed using a 60 meter capillary
column. Identification and quantitation of the PCB compounds is accomplished by comparison to
calibration standards containing a large number of PCB congeners in known concentration.
2.0 Definitions
The following terms and acronyms are associated with this procedure:
DCM Dichloromethane
GC-ECD Gas chromatography with electron capture detection
K-D Kuderna-Danish
NA-SO, Sodium Sultate
rpm Revolutions [vr minute
SRM Standard reference material
GFh Glass fiber filter (see description below)
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3.0 Responsible Staff
3.1 Project Manager. A Scientist responsible for 1) administration of the project; 2) providing project
specific quality control requirements to the laboratory, 3) defending the data in a Quality
Assurance Audit; and 4) reporting results to client.
3.2 Laboratory Supervisor. A Technical Specialist or Scientist having expertise in the principles
involved with this procedure and in the use of laboratory operations in general. Responsible for
1) ensuring that analysts are trained in the handling of solvents; 2) that appropriate quality control
samples are included with the sample analysis to monitor precision and accuracy of the analysis;
3) checking the analysts' work to ensure that samples are handled appropriately and that data are
collected and interpreted correctly; 4) making decisions regarding problems with the analysis or
deviations from the SOP; 5) defending the data in a Quality Assurance Audit; and 6) reporting
results to project manager or client.
3.3 Anal\st. A Technician, Technical Specialist, or Scientist assigned to conduct analyses using this
procedure. Responsible for 1) understanding the proper handing of samples and solvents;
2) recording information regarding extractions and any deviations from the SOP in the appropriate
log books; 3) analyzing the appropriate number of quality assurance samples for each batch of
samples analyzed; 4) reporting results to the Project Manager; and 5) participating in QA audits.
3.4 Quality- Assurance Representative. A qualified staff member assigned to the Quality Assurance
Unit. Responsible for monitoring the project activities and conducting Quality Assurance Audits
to ensure that 1) analysts have conducted the analysis according to the SOP and that deviations
from the SOP have been noted in project files; 2) instrument use and maintenance records are kept
correctly; and 3) data have been reported and presented accurately.
4.0 Procedure
4.1 Apparatus and Reagents
4.1.1 Separator/ funnels (sized to fit sample)
4.1.2 Erlenmeyer flasks, various sizes
4.1.3 Chromatography column, 15 x 250 mm with 250 mL reservoir and No. 2 Teflon stopcock
(Kontes #42080-0222)
4.1.4 Roller apparatus capable of rolling 250 mL Qorpak jars
4.1.5 Kudema-Danish (K-D) evaporator apparatus: 250 mL and/or 500 mL reservoir; 3 ball
macro Snyder column: 2 or 3 ball micro Snyder column: 10 mL or 25 mL concentrator
tube
4.1 .fi Hot water bath capable of reaching 100DC, located in a fume hood
4 I Vv'uter aspirator vacuum source (Bucchi #B169 or equivalent:
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Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
4.1.8 0.5 Liter Soxhlet extraction apparatus complete with flask and condenser (Ace #6810-10
or equivalent)
4.1.9 1 Liter round-bottom boiling flasks (Ace #6887-53 or equivalent)
4.1.10 Reducing Glass Joint Bushings 34/45 to 24/40 (Ace #5023-21 or equivalent)
4.1.11 Pre-Cleaned 293 mm Whatman OFF, 0.?A/m pore size, Whatman # 1825-293 or
equivalent
4.1.12 Boiling chips, carborundum, soxhlet extracted or baked at >400° C
4.1.13 Glass wool, soxhlet extracted >4hrs in 50:50 hexane-acetone
4.1.14 Nitrogen evaporation apparatus, N-Evap or equivalent, heated with a water bath
maintained at 25-35°C
4.1.15 RapidVap Evaporation System (Labconco)
4.1.16 Glass graduated cylinders
4.1.17 Stainless steel and teflon forceps
4.1.18 Steel rod, 3 mm x 50 cm
4.1.19 Microliter syringes or micropipets
4.1.20 Concentrated sulfuric acid
4.1.21 Solvents-pesticide grade or equivalent
Dichloromethane (DCM)
Hexane
Acetone
Reagent Water (Barnstead Organic-Free or equivalent)
4.1.22 Sodium sulfate-anhydrous, reagent grade, heated to 400°C for >4 hr, then cooled to room
temperature and stored in a desiccator
4.1.23 Alumina, Sigma F-20 or equivalent, 80-200 mesh
4.1.24 Silica, Amicon Matrix Silica pore diameter 60A , particle size 105 ^m
4.1.25 Surrogate solution- a hexane solution containing 200 ng/mL PCB 14. 50 ng/mL PCB 65,
50 ng/mL PCB 166, and 100 ng/mL dibutyl chlorendate (DEC). PCB numbers refer to
their IUPAC designations
4 I 2<> Internal standard solution- a hoxane solution containing 100 ng/mL nl PCB 30. PCB 204.
and PCB 103
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Biphenyls and Trans-Nonachlor Volume 2, Chapter^
4.1.27 Matrix spiking solution- a hexane solution having a nominal concentration of 1830 ng/mL
as total PCBs (prepared from the 1994 Aroclor mixture provided by M. Mullins) and
100 ng/mL trans Nonachlor
4.1.28 Acid silica gel - 30% w/w sulfuric acid
4.2 Sample Handling
Samples shall be kept frozen (-10 to -30°C) until analysis. Samples shall be extracted within
9 months of receipt at the lab unless specified otherwise by the Project Manager or project-specific
plans. Refer to the project-specific sampling plan for sample collection, preservation, and
handling methods.
4.3 Labware Preparation
Prior to use, all glassware, Teflon, and other labware should be washed with hot, soapy water and
rinsed with tap water, followed by deionized distilled water. Teflon should be solvent rinsed,
teflon stopcocks are sonicated for 30 minutes in dichloromethane. Additionally, glassware must
be baked at 450°C for at least 4 hours.
4.4 Filter Preparation
Prior to use, the filters must be muffled at 450 ± 20°C for 4 hrs to remove manufacturing
impurities. This process is fully described in Battelle SOP MSL-M-090-00.
4.5 Pentaplate Filtration Apparatus Preparation
A detailed description of how the "Pentaplate" apparatus is loaded with filters and used to collect
samples is contained in the project specific sampling QAPjP.
4.6 Filter Sample Extraction
4.6.1 A small wad of pre-cleaned glass wool is first placed over the siphon tube entrance port in
the soxhlet extractor body to prevent any paniculate filter material from escaping.
Note: Soxhlet body used for the resin extract, SOP# MSL-M-091 -00 is a modified
soxhlet with a capacity greater than 500 mL, using this soxhlet may cause "bumping'1 or
other problems with the filters.
The filters pertaining to a single sampling site are allowed to thaw, removed from their foil
pouches, folded, and placed in the soxhlet extractor body using clean teflon or stainless
steel forceps. Any water associated with the thawed samples is also added to soxhlet. The
extractor body containing the filters is then connected to a I liter boiling flask containing
several boiling stones, and 800-850 mL of acetone is added and allowed to siphon over
into the boiling flask below. Next. 100 ...L of surrogate solution is added to the filters in
the extractor body and. if the sample is to be spiked, 100 .-.L of matrix spikins solution is
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Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
also added. The condenser is then connected, the cooling water and heating mantle are
activated, and the filters are extracted for 4 hours. During extraction the soxhlet should
cycle 4 times per hour. The acetone extraction step is important in removing water from
the filters as it is critical to ensuring efficient extraction during the next extraction step.
4.6.2 The soxhlet apparatus is removed from the condenser and tilted to force the acetone
extract to siphon into the I liter boiling flask. The extract is then transferred from the
boiling flask into a 2 liter separator/ funnel.
Note: To avoid contamination of the sample from material on the exterior of the neck,
before transferring extracts from containers with ground glass joints always rinse the
exterior of the necks with the same solvent as that being transferred.
At this point fresh boiling stones and 800-850 mL of 50:50 hexane-acetone are added to
the boiling flask, the flask is connected to the soxhlet body, the condenser is connected
and the cooling water and heating mantle are activated. The filters are extracted with this
solvent system for > 16 hrs, after which the mantle is shut off and the system is allowed to
cool to room temperature. During extraction the soxhlet should cycle at least 4 times per
hour. After extraction the soxhlet body is siphoned over to remove the extraction solvent
into the boiling flask where it will be evaporated.
4.7 Acetone Extract Back Extraction
To accomplish this step 300 mL of reagent water is added to the acetone extract contained in the
separator/ funnel and the mixture is then extracted once with 200 mL hexane and twice more with
100 mL hexane. This step is performed in order to reduce the amount of polar or water soluble
interferences being carried through the procedure and to remove water from the acetone prior to
evaporation. The hexane extracts are collected in a 1 liter boiling flask for evaporation. If
emulsions are encountered or the acetone:water and hexane phases do not cleanly separate,
additional reagent water and/or a small amount of baked sodium chloride may be added to
facilitate separation of the phases.
4.8 Extract Evaporation
4.8.1 Several fresh boiling stones are added to the hexane-acetone extract contained in the
boiling flask, a 24/40 3-ball macro Snyder column is connected to the flask by means of a
reducing bushing, the Snyder column is wetted with hexane, and the extract is evaporated
to approximately 300 mL. This extract is then quantitatively transferred with several
hexane rinses (see note in Sec. 4.6.2) to the boiling flask containing the hexane back-
extract of the acetone rinse/extract. Several fresh boiling stones are added, a 3-ball macro
Snyder column is attached and wetted, and the solvent is evaporated to a volume of
approximately 200 mL. The contents is then transferred to a 250 mL K-D apparatus, fresh
boiling stones are added, and the extract is reduced to an apparent volume of 1-5 mL. The
K-D is allowed to cool and the 250 mL flask and macro Snvder column are removed from
the concentrator tube. Fresh boiling stones are added, a micro Snyder column is attached
and wetted with hexane. and the volume is reduced to approximately 1 mL. To effect a
complete exchange into hoxaiic fresh boiling stones and 10 ml. nt hexane is added and tlu1
volume is again reduced to one mL. This final step is then repeated once more and the
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Biphenyls and Trans-Nonachlor Volume 2, Chapter^
volume brought to approximately 1 mL. Any evaporation device (such as the Labconco
Rapidvap) that can he demonstrated to yield acceptable spike recoveries, acceptable
precision, and acceptable levels of contamination can be used in place of the K-D
apparatus.
4.9 Column Chromatography Clean-up of Extract
A silica/alumina cleanup column is used to remove polar interfering compounds remaining in the
extract prior to GC analysis.
4.9.1 Prepare 10% deactivated Alumina and 67c deactivated silica by activating a portion of
each by heating to 400°C for at least 4 hours and allowing to cool to room temperature in
a desiccator. Weigh a portion of the alumina or the silica into a glass jar with TFE-lined
lid. Add a weight of water equal to the percent deactivation desired (either 10% or 67c)
based upon the weight of he portion used. Place jar on roller for 30 minutes. Store in a
sealed glass container. The deactivated material must be used within 24 hours of
preparation or this procedure must be repeated. After initial heating to 400°C both the
alumina and silica gel must be stored either at approximately 130°C or in a desiccator
prior to deactivation.
4.9.2 Prepare acid silica gel (407c w/w) by thoroughly mixing the appropriate portions of
concentrated sulfuric acid and activated silica gel together in a clean container. The
amount of concentrated sulfuric acid to be used for any weight of activated silica gel can
be calculated by using the following equation:
136) X (gms Silica) = mLs cone. H2SO4
(0
Break up aggregates with a stirring rod or place the container on a roller table until a
uniform mixture is obtained. Store in a glass jar with the TFE-lined lid.
4.9.3 Prepare a column by placing a small portion of glass wool at the bottom of a
chromatography column. Pour 70 mL hexane into the column filling it to a level that is
approximately 1/3 the volume of the reservoir. Place a powder funnel in the column and
pour 10 g of the 107r deactivated alumina into a column, swirling the hexane and the
alumina allowing the alumina time to completely settle. Add 3 g of 6% deactivated silica
to the column in the same manner. Before adding the sodium sulfate open the stopcock
and allow the hexane to slowly drain, then add enough sodium sulfate to result in a plug
approximately 1 cm high. The acid silica gel must be slurry packed in order to prevent
trapping bubbles in the column. Into a small beaker containing 8 g of acid silica gel, pour
a sufficient amount of hexane to immerse the adsorbent. Swirl the beaker to release most
of the bubbles, then with the aid of a squeeze bottle of hexane, pour the slurry into the
column, swirling it to aid settling. Again using the same technique as described above,
add enough sodium sulfate .to result in a plug approximate^ 1 cm high. Drain the solvent
level down to just above the top of the sodium sulfate. Place a 250 mL K-D apparatus
under the column to collect the eluent.
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Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
4.9.4 The I mL sample extract from 4.8.1 is carefully transferred to the column with several
• small hexane rinses. A small portion of hexane should be used to rinse down the sides of
the column. Allow each rinse to drain to just above the sodium sulfate layer. Carefully
add a total of 60 mL of hexane to the column (including rinses) and allow the column to
drain completely, collecting all the eluent.
4.9.5 Add boiling stones to the K-D containing the eluent and reduce in volume to 0.9 mL (as
measured in the 10 mL concentrator tube) using macro and micro Snyder columns
followed by nitrogen evaporation using a stream of ultra high punty nitrogen. At this
point add 100 /uL of internal standard solution to bring the volume to 1.0 mL and transfer
to an autosampler vial for analysis.
4.10 Quality Control Sample Frequency
Samples prepared using this procedure should be processed in batches sized in accordance with
the project analytical QAPjP. The QA/QC samples described below and their frequency are
guidelines; the project specific QAPjP should be consulted prior to beginning any analysis of
sample preparation. Additional QA/QC samples may be required as specified in the project
specific QAPjP
4.10.1 Lab Procedural Blank - prepare one per batch. Prepared by working through the sample
preparation procedure using only solvents and reagents.
4.10.2 Lab Matrix Blank - prepare one per batch. This is a non-field exposed filter sample
prepared in a manner identical to that used for field samples.
4.10.3 Spiked Matrix Blank- prepare one per batch. This is a lab matrix blank fortified with
target analytes and prepared in a manner identical to that used for field samples.
4.10.4 Spiked Procedural Blank - prepare one per batch. This is a lab procedural blank which is
fortified with target analytes and prepared in a manner identical to that used for field
samples.
4.10.5 Sample Replicates - analyze one sample per batch in duplicate.
4.11 Data Recording and Storage
All standard preparation data will be recorded in accordance with SOP MSL-M-056.
All extraction data and sample extraction information will be recorded on the XAD-2 Resin and
Filter Extraction Data Sheet (Attachment I).
All transfers of data to forms and data reductions (e.g., concentration calculations, means, standard
deviations) will be checked by the analyst and approved by the project manager. Hard copies of GC
printouts of calibrations and sample data and spreadsheet reports will be kept in the Chemistry Group
Central Files. Analytical electronic data will be archived on magnetic tape.
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Biphenyls and Trans-Nonachlor Volume 2, Chapter^
5.0 Quality Control
5.1 Results of quality control samples (e.g.,blanks, spikes, intercomparison samples, and replicate
samples) prepared using this procedure will meet the criteria given in the project specific QAPjP.
Recovery of the surrogates will be used to monitor for extraction efficiency, unusual matrix effects.
or sample processing errors. Surrogate recovery criteria will be given in the project specific QAPjP
5.2 Solvents, reagents, glassware, and other sample processing hardware may cause artifacts or
interferences to sample analysis. The analyst must demonstrate that these materials are free from
interferences under the conditions of the procedure by analyzing method blanks.
6.0 Safety
All analysts following this procedure should be aware of routine laboratory safety concerns,
including the following:
Protective clothing and eyeglasses must be worn at all times when handling samples and chemicals
Proper care must be exercised when handling solvents and acids, and when using syringes.
Extractions of filter samples are only to be performed in the walk-in fume hood located in Room
MSL 114 or in the fume hood in Room MSL 231. Both of these hoods are equipped with heating
mantles that are equipped with safety earthed ground screens and with recirculating water chillers
that have been equipped with flow sensing devices.
The purpose of the safety earthed ground screens is to shut off power to the heating mantle unit
should the screen (which is located just above the heating element) become electrically connected to
the mantle housing, as in the case of a boiling vessel rupturing.
The flow sensing device on the recirculating chiller will shut off the chiller and the heating mantle
unit should coolant water flow be interrupted, as in the case of a hose breaking. This will prevent the
boiling flask from boiling dry.
In addition to these features each hood is equipped with a liquid sensor, which if it detects liquid
present in the hood or in the containment trays (used in the walk-in fume hood), will shut down
power to both the chiller and the heating mantles.
If overnight unattended extractions are to be performed the project manager must make arrangements
with the Building Director to ensure that at least once during the off-shifts a security guard checks for
any problems in the extraction areas.
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Extraction and Cleanup of
Glass Fiber Filters for Polychlorinated
Volume 2, Chapter 1 Biphenyls and Trans-Nonachlor
7.0 Training Requirements
All staff performing extractions of OFF filter samples for analysis of PCBs and trans-Nonachlor must
first read this SOP and then demonstrate proficiency in the process prior to performing the work.;
Proficiency will include demonstrating that I) a blank having an acceptable low level of
contaminants can be produced and 2) that blank spike recoveries are within acceptable recovery
range. Documentation of training will be recorded on training assignment and on-the-job training
forms from SOP MSL-A-006. Records of this training will be kept by the laboratory Quality
Assurance Representative.
8.0 References
8.1 J. I. Gomez-Bellinchon, Grimalt, J. O., and Albaiges, J., "Intercomparison Study of Liquid-Liquid
Extraction and Adsorption on Polyurethane and Amberlite XAD-2 for the Analysis of Hydrocarbons,
Polychlorobiphenyls, and Fatty Acids Dissolved in Seawater," Environ. Sci. Technol. 1988, 22,
677-685.
8.2 Quality Assurance Plan Green Bay Mass Balance Study "Cleaning Methods for XAD-2 Resin and
Filters" U.S. Environmental Protection Agency (EPA). 1986.
8.3 Analytical Quality Assurance Project Plan (QAPjP) for the EPA Lake Michigan PCB Mass Balance
Study, DRAFT, dated October 25, 1994.
8.4 MSL-D-001. Recording Data on Data Sheets and Laboratory Notebooks.
8.5 MSL-A-006. MSL Training.
8.6 MSL-M-056. Stock and Standard Solution Preparation.
8.7 MSL-M-093 PCB Congener Analysis of XAD Resins and GFIF Filters Using GC/ECD.
8.8 ASTM Method D4059-91, "Standard Test Method for Analysis of Polychlorinated Biphenyls in
Insulating Liquids by Gas Chromatography."
8.9 EPA 660/4-81-045, "The determination of Polychlorinated Biphenyls in TQuality Assurance Plan
Green Bay Mass Balance Study" "Cleaning Methods for XAD-2 Resin and Filters" U.S.
Environmental Protection Agency (EPA). 1986.
8.10 "Analytical Quality Assurance Plan for the Lake Michigan PCB Mass Balance Study."
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ro
oo
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DATF ANALYST
PROJECT/CF*
CRIUSE
BATCH*
RESIN/HLTER
SOP*
ALUMINA LOT #
GLASS WOOL LOT* and MFC
HEXANE LOT# and MFG
ACETONE LOT# and MFG
SILICA LOT#
Na2S04 LOT*
SURROGATE STD. no/vol
INTERNAL STD, no/vol
SPIKE SOLN A, no/vol ^
SAMPLE EXTRACTION
SAMPLE
ID
EXTRACTION
DATE
COLUMN
BATCH ID
NO OF
FILTERS
WATER
VOLUME (L)
SPIKE SOLN B, no/vol
COMMENTS
*»•
O
10
Tl
5S
>— •
^
Attachment 1 .
and Filter Extraction Data Sheet,
DCB Mass Balance Study
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-------
PCB Congener Analysis of XAD-2 Resins
and GFF Filters Using GC/ECD
Eric Crecelius and Lisa Lefkovitz
Pacific Northwest National Laboratory
Battelle Marine Sciences Laboratory
1529 West Sequim Bay Road
Sequim, WA 98382
Standard Operating Procedure MSL-M-093-00
June 1994
-------
PCB Congener Analysis of XAD-2 Resins
and GFF Filters Using GC/ECD
1.0 Application and Scope
1.1 This SOP is applicable to the analysis of GFF filter and XAD-2 resin extracts prepared according
to SOPs MSL-M-091 and MSL-M-092 for polychlonnated biphenyls (PCB) and trans-nonachlor
by capillary gas chromatography with 63Ni electron-capture detection.
1.2 This procedure provides typical gas chromatography (GC) conditions for the detection of trace
levels of PCBs and trans-nonachlor, methods for identifying the analytes, and methods for analyte
quantitation using the internal standard method. Attachment 1 lists the most frequently analyzed
compounds. However, this list may be amended to meet requirements of specific projects.
2.0 Definitions
The following terms and acronyms may be associated with this procedure:
ECD Electron capture detector or detection
GC Gas chromatography
PCB Polychlorinated biphenyl
RF Response factor
RRF Relative response factor; response factor of analyte normalized to the response
factor of the internal standard.
RSD Relative standard deviation (%)
RT Retention time
IS Internal standard - compound(s) added just prior to analysis on instrument.
Surrogate Compound(s) added prior to extraction to assess efficiency of method.
In addition, it should be noted that the numbering scheme used for PCB congeners, e.g. PCB 3, is
that used by Ballschmiter and Zell.
3.0 Responsible Staff
Project Manager. A Scientist responsible for 1) administration of the project; 2) providing project
specific quality control requirements to the laboratory; 3) defending the data in a Quality
Assurance Audit; and 4) reporting results to client.
Laboratory Supervisor: A Technical Specialist or Scientist having expertise in the principles
involved with this procedure and in the use of the GC. Responsible for 1) ensuring that analysts
are trained in operation of the GC; 2) appropriate quality control samples are included with the
sample analysis to monitor precision and accuracy of the analysis; 3) checking the analysts' work
to ensure that data are collected and interpreted correctly; 4) making decisions regarding problems
with the analysis or deviations from the SOP; 5) defending the data in a Quality Assurance Audit;
and 6) reporting results to project manager or client.
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PCS Congener Analysis ofXAD-2
Resins and GFF Filters Using GC/ECD Volume 2, Chapter^
Analyst: A Technician, Technical Specialist, or Scientist assigned to conduct analyses using this
procedure. Responsible for 1) understanding the proper use and maintenance of the GC;
2) recording information regarding instrument use and maintenance in the appropriate log books;
3) analyzing the appropriate number of quality assurance samples for each batch of samples
analyzed; 4) tabulating all sample and QC data and reviewing the quality of the data based on QC
guidelines presented in this SOP and any other project-specific QC guidelines; 5) reporting results
to the Project Manager; and 6) defending the data during an audit.
Quality Assurance Representative: A qualified staff member assigned to the Quality Assurance
Unit. Responsible for monitoring the project activities and conducting Quality Assurance Audits
to ensure that 1) analysts have conducted the analysis according to the SOP and that deviations
from the SOP have been noted in the appropriate log book or in the project files; 2) instrument use
and maintenance records are kept correctly; and 3) data have been reported and presented
accurately.
4.0 Procedures
The GC must be maintained and operated as described in Battelle SOP No. MSL-M-075.
4.1 GC Preparation
The primary quantitation GC column is a 60 meter x 0.25 mm (i.d.) fused silica capillary column
coated with a 5% phenyl-, 95% methyl-polysiloxane film of 0.25 u.m thickness. (J&W Scientific,
Inc., 60 meter DB-5 or equivalent). The confirmation column is a 60 m x 0.25 mm (i.d.) fused
silica capillary column coated with a 14% cyanopropylphenyl-86% methyl polysiloxane film of
0.25 m thickness (J&W Scientific, Inc. 60 meter DB-1701 or equivalent). Both columns are
installed in a single splitless injection port using a 2-hole ferrule.
4.2 Sample Collection, Preservation, and Handling
To conduct this analysis, the analyst should receive the samples as solvent extracts reduced to an
appropriate volume (as specified in SOP MSL-M-091 or MSL-M-092). Holding times to be
followed are those specified in the project specific QAPjP; normally the holding time for extracts
is 40 days from date of extraction. If holding times have been exceeded, the Project Manager
should be notified immediately. Refer to project-specific plans or protocols for sample collection,
preservation, and handling methods. .
4.3 Sample Specifications
Sample preparation methods may vary depending on the sample matrix and project needs; refer to
project-specific protocols. Normally, this method is used to analyze extracts prepared according to
Battelle SOPs MSL-M-091 and MSL-M-092. Samples and standards for analysis using this SOP
should be prepared in hexane.
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Volume 2, Chapter
PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD
Table 1: Suggested Instrument Conditions for PCB and Chlorinated Pesticide Analysis
Primary Column (DB-5) Conditions:
Injection port type Splitless
Injection port temperature 250°C
Detector temperature 300°C
Initial temperature 50°C
Initial hold 1.5 min
Ramp 1 rate 10°C/min
Final tempi 105°C
Final time 1 0.0 min
Ramp 2 rate 1 °C/min
Final temp 2 225°C
Ramp 3 rate 10°C/mm
Final temp 3 280°C
Final time 3 20 min
Carrier gas Hydrogen
Carrier gas velocity 60 cm/sec
ECD make-up gas Nitrogen
Make-up gas flow 30 cc/min
Split vent flow 215 cc/min
Split vent on-time 1.5 min
Injection volume 5 uL
Injection speed lOuL/sec
Hot needle time 0.07 min
Needle residence time 0.2 min
4.4 Analyte Identification
Prior to sample analysis, the elution order of the analytes of interest must be determined by
analyzing the analytes individually or in combination with other analytes having known or
predetermined retention times. The retention times of the analytes have all been verified on both
the quantitation (DB-5) and confirmation columns (DB-1701) specified above under the GC
conditions listed in Table 1, and are listed in Attachments 1 and 2. The elution order of the
congeners was determined from data provided by another laboratory using similar GC conditions
(Mullins et al. 1985, 1994 personal communications). Additional information on analyte
identification is discussed in Section 4.6.1.
4.5 Instrument Calibration
Before the sample is injected into the GC. the detectors must be calibrated to determine the
response of the detector to the analues of interest. Demonstration of linearity of detector response
is required before sample analysis. Calibration checks must be anaK/ed at a minimum frequency
of once every 10 samples during sample analysis.
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PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD Volume 2, Chapter^
4.5.1 Initial Calibration: Prior to initiating any sample analyses initial calibration is performed
by analyzing calibration standards at a total of seven levels spanning the concentration
range of 9.1 ng to 1830 ng as "Total PCBs" Not all the congeners respond or are clearly
resolved from other congeners at all these levels. On the DB-5 quantitation column,
eleven congeners (BZ #s 12, 13, 100, 147 & 124, 134, 130, 129, 167, 173, and 189) are
only determinable at three of these concentration levels. The calibration curves for these
consist of the three concentrations plus zero. All other congeners are calibrated using a
minimum of four concentrations. Calibration standards include the appropriate surrogates
(Surr.) and internal standards (IS) at concentrations identical to their concentrations in the
samples (see MSL-M-091 and MSL-M-092). An initial calibration must also be run if an>
GC conditions have changed significantly or if continuing calibration acceptance criteria
(CCAC) are not met. Initial calibration acceptance criteria are a correlation coefficient (r)
of 0.95 for a minimum 4 point curve using a quadratic or linear fit.
4.5.2 Continuing Calibration (CCAC): A mid-level calibration solution is analyzed as a
calibration check minimally every 10 samples while samples are being analyzed. All
sample analyses must be bracketed by two calibration check standards that meet
calibration criteria. Continuing calibration acceptance criteria for the primary quantitation
column are a 75-125% recovery relative to the total PCB value expected.
4.5.2.1 Performance Criteria: Additional performance criteria that will be evaluated
within each CCAC include: a recovery of 50-150% for PCBs 6 & 205 (which
represent small peaks), and a recovery of 75-125% for PCBs 101, 185, 44, & 180
(which represent average and large peaks). To ensure proper identification, the
retention time of the internal standard reference peaks, PCB 30 and PCB 204,
cannot shift by more than ±0.4 min (see Section 4.6.1).
4.5.2.2 Internal Standard Criteria: To ensure that the internal standards are not
interfered with the area or height ratios between the internal standards PCB 30
and PCB 204 in the samples are monitored. If the area or height ratios observed
in the samples differ from those observed during initial calibration by more than
±15% relative percent difference (RPD), all congeners must be quantitated
relative to the uncontaminated internal standard and the data flagged accordingly.
4.6 Sample Analysis Procedure
Samples are analyzed under the same analytical conditions as the calibration standards. Samples
must be bracketed by acceptable calibrations (see acceptance criteria in Section 4.5.2). Criteria for
accepting peaks as analytes of interest are explained in Sections 4.6.1 through 4.6.4.
4.6.1 Retention Time Windows: Analytes are identified by the data system by setting allowable
time windows for reference peak and analyte peak retention times. Initial retention times
are set during the initial calibration of the instrument. From that point forward, the system
utilizes retention time windows in which to "look" for peaks of interest and reference
peaks. The reference peak \sindows are set at ±0.4 min. and the analyte peak windows art-
set at ±0.10 min. Since the internal standard reference peaks are laree and clearly resolved
using a larger window for recognizing them is justified. The data s\stem then uses the
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PCB Congener Analysis ofXAD-2
Volume 2, Chapter 1 Resins and OFF Filters Using GC/ECD
actual retention time of the internal standard reference peaks to adjust the position of the
analyte peak windows, which are much narrower, to compensate for any minor
chromatographic drift. One internal standard reference peak shall also be designated an
RRT reference peak to further assist in proper peak identification through the use of
relative retention times.
4.6.2 Second Column Confirmation. Since each injection is split between the quantitation
column (DB-5) and confirmation column (DB-1701) all sample results will be confirmed
by whether or not a peak is observed at the appropriate retention time on both columns.
The retention time criteria described in Section 4.6.1 apply to the confirmation column
also. Trans-nonachlor can only be quantitated on the DB-1701 since it is interfered with
by PCB 99 on the DB-5. As a result of this, second column confirmation of trans-
nonachlor is not possible except as PCB 99 and trans-nonachlor on the DB-5. When
trans-nonachlor is confirmed in this manner the PCB 99 value must be flagged
accordingly.
4.6.3 Resolution. Resolution on the primary (DB-5) column should be sufficient to separate
congeners #17 and #18 into two peaks with a valley less than half the height of PCB #17.
If this cannot be accomplished a new column must be installed or the instrument
conditions optimized further.
4.6.4 Minimum Area or Height: Peaks with a signal-to-noise ratio of three or less should be
regarded as not detected unless otherwise noted in a specific project plan and/or
documented by project management.
5.0 Data Analysis and Reporting
5.1 Data Recording
Data quantitation and calculations will be performed on personal computers using commercial
software such as Varian Star Chromatography Software (Version 4.0 or higher), Microsoft Excel
(Version 4.0 or higher) or database software (Access Version 2.0). All transfers of data to forms
and data reductions (e.g., concentration calculations, means, standard deviations) will be checked
by the analyst and approved by the Laboratory Supervisor. Hard copies of GC printouts of
calibrations and sample data and spreadsheet reports will be kept in the GC/ECD files. A copy of
the summary sheets and extraction logs will be placed in the appropriate project file in the
Chemistry Group Central Files. Hard copies of chromatograms from each sample and all
calibrations will be kept in the GC/ECD files unless otherwise noted in a specific project plan. All
GC data files will be archived on magnetic tape.
5.2 Sample Quantitation
The internal standard method is used for quantitation. PCB 30 is used to quantify all PCB
congeners with retention times up to and including PCB 110, all other congeners are quantitated
vs. PCB 204. PCB 30 is used to quantitate the surrogates PCB 14 and PCB 65 and PCB 204 is
used to quantitate the surrogate PCB 166. In addition, the results reported arc corrected tor the
reco\er\ of the surrogates PCB 65 and PCB 166: the surrogate PCB 14 is not used because it is
oftentimes interfered with. The recovers of PCB 65 is used to correct the congeners quantitated
s v PCB 30 and the recovery of PCB 166 is used to correct the congeners quantitated vs. PCB 204.
2-291
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PCB Congener Analysis of XAD-2
Resins and OFF Filters Using GC/ECD _ Volume 2, Chapter/
If the ratio of internal standard areas exceeds the criteria set in Section 4.5.2.2, all congeners must
be quantitated relative to the uncontaminated internal standard and the data must be flagged
accordingly.
The concentration of a specific analyte in a sample is calculated by the Vanan Star
chromatography workstation. The system uses the concentration amounts for the individual
analytes entered into the peak table and the results from the calibration standards to generate the
coefficients of a polynomial curve fit that is. in turn, used to calculate the analytical
concentrations. This result is then adjusted for internal standard and surrogate recoveries in an
Excel spreadsheet:
Star result = Amt,,,url
Where:
Amt,,,arl is obtained by solving:
Area(or height ) + C)
Where:
A, B & C = coefficients of the polynomial equation
Amthlar) = amount of compound present in extract unadjusted for internal or surrogate
stds
Area(or height)lunkt = area or height of the peak for the selected compound found in the
analysis run.
At this point the result is not corrected by any internal standards. The results are then imported to
an Excel spreadsheet using various star macros and adjusted for internal and surrogate standard
recoveries.
Result = Amtu.al/ (Sample Volume)
Where:
expectedlnkll
Reclmn =
Amt,ailll=Amt,Mrl/Rec,nlill
Amtlial = AmtIMlll/Rec,,liril
Where:
Amt,iul = Amount of compound present in analysis run
Amt,Mlll = Amount of internal standard present in extract, calculated h\ Varian Star
Ann expected, ,^h = Amount of internal standard added to extract
•\'"'.,,,.-/ = Amount of surrogate standard present in extract, calculated h\ Varian Star
Amr expected, ^llin = Amount t/l surrogate standard added to cxirai 1
2-292
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PCS Congener Analysis ofXAD-2
Volume 2, Chapter 1 _ Resins and GFF Filters Using GC/ECD
5.3 Confirmation Data
The identification of particular PCB congeners is confirmed in ail the samples by analysis on a column
of dissimilar polarity (DB-1701 ). Confirmation is strictly by retention time only. The same criteria for
retention time windows as described for the quantitation column (Section 4. 6. 1 ) are applied to the
confirmation column.
5.4 Surrogate and Spike Recovery Calculation
Surrogate and Spike recoveries are calculated from the quantitation column. Calculations are as
follows:
Surrogate % Recovery = Qj.\ 100
Q,i — Quantity determined by analysis
Qa = Quantity added
Matrix Spike Recover = (SSR-SR) x 100
ESR
SSR = Spike sample result
SR = Sample result
ESR = Expected sample result = QJDivisor, where Divisor = amount sample analysed
The relative percent difference (RPD) between replicates is calculated as follows:
RPD = I SR - SDR\ A 100
1/2 (SR + SDR)
SDR = Sample Duplicate Result
6.0 Quality Control
Some quality control considerations associated with this SOP are described in the individual sections to
which they apply. The following additional quality control criteria apply unless otherwise specified in
a project specific QAPjP:
Field and Method Blanks < 1 0 ng Total PCB
Surrogate Recoveries 40-140% (excluding PCB 14)
Matrix Spike Recoveries 50 to 1 50% Total PCB & 70% of analytes within 50- 1 50% range
Blank Spike Recoveries 70-130% Total PCB, & 70% of analytes within 60-140% range
Sample Duplicate RPD <50% Total PCB and < 100% for analytes >5X MDL
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PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD Volume 2, Chapter^
7.0 Safety
All analysts following this procedure should be aware of routine laboratory safety concerns, including
the following:
1. Protective clothing and eyeglasses should be worn when appropriate
2. Proper care must be exercised when using syringes
3. Certain areas of the GC system are heated. Avoid bodily contact with these areas and use care
in handling flammable solvents in and around the GC system.
8.0 Training
All analysts following this procedure will be directly supervised by the Principal Investigator, qualified
analyst, or laboratory supervisor until they have demonstrated to the satisfaction of the supervisor that
they are capable of operating the GC independently. At a minimum, the analyst trainee should be
competent in operation and maintenance of the GC (SOP No. MSL-M-075). The analyst trainee
should also be able to analyze and quantify a multi-point calibration and quantitate a sample of known
concentration (e.g., a reference material or matrix spike) within established control limits.
Documentation of training will be recorded on training assignment and on-the-job training forms from
SOP MSL-A-006. Records of this training will be kept by the laboratory Quality Assurance
Representative.
9.0 References
9.1 MSL-A-006 Marine Sciences Laboratory Training.
9.2 MSL-M-075 Routine GC Maintenance.
9.3 MSL-M-091 Extraction and Cleanup of Resin Cartridges for Polychlorinated Biphenyls and trans
Nonachalor.
9.4 MSL-M-092 Extraction and Cleanup of Glass Fiber Filters for Polychlorinated Biphenyls and
Nonachlor.
9.5 Quality Assurance Plan Green Bay Mass Balance Study "Cleaning Methods for XAD-2 Resin and
Filters," U.S. Environmental Protection Agency (EPA). 1986.
9.6 Analytical Quality Assurance Project Plan (QAPjP) for the EPA Lake Michigan PCB Mass Balance
Study, DRAFT, dated October 25, 1994.
9.7 K Ballschmiter and Zell, "Analysis of Polychlorinated Biphenyls by Capillary Gas
chromatography." Fresenius Z. Analytical Chemistry, #302 pp. 20-31 (1980)
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Volume 2, Chapter 1
PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD
Attachment 1: Mixed Congener Standard on DB-5
(Page 1 of 5)
Title 7 pt pp-376 GLNPO June using height, for cal
Run File C:\STAR\MODULE16\728JN4\7284013.RUN
Method File : C:\STAR\MOOULE16\728JN4\72B4014.MTH
Sanple ID . PP-376C 183
Injection Date: 26-MAY-95 4:51 P« Calculation Date: 22-JUN-95 2:56 PM
Operator TJF
workstation:
Instrument : Varian Star 111
Channel : A - 5
Detector Type: ADC8 (10 Volts)
Bus Address 16
Sample Rate 5.00 Hz
Run Time 152.503 mm
Star Chromatography Software
Version 4.01
Chart Speed •
Start Time -
0.39 cm/nun
10.000 nun
Attenuation
End Time
39.012 39.119
40.253 40.409
58.510
57.084
59.395
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PCS Congener Analysis ofXAD-2
Resins and GFF Filters Using GC/ECD
Volume 2, Chapter 1
Attachment 1: Mixed Congener Standard on DB-5
(Page 2 of 5)
116.694
=$- 121.396
2-296
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Volume 2, Chapter 1
PCS Congener Analysis ofXAD-2
Resins and OFF Filters Using GC/ECD
Attachment 1: Mixed Congener Standard on DB-5
(Page 3 of 5)
Title : 7 pt pp-376 GLNPO June using height for cal
Run File : C:\STAR\MODUIE16\728JN4\7284013.RUN
Method File : C:\STAR\MODULE16\728JN4\7284014.HTH
Sample ID : PP-376C 183
Injection Date: 26-MAY-95 4:51 PM Calculation Date: 22-JUN-95 2:56 PM
Operator : TJF Detector Type: ADCB (10 Volts)
Workstation: Bus Address : 16
Instrument : Varian Star II Sample Rate : 5.00 Hz
Channel : A « 5 Run Time : 152.503 nin
Star Chronatography Software
********** version 4.01 •*••••***•*••*
Run Mode : Analysis
Peak Measurement: Peak Height
Calculation Type: External Standard
Peak
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
33
Peak
Home
1
3
4+10
7+9
6
8+5
14(SURR)
19
30{ISTD]
12
13
18
15+17
24
27
16
32
29
26
25
31+28
21
33
53
51
22
45
46
52
43
49
48+47
65(SURR)
44
42*37
41 + 71
64
40
Result
(NG/L)
12
7
3
1
1
14
19
0
10
0
0
3
3
0
0
2
2
0
0
0
10
0
3
0
0
2
0
0
4
0
2
1
4
4
.452705
.748489
.673396
.111898
.810442
.648691
.450569
.278382
.014711
.075297
.178523
.918240
.817946
.052955
.200533
.078020
.000064
.051388
.683344
.299107
.189798
.031160
.577584
.672404
.179196
.948125
.937357
.377848
.857748
.266150
.580539
.112766
.909873
.793020
1.452151
2
1
.459767
.899905
0.956243
Ret. Tiae Width
Time Offset Height Sep. 1/2 Status
(min) (min) (counts) Code (sec) Codes
18.
22.
25.
28.
30.
31.
33.
33.
35.
36.
36.
37.
37.
39.
39.
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43.
578
887
507
924
360
133
155
893
261
707
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545
760
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119
253
409
527
543
43.835
44.
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48.
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056
341
558
259
523
246
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451
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106
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257
437
269
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6.
11.
7.
7.
7.
6.
7.
1.
10.
7.
7.
7.
8.
7.
8.
5.
7.
8.
12.
9.
7.
9.
6.
7.
6.
6.
7.
2
1
6
9
5
3
4
3
3 R
8
1
0
8
1
5
3
7
6
4
1
0
7
8
7
9
8
8
,5
.5
8.0
7,
,4
0.0
7
7
9
8
10
7
.5
.1
.1
.2
.2
.5
2-297
-------
PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD
Volume 2, Chapter 1
Attachment 1: Mixed Congener Standard on DB-5
(Page 4 of 5)
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
103(ISTD)
100
63
74
70+76
66
95
91
56+60
92+84
89
101
99
83
97
81
87
85
136
110+77
82
151
135+144
147+124
107
123+149
118
134
114+131
146
132+153+105
141
137+176
130
163+138
158
129
178
166(SURR)
175
187+182
183
128
167
185
174
177
202+171
156
173
157+200
204(ISTD)
172+197
180
193
191
199
170+190
DBC(SURR)
198
9.
0.
0.
2.
3.
5.
2.
0.
3.
1.
0.'
1.
0.
0.
0.
0.
1.
0.
0.
2.
0.
1.
0.
0.
0.
3.
1.
0.
0.
0.
4.
1.
0.
0.
2.
0.
0.
1.
4.
0.
4.
1.
0.
980792
102683
205672
026874
643651
537148
298095
537131
821698
896827
109377
932718
758792
143149
568289
184326
073545
709927
778711
019769
469813
803968
948432
395914
124099
043263
2SS572
068224
141561
400207
593065
841368
271340
071601
926401
2S2452
015677
193907
808515
194360
078599
905587
094070
0.041623
0.494288
3.529921
1.909970
0.871405
0.065165
0.034467
0.439246
9.931970
0.595701
6.918325
0.444705
0
0
1
10
0
.110430
.430443
.942022
.149161
.125405
57.
58.
59.
59.
60.
61.
61.
62.
63.
64.
64.
65.
66.
67.
68.
69.
69.
70.
70.
71.
72.
73.
74.
74.
75.
75.
75.
77.
77.
78.
79.
81.
83.
83.
84.
84.
85.
85.
86.
86.
87.
88.
584
510
395
972
712
166
326
311
796
590
973
514
274
975
821
423
704
280
625
348
937
559
298
639
149
576
882
027
481
990
954
885
050
388
228
586
365
885
215
738
260
047
88.478
89.243
89.556
90.910
91.710
92.411
92.687
93.
.124
93.709
93.936
94
95
96
96
97
100
100
101
.732
.748
.160
.770
.250
.228
.727
.285
0.
0.
0.
0.
0.
0.
-0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
-0.
0.
0.
0.
0.
0.
0.
0.
0.
-0.
039
003
015
021
018
017
000
005
023
004
012
007
008
004
006
010
013
005
005
013
006
008
000
028
013
008
020
006
020
016
013
009
005
015
009
010
003
006
000
002
001
002
023
014
007
001
0.005
0.005
0.011
-0.000
-0.002
0.050
0.004
0
0
0
-0
0
-0
-0
.012
.003
,002
.007
.006
.013
.002
2275
33
68
767
1401
1361
719
164
1497
391
43
625
358,
57
327
81
551
328
210
911
273
801
333
23
51
1224
597
40
55
204
1858
1285
236
33
1103
160
43
518
3252
100
2366
1101
126
15
605
1802
934
731
82
35
326
5983
385
4264
323
70
388
1556
5274
179
VB
BB
BV
W
W
W
VB
BV
BV
W
VB
BB
BB
BB
BV
W
W
W
VB
BB
BB
BB
BV
VB
BV
W
VB
BB
BV
BB
BB
BB
BV
VB
BV
VB
VP
PV
W
TS
VB
BV
VB
BV
VB
BB
BB
BV
VB
BB
BV
VB
BB
BV
VB
BB
BB
BV
VB
TS
7.6 R
9.2
8.2
7.7
8.
9.
11.
7.
8.
7.
6.
8.
7.
7.
7.
7.
8.
8.
7.
8.
7.
8.
8.
7.
7.
8.
8.
6.
8.
7.
9.
8.
7.
7.
10.
8.
17.
8.
8.
0.
8.
0
2
5
2
2
7
5
0
6
6
9
9
3
0
4
4
5
0
1
6
6
1
3
4
3
8
5
1
9
7
6
8
4
1
4
0
4
8.1
9.
5
6.6
8.0
8.6
8.
0
8.0
8.2
7.0
8
.4
8.3 R
8.2
8.5
8
6
8
9
8
0
.4
.9
.1
.1
.4
.0
2-298
-------
Volume 2, Chapter 1
PCS Congener Analysis of XAD-2
Resins and OFF Filters Using GC/ECD
Attachment 1: Mixed Congener Standard on DB-5
(Page 5 of 5)
99
100
101
102
103
104
105
106
107
108
201
203
196
189
2081-195
207
194
205
206
209
4 .
2.
1.
0.
0.
0.
2.
0.
0.
0.
.806856
.407035
.977705
.039184
.896954
.093248
.084478
. H2808
.777460
.013712
101
102
102
105
107
108
no
111
116
121
.905
.656
.750
.148
.209
.476
.864
.538
.694
.396
-0
-0
-0
-0
0
-0
-0
-0
-0
0
.002
.012
.013
.020
.005
.015
.002
.003
.007
.000
2272
1611 *
1323
26
1064
133
2088
112
849
48
B8
BV
VB
BB
BB
BB
PB
BB
BB
BB
8.
8.
10.
8.
7
7.
8.
7.
7.
7.
.3
.3
.8
.1
.9
.4
.2
.7
.9
.2
0.826 82548
Totals: 249.217190
i
Status Codes:
R - Reference peak
Total Unidentified Counts . 5777 counts
Detected Peaks: 196 Rejected Peaks: 0 Identified Peaks: 108
Amount Standard: N/A Multiplier: 1.000000 Divisor: 1.000000
Baseline Offset: -11 microvolts
Noise (used): 140 microvolts monitored before this run
Rack: 1 Vial: 12 Injection Number: 1 Injection Volume: 5.0 ul
Original Notes:
MULLIH CAL STD W SINGLE LEVEL SURROGATES FOR SURROGATE
INTERNAL STD CALIBRATION
Appended Notes:
2-299
-------
Volume 2, Chapter 1
PCS Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD
Attachment 2. Mixed Congener Standard on DB-1701
(Page 1 of 5)
Title : June cal DB-1101 5 ul inj using height for cal
Run File : C:\STAR\MODULE16\728JN4\7284013.RUH
Method File : C:\STAR\HOtXJLElS\728JN4\72840H.HTH
Sanple ID : PP-376C 183
Injection Date: 26-MAY-95 4:51 PM Calculation Date: 22-JUN-95 3:05 PM
Operator : TJF
Workstation:
Instrument : Varlan Star 11
Channel : B - 01
Detector Type: ADCB (10 Voltsl
Bus Address : 16
Stopl* Rate : 5.00 Hi
Run Tine : 152.503 ain
Star Chronatoqraphy Software
Version 4.01
Chart Speed
Start Tine
0.42 CM/Bin Attenuation - 12 Zero Offset • 251
30.000 Bin End Tine • 140.000 Bin Kin / Tick - 5.00
10
'20
mVolts
57.367
60.958
8:8! «•'
— 64.585
(•^—
- 67.942
65.381
•69.381
74.M9
2-301
-------
PCS Congener Analysis ofXAD-2
Resins and GFF Filters Using GC/ECD
Volume2, Chapter?
