United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-96/018
September 1996
&EPA Trace Metal Cleanroom
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EPA/600/R-96/018
September 1996
Trace Metal Clean room
by
Margaret M. Goldberg
Analytical and Chemical Sciences
Research Triangle Institute
P. O. Box 12194
Research Triangle Park, NC 27709
Contract Number: 68-C5-0011
Work Assignment Number: 04
Billy B. Potter, Work Assignment Manager
Angela Moore, Project Officer
National Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Printed on Recycled Paper
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Disclaimer Notice
Although the information in this document has been funded wholly or in
part by the United States Environmental Protection Agency under Contract
Number 68-C2-0103 and 68-C5-0011, it does not necessarily reflect the
views of the Agency and no official endorsement should be inferred.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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Foreword
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The National Exposure
Research Laboratory - Cincinnati (NERL-Cincinnati) conducts research to:
Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in drinking waters, surface waters,
groundwaters, wastewaters, sediments, sludges, and solid wastes.
• I nvestigate methods for the identification and measurement of viruses,
bacteria and other microbiological organisms in aqueous samples and
to determine the responses of aquatic organisms to water quality.
Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants in
drinking water, surface water, groundwater, wastewater, sediment and
solid waste.
Develop methods and models to detect and quantify response in
aquatic and terrestrial organisms exposed to environmental stressors
and to correlate the exposure with effects on chemical and biological
indicators.
This publication, 'Trace Metal Cleanroom," was prepared as a part of
the Regional Applied Research Effort (RARE) Program at the request of
EPA Region II. This publication documents current cleanroom designs,
specifications and protocols for ultra low level analysis of arsenic, lead,
mercury and selenium. It provides guidance for regional environmental
laboratories in the special practices used in the analysis of ultra low trace
metal. NERL-Cincinnati is pleased to provide this manual and believes that
it will be of considerable value to many public and private laboratories that
wish to perform trace metal analyses in water matrices for regulatory or other
reasons.
Alfred P. Dufour, Director
Microbiological & Chemical Exposure Assessment
Research Division
National Exposure Research Laboratory - Cincinnati
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Abstract
Accurate chemical analysis of inorganic elements and their species at
ultra-trace concentrations is essential for understanding and modeling
environmental systems and determining health effects. In order to maintain
sample integrity and prevent contamination during sample preparation or
analysis, a high purity cleanroom or clean zone is required. Proper design,
construction, use, and maintenance of a trace element cleanroom are
necessary to achieve a high cleanliness environment, but guidelines and
protocols specific to trace element cleanrooms are not available in federal
or international cleanroom standards or in published literature. This work
developed practical design and construction options and use and
maintenance protocols based on several sources: review of existing
guidelines and standards, tours of six trace element cleanrooms, interviews
with designers and users, and experiences with the trace element cleanroom
suite at Research Triangle Institute. Several design options are possible
depending on technical factors such as the elements being analyzed and the
sample preparation and analysis methods used, and non-technical factors
such as the number of samples and analysts. Some of the most important
design features of any trace element cleanroom are the use of high
efficiency air filters, laminar air flow, acid-tolerant and low paniculate
construction materials, and vents for removal of acid-laden or toxic vapors.
However, it is equally important that the cleanroom have its access restricted
to essential technical staff only, and that the technical staff be thoroughly
trained in cleanroom use, behaviors, and maintenance.
IV
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Contents
Disclaimer Notice ii
Foreword iii
Abstract iv
Figures vii
Tables , viii
Abbreviations and Symbols ix
1.0 Introduction 1
2.0 Summary and Conclusions 2
3.0 Recommendations , 3
4.0 Technical Activities 4
Review of Standards 4
Tour of Laboratories 4
Consultation with Designers and Users 4
Review of Currently Used Procedures 4
Preparation of Protocols 5
5.0 Cleanroom Theory and Standards , 6
Definitions 6
Review of Cleanroom Theory 6
Review of Existing and Proposed Standards 13
6.0 Design specifications 17
General Considerations 17
Cleanroom Laboratory Designs 18
Construction Materials 25
7.0 Cleanroom Use 29
Laboratory Operations 29
Personnel 30
Cleanroom Garb and Gowning Procedures 31
Labware Use and Cleaning Procedures 31
8.0 Cleanroom Maintenance , 33
Initial Certification 33
Monitored Parameters 33
Maintenance Activities , 33
9.0 Reagent Purification 34
Procedures 34
Storage , 34
References 35
Bibliography 36
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Contents (Continued)
Appendices
A. RTI/ACS/SOP-174-001: Standard Operating Procedures for the ACS
Inorganic Class 100/10,000 Clean Lab Facility
B. RTI/ACS/SOP-174-002: Standard Operating Procedures for Cleaning
Labware in the ACS Inorganic Class 100/10,000 Clean Lab Facility
C. RTI/ACS/SOP-174-005: Standard Operating Procedures for Monitoring
and Maintaining Cleanliness of the ACS Inorganic Clean Lab Facility
D. RTI/ACS/SOP-174-007: Standard Operating Procedures for Purification
of Reagents inthe ACS Inorganic Clean Lab Facility for Trace/Ultratrace
Metal Analysis
E. Florida International University: Standard Operating Procedures for
Mercury Analysis in Water, Sediment and Tissue
F. Battelle Pacific Northwest Laboratories, Marine Sciences Laboratory,
Standard Operating Procedure MSL-M-027-01: Total Mercury in
Aqueous Samples by Cold Vapor Atomic Fluorescence
G. U.S. Environmental Protection Agency, Method 1631: Mercury in Water
by Oxidation, Purge and Trap, and CVAFS
H. RTI/6302/04-SOP: Standard Operating Procedures for the Operation
and Maintenance of a Trace Metal Cleanroom
vi
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Figures
Number Page
1 Conventional room with single air inlet and outlet and
turbulent air flow patterns 9
2 Horizontal laminar flow cleanroom , 10
3 Horizontal laminar flow cleanroom with HEPA fume hood 11
4 Vertical laminar flow cleanroom ...T 12
5 Vertical laminar flow cleanroom design used at RTI 14
6 Horizontal laminar flow clean air cabinets within an
enclosed laboratory 19
7 Vertical laminar flow fume hood 21
8 Mixed vertical laminar flow and turbulent flow cleanroom 22 •
9 ACS inorganic class 100/10,000 clean lab facility .....24
vii
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Tables
Number Page
1 Typical air particle emission rates from people ,.. 6
2 Typical particle sizes ,... 7
3 Airborne paniculate cleanliness classes .... 15
4 Typical airborne particulate CEN cleanliness classes for clean
rooms and clean air controlled spaces (final draft) 15
5 IES recommended practice documents , , 16
6 Chemical resistance of GLID-GUARD (Glidden Co.) epoxy
paint 26
7 Typical chemical resistance for MIPOLAM (Huls America Inc.)
floor covering 27
8 Plumbing materials for trace element cleanrooms 28
9 Operations performed in class 100 laboratory 29
10 Operations performed in class 10,000 laboratory 30
11 Operations performed in the anteroom 30
12 Operations performed in service room *30
viii
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List of Abbreviations and Symbols
Abbreviation
ACS
AsHs
ASV
CEN
D
EMSL*
GFAA
HCI
HOPE
HEPA
Hg(0)
HNO3
H2Os
H2SC-4
HVAC
ICP
ICP-MS
IES
mph
NA
NaOCI
NIST
NS
ppb
ppm
ppt
PVC
RTI
SOP
U
ULPA
Meaning
Analytical and Chemical Sciences (unit of RTI)
arsine
anodic stripping voltammetry
Comite' European de Normalisation
downstream particle concentration (particles/unit
volume).
Environmental Monitoring Systems Laboratory
graphite furnace atomic absorption
hydrochloric acid
high density polyethylene
High Efficiency Paniculate Air
metallic mercury
nitric acid
hydrogen peroxide
sulfuricacid
Heating, ventilating, and air conditioning
inductively coupled plasma
inductively coupled plasma-mass spectrometry '
Institute for Environmental Sciences
mega ohm
miles per hour
Not applicable
sodium hypoehlorite
National Institute of Standards and Technology
Not specified
part per billion
part per million
part per trillion
poly(vinyl chloride)
Research Triangle Institute
Standard Operating Procedure
upstream particle concentration (particles/unit volume)
Ultra Low Penetration Air
micron; 10-6 meters
EMSL-Cincinnati was reorganized in 1995 and is now part of the National Exposure Research Laboratory (NERL-Cincinnati).
IX
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Section 1.0
Introduction
In order to develop an accurate understanding and
model of trace element speciation and cycling in the
environment, it is necessary to protect environmental
samples from contamination during sample preparation
and analysis stages. In many cases, air-borne particu-
lates have high metal concentrations and are a major
source of sample contamination. In order to eliminate
this problem, specially designed trace metal cleanrooms
are used. Cleanrooms are rooms that have a high flow
rate of purified air which continuously blankets samples
and materials in a clean atmosphere. They are equipped
with High Efficiency Paniculate Air (HEPA) filters and/or
Ultra Low Penetration Air (ULPA) filters that remove
almost all particles from the air. These cleanrooms are
continually flushed with purified air so that samples can
be processed without atmospheric contamination. Use
of cleanrooms has enabled metal quantitation in the
parts per trillion range for some elements.
The Environmental Monitoring Systems Laboratory -
Cincinnati (EMSL - Cincinnati)* requested cleanroom
EMSL - Cincinnati was reorganized in 1995 and is now part of the
National Exposure Research Laboratory (NERL - Cincinnati).
design specifications and use protocols for cleanrooms
to be used for analysis of arsenic, lead, mercury, and
selenium. The sample types include water, wastewater,
seawater, and related matrices, and the expected el-
emental concentrations are in the parts per trillion to
parts per billion range. The specifications and protocols
will ultimately be used by EMSL - Cincinnati to provide
guidance to contract laboratories that perform inorganic
environmental analyses at trace and ultra-trace concen-
trations. Thus the goals of the work presented in this
report were to evaluate existing options and standards
for cleanroom designs and materials, develop functional
specifications for trace metal cleanrooms,, and develop
protocols for cleanroom use and reagent purification
procedures.
This report contains a summary of technical activities
undertaken, an overview of cleanroom theory and de-
sign options, a summary of existing and proposed stan-
dards issued by the federal government and private
organizations, recommendations for design, use, and
maintenance of a trace metal cleanroom, and protocols
developed for use in the Research Triangle Institute
(RTI) Inorganic Cleanroom Facility.
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Section 2.0
Summary and Conclusions
The goals of the Work Assignment were to develop
functional specifications for trace metal cleanrooms,
cleanroorn use protocols, and reagent purification pro-
tocols. In order to accomplish this, a review of existing
cleanroorn standards and recommendations was per-
formed, six trace metal cleanrooms were visited, and
interviews with the laboratory designers, principal us-
ers, and authors of cleanroorn standards were con-
ducted. In addition, we installed and started to use our
own trace metal cleanroorn at RTI during the period of
this Work Assignment.
One important conclusion of this report is that even with
a field as narrowly defined as trace element environ-
mental analytical chemistry, there are many different
needs and acceptable solutions for cleanroorn designs.
No one design will satisfy the needs of all users, in part
because not all users analyze the same suite of ele-
ments. The differences in design requirements stem
from technical factors such as the chemical behavior of
different elements, and the different preparation and
storage procedures required to best analyze those ele-
ments, and from non-technical factors such as the size
of the facility, number of analysts, local safety and utility
codes, etc.
Federal standards are available for cleanroorn class
designation and particle counting methods, but they are
not available for cleanroorn designs, construction mate-
rials, personnel garments, activities, or training, etc.
Guidelines are available from the Institute for Environ-
mental Sciences (I£S) in several of these areas, but
they are designed to be general guidelines that are not
specific to any one industry or application. As a result,
the guidelines represent a starting point for trace ele-
ment cleanroorn considerations, but do not provide in-
formation that can be comprehensively applied. This
report presents design, construction, use, and mainte-
nance options with specific application to trace element
cleanroorn laboratories used for environmental analyti-
cal chemistry. Advantages and disadvantages for each
cleanroorn design option are presented so that the
reader may have guidance for their own application.
Finally, standard operating procedures used at RTI,
Florida International University, Battelle Pacific North-
west, and Frontier Geosciences are included as appen-
dices to provide specific protocols used at those facili-
ties.
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Section 3.0
Recommendations
This report contains practical cleanroom design options
and Standard Operating Procedures (SOP) with specific
application to trace element analysis for environmental
samples. Theoretical aspects of air filtration and dynam-
ics are also presented as background information for the
reader. The primary recommendation pf this report is
that the reader use the design options presented, or
modify them for their application in a manner that is
consistent with efficient air filtration and laminar air flow
dynamics. '-,,.-••
The Standard Operating Procedures (SOP) included in
the appendices provide specific guidance in areas of
cleanroom use, maintenance, operations, personnel,
garments, and reagent purification procedures. These
documents are the most recent versions available but
are continuously revised to include the most appropriate
procedures. In some cases, such as recommended
cleanroom garment use or frequency of maintenance
activities, recommendations are made in an heuristic
manner. It should be recognized that as standard proce-
dures are rigorously applied and correlations made be-
tween cleanroom cleanliness levels and specific proce-
dures, the procedures will be modified as needed. Ini-
tially, it is prudent to err on the side of excess restrictions
and over-zealous contamination control rather than to
assume a more casual approach that could jeopardize
sample integrity. Thus, a secondary set of recommenda-
tions are to track the performance of the laboratories
using the various design options and SOPs presented,
and determine the effect of specific procedures and
designs on the cleanliness levels of the laboratories and
ultimately on the contamination levels of the samples.
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Section 4.0
Technical Activities
In order to accomplish the goals listed in Sections 1 and
2, several technical activities were performed. The fol-
lowing list summarizes the activities; they are briefly
described in this section and a more complete discus-
sion of the results of each activity is presented in subse-
quent sections of this report.
1. Review of federal standards and Institute of
Environmental Sciences (IES) standards for
cleanroom use;
2. Tour of six trace metal cleanrooms;
3. Consultation with cleanroom laboratory designers
and users;
4. Review of current cleanroom practices, sample
handling procedures, and reagent purification
procedures for inclusion in protocols; and
5. Preparation of protocols.
Review of Standards
Because there was no document available to describe
the unique design and material considerations required
for a trace metal cleanroom, only general guidance
could be gleaned from the guidelines published in Fed-
eral Standard 209E (FED-STD-209E; 1992) for
cleanrooms. Similarly, the Institute of Environmental
Sciences "Recommended Practice" documents and the
draft European standard, CEN/TC 243/WG 1 [Geilleit,
1992], which is based on Federal Standard 209, were
also reviewed for definitions and general guidelines.
Tour of Laboratories
Six laboratories were toured to learn how cleanroom
guidelines have been implemented and how successful
they are. The six laboratories were:
1. Florida International University, Miami, FL
Dr. Ronald Jones, Laboratory Designer and Director
Class 100 mercury analysis facility
2. National Institute of Standards and Technology,
Gaithersburg, MD
Dr. John Moody, Laboratory Designer and Director
Class 100 general metal analysis facility
3. University of North Carolina, Chapel Hill, NC
Dr. Steven Goldberg, Laboratory Designer and
Director
Class 100 general metals analysis facility
4. Brooks Rand Corp., Seattle, WA
Mr. Richard Brooks, President
Dr. Lian Liang, Laboratory Director
Class 100 mercury and general metals analysis facility
5. Frontier Geosciences, Seattle, WA
Mr. Nicolas Bloom, Laboratory Designer and Director
Mercury analysis facility
6. Battelle Pacific Northwest, Sequim, WA
Ms. Brenda Lasorsa, Laboratory Director
Mercury analysis facility
Consultation with Designers and Users
Each of the individuals listed above was interviewed at
length regarding the design considerations of their labo-
ratory and the procedures used to avoid sample and
reagent contamination. In some cases, additional work-
ers in the laboratory were also interviewed to provide
information about other sample handling and prepara-
tion procedures used. When possible, protocols and/or
publications were collected that detail the sample collec-
tion, preparation, and analysis procedures, the cleanroom
operation and maintenance procedures, and reagent
and labware purification procedures.
Review of Currently Used Procedures
The sample handling and preparation procedures pro-
vided by each of the consultants were reviewed along
with procedures available in the literature in order to
assess the relative utility of various techniques. Several
of these were then experimentally assessed in the RTI/
Analytical and Chemical Sciences (ACS) Inorganic Clean
Lab Facility. Any modifications suggested by our expe-
rience were incorporated into protocols and recommen-
dations.
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Preparation of Protocols RTI-ACS/SOP-I 74-005
Monitoring and Maintaining Cleanliness of the
The following standard operating procedures (SOP) were ACSa Inorganic Clean Lab Facility
prepared for use in the RTI/Analytical and Chemical
Sciences Inorganic Clean Lab Facility; copies of the RTI-ACS/SOP-174-007
protocols are contained in Appendices A through D: Purification of Reagents in the ACSa Inorganic Clean Lab
Facility for Trace/Ultratrace Metal Analysis
RTI-ACS/SOP-174-001
ACS3 Inorganic Class 100/10,000 Clean Lab Facility
RTI-ACS/SOP-174-002
Cleaning Labware in the ACS3 Inorganic Class 10O/ * ACS = Analytical and Chemical Sciences unit of Research
10,000 Clean Lab Facility Triangle Institute.
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Section 5.0
Clean room Theory and Standards
Definitions
A general definition of a cleanroom is a room which has
a gentle shower of highly filtered air for the purposes of
transporting airborne paniculate contaminants away from
sensitive samples or products, and maintaining a clean
environment with low particle concentrations. A
cleanroom is defined in U.S. Federal Standard 209E as
"a room in which the concentration of airborne particles
is controlled and which contains one or more clean
zones." A clean zone is defined as "a defined space in
which the concentration of airborne particles is con-
trolled to meet a specified airborne particulate cleanli-
ness class." Thus a cleanroom may have one or more
regions within the room in which the concentration of
airborne particles is maintained less than or equal to
specified limits.
Review of Cleanroom Theory
The theoretical aspects of particle dynamics, electro-
statics, filtration mechanisms, and flow velocity model-
ing are extremely important components of cleanroom
design, maintenance, and improvement. However, the
technical details involved are beyond the scope of this
report. The interested reader is referred to recent publi-
cations including Handbook of Contamination Control in
Microelectronics edited by Tolliver (1988), Particle Con-
trol for Semiconductor Manufacturing edited by Donovan
(1990), The Future Practice of Contamination Control
(Proceedings of the 11th International Symposium on
Contamination Control, London, 1992), and publications
presented in the Journal of the IES. The review pre-
sented below is intended to provide a basic level of
understanding required for discussion of cleanroom de-
sign options. It discusses sources of particulate con-
tamination in general, and the basic theory of air filtra-
tion, flow patterns and velocities.
Sources of Contamination
The ultimate goal of any cleanroom is to prevent con-
tamination of samples or products by airborne particu-
lates. Ideally, the designer has knowledge of the source
or sources of the particles, as well as their relative
number and size, and can thus be sure that the design
will achieve the cleanliness objectives. In some cases it
is necessary to know the chemical composition of par-
ticles contributed by the source(s). However, even with-
out a detailed knowledge of the number and type of
particles present in a given laboratory, the designer can
proceed using some general estimates. For example, a
typical non-smoking office environment has 100,000 to
200,000 particles that are 0.5 micron or larger per cubic
foot of air (Matthews, 1994). Similarly, ordinary activities
performed by people, such as sitting or walking, gener-
ate millions of particles every minute.
Table 1 lists typical air particle emission rates from
people engaged in various activities. The values are
presented as general guides only; individual circum-
stances may differ significantly.
In order to determine the source of particulate contami-
nation, some knowledge of other operations performed
in the building or in adjacent buildings is helpful. In
general, there are at least three major sources of air-
borne particulate contamination in any laboratory:
Airborne particles are brought into the laboratory from
sources exterior to the laboratory. Outdoor air with a
high particulate concentration is brought in through
the air handling system, and indoor air from corridors
and other laboratories is brought in through doorways
and inter-laboratory air passages.
Particles are generated within the laboratory by
personnel, equipment, and processes. Movement of
personnel within the laboratory not only generates
Table 1. Typical Air Particle Emission Rates from People
1.
Activity
Number of Particles 0.3
Micron or Larger Emitted
from Each Person/Minute
Standing or sitting; no movement
Standing or sitting, average body
movement, toe tapping
Changing positions, sitting to standing
Average walking (3.57 mph)
Fast walking (5 mph)
Calisthenics
100,000
1,000,000
2,500,000
7,500,000
10,000,000
15,000,000 to 30,000,000
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particles, but also generates air currents and eddies
that transport particles from one region to another.
3. Particles are brought into the laboratory on workers'
apparel and supplies. Clothing fibers abrade and
shed particles; shoes transport dirt and smaller
particles; books, papers, reagent bottles all transport
particles into a laboratory.
Proper design of the cleanroom can minimize the first
two of these sources, i.e., it can reduce the importation
of particles from sources external to the laboratory and it
can reduce the impact of particles generated within the
laboratory. This requires judicious choice of room size
and shape, mechanism of air filtration, cleanliness level,
volume and flow rate of air through the room, and
direction of air movement in all regions of the room. A
general discussion of these factors is presented below.
In order to minimize the contamination due to the third
source of airborne particulates, i.e., workers' apparel,
supplies, and operations, it js necessary to implement
stringent cleanroom procedures for personnel and re-
strict apparel to special cleanroom garb. These proce-
dures are highly specific to the operations performed in
the cleanroom, and are discussed in Section 7,
Cleanroom Use. .
Air Filtration and Recirculation
Contaminated air can be filtered to remove most of the
particles prior to entry into the cleanroom. A typical
cleanroom has pre-filters to remove large particles, tem-
perature and humidity controls for personnel comfort
and equipment function, and high performance filters to
remove submicron sized particles. Table 2 presents a
list of common particle types and their size range. There
are several definitions of particle size used for different
purposes, but this report will use the definition provided
by the Institute of Environmental Sciences [IES-CC-011-
85-T, 1985] which defines particle size as "the maximum
linear dimension of a particle as observed with an optical
microscope or the equivalent diameter of a particle
detected by an instrument. The equivalent diameter is
the diameter of a reference sphere having known prop-
erties and producing the same response in the sensing
instrument as the particle being measured."
The high efficiency filters used to remove the submicron
particles are typically High Efficiency Particulate Air
(HEPA) filters or Ultra Low Penetration Air (ULPA) fil-
ters. Specifications for HEPA filters require that they
have a minimum particle-collection efficiency of 99.97%
for 0.3 p,m particles, and ULPA filters have a minimum
particle-collection efficiency of 99.999% for particles in
the size range of 0.1 to 0.2 urn [IES-RP-CC001.3,1993].
Efficiency is defined as the ratio of the difference in
concentrations (upstream - downstream) to the upstream
concentration:
Efficiency {%) = U-D x 100
where U = upstream particle concentration
(particles/unit volume)
D = downstream particle concentration (particles/
unit volume).
This means that if the particle count upstream of a
HEPA filter is 100,000 particles per cubic foot (0.3 p.m)
then the count downstream of the filter will be a maxi-
mum of 30 particles per cubic foot. Similarly for the
ULPA filters, if there are 100,000 particles per cubic foot
(0.12 urn) upstream of the filters, then the count down-
stream will be a maximum of 1 particle per cubic foot.
While it might naively be assumed that filters remove all,
or nearly all, particles larger than a specified size and
are penetrated by particles smaller than that size, that is
not the case for HEPA or ULPA filters due to the effect of
diffusion of particles in the submicron size range [Ensor
and Donovan, 1988]. HEPA and ULPA filters exhibit a
maximum penetration by particles of approximately 120
nm size (Q.I 2 urn). Alternately stated, the filters have
higher collection efficiencies for particles both larger and
smaller than 0.12 join and have poorest performance for
particles approximately 0.12 jim.
After air has passed through the HEPA or ULPA filters it,
enters the cleanroom as highly purified air. Once inside)
the purified air can become contaminated with particles
from personnel, containers, implements, etc., but is
generally still cleaner than air external to the cleanroom.
Table 2. Typical Particle Sizes
Particle Description
Approximate Particle Size
Rangeftim)'
Gas molecules
Metallurgical dusts and fumes
Atmospheric dust
Viruses'
Rosin smoke
Tobacco smoke
Oil smoke
Zinc oxide fumes
Combustion nuclei
Sea salt nuclei
Carbon black
Colloidal silica
Paint pigments
Alkali fumes
Ammonium chloride fumes
Bacteria
Insecticide dusts
Ground talc.
Sulfuric concentrator mist
Coal dust
Fly ash
Cement dust
Red blood cell (adult human)
ant spores
Pollens
Beach sand
0.0005-0.01
0.001-100
0.001-30
0.003-0.06
0.01-1.0
0.01-i.O
0.03-1.0
0.01-0.3
0.01-0.1
0.03-0.6
0.01-0.3
0.02-0.05
0.1-5
0.1-5
0.1-3
0.3-30
0.5-10
0.5-50
1-20
1-100
1-200
3-100
7.5±0.3
10-50
10-100
90-3000
"Values taken from Austin (1970).
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For that reason, the air is recirculated back through the
filters and again enters the cleanroom as high purity air.
Recirculation has several important technical and eco-
nomic ramifications:
1. Recirculation increases the efficiency and longevity
of the filters. Because the recirculated air upstream of
the filters has a relatively low particle concentration,
it presents a minimum burden to the filter substrate
and prevents premature clogging of the pores. The
cost of replacing HEPA or ULPA filters is substantial
and thus maximizing filter life is an important economic
goal. Recirculation also produces cleaner air with
each successive pass through the filters, so that the
cleanroom is made "cleaner" with each iterative cycle.
2. Recirculationincreasestheenergyefficiency. Because
outdoor air generally needs to be conditioned with
respecttotemperature and humidity, while recirculated
air requires little to no additional conditioning, energy
costs are much less for conditioning recirculated air
than they are for outdoor air.
3. In a laboratory environment, recirculated air may be
hazardous and/or toxic. Neither HEPA nor ULPA
filters remove vapors from the air. Thus if hazardous
or toxic vapors are generated in a laboratory
cleanroom, they must be neutralized, captured, or
vented to the outside in order to protect the health and
safety of personnel in the cleanroom. Careful attention
must be paid during the cleanroom design phase so
that adequate space and facilities are designed for
removal of hazardous or toxic vapors from the
recirculating air. During subsequent laboratory usage,
it is essential that personnel are trained in the use of
the facility so that it is properly and safely used.
Air Flow Patterns
Conventional rooms and laboratories have air inlets
located in one or a few regions of the room and the air
disperses to other regions of the room in a turbulent flow
pattern, as depicted in Figure 1. One consequence of
turbulent flow is that it resuspends settled particles and
transports them to different regions of the room. This
represents a major source of contamination in many
laboratories.
In order to prevent this contamination, unidirectional
laminar air flow is used. Laminar flow means that the air
moves in parallel planes from its plane of entry into the
room to its plane of exit. Air flowing in laminar planes is
stratified such that there is very little transfer of particles
between plane boundaries. Thus, particles in one plane
of air will remain in that plane until they are removed
from the room.
In practice, it is impossible to achieve 100% laminar flow
with personnel and equipment in the room because their
presence is an obstruction to the air flow and turbulence
is generated in the region of any obstruction. Thus some
compromises to truly laminar flow are always neces-
sary. Other compromises to laminar flow are dictated by
safety, economics, or other practical factors, and have
resulted in use of several different cleanroom designs
with limited regions of laminar air flow. Figures 2 to 4
illustrate air flow patterns and particle movement for the
cases of totally horizontal laminar flow and totally verti-
cal laminar flow. A more thorough discussion of
cleanroom design Options is presented in Section 6.
Figure 2 depicts a horizontal laminar flow cleanroom in
which HEPA filters are located in one wall of the room.
Clean air sweeps through the room in horizontal sheets,
entraining any particles enroute, and carrying them out a
perforated side wall opposite the HEPA filters. The
advantages of this design are that truly laminar flow is
achieved in all work areas, i.e., bench top level and
higher, and that the cost is relatively low. However, if
people and/or equipment are present, laminar flow is
disrupted and particles and vapors are transported hori-
zontally. This results in contamination of samples and
products downstream, closer to the exit wall. In practice,
this design is useful for long, narrow cleanrooms with
only one work bench, or cleanrooms with only one
person present, but not in the configuration shown in
Figure 2. A design similar to Figure 2 with multiple work
benches and chemists was used during the 1970s at the
National Institute of Standards and Technology (NIST)
as a trace element cleanroom and was found to work
poorly because of cross-contamination [Moody, 1982].
In some laboratories that generate toxic vapors or bio-
logical agents, the horizontal flow design would present
a health and safety hazard because personnel would
always be in the path of the generated species. Thus,
the design has significant limitations, but in some cases
may be useful.
A modification to the design in Figure 2 is to include one
or more vertical flow, HEPA filtered fume hoods on the
wall opposite the HEPA banks, as shown in Figure 3.
This provides clean air in the region that previously was
turbulent, and permits operations such as hot acid di-
gestions to be performed. None of the cleanrooms vis-
ited uses this design, but it could potentially be useful.
Figure 4 shows a cleanroom with totally vertical laminar
flow. In this design, the HEPA filters are located in a
dropped ceiling and clean air sweeps downward through
the laboratory, transporting any particles down through
a perforated floor grid. Air is returned to the HEPA filters
through a plenum wall space, refiltered, and recirculated
through the cleanroom. The advantages of this design
are that the cross-contamination of samples or products
is minimized, particles swept off of personnel are trans-
ported down through the floor rather than onto samples,
and nearly laminar flow is maintained throughout the
room. Totally vertical laminar flow is generally recog-
nized as the most effective design for achieving cleanli-
ness classes 100 or lower, and has been successfully
employed in ultra-trace element laboratories such as the
lead isotope geochronology lab at the University of
North Carolina [Su et al., 1994] as well as at other
installations. The disadvantages of this design are the
construction costs and engineering complications in-
volved with the raised, chemical resistant, high strength,
perforated floor grids and supports.
-------
Supply air
Figure 1. Conventional room with single air inlet and outlet and turbulent air flow/patterns.
9
-------
Supply
HEPA Filter
Plenum wall
Rgure 2. Horizontal laminar flow cleanroom.
10
-------
Supply air
Return air supply
HEPA filter bank
Plenum wall
Figure 3. Horizontal laminar flow cleanroom with HEPA fune hood.
11
-------
f
Raised
floor
Figure 4. Vertical laminar flow cleanroom.
12
-------
A modified vertical laminar flow design is used in the
Class 100 cleanroom at RTI and is shown in Figure 5. It
contains ULPA filters in a ceiling grid that disperse clean
air downward in vertical laminar flow lines. One wall
contains a row of polypropylene benches for sample and
reagent preparation procedures that dp not involve hot
or concentrated acids. A small return air vent is located
at hand level for recirculating air back through a plenum
wall to the ULPA filters. This helps to remove any
particles generated by turbulence at the bench top.
Larger air return vents are located approximately 6
inches above floor level. Air travels through perforated
grills at the front of the polypropylene benches, sweeps
under the benches, and enters the return air vent at the
wall. This helps to remove particles brought in on
cleanroom foot covers as well as any settled particles
generated in the laboratory. The flooring in the Class
100 laboratory is chemical resistant PVG (MIPOLAM;
Huls America Inc.) with heat-welded seams, 6 inch
covered edges at walls to prevent seepage of chemicals
underneath the flooring, ;and a PVC floor drain leading to
an emergency chemical spill tank" outdoors; Thus, the
flooring should not generate'acid degradation products
(volatile or particulate) even in trie event of a chemical
spill, Tyvo fume hoods (one ULPA-filtered; one' not fil-
tered) are located on the opposite wall and provide
space to perform acid digestions and other work with
corrosive agents. Vapors and particles generated within
the fume hoods are exhausted to the outdoors and npt
recirculated.
Air Flow Velocity
Clean, filtered air is the agent that transports particles
out of a cleanroom and envelops samples in a contami-
nant-free environment. In achieving and maintaining a
high level of cleanliness, two general rules are "the more
clean air the better", and "the higher the flow rate the
better," However, there are limits to those rules. If flow
rate becomes too rapid, the airflow becomes turbulent
rather than laminar, the environment is no longer com-
fortable to workers, operations become inefficient, and
the cleanliness level decreases due to scouring of par-
ticles off of personnel or equipment. A minimum air flow
velocity of 70 ft/min is required to maintain laminar flow.
Most cleanropms have air flow velocities between 70
and 110 ft/min.
to any specific industry group. For example, they do not
provide any information regarding cleanroom design,
materials, construction, or use. However, the Institute of
Environmental Sciences publishes a series of "Recom-
mended Practices" that provide significant guidance to
users of cleanrooms. While they are not federal stan-
dards, they have been thoughtfully compiled by experts
in the contamination control industry. A listing of publica-
tions is available in the "Compendium of Standards,
Practices, Methods, and Similar Documents Relating to
Contamination Control" [IES-RD-CC009.2,1993].
U,S. Federal Standard 209E
The U.S. Federal Standard 209E (dated September 11,
1992) consists of eight sections: (1) Scope and limita-
tions; (2) Referenced documents; (3) Definitions; (4)
Airborne particulate cleanliness classes and U descrip-
tors; (5) Verification and monitoring of airborne particur
late cleanliness; (6) Recommendation for change; (7)
Conflict!with referenced documents; and (8) Federal
agency jnterests. the document provides a basis for
establishing a common; understanding of air cleanliness
based on the number concentration of particles of a
defined size in a defined volume of air over a defined
length of time, as determined using approved particle
counting techniques. ' ;
The intent of the document is to establish standard
classes of air cleanliness for cleanrooms and clean
zones and to prescribe methods for monitoring and
Verification. Tables and equations are provided to en-
able calculation of particle concentrations, the number
of sampling locations required, the volume of air to be
sampled, the cleanliness class, as well as other param-
eters. The document is used by cleanroom designers,
builders, and users as a common set of definitions and
procedures for determination and verification of air clean-
liness class.
The particle concentration limits specified by the stdn-
dard are a function of both the particle size and the
cleanroom class. Particle concentration limits are sum-
marized in Table 3 in both the U.S. standard units
(particles per cubic foot) and the European (metric)
standard units (particles per cubic meter), '
Review of Existing and Proposed Standards CEN/TC243
Existing cleanroom standards provide not only cleanli-
ness class designations, but also guidelines and proce-
dures used to monitor airborne particles, operate par-
ticle counting equipment, calculate the particle concen-
tration, and verify the cjeanroom class designation. How-
ever, because there are so many diverse applications of
cleanrooms. (Pharmaceuticals, semicpnductprs, textiles,
hospital operating rooms, chemical analysis), the'au-
thors of existing guidelines have chosen to provide
general guides that are appropriate for any cleanroom
application and have avoided making recommendations
The European committee charged with establishing
cleanroom standards is the Comite' ^European de
Normalisation (CEN). The committee drafted a docu-
ment in 1992, CEN/TC243, which is similar in scope and
intent to Federal Standard 209E. Equations and tables
were ..included' to enable determination ,Qf cleanrqorn
cleanjiness classes. However, in anticipation of the in-
ternation^l standard, (SO 209, the document was not
actively put into use. The class designations are pre-
sented in Table 4 for comparison with those in Federal
Standard'209E.
13
-------
Floor
Drain
Figure 5. Vertical laminar flow cleanroom design used at RTI.
14
-------
Table 3. Airborne Particulate Cleanliness Classes
Class limits are given for each class name. The limits designate specific concentrations (particles per unit volume) of airborne particles with sizes
equal to and larger than the particle sizes shown."
