United States
Environ marital
Protection Agency
Great Lakes
National Proqram
Office
EPA905-R-97-012c
June 1997
Lake Michigan Mass Balance Study
(LMMB) Methods Compendium
Volume 3: Metals, Conventional, Radiochemistry,
and Biomonitoring Sample
Analysis Techniques
Printed on focycbd faptr
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United States Office of Water EPA-905-R-97-Q12c
Environmental Protection 4303 October 1997
Agency
Lake Michigan Mass Balance Study (LMMB)
Methods Compendium
Volume 3: Metals, Conventionals,
Radiochemistry, and Biomonitoring Sample
Analysis Techniques
U.S. EPA
MID-CONTINENT ECOLOGY DIVISION
LSBRARY
DULUTH, MN 55804
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Lake Michigan Mass Balance Study
(LMMB) Methods Compendium
Volume 3: Metals, Conventionals, Radiochemistry,
and Biomonitoring Sample
Analysis Techniques
i Printed on Recyc/ed 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 for 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 Atrazme and Atrazine
Metabolites (Eisenreich, S., Schottler, S., and Mines, 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 14C: 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: FISH
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
LMMB 029
LMMB 030
LMMB 031
LMMB 032
LMMB 033
LMMB 034
LMMB 035
LMMB 036
LMMB 037
LMMB 038
LMMB 039
LMMB 040
LMMB 041
Instrumental Analysis and Quantitation of Polycyclic Aromatic Hydrocarbons
and Atrazine: IADN Project (Cortes, D. and Brubaker, W.) 2-3
Analysis of PCBs and Pesticides in Air and Precipitation Samples : IADN
Project - Gas Chromatography Procedure (Basu, I.) 2-23
Analysis of PCBs, Pesticides, and PAHs in Air and Precipitation Samples:
IADN Project - Sample Preparation Procedure (Basu, I.) 2-61
Analysis of PCBs, Pesticides, and PAHs in Air and Precipitation Samples:
Sample Preparation Procedures (Harlin, K. and Surratt, K.) 2-115
Standard Operating Procedure for the Analysis of PAHs and Atrazine by
GC/lon Trap MS (Peters, C. and Harlin, K.) 2-165
Standard Operating Procedure for the Analysis of PCBs and Organochlorine
Pesticides by GC-ECD (Harlin, K., Surratt, K., and Peters, C.) 2-189
Standard Operating Procedure for Isolation, Extraction and Analysis of
Atrazine, DEA and DIA (Eisenreich, S., Schottler, S., and Hines, N.) 2-243
Standard Operating Procedures for Semivolatile Organic Compounds in Dry
Deposition Samples (Eisenreich, S. and Franz, T.) 2-251
Extraction and Cleanup of XAD-2 Resin Cartridges for Polychlorinated
Biphenyls and Trans-Nonachlor (Crecelius, E. and Lefkovitz, L.) .
2-257
Extraction and Cleanup of Glass Fiber Filters for Polychlorinated Biphenyls
and Trans-Nonachlor (Crecelius, E. and Lefkovitz, L.) ... 2-271
PCB Congener Analysis of XAD-2 Resins and GFF Filters Using GC/ECD
(Crecelius, E. and Lefkovitz, L.) 2-285
PCBs and Pesticides in Surface Water by XAD-2 Resin Extraction (Wisconsin
State Lab of Hygiene) 2-307
Extraction and Cleanup of Sediments for Semivolatile Organics Following the
Internal Standard Method (Van Hoof, P. and Hsieh, J.) 2-325
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.
VI
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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 Metala 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
103-105°C) Volatile Suspended Solids (Ignited at 550°C) (Wisconsin State Lab
of Hygiene) . 3-187
VII
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Table of Contents
LMMB066 Outline of Standard Protocols for DOC Analyses (Shafer, M.) 3'193
LMMB 067 Outline of Standard Protocols for Paniculate 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
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
VIII
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Table of Contents
CHAPTER 3: RADIOCHEMISTRY
LMMB 082 Standard Operating Procedure for Primary Productivity Using UC: 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
IK
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Volume 3
Chapter 1: Metals
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Laboratory Methods for ICP-MS Analysis
of Trace Metals in Precipitation
Jon Talbot and Aaron Weiss
Hazardous Materials Lab
Hazardous Waste Research and Information Center
1 East Hazelwood
Champaign, IL61820
March 1994
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Laboratory Methods for ICP-MS
Analysis of Trace Metals in Precipitation
1.0 Preparation of 20% Nitric Acid for Cleaning Purposes
1.1 Preparation of Nitric Acid Bath
This bath, located in the Air Toxic Metals (ATM) Preparation Lab (see Section 20.2), is used for
cleaning those items which must be soaked, i.e. centrifuge tubes, Teflon bottles, beakers and
graduated cylinders. (See Sections 2.0 and 3.0)
1.1.1 Supplies
Deionized water
Reagent grade HNO3
Polyethylene gloves
1.1.2 Equipment
Polyethylene tank (Nalgene 12 x 12 x 12)
1.1.3 Acid Bath Preparation Procedure
a) Rinse polyethylene tank twice with deionized water.
b) Fill tank with 21 L of deionized water.
c) Measure 6 L of reagent grade HNO3 and add to tank.
(Formula is 1 L deionized water: 286 ml HNO3)
1.1.4 Comments
The acid bath should be remade every two months. Discard old acid bath appropriately.
1.2 Preparation of Nitric Acid to Fill 10 L Polyethylene Carboy
The carboy is kept in the ATM Prep Lab, and the acid is is used to fill volumetric glassware when
cleaning. (See Section 4.0)
1.2.1 Supplies
Deionized water
Reagent grade HNO3
Polyethylene gloves
1.2.2 Equipment
10 L Polyethylene Carboy with spigot
1.2.3 Acid Preparation Procedure
a) Rinse polyethylene carboy twice with deionized water.
b) Initially, fill carboy with 7 L of deionized water.
3-5
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Laboratory Methods for ICP-MS Analysis
of Trace Metals in Precipitation Volume 3, Chapter 1
c) Add 2 L of reagent grade HNO3.
d) As the level of 20% nitric acid in the carboy decreases, replenish it according to
the formula 1 liter deionized water: 286 mL HNO3.
2.0 Cleaning Sample Tubes
Due to problems of zinc contamination, new tubes should be acid-cleaned before use. Although
new tubes are preferable, used tubes can also be cleaned using this method. New tubes should not
be acid-cleaned but rather be used directly from the package if they are to contain samples to be
analyzed for either sodium or aluminum.
2.1 Supplies
ASTM Type I water
Deionized water
20% HNO3 (see Section 1.1)
Polyethylene gloves
2.2 Equipment
Nitric Acid bath (see Section 1.1)
Tube racks
2.3 Cleaning Procedure
2.3.1 Rinse tubes and caps with deionized water, filling each fully and discarding water.
2.3.2 Place tubes and caps in 20% acid bath, making sure that each is fully submerged in the
bath. Soak for no less than 24 hours.
2.3.3 Rinse tubes and caps with ASTM Type I water three times.
2.3.4 Shake off excess water and allow tubes and caps to dry fully using tube racks.
2.3.5 Store tubes and caps in the styrofoam racks in which they are shipped; they can be stored
indefinitely.
3.0 Cleaning Teflon Bottles, Graduated Cylinders and Beakers
3.1 Supplies
ASTM Type I water
Deionized water
20% HNO3 (see Section 1.1)
Polyethylene gloves
3.2 Equipment
Nitric Acid bath (see Section 1.1)
3-6
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Laboratory Methods for ICP-MS Analysis
Volume 3, Chapter 1 of Trace Metals in Precipitation
3.3 Cleaning Procedure
3.3.1 Rinse all items with deionized water, filling each fully and discarding water. Repeat once.
3.3.2 Place all items in the 20% nitric acid bath, making sure each is fully submerged. Soak for
no less than 24 hours.
3.3.3 Rinse each item with deionized water 2-3 times.
3.3.4 Final rinse each item with ASTM Type I water three times.
3.3.5 Completely fill each bottle with ASTM Type I water, cap and store for use in the
designated cabinet in the Air Toxic Metals (ATM) preparation laboratory.
3.3.6 Let all other items dry completely, cover with parafilm, and store for use in the designated
cabinet in the ATM preparation laboratory.
4.0 Cleaning Volumetric Glassware
4.1 Supplies
ASTM Type I water
Deionized water
20% HNO3 from carboy (see Section 1.2)
Polyethylene gloves
4.2 Cleaning Procedure
4.2.1 Rinse all volumetric glassware with deionized water, filling each item fully and discarding
water. Repeat once.
4.2.2 Fill each item with 20% nitric acid and let soak in the hood in the ATM preparation
laboratory for at least two to three hours.
4.2.3 Rinse each item with deionized water two to three times.
4.2.4 Final rinse each item with ASTM Type I water three times.
4.2.5 Completely fill each item with ASTM Type I water, stopper and store for use in the
designated cabinet in the Air Toxic Metals (ATM) preparation laboratory.
5.0 Preparation of 2% Ultrapure Nitric Acid
5.1 Supplies
ASTM Type I water
Ultrapure concentrated HNO3
PoKethylene gloves
3-7
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Laboratory Methods for ICP-MS Analysis
of Trace Metals in Precipitation Volume 3, Chapter 1
5.2 Equipment
500 mL Teflon bottle
25 mL graduated cylinder
5.3 2% HNO, Preparation Procedure
5.3.1 Rinse 500 rnL Teflon bottle twice with ASTM Type I water.
5.3.2 Fill with 500 mL ASTM Type I water.
5.3.3 Measure 14 mL ultrapure concentrated HNO3 and add to bottle.
5.3.4 Invert bottle and mix well. Store in prep area and replenish as needed.
6.0 Multi-element Calibration Standards Preparation
6.1 Supplies
Certified elemental standards (Spex 1000 ug/mL Plasma Standards)
2% Ultrapure HNO3 (see Section 5.0)
Pipet tips
Internal standard solution (see Section 16.0)
Acid cleaned polypropylene 15 mL centrifuge tubes (see Section 2.0)
6.2 Equipment
Pipettes
Eppendorf repeater pipet
Two Acid cleaned 125 mL Teflon bottles (see Section 3.0)
Two Acid cleaned 100 mL volumetric flasks (see Section 4.0)
6.3 Calibration Standards Preparation Procedure
6.3.1 Prepare a 10 |Jg/mL mixed stock standard containing the following elements: Arsenic
(As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Manganese (Mn), Nickel
(Ni), Selenium (Se), Titanium (Ti), Vanadium (V), and Zinc (Zn). Pipet 1 mL of each of
the 1000 ug/mL certified standards into a clean 100 mL volumetric flask. Dilute to
100 mL with ASTM Type I water containing 2% (w/w) ultrapure nitric acid (see
Section 5.0). Invert flask and mix well. Transfer to a clean 125 mL Teflon bottle.
6.3.2 Prepare a 100 ng/mL mixed stock standard. Pipet 1 mL of the prepared 10 ug/mL stock
standard into a clean 100 mL volumetric flask. Dilute to 100 mL with ASTM Type I
water containing 2% (w/w) ultrapure nitric acid. Invert flask and mix well. Transfer to a
clean 125 mL Teflon bottle.
6.3.3 Prepare Blank, 0.1, 0.3, 0.5. 0.7, 1.0, 3.0, 5.0, 7.0 and 10.0 ng/mL calibration standards
daily.
3-8
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Laboratory Methods for ICP-MS Analysis
Volume 3, Chapter 1 of Trace Metals in Precipitation
6.3.4 Into ten cleaned and labelled polypropylene 15 mL centrifuge tubes, pipet 0, 10, 30, 50,
70, 100, 300, 500, 700 and 1000 ^L of the 100 ng/mL multi-element standard
respectively.
6.3.5 Add 10.00, 9.99, 9.97, 9.95, 9.93, 9.90, 9.70, 9.50, 9.30 and 9.00 mL ASTM Type I water
containing 2% (w/w) ultrapure nitric acid (see Section 5.0) to each tube respectively.
6.3.6 Pipet 100 uL internal standard into each tube using an Eppendorf repeater pipet (see
Section 16.0). Cap tubes and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly to
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
7.0 Calibration Standards Preparation for Sodium and Aluminum
7.1 Supplies
Certified elemental standards (Spex 1000 ug/mL Plasma Standards
2% Ultrapure HNO3 (see Section 5.0)
Pipet tips
Internal standard solution (see Section 16.0)
New, unwashed polypropylene 15 mL centrifuge tubes
7.2 Equipment
Pipettes
Eppendorf repeater pipet
One Acid cleaned 125 mL Teflon bottle (see Section 3.0)
One Acid cleaned 100 mL volumetric flask (see Section 4.0)
7.3 Calibration Standards Preparation Procedure
7.3.1 Prepare a 1000 ng/mL mixed stock standard containing Aluminum (Al) and Sodium (Na).
Pipet 100 uL of both the Al and Na 1000 ug/mL certified standards into a clean 100 mL
volumetric flask. Dilute to 100 mL with ASTM Type I water containing 2%(w/w)
ultrapure nitric acid (see Section 5.0). Invert flask and mix well. Transfer to a clean
125 mL Teflon flask.
7.3.2 Prepare Blank, 1.0, 3.0, 5.0, 7.0, 10.0, 30.0, 50.0, 70.0 and 100.0 ng/mL calibration
standards daily.
7.3.3 Into 10 new, unwashed, labelled polypropylene 15 mL centrifuge tubes, pipet 0, 10, 30,
50, 70, 100, 300, 500, 700 and 1000 uL of the 1000 ng/mL Na and Al standard
respectively.
7.3.4 Add 10.00, 9.99, 9.97, 9.95, 9.93, 9.90, 9.70, 9.50, 9.30 and 9.00 mL ASTM Type I water
containing 2% (w/w) ultrapure nitric acid (see Section 5.0) to each tube respectively.
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7.3.5 Pipet 100 uL internal standard into each tube using and Eppendorf repeater pipet (see
Section 16.0). Cap tubes and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly to
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
8.0 Independent Check Standard Preparation
8.1 Supplies
Certified elemental standards (Spex 1000 Mg/mL Plasma Standards)
2% Ultrapure HNO, (see Section 5.0)
Pipet tips
Internal Standard solution (see Section 16.0)
Acid cleaned polypropylene 15 mL centrifuge tubes (see Section 2.0)
8.2 Equipment
Pipettes
Eppendorf repeater pipet
One Acid cleaned 125 mL Teflon bottle (see Section 3.0)
Three Acid cleaned 1000 mL Teflon bottles (see Section 3.0.)
One Acid cleaned 100 mL volumetric flask (see Section 4.0)
Three Acid cleaned 1000 mL volumetric flasks (see Section 4.0)
8.3 Independent Check Standard Preparation Procedure
8.3.1 Prepare a 1 (ig/mL mixed stock standard containing the following elements: Arsenic (As),
Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Manganese (Mn), Nickel (Ni),
Selenium (Se), Titanium (Ti), Vanadium (V), and Zinc (Zn). Pipet 100 uL of each of the
1000 ug/mL elemental standards into a clean 100 mL volumetric flask. Be sure that these
standards are from different lots than those used to prepare the calibration standards.
Dilute to 100 mL with ASTM Type I water containing 27c (w/w) ultrapure nitric acid (see
Section 5.0). Transfer to a clean 125 mL Teflon bottle.
8.3.2 Prepare nominal 0.45, 2.5 and 7.5 ng/mL check standards by pipetting 450, 2500 and
7500 uL mixed stock standard into acid cleaned 1000 mL volumetric flasks respectively.
Dilute to 1000 mL with ASTM Type I water containing 2% (w/w) ultrapure nitric acid
(see Section 5.0). Invert flasks and mix well. Transfer to acid washed, labelled 1000 mL
Teflon bottles. Store bottles a maximum of 6 months in cold room when not in use.
8.3.3 Alternatively prepare nominal 0.3, 0.8, and 5.0 ng/mL check standards by pipetting 300,
800, and 5000 uL of mixed stock standards into a acid cleaned 1000 mL volumetric
flasks. Dilute to 1000 mL with ASTM Type 1 water containing 2% (w/w) ultrapure nitric
acid (see Section 5.0). Invert flasks and mix well. Transfer to acid washed, labeled
1000 mL Teflon bottles. Store bottles a maximum of 6 months in cold room when not in
use.
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Laboratory Methods for ICP-MS Analysis
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Note: Concentrations for selenium should be 3.0, 8.0, and 50 ng/mL in this check
standard rather than 0.3, 0.8, and 5.0 ng/mL respectively. For selenium appropriate
aliquots should be taken to prepare these levels.
8.3.4 Into three cleaned and labelled 15 mL polypropylene centrifuge tubes, pipet 10 mL of one
of the sets of check standard solutions respectively. Repeat for as many sets of check
standards as are needed (see Section 18.4).
8.3.5 Add 100 uL internal standard to each tube using an Eppendorf repeater pipette (see
Section 16.0). Cap tubes and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly to
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
9.0 Independent Check Standard Preparation for Aluminum and
Sodium
9.1 Supplies
Certified elemental standards (Spex 1000 ug/mL Plasma Standards)
2% Ultrapure HNO3 (see Section 5.0)
Pipet tips
Internal Standard solution (see Section 16.0)
New, unwashed polypropylene 15 mL centrifuge tubes
9.2 Equipment
Pipettes
Eppendorf repeater pipet
One Acid cleaned 125 mL Teflon bottle (see Section 3.0)
Three Acid cleaned 1000 mL Teflon bottles (see Section 3.0)
One Acid cleaned 100 mL volumetric flask (see Section 4.0)
Three Acid cleaned 1000 mL volumetric flasks (see Section 4.0)
9.3 Independent Check Standard Preparation Procedure
9.3.1 Prepare a 10 (Jg/mL mixed stock standard containing Aluminum (Al) and Sodium (Na).
Pipet 1 mL of both the Na and Al 1000 ug/mL elemental standards into a clean 100 mL
volumetric flask. Be sure that these standards are from different lots than those used to
prepare the calibration standards. Dilute to 100 mL with ASTM Type I water containing
2% (w/w) ultrapure nitric acid (see Section 5.0). Transfer to a clean 125 mL Teflon
bottle.
9.3.2 Prepare nominal 7.5, 25.0 and 75.0 ng/mL check standards by pipetting 750, 2500 and
7500 uL of the Na and Al standard into acid cleaned 1000 mL volumetric flasks
respectively. Dilute to 1000 mL with ASTM Type I water containing 2% (w/w) ultrapure
nitric acid (see Section 5.0). Invert flasks and mix well. Transfer to acid washed, labelled
1000 mL Teflon bottles. Store indefinitely in cold room when not in use.
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9.3.3 Into three new, labelled, unwashed 15 mL polypropylene centrifuge tubes, pipet 10 mL
7.5, 25.0, and 75.0 ng/mL check standard solutions respectively. Repeat for as many sets
of check standards as are needed (see Section 18.4).
9.3.4 Add 100 uL internal standard to each tube using an Eppendorf repeater pipet (see Section
16.0). Cap tubes and shake well.
10.0 Sample Preparation
10.1 Supplies
Pipet tips
Internal standard solution (see Section 16.0)
Acid cleaned polypropylene 15 mL centrifuge tubes (see Section 2.0)
Note: The best results are obtained if these tubes are acid-washed before they are used to rid them
of zinc contamination. DO NOT, however, use acid-washed tubes to analyze for sodium! (see
Sections 2.0 and 11.0)
10.2 Equipment
Pipettes
Eppendorf repeater pipet
10.3 Sample Preparation Procedure
10.3.1 Pipet 10 mL of sample into a labelled, acid cleaned 15 mL centrifuge tube. Repeat for
each sample.
10.3.2 Add 100 pL internal standard to each centrifuge tube using an Eppendorf repeater pipet
(see Section 16.0). Cap tubes and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly Lo
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
• Note: The concentration of most metals in precipitation is-expected to be at sub-ng/mL
levels. The ICP-MS will be calibrated to 10 ng/mL for all elements except Sodium and
Aluminum (see Sections 6.0 and 7.0.). This should be a sufficient calibration range in
which to analyze the metals of interest. If any result should fall above 10% of the upper
calibration limit, the sample will be diluted appropriately and reanalyzed.
11.0 Sample Preparation for Analysis of Sodium and Aluminum
11.1 Supplies
Pipet tips
Internal standard solution (see Section 16.0)
New, unwashed polypropylene 15 mL centrifuge tubes
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Note: The centrifuge tubes must be new and unwashed when analyzing samples for Sodium and
Aluminum. Acid-washing actually contaminates the centrifuge tubes with these elements, thus
making it difficult to get consistent results.
11.2 Equipment
Pipettes
Eppendorf repeater pipet
11.3 Sample Preparation Procedure
11.3.1 Pipet 10 mL of sample into a new, labelled, unwashed 15 mL centrifuge tube. Repeat for
each sample.
11.3.2 Add 100 uL internal standard to each centrifuge tube using an Eppendorf repeater pipet
(see Section 16.0). Cap tubes and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly to
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
Note: The concentration of sodium and aluminum in precipitation is expected to be at
the ng/mL level. The ICP-MS will be calibrated to 100 ng/mL for these two elements (see
Section 7.0.) This should be an appropriate calibration range in which to analyze the
metals of interest. If any result falls outside 10% of the upper calibration limit, the sample
will be diluted appropriately and reanalyzed in another run.
12.0 Reagent Blank Preparation
12.1 Supplies
Pipet tips
2% Ultrapure HNO3 (see Section 5.0)
Internal standard solut'on (see Section 16.0)
Acid cleaned 15 mL polypropylene centrifuge tubes (see Section 2.0)
12.2 Equipment
Pipettes
Eppendorf repeater pipet
12.3 Reagent Blank Preparation
12.3.1 Pipet 10 mL ASTM Type I water containing 2% (w/w) ultrapure nitric acid (see
Section 5.0) into as many clean 15 mL polypropylene centrifuge tubes as needed (see
Section 18.4).
12.3.2 Add 100 uL internal standard to each tube using an Eppendorf repeater pipet (see
Section 16.0). Cap tubes and shake well.
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Laboratory Methods for ICP-MS Analysis
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Note: Alternative to pipette addition, internal standards can be added on-line directly to
sample and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
13.0 Reagent Blank Preparation When Analyzing for Sodium and
Aluminum
13.1 Supplies
Pipet tips
2% Ultrapure HNO3 (see Section 5.0)
Internal standard solution (see Section 16.0)
New, unwashed 15 mL polypropylene centrifuge tubes
13.2 Equipment
Pipettes
Eppendorf repeater pipet
13.3 Reagent Blank Preparation
13.3.1 Pipet 10 mL ASTM Type I water containing 2% (w/w) ultrapure nitric acid (see
Section 5.0) into as many new, labelled 15 mL polypropylene centrifuge tubes as needed
(see Section 18.4).
13.3.2 Add 100 uL internal standard to each tube using an Eppendorf repeater pipet (see
Section 16.0). Cap tubes and shake well.
14.0 Sample Spike Preparation
14.1 Supplies
Pipet tips
Internal standard solution (see Section 16.0)
100 ng/mL Mixed stock standard calibration solution (see Section 6.0)
Acid cleaned 15 mL polypropylene centrifuge tubes (see Section 2.0)
14.2 Equipment
Pipettes
Eppendorf repeater pipet
14.3 Sample Spike Preparation
Spikes should be prepared such that the spike concentration is close to the sample concentration,
i.e. a I ng/mL sample should be spiked with 1 ng/mL. However, the spike should be prepared at
suffiently high concentrations (usually 5 times MDL) such that instrument sensitivity does not
affect recoveries of the matrix spike. Below is the procedure for preparing a 1 ng/mL spike; adjust
it as necessary to achieve the proper spike level.
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14.4 Pipet 10 mL selected sample into a labelled, acid cleaned 15 mL centrifuge tube.
Note: Diluted samples should be spiked after the dilution is made.
14.5 Pipet 100 uL of the 100 ng/mL mixed stock standard calibration solution into the centrifuge tube.
This adds 10 ng of each of the spike elements, creating a final concentration for each element of
1 ng/mL. The spike elements include Aluminum (Al), Arsenic (As), Cadmium (Cd), Chromium
(Cr), Copper (Cu), Lead (Pb), Manganese (Mn), Nickel (Ni), Selenium (Se), Sodium (Na),
Titanium (Ti), Vanadium (V), and Zinc (Zn).
14.6 Pipet 100 uL internal standard into the centrifuge tube using an Eppendorf repeater pipet (see
Section 16.0). Cap the tube and shake well.
Note: Alternative to pipette addition, internal standards can be added on-line directly to sample
and standards using the appropriate tubing, a mixing tee, and the peristalic pump.
15.0 Sample Spike Preparation for Sodium and Aluminum
15.1 Supplies
Pipet tips
Internal standard solution (see Section 16.0)
1000 ng/mL Na and Al stock standard calibration solution (see Section 7.0)
New, unwashed 15 mL polypropylene centrifuge tubes
15.2 Equipment
Pipettes
Eppendorf repeater pipet
15.3 Sample Spike Preparation
Spikes should be prepared such that the spike concentration is close to the sample concentration,
i.e. a 10 ng/mL sample should be spiked with 10 ng/mL. Below is the procedure for preparing a
10 ng/mL spike; adjust it as necessary to achieve the proper spike level.
15.3.1 Pipet 10 mL selected sample into a new, labelled 15 mL centrifuge tube.
Note: Diluted samples should be spiked after the dilution is made.
15.3.2 Pipet 100 uL of the 1000 ng/mL Na and Al stock standard calibration solution into the
centrifuge tube. This adds 100 ng of each of the spike elements creating a final
concentration for each element of 10 ng/mL. The spike elements are Sodium (Na) and
Aluminum (Al).
15.3.3 Pipet 100 pL internal standard into the centrifuge tube using an Eppendorf repeater pipet
(see Section 16.0). Cap the tube and shake well.
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Laboratory Methods for ICP-MS Analysis
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16.0 Internal Standard Preparation
16.1 Supplies
Certified elemental standards (Spex 1000 ug/mL Plasma Standards)for Lithium (Li), Yttrium (Y)
and Thallium (Tl)2% Ultrapure HNO3 (see Section 5.0)
Pipet tips
16.2 Equipment
Pipettes
One Acid cleaned 100 mL Teflon bottle (see Section 3.0)
One Acid cleaned 100 mL volumetric flask (see Section 4.0)
16.3 Internal Standard Preparation, 1 mL = 10 jag
16.3.1 Pipet 1 mL of 1000 |ag/mL certified standard for each internal standard element (Li, Y and
Tl) into a cleaned 100 mL volumetric flask.
16.3.2 Dilute to 100 mL with ASTM Type I water containing 2% (w/w) ultrapure nitric acid (see
Section 5.0). Invert flask and mix well. Transfer to a cleaned and labelled 100 mL Teflon
bottle. Store indefinitely in cold room for use throughout the project.
16.4 Comments
Internal standards must be present in all samples, standards and blanks at identical levels. This is
achieved by directly adding an equal aliquot of the above internal standard solution to all solutions
to be analyzed. The concentration of the internal standard should be sufficiently high in order to
obtain good precision in the measurement of the isotope used for data correction and to minimize
the possibility of correction errors if the internal standard is naturally present in the sample. The
normal intensity range for an internal standard is between 100,000 and 500,000 ions/second. A
concentration of 100 ng/mL is used for each standard in this protocol (100 uL of a 10 |ig/mL stock
solution). Internal standards should be added to all solutions in a like manner, in this case by
using an Eppendorf repeater pipet, so that dilution effects resulting from the addition may be
disregarded.
An alternative approach to adding internal standards to all samples standards, and blanks at a
constant level is by using on-line addition. The internal standards maybe added directly on-line to
all samples, standards, and blanks using the appropriate tubing, a mixing tee, and the peristalic
pump.
17.0 Sensitivity Check Solution Preparation
This solution is used for checking instrument sensitivity prior to analysis. Internal standards are
not added to this solution.
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17.1 Supplies
Certified elemental standards (Spex 1000 ug/mL Plasma Standards) for Magnesium (Mg),
Rhodium (Rh) and Lead (Pb)
Ultrapure concentrated HNO3
Pipet tips
17.2 Equipment
Pipettes
One Acid cleaned 1000 mL Polypropylene bottle (see Section 3.0)
One Acid cleaned 1000 mL volumetric flask (see Section 4.0)
17.3 Sensitivity Check Solution Preparation, 1 mL = 10 ng
17.3.1 Pipet 10 uL of each 1000 Mg/mL certified standard (Mg, Rh and Pb) into a cleaned
1000 mL volumetric flask.
17.3.2 Dilute to 1000 mL with ASTM Type I water containing 2% (w/w) ultrapure nitric acid
(see Section 5.0).
17.3.3 Invert flask and mix well. Transfer to a cleaned and labelled 1000 mL polypropylene
bottle. This solution can be stored near the ICP-MS for easy access; it need not be kept in
a cold room.
18.0 ICP-MS Instrument Operation
18.1 Supplies
ICP-MS sensitivity check solution (see Section 16.0)
Manifold tubing for peristaltic pump
18.2 Equipment
Perkin-Elmer Elan 5000 ICP-MS
Argon gas supply, high-purity grade, 99.99%
Perkin-Elmer AS 90 Autosampler
IBM PS2 Model 70 microcomputer
Xenix operating system
Microsoft Windows 286
Perkin-Elmer Elan 5000 software version 2.0
Gilson Peristaltic Pump
18.3 ICP-MS Instrument Operation Procedure
18.3.1 Connect waste and sample manifold tubing to peristaltic pump. This tubing should be
changed daily.
18.3.2 Light plasma by pressing ignition button on front panel.
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18.3.3 When plasma is ignited, start peristaltic pump.
18.3.4 Turn on autosampler.
18.3.5 Turn on computer monitor and then computer. Wait for ":" prompt and press return.
When password prompt appears, press "Control D" Correct date if necessary; otherwise
press "Return" At login prompt, type "Elan".
18.3.6 Initialize autosampler to include 90 second read delay and a 30 second wash time. Fill the
wash reservoir with ASTM Type I water. This wash serves as a rinse blank to flush the
system between samples and minimize carry-over.
18.3.7 Consult ICP-MS manual for normal operation.
18.3.8 In graphics mode, perform a sensitivity check. Aspirate sensitivity solution (see
Section 17.0) and press "read" button on computer keyboard. Record ion counts, power,
nebulizer gas flow and base pressure in instrument log. Compare the present ion counts
versus those previously recorded. If sensitivities are 10-15% below previous counts, stop
and troubleshoot as specified in instrument manual. Otherwise, continue with analysis.
18.3.9 Place calibration standards and samples in autosampler tube rack. See below for sequence
of standards and samples.
18.3.10 Set up the analysis routine in quantitative analysis mode on the computer as described in
ELAN 5000 software manual using the following settings in the parameter file:
For Time Factor 1
Replicate Time (ms) 1000
Dwell Time (ms) 10
Scanning Mode Peak Hop
Sweeps/Reading 100
Readings/Replicate 1
Number of Replicates 3
Points/Spectral Peak 3
Resolution Normal
Transfer Frequency Replicate
Baseline Time (ms) 0
Polarity +
18.3.11 Analyze calibration standards and print calibration report. If r >/= 0.999, continue with
analysis of samples. Otherwise, stop analysis, determine source of error, correct problem
and repeat analysis of calibration standards.
18.4 Analysis sequence:
Blank
Calibration standards
Reagent blank
Check standards
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Volume 3, Chapter 1
Laboratory Methods for ICP-MS Analysis
of Trace Metals in Precipitation
SLRS-2 or SLRS-3 standard (certified performance standard)
EPA-1 or EPA-2 (diluted certified performance standard)
Samples 1-10
Duplicate sample
Spiked sample
Reagent Blank
Check standards
SLRS-2 or SLRS-3 standard (certified performance standard)
EPA-1 or EPA-2 (diluted certified performance standard)
Samples 11-20
Duplicate sample
Spiked sample
Reagent blank
Check standards
SLRS-2 or SLRS-3 standard (certified performance standard)
EPA-1 or EPA-2 (diluted certified performance standard)
18.5 Isotopes Analyzed
Element Symbol
Sodium
Aluminum
Titanium
Vanadium
Chromium
Manganese
Nickel
Nickel
Copper
Zinc
Arsenic
Selenium
Cadmium
Lead
Na
Al
Ti
V
Cr
Mn
Ni
Ni
Cu
Zn
As
Se
Cd
Pb
Isotope Corrections Programmed*
23
27
48 Ca
51
52
55
58 Fe
60 (used later)
63 TiO*
66
75
82 Kr
114 Sn
208
+ The Perkin-Elmer Elan 5000 software has pre-programmed elemental equations which are
applied to the indicated elements to correct for the isobaric interferences. (See Section 19.1)
* See Section 19.3 for the elemental equation entered manually for the TiO correction.
18.6 Certified Standard Concentrations
18.6.1 SLRS-2 standard (certified performance standard) concentrations:
ng/mL
ng/mL
ng/mL
ng/mL (below lowest standard)
ng/mL
ng/mL
ng/mL
ng/mL
Sodium (Na)
Aluminum (Al)
Arsenic (As)
Cadmium (Cd)
Chromium (Cr)
Copper (Cu)
1860
84
0.77
0.028
0.45
2.76
+/- 110
+/- 3.4
+/- 0.09
+/- 0.04
+/- 0.07
+/- 0. 1 7
Lead(Pb)
0.129
Manganese (Mn) 10.1
+/- 0.011
+/- 0.3
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Nickel (Ni)
Vanadium (V)
Zinc (Zn)
1.03
0.25
3.33
+/- 0.10
+/- 0.06
+/- 0.15
ng/mL
ng/mL
ng/mL
18.6.2 SLRS-3 standard (certified performance standard) concentrations:
Arsenic (As) 0.72 +/- 0.08 ng/mL
Chromium (Cr) 0.30 +/- 0.06 ng/mL
Copper (Cu) 1.35 +/- 0.11 ng/mL
Manganese (Mn) 3.9 +/- 0.45 ng/mL
Nickel (Ni) 0.83 +/- 0.12 ng/mL
Vanadium (V) 0.30 +/- 0.03 ng/mL
Zinc(Zn) 1.04 +/- 0.14 ng/mL
18.6.3 EPA-1 standard (diluted certified performance standard) concentrations:
Arsenic (As) 5.0 ng/mL
Cadmium (Cd) 5.0 ng/mL
Chromium (Cr) 5.0 ng/mL
Copper (Cu) 5.0 ng/mL
Lead (Pb) 5.0 ng/mL
Nickel (Ni) 5.0 ng/mL
Selenium (Se) 5.0 ng/mL
Titanium (Ti) 5.0 ng/mL
Vanadium (V) 5.0 ng/mL
Zinc (Zn) 5.0 ng/mL
Manganese (Mn) 5.0 ng/mL
Note: Also contains the following elements at 5.0 ng/mL each which are not being
analyzed for this project: Antimony, Beryllium, Calcium, Cobolt, Iron, Lithium,
Magnesium, Molybdenum, and Strontium. Although Lead is also present in EPA-1, the
quantitative results for it are inaccurate because Thallium is also present in the solution,
and Thallium is the internal standard used to quantitate Lead.
18.6.4 EPA-2 standard (diluted certified performance standard) concentrations:
Aluminum (Al) 5.0 ng/mL
Sodium (Na) 5.0 ng/mL
Note: Also contains the following elements at 5.0 ng/mL each which are net being
analyzed for this project: Barium, Boron, Potassium, Silicon and Silver.
18.6.5 Independent Check Standard at 0.8 ng/mL maybe used as the certified performance
standardfor these elements not certified or below the method detection limit in the SLRS-2
and SLRS-3 standards. The acceptance range is +/- 10% so that the acceptable ranges are:
Titanium (Ti) 0.8 +/- 0.8 ng/mL
Selenium (Se) 8.0 +/- 0.8 ng/mL
Cadmium (Cd) 0.8 +/- 0.08 ng/mL
Lead(Pb) 0.8 +/- 0.08 ng/mL
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19.0 Interferences
Several interference sources may cause inaccuracies in the determination of trace metals by ICP-
MS. The following descriptions are taken in large part from the Environmental Monitoring
Systems Laboratory Office of Research and Development, U.S. Environmental Protection Agency
Method 200.8 for the "Determination of Trace Elements in Waters and Wastes by Inductively
Coupled Plasma Mass Spectrometry", Revision 4.3, August 1990.
19.1 Isobaric elemental interferences are caused by isotopes of different elements which form singly or
doubly charged ions of the same nominal mass-to-charge ratio and which cannot be resolved by
the mass spectrometer in use. All data obtained under such conditions must be corrected by
measuring the signal from another isotope of the interfering element and subtracting the
appropriate signal ratio from the isotope of interest. This is done automatically by pre-
programmed correction factors in the Perkin-Elmer Elan 5000 software.
19.2 Abundance sensitivity is a property defining the degree to which the wings of a mass peak
contribute to adjacent masses. The abundance sensitivity is affected by ion energy and quadrupole
operating pressure. Wing overlap interferences may result when a small ion peak is being
measured adjacent to a large one. Such interferences can be minimized by adjusting the
spectrometer resolution appropriately.
19.3 Isobaric polyatomic ion interferences are caused by polyatomic species which have the same
nominal mass-to-charge ratio as the isotope of interest, and which cannot be resolved by the mass
spectrometer in use. These ions are commonly formed in the plasma or interface system from
support gases or sample components, and are therefore highly dependent on the sample matrix and
chosen instrument conditions. Such interferences must be recognized, and when they cannot be
avoided by the selection of alternative analytical isotopes, appropriate corrections must be made by
programming in a correction factor for the elements which are affected. In this study, elemental
correction equations will be applied to copper whose mass is overlapped by titanium oxide
(Ti47O16):
corrected Cu6J = Cu"' - (0.06 * Ti")
19.4 Physical interferences are associated with the physical processes which govern the transport of
sample into the plasma, sample conversion processes in the plasma, and the transmission of ions
through the plasma-mass spectrometer interface. These interferences may result in differences
between instrument responses for the sample and the calibration standards. Physical interferences
may occur in the transfer of solution to the nebulizer (e.g. viscosity effects), at the point of aerosol
formation and transport to the plasma (e.g. surface tension), or during excitation and ionization
processes within the plasma itself. High levels of dissolved solids in the sample may contribute
deposits of material on the extraction and/or skimmer cones reducing the effective diameter of the
orifices and therefore ion transmission. Precipitation samples are expected to be well within the
recommended limit of 0.2% (w/v) dissolved solids which will help to minimize such effects.
Internal standardization may be effectively used to compensate for many physical interference
effects. Internal standards ideally should have similar analytical behavior to the elements being
determined. In this study, the internal standards include Lithium (mass 7) for Sodium (23) and
Aluminum (27). Thallium (205) for Lead (208). and Yttrium (89) for the remaining elements in
the center of the mass range.
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Laboratory Methods for ICP-MS Analysis
of Trace Metals in Precipitation Volume 3, Chapter 1
19.5 Memory interferences result when isotopes of elements in a previous sample contribute to the
signals measured in a new sample. Memory effects can result from sample deposition on the
sampler and skimmer cones and from the buildup of sample material in the plasma torch and spray
chamber. The site where these effects occur is dependent on the element. Memory interferences
can be minimized by keeping the cones, torch and spray chamber clean and by flushing the system
with a rinse blank between samples (see Section 18.3.6).
20.0 Quality Control Information
20.1 QA/QC Objectives:
20.1.1 Calibration standards are to be prepared each day of analysis (See Section 6.0).
20.1.2 Appropriate check standards should be prepared for the duration of the project (See
Section 3.6.). Check standards will be control-charted for monitoring purposes.
20.1.3 QC standards shall include: reagent blank, check standards, duplicate, spike, and
certified standards.
20.1.4 No more than 10 samples shall be run between sets of QC standards.
20.1.5 Calibration coefficients shall have at least three 9's before proceeding with samples
(r = 0.999).
20.1.6 For every 10 samples or fraction thereof, one sample is randomly selected to be duplicated
and one sample is randomly selected to be spiked for QC purposes. The sample spike
should be prepared such that the spike concentration is close to the sample concentration
but still be within the calibration range.
20.1.7 The following limits shall be met before data is deemed acceptable and pa ssed on to the
QA/QC officer.
20.1.7.1 Duplicates: <10% difference or percent difference less than the method
detection limit, whichever is greater percent difference as calculated using
the following equation:
(C° measured ~~ C measure,!* / ^C "measured + C ^.v,,™/)/2] * 10° = % Difference
Where Cmraimd = measured concentration
o = the original sample
d - the duplicate sample
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Laboratory Methods for ICP-MS Analysis
Volume 3, Chapter 1 of Trace Metals in Precipitation
20.1.7.2 Spikes: 85-115% recovery as calculated using the following equation:
/ * V) - (C_f, * V'Wmoss of spike * 100 = % Spike Recovery
Where Cmmmni= measured concentration
V - volume and an askerisk
(*) = the spiked sample
20.1.7.3 Check standards: <10% relative standard deviation (RSD) within run (or
relative percent difference for only two data points) <15% RSD of all data
points in the project.
20.1.7.4 Certified standards: Must be within certified range (See Section 3.14.3
for current certified standard values).
If any one of the above criteria is not met and an explanation can be given
for deviation from the above limits, the data can be deemed acceptable.
Otherwise, all samples must be reanalyzed.
20.2 Laboratory Preparation Area
A laboratory in the high hazard section of the Hazardous Waste Research and Information Center
(HWRIC) has been designated exclusively for all preparation and glassware cleaning associated with
this project. Although not a clean room in the strict sense, it is kept as clean as possible and no other
preparation work is done in that room. Gloves and lab coats are required at all times. Individual work
spaces are lined with absorbent protective paper which is changed periodically. All glassware and
teflon is washed in a special tank in the hood in this same room (see Sections 2.0, 3.0, 4.0).
Additionally, all equipment (glassware, pipets, consumable supplies, etc.) for this project are also
stored in this room.
20.3 Table of Detection Limits and Analytical Ranges
Analytical Range
fog/U
0.1 1000
0.1 1000
0.1 1000
0.1 1000
0.1 1000
0.1 1000
0.1 1000
0.1 1000
0.1 1000
1.0- 1000
0.1 1000
0.1 1000
0.1 1000
3-23
Monitored Method Detection
Element Analytical Mass Limit (ug/L)
Aluminum
Arsenic
Cadmium
Chromium
Copper
Lead
Manganese
Nickel
Nickel
Selenium
Sodium
Titanium
Vanadium
Zinc
27
75
114
52
63
208
55
58
60 (used later)
82
23
48
51
66
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
1.0
0.1
0.1
0.1
-------
Standard Operating Procedures for
Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
Regendra D. Paode and Thomas M. Holsen
Department of Chemical and Environmental Engineering
Illinois Institute of Technology
Air Quality Laboratory
10 W 33rd Street
Chicago, IL 60616
February 10,1996
Revision 2
-------
Standard Operating Procedures for
Preparation, Handling and Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
1.0 Introduction
Dry deposition plates are used to measure the mass flux of particles and metals. This standard
operating procedure (SOP) addresses the protocol for preparation, handling and acid extraction of
these plates. The SOP also discusses quality assurance and quality control measures, and
performance criteria.
A schematic of the dry deposition plate is presented in Figure 1. The plate is made of PVC and is
21.5 cms long, 7.6 cms wide, and 0.65 cms thick with a sharp leading edge (<10 degree angle) to
ensure laminar flow. The plate is pointed into the wind by a wind vane. Each plate is covered
with 4 Mylar strips (7.6 cm x 2.5 cm) coated with approximately 8 mg of Apezion L grease
(thickness = 8 jam) to collect impacted particles (123 cm2 total exposed surface). The film is
placed on the plate and held down on the edges with a 5 mil thick Teflon or Mylar template, which
is secured at each end by spring clips. The strips are weighed before and after exposure to
determine the total mass of particles collected. The mass flux is determined by dividing the
collected mass by the exposure time and the exposed surface area.
2.0 Preparation of Dry Deposition Plates
Preparation and collection of accurate and reliable data on mass and metal fluxes with dry
deposition plates requires that proper laboratory procedures be used during preparation.
Laboratory equipment and reagents are listed in Appendix A. The various activities which have
been sequentially described in this section include cleaning of glassware used for preparation of
dry deposition plates; and cleaning, greasing, equilibrating, and weighing of the sampling media.
2.1 Cleaning of Glassware
Particle-free nylon gloves are used during all cleaning steps. All glassware (e.g., petri dishes,
beakers) used in connection with this research will be scrubbed with soap and rinsed in hot tap water.
Next, the glassware is rinsed three times in distilled water. Subsequently, it is soaked in a nitric acid
bath (5%) for at least 12 hours. After being removed from the bath, the glassware is rinsed three times
in distilled water. The final cleaning step involves rinsing the items three times in deionized water.
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
Volume 3, Chapter 1
10 cm
o
o
5.7 cm
1.8cm
^
7.6 cm
21.6cm
4 k-l
0.65 cm
Figure 1. Top View of a Dry Deposition Plate
2.2 Cleaning of Plates
The first step in the cleaning procedure involves wiping the dry deposition plate with a particle free
wipe (S/P Brand S/Pec-Wipe) wetted with double distilled methanol. The plates are subsequently
placed in a clean plastic wash tray. The second step involves rinsing the plates with deionized water.
Finally the plates are dried in a laminar flow clean bench.
2.3 Cleaning of Strips
Mylar (0.002 inches thick) is cut into 1 -inch x 3-inch pieces. The area to be greased is marked on each
strip with a scratch pen. Prior to being coated with grease the strips are cleaned. The first step in the
cleaning procedure involves dipping the strips in glass petri dish containing double distilled methanol
and scrubbing both sides with particle free wipe (S/P Brand S/Pec-Wipe). The strips are subsequently
dipped in a second petri dish containing deionized water and both sides are again scrubbed with a
particle free wipe. The third and fourth steps involve dipping the strips againln deionized water.
However, in these steps the strip is not scrubbed. Finally, the strips are put in a storage box for drying.
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Volume 3, Chapter 1
SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
2.4 Greasing and Equilibrating of Strips
After the strips dry, they are given a thin coat of L-Apiezon grease on the marked area. This is
accomplished by melting the grease in a small glass petri dish on a hot plate. A small paint brush is
used to coat the strips, which are also warmed on the hot plate. The brush is cleaned prior to use with
pure Hexane, followed by Double-Distilled-Methanol. After the strips are coated with the grease, they
are put into a dust-free storage box to equilibrate for at least 24 hours before weighing
2.5 Weighing and Mounting of Strips
The initial weight of strips is recorded using a micro balance able to measure at least 0.01 mg. After
initial weighing, the strips are mounted on the clean, dry deposition plates. The un-greased edges of the
strips are covered with a template made of a thick Mylar film. The Mylar templates are subsequently
held down with spring clips. The Mylar templates are cleaned with the same procedures used for the
strips. Four strips are mounted on each plate. The dry deposition plates with the mounted strips are put
in a dust-free plastic storage box in preparation for field sampling.
3.0 Field Sampling, Labeling, Shipping, and Post-sampling
Equilibration and Weighing of Plates
3.1 Field Sampling
The sample box is not opened until all the other preparations are made for field measurements. A list of
equipment and supplies for field investigations is provided in Appendix B. The plates and strips are
handled with particle free gloves to ensure that there is no physical contact with the greased surface.
After sampling, the plates are taken off by unscrewing the hold down nuts, and put in the storage
container. The plates are slid sideways into the slots, with the sharp edge into the thin slot. The total
sampling time is recorded. Details of field sampling are available in the field sampling SOP.
Each sample set includes field blanks. Field blanks are obtained by mounting four pre-weighed grease-
coated Mylar strips on a dry deposition plate. This plate is placed in the storage container along with
the sample plates and remain there.
3.2 Labeling/Tracking
All samples will be tagged in indelible ink to indicate the site, the sequence/number of sample, and the
status of sample (e.g., field blank). Every sample is assigned a unique identification code which follows
the sample through analysis and logging of all data. The label should follow the following format:
3.3 Site-Number/Status
Status would communicate whether the sample is a field blank, a regular sample, or a duplicate sample.
Field blanks are designated BK, while duplicates are labeled A and B. For example, the field blank
associated with the first sample will be labeled as -01BK. Samples are logged in the sample log
sheet. An example of the sample log sheet is presented in Appendix C. One copy of the sample data
sheet should be kept in the three-ring binder and the original returned with the sample.
The project evidence will be under the custody of the Principal Investigator and the sample custodian is
the Laboratory Coordinator. The project evidence will contain all sample log sheets and results of
laboratory analyses. All pertinent information from the data sheets is transferred to electronic media via
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles Volume 3, Chapter 1
computerized spreadsheets. The computer files are backed-up whenever new data is added and two
disk or tape copies are kept in separate secure areas at all times. Data generated by the analytical
instrument are stored in both electronic and hard copy formats.
If sample integrity is questionable, the PI will decide whether to discard the sample
3.4 Shipping
The samples are transported to the HTAQL immediately after sampling. If it is not possible to ship the
samples to ITTAQL immediately after sampling, they must be stored at room temperature away from
any sources of contamination.
The samples are shipped to the following address:
Dr. Thomas M. Holsen
10 West 33rd Street
Department of Chemical and Environmental Engineering
Illinois Institute of Technology
Chicago, IL 60616-3793
3.5 Equilibration/Weighing
At HTAQL, strips are unloaded from plates, and put back into the storage box for a 24 hour
equilibration period before the strips are again weighed.
4.0 Extraction Procedure and Analysis
Extraction is conducted in a Class 100 clean room on the Campus of the University of Michigan. The
procedure begins with washing the greased mylar strips with 10-20 mL of hexane in a Teflon vessel.
The hexane is subsequently evaporated with a stream of ultra-pure nitrogen. Twenty mL of 10% (v/v)
ultra-pure nitric acid is then added to the Teflon container and the container placed in a digestion bomb
and loaded into the microwave oven. Acid digestion is carried out for 30 minutes at 160°C and
approximately 160 psi. Following digestion, the bomb is allowed to cool for a period of 1 hour. The
samples will be analyzed on the ICP-MS.
Method detection limits (MDLs) will be calculated by injecting a low concentration sample 7 times into
the ICP MDL is defined as three times the standard deviation of the concentrations obtained in the
seven runs.
Field blanks (unexposed mylar strips) will be monitored to determine whether the sample preparation
and transport, the Apezion L grease coating on the mylar film, and the hexane wash contribute to
contamination. It is anticipated that the field blanks will have trace concentrations of metals due to the
grease, the hexane used for extraction, and the acid used for digestion. Sample concentrations will be
corrected by subtracting the concentration obtained for the field blank.
Extraction efficiencies will be calculated by measuring metal concentration after spiking a 10% nitric
acid solution with NIST Urban Paniculate Matter (UPM). The ICP-MS will be calibrated daily. A
standard curve will be deemed acceptable only if the r (coefficient of determination) is greater than
95%. After every 10 samples a standard will be analyzed as a sample. If the variation between sample
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Volume 3, Chapter 1 Dry Deposition of Atmospheric Particles
and standard concentration is more than 5% the instrument will be recalibrated. Instrument accuracy
will be checked daily by analyzing a 2% NIST standard, to ensure that the % recovery is between 70 to
120%. Precision will be estimated by analyzing split samples (e.g., two separate strips from the same
plate), and replicate sample extract analysis (same sample analyzed at different times).
Working standards are prepared daily by dilution of commercially available stock solution.
Standardization is accomplished with a four point calibration curve (one blank and three standards) that
bracket the expected concentration of the samples. To validate the accuracy of the calibration the four
standards are injected again to ensure that the relative percent deviation is within 15%. If the
concentration is out of range, the calibration curve is recalculated till the criteria are met. Refer to the
following section for further details on quality assurance/quality control (QA/QC).
5.0 Performance Criteria, Quality Assurance and Quality Control
The issues which need to be addressed in connection with quality control are as follows:
• Precision
• Accuracy
• Completeness
• Blanks
The QA/QC and performance criteria are illustrated in Table 1. In Appendix C, the statistical
parameters that are used during QA/QC are defined.
5.1 Precision
A measure of the reproducibility among multiple measurements of the same property, usually under
prescribed similar conditions. Quantitative measurements of precision include replicate field samples,
replicate laboratory samples, and analysis by different methods for comparison. The applicability of
these measurements is parameter dependent. In this protocol, at least 5 percent of the samples will be
split and analyzed. If the relative standard deviation falls below 20% the samples will be re-extracted
and analyzed.
5.2 Accuracy
Accuracy is a measure of the degree to which a measurement or computed value reflects the true value
of analyte present. Accuracy will be assessed as the recovery of a standard reference material or
surrogate/matrix spikes for organic analytes.
5.3 Blanks
Field blanks (FB) will be used to assess the extent of background contamination present in the field.
Process blanks (PB) are used to monitor the degree of background contamination introduced during the
laboratory analysis and must meet the criteria of mass < MDL.
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles Volume 3, Chapter 1
5.4 Completeness
Completeness is the measure of the number of valid samples (meeting all QA requirements) obtained
compared to the number required to achieve the objectives of the study. Overall completeness in the
number of valid samples compared to the number planned. Laboratory completeness is the number of
valid samples obtained compared to the number analyzed. Both types of completeness will be
reported. As with the other data quality attributes, completeness can be controlled through adherence
to the SOPs in order to minimize contamination and sampling errors.
6.0 References
6.1 EPA. 1994. Quality Assurance Project Plan - Atmospheric Monitoring for Lake Michigan
MassBalance and the Lake Michigan and Superior Loading Studies. Revision 1. EMP-A-QAPP.
6.2 University of Michigan Air Quality Laboratory. 1994. Draft Sampling and Analysis of Vapor Phase
Mercury in Ambient Samples, Revision 7.
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Volume 3, Chapter 1
SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
Table 1. Data Quality Objectives for Dry Deposition Plate - Metals
QA Criteria
arecision
accuracy
blanks
completeness
calibration
Sample Type
method: split samples (collocated
field samples)
instrument: replicate sample extract
analysis (different times)
NIST certified reference samples
field
procedural
field samples
std curve
blank + at least 3 stds
Frequency
5%
10%
5%
I/set
I/set
daily
Criteria
RSD<
20%
RSD<
15%
70% 95%
Control Action
re-extract and analyze*
repeat measurement
until criteria met
re-extract and analyze
until criteria met &/or
recalibrate
re-extract and analyze
re-extract and analyze
reoptimize instrument,
repeat calibration
Units
%
%
%
%
Field blanks: Anthropogenic metals: Not exceed the MDL by more than 0.3 ppb.
Crustal metals: Not exceed MDL by more than 3 ppb.
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Volume 3, Chapter 1 Dry Deposition of Atmospheric Particles
Appendix A. Laboratory Facilities, Equipments and Reagents
A.1 Preparation of Strips
1. Particle-free nylon gloves.
2. Balance.
3. Double-distilled methanol.
4. Plastic wash tray.
5. Laminar hood.
6. Plates.
7. Mylar strips.
8. Apezion L grease.
9. Deionized water.
10. Scratch pen.
11. Particle free wiper.
12. Storage box.
13. Glass petri dish.
A.2 Extraction of Strips
1. Teflon beaker.
2. Nitric acid (trace metal grade)
3. Ultrasonic bath.
4. Deionized water
5. Hot plate.
6. Volumetric flask (25 mL)
7. Polyethylene bottle.
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Volume 3, Chapter 1 Dry Deposition of Atmospheric Particles
Appendix B. Equipment and Supplies for Field Investigations
i. SOP
2. Plate holder (PVC).
3. Dry deposition plates.
4. Pre-weighed grease coated Mylar strips.
5. Mylar strip covers.
6. Teflon coated clips.
7. Particle free gloves.
8. Labelling tape.
9. Sample and field blank tracking forms.
10. Teflon tape.
11. Rubbermaid plate container.
12. Teflon coated tweezers.
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Volume 3, Chapter 1
SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
Appendix C. Sample Log Sheet
EAGLE SAMPLE LOG SHEET
SAMPLE NUMBER
SAMPLE LOCATION
WEATHER CONDITIONS
(CIRCLE ONE)
COVER STATUS
(CIRCLE ONE)
OPEN TIME, MIN
TOTAL TIME,MIN
RESET TIMER?*
WET TEST RESULTS
(CIRCLE ONE)
DATE
SUNNY
OPEN
YES
COVER THEN
UNCOVER
RAINY
CLOSED
NO
NO RESPONSE
CLOUDY
OTHER
(EXPLAIN
BELOW)
* - RESET TIMER ONLY WHEN STARTING A NEW SAMPLE
COMMENTS
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Volume 3, Chapter 1
SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles
Appendix D
QA Definitions
D.1 Precision
The precision will be evaluated by performing multiple analyses. Precision will be assessed by the
following three methods:
1.0 Difference
Difference = X, - X2
Where: X: - larger of the two observed values
X2 — smaller of the two observed values
This formula is used for parameters with concentrations below some established value.
2.0 Relative Percent Difference (RPD)
RPD = (XrX2)*100/(X,+X2)/2
This formula is used for duplicate measurements.
3.0 Relative Standard Deviation (RSD)
RSD = (s/y) x 100
Where: s = standard deviation
y — mean of replicate analyses
This formula is used for three or more replicate values and may be used when reporting precision on
aggregated data.
Standard deviation is defined as follows:
S =
n
£
n--l
(Y,
(n
-Y)2
1)
Where: y{ = measured value of the I th replicate
y = mean of replicate analyses
n = number of replicates
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SOP for Preparation, Handling and
Extraction of Dry Deposition Plates:
Dry Deposition of Atmospheric Particles Volume 3, Chapter 1
Appendix D.
QA Definitions (Cont'd)
D.2 Accuracy
Percent recovery, R, is used to assess accuracy for surrogate spikes, matrix surrogate spikes, and
standard reference materials. Recovery is calculated as:
R = (Measured mass/Actual mass) * 700
D.3 Completeness
Completeness is defined:
Completeness = (v/n) * 100
Where: V = number of samples judged valid
n = total number of measurements necessary to achieve project objectives
The completeness will be reported on an annual basis.
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Standard Operating Procedure for
EPA's LBL Energy Dispersive X-Ray
Fluorescence Spectrometry
Robert B. Kellogg
ManTech Environmental, Inc.
P.O. Box12312
Research Triangle Park, NC 27709
August 4,1992
-------
Standard Operating Procedure for EPA's LBL
Energy Dispersive X-Ray Fluorescence Spectrometry
1.0 Introduction
1.1 Scope and Application
This SOP applies to XRF analysis of ambient aerosols sampled with fine particle (<2.5 ^)
samplers, dichotomous samplers, and the VAPS (versatile air pollution sampler). The data are
intended for use in source apportionment research only.
1.2 Description of Spectrometer
The x-ray analyzer is an energy dispersive spectrometer custom made by Lawrence Berkeley
Laboratory and possesses some features not found on commercially available machines. The tube
is operated in a pulsed mode; it is actually turned off for 83 /^sec after an x- ray is detected. This
limits the maximum count rate to about 6.5 kHz - the optimum for the amplifier. This low count
rate also reduces pulse pile-up, a phenomenon to be minimized which occurs in the detector at
high count rates. The detector is a cryogenically cooled lithium-drifted silicon detector with an
electronic guard ring for electronic collimation of x-rays. In addition to these unique features
optimum excitation conditions are made possible by four fluorescers or secondary targets
providing analysis capability for Al to Pb. The four fluorescers and the elements which they excite
are: Ti, (Al to Ca); Co, (S to Mn); Mo, (Mn to Sr plus W, Au, Hg, and Pb), and Sm (Sr to La).
The machine is operated under control of an IBM PC/AT personal computer with a Nucleus Port
PCA card which provides complete data acquisition and operation of the sample changer. All
operational functions are controlled by computer menu (Appendix 9.4) allowing the operator
control with only a few keystrokes.
1.3 Personnel Requirements
The minimum training required is a Master's degree in chemistry or physics with five years
experience in energy dispersive x-ray fluorescence analysis of atmospheric aerosols and its
associated data processing. Proficiency in using the DOS operating system, Fortran programming,
and Lotus is required.
1.4 Precision and Accuracy
Precision varies with the element and concentration. At high concentrations (greater than
1 pg/cm2) a precision of 6% can be expected for elements analyzed by two fluorescers (S, Cl, K,
Ca, Mn, and Sr). For all other elements at high concentrations a precision of 8.6% can be
expected.
Based upon the analysis of NIST SRMS the accuracy is ±10%. See Appendix 9.2 for the elements
certified in these SRMs and Appendix 9.3 for typical analysis results of the SRMS.
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SOP for EPA's LBL Energy Dispersive
X-flay Fluorescence Spectrometry Volume 3, Chapter 1
1.5 Caveats
The spectrometer has an inherent contamination due to Sn (tin) which prevents quantitative
analysis of this element at low (<225 ng/cnr) concentrations. This element may be reported as
detected but if the concentration is less than the above stated amount the investigator should
disregard it.
The type of samplers mentioned in Scope and Application must be operated in accordance with
their instructions or severe errors in x-ray analysis may occur. For example, errors in flow rate will
not just give erroneous volumes but will cause a more serious condition of altering the cut points
upon which the coarse factor x-ray attenuations are based. If samples are intended for x-ray
analysis then the sampling protocol must conform to the constraints inherent within the method.
2.0 Sample Preparation, Storage, and Tracking
2.1 Sample Log-in Procedures
When samples are received for analysis they are assigned an XRFID which is logged in the form
entitled ASSIGNMENT OF XRF IDs. (See Appendix 9.8 for an example of this form) The IDs
are structured so that the first three digits represent the study name and the fourth digit represents a
sub-study. A sub-study can accommodate a maximum of 72 samples and there are 10 possible
sub-studies in each study. Each study name is assigned an archive ID which refers to a physical
Bernoulli disk on which the data are archived.
2.2 Sample Preparation
Filter samples are received in individual plastic containers packaged for delivery in the postal
delivery system. After receipt the filters are unpacked and arranged on the sample preparation
table so as to match the physical samples with the accompanying field data sheets. This will
ascertain that a complete data set has been received and will check for missing samples. The
individual samples are then unpacked and checked for any invalidating conditions such as holes,
tears, or a non-uniform deposit, any of which would prevent quantitative analysis. If such a
condition is found the sample is invalidated and noted on the appropriate xrf data entry form
corresponding to the type of sample. See Reference 2 in Section 8 for an explanation of the types
of data entry forms used.
All filter samples received for analysis are removed with tweezers from their container and placed
in a two-part sample frame (see drawing in Appendix 9.11) with the deposit side facing away from
the retaining ring. The spacer ring shown in the drawing is needed to provide the correct spacing.
When mounting filters such as Teflo which are already bonded to a supporting ring the spacer ring
is not needed. The retaining ring is snapped into place to firmly hold the filter. Note that in the
above described geometry the sample deposit is facing FM during analysis. The sample ID is
written on a pressure sensitive label and fixed to the recessed portion of the retaining ring and the
assembly is placed in the slot of an Argus slide tray corresponding to an entry line on the field data
entry form (see Section 4.1 on Field Data Entry). A pair of slide trays is then placed in the
spectrometer sample changer for analysis.
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SOP for EPA's LBL Energy Dispersive
Volume 3, Chapter 1 X-Ray Fluorescence Spectrometry
2.3 Sample Status and Tracking
After the sample IDs have been placed on the frame and the trays loaded the samples are ready for
analysis. A scheme has been devised whereby the run progress can be tracked through the process
and afterwards its storage location identified. Since several runs of samples may simultaneously
be in different stages of the analysis process an XRF RUN STATUS LOG form is used. This
form consists of a check list in which the completion dates are entered for the various steps in the
process (See Appendix 9.8 for an example of this form). Briefly, the check list is: XRFID (the
four-digit number identifying the run of samples); STUDY NAM (a descriptive name assigned by
the operator); XRF DATE (the date the XRF measurement was started); CARD DATE (the date
the field data entry was completed and verified); LSO DATE (the date the least squares analysis
was performed on the spectral data); QC CHECKS (the later of the dates of checking the run-time
QC data and the analytical results on the SRMS); IPAB XFER (date of uploading results to the
SARB data base); HARD COPY LIB (the type.format of data on file in the data library, either
ng/m3 or ng/cm2 format); HARD COPY FIL (type of data format on file in run-time I/O file
cabinet); ARCHIVE DATE (date data were archived).
After completion of analysis the trays of samples and a copy of the field data are placed in plastic
bags and stored by XRFID for an indefinite period of time in the wall cabinets located in S242J. If
samples are to be removed for scanning electron microscopy or returned to investigators there is a
log form entitled SAMPLE CHECK OUT LOG in which the IDs of the samples removed are
recorded along with date and signature. (See Appendix 9.8 for this form).
3.0 Spectrometer Operation
3.1 Preparation for Operation and Shutdown
All spectrometer operations are performed by an IBM PC/AT computer controlled interface.
When the computer is first turned on each day, two procedures must be performed in order to
initialize the spectrometer. These are: (1) position the first flourescer in proper alignment with the
sample and detector and (2) position the sample rotor in the load position to accept the first sample
from the sample changer. To execute these procedures do the following:
To locate fluorescer:
1. Select 2 from menu (see Appendix 9.4).
2. Press ENTER three times on keyboard and wait until resulting process is complete
3. Press ESC twice on keyboard.
4. Enter Y at prompt. Menu reappears
(Fluorescer is now located)
To find load position:
1. Select 1 from menu
2. Wait for FPLOT screen and enter 8
(Load position is now located)
The settings for the helium flow, the front panel controls, and the shutdown sequence are
explained in Appendix A of Reference 1 (see Section 8).
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3.2 Gain and Baseline Adjustment
Before the run of unknowns the gain and baseline must be checked. This will assure that the x-ray
energies are assigned the correct channel numbers. Refer to Appendix A of Reference I for this
procedure.
3.3 Run Options
Procedure 5 from the menu (see Appendix 9.4) is used to run samples. There are four run options
depending upon what kind of samples are being run. These options are:
I. Unknowns and QC standards
2. Unknowns only
3. Blanks and QC standards
4. Shapes and QC standards
Option 1 is generally used to measure all unknowns. The least squares spectral processing
program requires that QC standards and unknowns be processed in the same run so this option is
essential if subsequent spectral analysis is desired. Option 2 is used in special cases in which it is
necessary to add sample spectra to an existing run. Option 3 measures blanks for background
spectra. Option 4 measures shape standards for calibration purposes (See Section 5.4). The
keyboard input response required for each of these options is addressed in Appendix E of
Reference 1 and will not be elaborated upon in this document.
After the completion of Options 1, 3, or 4 the run-time QC files shall be updated (See Section 6.2
for a description of run- time QC). To update the files containing the data from run-time QC
follow the instruction below.
1. Make sure the run-time quality control criteria are met. (See section 6.4)
2. Select Procedure 21 from the menu (Appendix 9.4)
This will put the run-time QC data from the run into cumulative files named
C:\XRF\XRFRUN\XQC\ARCHxx.NEW where xx is the element name. See Section 6.6 on
Control Charts for the use of these files. The hard copy of the run-time printout and a copy of the
field data (see Section 4.1) are filed by XRFID in the run-time I/O file cabinet.
4.0 Data Analysis and Reporting
4.1 Field Data Entry
Data entry is accomplished by using one of four Lotus spreadsheets depending upon which kind of
sampler was used to collect the sample. Use of these spreadsheets is explained in Reference 2 (see
Section 8.0). Great emphasis shall be put on careful field data entry because it is the most error
prone step in the analysis process. After data entry is completed and verified (see Section 6.5) two
hard copies are made of the card file. One copy is filed in the run-time I/O file cabinet, one copy is
put in the plastic bag with the trays of samples, and the original is filed in the XRFID Assignment
notebook.
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4.2 Least Squares Analysis
The pulse height spectrum for each sample is deconvoluted into its constituent elemental spectra
by a linear least squares algorithm. In this process standard elemental shape spectra determined
during calibration (see Section 5.4) are fitted to the unknown spectra. A set of coefficients
determined by minimizing chi-square are proportional to the concentration of a given element.
Thus, through the calibration sensitivity the concentrations are determined. It is not within the
scope of this SOP to describe this process in any detail. Readers wishing a more detailed account
should refer to Reference 3 in Section 8.
All of the processing options for least squares analysis such as selection of the shapes, background,
attenuation factors, sensitivities, and field data are contained in the card files. To perform least
squares analysis on a run of unknowns follow the three instructions below:
1. Select Procedure 6 from menu (see Appendix 9.4 ).
2. Enter four digit xrfid as called for. (Processing of spectra will take up to 20 minutes for a
full 72 sample run).
3. Select Procedure 22 from menu to check SRM data results.
The three output files LSOnnnn.NG3, LSQnnnn.CM2, and IPABnnnn.DAT are created in
the same directory as the unknowns. (Refer to Reference 2 for more details on naming
conventions and file structure). Since SRM spectral data is processed with the unknowns
the operator should at this time add the current run's SRM data to the SRM data archive
(in Step 4 below) if the SRM data passed the acceptance criteria.
4. Select Procedure 10 from menu to archive SRM data.
This will update the SRM data in a cumulative file called
D:\XDATA\ARCHIVE\SRM.DAT for charting purposes described in Section 6.6.
4.3 Detection Limits
The detection limits are determined by propagation of errors. The sources of random error which
are considered are: (1) calibration uncertainty (±5%); (2) long-term system stability (±7%);
(3) peak and background counting statistics; (4) uncertainty in attenuation corrections;
(5) uncertainty in overlap corrections; (6) uncertainty in flow rate; and (7) uncertainty in coarse
fraction due to flow fraction correction (dichotomous samples only). For typical lo (68%
confidence level) detection limits on a Teflo blank for fine particles and a Nuclepore blank for
coarse (2.5 fj. - 10,u) particles see Appendix 9.6. These detection limits are defined in terms of the
uncertainty in the blank. This ignores the effect of other elements which generally is small except
for the light elements (potassium and lower) where overlapping spectral lines will increase the
detection limit. Note: The difference in the detection limits between the two filters is due more to
the difference in sensitivity to fine and coarse particles and less to the difference in filter material.
Higher confidence levels may be chosen for the detection limits by multiplying the lo limits by 2
for a 2o (or 95% level) or by 3 for 3o (or 99.7% level).
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4.4 Data Reporting
There are five output files created for data reporting depending upon the reported concentration
units and format. Three are created by the least squares analysis program (see Section 4.2) and
two are created upon request. The f iles are named so as to reveal their contents. They are:
(1) the LSQnnnn.NG3 file containing data as ng/m3, (2) the LSQnnnn.CM2 file containing data as
ng/cm2, (3) the IPABnnnn.DAT file which is a special format for uploading data to the branch data
base, (4) the LSQnnnn.SEM file which is a companion file to scanning electron microscopy in
which the elemental concentration in ng/cm2 has been converted to that of the most likely chemical
species, and (5) a Lotus spreadsheet created by the end user from the LSQnnnn.NG3 file.
Reference 2, Section 8.0 gives the procedures for creating hard copies of the first two files and the
uploading procedure for the third file. The fourth file is created from the LSQnnnn.CM2 file for
fine/coarse paired filters and so is available only in concentration units of ng/cm2. To create this
file follow the instructions below.
1. Run program SEMOUT. (This will execute from any directory).
2. Enter four-digit XRFID
3. Output file is in directory D:\XDATA\Xnnnn, with name LSQnnnn.SEM.
The fifth type of output file is intended for creation by the end user because there are some
customized choices which one rust make and it is assumed that the end user is the appropriate
person to make these choices. See Appendix 9.12 for instructions for creating the Lotus
spreadsheet version of the LSQnnnn.NG3 file.
Hard copies of LSQnnnn.NG3 and LSQnnnn.CM2 are filed in the data library located in room
S242J for use by branch personnel. (See Appendix 9.5 for selected examples of reports).
The uncertainty reported with each concentration is a lo (68% confidence level) uncertainty and is
determined by error propagation described in Section 4.3. Elements with concentrations below
three times the uncertainty are flagged with an asterisk (*) on the printed record. If the true
elemental concentration is zero then the fitting procedure implies that negative and positive results
are equally probable. Therefore negative numbers may be reported.
4.5 Data Archiving
A directory is created for each XRFID at run-time. In these directories are located all the raw
spectra files, field data or card files, and final data processed into report format. For a run
consisting of a full 72 samples the directory size is considerably larger than typical diskette size of
1.2 megabytes. To store all the contents of the directory and remove it from the hard disk, an
archiving procedure is used which compresses the data on Bernoulli cartridges and backs up the
same on diskettes. Refer to Reference 2, Section 8.0 for the archiving and de-archiving
procedures.
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5.0 Calibration
Calibration is by far the most complex task in the operation of the XRF facility. There are many
steps in the process which are best left to personnel with sufficient experience in energy dispersive
x-ray fluorescence analysis to understand the process. Heavy use is made of References 1 and 5 in
Section 8.0; it is the intent of this section to present the steps in a sequential organized manner and
refer the operator to the references for details.
There are several steps in the process which require manual entry of data (see Sections 5.7 and
5.8). These are error prone procedures which require careful attention. Gross entry errors may
eventually become obvious but small ones may go undetected so it is essential that the entered data
b^ carefully checked against the original records.
Calibration is performed only when a change in fluorescors is made or a serious malfunction
occurs requiring significant repairs. The spectrometer has gone as long as two years between
calibrations without persistent failures in the parameters monitored by the quality control
procedures. It takes approximately two weeks to complete a calibration.
5.1 Source and Description of Calibration Standards
There are three types of calibration standards. One type consists of thin films deposited on
Nuclepore film substrates (Micromatter Co., Eastsound, WA). These standards are available for
almost all the elements analyzed ranging in atomic number from 13 (Al) to 82 (Pb) with deposit
masses gravizatrically determined to ±5%. Another type consists of polymer films that contain
known amounts of two elements in the form of organo-metallic compounds dissolved in the
polymer (Reference 4, Section 8.0). These standards are available for elements with atomic
numbers above 21 (titanium and heavier). The third type are sulfur thin film Standard Reference
Materials available from NIST and certified for sulfur only. They are used only for calibration and
not for quality control. Some standards have high inherent volatility and do not serve well as
calibration standards. These are Se, Br, Hg, and elemental As. See Section 5.7 for the calibration
approach to determining the sensitivity for these elements. All standards are mounted in frames in
the same manner as unknowns, stored in Argus slide trays, and sealed in plastic bags until needed
for calibration.
The same set of standards is used every time the spectrometer is calibrated. The standards are
sufficiently durable to last many years, however occasionally one must be replaced due to
accidents in handling. To check standards against degradation we periodically'participate in
audits. (Sec Appendix 9.14 for an audit report). A listing of the set used in the last calibration
(October 1991) is given in Appendix 9.9.
5.2 Gain and Baseline Adjustments
To begin the calibration the gain and baseline are adjusted just as is done before any measurements
(See Section 3.2). However, this adjustment of the gain and baseline must not be changed until
all measurements of background and shapes standards are completed! This may take five days,
so it is wise for the operator to ascertain that the gain and baseline are stable prior to beginning
calibration by making several short runs of samples (Procedure 5 in menu, Appendix 9.4) over a
24 hour period. If the run-time quality control results are stable, calibration can begin.
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During the period that the background and shapes are being measured (see Sections 5.3 and 5.4)
the run-time quality control results are generated in hard copy form. The operator should have
about seven to 10 runs of the run-time QC results accumulated over the period of background and
shapes measurement during which no adjustments were made to the gain and baseline. The
results for all parameters measured are entered into the spreadsheet C:\LOTUS\QCTGT91.WK1
where the averages are computed. These averages form the new basis for run-time quality control
target limits for future measurements. To instate the new limits the operator runs the program
C:\XRF\XRFRUN\CALDB.EXE and follows the instructions to enter the data. (See Reference 5
pages 47, 59, and 73 found in Section 8.0). The data are then entered manually into file
C:\XRF\XRFRUN\QCTOLOO1.DAT.