Attachment 2: Mixed Congener Standard on DB-1701
(Page 2 of 5)
76.761
42.020
2-302
-------
Volume 2, Chapter 1
PCS Congener Analysis ofXAD-2
Resins and GFF Filters Using GC/ECD
Attachment 2: Mixed Congener Standard on DB-1701
(Page 3 of 5)
Title : June cal DB-1701 5 ul inj using height for cal
Run File : C:\STAR\HODULEl6\728JN4\7284013.Rtni
Method File : C-.\STAR\HODULE16\726JIM\72840U.KTH
Saaple ID : PP-376C 183
Injection Date: 26-HAY-95 4:51 PH Calculation Date: 22-JUN-95 3:05 PM
Operator : TJF
Workstation:
Instrument : Varian Star II
Channel : B » 01
Detector Type: ADCB (10 Volts)
Bus Address : 16
Sanple Rate : 5.00 Hi
Run Tin : 152.503 «in
Star ChroMtography Software «*«*•••••• Version 4.01
Run Mode : Analysis
Peak Heasureaent: Peak Height
Calculation Type: External Standard
Peak Peak
No. Haoe
1 10
2 4
3 7
4 9
5 6
6 6
7 5
8 14SURR
9 30ISTD
10 19
11 12+13+17+18
12 24
13 15
14 27
15 32
16 16+29
17 26+25
18 28+31
19 21+33+53
20 51
21 22
22 45
23 43+46+52
24 65+47+49
25 48
26 44
27 42
28 103ISTD
29 41+71+100+64
30 40+63
31 74
32 70
33 66+95
34 91
35 60+101
36 56+99
37 89+84
38 83
Result
(NG/L)
0.176037
3.018902
0.265841
0.335552
1.816117
14.159795
0.278926
5.03(743
10.156128
0.281359
6.391715
0.052619
1.490831
0.203341
1.854917
2.099022
1.021916
10.125882
4.102441
0.023595
2.925104
0.928908
4.576473
3.705014
0.847172
4.734031
1.447829
9.717182
3.625286
1.256547
2.065444
3.283874
5.817988
0.533801
5.634208
3.559681
1.446028
0.158861
Ret. Tine Width
Tine Offset Height Sep. 1/2 Status
(min) (nin) (counts) Code (sec) Codes
45.261
45.844
49.117
49.281
52.143
53.171
53.431
54.919
56.083
57.367
60.958
62.559
62.831
63.221
64.585
6S.381
67.942
69.381
71.875
72.245
73.676
74.149
76.102
76.477
76.761
80.575
80.786
82.020
82.314
85.098
85.856
87.639
87.991
88.717
91.523
92.018
92.557
95.090
0.000
0.014
0.018
0.010
0.011
0.005
-0.002
0.005
0.039
0.004
0.010
0.017
0.014
0.006
0.004
0.011
0.008
0.022
0.018
-0.019
0.015
0.016
0.011
0.004
0.003
0.016
0.009
-0.002
0.016
0.004
0.014
0.017
0.017
0.009
O.OOS
0.014
0.001
0.004
65
50
95
57
174
593
77
841
2043
59
643
49
54
51
223
273
128
1314
542
10
402
156
476
1378
172
803
296
1354
534
260
494
864
911
90
760
572
174
46
BB
BB
BB
BB
BB
BV
VB
BB
BP
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BV
VB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
6.2
6.1
7.8
9.7
6.8
7.3
7.2
7.5
7.2 R
7.2
8.7
7.0
8.2
7.3
7.5
7.6
13.1
9.0
8.1
11.4
7.8
7.6
8.1
9.5
8.0
8.2
9.7
7.7
11.7
7.5
7.9
8.0
9.9
7.7
8.2
9.8
7.9
8.1
2-303
-------
PCS'Congener Analysis of XAD-2
Resins and GFF Filters Using GC/ECD
Volume 2, Chapter^
Attachment 2: Mixed Congener Standard on DB-1701
(Page 4 of 5)
39 92+97
40 trans nonach
41 87
42 85
43 136
44 110
45 151
46 144
47 77
48 82
49 135
SO 149
51 107+123
52 118
53 114
54 134
55 131+137
56 146
57 153
58 132
59 141+176+105
60 129
61 130
62 163+138+158+
€3 182
64 187
65 166SXS
66 183
67 185
68 167
69 202+167+128
70 174+200+2041
71 177
72 171+197
73 173
74 156
75 172
76 157+180+199
77 193
78 191
79 198
80 170+190+201
81 196+203
82 208
83 207
84 189
85 195
86 194
87 205
88 206
89 20.9
Totals:
1.070954 95.977
0.006340 96.512
1.052906 97.292
0.695014 97.675
0.726305 97.945
2.009048 99.362
1.796553 99.955
0.630569 100.531
0.228873 101.073
0.464388 101.797
0.156599 102.165
3.115006 102.432
0.071140 102.667
1.281861 103.558
0.356901 103.899
0.076659 104.496
0.035547 105.067
0.403681 105.455
2.840417 106.450
0.684852 108.216
2.702293 109.348
0.014328 110.015
0.081114 111.250
4.417425 112.199
0.697081 112.930
3.900236 113.722
4.921273 113.888
1.816817 114.360
0.465152 116.771
0.037056 117.391
0.311932 117.624
11.663553 118.659
1.840270 119.537
0.363120 120.146
0.037543 121.310
0.070713 121.833
0.576366 122.556
7.206979 123.651
0.435179 124.422
0.124112 124.824
0.068043 128.187
0.939771 128.836
4.480081 129.174
0.101339 130.523
0.095886 131.078
0.036086 131.360
0.703326 131.705
1.901737 133.084
6.093067 133.394
0.724105 134.676
0.010731 135.533
187.695437
-0.002
-0.013
0.005
0.004
0.001
0.009
0.003
0.002
-0.003
0.015
0.014
0.011
0.011
0.019
-0.008
0.006
0.012
0.019
0.015
0.016
-0.009
0.013
0.007
0.010
0.018
0.023
-0.006
0.019
0.018
-0.003
-0.006
0.045
0.014
0.008
0.023
0.019
0.028
0.021
0.021
0.021
0.003
0,007
-0.008
-0.013
-0.015
-0.016
-0.016
-0.020
-0.020
-0.020
-0.019
245
2
296
187
104
530
458
92
133
161
10
686
22
385
24
28
45
106
895
555
515
16
20
757
48
1328
2041
569
302
9
191
3932
492
214
19
54
204
2487
159
41
109
209
1660
206
148
47
1035
2564
146
995
55
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BV
W
VB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
BB
8.7
2.6
7.8
7.8
7.9
8.0
7.6
7.2
8.4
7.6
8.0
8.1
11.3
7.8
8.1
8.3
7.7
7.9
8.3
8.3
14.4
7.1
6.9
10.6
8.5
9-6
8.8
8.3
8.2
6.7
8.0
9.5 R
8.3
8.2
8.2
7.5
7.6
8.1
8.2
7.6
0.0
6.4
6.6
4.3
4.7
0.0
3.5
3.2
0.0
3.6
3.3
0.651
Status Codes:
R - Reference peak
Total Unidentified Counts
Detected Peaks: 293
42619
6147 counts
Rejected Peaks: 6
Identified Peaks: 89
2-304
-------
PCS Congener Analysis of XAD-2
Volume 2, Chapter 1 Resins and GFF Filters Using GC/ECD
Attachment 2: Mixed Congener Standrd on DB-1701
(Page 5 of 5)
Amount Standard: N/A Multiplier: 1.000000 Divisor: l.OOOOOO
Baseline Offset: -14 microvolts
Noise (used): SO microvolts - monitored before this run
Rack: 1 Vial: 12 Injection Number: 1 Injection Volume: 5.0 ul
2-305
-------
PCBs and Pesticides in Surface Water
by XAD-2 Resin Extraction
Environmental Sciences Section
Organic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised January 1, 1996
-------
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
1.0 Application
l.l This method is used to determine congener specific PCB and pesticide concentrations at trace
levels in surface water. A multi-plate filtration system for collection of particulates, and an
XAD-2 resin column for collection of dissolved PCBs is used. Water volumes of 80 L to 160 L
can be analyzed by this method.
1.2 The following limits of detection (method detection limits) were determined for the resin
("dissolved") PCBs and the filters ("paniculate") PCBs for sample sizes of 80 L and 160 L:
BZ#(1)
#3
#4/10
#7/9
#6
#8/5
#19
#18
#15/17
#24/27
#16/32
#26
#25
#28/31
#33
#53
#51
#22
#45
#46
#52
#49
#47/48
#44
#37/42
#41/71/64
#40
#63
#74
#70/76
SOL
LOD (MDL)
ng/L
0.43
0.050
0.011
0.022
0.049
0.0070
0.014
0.030
0.0070
0.022
0.014
0.012
0.040
0.015
0.0080
0.0070
0.022
0.0090
0.0090
0.015
0.010
0.018
0.013
0.020
0.020
0.010
0.025
0.013
0.025
LOQ
ng/L
1.4
0.17
0.037
0.073
0.16
0.023
0.047
0.10
0.023
0.073
0.047
0.040
0.13
0.050
0.027
0.023
0.073
0.030
0.030
0.050
0.033
0.060
0.043
0.067
0.067
0.033
0.083
0.043
O.OS3
160 L
LOD (MDL)
ng/L
0.22
0.025
0.0055
0.011
0.024
0.0035
0.0070
0.015
0.0035
0.011
0.0070
0.0060
0.020
0.0075
0.0040
0.0035
0.011
0.0045
0.0045
0.0075
0.0050
0.0090
0.0065
0.010
0.010
0.0050
0.012
0.0065
0012
LOQ
ng/L
0.72
0.083
0.018
0.037
0.080
0.012
0.023
0.050
0.012
0.037
0.023
0.020
0.070
0.025
0.013
0.012
0.037
0.015
0.015
0.025
0.017
0.030
0.022
0.033
0.033
0.017
0.040
0.022
0.040
2-309
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PCSs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Volume2, Chapterl
BZ#(1)
#66
#95
#91
#56/60
#92/84
#89
#101
#99
#83
#97
#87
#85
#136
#77/110
#82
#151
#135/144
#123/149
#118
#146
#132/153/105
#141
#137/176
#163/138
#158
#178
#187/182
#183
#128
#167
#185
#174
#177
#202/171
#172
#180
#193
#199
#170/190
#198
#201
#203/196
SOL
LOD (MOD
ng/L
0.023
0.012
0.011
0.016
0.024
0.0060
0.011
0.0080
0.0090
0.0060
0.010
0.011
0.030
0.022
0.0070
0.010
0.013
0.010
0.016
0.011
0.020
0.0080
0.013
0.022
0.015
0.014
0.010
0.011
0.0090
0.012
0.0070
0.011
0.012
0.0080
0.015
0.013
0.015
0.0090
0.01 1
0.015
0.018
0.028
LOQ
ng/L
0.076
0.040
0.037
0.053
0.080
0.020
0.037
0.027
0.030
0.020
0.033
0.037
0.10
0.073
0.023
0.033
0.043
0.033
0.053
0.037
0.067
0.027
0.043
0.073
0.050
0.047
0.033
0.037
0.030
0.040
0.023
0.037
0.040
0.027
0.050
0.043
0.050
0.030
0.037
0.050
0.060
0.093
160 L
LOD (MDL)
ng/L
0.012
0.0060
0.0055
0.0080
0.012
0.0030
0.0055
0.0040
0.0045
0.0030
0.0050
0.0055
0.015
0.01 1
0.0035
0.0050
0.0065
0.0050
0.0080
0.0055
0.010
0.0040
0.0065
0.011
0.0075
0.0070
0.0050
0.0055
0.0045
0.0060
0.0035
0.0055
0.0060
0.0040
0.0075
0.0065
0.0075
0.0045
0.0055
0.0075
0.0090
0.014
LOQ
ng/L
0.040
0.020
0.018
0.027
0.040
0.010
0.018
0.013
0.015
0.010
0.017
0.018
0.050
0.037
0.012
0.017
0.022
0.017
0.027
0.018
0.033
0.013
0.022
0.037
0.025
0.023
0.017
0.018
0.015
0.020
0.012
0.018
0.020
0.013
0.025
0.022
0.025
0.015
0.018
0.025
0.030
0.047
2-310
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Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
BZ#(1)
#208/195
#207
#194
#206
SOL
LOD (MDL)
ng/L
0.0080
0.0070
0.011
0.0070
LOQ
ng/L
0.027
0.023
0.037
0.023
160 L
LOD (MDL)
ng/L
0.0040
0.0035
0.0055
0.0035
LOQ
ng/L
0.013
0.012
0.018
0.012
1.3 Pesticide LODs and LOQs: 80 L and 160 L Water
Compound
alpha-BHC
gamma-BHC (Lindane)
oxychlordane
gamma-chlordane
alpha-chlordane
trans-nonachlor
p,p'DDD
cis-nonachlor
p,p'DDT
toxaphene
hexachlorobenzene (HCB)
p,p'DDE
SOL
LOD (MDL)
ng/L
0.050
0.050
0.019
0.022
0.021
0.017
0.050
0.021
0.050
10.0
0.0060
0.030
LOQ
ng/L
0.16
0.16
0.063
0.073
0.070
0.057
0.16
0.070
0.16
33.0
0.020
0.10
160 L
LOD
(MDL)
ng/L
0.025
0.025
0.010
0.011
0.010
0.0085
0.025
0.010
0.025
5.0
0.0030
0.015
LOQ
ng/L
0.082
0.082
0.033
0.037
0.033
0.028
0.082
0.033
0.082
16.0
0.010
0.050
2.0 Sampling
2.1 Water samples are filtered and pumped through XAD-2 resin columns in the field. (See the field
standard operating plan.)
2.2 Foil-wrapped filters and resin columns received in the lab from the field are refrigerated at about
4°C until time for laboratory extraction.
3.0 Reagents
3.1 Hexane, acetone, ethyl ether, methylene chloride, methanol Pesticide Grade
3.2 Sodium sulfate ACS granular; stored at I30:C.
3.3 Silica Gel Davison Grade 923, 100-200 mesh activated at 130°C. deactivated with 3.5% water
for an hour prior to use.
3.4 Florisil PR Grade 60-100 mesh. Dried at 1 30CC. stored in air tight container at room
temperature.
3.5 Glass uool Soxhlet extracted in acetone/he\ane 50:50 for eight hours.
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PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction Volume 2, Chapter?
3.6 Glass fiber filters: 293 mm diameter, 0.7 micron mesh from Microfiltration system, Dublin, CA;
wrapped in aluminum foil and heated for four hours at 450°C, stored and sent to the field in the
foil packets.
3.7 Resin columns: 5.0 cm X 30.0 cm glass chromatographic columns, with threaded ends, 50 mm
thread size; heated for four hours at 450°C.
3.8 Nylon plugs, two per column, 50 mm thread size, and nylon adaptor plugs with swagelock fittings:
soaked overnight in a 50/50 mixture of acetone/hexane prior to use. O-rings are soaked in hexane
overnight.
3.9 Amberlite XAD-2, 20-60 mesh, Sigma Chemical Company, cleaned as described below.
3.10 HCl-Reagent grade, diluted to 50% and extracted with hexane three times.
4.0 XAD-2 Resin Column Preparation
4.1 The XAD-2 resin is cleaned in the lab by a series of solvent extractions in a large Soxhlet
apparatus. Approximately 2.5 kg of resin is extracted sequentially for 24 hrs each in methanol,
acetone, hexane, and methylene chloride. This is followed by sequential six hour extractions in
acetone, hexane, and acetone. This sequence cycles the resin back to a water-miscible solvent,
which is displaced from the resin by rinsing with several volumes of organic free water. Cleaned
resin is stored under organic free water in amber bottles for one to three months, until column
preparation. The hexane from the 6 hour extraction is used as a resin quality control blank. The
final six hour acetone extract can be used as the first acetone on the next batch of resin.
4.2 XAD-2 resin columns are prepared by first attaching one nylon adaptor with a Swagelock fitting
and a 3" length of latex tubing to one end of the glass column, and pushing a large plug of cleaned
glass wool into the bottom. The column is filled about Vi full with organic free water and clean
resin is poured into the column in a slurry to a final packed length of 19.5 cm (400 mL). The resin
is packed by pumping excess water out from the bottom using a water aspirator or peristaltic pump
but maintaining enough water in the column to cover the resin. The column should not contain air
bubbles or channels. The top of the column is plugged with wet glass wool and a nylon plug. The
nylon adaptor at the bottom is replaced with a nylon plug. Columns are wrapped for shipping and
stored in the lab until picked up by United States Geological Survey sampling crews.
4.3 A log is kept of resin batches as they are being cleaned, and of columns as they are prepared and
sent to the field so that traceability of samples to individual columns and to batches of cleaned
resin is maintained.
5.0 Surrogate and Matrix Spikes
5.1 Surrogate standards are added to each sample and blank prior to extraction to monitor analytical
reanenes of PCB congeners. The surrogates are PCB congeners #14. #65. and #166 at nominal
concentrations of 20. 5, and 5 ng/mL respectively They are added to the Soxhlet extractor of
even, sample and blank at the beginning of the analytical procedure.
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PCBs and Pesticides in
Volume 2, Chapter 1 Surface Water by XAD-2 Resin Extraction
5.2 The matrix spike solution consists of the following Aroclor mixture: Aroclors 1232, 1248, and
1262 at 0.25. 0.1 8, and 0.18 mg/L, respectively, in acetone. It does not contain internal standards.
With each batch of samples analyzed, an appropriate amount of this spike solution is added by
Class A volumetric pipet to a Soxhlet containing clean resin and to a Soxhlet containing a clean
filter. Surrogates are also added. These spikes are extracted and analyzed along with the samples.
5.3 With each batch of samples, a separate spike of chlorinated pesticides is added to a Soxhlet
containing clean resin and to a Soxhlet containing a clean filter.
6.0 Sample Extraction - Resin and Filters
6.1 The resin, representing the dissolved portion of a surface water sample, is analyzed by extracting
the resin and glass wool plugs in two Soxhlets, each with a 500 mL mixture of 50% acetone/50%
hexane, for 16 hours. Excess water at the top of the resin column is first poured off into an
Erlenmeyer flask. Resin is transferred to the Soxhlets in an acetone slurry, and rinsed at least
twice with acetone to remove as much water as possible. This acetone-water "rinsate" (400 to
450 mL) is added to the water in the Erlenmeyer and set aside. Surrogate spike solution is added
to each resin Soxhlet and to the rinsate.
6.2 The Soxhlet extract will still contain some water which will result in a two-layer
acetone-and-water/hexane system. After extraction is complete, the extract is reduced in volume
on a rotary evaporator to approximately 300 mL and transferred to a 500 mL separator/ funnel.
The water layer is drawn off and combined with the rinsate from that sample in a 1 L or 2 L
separatory funnel. The hexane layer is saved. Then 300 mL of organic-free water is added to the
rinsate. Five (5) mL of 50% HC1 is added to minimize emulsion. The rinsate is then extracted
three times with 100 mL, 75 mL and 75 mL of hexane. The hexane extracts are combined with
the hexane layer from the Soxhlet extract and concentrated to approximately 5 mL using 15 mL of
iso-octane as a keeper. Sodium sulfate (approximately lOg) is added to absorb residual water.
6.3 Filters from each sample are combined and extracted in a Soxhlet (separately from the resin) with
a 600 mL mixture of 50% acetone/50% hexane for 16 hours. Surrogate spike is added to the
Soxhlet at the beginning of the extraction.
6.4 The filter extract also contains some water which will form a separate layer. The filter extract is
concentrated to approximately 300 mL on a rotary evaporator, and transferred to a 500 mL
separatory funnel. The water layer is drawn off, transferred to a second separatory funnel, and
100 mL of organic-free water is added. Five (5) mL of 50% HC1 is added to minimize emulsion.
The rinsate is then extracted three times with hexane (75 mL, 50 mL, 50 mL). The hexane
extracts are combined with the filter Soxhlet hexane layer and concentrated on a rotary evaporator
to approximately 5 mL for clean-up, using I 5 mL of iso-octane as a keeper. Sodium sulfate
(approximately lOg) is added to absorb residual water.
7.0 Sample Clean-up and Fractionation
7.1 Flonsil and silica gel column chromatography are employed as clean-up techniques prior to
GC-EC unaKsis. The fractionations arc required to separate PCBs from as mun\ other parameters
as possible. This facilitates identification and analysis by GC-EC. The flonsil procedure is
performed first, followed by silica gel.
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PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction Volume 2, Chapter^
7.2 Florisil columns are prepared by placing 1 cm of anhydrous sodium sulfate in a 1 cm i.d. X 30 cm
chromatography column fitted with a 75 mL reservoir. The column should be previously filled to
slightly above the reservoir base with hexane. Eight grams of 60/100 mesh Florisil (Floridin
Company), activated at 130°C for 16 hours, is then added and topped with another 1 cm layer of
sodium sulfate. Avoid entrapping air bubbles when pouring the column. Adjust the hexane level
to within a few mm of the top layer of sulfate and discard the excess solvent (this also serves as a
column wash).
7.3 When hexane reaches the top of the upper sodium sulfate layer, the sample extract is quantitatively
transferred to the column and allowed to drain onto the bed of Florisil. The sample container is
washed with 5 mL of hexane and added to the column as the original extract has just reached the
top layer of sulfate. This also serves to wash down the walls of the column. When the hexane
rinse reaches the top of the Florisil, the elution solvent is added, and the eluate is collected for
further separation. The volume and makeup of the elution solvent is determined from the Florisil
elution check. Currently 50 mL of 94/6 hexane/ethyl-ether is used. This eluate is concentrated
under a gentle stream of air to about 5 mL and cleaned up on silica gel. See Section 7.4
7.4 The eluate from the Florisil column must be further fractionated through silica gel to separate
PCBs and chlorinated pesticides. Prepare the silica gel by heating at 130°C overnight and
deactivating before use by equilibrating one hour with 3.5% distilled water. (The percentage of
deactivation may change with different lots of silica gel.) Prepare silica gel columns (1cm i.d. x
30 cm) by first filling with hexane. Add 1 cm of anhydrous sodium sulfate, 5 gm of deactivated
silica gel and another 1 cm of sodium sulfate layer and quantitatively add the first florisil fraction.
Start collecting the eluate and elute the PCBs, HCB, and p,p'DDE with 50 mL of hexane. Two to
3 mL of iso-octane is added to this SGI fraction, and it is concentrated down under a gentle stream
of air to approximately 5 mL, then transferred to a centrifuge tube and further concentrated to
1.0 mL for GC analysis. A final clean-up is done by adding 1.0 mL concentrated sulfuric acid to
the SGI extract.
7.5 A second fraction is eluted from the silica gel column using 60 mL of 25% ethyl ether in hexane.
This will contain alpha-BHC, lindane, the chlordanes, nonachlors, p,p'DDD, p.p'DDT and
toxaphene. Two to 3 mL of iso-octane is added to this SG2 fraction and it is concentrated as in
Section 7.4 above, transferred to a centrifuge tube and further concentrated to 1.0 mL. A final
clean-up of this fraction is done by adding 1 mL of concentrated sulfuric acid to the extract,
mixing thoroughly, and allowing to sit for up to 24 hours.
8.0 Gas Chromatography for PCB Congeners, HCB, and p,p'DDE
8.1 GC Conditions
HP 5890-11 Gas Chromatograph
60M DB5 column. 0.2 mm ID. 0.1 urn film
Hydrogen carrier gas
Electron Capture Detector; 300°C
Pressure Programmable Injector; 265"C
Initial Pressure 40 psi. 1.0 mm hold
Programmed from 40 psi to 20 psi at 20 psi/min. then go to constant flow mode for remainder of run
Splitlcss miection: purge on at 0.70 min
Injector volume I uL
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Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Oven Temperature Profile:
Initial Temp 100°C, hold for 1.0 min
100GCto 150;Cat3°C/min
150°Cto220°Cat 1 °C/min
220 C to 280 C at 5°C/min, hold for three min
8.2 Standards
The single point calibration standard consists of a dilution of a stock solution of Aroclors 1232,
1248,and 1262 at 183 ^g/mL which was supplied by M. Mullein in June 1994. See
Table 1293.851 for congener composition of the stock solution. The diluted standard contains
Aroclors 1232, 1248, and 1262 at .225, .162, and .162 ng/L for a total of .549 ^g/mL PCB.
Quantitation of congeners #128 and #167 requires the addition of individual standards of these
congeners to the calibration mix, at nominal concentrations of 4 ng/mL and 2 ng/mL, respectively.
The total concentration of these congeners in the calibration mix must also include the contribution
from the Aroclors. This contribution is 0.30 ng/mL of #128 and 0.15 ng/mL of #167. This
standard also contains PCB congener #30 at a nominal concentration of 0.012 mg/L (12. ng/mL),
and PCB congener #204 at 0.013 mg/1 (13. ng/mL) which are used as retention time reference
peaks and as internal standards for quantitation. Congeners eluting prior to and including #77/110
use congener #30 as internal standard, those eluting after #77/110 use congener #204 as internal
standard. The calibration table contains the concentration in ng/mL of each congener in the mix,
including internal standards, as well as surrogates #14, #65, and #166 at nominal concentrations of
32, 7, and 8 ng/mL. See Table 1293.8b2.
8.3 A three-level calibration is performed yearly to verify detector response linearity, using the
single-point standard and standards at 0.5x and at 2x the single point standard. The RSD of the
three response factors for each congener shall be less than 25%. Alternatively, linearity may be
demonstrated by a correlation coefficient of at least 0.95.
8.4 Pesticide standards containing HCB, trans-nonachlor, and p,p'-DDE at concentrations of
4-8 ng/mL and containing internal standards #30 and #204 are also run with each batch of PCB
extracts.
8.5 Instrument Performance
Congener response factors are generated daily from a run of the single point calibration standard.
This standard will also be run every 12 hours as a performance standard and evaluated for
resolution, reproducibility, and sensitivity. In addition, a PCB performance standard at either the
,5x or 2x concentration will be run at a nominal frequency of every other sample batch. The
calculated concentrations of congeners #44, #101, #1 85 and #180 shall not differ from their
known concentrations by more than 25%. The calculated concentrations of congeners #6 and
#198 shall not differ from their known concentrations by more than 50%. If these limits are
exceeded, response factors will be regenerated, or the necessary instrument maintenance will be
performed.
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PCSs and Pesticides in
Surface Water by XAD-2 Resin Extraction Volume 2, ChapterJ
8.6 Samples
Some samples may need to be screened by packed column GC-EC. This is to insure that there has
been adequate clean-up, and that samples are diluted or concentrated to an appropriate volume for
injection onto the capillary column. Exactly known amounts of internal standards are added to the
cleaned-up sample extract just prior to capillary column gas chromatography. An appropriate
amount (usually 25 |uL) of a standard containing congeners #30 and #204 is added to the sample
extract to bring their concentrations in the extract to approximately the same as they are in the
calibration standard.
8.7 Calculations
Calculations for PCB Congeners are done by the HP3396 Integrator using the formula for internal
standard quantitation:
Conc.= Height (v) x RF (y) x Amount (IS) x Mult.
Height(IS) RF(IS)
Where: \ = analyte
IS = internal standard
RF = response factor - mass/peak hi.
Amount(IS) = mass of internal standard added to the sample
Mult. = multiplier = I/sample volume
Response factors are generated from a daily run of the calibration standard. Calculations for HCB
and p,p'-DDE are done manually using the same internal standard formula, and using congener
#30 as internal standard.
8.8 Confirmation
Confirmation of correct PCB and pesticide identification is done on 5% of the SGI extracts by
retention time agreement on a 60M DB-1 column, using the same GC conditions and standards as
given in Sections 8.1 and 8.2. Table 1293.8hl contains the concentration in ng/mL of each
congener in the mix.
9.0 Gas Chromatography for Pesticides in the Second Silica Gel
Fraction
9.1 GC Conditions
HP 5890 Gas Chromatograph or equivalent
60M DB1 column. 0.2 mm i.d., 0.1 urn film
Hydrogen carrier gas
Electron Capture Detector
Oven Temperature Profile:
90 C Initial temperature
90 C to I2():C at IO:C/min
120 C in 245 Cat4=C/min
245 C to 2X0: Cat l5:C/min. hold for 6 nun
2-316
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Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Injector temperature 265°C
Detector temperature 320°C
Pressure Programmable Injector
Initial Pressure 25 psi, I min hold
Programmed from 25 psi to 16.5 psi at
10 psi/min, hold at 16.5 psi 42 min
Splitless injection, I uL; purge on at 0.80 min
Retention times are given in the following table:
Compound
alpha-BHC
gamma-BHC (lindane)
#30 (Int. Std)
oxychlordane
gamma-chlordane
alpha-chlordane
trans-nonachlor
p,p'DDD
cis-nonachlor
p,p'-DDT
#204 (RT Reference)
toxaphene compounds
RT, min.
15.90
17.36
17.80
25.08
25.94
26.76
27.20
29.57
29.87
31.49
34.61
27 to 36 min.
RRT vs #204
.459
.502
.514
.725
.749
.773
.786
.854
.863
.910
1.000
—
9.2 Standards
A mixed standard containing the chlorinated pesticides at 4 to 8 ng/mL and also containing
congeners #30 and #204 as internal standards and retention time reference peaks is run with each
set of samples. Standards at 2x or 3x those concentrations will be run if high concentrations are
expected in samples. A standard containing toxaphene at 0.80 mg/L is also run with each batch of
samples.
9.3 Instrument performance
Response factors for pesticides are generated daily from a run of the 4 to 8 ng/mL standard. This
standard, or the 2x or 3x standard, will be run daily as a performance standard.
Calculated concentrations of pesticides in the performance standard should not differ from the
known concentration by more than 20%.
9.4 Samples
Internal standards PCB #30 and #204 are added to the second silica gel fraction, which has been
cleaned up \\ith MI If uric acid (See Section 7.5). just prior to capillary column gas chromatography.
2-317
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PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Volume 2, Chapter 1
9.5 Calculations for pesticides
Calculations for pesticides are done using peak heights, and the same internal standard formula
given in Section 8.7. PCB congener #30 is used as the internal standard for all pesticides; there is
often an interference in this fraction which co-elutes with congener #204.
Note: Trans-nonachlor may split on the silica gel and be found in both fractions. If this occurs it
is quantitated in both fractions and the sum is reported.
9.6 Calculations for toxaphene
Toxaphene is a multi-component mixture of chlorinated camphenes. Identification of toxaphene
in a sample requires a minimum of five sample peaks matching the retention times of standard
peaks. Quantitation is done by summing peak heights of five or more matching peaks in the
sample and in the standard, and using the internal standard formula:
Sum of peak heights
Cone = (sample)
Ht IS (sample)
x Amount IS x I
sample vol. L
Where \ = toxaphene
RF<\) = mass of standard
Sum of peak heights (std)
9.7 Confirmation
Confirmation of SG2 pesticides is done on 5% of the samples by retention time agreement on a
second column (e.g. 60M DB-5), or by GC/MS.
10.0 References
10.1 Ballschmiter. K. and Zell, M., 1980. Fresenius Z. Anal. Chem., 302, 20-31.
10.2 M. Mullein, File=C:\QPR04\QC\LMMBPCBl.WQI 21-June 1994 (Table 1293.8bl).
Table 1293.8bl PCB Stock Solution Concentrations 183 ue/mL
FILE=C:\QPRO4\QC\LMMBPCB1.WQ1
21-Jun-942Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
PCB-000
PCB-001
PCB -003
PCB-004+010(SUVl)
PCB -OOd
Congener
Conc'ns
ug/mL
4.1
12
" 0
3.4
1.9
FILE=C:\QPRO4\QC\LMMBPCB1.WQ1
21-Jun-942Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
PCB -097
PCB -099
PCB -100
PCB-101
PCB -107
Congener
Conc'ns
Mg/niL
0.56
0.74
0.11
l.S
0.13
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Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Table 1293.8bl PCB Stock Solution Concentrations 183 Mg/mL
!,E=C:\QPRO4\QC\LMMBPCB1.WQ1
21-Jun-94 2 Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
B-007+009(SUM)
B-008+005 (SUM)
B-012
B-013
B-015+017(SUM)
B-016
B-018
B-019
B-021
B-022
B-024+027 (SUM)
PCB-025
PCB-026
PCB -029
!CB-03 1+028 (SUM)
CB-032
CB-033
CB-037
CB-040
CB-041+071 (AVE)
CB-042
CB-043
CB-044
CB-045
PCB-046
PCB-047
PCB-048
PCB-049
PCB-05 1
PCB-052
PCB-053
PCB-056+060iAVHi
PCB -06 3
Congener
Conc'ns
Mg/mL
1.2
14
0.17
0.097
3.7
2.0
3.7
0.28
0.032
2.9
0.26
0.32
0.72
0.053
9.4
1.9
3.3
1.2
0.94
2.3
1.4
0.27
4.3
0.89
0.40
1.0
1.0
~> i
0.18
4.5
0.64
3.5
0.21
FILE=C :\QPRO4\QC\LMMBPCB 1 . WQ1
21-Jun-94 2 Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
PCB-110
PCB-1 14+131 (SUM)
PCB-118
PCB-1 19
PCB-123+149(SUM)
PCB-128
PCB-1 29
PCB- 130
PCB-1 32+1 53+105 (SUM)
PCB-134R
PCB-135+144(SUM)
PCB- 136
PCB-1 37+1 76 (AVE)
PCB-141
PCB -146
PCB-1 51
PCB- 156
PCB- 157+200 (AVE)
PCB- 158
PCB-163+138(SUM)
PCB- 167
PCB-170+190(SUM)
PCB-172
PCB- 173
PCB-174
PCB- 175
PCB-177
PCB- 178
PCB-1 80
PCB -183
PCB-1S5
PCB-IS7+182(AVE)
PCB-1S9
Congener
Conc'ns
Mg/mL
1.9
0.14
1.2
0.028
2.8
0.10
0.013
0.075
4.3
0.072
0.89
0.75
0.26
1.7
0.39
1.7
0.066
0.39
0.25
2.7
0.049
1.7
0.56
0.038
3.2
0.20
1.7
1.1
6.1
1.7
0.47
3.6
0.040
2-319
-------
PCSs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Volume 2, Chapter 1
Table 1293.8bl PCB Stock Solution Concentrations 183 ug/mL
FILE=C :\QPR04\QC\LMMBPCB 1 .WQ1
21-Jun-94 2 Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
PCB-064
PCB-066
PCB-070+076 (SUM)
PCB-074
PCB-077
PCB-081
PCB-082
PCB-083
PCB-085
PCB-087
PCB-089
PCB-091
PCB-092+084 (SUM)
PCB-095
Congener
Conc'ns
ug/mL
1.8
5.2
3.4
1.9
0.23
0.16
0.44
0.15
0.70
1.0
0.10
0.51
1.8
2.0
FILE=C:\QPRO4\QC\LMMBPCB1.WQ1
21-Jun-94 2 Sig.Fig.
15:01 LMMB
CALCULATED AVERAGE Calc'd
Peak Name
PCB-191
PCB- 193
PCB- 194
PCB- 197
PCB- 198
PCB- 199
PCB-201
PCB-202+171 (AVE)
PCB-203+196(SUM)
PCB-205
PCB-206
PCB-207
PCB-208+195 (SUM)
PCB-209
Congener
Conc'ns
Mg/mL
0.12
0.42
1.8
0.11
0.12
0.43
4.2
0.79
4.3
0.11
0.68
0.093
0.80
0.012
Table 1293.8b2 PCB Congener Calibration Concentrations - DB-5 Column
CAL
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Name
#1
#3
#4/10
#7/9
#6
#8/5
#14
#19
ISTD1#30
#18
#15/17
#24/27
# 1 (V 1 2
#26
#25
Amount
ng/mL
3.6000E+01
2.1000E+01
1.0200E+01
3.6000E+00
5.7000E+00
4.2000E+01
3.1600E+01
8.4000E-01
1.4200E+01
1.1100E+01
1.1100E+01
7.8000E-01
1.1700E+01
2.1600E+00
9.6000E-01
CAL
#
42
43
44
45
46
47
48
49
50
51
52
53
^4
X"i
56
Name
#83
#97
#87
#85
#136
#77/110
#82
#151
#135/144
#123/149
#118
#146
^132/153/105
#141
#137/176
Amount
ng/mL
4.5000E-01
1.6800E+00
3.0000E+00
2.1000E+00
2.2500E+00
6.4000E+00
1.3200E+00
5.1000E+00
2.6700E+00
8.4000E+00
3.6000E+00
1 . 1 700E+00
1 2900E+01
5.1000E+00
7.SOOOE-01
2-320
-------
Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Table 1293.852 PCB Congener Calibration Concentrations - DB-5 Column
CAL
#
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Name
#28/3 1
#33
#53
#51
#22
#45
#46
#52
#49
#47/48
#65
#44
#37/42
#41/71/64
#40
#63
#74
#70/76
#66
#95
#91
#56/60
#92/84
#89
#101
#99
Amount
ng/mL
2.8200E+01
9.9000E+00
1.9200E+00
5.4000E-01
8.7000E+00
2.6700E+00
1 .2000E+00
1.3500E+01
6.9000E+00
6.0000E+00
7.8000E+00
1.2900E+01
7.800E+00
1.2300E+01
2.8000E+00
6.3000E-01
5.7000E+00
1.0200E+01
1.5600E+01
6.0000E+00
1.5300E+00
1.0500E+01
5.4000E+00
3.0000E-01
5.4000E+00
2.2200E+00
CAL
#
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Name
#163/138
#158
#178
#166
#187/182
#183
#128
#167
#185
#174
#177
#202/171
#157/200
#ISTD 2 #204
#172
#180
#193
#199
#170/190
#198
#201
#203/196
#208/195
#207
#194
#206
Amount
ng/mL
8.1000E+00
7.5000E-01
3.3000E+00
8.4000E+00
1.0800E+01
5.100E+00
4.3000E+00
1.9000E+00
1 .4000E+00
9.6000E+00
5.1000E+00
2.3700E+00
1 . 1 700E+00
1.5600E+01
1 .6800E+00
1.8300E+01
1.2600E+00
1.3000E+00
5.1000E+00
3.6000E-01
1.3000E+01
1.3000E+01
2.4000E+00
2.8000E-01
5.4000E+00
2.0000E+00
Table 1293.8hl PCB Congener Calibration Concentrations - DB-1 Column
CAL
#
1
2
3
4
5
6
7
Name
#1
#3
#4/10
#7/9
#6
#8/5
#14
Amount
ng/mL
3.6000E+01
2.1000E+01
l.OOOOE+01
3.6000E+00
5.7000E+00
4.2000E+01
3.1600E+01
CAL
#
44
45
46
47
48
49
50
Name
#97
#87
#85
#136
#1 10
*s:
#151
Amount
ng/mL
I.7000E+00
3.0000E+00
2.1000E+00
2.3000E+00
5 7000E+00
1 3000H+00
5.1000H+00
2-321
-------
PCSs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Volume 2, Chapter 1
Table 1293.8H1 PCB Congener Calibration Concentrations - DB-1 Column
CAL
#
8
9
10
11
12
13
14
15
16
17
18
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Name
ISTD1 #30
#18
#15/17
#24/27
#16
#32
#26
#25
#31
#28
#33/53
#22
#45
#46
#52
#49
#48
#47
#65
#44
#42
#41/71/64
#40
#63
#74
#70/76
#66
#95
#91
#56/60
#84
#89
#101
#99
#83
Amount
ng/mL
1.4200E+01
1.1000E+01
1.1000E+01
7.8000E-01
6.0000E+00
5.7000E+00
2.2000E+00
9.6000E-01
1.4000E+01
1.3000E+01
1 . 1 800E+00
8.7000E+00
2.7000E+00
1.2000E+00
1.3500E+01
6.9000E+00
3.0000E+00
3.0000E+00
7.8000E+00
1.3000E+01
4.2000E+00
1.2300E+01
2.8000E+00
6.3000E-01
5.7000E+00
l.OOOOE+01
1.6000E+01
6.0000E+00
1 .5000E+00
l.OOOOE+01
3.9000E+00
3.0000E-01
5.4000E+00
2.2000E+00
4.5000E-01
CAL
#
51
52
53
54
56
57
58
59
60
61
62
64
65
66
67
68
69
70
71
72
73
74
76
77
78
79
80
81
82
83
84
85
86
88
Name
#135
#144
#123/149/118
#105/146/132
#153
#141
#137/130
#138/168
#158
#166
#178
#182/187/128
#183
#167
#185
#174
#177
#171/156
#173
#200
#ISTD 2 #204
#172
#180
#193
#199
#170
#190
#198
#201
#203/196
#195
#207
#194
#206
Amount
ng/mL
8.4000E-01
1.8000E+00
1.2000E+01
5.3000E+00
8.1000E+00
5.1000E+00
1 .OOOOE+00
8.1000E+00
7.5000E-01
8.4000E+00
3.3000E+00
1.5000E+01
5.1000E+00
1 .9000E+00
1 .4000E+00
9.6000E+00
5.1000E+00
1 .5000E+00
1.1000E-01
1 .5000E+00
1.5600E+01
1.7000E+00
1.8000E+01
1 .3000E+00
1 .3000E+00
3.6000E+00
1 .4000E+00
3.6000E-01
1.3000E+01
1.3000E+01
2.0000E+00
2.8000E-01
5.4000E+00
2. OOOOE+00
2-322
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Volume 2, Chapter 1
PCBs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Lake Michigan Tributary Study Measurement Quality Objectives
QC Description
PCBs QC Objective
Trans-Nonachlor
QC Objective
Holding Time & Storage
To extraction
After Extraction
Storage Conditions
NA
NA
4°C.
NA
NA
4CC.
Sample Set
<8
Reporting Units
ng/L Dissolved and Paniculate
ng/L Dissolved and
Particulate
XAD Cleanliness Check
Frequency
Criteria
I per resin batch
-------
PCSs and Pesticides in
Surface Water by XAD-2 Resin Extraction
Volume 2, Chapter 1
Lake Michigan Tributary Study Measurement Quality Objectives
QC Description
PCBs QC Objective
Trans-Nonachlor
QC Objective
Blanks
Field Blanks (FRB)
Frequency
Criteria
Lab Reagent Blank (LRB)
Frequency
Criteria
Trip Blank (FTB)
Frequency
Criteria
Lab Dry/Procedural Blank
(LDB)
Frequency
Criteria
1:20
5\ MDL.
Confirmation (CON i
5% bv DB-1 C.C/HC
5% bv DB-5 GC/hC
2-324
-------
Extraction and Cleanup of Sediments for
Semivolatile Organics Following
the Internal Standard Method
Patricia Van Hoof and Jui-Lan Hsieh
Great Lakes Environmental Research Laboratory
National Oceanic and Atmospheric Administration
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105
Standard Operating Procedure GLERL - M - 401 - 01
May 10, 1996
Version 1.0
-------
Extraction and Cleanup of Sediments for Semivolatile Organics
Following the Internal Standard Method
1.0 Scope and Application
This SOP is applicable to the extraction of semivolatile organic compounds from sediment
matrices for analysis by gas chromatography (GC). Samples processed using this extraction
method can be analyzed for semivolatile organic compounds listed in EPA Methods 608, 610, or
625, 8080, 8270, 8310 (EPA 1984, 1986). This method may be applied to other analytes once
acceptable extraction efficiency has been demonstrated.
In this procedure, approximately 15-30 g wet weight sediment is extracted with dichloromethane
(DCM) in a 30°C sonication bath. The extracts are dried over sodium sulfate and passed through
a cleanup column. Column chromatography fractionation allows for separation of pesticides
(PESs) from the majority of polychlorinated biphenyls (PCBs). Polyaromatic hydrocarbons
(PAHs) elute along with the PESs. After a concentration step, internal standards are added and the
sample is ready for analysis.
2.0 Responsible Staff
Project Manager. A Scientist responsible for 1) administration of the project; 2) providing project
specific quality control requirements to the laboratory; 3) defending the data in a Quality
Assurance Audit; and 4) reporting results to the client.
Laboratory Supervisor: A Technical Specialist or Scientist having expertise in the principles
involved with this procedure and in the use of laboratory operations in general. Responsible for 1)
ensuring that analysts are trained in the handling of solvents; 2) that appropriate quality control
samples are included with the sample analysis to monitor precision and accuracy of target
compound concentrations; 3) checking the analysts' work to ensure that samples are handled
appropriately and that data are collected and interpreted correctly; 4) making decisions regarding
problems with the analysis or deviations from the SOP; 5) defending the data in a Quality
Assurance Audit; and 6) reporting results to project manager or client.
Analyst: A Technician, Technical Specialist, or Scientist assigned to conduct analyses using the
procedure. Responsible for 1) understanding the proper handling of samples and solvents; 2)
recording information regarding extractions and any deviations from the SOP in the appropriate
log books; 3) analyzing the appropriate number of quality control samples for each batch of
samples analyzed; 4) reporting results to the Project Manager; 5) participating in QA Audits.
3.0 Procedure
3.1 Apparatus and Reagents
3.1.1 Branson model S210 ultrasonic cleaner with variable temperature water bath
2-327
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Extraction and Cleanup of Sediments
for Semivolatile Organics
Following the Internal Standard Method Volume 2, Chapter 1
3.1.2 Nitrogen evaporator, N-Evap or equivalent, heated with a water bath maintained at
35D±PC
3.1.3 Rotary evaporator heated with a water bath maintained at 30°±1 °C
3.1.4 Glassware
3.1.4.1 250 mL Erlenmeyer flasks
3.1.4.2 100 mL graduated cylinders
3.1.4.3 1000 mL separatory funnels
3.1.4.4 50 mL, 150 mL, and 250 mL beakers
3.1.4.5 Glass rod, 20" long
3.1.4.6 500 mL round bottom flasks
3.1.5 Apparatus for determining sample wet and dry weight
3.1.5.1 Top-loading balance, accurate to 0.001 grams
3.1.5.2 Drying oven maintained at 90°C
3.1.5.3 Aluminum weighing pans
3.1.5.4 Stainless steel spatulas
3.1.6 Long-tipped Pasteur pipettes (9.5" long)
3.1.7 Wide-bore Pasteur pipettes
3.1.8 Chromatography columns, 310 x 11 mm, with Teflon stopcocks
3.1.9 Drying oven maintained at 130°C
3.1.10 Desiccator
3.1.11 Muffle furnace
3.1.12 Sample vials, 2 mL capacity
3.1.13 Purified water (Millipore filtration system)
3.1.14 Saturated sodium chloride solution
3.1.15 Microliter syringes with replaceable needles
2-328
-------
Extraction and Cleanup of Sediments
for Semivolatile Organics
Volume 2, Chapter 1 Following the Internal Standard Method
3.1.16 Glass wool, cleaned by soxhlet extraction using DCM
3.1.17 Sodium sulfate, anhydrous (Mallinkrodt- AR), heated to 450°C for at least 10 hrs, stored in
130°Coven
3.1.18 Silica gel (Aldnch grade 634, 100 - 200 mesh, 60 A, 99+%-), stored in a 130°Coven
3.1.19 Aluminum foil
3.1.20 Solvents: pesticide grade or equivalent
3.1.20.1 Acetone (DMK)
3.1.20.2 Methylene chloride (DCM)
3.1.20.3 Hexane
3.1.20.4 Diethyl Ether
3.1.21 Copper, activated by treating with concentrated HC1 and washing with purified water
twice, methanol twice, methylene chloride twice, and hexane twice, then storing in
hexane.
3.1.22 Ring weights for securing flasks in water bath
3.2 Standards
3.2.1 PAH surrogate standard - containing 2-4 mg/mL each of naphthalene-d8, anthracene-dm,
benz(a)anthracene-di:, benzo(e)pyrene-d,:, and benzo(ghi)perylene-dn in DMK.
3.2.2 PAH internal standard - containing 20 mg/mL each of acenapthylene-dlo, fluorene-d,,,.
chrysene-dl2, and perylene-d,2 in hexane.
3.2.3 PCB surrogate standard - containing 400 ng/mL of PCB 14, 100 ng/mL PCB 65,
and 80 ng/mL of PCB 166 in DMK.
3.2.4 PCB internal standard - containing 170 ng/mL of PCB 30, and 130 ng/mL of PCB
204 in hexane.
3.2.5 Matrix spiking standard - containing 2 mg/mL NIST SRM 1491 PAH standard mixture.
730 ng/mL total PCBs (Mullin's 1994 Aroclor mix). 660 ng/mL trans nonachlor. and
800 ng/mL of other individual pesticides in DMK.
3.3 Sample Handling
Samples shall be kept frozen at approximately -15'C until extraction, unless specified differently
on a project-specific huMs All extracts held overnight sh.ill he covered and refrigerated at -1 ('
Extracts shall be sa\ed and stored at 4'C in ca.se re-anal\Ms is required.
2-329
-------
Extraction and Cleanup of Sediments
for Semivolatile Organics
Following the Internal Standard Method Volume 2, Chapter^
3.4 Labware Preparation
Prior to use, all glassware, Teflon, and other labware should be washed with hot, soapy water and
rinsed with tap water, followed by purified water. Additionally, all non-volumetric glassware
should be combusted in a muffle furnace @ 450°C for at least 16 hours. All volumetric glassware
(i.e., graduated cylinders) should be solvent rinsed twice prior to use.
3.5 Dry Weight Determination
Dry-to-wet weight ratios of sediment samples should be analyzed in triplicate at the time of
sediment sample extractions. Aluminum weighing pans are weighed, a sample of wet sediment is
added, and the pans are weighed again. All samples are dried in the 90°C oven overnight or until
a constant weight is obtained (no change in weight for a period of 3 minutes). Dry-to-wet weigh!
ratios are determined as follows:
Dry Wt = (K dry sample + pan) - (g pan)
Wet Wt (g wet sample + pan) - (g pan)
The dry weight of the sample to be extracted is determined as follows:
Sample Dry Wt = Sample Wet Wt x Mean Dry-to-Wet Wt Ratio
Information for dry weight determinations shall be recorded in the Sediment Extraction Data Sheet
(Attachment 1) for the individual samples. The following criteria for the coefficient of variation
for four measurements were used: if dry/wet ratio < 20%, then c.v. < 25%
if dry/wet ratio > 20%. then c.v. < 15%.
3.6 Sample Extraction
Weigh the appropriate amount of well-mixed wet sediment onto a piece of tared alumina foil to the
nearest 0.01 g. Fifteen to 30 g wet weight sediment shall be weighed out when trace (nanogram
per gram dry weight) quantities of contaminants are expected. Mix sediment with appropriate
amount of sodium sulfate and then scoop mixture into a 250 mL Erlenmeyer flask. One hundred
microliters of the PAH surrogate standard and 62 microliters of the PCB surrogate standard are
pipetted directly onto the sediment sample. Sample weights and standard volumes shall be
recorded in the Sediment Extraction Data Sheet (Attachment 1).
Add 150 mL DCM to the flask, mixing thoroughly. Place ring weight around flask, and cover
opening with foil. Place all batch samples in 30°C ultrasonic bath and sonicate for 60 minutes.
After sonication, let stand in 30°C bath overnight (24 hours). The next day, sonicate samples
again for 60 minutes at 30 "C. Remove flasks from bath.
Set up filtration column as follows: plug the inside of a wide-bore Pasteur pipette with a piece ol
clean glass wool (3 cm in length). Clamp pipette filter above a 500 mL round bottom flask. Filter
solvent phase from the Erlenmeyer flask through the glass \\ool via transfer Pasteur pipette.
2-330
-------
Extraction and Cleanup of Sediments
for Semivolatile Organics
Volume 2, Chapter 1 Following the Internal Standard Method
3.7 Extract Evaporation
Reduce solvent in the flask to 15 mL using the rotary evaporator apparatus. Transfer the extract
to a conical centrifuge tube and further reduce solvent to approximately 1 mL using the nitrogen
evaporator apparatus. Exchange solvent by adding 5 mL hexane, then evaporate to approximately
1 mL and repeat this step twice more. Transfer extract from the centrifuge tube to a
pre-combusted 2 mL sample vial, rinse with 1 mL hexane, add to vial, and cap securely.
3.8 Pre-Column Preparation and Extract Clean-up
3.8.1 Preparation of 37r deactivated silica gel and 109?- deactivated alumina
Day I
Place silica in a 130°C oven overnight (at least 18 hrs). Place alumina in a shallow
ceramic dish and activate it in a muffle furnace @ 450°C for at least 16 hours.
Day 2
Remove silica and alumina from oven and let cool on counter top until room temperature
is reached (approximately 5-10 minutes). When silica and alumina have reached ambient
temperature, deactivate it as follows:
--Working quickly, weigh out desired amount of silica and alumina in separate
round-bottom flasks. Stopper immediately.
—Add 3% and 10% weight/volume of deionized water to silica and alumina respectively,
using the following equation:
% deactivation ~ mL DI water
100 - % deactivat. weight of silica (gm)
-Shake for 10 minutes. Store in desiccator overnight for equilibration. Use deactivated
silica and alumina within three days. Any unused silica and alumina may be reused after
re-activating and re-deactivating.
3.8.2 Preparation of Sodium Sulfate
Pour aliquot of sodium sulfate into a shallow ceramic dish. Heat at 450°C for 16 hrs.
Cool, then store in 130°C drying oven until ready for use.
3.8.3 Column Preparation
Assemble stopcock on column. Stuff glass wool plug (approximately 1 cm) into lower
end of the column with a glass rod. Clamp column securely onto stand. Place empts
150 mL beaker under column. Close stopcock; fill column half full with hexane. Make a
slurry of hexane and 3-grams deactivated silica gel. Open stopcock to partially drain
excess hexane dislodging any trapped bubbles in glass wool plug.
Pour slurry into column tapping column gentl\ u ith a glass rod or spatula. Rinse column
and beaker \\ ith hexane via Pasteur pipette. Make a slurr\ of hexane and 10 grams ol
2-331
-------
Extraction and Cleanup of Sediments
for Semivolatile Organics
Following the Internal Standard Method Volume 2, Chapter_?
deactivated alumina. Pour slurry into column. Cap column with 2 inches sodium sulfate.
Wash column with 25 mL hexane for equilibration. When hexane level reaches 1 cm
above top of sodium sulfate, close stopcock to prevent further dripping. Never let column
run dry. If column is not going to be used immediately, cover column and tip with foil.
3.8.4 Extract Clean-Up
First fraction: PCBs, HCB, 4,4'-DDE, aldrin, and heptachlor
Sonicate sample for 10 seconds before loading onto column. Open stopcock and let drip
until hexane level is at the top of the sodium sulfate. Place a conical centrifuge tube under
the column to catch eluate of first fraction. Measure 35 mL of hexane into a graduated
cylinder. Load sample onto column via Pasteur pipette. Open stopcock and let sample
flow to just the top of the sodium sulfate. Rinse sample vial with approximately 5-mL
hexane in the cylinder and load onto column. Set drip rate to approximately 2 drops per
second. When rinse reaches the top of the sodium sulfate, add the rest of hexane to the
column.
Second fraction: Alpha-BHC, gamma-BHC, heptachlor epoxide, alpha-chlordane.
gamma-chlordane, trans-nonachlor, dieldrin, cis-nonachlor, 4,4'-DDT, 4,4'-DDD and all
PAHs.