Class Nameb
SI English"
0.1 urn
Volume Units
(m3) (ft3)
0.2jim
Volume Units Volume
(m3) (ft3) (m3)
Class Limits
O.Sjim
Units
(ft3)
Volume
(m3)
Units
(ft3)
Volume
(m3)
Units
(ft3)
M1
M1.5
M2
M2.5
M3
M3.5
M4
1
10
100
350
1240
3500
12400
35000
9.91
35.0
99.1
350
991
75.7
265
757
2650
7570
26500
75700
2.14
7.50
21.4
75.0
214
750
2140
30.9
106
309
1060
3090
10600
30900
0.875
3.00
8.75
30.0
87.5
300
875
10.0
35.3
100
353
1000
3530
10000
0.283
1.00
2.83
10.0
28.3
100
283
M4.5
M5
M5.5
M6
M6.5
M7
1000
-
10000
-
100000
-
35300
100000
353000
1000000
3530000
10000000
1000
2830
10000
28300
100000
283000
247
618
2470
6180
24700
61800
7.00
17.5
70.0
175
700
1750
The class limits shown in Table 3 are defined for classification purposes only and do not necessarily represent the size distribution to be
found in any particular situation.
Concentration limits for intermediate classes can be calculated, approximately, from the following equations:
particles/m3 = 10M (0.5/d)2-2
where M is the numerical designation of the class based on SI units, and d is the particle size in micrometers, or particles/ft3 = N (0.5/d)2-2
where Np is the numerical designation of the class based on English (U.S. customary) units, and d is the particle size in micromeiers.
For naming and describing the classes, SI name sand units are preferred; however, English (U.S. customary) units may be used.
Table 4. Typical Airborne Particulate Cen Cleanliness Classes for Clean Rooms and Clean Air Controlled Spaces (Final Draft)
CEN
Maximum permitted number of particles/ms of a size equal to, or greater than, the
Class
0
1
2
3
4 '
5
6
7
NS
NA
()
*
0.1 jim
25
250
2,500
25,000
NS
NS
NS
NS
- Not specified
- Not applicable
- For reference only
- Rounded values
0.2 urn
6*
63*
625
6,250
62,500
NS
NS
NS
0.3 jim
NA
28*
278*
2,778*
27,778*
NS
NS
NS
considered size.
0.5 (im
(D
10
100
1,000
10,000
100,000
1 ,000,000
(10,000,000)
1 p.m
NA
NA
25
250
2,500
25,000
250,000
2,500,000
Sum
NA
NA
NA
10
100
1,000
10,000,
100,000
10fim
NA
NA
NA
NA
25
250
2,500
25,000
ISO/TC209
An international standard, ISO/TC209, is currently be-
ing prepared that will become the cleanroom standard
for European, Asian, and other countries. While it is
anticipated that the United States will also agree to
adopt the standard, it is not certain at this time that it will
replace Fed. Std. 209E. According to Richard Matthews,
Chairman of the ISO/TC209 committee, the ISO stan-
dard will redefine cleanliness classes using a formula
that is slightly different than that in Fed. Std. 209E and it
will present acceptable measurement and verification
methods in a manner very similar to Fed. Std. 209iE.
However, the ISO'standard will be much broader in
scope, and will also provide guidance and specifications
in areas of biocontamination control, design and con-
15
-------
struction, personnel behaviors, support services, and
other topics. While the intent is to provide a general
document suitable for all cleanroom users and not spe-
cific to any one industry segment, the standard will
venture into the area of biocontamination, which will be
used to regulate the food, pharmaceutical, and medical
industries. On the other hand, the guidance it will pro-
vide in cleanroom construction materials and protocols
will be very general, and will not include issues specific
to trace element laboratories, for example.
It is anticipated that the standard will be released in
segments, with the cleanroom class designations and
measurement protocols included in the first release in
early 1996. The other segments are scheduled for re-
lease later that year.
Institute of Environmental Sciences
Recommended Practices
The Institute of Environmental Sciences (IES) has pub-
lished a series of "Recommended Practices" that pro-
vide guidance to cleanroom users in many areas of
contamination control. The documents are not federal
standards, but are fully compatible with Federal Stan-
dard 209E. One function of the documents is to provide
definitions and cleanroom product and performance
specifications that are used for a common basis of
agreement among cleanroom designers, builders, and
users. In addition, the documents provide recommenda-
tions for cleanroom garments (designs and fabrics) and
gowning procedures, glove and wipe materials,
cleanroom cleaning procedures and schedules, and other
practices intended to maintain the cleanliness of the
facility. An abbreviated listing of IES documents is pre-
sented in Table 5.
Table 5. IES Recommended Practice Documents
IES Document Number Title
Date
IES-RP-CC-001.3
IES-RP-CC-002-86
IES-RP-CC-003.2
IES-RP-CC-004.2
IES-RP-CC-005.2
IES-RP-CC-006.2
IES-RP-CO007.1
IES-RP-CG-008-84
IES-CC-009,2
IES-RP-CC-011-85-T
IES-RP-CC-012.1
IES-RP-CC-016.1
IES-RP-CC-018.2
IES-RP-CC-020-88-T
HEPA and ULPA Filters
Recommended Practice for Laminar Flow Clean Air Devices
Garments Required in Cleanrooms and Controlled
Environments
Evaluating Wiping Materials Used in Cleanrooms and Other
Controlled Environments
Cleanroom Gloves and Finger Cots
Testing Cleanrooms
Testing ULPA Filters
Recommended Practice for Gas-Phase Adsorber Cells
Compendium of Standards, Practices, Methods, and Similar
Documents Relating to Contamination Control
A Glossary of Terms and Definitions Related to Contamination
Control
Considerations in Cleanroom Design
Recommended Practice for the Rate of Deposition of
Nonvolatile Residue in Cleanrooms
Recommended Practice for Cleanroom Housekeeping-Operating and
Monitoring Procedures
Recommended Practice for Substrates and Forms for
Documentation in Cleanrooms
1993
1986
1993
1992
1994
1993
1992
1984
1993
1985
1993
1992
1992
1988
16
-------
Section 6.0
Design Specifications
General Considerations
The purpose of a trace metal cleanroom is to protect
samples and materials from airborne contamination dur-
ing sample preparation and analysis. The design of the
cleanroom must obviously be compatible with this pur-
pose, but it must also be compatible with the specific
operations performed in the laboratory, the safety needs
of the personnel, the chemical nature of the reagents
and samples, and the economic constraints of the user.
This chapter discusses the design specifications inher-
ent in those needs and the options available to meet
them. Several cleanroom designs that are currently
used are presented along with the options and compro-
mises required in each case.
Initially, the designer must determine the intended uses
of the trace element laboratory including the specific
elements to be determined, operations to be performed,
reagents to be used, and waste handling requirements.
Design options are significantly different for laboratories
that analyze vapor phase metals such as mercury than
for those that analyze metals that are subject to con-
tamination primarily from air particulates, such as lead
and zinc. Both types require high purity air that is free of
the analyte of interest, but analysis of vapor phase
metals does not necessarily require the particulate clean-
liness level that analysis of other metals does. Instead, it
may require the use of vapor sorbent traps or other
forms of gas scrubbers. Similarly, laboratories that spe-
cialize in the analysis of water samples or perform
sample digestions only in sealed ampules will not have
the same restrictions on construction materials, safety
requirements, or waste disposal that most trace element
laboratories have, due to the use of high quantities of
hot, corrosive acids and oxidizing agents. The primary
mandates for. design of a trace element laboratory that
uses hot acids are to eliminate the use of metal in
construction materials to the extent, possible, and to
exhaust all corrosive vapors,to the outdoors. Thus, if the
intended use of the laboratory is analysis of mercury,
then a relatively;simple room* design and,traditional
cleanroom construction materials can be used, perhaps
in combination with vapor sorbent traps. However, if the
intended use of the laboratory is analysis of other met-
als, then the room design must include laminar flow
exhausting hoods and must eliminate, or at least mini-
mize, metallic construction materials.
Amplification and re-emphasis are provided here re-
garding the need for non-metallic construction materials
in trace element laboratories. These labs use large
quantities of hot mineral acids and oxidizing agents for
sample dissolution, generate corrosive vapors that need
to be vented, and generate toxic vapors that need to be
contained or vented. The acids and oxidizers (primarily
HNO3, H2SO4, aqua regia, H2O2, NaOCI, and alkaline
fluxes) wreak havoc with most construction materials,
such as steel, aluminum, cement, and paint. In fact, the
method of choice/for dissolving most of those materials
is to subject them to hot mineral acids and oxidizing
agents. The rust, corrosion, and dust level observed in
most traditional trace element laboratories-are, testa-
ment to the effectiveness of this process and to the need
to use different construction materials in a laboratory
where particle counts are to be minimized. It is held as
axiomatic that any metals present in a trace element
laboratory will eventually be corroded by the acids and
oxidizers, and that any corrosion-particle sources present
in the laboratory or air system will eventually contami-
nate samples/Thus, use of non-metallic construction
materials is essential for long-term viability of the trace
element cleanroom.
The most acid-resistant materials are plastics, silicates,
and precious metals, and of these, plastics are the
obvious choice for an affordable construction material.
For that reason, plastics are used wherever possible in
trace element cleanrooms and great pains are taken to
totally exclude metallic components. However, because
of limited demand, plastic construction components are
generally more expensive than their metallic counter-
parts; and the cost of building a plastic cleanroom
laboratory can become extremely high. This fact compli-
cates the design choices for trace element cjeanrooms
and:has prompted most designers to opt.fpr some forms
of compromise ^between ideal cleanroom conditions,
affordable materials, and designs and materials that can
be maintained for an extended period of time without
corrosion or contamination.
17
-------
After the designer has established the intended analyti-
cal uses of the cleanroom and compatible construction
materials, the designer must then determine the level of
cleanliness required and the engineering and construc-
tion requirements necessary to meet that cleanliness
class. This includes the number, type, and arrangement
of filters and air returns, the air flow rate, the volume and
rate capacity of the air handling system, the heating and
cooling requirements, etc. The level of cleanliness typi-
cally sought for trace element laboratories is Class 100,
which means that there are no more than 100 particles
of 0.5 urn diameter or larger per cubic foot of air. This
level of air cleanliness is arbitrary because the true
relationship between airborne particle concentration and
airborne metal concentration has not been established,
and indeed, will certainly vary from location to location.
Nevertheless, Class 100 has become the unofficial stan-
dard for trace metal cleanrooms. This view is likely to
persist due to the fact that in well maintained Class 100
laboratories where analytical blanks are carefully tracked
and evaluated, the cause of high blanks is nearly univer-
sally attributable to contaminated reagents, insufficiently
clean labware, or analyst error, and not attributable to air
contamination.
The next section of this chapter presents cleanroom
design options suitable for Class 100 cleanrooms or
clean zones that are compatible with the analytical
requirements for ultratrace element analysis.
Cleanroom Laboratory Designs
There are five categories of Class 100 cleanroom labo-
ratory designs that can be used for trace metal analysis.
From the simplest and least expensive to the most
complex and expensive, they are: clean air cabinets or
benches, transportable enclosures, class 100 nonmetallic
fume hoods, class 100 non-metallic laboratories, and
cleanroom suites. The choice of laboratory design is
dictated by several technical factors, including the met-
als being analyzed, the types of laboratory operations
required, and the instrumental analysis methods used.
Nontechnical factors include cost and size of the opera-
tion (number of samples, number of analysts, etc).
Clean Air Cabinets
The simplest, least expensive, and most limited purpose
category of clean air enclosure is a clean air cabinet.
Clean air cabinets typically consist of bench top boxes
that contain a prefiiter, HEPA filter, and blower motor.
They have sufficient space to perform limited operations
such as cleaning or preparation of labware or sample
containers, or some sample preparation procedures. If
constructed of non-metallic materials, they can be suit-
able for use with any metal. Commercially available
units are non-exhausting, hence they are not suitable for
operations that generate toxic or hazardous vapors,
such as acid digestion, solvent extraction, or generation
of Hg(0) or AsH3 vapors. For reasons of size, economy,
and historical use, most commercially available cabinets
provide horizontal laminar flow of clean air. As dis-
cussed previously, horizontal laminar flow is less desir-
able than vertical laminar flow for trace metal analysis
because of sample and material cross-contamination.
An alternate form of clean air cabinet is a glove box
which is designed to flush high purity gases through the
enclosure and out through a vented exit. In some situa-
tions, glove boxes are an economical approach to con-
tamination prevention. If the goal of the glove box is to
prevent samples from becoming contaminated with dirty
air, and if limited preparation of samples is required for
analysis, then placing samples in a glove box is much
more economical than treating and recirculating a room
full of air. However, the glove box approach to sample
handling has very limited application in most laborato-
ries because of several factors:
1. Gas flow is typically horizontal and rarely, if ever
laminar. Thus chances for cross-contamination of
open samples or reagents are very high.
2. Additional apparatus is required to verify and maintain
the purity of the gas with respect to the analyte. This
can be expensive and time-consuming, which defeats
the original purpose of the glove box approach.
3. Because access to samples and equipment inside
the glove box is slower and more difficult than in other
laboratory settings, the types of operations that can
be performed need to be considered carefully to avoid
unsafe operating conditions. Thus many operations
that might be performed in a fume hood, for example,
would pose a safety hazard if performed in a glove
box.
Clean air cabinets can be used either as stand-alone
enclosures within an ordinary trace element laboratory
or as a clean zone within an enclosed cleanroom. A
caution is included here regarding use of clean air
cabinets as stand-alone enclosures. While some mod-
els have sufficient motor size to maintain laminar air
flow, others are marginally sufficient and the air flow
lines degrade into turbulent flow when the front sash is
opened to allow the analyst to work in the enclosure.
Use of a clean air cabinet in an otherwise unclean
laboratory should be carefully evaluated before the cabi-
net is commissioned with samples.
Figure 6 illustrates the use of clean air cabinets within an
enclosed cleanroom. A small cleanroom was built within
an existing laboratory for the purpose of Ultratrace mer-
cury analysis. The cleanroom contains two commercial
horizontal laminar flow benches where all high cleanli-
ness tasks are performed, and a workbench opposite
them where other tasks with lesser requirements are
performed. Air intake for the HEPA filters is at the top of
the laminar flow benches. A sorbent trap for mercury
vapors is located between the prefiiter and HEPA filter; it
consists of gold-coated activated carbon held stationary
between layers of air conditioning felt. Room air is
recirculated from within the room rather than through
18
-------
Gold/carbon
filter system
Air intake
Prefilter
ysiisiKiaxeimf^ssms^mmxmismmiiiiiim
Bench
Figure 6. Horizontal laminar flow clean air cabinets within an enclosed laboratory.
19
-------
plenum spaces, and samples and sensitive operations
are protected from the turbulent flow by keeping them
inside the laminar flow benches. The advantages of this
design are that it is an inexpensive laboratory to build
and maintain, and that it is very convenient to use. The
disadvantages of the design are in the small size, limited
region of laminar flow air, and potential cross-contami-
nation inherent to horizontal flow designs. It should be
pointed out that although this design would not achieve
the same cleanliness class rating as a totally vertical
laminar flow design and could not be used for open
vesse! acid digestion procedures, the horizontal design
was adequate for the ultra low mercury analysis.
Transportable Enclosures
Larger clean air enclosures can be purchased that con-
sist of two or more HEPA filters arranged in a rigid,
overhead, aluminum grid which is supported on four
aluminum legs. This design provides a vertical laminar
flow column of clean air which is enclosed either by rigid
walls (typically Plexiglass® or polypropylene) or by soft
walls (typically thick sheets of clear polymeric materi-
als). Prefilters and blower motors are compactly at-
tached to the filter housing. The entire enclosure is
transportable and can be used in different laboratories
as needed or even shipboard on research vessels. The
design is especially well suited to some types of analyti-
cal instrumentation. If the entire instrument, or at least
the sample introduction region of the instrument, can be
enclosed in a soft-walled, clean air environment, then
sample contamination can be prevented at a critical
point of analysis.
There are two significant limitations to this design: (1) it
Is non-exhausting, and so only operations that do not
generate toxic or hazardous vapors can be performed;
and (2) all commercially available units use aluminum or
other metals for structural support and filter and motor
housing, and so are not suitable for analysis of various
metals. Theoretically, they could be constructed of
polypropylene and thus eliminate the second limitation,
but no polypropylene units are commercially available
yet
Class 100 Non-Metallic Fume Hoods
For laboratory operations that require use of acids or
solvents, exhausting fume hoods are necessary to pro-
tect laboratory personnel and prevent corrosion within
the laboratory. Class 100 HEPA-filtered hoods are com-
mercially available in vertical laminar flow designs, and
in non-metallic construction materials. Typically either
polypropylene or fiberglass is used, with optional con-
tainment wells of heat-resistant or solvent-tolerant poly-
meric materials. These hoods are relatively expensive
(56,000 to $25,000 typically, depending on size and
options), are not transportable, and require training for
proper use and maintenance, but are suitable for all
types of sample and labware preparative activities. Com-
mercial models are available from several manufactur-
ers in lengths of 4 to 8 feet. These units can be used as
stand-alone clean zones or included as components of a
larger laboratory design.
A typical hood design is shown in Figure 7 illustrating air
intake through prefilters and HEPA filters, vertical lami-
nar flow through the work surface, and venting of fumes.
Room air intake is minimized by maintaining the sash at
a height of 4 to 6 inches above the work surface and
ducting room air through a perforated grill that extends
to the front of the hood. In this way, samples and
materials inside the hood are exposed to clean, HEPA
filtered air only.
Class 100 Non-Metallic Laboratories
Currently available options for design and construction
materials for Class 100 nonmetallic laboratories are
presented below along with the compromises that each
choice implies.
One of the first choices for the designer of a trace
element cleanroom laboratory is whether to use a totally
vertical laminar flow design or a design that includes
regions of vertical laminar flow and regions of horizontal
or turbulent flow. In most cases, the totally vertical
laminar flow design will provide a better cleanliness
rating, but it is substantially more expensive and carries
a long-term risk associated with maintenance of the floor
material. As discussed previously, a floor grid is used in
combination with a raised floor to enable return air to
pass under the floor grid. However, structural supports
for the floor grid are necessarily composed of metal
(typically, steel), and although these can be epoxy painted
to make them resistant to acid vapors, they are not
resistant to spills of acids or oxidants. Thus a major risk
with this design is that the floor supports will become
sites of corrosion and particle generation.
In the case of the cleanroom with only regions of vertical
laminar flow, all exposed surfaces within the room can
be non-metallic. However, there will be regions of turbu-
lent flow, and hence particle resuspension, within the
room. The vertical laminar flow regions are the cleanest
regions, and so the most sensitive operations are per-
formed in them. The turbulent regions must be carefully
considered and designated a less critical function. An
example of such a design is shown in Figure 8, and is
the design currently used at NIST for trace element
sample preparation. The laboratory is a hybrid design
that combines the effectiveness of a vertical laminar flow
design and the economy of a horizontal laminar flow
design. Modular HEPA filters and blower units are lo-
cated in or just below the ceiling and clean air is passed
downward in a vertical laminar flow over a workbench
area used for tasks that require the highest cleanliness
level. The air then passes across an aisle and a second
workbench where tasks of lesser cleanliness require-
ments are performed. The air is returned through a
plenum wall at foot, hand, and ceiling levels and recircu-
lated through the HEPA filters. Clear Plexiglass screens
are hung from the HEPA frame to provide isolation of the
clean, filtered air from that of the turbulent, less clean air
20
-------
20" x 20" prefilters
Cabinet
storage
Perforated polypropylene
work surface
12"x 12" polypropylene sink
Room air pre-filters (2)
Drain'
u_
O
"en
i
§
t
V
*-«s
s*
I I i
J L \
'••
i.":.."^« .^- •*!-!
1 i i 1
Clean air 1
6O FPM i
downflow
v;
~^f~
Return air plenum
under work surface
fully seam welded
HEPA
80 FPM
Inflow velocity
Side view
Figure 7. Vertical laminar flow fume hood.
21
-------
Plexiglass shield
Figure 8. Mixed vertical laminar flow and turbulent flow cleanroom.
22
-------
in the aisle and elsewhere in the room. The screens also
protect samples from particles shed by personnel. Only
a minimum of open space is left between the bench top
and the Plexiglass shield for the chemists' hands to
manipulate the samples. In this way, the samples are
maintained in a clean air stream and shielded from
contamination. The advantage of this design is that a
large cost savings is realized in using only zones of
HEPA filtered air rather than an entire room. The disad-
vantage is that only one workbench is maintained as a
clean zone. The other is suitable only for tasks with less
stringent cleanliness requirements such as notebook
recording, logging unopened samples and containers,
performing "rough cleaning" procedures, etc.
Fume hoods are located along the less clean wall and
may be HEPA-filtered or non-filtered designs.
Cleanroom Suites
For laboratories that analyze a variety of metals at ultra-
trace concentrations, the best choice of design is un-
doubtedly a cleanroom suite. The suite consists of sev-
eral rooms including one or more of each of the follow-
ing: ' '
1. A non-metallic Class 100 or lower cleanroom
laboratory where sample preparations and digestions
are performed; ' .
2. A Class 100 anteroom for gowning and isolation;
3. A Class 10,000 or lower instrument room where
instrumental analysis is performed;
4. A service room forsupplies, gas cylinders, refrigerators
and freezers, etc.; and
5. Office areas adjacent to the laboratories.
An example design of a cleanroom suite is shown
schematically in Figure 9. The five areas listed above
are shown with their relationship to each other. The
Class 100 cleanroom is the inner-most room of the suite
so that it is the most isolated from the air and activities
elsewhere in the building. The design of the Class 100
cleanroom was shown in Figure 5. In order to enter the
Class 100 cleanroom, the chemist must first enter the
service room, the Class 10,000 Instrument Room, the
anteroom, and finally the Class 100 cleanroom. En-
trance to each room requires additional layers of protec-
tive garb, and is explained in more detail below. Three
emergency exit doors are shown; they are not used for
access into or out of the suite except in the case of an
emergency.
The philosophy underlying the design of the cleanroom
suite is that the operations required for sample receipt,
preparation, storage, instrumental analysis, and data
analysis can be separated and performed in designated
areas. In that way, operations that require high purity air
and a metal-free environment can be performed in the
Class 100 region, those that will generate significant
particulate matter can be performed in the Service Room
or Office, etc. Similarly, personnel and chemicals asso-
ciated with those tasks are permitted to enter only the
rooms associated with their tasks.
Class 100 Cleanroom Laboratory
The Class 100 laboratory is used for sample and labware
preparation and was designed to tolerate large quanti-
ties of acids and oxidants. Ultra-clean air is supplied
through ULPA filters located in the ceiling grid; over 90%
of the ceiling is covered with 99.99975% ULPA filters.
High purity air is forced downward in a laminar flow and
bathes work benches with particle-free air. Air is then
returned to a recirculation unit at two heights along a
plenum wall: the primary air return is a baseboard return
extending from the floor up to a height of 18 inches; the
secondary return is at hand level extending from the
benchtop surface to a height of six inches above the
benchtop. This air is recirculated through the ceiling
ULPA filters to achieve continuous removal of particles.
Metal-free construction materials were used to the ex-
tent possible to build the Class 100 laboratory. The
walls, floors, benches, fume hoods, acid bath cabinet,
drawers, and sinks are made of polypropylene, and all
plumbing materials are either polypropylene or PVC. All
other service lines (electrical, gas, etc.) are encased in
PVC tubing.
The Class 100 laboratory contains several features in
addition to the clean air supply that facilitate ultra-trace
level metal analysis. The laboratory is equipped with
three areas of one-pass, air exhaust: an 8-foot fume
hood with a vented base cabinet; a 4-foot fume hood
with vented base cabinet; and a 6-foot vented acid bath
cabinet. The fume hoods are used for acid digestion,
evaporation, or extraction of samples and prevent expo-
sure of personnel or equipment to acid vapors. The acid
bath cabinet houses multiple plastic tubs for acid leach-
ing of glassware and plasticware. The interior of the
cabinet is flushed horizontally with Class 100 air and
vented to the outside of the building in order to minimize
exposure of personnel to acid vapors even when the
baths are open.
Class 100 Anteroom
The Class 100 anteroom is a small room located be-
tween the Class 100 laboratory and the Class 10,000
instrument room. The purpose_ of the anteroom is to
provide a space for gowning in cleanroom garb and
enveloping personnel, samples, and materials in a clean
atmosphere prior to .admittance to the Class 100 labora-
tory. The anteroom is maintained as a Class 100
cleanroom by a ceiling ULPA filter, but at an air pressure
intermediate between that of the Class 100 laboratory
(highest 'pressure) and the Class 10,000 instrument
room. In that way, air moves from the Class 100 labora-
tory to the anteroom to the instrument room, and thus
particles and contaminants always move from a more
clean region to a less clean one and contamination of
clean regions is prevented. Some cleanroom suites use
an air shower as a passageway into the Class 100 room.
23
-------
29'-
i
h
g
f
u
Instrument
laboratory
b ""'"^
Service
a room
miim
[~~| Class 100 [2
[U Non-filtered ^
a. Gas Cylinders
b. D.I. Water
:. Refrigerator
i Freezer
3. Clean Room Supplies
/FI
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'•^ t^tfyfjf /f 'f j^i,vj,
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.
PE5100ZL k. 8'HEPAHood
4' HEPA Hood I. Non-HEPA 4' Exhaust ng Hood
Bench m. 6' Acid Baths
Sink n. Desk
ICP o. Questron V6 AFS
Figure 9. ACS Inorganics Class 100/10,000 Clean Lab Facility.
24
-------
Their use, however, is controversial, as some experts
believe that the air shower scours additional particles off
of personnel and materials and suspends them, causing
higher airborne particle concentrations.
Class 10,000 Instrument Room
The instrument room is a class 10,000 area, with par-
tially ULPA-filtered air supply. The filters are located in
areas of sample handling and sample introduction to
instruments. The design philosophy of this room has
three principal tenets:
1. Prevent the corrosion of metal-based instruments
and construction components by minimizing the
presence of acid in the room. A low concentration of
acid is typically required in samples and standards to
enhance analyte stability prior to analysis, but the
acid concentration should be kept low and confined to
closed bottles or flasks prior to analysis.
2. Locate HEPA/ULPA filters where clean air is most
needed in this room. Complete ceiling coverage is not
required in the instrument room, but filters should be
located in the ceiling above autosamplers, locations
where samples are opened, and other sensitive areas.
3. Include one small HEPA-filtered, polypropylene,
exhausting fume hood in the instrument room so that
simple manipulations can be performed on samples
and standards in that room. In that way, the analyst
can quickly and efficiently prepare dilutions or spikes,
for example, without being required to take the samples
to the anteroom, dress in cleanroom garb, perform
the manipulations in the Class 100 Lab, remove the
cleanroom garb, and return to the instrument room.
No digestions or hot acids are permitted in the
instrument room fume hood, but room temperature
dilute acids are permitted.
Minimal particulate control measures are used: autho-
rized personnel only are permitted to enter, cleanroom
booties are required, and a tacky mat is located near the
entrance.
Service Room
A •service room is an essential part of the cleanroom
suite because there are many items necessary for op-
eration of the laboratories that cannot be made clean
enough to bring them directly into the laboratories. For
example, many analytical instruments require cylinders
of compressed gas for their operation. These cylinders
are dirty, many have corrosion products or peeling paint
on their surfaces, and cannot be allowed to enter the
Class 100 or 10,000 laboratories. Thus a service room
in close proximity to the laboratories is required to house
the gas cylinders. Gas plumbing lines should be brought
through the walls to all necessary locations with small,
final stage regulator valves located near the instru-
ments.
Similarly,, water deionizing equipment, air driers, refrig-
erators, freezers, cleanroom supplies, HVAC controls,
and other miscellaneous items necessary for proper
function of the laboratories or instruments should be
located in the service room.
Construction Materials
There are two fundamental properties that construction
materials must have in any cleanroom: they must pro-
vide the structural support necessary, and they must not
contribute particles to the environment. In addition, ma-
terials used in a trace element cleanroom must be
compatible with the chemicals, operations, and analytes
used in the laboratory. That is, they must not degrade,
corrode, or alter their surface properties as a result of
contact with chemicals (acids, oxidants, alkaline fluxes,
solvents) used in the laboratory or as a result of the
operations (digestions, extractions, refluxing) performed
there, and they must not contribute analyte(s) to the
environment by outgassing or extraction. This list of
requirements is highly restrictive and in practice neces-
sitates the use of plastics whenever possible. However,
even plastics should not be used indiscriminately. Poly-
urethane may contain high concentrations of mercury,
fiberglass may contain significant cobalt, and PVC may
contain lead and other metals. Many plastics are not
compatible with all mineral acids. Thus the choice of
plastic must be,made with an understanding of the
limitations of the material not only for strength, but also
for durability and contamination of the cleanroom envi-
ronment. A discussion of materials available for each
section of a trace element cleanroom is presented in the
following sections.
Ceilings
In a Class 100 cleanroom using vertical laminar flow, the
HEPA/ULPA filters are located in a ceiling grid. The
filters are the most important component of the ceiling,
and so other components of the ceiling should be se-
lected after determining the availability and limitations of
the filters. While the most commonly used HEPA and
ULPA filters are not suitable for a trace element labora-
tory, other designs of the filters are available and are
suitable. For example, the pleated filter material is typi-
cally supported with aluminum separators between the
folded pleats, and the filters are framed in an aluminum
support. For a trace element cleanroom, the use of
exposed aluminum is not desirable because it's surface
can be readily oxidized and generate corrosion par-
ticles. However, HEPA and ULPA filters can be pur-
chased without aluminum if the purchaser specifies
separatorless filters housed in wooden supports. As an
extra precaution against particles shed from wood, the
supports should be painted with a chemical resistant
epoxy paint. A list of chemical spot test results for an
epoxy paint from one manufacturer is presented in
Table 6,
Commercially available ceiling grids used to support the
HEPA/ULPA filters consist of a T-grid system typically
made of aluminum. A plastic T-grid system is available,
but at a very high price. One option used in Class 100
25
-------
Table 8. Chemical Resistance of Glid-Guard (Glidden Xo.)
Epoxy Paint*
Chemical
Hydrochloric acid
Sulfuric acid
Phosphoric acid
Nitric acid
Hydrogen peroxide
Acetic acid
Oleic acid
Lactic acid
Sodium hypochlorite
Chromic acid
Ammonium hydroxide
Sodium hydroxide
Xytena
Ethyl alcohol
Trtehloroethylene
Methyl ethyl ketone
Gasoline
Sodium chloride
Copper chloride
Mineral oil
Formaldehyde
Phenol
Concentration (%)
5
5 and 50
10
5
30
5 and 50
ML"
NL
6
20
30
5 and 50
neat
NL
neat
neat
neat
5 and 50
20
neat
37
saturated
Result of 48
Hour Spot Contact
No change
No change
No change
No change
No change
No change
No change
Slight softening
after 48 hours
Discoloration
after 24-48 hours
Discoloration
after 24-48 hours
No change
No change
No change
No change
No'change
No change
No change
No change
No change
No change
No change
Slight discoloration
and softening
* Information from The Glidden Company, GLID-GUARD product
Information sheet.
* NL » not listed by paint manufacturer.
trace element cleanrooms is to paint the aluminum with
an epoxy paint that is acid resistant. The epoxy paints
typically perform well in the presence of acid vapors, but
are not able to withstand contact with concentrated
(liquid) acids. Thus, for the purpose of coating a ceiling
grid, epoxy paints are a suitable option. Great care must
be taken however to insure that all surfaces are cov-
ered. If there are holes in the coverage, the acid vapors
can readily attack the aluminum and start to corrode the
ceiling grid. In a Class 10000 instrument room, the
aluminum grid can be used without epoxy painting, but it
is prudent to paint the aluminum ceiling grid.
Two notes of caution are included here regarding paints.
First, the paints recommended for use in a cleanroom
are epoxy paints rather than latex paints because epoxy
provides a better sealant that prevents emission of
particles from the underlying material into the air. Sec-
ondly, while none of the epoxy paints tested during
construction of the RTI cleanroom contained significant
mercury or lead, and paint manufacturers said that
those elements have not been used historically in epoxy
paints, it is prudent to analyze all paints before applica-
tion in a cleanroom. Older latex paints may contain both
mercury and lead and represent a continual source of
elemental contamination. Cleanrooms located in previ-
ously-built laboratories may require paint removal be-
fore cleanroom construction can proceed.
Cleanroom lighting fixtures are available that are com-
patible with the air streamlines in a vertical laminar flow
cleanroom and with the requirements of no exposed
metal surfaces. Teardrop shaped lights that attach to the
T-grid system are available with plastic exteriors and no
exposed metal. In a mixed vertical laminar flow and
turbulent flow cleanroom such as the one used at NIST,
traditional light fixtures are located above the ceiling,
and plastic light diffusers are used flush with the ceiling.
No metal is exposed in the path of the air.
Walls and Supports
Wood, steel, and cement structures are needed in any
facility to provide strength and structural support, and
laboratory walls are typically made of gypsum, wood
board, or cinder blocks. Unfortunately, none of these
materials is suitable for an exposed surface in a trace
element cleanroom. Most shed particles even when
newly installed, and all shed particles after extended
interaction with acid vapors. Therefore, in order to use
these construction materials in a trace element
cleanroom, even inside a plenum wall, they must be
covered in such a way as to prevent generation and
transport of particles. The best solution is to cover all
walls and supports with a chemical-resistant plastic,
such as some polypropylene or PVC products. The
chemical resistance for one such product, MIPOLAM
(Huls America Inc.), is summarized in Table 7. The
nature of these plastics requires that all seams be
welded to provide complete coverage of the underlying
materials. This is an expensive option in the short term,
but will provide long term protection against acid-gener-
ated particles.
A less expensive, but less permanent, option is to epoxy
paint all surfaces to a "no pinholes evident" finish. That
is, the paint should be applied in enough coats that a
complete barrier is provided and no gaps, or pinholes,
are evident in the epoxy surface. This surface is suscep-
tible to chipping and chemical dissolution, and is thus
not likely to provide the length of service that a plastic
laminate will. Some form of acid-resistant splash or spill
guard will always be necessary with this option. A painted
surface inside a cleanroom will require continual moni-
toring, "touch up" painting, and eventually may require
complete resurfacing.
This option is presented because of the use of seam-
welded plastic laminate is so expensive. However, it can
only be recommended if the cleanroom manager is
extremely diligent and the labor for painting (and re-
painting) is inexpensive.
Floors
The choice for flooring materials depends on the design
of the cleanroom and the operations performed in it. For
a totally vertical laminar flow Class 100 or lower
cleanroom, a plastic or fiberglass grid is used for the
raised flooring. In general, plastic is preferred over fiber-
glass because fiberglass abrades more easily and gen-
erates particles after a period of time. The flooring in the
26
-------
Table 7. Typical Chemical Resistance for Mipolam (HULS America Inc.) Floor Covering3
Chemical Concentration (%) Tomnorat
Acetic acid, aqueous
Ammonia, aqueous
Boric acid, aqueous
Carbon dioxide
Caustic soda, aqueous
Chromic acid, aqueous
Copper sulfate, aqueous
Exhaust gas, carbon dioxide
Exhaust gas, hydrochloric acid
Ferric chloride, aqueous
Glucose, aqueous
Hydrochloric acid
Hydrogen bromide, aqueous
Hydrogen peroxide
Nitric acid, aqueous
Oxygen
Phosphoric acid, aqueous
Photo fixing baths
Potassium hydroxide solution
Potassium salts, aqueous
Sea water
Silver nitrate
Sodium chloride
Su If uric acid
Urea, aqueous
Urine
Acetic acid
Acetylene
Butyric acid, aqueous
Diesel oils, pressure oil
Ethylene glycol
Glycol
Lead acetate, aqueous
Potassium hydroxide solution
Acetone
Carbon disulfide
Ethyl acetate
Methylene chloride
6
saturated
any
any
4
0.5...10
any
any
any
any
saturated
any
any
up to 80
15
any
any
common
concentration
15
any
normal
10
any
up to. 60
any
50
100
20
100
100
100
concentrated
any
100
100
100
70
100
140
100
100
70
140
140
140
140
70
140
100
70
70
140
140
100
70
140
100
140
140
140
140
70
70
70
70
100
100
100
70
70
70
70
70
70
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
resistant
limited resistance
limited resistance
limited resistance
limited resistance ;
limited resistance
limited resistance
limited resistance
limited resistance
unstable
unstable
unstable
unstable
Information from the Huls America Inc., MIPOLAM product information sheet.