5.3 Background Measurement
Thirty-six clean Teflo blanks are kept sealed in a plastic bag and are used exclusively for
background measurement. Blanks are analyzed using Procedure 5 from the menu (Appendix 9.4)
with run Option 3. (see Section 3.3). This will put the spectra in the directory
D:\XDATA\BLANK\ with names Blnnnnpp.XRO. Procedure 11 from the menu is used to sum
blanks on a channel by channel basis. This procedure is self explanatory but for more information
refer to Section 4.0 of Reference 1 found in Section 8.0 of this SOP for instructions. Note: the
operator may find that it is best to preview the spectra for each fluorescer before selecting the
spectra to be included in the sum in order to know which filters are contaminated. This can be
done with Procedure 11 - just execute the procedure twice, once for a preview and again for final
selection. If contaminated filters are found they are excluded from the sum. When the procedure
is complete there are four background spectra files called 'SUMBLANK' files - one for each
fluorescer and with names SBnnnn.BFj where "j" is the fluorescer number. These files reside in
the D:\XDATA\BLANK\ sub-directory.
5.4 Shape Standards
The shapes standards are thin film standards consisting of ultra pure elemental materials for the
purpose of determining the physical shape of the pulse height spectrum. For this purpose it is not
necessary for the concentration of the standard to be known - only that it be pure. A slight
contaminant in the region of interest in a shape standard can have serious effect on the ability of
the least squares fitting algorithm to fit the shapes to the unknown. For this reason the Se, Br, Hg,
and elemental As standards, whose compounds are volatile, are kept in separate plastic bags to
prevent contamination of other standards. For most of the shape standards calibration standards
are used because the calibration standards are quite pure. However, a few of the calibration
standards have impurities which render them ineffective as shapes standards even though they are
adequate for calibration. In these cases aerosol standards have been prepared for exclusive use as
shape standards. The shape standards are kept in trays and sealed in plastic bags until needed.
The shape standards are analyzed using Selection 5 from the computer menu (see Appendix 9.4)
with Option 4. This will store the raw spectra in the sub-directory D:\XDATA\SHAPE\ with the
file names STnnnnpp.XRO. After shapes measurement the shape spectra must have the
background subtracted from them using Selection 12 from the computer menu. This is an
interactive procedure and is described in Section 4.0 of Reference 1. found in Section 8.0 of this
SOP Guidelines for subtraction of the SUMBLANK f iles from the shape spectra are also given
in Reference 5, page 20. The background subtracted spectra are given file names Bknnnn.SDj,
where "j" refers to the fluorescor number.
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The channel limits which define the location of the elemental peaks in the spectrum are selected
next. This is done by viewing the spectra BKnnnn.SDJ with Selection 13 from the computer
menu. Guidelines for choosing the channel limits are given in Reference 5, page 20. Upon
selection of the limits they are put into the file C:\XRF\CALIB\SHCARD.CD manually via an
editor. Refer to Reference 5, page 23 for the structure of this file.
The shapes files C:\XRF\CALffi\SHAPES.SHn are created next by running the program
C:\XRF\CALEB\SHAPES.FOR using file SHCARD.CD as the input file. In addition to shapes
data the shapes files contain the elemental sensitivities which at this point in the calibration
process are all set equal to 1000. They will be changed to correct values once the calibration
standards are measured. These shapes files must be copied to the D:\XDATA\SHAPE sub-
directory.
5.5 Determine Fraction of Measured K Lines to Total K Lines
In the calculation of the sensitivity, a physical model is used in which it is assumed that all of the
Kcc, K6 and escape peak x-rays are measured. This is not always the case as sometimes the K6
and escape peaks are excluded as is typical at fluorescer boundaries. In such cases as these, the
ratio of measured lines to total lines must be determined for each element. This is done by
measuring each applicable element using FPLOT from the computer menu (Appendix 9.4) and is
described in Appendix A of Reference 1 found in Section 8.0 of this SOP Reference 5, pages 52
to 57 gives a detailed account of which elements require this measurement and the results from the
last calibration.
5.6 Measure Calibration Standards
The calibration standards are measured as described in Section 3.3 using run Option 1. At this
point in the calibration the new quality control tolerance limits have been determined and instated
in the proper file and the background and shapes standards have been measured, so the gain and
baseline can now be adjusted if necessary to meet the new criteria.
5.7 Calculate Sensitivities
The sensitivities are calculated using a model based on the fundamentals of the x-ray physics
process as well as measurements on the calibration standards. This approach allows the
calculation of sensitivities for elements for which there are poor or no standards such as volatile
ones like Se, Br, Hg, and elemental As as well as improving on elements with good standards. See
Section 4.0 of Reference 1 found in Section 8.0 of this SOP for more details on the model and
approach.
The spectra from the calibration standards are analyzed as described in Section 4.2 using the newly
determined shapes files D:\XDATA\SHAPE\SHAPES.SHn. With the sensitivities set equal to
1000 in these f iles the results of analysis will be in terms of intensity rather than concentration in
the output file LSQnnnn.CM2 under the coltimns entitled "RAW DATA". A hard copy of this file
must be printed because the intensity data must be entered by hand into file
C:\XRF\CALIBVFACTOR\XXXXXX.YYY where XXXXXX.YYY is an operator-defined file
name. (Refer to file CAL91C.DAT in the same directory-for an example of the format of the
data). The operator then runs program C:\XRF\CALEB\FACTOR\XRFCAL.EXE and specifies
XXXXXX.YYY as the input file to calculate the sensitivities and write them to file
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XRFCAL.OUT. Refer to Reference 6, Section 8.0 for the contents of XRFCAL.OUT from the
latest calibration period. This file shows the deviation of each standard from the fitted curve and a
value of chi-square for each fluorescer. If the deviations and chi-squares differ significantly from
previous values then the operator must determine which standards are causing the effect and assign
a higher uncertainty (or weighting) to thou, or replace or remove them if necessary. The fitted
sensitivities must then be entered by hand to the shapes files D:\XDATA\SHAPE\SHAPES.SHj to
create the final version of the shapes files for subsequent runs. Refer to Reference 5, page 29 for
an example of a shapes file.
5.8 Overlap Coefficients
The final step in calibration is assembling the necessary data to compute the overlap coefficients.
To obtain this data the operator reprocesses the calibration spectra with the newly fitted
sensitivities in the shapes files. The elements which are affected by overlapping elements are
contained in spreadsheet file C:\LOTUS\CAL91\OLAP91.WK1 along with all the necessary data
on each standard. Refer to Reference 6, Section 8.0 for the contents of the spreadsheet from the
latest calibration data. The data from the least squares analysis of the calibration standards which
is to be entered manually into this spreadsheet is contained under the column "RAW DATA" in
the LSQnnnn.CM2 file. The calculated overlap coefficients and their uncertainties are then
manually entered into the file D:\XDATA\ATTEN\OLAP91.LBL and
D:\XDATA\ATTEN\UOLAP91 .LBL.
6.0 Quality Control
6.1 Description of QC Standards
Along with the two trays of samples analyzed in each run there are two sets of six quality control
standards that are permanently mounted in the sample changer. One set, called the bottom
standards is analyzed at the beginning of each run and the other set, called the top standards is
analyzed at the end of each run. One standard in each set, is an NIST SRM on which a
quantitative analysis is reported and all the others are used as a run-time evaluation of the
operating condition of the spectrometer. A description of these standards follows:
Bottom standards
ID Description
Al 58 MicroMatter Al film on Nuclepore filter
S NTHP20 Sulfate aerosol on Teflo filter
VK AER01 Vanadium & potassium aerosol on Teflo filter
FePb 39b Polymer film containing Fe & Pb
ZrCd 38y Polymer film containing Zr & Cd
SRX 1833 NIST SRH certified for Si, K, Ti, Fe, Zn, Pb
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Top standards
ID Description
Al 50 MicroMatter Al film on Nuclepore filter
S NTHP19 Sulfate aerosol on Toflo filter
VK AER02 Vanadium & potassium aerosol on Teflo filter
FePb 31 a Polymer film containing Fe & Pb
ZrCd 40a Polymer film containing Zr & Cd
SRM 1832 NIST SRM certified for Al, Si, Ca, V, Mn, Co, Cu
6.2 Run-time Quality Control
During a run of samples one gets a hard copy printout of the results of the measurements on the
top and bottom QC standards (see Appendix 9.7 for a typical run-time printout). The parameters
which are checked and their significance are: peak areas (monitors change in sensitivity),
background areas (monitors contamination or background changes), CHAN, or centroid (monitors
gain and baseline adjustment to insure that spectra are assigned the correct channel), and FWHM,
(monitors degradation of the detector resolution). These four parameters are measured for
elements ranging from aluminum to lead and include atmospheric argon. The acceptable ranges
for these parameters are based on averages and allowable uncertainties determined during
calibration. The allowable uncertainties for elements other than argon are:
Peak area: ±
Background area: ±
Centroid: ±
FWKM: ±
7%
30%
3% of FWHM
6%
Any deviation from these established limits is automatically flagged at run-time for rapid and easy
recognition. This process results in 48 measurements made both before and after unknowns are
analyzed for a total of 96 measurements to assure proper operating condition of the XRF
spectrometer.
6.3 Quality Control with Standard Reference Materials
In addition to the run-time quality control procedure above the analysis results of the SRMs are
included in all data reports. The value reported is to be compared to the NIST effective value.
.(see Appendix 9.2 for explanation of effective value and 9.3 for results of SRM analysis). This
provides an overall check of the spectral processing program for the elements which are certified
in the standards. The sole purpose of the SRMs is to provide a quality control measure; the
standards are not used for calibration.
6.4 Acceptance Criteria and Procedures for Corrective Action
An entire XRF run is invalidated if more than two of the measurements on the 12 QC standards
exceed the allowable uncertainties as described under the Run-time Quality Control section or any
one measurement lies more than 20% outside of the limits. There is a special case in which three
failures are allowed and that is if one of the failures is due to arithmetic rounding off. The
acceptance criteria of results 4, for the elements certified in the SRMs is that the uncertainty
intervals for the analytical results and the certified values should overlap each other.
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If a run is invalidated due to centroid failure it can be corrected by adjusting the gain or baseline.
Correction for this is described under Gain and Baseline Adjustment in Spectrometer Operation
(Section 3.2). Failure due to a decrease in peak areas is usually due to dust on the detector
window which is remedied by gently blowing the area clean. Persistent high or low peak areas not
responding to the above measures indicate need for recalibration. Failure due to a high Ar peak
indicates that the helium flushing system is low on helium and atmospheric Ar has entered the
measurement cavity. Replacing the empty helium bottle will remedy this. After the necessary
adjustments are made the run is repeated.
6.5 Field Data Entry Checking
Field data consists of sample IDs, flow rates, sampling times, sites IDs, and other vital
information. These data are essential for proper data processing to produce the necessary
information for the investigators. To this end it is essential that a method be in place to insure that
field data is accurately entered into the data processing program.
There are four LOTUS spreadsheets which are used for data entry depending on which type of
sampler was used. These spreadsheets and their use are explained in Reference 2 (see
Section 8.0). Data are entered into these spreadsheets from the corresponding field data sheet
either by manual or electronic transfer. After manual entry the spreadsheet data are compared with
the data on the entry form. If the data agree with that on the data form, a printed copy of the
spreadsheet is signed to indicate its validity and is filed in the XRF ID assignment notebook.
Electronic field data entry is used when field data is submitted on diskettes. For sufficiently large
field data sets it is usually advantageous to write a computer program to extract the data and render
it in a format suitable to the corresponding LOTUS spreadsheet. There is no protocol for such
data transfer because the field data may come in a different format for each study which means that
a different program must be written for each case. Nonetheless such electronic transfer is still less
error prone than direct manual entry. For electronic field data entry the data for the first and last
samples in the spreadsheet and randomly selected data from the middle of the spreadsheet are
compared to the samples in the corresponding submitted data. If the data agree then a printed copy
of the spreadsheet is filed in the XRF ID assignment notebook.
6.6 Control Charts
Control charts are maintained on both Standard Reference Materials and the top QC standards.
The top QC standards are chosen for plotting because they are measured at the end of the run. Use
of the bottom standards may bias the results because they are analyzed at the beginning of the run
immediately after the gain and baseline adjustment (see Section 3.2) and therefore may have a
greater probability of being in control.
Two sets of control charts are maintained, one set is based on the run-time QC data and consists of
plots of peak area, background area, centroid, and FWHM for the following elements: Al, Si, S,
K, Ca, V, Fe, Zr, Cd, and Pb. Each parameter is divided by its mean which was determined during
calibration and this normalized value is plotted against a chronological run number to produce an
historical record of performance. The control chart upper and lower limits are based on experience
and are: peak (±7%); background (±30%); FWHM (±6%); centroid (±3% of FWHM). The
second set of control charts is based on actual least squares analysis of SRM Spectra for the
elements certified in both SRKs (Al, Si, K, Ca, Ti, V, Mn, Fe, Co, Cu, Zn, and Pb). Here the
measured concentration is divided by the effective value and this ratio is plotted as a function of
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time. The control limits are not set at a fixed level but are based on overlapping uncertainties and
are computed in the following manner: Assume an element in the standard is certified at a
concentration of 1000 ±120 ng/cm2 and upon analysis the concentration was determined to be
850 ± 100. The upper and lower control limits are (1000+120+100)71000 or 1.22 and (1000-120-
100)71000 or .78, respectively. The control limits vary slightly because the uncertainty of the
analytical results vary too. The plots for both sets of control charts will be in effect until
recalibration. Ratios rather than absolute magnitudes are plotted to allow a more rapid assessment
of relative change.
The collection of the run-time QC data is explained in Section 3.3 (Run Options) and the saving of
SRM analysis results is explained in Section 4.2 (Least Squares Analysis). The instructions for
creating the control charts for these data sets for subsequent viewing and retrieval are given below.
1. Copy files C:\XRRXRFRUN\XQC\ARCHxx.NEW to C:\QCXRF directory
2. Copy file D:\XDATA\ARCHIVE\SRM.DAT to C:\QCXRF directory
3. Run LOTUS 123
4. Retrieve C:\QCXRF\START.WK1
This automatically creates all graphs as .PIC files for viewing or printing with LOTUS
PRLNTGRAPH. See Appendix 9.10 for selected examples of control charts and Section 6.2 for
the significance of the parameters plotted.
6.7 Self Consistency Checks
There are certain properties that ambient aerosols possess which can be checked to ascertain the
validity of the analysis. One of these properties is that coarse fraction calcium is expected to be
greater than fine fraction calcium. If analysis results do not confirm this then it is indicative of a
sampler malfunction and such is automatically noted on the data report.
A second self-consistency check involves mass balance between the xrf-deternined concentration
and the gravimetric measurements. The elemental concentrations are converted to chemical
compound concentrations based on a species most likely to be present. The mass determined by
XRF plus the light element mass such as carbon and nitrogen determined by companion methods
should closely match the gravimetric mass. This information is useful to the investigators but is
not routinely included in the data report.
6.8 Goodness-of-Fit Measurements
The fitted spectrum and the measured spectrum are compared and a value for chi-square is
calculated and reported with the data. Chi-square values that are much larger than 1.0 indicate a
problem in the fitting procedure. Changes in detector resolution or gain in the amplifier produce
large values for chi-square; however such changes vould be detected by their run-time quality
control procedure (see Section 6.2). Also, large chi-square values can accompany results for
heavily loaded filters even though the relative errors are typical. In addition, elements analyzed by
the titanium fluorescer may experience large chi-square values due to interferences from
overlapping elements. Chi-square is a more useful measure of goodness-of-fit for the other
fluorescers for this reason.
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To acquire more information about fitting problems the fitted spectra can be viewed on the screen
or a hard copy printed. Such plots can be compared to the unknown spectra, background spectra,
or to the library shape standards to help elucidate the suspected problem. Various statistics such as
the correlation coefficient can be calculated on the fitted and measured spectra as a additional
measure of the goodness-of-fit. See Appendix 9.13 for an example of the fitted spectrum
superposed on its measured spectrum along with the associated statistics. The fitted spectra are
stored in the directory D:\XDATA\LSQFIT. Refer to Section 3.0 of Reference 1 found in
Section 8.0 of this SOP for instructions for plotting spectra.
6.9 Audit Reports
From time to time the x-ray facility is audited by in-house Quality Assurance personnel. Refer to
Appendix 9.14 for the latest report.
7.0 Preventive Maintenance
7.1 Liquid Nitrogen Filling Procedure
The liquid nitrogen dewar shall be filled weekly. A check of the liquid nitrogen log will show the
last time it was filled as well as the purchase order number and vendor from whom to order a
replacement tank. A filled dewar will last approximately eight to nine days but safe practice calls
for weekly filling.
1. Make sure x-rays are off (refer to shutdown procedure in Appendix A, Reference 1 found
in Section 8.0 of this SOP) and open rear cabinet behind the sample changer.
2. Remove argon bag and place temporarily in safe place where it will not attract dust.
3. Open black-handled valve.
4. Enter time of day in log book under "BLOW" and quickly proceed to liquid nitrogen tank
outside lab and open the valve marked "LIQUID".
5. Go behind spectrometer and wait three to four minutes and open blue-handled brass valve
to allow liquid nitrogen to flow into the dewar. Then close the black-handled valve. Enter
the time at which the blue-handled valve was opened under "FILL".
6. The filling will take approximately six to nine minutes. During this time locate the
protective glove near the log book and wait until overflow occurs from the neck of the
dewar. When overflow occurs turn off the blue-handled valve and quickly go (with glove)
to the liquid nitrogen tank and turn off the valve marked "LIQUID". Enter time at which
blue-handled valve was turned off in log book under "STOP" and the total filling time
under "t" (Should filling take longer than 10 minutes turn off the blue-handled valve and
proceed as if overflow has occurred.)
7. Wait approximately two hours for the frozen condensation on the lines to evaporate and
reinsert argon bag and close and lock rear cabinet.
3-58
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SOP for EPA's LBL Energy Dispersive
Volume 3, Chapter 1 X-Ray Fluorescence Spectrometry
7.2 Argon Bag Filling Procedure
The argon bag on top of the conical x-ray shield shall be filled as needed - usually once or twice a
week.
1. Make sure x-rays are off (refer to shutdown procedure in Appendix A, Reference 1 found
in Section 8.0 of this SOP) and take argon bag to argon tank.
2. Attach hose and fill. Rate of filling and amount of fill is not important.
3. Replace argon bag in spectrometer. Bleed excess argon from bag if necessary. Close and
lock rear cabinet.
7.3 Preparation for Power Outage
To prevent damage during power outages to the x-ray tube certain precautions shall be taken.
Refer to EPA XRF SPECTROMETER USER'S GUIDE, VOLUME 1, Appendix A for this
procedure.
7.4 Lab Environment
The laboratory space housing the spectrometer is temperature controlled to 70 ± 2.0°F with a
relative humidity of 45%. These conditions are maintained by equipment located in the room. If
malfunctions occur so as alter these conditions the spectrometer shall not be operated. The proper
maintenance personnel shall be notified for repairs.
8.0 References
8.1 Barlow, P.M., LBL XRF Spectrometer User's Guide, Volume 1.
8.2 Waldruff, P., LBL XRF Spectrometer User's Guide, Volume 11.
8.3 Drane, E.A., et. al., Data Processing Procedures for Elemental Analysis of Atmospheric Aerosols
by X-ray Fluorescence, Document TR-83-01 submitted under contract 68-02-2566.
8.4 Dzubay, T.G., et. al., Polymer Film Standards for X-ray Fluorescence Spectrometers, J. Trace and
Microprobe Techniques, 5(4), 327-341, 1987-88.
8.5 Laboratory Notebook No. 888242, Issued to Thomas G. Dzubay, March 28, 1988.
8.6 Calibration Notebook, Located in S242J.
3-59
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Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.0 Appendix
9.1 Definitions
card files
chi-square
fluorescer
fwhm
NIST
nnnn
PP
run-time
SARB
shape
SRMs
Teflo
unknown
an ASCII file created by LOTUS containing the field data and data
processing options. Used in least squares analysis of spectra.
a statistic which is a function of the sun of squares of the differences of
the fitted and measured spectrum.
a secondary target excited by the x-ray source and in turn excites the
sample.
full width at half maximum, a measure of spectral resolution LBL -
Lawrence Berkeley Laboratory.
National Institute of Standards and Technology.
representation of an XRFID in a file name.
representation of slot position in Argus slide tray.
during the operation of the spectrometer.
Source Apportionment Research Branch.
the actual shape of a background corrected pulse height spectrum for an
element.
standard reference materials.
trade name of a Teflon filter.
a sample submitted for analysis whose elemental concentration is not
known.
xrf x-ray fluorescence.
9.2 Derivation of -Effective Mass from NIST Certified Values
When NIST standards are analyzed no attenuation corrections are made even though such
corrections are routinely made for aerosol samples. Also the NIST standards could not be
mounted in the same plane as the calibration standards so an empirically determined geometric
correction factor was determined to correct for this. Therefore, the attenuation and spacing
corrections are applied to the standards by means of the below expression.
E=IOOOO"-MP/(AFZ)
3-61
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SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volume 3, Chapter 1
9.3
Where:
E
M
P
A
F
Z
effective mass (ng/cm2)
total mass of standard film as measured by NIST (mg)
% abundance supplied by NIST
area of deposit (cm2) supplied by NIST
attenuation correction supplied by NIST
spacing correction measured in XRF lab
SRM 1833
Serial No.: 882 Area (cm2): 10.06
Element Certified % F Z
Si
K
Ti
Fe
Zn
Pb
Serial No.
Element
Al
Si
Ca
V
Mn
Co
Cu
21.63 +- 1.4
11.48 +- 1.1
8.91 +- 1.2
9.62 +- 0.3
2.16 +- 0.2
9.03 +- 0.5
1 502 Area (cm
Certified %
9.27 +- 0.6
21.39 +- 0.7
12.15 +- 0.8
2.96 +- 0.3
2.83 +- 0.3
0.65 +- 0.04
1.49 +- 0.1
.14
.07
.04
.02
.01
.00
SRM
2): 10
F
.17
.14
.04
.02
.02
.01
.01
.043
.050
.061
.153
.153
1.153
1832
06
Z
1.043
1.043
1.050
1.061
1.110
.153
1.153
Results of SRM Analysis on 4/29/1992
SI
K
TI
FE
ZN
PB
SRM 1833
30153.7 +- 2650.4
16289.8 +- 991.6
13375.8 +- 1151.0
13376.1 +- 1151.8
2932.4 +- 252.9
13277.6 +- 1142.7
/
C
IV
C
AL
SI
:A
v
IN
:o
cu
Film Weight (mg): 1.658
Effective mass (ng/cm2)
29982 +-
16841 +-
13308 +-
13481 +-
3057 +-
12908 +-
Film Weight
Effective mass
12459 +-
29506 +-
18249 +-
4486 +-
4100 +-
915 +-
2099 +-
SRM 1832
13353.6 +-
27852.2 +-
18825.4 +-
4591.4 +-
4421.0 +-
922.1 +-
2027.5 +-
1940
1614
1792
420
283
715
(mg): 1.650
(ng/cm2)
806
966
1202
455
435
56
141
1171.4
2403.0
1145.5
395.3
270.0
80.3
175.0
3-62
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Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.4 Computer Menu
MAKE
SELECTION
AND
PRESS
ENTER
SELECTIONS
1. RunFPLOT
2. Run DEBUG
3. Run CMD for testing basic XRF commands.
4. Test frames for sample changer compatibility.
5. Run XRF analyzer.
6. Least Squares Analysis of XRF Spectra
7. Format diskette on Drive A: (1 213 952 bytes)
8. Format diskette on Drive B: (362 496 bytes)
9. Check fixed disk
10. Add SRM 1833&1832to archive
11. Run SUMBLK on blanks
12. Run BAKSUB on shapes standards
13. Plot spectra on screen
14. Convert spectra binary -> decimal & vice versa
15. Determine calibration factors
16. Run S & Cd QC standards
17. Archive XRF data
18. De-archive XRF data
19. Cross talk (Xtalk)
20. Upload data to VAX and print
21. Add last run's QC standards to archive (non-SRMs)
22. Check SRM data on selected XRFID
23. Print x-ray data to lab HP laser printer
3-63
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SOP for EPA's LBL Energy Dispersive
Volume 3, Chapter 1
9.5 Data Report on a Sample from a VAPS Sampler
SUMMARY: TELPLICE: 3/16-3/30
SITE
DURATION (MIN)
FLOW
FRAC
XRF ID =
SAMPLE ID
MASS
*AL
SI
*P
S
CL
K
CA
*SC
*n
*v
*CR
MN
FE
*CO
MI
CU
ZN
*GA
*GE
AS
SE
BR
*RB
SR
*Y
*ZR
*MO
*RH
*PD
*AG
*CD
SN
*SB
*TE
*I
*CS
*BA
*LA
*W
*AU
*MG
PB
DTEP
714.0
.0869
148206
T0033
FINE, MG/M3
77912. +-
162.2 +-
213.4 +-
12.1 +-
2653.4 +-
1164.4 +-
193.6 +-
43.4 +-
3.6 +-
17.6 +-
4.6 +-
2.0 +-
10.0 +-
243.7 +-
2.8 +-
3.8 +-
14.3 +-
167.5 +-
2.4 +-
3.3 +-
24.7 +-
4.7 +-
29.0 +-
1.7 +-
2.9 +-
12.4 +-
2.9 +-
7.3 +-
.0 +-
-3.6 +-
-6.4 +-
8.5 +-
54.3 +-
-1.6
2.5 +-
25.0 +-
-4.0 +-
-7.7 +-
-4.8 +-
-1.1 +-
-.9 +-
-.4 +-
221.6 +-
SAMPLE
DATE =
FLOW (L/MIN)
XRF ID
SAMPLE
1962.
74.1
40.4
18.5
183.7
79.3
13.8
5.6
4.1
6.6
2.3
1.0
1.4
21.9
1.8
1.2
1.9
14.9
1.0
1.3
3.6
.8
2.8
.8
.9
6.1
4.8
4.8
3.2
3.1
3.4
• 4.5
9.4
6.4
7.5
9.6
11.2
13.7
34.5
2.6
1.8
1.9
19.7
ID
MASS
AL
SI
*p
7S
*CL
K
CA
*SC
Tl
*v
CR
MN
FE
*CO
*MI
CU
ZN
*GA
*GE
*AS
*SE
BR
*RB
SR
*Y
*ZR
*MO
*RH
*PD
*AG
*CD
*SN
*SB
*TE
*I
*CS
BA
*LA
*W
*AU
*MG
PB
3/20/92 AND 1900 HOURS
= +- .500
= 148256
= MU0033
COARSE, MG/M3
11347. +-
539.9 +-
909.5 +-
-5.5 +-
285.7 +-
34.8 +-
63.5 +-
181.7 +-
-1.3 +-
54.7 +-
3.2 +-
9.8 +-
10.1 +-
783.5 +-
4.8 +-
.3 +-
8.8 +-
27.6 +-
-.0 +-
.0 +-
1.8 +-
.7 +-
7.9 +-
1.0 +-
2.2 +-
3.9 +-
4.3 +-
-3.2 +-
-1.2 +-
-1.0 +-
1.2 +-
-.7 +-
2.3 +-
-.6 +-
-7.2 +-
2.4 +-
12.4 +-
25.1 +-
22.6 +-
1.5 +-
.2 +-
1.5 +-
46.0 +-
8!2.
173.8
232.7
11.3
84.9
24.6
8.9
13.9
2.2
9.6
1.7
1.6
1.3
78.2
1.7
.6
1.3
4.9
.4
.6
1.2
.4
1.1
.4
.5
2.9
2.6
2.2
1.6
1.7
1.9
2.2
3.9
3.3
3.8
4.7
3.9
7.4
17.9
1.3
.9
1.0
6.2
INDICATES THAT THE CONCENTRATION IS BELOW THREE TIME THE UNCERTAINTY.
XRF DATE = 04/29/1992 16:35 RBK (F): 04/29/1992 20:35 RBK
(C) SPECTRAL ANALYSIS DATE = 5/20/1992
3-64
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Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
9.6
Example SEM Format
FINE FILTER
XRFID
SAMPLE ID
FINE MASS (UG) =
FINE AREA (CM') =
Data Report
148602
T3001
240.0
11.95
COARSE FILTER
XRFID
SAMPLE ID
COARSE MASS (UG) =
COARSE AREA (CM3) =
M300I
270.0
11.95
148652
.
FINE LOADING (UG/CM') = 20.
COARSE LOADING (UG/CM:) = 22.6
ELEMENT NO/CM' ASSUMED WEIGHTS
COMPOUND
ELEMENT NG/CM2 ASSUMED WEIGHT1
COMPOUND
*AL
SI
*P
S
*CL
K
CA
*SC
*TI
*V
*CR
*MN
FE
*CO
*NI
CU
ZN
*GA
*GE
*AS
*SE
BR
*RB
*SR
*Y
*ZR
*MO
*RH
*PD
*AG
*CD
SN
*SB
*TE
1
*CS
*BA
*LA
*W
*AU
*HG
PB
Fraction of tine mass
-57.1
95.6
12.6
324.3
15.9
20.3
55.9
-11.8
8.4
.9
2.9
.8
218.7
1.5
1.1
15.8
20.2
-1.7
-1.9
6.3
-1.4
113.0
-2.1
.9
14.6
-7.8
2.3
-1.9
-4.2
-3.3
7.0
126.0
-11.3
6.1
49.8
-3.8
9.8
-1.8
-7.1
5.0
.1
390.7
ai. a >u m
* INDICATES THAT THE
+-58.4
+-25.6
+-12.9
+-42.9
+- 7.8
+- 5.1
+- 7.7
+- 4.8
+- 6.8
+- 2.3
+- 1.1
+- 1.1
+-20.2
+- 2.6
+- 1.5
+- 2.4
+- 2.6
+- 1.4
+- 1.7
+- 5.2
+- .9
+-10.1
+- 1.4
+- 1.3
+- 6.7
+- 4.7
+- 4.8
+- 3.4
+- 3.4
+- 3.7
+- 4.8
+-14.3
+- 6.6
+- 8.1
+- 1 1 . 1
+-11. 8
+-14. 6
+-36. 1
+- 2.8
+- 2.6
+- 2.8
+-34.5
;cd for =
A1203
Si02
P04
(NH4)2S04
NaCl
K.20
CaCO3
Sc203
Ti02
V205
Cr203
Mn02
FeO
CoO
NiO
CuO
ZnO
Ga
Ge
As
Se
Br
Rb
Sr
Y
Zr
Mo
Rh
Pd
Ag
CdO
SnO
Sb
Te
I
Cs
Ba
La
W
Au
Hg
PbO
.14
CONCENTRATION
-.40 +-
1.02+-
.19 +-
6.66 +-
.20+-
.12 +-
.70+-
-.09 +-
.07+-
.00+-
.02+-
.00+-
1 .40 +-
.00+-
.00+-
.10+-
.13 +-
-.00 +-
-.00 +-
.03 +-
-.00 +-
.56+-
-.01 +-
.00+-
.07+-
-.04 +-
.01 +-
-.00 +-
-.02 +-
-.02 +-
.04 +-
.71 +-
-.06 +-
.03 +-
.25 +-
-.02 +-
.05 +-
-.00 +-
-.04 +-
.02+-
.00+-
2. 10+-
.41
.27
.20
.88.1
0
.03
.10
.04
.06
.02
.00
.00
.13
.02
.00
.01
.02
.00
.00
.03
.00
.05
.00
.00
.03
.02
02
.02
.02
.02
.03
.08
.03
.04
.06
.06
.07
.18
.01
.01
.01
.19
1S BELOW Th
*AL
SI
S
CL
K
CA
*SC
TI
*V
*CR
MN
FE
*CO
*NI
CU
ZN
*GA
*GE
*AS
*SE
BR
*RB
*SR
*Y
*ZR
*MO
*RH
*PD
*AG
*CD
SN
*SB
*TE
*CS
BA
*LA
*W
*AU
*HG
PB
408.2 +-182.7
2198.0+-542.0
53.0
128.4
162.9
172.9
816.8
-7.1
55.8
2.7
.0
14.1
+-29.2
+-34.2
+-21.2
+-17.7
+-59.2
.+- 5.5
+-15.7
+- 3.9
+- 1.8
+- 2.2
1570.6+-151.6
-.5 +- 4.6
3.0
94.3
59.1
1.2
-3.7
2.6
-1.9
53.1
2.0
4.0
4.7
-3.5
4.2
-6.5
.2
6.9
7.0
101.1
14.7
-10.7
32.1
2.2
92.7
46.1
-3.9
-.4
2.2
219.3
+- 2.0
+-10.3
6.7
1.5
1.7
4.1
1.0
5.1
1.4
1.5
6.2
5.0
5.2
3.5
3.8
4.4
5.0
+-12.9
+- 7.8
+- 8.5
+-11.1
+-12.7
+-18.3
+-39.8
+- 3.4
+- 2.7
+- 3.1
+-19.9
A1203
Si02
P04
S04
NaCl
K20
CaC03
Sc203
Ti02
V205
Cr203
Mn02
FeO
CoO
NiO
CuO
ZnO
Ga
Ge
As
Se
Br
Rb
Sr
Y
Zr
Mo
Rh
Pd
Ag
CdO
SnO
Sb
Te
I
Cs
Ba
La
W
Au
Hg
PbO
2.56 +-
20.81 +-
.72+-
I.70+-
1.83+-
.92+-
9.03 +-
-.05 +-
.41 +-
.02+-
.00+-
.10+-
8.94 +-
-.00 +-
.02+-
.52+-
.33+-
.00+-
-.02 +-
.01 +-
-.00 +-
.24+-
.00+-
.02+-
.02+-
-.02 +-
.02+-
-.03 +-
.00+-
.03+-
.04+-
.51 +-
.07+-
-.05 +-
.14+-
.00+-
.41 +-
.20+-
-.02 +-
-.00 +-
.00+-
1.05 +-
1.15
5.13
.40
.45
.24
.09
.65
.04
.12
.03
.01
.02
.86
.03
.01
.06
.04
.00
.00
.02
.00
.02
.00
.00
.03
.02
.02
.02
.02
.02
.03
.06
.03
.04
.05
.06
.08
.18
.02
.01
.01
.09
Fraction of coarse mass
iunted for = .47
3-65
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SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volume 3, Chapter 1
9.7 Detection Limits
Element
AL
SI
P
S
CL
K
CA
SC
TI
V
CR
MN
FE
CO
NI
CU
ZN
GA
GE
AS
SE
BR
RB
SR
Y
ZR
MO
RH
PD
AG
CD
SN
SB
TE
I
CS
BA
LA
W
AU
HG
PB
( I o) for Teflo and Nuclepore
Teflo - fine
DL
ng/cnr
55.0
17.3
10.8
6.0
4.8
3.1
3.4
3.3
5.0
.9
.0
.1
2.2
.5
.2
.0
.8
.7
1.1
1.1
.6
.8
.8
.9
4.1
3.7
3.6
2.6
2.6
2.8
3.3
6.1
5.0
6.3
7.1
8.9
10.7
26.7
2.0
1.7
1.9
2.0
Blank Filters
Nuclepore
Element
AL
SI
P
S
CL
K
CA
SC
TI
V
CR
MN
FE
CO
NI
CU
ZN
GA
GE
AS
SE
BR
RB
SR
Y
ZR
MO
RH
PD
AG
CD
SN
SB
TE
I
CS
BA
LA
W
AU
HG
PB
- coarse
DL
ng/,cm
95.5
34.6
16.2
9.1
7.2
3.4
3.6
3.7
5.2
2.3
1.7
1.4
3.7
1.7
1.6
1.3
.9
.7
1.2
1.2
.7
.9
.8
1.0
4.0
3.7
3.9
2.6
2.6
2.9
3.5
6.1
5.1
6.3
7.3
8.8
10.7
27.6
2.0
1.8
1.9
2.1
3-66
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Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
These detection limits are interference-free and therefore ignore the effect of overlapping spectral
lines on the light elements (postassium and lower). On an actual sample, the detection limit may
be higher for these elements.
The difference in detection limits between the two filters is due more to the difference in
sensitivity to fine and coarse particles and less to the difference in filter material.
Higher confidence levels may be chosen for the detection limits by multiplying the I o Imits by 2
for a 2o (or 95% level) or by 3 for 3o (or 99.7% level).
To convert the detection limits to more useful units, one can use the typical deposit areas for
37 mm and 47 mm diameter filters of 6.5cm3 and 12.0 cm2, respectively.
9.8 Typical Run-time QC Results
08/14/1992 13:12rbk
RESULTS FOR BOTTOM STANDARDS:
S F
1 1 AL58
LOWER LIMITS:
UPPER LIMITS:
151137. SUMMARY:
2 1 SNTHP20
LOWER LIMITS:
UPPER LIMITS:
151138. SUMMARY:
3 2 VKAERO1
LOWER LIMITS:
UPPER LIMITS:
151139. SUMMARY:
4 3 FEPB 39B
LOWER LIMITS:
UPPER LIMITS:
151140. SUMMARY:
5 4 ZRCD 38Y
LOWER LIMITS:
UPPER LIMITS:
151141. SUMMARY:
6 8 SRM 1833
LOWER LIMITS:
UPPER LIMITS:
151142. SUMMARY:
AREA BACK CHAN FWHM
AREA BACK CHAN FWHM
35178.
32640.
37554.
OK
22416.
20240.
23287.
OK
26838.
24551.
28247.
OK
39328.
34571.
39775.
OK
37962.
33844.
38938.
OK
18361.
17207.
19797.
OK
5806.
3944.
7325.
OK
5850.
4012.
7450.
OK
4336.
2754.
5115.
OK
1974.
1245.
2312.
OK
3152.
2138.
3971.
OK
7718.
5304.
9850.
OK
28.88
28.64
29.00
OK
54.86
54.54
54.92
OK
86.91
86.61
87.09
OK
182.09
181.85
182.27
OK
700.67
700.42
701.10
OK
36.79
36.53
36.89
OK
6.1
5.7
6.5
OK
6.3
5.9
6.7
OK
8.1
7.6
8.6
OK
6.9
6.5
7.3
OK
11.3
10.6
12.0
OK
6.2
5.7
6.5
OK
-942.
1107.
-3322.
OK
17.
-268.
803.
OK
24235.
22071.
25393.
OK
50458.
45280.
52096.
OK
28069.
25190.
28982.
OK
213058.
195242.
224633.
OK
4277.
2089.
6267.
OK
3052.
1485.
4456.
OK
2695.
1766.
3280.
OK
13314.
8602.
15975.
OK
1485.
1031.
1915.
OK
23562.
15604.
28978.
OK
79.43 .0
65.00 .0
85.00 .0
OK OK OK
.00 .0
65.00 .0
85.00 .0
OK OK
137.256.1
137.01 5.7
137.37 6.5
OK OK OK
375.347.7
375.13 7.2
375.59 8.2
OK OK OK
472.35 8.8
472.13 8.3
472.65 9.3
OK OK OK
123.41 6.1
123.18 5.7
123.546.5
OK OK OK
3-67
-------
SOP for EPA's LBL Energy Dispersive
Volume 3, Chapter 1
RESULTS FOR BOTTOM STANDARDS:
S F
1 1 AL50
LOWER LIMITS:
UPPER LIMITS.
151187. SUMMARY:
2 1 SNTHP19
LOWER LIMITS:
UPPER LIMITS:
151188. SUMMARY:
3 2 VKAERO2
LOWER LIMITS:
UPPER LIMITS:
151189. SUMMARY:
4 3 FEPB31A
LOWER LIMITS:
UPPER LIMITS:
151190. SUMMARY:
5 4 ZRCD40A
LOWER LIMITS:
UPPER LIMITS:
151 191. SUMMARY:
6 8 SRM 1832
LOWER LIMITS:
UPPER LIMITS:
151192. SUMMARY:
AREA
31159.
29098.
33478.
OK
21232.
19693.
22658.
OK
43761.
40352.
46427.
OK
38593.
34921.
40178.
OK
41860.
37480.
43122.
OK
14358.
13773.
15846.
OK
BACK
4361.
3045.
5655.
OK
4941.
3323.
6172.
OK
5716.
3807.
7070.
OK
1953.
1271.
2360.
OK
3548.
2380.
4419.
OK
10651.
7304.
13564.
OK
CHAN
28.93
28.64
29.00
OK
54.81
54.54
54.92
OK
86.87
86.61
87.09
OK
182.10
181.85
182.27
OK
700.74
700.42
701.10
OK
36.57
36.31
36.65
OK
FWHM
6.1
5.7
6.5
OK
6.4
5.9
6.7
OK
8.1
7.6
8.6
OK
6.9
6.5
7.3
OK
11.3
10.6
12.0
OK
5.8
5.5
6.1
OK
AREA BACK
-143.
-41.
122.
95.
-730.
2190.
OK
39176.
36052.
41479.
OK
50043.
45828.
52727.
OK
31100.
28100.
32330.
OK
160051.
147811.
170062.
OK
3080.
1506.
4518.
OK
2667.
1274.
3822.
OK
3828.
2430.
4514.
OK
13209.
8743.
16237.
OK
1755.
1069.
1986.
OK
45676.
31874.
59194.
OK
CHAN FWHM
.00207 .9
65.00 .0
85.00 .0
OK OK
.00148.2
65.00 .0
85.00 .0
OK OK
137.25 6.1
137.01 5.7
137.37 6.5
OK OK OK
375.39 7.7
375.13 7.2
375.59 8.2
OK OK OK
472.38 8.8
472.13 8.3
472.65 9.3
OK OK OK
98.37 6.3
98.10 5.9
98.48 6.7
OK OK OK
3-68
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.9 Assignment of XRFIDs Form
ASSIGNMENT OF XRFlDs
FIRST 3
DIGITS
STUDY NAME
& ARCHIVE ID
SUB-STUDIES
01234567 8 9
Assigner must enter data and initials each time a sub-study assignment is made.
3-69
-------
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volume 3, Chapter 1
9. \ 0 XRF Run Status Log Form
XRF RUN STATUS LOG
XRFID
STUDY NAME &
NO. OF SAMPLES
XRF
DATE
CARD
DATE
.
LSQ
DATE
QC
CHECKS
IPAB
XFER
HARDCOPY
LIB FILE
ARCHIVE
DATE
3-70
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.11 Sample Check-Out Form
SAMPLE CHECK-OUT LOG
XRFID
SLOT NUMBERS
DATE
SAMPLE IDs, COMMENTS, INITIALS
3-71
-------
SOP for EPA's LBL Energy Dispersive
Volume 3, Chapter 1
9.12 Calibration Standards
Standard
ID
A157
A154
A163
A129
A143.2
A162
A175
SiO46
SJO47
SiO51A
SiO51B
SJO56
SiOSO
SiO27.6
SiO46.1
SiO72.2
GaP34
GaP40
GaP70
GaP 105
CuS 13
CuS33
NIST135
NIST137
NIST138
NIST140
NIST141
NIST143
NIST142
NIST139
NIST136
NIST134
NaCl 57
NaCl 87
NaCl 45
NaC! 72
KC145
KC153
KC170
KC145
KC153
KC170
CaF237
CaF229
CaF290
Element
Al
Al
Al
Ai
Al
Al
Al
Si
Si
Si
Si
Si
Si
Si
Si
Si
P
P
P
P
S
s
S
s
s
s
s
s
s
s
s
s
Cl
Cl
Cl
Cl
Cl
Cl
Cl
K
K
K
Ca
Ca
Ca
and Concentrations
jag/cm2
57.0
54.0
63.0
29.0
43.2
62.0
75.0
29.3
29.9
32.5
32.5
35.7
51.0
17.6
29.4
46.0
10.5
12.3
21.5
32.3
13.0
33.0
1.91
2.04
2.14
2.31
2.35
2.30
2.26
2.30
1.76
1.77
34.6
52.8
27.1
43.4
21.4
25.4
33.3
23.6
28.0
36.7
19.0
14.9
46.2
Standard
ID
TiGe29x
V45
V53
NiV21c
Cr30
Cr53
Cr85
Cr84
Cr75
Cr74
Crl22
CrCu32a
CrCu26g
MnZn24b
Mn57
Mn 183
MnZn27x
FePb37y
Fel07
Fe 127
Fe46
Fe88
FePb38y
Co45a
Co45b
RbCo29c
RbCo25b
Ni54
Ni88
NiV21c
Ni 101
Cu96
Cu 104
Cu 128
CrCu26g
CrCu32a
Cu38
Zn51
Zn 125
MnZn27x
MnZn24b
GaP 34
GaP 40
GaP 70
GaP 105
Element
Ti
V
V
V
Cr
Cr
Cr
Cr
Cr
Cr
Cr
Cr
Cr
Mn
Mn
Mn
Mn
Fe
Fe
Fe
Fe
Fe
Fe
Co
Co
Co
Co
Ni
Ni
Ni
Ni
Cu
Cu
Cu '
Cu
Cu
Cu
Zn
Zn
Zn
Zn
Ga
Ga
Ga
Ga
(ig/cm2
2.36
45.0
53.0
6.64
30.0
53.0
85.0
84.0
75.0
74.0
122.
9.19
8.14
8.57
57.0
183.
9.10
7.72
107.0
127.0
46.0
88.0
7.71
45.0
45.0
7.43
7.65
54.0
88.0
5.77
101.0
96.0
104.0
128.0
7.65
8.63
38.0
51.0
125.0
8.46
7.97
23.5
27.7
48.5
72.7
Standard
ID
SrF2 13
SrF2 92
SrF2103
YF346
ZrCd24c
ZrCd20w
MoO3145
MoO3106
MoO3110
MoO3 59
Mo03 54
Rhl6
Pd33
Pd 198
Ag35
Agl32
Cd83
ZrCd20w
ZrCd24c
Cd77
Sn40
Sn 185
Sn97a
Sn97b
Sn79
Sb 194
Sb47
Sb 147
Sb42
SbSr29z
SbSr31y
Te53
KI46
CsBr53
CsBr54
CsBr51
BaF2108
BaF248
BaF260
BaF257
BaF2143
BaF2114
BaAs23y
BaAs36w
LaF3157
Element
Sr
Sr
Sr
Y
Zr
Zr
Mo
Mo
Mo
Mo
Mo
Rh
Pd
Pd
Ag
Ag
Cd
Cd
Cd
Cd
Sn
Sn
Sn
Sn
Sn
Sb
Sb
Sb
Sb
Sb
Sb
Te
I
Cs
Cs
Cs
Ba
Ba
Ba
Ba
Ba
Ba
Ba
Ba
La
jug/cm2
12.8
64.2
71.8
28.0
9.85
10.77
96.7
70.7
73.3
39.3
36.0
16.0
33.0
198.0
35.0
132.0
83.0
9.15
8.38
77.0
40.0
185.0
97.0
97.0
79.0
194.0
47.0
147.0
42.0
5.01
5.18
53.0
35.2
33.1
33.7
31.9
84.6
37.6
47.0
44.7
112.0
89.36
4.98
4.911
111.3
3-72
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Standard
ID
CaF291
CaF2102
CaF266
CaF2 28
CaF233
CaF239
CaF254
CaF229
CaF230
CaF252
CaF248
CaF245
CaF236
CaF2134
CaF2110
ScF3 57
Ti39
Ti95
TiGe33d
Element
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Ca
Sc
Ti
Ti
Ti
|ug/cnr
46.7
52.4
33.9
1 4.4
16.9
20.0
27.2
1 4.9
15.4
26.7
24.6
23. 1
18.5
68.6
56.5
25. 1
39.0
95.0
2.46
Standard
ID
Ge37
TiGe29x
TiGe33d
Ge 140
BaAs23y
BaAs36w
CsBr53
CsBr 54
CsBrSl
RbNO346
RbCo25b
RbCo29c
RbNO311
RbNO313
SrF2 57
SbSr29z
SrF2 50
SbSr31y
SrF2137
Element
Ge
Ge
Ge
Ge
As
As
Br
Br
Br
Rb
Rb
Rb
Rb
Rb
Sr
Sr
Sr
Sr
Sr
ug/cnr
37.0
5.94
6.22
140.0
5.60
5.521
19.9
20.3
19.1
26.6
7.88
7.65
69.0
12.9
39.8
4.97 '
34.9
5.14
95.6
Standard
ID
LaF362
WO352
W0370
WO358
Au33
Au89
Au62
Au55
Au45
Pb67
Pb55
Pb 118
Pb 133
Pb 138
Pb64
FePb37y
FePb38y
Element
La
W
W
W
Au
Au
Au
Au
Au
Pb
Pb
Pb
Pb
Pb
Pb
Pb
Pb
ug/cm2
44.0
52.0
70.0
58.0
33.0
89.0
62.0
55.0
45.0
67.0
55.0
118.0
133.0
138.0
64.0
7.47
7.46
3-73
-------
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volume 3, Chapter 1
9.13 QC Chart for Pb in SRM 1833
XRF Analysis of PB in SRM1833
1.18
1.16
1.14
1.12
1.1
1.08
1.06
1.04
1.02
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86
0.84
_
-
-
-
-
-
_
"
-
--:-
n
j 5E~_ ,k ^ \5 uT ^^^'X^ -- -
/ X^f ~~"~^>i ' /"""^--^-J^- ^^"W^ -if^' '' "£ r ' \~ r~
''•' V] "^^ ~ .T^a\^ LJ^_ -*• '^ \ ^
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.14 QC Chart for Fe Peak
IRON PEAK
w
s
0
w
S
oi
0
1.08
1.07
1.06
1.05
1.04
1.03
1.02
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94
0.93
0.92
_
-
-
_
-
-
-
-
-
-
•
T T
t : <
\ -i - (
r ; V
, , i f ;';.i.
1 ' ' /' : T
-M | ' . ' - ; -
;•
-'•
1
i i : i I
20
40
RUN NUMBER
LOW LIM IRON
60
U?UM
80
3-75
-------
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volumes, ChapteM
9.15 QC Chart for S Background
SULFUR BACKGROUND
w
a
<
Q
w
s
ei
0
1.4
1.3
1.2
1.1
0.9
0.8
0.7
0.6
20
LOWLIM
40
RUN NUMBER
SULFUR
60
80
3-76
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.16 QC Chart for AL fwhm
ALUMINUM FWHM
u
5
Q
u
a
si
0
55
1.08
1.07
1.06
1.05
1.04
1.03
1.02
1.01
1
0.99
0.98
0.97
0.96
0.95
0.94
0.93
-
-
-
-
-
-
-
-
'I I '"""
.. , , . , ,. .1. .,._..,., „,.„ .; , — . i . , .. .- . *-. 1 ! „.__<....
.jwt i ! : i -1- 1 I i i :-;. i i :
1 I i 1 1
20
LOW LIM
40
RUN NUMBER
ALUMINUM
60
80
3-77
-------
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
Volume3t Chapter 1_
9.17 QC Chart for Cd Centroid
W
a
Q
W
J
s
*
0
is
1.0006
1.0005
1.0004
1.0003
1.0002
1.0001
0.9999 _
0.9998
0.9997
0.9996
0.9995 _
CADMIUM CENTROID
20
LOW LIM
40
RUN NUMBER
CADMIUM
60
UP
so
3-78
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.18 Exploded View of Filter and Sample Frame Assembly
Retaining Ring
Filter
Spacer Ring
Sample Fran*
3-79
-------
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry Volume 3, Chapter 1
9.19 Creation of Lotus Spreadsheet Data
All data generated after Feb 27, 1992 and reported under the name LSQnnnn.NGS can be put into
spreadsheet form and reorganized under a different format. Reported with the data are two
external files, NG3XTR8.EXE (a FORTRAN executable program requiring a math coprocesser)
and REORG1.WK1 (a Lotus file Containing a macro). The program asks the user for the
necessary information as needed. A hypothetical XRFID of 1111 will be used for illustration. To
create the spreadsheet follow the instructions below. (User responses are in single quotes).
1. Type NG3XTRS from any directory
2. Enter file name 'LSQ1111 .NG31
3. Enter output file 'EXAMPLE.DAT'
4. Extract samples or blanks ISO (Here one makes a choice because both cannot be in same
spreadsheet)
5. Enter output format 'S'
6. Uncertainty multiplier '3'
(Others may be chosen - see instructions in program. Remember the decimal. Program
now begins extracting data from the file and creating output file).
7. Run Lotus and retrieve REORG1 .WK1
8. Enter home. 'HOME'
9. Perform FILE/IMPORT/NUMBERS and import 'EXAMPLE.DAT1
10. Enter 'ALT S' and wait until execution finishes
11. Remember to save spreadsheet under a different name so as to not alter the original
spreadsheet containing the macro.
3-80
-------
Volume 3, Chapter 1
SOP for EPA's LBL Energy Dispersive
X-Ray Fluorescence Spectrometry
9.20 Superposition of Fitted and Measured Spectrum
Regression Output:
9.13 Superposition of Spectra
FitVB. Measured forTi Flnorescer
ray Intensity
*
240
230
220
210
200
A.
_ / \
/' 1
190 1_ r ',
180 |_ \
170 L . i i
160 L / \ !•
150 i_ r I r-/*
140 |_ / /
130 i / ' /
120 L /' ': /•
110
100
90
80
70
60
50
40
1 ', /
— 1 ' ,'
/
7 V- /-
/ y ^ ^^^
/
/
Vv/V"X'""^"— N y~A_y
II' ' 1 1 ! 1 1
10
30
50
70
90
no
Channel Number
LSQ Fit Measured
Constant
Std Err of Y Est
R Squared
No. Of Observations
Degrees of Freedom
X Coefficients(s)
Std Err of Coeff.
4.50
9.34
.956
85
83
.960
.022
3-81
-------
Analysis of Surface Waters for Trace
Elements by Inductively-Coupled
Plasma Mass Spectrometry
Martin Shafer and Joel Overdier
Water Chemistry Program
University of Wisconsin-Madison
Madison, Wl 53706
November 1995
Revision 4
-------
Analysis of Surface Waters for Trace Elements by Inductively-Coupled
Plasma Mass Spectrometry
1.0 Introduction
This document outlines a complete method for the determination of a suite of trace metals in
surface waters. All phases of the process are discussed, from equipment preparation and sampling
techniques, to instrumental analysis, and field and laboratory QA/QC. Each phase is of equal
importance in producing quality data, and failure to strictly adhere to the protocols at each step of
the process can severely compromise data integrity. The method describes the use of inductively-
coupled plasma mass spectrometry (ICP-MS) for the determination of Aluminum (AI), Arsenic
(As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Lead (Pb), Silver (Ag), and Zinc (Zn).
ICP-MS protocols generally follow methods 200.8 and 6020 CLP-M, with modifications to allow
quantification at low ng L"1 levels and to prevent contamination of the instrument. Detailed
descriptions of field methods are not presented in this manuscript. The primary focus of this
document is on analytical methods and quality assurance. Separate documents are available which
describe in great detail field methods employed in studies of a variety of surface water systems.
2.0 Equipment Preparation
2.1 General
Most stages of field apparatus cleaning and preparation are performed within a clean lab. The
most critical steps of preparation are carried out within laminar flow benches, located in the clean
lab. The sampling equipment (sampler, sample bottles, filtration system, acidification vials,
tubing) are fabricated from Teflon PFA or Teflon FEP. These materials were selected as most
suitable for sample contact because of their generally low trace metal content, resistance to
degradation during rigorous cleaning procedures, and hydrophobic character. Trace metal grade
acids are used in all final cleaning and storage stages. Field manipulations and potential for
contamination are minimized by prepackaging apparatus in the clean lab. Polyethylene gloves are
used at all times when handling sample bottles and filtration apparatus in the lab and field.
Samplers, bottles, and acidification acid are "blanked" before use in the field. Dedicated blank
studies have demonstrated that contamination from the samplers and filtration apparatus are minor
components of the method blank. For a detailed description of field methods refer to the field
methods document specific to a given study.
2.2 Cleaning and Packaging
2.2.1 Teflon Bottle Preparation Procedure
Revisions. November 1994
Never place a Teflon bottle directly onto a counter top. Always place a new piece of
plastic down on counter before placing bottles on counter.
3-85
-------
Analysis of Surface Waters for Trace
Elements By ICP-MS Volume 3, Chapter 1
(a) Bottle ID
Verify that the Teflon bottle has an ID number etched into its side. If the bottle
has not been etched, set aside and verify with a supervisor the appropriate number
to etch at a later date.
(b) Acetone Wash
Fill bottles with acetone (ACS Reagent), leach for two hours, remove acetone,
rinse twice with milli-Q water. Record date on cleaning log. The used acetone is
placed into glass containers labeled Use Acetone For Bottle Cleaning, and may be
reused five times before disposing. Note on acetone bottle number of uses.
(c) 50% HC1 Leach
Fill bottle to top of neck with 50% HC1 (ACS Reagent), leach for three to four
days at room temperature, remove HC1, rinse three times with milli-Q water. The
50% acid is prepared and stored in 2.5 L acid bottles - Clearly Labeled 50% HCl
For Teflon Bottle Cleaning. The acid may be reused five times before disposing.
Note on acid bottle number of uses. The acid may be added to the Teflon bottle
directly from the acid bottle, with or without the aid of a funnel; or may first be
placed into a plastic beaker. Do not touch lip of acid bottle to lip of Teflon bottle.
After filling a batch of bottles, place into a large plastic bag. Seal bag, label bag
with date, type of acid, and your name.
(d) 50% HNO3 Leach
Fill bottle to top of neck with 50% HNO3 (ACS Reagent), place in 20% HNO3
acid bath, leach inside and out for three to four days at room temperature.
Remove bottle from bath and thoroughly rinse outside of bottle with milli-Q
water. Remove 50% HNO3 acid, and rinse three times with milli-Q water. The
50% acid is prepared and stored in 2.5 L acid bottles - Clearly Labeled 50%
HNO3 For Teflon Bottle Cleaning. The acid may be reused five times before
disposing. Note on acid bottle number of uses. The acid may be added to the
Teflon bottle directly from the acid bottle, with or without the aid of a funnel; or
may first be placed into a plastic beaker. Do not touch lip of acid bottle to lip of
Teflon bottle. Record on the 20% acid bath the date the bottles went in.
(e) I % High Punty HNO3 Leach
Fill bottle with 1.0% HNO3 (Baker Trace Metal Grade; TMA), and store in this
manner until bottle is required, but at least three days. The diluted acid is best
prepared in original, clean 2.5 L acid bottles. Fill to just below neck with milli-Q
water, add 25 mL concentrated TMA (use Teflon Beaker), cap, mix, and dispense.
A 2.5 L bottle should be Clearly Labeled 1% TMA For Teflon Bottle Cleaning
and used exclusively for preparation of dilute acid. Discard any unused dilute
acid. An alternate filling method is to fill bottles '/z full with MQ from 20 L
carboy; dispense concentrated acid via a Teflon beaker and a Teflon measuring
vial; top off bottle with MQ.
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(f) Drying
Remove and discard dilute acid, rinse four times with milli-Q water, dry bottle
and cap under laminar flow hood. Sign out hood for this step, and make sure it is
relatively free of other apparatus. Be extremely careful not to Contaminate the
Cap and Bottle. Do not leave bottle/cap in hood for longer than it takes to dry
them and never longer than six hours.
(g) Taring
Assemble bottle and cap under hood and obtain bottle tare weight (±0.02 g) using
top-loading balance next to clean bench. Bottle weights are recorded in the
Teflon Bottle Weigh Log. Cap all bottles first; use your clean gloved hand to
handle bottles; use dirty gloved hand to record data on cleaning log sheet. Make
sure a clean piece of plastic is covering balance tray. After weighing, return
bottles to laminar flow hood for bagging.
(h) Double Bagging
(i) Notes
Under laminar flow hood, place bottle in appropriately sized polyethylene (PE)
zip-lock bag. Double bag with another PE zip-lock bag labeled with sample
bottle ID. Use a new clean pair of PE gloves for these steps. A black Sharpie is
used to label outer bag with bottle ID. Double check that bottle ID corresponds to
bag ED.
1. All preparation steps must be performed in the clean room.
2. Acetone and 50% acid use must be under a fume hood.
3. Do not place Teflon bottles onto an uncoated lab bench. Make sure the
bench has a clean plastic surface (place new plastic even over Teflon
overlay).
4. You must wear clean polyethylene gloves when handling Teflon ware.
5. Follow all clean room protocols when cleaning Teflon ware (clean lab
coat, shoe covers, etc.).
6. All acid dilutions are performed with milli-Q water.
7. Tighten bottle caps thoroughly to prevent acid leakage.
8. Use only designated Teflon beaker for TMA.
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Experiments have shown that Teflon bottles prepared in this manner introduce
undetectable levels of Al, Cd, Cr, Cu, Fe, Pb, and Zn to samples stored at 4°C for
periods of at least nine months. In parallel experiments, no loss of trace metal was
observed in spiked sample pairs.
2.2.2 Sampler, Filtration Apparatus, Acid Vial, Preparation
Revisions. November 1994
All components of samplers (except polyethylene extension pole), filtration apparatus, and
acid vials are cleaned as follows:
(a) Leach, in acid bath, three to four days in 50% HC1 (ACS Reagent) at room
temperature, rinse three times with milli-Q.
(b) Leach, in acid bath, three to four days in 50% HNO3 (ACS Reagent) at room
temperature, rinse three times with milli-Q.
(c) Leach, in acid bath, four to five days in 1 % HNO3 (Baker Trace Metal Grade),
rinse four times with milli-Q water.
(d) Dry under laminar-flow hood, assemble (if required) components under clean
bench, double bag in clean polyethylene bags. Do not leave vials under clean
bench for longer than two hours.
Acidification vials are cleaned in 1 L wide-mouth Teflon bottles.
Polyethylene extension poles are cleaned by scrubbing pole with clean room
wipers soaked in 10% HNO3, and thoroughly rinsing with milli-Q water. After
cleaning the poles are sealed in ultra high molecular weight polyethylene
(UHMWPE) bags.
2.2.3 ICP-MS Sub-Sample Tube Preparation
Polypropylene tubes with polypropylene snap caps (17 x 100 mm) are used 'o contain
sample during ICP-MS analysis.
(a) Leach, cap and body, two to three days in 10% HNO3 (ACS Reagent) at room
temperature, rinse three times with milli-Q.
(b) Leach, cap and body, two to three days in 2% HNO3 (Baker Trace Metal Grade),
rinse four times with milli-Q water.
(c) Dry under laminar-flow hood, assemble, and store in clean polyethylene bag.
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2.2.4 Revision 3. November 1994
Procedures For Recycling Field Supplies
(a) Acidification Vials
1. Rinse vials/caps inside and outside with MQ three to four times.
2. Place in 50% reagent nitric acid bath in Teflon bottle for two days.
3. Rinse vials/caps with MQ three to four times.
4. Place vials/caps in 1-2% Trace Metal Nitric Acid (TMA) in Teflon bottle
for three days.
5. Rinse vials/caps four times with MQ, shake off excess water.
6. Dry under laminar flow hood for no longer than two hours.
7. Fill with acidification acid, or cap vials and place in labeled zip-lock to be
filled later.
Note: All steps to be performed in clean lab, with gloved hands, and
clean lab coat. If vials are placed onto a lab bench, it must be covered
with new plastic.
(b) SPM/DOC Bottles
1. Rinse 1 L bottle/cap inside and outside with MQ three to four times.
2. Fill bottle with new 10% reagent nitric acid.
3. Leach for a minimum of two days.
4. Dump acid and rinse bottle/cap four times with MQ.
5. Shake out excess water and dry under laminar flow hood for no longer
than four to five hours.
6. Cap bottles and place in labeled bag.
Note: All steps to be performed in clean lab, with gloved hands, and
clean lab coat. If bottles are placed onto a lab bench, it must be covered
with new plastic.
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(c) Teflon Sample Bottles
I. Refer to complete cleaning protocol.
2. Start procedure from 50% ACS reagent nitric step.
3. Follow protocol to end.
Note: All steps to be performed in clean lab, with gloved hands, and
clean lab coat. If bottles are placed onto a lab bench, it must be covered
with new plastic.
(d) TTAF and PCR (TCR)
I. Rinse with MQ.
2. Place in 50% reagent nitric acid bath for two days.
3. Rinse with MQ.
4. Place in 1 -2% Trace Metal Acid bath for three days.
5. Rinse with MQ four times.
6. Dry under Laminar Flow Hood for no more than two hours.
7. Double-bag in labeled zip-lock bags
8. Record cleaning date on outer zip-lock.
9. Steps 2 through 4 may be replaced with a 24-hour near-boiling
concentrated nitric acid bath leach following Hg bottle cleaning protocols.
Note: All steps to be performed in clean lab, with gloved hands, and
clean lab coat. If adapters are placed onto a lab bench, it must be covered
with new plastic.
(e) Tubing Rinse Acid Carboy
1. Dump out any remaining acid.
2. Rinse carboy with MQ three times.
3. Fill carboy to 2" below neck with MQ.
4 Add 400 mL concentrated Trace Metal Nitric Acid (TMA). Use a Teflon
beaker dedicated for TM A to deliver acid.
5. Cap carboy and bag with large poly bag.
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Note: All steps to be performed in clean lab, with gloved hands, and
clean lab coat.
(0 ICP-MS Sub-Sample Tubes
Tubes are not reused.
2.2.5 Filling of Field Acidification Vials
(a) Field Acidification Solution Preparation
I. Prepare a 25 mg L'1 solution of Rare Earth Elements (Y, Yb, Ho, Th)
REE (1 mL each/40 mL (final volume) of 2% Ultrex HNO,) using the
1000 ppm stock solutions from High Purity Standards.
2. Prepare a 50% Ultrex HNO, Acid solution (v/v). Allow the solution to
cool to room temperature. Calculate the density (g/mL) of the 50% acid
by weighing three replicates of 1.000 mL. Use the average of the three
values. Record the values on the Field Acidification Preparation data
sheet.
3. To make the final solution, add 2.000 mL of the 25 mg L ' REE stock to
the mass equivalent of 298.00 mL of the 50% Ultrex Acid Solution. This
yields a solution containing Y, Ho, Yb, Th = 166.7 ug L-l (scale the
recipe as necessary).
4. Label the new acidification solution with the date and batch ID. The
batch ID is assigned sequentially beginning with FS9501 for the 1995
sampling year. Check the Trace Metal Preservative Log Book for the
most recent batch. Place the data sheet into the QC log.
5. A dilution of the acidification solution must be analyzed by ICP-MS to
verify the REE concentrations and the absence of elements of interest. A
5.0 mL (approx.) volume of the solution also needs to be archived.
6. Use 3.000 mL of acidification solution for preserving both 250 mL Total
and 250 mL Filtrable metal samples (Final concentration of 0.6%).
(b) Dispensing Acidification Solution
Wear poly gloves and clean-room suit at all steps.
1. Arrange Teflon vials under the laminar flow bench on a new plastic sheet.
2. Set up digital pipet with a leached tip and set to deliver 1.500 mL. Do not
let clean tip touch any surface during filling procedure.
3. Pour an aliquot of acid solution into the designated clean Teflon beaker.
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4. Rinse tip twice by dispensing acid into a waste container, and then begin
filling Teflon vials with 3.000 mL of acid solution (2 x 1.500).
5. Replace vial caps and wrench tight with green plastic wrenches.
6. Place vials into zip lock bag and then insert bag into a labeled zip-lock
bag.
2.2.6 Preparation of Polycarbonate Filters for Trace Metal Filtration Revision 3
November 1994.
Note: This procedure was not used for the LMMB Tributary Study.
Polycarbonate track-etched filters (47mm or 90mm diameter, 0.4 jam pore size) are used to
obtain paniculate samples and suspended mass levels. They are prepared as follows:
(a) Petri-Dish Preparation
Filters are placed individually into polystyrene petri dishes. The dishes are pre-
cleaned by soaking tops and bottoms separately in 10% HNO3 (ACS Reagent) for
24 hours, followed by rinsing with milli-Q water. Dishes are then dried under a
laminar flow clean bench. Dishes are then assembled under the clean bench and
bagged for later use.
(b) Filter Weighing
Clean petri dishes are labeled top and bottom with a sequence number and pore
size using a black Sharpie. Filters are placed into the dishes and allowed to
equilibrate in the balance room for at least two hours before taring. Filters are
handled only with all plastic forceps. While equilibrating the partially opened
dishes are loosely covered with a clean plastic bag. Every seventh filter is
designated as a temperature and humidity control, and is so designated in the filter
log book and on the petri dish. Filters are tared on a Perkin Elmer microbalance
(AD-4) to a significance level of 1 jjg after equilibration for 60 seconds. Balance
calibration is recorded in the filter log book, and is performed with a CLASS M
weight before weighing each batch of filters, and is cherked after ever tenth filter.
10% of the filters are re-weighed. Polonium ionization sources are used to
eliminate static charges. Refer to Filter Weighing SOP for details.
(c) Filter Leaching
Tared filters are leached individually in their previously cleaned polystyrene petri
dishes. A 1 molar (63 mL or 89.5 g concentrated reagent per liter of milli-Q
water) solution of HNO3 (Baker Ultrex II Grade) is prepared in a dedicated
polyethylene bottle. The 1M solution is transferred to a clean polyethylene wash
bottle and then added to the bottom portion of the petri dish to nearly fill the
contained volume (approx. 10 mL per dish). The top half of the petri dish is then
replaced and the filter leached for 48 hours at room temperature. All filter
leaching and rinsing is performed in the clean lab.
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(d) Filter Rinsing
After leaching for the required time the acid solution is poured off into a plastic
waste beaker. The filters are then rinsed 10 times with milli-Q water by squirting
in approx. Ten mL with a polyethylene wash bottle, discarding rinse, and
repeating this process 10 times. The rinsing process is performed under the
laminar flow clean bench. The leached and rinsed filters are stored in a few mL's
of milli-Q water until used.
2.2.7 Loading of Teflon Filter Columns
Note: This procedure was not used for the LMMB Tributary Study.
Pre-cleaned and rinsed filters (see [4]) are loaded into all-Teflon filter columns for use in
the field. The columns consist of a Teflon filter support base and a threaded Teflon
column segment with Teflon cap. The segment will contain approx. 150 mL of sample.
Filters are loaded into the Teflon columns under the laminar clean bench. After loading,
the filter columns are individually doubled bagged with polyethylene zip-lock bags.
3.0 Clean Room Protocols
Note: Refer to the Clean Room Operations SOP for more detailed descriptions of procedures. A
few specific points are outlined below.
3.1 All personnel entering clean rooms must wear full clean-room garb (coveralls, eyes-only hoods,
and foot covers.
3.2 Particle counts (>0.3 urn) in the room are to be monitored with a portable laser-based counter and
logged on a daily basis (ICP-MS clean-room only) [Two locations, 10 min integrations, sampled
every hour for four hours].
3.3 Floors are to be wiped with a clean room tacky mat mop on a weekly basis; more often if judged
necessary.
3 4 Paper towels and wipers must not be used in clean room; only clean room wipers are allowed.
3.5 Shipping containers (cardboard, paper, etc.) are not allowed in the room.
3.6 Tacky mats are to be positioned at entrance to clean room.
3.7 Food and drinks are not permitted in the clean room.
3.8 Positive pressure is to be maintained in the clean room at all times. Daily readings of the
magnehellic gauge are to be taken and recorded in the particle count log book.
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4.0 Trace Metal Sampling
Note: The procedures outlined in Sections 4.1 through 4.3 are not those used in the LMMB
Tributary loading study. Refer to the separate document outlining Field Trace Metal Protocols for
LMMB Tributary Monitoring.
4.1 General
Trace metal samples will be obtained by personnel trained in trace level sample collection. The
procedures that they will be following will be fully documented, and available for review in the
field. The two-person trace metal team must wear trace metal - compatible garments and clean
polyethylene gloves during the sample collection period or whenever handling trace metal
samples.
Two "blanked" trace metal clean samplers will be used. Our grab sampler will be used in flowing
river systems where depth profiling is not practical or necessary. Where depth and longitudinal
integration is required, a modified, all Teflon USGS DH-81 sampler is applied. Ease of use under
less than optimum field conditions was an important design criterium. The grab sampler consists
of a heavy Teflon collar affixed to the end of a 2 meter long polyethylene pole, which serves to
remove operator from immediate vicinity of sampling point. The collar was designed to securely
hold a 500 mL FEP Teflon bottle.
A Teflon closing mechanism, threaded onto the bottle, enables the operator to open and close the
bottle under water, thereby avoiding surface microlayer contamination. A Teflon pull cord,
attached to the seal device, allows operator to remotely select to bring seal plate position.
Rigorous "clean-hands" "dirty-hands" procedures are an integral part of sampling protocol. The
bottle and clean end of sampler is handled only by personal with clean shoulder-length PE gloves
and full clean suit. The sample bottle, immediately upon recovery, is placed in the inner trace
metal clean zip-lock bag by the "clean-hands" person, and then double bagged. Loading and un-
loading of samplers with bottles is performed within large polyethylene bags.
4.2 Filtration
The field filtration apparatus consists of a 1 L Teflon PFA vacuum jar, a Teflon lid with three 1A"
fittings, and pre-loaded 47 mm Teflon PFA filter holders mated with 150 mL Teflon PFA
columns. To use, a filter unit/column is attached to vacuum jar and a representative aliquot of
total metal sample is added directly to the column. Suction from a peristaltic pump is applied to
the jar, and filtrate collected directly in 125 mL Teflon bottle after a rin^e step. The complete
filtration procedure is performed within an argon-flushed plexiglass glove box. This type of
filtration system has significant advantages over other designs in that contact surfaces are
minimized, all critical components can be pre-packaged in a clean lab, and exposure to ambient air
is eliminated by use of sealed columns and glove box.
The apparatus can be operated in a pressure mode if required. Filtration is initiated as soon as
possible after sample collection, typically within 15 minutes. After filtration is complete the
125 mL bottle is double bagged.
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4.3 Acidification
Total (500 mL bottle) and filtrate (125 mL bottle) samples are acidified within the glove box using
50% Ultrex nitric acid contained in pre-packaged, individually dosed, Teflon PFA vials. Vials are
opened with plastic hex wrenches. Acidification is at a rate of 5 mL concentrated nitric acid per
liter. Alternatively, if filtration is carried out on a boat, acidification can be performed in the open
air immediately after sample collection using strict clean-hands-dirty hands protocols. In this case,
both field personnel must be garmented in clean suit coveralls. Sub-samples (20 mL) of
acidification acid are saved at the time of preparation in Teflon vials, and trace element levels
determined by ICP-MS. If the level of any element of concern in a 2% MQ solution of this acid is
greater than five times the average acid MQ calibration blank metal concentration, then that acid
batch is rejected.
5.0 Field Quality Assurance Protocols
5.1 Recovery Spikes
5.1.1 Rare Element Spike
All samples are acidified with 50% Ultrex HNO, containing four rare elements, Yttrium
(Y), Holmium (Ho), Ytterbium (Yb), and Thorium (Th). Teflon PFA vials containing pre-
measured quantities of acid-spike solution is used to deliver the spike with acidification
acid. Indigenous levels of these elements in each river system are quantified before
monitoring begins. Spiking levels of approx. 3 |jg L ' are employed, a level easily
measured and of similar concentration to many of the trace elements of concern, yet well
above extremely low indigenous levels. The recovery control range for all rare elements
(except Thorium) is 70-125%. If all three elements (Y, Ho, Yb) fail, the sample results
are flagged as estimated, and the sample should be diluted and reanalyzed. If any two
elements fail, the sample results are also flagged as estimated and dependent upon the
degree of failure, these samples should also be reanalyzed. Sample data will not be
flagged if just one surrogate element falls outside the control range.
5.1.2 Trace Metal Spike
Replicate Total and Filtrate samples both taken at a frequency of 10%, will be spiked with
an acid-mix of the metals of concern. The spike will be delivered as described in [ 1 ]
above. The spike is designed to approx. double ambient concentrations of each metal of
concern, or elevate ambient levels by 15 ng L ', whichever is larger. All trace element
spikes must recover in the range of 70-125 %. If any element fails, the sample will be
reanalyzed after dilution or other matrix modification technique, only after ruling out field
conditions (field technician error, high ambient metals levels, etc.) as a reason for potential
bias.
5.1.3 Blank Spike
A field MQ-blank will be spiked with trace metals whenever a sample spike is performed.
Frequency of 5r/r. Procedure is identical to [2] above.