While the hexane is dripping from the first fraction, measure 50 mL of 10% diethyl ether
in hexane into the graduated cylinder. When the hexane level from the first fraction
reaches the top of the sodium sulfate layer, add the 10% solution to the column. Replace
conical centrifuge tube with another for collection of the second fraction. Once the column
has stopped dripping, remove the tube and evaporate the solvent.
3.8.5 Evaporation of First and Second Solvent Fractions and Sample Preparation
Place the centrifuge tubes from the first and second fractions in the nitrogen evaporator
water bath. Reduce solvent in the flasks to 1 mL using the nitrogen evaporator apparatus.
The second fraction requires solvent exchange into hexane by adding 5 mL hexane. Rinse
sample tubes with a small amount of hexane and transfer to sample vials, keeping volume
of solvent less than I mL. Add the appropriate amount of internal standards. Record the
amount and ID of the IS in the Sediment and Tissue Extraction Data Sheet. The first
fraction requires sulfur cleanup by adding activated copper beads. Add more if all the
copperheads in sample vial turn black. Cap sample vials, mix, and store at -15 C (first
fraction samples) or at 4:C (second fraction samples) until gas chromatography analysis
4.0 Quality Control Sample Frequency
Samples prepared using this procedure should be processed in batches of approximately
10 samples plus quality control samples.
Laboratory Reagent Blank i LRB) - analyze one per batch Prepare by working through the
sample procedure without a sample matrix, but including \a SO,, extraction solvents, and
appropriate surrogates and internal standards.
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Extraction and Cleanup of Sediments
for Semivolatile Organics
Volume 2, Chapter 1 Following the Internal Standard Method
Laboratory Matrix Spike (LMS) - analyze one per batch. Prepare by fortifying a matrix known to
be below detection limits for an analyte(s) (e.g. pre-industrial sediment) with known amount of
surrogates, target analyte(s), and internal standards.
Laboratory Environmental Matrix Blank (MSB) - analyze less frequently, once or twice per field
trip. Prepare by working a matrix known to be below detection limits for an analyte(s) through the
sample procedure including surrogate and internal standard additions.
Laboratory Duplicate (LDl) - analyze one per batch. Prepare by splitting a sample and treating
identically throughout the analytical procedure.
Laboratory Performance Check (LPC) - run one per batch. A calibration solution used to verify
whether the initial calibration data are currently valid.
Field Duplicate (FDl) - analyze one per batch if possible, otherwise as often as logistics allow.
Field Reagent Blank (FRB) - collect one per field trip. Collect sediment of pre-industrial age for
subcores from several sites (~ 7). This "clean" older sediment will then be run through the entire
collection and analytical systems just as routine field samples.
2-333
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Analysis of Polychlorinated Biphenyls
and Chlorinated Pesticides by
Gas Chromatography with
Electron Capture Detection
Patricia Van Hoof and Jui-Lan Hsieh
Great Lakes Environmental Research Laboratory
National Oceanic and Atmospheric Administration
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105
Standard Operating Procedure GLERL - M - 501 - 02
May 10,1996
Version 2.0
-------
Analysis of Polychlorinated Biphenyls and
Chlorinated Pesticides by Gas Chromatography
with Electron Capture Detection
1.0 Scope and Application
This SOP is applicable to the analysis of environmental sample extracts for polychlorinated
biphenyls (PCBs) as Aroclors and individual congeners and chlorinated pesticides by capillary gas
chromatography with 61Ni electron-capture detection.
This procedure provides typical gas chromatography (GC) conditions for the detection of trace
levels of PCBs and pesticides, methods for identifying the analytes, and methods for analyte
quantification using the internal standard method. Tables 1 and 2 list the most frequently analyzed
compounds and formulations. However, this list may be amended to meet requirements of specific
projects.
2.0 Definitions
The following terms and acronyms may be associated with this procedure:
ECD Electron capture detector or detection
GC Gas chromatography
PCB Polychlorinated biphenyl
RF Response factor
RRF Relative response factor; response factor of analyte normalized to the response factor of
the internal standard.
RSD Relative standard deviation (%)
RT Retention time
IS Internal standard - compound(s) added just prior to analysis on instrument.
SS Surrogate standard - Compound(s) added prior to extraction to assess efficiency of
method.
3.0 Responsible Staff
Project Manager. A Scientist responsible for 1) administration of the project; 2) providing project
specific quality control requirements to the laboratory; 3) defending the data in a Quality
Assurance Audit; and 4) reporting results to client.
Laboratory Supervisor: A Technical Specialist or Scientist having expertise in the principles
involved with this procedure and in the use of the GC. Responsible for 1) ensuring that analysts
are trained in operation of the GC; 2) appropriate quality control samples are included with the
sample analysis to monitor precision and accuracy of the analysis; 3) checking the analysts' work
to ensure that data are collected and interpreted correctly: 4) making decisions regarding problems
with the anaKsis or dev lations from the SOP: 5) defending the data in a Quality Assurance Audit.
and 6) reporting results to project manager or client.
2-337
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Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD Volume 2, Chapter 1
Analyst: A Technician, Technical Specialist, or Scientist assigned to conduct analyses using this
procedure. Responsible for 1) understanding the proper use and maintenance of the GC: 2)
recording information regarding instrument use and maintenance in the appropriate log books; 3)
analyzing the appropriate number of quality assurance samples for each batch of samples analyzed;
4) tabulating all sample and QC data and reviewing the quality of the data based on QC guidelines
presented in this SOP and any other project-specific QC guidelines; 5) reporting results to the
Project Manager; and 6) defending the data during an audit.
Table 1. PCB and Chlorinated Pesticide Analyte List
PCBs (Aroclors) Suggested Internal Standards
Aroclorl232 PCB-030
Aroclorl248 PCB-204
Aroclor 1262
Aldrin Suggested Surrogate Standards
alpha-BHC PCB-014
beta-BHC PCB-065
gamma-BHC (Lindane) PCB-166
delta-BHC PBB-153
4,4'-DDE
4,4'-DDD
4,4'-DDT
(cis)alpha-Chlordane
(trans)gamma-Chlordane
Tech. Chlordane
Dieldrin
Endosufan I
Endosufan II
Endrin
Endrin Aldehyde
Endrin ketone
Heptachlor
Heptachlor Epoxide
Endosulfan Sulfate
Hexachlorobenzene
Mirex
Trans-Nonachlor
Cis-nonachlor
2-338
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Volume 2, Chapter 1
Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD
Table
2. PCB Congener List
CB Numbera CAS Nomenclatureb
8
18
28
29
44
49
50
52
66
77
87
101
104
105
118
126
128
138
153
154
170
180
183
184
187
188
195
200C
206
209
a
b
(.
2,4'-dichlorobiphenyl
2,2',5-trichlorobiphenyl
2,4,4'-trichlorobiphenyl
2,4,5-trichlorobiphenyl
2,3',3,5'-tetrachlorobiphenyl
2,2',4,5'-tetrachlorobiphenyl
2,2',4,6-tetrachlorobiphenyl
2,2',5,5'-tetrachlorobiphenyl
2.3',4,4'-tetrachlorobiphenyl
3,3',4,4'-tetrachlorobiphenyl
2,2',3,4,5'-pentachlorobiphenyl
2,2',4,5,5'-pentachlorobiphenyl
2,2',4,6,6'-pentachlorobiphenyl
2,3,3',4,4'-pentachlorobiphenyl
2,3',4',4',5-pentachlorobiphenyl
3,3',4,4',5-pentachlorobipheny!
2,2',3,3',4,4'-hexachlorobiphenyl
2,2',3',4,4',5-hexachlorobiphenyl
2,2',4,4',5,5'-hexachlorobiphenyl
2,2',4,4',5,6'-hexachlorobiphenyl
2,2',3,3'A4',5-heptachlorobiphenyl
2,2',3,4,4',5,5'-heptachlorobiphenyl
2,2',3,4,4',5',6-heptachlorobiphenyl
2,2',3,4,4',6,6'-heptachlorobiphenyl
2,2',3,4',5,5',6-heptachlorobiphenyl
2,2',3,4',5,6,6'-heptachlorobiphenyl
2,2',3,3',4,4',5,6-octachlorobiphenyl
2,2',3,3',4,5',6,6'-octachlorobiphenyl
2,2',3,3',4,4',5,5',6-nonachlorobiphenyl
2,2',3,3',4,4',5,5',6,6'-decachlorobiphenyl
Ballschmiter and Zell numbering scheme.
Chemical Abstracts, Tenth Collective Inex, Index Guide, American
Columbus, Ohio, 1982.
CB 200 in the Ballschmiter and Zell numbering scheme.
CAS Registry Number5
34883-43-7
37680-65-2
7012-37-5
15862-07-4
41464-29-5
41464-40-8
62796-65-8
35693-99-3
32598-10-0
32598-13-3
38380-02-8
37680-73-2
56558-16-8
32598-14-4
31508-00-6
57465-28-8
38380-07-3
35065-28-2
35065-27-1
60145-55-4
35065-30-6
35065-29-3
52663-69-1
74472-48-3
52663-68-0
74487-85-7
52663-78-2
40186-71-8
40186-72-9
205 1 -24-3
Chemical Society,
2-339
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Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD Volume 2, Chapten
Table 3. Suggested Instrument Conditions for PCB and Chlorinated Pesticide Analysis
Injection port temperature 250°C
TOCO/-1
Detector temperature J^-> u
IOO°P
Initital temperature JUU ^
Initial hold °™in .
t 1 C/mmto265 C
rat£ 20'C/min to 300°C
IT 0 min
Final hold , _ ,
Carrier gas flow (linear velocity) c sec
. . . & „ 40 mL/min
Makeup gas tlow ...
,, " 3 mL/min
Purge Vent ,„ , , .
„ .:- 60 mL/min
Split vent
Purge on after 1 min
4.0 Procedures
4.1 GC Preparation
The GC is typically fitted with one column; a DB-5 60-m x 0.25 mm (i.d.) fused silica capillary
column with a 0.1-^m film thickness (J&W Scientific, Inc.). Suggested instrumental conditions
are listed in Table 3. Other columns or GC conditions may be specified in individual project
plans.
4.2 Sample Collection, Preservation, and Handling
To conduct this analysis, the analyst should receive the samples as solvent extracts reduced to an
appropriate volume. All organic extracts are normally analyzed within 40 days from extraction.
Refer to project-specific plans or protocols for sample collection, preservation, and handling
methods. If holding times have been exceeded, the Project manager should be notified
immediately.
4.3 Sample Specifications
Sample preparation methods may vary depending on the sample matrix and project needs; refer to
project-specific protocols. Samples and standards for analysis using this SOP should be prepared
in hexane unless otherwise specified. Methylene chloride injected into the GC/ECD system
should be limited, as it may damage the detector. Unless otherwise specified, standard and sample
aliquots of 2-uL volumes will be injected.
4.4 Analyte Identification
Prior to sample analysis, the elution order of the analytes of interest must be determined by
analyzing the analytes individually or in combination \\ith other analytes having known or
predetermined retention times.
2-340
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Volume 2, Chapter 1
Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD
4.5 Instrument Calibration
Before the sample is injected into the GC, the detector must be calibrated to determine the
response of the detector to the analytes of interest. Demonstration of linearity of detector response
is required before sample analysis. Calibration checks must be analyzed at a minimum frequency
of once every 10 samples during sample analysis.
4.5.1 Initial Calibration
The initial calibration consists of the analysis of a minimum of five calibration solutions.
each at different concentrations that span the expected concentration range of the samples.
These standards include the analytes of interest as well as the appropriate surrogates (SS)
and internal standards (IS). The concentration of the LOW standard should be
approximately 2-5 times the detection limit of the instrument. The MID-range calibration
standard should be near the expected concentration of the samples. The HIGH
concentration standard should be approximately 4-10 times the concentration of the MID
standard, or high enough to span the expected concentration range. The range of
concentrations of these standards are as follows:
Pesticides
1.0- lOOng/mL
PCB Congener Mixture
1.0-100 ng/mL
The concentrations of IS and SS should be the same in all calibration solutions and in the
same concentration range as they are spiked in the samples, typically at a concentration
2 to 5 times below the highest calibration standard. Initial calibration standards must be
analyzed prior to initiating sample analyses. An initial calibration should also be run if
any GC conditions have changed. If GC conditions have not changed since the previous
initial calibration, a continuing calibration standard may be analyzed and if it falls within
acceptable criteria, the previous initial calibration may be used.
4.5.2 Continuing Calibration
The upper mid-level calibration solution is analyzed as a calibration check minimally
every 10 samples while samples are being analyzed. All sample analyses must be
bracketed by two calibration check standards that meet calibration criteria (see acceptance
criteria in section 4.5.4.3, Relative Response Factors).
4.5.3 Calibration for Analysis of Aroclors and Multi-Peak Pesticides
A multilevel calibration is analyzed when samples are to be quantified for multicomponent
mixtures such as Aroclors, or toxaphene. Calibration solutions are analyzed minimally at
the beginning of each analysis run or sample batch unless otherwise noted in project
protocols.
2-341
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Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD Volume 2, Chapter_r
4.5,4 Relative Response Factors
The relative response factor (RRF) of each analyte is calculated as follows:
RRFA = (HA) x (C/S)/(HIS) x (CA)
where: HA = Analyte Peak Height
H/s = Internal Standard Peak Height
C,s= Concentration of Internal Standard
C± - Concentration of Analyte
4.5.4.1 Initial Calibration Response Factors
Individual Relative Response factors are generated for each analyte at each
calibration level. A weighted average RRF (XRRF) and correlation coefficient are
calculated from the linear regression of the ratio of responses (HA/H1S) versus the
ratio of amounts (CA/C]S) for a multipoint calibration. The correlation coefficient
must be >0.95 for each individual analyte unless otherwise specified in project
plans. If any correlation coefficient does not meet the acceptable criteria, the
initial calibration must be repeated and all samples associated with that calibration
re-run (unless otherwise specified in a specific project plan and/or documented by
the Project Manager).
4.5.4.2 Response Factors for Six Mixture Components
For multi-peak analytes such as polychlorinated biphenyls, up to 110 significant
peaks or specific predetermined components are chosen. An RRF is calculated
for each peak as described above. A similar method is used for quantifying the
technical mixture of chlordane. The acceptance criteria for the multicomponent
mixture RRF linear regression is the same as stated for single peak components
(r>0.95).
4.5.4.3 Continuing Calibration RFs
Continuing calibration checks are considered acceptable if the 7c difference
between the concentration of the analyte and the known calibration concentration
is less than 25% for four selected medium to large peaks and less than 50% for
two selected small peaks. If the newly generated concentrations are acceptable.
the initial calibration is still valid and sample analysis may continue. If the
percent difference exceeds the acceptable criteria, remedial action should be taken
and the continuing calibration check solution should be reanalyzed. If the
calibration fails again, the analyses should be terminated, remedial action taken, a
new initial calibration should be performed, and the affected samples reanalv/cd.
The percent difference is calculated as follows:
% difference = <€„ -CJ/C,,.v 100c/c
where: Cw = Concentration of the anal\te from initial calibration
C ^Concentration of the ana/vie tmm continuing calibration check
2-342
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Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
Volume 2, Chapter 1 GC/ECD
4.6 Evaluation of DDT and Endrin Degradation
DDT and endrin are easily degraded in the injection port, if the injection port or front of the
column is contaminated with buildup of high boiling residue from sample injection. Check for
degradation problems by injecting a mid-concentration standard containing only 4,4'-DDT and
endrin prior to sample analyses. Look for the degradation products of 4,4'-DDT (4,4'-DDE and
4,4'-DDD) and endrin (endrin ketone and endrin aldehyde).
If degradation of either DDT or endrin exceeds 20% or the combined breakdown of DDT and
endrin is greater than 20%, then take corrective action before proceeding with calibration.
Corrective action includes cleaning and deactivating the injection port, breaking off at least 0.5 m
of column and remounting it. Lowering the injection port temperature may also be an option.
This should be determined by the laboratory Supervisor. Calculate percent breakdown as follows:
% breakdown = Response (peak height) (DDE + DDD) x 100
far 4,4'-DDT Response (peak height) DDT injected
% breakdown = Response (peak height) endrin aldeh\de + endrin ketone x WO
for endrin Response (peak height) endrin
Combined % breakdown = % breakdown DDT + % breakdown endrin
4.7 Sample Analysis Procedure
Samples are analyzed under the same analytical conditions as the calibration standards. Samples
must be bracketed by acceptable calibrations. Criteria for accepting peaks as analytes of interest
are explained in Sections 4.7.1 through 4.7.2.
4.7.1 Relative Retention Time
Retention time (RT) windows for each analyte may be determined daily or by batch of
samples. Relative retention time (RRT) for each analyte shall be determined from the
ratio of the RT of the analyte and the RT of a time reference compound, usually the
internal standard for a designated time interval. RRT for a particular analyte shall be
within 1 % of RRT determined during initial calibration for a peak to be identified.
4.7.2 Minimum Height
Peaks with a signal-to-noise ratio of three or less should be regarded as not detected unless
otherwise noted in a specific project plan and/or documented by project management.
5.0 Data Analysis and Reporting
5.1 Data Recording
Data quantification and calculations will be performed on personal computers using commercial
spreadsheet software such as HP CHEM version A03.01 and Microsoft Excel. All transfers of
data to forms and data reductions (e.g., concentration calculations, means, standard deviations)
will be checked by the analyst and approved by the Laboratory Supervisor. Hard copies of GC
printouts of calibrations and sample data and spreadsheet reports will be kept in the GC/ECD files
A copy of the summary sheets and extraction logs will be placed in the appropriate project file in
the Laboratory Supervisor's Central Files. Hard copies of chromatograms from each sample and
all calibrations will be kept in the GC/ECD files unless otherwise noted in a specific project plan
2-343
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Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD Volume 2, Chapter^
5.2 Sample Quantification
The internal standard method is used to quantify PCBs and chlorinated pesticides in environmental
samples. The internal standards added to the samples prior to GC analysis are the basis for sample
quantification.
5.2.1 Single-Peak Analytes
The concentration of a specific analyte in a sample is calculated as follows:
Concentration = [HA x Amt[S /[H[S x XRRf]]/SampJe Amt
(ng/sample amt)
where: H^~ Peak Height ofanalvte in sample
HIS = Peak Height of IS '
XKRf = Relative response factor of the analyte based on the linear
regression of the initial calibration
AmtK = Amount of the IS added (ng)
Sample Amt - g (sediment) or L (water)
5.2.2 Multicomponent Analytes
The same calculation as above is used for quantifying multi-peak analytes.
Multicomponent analyses are performed as follows:
Quantification
1. Analyze samples as described previously.
2. Using RRF identify the peaks of interest in the samples.
3. Calculate concentration for each identified peak in the mixture as described above
(Section 5.2.1).
5.3 Dual Column Confirmation Data
Data from the confirmation column is treated exactly the same as that obtained from the primary
column as described above. QA criteria outlined in Section 4.0 also applies. Quantitative
comparison of the values obtained from both columns should be performed for the single peak
chlorinated pesticides. In the absence of interferences, values obtained from each column should
be within approximately a factor of two of each other to be considered acceptable. If the criteria
are not met, the value from the primary column is reported with a "G" flag. This criteria is only a
guideline and should not be applied as an absolute rule, especially as concentration values
approach detection limits. The value reported should be that obtained from the primary column
unless chromatographic interferences would indicate that a more accurate value has been obtained
from the confirmation column.
In addition, a number of compounds may co-elute when multi-component mixtures are present in
the samples. The quantitative confirmation/comparison guidelines cannot be strictly adhered to in
these cases. Likewise, if chromatographic interferences (i.e.. poor peak shape, baseline drift etc.)
are present on one of the columns this quantitative comparison is not required. These decisions
are based in a large part on the judgement of the anaKst and ain decisions made in reeards to thi
should be noted on the analytical reports and on copies of the chromatograms.
2-344
s
-------
Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
Volume 2, Chapter 1 GC/ECD
5.4 Surrogate and Matrix Spike Recovery Calculations
Calculation of Surrogate recovery is as follows:
% Surrogate Recover = Qt,/Qu x 100
Qj = Quantity determined by analysis
Q« - Quantity added
The matrix spike recovery is determined as follows:
Matrix Spike Recovery = (SSR/SR) x 100/SA
SSR = Spike sample result
SR = Fraction surrogate recovery
SA = Spike added
Note that the matrix spike recovery is a surrogate corrected calculation.
The Relative Percent Difference (RPD) between spike and spike duplicates is calculated as follows:
RPD = I MSR - MSDR x 100
-------
Analysis of Polychlorinated
Biphenyls and Chlorinated Pesticides by
GC/ECD . Volume 2, Chapter?
Minimum requirements for quality control samples, such as method blanks, matrix spikes, and
standard reference materials (SRM), intended to monitor precision and accuracy of the analytical
method, are specified in the extraction SOPs. In addition, project specific guidelines may be
specified which differ from those outlined in the SOPs.
7.0 Safety
All analysts following this procedure should be aware of routine laboratory safety concerns,
including the following:
1. Protective clothing and eyeglasses should be worn when appropriate
2. Proper care must be exercised when using syringes
3. Certain areas of the GC system are heated. Avoid bodily contact with these areas and use
care in handling flammable solvents in and around the GC system.
8.0 Training
All analysts following this procedure will be directly supervised by the Principal investigator,
qualified analyst, or laboratory supervisor until they have demonstrated to the satisfaction of the
supervisor that they are capable of operating the GC independently. At a minimum, the analyst
trainee should be competent in operation and maintenance of the GC. The analyst trainee should
also be able to analyze and quantify a multi-point calibration and quantitate a sample of known
concentration (e.g., a reference material or matrix spike) within established control limits
9.0 References
EPA Method 8000. U.S. Environmental Protection Agency (EPA). 1988. Test Methods for
Evaluating Solid Wastes: Physical/Chemical Methods. EPA-600-4-79-020. 3rd Edition.
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
2-346
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Standard Operating Procedure for
the Analysis of PCB Congeners
by GC/ECD and Trans-Nonachlor
by GC/MS/ECNI
Deborah L. Swackhamer and Annette G. Trowbridge
Division of Environmental and Occupational Health
School of Public Health
Box 807 Mayo Building
University of Minnesota
Minneapolis, MN 55455
and
Edward A. Nater
Department of Soil, Water, Air, and Climate
439 Borlaug Hall
University of Minnesota
St. Paul, MN 55108
May 13, 1996
Revision 3
-------
Standard Operating Procedure for
the Analysis of PCB Congeners by GC/ECD and
Trans-Nonachlor by GC/MS/ECNI
1.0 Scope and Application
l.l Scope
This method is used to determine the concentrations of PCB congeners and trans-nonachlor in
extracts from phytoplankton. zooplankton, Mysis, Diporeia, detritus and dissolved phase of lake
water samples. As the detritus and dissolved phase parameters are not funded by this project.
procedures relating to these parameters are provided as information only. The following analytes
are measured by this Standard Operating Procedure (SOP):
Analyte
trans-nonachlor
PCB Congener #
1
3
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
21
~>~>
24
25
CAS#
39765-80-5
CAS#
2051-60-7
2051-62-9
13029-08-8
16605-91-7
25569-80-6
33284-50-3
34883-43-7
34883-39-1
33146-45-1
2974-92-7
2974-90-5
34883-41-5
2050-68-2
38444-78-9
37680-66-3
37680-65-2
38444-73-4
55702-46-0
38444-85-8
55702-45-9
55712-37-3
2-349
-------
SOP for the Analysis of PCS
Congeners by GC/ECD and
Trans-Nonachlor by GC/MS/ECNI
Volume 2, Chapter 1
PCB Congener #
26
27
28
29
30
31
32
33
37
40
41
42
43
44
45
46
47
48
49
51
52
53
56
60
63
64
65
66
70
71
74
76
77
81
82
83
CAS#
38444-81-4
38444-76-7
7012-37-5
15862-07-4
35693-92-6
16606-02-3
38444-77-8
38444-86-9
38444-90-5
8444-93-8
52663-59-9
36559-22-5
70362-46-8
41464-39-5
70362-45-7
41464-47-5
2437-79-8
70362-47-9
41464-40-8
68194-04-7
35693-99-3
41464-41-9
41464-43-1
33025-41-1
74472-34-7
52663-58-8
33284-54-7
32598-10-0
32598-11-1
41464-46-4
32690-93-0
70362-48-0
32598-13-3
70362-50-4
52663-62-4
60145-20-2
2-350
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Volume 2, Chapter 1
SOP for the Analysis of PCB
Congeners by GC/ECD and
Trans-Nonachlor by GC/MS/ECNI
PCB Congener #
84
85
87
89
91
92
95
97
99
100
101
105
107
110
114
118
119
123
124
128
129
130
131
132
134
135
136
137
138
141
144
146
147
149
151
153
CAS#
52663-60-2
65510-45-4
38380-02-8
73575-57-2
68194-05-8
52663-61-3
38379-99-6
41464-51-1
38380-01-7
39485-83-1
37680-73-2
32598-14-4
70424-68-9
38380-03-9
74472-37-0
31508-00-6
56558-17-9
65510-44-3
70424-70-3
38380-07-3
55215-18-4
52663-66-8
61798-70-7
38380-05-1
52704-70-8
52744-13-5
38411-22-2
35694-06-5
35065-28-2
52712-04-6
68194-14-9
51908-16-8
68194-13-8
38380-04-0
52663-63-5
35065-27-1
2-351
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SOP for the Analysis of PCS
Congeners by GC/ECD and
Trans-Nonachlor by GC/MS/ECNI
Volume2, Chapterl
PCB Congener #
156
157
158
163
166
167
170
171
172
173
174
175
176
177
178
180
182
183
185
187
189
190
191
193
194
195
196
197
198
199
200
201
202
203
204
205
CAS#
38380-08-4
69782-90-7
74472-42-7
74472-44-9
41411-63-6
52663-72-6
35065-30-6
52663-71-5
52663-74-8
68194-16-1
38411-25-5
40186-70-7
52663-65-7
52663-70-4
52663-67-9
35065-29-3
60145-23-5
52663-69-1
52712-05-7
52663-68-0
39635-61-9
41411-64-7
74472-50-7
69782-91-8
35694-08-7
52663-78-2
42740-50-1
33091-17-7
68194-17-2
52663-75-9
52663-73-7
40186-71-8
2136-99-4
52663-76-0
74472-52-9
4472-53-0
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Congeners by GC/ECD and
Trans-Nonachlor by GC/MS/ECNI
PCB Congener #
206
207
208
209
CAS#
4U I 86-72-9
52663-79-3
52663-77-1
205 1 -24-3
1.2 Method Optimization
The analyst selects columns and calibration procedures most appropriate for the specific analytes
of interest in a study. Matrix-specific performance data are established and the stability of the
analytical system and instrument calibration are established for each new matrix.
1.3 Resolution
The analytes listed in Section 1.1 are detected in a clean matrix. Some analytes co-elute with other
analytes so that one peak is identified as more than one PCB congener. Analytes that can be
detected and quantified in individual samples may differ. This is due to the possible chemical and
chromatographic behavior of many of these analytes in real environmental matrices.
Cleanup/fractionation schemes are provided in this method.
1.4 Compound Identification
Identification based on single column GC analysis should be supported by one other qualitative
technique. Qualitative support for PCB identification will be provided by GC/MS/ECNI.
Confirmation of selected PCB congeners with more than four chlorines will be conducted on 5%
of the samples. The GC/MS/ECNI method for the analysis of trans-nonachlor directly confirms
the identity of the compound, so external confirmation is not needed.
1.5 Method Usage
This method is restricted to use by or under the supervision of analysts experienced in the use of a
gas chromatograph (GC), mass spectrometer (MS) and in the interpretation of gas chromatograms
and mass spectra. Each analyst must demonstrate the ability to generate acceptable results with
this method.
1.6 Working Linear Range
It is important to ensure linearity between a range of PCB concentrations. To demonstrate
linearity, five calibration standard solutions ranging from approximately 300 ng/mL to 3000
ng/mL will be run on the GC and compared on a congener basis.
1.7 Limit of Detection Terminology
This study will incorporate three terms describing detection limits - Instrument Detection Limit
(IDL). Method Detection Limit (MDL) and System Detection Limit (SDLi - see Section 13.2 for
specific calculations. Definitions for each term follows:
IDL = y-intercept of initial calibration cur\e
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Trans-Nonachlor by GC/MS/ECNI : Volume 2, Chapter^
MDL = three standard deviations of seven injections of low level homolog spikes, adjusted for
congener specific relative response factors (RRF)
SDL = three standard deviations of seven injections of field matrix blanks
2.0 Summary of Method
All samples will undergo methanol rinse, four hour Soxhlet extraction with methanol and 16 to
24 hour Soxhlet extraction with dichloromethane (DCM). Surrogate compounds (congeners 14,
65, 166 for PCBs and "C-chlordane or "Cl-nonachlor for trans-nonachlor) are added at the
beginning of the extraction to monitor the efficiency of the extraction process. The methanol
fraction is batch extracted with hexane. Water/methanol phase is discarded. The hexane fraction
containing PCBs is saved, combined with the DCM fraction, solvent exchanged to hexane and
volume reduced to approximately 15 mL.
Extracts have lipids removed by passing them over a column containing:
lgNa2SO4
13g 6% deactivated Al
lgNa:SO4
Washed with 2 x 60 mL hexane
The columns are eluted with 150 mL hexane, and the extracts are volume reduced to
approximately 10 mL.
The extracts undergo column chromatography to separate PCBs from nonachlor and toxaphene
and to aid in further cleanup. The extracts are loaded onto the following column:
3g Na,SO4
4.5g 0% deactivated Si
lgNa:SO4
6g 1 % deactivated Al
lgNa:SO4
Washed with 2 x 50 mL 2% DCM/hexane, 2 x 50 mL 40% DCM/hexane, 2 x 60 mL 100%
hexane.
The columns are then eluted with 95 mL 100% hexane followed by 105 mL 40% DCM/hexane.
Both fractions are solvent exchanged and volume reduced to approximately 1 mL. The final
extracts are stored in amber vials in the freezer until analysis. Just prior to instrumental analysis,
extracts are reduced to approximately 200-300 uL by a gentle stream of nitrogen and internal
standards are added. Internal standards used are PCB congeners 30 and 204 for PCBs and 204 for
trans-nonachlor.
PCBs \\ill be quantified by individual congener using the method of Mullin (1985). The 1994
congener standard supplied by Mullin will be used as the quantitation, performance, and spiking
standard. Analysis will be accomplished using a Hewlett Packard GC equipped with an MNi
electron capture detector (ECD) and a 60 m high resolution capillur) column (DB-5) with H,
carrier gas. Individual congener data are processed by Millennium Chmmatoeraphic Management
Sv^tein (Waters Corporation). Concentrations will be reported by ind\\\dual congener, homologe
distributions and total PCBs.
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Volume 2, Chapter 1 Trans-Nonachlor by GC/MS/ECNI
Trans-nonachlor will be analyzed by electron capture negative ionization (ECNI) GC/MS with
selected ion monitoring (SIM). The GC utilizes a 60m DB-5 MS column with He carrier gas.
Methane is the reagent gas. Further instrument specifics can be located in Section 5.19. Mass
spectra are acquired and processed by Hewlett Packard (HP) RTE-A and Aquarius software.
3.0 Interferences and Corrective Action
3.1 Sources of Interference
Sources in this method can be grouped into three broad categories: contaminated solvents,
reagents, XAD resin, or sample processing hardware; contaminated GC carrier gas, parts, column
surfaces or detector surfaces; and the presence of coeluting compounds in the sample matrix to
which the ECD will respond. Interferences coextracted from the samples will vary considerably
from sample to sample. While general cleanup techniques are provided as part of this method,
unique samples may require additional cleanup approaches to achieve desired degrees of
discrimination and quantitation.
3.2 Interferences by Phthalate Esters
These interferences which are introduced during sample preparation can pose a major problem in
PCB determinations. These materials may be removed prior to analysis using the silica
gel/alumina cleanup. Common flexible plastics contain varying amounts of phthalate esters which
are easily extracted or leached from materials during laboratory operations. Cross-contamination
of clean glassware routinely occurs when plastics are handled during extraction steps, especially
when solvent-wetted surfaces are handled. Interferences from phthalate esters can best be
minimized by avoiding contact with any plastic materials. Exhaustive cleanup of solvents,
reagents and glassware may be required to eliminate background phthalate ester contamination.
3.3 Glassware
Glassware must be scrupulously cleaned. Used glassware is cleaned with Alconox detergent in hot
water, rinsed with tap water followed by deionized water. The glassware is allowed to dry, is foil
wrapped, and ashed for a minimum of four hours at 450°C. It is then stored in a clean
environment.
3.4 Toxaphene
The presence of toxaphene will result in a series of peaks that interfere with the detection of PCBs.
Toxaphene and PCBs will be separated by column chromatography using a silica gel/alumina
column. PCBs will be recovered in the first elution fraction while toxaphene will elute in the
second fraction.
4.0 Safety Precautions
4.1 Safety Attire
Latex disposable gloves and lab coats a:v worn when making up stock solutions of standards. Lab
coats are preferred but optional during other activities. Gloves will not be vvorn during other steps
in the method due to the possibility of sample contamination resulting from materials in the gloves.
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Trans-Nonachlor by GC/MS/ECNI Volume 2,
4.2 Equipment Testing
Testing of the MNi ECD is performed bi-annually by the University of Minnesota Radiation
Protection Division. Testing of the hoods is performed by the University of Minnesota
Environmental Health and Safety Division on a bi-annual basis.
5.0 Apparatus and Materials
5.1 Soxhlet Extractor
Soxhlet extractors used are 50 mm ID with 500 mL round bottom flask. This will be used with all
matrices except for XAD which will use 68 mm ID with 1000 mL round bottom flasks. A plug of
ashed glass wool is placed in the bottom of the Soxhlet before the sample is introduced.
5.2 Kuderna-Danish (KD) Concentrator
The KD apparatus consists of three parts as described below.
5.2.1 Receiver
Either 10 mL or 15 mL. Ground glass stoppers of the appropriate size are used to prevent
evaporation of extracts after volume reduction.
5.2.2 KDBody
Either 500 mL or 250 mL. The evaporation flask is attached to the receiver with delrin
clamps.
5.2.3 Snyder column
Three ball macro. Ground glass stoppers of the appropriate size are used to prevent
contamination if volume reduction must be interrupted before completion.
5.3 Lipid Removal
An initial cleanup column (1 x 50 cm) is used to remove lipids comprised of 6% deactivated
alumina and anhydrous sodium sulfate.
5.3.1 Sodium sulfate
Anhydrous granular. Mallmckrodt. pre-ashed at 450°C for four hours.
5.3.2 Alumina
60-325 mesh. Fisher Scientific, pre-ashed at 450:C for four hours.
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5.4 Cleanup Columns
Cleanup columns I cm x 50 cm. Columns are comprised of a small plug of ashed glass wool,
ashed anhydrous sodium sulfate, I 7c deactivated alumina and 09r deactivated silica gel.
5.4.] Sodium sulfate
Anhydrous granular, Mallinckrodt, pre-ashed at 450°C for four hours.
5.4.2 Alumina
60-325 mesh, Fisher Scientific, pre-ashed at 450°C for four hours.
5.4.3 Silica gel
60-200 mesh. Baker Analyzed, pre-ashed at 300°C for four hours.
5.5 Nitrogen Evaporation Apparatus
Pierce Reacti Therm Model 18780 Evaporating Unit. Ultra-pure Carrier grade nitrogen is used at
a flow setting of 7 psi.
5.6 Boiling Chips
Eight to 12 mesh, Cargille Laboratory, ashed at 450°C for a minimum of four hours.
5.7 Heating Mantle
Electrothermal Soxhlet Apparatus heater. Individual solid-state controllers control the heat for
each of six individual heating bays.
5.8 Steam Bath
Heated, concentric ring cover. Steam bath is used in the hood.
5.9 Extract Vials
Extracts are stored in 4 mL amber vials which have been wrapped in aluminum foil and ashed at
450'C for a minimum of four hours.
5.10 Drummond Pipets
Drmnmond pipettes are used to deliver surrogates and internal standards in amounts of 10 uL,
25 uL. 50 uL. and lOOuL.
5.1 I Vorlex Mixer
Deluxe Mixer bv Scientific Products
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5.12 Apparatus for Determining Percent Dry Weight:
5.12.1 Sartorius MCI Balance
Self calibrating.
5.12.2 Drying oven
Precision Scientific, drying is performed at 60°C.
5.13 Apparatus for Determining Percent Lipid:
5.13.1 Sartorius MCI Balance
Self calibrating.
5.13.2 Drying oven
Precision Scientific, drying is performed at 60°C.
5.13.3 Aluminum weighing tins
Fisherbrand.
5.14 SPM Determination:
5.14.1 0.4 (am Nuclepore filters, 47 mm diameter
5.14.2 Sartorius MCI Balance
5.15 POC Apparatus
Leko Total Carbon Analyzer
5.16 DOC Analyzer
Ionics Model 555, Thermal Combustion, Total Carbon Analyzer.
5.17 Gas Chromatograph
Following are the gas chromatograph parameters.
Model: HP 5890A
Injector: Splitless
Injector Temp.: 225°C
Detector Temp.: 325°C
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Trans-Nonachlor by GC/MS/ECNI
Temperature Program: 100°C hold for 10 minutes
100°C-!300Cat 10°/min.
130°C-255°Cat l°/min.
255°C-285°Cat 10°/min.
160 minutes for a run
luL
Millennium 2010 Chromatography Manager
Injection Volume:
Carrier Gas:
Processing System:
5.18 Narrow-bore columns:
5.18.1 GC Column 1
60 m x 0.25 mm internal diameter (ID) fused silica capillary column DB-5 (J & W
Scientific) chemically bonded with 5%-Phenyl Methylpolysiloxane, 0.25 urn film
thickness.
5.18.2 GC/MS/ECNI Column
60m x 0.25 mm ID fused silica capillary column DB-5 MS (J & W Scientific), 0.25 urn
film thickness.
5.19 GC/MS/ECNI
Following are the parameters for the MS system.
Model:
Autosampler:
Mode:
Injector:
Injector Temp.:
Temperature Program:
Injection Volume:
GC Carrier Gas:
NI Reagent Gas:
Transfer Line Temp:
Source Temp:
Source Pressure:
Electron Energy:
Emission Current:
Mass Range:
Mass Accuracy:
Scan Start Time:
Hewlett Packard 5988A
HP 7637
Single Ion Monitoring (SIM), Negative Ion Mode
Splitless
270°C
80°C hold for one minute
80°C-210°Cat 10°/min.
2100C-250°Cat0.8°/min.
250°C-290°C at 10°/min.
1 uL
He
Methane
290°C
100°C
1.2Torr
240 EV
300 A
l-800m/z
0.3 ± dalton
24 minutes
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Trans-Nonachlor by GC/MS/ECNI Volume 2, Chapter^
6.0 Reagents
6.1 Chemicals
Reagent or pesticide grade chemicals shall be used in all tests. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without
lessening the accuracy of the determination.
Note: Standard solutions (stock, calibration, internal, and surrogate) are stored at -16 ±4"C in
amber glass containers. When stock standard solutions are prepared, it is recommended that
aliquots of that stock solution be stored in individual small vials for use as working solutions.
Standard solution must be replaced if routine QC indicates a problem.
6.2 Solvents
All solvents should be pesticide quality or equivalent. Solvents must be exchanged to hexane
prior to analysis.
6.2.1 Hexane
Fisher Optima.
6.2.2 DCM
Fisher Optima.
6.2.3 Methanol
Fisher Optima.
6.2.4 Acetone
Fisher Optima.
6.3 Stock Standard Solutions
6.3.1 Mullins mix: 183ug/mL
Aroclors 1232 = 75ug/mL
1248 = 54 ug/mL
1 262 = 54 ug/mL
6.3.2 L'ltra mix:
Congeners 001 = 12ug/mL
006 =14.2 ug/mL
029 = 6.3 ug/mL
049 = 5.86 ug/mL
101 =4.93 uti/inL
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Trans-Nonachlor by GC/MS/ECNI
I4l = 2.l9ug/mL
180 = 2.21 ug/mL
194= 1.69 ug/mL
206 = 2.05 ug/mL
209= 1.36 ug/mL
Additional Congener 001 will be added to the ultra mix due to it's low ECD response.
Congener 001 was supplied by Ultra Scientific.
6.3.3 Trans-nonachlor
Accustandard solid.
6.3.4 Stable isotopes
Liquid, as is from Cambridge Isotopes.
6.3.5 Initial calibration standards
Dilutions of the Mullins mix listed above. Concentrations are listed under Section 6.8.
6.4 Sodium Sulfate
Anhydrous, granular - Mallinckrodt. Ash at 450°C for a minimum of four hours.
6.5 Alumina
60-325 mesh - Fisher Scientific. Ash at 450°C for a minimum of four hours. Store in oven at
105°C. When ready to use cool to room temperature. Weigh out needed mass into a round
bottom flask. Add water to provide the necessary deactivation on a mass to mass basis. Stopper
the flask with the appropriate sized ground glass stopper. Shake for two minutes. Wrap stopper
and top of flask with parafilm and set in desiccator for 24 hours.
6.6 Silica Gel
60-200 mesh - Baker Analyzed. Ash at 300 :C for a minimum of four hours. Store in oven at
105CC. When ready to use cool to room temperature. No deactivation is required for the
procedure used in this method.
6.7 Calibration Standards
Standards are prepared at five concentrations by dilution of the stock standard with hexane.
Concentrations correspond to the expected range of concentrations found in real samples and are
in the linear range of the detector. Concentrations for the initial calibration standards are:
366 ng/mL. 586 ng/mL, 732 ng/mL, 1464 ng/mL. and 2928 ng/mL. The continuing calibration
standard is 732 ng/mL.
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Trans-Nonachlor by GC/MS/ECNI Volume 2, Chapter^
6.8 Internal Standards
6.8.1 Source
Solids purchased from Ultra Scientific.
6.8.2 Concentration
#030 = 82.73 ng/mL
#204 = 59.16ng/mL
6.8.3 Preparation
Individual stock solution of Congeners 30 and 204 were prepared in concentrations of
16,545 ng/mL and 11,831 ng/mL respectively in hexane. To a 100 mL volumetric flask
0.5 mL of each solution was dispensed via a 0.5 mL volumetric pipet. Hexane was added
to the flask to bring to volume.
6.8.4 Procedure for addition to extracts
The 4 mL vial of internal standard solution will be removed from the freezer and allowed
to come to room temperature. A drummond pipet of appropriate size will be cleaned and
used in the transfer of internal standard to extracts. All extracts will be vortexed to ensure
proper mixing.
6.8.5 Storage
Internal standard solutions are stored in amber glass bottles in a -16 ±4°C freezer.
6.9 Surrogate Standards
6.9.1 Source
PCB congener solids purchased from Ultra Scientific; stable isotope solutions purchased
from Cambridge Isotopes.
6.9.2 Concentration
#014 = 439.2 ng/mL
#065 = 106.4 ng/mL
#166= 125.04 ng/mL
"Cchlordane= 1317 ng/mL
<7C1 nonachlor= 1300ns/mL
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6.9.3 Preparation
Individual stock solution of Congeners 14, 65, and 166 were prepared in concentrations of
5.490 |ag/mL, 5.320 ug/mL and 5.210 ug/mL respectively in hexane. To a 50 mL
volumetric flask 4.0 mLof#14, 1.0 mLof#65 and 1.2 mLof#166 was dispensed via
volumetric pipets. Hexane was added to the flask to bring to volume. The stable isotope
solutions were diluted in hexane to appropriate concentrations in hexane.
6.9.4 Procedure for addition to samples
The 4 mL vial of surrogate solution will be removed from the freezer and allowed to come
to room temperature. A drummond pipet of appropriate size will be cleaned and used in
the transfer of surrogate solution to samples prior to extraction.
6.9.5 Storage
Surrogate standard solutions are stored in amber glass bottles in a -16 ±4°C freezer.
6.10 Matrix Spike Standard
6.10.1 Source
Mullins Mix.
6.10.2 Concentration
2928 ng/mL stock solution.
6.10.3 Preparation
The 2928 ng/mL Stock Spiking Solution will be diluted with hexane in the preparation of
matrix spikes with concentrations that are within a factor of five of the media of interest.
An appropriate amount of nonachlor will be added during this dilution step to be within a
factor of five of the media of interest.
6.10.4 Procedure for addition to samples
The 4 mL vial of matrix spike solution will be removed from the freezer and allowed to
come to room temperature. A drummond pipet of appropriate size will be cleaned and
used in the transfer of matrix spike solution to the matrix prior to extraction.
6.10.5 Storage
Matrix spike standard solutions are stored in amber glass bottles in a -16 ±4°C freezer.
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7.0 Sample Collection, Preservation and Handling
7.1 Collection
Details of sample collection are found in the SOP "Sampling Procedures for the Lake Michigan
Lower Pelagic Foodchain for PCBs, Nonachlor and Mercury", Revision 1,31 August, 1994.
7.2 Zooplankton
7.2.1 Definition
The zooplankton fraction is operationally defined as particulate matter greater than 100
l^m (excluding fish). Approximately 10 g (wet weight) is needed for the analyses.
7.2.2 Collection
Wet zooplankton is removed from the net container and transferred to a clean glass bottle.
The slurry in the glass bottle is poured through a piece of 100 (am Nitex netting supported
by either a funnel or seive. The material remaining on the netting is removed by a spatula
to an ashed glass jar.
7.2.3 Storage
Jars containing zooplankton are labeled and frozen until extraction.
7.3 Phytoplankton
7.3.1 Definition
The phytoplankton fraction is operationally defined as particulate matter between 10 and
100 urn. Approximately 10 g (wet weight) is needed for the analyses.
7.3.2 Collection
Wet phytoplankton will be quantitatively transferred from the net container to a clean
glass graduated bottle. After a small subsample is provided for Hg analysis the remaining
slurry is taken to the next nearest hundred mL with filtered lake water (i.e. slurry volume
reads between 600 mL and 700 mL the volume is increased to 700 mL.).
7.3.3 Subsamples
The suspension is homogenized and subsampled for mass, taxonomic identification,
organic carbon and stable isotopes. The remainder of the suspension is quantitatively
transferred using filtered lake water and filtered through an ashed 125 mm GF/F glass
fiber filter in a Buchner funnel bv a gentle vacuum.
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7.3.4 Storage
The glass fiber filter containing the phytoplankton will be folded in quarters, wrapped in
foil, sealed in a freezer bag, labeled and frozen until extraction.
7.4 Detrital Fraction
7.4.1 Definition
The detrital fraction is operationally defined as the material collected between 0.7 and
10 um, as isolated by a 293 mm GF/F glass fiber filter after passing through a piece of
10 um Nitex netting. Approximately 1000 L of water must be processed for this size
fraction.
7.4.2 Storage
The GF/F filters containing the detrital sample will be individually stored. Each filter will
be folded in quarters, wrapped in aluminum foil, sealed in a freezer bag, and labeled and
frozen until extraction.
7.5 Mysis Relicta
7.5.1 Samples of Mysis relicta are hand picked to ensure clean collections. Approximately 10 g
(wet weight) is needed for the analyses.
7.5.2 Collection
Mvsis are collected by vertical tows with 500 um nets or by benthic sled tows. Material
from these methods of collection are transferred to a pan for hand-picking of organisms.
Organisms are placed in ashed glass jars.
7.5.3 Storage
Jars containing Mysis are labeled and frozen until extraction.
7.6 Diporeia sp.
7.6.1 Samples of Diporeia sp. are hand picked to ensure clean collections. Approximately 10 g
(wet weight) is needed for the analyses.
7.6.2 Collection
Diporeia sp. are collected by benthic sled tows. Material from this method of collection
are transferred to a pan for hand-picking of organisms. Organisms are placed in ashed
glass jars.
7.6.3 Storage
Jars containing Diporeia sp. are labeled and frozen until extraction.
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7.7 Holding Times
Samples are to be extracted within one year of collection, beginning after approval of the QAPjP.
Extracts are stored in the freezer at -16 ±4°C in the dark and analyzed within three years after
extraction.
8.0 Sample Preparation Procedure
8.1 Sample Selection
An appropriate sample is removed from the freezer and allowed to thaw. For biota samples
aliquots are taken for weight wet and dry weight determinations.
8.2 Wet Weight Determination
An ashed glass beaker is placed on the balance and tared. An appropriate amount of sample is
weighed into the beaker (between 5 and 10 g). The wet weight of the sample is recorded in the
Lake Michigan Pelagic Foodchain notebook.
8.3 Dry Weight Determination
8.3.1 A small aliquot is taken from the remaining sample and placed in a tared aluminum foil
cup. The wet weight is recorded. These aliquots are placed in a drying oven until they
reach a consistent weight which is recorded in the notebook.
8.3.2 Phytoplankton dry weights are determined from SPM subsamples collected in the field.
Nuclepore filters (0.4 jam) are allowed to air dry and their final consistent weight is
recorded in the SPM field notebooks.
8.4 Methanol Wash
8.4.1 Set up a 1000 mL separatory funnel with a conical funnel on top. The conical funnel will
hold a plug of ashed glass wool.
8.4.2 Transfer the whole wet sample from the beaker onto the glass wool with methanol
(MeOH). Wash the sample with approximately 5-20 mL MeOH.
8.5 Methanol Extraction
8.5.1 Transfer both the sample and glass wool from the conical funnel to a Soxhlet extractor
apparatus containing a plug of ashed glass \\nol. Sample is transferred with methanol
8.5.2 In the case of filters, the filters will be carefully cut into pieces and the pieces will be
added to the Soxhlet extractor. Cutting uill be done over the aluminum foil the filters
were trapped in and the aluminum foil \\ill he rinsed with methanol with the methanol
rinse added to the Soxhlet.
8.5.3 Add approximately 1 tablespoon ashed hoi line stones to a 500 mL round bottom flask.
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8.5.4 Connect the round bottom to the Soxhlet extractor.
8.5.5 Add approximately 300 mL MeOH through the Soxhlet so it completely covers the sample
and drains to the round bottom flask.
8.5.6 Add the appropriate amount of surrogate standard solution. This is dependent on the
matnx being extracted - it will vary from 50-200 L. The lab procedural blank will be
spiked with an analogous amount of surrogate.
8.5.7 Connect the Soxhlet extractor to the condenser.
8.5.8 Turn on the heating mantel. Let cycle for four hours.
8.5.9 After four hours turn the heating mantel off and let sample cool until round bottom flasks
are warm.
8.5.10 Quantitatively transfer the MeOH extract from the round bottom flask to the separator/
funnel set up in Section 8.4.
8.6 Dichloromethane Extraction (DCM)
8.6.1 Recharge the round bottom flask with approximately 300 mL DCM.
8.6.2 Reattach the round bottom to the Soxhlet extractor.
8.6.3 Cycle for 16-24 hours.
8.6.4. Allow the extract to cool to room temperature after the extraction is complete.
8.7 Batch Extraction of Methanol Fraction
8.7.1 To the separator/ funnel containing the MeOH fractions add the following:
100 mL Barnstead Nanopure water
50 mL saturated NaCl solution
50 mL hexane
8.7.2 Shake separatory funnel for three minutes. Vent funnel often.
8.7.3 Drain lower water layer to a large bottle.
8.7.4 Drain hexane layer into a KD body connected to a receiver holding a funnel containing
approximately 150 g sodium sulfate (Na:SO,( on top of a plug of ashed glass wool. Wash
the N'a.SO, with approximately 2x15 mL hexane.