Results after 1 month of storage and subsequent drying during not less than 18 days.
air return beneath the raised flooring should be a seam-
welded, acid resistant plastic, such as MIPOLAM (Huls
America Inc.). For other styles of Class 100 or lower
cleanrooms, a seam-welded acid resistant plastic should
also be used.
The flooring materials used in an instrument room,
anteroom, or service room do not generally need to
conform to the same chemical resistance requirements
that those in the Class 100 laboratory do, because wet
chemical operations are not performed in those areas.
However, the floor must still be easily cleaned and not
generate particles, and thus vinyl flooring is recom-
mended.
Exhausting Fume Hoods and Acid Baths
Large quantities of acids are used in fume hoods for
sample digestions, and iri acid baths for leaching metals
out of labware. Any metal in these areas would be
rapidly corroded and contribute metal contamination
and particles to the laboratory environment. Thus, only
plastic, fiberglass, or glass are permissible in those
areas. Both polypropylene and fiberglass fume hoods
are commercially available with several different design
options. The polypropylene hoods can be purchased in
ULPA- or HEPA-filtered options that provide Class 10 or
100 vertical laminar flow environments, respectively. It is
essential that air be continuously exhausted from fume
hoods and acid baths so that the acid vapors do not
enter the recirculation path where they could damage
filters or pose a health threat to personnel.
A note of caution is presented regarding commercially
available fume hoods. Some manufacturers use polypro-
pylene only in parts of the hood that are visibly in the
path of acid vapors, and thus the purchaser must be
careful to specify that all surfaces, sash guides,and
runners, pulley weight enclosures, fan blades, exhaust
ducts, etc. be composed of plastic or fiberglass. Any
metal present in the path of hot acid fumes will eventually
corrode and/or dissolve.
27
-------
Storage Cabinets
Storage cabinets for labware and supplies are commer-
cially available in polypropylene, fiberglass, and wood.
In a Class 10,000 laboratory, wooden cabinets are
suitable after they are painted to minimize particle gen-
eration. In a Class 100 or lower laboratory, polypropy-
lene or fiberglass should be used. As is the case for floor
grids, polypropylene is preferred over fiberglass be-
cause it has less potential to generate particles. Some
deanroom laboratories use epoxy-painted wooden cabi-
nets and drawers, but these should be used with the
greatest of caution and the understanding that they will
need to be repainted frequently.
Utilities
Electrical and plumbing utilities present a special chal-
lenge to the designer of a trace element cleanroom
because the customary construction materials include
extensive use of metal components and solders. How-
ever, non-metallic substitutes are available for most
materials. Some examples for plumbing and electrical
materials are presented in the following sections.
Plumbing Materials
Table 8 presents both customary plumbing materials
and substitute materials that are suitable for use in trace
element cleanroom laboratories. All of the materials are
commonly available and meet plumbing code require-
ments.
The use of copper, stainless steel, or other metal pipes
for water or gas lines should be prohibited to the extent
possible. For tap water and aspirators, PVC pipes are
available In both ambient and high temperature variet-
ies. Delonlzed water should be transported in polypropy-
lene or teflon tubing if a recirculating system is used. For
gas lines, it is possible to substitute PVC, teflon, or
polypropylene for some inert gases; for other reactive
gases, such as acetylene, it is necessary to use stain-
less steel tubing inside PVC conduit. The use of metallic
solder can also be avoided with plastic tubing. Polypro-
pylene joints can be heat welded and PVC joints are
generally solvent welded. Safety equipment, such as
emergency showers and eye wash stations, are avail-
able in plastic versions. Any metallic parts should be
coated with epoxy paints.
Electrical
In the trace element laboratory, the corrosive effect of
acids and their vapors on electrical components is a
serious concern, but use of plastic components where
possible reduces problems greatly. Electrical wiring can
easily be made safe from acid vapors and spills by
encasing it in PVC conduit. If one or both ends of the
conduit is exposed to acid vapors, rubber grommets or
other acid-resistant fittings can be used for isolation.
Similarly, plastic junction boxes can be substituted for
metal ones and plastic outlet covers can be used to
protect duplexes and quadriplexes when not in use. A
note of warning is included here regarding local electri-
cal code requirements. The substitution of plastic for
metal components may be a problem in some regions
due to local electrical code requirements. For example,
during the construction of the RTI cleanroom, it was
learned that one style of plastic junction box that was
permissible in North Carolina was not permissible in
Minnesota. In some cases, substituting components of
difference size from the original design was all that was
necessary to meet local code requirements. Thus, it is
necessary to consider local codes and make design
changes where necessary.
Table 8. Plumbing Materials for Trace Element Cleanrooms
Application
Cold tap water
Hot tap water
Deionized water
Customary Material
copper pipe
copper pipe
high density poly-
Substitute Material
for Trace Element
Cleanroom
PVC pipe
high temperature PVC
polypropylene
Aspirators
Safety shower
and eye wash
Plumbing
ethylene (HOPE) or
polypropylene
copper pipe; brass,
copper of stainless
steel nozzle and
handle
assorted metals
metallic solder
PVC pipe; plastic
nozzle and handle
assorted plastics;
epoxy-coated metal
pull chain
heat welded poly-
propylene; solvent
welded PVC
28
-------
Section 7.0
Cleanroom Use
Laboratory Operations
In any trace element laboratory, there are a great variety
of operations that must be performed from receipt of
samples and supplies, to preparation of labware and
samples, instrumental analysis, data reduction, report
preparation, cleanup, and archiving activities. Each op-
eration has its own requirements for the level of cleanli-
ness, chemical resistance, vapor exhaust, etc., and thus
each operation should be performed in a designated
room or zone to best meet those requirements. In a
trace element cleanroom setting, the segregation of
"dirty" from "clean" operations is even more important
than in non-cleanroom laboratories because the analyte
concentrations are significantly lower and hence the air
cleanliness requirements are significantly greater in the
cleanroom laboratories. Performance of "dirty" opera-
tions in a cleanroom environment would immediately
contaminate any open samples or reagents in the re-
gion, and would eventually overwhelm the ability of the
air filtration system to remove the particle load. Thus
laboratory operations are strictly segregated in trace
element cleanrooms and personnel need to be instructed
in the theory and practice of maintaining the cleanroom
environment.
In general, laboratory operations that involve acids should
be performed in clean rooms or clean zones that are
constructed of acid-resistant materials and that effi-
ciently vent vapors. Laboratory operations that require
analytical instrumentation should be performed in re-
gions or rooms that have clean, purified air, but are
removed from all acid vapors.
IMPORTANT - Operations that are inherently dirty, par-
ticulate-generating, or metal vapor-generating should be
performed in a service room or hazardous materials
laboratory separate from the cleanroom laboratories.
Tables 9-12 list some of the commonly performed labora-
tory operations, a suggested location for the operation,
and the facility considerations required. Examples of
materials handling and operation segregation procedures
are presented in Appendix A, Standard Operating Proce-
dures for the RTI-ACS Inorganic Class 100/10,000 Clean
Lab Facility.
Procedures are also required for transferring samples
and supplies from one region or room to another. In
general, decontamination procedures are required for
transfer of materials from a less clean region to a more
clean one. The procedures typically involve removing
Table 9. Operations Performed in
Operation
Class 100 Laboratory
Location
Special Considerations
Acid digestions
Microwave digestions
Glassware soak
Weighing
Filtration
Centrifugation
Sample preparation
Glassware rinsing and drying
Micro-ware rinsing and drying
Ion exchange
Solvent extraction
Polypropylene, HEPA exhaust hood
Polypropylene exhaust hood
Acid bath cabinet
Semi-micro balance on bench top
Bench top aspirators
Polypropylene exhaust hood
Bench top;
Sink
Bench top
HEPA exhaust hood
No metal: corrosion from hot acid. Air: Once through. Special
plumbing and air
No metal in hood. Vent exhaust from microwave directly to
hood exhaust. Air: Once through.
No metal. Air: Once through; intake at hand level; vented at top
back.
Vibration damping needed. Air: Laminar downflow; recirculating
No metal. Air: Laminar downflow; recirculating. Water: tap with
splash guards
Air: Laminar downflow; once through.
No metal
Air: Laminar downflow; recirculating. Access to deionized water.
All plumbing (in and out) through plastic pipes: No glass; No
metal.
Air: Laminar downflow; recirculating
Chemical resistant materials: solvent and acid tolerant; Air:
Laminar downflow; exhausting.
29
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external cartons or shipping materials in the service
room, wiping all surfaces of bottles and containers with
deionized water on cleanroom wipers under HEPA-
(iltered air in the Class 10,000 room or anteroom as
appropriate, allowing sufficient time in the anteroom for
the container to be well flushed with Class 100 air, and
finally entry into the Class 100 cleanroom laboratory.
These procedures are very important because particles
and analytes are readily transported on shipping car-
tons, exteriors of containers and sample vials, paper-
work, etc., and can contaminate materials in the
cleanroom.
Table 10. Operations Performed in Class 10,000 Laboratory
Operation Location Special Considerations
Instrumental
Analysis
Sample Dilutions
Rough deanup
GFAA; ICP;
ASV
HEPA exhaust
hood
Sink
Air: HEPA over critical
regions.
Air: Laminar downflow;
once through.
Plastic plumbing
Tabta 11. Operations Performed In the Anteroom
Operation Location Special Considerations
Storage of
cleanroom
garb and
special
supplies
Gowning
cleanroom
Perforated New garb is stored on perforated
plastic shelves; shelves inside cleanroom plastic
Plastic clips wrapping until ready for use. Used
frocks are stored upright, attached
by plastic clips. Plastic shelves are
perforated to allow air to flush
vertically and prevent settling of
particles.
Under ULPA Reusable, low paniculate,
filter
Final cleaning Under ULPA
of supplies. filter
garb; Talc-free plastic gloves; Plastic
trash can with lid for disposing of
garb after use.
Cleanroom wipers; deionized
water.
Table 12. Operations Performed in Service Room
Operation Location Special Considerations
Gas cylinder
storage
Supply storage
Water deionization
Sample storage Refrigerator,
freezer
HVAC controls
Metal gas lines encased in
PVC conduit are routed through
walls to specified locations in
labs.
None
Polypropylene plumbing;
Recirculating system; 18 MQ
None
Continuous readout of air temp.,
pressure, humidity, alarm status
for each room.
Although a list of possible routes or mechanisms of
sample contamination is nearly infinite, a short list of the
most common causes would include analyte-bearing
particles emitted by personnel, clothing, and supplies,
and contaminated labware and reagents. The proce-
dures used by cleanroom personnel to minimize those
sources of contamination are discussed included in
Appendices A through D.
Personnel
Authorization for Entry
One of the most significant sources of particles and
contamination in a cleanroom laboratory is personnel.
Particles are transported on shoes, clothing, supplies,
and hair; and as illustrated earlier, millions of particles
are generated every minute by people performing ordi-
nary movements. Thus, one of the simplest methods
used to reduce airborne particle concentrations in the
laboratory is to restrict entrance to the laboratory to only
those personnel who need to work there. Cleanrooms
are restricted access areas; appropriate signs should be
posted outside the rooms so that unauthorized person-
nel do not enter.
Laboratory Behaviors and Training Programs
All personnel who work in a cleanroom need to be
trained in the theoretical and practical aspects of
cleanroom procedures in order to ensure a high level of
cleanliness and minimization of contamination.
Cleanroom training courses are commercially available
both as formal instruction sessions and as video taped
lectures. However, this form of training is designed for
the microelectronics and pharmaceutical industries. While
the general concepts are the same, specific training for
trace element laboratory use is not presented. The types
of behaviors that require instruction are listed below and
discussed more fully in the following sections:
1. Movement inside the cleanroom facility. All personnel
must understand the location of the clean air source,
the direction of air flow, return air locations, and
relative air cleanliness in all regions of the facility in
order to properly use the facility. In addition, they
need to be made aware of the relative number of
particles generated and the direction of their movement
as a result of walking, reaching, manipulating samples,
etc. Special features of the laboratory, such as those
designed to prevent work bench contamination or
degradation of materials, need to be thoroughly
understood so that they can be properly used.
2. Gowning procedures. The policy adopted for
cleanroom garb, use of tacky mats, use of cosmetics,
etc. needs to be understood and followed by all who
enter the cleanroom areas.
3. Sample and reagent handling procedures. In vertical
laminar flow regions, it is imperative that nothing be
placed upstream of an open sample or reagent
30
-------
because contamination will be swept directly into the
container. This is a difficult requirement for chemists
who have become accustomed to manipulating
several samples on a hot plate or bench in a non-
cleanroom setting. Their hands, arms, faces, pipettes,
etc., can no longer pass over any container or
apparatus, but must always approach/observe from
the side where any particles shed will be carried away
in a separate air flow stream.
4. Labware use and cleaning procedures. In general,
labware used in the cleanroom for ultra-trace analysis
has a designated function and is used solely for that
purpose.
5. Housekeeping procedures. The type of cleaning
activities, frequency of performance, and personnel
responsible for cleanroom cleaning need to be clearly
stated and understood in order to maintain the
cleanliness level desired.
6. Facility maintenance procedures. Parameters that
should be monitored to ensure that the air filtration,
recirculation, and exhaust systems are functioning
properly might include pressure drop across HEPA
filters, relative static pressures in different rooms of
the facility, air flow rates, particle counts, temperature,
and humidity. In addition, analyte concentrations
should be measured in air, particle traps, acid baths,
deionized water, etc., to ensure that the facility has
adequate cleanliness for ultra-trace analysis
procedures. The types of measurements, frequency
of performance, and personnel responsible need to
be clearly stated and understood.
Cleanroom Garb and Gowning Procedures
Probably no topic in trace element cleanroom use is
more controversial than that of garb requirements. While
it is generally recognized that protection of samples from
personnel generated particulates is required and that
appropriate cleanroom garb can effectively minimize
emission of particles from personnel, it is also recog-
nized that excessively elaborate cleanroom gowning
procedures can be counter-productive in terms of lost
productivity, safety hazards in a chemical laboratory,
and labor and supply, monies associated with cleanroom
gowning. The IES recommended practices for person-
nel garments recommend that personnel wear the fol-
lowing garments: a cleanroom frock, shoe covers, and
hair cover in Class 10,000 rooms; cleanroom coverall,
boots, hair cover, hood, and facial cover in Class 100
rooms; and cleanroom coverall, boots, hair cover, hood,
facial cover, barrier gloves, and inner suit in Class 10
rooms. Of the operating trace element cleanrooms vis-
ited during the course of this project, none adheres
completely to the IES recpmrnendations, four require
some level of cleanroom gowning, and three (mercury
laboratories) do not require any cleanroom attire.
Each laboratory has its own set of needs, personnel
abilities, facility design, and philosophy of achieving
required cleanliness. Laboratories that analyze mercury
only are not greatly concerned about airborne particu-
late contamination even though that has been shown to
be a significant route of global mercury transport through
the environment. They typically require either no special
gowning or simply foot covers. Mercury-bearing particu-
lates from clothing and personnel have not historically
been a problem in these laboratories even though they
are determining mercury at sub parts per trillion levels.
Laboratories that analyze other metals are more cau-
tious about gowning prior to entering a cleanroom. All
require foot cover at a minimum, and most also require
cleanroom frocks, head covers, and barrier gloves. The
general belief among trace element cleanroom manag-
ers, however, is that contamination from personnel who
follow proper behavioral procedures in the cleanroom is
minor relative to contamination from labware and re-
agents.
Labware Use and Cleaning Procedures
If a cleanroom facility is well designed and functioning
properly, and if laboratory personnel, wear protective
cleanroom garments and follow proper behavioral pro-
cedures, then the major sources of contamination re-
maining are labware and reagents. Both of these sources
can be effectively minimized, but frequent analysis of
labware and reagent blanks is mandatory to ensure that
contamination levels are not rising.
Routine Labware
The types of labware used in the Class 100 cleanroom
include typical glass, quartz, teflon, polyethylene, poly-
styrene, and polypropylene containers and pipettes. With
the exception of micropipettors, metal implements are
rarely used in the trace element cleanroom. The first
major difference between cleanroom labware and non-
cleanroom labware is that cleanroom labware is divided
into "low level" and "high level" labware. "Low level"
labware is used only with solutions that have metal ions
at concentrations below 1 ppm. In this way, the surface
Sites of the labware should never be exposed to high
concentrations of analytes. "High level" labware is used
for solutions that have analyte concentrations greater
than 1 ppm, such as stock reagents, matrix modifiers, or
concentrated metal standards used for preparing more
dilute standards. Because glassware tends to exhibit
"memory" effects (i.e., strong, slowly reversible adsorp-
tion) from previous solutions, it is very important to
maintain the separation between the "low level" and
"high level" glassware. Further segregation of labware is
strongly advised for specific metals and ultra-low con-
centration ranges,-For example, lead, chromium, and
boron are highly sorbed by silicate surfaces of glass or
quartz, and to a lesser extent by plastics. When pos-
sible, labware that is used for those metals should be
dedicated to those metals only. Also, analytes that are
present in the parts per trillion concentration range
should have dedicated ultra-low level labware and acid
soaking baths.
31
-------
If the uptake of analyte by the labware is not permanent,
the labware is subjected to rigorous cleaning proce-
dures and stored in a clean manner appropriate to the
analytical use. Labware cleaning procedures are in-
cluded in Appendix B, "Standard Operating Procedures
for Cleaning Labware in the ACS Inorganic Class 1007
10,000 Clean Lab Facility." In brief, the procedures
involve the following steps:
* separation of labware into "low level" and "high
level11 basins
* pre-treatment (removing labels, rinsing with tap wa-
ter)
• detergent washing
• rinsing with tap water followed by deionized water
* acid leaching
* final rinse with deionized water
* drying in clean air stream
• storage
Teflon labware used for mercury analysis require an
additional cleaning step to oxidize and bind any residual
mercury. One cleaning recipe is to fill cleaned teflon
containers with 1% HNO3 plus a brominating solution
(KBr +KBrO3), store the containers until ready for use,
discard the solution just prior to reuse, and rinse with low
mercury deionized water (see Appendix E: Standard
Operating Procedures for Mercury Analysis in Water,
Sediment and Tissue). Another cleaning procedure in-
volves boiling cleaned containers in concentrated HNO3
for 24 hours in a large teflon vat, rinsing with low
mercury water, filling with 1% HCI and warming for 24
hours, discarding the HCI and refilling with fresh 1% HCI
for storage until use (see Appendix F: Standard Operat-
ing Procedure for Total Mercury in Aqueous Samples by
Cold Vapor Atomic Fluorescence, and Appendix G:
Total and "Acid-Labile" Mercury in Aqueous Media).
Glassware used for mercury analysis can be heated in
an oven prior to use to remove any residual reduced
forms of mercury.
Microwave Vessels
Teflon microwave vessels are treated differently for
some analytes. While teflon is generally an extremely
inert material with minimal adsorptive or surface-active
properties, it has been found to exhibit a memory effort
for some metals, such as indium. In those cases, routine
cleaning and acid leaching procedures are not adequate
to quantitatively remove the metal. A high pressure, acid
"steam cleaning" procedure is used for these vessels in
which high purity acids are added to the pre-cleaned
teflon vessels, and the vessels are carried through a
microwave cycle to extract the teflon with acid at high
temperatures and pressures. The extract is analyzed for
the metal of interest and the vessels are reused only if
the extract is sufficiently clean.
Deionized Water
A high quality deionization system is essential for per-
forming ultra-trace analysis of metals. In general, the
existing literature for water deionization for the micro-
electronics industry is appropriate for trace metal analy-
sis as well. Both groups require 18 MGi resistivity with
ultra-low concentrations of metal ions. However, the
metal ions of concern for the two groups are significantly
different, and while vendors of water deionization sys-
tems can produce ICPMS analyses of metals of interest
to semiconductor manufacturers, they are generally ill-
prepared for the needs of the trace element chemistry
laboratory. For example, ultrarlow sodium and potas-
sium ion concentrations are required for semiconductor
manufacture, but are generally of little concern for ultra-
trace level environmental analyses. However, sub-parts
per billion levels of lead and arsenic are of great impor-
tance for environmental analysis, but relatively little for
the semiconductor industry.
Water deionization systems are not generally optimized
for ultra-trace mercury analysis and in several cases
actually contribute mercury to the water. Deionization
resins that are recycled using a caustic treatment are
particularly susceptible to mercury contamination. Deion-
ized water should be routinely analyzed for contamina-
tion of metals of interest. Water that is 18 MQ resistivity
is not necessarily sufficiently pure in any'one metal.
32
-------
Section 8.0
Cleanroom Maintenance
Initial Certification
Once the cleanroom has been completed and is ready
for occupancy, testing should be performed to verify its
cleanliness and performance. The Institute for Environ-
mental Sciences has published guidelines for testing
cleanrooms and made recommendations for the tests to
be performed [IES-RP-CC-006-84-T, 1984]. The docu-
ment includes tests for airflow velocity and uniformity,
HEPA filter leaks, airflow parallelism, room recovery,
airborne particle count, particle fallout count, room pres-
surization, noise, vibration, and other tests.
Monitored Parameters
Following initial certification of the cleanroom, a sched-
ule should be established for continued monitoring of air
quality parameters. In the RTI cleanroom, various pa-
rameters are monitored on a continuous, weekly, quar-
terly, or semi-annually basis as explained in Appendix C
(Standard Operating Procedure for Monitoring and Main-
taining Cleanliness of the ACS Inorganic Clean Lab
Facility).
The parameters that are monitored most frequently are
those that indicate failure of the air handling equipment
or emergency situations (smoke, excess heat). Continu-
ous display, real time monitors display static pressure,
temperature, and relative humidity in each room of the
cleanroom suite, and a computer interface to the display
updates a record of these parameters every 12 hours. In
a properly functioning cleanroom, the cleanest room
should have the highest static pressure, the next most
clean room should have the next highest pressure, and
so on, so that air will travel from the more clean rooms to
the less clean rooms. If pressures are inverted between
rooms, the direction of air travel will be reversed and
contaminated air will enter the cleaner rooms.
Parameters that are monitored on a daily basis include
deionized water resistivity, HEPA filter pressure drop,
and refrigerator and freezer temperatures. Less fre-
quently monitored parameters include particle counts
(every three to six months), air flow rates (annually),
acid bath concentrations of analytes of interest (semi-
annually or as needed), and "room blank" concentra-
tions of analytes. The "room blanks" are non-volatile
particles that settle in open teflon beakers placed in
specific regions of the laboratory. Control teflon beakers
with lids in place accompany the "room blank" beakers
to enable subtraction of the teflon contribution to deter-
mined background analyte concentrations.
Maintenance Activities
Housekeeping and maintenance activities in the
cleanroom include wiping work surfaces, mopping floors,
replacing sticky mats, and removing trash. These activi-
ties are performed by the technical staff rather than the
janitorial staff because entrance is restricted to cleanroom
trained personnel only. Monthly charts are used to record
performance of each activity and note any problems. In
the Class 100 room, work surfaces (benches and hoods)
are wiped daily with deionized water on cleanroom
wipes. The floor is mopped with .tap water weekly. A
metal-free detergent may be used if needed, but is
generally avoided because of the potential of a particle-
generating residue on the floor. Sticky mats at the
entrance to the Class 100 and 10,000 rooms are changed
weekly or more often if needed. Trash is removed from
the cleanroom daily.
Non-routine maintenance activities include semi-annual
replacement of bag filters and pre-filters, checks on
motor drive belts, and examination of HVAC equipment
for signs of wear or stress.
33
-------
Section 9.0
Reagent Purification
Procedures
Reagents commonly used in trace element analysis
Include deionized water, acids, bases, oxidizing agents,
reducing agents, buffers, complexing agents, and to a
lesser extent, solvents. The demand for high purity
reagents has been met by commercial chemical manu-
facturers in some cases. However for many others,
specific purification procedures need to be developed to
adequately control contamination.
Acids are prepared by triple distillation in sub-boiling
teflon stills. Commercially available high purity mineral
acids are generally excellent quality (ULTREX,
SUPRAPUR, OPTIMA), but at a relatively high price.
Equal or better quality acids can be prepared in a
cleanroom setting using custom-designed teflon stills
such as those at NIST or the University of North Caro-
lina Geochronology Lab. These stills require daily atten-
tion and maintenance, but if properly tended yield a
more economical source of high purity acids.
In general, reagents are purified by repetitive distillation,
repetitive crystallization, ion exchange, or complexation
followed by solvent extraction. Volatile impurities, such
as mercury, can often be eliminated by heating or inert
gas purge. Many examples and recipes are available in
publications on trace element contamination control
[Zief, 1976; Moody and Beary, 1982; Mitchell, 1982].
Standard Operating Procedures used for purification of
graphite furnace matrix modifiers used at RTI are pre-
sented in Appendix D (Standard Operating Procedures
for Purification of Reagents in the ACS Inorganic Clean
Lab Facility for Trace/Ultratrace Metal Analysis). Proce-
dures for purification of reagents used for mercury
analysis are presented in Appendices E, F, and G.
Storage
Storage of high purity reagents and standards requires
careful consideration. Ultra-low concentration standards
and samples need to be contained in materials that do
not adsorb the analyte and hence cause loss of analyte
overtime, and the container materials must not leach or
otherwise contaminate the solution with the analyte and
thus cause elevated analyte concentrations. In general,
teflon is the material of choice due to its highly inert
nature, but other plastics, such as polyethylene, are well
suited for storage of most trace element solutions.
Glass is generally avoided because of its high affinity for
adsorption and because it contains a variety of metallic
impurities in its matrix which can slowly leach into solu-
tion. For volatile analytes such as reduced forms of
mercury, arsenic, selenium, etc., the containers must be
dense enough that vapors do not permeate through the
container walls causing loss of analyte from the sample
or contamination by analyte in the atmosphere. Similarly,
the caps must be able to be closed very tightly to prevent
exchange of vapors in or out of the bottles. Nicolas
Bloom recommends teflon bottles that can be torqued
closed with a wrench for aqueous mercury samples. As
an additional precaution against atmospheric contami-
nation of samples, mercury analysts typically include a
mercury vapor phase sorbent trap, such as gold coated
activated carbon or gold impregnated fabric, inside the
cleanroom and also inside refrigerators and storage
areas.
Storage of high concentration standards or samples
represents a different problem. In the case of volatile
analytes, loss of analyte from the solution to the atmo-
sphere and eventually to other samples is a major con-
cern. One mercury analyst reported that storage of a
1000 ppm mercury standard in a refrigerator contami-
nated the refrigerator so badly that it had to be removed
from service. High concentration standards and samples
need to be stored in vaportight containers in regions that
are remote from low level analytes. Several of the
cleanroom designers/operators interviewed stated that
they do not permit high concentrations of volatile analytes
to enter the cleanroom areas. The maximum permissible
mercury concentration that is stored in the cleanroom is
typically 1 ppm for laboratories that conduct analyses in
the ppt range.
Vapor exchange is not a problem for storage of non-
volatile reagents and standards, but spills of high con-
centration solutions are still a problem. Some cleanrooms
do not allow storage of any standard at a concentration
greater than 1 ppm. Others allow storage of higher
concentration standards and reagents but in designated
regions of the cleanroom only. For example, in the RTI
cleanroom, all work performed with solutions containing
analytes at concentrations greater than 1 ppm must be
performed on specified "high level" laboratory benches
using designated "high level" labware. Those regions
and labware are never used for "low level" work and thus
chances of cross-contamination are kept low.
34
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References
Austin, Philip R. Design and Operation of Clean Rooms;
Business News Publishing Company: Detroit, 1970
Donovan, Robert P., ed., Particle Control for
Semiconductor Manufacturing, Marcel Dekker, Inc.:
New York, 1990, 464 pp.
Ensor, David and Robert Donovan, "Aerosol Filtration
Technology." In Handbook of Contamination Control
in Microelectronics, Tolliver, Donald L, ed. Noyes
Publications: Park Ridge, NJ, 1988, 1-67.
Federal Standard 209E: Airborne Particulate Cleanliness
Classes in Cleanrooms and Clean Zones, FED-STD-
209E, U.S. Government Printing Office: Washington,
D.C., 1992.
Geilleit, R.A.H.M., "Present status of the European 209-
WG 1 of TC 243." In The Future Practice of
Contamination Control, Mechanical Engineering
Publications: London, 1992, 373-376.
Institute of Environmental Sciences. IES-CC-011-85-T:
A Glossary of Terms and Definitions Related to
Contamination Control. Mount Prospect, IL, 1985, 16
PP-
Institute of Environmental Sciences. IES-RD-CC009.2:
Compendium of Standards, Practices, Methods, and
Similar Documents Relating to Contamination Control.
Mount Prospect, IL , 1993, 70 pp.
Institute of Environmental Sciences. IES-RP-CC001.3:
HEPA and ULPA Filters. Mount Prospect, IL, 1993,22
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Institute of Environmental Sciences. IES-RP-CC-006-
84-T: Testing Clean Rooms. Mount Prospect, IL, 1984,
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Matthews, Richard A., "Cleanroom basics: A guide for
the perplexed", Medical Device and Diagnostic Industry,
February 1994, 57-62.
Mielke, R.L., "The status and future of FED-STD-209E
of the United States of America." In The Future
Practice of Contamination Control, Mechanical
Engineering Publications: London, 1992, 343-344.
Mitchell, J.W., "Purification of Analytical Reagents",
Talanta, 1982,29:993.
Moody, John R., "NBS Clean Laboratories for Trace
Element Analysis", Anal.Chem. 1982, 54:1358A.
Moody, John R. and E.E. Beary, "Purified Reagents for
Trace Metal Analysis", Talanta 1982, 29:1003.
Su, Q., Goldberg, S.A., and Fullagar, P.O., "Precise U-
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Appalachian Blue Ridge and Their Implications for the
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Tolliver, Donald L., ed., Handbook of Contamination
Control in Microelectronics, Noyes Publications: Park
Ridge, NJ, 1988,488pp.
Zief, M. and J.W. Mitchell, "Contamination Control in
Trace Element Analysis"; In Chemical Analysis 1976,
Vol. 47, Chapter 6.
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36
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Appendix A
RTI/ACS/SOP-174-001
Revision 0
Standard Operating Procedures for the ACS Inorganic
Class 100/10,000 Clean Lab Facility
Research Triangle Institute
Analytical and Chemical Sciences
Post Office Box 12194
Research Triangle Park, NC 27709
May 1994
-------
AGS/SQP-1:74-00,1
May 1.994
Revision Of
Table of Contents
page
1.0 Introduction A_3
2.0 Laboratory Description L.IIZZ A-3
3.0 Laboratory Apparel !™"™Z™A-6
4.0 Materials Handling and Exchange . ...1" A-8
A-2
-------
ACS/SOP-174-001
May 1994
Revision 0
Standard Operating Procedures for the
ACS Inorganic Class 100/10,000 Clean Lab Facility
RTI/ACS-SOP-174-001
1.0 Introduction
The ACS Inorganic Class 100/10,000 Clean Lab Facility
is a controlled access laboratory designed for minimiz-
ing paniculate contaminants for the purposes of ultra-
trace metal analysis. The facility is restricted to Inorganics
Research personnel and escorted visitors trained in the
protocols necessary to maintain Class 100 conditions.
Appropriate garments must be worn and protocols fol-
lowed for admittance into the Class 100 Clean Lab area.
This document outlines the proper procedures and equip-
ment for use and maintenance of the ACS Inorganic
Class 100/10,000 Clean Lab Facility.
2.0 Laboratory Description
The ACS Inorganic Class 100/10,000 Clean Lab Facility
is a suite of rooms within Dreyfus 193. Figure 1 shows
the layout of each room; a description of each is pro-
vided in the following sections.
2.1 Office Area
The 193 Office is accessed from the Dreyfus hallway
and has no entrance restrictions. There is a door from
the Office into the Inorganic Class 100/10,000 Clean
Lab Anteroom; this door remains locked from the Office
side, but is unlocked from the laboratory side. This
arrangement permits emergency egress from the Inor-
ganic Class 100/10,000 Clean Lab and anteroom, but
prohibits routine entrance from the office. The key to this
door is kept in the Office.
2.2 Service Room
The Service Room is a small, general access room that
contains supplies, gas cylinders, water deionizer, air
purification system, refrigerator, and freezer. It is acces-
sible from the Dreyfus hallway and has no entrance
restrictions. The door from the Service Room into the
Instrument Lab is a restricted passage. Only authorized
personnel may enter the Instrument Lab. Both the Office
and Service Room are supplied with air filtered through
standard 95% efficiency filters, but not HEPA filters.
2.3 Instrument Lab
The Instrument Lab is a Class 10,000 area, with a
partially HEPA-filtered air supply (HEPA-filters are lo-
cated in areas of sample handling). Minimal particulate
control measures are used ('lacky-mat" flooring at the
entrance). The lab contains instruments for trace and
ultra-trace metal analysis (GFAA, ICP, HGAF, etc.). The
Instrument Lab is a restricted area accessible only to
authorized personnel. It is entered via the 193 Service
Room and may be exited to the Service Room, Ante-
room, or through an emergency exit door to the outside
of the building.
2.4 Anteroom
The Anteroom is the transition zone from the Instrument
Lab to the Class 100 Clean Lab and is a restricted area,
limited to trained personnel only. The Anteroom is sup-
plied with HEPA-filtered air and provided with "tacky
mat" flooring to reduce particulates carried into the
Class 100 area. The Anteroom is used to put on Clean
Lab garments before entering the Class 100 area (clean-
lab coats, lab shoes or shoe covers, head covers) and to
store Clean Lab cleaning supplies (lab cart, mop, bucket,
clean wipes).
The Anteroom is the sole access to the Class 100 Clean
Lab. The Anteroom has a door which opens to the Office
area, which is to be used as an emergency exit only.
2.5 Class 100 Clean Lab
The Class 100 Clean Lab is an inorganic analysis labo-
ratory designed to enable contaminant-free preparation
of samples for metal analysis at the parts-per-trillion
level. The two most unique features of the room are the
ultra-clean air supply and the metal-free construction.
Ultra-clean air is supplied through HEPA filters located
in a ceiling grid; over 90% of the ceiling is covered with
99.99759/0 efficient HEPA filters. As depicted in Figure 2,
Class 100 air is forced downward in a laminar flow and
bathes work benches with particle-free air. Air is re-
A-3
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ACS/SOP-174-001
May 1994
Revision 0
29'-
Service
room
iment
atory
o
j
1
1
1
1
. tt
k ,-
-
"" h
I
«
Class 10O \ \ Class 10.OOO
Non-filtered ^ Plenum wall
I
Class 100
clean
room
''V-,
Office
44'
a Gas Cylinders
b. D.(.Water
c. Refrigerator
d. Freezer
e. Cleanroom Supplies
f. PE5100ZL
g. 4'HEPAHood
h. Bench
i. Sink
j. ICP
k. 8'HEPAHood
I. Non-HEPA 4' Exhausting Hood
m. 6' Acid Baths
n. Desk
o. Questron V6 AFS
Figure 1. ACS Inorganics Class 100/10,000 Clean Lab Facility.
A-4
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ACS/SOP-174-001
May 1994
Revision 0
Floor
Drain
Figure 2. Vertical laminar flow cleanroom design used at RTI.
A-5
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ACS/SOP-174-001
May 1994
Revision 0
turned to a recirculation unit at two heights along a
plenum wall: the primary air return is a baseboard return
extending from the floor up to a height of 18 inches; the
secondary return is at hand level extending from the
benchtop surface to a height of six inches above the
benchtop. This air is recirculated through the ceiling
HEPA filters to achieve continuous removal of particles.