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5.2
Blanks
5.2.1 Bottle Blanks
Every batch of 20 field bottles will include one field bottle blank (5% Frequency). These
bottles are prepared in an identical manner to sample bottles except that prior to final
packaging, they are filled with MQ-blank water. An additional lab bottle blank
(5% Frequency) is prepared at the same time. Field blanks are brought to sampling sites
and acidified in the field using trace metal clean protocols as described in Section 4.3.
5.2.2 Filtration Blanks
Filter and tubing blanks will be performed at a frequency of about one in every 40 samples
(2.5%). Eight liters of MQ water are sent to the field in two large Teflon Bottles as part of
a dedicated Field Blank Kit. This water is used to blank, separately, Calyx filters, and the
Teflon Tubing Sampling Line. A sub-sample of the feed MQ is also obtained to serve as a
reference level.
5.2.3 Spike Blanks
Every batch of 20 field bottles will include one blank for field spiking (5% Frequency).
See Section 5.1.3.
5.3 Replication
Our goal for the Lake Michigan Tributary Monitoring Project / Lake Michigan Mass Balance
Project is 15% to 20% replication of field samples, excluding QC samples. This will be
accomplished by completely duplicating individual sites on a frequency of one in five
revisitations. Our goal should be to limit field "replicate" error to ±15% for analytes greater than
five times the analytical reporting limit.
5.4 Interlaboratory Comparison
At least once during the duration of the study, field samples will be collected and split for
shipment to a cooperating laboratory.
5.5 Field Quality Assurance Summary
Type
Recovery Spikes
-— rare element
— analyte in sample
—- analyte in blank
Frequency
all samples
10%
5%
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Type
Blanks
— bottle (field)
— bottle (lab)
-— filter
— tubing
Replication
— samples (ex. QC)
Interlab Comparison
— split samples/method comparison
Audit
- — internal field
— external field
Frequency
5%
5%
2.5%
2.5%
15-20%
once
twice
once
6.0 Laboratory Sample Handling
6.1 Sample Logging
Upon arrival at laboratory, the field data sheet will be recovered, copied and filed, and samples
logged into the appropriate databases. The following data are logged into the Trace Metal sample
database upon arrival of samples at laboratory:
1. Site coding
2. Sample bottle coding
3. Sample type
(a) Blank
i. Bottle
ii. Filter
iii. Tubing
iv. Feed
(b) Replicate
(c) Spike
i. Sample
ii. Blank
(d) Sample
i. Unfiltered
ii. Filtered
4. Sampling date and time
5. Shipping date
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6. Arrival date
7. Acidification acid batch
8. Spike acid batch
9. Calyx filter batch
10. Pump-head tubing batch
After log-in the double-bagged Trace Metal samples are placed in plastic egg-crates in a cold room
(2-6°C).
6.2 Sample Taring
The double bagged bottles are taken into the clean lab after placing them into large, clean HOPE
bags. In the clean lab, garmented (gloves, frock, booties) personnel will remove and discard the
outer sample bags after verifying that the etched bottle ID corresponds with bag designation. After
re-gloving the sample handler weighs the bottles after temporarily removing them from the inner
bag. Bottle weights are recorded in the sample bottle database, and sample volume calculated.
Batches of sample bottles (in inner zip-lock bags) are then doubled bagged into two new large
UHMWPE bags and refrigerated (2-6°C).
6.3 Spiking
Batch runs of approximately 20 samples are developed, and on the day prior to ICP-MS analysis,
the designated samples are retrieved from storage. Clean-room personnel working under a laminar
flow clean bench remove sample bottles from inner bag, and spike a previously calculated volume
of mixed internal standard solution into the sample bottle. Sample volumes are determined after
bottle and sample weights have been recorded. The sample is mixed and allowed to equilibrate for
at least one hour. An approx. 12 mL subsample for ICP-MS analysis is then removed by pouring
into a trace metal clean polypropylene capable tube (17 x 100 mm). Tubes are capped, mixed by
gently shaking, and placed into polypropylene racks. Racks of tubes are sealed in UHMWPE bags
and kept refrigerated in the clean lab until actual analysis begins.
6.4 Holding Times
ICP-MS analysis of any given sample will be completed within a maximum holding period of
six months. If holding times are exceeded, the sample(s) in question will be flagged as estimated.
6.5 Pre-treatment
6.5.1 Filtrates
Field filtered samples acidified to a level of 0.6% with Ultrex HNO3 are analyzed without
further pre-treatment.
6.5.2 Totals
Acid-recoverable metals are determined on "total" samples as follows. Un-filtered
samples are acidified in the field to a level of 0.6% Ultrex HNO3. In the clean-laboratory,
after taring, the samples are spiked with an additional 2.5 mL of concentrated Ultrex
HNO-,, bringing the acid concentration to 1.6%. Sample bottles are placed in a laboratory
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oven maintained at 60CC, after replacement of field outer bags with designated oven outer
bags. Samples are heated for a period of 12 hours, after which they are allowed to cool at
room temperature. Bottle seal integrity is checked before placing samples in temporary
refrigerated storage. The samples are not filtered after heating.
7.0 Instrumental Analysis
7.1 Mass Selection
7.1.1 Analyses
Aluminum (Al): monoisotopic, mass 27
Chromium (Cr): masses 50, 52 and 53 monitored.
Copper (Cu): masses 63 and 65 monitored.
Zinc (Zn): masses 64, 66, and 68 monitored.
Arsenic (As): monoisotopic, mass 75
Silver (Ag): masses 107 and 109 monitored.
Cadmium (Cd): masses 114, 111, and 110 monitored.
Lead (Pb): masses 206, 207, and 208 monitoied.
Note: Elements Aluminum and Silver are not included in the Lake Michigan Tributary Loading
Study, but are monitored for research studies.
7.1.2 Internal Standards
(a) Field Spiked (b) Lab Spiked
Yttrium (Y): mass 89 Gallium (Ga): .mass 71
Holmium (Ho): mass 165 Indium (In): mass 115
Ytterbium (Yb): mass 174 Bismuth (Bi): mass 209
Thorium (Th): mass 232
7.1.3 Isobaric and Spectral Interference
For those analyses with multiple isotopes, several isotopes are monitored to facilitate
spectral corrections. In addition, a suite of masses of potential elemental and molecular
interferences are monitored to allow for spectral corrections at the extremely low ambient
analyte levels. The spectral correction equations used will be reported with each run
A summary of molecular and elemental interferant masses to be evaluated for each analyte
element are as follows:
Aluminum: none
Arsenic: masses 77, 82
Cadmium: masses 98, 106, 108, 118, 120
Chromium: masses 13
Copper: 60,61,62
Indium: mass 118
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Lead: none
Silver: 90,98
Zinc: none
Interference equations for the analyses of interest are summarized below:
?%BKGD = (I101 + 1125 )/2
?AL27 = I27 ?%BKGD
7CR52 = 152 - ?%BKGD
?%CLO51 =151 -(415*150)
?%CLO53 = 153 - (0.1133652 * 7CR52)
?%ARCL77 = 177 - (0.84842 * 182) - ?%BKGD
?%ARCL75 = ?%ARCL77 * 3.06
?%CAOO76 = 176 - 5.368 * (I78-2.669*(I82-I83))
?%CAOO75 = 0.0671 * (?%CAOO76 - ?%BKGD)
7CR53 = 153 - (0.328 * ?%CLO51) - ?%BKGD - (0.0031 * 113)
7CU63 = 163 - ?%BKGD
7CU65 = 165 - ?%BKGD
7ZN66 = 166 - ?%BKGD
?AS75 = I75 ?%BKGD
?CD110 = II10 - (0.00079 * 198) - ?%BKGD
?CD111 = II11 - (0.0014 * 198) - ?%BKGD
?CD114 = 1114- (0.0268 * (II18 - ?%BKGD)) - ?%BKGD
7AG107 = 1107 - (0.0006 * 198) - (0.001 * 190) - ?%BKGD
7AG109 = 1109 - (0.0004 * 198) - ?%BKGD
?IN115 = 1115-(0.0149* II18)
?PB206 = I206 1211
7PB 207 = 1207 1211
7PB208 = (1208 -1211) + 7PB206 + 7PB207
?%CAOH59 = 0.312 * (161 - (0.0478 * 160)) ?%BKGD
?%AROH59 = 0.604 * (153 - 0.113 * (152 - ?%BKGD))
7CO59 = 159 - ?%CAOH59 - ?%BKGD
I = Intensity at a given mass.
BKGD = Background intensity.
7.1.4 Oxide Formation
At least one of the following three sets of parent/oxide/hydroxide masses will be
monitored to assess the extent of oxide formation:
Yttrium (Y,89)
Yttrium Oxide (YO, 105)
Yttrium Hydroxide (YOH, 106)
Cerium (Ce, 140)
Cerium Oxide (CeO, 156)
Cerium Hydroxide (CeOH, 157)
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Analysis of Surface Waters for Trace
Volume 3, Chapter 1 Elements by ICP-MS
Thorium (Th, 232)
Thorium Oxide (Th, 248)
Thorium Hydroxide (Th, 249)
7.1.5 Instrument Background
The following masses will be monitored to assess background instrumental noise:
99, 101, 125, and 211.
7.1.6 Element Menu
An example element menu is outlined in Appendix 5.
7.2 Contamination Reduction
7.2.1 Glassware
Sample introduction components (front-end) of the ICP-MS are regularly decontaminated
to ensure optimum performance at ultra-trace levels. Ail glassware (torch, spray chamber,
elbow, nebulizer, tube adaptor) is soaked in 25% reagent grade nitric acid overnight,
rinsed with MQ water, installed, and flushed (by nebulization) with 3% trace metal grade
nitric acid before use. The glassware is re-cleaned after every run sequence/batch.
Change of glassware is noted on the ICP-MS operational log.
7.2.2 Tubing
All tubing from peri-pump to pneumatic nebulizer, from peri-pump to ultrasonic
nebulizer, and from ultrasonic nebulizer to torch is Teflon. The Teflon tubing is initially
cleaned in the same manner as all other Teflon ware [acetone, 50% HC1, 50% HNO3, 1 %
HNO3 (see Equipment Preparation)]. Flexible peri-pump tubing is changed at least once
per week and is initially prepared by pumping 1 L of 3% trace metal grade HNO3 through
tubing. Tubing changes are noted on the ICP-MS operational log.
7.2.3 Cones
Instrument cones (sampler and skimmer) are changed after every run sequence. They are
cleaned by polishing with POLARIS powder, followed by sonication for two 15 minute
periods in MQ water (water is changed between periods) and a final 15 minute sonication
in acetone. Clean cones are placed in polyethylene gloves and stored in small plastic jars.
7.2.4 Handling
Cleaning of components is performed in the clean room, and clean cones, glassware, and
tubing is handled only with PE gloved hands.
Extreme care must be taken to minimize the possibility that the ICP-MS could become
contaminated by samples with high metal concentrations. Samples with transition metal
levels greater than 25 ug L ' should either be diluted or run on a different instrument. To
achieve optimum performance it may be required to group samples of similar matrix.
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Analysis of Surface Waters for Trace
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7.3 ICP-MS Setup and Pre-qualification
A QA notebook containing dedicated forms for each of the control procedures is maintained, and
where practical a parallel electronic database is also kept (See Summary Table I).
7.3.1 Warm up
Instrument shall warm up in analyze, mode (multiplier on-line) while nebulizing an
acidified (2-3% HNO3 Trace Metal Grade ) MQ rinse solution for an minimum of
30 minutes before calibration and blank checks are performed.
7.3.2 Mass Calibration
A 5 ug L"' (pneumatic) or 1 ug L ' (ultrasonic) tuning solution [Be(9); Co(59); In(l 15);
La(139) or Ce(140); Bi(209); U(238)] is used for instrument mass calibration. The tuning
solution is run prior to analyte calibration, and whenever response/resolution is altered.
Masses must agree within 0.1 amu of actual before proceeding with analyses. A record of
tuning solution preparation is kept in the standard solution log. Tuning data will be
logged on a dedicated form in the QA document.
7.3.3 Response Calibration-Mass Linearity
Linearity of response as a function of mass is examined by performing a response
calibration with a tuning solution. The guidance criteria for response of Be, Co, La, and
Bi with respect to In are: 9Be/U5In = 0.25-1.0; 59Co/ll5In = 0.75-1.50; 139La/u5In =
0.75-1.25; 209Bi/"5In = 0.5-1.1. These criteria are used as predictors of possible sensitivity
problems with analyte elements. As long as analyte sensitivity criteria are met, failure of
response calibration criteria is considered as a warning to check instrument, but does not
invalidate run sequence. Response calibration data are logged in QA document.
7.3.4 Detector Cross-Calibration
Pulse-counting and analog modes of detector are cross-calibrated by running a higher level
tuning solution. The procedure is performed at least twice a week and when ever
multiplier settings are altered.
7.3.5 Resolution Check
Appropriate resolution is confirmed from a scan of a 5 ug L ' multi element tuning
solution. The baseline between isotope peaks of a high mass element (Pb: 206, 207, 208)
and low mass element (Mg: 25, 26) are examined. Baseline must be resolved within at
least 10 raw counts. Resolution data, and confirmation (hard copy of scan) is logged in
QA document. If mass calibration or resolution are out of control then the instrument
must be tuned or otherwise adjusted to meet criteria before analyses can proceed.
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7.3.6 Memory Check
A memory check solution containing all the analyte elements at 25 ug L"1, followed by
two MQ blanks, is run to check rinse out performance. Currently our protocols call for an
eight minute rinse between samples. Rinse out performance will be checked once a day
on a tuned and calibrated instrument before actual samples are run. Rinse levels in second
MQ blank (mean of seven replicate acquisitions) may not exceed reporting limits. If rinse
levels are out of control, then the Memory Check test is repeated. If second test fails then
the source of contamination must be isolated and corrected before samples are run.
7.3.7 B lank Acceptance
After mass, resolution, and response calibrations, and memory check have been executed,
a calibration blank is run for 10 replicate acquisitions. Interference corrected analyte blank
levels in either (a) calibration blank, or (b) second MQ rinse of Memory Check must be
below previously established limits. If blank criteria are not met, the source of
contamination must be isolated before proceeding with analyses. Blank criteria are shown
in the table below:
Blank Criteria (ng L ')
Element
Aluminum
Arsenic
Cadmium
Chromium
Copper
Lead
Silver
Zinc
Pneumatic
0.4
0.060
0.007
0.2
0.060
0.007
0.007
0.060
Ultrasonic
0.4
0.040
0.004
0.1
0.040
0.005
0.004
0.040
Note: Elements Aluminum and Silver are not included in the Lake Michigan Tributary
Loading Study, but are monitored for research studies.
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Volume 3, Chapter 1
7.3.8 Calibration - Sensitivity
High purity, individual metal, NIST traceable, metal standards (prepared with HNO3) are
obtained from High Purity Standards Corporation. Diluted, multi-element calibration
solutions are prepared with MQ water, stabilized with 1-2% Ultrex HNO3, and are stored
in FEP bottles. These standards are coded and recorded in standard solution log.
Working solutions are prepared approximately bi-weekly. Linear ranges are established in
dedicated studies run prior to sample analysis, and results logged in QA document.
Analyte calibration is established using one standard and a calibration blank during
routine sample analysis. The following calibration concentrations are used:
Calibration Standard Concentrations
Element
Ag
As
Al
Cd
Cr
Cu
Pb
Zn
Pneumatic
Check
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
High
1.0
5.0
20.
1.0
5.0
5.0
5.0
5.0
Ultrasonic
Check
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
High
2.0
2.0
10.
2.0
2.0
2.0
2.0
2.0
The calibration slope is determined by fitting a line from 0,0 to the blank subtracted
standard. Calibration information for each run is summarized in a dedicated form which
becomes part of the QA document.
Sensitivity criteria have been established and must be met before sample analysis can
proceed. Sensitivity is verified during the analyte calibration procedure. If the initial
sensitivity test fails, then both cones should be replaced. If cone replacement does not
restore necessary sensitivity, and other front-end factors have been checked, then
multiplier voltage level should be adjusted to correct sensitivity. Sensitivity factors and
multiplier settings are recorded on a dedicated form in the QA document.
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Volume 3, Chapter 1
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Sensitivity threshold criteria are given below:
Sensitivity Criteria
CPS/ppb
Element
Aluminum (27)
Arsenic (75)
Cadmium (114)
Chromium (52)
Copper (63)
Lead (208)
Silver (107)
Zinc (66)
Pneumatic
60,000
8,000
15,000
50,000
30,000
60,000
25,000
7,000
Ultrasonic
400,000
30,000
60,000
400,000
150,000
350,000
150,000
30,000
Note: Elements Aluminum and Silver are not included in the Lake Michigan Tributary
Loading Study, but are monitored for research studies.
7.3.9 Stability
Stability is verified with the analyte calibration solution. RSD's (minimum of four
replicates) for each element must be better than control specification (currently 5%) before
continuing with analyses. Stability data is logged on dedicated form in QA document.
7.3.10 ICP-MS Operation Log
Significant ICP-MS operating parameters, gas flows, vacuum levels, lens settings, power
levels, operators, etc are recorded for each run sequence in the instrument log book.
7.4 Data Acquisition
7.4.1 General
Elemental quantification will be in the peak-jumping mode. A minimum of four replicate
integrations are obtained during each sample analysis. Samples are aspirated for at least
30 seconds after appearance of peak before collecting data. Metal concentrations in units
of ug L' are recorded in the instrument data report for each integration. The mean value
will be entered into an electronic spreadsheet/database. The RSD of replicate integrations
for any given sample analysis must be <15% for analyses less than five times reporting
limit. If RSD criteria fails then the sample must be re-run. If upon re-run, the RSD
criteria is still not met, then the data will be judged estimated. Failed samples are recorded
in the QA document. Data from calibration blanks, background noise evaluation,
sensitivity, and high-purity water analysis will be used in determining appropriate blank to
subtract for any given run.
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Analysis of Surface Waters for Trace
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7.4.2 Internal Standards (IS)
A minimum of three internal standards are used to correct for matrix suppression and
sensitivity variations. The internal standards used are: Ga (71), In (115), and Bi (209),
added at a concentration of 5 pg L ' for ultrasonic and pneumatic nebulization. Internal
standards are added to the sample with electronic digital pipets, not with the peri-pump.
For samples, our internal standard response criteria is >30% and <140% of the internal
standard response in the calibration blank. Samples are re-run with new cones and/or a
1 + 1 dilution if IS response criteria are not met. Failed samples are recorded in the QA
document. The criteria for standards in 1 -2% HNO3 is an internal standard response
between 50% and 125% of calibration blank. If standards fail then the previous sample
results are marked as estimated, and are later re-run with new cones. Selected samples
from each of the tributaries are screened for the presence of indigenous internal standard
elements. The internal standard spiking solution is scanned for the presence of
contaminants and stability.
8.0 ICP-MS QA/QC
8.1 ICV
The initial calibration verification solution (ICV) is run immediately after the first calibration and
must agree within ±10/15% (PNU/USN) before proceeding with analyses. If the ICV fails, the run
is stopped, the instrument recalibrated, and the ICV re-run with a new solution. The ICV is
prepared separately from the calibration solution and at a different concentration so that response
linearity can be verified. ICV results are recorded on a dedicated form in the QA document.
8.2 Check Standard
The continuing calibration verification solution (CCV) and continuing calibration blank (CCB) are
run after a maximum of 10 samples and at the end of a run. Calibration slopes (blank corrected)
must agree within 10/15%, (PNU/USN) for all metals except Al, and Cr, which must agree within
±15% (PNU). If calibration is out of control, then either the instrument must be recalibrated and
the set of out of control samples re-run, or sample results resloped after consultation with QA
manager. If a sample re-run as a result of a CCV failure, again falls under a CCV failure then the
sample data will be judged acceptable if the CCV falls within the range of ±15% (±20% Al, Cr);
and judged estimated if CCV falls within the range of ±25% (±30% Al,Cr). CCV results are
recorded on a dedicated form in the QA document.
8.3 IDL
An IDL subset determination is performed on a daily basis by running a spiked blank, [three times
the average of the standard deviations of seven consecutive measurements of a spiked blank
(5x-15x IDL) obtained on three nonconsecutive days]. Typical spike concentrations are
200 ng L'1 Run sequence results are logged on a dedicated form in the QA document. Metal
concentrations in this standard are well below levels in the high calibration solution so that
intensity linearity may be confirmed at near reporting limit levels. An additional IDL estimate can
be obtained from the 10 replicates of the calibration blank. Blank spikes must recover within
±1 5% (As, Cd, Pb, Zn); ±20% (Cu); and ±25% (Cr) of accepted values.
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Volume 3, Chapter 1
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8.4 Check Blanks
Check blanks are run before the ICV (ICB) and paired with each of the three CCV's (CCB's).
Analyte levels in check blanks and calibration blank are considered acceptable if they do not
exceed Blank Acceptance Criteria (see Table) or do not represent more than 5% of actual sample
signal. Sample and QC data may be blank corrected if a definite trend in CCB data above
background levels is observed.
SRM's and LCS
One certified standard reference solution (SRM) and one laboratory control sample (LCS) are run
with each batch sequence. The SRM is run three times during the run sequence. Our current
SRM is the Canadian trace element standard, SLRS-3. The LCS is a pooled Lake Michigan
tributary sample, amended with certain metals. LCS "certified" concentrations will be established
by seven replicate ICP-MS analyses. SRM and LCS data from each run are logged on a dedicated
form and kept in the QA document. Control limits for the SRM and LCS standards are given in
the table below. If at least one of the three SRM controls run during the batch sequence falls
within the control limits, then the sample data will be considered in control. Samples must be re-
run if all three SRM's are out of control. Sample data will be judged estimated if upon re-run, all
three SRM samples are again out of control.
SRM - Criteria
MgL'
Element
Aluminum
Arsenic
Cadmium
Chromium
Copper
Lead
Silver
Zinc
SLRS-3*
Certified Levels
31 ±3
0.72 + 0.05
0.01 3 ±0.002
0.30 ±0.04
1.35 ±0.07
0.068+0.007
not certified
1.04 ±0.09
Control Range
25-37
0.58-0.86
0.009-0.017
0.21 -0.39
1.15 1.55
0.054-0.082
not certified
0.94- 1.36
"Canadian Riverine Water Reference Material for Trace Metals.
National Research Council Canada - Institute for Environmental Chemistry
Note: Elements Aluminum and Silver are not included in the Lake Michigan Tributary Loading
Study, but are monitored for research studies.
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Analysis of Surface Waters for Trace
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Volume 3, Chapter 1
LCS - Criteria
Element
Aluminum
Arsenic
Cadmium
Chromium
Copper
Lead
Silver
Zinc
LCS (0524)
Certified Levels
24 ±2.5
0.35 ±0.03
0.003 ±0.001
0.41 ±0.05
0.14 ±0.02
0.088 ± 0.007
not certified
0.40 ± 0.04
—
Control Range
19-29
0.28-0.42
0.002 - 0.004
0.29-0.53
0.11 -0.17
0.070-0.106
not certified
0.32-0.48
8.6 Replication
At a minimum, 20% of actual samples are replicated. The acceptance criteria (RPD) for within
run sample duplicates for sample values >5x reporting limit are ±10% for As, Cu, Pb, and Zn;
±15% for Al, Cd and Cr. If a given replicate does not fall within control limits then it must be re-
run. If after re-run, the relative standard deviation of the three determinations is >10% (As, Cu,
Pb, Zn), >15% (Al, Cd, Cr) then the data is judged as estimated. Steps are taken at this point to
determine the reason for the variance: Variation of individual integrations within a given analysis
are examined and sample matrix is scrutinized. At least two sample - replicate pair are run within
every 20 sample batch run. The acceptance criteria (RPD) for sample replicates run in different
batches for sample values >5x reporting limit are ±15% for As, Cu, Pb, and Zn; ±20% for Al, Cd
and Cr. If a given replicate does not fall within control limits then it must be re-run. If after re-
run, the relative standard deviation of the three determinations is >15% (As, Cu, Pb, Zn), >20%
(Al, Cd, Cr) then the data is judged as estimated.
8.7 Matrix Spike
Matrix spikes must recover within 70% to 125% of accepted value. At least two spike sample
pairs are run with each batch of 20 actual samples (typically two lab spike pairs and one field spike
pair). Representative spiking levels are: 0.050 (ag L ' Cd; 1.00 ug L ' As, Cr, Cu, Pb, and
2.00 ug L ' Zn. If any element fails, the sample must be reanalyzed after dilution or other matrix
modification technique. Spiked blanks are also run. If recovery of analyte is outside of control
limits on re-analysis then results are qualified as estimated.
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Volume 3, Chapter 1
Analysis of Surface Waters for Trace
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8.8 Interference Check
An interference check solution along with a mixed trace element standard is run at least once per
week at the end of a run sequence to verify interference correction equations. Levels of
interferents in the test solution are listed in the table below. The analyses of concern are run at a
level of 5.0 |jg L'1. Acceptance criterium is ±20% of established value.
Interference Check Solution
Levels in mg L"1
Element
Aluminum
Calcium
Carbon
Chloride
Iron
Magnesium
Molydenum
Phosphorus
Potassium
Sodium
Sulfur
Concentration
100
100
50
500
100
100
0.005
5
20
50
50
8.9 ICP/MS Sequence, Sample Order Summary
1 CALEB BLANK
2 "HIGH STD - 5 ppb Cr, Cu, As. Pb; 10 ppb Zn; 1 ppb Cd"
3 ICB
4 ICV 2 ppb ALL
5 SLRS-3 #1
6 LCS
7 SAMPLE 1
8 SAMPLE 1 DUP
9 "SAMPLE 1 LAB SPIKE ( 1.000 pg/L Cr, Cu, As, Pb; 2pg/L Zn; 0.050 jig/L Cd)"
10 SAMPLE 2
11 SAMPLE 2 LAB SPIKE
12 SAMPLES
13 SAMPLE 4
14 SAMPLE 4 DUP
15 CCB#1
16 CCV#1 - 2PPB SOLN
17 BLANK SPIKE 0.200 ug/L
18 SAMPLE 5 (METHOD BLANK)
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Analysis of Surface Waters for Trace
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] 9 SAMPLE 6 (METHOD BLANK)
20 SAMPLE 7
21 SAMPLE 8 (FIELD SPIKE OF SAMPLE 7)
22 SAMPLE 9
23 SAMPLE 10
24 SAMPLE 11
25 SAMPLE 12
26 SAMPLE 13
27 CCB#2
28 CCV#2
29 SLRS-3 #2
30 SAMPLE 14
31 SAMPLE 15
32 SAMPLE 16
33 SAMPLE 17
34 SAMPLE 18
35 SAMPLE 19
36 SAMPLE 20
37 CCV#3
38 CCB#3
39 SLRS-3 #3
40 RINSE
41 HIGHSTD
42 INTERFERENCE CHK
43 RINSE
8.10 Quality Assurance Documentation Summary
ICP-MS Prequalification Logs
10. Instrument log of operating settings and conditions.
11. Mass calibration data.
12. Mass resolution data.
13. Response calibration - linearity.
14. Short term stability.
15. Blank data [acceptance test].
16. Sensitivity summary.
17. IDL summary.
18. Memory check results.
19. Internal standard purity scan.
ICP-MS Calibration Logs
20. Calibration solutions - dates of preparation and scans of purity.
21. Calibration linear ranges.
22. Calibration data - slopes.
23. Interference equations applied.
ICP-MS Run QC
24. Batch design.
25. CCV, CCB run summary.
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Volume 3, Chapter 1
Analysis of Surface Waters for Trace
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26.
27.
28.
29.
LCS, SRM run summary.
Run replicate summary.
Run spike summary (lab).
Internal standard recovery summary.
Field QC
30. Field spike summary - rare element recovery.
31. Field spike summary - metals of concern.
32. Field blank summary - bottle blanks.
33. Field blank summary - filtration blanks.
34. Field blank summary - tubing - sampler blanks.
35. Field replicate summary.
9.0 Detection Levels
ICP-MS detection limits for ultrasonic and pneumatic nebulization for the elements of concern are
shown in the table below.
Detection Limits*
ngL1
Element
Aluminum
Arsenic
Cadmium
Chromium
Copper
Lead
Silver
Zinc
Pneumatic
25
15
2.5
20
8
3
1.5
10
Ultrasonic
15
10
0.5
8
4
0.5
0.3
2.5
"Three times the standard deviation of seven consecutive measurements of a spiked blank
(5x-15xIDL).
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Volume 3, Chapter 1
Analysis of Surface Waters for Trace
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Appendix 1.
ICP-MS Batch Analysis QA Outline
15-18 Samples per Batch
Sample Type
Frequency
ICP-MS Qualification
-Blank Levels
-Stability
-Sensitivity
-Resolution
-Interference Check
Blanks Levels During Run
Calibration Blank
Check Blanks
Memory Check
Recovery
Lab Analyte Spike, Blank Matrix
Lab Analyte Spike, Sample Matrix
Internal Standards, 3-metals
Precision
Replicate Sample Acquisitions
Lab Sample Replicates (within batch)
Lab Sample Replicates (different batch)
Accuracy
Standard Reference Material (SLRS-3)
Laboratory Control Sample (Trib Matrix)
Before each sample batch
Before each sample batch
Before each sample batch
Before each sample batch
Once per week
One per batch
Four per batch
One per batch
One per batch
Two per batch
All Samples
Four per sample
Two per batch
20%
Three per batch
One per batch
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Appendix 2.
Summary of ICP-MS QA/QC Protocols
I
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9
B>
tJ
w
en
Summary of ICP-MS QC Protocol
Pre-Qualification A
Type
Warm - Up
Mass
Calibration
Response
Calibration
Detector Cross
Calibration
Resolution
Check
Internal
Standards IS
Frequency
Daily
Before Each Batch
Tuning Solution
Before Each Batch
Tuning Solution
Twice Per Week
Before Each Batch
Tuning + Mg, Pb
Every Acquisition
7lGa, "5In,2MBi
Concentration
2-3% HNO
5 (ag L"' Pneumatic
1 |agL~' Ultrasonic
5 |Jg L"' Pneumatic
1 jag L"1 Ultrasonic
50 jag L"' Pneumatic
5 |ag L~' Ultrasonic
5MgL-'
5 Mg L-'
Acceptance Criteria
> 30 minutes in Analyze Mode
< 0. 1 amu from actual value.
'Be, MCo, "sln, 139U ^Bi, aKU
9Be/1LSIn = .25-1.0; 5l)Co/"5In = .75-1.5
IMLa/"5In = .75-1.25; -™Bi/"sIn = .5-1.1
For "-In: Pulse/Analog >200
Baseline resolved to < 10 raw counts for
Mg(25,26) and Pb(206,207,208)
50-125% of Cal. Blk. For Standards
30-140% of Cal. Blk. For Samples
# Informational Test - other checks of performance take precedence (see text).
'
o
en
rn
B"
D
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T
t>
s
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Appendix 2.
Summary of ICP-MS QA/QC Protocols - Continued
03 *
li
•3 -T^
§1.
en
w
en
Summary of ICP-MS QC Protocol
Pre-Qualification B
Type
Memory Check
25 ug L-'
Stability
Check
Blank
Qualification
Sensitivity
Qualification
Interference
Check
Frequency
Before
Each Batch
Before
Each
Batch
Before
Each Batch
Before
Each Batch
End of Run
2 per week
Units
ngL'1
RSD
ngU'
CPS/
ppb
%of
true
Acceptance Criteria (Pneumatic /Ultrasonic)
7SAs
60
4% @
5 ppb
60/40
or<5%
8,000 /
30,000
±20%
114Cd
7
4% @
1 ppb
7/4 or
<5%
1 5,000 /
60,000
±207o
52Cr
200
4% @
5 ppb
200 / 100
or <5%
50,000 /
400,000
±20%
MCu
60
4% @
5 ppb
60/40
or<5%
30,000 /
1 50,000
±20%
208pb
7
4% @
5 ppb
7/5
or<5%
60,000 /
350,000
± 20%
66Zn
60
4% @
5 ppb
60/40
or <5%
7,000 /
30,000
±20%
n
3
3
-------
Appendix 2.
Summary of ICP-MS QA/QC Protocols - Continued
CO
I
iff
Summary of ICP-MS QC Protocol
Run Sequence Part A
Type
Initial Check
Blank, ICB
Initial. 1CV
Calib. Verif.
2 or 1 (jgL'1
Check, CCV
Standard
2 or 1 (ag L'1
Check Blank
CCB
Method
Blanks
Frequency
1 per Batch
Before ICV
1 per Batch
Before Samples
Every 10 Samples
3 per Batch
Every 10 Samples
3 per Batch
2 per Batch
Units
ngL1
%
%
ngL1
ngL'1
Acceptance Criteria
75As
60/30
or<5%
±10/15%
±10/15%
60/40
or < 5%
100
I14Cd
7/4
or<5%
±10/15%
±10/15%
7/4
or<5%
16
52Cr
200/100
or < 5%
±10/15%
±15%
200/100
or<5%
160 .
63Cu
60/20
or < 5%
±10/15%
±10/15%
60/40
or<5%
80
208pb
7/5
or < 5%
±10/15%
±10/15%
7/5
or<5%
16
66Zn
60/25
or < 5%
±10/15%
±10/15%
60/40
or < 5%
120
11
0 -^
cr »
t 3"
§ o
CO
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Appendix 2.
00
00
Summary of ICP-MS QA/QC Protocols - Continued
Summary of ICP-MS QC Protocol
Run Sequence Part B
Type
Analyte
Spike Blank -
Lab
Analyte
Spike Matrix
-Lab
Analyte
Spike Matrix
- Field
Analyte
Spike Blank -
Field
SRM SLRS-3
LCS# Trib.
Mix
Frequency
1 per Batch
MDL
2 per Batch
1 per
Batch**
1 per
Batch**
3 per Batch
1 per Batch
Units
% of true
% of true
% of true
% of true
MgL"'
M§L-'
Acceptance Criteria (Pneumatic/Ultrasonic)
7SAs
± 1 5 % @
0.2 ppb
70- 125%
@ 1 ppb
70- 125%
± 15%
.58-.S6
1.02-1.83
(1.46)
I14Cd
± 1 5 % @
0.2 ppb
70- 125%
@ 0.5 ppb
70- 125%
±15%
.009-.017
0.033-
0.059
(.047)
52Cr
± 25 % @
0.2 ppb
70- 125%
@ 1 ppb
70- 125%
±25 %
.21-.39
0.90-1.61
(1.29)
63Cu
± 20 % @
0.2 ppb
70- 125%
@ 1 ppb
70- 125%
± 20 %
1.15-1.55
1.20-2.15
(1.72)
208pb
± 1 5 % @
0.2 ppb
70 - 1 20 %
@ 1 ppb
70- 125%
±15%
.054 - .082
.53 - .94
(-75)
66Zn
± 1 5 % @
0.2 ppb
70 - 1 20 %
@ 2 ppb
70- 125%
±15%
.94- 1.36
3.00-5.30
(4.24)
.
- in
°
V>
** One field spiked sample is run with each batch, either a blank or river sample.
# LCS samples will vary over duration of study: values shown are for LCS-2-(a new LCS undergoing certification)
Values in parentheses are mean level of analyte in new LCS.
"
8
tj
-------
Appendix 2.
(B
Co
I
ff
w
-^
(O
Summary of ICP-MS QA/QC Protocols - Continued
Summary of ICP-MS QC Protocol
Run Sequence Part C
Type
Replicate
Acquisitions
Duplicate
Samples
Within Run
Duplicate
Samples
Overall
Frequency
4 per Sample
2 per Batch
20%
Units
RPD
RPD
RPD
Acceptance Criteria
7SAs
±10%
>5xRL
±10%
>5xRL
±15%
>5xRL
1HCd
±15%
>5xRL
±15%
>5xRL
±20%
>5x RL
52Cr
±15%
>5xRL
±15%
>5xRL
±20%
>5\ RL
63Cu
±10%
>5xRL
±10%
>5xRL
±15%
>5x RL
20Spb
±10%
>5x RL
±10%
>5xRL
±15%
>5x RL
MZn
±10%
>5xRL
±10%
>5xRL
±15%
>5x RL
# LCS samples will vary over duration of study
(values shown are outdated - a new LCS is under certification)
RL = Reporting Limit
0)
OS
n> CD
(n to
-------
Analysis of Surface Waters for Trace
Volume 3, Chapter 1 _ Elements by ICP-MS
Appendix 3.
Summary of Calculation Procedure
Outline of calculation methods used by UW-Water Chemistry
Program to generate concentration data from
ICP-MS instrumentation for LMMB study
1. Count rates are obtained by peak-jumping to specific isotopes as the quadrapole "sweeps" the
mass range. We look at three points (0.05 AMU apart) on the peak for each selected mass, which
are subsequently averaged. Dwell times on each peak vary (see attached isotope list), but range
from 10-200 msec. For an acquisition time of 90 seconds, approximately five "sweeps" are made.
2. The initial calibration, run just prior to the start of a batch, and after the instrument has passed a
series of pre-qualification tests (see our SOP) involves running:
a. An acidified mixed-metal calibration standard (see SOP for levels)
b. An acidified high-purity water calibration blank
The isotope count rates obtained from these analyses are processed through the correction
equations (see below) and a line is fit through the blank-subtracted count rate and 0,0 to generate
an initial response slope for each isotope.
Linearity of response is determined in dedicated studies and is verified occasionally.
3. Three internal standards are spiked into each sample just before analysis, and used to evaluate
changes in the response characteristics of the instrument over the course of a run. For each
individual acquisition four response regions are defined over the mass spectrum by the internal
standards. Linear interpolation between regions is used to generate a isotope specific response
factor, which is ratioed with the response from the internal standards in the calibration blank. This
ratio is applied to the raw count data before processing by correction equations.
4. We apply a series of equations to the internal standard processed count rate data to correct for
electron multiplier noise, and isobaric and spectral interferences (see attached list). Multiplier
noise (BKGD in interference equations) is obtained by collecting count rates at masses where no
known isotopes/species exist. The equation corrected, response normalized data from one 90
second aspiration is stored and printed as one acquisition.
5. Three additional acquisitions are performed, stored and printed, and the mean of these four
analyses is what we are reporting as the sample concentration.
3-121
-------
Analysis of Surface Waters for Trace
Elements By ICP-MS Volume 3, Chapter 1
6. In general no "blank" is subtracted from the sample results reported in Step 5. If the four to five
check blanks run over the course of an analysis sequence indicate that a specific analyte was being
picked up from the front-end of the instrument (e.g. cones or nebulizer), then a blank correction
may be applied to the sample results after review in light of other QC checks and consultation with
QA manager. If a sample is "instrument blank" corrected it is flagged and the magnitude of the
correction reported. Specific instrument blank limits have been established and are given in our
ICP-MS analysis SOP.
7. The contribution of analytes from the sample acidification solution that we use (50% Ultrex
HNO3, refer to SOP for amounts added to samples) is negligible, and therefore we do not report a
blank subtraction for this contribution.
8. Other potential blank components, such as Teflon bottles, field handling, filter, sampling line, etc.
were routinely quantified as part of the field QA program. In general the quantity of added analyte
from these sources is extremely small and therefore no blank corrections have been applied to
actual samples.
3-122
-------
Volume 3, Chapter 1
Analysis of Surface Waters for Trace
Elements by ICP-MS
Appendix 4.
Example of Element Menu
File Name :
Date Created :
Date Last Used :
Scan Parameters
ELEMENT MENU
TRACE ELEMENT USN 021795
Wed 1 Dec 1993
Wed 1 Dec 1993
Time Created
Tine Last Used
15:32:30
15:32:30
Channels per AMU : 20
PC Dwell time (is) : 320
Analog Dwell Time (is) : 320
Collector Type : DUAL
Mass Range for Scan : 5.60
Skipped Mass Regions ...
249.54 amu
PC ...
Automatic
Analog . . .
From To
11.40 12.60
13.40 22.60
27.40 41.60
79.40 80.60
Isotopes Selected/Peak Jump Parameters . . .
Element Name
Aluminium
so
Chromium
Vanadium
Chromium
Chromium
Cobalt
Nickel
MgCl
Nickel
Copper
Zinc
Copper
Zinc
Ba++
Gallium
Germanium
Arsenic
CaO2
CaO2
CaO2
Selenium
Krypton
Yttrium
Zirconium
Niobium
Molybdenum
Molybdenum
Ruthenium
Ruthenium
RuHe
symbol
Al
M*
Cr
V
Cr
Cr
Co
Ni
M*
Ni
CU
Zn
CU
Zn
M*
Ga
Ge
As
M*
M*
M*
Se
Kr
Y
Zr
Nb
Mo
MO
Ru
Ru
M*
Maes
27
43
50
51
52
53
59
60
61
62
63
64
65
66
68
71
72
75
76
77
78
82
83
89
90
93
95
98
99
101
104
Abundance
10O.O
94.8
4.3
99.8
83.8
9 .5
100.0
26.2
27.8
3.7
69. 1
4P.9
30.9
27.8
11.3
39.8
27.4
100.0
0.0
0.0
0.0
8.8
11.5
100.0
51.5
100.0
14.8
24.0
12.8
17.0
14.9
Dwell Time
2000
10000
20000
50000
50000
20000
10000
20000
20000
20000
40000
20000
40000
50000
20000
25000
50000
50000
20000
20000
20000
50000
50000
10000
50000
50000
20000
20000
50000
50000
50000
Points/peak
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Collector
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
3-123
-------
Analysis of Surface Waters for Trace
Elements By ICP-MS
Volume 3, Chapter 1
Appendix 4. (cont'd)
Example of Element Menu
YO
YOH
Silver
Palladium
Silver
Cadmium
Cadmium
Cadmium
Indium
Tin
Tin
Antimony
Tellurium
Barium
Cerium
CeO
CeOH
Holmium
Ytterbium
Mercury
Mercury
Lead
Lead
Lead
Bismuth
PbHe
Thorium
Uranium
ThO
ThOH
M*
M*
Ag
Pd
Ag
Cd
Cd
Cd
In
Sn
Sn
Sb
Te
Ba
Ce
M*
M*
Ho
Yb
Hg
Hg
Pb
Pb
Pb
Bi
M*
Th
u
M*
M*
105
106
1O7
108
1O9
110
111
114
115
118
12O
121
125
138
14O
156
157
165
174
202
204
206
2O7
208
209
211
232
238
248
249
99.8
99.7
51.3
26.7
48. 6
12.4
12.9
28.8
95.8
24.0
33.0
57.2
7.0
71.7
88.5
88.3
88. 3
100. O
31. 8
29.8
6.8
25. 1
21.1
52.4
100.0
21.1
100.0
99.3
99.8
99.7
20000
20000
2OOOOO
50000
2OOOOO
100000
100000
50000
20000
5OOOO
50000
20000
50000
2000
10000
20000
2000O
10000
10000
50000
100000
50000
5OOOO
5OOOO
20000
50000
50000
50000
2OOOO
2OOOO
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
DUAL
3-124
-------
Volume 3
Chapter 2: Conventionals
-------
ESS Method 130.1:
General Auto Analyzer Procedures
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 130.1:
General Auto Analyzer Procedures
1.0 Scope and Application
The continuous flow analysis method may be used to determine many chemical constituents in
drinking and surface waters and wastes.
2.0 Apparatus and Summary of Method
2.1 The Auto Analyzer II system is comprised of five separate modules interconnected by tubing and
electrical cables. The typical system includes: 1) Sampler; 2) Proportioning Pump; 3) Manifold;
4) Colorimeter; and 5) Printer/Plotter.
2.2 The Proportioning Pump uses flow-rated tubing to proportion the flow of samples and reagents
through the system. Samples are separated by segments of wash solution. Segments of air are
introduced at two second intervals to help separate samples, mix reagents, and cleanse tubing.
Each parameter has a unique Manifold for introducing reagents, mixing, heating and diluting as
needed. The Printer/Plotter is used to record the concentrations of constituents determined by the
Colorimeter response.
3.0 Sample Handling, Preservation and Pretreatment
3.1 Samples are collected in specified containers and preserved according to Method 100.1 - Sample
Preservation and Holding Times.
3.2 Samples must be free of paniculate matter when introduced into the system. To accomplish this,
samples are filtered according to Method 100.2. Total phosphorus and total Kjeldahl nitrogen
samples should be centrifuged or held overnight after digestion to allow particulates to settle.
4.0 General Operating Procedures
4.1 Check maintenance log for any needed instrument care.
4.2 Turn on Colorimeter lamps at beginning of a work week and leave on until the end of the week.
Allow to warm up for 30 minutes.
4.3 Check heating baths' temperatures. Clean platen with alcohol and install. Start Proportioning
Pump.
4.4 Hydraulic Check
4.4.1 Pump Milli-Q water with appropriate wetting agent through the system. TKN(NH3) and
sulfate washes require Brij-35. Dissolved phosphorus and silica washes need sodium
lauryl sulfate.
3-129
-------
ESS Method 130.1: General Auto Analyzer Procedures Volume 3, Chapter 2
4.4.2 Check for leaks and pinched lines.
4.4.3 Establish a good bubble pattern.
4.5 Prepare any needed reagents and standards.
4.6 Check colorimeter output for each channel used.
4.6.1 On Colorimeter, turn Display Rotary Switch to Zero. Using a screwdriver, adjust Zero
control to obtain zero on the voltmeter.
4.6.2 Turn Display Rotary Switch to Full Scale. Using a screwdriver, adjust Full Scale control
to obtain full range (5.00 volts on the voltmeter).
4.7 Baseline Checks
4.7.1 On Colorimeter, turn Display Rotary Switch to Damp 1, set Std Cat at 1.0, and set
reversing switch to "D".
4.7.2 When system is thoroughly washed with water and wetting agent, adjust Baseline control
to obtain zero on the voltmeter. Check for straight baseline.
4.7.3 Introduce appropriate reagents into the system as directed in different methods and allow
reagents to flow until a straight baseline is obtained. Using Baseline control, reset meter
to Zero. This is correcting for background contamination in reagents.
4.8 Calibration Procedures
4.8.1 Load sample tray with standards specified in various method Tray Protocols. Glass dispo
culture tubes (10 mL) are used for dissolved P, TKN, TOT-P, and low level TOT-P.
Polystyrene dispo beakers (4 mL) are used for dissolved Silica and Sulfates. Fill
remaining cups with unknown samples, duplicates, spikes, and mid-range standard checks
according to Tray Protocol.
4.8.2 Place red peg at last cup. When an analysis requires use of the B wash solution receptacle,
the red peg is placed at the second to last cup.
4.8.3 For each channel used, set Std Cal control on the Colorimeter to an approximate value
expected (the approximate value can be obtained from previous day's run as recorded on
the chart for Baseline and Std Cal settings). Raise baseline about 10% (approximately .14
on voltmeter).
4.8.4 Start Sampler.
4.8.5 When the first standard (primer) comes through, adjust the Std Cal control on the
Colorimeter so that the top of the peak registers about 95^ of full range (approximately
4.80 on voltmeter). Record Std Cal values on appropriate Baseline and Std Cal Settings
chart.
3-130
-------
Volume 3, Chapter 2 ESS Method 130.1: General Auto Analyzer Procedures
4.9 Shutdown Procedures
4.9.1 After last cup has been sampled, turn off Sampler.
4.9.2 Check the return to 10% baseline after last sample value has been printed, to check on
baseline drift.
4.9.3 Connect reagent tubes to wash bottles and flush at high speed, if available. Continue to
wash with water and wetting agents until system is rinsed completely. Reset Sid Cat to
1.0, and adjust baseline to zero.
4.9.4 Remove chart from Printer/Plotter, Initial, record Std Cal, & calculate correlation
coefficient(r).
4.9.5 Shut off pump. Lift platen off and store upside down. Adjust pump so that the air bar is
up. Disconnect wash water tubing from containers. Caution: Wash solution will siphon
onto the laboratory bench if the wash water tubing is not removed from the containers.
4.9.6 Discard all used sample cups.
5.0 Quality Assurance Procedures
5.1 Baseline and Std Cal Settings Chart.
5.1.1 The Std Cal values for each nutrient and each range used are recorded.
5.1.2 Included in this chart are the date and analyst's initials.
5.2 Pump Tubing Chart
The lot numbers of all flow-rated pump tubing and date the packages are opened are recorded.
5.3 A calibration curve as described in each method is run at the beginning of each range to establish
system linearity. Subsequently, mid-range standards are included after every 10-20 samples
(depending on method) to verify the curve and at the end of the run.
5.4 Precis;on is checked every day by analyzing 10% of all samples in duplicate. When filtered
samples are analyzed in duplicate, the sample and its duplicate must be filtered separately and be
treated as independent samples. The absolute differences between duplicates are plotted on
Shewhart Charts to verify that the analyses are within the quality control limits.
5.5 Accuracy is verified daily by analyzing a sample spiked with a standard solution.
5.5.1 For soluble chemical constituents, a spike sample is made by mixing an equal volume of
sample with an equal volume of a standard solution of approximately the same
concentration. The calculations are as follows:
Spiked Sample Concentration - '/i sample cone. X 100 = % Recover
'/2 Standard cone.
3-131
-------
ESS Method 130.1: General Auto Analyzer Procedures Volume 3, Chapter 2
5.5.2 For total phosphorus and total Kjeldahl nitrogen digested on the Block Digestor, a volume
of sample (10 mL or less) is pipetted into the digester tube and a volume of standard
(10 mL or less) is added and run through the digestion procedure with samples and
standards. The standard added can be Nicotinic Acid for TKN or AMP for TP separately
or a combination of Glutamic Acid and KH2PO4 for a dual spike. The calculations are as
follows:
Spiked Sample Concentration - Sample Background Concentration X 100 = % Recovery
Spike Concentration
5.5.3 The % Recoveries are plotted on Shewhart Charts to verify that the analyses are within the
quality control limits.
5.6 Daily worksheets are stamped with "Q.C. Audit Date " whereby another chemist can
verify, initial and date that the analyses meet the Q.C. criteria designated for the lab and the
particular parameter measured.
5.7 Reagents are dated and initialed when they are prepared.
6.0 Preventive Maintenance
6.1 A log is kept for dating maintenance procedures performed on any module. Figure 2.
6.2 Daily
6.2.1 Clean surfaces of entire system and area.
6.2.2 Check surface of pump platen and rollers. Clean with alcohol, if necessary.
6.3 Weekly
6.3.1 Remove, clean with alcohol, and lightly lubricate side rails with Semi-Fluid Lubricant.
6.3.2 Clean pump rollers and platen with alcohol.
6.4 Monthly
6.4.1 Change pump tubing monthly or when deemed necessary.
6.4.2 Adjust silicone tubing under air bar to a new position.
6.4.3 Oil air bar linkage with one drop Prolonged Service oil.
6.4.4 Oil two felt pads with two drops oil.
6.4.5 Oil needle bearings of main drive shaft b> putting one drop oil in each of two holes.
3-132
-------
Volume 3, Chapter 2 ESS Method 130.1: General Auto Analyzer Procedures
6.5 Three Months
6.5.1 Replace sample tubing.
6.5.2 Clean sample probe with wire stylet.
6.5.3 Clean sampler pole with freon and oil lightly.
6.6 Six Months
6.6.1 Put one drop oil on each end of each pump roller and rhain interface. Rotate rollers anu
wipe off excess oil with alcohol.
6.6.2 Clean Colorimeter flowcell and filters.
6.6.3 Clean Colorimeter lamp and socket controls.
6.7 Eighteen Months
Lubricate four spots on Sampler as directed in Instrument Manual.
7.0 Miscellaneous Maintenance
7.1 Clean heating bath with cleaning acid.
7.2 Clean dilution coils and debubblers with 50% HC1.
7.3 Change various tubing and connections and clean glass connections.
7.4 Clean sample splitter.
7.5 Clean color reagent line on phosphorus Auto Analyzer with 20% NaOH and H2O:. This is done as
follows:
7.5.1 MQ line in MQ H2O (No Levor) and Color Reagent line in 20% NaOH for 20 minutes.
7.5.2 MQ line in MQ H2O (No Levor) and Color Reagent line in H2O2 for 10 minutes.
7.5.3 Both lines in MQ H2O (No Levor) for 10 minutes.
7.5.4 Both lines in MQ H2O with Levor (4 mL Levor/125 mL MQ) for 10 minutes.
7.5.5 Both lines in MQ Levor wash solution (3.0 rnL/L MQ) until stable baseline is obtained.
3-133
-------
ESS Method 130.1: General Auto Analyzer Procedures Volume 3, Chapter 2
8.0 Peaking FlowCell
Whenever any maintenance has been performed on the Colorimeter it is necessary to peak the flowcell as
follows:
8.1 Turn Display Rotary Switch to Normal
8.2 SetStdCalat 1.0.
8.3 Set reversing switch at "D"
8.4 Set Baseline control at mid-point. (Control has 10 complete turns, so set at 5 turns from either
extreme.)
8.5 Set voltmeter at half scale (2.50) by using both sample and reference apertures.
8.6 Rotate the peaking screw on the sample phototube housing assembly to obtain minimum deflection
on voltmeter.
8.7 Rotate the peaking screw on the reference phototube housing to obtain maximum deflection on the
voltmeter.
8.8 Open both apertures completely clockwise.
8.9 Note voltmeter reading:
8.9.1 If value is below zero, more light is reaching the sample phototube than the reference.
Correct by closing sample aperature (A) to adjust value to zero.
8.9.2 If value is above zero, less light is reaching the sample phototube than the reference.
Correct by closing the reference aperature (B) to adjust to zero.
8.9.3 One aperature should be Fully Open at all times.
8.9.4 Fine adjust by using Baseline control.
3-134
-------
ESS Method 200.5:
Determination of Inorganic Anions in
Water by Ion Chromatography
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 200.5:
Determination of Inorganic Anions in Water
by Ion Chromatography
1.0 Scope and Application
1.1 This method covers the determination of the following inorganic anions: Chloride, Nitrate-N,
Sulfate.
1.2 This is an ion chromatographic (1C) method applicable to the determination of the anions listed
above in drinking water, surface water, and mixed domestic and industrial wastewater.
2.0 Summary of Method
A small volume of sample, typically 5 mL, is introduced into an ion chromatograph. The anions of
interest are separated and measured, using a system comprised of a guard column, separator
column, suppressor column, and conductivity detector.
3.0 Interferences
3.1 Interferences can be caused by substances with retention times that are similar to and overlap those
of the anion of interest. Large amounts of an anion can interfere with peak resolution of an
adjacent anion. Sample dilution and/or spiking can be used to solve most interference problems.
3.2 The water dip or negative peak that elutes near and can interfere with the chloride peak can be
eliminated by the addition of the equivalent of 1 mL of concentrated eluent (6.3 100X) to 100 mL
of each standard and sample.
3.3 Method interferences may be caused by contaminants in the reagent water, reagents, glassware,
and other sample processing apparatus that lead to discrete artifacts or elevated baseline in ion
chromatograms.
3.4 Samples that contain particles larger than 0.45 microns and reagent solutions that contain particles
larger than 0.20 microns require filtration to prevent damage to instrument column and flow
systems.
4.0 Sample Collection, Preservation and Storage
4.1 Samples should be collected in scrupulously clean 60 mL polyethylene bottles.
4.2 Sample preservation and holding times for the anions that can be determined by this methods are
as follows:
Anal\te
Chloride
Nitrate-N
Sulfate
Preservation
None required
Cool to 4°C
Cool to 4°C
Holding Time
28 days
48 hours
28 days
3-137
-------
ESS Method 200.5: Determination of Inorganic
Anions in Water by Ion Chromatography Volume 3, Chapter 2
4.3 The method of preservation and the holding time for samples analyzed by this method are
determined by the anions of interest. In a given sample, the anion that requires the most
preservation treatment and the shortest holding time will determine the preservation treatment and
holding time for the total sample.
4.4 Samples should be filtered through a 0.45 m filter to remove particulate matter.
5.0 Apparatus and Materials
5.1 Balance - Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.2 Ion chromatograph - Dionex Model 4000i complete with all required accessories including
analytical columns, compressed air, detector, and integrator.
5.2.1 Anion guard column: 4 x 50 mm, Dionex P/N 37042, or equivalent.
5.2.2 Anion separator column: 4 x 250 mm, Dionex P/N 37041, or equivalent.
5.2.3 Anion suppressor column: Membrane Suppressor, Dionex P/N 43074, or equivalent.
5.2.4 Detector - Conductivity cell: approximately 6 uL volume, Dionex, COM2, or equivalent.
5.3 Integration System
5.3.1 Dionex Model 4270 Integrator (Spectraphysics, Inc.)
5.4 Automation Accessories
5.4.1 Dionex Automated Sampler
5.4.2 Dionex Automation Interface
5.4.3 Autosampler vials with filter caps, 5 mL capacity
6.0 Reagents and Consumable Materials
6.1 Sample bottles: 60 mL polyethylene.
6.2 Reagent water: Milli-Q water, Millipore Corp., Bedford, Mass.
6.3 Eluent solution: Sodium bicarbonate 0.75 mm, sodium carbonate 2.00 mm. Dissolve 0.25 g
sodium bicarbonate (NaHCO,) and 0.933 g of sodium carbonate (Na,COO in reagent water and
dilute to 4 L.
6.4 Regeneration solution (membrane suppressor): Sulfuric acid 0.025N. Dilute 2.8 mL cone.
sulr'uric acid (H2SO4) to 4 L with reagent water.
3-138
-------
ESS Method 200.5: Determination of Inorganic
Volume 3, Chapter 2 Anions in Water by Ion Chromatography
6.5 Stock standard solutions, 1000 mg/L (1 mg/mL): Stock standard solutions may be purchased as
certified solutions or prepared from ACS reagent grade materials (dried at 105°C for 30 min.)
6.5.1 Chloride (CL') 1000 mg/L: Dissolve 1.6485 g sodium chloride in reagent water and dilute
to 1 L.
6.5.2 Nitrate (NO,-N) 1000 mg/L: Dissolve 6.0679 g sodium nitrate in reagent water and dilute
to 1 L.
6.5.3 Sulfate (SO4) 1000 mg/L: Dissolve 1.8141 g potassium sulfate in reagent water and dilute
to 1 L.
6.5.4 Stability of standards: Stock standards (6.5) are stable for at least six months when stored
at 4°C. Dilute working standards should be prepared weekly.
7.0 4000i Ion Chromatograph (1C) Operation Procedure
7.1 Check the level of the various eluant bottles on top of the 40001. Each should contain more than
enough for the planned runs.
7.2 Turn gas (Loading Dock) on. About 80 psi should be delivered to the 1C. Open line toggle (up)
behind instrument.
7.3 Eluent Degas Module (EDM):
7.3.1 If the 1C is off, push POWER button to turn on (It is normally kept on.)
7.3.2 Turn system switch on. Mode switches should be on Pressure.
7.3.3 Turn on eluent reservoir switch (#1). EGM pressure should be about 15 psi. The
black/red dial adjusts the pressure.
Eluent Reservoir Numbers:
#1 = NaHCO3/Na2CO3 buffer. For strong acids and used with program #1/STD #1.
#2 = Na2B4O7 buffer. For weak acids. Used with program #2/STD #2.
#3 = Various unusual buffers.
#4 = Deionized water. Use with program 6 to rinse columns.
7.4 Turn gas valve located on the left of the 1C until the gauges reads 15 psi. This turns the column
regenerating system on.
7.5 Conductivity Detector (CD): Be sure the proper column is in place. Then Cell On. Output range
is usually set to 10.
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ESS Method 200.5: Determination of Inorganic
Anions in Water by Ion Chromatography Volumes. Chapter 2
7.6 Gradient Pump (GP):
7.6.1 Press PGM, enter program number (1 for strong acid, 2 for weak acids) Stop - Start to
Start.
7.6.2 Let the 1C run until the conductivity readout stabilizes to ±0.1 units (usually 15 to
30 minutes)
7.6.3 To list program #1 in the GP press the following:
List PGM 1 List (to get time), List (to get time 2) etc. Each push of list will give
an event and the time it occurs.
7.7 Programming the Integrator (INT):
7.7.1 In using the INT, each key has three meanings: They are rotated by the Shift Key.
Red light on = blue command under key
(shift) - Slow red blink = number upper left
(shift) - Fast red blink = letter, upper right
(shift) - Red light on, etc.
7.7.2 Use File I enter will load file #1 into active status.
Prfile gives a printout of the file in active status.
7.7.3 Editing a Program
Sometimes it is necessary to change a program, often a retention time changes enough that
the integrator does not recognize it. In that case you must delete the entry and replace it
with the new time.
Dialog puts integrator in editing mode
To delete an entry for a time (example 13 minutes)
-13 enter
To repace the entry for time 13 minutes
TT =13 enter etc.
Enter Escape Escape to exit from dialog.
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ESS Method 200.5: Determination of Inorganic
Volume 3, Chapter 2 Anions in Water by Ion Chromatography
7.8 To Standardize
Standard 1 strong acids. This is run using Program 1 on the GP and Use File 8 on the integrator.
7.8.1 Pour the standard to be used into a tube and cap. Load into rack (white dot on the right).
7.8.2 On Integrator: TFN T (shift) 3 Enter TV = 1 Enter
7.8.3 On T3 Auto Sampler: Hold - Run
7.8.4 (shift) (shift) R N (shift) = 0 Enter
(shift) (shift) Z Z (shift) = Number of STDS, typically 1, Enter
Calib 1 enter (note 1 = calibration On.; = calibration Off)
Wait for status light on T3 to flash Load, then on integrator:
Inject A (After flashing Load, the status light changes to Ready)
7.9 To Run Samples (or QC's): (See Section for 10 Sample Preparations)
On Integrator:
7.9.1 Use File = 1 enter (Note: to get back to File Ifrom File 9)
7.9.2 TFN T (shift) 3 enter TV = / Enter
on T3 Autosampler: Hold - Run
*Back to integrator:
7.9.3 (shift) (shift) R N (shift) = 0 enter
(shift) (shift) Z Z (shift) = # of injection enter
(Note: ZZ = 2forQCs)
Wait for status light to flash LOAD, or READY on T3
Inject A
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ESS Method 200.5: Determination of Inorganic
Anions in Water by Ion Chromatography Volume 3. Chapter 2
8.0 Shut Down Procedure
8.1 Rinse the column with water, as follows:
* On EDM: Reservoir switch #4 On (i.e. UP)
* OnGP: PGM 6 Stop - Stan
This pumps H2O thru the column. Let this run for about five minutes then continue.
8.2 Complete Shutdown as follows:
* On CD: Cell Off
* OnGP: Start -Stop
* Valve on side counter clockwise until dial reads 0 * Turn N2 off at cylinder (loading dock)
* Loosen cap on eluent reservoir #1 to let N2 escape.
(The pressure should drop on the main gauge and then on the other two). Wait until both gauges
read 0.
* On EGM: turn all EDM toggle switches off (i.e. down )turn system pressure off (down).
* Tighten cap on reservoir #1.
* Turn off (down) main toggle valve (behind instrument).
* Turn main power off (blue button on CD).
9.0 Calibration and Standardization (High level method)
9.1 For each analyte of interest prepare calibration standards at a minimum of three concentration
levels and a blank by adding accurately measured volumes of one or more stock standards (6.5) to
a volumetric flask and diluting to volume with reagent water. Typically, the working standard
range for the high level I.C. method will be 0-2 mg/L for Chloride and Nitrate and 0-10 mg/L for
Sulfate. If the working range exceeds the linear range of the system, a sufficient number of
standards must be analyzed to allow an accurate calibration curve to be established. One of the
standards should be representative of a concentration near, but above, the method detection limit if
the system is operated on an applicable attenuator range. The other standards should correspond to
the range of concentrations expected in the sample or should define the working range of the
detector. Unless the attenuator range settings are proven to be linear, each setting must be
calibrated individually.
9.2 Using injection of 0.2 mL of each calibration standard, tabulate peak height or area responses
against the concentration. The results are used to prepare a calibration curve for each analyte.
This procedure will be automatically performed by the integration system.
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ESS Method 200.5: Determination of Inorganic
Volume 3, Chapter 2 Anions in Water by Ion Chromatography
9.3 The working calibration curve must be verified on each working day, or whenever the anion eluent
is changed, and after every 10 samples. If the peak concentration response for any analyte varies
from the expected values by more than ±10%, the tests must be repeated, using fresh calibration
standards. If the results are still more than ±10%, an entire new calibration curve must be
prepared for that analyte.
9.4 Nonlinear response can result when the separator column capacity is exceeded (overloading).
Maximum column loading (all anions) should not exceed about 400 ppm.
10.0 Calibration and Standardization (Microlevel Method).
Calibration and operating conditions are the same for this method as the high-level method except
for the following:
10.1 The working standard range is 0-0.4 mg/L for Nitrate and Chloride but 0-4 mg/L for Sulfate.
10.2 A separate anion separator column is used for the micro-level method.
10.3 The attenuation setting on the 1C is changed from 10 us to 3 (as.
11.0 Procedure
11.1 Operating conditions: columns as specified in Section 5.2; detector as specified in Secction 5.2;
eluent as specified in Section 6.3; sample loop - 200 uL; pump volume - 2.30 mL/min; full scale -
10 mhos/cm.
Note: The operating conditions may need to be changed to meet specific applications.
11.2 Check system calibration daily and, if required, recalibrate as described in Section 7.0.
11.3 Load and inject a fixed amount of well mixed sample. Flush injection loop thoroughly, using each
new sample.
11.4 The width of the retention time window used to make identifications should be based upon
measurements of actual retention time variations of standards over the course of a day. Three
times the standard deviation of a retention time can be used to calculate a suggested window size
for a compound.
11.5 If the response for the peak exceeds the working range of the system, dilute the sample with an
appropriate amount of reagent water and reanalyze.
11.6 If the resulting chromatogram fails to produce adequate resolution, or if identification of specific
anions is questionable, spike the sample with an appropriate amount of standard and reanalyze.
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ESS Method 200.5: Determination of Inorganic
Anions in Water by Ion Chromatography Volumes, Chapter 2
12.0 Automated Calculation
12.1. Both the processes of generating a calibration curve and calculating unknown sample
concentrations are performed by the Dionex Model 4270 integration system.
12.2 A report including calibration coefficients is printed following the last standard run in a calibration
curve.
12.3 Results for the unknown samples are printed in a report following each sample run.
12.4 Report results in mg/L.
13.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
14.0 References
14.1 Annual book of ASTM Standards, Part 31 Water, proposed test method for "Anions in Water by
Ion Chromatography," p. 1485-1492 (1982).
14.2 Standard Methods for the Examination of Water and Wastewater, Method 429, 16th Ed.,
p. 483-488(1985).
14.3 Dionex, System 4000i Operators Manual, Dionex Corp., Sunnyvale, California 94086.
14.4 The Determination of Inorganic Anion in Water by Ion Chromatography - Method 300.0, United
States Environmental Protection Agency, EPA-600/4-84-017, March 1984.
14.5 Spectra-physics SP4270/Sp4290 Integrator User's Guide Spectra Physics, Inc., San Jose,
CA 95134.
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ESS Method 140.4:
Chloride - Automated Flow
Injection Analysis
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised April 1993
-------
ESS Method 140.4:
Chloride - Automated Flow Injection Analysis
1.0 Scope and Application
1.1 This automated method is applicable to drinking, surface, and saline waters, domestic and
industrial wastes.
1.2 Samples with concentrations in the range of 1.0-100 mg Cl/L can be analyzed directly.
However, the range may be extended through the use of a digital diluter. Approximately
100 samples per hour can be analyzed.
2.0 Summary of Method
2.1 The automated procedure for the determination of chloride is based on the liberation of the
thiocyanate ion (SCN) from mercuric thiocyanate, through sequestration of mercury by the
chloride ion, to form un-ionized, but soluble mercuric chloride. The liberated SCN then
reacts with the ferric ion to form highly colored ferric thiocyanate. This is measured
colorimetrically. However, since the chemistry does not follow Beer's law, a straight line
calibration curve is not obtained, necessitating a greater number of standards.
2.2 The reaction may be written as follows:
Hg(SCN)2+ 2 Ct - HgCl2 + 2(SCN) (SCN)- + Fe3 - (Fe[SCN])+2
3.0 Sample Handling and Preservation
There are no special requirements, however, the maximum holding time is 28 days.
4.0 Interferences: Interferents belong to two classes:
4.1 Substances which reduce iron(III) to iron(Il) and mercury(III) to mercury(II). (e.g.. sulfite,
thiosulfate).
4.2 Other halides which also form strong complexes with mercuric ion (e.g., Br, I).
If any question of interferences arise, calibration curves should be prepared in water and in
the suspected interfering matrix. If the two curves differ significantly, then there is
interference and the standards must be prepared in the interfering matrix instead of in
water.
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ESS Method 140.4: Chloride - Automated Flow Injection Analysis Volume 3, Chapter 2
5.0 Apparatus: Lachat QuikChem Automated Flow Injection
Analyzer which includes:
5.1 XYZ Automatic Sampler
5.2 Proportioning Pump
5.3 Injection Module with a 20 cm 0.8 mm i.d. sample loop.
5.4 Colorimeter
5.4.1 Flow cell, 10mm, 80 nL
5.4.2 Interference filter wavelength, 480 nm
5.5 Reaction Module 10-117-07-1-B
5.6 Automated Digital Diluter
5.7 IBM Personal System 12 computer
5.8 QuikChem AE System Unit
5.9 Recorder or Quik-Calc II Software System
6.0 Reagents
6.1 Milli-Q: All reagents must be made with Milli-Q water. Millipore Corp., Bedford, MA
6.2 Chloride Color Reagent (Technicon No. TO 1 -0352).
7.0 Standards
7.1 Stock Standard 1000 mg C17L
7.1.1 Chloride stock solution A, lOOOmgCI/L: Dissolve 1.6482 g of sodium chloride
(NaCl) (dried at 105°Cfor 1 h) in Milli-Q water and dilute to 1 L.
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Volume 3, Chapter 2 ESS Method 140.4 : Chloride - Automated Flow Injection Analysis
7.1.2 High level working standards, 20-100 mg Cl/L: Prepare the high level working
standards by diluting the following volumes of chloride stock solution A (7.1.1) to
500 mL with Milli-Q water:
mL Stock
Cone, mg Cl/L Standard (7.1.1) 500 mL
100.0 50.0
80.0 40.0
60.0 30.0
40.0 20.0
20.0 10.0
7.2 Stock Standard 100 mg Cl/L
7.2.1 Chloride stock solution B, 100 mg Cl/L: Dilute 50 mL of chloride stock solution
A (7.1.1) to 500 mL with Milli-Q water.
7.2.2 Low level working standards 1.0-10 mg Cl/L: Prepare the low level working
standards by diluting the following volumes of chloride stock solution B (7.2.1) to
500 mL with Milli-Q water:
mL Stock
Cone, mg Cl/L Standard (1.2) 500 mL
10.0 50.0
5.0 25.0
2.0 10.0
1.0 5.0
8.0 Injection Timing
Pump speed: 35
Cycle period: 30 s
Load period: 15 s
Inject period: 15 s
Inject to start of peak period: 8 s
Inject to end of peak period: 30 s
9.0 System Operation:
9.1 Start-up
9.1.1 Turn on and check diagnostics.
9.1.2 Attach reagent lines.
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ESS Method 140.4: Chloride - Automated Flow Injection Analysis Volumes, Chapter 2
9.2 Procedure
Follow directions in General Operating Procedures.
10.0 Notes
10.1 Collect the effluent from the chloride channel in a separate waste container of known
volume.
10.2 When the container is filled, place it in a hood and add 20 mL of 13% thioacetamide (6.8)
for each liter of chloride waste.
10.3 Mix thoroughly and allow the solution (which has a very strong skunk smell) to stand in a
hood 24 hours. The container should be capped. Mercuric sulfide precipitate is formed
during this time.
10.4 After 24 hours, filter (in a hood) the solution through a Buchner funnel. The clear filtrate
can be discarded in a sink in a hood. The residue containing the mercuric sulfide can be
stored indefinitely in a glass container and eventually disposed of as a hazardous waste.
11.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality
Assurance Manual.
12.0 References
12.1 U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water and
Wastes, EPA-600/4-79-020, Method 325.2, (1979).
12.2 Lachat Instruments, Method 10-117-07-1-B, 1991.
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Volume 3, Chapter 2
ESS Method 140.4 : Chloride - Automated Flow Injection Analysis
-PUHl'-FtrOK-
fPON WAteP
fro* wash
bath drain
9 ra.u
CflRRHR
green
SftMPLE
vrcen
i to wash
"f bath till
y to wistc
2.5"
Color Reagent A A
V 1
• / A A \ A SaMplB Loop = 20 CH +o
A( — •/\f*~ y*
\ v / L to port 6 or Filter: 400 n« How
ON /5 " next va.lw or waste ceil
Carrier is Milli-Q Water.
1" is 70.0 cm of tubing on a 1" coil support.
2.5" is 168 cm of tubing on a 2.5 " coil support.
All manifold tubing is 0.8 mm (0.032") i.d. This is 5.2 uL/cm.
Sample Loop:
Cycle Period:
Number of Standards:
Segmented between each Standard
Check Standard:
20 cm
30 sec
9
40.0 mg/L
Figure 1. Manifold Diagram
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ESS Method 220.3:
Ammonia Nitrogen and Nitrate + Nitrite
Nitrogen, Automated Flow Injection
Analysis Method
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised September 1991
-------
ESS Method 220.3:
Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method
1.0 Scope and Application
1.1 This method pertains to the simultaneous determination of ammonia and nitrate in surface,
drinking and ground waters, and domestic and industrial wastes samples which have been
preserved with H,SO4.
1.2 The applicable range of the ammonia channel is 0.02-10.0 mg NH,-N/L. The applicable range of
the nitrate channel is 0.02-35.0 mg NO,+NO2-N/L. The ranges may be extended with the digital
diluter.
2.0 Summary of Method
2.1 NHj-N: Alkaline phenol and sodium hypochlorite react with ammonia to form a blue indophenol
compound which is proportional to the ammonia concentration. The presence of EDTA in the
buffer prevents precipitation of calcium and magnesium. The color is intensified by adding
sodium nitroprusside. The resulting water soluble colored dye is measured colorimetrically at
630 nm.
2.2 NO3+NO2-N: The same sample is passed through a copperized cadmium column which reduces
nitrate quantitatively to nitrite. The total nitrite (reduced nitrate plus original nitrite) is then
determined by diazotizing with sulfanilamide followed by coupling with N-(l-naphthyl)
ethylenediamine dihydrochloride. The resulting water soluble magenta colored dye is measured
colorimetrically at 520 nm. Nitrite alone can also be determined by removing the cadmium
column.
3.0 Sample Handling and Preservation
3.1 The samples are collected in 250 mL high density polyethylene containers.
3.2 Samples are preserved in the field with 2 mL of 12.5% H2SO4/250 mL (1 mL of cone. H2SO4/L,
pH <2) and stored at 4°C.
4.0 Interferences
4.1 Calcium, magnesium, iron and copper ions, or other metals may precipitate if present in sufficient
concentration. EDTA is added to the sample in-line in order to prevent this problem.
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ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method Volume 3, Chapter 2
4.2 Color, turbidity, and certain organic species may interfere.
4.2.1 Sample color may be corrected for by running the samples through the manifold with all
reagents pumping except hypochlorite, which is replaced by Milli-Q water. The resulting
absorbance readings are then subtracted from those obtained for samples determined with
color formation in addition to sample color.
4.2.2 Turbidity is removed by manual filtration. Build up of suspended matter in the reduction
column will restrict sample flow.
4.2.3 Samples that contain large concentrations of oil and grease will coat the surface of the
cadmium. This interference is eliminated by pre-extracting the sample with an organic
solvent, such as Freon.
5.0 Apparatus
Lachat QuikChem AE Automated Flow Injection Ion Analyzer consisting of:
5.1 XYZ Sampler.
5.2 Peristaltic Pump.
5.3 Two QuikChem AE Sample Processing modules with Alpha and Beta detectors.
5.3.1 Interference filters: two 520 nm for NO3-N and two 630 nm filters
for NH3-N.