8.7.5 Pour the \\ater from Section 8.7.3 back into its corresponding separatory funnel
8.7.6 Rinse the bottle with 50 mL hexane and add to separatory funnel.
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8.7.7 Shake separatory funnel for three minutes, and drain lower layer as above. Add the
hexane layer to the KD from Section 8.7 4. Repeat one more time starting at
Section 8.7.5. Wash separatory funnel with 3x15 mL hexane on the last extraction.
8.8 Volume Reduction and Solvent Exchange
8.8.1 Attach a three-ball Snyder column to the KD assembly. Place on steam bath and adjust
the vertical position of the assembly so that the appropriate flux is accomplished At the
proper rate of distillation, the balls of the column will actively chatter, but the chambers
will not flood.
8.8.2 Reduce the MeOH fraction to approximately 10-15 mL.
8.8.3 Quantitatively transfer the DCM extract obtained from Section 8.6 to the KD apparatus.
8.8.4 Volume reduce to approximately 15 mL.
8.8.5 Solvent exchange the extract to hexane by adding 30 mL hexane, reducing volume to
15 mL, adding another 30 mL hexane, reducing volume to 15 mL and adding a final
15 mL hexane.
8.8.6 Reduce the volume to 10-15 mL. Turn off the steam tables, cool the KD assembly to
room temperature and stopper with a ground glass stopper of appropriate size.
8.8.7 Remove the KD body from the receiver - rinse the ground glass joint with hexane into the
receiver.
8.8.8 Volume reduce the extract to less than 10 mL using a Nitrogen evaporation apparatus.
Needles are sonicated in hexane prior to use.
8.8.9 Transfer, with hexane, the extract to a 25 mL graduated cylinder.
8.9 Lipid Content Determination
8.9.1 Add hexane to the graduated cylinder to bring extract to 10 mL. Using a 1 mL volumetric
pipet, transfer 1 mL of the extract to a pre-weighed aluminum tin.
8.9.2 Evaporate solvent from the aluminum tin by air exposure.
8.9.3 After solvent has evaporated, weigh the aluminum tin to constant weight using the
Sartorius MCI balance. Residue remaining in the tin is the lipid.
8.10 Lipid Removal
8.10.1 Column is assembled in the following fashion from top to bottom.
2 y allied sodium sultate
13 g (v < deactivated alumina
I e ashed sodium sulfate
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Plug of ashed glass wool
Wash columns with 2 x 60 mL hexane
8.10.2 Deactivation
See Section 6.6 for alumina deactivation procedure.
8.10.3 Columns
1 x 50 cm columns.
8.10.4 Quantitatively load the extract to the top of the column using hexane.
8.10.5 Elute with 3 x 50 mL hexane and collect in a 250 mL KD body attached to a receiver
containing two to three ashed boiling chips.
8.10.6 Attach three ball Snyder columns to the KD body and place the assembly on the steam
table. Adjust vertical height for appropriate flux.
8.10.7 Reduce the extracts to approximately 5-10 mL using the same procedure described in
Sections 8.8.4 through 8.8.7.
8.11 Column Cleanup/Fractionation
8.11.1 Cleanup/fractionation column is assembled in the following fashion from top to bottom.
3 g ashed sodium sulfate
4.5 g 0% deactivated silica
1 g ashed sodium sulfate
6 g 1 % deactivated alumina
1 g ashed sodium sulfate
Plug of ashed glass wool
Wash columns with 2 x 50 mL 2% DCM/hexane, 2 x 50 mL 40%
DCM/Hexane, 2 x 60 mL 100% hexane
The silica is made into a slurry using hexane before it is poured into the column. Pouring
silica in this fashion results in columns that pack better and have fewer air channels
8.11.2 Deactivation
See Section 6.6 for alumina deactivation procedure.
8.11.3 Columns
1 x 50 cm columns.
8.1 1.4 Quantitati\el\ load the extract to the top of the column using hexane.
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8.11.5 Elute with 95 mL 100% hexane and collect in a 250 mL KD body attached to a receiver
containing two to three ashed boiling chips. This is labeled the Fl fraction containing
PCBs.
8.11.6 Elute again with 105 mL 40% DCM in hexane and collect in a different 250 mL KD body
attached to a receiver containing two to three ashed boiling chips. This is labeled the F2
fraction containing trans-nonachlor and toxaphene.
8.12 Final Solvent Exchange
8.12.1 Attach three ball Snyder columns to the KD body and place the assembly on the steam
table. Adjust vertical height for appropriate flux.
8.12.2 Reduce the extracts to approximately 5-10 mL using the same procedure described in
Sections 8.8.4 through 8.8.7.
8.12.3 Reduce to a final volume of 2-3 mL using the procedure described in Section 8.8.8.
8.12.4 Quantitatively transfer the extract using hexane from the receiver to ashed 4 mL amber
vials.
8.13 Storage
Store extract in the freezer at -16 ±4°C until internal standards addition and analysis.
8.14 Internal Standards Addition
8.14.1 Remove extracts from the freezer.
8.14.2 Remove internal standards solution from freezer and allow to come to room temperature.
8.14.3 Reduce extract to approximately 300 uL using nitrogen evaporation apparatus.
8.14.4 Add 50-200 uL internal standard solution (#30, #204) depending on the matrix to the PCB
extract fraction using a Drummond pipet and 50-200 uL of chlordane (matrix dependent)
to the trans-nonachlor extract fraction via Drummond pipet.
8.14.5 Vortex the extracts. The extract are now ready for instrumental analysis.
9.0 Instrument Calibration and Quantitation
9.1 GC/ECD Analysis for PCB Congeners
9.1.1 Annual Initial Calibration
The GC/ECD \\ill be calibrated at the beginning of the project (or after any major
instrument repairs such as replacements of column, detector, or injector assemhK i h\
using calibration standards prepared at fi\e different concentrations as listed in
Section 6.S. The concentrations correspond to the expected range of concentrations m the
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samples or define the working range of the detector. Each calibration standard is injected
using the technique that will be used for the injection of environmental extracts. Peak
areas are tabulated against the mass injected for each congener and associated internal
standard area and mass.
Note: Because of the sensitivity of the electron capture detector, the injection port and
column should always be cleaned prior to performing the initial calibration.
9.1.1.1 Relative Response Factor (RRF)
Millennium Chromatographic Management System automatically calculates the
RRF each time it calibrates a calibration standard. The calculation used for this is
as follows:
RRF = (CJA} I (CJAJ
Where A., = area of the congener
A^ = area of the internal standard
C,, = mass (ng) of the internal standard
C, = mass (ng) of the congener
9.1.1.2 Relative Standard Deviation (RSD)
The math for this calculation across all five RRFs is as follows:
RSD = sd I XRRF x 100
Where sd = standard deviation of the five RRF measurements
XRRF = mean RRF across all five RRFs.
9.1.1.3 If RSD value for more than 5% of the congeners exceeds 25%, the GC system
will be reoptimized and calibration will be performed under the new conditions.
9.1.2 For calibration verification a midrange concentration standard containing all congeners,
surrogates and internal standards will be injected as a Continuing Calibration Standard
with each set of samples run on the GC.
9.1.2.1 Each congener RRF calculated by Millennium is compared to the mean RRF from
the initial calibration. A relative percent difference (RPD) of the continuing
calibration RRF from the initial calibration mean RRF for each congener is
determined as follows:
RPD (RRF X /XAWii> - 100
RRFi: = RRF for continuing calibration standard
XKK/.,( = mean RRF from the initial calibration
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9.1.2.2 If the c/c difference exceeds 100% for more than 30% of the congeners, the GC
system will be inspected to determine the cause. Any required maintenance will
be performed and calibration will be reverified. If routine maintenance does not
correct the GC performance based on the annual initial calibration, a new initial
calibration will be performed.
9.2 Retention Time Windows
9.2.1 Retention time (RT) windows can be established only when the GC is operating under
optimum conditions. Any drift in retention times result in meaningless RT windows. The
width of RT windows is established on a run by run basis. Each calibration standard run
at the beginning of a sample set is used to establish RT windows. These RT windows are
confirmed on the performance standard run at the end of the sample set.
9.2.2 If the RT windows determined from the calibration standard fail to identify peaks correctly
in the performance standard, it will be necessary to perform maintenance steps.
9.2.3 Check for leaks throughout the GC system. This problem can cause large and continuing
shifts of all peak retention times. Check for a column blockage leading to skewed and/or
deformed peaks.
9.2.4 Once maintenance has been performed the sample set will be rerun. If the problem is still
not corrected it may be necessary to change the column.
9.2.5 Retention time windows are recalculated when columns are clipped or new columns are
installed.
9.3 GC Analysis of Samples
9.3.1 Samples are analyzed in a set referred to as a sample set. The sequence will always begin
with a hexane injection followed by a continuing calibration check, sample extracts, field
blanks, procedural blank and a performance standard. A typical run sequence would look
like this:
Vial Description
1 Hexane
2 Standard
3 Sample
4 Sample
5 Sample
6 Sample
7 Sample
8 Proced. Blank
9 Performance Standard
9.3.2 Continuation of sample injection may continue for as long as the daily continuing
calibration standard and Mulhn mix standard interspersed with the samples meet OC
requirements. It is recommended that standards be analyzed at least after every 20
environmental samples and at the end of a set.
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9.3.3 Each sample set will be bracketed with an acceptable continuing calibration standard and a
performance standard.
9.3.4 Baselines for the continuing calibration standard are carefully drawn, peaks are identified
and daily absolute RT windows for each congener are established. Congener
identification occurs when a peak from a sample extract falls within the daily retention
time window.
9.3.5 Millennium automatically performs calculation of peak mass using the internal standard
calibration procedure as follows:
* RRF ^
Where PCfim,,() = pg PCB congener detected in sample
AreaPCB = area of PCB congener detected in sample
RRF = RRF of PCB congener from continuing calibration std
massnt[1 = pg applicable internal standard added to sample
areanld = area of internal standard response in sample
9.3.6 If the peak response is less than the SDL the validity of the quantitative result may be
questionable. The sample will be analyzed to determine if further concentration is
warranted.
9.4 GC/MS/ECNI Analysis of Trans-nonachlor
9.4.1 Tuning
The GC-MS is tuned approximately every two to three weeks. The decision to re-tune the
instrument is based on evaluating a daily injection of the performance standard
octafluoronaphthalene (OFN). Peak area, shape, and electron multiplier setting
(sensitivity) are evaluated by a trained operator. If re-tuning is necessary the instrument is
re-tuned in negative chemical ionization mode.
9.4.2 Initial calibration
Consists of three solutions of perfluorotributylamine (PFTBA) at concentrations ranging
from 10 ng/mL to 75 ng/mL. Criteria include m/z 633 = 50,000 and m/z 452 should equal
3-15% of m/z 633. The calibration is done at the beginning of the project, and anytime
after source vacuum has been lost and re-gained due to cleaning or repairs.
9.4.3 Continuing Calibration
A trans-nonachlor standard will be run with each sample set.
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9.4.4 RT Window and SIM criteria - retention time window is set based on the trans-nonachlor
standard run as the continuing calibration standard. The standard is run under full scan
and the retention time for trans-nonachlor is determined. Within this retention time the
program monitors for ions corresponding to the mass of trans-nonachlor - 444 m/z and
442 m/z. The m/z 444 is the quantitation ion, m/z 442 is the confirmation ion. Samples
will be checked within the retention time window for the appropriate ions.
9.4.5 Analysis sequence
Samples are analyzed in sample sets. A typical run sequence appears as follows:
Vial Description
1 Hexane
2 Standard
3-8 Samples
9 Standard
10-15 Samples
16 Standard
9.4.6 Baselines
Baselines are manually set using the HP Aquarius software program.
9.4.7 Quantitation
Areas are determined by Aquarius, and the mass of trans-nonachlor is calculated using the
internal standard method as described in Section 9.3.5.
10.0 Preventative Maintenance
lO.l GC
10.1.1 Columns
When installing a column, it is very important to scrutinize the cut very carefully as jagged
or angled cuts can result in poor chromatography or gas leaks.
10.1.2 Gases
Gases go through a series of filters before reaching the GC including; moisture trap,
hydrocarbon trap, oxygen trap, indicating oxygen trap and chemical filter. Only Ultra-
Pure Carrier grade hydrogen and helium with purity of 99.999% should be used.
10.1.3 Septa
Septa should be changed with each run. Only teflon septa should be used with the
GC/ECD.
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10.1.4 Injector
If chromatography indicates an injection side problem it will be necessary to clean the
injector port and replace the injector liner. At this time it is also a good idea to clip the
column and reinstall.
10.1.5 ECD
Since the ECD contains a radioactive compound (63Ni) it is regulated by the Nuclear
Regulatory Commission. All maintenance relating to the ECD is handled by the
University of Minnesota Radiation Protection Division.
10.2 MS
10.2.1 Mechanical pump oil is changed bi-annually.
10.2.2 Refrigerant coolant level is checked once every three months.
10.2.3 The source is cleaned as needed determined by chromatography quality.
10.2.4 Poles and ceramics are cleaned as needed.
10.2.5 Fan screens are cleaned and the area surrounding the instrument is vacuumed on a
monthly basis.
10.2.6 Cleaning contacts on the component boards and vacuuming component boards is covered
though an HP service contract for the instrument.
10.3 Balances
10.3.1 Sartorius MCI balance has internal calibration ability. Before weighing, an internal
calibration is performed. Weights ranging from 2 mg - 1 g are used to double check the
calibration.
10.3.2 Balances are checked before weighing for cleanliness. Solids wedged under the weighing
pan can result in unstable readings.
11.0 Quality Control Requirements
11.1 Surrogate Recovery
11.1.1 Surrogate standard recovery in all samples, blanks, and spikes will be calculated. Samples
with average surrogate spike recoveries <50% or > 125% will be re-analyzed or flagged.
The following steps will also be investigated.
11.1.2 Confirm that there are no errors in amounts of surrogate solutions or internal standards
added to the solution.
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11.1.3 Examine chromatograms for baseline determination and interfering peaks.
11.1.4 Recalculate the data if any of the above checks reveal a problem.
11.1.5 Reextract and reanalyze the sample if none of the above are a problem. If an individual
surrogate standard exceeds the stated limits, the surrogate will be flagged as FSS. If two
of three surrogates exceed the QA limits, all data for that sample will be flagged FSS.
11.2 Calibration QC
11.2.1 Initial Calibration
An initial calibration will be performed annually. Five concentrations of all congeners
will be used and the RRF RSD across all five concentrations will be <25% for 95% of the
congeners in order for the calibration to be considered valid. If this criteria is not met,
recalibration must occur until it is. If problems persist, the stability of the standards used
and the dilution techniques should be checked.
11.2.2 Continuing Calibration
A continuing calibration standard will be analyzed and compared against the initial
calibration with each run. The RRF RSD of each congener from the initial calibration
RRF must be <100% in order for sample analyses to occur. If this criteria is not met,
action must be taken to achieve an acceptable calibration prior to resuming sample
analysis.
11.3 Internal Standards Performance
Internal standard RF and 204/30 ratio in each sample, blank and standard will be evaluated for
acceptance. The change in each internal standard's RF in each sample compared to the continuing
calibration RF must be <50%. The change in ratio of 204/30 area response for each sample
compared to the continuing calibration ratio must be < 50%. If the ratio fails this criteria, then the
absolute RF of each internal standard is examined to determine of one or both internals standards
are compromised (change in RF >50%). If one internal standard in the PCB analysis is
compromised, the other internal standard should be used for quantitation. If both fail the sample
will be rerun or all sample data will be flagged FIS. If #204 for GC/MS/ECNI analysis of trans-
nonachlor fails the criteria of the change in RF compared to the continuing calibration (RF <50%)
the sample will be rerun or all data will be flagged FIS.
11.4 Blanks
11.4.1 Lab Procedure Blank
A lab procedure blank will be prepared with every six samples. This blank consists of all
reagents, surrogates and internal standards used in the extraction of environmental samples
at the volumes used in these extractions. The procedure blank is carried through the entire
analytical procedure in the same manner as a sample. Any detected congeners at a
concentration
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SOP for the Analysis of PCB
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the data by the analyst. Also, all reagents will be checked before proceeding with
additional analyses and the associated sample sets will be checked against previous ones
for self consistency. If the sample data or reagent purity are questionable, samples will be
re-extracted or flagged FKB if no further sample is available. If sample data are consistent
with previous data and reagent blanks are acceptable then the data will be accepted
without flagging.
11.4.2 Field Blanks
Collection of field blanks will occur at a frequency of two blanks per 11
phytoplankton/detrital/XAD fraction samples collected. Any detected congeners at a
concentration
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11.8 Retention Time Window
Analytes reported as detected must be within the retention time window as described in
Section 9.2. If the analyst believes that a retention time window shift has occurred and the
analytes are present outside the statistically established window, maintenance on the instrument
will be performed and the sample set rerun.
12.0 Data Reporting
12.1 Units
All PCB congeners and trans-nonachlor will be reported as pg/g dry weight calculated as:
pglg PCB or trans-nonachlor = [Av x RRF x A/J / [A^ x Wv]
Where A^ - area counts of the analyte in the sample
RRF = relative response factor of the analyte based on the continuing calibration
standard
A/(( = pg of internal standard added to the sample
/4M = area counts of internal standard in the sample
W^ = weight of sample extracted, in g dry weight
12.2 PCBs
12.2.1 Total PCBs as a sum of all congeners, the sum of each homologe series and each congener
concentration will be reported.
12.2.2 A surrogate correction value will be reported in the appropriate field of the "Laboratory
Reporting Standard" Raw data will be divided by this value to provide the reported result
value.
Surrogate Recovery Correction: Each congener concentration will be corrected to the
recovery of the surrogate that best represents that congener. This is determined by running
duplicate procedural spikes, and correcting the recoveries to each of the three surrogate
standards. The region of the chromatogram that is corrected closest to 100% by a given
surrogate would be the most appropriate surrogate for those congeners. In past projects.
the first third of the chromatogram was best corrected to Congener 14, the middle third to
Congener 65, and the final third to Congener 166 (see Table 6, QAPjP). This study will he
conducted specifically for this project.
12.1 Data
Data from Millennium and GC/MS/ECNI data system Aquarius \\ill be electronically transferred
to spreadsheets in Excel for QA review and data reduction/calculation.
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12.4 Associated QC
Results of lab/field blanks, lab/field duplicates, matrix spike recoveries, surrogate spike recoveries,
and performance standard recoveries will be reported with each sample batch.
13.0 Method Validation Procedures
13.1 Analyst Proficiency
Analysts associated with this project have reviewed the Sampling SOP. the Sample and Analysis
Quality Assurance Project Plan and this Analysis SOP Analysts working with either the GC or
the GC/MS/ENCI have extensive experience in operating, maintenance, and repair of the
instrument. Additionally, analysts have a strong working knowledge of the software used in
analyzing the data associated with each instrument.
13.2 Detection Limit Determinations
13.2.1 IDL
The annual initial five-point calibration curve will be constructed and extrapolated to
determine the y axis intercept. This intercept will be considered the Instrument Detection
Limit (IDL).
13.2.2 MDL
The Method Detection Limit (MDL) for each homologue series will be determined once
during the project using the Ultra standard mix at a low level concentration in a procedural
spike. A mean value and standard deviation for each congener in the Ultra mix will be
established across seven spikes. The Ultra MDLS are equal to three times the standard
deviation. The Ultra MDL for a given homolog will be proportioned against each
congener's RRF within a homolog to establish a congener specific MDL. The congener
specific RRF will be determined from PCB calibration standards run on the GC the same
day as the MDL spiked samples. These MDLs will be used in data reporting.
13.2.3 SDL
The System Detection Limit (SDL) will be calculated annually. This will occur once
seven field blanks have been analyzed. The SDL will be set as +3sd of the mean field
blank concentration.
14.0 References
I4.I Mullin, M. 1985. PCB Congener Workshop, Large Lakes Research Station, U.S. EPA, Grosse
He. Michigan.
14 1 Pearson, R.F.. K.C. Hornbuckle. K.A. Golden, S.J. Eisenreich. and D.L. Swackhamer. 1996.
PCBs in Lake Michigan Water' Comparison to 1980 and mass budget for 1991.
Environ. Sci. Tec/in,it. 30'142lM43(i.
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Extraction and Lipid Separation of
Fish Samples for Contaminant
Analysis and Lipid Determination
Standard Operating Procedure SOP No. HC521A
Larry J. Schmidt
U.S. Geological Survey
Great Lakes Science Center
1451 Green Road
Ann Arbor, Ml 48105-2899
January 12,1995
Version 1.0
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Extraction and Lipid Separation of Fish Samples for
Contaminant Analysis and Lipid Determination
1.0 Scope and Application
This method covers the extraction of fish samples for organic analysis by gas
chromatography (GC) or gas chromatography/mass spectrometry (GC/MS). For analysis
of PCBs and the chlorinated pesticides currently reported by this laboratory (as of the
above date), a 90/10 mixture by volume of petroleum ether/ethyl acetate is used for the
extraction. If the samples are to be analyzed for general contaminants a 80/20 mixture of
the same solvents is used. If more polar pesticides (such as atrazine) are analyzed by this
laboratory in the future, a more polar extracting solvent mixture will probably be needed.
2.0 Summary of Method
This method covers only the extraction and cleanup portions of the testing procedure and
is applicable to fish only. Analytical procedures for PCBs and pesticides in fish are
already in place and are covered by the appropriate NBS/GLSC methods.
3.0 Interferences
3.1 Interferences from sample preparation glassware and reagents are routinely monitored by
running method blanks. The method blank is run through the entire extraction process
along with the samples, except that it consists only of sodium sulfate, the compound that is
mixed with fish tissue before extraction.
3.2 All glassware is cleaned as soon as possible after use by rinsing with the last solvent used
in it. Solvent rinsing is followed by detergent washing with hot water, and rinses with tap
water and distilled water. The glassware is then drained dry and heated in a muffle
furnace at 400°C for two hours. The glassware is solvent rinsed with acetone, hexane,
and the solvent or solvent mixture used for a given operation immediately prior to
glassware use. Volumetric ware is not heated in a muffle furnace. After cooling and
drying, glassware is sealed and stored in a clean environment to prevent accumulation of
dust and other contaminants. Glassware is stored inverted or capped with aluminum foil.
4.0 Safety
PCBs and pesticides have been tentatively classified as known or suspected, human or
mammalian carcinogens. The toxicity or carcinogenicity of each chemical and reagent
used in this method has not been precisely defined, although each chemical compound
should be treated as a potential health hazard. The NBS Great Lakes Science Center
maintains a current awareness file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of material safct\ data sheets is also
available to all personnel m\ol\cd in Chemical anaKsis.
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5,0 Apparatus
5.1 Glass chromatography columns - 330 mm x 23 mm I.D., each fitted with a removable
teflon stopcock. Kontes K-420540-0234 or equivalent.
5.2 Beakers -- 150 mL Pyrex Berzelius beakers.
5.3 Glass wool -- Corning Pyrex brand #3950 or equivalent. Solvents - Petroleum ether, ethyl
acetate, and iso-octane — Pesticide quality or equivalent.
5.4 Zymark Turbovap II Concentrator.
5.5 Zymark Turbovap Concentration Tubes, 200 mL with 1 mL endpoint.
5.6 Zymark Benchmate Workstation robot equipped with Rheodyne 700 L switch valve with a
2 mL loop.
5.7 HPLC solvent pump, Waters, or equivalent.
5.8 ISCO Foxy 200 fraction collector.
5.9 Glass GPC column (51x1.5 cm i.d) packed with 200-400 mesh SX-3 biobeads.
5.10 Jones disposable 0.5 g micro silica gel columns.
5.11 Concentration Tubes, Kadurna-Danish - 10 mL. graduated. Kontes k-570050-1025 or
equivalent.
5.12 Organomation N-Evap Concentrator Model III.
5.13 Argon gas, purified grade.
5.14 Vials, 15 mL glass, with Teflon-lined screw cap.
5.15 Sodium sulfate - (ACS) Granular, anhydrous. Purified by heating at 400°C. for 4 h in a
shallow tray. Supelco 2-0296 or equivalent.
6.0 Extraction Procedure
6.1 Sample Preparation
Samples are processed in sets of about 5-14 samples including all the necessary QA/QC
samples specified in the QAPjP for this work. Fish tissue, previously homogenized
according to the fish processing method HC500A.SOP, is thawed and thoroughly mixed.
Ten g of tissue is weighed into a contaminant-free beaker and mixed with 40.0 g of
\a2SO4 \\hich has been pre\ioiis|\ dried h\ heating to 140 C overnight. The mixture is
stirred frequently until it is dry and free flowing, containing no large lumps. The beaker is
then labelled with the appropriate sample number and weight. Necessar\ surrogates
specified in the QAPjP Plan for this study
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6.2 Extraction
6.2.1 The extraction columns are prepared by inserting a small glass wool plug into the
bottom of each chromatography column, and then the column is rinsed twice with
15 mL of petroleum ether. Air is removed from the glass wool by lightly tapping
it with a clean glass stirring rod. A Zymark concentration tube is placed under
each column and the appropriate labels are transferred to these tubes. The sample
mixture is then poured into the column, after which 50 mL of the appropriate
solvent is added to the sample beaker, stirred, and transferred to the column. The
solvent is allowed to pass through the column, but as it begins to elute into the
concentration tube, the stopcock is closed. At this point the column is lightly
stirred with a glass rod to remove trapped air. Elution is then continued at the rate
of I-2 mL/min. until the solvent level reaches the beginning of the sample
mixture.
6.2.2 Another 50 mL of the appropriate solvent mixture is added and elution is
continued at the same rate. The columns are allowed to drain completely after the
second 50 mL of solvent is added. The stopcock tips are rinsed with ethyl acetate
to wash any residual lipid and analytes into the concentration tube.
6.3 Extract Concentration
The eluant is concentrated by placing the Zymark concentrator tubes in the Turbovap
concentrator. The concentrator's water bath is kept at 40°C, and the argon sweep gas
pressure is set to 10-12 psi with the Turbovap's control knob. Samples are concentrated to
1 mL or the minimum amount allowed by a sample's lipid content, whichever volume is
greater. The residual solvent from this process should be mostly ethyl acetate. Each
extract is then transferred to a graduated (or marked) culture tube and diluted to volume
(10 or 20 mL depending on lipid and contaminant concentration) with ethyl acetate. The
tubes are sealed with a teflon-lined screw cap and the labels are transferred to the
appropriate tubes.
6.4 Lipid Determination
Prior to cleanup by gel permeation chromatography, a volume of sample extract equivalent
to 1 g of tissue is pipetted into a preweighed (after acetone rinsing and drying) aluminum
drying pan. Preweighing is to the nearest 0.1 milligram. The extract is allowed to
evaporate under static conditions in a fume hood for 2 hours. The pan is again weighed to
the nearest 0.1 milligram and the percent extractable lipid is computed as 100 x (1-weight
of residual lipid).
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7.0 Lipid Separation/Removal
7.1 The GPC column is initially calibrated by taking 5 mL fractions as the elution proceeds
through the dump cycle, during which the lipids are routed to a waste bottle, and then
throueh the collect cycle, during which the compounds of interest are routed to the
appropriate collecting station. The endpoint of lipid removal is monitored by the
gravimetric procedure detailed in Section 6.4. The starting point of compound collection
is monitored by GC/GCMS analysis of the 5 mL fractions. The normal dump fraction is
about 43 mL, followed by a collect fraction of 45 mL.
7.2 The status of the GPC column is monitored by either a pre- or post-run procedure for each
batch extraction. While a batch is proceeding, or processing put on hold until the spike
sample (run first in a batch) is brought from 45 to 50 mL and 2 ^L of this are injected into
a GC/EC to estimate recovery before advancing to the next sample. The peak height ratios
of transnonachlor (elutes at beginning of collect cycle) and p,p'-DDE (elutes toward the
end of collect cycle) should closely match that in the spike standard. The GPC can be
prevented from advancing to the next sample or stopped at any point in the sample set.
But once a sample is loaded on the column that sample will need to be rerun in case of a
problem. If the original calibration conditions are changed after the spike sample any
samples in the batch processed by GPC before correcting the problem must be rerun.
Usually the GPC monitoring will not indicate a problem so frequently the spike will be
checked during or after the entire batch has been processed or when recoveries are
determined from previous sample sets to be low.
7.3 For PCB and/or pesticides analysis, a 2 mL portion of sample extract is loaded into the
calibrated loop of the gel permeation system (attachment to the Benchmate Robot) and
lipid/contaminants are separated with ethyl acetate through 200-400 mesh SX-3 Biobeads.
The first fraction contains the lipids is discarded and the second fraction containing the
contaminants is collected in a clean, appropriately labelled Zymark concentration tube.
This fraction will be greater than 93% lipid-free. Consecutive loops of each extract can be
processed when greater amounts of contaminants are needed (i.e. GC/MS). Refer to GPC
technical manual for complete details of GPC setup and operation. The extracts are
concentrated with the Zymark Turbovap set to the same conditions as specified in
Section 6.3 above. The extracts are brought to 1 mL with the Zymark concentrator and
then brought up to final volume with iso-octane.
7.4 Each sample is passed through a 0.5 g acidified micro-column of silica gel to remove the
last lc/c of the lipid. The silica gel column is designed to work with the Zymark
Benchmate Workstation Robot. Fifty ^L of 1:1 concentrated sulfuric and water is added
manually to a silica gel column for each sample in the set being processed. Thereafter, the
Benchmate will automatically rinse, load the sample (extract from 1 g of fish), weight
amount loaded on the column, and elute the desired analytes from the column. Samples
are then transferred to a graduated culture tube and evaporated using the Organomation
N-Evap Concentrator to about 0.5 mL. The sample is then diluted to volume (usually
1 -5 mL) with iso-octane and then transferred to a GC or GC/MS autosampler vial and
stored until readv for analvsis.
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Extraction and Lipid Separation of
Fish Samples for Contaminant Analysis and
Volume 2, Chapter 1 Lipid Determination
7.5 This is a manual method for removal of remaining lipids after GPC and was used for most
of the mass balance fish samples hesinnins because with the automated method lipid was
causing problems quantitating several PCB congeners. One mL of iso-octane is added to
the GPC extract (equivalent to g fish tissue) and samples are concentrated to a final
volume of I mL with the Zymark TurboVap set to conditions as specified in Section 6.3.
Each sample is passed through a borosilicate glass column, 30 cm X 1.5 cm i.d.,
assembled as follows from bottom to top:
a. Pyrex wool plug
b. 1.0 g dried sodium sulfate
c. 7.0 g of a 10% (w/w) mixture sodium bicarbonate and sodium sulfate
d. 3.6 g 2% deactivated silica gel
e. 0.2-0.3 mL concentrated sulfuric acid
Each column is conditioned with 5 mL of the elution solvent (5:95 ethyl acetate:hexane)
before loading sample onto the column. Zymark tubes are rinsed with 5 mL of elution
solvent which is passed through the eluant from the column is collected in 12 mL
calibrated centrifuge tube. Three additional 2 mL aliquots are passed through the column
and collected in the same receiving tube. Each sample is then concentrated to the final
volume necessary for analysis using a stream of argon gas, approx. 10 psi, and a water
bath at 38°C.
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Analysis of Total PCBs and PCB
Congeners and Trans-nonachlor in
Fish by Gas Chromatography/
Negative Chemical lonization
Single Ion Mass Spectrometry
Standard Operating Procedure SOP No. HC 519.D
(Replaces: No. HC 519C)
Larry J. Schmidt
U.S. Geological Survey
Great Lakes Science Center
1451 Green Road
Ann Arbor, Ml 48105-2899
May 16, 1996
Version 2.0
-------
Analysis of Total PCBs and PCB Congeners and Trans-nonachlor
in Fish by Gas Chromatography/Negative Chemical lonization
Single Ion Mass Spectrometry
1.0 Scope and Application
I. I This method covers the determination of total PCBs, PCB congeners and Trans-nonachlor
developed at the National Biological Service/Great Lakes Science Center (NBS/GLSC) by gas
chromatography using negative chemical ionization single ion monitoring mass spectrometry
(GC/NCI/SIM) for the U.S. EPA Mass Balance Study. The parameters presently reported by this
method are given in Table 1. The parameters listed are qualitatively and quantitatively
determined as target compounds by this method. The NCI reagent gas used is methane.
1.2 The method detection limits (MDL) for selected congeners are also listed in Table 1. These were
determined according to EPA rule Appendix B of 40 CFR Part 136 on method blanks spiked with
one congener at each chlorine level (one through ten) at very low levels (3-10X signal to noise
ratio) on the GC/MS just before extraction. The value for the other congeners at a given level of
chlorination were calculated using the ratio of the normal response factor for the congener used in
the MDL study for that level of chlorination to the normal response factor of the congener in
question, multiplied by the detection limit of that congener representing the chlorine level.
2.0 Summary of Method
This method covers only the analytical portion of the testing procedure applicable to fish.
Sampling and sample preparation procedures for PCBs and trans-nonachlor in these matrices are
already in place and are covered by the appropriate NBS/GLSC methods. Qualitative
identification the parameters in the resulting extracts is performed using the retention time and the
relative abundance of two characteristic masses (m/z). Quantitative analysis is performed using an
internal standard technique with a single characteristic m/z.
3.0 Interferences
3.1 Interferences from sample preparation glassware and reagents are routinely monitored by running
method blanks. The method blank is run through the entire extraction process along with the
samples, except that it consists only of sodium sulfate, the compound that is mixed with fish tissue
before extraction.
3.2 Matrix interferences may be caused by compounds that are co-extracted from the sample, and may
vary considerably from source to source. The level of interference using GC/NCI/SIM is far less.
however, than when using standard positive electron-impact (F.I) mass spectrometry.
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Negative Chemical lonization Single Ion Mass Spectrometry Volume 2, Chapter_f
3.3 Two congeners of interest, numbers 77 and 126, are not separated completely from interfering
PCBs. For the Mass Balance Study, they will be quantitated by subtracting out the typical
contribution of the interfering PCB and will thus be quantitated somewhat less accurately than the
other target congeners. Neither compound contributes significantly to total PCBs in biota.
4.0 Safety
4.1 PCBs have been tentatively classified as known or suspected, human or mammalian carcinogens.
Primary standards of these toxic compounds must be handled in a manner to avoid direct contact
4.2 The toxicity or carcinogenicity of each chemical and reagent used in this method has not been
precisely defined, although each chemical compound should be treated as a potential health
hazard. The NBS/GLSC maintains a current awareness file of OSHA regulations regarding the
safe handling of the chemicals specified in this method. A reference file of material data handling
sheets is also available to all personnel involved in chemical analysis.
5.0 Apparatus
5.1 Gas Chromatograph The NBS/GLSC uses an HP5890 gas chromatograph (GC) equipped with an
HP7673A robotic autosampler. All data are acquired using computer controlled batching of
sample, standard, and quality control runs. This approach is critical to obtaining the retention time
reproducibility needed for doing PCB congener work, even though relative retention times are
used. Members of a given congener elute closely enough together that tight control of
chromatographic conditions is necessary to avoid misidentification, as the ion ratios for a given
congener series are very similar. The HP gas chromatograph is capable of multi-stage temperature
programming (ramping) and is equipped for splitless/split capillary injection.
5.2 Column - A 30 meter DB-5 fused silica capillary column is used, with an I.D. of .25 mm and a
coating thickness of .25 micron.
5.3 Mass Spectrometer - The NBS/GLSC uses an HP5988A research-grade low resolution mass
spectrometer (MS) equipped with positive and negative chemical ionization capability. The
instrument can perform single ion monitoring (SIM), analyzing up to 999 ion groups of 20 ions per
group during each run. The GC capillary column is interfaced directly into the MS source with no
splitting of carrier gas.
5.4 Data System The HP5988A mass spectrometer is equipped with an RTE-A data system, capable
of doing automatic identification and quantification of target compounds using a reverse search for
identification and an internal standard method for quantitation. This system also controls data
acquisition, including automatic operation of the GC. MS, and autosampler, and any other required
manipulation of the raw data or processed files. The HP Enviroquant software is used for data
reduction, which will necessitate transfer of raw data files from RTF to PC/DOS by RS-232 using
a procedure file.
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Analysis of Total PCBs and PCS Congeners and
Trans-nonachlor in Fish by Gas Chromatography/
Volume 2, Chapter 1 Negative Chemical lonization Single Ion Mass Spectrometry
6.0 Analytical Procedure
6.1 Tuning and Calibration--The source is operated in the CI mode using methane reagent gas. When
the source pressure has stabilized at a value known to produce satisfactory results (.5 to .8 Torr),
the instrument is manually tuned and calibrated using an NCI customized tuning file. Pressure
stabilization usually takes about 45 minutes. Tuning is checked daily by using at least two ions
which span the approximate range of interest for PCBs/trans-nonachlor. Using FC-43, the ions
currently selected are 235 and 452. A third ion, 633, is always present in the display, but is not
used for tuning. The parameter ramp program is run for purposes of maximizing NCI response,
with the HP lenses known as repeller, drawout, ion focus, and entrance lens being adjusted so that
their settings are near the displayed curve maxima for the two ions. Since the lens settings are
interactive, the lens sequence should be rechecked until no changes to the settings are needed. The
manufacturer's manual contains detailed instructions on manual tuning in NCI. Once tuning is
accomplished, a half-page profile scan is printed out. The source pressure and any unusual
conditions is noted on this display, and the exact masses of ion 452 is checked to assure that it has
not changed more than 0.15 amu from 452.0. Any centroid change greater than this may require
alteration of the exact masses as given in the ion groupings (presented in Table 3) of this method.
It is much simpler to adjust the centroid in the tuning file to within 0.15 of 452.0 with the Mass
Offset (B/b) control in the manual tuning procedure. Once proper tuning and calibration have
been obtained, the manual tune is saved before exiting the manual tuning program. Note that
tuning is most needed the first few weeks after the source has been cleaned. The system becomes
very stable thereafter, and usually no tuning operations are necessary other than printing out and
archiving a copy of the tuning display.
6.2 Calibration and Linear Range
6.2.1 GC and MS conditions are set by the analytical method. These conditions are given in
Table 2 for the GC parameters and in Table 3 for the MS parameters. It is important to
note that the method file controls if there is a disparity between it and the tuning file with
respect to source temperature, multiplier voltage, and/or emission current. The column
head pressure is manually controlled using the GC oven pressure controller and the oven
may be baked by setting the oven temperature from the keyboard if no method is running.
Source pressure (reagent gas flow) is controlled manually using the CI reactant gas flow
controller. All other parameters are established through the terminal and keyboard.
•6.2.2 Standards of a 25:18:18 ratio of Aroclor 1232, 1248, and 1262 are run at concentrations of
500, 2500, and 5000 ng/g to demonstrate linear response over this range. Using these
concentrations, we obtain three concentrations for all congeners which do not saturate at
the highest concentration and do not go below the detection limit for the least concentrated
congener. A linear three-point curve is established when the standard deviation of the
relative response ratios at the three concentrations is less than 257c.
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Negative Chemical lonization Single Ion Mass Spectrometry Volume 2, Chapter 1
6.2.3 The 2500 PPB concentration is designated as the calibration standard. The concentrations
in the ID file are based on this standard. It is run at the beginning of each batch run. The
performance standard is be run immediately following the calibration standard. Congener
concentrations in the performance standard are calculated from the calibration standard
response factors. A subset of six congeners are used to evaluate the current response
factors. These include #44, #207 (small peaks), #101, #185 (average peaks, and #151,
#180 (large peaks). Calculated concentrations of these congeners are compared to their
known concentrations. Deviations from actual concentrations of greater than 50% for the
small peaks and greater than 10% for the average and large peaks result in flagging of all
samples in the data set for the failed congener.
6.2.4 Because a single calibration standard is being used to generate RRFs, the calibration
standard concentration should be within a factor of five of the concentrations of PCBs in
the sample extracts. Sample extracts that fall outside this range are either diluted or
concentrated to bring them to within this range.
6.3 Sample Run
Once the source pressure has stabilized at the desired value and a manual tune has been performed
(or checked), a batch data acquisition (sample run) can be initiated. A batch data acquisition is
begun by accessing the batch sequence menu. Every batch includes a method blank, standards, a
spike standard, a surrogate standard, a check sample, a background fish extract, then actual
samples and spikes. The latter will vary with the number of samples extracted at one time.
Information on each sample is entered sequentially into the menus as they come up until the batch
edit. The proper tuning file, method file, allowable space per run, and total cartridge space
(minimum 600 tracks for a normal batch) for the batch are also inputted to the batch file. When
finished, the batch sequence listed and checked for correctness, the batch is started. Before the
start command is given, a final check is made as to condition of column, solvent wash vials,
septum, injection port liner, and leak-free status of the injection area. Internal standards should be
spiked into the samples and spikes just before the beginning of the batch data acquisition run.
6.4 Data Reduction and Reporting
6.4.1 Quantification of PCBs is congener specific and done by the internal standard method.
The internal standards that will be used are congeners #136 and #204. Congeners eluting
prior to and including #110 are quantitated relative to internal standard #136, and those
after #110 are quantitated relative to #204.
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Analysis of Total PCBs and PCB Congeners and
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Volume 2, Chapter 1 Negative Chemical lonization Single Ion Mass Spectrometry
6.4.2 Data analysis begins with the execution of AUTSFX (Single ion monitoring background
subtraction program) for the entire batch and then BATCH3 using ID file MBSUR for the
entire batch. This will provide assurance that the batch completed without gross problems
such as syringe lugging or failure of GC temperature controlled zones. The CENTROIDS
procedure file is the first injection (DOSE) to verify calibration of mass axis. A procedure
file is then run on the standard PCB solution calibration standard to check source
condition and GC resolution. The appropriate data are recorded in a log book. The batch
program is then executed to produce the QT output file needed to perform a calibration
check. If many compounds are absent, it usually means the internal standard retention
times must be adjusted in the ID file. Ion ratios almost never have to be adjusted. Even if
all compounds are present after adjusting the ID file, the retention times should be closely
checked to assure that they are no more than 0.2 minute from expected. Once the QT
output file is acceptable for all standards runs, the calibration is checked.
6.4.3 At this time, spike and surrogate recoveries are calculated and examined for acceptability
insofar as method performance is concerned. Surrogates (PCBs #65 and #166) are spiked
into every sample and blank. The matrix spike is spiked into a hatchery fish at 30 times
background PCB levels. These spikes are made directly into the fish/sodium sulfate
mixture immediately before solvent extraction. For a given sample set, acceptance values
for spike recoveries are specified in Table 7.1 of the Mass Balance QAPjP If matrix
spike recoveries do not meet these standards, then data from that sample set are flagged.
If surrogate spike recoveries do not meet these standards, then that sample must be
evaluated according to the QAPjP If the flags are determined to be serious samples must
be re-extracted and analyzed. The spike ID file is MBSPK. The surrogate ID file is
MBSUR.
6.4.4 Once checking of standards runs is satisfactorily completed, the ID file is updated for a
final time using the response factors from the calibration standard by executing the QCAL
command file. The final QT reports are then produced. The value for the internal
standards in the ID file may have to be adjusted to reflect differing final volumes or initial
weights.
6.4.5 At this point a comparison of duplicates is made, and results for the check fish run during
the batch are compared to values representing an average of six or more check fish ran
previously. The acceptance values for duplicates and for the check fish are specified in
Table 7.1 of the NBS/CLSO QAPjP.
7.0 Method Performance
As part of NBS-GLSC internal ongoing quality control and performance monitoring, check fish
samples are run as deemed to be necessary (usually one per sample batch). These are run as
ordinary samples and the results are tracked for consistency over time. Baseline data for check
fish samples are generated by a one time extraction of six replicate lab reference fish samples. The
replicates are processed through sample preparation and analyzed in the normal manner. The
concentration and standard deviations (N=6) are calculated for the parameters routinely analyzed.
Variation in the check fish samples over the course of many extraction batches can be expected to
be no better, at best, than this baseline data.
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Analysis of Total PCBs and PCB Congeners and
Trans-nonachlor in Fish by Gas Chromatography/
Negative Chemical lonization Single Ion Mass Spectrometry
Table 1. PCB Congeners/trans-nonachlor to be Determined by GC/NCI/SIM.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42
43
Compound
PCB Congener #3 1 +#28
PCB Congener #33
PCB Congener #22
PCB Congener #52
PCB Congener #49
PCB Congener #47+#48
PCB Congener #44
PCB Congener #42
PCB Congener #41 +#71
PCB Congener #64
PCB Congener #40
PCB Congener #63
PCB Congener #74
PCB Congener#70 + #76
PCB Congener #66
PCB Congener #95
PCB Congener #91
PCB Congener #56+#60
PCB Congener #84+#92+#89
PCB Congener #101
PCB Congener #99
Trans-nonachlor
PCB Congener #11 9
PCB Congener #83
PCB Congener #97
PCB Congener #81 +#87
PCB Congener #85
PCB Congener #77
PCB Congener #110
PCB Congener #82
PCB Congener #151
PCB Congener #144 + #135
PCB Congener #107
PCB Congener #123
PCB Congener #149
PCB Congener #11 8
PCB Congener #134
PCB Congener #1 14
PCB Congener #131
PCB Congener #146
PCB Congener #132 + #153
PCB Congener #105
PCB Congener #141
Instrument Detection Limit
using 1 g sample (ng/g)
9
4
4
12
18
6
25
4
18
4
7
0.4
2
1
2
6
7
1
1
0.2
0.4
0.08
0.1
0.6
0.9
0.6
0.3
0.2
0.5
1
0.02
0.03
0.3
0.1
0.04
0.3
0.02
0.4
0.01
0.01
0.02
0.02
0.1
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Analysis of Total PCBs and PCB Congeners and
Trans-nonachlor in Fish by Gas Chromatography/
Volume 2, Chapter 1
Table 1. PCB Congeners/trans-nonachlor to be
Determined by GC/NCI/SIM. (Cont'd)
Compound Instrument Detection Limit
using 1
44. PCB Congener #137 + #176
45. PCB Congener #138 + #163
46. PCB Congener #158
47. PCB Congener #129
48. PCB Congener #126
49. PCB Congener #178
50. PCB Congener #175
51. PCB Congener #187 + #182
52. PCB Congener #183
53. PCB Congener #128
54. PCB Congener #167
55. PCB Congener #185
56. PCB Congener #174
57. PCB Congener #177
58. PCB Congener #202
59. PCB Congener #171
60. PCB Congener #156
61. PCB Congener #173
62. PCB Congener #157
63. PCB Congener #200
64. PCB Congener #172
65. PCB Congener #197
66. PCB Congener #180
67. PCB Congener #193
68. PCB Congener #191
69. PCB Congener #199
70. PCB Congener #170 + #190
71. PCB Congener #198
72. PCB Congener #201
73. PCB Congener #203 + #1 96
74. PCB Congener #189
75. PCB Congener #195
76. PCB Congener #208
77. PCB Congener #207
78. PCB Congener #194
79. PCB Congener #205
80. PCB Congener #206
81. PCB Congener #209
g sample (ng/g)
0.08
0.04
0.03
0.01
0.03
0.1
0.1
0.08
0.06
0.02
0.03
0.04
0.09
0.1
0.2
0.1
0.04
0.06
0.03
0.2
0.04
0.04
0.07
0.08
0.1
0.2
0.09
0.1
0.3
0.4
0.1
0.1
0.07
0.1
0.1
0.2
0.2
0.07
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Analysis of Total PCBs and PCS Congeners and
Trans-nonachlor in Fish by Gas Chromatography/
Negative Chemical lonization Single Ion Mass Spectrometry Volume 2, Chapten
Table 2. GC and Autosampler Operating Parameters
1. Column - 30 meter DB-5 (J&W Scientific), .25 mm I.D., .25 micron film thickness.
2. GC Temperature Program Initial temperature 80°C, hold for one minute, then program to 150° at
20°/minute, then program to 250° at 2°/minute, hold five minutes. Post-run bakeout is 300° for
six minutes.
3. Oven Equilibration Time - three minutes
Total Run Time - 59 minutes
Scanning Start Time - four minutes
Splitless Operation Time - two minutes
4. Injection Port Temperature - 280°C
GC/MS Interface Temperature - 280°C
5. Sample Injection Volume 2 microliters. Data Cartridge Space - Minimum 600 tracks available
space for a 24-run batch. Minimum Blocks Reserved for CR Check 3000.
6. Carrier Gas - Helium at 10-15 psi column head pressure. This can vary, depending mostly on
column aae.
Table 3. Mass Spectrometer Operating Parameters
1. Source Temperature - 110°C
2. Multiplier Voltage - 1400-2800V (depending on stage of multiplier life)
3. Emission Current - 300 uA
4. Electron Energy - 200 eV
5. Reagent Gas Methane
6. Source Pressure - (0.5 to 0.8 Torr).
7. SIM Groupings - Group 1, run from 4.0 to 16.3 minutes
Group 2, run from 16.3 to 21.5 minutes
Group 3, run from 21.5 to 26.0 minutes
Group 4. run from 26.0 to 30.0 minutes
Group 5, run from 30.0 to 35.5 minutes
Group 6, am from 35.5 to 39.85 minutes
Group 7, run from 39.85 to 45.6 minutes
Group 8, run from 45.6 to 46.3 minutes
Group 9. run from 4ft ^ to 50.0 minutes
Group 10. run from 50 0 io 59.0 minutes
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Volume 2, Chapter 1 Negative Chemical lonization Single Ion Mass Spectrometry
Table 3. Mass Spectrometer Operating Parameters (Cont'd)
Group I Exact masses 254.8, 254.9, 255, 256.8, 256.9, 257 (Lindane) each at dwell times of
150 milliseconds.
Group 2 Exact masses 255.86, 255.87, 255.88, 255.89, 256.9 256.91, 256.92, 256.93, 256.94,
256.95, 257.86, 257.87, 257.88, 257.89, 257.9, 257.91, 257.92, 257.93, 257.94, 257.95
(Trichlorobiphenyls) each at dwell times of 45 milliseconds.
Group 3 Exact masses 289.86, 289.87, 289.88, 289.89, 289.9 289.91, 289.92. 289.93, 289.94,
289.95, 291.86, 291.87, 291.88, 291.89, 291.9, 291.91, 291.92, 291.93, 291.94, 291.95
(Tetrachlorobiphenyls) each at dwell times of 45 milliseconds.
Group 4 Exact masses 289.79, 289.8, 289.9, 291.79, 291.8, 291.9 (Tetrachlorobiphenyls), Exact
masses 325.79, 325.8, 325.9, 327.79, 327.8, 327.9 (Pentachlorobiphenyls), Exact masses
441.6, 441.7, 443.6, 443.7 (Trans-nonachlor) each at dwell times of 55 milliseconds.
Group 5 Exact masses 289.79, 289.8, 291.79, 291.8 (Tetrachlorobiphenyls), Exact masses 315.8,
315.9, 317.8, 317.9(4,4'-DDE), Exact masses 325.8, 325.9, 327.8, 327.9
(Pentachlorobiphenyls), Exact masses 359.7, 359.8, 358.9, 361.8 (Hexachlorobiphenyls),
Exact masses 379.7, 379.8, 381.7.381.8 (Dieldrin) each at dwell times of 45 milliseconds.
Group 6 Exact masses 325.77, 325.78, 325.79, 325.8, 325.81, 325.82, 327.77, 327.78, 327.79,
327.8, 327.81, 327.82, (Pentachlorobiphenyls), Exact masses 359.7, 359.8, 361.7, 361.8
(Hexachlorobiphenyls), Exact masses 393.6, 393.7, 395.6, 395.7 (Heptachlorobiphenyls)
each at dwell times of 45 milliseconds.
Group 7 Exact masses 359.69, 359.7, 359.8. 361.69, 361.7, 361.8, (Hexachlorobiphenyls), Exact
masses 393.59, 393.6, 393.7, 395.59, 395.6, 395.7 (Heptachlorobiphenyls), Exact masses
427.59, 427.6, 427.7, 429.59, 429.6, 429.7 (Octachlorobiphenyls) each at dwell times of
50 milliseconds.