Metal-free construction materials were used to the ex-
tent possible to build the Class 100 Clean Lab. This was
done to prevent acid corrosion and resulting metal con-
tamination. The walls, floors, benches, fume hoods, acid
bath cabinet, drawers, and sinks are made of polypropy-
lene, and all plumbing materials are either polypropy-
lene or PVC. All other service lines (electrical, gas, etc.)
are encased in PVC tubing.
The Class 100 Clean Lab contains several features in
addition to the clean air supply that facilitate ultra-trace
level analysis. The lab is equipped with three areas of
one-pass, air exhaust: (1) an 8-foot wide Class 100
fume hood with vented base cabinet; (2) a 4-foot wide
fume hood with vented base cabinet; and (3) a 6-foot
wide vented acid bath cabinet. The fume hoods are
used for acid digestion, evaporation, or extraction of
samples and prevent exposure of personnel or equip-
ment to acid vapors. Diagrams showing the design and
air flow patterns in the 8-foot hood are presented in
Figure 3. A microwave digestion system is located in the
4-foot hood, and two remote control ceramic hot plates
are located in the 8-foot hood. The acid bath cabinet
houses multiple tubs for acid leaching of glassware and
plasticware. The interior of the cabinet is flushed hori-
zontally with Class 100 air and vented to the outside of
the building in order to minimize exposure of personnel
to acid vapors even when the baths are open.
The Class 100 Clean Lab is the most restricted area of
the Inorganic Class 100/10,000 Clean Lab facility, re-
quiring appropriate apparel for entry (cleanroom shoes
or shoe covers, cleanroom lab coats, etc.). Access to
the Class 100 Clean Lab is through the anteroom via the
Instrument Lab. The Class 100 Clean Lab also has an
emergency door escape route leading to the outside of
the building.
3.0 Laboratory Apparel
3.1 Off ice Area
The Dreyfus 193 Office area is non-restricted, non-
laboratory area with no dress restrictions.
3.2 Service Room
The Dreyfus 193 Service Room is a non-restricted area
with no dress restrictions.
3.3 Instrument Lab
The Inorganic Class 100/10,000 Clean Lab Instrument
Lab is designated as a Class 10,000 environment; lab
apparel requirements are set up to maintain this envi-
ronment and for the safety of laboratory workers; re-
quirements are described in the following sections.
3.3.1 Clean Garments
Cleanroom shoes or shoe covers are required for en-
trance to the Instrument Lab. Laboratory coats and
powder-free gloves are available. A "tacky-mat" at the
entrance door will reduce footborne contamination. The
lab personnel should always be conscious of minimizing
contamination when entering the Instrument Lab, and
when passing from the Instrument Lab into the Class
100 Clean Lab.
3.3.2 Safety Garments
Working in the Instrument Lab will entail exposure to a
variety of chemicals including dilute acids, sample di-
gests, and standard metal solutions. The required pro-
tective measures are safety glasses and closed-toe
shoes. Lab coats and acid-resistant gloves are recom-
mended when handling samples, standards, reagents
and wastes.
Visitors to the Instrument Lab will be required to wear
appropriate shoes and safety glasses.
3.4 Class 100 Clean Lab
The Class 100 Clean Lab is designated as a Class 100
facility; the procedures and lab apparel required are
designed to maintain the Class 100 conditions and for
the safety of the lab personnel.
3.4.1 Clean Garments
There are strict requirements for garments to minimize
particulates and contaminants carried into the Clean lab.
Entry to the Class 100 Clean Lab is through the Ante-
room, where cleanroom garments will be kept for labora-
tory personnel. Extra cleanroom garments will be avail-
able for visitors on the Service Room storage shelves.
• All personnel will remove or cover street shoes (labo-
ratory workers will have shoes designated for
cleanroom use only, visitors will use disposable shoe
covers).
• All personnel will wear low-particulate, Class 100 lab
jackets in the Class 100 Clean Lab.
• All personnel will wear disposable head covers.
A-6
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20" x 20" prefilters
12"x 12" polypropylene sink
Room air pre-filters (2)
I
Cabinet
storage
Perforated polypropylene
work surface
o
Drain1
T T T
Clean air
60FPM
downflow
Return air plenum
under work surface
fully seam welded
ACS/SOP-174-001
May 1994
Revision 0
HEPA
80FPM
Inflow velocity
Side view
Figure 3. Vertical laminar flow fume hood.
A-7
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ACS/SOP-174-001
May 1994
Revision 0
• Class 100 powder-free gloves are available for work-
Ing in the Class 100 Clean Lab. Gloves worn in the
Instrument Lab should be rinsed or discarded before
entry into the Class 100 Clean Lab.
3.4.2 Safety Garments
Safety garments are required in the Class 100 Clean
Lab to protect the lab workers from exposure to concen-
trated acids and oxidizers used in sample digestion and
from exposure to sample materials which may be partly
or totally uncharacterized. Safety glasses, lab coat and
appropriate shoes are minimally required. Gloves are
recommended for handling toxic chemicals, acids or
unknowns.
Additionally, lab personnel may use a face shield, lab
apron, dust mask or respirator or other safety equipment
for certain tasks. Routine safety equipment must meet
Class 100 standards of cleanliness before being brought
Into the Class 100 Clean Lab, either purchased pre-
packaged for Class 100 use from a cleanroom supplier,
or cleaned and packaged in the Class 100 hood in the
Instrument Lab. However, personnel safety is always
the primary consideration and in some cases it may be
necessary to use non-Clean Lab equipment or supplies.
4.2 Service Room Materials Handling
Protocols
The Service Room acts as a storage area for the Instru-
ment Lab and the Class 100 Clean Lab. Materials must
pass from the Service Room to the Instrument Lab, then
through the Anteroom into the Class 100 Clean Lab,
with appropriate decontamination procedures in the In-
strument Lab and Anteroom.
4.3 Instrument Lab Materials Handling
Protocols
The Instrument Lab is designed for instrumental analy-
sis and will have minimal reagent handling procedures.
Reagents and samples will be brought into the Instru-
ment Lab only for analysis purposes (samples, calibra-
tion standards, matrix modifiers, acids, blanks, etc.) or
for cleaning procedures before transfer to the Class 100
Clean Lab.
4.3.1 Acid exposure in the Instrument Lab will be
limited. No concentrated acids will be stored in the
Instrument Lab.
4.3.2 Acid solutions used for ICP or AA flushing solu-
tions will generally be <25% concentration and <1.0 L
volume. Instrument reservoirs for acid solutions will be
closed or covered with parafilm or similar sealant to
minimize acid vapors in the Instrument Lab.
4.0 Materials Handling and Exchange 4.4
The working areas of Dreyfus 193 are laid out so that
personnel pass from the "dirty" areas (i.e., the hallway,
the office, the Service Room) to increasingly cleaner
areas (the Instrument Room, the Anteroom, the
Cleanroom). Appropriate measures are taken to reduce
contamination in each progressively cleaner area (see
Sections 3.3-3.4.)
To minimize contamination in the cleaner areas, each
area will be provided with its own supplies according to
the tasks performed there. To this end, procedures are
in place regarding supplies, cleaning procedures, sample
storage and waste disposal for the Inorganic Class 10O/
10,000 Clean Lab Facility.
4.1 Office Materials Handling Protocols
No reagents or samples will be permitted in the Office
area. Any documents, notes, notebooks or raw data
brought into the Office area from the Instrument Lab or
Class 100 Clean Lab will be as contamination free as
possible.
Class 100 Clean Lab Materials
Handling Protocols
4.4.1 High Level Materials Handling Protocols
Specific lab bench space and lab supplies will be desig-
nated for High Level Operations (> 1 ppm metals con-
centration) within the Class 100 Clean Lab. High Level
Operations are defined as the handling and dilution of
concentrated metal standard solutions and for preparing
reagents for sample preparation or analytical applica-
tions.
4.4.2 New supplies and equipment for High Level
Operations will be initially washed and/or rinsed with
deionized water and dried in a Class 100 environment.
4.4.3 High Level Operations labware will be washed
and acid leached according to ACS-SOP-174-002 "Stan-
dard Operating Procedure for Cleaning Labware in the
ACS Inorganic Class 100/10,000 Clean Lab Facility"
then stored in cabinets designated for High Level Op-
erations equipment. Washing basins, baskets and acid
leaching baths will be designated for High Level Opera-
tions.
4.4.4 In general, High Level Operations equipment
and supplies will not be used for other applications.
Exceptions may be made on the judgement of the
Laboratory Supervisor, or Project Officer.
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ACS/SOP-174-001
May 1994
Revision 0
4.4.5 The High Level Operations bench space will be
cleaned and equipment and chemicals put away after all
operations.
4.5 Low Level Operations Materials
Handling Protocols
The Class 100 Clean Lab is supplied with designated
labware (glassware, teflon and plasticware, pipettors
and tips, volumetric pipets and bulbs) for exclusive use
in Low Level Operations.
4.5.1 Supplies will be initially cleaned and decontami-
nated with stringent measures (see SOP 174-002, "SOP
for Cleaning Labware in the ACS Inorganic Class 1007
10,000 Clean Lab Facility") before being brought into the
Class 100 Clean Lab.
4.5.2 Low Level Operation labware will be rinsed in
the Class 100 Clean Lab or, if necessary, washed in the
Instrument Lab, then acid leached in acid baths desig-
nated for Low Level Operations.
4.5.3 Under no circumstances will Low Level Opera-
tions equipment or supplies be removed from the Class
100 Clean Lab Facility or used in High Level operations.
4.5.4 Exposure of Low Level Operations labware to
metal concentrations >1ppm will be strictly avoided.
Metal solutions or reagents brought into the Low Level
Operations working areas will be of the lowest working
concentrations possible.
4.5.5 New or replacement supplies (including reagents,
acids, and Samples) brought into the Class 100 Clean
Lab will be in Class 100 packaging, or cleaned by
laboratory personnel in the Class 100 hood in the Instru-
ment Lab (see SOP 174-002, "SOP for Cleaning Labware
for the Class 100 Clean Lab Facility") before being
transferred into the Class 100 Clean Lab.
Supplies packed in Class 100 packaging will be brought
into the Anteroom, where external packaging can be
discarded, then brought into the Class 100 Clean Lab,
where internal packaging can be opened and discarded.
4.6 Waste Disposal
4.6.1 Office Area
Trash from the Office area will be recycled or disposed
of by Housekeeping personnel according to RTI guide-
lines.
4.6.2 Service Room
Non-chemical trash from the Service Room will be dis-
posed of by Housekeeping personnel according to RTI
guidelines.
4.6.3 Instrument Lab
Non-contaminated trash from the Instrument Lab will be
disposed of by Housekeeping personnel according to
RTI guidelines. The trash can will be placed in the
Service Room or the hallway for pickup.
Chemical wastes from the Instrument Lab will be col-
lected in plastic or glass containers. When possible,
wastes will be neutralized and flushed down the drain.
Otherwise, wastes will be labelled as to metals and
matrix, and approximate concentrations (if >5ppm) and
collected by RTI Safety personnel.
If necessary, chemical wastes will be transferred to Lab
169 for characterization, then collected by RTI Safety
personnel. Lab personnel will be responsible for dis-
posal of waste generated by their projects.
4.6.4 Class 100 Clean Lab
RTI Maintenance and Safety personnel will not need
access to the Class 100 Clean Lab or Anteroom. Labo-
ratory personnel will be responsible for all cleaning and
waste collection in these restricted areas.
Non-contaminated trash from the Class 100 Clean Lab
(and Anteroom) will be collected by the lab worker from
lined, plastic trash cans in the Class 100 Clean Lab. The
trash bag will be collected daily and disposed of with
trash from other lab areas.
Chemical wastes from the Class 100 Clean Lab will be
collected in plastic containers and transferred to the
Instrument Lab for treatment and disposal.
Chemical wastes will not be generated or stored in the
Anteroom.
A-9
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Appendix B
RTI/ACS/SOP-174-002
Revision 0
Standard Operating Procedures for Cleaning Labware in the
ACS Inorganic Class 100/10,000 Clean Lab Facility
Research Triangle Institute
Analytical and Chemical Sciences
Post Off ice Box 12194
Research Triangle Park, NC 27709
May 1994
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RTI/ACS/SOP-174-002
May 1994
Revision 0
Table of Contents
Page
1.0 Introduction [[[ B-3
2.0 Summary of Procedure [[[ B-3
3.0 Routine Labware Cleaning Procedure [[[ B-3
4.0 Maintenance of Acid Leaching Baths [[[ B-4
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RTI/ACS/SOP-174-002
May 1994
Revision 0
Standard Operating Procedures for Cleaning Labware in the
ACS Inorganic Class 100/10,000 Clean Lab Facility
1.0 introduction
This document describes the cleaning procedures nec-
essary for labware used exclusively in the ACS Inor-
ganic Class 100/10,000 Clean Lab Facility. The ACS
Inorganic Class 100/10,000 Clean Lab Facility (Dreyfus
193, A-D) is supplied with separate, dedicated sets of
labware for High Level Operations (> 1 ppm) and Low
Level ultra-trace metal applications.
Cleaning procedures will be done by ACS technical
support personnel or laboratory personnel within the
ACS Inorganics Class 100/10,000 Clean Lab Facility
(when possible), using the sinks and designated basins,
deionized water lines and the acid-leaching baths.
2.0 Summary of Procedure
A general, routine cleaning procedure for Clean Lab
labware can be summarized in the following steps:
• separation of High Level and Low Level labware
• pre-treatment (removing labels, rinsing with tap wa-
ter)
• detergent wash (soaking or scrubbing)
• thorough rinse (tap water and deionized water)
• acid leaching
• final rinse in deionized water
• drying (in Clean Lab hood)
• storage (if required)
The steps outlined above will be adequate for most
Clean Lab applications. For some sensitive, non-routine
applications, additional hot acid leaching may be re-
quired. All procedures are detailed below.
3.0 Routine Labware Cleaning RTI/ACS/
Procedure
The routine labware cleaning procedure is applicable to
most High Level or Low Level Operations. For some
applications an alternate cleaning procedure may be
used (i.e. eliminate the detergent soak in order to reduce
contamination risks from the detergent or employ other
soaking agents).
3,1 Laboratory technical personnel will be respon-
sible for preparing the labware for cleaning:
• empty the dirty labware of any chemical residue and
rinse well with tap water.
• remove label tape or lab marker labelling (acetone
or methanol is usually suitable for removing lab
marker or tape residues from teflon, glass and most
plastics.)
• segregate labware as to High Level or Low Level
applications.
3.2 ACS support personnel (dishwashers) or labo-
ratory personnel will be responsible for washing and
rinsing procedures:
• prepare a solution of a suitable laboratory detergent
(Alconox, Liquinox etc.) in the designated basin
(Low Level or High Level) for soaking the labware.
• submerge the labware, careful to wet all surfaces.
• soak for as long as necessary (to remove any
organic residue).
• rinse the labware with tap water until all detergent
residue is gone.
• rinse with deionized water three times.
• place the labware onto the appropriate lab cart
(High Level or Low Level Operations) for return to
the lab.
3.3 Laboratory technical personnel are responsible
for all acid leaching procedures: submerge rinsed labware
into an appropriate acid leaching bath set up in the acid
bath cabinet, or in the Clean Lab hood. All surfaces
(interior and exterior) must be exposed to the leaching
solution. Labware will be soaked for at least 8 hours. .
3.4 For some Low Level ultra-trace applications,
only the interior of the labware will be leached in order to
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RTI/ACS/SOP-174-002
May 1994
Revision 0
prevent contamination from the printed labels on the
exterior. This labware will be filled with the leaching
solution and allowed to sit at room temperature for an
appropriate length of time (2-4 hours). Alternately, the
acid-filled labware will be soaked in a warm water bath.
CAUTION
1. Laboratory personnel must take safety precautions
when working with acid leaching baths: lab coat,
safety glasses or face shield, Playtex-type latex
gloves, sleeve covers, use of plastic tongs etc.
Gloves should be used exclusively for acid-bath
tasks.
2. All laboratory surfaces exposed to acid solutions
should be rinsed and if necessary neutralized.
3.5 Labware will be retrieved from the acid bath,
drained of excess leaching solution, and rinse with
copious deionized water (5-10 rinses.)
3.6 The labware will be dried on a drying rack or on
Class-100 lab wipes on the lab bench or in the hood (if
necessary).
3.7 Labware not used immediately will be stored in
the acid baths until needed, or placed in plastic ziplock
bags and stored in appropriate cabinets designated for
High Level or Low Level Operations.
3.8 Volumetric Pipets
3.8.1 High Level Operations
High Level Operations include diluting calibration stan-
dards for use in the Instrument Lab, and diluting or
transferring reagent solutions for sample preparation or
analytical uses.
Volumetric pipets used in High Level Operations will be
copiously rinsed with an appropriate rinse solution after
use, then rinsed with at least 5 volumes of deionized
water before being returned to the pipet acid bath desig-
nated for High Level Operations labware. Pipets should
be cleaned with detergent (or alcoholic KOH) only when
poor wetting and drainage characteristics compromise
volumetric performance.
3.8.2 Low Level Operations
Pipets used for low level operations will be etched with
an "L" to designate low level applications only. Low
Level Operations include transferring or diluting sample
preparations and dilute, decontaminated reagent solu-
tions. Volumetric pipets used in Low Level Operations
will be copiously rinsed with an appropriate rinse solu-
tion after use, then rinsed with at least 5 volumes of
deionized water before being returned to the pipet acid
bath designated for Low Level Operations labware. Pi-
pets should be cleaned with detergent (or alcoholic
KOH) only when poor wetting and drainage characteris-
tics compromise volumetric performance.
3.9 Microwave Digestion Vessels
Microwave digestion vessels will be numbered and seg-
regated according to the application and analyte. Be-
tween uses the vessels will be washed or soaked in
detergent, rinsed and acid-extracted according to the
needs of the project and analyte (see Section 6.0)
4.0 Maintenance of Acid Leaching Baths
The Clean Lab acid baths will be prepared as 20%
HNO3 (reagent grade concentrated nitric acid) in deion-
ized water. Separate acid baths will be prepared and
designated for High Level Operations labware and Low
Level Operations labware.
Contamination levels of the baths will be monitored by
sampling the leaching solution from each bath for analy-
sis. The acid leaching solutions will be sampled and
analyzed when the baths are first prepared and once
every month thereafter (see ACS/SOP-174-005 "Moni-
toring and Maintenance of the Cleanliness of the Clean
Lab Facilities"). The samples will be analyzed for cad-
mium, lead and arsenic (Cd, Pb, As) or for the current
analyte of interest.
The acid leaching baths will be changed when any of the
analytes of interest reach a concentration > 1 ppm or in
the event of known accidental contamination. If any
analyte exceeds the 1 ppm threshold but is not currently
being determined for a project, it is not necessary to
change the acid bath until that analyte is being deter-
mined for a project.
To prevent accidental contamination, laboratory person-
nel will check that High Level and Low Level labware are
kept separate and are leached in the designated baths.
All labware will be rinsed with deionized water before
submerging in the acid leaching baths.
Lab personnel will have Playtex (or similar) gloves spe-
cifically designated for use in the acid leaching baths.
These gloves will be rinsed with deionized water before
each use in the acid leaching baths. After use in the acid
baths, the gloves will be rinsed copiously with tap water
and hung to dry. These gloves will not be used for
handling samples, reagents or waste.
5.0 Non-Routine Cleaning Procedure
For sensitive Low Level applications (ultra-trace Pb, Hg
etc.) an alternate hot acid leaching procedure may be
used for Low Level labware.
B-4
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5.1 A leaching bath will be prepared in a 1.0 or 4.0 L
beaker dedicated for use as an ultra-trace level leaching
bath. Usually a 20% HCL or 20% HCI + 20% HNO
solution is adequate, though specific methods may re-
quire other leaching treatments. Acids of trace-metal
grade or better will be used to prepare the leaching
solution. The minimum volume of leaching solution suffi-
cient for the labware being treated will be prepared. The
leaching bath will be kept in a Class 100 hood during
preparation and during the leaching process.
5.2 Labware will be submerged into the ultra-trace
leaching bath, taking care to expose all interior and
exterior surfaces to the leaching solution. The bath will
be heated on a hotplate for at least two hours.
NOTE: Some plastics will discolor, soften or become
brittle with exposure to hot, oxidizing acids. Lab person-
nel must make sure labware is compatible with hot acid
leaching.
5.3 The ultra-trace leaching bath will be allowed to
cool and the labware will be retrieved and rinse with
copious volumes of deionized water.
5.4 The labware will be allowed to dry in the hood or
on the benchtop on Class 100 lab wipes. The volumetric
flasks will be stored with deionized water in them until
use, other labware will be stored dry in plastic bags.
6.0 Microwave Digestion Vessels
6.1 Microwave vessels will be labelled with unique
numbers and segregated for use with trace 01 ultra-trace
level analyses. Databases will be maintained to keep
records for the vessels, including sample types, acids
and oxidants, acid-extraction and any contamination of
analytes for each vessel.
6.2 For sensitive analyses, the high-pressure acid-
extraction will be performed and the extract analyzed for
analyte carry-over. (Specific acids, extraction times and
programs, analytes and analysis methods will vary ac-
cording to the needs of the project.)
An example of a general-use high-pressure extraction js
as follows:
• add 10-mL of 10% HNO3 to each detergent-cleaned
microwave vessel, cap and seal.
RTI/ACS/SOP-174-002
May 1994
Revision 0
• microwave for a total 30 minutes at 100% power
{alternating 3 minutes at 100% power, 2 minutes at
0% power.)
• cool and vent the vessel.
• dilute the extract 1-to-2 with deionized water.
• analyze for the analyte of interest by GFAA, FAA,
HGAF, ICP, etc.
For GFAA analyses, a signal of <0.002 absorbance
units will be acceptable for the extracts and the vessel
will be considered suitable for use. Suitability thresholds
will be established for analyses as needed.
6.3 The extract solutions will be neutralized and
discarded. The microwave vessels will be rinsed copi-
ously with deionized water and dried in a Class 100
environment (benchtop or hood.) If not used immedi-
ately, the microwave vessels will be capped and stored
in sealed plastic bags until needed.
6.4 Vessels that do not meet the suitability criterion
must be re-extracted.
7.0 Contamination
If any Low Level Operations labware is inadvertently
exposed to high levels of metals of interest (e.g. from
highly contaminated samples, or high metals in a sample
matrix) the labware will be removed from Low Level
applications and replaced, or decontaminated by non-
routine methods until its suitability for Low Level applica-
tions is verified.
8.0 Cleaning Expendable Supplies
Accurate analysis of trace and ultra-trace metals re-
quires minimizing all contamination sources, including
disposable and expendable lab supplies. Pasteur pi-
pets, pipettortips, disposable microbeakers, plastic weigh
boats and other supplies with which samples or re-
agents will come into contact will be rinsed with deion-
ized water and (if necessary) dried in Class 100 air.
For sensitive Low Level methods requiring non-routine
cleaning of labware, these expendables will be soaked
in or rinsed with dilute acid (approximately 10% HNO3or
HCI) before the deionized water rinse.
B-5
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Appendix C
RTI/ACS/SOP-174-005
Revision 0
Standard Operating Procedures for Monitoring and Maintaining
Cleanliness of the ACS Inorganic Clean Lab Facility
Research Triangle Institute
Analytical and Chemical Sciences
Post Office Box 12194
Research Triangle Park, NC 27709
August 1994
-------
ACS/SOP-174-005
August 1994
Revision 0
Standard Operating Procedures for Monitoring and
Maintaining Cleanliness of the ACS Inorganic Clean Lab Facility
1.0 Introduction
Cleanrooms are designed to control and limit the air-
borne particles in the working environment. The level of
cleanliness is specified by the maximum allowable par-
ticles per cubic foot of air as determined by statistical
methods (Table 1). The class designation is taken from
the maximum allowable number of particles, 0.5 jam or
larger, per cubic foot. This document concerns the Class
100 and Class 10,000 clean labs located in room 193 in
the Dreyfus building. The floor diagram of the facility is
given in Figure 1. This Clean Lab Facility is specially
designed to eliminate the contamination problems in
trace and ultra trace metal analysis.
The purpose of this SOP is to provide guidelines to all
laboratory personnel for proper maintenance of the facil-
ity to preserve its cleanliness over time.
2.0 Initial Testing and Certification of
the Clean Lab Facility
The certification of the Clean Lab Facility was performed
by an independent company (Contamination Control
Technologies, Inc.) certified by the National Environ-
mental Balancing Bureau. A copy of the certified docu-
ment is attached (Appendix I). The certification proce-
dure involved the measurement of particle counts that
would reflect the performance of the air handling system
of the Clean Lab Facility. Particle counts will be moni-
tored periodically to ensure that the initial standards are
maintained.
Table 1. Airborne Participate Cleanliness Classes
Class Name"
Class Limits"
0.5 tim (particles/ft3)
1
10
100
1,000
10,000
100.000
1.00
10.0
100
1,000
10,000
100,000
Concentration limits for intermediate classes can be calculated,
approximately, from the following equation:
Parttoles/m3 = Nc(0.5/d)"
Where "N" is the numerical designation of the class based on
English (U.S. customary) units, and "d" is the particle size in jim.
Class limits designate specific concentrations (particles per unit
volume) of airborne particles with sizes equal to and larger than
0.5 |im diameter.
3.0 Monitoring the Cleanliness of the
Clean Lab Facility
3.1 More Frequently Monitored
Parameters
The parameters that indicate the proper operation of the
air handling system will be monitored on a weekly basis.
A proper air handling system will ensure that no flow of
air occurs from a less clean area to a more clean area,
for example, the flow of air should not proceed from the
anteroom to the Class 100 room or from the instrument
room to the anteroom. It also helps in rapid exhaust of
any particulate matter that is created by the movement
of people, operation of equipment, etc., so that they do
not contribute to sample contamination.
3.1.1 The air pressure of each room in the Clean Lab
Facility will be monitored on a weekly basis by lab
personnel to ensure that the following gradient exists.
From most positive pressure to least positive pressure
the order should be:
Class 100 > Anteroom > Class 10,000 > Service Room
= Office Room.
In the event the measured gradient is different from the
above gradient, the laboratory manager will contact
John Berkley or the person in-charge at RTI Heating,
Ventilation and Air Conditioning.
3.1.2 The temperature in the Clean Lab Facility will be
monitored by lab personnel continuously through an
electronic monitoring system installed in the lab. Data
will be printed out on a daily basis. In the event the
temperature makes the facility uncomfortable to the
working personnel, HVAC will be contacted by the labo-
ratory manager for corrective actions (refer to section
3.1.1 for a contact person).
3.2 Less Frequently Monitored
Parameters
These include physical and chemical properties that are
measured less frequently.
3.2.1 Particle measurements will be made in both
Class 100 and Class 10,000 areas once every three
months and will 'be compared to those obtained at the
initial certification. The laboratory manager is respon-
sible for making necessary arrangements to have the
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29'-
44'
|^J Class 10,000
Plenum wall
ACS/SOP-174-005
August 1994
Revision 0
a. Gas Cylinders
b. D. I. Water
c. Refrigerator
d. Freezer
e. Clean Room Supplies
f. PE5100ZL
g. 4'HEPAHood
h. Bench
i. Sink
j. ICP
k. 81 HEPA Hood
I. Non-HEPA 4' Exhausting Hood
m- 6' Acid Baths
n. Desk
o. Questron V6 AVS
Figure 1. ACS Inorganic Class 100/10,000 Clean Lab Facility.
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particle measurements made as specified. The mea-
surements must be made at locations where an open
sample would be located, and will include, but not
Ifmited to, inside the 8' hood and on top of the 11' bench
in the Class 100 room.
3.2.2 All of the parameters mentioned above (under
sections 3.1 and 3.2.1) are good parameters for moni-
toring the air handling efficiency of the Clean Lab Facil-
ity. However, they do not provide direct information on
contamination levels of various metals of interest within
the laboratory. Lead (Pb), arsenic (As) and cadmium
(Cd) are chosen as the contamination monitoring agents.
These three elements will be measured in laboratory
blanks and controls on a monthly basis to establish the
cleanliness of the Clean Lab Facility with respect to
trace metals. Suitable sample locations will be chosen
and two samples will be taken from each location along
with blanks. One sample will be tested for As by hydride
generation atomic fluorescence spectrometry and the
other will be tested for Pb and Cd by GFAAS. Sampling
will be done by placing four clean Teflon beakers, two
open (samples) and two covered (blanks) at each sam-
pling location. The sampling locations and acceptable
limits are given in Tables 2 and 3.
Table 2. Sampling Locations
Sampling Locations
No. Samples
Class 100 Room
11'Bench
8'Hood
Class 10,000 Room
4'Hood
On top of the GFAA Autosampler
2
2
4.0 Maintenance of the Clean Lab
Facility
All routine maintenance performed will be recorded in
clean lab maintenance log books. All periodic mainte-
nance schedules are prepared as checklists and are
given in Appendix II.
4.1 All exposed bench surfaces and hood surfaces
in the Class 100 room will be wiped with a lint free, static
free wipe wetted with deionized water on a daily basis
prior to any laboratory activities. (Appendix II, RTI/ACS/
174-005/94-01.)
4.2 The floor in the Class 100 room will be moped
with tap water on the first working day of every week. A
suitable metal free detergent may be used when neces-
sary. (Appendix II, RTI/ACS/174-005/94-01.)
4.3 The cleaning procedure described in section 4.1
will be carried out in the Class 10,000 area as well, but
with the exception that it will be performed every 4-5
weeks. (Appendix II, RTI/ACS/174-005/94-02.)
4.4 The cleaning procedure described in section 4.2
will be carried out in the Class 10,000 area as well, but
with the exception that it will be performed only once
each month. (Appendix II, RTI/ACS/174-005/94-02.)
4.5 Sticky mats that are kept at entry doors of Class
100 and 10,000 areas must be replaced when neces-
sary. Proper maintenance of these sticky mats will mini-
mize the entry of dirt into the clean area from chemist's
feet. (Appendix II, RTI/ACS/174-005/94-03.)
4.6 All fume hoods will be inspected by the custo-
dian for their proper operation regularly. The static pres-
sure through the filter(s) in the fume hood will be moni-
tored once a week and recorded in the log book (Appen-
dix II, RTI/ACS/174-00/94-04, 05, 06.)
4.7 The entrance and exit procedures and the trans-
fer of items (samples, reagents, labware, apparatus,
etc.) from one area to another also play an important
role in maintaining the cleanliness of the Cleanroom
Facility. These issues will be addressed by separate
documents.
Table 3. Acceptable Contaminant Limits
Limit"
Element (ng/cmz/24 h)
Pb
As
Cd
1.0
0.1
0.1
Concentrations are given In mg of analyte per cm2 of exposed
area per 24 hours of sampling.
5.0 Corrective Actions
If any of the measured physical or chemical parameters
indicate any sign of degradation of the cleanliness of the
Clean Lab Facility it should be reported to the laboratory
manager immediately. Once any measured physical or
chemical parameters exceed the recommended level,
necessary steps will be taken to ascertain the source
and appropriate corrective actions will be taken to rees-
tablish the cleanliness of the Clean Lab Facility.
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Appendix!
Airborne Particle Count
Laser Particle Counter Method
Purpose:
This test is performed to measure the airborne panicu-
late levels within the Class 100 and 10,000 Cleanrooms,
the Anteroom, the two HEPA filtered workstations and
the one exhaust hood and to identify potential problem
areas.
Instrumentation:
Laser Particle counter with Built-in Recorder
Manufacturer: Particle Measuring Systems, Inc.
Model: LPC-525A
Calibration: November 15, 1993
SN: 12005-1287-14
Procedure:
Sampling duration for each count is one minute, with a
total sample volume of 1.0 cubic foot. Sampling height is
approximately 46 inches above the floor. Three counts
are taken to each sampling location. Counts are re-
corded for alt particles greater than or equal to 0.5
micrometers and for all particles greater than or equal to
5.0 micrometers. A 95% upper confidence levels (UCL)
is calculated for each room for counts greater than or
equal to 0.5 micrometers and for all particles greater
than or equal to 5.0 micrometers as described in Federal
Standard 209E.
Results:
The rooms were tested in an "as built" condition, i.e.,
only testing personnel were present during the particle
count sampling.
The Class 100 Cleanroom meets requirements for an
English (U.S. customary) Class 100 (at 0.5 um and 5.0
um) or a SI (metric) class M3.5 (at 0.5 um and 5.0 urn)
classification under "as built" conditions.
The Class 10,000 Cleanroom meets requirements for an
English (U.S. Customary) Class 10,000 (at 0.5 urn and
5.0 um) or a SI (metric) Class M5.5 (at 0.5 urn and 5.0
um) classification under "as built" conditions.
Particle count locations, particle counts, and the statisti-
cal analyses of the counts are provided on the following
pages.
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Appendix It
RT1/ACS/174-005/94-01
Routine Maintenance of the Clean Lab
1. Wiping of all surfaces (bench tops and hood areas) with a wetted (with deionized water) cleanroom wipe must be done at the
beginning of each working day..
2. Mopping of the clean lab floor with tap water must be done on the first working day of every week.
Please sign and put check marks where appropriate after performing duties.
DATE
NAME
WIPE SURFACES MOP THE FLOOR
INITIAL
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Appendix II
RTI/ACS/174-005/94-01
Routine Maintenance of the Instrument Room
1. Wiping of all surfaces (bench tops and hood areas) with a wetted (with deionized water) wipe must be done on the first working day of
every week.
2. Mopping of the clean lab floor with tap water must be done once every 5 weeks.
Please sign and put check marks where appropriate after performing duties.
DATE
NAME
WIPE SURFACES
MOP THE FLOOR
INITIAL
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Appendix II
RTI/ACS/174-005/94-03
Routine Maintenance of the Clean Lab Facility
Sticky Mat Replacement
ACS/SOP-174-005
August 1994
Revision 0
Please record the date when a fresh sticky mat is exposed.
Location: Inst. Room
Location: Anteroom
Date
Initial/Comments
Date
Initial/Comments
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Appendix II
RTI/ACS/174-005/94-04
Routine Maintenance Procedure for Hoods in the Cleanroom Facility
Monitoring of hoods must be done at the beginning of each week.
Please notify R. Fernando (Room 181, Ext. 6730) in the event of any problem or malfunction of the hood operation.
Period: MM/YY - MM/YY
Hood Tvoe: 8' hood in the Clean Lab.
Pate
Pressure (i.w.g.)
Indicator Light
Normal Caution/Alert
Comments
Initial
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Appendix II
RTVACS/174-005/94-05
Routine Maintenance Procedure for Hoods in the Cleanroom Facility
Monitoring of hoods must be done at the beginning of each week.
Please notify R. Fernando (Room 181, Ext. 6730) in the event of any problem or malfunction of the hood operation.
Hood Tvoe: 4' hood in the Clean Lab. Period: MM/YY - MM//YY
Date
Pressure (i.w.g.)
Indicator Light
Normal Caution/Alert
Comments
Initial
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Appendix II
RTI/ACS/174-005/94-06
Routine Maintenance Procedure for Hoods in the Cleanroom Facility
Monitoring of hoop's must be done at the beginning of each week.
Please notify R. Fernando (Room 181, Ext. 6730) in the event of any problem or malfunction of the hood operation.
Hood Type: 4' hood in the Instrument Lab.
Date
Pressure (i.w.g.)
Indicator Light
Normal Caution/Alert
Comments
Initial
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Appendix D
RTI/ACS/SOP-174-007
Revision 0
Standard Operating
Reagents in the
Procedures for Purification of
ACS Inorganic Clean Lab Facility for
Trace/Ultratrace Metal Analysis
Research Triangle Institute
Analytical and Chemical Sciences
Post Office Box 12194
Research Triangle Park, IMC 27709
September 1994
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ACS/SOP-174-007
September 1994
Revision 0
RTI/ACS-SOP-174-007
Standard Operating Procedures for
Purification of Reagents in the ACS Inorganic Clean Lab Facility for
Trace/Ultratrace Metal Analysis
1.0 Scope and Application
1.1 Analysis of trace element concentrations at and
below 1 mg/mL (ppb) is often required. Contamination is
the major problem in getting high quality data at these
levels. Contamination in trace and ultratrace analysis is
understood as the increase in the measured amount or
concentration of a component, resulting from its intro-
duction at various stages of the analytical procedure
from sources other than the sample. Several indepen-
dent sources, besides the sample itself, add to the final
signal for a particular analyte. These are the laboratory
atmosphere and working areas, tools and apparatus
associated with sampling, sample preparation, labora-
tory ware, and reagents. Compared to other sources of
contamination, contribution from reagents can often be
measured quantitatively and can also be reduced effec-
tively. Purification of reagents as a means to prevent
contamination from reagents is discussed here along
with the purification procedures.