5.3.2 Flow cells: 2 Alpha 0.1 cm, 8.0 |aL and 2 Beta 1.0 cm., 80 uL.
5.3.3 Sample Loops: Ammonia 180 cm loop, Nitrate 59 cm loop.
5.4 Reaction Module 10-107-06-1-Z with heating unit.
5.5 Reaction Module 10-107-04-1 -Z with cadmium column.
5.6 Automated Digital Diluter.
5.7 IBM Personal System 12 Computer.
5.8 QuikChem AE System Unit.
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ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Volume 3, Chapter 2 Automated Flow Injection Analysis Method
6.0 Reagents
Ammonia
6.1 Milli-Q water: Millipore Corp., Bedford, MA. All reagents must be made with NH3-free Milli-Q
water.
6.2 Dilution water: Add 1 mL H:SO4 to 1 L Milli-Q water.
6.3 Alkaline phenol: Dissolve 83 g phenol in a 1 L Erlenmeyer flask containing about 500 mL Milli-
Q water. While stirring, slowly add 32 gm NaOH. Cool, dilute to 1 L, and filter through a glass
fiber filter if necessary. Two liters can be made at one time. Store in a dark bottle.
6.4 Sodium hypochlorite solution: Dilute 500 mL of commercial bleach containing 5.25% available
chlorine (e.g. Clorox) to 1 L with Milli-Q water, and filter through a 0.45 |um membrane filter, if
necessary. Store at 4°C.
6.5 Buffer: Dissolve 50 g disodium ethylenediamine-tetraacetate (Na: EDTA) and 12.5 g NaOH in
900 mL of Milli-Q water. Dilute to 1 L.
6.6 Sodium nitroprusside: Dissolve 7 g of Na2Fe(CN)3NO-2H,O (alternate name: sodium
nitroferricyanide) in 900 mL of Milli-Q water and dilute to 1 L. Reagent is light sensitive, store in
dark container.
Nitrate
6.7 Ammonium chloride buffer, pH 8.5: In a hood, to a 1 L volumetric flask, add 500 mL Milli-Q
water, 105 mL concentrated HC1, 95 mL concentrated ammonium hydroxide (NH4OH) and 1.0 g
disodium EDTA. Dissolve and dilute almost to volume. Allow to cool overnight. Adjust the pH
to 8.5 ± 0.1 with either cone. HC1 or cone. NH4OH. Dilute to volume. 2 L can be made at one
time.
6.8 Sulfanilamide color reagent: To approximately 1500 mL of Milli-Q water, add 200 mL 85%
phosphoric acid (H3PO4), 80 g sulfanilamide (C6H8N,O2S), and 2.0 g N-(l-naphthyl)
ethylenediamine dihydrochloride (C|;HMN2-2HC1). Dissolve and dilute to 2 L. Store in brown
bottle and keep in a cool, dark place. This solution is stable for several months.
6.9 Cadmium column: Use prepacked column from Lachat. The efficiency should be above 90%. To
check this:
6.9.1 Have system running with all reagents, but no cadmium column.
6.9.2 Run calibration curve with nitrite standards: 20, 10, 5. 1.0 mg NO2-N/L.
6.9.3 Attach cadmium column and run calibration curve with nitrate standards: 20, 10, 5, 1 mg
NOj-N/L.
6.9.4 Calculate percent recovery of standards.
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ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method
Volumes, Chapter2
7.0 Stock Standards
7.1 Ammonia standard solution A (1000 mg NH3-N/L): Dissolve 3.819 g of anhydrous ammonium
chloride (NH4C1), dried at 105°C for 1 hr, in 900 mL Milli-Q water. Add 1 mL cone. H2SO4 and
dilute to 1 L (1.0 mL = 1.0 mg NHrN).
1.1 Ammonia standard solution B (100 mg NH3-N/L): Dilute 100 mL standard solution A to 900 mL
7.3
Milli-Q water. Add 1 mL cone. H2SO4 and dilute to 1 L (1.0 mL = 0.1 mg NH,-N).
Ammonia standard solution C (10 mg NH3-N/L): Dilute 25 mL standard solution B to 250 mL
(1.0mL = 0.01 mgNHj-N).
7.4 Nitrate standard solution A (1000 mg NO3-N/L): Dissolve 7.218 g potassium nitrate (KNO3) in
900 mL Milli-Q water. Add 2 mL chloroform and dilute to 1 L (1.0 mL = 1.0 mg NO3-N).
7.5 Nitrate standard solution B (100 mg NO3-N/L): Dilute 100 mL standard solution A to 900 mL
Milli-Q water. Add 2 mL chloroform and dilute to 1 L (1.0 mL = 0.1 mg NO3-N).
7.6 Nitrate standard solution C (10 mg NH3-N/L): Dilute 25 mL standard solution B to 250 mL (1.0
mL + 0.01 mg NO3-N).
8.0 Mixed Working Standards
Prepare the following standards by adding appropriate amounts of stock standards to 500 mL
Milli-Q water. Add 1 mL cone. H2SO4 and dilute to 1 L.
Std.
ID
C
B
A
Cone.
mg NH,-N/L
5.0
7.5
10.0
mg NO,-N/L
10.0
20.0
35.0
mL Stock Standard A
mL(1000
mgNH,-N/L)
5.0
7.5
10.0
mL(1000
mg NO3-N/L)
10.0
20.0
35.0
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Volume 3, Chapter 2
ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method
Std.
ID
E
D
SP
CK
Cone.
mg NH,-N/L
0.5
1.0
2.0
3.0
mg NO,-N/L
0.5
1.0
5.0
5.0
mL Stock Standard B
mL(100
mg NH3-N/L)
5.0
10.0
20.0
30.0
mL(100
mg NO,-N/L)
5.0
10.0
50.0
50.0
Std.
ID
H
G
F
CK
Cone.
mg NHj-N/L
0.02
0.05
0.10
0.30
mg NO,-N/L
0.02
0.05
0.10
0.30
mL Working Standard C
mL(10mg/NH,-N/L)
2.0
5.0
10.0
30.0
mL(10mgNO,N/L)
2.0
5.0
10.0
30.0
9.0 Setup for Both Channels
9.1 Use alpha and beta detectors for optical dilution: Place the 0.1 cm flowcell in the alpha detector
and the 1.0 cm flowcell in the beta detector for each channel.
9.2 Sample loops - ports 1 and 4.
a. Ammonia - 180 cm loop.
b. Nitrate - 59 cm ioop.
9.3 Connect the sample line to port 6 of the NH3-N channel.
9.4 Connect the port 5-6 tube from port 5 of the NH,-N channel to port 6 of the NO3-N channel.
9.5 Attach pump tubes according to flow diagram.
a. Sample line: Green, cut to 2 cm at each end.
b. Wash line: Green.
c. Nitroprusside: Orange.
d. Hypochlorite: Black.
e. Alkaline phenol: Orange.
f. EDTA buffer: Red.
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ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Automated Flow Injection Analysis Method Volumes, Chapter 2
g. NH, Carrier water: Blue.
h. NH4C1 buffer: Yellow-blue.
i. Sulfanilamide: White.
j. NO3 carrier water: Orange.
9.6 Degas all ammonia reagents, except phenol, with helium for 2 to 3 minutes just prior to attaching
lines to system.
10.0 Start-up
10.1 Turn on and check diagnostics.
10.2 Attach reagent lines.
10.3 Attach cadmium column.
10.3.1 Turn pump down to 05.
10.3.2 First remove line from column.
10.3.3 Remove line from * connection (buffer + sample inlet).
10.3.4 Attach * line to column.
10.3.5 Attach column line to *.
10.3.6 To remove column, reverse procedure.
10.3.7 Turn pump speed to 35.
10.4 Pour standards.
11.0 Procedure
11.1 Follow directions in General Operating Procedures.
11.2 Clean both channels with 10% HC1 during final shutdown.
12.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
3-160
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ESS Method 220.3: Ammonia Nitrogen and Nitrate + Nitrite Nitrogen,
Volume 3, Chapter 2 Automated Flow Injection Analysis Method
13.0 References
13.1 U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water and Wastes,
EPA-600/4-79-020, Method 350.1, (1979).
13.2 U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water and Wastes,
EPA-600/4-79-020, Method 353.2, (1979).
13.3 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of Water Resources Inv., Book 5, Ch. Al, (1979).
13.4 Lachat Instruments, Method 10-107-06-1 -Z, March 1990.
13.5 Lachat Instruments, Method 10-107-04-1 -Z, March 1990.
3-161
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ESS Method 230.1:
Total Phosphorus and Total Kjeldahl
Nitrogen, Semi-Automated Method
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 230.1:
Total Phosphorus and Total Kjeldahl Nitrogen,
Semi-Automated Method
1.0 Scope and Application
l.l This method covers the determination of total Kjeldahl nitrogen and total phosphorus in drinking,
surface and waste waters. The operating range is 0.1 to 10.0 mg N/L and 0.02 to 2.00 mg P/L.
1.2 The digestion converts nitrogen compounds such as amino acids, proteins and peptides to
ammonia, but may not convert all amines, nitro compounds, hydrazones, oximes, semicarbazones,
and some refractory tertiary amines.
2.0 Summary of Method
2.1 Organic nitrogen and phosphorus compounds are digested with a sulfuric acid solution containing
potassium sulfate and using mercuric sulfate as a catalyst:
H2SO4 + organic nitrogen tig (NH4)2SO4
K2SO4
H2SO4 + organic phosphorus Hg K3PO4
K2SO4
2.2 The digested solution is analyzed spectrophotometrically as ammonia and phosphate using an
automated system with an internal neutralization step.
2.2.1 In the TKN determination, the NH, is treated with sodium hypochlorite and sodium
phenolate to form indophenol blue. Sodium nitroprusside is used to intensify the color.
The intensity of the color is directly related to the concentration of TKN.
2.2.2 In the TP determination, the PO4 reacts with ammonium molybdate in the presence of
H,SO4 to form a phosphomolybdenum complex. Potassium antimonyl tartrate and
ascorbic acid are used to reduce the complex, forming a blue color which is proportional
to the TP concentration.
3.0 Sample Handling and Preservation
3.1 The samples are collected in 250 mL high density polyethylene containers.
3.2 Samples are preserved in the field with 2 mL of 12.5% H2SO4/250 mL (1 mL of cone. H2SO4/L,
pH<2) and stored at 4°C.
3.3 Samples such as sewage, paper mill wastes, etc., which contain settleable materials must be
homogenized before withdrawing an aliquot for analysis.
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ESS Method 230.1: Total Phosphorus and
Total Kjeldahl Nitrogen, Semi-Automated Method Volumes, Chapter 2
4.0 Interferences
4.1 A sodium citrate - sodium potassium tartrate complexing reagent is used in the TKN manifold to
minimize the interference caused by the precipitation of metal ions.
4.2 A sodium chloride reagent is used to prevent the reduction of mercuric ions in the TP manifold.
5.0 Apparatus
5.1 Technicon BD-40 Block Digestor (two units)
5.2 Technicon #114-0024-02 glass tubes
5.3 Technicon rack #114-0009-02
5.4 Teflon boiling chips
5.5 Sonicator Cell Disrupter (Heat Systems Ultrasonics, Inc., Plainview, NY)
5.6 Oxford 500 and 1000 uL pipet with disposable polypropylene tips
5.7 Vortex-genie mixer
5.8 Labindustries 10 mL capacity Repipet (2 units)
5.9 Culture tubes, 15 x 85 mm disposable glass
5.10 Technicon AutoAnalyzer II system consisting of:
5.10.1 Sampler IV with a 30/h (2:1) Cam
5.10.2 Proportioning Pump III with dilution manifold
5.10.3 Modified ammonia manifold
5.10.4 Modified orthophosphate manifold
5.10.5 Colorimeter equipped with 15 mm flowcells and 630 nm interference filters for TKN
5.10.6 Colorimeter equipped with 50 mm flowcells and 880 nm interference filters for TP
5.10.7 Printer/Plotter
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ESS Method 230.1: Total Phosphorus and
Volume 3, Chapter 2 Total Kjeldahl Nitrogen, Semi-Automated Method
6.0 Reagents
6.1 Digestion acid solution
6.1.1 Sulfuric acid, 6 N: Dilute 167 mL of concentrated H2SO4 to 1 L with Milli-Q water (Milli-
Q reagent grade water system, Millipore Corp.).
6.1.2 Mercuric oxide solution: Dissolve 2.0 g of HgO in 25 mL of 6 N H:SO4 (Section 6.1.1).
6.1.3 Potassium sulfate solution: Partially dissolve 134 g of K2SO4 in 500 mL of Milli-Q water.
6.1.4 Add 200 mL of concentrated H2SO4 to the K2SO4 solution (Section 6.1.3) and stir until
K,SO4 is dissolved.
6.1.5 Add HgO solution (Section 6.1.2) to the K2SO4 acid solution (Section 6.1.4), cool slightly,
dilute to 1 L and store above 20°C.
6.2 Digestion tube dilution water: Use Milli-Q water that is N and P free.
6.3 Sampler wash solution: Add 70 mL of cone. H2SO4 to 1500 mL of Milli-Q water and
dilute to 2 L.
6.4 Dilution loop solution: Add 50 mL of 20% w/v NaOH to 1500 mL of Milli-Q water and
dilute to 2 L.
6.5 TKN Reagents
6.5.1 Complexing reagent: Dissolve 33 g of sodium potassium tartrate (NaKC4H4O6«4H2O) and
24 g of sodium citrate (Na3C6H5O/2H2O) in 900 mL of Milli-Q water and dilute to 1 L.
Add0.25mLofBrij-35.
6.5.2 Alkaline phenol: Using a 1 L Erlenmeyer flask, dissolve 83 g of phenol in about 50 mL
Milli-Q water. Cautiously add with mixing 180 mL of 20% w/v NaOH. Cool, dilute to
1 L, and filter througn a glass fiber filter. 2 L can be made at one time.
6.5.3 Sodium hypochlorite solution: Dilute 200 mL of commercial bleach containing 5.25%
available chlorine (e.g. Clorox) to 1 L with Milli-Q water and filter through a 0.45 (am
membrane filter. Store at 4°C.
6.5.4 Sodium nitroprusside: Dissolve 0.5 g of (Na2Fe(CN)sNO-2H,O) in 900 mL of Milli-Q
water and dilute to 1 L. Reagent is light sensitive, store in dark containers.
6.6 Phosphorus Reagents
6.6.1 Diluent water solution: Dissolve 5 g of sodium chloride in 900 mL of Milli-Q water and
dilute to 1 L. Add 0.25 mL uf Levor IV
6.6.2 Stock Solution A, 4.9 N Sulfuric acid: Add 136 mL of cone. H,SO, to 800 mL of Milli-Q
water. Cool and dilute to 1 L.
3-167
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ESS Method 230.1: Total Phosphorus and
Total Kjeldahl Nitrogen, Semi-Automated Method Volume 3, Chapter 2
6.6.3 Stock Solution B, Ammonium molybdate: Dissolve 40 g of (NH4)6Mo7O24»4H2O in
900 mL of Milli-Q water and dilute to I L. Store at 4°C.
6.6.4 Stock Solution C, Ascorbic acid: Dissolve 9 g of ascorbic acid (C5H8O6) in 400 mL of
Milli-Q water and dilute to 500 mL. Store at 4°C. Keep well stoppered. Prepare fresh
monthly or as needed.
6.6.5 Stock Solution D, Antimony potassium tartrate: Dissolve 3.0 g of K(SbO)C4H4O6»l/2H:O
in 800 mL of Milli-Q water and dilute to I L. Store at 4°C.
6.6.6 Combined color reagent: Combine the following solutions in order, mixing after each
addition: 50 mL of Stock A, 15 mL of Stock B, 30 mL of Stock C and 5 mL of Stock D.
Prepare fresh daily.
6.7 Standard Solutions
6.7.1 Stock nitrogen standard: Dissolve 1.050 g of glutamic acid (dried at !05°C for I h) in
900 mL of Milli-Q water. Add 2 mL of cone. H2SO4 and dilute to I L. 1.0 mL =
0.lOOmgN(lOOmgN/L).
6.7.2 Stock phosphorus standard: Dissolve 0.4394 g of potassium phosphate monobasic
(KH2PO4) (dried at 105°C for I h) in 900 mL of Milli-Q water. Add 2 mL of cone. H2SO4
and dilute to 1 L. 1.0 mL = 0.100 mg P (100 mg P/L).
6.7.3 Stock nitrogen spike solution (Nicotinic acid, NA): Dissolve 0.8790 g of NA (dried at
105°C for 1 h) in 900 mL of Milli-Q water. Add 2 mL of cone. H:SO4 and dilute to 1 L.
1.0 mL = 0.1 mg N (100 mg N/L).
6.7.3.1 Working NA solution (5.0 mg N/L): Add 50 mL stock NA solution
(Section 6.7.3) to 900 mL Milli-Q water. Add 2 mL of cone. H,SO4 and dilute to
1 L.
6.7.4 Stock phosphorus spike solution (Adenosine 5'-monophosphate, AMP): Dissolve
0.2242 g AMP (dried at 105°C for I h) in 900 mL of Milli-Q water. Add 2 mL of cone.
H:SO4 and dilute to 1 L. 1.0 mL = 0.02 mg P (20 mg P/L).
6.7.4.1 Working AMP solution (1.0 mg P/L): Add 50. mL stock AMP solution
(Section 6.7.4) to 900 mL Milli-Q water. Add 2 mL of cone. H,SO4 and dilute to
1 L.
6.7.5 Working standards: Prepare the following standards by adding appropriate amounts of the
stock standards to 500 mL of Milli-Q water. Add 2 mL of cone. H:SO4 and dilute to L:
\ Cone. mL Standard Soln./L
mg P/L mg N/L P stock N stock
0.40
1.00
2.00
2.00
5.00
10.00
4.00
10.00
20.00
20.00
50.00
100.00
3-168
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ESS Method 230.1: Total Phosphorus and
Volume 3, Chapter 2 Total Kjeldahl Nitrogen, Semi-Automated Method
7.0 Procedure
7.1 All glassware must be rinsed with I: I HCl to prevent phosphorus contamination. No commercial
detergents may be used.
7.2 Put four to eight Teflon boiling chips in each tube.
7.3 Homogenize any non-uniform samples, such as sewage, paper mill wastes, farm wastes, etc. with
the Sonicator Cell Disrupter for about 30 seconds.
7.4 Transfer sample to the digestion tube using a large orifice pipet. Determine the sample volume
from the NH_,-N and Diss-P concentrations and the following guide. Do not use more than 20 mL.
Sample Volume TKN Range TP Range
(mL) (mg N/L) (mg P/L)
20 0.05 5 0.01 1
10 0.1 10 0.02 2
5 0.2 20 0.04- 4
2 0.5 50 0.1 10
1 1.0 -100 0.2 20
7.5 Quick Phosphorus Test: When the concentration of dissolved phosphorus is unknown, a quick test
should be performed to determine the volume needed for the total phosphorus analysis.
7.5.1 Reagents: (A) Ammonium molybdate reagent: Dissolve 25 g (NH4)(,Mo7O24'4H2O in
175 mL Milli-Q water. Cautiously add 280 mL concentrated H2SO4 to 400 mL Milli-Q
water. Cool, add the molybdate solution, and dilute to 1 L. (B) Stannous chloride
reagent: Dissolve 2.5 g of fresh SnCl2*H2O in 100 mL glycerol. Heat in a water bath and
stir with a glass rod until dissolved.
7.5.2 Procedure: Dilute a test volume to 25 mL with Milli-Q water. Add 1 mL of ammonium
molybdate reagent (A) and mix well with vortex mixer. Add 1 drop of the stannous
chloride reagent (B) and mix well. A pale blue color denotes that the test volume is a
suitable approximate volume of sample. A medium to dark blue color means a smaller
volume should be used. Another quick test, using a smaller volume, can be performed if
uncertain about the dilution needed.
7.6 Each set of 40 samples should include the following: two blanks (10 mL Milli-Q water), five
intercalibration standards, one spiked sample, and three duplicate samples.
3-169
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ESS Method 230.1: Total Phusphorus and
Total Kjeldahl Nitrogen, Semi-Automated Method Volumes, Chapter 2
7.6.1 A typical run pattern is as follows:
I. Standard, TKN = 10.0, TP = 2.0 as Primer
2. Standard, TKN = 10.0, TP = 2.0
3. Standard, TKN = 7.5, TP = 1.5
4. Standard, TKN = 5.0, TP = 1.0
5. Standard, TKN = 5.0, TP = 1.0
6. Standard, TKN = 2.0, TP = 0.5
7. Blank
8. Blank
9-40. Samples, spiked sample, duplicate samples, NA and AMP, randomly distributed.
7.7 Add 2 mL of digestion acid solution (Section 6.1) to each tube using the repipet and mix
thoroughly with vortex mixer.
7.8 Transfer the tubes to the rack provided with the Block Digester.
7.9 Place the rack of 40 tubes in the first Block Digester, preheated to 200°C, for about 60 minutes or
until all water has evaporated.
7.10 When evaporation is complete, transfer the rack of tubes to the second Block Digestor, preheated
to 380°C, and time the digestion for 75 minutes. Remove the tubes from the digestor and allow to
cool for 10-15 minutes.
7.11 Using a second Repipet dispenser, add 10.0 mL of Milli-Q water to each tube and mix well using
the vortex mixer. Transfer the solution to a clean 15 x 85 mm test tube, and cover with Parafilm
(American Can Co., Greenwich, CT). If the sample contains clay-like particulates, allow to settle
overnight or centrifuge until clear. If the sample contains dark material, the sample must be
redigested at a greater dilution.
7.12 Place the tubes in the Sampler IV tray.
7.13 Set up the manifolds. Allow the colorimeters, and printer to warm up for one-half hour.
7.14 Load Sampler according to the CFDA Tray Protocol.
7.15 Analyze according to procedures described in the LIMS-CFDA Methods manual and General
AutoAnalyzer Procedures.
8.0 Calculation
8.1 The total phosphorus and total Kjeldahl nitrogen concentrations are obtained directly from the
LIMS plotter.
8.2 If a sample is outside the optimal operating range, select an appropriate sample volume
(Section 7.4) and repeat the analysis.
3-170
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ESS Method 230.1: Total Phosphorus and
Volume 3, Chapter 2 Total Kjeldahl Nitrogen, Semi-Automated Method
9.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
10.0 References
10.1 Jirka, A.M., Carter, M.J., May, D., and Fuller, F.D., "Ultramicro Semiautomated Method for
Simultaneous Determination of Total Phosphorus and Total Kjeldahl Nitrogen in Waste waters",
Environ. Science and Technology. 10:1038-1044, (1976).
10.2 Bowman, G.T and Delfino, J.D., "Determination of Total Kjeldahl Nitrogen and Total Phosphorus
in Surface Water and Wastewater", JWPCF. 54,1324 (1982).
3-171
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ESS Method 310.1:
Ortho-Phosphorus, Dissolved
Automated, Ascorbic Acid
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 310.1:
Ortho-Phosphorus, Dissolved
Automated, Ascorbic Acid
1.0 Scope and Application
1.1 This method may be used to determine concentrations of orthophosphate in most waters and
wastewater in the range from 0.002-0.200 mg P/L. The concentration range may be extended to
0.2-2.00 mg P/L by utilizing a dilution loop.
1.2 Approximately 30 samples per hour can be analyzed.
2.0 Summary of Method
Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute
solutions of orthophosphate-phosphorus to form an antimony-phospho-molybdate complex. This
complex is reduced to an intensely blue-colored complex by ascorbic acid. The color is
proportional to the phosphorus concentration.
3.0 Sample Handling and Preservation
Samples must be filtered through a 0.45 pm filter, cooled to 4°C and analyzed as soon as possible.
4.0 Interferences
4.1 Barium, lead, and silver interfere by forming a precipitate.
4.2 The interference from silica, which forms a pale-blue complex is small and can usually be
considered negligible.
4.3 Arsenate is determined similarly to phosphorus and should be considered when present in
concentrations higher than phosphorus.
5.0 Apparatus
Technicon AutoAnalyzer II system consisting of:
5.1 Sampler IV with a 30/h (2:1) Cam
5.2 Analytical manifold (orthophosphate in seawater) with internal heating bath at 37.5°C and
dilution loop
5.3 Proportioning pump IEI
5.4 Colorimeter equipped with 50 mm flowcells and 880 nm interference filters
5.5 Printer/Plotter
3-175
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ESS Method 310.1: Ortho-Phosphorus, Dissolved
Automated, Ascorbic Acid Volume 3, Chapter 2
6.0 Reagents
6.1 Stock Solution A; Sulfuric acid solution, 4.9 N: Add 136 mL concentrated H2SO4 to 800 mL
Milli-Q water. Cool and dilute to 1 L with Milli-Q water.
6.2 Stock Solution B; Ammonium molybdate solution: Dissolve 40 g of ((NH4)6 Mo7O24»4H2O) in
900 mL Milli-Q water and dilute to 1 L. Store at 4°C.
6.3 Stock Solution C; Ascorbic acid: Dissolve 9 g of ascorbic acid (C6H8O6) in 400 mL Milli-Q water
and dilute to 500 mL. Store at 4°C. Keep well stoppered. Prepare fresh monthly or as needed.
6.4 Stock solution D; Antimony potassium tartrate: Dissolve 3.0 g of (K(SbO)C4H4O6»'/2H:O) in
800 mL Milli-Q water and dilute to 1 L. Store at 4°C.
6.5 Combined color reagent: Combine the following solutions in order, mixing after each addition:
(Prepare fresh daily)
Stock A, 6.1 (4.9 N H:SO4) 50 mL
Stock B, 6.2 (Ammonium molybdate solution) 15 mL
Stock C, 6.3 (Ascorbic acid solution) 30 mL
Stock D, 6.4 (Antimony-tartrate solution) 5 mL
6.6 Water diluent solution: Add 4.0 g sodium lauryl sulfate and 5 g NaCl per L of Milli-Q water.
6.7 Stock phosphorus standard: Dissolve 0.4394 g of Potassium phosphate monobasic (KH:PO4)
(dried at 105°C for one hour) in 900 mL Milli-Q water. Add 2 mL of concentrated H2SO4 and
dilute to 1 L. 1.0 mL = 0.100 mg P (100 mg P/L).
6.8 Standard phosphorus solution 1: Dilute 100.0 mL of stock solution (6.7) to 500 mL with Milli-Q
water. 1.0 mL = 0.020 mg P (20 mg P/L).
6.9 Standard phosphorus solution 2: Dilute 10.0 mL of stock solution (6.7) to 1 L. 1.0 mL =
0.001 mgP(1.0mgP/L).
6.10 Working standard solutions:
6.10.1 Low Range (0.002-0.200 mg P/L): Prepare the following standards by diluting suitable
volumes of standard solution 2 (6.9) to appropriate volumes with Milli-Q water:
mgP/L
0.005
0.050
0.100
0.150
0.200
mL of standard
solution 2
1 .0/200 mL
5.0/1 00 mL
50/500 mL
15/100mL
40/200 mL
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ESS Method 310.1: Ortho-Phosphorus, Dissolved
Volume 3, Chapter 2 Automated, Ascorbic Acid
6.10.2 High Range (0.02-2.00 mg P/L): Prepare the following standards by diluting suitable
volumes of standard solution 1 (6.8) to 200.0 mL with Milli-Q water:
mL of standard
mgP/L solution 1/200.0 mL
0.50 5.0
1.00 10.0
1.50 15.0
2.00 20.0
7.0 Procedure
7.1 Set up the manifold as shown in Figure 1. For the high concentration range, use the dilution
manifold (Figure 1.).
7.2 Allow the colorimeter, and printer to warm up for 30 minutes. Obtain a stable baseline with all
reagents, feeding Milli-Q water through the sample line.
7.3 Load the autosampler according to the CFDA Tray Protocol.
7.4 Analyze according to procedures in the LIMS-CFDA Methods Manual and General AutoAnalyzer
Procedures.
8.0 Calculations
The phosphorus concentration is obtained directly from the LIMS plotter.
9.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
10.0 References
10.1 Methods for Chemical Analysis of Water and Wastes, U.S. Environmental Protection Agency,
EPA 600/4-79-020, p 365.1, (1979).
10.2 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of WaterResources Inv., Book #5, Ch.Al, p 514, (1985).
10.3 Ortho Phosphate in Water and Seawater, Industrial Method No. 155-71W, Technicon Instruments
Corporation, Tarrytown, NY (1973).
3-177
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ESS Method 310.1: Ortho-Phosphorus, Dissolved
Automated, Ascorbic Acid
Volume 3, Chapter 2
Figure 1. Manifold Set Up
ORTHO PHOSPHATE IN WATER AND SEAWATER
RANGE: 0 - 0.2 eg P/l
MANIFOLD NO. 116-0221-01
To Sampler IV
157-8273-03 Wash Receptacle
37.5 °C 5 Turns 5 Turns
7.7 ml 170-0103 170-0103
OQQO "0 QQQO
o
faste
CRN/CRN (2.00) WATER
BLK/BLK (0.32) AIR
BLK/BLK (0.32) WATER
ORN/ORN (0.42) SAMPLE " f
ORN/WHT (0.23) REAGENT
WHT/WHT (0.60) FROM F/C
COLORIMETER
330 nm
SO mm F/C x 1.5 mm 10
193-B023-01
To F/C
Pump Tuba
••POLYETHYLENE 0.034 10
SAMPLER IV
30/HR
2:1
NOTE: FIGURES IN PARENTHESES SIGNIFY
FLOW RATES IN ML/MIN.
3-178
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ESS Method 310.2:
Phosphorus, Total, Low Level
(Persulfate Digestion)
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 310.2:
Phosphorus, Total, Low Level
(Persulfate Digestion)
1.0 Scope and Application
This method is applicable to the determination of total phosphorus in surface waters in the range of
0.002 to 0.200 mg P/L.
2.0 Summary of Method
Samples are digested in an autoclave for 30 minutes at 121 °C with ammonium persulfate and
sulfuric acid to convert all phosphorus to orthophosphate. The orthophosphate is then analyzed
with the Technicon AAII using the ascorbic acid procedure (Method 310.1).
3.0 Sample Handling and Preservation
Samples are preserved in the field by the addition of 2 mL of 12.5% H2SO4 per 250 mL sample.
They are refrigerated at 4°C until analysis is performed.
4.0 Apparatus
4.1 Digestion tubes, 20 x 150 mm, disposable borosilicate glass.
4.2 Autoclave.
4.3 Technicon AutoAnalyzer II system consisting of:
4.3.1 Sampler IV with a 30/h (2:1) Cam
4.3.2 Analytical manifold (Orthophosphate in Seawater) with internal heating bath at 37.5°C
4.3.3 Proportioning pump III
—. j-
4.3.4 Colorimeter equipped with 50 mm flow cells and 880 nm interference filters
4.3.5 Recorder/Printer
4.4 8 mL and 4 mL volumetric pipettes.
4.5 Culture tubes: 15 x 85 mm disposable glass.
4.6 Caps, Polypropylene, for disposable culture tubes (4.5).
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ESS Method 310.2: Phosphorus, Total, Low Level
(Persulfate Digestion) Volume 3, Chapter 2
5.0 Reagents
5.1 Stock acid solution, 6 N Sulfuric Acid: Dilute 166 mL of concentrated H2SO4 to 1 L with Milli-Q
water. This is equivalent to the procedure used in Methods for Chemical Analysis of Water and
Wastes, p. 365.3 (Section 10.0), if 1 mL/L sulfuric acid is used to preserve the samples.
5.2 Stock persulfate solution: Dissolve 32 g ammonium persulfate ((NH4)2S2O8) in Milli-Q water and
dilute to 100 mL (Stable two weeks at 4°C).
5.3 Working digestion acid solution: Combine equal volumes of stock acid (Section 5.1) and stock
persulfate (Section 5.2) solutions. Prepare daily.
5.4 Color reagent:
5.4.1 Stock Solution A; Sulfuric acid solution, 4.9 N: Add 136 mL concentrated H:SO4 to
800 mL Milli-Q water. Cool and dilute to 1 L with Milli-Q water.
5.4.2 Stock Solution B; Ammonium molybdate solution: Dissolve 40 g of (NH4)6Mo7O24*4H20
in 900 mL Milli-Q water and dilute to 1 L. Store at 4°C.
5.4.3 Stock Solution C; Ascorbic acid: Dissolve 9 g of ascorbic acid (C6H8O6) in 400 mL Milli-
Q water and dilute to 500 mL. Store at 4°C. Keep well stoppered. Prepare fresh monthly
or as needed.
5.4.4 Stock Solution D; Antimony potassium tartrate: Dissolve 3.0 g of K(SbO)C4H4O6'!/2H2O
in 800 mL Milli-Q water and dilute to 1 L. Store at 4°C.
5.4.5 Combined color reagent: Combine the following solutions in order, mixing after each
addition: (Prepare fresh daily)
Stock A, 5.4.1 (4.9 N H2SO4) 50 mL
Stock B, 5.4.2 (Ammonium molybdate solution) 15 mL
Stock C, 5.4.3 (Ascorbic acid solution) 30 mL
Stock D, 5.4.4 (Antimony-tartrate solution) 5 mL
5.5 Sampler wash solution: Dilute 6 mL of concentrated sulfuric acid to 1 L with Milli-Q water.
5.6 Diluent water solution: Add 4.0 g sodium lauryl sulfate and 5 g NaCl per L of Milli-Q water.
5.7 Stock phosphorus standard: Dissolve 0.4394 g of potassium phosphate monobasic (KFLPO4)
(dried at 105°C for 1 h) in 900 mL Milli-Q water. Add 2.0 mL of concentrated H,SO4 and dilute
tolL. 1.0 mL = 0.100 mgP (100 mgP/L).
5.8 Standard phosphorus solution: Dilute 10.0 mL of stock phosphorus standard (5.7) to 1 L.
1.0 mL = 0.001 mg P (1.0 mg P/L).
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ESS Method 310.2: Phosphorus, Total, Low Level
Volume 3, Chapter 2 (Persulfate Digestion)
5.9 Working standard solutions: Prepare the following standards by diluting suitable volumes of
standard solution (5.8) to 200.0 mL with Milli-Q water (Add 20 mL of l% H,SO4 before diluting
to 200.0 mL):
mL of standard
mg P/L solution (5.8)7200.0 mL
0.005 l.O
0.050 10.0
0.100 20.0
0.150 30.0
0.200 40.0
5.10 Stock Adenosine 5'-Monophosphate (AMP) solution: Dissolve 0.2242 g of AMP (dried at 105 JC
for 1 h) in 900 mL of Milli-Q water. Add 2 mL of cone. H2SO4 and dilute to 1 L. 1.0 mL = 0.02
mg P (20 mg P/L).
5.11 Working AMP solution: Dilute 5 mL of stock AMP (5.10) to 1 L. (0.100 mg P/L).
6.0 Procedure
6.1 Load test tube racks with disposable digestion tubes and add samples, standards, duplicates, spikes
and blanks according to CFDA Tray Protocol.
6.1.1 Prepare a standard curve by pipetting 8 mL of standards and blanks (Milli-Q water) using
a Class A volumetric pipet.
6.1.2 Transfer 8 mL of each sample to a digestion tube using an 8 mL cut-off (large bore)
volumetric pipet.
6.1.3 A 0.100 mg P/L standard with a following Milli-Q water blank should be inserted after
every 20 samples.
6.1.4 Prepare a minimum of 10% of the samples in duplicate, and spike 5% or at least two
samples per digestion. Spikes are prepared by mixing 4 mL of a sample with 4 mL of
AMP solution (5.11) or 4 mL of 0.050 mg P/L working standard solution.
6.2 All digestion tubes should have 8 mL of liquid before the addition of digestion acid. Add 0.5 mL
of working digestion acid solution (5.3) to each tube, mix and cover with Caps.
6.3 Autoclave the digestion tubes for 30 minutes at 121 °C, 15-20 psi (specify manual autoclave).
6.4 Remove the tubes from the autoclave, cool, mix, transfer to the 15 x 85 mm disposable glass
culture tubes and cover with parafilm.
6.5 Allow any particulate matter to settle overnight.
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ESS Method 310.2: Phosphorus, Total, Low Level
(Persulfate Digestion)
Volume 3, Chapter 2
6.6 Sample analysis
6.6.1 Set up manifold as shown in Figure 1.
ORTHO PHOSPHATE IN WATER AND SEAV/ATER
RANGE: 0 - 0.2 Kg P/l
MANIFOLD NO. 116-0221-01
* c-, =,-,-,<> M T° Sampler IV ^ s-\ CRN/CRN (2.00) WATER
157-8273-03 Wash Receptacle •< (J -—.—i .
37.S°C 5 Turns 5 Turns BLK/BLK (0.32) AIR
7.7 ml 170-0103 170-0103 |—\_/ •
OQQQ A1° QQQQ s-\ BLK/BLK (0.32) WATER
/aste
116-0433-01
O
o
o
ORN/ORN (0.42) SAMPLE " f
Wast*
ORN/WHT (0.23) REAGENT
WHT/V/HT (0.60) FROM F/C
To F/C
SAMPLER IV
COLORIMETER Pump Tuba "POLYETHYLENE 0.034 ID 3°/HR
880 nm
SO mm F/C x 1.S mm 10 NOTE: FIGURES IN PARENTHESES SIGNIFY
199-B023-01 FLOW RATES IN ML/MIN.
Figure 1. Manifold Set Up
6.6.2 Allow the colorimeter, recorder and printer to warm up for 30 minutes.
6.6.3 Obtain a stable baseline with all reagents, feeding Milli-Q water through die sample line.
6.6.4 Place culture tubes in the sampler and remove the parafilm.
6.6.5 Analyze according to procedures described in General AutoAnalyzer Procedures and
LIMS-CFDA Methods manual.
7.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
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ESS Method 310.2 : Phosphorus, Total, Low Level
Volume 3, Chapter 2 (Persulfate Digestion)
8.0 References
8.1 Central Regional Laboratory Procedure for the Analysis of Total Phosphorus, U.S. Environmental
Protection Agency, Region V, 4 p., (1978).
8.2 Methods for Chemical Analysis of Water and Wastes, U.S. Environmental Protection Agency,
EPA 600/4-79-020, p. 365.1, (1983).
8.3 Ortho Phosphate in Water and Seawater, Industrial Method 155-71W, Technicon Instruments
Corporation, Tarrytown, NY (1973).
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ESS Method 340.2:
Total Suspended Solids, Mass Balance
(Dried at 103-105°C)
Volatile Suspended Solids
(Ignited at 550°C)
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised June 1993
-------
ESS Method 340.2:
Total Suspended Solids, Mass Balance (Dried at 103-105°C)
Volatile Suspended Solids (Ignited at 550°C)
1.0 Scope and Application
l.l This method is applicable to drinking, surface, and saline waters, domestic and industrial wastes.
1.2 The practical range of the determination is 2 mg/L to 20,000 mg/L.
1.3 This method was used in the Wisconsin Green Bay Mass Balance Study, and was intended for use
in sediment transport/loading work.
2.0 Summary of Method
A well-mixed sample is filtered through a standard GF/F glass fiber filter, and the residue retained
on the filter is dried to constant weight at 103-105°C.
3.0 Definitions
Total Suspended Solids is defined as those solids which are retained by a glass fiber filter and
dried to constant weight at 103-105°C.
4.0 Sample Handling and Preservation
4.1 Non-representative particulates such as leaves, sticks, fish, and lumps of feca! mater should be
excluded from the sample if it is determined that their inclusion is not desired in the final result.
4.2 Preservation of the sample is not practical; analysis should begin as soon as possible.
Refrigeration or icing to 4°C, to minimize microbiological decomposition of solids, is required.
5.0 Interferences
5.1 " Filtration apparatus, filter material, pre-washing, post-washing, and drying temperature are
specifed because these variables have been shown to affect the results.
5.2 Samples high in Total Dissolved Solids, such as saline waters, brines and some wastes, may be
subject to a positive interference. Care must be taken in selecting the filtering apparatus so that
washing of the filter and any dissolved solids in the filter minimizes the potential interference.
6.0 Apparatus
6.1 Glass microfiber filters discs, 5.5 cm, without organic binder, Whatman type GF/F (0.7
6.2 Disposable aluminum dishes
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ESS Method 340.2: Total Suspended
Solids, Mass Balance, Volatile Suspended Solids Volumes, Chapter 2
6.3 Tweezers
6.4 Suction flask, 1000 mL
6.5 47 mm glass microanalysis filter holder (funnel, clamp, and base)
6.6 Drying oven for operation at 103-105°C
6.7 Muffle furnace for operation at 550 ± 50°C
6.8 Desiccator
6.9 Analytical balance, capable of weighing to 0.1 mg, an RS232C interface and a personal computer
6.10 Milli-Q reagent grade water (ASTM Type I water), Millipore Corp, Bedford, MA
7.0 Procedure for Total Suspended Solids
7.1 Preparation of the glass fiber filter disk: Insert the filter disk onto the base and clamp on funnel.
While vacuum is applied, wash the disk with three successive 20 mL volumes of Milli-Q water.
Remove all traces of water by continuing to apply vacuum after water has passed through.
Remove funnel from base and place filter in the aluminum dish and ignite in the muffle furnace at
550°C ± 50°C for 30 minutes. Rewash the filter with an additional three successive 20 mL
volumes of Milli-Q water, and dry in an oven at 103-105°C for one hour. When needed, remove
dish from the oven, desiccate, and weigh.
7.2 Select a sample volume (max. of 200 mL) that will yield no more than 200 mg of total suspended
solids.
7.3 Place the filter on the base and clamp on funnel and apply vacuum. Wet the filter with a small
volume of Milli-Q water to seal the filter against the base.
7.4 Shake the sample vigorously and quantitatively transfer the sample to the filter using a large
orifice, volumetric pipet. Remove all traces of water by continuing to apply vacuum after sample
has passed through.
7.5 Rinse the pipet and funnel onto the filter with small volume of Milli-Q water. Remove all traces
of water by continuing to apply vacuum after water has passed through.
7.6 Carefully remove the and filter from the base. Dry at least one hour at 103-105°C. Cool in a
desiccator and weigh.
7.7 Retain the sample in the dish for subsequent ignition at 550°C if volatile suspended solids is
desired.
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ESS Method 340.2: Total Suspended
Volume 3, Chapter 2 Solids, Mass Balance, Volatile Suspended Solids
8.0 Calculation of Total Suspended Solids
Calculate Total Suspended Solids as follows:
Total Suspended Solids, mg/L = (A-B)x 1,000/C
Where: A = weight of filter and dish + residue in mg
B - weight of filter and dish in mg
C = volume of sample filtered in mL
9.0 Procedure for Volatile Suspended Solids
9.1 After determining the final weight in the total suspended solids analysis (7.6), place the filter and
dish in the muffle furnace and ignite at 550°C ± 50°C for 30 minutes.
9.2 Allow to partially air cool, desiccate and weigh.
10.0 Calculation of Volatile Suspended Solids
Volatile Suspended Solids, mg/L = (A -B) x 1,000/C
Where: A — weight of residue + filter and crucible in mg from Total Suspended Solids test (7.7)
B = weight of residue + filter and crucible in mg after ignition (9.2)
C = volume of sample filtered in mL
11.0 Precision and Accuracy
Precision data are available in the Inorganic Chemistry Quality Assurance Manual.
12.0 References
12.1 Methods for the Chemical Analysis of Water and Waster, U.S. Environmental Protection Agency,
EPA 600/4-79-020, p. 160.2, (1979).
12.2 Standard Methods for the Examination of Water and Wastewater, 16th Edition, p. 96,
Method 209C. (1985).
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Outline of Standard Protocols
for DOC Analyses
Martin Shafer
Water Chemistry Program
University of Wisconsin-Madison
February 1995
Revision 2
-------
Outline of Standard Protocols
for DOC Analyses
Martin Shafer
Water Chemistry Program
University of Wisconsin-Madison
February 1995
Revision 2
-------
Outline of Standard Protocols for DOC Analyses
1.0 Introduction
This document outlines a procedure for the analysis of organic carbon in surface and ground
waters.
Organic carbon is determined on a Shimadzu TOC-5000 analyzer with ASI-5000 autosampler and
Balston 78-30 high purity TOC gas generator. Detailed maintenance and trouble-shooting
information for this instrument can be found in the TOC-5000 Instruction Manual (1991) P/N 638-
90216.
Organic carbon is measured (after removal of inorganic carbon by acidification and purging) by
conversion to CO, (high temperature (680°C) catalytic oxidation) and quantification by a non-
dispersive infrared detector.
The method as described is applicable to organic carbon levels in the range of 0.2 - 50 mg L ', and
inorganic carbon levels less than 1000 mg L '
Field filtered samples in borosilicate glass vials with Teflon-faced septa are placed in a cooler, on
ice, and shipped to the lab by overnight FEDEX delivery. No preservatives are added in the field.
Samples are frozen after arrival at the lab.
2.0 Preparation
2.1 Use glassware (as opposed to plasticware) wherever possible.
2.2 All new and used glassware must be prepared by first being rinsed with MQ 3-4x, dried, wrapped
in foil (shinny side out), and ashed at 475°C for at least 8 hours. Cool to room temp before use.
2.3 Stock standard solution of Potassium Biphthalate (KHP) is prepared in a 250 mL ashed
volumetric flask. This solution must be stored in the cold room and made fresh after 30 days.
To prepare, dissolve 0.3321 g KHP in MQ (approximately 200 mL in volumetric flask), dilute to
line, and mix. Concentration = 625.1 mg L ' carbon.
2.4 2N HC1 is prepared in a 1000 mL ashed volumetric flask. Add 166 mL of trace metal grade HCL
to approximately 600 mL of MQ in the volumetric flask. Mix, dilute to line, and mix again.
2.5 Ash pieces of foil to cap autosampler tubes with.
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Outline of Standard Protocols for DOC Analyses
Volumes, Chapter2
3.0 Setup
3.1 Blanks and diluted standards for the calibration curve are prepared in ashed 250 mL ground glass
stoppered BOD bottles. Tare the bottle, weigh in the requisite amount of fresh MQ (you may use
a plastic wash bottle), then add the proper volume of stock standard with an eppendorf pipet. [do
not take directly from stock flask - pour an aliquot into an ashed glass beaker]. Mix well. Label
diluted standards. The following standards must be prepared:
mgL1
0.0
3.125
6.25
12.5
mL (g) MQ
100.00
99.50
99.00
98.00
mL Stock
0.000
0.500
1.000
2.000
3.2 All samples and standards are run in ashed, glass autosampler tubes. Samples in 9 mL tubes, and
standards in 40 mL tubes. The large tubes fit in the inner ring of the autosampler (S1-S8), and
samples in outer rings (1-72). Add standards to tubes after you have completed setting up the
samples.
3.3 Samples are normally collected in 20 mL glass vials with Teflon lined caps, and then kept frozen
until just before analysis. You can thaw samples in one of two ways: (a) the day prior to scheduled
analysis, pull from the freezer the exact number of samples you will be running, place in a cold
room and let thaw overnight, (b) on the day of analysis, retrieve the samples from the freezer, place
in a 40 °C oven, and warm until all traces of ice disappear.
3.4 Line up the samples, and group into sets of eight. Fill out the Sample Analysis Form with the
complete names of all samples to be run. Notice that groups of eight tubes are run bracketed
before and after with a sample of MQ, and then the instrument is recalibrated. Run the replicate
pairs of each sample back to back (4x2 = 8), and then duplicate 10% of samples at end of run
sequence.
3.5 Set up a test tube rack with all the required tubes. Lay a small plastic bag over the tubes while
filling to prevent contamination. Mix the sample vial very throughlj (It is extremely important to
transfer a representative sample), and carefully pour sample into the autosampler tube until the
level is even with label on the tube (approx. 7 mL). You may fill MQ tubes with a wash bottle,
either all together, or as you proceed in sequence.
3.6 When all tubes have been filled they must be acidified with 100 /nL of 2N HC1 to remove
inorganic carbon. Pour an aliquot of acid into an ashed glass beaker, and add acid to each of the
filled tubes in sequence. DO NOT SKIP ANY.
3.7 When all tubes have been acidified, they must be capped with a small piece of ashed foil.
Tear or cut up approximately 1.5 x 1.5 cm sections of foil and tightly cap top of tube. Do not
extend foil beyond tube ridge or tube may not fit properly in autosampler rack.
3-196
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Volume 3, Chapter 2
Outline of Standard Protocols for DOC Analyses
3.8 After all tubes are capped, they must be thoroughly mixed. Place finger on top of foil cap and
gently invert 3-4 times to homogenize.
3.9 Set tubes in numerical sequence in autosampler rack. Fill blank and standard tubes and place in
autosampler rack. Replace autosampler lid, making sure that all three legs are securely settled in
place.
3.10 Run two MQ blanks as an extra group, at the end of the run, so that:
a. you can check the calibration slope at the end of the last sample batch
b. you provide an extra rinse of system lines
3.11 Do not leave the instrument unattended until you have:
a. verified that the autosampler is sampling properly
b. verified that the blanks are not unusually high (>1000-1500)
c. verified that standards give an acceptable response
3.125 mg L-l : 7,000 - 9,000 (blank corrected)
6.25 mg L-l : 14,500 - 18,000 (blank corrected)
12.5 mg L-1 : 29,000 - 36,000 (blank corrected)
3.12 Re-freeze remaining sample in original sample vial.
4.0 Instrument Variables
4.1
General Conditions
TC Catalyst
Syringe Size
Number of Washes
Unit of Concentration
Auto Range and Injection
Auto Regen. of 1C
Auto Printout
Buzzer
TC Furnace
4.2 ASI Sample Measurement Conditions
Type
Calibration Curve
Range
Injection Volume
Washes
Number of Injections
Max Injections
Standard Deviation
CV
Sparge Time (SP)
Shift to Origin
High Sensitivity (2)
250 MUD
4
mg/L (3)
on(l)
off (2)
data and peak plot (2)
used (1)
on(l)
NPOC
Cl =8
xl
50 ^L
4
4
5
200
29c
4 min
on(l)
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Outline of Standard Protocols for DOC Analyses Volume 3, Chapter 2
4.3 ASI Conditions
Rinse No Rinse (2)
# of Needle Washes 0
Flow Line Washes 3
Calibrate Before Each Sample Group (2)
Print Info Cal and Data (3)
Auto Addition of Acid Off (2)
Acid Volume 0
Rinse After Addition No Rinse (2)
Finish/Running No Change (3)
Key Lock No (2)
5.0 Pre-Run Check-Out
5.1 Verify that TC furnace is turned on. From General Condition Menu choose TC Furnace on.
5.2 Verify that TC furnace is at proper temperature (680 °C) and that Baseline Position, Fluctuation,
and Noise read OK. From Main Menu choose Monitor to get data display.
5.3 Verify from flowmeter that carrier gas flow is between 140 and 160. Adjust needle valve if out of
range.
5.4 Verify that range is set on "1". If not, go to Maintenance Menu and choose Range Set.
5.5 Regenerate the column 3x with 0.02N HCL before each run. From Maintenance Menu
choose TC Regeneration. Place acid in autosampler position S1.
5.6 Fill humidifier water container to level of etched line. Open front panel, container is on right side
of instrument. Fill using a wash bottle after unscrewing black vent cap. Ensure that Teflon tape is
in good condition and, if not, re-tape.
5.7 Fill Inorganic Carbon (1C) acid reservoir with 25% reagent grade phosphoric acid to at least 1/2
full. Open front panel, container is on left side of instrument. Take care to not over tighten th^
cap or you run the risk of splitting the hard plastic black ring.
5.8 Fill the autosampler needle rinse port with fresh MQ. From Autosampler Menu choose ASI Pause,
and then select Rinse Maintenance.
5.9 Verify that the Inorganic Carbon (1C) reaction chamber is draining properly. It should be 1/3 to 1/2.
full and bubbling from passage of carrier gas purge.
5.10 Verify that there is enough printer paper remaining to complete vour run. If not, install a new roll.
5.11 Verify that the autosampler lid is on properly.
5.12 Do not bump or move the autosampler arm manually.
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Volume 3, Chapter 2 Outline of Standard Protocols for DOC Analyses
6.0 Post-Analysis Procedures
6.1 Dump sample from autosampler vial and rinse vial 4x with MQ water. Place vial in 10% HC1 acid
bath for 24 hours. Remove vials, rinse 4x with MQ, dry, wrap in foil, and ash.
6.2 Data printouts are recovered from the instrument and placed in designated storage drawer.
7.0 Data Reduction
7.1 The mean peak area of the four analyses is entered into an electronic copy (EXCEL spreadsheet)
of the sample analysis form.
7.2 Calibration slopes are generated from linear regression of the blank corrected standard data.
Data is summarized in the DOC Standard Analysis Form.
7.3 A unique slope for each sample is generated by linearly interpolating between calibrations.
7.4 Sample data is not blank corrected.
7.5 The ratio of sample peak area and corresponding slope is used to generate a concentration.
7.6 Concentration data is copied to site specific spreadsheets.
7.7 Hard copies of sample analysis forms are copied and filed in DOC batch log book. Electronic
copies of sample analysis forms are backed-up on floppies. Original instrument printouts are filed
in archive box.
8.0 QA/QC
8.1 MQ blanks are run before and after each group of 8 samples (20%). Control limits for check
blanks are 0.5 mg L ' , or 0.2 mg L"' greater than calibration blank. If more than 50% of the check
blanks in the analysis batch exceed this limit, then the sample values will be estimated.
8.2 The instrument is re-calibrated after every 10 samples. Slopes must exceed 2300. If response falls
below this value, then samples influenced by that slope are estimated. Correlation coefficients
must be better than 0.99. If linearity falls below this value, then samples influenced by that slope
are estimated.
8.3 Four replicate analyses are run on each sample. If RSD is >2.0%, then one additional analysis is
performed on that sample.
8.4 10% of samples are replicated at the end of each batch. No control limits have been set.
8.5 Over-range samples are automatically re-pipetted with a smaller volume. If this volume is less
than 10 /J.L, then the data is considered unacceptable and the sample must be re-run.
8.6 Field blanks are run periodically to detect contamination problems. If filter blanks exceed feed
water by more than 1.0 mg L '. then steps are taken to isolate and control the source of
contamination.
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Outline of Standard Protocols for DOC Analyses Volume 3, Chapter 2
8.7 Duplicate filtrations are performed at every site which generates duplicate DOC samples for each
site occupation. We are using an RSD of 15% as an action level to investigate both analytical and
field problems.
8.8 Data will be reported to EPA with three (3) significant digits.
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Outline of Standard Protocols for
Particulate Organic Carbon (POC) Analyses
Rich Baldino
Water Chemistry Program
University of Wisconsin-Madison
May 2,1995
Revision 1
-------
Outline of Standard Protocols for
Particulate Organic Carbon (POC) Analyses
1.0 Introduction
This method is for the determination of organic carbon on filter borne particles in the presence of
inorganic carbon. The method detection limit for this procedure is 5 yug of organic carbon
remaining on a GF/F filter. The maximum amount of carbon measurable is approximately 5 mg of
carbon.
2.0 Selection and Pre-treatment of POC samples
2.1 Selection of Samples
Particulate organic carbon (POC) analysis begins with the selection of samples for analysis. To
select POC samples, consult the complete list of POC samples compiled from the "TMLOGIN"
database (DOC Login table) using the report "POC Analysis" This report contains all the POC
results up to the date of printing. The next ten samples in order of collection date, which have not
yet been analyzed, are selected as the next analysis batch. These ten samples are pulled from the
POC freezer for analysis.
2.2 Sample Treatment
2.2.1 Sample treatment begins by placing twelve ashed aluminum planchets in the hood on a
plastic tray. Two planchets are designated as blanks and an ashed GF/F glass fiber filter is
placed in each. The blank filters are then treated with 200 //L sulfurous acid. The first
sample is pulled and the planchet tab is labeled, using a black Sharpie, with the last three
characters of the POC sample number (such as "AO3" for TMENOAO3) which assures a
unique sample number for each POC filter. The GF/F filter is folded in half and 200 ^L
of sulfurous acid is added directly onto the filter, while being held in the folded position
by a pair of stainless steel tweezers. If any visible residue is retained on the aluminum foil
used to protect the filter during storage, then the sections coated with the residue are
removed and placed in an ashed planchet. Any foil sections are labeled with the same
identifier as the filter, with the addition of a second number (e.g. AO31). The section(s) of
foil are then also treated with 200 ^L of sulfurous acid. The following nine samples are
treated in the same manner.
2.2.2 After all filters and foil sections have been treated with 200 fj.L of acid, the planchets are
placed in a 60 °C oven for 20 to 30 minutes. Following drying, all filters and foil sections
are treated with an additional 200 ,uL of sulfurous acid and dried for approximately one
hour.
3.0 Instrumental Analysis
3.1 Carbon is quantified on a Perkin-Elmer 2400 CHN elemental analyzer. Details of instrument
operation and maintenance can be found in the PE 2400 CHN Elemental Analyzer Instruction
Manual (Part number 0993-7147). While the samples are drying, the CHN analyzer is calibrated
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Outline of Standard Protocols for
Particulate Organic Carbon (POC) Analyses Volume 3, Chapter 2
and a check standard is run according to the manufacturer's instructions. When the filters are
almost dry, clean tin disks are added to the planchets containing filters (do not cover filters) to
allow the tin to dry (tin disks are stored in Milli-Q water to minimize contamination). After the
filters and disks are completely dry, the planchets are removed from the oven and the filters are
rolled inside the tin disks. Just before analysis, the rolled filters/tin are compressed using an
aluminum tube and two stainless steel rods so that the samples do not unravel inside the instrument
and cause the autosampler to jam.
Samples are analyzed in groups of five, with a treated filter blank, an analysis blank, and a check
standard run afterward. Treated filter blanks must have no more than 8.8 ±1.5 /^g carbon.
Analysis blanks must agree with the blank runs used to establish the baseline, within ten analysis
counts. Check standard carbon results must agree with the true amounts to within two percent.
3.2 Outline of 2400 POC Procedure
3.2.1 Starting up the machine
3.2.1.1 Turn on Oxygen (10-20 psi) and Nitrogen (42.5 psi) tanks.
3.2.1.2 Increase Helium tank to 20 psi.
3.2.1.3 Press Standby to activate purge.
3.2.1.4 Purge.
a. Press purge gas button
b. He-yes
c. Enter time of 300s
d. O - no
3.2.1.5 Run Blank.
a. Press single run button
b. Press 1 for blank
c. Press 1 again for one run
d. Press enter
e. Press start
3.2.1.6 Read Normals.
a. Carbon is usually around -20 to -30
b. Nitrogen is usually around +30 to + 40
c. If numbers aren't close, purge again for 200s with 1 run
d. If numbers are still not close, purge again for 100s with 2 runs
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Outline of Standard Protocols for
Volume 3, Chapter 2 Paniculate Organic Carbon (POC) Analyses
3.2.2 Making Standards
3.2.2.1 Use acetanilide.
3.2.2.2 Calibrate ju-balance on 20 mg range, using 10.000 mg calibration weight.
3.2.2.3 Use two thimbles to tare balance.
3.2.2.4 Take left thimble and place about 0.6 to 1.2 mg of acetanilide inside with metal
spatula.
3.2.2.5 Make three standards and a check standard for the beginning of the run. Then
make a standard for every five samples.
3.2.2.6 Place thimble in plastic container after it has been folded.
3.2.3 Running Standards
3.2.3.1 Place standards into carousel in order weighed.
3.2.3.2 Press autorun button. Run standards in single runs.
3.2.3.3 Leftmost number should appear as 1. If not, press 4, and then 1 to reset.
3.2.3.4 For standards, press 2 for K factor.
3.2.3.5 Press 1. Punch in weight of first standard. Press Enter. Press Start.
3.2.3.6 For check standard, press 3 for sample.
3.2.3.7 For samples, create an ID#.
3.2.3.8 Press start. Watch and verify carousel alignment.
3.2.4 Run Order
3.2.4.1 Three standards, a check standard, five samples, a foil blank, a method blank, a
check standard, five samples, a foil blank, a method (empty run) blank, and a
check standard.
3.2.5 Turning Off the Machine
3.2.5.1 Press auto run.
3.2.5.2 Press standby button.
3.2.5.3 Shut off the Oxygen and Nitrogen tanks.
3.2.5.4 Decrease Helium pressure to 10 psi.
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ESS Method 360.2:
Silica Dissolved, Automated, Colorimetric
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 360.2:
Silica Dissolved,
Automated, Colorimetric
1.0 Application
1.1 This method may be used to determine concentrations of dissolved reactive silica in surface waters
in the range from 0.1-10 mg SiO2/L by utilizing a dilution loop and a 30/h (2:1) Cam.
1.2 Approximately 50 samples per hour can be analyzed in the low range, and 25 samples per hour in
the high range.
2.0 Summary of Method
Silica reacts with molybdate reagent in acid media to form a yeilow silicomolybdate complex.
This complex is reduced by ascorbic acid to form the molybdate blue color. The color intensity is
proportional to the silica concentration.
3.0 Sample Handling and Preservation
Samples must be filtered through a 0.45 (am filter, cooled to 4°C and analyzed within 28 days.
4.0 Interferences
4.1 Interference from phosphate, which forms a phosphomolybdate complex is eliminated by the
oxalic acid introduced to the sample stream before the addition of the ascorbic acid reagent.
4.2 Tannin interference may also be eliminated by the addition of oxalic acid.
4.3 Hydrogen sulfide is an interference which must be removed by boiling an acidified sample before
analysis.
4.4 Large amounts of iron and color may also interfere.
5.0 Apparatus
Technicon AutoAnalyzer II system consisting of:
5.1 Sampler IV with 50/h (2:1) Cam or 30/h (2:1) Cam
5.2 Analytical Manifold
5.3 Proportioning Pump III
5.4 Colorimeter equipped with 15 mm flow cells
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ESS Method 360.2: Silica Dissolved,
Automated, Colorimetric Volume 3, Chapter 2
5.5 660 nm interference filters
5.6 Recorder/Printer
6.0 Reagents
6.1 Ammonium molybdate reagent: Dissolve 5 g (NH4)6Mo7O24»4H,O in 0.1 N sulfuric acid (2.8 mL
concentrated sulfuric acid/L Milli-Q water) and dilute to 500 mL with the same. Store in an amber
plastic container at 4°C. Stable for two months usually. If STDCAL value is higher than normal,
make new.
6.2 Ascorbic acid reagent: Dissolve 8.8 g ascorbic acid in 250 mL Milli-Q water containing 25 mL
acetone and dilute to 500 mL with Milli-Q water. Add 0.25 mL Levor IV solution. Store in an
amber plastic container at 4°C.
6.3 Levor IV solution: Technicon No. 21-0332 or equivalent.
6.4 Oxalic acid solution: Dissolve 25 g oxalic acid in Milli-Q water and dilute to 500 mL. Store in a
plastic bottle.
6.5 Milli-Q water: ASTM Type I reagent water, Millipore Corp., Bedford, MA.
6.6 Silica stock standard solution, 100 mg SiO2/L
6.6.1 Dilute 100 mL of Ricca or Banco 1000 mg/L standard solution (1 mL = 1.0 mg SiO2)
to 1 L with Milli-Q water. (1 mL = 0.10 mg SiO2)
6.6.2 Transfer the stock standard solution to a 1 L polyethylene bottle and store at 4°C.
6.7 Low level working standards (0.1-10 mg SiO,/L): Prepare the low level working standards by
diluting the following volumes of stock standard solution (6.6) to 100 mL with Milli-Q water.
Transfer the working standard solutions into polyethylene bottles and store at 4°C.
mL Stock
Cone, mg SiCVL Standard (6.6)7100 mL
1.0 1.0
1.5 1.5
2.5 2.5
5.0 5.0
7.5 7.5
10.0 10.0
3-210
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ESS Method 360.2: Silica Dissolved,
Volume 3, Chapter 2 Automated, Colonmetric
6.8 High level working standards (0.3-30 mg SiOTL): Prepare the high level working standards by
diluting the following volumes of stock standard solution (6.6) to 100 mL with Milli-Q water.
Transfer the standard solutions into polyethylene bottles and store at 4°C.
mL Stock
Cone, mg SiO2/L Standard (6.6)7100 mL
5.0 5.0
10.0 10.0
15.0 15.0
20.0 20.0
25.0 25.0
30.0 30.0
7.0 Procedure
7.1 Set up the manifold as shown in Figure 1. For concentrations greater than 10 mg SiOTL, use the
dilution loop with appropriate standards.
7.2 Allow the colorimeter, recorder and printer to warm up for 30 minutes. Obtain a stable baseline
with all lines in Milli-Q water containing 0.5 mL Levor/500 mL. Then attach reagents, feeding
Milli-Q water through the sample line.
7.3 Load sampler according to CFDA Tray Protocol.
7.4 Analyze according to procedures described in LIMS-CFDA Methods manual and General Auto
Analyzer Procedures.
8.0 Calculations
The silica concentration is obtained directly from the LIMS plotter.
9.0 Precision and Accuracy
Precision and Accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
"Manual.
10.0 References
10.1 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of Water-Resources Inv. Book #5 Ch. A1, p. 555 (1985).
10.2 Silicates in Water and Wastewater, Industrial Method No. 105-71W, Technicon Instruments
Corporation. Tarrytown, NY (1973).
3-211
-------
ESS Method 360.2: Silica Dissolved,
Automated, Colorimetric
Volume 3, Chapter 2
DISSOLVED SILICA
Range: 0.030-10
To Sampler IV
Wash Receptacle
22 Turns 20 Turns
157-0370 157-B089
0000 0000 0000
157-8095
20 Turns
",16-0489-01
0
o
•o
o
o
GRN/GRN f2.00) WATER
BLK/BLK (0.32) AIR
Waste
Waste
ORN/ORN (0.42) MOLYBDATE
BLK/BLK (0.32) SAMPLE
BLK/BLK (0.32) OXALIC ACID
ORN/ORN (0.42) ASCORBIC ACID
GRY/GRY (1.00) FROM F/C
SAMFLEfUV
NOTE: FIGURES IN PARENTHESES
SIGNIFY FLOW RATES IN
ML/MIN-
2:1
COLORIMETER
830 nm
15 mm F/C
To F/C
Pump Tube
Figure 1.
3-212
-------
ESS Method 360.3:
Silica, Dissolved, Micro Level
Automated, Colorimetric
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 360.3:
Silica, Dissolved, Micro Level
Automated, Colorimetric
1.0 Application
l.l This method may be used to determine concentrations of dissolved reactive silica in surface waters
in the range from 0.05-2.00 mg SiO2/L.
1.2 Approximately 25 samples per hour can be analyzed.
2.0 Summary of Method
Silica reacts with molybdate reagent in acid media to form a yellow silicomolybdate complex.
This complex is reduced by ascorbic acid to form the molybdate blue color. The color intensity is
proportional to the silica concentration.
3.0 Sample Handling and Preservation
Samples must be filtered through a 0.45 urn filter, cooled to 4°C and analyzed within 28 days.
4.0 Interferences
4.1 Interference from phosphate, which forms a phosphomolybdate complex is eliminated by the
oxalic acid introduced to the sample stream before the addition of the ascorbic acid reagent.
4.2 Tannin interference may also be eliminated by the addition of oxalic acid.
4.3 Hydrogen sulfide is an interference which must be removed by boiling an acidified sample before
analysis.
4.4 Large amounts of iron and color may also interfere.
5.0 Apparatus
Technicon AutoAnalyzer II system consisting of:
5.1 Sampler IV with 30/h (6:1) Cam
5.2 Analytical Manifold
5.3 Proportioning Pump III
5.4 Colorimeter equipped with 2.0 x 50 mm flow cells
3-215
-------
ESS Method 360.3: Silica, Dissolved, Micro Level
Automated, Colorimetric Volume 3, C/?agter£
5.5 660 nm interference filters
5.6 Recorder/Printer
6.0 Reagents
6.1 Ammonium molybdate reagent: Dissolve 5 g (NH4)6Mo7O24»4H2O in 0.1 N sulfuric acid (2.8 mL
concentrated sulfuric acid/L Milli-Q water) and dilute to 500 mL with the same. Store in an amber
plastic container at 4°C. Stable for two months usually. If STDCAL value is higher than normal,
make new.
6.2 Ascorbic acid reagent: Dissolve 8.8 g ascorbic acid in 250 mL Milli-Q water containing 25 mL
acetone and dilute to 500 mL with Milli-Q water. Add 0.25 mL Levor IV solution. Store in an
amber plastic container at 4°C.
6.3 Levor IV solution: Technicon No. 21-0332 or equivalent.
6.4 Oxalic acid solution: Dissolve 25 g oxalic acid in Milli-Q water and dilute to 500 mL. Store in a
plastic bottle.
6.5 Milli-Q water: ASTM Type I reagent water, Millipore Corp., Bedford, MA.
6.6 Silica stock standard solution, 100 mg SiO2/L
6.6.1 Dilute 100 mL of Ricca or Banco 1000 mg/L standard solution (1 mL = 1.0 mg SiO2) to
1L with Milli-Q water. (1 mL = 0.10 mg SiO2)
6.6.2 Transfer the stock standard solution to a 1 L polyethylene bottle and store at 4°C.
6.7 Working standards (0.02-2.00 mg SiO,/L): Prepare the working standards by diluting the
following volumes of stock standard solution (6.6) to the volume listed with Milli-Q water.
Transfer the working standard solutions into polyethylene bottles and store at 4°C.
mL Stock
Cone, mg SiO2/L Standard (6.6)
0.02 0.20mL/lL
0.05 0.50mL/lL
0.15 1.50 mL/L
0.20 0.20mL/100mL
0.50 0.50mL/100mL
1.00 l.OOmL/lOOmL
1.50 I.SOmL/lOOmL
2.00 2.00mL/100mL
6.8 The 0.20 mg SiO/L working standard is used for carryover correction.
3-216
-------
ESS Method 360.3: Silica, Dissolved, Micro Level
Volume 3, Chapter 2 Automated, Colorimetric
7.0 Procedure
7.1 Set up the manifold as shown in Figure 1.
7.2 Allow the colorimeter, recorder and printer to warm up for 30 minutes. Obtain a stable baseline
with all lines in Milli-Q water containing 0.5 mL LEVOR/500 mL. Then attach reagents, feeding
Milli-Q water through the sample line.
7.3 Load sampler according to CFDA Tray Protocol.
7.4 Analyze according to procedures described in LIMS-CFDA Methods manual and General Auto
Analyzer Procedures.
8.0 Calculations
The silica concentration is obtained directly from the LIMS plotter.
9.0 Precision and Accuracy
Precision and Accuracy data are available in the Inorganic Chemistry Unit Quality Assurance
Manual.
10.0 References
10.1 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of Water-Resources Inv. Book #5 Ch. Al, p. 555 (1985).
10.2 Silicates in Water and Wastewater, Industrial Method No. 105-71W, Technicon Instruments
Corporation, Tarrytown, NY (1973).
3-217
-------
ESS Method 360.3: Silica, Dissolved, Micro Level
Automated, Color/metric
Volume 3, Chapter 2
DISSOLVED SILICA
Range: O.Ofl0-iO
To Sampler IV
Wash Receptacle
22 Turns 20 Turns
157-0370 157-8089
0000 0000 0000
1S7-BQ9S
20 Turns
Waste
COLORIMETER
650 nm
IS mm F/C
115-0489-01
O
o
GRN/GRN (2.00) WATER
BLK/BLK (0.32) AIR
ORN/ORN (0,42) MOLYBDATE
BLK/BLK (0.32) SAMPLE
o
o
Waste ^ Q
BLK/BLK (0.32) OXALIC ACiD
ORN/ORN (0.42) ASCORBIC ACID
GRY/GRY (1.00) FROM F/C
SAMPLER IV
NOTE: FIGURES IN PARENTHESES
SIGNIFY FLOW RATES IN
ML/MIN.
2:1
To F/C
Pump Tube
Figure 1. Manifold Set Up
3-218
-------
ESS Method 370.2:
Sulfates Colorimetric,
Automated, Methylthymol Blue
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised October 1992
-------
ESS Method 370.2:
Sulfates Colorimetric,
Automated, Methylthymol Blue
1.0 Scope and Application
1.1 This method is applicable to the determination of sulfate in drinking and surface waters, domestic
and industrial wastes.
1.2 Samples with concentrations in the range of 10 to 100 mg SO4/L cin be analyzed directly.
However, the range may be extended by diluting samples prior to analysis. The sensitivity can be
increased to analyze samples in the range of 1.0 to 30 mg SO4/L. Approximately 30 samples per
hour can be analyzed.
2.0 Summary of Method
2.1 The sample is first passed through a sodium form cation exchange column to remove multivalent
metal ions. The sample containing sulfate then reacts with an alcohol solution of barium chloride
and methylthymol blue (MTB) at a pH of 2.5-3.0 to form barium sulfate. The combined solution
is raised to a pH of 12.5-13.0 so that excess barium reacts with MTB. The uncomplexed MTB
color is gray; if it is at all chelated with barium, the color is blue. Initially, the barium and MTB
are equimolar and equivalent to 300 mg SO/L; thus the amount of uncomplexed MTB is equal to
the sulfate present.
2.2 The reactions are:
At pH 2.5: X SO42- + Y BaCl2 - X BaSO4 + (Y-X) Ba*+ (excess)
At pH 12.5: (Y-X) Ba" + Y MTB - (Y-X) MTB'Ba + X MTB
3.0 Sample Handling and Preservation
All samples should be refrigerated at 4°C.
4.0 Interferences
4.1 The ion exchange column eliminates interferences from multivalent cations, e.g. Ca, Al, Fe. A
mid-scale sulfate standard containing Ca""" should be analyzed periodically to insure the column's
performance.
4.2 Turbid samples should be filtered to remove particulates.
4.3 Samples with a pH below 2 should be neutralized because high acid concentrations elute cations
from the ion exchange resin.
3-221
-------
ESS Method 370.2: Sulfates Colorimetric,
Automated, Methylthymol Blue Volume 3, Chapter 2
5.0 Apparatus
5.1 Technicon AutoAnalyzer consisting of:
5.1.1 Sampler IV with a 30/h (2:1) Cam for both concentration ranges
5.1.2 Analytical manifold - for both ranges
5.1.3 Proportioning pump III
5.1.4 Colorimeter equipped with 15 mm flowcells and solvaflex tubing and 460 nm interference filters.
5.1.5 Printer/Plotter
5.2 Column glass, 7.5" long with a 2.0 mm ID and 3.6 mm OD. Alternatively a 7.5 in piece of purple-
purple pump tube may be used.
6.0 Reagents
6.1 Barium chloride: Dissolve 1.526 g of barium chloride dihydrate (BaCl2»2H,O) in 500 mL of
Milli-Q water and dilute to 1 L.
6.2 Methylthymol blue: Dissolve 0.1182 g of methylthymol blue (3'3"-bis-N, N-bis (carboxymethyl)-
amino methylthymolsulfonephthalein pentasodium salt) in 25.0 mL of barium chloride solution
(6.1). Add 4 mL of 1.0 N hydrochloric acid, which changes the color to bright orange. Add
71 mL of Milli-Q water and dilute to 500 mL with ethyl alcohol (Aldrich Chemical Co.,
spectrophotometric grade). The pH of this solution is 2.6. Store in a brown glass bottle overnight
at 4°C. Prepare new reagent for each use.
6.3 Buffer, pH 10.5 ± 0.5: Dissolve 6.75 g of ammonium chloride in 500 mL of Milli-Q water. Add
57 mL of concentrated ammonium hydroxide and dilute to 1 L with Milli-Q water.
6.4 Buffered EDTA: Dissolve 40 g of tetrasodium EDTA in pH 10.5 buffer (Section 6.3\ and dilute
to 1 L with buffer.
6.4.1 Alternative method for making Buffered EDTA: Dissolve 6.75 g NH4C1 and 40 g
tetrasodium EDTA in 500 mL Milli-Q water and 57 mL concentrated NH4OH. Dilute to
1 L with Milli-Q water.
6.5 Sodium hydroxide, 0.18N: Dissolve 7.2 g sodium hydroxide in 900 mL of Milli-Q water. Allow
to cool and dilute to 1 L with Milli-Q water.