Group 8 Exact masses 359.66-359.74 (10 ions) and exact masses 359.76-359.84 (10 ions), each at
dwell times of 45 milliseconds (PCB #169).
Group 9 Same exact masses and dwell times as Group 7, except add exact masses 463.6, 465.6
(Nonachlorobiphenyls) and reduce each dwell time to 45 milliseconds.
Group 10 Exact masses 427.68, 427.69, 427.7. 427.71, 427.72, 429.68, 429.69. 429.7, 429.71,
429.72 (Octachlorobiphenyls). Exact masses 463.59, 463.6, 463.61. 465.59, 465.6, 465.61
(Nonachlorobiphenyls). Exact masses 497.49, 497.50, 499.49. 499.50
(Decachlorobiphenyl), each at dwell times of 55 milliseconds.
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Volume 2
Chapter 2: Mercury Analysis
-------
Standard Operating Procedure for
Analysis of Vapor Phase Mercury
Gerald J. Keeler and Matthew S. Landis
University of Michigan
Air Quality Laboratory
109 South Observatory Street
Ann Arbor, Ml 48109-2029
June 1,1994
Version 2.0
-------
Standard Operating Procedure for
Analysis of Vapor Phase Mercury
1.0 Introduction to Principals of Vapor Phase Mercury Sampling and
Analysis
Mercury in the atmosphere exists predominantly in the gas phase in the form of elemental mercury
(Schroeder, 1982). Other species of mercury found in the gas phase include methyl and
dimethylmercury, and mercuric chloride, mercuric hydroxide and free divalent mercury. Vapor
phase mercury is quantitatively removed from an air stream by amalgamation onto gold. While the
amalgamation process is believed to remove most vapor phase mercury species with >99%
efficiency, the analytical procedure employed determines whether or not 'total mercury' or
predominantly elemental mercury is quantified. At the University of Michigan Air Quality
Laboratory (UMAQL) vapor phase mercury is collected onto gold-coated borosilicate glass bead
traps by drawing air at a low flow rate through a baked glass fiber pre-filter followed by the gold-
coated borosilicate glass bead trap. The air is prefiltered to eliminate particles from the gas phase
collection traps. After sampling, vapor phase mercury is quantified by cold vapor atomic
florescence spectrometry (CVAFS).
In the past, methods for collection of vapor phase mercury have dictated long sampling duration,
often from 24 hours up to a week. The collection method employed for the Lake Michigan
Loading Study and described in this protocol uses gold-coated borosilicate glass bead traps, which
UMAQL has determined to be >99% efficient at collection of vapor phase mercury (at a flow rate
<1 1pm). Dual-amalgamation and subsequent analysis by cold-vapor atomic florescence, allows
detection of mercury at picogram levels. After thermal desorption, gold-coated bead traps are re-
used since they do not exhibit memory effects. Due to the collection efficiency of gold-coated
beads and the ability to detect picogram amounts of mercury, sampling strategies using gold-
coated bead traps can employ much shorter duration samples than have previously been possible.
Short sampling duration provides the resolution necessary to use receptor models in determining
sources and source contributions of measured vapor phase mercury.
Preparation and collection of accurate and reliable data on mercury concentrations in
environmental samples requires that ultra clean procedures are used. All sampling supplies with
which a sample will come into contact must be acid cleaned in a Class 100 Clean Room. At the
sampling site, precautions taken to avoid contamination of the sample include storing samples at
an outdoor staging area and special operator handling. These and other techniques employed to
minimize contamination of the samples are described in detail in this protocol.
2.0 Sample Preparation
2.1 Acid Cleaning Procedure
All Teflon filter packs. Teflon jars. Teflon tubing, gold trap fittings and end plugs (referred to
below as 'supplies'! are cleaned iisinu an 1 1 day procedure described h\ Rossmann and Barres
(1991).
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Supplies to be acid cleaned are first rinsed in reagent grade acetone under a fume hood, then
washed in hot tap water and diluted Alconox. Supplies are rinsed five times in cold tap water then
rinsed three times in DI water. The supplies are then heated in 3M hydrochloric acid (EM Science
Tracepur HCI in Milli-Q water (18.2 MQ/cm)) for six hours at 80° C. One liter of 3M HCI is
prepared by adding 750 mL of Milli-Q water to 250 mL of concentrated EM Science Tracepur
HCI. The 3M HCI can be used several times and is stored for reuse in a polyethylene carboy
dedicated for this purpose. The supplies are placed into clean polyethylene tubs which are then
filled with the 3M HCI, making sure that all of the surfaces are submersed in the HCI. The tubs
are covered and placed in a water bath which is heated to 80°C in a fume hood. The water in the
bath is maintained at the level of the acid inside the tubs. After the water in the bath reaches SOT,
the supplies in the tubs are allowed to soak for 6 hours.
After the 6 hour, 80°C soak, the tubs are removed from the water bath and allowed to cool in the
fume hood. When cool, the 3 M HCI is poured back into its polyethylene carboy. The supplies
are rinsed in the tubs three times with Milli-Q water. The supplies are then soaked in a 0.56M
nitric acid solution (Baker Instra-Analyzed HNO3 in Milli-Q water) for 72 hours at room
temperature in the same polyethylene tubs in which they were heated with HCI. The nitric acid
solution is made by adding 35 mL Baker Instra-Analyzed HNO, to 965 mL of Milli-Q water.
Nitric acid is reused for up to 6 months and is stored in a carboy dedicated for HNO,. At the end
of the 3 day soak, the supplies being cleaned are rinsed three times with Milli-Q water and
transferred into a Class 100 Clean Room.
Inside the clean room, the supplies are again rinsed three times with Milli-Q water. The tubs
containing the supplies are filled with 0.56 M Baker Instra-Analyzed HNO, that is kept in the
clean room and is dedicated for this final step only. The supplies are then allowed to soak in this
acid for 7 days. This acid is prepared by adding 35 mL of the Instra-Analyzed HNO, to 965 mL of
Milli-Q water. At the end of the seven day acid soak inside the clean room, the supplies are rinsed
five times with Milli-Q water and allowed to air dry on a clean surface. When the supplies are dry,
they are triple bagged in new polyethylene bags and removed from the clean room, ready for use in
sampling.
2.2 Preparation of Gold-Coated Bead Traps and Pre-Filters
Gold-coated borosilicate glass bead traps are constructed at The University of Michigan Air
Quality Laboratory and tested prior to use in the field. The gold-coated beads used in the traps are
made by generating a gold plasma under vacuum conditions that uniformly deposits onto the
surface of the beads. The thickness of the coating generated using this process is about 300 A.
The gold-coated beads are contained in a quartz tube which is 10 cm long with an inner diameter
of 5 mm and an outer diameter of 7 mm. Teflon heat-shrink tubing is attached to both ends of the
tube into which Teflon endplugs are placed when the trap is in storage or connectors when the trap
is being used to collect a sample. Fach trap contains approximately 0.7 g of gold-coated
borosilicate glass beads which are held in place using quartz wool and two sets of three radial
indentations in the quartz tube The gold-coated beads, quartz tubes and quartz wool are baked at
600°C for one hour prior to making the trap. In addition. Teflon endplugs and heat shrink tubing
are acid cleaned as previously described. After each trap is made, it is given a unique number
identifier in order to chart the history and performance of the trap New traps are first conditioned
by drawing approximately 0.4 m nl air through the trap then heating the trap to 500°C for 5
minutes. Inert gas is purged through the traps at 300 cc/min during heating procedure to remove
moisture and other volatile constituents. The conditioning procedure is performed twice prior to
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testing the trap. The trap is then tested by injecting a known amount of elemental mercury vapor
and comparing the result to an analytical standard (standard generation is described on page 9).
The trap must exhibit duplicate measurements that are within 5% of the standard and the replicate
measurements must also be within 5% of each other. Following this test, the trap is then blanked
(described below) and stored for 7 days. After 7 days, the trap is analyzed for a storage blank
(sample analysis is described in Section 3.0). The storage blank must be less than 15 pg for the
trap to be accepted for use in field sampling. Gold traps are stored with endplugs in place, triple
bagged in polyethylene before and after sampling.
Just before going into the field to collect vapor phase mercury samples, gold-coated bead traps are
blanked again. Blanking a trap removes all mercury from the gold-coated bead surface and will
also remove water vapor and other unwanted constituents. Traps are blanked by placing them in
the analytical train and heating them to 500°C for 2 minutes, identical to a normal sample analysis.
Vapor phase mercury samples collected onto gold-coated borosilicate glass bead traps must be
prefiltered to exclude particles. Glass fiber filters (Gelman Sciences) are pre-treated to remove all
mercury prior to use in sampling. Glass fiber filters, 47 mm in diameter, are placed in a clean
crucible with a lid. The crucible is placed in a muffle furnace which is heated to 500°C and the
filters are allowed to bake at this temperature for one hour. While hot, filters are removed from the
crucible with acid-cleaned Teflon-coated forceps and placed in an acid-cleaned Teflon jar which is
closed and sealed with Teflon tape. The Teflon jar is triple bagged and stored at -40°C until use.
Filters are stored no more than 3 months prior to use and frequent blanks are taken to
ensure the filters remain clean.
3.0 Sample Analysis
3.1 Summary
Vapor phase mercury collected onto a gold-coated bead trap is thermally desorbed and carried by a
mercury-free helium stream into a cold-vapor atomic fluorescence (CVAFS) analyzer where the
mercury atoms are excited by light of wavelength 253.7 nm. These excited atoms fluoresce in the
same wavelength which is detected by a photomultiplier tube and the signal is converted to voltage
which is sent to an integrator.
Gold-coated bead traps are >99% efficient at collecting mercury in its various forms in the
atmosphere. In sampling locations away from local sources the predominant form of mercury in
the atmosphere is elemental mercury, however, in source regions, there may be an important
fraction of other mercury species present. The temperature used to desorb mercury from the traps
determines whether or not a mercury compound (other than elemental mercury) released from the
gold is in elemental form or a molecular form. Typically, when gold-coated bead traps are
employed, a desorption temperature of 500° C or less is used since the melting point of borosilicale
glass is typically about 650° C (rendering the trap unusable). At 500° C mercury compounds will
not quantitatively be converted into elemental mercury. Published values for temperatures
required to break methyl mercury bonds approach 1000° C. Therefore, in environments in which
there may be mercury species other than elemental mercury, in the atmosphere, it is incorrect to
term this CVAFS procedure an anaKsis of "total gaseous phase mcrcun" At UMAQL a
desorption temperature of 500° C is employed and results of this anaKsis are referred to simply as
"\apor phase mercury"
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3.2 Sample Analysis and Data Acquisition
The CVAFS analyzer used for vapor phase mercury analysis is operated in a normal laboratory
setting (outside of the clean room) because the risk of contamination of the samples is minimal.
The instrument is keep on at all times, since this has been shown to stabilize the UV lamp and
maintain consistency from one day to the next. The power supplied to the CVAFS analyzer is
modulated by a line tamer (Shape Magnetronics) to prevent power fluctuations. It is imperative
that the mercury lamp not experience wide temperature fluctuations or power surges since both of
these drastically affect the sensitivity of the instrument. During operation of the instrument the
helium carrier gas flow rate is regulated upstream of the analyzer using a mass flow controller
(Tylan) which is set to maintain a 35 cc/min flow rate. This flow rate has been determined by
UMAQL to yield the optimal peak characteristics for mercury standards. The regulator on the
helium cylinder is set at 50 Kilopascals. The helium stream is prefiltered using a gold-coated trap
before entering the analytical train in order to remove any mercury. In the analytical train, mercury
is thermally desorbed from the sample trap, and amalgamated onto the analytical trap which is
subsequently thermally desorbed into the CVAFS analyzer where the mercury atoms are detected.
Traps are desorbed by heating a nichrome coil which is wrapped around the trap covering the
gold-coated beads. Application of 12 volts of current to the coil is sufficient to achieve a
temperature of 500° C inside the gold bead trap (voltage may vary due to variations in length and
thickness of nichrome wire). Two fans supply cool air to the sample and analytical trap separately
in order to speed analysis time.
For comparability between laboratories UMAQL sets the gain on the CVAFS analyzer to yield an
approximate 1000 mV net response for a 1 ng mercury standard. The background on the CVAFS
analyzer is set at 5.0 and maintained in that position in order to track the drift in the baseline of the
analyzer. A Hewlett Packard Integrator is connected to the analyzer to convert output signal into
an integrated area of the detected response. Area units are used for all sample calculations since
area is much more reliable than peak height.
Particle-free clean room gloves are worn during all procedures. To analyze a sample trap, the trap
is placed snugly into the analytical train using friction fit Teflon connectors and Teflon sleeves.
The nichrome coil used specifically for the sample trap is slid over the trap and moved to
completely cover the quartz wool plugs and the gold-coated beads contained between the plugs.
Helium is allowed to flow through the sample trap for 2 minutes before analysis begins in order to
purge air and water vapor from the analytical train. A circuit controller (ChonTrol) is employed
which is programmed to turn on the variable transformers and fans in a precise and reproducible
manner. First, the sample coil is heated for 2 minutes, then it is cooled while the analytical trap is
heated for 2 minutes. The analytical trap is then cooled for 2.5 minutes and the fan to the sample
trap is turned off. While the analytical trap is cooling, a new sample trap is installed in the
analytical train and helium is passed through this trap until the analytical trap is cool and ready for
another sample. When the analytical trap begins heating, the integrator is turned on and the
ambient temperature, time and base mV are recorded in a log book and the LCD display on the
analyzer is set to record the peak mV (by depressing the Peak button on the face of the analyzer).
After the sample is analyzed and the peak height and area reported b\ the CVAFS and integrator
respectively, these values are recorded in the log book.
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A standard curve is analyzed at the beginning of each day of analysis and a control standard which
yields a response in the range of the samples being analyzed is run every six samples. Criteria for
the standard curves and control standards are described below in Section 3.3. All sample analysis
is recorded in a log book specific to the analyzer with which samples are being quantified and also
in a lab notebook specific to the study for which the samples were collected. At the end of the day
of analysis all results from the log sheet are entered into an Excel spreadsheet for subsequent
checking and processing by a statistical software program, SAS (Gary, NC).
3.3 Standard Curve and Control Standards
A standard curve, generated by injecting different volumes of mercury saturated air, is analyzed
before each day of analysis. The amount of mercury per volume of mercury saturated air depends
on the temperature of the mercury saturated air. This relationship is described by the Ideal Gas
Law. The amount of mercury injected for the calibration curve is tailored to the expected value of
the samples to be analyzed. At UMAQL, a typical calibration curve consists of five injection
volumes: 0 mL, 20 mL, 40 mL, 80 mL and 160 mL of mercury saturated air, which represent
mercury masses shown in Table 1.
Standards for the calibration curve are generated by withdrawing known volumes of gaseous
elemental mercury from the headspace of a closed 150 mL flask containing about 2-3 mL of
metallic mercury. The flask is maintained below ambient temperature by being immersed in a
constant temperature (+\- 0.1° C) circulating water bath. The temperature of the circulating water
bath and thus the flask of mercury saturated air is usually maintained at a temperature of 16.6° C
while ambient temperature is about 22-24° C. The temperature of the flask must be maintained
below ambient temperature otherwise, the mercury injected will condense on the walls of the
injection apparatus. Also, the flask is purged periodically with N: in order to displace oxygen
which may oxidize the surface of the mercury. The mercury is withdrawn from the flask using a
gas tight syringe (Hamilton) and is injected onto and quantitatively captured by a gold coated bead
trap. The trap is then thermally desorbed and analyzed using the CVAFS analyzer.
At the start of each day of analysis, the syringe is "conditioned" for 15 minutes. The syringe is
conditioned by flushing it three times with mercury saturated air, then the maximum amount of
mercury saturated air is pulled into the syringe and the needle is left inside the flask for 15
minutes. While the syringe is conditioning, the injection port is inserted into the analytical train
and is connected to a gold coated bead test trap using friction fit Teflon connectors. The injection
port is fitted with a Minnert valve and Teflon coated silicon septum. After 15 minutes, the syringe
is flushed with mercury saturated air three times and 80 mL is withdrawn and injected onto the test
trap to "condition" the injection port. The injection port is conditioned for a total of three times
before the syringe and injection port are ready to be used for a calibration curve. This
conditioning process is necessary before each day of analysis to ensure precise and reproducible
results.
The standard curve is generated starting with the zero point (0 mL) and continued in ascending
order to the highest volume, usually 160 mL. First, a blanked gold-coated bead trap is attached to
the downstream end of the injection port. Prior to withdrawing the necessary volume of mercury
saturated air for each injection, the temperature of the flask is recorded and the syringe is flushed
three times with mercury saturated air After flushing the s\nnge. the required \olume of mercur\
saturated air is withdrawn. The s\rmge is then removed from the flask and the mercury saturated
air is injected onto the gold trap \ia the ui|cction port as quickh as possible to avoid diffusion ol
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SOP for Analysis of
Vapor Phase Mercury Volume 2, Chapter 2
mercury from the needle tip. After the injection is complete, the syringe is returned to the flask
and the maximum amount of mercury saturated air is pulled into the syringe and allowed to sit
until the next injection. After the trap is loaded with a standard amount of mercury, the injection
port is removed from the analytical train and the trap is analyzed as described above. The injection
port is kept sealed in a polyethylene bag between injections.
The zero point of the calibration curve (0 mL) is generated in the same manner described above
except that no mercury saturated air is withdrawn into the syringe. Instead, the syringe is flushed
three times and fully depressed prior to removing the syringe from the flask and the needle is
promptly inserted and removed from the injection port. The zero point indicates the amount of
mercury that comes from the needle tip and the injection apparatus and is usually between 1-6 pg
The 0 mL injection is also a good indication of when the septum in the injection port needs to be
changed. If the 0 mL injection is higher than 10 pg then the septum is changed. Otherwise, the
septum is changed after loading 30 traps for standards or controls.
At any given temperature the vapor density of mercury can be calculated using the Ideal Gas Law
and the saturation vapor pressure of mercury. A table of associated vapor densities versus
temperature has been compiled and is used to determine the amount of mercury injected for each
standard. Table 1 lists the amount of mercury delivered for the 5 injection volumes used to
generate the standard curve at a temperature of 16.6° C.
Table 1. Amount of Mercury Injected for a Typical Calibration Curve
Volume of Mercury Saturated Amount of Mercury
Air Injected (mL) Injected (ng)
0 0
20 0.198
40 0.396
80 0.793
120 1.190
Flask temperature = I6.6°C
Vapor density = 9.912 ng/cnr
/ cm' = 1000 mL
After the standards for the calibration curve have been analyzed, a linear regression is calculated to
establish the coefficient of determination (r), the slope of the line and how well the slope of the
curve predicts each of the points in the calibration curve. The slope of the line is forced through
zero and the 0 mL injection area is subtracted from each of the points on the curve. The r must be
>0.999 and each of the points on the curve must be predicted by the slope within 57c of their true
value (Table 2). If these criteria are not met, specific points which are errant are repeated and the
linear regression recalculated.
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SOP for Analysis of
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Table 2. Example Calibration Curve and Calculation of Slope:
Standard Response Response - Predicted Value
(ngHg) Area Units Zero Point (ng Hg)
0 71,219 0 0
0.198 2,286,400 2,215,181 0.197
0.396 4,495,200 4,423,981 0.393
0.793 9,089,300 8,946,862 0.794
1.190 13,597,000 13,383,343 1.190
Slope = 8.S886E-8 ng/AU
Slope1 = 11,250,357 AU/ng
r = 0.9999
This curve is accepted and sample analysis commences.
Control standards are analyzed every sixth sample. The control standards are generated in the
same manner as described above and are chosen to be representative of the samples being
analyzed, usually an 80 mL injection. The integrated area from each of the control standards must
be within 57c of the slope of the calibration curve in order to continue analyzing. If this is not the
case, a second control is analyzed immediately. If the second control indicates that analyzer
sensitivity has changed a second calibration curve is generated and sample analysis is continued.
The mercury flask is checked weekly to determine if the surface of the mercury is oxidized. If
there is visible discoloration on the surface of the mercury then the" flask is purged for 15 minutes
with N2 to reduce the oxidized layer. Also, to determine if water has entered the flask the mercury
is gently swirled around the bottom of the flask. If the mercury moves around the flask without
adhesion to the glass then there is no water in the flask.
3.4 Calculation of Mercury Concentration in Sample
The vapor phase mercury concentration from a set of gold-coated bead traps is calculated in ng/nr
First, the amount of mercury detected on each of the sample traps (A & B) is calculated by
multiplying the integrator response from the individual trap by the slope of the calibration curve
which is in ng/Area Unit (AU). The product is ng of mercury (Table 3). Each trap is field blank
corrected and the results are summed to yield the total mercury mass collected. The calculated
value, in nanograms of mercury is converted to ng Hg/W by calculating the total volume of air
drawn through the sample and dividing the ng of mercury by the cubic meters of air sampled.
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Table 3. Calculation of ng Hg/mJ in a Vapor Phase Sample.
1. Calculation of ng of mercury recovered from a set of sample traps
ng Hg on the Sample Trap (A) = Response (AU) x Slope of the calibration curve (ng/AU)
7,735,900 X 8.8886E -8 = 0.634 ng Hg
ng Hg on the Sample Trap (B) = Response (AU) x Slope of the calibration curve (ng/AU)
287,560 X 8.8886E -8 = 0.025 ng Hg
2. Field blank correction and summation of traps
Corrected ng Hg on Sample Traps = (ng on trap A + ng on trap B) - 2(mean ng of field blanks)
(0.634+ 0.025)-2(0.016) = 0.627
3. Calculation of nr1 sampled at a flow rate of 300 cc/min and a sample duration of 24 hours:
Flow Rate x Duration of Sample = Volume of Air Sampled
300ml 1,440 min m3 ,
x 24 hrx x = 0.432 w3
min 24 hr 1,000,000 ml
4. Concentration of Vapor Phase Mercury in Sample = ng Hg/m3
0.627 ng Hg ,
~- = 1.451 ngHg/m3
0.432m3
3.5 Trouble-Shooting
A source of irreproducible results may be due to faulty gold-coated bead traps. These traps are
numbered with discrete identifiers. All samples must be identified with a particular trap number
and trap performance must be tracked. Contact with halogen fumes, organic fumes or overheating
of the trap during analysis can damage the trap, rendering it unusable. If performance of a gold
trap is suspect, at least two consecutive standards are analyzed from this trap to determine its
ability to amalgamate and release mercury.
Another source of irreproducible results could be from water or water \apor seeping into the flask
containing mercury. If this is the case, the mercury in the flask is properly discarded and the flask
is rinsed with 0.56M HNO, and allowed to thoroughly dry. After dr\ing, the flask is replenished
uith about 2-3 mL of triple distilled metallic mercury and purged with clean N, for 5 minutes.
The flask is then be returned to the constant temperature water bath and allowed to thermally
euuihh'ate and to reach saturation for at least 3 hours.
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Volume 2, Chapter 2 yapor phase Mercury
It a low response is observed, the trap loading apparatus is checked for leaks, especially the
plunger tip in the syringe or the where the needle contacts the plunger tip. If the plunger tip is
worn, the syringe will no longer be gas tight and needs to be carefully replaced without damaging
the plunger tip. If there is any material (Teflon shavings, etc.) in the base of the needle where it
contacts the plunger tip, the material is wiped away with a particle-free clean wipe. Also, the side
port hole in the needle tip is checked to make sure that it is not clogged with any foreign material
(Teflon from the septum, etc.).
If peak-broadening is observed or no peak is detected in a sample, the analytical train is checked
for leaks. Peak broadening is often the result of low gas flow, water vapor on the gold-coated bead
trap, inadequate heating of analytical trap or an analytical trap damaged by exposure to halogen
fumes or overheating. Analytical traps are replaced only when a potential problem is suspected
with the trap.
If a broad peak is observed directly after the sharp mercury peak then the problem is most likely a
thermally or chemically damaged trap and the line from the analytical trap to the detector is
replaced as gold atoms may have been liberated from the trap and migrated down stream.
If the baseline drifts more than 10% the UV lamp is replaced. After replacement, the analyzer is
allowed to equilibrate for 24 hours. If the problem persists, sources of power fluctuation, drafts or
air currents that may be changing the temperature of the UV lamp are investigated.
Room temperature in which the CVAFS is located is maintained between 20-22 C. however, if the
temperature exceeds 26° C analysis is stopped, since instrumental noise increases significantly.
4.0 Performance Criteria, Quality Assurance and Quality Control
4.1 Field operators are carefully instructed in the techniques of contaminant-free vapor phase mercury
collection. All of the operators are currently operating sampling equipment for either the National
Dry Deposition Network, the National Atmospheric Deposition Program, the Integrated
Atmospheric Deposition Network or the Great Lakes Acid Deposition Network.
4.2 Every 6 months UMAQL personnel will inspect the sampling sites to audit the sampling
equipment and make all necessary repairs or adjustments.
4.3 Co-located samples are collected from one sampling site during the study to quantify method
precision. Reported concentrations are based on the mean of the two co-located samples.
4.4 Precision and accuracy levels will be set and maintained for each type of analysis. A relative
precision for total mercury of less than 109r is maintained for samples with values at least 3
standard deviations greater than the detection limit. Analysis of standards and controls is within
5% of the stated value.
4.5 A minimum of 25% of all samples analyzed are field blanks or analytical blanks to ensure that
samples are collected in a contaminant-free manner.
4.6 H\cr\ ^ months maintenance on me C\ -\KS anukzer is conducted. iiKludiiiL1 icplacement ol the
I'V lamp, the Teflon tubing, and the detection cell.
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SOP for Analysis of
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4.7 Gold traps are checked prior to every sample with 0.8 ng of mercury in order to ensure that their
use during the previous sample collection has not diminished trap performance.
5.0 References
5.1 Bloom, N.S. and Fitzgerald, W.F (1988) Determination of Volatile Mercury Species at the
Picogram Level by Low-Temperature Gas Chromatography with Cold-Vapor Atomic Fluorescence
Detection. Anal. Chem. Acta. 208, 151.
5.2 Dumarey, R., Temmerman, E., Dams, R. and Hoste, J. (1985) The Accuracy of the Vapour-
Injection Calibration of Mercury by Amalgamation/Cold-Vapour Atomic Absorption
Spectrometry. Anal. Chem.. Acta. 170, 337-340.
5.3 Dumarey, R., Dams, R., and Hoste, J. (1985) Comparison of the collection and desorption
efficiency of activated charcoal, silver, and gold for the determination of vapor-phase atmospheric
mercury. Anal. Chem. 57, 2638-2643.
5.4 Fitzgerald, W.F., and Gill, G.A. (1979) Sub-Nanogram Determination of Mercury by Two-Stage
Gold Amalgamation and Gas Phase Detection Applied to Atmospheric Analysis. Anal. Chem. 15,
1714.
5.5 Lindberg, S.E. (1981) Author's Reply 'Mercury partitioning in a power plant plume and its
influence on atmospheric removal mechanisms.' Atmos. Environ. 15, 631-635.
5.6 Rossmann, R. and Barres, J. (1991) Trace element concentrations in near-surface waters of the
Great Lakes and methods of collection, storage, and analysis J. Great Lakes Res. \ 4,: 188.
5.7 Schroeder, W.H. (1982) Sampling and analysis of mercury and its compounds in the atmosphere.
Environ. Sci.. Technol. 16, 394-400.
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APPENDIX A: FACILITIES, EQUIPMENT AND REAGENTS
Following is a list of the required facilities, equipment, supplies and reagents for sample preparation,
sample collection and sample analysis that are outlined in this document. The make and model of the
following items are those used at The University of Michigan Air Quality Laboratory. Many of these items
are available from a variety of sources.
1. Preparation of Field Supplies
Class 100 Clean Room, Work Stations
Clean Room Gloves
Particle-free Wipes
Clean Room Cap, Gown and Boots
Milli-Q Water (18.2MQ/cm)
Exhaust Hood
Acetone
Alconox
Polyethylene Tubs
EM Science Tracepur and Suprapur Hydrochloric Acid
Polytherm Water Bath (Science/Electronics)
Baker Instra-Analyzed or EM Science Suprapur Nitric Acid
New Polyethylene Bags
20 Liter Polyethylene Carboys
2. Sample Collection
Mass Flow Controlled Vacuum Pump (URG, Model 3000-02M)
Calibrated 300 cc/min Rotameter (Matheson)
HOPE Tubing with quick connects
Black Latex Tubing
Mercury Sampling Box (UMAQL, See Appendix B)
Acid-Cleaned 47 mm Teflon Filter Holders (Savillex, PFA Labware)
47 mm Preheated Glass Fiber Filters (Gelman Sciences A/E)
Acid-Cleaned Teflon Jars (Savillex, PFA Labware)
Teflon-Coated Forceps
'Blanked' Gold-Bead Traps (UMAQL)
Teflon Endplugs
Trap Heater & Variable Transformer
Acid-Cleaned Teflon Tubing
Particle-Free Gloves
Teflon Tape
Sample Labels
Field Operator Log Book
Shipping Boxes
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3. Sample Analysis
Cold Vapor Atomic Florescence Detector (Brooks Rand, LTD.)
Line Tamer/Conditioner (Shape Magnetronics Model PCLT 150)
Integrator (Hewlett-Packard Model 3390A)
Helium, Ultra High Purity Grade (99.999%)
Mass Flow Controller (Tylan)
Nichrome Coils (UMAQL)
Electric Leads
Variable Transformers (Staco Energy Products Co. Type 3PN1010)
Cooling Fans
Gold-Coated Glass Bead Traps (UMAQL)
Gas Tight Syringe (Hamilton series 1800)
Injection Port (UMAQL)
Constant Temperature Circulating Water Bath (Fisher Model 901)
Instrument Grade Metallic Mercury (Triple Distilled)
Mercury Flask (UMAQL)
Certified Immersion Thermometer (Kessler Instruments, Inc. 15041 A)
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Standard Operating Procedure for
Analysis of Mercury in Precipitation
Gerald J. Keeler and Matthew S. Landis
University of Michigan
Air Quality Laboratory
109 South Observatory Street
Ann Arbor, Ml 48109-2029
June 1,1994
Version 2.0
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Standard Operating Procedure for
Analysis of Mercury in Precipitation
1.0 Introduction/Overview
The objective of the Lake Michigan Loading Study is to assess the contribution of atmospheric
deposition to the concentration of mercury and other toxic trace species found in Lake Michigan
The atmosphere has been implicated as one of the dominant sources of mercury and trace elements
to bodies of water and it is clear from investigations in remote regions of the globe that long range
transport of mercury and other toxics from source regions is occurring. By quantifying the wet
deposition and ambient concentrations of mercury it will be possible to determine the relative
importance of precipitation and dry deposition in accounting for the atmospheric loading of
mercury to Lake Michigan. In addition, investigating other ambient trace species will aid in the
identification of significant mercury sources.
2.0 Sample Handling and Processing
2.1 Sample Handling
Precipitation samples contain exceptionally low levels of trace metals and therefore these samples
are processed with careful attention to avoid contamination. Blanks are routinely analyzed to
ensure extremely low analyte levels and operating conditions.
When samples are received at UMAQL they are logged in, the tracking form sent with the sample
is catalogued and the sample is taken to a staging area to be moved into the clean room. At the
staging area outside the clean room, the outer polyethylene bag is removed from the sample. The
double-bagged sample is placed in the outer room of the clean room, and the analyst suits up to
enter the clean room according to laboratory protocol. As the sample is brought into the clean
room the second outer bag is removed and left in the outer room of the clean room. The inner bag
is removed inside the clean room and discarded.
2.2 Sample Processing
The precipitation volume of each sample is determined gravimetncally. Mercury samples are
oxidized to 17c bromine monochloride volume/volume and are stored in a dark cold room
overnight before being analyzed according to the following procedure.
3.0 Sample Analysis and Data Acquisition
3.1 Summary
Mercury is purged from solution in a mercury-free nitrogen stream after appropriate sample
pretreatment and reduction. Mercury liberated from solution IN concentrated on a gold-coated lx\ul
trap. A mercury-free pretreated soda lime trap is utili/ed in the purge system to capture acid gases
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter^
that may damage the gold-coated bead trap. The gold-coated bead trap is subsequently thermally
desorbed and carried by a mercury-free helium stream into a cold-vapor atomic fluorescence
(CVAFS) analyzer where the mercury atoms are excited by light of wavelength 253.7 nm. These
excited atoms fluoresce in the same wavelength which is detected by a photomultiplier tube and
the signal is converted to voltage which is sent to an integrator.
All analytical procedures for determination of mercury in precipitation are carried inside a Class
100 Clean Room. Volatilization/Recapture (Section 3.3.1) is carried out in a class 100 laminar
flow exhaust hood inside the clean room. Nitrogen utilized for purging is 99.998% pure and is
stripped of any mercury using a gold coated trap before use in the purge system. Clean room
gloves are worn at all times and all labware with which the samples and reagents comes into
contact is cleaned weekly using the acid cleaning procedure described in Standard Operating
Procedure for Sampling of 'Mercury in Precipitation, Section 2.1.
3.2 Reagents and Materials
All reagent lot numbers, preparation dates and procedures are recorded for each new batch of
reagent used. A reagent blank is obtained after each new batch of reagent has been prepared.
Bromine monochloride (BrCl), stannous chloride (SnCK) and hydroxylamine hydrochloride
(NH:OH'HC1) are prepared fresh monthly.
Solid reagents (potassium bromide, potassium bromate, hydroxylamine hydrochloride and
stannous chloride) are stored in the clean room in a desiccator containing silica gel and an open
bed of activated charcoal. The caps of all reagent bottles are Teflon taped to reduce entry of vapor
phase compounds. Even with these precautions, reagents will nevertheless absorb mercury over
time and must be replaced. All reagents are made in the clean room, except the working standard
solution.
3.2.1 Hydrochloric Acid
EM Science Suprapur hydrochloric acid is used to prepare BrCl and SnCL This acid
characteristically has a very low blank value (20 pg/mL).
3.2.2 Bromine Monochloride
Bromine monochloride is prepared in a class 100 laminar flow exhaust hood by adding
11.0 mg KBr per mL of HC1 while the solution is stirred using an acid-cleaned Teflon-
coated magnestir. When the KBr is dissolved, 15.0 mg KBrO-, per mL of HC1 is added
slowly and the solution is allowed to continue stirring. This process produces chlorine and
bromine gas and must be performed slowly in a functioning fume hood. After addition of
the salts the solution is a deep yellow color. If there is no color (or very faint) then the
BrCl has been substantially reduced and will not have enough oxidizing power for use In
this case, the solution is remade. Bromine monochloride is stored at room temperature in
the clean room. Fresh bromine monochloride is be prepared monthly or as needed.
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Volume 2, Chapter 2 SOP for Analysis of Mercury in Precipitation
3.2.3 Hydroxylamine Hydrochloride
30 grams of XFLOH.HCl is dissolved in MQ-uater to make 100 mL in an acid-cleaned
100 mL volumetric flask. This solution is purified by adding 0.5 mL of SnCK and purging
overnight with Hg-free N:. The solution is stored in an acid-cleaned, dark Teflon bottle in
the refrigerator. Fresh hydroxylamine solution is prepared every month or as needed.
3.2.4 Stannous Chloride
20.0 grams of SnCl:.H:O is placed into an acid-cleaned 100 mL volumetric flask.
Working in a fume hood, 10 mL of concentrated HC1 is added and the solution is then
brought to 100 mL with Milli-Q water. The solution is stored in an acid-cleaned, dark
Teflon bottle in the refrigerator. Fresh stannous chloride is prepared every month or as
needed.
3.2.5 Milli-Q Water
Deionized water, with a resistivity of 18.2 MQ/cm, is prepared using a Milli-Q system
from a pre-purified (reverse osmosis) water source. Milli-Q water is used for reagent
preparation.
3.2.6 Soda Lime Traps
High purity grade soda lime (EM Science) is utilized in an acid-cleaned glass tube with
glass wool endplugs and Teflon connectors. After packing, this trap is conditioned by
purging a 0.5 M HC1 solution through the trap for 30 minutes. The soda lime trap is
changed after analysis of 30 samples.
3.2.7 Preparation of Working Standard Solution
100 uL of the stock Hg solution (1 mg/mL in nitric acid) is pipetted into a 1 L volumetric
flask. 5 mL of concentrated BrCl is added and the flask is brought up to volume with
MQ-water and thoroughly mixed. This is the Secondary Standard solution (100 ng
Hg/mL). Replace this solution as needed (it is stable for at least one year).
The Working Standard (2 ng Hg/mL) is prepared from the Secondary Standard solution by
placing 2 mL of Secondary Standard into a 100 mL volumetric flask, adding 1 mL of BrCl
and bringing the solution to volume with MQ. The Working Standard is replaced
monthly.
3.3 Sample Analysis and Data Acquisition
3.3.1 Volatilization/Recapture
Volatilization of mercury from solution is accomplished using a glass impinger assembly
manufactured at the University of Michigan. A 100 mL graduated bubbler attaches to an
impinger via a ground glass fitting. N, flow is regulated using a Teflon stopcock. A sod;i
lime trap is incorporated into the system to pre\ent damage ot the gold-coated bead traps
by capturing acid gases liberated during the purging procedure.
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter^
Total mercury is quantified by oxidizing all mercury forms using bromine monochloride.
Bromine monochloride is a strong oxidizing agent, capable of breaking organic bonds
with mercury, thus liberating the divalent form of mercury. Approximately 10 mL of the
oxidized precipitation sample to be analyzed is poured into an acid-cleaned or previously
purged glass bubbler. The bubbler is rinsed by gently swirling the solution. After rinsing,
the solution is discarded. 100 mL of the oxidized precipitation sample is carefully poured
into the graduated glass bubbler and 250 |aL of hydroxylamine hydrochloride is added. A
stopper is then inserted into the bubbler, its swirled briefly and allowed to react for 5
minutes to reduce the excess bromine monochloride from solution. Bromine
monochloride is reduced from solution since halogens liberated from solution will quick!)
damage the gold-coated bead traps onto which the purged elemental mercury is
amalgamated.
A blanked gold-coated bead sample trap is affixed to the end of the soda lime trap. The
bubbler is opened, 500 uL of stannous chloride is added, and the bubbler is quickly
attached to the impinger. The N2 flow is adjusted to 450 cc/min using a calibrated
rotameter and the solution is purged for 7 minutes. The stannous chloride reduces the
divalent mercury to Hg° which is quantitatively captured on the goad-coated bead trap.
3.3.2 Analysis of Total Mercury
The CVAFS analyzer used for mercury in precipitation determinations is kept on at all
times, since this has been shown to stabilize the UV lamp and maintain consistency from
one day to the next. The power supplied to the CVAFS analyzer is modulated by a line
tamer (Shape Magnetronics) to prevent power fluctuations. It is imperative that the
mercury lamp not experience wide temperature fluctuations or power surges since both of
these drastically affect the sensitivity of the instrument. During operation of the
instrument the helium carrier gas flow rate is regulated upstream of the analyzer using a
mass flow controller (Tylan) which is set to maintain a 35 cc/min flow rate. This flow rate
has been determined by UMAQL to yield the optimal peak characteristics for mercury
standards. The regulator on the helium cylinder is set at 50 Kilopascals. The helium
stream is prefiltered using a gold-coated trap before entering the analytical train in order to
remove any mercury. In the analytical train, mercury is thermally desorbed from the
sample trap, and amalgamated onto the analytical trap which is subsequently thermally
desorbed into the CVAFS analyzer where the mercury atoms are detected. Traps are
desorbed by heating a nichrome coil which is wrapped around the trap covering the gold-
coated beads. Application of 12 volts of current to the coil is sufficient to achieve a
temperature of 500 :C inside the gold bead trap (voltage may vary due to variations in
length and thickness of nichrome wire). Two fans supply cool air to the sample and
analytical trap separately in order to speed analysis time.
For comparability between laboratories UMAQL sets the gam on the CVAFS analyzer to
yield an approximate 1000 mV net response for a 1 ng mercury standard. The background
on the CVAFS analyzer is set at 5.0 and maintained in that position in order to track the
drift in the baseline of the analyzer. A Hewlett Packard Integrator is connected to the
analyzer to convert output signal into an integrated area of the detected response. Area
units (AL") are used for all sample calculations since area is much more reliable than peak
height.
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Volume 2, Chapter 2 SOP for Analysis of Mercury in Precipitation
To analyze a sample trap, the trap is placed snugly into the analytical train using friction
fit Teflon connectors and Teflon sleeves. The nichrome coil used specifically for the
sample trap is slid over the trap and moved to completely cover the quartz wool plugs and
the gold-coated beads contained between the plugs. Helium is allowed to flow through the
sample trap for 2 minutes before analysis begins in order to purge air and water vapor
from the analytical train. A circuit controller (ChonTrol) is employed which is
programmed to turn on the variable transformers and fans in a precise and reproducible
manner. First, the sample coil is heated for 2 minutes, then it is cooled while the
analytical trap is heated for 2 minutes. The analytical trap is then cooled for 2.5 minutes
and the fan to the sample trap is turned off. While the analytical trap is cooling, a new
sample trap is installed in the analytical train and helium is passed through this trap until
the analytical trap is cool and ready for another sample. When the analytical trap begins
heating, the integrator is turned on and the ambient temperature, time and base mV are
recorded in a log book and the LCD display on the analyzer is set to record the peak mV
(by depressing the Peak button on the face of the analyzer). After the sample is analyzed
and the peak height and area reported by the CVAFS and integrator respectively, these
values are recorded in the log book.
A standard curve is analyzed at the beginning of each day of analysis and a control
standard which yields a response in the range of the samples being analyzed is run every
six samples. Criteria for the standard curves and control standards are described below in
Section 3.3.4. All sample analysis is recorded in a log book specific to the analyzer with
which samples are being quantified and also in a lab notebook specific to the study for
which the samples were collected. At the end of the day of analysis all results from the
log'Sheet are entered into a computer spreadsheet file for subsequent checking and
processing by a statistical software program, SAS (Gary, NC).
3.3.3 System Purge and Blanks
At the start of each day of analysis, each impinger system is purged after the soda lime
trap is conditioned. First 25 mL of Milli-Q water is added to an acid cleaned bubbler, then
1.0 mL of SnCU is added and the solution is purged at 450 cc/min for 15 minutes. After
each system is purged a System Blank is generated to ensure the impinger assembly is free
of contamination.
System Blank (Bubbler Blank): This blank is generated by adding 1.0 mL of SnCK to the
system purge solution and purging the solution onto a blanked gold-coated bead trap at
450 cc/min for 5 minutes. After the System Blanks have been completed, one of the
purged bubblers is dedicated for generation of standards.
A Total Reagent Blank is generated on each day of analysis for each analytical aliquot
volume. The Total Reagent Blank is used to calculate the method detection limit
(presently 0.1 ng/L for a 100 mL aliquot) and to calculate sample concentration (Section
3.3.5).
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter 2
Total Reagent Blank: A Total Reagent Blank is generated in sample solution that has
been purged of mercury. Bromine monochloride is pipetted into the solution, allowing
reduction of halogens by hydroxylamme for 5 minutes then mercury is purged from the
solution after adding SnCl2. Purged mercury is captured on a gold-coated trap and
analyzed as described above in Section 3.3.2. For a 100 mL aliquot the following reagent
volumes are used: 1.0 mL of BrCl, 0.25 mL NH:OH-HCI, and 0.5 mL of SnCl:.
3.3.4 Standard Curve and Control Standards
A standard curve, generated by bubbling different volumes of mercury working standard
solution, is analyzed before each day of analysis. The amount of mercury bubbled for the
calibration curve is tailored to the expected value of the samples to be analyzed. At
UMAQL, a typical calibration curve consists of five standards: 0 ng, 0.1 ng, 0.2 ng, 0.5 ng
and 1.0 ng. The volumes of working standard solution required to achieve the five
standard concentrations are shown in Table 1.
Table 1. Volumes of Working Standard Required to Generate Aqueous Standards
Hg Delivered to Trap Volume of Hg Standard
0 ng 0 mL
0.1 ng 0.05 mL
0.2 ng O.IOmL
0.5 ng 0.25 mL
1.0 ng 0.50 mL
Standards for the calibration curve are generated starting with the zero point (0 mL of
working standard solution) and continued in ascending order to the highest volume,
usually 0.50 mL of working standard solution. First, a blanked gold-coated bead trap is
attached to the end of the soda lime trap. Then 1.0 mL of SnCI: is added to the standard
bubbler followed by the appropriate volume of working standard solution. The standard
bubbler is quickly attached to the impinger and the solution is purged at 450 cc/min for 5
minutes. The gold-coated bead trap is analyzed immediately after purging.
After each of the standards for the calibration curve has been analyzed, a linear regression
is calculated to establish the coefficient of determination (r), the slope of the line and how
well the slope of the curve predicts each of the points in the calibration curve. The 0 mL
standard area is subtracted from each of the other points which are then regressed against
the expected values using no intercept (line is forced through zero). The r must be
>0.995 and each of the points on the curve must be predicted by the slope within 10rr of
their true value (Table 2). If these criteria are not met. specific points which are errant are
repeated and the linear regression recalculated.
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Volume 2, Chapter 2 SOP for Analysis of Mercury in Precipitation
Table 2. Example Calibration Curve and Calculation of Slope
Standard Response Response (AD)- Predicted
(ng Hg) (AD) Zero Point (AU) Value (ng Hg)
0 56,714 0 0
0.100 1,364,600 1,307,886 0.103
0.200 2,571,000 2,514,286 0.199
0.500 6,357,800 6,301,086 0.498
1.000 12,709,000 12,652,286 1.001
Slope = 7.909E-8 ng/AU
Slope'1 = 12,643,823 AU/ng
r = 0.9999
This curve is accepted and sample analysis commences.
Control standards are analyzed every sixth sample. The control standards are generated in
the same manner as described above and are chosen to be representative of the samples
being analyzed. The integrated area from each of the control standards must be within
10% of the slope of the calibration curve in order to continue analyzing. If this is not the
case, a second control is analyzed immediately. If the second control indicates that
analyzer sensitivity has changed a second calibration curve is generated and sample
analysis is continued.
3.3.5 Calculation of Mercury Concentration
Mercury concentration in precipitation is calculated in ng/L. The total reagent blank
response is subtracted from the analytical aliquot response and the difference is multiplied
by the slope of the calibration curve which is in ng/AU. The mass of Hg for the entire
sample is then calculated by dividing the analytical aliquot Hg mass by the analytical
aliquot volume and multiplying the result by the total volume of sample. Once the ng of
mercury in the entire sample is determined, the concentration is then adjusted for the
(i) Teflon sample bottle blank, (ii) HC1 preservative blank, (iii) HC1 preservative dilution
factor and finally converted to ng/L for reporting purposes (Table 3). The Hg
concentration is calculated using Eqn. (1).
(1) Hgpcp= {(CAU*Slope*TV/AV) (AB+BB)}*(1000/PV)
where: Hgpcp = Hg concentration in precipitation (ng/L)
CAU - Total reagent blank corrected sample area units
Slope = Slope of the five point calibration curve (forced through zero)
TV - Total volume of sample (precipitation volume + HCl volume)
AV = Volume of analytical aliquot
AB = HCl .icid preservative blank
BB = Mean Teflon sample bottle check blank
PV = \ i>Uimc ni' precipitation collected
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter^
Table 3. Calculation of ng Hg/L in a Precipitation Sample.
1. Calculation of ng of mercury recovered from the analytical aliquot
ng Hg = (sample response (AU)-total reagent blank (AU)) * slope of calibration curve (ng/AU)
(8,657,600 - 74,543) ' 7.909E-8 = 0.679 ng Hg
2. Calculation of ng of mercury for total sample volume
ng Hg = (ng Hg from analytical aliquot" total volume of sample)/ volume of analytical aliquot
(0.679 ng Hg ' 439 mL) /100mL= 2.980 ng Hg
3. HCI acid preservative blank and Bottle blank correction
ng Hg = ng Hg for total sample volume - (HCI acid blank + Bottle blank)
2.980 - (0.004 + 0.020) = 2.956 ng Hg
4. Conversion to ng Hg/L
ng Hg/L = ng Hg for total sample volume * (1000 /precipitation volume collected)
2.956 ' (1000/419) = 7.1 ng Hg/L
3.3.6 Trouble-Shooting
• A source of irreproducible results may be due to faulty gold-coated bead traps.
These traps are numbered with discrete identifiers. Contact with halogen fumes,
organic fumes or overheating of the trap during analysis can damage the trap,
rendering it unusable. If performance of a gold trap is suspect, at least two
consecutive standards are analyzed from this trap to determine its ability to
amalgamate and release mercury.
• If a low response is observed, the impinger assembly is checked for leaks. Teflon
compression fittings on the soda lime trap and the Teflon nut on the Teflon
stopcock are the most common location of leaks.
• If peak-broadening is observed or no peak is detected in a sample, the analytical
train is checked for leaks. Peak broadening is often the result of low gas flow.
water vapor on the gold-coated bead trap, inadequate heating of analytical trap or
an analytical trap damaged by exposure to halogen fumes or overheating.
Analytical traps are replaced only when a potential problem is suspected with the
trap.
If the baseline drifts more than 10% the UV lamp is replaced. After replacement.
the analyzer is alkmed to equilibrate for 24 hours. If the problem persists, sources
ot po\\er tluciuatmn sources, drafts or air currents that ma\ be changing the
temperature of the IV lamp are investigated.
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Volume 2, Chapter 2 SOP for Analysis of Mercury in Precipitation
• Room temperature in which the CVAFS is located is maintained between 20-
22°C, however, if the temperature exceeds 26°C analysis is stopped, since
instrumental noise increases significantly.
4.0 Performance Criteria, Quality Assurance and Quality Control
4.1 Field operators are carefully instructed in the techniques of contaminant-free precipitation sample
collection for mercury determination. All of the operators are currently operating sampling
equipment for either the National Dry Deposition Network, the National Atmospheric Deposition
Program, the Integrated Atmospheric Deposition Network or the Great Lakes Acid Deposition
Network and are experienced with other precipitation collectors.
4.2 Every 6 months UMAQL personnel inspect each of the sampling sites to audit the performance of
the equipment and to make all necessary repairs or adjustments.
4.3 Co-located samples are collected from three sampling sites during the study to quantify method
precision. Reported concentrations for co-located samples are based on the mean of the two
samples.
4.4 In order to confirm that the collection funnel assemblies are free of mercury, funnel blank samples
are collected throughout the study.
4.5 Precision and accuracy levels will be set and maintained for each type of analysis. A relative
precision for total mercury of less than 15% is maintained for samples with values at least 3
standard deviations greater than the detection limit. Analysis of standards and controls is within
1 0% of the stated value.
4.6 A minimum of 25% of all samples are analyzed in duplicate. Reported concentrations will be
based on the mean of the replicates. Analytical precision averages better than 7%.
4.8 Every 3 months maintenance on the CVAFS analyzer is conducted, including replacement of the
UV lamp, the Teflon tubing, and the detection cell.
4.9 The analytical trap is not changed unless it begins to demonstrate poor recovery or release of
amalgamated mercury.
4.10 Teflon sleeves heat-sealed onto the gold trap are replaced frequently to maintain a gas tight seal.
5.0 Clean Room Procedures
5.1 Entering the Clean Room
Shoes are taken off outside the clean room by all UMAQL personnel. Personnel then enter the
outer \estibule (changing room). Once inside, the hood is put on followed by the clean room suit
and clean room boots. The boots are snapped to the suit at the back of the leg (to hold up the
boots) and are buckled in the front. Personnel then step over a dividing bench where they put on
clean room gloves and snap the clean room suit at the wrist. Now fully clothed they enter the
clean room making sure to secureK close (he door behind.