1.2 A list of most commonly used reagents in trace/
ultratrace metal analysis is given in Table 1. These
include water, acids, bases, buffers, oxidants, reduc-
tants and other reagent chemicals. The demand for high
purity reagents has been met by commercial chemical
manufacturers in some cases. However for many oth-
ers, specific purification procedures need to be devel-
oped to adequately control the contamination level.
2.0 Summary
The procedures given here are used to purify the re-
agents that are not available from the manufacturer at
the required purity. These procedures are tested in the
cleanroom environment for their performance in produc-
ing high purity reagents and will be modified as neces-
sary.
3.0 Interferences
3,1 Extreme care must be taken to avoid the con-
tamination during the purification process and also not
to recontaminate the purified reagents. The primary
sources of contamination are particulates in air, impuri-
Table 1. Reagents that are Commonly Used in Trace/Ultratrace
Metal Analysis by Graphite Furnace Atomic Absorption
Spectrometry and Hydride Generation Atomic
Fluorescence Spectrometry
Acids and Bases
Analysis
Reagents for As and Hg
Nitric acid
Sulfuric acid
Hydrochloric acid
Perchloric acid
Sodium hydroxide
Matrix Modifiers for GFAA
Potassium iodide
Potassium bromide
Sodium borohydride
Potassium bromate
Tin (II) chloride
General Purpose Reagents
Palladium
Magnesium nitrate
Ammonium dihydrogen phosphate
Diammonium hydrogen phosphate
Hydroxylamine hydrochloride
Nickel nitrate
Triton X-100
Ascorbic acid
Hydrogen peroxide
ties in reagents that are used to purify the component of
interest, trace elements from containers, sample han-
dling by analysts, etc. With the use of Class 100
cleanroom environment and by following proper proce-
dures for sample handling, it may be possible to keep
the contaminants at a level below the instrument detec-
tion limit.
3.2 Storage vessels, storage conditions (tempera-
ture, humidity, etc.), and storage locations must be
chosen appropriately to maintain the quality of the puri-
fied reagents.
4.0 Safety
Since the toxicity of the chemicals used in these proce-
dures is not clearly defined, they should be treated as
potential health hazards at all times and personal expo-
sure to these chemicals should be minimized.
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5.0 Equipment
5.1 Graphite Furnace Atomic
Absorption Spectrometer (GFAA)
Perkin-Elmer 5100 atomic absorption spectrometer with
a transversely heated graphite furnace and Zeeman
background correction is used to analyze the purified
reagents to established their purity. The procedures for
the operation of PE 5100ZL are given in ACS/SOP-171-
005.
5.2 Hydride Generation A tomic
Fluorescence Spectrometer (HGAF)
PS Analytical hydride generation atomic fluorescence
spectrometer (HGAF) is used to analyze the purified
reagents for As, and thus establish their purity. The
procedure for the operation of the HGAF is given in
ACS/SOP-174-008.
6.0 Storage
Once purified, the purity of the reagent is critically de-
pendent upon the storage conditions and the duration of
storage, container material, storage location and tem-
perature are particularly important.
Purified reagents are stored in thoroughly cleaned ves-
sels (Sections 7.1.2.1 and 7.2.2.1) and prior to transfer
of the purified reagent, the vessel must be rinsed with
the reagent being stored. Storage vessels must be
clearly labelled with the reagent name, concentration,
date prepared and other relevant information. If the
reagent requires specific storage conditions, it will be
stored accordingly, otherwise it is placed inside an air-
tight polyethylene bag and stored in metal free cabinet in
the Clean Lab.
7.0 Reagent Purification Procedures
7.1 Ammonium Dihydrogen Phosphate
(Ammonium Phosphate, Monobasic)
-NH4H2P04
7.1.1 Reagents
All reagents must be of recognized analytical grade,
unless specified otherwise.
• Ammonium dihydrogen phosphate -1.667 g
• Ammonium hydroxide - about 100 ml
• Chelex-100 chelating ion exchange resin
• Ethylenediaminetetraacetic acid diammonium salt
(EDTA) - 0.556 g.
7.1.2 Equipments
7.1.2.1 Laboratory Ware
All labware (glassware/plasticware) must be thoroughly
cleaned by washing with detergent and water followed
by rinsing with deionized water. Then they are acid
leached in warm 20% nitric acid for at least 12 hrs,
drained, thoroughly rinsed with deionized water and are
dried under HEPA filtered air.
The storage bottles that will be used to store the purified
reagent must be cleaned by warming in 50% HNO3 for
12-24 hrs followed by rinsing with deionized water sev-
eral times.
• 50 ml Teflon or polypropylene beakers (2)
• 600 mL Teflon or polypropylene beaker
• 500 mL Teflon or polypropylene storage bottle
• Plastic column
7.1.2.2 Apparatus
• Analytical balance
7.1.3 Procedure
• Weigh out 1.667 g of ammonium dihydrogen phos-
phate into a 600 mL Teflon or polypropylene beaker
and add sufficient deionized water to dissolve the
material.
• Add 5.0 mL of 14 M ammonium hydroxide solution
to the beaker containing ammonium dihydrogen
phosphate and dilute to 500 mL with deionized
water.
» Prepare a column of chelex-100 and pass a solution
of ammonium hydroxide through the column to con-
vert the chelating resin to ammonium form.
• Pass the ammonium dihydrogen phosphate solution
through the column at a flow rate of 0.5 mL/min.
• Add 0.556 g of ethylenediaminetetraacetic acid
diammonium salt to the eluent and.dilute to 500 mL
with deionized water. Store the solution in an acid
leached storage bottle. The concentration of
NH4H2PO4 in the final solution is 0.03 M.
7.2 Magnesium Nitrate - Mg(NO^2
7.2.1 Reagents
All reagents must be of recognized analytical grade,
unless specified otherwise.
• Magnesium nitrate - 4 g
• Ammonium pyrrolidinedithiocarbamate (APDC) 0.25g
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• Nitric acid - several drops
• Methyl isobutyl ketone (MIBK) - 750 mL
7.2.2 Equipment
7,2,2.1 Laboratory Ware
All labware (glassware/plasticware) must be thoroughly
cleaned by washing with detergent and water followed
by rinsing with deionized water. Then they are acid
leached in warm 20% nitric acid for at least 12 hrs,
drained, rinsed thoroughly with deionized water and are
dried under HEPA filtered air.
The storage bottles that will be used to store the purified
reagent must be cleaned by warming in 50% HNO3 for
12-24 hrs followed by rinsing with deionized water sev-
eral times.
* 1 L Teflon or polypropylene volumetric flask
• 1 L Teflon or polypropylene beaker
• 2 L Teflon or polypropylene separatory funnel
* 100 mL graduated cylinder (glass or plastic)
• 1 L Teflon or polypropylene storage bottle
7.2.2.2 Apparatus
• Analytical balance
* pH meter
7,2.3 Procedure
* Dissolve 4 g of Mg(NO3)2 in 1 L of deionized water in
1 L volumetric flask.
• Transfer the solution to 1 L beaker and add 0.25 g of
APDC.
• Adjust the pH of the solution to 5.8 with HNO3.
• Transfer one half of the solution to a 1 L separatory
funnel and add 125 mL of MIBK.
• Extract the metal complexes with MIBK.
• Repeat the extraction 2 more times with a fresh
portion of MIBK each time.
• Repeat the extraction procedure for the second half
of the solution.
• Combine the aqueous phases and boil for 20 mins.
to remove any MIBK.
• Store in a clean Teflon or polypropylene storage
bottle.
8.0 References
1. Purification of Matrix Modifiers Used in GFAA,
Fishman, et al., J. Assoc. of Anal. Chem., 69, 706
(1986).
2. Purification of Analytical Reagents, Mitchell.J.W.
Talanta, 29, 993(1982).
3. Purified Reagents for Trace Metal Analysis, Moody,
J.R. and Beary, E.S., Talanta, 29,1003 (1982).
4. Purification of Analytical Reagents and Other Liquids
by Low Temperature Vacuum Sublimation, Mitchell,
J.W., Anal. Chem., 50, 194 (1978).
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Appendix E
Standard Operating Procedures for Mercury Analysis in
Water, Sediment and Tissue
Southeast Environmental Research Program
Florida International University
University Park
Miami, Florida 33199
July 27,1993
Revised:
September 13,1993
September 21,1993
November 4, 1993
-------
Mercury
Collection and Storage
Field Collection of Samples
Water samples are collected in Teflon (FEP) bottles.
Collection is done while wearing vinyl gloves (Polyethyl-
ene shoulder length PPE glove, OakTech). Samples are
then placed in zip-lock polyethylene bags and placed in
an additional bag in a plastic ice chest/cooler. In the
laboratory 1 ml of trace metal grade HCI is added per
100 ml of sample. These additions are done in a "Hg-
free1' room (described below). Solid (soil) samples are
collected in polyethylene specimen cups (Elkay non-
sterile wide-mouth specimen cups with screw caps) -
128 ml volume) and placed in polyethylene zip-lock
bags. All field samples are kept in a cooler until they are
returned to the laboratory. These coolers are used ex-
clusively for low level Hg samples.
Sample Storage
A number of storage tests have been done in this
laboratory to determine the type(s) of bottles which are
best suited for long term storage of low level samples.
Acidified water samples (1 ml 12 N HCI/100 ml sample)
may be stored in Teflon (Nalgene FEP) bottles in either
a refrigerator or outside the mercury-free room with no
effects on the mercury concentration of the sample.
Refrigerated storage and FEP bottles are recommended.
Polyethylene (Nalgene LDPE) bottles stored in the re-
frigerator show minor accumulation and cannot be used
for storage of low level samples unless refrigerated. The
plastic itself leaches mercury into the solution. This
effect is facilitated by acid washing. Mercury accumu-
lated in acid washed bottles to approximately 70-80 ppt
with accumulation in non-washed bottles at 15 ppt in 30
days. For higher level samples the storage vessel type
is not as critical.
Analytical Methods
Cleanroom
All glassware, acids, reagents, etc. Are stored in this
room. It is equipped with a bank of laminar flow hoods, a
separate water supply and gold-charcoal filter appara-
tus, refrigeration unit, oven, analytical balance, and a
"flypaper" covered floor which is changed when needed.
Contamination is checked weekly by monitoring acidi-
fied (1% HCI) replicate water samples which are stored
open in and outside the laminar flow hoods. Data on this
quality control monitoring is stored in both as lotus file on
computer and as hard copy in a data notebook. If
significant levels of Hg are found (>20ppt) the source of
this Hg will be located and if necessary, gold and
charcoal filters will be reconditioned.
All new glassware and teflon used in analysis has been
previously monitored for Hg content. In addition, acid-
water blanks are run in glassware each analysis. No
glassware is reused. All pipettes, reusable teflon bea-
kers, and constantly used lab items are rinsed 0.5 N
HCI, followed by Dl water (described in sample prepara-
tion section) directly prior to use.
Teflon bottles which have been previously used for
samples are rinsed twice with Dl water and filled with a
1% HCI. After filling, 1 ml of mixed brominating agent
(see reagent section for preparation) for every 50 ml of
acid water is added and the bottle is shaken. This
mixture remains in the bottles until it is used. The bottle
is then rinsed 3X in Dl water.
Sample Preparation
Water
Water samples may be analyzed for inorganic Hg and
for total Hg. Inorganic samples are acidified as men-
tioned above. They are then ready for analysis.
Water Sample Preparation for Total Hg
Analysis
Reagents/equipment list
Mercury-free Water:
Tap water is first filtered through a Culligan system
consisting of activated charcoal and two mixed bed ion
exchange cartridges and then piped to the mercury-free
room. It is then passed through a Barnstead Mega-ohm
B Pure System. This system is fitted with two filters
(Thermolyne: cplloid/organic-D0835, and ultrapure-
D0809) in line with a 0.22 micron pleated particle filter.
Mercury levels are not detectable by both our methods
and independent lab analysis (<0.1 ppt). The only water
available for use in the Hg laboratory is this Hg-free
water. All reference to Dl water in this "SOP" should be
assumed to be Hg-free water as described above.
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Bromination Reagents:
0.1 M Potassium Bromate:
Heat 8.385 g KBrO3 overnight in a glass scintillation vial
(Kimble 74511) at 250°C +/- 20°C in a furnace to re-
move mercury. After cooling dissolve the potassium
bromate in 500 ml of deionized water and store in a
glass bottle. Prepare weekly.
0.2 M Potassium Bromide:
Heat 11.9 g KBr overnight in a glass scintillation vial at
250°C +/- 20°C to remove mercury. After cooling dis-
solve the potassium bromide in 500 ml of deionized
water and store in a glass bottle. Prepare weekly.
0.05 M Potassium Bromide (KBr): -0.1 M Potassium
Bromate (KBrOJ
Mix equal volumes (100 ml) of bromate and bromide in a
borosilicate 150 ml screw cap glass bottle. Prepare
daily.
Hydroxylamine Hydrochloride:
Dissolve 2.4 g of NH2OH.HCI in 20 ml of deionized water
in a glass scintillation vial. Prepare weekly.
Stannous Chloride:
To 20 grams of Stannous Chloride (SnCI2) add 165 ml of
12 N HCI. Bring to 1000 ml using Hg-free deionized
water in the borosilicate glass bottle used to hold the
reagent during analysis. NOTE: no special treatment is
needed for low level Hg ( <10ppt). The instrument
analysis compensates for background levels in the re-
agent.
12 N HCI:
Concentrated HCI (12 N HCI) is dispensed via a pipette
or poured into a graduate cylinder either which has been
previously acid washed and rinsed three times with Dl
water. 5% acid (0.6 N HCI) is contained in a Nalgene
"low-boy" bottle and dispensed through a tube con-
nected to the spigot.
Nitric Acid:
16 N Nitric acid is dispensed through a repipette which
has been previously washed and rinsed three times with
Dl water.
Water sample digestion:
125 ml of acidified sample (1 ml 12 N HCI/100 ml
sample) is brominated (2.5 ml KBrCyKBrO mixed re-
agent as described above). After that time, 500 pi of
hydroxylamine hydrochloride is added to the solution to
inhibit further reaction. Samples are permitted to settle
for 30 min. before analysis.
Soils and Sediments (Carbonate and
Clastic)
Sediment samples are homogenized and slurried using
a glass bottled blender. 120 ml of sediment is slurried
with 50 ml of distilled water. This mixture is then blended
for 3 min. Using a syringe, 5 ml of slurry are removed,
placed in a polyethylene specimen cup and diluted by
adding 45 ml of 5% HCI. (The HCI acts to neutralize
carbonate sediments prior to digestion. It is necessary to
prevent a violent reaction when the vessel is subse-
quently sealed and autoclaved.) After mixing, 1 ml of this
solution is transferred to a 10 ml ampule. Nitric acid (2
ml cone. HNO3) is added to the ampule and it is left to
stand for 20 min. The ampule is then sealed and auto-
claved for 1 h at 150°C. Ampules must be cooled,
completely before further processing.
To process ampule contents pipette 0.5 ml of the di-
gested solution into a 20 ml polyethylene scintillation vial
(Kimble #58504) containing 19.5 ml of 0.12 N HCI
solution. ; ''.,'.;
Plant and Animal Tissue
Animal and plant tissue are treated similarly. Initial dilu-
tions of homogenate vary with the type of tissue. In
addition, the HCI step used to neutralize carbonates is
not used for tissue analysis. Additional details of the
tissue processing procedure will be added in future
drafts.
Digestion of Standard Reference Material
and Spiked Material
A series of method tests have been run both with spiked
tissue, spiked sediment, and NBS or NIST certified
samples to test for digestion efficiency. NBS oyster
tissue (566a), NIST sediment nominal 50 jig/g (8407)
and 60 jig/g (8406) were used in these tests. In addition,
a sample of certified material is digested and run with
each analysis of tissue or sediment. Digestion efficiency
is 100% in all cases.
Sample Storage After Preparation
Samples may be stored, ampulated for an indefinite time
until ready to be analyzed.
Standard Preparation
All preparation and storage of secondary standards is
done in a Hg-free room. Primary working standard is
prepared and stored outside the Hg-free room. The
prima'ry stock standard is made by addition of 1 ml of
NBS certified primary Hg standard (SEPEX PLHG4-2X)
1000 |ig/ml) to 1000 ml of filtered deionized water plus 5
ml of trace metal, grade HCI. This standard Is good for
two days. Secondary standards are made in 500 ml
nalgene FEP bottles. Concentrated HCI (5 ml) is added
to 495 ml of filtered deionized water in a Polyethylene
E-3
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11.
bottle. When acids and brominating agents are added,
the external laboratory hood is turned on creating a
negative pressure in the area where acid addition is
being done. The primary stock is then brought into the
Hg-free room and 5 ni-25 \i\ (depending on final concen-
tration, i.e. 10ppt-50ppt) is added to the bottles contain-
ing the water-acid mixture. Make up daily. All pipettes,
micropipettes and pipette tips are calibrated before use
using analytical balance. The temperature of the
cleanroom is assumed to approximate 25°C and Dl
water is used as a standard to weigh.
Analytical Instrumental Technique
Cold Vapor Atomic Fluorescent Spectrometry (AFS) is
the method used for Hg determination. The system used
Is a PSA Merlin plus supplied by Questron Corporation,
Princeton, New Jersey 08543. This system contains an
autosampler, vapor generator, fluorescence monitor and
an IBM compatible computer system as the electronic
data interface. In the AFS method, SnCI2 is mixed with
the liquid sample fed by the auto sampler, which then
enters a gas liquid fritted separator (no maintenance has
been necessary for this separator to date). The sample
flows through peristaltic pump tubing (changed once
every two weeks). As mercury enters the vapor phase it
is stripped and carried along a gas stream (Argon- Zero
grade) to the detector. The detection limit is better than
0.3 pg/1 (SD X 100/mean) of the baseline variation.
Baseline noise translates into variation of between 0.087-
0.185ppt.
The only modifications we have made in the apparatus
are:
1. Modification of the tubing in the hydride generator
pump to prevent it from being pulled through the
pump (a small rubber-banding is wrapped on
approximately 5mm of the tubing at the left side of
hydride generator pumping system).
2. Modification of the computer output using lotus to
permit more accurate representation of the peak
height data.
3. Placing the entire sampling system in a vented
hooded area.
The procedure for running the instrument is as
follows:
1. Tighten the three tubes in the hydride generator by 13
clamping.
2. Tighten the peristaltic pump (pumps wash water
and waste water).
3. Turn on the wash water to the system.
4. Turn on the computer.
5. Turn on the gas to the system. The argon (Zero
grade) is flowed through two gas purifiers (charcoal
and gold) before reaching the instrument.
6. Turn on the line stabilizer/conditioner.
7. Check to make sure no tubes are crimped, and that
flow is smooth in all tubes before proceeding.
8. Allow the system to run on water for 15 minutes.
NOTE: Depending on the age of the HG lamp, there
may be considerable drift encountered in the first
two hours of running. If drift problems arise, permit
the lamp to stabilize before running samples.
9. Check gas flow at gas controller. The Argon is more
precisely controlled through mass flow controllers.
The optimum level for the carrier gas is 125 cc/min,
with a shield gas level optimized at 140 cc/min. This
flow controller is installed in front of the instrument,
therefore, flow controllers on the instrument itself
are open to full capacity.
10. After 15 min. switch the instrument on the SnCfe.
12.
NOTE: the sensitivity dial on the instrument is run at
highest sensitivity for water but may be lowered for
running of sediment, soil and tissue samples. This
method is adequate for samples of the range we
have run to date (final concentration 0.5-1 OOppt).
When the instrument is ready, zero the fluorescence
detector and run D.I. acidified water to check
baseline response of the instrument and guard
against unexplained contamination from reagent
preparation. When peak height of D.I. is 0.0-0.3 a
standard may be run. Initially, one high standard is
run to test for consistency of standard preparation
and machine function. The range of standards will
reflect the concentration of samples to analyze. Our
water standards range from 0 ppt, to 50ppt while
our soil sample standards will include a 10Oppt
standard. Eight standards (four concentrations,
two replicates) are run for each standard curve.
Standards run for low level samples are 10,20, and
30 ppt. Standards, blanks, and high level samples
(generally fish, sediments and soil) may be run in
plastic scintillation vials. Water samples for total Hg
are digested and run in glass scintillation vials.
Digested acidified D.I. water samples are analyzed
along with the samples as "digestion" blanks. This
number is subtracted from sample values.
After running the standards, run four water blanks,
and then run samples. All samples are run in
replicate. One run of fifty samples plus standards
takes 2 hours and uses 10ml of SnCIs per sample.
A new standard curve is run when the SnCIa is
replaced. In addition to running afull setof standards
at the beginning of the analysis for each bottle of
stannous chloride, replicate 50 ppt and zero ppt are
E-4
-------
run after every 12 samples. NOTE: when running in
125 ml teflon bottles, remove the auto sampler and
run three analyses from each bottle. After 5 bottles
analyze the high (SOppt) standard and a D.I. Blank
twice each. Atthe end of the run analyze 3 replicates
of the SOppt standard.
To turn off the instrument:
1. If youareusingtheresultsdirectlyfromthecompany
supplied computer program, make sure you have
printed and/or saved results. This program does
not reliably transfer files to ascii or Lotus although
it has functions for these tasks.
2. Replace the SnCb solution with water and flush the
instrument for 5 min.
3. Turn off the wash water.
4. Turn off the gas.
5. Run the pump until no more liquid is present in the
pump tubing.
6. Turn off the line stabilizer and the computer.
7. Release tubing in the Hydride generator.
8. Check the waste water container and empty if
necessary.
The procedure for running the computer is as fol-
lows:
1. Choose library, press select to choose methods
and to see methods stored.
The method we use is:
2. If you wish to analyze samples or run blanks choose
"analyze", "batch". Specify batch (sample) size.
The computer will ask you whether the sample tray
is in position and if you wish to change the sample
tray. If you respond no twice, the instrument will
then align the sample tray to the run you have
3.
specified. It will then be necessary to return to the
analyze menu and check batch etc. You may then
respond "YES" to the questions about tray position.
This response will initiate the run. The instrument
will analyze 50 samples. If you have more than 50
samples you must re-select analyze and you can
choose a reference number which reflects the
actual number of samples you are running.
If you wish to run standards, select "calibration".
You may then choose to select a new curve, modify
the old curve, etc.
Data Transfer
We do not find the program supplied with the AFS
adequate for our needs. Specifically, there is not a direct
method to correct for drift. In addition, the curve fitting
function is not adequate for low level samples (< 1 ppt).
We therefore after printing results from the machine,
save the data as an asci file and transfer it into a Lotus
spread sheet.
Data Handling
Calculation Standard Curve
Currently the NBS standard available for Hg is 1000|ig/
ml. With appropriate dilutions, standards can be made
repeatable and reliably to 10ppt using standard dilution
and pipetting procedures. The intercept location in the
standard calculation becomes critical to proper calcula-
tion of concentrations. We have found the most reliable
and reproducible results are generated by running a set
of standards, and then assuming the standard regres-
sion is acceptable, ignoring all but the SOppt standard
and forcing the intercept through zero to generate the
regression for linear estimate. This method is used for
water samples and has also been found to be compa-
rable to traditional estimating procedures used in sedi-
ment, soil and tissue analysis. All data is printed as hard
copy and stored on computer disks. We maintain a
back-up copy for each disk.
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Appendix F
MSL/M-027-01
Standard Operating Procedures for
Total Mercury in Aqueous Samples by Cold Vapor Atomic Fluorescence
Battelle Pacific Northwest Laboratories
Marine Sciences Laboratory
May 28, 1993
-------
SOP MSL/M/027-01
Total Mercury in Aqueous Samples by Cold Vapor Atomic Fluorescence
1.0 Scope and Application
This Is a peer-reviewed, published procedure and is
applicable to the determination of total mercury in aque-
ous samples. All samples must be subjected to a BrCI
UV oxidation step prior to analysis.
2.0 Summary of Method
The method is a cold vapor atomic fluorescence tech-
nique, based upon the emission of 254 nm radiation by
excited Hg° atoms in an inert gas stream. Mercuric ions
in the oxidized samples are reduced to Hg° with SnCI2,
and then purged onto gold-coated sand traps as a
means of preconcentration and interference removal.
Mercury vapor is thermally desorbed to a second "ana-
lytical" gold trap, and from that into the fluorescence cell.
Fluorescence (peak area) is proportional to the quantity
of mercury collected, which is quantified using a stan-
dard curve as a function of the quantity of sample
purged.
Typical detection limit for the method is 0.200 ng/L as
Hg.
3.0 Sample Collection, Preservation and
Handling
3.1 Samples should be collected in teflon bottles.
All teflon bottles should be washed and then boiled in
concentrated HNO3 for 24 hours. Vessels are thoroughly
rinsed in tap water shown to contain negligible concen-
trations of mercury, then filled with 1% HCI in tap water
and heated to 45±5° for 24 hours. This water is poured
off and the vessels are refilled with 1% HCI and stored
full until use. Just prior to use, vessels are emptied and
dried in a clean drying oven at 75±5°C. Vessels to be
shipped are packed in clean polyethylene bags. Samples
can be collected in ashed or acid-cleaned borosilicate or
quartz glass bottles with teflon lids. Under no circum-
stances should polyethylene, polypropylene, or vinyl
containers be used.
3.2 Sample bottles should be rinsed once with
sample water, and then filled. Samples should be pre-
served with 0.5% by volume of high purity HCI and
refrigerated at 4±2°C, but not frozen.
4.0 Definitions
4.1 Atomic Fluorescence - detection based on fluo-
rescent emission from excited atoms of a particular
element at a characteristic wavelength. The amount of
fluorescence, quantified by integration of peak area, is
proportional to the concentration of the atom of interest.
4.2 Acid-cleaned - cleaned in nitric acid of the high-
est concentration and temperature which can be with-
stood by the item being cleaned. Glass and teflon con-
tainers are boiled in concentrated nitric acid for 48
hours.
5.0 Potential Interferences
5.1 Due to the strong oxidation step, followed by
dual gold amalgamation, there are no observed interfer-
ences with the method. The potential exists for destruc-
tion of the gold traps (and consequently low values) if
free halogens are purged onto them or if they are
overheated (<500°C). When these instructions are fol-
lowed, neither of these problems is likely to occur.
5.2 Water vapor may be collected on the gold traps,
and be released on to the fluorescence cell where it
condenses, giving a false peak due to scattering of the
excitation radiation. This can be avoided by pre-drying
the gold trap and by discarding traps which tend to
absorb large quantities of water vapor.
5.3 As always with atomic fluorescence, the fluores-
cence intensity is strongly dependent upon the inertness
of the carrier gas. The dual amalgamation technique
eliminates quenching due to trace gases, but it still
remains the analyst's responsibility to ensure high purity
inert carrier gas and a leak-free analytical system,
6.0 Responsible Staff
6.1 Technician - sample prep, digestions
6.2 Analyst - analysis, calculations
6.3 QA/QC staff - data checking
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SOP MSL/M/027-01
7.0 Apparatus and Reagents
7.1 Apparatus
7.1.1 Cold Vapor Atomic Fluorescence Spectro-
photometer (CVAF). The components of this detector
include a four-watt low pressure mercury vapor lamp, a
far UV quartz flow-through fluorescence cell (12 mm x
12 mm x 45 mm) with a 10 mm path length, and a UV-
visible photomultiplier, sensitive to <230 nm isolated
from outside light with a 254 nm interference filter. The
carrier gas flow is controlled using a flowmeter with
needle valve capable of keeping a constant flow of 25
ml/min.
7.1.2 Flowmeter/needle Valves. Flowmeter capable
of controlling and measuring gas flow to the purge
vessel at 200-500 ml/min.
7.1.3 Teflon Fittings. Connections between compo-
nents and columns are made using 6.4 mm O.D. Teflon
FEP tubing, and teflon friction-fit or threaded tubing
connectors. Connections between components requir-
ing mobility are made with 3.2 mm O.D. Teflon tubing
due to its greater flexibility.
7.1.4 Acid Fume Pretrap. A 10 cm x 0.9 cm diameter
teflon tube containing 2-3g of reagent grade, non-indi-
cating 8-14 mesh soda lime, packed between silanized
glass wool plugs. This trap is purged of Hg by placing it
on the output of a clean cold vapor generator, filled with
1% HCI, and purging overnight with N2 at 100 ml/min.
7.1.5 Cold Vapor Generator. A 250 ml or 125 ml
florence flask with standard taper 24/40 neck, fitted with
a purging stopper having a coarse glass fit which ex-
tends to within 0.2 cm of the flask bottom.
7.1.6 Gold-coated Sand Column. Made from 10 cm
lengths of 6.5 mm O.D. X 4 mm I.D. Quartz tubing, with
a coarse quartz frit or crimp 2.0 cm from one end. The
tube is filled with 3.4 cm of gold-coated ashed (800°C for
6 hours) quartz sand (60/80 mesh). The end is then
plugged with quartz wool. Gold is applied to the sand as
a coating several atoms thick using as ion exchange
gilding apparatus. The columns are heated to 450-
500°C with a coil consisting of 24 gauge nichrome wire
at a potential of 10 VAC.
7.1.7 Oxidation Bottles. Acid-cleaned, 135 ml teflon
bottles and caps.
7.1.8 Pipetters. All plastic pneumatic fixed and vari-
able volume pipetters in the range of 10 p.! to 5 ml
(calibrated).
7.1.9 Recorder. Multi-range chart-recorder/integrator
with 0.1 - 5.0 mV input and variable speed.
7.2 Reagents
7.2.1 Water. Deep well tap water which has been
determined to be very low (<0.02 ng/l) in mercury. The
water is continuously monitored for mercury.
7,2.2 10% Stannous Chloride (SnCI2). A solution
containing 200 grams of SnCI2«2H20 and 100 ml of
concentrated HCI, brought to a final volume of 1 liter with
mercury-free water. This solution is purged overnight
with mercury-free nitrogen at 500 ml/min to remove all
traces of mercury. Store tightly capped and in the dark.
7.2.3 5% Bromine Monochloride (BrCI). 27 g of KBr
are added to a 2-liter bottle of concentrated HCI (<5 ng/
Hg). A clean magnetic stir bar (teflon coated) is placed in
the bottle and the solution is stirred for one hour in a
fume hood. Next 38 g of pre-analyzed, low Hg KBr03 are
slowly added to the acid as stirring continues. CAU-
TION: This process should always be carried out in a
fume hood. The fumes from this reagent are very corro-
sive and a strong irritant. When all of the KBrO has been
added, the solution should have gone from yellow to red
to orange. Loosely cap the bottle and allow to stir
another hour before tightening the lid.
7.2.4 30% Hydroxylamine Hydrochloride. Dissolve
1 of NH2OH'HCI in low Hg water to make 500 ml. This
solution may be purified by adding 1.0 ml SnCI2 reagent
and purging overnight at 500 ml/min with Hg-free N2.
7.2.5 Stock Mercury Standard. A commercially avail-
able 1000 mg Hg/l atomic absorption standard is used.
7.2.6 Secondary Standard Solution. 0.100 ml is
diluted with Hg-free water containing 5 ml BrCI, to a final
volume of 100 ml in a teflon bottle. This solution contains
1000 ng/liter and should be restandardized or replaced
annually.
7.2.7 Working Standard Solution. Dilute 1.0 ml of
the 2° mercury standard to 100 ml with Hg-free water
containing 1% (by volume) bromine monochloride, using
a 100 ml class A volumetric flask. This solution has
a[Hg] 10.0 ng/ml and should be replaced semi-annually.
7.2.8 Nitrogen. Grad 4.5 nitrogen which has been
further purified by theremoval of Hg using an in-line gold
coated sand trap.
7.2.9 Helium or Argon. Grade 5.0 inert gas which
has been further purified bythe removal of Hg using an
in-line gold coated sand trap.
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SOP MSL/M/027-01
8.0 Procedure
8.1 Sample Preparation
8.1.1 Place a 50±5 ml aliquot of the acidified sample
in a 125 ml teflon bottle, either by a pipette or by weight
difference, recording the volume on a mercury datasheet.
Add 0.5 ml of BrCI reagent to each sample, and place
the bottle in the UV oxidation booth for 2 hours.
8,2 Analysis
8.2.1 Add 75 ml of low Hg water to each bubbler,
followed by 1 ml of cone. HCI and 0.500 ml of SnCI2
solution. The bubbler is purged with N2 at 350 ml/min for
10 minutes, then a gold-coated sand trap is attached to
the soda lime pretrap and purged for 20 minutes. This
value is the bubbler blank. Just prior to analysis, add
0.250 ml NH2OH-HCI, or half volume of NH OH-HCI as
BrCI, to the samples and let react for 5 minutes. The
yellow color should disappear as the NH OH-HCI reacts
with the BrCI. To analyze samples, attach a fresh gold-
coated sand trap to the soda lime trap, then add 30±5ml
of digestate to each bubbler (by weight difference).
Record the sample number and the weight of the sample
bottle before and after adding an aliquot to the bubbler
on a mercury datasheet. Weigh the samples on a bal-
ance which is gently swirled, and the sample is purged
for 20 minutes. New samples may be sequentially added
to the bubblers, up to a maximum of 3 consecutive
samples. After 3 samples, rinse the bubblers with clean
low Hg water and the above sequence is repeated.
8.2.2 To analyze the mercury contained on the gold
trap, the gold trap is placed inside a coil of nichrome wire
and then inserted in-line with a mercury analyzer incom-
ing Hg-free He and the second (analytical) gold-coated
sand trap. The He flow rate should be about 30 ml/min.
The system is purged for 2 minutes to dry off any
condensed water vapor. 10 VAC is applied to the nicrome
coil on the working gold-coated trap for 4 minutes,
thermally desorbing the Hg as Hg° which is then re-
sorbed by the analytical gold-coated sand column. The
voltage to the working gold-coated sand trap is turned
off, and a cooling stream of compressed air is directed
towards the trap. 10 VAC is applied to the analytical
gold-coated sand trap, and the integrator is activated.
The analytical traps heated for 4.0 minutes, or 1 minute
beyond the point where the mercury peak returns to
baseline.
8.2.3 Following the integration of the mercury peak,
the voltage to the analytical trap is turned off. A stream
of cooling compressed air is directed towards the ana-
lytical trap. The sample gold-coated sand trap is re-
moved from the gas stream, and the teflon end plugs are
replaced until it is needed to collect another sample. The
next sample gold-coated sand trap is placed inside the
nichrome wire coil, placed in-line with the mercury ana-
lyzer incoming Hg-free, and the procedure is repeated.
Under no circumstances should a sample gold-coated
sand trap be heated while the analytical column is still
warm, or analyte may be lost by passing through the
analytical column.
8.2.4 Peaks generated using this technique should be
very sharp and almost symmetrical. The peak comes off
at approximately 1 minute and has a half-height width of
about 4 seconds. Broad or asymmetrical peaks are
indicative of an analytical problem possibly including:
low gas flow, water vapor on the column, or an analytical
column damaged by chemical fumes or overheating. If
the analytical column has been damaged, replace the
column and the tubing downstream due to the possibility
of gold migration on the downstream surfaces.
8.2.5 Cold vapor atomic fluorescence for mercury is
linear over at least five orders of magnitude (Bloom and
Fitzgerald, 1988). This method is virtually interference
free, so the method of standard additions is not routinely
applied. To run standards, an aliquot of working stan-
dard solution in the range of 1 ng Hg is. pipetted into a
purged bubbler containing 0.5 ml of SnCI2 solution, and
analyzed as one would a sample.