6.6 Dilution water
6.6.1 High range: Add 0.75 mL of 1000 mg/L sulfate standard and 1.0 mL Brij-35 (30%) to
2Lof Milli-Q water.
3-222
-------
ESS Method 370.2: Sulfates Colorimetric,
Volume 3, Chapter 2 Automated, Methylthymol Blue
6.6.2 Low range: Add 4 mL of 1000 mg/L suifate standard and 0.5 mL Brij-35 to 1 L of Milli-
Q water.
6.7 Ion exchange resin: Bio-Rex 70, 20-50 mesh, sodium form, Bio-Rad Laboratories, Richmond,
California. Free from fines by stirring with several portions of Milli-Q water and decanting the
supernatant before settling is complete.
6.8 Suifate stock solution, 1000 mg/L: Dissolve 1.479 g of anhydrous sodium suifate (Na,SO4) (dried
at 105°C for one hour) in Milli-Q water and dilute to 1 L.
6.9 High level working standards, 10-100 mg SO4/L: Prepare the high level working standards by
diluting the following volumes of stock standard solution (Section 6.8) to 100 mL with Milli-Q
water. Use 10 mL buret.
mL Stock
Cone, mg SO4/L Standard/100 mL
10.0 1.0
30.0 3.0
40.0 4.0
50.0 5.0
60.0 6.0
70.0 7.0
80.0 8.0
90.0 9.0
100.0 10.0
6.10 Low level working standards, 1.0-30 mg SO4/L: Prepare the low level working standards by
diluting the following volumes of stock standard solution (Section 6.8) to 500 mL with Milli-Q
water:
mL Stock
Cone, mg SO4/L Standard/500 mL
1.0 0.5
3.0 1.5
6.0 3.0
10.0 5.0
15.0 7.5
18.0 9.0
22.0 11.0
26.0 13.0
30.0 15.0
6.11 Calcium hardness solution for column efficiency check, approximately 1000 mg/L as CaCo3:
Dissolve 1.5 g calcium chloride dihydrate (CaCK*2 H:O) in 1 L with Milli-Q water. (Illinois EPA
Method).
3-223
-------
ESS Method 370.2: Sulfates Colorimetric,
Automated, Methylthymol Blue Volume 3, Chapter 2
7.0 Procedure
7.1 Set up the manifold for high level (10-100 mg SO4/L) or low level (1.0-30 mg SCyL) as described
in Figure 1. Be sure to use silicone tubing and silicone pump tubing where noted **
7.2 Prepare the ion exchange column by dropping a room temperature slurry of the resin into the
column. This is conveniently done by using a small funnel attached with tubing to the glass
U-shaped column. Place a glass wool plug at the end of the column to prevent the resin from
passing through the column. Fill the column with water and pour the resin slurry into the funnel.
Care should be taken to avoid air bubbles entering the column. If air bubbles become trapped,
prepare the column over again. Insert the column in the manifold after the dilution water reagent
has been pumped through the system. The column can exchange the equivalent of 35 mg of
calcium. The column should be prepared as often as necessary to assure that no more than 50% of
its capacity is used. To check the column efficiency: analyze as Reagent Blank a 1:1 mixture of
mid-range standard (50 mg SO4/L) and calcium hardness solution (6.11).
7.3 Allow the colorimeter and printer to warm up for 30 minutes while pumping a Brij-35 solution
(5 mL Brij-35 (30%)/200 mL) through the NaOH and MTB reagent lines. This coats the tubing
and helps prevent Bad, from precipitating inside the system. Pump the reagents until a stable
baseline is achieved and allow to run about 30 minutes. Follow an air segment from sampler to
mixing coil. If it breaks up, replace tubing and clean connections.
7.4 Load the sampler according to the CFDA Tray Protocol.
7.5 Analyze according to procedures described in the LEVIS-CFDA Methods manual and General
AutoAnalyzer Procedures.
7.6 At the end of each day, wash the system by placing the methylthymol blue and sodium hydroxide
lines in water for a few minutes and then in the buffered EDTA solution (6.4) for at least 15
minutes. The dilution water line should be in Milli-Q water. Insert all waste lines in 10% (v/v)
HC1 while washing with the EDTA solution to prevent NH3 gas from being evolved into the
laboratory. After washing with the EDTA solution, place all lines in Milli-Q water and rinse for
15 minutes before shutting down.
Note: The system must be thoroughly cleaned with the EDTA solution and rinsed to prevent
hydrolic problems on subsequent analytical runs.
8.0 Calculations
The sulfate concentration is obtained directly from the LIMS plotter.
9.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Methods file.
3-224
-------
ESS Method 370.2: Sulfates Colorimetric,
Volume 3, Chapter 2 Automated, Methylthymol Blue
10.0 References
10.1 Methods for Chemical Analysis of Water and Wastes, U.S. Environmental Protection Agency,
EPA 600/4-79-020, p 375.2, (1979).
10.2 Sulfate (Automated Methylthymol Blue Method), U.S. Environmental Protection Agency, Central
Region Laboratory, Region V, Chicago, IL, (1978).
10.3 Sulfate in Water and Wastewater, Technicon Industrial Systems, Tarrytown, NY. Industrial
Method No. 118-71W/TENTATIVE.
10.4 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of Water Resources Inv. Book #5, Ch. Al, p 501 (1979).
3-225
-------
ESS Method 370.3:
Sulfates Colorimetric, Automated
Flow Injection, Methylthymol Blue
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised March 1993
-------
ESS Method 370.3:
Suifates Colorimetric,
Automated Flow Injection, Methylthymol Blue
1.0 Scope and Application
]. 1 This method is applicable to the determination of sulfate in drinking and surface waters, domestic
and industrial wastes.
1.2 Samples with concentrations in the range of 10 to 100 mg SO4/L can be analyzed directly.
However, the range may be extended by diluting samples prior to analysis.
2.0 Summary of Method
2.1 The sample is first passed through a sodium form cation exchange column to remove multivalent
metal ions. The sample containing sulfate then reacts with an alcohol solution of barium chloride
and methylthymol blue (MTB) at a pH of 2.5-3.0 to form barium sulfate. The combined solution
is raised to a pH of 12.5-13.0 so that excess barium reacts with MTB. The uncomplexed MTB
color is gray; if it is at all chelated with barium, the color is blue. Initially, the barium and MTB
are equimolar and equivalent to 300 mg SO4/L; thus the amount of uncomplexed MTB is
proportional to the sulfate concentration present.
2.2 The reactions are:
At pH 2.5: X SO42 + Y BaCl2 - X BaSO4 + (Y-X) Ba++ (excess)
At pH 12.5: (Y-X) Ba++ + Y MTB - (Y-X) MTB'Ba + X MTB
3.0 Sample Handling and Preservation
All samples should be refrigerated at 4°C.
4.0 Interferences
4.1 The cation exchange column eliminates interferences from multivalent cations, e.g. Ca, Al, Fe. A
mid-scale sulfate standard containing Ca++ should be analyzed periodically to insure the column's
performance.
4.2 Turbid samples should be filtered to remove particulates.
4.3 Samples with a pH below 2 should be neutralized because high acid concentrations elute cations
from the ion exchange resin.
3-229
-------
ESS Method 370.3: Sulfates Colorimetric,
Automated Flow Injection, Methylthymol Blue Volumes, Chapter 2
4.4 Orthophosphate: Orthophosphate forms a precipitate with barium at high pH. If samples are
known to be high in Orthophosphate, a recovery study, using added amounts of sulfate, should be
done, or a sample blank containing only the Orthophosphate matrix should be run.
5.0 Apparatus
Lachat QuikChem Automated Flow Injection Ion Analyzer which includes:
5.1 Automatic Sampler
5.2 Proportioning Pump
5.3 Injection Module with a 20 cm x 0.8 mm i.d. sample loop
5.4 Colorimeter
5.4.1 Flow Cell, 10mm, 80 uL
5.4.2 Interference Filter, 460 nm
5.5 Reaction Module 10-116-10-2-C
5.6 Automated Digital Diluter
5.7 QuikCalc II Software System or Recorder
5.8 QuikChem AE System Unit
5.9 IBM Personal System 12 Computer
6.0 Reagents
Use deionized water (10 megohm) for all solutions.
6.1 Degassing with Helium: To prevent bubble formation, degas all solutions except the standards
with helium. Use He at 20 lb/in2 through a gas dispersion tube. Bubble He vigorously through the
solution for at least one minute.
6.2 Barium chloride (6.24 mm): Dissolve 1.526 g of barium chloride dihydrate (BaCl:»2H,O) in
500 mL of Milli-Q water and dilute to 1 L.
6.3 Methylthymol blue: Dissolve 0.1 182 g of methylthymol blue (3'3"-bih-N, N-bis (carboxymethyl)-
amino methylthymolsulfonephthalein pentasodium salt) in 25.0 mL of barium chloride solution
(6.2). Add 4 mL of 1.0 N hydrochloric acid, which changes the color to bright orange. Add
71 mL of Milli-Q water and dilute to 500 mL with ethyl alcohol (Aldrich Chemical Co.,
spectrophotometric grade). The pH of this solution is 2.6. Store in a brown glass bottle overnight
at 4 C. Prepare new reagent for each use. Allow to warm to room temperature before using, then
degas with helium.
3-230
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ESS Method 370.3: Sulfates Colorimetric,
Volume 3, Chapter 2 Automated Flow Injection, Methylthymol Blue
6.4 Buffer, pH 10.5 ±0.5: Dissolve 6.75 g of ammonium chloride in 500 mL of Milli-Q water. Add
57 mL of concentrated ammonium hydroxide and dilute to 1 L with Milli-Q water.
6.5 Buffered EDTA: For cleaning manifold. Dissolve 40 g of tetrasodium EDTA in pH 10.5 Buffer
(6.4), and dilute to 1 L with buffer.
Alternative method for making Buffered EDTA: Dissolve 6.75 g ammonium chloride (NH4C1)
and 40 g tetrasodium EDTA in 500 mL Milli-Q water and 57 mL concentrated ammonium
hydroxide (NH4OH). Dilute to 1 L with Milli-Q water. (Caution: Fumes!)
6.6 Sodium hydroxide, 0.18N: Dissolve 7.2 g sodium hydroxide in 900 mL of Milli-Q water. Allow
to cool and dilute to 1 L with Milli-Q water. Store in plastic bottle. Degas with helium.
6.7 Carrier - 0.30 mg SO4 2/L
To a 1 L volumetric flask, add 0.30 mL of 1000 mg/L stock sulfate solution. Dilute to 1 L with
Milli-Q water. Degas with Helium.
6.8 Hydrochloric Acid, 1.0 M: Add 83 mL of concentrated Hydrochloric Acid (HC1) (specific gravity
1.20, 37%) to 800 mL Milli-Q water. Dilute to 1 L. (Caution: Fumes!)
6.9 Calcium hardness solution (1000 mg/L CaCO3) for column efficiency check. Dissolve 1.5 g
calcium chloride dihydrate (CaCl:'2 H,O) in 1 L with Milli-Q water. (Illinois EPA Method).
6.10 Cation Exchange Column Preparation:
6.10.1 Prepare approximately 0.5 g of BioRex 70 ion exchange resin, 50 - 100 mesh, by mixing
with sufficient water to make a slurry.
6.10.2 Remove one end fitting and foam plug from the glass column (Lachat part no. 5000-232).
Fill the column with water then add the slurry and allow it to settle by gravity to pack the
column. Take care to avoid trapping air bubbles in the column or its fittings at this point
and during all subsequent operations.
6.10.3 When the resin has settled, replace the end fitting and foam plug. To ensure a good seal
take care to remove any resin particles from the threads of the glass, the column end, and
the end fitting. To store the column, the ends of the Teflon tubing may be joined with a
union.
6.10.4 If desired, the spent resin may be regenerated using the following procedure: Collect the
used resin in a small beaker or flask. Wash with dilute HCI until the wash tests free of
calcium and magnesium: a calmagite solution will be wine-red in the presence of these
cations and a lighter red in their absence. This procedure removes the divalent cations by
protonating the carboxylate exchange group (-COOH). Convert the resin back to the
sodium form by neutralizing with washes of 0.5 M NaOH until the wash has a pH of 9 or
greater. Rinse with water and store.
3-231
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ESS Method 370.3: Sulfates Colorimetric,
Automated Flow Injection, Methylthymol Blue Volumes, Chapter 2
7.0 Standards
7.1 Sulfate stock solution, 1000 mg SO4/L: Dissolve 1.479 g of anhydrous sodium sulfate (Na2SO4)
(dried at 105°C for one hour) in Milli-Q water and dilute to 1 L.
7.2 Intermediate stock standard, 100 mg SO427L: In a 500 mL volumetric flask, dilute 50.0 mL of the
stock sulfate solution (7.1) to the mark with Milli-Q water.
7.3 High Level working standards, 20-100 mg SO4/L: Prepare the high level working standards by
diluting the following volumes of stock standard solution (7.1) to 200 mL with Milli-Q water. Use
25 mL buret.
mL Stock
Cone, mg SOVL Standard/200 mL
20.0 4.0
60.0 12.0
80.0 16.0
100.0 20.0
mL Stock
Cone, mg SO_/L Standard/500 mL
50.0 25 mL
7.4 Low level working standards, 2.0-10.0 mg SO4/L: Prepare the low level working standards by
diluting the following volumes of stock standard solution (7.2) to 200 mL with Milli-Q water. Use
25 mL buret.
mL Stock
Cone. mgSO/L Standard/200 mL
2.0 4.0 mL
5.0 lO.OmL
10.0 20.0 mL
8.0 Injection Timing
Cycle period: 50 s
Load period: 30 s
Inject period: 30 s
Inject to peak start period 17 s
Inject to peak end period: 54 s
Sample loop length: 20 cm
9.0 System Operation
9.1 Inspect modules for proper connections.
9.2 Turn on power to all modules and check diagnostics.
3-232
-------
ESS Method 370.3: Sulfates Colorimetric,
Volumes, Chapter 2 Automated Flow Injection, Methylthymol Blue
9.3 Follow directions in General Operating Procedures.
9.4 Pump the reagents onto the manifold with a short piece of manifold tubing in place of the column.
When all air has passed and the baseline is steady, turn off the pump and place the column in line.
To prevent air from entering the column when it is added to the manifold, always connect the
column to the valve first and to the manifold second. When the column is in place, resume
pumping.
9.5 Pump system until a stable baseline is attained.
9.6 To check the column efficiency, analyze as Reagent Blank a 1:1 mixture of mid-range standard
(50 mg SO4/L) an calcium hardness solution (6.9). Calculate the percent
True Value - 25 mg/L x 100 = Percent Recovery.
25 mg/L
9.7 Include in your run a Reagent Blank (Milli-Q water) and a known reference sample.
9.8 At end of run, turn the pump off and remove the column. To prevent air bubbles from entering the
column when removing the column from the manifold, disconnect the column from the manifold
first, then disconnect it from the valve and reconnect the column ends with a union. Replace the
column with a short piece of manifold tubing.
9.9 Rinse the manifold with water, then with buffered EDTA, then water, and finally pump dry.
9.10 Turn off pump, all modules, and release pump tube cassettes.
10.0 System Notes
10.1 If the baseline is noisy even without the column in line, degas all reagents thoroughly, especially
the carrier (see 1. Reagents, above). Also check to see that the back-pressure coil (255 cm on a 4"
coil support) is in place on the outlet of the flow cell. If these measures do not solve the problem,
check all hydraulic connections on the manifold and valve module for blockages, leaks, etc.
The red silicone pump tube used for the color reagents wears faster than the standard PVC tubes
and should be changed once a week.
If the baseline is good without the column in line but noisy with the column, repack the column.
Even small air bubbles in the column can cause pulsing and noise. Also check the column fittings
for blockages and leaks.
If the baseline drifts badly, clean the manifold with the buffered EDTA (8. Reagents, above).
10.2 If the sensitivity of the method is poor as indicated by the need for an extremely high gain, check
to see that the pump tube for the color reagent is silicone and not the standard PVC. Also, be sure
that the transmission line for this tube is Teflon and not the standard tygon tube. The alcohol in
the color reagent extracts plasticizer from the PVC pump tubes and transmission lines which then
produces marked turbidity when mixed with the sodium hydroxide on the manifold. This turbidity
results in a high baseline and lack of sensitivity.
3-233
-------
ESS Method 370.3: Sulfates Colorimetric,
Automated Flow Injection, Methylthymol Blue Volumes, Chapter 2
10.3 The balance between the MTB concentration and that of the Ba2* ion is critical to the proper
operation of this method. If the barium concentration is too high, the detection limit will be
adversely affected, while if the MTB concentration is too high, the baseline will be raised and the
signal from the sulfate ions will be lowered. Thus the purity of the MTB used must either be
known or be determined for each lot of material used. As a service to our users, Lachat makes
available preassayed MTB (Part No. 5000-237) with a lot-specific recipe for the Barium-MTB
Color Reagent (5. Reagents, above).
11.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Unit Methods file.
12.0 References
12.1 Methods for Chemical Analysis of Water and Wastes, U.S. Environmental Protection Agency,
EPA 600/4-79-020, p 375.2, (1979).
12.2 Sulfate (Automated Methylthymol Blue Method), U.S. Environmental Protection Agency, Central
Region Laboratory, Region V, Chicago, IL, (1978).
12.3 Sulfate in Water and Wastewater, Technicon Industrial Systems, Tarrytown, NY. Industrial
Method No. 118-71W/TENTATIVE.
12.4 Methods for Determination of Inorganic Substances in Water and Fluvial Sediments, U.S.
Geological Survey Techniques of Water Resources Inv. Book #5, Ch. Al, p 501 (1979).
12.5 U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water and Wastes,
EPA-600/4-79-020, March 1983, Method 375.2.
12.6 Standard Methods for the Examination of Water and Wastewater (1985) 16 Edition, APHA-
AWWA-WPCF, Part 426D, pp. 468-470.
12.7 Colovos, G., et al., Anal. Chem. (1976) 48, 1693-1696 (1976).
3-234
-------
Standard Operating Procedure for
Chloride and Silica in Lake Water
(Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
April 15,1994
-------
Standard Operating Procedure for
Chloride and Silica in Lake Water
(Lachat Method)
1.0 Scope and Application
1.1 This method covers the determination of chloride and silica in lake water.
1.2 The approximate working range is 0.03-30.0 mg-Cl/L and 0.01 -2.00 mg-Si/L. The method
detection limits are 0.030 mg-Cl/L and 0.010 mg-Si/L.
2.0 Summary
2.1 Thiocyanate ion is liberated from mercuric thiocyanate by the formation of soluble mercuric
chloride. In the presence of ferric ion, free thiocyanate ion forms the highly colored ferric
thiocyanate, of which the absorbance is proportional to the chloride concentration. Ferric
thiocyanante absorbs strongly at 480 nm. The calibration curve is non-linear.
2.2 Soluble silica species react with molybdate under acidic conditions to form a yellow
silicamolybdate complex. This complex is subsequently reduced with l-amino-2-napthol-
4-sulfonic acid (ANSA) and bisulfite to form a heteropoly blue complex which has an absorbance
maximum at 820 nm.
3.0 Sample Handling and Preservation
3.1 Samples are collected in clean plastic containers
3.2 Samples should be stored at 4°C.
4.0 Interferences
4.1 Chloride
4.1.1 Substances which reduce iron(III) to iron(II) and mercuryiIII) to mercury(II). (e.g. sulfite,
thiosulfate).
4.1.2 Halides which also form strong complexes with mercuric ion (e.g. Br, I) give a positive
result.
4.2 Silica
4.2.1 The interference due to phosphate is reduced by the addition of oxalic acid. An 8"
reaction coil after the oxalic acid may be substituted for the 4" coil if found to be
necessary.
3-237
-------
SOP for Chloride and Silica in Lake Water (Lachat Method) Volume 3, Chapter 2
4.2.2 Tannin and large amounts of iron or sulfides are interferences.
4.2.3 Silica contamination may be avoided by storing samples, standards, and reagents in
plastic.
5.0 Apparatus
5.1 13 X 100 mm plastic test tubes.
5.2 Lachat QuikChem AE
5.2.1 XYZ Sampler
5.2.2 Chloride manifold (Lachat Method # 10-1 17-07-1-C)
5.2.3 Silica manifold (Lachat Method # 10-114-27-1-B)
5.2.4 Printer
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following information:
Identity: (Oxalic Acid)
Date: (mm/dd/yy)
Initials of Preparer: (M.S.)
All standards shall be stored in appropriate bottles and labeled as above with the following also
included:
Concentration: (1000 mg-Cl/L)
6.2 Use deionized water for all solutions.
6 3 Chloride
6.3.1 Stock Mercuric Thiocyanate Solution: In a 1 L volumetric tlask, dissolve 4.1.7 g mercuric
thiocyanate (Hg(SCN), in approximately 500 mL methanol. Dilute to the mark with
methanol and invert three times to mix.
Caution: Mercury is a very toxic metal! Wear gloves!
6.3.2 Stock Ferric Nitrate Reagent, 0.5 M: In a 1 L volumetric flask dissolve 202 g ferric nitrate
Fe(NO3)3<9H20 in approximately 800 mL water. Add 25 mL concentrated nitric acid and
dilute to the mark. Invert three times to mix.
3-238
-------
Volumes, Chapter 2 SOP for Chloride and Silica in Lake Water (Lachat Method)
6.3.3 Combined Color Reagent: In a 500 mL volumetric flask, mix 75 mL stock mercuric
thiocyanate with 75 mL stock ferric nitrate reagent and dilute to the mark with water.
Invert three times to mix. Vacuum filter through a 0.45 micrometer membrane filter.
6.4 Silica
6.4.1 Molybdate Reagent: In a 500 mL volumetric flask dissolve 20.0 g of ammonium
molybdate tetrahydrate [(NH4)6Mo7O24»4H,O] in approximately 400 mL of water. When
all solid material has dissolved, add 8.0 mL of concentrated sulfuric acid. Dilute to
500 mL and invert three times to mix. Store in plastic and refrigerate. Degas with helium.
Prepare this reagent monthly. Discard if precipitate or blue color is observed.
6.4.2 Oxalic Acid: In a 500 mL volumetric flask, dissolve 50.0 g of oxalic acid
[HO,CCO2H*2H2O] in approximately 450 mL of water. Dilute to the mark and stir to
dissolve. Store in plastic. Do not refrigerate. Degas with helium.
6.4.3 ANSA Reducing Agent: In a 100 mL volumetric flask dissolve 2.0 g of sodium sulfite
(Na:SO,) in approximately 80 mL of water. Add 0.25 g of l-ainmo-2-napthol-4-sulfonic
acid. Dissolve and dilute to the mark. Prepare a second solution by dissolving 15 g of
sodium bisulfite (NaHSOO in 300 mL water. In a dark plastic container mix the two
solutions. Add 4 mL glycerol. Degas with helium. Store refrigerated and discard when it
becomes dark.
6.5 Preparation of Standards
6.5.1 10,000 mg-Cl/L Stock Calibration Solution: In a 500 mL volumetric flask dissolve
8.240 g of Sodium Chloride (NaCl) (dried at 105°C for two hours). Dilute to the mark
with water.
6.5.2 1,000 mg-Si/L Stock Calibration Solution: Purchased commercially.
6.5.3 Intermediate Chloride Calibration Standard (1,000 mg-Cl/L): To a 500 mL volumetric
flask, addSOmL 10,000 mg/L Chloride Solution (6.5.1). Dilute to the mark.
6.5.4 Intermediate Silica Calibration Standard (200 mg-Si/L): To a 500 mL volumetric flask,
add 100 mL of 1,000 mg/L Silica Solution (6.5.2). Dilute to the mark.
3-239
-------
SOP for Chloride and Silica in Lake Water (Lachat Method) Volumes, Chapter 2
6.5.5 Combined Working Calibration Standards: Prepare standards over the range of analysis.
For the working range 0-30 mg-Cl/L and 0-2.00 mg-Si/L, the following standards may be
used:
mL Intermediate mL Intermediate Concentration Concentration
Standard (6.5.3) Standard (6.5.4) mg-Cl/L mg-Si/L
Diluted to 1 L Diluted to 1 L
0.0 — 0.00
1.5 - 1-50
3.0 — 3.00
5.0 0.0 5.00 0.00
10.0 0.5 10.00 0.10
15.0 1.0 15.00 0.20
20.0 2.5 20.00 0.50
25.0 5.0 25.00 1.00
30.0 10.0 30.00 2.00
Note: Use volumetric flasks.
6.5.6 Chloride High Check Control Standard Stock (1,730 mg-Cl/L): In a 1 L volumetric flask
dissolve 3.6382 g of Potassium Chloride (KC1) and dilute to the mark.
6.5.7 Chloride Low Check Control Standard Stock (560 mg-Cl/L): In a 1 L volumetric flask
dissolve 1.1777 g of Potassium Chloride (KCL) and dilute to the mark.
6.5.8 Silica Control Standard Stock (467 mg-Si/L): In a 1 L volumetric flask dissolve 3.13 g of
Sodium Fluorosilicate (Na2SiF6) and dilute to the mark.
6.5.9 Silica Intermediate High Check Control Standard (46.7 mg-Si/L): To a 500 mL
volumetric flask add 50 mL of Silica Central Standard Stock (6.5.8) and dilute to the
mark.
6.5.10 Silica Intermediate Low Check Control Standard (9.3 mg-Si/L): To a 500 mL volumetric
flask add 10 mL of Silica Control Standard Stock (6.5.8) and dilute to the mark.
6.5.1 I Combined Working High Control Standard (CH): In a 200 mL volumetric flask, add
2 mL of Chloride High Check Control Standard Stock (6.5.6) anu 2 mL of Silica
Intermediate High Check Control Standard (6.5.9) and dilute to the mark. The
concentrations of the High Control Standard are 17.3 mg-Cl/L and 0.467 mg-Si/L.
6.5.12 Combined Working Low Control Standard (CL): In a 200 mL volumetric flask combine
2 mL of Chloride Low Check Control Standard Stock (6.5.7) and 2 mL of Silica
Intermediate Low Check Control Standard (6.5.10) and dilute to the mark. The
concentrations of the Low Control Standard are 5.6 mg-Cl/L and 0.093 mg-Si/L.
3-240
-------
Volume 3, Chapter 2 SOP for Chloride and Silica in Lake Water (Lachat Method)
7.0 Procedure
Follow the Lachat Procedural SOP (Typical Daily Operation Section).
8.0 Calculations
The computer yields results directly in mg-Cl/L and mg-Si/L.
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient for both parameters (r) = 0.995.
9.2 The following items are required with the minimum frequency indicated. Any audit out-of-control
requires corrective action.
Audit Type Frequency Limits
Chloride:
CH
CL
Reagent Blank (RB)
Silica:
CH
CL
Reagent Blank (RB)
10.0 Waste Disposal
10.1 The effluent from the chloride channel contains mercuric thiocyanante, which is toxic. This waste
should be collected and discarded in the orange labeled (corrosive) waste container.
10.2 The effluent from the silica channel is an acidic waste and should be disposed of in the yellow
labeled (acidic) waste container.
11.0 Preventative Maintenance
Required maintenance is described in the Lachat Procedural SOP
12.0 Troubleshooting
The most common problem is air bubbles in the lines due to insufficient purging of reagents with
helium.
Method
Method
Method
Method
Method
Method
Beg, End, 1/40
Beg, End, 1/40
Beg, End, 1/40
Beg, End, 1/40
Beg, End, 1/40
Beg, End, 1/40
I7.3± 1.2
5. 6 ±0.6
0.0 ±0.2
0.467 ±0.053
0.093 ±0.0 18
0.000 ±0.021
3-241
-------
SOP for Chloride and Silica in Lake Water (Lachat Method)
Volume 3, Chapter 2
13.0 References
13.1 Lachat Instruments, Method Number 10-117-07-1-B, Chloride in waters, Revision Date June
1993.
13.2 Lachat Instruments, Method Number 10-114-27-1-B, Silica as silicon dioxide (SiO2), Revision
Date February 1989.
Green/Green Tube
Gray/Gray Tube
Yellow/Yellow Tube -
Green/Green Tube -
Color Reagent
-» From sampler wash to wash bath fill
2 5"
-, \ \ \ • -,
Carrier
1.0"
\\\ —
Sample
6 5
•"• To port 6 of
next valve** or
to waste
To Flow Cell
Legend
1.0"
\\\ 1.0" Mixing coil (there is 70 cm Of -Juing on the 1.0" coil support)
2.5"
\\\ 2.5" Mixing coil (there is 168 cm of :joing on the 2.5" coil support)
1 3
l| V §4 6 Port Valve
5 6
Figure 1. Chloride Analytical Manifold (Lake Water Analysis)
Comments
1. Filter used is 480 nm.
2. Sample loop length is 100 cm.
3. All manifold tubing is 0.8 mm (0.032") ID. This relates to a flow of 5.2 uL/cm.
4. The Carrier is helium degassed DI Water.
** This will occur if more than one parameter is he ing run simultaneously.
3-242
-------
Volume 3, Chapter 2
SOP for Chloride and Silica in Lake Water (Lachat Method)
Orange/Orange Tube
Black/Black Tube —
Orange/Orange Tube
Yellow/Yellow Tube•
Green/Green Tube•
-"• From sampler wash to wash bath fill
ANSA
Oxalic Acid
Molybdate
• Note •
4.0" | #1 | 40"
-\\\ 1 \\\ ' \\\-
Carrier
Sample
6 5
To port 6 of
next valve* * or
to waste
To
Flow
Cell
4.0"
\\\ 4.0" Mixing coil (there is 255 cm of tubing on the 4.0" coil support)
2 3
l| V|4
6 Port Valve
5 6
Figure 2. Silica Analytical Manifold (Lake Water Analysis)
Comments
1. Filter used is 820 nm.
2. Sample loop length is 150 cm.
3. All manifold tubing is 0.8 mm (0.032") ID. This relates to a flow of 5.2 uL/cm.
4. The Carrier is helium degassed DI Water.
** This will occur if more than one parameter is being nan simultaneously.
Note 1: The manifold will come with a 4.0" coil here. This can be replaced with an 8" coil if found to be
necessary. See Interferences Section.
3-243
-------
NUTRIENTS SECTION QUALITY CONTROL SHEET
8
-
§
ANALYTK: SILICA
PROGRAM: LIMNOLOGY
DATA SET:
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(0.4 14 to 0.520)
CL
(0.075 to 0. 1 1 1 )
BLANK AUDIT
REAGENT BLANK (LB)
(-0.021 to 0.021)
a
to
I
I
s
Q>
I
O
0.
CXJMMENTS:
ANALYST:.
DATE: / /
TEAM LEADER:
__DATE: / /_
-------
NUTRIENTS SECTION QUALITY CONTROL SHEET
ANALYTE: CHLORIDE
PROGRAM: LIMNOLOGY
DATA SET:
w
f\J
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(16.1 to 18.5)
CL
(5.0lo 6. 2)
BLANK AUDIT
REAGENT BLANK (LB)
(-0.2 to 0.2)
COMMENTS:.
ANALYST.,
DATE: / /.
TEAM LEADER:
_DATE: / /
-------
Standard Operating Procedure for
Dissolved Reactive Phosphorous
(Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
Januarys, 1995
Revision 1
-------
Standard Operating Procedure for
Dissolved Reactive Phosphorous (Lachat Method)
1.0 Scope and Application
1.1 This method covers the determination of dissolved reactive phosphorous (DRP) in lake water.
1.2 The approximate working range is 1 to 25 ug/L. The method detection limit is 1 (Jg/L.
2.0 Summary
The orthophosphate ion (PGy') reacts with ammonium molybdate and antimony potassium tartrate
under acidic conditions to form a complex. This complex is reduced with ascorbic acid to form a
blue complex which absorbs light at 880 nm. The absorbance is proportional to the concentration
of orthophosphate in the sample.
3.0 Sample Handling and Preservation
3.1 Samples are collected in new glass or plastic containers.
3.2 Samples are filtered and frozen until analysis.
4.0 Interferences
4.1 Silica forms a pale blue complex which also absorbs at 880 nm. This interference is generally
insignificant. A silica concentration of 50 mg SiO2/L is required to produce a 0.0008 mg P/L
positive error in orthophosphorous.
4.2 Glassware contamination is a problem in low level phosphorous determinations. Glassware should
be washed with 1:1 HC1 and rinsed several times with diH,O. Special glassware (volumetric
flasks, graduated cylinders, etc.) has been designated for DRP ONLY use.
4.3 High concentration of ferric ion or arsenate ion can cause error due to competition with the
complex for ascorbic acid. Such concentrations are highly unlikely in lake water.
5.0 Apparatus
Lachat QuikChem AE
5.1 Phosphate Manifold (Lachat Manifold #30-115-01-1-8).
5.2 Printer
5.3 XYZ Sampler
3-249
-------
SOP for Dissolved Reactive Phosphorous (Lachat Method) Volume 3, Chapter 2
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following
information:
Identity: Ascorbic Acid
Date: mm/dd/yy
Initials of Preparer: M.S.
All standards should be stored in the appropriate bottles and labeled as above with the following
also included:
Concentration: 100 mg/L
6.2 Use deionized water for all solutions.
6.3 Stock Antimony Potassium Tartrate Solution: In a 1 L volumetric flask, dissolve 3.0 g of
antimony potassium tartrate [K(SbO)C4H4O6»'/2H2O] in approximately 800 mL of water. Dilute to
the mark and invert three times to mix. Store in a dark bottle.
6.4 Stock Ammonium Molybdate Solution: In a I L volumetric flask dissolve 40.0 g of ammonium
molybdate tetrahydrate [(NH4)Mo7O24] in about 400 mL of water. Dilute to the mark and invert to
mix.
6.5 Molybdate Color Reagent: In a 1 L volumetric flask containing about 500 mL of water, add
35 mL concentrated sulfuric acid. Swirl to mix. (Caution: The solution will get hot!) Add
72.0 mL of the Stock Potassium Tartrate Solution and 213 mL of the Stock Ammonium
Molybdate Solution. Dilute to the mark and invert three times to mix. De-gas with helium.
6.6 Ascorbic Acid: In a 1 L volumetric flask dissolve 60.0 g ascorbic acid in about 700 mL water.
Dilute to the mark and invert three times to mix. Degas thoroughly!! Add 1.0 g sodium dodecyl
sulfate [CH,(CH2)MOSO3Na]. Mix gently with stir bar; do not shake to mix. Prepare fresh
weeklv.
6.7 Sodium Hydroxide - EDTA Rinse: Dissolve 65 g sodium hydroxide (NaOH) and 6 g tetrasodium
ethylenediamine tetraacetic acid (NA4EDTA) in 1 L of water.
6.8 Preparation of Standards
6.8.1 Stock 100 mg P/L Calibration Standard: Dry a small amount of potassium
dihydrogen phosphate (KH;PO4) in an oven at 105 C to constant weight. In a 1 L
volumetric flask, dissolve 0.4394 g of dried standard in about 500 mL diH:O.
Dilute to the mark and invert to mix. Store at 4 = C.
6.8.2 Intermediate 1.0 mg P/L Calibration Standard: Using a volumetric pipet, pipet
10 mL of the Stock Calibration Standard (6.8.1) into a 1 L volumetric flask.
Dilute to the mark and in\ert to mix. Store at 4=C.
3-250
-------
Volume 3, Chapter 2 SOP for Dissolved Reactive Phosphorous (Lachat Method)
6.8.3 Working Calibration Standards: Prepare standards over the range of analysis. For
the working range of 0-25 pg/L; the following standards may be used:
mL Intermediate
Solution (6.8.2) Concentration
diluted to 1 L ug P/L
0.0 0.00
2.5 2.50
5.0 5.00
7.5 7.50
10.0 10.00
15.0 15.00
25.0 25.00
Note: Use volumetric flask. Store at 4°C.
6.8.4 Stock 100 mg P/L Control Standard: Dry a small amount of Sodium Phosphate,
dibasic anhydrous (Na2HPO4) in an oven at 105°C to constant weight. In a 1 L
volumetric flask, dissolve 0.458 g of dried standard in about 500 mL water. Store
at4°C.
6.8.5 Intermediate 1.0 mg P/L Control Standard: Using a volumetric pipet, transfer
10.0 mL of the Stock Control Standard (6.8.4) into a 1 L volumetric flask. Dilute
to the mark and invert to mix. Store at 4°C.
6.8.6 Working Control Standards: The following concentrations are typical:
mL Intermediate
Standard (6.8.5) Concentration
diluted to 1 L ug P/L
CS-1 9.0 9.00
CS-2 3.0 3.00
Note: Use volumetric flask. Store at 4°C,
7.0 Procedure
7.1 Allow at least 15 minutes for the heating block to warm up to 37 °C.
7.2 Samples are pre-filtered and frozen. They should be brought to room temperature prior to
analysis.
7.3 Follow the Lachat Daily Operation Procedural SOP
7.4 At the end of a run, place all lines into the NuOH-EDTA solution (Section 6.7). Pump this
solution tor approximately five minutes. Follow with a thorough water rinse
3-251
-------
SOP for Dissolved Reactive Phosphorous (Lachat Method) Volume 3, Chapter 2
8.0 Calculations
The computer yields results directly in ug P/L.
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient (r) is 0.995.
9.2 The following items are required with the minimum frequency indicated:
Audit Type Frequency Limits
CS-1 Method Beg,End, 1/40 Samp. 9 ±4
CS-2 Method Beg.End, 1/40 Samp. 3 ±2
Reagent Blank Method Beg.End, 1/40 Samp. 0±1
10.0 Waste Disposal
Effluent from this channel as well as the sample effluent is acidic. It should be disposed of in a
yellow labelled waste container.
11.0 Preventive Maintenance
Required maintenance is described in the Lachat Procedural SOP.
12.0 Troubleshooting
12.1 If the baseline drifts and cleaning the system in the prescribed manner does not help, the heating
coil tubing may need to be changed.
12.2 An unusually noisy baseline may be due to insufficient purging of air from the reagents. Tiny
bubbles tend to develop in the heated tubing and become trapped in the flow cell causing baseline
problems.
13.0 References
13.1 Lachat Instruments, Method Number. 10-1 15-01-1-B, Orthophosphate in water, Revision Date
April 1992.
13.2 Lachat QuikChem AE Operation Manual.
13.3 GLNPO Soluble Reactive Phosphorous (Orthophosphate). August 1990.
3-252
-------
Volume 3, Chapter 2
SOP for Dissolved Reactive Phosphorous (Lachat Method)
DRP Analytical Manifold
fill
Orange/Orange
Orange/Orange
Yellow/Yellow
Green/Green Tube
From sampler wash to wash bath
Molybdate Color Reagent
Ascorbic Acid
Carrier
Sample
i •
2"
\\\
2 3
6 5
37°C
To flow
cell
To port 6 of
next valve** or
to waste
Legend
2.0" \\\: 2.0" Mixing coil (there is 135 cm of tubing on the 2.0" coil support)
2 3
V
5 6
4 : 6 Port Valve
\\\
37°C
The box shows 175 cm
of tubing wrapped
around the block
heater.
Comments
1. Filter used is 880 nm.
2. Sample loop length is 125 cm.
3. All manifold tubing is 0.8 mm (0.032") ID. This relates to a flow of 5.1 uL/cm.
4. The cmricr \^ helium degassed diFLO.
5. Timing: Cycle Period is 36 sec. Inject to start of peak period is 9 sec.
** If more than one channel is being used.
3-253
-------
8
NUTRIENTS SECTION QUALITY CONTROL SHEET
ANALYTE: DISSOLVED REACTIVE PHOSPHOROUS PROGRAM: LIMNOLOGY
DATA SET:
CO
en
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(13 to 5)
CL
(\ 10 5)
BLANK AUDIT
REAGENT BLANK (LB)
(-1 to I)
(A
(n
O
I
Q.
Is
Q)
I
8
o
A
COMMENTS:.
ANALYST:.
DATE: / / TEAM LEADER:
.DATE: / /
(&
3
-------
II
Standard Operating Procedure for
Ammonia (Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
April 15,1994
-------
Standard Operating Procedure for
Ammonia (Lachat Method)
1.0 Scope and Application
1.1 This method covers the determination of ammonia in lake water.
1.2 The approximate working range is 0.02 to 2.00 mg-N(as NH3)/L. The method detection limit is
0.02 mg-N/L.
2.0 Summary
When ammonia is heated with salicylate and hypochlorite in an alkaline phosphate buffer, an
emerald green color is produced which is proportional to the ammonia concentration. The color is
intensified by the addition of sodium nitroprusside.
3.0 Sample Handling and Preservation
3.1 Samples are collected in clean glass or plastic containers.
3.2 Samples are preserved by the addition of 1 mL of concentrated sulfuric acid per liter of sample.
4.0 Interferences
4.1 In alkaline solutions, calcium and magnesium will interfere by forming a precipitate which scatters
light. EDTA is added to the buffer to prevent this interference.
4.2 Sample turbidity may interfere. Turbid samples may be decanted or filtered prior to analysis.
5.0 Apparatus
5.1 13 X 100 mm Test Tubes
5.2 Lachat QuikChem AE
5.2.1 Ammonia manifold (Lachat method number 10-107-06-2-C)
5.2.2 XYZ Sampler
5.2.3 Printer
3-257
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SOP for Ammonia (Lachat Method) Volume 3, Chapter 2
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following information:
Identity: (Buffer)
Date: (mm/dd/yy)
Initials of Preparer: (M.S.)
All standards should be stored in appropriate bottles and labeled as above with the following also
included:
Concentration: (lOOOmg-N/L)
6.2 Use deionized water for all solutions.
6.3 Buffer: In a 1 L volumetric flask dissolve 30.0 g sodium hydroxide (NaOH), 25.0 g
ethylenediaminetetraacetic acid, disodium salt dihydrate, and 67 g sodium phosphate dibasic
heptahydrate (Na,HPO4»7H2O) in about 900 mL of water. Dilute to the mark and invert to mix.
De-gas with helium.
6.4 Salicylate-Nitroprusside Color Reagent: In a 500 mL volumetric flask, dissolve 72 g sodium
salicylate (salicylic acid sodium salt, [C6H4(OH)(COO)Na]) and 1.75 g sodium nitroprusside
(sodium nitroferricyanide dihydrate, [Na2Fe(CN)5NO*2H2O), in about 400 mL water. Dilute to the
mark. Stir or shake to dissolve. Refrigerate. Prepare fresh weekly. De-gas with helium.
6.5 Hypochlorite Reagent: In a 1 L volumetric flask, dilute 60 mL Regular Clorox Bleach [5.25%
sodium hypochlorite (NaCIO), The Clorox Company, Oakland, CA] to the mark with water.
Invert three times to mix. De-gas with helium. Refrigerate.
6.6 Preparation of Standards
6.6.1 Stock 1000 mg-N(as NH,)/L Calibration Standard: In a 1 L volumetric flask, dissolve
3.819 g of ammonium chloride (NH4CI), dried for one hour at 105°C, in about 500 mL
water. Add 1 mL concentrated H:S04 and dilute to the mark.
6.6.2 Intermediate 100 mg-N(as NH,)/L Calibration Standard: In a 1 L volumetric flask, dilute
100.0 mL of stock calibration standard (6.6.1) in about 500 mL water. Add I mL H,SO4
and dilute to the mark with water. This solution is also the spike solution.
3-258
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Volume 3, Chapter 2 SOP for Ammonia (Lachat Method)
6.6.3 Working Calibration Standards: Prepare standards over the range of analysis. For the
working range of 0-2.00 mg-N(as NH,)/L, the following standards may be used:
mL Intermediate Standard Concentration
(6.6.2) diluted to 1L mg/L
0.0 0.00
0.2 0.02
2.5 0.25
5.0 0.50
7.5 0.75
10.0 1.00
20.0 2.00
Note: Use volumetric flasks and preserve the working standards by addition of 1 mL of
concentrated H2SO4.
6.6.4 Stock 100 mg/L Ammonia Control Standard: (Any ammonia compound may be used for
the control standards. They should be prepared by someone other than the analyst.) In a
1 L volumetric flask, dissolve 0.4716848 g of ammonia sulfate [(NH4),SO4], dried at
105°C for one hour, in about 500 mL water. Add 1 mL of concentrated H2SO4 and dilute
to the mark.
6.6.5 Working Control Standards: The following concentrations are typical:
mL Stock Control Standard Concentration
(6.6.4) diluted to 1 L mg/L
CS-1 2.0 0.20
CS-2 6.0 0.60
Note: Use volumetric flasks. Preserve the control standards by addition of 1 mL of
concentrated H,SO4.
7.0 Procedure
7.1 Allow at least 15 minutes for the heating block to warm up to60:C before beginning the analysis.
7.2 Follow the Lachat Procedural SOP (Typical Daily Operation Section) for the remainder of the
analysis.
7.3 This method can be run simultaneously with the Nitrate/Nitrite method. Combined standards
should then be prepared.
8.0 Calculations
The computer yields results directly in mg-N(as NH.)/L.
3-259
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SOP for Ammonia (Lachat Method)
Volumes, Chapter2
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient (r) = 0.995.
9.2 The following items are required with the minimum frequency indicated:
Audit
Type
Frequency
Limits
CS-1
CS-2
Reagent Blank
Lab Blank"
Duplicate"
Spike"
Method
Method
Method
Method
Method
Method
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
1/40 Sample
1/40 Sample
0.60 ± 0.04
0.20 ±0.03
0.00 ± MDL
0.00±MDL
A < 0.02
IOC ± 12
'These audits are not included in Lake Water Analysis.
10.0 Waste Disposal
Effluent from this channel is basic. It should be disposed of in a blue labeled waste container.
11.0 Preventive Maintenance
Required maintenance is described in the Lachat Procedural SOP.
12.0 Troubleshooting
It is very important to thoroughly purge all reagents of air before they are used. Insufficient
purging will result in a noisy baseline and air spikes in the peaks.
13.0 References
13.1 Lachat Instruments, Method Number 10-107-06-2-C, Ammonia in surface water, wastewater.
Revision Date, August 1992.
13.2 Lachat QuikChem Operating Manual.
13.3 GLAS Standard Operating Procedure, Ammonia Nitrogen, February 1993.
3-260
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Volume 3, Chapter 2
SOP for Ammonia (Lachat Method)
-•• From sampler wash to wash bath
fill
r?oH / RnH
Hypochlorite
Sal icy late /Nitroprus side
Buffer 1.0" 1.0"
r v v v ' \ \ \ \ \ S
60'C
2 3
Carrier To fl
^ D
6 5 next valve** or
to waste
Legend
1.0" : 1.0" Mixing coil (there is 70 cm of tubing on the 1.0" coil support)
\\\
2 3
l| V |4 : 6 Port Valve
5 6
\\\,
: The box shows 650 cm
of tubing wrapped
60°C around the block
heater.
Figure 1. Ammonia Analytical Manifold
Comments:
2.
3.
4.
5.
**
Filter used is 660 nm.
Sample loop length is 25 cm.
All manifold tubing is 0.8 mm (0.032") ID. This relates to a now of 5.2 ^L/cm.
The Carrier is helium degassed DI Water.
Timing: Cycle period is 40 seconds. Inject to start of peak is 26 seconds.
If more than one channel is being used.
3-261
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Standard Operating Procedure for
Nitrate, Nitrite (Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
Aprils, 1995
Revision 2
-------
Standard Operating Procedure for
Nitrate, Nitrite (Lachat Method)
1.0 Scope and Application
I.! This method covers the determination of nitrate and nitrite in lake/rain water.
1.2 The approximate working range is 0.03 to 2.00 mg N(as NO, + NO,)/L. The method detection
limit is 0.03 mg N/L.
2.0 Summary
Nitrate is quantitatively reduced to nitrite by passage of the sample through a column containing
copper coated cadmium. The nitrate (reduced nitrate plus original nitrite) is determined by
diazotizing with sulfanilamide dihydrochloride. The resulting water soluble dye has a magenta
color which is read at 520 nm.
3.0 Sample Handling and Preservation
3.1 Samples are collected in clean glass or plastic containers. Flexidome and phenolic resin (black)
caps, or caps with glued plastic liners may contaminate the samples. Polypropylene caps should be
used.
3.2 Samples are preserved by addition of 1 mL of concentrated sulfuric acid per liter of sample.
4.0 Interferences
4.1 Residual chlorine can interfere by oxidizing the cadmium column.
4.2 Low results would be obtained for samples that contain high concentrations of iron, copper, or
other metals. In this method, EDTA is added to the buffer to reduce this interference.
5.0 Apparatus
5.1 13 x 100 :nm test tubes
5.2 Lachat QuikChem AE
5.2.1 XYZ Sampler
5.2.2 Nitrate/Nitrite Manifold (Lachat Method # 10-107-04-1-C)
5.2.3 Cadmium-Copper Reduction Column
5.2.4 Printer
3-265
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SOP for Nitrate, Nitrite (Lachat Method) Volume 3, Chapter 2
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following information:
Identity: (15 N Sodium hydroxide)
Date: (mm/dd/yy)
Initials of Preparer. (M.S.)
All standards will be stored in appropriate bottles and labeled as above with the following also
included:
Concentration: (1000 mg- N/L)
6.2 Use deionized water for all solutions.
6.3 15 N Sodium hydroxide: Add 150 g of NaOH very slowly to 250 mL of water.
Caution: The solution will get very hot! Swirl until dissolved. Cool and store in a plastic bottle.
6.4 Ammonium chloride buffer, pH 8.5: In a 1 L volumetric flask, dissolve 85.0 g ammonium
chloride (NH4C1) and 1.0 g disodium ethylenediamine tetraacetic acid dihydrate (Na:EDTA«2H,0)
in about 800 mL water. Adjust pH to 8.5 with 15 N NaOH. Degas with helium.
6.5 Sulfanilamide color reagent: To a 1 L volumetric flask add about 600 mL water. Then add
100 mL of 85% phosphoric acid (H,PO4) 40.0 g sulfanilamide, and 1.0 g N-(l-naphthyl)
ethylenediamine dihydrochloride (NED). Shake to wet, and stir to dissolve for 30 min. Dilute to
the mark, and invert to mix. Store in a dark bottle. This solution is stable for one month. Degas
with helium.
6.6 Preparation of Standards
6.6.1 Stock 1000 mg-N/L Nitrate Solution: Dissolve 7.218 g of potassium nitrate (KNO,), dried
for one hour at 105°C, in 500 mL of DI water. Add 1 mL of concentrated sulfunc acid
(H:SO4) and dilute to I L.
6.6.2 Intermediate 100 mg-N/L Nitrate Standard Solution (Spike): Dilute 100 mL of stock
nitrate solution (6.6.1) to 500 mL. Add 1 mL of concentrated sulfuric acid and dilute to
I L with DI water. This solution is also the spike solution.
3-266
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Volume 3, Chapter 2 SOP for Nitrate, Nitrite (Lachat Method)
6.6.3 Working Standards: Prepare standards over the range of analysis. For the working range
of 0-2.00 mg N03-N/L, the following standards may be used:
mL Intermediate Standard Concentration
Solution (6.6.2) mg-N/L
diluted to 1 L
0.0 0.00
0.2 0.02
2.5 0.25
5.0 0.50
7.5 0.75
10.0 1.00
20.0 2.00
Note: Use volumetric flasks. Preserve the working standards by addition of 1 mL of
concentrated sulfuric acid.
6.6.4 Stock 100 mg-N/L Nitrate Control Standards: Any nitrate compound may be used for
control standards. They should be prepared by someone other than the analyst. Weigh
0.6068146 g of sodium nitrate (NaNO3) (dried at 105°C for one hour) and dissolve in
500 mL of DI water. Add 1 mL concentrated H2SO4. Dilute to 1 L in volumetric flask
with DI water.
6.6.5 Prepare the control standards using solution (6.6.4).
mL Control Standard Concentration
Solution (6.7.4) mg-N/L
diluted to 1 L
CS-l(LPCl) 6 0.600
CS-2(LPC-2) 2 0.200
Note: Use volumetric flasks. Preserve the control standards by addition of 1 mL of
concentrated sulfuric acid.
7.0 Procedure
Follow the Lachat Procedural SOP (Typical Daily Operation Section). Remember to establish
reagent flow through entire system before diverting flow through cadmium column.
8.0 Calculations
The computer yields results directly in mg-N(as NO2+NO3)/L.
3-267
-------
SOP for Nitrate, Nitrite (Lachat Method)
Volume 3, Chapter 2
9.0 Quality Control
The following items are required with the minimum frequency indicated.
Audit
Type
Frequency
Rain:
CS-l(LPC-l)
CS-2(LPC-2)
Reagent Blank(LCB)
Lab. Blank(LRB)
Duplicate(LD)
Spike(LCO)
Method
Method
Method
Method
Method
Method
Beg, End, 1/40 Samp
Beg, End, 1/40 Samp
Beg, End, 1/40 Samp
Beg, End, 1/40 Samp
1/40 Samp
1/40 Samp
Lake:
CS-l(LPC-l) Method
CS-2(LPC-2) Method
Reagent Blank(LCB) Method
Beg + End, 1/40 Samp
Beg + End, 1/40 Samp
Beg + End, 1/40 Samp
Limits
0.60 ±0.09
0.20 + 0.03
0.00 ± 0.03
0.00 ± 0.03
A < 0.03
100 ± 12%
0.60 ± 0.09
0.20 + 0.03
0.00 + 0.03
10.0 Waste Disposal
Effluent from this channel should be neutralized with sodium hydroxide to a pH of 6-9 and then
washed down the laboratory drain with plenty of water.
11.0 Preventive Maintenance
Required maintenance is described in the Lachat Procedural SOP
12.0 Troubleshooting
The most common problem is deactivation of the cadmium column which results in low values
and non-linear calibration curves. The deactivation of the column is quantified by a column
having a :89% efficiency factor. The only solution is replacement of the column. This procedure
is outlined in the following section.
13.0 Cadmium Column Preparation
Note: Prepacked cadmium columns are available from Lachat Instruments.
13.1 Preparation of Reagents for Cadmium Column
13.1.1 IN Hydrochloric acid (HCI): In a 100 mL container, add 8 mL concentrated HC1 to
92 mL water. Stir to mix.
3-268
-------
Volume 3, Chapter 2 _ SOP for Nitrate, Nitrite (Lachat Method)
13.1.2 2% Copper Sulfate Solution: In a 1 L volumetric flask dissolve 20 g copper sulfate
(CuSO4-5H20) in about 800 mL water. Dilute to mark with water. Invert to mix
thoroughly.
13.2 Cadmium Preparation
Place 10-20 g of coarse cadmium granules (0.3-1.5 mm diameter) in a 250 mL beaker. Wash with
50 mL of acetone, then water, then two 50 mL portions of 1 N HCI. Rinse several times with
water.
13.3 Copperization
Add a 100 mL portion of 2% Copper Sulfate Solution to the cadmium prepared above. Swirl for
about five minutes, then decant the liquid and repeat with a fresh 100 mL portion of the 2% copper
sulfate solution. Continue this process until the blue aqueous copper color persist. Decant and
wash with at least five portions of ammonium chloride buffer to remove colloidal copper. The
cadmium should be black or dark gray. The copperized cadmium granule may be stored in a
stoppered bottle under ammonium chloride buffer.
13.4 Packing the Column
Wear gloves and do all cadmium transfers over a special tray or beaker. Clamp the empty column
upright so that your hands are free. Unscrew one of the colored fittings from an end of the
column, and pull out and save the foam plug. The column and threads are glass so be careful not
to break or chip them. Fasten this fitting up higher than the open end of the column and
completely fill the column, attached fittings, and tubing with ammonium chloride buffer. Scoop
up prepared copperized cadmium granules with a spatula and pour them unto the top of the filled
column so that they sink down to the bottom of the column. Continue pouring the cadmium in and
tapping the column with a screw driver handle to dislodge andy air bubbles and to prevent gaps in
the cadmium filling. When the cadmium granules reach to about 5 mm form the open end of the
column, push in the foam plug and screw on the top fitting. Rinse the outside of the column with
DI water.
If air remains in the column or is introduced accidentally, connect the column into the manifold,
turn the pump on maximum, and tap firmly with a screwdriver handle, working up the column
until all air is removed.
13.5 Cadmium Column Insertion Procedure
13.5.1 Before inserting the column, pump all reagents into manifold.
13.5.2 Turn the pump off.
13.5.3 On the column, disconnect the center tubing from one of the union connectors and
immediately connect to the outlet tubing of the buffer mixing coil.
13.5.4 Connect the open tubing on the column to the tee fitting where the color reagent is added.
Do not let air enter the column.
3-269
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SOP for Nitrate, Nitrite (Lachat Method) Volume 3, Chapter 2
13.5.5 Return the pump to normal speed.
13.5.6 The direction of reagent flow through the column is not relevant.
14.0 References
14.1 Lachat Instruments, Method Number 10-107-04-1-C, Nitrate/Nitrite in Surface Water,
Wastewater, Revision Date November 1992.
14.2 Lachat QuikChem AE Operating Manual.
3-270
-------
Volume 3, Chapter 2
SOP for Nitrate, Nitrite (Lachat Method)
Green/Green Tube
White/White Tube
-•• From sampler wash to wash bath fill
Sulfanilamide Color Reagent
I 2.0"
Yellow/Blue Tube -
——I \ \ \ ••-.
Ammonia Buffer
2.0"
-\\\ —
Carrier
Orang/Orang Tube
Green/Green Tube
2 3
V • 4
—1 =
CADMIUM
COLUMN
Sample
Tf
6 5
To port 6 of
next "alve ** or
to waste
To Flow Cell
Legend
2.0"
\\\
2.0" Mixing coil (there is 135 cm of tubing on the 2.0" coil support)
6 Port Valve
5 6
Figure 1. Nitrate Analytical Manifold (Lake and Rain Water Analysis)
Comments
* 1. This is a 1 state switching valve used to place the cadmium column in-line with the manifold.
State One: Nitrate + Nitrite State Two: Nitrite
Solution flow is through the
cadmium column.
Solution flow by-passes the cadmium
column.
1. Filter used is 520 nm.
2. Sample loop length is 25 cm.
3. All manifold tubing is 0.8 mm (0.032") ID. This relates to a flow of 5.2 uL/cm.
4. The Carrier is helium degassed DI Water.
** This will occur if more than one parameter is being run simultaneously.
3-271
-------
NUTRIENTS SECTION QUALITY CONTROL SHEET
ANALYTE: NITRATE-NITRITE
PROGRAM: LIMNOLOGY
DATA SET:
OJ
IV)
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CS-1 (LPC-I)
(0.51 lo 0.69)
CS-2 (LPC-2)
(0.1 7 to 0.23)
BLANK AUDIT
REAGENT BLANK (LB)
(-0.03 to 0.03)
COMMENTS:.
ANALYST:.
DATE: / /
TEAM LEADER:
__DATE: / /_
-------
I
NUTRIENTS SECTION QUALITY CONTROL SHEET
9
ANALYTE: NITRATE-NITRITE
PROGRAM: ATMOSPHERIC WEEKLY
DATA SET:.
CJ
r\3
JATE
SAMPLE
FROM
TO
CHECK STANDARD
AUDIT
CS-I
(LPC-1)
(0.51 to 0.69)
CS-2
(LPC-2)
(0.17 to 0.23)
BLANK
AUDIT
R.BLK.
(LCB)
L.BLK.
(LRB)
(-0.03 to 0.03)
DUPLICATE
AUDIT
SAMP.
#
DUP.
(LD)
<0.03
SPIKE AUDIT C2(VI +V2)-CIVI X 100%
T2V2
MEASURED
SAMPLE
CONC.
Cl
MEASURED
SPIKED
SAMPLE
CONC.
C2
SAMPLE
VOLUME
(mL)
VI
SPIKE
VOLUME
(mL)
V2
ORIG.
SPIKE
CONC.
T2
%REC
(LSO)
88 to 1 1 2%
C/)
O
TJ
COMMENTS:,
0)
iff
ANALYST:.
DATE:.
TEAM LEADER:
_DATE:
to
I
a.
-------
II
Standard Operating Procedure for
Total Kjeldahl Nitrogen
(Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
April 15,1994
Revision 2
-------
Standard Operating Procedure for
Total Kjeldahl Nitrogen (Lachat Method)
1.0 Scope and Application
1.1 This method covers the determination of Total Kjeldahl Nitrogen (TKN) in lake and rain water.
1.2 The approximate working range is 0.10 to 2.50 mg-N/L. The method detection limit is
O.lOmg-N/L.
2.0 Summary
2.1 Samples are digested in sulfuric acid in the presence of a mercuric oxide catalyst. The Kjeldahl
nitrogen present is converted to ammonium cation. Potassium sulfate helps speed the conversion
to ammonium.
2.2 After injection onto the manifold, the samples pH is raised to a known, basic pH with a
concentrated buffer. This neutralization converts the ammonium to ammonia. The ammonia is
heated with salicylate and hypochlorite to produce a blue color which is proportional to the
ammonia concentration. The color is intensified by adding sodium nitroprusside. The presence of
tartrate in the buffer prevents precipitation of calcium and magnesium.
3.0 Sample Handling and Preservation
3.1 Samples are collected in new or acid-washed glass or plastic containers.
3.2 Samples are preserved by addition of 1 mL of H2SO4 per liter of samples. Store at room
temperature.
4.0 Interferences
4.1 Samples must not consume more than half of the sulfuric acid during digestion. The buffer will
accommodate a range of 2-4% (v/v) H,SO4 in the diluted digested sample with no change in signal
intensity.
4.2 Incomplete digestion, evident by dark particles in digested samples may cause low results. When
this occurs, the original sample must be diluted and redigested.
5.0 Apparatus
5.1 Digestion tubes: l"x 8" heavy-walled pyrex tubes.
5.2 Block Digester.
5.3 Adjustable pipets with disposable tips capable of delivering 10 mL and 2 mL volumes.
3-277
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SOP for Total Kjeldahl Nitrogen (Lachat Method) Volumes, Chapter 2
5.4 13 x 100 mm disposable test tubes.
5.5 Lachat QuikChem AE
5.5.1 XYZ Sampler
5.5.2 TKN Manifold (Lachat Manifold #10-107-06-2-E)
5.5.3 Printer
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following information:
Identity: (Buffer)
Date: (mm/dd/yy)
Initials of Preparer: (M.S.)
All standards will be stored in appropriate bottles and labeled as above with the following also
included:
Concentration: (100 mg-N/L)
6.2 Use deionized water for all solutions.
6.3 Digestion Solution: Add 4 mL of concentrated H,SO4 to 21 mL water. Dissolve 2.0 g mercuric
oxide (HgO) in the solution. Set this aside.
In a 1 L volumetric flask carefully add 200 mL of concentrated H,SO4 to about 500 mL water.
While this solution is still hot, dissolve 134 g of potassium sulfate (K-,SO4) in it. Add the HgO
solution. Cool and dilute. Store at room temperature. Do not allow salt to precipitate. If
precipitation does occur, put reagent bottle in warm water bath for about 30 minutes. Stir on stir
plate until precipitation is no longer evident.
6.4 Buffer: In a 1 L volumetric flask dissolve 65 g sodium hydroxide (NaOH), 20.0 g disodium
EDTA (ethylenediaminetetraacetic acid disodium salt), and 35.0 g sodium phosphate dibasic
heptahydrate (Na:HPOj*7FLO) in about 900 mL water. Dilute to the mark and invert to mix.
De-gas with helium.
6.5 Salicylate - Nitroprusside Reagent: In a 1 L volumetric flask dissolve 150.0 g sodium salicylate
[salicylic acid sodium salt. C,,H4(OH)(COO)Na], and 1.00 g sodium mtroprusside [sodium
nitroferricyanide dihydrate, Na,Fe(CN)5NO*2H2O] in about 800 mL water. Dilute to the mark and
invert to mix. Store in a dark bottle and prepare fresh monthly. De-gas with helium.
6.6 Hypochlorite Solution: In a 500 mL volumetric flask, dilute 30 mL Regular Clorox Bleach
(5.25% sodium hypochlorite) to the mark with water. Invert to mix. De-gas with helium.
3-278
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Volume 3, Chapter 2 SOP for Total Kjeldahl Nitrogen (Lachat Method)
6.7 Diluent: Note: Diluent is prepared for use in the auto-dilutor to dilute off-scale samples. This
reagent is not used on-line. In a 1 L volumetric flask containing approximately 600 mL water, add
240 mL Digestion Solution (6.3). Dilute to the mark and invert to mix.
6.8 Preparation of Standards
6.8.1 Stock 100 mg-N/L Nitrogen Standard: In a 1 L volumetric flask, dissolve 1.050 g dried
L-(+)-glutamic acid in 500 mL water. Add 1 mL of concentrated H,SO4 and dilute to the
mark.
6.8.2 Working Standards: Prepare standards over the range of analysis. For the working range
of 0-2.50 mg-N/L, the following standards may be used:
mL Stock
Solution(6.8.1)
diluted to 1 L
0.00
1.00
2.50
5.00
7.50
10.00
25.00
Concentration
mg-N/L
0.00
0.10
0.25
0.50
0.75
1.00
2.50
Note: Use volumetric flasks. Preserve the working standards by addition of 1 mL of
concentrated H,SO4.
6.8.3 Stock 227 mg-N/L Nitrogen Control Standard: In a 1L volumetric flask dissolve 1.3845 g
of adenosine-5-monophosphoric acid disodium salt in 500 mL water. Add 1 mL of cone.
FLSO., and dilute to the mark.
6.8.4 Working Control Standards: The following concentrations are typical:
mL Stock Control
Standard (6.8.3) Concentration
diluted to 1 L mg-N/L
CH 5.0 1.15
CL 2.0 0.45
Note: Use volumetric flasks. Preserve the control standards by addition of 1 mL of
concentrated H:SO4.
3-279
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SOP for Total Kjeldahl Nitrogen (Lachat Method) Volumes, Chapter 2
7.0 Procedure
7.1 Digestion
7.1.1 Rinse all glassware once with 1:1 HC1, and then three times with deionized water. Do not
use commercial detergents.
7.1.2 Using an automatic pipet with disposable tips, withdraw a 10 mL aliquot of sample.
Discard this first portion. Withdraw another 10 mL aliquot and transfer to a digestion
tube.
7.1.3 Add 2.0 mL of digestion solution (6.3) and several (two to three) boiling chips.
7.1.4 Prepare all samples, calibration standards, blanks, control standards, spikes, and duplicates
in the same manner.
7.1.5 Place the rack of tubes in a pre-heated block digester at 200°C for 60 minutes. Be sure to
place the end plates in position on the racks so heating occurs evenly.
7.1.6 Transfer the rack to a pre-heated high temperature block. Heat at 370°C for 30 minutes.
7.1.7 Remove the tubes from the block and allow to cool for about 15 minutes.
7.1.8 Add a 10 mL aliquot of deionized water to each tube. Mix the samples well using a
Vortex mixer. Transfer to 13 x 100 mm test tubes for analysis. Samples may also be
covered with aluminum foil and held for later analysis.
7.2 Analysis of Digested Samples
7.2.1 Allow at least 15 minutes for the heating unit to warm up to 60°C.
7.2.2 If the salicylate reagent is merged with a sample containing sulfuric acid in the absence of
the buffer solution, the salicylate reagent will precipitate. To prevent this, prime the
system by first placing the buffer transmission line in the buffer. Pump until the air
bubble introduced during the transfer reaches the "T" fitting on the manifold. Then place
all other lines in the proper containers. If precipitation does occur, all teflon tubing
should be replaced.
7.2.3 It is very important that all reagents be purged thoroughly with helium before beginning
analysis. Usually two to three minutes will suffice for each reagent.
7.2.4 Follow the Lachat Procedural SOP for the remainder of the analysis.
7.2.5 The diluent in the auto dilutor is reagent 6.7 not deionized water.
7.2.6 In normal operation, the digested blank will result in a slight peak. This is due to the acid
in the digest and is present in every injection. Since this blank is constant for all samples
and standards it will not effect data quality.
3-280
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Volume 3, Chapter 2 SOP for Total Kjeldahl Nitrogen (Lachat Method)
8.0 Calculations
The computer yields results directly in mg-N/L.
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient (r) for TKN calibration curve is 0.995.
9.2 The following items are required with the minimum frequency indicated:
Audit Type Frequency Limits
Rain:
CH
CL
Reagent Blank(LB)
Lab Blank(RB)
Duplicate(LD)
Spike(LSF)
Lake:
CH
CL
Reagent Blank(RB)
Method
Method
Method
Method
Method
Method
Method
Method
Method
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
1/40 Samp.
1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
Beg, End, 1/40 Samp.
1.15 ±0.15
0.45 ±0.1 2
0.00 ±0.10
0.00 ±0.10
A < 0.10
100 ±24
1.15 ±0.15
0.45 ±0.1 2
0.00 ±0.10
10.0 Waste Disposal
Effluent from this channel is acidic and should be disposed of in a yellow labeled waste container.
11.0 Preventive Maintenance
Required maintenance is described in the Lachat Procedural SOP
12.0 Troubleshooting
12.1 If the baseline drifts, peaks are too wide, or problems with precision arise, clean the manifold by
the following procedure:
12.1.1. Place all reagent transmission lines in water and pump to clear reagents (two to five
minutes).
12.1.2. Place reagent lines and earner in 1 M HC1 (one volume of HC1 added to 11 volumes of
water) and pump for several minutes.
12.1.3. Place all transmission lines in water and pump for several minutes.
12.1.4. Resume pumping reagents.
3-281
-------
SOP for Total Kjeldahl Nitrogen (Lachat Method) Volume 3, Chapter 2
13.0 References
13.1 Lachat Instruments, Method Number 10-107-06-2-E, Total Kjeldahl Nitrogen in waters, Revision
Date July 1993.
13.2 Lachat QuikChem AE Operating Manual.
13.3 GLAS Standard Operating Procedure, Total Kjeldahl Nitrogen. July 1992.
3-282
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Volume 3, Chapter 2
SOP for Total Kjeldahl Nitrogen (Lachat Method)
fill
From sampler wash to wash bath
Hypochlorite
i
Sal icy late /Nitroprusside
\
Buffer 4.0" 1.0"
* • • ~r — - \ \ \ \ \ \ • • 1 \ \ \
60'C
2 3
Carrier 1 To fl
— O
6 5 next valve** or
to waste
Legend
4.0"
\\\ : 4.0" Mixing coil (there is 255 cm of tubing on the 4.0" coil
support)
1.0" : 1.0" Mixing coil (there is 70 cm of tubing on the 1.0" coil
support)
\\\
2 3
l| V |4 : 6 Port Valve
5 6
\\\
60°C
The box shows 650 cm
of tubing wrapped
ground the large block
heater.
Figure 1. TKN Analytical Manifold (Lake and Rain Water)
Comments
a.
b.
c.
d.
e.
**
Filter used is 660 nm.
Sample loop length is 25 cm.
All manifold tubing is 0.8mm (0.032")ID. This relates to a flow of ^ 2 uL/cm.
The Carrier is helium degassed DI Water.
Timing: Cycle period is 49 seconds. Inject to start of peak is 47 seconds.
It more than one channel is beine used.
3-283
-------
8
NUTRIENTS SECTION QUALITY CONTROL SHEET
o
g
5
D'
ANALYTE: TKN
PROGRAM: LIMNOLOGY
DATA SET:
1
w
CD
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(1.00 lo 1.30)
CL
(0.33 to 0.57)
BLANK AUDIT
REAGENT BLANK (LB)
(-0.10 to 0.10)
o
Q.
COMMENTS.
ANALYST:.
DATE: / /
TEAM LEADER:
_DATE: /
I
n>
Cj
I
-------
Standard Operating Procedure for
Total and Dissolved Phosphorous
(Lachat Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
June 13,1994
Revision 1
-------
Standard Operating Procedure for
Total and Dissolved Phosphorous
(Lachat Method)
1.0 Scope and Application
1.1 This method covers the determination of total phosphorus and total dissolved phosphorous in
lake/rain water.
1.2 The approximate working range is 1 to 50 ug/L. The method detection limit is 1 |ug/L.
2.0 Summary
2.1 Samples are digested in the presence of sulfuric acid and persulfate to convert or "hydrolyze"
polyphosphates and organic phosphorous to orthophosphate.
2.2 The orthophosphate ion (PO43~) reacts with ammonium molybdate and antimony potassium tartrate
under acidic conditions to form 12-molybdophosphoric acid. This complex is reduced with
ascorbic acid to form a blue complex which absorbs light at 880 nm. The absorbance is
proportional to the concentration of orthophosphate in the sample.
3.0 Sample Handling and Preservation
3.1 Samples are collected in new or acid-washed glass or plastic containers.
3.2 Samples are preserved by addition of 1 mL of H2S04 per liter of sample.
3.3 The preserved samples are stable for at least 28 days when stored at room temperature.
4.0 Interferences
4.1 Silica forms a pale blue complex which also absorbs at 880 nm. This interference is generally
insignificant. A silica concentration of 50 mg SiO/L is required to produce a 0.008 mg P/L
positive error in orthophosphorous.
4.2 Glassware contamination is a problem in low level phosphorus determinations. Glassware should
be washed with 1:1 HC1 and rinsed several times with diHUO. Special glassware (volumetric
flasks, graduated cylinders, etc.) has been designated for TP ONLY use.
4.3 High concentrations of ferric iron or arsenate ion can cause error due to competition with the
complex for ascorbic acid. Such concentrations are highly unlikely in lake water.
3-287
-------
SOP for Total and Dissolved
Phosphorous (Lachat Method) Volume 3, Chapter 2
5.0 Apparatus
5.1 Digestion tubes: Borosilicate Glass 16 x 100 mm Culture Tubes, and digestion caps: White
polypropylene screw caps.
5.2 Autoclave
5.3 Automatic pipets with disposable tips calibrated to deliver 8.0 mL and 1.0 mL.
5.4 Lachat QuikChem AE
5.4.1 Phosphate Manifold (Lachat Manifold # 30-1 15-01 -1 -B)
5.4.2 Printer
5.4.3 XYZ Sampler
6.0 Reagents and Standards
6.1 All reagents should be stored in the appropriate bottles and labeled with the following information:
Identity: (Ascorbic Acid)
Date: (mm/dd/yy)
Initials of Preparer: (M.S.)
All standards will be stored in appropriate bottles and labeled as above with the following also
included:
Concentration: (lOOmgP/L)
6.2 Use deionized water for all solutions.
6.3 0.9 M H2SO4: To a 500 mL volumetric flask containing about 400 mLs of diFLO add 25 mL of
concentrated sulfuric acid. Dilute to the mark and invert three times to mix.
6.4 0.28 M Ammonium Persulfate: In a 500 mL volumetric flask, dissolve 31.5 g ammonium
persulfate [(NH4)2S:OH] in about 400 ml of water. Dilute to the mark ;>nd invert to mix.
6.5 Stock Ammonium Molybdate Solution: In a 1 L volumetric flask dissolve 40.0 g ammonium
molybdate tetrahydrate [(NH4)Mo7O24] in approximately 800 mL water. Dilute to the mark and
invert three times to mix. Store in plastic and refrigerate.
6.6 Stock Antimony Potassium Tartrate Solution: In a 1 L volumetric flask, dissolve 3.0 g antimony
potassium tartrate [K(SbO)C4H4O6-'/2H2O] in approximately 800 mL of water. Dilute to the mark
and invert three times to mix. Store in a dark bottle and refrigerate.
3-288
-------
SOP for Total and Dissolved
Volume 3, Chapter 2 Phosphorous (Lachat Method)
6.7 Molybdate Color Reagent: In a 1 L volumetric flask containing about 500 mL water, add 20.9 mL
concentrated sulfuric acid. Swirl to mix. (Caution: The solution will get hot!) Add 72.0 mL of
the Stock Antimony Potassium Tartrate Solution and 213 mL of the Stock Ammonium Molybdate
Solution. Dilute to the mark and invert three times to mix. De-gas with helium.
6.8 Ascorbic Acid Reducing Solution: In a 1 L volumetric flask dissolve 60.0 g ascorbic acid in about
700 mL water. Dilute to the mark and invert three times to mix. Degas. Add 1.0 g dodecyl
sulfate, sodium salt (CH,(CH2)HOSO,Na). De-gas with helium. Prepare fresh weekly.