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter 2
5.2 Taking Supplies into the Clean Room
All supplies to be taken into the clean room are double bagged in polyethylene. The supplies to be
taken into the clean room are placed in the outer dressing room. Upon entering the clean room, the
outer bag is removed and left in the entry room. All supplies that enter the clean room that have
not been bagged are rinsed with MQ-water and wiped off with particle-fee wipes.
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Volume 2, Chapter 2 SOP for Analysis of Mercury in Precipitation
Appendix A: Facilities, Equipment and Reagents
Following is a list of the required facilities, equipment, supplies and reagents for sample preparation,
sample collection and sample analysis that are outlined in this document. The make and model of the
following items are those used at The University of Michigan Air Quality Laboratory. Many of these items
are available from a variety of sources.
1. Preparation of Field Supplies
Class 100 Clean Room. Work Stations
Clean Room Gloves
Particle-free Wipes
Clean Room Cap, Gown and Boots
Milli-Q Water (18.2MQ/cm)
Exhaust Hood
Acetone
Alconox
Polyethylene Tubs
EM Science Tracepur and Suprapur Hydrochloric Acid
Polytherm Water Bath (Science/Electronics)
Baker Instra-Analyzed or EM Science Suprapur Nitric Acid
New Polyethylene Bags
20 Liter Polyethylene Carboys
2. Sample Collection
MIC-B Wet-Only Precipitation Collector (MIC)
UMAQL Modified Acrylic Insert
Digital Indoor/Outdoor Recording Thermometer
BSG Collection Funnels
Polypropylene Collection Funnels
Teflon & Polypropylene Precipitation Adapters
Glass P-trap Vapor Lock
1 Liter Teflon & Polypropylene Sample Bottles
Funnel Heat Tape Nests & Variable Transformer
Ceramic Space Heater
2 Gallon HOPE Bucket
Particle-Free Gloves
Teflon Tape
Sample Labels
Permanent Label Markers
Field Operator Log Book
Shipping Crates
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SOP for Analysis of Mercury in Precipitation Volume 2, Chapter 2
3. Sample Analysis
Cold Vapor Atomic Florescence Detector (Brooks Rand, LTD.)
Line Tamer/Conditioner (Shape Magnetronics Model PCLT 150)
Integrator (Hewlett-Packard Model 3390A)
Helium, Ultra High Punty Grade (99.999%)
Mass Flow Controller (Tylan)
Nichrome Coils (UMAQL)
Electric Leads
Variable Transformers (Staco Energy Products Co. Type 3PN1010)
Cooling Fans
Gold-Coated Glass Bead Traps (UMAQL)
Pre-purified (99.998%), Analyzed Nitrogen
Glass Impingers
Resin-coated Ring Stand
100 mL Graduated Glass Bubblers
12.7 cm lengths of 1.27 cm OD Glass Tubing (Soda Lime Trap)
Automatic Pipettes
Teflon Reagent Bottles (Clear and Opaque)
Teflon Reagent Vials
Resin-coated Wire Rack (Support Bubblers)
Refrigerator
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Standard Operating Procedure for
Analysis of Particulate Phase Mercury
Gerald J. Keeler and Matthew S. Landis
University of Michigan
Air Quality Laboratory
109 South Observatory Street
Ann Arbor, Ml 48109-2029
June 1,1994
Version 2.0
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Standard Operating Procedure for
Analysis of Particulate Phase Mercury
1.0 Introduction/Overview
The objective of the Lake Michigan Loading Study is to assess the contribution of atmospheric
deposition to the concentration of mercury and other toxic trace species found in Lake Michigan
The atmosphere has been implicated as one of the dominant sources of mercury and trace elements
to bodies of water and it is clear from investigations in remote regions of the globe that long range
transport of mercury and other toxics from source regions is occurring. By quantifying the wet
deposition and ambient concentrations of mercury it will be possible to determine the relative
importance of precipitation and dry deposition in accounting for the atmospheric loading of
mercury to Lake Michigan. In addition, investigating other ambient trace species will aid in the
identification of significant mercury sources.
Particle-phase mercury, Hg(p), generally represents a small but significant fraction of total
atmospheric mercury. Recent advances in analytical chemistry have made quantification of the
extremely low levels of Hg(p) possible, however, tremendous care must be exercised in all phases
of sample handling and analysis. This protocol describes analysis of 'acid-extractable' total
mercury from atmospheric paniculate samples.
2.0 Sample Analysis
2.1 Summary
The technique described by this protocol is designed for use with glass-fiber or quartz fiber filter
media. When used in conjunction with an open-faced filter pack, these media demonstrate a low
pressure drop and have a very low background level of Hg with proper pretreatment. Sample
filters are stored at -40°C before analysis to prevent volatilization of the collected Hg(p).
Particulate mercury is extracted into a 1.6 M nitric acid solution utilizing a microwave digestion
technique. The mercury forms are then oxidized with bromine monochloride, to Hg:+ Oxidized
mercury forms are subsequently reduced to HgO with stannous chloride (SnCU). In this volatile
form, the metal is purged from solution using an Hg-free nitrogen stream and collected on a gold-
coated bead trap. A mercury-free pretreated soda lime trap is utilized in the purge system to
capture acid gases that may damage the gold-coated bead trap. Quantification is accomplished
using a dual amalgamation technique followed by cold vapor atomic fluorescence spectroscopy
(CVAFS).
All analytical procedures for determination of particulate phase mercury are carried out in a class
100 laminar flow exhaust hood inside a Class 100 Clean Room. Nitrogen utilized for purging is
99.9989c pure and is stripped of any mercury using a gold coated trap before use in the purge
system. Clean room gloves are worn at all times and all labware with which the samples and
reagents comes into contact is cleaned weekly using the acid cleaning procedure described in
Standard Operating Procedure for Sampling of Paniculate Phase Mercury, Section 2.1.
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SOP for Analysis of Paniculate Phase Mercury Volume 2, Chapter^
2.2 Reagents and Materials
All reagent lot numbers, preparation dates and procedures are recorded for each new batch of
reagent used. A reagent blank is obtained after each new batch of reagent has been prepared.
Bromine monochloride (BrCl), stannous chloride (SnCl:) and hydroxylamine hydrochloride
(NH:OH»HC1) are prepared fresh monthly.
Solid reagents (potassium bromide, potassium bromate, hydroxylamine hydrochloride and
stannous chloride) are stored in the clean room in a desiccator containing silica gel and an open
bed of activated charcoal. The caps of all reagent bottles are Teflon taped to reduce entry of vapor
phase compounds. Even with these precautions, reagents will nevertheless absorb mercury over
time and must be replaced. All reagents are made in the clean room, except the working standard
solution.
2.2.1 Hydrochloric Acid
EM Science Suprapur hydrochloric acid is used to prepare BrCl and SnCl,. This acid
characteristically has a very low blank value (20 pg/mL).
2.2.2 Bromine Monochloride
Bromine monochloride is prepared in a class 100 laminar flow exhaust hood by adding
11.0 mg KBr per mL of HC1 while the solution is stirred using an acid-cleaned Teflon-
coated magnestir. When the KBr is dissolved, 15.0 mg KBrO, per mL of HC1 is added
slowly and the solution is allowed to continue stirring. This process produces chlorine and
bromine gas and must be performed slowly in a functioning exhaust hood. After addition
of the salts the solution is a deep yellow color. If there is no color (or very faint) then the
BrCl has been substantially reduced and will not have enough oxidizing power for use. In
this case, the solution is remade. Bromine monochloride is stored at room temperature in
the clean room. Fresh bromine monochloride is be prepared monthly or as needed.
2.2.3 Hydroxylamine Hydrochloride
30 grams of NH2OH-HC1 is dissolved in MQ-water to make 100 mL in an acid-cleaned
100 mL volumetric flask. This solution is purified by adding 0.5 mL of SnCU and purging
overnight with Hg-free N2. The solution is stored in an acid-cleaned, dark Teflon bottle in
the refrigerator. Fresh hydroxylamine solution is prepared every month or as needed.
2.2.4 Stannous Chloride
20.0 gm of SnCl:»H:O is placed into an acid-cleaned 100 mL volumetric flask. Working
in a fume hood, 10 mL of concentrated HCI is added and the solution is then brought to
100 mL with Milli-Q water. The solution is stored in an acid-cleaned, dark Teflon bottle
in the refrigerator. Fresh stannous chloride is prepared every month or as needed.
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Volume 2, Chapter 2 SOP for Analysis of Particulate Phase Mercury
2.2.5 Milli-Q Water
Deionized water, with a resistivity of 18.2 MQ/cm, is prepared using a Milli-Q system
from a pre-purified (reverse osmosis) water source. Milli-Q water is used for reagent
preparation.
2.2.6 Soda Lime Traps
High purity grade soda lime (EM Science) is utilized in an acid-cleaned glass tube with
glass wool endplugs and Teflon connectors. After packing, this trap is conditioned by
purging a 0.5 M HC1 solution through the trap for 30 minutes. The soda lime trap is
changed after analysis of 30 samples.
2.2.7 Preparation of Working Standard Solution
100 |aL of the stock Hg solution (1 mg/mL in nitric acid) is pipetted into a 1 L volumetric
flask. 5 mL of concentrated BrCl is added and the flask is brought up to volume with
MQ-water and thoroughly mixed. This is the Secondary Standard solution (100 ng
Hg/mL). Replace this solution as needed (it is stable for at least one year).
The Working Standard (2 ng Hg/mL) is prepared from the Secondary Standard solution by
placing 2 mL of Secondary Standard into a 100 mL volumetric flask, adding 1 mL of BrCl
and bringing the solution to volume with MQ. The Working Standard is replaced
monthly.
2.2.8 Nitric Acid Extraction Solution.
The extraction solution is a 10% dilution of concentrated nitric acid (1.6M). A 1000 mL
volumetric flask is filled with about 800 mL of Milli-Q water. In a hood, 100 mL of
suprapur HNO, (EM Science) is measured using a graduated cylinder and poured into the
flask. The solution is mixed completely and allowed to cool in the hood with a glass
stopper closing the top. When cool, the flask is brought up to volume with Milli-Q water.
After the extraction solution is thoroughly mixed it is poured into an acid-cleaned re-
pipetting dispenser.
2.3 Sample Handling and Preparation
2.3.1 Sample Handling
If samples are not analyzed immediately, they are stored triple bagged in a dark freezer
(-40°C) to avoid exposure to laboratory air. Particle free gloves are worn whenever vials,
filters or dishes are handled or transferred.
2.3.2 Sample Preparation
Eighteen samples, five standards and one vessel check arc prepared for each day of
particulate mercur\ nnulvsiv Eighteen acid-cleaned Teflon \essel liners are inserted into
outer vessel bodies and arc placed in a laminar flou \\ork -.union. The vessel bodies arc
then labeled uith complete sample identifications. The pcin dishes containing the
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SOP for Analysis of Particulate Phase Mercury Volume 2, Chapter 2
samples to be analyzed are taken out of their polyethylene bags and the Teflon sealing tape
is removed. Each sample filter is carefully folded into quarters and placed in its
corresponding vessel using Teflon-coated forceps. The forceps are rinsed in a beaker of
HNO, extraction solution followed by a beaker of Milli-Q water and are dried using a
particle-free wipe before handling the next filter. After each filter has been placed in a
vessel it is capped and is moved into a laminar flow exhaust hood.
2.4 Sample Filter Extraction
The HNO, extraction solution is made on the same day that it is used and is dispensed using a
calibrated Repipet II dispenser. 20 mL of extraction solution is dispensed into each of the Teflon
vessels containing the sample filters. The Teflon vessel liners are then weighted and inserted back
into their digestion vessel body and tightly capped. An acid-cleaned rupture disc membrane is
placed into an acid-cleaned vent stem which is attached to the top of each microwave vessel cap.
Each capped vessel is swirled lightly.
Note: Vigorous shaking of the vessel will cause the filter to disperse in solution which will make
it very difficult to pipette solution for analysis.
Only twelve samples and/or standards can be microwave digested at a time. One vessel from each
of the two digestion runs is outfitted with a Teflon thermowell cap into which a Teflon-coated
Pyrex thermowell is inserted. Eleven regular vessels and one thermocouple vessel are loaded in
the carousel tray. The carousel tray is then removed from the clean room and is placed in the
microwave digester. The fiber optic temperature probe is carefully inserted into the vessel with the
thermowell. A pressure monitoring and control probe is also attached to the same vessel. The
digestion program for the particulate mercury filters is then initiated. The program heats the
samples to 160=C (approximately 70 psi) for 20 minutes.
After the samples are heated, the microwave digester fan will remain on to help cool the Teflon
vessels. The vessels are allowed to cool until the pressure inside the control vessel is 1-2 psi
(approximately 60 minutes). The fiber optic probe and pressure probe are carefully removed and
the carousel is transferred back into the clean room. The vessels are then reweighed to confirm no
loss during digestion. 0.5 mL of BrCl is added to each vessel. The vessels are then gently swirled
to collect liquid droplets from the side of the liner. The vessels are allowed to react for one hour
prior to analysis.
2.5 Sample Analysis and Data Acquisition
2.5.1 Volatilization/Recapture
Volatilization of mercury from solution is accomplished using a glass impinger assembly
manufactured at the University of Michigan. A 25 mL graduated bubbler attaches to an
impinger via a ground glass fitting. N-, flow is regulated using a Teflon stopcock. A soda
lime trap is incorporated into the system to prevent damage of the gold-coated bead traps
by capturing acid gases liberated during the purging procedure.
Total mercury is quantified b> oxidizing all mercurv forms using bromine monochloride.
Bromine monochloride is u strong oxidizing agent, capable of breaking organic bonds
with mercury, thus liberating the divalent form of mercur\ iHsr" i A 5 mL aliquot of the
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Volume 2, Chapter 2 SOP for Analysis of Particulate Phase Mercury
oxidized filter sample solution is carefully pipetted into a graduated glass bubbler
containing 20 mL of a previously purged sample and 100 uL of hydroxylamine
hydrochloride is added. A stopper is then inserted into the bubbler, its swirled briefly and
allowed to react for 5 minutes to reduce the excess bromine monochloride from solution.
Bromine monochloride is reduced from solution since halogens liberated from solution
will quickly damage the gold-coated bead traps onto which the purged elemental mercury
is amalgamated.
A blanked gold-coated bead sample trap is affixed to the end of the soda lime trap. The
bubbler is opened, 500 uL of stannous chloride is added, and the bubbler is quickly
attached to the impinger. The N, flow is adjusted to 450 cc/min using a calibrated
rotameter and the solution is purged for 7 minutes. The stannous chloride reduces the
divalent mercury to Hg° which is quantitatively captured on the gold-coated bead trap.
2.5.2 Analysis of Total Mercury
The CVAFS analyzer used for particulate mercury analysis is kept on at all times, since
this has been shown to stabilize the UV lamp and maintain consistency from one day to
the next. The power supplied to the CVAFS analyzer is modulated by a line tamer (Shape
Magnetronics) to prevent power fluctuations. It is imperative that the mercury lamp not
experience wide temperature fluctuations or power surges since both of these drastically
affect the sensitivity of the instrument. During operation of the instrument the helium
carrier gas flow rate is regulated upstream of the analyzer using a mass flow controller
(Tylan) which is set to maintain a 35 cc/min flow rate. This flow rate has been determined
by UMAQL to yield the optimal peak characteristics for mercury standards. The regulator
on the helium cylinder is set at 50 Kilopascals. The helium stream is prefiltered using a
gold-coated trap before entering the analytical train in order to remove any mercury. In
the analytical train, mercury is thermally desorbed from the sample trap, and amalgamated
onto the analytical trap which is subsequently thermally desorbed into the CVAFS
analyzer where the mercury atoms are detected. Traps are desorbed by heating a nichrome
coil which is wrapped around the trap covering the gold-coated beads. Application of 12
volts of current to the coil is sufficient to achieve a temperature of 500°C inside the gold
bead trap (voltage may vary due to variations in length and thickness of nichrome wire).
Two fans supply cool air to the sample and analytical traps separately in order to speed
analysis time.
The gain on the particulate phase mercury CVAFS analyzer is set to yield approximately
2000 mV of net response for a 1 ng mercury standard. The background on the CVAFS
analyzer is set at 5.0 and maintained in that position in order to track the drift in the
baseline of the analyzer. A Hewlett Packard Integrator is connected to the analyzer to
convert output signal into an integrated area of the detected response. Area units are used
for all sample calculations since area is much more reliable than peak height.
To analyze a sample trap, the trap is placed snugly into the analytical train using friction
fit Teflon connectors and Teflon sleeves. The nichrome coil used specifically for the
sample trap is slid over the trap and moved to completely cover the quartz wool plugs and
the gold-coated beads contained between the plugs. Helium is allowed to flow through the
sample trap for 2 minutes before analysis begins in order to purge air and water vapor
from the analytical train A circuit controller (ChonTrol) is employed which is
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SOP for Analysis of Particulate Phase Mercury Volume 2, Chapter 2
programmed to turn on the variable transformers and fans in a precise and reproducible
manner. First, the sample coil is heated for 2 minutes, then it is cooled while the
analytical trap is heated for 2 minutes. The analytical trap is then cooled for 2.5 minutes
and the fan to the sample trap is turned off. While the analytical trap is cooling, a new
sample trap is installed in the analytical train and helium is passed through this trap until
the analytical trap is cool and ready for another sample. When the analytical trap begins
heating, the integrator is turned on and the ambient temperature, time and base mV are
recorded in a log book and the LCD display on the analyzer is set to record the peak mV
(by depressing the Peak button on the face of the analyzer). After the sample is analyzed
and the peak height and area reported by the CVAFS and integrator respectively, these
values are recorded in the log book.
A standard curve is analyzed at the beginning of each day of analysis and a control
standard which yields a response in the range of the samples being analyzed is run every
six samples. Criteria for the standard curves and control standards are described below in
Section 2.5.4. All sample analysis is recorded in a log book specific to the analyzer with
which samples are being quantified and also in a lab notebook specific to the study for
which the samples were collected. At the end of the day of analysis all results from the
log sheet are entered into a computer spreadsheet file for subsequent checking and
processing by a statistical software program, SAS (Cary, NC).
2.5.3 System Purge and Blanks
At the start of each day of analysis, each irnpinger system is purged after the soda lime
trap is conditioned. First 20 mL of Milli-Q water is added to an acid-cleaned bubbler,
then 1.0 mL of SnCK is added and the solution is purged at 450 cc/min for 15 minutes.
After each system is purged a System Blank is generated to ensure the irnpinger assembly
is free of contamination.
System Blank (Bubbler Blank): This blank is generated by adding 500 uL of SnCK to the
system purge solution and purging the solution onto a blanked gold-coated bead trap at
450 cc/min for 5 minutes. After the System Blanks have been completed, one of the
purged bubblers is dedicated for generation of standards.
Total Reagent Blank: This blank is synonymous with the 0 pg filter standard (Section
2.5.4), which is generated on each day of analysis. The Total Reagent Blank is used to
calculate the method detection limit (presently 1.0 pg/nr') and to calculate sample
concentration (Section 2.5.5).
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Volume 2, Chapter 2 SOP for Analysis of Particulate Phase Mercury
2.5.4 Standard Curve and Control Standards
A standard curve, generated by bubbling five different filter standard solutions, is
analyzed before each day of analysis. The concentration of the 5 mL filter standard
solution aliquots for the calibration curve are tailored to the expected value of the samples
to be analyzed. At UMAQL, a typical calibration curve consists of five filter standards: 0
pg, 100 pg, 200 pg, 500 pg and 1000 pg. Because of the BrCl dilution factor imparted
onto the filter standards, the nominal pg of mercury delivered by each 5 mL aliquot is
slightly less than standard name indicates. The volumes of standard working solution
added to each filter standard vessel to achieve the five standard concentrations in each 5
mL aliquot are shown in Table 1.
Table 1. Calibration Curve for Bubbled Hg Standards
Hg in 5 mL of Filter Standard Solution Volume of Standard Working Solution
Opg OuL
96.6 pg 200 uL
191.4 pg 400 uL
465.1 pg lOOOuL
888.9 pg 2000 uL
The filter standard solutions are prepared at the same time that the field samples are
extracted. Baked glass fiber filters are folded and placed into acid-cleaned Teflon vessel
liners and the appropriate volume of standard working solution is pipetted directly onto
the filter. The filters are then extracted in the same manner as the sample filters (described
in Section 2.4)
Standards for the calibration curve are generated starting with the zero point and continued
in ascending order to the highest, usually 1000 pg. First, a blanked gold-coated bead trap
is attached to the end of the soda lime trap. Then 5.0 mL of filter standard solution is
pipetted into the standard bubbler followed by 100 uL of NH2OH«HC1. After the solution
has reacted for 5 minutes 500 uL of SnCU is added. The standard bubbler is quickly
attached to the impinger and the solution is purged at 450 cc/min for 5 minutes. The gold-
coated bead trap is analyzed immediately after purging.
After each of the standards for the calibration curve has been analyzed, a linear regression
is calculated to establish the coefficient of determination (r), the slope of the line and how
well the slope of the curve predicts each of the points in the calibration curve. The 0 pg
standard area is subtracted from each of the other points which are then regressed against
the expected values usint! no intercept (line is forced through zero). The r must be
>0.995 and each of the points on the curve must be predicted by the slope within 10% of
their true value (Table 2). If these criteria are not met, specific points which are errant are
repeated and the linear regression recalculated.
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SOP for Analysis of Particulate Phase Mercury
Volume 2, Chapter 2
Table 2. Example Calibration Curve and Calculation of Slope
Standard
(pg Hg)
0
96.6
191.4
465.1
Response
(AU)
141,510
2,156,000
4,126,400
10.214,000
19,548,000
Response (AU)-
Zero Point (AU)
0
2,010,490
3,984,890
10.072.490
19,406,490
Predicted Value (pg
Hg)
0
92.4
183.2
463.0
892.1
Slope = 4.5969E-5ng/AU
Slope'1 = 21,754 AU/ng
r = 0.9998
This curve is accepted and sample analysis commences.
Control standards are analyzed every sixth sample. The control standards are generated in
the same manner as described above and are chosen to be representative of the samples
being analyzed. The integrated area from each of the control standards must be within
10% of the slope of the calibration curve in order to continue analyzing. If this is not the
case, a second control is analyzed immediately. If the second control indicates that
analyzer sensitivity has changed a second calibration curve is generated and sample
analysis is continued.
2.5.5 Calculation of Particulate Phase Mercury Concentration
The paniculate phase mercury concentration from a glass fiber or quartz fiber filter is
calculated in pg/m3 The total reagent blank response is subtracted from the analytical
aliquot response and the difference is multiplied by the slope of the calibration curve
which is in pg/AU. The mass of Hg for the entire sample is then calculated by dividing
the analytical aliquot Hg mass by the analytical aliquot volume and multiplying the result
by the total volume of sample. The calculated value, in picograms of mercury is converted
to pg Hg/m3 by calculating the total volume of air drawn through the filter and dividing
the pg of mercury by the cubic meters of air sampled (Table 3).
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Volume 2, Chapter 2 SOP for Analysis of Particulate Phase Mercury
Table 3. Calculation of pg Hg/m1 in a Particle Phase Sample
1. Calculation of pg of mercury recovered from the analytical aliquot
P8 H8 = (sample response (AU)-total reagent blank (AU)) * slope of calibration curve (pg/AU)
(5,944,100- 141,510) * 4.5969E-5 = 266.7pg Hg
2. Calculation of ng of mercury in entire sample volume
pg Hg = (pg Hgfrom analytical aliquot * total volume of sample)/volume of anal\ticai aliquot
(266.7pg Hg * 20.5 mL)/5.0 mL = 1093.4 pg Hg
3. Calculation of m3 sampled at a flow rate of 30 1pm and a sample duration of 24 hours:
Volume of Air Sampled = (DTM Reading Off- DTM Reading On) * DTM Calibration Curve
(1982.597 1937.864) * 0.97886 -0.00024 = 43.787m1
4. Calculation of Particle Phase Mercury Concentration in Sample = pg Hg/m1
1093.4 pg Hg / 43.787 mj = 25 pg Hg/m'
2.5.6 Trouble-Shooting
A source of irreproducible results may be due to faulty gold-coated bead traps. These
traps are numbered with discrete identifiers. Contact with halogen fumes, organic fumes
or overheating of the trap during analysis can damage the trap, rendering it unusable. If
performance of a gold trap is suspect, at least two consecutive standards are analyzed from
this trap to determine its ability to amalgamate and release mercury.
If a low response is observed, the impinger assembly is checked for leaks. Teflon
compression fittings on the soda lime trap and the Teflon nut on the Teflon stopcock are
the most common location of leaks.
If peak-broadening is observed or no peak is detected in a sample, the analytical train is
checked for leaks. Peak broadening is often the result of low gas flow, water vapor on the
gold-coated bead trap, inadequate heating of analytical trap or an analytical trap damaged
by exposure to halogen fumes or overheating. The analytical trap is not replaced unless it
begins to demonstrate poor recovery or release of amalgamated mercury.
If the baseline drifts more than ICK-f the UV lamp is replaced. After replacement, the
analyzer is allowed to equilibrate for 24 hours. If the problem persists, sources of power
fluctuation, drafts or air currents that may be changing the temperature of the UV lamp are
investigated.
Room temperature in which the CYAFS is located is maintained between 20-22 C.
however, if the temperature exceeds 26 "C analysis is stopped. SUKC instrumental noise
increases significantly.
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SOP for Analysis of Particulate Phase Mercury Volume 2, Chapter 2
3.0 Performance Criteria, Quality Assurance and Quality Control
3.1 Field operators are carefully instructed in the techniques of contaminant-free particulate phase
mercury sample collection. All of the operators are currently operating sampling equipment for
either the National Dry Deposition Network, the National Atmospheric Deposition Program, the
Integrated Atmospheric Deposition Network or the Great Lakes Acid Deposition Network.
3.2 Every 6 months UMAQL personnel inspect each of the sampling sites to audit the performance of
the equipment and to make all necessary repairs or adjustments.
3.3 Co-located samples are collected from one sampling site during the study to quantify method
precision. Reported concentrations for co-located samples are based on the mean of the two
samples.
3.4 Precision and accuracy levels will be set and maintained for each type of analysis. A relative
precision for total mercury of less than 15% is maintained for samples with values at least 3
standard deviations greater than the detection limit. Analysis of standards and controls is within
10% of the stated value.
A minimum of 25% of all samples are analyzed in duplicate. Reported concentrations are based
on the mean of the replicates. Analytical precision averages better than 6%.
3.5 Every 3 months maintenance on the CVAFS analyzer is conducted, including replacement of the
UV lamp, the Teflon tubing, and the detection cell.
4.0 Clean Room Procedures
4.1 Entering the Clean Room
Shoes are taken off outside the clean room by all UMAQL personnel. Personnel then enter the
outer vestibule (changing room). Once inside, the hood is put on followed by the clean room suit
and clean room boots. The boots are snapped to the suit at the back of the leg (to hold up the
boots) and are buckled in the front. Personnel then step over a dividing bench where they put on
clean room gloves and snap the clean room suit at the wrist. Now fully clothed they enter the
clean room making sure to securely close the door behind.
4.2 Taking Supplies into the Clean Room
All supplies to be taken into the clean room are double bagged in polyethylene. The supplies to be
taken into the clean room are placed in the outer dressing room. Upon entering the clean room, the
outer bag is removed and left in the entr\ room. All supplies that enter the clean room that have
not been bagged are rinsed with MQ-\\ater and wiped off with particle-free wipes.
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Volume 2, Chapter 2 SOP for Analysis of Paniculate Phase Mercury
Appendix A: Facilities, Equipment and Reagents
Following is a list of the required facilities, equipment, supplies and reagents for sample preparation, sample
collection and sample analysis that are outlined in this document. The make and model of the following items
are those used at The University of Michigan Air Quality Laboratory. Many of these items are available from
a variety of sources.
1. Preparation of Field Supplies
Class 100 Clean Room, Work Stations
Clean Room Gloves
Particle-free Wipes
Clean Room Cap, Gown and Boots
Milli-Q Water (18.2 MQ/cm)
Exhaust Hood
Acetone
Alconox
Polyethylene Tubs
EM Science Tracepur and Suprapur Hydrochloric Acid
Polytherm Water Bath (Science/Electronics)
Baker Instra-Analyzed or EM Science Suprapur Nitric Acid
New Polyethylene Bags
20 Liter Polyethylene Carboys
2. Sample Collection
Vacuum Pump (URG, Model 3000-02M)
Calibrated Dry Test Meter (DTM)
Calibrated 30 1pm Rotameter (Matheson)
HOPE Tubing with quick connects
Black Latex Tubing
Mercury Sampling Box (UMAQL, See Appendix B)
Acid-Cleaned 47 mm Teflon Filter Holders (Savillex, PFA Labware)
47 mm Preheated Glass Fiber Filters (Gelman Sciences A/E)
Acid-Cleaned Teflon Jars (Savillex. PFA Labware)
Teflon-Coated Forceps
Particle-Free Gloves
Teflon Tape
Sample Labels
Field Operator Log Book
Sample Tracking Forms
Shipping Boxes
3. Sample Analysis
Cold Vapor Atomic Florescence Detector (Brooks Rand. LTD.)
Line Tamer/Conditioner (Shape Magnetronies Model PCLT 150)
Integrator (Hewlett-Packard Model
Helium, Ultra Hiah Purity Grade i
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SOP for Analysis of Paniculate Phase Mercury Volume 2, Chapter 2
Mass Flow Controller (Tylan)
Nichrome Coils (UMAQL)
Electric Leads
Variable Transformers (Staco Energy Products Co. Type 3PN1010)
Cooling Fans
Gold-Coated Glass Bead Traps (UMAQL)
Pre-punfied (99.998%), Analyzed Nitrogen
Glass Impingers
Resin-coated Ring Stand
25 mL Graduated Glass Bubblers
12.7 cm lengths of 1.27 cm OD Glass Tubing (Soda Lime Trap)
Automatic Pipettes
Repipet II Dispenser ( Labindustnes)
Reagents (Section 4.2)
Magnetic Stir Plate
Class A Volumetric Flasks
Teflon Reagent Bottles (Clear and Opaque)
Teflon Reagent Vials
Resin-coated Wire Rack (Support Bubblers)
Refrigerator
Freezer (-40°C)
Microwave Digester (CEM)
Microwave accessories:
24 Teflon Lined Vessels
Rupture Disks
Thermowell
Fiber Optic Temperature Probe
Pressure Tubing
Carousel
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Standard Operating Procedure for
Mercury Analysis
Robert P. Mason and Kristin A. Sullivan
Chesapeake Biological Laboratory
University of Maryland
P.O. Box 38
Solomons, MD 20688
June 26, 1996
-------
University of Maryland Standard Operating Procedures
for the Lake Michigan Mass Balance Project
Calibration Frequency And Procedures (Taken from QAPjP)
The detection limits of validated EPA methods for mercury quantification are orders of magnitude too high
for Lake Michigan waters. For this reason, a peer reviewed method will be employed.
University of Maryland lab personnel will employ a Brooks-Rand Cold Vapor Atomic Fluorescence
Spectrophotometer (CVAFS) Model-2 (Brooks-Rand, Ltd, Seattle, WA) to measure mercury.
Commercially available, traceable HgNO, standard (from Fisher Scientific or comparable supplier), diluted
using Class A volumetric equipment, will be used to calibrate the CVAFS. Working standards will be
replaced at least monthly. An intercalibration with outside laboratories will be performed yearly to check
our analytical procedure.
During each analysis session, the CVAFS will be initially calibrated with a four point curve spanning the
anticipated range of sample signals. This calibration is achieved using a vapor mercury standard. As
mercury is a liquid at room temperature it has a well-defined vapor pressure, which is temperature
dependent. A temperature-controlled sealed container containing liquid mercury will therefore have a
defined air concentration of mercury. This mercury-saturated air can be subsampled via a septum port
using a gas tight chromatography syringe. Known aliquots of the air are then injected into the gas stream
of the dual gold trap-CVAFS system, and trapped on the gold column just prior to the analyzer. Heating
the column releases the Hg into the analyzer allowing quantification of the injected concentration. This
method allows calibration of the instrument independent of the wet chemical manipulation-sparging
techniques associated with mercury determination in water samples and allows a calibration curve to be
established based on instrument sensitivity and linearity. Changes in CVAFS sensitivity will be monitored
with single point standard injections after every five sample analyses. If CVAFS sensitivity drifts more
than 10% during an analysis session - this would be atypical of the instrument - these single point
standards will be used to recalibrate the analyzer.
Due to infrequent rapid temperature changes in the laboratory, obtaining a standard curve using the
temperature dependent vapor mercury standard may be difficult. The technician under these circumstances
is advised to proceed with the calibration verification (liquid standard) and sample analysis and wait until
later in the day when the temperature has stabilized to run a vapor mercury standard curve. The calibration
verification standard results are then checked using this curve. If specifications are not met, samples
analyzed prior to the curve will be reanalyzed.
If the calibration QC requirement is not met, corrective measures will be taken. These include checking
the integrity of the septum injection port and replacing if necessary; changing the injection needle to insure
that this is not a source of error: check the stability of the gas tlow u ithin the analyzer and check and
replace any tubing that might have become contaminated and might be leaching mercury into the system.
The lamp and photomultiplier do deteriorate over the long-term (years) and this could be another source of
variability. If none of these procedures results in a sufficient improvement in the calibration, the
instrument will be returned to the manufacturer for checking and recalibration. All maintenance.
calibration and repair will be logged in an equipment notebook As the analyzer is kept on constantly, no
specific recording of usage is maintained. Field equipment \\ill be checked before and at the end of each
cruise. The pump will be calibrated for flowrate at the beginning of the study. As filtration volume \\ill be
determined volumetrically. small changes in the pump flovnutc arc not critical. The pump head and
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SOP for Mercury Analysis Volume 2, Chapter 2
peristaltic tubing will be checked weekly during the cruises, and additional supplies will be on board. For
the Go-Flo bottles, O-rings and the rubber tubing and the associated closing mechanisms are checked on a
deployment basis. Spares are kept on board as maintenance can be performed, in the majority of cases, on
board. In addition, there will be at least one additional Go-Flo, besides those being used, as a spare.
Analytical Procedures (Taken from QAPjP)
See Appendix 1 of our QAPjP for more detailed laboratory protocol for total mercury analyses. The
protocol as written by Steve Claas for the University of Wisconsin-Madison Water Chemistry Program is
essentially the same as that to be used at the University of Maryland. As this is the most recent detailed
method, this should be considered the reference method. The CVAFS techniques have been developed
based on techniques first initiated through the University of Connecticut (Bloom and Fitzgerald, 1988) and
are generally used throughout the mercury research community for the analysis of mercury in
environmental samples. The PI has been using similar techniques for the analysis of open ocean seawater,
estuarine and freshwater samples for the last six years. The methods used are based on sample digestion
methods described in Bloom and Crecelius (1983) and the CVAFS total mercury method of Bloom and
Fitzgerald (1988). Deviations from these methods are described below.
1) The analyzer is kept on usually, so no warmup time is required.
2) A Hewlett-Packard integrator will be used instead of a strip chart recorder.
3) The calibration procedure is different from that used by the University of Wisconsin. See details
above for the calibration procedure.
4) Analysis of water samples: Subsamples to be analyzed will be decanted from the 2 L Teflon
bottle into a smaller, preweighed Teflon bottle for analysis. After tarring the sample, it will be wet
oxidized with 100 uL of BrCl solution for every 100 mL of sample. Samples will be sealed in
double polyethylene bags and heated for at least an hour, and preferably overnight, at 70°C before
prereduction and analysis. Aliquots of 125-500 mL, depending on expected mercury
concentration, will be purged and trapped. Argon will be used as the carrier gas and the purge
time will be such that 20 volumes of argon are purged through each sample volume.
5) Filters containing paniculate will be unfrozen. Two mL of MilliQ water and 2 mL of BrCl reagent
will be added and the bottle sealed. From this point the samples are treated in a similar fashion to
the water sample, except 2 mL of hydroxylamine HC1 is used to produce samples. The detection
limit for both methods are similar, as the analytical techniques are comparable and the BrCl
reagent is the primary source of the blank. For similar amounts of added BrCl, the blank should be
comparable. The method has a mean detection limit of approximately 0.1 ng/L. Detection limits
depend to a large extent on volume of sample analyzed and can be lowered by increasing the
volume of the purged aliquot.
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Standard Operating Procedure for Total Hg
(Mason Lab Detailed Procedure) Analytical System
1.0 At start of day, turn on argon tanks for bubblers and analyzer. Pressure into analyzer should be
b/w 50-60 units.
2.0 A column should be connected in line with crimped side facing integrator (First on, First off
concept). Make sure that coils are covering gold and quartz wool or improper heating will result.
3.0 Run a standard curve by spiking you sample column with 20, 30, and 50 cc of mercury. Turn the
integrator on by pressing before each spike.
4.0 Turn on heating program by pressing , <1>, on the Control Programmer. It will
run through the heating program for 8 min.
5.0 Spikes of 20 to 50 cc should be run for most low level detection. For each spike, you must record
the temperature and convert temperature to vapor pressure with the conversion chart on wall. A
standard curve including 0 as a point should yield a correlation of 0.999 or better.
6.0 When done analyzing a sample, integrator is shut off by pressing .
7.0 Hg Collection System (Bubbler System)
7.1 While standard curve is being run, the bubbler blanks can be run with the second set of
columns.
7.2 Rinse bubblers two to three times with Q-water, then fill to mark. Rinse spargers with
Q-water, then replace bubblers.
7.3 Stannous Chloride: The SnCK pipette is labeled and should only be used for this reagent.
Rinse the tip three to four times in 6N HC1 then rinse one time with SnCU. All waste
should be placed in labeled waste container. Spike 0.5 mL into each bubbler, then rinse
tip again three to four times with HC1. Rinsing keeps the pipette tip clean (we do not
change this tip) and it keeps the SnCl, from precipitating (it dissolves under acid
conditions).
7.4 Place lids firmly on bubblers so that no leaking occurs. Place clean gold traps onto
bubblers, next to soda lime columns. (Columns should have crimped end facing
bubbler-First on. First off). Turn the gas on to 60 units for larger bubblers and ~ 20 for
small bubblers. Samples should bubble for about 20 min.
7.5 After running Q-water blanks, a standard Hg spike should be am using the now blanked
water. Standard Hg (~lng/mL) is kept in the dark in the refrigerator. Only remo\e for
use. Use 1 mL pipette labeled Hg Std only and take a pipette tip from the acid bath.
Rinse with Q-water. then rinse once with standard before dispensing into bubblers. Hg
waste and tip should go in flask labeled Hg waste. Ultimately, any Hg waste should be
placed in labeled aeid bottle at back of hood.
7.6 After spiking wiih standard, spike bubblers with SnCU.
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SOP for Mercury Analysis Volume 2, Chapter^
7.7 After standard is run, a blank should be run using the same blanked water. Only SnCl,
should be added.
7.8 If standard and blank values are acceptable, then you are ready to run samples.
8.0 Water samples
8.1 For Reactive Hg, just add a known volume to each bubbler and spike with SnCI2.
8.2 For Total Hg. rinse out acid cleaned 500 mL bottles with Q-water. (I usuall} rinse out
about six and re-use them over again). This is done if your sample is in a 2 L bottle, as
you only need 500 mL for analysis. If you are using smaller bottles this step is
unnecessary. When running a duplicate, you will need two bottles per sample.
8.3 Rinse bottles once with sample, then fill with sample. Spike with BrCl. (Use pipette
labeled BrCl and keep same tip; rinse with Q-water several times then one time with
reagent prior to use). The volume of BrCl used will depend on how much mercury is in
the sample (ie. rain samples usually get 1 mL BrCl). Tighten lids on samples, shake, then
let sit for at least l/2 hour.
8.4 After time has expired, add the same volume of Hydroxylamine HC1 as BrCl to your
samples, rinsing the tip in the same manner as your BrCl addition. Shake well; samples
are ready for analysis.
8.5 To analyze samples, empty bubblers of old water. All waste from bubblers must be placed
in container labeled SnCK. Rinse bubbler with sample. Record sample volume using
balance. Again, samples need to be treated with SnCl, prior to analysis.
9.0 Filter samples
9.1 Filters should be placed into acid cleaned vials prior to analysis.
9.2 To each vial add 2 mL Q-water and 2 mL BrCl. Use Teflon coated forceps to push an\
unexposed filters into reagent and tighten caps. Shake gently and let stand for Vi hour.
9.3 Add 2 mL of Hydroxylamine HC1 and shake well to neutralize.
9.4 To blanked bubblers, add 2 mL of sample (try to avoid getting filter paper pieces in pipette
tip). Spike with SnCL
10.0 End of the day
10.1 When finished running samples, empty bubblers into SnCK waste container and rinse-
several times with Q-water. Fill again with Q-uater and replace lids. If you are not going
to use bubblers lor an extended time period, top off with concentrated acid to clean.
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Volume 2, Chapter 2 SOP for Mercury Analysis
10.2 All SnCK waste should be placed in labeled flask in hood. When flask is full add some
dissolved NaOH pellets to precipitate the SnCk Let stand overnight. Before next use.
decant clear liquid into sink being careful not to let any precipitate escape. When a
substantial amount of precipitate has accumulated, empty flask into Hg/SnCl: bottle for
proper waste disposal.
10.3 Shut off gas cylinders.
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Total Mercury Analysis
in Aqueous Samples
James P. Hurley
Bureau of Integrated Science Services
Wisconsin Department of Natural Resources
April 1994
Revision 2
-------
Total Mercury Analysis in Aqueous Samples
1.0 Introduction
This manual contains instructions for measuring total mercury in aqueous samples. Both
experienced chemists and neophyte technicians will find these protocols understandable and
useful. Besides dictating each step in the total mercury analysis, this manual provides
explanations, suggestions, precautions, definitions, tips and helpful background information.
Although the stepwise protocols are complete, this manual cannot replace a knowledgeable,
experienced, and friendly human instructor.
Environmental Mercury Pollution
After new sampling and analytical techniques were developed and applied during the mid 1980s,
researchers discovered environmental mercury concentrations were up to 1000 times lower than
previously reported. For many years, scientists had unknowingly contaminated their samples
during collection and analysis. Scientists now believe the concentration of mercury in our
atmosphere, oceans, and lakes is often quite low. In some cases, concentrations are almost too low
to measure.
Because mercury is found in paints, batteries, fluorescent lights, electric switches, and many other
human inventions, concentrations of the element tend to be quite high inside and near buildings.
Unfortunately, the situation is often worse in laboratories which frequently house mercurial
reagents and mercury equipped instruments. Mercury's ubiquity, along with some of its chemical
properties, make contaminating environmental samples virtually inevitable. For this reason,
special techniques must be implemented when analyzing total mercury samples.
2.0 Clean Techniques
The following guidelines were developed to minimize the probability of sample contamination.
These Clean techniques should be followed when analyzing mercury samples.
2.1 Never open mercury sample bottles indoors unless working in a trace-metal clean lab.
2.2 Keep sample bottles sealed in plastic bags except when adding to or taking from the bottles.
2.3 Minimize sample contact time with atmosphere. Avoid breathing into samples.
2.4 Wear a lint free suit.
2.5 Wear plastic gloves when handling mercury samples and analytical equipment such as gold traps
and bubblers.
2.6 Do not place potentially contaminated objects such as pipettors, balances, and lab benches into
contact with mercury sample bottles. Cover equipment and benches with plastic bags, gloves or
particle free towels if necessary.
2.1 Work in a laminar flow or HEPA-filtered hood whenever possible.
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Total Mercury Analysis in Aqueous Samples Volume 2, Chapter 2
2.8 Keep lab work areas uncluttered.
2.9 Clean the trace-metal clean lab frequently.
3.0 Definitions
3.1 Analytical Trap: This is the gold trap that captures the mercury from a sample trap and
subsequently releases that mercury into the analyzer. It is the trap farthest down-stream and is not
routinely removed from the analytical system.
3.2 Analyzer: The mercury analyzer is a cold vapor atomic fluorescence spectrophotometer (CVAFS).
In the CVAFS, light from a small mercury vapor lamp is shined through a quartz flow cell that
contains mercury in a stream of argon carrier gas. This light excites the mercury atoms which
subsequently emit more light. The amount of light emitted by the mercury is proportional to the
amount of mercury passing through the cell. The light emitted by the mercury atoms passes
through a filter (254nm) and into a photomultiplier tube (PMT) which converts the light into an
electrical signal. This signal is received and plotted by a stripchart recorder or integrator.
3.3 Blank (noun): The mercury that is associated with equipment and reagents must be measured; this
quantity of mercury must subsequently be subtracted from the mercury measured in samples. This
measured quantity of mercury from background levels is called a blank. Sometimes blanks are
associated with specific objects, such as a bubbler blank or a BrCl blank. Sometimes blanks are
associated with specific processes, such a traveling blank or a digestion blank. The term blank can
also be used in a more generic sense to refer to the sum of background contamination from all
potential sources.
3.4 Blank (verb):
1) The process of measuring the quantity of mercury that is associated with equipment and
reagents is called blanking. For example, during an analysis session, the analyst must
blank the bubblers.
2) The process of thermally desorbing mercury from a gold trap is called blanking. This is
sometimes also referred to as analyzing a trap.
3.5 Bubbler: A flask and stopper system used to purge aqueous samples. Typically, 250 mL, flat
bottomed, spherical, 24/40 jointed, boiling flasks are used for total mercury analyses.
3.6 Bubbler Stopper: This is a modified ground glass joint which fits into the bubbler flask. The
stoppers have a glass tube which extends from a vertical gas inlet on the top of the stopper and
terminates in a frit near the bottom of the bubbler flask. An outlet extends horizontally near the
top of the stopper.
3.7 Bubbling Matrix: An aqueous mixture which may contain any combination of deionized water,
HC1, SnCK. BrCl, NH:OH-HC1, total mercury standard, and/or water sample. This is the solution
out of which elemental mercury is purged.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
3.8 Coil: Coils are made from nichrome wire. These coils are used to desorb mercury from gold
traps. Variable autotransformers are used to apply a potential of approximately 10 VAC to the
coils; this heats the gold traps desorbing elemental mercury.
3.9 FEP: A type of Teflon (Fluorinated Ethylene-Propylene).
3.10 Glass Wool: Fibrous strands of silanized glass. Plugs of glass wool are used to hold soda-lime
chips in soda-lime traps.
3.11 Gold Trap: Gold traps are made from a 9 cm length of 4 mm I.D. quartz tube. The tubes contain
approximately 1 g of gold coated beads held in place with silanized glass wool plugs. A
constriction in the quartz tube holds all the packing materials in place. Because elemental mercury
forms an amalgam with gold, these gold coated bead traps are used to preconcentrate mercury
purged from aqueous samples.
3.12 Prereduce: The addition of NH,OH«HC1 to a brominated sample. Chemically reduces excess
BrCl which could damage the gold traps.
3.13 PTFE: A type of Teflon (Poly Tetra Fluoro Ethylene).
3.14 Purge: To pass N2 bubbles through an aqueous matrix to remove and trap the elemental mercury
contained within that matrix.
3.15 Regulator Units: These consist of the single or two stage regulators on the N2 and Ar cylinders
and any needle valves and other brass or Teflon fittings connected to these regulators.
3.16 Sample Traps: These are the gold traps that are first attached to the purging apparatus to capture
elemental mercury and then connected to the analytical system for mercury measurement. These
traps are connected to the Tenax TA® pretraps or soda-lime traps during bubbling and to the
argon line and analytical trap during blanking.
3.17 Scrubbing Traps/These are the gold traps attached to the purging and analytical systems that are
intended to remove contaminant mercury from the N: and Ar gases.
3.18 Soda-lime Trap: Soda-lime traps are made from 10 cm lengths of 0.9 cm I.D. Teflon tubing.
These traps are packed with non-indicating, 8-14 mesh, reagent grade soda-lime; the ends are
plugged with glass wool. These traps neutralize acid fumes and trap water vapor during the
purging process.
3.19 Tenax TA®: A porous polymer based on 2,6-diphenyl-p-phylene oxide used to trap matrix-
interferents associated with some tributary samples. Placed between the gold trap and soda lime
trap.
3.20 Timer Controller: A device used to switch coils and fans on and off at appropriate times during
gold trap blanking.
3.21 Total Mercnr\: The sum of all the different chemical forms of mcrcun (includes inorganic and
oreanic forms).
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Total Mercury Analysis in Aqueous Samples Volume 2, Chapter 2
3.22 Working Standard: A solution with a known concentration of Hg:+ This solution, mixed form
more concentrated primary and secondary standards, is used to calibrate the analyzer. It typically
has a concentration of 10 ng/mL.
4.0 Cautions
The following list of warnings and prescriptions is incomplete. Consult the material safety data
sheets that are shipped with all hazardous chemicals.
4.1 BrCl: Extremely corrosive. Contact with any body part will cause severe injury. The BrCI
solution releases toxic and extremely caustic CU, Br, and BrCl fumes which will cause severe
damage to the respiratory system if inhaled. Flush affected areas with water and mild soap.
Always use BrCl under a well operating fume hood.
4.2 Glass Wool: Harmful if inhaled. May irritate skin. Handle with gloves.
4.3 HCl: Can cause severe bums. Fumes can cause severe damage to respiratory system. Flush
affected areas with large amounts of water. Always work with concentrated HCl under a fume
hood.
4.4 NH2OH-HCl: Harmful if inhaled or swallowed. Avoid contact with eyes and skin. Flush affected
areas with water and mild soap. Has caused mutagenic effects in laboratory animals.
4.5 Nichrome Coils: Coils heat up to 450-500°C during a blanking cycle. Under normal room light,
hot coils look no different than cold coils. Always approach coils tentatively.
4.6 SnCl:: Can cause eye and skin irritation. Rinse affected areas with large amounts of water and
mild soap. Persons with a history of skin disease may be at an increased risk from exposure.
4.7 Soda-Lime: Can cause burns. Avoid contact with skin and eyes. Rinse affected areas with large
amounts of water.
4.8 Total Mercury Standard: Mercury in the standard can damage the nervous system. Avoid contact
with skin. The working standard contains 1 to 5% BrCl.
5.0 Total Mercury Analysis Sessions
A typical total mercury analysis session often lasts from 8 to 1 2 hours. Depending on the number
of standards, blanks, and replicates that are analyzed, 9 to 18 samples can be analyzed during one
session.
Although instructions in this manual are written as linear, stepwise protocols, procedures must
often be performed concurrent!}. Below is a suggested sequence of events for one analysis
session.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
5.1 Begin each analysis session by removing any mercury that may have accumulated on the six
sample traps since the last analysis session. Follow the trap blanking procedure in Section 8.0.
While the traps are being blanked, prepare the bubblers for blanking and analysis as prescribed in
Section 8.3.
5.2 After the bubblers have been filled with an initial matrix and have started bubbling, install the
soda-lime traps (Section 9.3.3) and Tenax TA® pretraps (Section 8.3.5). Note that no sample
traps are attached at this time; this initial 20 minute purging serves to preclean the matrix, soda-
lime and Tenax TA® pretraps. Continue the initial sample trap blanking procedure while
bubbling.
5.3 After the bubblers have purged for at least 20 minutes, blank the bubblers (Section 8.4). Continue
the initial sample trap blanking procedure if not already completed.
5.4 After the 20 minute purging period, analyze the gold traps from the bubbler blanks as prescribed in
Section 10.0. By now the last of the sample traps should have undergone an initial blanking.