8.2.6 Gold-coated sand traps should be kept track of
by unique identifiers, so that any trap producing poor
results can be quickly recognized and discarded. Occa-
sionally due to inadvertent contact with halogen fumes,
bubbler solution, organic fumes, or overheating, a sample
gold-coated sand trap may become damaged
irreproducible results. Suspect gold-coated sand traps
should be checked with at least two consecutive stan-
dard runs before continued use.
8.2.7 The major cause of analytical problems with this
method is from using the soda lime pretraps too long.
These traps should be purged overnight as described
and then used for only one day's analytical work. Longer
use risks irreproducibility, as the traps may begin retard-
ing the flow of Hg°. Also, as the traps become very wet
there is a risk of NaOH-saturated water drops coming off
onto the gold-coated sand traps.
9.0 Quality Control
9.1 All quality control data should be maintained
and available for each reference or inspection.
9.2 Quality assurance data must be composed of a
minimum of 2 blanks and 2 certified reference materials.
Such checks should be run at least twice (1 standard
and 1 blank) per day, or every 20 samples, whichever is
first.
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SOP MSL/M/027-01
9.3 Samples containing high analyte concentrations
may be run either following dilution, or on a separate run
at a lower instrumental sensitivity.
9.4 Duplicate or triplicate analyses (depending upon
client preference) should be run once every 10 samples
or once per batch, whichever comes first.
9.5 No certified materials exist for Hg in water near
ambient levels. NRCC or NBS certified standard mate-
rials for mercury in tissues and sediments should be
analyzed as a QA/QC measure in lieu of these.
9.6 Procedural spike recoveries should be run once
per 10 samples or once per batch, whichever comes
first, in the absence of a suitable certified standard
tissue, or at the request of the client.
10.0 Calculations
10.1 Calculations may be made using either a best fit
linear regression analysis of the standards and blanks or
by using the average response factor method.
10.1.1 Average Response Factor Method:
Ave Response Factor (RF) =
^T ((std peak area - blk area)/[Hg]ng
[Hg]ng/L
#std
sam pk area
RF*V
- blk
(Where std peak area is the standard peak area, blk
area is the average blank area, [Hg] is the Hg concentra-
tion in ng/L, sam pk area is the sample peak area, v is
the sample aliquot analyzed in liters, and RF is the
average response factor in area/ng and blank in ng/L)
10.1.2 Linear Regression Method:
[Hg]ng/L
sam pk area
slope * v
(Where slope is the slope of the standard regression line
in area/ng. For an explanation of the other variables,
refer to the average response method above.)
The final sample concentrations must be corrected for
dilution by reagents as follows:
[Hg]ng/L = [Hg]ng/L(analytical)*
(total volume/initial volume)
. where: total volume = volume of sample + reagents
initial volume = volume of sample only
10.2 Method Detection Limit (MDL):
The MDL is calculated by recording the results of a
standard analyzed seven times, whose concentration is
within 10 times of the actual method detection limit.
MDL[Hg]ng = SD*t(0jOI(1Xll.1))
(Where SD is the standard deviation of the [Hgjng of the
standards analyzed multiplied by the student t value for
99% one tailed confidence interval with n-1 degrees of
freedom.)
Detection Limit [Hg]ng/L = MDL/sam vol (L)
(Where MDL is the method detection limit [Hg]ng/L and
sam vol is the volume of the sample analyzed in liters.)
11.0 References
Bloom, N.S. 1983. "Determination of Silver in Marine
Sediments by Zeeman-Corrected Graphite Furnace
Atomic Absorption Spectroscopy." Atomic
Spectroscopy. 47204.
Bloom, N.S. 1989. "Determination of Picogram Levels of
Methylmercury by Aqueous Phase Ethylation,
Followed by Cryogenic Gahy with Cold Vapor Atomic
Fluorescence Detection." Can. J. Fish Aa. Sci.
7:1131.
Bloom, N.S., and E.A. Crecelius. 1983. "Determination
Of Mercury in Seawater at Subnanogram per Liter
Levels." Mar. Chem. 14:49.
Bloom, N.S., and E.A. Crecelius. 1987. "Distribution of
Silver, Mercury, Lead, Copper and Cadmium inound
Sediments." Mar. Chem. 21:377.
Bloom, N.S., and W.F. Fitzgerald. 1988. "Determination
of Volatile Mercury Species at the Picogram Level
by Low-Temperature Gas Chromatography with
Cold-Vapor Atomic Fluorescence Detection." Anal.
Chem. Acta. 208:151.
Fitzgerald, W.F., and G.A. Gill. 1979. "Sub-Nanogram
^Determination of Mercury by Two-Stage Gold
Amalgamation and Gas Phase Detection Applied to
Atmospheric Analysis." Anal. Chem. 15:1714.
F-5
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SOP MSL/M/027-01
Attachment 1 (page 1 of 1)
MERCURY DATA SHEET
DATE: CALIBRATION:
TYPE: ER TOTAL METHYL CALIBRATION TYPE:
ANALYST: (SEE OVER)
RLE NAME: PROJECT NAME(S):
Roport*
Sample ID
Bubbler
Sample Volume
Units
[Hg]( )
Comments
F-6
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Appendix G
Method 1631
Mercury in Water by Oxidation, Purge and Trap, and CVAFS
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Engineering and Analysis Division (4303)
401 M Street SW
Washington, DC 20460
DRAFT
January 19§6
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Method 1631
Mercury in Water by Oxidation, Purge and Trap, and CVAFS
Acknowledgments
This method was prepared under the direction of William
A. Telliard of the Engineering and Analysis Division
(EAD) within the U.S. Environmental Agency's (EPA's)
Office of Science and Technology (OST). The method
was prepared by Nicholas Bloom of Frontier GeoSciences
under EPA Contract 68-C3-0337 with the DynGorp Envi-
ronmental Programs Division. Additional assistance in
preparing the method was provided by Interface, Inc.
Disclaimer
This sampling method has been reviewed and approved
for publication by the Analytical Methods Staff within the
Engineering and Analysis Division of the U.S. Environ-
mental Protection Agency. Mention of trade names or
commercial products does not constitute endorsement
or recommendation for use.
Questions concerning this method or its application
should be addressed to:
W.A. Telliard
Engineering and Analysis Division (4303)
U.S. Environmental Protection Agency
401 M Street SW
Washington, DC 20460
Phone: 202/260-7134
Fax: 202/260-7185
Introduction
This analytical method supports water quality monitoring
programs authorized under the Clean Water Act. Sec-
tion 304(a) of the Clean Water Act requires EPA to
publish water quality criteria that reflect the latest scien-
tific knowledge concerning the physical fate (e.g., con-
centration and dispersal) of pollutants, the effects of
pollutants on ecological and human health, and the
effect of pollutants on biological community diversity,
productivity, and stability.
Section 303 of the Clean Water Act requires states to
set a water quality standard for each body of water
within its boundaries. A state water quality standard
consists of a designated use or uses of a waterbody or a
segment of a waterbody, the water quality criteria that
are necessary to protect the designated use or uses,
and an antidegradation policy. These water quality stan-
dards serve two purposes: (1) they establish the water
quality goals for a specific waterbody, and (2) they are
the basis for establishing water quality-based treatment
controls and strategies beyond the technology-based
controls required by Sections 301 (b) and 306 of the
Clean Water Act.
In defining water quality standards, the state may use
narrative criteria, numeric criteria, or both. However, the
1987 amendments to the Clean Water Act required
states to adopt numeric criteria for toxic pollutants (des-
ignated in Section 307(a) of the Act) based on EPA
Section 304(a) criteria or other scientific data, when the
discharge or presence of those toxic pollutants could
reasonably be expected to interfere with designated
uses.
In some cases, these water quality criteria are as much
as 280 times lower than those achievable using existing
EPA methods and required to support technology-based
permits. Therefore, EPA developed new sampling and
analysis methods to specifically address state needs for
measuring toxic metals at water quality criteria levels,
when such measurements are necessary to protect
designated uses in state water quality standards. The
latest criteria published by EPA are those listed in the
National Toxics Rule (58 FR 60848) and the Stay of
Federal Water Quality Criteria for Metals (60 FR 22228).
These rules include water quality criteria for 13 metals,
and it is these criteria on which the new sampling and
analysis methods are based. Method 1631 was specifi-
cally developed to provide reliable measurements of
mercury at EPA WQC levels.
In developing these methods, EPA found that one of the
greatest difficulties in measuring pollutants at these
levels was precluding sample contamination during col-
lection, transport, and analysis. The degree of difficulty,
however, is highly dependent on the metal and site-
specific conditions. This analytical method, therefore, is
designed to provide the level of protection necessary to
preclude contamination in nearly all situations. It is also
designed to provide the procedures necessary to pro-
duce reliable results at the lowest possible water quality
criteria published by EPA. In recognition of the variety of
situations to which this method may be applied, and in
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Method 1631
recognition of continuing technological advances, the
method is performance based. Alternative procedures
may be used as long as those procedures are demon-
strated to yield reliable results.
Requests for additional copies of this method should be
directed to:
U.S. EPA NCEPI
11209 Kenwood Road
Cincinnati, OH 45242
1-800-490-9198
Note: This method is intended to be performance based, and the laboratory
is permitted to omit any step or modify any procedure provided that all
performance requirements set forth in this method are met. The laboratory is
not allowed to omit any quality control analyses. The terms "must," "may,"
and "should" are included throughout this method and are intended to
illustrate the importance of the procedures in producing verifiable data at
water quality criteria levels, The term "must" is used to indicate the steps
that are critical to production of reliable results; however, these procedures
may be modified or omitted if the laboratory can demonstrate data quality is
not affected.
1.0 Scope and Application
1.1 This method is for determination of total mer-
cury (Hg) in filtered and unfiltered water by oxidation,
purge and trap, desorption, and cold-vapor atomic fluo-
rescence detection. This method is for use in EPA's data
gathering and monitoring programs associated with the
Glean Water Act, the Resource Conservation and Re-
covery Act, the Comprehensive Environmental Re-
sponse, Compensation and Liability Act, and the Safe
Drinking Water Act. The method is based oh a contrac-
tor-developed method (Reference 1) and on peer-re-
viewed, published procedures for the determination of
mercury and in aqueous samples, ranging from sea
water to sewage effluent (References 2-5).
1.2 This method is accompanied by Method 1669:
Sampling Ambient Water for Determination of Trace
Metals at EPA Water Quality Criteria Levels (Sampling
Method). The Sampling Method is necessary to ensure
that contamination will not compromise trace metals
determinations during the sampling process.
1.3 This method is designed for measurement of
total Hg in the range of 0.2-100 ng/L and may be
extended to higher levels by selection of a smaller
sample size. This method is not intended for determina-
tion of metals at concentrations normally found in treated
and untreated discharges from industrial facilities. Exist-
ing regulations (40 CFR Parts 400-500) typically limit
concentrations in industrial, discharges to the part-per-
billipn (ppb) range, whereas ambient mercury concen-
trations are normally in the low part-per-trillion (ppt)
range.
1.4 The ease of contaminating ambient water
samples with the metal(s) of interest and interfering
substances cannot be overemphasized. This method
includes suggestions for improvements in facilities and
analytical techniques that should maximize the ability of
the laboratory to make reliable trace metals determina-
tions and minimize contamination. Section 4.0 gives
these suggestions.
1.5 The detection limits and quantitation levels in
this method are usually dependent on the level of back-
ground elements rather than instrumental limitations.
The method detection limit (MDL; 40 CFR 136, Appen-
dix B) for total mercury has been estimated to be 0.05
ng/L when no background elements or interferences are
present. The minimum level (ML) has been established
as 0.2 ng/L.
1.6 Clean and ultraclean—The terms "clean" and
"ultraclean" have been applied to the techniques needed
to reduce or eliminate contamination in trace metals
determinations. These terms are not used in this method
because they lack an exact definition. However, the
information provided in this method is consistent with
the summary guidance on clean and ultraclean tech-
niques.
1.7 This method follows the EPA Environmental
Methods Management Council's "Format for Method
Documentation."
1.8 This method is "performance based." The ana-
lyst is permitted to modify the method to overcome
interferences or lower the cost of measurements if all
performance criteria are met. Section 9.1.2 gives the
requirements for establishing method equivalency.
1.9 Any modification of this method, beyond those
expressly permitted, shall be considered a major modifi-
cation subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.10 This method should be used only by analysts
who are experienced in the use of CVAF analysis and
who are thoroughly trained in the sample handling and
instrumental techniques described in this method. Each
analyst who uses this method must demonstrate the
ability to generate acceptable results using the proce-
dure in Section 9.2.
1.11 This method is accompanied by a data verifica-
tion and validation guidance document, Guidance on the
Documentation and Evaluation of Trace Metals Data
Collected for CWA Compliance Monitoring. Data users
should state data quality objectives (DQOs) required for
a project before this method is used.
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Method 1631
2.0 Summary of Method
2.1 A 100-2000 mL sample is collected directly into
specially cleaned, pretested, fluoropolymer bottle(s) us-
ing sample handling techniques specially designed for
collection of mercury at trace levels (Reference 6).
2.2 The sample is either field or laboratory-pre-
served by the addition of 5 mL of pretested 12 N HCI per
liter of sample, depending on the time between sample
collection and arrival at the laboratory.
2,3 Sample preparation and analysis are conducted
at laboratory facilities specially designed for determina-
tion of mercury at 0.2-100 ng/L concentration. At this
facility, a 100-mL sample aliquot is placed in a specially
designed purge vessel.
2.4 Before analysis, 0.2 N BrCI solution is added to
oxidize all Hg compounds to Hg(ll).
2.5 After oxidation, the sample is sequentially
prereduced with NH2OH-HCI to destroy the free halo-
gens, and then reduced with SnCI, to convert Hg(ll) to
volatile Hg(0).
2.6 The Hg(0) is separated from solution by purging
with nitrogen onto a gold-coated sand trap.
2.7 The trapped Hg is thermally desorbed from the
gold trap into an inert gas stream that carries the re-
leased Hg(0) into the cell of a cold-vapor atomic fluores-
cence spectrometer (CVAFS) for detection.
2.8 Quality is ensured through calibration and test-
ing of the oxidation, purging, and detection systems.
3.0 Definitions
3.1 Total mercury as defined by this method means
all BrCI-oxidizable mercury forms and species found in
aqueous solution. This includes but is not limited to
Hg(H), Hg(0), strongly organocomplexed Hg(ll) com-
pounds, adsorbed paniculate Hg, and several tested
covalently bound organomercurials (i.e., CH.HgCI,
(CHjLHfl, and C6H HgOOCCH3). The recovery of Hg
bound within microbial cells may require the additional
step of UV photo-oxidation. In this context, "total" mer-
cury refers to the forms and species of mercury, not to
the total recoverable or dissolved fraction normally de-
termined in an unfiltered or filtered sample, respectively.
In this method, the total recoverable fraction will be
referred to as "total recoverable" or "unfiltered."
3.2 Definitions of other terms used in this method
are given in the glossary at the end of the method.
4.0 Contamination and Interferences
4.1 Preventing ambient water samples from becom-
ing contaminated during the sampling and analytical
process constitutes one of the greatest difficulties en-
countered in trace metals determinations. Over the last
two decades, marine chemists have come to recognize
that much of the historical data on the concentrations of
dissolved trace metals in seawater are erroneously high
because the concentrations reflect contamination from
sampling and analysis rather than ambient levels. There-
fore, it is imperative that extreme care be taken to avoid
contamination when collecting and analyzing ambient
water samples for trace metals.
4.2 Samples may become contaminated by numer-
ous routes. Potential sources of trace metals contamina-
tion during sampling include: metallic or metal-contain-
ing labware (e.g., talc gloves that contain high levels of
zinc), containers, sampling equipment, reagents, and
reagent water; improperly cleaned and stored equip-
ment, labware, and reagents; and atmospheric inputs
such as dirt and dust. Even human contact can be a
source of trace metals contamination. For example, it
has been demonstrated that dental work (e.g., mercury
amalgam fillings) in the mouths of laboratory personnel
can contaminate samples that are directly exposed to
exhalation (Reference 5).
4.3 Contamination Control
4.3.1 . Philosophy—The philosophy behind contami-
nation control is to ensure that any object or substance
that contacts the sample is metal free and free from any
material that may contain metals.
4.3.1.1 The integrity of the results produced cannot be
compromised by contamination of samples. This method
and the Sampling Method give requirements and sug-
gestions for control of sample contamination.
4.3.1.2 Substances in a sample cannot be allowed to
contaminate the laboratory work area or instrumentation
used for trace metals measurements. This method gives
requirements and suggestions for protecting the labora-
tory.
4.3.1.3 Although contamination control is essential, per-
sonnel health and safety remain the highest priority. The
Sampling Method and Section 5 of this method give
requirements and suggestions for personnel safety.
4.3.2 Avoiding contamination—The best way to con-
trol contamination is to completely avoid exposure of the
sample to contamination in the first place. Avoiding
exposure means performing operations in an area known
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Method 1631
to be free from contamination. Two of the most impor-
tant factors in avoiding/reducing sample contamination
are (1) an awareness of potential sources of contamina-
tion and (2) strict attention to work being done. There-
fore, it is imperative that the procedures described in this
method be carried out by well-trained, experienced per-
sonnel.
4.3.3 Use a clean environment—The ideal environ-
ment for processing samples is a class 100 cleanroom
(Section 6.1.1). If a cleanroom is not available, all sample
preparation should be performed in a class 100 clean
bench or a nonmetal glove box fed by mercury- and
particle-free air or nitrogen. Digestions should be per-
formed in a nonmetal fume hood situated, ideally, in the
cleanroom.
4.3.4 Minimize exposure—The Apparatus that will
contact samples, blanks, or standard solutions should
be opened or exposed only in a cleanroom, clean bench,
or glove box so that exposure to an uncontrolled atmo-
sphere is minimized. When not being used, the Appara-
tus should be covered with clean plastic wrap, stored in
the clean bench or in a plastic box or glove box, or
bagged in clean zip-type bags. Minimizing the time
between cleaning and use will also minimize contamina-
tion.
4.3.5 Clean work surfaces—Before a given batch of
samples is processed, all work surfaces in the hood,
clean bench, or glove box in which the samples will be
processed should be cleaned by wiping with a lint-free
cloth or wipe soaked with reagent water.
4.3.6 Wear gloves—Sampling personnel must wear
clean, nontalc gloves (Section 6.9.7) during all opera-
tions involving handling of the Apparatus, samples, and
blanks. Only clean gloves may touch the Apparatus. If
another object or substance is touched, the glove(s)
must be changed before again handling the Apparatus.
If it is even suspected that gloves have become con-
taminated, work must be halted, the contaminated gloves
removed, and a new pair of clean gloves put on. Wear-
ing multiple layers of clean gloves will allow the old pair
to be quickly stripped with minimal disruption to the work
activity.
4.3.7 Use metal-free Apparatus—All Apparatus used
for determination of metals at ambient water quality
criteria levels must be nonmetalNc, free of material that
may contain metals, or both.
4.3.7.1 Construction materials—Only fluoropolymer
containers should be used for samples that will be
analyzed for mercury because mercury vapors can dif-
fuse in or out of the other materials, resulting either in
contamination or low-biased results. All materials, re-
gardless of construction, that will directly or indirectly
contact the sample must be cleaned using the proce-
dures in this method and must be known to be clean and
metal free before proceeding.
4.3.7.2 Serialization—It is recommended that serial
numbers be indelibly marked or etched on each piece of
Apparatus so that contamination can be traced, and
logbooks should be maintained to track the sample from
the container through the labware to injection into the
instrument. It may be useful to dedicate separate sets of
labware to different sample types; e.g., receiving waters
vs. effluents. However, the Apparatus used for process-
ing blanks and standards must be mixed with the Appa-
ratus used to process samples so that contamination of
all labware can be detected.
4.3.7.3 The laboratory or cleaning facility is responsible
for cleaning the Apparatus used by the sampling team. If
there are any indications that the Apparatus is not clean
when received by the sampling team (e.g., ripped stor-
age bags), an assessment of the likelihood of contami-
nation must be made. Sampling must not proceed if it is
possible that the Apparatus is contaminated. If the Ap-
paratus is contaminated, it must be returned to the
laboratory or cleaning facility for proper cleaning before
any sampling activity resumes.
4.3.8 Avoid sources of contamination—Avoid contami-
nation by being aware of potential sources and routes of
contamination.
4.3.8.1 Contamination by carryover—Contamination
may occur when a sample containing low concentra-
tions of metals is processed immediately after a sample
containing relatively high concentrations of these met-
als. To reduce carryover, the sample introduction sys-
tem may be rinsed between samples with dilute acid and
reagent water. When an unusually concentrated sample
is encountered, it is followed by analysis of a laboratory
blank to check for carryover. Samples known or sus-
pected to contain the lowest concentration of metals
should be analyzed first followed by samples containing
higher levels.
4.3.8.2 Contamination by samples—Significant labo-
ratory or instrument contamination may result when
untreated effluents, in-process waters, landfill leachates,
and other samples containing high concentrations of
inorganic substances are processed and analyzed. This
method is not intended for application to these samples,
and samples containing high concentrations should not
be permitted into the cleanroom and laboratory dedi-
cated for processing trace metals samples.
4.3.8.3 Contamination by indirect contact—Appara-
tus that may not directly come in contact with the samples
may still be a source of contamination. For example,
clean tubing placed in a dirty plastic bag may pick up
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Method 1631
contamination from the bag and subsequently transfer
the contamination to the sample. Therefore, it is impera-
tive that every piece of the Apparatus that is directly or
indirectly used in the collection, processing, and analy-
sis of ambient water samples be cleaned as specified in
Section 11.
4.3.8.4 Contamination by airborne paniculate matter—
Less obvious substances capable of contaminating
samples include airborne particles. Samples may be
contaminated by airborne dust, dirt, particles, or vapors
from unfiltered air supplies; nearby corroded or rusted
pipes, wires, or other fixtures; or metal containing paint.
Whenever possible, sample processing and analysis
should occur as far as possible from sources of airborne
contamination.
4.4 Interferences
4,4.1 Because all forms of Hg are oxidized in the BrCI
oxidation step, there are no observed interferences with
this method.
4.4.2 The potential exists for destruction of the gold
trap if it is exposed to free halogens or if the trap is
overheated (> 500°C).
4.4.3 Water vapor may collect in the gold trap and
subsequently condense in the fluorescence cell upon
desorption, giving a false peak due to scattering of the
excitation radiation. Condensation can be avoided by
predrying the gold trap, and by discarding those traps
that tend to absorb large quantities of water vapor.
4.4.4 The fluorescence intensity is susceptible to the
presence of foreign species in the carrier gas, which
may cause "quenching" of the excited Hg atoms. The
dual-trap technique in this method eliminates some
quenching due to impurities in the carrier gas, but it
remains the analyst's responsibility to ensure high-purity
inert carrier gas and a leak-free analytical train.
5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical
used in this method has not been precisely determined;
however, each compound should be treated as a poten-
tial health hazard. Exposure to these compounds should
be reduced to the lowest possible level.
5.5.1.1 Chronic mercury exposure may cause kidney
damage, muscle tremors, spasms, personality changes,
depression, irritability and nervousness. Organo-
mercurials may cause permanent brain damage. Be-
cause of the available toxicological and physical proper-
ties of the Hg, pure standards should be handled only by
highly trained personnel thoroughly familiar with han-
dling and cautionary procedures and the associated
risks.
5.5.1.2 It is recommended that the laboratory purchase
a dilute standard solution of the Hg in this method. If
primary solutions are prepared, they shall be prepared
in a hood, and a NIOSH/MESA-approved toxic gas
respirator shall be worn when high concentrations are
handled.
5.2 This method does not address all safety issues
associated with its use. The laboratory is responsible for
maintaining a current awareness file of OSHA regula-
tions for the safe handling of the chemicals specified in
this method. A reference file of material safety data
sheets (MSDSs) should also be made available to all
personnel involved in these analyses. It is also sug-
gested that the laboratory perform personal hygiene
monitoring of each analyst who uses this method and
that the results of this monitoring be made available to
the analyst. Additional information on laboratory safety
can be found in References 7-10. The references and
bibliography at the end of Reference 10 are particularly
comprehensive in dealing with the general subject of
laboratory safety.
5.3 Samples suspected to contain high concentra-
tions of Hg are handled using essentially the same
techniques employed in handling radioactive or infec-
tious materials. Well-ventilated, controlled access labo-
ratories are required. Assistance in evaluating the health
hazards of particular laboratory conditions may be ob-
tained from certain consulting laboratories and from
State Departments of Health or Labor, many of which
have an industrial health service. Each laboratory must
develop a strict safety program for handling Hg.
5.3.1 Facility—When samples known or suspected
of containing high concentrations of mercury are handled,
all operations (including removal of samples from sample
containers, weighing, transferring, and mixing) should
be performed in a glove box demonstrated to be leaktight
or in a fume hood demonstrated to have adequate
airflow. Gross losses to the laboratory ventilation system
must not be allowed. Handling of the dilute solutions
normally used in analytical and animal work presents no
inhalation hazards except in an accident.
5.3.2 Protective equipment—Disposable plastic
gloves, apron or lab coat, safety glasses or mask, and a
glove box or fume hood adequate for radioactive work
should be used. During analytical operations that may
give rise to aerosols or dusts, personnel should wear
respirators equipped with activated carbon filters.
5.3.3 Training—Workers must be trained in the proper
method of removing contaminated gloves and clothing
without contacting the exterior surfaces.
5.3.4 Personal hygiene—Hands and forearms should
be washed thoroughly after each manipulation and be-
fore breaks (coffee, lunch, and shift).
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Method 1631
5.3.5 Confinement—Isolated work areas posted with
signs, segregated glassware and tools, and plastic ab-
sorbent paper on bench tops will aid in confining con-
tamination.
5.3.6 Effluent vapors—The effluent from the CVAFS
should pass through either a column of activated char-
coal or a trap containing gold or sulfur to amalgamate or
react mercury vapors.
5.3.7 Waste handling—Good technique includes
minimizing contaminated waste. Plastic bag liners should
be used in waste cans. Janitors and other personnel
must be trained in the safe handling of waste.
5.3.8 Decontamination
5.3.8.1 Decontamination of personnel—Use any mild
soap with plenty of scrubbing action.
5.3.8.2 Glassware, tools, and surfaces—Sulfur pow-
der will react with mercury to produce mercuric sulfide,
thereby eliminating the possible volatilization of Hg.
Satisfactory cleaning may be accomplished by dusting a
surface lightly with sulfur powder, then washing with any
detergent and water.
5.3.9 Laundry—Clothing known to be contaminated
should be collected in plastic bags. Persons who convey
the bags and launder the clothing should be advised of
the hazard and trained in proper handling. If the laun-
derer knows of the potential problem, the clothing may
be put into a washer without contact. The washer should
be run through a cycle before being used again for other
clothing.
5.3.10 Wipe tests—A useful method of determining
cleanliness of work surfaces and tools is to wipe the
surface with a piece of filter paper. Extraction and analy-
sis by this method can achieve a limit of detection of less
than 1 ng per wipe. Less than 0.1 pg per wipe indicates
acceptable cleanliness; anything higher warrants further
cleaning. More than 10 jig on a wipe constitutes an
acute hazard and requires prompt cleaning before fur-
ther use of the equipment or work space, and indicates
that unacceptable work practices have been employed.
6.0 Apparatus and Materials
Disclaimer: The mention of trade names or commercial
products in this method is for llustrative purposes
only and does not constitute endorsement or
recommendation for use by the Environmental
Protection Agency. Equivalent performance may be
achievable using apparatus and materials other than
those suggested here. The laboratory is responsible
for demonstrating equivalent performance.
6.1 Sampling equipment
6.1.1 Sample collection bottles-Fluoropolymer, 125-
to 1000-mL, with fluoropolymer or fluoropolymer-lined
cap.
6.1.2 Cleaning—New bottles are cleaned by heating
to 65-75°C in 4 N HCI for at least 48 h. The bottles are
cooled, rinsed three times with reagent water, and filled
with reagent water containing 1 % HCI. These bottles are
capped and placed in a clean oven at 60-70°C over-
night. After cooling, they are rinsed three more times,
filled with reagent water plus 0.4% (v/v) HCI, and placed
in a mercury-free class 100 clean bench until dry. The
bottles are then tightly capped (with a wrench) and
double-bagged in new polyethylene zip-type bags until
needed. After the initial cleaning, bottles are cleaned as
above, except with only 6-12 h in the hot 4 N HCI step.
6.1.3 Filtration Apparatus
6.1.3.1 Filter—Gelman Supor 0.45-jim, 15-mm diam-
eter capsule filter (Gelman 12175, or equivalent)
6.1.3.2 Peristaltic pump—115-V a.c., 12-V d.c., internal
battery, variable-speed, single-head (Cole-Parmer, por-
table, "Masterflex US," Catalog No. H-07570-10 drive
with Quick Load pump head, Catalog No. H-07021-24,
or equivalent).
6.1.3.3 Tubing for.use with peristaltic pump—sty-
rene/ethylene/butylene/silicone (SEBS) resin, approx 3/
8-in i.d. by approximately 3 ft (Cole-Parmer size 18,
Catalog No. G-06464-18, or approximately 1/4-in i.d.,
Cole-Parmer size 17, Catalog No. G-06464-17, or equiva-
lent). Tubing is cleaned by soaking in 5-10% HCI solu-
tion for 8-24 h, rinsing with reagent water in a clean
bench in a cleanroom, and drying in the clean bench by
purging with metal-free air or nitrogen. After drying, the
tubing is double-bagged in clear polyethylene bags,
serialized with a unique number, and stored until use.
6.2 Equipment for bottle and glassware
cleaning
6.2.1 Vat, 100-200 L, high-density polyethylene
(HOPE), half filled with 4 N HCI in reagent water.
6.2.2 Panel immersion heater, 500-W, all-
fluoropolymer coated, 120 vac (Cole-Parmer H03053-
04, or equivalent)
NOTE: Safety note: Read instructions carefully!! The
heater will maintain steady state, without temperature
feedback control, of 60-75°C in a vat of the size
described. However, the equilibrium temperature
will be higher (up to boiling) in a smaller vat. Also,
the heater plate MUST be maintained in a vertical
position, completely submerged and away from the
vat walls to avoid melting the vat or burning out!
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Method 1631
6.2.3 Laboratory sink in class 100 clean area, with
high-flow reagent water (Section 7.1) for rinsing.
6.2.4 Clean bench, class 100, for drying rinsed bottles.
6.2.5 Oven, stainless steel, in class 100 clean area,
capable of maintaining ± 5°C in the 60-70°C tempera-
ture range.
6.3 Cold vapor atomic fluorescence spectrometer
(CVAFS): The CVAFS system used may either be pur-
chased from a supplier, or built in the laboratory from
commercially available components.
6.3.1 Commercially available: Tekran (Toronto, ON)
Model 2357 CVAFS, or BrooksRand (Seattle, WA) Model
3 CVAFS, or equivalent.
6.3.2 Custom-built CVAFS (Reference 11). Figure 1
shows the schematic diagram. The system consists of
the following:
6.3.2.1 Low-pressure 4-W mercury vapor lamp.
6.3.2.2 Far UV quartz flow-through fluorescence
cell—12 mm x 12 mm x 45 mm, with a 10-mm path
length (NSG cells or equivalent).
6.3.2.3 UV-visible photomultiplier (PMT)—sensitive
to < 230 nm. This PMT is isolated from outside light with
a 253.7-nm interference filter (Oriel Corp., Stanford, CT
or equivalent).
6.3.2.4 Photometer and PMT power supply (Oriel Corp.
or equivalent), to convert PMT output (nanoamp) to
millivolts
6.3.2.5 Black anodized aluminum optical block—
holds fluorescence cell, PMT, and light source at per-
pendicular angles, and provides collimation of incident
and fluorescent beams (Frontier Geosciences Inc., Se-
attle, WA or equivalent).
6.3.2.6 Flowmeter, with needle valve capable of repro-
ducibly keeping the carrier gas flow rate at 30 mL/min.
Helium
in
0-1000 volt DC
power supply
Current-to-
voltage
converter
110°C
Figure 1. Schematic Oiapor Atomic Fluorescence Spectrometer (CVAFS) Detector.
G-8
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Method 1631
6.3.2,7 Ultra high-purity argon (grade 5.0)
6.4 Equipment for Hg purging system—Figure
2a shows the schematic diagram for the purging system.
The system consists of the following:
6.4.1 Flow meter/needle valve—capable of control-
ling and measuring gas flow rate to the purge vessel at
350 (± 50) mL/min.
6.4.2 Fluoropolymer fittings—Connections between
components and columns are made using 6.4-mm o.d.
fluoropolymer tubing and fluoropolymer friction-fit or
threaded tubing connectors. Connections between com-
ponents requiring mobility are made with 3.2-mm o.d.
fluoropolymer tubing because of its greater flexibility.
6.4.3 Acid fume pretrap—10-cm long x 0.9-cm i.d.
fluoropolymer tube containing 2-3 g of reagent grade,
nonindicating, 8-14 mesh soda lime chunks, packed
between wads of silanized glass wool. This trap is
cleaned of Hg by placing on the output of a bubbler and
purging for 1 h with N2 at 350 mL/min.
6.4.4 Bubbler—200-mL borosilicate glass (15 cm high
x 5.0 cm diameter) with standard taper 24/40 neck, fitted
with a sparging stopper having a coarse glass frit that
extends to within 0.2 cm of the bubbler bottom.
6.5 Equipment for the Dual-trap Hg°
Preconcentrating System
6.5.1 Figure 2b shows the schematic for the dual-trap
amalgamation system (Reference 5).
He gas
Soda lime pre-trap Gold sample trap
J=L
He gas
©
Aqueous sample + SnCI2
Gas phase syringe injection port
Gold sample trap
Quartz detection
ce" \ Photomultiplier tube
l~ 1
II
Hg free
He gas
wfflflffiW(a
\4bf&F,J
WSSSSSSSSSSSSSSA
I
Nichrome coil ' / Nichrome coil
Gold analysis trap C^\
Signal to
: recorder/
integrator
\
Hg lamp
I He gas
Figure 2. Schematic diagram of bubbler setup (A), and dua(-amalystem (B), showing proper orientation of gold traps and soda lime
pretraps.
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Method 1631
6.5.2 Gold-coated sand trap—10-cm x 6.5-mm o.d.
x 4-mm i.d. quartz tubing. The tube is filled with 3.4 cm
of gold-coated 45/60 mesh quartz sand (Frontier Geo-
sclences Inc., Seattle, WA or equivalent). The ends are
plugged with quartz wool.
6.5.2.1 Traps are fitted with 6.5-mm i.d. fluoropolymer
friction-fit sleeves for making connection to the system.
When traps are not in use, fluoropolymer end plugs are
inserted in trap ends to preclude contamination.
6.5.2.2 At least six traps are needed for efficient opera-
tion: one as the "analytical" trap, and the others to
sequentially collect samples on.
6.5.3 Heating of gold-coated sand traps—To blank
traps and desorb Hg collected on the traps, heat for 3.0
mln to 450-500°C (a barely visible red glow when the
room is darkened) with a coil consisting of 75 cm of 24-
gauge Nichrome wire at a potential of 10 vac. Potential
is applied and finely adjusted with an autotransformer.
6.5.4 Tinners—The heating interval is controlled by a
timer-activated 120-V outlet (Gralabor equivalent), into
which the heating coil autotransformer is plugged. Two
timers are required, one each for the "sample" trap and
the "analytical" trap.
6.5.5 Air blowers—After heating, traps are cooled by
blowing air from a small squirrel-cage blower positioned
immediately above the trap. Two blowers are required,
one each for the "sample" trap and the "analytical" trap.
6.6 Recorder/integrator—Any integrator with a
range compatible with the CVAFS is acceptable.