6.9 Sulfuric Acid Carrier Solution: In a 1 L volumetric flask containing about 900 mL water, udd
9 mL concentrated sulfuric acid (H2SO4). Dilute to the mark with water. Invert three times to mix.
De-gas thoroughly.
6.10 Sodium Hydroxide EDTA Rinse: Dissolve 65 g sodium hydroxide (NaOH) and 6 g tetrasodium
ethlenediamine tetraacetic acid (Na4EDTA) in 1 L of water.
6.11 Hydrochloric Acid Rinse: Combine equal parts water and concentrated hydrochloric acid (HC1).
6.12 Preparation of Standards
6.12.1 Stock 100 mg P/L Calibration Standard: Dry a small amount of potassium dihydrogen
phosphate (KH:PO4) in an oven at 105°C to constant weight. In a 1 L volumetric flask,
dissolve 0.4394 g of dried reagent in about 500 mL diH:O. Add 1.0 mL of concentrated
sulfuric acid and dilute to the mark. Store at 4°C.
6.12.2 Intermediate 1.0 mg P/L Calibration Standard: Using a volumetric pipet, pipet 10 mL of
the Stock Calibration Standard (6.12.1) into a 1 L volumetric flask. Add 1.0 mL of
concentrated sulfuric acid and dilute to the mark. Store at 4°C.
6.12.3 Working Calibration Standards: Prepare standards over the range of analysis. For the
working range of 0-50 ug/L, the following standards may be used:
mL Intermediate Concentration
Solutiqn(6.12.2) T
diluted to 1 L
0.0 0.00
2.5 2.50
5.0 5.00
7.5 7.50
10.0 10.00
25.0 25.00
50.0 50.00
Note: Use volumetric flasks. Preserve the working standards by addition of 1.0 mL of
concentrated sulfuric acid and store at 4°C.
3-289
-------
SOP for Total and Dissolved
Phosphorous (LachatMethod) Volumes, Chapter2
6.12.4 Stock 100 mg P/L Control Standard: Dry a small amount of Adenosine-
5-Monophosphoric Acid, Disodium salt, [(CloH,2N5O7PNa,'2H,O), F.W - 427.236g/mole,
Fluka] in an oven at 105°C to constant weight. Allow 10 cool to room temperature in a
desiccator. In a 1 L volumetric flask, dissolve 1.3793 g of the dried reagent in about '
500 mL of water. Add 1.0 mL of concentrated sulfuric acid and dilute to the mark. Store
at4°C.
6.12.5 Intermediate 1.0 mg P/L Control Standard: Using a volumetric pipet, transfer 10.0 mL of
the Stock Control Standard (6.12.4) into a 1 L volumetric flask. Add 1.0 mL water, dilute
to the mark,and invert to mix. Store at 4°C.
6.12.6 Working Control Standards: The following concentrations are typical:
mL Intermediate
Standard (6.12.5) Concentration
diluted to 1 L ug P/L
CS-1 15.0 15.00
CS-2 3.0 3.00
Note: Use volumetric flasks. Preserve the control standards by addition of 1 mL of
concentrated H2SO4. Store at 4°C.
7.0 Procedure
7.1 Digestion
7.1.1 Do Not Use Commercial Detergents. Soak digestion tubes in 1:1 HC1 for one hour, rinse
thoroughly with diH2O and allow to dry completely before use.
7.1.2 Using an automatic pipet with disposable tip, withdraw a 8 mL aliquot of sample. Discard
this first portion. Withdraw another 8 mL aliquot and transfer to a digestion tube.
7.1.3 Add 1.0 mL of 0.9M H2SO4 (Reagent 6.3), and 1.0 mL of 0.28M Ammonium Persulfate
(Reagent 6.4).
7.1.4 Cap the tube tightly and place in metal digestion rack.
7.1.5 Prepare all samples, calibration standards, blanks, and control standards in the same
manner.
7.1.6 Place the rack of tubes in an autoclave at 1213C for 30 minutes.
7.1.7 Allow the samples to cool to room temperature before analysis. Redigest any tubes that
gain or lose volume.
3-290
-------
SOP for Total and Dissolved
Volume 3, Chapter 2 Phosphorous (Lachat Method)
7.2 Analysis
7.2.1 Allow at least 15 minutes for the heating block to warm up to 37°C.
7.2.2 Follow the Lachat Procedural SOP (Typical Daily Operation Section) for the remainder of
the analysis.
7.2.3 At the end of a run, place all lines into the NaOH-EDTA solution (6.10). Pump this
solution for approximately five minutes. Rinse lines in water and then pump for another
five minutes in 1:1 HC1 (Reagent 6.11). Follow with a thorough water rinse.
8.0 Calculations
The computer yields results directly in ug P/L.
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient (r) is 0.995.
9.2 The following items are required with the minimum frequency indicated:
Audit Type Frequency Limits
Rain:
CS-1
CS-2
Reagent Blank
Lab Blank
Duplicate
Spike
Lake:
CH
CL
Reagent Blank
Method
Method
Method
Method
Method
Method
Method
Method
Method
Beg,
Beg,
Beg,
Beg,
1/40
1/40
Beg,
Beg,
Beg,
End, 1/40
End, 1/40
End, 1/40
End, 1/40
Sample
Sample
End, 1/40
End, 1/40
End, 1/40
Samp.
Samp.
Samp.
Samp.
Samp.
Samp.
Samp.
15
3
0
0
A
100
15
3
0
±3*
±2*
± 1
± 1
< 1
± 19%
±3*
±2*
± 1
*These limit ranges are performance estimates based on data obtained during MDL study.
10.0 Waste Disposal
Effluent from this channel as well as the sample effluent is acidic. It should be disposed of in a
yellow labeled waste container.
11.0 Preventive Maintenance
Required maintenance is described in the Lachat Procedural SOP
3-291
-------
SOP for Total and Dissolved
Phosphorous (Lachat Method) Volume 3, Chapter^
12.0 Troubleshooting
12.1 If the baseline drifts and cleaning the system in the prescribed manner does not help, the heating
coil tubing may need to be changed.
12.2 If negative peaks are observed in some or all of the samples or standards, it is probably due to
matrix difference between the carrier and the samples. Check to be sure the carrier was made up
properly and that the sulfuric acid addition to the digestate was not unintentionally omitted.
Re-digest those samples that exhibited the negative peaks.
12.3 An unusually noisy baseline may be due to insufficient purging of air from the reagents. Tiny
bubbles tend to develop in the heated tubing and may become trapped in the flow cell causing
baseline problems.
13.0 References
13.1 Lachat Instruments, Method Number 10-115-01-1-F, Total Phosphorus in Persulfate Digest,
Revision Date May 1992.
13.2 Lachat QuikChem AE Operating Manual.
13.3 GLAS Standard Operating Procedure, Total Phosphorus, Low-Level Micro-persulfate digestion.
August 1990.
3-292
-------
Volume 3, Chapter 2
SOP for Total and Dissolved
Phosphorous (Lachat Method)
-»• From sampler wash to wash bath
fill
Orange/Orange
Orange/Orange
Yellow/Yellow
Green/Green Tube
Molybdate Color Reagent
Ascorbic Acid
2"
\\\
2 3
Carrier
\\\|—|
Sample
6 5
37°C
To flow
cell
•• To port 6 of
next valve** or
to waste
Legend
2.0"
\\\ :
2 3
2.0" Mixing coil (there is 135 cm of tubing on the 2.0" coil
support)
l| V |4 : 6 Port Valve
5 6
\\\
37°C
The box shows 175 cm
of tubing wrapped
ctround the block
heater.
Figure 1. TP/TDP Analytical Manifold
Comments
1. Filter used is 880 nm.
2. Sample loop length is 1 25 cm.
3. All manifold tubing is 0.8 mm (0.032") ID. This relates to a flow of 5.2 pL/cm.
4. The carrier is Reagent 6.9.
5. Timing: Cycle Period is 44 sec. Inject to start of peak period is 1 1 sec.
** If more than one channel is beinn used.
3-293
-------
NUTRIENTS SECTION QUALITY CONTROL SHEET
ANALYTE: TOTAL PHOSPHOROUS
PROGRAM: LIMNOLOGY
DATA SET:
CO
ro
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(12 to \X)
CL
(1 to 5)
BLANK AUDIT
REAGENT BLANK (LB)
(-1 to 1)
COMMENTS
ANALYST:,
DATE: / /
TEAM LEADER:
_DATE: / /
. en
1°
^?
9 ^
-------
NUTRIENTS SECTION QUALITY CONTROL SHEET
ANALYTE: TOTAL DISSOLVED PHOSPHOROUS PROGRAM: LIMNOLOGY
DATA SET:
CO
Ul
DATE
SAMPLE
FROM
TO
CHECK STANDARD AUDIT
CH
(12 to 8)
CL
(1 to 5)
BLANK AUDIT
REAGENT BLANK (LB)
(-1 to 1)
C'OMMENIS •_
ANALYST:,
_DATE: / / TEAM LEADER:.
.DATE: / /
-------
Analysis of Total Suspended
Particles (TSP) and Total Organic
Carbon (TOC) in Air Samples:
Integrated Atmospheric
Deposition Network (IADN)
TSP/TOC Procedure
Michael Wassouf and llora Basu
School of Public and Environmental Affairs
Indiana University
Bloomington, IN 47405
November 28,1995
Version 1.0
-------
Analysis of Total Suspended Particles (TSP)
and Total Organic Carbon (TOC) in Air Samples:
Integrated Atmospheric Deposition Network
(IADN) TSPTOC Procedure
1.0 Introduction
Air particulates are collected from three sites: Eagle Harbor, 100 meters from Lake Superior,
Michigan on the Keweenan Peninsula; Sleeping Bear Dunes, on Lake Michigan 5 km south of
Empire, Michigan; and Sturgeon Point, 25 km southwest of Buffalo, New York and 100 meters
from Lake Erie. Air is drawn through the Whatman quartz microfibre filter, 20.3 x 25.4 cm at a
flow rate of 68 m3/hr for 24 hours using an Anderson Hi-Vol air sampler. All particles greater
than two microns are retained by the filter. The filters are then transported to Indiana University
where they are analyzed for the total suspended particle and total organic carbon.
2.0 Supplies and Equipment:
2.1 Supplies
2.1.1 Quartz Microfibre Filters (Whatman 20.3 x 25.4 cm)
2.1.2 LiNO3
2.1.3 Aluminum foil
2.1.4 Tweezers
2.1.5 Hexane (EM Science, Omnisolv)
2.1.6 Plastic Zip-Lock bags
7.2 Equipment
2.2.1 Balance (Mettler AE 50)
2.2.2 Mettler Balance GD Hanger
2.2.3 Humidity Chamber (Lab-Line Desicab No. 1477) with wet LiNO3 in a tray.
2.2.4 Leco Total Carbon Analyzer
2.2.5 Muffle Furnace (Thermolyne Type 30400)
3-299
-------
Analysis of Total Suspended Particles (TSP) and
Total Organic Carbon (TOO) in Air Samples
IADN: TSP/TOCProcedure Volumes, Chapter2
3.0 Balance Calibration
3.1 The balance must be connected to the power supply for at least 60 minutes before calibrating.
3.2 Press and hold the single control bar until -CAL- appears in the display, then release the control
bar. The display changes to CAL-—, followed by CAL 50 (blinks).
3.3 Move calibration lever all the way to the rear; the display changes to CAL—, followed by
50.0000, then to CAL 0 (blinks).
3.4 Move calibration lever all the way back to the front of the balance; the display changes to —,
followed by 0.0000.
4.0 Filter Preparation Before Sampling
3.1 Wrap filters in aluminum foil (shiny side out).
4.2 Muffle filters for four to six hours @ 450°C, store in freezer until use.
4.3 Open foil slightly and place filters in desiccator (50% humidity via LiNO,) for 24 hours.
4.4 Re-zero balance.
4.5 Write Filter ID# in top right corner of filter with pencil.
4.6 Open balance hanger and insert filter (unwrapped) using tweezers rinsed in hexane; close door.
4.7 Wait until balance equilibrates and record mass in Filter Log Book as Initial Weight.
4.8 Remove filter from balance and re-zero.
4.9 Weigh filter again; if mass is within 0.1 mg of first mass go on, if not repeat weighing until two
measurements within 0.1 g are taken and record the average as Initial Weight.
4.10 Re-wrap filter in original piece of aluminum foil .
4.11 Write Filter ID# on foil with magic maker.
4.12 Place in plastic zip-lock bag.
4.13 Repeat 1-12 for each filter.
3-300
-------
Analysis of Total Suspended Particles (TSP) and
Total Organic Carbon (TOO) in Air Samples:
Volume 3, Chapter 2 IADN - TSP/TOC Procedure
5.0 TSP Measurement on Filter
5.1 Remove filter from plastic bag; open foil slightly and place in desiccator for 24 hours.
5.2 Record Sample ID Code with corresponding Filter ID# in Filter Log Book and on plastic bag.
5.3 Weight filter as above.
5.4 Record mass as Final Weight
5.5 Calculate and record TSP (Final Weight - Initial Weight).
5.6 Re-wrap filter in original foil and plastic bag.
5.7 Place in cold room or freezer until TOC analysis.
6.0 Preparing Filters for TOC Analysis
6.1 Remove filter from bag and foil.
6.2 Cut six discs, 1.9 cm diameter, from filter with cork borer and place discs in a plastic petri dish.
6.3 Record Sample ID Code on petri dish.
6.4 Record TSP from Filter Log Book in TSP/TOC Log as TSP on Filter.
6.5 Calculate and record TSP on Circle (TSP on Filter x 2.84/404). The area of the whole filter is
404 sq. cm. The area of the circle is 2.84 sq. cm.
6.6 Multiply TOC by six, this is the number you will enter into the Leko Carbon Analyzer.
3-301
-------
Analysis of Total Suspended Particles (TSP) and
Total Organic Carbon (TOO) in Air Samples
IADN: TSP/TOC Procedure Volume 3, Chapter 2
7.0 TOC Analysis: Using LEKO Total Carbon Analyzer
See following pages (from Leco operator's manual).
CALIBRATION
Leco needs calibration for both the balance and the standards. This is performed at the beginning of each
day that Leco will be used.
To calibrate the balance:
1. Press the "system update" key.
2. Press the "5" key.
3. Place a crucible on the balance.
4. Press the "tare" key.
5. Place a 1 gram weight in the crucible; the readout will flash.
6. Remove the 1 gram weight.
Leco needs calibration with carbon and sulfur standards before combusting samples. To calibrate for
carbon and sulfur, the standards will be combusted using the same procedure used for samples (see Sample
Combustion). A minimum of 5 standard combustions are used for the calibration. The values for these
combustions should be within O.OSg of each other. When 5 consistent values have been obtained, perform
the following procedure for calibration:
1. Press the "system update" key. Message center will display "display contents?" Press the
'T'key.
2. "Auto Calibrate By Stack" will be displayed on message center. Press the "Yes" key.
3. "Carbon recalibrate" will be displayed on the message center. If you are calibrating for
carbon, press the "Yes" key. If you are calibrating for sulfur, press the "No" key. The
message center will now display "Sulfur Recalibrate." Press the "Yes" key.
4. Message center will now display "Calibrate By Standard." Press the "Yes" key.
Message center will ask the value of the standard. Press the "Enter" key.
5. A complete answer stack for the last 10 analyses will be printed.
6. The print out list will be displayed on the message center one value at a time. Press the
"Yes" key for values to be included, and the "No" key for values to be excluded.
7. A revised answer list will print out at the end of calculations. Check this list against the
original answer list to be sure Leco has recalibrated.
3-302
-------
Analysis of Total Suspended Particles (TSP) and
Total Organic Carbon (TOO) in Air Samples:
Volume 3, Chapter 2 . IADN - TSP/TOC Procedure
SAMPLE COMBUSTION, STANDARD PROCEDURE
Leco must warm up for one hour before use. Both the furnace and measurement units need power switches
turned on. Be sure oxygen and compressed air valves are open before beginning combustions.
To combust samples, you will need:
1. Crucibles
2. Lecocel
3. Iron Chip Accelerator
To combust samples, perform the following:
1. Place a crucible on the balance. Do not touch the crucible with fingers.
2. Measure 250 mg of the sample into the crucible. Mass will be displayed on message
center.
3. Press the "Enter" key. Mass will be moved to the left side of the display.
4. Using tongs, remove the crucible from the balance. Tap to distribute sample evenly on the
bottom of the crucible.
SAMPLE COMBUSTION, MANUAL WEIGHT PROCEDURE
Leco must warm up for one hour before use. Both the furnace and measurement units need power switches
turned on. Be sure oxygen and compressed air valves are open before beginning combustions.
To combust samples, you will need:
1. Lecocel
2. Iron Chip Accelerator
To combust samples, perform the following:
1. Press the "Manual Weight" key.
2. Enter the sample weight using the keyboard.
3. Press the "Enter" key. Mass will be moved to the left side of the display.
4. Using tongs, remove the crucible from the balance. Tap to distribute sample evenly on the
bottom of the crucible.
3-303
-------
II
Standard Operating Procedures for
Determining Total Phosphorus, Available
Phosphorus, and Biogenic Silica
Concentrations of Lake Michigan Sediments
and Sediment Trap Material
Tom Johengen
NOAA/Great Lakes Environmental Research Lab
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105-1593
GLERL - SED NUTRIENT - 96
January 1996
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Standard Operating Procedures for Determining Total Phosphorus,
Available Phosphorus, and Biogenic Silica Concentrations of Lake
Michigan Sediments and Sediment Trap Material
1.0 Available Phosphorus
l.l Available phosphorus is determined by extracting sediments with 0. IN NaOH, following the
analytical procedures described by Williams et al. (1967; 1980). This chemical extraction
procedure has compared favorably with algal assay estimates of available phosphorus (Sagheret
al., 1975; Williams et al., 1980; Sonzogni et al., 1982; Dorich et al., 1985).
Prior to Analysis:
1.1.1 Weigh 25-35 mg (for box cores) or 75-100 mg (for ponars or sandy samples) of freeze-
dried, homogenized, sediment on the Mettler AT250 balance. Calibrate balance at
beginning and end of session with standard weights. Weigh sediment onto pre-tared
glassine weigh paper. Transfer to pre-labeled, 50 mL polypropylene tubes. Record
sample weight and vial number in log book.
1.1.2 Include one procedural blank (no sediment), and two reference sediment samples with
each batch.
1.1.3 Add 30 mL of 0.1N NaOH using the Brinkman repeator pipet. Cap tightly and mix well.
1.1.4 Place tubes into a25°C shaking water bath and shake for 17 hours.
On day of Analysis:
1.1.5 Add 3 mL of 1.0 N HC1 to neutralize sample using Eppendorf pipet.
1.1.6 Cap and mix well. Record the sample extract volume, (i.e. 33 mL)
1.1.7 Allow samples to settle for one-half to one hour.
1.1.8 Pipet 10 mL of DDW into pre-labeled Falcon polypropylene sampling tubes using a
Brinkman repeator pipet. Next, pipet 1.0 mL of sample into each tube of DDW, rinsing
the pipet tube once with each new sample before dispensing. These volumes will give a
dilution factor of 11 (11/1) for the extract concentration. Record dilution volumes used on
the trace and spreadsheet.
1.1.9 Analyze P concentration using standard procedures outlined in Davis & Simmons (19)
lab manual.
1.1.10 Standards are made up in sample matrix and should account for any P-contamination
introduced by the reagents. Matrix, and procedural blanks are included with each run to
test for contamination or interferences. Matrix and procedural blanks should ideally be the
same and should approximate the intercept value of the standard regression.
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SOPs for Determining Total Phosphorus, Available Phosphorus,
and Siogenic Silica Concentrations of Lake Michigan
Sediments and Sediment Trap Material Volume 3, Chapter^
1.1.11 Determine P concentration of sample extract (applying dilution factor) against standard
regression, then calculate P mass for the extract volume.
1.1.12 Calculate Available P-content of sediment by dividing mass by sample weight. Report
units as ug P/mg DW.
1.2 Standards
1.2.1 Standards are made in the same matrix as samples. The 30 mL NaOH/3 mL HC1 sample
is diluted 1/11 before analysis, so for standards in 100 mL flask add 8.2 mL 0. IN NaOH
and 0.82 mL 1.0 N HC1 and bring up to volume.
1 1.2 Create standards from two stock of 1000 ug P/L. Make up in designated 100 mL flasks.
1.2.3 Expected Range for samples using the extract sample dilution of 11 is 2-20 ug/L.
Suggested Stds: 2,4,8, 12, 16,20.
1.3 Glassware
Standards are created in dedicated 100 mL volumetric flasks. Flasks are acid washed with 10%
HCL and rinsed six times with DDW between each use. Sediment samples are extracted in new,
used once only, polypropylene centrifuge tubes which have been shown to be free of contaminants.
Extracts are neutralized and diluted in Falcon polypropylene test tubes which have been shown to
be free of contaminants and then analyzed from these tubes
1.4 Waste
All sample wastes are collected and neutralized prior to disposal down the drain. There are no
known toxic wastes generated by these procedures which would require special handling and
disposal.
2.0 Total Phosphorus
2.1 Total phosphorus content is determined using a modification of the combustion and hot HC1
extraction procedure of Andersen (1976).
Prior to Anul\sis:
2.2.1 Weigh 25-35 mg (for box cores) or 75-100 mg (for ponars or sandy samples) of freeze-
dried, homogenized, sediment on the Mettler AT250 balance. Pre-calibrate balance with
standard weights. Weigh material onto pre-tared glassine weigh paper. Transfer to pre-
numbered (etched), acid-washed Pyrex test tube. Record sample weight and vial number
in log book.
2.2.2 Include one procedural blank (no sediment), and two reference sediment samples with
each batch.
2.2.3 Remove caps and cover tubes uith Al-foil. Combust Pyrex tubes vuth sediment at 500°C
for two hours.
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SOPs for Determining Total Phosphorus, Available Phosphorus,
and Biogenic Silica Concentrations of Lake Michigan
Volume 3, Chapter 2 Sediments and Sediment Trap Material
On Day of Analysis:
2.2.4 Add 25 mL of 1.0 N HC1 using the Eppendorf pipet. Cap tightly and mix well.
2.2.5 Place tubes into a boiling water bath (99°C) for 30 minutes.
2.2.6 After cooling, add 25 mL of DDW to bring sample volume to 50 mL. Mix well and let
samples sit for one hour to cool and settle. Record the sample extract volume.
2.2.7 Pipet 10 mL of DDW into pre-labeled Falcon polypropylene sampling tubes using a
Brinkman repeator pipet. Next, pipet 0.4 mL of sample into each tube of DDW, rinsing
the pipet tube once with each new sample before dispensing. These volumes give a
sample dilution factor of 26 (10.4/0.4) for the extract concentration. Record dilution
volumes on the trace and in the spreadsheet.
2.2.8 Analyze P concentration on the Auto Analyzer II using the standard molybdate/ascorbic
acid procedures described by Davis and Simmons (1979). There is no need to neutralize
the sample prior to analysis. Blanks are included to test for contamination or
interferences.
2.2.9 Determine P concentration of sample extract (applying dilution factor) against standard
regression, then calculate P mass for the extract volume.
2.2.10 Calculate P-content of sediment by dividing mass by sample weight. Report units as
ugP/mg DW.
2.3 Standards
2.3.1 Standards are made in the same matrix as samples. The 50 mL sample extract is in 0.5N
HC1 and is diluted by a factor of 26 to make a final sample matrix of 0.019N HC1. For
standards, add 1.9 mL 1 .ON HC1 to the 100 mL flask and bring up to volume.
2.3.2 Create standards from 2° stock of 1000 ug P/L. Make up in designated 100 mL flasks.
2.3.3 Expected Range for samples using the extract sample dilution of 26 is 10-100 ug/L.
2.4 Glassware
Standards are created in dedicated 100 mL volumetric flasks. Flasks are acid washed with 10%
HC1 and nnsed six times with DDW between each use. Sediment samples are extracted in acid-
washed Pyrex test tubes. Between analyses the Pyrex tubes are nnsed with DDW three times to
remove any sediment or acid residue and then soaked in a 25% HCI batch for at least 24 hrs.
Tubes are rinsed six times with DDW and inverted on clean paper towels to dry. Extracts are
diluted m Falcon polypropylene test tubes which have been shown to be free of contaminants and
then analyzed from these tubes.
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SOPs for Determining Total Phosphorus, Available Phosphorus,
and Biogenic Silica Concentrations of Lake Michigan
Sediments and Sediment Trap Material Volumes, Chapter 2
2.5 Waste
All sample wastes are collected and neutralized prior to disposal down the drain. There are no
known toxic wastes generated by these procedures which would require special handling and
disposal.
3.0 Biogenic Silica
3.1 Biogenic silica refers to silica which has been assimilated by diatoms and incorporated into their
frustules as an amorphous polymorph of silica (Krausse et al., 1983). Biogenic silica is
determined using a wet alkaline digestion with 1 % Na2CO, at 85°C. Mineral contributions to the
silica pool are corrected for by using a timed extraction procedure and the differential rates of
extraction for biogenic bersus mineral forms (DeMaster 1981).
Prior to Analysis:
3.1.1 Weigh 25-35 mg for clay/silty sediment or 50-70 mg for sandy sediment of freeze-dried,
homogenized, sediment on the Mettler AT250 balance. Calibrate balance at beginning
and end of session with standard weights for 10, 30, 50 mg. Weigh material onto pre-
tared glassine weigh paper. Transfer to pre-labeled, 50 mL polypropylene tubes. Record
sample weight and vial number in log book.
3.1.2 Include one procedural blank (no sediment), and two reference sediment samples with
each batch.
On day of Analysis:
3.1.3 Pre-label Falcon sample tubes (five tubes for each sample: Tl, T2, T3, T4, T5).
3.1.4 Fill each sample tube with 0.19 mL IN HCI and then add 10 mL DDW.
3.1.5 Add 40 mL of 1 % Na:CO3 to each sediment sample using the Brinkman repeater pipet.
Cap tightly and mix well.
3.1.6 Place centrifuge tubes into a 85 °C shaking water bath (100 rpm) and shake for six hours
taking 1.0 mL sub-samples at intervals of 2, 3, 4, 5, and 6 hours. Set up samples in
batches of 10-12 for subsampling. Stagger a 2nd batch 15 minutes apart from 1"
3.1.7 Sub-sample each sample as follows: Stop shaker. Remove samples. Mix well and then let
settle for five minutes. Remove I mL of extract from sample and add it to sample tube
with 10mLmLDDWand0.19mLof IN HCL. NB: Be careful to get consistent
volumes when pipetting the hot samples. Keep tip at constant temp. Need to calibrate
actual volume withdrawn using 1.0 mL pipet. Return sediment samples to bath ASAP and
re-start shaker.
3.1.8 Analyze SiO; concentration using standard procedures. Blanks are included to test for
contamination or interferences.
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SOPs for Determining Total Phosphorus, Available Phosphorus,
and Biogenic Silica Concentrations of Lake Michigan
Volume 3, Chapter 2 Sediments and Sediment Trap Material
3.1.9 Determine SiO: concentration of sample extract against regression for 10-50 ppm
standards. Don't multiply by any sample dilution factor. It is already accounted for by
treating standards and samples identically. Calculate SiO, mass for the extract volume
(40 mL).
3.1.10 Calculate SiO,-content of sediment by dividing mass by sample weight. Report units as
mg SiO:/g DW.
Standards:
3.1.11 Make up five SiO, standards and a reagent blank in 50 mL of 1 % Na,CO,.
Suggested range is 10-50 mg/L for BOX CORES and 2-20 mg/L for PONARS.
Create standards from primary SiO: stock of 1000 mg/L.
Make standards in designated 50 mL polypropylene tubes.
3.1.12 Standards get diluted identically to samples so there is no dilution factor for samples.
Dilute 1 mL of each standard with 0.19 mL IN HC1 and 10 mL DDW. NB: Standards
are not heated.
3.2 Glassware
Standards are created in polypropylene centrifuge tubes which have been shown to be free of
contaminants. Tubes are dedicated for each specific standard concentration. Tubes are acid
washed with 10% HC1 and rinsed 6 times with DDW between each use. Sediment samples are
extracted in new, used once only, polypropylene centrifuge tubes which have been shown to be
free of contaminants. Extracts are neutralized and diluted in Falcon polypropylene test tubes
which have been shown to be free of contaminants and then analyzed from these tubes
3.3 Waste
All sample wastes are collected and neutralized prior to disposal down the drain. There are no
known toxic wastes generated by these procedures which would require special handling and
disposal.
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Standard Operating Procedure for
Perkin Elmer CHN Analyzer (Model 2400)
Brian J. Eadie
NOAA/Great Lakes Environmental Research Lab
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105-1593
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Standard Operating Procedure for
Perkin Elmer CHN Analyzer (Model 2400)
This is an appendix to the EPA Lake Michigan Mass Balance QAPP for GLERL Sediment trap and
Sediment samples
Items in italics are instrument control panel functions
For more detailed information, see the instrument manual provided by Perkin Elmer.
1.0 Initialization
I. I Check that furnace is on (parameter 12)
1.2 Check oven temperatures
1.2.1 Combustion oven 925° C (parameter 7)
1.2.2 Reduction oven 640° C (parameter 8)
1.3 Check pressure gauges
O2 15psi
He 20 psi
Air 60 psi
1.4 Purge He and O2 gases when:
1.4.1 Gases have been changed
1.4.2 Reduction or combustion tubes have been changed
1.4.3 Instrument has been down a long time
He 120 seconds
02 20 seconds
1.5 Run a combustion zone leak test
Enter diagnostics
Gas
Leak test
Combustion zone leak test -2
1.5.1 Pressure will rise to above 760 psi and stabilize. To pass, it should stay above 760 psi.
Enter diagnostics to get out of diagnostic mode, when the test has been completed.
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SOP for Perkin Elmer CHN Analyzer (Model 2400) Volume 3, Chapter 2
1.6 Release H-valve to reduce pressure caused by the combustion zone leak test.
Enter Diagnostic
Gas
Valve
7-on ( wait approx. one minute )
Enter diagnostic to close valve and get out of diagnostic mode.
1.7 List parameters
Hit Monitor key, press monitor key again to go to standby.
1.8 Calibrate autobalance - repeat three times, see autobalance manual.
1.9 Sediment trap Sample Preparation (after freeze drying)
1.9.1 Separate each interval of the core or sediment trap sample/composite into an unacidified
(total carbon and nitrogen) segment, and acidified (organic carbon and nitrogen) segment.
1.9.2 Unacidified segment - grind and dry @ 80-90°C.
1.9.3 Acidified segment - add IN HC1, shake overnight, dry and regrind.
1.9.4 Weigh each sediment interval within a tin capsule, which must be tared before weighing
the sediment.
1.9.5 It's critical to follow sterile handling procedures with the tweezers (all handling
instruments), and the aluminum foil workspace. Wipe instruments and workspace with
tissue paper between samples.
1.9.6 Fold tin capsule so as to easily pass through the CKN entry chamber hole without getting
caught by its edges.
1.10 Run five instrument blanks
1.10.1 Hit single run - press blank then 5.
1.10.2 Blanks should reproduce within the following range:
Carbon ±30
Hydrogen ±100
Nitrogen ±16
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Volumes, Chapter 2 SOP for Perkin Elmer CHN Analyzer (Model 2400)
1.11 Standardize instrument - sets groundwork for the K-factors.
1.11.1 Weigh four acetanilide samples between 2-3 mg
1.11.2 Run three as K-factors; should get three K-factors within tolerance.
Hit single run, K-factor then SI and weight.
Carbon 16 ±3.5
Hydrogen 50 ± 20
Nitrogen 6 ± 3
1.11.3 Run acetanilide as a sample. Certification that instrument is within tolerances. (±2%
acetanilide ). Run over if not within tolerance.
Carbon 71.09 %
Hydrogen 6.7 %
Nitrogen 10.36 %
1.11.4 Run two tin blanks.
1.12 Single run vs Autorun
1.12.1 Single run mode: Run samples one at a time. Hit single run, then sample; enter ID and
corresponding weight.
1.12.2 Autorun mode: Run up to 60 consecutive samples in a carousel. Load samples within
carousel, hit autorun key. Press 4 RP (reset-print), then 1 Reset; this will reset internal
counter to 1 which coincides with the first slot in the carousel. Enter the ID number and
weight of the first sample, press enter then start. This starts the autocarousel, then
continue to enter ID and weight for the remaining samples.
1.13 Criteria for acceptance of data
1.13.1 Run standard as samples every 10 samples, result should be within 2 sd of average value.
1.13.2 Number of counts for Nitrogen (NR-ZR) >100 or sample wns too small and the
performance of the instrument will not be acceptable.
1.14 Shut off the He tank at the end of the day.
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Quality Assurance Plan for
the Use of Sediment Traps
Brian J. Eadie
NOAA/Great Lakes Environmental Research Lab
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105-1593
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Quality Assurance Plan for the Use of Sediment Traps
1.0 Project Title
Use of Sediment Traps for Environmental/Ecosystem Indicator Measurements in the Great
Lakes.
2.0 General Overview
Sediment traps, which passively collect paniculate material settling out of the water
column, have been used with success in the Great Lakes and elsewhere. Traps provide an
efficient tool for the collection of integrated samples for detailed analysis. Measuring the
mass collected allows us to calculate the gross downward flux of paniculate matter and
associated constituents and to calculate settling velocities. The difference between this
measurement of gross downward flux and the net sediment accumulation rate is a good
long term approximation of the flux due to sediment resuspension.' Under stable, stratified
conditions, shorter term resuspension fluxes can be estimated from trap flux profiles at a
single station.
In the Great Lakes, as in most aquatic systems, the rapid and efficient processes of
sorption and settling scavenge contaminants from the water column with the result that the
largest fraction of persistent trace contaminant inventories presently reside in sediments.
However, studies of the long-term behavior of certain fallout radionuclides and stable
contaminants in the Great Lakes have shown that higher levels persist in the lakes than
expected if settling and burial were the sole transport process. Materials return from
sediments due primarily to resuspensipn. Constituents initially transferred to sediments
are homogenized via bioturbation creating a mixed layer corresponding to a decade or
more of accumulation. These are resuspended back into the water column during the
isothermal period and are available for uptake by pelagic biota. It is now accepted that the
internal recycling caused by the coupled processes of bioturbation and resuspension are
responsible for the continuing elevated concentrations of trace contaminants (e.g. PCB,
DDT) in fish and the lag in lake response to nutrient abatement.
Since 1977, GLERL has been examining the processes of particle flux and resuspension
through the use of sediment traps, passive cylinders deployed to intercept materials
settling to the bottom. We have learned much about the transport of mass, contaminants
and tracers and the results are now routinely incorporated into program sampling and
modeling strategies and management considerations. Although the traps themselves are
relatively inexpensive, the logistics of deployment and retrieval are quite expensive
restricting both where and how frequently we can sample.
Simple next generation traps, that have sequencing capability for multiple samples per
deployment, were developed by several investigators but we were not able to identify
sufficient funds until recently. After discussions with a number of scientists around the
world that use traps, careful consideration of working designs, our own experience with
trapping in the lakes, and the types of experiments that we wanted to conduct, we settled
on a design with 23 sampling intervals per deployment. After almost a \ear of effort, the
prototype was completed in July, 1990. The trap was subsequent!) deployed for three
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QAP for the Use of Sediment Traps Volume 3, Chapter^
long tests, including an overwinter deployment. All deployments were completely
successful. Six slightly modified copies of this trap were subsequently constructed. We
see this instrument as a major tool in our future investigations of lake processes (such as
short term sediment transport immediately after ice-out, high frequency bottom boundary
fluxes and other logistically difficult experiments) and a valuable integrating sampler for
the EMAP program.
3.0 Objectives
3.1 To quantify the seasonal flux of paniculate matter.
3.2 To provide subsamples for diatom analysis to EPA-EMAP
4.0 Experimental Design Features
Since the lake is shallow, many nuisance and toxic constituent concentrations and removal
rates are mediated by internal recycling dominated by episodic sediment resuspension.
Sediment traps that collect sufficient mass of settling paniculate matter over relatively
short time scales (weeks) have been built and successfully tested by our laboratory. These
have been deployed in southern Lake Michigan and subsamples will be made available for
EMAP analysis. Initial analysis is restricted to diatom counting, but other EMAP-useful
parameters may be developed.
GLERLs 7 autosequencing traps were deployed at its long term station (35 Km offshore in
southeastern Lake Michigan; 100 m total depth) in mid-October, 1991. The 23 samplers
in each trap were programmed for equal time intervals of 15 days with retrieval scheduled
for late September 1992. The traps were deployed on a single line at depths of 15, 35
(duplicate), 75, 90, and 95 (duplicate) meters. There will be (7*23=161) samples,
including 46 duplicates.
Upon retrieval, the samples will be allowed to settle, the overlying water siphoned off and
the slurry will be freeze dried in an ultra clean freeze drier. Samples will be weighed and
fluxes calculated. The samples will be split and a portion made available for diatom
analysis by Dr E. Stoermer (U MI), who will be quantifying diatoms for EMAP. The
major fraction of these samples will be analyzed by GLERL or distributed to other
collaborating investigators as arranged prior to any EMAP discussions.
The only analytical measurement being proposed by GLERL is for the calculation of mass
flux: the procedure for this is well developed and frequently reported on by GLERL.
Briefly, 8" diameter cylindrical sediment traps of aspect ratio 5:1 will be deployed and
retrieved after 23 equally spaced 15 day intervals. The collection efficiency of these traps
is close to 100% and precision, as represented by a coefficient uf variation, is
approximately 10%. Trap samples will be stored at 4°C in transport. After settling for
24 hours, overlying water will be carefully siphoned off and the remainder will be freeze
dried, weighed and the mass flux calculated. Subsamples will he separated for diatom
analysis with the help of Dr. Stoermer (UMI).
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Volume 3, Chapter 2
QAP for the Use of Sediment Traps
5.0 Workshop - Longer Term EMAP Strategy
Sediment traps and sediments are sample matrices that are important in the EMAP
program but have not been well thought through. Much has been learned over the past
20 years about how to interpret sediment trap samples and the information stored in
sediments. Since similar analyses will be made on both sediments and traps, GLERL will
convene a small workshop to develop a sampling protocol for these media that meets the
goals of the program and is technically defensible. Core participants should include:
David Edgington
John Robbins
Alena Mudroch
] Val Klump
Rick Bourbonniere
Steve Eisenreich
Anders Andren
Gene Stoermer
Another Biologist (Russ Kreis ?)
Brian Eadie NOAA/GLERL
Fernando Rosa CCIW/NWRI
U Wisconsin-Milwaukee
NOAA/GLERL
CCIW/NWRI
U Wisconsin-Milwaukee
CCIW/NWRI
U Minnesota
U Wisconsin-Madison
U Michigan
radionuclides
radionuclides
geochemistry
geochemistry
organics
contaminants
contaminants
diatoms
traps, carbon
traps, nutrients
6.0 Project Timetable
This project will begin on May 1, 1992 and continue through September 30, 1993.
Item
Proposal
Funding
Initial Deployment
Retrieval
Redeployment undecided
Mass Measurements
Sample Distribution
Deployment Final Report
Workshop
Workshop Report/Recommendations
1991 1992
Oct Jan Apr Jul
1993
Oct Jan Apr Jul Oct
X
X
XX
XX
XX
XX
XX
7.0 Project Responsibilities
Brian J. Eadie (313) 668-2281 will be the principal investigator. A student will be hired
to assist in the field work during the summer and sample preparation during Fall and
Winter.
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QAP for the Use of Sediment Traps Volume 3, Chapter?
8.0 Communications
Regular, daily contact will be maintained between Dr. Eadie and others working on this
project. Progress will be discussed with the EPA-EMAP contract officer at quarterly
intervals. A final report will be submitted at the completion of the project.
9.0 Quality Control/Quality Assurance
9.1 Trap Collections: Duplicate traps have been deployed at two depths in this study.
GLERL has completed a study of trap precision and has found the sample
collection/handling coefficient of variation to be less than 10%.
9.2 Sample Tracking Procedure: Trap samples will be stored prior to freeze drying in
precleaned 60 mL poisoned (5 mL CHCI3) polyethylene bottles. After drying, they will
be weighed and transferred into precleaned scintillation vials for storage in a freezer. Data
sheets will be kept by Dr. Eadie and all data will be entered into a PC for ready availability
by interested parties.
9.3 Calibration Procedures and Preventive Maintenance: All trap samples will be weighed on
a GLERL analytical balance, regularly maintained by a service contract and calibrated
with known standard weights.
9.4 All pertinent data will be entered into computer files for easy access. Data reduction
procedures established at GLERL will be used. The PI will review all data for validity.
Data will be reported to the EPA and to other EMAP participants upon request.
Parameter Reference Conditions Precision Accuracy
Mass Flux Eadie, et Post Collection ±10% ±10%
al., 1984
10.0 Reporting
A report will be generated on the biweekly mass fluxes at the collection site. A separate
report, with recommendations for sample strategy using traps/sediments for EMAP goals,
will be a product of the proposed workshop.
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Volume 3
Chapter 3: Radiochemistry
-------
II
Standard Operating Procedure for
Primary Productivity Using 14C:
Laboratory Procedures
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
April 13,1994
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Standard Operating Procedure for
Primary Productivity Using 14C:
Laboratory Procedures
1.0 Scope and Application
This method is intended to determine primary productivity from Great Lakes waters.
2.0 Summary of Method
The radioactivity of the filter containing the algal cells is determined by liquid scintillation
counting. Calculation of the productivity parameter requires information about the total inorganic
carbon available in the incubation vessel, the length of time of incubation, the chlorophyll content
of the incubated sample and specific activity of the radiotracer.
3.0 Safety and Waste Handling
3.1 14C is classified as a low-level beta emitter. Wearing personal protective laboratory gear at all
times when in contact with the inoculated vials, can effectively prevent any exposure.
3.2 All spills of radioactive or suspected radioactive materials must be immediately reported to the
CRL Safety and Health Officer and decontaminated immediately.
3.3 All radioactive samples and standards should be properly labeled with the isotope and activity
indicated and properly stored in designated locations.
3.4 Under the Atomic Energy Act of 1954 a license is required designating the radioactive source, it's
use as applicable to the laboratories and conditions by which the licensed material should be used.
The current license (#12-10243-01) expires on March 31, 1995.
4.0 Apparatus
4.1 Packard TRI-CARB 4430 Liquid Scintillation Counter
4.2 Acetone
4.3 Kimwipes
4.4 Nucleopore filters 0.2 (am pore size, 2.5 cm diameter
4.5 Rubber bulbs (2)
4.6 9" Pasteur disposable pipets (sterilized)
4.7 200 mL volumetric flask
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SOP for Primary Productivity Using '"C:
Laboratory Procedures Volume 3, Chapter 3
4.8 250 mL Flask with sidearm
4.9 Gelco 250 cc filtering system
4.10 Plastic filter plate
4.11 9 mm diameter rubber tubing
4.12 Glass funnel
4.13 Filter forcepts
4.14 1 mL volumetric pipet
4.15 Adjustable 10 mL Macropipettor
4.16 Gas-tight 20 mL vials
5.0 Reagents
5.1 Buffered water: Using Super Q water available in the chemistry lab, add 0.18 N NaOH dropwise
until pH increases to 9.5. Prepare at least 400 mL of buffered water for every ampoule of I4C one
expected to use. Store buffered water in refrigerator at 5°C until ready to use in order to be at the
same temperature as the I4C solution.
5.2 Filtered water: (This procedure may be performed in the field)
5.2.1 After ensuring that all glassware has been properly cleaned, wrap in brown paper and
autoclave on high temperature setting (wrap cycle). Keep glassware in wrapping paper
until directly before use to avoid contamination.
5.2.2 Set up filtration apparatus using Nucleopore 0.2 |am filters and cover filter funnel until
ready to use.
5.2.3 Pour cooled buffered water (Section 5.1) into filtration funnel and turn on vacuum
pressure. Cover the funnel while the solution is being filtered.
5.2.4 Replace the filter as necessary and prepare at least 400 mL of filtered water per ampoule
14C.
5.2.5 Following the filtration, disassemble the filtration apparatus and cover the filter flask with
parafilm until ready to use.
5.2.6 Decant approximately 20 mL of the filtered water into a second beaker to serve as wash
water. Cover the beaker with parafilm.
5.3 0.5 N Hydrochloric Acid Solution: Add 4.25 mL of concentrated hydorchloric acid to 800 mL of
deionized water in a 1 L volumetric flask Adjust volume to one lifer with deionized water.
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SOP for Primary Productivity Using 14C:
Volume 3, Chapter 3 Laboratory Procedures
6.0 Stock Solution
6.1 Ensure all the liquid in the top portion of the I4C ampoule has been shaken down into the lower
Section. Using a diamond pencil or a rough-sided file, score the weak edge of the UC ampoule.
6.2 Carefully, placing the ampoule between thumb and forefinger, break off the top at the weakened
neck. Pipette the contents into a 200 mL volumetric flask using the sterilized Pasteur pipettes.
6.3 Using a disposable glass pipet, carefully rinse out both the top and bottom portions of the ampoule
using the filtered water and add wash solution to the flask. Repeat three times.
6.4 Adjust volume in the flask to 200 mL using filtered water. With stopper firmly in place, vortex for
several seconds to mix solution.
6.5 Pour the stock solution into a cleaned and autoclaved Nalgene container with an opening wide
enough to accommodate a macropipettor.
6.6 Dispense 13 mL of the stock solution into a 20 mL gas tight vial and screw cap on tightly. Fifteen
vials can be prepared from each ampoule which is diluted. For each set of incubated bottles, one
vial will be used for inoculation.
6.7 Label each vial with "Stock #" Assign a chronological number beginning with number one,
indicating the first ampoule diluted for that year. Store in a radioactively labeled (specific activity)
container at 5°C until ready for analysis.
6.8 For quality control measures, using a volumetric pipet, dilute a 1 mL sample of the freshly made
stock solution into 300 mL water (pH 8.5).
6.9 Vortex dilution several times and dispense a 1 mL sample into a scintillation vial.
6.10 Add 20 mL of Ecoscint plus 1 mL phenoethylamine. Clearly label cap with year and the stock
number which the sample represents. This number will correspond with each stock solution which
is subsequently made. This v ill be analyzed along with the other samples to determine the
beginning activity of the stock solution.
6.11 If possible, set aside one vial from each stock solution to determine the final activity, repeating
Steps 6.5 through 6.7.
7.0 Instrument Calibration Procedure
7.1 The Liquid Scintillation counter should be calibrated once per month and directly prior to sample
analysis. The results of the monthly calibration are included in the biology report.
7.2 Refer to the Standard Operating Procedures for calibration instruction.
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SOP for Primary Productivity Using "C:
Laboratory Procedures Volume 3, Chapters
8.0 Analytical Procedures
8.1 After 24 hours, remove scintillation vials and clean the outside of each vial by holding the vial by
the cap and wiping the outside walls with an acetone-impregnated tissue and drying with a clean
dry tissue. Due to the high flammability of acetone, this procedure should be performed
underneath a hood.
8.2 Remove the scintillation vial from sample storage and place them in the scintillation trays and
allow the samples to dark adapt for 24 hours.
8.3 Ensure all that the numbers on the caps are readable and in chronological order.
8.4 Place the vials, handling only the caps, into the counter.
8.5 Each series of samples, field standards and background (laboratory blanks) should be counted for
20 minutes. Check output screen on scintillation counter for the value of two sigmas.
8.6 Counting efficiency should be obtained to obtain results as DPM. Most scintillation counters
output results as DPM if a set of quenched standards are provided.
9.0 Sample Calculations
9.1 The carbon uptake can be calculated as follows:
C
12 _ C'4U xC'2A x 1.06
Cl4Axt
Where: t = exposure time (hours)
Cn = carbon uptake rate (mg/C/L/Hr)
CI4U = sample activity (DPM)
C/4A = added activity (DPM)
CnA - inorganic carbon available (mg/L) determined by means of pH
and alkalinity or by direct determination of total inorganic
carbon
1.06 = isotope effect constant
9.2 Normalize the carbon uptake rate to chlorophyll content:
C/: uptake
CHI
Where: CHI = Chlorophyll concentration mg/l
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SOP for Primary Productivity Using "C:
Volume 3, Chapter 3 Laboratory Procedures
9.3 For each sample incubated, report
9.3.1 Unadjusted production rate, mg C/L/hr.
9.3.2 Normalize production rate, mg C/L/hr/mg chlorophyll
9.3.3 Light intensity at which sample was incubated
9.3.4 Length of incubation
10.0 Waste Calculations
10.1 While in the field, it is possible to make a rough estimate of the activity for each waste container
(See Standard Operating Procedure for Primary Productivity Using I4C Field Procedure
Section 2.2.2). This estimate is necessary for shipping and storage purposes and can be
approximated in uCi/mL.
10.2 A 14C waste form must be properly filled out for each survey. A example of this form can be
found in the appendix. The actual waste values are required to be reported in total uCi and the
form submitted to the CRL Health and Safety Officer.
10.3 Following analysis at CRL in the Scintillation Counter, the DPM values for the waste are
attainable, thus allowing the actual uCi values to be determined.
10.4 For each cubic, multiply the DPM value by the waste volume, V, to obtain the total DPM per
cubic.
10.5 Average the DPM values for all Total Activity vials. This factor corresponds with 0.01667 uCi,
the activity of 1 mL (5 uCi) of the stock solution added to a 300 mL sample. The Total Activity is
the actual specific activity of a 1 mL sample from the incubation bottles.
10.6 Multiply V by 0.01667 uCi and divide this by the Total Activity average. Repeat for each waste
cubie.
10.7 Waste calculations should show individual uCi values as well as the total amount of waste
generated per survey. See attached Appendix 1 for sample calculations.
11.0 Quality Control
Prior to the sample analysis in the Liquid Scintillation Counter, the background and efficiency of
the counter is calculated. See Standard Operating Procedures for the Liquid Scintillation
Counter.
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SOP for Primary Productivity Using "C:
Volume 3, Chapter 3 Laboratory Procedures
Appendix 1.
Radioactivity calculations for14C waste water (approximate)
I mL stock solution = 5 jaCi
1 mL added to 300 mL HO
5 |aCi/300mL = 0.016671nCi/mL
#1 (46547.4 DPM/mL)(18000mL) = 8.38xl08DPM
#2(41184.7 DPM/mL)(18000mL) = 7.41xl08DPM
#3(45052.6DPM/mL)(14400mL) = 6.48xl08 DPM
#4 (51063.5 DPM/mL)(14400mL) = 7.35xl08DPM
#5 (4534.88 DPM/mL)(7200mL) = 3.26xl07 DPM
#6(13642.3 DPM/mL)(18000mL) = 2.45xl08DPM
#7 (14388.6 DPM/mL)(16000mL) = 2.30xl08 DPM
0.01667|aCi corresponds to 36117.92 DPM (avg. of all vials)
#1 (8.38xlOs DPM)(O.O1667^Ci/mL)/36117.92 = 386.70 ^Ci
#2 (7.41xl08 DPM)(O.O1667nCi/mL)/36117.92 = 342.15 jiCi
#3 (6.48x108 DPM)(O.O1667|jCi/mL)/36117.92 = 299.43 ^Ci
#4(7.35xl08 DPM)(O.O1667nCi/mL)/36117.92 = 339.38 ^Ci
#5 (3.26xl07 DPM)(O.O1667^iCi/mL)/36117.92 = 15.07 ^Ci
#6 (2.45xl08 DPM)(O.O1667|aCi/mL)/36117.92 = 113.34 jiCi
#7 (2.30xl08 DPM)(O.O1667|uCi/mLV36117.92 = 106.26 pCi
Total: 1602.33 pCi
1.60 mCi -Total waste
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Protocol for Standard Analysis for
Cesium-137
John A. Robbins
Great Lakes Environmental Research Laboratory
2205 Commonwealth Boulevard
Ann Arbor, Ml 48105
and
David N. Edgington
Center for Great Lakes Studies
University of Wisconsin
600 East Greenfield Avenue
Milwaukee, Wl 53204
June 1994
-------
Protocol for Standard Analysis for Cesium-137
1.0 Sample Preparation and Characteristics
The following comments apply principally to the fine-grained, highly inorganic sediments (organic
matter <5%) recovered from box or gravity cores from the Great Lakes. Preferably samples should
be freeze-dried and disaggregated lightly using mortar and pestle so as to pass through a 0.50 mm
sieve. In rare instances where there are pieces resistant to disaggregation, such as shells, fibrous
organic matter, cinders or stones, they should be removed anu their removal noted in comment
spaces of final records.
2.0 Sample Geometry
For sample weights above 6 grams, use the standard (150 ml) snap cap counting vials ("standard
geometry") which have been cleaned, rinsed with distilled water and dried. When possible load
vials with 20.0 + 0.2 g of dry sediment. When less sediment is available, use the entire portion for
counting. When less than 6 g of sediment is available, use plastic scintillation vials ("small sample
geometry"). In either case, record net weights to at least three digits to the right of the decimal
point. Level out the surface of the sediments in the vials by tapping them on a counter or by other
effective techniques. The height of the surface above the outside bottom of the vial should vary by
no more than 1 mm. Either before or after counting, estimate the height of the surface above the
outside bottom of the container (to ± 0.05 cm).
3.0 Sample Counting
Check the condition of the polyethylene protective sheet over the top of the detector housing.
Replace if dirty. Place the appropriate retainer collar for standard or small sample geometry over
the detector housing. Gently place vial in the center of the retainer collar making sure that it rests
on the top of the detector housing. Do not force the sample on to the housing top since it is made
of thin aluminum and can easily be damaged. If the sample is not seating properly, remove it and
determine the cause of the problem. Gently close the clam-shell top of the shield. Zero (ALT-3)
the appropriate multichannel analyzer (MCA) and initialize counting (ALT-1). Each sample
should generally be counted from 12 to 24 hours.
4.0 MCA Data Processing and File Storage
On completion of counting, stop the MCA (ALT-2) and transfer the spectrum from the
Multichannel Buffer (MCB) to the computer (ALT-5). Switch to the computer-based spectrum for
further data processing (ALT-6). Select the appropriate region of interest (ROI) file (usually
DET*.ROI) using ALT-R and ALT-S. This will illuminate about 1 1 regions of which two should
be manually reset after each counting to take care of the possibility of small system gain shifts.
The two regions are for Cs-137 (661.6 KeV) and K-40 (1460.8 KeV). Locate these regions by
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Protocol for Standard Analysis for
Cesium-137 Volume 3, Chapter 3
first holding down the shift key while depressing the left or right arrow key to locate the left or
right edge of the appropriate ROI. Then hole down the CRTL key while using the left or right
arrow keys to locate the photopeak maximum. Use the DEL key to remove the existing ROI for
the peak and the INS key to redefine it for the present spectrum. Having established correct ROIs,
insert the current 1.4 mb floppy disk into the appropriate hard drive, and instruct the system to
save the MCA file (ALT-F then ALT-S). In response to the resultant query, enter the current file
name for the spectrum. This will always have the form A:D*_xxxx.CHN where * is the detector
index 1-4 and xxxx is the record number (for example D1_0626.CHN). This number can be
inferred from inspection of the printed reports in the counting lab. Enter this information.
Following entry (using RETURN) enter requested information on the sample analyzed. This
generally has the form: lake abbreviation and year, sample code, section interval, net sample
weight and height of sample above bottom of the vial (for example LM94 41A 10-12 CM 20.0265
G H=l .5 cm). In addition to filing the entire spectral record, produce a hard copy report by using
ALT-F then ALT-T and entering PRN in response to the query. The top of the printed output
should be labeled with the file name of the report (D1_0626.RPT). The report may be stored on
the floppy using ALT-F the ALT-T and entering A:D*_xxxx.RPT to the query.
5.0 Detector Stability Check
Count the radiocesium standard sediment (standard geometry AMS-86-1 20G) on detectors #1 and
#2 on alternate weeks for at least three hours. This standard has three detectable gammas, the one
from Cs-137 (661.6 KeV) and two from Co-60 (1173.2 and 1332.5 KeV). The appropriate ROIs
can be loaded from files AMS1 .ROI or AMS2.ROI. Files should be saved on the appropriate
floppy for the detector numbered in sequence with the samples. A report should be printed as well
and the counting time plus net counts and error for each of the three photopeaks entered in
GAM 1.DAT or GAM2.DAT files for use with stability monitoring programs.
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Determination of the Activity of
Lead-210 in Sediments and Soils
David N. Edgington
Great Lakes Water Institute
University of Wisconsin
Milwaukee, Wl
and
John A. Bobbins
Great Lakes Environmental Research Laboratory
NOAA
Ann Arbor, Ml
1975
-------
Determination of the Activity of
Lead-210 in Sediments and Soils
1.0 Theory
Geochronology with the naturally occurring Pb-210 is based on the principle that the isotope has
been continuously delivered to the earth's surface and undergoes continuous radioactive decav
following incorporation into steadily accumulating sediments. The activity of Pb-210 in sections
from sediment cores taken from lakes is used to determine the rate of that sediment accumulation
with time Anthea lake. In this method the activity of the Pb-210 granddaughter, Po-210, is
actually measured, as Pb-210 is a weak beta emitter and is not readily detected. Po-210 is the
alpha emitting granddaughter of Pb-210, and can be used to represent the actual Pb-210
concentration in each sample because the two isotopes are assumed to be in seqular equilibrium.
The daughter is used because in an acidic solution it will spontaneously plate on to a copper disk,
which can then be counted on a high resolution alpha specirometry system. A yield monitor,
Po-208, is added to each sample so that the exact activity of Po-210 can be determined.
Sediment cores are collected with a gravity or box corer. The samples are extruded at known
intervals, usually 1-2 cm, and placed into preweighed bottles. The bottles are weighed again and
placed in a 60&C oven and dried to constant weight. The difference in wet and dry weight is used
to calculate the porosity of the sediment. The samples are then ground to a fine powder and stored
until used.
2.0 Counting
The Po-210 and Po-208 concentration on each disk is determined by alpha spectrometry using
silicon surface barrier detectors and a multi-channel analyzer system. The disks are counted in
vacuum chambers to enhance the resolution of the Po-210 and Po-208 alpha peaks. The outputs
from the silicon detectors are amplified and transmitted through a multiplexer system into a
computer based multi-channel analyzer. The Po-210 and Po-^OS peaks are displayed on screen
across a 4000-6000 Kev energy spectrum, separated by cursors and recorded. The relative
concentrations are determined from the number of counts within each region of interest, the
counting time is 60,000 sec. The concentration of Pb-210 at the time of sediment sampling is
calculated from the count rates corrected for counting background, growth and decay, counting
efficiency and recovery of the Po-208 yield monitor.
3.0 References
Robbins, J.A. and Edgington (1975). Determination of recent Sedimentation rates in Lake
Michigan using Pb-210 and Cs-137. Geochim. Cosmochim. Acta. 39, 285-304.
Robbins, J.A. (1978). Geochemical and Geophysical Applications of Radioactive Lead. In:
J.O. Nriagu (ed.), The Biogeochemistry of Lead in the Environment, Elsevier/North-Holland
Biomedical Press New York, N.Y. pp. 285-393.
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Determination of the Activity of Lead-210
in Sediments and Soils Volumes, Chapters
4.0 Digestion
4.1 Weigh 0.50 gms of dried sediment into a 125 mL Erienmeyer flask. Record sample ID and date.
4.2 Pipette 1.0 mL of Po-208 standard into flask. Check the delivery of the pipette be for use. Record
the activity of the standard and the date.
4.3 Add 50 mL of 6 N Hydrochloric Acid (1:1, water: Cone. Acid always add the acid slowly to the
water with mixing) to the flask. Add 1 mL of 30% Hydrogen Peroxide and 1 drop of Octanol.
4.4 Place the flask on a hot plate and heat to 90-95°C. Heat for 30 min. and remove from the hot plate
and cool slightly. Add one drop of octanol and 1 mL of 30% hydrogen peroxide, if the samples
foam vigorously add more octanol. Return the samples to heat for 30 min. Repeat the addition of
peroxide at least two more times. If the samples continue to foam add additional peroxide until
foaming subsides. Continue to heat for a total of four hours.
4.5 Remove the samples from heat, cover with a watch glass, and let stand over night.
4.6 Label the back of a copper disk with a water proof marker, spray with urethane, use three light
coats. Label should contain sample ID (Lake, Station and Depth)
5.0 Filtration
5.1 Filter the sample through a Whatman No. 42 filter paper into another Erienmeyer flask. A
Buchner funnel attached to vacuum is best for this step.
5.2 Rinse the digestion flask three times with small portions of Type 1 water and add to the filter.
6.0 Plating
Po-210 Plating Procedure-Caution!! This procedure uses Concentrated Hydrochloric acid and
307r Hydrogen Peroxide both of which can cause severe burns - Safety glasses, protective gloves
and a lab coat must be worn while performing this procedure.
6.1 Place the flask on a hot plate, carefully reduce the volume to approx. 5 mL. Do not let the sample
go to dryness. Cool.
6.3 Measure the pH, adjust to between 0.5 and 1.0, use HC1 or NaOH.
6.4 Add 0.1 to 0.2 gms of Ascorbic acid to each sample and dissolve. The ascorbic acid is added to
form a complex with ferric iron, thereby preventing its possible interference with the Po-210
plating.
6.5 Transfer the sample to a 125 mL plastic bottle, rinse the flask three times.
6.6 Polish the previously labeled disk with polishing, rub it off with a Kimwipe.
6.7 Add the disk to the sample in the plastic bottle, make sure the polished side is up. Cap the bottle.
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Determination of the Activity of Lead-210
Volume 3, Chapter 3 in Sediments and Soils
6.8 Place the bottle in a 95°C oven. Heat overnight.
6.9 Remove the samples from the oven. To remove the copper disk, tighten the cap on the bottle and
turn it upside down, the copper disk should be in the cap. Slowly turn the bottle over, the disk
should remain in the cap.
6.10 Remove the disk, rinse with Type I water then with ethanol, pat dry (do not rub), place in a plastic
zip lock bag. The bag should be labeled with the sample ID, date digested and the date plated.
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Volume 3
Chapter 4: Biomonitoring
-------
Standard Operating Procedure for
Chlorophyll-a and Pheophytin-a
(Turner Designs Method)
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
November 1,1995
-------
Standard Operating Procedure for
Chlorophyll-aand Pheophytin-a (Turner Designs Method)
1.0 Scope and Application
This method is applicable to waters from the Great Lakes and Tributary streams. The description
is for 90% buffered acetone and fine mesh glass fiber filters.
2.0 Summary of Method
A representative sample of algae is collected on a filter by vacuum filtration in dim light. The
filter is then placed in a screw cap culture tube in the dark. The tube is stored, in the dark, at sub-
freezing temperatures until the time of analysis. At the time of analysis, 10 mL of 90% buffered
acetone is added to the tube. Each acetone filled tube is placed in an ultrasonic bath, filled with
ice and sonicated, for 20 minutes after which the tube is steeped, at 0°C, between 16 to 24 hours.
The tube is centrifuged prior to determination of the fluorescence. Prescribed optical filters are
used to determine the excitation and emission wavelengths (approx. 420 nm and 670 nm). The
assay of chlorophyll-a is calculated from the decrease in fluorescence caused by acidification. The
pheophytin-a is calculated from the residual fluorescence after accounting for that produced by the
chlorophyll-fl.
3.0 Sample Handling and Preservation
3.1 The entire procedure should be carried out as much as possible in subdued light (Green) to prevent
photodecomposition. The frozen samples should also be protected from light during storage for
the same reason.
3.2 To prevent a chiorophyll-a degredation product, pheophytin, all glassware should be clean and
acid-free.
4.0 Interferences
Though chlorophyll-b, -c, pheophytin-/?, -c and other organic materials interfere they are assumed
to be at concentrations not considered significant.
5.0 Equipment Required
Turner Designs model 10-AU filter fluorometer with appropriate filters
Plastic filter funnel, Gelman
Vacuum system (3-4 psi)
GF/F filters, Whatman (47 mm)
16 X 100 mm screw cap culture tubes
1 3 X 100 mm culture tubes, disposable
250 mL filter flask with sidearm
Nalgene Tubing
(1) 200 mL volumetric flask
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SOP for Chlorophyll-a and Pheophytin-a
(Turner Designs Method) Volume 3, Chapter 4
(5) 100 mL volumetric flask
Aluminum Foil
Parafilm
Spectrophotometer
Disposable glass pipets
Chlorophyll-a standard (substantially free of chlorophyll b)
High purity grade acetone (1 L)
Magnesium Carbonate
Concentrated HCL
Plotter
Filter forceps
Vacuum pressure
6.0 Reagents
6.1 Saturated Magnesium Carbonate Solution: Add 10 gram magnesium carbonate to 1000 mL of
deionized water. The solution is allowed to settle for a minimum of 24 hours. Only the clear
"powder free" solution is used during subsequent steps.
6.2 90% (v/v) Buffered Acetone: Add 100 mL of the Magnesium Carbonate solution (Section 6.1) to
900 mL of Acetone in 1 L volumetric flask.
6.3 0.1 N Hydrochloric Acid solution: Add 8.5 mL of concentrated hydrochloric acid to 800 mL of
deionized water in 1 liter volumetric flask. Adjust volume to 1 L with deionized water.
7.0 Calibration and QC Check Standards
7.1 Chlorophyll-a Calibration Stock Standard: In subdued light, before breaking the tip of the
ampule. Weigh the ampule and its contents to the nearest 0.1 mg. Carefully break the tip of the
ampule. Transfer the entire contents of the ampule into a 200 mL volumetric flask. Carefully
rinse the ampule and inside of tip with 90% Buffered Acetone (Section 6.2), at least three times,
into the 200 mL volumetric flask. Adjust volume to 2CO mL in flask with buffered acetone
(Section 6.2).
Store the broken ampule and tip until all of the residual acetone has evaporated. Reweigh the
empty ampule and tip. Determine by difference the weight of chlorophyll-a added to the flask.
7.1.1 Determine the purity of the chlorophyll solution spectrophotometrically. Measure the
Optical Density (O.D.) of the Stock solution (Section 7.1) on the spectrophotometer at
663 nm. Use the equation below to calculate chlorophyll-a concentration.
Chlorophyll-a (mg/L) = 11.42 X O.D.663
11.42 = extinction coefficient of chlorophyll-a at 663 nm
Note: If the concentration is 5 mg/L (1 mg/200 mL) and the purity is 100% then the O.D.
should be 0.4378.
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Volume 3, Chapter 4
SOP for Chlorophyll-a and Pheophytin-a
(Turner Designs Method)
7.2 Chlorophyll-a Intermediate Calibration Standard (2000 |Jg/L)
Dilute the Chlorophyll-a Stock Standard to a concentration of 2000 ug/L. Prepare at least 50 mL
of solution.
7.3 Chlorophyll-a Working Calibration Standards:
mL of Intermediate Solution
Std. (Section 7.2.1) diluted
to 100 mL
4.0
0.0
Concentration of
Calibration Std.
ug/L Chlorophyll-a
80
0
7.4 Chlorophyll-a Working QC Check Standards:
mL of Intermediate Solution
Std. (Section 7.2) diluted
to JOOmL
5.0
0.5
Concentration of
QC Check Std.
ug/L Chlorophyll-a
100
10
8.0 Calibration and Standardization
8.1
8.2
Calibration should be done each time a batch of samples are analyzed. Allow the instrument to
warm-up for at least 15 minutes. See the Turner Design's Model 10-AU-005 Field Fluorometer
User's Manual Section 3 (Method B) for a full discussion of instrument calibration instructions.
Samples and standards are to be maintained at the same temperature by using a cooler filled with
ice.
To measure pheophytin-a, it will be necessary to obtain before-to after acidification response rn'ios
" 'r»u/c'
To measure pheophytin-a, it will be necessary to obta
of the chlorophyll-a calibration standards as follows:
> Measure the fluorescence of each standard.
> Remove the cuvette from the fluorometer.
> Acidify standard by adding 0.15 mL of 0.! N HCL (using an autopipet) for every 5 mL of
standard solution used.
> Carefully mix solution by vortexing at speed "9" for 10 seconds and measure fluorescence of the
standard solution again.
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SOP for Chlorophyll-a and Pheophytin-a
(Turner Designs Method) Volume 3, Chapter 4
Calculate the ratio, r, as follows:
Where Rb = Fluorescence of pure chlorophyll-a standard solution before acidification.
Ra = Fluorescence of pure chlorophyll-a standard solution after acidification.
9.0 Procedure
9.1 Sample Preparation
9.1.1 Add 10 mL of 90% buffered acetone (Section 6.2) to the tube containing the filter. Recap
tube and invert tube three times making sure that the filter is totally submerged in buffered
acetone solution.
9.1.2 Place each tube in an ultrasonic bath, that had been previously filled with water and ice,
for 20 minutes.
9.1.3 After 20 minutes, return sample tube to freezer to steep for 16 to 24 hours.
9.2 Sample Analysis
9.2.1 Samples and standards should all be maintained at the same temperature by using a cooler
filled with ice.
9.2.2 After the fluorometer has wanned up for at least 15 minutes, use the 90% buffered acetone
solution to zero the instrument on the sensitivity setting that will be used for sample
analysis.
9.2.3 Following calibration, verify that the flourometer is set at AUTO RANGE" setting.
9.2.4 Invert sample cuvette four times to mix.
9.2.5 Using a filter flask with a sidearm attached to a vacuum unit, filter entire contents of
sample through a GF/F (47 mm) filter, directly into cuvette used for analysis.
Note: Do not let vacuum pressure exceed 1-2 psi or sample volume will be affected
9.2.6 If the concentration of chlorophyll-^ in the sample is > 90% of the highest calibration
standard, then dilute the sample with the 90% buffered acetone solution and reanalyze.
9.2.7 Record the fluorescence measurement and sensitivity reading used for the sample.
9.2.8 The volume of sample that is to be used for analysis must be known so that correct amount
of acid can be added in the pheophytin determination step.
Add 0.15 niL of O.I /V HCL solution fur fvrrv 5 nil. of extraction solution.
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SOP for Chlorophyll-a and Pheophytin-a
Volume 3, Chapter 4 (Turner Designs Method)
9.2.9 Remove the tube from the fluorometer and acidify the extract using 0.1 N HCL solution.
9.2.10 Mix solution for 10 seconds using a vortex set at speed "9" before measuring fluorescence
again.
10.0 Calculations
Chlorophyll-a (\iglL) = (r/r-l) (Rh - tf()
Pheophytin-a (\iglL) - (rlr-\) (rRu /?,,)
I O.I Determine the chlorophyll-a concentration in the sample extract and the pheophyun-a
concentration in ug/L as follows:
Where Rh = Fluorescence of sample extract before acidification.
Ra = Fluorescence of sample extract after acidification.
r = The before-after acidification ratio of a pure chlorophyll-a solution (Section 8.2).
10.2 The concentration of chlorophyll-a and pheophytin-a in the lake water sample is calculated by
multiplying the results obtained above by 10 mL (the extraction volume) and dividing this answer
by the volume (mL) of the lake water sample that was filtered on the boat. Any other dilution
factors should be incorporated accordingly.
11.0 Quality Control
The following audits are to performed:
Audit Frequency Limit***
High Check
Low Check
Lab Blk.
Lab Dupl.
Field Dupl.
Field Blk.
Once/batch
Once/batch
Once/batch
Once/batch
Once/batch
Once/batch
100 ug± 15
10ug± 1.5
0.00 ug± 0.11
RPD 15%
RPD 15%
0.00 ug± 0.11
*** These limits are estimates based upon data taken from original method and do not pertain to
performance data done at CRL. These limits are guidelines. Actual performance limits will still
need to be calculated when enough data is available.
12.0 Waste Disposal
Follow all laboratory waste disposal guidelines regarding the disposal of acetone solutions.
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SOP for Chlorophyll-a and Pheophytin-a
(TurnerDesigns Method) Volumes, Chapter4
13.0 References
13.1 Arar, Elizabeth J. and Collins, Gary B., "In Vitro Determination of Chlorophyll-a and
Pheophytin-a in Marine and Freshwater Phytoplankton by Fluorescence", Environmental
Monitoring and Support Laboratory. U.S. EPA 1992.
13.2 Turner Designs Model 10-AU-005 Field Fluorometer User's Manual/November 1992 (P/N 10-
AU-075).
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ESS Method 150.1:
Chlorophyll - Spectrophotometric
Environmental Sciences Section
Inorganic Chemistry Unit
Wisconsin State Lab of Hygiene
465 Henry Mall
Madison, Wl 53706
Revised September 1991
-------
ESS Method 150.1:
Chlorophyll - Spectrophotometric
1.0 Application
1.1 Chlorophyll a, a characteristic algal pigment, constitutes approximately 1 % to 2% (dry weight) of
planktonic algal biomass. This feature makes chlorophyll a a convenient indicator of algal
biomass.
1.2 This method is applicable to most surface waters.
2.0 Summary of Method
2.1 Algal cells are concentrated by filtering a known volume of water through a membrane filter
(47 mm, 5.0 (am pore size). The pigments are extracted from the concentrated algal sample in an
aqueous solution of acetone. The chlorophyll a concentration is determined
spectrophotometrically by measuring the absorbance (optical density - OD) of the extract at
various wavelengths. The resulting absorbance measurements are then applied to a standard
equation.
3.0 Sample Preservation and Preparation
3.1 Chlorophyll a samples should be placed in a dark cooler and packed in ice at the time of
collection.
3.2 Filter from 50 to 2000 mL of sample through a 5 (am membrane filter, applying vacuum until the
sample is dry.
3.2.1 Add 0.2 mL of MgCO3 suspension during the final phase of the filtration.
3.2.2 Fold the filter into quarters, wrap in aluminum foil; place in a desiccator and freeze.
3.2.3 Samples may be held frozen for up to 30 days if taken from waters of pH 7 or greater.
Samples from acidic waters should be processed promptly.
4.0 Comments
4.1 Pheophytin, a natural degradation product of chlorophyll, has an absorption peak in the same
spectral region as chlorophyll a. It may be necessary to make a correction when pheophytin
concentration becomes significantly high.
4.1.1 Corrected chlorophyll a refers to the method with the pheophytin correction (acidification
method).
4.1.2 Uncorrected chlorophyll a refers to the method without the pheophytin correction
(Trichromatic method).
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Chlorophyll - Spectrophotometric Volume 3, Chapter*
4.2 Handle samples in subdued light to prevent photochemical breakdown of the chlorophyll.
4.3 Protect the acetone extract from more than momentary exposure to light.
5.0 Apparatus
5.1 Sonicator cell disrupter, Heat Systems-Ultrasonics Inc., Model W-220F, equipped with a microtip.
5.2 Beckman Model DU-6 scanning spectrophotometer, 2.0 nm slit or narrower.
5.2.1 Printer.
5.2.2 1.0, 5.0, 10.0cm spectrophotometer cells.
5.3 Calibrated 15 mL centrifuge tubes with teflon lined caps.
5.4 Centrifuge capable of attaining 500 g.
5.5 Dark box: Light tight box capable of holding a small test tube rack.
5.6 Standard laboratory glassware including membrane filtration apparatus.
5.7 Millipore SM 5.0 pm membrane filters (47 mm).
5.8 Vacuum source.
6.0 Reagents
6.1 Aqueous acetone solution: Mix 90 parts reagent grade acetone with 10 parts Milli-Q water
(Millipore Reagent Grade Water System).
6.2 0.1 N Hydrochloric acid: Add 8.3 mL of reagent grade hydrochloric acid and dilute to 1000 mL
with Milli-Q water.
6.3 1% Magnesium carbonate suspension: Add 1.0 g of magnesium carbonate powder to 100 mL of
Milli-Q water.
7.0 Procedure
7.1 Place the filter containing the concentrated algal sample in a centrifuge tube.
7.1.1 Add about 10 mL of aqueous acetone solution; cap tightly and place in the dark box.
7.2 Repeat Step 7.1 until the desired number of samples have been processed.
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7.3 Remove the cap from the centrifuge tube, insert the microtip, and sonify for 20 seconds at the
5 setting.
7.3.1 Rinse the microtip into the centrifuge tube with approximately 1 mL of aqueous acetone
solution.