Purge and trap standards to calibrate the analyzer (Section 8.4) while analyzing the bubbler blank
traps. You should also prereduce the first set of three samples. (Prereduce fewer if you want to
replicate analyses, of course.)
5.5 After the bubbler blank traps have been analyzed and the standards have finished purging, analyze
one or two gold traps with standards. Assess the analytical and purging systems. Are the bubbler
blanks low? Do the standards indicate sufficient analyzer sensitivity? If all is well, run another set
of standards so trap efficiency for all traps can be assessed and also run a set of second set of
bubbler blanks. If traps are behaving similarly and bubbler blanks are low and seem stable, purge
and trap the prereduced samples (Section 9.2).
5.6 By this time you should have established a cycle in which one set of samples can be purged and
trapped while another set is being analyzed. Continue this cycle for two to four more rounds.
Don't forget to prereduce samples a few minutes before pouring them into the bubblers.
5.7 Continue to purge and analyze samples. At minimum, a set of standards and bubbler blanks
should be run midsession and at the end of an analysis session.
6.0 Protocol Organization
The stepwise protocols are organized as follows.
6.1 Step 1 These sections describe major tasks
Caution: These sections list dangerous equipment and reagents
6.2 These sections provide detailed prescriptions on equipment use and sample manipulation.
These sections expand on the instructions given above and include precautions, reminders, and
helpful tips. Background information and explanations are sometimes also included in this section
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Volume 2, Chapter 2
7.0 How to Prepare the Analytical System
7.1 Step 1 Analyzer is normally left on at all times.
7.1.1 The analyzer should be switched on at least 24 hours before beginning analyses. For a
Brooks Rand Model 2 analyzer, turn on by the red rocker switch on the front panel.
Analyzer sensitivity is correlated to operating temperature. Turning the analyzer on in
advance allows electronic components to warm and stabilize. If the analyzer is not
switched on in advance, be certain to calibrate frequently between analyses.
Sarrple Coil
Transformer
Analytical Coil
Transformer
Nsedle Valve
Rowrreter
Analyzer
NottoSode
Timer
Controller
Integrator
and/or
Stripe hart
Figure 1. The Total Mercury Analytical System
7.2 Step 2 Adjust the systems settings
7.2.1 Switch the variable transformers that power the sample and analytical nichrome coils to
the 140 V position. The transformer dials should be set at approximately 10%.
These settings control the temperature ramp used to thermally desorb mercury from the
gold traps. Low coil temperatures allow mercury to remain on the trap. High
temperatures can compromise trapping efficiency and may vaporize the gold: which then
plates out further downstream destroying tubing, fittings and the analyzer's quartz cell. If
a coil emits a slight reddish glow in a dimly lighted room but does not glow under normal
room light, the coil temperature is likely within the acceptable range. When in doubt, test
the traps with Hg micctions to determine efficiency and reburn the traps to assess
carryover.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
1.2.2 Turn on the Ar gas flow with the small needle valve feeding into the gold trap on the
regulator unit. If necessary, adjust the Ar flow to about 60 units using the needle valve on
the analyzer flow-meter.
The rate of Ar flow controls peak shape which should be symmetrical. If a suitable Ar
flow cannot be established using the flow-meter, carefully adjust the Ar regulator valve.
Adjust carefully since high pressures can cause friction connections to leak or burst.
7.2.3 Make necessary adjustments to the system settings. Consult the Appendix for appropriate
total mercury analysis settings.
Some settings, such as analyzer gain, rarely need adjustment. Others, such as stripchart
settings, change for various mercury species and average sample concentrations. Always
check every setting before beginning analyses. Figure 1 illustrates the critical components
of the analytical system.
8.0 How to Blank Traps & Bubblers and Calibrate the Analyzer
8.1 Step 1 Blank the gold traps
8.2 Caution: Nichrome coils
8.2.1 Remove the end-plugs from a gold trap and orient the trap so the constriction is on the
downstream or right side of the gold beads. Slip the sample nichrome coil around the
trap's quartz barrel. See Figure 1.
It is important to properly orient the trap before inserting it into the coil. Twisting the trap
after it is nested in the coil can break the coil or connections.
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Volume 2, Chapter 2
Silanized Glass Wool
Plugs
Gold Coated Beads Quartz Barrel
Teflon Sleeves
During Blanking and Trapping
Direction of Gas Flow
Figure 2. Gold Coated Bead Mercury Amalgamation Trap.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
8.2.2 First connect the trap to the analytical (right) side of the system, then plug the Ar line into
the leftside of the trap.
Improperly connecting a trap to the Ar line can cause the packing material to shift or be
blown out of the quartz tube.
8.2.3 Carefully adjust the position of the nichrome coils so they are centered over the gold bead
region of the traps.
The coils should be placed in precisely the same position every time a trap is heated.
Mercury can plate out on the inside of the traps barrel during normal use. This plated
mercury is subsequently desorbed into the sample stream if the coil extends over a section
of the trap that is not normally heated.
8.2.4 Press the sequence <1> on the timer controller.
This program switches the sample coil on for 4 minutes (Figure 3). This blanking
procedure is intended only to clean residual mercury from the traps which may have
accumulated since last use. Because this is not a quantitative step, the analytical trap may
be purged just twice while blanking the last two traps in the set of sample traps. This will
be explained in G.
8.2.5 After the 4-minute sample trap heating cycling is complete, press the sequence
<3> on the timer controller.
This switches on the sample trap cooling fan. Allow the coil and trap to cool for about 2
minutes before proceeding.
8.2.6 When the sample nichrome coil and trap are cool, press the sequence <3>
on the timer controller. Remove the sample gold trap: disconnect the analvtical
(right) side first.
This switches off the sample trap cooling fan. Improperly disconnecting a trap from the
Ar line can cause the packing material to shift or be blown out of the quartz tube.
8.2.7 Repeat Step 1 for four traps. For the last two traps, follow the above blanking procedures
through C, then press the sequence <8> on the timer controller.
Program 8 appropriately controls coils and fans over an 8 minute period; both the
sample and analytical traps are blanked. See Figure 3.
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Total Mercury Analysis in Aqueous Samples
Volume 2, Chapter 2
Total Rn
Integrator
SarrpleCoil
Sample Fan
Analytical Coil
Analytical Fan
Tirre (Mnutes) 0
8
Figure 3. Total Mercury Analysis Event Sequence. Shaded Bars Indicate the Time Periods
When Each Device Is Switched On.
inlet
N2 Flow
Bubbler
Stopper
Nitrogen
Scrubbing
Vertical Gold
Trap
Soda-Lime
Tenax TA®
\
Sample Gold
PTFE Connectors
Bubbler
N, Flow
Bubbling
Matrix
Figure 4. Total Mercury Purging and Trapping System.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
8.3 Step 2 - Prepare bubblers for blanking and analyses
Caution: HC1, SnCU
8.3.1 Using clean deionized water, thoroughly rinse and partially fill each bubbler (about half
full, or 125 mL).
Deionized water from a Milli-Q system is quite clean (0.1-0.3 ng/L) if the plumbing has
been completely flushed.
8.3.2 Clear the HC1 repipetor by pumping a few strokes into a small beaker and disposing this
acid. Dispense 5 mL of clean acid into each bubbler.
Avoid producing acid fumes by filling the beaker halfway with water before pumping the
repipetor.
8.3.3 Dispense 0.5 mL of SnCl2 into each bubbler.
Remember to rinse the pipette tip.
8.3.4 After removing the end-plugs, attach the gold traps to the vertical inlets on the bubbler
stoppers. Plug the N7 lines into the gold traps. See Figure 4.
These gold traps scrub residual elemental mercury from the N2. Remove the Teflon
sleeve and plug from the end of the trap nearest the constriction; carefully insert the trap
into the sleeve on the bubbler top. Remove just the plug from the other end of the trap
and connect the N9 line.
8.3.5 Attach soda lime traps - see Section 9.3.3 for description of soda lime traps.
8.3.6 Attach Tenax TA® pretraps (if used) downstream of the soda lime traps.
Some tributary samples, particularly early spring samples, exhibited a pronounced matrix
effect which is eliminated by using these pretraps. It is recommended that the pretraps be
used for all tributary samples.
8.3.7 Turn on the N-> with the small needle valve feeding into the gold trap attached to the
regulator unit. Adjust the N-, flow into the bubblers if necessary. All flow rates should be
about 50 units. See Figure 5.
The exact flow rate is unimportant. Make certain that all bubblers have approximately the
same flow rate. If an appropriate N, flow cannot be established with the flow-meters,
carefully adjust the N-, regulator valve. Monitor the back pressure. Excessive pressure
will cause the bubbler stoppers to pop off.
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Flowmeters
Bubblers
Not lo Scale
Figure 5. Total Mercury Purging and Trapping System
8.4 Step 3 Blank the bubblers
Caution: SnCl:
8.4.1 Dispense 0.5 mL of SnC^ into each bubbler containing the pre-bubbled solution from
Steps 2 and 3. After removing the end-plugs, attach a gold sample trap to each Tenax
TA® pretrap (if used) or directly to each soda-lime trap.
Because 0.5 mL of SnCUis added to every sample independent of sample volume, the
SnCI, is considered part of the bubbler blank. Attach the gold trap so the constriction is
farthest from the Tenax TA® pretrap or soda lime trap i.e., on the down-stream end.
8.4.2 Bubble for 20 minutes.
This process purges and traps the ubiquitous residual mercury' from the glassware and
bubbling matrix. Assume this amount of mercury is released even, time a sample is
purged. Because it is an artifact, this quantity of mercury must be subtracted from the
standard and samples.
8.4 3 Remove and plug the sample traps: analyze each gold trap.
Turn to How to Analyze Gold Traps on Section 10.0 to continue
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Volume 2, Chapter 2 _ __ _ Total Mercury Analysis in Aqueous Samples
8.5 Step 4 - Calibrate the Analyzer
Caution: SnCI2 total mercury standard
8.5. 1 Add 0.5 mL SnCl2 and a series of volumes (usually 25, 50 and 100 ^L) of total mercury
working standard to the pre-bubbled matrix from Steps 2, 3 and 4. Attach a gold sample
trap to each of the soda-lime traps connected to the bubblers.
s
The concentration of the total mercury working standard is 10 mg/mL; a 100 pL aliquot has
1 mg of mercury in it. If you suspect that bubbled samples will have significantly more or
less mercury choose a volumes of standard that will more closely match the sample
concentrations.
8.5.2 Bubble for 20 minutes.
This process purges and traps a known mass of mercury.
8.5.3 Turn off N2; twist bubbler stopper to release seal; remove and plug the gold traps.
Analyze each gold trap.
To prevent water from being sucked into the traps that scrub mercury from the N7, always
turn off the N9 before removing the sample traps. Turn to How to Analyze Gold Traps on
Section 10.0 to continue.
9.0 How to Prepare Water Samples for Analysis
9.1 Step 1 Digest the water samples
Caution: BrCl
9.1.1 Add excess BrCl to every water sample. This will be indicated by a persistent yellow
color. For tributary samples generally 5 to 8 mL per 500 mL bottle are required. Record
the amount of BrCl added to the sample.
Mercury in any of its chemical forms is oxidized to Hg2+ by BrCl. Remember to record
the amount of BrCl in each sample so the appropriate reagent blank can be subtracted.
9.1.2 Repackage sample bottles in two plastic bags. Place in a 70 ~C oven overnight. If the
yellow color disappears, continue to add BrCl until the yellow color remains. Always
wrench tighten bottles after they have been removed from the oven and cooled.
Some samples contain compounds that compete with mercury for the BrCl. These
compounds effectively neutralize all the BrCl before the mercury is oxidized. For this
reason, BrCl should be added in excess, indicated by a persistent yellow color. Wrench
tightening is necessary, since the bottle caps become loose during the heating and cooling
process.
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Total Mercury Analysis in Aqueous Samples Volume 2, Chapter^
9.2 Step 2 Prereduce samples
Caution: NH:OH-HC1
9.2.1 To each digested sample, add 30 ^L of NI^OH-HCl solution for every 1 mL of BrCl
added in Step 1. Swirl the sample.
The NFLOH-HC1 reduces (i.e. neutralizes) the excess BrCl in the sample.
9.2.2 Allow the sample to react for 5 minutes.
The yellow color from the excess BrCl should disappear.
9.3 Step 3 - Purge samples and capture mercury on gold traps
Caution: SnCU
9.3.1 Place a bubbler on a pan balance and tare. The bubbler may be empty or contain a purged
solution from bubbler blanks, standards or a previous sample. Dispense about 100 to 125
mL of sample into the bubbler. Try to keep the same overall volume in the bubbler during
the day. Record the exact volume dispensed.
Because the matrix from the blanks, standards and bubbled samples has been purged of
mercury, sometimes it is more efficient to simply add another sample without emptying
the bubbler. Make sure, however, that the new sample will fit into the bubbler before
pouring. Of course the volume of sample dispensed into the bubbler can be more or less
than prescribed here, depending on the suspected mercury concentration. By keeping the
bubbled volume within a 1 mL range for an entire analysis session, you can facilitate easy
concentration calculations. Also, by keeping the same overall liquid level in the bubbler
constant you minimize the possibility of cleaning bubbler surfaces not cleaned initially at
the beginning of the day.
9.3.2 Add 0.5 mL of SnCl2 to the sample and cap.
The SnCl: reduces the Hg2+ in the sample to Hg°. Hg° is volatile and can be purged from
the water sample onto gold traps.
9.3.3 Attach a gold sample trap to each soda-lime trap or Tenax TA ® pretrap. Reconnect the
NI lines.
Attach the gold trap so the constriction is farthest from the soda lime or Tenax TA®
pretrap. t.e,. on the down-stream end.
9.3.4 Bubble for 20 minutes.
This process purges mercury from the sample water; the mercury is trapped on the gold
heads.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
9.3.5 Turn off N^, twist bubbler stoppers to release seal; remove and plug the sample traps.
Analyze each gold trap.
To prevent water from being sucked into the vertical gold traps that scrub mercury from
the N2, always disconnect the N2 lines before removing the sample traps. Turn to How to
Analyze Gold Traps on Section 10.0 to continue
10.0 How to Analyze Gold Traps
lO.l Caution: Nichroine coils
10.1.1 Remove the end-plugs from a gold trap and orient the trap so the constriction is on the
downstream or right side of the gold beads. Slip the nichrome coil around the trap's quartz
barrel.
It is important to properly orient the trap before inserting it into the coil. Twisting the trap
after it is nested in the coil can break the coil or connections.
\OA.2 First connect the trap to the analytical (right) side of the system, then plug the Ar line into
the left end of the trap
Improperly connecting a trap to the Ar line can cause the packing material to shift or be
blown out of the quartz tube.
10.1.3 Carefully adjust the position of the nichrome coils so they are centered over the gold bead
region of the traps.
The coils should be placed in precisely the same position every time a trap is heated.
Mercury can plate out on the inside of the traps barrel during normal use. This plated
mercury is subsequently desorbed into the sample stream if the coil extends over a section
of the trap that is not normally heated.
10.1.4 Press the sequence <8> on the timer controller. If you are using an
integrator.
Program 8 appropriately controls coils and fans over an 8 minute period; both the
sample and analytical traps are heated. See Figure 3.
10.1.5 When the cooling fans turn off (after about 8 minutes), write the sample identification next
to the peak on the chart or integrator paper. Remove the sample gold trap; disconnect the
analytical (right) side first.
Improperly disconnecting a trap from the Ar line can cause the packing material to shift or
be blown out of the quartz tube.
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Total Mercury Analysis in Aqueous Samples Volume 2, Chapter 2
10.1.6 Repeat Step 1 for the other two traps.
With a system of three bubblers, sample bubbling time is approximately equal to the time
needed to analyze three traps. Establish a cycle where one set of three traps is being
analyzed while the other set is capturing purged mercury.
11.0 How to Prepare Soda Lime Traps
11.1 Caution: Glass wool, soda-lime
11.1.1 Assemble materials on the lab bench before beginning. You'll need FEP tubes, PTFE
machined connectors (10 mM to 5 mM). glass wool, teflon tape and soda-lime.
The FEP tubes and PTFE connectors can be used more than once between
cleanings. Repack the traps with fresh soda-lime at least every other day of
analysis.
11.1.2 Create six small balls of glass wool to plug the ends of the traps.
The balls of glass wool should be about 0.5 cm in diameter. They need not be very dense.
11.1.3 Place one of the glass wool balls into an FEP tube. Carefully wrap a piece of teflon tape
around a machined connector. Make sure the tape is free of wrinkles, as this can be a
conduit for leaks, and is not covering the bore hole for gas flow. Insert this machined
connector carefully so the teflon tape is not bunched or wrinkled.
The connector should fit tightly in the tube. Take care not to pinch an excess of glass
wool fibers between the outside of the connector and the inside of the tube. This can
create also leaky connections.
11.1.4 Holding the connector, scoop soda-lime into the tube. Fill the tube to 0.25 cm of the
unplugged end.
To avoid spilling soda-lime around the Clean Lab, stand over a trash can while filling
the traps.
11.1.5 Place another glass wool plug into the tube and insert a connector wrapped with teflon
tape. Wipe any soda-lime dust off the outside of the trap with a towel.
Both the soda-lime and the glass wool will compress to allow insertion of the connector.
11.1.6 After building 2 more traps, place one trap on the horizontal outlet of each bubbler.
The soda-lime traps are symmetrical so orientation is not important.
11.17 After the soda-lime traps have been installed, bubble overnight in a VlilliQ matrix with
nil. HCI and a few mL SnCK at a I Km IMIC of 5 to 10 units.
This process cleans mercury from the soda-lime traps and the matrix in the bubblers.
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Volume 2, Chapter 2 Total Mercury Analysis in Aqueous Samples
Note: Soda lime may be reused once. After the first use, dry the traps by attaching directly to
N: pretraps overnight applying a N, flow rate of about 5 units
12.0 How to Prepare Reagents
12.1 Stannous Chloride
To prepare the stannous chloride solution, 200 g of SnCK is measured using a large weighing boat
and Teflon spatula. The crystals are poured into a clean I L Teflon bottle and then 100 mL of
concentrated Tracepure HCl is added to the I L bottle with the SnCK crystals. This can be done
either using the HCl repipetor or a type of clean volumetric glassware. Milli-Q water is then added
to bring the solution to 1 L. The solution is purged with N2 overnight and then labeled SnCK and
the date the solution was purged. The SnCl, solution is stored doubled-bagged in the refrigerator.
12.2 Hydroxylamine Hydrochloride
To prepare NH:OH-HC1 solution, 300 g of NH:OH-HC1 crystals are measured using a large
weighing boat and Teflon spatula. The crystals are poured into a clean Teflon 1 L bottle and then
filled to 1L with Milli-Q water. (To make 125 mL of solution use 37.5 g of NH:OH-HC1 crystals
and 125 mL bottle.) Cap the bottle and shake until all crystals are dissolved. For each 1 L of
solution, add 1 mL of SnCU and purge the solution overnight with N,.
12.3 Bromine Monochloride
Caution: To prepare BrCI solution, all work must be done in the fume hood and wearing safety
goggles.
First 8.6 g of KBr is measured in the hood using a weighing boat and Teflon spatula. The KBr is
poured in the 1L Teflon bottle (BrCI stock solution bottle) and the bottle is then filled with 800
mL of concentrated Tracepure HCl and a clean magnetic stir bar added. For approximately an
hour the solution is stirred in the fume hood with a stir plate. After the hour is up, ver\ slowly add
the KBrO4 crystals while stirring slowly. Add small amounts of crystals and only add more when
fizzing has stopped. Then allow the solution to stir with the cap on loosely for another hour.
Smaller amounts of this stock solution (about 50 mL) are purified in a teflon, sub-boiling
distillation apparatus (Savillex®).
12.4 Mercury Standards
To prepare the mercury secondary standard use a stock mercury standard of 1000 mg/L. In a
clean 100.0 mL class A volumetric flask, pipette 100.0 uL of the stock solution and 5 mL of BrCI
solution into the volumetric flask and dilute to 100.0 mL with Milli-Q water. This pipetting must
be done extremely accurately and should be redone if pipette error or contamination occurs. After
mixing, the solution is poured into a clean 125 mL Teflon bottle and labeled with Hg 2. ID of
stock solution, the date, and your initials. This can be stored in a refrigerator for up to one year.
To prepare the working standard, dispense 1.00 mL of the mercury secondary standard and 1 to 5
mL of BrCI solution into another clean 100 mL class A volumetric flask and bring to volume with
MilliQ water This pipetting must also be done with great accuracy. After mixing the solution,
pour it into a clean 1 25 mL Teflon bottle and label with Hg Working Standard, the date, and your
initials. This solution should be replaced inonthK.
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Standard Operating Procedure for
Analysis of Sediment for Total Mercury Using
the Cold Vapor Technique with the Leeman
Labs, Inc. Automated Mercury System
Theresa Uscinowicz, A & O Chemical Company1
and
Ronald Rossmann, USEPA
Large Lakes Research Station
9311 Groh Road
Grosselle, MI48138
Mid-Continent Ecology Division - Duluth
National Health and Environmental Effects Research Laboratory
Octobers, 1996
Revision 1
Current aflilution is SoBran, Inc./Pathology Associates International.
-------
Standard Operating Procedure for Analysis of Sediment for Total
Mercury Using the Cold Vapor Technique with the Leeman Labs, Inc.
Automated Mercury System
1.0 Introduction
Elemental concentrations of mercury in sediment and water are determined by the PS200 system,
and its operation is based upon cold vapor AAAS. The prepared sample enters the system in the
divalent phase, and is mixed with stannous chloride to form elemental mercury vapor. This
mixture-moves to the liquid gas. separator, and argon carries the mercury vapor through a drying
tube for vapor removal. The vapor enters one path of the cell optimized. The mercury lamp emits
light at 254 nm, and absorbance is measured by the detector.
2.0 Materials Required
2.1 Chemicals
Reagents needed include the following ultra pure grade chemicals:
2.1.1 Leeman Labs lOOppm Mercury Standard
2.1.2 Leeman Labs Hydrochloric, Nitric Acids
2.1.3 Leeman Labs Ultra Pure Water
2.1.4 Liquinox
2.1.5 J.T. Baker Brand Hydrochloric, Nitric Acids
2.1.6 J.T. Baker Brand Stannous Chloride or Leeman Labs Stannous Chloride
2.1.7 Hydroxylamine Hydrochloride
2.1.8 Magnesium Perchlorate 10-20 size mesh
2.1.9 Potassium Permanganate
2.2 Equipment and Supplies
2.2.1 Supplies Needed for analyses include:
2.2.1.1 PS200 Automated Mercur> Analyzer with autosampler. \\ ith
associated data acquisition system
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter^
2.2.1.2 AP200 Automated Preparation System with associated data
acquisition system
2.2.1.3 Analytical Balance in Biology Lab, Mettler 2100T
2.2. [ .4 EDP pipettors and associated disposable tips
2.2.1.5 Associated pump tubing for sample drainage, tin chloride
2.2.1.6 Polyethylene tubes 12 mL capacity, and caps
2.2.1.7 Polyethylene tubes 45mL capacity
2.2.1.8 Teflon or polyethylene beakers
2.2.1.9 Teflon wash bottles
2.2.1.10 Teflon bottles (60 mL)
2.2.1.11 Electronic balance
2.2.1.12 PVC gloves
2.2.1.13 Paper towels, clean wipes
2.2.1.14 Lubricating oil for autosampler
2.2.1.15 Quartz wool and quartz glass drying tubes
2.2.1.16 Teflon spatula
2.2.2 Supplies needed before sample analyses if not using automated preparation system:
2.2.2.1 CEM microwave digester with associated Teflon PFA vessels
2.2.2.2 Low density 30-mL polyethylene bottles for sample storage
2.3 Reference Documents
The user of this method must be familiar with the following established standard
operation procedures:
LLRS-MET-SOP-001 Standard Operating Procedures for the Preparation ot Materials
used for Ultra-low I"iucc Hement AnuKses
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
LLRS-MET-SOP-003 Standard Operating Procedures for the Maintenance of the LLRS
Trace Metal Laboratories
LLRS-MET-SOP-010 Standard Operating Procedures for Analysis of Total Mercury in
Tissue and Sediment using the Cold Vapor Technique with the
Perkin-Elmer Model MHS-20 Gold Amalgam System
LLRS-QA-001 Minimum Analytical Quality Assurance Objectives for U. S. EPA
Large Lakes Research Station
LLRS-QA-SOP-001 Standard Operating Procedures for the Release of Data
LLRS-QA-SOP-002 Standard Operating Procedures for the Routine Review of Data
Quality and Quantity
3.0 Reagent Preparation
3.1 5% Nitric Acid Solution
This acid solution is used for rinsing materials used for cleaning materials used in the
analysis of samples.
3.4.1 Rinse a pre-cleaned graduated cylinder (100 mL) with three rinses of MSQ.
3.4.2 To the graduated cylinder, add 95 mL of MSQ.
3.4.3 Carefully add 5 mL of concentrated reagent grade nitric acid.
3.4.4 Transfer the solution to a squirt bottle.
3.4.5 Repeat steps 3.4.1 through 3.4.3 but add five milliliters of reagent grade acid.
(For preparation of 5% nitric acid rinses).
3.4.6 Repeat step 3.4.4 but add to separate.precleaned teflon bottle.
3.2 10% Hydrochloric Acid Rinse Solution
Use reagent grade J.T. Baker or Leeman Labs hydrochloric acid. The amount needed for
a one day run is 300 mL. Approximately 2 L will be needed to be prepared weekly.
WARNING
Hydrochloric acid is highly corrosive and incompatible with metals, hydroxides,
amines, and alkalis. Handle it while wearing personal protection gear. A full face
shield is recommended. Always use the concentrated acid under a fume hood. Store it
in an appropriate place. Store concentrated acid in a corrosives cabinet.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
3.2.1 Use a precleaned 1000 mL polyethylene graduated cylinder. Carefully rinse the
inside of the cylinder with 5% reagent grade nitric acid using a Teflon
(precleaned) squirt bottle. Follow this rinse with three rinses of Millipore Super-
Q water (MSQ).
3.2.2 Add 900 mL of MSQ to the graduated cylinder.
3.2.3 Carefully add 100 mL of reagent grade hydrochloric acid to the cylinder.
3.2.4 Transfer the mixture to a high density polyethylene bottle from Leeman
Labs.
3.3 1:1 Nitric Acid Solution
As recommended by the manufacturer, polyethylene autosampler cups must soak in 1:1
nitric acid before running to ensure acceptable results. Soak the cups for at least two
hours. Use J.T. Baker trace metal grade nitric acid for preparation. This solution can be
recycled, provided the autosampler cups are adequately rinsed with MSQ a minimum of
ten times before and after use. Dispose of 1:1 nitric after one month to eliminate the
possibility of any residual contamination.
WARNING
Nitric acid is corrosive and incompatible with combustible materials, metallic powders,
hydrogen sulfide, carbides, and alcohols. Handle it with personal protection. A full
face shield is recommended for the concentrated acid. Always use the acid under a
fume hood. Store the concentrated acid in a corrosives cabinet.
3.3.1 Use an appropriate pre-cleaned container. An empty Suprapure hydrochloric acid
bottle has been used. Rinse the container at least three times with MSQ before
addition of nitric acid.
3.3.2 Using the graduations on the glass bottle, add 400 mL MSQ.
3.3.3 Carefully add 400mL of J.T. Baker nitric acid to the glass bottle.
3.3.4 Swirl the contents of the bottle and , if necessary, label.
3.4 10%(w/v) Stannous Chloride Solution
The volume of tin chloride solution required to run is dependent upon the daily run time.
To run for six hours approximately 250 mL are required.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
WARNING
Stannous chloride should be handled with care. Avoid contact with eyes, skin, and
clothing. Avoid breathing its dust. Handle solid chemical under a fume hood. Handle
while wearing personal protection gear for eyes and skin.
3.4.1 Rinse a precleaned wide mouth teflon bottle three times with quartz distilled
water.
3.4.2 Tare the bottle and add 25 g of tin chloride to the bottle.
3.4.3 Rinse a precleaned graduated cylinder with 5% reagent grade nitric acid solution
followed by three rinses of MSQ.
3.4.4 Carefully add 25 mL of reagent grade hydrochloric acid to the graduated cylinder.
Carefully pour the cylinder's contents into the bottle containing the stannous
chloride.
3.4.5 Swirl the contents of the bottle vigorously to dissolve the stannous
chloride.
3.4.6 After the stannous chloride has been dissolved, add 200 mL of MSQ using the
same graduated cylinder.
3.4.7 Replace the cover and vigorously shake the bottle to ensure all of the tin chloride
is dissolved.
3.5 10% Nitric Acid Solution
This acid solution is used for preparation of standards and for dilution of samples.
Historically, this solution is used for samples that have undergone microwave digestion.
3.5.1. Rinse a pre-cleaned graduated cylinder (100 mL) with 5% reagent grade nitric
acid solution followed by three rinses of MSQ.
3.5.2 To the graduated cylinder, add 90 mL of MSQ.
3.5.3 Carefully add 10 mL of concentrated Seastar or reagent grade nitric acid
(whichever matches the matrix of samples).
3.5.4 Transfer the solution to a teflon bottle.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
3.6 Working Mercury Standard Solution
Commercial 100 ppm Leeman Labs mercury standard is used to prepare the working
standard. The working standard is prepared at a concentration of 100 ppb or 0.1 ug
Hg/mL and is made fresh weekly.
WARNING
Mercury is a poison. Handle it, its compounds, and its solutions with personal
protection. Mercury can form a vapor and be inhaled. It also is absorbed through the
skin. Use this material in a fume hood. Always wear personal protection gear.
3.6.1 Rinse a pre-cleaned teflon bottle (LLRS-MET-SOP-001) three times with MSQ
and air dry.
3.6.2 Using a precleaned graduated cylinder, rinse three times with 5% nitric
and three times with MSQ.
3.6.3 Using two EDP separate automatic pipettors, rinse 2-1000 mL tips three times
with a 5% reagent grade nitric solution followed by three rinses with MSQ.
3.6.4 Tare a dry bottle on the electronic scale.
3.6.5 Using an additional EDP pipettor, rinse a 100 uL tip in the same manner as 3.6.2
3.6.6 Using the cleaned 100 uL tip, carefully add 50 uL of the commercial 100 ppm
Leeman Labs mercury standard solution to the bottle.
3.6.7 Transfer 45mLs of the 10% nitric acid solution using the cleaned graduated
cylinder.
3.6.8 Using an EDP pipettor, with a 1000 uL precleaned tip, add 10% nitric acid
solution to the bottle until the weight is 50 g.
3.7 Preparation of Recommended Range of Calibration Standards
The calibration standards are prepared fresh semiweekly.
3.7.1 Microwave Digestion Standard Range
The following range of standards has been used for analyses of Green Bay
Sediment samples and bracket the samples well. 0.250 ppb. 0.500 ppb. 1.00 ppb
2.00 ppb. 5.00 ppb are used (0.00025 ug/mL. 0.00050 ug/mL. 0.001 ug/mL.
0.002 ug/mL. 0.005 ug/mL). It may be possible to go below 0.250 ppb depending
upon instrument performance. The lowest concentration above background noise
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SOP for Analysis of Sediment (or Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
is approximately 0.087 ppb (0.000087 ug/ mL). 60 mL of each calibration
standard will last for two daily runs. Prepare double of the desired standard that
will be run as a check standard. The autosampler cups must be filled to at least 40
milliliters, (60 mL total capacity).
3.7.1.1 Rinse six pre-cleaned teflon bottles three times with MSQ and allow to air
dry.
3.7.1.2 Prepare two separate EDP pipettors each with a precleaned 1000 pL tip.
(Rinse each pipette tip three times with 5% nitric acid followed by three
rinses with MSQ) .
3.7.1.3 Rinse a precleaned graduated cylinder with 5% nitric acid followed by
three rinses of MSQ.
3.7.1.4 For each standard, tare each bottle individually.
3.7.1.5 For the 0.250 ppb standard, pipette 150 uL of the 100 ppb working
standard to the bottle.
3.7.1.6 Using the graduated cylinder, bring the total volume up to 55 mL by the
careful addition of prepared Seastar 10% nitric acid. The weight of the
bottles contents should now be roughly 55 g.
3.7.1.7 Add prepared Seastar 10% nitric acid with the other EDP pipettor and
precleaned tip until the total weight is 60 g.
3.7.1.8 Repeat steps 3.7.1.6 3.7.1.9 for the remaining standards by the
addition of 300 uL of the 100 ppb standard for a 0.500 ppb calibration
standard, 600 uL of the 100 ppb standard for the 1.00 ppb calibration
standard, 1200 uL of the 100 ppb standard for the 2.00 ppb calibration
standard, and 3000 uL of the 100 ppb standard for the 5.00 ppb calibration
standard.
3.7.1.9 Do not recycle the standards remaining in the autosampler cups at the end
of the day. Properly dispose of these daily. Attempts to recycle the
standards from autosampler cups have given diminished intensities.
3.7.2 Automated Digester Standard Range
Use the following range of standards: 0.000 ppb. 0.125 ppb, 0.250 ppb. 0.500 ppb,
1.00 ppb. 2.00 ppb. Prepare in a 2% hydrochloric acid matrix in precleaned
Teflon bottles. L sc Leeman Labs or J.T. Baker Indrochloric acid. Prepaie 5()mL
of each standaid weeklv or as needed.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
3.7.2.1 0 ppb Standard
3.7.2.1.1 Rinse a precleaned graduated cylinder with 5% nitric acid
followed by three rinses of MSQ. Dispose of waste in an
appropriate container.
3.7.2.1.2 Rinse a precleaned 500mL teflon bottle three times with MSQ.
3.7.2.1.3 Add 245 mL MSQ to the bottle.
3.1.2.1A Carefully add 5 mL of concentrated hydrochloric acid to the
bottle.
3.7.2.1.5 Transfer the solution to a teflon bottle.
3.7.2.2 0.125 ppb Standard
3.7.2.2.1 Prepare one EDP pipettor with a precleaned 100 uL
tip.
3.7.2.2.2 Prepare two separate EDP pipettors each with a precleaned 1000
uL tip.
3.7.2.2.3 Rinse a teflon bottle three times with MSQ that will be used for
each standard.
3.7.2.2.4 Tare the bottle on the balance.
3.7.2.2.5 Pipette 62.5 uL of the 100 ppb working standard into the bottle
3.7.2.2.6 Use the rinsed graduated cylinder to transfer 45 mL of the 0 ppb
standard to the bottle.
3.7.2.2.7 Slowly pipette 5 mL of 0 ppb standard to the bottle until the
bottle contents weight 50 g.
3.7.2.3 0.250 ppb Standard
For the 0.250 ppb std, follow steps 3.7.2.2.1 through 3.7.2.2.4. In step
3.7.2.2.5, substitute 125 |jL of the 100 ppb working standard using a new
1000 uL precleaned tip. Follow steps 3.7.2.2.6 through 3.7.2.2.7.
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Volume 2, Chapter 2
SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
3.7.2.4 0.500 ppb Standard
For the 0.500 ppb std, follow steps 3.7.2.2.1 through 3.7.2.2.4. In step
3.7.2.2.5, substitute 250 uL of the 100 ppb working standard using a new
1000 uL precleaned tip. Follow steps 3.7.2.2.6 through 3.7.2.2.7.
3.7.2.5 1.00 ppb Standard
For the 1.00 ppb std, follow steps 3.7.2.2.1 through 3.7.2.2.4. In step
3.7.2.2.5, substitute 500 uL of the 100 ppb working standard using a new
1000 uL precleaned tip. Follow steps 3.7.2.2.6 through 3.7.2.2.7.
- 3.7.2.6 2.00 ppb Standard
For the 2.00 ppb std, follow steps 3.7.2.2.1 through 3.7.2.2.4. In step
3.7.2.2.5, substitute 1000 uL of the 100 ppb working standard using a new
1000 uL precleaned tip. Follow steps 3.7.2.2.6 through 3.7.2.2.7.
3.8 Reagents Needed for Preparation of Sediment Samples using Protocol PRP7471
3.8.1 50% Aqua Regia (3:1 Hydrochloric : Nitric)
3.8.1.1 Use the glass container that previously held the Suprapure hydrochloric
acid, this is supplied with graduations. Rinse three times with MSQ to
eliminate any residual contamination.
3.8.1.2 To this glass container add 400 mL of MSQ water.
3.8.1.3 To the glass container add 300 mL of Leeman Labs hydrochloric acid to
the bottle.
3.8.1.4 Finally add to the bottle 100 mL of Leeman Labs nitric acid.
3.8.1.5 Carefully transfer solution to instrument bottle.
3.8.2 Potassium Permanganate Solution
3.8.2.1 Rinse a 100 mL precleaned finely graduated cylinder three times with V,
reagent grade nitric acid followed by three rinses of MSQ.
3.8.2.2 Dispose of the instrument bottle's contents if applicable and rinse three
times with MSQ.
3.8.2.3 Add 40mLs of the Leeman Labs potassium permanganate solution to the
rinsed 100 mL graduated cylinder.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
WARNING
Potassium permanganate is a strong oxidizer. Keep it from contact with clothing or
combustible materials. Avoid contact with eyes or skin. Avoid breathing the dust.
Handle solid chemical under a fume hood. Handle it while wearing personal
protection gear.
3.8.2.4 Transfer to the instrument bottle.
3.8.2.5 Add 760 mL of MSQ to the bottle.
3.8.2.6 Swirl the contents of the bottle to ensure a mixed solution.
3.8.2.7 Attach to the instrument.
3.8.3 Hydroxylamine Sulfate Solution
3.8.3.1 Rinse a 100 mL precleaned finely graduated cylinder three times with 5%
reagent grade nitric acid followed by three rinses of MSQ.
3.8.3.2 Dispose of the instrument bottle's contents if applicable and rinse three
times with MSQ.
WARNING
Hydroxylamine sulfate is an eye, skin, inhalation, and ingestion hazard. It will
cause skin irritation and may be absorbed through the skin. Always wear eye
and skin protection.
3.8.3.3 Add 96mL of the Leeman Labs Hydroxylamine Sulfate solution to the
lOOmL graduated cylinder.
3.8.3.4 Transfer the Hydroxylamine Sulfqte to the instrument bottle and continue
to add 704 mL of MSQ to the instrument bottle.
3.8.3.5 Swirl bottle to ensure a mixed solution.
3.8.3.6 Attach bottle to instrument.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
NOTE
This is a modification of the protocol that calls for full strength concentrations
of Potassium Permanganate and Hydroxylamine Sulfate. Full strength
concentrations of these chemicals were attempted and the fumes generated were
very strong. There was no difference in instrument performance in using some
higher concentrations of reagents in the protocol. Both gave acceptable
recoveries on the SRM material and similar instrumental intensities.
4.0 Microwave Sample Preparation
4.1 Preparation of Teflon Digester Vessels
Teflon digester vessels are cleaned following the procedure in section 4 of LLRS-MET-
SOP-010. An alternate method proposed by the manufacturer to increase the life of the
digestion vessels is under consideration. It may not be used until it is verified that blanks
are equally low for the two methods. Upon verification, the following alternate steps may
be substituted for steps 4.4 through 4.7 in LLRS-MET-SOP-010. Steps are taken directly
from CEM manual with slight variations to fit constraints of laboratories (Oilman 1988).
4.1.1 Add 20 mL of concentrated nitric acid to the digestion flask. Place the safety disk
on the vessel and tighten finger tight only. Place the vessel in the turntable, and
attach a venting tube.
4.1.2 Repeat step until the turntable contains 12 vessels.
4.1.3 Turn the MDS-81D exhaust onto the maximum fan speed. Ensure the turntable is
rotating.
4.1.4 Program the instrument for five minutes and 100% power. Depress the start key
and allow the acid to heat.
4.1.5 Allow the acid to cool to room temperature and manually vent each vessel. Open
vessels and pour the acid into an appropriate waste container.
4.1.6 Rinse the vessels three times with MSQ water and allow them to dry in a clean
area.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
4.2 Extraction of Samples
The maximum weight of samples extracted in CEM vessels without venting and still
obtaining acceptable recoveries for all metals has been two grams. For mercury, an
average weight of 0.300g has been used. The total volume is 25mL in 10% Seastar nitric
acid. Follow steps 5.1.1 through 5.1.11 in LLRS-MET-SOP-010 for preparation of
sediments
5.0 AUTOMATED DIGESTER (AP200) SAMPLE PREPARATION
5.1 Daily Instrument Setup
5.1.1 Clean autosampler rails with isopropyl alcohol.
5.1.2 Lubricate rails with oil daily
NOTE
Only if reagents are low or instrument was in shutdown will following steps
need to be followed (5.1.3 through 5.)
5.1.3 Open cover and carefully disconnect each bottle separately from the interior of
the instrument.
5.1.4 Rinse each bottle three times with MSQ
5.1.5 Fill bottles numbered 1,5,6 with MSQ up to the 800ml mark.
5.1.6 Prepare and fill bottle #3 with the Potassium Permanganate Solution.
5.1.7 Prepare and fill bottle #4 with Hydroxylamme Sulfate Solution
5.1.8 Carefully prepare and fill bottle #2 with the 3:1 Aquaregia solution
5.1.9 Check conditions of all fittings, caps and bottles
5.1.10 Close cover and pressurize system (turn gas on).
5.1.11 Set gas pressure = 20 psi
CAUTION
Do not exceed 25 psi
5.1.12 Turn instrument on
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
5.1.13 Turn computer and monitor on.
5.1.14 Turn printer on and press the online button.
5.1.15 After the computer has booted up, at the C: prompt, type apps.
5.1.16 Follow instructions on p4-4 of manual revision c.
5.2 Software and Instrument Setup
Follow the instruction in system startup section 4-3, What follows is a summary. The
user should review the following sections prior to analyses.
References to AP200 Manual
Section 3- System Testing
Section 4- System Operation
Section 5- Routine Maintenance
Section 6- Troubleshooting
5.2.1 Select Protocal and Get PRP7471, defines method, (sec 4-4 of manual)
5.2.2 Check reagent pressure of system, ensure it is > then 5.5psi and within 6.5.
(section 2-10 of manual)
5.2.3 Go to menu, Fl, select Utility, select Diagnostics
5.2.4 Select Reagent Pressure
5.2.5 Run the change rinse solution macro, @CHRINS
5.2.6 Rinse the autosampler rinse tray 3-4 times with MSQ
5.2.7 Replace the rinse tray
5.2.8 Run the wake up macro @WAKEUP (section 4-3 of manual)
5.2.9 Check centering of autosampler tip over cups, adjust if necessary (section 3-1)
5.2.10 Check precision of dispenser IX/week (section 3-3). or if there is a pressure
change of .2 psi in system at step 14.3.3
5.2.1 1 Set up autosampler sequence, and start finish sequence (section 4.6)
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
5.3 Preparation for Sample Digestion
5.3.1 Soak the 45mL standard cups and sample cups in 1:1 HNO3 for at least 2.0 hours.
5.3.2 Recycle the acid from the cups back into the Suprapure Acid bottle.
5.3.3 Rinse each cup three to six times with MSQ and allow to air dry.
5.4 Preparation of Standards for Automated Digestion
5.4.1 Prepare an EDP pipettor with a precleaned lOOOul tip.
5.4.2 Using the electronic scale, tare the beaker, and then the standard cup within the
beaker.
5.4.3 Record the weight of the empty cup on the extraction sheet.
5.4.4 Under the laboratory hood, pipette 5mls of the 0 ppb std to the cup.
5.4.6 Weigh the cup to confirm volume delivered is 5mls, and adjust accordingly with
the EDP pipettor.
5.4.7 Repeat steps 16.2 16.5 to extract multiple 0 standards.
5.4.8 Repeat steps 16.1 to 16.5 for the 0.125ppb std, 0.250ppb std, O.SOOppbstd.
l.OOppb std, and 2.00ppb std. Each time use a new precleaned lOOOul tip.
5.4.9 Rinse polyethylene vapor covers 3x with MSQ and place over sample cups.
5.4.10 Snap in place aluminum guard over polyethylene vapor barrier.
5.5 Preparation of Sediment Samples for Automated Digestion
Note
Please read the instruction manual for the Mettler Analytical Balance before
proceeding.
5.5.1 Rinse a precleaned teflon spatula with 5% nitric acid.
5.5.2 Rinse the spatula three tunes with MSQ.
5.5.3 Use the analytical balance in the Biologv Lab. Meulci "KK)T
5 5 4 Tare the lOOmL poKctln Icne beaker provided.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
5.5.5 Add the 45mL sample cup to the beaker and tare the scale again.
5.5.6 Record the weight of the sample cup on the digestion sheet.
5.5.7 Carefully open the Whirlpak bag of sediment.
5.5.8 Open the Whirlpak bag partially, so only a small circular opening exists (about the
size of a nickel). Caution: if the bag is open all the way, dust from the bag tends
to migrate upwards.
5.5.9 Using the precleaned teflon spatula, carefully scoop an aliquot of sample =
0.1 OOg.
5.5.10 Transfer this aliquot to the sample cup. Note: sand like samples will require a
very small aliquot, and silty samples will require a larger aliquot.
5.5.11 Record the weight of the sample in the most significant digits available on the
extraction log sheet.
5.5.12 Place the cup in its designated slot in a sample rack.
5.5.13 Zero the scale with the polyethylene beaker on it.
5.5.14 Rinse the teflon spatula with MSQ water three times.
5.5.15 Wipe teflon spatula dry with a fresh clean wipe square between samples.
5.5.16 Repeat steps 16.6.5 till 16.6.15 for the desired amount of samples, usually
fourteen.
5.5.17 Load dummy cups into any space not occupied by actual sample.
5.5.18 Rinse polyethylene vapor covers 3x with MSQ and place over sample cups.
5.5.19 Snap in place aluminum guard over polyethylene vapor barrier.
5.6 Initiation of the Digestion Procedure
5.6.1 Load autosampler racks into PS200
5.6.2 Confirm autosampler start to finish sequence is correct.
5.6.3 Go to Mam Menu. Fl. picss MACRO key. and t\pe in (•' PRP7471
5.6.4 Method will begin to run at this point.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
5.6.5 User will be prompted the following: Wait before reducing with KMNO4.
answer Y
CAUTION
User will be prompted near end of 5 hr procedure: Add Hydroxylamine Sulfate, answer Y.
Method will not proceed without this input.
5.7 Shutdown of AP200
5.7.1 After protocol is completed, wipe down bath to remove any remaining water.
5.7.2 Run an @APNAP two or three times to clean the dispenser with water.
5.7.3 Remove chemicals and replace reagents with MSQ if it will not be run for an
extended period, and run an ©CLEAN twice. This cleans not only the dispenser,
but all reagent lines.
6.0 Automated Analysis of Digested Extracts
6.1 Preparation of the Instrument
6.1.1 Preparation of Drying Tube
6.1.1.1 Rinse a quartz drying tube three times with MSQ, followed by a dilute
rinse of Liquinox, if it previously contained perchlorate.
6.1.1.2 Rinse several times with MSQ to eliminate Liquinox residuals.
6.1.1.3 Allow tube to air dry.
6.1.1.4 Rinse the teflon spatula three times with MSQ and dry with a fresh clean
wipe.
6.1.1.5 Gently place a small plug of quartz wool into one end of drying tube.
6.1.1.6 Carefully pour the Leeman Labs Magnesium Perchlorate into the
plugged drying tube. Try to fill with coar.se grained perchlorate. Do not
overfill the drying tube. Overfilling will cause the drying tube to become
blocked more easily once it becomes moistened by the gas stream. When
filled, the perchlorate should be able to move within the tube when
sentlv movine the drvine tube from side to side.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
WARNING
Magnesium perchlorate is moderately toxic and a strong oxidizing material. It
is a dangerous fire and explosion risk in contact with organic materials. It is
an inhalation hazard, and contact with skin or eyes can cause irritation. Work
with it in a fume hood while wearing skin and eye protection.
6.1.2 Autosampler Tray Rinse with 10% Hydrochloric Acid
6.1.2.1 Rinse the autosampler rinse tray three times with MSQ.
6.1.2.2 Fill the tray with the 10% hydrochloric rinse prepared in step 3.1.
6.1.3 Tin Chloride Rinse
6.1.3.1 Rinse the tin chloride bottle out three times with MSQ.
6.1.3.2 Fill it with the 10% tin chloride prepared in step 3.3.
6.1.4 Check Tubing Condition and Adjust to Appropriate Tension
6.1.4.1 Check condition of tubing for flattening, abrasion or other signs of wear.
If flattened, replace it.
6.1.4.2 Adjust tension on clamps to a halfway point. The sample line should be
halfway minus one notch.
CAUTION
i
Do not over tighten clamps. Too much tension will cause tube flattening and a
decrease in overall sensitivity.
6.1.5 Clean and Oil Autosampler Rails
6.1.5.1 Using a clean wipe or clean a paper towel, wipe the autosampler rails
with isopropyl alcohol.
6.1.5.2 Place a small amount of oil on bottom of each rail.
6.1.5.3 Complete a high stress maintenance cleaning monthly.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
6.1.6 Warmup Period
6.1.6.1 Allow the instrument to warmup for at least one hour time before
analyses. If the instrument was in SHUTDOWN mode for an extended
period allow it to warm up for several hours.
6.1.6.2 Software commands for warmstrt or coldstrt are found on page 16 of the
manual.
6.1.6.3 Perform a COLDSTRT or WARMSTRT
6.1.7 Optimize Optics with an Aperture Test
6.1.7.1 Go to Main Menu, Fl
6.1.7.2 Select Diagnostics, Select Aperture Test
6.1.7.3 Unscrew screw which is furthest out until the minimum absorbance is
obtained. An acceptable value is 0-100.
6.1.7.4 Select Test Optics from Diagnostics menu, and confirm gain, or
intensities are in the range of 500000-1100000 (= to voltage on lamp).
6.1.7.5 Difference between the two beams must be less than 100,000.
6.2 Software Setup for Routine Analysis
Consult Leeman Labs Automated Mercury Analyzer Manual pp 17-29 for guidance on
the software setup. What follows are the software and instrumental parameters used to
date. Complete the following steps before analyses.
6.2.1 Establishing a Protocol = Method file that Contains All Instrumental Parameters
6.2.1.1 From the Main Menu, select Protocol and then select Get.
6.2.1.2 To create a new Protocol, enter its name.
6.2.1.3 Suggested protocol naming is as follows: YYMMDD (Year. Year,
Month, Month. Day. Day. Example 960916F = September 16. 1996.
Note
Limitation is 8 Characters
6.2.1.4 Computer will guide you through prompts (PS200 Manual pp. 18-19).
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Volume 2, Chapter 2
SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
6.2.2 Creating a Folder=Data File
6.2.2.1 Press Fl, to be at the Main Menu.
6.2.2.2 Select Data output and select Open Folder.
6.2.2.3 Type in a folder name, suggested name = same name as protocol to avoid
confusion.
6.2.2.4 Follow instructions on page 19 of the manual.
6.2.3 Entering Instrumental Parameters
6.2.3.1 Follow instructions in PS200 instrument manual on pp 20-21.
6.2.3.2 The following are parameters used to date:
Integrations: 1
Uptake Time: 10
Weight: Y
Dilution: Y
Percent Recovery: N
On/Off: 10 (higher integration time generates a nonlinear
curve)
Flow Rate: 0.30 L/min
6.2.4 Standard Concentration Calculations
For those standards that are prepared with the automated digestion system, the
appropriate concentration to be used for keying in standard concentration data
must be calculated. Do not do this for microwave digested samples.
6.2.4.1 Tare a 100ml polyethylene beaker on the electronic scale.
6.2.4.2 Weigh a sample cup.
6.2.4.3 Record the weight on the extraction log, and zero the scale.
6.2.4.4 Repeat 6.1.1 through 6.1.3 for all sample cups.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
6.2.4.5 Apply the following formula to calculate the final concentration of the
extracted standards:
C, = C, * V,/V, ;
where,
C, is the prepared standard concentration,
C, is the final concentration in the standard.