6.7 Pipettors—All-plastic pneumatic fixed-volume
and variable pipettors in the range of 10 uL to 5.0 mL.
6.8 Analytical balance capable of weighing to the
nearest 0.01 g.
7.0 Reagents and Standards
7.1 Reagent water—Water in which mercury is not
detected by this method; 18-Mn ultrapure deionized
water starting from a prepurified (distilled, R.O., etc.)
source.
7.2 Air—It is very important that the laboratory air
be low in both paniculate and gaseous mercury. Ideally,
mercury work should be conducted in a new laboratory
with mercury-free paint on the walls. Outside air, which
is very low in Hg, should be brought directly into the
class 100 clean bench air intake. If this is impossible, air
coming into the clean bench can be cleaned for mercury
by placing a gold-coated cloth prefilter over the intake.
7.2.1 Gold-coated cloth filter: Soak 2 m2 of cotton
gauze in 500 mL of 2% gold chloride solution at pH 7. In
a hood, add 100 mL of 30% NH2OH-HCI solution, and
homogenize into the cloth with gloved hands. As colloi-
dal gold is precipitated, the material will turn black. Allow
the mixture to set for several hours, then rinse with
copious amounts of deionized water. Squeeze-dry the
rinsed cloth, and spread flat on newspapers to air-dry.
When dry, fold and place over the intake prefilter of your
laminar flow hood.
CAUTION: Great care should be taken to avoid
contaminating the laboratory with gold dust. This
could cause interferences with the analysis if gold
becomes incorporated into the samples or
, equipment. The gilding procedure should be done in
a remote laboratory if at all possible.
7.3 Hydrochloric acid—trace-metal purified reagent
HCI containing less than 5 pg/mL Hg.
7.4 Hydroxylamine hydrochloride—Dissolve 300
g of NH2OH-HCI in reagent water and bring to 1.0 L. This
solution may be purified by the addition of 1.0 mL of
SnCI2 solution and purging overnight at 500 mL/min with
Hg-free N2.
7.5 Stannous chloride—Bring 200 g of SnCI2-2H2O
and 100 mL concentrated HCI to 1.0 L with reagent
water. Purge overnight with mercury-free N2 at 500 mL/
min to remove all traces of Hg. Store tightly capped.
7.6 Bromine monochloride (BrCI)—Dissolve 27 g
of reagent grade KBr in 2.5 L of low-Hg HCI. Place a
clean magnetic stir bar in the bottle and stir for approxi-
mately 1 h in a fume hood. Slowly add 38 g reagent
grade KBrO3 to the acid with stirring. When all of the
KBrO3 has been added, the solution color should change
from yellow to red to orange. Loosely cap the bottle, and
allow to stir another hour before tightening the lid.
CAUTION: This process generates copious quantities
of free halogens (Cl, Br, BrCI), which are released
from the bottle. Add the KBrO3 SLOWLY in a fume
hood!
7.7 Stock mercury standard—NIST-certified
10,000-ppm aqueous Hg solution (NBS-3133). This so-
lution is stable at least until the NIST expiration date.
7.8 Secondary Hg standard—Dilute 0.100 mL of
the stock solution to 1.00 L of water containing 5 mL of
BrCI. This solution contains 1.00 jig/mL (1 -00 ppm) Hg.
Keep in a tightly closed fluoropolymer bottle. This solu-
tion is stable indefinitely.
7.9 Working Hg standard—Dilute 5.00 mL of the
secondary Hg standard to 1.00 L in a class A volumetric
G-10
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Method 1631
flask with reagent water containing 0.5% by volume BrCI
solution. This solution contains 5.0 ng/mL and should be
replaced monthly.
7.10 Calibration solutions—Using the secondary
Hg standard (Section 7.8), prepare five calibration solu-
tions to contain Hg at a concentration of 0.2,1.0,5,25,
and 100 ng/L in reagent water (Section 7.1).
7.11 Nitrogen—Grade 4.5 (standard laboratory
grade) nitrogen that has been further purified by the
removal of Hg using a gold-coated sand trap.
7.12 Argon—Grade 5.0 (ultra high-purity, GC grade)
inert gas that has been further purified by the removal of
Hg using a gold-coated sand trap.
8.0 Sample Collection, Preservation,
and Storage
8.1 Before samples are collected, consideration
should be given to the type of data required, (i.e.,
dissolved or total recoverable), so that appropriate pres-
ervation and pretreatment steps can be taken. The pH of
all aqueous samples must be tested immediately before
aliquotting for processing or direct analysis to ensure the
sample has been properly preserved. If properly acid-
preserved, the sample can be held up to 6 months
before analysis.
8.2 Samples are collected only into rigorously
cleaned fluoropolymer bottles with fluoropolymer or
fluoropoiymer-lined caps. It is critical that the bottles
have tightly sealing caps to avoid diffusion of atmo-
spheric Hg through the threads (Reference 4). Clean
bottles filled with high-purity 0.4% (v/v) HCI are dried,
capped, and double bagged in new zip-type bags in the
cleanroom, and stored in wooden or plastic boxes until
use.
8.3 Collect samples using the Sampling Method
(Reference 6). Procedures in the Sampling Method are
based on rigorous protocols for collection of samples for
mercury (References 4 and 11).
8.4 Sample filtration—For dissolved Hg, samples
and field blanks are filtered through a 0.45-fim capsule
filter at the field site. The Sampling Method describes
filtering procedures. For the determination of total recov-
erable Hg, samples are filtered before preservation,
8.5 Preservation—Samples may be preserved by
adding 5 mL/L of concentrated HCI (to allow both total
and methyl Hg determination) or 5 mL/L BrCI solution, if
total mercury only is to be determined. Acid- and BrCI-
preserved samples are stabile for a minimum of 6 months.
8.5.1 Samples may be shipped to the laboratory
unpreserved if they are (1) collected in fluoropolymer
bottles, (2) filled to the top with no head space, (3)
capped tightly, and (4) maintained at 0-4°C from the
time of collection until preservation. The samples must
be acid-preserved within 48 h after sampling.
8.5.2 Samples that are acid-preserved may lose Hg
to coagulated organic materials in the water or the Hg
may be condensed on the walls (Reference 12). Add
BrCI directly to the sample bottle at least 24 h before
analysis to prevent coagulation, condensation, or both.
Aliquots for determination of other Hg species must be
removed before BrCI is added. If BrCI cannot be added
directly to the sample bottle, the bottle should be vigor-
ously shaken before subsampling.
8.5.3 All handling of the samples in the laboratory
should be undertaken in a mercury-free clean bench,
after rinsing the outside of the bottles with reagent water
and drying in the clean air hood.
8.5.4 If preserved in the laboratory, preserve a blank
and OPR with each sample batch.
8.6 Storage—Sample bottles should be stored in
polyethylene bags at 0-4°C until analysis.
9.0 Quality Control
9.1 Each laboratory that uses this method is re-
quired to operate a formal quality assurance program
(Reference 13). The minimum requirements of this pro-
gram consist of an initial demonstration of laboratory
capability, ongoing analysis of standards and blanks as
a test of continued performance, and the analysis of
matrix spikes (MS) and matrix spike duplicates (MSD) to
assess accuracy and precision. Laboratory performance
is compared to established performance criteria to de-
termine that the results of analyses meet the perfor-
mance characteristics of the method.
9.1.1 The analyst shall make an initial demonstration
of the ability to generate acceptable accuracy and preci-
sion with this method. This ability is established as
described in'Section 9.2.
9.1.2 In recognition of advances that are occurring in
analytical technology, the analyst is permitted certain
options to improve results or lower the cost of measure-
ments. These options include automation of the dual-
amalgamation system, direct electronic data acquisition,
changes in the bubbler design (including substitution of
a flow-injection system) to maximize throughput, and
changes in the detector (i.e., CVAAS), where less sensi-
tivity is acceptable or desired. Changes in the principle
G-11
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Method 1631
of the determinative technique, such as the use of
colorimetry, are not allowed. If an analytical technique
other than the techniques specified in the method is
used, that technique must have a specificity equal to or
better than the specificity of the techniques in the method
for the analytes of interest.
9.1.2,1 Each time this method is modified, the analyst is
required to repeat the procedure in Section 9.2. If the
change will affect the detection limit of the method, the
laboratory is required to demonstrate that the MDL (40
CFR Part 136, Appendix B) is lower than one-third the
regulatory compliance level or lower than the MDL of
this method, whichever is higher. If the change will affect
calibration, the analyst must recalibrate the instrument
according to Section 10.
9.1.2.2 The laboratory is required to maintain records of
modifications made to this method. These records in-
clude the following, at a minimum:
9.1.2.2.1 The names, titles, addresses, and telephone
numbers of the analyst(s) who performed the analyses
and modification, and the quality control officer who
witnessed and will verify the analyses and modification.
'9.1.2.2.2 A narrative stating the reason(s) for the
modiflcation(s).
9.1.2.2.3 Results from all quality control (QC) tests
comparing the modified method to this method, includ-
ing the following:
(a) Calibration (Section 10)
(b) Calibration verification (Section 9.5)
(c) Initial precision and recovery (Section 9.2)
(d) Analysis of blanks (Section 9.4)
(e) Accuracy assessment (Section 9.3)
(f) Ongoing precision and recovery (Section 9.6)
9.1.2.2.4 Data that will allow an independent reviewer to
validate each determination by tracking the instrument
output to the final result. These data are to include the
following:
(a) Sample numbers and other identifiers
(b) Processing dates
(c) Analysis dates
(d) Analysis sequence/run chronology
(e) Sample weight or volume
(f) Copies of logbooks, chart recorder, or other raw
data output
(g) Calculations linking raw data to the results reported
9.1.3 Analyses of MS and MSD samples are required
to demonstrate the accuracy and precision and to moni-
tor matrix interferences. Section 9.3 describes the pro-
cedure and QC criteria for spiking.
9.1.4 Analyses of laboratory blanks are required to,
demonstrate acceptable levels of contamination. Sec-
tion 9.4 describes the procedures and criteria for analyz-
ing a blank.
9.1.5 The laboratory shall, on an ongoing basis, dem-
onstrate through analysis of the ongoing precision and
recovery (OPR) sample and the quality control sample
(QCS) that the system is in control. Sections 9.5 and 9.6
describe these procedures respectively.
9.1.6 The laboratory shall maintain records to define
the quality of the data that are generated. Sections 9.3.7
and 9.6.3 describe the development of accuracy state-
ments.
9.1.7 The determination of total Hg in water is con-
trolled by an analytical batch. An analytical batch is a set
of samples oxidized with the same batch of reagents,
and analyzed during the same 12-hour shift. A batch
may be from 1 to as many as 10 samples. Each batch
must be accompanied by at least three bubbler blanks
(Section 9.4), an OPR sample, and one MS and one
MSD. If more than 10 samples are run during one 12-
hour shift, an additional bubbler blank, OPR sample,
and MS/MSD must be analyzed for each additional 10 or
fewer additional samples. Reagent blanks for this deter-
mination are required when the batch of reagents (bro-
mine monochloride plus hydroxylamine hydrochloride)
are made, with verification in triplicate each month until
a new batch of reagents is needed.
9.2 Initial demonstration of laboratory
capability
9.2.1 Method detection limit—To establish the abil-
ity to detect Hg, the analyst shall determine the MDL
determined according to the procedure in 40 CFR 136,
Appendix B using the apparatus, reagents, and stan-
dards that will be used in the practice of this method.
The laboratory must produce an MDL that is less than or
equal to the MDL listed in Section 1.3 or one-third the
regulatory compliance limit, whichever is greater. The
MDL should be determined when a new operator begins
work or whenever, in the judgment of the analyst, a
change in instrument hardware or operating conditions
would dictate that the MDL be redetermined.
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Method 1631
9.2.2 Initial precision and recovery (IPR)—To es-
tablish the ability to generate acceptable precision and
accuracy, the analyst shall perform the following opera-
tions:
9.2.2.1 Analyze four replicates of the working Hg stan-
dard (Section 7.9) according to the procedure beginning
in Section 11.
9.2.2.2 Using the results of the set of four analyses,
compute the average percent recovery (X), and the
standard deviation of the percent recovery (s) for total
Hg.
9.2.2.3 Compare s and X with the corresponding limits
for initial precision and recovery in Table 1. If s and X
meet the acceptance criteria, system performance is
acceptable and analysis of samples may begin. If, how-
ever, s exceeds the precision limit or X falls outside the
acceptance range, system performance is unaccept-
able. Correct the problem and repeat the test (Section
9.2.2.1).
9.3.1,2 If the concentration of total Hg in a sample is not
being checked against a limit, the spike shall be at the
7,9) or at 1-5 times the background concentration,
whichever concentration is higher.
9.3.2 To determine the background concentration (B),
analyze 1 sample aliquot from each set of 10 samples
from each site or discharge according to the procedure
in Section 11. If the expected background concentration
is known from previous experience or other knowledge,
the spiking level may be established a priori.
9.3.2.1 If necessary, prepare a standard solution ap-
propriate to produce a level in the sample at the regula-
tory compliance limit or at 1-5 times the background
concentration (Section 9.3.1).
9.3.2.2 Spike two additional sample aliquots with the
spiking solution and analyze these aliquots to determine
the concentration after spiking (A).
9.3.3 Calculate the percent recovery (P) in each ali-
quot using the following equation:
Table 1. Acceptance Criteria For Performance Test
100
(A-B)
Acceptance Criterion
Section
Limits
Method Detection Limit
Initial Precision and Recovery
Precision (s)
Recovery (X)
Interlaboratory Intercomparison
Matrix Spike/Matrix Spike Duplicate
Recovery
Relative Percent Difference
Bubbler Blanks
Maximum
Mean
Ongoing Precision and Recovery
9.2.1
9.9.2
9.2.2.3
9.2.2.3
9.2.2.2
9.3
9.3.4
9.3.6
9.4
9.4.1.2
9.4.1.3
9.5
<0.2ng/L
±21%
79-121%
75-125%
75-125%
± 24%
<50pg
<25pg
77-123%
9.3 Matrix spike (MS) and matrix spike duplicate
(MSD)—To assess the performance of the method on a
given sample matrix, the laboratory must spike, in dupli-
cate, a minimum of 10% (1 sample in 10) from a given
sampling site or, if for compliance monitoring, from a
given discharge. Blanks (e.g., field blanks) may not be
used for MS/MSD analysis.
9.3.1 The concentration of the spike in the sample
shall be determined as follows:.
9.3.1.1 If, as in compliance monitoring, the concentra-
tion of total Hg in the sample is being checked against a
regulatory concentration limit, the spiking level shall be
at that limit or at 1-5 times higher than the background
concentration of the sample (determined in Section 9.3.2),
whichever concentration is higher.
where: ,
A = Measured Concentration of analyte after spiking
B = Measured concentration of analyte before spiking
C = True concentration of the spike
9.3.4 Compare the percent recovery (P) with the QC
acceptance criteria in Table 1.
9.3.4.1 If the results of spike fail the acceptance crite-
ria, and recovery for the OPR standard (Section 9.6) for
the analytical batch is within the acceptance criteria in
Table 1, an interference may be present. The result may
not be reported for regulatory compliance purposes. If
the interference can be attributed to sampling, the site
or discharge should be resampled. If the interference
can be attributed to a method deficiency, the analyst
must modify the method, repeat the test required in
Section 9.1.2, and repeat analysis of the sample and
MS/MSD. However, when this method was written,
there were no known interferences in the determination
of total Hg using this method. If such a result is ob-
served, the analyst should investigate it thoroughly.
9.3.4.2 If the results of both the spike and the OPR test
fail the acceptance criteria, the analytical system is
judged to be out of control. The analyst must identify
and correct the problem and reanalyze the sample
batch.
9.3.5 Relative percent difference between dupli-
cates—Compute the relative percent difference (RPD)
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Method 1631
between the MS and MSD according to the following
equation using the concentrations found in the MS and
MSD, Do not use the recoveries calculated in Section
9.3.3 for this calculation because the RPD is inflated
when the background concentration is near the spike
concentration.
RPD - 200*
D2)
where:
D1 « concentration of Hg in the MS sample
D2 « concentration of Hg in the MSD sample
9.3.6 The RPD for the MS/MSD pair shall meet the
acceptance criterion in Table 1. If the criterion is not met,
the system is judged to be out of control. The problem
must immediately be identified and corrected, and the
analytical batch reanalyzed.
9.3.7 As part of the QC program for the laboratory,
method precision and accuracy for samples should be
assessed and records maintained. After analyzing five
samples in which the recovery passes the test in Section
9.3.4, compute the average percent recovery (Pa) and
the standard deviation of the percent recovery (sj.
Express the accuracy assessment as a percent recov-
ery interval from P - 2sp to Pa+ 2sp. For example, if Pa =
90% and sp « 10% for five analyses, the accuracy
interval is expressed as 70-110%. Update the accuracy
assessment regularly (e.g., after every five to ten new
accuracy measurements).
9.4 Blanks—Blanks are critical to the reliable deter-
mination of Hg at low levels. The sections below give the
minimum requirements for analysis of blanks. However,
it is suggested that additional blanks be analyzed as
necessary to pinpoint sources of contamination in, and
external to, the laboratory.
9.4.1 Bubbler blanks—Bubbler blanks are analyzed
to demonstrate freedom from system contamination.
9.4.1.1 Immediately after analyzing a sample for total
Hg, place a clean gold trap on the bubbler, analyze the
sample a second time using the procedure in Section
11, and determine the amount of Hg remaining in the
system.
9.4.1.2 If the bubbler blank is found to contain more
than 50 pg Hg, the system is out of control. The problem
must be investigated and remedied, and the samples
run on that bubbler must be reanalyzed. The remedy for
a contaminated bubbler usually involves cleaning the
bubbler, changing the soda lime trap on the affected
bubbler, or both. If the blank from another bubbler
contains less than 50 pg Hg, the data associated with
that bubbler remain valid.
9.4.1.3 The mean result for all bubbler blanks (from
bubblers passing the specification in Section 9.4.2) in an
analytical batch (at least three bubbler blanks) is calcu-
lated at the end of the batch. The mean result must be <
25 pg with a standard deviation of < 10 pg for the batch
to be considered valid. If the mean is < 25 pg, the value
is subtracted from all raw data before results are calcu-
lated.
9.4.2 Reagent blanks—Since even reagent water
often contains measurable 'Hg, blanks must be deter-
mined on solutions of reagents by adding these re-
agents to previously purged reagent water in the bub-
bler.
9.4.2.1 Add aliquots of BrCl (0.5 ml), NH2OH (0.2 mL)
and SnCI2 (0.5 mL) individually to previously purged
reagent water in the bubbler.
9.4.2.2 The presence of more than 25 pg of Hg indi-
cates a problem with the reagent solution. The purging
of reagent solutions with mercury-free nitrogen or argon
can reduce Hg to acceptable levels.
9.4.3 Field blanks
9.4.3.1 Analyze the field blank(s) shipped with each set
of samples (samples collected from the same site at the
same time, to a maximum of 10 samples). Analyze the
blank immediately before analyzing the samples in the
batch.
9.4.3.2 If Hg or any potentially interfering substance is
found in the field blank at a concentration equal to or
greater than the ML (Table 1), or greater than one-fifth
the level in the associated sample, whichever is greater,
results for associated samples may be the result of
contamination and may not be reported for regulatory
compliance purposes.
9.4.3.3 Alternatively, if a sufficient number of field blanks
(three minimum) are analyzed to characterize the nature
of the field blank, the average concentration plus two
standard deviations must be less than the regulatory
compliance level or less than one-half the level in the
associated sample, whichever is greater.
9.4.3.4 If contamination of the field blanks and associ-
ated samples is known or suspected, the laboratory
should communicate this to the sampling team so that
the source of contamination can be identified and cor-
rective measures taken before the next sampling event.
9.4.4 Equipment blanks—Before any sampling equip-
ment is used at a given site, the laboratory or cleaning
facility is required to generate equipment blanks to
demonstrate that the sampling equipment is free from
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Method 1631
contamination. Two types of equipment blanks are re-
quired: bottle blanks and sampler check blanks.
9.4.4.1 Bottle blanks—After undergoing the cleaning
procedures in this method, bottles should be subjected
to conditions of use to verify the effectiveness of the
cleaning procedures. A representative set of sample
bottles should be filled with reagent water acidified to pH
< 2 and allowed to stand for a minimum of 24 h. Ideally,
the time that the bottles are allowed to stand should be
as close as possible to the actual time that the sample
will be in contact with the bottle. After standing, the
water should be analyzed for any signs of contamina-
tion. If any bottle shows signs of contamination, the
problem must be identified, the cleaning procedures
corrected or cleaning solutions changed, and all af-
fected bottles recleaned.
9.4.4.2 Sampler check blanks—Sampler check blanks
are generated in the laboratory or at the equipment
cleaning contractor's facility by processing reagent wa-
ter through the sampling devices using the same proce-
dures that are used in the field (see Sampling Method).
Therefore, the "clean hands/dirty hands" technique used
during field sampling should be followed when preparing
sampler check blanks at the laboratory or cleaning
facility.
9.4.4.2.1 Sampler check blanks are generated by filling
a large carboy or other container with reagent water
(Section 7.1) and processing the reagent water through
the equipment using the same procedures that are used
in the field (see Sampling Method). For example, manual
grab sampler check blanks are collected by directly
submerging a sample bottle into the water, filling the
bottle, and capping. Subsurface sampler check blanks
are collected by immersing the sampler into the water
and pumping water into a sample container.
9.4.4.2.2 The sampler check blank must be analyzed
using the procedures in this method. If any metal of
interest or any potentially interfering substance is de-
tected in the blank, the source of contamination or
interference must be identified, and the problem cor-
rected. The equipment must be demonstrated to be free
from the metal(s) of interest before the equipment may
be used in the field.
9.4.4.2.3 Sampler check blanks must be run on all
equipment that will be used in the field. If, for example,
samples are to be collected using both a grab sampling
device and a subsurface sampling device, a sampler
check blank must be run on both pieces of equipment.
9.5 Ongoing precision and recovery (OPR)—To
demonstrate that the analysis system is in control and
that acceptable precision and accuracy is being main-
tained within each analytical batch, the analyst shall
perform the following operations:
9.5.1 Analyze the low-level Hg working standard (Sec-
tion 7.9) and a bubbler blank before analysis of each
analytical batch according to the procedure beginning in
Section 11. Subtract the peak area of the bubbler blank
from the area for the standard and compute the concen-
tration for the blank-subtracted standard.
9.5.2 Compare the concentration with the limits for
ongoing precision and recovery in Table 1. If the con-
centration is in the range specified, the analysis system
is in control and analysis of samples and blanks may
proceed. If, however, the concentration is not in the
specified range, the analytical process is not in control.
Correct the problem and repeat the ongoing precision
and recovery test.
9.5.3 The laboratory should add results that pass the
specification in Section 9.5.2 to IPR and previous OPR
data and update QC charts to form a graphic represen-
tation of continued laboratory performance. The labora-
tory should also develop a statement of laboratory data
quality for each analyte by calculating the average per-
cent recovery (R) and the standard deviation of the
percent recovery (sr). Express the accuracy as a recov-
ery interval from R - 2sr to R + 2sr. For example, if R =
95% and sr = 5%, the accuracy is 85-105%.
9.6 Quality control sample (QCS)—It is suggested
that the laboratory obtain a QCS from a source different
from the Hg used to produce the standards used rou-
tinely in this method (Sections 7.7-7.10), and that the
QCS be analyzed periodically to verify the concentration
of these standards.
9.7 Depending on specific program requirements,
the laboratory may be required to analyze field dupli-
cates and field spikes collected to assess the precision
and accuracy of the sampling, sample transportation,
and storage techniques. The relative percent difference
(RPD) between field duplicates should be less than
20%. If the RPD of the field duplicates exceeds 20%, the
laboratory should communicate this to the sampling
team so that the source of error can be identified and
corrective measures taken before the next sampling
event.
10.0 Calibration and Standardization
10.1 Establish the operating conditions necessary to
purge Hg from the bubbler and to desorb Hg from the
trap in-a sharp peak. The system is calibrated using the
external standard technique as follows:
10.1.1 Initial calibration—Analyze each calibration
standard (Section 7.10) according to the procedure in
Section 11. After the analysis of each standard, analyze
a bubbler blank (Section 9.4.1) on the same bubbler
used for the standard. Subtract the peak area of the
bubbler blank from the area of each respective stan-
G-15
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Method 1631
dard. Tabulate the resulting peak area against the re-
spective concentration of each solution to form five
calibration factors. Calculate the relative standard devia-
tion (RSD) of the calibration factor over the five-point
range.
10.1.2 Linearity—If the calibration factor is constant (<
20% RSD) over the five-point calibration range, linearity
through the origin can be assumed and the average
calibration factor can be used; otherwise, a complete
calibration curve must be used over the five-point range.
10.2 Calibration verification and ongoing precision
and recovery—The ongoing precision and recovery stan-
dard (Section 9.5) is used to verify the working calibra-
tion curve or calibration factor at the beginning of each
12-hour working shift on which samples are analyzed.
11.0 Procedure
11.1 Sample Preparation
11.1.1 Pour a 100-mL aliquot from a thoroughly shaken,
acidified sample, into a 125-mL fluoropolymer bottle.
Add bromine monochloride (BrCI), cap the bottle, and
digest at room temperature for 12 hs minimum.
11.1.1.1 For clear water and filtered samples, add 0.5
ml of BrCI; for brown water and turbid samples, add 1.0
mi. of BrCI. If the yellow color disappears because of
consumption by organic matter or sulfides, more BrCI
should be added until a permanent (12-h) yellow color is
obtained.
11.1.1.2 Some highly organic matrices, such as sewage
effluent, will require high levels of BrCI (i.e., 5 mL/100
ml of sample), and longer oxidation times, or elevated
temperatures (i.e.; place sealed bottles in oven at 50°C
for 6 h). The oxidation always must be continued until a
permanent yellow color remains.
11.1.2 Matrix spikes and matrix spike duplicates—
For each 10 or fewer samples, pour two additional 100-
mL alfquots from a randomly selected sample, spike at
the level specified in Section 9.3, and process in the
same manner as the samples.
11.2 Hg reduction and purging—Place 100 mL of
reagent water in each bubbler, add 1.0 mL of SnCI2, and
purge with Hg-free N2 for 20 min at 300-400 mL/min.
11.2.1 Connect a gold/sand trap to the output of the
soda lime pretrap, and purge the water another 20 min
to obtain a bubbler blank. Discard the water in the
bubbler.
11.2.2 Add 0.2 mL of 30% NH2OH to the BrCI-oxidized
sample in the 125-mL fluoropolymer bottle. Cap the
bottle and swirl the sample. The yellow color will disap-
pear, indicating the destruction of the BrCI. Allow the
sample to react for 5 min with periodic swirling to be sure
that no traces of halogens remain.
NOTE: Purging of halogens onto the gold trap will result
in damage and low or irreproducible results.
11.2.3 Connect a fresh trap to the bubbler, pour the
reduced sample into the bubbler, add 0.5 mL of 20%
SnCI2 solution, and.purge the sample with N2 for 20 min.
11.3 Desorption of Hg from the gold trap
11.3.1 Remove the gold (sample) trap from the bub-
bler, place the Nichrome wire coil around the sample
trap and connect the sample trap into the analyzer train
between the incoming Hg-free argon and the second
gold-coated (analytical) sand trap (Figure 1a).
11.3.2 Pass argon through the sample and analytical
traps at a flow rate of approximately 30 mL/min for
approximately 2 min to drive off condensed water vapor.
11.3.3 Apply electrical current to the coil around the
sample trap for 3 minutes to thermally desorb the mer-
cury (as Hg°) from the sample trap onto the analytical
gold trap.
11.3.4 After the 3-min desorption time, turn off the
current to the Nichrome coil, and cool the sample trap
using the cooling fan.
11.3.5 Apply electrical current to the Nichrome wire coil
around the analytical trap and begin data collection.
Heat the analytical trap for 3 min or for 1 min beyond the
point at which the peak returns to baseline, whichever is
greater.
11.3.6 Stop data collection, turn off the current to the
Nichrome coil, and cool the analytical trap to room
temperature using the cooling fan.
11.3.7 Place the next sample trap in line and proceed
with analysis of the next sample.
NOTE: The analytical trap must be at or near room
temperature when the sample trap is heated;
otherwise, Hg° may be lost by passing through the
analytical trap.
11.4 Peaks generated using this technique should be
very sharp and almost symmetrical. Mercury elutes at
approximately 1 min and has a width at half-height of
about 5 seconds.
11.4.1 Broad or asymmetrical peaks indicate a prob-
lem with the desorption train, such as low gas flow rate,
water vapor on the trap(s), or an analytical column
damaged by chemical fumes or overheating.
G-16
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Method 1631
11,4.2 Damage to an analytical trap is also indicated
by a sharp peak, followed by a small, broad peak.
11.4.3 If the analytical trap has been damaged, the
trap and the fluoropolymer tubing should be discarded
because of the possibility of gold migration onto down-
stream surfaces of the instrument tubing and analytical
system.
11.4.4 Gold-coated sand traps should be tracked by
unique identifiers so that any trap producing poor results
can be quickly recognized and discarded,
12.0 Data Analysis and Calculations
12.1 Subtract the peak area of the mean of a mini-
mum of three bubbler blanks (Section 9.4.1.3) from the
peak area of each sample. ,
12.2 Using the blank-subtracted area, calculate the
concentration of Hg in each sample directly from the
mean calibration factor if a linear calibration is used, or
from the calibration curve if the calibration factor does
not meet the criterion in Section 10.1.2.
12.3 Reporting—Report results for samples in ng/L
to three significant figures for total Hg found above the
ML (Section 1.3). Report results below the ML as < 0.2
ng/L, or as required by the permitting authority or in the
permit.
13.0 Method Performance
The data in Table 2 gives an example of the perfor-
mance of the method under actual operating conditions
by several different analysts over a period of 1 year. In
addition to such data, this methodology has been
intercompared with other techniques for low-level mer-
cury determination in water under a variety of studies,
including ICES-5 (Reference 14) and the International
Mercury Speciation Intercomparison Exercise (Refer-
ence 15).
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of
waste at the point of.gerieration. Many opportunities for
pollution prevention exist in laboratory operation. EPA
has established a preferred hierarchy of environmental
management techniques that places pollution preven-
tion as the management option of first choice. Whenever
feasible, laboratory personnel should use pollution pre-
vention techniques to address their waste generation.
When wastes cannot be feasibly reduced at the source,
the Agency recommends recycling as the next best
option. The acids used in this method should be reused
as practicable by purifying by electrochemical tech-
niques. The only other chemicals used in this method
are the neat materials used in preparing standards.
These standards are used in extremely small amounts
and pose little threat to the environment when managed
properly. Standards should be prepared in volumes
consistent with laboratory use to minimize the disposal
of excess volumes of expired standards.
14.2 For information about pollution prevention that
may be applied to laboratories and research institutions,
consult Less is Better: Laboratory Chemical Manage-
ment for Waste Reduction, available from the American
Chemical Society's Department of Governmental Rela-
tions and Science Policy, 1155 16th Street NW, Wash-
ington DC 20036, 202/872^477,
15.0 Waste Management
15.1 The laboratory is responsible for complying with
all federal, state, and local regulations governing waste
management, particularly hazardous waste identifica-
tion rules and land disposal restrictions, and for protect-
ing the air, water, and land by minimizing and controlling
all releases from fume hoods and bench operations.
Compliance with all sewage discharge permits and regu-
lations is also required.
15.2 Acids, samples at pH < 2, and BrCI solutions
must be neutralized before being disposed of, or must
be handled as hazardous waste.
15.3 For further information on waste management,
consult The Waste Management Manual for Laboratory
Personnel and Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, both available from the
American Chemical Society's Department of Govern-
ment Relations and Science Policy, 1155 16th Street
NW, Washington, DC 20036.
16.0 References
1 Frontier Geosciences, Inc., Purchase Order 8762
from DynCorp Viar, Inc., August 22, 1994.
2 Fitzgerald, W.F.; Gill, G.A. "Sub-Nanogram
Determination of Mercury by Two-Stage Gold
Amalgamation and Gas Phase Detection Applied to
Atmospheric Analysis," Anal. Chem. 1979, 15,1714.
3 Bloom, N.S; Crecelius, E.A. "Determination of
Mercury in Sea water at Subnanogram per Liter
Levels," Mar. Chem. 1983, 14, 49.
4 Gill, G.A.; Fitzgerald, W.F. "Mercury Sampling of
Open Ocean Waters at the Picogram Level," Deep
Sea Res 1985, 32,287.
5 Bloom, N.S; Fitzgerald, W.F. "Determination of
Volatile Mercury Species at the Picogram Level by
Low-Temperature Gas Chromatography with Cold-
Vapor Atomic Fluorescence Detection," Anal. Chim.
Acta. 1988, 208, 151.
G-17
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Method 1631
Tablo 2. Typical QC Results for Routine Water Analysis (Frontier
Geosciences Inc., February-August 1993)
Parameter
Units
Mean
SD
Reagent Blanks
Matrix Spske Recoveries
Laboratory Duplicates
Field Duplicates
Intercomparison Exercise
ng/L
%
RPD
RPD
% Diff
0.14
99.6
4.9
13.3
0.0
0.04
6.3
6.6
14.6
13.3
36
60
49
33
18 labs
6 Method 1669, "Method for Sampling Ambient Water
for Determination of Metals at EPA Ambient Criteria
Levels," U.S. Environmental Protection Agency,
Office of Water, Office of Science and Technology,
Engineering and Analysis Division (4303), 401 M
Street SW, Washington, DC 20460, April 1995 with
January 1996 revisions.
7 "Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service.
Centers for Disease Control. NIOSH Publication 77-
206, Aug. 1977, NTIS PB-277256.
8 "OSHA Safety and Health Standards, General
Industry," OSHA 2206, 29 CFR1910.
9 "Safety in Academic Chemistry Laboratories," ACS
Committee on Chemical Safety, 1979.
10 "Standard Methods for the Examination of Water
and Wastewater," 18th ed. and later revisions,
American Public Health Association, 101515th Street
NW, Washington, DC 20005. 1-35: Section 1090
(Safety), 1992.
11 Bloom, N.S. Trace Metals & Ultra-Clean Sample
Handling," Environ. Lab. 1995, 7,20.
12 Bloom, N.S. "Influence of Analytical Conditions on
the Observed 'Reactive Mercury,' Concentrations in
Natural Fresh Waters." In Mercury as a Global
Pollutant; Huckabee, J. and Watras, C.J., Eds.;
Lewis Publishers, Ann Arbor, Ml: 1994.
13 "Handbook of Analytical Quality Control in Water
and Wastewater Laboratories," U.S. Environmental
Protection Agency. Environmental Monitoring
Systems Laboratory, Cincinnati, OH 45268, EPA-
600/4-79-019, March 1979.
14 Cossa, D.; Couran, P. "An International
Intercomparison Exercise for Total Mercury in Sea
Water," /4pp. Organomet. Chem. 1990, 4,49.
15 Bloom, N.S.; Horvat, M.; Watras, C.J. "Results of
the International Mercury Speciation Intercomparison
Exercise," Wat. Air. Soil Pollut, in press.
17.0 Glossary
The definitions and purposes below are specific to this
method, but have been conformed to common usage as
much as possible.
17.1 Ambient Water—Waters in the natural
environment (e.g., rivers, lakes, streams, and other
receiving waters), as opposed to effluent discharges.
17.2 Analytical Shift—All of the 12-hour period during
which analyses are performed. The period begins
with the purging of the OPR standard and ends
exactly 12 hours later. All analyses both started and
completed within this 12-hour period are valid.
17.3 Bubbler Blank—The process of analyzing water
in the bubbler, including purging Hg from the water,
trapping the Hg purged on a sample trap, desorbing
the Hg onto an analytical trap, desorbing the Hg
from the analytical trap, and determining the amount
of Hg present. The blank is somewhat different
between days, and the average of a minimum of the
results from three bubbler blanks must be subtracted
from all standards and samples before reporting the
results for these standards and samples.