7.3.2 Bnng the extract to a volume of 13.0 mL with the acetone solution, cap, mix and return to
the dark box.
7.3.3 Repeat the steps outlined in Step 7.3 until all of the samples have been sonified.
7.4 Place the dark box in the 4°C cold room and allow the extract to steep overnight.
7.5 Clarify the extract by centrifuging the extract for 20 minutes at approximately 500 g. (Mix the
extract thoroughly before centrifuging.)
7.6 Carefully transfer the clear extract to a 5.0 cm cell and using the multi wavelength mode on the
spectrophotometer, measure the absorbance at: 750, 663, 645, and 630 nm (if unconnected
chlorophyll is desired) or at 750, 665, 663, 645, and 630 nm if both corrected and uncorrected
chlorophyll are desired).
7.6.1 Use a shorter or longer cell as necessary to maintain absorbance between approximately
0.1-1.0at663nm.
7.6.2 Note: Operate the spectrophotometer at a slit width no wider than 2 nm for maximum
resolution.
7.7 For corrected samples: Immediately after measuring the absorbance, add 0.1 mL of 0.1 N HC1 to
the spectrophotometer cell, mix, wait 90 seconds and measure the absorbance specified in
Step 7.6.
7.8 Discard the sample, rinse the cell two times with 5 mL portions of aqueous acetone solution.
7.9 Repeat Steps 7.6 through 7.8 until all of the samples have been measured.
8.0 Calculations (manual)
8.1 Determine the absorbance at 750, 663, 645, and 630 nm directly from the printout.
8.2 Subtract the absorbance at 750 nm from the 630, 645, and 663 nm values (turbidity correction).
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8.3 Calculate the uncorrected chlorophyll a concentration by inserting the corrected absorbance values
in the following equation.
Uncorrected [11.64 (Abs663)-2.16 (Abs645)+0.lO (Abs630)} E(F)
Chlorophyll a fag/L = V(L)
Where F = Dilution Factor (i.e., if the 663 Abs is >0.99 with the I cm cell, dilute,
re-analyze and insert the dilution factor in the equation)
E = The volume of acetone used for the extraction (mL)
V = The volume of water filtered (L)
L = The cell path length (cm)
8.4 For corrected samples, determine the absorbance at 665 nm and 750 nm after acidification.
8.5 Subtract the absorbance at 750 nm from the absorbance at 665 nm (turbidity correction).
8.6 Calculate the corrected chlorophyll a and Pheophytin a concentration by inserting the turbidity
corrected absorbance readings in the following equations.
26.73(663,, -665a) E(F)
Corrected Chlorophyll a (ug/l) = ' y77~\
26.73(1.7 x [665J-663fc) £(F)
Pheophytin a (ug/l) =
V(L,)
Where F = Dilution Factor (if the extract requires dilution)
E = The volume of acetone used for the extraction (mL)
V = The volume of water filtered (L)
L - The cell path length (cm)
665 a = The turbidity corrected Abs at 665 nm after acidification
663h = The turbidity corrected Abs at 663 nm before acidification
9.0 Computer Automated Calculations
9.1 A personal computer may be used to calculate the chlorophyll concentrations, and to evaluate the
process quality control data. This technique greatly increases the speed of the analyses and
significantly reduces computational and transcription errors.
9.1.1 Detailed, step by step instructions for the computer automated method are available in the
PC Laboratory Automation Manual.
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9.2 Equipment needed
9.2.1 IBM or IBM compatible PC with an RS232C board.
9.2.2 Beckman DU-6 communication software or other data acquisition software (Procomm,
Lotus (Lotus measure), Measure, etc.).
9.2.3 Spreadsheet software; Lotus 1,2,3.
9.3 Data transfer
9.3.1 Connect the PC to the Beckman DU-6 via the RS232 ports.
9.3.2 Install the Beckman data capture software and select the appropriate setup from the main
menu. Create a file for the transferred absorbance values.
9.3.3 On the spectrophotometer, select 'output' from 'Data I/O'. Run the samples with the
appropriate wavelengths.
9.3.4 After completing the analyses, transfer the file containing the absorbance data to a
Lotus 1,2,3 spreadsheet (programmed to perform the calculations in Section 8.0) to
calculate the final chlorophyll results.
9.3.5 Evaluate the quality control data and print a final report for subsequent review by another
qualified analyst.
9.3.6 After the data review, transfer the result to the Laboratory Information System (LEMS)
database.
10.0 Precision and Accuracy
Precision and accuracy data are available in the Inorganic Chemistry Quality Assurance manual.
11.0 References
11.1 Biological Field and Laboratory Methods for the Quality of Surface Waters and Effluents, U.S.
Environmental Protection Agency, EPA-670/4-73-001, p. 14, (1973).
11.2 Nelson, D.H., "Improved Chlorophyll Extraction Method", Science, 132, p. 351, (1960).
11.3 Rai, H., "Methods Involving the Determination of Photosynthetic Pigments using
Spectrophotometry", Verh, Internal. Verein. Limnol. 18, pp. 1864-1875, (1973).
11.4 Standard Methods for the Examination of Water and Wastewaier, 14th Ed. pp. 1029-1033, (1975).
11.5 Standard Methods for the Examination of Water and Wastevvater, 17th Ed. pp. 10-31-10-39,
(1989).
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Standard Operating Procedure for
Phytoplankton Analysis
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
December 15,1994
-------
Standard Operating Procedure for
Phytoplankton Analysis
1.0 Scope and Application
This method is utilized to identify, enumerate and measure phytoplankton taxa in samples
collected from the Great Lakes. Algal taxa are identified to the lowest taxonomic rank possible. A
listing of all organisms identified and their respective density and morphometric measurement for
biovolume calculation is reported.
2.0 Summary of Method
The method consists of two parts - analysis of phytoplankton (excluding most diatoms) and
analysis of diatom. For operational reasons, the first part of the analysis is also called "soft algae"
analysis. The "soft algae" are defined as those that are either naked or have a cellulosic cell wall
and cannot withstand acid digestion treatment. In contrast, diatoms have relatively "hard" silicious
valves and the valves can tolerate harsh acid treatment. Initially a preliminary scan is made of a
settled 10 mL sample in order to determine the volume to be used for each of the two analyses.
For the soft algae analysis, organisms are enumerated in a settling chamber using an inverted
microscope at 500x magnification. For diatom analyses, the samples are pretreated with strong
oxidants and the cleaned samples are mounted on glass slides and enumerated using a compound
microscope at 1250x magnification.
3.0 Sample Collection and Preservation
3.1 See United States Environmental Protection Agency Great Lakes Analytical Contract Operation
Procedure for phytoplankton sample collection and preservation.
3.2 After the preserved phytoplankton samples arrive at the laboratory from the survey, an additional
10 mL of Formalin is added to each sample to enhance the storage life of the sample.
3.3 All sample containers and diatom slides must be properly labeled as follows:
a. Sample containers: Lake: Station, CRL and LAB Number; Sampling Date; Sample Type
(Integrated, B-l, B-2..etc.).
b. Diatom slides: Lake; Station, CRL and LAB Number.
Note: CRL numbers are assigned by GLNPO to all samples-collected in the field, LAB numbers
are assigned by the contractor in the laboratory, for internal use only, to facilitate the
sample log-in and identification procedure. Each sample has its own CRL Number that
corresponds to a specific LAB Number (see United States Environmental Protection
Agency Phytoplankton and Zooplankton Sample Log-in Standard Operating Procedure).
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Volume 3, Chapter 4
L PHYTOPLANKTON SAMPLE
1
10 mL PRELIMINARY COUNT
SOFT ALGAE ANALYSIS
DATA SUMMARY
1
CELL DENSITY
CALCULATION
DIATOM ANALYSIS
DATA SUMMARY
CELL DENSITY
C A LC U L AT1O N
DATA REV IEW
SAMPLE ARCHIVE
4.0 Determination of Sample Volume Required for Analyses
4.1 10 mL Preliminary Investigation
The 10 mL preliminary investigation is usually performed by the soft algae analysts in order to
determine the appropriate volume of sample required for both soft algae and diatom analyses.
4.1.1 Apparatus
4.1.1.1 Inverted microscope with an objective system for magnification up to 150x (Leitz
Diavert or another equal quality inverted microscope).
4.1.1.2 Tubular plankton chamber or combined plate chamber 10 cc.
4.1.1.3 Cover plate for plankton chamber, 33 mm dia.. 2 mm thick.
4.1.1.4 Base plate for plankton chamber, 27.5 mm dia., 0.2 mm thick.
4.1.1.5 10 mL automacropipette.
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4.1.2 Procedure
This procedure is done by settling lOmL of each sample and counting the total number of
organisms and number of diatom cells within a 10 mm2 area. No identifications are done
at this time but any irregularities such as excessive sediment in the sample are noted. All
information from the 10 mL preliminary count is recorded in a pre-printed data form
(Appendix 1). This includes unusual observations such as poor sample preservation, high
bacterial or fungal populations, occurrence of special or rare phytoplankton taxa ... etc.
Note: The definition of an organism for 10 mL preliminary counts is as follows:
A colony, a filament, or a single cell. The units of a colony or a filament are not counted
as organisms at this time but the whole aggregate is counted as one organism.
Note: 10 mnr = One transect from edge of chamber to edge of chamber at 250x.
4.2 Determination of Sample Volume Settled
4.2.1 There is no exact limit set for determining the volume needed, each sample is examined
for the number of organisms present, amount of debris in the sample and its distribution
pattern. Large amount of debris often require that smaller then optimal volumes be
settled.
4.2.2 Most samples are settled at 10 or 25 mL, with 25 mL being the usual volume. Only when
samples are difficult or impossible to count are 5 mL or 2.5 mL samples used. The 50 mL
samples are used when very low number of organisms are found in the samples.
4.2.3 The volume needed for setting (soft algae analysis) and for digestion (diatom analysis) is
determined from the number of all organisms counted during the 10 mL preliminary
investigation. However, the minimum volume for digestion is recommended to be
500 mL. For example:
10 mL preliminary counts
1) 101 organisms total
2) 103 diatom cells (Note: 1 cell has two frustules or valves)
Count needed (minimum)
1) 250 organisms total
2) 500 diatom frustules (250 cells)
Final volumes
1) 25 mL sample for sedimentation
2) 500 mL sample for digestion
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SOP for Phytoplankton Analysis Volume 3, Chapter 4
4.2.4 The final volume may be slightly over-estimated to ensure that the minimum counts
required are met. The preliminary count also helps to ensure that there is enough sample
for both final investigations.
5.0 Sample Analyses
Samples are analyzed by data set, and a QC count is chosen for 10% of the samples in each set.
The QC is chosen by the Team Leader who takes into account the 10 mL preliminary data and the
diatom counts, if available.
5.1 Soft Algae Sample Analysis
Organisms are identified to the lowest taxonomic rank possible. Characteristics such as size,
shape, color and the presence of flagella are used in the identification process. Any obscure or
unidentifiable organisms are checked by the Team Leader or one other analyst. Drawings are
made of the organism, complete with all sample identifiers (i.e. LAB and CRL numbers, Station
number. Survey number, and analyst's initials). The drawing is then added to the permanent card
file in the lab, and may also be sent out to other specialists for identification or verification. The
card file is reviewed frequently and any additional information is added as received.
5.1.1 Apparatus
5.1.1.1 Inverted microscope with an objective system for magnification up to 600x (Leitz
Diavert or another equal quality inverted microscope)
5.1.1.2 Tubular plankton chamber or combined plate chamber lOcc.
5.1.1.3 Cover plate for plankton chamber, 33 mm dia., 2 mm thick
5.1.1.4 Base plate for plankton chamber, 27.5 mm dia., 0.2 mm thick
5.1.1.5 10 mL automacropipette
5.1.1.6 Syringe 20 mL with cannula, 14 gauge 4 inch
5.1.1.7 Long-neck disposable pipettes
5.1.1.8 Rubber bulbs for pipettes
5.1.2 Analytical procedures
5.1.2.1 Sample Sedimentation
The phytoplankton sample is mixed by gently inverting the sample bottle for
60 seconds. The predetermined sample volume (see Section 3.0) is loaded into a
sedimentation chamber of appropriate volume. Samples should be added to the
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chamber with a syringe (less than 10 mL) or macropipettor (10 mL or more). The
sample bottle should be inverted at least once between each addition. This is done
because larger organisms settle quickly and may remain in the bottle if the sample
is simply poured. The chamber is topped with a round glass top plate.
5.1.2.2 Sample Settling
Algae are allowed to settle onto the base of the settling chamber. Since oil
immersion may be used in the course of identification, the coverglass at the
bottom of the chamber should not be thicker than 0.2 - 0.3 mm in thickness (or
No. 1 coverglass). The time recommended for complete sedimentation varies
with the height of the chamber, i.e. 8 cm/day to 4 cm/day depending on accuracy
required in enumeration (Furet & Benson-Evans. 1982).
Approximate settling times necessary are as follow:
lOOmL 100 hours
50 mL 50 hours
25 mL 25 hours
lOmL 10 hours
5 mL 5 hours
2 mL 2 hours
5.1.2.3 Sedimented Sample Analysis
Only "live" forms (chloroplast containing organisms) are counted and identified at
500x. Higher magnification may be used for identification when necessary.
5.1.2.3.1 The chamber of settled material is scanned and the dominant
(four or five most common organisms) as well as subdominant
taxa are determined. This is to give the analyst an idea of the
sample composition as well as to insure that the sample is evenly
settled.
5.1.2.3.2 Enumeration and identification are done by scanning parallel
strips of 10 mm per strip (each strip has a width of 0.2 mm which
gives an area of 2 mm2). A minimum of three strips (30 mm or
6 mm2) is required, including no less than 250 "live" organisms.
If 250 organisms are not observed within the three strips,
identification and enumeration are continued in strips until at
least 250 are counted. The area counted is recorded as it is
needed for cells per mL calculation.
5.1.2.3.3 The number of "live" cells are identified and enumerated to the
lowest taxonomic rank possible. All "emptied" lorica from
Chrysophyta are also identified and enumerated.
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5.1.2.3.4
5.1.2.3.5
At least 10 specimens of each taxa are measured for cell volume
calculations. When fewer than 10 specimens are present those
present are measured as they occur. The measurements required
are those which are necessary for the volume calculation of a
solid which best approximates the shape of any particular
organism. For most organisms the measurements are taken from
outside wall to outside wall.
Those forms which are loricate (e.g., selected members of
Chlorophyta, Euglenophyta and Chrysophyta) must have the
active portion, i.e. protoplast, measured. Empty lorica are also
counted, but not measured. Filamentous and colonial forms
require measurements of the individual components.
Diatom cells are counted while making the strip counts at 500x.
At this magnification the diatoms are enumerated and identified
only as live pennates, empty pennates, live Gentries, and empty
Gentries. Actual identification of diatoms and cell volume
measurements are done under oil immersion (1250x) by another
method (see Section 5.2). The only diatoms which must be
counted at 500x are: Asterioneila formosa, Fragilaria capucina,
Fragilaria crotonensis, Tabellaria flocculosa, Rhizosolenia
eriensis and species of Rhizosolenia longiseta.
5.1.3 Archiving
Soft algae samples are to be archived one data set at a time.
5.1.3.1 Gently mix the remainder of the phytoplankton sample by repeatedly inverting the
bottle for about one minute. Carefully empty the sample into a 500 mL graduated
cylinder and cover the cylinder with a plastic Petri plate. Record the volume of
sample settled on a pre-printed phytoplankton archive form (Appendix 6). A
larger and/or smaller graduated cylinder may be used depending on the volume
remaining in phytoplankton sample bottle.
5.1.3.2 Rinse the sample bottle three times with a small amount of RO/DI or distilled
water (about 5 mL). Empty the rinse water into the graduated cylinder.
5.1.3.3 Settle the sample for a minimum of seven days, but not more than 14 days. Do
not disturb the cylinder.
5.1.3.4 At the end of the settling period, carefully siphon off the top of the water column
without disturbing the settled materials. Generally, about 18-22 mL of the sample
should be remaining in the cylinder.
5.1.3.5 Decant the remaining sample from the graduated cylinder into a pre-labeled
25 mL glass liquid scintillation vial. Rinse the cylinder two times with about
2 mL of RO/DI or distilled water and empty the rinse water into the vial. This is
the archived sample.
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5.1.3.6 Add about 0.5 mL of Formalin solution to the archived sample before putting the
cap on the vial.
5.1.3.7 Store the archived sample in a pre-labeled tray/box.
5.1.3.8 Record the archived sample information (CRL number, lab number, station
number, original volume and concentrated volume) into the computer using
DBASE III + data management program.
5.2 Diatom Sample Analysis
Diatom identifications and enumerations are performed on prepared slides. Because the cellular
contents of diatoms obscure the wall markings on which the taxonomy is based, the organic
matters inside the cell must be removed (oxidized) prior to identification.
5.2.1 Apparatus
5.2.1.1 Research quality compound microscope with an objecuve system of magnification
up to 1400x (Letiz Dialux or another equal or better quality compound
microscope).
5.2.1.2 Beakers - 300 and 600 mL
5.2.1.3 Hotplate
5.2.1.4 Centrifuge
5.2.1.5 Centrifuge tubes, graduated 15 mL
5.2.1.6 Cover slips, round, #1 thickness, 22 mm diam.
5.2.1.7 Precleaned microscope slides, 25 X 75 mm.
5.2.1.8 Long-neck disposable pasteur pipettes
5.2.1.9 Rubber bulbs for pipettes
5.2.1.10 Slide Warmer
5.2.2 Reagents
5.2.2.1 HNO3 Nitric Acid (concentrated)
5.2.2.2 H2O, Hydrogen peroxide (30% solution)
Hydrogen peroxide must be kept in an air-tight container, store in dim light or in
the dark, and in a refrigerator.
3.2.2.3 K,Cr,O- Potassium dichromate
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5.2.2.4 Hyrax™ mounting media
5.2.2.5 Toluene or Xylene
5.2.2.6 "Leitz" immersion oil
5.2.3 Cleaning Of Diatoms Valves and Slides Preparation
This section describes a method for cleaning diatom valves and preparing permanent
diatom slides.
5.2.3.1 Cleaning of Diatoms
The first three steps of the diatom cleaning procedure must be carried out under
the hood.
5.2.3.1.1 A specified volume (see Section 4.0) of uniformly mixed sample
is poured into a 600 mL beaker. The recommended minimum
volume is 500 mL. Mix the sample by gently inverting the
sample bottle for a minimum of one minute.
5.2.3.1.2 Add 20 mL of concentrated HNO, to digest organic matter in the
sample. Place beaker on a hot plate and concentrate sample to
approximately 20 mL by heat evaporation. Allow sample to cool
and transfer to a 300 mL beaker. Rinse the side of the 600 mL
beaker several times with DI/RO water and transfer the rinse
water to the digested sample.
5.2.3.1.3 Adjust the volume of the digested sample to 150 mL with DI/RO
water. Further oxidize the sample with 25 mL of 30% H2O2.
Accelerate the process by adding a few grains of K2Cr207. Place
beaker on a hot plate and concentrate sample to approximately
10 mL by heat evaporation. Allow the sample to cool and
transfer to a 15 mL graduated centrifuge tube.
5.2.3.1.4 Rinse the side of the beaker several times with DI/RO water and
transfer the rinse water to the centrifuge tube. Fill the tube with
DI/RO water and centrifuge at low speed (1500 rpm) for
30 minutes.
5.2.3.1.5 Draw off all but 0.5 mL of supernatant in the centrifuge tube
using a vacuum system. Take care not to disturb the pellet at the
bottom of the tube. Add approximately 10 mL of DI/RO water to
the tube and gently shake the sample using a vortex mixer.
Recentrifuge the sample for 30 minutes at low speed (1500 rpm).
Repeat Step 5.2.3.1.5 10 times.
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5.2.3.1.6 Upon final centrifugation draw off all but 0.5 mL of supernatant.
Bring volume up to approximately 5 mL with DI water. This is
the "cleaned" sample to be used to prepare diatom slide for
analysis.
5.2.3.2 Diatom Slide Preparation
Where possible, two duplicate slides should be made from each sample. The
second slide will be sent to a repository at a later date.
5.2.3.2.1 Place a clean coverslip (thickness: No. 1; size 22 mm, circular)
on a slide warmer (150-200°F).
5.2.3.2.2 Gently mix the sample and pipette about 0.25 mL aliquot of the
sample on a coverslip and let dry. Examine the dried coverslip
under the microscope. If the diatom density is not sufficient for
counting, dry more sample on to the coverslip.
5.2.3.2.3 Add a small drop of Hyrax mounting medium to the center of a
clean prelabeled slide (75 X 25 mm). If the Hyrax mounting
medium is too viscous, add a few drops of toluene and/or xylene
to dilute the medium.
5.2.3.2.4 Mount the coverslip, diatom side down, on the slide and place on
hotplate.
5.2.3.2.5 Allow solvent to evaporate until bubbles are no longer formed
under the coverslip. Remove from the hotplate.
5.2.3.2.6 Press coverslip gently with pencil eraser to extrude excess Hyrax
immediately after removing from heat as the medium sets up very
quickly.
5.2.3.2.7 Allow the slide to cool and remove excess Hyrax before
examining. It will scrape away easily with a razor blade if all of
the solvent is removed: if it is sticky, return to the hotplate to
remove any remaining solvent.
5.2.3.2.8 Clean and label (CRL number, LAB number, Station number) the
slide.
5.2.4 Diatom Enumeration and Identification
Diatoms are identified and enumerated to lowest taxonomic rank possible at I250x.
5.2.4.1 A minimum of 500 frustules is counted (2 frustules = 1 diatom cell) per sample
(slide).
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5.2.4.2 At least 10 specimens of each taxa are measured (wall to wall) for cell volume
calculations. When fewer than 10 specimens are present, those present are
measured as they occur (Appendices 4 & 5).
5.2.5 Archiving
5.2.5.1 After the diatom slides are made, transfer the remainder of 'cleaned" sample to a
pre-labeled 9 mL glass vial.
5.2.5.2 Store the diatom archived sample in a box for future reference.
6.0 Calculations
6.1 Report the results of the sample sedimentation procedure as cells per mL which is calculated as
follows:
C x TA
cellslmL =
L x W x V x S
Where: C = cell count
L = length of strip (mm)
W = width of strip (mm)
V = volume of chamber (mL)
S = number of strips counted
TA - total area of chamber bottom (mm2)
Note: Calculation factor listed at the bottom of Appendix 3 is equal to:
TA
L x W x V x S
6.2 Reasonable approximations of geometric shape and mean dimensions will be reported so that cell
volume estimates can be determined.
6.3 The data from the diatom slides is reported as percent composition of the 1250x count. This
percent is applied back to the diatom counts at 500x to determine a cells/mL count for each
species.
6.3.1 Calculate the total live diatom cells/mL as per formula in Step 6.1.
6.3.2 Calculate the percent composition of the pennate and centric diatom taxa on the prepared
slide by dividing the number observe by the total pennate and total centric diatom values
enumerated respectively.
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6.3.3 Calculate the cells/mL for each diatom taxon by multiplying the total live pennate and
centric diatom cells/mL (from the soft algae analysis) by the percent pennate or centric
diatom counts respectively (from the diatom analysis).
7.0 Quality Control and Method Precision
7.1 Ten percent of all samples collected are analyzed in duplicate. At least one duplicate count is done
per data set if the data set contains less than 10 samples. This includes identification, and
tabulation of data. Data shall be calculated for the groups below:
Cyanophyta Other minor divisions
Chlorophyta Indeterminable forms
Chrysophyta Pennate Diatoms
Cryptophyta Centric Diatoms
Total phytoplankton
The relative percent difference (RFC)) between duplicate determinations shall be compared to the
guidelines listed below:
Cyanophyta (Picoplankton + Cyanophyta) 56%
Chlorophyta 82 *
Chrysophyta 87 *
Cryptophyta 52
Others 22
Unidentified 75
Pennales (Live + Empty) 80 ***
Centrales (Live + Empty) 72 ****
Total 48
* Cells must number >140 before RPO guideline can be applied
** Cells must number >198 before RPD guideline can be applied
*** Cells must number >98 before RPO guideline can be applied
**** Cells must number >274 before RPD guideline can be applied
value) - (smaller value)
Average value
7.2 Determinations which exceed the control guidelines listed above may require re-analysis unless:
7.2.1 The RPO value is the result of low density (especially true for the other minor divisions
category).
7.2.2 The RPO value is the result of chance occurrence of colonial forms which are enumerated
as individuals thus skewing the population estimate.
7.2.3 Other reasonable explanations can be provided to explain the differences between counts.
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SOP for Phytoplankton Analysis
Volume 3, Chapter 4
7.3 Previously calculated RPD values are used to determine the consistency of the identifications
between analysts at the division level, as they only compare total cell numbers and not actual
species identifications.
7.3.1 If the calculated values fall outside RPD Guidelines and no explanation can be found, the
sample may be reanalyzed by either or both analysts or a third analyst, where necessary.
7.3.2 If the sample data are accepted by the analysts, they are then submitted to the Team
Leader for his or her approval.
7.4 Photographic and Line-drawing Record
Photographic records of diatom and other phytoplankton taxa should be taken. Resulting positive
prints should be enlarged to a specific diameter (i.e. lOOOx) and attached to 5 x 8 index cards or
8l/2 x 11" sheets. The card must contain the following information:
a) Taxon name with dimensions and magnification
b) Photograph with negative reference number (if any)
c) Sampling date and location
d) Location of specimen on slide (diatoms only)
e) Slide identification number (diatoms only)
f) Comments
g) Name of analyst
Example format:
Taxon:
Photograph(s)/Line Drawing(s)
Dimensions:
Comments:
X urn
Slide ID:
Sampling date:
Location:
Analyst: _
This continuously updated file serves as the quality control reference document for diatom and
other phytoplankton taxa. The file also serves as reference standard for the questionable and
unidentifiable forms.
8.0 Safety and Waste Disposal
Proper PPE should be worn in the laboratory while handling and preparing samples for analysis,
especially during the digestion process. Follow all laboratory waste disposal guidelines regarding
the disposal of acid waste. Do not discard samples containing acid into the sink. All waste should
be placed in a designated, and labeled, waste drum.
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Volume 3, Chapter 4 SOP for Phytoplankton Analysis
9.0 References
9.1 Standard Operating Procedure For The Analysis Of Phytoplankton - U.S. E.P.A. - GLNPO 1987.
Prepared by the Bionetics Corporation.
9.2 Furet, J.E. and K. Benson-Evans. 1982. An evaluation of the time required to obtain
sedimentation of fixed algal particles prior to enumeration. Br. Phycol. J. 17: 253-258.
9.3 U.S. E.P.A. Great Lakes Program Office. 1987. Analytical Contract Operation Procedure for
Phytoplankton Sample Collection and Preservation.
9.4 Utermohl, H. 1958. Zur vervoilkommnung der quantitativen phytoplankton-methodik.
Mitt. Int. Ver. Limnol. 9. 38 pp.
9.5 Phytoplankton Sampling And Preservation Standard Operating Procedure - U.S. E.P.A. 1994.
Prepared by the Enviroscience Corporation.
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 1. Great Lakes - Phytoplankton Samples
Preliminary Investigation
# Organisms in 10 mL - Min. Area = 15 mnv
Sample
Number
Station #
& Depth
# Organisms
um
Total Vol.
Needed
Comments
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 2. Phytoplankton Bench Sheet
Sample Number
Lab Number
Station & Depth _
Date Collected
Data Set Number
Lake
Analyzed by
Date Analyzed
Method
Volume Analyzed
Cell Tally
PICOPLANKTON - spheres Sweep 1 =
Sweep 2 =
Sweep 3 =
PICOPLANKTON rods
UNIDENTIFIED OVOID - flagellates
UNIDENTIFIED SPHERICAL - flagellates
RHODOMONAS MINUTA VAR. MANNOPLANCTICA
COCCOID - OVOID
COCCOID - SPHERES
ANACYSTIS MONTANA f MINOR
HAPTOPHYTES SPP
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 3. Phytoplankton Analysis
Lake
Analyzed by
Data analyzed
Method
TOTALS
Picoplankton
Cyanophyta (Blue-greens)
coccoids
filaments
Chlorophyta (Greens)
coccoids
filaments
flagellates
Desmids
Chysophyta (Golden Browns)
coccoids
flagellates
Haptophytes
colorless flagellates
Cryptophyta
Pyrrhopnyta (dinoflagellates)
Euglenophyta (Euglenoids)
Xanthophyta (Yellow greens)
Chloromonadocnyta (Chloromonads)
Unidentified flagellates and coccoids
Eacillanophyta (Diatoms-Live Cells)
Rhizosomia spp
live pennates
empty pennates
live centrics
empty centrics
diatom valves (@ 1250x)
Sample Number
Lab Number
Station & Depth
Date Collected
@ 500x
_cells/mL
_cells/nL
cells/mL
cells/mL
_cells/mL
_cells/mL
_cells/mL
_cells/mL
_cells/mL
_cells/mL
_cells/mL
_cells/mL
li\e cells/mL
jjmply cells/mL
frustulcs
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SOP for Phytoplankton Analysis
Volume 3, Chapter 4
Dominant species
Area scanned
Volume settled
Calculation factor
mm2
mL
Total
cells/mL
Calculation factors for phytoplankton samples
Volume of Sample Settled
Strips
1
2
3
4
5
6
7
5 mL
49.0875
24.5438
16.3625
12.2719
9.8175
8.1813
7.0125
10 mL
24.5438
12.2719
8.1813
6.1359
4.9088
4.0906
3.5063
25 mL 50 mL
9.8175 4.9088
4.9088 2.4544
3.2725 1.6363
2.4544 1.2272
1.9635 0.9818
1.6363 0.8181
1.4025 0.7013
lOOmL
2.4544
1.2272
0.8181
0.6136
0.4909
0.4091
0.3506
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 4. Phytoplankton Analysis
Lake
Analyzed by
Data analyzed
Method
TAXON
Sample Mum
Lab Number
Station Num
Date Collect
Cell
Tally
Cells
per niL
Cell
Shape
ber
ber
:d
A veruge Cell Dimensions
Lentil
Width
Depth
Dktineler
Cells
Measured
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 5. Quality Control Data Sheet
Relative Percent Difference
Phytoplankton
CRUISE
STATION #
D.S.#
CYANOPHYTA
CHLOROPHYTA
CHRYSOPHYTA
CRYPTOPHYTA
others
unidentified
PENNALES
CENTRALES
total
CRL #
LAB #
DIVISION
COUNT 1
COUNT 2
RPD
LIMITS
L
56
82*
87**
52
<23
75
80***
48
* Cells must number > 140
** Cells must number >198
*** Cells must number >98
**** Cells must number >274
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Volume 3, Chapter 4
SOP for Phytoplankton Analysis
Appendix 6. Phytoplankton Archive Data
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I
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orig. Vol. 1 L
8 0
8G I__
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orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. I L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I
cone, from mL
orig. Vol. 1 L
8 0
8G I__
cone, from mL
orig. Vol. 1 L
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Volume 3, Chapter 4
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Appendix 7. Phytoplankton Archive Labels
89-0081
89GA20I12
LM 17
cone, from
orig. vol.
89-0084
89GA20I72
LM 19
cone, from
orig. vol.
89-0088
89GA21I12
LM32
cone, from
orig. vol.
00-0000
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XX 00
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orig. vol.
00-0000
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XX 00
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orig. vol.
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orig. vol.
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orig. vol.
00-0000
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XX 00
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orig. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
orig. vol.
41 mL
1 L
435 mL
1 L
487 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
89-0082
89GA20I32
LM 11
cone, from
orig. vol.
89-0085
89GA20I92
LM23
cone, from
orig. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
orig. vol.
00-0000
OOXXOOXOO
XX 00
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orig. vol.
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XX 00
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orig. vol.
00-0000
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XX 00
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orig. vol.
00-0000
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XX 00
cone, from
orig. vol.
00-0000
OOXXOOXOO
XX 00
eonc. from
orig. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
orig. vol.
522 mL
1 L
445 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
89-0083
89GA20I52
LM 18
cone, from
orig. vol.
89-0086
89GA21I12
LO27
cone, from
orig. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
orig. vol.
00-0000
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XX 00
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orig. vol.
00-0000
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XX 00
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orig. vol.
00-0000
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XX 00
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orig. vol.
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XX 00
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ong. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
ong. vol.
00-0000
OOXXOOXOO
XX 00
cone, from
ong. vol.
477 mL
1 L
505 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
000 mL
1 L
3-393
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Standard Operating Procedure for
Zooplankton Analysis
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
December 14,1994
-------
Standard Operating Procedure for
Zooplankton Analysis
1.0 Scope and Application
This method is utilized to identify and enumerate zooplankton populations from the Great Lakes.
Zoopiankton taxa are identified to the lowest taxonomic rank possible. A listing of all organisms
identified and their respective densities and morphometric measurements for biovolume
calculations are generated and reported.
2.0 Summary of Method
The method, as developed from Gannon (1971), Stemberger (1979) and Evans et al. (1982), is
used to examine a preserved zooplankton sample from a conical net towed vertically through a
water column. Microcrustacea are examined in four stratified aliquots under a stereoscopic
microscope at 20x magnification. The Rotifera are examined in two equal volume subsamples
under a compound microscope at lOOx magnification,
3.0 Sample Collection and Preservation
See United States Environmental Protection Agency Central Regional Laboratory Standard
Operation Procedure for Zooplankton Sample Collection And Preservation.
4.0 Apparatus
4.1 Stereozoom stereoscopic microscope with lOx to 70x magnification (Bausch and Lomb or another
equal quality stereoscopic microscope)
4.2 Compound microscope with lOOx to 400x magnification (Bausch and Lomb or another equal
quality compound microscope)
4.3 Calibrated Hensen-Stempel pipettes or large bore calibrated automatic pipettes: 1, 2, and 5 mL
sizes
4.4 Graduated cylinders: 100, 250 and 500 mL
4.5 Folsom plankton splitter
4.6 Ward counting wheel
4.7 Sedgwick-Rafter counting cell
4.8 Cover glass for Sedgwick-Rafter counting cell
4.9 Microscope slide, 1 x 3 inch
4.10 Cover slip, thickness: #1, 22 mm diameter
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SOP for Zooplankton Analysis Volume 3, Chapter 4
4.11 Condenser tubes with 64 pm mesh - over end
4.12 Rubber bulb for condenser tubes
4.13 Microprobe
4.14 Micro-transfer loop
4.15 Micro-forcep
4.16 400 mL glass jars with split fractions written on labels
4.17 2 L waste container
Note: Condensei tube is constructed of a 30 cm long glass tube with an inside diameter of 1.1 cm.
A small piece of "Nytex" mesh (5x5 cm, and 64 (am pore size) is used to cover one end of the tube
and mesh is secured by an 0-ring or a rubber band. A 150 mL heavy duty rubber bulb is attached
at the other end of the glass tube to apply suction.
5.0 Reagents
5.1 Formalin (= 37-40% formaldehyde solution)
5.2 5% Sodium hypochlorite solution (Chlorox bleach)
6.0 Analytical Procedure
A complete zooplankton analysis consists of two parts. In the first part, four subsamples (A, B, C,
and D Counts) are examined for microcrustaceans at 10-70x magnification and in the second, two
subsamples are examined for rotifers at lOOx magnification.
6.1 Microcrustacean Sample Analysis
The microcrustacean stratified counting method is described in 6.1.1 and the splitting procedure is
summarized in Figure 1.
Note: All containers from which zooplankton are transferred are to he rinsed thoroughly with
RO/Dl/distilled water to remove any residual organisms adhering to the container. This includes
the Folsom splitter, glass jars, and Ward counting wheels.
6.1.1 Microcrustacean Stratified Counts
6.1.1.1 Sample is divided into two 'equal" portions using a Folsom plankton splitter. One
subsample from the split is saved in a labeled jar indicating the fraction of total
original volume it contains O/i).
6.1.1.2 The second subsample from the split is placed in the Folsom plankton splitter and
divided again. One subsample is saved in a labeled jar indicating the fraction of
the total original volume it contains (V*).
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Volume 3, Chapter 4 SOP for Zooplankton Analysis
6.1.1.3 Repeat Steps 6.1.1.1 and 6.1.1.2 as many times as necessary until the last two
subsamples contain at least 200 and no more than 400 Microcrustacea each (not
including nauplii). These two subsamples of equal fraction are saved in
appropriately labeled jars.
6.1.2 Four subsamples are to be examined and enumerated. Remove the aqueous portion of the
sample with the condensing tube and transfer the remaining organisms in the counting
wheel. All Microcrustacea are identified and enumerated under a stereozoom microscope.
The four subsamples are listed below in 6.1.2.1, 6.1.2.2 and 6.1.2.3.
6.1.2.1 The final two subsamples which contain 200-400 organisms (see 6.1.1.3) are to be
counted first. These are referred to as the A and B Counts. All microcrustaceans
are examined and enumerated.
6.1.2.2 A third sample equal in fraction to the sum of the first two (A & B) samples is
examined for subdominant taxa ^taxa numbered less than 40 in both A and B
counts combined). This is the C Count.
6.1.2.3 A fourth subsample equal in fraction to the sum of the first three (A, B and C)
counts is examined for large and rare taxa. This is the D count.
ORIGINAL
SAM RLE
RRST SAMPLE SPLIT ' '- "* T H IS S A M P L E IS H E L D
L'NTIL NEEDED
111" - THIS SAMPLE PORTION IS COUNTED
D'SAMPLE
THIS SAMPLE PORTION1 IS COUNTED
C SAMPLE
mh SAMPLE SPLIT 111" l/i" THIS SAMPLE PORTION IS COUNTED
_\ AND JJ S AMPLE THESE SAMPLE- PORTIONS ARE THE T U, 0 FINAL •> A M P L E V 0 L L M h S
C THE FIRST PROCEEDING SAMPLE DIVISION
U T H I. S t I O N 0 PR f) C E E D I I N C, i \ M PI h DIVISION
••• NOTE THEAlTUAL FINAL SAMPLF DIVISION, A AND B ( O I N T S , U IL L B E DETER M I S t D B 1 THE
DENSITY O F O R G A N I S M S I N T H F O R I C, I N \ L S A M P L E T II E F I N A L S A M P L E D I V I S I O N \ O L L M t M I S T II A V E
AT LEAST :o,I ORGANISMS B L T NOT MORE THAN J 00 ORGAN IS MS IN IT
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6.1.3 Those organisms requiring higher magnification (100-lOOOx) for identification are
mounted on slide and examined under a compound microscope.
6.1.4 When adding the Microcrustacea to the counting wheel make sure that all organisms are
settled to the bottom. It is possible to sink the floating Microcrustacea by gently pressing
them down using the microprobe.
6.1.5 It is necessary to identify the sex of all mature Copopods encountered.
6.1.6 When duplicate samples are collected in the field, both original and duplicate sample
should be analyzed by the same analyst.
6.1.7 If a sample cannot be archived immediately, a few drops of formalin should be added to
the sample in order to prevent organisms from clumping.
6.1.8 In order to check for consistency of identification and enumeration, analysts should
compare their microcrustacean and rotifer results with historical data. In some occasions,
analysts may choose to re-examine archived sample(s) in order to confirm identifications
or to clarify some taxonomic problem(s).
6.1.9 After the taxonomic status of a new or unknown organism is decided, the organism should
be isolated and placed in a relabeled vial and preserved with 4-6% Formalin. This will
serve as the voucher specimen. The label on the vial should include the name of the
taxon, date preserved and initials by analysis.
6.1.10 It is important that the voucher specimens are checked periodically so that the lost or
damaged ones can be replaced. At least one 'representative' specimen should be available
in a vial at all times for examination.
Note: Adult Calanoid and Cyclopoid copepods are identified according to Wilson (1959)
and Yeatman (1959) respectively. Adult Harpacticoids are identified to species where
possible with the use of Wilson and Yeatman (1959). Immature Copepods are identified
at least to suborder (Calanoid, Cyclopoid. or Harpacticoid) and to genus where possible.
Nauplii are combined as a group and counted with the rotifers (see Section 6.2).
Cladocerans are identified to species except D'mphanosoma. Brooks (1957) and Evans
(1985) are used for Daphnia and Balcer et al. (1984) for Eubosmina. The Chydoridae and
the remaining Cladocera are identified according to Pennak (1953), Brooks (1959) and
Balcer et al. (1984).
6.2 Rotifer Sample Analysis
6.2.1 A jar is selected based on its rotifer density estimated from 6.1.1, The sample is throughly
mixed, and a 1 mL subsample is withdrawn with a Hensen-Stempel pipette (or other
precalibrated large-bore pipette). The I mL subsample described above should contain at
least 200 but no more than 400 rotifers and crustacean nauplii. If the subsample contains
fewer than 200 organisms, another subsample is taken from ajar with a larger fraction of
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Volumes, Chapter 4 SOP for Zooplankton Analysis
the original sample volume. If the subsample contains more than 400 organisms, another
subsample from ajar with a smaller fraction is used. If a 1 mL aliquot of the original
sample (unsplit) has fewer than 200 organisms, a second 1 mL aliquot reexamined and the
results are combined. The volume of the split sample of the jar is then measured in the
graduated cylinder.
6.2.2 The subsample is placed in a Sedgwick-Rafter cell and covered with a glass cover slip.
All rotifers, microcrustacean nauplii and Dreissena veliger and post-veligers are identified
and enumerated under a compound microscope at lOOx magnification.
6.2.3 After the first rotifer count is completed, a second "duplicate" count from the same jar
(6.2.1) equal in volume to the first, is enumerated.
Note: Rotifers are identified to genus and to species where possible according to Pennak
(1953), Edmonson (1959), Rutner-Kolisko (1974) and Stemberger (1979). Some rotifers
may be indistinguishable by their gross morphology because of their contracted state;
therefore, identification of these organisms is determined by examination of their chitinous
mouthparts after using sodium hypochlorite bleach as a clearing agent (Stemberger 1979).
6.3 Archiving Microcrustacean And Rotifer Samples
All zooplankton samples are archived after they have been analyzed. These archived samples are
an integral part of the quality assurance program of the contract. These samples can be reanalyzed
by the contractor (internal) or send to an outside laboratory (external) for quality assurance
check(s) or confirmation of identification(s).
6.3.1 All crustacean and rotifer subsamples are combined into a single jar. Depending on the
amount of algal materials suspended in the water column, the organisms are allowed to
settle (usually from 15 minutes to 1 hour) and the surface water is siphoned effusing a
condenser tube. The remaining combined sample is transferred to a 125 mL glass
"Qorpak" bottle. Fill the sample bottle close to the top with distilled water and add
approximately 5 mL of formalin solution to the sample. Label the bottle and the storage
box with lake, cruise, station, sampling depth and sample number. All archiving
information is computerized using a Word Perfect (Version 5.1) word processing program.
6.3.2 When archiving the sample, the excess water from the splitting jars are placed into a 2 L
waste container.
Note: Glass jars and the Folsom plankton splitter should be kept clean to avoid residue
buildup.
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SOP forZooplankton Analysis Volumes, Chapter4
7.0 Measurements of Microcrustaceans and Rotifers
For the integrated samples (20 m) and B-l (Lake Erie) samples, it is necessary to take length
measurements of microcrustaceans and rotifers:
7.1 Microcrustaceans
The first 20 encounters per species per sample are measured:
Cladocera: Length from the top of the head to the base of the caudal spine.
Copepoda: Length from tip of the head to the insertion of spines into the caudal ramus.
Mysis: Carapace length, or the length from the tip of the head to the cleft in the telson.
Bythotrephes: Body length, excluding the spine.
7.2 Rotifers
The first 20 encounters per species per survey per lake are measured:
Rotifers:
1) Loricate forms: body length from corona to the opposite end at the base of spine (if present).
2) Non-loricate forms: body length from corona to the opposite end, excluding spines, paddles,
"toes" or other extensions.
8.0 Calculations
Zooplankton data are reported as number of organisms per cubic meter which are calculated as
follows:
8.1 Volume of water filtered
V = aNRA where;
V = Volume of water filtered (m')
a = Flow meter calibration factor (read from the manufacturers calibration graph -vanes with flow
meter and their respective calibration constants. Some examples of previously used factors:
Meter No.
4738
4473
3272
3266
3099
0.1408
0.1475
0.1520
0.1465
0.1535
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Volume 3, Chapter 4 SOP for Zooplankton Analysis
NR= number of revolutions (read from the flow meter d>al)
A = area of the mouth of the net (M2)
= 0. 1 963 nr for 0.5 m diameter net
8.2 Microcrustacean densities
Where D = density of organisms in numbers per cubic meter
N = number of organisms
S = split factor
V = volume of water filtered (from 8.1)
8.3 Rotifer (and nauplii) densities
N x V x S
D =
NAxV
Where D - density of organisms in number per cubic meter
N = number of organisms
NA= number of I mL aliquot examined
Vt = volume of subsamples from which aliquot were removed
S = split factor
V = volume of water filtered (from 8.1)
8.4 Data entry
All microcrustacean and rotifer calculations are made using a Lotus 123 ver. 2.01 worksheet
program. Raw data are entered using template file ZOOPLNK.WK1. The following data taken
from bench sheets must be entered as "numerical values" (not "labels") onto the worksheet:
Number of revolutions; Flow meter calibration factor; Working volumc/Subsample volume and
Split/Split factor. The following items are to be submitted for data review;
8.4.1 A hard copy of all data entered as well as the calculated results and,
8.4.2 A floppy disk containing all information described in 8.4.1.
Note: Backup/duplicate disks must be made of all data disks submitted to EPA. These
disks are to be kept in CRL biology laboratory.
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SOP for Zooplankton Analysis Volume 3, Chapter 4
9.0 Quality Control and Methods Precision
9.1 In general, 10% of all samples analyzed are analyzed in duplicate by a second analyst. If a data set
has less than 10 samples, at least one sample from that data set is also analyzed in duplicate.
Duplicate analyzes include identification and tabulation of data. Data are calculated for the
following groups: total immature Copepoda, total mature Copepoda, total Cladocera, total Rotifer
and total zooplankton.
The relative percent difference (RPD) between duplicate determinations are compared to
guidelines listed below:
Total immature Copepoda RPD <21 %
Total mature Copepoda RPD <34%
Total Cladocera RPD <39%
Total Rotifer RPD <30%
Total Zooplankton RPD < 19%
RPD is calculated as follows:
RpD = (Larger Value) -(Smaller Value) ^ )OQ%
Average Value
9.2 Determinations which exceed control guidelines may require re-analysis unless:
9.2.1 The RPD values are the result of low population for which fewer than 100 individuals
have been counted.
9.2.2 The RPD values are the result of a chance occurrence of colonial forms which are
enumerated as individuals and which skew the results.
10.0 Safety and Waste Disposal
Proper PPE should be worn in the laboratory while handling and preparing samples for analyses.
Follow all laboratory waste disposal guidelines regarding the disposal of Formalin (37%
formaldehyde) solutions. Everyday waste should be emptied into a pre-labeled designated waste
drum for Formalin waste. Do not discard samples containing Formalin solutions into the sink.
11.0 References
11.1 Balcer, M.D., N.L. KordaandS.I. Dodson. 1984. Zooplankton of the Great Lakes. A guide to
the identification and ecology of the common crustacean species, 174p. Univ. Wise. Press.
Madison.
11.2 Brooks, J.L. 1957. The systematics of North American Daphnia. Mem. Connecticut Acad. Arts
and Sci., 13: 1-180.Can. J. Zool. 50: 1373-1403.
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Volume 3, Chapter 4 SOP for Zooplankton Analysis
11.3 Brooks, J.L. 1959. Cladocera, p. 587-656. In: W.T. Edmondson (ed.) Fresh-water Biology, 2nd
Ed., Wiley, New York, pp. 1248.
11.4 Edmondson, W.T. 1959. Rotifers, p. 420-494. In: W.T. Edmondson (ed.) Fresh-water Biology,
2nd Ed., Wiley, New York, pp. 1248.
11.5 Evans, M. 1985. The morphology of Daphnia pitlicaria, a species newly dominating the offshore
southeastern Lake Michigan summer Daphnia community. Trans Amer. Micro. Soc, 104:
223-231.
11.6 Evans, M.S., D.W. Sell and D.I. Page. 1982. Zooplankton studies in 1977 and 1978 at the Donald
C. Cook Nuclear Power Plant: Comparisons of preoperational (1971 -1974) and operational
(1975-1970) population characteristics. Univ. Michigan. Great Lakes Res. Div. Spec. Rep. 89.
11.7 Gannon, J.E. 1971. Two counting cells for the enumeration of Zooplankton micro-Crustacea.
Trans Amer. Micros. Soc. 90: 486-490.
11.8 Pennak, R.W. 1953. Freshwater invertebrates of the United States. The Ronald Press Co., N.Y.,
pp. 769.
11.9 Rutner-Kolisko, A. 1974. Planktonic rotifers: Biology and taxonomy. Die Binnengewasser 26(1)
Supplement: 146 pp.
11.10 Stemberger, R.S. 1979. A guide to rotifers of the Laurentian Great Lakes, U.S. Environmental
Protection Agency, Rept. No. EPA 600/4-79-021, 185 pp.
11.11 U.S.E.P.A. 1994. United States Environmental Protection Agency Central Regional Laboratory
Standard Operation Procedure for Zooplankton Sample Collection and Preservation.
11.12 Wilson, M.S. 1959. Calanoida, P. 738-794. In: W.T, Edmondson (ed.) Fresh-water Biology, 2nd
Ed., Wiley, New York, pp, 1248.
11.13 Wilson, M.S. and H.C. Yeatman. 1959. Harpacticoida, p. 815-861. In: W.T. Edmondson (ed.)
Fresh-water Biology, 2nd Ed., Wiley, New York, pp. 1248.
11.14 Yeatman, H.C. 1959. Cyclopoida, p. 795-814. In: W.T. Edmondson (ed,) Fresh-water Biology,
2nd Ed., Wiley, New York, pp. 1248.
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Volume 3, Chapter 4
SOP for Zooplankton Analysis
Appendix 1.
Date
deceived
Data
Set
Sample
Type
Lab
Number
River or
Lake
Station
Depth
CRL Number
Notes
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Volume 3, Chapter 4
SOP for Zooplankton Analysis
Appendix 2.
ZOOPLANKTON ANALYSIS: ROTIFERS
Lake:
Date Collected: _
Depth of tsw (m):
Analyzed by:
Working Volume (mL):
Mililiters in subsample:
ORGANISM
Ascomorena ovalis
Aspianenna priodonta
Bdailoid Rotifera
Bracniamus
Collethaca
Conocailcicas
Conocailas unicornis
Ftlinia longiseta
Gastrosus stylifar
Kelliserssia longispina
Keratalla cocalearsis
Keratalla crassa
Keratalla eartinae
Keratalla nimnalis
Keratalla quadrats
Nothsica folicae
Nothsica laurantiia
Nothsica squmula
Plossoma truncarum
Polyarenra colicrsstari
Polyarena major
Polyarena remaia
Polyarena vulgaris
Syncnista spp.
Trisassarci similis
Trisassarci cylinanca
Trisassarci multisariais
Copepod nauplii:
Braissana polymorena:
Veligar
Post-Veligar
Sample No.:
Station:
Revs:
Analyzed:
Lab No.:
St. Captain:
FM#:
Split:
A
B
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Volume 3, Chapter 4 SOP for Zooplankton Analysis
Appendix 3.
MICROCRUSTACERAS
Sample no.: Lab no.:
Split factor.
FA MA FB MB FC MC FD MD
Cyclop.s bicuipidanius tn
Cyclops vernahs
Eucyclops agalis
Eurycamera aftinis
Mesocyclops edax
Tropocyclops orasims m
Diaptomas ashlandi
Diaptomas mmnon.s
Diaptomas oragonansis
Diaptomas sicillis
Diaptomas siciloidas
Epischura lacustens
Limnocaalanus macruras
Senecaila calandidas
TOTAL MATURE COPEPODA
Sosmina longirostna
Chycorus spasanous
Dapania longiramis
Dapania puhcana
Dapania retrucurva
Diapnanosoma birgil
Eubosmina corageni
Holopadium gibberum
Lapeseora kingstil
Polypnamus pediculus
Eythessrapnes cadarstream
Capnnia scnoadlar
TOTAL CLADOC1RA:
Cyclops copepoditas
Mesocyclops copepoditas
Tropocyclops copepoditas
Uiatemus copepoditas
Episcnura copepoditas
I "nnocaianus copepoditas
Senecaila copepoditas
Eurytamora copepoditas
TOTAL IMMATURE COPEPODA (including naupilli)
Other organisms
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SOP for Zooplankton Analysis
Appendix 4.
Sample #•
Lake:
Cruise
_Analyst:_
_Station:_
Run:
Microcrustacean Measurements
Taxon
Cyclops biscuspidieus (homas) 9
ij
Cyclops vernalis 9
cf
Eucyclops agiiis 9
o1
Vlesocyclops edas 9
o-
Tropocyclops prasimus mesicams 9
o"
Diaplomus aslandi 9
tf
Diaplomas minmims 9
o-
Diapolmas oregonensis 9
cT
Diaplomus sicilis 9
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Volume 3, Chapter 4
SOP for Zooplankton Analysis
Appendix 5.
Sample #:
Microcrustacean Measurements
Taxon
Daphnia pulicaria ?
0"
Daphnia retrocurpe ?
d"
Eubesmina coregena
Ileopedium gibberum
Laptodera
Polyphemus pediculus
Cyclops copepoditas
Mesocyclops copepoditas
Tropocyclops copepodites
Diacyclops copepodites
Epishara copepoditas
Limnocalanus copepodites
Senacotta copepodites
i
2
.1
4
5
6
7
8
9
10
II
12
1.1
14
15
16
17
18
19
20
°Cal
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Quality Assurance Project Plan:
Diet Analysis for Forage Fish
Bruce M. Davis and Jacquiline F. Savino
U.S. Geological Survey
Great Lakes Science Center
1451 Green Road
Ann Arbor, Ml 48105-2899
May 1994
Version 1.0
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Quality Assurance Project Plan:
Diet Analysis for Forage Fish
1.0 Project Description
1.1 Introduction
The Great Lakes National Program Office (GLNPO) of the US EPA has initiated a Mass Balance
Study for selected toxic contaminants in Lake Michigan. The mass balance effort will be part of a
"Lake Michigan Enhanced Monitoring Program," which includes tributary and atmospheric load
monitoring, source inventories, and fate and effects evaluations. In general, the primary goal of
this enhanced monitoring program is to develop a sound, scientific base of information to guide
future toxic load reduction efforts at the Federal, State, and local levels.
A modeling team will construct a mass budget and mass balance model for a limited group of
contaminants which are present in Lake Michigan at concentrations which pose a risk to aquatic
and terrestrial organisms (including humans) within the ecosystem. Components to the mass
balance model will be designed to predict contaminant concentrations in the water column and
target fish species over a two year period, relative to loadings. Predictions of contaminant levels in
three species of fish will be calculated as final output of the model. The target fish species
include:
Lake trout (Salvelinus namaycush)
Coho salmon (Oncorhynchus kisutch)
Bloater chub (Coregonus hovi)
The calibration of the food web model(s) for these target species requires data on contaminant
concentrations and fluxes (diet) not only in these species, but also in the supporting trophic levels.
The contaminant burden of each prey species varies based on feeding patterns at lower trophic
levels.
The basic design and data requirements for the fish samples have been outlined in Tables 5 and 6
and in Appendix 4 of the Lake Michigan Mass Budget/Mass Balance (LMMB) work plan of
October 14, 1993. This project addresses a subset of the work objectives for the lower trophic
levels (forage fish diets and zooplankton abundance). The forage fish studied in this project
include:
Bloater chub
Rainbow smelt (Osmerus mordax)
Alewife (Alosa pseudoharengus)
Slimy sculpin (Cottus cognatus)
Deepwater sculpin (Myocephalus thompsoni)
The study starts in May 1994, the field season lasts through November 1994, and the data analyses
lasts nine months after the last field collection.
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Volume 3, Chapter 4
The specific objective of this study is to
1) determine the diets of these forage Fish at each site and season.
2) determine zooplankton availability at each site and season.
The diet information of forage fish and zooplankton abundance sampled by this project will enable
the modelers to quantify the movement of contaminants from their source, through the food web,
and ultimately the body burden in the target fish species.
1.2 Experimental Design
Three sites (Sturgeon Bay, Port Washington, and Saugatuck) and three seasons (spring, summer,
and fall) will be sampled to determine spatial and temporal effects on feeding by forage fish and
availability of zooplankton (Table 1.1).
Table 1.1. Summary of critical and noncritical measurements for the evaluation of diets of
forage fish and zooplankton availability.
Parameter
Location
Sample Date
Fish Length
Fish Weight
Diet Species
Diet Item
^ength
Zooplankton
Species
Zooplankton
^ength
Sampling
Instrument
GPS Loran
NA
NA
NA
NA
NA
NA
NA
Sampling
Method
SOP
NA
NA
NA
SOP
SOP
SOP
SOP
Analytical
Instrument
NA
NA
measuring
board ruler
spring or
electronic
balance
dissecting
microscope
ocular
micrometer
dissecting
microscope
ocular
micrometer
Analytical
Method
NA
NA
SOP
SOP
SOP
SOP
SOP
SOP
Reporting
Units
Lake
Regions
mo/day/yy
xx/xx/xx
mm
Kg
total
number
mm
total
number
mm
LOD
Sturgeon
Bay, Port
Washington,
Saugatuck
day
2 mm
lg
Genus or
species for
common
taxa
0.1 mm
Genus or
species for
common
taxa
0.1 mm
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2.0 Project Organization and Responsibilities
Paul Bertram John Gannon Lou Blume
EPA Project Officer NBS EPA QA Manager
Biota Co-Chair Biota Co-Chair
Jacqueline Savino
NBS
Project Manager
Bruce Davis
NBS
Field Manager
Two technical positions
NBS
Laboratory Analysis
2.1 GLNPO Project Officer and Biota Co-Chair
The GLNPO Project Officer is the Agency official who initiates the grant, evaluates the proposal,
and is the technical representative for EPA. The Project Officer is responsible for:
Budgeting
Program planning, scheduling, and prioritization
Developing project objectives and data quality objectives
Ensuring that project meets GLNPO missions
Technical guidance
Program and data reviews including audits
Data quality
Final deliverables
2.2 GLNPO QA Manager
The GLNPO QA Manager (QAM) is responsible for ensuring that each project funded by EPA
satisfies the Agency's requirements for QA programs. The QAM is responsible for:
Offering guidance on QA techniques
Evaluating QA Project Plans (QAPjPs) and approving QAPjPs for the Agency Assisting in the
coordination of audits
2.3 NBS Biota Co-Chair
The Biota Co-Chair from NBS works in partnership with the GLNPO QA Project Leader to
implement the Biota portion of the Lake Michigan Mass Balance Project. Duties are:
Program planning, scheduling, and prioritization
Developing project objectives and data quality objective
Ensuring that project meets GLNPO missions
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Diet Analysis for Forage Fish Volume 3, Chapter 4
2.4 NBS Project Manager
The Project Manager is the NBS official who initiated the proposal to perform the forage fish diet
portion of the LMMB project and is responsible for:
Developing the sampling plan for forage fish diet and zooplankton collection.
Administration of the forage fish diet segment of the biota objectives.
Overall supervision of field and laboratory work.
Ensures OA objectives are met
Technical supervision
Final deliverables
Data quality assessment
2.5 NBS Field Manager
The Field Manager is the NBS position that will provide daily supervision of the field collection
activities and laboratory analyses and the achievement of the QA objectives. This person is
responsible for:
Collecting field data
Directly supervise the field crew activities
Reviews progress toward QA objectives
Develops and implements sampling and analytical procedures
Prepares reports and deliverables
Trains field crews on sampling and analytical procedures
Data quality assessment and audits for laboratory and field segments
2.6 Field Sampling, and Analysis Personnel
The positions are responsible for the majority of the field sampling and laboratory identification.
They will receive training and guidance from the Project and Field Managers, who will also audit
their work to ensure QA objectives are met. Minimum qualifications are B.S. in the biological
sciences or two years undergraduate experience in biological sciences and work experience.
3.0 Quality Assurance Objectives
As outlined in the Lake Michigan Mass Budget/Mass Balance Work Plan, the proposed model
output should be within a factor of two of the observed concentrations in the water column and
target fish. It is also estimated that the required level of model accuracy can be achieved if
loadings and contaminant mass in significant environmental compartments are determined to
within ±20% to 30% of the actual value.
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3.1 Objectives:
1) Within each season/region strata, collect as representative a sample of coho salmon as
possible so as to minimize the spatial and temporal population uncertainty (Sp2) to the
extent possible (given the sample size that can be collected with the financial logistic, and
biological constraints of this project).
2) To collect, package, and transport each sample, and to record, summarize, and report each
physical measurement with a level of precision, accuracy, detectability, and completeness
that will ensure that Measurement Uncertainty (Sm2) will be nominal compared to Sp2
and therefore not affect the interpretation of the results.
Variability in the diet of Lake Michigan forage Fish can be great, especially when examined from a
lakewide perspective encompassing large scale spatial and temporal gradients. The desired sample
size for determining diet is to a large degree constrained by the difficulty and time required to
analyze the samples.
3.2 Measurement Quality Objectives
Measurement quality objectives are designed to control various phases of the measurement process
and to ensure that total measurement uncertainty is within ranges prescribed by the DQOs
(Table 3.1). The MQOs can be defined in terms of data quality attributes: precision, accuracy,
completeness, detectability, representativeness, and comparability. The first four can be defined in
quantitative terms, while the later two are qualitative.
Precision. A measure of mutual agreement among multiple measurements of the same property,
usually under prescribed similar conditions. Precision will be evaluated through auditing of data
collection activities to determine whether activities are performed in a consistent manner, and by
established protocol.
Accuracy. The degree of agreement between a measurement (or an average of measurements of
the same thing), and the amount actually present.
Completeness. For 'his QAPjP, completeness is the measure of the number of valid samples
obtained compared to the amount that is needed to meet the DQOs. The EMP-A completeness
goal is 90%.
Detectability. The determination of the low-range critical value of a characteristic that a method-
specific procedure can reliably discern or is necessary to meet program objectives.
Representativeness. Express the degree to which data accurately and precisely represent
characteristics of a population, parameter variations at a sampling point, a process condition, or an
environmental condition.
Comparability. Express the confidence with which one data set can be compared to another.
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Quality Assurance Project Plan:
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3.3 Laboratory MQOs
The following information describes the procedures used to control and assess measurement
uncertainty occurring during laboratory analyses. Laboratory parameters in this section will
include fish length, fish weight, prey number, and prey length. The majority of the uncertainties
occurring in the laboratory can be alleviated by the development of detailed standard operating
procedures (SOPs), and adequate training program at appropriate frequency, and a laboratory audit
program. SOPs have been developed and training has occurred. Laboratory audits will be
implemented during the course of the program implementation.
3.4 Precision
Another term for precision is repeatability. Repeatability in the laboratory is very important to
precision, as well as data comparability. Repeatability is controlled by the development of detailed
SOPs and adequate training in those SOPS. Laboratory precision can also be evaluated through
the implementation of laboratory technical systems audits. These audits will be used to evaluate
the adherence to the SOPS. Audits are discussed in Section 8.0.
3.5 Accuracy
As stated earlier, accuracy is based on the difference between an estimate, derived from data, and
the true value of the parameter being estimated. For the laboratory measurements, the true value is
dependent on the calibration of the instrument (ruler or scale). Following calibration procedures
and precision requirements will provide an indication of accuracy. Following SOPs as written
should reduce contamination as much as possible. Accuracy is also based on training. Therefore,
during audits the trainer will remeasure 5% of the samples to determine accuracy. If accuracy
requirements are not met, the trainer will review the methods with the sampler until agreement is
reached.
3.6 Detectability
Detectability in this study is a function of how accurate and repeatable the measuring instruments
can be maintained. Rulers or micrometers, unless broken, will be considered accurate. Therefore,
detectability of length is a function of following the SOPs. Similarly, scales, if calibrated properly,
should reflect an accurate weight. The SOPs will discuss ways to measure samples within the
detectability requirements.
3.7 Completeness
Completeness for the field is defined as the successful collection of all viable samples in the
appropriate time frame. A viable sample would be defined as any single sample whose integrity
has not been effected during the collection process and would therefore not be flagged with a field
qualifier.
In any case the DQOs are based on the evaluation of a statistically relevant number of samples
which are effected by all errors occurring in the field and laboratory Therefore, the overall goal is
a completeness of 90%. The completeness objective for the measurement phase is 100%. As with
the other data quality attributes, completeness can be controlled through the adherence to the SOPs
in order to minimize contamination and sampling errors.
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3.8 Representativeness
Representativeness, with respect to the overall program objectives is a function of the statistical
sampling design and how well this design estimates the measurement parameters to this project.
Variation in forage fish diet is expected seasonally but also from year-to-year, depending on the
abundance of prey and environmental factors that might affect feeding behavior. Since the
sampling period for this project is only one year, the review of past forage fish data will assist in
determining how representative the 1994 diet of forage fish is to the yearly variation that can be
expected.
3.9 Comparability
Comparability will be maintained by the adherence of the SOPs. Adherence of these SOPs by all
samplers will allow for comparability of data among sites and throughout the project. Evaluation
of comparability occurs through the implementation of the training program and the field technical
systems audits.
Table 3.1. Measurement Quality Objectives for Forage Fish Diets and Zooplankton
Parameters
^ocation
:ish Length
Precision
Accuracy
Completeness
rish Weight
Precision
Accuracy
Completeness
Sample Type
Remeasurement
Independent
remeasurement
Remeasurement
Independent
remeasurement
Frequency
5%
5%
NA
5%
5%
NA
Acceptance: Other Corrective Action
The accuracy required is to regions of lake.
2 mm of original measurement- recalibrate
remeasure sample to compare to closest; add
appropriate flags if unable to remeasure
samples.
2 mm of original measurement - review
protocols and remeasure another sample; add
appropriate flags if unable to remeasure
samples.
90%
1 g of the original measurement - recalibrate
and remeasure sample to compare to closest;
add appropriate flags if unable to revveigh
samples.
1 g of original measurement - review protocols
and remeasure another sample; add appropriate
flags if unable to reweigh samples.
90%
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Volume 3, Chapter 4
Table 3.1. Measurement Quality Objectives for Forage Fish Diets and Zooplankton
Parameters
Sample Type
Frequency
Acceptance: Other Corrective Action
Zooplankton
Species
Precision
Accuracy
Completeness
Re-identify,
inspection
Independent
re-identify,
inspection
5%
5%
NA
95% identification, precision will be maintaine
through training and periodic audits to verify
accuracy of identification prey items; add
appropriate flags if unable to re-identify
samples.
See SOPS; add appropriate flags if unable to
re-identify samples.
90%
Zooplankton
Length
Precision
Remeasurement
5%
Accuracy
Independent
re-identify,
inspection
5%
Completeness
NA
+ 0.1 mm of original measurement - recalibrate
instrument remeasure sample and compare to
closest; add appropriate flags if unable to
remeasure samples.
+ 0.1 mm of original measurement - review
remeasurement protocols and remeasure
another sample, add appropriate flags if unable
to remeasure samples.
90%
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4.0 Site Selection and Sampling Procedures
Forage fish and zooplankton samples will be taken at three regions (Sturgeon Bay, Saugatuck and
Port Washington) in spring, summer, and fall of 1994. Table 4.1 outlines the anticipated sampling
regimes.
Table 4.1. Sampling Regimes
Biotic
Element
Bloater
Alewife
Smelt
Sculpin
Total fish
Zooplankton
Group
0-2 yr
4+ yr
60- 120 mm
>120mm
>100mm
slimy
deepwater
Number
Collected/
Sample
20
20
20
20
20
20
20
3 depth strata
(hypolimnion,
epilimnion,
metalimnion)
Number
Analyzed/
Sample
10
10
10
10
10
10
10
3
Collections
9 (=3 seasons x 3 regions)
9
9
9
9
9
9
54 =
3 seasons, 3 regions,
3 sites (within a region),
2 replicates/sites
Total
Analysis
90
90
90
90
90
90
90
630
162
Ten extra fish will be collected for each sample when possible to allow for empty stomachs. The
extra fish can also be used to confirm diets if anomalous results are found in an area.
Formal chain of custody procedures are not required. However, records must be kept of sample
collection, labeling, handling, transport, and laboratory analysis. Field sheets will be used to track
integrity of sample from field to laboratory (Appendix). The unique sample I.D. assigned at
collection will be carried through to data tabulation.
5.0 Analytical Procedures and Calibration
Standard Operating Procedures for field sampling and laboratory analyses are attached.
Measurements of length and weight are the basic analytical procedures conducted for this project.
6.0 Data Reduction, Validation, and Reporting
The responsibility for data reduction, validation and reporting will be shared between Jacqueline
Savino and Bruce Davis. All samples will be given a unique labeling code that identifies sample
type, location, time of collection, replicate, and any other necessary information. Log books will
be kept that record the sample I.D. code, pertinent collection site characteristics, and taxon (fish or
zooplankton sample). All information gathered from fish preparation in the laboratory will be
added to information in the log book.
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Standard forms will be developed for laboratory data entry. Forms will be collected at the end of
each week and checked for completeness. All data will be kept as "hard copy" and in computer
files entered in ASCII data sets. Data set validation will be accomplished through comparison of
hard copy with output of computer files.
Raw data will be permanently archived in mainframe computer files at the National Biological
Survey - Great Lakes Science Center so that it can be referenced in the case of data entry error.
Copies of all files will be held separately at the NOAA Great Lakes Environmental Research
Laboratory as a means of protection against fire, vandalism, and computer failure.
The raw data will be reduced so that 1) average size of each forage fish species and their diet by
taxon within a given strata (age-season-region) and 2) the average zooplankton abundance by
taxon within a given strata (age-season-region-depth) can be reported (Table 6.1). The primary
descriptive statistics calculated and reported will include means, frequency of occurrence, and
sample sizes. The range and standard error associated with each mean will indicate the variance
associated with these data.
Table 6.1. Reported Statistics Associated with Each Biotic Element
Biotic Element
Forage fish
Forage fish diet
Zooplankton
Strata
age, season, region
age, season, region,
diet taxon
age, season, region,
depth, taxon
Measurement
length, weight
number
length
number
length
Statistics
mean, standard error, range,
sample size
mean, frequency of
occurrence, standard error,
range, sample size
mean, standard error, range,
sample size
mean, frequency of
occurrence, standard error,
range, sample size
mean, standard error, range,
sample size
7.0 Internal Quality Control Checks
Quality assurance for this project will be achieved primarily through specific training both prior to
sampling and during the sampling season. Bruce Davis on the NBS staff is experienced in diet
sampling and will provide training sessions on procedures before the sampling begins and while in
progress. Personal observation of sample under magnification is required to provide identification
of zooplankton to lowest possible taxon. Differences among observers will be checked at
beginning of samples taken from each new site and season and for every 20 samples (5%) after
initial checks. Replicate counts, identifications, and measurements taken by different individuals
for a sample must agree within acceptance criteria in Table 3.1.
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Measurements of length and weight required for this project are straightforward and their variation
will be a function of the ruler or weight scale used than the person taking the measurement. The
rulers or measuring boards will be examined prior to the field season to ensure the error between
them is less than ±2 mm. The weight scales used for this project will be standardized against
standard weights at the beginning of the project and compared to each other throughout the
sampling period. The readability of the scales used is 1 g for forage fish. Size of prey individuals
will be determined using dissecting microscope with an ocular micrometer. Other methods will be
acceptable provided that precision requirements are met.
The Pis will review and verify all raw data. The Pis will have responsibility for all statistical
analyses.
8.0 Performance and Systems Audits
Specific Audits will not be conducted as part of this sampling project. Procedures required for this
project are straightforward and not complicated. The duration of the project is also short enough
that the yearly checks on performance of the field and laboratory staff will serve as audit checks
for this project. In yearly checks, we will use acceptance criteria in SOPs. The amount of staff
involved in this project will be few, therefore, the ability to control the quality of the project will
not require elaborate auditing procedures. Quality control audits at each stage of the field
sampling and analysis will be conducted by the Project Manager, the Field Manager, or the EPA
QA Manager. Audit reports will be kept on file at the NBS-GLC and available for review at any
time.
Inadequacies in sampling procedures or the quality of the data collected will be addressed
immediately by the Project Manager or Field Manager when discovered. All previous and current
data collected by the person when the inadequacies will be review for accuracy.
An audit form for this project will be developed.
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9.0 Calculation of Data Quality Indicators
9.1 Precision
For QA reporting we will use relative standard deviation to report precision.
RSD - (s/y) x 100%
Where: RSD = relative standard deviation
s - standard deviation
y = mean of replicate analyses
n
E Or^/fo-
Where: s = standard deviation
y, — measured value of the ith replicate
y~ = mean of replicate measurements
n - number of replicates
However, on a case by case reporting we will use absolute differences between measurements to
insure that they are within criteria stated in MQOs (Table 3.1).
9.2 Accuracy
Accuracy will be based upon expert remeasurements of a percentage of samples.
Accuracy will be evaluated by determining whether the measurements are within the acceptance
limits (Table 3.1). Deviations beyond the acceptance criteria could be justification for retraining
technicians.
Bias can be estimated from the theoretical "true" value of the expert measurement. "System" bias
for the study may be calculated from individual samples and is defined:
Bias {£ (Yik - /?,)} /„
Where: Ylk = the average observed value for the ith audit sample and k observations
/?, = is the theoretical reference value
n = the number of reference samples used in the assessment
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9.3 Completeness
Completeness is defined as follows for all measurements:
%C = 100% x (Vln)
Where: %C - percent completeness
V = number of measurements judged valid
n = total number of measurements necessary to achieve a specified level of confidence in
decision making.
9.4 Representativeness
Based upon the objectives, the three seasonal collections (spring, summer, fall) represent different
forage fish diet conditions. In order to determine whether a change is statistically significant, the
samples must be representative of the population, and the samples must be collected and analyzed
in a consistent manner. Based on our sampling design (Table 4.1), ws assume that we are getting
a representative sample of fish and zooplankton within a region and season. We will evaluate
representative through qualitative comparisons of past samples from Lake Michigan.
9.5 Comparability
Comparability is very similar to representativeness in that comparability is ensured through the use
of similar sampling and analytical techniques. Comparability will be assessed through the
evaluation of precision and accuracy measurements and technical systems audits.
10.0 Corrective Action
Table 3.1, Table 10.1, internal consistency sections, SOPs, and audit section discuss the corrective
action plan. Jacqueline Savino and Bruce Davis will initiate corrective actions. Audit reports will
document corrective actions through data flags. Will revise QA plans if methods change.
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Volume 3, Chapter 4
Table 10.1 provides an initial list of flags. Pis will develop flags as conditions warrant.
Table 10.1. List of Flags.
LAC
FAC
[SP
UNK
EER
OIL
RET
REN
REJ
BAG
laboratory accident
field accident
improper sample
preservation
unknown sex
entry error
data point outlier
returned for re-analysis
re-analyzed
rejected
background correction
There was an accident in the laboratory that either
destroyed the sample or rendered it not suitable for analysis
There was an accident in the field that either destroyed the
sample or rendered it not suitable for analysis.
Due to improper preservation of the sample, it was
rendered not suitable for analysis.
In the case of species, indicates undetermined sex.
The recorded value is known to be incorrect but the correct
values cannot be determined to enter a correction.
When a series of data are plotted and analyzed, this point is
obviously not within the normal distribution of the data,
and eliminated from further analysis.
The analysis result is not approved by laboratory
management and re-analysis is required by the bench
analyst with no change in the method.
The indicated analysis results were generated from a
re-analysis of the same sample.
The analysis results have been rejected for an unspecified
reason by the laboratory. For any results where a mean is
being determined, this data was not utilized in the
calculation of the mean.
Background correction has been applied to this value.