V, is the volume of prepared standard used,
and V-, is the total volume of the extract
For example if 5 mL of 0.500 ppb standard was added to the digestion
tube and the final volume of the extract is 43 mL, the resulting standard
concentration is as follows:
C, = 0.500 ppb * 5 mL/43 mL = 0.058 ppb.
6.2.4.6 Record this concentration on the extraction log.
6.2.5 Entering Standard Concentrations
6.2.5.1 Follow instructions on p. 21 of the manual. Enter units in ppb, not ppm
(i.e. 0.500 not 0.00050).
6.2.5.2 Calculations are only carried out to three decimal points, 0.00050 will be
truncated to 0.000.
6.2.5.3 Do not enter terms of units, i.e. ppb, ppm. The final calculation will be in
6.2.5.4 The following are ranges of standards used for analysis. These ranges
have been successful in bracketing low level samples.
Microwave Digestion Standards (ppb) Automated Prepared Standards (ppb)
(Dependent upon total volume of extract.
See section 6.2.4)
0.000 0.0000
0.250 -0.0140
0.500 -0.0280
1.000 -0.0570
2.000 -0.1140
4.000 -0.2280
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Volume 2, Chapter 2
SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
6.2.6 Reset Calibration Intensity Data
Follow instructions on page 22 of manual.
6.2.7 Autosampler Rinse Time
6.2.7.1 Follow instructions on page 23 of manual.
6.2.7.2 Use a rinse time of 60, not 50 seconds. This is the rinse time between
samples in the analyses mode.
6.2.8 Autosampler Rack Entry
6.2.8.1 For basic entry information, refer to page 25 of the manual. For extended
information on macros and advanced command see reference section A-
B-7.
6.2.8.2 What follows is an example of an autosampler rack file. It was used for
analyses of Green Bay sediment. Prepare the file before analyses.
NOTE
Actual sample weight must be multiplied by 1000 to obtain results in ug/g.
Total volume = extraction volume * dilution factor.
6.3
1 OPPB 1.000
2GII25F102SQ* 323.00
250.00
SRM diluted lOx
3GB88-71
4 GB89-73
5 GB89-74
280.00
241.00
5.700
125.00
125.00
125.00
extracted in 25mls
Sample diluted 5x extracted
in 25mls
A microwave digested SRM will need to be diluted 10 to 20 times
depending upon the weight of the sample to be within range of standards.
Certified value for SRM2704 = 1.47 ug/g. For a sample that has a
extraction weight of 0.250g in 25mL, (1.47*.250)725=0.018375
ppm=18.37 ppb. This is diluted 20X = .918 ppb
Analysis of Extracts
6.3.1 Filling Autosampler Cups
6.3.1.1 Recycle the 50c/r acid rinse used in the autosampler cups. Place it in the
skiss SUPRAPURE acid bottle.
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Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
6.3.1.2 Rinse each cup 5-10 times with MSQ.
6.3.1.3 Allow cups to air dry
6.3.1.4 Fill each standard cup with its designated standard.
6.3.1.5 Using two separate EDP pipettors, prepare a clean lOmL tip and lOOOuL
tip. Rinse each tip three times with 5% nitric acid followed by three
rinses of MSQ.
6.3.1.6 For samples and SRMS requiring dilution, dilute to at least half their
capacity of the autosampler tips (6mL). First add the required volume of
diluent with the lOmL tip and then the required amount of the sample.
An SRM will need to be diluted 10 to 20 times depending on weight of
sample. For dilutions of auto-digested samples use 0 ppb standard that
has undergone digestion. For microwave digested samples use 10%
Seastar nitric acid.
6.3.1.7 Mix the sample 3-5 times with the lOOOuL tip.
6.3.1.8 Use a new precleaned lOOOuL tip for each sample.
6.3.2 Calibrate the Instrument
Calibrate the instrument using the Macro CAL245 (p. 26 in PS200 instrument
manual). Use a 5-point calibration curve that includes a zero standard. If an
acceptable correlation coefficient is obtained (0.995) and a standard's intensity is
within a the range expected, continue with SRM analyses. See Appendix A for
historic performance of the instrument
6.3.3 Analyze the SRM
Analyze the SRM. Refer to page 25 in manual for autosampler start to finish
sequence and reference section A-B 7.
6.3.4 Check Standards
6.3.4.1 Run check standards every 10 samples to ensure the instrument has not
drifted from its calibration ranse.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
6.3.4.2 Acceptable Check Standard Ranges are:
Microwave Digestion Automated Digestion
0.250ppb = 15% ~0.014ppb = 15-20%
0.500ppb=10% -0.028 ppb = 15%
1.00ppb=10% ~0.058ppb= 10%
2.00ppb=10% -0.115 ppb = 10%
6.3 4.3 Refer to reference section c-1 1 for more information.
CAUTION
If check standards fail, recalibrate the instrument. Do not use update slope or
intercept.
6.4 Data File Preparation
Refer to the reference sections D-l, E-l-5, R-3, and R-5 for preparation of post-run data
and computer files in the AP200 Manual. These data references apply to digested and
samples prepared on the Automated Digester.
6.5 Instrument Shutdown
6.5.1 Dispose of 10% Hydrochloric acid rinse in an appropriate container.
6.5.2 Rinse the tray out three times with MSQ.
6.5.3 Transfer remaining tin chloride to teflon bottle in which it was earlier prepared.
6.5.4 Rinse out tin chloride bottle three times with MSQ.
6.5.5 Fill autosampler tray and tin chloride bottle with MSQ, flush for ten minutes.
6.5.6 Use OVERNITE, or SHUTDOWN modes to shutdown the instrument.
6.5.7 If using OVERNITE MODE, check condition of drying tube, to ensure it is not
saturated with moisture.
6.5.8 Repack a new drying tube if necessary.
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Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System Volume 2, Chapter 2
7.0 Suggestions for Successful Analyses
7.1 Allow the autosampler cups and standard cups to rest in 50% nitric acid for at least two hours
before analyses.
7.2 For best results prepare tin chloride and standards as described.
7.3 Prepare a loosely packed drying tube daily. After use, dispose of perchlorate in an appropriate
container. Rinse a drying tube with MSQ and flush lightly with Liquinox. Rinse several times to
eliminate any residual Liquinox.
7.4 Periodically check tin chloride line and liquid gas separator for any blockage.
7.5 Check at seals of teflon tubing of drying tube connection for gas leaks.
7.6 Change tin chloride line and sample line weekly or after four days of continuous use.
7.7 Change drain tubing every two weeks as needed.
7.8 Clean autosampler rails with isopropyl alcohol weekly and oil rails daily. If not sufficiently
lubricated, the autosampler arm will encounter snags or stops.
7.9 Biweekly calibrate the autosampler tip to ensure it is picking up more than three milliliters.
8.0 Literature Cited
Oilman, L. B., 1988. General Guidelines for Microwave Sample Preparation. Revision of July
1988. CEM Corporation, Matthews, NC.
Leeman Labs, Inc., 1991. PS200 Automated Mercury Analyzer Set-up and Operating Manual.
Version of November 1991.
Leeman Labs, Inc., 1993. AP200 Automated Mercury Preparation System Manual. Revision C
(11/20/93).
Rossmann, R. And K. A. Rygwelski, 1996. Standard Operating Procedures for the Release of
Data. Revision 1 (1/18/96) LLRS-QA-SOP-001.
Rossmann. R.. 1992. Standard Operating Procedures for the Routine Review of Data Quality
and Quantity. Revision 0 (12/2/92) LLRS-QA-SOP-002.
Rossmann. R., 1992. Minimum Analytical Quality Assurance Objectives for U. S. EPA Large
Lakes Research Station. Revision 1 (7/28/92) LLRS-QA-001.
Rossmann. R., 1993. Standard Operating Procedures for the Preparation of Materials used for
Ultra-low Trace Element Analyses. Revision 1 (8/9/93) LLRS-MET-SOP-001
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Using the Cold Vapor Technique with the
Volume 2, Chapter 2 Leeman Labs, Inc. Automated Mercury System
Rossmann, R., 1993. Standard Operating Procedures for the Preparation of Materials used for
Ultra-low Trace Element Analyses. Revision 1 (8/9/93) LLRS-MET-SOP-001
Rossmann, R., 1994. Standard Operating Procedures for the Maintenance of the LLRS Trace
Metal Laboratories. Revision 1 (5/4/94) LLRS-MET-SOP-003.
Rossmann, R. and T. Uscinowicz, 1994. Standard Operating Procedures for Analysis of Total
Mercury in Tissue and Sediment using the Cold Vapor Technique with the Perkin-Elmer
Model MHS-20 Gold Amalgam System. Revision 1 (5/6/94) LLRS-MET-SOP-010.
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
APPENDIX: HISTORIC PERFORMANCE FOR STANDARDS ON PS200.
Daily Standard Intensities and Percent Drift for Standards. Italicized dates are check standards.
Date of
Analysis
01/17/96
01/18/96
01/24/96
01/25/96
01/29/96
01/31/96
02/01/96
02/05/96
02/05/96
02/06/96
2/6/96
2/6/96
2/6/96
02/08/96
2/8/96
2/8/96
02/13/9
2/13/96
2/13/96b
2/13/965
02/15/96
2/15/96
2/15/96b
Begin New Lamp
03/04/96
03/04/96
03/04/96
03/05/96
03/05/96
03/06/96
0.250
11567
15405
14521
12899
12153
13360
15429
13614
13675
13628
12642
32237
31507
299 1 8
22484
22151
Standard Concentration (ppb)
0.500 1.000 2.000
30807
24577
27507
25177
30207
25133
26287
28271
28294
21563
25966
24807
23963
31433
32989
31222
27169
30254
26879
28977
24557
21928
44465
46352
41993
65754
64815
45823
62450
52846
62220
55494
54795
47097
52552
55116
57208
43415
47569
49621
48835
62122
62558
60391
59876
63307
58308
60081
57516
54759
53348
277 1 3
30143
19879
24625
14069
90596
128462
112763
127897
117531
108733
110427
113649
107236
116260
95356
103997
102217
99624
124122
121403
120489
121738
125174
121548
122166
118139
111736
112076
233412
226759
217858
264984
259107
180965
5.000
319499
289297
309450
279308
283705
280385
286800
262538
287149
258884
263870
259967
251241
312746
316603
312282
299635
304689
297642
278297
286801
268327
270167
620712
626601
598765
628674
609 1 1 3
422997
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SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
Volume 2, Chapter 2
APPENDIX: Continued.
Daily Standard Intensities and Percent Drift for Standards. Italicized dates are check standards.
Date of
Analysis
03/07/96
03/07/96
03/1 2/96
03/14/96
03/14/96
03/14/96
03/14/96
03/15/96
03/15/96
03/19/96
03/22/96
03/26/96b
03/28/96'
03/28/962
03/29/96
03/29/96
03/29/96
03/29/96
04/03/96
04/09/96
04/09/96
04/09/96
04/11/96
0.250
21132
19282
23058
24152
24206
24008
25333
23274
23430
22930
23663
27619
22912
21975
20843
20494
42012
40964
21043
Standard Concentration (ppb)
0.500 i.OOO
44232
39037
37940
46864
47468
47189
47491
48936
48272
35727
43189
45156
44270
53606
47100
44362
44504
42173
41875
42323
95743
79923
79106
96048
95645
96993
94850
98592
98146
81247
90404
84083
79330
109573
96970
90593
85748
83552
81646
89779
2.000
194749
202894
166230
192849
188087
188049
187538
195526
197376
178190
194519
188764
184093
212636
194573
191256
196257
167910
169354
167949
160894
178299
5.000
478754
411516
457716
469761
454648
453447
485709
473520
469406
493374
465947
457756
523307
483750
474460
429912
419310
401227
444557
Old Tubing
: New Tubing
2-502
-------
Volume 2, Chapter 2
SOP for Analysis of Sediment for Total Mercury
Using the Cold Vapor Technique with the
Leeman Labs, Inc. Automated Mercury System
APPENDIX: Continued.
Daily Standard Intensities and Percent Drift for Standards Prepared with Automated Digestion
System. Italicized dates are check standards.
Date of
Analysis
06/11/96
06/13/96
06/18/96
07/03/96
07/09/96
07/12/96a
07/12/96b
07/16/96
07/17/96
07/24/96
07/25/96
08/01/96
08/15/96
08/16/96
09/18/96
-0.014
927
1175
980
600
895
1069
816
Standard Concentration (ppb)
-0.028 -0.056 -0.114
1937
2162
1867
1973
2124
1923
1728
1678
1819
4278
4275
4138
5043
4098
4308
4457
4146
4492
3989
3910
4189
360
8331
8689
8979
8057
8369
8088
8118
8488
8454
8043
7546
7908
7977
8468
7382
-0.228
17993
17995
15743
18117
17318
17009
17285
16962
16190
17072
15964
15077
2-503
-------
Mercury in Plankton
Edward A. Nater and Bruce D. Cook
Department of Soil, Water, and Climate
439 Borlaug Hall
University of Minnesota
St. Paul, MN 55108
Octobers, 1996
-------
Mercury in Plankton
1.0 Subsample Collection
Samples were collected from the sampler cup at the base of the plankton net or the phytovibe nets
by backwashing with lake water. The portion of the plankton suspension to be analyzed for
mercury was then transferred to a 500 mL PFA Teflon jar and covered with its screw-on lid. This
sample was carried into the clean room where a 20-30 mL subsample of the plankton suspension
was collected using cleunroom techniques. A 10 mL automatic pipetter was used to transfer the
sample to a cleaned, tared 30 mL PFA Teflon sample vial, which was sealed in a pre-marked
polyethylene zipper bag. The bagged sample was then placed in a second polyethylene zipper bag
which had been marked with the sample identifier. Once the log book had been filled out with
sample number, location, type, date, initials, and observations, the sample was frozen.
2.0 Sample Containers
Samples were collected and stored in PFA Teflon vials and jars. These containers produce
negligible Hg contamination of samples and can withstand extremely rigorous cleaning methods.
New containers were purchased for this study.
3.0 Container Labeling
PFA Teflon is difficult to mark: laboratory ink markers do not produce permanent markings and
adhesive labels do not stick well to PFA Teflon. Consequently, sample bottles were marked by
engraving a unique sample number on both the bottle and the cap. These unique identifying
numbers consisted of a letter followed by a two digit number (e.g., A07). The unique identifying
number was used to track samples through all phases of sample collection, storage, processing, and
analysis, and was cross-referenced to all sample attributes.
Polypropylene containers were used for storage of some reagents and solutions, and polyethylene
bags were used for sample bottle storage; all were marked with permanent laboratory markers.
4.0 Cleaning of Sample Containers
Sample containers and other plasticware that may come in contact with samples were cleaned by
one of the following two methods, depending on their composition.
4.1 Cleaning Procedure A (PFA Teflon and Other FluoropolymerM
Items (sample bottles, stirring rods, large sample containers) were:
Washed in hot tap water with a laboratory detergent (Alconox or similar product)
combination with a laboratory surfactant (Vers-A-Kleen or similar product).
Rinsed repealed!) in hot tap \\ater.
Rinsed thorousihK in doublv-deionized water
2-507
in
-------
Mercury in Plankton Volume 2, Chapter 2
• Acid-washed in 507r HC1 maintained at 70°C overnight.
• Rinsed thoroughly in doubly-deiomzed water.
• Soaked for four hours (minimum) in doubly-deionized water.
• Either:
- dried in an inverted position on clean polystyrene racks in a dust-free
environment; or
filled with 1 % (v/v) HC1 and sealed until use.
• Sealed with its own cap.
• Sealed in a polyethylene zipper bag subjected to cleaning Procedure B below. This bag
was marked with the unique sample bottle identifier.
• Once sealed in the "inner" bag, the sealed sample bottle plus inner bag was sealed in a
second, "outer", zipper bag subjected to cleaning Procedure B. This bag was also marked
with the unique sample bottle number.
• Sample bottles sealed in inner and outer bags were stored in large polyethylene bags in
cabinets in the laboratory. They were stored in clean coolers (ice chests) or PVC dry bags
(the kind kayakers use) for transfer to, and during use on, the EPA lakes sampling vessel.
4.2 Cleaning Procedure B (Polypropylene, Polyethylene)
Items (pipette tips, polyethylene zipper bags) were:
• Washed in hot tap water with a laboratory detergent (Alconox or similar product) in
combination with a laboratory surfactant (Vers-A-Kleen or similar product).
• Rinsed repeatedly in hot tap water.
• Rinsed thoroughly in doubly-deionized water.
• Acid-washed overnight in room temperature 30r/r HCI.
• Rinsed thoroughly in doubly-deionized water.
• Soaked four hours minimum in reagent-grade vsater.
Dried in an inverted position on clean polystyrene racks in a dust-free environment.
• Sealed in polyethylene bags that have been similarly cleaned.
2-508
-------
Volume 2, Chapter 2 Mercury in Plankton
5.0 Example Sample Label
Samples were uniquely coded at the time of collection including the unique sample bottle number,
date, location, phase collected, sample volume, and whether they are duplicates or field blanks.
An example of a sample label would be the following:
A07 LM 5 6-16-94 Z 30 mL a
identifier lake station date phase volume replicate
The above example indicates a sample placed in sample bottle A07. from lake Michigan.
Station 5, taken on June 16, 1994, zooplankton, a 30 mL subsample, and is replicate "a1 All
pertinent station information were cross-referenced and recorded in the field notebook.
Identification of preservation methods on the label were not necessary as all samples were
preserved in the same manner.
6.0 Calibration Procedures and Frequency
All field equipment, with the exception of the plankton nets, was maintained and cleaned by U of
MN personnel. Field notebooks noted problems with field equipment. The cold vapor atomic
fluorescence spectrometer (CVAFS) used for Hg analysis in this study was subjected to checks for
resolution, sensitivity, and reproducibility of response factors (mV/mass Hg) before and during
analysis of each batch of samples. Instrument calibration curves were produced before and during
analysis of each batch of samples. All standards were obtained from commercial sources.
Logbooks recorded dates of calibration, names and concentrations of standards used, result of
calibration, and any corrective action needed and taken.
7.0 Sample Preparation
Prior to analysis, samples were prepared by lyophilization (freeze-drying). The operational status
of the freeze-dryer was checked before sample preparation began. Double-bagged sample bottles
were removed from the freezer and carried into the cleanroom. Label information was recorded
for each sample placed in the freeze-dryer. Using clean-hands/dirty-hands techniques, the samples
were removed from their outer bags and the sample and its inner bag were placed in a sample tray.
In the cleanroom the inner bags were opened and the lids of the samples were loosened but
remained on top of the samples. The status of the dryer and the samples was checked at least once
during the first half hour of the process and then daily thereafter.
Upon removal from the freeze-dryer (usually five days) the sample bottles were tightly sealed and
the inner bags closed. The samples were prepared for digestion back in the cleanroom.
8.0 Sample Digestion
A portion of the lyophilized sample (20 ± 10 mg) was transferred to a clean, pre-weighed 5 mL
conical-bottom PFA Teflon digestion vessel and the weight recorded to ±0.1 mg. For zooplankton
samples. 4 mL of a 1 • 1 concentrated sulfuric (H:SO,) and nitric ( HMD,) acid mixture was added to
the digestion vessel which was tightly sealed. For ph\topLmkton samples and zooplankton
samples weishins less than 10 me. 2 mL of a 1:1 acid mixture was added. The digestion \essds
were triple-bagged, and then placed in a hot (70 "Cl water haih mernight.
2-509
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Mercury in Plankton Volume 2, Chapter 2
9.0 Sample Analysis
Samples were analyzed by cold vapor atomic fluorescence spectroscopy (CVAFS) using the
double amalgamation technique of Bloom and Crecelius (1983) as described in Claas (1996). The
oxidizing acids, H,SO4 and HNO,. were pre-reduced by reaction with hydroxylamine
hydrochloride, which was added to the bubbler flasks prior to the introduction of the sample.
One mL of the digestate was added to the bubbler and allowed to react with the hydroxylamine
hydrochloride for five minutes prior to the addition of 0.5 mL of the stannous chloride (SnCK)
reducing agent. A sample gold trap was placed on the bubbler behind a soda-lime trap and 0.5 mL
of stannous chloride to the sample. The bubbler was sealed and the N2 gas was turned on at
350 mL min'1 The sample was then allowed to bubble for 20 minutes.
The sample gold trap was then removed from the bubbler and placed in the analytical system
upstream of the analytical gold trap. The argon (Ar) flow was turned on and calibrated to 50 mL
min ' The automatic sequence controller was then turned on, which heats the sample trap to
thermally release its Hg onto the analytical trap, then heated in turn to release the Hg from the
analytical trap into the CVAFS analyzer. The peak height and area were recorded for each sample.
10.0 QA
Standards, blanks, and bubbler blanks were run at the beginning and during each set of samples.
Knowns (NIST standard reference materials, generally citrus leaves or apple leaves) were run
along with the standards. Blanks, duplicates, and spiked samples were also interspersed with the
samples such that the total number of QA samples constituted 25% of all samples run.
11.0 Data Reduction
Bubbler blank peak areas were subtracted from the peak areas of the standards and a response
factor (ng peak area'1) was calculated for each of the standards (0.2, 0.4, and 0.8 ng Hg). The
mean response factor was then used to convert peak areas for the samples into quantities of Hg
analyzed. Values were then converted to concentrations of Hg per dry weight of plankton (ns a ')
by using the dilution factors and sample weights.
2-510
-------
Versatile Combustion-Amalgamation
Technique for the Photometric
Determination of Mercury in Fish
and Environmental Samples
Wayne A. Willford and Robert J. Hesselberg
Great Lakes Fishery Laboratory, Bureau of Sport Fisheries and
Wildlife, Fish and Wildlife Service, U.S. Department of the Interior
Ann Arbor, Ml 48107
and
Harold L. Bergman
Department of Fisheries and Wildlife, Michigan State University
East Lansing, Ml 48823
1973
-------
Acknowledgments
This method was originally published as:
Willford, W.A., Hesselberg, R.J., and Bergman, H.L., "Versatile Combustion-Amalgamation Technique
for the Photometric Determination of Mercury in Fish and Environmental Samples", Journal of the
Association of Official Analytical Chemists. Vol. 56, No. 4 (1973).
Permission has been granted by AOAC International to reprint this method as a part of the Lake Michigan
Mass Balance Methods Compendium.
-------
Versatile Combustion-Amalgamation Technique for the Photometric
Determination of Mercury in Fish and Environmental Samples
1.0 Overview
Total mercury in a variety of substances is determined rapidly and precisely by direct sample
combustion, collection of released mercury by amalgamation, and photometric measurement of
mercury volatilized from the heated amalgam. Up to 0.2 g fish tissue is heated in a stream of CK
(1.2 L/min) for 3.5 min in one tube of a two-tube induction furnace. The released mercury vapor
and combustion products are carried by the stream of O-, through a series of traps (6<7c NaOH
scrubber, water condenser, and Mg(CIO4)2 drying tube) and the mercury is collected in a 10 mm
diameter column of 24-gauge gold wire (8 g) cut into 3 mm lengths. The resulting amalgam is
heated in the second tube of the induction furnace and the volatilized mercury is measured with a
mercury vapor meter equipped with a recorder integrator. Total analysis time is approximately
8 min./sample. The detection limit is less than 0.002 ug and the system is easily converted for use
with other biological materials, water, and sediments.
Concern over mercury contamination in the environment has resulted in a rapid proliferation of
methods in which the principle of "flameless atomic absorption" is used for the determination of
mercury (I-4). Common to all of the flameless methods is the production of an elemental mercury
vapor which can be measured photometrically. Methods generally differ only in the means by
which the mercury is released from the sample and in the steps taken to remove interferences.
Mercury is commonly released by acid digestion, followed by reduction and aeration (5, 6) or
amalgamation and heating (7, 8); direct combustion (9, 10); or various combinations of these and
other less common techniques (11-15). Our experience has shown that present flameless methods
tend to be deficient in one or more of the following areas: sensitivity, accuracy and precision,
effort or time required for analysis, ease of adaptability to various sample matrices, freedom from
error due to sample or reagent contamination, and safety of operation.
This paper describes a method in routine use at the Great Lakes Fishery Laboratory for the
determination of total mercury in fish and other environmental samples. The method combines
several proven techniques into a unit that is simple in design and operation and adequately meets
the criteria defined above. Mercury is volatilized from the sample in a stream of oxygen by means
of combustion in a high frequency induction furnace. The mercury vapor is carried along with
combustion products and other volatilized materials from the sample by a gas stream through a
series of traps (sodium hydroxide, water condenser, and magnesium perchlorate) to reduce
interferences. Final separation of mercury from possible interferences is accomplished by
amalgamation on gold. The amalgam is then heated in the induction furnace and the released
mercury is measured in a mercury vapor meter. Total analysis time is about 8 mm./sample and a
single analxst can make up to 40 determinations in eight hours.
2-515
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples Volume 2, Chapter 2
2.0 Method
2.1 Apparatus and Reagents
2.1.1 High frequency induction furnace.--Laboratory Equipment Corp. Model 523000 (Leco)
two-tube induction furnace equipped with Type "L" conversion for combustion sulfur
analyses of hydrocarbons and Type "C" conversion for gasometric or gravimetric carbon
analyses of iron and steel (Type "C" combustion tube removed). See Fig. 1.
2.1.2 Induction furnace accessories.--Leco No. 84 variable transformer for control of power
input to induction furnace. Ceramic crucibles (Leco No. 528035) and Vycor insert
crucibles (Leco No. 550183) with silicon carbide crucible covers (Leco No. 763212).
2.1.3 Spectrophotometer.-Laboratory Data Control Model 1235 'mercury monitor, with
Beckman Model 1005, 10 mv recorder equipped with Disc integrator.
2.1.4 Sodium hydroxide gas wash trap.-125 mL gas washing bottle (Corning No. 31770)
containing ca 35 mL 6 % (w/v) NaOH in distilled water. Renew daily. Solution should
be prepared before use (1 L) and aerated to remove traces of mercury found in reagent
NaOH.
2.1.5 Water condenser-.-28 X 200 mm od separable vacuum trap (Corning No. 7729) with tube
portion immersed in ice bath. Empty as needed to prevent bubbling.
2.1.6 Drying tube.-\8 X 150 mm od drying tube filled with anhydrous magnesium perchlorate
(Mg(CIO4):. Replace daily.
2.1.7 Amalgamator.-Quartz tube (Fig. 2) containing 8 g 24-gauge gold wire cut into short
lengths (ca 3 mm) supported by a very coarse glass disk over a layer of silicon carbide
chips (used to preheat gas stream).
2.1.8 Flow meters.-Two gas controller-flow meters with 0-5 L/min. capacity (Matheson Model
62OBBU).
2.1.9 Heating tape.-2.5 X 610 cm "Briskeat" heating tape (96 watts) connected to
STACO, Inc., Type 500B (120 v) variable transformer.
2.1.10 Mercury standard solution.-! 1) Stock solution.-1000 ug/mL. Dissolve 0.1 358 g HgCI, in
100.0 mL water or use commercially available standard solution. (2) Working solntion.-
As required to give desired ug in 0.05-0.2 mL water for particular range of sensitivity.
2-516
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2, Chapter.
Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples
2-517
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples
Volume 2, Chapter 2
11 mm I.D.
13 mmO.D.
Gold
Wire
Silicon Carbide
Chips
Quartz Disks
Glasswool
6mmO.D.
Fig. 2—Detailed drawing of gold amalgamator.
2-518
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Volume 2, Chapter 2 Environmental Samples
3.0 Assembly of Apparatus
Connect a regulated cylinder of compressed oxygen, using flexible tubing so that the gas stream
passes through controller-flow meter, the reference cell of the mercury vapor meter, and the gas
inlet post of the tray assembly on the Type "L" arm of the induction furnace (Fig. 3). Connect the
Type "L" tube, with ignitor installed, to the NaOH gas wash bottle with Teflon or glass tubing.
Wrap exposed portion of combustion tube and connecting tube to gas wash with heat tape
controlled by a 1 lOv variable transformer. Maintain internal temperature of connecting tube in
excess of 100 :C to prevent condensation in line leading to the NaOH gas wash. Connect NaOH
gas wash in series to the water condenser, drying tube, and bottom of the amalgamator, using
Tygon tubing.
Remove lower guard, tray assembly, and combustion tube from Type "C" arm (carbon analysis) of
induction furnace. Install amalgamator in modified arm by attaching it to center post of raising-
and-locking assembly for that arm. Curved metal spatula ("Scoopula") bent at 90° and inserted
into center post serves as adequate bed for attachment of amalgamator. Adjust height of
amalgamator on center post so that entire column of gold and silicon carbide is completely within
induction coil when locked in raised position. Connect top of amalgamator to absorption cell of
mercury analyzer with flexible tubing and run exhaust tube from outlet of absorption cell to fume
hood; install second flow meter in this exhaust tube to detect possible leaks in system.
Before using system, ensure that all connecting lines and glassware are free of mercury
contamination or residues of acid. Flush sample combustion tube and connecting line to NaOH
gas wash bottle with 6% (w/v) NaOH daily. This precaution is required to prevent accumulation
of acids in line which may trap mercury and reduce recoveries.
4.0 Determination
Establish 1.2 L/min flow of oxygen in system, using flow meter on gas inlet line. Check for gas
leaks by comparing readings with exhaust flow meter. Weigh 0.05-0.1 g sample into silica
crucible and place silicon carbide cover over crucible. Place covered crucible on ceramic pedestal
of Type "L" arm of induction furnace. Adjust setting on variable transformer that controls power
to induction furnace to read 80. Lift raising mechanism on Type "L" arm, locking sample into
position in induction coil, and heat for 3.5 min. Second arm of furnace (amalgamator) must be in
lowered position with gold out of induction coil during this period. After combustion of the
sample, lower crucible from coil and place crucible and cover in safe place to cool.
Re-establish gas flow in system by locking raising mechanism of Type "L" arm into raised position
without crucible. Adjust variable transformer to setting of 60, raise and lock gold amalgamator
into position, and heat until recorder peak returns to baseline (1-2). Lower gold amalgamator and
allow to cool (ca 1 min). Count integrator sweeps under peak and compare with standard curve
prepared by heating standard solutions, using same operating procedures as above. Because of
porosity of silica crucibles, use of Vycor liner is required for standard solutions and other liquid
samples. It is recommended that volume of standards used in system not exceed 0.2 mL.
2-519
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples
Volume 2, Chapter 2
Fum* Hood
or Vint
>-
Floo
Mel.r
sompl* Combustion Tub«|
T»o-Tub«
Induction Furnace
j Gold Amotgomolor
Fig. 3—Combustion amalgamation system for the photometric determination of mercury.
Silicon carbide covers can be used for an indefinite number of samples. For rapid analysis of
several samples, two covers are alternated to allow adequate time for cooling between samples.
Silica crucibles are also reusable; however, it is recommended they be fired at 800°C for two
hours in a muffle furnace between uses, to prevent accumulation of organic residue.
5.0 Results and Discussion
Operating Procedures
The described rate of oxygen flow and settings used on the variable transformer that controls the
rate of heating in the induction furnace give a good balance between sensitivity, precision, and
time of analvsis for samples of fish containing 0.02-5.0 ppm mercury. Samples in this range are
analyzed by attenuating the mercury analyzer and adjusting the sample size. Typical analytical
ranges used are 0.002-0.01, 0.01-0.05, and 0.05-0.25 ug. Fish samples larger than 0.2 g can
overload the system and greatly reduce accuracy of the results.
For analysis of samples outside the range of 0.02-5.0 ppm a change of operating procedure is
required. Sensitivity can be increased by reducing the rate of oxygen flow or reduced by
increasing the rate. This technique, within limits, effectively increases or decreases the
concentration arid the retention time of the mercury vapor in the absorption cell of the mercury
analyzer, resulting in the altered sensitivity. Installation of a stream splitter in the gas line between
the sample combustion tube and sodium hydroxide gas wash also effectively reduces sensitivity.
Precision may be reduced and time required for analysis increased, however, at altered flow rates.
The heating rate and temperature of the gold amalgamator can be varied by adjusting the variable
transformer attached to the induction furnace; this results in a large alteration of observed peak
height on the recorder tor a given amount of mercury atid oxygen flow rate. The integrator counts
vary only slightly, however, for settings on the transformer between the values of 50 and \(W7< It
is this characteristic \\hich makes it mandatory to use area measurement (integrator counts) m
place of peak height to ensure reproducible results. The heating rate and temperature obtained in
the induction field at a given power setting are functions of geometry, mass, density, and puriu of
the amalgamator. After repeated use, the heating characteristics of the amalgamator slowly
2-520
-------
Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Volume 2, Chapter 2 Environmental Samples
change, as evidenced by increased peak width and lowered height. This change is probably due to
a gradual accumulation of oxides and organics on the amalgamator (8. 13). Periodic removal of
the amalgamator (semi-monthly) and firing of the gold in a muffle furnace at 800°C for two hours
generally restores the amalgamator to its original characteristics. The use of an integrator
compensates for the changing characteristics of the amalgamator while it is in use.
6.0 Precision and Accuracy
The average recoveries of mercury (HgCU) from fortified samples offish muscle, lake sediment,
aquatic vegetation, and bituminous coal were 94-105% for fish muscle and 94-1 129? for all
materials tested (Table 1). The average recovery for all materials and levels tested was 102.7%
(coefficient of variation, 5.5%). Our inability to obtain truly homogenous sediments of constant
mercury background undoubtedly contributed to the wider variation in recoveries from sediment.
Limited testing of fish samples fortified with methyl mercuric chloride gave recoveries within the
range obtained with mercuric chloride. The accuracy of the method was determined on samples of
fish flesh and sediments analyzed concurrently by several laboratories (Table 2). Values obtained
by the described method (Method CA) agree well with average values obtained by other
laboratories using acid-digestion, flameless atomic absorption, and neutron activation techniques.
To determine the reproducibility of the method over a period of time, we collected, composited,
and homogenized large amounts offish muscle containing different levels of mercury
contamination to form a source of uniform reference material for analysis. Samples of this
material were analyzed daily along with unknown samples. Table 3 presents a statistical treatment
of data accumulated from these repeated analyses. The values given should include nearly every
source of routine error that can be expected when this method is used, since they represent over
100 days of testing. Sample replication on any given day generally gave a coefficient of variation
less than 10% and routinely 5% or less, as indicated in Table 1. Failure of standard solutions or
check samples to give at least ±10% agreement indicates that the analytical system should be dis-
mantled and cleaned before routine analyses are performed.
2-521
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples
Volume 2, Chapter 2
Table 1. Recoveries of mercury (HgCl,) added to samples of fish
muscle, lake sediment, vegetation, and coal
Sample and
amount of
mercury added.
No. of
replicates
Av. amount
recovered."
Coeff. of
van, %
Fish muscle (coho salmon)
0.01
0.03
0.06
0.20
6
6
6
6
94.4
104.9
98.5
104.0
5.8
3.7
3.1
4.7
Sediment (Lake Michigan)
0.03
0.06
112.2
96.2
2.1
11.5
Vegetation (chara)
0.03
0.06
102.2
106.8
5.8
0.6
Coal (bituminous)
0.03
0.06
109.8
99.3
2.4
3.1
J Values corrected for following background (ppm) in samples: fish muscle. 0.093; sediment, 0.032; vegetation.
0.016; and coal, 0.081.
2-522
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Volume 2, Chapter 2
Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples
Table 2. Average mercury content (ppm) in fish flesh and sediments as determined by the described
method and other methods of analysis
Sample"
CA
Method"
FAAS
NAA
Fish flesh
1
9
Sediment
0.22(1)
0.83(1)
2.1 (1)
0.17(12)
0.76(12)
2.4 (12)
0.92(2)
2.5 (2)
4
5
6
0.1*
107
45
5(1)
(1)
(1)
0.17
110
45
(9)
(20)
(20)
—
120
44
(1)
(1)
a Samples ]-4: Mercury in Fish and Sediment Round-Robin-1971, Ontario Water Resources Commission, Division
of Laboratories. Samples 5 and 6: Mercury in Sediment Round-Robin-1972, Environmental Protection Agency,
Region IV, Surveillance and Analysis Division.
b Methods used: CA, combustion-amalgamation; FAAS, flameless atomic absorption (variety of digestion mixtures),
NAA, neutron activation analysis. Numbers in parentheses show the number of laboratories participating.
Table 3. Accumulated data on the precision of replicate analyses
of fish flesh during several weeks of testing
Sample
A
B
C
D
No. of
Analyses
r
258
60
65
186
No. of
Days
58
16
16
49
Av. Concn,
PPM
0.0883
0.399
0.774
2.82
Std dev.
0.0123
0.0485
0.0837
0.214
Range
0.0563
0.310
0.596
2.24
-0.141
0.569
1.09
3.20
Coeff.
var c/<
1 3 9
122
10 S
7.6
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples Volume 2, Chapter 2
7.0 Sample Adaptation
The data given in this paper were derived primarily from "clean" samples of fish muscle,
vegetation, lake sediments, and coal. We found, however, that some samples of individual tissues,
as well as of whole fish, caused an acidification in the lines, resulting in low recoveries of
mercury. The problem was eliminated by air-drying the samples overnight at room temperature to
remove most of the water. We have adopted this practice for all samples of whole fish. Care must
be taken, however, to ensure that no mercury contaminated air or dust reaches the samples during
this drying period.
Since the heat supplied by the silicon carbide cover used during sample combustion may not be
adequate for samples other than those tested (e.g., geological materials or metals), we also tested a
quartz-enclosed silicon carbide crucible (Leco No. 550182) in place of the silica crucible. This
crucible was satisfactory for samples of low organic content but not for biological materials
because combustion was apparently too rapid and caused erratic results. The addition of
"activators" of metal (copper and iron) to the sample gave the same results; they, too, were
unsatisfactory for biological materials and, in addition, had high and variable background levels of
mercury.
Water can be analyzed directly in the system, with a sensitivity as low as about 10 P.B., by using
the same analytical procedure used for standard solutions. However, a superior and much more
sensitive method is a modification of the procedure described by Kala (13). In this modification, a
100 mL water sample is heated with sulfuric acid, nitric acid, potassium permanganate, and
potassium persulfate; decolorized with hydroxylamine hydrochloride; reduced with stannous
chloride; and aerated in a gas wash bottle (16). The released mercury vapor is passed through
magnesium perchlorate and amalgamated on gold as described earlier in this paper. A sensitivity
of less than 0.02 P.B. can be obtained with this technique.
Of the various materials we have tried in the system, only those which are volatile or explosive in
nature could not be analyzed. Samples of volatile solvents have been analyzed after evaporation to
dryness, but this technique is subject to loss of mercury from the sample. Extraction and
concentration of the mercury into an aqueous system which can be analyzed similarly to standard
solutions is a useful technique.
8.0 Conclusions
The described method has been found to be a reliable and rapid technique for the precise
determination of total mercury in a variety of samples. Materials that do not lend themselves to
direct analysis by the method as described can be accommodated by minor changes in equipment
or sample preparation. Routine use of the method at the Great Lakes Fishery Laboratory has
shown it to be superior to previously tested methods for use with fish tissue, and preliminary work
strongly suggests that it is superior for numerous other t\pes of samples as well.
Major advantages of the method over the normal acid-digestion, flameless atomic absorption
techniques include: siinplicit\ of operation; speed of complete analysis; high sensitivity, precision.
and accuracy; small sample si/.e required; freedom from rigorous and sometimes hazardous acid
digestion procedures, lixvdom from reagent and glassuare contamination: and comparati\cl\ Km
cost of equipment.
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Volume 2, Chapter 2 Environmental Samples
Disadvantages of the method as described include: somewhat limited usable range of sensitivity
(0.02-5.0 ppm); a general inability to analyze highly contaminated samples (>5.0 ppm) without the
use of a gas stream splatter or an extremely small sample; necessity for frequent changes in
attenuation of mercury vapor meter, unless previous knowledge permits grouping of samples
within ranges having less than a five-fold difference, in concentration; and increased emphasis on
the need for well homogenized representative samples because of the small sample size used. It is
expected that minor changes in the system or substitution of a less sensitive mercury vapor meter
would overcome many of these disadvantages when they are restrictive for a particular use.
9.0 Acknowledgments
We thank Richard A. Stone and James R. Olson for their technical assistance and Phillip T.
Lunsford, Laboratory Equipment Corporation, St. Joseph, Mich., for his many helpful suggestions.
This work was funded in part by the Environmental Protection Agency (formerly the Federal
Water Quality Administration).
10.0 References
10.1 Manning, D.C. (1970) At. Absorption Newsletter 9, 97-99
10.2 Slavin, S. (1971) At. Absorption Newsletter 10, 17-39
10.3 Slavin, S. (1972) At. Absorption Newsletter 11, 7-32
10.4 Slavin, S. (1972) At. Absorption Newsletter 11, 74-88
10.5 Hatch, W.R., and Ott, W.L. (1968) Anal. Chem. 40, 2085-2087
10.6 Munns, R.K., and Holland, D.C. (1971) JAOAC 54@ 202-205
10.7 Fishman, M.J. (1970) Anal. Chan. 42, 14621463
10.8 Okuno, I., Wilson, R.A., and White, R,. E. (1972) JAOAC 55. 96-100
10.9 Herrmann, W.J., Jr., Butler. J.W., and Smith,
R.G. (1970) in Laboratory- Diagnosis of Diseases Caused by Toxic Agents, F. W Sunderman and
F W. Sunderman, Jr. (eds.). Warren H. Green, Inc., St. Louis. Mo., pp. 379-386
10.10 Thomas. R.J., Hagstrom, R.A., and Kuchar,
E. (1972) J. Anal. Chem. 44. 512-515
10.1 1 Thilliez, G.. (1968) Chim. Anal. 50. 226-232
10.12 Lidums, V.. and Ulfvarson. U. (1968) Acta Chem. Scaml. 22. 2150-2156
10 13 Kala. G.W (1970) At. .\/>wi/y>m<» AVu.s/c'mv 9. 84-S7
Id 14 Ukita, T.. Osa\\a. T . Imura. \ . Tonomura. M.. Sa\ato. Y N.iLnmira. K.. Kanno. S., Fukui. S .
Kaneko. M.. Ishikura. S.. Yunaha. M.. and Nakamura. T ( I97()i./. Hyg. Chem. 16. 258-266
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Versatile Combustion-Amalgamation Technique for the
Photometric Determination of Mercury in Fish and
Environmental Samples Volume 2, Chapter 2
10.15 Joensuu, O.I. (1971) Appl. Spectry. 25. 526-528
10.16 Methods for Chemical Analysis of Water and Waste, (1971) Environmental Protection Agency,
National Environmental Research Center, Analytical Quality Control Laboratory, Cincinnati,
Ohio, pp. 121-130
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Analysis of Fish for Total Mercury
Standard Operating Procedure SOP No. HC520B.SOP
Jerome O. Nriagu
University of Michigan
Department of Environmental and Industrial Health
109 Observatory Street
Ann Arbor, Michigan 48109
In Cooperation with:
U.S. Geological Survey
Great Lakes Science Center
1451 Green Road
Ann Arbor, Michigan 48105-2899
May 1, 1996
Version 1.0
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Analysis of Fish for Total Mercury
SOP # HC520B.SOP
1.0 Scope and Application
This method is only tor total mercury measurement after dissolving the fish tissue in concentrated
nitric acid under high pressure and temperature using microwave digestion system. The mercury
in solution will be analyzed using a nondispersive atomic fluorescence spectrometer. Digestion
procedure for samples is described by Feng et al. (1994) and the procedure for instrumental
measurement of mercury is given by Bloom and Fitzgerald. Procedures for collection,
homogenization, and data reporting are covered by other appropriate NBS/GLSC SOPs.
Note: This method replaces HC520A.SOP
2.0 Summary of Method
This method is only for total mercury measurement after dissolving the fish tissue in concentrated
nitric acid under high pressure and temperature using a microwave digestion system. The mercury
in solution will be analyzed using a nondispersive atomic fluorescence spectrometer. Digestion
procedure for samples is described by Feng et al. (1994) and the procedure for instrumental
measurement of mercury is given by Bloom and Fitzgerald. Procedures for collection,
homogenization, and data reporting are covered by other appropriate NBS/GLSC methods
3.0 Interferences
3.1 Contamination in the laboratory will be minimized by processing the samples in an epoxy-coated
plastic chamber equipped with HEPA filers to achieve Class 100 laminar flow conditions. Milli-
Q water and quartz-redistilled acids will be used for sample digestion.
3.2 All the labware (glass or teflon will be decontaminated using the 9-step procedure described by
Nriagu et al. (1993). Some of the key steps include degreasing with soap, sequential washing with
acetone, concentrated nitric and hydrochloric acid, soaking in warm (40-50° C) 2 M nitnc acid for
3 days, and rinsing thoroughly with Milli-Q water. After use, the labware is soaked in 6 M
hydrochloric acid for three days, followed by warm nitric acid for three days and then rinsed with
Milli-Q water. The final rinse is done in the HEPA-equipped chamber, and all the containers are
stored in acid-washed, triple plastic bags. Cleaned volumetric flasks will be filled with 1.0 M
nitric acid and triple bagged for storage.
4.0 Safety
Mercury in the pure form is toxic. Both nitric and sulfuric acid in the concentrated form \vill cause
severe chemical burn on tissue and require use of safety glasses when handling, even at dilutions
containing 20£r of the concentrated form. The hazards of each chemical and reagent used in this
method have been generally defined, but each chemical compound used should he treated as a
potential health ha/ard. A reference file of material safety data sheets is available at the U of M
Lab and in NBS to all personnel involved in chemical analysis.
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Analysis of Fish for Total Mercury
SOP No. HC520B.SOP Volume 2, Chapter 2
5.0 Apparatus and Chemicals
5.1 Teflon-lined pressure bombs, 90 mL
5.2 Sphex CDS 7000 Microwave digestion unit
5.3 Cold vapor atomic fluorescence spectrometer, Rand Corp..Seattle, WA.
5.4 Freeze dryer, model 25 SRC, Virtis, Gardiner, NY
5.5 Balance, Top loading, Sartorius, 1204 (0.01 g) or better
5.6 Ultra pure acids (nitric and hydrochloric)
5.7 Milli-Q water
5.8 Stannous chloride, ACS grade, Fisher Scientific
5.9 Mercury standard stock solution, Perkin Elmer
5.10 Aluminum weighing pan (6 X 2 cm)
5.11 Whatman quartz 47 mm filter membrane, acid washed, #1851-047
6.0 Digestion Procedure
6.1. Homogenized study fish samples are stored frozen in glass screw cap containers until analyzed.
Fish samples are thawed just prior to being weighed for digestion. For each sample from the study,
check, duplicate, or spiked samples about one gram of homogenized tissue is weighed into a pre-
weighed digestion tube. Record both empty weighing pan and weighing pan plus wet tissue
weights which will be used to determine dry weight.
6.2 Digestion
6.2.1 Add 10 mL of concentrated nitric acid to the sample in the digestion vessel and leave the
mixture at room temperature for 30 minutes. Then place the teflon vessel containing the
sample into the double outer liner of the digestion bomb, cap with a sensor head and
pressure rupture disc. Place the sealed vessel in the microwave carousel. Prepare the
remaining samples of the set in the same manner The set must also contain a blank.
spike, duplicate, and reference samples.
6.2.2 After connecting the sensor cables to a port in the oven cavity, the power level and time
for each digestion stage are programmed into the computer controller. To minimize
violent reactions the oven temperature is slowly ramped to the set temperature oxer 20-30
minutes. When the temperature reaches 190 C and pressure 180 psi, heat the sample for
an additional 15 minutes. After the digestion step is completed digestion bombs arc
cooled to room temperature.
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Analysis of Fish for Total Mercury
Volume 2, Chapter 2 SOP No. HC520B.SOP
6.2.3 Each cooled digestion bomb is opened and the clear liquid (fish dissolved in nitric acid) is
diluted to 25 mL using mercury free Milli-Q water. Residues in sample vessel after
digestion is often indicative of incomplete or improper digestion and the sample batch are
re-digested. Sample filtration is not required in completely digested fish tissues.
7.0 Analysis
7.1 An aliquot (0.5 to 1.0 mL) of the digested sample is added to 10 mL of Milli-Q water in a glass
reduction chamber. The mixture is purged with ultra-pure argon for 10 minutes. One mL of
stannous chloride is then added to the mixture to reduce the mercury, and the elemental mercury
formed is stripped (with argon) and collected on gold-coated quartz grains. The trapped mercury
is subsequently desorbed thermally and measured on a Tekran CVAFS Mercury Analyzer Model
2500. The output, as peak height or peak area, is recorded by means of an HP 3396A Integrator.
7.2 Calibration
7.2.1 The calibration curve is prepared by reducing standard solutions containing 1, 2. 4 and 6
ng of mercury. This is done by using 0.5, 1.0, 2.0 and 3.0 mL of standard solution
containing 2.0 ^g/L (or 2.0 ng/mL) Hg. Each aliquot of the standard solution is added to
10 mL of Milli-Q water in a glass reduction chamber. The mixture is purged with ultra-
pure argon for 10 minutes. One mL of stannous chloride is then added to the mixture to
reduce the mercury, and the elemental mercury formed is stripped (with argon) and
collected on gold-coated quartz grains. The trapped mercury is subsequently desorbed
thermally and measured on a Tekran CVAFS Mercury Analyzer Model 2500. The outputs
for the four standard samples, recorded by an HP 3396A Integrator, are used to derive the
average slope for the standard curve in terms of atomic fluorescence units per 1.0 ng of
Hg. "
7.2.2 Calibration of the spectrometer is with a minimum of four serial diluted standards of
mercury covering the range expected in the samples that have been taken through the
digestion and reduction procedure. The calibration range for most samples is expected to
be from about 0.1 3 ng of Hg. If concentrations in the fish are lower the calibration
range will be moved downward since the detection limit of the instrument is 0.01 ng/L of
dissolved Hg.
7.3 Determining the method detection limit
7.3.1 The method detection limit will be determine using the USEPA method of seven
(40 CFR) using spiked fish tissue or other acceptable matrix containing less than 1 ng of
mercury.
7.3.2 When a peak is visible below the established method detection limit a concentration will
be reported but result will be flagged using established EPA codes. When no peak is
detected for mercury the results will be reported as zero.
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Analysis of Fish for Total Mercury
SOP No. HC520B.SOP Volume 2, Chapter 2
7.4 Result
7.4.1 Results will be reported in ng/g (wet weight) or as stated in the data standards being
established by USEPA. Mercury data, including results from calibration, check.
duplicates, spikes, and reference samples will be submitted to NBS-GLSC where they will
be checked by Jim Hickey for consistency with EPA standards and completeness.
Acceptance criteria is shown in Tables 7.1 and 7.2 of the NBS Analytical Quality
Assurance Project Plan for IAG DW14947692-02-0. NBS will submit mercury results to
USEPA using data standards that are currently being finalized.
8.0 References
8.1 Bloom, N.S. and W.F. Fitzgerald. 1988. Determination of volatile mercury species at the
picogram level by low-temperature gas chromatograph with coldrvapor atomic fluorescence
detection. Anal. Chim. Acta208: 151-161
8.2 Feng, Y. and R.S. Barratt. 1994. Digestion of dust samples in a microwave oven. Sci. total
Environ. 143: 157-161
8.2 Nriagu, J.O., Lawson. G., Wong, H.K.T. and J.M. Azcue. 1993. A protocol for minimizing
contamination in the analysis of trace metals in Great Lakes waters. J. Great Lakes Res. 19: 175-
182
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