17.4 Intercomparison Study—An exercise in which
samples are prepared and split by a reference
laboratory, then analyzed by one or more testing
laboratories and the reference laboratory. The
intercomparison, with a reputable laboratory as the
reference laboratory, serves as the best test of the
precision and accuracy of the analyses at natural
environmental levels.
17.5 Matrix Spike (MS) and Matrix Spike Duplicate
(MSD)—Aliquots of an environmental sample to
which known quantities of the analyte(s) of interest
is added in the laboratory. The MS and MSD are
analyzed exactly like a sample. Their purpose is to
quantify the bias and precision caused by the sample
matrix. The background concentrations of the
analytes in the sample matrix must be determined in
a separate aliquot and the measured values in the
MS and MSD corrected for these background
concentrations.
17.6 Must—This action, activity, or procedural step
is required.
17.7 Quality Control Sample (QCS)—A sample
containing Hg at known concentrations. The QCS is
obtained from a source external to the laboratory, or
is prepared from a source of standards different
from the source of calibration standards. It is used to
check laboratory performance with test materials
prepared external to the usual preparation process.
G-18
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Method 1631
17.8 Reagent Water—Water known not to contain
the analyte(s) of interest at the detection limit of this
method. For this method, the Hg level is made as
low as possible in mercury usually by double
deionization. The reagent water is used to wash
bottles and as trip and field blanks.
17.9 Should—This action, activity, or procedure is
suggested, but not required.
17.10 Stock Solution—A solution containing an
analyte that is prepared from a reference material
traceable to EPA, NIST, or a source that will attest to
the purity and authenticity of the reference material.
17.11 Ultraclean Handling—A series of established
procedures designed to ensure that samples are not
contaminated for Hg during sample collection,
storage, or analysis.
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Appendix H
RTI/6302/04
Standard Operating Procedures for the Operation and
Maintenance of a Trace Metal Cleanroom
Margaret M. Goldberg
Analytical and Chemical Sciences
Research Triangle Institute
P.O. Box 12194
Research Triangle Park, NC 27709
Contract Number: 68-C5-0011
Work Assignment Number: 04
October 16, 1995
-------
Operation and Maintenance of a Trace Metal Cleanroom
1.0 Scope and Application
1.1 This method is for the operation and maintenance
of a Trace Metal Cleanroom facility. The ultimate goal of
cleanroom utilization is to prevent contamination of
samples or products by airborne particulates.The method
may be used to establish and maintain the cleanliness of
a cleanroom facility in which samples are prepared and
analyzed for metals at EPA water quality criteria (WQC)
levels. It is also suited for analysis of other types of envi-
ronmental samples that require a high degree of protec-
tion from ambient air contamination.
1.2 Typically, airborne particulates have high metal
concentrations and are a major source of sample con-
tamination. In order to eliminate this problem, specially
designed trace metal cleanrooms are used. Cleanrooms
are rooms that have a high flow rate of purified air which
continuously blankets samples and materials in a clean
atmosphere.They are equipped with High Efficiency Par-
ttoulate Air (HEPA) filters and/or Ultra Low Penetration
Air (ULPA) filters that remove almost all particles from
the air. These cleanrooms are continually flushed with
purified air so that samples can be processed without
atmospheric contamination. Use of cleanrooms has en-
abled metal quantitation in the parts per trillion range for
some elements.
1.3 This method addresses minimization of contami-
nation of samples, reagents, and labware from metals
found in air, construction materials, laboratory appara-
tus, and sample and reagent containers, and from air-
borne metals generated by laboratory personnel or op-
erations.
1.4 This method is applicable to all metallic elements
since all are susceptible to atmospheric transport to some
extent. While most metallic elements are transported
through the atmosphere attached to particles, some ele-
ments (such as mercury) are transported in the vapor
phase. Different cleanroom operations that are required
as a result of differences in the chemical behavior of par-
ticle-borne and vapor phase metals are included.
1.5 The method is intended for analysis of metals at
WQC concentrations, which are typically in the parts-per-
trillion (ppt) to low parts-per-billion (ppb) range. It does
not apply to metals at high concentrations such as those
associated with discharges from industrial facilities, but
does apply to industries that have permits based on wa-
ter quality guidelines.
1.6 The method is based on the research and expe-
rience of analysts who determine metal concentrations
at "ultra-trace" concentrations using cleanroom laborato-
ries. Selected references are provided (1-4). Additional
information about ultraclean techniques may be found in
EPA technical guidance (Reference 5) and about
Cleanroom design, use, and maintenance in References
6 and 7.
1.7 This method includes procedures to be used to
maintain a trace metal cleanroom, and to prepare labware,
sampling supplies, and apparatus for analysis of metals
at WQC levels.
Other EPA methods and guidance documents that ad-
dress clean sampling and analysis techniques for trace
metals in ambient waters are listed in Table 1.
2.0 Summary of Method
2.1 This method presents restrictions for personnel
entrance to a cleanroom facility, requirements for
cleanroom garb, procedures for handling and transport-
ing materials into the cleanroom, labware cleaning refer-
ences, and procedures for maintaining and monitoring
cleanliness in the cleanroom facility.
2.2 Definitions of cleanroom terminology, descrip-
tions of cleanroom facilities, and minimum requirements
for cleanrooms are presented.
3.0 Definitions
3.1 A general definition of a cleanroom is a room
which has a gentle shower of highly filtered air for the
purposes of transporting airborne particulate contami-
nants away from sensitive samples or apparatus, and
maintaining a clean environment with low atmospheric
metal concentrations.
3.2 A cleanroom is defined in U.S. Federal Standard
209E as "a room in which the concentration of airborne
particles is controlled and which contains one or more
clean zones" (Reference 8).
H-2
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Table 1.
EPA
Method
Title
Document
Number
1631
1636
1637
1638
1639
1640
1669
Total Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor
Atomic Fluorescence Spectrometry
Determination ofHexavalent Chromium by Ion Chromatography
, Determination of Trace Elements in Ambient Waters by Chelation
Preconcentration and Graphite Furnace Atomic Absorption
Determination of Trace Elements irt Ambient Waters by
Inductively Coupled Plasma-Mass Spectrometry
Determination of Trace Elements in Ambient Waters by Stabilized
Temperature Graphite Furnace Atomic Absorption
Determination of Trace Elements in Ambient Waters by
On-Line Chelation Preconcentration and Inductively
Coupled Plasma-Mass Spectrometry
Sampling Ambient Water for Determination of Trace
Metals at EPA Water Quality Criteria Levels
Guidance on the Documentation and Evaluation of Trace Metals
Data Collected for Clean Water Act Compliance Monitoring
Guidance on Establishing Trace Metal Cleanrooms in
Existing Facilities
EPA821-R-95-027
EPA-821-R-95-029
EPA821-R-95-030
EPA821-R-95-031
EPA821-R-95-032
EPA821-R-95-033
EPA821-R-95-034
EPA821-B-95-002
EPA821-B-95-001
3.3 A clean zone is defined in U.S. Federal Standard
209E as "a defined space in which the concentration of
airborne particles is controlled to meet a specified air-
borne particulate cleanliness class" (Reference 8).
3.4 Particulate cleanliness classes are defined at a
maximum number of particles of a given size per unit
volume of air. For example, a class 100 cleanroom has a
maximum of 100 particles, 0.5 urn or larger, per cubic
foot of air. Table 1 lists maximum particle concentrations
permissible for different cleanliness class designations
(References).
3.5 High efficiency filters used to remove submicron
particles are typically High Efficiency Particulate Air
(HEPA) filters or Ultra Low Penetration Air (ULPA) filters.
3.6 HEPA filters have a minimum particle-collection
efficiency of 99.97% for 0.3 |xm particles (Reference 9),
3.7 ULPA filters have a minimum particle-collection
efficiency of 99.999% for particles in the size range of
0.1 to 0.2 jim (Reference 9).
3.8 Efficiency is defined as the ratio of the difference
in particle concentrations (upstream - downstream) to the
upstream particle concentration:
Efficiency (%)
U - D
U
xlOO
where
U = upstream particle concentration (particles/unit vol-
ume)
D = downstream particle concentration (particles/unit
volume).
This means that if the particle count upstream of a HEPA
filter is 100,000 particles per cubic foot (0.3 urn), then the
count downstream of the filter will be a maximum of 30
particles per cubic foot. Similarly for the ULPA filters, if
there are 100,000 particles per cubic foot (0.12 urn) up-
stream of the filters, then the count downstream will be a
maximum of 1 particle per cubic foot (Reference 10).
3.9 Laminar air flow is defined as the movement of
air in parallel sheets or columns which are separate from
adjacent sheets or columns of air. Air in one laminar flow
sheet does not mix or interact with air in any other lami-
nar flow sheet.
4.0 Contamination and Interferences
4.1 Sources of contamination for metals at WQC lev-
els include three primary routes:
1. contamination from airborne particles and metal-
containing vapors;
2. contamination from particles generated by person-
nel, clothing, equipment, or apparatus, and
3. improperly cleaned labware, sample containers, ap-
paratus, and reagents. A properly designed and
functioning cleanroom minimizes contamination
from airborne particles and vapors, but use of a
cleanroom does guarantee that samples will be
uncontaminated. The behaviors of personnel, pro-
cedures used to clean and store labware and ap-
paratus, and the purity of reagents are also very
important in providing contaminant-free analyses.
H-3
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4.2 Contamination of samples from elements asso-
ciated with airborne particles or vapors can be substan-
tially reduced in a cleanroom facility by high efficiency air
filtration combined with vapor phase sorption. Most par-
ticles and metal-containing vapors are removed from the
air, and the resulting clean, filtered air continuously bathes
samples and apparatus.
4.3 In a properly functioning cleanroom with sufficient
flow of clean, filtered air to maintain laminar flow condi-
tions, the major sources of contamination remaining are
personnel, labware, and reagents. Personnel must be
trained to understand the air flow patterns, cleanliness
regions and requirements, and material handling proce-
dures and requirements in addition to the analytical pro-
cedures they must use. Great care must be paid by per-
sonnel to avoid sources of contamination, to decontami-
nate materials before allowing them to enter the
cleanroom, and to continually maintain the clean status
of their environment. The importance of training and vigi-
lance should not be underestimated.
4.4 A compendium of standards, practices, and meth-
ods approved by the Institute of Environmental Sciences
for contamination control is provided in Reference 11 .This
compendium provides much useful information for
cleanrooms in general and for those used in the micro-
electronics industry in particular. It does not contain in-
formation specific to trace metal cleanrooms, but does
provide approaches that have been demonstrated to re-
duce particle contamination in cleanrooms.
5.0
5.1
Safety
Acid Baths
5.1.1 Acid baths are used in trace metal laboratories
for leaching labware.These baths may contain HNO3, HCI,
HBr, H?SO4, HF or mixtures of these acids at high con-
centrations and may be used at room temperature or el-
evated temperature. The baths should be constructed of
a material that is suited to the type of acid contained so
that it will not become brittle or leak after continued expo-
sure to the acid. Check chemical compatibility lists for
materials provided by the vendor. Additionally, acid baths
should have lids to contain fumes and be located in a
polypropylene exhaust cabinet or fume hood.
5.1.2 Care must be taken to avoid any acid spill when
removing the acid-leached labware from acid baths. In
the event of accidental spill, a proper cleanup procedure
should be followed.
5.1.3 Acid baths must be properly labeled to indicate
the type of acid(s) and the concentration of the acid(s).
5.2 Hot Plates
5.2.1 Most trace metal cleanrooms use hot plates to
heat acid baths and to regulate the digestion of samples.
The large amounts of acid used and acid vapors gener-
ated can create a safety hazard for electrical components,
such as hot plates.
5.2.2 Hot plates should be inspected regularly by labo-
ratory personnel to ensure the absence of corrosion or
other damage to electrical leads and connections. In some
commercially available hot plates, an acid-corroded elec-
trical lead can result in the hot plate cycling to its highest
setting, potentially leading to a fire. In others, corroded
electrical leads can result in electrical shock. Any defects
in a hot plate or controller should be corrected immedi-
ately and the unit replaced if necessary.
5.2.3 Hot plates in polypropylene hoods should never
be operated above a temperature of 400°C, as the heat
may damage the hood material. Excessive heat can lead
to a fire.
5.3 Handling Chemicals
5.3.1 When handling chemicals, guidelines given in the
material handling data sheets (MSDS) should be followed
at all times. MSDS sheets are available through the manu-
facturer.
5.3.2 Because the room air inside a cleanroom is re-
circulated (to increase particle removal efficiency), a safety
hazard could rapidly be created if toxic fumes were
present in the laboratory. It is therefore very important
that an exhausting fume hood be used for all volatile
chemicals and vapor-generating procedures.
5.3.3 All prepared solutions should be properly labeled
to reflect their contents (chemical name, matrix, solvent,
preparation date, storage conditions, preparer).
5.3.4 Wastes that are generated must be stored in
appropriate containers. Waste containers must be labeled
to indicate their contents and approximate concentration
whenever possible.
5.4 Safety Garments
5.4.1 Safety garments are required in the cleanroom
to protect personnel from exposure to concentrated ac-
ids and oxidizers used in sample digestion and from ex-
posure to sample materials which may be partly or totally
uncharacterized. Safety glasses, lab coat, appropriate
shoes, and gloves are required.
5.4.2 Additionally, lab personnel may use a face shield,
lab apron, respirator or other safety equipment for cer-
tain tasks.
5.4.3 Routine safety equipment must meet Class 100
standards of cleanliness before being brought into the
cleanroom. However, personnel safety is always the pri-
mary consideration and in some cases it may be neces-
sary to use non-cleanroom equipment or supplies.
6.0 Facility, Equipment, and Supplies
6.1 Ideally, the Cleanroom Facility will consist of a
suite of rooms, each provided with high purity laminar-
flow air of designated cleanliness classes, including a
laboratory and anteroom at class 100 or better. Minimally,
H-4
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it will consist of two rooms, an anteroom and a cleanroom
laboratory, both provided with high purity laminar-flow air
at class 100 or better.
6.2 Ideally, the cleanroom laboratory used for prepa-
ration of samples and apparatus will contain one or more
banks of HE PA or ULPA filters in the ceiling providing
vertical down-flow laminar air. Minimally, the cleanroom
will contain a series of clean zones in which HEPA or
ULPA-filtered air is supplied in laminar flow at a desig-
nated cleanliness class.
6.3 Particulate Cleanliness Class: Class 100 air is
the minimum particulate cleanliness class appropriate to
preparation of apparatus and samples for metal analysis
at WQC levels.
6.4 Fume hoods:
6.4.1 Non-metallic exhausting fume hoods are required
for all operations with acids. Suitable construction mate-
rials are polypropylene (preferred) or fiberglass (alter-
nate).
6.4,2 Class 100 (or better) HEPA-filtered non-metallic
exhausting fume hoods are required for acid digestion of
samples or other operations in which samples are present
in open containers and acid vapors are generated.
6.4.3 Operations using volatile organic compounds
should be performed in an exhaust hood with appropri-
ate solvent resistance. When a stainless steel hood is
required, the hood should be located in a cleanroom labo-
ratory where acids are not used.
6.4.4 Operations involving hot plates must be per-
formed within the thermal limitations of the fume hood
construction materials. Heat-tolerant polypropylene and
teflon inserts are commercially available for hot plate re-
gions in polypropylene fume hoods. Heat-tolerant ceramic
inserts and plates are also useful for cooling samples
while they continue to evolve corrosive fumes.
6.4.5 The pattern of air flow in HEPA-filtered fume
hoods is critically important. Clean, HEPA-filtered air
should flow downward in a laminar flow to protect samples
from contamination. All air inside the hood should be one-
pass air, vented to the outside of the building. If signifi-
cant acid vapors are generated in the hood, it should be
equipped with a water wash-down for the exhaust ple-
num or an acid scrubber at the outlet of the exhaust. Air
exhaust should be initiated at hand level at both the front
of the work surface and the back of the work surface in-
side the hood. In this way, samples are exposed to clean,
HEPA-filtered air only, and acid vapors are efficiently re-
moved.
6.5 Clean Benches
6.5.1 Clean benches are HEPA-filtered non-exhaust-
ing enclosures located within a laboratory. Alternately they
may be non-exhausting work surface regions within
HEPA-filtered rooms.
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6.5.2 The minimum cleanliness designation for a clean
bench suitable for metal analysis at WQC levels is class
100.
6.5.3 Vertical laminar flow of air is the preferred flow
pattern for all cleanroom operations, but in some cases,
horizontal flow may be adequate. However, the likelihood
of cross-contamination of samples or standards is much
higher with a horizontal laminar flow design than with a
vertical laminar flow design, and the analyst must be spe-
cifically trained to understand the limitations to the use of
a horizontal flow clean bench.
6.5.4 Construction materials must be able to resist
acids used in sample and standard preparation. In prac-
tice, polypropylene is typically the preferred material.
6.6 Anteroom:
6.6.1 The anteroom is a changing room for personnel
and equilibration room for materials entering the class
100 laboratory. The anteroom should be maintained at
class 100.
6.6.2 Cleanroom garb should be stored in the ante-
room so that personnel can dress in clean, non-shed-
ding jackets, head covers, foot covers, gloves, and if nec-
essary face masks, before entering the class 100 labora-
tory.
6.6.3 Cleanroom garb should never be allowed to en-
ter a region of dirtier air.
6.6.4 Vertical, down-flow, HEPA-filtered air should be
provided in the anteroom to wash garb, personnel, and
materials before admittance into the class 100 labora-
tory.
6.6.5 Storage and equilibration areas within the ante-
room should be designed to provide minimal disruption
to the laminar flow of air. Perforated shelves, hangers for
lab coats, clips for head covers, etc, should be made of
non-shedding, non-corroding material, such as plastic,
and arranged within the anteroom to permit continued
down-flow of air with minimal turbulence.
6.7 Construction Materials
6.7.1 A|l construction materials must be non-shedding,
non-corroding materials. Due to the quantity and concen-
tration of acids used in trace metal sample preparation
and analysis, the preferred material for most components
is plastic. Polypropylene wall laminates, flooring, benches,
hoods, and ceiling grids are commercially available.
Polypropylene, poly(vinylchloride), and teflon are suitable
for specific plumbing purposes. Seams and joints should
be heat-welded, to the extent possible, to ensure con-
tinuous seal without outgassing of solvents. Other plas-
tics may be suitable for specific components, but should
be tested for acid and solvent resistivity, thermal toler-
ance, outgassing, and metal leachability prior to use.
6.7.2 Epoxy paints may be used to provide a non-cor-
roding coating for traditional construction materials (such
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as metal, wood, cinder block, etc.) in the cieanroom.
However, use of epoxy paints is considered a temporary
solution to the problem of particle generation. Two warn-
ings are presented regarding the use of epoxy paints:
6.7.2.1 Epoxy paints dp not provide as permanent a bar-
rier to particle generation as do plastics. Acids will cor-
rode epoxy paints and the paints will degrade with age.
Surfaces coated with epoxy paints will need to be checked
frequently for signs of corrosion. At the first sign of corro-
sion, painted metal components (such as door hinges,
drawer pulls, etc.) must be removed from the lab and re-
placed or re-finished with fresh epoxy paint. Painted wall
surfaces and wooden components present less obvious
signs of the onset of degradation, but must be repainted
at a frequency that will prevent particle generation within
the laboratory.
6.7.2.2 Laboratories that analyze mercury or other va-
por-phase metal species must test the epoxy paints for
their metal concentration prior to use. The volatile metal
species will outgas and contaminate the laboratory if
present in the paints.
6.8 Additional information about cieanroom philoso-
phy, design, construction materials, components, etc. may
be found in References 1-8 and in guidance documents
and standards published through the Institute of Environ-
mental Sciences (References 9-12).
7.0 Reagents and Standards
7.1 Reagents and standards appropriate to the EPA
sampling and analysis method(s) followed and analyte(s)
determined should be used.This method does not specify
the analysis procedure, reagents, or standards.
8.0 Sample Collection, Preservation,
and Storage
8.1 Sample collection, preservation, and storage pro-
cedures specitied in the appropriate EPA method should
be followed. This method does not specify the types of
samples to be collected nor the preservation and stor-
age procedures to be followed.
9.0 Quality Control
9.1 Quality control procedures appropriate to the EPA
method(s) followed and analyte(s) determined should be
used.This method does not specify the analysis or qual-
ity control procedure(s).
10.0 Calibration and Standardization
10.1 Calibration and standardization procedures
specified in the appropriate EPA method should be fol-
lowed. This method does not specify the analytical in-
strumentation nor procedures to be used.
11.0 Procedures
11.1 Personnel Entry and Garb
11.1.1 One of the most significant sources of particles
and contamination in a cieanroom laboratory is person-
nel. Particles are transported on and generated by shoes,
clothing, hair, skin, and supplies. Thus one of the sim-
plest methods used to reduce airborne particle concen-
trations in the laboratory is to restrict entrance to the labo-
ratory to only those personnel who need to work there.
At the discretion of the laboratory manager, escorted visi-
tors who are trained in cieanroom protocols and appro-
priately garbed may be allowed entrance.
11.1.2 Cleanrooms are restricted access areas. Appro-
priate signs should be posted outside the rooms so that
unauthorized personnel do not enter.
11.1.3 Appropriate cieanroom garb must be worn by all
personnel in the facility. Cieanroom shoe covers, jackets,
hair covers, face masks, gloves, etc. are commercially
available through cieanroom vendors.
11.1.3.1 All personnel must either cover street shoes
with disposable cieanroom shoe covers or wear shoes
designated and reserved for cieanroom use only. If there
are multiple Cleanrooms within the facility at different
cleanliness designations, such as class 10,000 and class
100, then shoe covers should be changed between rooms
such that the cleanest covers are worn in the room with
the lowest class designation.
11.1.3.2 AH personnel must wear non-shedding,
cieanroom lab jackets or coveralls in a class 100 (or lower)
cieanroom or clean zone.
11.1.3.3 All personnel must wear disposable, non-
shedding, cieanroom head covers in a class 100 (or lower)
cieanroom or clean zone.
11.1.3.4 Clean, powder-free gloves must be worn in a
class 100 (or lower) cieanroom or clean zone.
11.1.3.5 Laboratories that analyze only mercury do not
have the same restrictions as those that analyze other
metals. Because mercury is primarily transported in the
vapor phase and not attached to airborne particles, it is
not necessary to wear all of the particle-barrier layers
described above. Nevertheless, cieanroom foot covers
and powder-free gloves should be worn in mercury
Cleanrooms. In addition, breath and saliva may contain
high concentrations of mercury (Reference 13), and it may
be prudent to wear a cieanroom face mask.
11.2 Materials Handling and Exchange
Procedures
11.2.1 In order to prepare and analyze samples in a
cieanroom environment, it is necessary to transport
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samples, reagents, supplies, etc. from a "non-clean" en-
vironment to the cleanroom environment. The transfer
should be accomplished in a series of decontamination
steps with each step moving the materials to a progres-
sively cleaner area.
11.2.2 Each area of the facility should contain supplies
appropriate to the decontamination to be performed in
that region.
11.2.3 A service room or receiving area should be used
to receive materials, remove outer shipping containers,
and remove any outside dust or contamination.
11.2.4 Supplies and apparatus that cannot be ad-
equately cleaned to allow them in a cleanroom, such as
cylinders of compressed gas, air driers, refrigerators, etc.,
should be stored in an outer room such as a service room.
Gas and water lines can be plumbed from the outer room
to the cleanroom, as appropriate.
11.2.5 A HEPA-filtered room or zone should be used to
remove intermediate layers of wrap such as the outer
bag of double-bagged samples or supplies. Water and
lint-free wipes should be available in this region to rinse
and dry materials, such as reagent bottles or other non-
cleanroom packaged supplies.
11.2.6 In addition to laboratory reagents and supplies,
ordinary materials brought into the cleanroom, such as
notebooks, pens, calculators, written protocols, etc.,
should be appropriately decontaminated in a HEPA-fil-
tered room or zone before they are brought into the
cleanroom. All materials in ambient air are contaminated
with atmospheric dust that adheres loosely to their sur-
face. The purpose of decontaminating ordinary materials
is to remove this dust so that it is not transported into the
cleanroom. In practice, it is usually best to leave a supply
of cleaned, ordinary materials in the cleanroom.
11.2.7 Materials that are purchased from cleanroom
suppliers with "Class 100 packaging" should be brought
into a class 100 zone, such as an anteroom to a class
100 laboratory, to be opened.
11.2.8 Cleanroom garb and supplies should be stored
in a HEPA-filtered anteroom or region where their clean-
liness can be maintained.
11.2.9 In order to prevent contamination of an ultra-clean
region or the supplies kept in that region, separate areas
should be used for "high level" reagents and standards,
i.e. those containing more than 1 part-per-million (ppm)
of metals, and "low level" reagents and standards, con-
taining less than 1 ppm of metals. In practice, this may be
accomplished by keeping the chemicals and associated
labware in separate rooms or by designating separate
areas within a single cleanroom for "high level" and "low
level" operations. In either case, separate labware should
be maintained. For example, flasks and pipets that are
used for high concentrations of metals should never be
used for solutions that contain low levels of metals. Sepa-
rate wash basins, acid leaching baths, and storage cabi-
nets should be used for high level and low level labware.
H-7
11.3 Procedures for Cleaning Labware
and Apparatus
11.3,1 For preparing labware and apparatus for analy-
sis of metals other than mercury, follow the cleaning pro-
cedures specified in EPA Method 1640: Determination of
Trace Elements in Ambient Waters by On-Line Chelation
Preconcentration and Inductively Coupled Plasma-Mass
Spectrometry, Section 11.0.
11.3.2 For preparing labware and apparatus for analy-
sis of mercury, follow the cleaning procedures specified
in EPA Method 1631: Mercury in Water by Oxidation,
Purge and Trap, and Cold Vapor Atomic Fluorescence
Spectrometry, Section 6.0.
11.4 Procedures for Maintaining
Cleanliness
11.4.1 All exposed bench surfaces and hood surfaces
in the cleanroom must be wiped daily with a lint-free
cleanroom wiper wetted with deionized water. This should
be performed prior to initiating work each day and again
as needed during the day to maintain clean work sur-
faces.
11.4.2 The cleanroom floor should be mopped with tap
water on the first working day of every week, or .more
often if needed. A suitable metal free detergent may be
used if necessary, but generally, clean detergent-free
water is preferred so that no residual, particle-generating
film is left. The purpose of mopping this floor is to remove
the small accumulation of dust particles or laboratory
debris, rather than to remove any significant amount of
dirt or mud. The mop used for this .purpose should be
reserved for cleanroom use only, and should be con-
structed of plastic and sponge with a minimum of metal
parts. It should be stored in a class 100 anteroom or
equivalent space between uses so that it remains suffi-
ciently clean for use.
11.4.3 Large adhesive mats kept at the entrance to
cleanroom areas must be replaced periodically. The fre-
quency of replacement depends on the traffic through
the area, but should be sufficient to trap dirt and lint from
the shoes of personnel. Frequent replacement of these
mats will minimize the transport of dirt into clean areas.
11.4.4 If HEPA or ULPA filters are functioning properly,
the pressure differential across the filter face should be
within a range specified by the manufacturer. If the pres-
sure drop is outside of the target range, the filter should
be replaced. Pressure differential is routinely monitored
using a manometer.
11.4.5 Prefilters used to trap large particles should be
examined and changed periodically to ensure that the
relatively expensive HEPA and ULPA filters do not be-
come clogged prematurely.
11.4.6 Acid baths used to clean labware must be
changed periodically to ensure that the concentration of
metals is sufficiently low. It is recommended that the baths
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be changed when any of the analytes of interest reach a
concentration greater than 1 ppm or in the event of known
contamination. For routine use, only clean plastic tongs
should be inserted into an acid bath to retrieve labware.
11.4.7 All laboratory operations performed can poten-
tially impact the cleanliness of the facility by generating
or transporting particles and/or vapors, or by contami-
nating labware or acid baths. The location for each labo-
ratory activity should be selected carefully so that the
cleanliness of samples, labware, and apparatus is main-
tained and so that the area does not become contami-
nated or hazardous as a result of the operation.
1.5 Procedures for Monitoring Cleanli-
ness
11.5.1 Monitoring the cleanliness of the cleanroom fa-
cility requires measurement of two sets of parameters.
The first set are physical parameters that indicate if the
air handling system is functioning properly. It includes
measurement of temperature, pressure, relative humid-
ity, and the alarm status of various fans, motors, and
smoke detectors. The second set are chemical param-
eters that indicate if the cleanroom is sufficiently clean in
selected anaJytes to enable contaminant-free analyses.
It includes measurement of chemical concentrations in
acid baths and traps for settled particles.
11.5.2 Initial certification of the cleanliness class of a
cleanroom facility should be performed using standard
cteanroom tests specified by the Institute of Environmen-
tal Sciences (Reference 12). Independent testing agen-
cies have appropriate equipment and experience to con-
duct these tests and certify the cleanliness class. Certifi-
cation should be repeated periodically; annual re-certifi-
cation is recommended.
11.5.3 After the initial certification, proper functioning of
the air handling system should be ascertained at least
once daily.
11.5.3.1 In cleanrooms or clean zones equipped with a
manometer on each HEPA or ULPA filter, the pressure
differential should be noted for each filter. If the pressure
drop is not within the target range, the filter should be
replaced.
11.5.3.2 Because air moves from regions of higher pres-
sure to regions of lower pressure, airborne contaminants
will be transported along pressure differentials such that
the cleanest regions will be associated with the highest
pressure, and the dirtiest regions will be associated with
the lowest (ambient) pressure. Proper air flow in a
cleanroom facility can thus easily be monitored by mea-
suring static pressure in each room of the facility. The
cleanest region (lowest class designation) should have
the highest static pressure, and each progressively "dirtier"
region should have a correspondingly lower static pres-
sure. Using this method, the values of static pressure
measured in each room will depend on atmospheric con-
ditions, but the pressure trends should be maintained.
11.5.3.3 .The temperature and relative humidity in the
cleanroom facility may be monitored to ensure that the
air handling system is functioning properly, but unless
extreme conditions are observed indicating HVAC break-
down, they should not have a significant impact on the
cleanliness of the facility.
11.5.3.4 Particle concentration measurements should
be made annually and compared to those obtained dur-
ing the initial certification of the facility. High particle counts
in any region of the facility are indicative of either local-
ized particle generation or failure of the HEPA or ULPA
filter(s) in that region. In either case, corrective actions
should be taken to locate and remove the source'of the
particles.
11.5.4 In addition to the physical parameters used to
monitor the cleanliness of the facility, periodic measure-
ments should be made to determine the background con-
centration of specific metals within the laboratory. The list
of metals monitored should be selected according to the
analysis needs and schedule of the laboratory.
11.5.4.1 Sampling of facility background should be per-
formed by placing four clean teflon beakers, two open
(samples) and two covered (blanks), at each sampling
location within the cleanroom facility.
11.5.4.2 The sampling locations should be selected to
correspond to regions where sample preparation activi-
ties typically occur.
11.5.4.3 Sampling should be performed every six
months or more frequently.
11.5.4.4 A small amount of highest purity acid is added
to each beaker and warmed to dissolve any settled par-
ticles. The solution should then be diluted and analyzed
using the method most appropriate for the metals being
monitored.
11.5.4.5 Maximum acceptable background limits are
typically 0.1 ng cnrr2 day1. After a series of measurements
are made in a cleanroom facility, a more appropriate limit
value should be substituted for each element of interest.
12.0 Data Analysis and Calculations
12.1 Data analysis and calculation methods appropri-
ate to each metal determined and analysis method em-
ployed should be followed. Consult the appropriate EPA
method for specific guidance.
13.0 Method Performance
13.1 Consult the appropriate EPA method for an evalu-
ation of the performance of the analytical method.
14.0 Pollution Prevention
14.1 Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Many opportunities for pollu-
H-8
-------
tion prevention exist in laboratory operation. The EPA has
established a preferred hierarchy of environmental man-
agement techniques that places pollution prevention as
the management option of first choice. Whenever feasible,
laboratory personnel should use pollution prevention tech-
niques to address their waste generation. When wastes
cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option. The acids
used in this method should be reused as practicable by
purifying by electrochemical techniques. The only other
chemicals used in this method are the metal standards.
These standards are used in extremely smalt amounts
and pose little threat to the environment when managed
properly. Standards should be prepared in volumes con-
sistent with laboratory use to minimize the volume of ex-
pired standards to be disposed.
14.2 For information about pollution prevention that
may be applicable to laboratories and research institu-
tions, consult Less is Better: Laboratory Chemical Man-
agement for Waste Reduction, available from the Ameri-
can Chemical Society's Department of Government Re-
lations and Science Policy, 1155 16th Street NW, Wash-
ington DC 20036, 202/872-4477.
15.0 Waste Management
15.1 The Environmental Protection Agency requires
that laboratory waste management practices be con-
ducted consistent with all applicable rules and regula-
tions. The Agency urges laboratories to protect the air,
water, and land by minimizing and controlling all releases
from hoods and bench operations, complying with the
letter and spirit of any sewer discharge permits and regu-
lations, and by complying with all solid and hazardous
waste regulations, particularly the hazardous waste iden-
tification rules and land disposal restrictions. For further
information on waste management consult The Waste
Management Manual for Laboratory Personnel, available
from the American Chemical Society at the address listed
in Section 14.2.
16.0 References
1. Patterson, C.C.; Settle, D.M. "Accuracy in Trace Analy-
sis"; In National Bureau of Standards Special Publi-
cation 442; LaFleur, P.O., Ed., U.S. Government Print-
ing Office, Washington, D.C., 1976.
4.
5.
6.
7.
2. Moody, J.R. "NBS Clean Laboratories for Trace Ele-
ment Analysis," Anal. Chem. 1982, 54,1358A.
3. Zief, M.; Mitchell, J.W. "Contamination Control in Trace
Metals Analysis"; In Chemical Analysis 1976, Vol. 47,
Chapter 6.
Nriagu, J.O.; Larson, G.; Wong, H.K.T.; Azcue, J.M.
"A Protocol for Minimizing Contamination in the Analy-
sis of Trace Metals in Great Lakes Waters," J. Great
Lakes Research 1993,19,175.
Prothro, Martha G., "Office of Water Policy and Tech-
nical Guidance on Interpretation and Implementation
of Aquatic Life Metals Criteria," EPA Memorandum to
Regional Water Management and Environmental
Services Division Directors, Oct. 1,1993.
Guidance on Establishing Trace Metal Cleanrooms
in Existing Facilities; U.S. Environmental Protection
Agency. Office of Water; Engineering and Analysis
Division; Washington, DC. EPA 821-B-95-001, April
1995.
Goldberg, Margaret M., Trace Metal Cleanroom, Re-
port to EPA on Contract 68-C5-0011, Work Assign-
ment Number 4,1995.
8. Federal Standard 209E: "Airborne Paniculate Clean-
liness Classes in Cleanrooms and Clean Zones,"
FED-STD-209E, U.S. Government Printing Office:
Washington, D.C. 1992.
9. Institute of Environmental Sciences. IES-RP-
CC001.3: HERA and ULPA Filters. Mount Pros-
pect, IL, 1993, 22pp.
10. Institute of Environmental Sciences. IES-CC-011 -85-
T: A Glossary of Terms and Definitions Related to
Contamination Control. Mount Prospect, IL, 1985,
16pp.
11. Institute of Environmental Sciences. IES-RD-
CC009.2: Compendium of Standards, Practices,
Methods, and Similar Documents Relating to Con-
tamination Control. Mount Prospect, IL, 1993,70 pp.
12. Institute of Environmental Sciences. IES-RP-CC-006-
84-T:Testing Cleanrooms. Mount Prospect, IL, 1984,
16pp.
13. Araronsson, A.M.; Lind, B.; Nylander, M.; and
Nordberg, M. Biology of Metals 1989, 2, 25-30.
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