11.0 Quality Control Reports to Management
A progress report outlining the achievement of the Quality Assurance Objectives will be provided
to the Program Manager at the end of the project. The Project Manager will be notified
immediately, however, if substantive changes are made to the QAPjP The Quality Control Report
will include a summary of the results of audits that were conducted, data quality assessment, and
the corrective actions that were taken. The report will use statistical techniques defined in
Section 9.0 and will state whether quality was better or worse than expectations defined in
Table 3.1.
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Quality Assurance Project Plan:
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Appendix 1.
Standard Operating Procedures
1.0 Collecting Forage and Zooplankton
This SOP is intended to provide a step by step procedure for collecting forage fish and
zooplankton to use in determining forage fish diets and zooplankton abundance in the Enhanced
Monitoring Program Lake Michigan Mass Balance Study.
1.1 Overview
Forage fish and zooplankton will be collected at three regions and three seasons in Lake Michigan.
Specific details of the study are documented in the Lake Michigan Mass Balance workplan and in
the QA project plan. Critical and non-critical associated information, as follows, will be recorded:
Critical Non-Critical
Location Sample depth
Date of sample Time of sample
Sample length Sample weight
Age Water temperature
These samples will be collected by NBS personnel while on their vessels. Therefore, there is a
good chance that both critical and noncritical measurements will be taken.
Summary of Method
The following sampling activities will take place and are discussed in detail:
I) Collection of fish samples
2) Collection of zooplankton samples
1.2 Safety
In any field operation, emphasis must be placed on safety. Samplers must be aware of the
potential safety hazards to which they are subjected. Follow all safety protocols and equipment
guidelines, and be prepared for emergency situations. The sampler is responsible for his/her safety
from potential hazards.
1.3 Equipment Check and Calibration
1.3.1 Serviceable Equipment
Fishing vessel equipped with
Locational instrument (GPS, Loran)
Sampling gear (midwater, bottom trawl)
Plankton net
Ice chests, including appropriate amount of ice
Measuring board (mm markings required)
Spring scale (I-IO Kg)
Calibrating weight
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1.3.2 Consumable Equipment
Fish storage bag
Formalin
Phloxine B dye
Alka-seltzer
Sugar
Borax
Bucket
Sample labels
Reporting sheets
Marking equipment (pencils and permanent marker)
Scale envelopes
Cleaning sponge and brush
Rubber gloves for
preserving fish
handling fish
Glass sample jars (zooplankton)
1.3.3 Calibration and Standardization
Equipment necessary for calibration and the required frequency can be found in Table 1.1.
Record calibration information (date, standards, results, and corrective action) in
log books.
Table 1.1. Equipment Necessary for Calibration and Required Frequency.
Instrument
Thermometer
Locational Device
Measuring Board
Calibration
Technique
Ice bath or boiling
water
Record pier-head
position
Check against
second device
Frequency
Start and end of
year
Per trip
Start and end of
year
Acceptance
Criteria
±2°
Can be adjusted to
±0.25 Km
±2 mm
1.4
Procedures
Collection of fish samples
1.4.1.1 Fish distributions are determined using acoustic instrumentation aboard large
vessels, and fish are captured with a midwater or bottom trawl.
1.4.1.2 For each collection of fish captured, record all site and sample identification data
specified on the Field Data Sheet and I.D. labels.
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Note: Data recorded will include: Objective (forage diet), gear, lake, region,
lat./long. or statistical grid, species, date, I.D. number, lake depth/capture
depth, water temperature, time of capture/time of sampling, field qualifier
flag, collector's name).
1.4.1.3 Subsamples of targeted fish are taken as follows:
Within the constraints of the demarcation of forage fish for diet, sampling into the
age and size groups specified in the LMMB plan of October 14, 1993, special care
must be taken to assure that these fish are representative by size (and hence age)
of all fish caught of the various categories being sampled.
When the trawl catch is small, the entire catch is retained and sorted by species on
the sorting table in the bow of the vessel. When the catch is large, however, it is
first randomly subsampled in the stern of the boat after running it into plastic fish
boxes that hold about 50 Ib each. The randomization is accomplished by running
the fish box or boxes back and forth over a 5 gallon bucket or buckets while fish
are slowly "poured" from the box. The subsample m the buckets is sorted into
species in the laboratory, and each species is counted.
A further sample of the catch of fish in each diet group will be obtained by first
mixing and spreading all fish in a given group on the sorting table. All fish on a
section of the table will then be retained for the diet sample. This procedure is
intended to avoid the inevitable bias that occurs when the sorter picks fish
individually from the catch.
Because the age of bloater chubs will not be known in the field, a length cut-off
based on sampling in recent years will be used to obtain an approximate
separation by age into the specified age categories for chubs of 0-2 years and
4+ years of age.
1.4.1.4 Captured fish are identified to species and counted.
1.4.1.5 Each sample is placed in labeled plastic bags and then deep-frozen or placed on
ice.
1.4.1.6 Frozen fish are transported to NBS-Great Lakes Science Center on ice in coolers
to the laboratory freezer.
1.4.2 Collection of zooplankton samples
1.4.2.1 Zooplankton samples will be taken with stratified vertical tows.
1.4.2.2 For each collection of zooplankton, record all site and sample identification data-
specified on the Field Data Sheet and I.D. labels.
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Note: Data recorded will include: Objective (zooplankton), gear, lake, region,
site (within region), replicate, lat./long. or statistical grid, species, date,
I.D. number, lake depth/capture depth, water temperature, time of capture/time of
sampling, field qualifier flag, collector's name).
1.4.2.3 The outside of the net is backwashed with water after each haul to rinse all
zooplankters into the bucket.
1.4.2.4 Place the cod end of the net in a bucket of water and add an alkaseltzer tablet
(narcotizing and buffering agent).
1.4.2.5 Each sample is washed from the bucket, with distilled water, into a sample jar.
1.4.2.6 Add 4 g sucrose and 2 g Borax/100 mL water.
1.4.2.7 Add buffered formalin (with 8 mg Phloxine B dye/1 formalin added to enhance
visibility of zooplankton) such that each sample contains 5% formalin by volume.
1.4.2.8 Zooplankton samples are transported to the NBS-Great Lakes Science Center in
federal vehicles.
1.4.2.9 Integrity of samples checked upon arrival to laboratory and recorded on field
sampling data sheets.
2.0 Forage Fish Diets and Zooplankton Abundance
This SOP is intended to provide a step by step procedure for analyzing stomach contents of forage
fish and zooplankton availability.
2.1 Overview
Stomach contents of forage fish and zooplankton availability will be analyzed in the laboratory at
NBS-Great Lakes Science Center. Specific details of the study are documented in the Lake
Michigan Mass Balance workplan and in the QA project plan. Critical and non-critical associated
information, as follows, will be recorded:
Critical Non-critical
taxon identification taxon length
taxon number
Summary of Method
The following sampling activities will take place and are discussed in detail:
1) Preparing and analyzing fish samples
2) Analyzing zooplankton samples
3) Data reporting
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Volume 3, Chapter 4
Quality Assurance Project Plan:
Diet Analysis for Forage Fish
2.2 Safety
In any operation, emphasis must be placed on safety. Personnel must be aware of the potential
safety hazards to which they are subjected. Follow all safety protocols and equipment guidelines,
and be prepared for emergency situations. The laboratory personnel is responsible for his/her
safety from potential hazards.
2.3 Equipment Check and Calibration
2.3.1 Serviceable Equipment
Fume hood
Rinse water supply and rinsing bath
Rinse tray
Dissecting cray and tools (scalpel, forceps, scissors)
Dissecting microscope with ocular micrometer
Electronic balance and calibration weights
Plastic ruler (mm divisions)
Glass specimen jars
Computer and printer (with hard drive, disk drive, and necessary software)
2.3.2 Consumable Equipment/Supplies
Formalin
Rubber gloves
Paper toweling
Plastic bags
Reporting sheets and marking devices
2.3.3 Calibration and Standardization
Equipment necessary for calibration and the required frequency can be found in Table 2.1.
Table 2.1. Equipment Necessary for Calibration and Required Frequency.
Instrument
Plastic Ruler
Electronic Balance
Computer
Ocular Micrometer
Calibration
Technique
Check against
second device
Use calibration
weight (300 g) and
slope adjust
Virus scan
Check against
second device
Frequency
Start-end/season
Daily
Every boot-up
Start-end/season
Acceptance
Criteria
±0.5 mm
±0.1 g
No viruses
±0. 1 mm
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2.4. Preparing and Analyzing Stomach Contents of Fish
Proceed with the following steps in a well ventilated (fume hood operating if necessary) area
intended for work of this nature. Wear rubber gloves when handling preserved prey items, have
equipment set up, calibrated and ready for use, and start with and maintain a clean work area.
2.4.1 Fish are thawed under cool water and individually weighed to the nearest gram and
measured to the nearest millimeter.
2.4.2 Record lengths and weights for fish with unique I.D. labels in log books containing all
associated information.
2.4.3 Stomachs are removed using surgical scissors (from esophagus to pyloric caecum). The
stomach is then preserved in 10% formalin. At this time we also determine the sex of the
individual fish if possible.
2.4.4 At a later date the stomachs are opened and contents removed completely. Contents are
teased apart and assessed as to whether they can be completely counted or need to be
subsampled (all large prey are counted completely).
2.4.5 Contents to be subsampled are diluted to a known volume (usually 100 mL), gently
stirred, and a 10% subsample is removed.
2.4.6 The contents are then identified to the lowest possible taxon, enumerated, and measured
with aid of a Ward counting wheel under a dissecting microscope with an ocular
micrometer. Up to 10 individuals per taxon per fish are measured to the nearest micron.
2.4.7 Record data as indicated on record sheets.
2.5 Analyzing Zooplankton Samples
2.5.1 In the laboratory, each sample is strained and drained of formalin.
2.5.2 If subsampling is necessary, the sample is diluted with water of a known .'olume, stirred to
provide a consistent density of plankton, and then subsampled (4 mL). The subsample is
returned to the original sample after processing and the procedure is repeated for a total of
three subsamples. Certain taxa (such as Mysis, Bythotrephes, and amphipods) are
considered loo large to be subsampled; all are removed by hand using the naked eye or a
magnifying light, and then processed in the same manner.
2.5.3 The zooplankters are identified to lowest possible taxon, enumerated, and measured with
aid of a Ward counting wheel under a dissecting microscope with an ocular micrometer.
Most mature specimens can be identified to genus and species; most immatures can be
identified to family or genus. Specimens smaller than rotifers (<100 microns) will not be
counted. Up to 10 individuals per species per station are measured to the nearest micron.
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2.5.4 The three subsample counts are averaged and the resulting mean is used to calculate
number of organisms per liter (or cubic meter).
2.5.5 Record data as indicated on record sheets.
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Volume 3
Chapter 5: Shipboard Measurements
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Standard Operating Procedure for
GLNPO Turbidity:
Nephelometric Method
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
March 1992
-------
Standard Operating Procedure for GLNPO Turbidity:
Nephelometric Method
1.0 Scope and Application
1.1 This method is applicable to drinking, surface, and saline waters.
1.2 The working range is 0-20 NTU. Samples more turbid than 20 NTU can be determined
by appropriate dilution.
2.0 Summary of Method
The method is based upon a comparison of the intensity of light scattered by the sample
under defined conditions with the intensity of light scattered by a standard reference
suspension. The higher the intensity of scattered light, the higher the turbidity. The
design of the nephelometer is specified in the method. A standard suspension of Formazin
is used for calibration.
3.0 Sample Handling and Preservation
Samples are analyzed immediately or stored at 4°C. They are considered stable for at least 48 hrs
when stored at 4°C.
4.0 Interferences
4.1 The presence of floating debris and coarse sediments will give high readings.
4.2 Air bubbles will cause high results.
4.3 Colored samples will cause low results.
5.0 Apparatus
5.1 The turbidimeter shall consist of a nephelometer with light source for illuminating the
sample and one or more photo-electric detectors with a readout device to indicate the
intensity of light scattered at right angles to the path of the incident light. The
turbidimeter should be so designed that little stray light reaches the detector in the absence
of turbidity and should be free from significant drift after a short warm-up period.
5.2 The sensitivity of the instrument should permit detection of a turbidity difference of
0.02 unit or less in waters having turbidities less than 1 unit. The instrument should
measure from 0 to 20 units turbidity. Several ranges may be necessary to obtain both
adequate coverage and sufficient sensitivity for low turbidities.
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SOP for GLNPO Turbidity:
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5.3 The sample tubes to be used with the available instrument must be clear, colorless glass.
They should be kept scrupulously clean, both inside and out, and discarded when they
become scratched or etched. They must not be handled at all where the light strikes them,
but should be provided with sufficient extra length, or with a protective case, so that they
may be handled. Differences in physical design of turbidimeters will cause differences in
measured values for turbidity even though the same suspension is used for calibration. To
minimize such differences, the following design criteria should be observed.
5.4 Light source: Tungsten lamp operated at a color temperature between 2200-3000°K.
5.4.1 Distance traversed by incident light and scattered light within the sample tube: Total not
to exceed 10 cm.
5.4.2 Detector: Centered at 90° to the incident light path and not to exceed ±30° from 90°.
The Detector, and filter system if used, shall have a spectral peak response between 400
and 600 nm.
5.5 The Hach Turbidimeter Model 2100 and 2100A, is in wide use and has been found to be
reliable, however, other instruments meeting the above design criteria are acceptable.
6.0 Reagents
6.1 Reagent water: All reagents are prepared using water which has passed through at least
two ion exchange cartridges. Throughout this SOP, water is understood to mean reagent
water unless otherwise specified, and dilute, used as a verb, means dilute with reagent
water.
6.2 Stock formazin turbidity suspension:
Solution 1: Dissolve 1.00 g hydrazine sulfate, (NH2)2-H2SO4, in water and dilute to 100 mL in a
volumetric flask.
Solution 2: Dissolve 10.00 g hexamethylenetetramine in water and dilute to 100 mL in a
volumetric flask.
In a clean dry 100 mL volumetric flask, mix 5.0 mL (volumetric pipet) of solution 1 with 5.0 mL
(volumetric pipet) of Solution 2. Allow to stand 24 hours at 25 ±33C, then dilute to 100 mL and
mix. Prepare monthly.
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SOP for GLNPO Turbidity:
Volume 3, Chapter 5 Nephelometric Method
6.3 Standard formazin turbidity suspension:
Working standards can be prepared by dilution of the following quantities of the stock formazin
turbidity suspension (nominal 400 NTU) to 200 mL.
Dilute to 200 mL Resultant NTU
lOmL 20
5 mL 10
2 mL 4
0.5 mL 1
0.2 mL 0.4
0.0 mL 0
7.0 Procedure for Turner Designs Model 100 Nephelometer
7.1 The instrument must be switched on and allowed to warm up for at least one half hour
prior to use.
7.2 Monthly the I X range and the 10 X range should be correlated by initially adjusting the
calibrate knob so that a 10.0 standard reads 9.80 on the I X range. The range is then
switched to 10 X and the top screw inside the front door is adjusted until the reading is
9.8. Check the I X range to verify that the reading is still 9.80.
7.3 Monthly and as necessary to preclude zero readings for positive turbidity samples(the
meter will not display negative readings), zero turbidity must be set to assure positive
readings. With the cell removed from the holder and the range set to I X, adjust the lower
screw inside the front door so that the digital readout is between 0.01 and 0.05 units.
7.4 Initially and for each lake or weekly (whichever comes first) a geometric series of
calibration standards prepared as above must be used to define a calibration curve. A
sealed reference 20 NTU commercial standard is used to obtain a readout of 20.0 by
adjusting the calibrate knob. Readings are then made on the freshly prepared formazin
standards.
7.5 Prior to taking a series of readings, the reference 20 NTU commercial standard will be
used to set the readout to 20.0. If the reference 20 NTU standard is lost, prepare or
otherwise obtain a new 20.0 NTU reference and proceed to Step 7.4.
7.6 Except for the 20 NTU commercial reference standard all readings should be made using
the same sample cell. The sample cell should contain reagent water when not in use. It
should be handled in a manner to preclude touching it where the light strikes it. It should
be discarded and the machine re-standardized when it becomes scratched or etched.
7.7 Readings for samples and calibration standards over 6.0 NTU should be made on the High
Range and those under 6.0 NTU should be made on the Low Ranse.
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SOP for GLNPO Turbidity:
Nephelometric Method Volume 3, Chapter 5
7.8 An aliquot of the sample warmed to 25 °C is used for the turbidity measurements to
preclude condensation on a cold sample cell. The condensation would cause erroneous
readings.
7.9 Readings may be taken immediately. If the turbidity declines continuously such as with
waters from the Niagara plume it is assumed that the initial readings are as correct as any
that will be obtained. If a reading is variable, however, such as is found with a piece of
debris, a second aliquot can be used for the reading.
7.10 If the turbidity is over 20.0 it can be diluted 1:1, 1:3, 1:7, 1:15 etc. to obtain a reading and
the appropriate factor applied to the intermediate result to determine the actual turbidity.
8.0 Calculations
8.1 Use linear regression on the results from the calibration standards to generate a calibration
curve.
8.2 The results are not edited by the analyst. If the result is -0.02 then that result is reported.
The computer program rounds the results to 0.01 NTU.
9.0 Quality Control
9.1 Turbidity
Two Control Standards are run once per 12 hour shift, or once every two stations, whichever is
less. The check standards are 10 NTU and 0.5 NTU, presently obtained from Advanced Polymers
Systems. A reagent blank (reagent water processed through the sample storage container) is run
approximately once in every four stations.
10.0 Preventive Maintenance
10.1 The cuvet should only be handled at the top Vs and efforts should be made to preclude
spilling the sample or standard on the outside of cuvet, which will necessitate drying the
cuvet with a clean soft dry tissue.
10.2 A separate bottle of verified low turbidity reagent water should be maintained exclusively
for laboratory blanks and working standards preparation. When it shous signs of
deterioration it should be replaced.
11.0 Troubleshooting/Corrective Action
11.1 A dirty or scratched cuvet should not be used. If reagent water gives a reading 0.10 units
more than the empty compartment, then the water is turbid or the cell needs to be cleaned
or discarded. A blank reading that is 0.03 to 0.05 units more than the empty compartment
is not unusual.
I 1.2 The source of excessive background readings can sometimes be identified by opening the
front door of the instrument and observing the cuvet in place.
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SOP for GLNPO Turbidity:
Volume 3, Chapter 5 Nephelometric Method
12.0 References
12.1 EPA Publication, March 1979. "Methods for Chemical Analysis of Water and Wastes"
EPA #600/4-79-02.
12.2 Standard Methods for the Analysis of Water and Waste Water, 16th Edition
APHA-AWWA-WPCF
12.3 Instruction manual for Turner Designs Model 100 Nephelometer.
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Standard Operating Procedure for
GLNPO Total
Alkalinity Titration
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
February 1992
-------
Standard Operating Procedure for
GLNPO Total Alkalinity Titration
1.0 Scope and Application
I. I This method is applicable to drinking, surface, and saline waters; domestic and industrial wastes.
1.2 This method is configured for water in the range of 10 to 250 mJL/L total alkalinity as CaCO3.
2.0 Summary of Method
This procedure for total alkalinity is an adaptation of the technique outlined in Standard Methods
for the Analysis of Water and Waste Water. A measured amount of sample is titrated with acid to
a pH of 4.5.
3.0 Sample Handling and Preservation
Glass or plastic containers are suitable. A representative unaltered aliquot is used. Biological
activity could modify the nitrogen balance and therefore slightly alter the total alkalinity, if the
sample is not analyzed immediately.
4.0 Interferences
4.1 Oil and grease may interfere by coating the electrodes and causing a sluggish response.
4.2 High mineral content may interfere by altering the activity of the water.
5.0 Apparatus
5.1 pH meter with a combination electrode.
5.2 Buret 25 mL auto zero.
5.3 Variable speed stirring motor and glass stirring paddle. The speed of this stirring apparatus should
be adjusted each time it is used such that the solution is stirring rapidly, but not so rapid that the
surface is broken.
5.4 Beaker 150-200 mL.
6.0 Reagents
6.1 Standard titrant - 0.0200 N H2SO4 (commercial). Alternatively, a more concentrated commercial
standard( 1.0 N or 0.8 N) can be diluted volumetrically on site to 0.0200 N with reagent water.
6.2 Standard pH buffers 4.0 and 7.0. Prepare from concentrates or powders as described with the
product. Use graduated cylinders for dilutions.
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SOP for GLNPO Total
Alkalinity Titration Volumes, Chapters
6.3 Stock alkalinity control standard: Dissolve 4.24 gm of Na2CO3 (dried at 250°C for two hours and
cooled in a desiccator) in reagent water and dilute to one liter in a volumetric flask. Use this
solution to prepare control standards by dilution with volumetric labware.
6.4 Typical control standards for the working range of 10-250 mg alkalinity per liter may be prepared
as follows.
Concentration
mL stock diluted mg alk/per liter
to 1 L as CaCO3
20 80
25 100
7.0 Procedure
7.1 pH meter calibration.
7.1.1 Bring ph buffers 7.0 and 4.0 to 25°C ± 5°C. Set the temperature control knob to 25°C.
7.1.2 With pH 7.0 buffer on electrode and stirrer on, adjust calib. control so meter reads 7.0.
7.1.3 With pH 4.0 buffer on electrode and stirrer on, adjust amplification or gain on meter so
that meter reads 4.0.
7.1.4 Repeat Steps 7.1.2 through 7.1.3 above until no further adjustment is necessary.
7.2 pH meter daily check.
7.2.1 With pH 4.0 buffer on the electrode, and stirrer on, adjust the calib knob so meter reads
4.0.
7.2.2 With pH 7.0 buffer on the electrode and the stirrer on, check to be sure that the meter
reads 7.0. If not then perform the pH meter calibration procedure above.
7.3 Titrate 100 mL of sample or check standard (modified 100 mL volumetric flask) with the
0.0200 N H2S04 to pH 4.5 using moderately vigorous stirring action near the end of the titration.
The stimng action should be vigorous enough near the end of titration to break the surface to
allow rapid equilibrium of CO2 between the solution and the atmosphere.
8.0 Calculations
Total Alkalinity as CaC03 in mg/L= (ml of titrant) X 10
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SOP for GLNPO Total
Volume 3, Chapter 5 Alkalinity Titration
9.0 Quality Control
9.1 GLNPO Total Alkalinity
The two control standards described above are run once per 12 hour shift, or once every two
stations, whichever is less. Reagent blanks (reagent water processed through the sample storage
container) are run approximately once in every four stations.
10.0 Preventative Maintenance
10.1 This is described in the laboratory logbook.
10.2 Maintain pH 7.0 buffer on the electrode when not in use.
11.0 References
11.1 EPA Publication, March 1979. "Methods for Chemical Analysis of Water and Wastes"
EPA #600/4-79-02.
11.2 Standard Methods for the Analysis of Water and WasteWater, 16th Edition
APHA-AWWA-WPCF.
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Standard Operating Procedure for
Electrometric pH
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
February 1992
-------
Standard Operating Procedure for
Electrometric pH
1.0 Scope
l.l This method is applicable to drinking, surface, and saline waters; domestic and industrial wastes.
1.2 The working range is 6.0 to 10.0 pH units.
2.0 Summary of Method
The pH of a sample is determined electrometrically using a glass electrode in combination with a
reference electrode, or with a combination pH electrode.
3.0 Sample Handling and Preservation
3.1 Samples are collected in clean glass or plastic containers. They should be completely filled
whenever possible.
3.2 Sample are stored at 4°C. They are considered stable for at least 24 hours.
4.0 Interferences
4.1 Any sample constituent which coats the electrode can cause sluggish response. The electrodes
must be kept clean.
4.2 Temperature effects on the electrometric measurement of pH arise from two sources. The first
source is caused by change in electrode output at various temperatures. This can be avoided by
using an instrument with automatic temperature compensation. The second source is the change
of pH inherent in the sample at various temperatures. Both sources of variation are avoided by
conducting all measurements and standardization at 25°C.
5.0 Apparatus
5.1 pH meter, such as the Accumet 25
5.2 Electrodes
5.2.1 A glass electrode and a reference electrode may be used.
5.2.2 A combination pH electrode, such as Orion Ross type epox^ body.
3-459
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SOP for Electrometric pH Volume 3, Chapter 5
6.0 Reagents
6.1 Reagent Water
6.2 Calibration Standards: Standard buffers are commercially available. Buffers of 7.00 and 10.00
are used for calibrating the instrument. Graduated cylinders should be used for dilution of the
buffer concentrates.
6.3 Control Standards: Commercially available buffers 6.86 and 9.18 are used for low and high
control standards for lake water samples. Graduated cylinders should be used for dilution of the
buffer powder packets.
7.0 Calibration
Calibrate the pH meter according to the manufacturer's instructions.
7.1 All standards and samples are brought to 25°C before use.
7.2 All standardizations are preceeded by rinses with material to be used for calibration.
7.3 A pH 7.0 buffer is placed on the apparatus and the stand key is pressed.
7.4 When the meter so indicates, rinse the apparatus with reagent water and then buffer 10.
7.5 A pH 10.01 buffer is placed on the apparatus and the slope key is pressed.
7.6 The meter will indicate when the standardization is complete.
8.0 Analytical Procedure
8.1 All samples and standards are brought to 25°C before use.
8.2 Rinse the electrodes and other equipment contacting the sample with reagent water.
8.3 Pour an aliquot of sample into a suitable container. Place the sample onto the stirrer and electrode
and stir it moderately rapid without breaking the surface.
8.4 When the meter stabilizes, record the pH reading.
8.5 Analyze control standards in the same manner.
8.6 When all measurements are complete, store the electrode in pH 7.0 buffer.
9.0 Calculations
The pH values are determined directly from the meter readings.
3-460
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Volume 3, Chapter 5 SOP for Electrometric pH
10.0 Quality Control
10.1 GLNPO Electrometric pH
Two Control standards are run once per 12 hour shift, or once every two stations, whichever is
less. The check standards have values of 9.18 and 6.86.
11.0 Troubleshooting/Corrective Action
11.1 Problems associated with non-linearity can generally be traced to a defective electrode or one or
more defective buffer solutions.
11.2 A sluggish response may be due to a dirty electrode membrane or a plugged junction in the
reference electrode, or inadequate reference electrode solution. A dirty membrane can sometimes
be cleaned with ethanol or 1 N NaOH (three or four minutes with the stirrer running).
12.0 References
EPA Publication, March 1979. "Methods for Chemical Analysis of Water and Wastes"
EPA #600/4-79-02.
Standard Methods for the Analysis of Water and Waste Water, 16th Edition
APHA-AWWA-WPCF
3-461
-------
Standard Operating Procedure for
Meteorological Data Aboard
the RV/Lake Guardian
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
February 1992
-------
Standard Operating Procedure for
Meteorological Data Aboard the
RV/Lake Guardian
1.0 Scope and Application
1.1 This method, applicable to all surveillance cruises performed by the RV/Lake Guardian was in
effect during the calendar years 1994 and 1995.
1.2 These procedures are implemented while the vessel is underway and while occupying a sampling
station.
2.0 Summary of Method
The officer in charge of the bridge is responsible for implementing the procedures herein
described. On the hour, the following parameters are recorded in the ship's log: wind speed and
direction, wave height and direction, air temperature, barometric pressure, visibility, present
weather conditions and heading (when underway). For each significant event the time and
description is recorded. At each sampling station, the station identification, arrival time, departure
time, wind speed and direction, wave height and direction, barometric pressure, water depth, air
temperature, geographic location (loran and/or GPS), and final location (if vessel has drifted
during sampling) are recorded. The deviation of the ship time from Greenwich mean time is
recorded daily.
3.0 Apparatus
3.1 Wind Speed and Wind Direction meter.
Electric Speed Indicator Company, Cleveland Ohio.
U.S. Dept. of Commerce, Weather Bureau.
Built-in correcting device for the Ship heading.
3.2 Aneroid Barometer.
3.3 Electronic thermometer.
RMS Technology for the Weather Bureau.
3.4 Gyro-Compass, Sperry SR 130.
3.5 Magnetic Compass, Ritchie 5"
3.6 Fathometer, Furuno FE 881 Mk-11.
3.7 Loran, Northstar 800.
3.8 GPS StaNav, Furuno GP 500.
3.9 Doppler Speed Log, JEC JLN-203.
3-465
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Standard Operating Procedure for
GLNPO Specific Conductance:
Conductivity Bridge
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
February 1992
-------
Standard Operating Procedure for
GLNPO Specific Conductance:
Conductivity Bridge
1.0 Scope and Application
1.1 This method is applicable to drinking and surface waters.
1.2 The approximate working range is 10 to 500 mhos/cm.
2.0 Summary of Method
2.1 The specific conductance of a sample is measured using a self-contained conductivity meter,
Wheatstone Bridge type, or equivalent.
2.2 The conductivity is measured at 25°C.
3.0 Sample Handling and Preservation
3.1 Samples are collected in clean glass or plastic containers.
3.2 Samples are stored at 4°C. They are considered stable for 28 days.
3.3 When placing a beaker of standard or sample on the apparatus, care must be taken to assure that no
air bubbles are trapped inside the electrode.
3.4 The apparatus must be rinsed once with a portion of the solution (sample or standard) before
taking a reading or calibrating the meter.
4.0 Interferences
4.1 Oil, grease, algae, or dirt can interfere by coating the electrodes, causing sluggish response and
incorrect readings.
4.2 Sample temperatures other than 25°C will cause incorrect results.
5.0 Apparatus
5.1 Conductivity Meter, Wheatstone Bridge type or equivalent, with nominal 1 cm cell constant.
(YSI Model 35 with YSI probe 3403).
5.2 Variable speed stirring motor with glass stirring paddle.
5.3 Immersion heater with controller.
3-469
-------
SOP for GLNPO Specific Conductance:
Conductivity Bridge Volume 3, Chapter 5
6.0 Reagents
6.1 Reagent water: All reagents are prepared using reagent water that has passed through at least two
ion exchange cartridges. The specific conductance of the reagent water used for standard
preparation must be less than 1 u mho/cm.
6.2 Stock sodium chloride set standard solution. Dissolve 10.000 gm of dried NaCl in reagent water
and dilute to 1 L in a volumetric flask.
6.3 Working Calibration Standards
The following may be prepared with volumetric labware.
mL of 10 gm/L NaCl Specific Conductance
diluted to 1 L umhos/crn
20 415.8
15 313.5
10 210.3
5 106.1
6.4 Stock Control Standard Solution: Any salt solution with known specific conductance may be used
to prepare control standards. However it should be prepared entirely independently by someone
other than the analyst. The following is acceptable.
Dissolve 10.000 gm of dried KC1 (105°C for two hours) in reagent water and dilute to 1 L in a
volumetric flask.
6.5 Control Standards: The following may be prepared using volumetric labware.
mL 10.00 g/L KCI Specific Conductance
diluted to 1 L umhos/cm at 25°C
Hi Control 15 293.3
Lo Control 10 196.5
7.0 Instrument Calibration - YSI Model 35
7.1 The calibration procedure involves correlating the instrument reading to the known concentration
of the calibration standards and results in determining the cell constant correction.
7.2 Pour a used portion of 313.5 umho/cm NaCl standard into an appropriate receptacle and place this
on the apparatus. Turn on the stirrer to rinse the various components and then discard the solution.
3-470
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SOP for GLNPO Specific Conductance:
Volume 3, Chapter 5 Conductivity Bridge
7.3 Pour a fresh portion of 313.5 umho/cm NaCI standard into the same receptacle and place on the
apparatus. Turn on the stirrer and the heater and adjust the temperature to 25.0°C. Adjust the
umho calibration potentiometer so that the reading coincides with the true value. Check to be sure
the temperature is still 25.0°C. Save this solution for rinse for the next standardization.
7.4 Similarly, check the other set standards and a blank (Reagent Water) to verify proper operation
over the entire range (Do not readjust the umho calibration potentiometer).
8.0 Analytical Procedure
8.1 Rinse the apparatus with sample by filling the receptacle and putting it in place on the apparatus.
Discard this rinse and then fill the receptacle with sample and place it on the apparatus such that
there are no air bubbles inside the conductivity cell. For samples from the same station with about
the same conductivity, it is not necessary to rinse between samples.
8.2 Adjust the temperature to 25.0°C, and record the reading from the conductivity meter.
8.3 When the apparatus is not being used, the conductivity cell should be immersed in reagent water.
For extended periods of non-use, the cell may be thoroughly rinsed with reagent water and left to
dry. Before re-use, it must be soaked overnight in one of the standards.
9.0 Calculations
The specific conductance is determined directly from the proper meter readings and the range
indication, e.g.,:
Meter Reading X Range
umho/cm Specific Conductance
6.20X100 620
0.62 X 1 0.62
10.0 Quality Control
10.1 GLNPO Specific Conductance
The two control standards described above are run once every 12 hour shift, or once every two
stations, whichever is less. Reagent blanks (reagent water processed through the sample storage
container) are run approximately once in every four stations.
11.0 Preventive Maintenance
This is described in the system log book.
3-471
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SOP for GLNPO Specific Conductance:
Conductivity Bridge Volume 3, Chapters
12.0 Troubleshooting/Corrective Action
12.1 Non linear response may be caused by a non-conditioned cell, a dirty cell, inadequate care in
preparation of standards, inadequate attention to precluding bubbles from the cell during
standardization, a defective cell, inadequately precise control of temperature, or inadequate rinsing
of the apparatus between standards.
12.2 If the cell is so dirty that bubbles always form at the top of the cell when a receptacle of water is
placed on the apparatus, ethanol or 1 N NaOH may be used to attempt cleaning. Neither should be
left on the cell for more than two or three minutes.
13.0 References
13.1 "Methods for Chemical Analysis of Water and Wastes"; March, 1979. EPA Publication
#600/4-79-02.
13.2 "Operating Instructions, YSI Model 35 Conductivity Meter.
13.3 "Calibration of Conductance Cells at 25°C with Aqueous Solutions of Potassium Chloride";
April 1959. Journal of the American Chemical Society. 1557-1559
3-472
-------
Total Hardness Titration
Marvin Palmer
United States Environmental Protection Agency
Great Lakes National Program Office
Region 5
77 West Jackson 9th Floor
Chicago, Illinois 60604
July 1994
-------
Total Hardness Titration
1.0 Background
Hardness of water is a measure of the total concentration of the calcium and magnesium ions
expressed as calcium carbonate.
In this procedure, a water sample is buffered to pH 10.1 and indicator is then added to the buffered
sample. The indicator, when added to a solution containing Ca and Mg ions, turns red. EDTA,
the titrant, complexes with Mg and Ca cations, removing them from association with the indicator.
When all the Mg and Ca are complexed with EDTA, the indicator will turn blue.
The analysis must be performed on the mid depth sample during unstratified conditions, and on
the mid-epilimnion and mid hypo-limnion sample when stratification is present.
2.0 Procedure
2.1 A 100 mL water sample is measured into a plastic beaker containing a stirring bar. The water
should be at room temperature, so it is easiest to use the water warmed for specific conductance
measurement.
2.2 A 1 mL volume of buffer solution is added to the stirred water. This buffer solution is found in
the small bottle marked "Buffer Solution Hardness 1"
2.3 One packet of indicator "ManVer 2 Hardness Indicator Powder Pillows" is added to the buffered
sample. A red color will result. From this point, no more than five minutes should elapse to the
end of the analysis to prevent CaCO3 formation.
2.4 While stirring, the sample is titrated with 0.01M EDTA solution until the sample turns blue (no
tinge of red remains).
2.5 The volume of titrant is marked on the "board sheet."
2.6 The titrated sample, with a pH of approximately 10, is discarded into a holding container for future
neutralization.
2.7 Calculations: Total Hardness; mg/L as CaCO3 = 10 x mL of titrant.
2.8 These reagents and chemicals can be obtained from Hach Chemical Company and are described in
Standard Methods for the examination of Water and Wastewater, 14th Edition.
3-475
-------
Standard Operating Procedure for the
Analysis of Dissolved-Phase Organic Carbon
in Great Lakes Waters
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
December 19,1996
-------
Standard Operating Procedure
for the Analysis of
Dissolved-Phase Organic Carbon
in Great Lakes Waters
1.0 Scope and Application
This Procedure describes the analysis of filtrates from Great Lakes water samples for dissolved
organic carbon (DOC). After filtration, the analysis of the filtrates is byconversion of organic
carbon to CO2by an ultraviolet (UV) digester with detection of CO, by an infrared (IR) analyzer.
This SOP covers standard and instrument preparation, instrument calibration and maintenance,
analysis of carbon, and calculation of results.
2.0 Safety and Waste Handling
All applicable safety and waste handing rules are to be followed. These include the proper labeling
and disposal of chemical wastes. Over-board discharges of chemical wastes are forbidden. Refer to
the GLNPO Safety, Health, and Environmental Compliance Manual for specific rules.
3.0 Summary of Procedure
The determination of organic carbon requires the removal of inorganic carbon, which is present in
Great Lakes water samples as carbonate. Removal of inorganic carbon is achieved inside the
analyzer by acidifying the sample with 1.0 N sulfuric acid. A high-velocity stream of organic-free
air transforms the acidified filtrate into a thin, turbulent liquid film. The film is transported rapidly
through a large-bore coil which provides the necessary surface area for efficient CO, removal. At a
purge rate of 500 mL per minute, up to 500 mg of inorganic carbon can be removed with minimal
loss of volatile organic compounds. An aliquot of the carbonate-free filtrate is then segmented in
the automatic analyzer for analysis. The aliquot is mixed with a stream of 1.0 N sulfuric acid and
potassium persulfate, and subjected to ultraviolet radiation to assure complete oxidation of the
organic carbon. The resulting CO2 is then purged with a stream of CO, free air or nitrogen, and is
detected with a non-dispersive infra-red analyzer. The signal from the IR detector is output to a
strip chart recorder. The concentration of dissolved organic carbon in the filtrate is calculated
using the peak height method.
4.0 Description of Instrumentation
The instrumentation consists of a Technicon Auto Analyzer II system, including an Auto Sampler
IV, a Proportioning Pump III, a DOC manifold, an ultraviolet digester, and a CO, and non-
dispersive IR analyzer (Beckman Model 865). A source of CO, -free air with flow control,
indicators, and a strip chart recorder are also used.
3-479
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SOP for the Analysis of Dissolved-Phase
Organic Carbon in Great Lakes Waters Volume 3, Chapters
5.0 Preparation
5.1 Sample Handling and Preservation
Great Lakes water samples are filtered immediately after collection, transferred to clean glass
containers, and stored at 4 °C until analysis. Filtrates are stable for 24 hours if properly stored.
5.2 Interferences
5.2.1 Inorganic carbon is the only known interferant in this analysis. Inorganic carbon is
removed in the autoanalyzer through addition of 1.0 N sulfuric acid. Low results for this
analysis may be obtained on some volatile organic compounds.
5.2.2 Organic vapors, such as solvents, may contaminate the filtrates unless care is taken.
5.3 Preparation of Reagents
5.3.1 Organic-free, distilled, deionized water (from now on referred to as organic-free water) is
used for the preparation of all reagents and standards.
5.3.2 All reagents should be stored in appropriate glass bottles and labeled with reagent identity,
date of preparation, concentration, and the initials of the preparer.
5.3.3 1.0 N sulfuric acid reagent: Add 28 mL of concentrated sulfuric acid to about 800 mL of
organic-free water. Mix and dilute to one liter.
5.3.4 4% persulfate reagent: Dissolve 40 grams of potassium persulfate (K2S2O5) in organic-
free water. Mix and dilute to one liter.
5.4 Preparation of Calibration Standards
5.4.1 Stock 1000 mg/L carbon solution: Dissolve 2.125 grams of potassium biphthalate
(KHC8H4O4) in 500 mL of organic-free water. Add 1 mL of concentrated sulfuric acid
(H,SO4). Mix and dilute to one liter.
5.4.2 Working calibration standards: Prepare standards to cover the entire range of expected
concentrations of DOC. Working calibration standards should be prepared daily . For a
typical working range of 0 - 10 mg of carbon/L, the following standards may be used:
mL of stock carbon solution* Concentration (mg carbon/L)
2.0
1.0
0.5
0.2
0.1
0.0
10.0
5.0
2.5
1.0
0.5
0.0
*diluted to 200 mL in or»anic-free water.
3-480
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SOP for the Analysis of Dissolved-Phase
Volume 3, Chapter 5 Organic Carbon in Great Lakes Waters
5.4.3 Stock 514 mg carbon/L control solution: Dissolve 1.2604 grams of glutamic acid
(C5H9O4N), which has been dried for 2-3 hours at 70°C, in 500 mL of organic-free water.
Add 1 mL of concentrated sulfuric acid (H2SO4) and dilute to one liter.
5.4.4 Working Control Standards: Prepare the following control standards on a daily basis:
mL of stock control solution* Concentration (mg carbon/L)
High Check (CS-1) 2.0 mL 5.14
Low Check (CS-2) 0.5 mL 1.28
*diluted to 200 mL in organic-free water.
5.4.5 Label all calibration and control standard solutions with ID, date of preparation,
concentration, and initials of the preparer.
6.0 Analytical Procedures
6.1 Instrument Set-up
Assemble the Auto Analyzer DOC manifold following the diagram in Figure 1. Make sure there
are no leaks in the air system. Activate the IR detector by turning on the main power switch and
allowing it to warm up for a minimum of 2 hours prior to use. At the same time, run CO2-free air
through the IR detector and set the zero control read near zero. After the instrument is assembled
and checked against the diagram in Figure 1, switch on the reagent flows, the air flow, the UV
digester and the proportioning pump. Wait for a stable baseline from the IR detector before
starting the calibration procedures.
6.2 Procedures
6.2.1 Once a stable baseline from the IR detector has been achieved, run the highest calibration
standard (primer). Adjust the strip chart recorder to the appropriate range to keep the peak
on the chart paper.
6.2.2 Load the automatic sampler tray and run the remaining calibration standards, check
standards, blanks, and Great Lakes water sample filtrates in the following order:
1st: 10 mg carbon/L calibration standard (primer)
2nd: From the 5.0 mg carbon/L standard down to the 0.0 mg carbon/L standard
(from high to low)
3rd: a reagent blank
4th: CS-1
5th: CS-2
6th: up to 40 filtrates
7th: a reagent blank
8th: CS-1
9th: CS-2
3-481
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SOP tor the Analysis of Dissolved-Phase
Organic Carbon in Great Lakes Waters Volume 3, Chapter 5
6.3 Instrument Shut-down
6.3.1 After analysis is completed, some parts of the instrument are shut down. First put the
system on wash for at least 30 minutes. Then turn off the automatic sampler, proportioning
pump, and UV digester. The organic-free air supply should be allowed to run through the
IR detector.
6.3.2 The infrared detector should be left on.
REPEAT: LEAVE THE IR DETECTOR ON.
7.0 Calculations
Measure the peak heights of the calibration standards (manually). Calculate the regression
equation for the calibration curve using a second order regression with zero forcing. Apply this
regression equation to determine the DOC concentration in the filtrates from the peak heights.
8.0 Maintenance and Trouble-Shooting
An unstable baseline may indicate that the manifold system may need some tubing replacement or
there is a leak in the air system. Change the drierite trap between the phase separator and the IR
detector prior to complete exhaustion of the trap.
9.0 Quality Control
9.1 The minimum acceptable correlation coefficient (r) for the calibration curve is 0.995.
9.2 The following criteria are required to be met, with the minimum frequency indicated, for the
analysis to be considered in control.
Criterion Frequency Limits (mg carbon/L ± 3 std)
High Check (CS-1) Begin+End, 1/40 samples 5.14 + 0.90
Low Check (CS-2) Begin+End, 1/40 Samples 1.28 ± 0.60
Reagent Blank Begin+End, 1/40 Samples 0.00 ± 0.60
Lab. Blank Begin+End, 1/40 Samples 0.00 ± 0.60
3-482
-------
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SOP for the Analysis
Volume 3, Chapter 5 Oraanic Carbon in
r~ «>
•^
-------
Standard Operating Procedure for the
Analysis of Particulate-Phase Organic Carbon
in Great Lakes Waters
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
August 2, 1994
-------
Standard Operating Procedure for the
Analysis of Particulate-Phase Organic Carbon
in Great Lakes Waters
1.0 Scope and Application
This Standard Operating Procedure describes the analysis of glass fiber filters from particulate-
phase Great Lakes waters samples and associated quality control blanks and duplicate samples for
particulate organic carbon (POC). The analysis is by catalytic combustion followed by packed
column gas chromatographic separation with thermal conductivity detection. This SOP covers
standard and instrument preparation, instrument calibration and maintenance, elemental analysis of
carbon, and calculation of results.
2.0 Safety and Waste Handling
All applicable safety and waste handling rules are to be followed. These include the proper
labeling and disposal of chemical wastes. Over-board discharges of chemical wastes are forbidden.
Refer to the GLNPO Safety, Health, and Environmental Compliance Manual for specific rules.
3.0 Summary of Procedure
Filtered Great Lakes water samples are analyzed for POC in a ship-board or land-based laboratory.
Sub-samples of the exposed glass fiber filters are loaded into small tin capsules and placed in the
autosampler of a Carlo Erba Elemental Analyzer 1108. At preset intervals, the tin capsules are
dropped into a vertical quartz reactor tube inside a 1000°C furnace. After a capsule drops into the
reactor tube, the carrier gas is temporarily enriched with oxygen causing instantaneous oxidation
of the sample. Quantitative oxidation is achieved by passing the resulting mixture of gases over a
tungstic anhydride catalyst. The gas mixture then passes over elemental copper to remove excess
oxygen and to reduce nitrogen oxides to elemental nitrogen. The sample gases pass though a
packed chromatographic column, are separated, eluted, and detected by a thermal conductivity
detector (TCD). Organic carbon is quantified by the external standard method.
4.0 Description of Instrumentation
The Carlo Erba EA 1108 Elemental Analyzer is a commercially-available instrument comprised of
a combustion furnace, gas chromatographic oven, and thermal conductivity detector. It can be
configured to detect carbon, hydrogen, nitrogen, and sulfur simultaneously (an oxygen
determination mode is also possible). The instrument is equipped with a pneumatic autosampler
and a PC-based computer data system (Carlo Erba Eager 200). The analytical method uses one of
two available furnaces to house a catalytic reactor tube. The reactor tube is packed with an upper
part which functions as an oxidation catalyst (tungstic anhydride), and a lower portion which
functions as the reduction reactor (elemental copper). After exiting the reactor tube, the gas-phase
sample travels through a water trap (anhydrone), and then, into a packed chromatographic column.
The sample components are separated by the column as CO2, H:, N;, and H2S. These species are
detected by a thermal conductivity detector (TCD).
3-487
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SOP for the Analysis of Particulate-Phase
Organic Carbon in Great Lakes Waters Volume 3, Chapter 5
5.0 Preparation
5.1 Preparation of Calibration Standards
5.1.1 The calibration standard stock solution of potassium hydrogen phthalate (KHP, AR grade
or better) is prepared by dissolving 4.2509 grams of crushed, dried (110 °C for two hours)
KHC8H4O4in organic-free, distilled, deionized water (from now on referred to as organic-
free water). Add 0.2 mL of concentrated sulfuric acid (AR grade or better) and dilute to 1
liter in a volumetric flask. The concentration of the solution is 2000 mg of carbon/L.
5.1.2 The stock solution is stored at 4 °C in a clean glass bottle. Label the bottle with reagent
name, concentration, date prepared, expiration date, and analyst's initials.
5.1.3 Working calibration standards are prepared from the stock KHP solution. Use Class A
volumetric pipets and volumetric flasks. Store the working calibration standards in clean
glass bottles with Teflon-lined caps at 4 °C. Label the bottles with reagent name,
concentration, date prepared, expiration date, and analyst's initials. Prepare the working
standards following these directions:
mL Stock KHP,
diluted to 100 mL
5.0
10.0
25.0
50.0
75.0
100.0
(jg carbon/50 jaL spike
5.0
10.0
25.0
50.0
75.0
100.0
5.1.4 Stock and working calibration standards are prepared on a monthly basis.
5.2 Preparation of Calibration Check Standards
5.2.1 The calibration check standard stock solution of ethylenediamine tetra-acetic acid (EDTA,
di-sodium salt, AR grade or better) is prepared by dissolving 6.1983 grams of EDTA in
organic-free water. Add 0.2 mL of concentrated sulfuric acid (AR grade or better), taking
precaution to prevent precipitation, and dilute to 1 liter in a volumetric flask. The
concentration of the solution is 2000 mg of carbon/L.
5.2.2 The stock solution is stored at 4 °C in a clean glass bottle. Label the bottle with reagent
name, concentration, date prepared, expiration date, and analyst's initials.
3-488
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SOP for the Analysis of Particulate-Phase
Volume 3, Chapter 5 Organic Carbon in Great Lakes Waters
5.2.3 Working calibration check standards are prepared from the stock EDTA solution. Use
Class A volumetric pipets and volumetric flasks. Store the working calibration check
standards in clean glass bottles with Teflon-lined caps at 4 °C. Label the bottles with
reagent name, concentration, date prepared, expiration date and analyst's initials. Prepare
the working standards following these directions:
mL stock EDTA
15.0 diluted to 100 mL
20.0 diluted to 25 mL
jag carbon/50 |uL spike
15.0
80.0
5.2.4 Stock and working calibration check standards are prepared on a monthly basis.
5.3 Preparation of Elemental Analyzer
5.3.1 Read the operating manuals for the elemental analyzer and data system. The instrument is
initially installed following instructions in the manuals. Field assistance from the
manufacturer may be necessary. The data system requires the installation of a circuit board
into the PC (see the data system manual). If the instrument is to be used in a ship-based
laboratory, proper installation requires both vibration isolation and secure mounting.
5.3.2 Verify that the instrument has been properly and securely installed. Locate the main power
switch on the left side of the back panel. The instrument may be turned off at this switch
when not in use for extended time periods, or it can be left in stand-by for shorter periods.
The instrument power should be off during preparation.
5.3.3 The instrument operates using three pneumatic systems. Helium and oxygen must be UHP
grade (99.999%) or better. The gases required are:
Helium: Helium is used as the carrier gas. A combination oxygen/hydrocarbon trap (e.g.,
Supelco OMI-1) is installed in the helium line, as close to the instrument as possible. Set
the helium supply pressure to 200-300 kPa at the tank.
Oxygen: Oxygen is used during the combustion step in the reactor tube. Set the oxygen
supply pressure to 100 kPa at the tank.
Air: Air is used to operate pneumatic valves in the instrument and to operate the
autosampler. Set the air supply pressure to 350-400 kPa at the tank.
5.3.4 The instrument's analytical configuration is determined by the materials used to pack the
reactor tube. A CHNS configuration will be described here. Refer to the operating manual
for other possible configurations. Figure 1 shows the CHNS packing.
5.3.5 Pack the reactor tube by marking the sizes of the packing layers (see Figure 1) on the
outside of the quartz tube with a permanent marker and filling to the marks with the
appropriate material. The packing materials are held in place with quartz wool.
5.3.6 Place the Viton O-ring on the empty portion of the packed reactor tube, with the flat part
facing down.
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5.3.7 Lower the reactor tube into the left furnace of the instrument with the copper layer going
in first (towards the bottom). Adjust the O-ring so that it is approximately 1 inch from the
top of the reactor tube.
5.3.8 Attach the autosampler to the top of the reactor tube.
5.3.9 Install the nut and washer followed by a Viton O-ring with the flat end facing up. Attach
the coupling to the bottom of the reactor tube.
5.3.10 A water trap is used to keep water out of the detector. An unstable, high baseline will
result if a water trap is not used. Pack the water trap by filling the glass tube with
anhydrone (magnesium perchlorate). Use 5 mm of quartz wool to hold the material in
place.
5.3.11 Connect the fittings to the water trap and clip it into the mounting bracket in front of the
chromatographic oven. Check the water trap daily, and repack when needed.
5.3.12 A leak check of the pneumatic systems is necessary at the start of each day of analysis.
Turn on the main power switch. Depress the filament standby push-button, which is
located in the lower left corner of the instrument control panel.
5.3.13 Follow these steps to do a leak check:
A. Set the air pressure to 350 kPa using the pressure regulator on the front
panel of the instrument.
B. Wait 2 minutes.
C. Turn the tank pressure regulator off. If the system maintains the 350 kPa
pressure for 2 minutes, the air system is leak-tight.
D. Turn the helium and oxygen pressure regulators on the front panel to zero
and cap off the carrier gas vents (measuring and reference) and oxygen
vent located on the lower front panel with the caps provided with the
instrument.
E. Adjust the pressure regulators on the front panel to 100 kPa for helium
and 150 kPa for oxygen.
F Wait 2 minutes.
G. Turn the front panel valves for helium and oxygen off. If the system
maintains the 100 kPa. for helium, and 150 kPa, for oxygen, pressures for
3 minutes, the Systems are leak-tight. Replace the caps with the fittings.
5.3.14 If there are leaks, refer to the operating manual (section 3.2 in the Carlo Erba EA 1108
Manual). Leaks can be traced using a liquid leak detector (e.g., Snoop) to check for loose
connections.
5.3.15 When the leak check procedure is completed, release the filament standby button.
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Volume 3, Chapter 5 Organic Carbon in Great Lakes Waters
5.3.16 Set the helium and oxygen flow rates:
A. Set the helium analytical flow rate to 100 mL/min. using the front panel pressure
regulator. Measure the flow at VENT M on the lower front panel of the
instrument using a soap-bubble flowmeter.
B. Set the helium reference flow rate to 40 mL/min. using the front panel pressure
regulator. Measure the flow at the PURGE vent on the lower front panel of the
instrument using a soap-bubble flow meter.
C. Set the oxygen flow rate to 12 mL/min. using the front panel pressure regulator.
Measure the flow at the OXYGEN vent on the luwer front panel of the instrument
using a soap-bubble flowmeter.
5.3.17 Set the instrument control panel settings as follows:
A. Left furnace-1000 °C
B. Right furnace -500 °C
C. Oven temperature - 70 °C
D. Filament temperature - 180 °C
E. Cycle - 180 seconds: the time needed for the complete analysis of one sample
F. Sample start - 15 seconds: the time into the run when the sample is dropped
into the reactor tube
G. Sample stop - 40 seconds: the time into the run when the autosampler advances
to the next sample
H. Oxygen inject 35 seconds: the time into the run when the oxygen enrichment
will stop
I. Peak Enable - 0 seconds: the time at which data acquisition will start
5.3.18 The settings for "F" and "G" may need to be adjusted in order to optimize the system for
complete combustion of the sample. Combustion of the samples can be viewed through
the "view finder" located on the face of the autosampler. It is recommended to
occasionally observe the combustion of a sample to confirm that the control panel settings
are optimum. If incomplete combustion is suspected, increase the oxygen inject time. Set
the sample stop time to 5 seconds greater than the oxygen inject time.
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SOP for the Analysis of Particulate-Phase
Organic Carbon in Great Lakes Waters Volume 3, Chapter 5
6.0 Analytical Procedures
6.1 Instrument Start-up
6.1.1 Turn on the main power switch.
6.1.2 Turn on the gases at the tanks, and turn the instrument purge and carrier valves to the "on"
position. These valves are located below the pressure gauges on the front panel of the
instrument. Release the filament and furnace stand-by push-buttons on the instrument
control panel. Allow the furnace to reach the set point temperature (1000°C).
6.1.3 Perform a leak check on the gas systems (section 5.3.13) at the start of each day of
analysis. Check the gas flow rates (section 5.3.16).
6.1.4 Turn on the data system and launch the data acquisition software (Carlo Erba Eager 200).
6.2 Instrument Calibration
6.2.1 Cut out 6 filter discs from a muffled, 47 mm GF/F filter with a clean, 12 mm diameter
cork boring tool (#6).
6.2.2 Spike 50 mL of each level of KHP calibration standard (section 5.1) on a separate disc
using a micro-pipettor. Allow the spiked filter discs to dry. Rinse the micro-pipettor tip
with organic-free water 5 times between spikes, and initialize the tip with calibration
standard 5 times prior to spiking a filter disc.
6.2.3 While the spiked filter discs are drying, fill in the sample table in the data acquisition
software. Start the analysis sequence with a by-pass, which consists of an empty tin
sample container. Continue filling in the sample table by moving from the lowest to
highest calibration standard concentration. Enter the amount of carbon spiked in
milligrams in the "sample weight" column. Enter "standard" in the "sample type" column
and "POC" in the "standard type" column.
6.2.4 Verify that the analysis sequence listed in the sample table is correct. This instructs the
data acquisition software what samples are being run and where to save the data files. The
first and last discs must be properly noted in the sample table.
6.2.5 Using stainless steel forceps, fold the spiked discs into eighths and place them into
individual tin sample containers. Pack the spiked filters completely inside of the tin
containers.
6.2.6 Load the calibration standards into the autosampler in the same order as the analysis
sequence created in the sample table.
6.2.7 Start the instrument by selecting the "run" command in the menu. Nitrogen (N,) will elute
first, followed by carbon (CO:). Confirm that the retention time listed in the component
table of the data acquisition software is the same as the actual retention time. If the
difference between the actual and listed retention times is too large, the data system will
not identify the peaks properly. Enter the correct retention times in the table and re-start
the calibration if needed.
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SOP for the Analysis of Particulate-Phase
Volume 3, Chapters Organic Carbon in Great Lakes Waters
6.2.8 After all of the calibration standard discs have been analyzed, confirm that the correlation
coefficient for the six-level calibration plot is 0.995 or better. If this condition is not met,
repeat the calibration procedure (steps 6.2.1-6.2.8).
6.2.9 If the calibration plot is satisfactory, cut out three 12 mm discs from a muffled, 47 mm
GF/F filter. Spike 50 mL of the two calibration check standards onto separate discs and
50 mL of organic-free water onto the third disc for a filter blank. Allow the discs to dry.
6.2.10 Enter the information for the two calibration checks and one filter blank in the sample
table. Enter "unknown" under "sample type" and "100" for "sample weight". Verify that
the analysis sequence is correct. The first and last discs must be properly noted in the
sample table.
6.2.1 1 Load the two calibration check spikes and the blank in the autosampler. Analyze these
filter discs and determine if they are within tolerances. The calibration checks must be
within ±20% of the nominal spike mass. The blank must be less than 5.0 mg carbon,
which corresponds to the lowest calibration level. If the calibration check spikes or filter
blank are not within these tolerances, refer to section 9.0.
6.2.12 If the quality control spikes and blank are within tolerances, then proceed with the analysis
of POC lake water samples.
6.3 Analysis of Samples
6.3.1 Open the aluminum foil envelope containing a POC filter sample. Remove a small piece
of aluminum foil from the envelope. Place the piece of aluminum foil, with the dull side
facing up, into a 50 x 9 mm plastic Petri dish.
6.3.2 Cut out two 12 mm discs from the filter, while it is folded in half, with a 12 mm diameter
cork boring tool (#6). Take care to cut out the discs from the area of the 47 mm filter that
is coated with particles. Separate the four resulting discs. Place the discs, particle side up,
on the foil in the Petri dish. Cover the Petri dish to prevent dust particles from settling on
them, but loose enough to allow the discs to dry. Label the cover of the Petri dish with the
Great Lake sampling station identification number that is listed on the aluminum foil
envelope.
6.3.3 Repeat steps 6.3.1 and 6.3.2 for 4 more POC filters for a total of twenty 12 mm discs. Cut
out three more discs from a muffled, 47 mm GF/F filter and follow step 6.2.9 for the
calibration check standards and the filter blank.
6.3.4 Fill in the data acquisition software sample table for the twenty sample discs followed by
the three quality control samples. Enter "unknown" in the "sample type" column and
"100" in the "sample weight" column. Verify that the information in the sample table is
correct. The first and last discs must be properly noted in the sample table.
6.3.5 Allow the discs to dry, fold them into eighths, and place them into individual tin sample
containers. Pack the filters completely inside of the tin containers. Load the tin containers
into the autosampler in the same order as the analysis sequence created in the sample
table.
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SOP for the Analysis of Particulate-Phase
Organic Carbon in Great Lakes Waters Volume 3, Chapters
6.3.6 Analyze the sample discs by selecting the "run" command in the menu. If tolerances for
the two calibration check standards or the blank are not met, refer to section 9.0.
6.3.7 If quality control tolerances are met, then repeat steps 6.3.1 -6.3.6 for another batch of
sample filters.
6.4 Instrument Shut-down
6.4.1 Depress the furnace and filament stand-by push-buttons on the instrument control panel.
6.4.2 Turn off the oxygen and air at the tanks. Helium is turned off only for a long-term shut-
down.
6.4.3 For a short-term shutdown, steps 6.4.1 and 6.4.2 are sufficient. For a long-term shutdown,
continue with steps 6.4.4-6.4.6.
6.4.4 Remove the water trap and dispose of the contents properly. Connect the water trap inlet
and outlet fittings together with an adapter.
6.4.5 Remove the chromatographic column and cap both ends of the column. Connect the
column inlet and outlet fittings together with an adaptor.
6.4.6 Cap the three vents on the lower front panel of the instrument. Turn off the main power
switch.
7.0 Calculations
POC samples are collected by filtering the lake water through a 47 mm diameter glass fiber filter.
The glass filtering apparatus exposes only a 38 mm diameter area of the 47 mm filter to the sample
water. From this 38 mm diameter area, four 12.065 mm diameter discs (#6 cork boring tool) are
cut out and analyzed for organic carbon. The resulting mass of carbon from the analysis of four
discs per sample must be summed, multiplied by an area correction factor (x), arid divided by the
volume of water filtered to calculate the measured POC concentration in mg/L. The area correction
factor is calculated by a ratio of the effective filter and disc areas:
AREA38= 3.14159(19 mm)2 = 1134 mm2
AREA12= 3.14159(6.0325 mm)2= 114:33 mm2
CORRECTION FACTOR (X) = 1134/4(114.33) =2.48
8.0 Maintenance and Trouble-Shooting
8.1 The copper metal in the reactor tube will oxidize and must be replaced periodically. If the
detector response for nitrogen increases significantly in consecutive chromatograms, this is
a symptom of complete oxidation of the copper. Generally, about 75 analytical runs can be
made from one 80 mm packing of copper in the reactor tube.
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SOP for the Analysis of Particulate-Phase
Volume 3, Chapter 5 Organic Carbon in Great Lakes Waters
Follow these steps to repack the copper:
A. Depress the filament stand-by push-button on the instrument control panel.
B. Remove the coupling from the bottom of the reactor tube and disconnect the
autosampler from the top of the reactor tube.
C. Remove the reactor tube from the furnace. Remove the quartz wool plug and
the copper metal from the bottom of the reactor tube.
D. Repack the reactor tube with 80 mm of fresh copper wire pieces. Use fresh
quartz wool to hold the copper in piace.
E. Scrape out the tin dioxide residue from the top of the reactor tube. Replace the
quartz wool if needed.
F. Reconnect the top of the reactor tube to the autosampler and the bottom to the
coupling. Use new Viton O-rings on the reactor tube.
G. Perform a leak check of the pneumatic systems (section 5.3.13).
8.2 . An unstable baseline may indicate that the tungstic anhydride in the reactor tube needs to
be repacked. Follow the steps in section 8.1, except remove the upper contents (tungstic
anhydride) of the reactor tube.
8.3 Baseline problems may also be due to water in the detector. Water can also cause the flow
of the carrier gas (helium) to drop. Check the water trap (anhydrone) and repack if
necessary. If the water trap is not spent, check the gas lines and chromatographic column
for blockages. Refer to the instrument operating manual.
8.4 If gas leaks are suspected, see section 5.3.13 for the leak testing procedures.
9.0 Quality Control
9.1 The six-level calibration plot must have a correlation coefficient of 0.995 or better. A new
calibration plot must be generated at the start of each day of analysis.
9.2 An empty tin sample container is analyzed prior to calibration at the start of each day of
analysis.
9.3 Calibration check standards and filter blanks are run immediately after the generation of a
calibration plot and after the analysis of every twenty sample discs. If tolerances for
quality control standards and blanks are not met, prepare and analyze another set of quality
control standards and a filter blank. If the tolerances are not met again, the instrument
must be recalibrated.
9.4 Stock and working calibration (KHP) and calibration check (EDTA) solutions must be
prepared at the beginning of each Great Lakes survey.
9.5 POC filters are analyzed promptly, in a ship-board laboratory, during the course of a
survey.
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Volume 3, Chapter 5
10mm
80mm
20mm
80mm
40 nun
A. Quartz Wool
B. Tuivgjtic Anhydride
C. Reduced Copper Wire
Figure 1: CHNS Reactor Tube Configuration
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SOP for the Analysis of Particulate-Phase
Organic Carbon in Great Lakes Waters
Table 1: List of Equipment
(most quantities depend on number of samples)
EQUIPMENT
Tin sample containers
Reactor tube
Anhydrone
Quartz wool
Copper wire pieces
Viton O-ring for water trap
Viton O-ring for reactor tube
Tungstic anhydride
EDTA (di-sodium salt)
Potassium hydrogen phthalate
Concentrated sulfuric acid
47 mm GF/F filters
50 mL micro-pipettor
OMI-1 indicator tube
SOURCE OR EQUIVALENT
Fisons 24006400 (100/pack)
Fisons 46820000 (set of 2)
Fisons 33821900 (100 grams)
Fisons 33822200 (5 grams)
Fisons 33835310 (40 grams)
Fisons 29013603 (set of 2)
Fisons 29032910 (set of 10)
Fisons 33835420 (25 grams)
J.T.Baker 8993-01(500 grams)
Fisher P243-100 (100 grams)
Fisher A510-500 (500 mL)
Whatman 182547 (100/pack)
Daigger Scientific G20537F
Supelco 2-3900
Miscellaneous
- 50 x 9 mm plastic Petri dish
- 400 mL beaker
12 mm punch
- stainless steel forceps
- Class A volumetric flasks and pipets
- permanent markers
- aluminum foil
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Standard Operating Procedure for the
Sampling and Analysis of Total Suspended
Solids in Great Lakes Waters
Grace Analytical Lab
536 South Clark Street
10th Floor
Chicago, IL 60605
August 2, 1994
-------
Standard Operating Procedure for the
Sampling and Analysis of Total Suspended Solids
in Great Lakes Waters
1.0 Scope and Application
This Standard Operating Procedure describes the sampling and analysis of Great Lakes Waters for
total suspended solids (TSS). Samples of lake water are collected and filtered through a 0.7 uM
pore-size glass fiber filter. Total suspended solids are operationally defined as the mass retained on
the filter per unit volume of water.
2.0 Safety and Waste Handling
All applicable safety and waste handling rules are to be followed. These include the proper
labeling and disposal of chemical wastes. Over-board discharges of chemical wastes are forbidden.
Refer to the GLNPO Safety, Health, and Environmental Compliance Manual for specific rules.
3.0 Summary of Procedure
Great Lakes water samples are collected at pre-determined sampling stations and depths via either
a submersible pump or Rosette sampler. Sub-samples of water are then filtered under vacuum
through a 47 mm diameter glass fiber filter, which has been washed and dried to constant weight.
The suspended solids are retained on the filter and frozen at -10 °C until final weighing on an
analytical balance in a land-based laboratory.
4.0 Description of Apparatus
Glass fiber filters are pre-weighed on an analytical balance. Water samples (typically 2-4 liters for
open-lake locations) are collected from an over-board pump or Rosette sampler. The filters are
supported on a commercially-available, all-glass, 350 mL vacuum filtration apparatus. Two
filtration apparatuses are attached, side-by-side, to ring stands. Tygon tubing (3/8" ID) is used to
connect the filtration flasks to an oil-less vacuum pump. Final weights of the filters are determined
identically to the initial weights. The equipment needed are listed in Table 1.
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SOP for the Sampling and Analysis of
Total Suspended Solids in Great Lakes Waters Volume 3, Chapter 5
5.0 Preparation of Filters and Analytical Balance
5.1 Preparation of Filters
5.1.1 Filter preparation should take place as close to the start of the survey as possible.
5.1.2 Filters are to be handled only with stainless steel forceps. Filters that are mishandled after
preparation should be discarded.
5.1.3 Label the 47 mm diameter GF/F filters (0.7 \iM pore-size) using a permanent marker on
the outer edge of each filter. Label from 1 - "X" with "X" being the total number of filters
prepared. Allow the ink to dry for 5 minutes before proceeding to step 5.1.4.
5.1.4 Condition the filters using a 350 mL vacuum filtration apparatus. Pass 350 mL of organic-
free, distilled, deionized water (from now on referred to as organic-free water) through
each filter. Place the filters onto the filtration apparatus with the labeled side facing up.
5.1.5 Remove the filters from the filtration apparatus and place them into individual 50 mm
aluminum weighing pans. Dry the filters in a 105 °C oven for 2 hours.
5.1.6 Remove the filters from the oven and place them into a desiccator. Allow the filters to cool
for 5 minutes.
5.1.7 Prepare the analytical balance as described in section 5.2.
5.1.8 Remove the filters from the desiccator in small groups and weigh them on the analytical
balance.
5.1.9 Record the initial filter weights on the TSS Sampling Log Sheet and place them
individually into identically-numbered 50 mm diameter plastic petri dishes.
5.1.10 Every tenth filter must be redried in the 105 °C oven for uvo hours and re-weighed.
Record the second value on the log sheet.
5.1.11 If the second weight does not fall within +/- 0.1 mg of the initial weight, check if the
balance is zeroed correctly. If the balance has deviated, re-zero and re-weigh the filter. If
the weight still does not fall within +/- 0.1 mg, the previous group of 10 filters must be re-
dried and re-weigned.
5.2 Preparation of Analytical Balance
5.2.1 A top-loading analytical balance with a capacity of 200 mg and a resolution of 0.01 mg is
needed. The balance should be accompanied with a set of calibration weights, preferably
NIST traceable, (e.g., Mettler 22 balance with BA monitor.)
5.2.2 Allow sufficient time for the analytical balance to warm up to operating temperature. Then
zero the balance.
5.2.3 Record in the balance logbook the performance over the following range: 10 mg, 30 mg,
50 mg, 100 mg, and 150 mg. This range allows the use of filters weighing 1-150 mg.
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Volume 3, Chapter 5 Tote/ Suspended Solids in Great Lakes Waters
5.2.4 Record in the balance logbook the temperature of the balance room. Note any fluctuations
during use.
5.2.5 A new, dry 47 mm GF/F filter weighs approximately 120 mg. Tare the balance to
100.00 mg using the 100 mg weight from the set of calibration weights.
5.2.6 During the filter preparation and analysis procedures, the balance is tared to 100.00 mg
after every tenth filter. If the balance deviates more than +/- 0.03 mg, the balance is again
tared to 100.00 mg, and the previous group of 10 filters is re-weighed.
6.0 Filtration and Analysis Procedures
6.1 Filtration Procedure
6.1.1 Using stainless steel forceps, place one 47 mm GF/F filter onto the fitted glass support of
the sampling apparatus. Place the glass funnel on top of the filter and secure with the
clamp. Label the Great Lake name, station number, sampling depth, and date onto the
petri dish.
6.1.2 Collect the lake water sub-samples from the submersible pump hose or Rosette sampler.
Allow the overboard pump line to flush for 15-30 minutes. Collect the lake water into a 4
liter cubitainer or four, 1 liter bottles. Rinse the container(s) twice with approximately 1
liter of lake water before collecting the sample. If the lake water is to be collected from the
Rosette, rinse the container(s) with only 200-300 mL of lake water to insure there is
enough remaining to establish a significant paniculate load on the filter (see section 6.1.6).
6.1.3 Measure the volume of lake water to be filtered in a graduated cylinder, or mark four 1
liter Teflon bottles at the 1 liter level. Prior to filling, rinse the bottles or cylinder twice
with approximately 100 mL of lake water.
6.1.4 Connect the vacuum pump to the filtration flask. Pour a measured volume of lake water
into the glass filtration funnel. Turn on the vacuum pump. Maintain the vacuum between
5-10 inches of Hg during filtration.
6.1.5 Continue pouring lake water into the funnel until sufficient suspended solids have been
collected.
6.1.6 The volume of lake water required to produce a reliable TSS measurement will vary with
lake, station location, depth, and time of year. More than 1 mg of suspended solid material
is needed. For open-lake, oligotrophic conditions, typically 2-4 liters will provide enough
paniculate matter. For near-shore locations, or meso-eutrophic and eutrophic conditions,
lake water volumes in the range of 200-500 mL are typical. A filter that becomes visibly
covered with solids and a flow of water through the filter that drops significantly are
evidence that sufficient suspended solids have been collected.
6.1.7 After the lake water has been filtered, rinse the sides of the funnel with approximately
20 mL of organic-free water and filter this rinse. Turn off the vacuum pump.
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Total Suspended Solids in Great Lakes Waters Volumes, Chapters
6.1.8 Remove the funnel and, using stainless steel forceps, fold the filter in half and place it
back into the numbered petri dish. Place groups of petri dishes in a labeled Ziplock bag
and store at -10 °C. Record the Great Lake name, station number, sampling depth, volume
filtered, analyst, date, and time of day on the TSS Sampling Log Sheet.
6.1.9 Empty the filtrate from the filtration flask.
6.1.10 Rinse the filtration funnel, fitted glass support, flask, and the container(s) with organic-
free water.
6.1.11 Re-assemble the filtration apparatus.
6.1.12 Place aluminum foil covers over the filtration funnel.
6.2 Analysis Procedure
6.2.1 Remove the filters from the freezer and allow them to thaw. Using stainless steel forceps,
remove the filters from the petri dishes and place each in an individual 50 mm aluminum
weighing pan. When handling the filters, grasp only the outer edges with the forceps.
6.2.2 Dry the filters in a 105 °C oven for two hours.
6.2.3 Prepare the analytical balance (section 5.2).
6.2.4 Using the same analytical balance as the initial weighing procedure, follow steps 5.1.6 -
5.1.12 to determine the final weights of the filters.
6.2.5 Store the filters in a freezer after all of them have been weighed and the results recorded.
6.2.6 Calculate the total suspended solids (TSS) as:
Total Suspended Solids (mg/L) = [Final Weight - Initial Weight]
[sample volume in liters]
7.0 Quality Control
7.1 A duplicate sample will be filtered in parallel at least once during th. sampling of each Great
Lake.
7.2 A TSS matrix blank will be collected, in duplicate, at the beginning of each survey of the Great
Lakes and at least once during the sampling of each Great Lake. A TSS matrix blank is collected
by filtering 1 liter of organic-free water. The matrix blanks are processed identically to Great
Lakes water samples.
7.3 A TSS field blank will be collected, in duplicate, at the beginning of each survey of the Great
Lakes and at least once during the sampling of each Great Lake. A TSS field blank is prepared by
taking a filter out of the foil envelope, placing it onto the fitted glass support of a clean filtration
apparatus, wetting the filter with organic-free water and assembling the filtration apparatus. The
apparatus is disassembled, and the filter is lemoved and processed in the same manner as a sample.
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Volume 3, Chapter 5 Total Suspended Solids in Great Lakes Waters
7.4 Two TSS trip blanks will be processed after the survey has ended. This is accomplished by placing
two filters in their petri dishes into the Ziplock bag and processing these filters like samples.
7.5 Because TSS is an ancillary parameter to the determination of hydrophobic organic contaminants
(HOCs), the TSS samples during an organics survey are taken simultaneous to the HOC samples.
Therefore when a HOC matrix blank, field blank or duplicate sample is collected a TSS matrix
blank, field blank or duplicate will also be collected.
Table 1: List of Filtration Equipment
Quantity Equipment Source or Equivalent
2 Oil-less Vacuum Pump Schuco 5711-130
2 Teflon wash bottle Cole-Parmer N-06052-60
2 350 mL all-glass filtration apparatus Nucleopore
2 Stainless steel forceps
2 Support/ring stand for filtration apparatus
1 Toploading analytical balance
200 mg capacity
0.01 mg resolution
calibration weights
1 Dessicator
1 Drying oven
Miscellaneous (some quantities depend on number of samples)
- 47 mm GF/F filters (0.7 uM pore-size) Whatman 1825-47
- Cubitainers
Tygon tubing (3/8"ID)
- 50 mm diameter aluminum weighing pans
- 50 mm diameter plastic Petri dishes
- permanent markers
- Ziplock freezer bags
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